Volume 9 (2011)
  Special Issue II

International Scientific Conference
Food/Feed Quality, Safety and Risks in Agriculture (FLAVOURE Conference)
Tallinn, Estonia, 25 – 28 October, 2011

Conference and volume information – PDF (129 K)

Contents


Pages

383-388 S. Cerina, V. Krastina and I. Vitina
Production and Expenses of Enriched Composition Broiler Chicken Meat in Latvia
Abstract |

Production and Expenses of Enriched Composition Broiler Chicken Meat in Latvia

S. Cerina, V. Krastina and I. Vitina

Research Institute of Biotechnology and Veterinary Medicine “Sigra” of Latvia University of Agriculture, Institūta 1, Sigulda, Latvia; LV–2150, e–mail: sigra@lis.lv

Abstract:

Enriched composition of broiler chicken meat, in comparison with commercial mass production, contains increased levels of ω–6 and ω–3 fatty acids and carotenoids complex, which positively influence human health and prevent risk factors that cause various diseases. The aim of the investigations was to evaluate the possibility of obtaining an enriched composition broiler chicken meat and to evaluate the expenses of production in bio-economic aspects by using vegetable oils that contain an increased amount of ω–6 and ω–3 fatty acids level and an additive of carotenoids complex “Karotinas V”. The feeding trial was carried out with cross ROSS 308 broiler chickens ranging in age from 1–42 days (n  = 300). It was concluded that the combination of oils in broiler chicken feed for producing enriched composition meat is 1.0% flax seed, 1.0% rapeseed and 2.0% soybean oils and 0.1% carotenoids complex. Use of the composition resulted in broiler chicken meat with 27.4% ω–6 and with 8.3% ω–3 fatty acids in total lipids, which is about 3.9% and 3.2 % higher than in the commercial product. Poultry organism metabolic processes are essential factors that determine the carryover levels of fatty acids and carotenoids from feed to meat, and it is impossible to precisely evaluate and calculate these physiological processes in organisms, economically. In the trial, the expense of feed consumption per 1,000 broiler chickens was higher than by using commercial feed but increased the broiler chickens’ live weight, providing a possible 15% increase in total sales revenues for 1,000 broiler chickens. The tested combination of oils resulted in increased levels of ω–6 and ω–3 fatty acids in broiler chickens’ tissue: as a result, income was higher in the experimental group.

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389-394 I. Jansons, A. Jemeljanovs, I. H. Konosonoka, V. Sterna, B. Lujane
The Influence of Organic Acid Additive, Phytoadditive and Complex of Organic Acid Additive Phytoadditive on Pig Productivity, Meat Quality
Abstract |

The Influence of Organic Acid Additive, Phytoadditive and Complex of Organic Acid Additive Phytoadditive on Pig Productivity, Meat Quality

I. Jansons, A. Jemeljanovs, I. H. Konosonoka, V. Sterna, B. Lujane

Research Institute of Biotechnology and Veterinary Medicine “Sigra” of Latvia University of Agriculture, Instituta 1, Sigulda, Latvia, LV-2150; e-mail: sigra@lis.lv

Abstract:

A study was conducted to determine the efficiency of organic acids, phytoadditives and an organic acids and phytoadditive complex on pigs' growth processes and meat quality. Control group pigs (group 1) were fed with a complete ration (basic feed); the trial group pigs additionally received an organic acid additive (group 2), a phytoadditive (group 3), an organic acids and phytoadditive complex (group 4). The highest impact of 12% on the live weight gain of pigs was exercised by inclusion of the newly developed phytoadditive in the feed ration compared with the control group. The feed conversion ratio for pigs having received organic acid additives was by 4.2% higher, for animals having received the phytoadditive – by 8.1% and for animals having received a complex of both – by 7.45% higher than for the control group pigs where feed consumption was 3.09. The phytoadditive and the organic acids and phytoadditive complex as a pig feed supplement ensures a higher protein quality in muscle tissue, i.e., a higher nutritive value. The highest impact on the cholesterol level reduction in muscle tissue was exercised by the phytoadditive by 51.1 mg kg−1 in comparison with the control group.

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395-401 K. Kaseleht , T. Paalme and I. Nisamedtinov
Quantitative Analysis of Acetaldehyde in Foods Consumed by Children using SPME/GC-MS(Tof), On-fiber Derivatization and Deuterated Acetaldehyde as an Internal Standard
Abstract |

Quantitative Analysis of Acetaldehyde in Foods Consumed by Children using SPME/GC-MS(Tof), On-fiber Derivatization and Deuterated Acetaldehyde as an Internal Standard

K. Kaseleht¹ ², T. Paalme¹ ² and I. Nisamedtinov¹ ²

¹Department of Food Processing, Tallinn University of Technology, Ehitajate tee 5, EE12618 Tallinn, Estonia; e-mail: kristel.kaseleht@mail.ee, tpaalme@staff.ttu.ee
²Competence Center of Food and Fermentation Technologies, Akadeemia tee 15b, EE12618, Tallinn, Estonia; e-mail: inisamedtinov@lallemand.com

Abstract:

The aim of this study was to develop a precise quantitative method for acetaldehyde determination in solid food matrixes as, to the authors’ best knowledge, no such method was available. The method was applied for quantification of acetaldehyde in various foods consumed by children such as yoghurt, purees, curd creams etc. On-fiber derivatization of acetaldehyde with PFBHA was used to increase the method sensitivity and deuterated acetaldehyde was used as an internal standard for exact quantification. The article is mostly focused on method development, including sample preparation. The amount of acetaldehyde in foods was found to be rather negligible, with the highest concentration (up to 31.5 ± 0.05 mg – kg 1) detected in yoghurts.

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403-408 T. Klesment , J. Stekolštšikova and K. Laos
The Influence of Hydrocolloids on Storage Quality of 10% Dairy Fat Ice Cream
Abstract |

The Influence of Hydrocolloids on Storage Quality of 10% Dairy Fat Ice Cream

T. Klesment¹ ², J. Stekolštšikova¹ and K. Laos¹ ²

¹Competence Center of Food and Fermentation Technology, Akadeemia tee 15B, 12618, Tallinn, Estonia; e-mail: tiina.klesment@gmail.com, jelena.lillo@gmail.com
²Tallinn University of Technology, Ehitajate tee 5, 80035, Tallinn, Estonia; e-mail: katrin@tftak.eu

Abstract:

In the present study, texture and flavour attributes were used to evaluate hydrocolloids (guar gum, carrageenan, xanthan gum and locust bean gum) and their blends on the crystallization of ice cream during a 13 month storage period. Only certain stabilizers retard ice crystal growth. Guar gum and xanthan gum blends attained the better stabilizing effect, improving textural and taste quality.  Locust bean gum and carrageenan blends remarkably deteriorated ice cream shelf life.

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409-414 K. Koppel , E. Chambers IV, D. H. Chambers
Flavour and Acceptance of Estonian Cheeses
Abstract |

Flavour and Acceptance of Estonian Cheeses

K. Koppel¹ ², E. Chambers IV³, D. H. Chambers³

¹Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086, Tallinn, Estonia
²Competence Center of Food and Fermentation Technologies, Akadeemia tee 15B, 12618, Tallinn, Estonia, email: kadri@tftak.eu
³The Sensory Analysis Center, Kansas State University, Justin Hall, Manhattan, Kansas 66506-1407, email: eciv@ksu.edu, delores@ksu.edu

Abstract:

The flavour and acceptance of locally manufactured cheeses in Estonia were studied. The 36 cheeses, varying in texture, manufacturing technology, fat content, and additives, were described by 32 flavour attributes. Estonian cheese was described as milky and buttery, with sweet aromatics, occasionally with biting and butyric acid aromatics. The cheeses are usually not highly aged, and thus do not have dominant astringent or bitter sensations found in cheeses from other countries. Based on a cluster analysis of the flavour of the cheeses, four were chosen for an acceptance study. One hundred and eleven consumers in Estonia tested the four cheeses. Cluster analysis of the consumers’ liking scores indicated two clusters of consumers, one cluster preferring the younger cheeses and the second cluster preferring more aged cheeses. The study provides information concerning cheese flavour and preferences in an area of Eastern Europe which has been lacking in previous literature.

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415-420 M-L. Kütt, M. Malbe and J. Stagsted
Nanostructure-Assisted Laser Desorption/Ionization (NALDI) for Analysis of Peptides in Milk and Colostrum
Abstract |
Full text PDF (196 kB)

Nanostructure-Assisted Laser Desorption/Ionization (NALDI) for Analysis of Peptides in Milk and Colostrum

M-L. Kütt¹⋅², M. Malbe¹ and J. Stagsted³

¹Department of Agricultural Products, Estonian Research Institute of Agriculture,Teaduse 13, EE75501 Saku, Estonia; e-mail: maryliis.kytt@eria.ee; marge.malbe@eria.ee
²Department of Food Processing, Faculty of Chemical and Materials Technology,Tallinn University of Technology, Estonia
³ Department of Food Science, Aarhus University, Blichers Allé 20, Postboks 50D K-8830 Tjele, Denmark; e-mail: Jan.Stagsted@agrsci.dk

Abstract:

Several bioactive proteins have been identified in colostrum and milk. However there are needs for development of technologies to identify and purify low molecular weight (LMW) peptides with bioactivity. The most used method is Matrix Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS), but matrix suppression often prevents detection of LMW components. Our approach was to work out a suitable method for analysing small peptides in bovine milk and colostrum without extensive sample pre-treatment. Nanostructure-Assisted Laser Desorption/Ionization (NALDI) is a matrix-free method to identify such LMW components. We also made a comparison between MALDI and NALDI for detection of peptides from colostrum samples. Our results show that NALDI provides better intensity compared with MALDI. It allows us to sequence small peptides and to identify a fragment of β-casein from the colostrum sample. Further studies are needed for comprehensive identification and characterization of LMW bioactive peptides from colostrum and milk.

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421-426 O. Läänemets, A.-H. Viira and M. Nurmet
Price, Yield, and Revenue Risk in Wheat Production in Estonia
Abstract |

Price, Yield, and Revenue Risk in Wheat Production in Estonia

O. Läänemets¹, A.-H. Viira¹ and M. Nurmet¹ ²

¹Institute of Economics and Social Sciences, Estonian University of Life Sciences, Kreutzwaldi 1A, 51014 Tartu, Estonia; e-mails: ottlaanemets@gmail.com; ants.viira@emu.ee
²Faculty of Economics, University of Tartu; Narva Rd. 4, 51009 Tartu, Estonia; e-mail: nurmet@eau.ee

Abstract:

 In recent years, price risk has been increasingly acute for Estonian cereal growers due to increased volatility of commodity prices in the world market. Price risk is especially important due to long production cycle of the cereals. Inputs for growing wheat are bought months before the harvest, but the producers are unable to affect the output price. Price volatility and yield uncertainty increase income uncertainty. In the paper we analyse wheat price and yield variability and respective impact of these on sales revenue of wheat in Estonian conditions. The results show that the variability of yields and producer price of wheat are similar, while the variance of sales revenue of wheat per hectare indicates that production and price risk cumulate.

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427-432 K. Laos , E. Kirs , R. Pall and K. Martverk
The Crystallization Behaviour of Estonian Honeys
Abstract |
Full text PDF (100 kB)

The Crystallization Behaviour of Estonian Honeys

K. Laos¹ ², E. Kirs¹ ², R. Pall¹ and K. Martverk¹

¹Department of Food Processing, Tallinn University of Technology, Ehitajate tee 5, EE12086 Tallinn, Estonia; e-mails: katrin.laos@ttu.ee; evelinkirs@gmail.com; praili@hotmail.com; kaie.martverk@ttu.ee
²Competence Center of Food and Fermentation Technologies, Akadeemia tee 15B, EE12618 Tallinn, Estonia

Abstract:

The feasibility of water activity and viscosity measurement was studied to characterize the isothermal crystallization of Estonian honeys. In parallel, samples were observed by light microscopy. The most important phenomenon for crystallization is the fructose/glucose ratio in favour of glucose. The increase in water activity and viscosity was noticed during crystallization. Polarized light microscopy was more sensitive than water activity or viscosity for determining the crystallization time.

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433-438 A. M. Méndez, D. Castillo , A. del Pozo, I. Matus, R. Morcuende
Differences in Stem Soluble Carbohydrate Contents among Recombinant Chromosome Substitution Lines (RCSLs) of Barley under Drought in a Mediterranean–type Environment
Abstract |
Full text PDF (140 kB)

Differences in Stem Soluble Carbohydrate Contents among Recombinant Chromosome Substitution Lines (RCSLs) of Barley under Drought in a Mediterranean–type Environment

A. M. Méndez¹, D. Castillo² ³, A. del Pozo², I. Matus³, R. Morcuende¹

¹Institute of Natural Resources and Agrobiology of Salamanca, IRNASA–CSIC, Apartado 257, 37071 Salamanca, Spain; e–mail: rosa.morcuende@irnasa.csic.es
²Faculty of Agricultural Sciences, University of Talca, Casilla 747, Talca, Chile
³Instituto de Investigaciones Agropecuarias CRI-Quilamapu, Casilla 426, Chillán, Chile

Abstract:

Drought is one of the major abiotic stresses that dramatically threaten the global food supply and it is becoming an increasingly severe problem in many regions of the world, mainly in Mediterranean areas and/or climates. This study investigates the effect of drought on the stem soluble carbohydrate content and its role in grain filling in different barley genotypes –four recombinant chromosome substitution lines (RCSLs) and the recurrent parent cv. Harrington, which had been growing in two contrasting Mediterranean environments in central Chile. At anthesis, drought stress increased the stem glucose and fructose contents in lines 76 and 78 and fructans in all the genotypes. At maturity, in non-stressed plants the soluble carbohydrate content in the stem decreased, suggesting a mobilization of carbohydrates from the stem into the grain. Drought increased the stem content of fructose, sucrose and fructans in all genotypes. The accumulation of fructans was higher in RCSLs as compared to Harrington, providing evidence that the introgression of the wild ancestor (Hordeum vulgare ssp. spontaneum) into cv. Harrington increases the terminal drought tolerance of barley. Line 89 showed the maximal content of fructans and it could be considered as the most tolerant to terminal drought of all RCSLs. However, this genotype showed the lowest grain weight and yield, indicating that is the most susceptible line of those referred to as grain yield.

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439-442 J. Mičulis, A. Valdovska, V. Šterna and J. Zutis
Polycyclic Aromatic Hydrocarbons in Smoked Fish and Meat
Abstract |

Polycyclic Aromatic Hydrocarbons in Smoked Fish and Meat

J. Mičulis¹*, A. Valdovska², V. Šterna¹ and J. Zutis³

¹Research Institute of Biotechnology and Veterinary Medicine ”Sigra”, Latvia University of Agriculture, Instituta 1, LV–2150 Sigulda, Latvia; e–mail: sigra@lis.lv
²Faculty of Veterinary Medicine, Latvian University of Agriculture, K. Helmana 8, LV–3004 Jelgava, Latvia, e–mail: Anda.Valdovska@llu.lv
³Meat and Milk Industry Engineering Centre, Dzirnavu 42, LV–1010 Riga, Latvia; e- mail: gpric@snmail.lv

Abstract:

Polycyclic aromatic hydrocarbons (PAH`s) can significantly influence smoked meat quality and safety. Toxicological studies on individual PAHs in animals, mainly on the PAH benzo(a)pyrene, have shown various toxicological effects. One significant source of PAHs in the human food chain is the smoking of meat and fish. Smoke not only gives special taste, colour and aroma to food, but also enhances preservation due to the dehydrating, bactericidal and antioxidant properties of smoke. Therefore the aim of our investigation was to determine the contents of PAH4 (benzo(a)antracene, benzo(a)pyrene, benzo(b)fluorantene, chrysene) in a variety of industrially smoked meat and fish products. Results were summarized and compared with maximum acceptable levels set by Draft European Commission regulation (EC) planned to be in force beginning 1.9. 2012.

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443-450 unit of Rubisco (Drake et al., ), mediated by the increased level of carbohydrates in leaves under elevated CO or low nitrogen contents (Riviere-Rolland et al., ; Geiger et al., ; Pérez et al., 00).Nitrate reductase –NR– catalyses the first reaction in the pathway of nitrateassimilation and is regulated by a hierarchy of sophisticated mechanisms leading to changes in transcription, post-translational modification and protein turnover (Scheible et al., a). The expression of the enzyme is induced by nitrate and sugars (Krapp et al., ; Vincentz et al., ), and repressed by glutamine or closely related metabolites (Vincentz et al., ). Elevated CO leads to an accumulation of carbohydrates (Drake et al., ; Pérez et al., 00) and, as long as the rate of nitrate assimilation and amino acid synthesis are regulated in response to changes in the availability of carbohydrates, an increase of the enzyme activity and gene expression under elevated CO concentrations may be expected. However, there is no consistent evidence for an increase of NR activity in elevated CO. Although CO enrichment led to a small increase of NR activity (Fonseca et al., ; Geiger et al., ), sometimes it has also decreased (Ferrario-Mery et al., ; Geiger et al., ). The anomalous decline of NR activity could be explained because plants growing under such conditions might become nitrate limited, and nitrate is required for induction of gene expression for the enzyme (Crawford, ; Scheible et al., a). Alternatively, it could be a consequence of preferential assimilation of ammonium under elevated COconditions, resulting in formation of glutamine and repression and/or post–translational inactivation and degradation of NR (Scheible et al., a; Morcuende et al., ). Moreover, NR is subject to diurnal changes in the level of transcripts and activity (Scheible et al., a), and any change in the level of carbohydrates and nitrogen metabolites could play a role in its regulation (Scheible et al., a). It could be hypothesized that the increases of atmospheric CO concentration and temperature predicted with climate change could lead to changes in the pool of such metabolites and it is quite likely that the diurnal regulation of NR will be modified.The purpose of the present study was to assess whether NR activity and its diurnalchanges in flag leaves of wheat are affected by combined increases in atmospheric COand temperature and to ascertain whether nitrogen supply modifies these effects, as long as the response to enhanced CO depends on nitrogen availability. With this objective the NR activity and the amount of amino acids were determined. The leaf carbohydrate content previously reported by Pérez et al. (00) is also considered. The flag leaf at ear emergence was selected for this study as a stage when acclimation to elevated CO is more likely than in younger plants and the ear provides an active sink for assimilates. In order to approximate the natural environment of Mediterranean wheat crops, this experiment was conducted in the field under temperature gradient chambers.MATERIALS AND METHODSPlant cultivationThis field experiment was conducted in a clay-sand soil located at the farm of theCSIC Institute of Natural Resources and Agrobiology, in Salamanca, Spain (°N, 00 m above sea level). The climate corresponds to a Mediterranean type.
Nitrogen Modulates the Diurnal Regulation of Nitrate Reductase in Wheat Plants – Projections Towards Climate Change R. Morcuende, P. Pérez, R. Martínez-Carrasco and E. Gutiérrez Institute of Natural Resources and Agrobiology of Salamanca, IRNASA–CSIC, Apartado 257, 37071 Salamanca, Spain; e–mail: rosa.morcuende@irnasa.csic.es Abstract: This study investigates whether the diurnal regulation of nitrate reductase activity in the flag leaf of wheat is affected by combined increases of CO2 and temperature in the air and to ascertain whether the nitrogen supply modifies these effects. Spring wheat was grown at ambient (360 μmol mol−1) or elevated (700 μmol mol−1) CO2, under ambient and 4°C warmer temperatures, and with two levels of nitrogen supply in field temperature gradient chambers. At ear emergence, NR activity reaches a maximum in the early part of the light period and declines later in the light period and during the first part of the night. Although elevated CO2 did not increase NR activity, it led to a modification of the diurnal regulation. During the last part of the photoperiod the decline of the activity was faster in plants grown in ambient CO2, in which the accumulation of amino acids was higher. The maximum reached in the first hours of the light period in plants grown in elevated CO2 and nitrogen abundance was related to a higher accumulation of soluble carbohydrates. The dark inactivation of NR was prevented in plants grown in elevated CO2 with low nitrogen. Additionally, the higher decline of NR activation in plants grown with ample nitrogen supply and higher temperatures was related to the accumulation of amino acids. It is concluded that nitrogen plays a role in the activity and post-translational regulation of NR under the future climatic scenario. Key words: carbohydrates, diurnal rhythm, elevated CO2, nitrate reductase, nitrogen, temperature, wheat INTRODUCTION Due to anthropogenic activities the concentration of carbon dioxide in the atmosphere is increasing and is projected to double from its current level by the end of this century. As a consequence, the earth´s mean annual surface temperature is also rising and is predicted to increase as much as 1.5–4.5ºC within this century (Schneider, 2001). Both CO2 and temperature are key factors affecting photosynthesis, plant growth and development, and their predicted changes will have a significant impact on plant productivity. The atmospheric CO2 enrichment initially improves carbon fixation by plants. However, in the long term plants growing in elevated CO2 often show an acclimatory down-regulation of the photosynthetic capacity of leaves (Drake et al., 1997; Pérez et al., 2005; Martínez-Carrasco et al., 2005) characterized by a reduction in the amount and activity of ribulose–1,5–bisphosphate carboxylase –Rubisco– (Drake et al., 1997), which is often associated with decreased expression of genes encoding the small sub- 443
Abstract |

Nitrogen Modulates the Diurnal Regulation of Nitrate Reductase in Wheat Plants – Projections Towards Climate Change R. Morcuende, P. Pérez, R. Martínez-Carrasco and E. Gutiérrez Institute of Natural Resources and Agrobiology of Salamanca, IRNASA–CSIC, Apartado 257, 37071 Salamanca, Spain; e–mail: rosa.morcuende@irnasa.csic.es Abstract: This study investigates whether the diurnal regulation of nitrate reductase activity in the flag leaf of wheat is affected by combined increases of CO2 and temperature in the air and to ascertain whether the nitrogen supply modifies these effects. Spring wheat was grown at ambient (360 μmol mol−1) or elevated (700 μmol mol−1) CO2, under ambient and 4°C warmer temperatures, and with two levels of nitrogen supply in field temperature gradient chambers. At ear emergence, NR activity reaches a maximum in the early part of the light period and declines later in the light period and during the first part of the night. Although elevated CO2 did not increase NR activity, it led to a modification of the diurnal regulation. During the last part of the photoperiod the decline of the activity was faster in plants grown in ambient CO2, in which the accumulation of amino acids was higher. The maximum reached in the first hours of the light period in plants grown in elevated CO2 and nitrogen abundance was related to a higher accumulation of soluble carbohydrates. The dark inactivation of NR was prevented in plants grown in elevated CO2 with low nitrogen. Additionally, the higher decline of NR activation in plants grown with ample nitrogen supply and higher temperatures was related to the accumulation of amino acids. It is concluded that nitrogen plays a role in the activity and post-translational regulation of NR under the future climatic scenario. Key words: carbohydrates, diurnal rhythm, elevated CO2, nitrate reductase, nitrogen, temperature, wheat INTRODUCTION Due to anthropogenic activities the concentration of carbon dioxide in the atmosphere is increasing and is projected to double from its current level by the end of this century. As a consequence, the earth´s mean annual surface temperature is also rising and is predicted to increase as much as 1.5–4.5ºC within this century (Schneider, 2001). Both CO2 and temperature are key factors affecting photosynthesis, plant growth and development, and their predicted changes will have a significant impact on plant productivity. The atmospheric CO2 enrichment initially improves carbon fixation by plants. However, in the long term plants growing in elevated CO2 often show an acclimatory down-regulation of the photosynthetic capacity of leaves (Drake et al., 1997; Pérez et al., 2005; Martínez-Carrasco et al., 2005) characterized by a reduction in the amount and activity of ribulose–1,5–bisphosphate carboxylase –Rubisco– (Drake et al., 1997), which is often associated with decreased expression of genes encoding the small sub- 443

unit of Rubisco (Drake et al., ¹⁹⁹⁷), mediated by the increased level of carbohydrates in leaves under elevated CO² or low nitrogen contents (Riviere-Rolland et al., ¹⁹⁹⁶; Geiger et al., ¹⁹⁹⁹; Pérez et al., ²00⁵).Nitrate reductase –NR– catalyses the first reaction in the pathway of nitrateassimilation and is regulated by a hierarchy of sophisticated mechanisms leading to changes in transcription, post-translational modification and protein turnover (Scheible et al., ¹⁹⁹⁷a). The expression of the enzyme is induced by nitrate and sugars (Krapp et al., ¹⁹⁹³; Vincentz et al., ¹⁹⁹³), and repressed by glutamine or closely related metabolites (Vincentz et al., ¹⁹⁹³). Elevated CO² leads to an accumulation of carbohydrates (Drake et al., ¹⁹⁹⁷; Pérez et al., ²00⁵) and, as long as the rate of nitrate assimilation and amino acid synthesis are regulated in response to changes in the availability of carbohydrates, an increase of the enzyme activity and gene expression under elevated CO² concentrations may be expected. However, there is no consistent evidence for an increase of NR activity in elevated CO². Although CO² enrichment led to a small increase of NR activity (Fonseca et al., ¹⁹⁹⁷; Geiger et al., ¹⁹⁹⁸), sometimes it has also decreased (Ferrario-Mery et al., ¹⁹⁹⁷; Geiger et al., ¹⁹⁹⁹). The anomalous decline of NR activity could be explained because plants growing under such conditions might become nitrate limited, and nitrate is required for induction of gene expression for the enzyme (Crawford, ¹⁹⁹⁵; Scheible et al., ¹⁹⁹⁷a). Alternatively, it could be a consequence of preferential assimilation of ammonium under elevated CO²conditions, resulting in formation of glutamine and repression and/or post–translational inactivation and degradation of NR (Scheible et al., ¹⁹⁹⁷a; Morcuende et al., ¹⁹⁹⁸). Moreover, NR is subject to diurnal changes in the level of transcripts and activity (Scheible et al., ¹⁹⁹⁷a), and any change in the level of carbohydrates and nitrogen metabolites could play a role in its regulation (Scheible et al., ¹⁹⁹⁷a). It could be hypothesized that the increases of atmospheric CO² concentration and temperature predicted with climate change could lead to changes in the pool of such metabolites and it is quite likely that the diurnal regulation of NR will be modified.The purpose of the present study was to assess whether NR activity and its diurnalchanges in flag leaves of wheat are affected by combined increases in atmospheric CO²and temperature and to ascertain whether nitrogen supply modifies these effects, as long as the response to enhanced CO² depends on nitrogen availability. With this objective the NR activity and the amount of amino acids were determined. The leaf carbohydrate content previously reported by Pérez et al. (²00⁵) is also considered. The flag leaf at ear emergence was selected for this study as a stage when acclimation to elevated CO² is more likely than in younger plants and the ear provides an active sink for assimilates. In order to approximate the natural environment of Mediterranean wheat crops, this experiment was conducted in the field under temperature gradient chambers.MATERIALS AND METHODSPlant cultivationThis field experiment was conducted in a clay-sand soil located at the farm of theCSIC Institute of Natural Resources and Agrobiology, in Salamanca, Spain (⁴¹°N, ⁸00 m above sea level). The climate corresponds to a Mediterranean type.

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Abstract:

Spring wheat (Triticum aestivum L cv. Alcázar) was sown at a rate of 180 kg ha−1and 0.13 row spacing on 13 February. Before sowing, N (as NH4NO3), P and K fertilizers (80, 40 and 40 kg ha−1, respectively) were applied. The crop was watered weekly through a drip irrigation system providing amounts of water equivalent to the average rainfall in this area during the period of the experiment (198 mm between February and June).After seedling emergence, two temperature gradient chambers (Pérez et al., 2005),based on those described by Rawson et al. (1995), were mounted over the crop on 23 March. One chamber was kept at the ambient air CO2 concentration (360 μmol mol−1) and another at 700 μmol mol−1 (elevated CO2) by injecting pure CO2 at the two inlet fans during the light hours. The temperature difference between the extreme modules in a chamber was set at 4°C. Additional (40 kg ha−1) nitrogen was added to one of the longitudinal halves of each chamber 34 days after sowing, such that two levels of this nutrient (80 and 120 kg ha−1) were compared. The samplings were repeated in four consecutive sections within the two module halves.On day 3 after the beginning of ear emergence in the whole experiment (22 May)ear emergence was advanced about 3 days by warm temperatures – flag leaves (two per replicate) were harvested and immediately plunged into liquid nitrogen just before dawn, 4–6 h later, 1–2 h before dusk and 2–3 h into the dark period; light intensities were < 10, 1700, 100 and < 10 μmol m−2 s−1, respectively.Nitrate reductase activity and amino acids analysesNitrate reductase activity in the absence or presence of 10 mM Mg+2 wasdetermined as in Scheible et al. (1997a) with either 5 mM EDTA or 10 mM magnesium acetate in the assay buffer. The activation state of the enzyme is given by the ratio of its activity in the presence and the absence of 10 mM Mg+2 multiplied by 100%.Frozen leaf subsamples (100 mg fresh weight) stored in liquid nitrogen wereextracted three times in 1 ml 80% ethanol with 10 mM Hepes–KOH pH 7.5 at 80°C for 30 min and the extracts were pooled. Then, the residue was extracted three times in 1 ml water at 80°C for 30 min and the extracts were pooled. Amino acids were analysed in the ethanol extracts according to Hare (1977). The same extract was also used to measure the soluble carbohydrates as previously reported (Pérez et al., 2005), while the residue from the ethanol water extractions was used for the assay of starch (Pérez et al., 2005).Experimental design and statistical analysesAnalyses of variance were performed as in a nested design according to Snedecor& Cochran (1967), with temperature and nitrogen as a stratum included in CO2 and replicates as a stratum included in that for temperature and nitrogen. Time effects were evaluated by including the hour of the day as a further stratum in the analysis as described by Pérez et al. (2005).RESULTSThe study of the diurnal changes of NR activity, a key enzyme in the control ofnitrate assimilation rate, together with the amount of carbohydrates and amino acids,445

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451-454 et al., 00). Song & Thornalley (00) found that a glucosinolates level of 0. μmol g− in broccoli can be linked to a reduced cancer risk. Glucosinolates, which are secondary metabolites, are suggested to have an important role in plant resistance to herbivores and pathogens (Koroleva et al., 00).The object of the present study was to evaluate the effect of the nitrogen rate anddifferent application times to crude protein and glucosinolates content of winter turnip rape.MATERIALS AND METHODSThe trials were carried out at the Jõgeva Plant Breeding Institute (PBI) (N °’;E°0’) in the 00-0, 00-0 and 00-0 growing seasons. The soil was soddy-calcarous podzolic, soil texture sandy-clay. Chemical composition of the soil (analyses made by Estonian Agricultural Research Centre) is shown in Table .Table . Chemical composition of the soil of the trial area in 00–0.PKCaMgCuSOrganic matter,Year−pHmg kg %
Effect of Top-fertilizing of Raw Protein and Glucosinolates Content of Winter Turnip Rape L. Narits Jõgeva Plant Breeding Institute, J.Aamisepa 1, EE48309 Jõgeva, Estonia; e-mail: Lea.Narits@jpbi.ee Abstract. Rapeseed is a major oil–yielding crop, ranking third place after soybeans and oil palm in the world. Rapeseed contains as average 36–38% crude protein and content of anti–nutritional compounds, among which glucosinolates have received the major attention. The object of the present study was to evaluate the effect of the nitrogen rate and different application times to the crude protein and glucosinolate content of winter turnip rape. The trials were carried out at the Jõgeva Plant Breeding Institute in the 2007–08, 2008–09 and 2009–10 growing seasons. Ammonium sulfate (nitrogen content 21%, sulphur 24%) was used as top–fertilizer. Three different nitrogen rates, 120, 140 and 160 kg N ha−1 and three different application times were used: A) once at the beginning of spring growth (oilseed rape growing code 26), B) A + when the main stalk was 10 cm (code 33), C) B + start of flowering (code 60) (a total of nine different variants) in equal portions. The results indicate that the quantity of the fertilizer has not as strong an impact as application time on the glucosinolate content. The lowest glucosinolate content was obtained from the variant of one N application. The highest protein content was obtained from the variant of three times split-N. Key words: glucosinolates, fertilizer, protein, winter turnip rape INTRODUCTION Rapeseed, which includes winter turnip rape (Brassica rapa L. ssp. oleifera (DC.) Metzg), is a major oil-yielding crop, ranking third after soybeans (Glycine max) and oil palm (Elaeis sp.) in the world. On average, rapeseed meal contains on an as-fed basis (90% dry matter) 36–38% crude protein, 10–12% crude fibre, 1–2% lipids, 6–8% ash, less than 1% calcium, and 1.2% total phosphorus (Scarisbrick & Daniels, 1986). Rapeseed meal is a high protein-containing material that can be used as a feed for livestock and poultry (Rutkowski, 1970; Uruakpa & Arntfield, 2005). Rapeseeds contain levels of anti-nutritional factors that cause problems in all production animals. These factors include glucosinolates (goitrogenic), erucic acid (toxic), tannins, sinapine, phytic acid, and mucilage (Mavromichalis, 2010). The glucosinolates receive the most attention, because they have been shown to dramatically depress animal performance. Symptoms of poisoning in poultry may include depressed growth, poor egg production, enlarged thyroid in chick embryos, and liver damage. Symptoms of poisoning in swine include growth depression, goiters, and enlarged livers (Cornell University homepage). Interestingly, a lower level of glucosinolates content has been reported to have positive effect on human health (Tan 451
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Effect of Top-fertilizing of Raw Protein and Glucosinolates Content of Winter Turnip Rape L. Narits Jõgeva Plant Breeding Institute, J.Aamisepa 1, EE48309 Jõgeva, Estonia; e-mail: Lea.Narits@jpbi.ee Abstract. Rapeseed is a major oil–yielding crop, ranking third place after soybeans and oil palm in the world. Rapeseed contains as average 36–38% crude protein and content of anti–nutritional compounds, among which glucosinolates have received the major attention. The object of the present study was to evaluate the effect of the nitrogen rate and different application times to the crude protein and glucosinolate content of winter turnip rape. The trials were carried out at the Jõgeva Plant Breeding Institute in the 2007–08, 2008–09 and 2009–10 growing seasons. Ammonium sulfate (nitrogen content 21%, sulphur 24%) was used as top–fertilizer. Three different nitrogen rates, 120, 140 and 160 kg N ha−1 and three different application times were used: A) once at the beginning of spring growth (oilseed rape growing code 26), B) A + when the main stalk was 10 cm (code 33), C) B + start of flowering (code 60) (a total of nine different variants) in equal portions. The results indicate that the quantity of the fertilizer has not as strong an impact as application time on the glucosinolate content. The lowest glucosinolate content was obtained from the variant of one N application. The highest protein content was obtained from the variant of three times split-N. Key words: glucosinolates, fertilizer, protein, winter turnip rape INTRODUCTION Rapeseed, which includes winter turnip rape (Brassica rapa L. ssp. oleifera (DC.) Metzg), is a major oil-yielding crop, ranking third after soybeans (Glycine max) and oil palm (Elaeis sp.) in the world. On average, rapeseed meal contains on an as-fed basis (90% dry matter) 36–38% crude protein, 10–12% crude fibre, 1–2% lipids, 6–8% ash, less than 1% calcium, and 1.2% total phosphorus (Scarisbrick & Daniels, 1986). Rapeseed meal is a high protein-containing material that can be used as a feed for livestock and poultry (Rutkowski, 1970; Uruakpa & Arntfield, 2005). Rapeseeds contain levels of anti-nutritional factors that cause problems in all production animals. These factors include glucosinolates (goitrogenic), erucic acid (toxic), tannins, sinapine, phytic acid, and mucilage (Mavromichalis, 2010). The glucosinolates receive the most attention, because they have been shown to dramatically depress animal performance. Symptoms of poisoning in poultry may include depressed growth, poor egg production, enlarged thyroid in chick embryos, and liver damage. Symptoms of poisoning in swine include growth depression, goiters, and enlarged livers (Cornell University homepage). Interestingly, a lower level of glucosinolates content has been reported to have positive effect on human health (Tan 451

et al., ²0¹0). Song & Thornalley (²00⁷) found that a glucosinolates level of 0.⁶¹ μmol g−¹ in broccoli can be linked to a reduced cancer risk. Glucosinolates, which are secondary metabolites, are suggested to have an important role in plant resistance to herbivores and pathogens (Koroleva et al., ²0¹0).The object of the present study was to evaluate the effect of the nitrogen rate anddifferent application times to crude protein and glucosinolates content of winter turnip rape.MATERIALS AND METHODSThe trials were carried out at the Jõgeva Plant Breeding Institute (PBI) (N ⁵⁸°⁷⁶’;E²⁶°⁴0’) in the ²00⁷-0⁸, ²00⁸-0⁹ and ²00⁹-¹0 growing seasons. The soil was soddy-calcarous podzolic, soil texture sandy-clay. Chemical composition of the soil (analyses made by Estonian Agricultural Research Centre) is shown in Table ¹.Table ¹. Chemical composition of the soil of the trial area in ²00⁷–0⁹.PKCaMgCuSOrganic matter,Year−pHmg kg ¹%

20071041431,6701104.56,86.12.520082441951,710851.26,66.32.220092012031,670861.17.06.02.0Every year black fallow was used as the precrop. Before sowing the trial area wasfertilized by Kemira Power 5-10-25, 300 kg ha-1 (N – 15; P – 13.2; K – 62.3; S – 21; Fe – 6; B – 0.06 kg ha−1). In 2007 and 2008 sowing was carried out on August 15th, in 2009 on August 13th. The trial was established on 10 m² plots using NNA (nearest neighbour adjustment) randomised design in three replications. The sowing rate was 100 germinated seeds per m². The winter oilseed turnip rape varieties Largo and Prisma were used for testing. All the years winter hardiness of the variety was good. No chemical plant protection was used during the growing period.Ammonium sulfate (nitrogen content 21%, sulphur 24%) was used as top-fertilizer. Three different nitrogen rates, 120, 140 and 160 kg N ha−1 and three different application times were used: A) once at the beginning of spring growth (oilseed rape growing code 26), B) A + when the main stalk was 10 cm (code 33), C) B + start of flowering (code 60) (the total of nine different variants) in equal portions. Timing of the nitrogen application was based on the growth stages described by Paul (1988).The trials were harvested on August 7th (2008), August 11th (2009) and July 20th(2010). Seeds were dried to the moisture content of 7.5% and sorted. Raw protein and glycosinolates (GSL) content in seeds were analysed by Near Infrared Reflectance Spectroscopy (NIRS) at the laboratory of the Jõgeva PBI.The Least Significant Difference (LSD) procedure was used when the F-test wassignificant (P > 0.05) and correlation analysis was carried out (R < 0.05 and 0.001). The results were processed by the program Statistica 4.5.452

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RESULTS AND DISCUSSIONThe average GSL content in the trial was 14.1 μmol g−1, maximum was in 2008 –15.9 μmol g-1, minimum was in 2009 – 12.7 μmol g−1. The variety Prisma produced higher GSL content every year (Table 2). Söchtling & Verret (2004) have indicated that GSL content is not significantly affected by N fertilizer. Bilsborrow et al. (1993), Thakral et al. (1996) reported that GSL content increased with the increasing rate of N. In our trial significant positive correlation between GSL content and N application time (R = 0.40***) was found, which indicates that increasing the N applications increased GSL content in seeds. N rates had no significant influence on the GSL content of seeds. The highest GSL content was obtained in variety Prisma in the variant with N 160 kg ha−1 (split in three applications). The level of GSL in Brassica plants is highly dependent on genetic factors as well as environmental determinants, such as the available soil sulphur content (De Pascale et al., 2007). There was positive significant correlation (R = 0.31*) between GSL content and variety.Table 2. Glucosinolates content (μmol g−1) and raw protein content (%) of winter turnip rape in 2008–10 (in dry matter); application times: A) once at the beginning of spring growth, B) A + when the main stalk was 10 cm, C) B + start of flowering.VarietyNitrogenApplicationGlucosinolates content,Crude protein content, %rate,variantμ−mol g 1−kg ha 1200820092010200820092010Largo120

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455-460 L. Proskina, I. Vitina, A. Jemeljanovs, V. Krastina, B. Lujane
The Use of Rapeseed-oil Cake in the Rations of Farmed Red Deer (Cervus elaphus)
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The Use of Rapeseed-oil Cake in the Rations of Farmed Red Deer (Cervus elaphus)

L. Proskina, I. Vitina, A. Jemeljanovs, V. Krastina, B. Lujane

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461-468 kg- of cereal flours for DON; μg kg- for ZON in cereals intended for direct human consumption; and 000 μg kg- for FBs in maize intended for direct human consumption. Statutory regulations do not exist for T- and HT-, yet. In addition, it should be emphasized that the recent Serbian regulation (0) has established the maximum level for the following mycotoxins AFs, OTA, DON, ZON, FB and FB, and it is in line with mentioned EU regulations.The analysis of mycotoxins is challenging as they are detected in very lowconcentrations in complex sample matrices. Since most commodities are contaminated with several mycotoxins, there is a need to develop multi-mycotoxin analysis methods for different feed/food matrices. In recent years, liquid chromatography coupled with tandem mass spectrometry (MS/MS) has become the most universal approach for mycotoxin analysis (Krska et al., 00; Škrbić et al., 0); the extensive review article of Zollner & Mayer-Helm (00) gives an overview of the impact of modern LC/MS methodology in the field of mycotoxin research and analysis. Many routine laboratories use preparatory methods based on extraction/cleanup/pre-concentration steps regarding only single or small groups of similar mycotoxins (Manova & Mladenova, 00; Sokolović & Šimpraga, 00). Rarely, studies concern simultaneous determination of the mycotoxins produced by different fungal species; for instance, while analyzing Fusarium toxins, Biselli et al. (00) included the analysis of OTA and AFs in food and feed matrices for additional verification purposes. Although the existing methods for mycotoxin determination are well established, and in some cases interlaboratory validated, the current trend is to introduce simple (one step-), broad scope procedures which, thanks to the use of modern separation/detection instrumental technologies, allow accurate determination of as many as possible major mycotoxins even at low levels in crude extracts, not applying the labor/cost-demanding clean-up step (Škrbić et al., 0). Thus, in this paper a simple sample preparation technique with only one step of extraction was chosen to be used in order to allow fast analysis of eleven mycotoxins (Fusarium, Aspergillus and Penicillium) in crude extracts of wheat flour from Serbia. To our best knowledge, this is the first publication of a study on the coupling of UHPLC with HESI-MS/MS for determining simultaneously mycotoxins produced by Aspergillus, Fusarium, and Penicillium genera in wheat flour from the Serbian market.MATERIALS AND METHODSReagents and chemicalsIndividual standard stock solutions of aflatoxins B ( μg ml-), B (0. μg ml-),G ( μg ml-), and G (0. μg ml-), ochratoxin A (OTA, 000 μg ml-), HT- toxin (00, μg ml-), T- toxin (00 μg ml-), deoxynivalenol (DON, 00 μg ml-), zearalenone (ZON, 00 μg ml-), fumonisins B(FB, 0 μg ml-) and B (FB, 0 μg ml-) were purchased from Supelco Co. (Bellefonte, PA). All standards dissolved in acetonitrile were stored at −0oC in amber glass vials, and brought to room temperature before use. Two composite working standard solutions (solutions and ) were prepared by diluting the above-mentioned stock solutions in acetonitrile for all mycotoxins. The concentrations of each mycotoxin (ZON, HT-, T-, DON, FB and FB) in the working solution were 00 ng ml-. Working standard solution contained other mycotoxins (AFB, AFB, AFG, AFG and OTA) with concentration ng ml-. These
Multi-mycotoxin Analysis by UHPLC-HESI-MS/MS: A Preliminary Survey of Serbian Wheat Flour B. Škrbić1, M. Godula2, N. Đurišić-Mladenović1 and J. Živančev1 1Faculty of Technology, University of Novi Sad, Novi Sad, Serbia; e-mail: biljana@tf.uns.ac.rs; natasadjm@tf.uns.ac.rs; jelena.zivancev@tf.uns.ac.rs 2 Thermo Fisher Scientific, Prague, Czech Republic; michal.godula@thermofisher.com Abstract. The aim of this paper is to present a high throughput method for the determination of eleven Fusarium, Aspergillus and Penicillium mycotoxins in wheat flour from Serbia. Mycotoxins were extracted from samples by one-step solvent extraction (acetonitrile-water (86:14, v/v)) without any cleanup and directly injected into an ultra-high pressure liquid chromatography/heated electrospray ionization-triple quadrupole mass spectrometry (UHPLC-HESI-MS/MS) system. The chromatographic separation was achieved in only 4 min. Quantification was performed by external calibration with matrix-matched standard solutions. The method recovery, linearity, limit of detection (LOD), and limit of quantification (LOQ) were determined. The developed method has been applied to the analysis of samples of wheat flour collected from Serbian local markets. Key words: Multi-mycotoxin method, UHPLC-HESI-MS/MS, wheat flour, Serbia INTRODUCTION Mycotoxins are toxic natural secondary metabolites produced by several species of fungi on agricultural commodities in the field or during storage. The most predominant mycotoxins are the aflatoxins (AFs, among which AFB1, AFG1, AFB2 and AFG2 are the most analyzed ones) produced by Aspergillus species, ochratoxin A (OTA) produced by both Aspergillus and Penicillium species, and toxins from fungi belonging to the Fusarium, for example, deoxynivalenol (DON), zearalenone (ZON), T-2 and HT-2 toxins, and fumonisins (FB1 and FB2). Generally, mycotoxins are stable chemical compounds and can neither be completely removed from the food supply nor destroyed during processing and heat treatment, thus, monitoring of these contaminants in food and in feed are important issues associated with public health, agricultural production, food processing, and trade. The co-occurrence of different toxic compounds implies a potential risk of additional or even synergetic toxic effects after consumption of contaminated food/feed commodities. The Commission of the European Communities established the maximum level for several mycotoxins for cereals (Commission Regulation 1881/2006 (EC) amended by Commission Regulation 1126/2007 (EC)): 4 μg kg-1 for total AFs, and 2 μg kg-1 for AFB1 for all cereals and all products derived from cereals; 3 μg kg-1 for OTA for all products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption; 750 μg 461
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Multi-mycotoxin Analysis by UHPLC-HESI-MS/MS: A Preliminary Survey of Serbian Wheat Flour B. Škrbić1, M. Godula2, N. Đurišić-Mladenović1 and J. Živančev1 1Faculty of Technology, University of Novi Sad, Novi Sad, Serbia; e-mail: biljana@tf.uns.ac.rs; natasadjm@tf.uns.ac.rs; jelena.zivancev@tf.uns.ac.rs 2 Thermo Fisher Scientific, Prague, Czech Republic; michal.godula@thermofisher.com Abstract. The aim of this paper is to present a high throughput method for the determination of eleven Fusarium, Aspergillus and Penicillium mycotoxins in wheat flour from Serbia. Mycotoxins were extracted from samples by one-step solvent extraction (acetonitrile-water (86:14, v/v)) without any cleanup and directly injected into an ultra-high pressure liquid chromatography/heated electrospray ionization-triple quadrupole mass spectrometry (UHPLC-HESI-MS/MS) system. The chromatographic separation was achieved in only 4 min. Quantification was performed by external calibration with matrix-matched standard solutions. The method recovery, linearity, limit of detection (LOD), and limit of quantification (LOQ) were determined. The developed method has been applied to the analysis of samples of wheat flour collected from Serbian local markets. Key words: Multi-mycotoxin method, UHPLC-HESI-MS/MS, wheat flour, Serbia INTRODUCTION Mycotoxins are toxic natural secondary metabolites produced by several species of fungi on agricultural commodities in the field or during storage. The most predominant mycotoxins are the aflatoxins (AFs, among which AFB1, AFG1, AFB2 and AFG2 are the most analyzed ones) produced by Aspergillus species, ochratoxin A (OTA) produced by both Aspergillus and Penicillium species, and toxins from fungi belonging to the Fusarium, for example, deoxynivalenol (DON), zearalenone (ZON), T-2 and HT-2 toxins, and fumonisins (FB1 and FB2). Generally, mycotoxins are stable chemical compounds and can neither be completely removed from the food supply nor destroyed during processing and heat treatment, thus, monitoring of these contaminants in food and in feed are important issues associated with public health, agricultural production, food processing, and trade. The co-occurrence of different toxic compounds implies a potential risk of additional or even synergetic toxic effects after consumption of contaminated food/feed commodities. The Commission of the European Communities established the maximum level for several mycotoxins for cereals (Commission Regulation 1881/2006 (EC) amended by Commission Regulation 1126/2007 (EC)): 4 μg kg-1 for total AFs, and 2 μg kg-1 for AFB1 for all cereals and all products derived from cereals; 3 μg kg-1 for OTA for all products derived from unprocessed cereals, including processed cereal products and cereals intended for direct human consumption; 750 μg 461

kg-¹ of cereal flours for DON; ⁷⁵ μg kg-¹ for ZON in cereals intended for direct human consumption; and ¹000 μg kg-¹ for FBs in maize intended for direct human consumption. Statutory regulations do not exist for T-² and HT-², yet. In addition, it should be emphasized that the recent Serbian regulation (²0¹¹) has established the maximum level for the following mycotoxins AFs, OTA, DON, ZON, FB¹ and FB², and it is in line with mentioned EU regulations.The analysis of mycotoxins is challenging as they are detected in very lowconcentrations in complex sample matrices. Since most commodities are contaminated with several mycotoxins, there is a need to develop multi-mycotoxin analysis methods for different feed/food matrices. In recent years, liquid chromatography coupled with tandem mass spectrometry (MS/MS) has become the most universal approach for mycotoxin analysis (Krska et al., ²00⁸; Škrbić et al., ²0¹¹); the extensive review article of Zollner & Mayer-Helm (²00⁶) gives an overview of the impact of modern LC/MS methodology in the field of mycotoxin research and analysis. Many routine laboratories use preparatory methods based on extraction/cleanup/pre-concentration steps regarding only single or small groups of similar mycotoxins (Manova & Mladenova, ²00⁹; Sokolović & Šimpraga, ²00⁶). Rarely, studies concern simultaneous determination of the mycotoxins produced by different fungal species; for instance, while analyzing Fusarium toxins, Biselli et al. (²00⁴) included the analysis of OTA and AFs in food and feed matrices for additional verification purposes. Although the existing methods for mycotoxin determination are well established, and in some cases interlaboratory validated, the current trend is to introduce simple (one step-), broad scope procedures which, thanks to the use of modern separation/detection instrumental technologies, allow accurate determination of as many as possible major mycotoxins even at low levels in crude extracts, not applying the labor/cost-demanding clean-up step (Škrbić et al., ²0¹¹). Thus, in this paper a simple sample preparation technique with only one step of extraction was chosen to be used in order to allow fast analysis of eleven mycotoxins (Fusarium, Aspergillus and Penicillium) in crude extracts of wheat flour from Serbia. To our best knowledge, this is the first publication of a study on the coupling of UHPLC with HESI-MS/MS for determining simultaneously mycotoxins produced by Aspergillus, Fusarium, and Penicillium genera in wheat flour from the Serbian market.MATERIALS AND METHODSReagents and chemicalsIndividual standard stock solutions of aflatoxins B¹ (² μg ml-¹), B² (0.⁵ μg ml-¹),G¹ (² μg ml-¹), and G² (0.⁵ μg ml-¹), ochratoxin A (OTA, ¹000 μg ml-¹), HT-² toxin (¹00, μg ml-¹), T-² toxin (¹00 μg ml-¹), deoxynivalenol (DON, ¹00 μg ml-¹), zearalenone (ZON, ¹00 μg ml-¹), fumonisins B¹(FB¹, ⁵0 μg ml-¹) and B² (FB², ⁵0 μg ml-¹) were purchased from Supelco Co. (Bellefonte, PA). All standards dissolved in acetonitrile were stored at −²0oC in amber glass vials, and brought to room temperature before use. Two composite working standard solutions (solutions ¹ and ²) were prepared by diluting the above-mentioned stock solutions in acetonitrile for all mycotoxins. The concentrations of each mycotoxin (ZON, HT-², T-², DON, FB¹ and FB²) in the working solution ¹ were ²00 ng ml-¹. Working standard solution ² contained other mycotoxins (AFB¹, AFB², AFG¹, AFG² and OTA) with concentration ² ng ml-¹. These

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composite solutions were used to prepare matrix-matched calibration standards by adding them in appropriate dilution to the extract of the uncontaminated sample to obtain concentration ranges that cover the maximum allowable concentrations and also the expected mycotoxin occurrence (in accordance with the available literature data). Ultra-pure water was produced by Milli-Q purification system (Millipore, Molsheim, France). Methanol, acetonitrile and ammonium acetate (all LC-MS gradient grade) were supplied from J.T. Baker (Deventer, The Netherlands); glacial acetic acid (p.a.) was obtained from LTG Promochem (Wesel, Germany).SamplesIn January 2011, fifteen wheat flour samples were collected randomly fromdifferent supermarkets within Novi Sad, the capitol of the northern Serbian province of Vojvodina, where the biggest producers of flour in Serbia are located. Five different brand names were selected in order to have a market-representative sampling. Packs of the samples were 1000 g containing different "types" of wheat flour. In Serbia, commercially available flours are classified into standards (called "types") based on the mineral (ash) content corresponding to different extraction rates during milling. The milling companies in Serbia use the national regulation 52/1995 for grading flour (Serbian regulation, 1995), with the ash content being analyzed by the producers according to the regulation 74/1988, (Serbian regulation, 1988). Flour samples belonged to type 400 (T 400; n=5) and type 500 (T 500; n=10). Type 400 is defined by an ash content of up to 450 mg per 100 g (dry basis matter) flour. Type 500 is defined by an ash content between 460 and 550 mg per 100 g (dry basis matter) of flour. They were stored in their original packets at 4–5oC until analysis was carried out.Sample preparationThe method used to prepare the crude extracts of the cereal flours was previouslydescribed by Škrbić et al. (2011) for preparation of wheat extracts for Fusarium mycotoxins analysis by HPLC-MS/MS. Only slight modifications were made with respect to amount of sample, volume of solvent used for extraction and dilution of the extract. Five grams of homogenized wheat flour samples were extracted by shaking with 20 mL of acetonitrile/water mixture (86:14, v/v) for an hour using an automatic shaker (Promax 2020, Heidolph Instruments, Germany). This solvent mixture has been used widely for extraction of mycotoxins, but the majority of the methods described in literature are based on the subsequent clean-up of the obtained extracts (Biselli et al., 2004; Biselli & Hummert, 2005; Zolner & Mayer-Helm, 2006), while “purification” of the extract was omitted in this study and compensated for by usage of modern UHPLC-MS/MS technique. The suspensions were filtered and an aliquot (1 mL) of filtered crude extracts was transferred into glass vials and diluted with 3 ml of the UHPLC mobile phase of the initial content (95% A and 5% B; explanation of the composition of the eluent A and B is given in the subsequent section). Thus, the final extracts contained 0.0625 g sample per mL. Before injection into the UHPLC-HESI-MS/MS system, the extracts were passed through the 0.2 μm nylon syringe filter.463

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469-472 investigation was to evaluate the protein composition of game meat in Latvian farms and wildlife and to compare different game species.MATERIAL AND METHODSExperimental designThe chemical analyses of samples were made, i.e. wild deer (), farm deer(), roe deer (), elk (), wild boar meat () using samples collected after hunting in the Vidzeme and Latgale regions of Latvia. Meat samples (m. logissimus lumborum) were collected in the autumn-winter seasons from 00-0. The research was conducted at the laboratory of Biochemistry and Microbiology of the Research Institute of Biotechnology and Veterinary Medicine ‘Sigra’. In the studied samples, protein, amino acids and protein of connective tissue were determined. Sample preparation was made within hours after slaughtering or hunting. Meat samples of 00–00 g were homogenized with BŰCHI B-00.MethodsP r o t e i n c o n t e n t was determined as total nitrogen content by the Kjeldahlmethod, using coefficient . for the calculation (ISO :).A m i n o a c i d s : dried, defatted meat samples were treated with constant boiling
Evaluation of Protein Composition of Game Meat in Latvian Farms and Wildlife V. Strazdina, A. Jemeljanovs, V. Sterna and V. Vjazevica Research institute of Biotechnology and Veterinary medicine ‘Sigra’ of Latvian University of Agriculture, Institute str. 1, Sigulda, LV 2150, Latvia, e-mail: sigra@lis.lv Abstract. The meat of wild animals is highly favourable for human health because it has lower SFA content than domestic animals but higher protein content. In recent years consumption and assortment of game meat products has significantly increased. Deer farms are being established. There have been few investigations of the biochemical composition of game meat, therefore, the aim of the investigation was to evaluate protein composition of game meat in Latvian farms and wildlife. The investigations were carried out in different regions of Latvia. The chemical analyses of 76 samples were made, i.e. wild deer (18), farm deer (12), roe deer (16), elk (18), wild boar (12) meat samples were collected after hunting in the Vidzeme and Latgale regions of Latvia. Protein, amino acids and the content of connective tissue (4-hidroxiproline) were determined in the studied samples. Protein protein ranged from 22.21–23.59%. The content of connective tissue ranged from 2.22% in elk meat up to 3.09% in roe deer. The sum of essential amino acids in game meat samples was determined from 27.06–45.70 g 100 g−1. Elk meat had the highest protein content and lowest content of connective tissues among the game meat. Key words: amino acids, connective tissue, dietetic product, game meat, protein INTRODUCTION Protein varies among the meat animal species: its content ranges between 13 and 23% of the fresh weight (Honikel, 2009). The amino acid profile is important because some amino acids cannot be synthesized by human organisms and therefore must be supplied by the diet. Meat is rich in so-called essential or indispensable amino acids –lysine, leucine, isoleucine, and sulfur-containing amino acids – and in this sense meat has high-quality protein (Young et al., 2001). Consumers expect the meat products on the market to have the required nutritional value, to be wholesome, lean, and have adequate juiciness and tenderness. Connective tissue is an extracellular network of proteins, which is also decisive for meat tenderness. The most esteemed cuts of meat in a carcass are those that have a low content of connective tissue. That is why in many countries, in addition to protein content, a value for connective tissue is determined and used as a part of the quality characteristic of the meat. In recent years the assortment and consumption of game meat products have significantly increased, but investigations about the biochemical composition of game meat are few. Deer farms have been established, and the biochemical composition of farm deer meat has been analysed in recent years, but data about the nutritive value and composition of elk, roe deer or wild boar are not sufficient. Therefore the aim of the 469
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Evaluation of Protein Composition of Game Meat in Latvian Farms and Wildlife V. Strazdina, A. Jemeljanovs, V. Sterna and V. Vjazevica Research institute of Biotechnology and Veterinary medicine ‘Sigra’ of Latvian University of Agriculture, Institute str. 1, Sigulda, LV 2150, Latvia, e-mail: sigra@lis.lv Abstract. The meat of wild animals is highly favourable for human health because it has lower SFA content than domestic animals but higher protein content. In recent years consumption and assortment of game meat products has significantly increased. Deer farms are being established. There have been few investigations of the biochemical composition of game meat, therefore, the aim of the investigation was to evaluate protein composition of game meat in Latvian farms and wildlife. The investigations were carried out in different regions of Latvia. The chemical analyses of 76 samples were made, i.e. wild deer (18), farm deer (12), roe deer (16), elk (18), wild boar (12) meat samples were collected after hunting in the Vidzeme and Latgale regions of Latvia. Protein, amino acids and the content of connective tissue (4-hidroxiproline) were determined in the studied samples. Protein protein ranged from 22.21–23.59%. The content of connective tissue ranged from 2.22% in elk meat up to 3.09% in roe deer. The sum of essential amino acids in game meat samples was determined from 27.06–45.70 g 100 g−1. Elk meat had the highest protein content and lowest content of connective tissues among the game meat. Key words: amino acids, connective tissue, dietetic product, game meat, protein INTRODUCTION Protein varies among the meat animal species: its content ranges between 13 and 23% of the fresh weight (Honikel, 2009). The amino acid profile is important because some amino acids cannot be synthesized by human organisms and therefore must be supplied by the diet. Meat is rich in so-called essential or indispensable amino acids –lysine, leucine, isoleucine, and sulfur-containing amino acids – and in this sense meat has high-quality protein (Young et al., 2001). Consumers expect the meat products on the market to have the required nutritional value, to be wholesome, lean, and have adequate juiciness and tenderness. Connective tissue is an extracellular network of proteins, which is also decisive for meat tenderness. The most esteemed cuts of meat in a carcass are those that have a low content of connective tissue. That is why in many countries, in addition to protein content, a value for connective tissue is determined and used as a part of the quality characteristic of the meat. In recent years the assortment and consumption of game meat products have significantly increased, but investigations about the biochemical composition of game meat are few. Deer farms have been established, and the biochemical composition of farm deer meat has been analysed in recent years, but data about the nutritive value and composition of elk, roe deer or wild boar are not sufficient. Therefore the aim of the 469

investigation was to evaluate the protein composition of game meat in Latvian farms and wildlife and to compare different game species.MATERIAL AND METHODSExperimental designThe chemical analyses of ⁷⁶ samples were made, i.e. wild deer (¹⁸), farm deer(¹²), roe deer (¹⁶), elk (¹⁸), wild boar meat (¹²) using samples collected after hunting in the Vidzeme and Latgale regions of Latvia. Meat samples (m. logissimus lumborum) were collected in the autumn-winter seasons from ²00⁷-¹0. The research was conducted at the laboratory of Biochemistry and Microbiology of the Research Institute of Biotechnology and Veterinary Medicine ‘Sigra’. In the studied samples, protein, amino acids and protein of connective tissue were determined. Sample preparation was made within ⁴⁸ hours after slaughtering or hunting. Meat samples of ²00–⁴00 g were homogenized with BŰCHI B-⁴00.MethodsP r o t e i n c o n t e n t was determined as total nitrogen content by the Kjeldahlmethod, using coefficient ⁶.²⁵ for the calculation (ISO ⁹³⁷:¹⁹⁷⁴).A m i n o a c i d s : dried, defatted meat samples were treated with constant boiling

⁶N hydrochloric acid in an oven at around 110°C for 23 h. Hydrolyzate was diluted with 0.1% formic acid. The sample (2 ml) was filtered using a syringe filter with 0.45μm nylon membrane. Amino acids were detected using reversed-phase HPLC/MS (Waters Alliance 2695, Waters 3100, column XTerra MS C18 5 μm, 1 x 100 mm). Mobile phase (90% acetonitrile: 10% dejonized water) 0.5 ml min−1, column temperature. 40ºC. Data acquisition used programme Empower pro.C o n n e c t i v e t i s s u e p r o t e i n was calculated via determination of specificamino acid 4-hydroxiproline, which is exclusively present in collagen. Meat samples were hydrolyzed in acid (3.5 M H2SO4 at ~105ºC). The 4-hydroxiproline was oxidized with chloramine-T to a pyrrole. With 4- dimethylaminobenzaldehyde a red color develops, which is measured spectrometrically at 560 nm (ISO 3496:1994(E)). Collagen is calculated by 8x the concentration of 4-hydroxiproline and expressed as % of total protein (Honikel, 2009).The statistical analysis was performed using SPSS 17. One-way ANOVA was usedfor comparison mean values. Statistical significance was declared at P < 0.05.RESULTS AND DISCUSSIONThe calculated content of protein in samples of game meat was 22.21–23.59%; elkmeat samples were the richest. The protein content of farm deer meat samples has a wider interval of varieties than the wild deer meat samples, which could be the result of additional feed portions from October till March for farm deer. The results of the statistical analysis showed that the total protein content in the ruminants’ meat did not differ significantly (F = 1.286; P = 0.297 > 0.05). The results of our investigation are similar with other research findings, where protein content in raw deer meat samples was reported as 21.7%, in boar meat samples 21.9% (Paleari et al., 2003) Protein composition and fat content of game meat samples are compared in Table 1.470

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Table 1. Biochemical composition of meat samples.ProteinConnectiveFat content,SpeciesnMinimum Maximumcontent, %tissue content, %%Wild deer1822.3619.6423.942.501.60Farm deer1221.8419.7623.412.312.44Roe deer1622.8218.6125.403.091.59Elk 1822.7221.6923.27 2.221.31Wild boar1222.9218.1625.882.862.82Connective tissue is decisive for meat tenderness, therefore the connective tissuecontent in game meat samples was determined; it ranged from 2.22% in elk meat till 3.09% in roe deer meat. Meat protein generally contains 2.5–12% connective tissue protein (Honikel, 2009). Accordingly, game meat could be classified as high quality meat.Average contents of amino acids in game are shown in Table 2; it significantlydiffered among species (P < 0.05).Table 2. Content of amino acids in meat samples.Average content of amino acid, g kg−1Amino acidWild deerFarm deerRoe deerElkWild boarMean ± SDMean ± SDMean ± SDMean ± SD

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473-478 properties of milk gels, both chymosin and acid induced, are affected by the heat treatment applied to milk (Parnell et al., ), and can result in irreversible changes in milk protein structure. Some of the changes involved are whey protein denaturation and aggregation, interactions of whey proteins with casein micelles, reactions between lactose and proteins, changes in casein micelle structure, transfer of soluble calcium and phosphate to colloidal phase, changes in fat globule membranes, and decrease in pH (Singh & Waungana, 00).The thermophilic bacteria St. thermophilus are widely used in the dairy industryin the production of yoghurt and hard ‘cooked’ cheeses (Emmental, Gruyere, Grana). In the industrial implementations of St. thermophilus, fast growth of the bacteria is crucial in intense acidification of milk (Derzelle et al., 00).The species of Lb. casei/paracasei and Lb. plantarum are the main components ofmesophilic nonstarter microflora (Laht et al., 00) and become important as adjunct cultures for the production of fermented milk products (Dupont et al., 000). As the starter bacteria decrease in number, a secondary microbial flora grows in the maturing cheese and it becomes dominant after – months of ripening (Laht et al., 00).Calorimetry is especially helpful in the studies of the growth of the bacteria inopaque media and is a useful method to obtain kinetic and thermodynamic information on microbial growth (Kabanova et al., 00; Kriščiunaite et al., 0). The objectives of this investigation were to study the effect of milk heat treatment on the growth parameters of thermophilic starter and non-starter lactic acid bacteria using isothermal batch microcalorimetry.MATERIALS AND METHODSBacterial culture and preparation of growth inoculaThe strain of St. thermophilus ST used in this work was provided by Chr.Hansen (Denmark). Frozen cultures of St. thermophilus ST culture were thawed and pre-grown on Petri dishes with M Agar (LAB M, UK) for h at 0°C. One colony from a pre-grown Petri dish was used as an inoculum for a 0 mL culture in sterilized RSM (Kalev Paide Tootmine AS, Paide, Estonia) at 0°C and left till coagulation. % of pre-grown culture was used as inoculum for the next 0 mL of RSM, left until coagulation and further used for inoculation of differently heat-treated milk samples.A frozen Lb. paracasei SR culture isolated from Estonian cheese (Kask et al.,
The Effect of Milk Heat Treatment on the Growth Characteristics of Lactic Acid Bacteria I. Stulova 1, 2 , N. Kabanova 1, 2, T. Kriščiunaite 1, 2, T.-M. Laht 1, 2 and R. Vilu 1,2 1 Tallinn University of Technology, Ehitajate tee 5, 19086, Tallinn, Estonia 2 Competence Center of Food and Fermentation Technologies (CCFFT), Akadeemia tee 15B, 12618, Tallinn, Estonia; e-mails: irina.stulova@tftak.eu; natalja@tftak.eu; tiina@tftak.eu; tiiu@tftak.eu; raivo@tftak.eu Abstract. The ability to growth in milk is an important feature for lactic acid bacteria (LAB) used as starters for fermented milk products. Several decades ago the results of the studies varied widely: some of them showed that LAB grew better in raw milk and others demonstrated improved growth of the bacteria in heat-treated milk (Foster et al., 1952). The effectiveness of heat treatment of milk as a tool for modifying the functional properties of protein components has been extensively documented in the literature (Raikos, 2010), but the information on the influence of heat treatment of milk on the growth of LAB is not exhaustive. Peculiarities of growth of Streptococcus thermophilus ST12 and Lactobacillus paracasei S1R1 were studied using isothermal batch microcalorimeter TAMIII. Bacterial growth was monitored in pasteurized and ultra-high temperature (UHT) treated milk with different fat content, and also in reconstituted skim milk (RSM) prepared from low-heat skim milk powder (LHSMP). Heat produced during different growth stages (Qtot, Qexp), maximal specific growth rate (μmax) and lag–phase (λ) duration were determined by processing calorimetric curves, and detailed analysis of growth of the bacteria in differently pretreated milks were carried out on the basis of these data. The results of the experiments showed that primarily heat treatment and, to a minor extent, fat content of milk influenced the growth parameters of both bacterial strains, especially Lb. paracasei, growth of which was almost completely inhibited in UHT milk Key words: bacterial growth, Lactobacillus paracasei, microcalorimetry, milk treatment, Streptococcus thermophilus INTRODUCTION Milk as a raw material has a relatively short shelf life but it can be prolonged by heat treatment, which is an essential step adopted by the dairy industry (Raikos, 2010). For a high proportion of cheese varieties, pasteurization is the sole treatment applied to the cheese-milk (Kelly et al., 2008). The temperature/time combinations for the batch heat treatments used in yoghurt manufacture are 85°C for 30 min or 90–95°C for 5 min. However, very high temperature short time (100–130 °C for 4 to 16 s) or ultra-high temperature (UHT) 140°C for 4 to 16 s treatments are also sometimes used (Lee & Lucey, 2010). Heat treatment of milk during commercial processing operations not only inactivates the microorganisms (Odriozola-Serrano et al., 2007), but also results in a number of physico-chemical changes in the milk constituents. The rheological 473
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The Effect of Milk Heat Treatment on the Growth Characteristics of Lactic Acid Bacteria I. Stulova 1, 2 , N. Kabanova 1, 2, T. Kriščiunaite 1, 2, T.-M. Laht 1, 2 and R. Vilu 1,2 1 Tallinn University of Technology, Ehitajate tee 5, 19086, Tallinn, Estonia 2 Competence Center of Food and Fermentation Technologies (CCFFT), Akadeemia tee 15B, 12618, Tallinn, Estonia; e-mails: irina.stulova@tftak.eu; natalja@tftak.eu; tiina@tftak.eu; tiiu@tftak.eu; raivo@tftak.eu Abstract. The ability to growth in milk is an important feature for lactic acid bacteria (LAB) used as starters for fermented milk products. Several decades ago the results of the studies varied widely: some of them showed that LAB grew better in raw milk and others demonstrated improved growth of the bacteria in heat-treated milk (Foster et al., 1952). The effectiveness of heat treatment of milk as a tool for modifying the functional properties of protein components has been extensively documented in the literature (Raikos, 2010), but the information on the influence of heat treatment of milk on the growth of LAB is not exhaustive. Peculiarities of growth of Streptococcus thermophilus ST12 and Lactobacillus paracasei S1R1 were studied using isothermal batch microcalorimeter TAMIII. Bacterial growth was monitored in pasteurized and ultra-high temperature (UHT) treated milk with different fat content, and also in reconstituted skim milk (RSM) prepared from low-heat skim milk powder (LHSMP). Heat produced during different growth stages (Qtot, Qexp), maximal specific growth rate (μmax) and lag–phase (λ) duration were determined by processing calorimetric curves, and detailed analysis of growth of the bacteria in differently pretreated milks were carried out on the basis of these data. The results of the experiments showed that primarily heat treatment and, to a minor extent, fat content of milk influenced the growth parameters of both bacterial strains, especially Lb. paracasei, growth of which was almost completely inhibited in UHT milk Key words: bacterial growth, Lactobacillus paracasei, microcalorimetry, milk treatment, Streptococcus thermophilus INTRODUCTION Milk as a raw material has a relatively short shelf life but it can be prolonged by heat treatment, which is an essential step adopted by the dairy industry (Raikos, 2010). For a high proportion of cheese varieties, pasteurization is the sole treatment applied to the cheese-milk (Kelly et al., 2008). The temperature/time combinations for the batch heat treatments used in yoghurt manufacture are 85°C for 30 min or 90–95°C for 5 min. However, very high temperature short time (100–130 °C for 4 to 16 s) or ultra-high temperature (UHT) 140°C for 4 to 16 s treatments are also sometimes used (Lee & Lucey, 2010). Heat treatment of milk during commercial processing operations not only inactivates the microorganisms (Odriozola-Serrano et al., 2007), but also results in a number of physico-chemical changes in the milk constituents. The rheological 473

properties of milk gels, both chymosin and acid induced, are affected by the heat treatment applied to milk (Parnell et al., ¹⁹⁸⁸), and can result in irreversible changes in milk protein structure. Some of the changes involved are whey protein denaturation and aggregation, interactions of whey proteins with casein micelles, reactions between lactose and proteins, changes in casein micelle structure, transfer of soluble calcium and phosphate to colloidal phase, changes in fat globule membranes, and decrease in pH (Singh & Waungana, ²00¹).The thermophilic bacteria St. thermophilus are widely used in the dairy industryin the production of yoghurt and hard ‘cooked’ cheeses (Emmental, Gruyere, Grana). In the industrial implementations of St. thermophilus, fast growth of the bacteria is crucial in intense acidification of milk (Derzelle et al., ²00⁵).The species of Lb. casei/paracasei and Lb. plantarum are the main components ofmesophilic nonstarter microflora (Laht et al., ²00²) and become important as adjunct cultures for the production of fermented milk products (Dupont et al., ²000). As the starter bacteria decrease in number, a secondary microbial flora grows in the maturing cheese and it becomes dominant after ¹–³ months of ripening (Laht et al., ²00²).Calorimetry is especially helpful in the studies of the growth of the bacteria inopaque media and is a useful method to obtain kinetic and thermodynamic information on microbial growth (Kabanova et al., ²00⁹; Kriščiunaite et al., ²0¹¹). The objectives of this investigation were to study the effect of milk heat treatment on the growth parameters of thermophilic starter and non-starter lactic acid bacteria using isothermal batch microcalorimetry.MATERIALS AND METHODSBacterial culture and preparation of growth inoculaThe strain of St. thermophilus ST¹² used in this work was provided by Chr.Hansen (Denmark). Frozen cultures of St. thermophilus ST¹² culture were thawed and pre-grown on Petri dishes with M¹⁷ Agar (LAB M, UK) for ²⁴ h at ⁴0°C. One colony from a pre-grown Petri dish was used as an inoculum for a ¹0 mL culture in sterilized RSM (Kalev Paide Tootmine AS, Paide, Estonia) at ⁴0°C and left till coagulation. ¹% of pre-grown culture was used as inoculum for the next ¹0 mL of RSM, left until coagulation and further used for inoculation of differently heat-treated milk samples.A frozen Lb. paracasei S¹R¹ culture isolated from Estonian cheese (Kask et al.,

2003) was thawed and pre-cultured on Petri dishes with MRS Agar (LAB M) medium for 24 h at 35°C. One colony from a pre-grown Petri dish was used as an inoculum for a 10 mL culture in liquid sterilized MRS Broth (LAB M) at 35°C. 1 mL of bacterial suspension grown overnight was used as inoculum for the next liquid 10 mL of MRS Broth and further used for inoculation of differently heat-treated milk samples.Milk samplesLow-heat skim milk powder (Kalev Paide Tootmine AS, Paide, Estonia) wasreconstituted in distilled water with thorough mixing for 1 h to yield a final concentration of 10% (w/v) milk solids. Commercial pasteurized milk with 3.5%, 2.5% and 0.05% fat content (Tere AS, Tallinn, Estonia) and commercial UHT milk with 3.5% and 0.05% fat content (Kalev Paide Tootmine AS) used in the study were obtained from retail sellers.474

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Calorimetric equipment and measurementsAfter the addition of bacteria, samples were stirred and 2 mL were transferredinto the autoclaved ampoules. Isothermal batch microcalorimeter TAM III Thermal Activity Monitor (Thermometric, Järfälla, Sweden) equipped with 24 channels was used for the study of the growth of St. thermophilus ST12 and Lb. paracasei S1R1 in various milk substrates. Data analysis was accomplished using TAM Assistant program (v 0.9.1012.40, SciTech Software AB, Thermometric AB).Statistical analysisThe experimental data were submitted to single-factor analysis of variance(ANOVA), and the differences among means were determined by Fisher’s least significant difference (LSD) test.RESULTS AND DISCUSSIONThe power-time curves describing the growth of St. thermophilus ST12 at 40°Cand Lb. paracasei S1R1 at 35°C in differently pretreated milk samples at the same initial inoculation rate of 105 cfu mL−1 are presented in Fig. 1 and Fig. 2, respectively. Each curve is the average of three power-time curves, obtained with replicated samples.The power-time curves were processed as described by Kabanova et al., 2009 andthe numerical results were presented in Table 1. ANOVA of the data showed that both milk thermal processing and fat content significantly affected the growth characteristics of both bacteria (P < 0.05).Table 1. Parameters describing St. thermophilus ST12 growth in differently pretreated milk samples: means of maximum specific growth rate (μmax), heat evolved during the exponential growth phase (Qexp), total heat (Qtot) and time at maximum heat production rate (t(dQ/dt)max) obtained from microcalorimetric power-time curves.μ max, h−1Qexp, J mL−1t(dQ/dt)max, hSample1st exp.2nd exp.1st exp.2nd exp.Qtot, J mL−1end of 2ndphasephasephasephaseexp. phaseRSM 1.72a 1.38a 0.44a 2.43a 6.73a 6.69aPast 0.05%2.03b 1.80b 0.07b 1.89b 6.51a 5.88bPast 2.5%2.03b 1.55c 0.08c 2.05c 6.51a 5.88bPast 3.5%1.94c 1.47c 0.07b 2.01bc 6.58a 6.05bUHT 0.05%2.03b 1.22d 0.10d 2.39a 5.98b 15.87cUHT 3.5%1.99bc 0.98e 0.11e 2.41a 5.88b 16.02cThe dual-peak power-time curve of diauxic growth of St. thermophilus ST12 wasregistered in reconstituted milk prepared from LHSMP, also in pasteurized and UHT milk. Two peaks observed correspond to two growth phases: the first exponential growth phase (a shoulder of the curve), and the second (major) exponential growth phase. However, power-time curves in the milk subjected to various heat treatments were completely different. In RSM the heat evolved during the first exponential phase was 6.5% of the total, whereas the contribution of this phase was 1.1–1.9% in other475

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479-488 dairy cows. Grasslands are characterized by multiple functions and values. They provide forage for grazing and browsing animals, both domestic and wild, and support rural economies, functioning as the major source of livelihood for local communities. Grassland landscapes are aesthetically pleasing, provide recreation opportunities, open space and improve the quality of life of the whole society (Peeters, 00).Table . The percentage of grassland in agricultural land (AA) in different EU countries by EURASTAT, 00.Country% of AA
The Effect of Grassland-based Forages on Milk Quality and Quantity P. Stypinski Warsaw University of Life Sciences (SGGW), Department of Agronomy Nowoursynowska 159, 02-776 Warsaw, Poland, piotr_stypinski@sggw.pl Abstract: Grassland is the first land use in the agricultural areas (AA) of Europe, covering, with rangeland, 56 million ha (33% of AA in EU). Grasslands are characterized by multiple functions and values but one of the most important is forage production for ruminants. In the “grassland region” milk production is connected with grassland management and proper utilisation, whereas in other parts of Europe milk production is based on maize and concentrates. Unfortunately, grassland, particularly grazing, seems to be less important than in the past. Milk quality depends on animal feed. Milk and meat produced from grassland, particularly from botanically diverse pastures, have higher concentrations of those fatty acids and antioxidants which are considered to be of benefit to human health. Key words: fatty acid, grassland management, grassland potential, milk quality INTRODUCTION Grassland is the first land use in the agricultural areas (AA) of Europe. Grasslands and rangeland cover 56 million ha (33% of AA in EU), including about 17.5 million ha of rangelands (10% of AA), mainly in the mountain areas (EUROSTAT, 2008), (Peeters, 2009). However these numbers hide large differences among Member States of the EU: for example in the UK 65% of AA is covered by grassland, in Ireland more than 70%, while in Eastern Europe the proportion is lower, e.g. Poland (21%), Estonia (25%), Romania 33% (Table 1). The seasonality of production of grassland and forage is primarily influenced by temperature and soil moisture which limit the length and determine the intensity of the growing season. In most of Europe, temperature dictates the main seasonal trends in herbage growth but in southern and Eastern Europe, in particular, summer trends are conditioned by the availability of soil moisture (Laidlaw et al., 2006). Milk production per 1 ha of agricultural land is generally connected with the share of grassland in total agricultural lands; the best milk productivity is observed in the Atlantic zone of Europe (Smit et al., 2008). Dry matter production, forage quality, management, stocking rate and animal production differ in some European regions depending on many factors. Low production sward can only produce annually about 2–3 tonnes of dry matter (DM) per ha, while in contrast high production sward can yield as much as 10–12 t DM or even 15-20 t DM under good management and production conditions, and is usually used for 479
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The Effect of Grassland-based Forages on Milk Quality and Quantity P. Stypinski Warsaw University of Life Sciences (SGGW), Department of Agronomy Nowoursynowska 159, 02-776 Warsaw, Poland, piotr_stypinski@sggw.pl Abstract: Grassland is the first land use in the agricultural areas (AA) of Europe, covering, with rangeland, 56 million ha (33% of AA in EU). Grasslands are characterized by multiple functions and values but one of the most important is forage production for ruminants. In the “grassland region” milk production is connected with grassland management and proper utilisation, whereas in other parts of Europe milk production is based on maize and concentrates. Unfortunately, grassland, particularly grazing, seems to be less important than in the past. Milk quality depends on animal feed. Milk and meat produced from grassland, particularly from botanically diverse pastures, have higher concentrations of those fatty acids and antioxidants which are considered to be of benefit to human health. Key words: fatty acid, grassland management, grassland potential, milk quality INTRODUCTION Grassland is the first land use in the agricultural areas (AA) of Europe. Grasslands and rangeland cover 56 million ha (33% of AA in EU), including about 17.5 million ha of rangelands (10% of AA), mainly in the mountain areas (EUROSTAT, 2008), (Peeters, 2009). However these numbers hide large differences among Member States of the EU: for example in the UK 65% of AA is covered by grassland, in Ireland more than 70%, while in Eastern Europe the proportion is lower, e.g. Poland (21%), Estonia (25%), Romania 33% (Table 1). The seasonality of production of grassland and forage is primarily influenced by temperature and soil moisture which limit the length and determine the intensity of the growing season. In most of Europe, temperature dictates the main seasonal trends in herbage growth but in southern and Eastern Europe, in particular, summer trends are conditioned by the availability of soil moisture (Laidlaw et al., 2006). Milk production per 1 ha of agricultural land is generally connected with the share of grassland in total agricultural lands; the best milk productivity is observed in the Atlantic zone of Europe (Smit et al., 2008). Dry matter production, forage quality, management, stocking rate and animal production differ in some European regions depending on many factors. Low production sward can only produce annually about 2–3 tonnes of dry matter (DM) per ha, while in contrast high production sward can yield as much as 10–12 t DM or even 15-20 t DM under good management and production conditions, and is usually used for 479

dairy cows. Grasslands are characterized by multiple functions and values. They provide forage for grazing and browsing animals, both domestic and wild, and support rural economies, functioning as the major source of livelihood for local communities. Grassland landscapes are aesthetically pleasing, provide recreation opportunities, open space and improve the quality of life of the whole society (Peeters, ²00⁸).Table ¹. The percentage of grassland in agricultural land (AA) in different EU countries by EURASTAT, ²00⁸.Country% of AA

Country% of AAMalta 0,0

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France33.4Finland 1.3

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489-494 health of the Baltic Sea has changed remarkably during past decades, and it also influences the environment of the fish living in the Baltic Sea (Lankov et al., 00; Raid et al., 00; Ojaveer et al., 0).Sensory analysis gives a holistic and integrated picture of the fish whereasinstrumental methods generally measure only one specific compound or a set of attributes related to one set of properties (Nielsen, ). Sensory analysis can be more accurately interpreted by using various data from instrumental analysis. Partial least squares technique is used to show relationship between sensory attributes and chemical variables. Baltic sprat and Baltic herring composition is known to vary the most in lipid and water content (Krosing & Veldre, ; Kolakowska et al., ; Szlinder-Richert et al., 00). The variation in the chemical composition of Baltic sprat and Baltic herring is related to nutrition, catching season, fish size, seasonal and sexual variations. Variation in chemical composition might lead to changes in sensory attributes, including flavor, aroma, texture, and visual appearance which control the acceptability of fish as food (Flick & Martin, ). The aim of the current work was to evaluate the sensory properties and their relations to lipid content, water content and protein content of Baltic sprat and Baltic herring.MATERIALS AND METHODSSamplesTwenty-six samples of Baltic sprat and samples of Baltic herring were caughtfrom different locations in Estonia’s coastal waters (Gulf of Riga and Finland) from February 00 until March 00. Samples were taken only from spring-spawn fish populations. Samples of fish (about kg) were immediately frozen after landing and stored at −°C. The fish was analyzed during two months followed to catching. The frozen fish samples were thawed at °C hours before analysis. Thawed fish were de-headed, gutted, de-boned and washed. Samples for sensory analysis were packed by two in aluminum foil, steamed for 0 minutes at °C, and served immediately.Composition analysisSamples for composition analysis (minimum kg) were minced twice and kept at
Sensory and Chemical Properties of Baltic Sprat (Sprattus sprattus balticus) and Baltic Herring (Clupea harengus membras) in Different Catching Seasons L. Timberg1,2, K. Koppel1,2, R. Kuldjärv1,2 and T. Paalme1,2 1Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn Estonia; e-mail: loreida@tftak.eu, kadri@tftak.eu, rain@tftak.eu 2Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086, Tallinn, Estonia; e-mail: tpaalme@staff.ttu.ee Abstract. Baltic sprat (Sprattus sprattus balticus) and Baltic herring (Clupea harengus membras L) are two of the most caught fish species among the Estonian seacoast fishermen, and therefore it is important to understand catching season effects on sprat and herring sensory and nutritional quality. The aim of this study was to measure and compare sensory and chemical variability of Baltic sprat and Baltic herring during different catching seasons. Batches of Baltic sprat and Baltic herring were caught from different locations in Estonian coastal waters from February 2008 until April 2009. Water content, protein content, lipid and ash content were measured. Descriptive sensory evaluation of steamed fish was conducted and results analyzed using Partial Least Squares Regression. The results suggested differentiation possibilities between fish from different seasons. The variations lie in fat and water contents, hardness, characteristic flavor and sweetness of the fish flesh. Key words: Baltic herring (Clupea harengus membras L), Baltic sprat (Sprattus sprattus balticus), descriptive sensory evaluation INTRODUCTION Baltic sprat and Baltic herring and dishes of these fish have been eaten by people living around the Baltic Sea for centuries. Many different technologies like smoking, spicing, salting, marinating, fermenting etc. are used to produce fish products from Baltic sprat and Baltic herring for the Baltic countries and for the Eastern European markets. Some of these product types are very sensitive to fish quality and others are less, because of strong flavor and structure additives. Nutritional composition of Baltic sprat caught from coastal waters in Estonia has been previously monitored and reported by Krosing and Veldre (1973). Baltic herring composition has been monitored and reported by several authors (Kolakowska et al., 1992; Szlinder-Richert et al., 2010). Baltic sprat and Baltic herring are under strict surveillance when it comes to dioxins and other contaminants (Vuorinen et al., 2002; Simm, et al., 2006; Szlinder-Richert et al., 2009), but until now the composition and sensory quality of the Baltic sprat and Baltic herring from gulf of Riga and Finland have not been thoroughly studied. There is clear evidence that the ecological state of 489
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Sensory and Chemical Properties of Baltic Sprat (Sprattus sprattus balticus) and Baltic Herring (Clupea harengus membras) in Different Catching Seasons L. Timberg1,2, K. Koppel1,2, R. Kuldjärv1,2 and T. Paalme1,2 1Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15b, 12618 Tallinn Estonia; e-mail: loreida@tftak.eu, kadri@tftak.eu, rain@tftak.eu 2Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086, Tallinn, Estonia; e-mail: tpaalme@staff.ttu.ee Abstract. Baltic sprat (Sprattus sprattus balticus) and Baltic herring (Clupea harengus membras L) are two of the most caught fish species among the Estonian seacoast fishermen, and therefore it is important to understand catching season effects on sprat and herring sensory and nutritional quality. The aim of this study was to measure and compare sensory and chemical variability of Baltic sprat and Baltic herring during different catching seasons. Batches of Baltic sprat and Baltic herring were caught from different locations in Estonian coastal waters from February 2008 until April 2009. Water content, protein content, lipid and ash content were measured. Descriptive sensory evaluation of steamed fish was conducted and results analyzed using Partial Least Squares Regression. The results suggested differentiation possibilities between fish from different seasons. The variations lie in fat and water contents, hardness, characteristic flavor and sweetness of the fish flesh. Key words: Baltic herring (Clupea harengus membras L), Baltic sprat (Sprattus sprattus balticus), descriptive sensory evaluation INTRODUCTION Baltic sprat and Baltic herring and dishes of these fish have been eaten by people living around the Baltic Sea for centuries. Many different technologies like smoking, spicing, salting, marinating, fermenting etc. are used to produce fish products from Baltic sprat and Baltic herring for the Baltic countries and for the Eastern European markets. Some of these product types are very sensitive to fish quality and others are less, because of strong flavor and structure additives. Nutritional composition of Baltic sprat caught from coastal waters in Estonia has been previously monitored and reported by Krosing and Veldre (1973). Baltic herring composition has been monitored and reported by several authors (Kolakowska et al., 1992; Szlinder-Richert et al., 2010). Baltic sprat and Baltic herring are under strict surveillance when it comes to dioxins and other contaminants (Vuorinen et al., 2002; Simm, et al., 2006; Szlinder-Richert et al., 2009), but until now the composition and sensory quality of the Baltic sprat and Baltic herring from gulf of Riga and Finland have not been thoroughly studied. There is clear evidence that the ecological state of 489

health of the Baltic Sea has changed remarkably during past decades, and it also influences the environment of the fish living in the Baltic Sea (Lankov et al., ²0¹0; Raid et al., ²0¹0; Ojaveer et al., ²0¹¹).Sensory analysis gives a holistic and integrated picture of the fish whereasinstrumental methods generally measure only one specific compound or a set of attributes related to one set of properties (Nielsen, ¹⁹⁹⁷). Sensory analysis can be more accurately interpreted by using various data from instrumental analysis. Partial least squares technique is used to show relationship between sensory attributes and chemical variables. Baltic sprat and Baltic herring composition is known to vary the most in lipid and water content (Krosing & Veldre, ¹⁹⁷³; Kolakowska et al., ¹⁹⁹²; Szlinder-Richert et al., ²0¹0). The variation in the chemical composition of Baltic sprat and Baltic herring is related to nutrition, catching season, fish size, seasonal and sexual variations. Variation in chemical composition might lead to changes in sensory attributes, including flavor, aroma, texture, and visual appearance which control the acceptability of fish as food (Flick & Martin, ¹⁹⁹²). The aim of the current work was to evaluate the sensory properties and their relations to lipid content, water content and protein content of Baltic sprat and Baltic herring.MATERIALS AND METHODSSamplesTwenty-six samples of Baltic sprat and ³³ samples of Baltic herring were caughtfrom different locations in Estonia’s coastal waters (Gulf of Riga and Finland) from February ²00⁸ until March ²00⁹. Samples were taken only from spring-spawn fish populations. Samples of fish (about ² kg) were immediately frozen after landing and stored at −¹⁸°C. The fish was analyzed during two months followed to catching. The frozen fish samples were thawed at ⁴°C ⁴⁸ hours before analysis. Thawed fish were de-headed, gutted, de-boned and washed. Samples for sensory analysis were packed by two in aluminum foil, steamed for ¹0 minutes at ⁶⁵°C, and served immediately.Composition analysisSamples for composition analysis (minimum ¹ kg) were minced twice and kept at

4°C until analyzed within the same day. All measurements were carried out in triplicate. Water content of the fish samples was measured using a halogen analyzer (HR 83, Mettler Toledo, Switzerland). The protein content of the fish samples was measured by Kjeldhal method (Velp Scientifica UDK 142, Italy). The lipid content of fish was measured by Soxhlet method (Velp Scientifica SER 148 Solvent Extractor, Italy). All necessary reagents were purchased from Sigma-Aldrich, Germany.Sensory AnalysisDescriptive sensory analysis was performed by 5 trained panelists. Sensoryanalysis was conducted in a laboratory equipped with individual booths (ISO 8589–1988). The panelists were trained during five one-hour sessions to evaluate the appearance (skin color, meat color, gapping, broken skin, shape), flavor (off-flavor, characteristic flavor, off-aroma, characteristic aroma, sweetness), and texture (hardness, bone separation, cohesiveness, moistness, greasiness) attributes. All of the490

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panelists had at least 50 hours of previous testing experience in descriptive evaluation of fish products. The samples were coded with random three-digit numbers and evaluated in two repetitions on a 9-point numerical scale, anchored at both ends. The evaluations took place shortly after the samples were steamed. Unsalted crackers and purified water were available for palate cleansing.Statistical AnalysisThe data was analyzed using the Unscrambler 9.8 (Camo Software, Norway).Partial Least Squares (PLS1) regression was used to plot average sensory scores and composition data. The data matrix included humidity content and sensory attribute scores for data regression. XLSTAT (2009, Addinsoft, France) was used to calculate the correlations between sensory and chemical or biological properties (Pearson, P = 0.05).RESULTS AND DISCUSSIONBaltic spratComposition of Baltic sprat remained in range 57–73% water, 15–17% protein,10–24% lipid, and 2–4% ash (Fig. 1). Krosing and Veldre (1973) showed in their studies that Baltic sprat composition was 66–80% water, 15–17% protein and 3–18% lipid. Research results showed an inverse relationship between water and lipid – the lower the water content the higher the lipid content and vice versa, which has also been observed by other researchers (Rehbein & Oehlenschläger, 2009). Lipid content of the fish increased from 13 ± 1.6% in spring to maximum values in October and November, on average 22 ± 3%. As the lipid content increased, the water content decreased in8060, %geSpring Summer Autumn Wintertan 40ercPe20012.02.0822.05.0830.08.0808.12.0818.03.09B.H waterB.S waterB.H proteinB.S proteinB.H lipidB.S lipidB.H ashB.S ashFigure 1. Baltic sprat & Baltic herring composition, where B.H water – Baltic herringwater%, B.S water – Baltic sprat water%, B.H protein – Baltic herring protein%, B.Sprotein – Baltic sprat protein%, B.H lipid – Baltic herring lipid%, B.S lipid – Balticsprat lipid%, B.H ash – Baltic herring ash%, B.S ash – Baltic sprat ash%.491

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, , , ,




495-500 the following day. All measurements were carried out in three repetitions.The moisture content of the fish samples was measured using a halogen moistureanalyzer (HR , Mettler Toledo, Switzerland). The protein content of the fish samples was measured by Kjeldahl method (Velp Scientifica UDK , Italy). The lipid content of fish was measured by Soxhlet method (Velp Scientifica SER Solvent Extractor, Italy).The fatty acid profile of trout samples was determined as fatty acid methyl esters(FAMEs). The Bligh & Dyer () method was used for lipid extraction. The FAMEs were prepared according to the standard EVS–EN ISO 0:000. The prepared methyl esters samples were injected into the gas chromatograph (Agilent 0A GC System) equipped with a flame ionization detector (FID) at a split ratio of :0. Helium served as the carrier gas (flow ml per min). Agilent J&W GC Column HP– (0 m x 0. mm x 0. μm) was used for the separation of FAMEs. The analytical conditions were: injector port temperature −0°C and detector temperature −0°C. The oven was programmed from –0°C. Retention times of FAMEs of the standard mixture were used to identify chromatographic peaks of the sample. Supelco Component FAME mix was used as standard FAME mixture. Fatty acid content in the samples was calculated, based on the peak area ratio and expressed as g fatty acid per 00 g lipid.All necessary reagents were purchased from Sigma-Aldrich, Germany.Statistical AnalysisXLSTAT (00, AddInsoft, France) was used for lipid, moisture, protein, and FA(P = 0.0) Analysis of Variance (ANOVA) between samples. Principal Component Analysis (PCA) was used to visualize relations between samples. Statistically significant correlations (Pearson, P = 0.0) are given in this paper. Samples were clustered using K-means clustering according to the lipid content.RESULTS AND DISCUSSIONMoisture, lipid and protein composition of Estonian farmed and importedRainbow trout are shown in Table . The moisture content was in the range of .-.%, the lipid content was in the range of .-.%, and the protein ranged from .-.%. There was a strong negative correlation between water and lipid content of the Rainbow trout (R = −0., P = 0.0). According to the lipid content clustering analysis (k–means) was performed, which indicated that there were three Rainbow trout groups: group (G)–lipid content .–.%; group (G)–lipid content .–.%; and group (G)–lipid content .–.%.The PCA plot (Figure ) shows the location of the Rainbow trout in multivariatespace according to the first (PC) and second (PC) principal component. The first and second principal components explained % of the total variance between the samples. The PCA plot confirms Rainbow trout grouping into three groups as the first principal component divides the samples according to their lipid contents. Sample T was higher in protein content than the rest of the samples.The FA contents of Estonian farmed and imported Rainbow trout are shown inTable . In all samples, C:0, C:, and docosahexaenoic acid (DHA, C:n–) were dominant, which has also been observed by other researchers (Blanchet et al., 00; Suzuki et al., ). The linoleic acid (:n–) content in all analyzed Rainbow
Rainbow Trout Composition and Fatty Acid Content in Estonia L. Timberg1,2, R. Kuldjärv1,2, K. Koppel1,2 and T.Paalme1,2 1Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15B, 12618 Tallinn, Estonia; e–mail: loreida@tftak.eu; rain@tftak.eu; kadri@tftak.eu 2Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086 Tallinn, Estonia; e–mail: tpaalme@staff.ttu.ee Abstract. Rainbow trout (Oncorhynchus mykiss) is the most popular aquaculture species in Estonia. The aim of the present study was to examine and compare moisture, protein, lipid and fatty acid (FA) compositions in Rainbow trout from different fish farms in Estonia and that farmed in Finland and Norway. The total lipid content in different Rainbow trout varied more than 5.5 fold, but FA proportions were very similar in all Rainbowtrout. However, it is important to note that Estonian farmed Rainbow trout had generally lower lipid content and therefore also a lower amount of essential FAs. Key words: Fatty acid, lipid, Rainbow trout (Oncorhynchus mykiss) INTRODUCTION Natural resources of fish can no longer fulfil demands of fish consumers; the shortage is forcing the aquaculture sector to expand. There are about 15 fish farms in Estonia where Rainbow trout is cultured. The annual volume of Estonian farmed Rainbow trout is about 700 tons, but many fish farms are expanding; production is expected to double in the next few years. Therefore, the Estonian aquaculture sector is interested in producing Rainbow trout which has high nutritional value, stabile quality and is also compatible with Rainbow trout farmed in other countries. Fat is one of the most important components of fish meat. It attracts consumers’ attraction due to the fatty acid (FA) profile, especially n–3 and n–6 FAs (Ruxton et al., 2004; Breslow, 2006). Therefore, the main aim of the study was to characterize and compare the moisture, protein, lipid and FA profiles of Rainbow trout from different fish farms in Estonia and imported Rainbow trout available in Estonian supermarkets. Experiment design Rainbow trout samples from ten different aquaculture facilities in Estonia were acquired (samples E1–E10). Fish were gutted, packed in ice and transported to the laboratory on the day of slaughter; all analyses were performed the next day. Three samples of Rainbow trout (imported) cultured in other countries were purchased from Estonian supermarkets (sample T1–Finland, T2–Norway, and T3–Finland). The imported trout had been slaughtered 4–6 days before purchase and had already been gutted, packed in plastic bag, and transported to the laboratory within an hour after purchase, where the fish was immediately packed in ice. All analyses were performed 495
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Rainbow Trout Composition and Fatty Acid Content in Estonia L. Timberg1,2, R. Kuldjärv1,2, K. Koppel1,2 and T.Paalme1,2 1Competence Centre of Food and Fermentation Technologies, Akadeemia tee 15B, 12618 Tallinn, Estonia; e–mail: loreida@tftak.eu; rain@tftak.eu; kadri@tftak.eu 2Tallinn University of Technology, Department of Food Processing, Ehitajate tee 5, 19086 Tallinn, Estonia; e–mail: tpaalme@staff.ttu.ee Abstract. Rainbow trout (Oncorhynchus mykiss) is the most popular aquaculture species in Estonia. The aim of the present study was to examine and compare moisture, protein, lipid and fatty acid (FA) compositions in Rainbow trout from different fish farms in Estonia and that farmed in Finland and Norway. The total lipid content in different Rainbow trout varied more than 5.5 fold, but FA proportions were very similar in all Rainbowtrout. However, it is important to note that Estonian farmed Rainbow trout had generally lower lipid content and therefore also a lower amount of essential FAs. Key words: Fatty acid, lipid, Rainbow trout (Oncorhynchus mykiss) INTRODUCTION Natural resources of fish can no longer fulfil demands of fish consumers; the shortage is forcing the aquaculture sector to expand. There are about 15 fish farms in Estonia where Rainbow trout is cultured. The annual volume of Estonian farmed Rainbow trout is about 700 tons, but many fish farms are expanding; production is expected to double in the next few years. Therefore, the Estonian aquaculture sector is interested in producing Rainbow trout which has high nutritional value, stabile quality and is also compatible with Rainbow trout farmed in other countries. Fat is one of the most important components of fish meat. It attracts consumers’ attraction due to the fatty acid (FA) profile, especially n–3 and n–6 FAs (Ruxton et al., 2004; Breslow, 2006). Therefore, the main aim of the study was to characterize and compare the moisture, protein, lipid and FA profiles of Rainbow trout from different fish farms in Estonia and imported Rainbow trout available in Estonian supermarkets. Experiment design Rainbow trout samples from ten different aquaculture facilities in Estonia were acquired (samples E1–E10). Fish were gutted, packed in ice and transported to the laboratory on the day of slaughter; all analyses were performed the next day. Three samples of Rainbow trout (imported) cultured in other countries were purchased from Estonian supermarkets (sample T1–Finland, T2–Norway, and T3–Finland). The imported trout had been slaughtered 4–6 days before purchase and had already been gutted, packed in plastic bag, and transported to the laboratory within an hour after purchase, where the fish was immediately packed in ice. All analyses were performed 495

the following day. All measurements were carried out in three repetitions.The moisture content of the fish samples was measured using a halogen moistureanalyzer (HR ⁸³, Mettler Toledo, Switzerland). The protein content of the fish samples was measured by Kjeldahl method (Velp Scientifica UDK ¹⁴², Italy). The lipid content of fish was measured by Soxhlet method (Velp Scientifica SER ¹⁴⁸ Solvent Extractor, Italy).The fatty acid profile of trout samples was determined as fatty acid methyl esters(FAMEs). The Bligh & Dyer (¹⁹⁵⁹) method was used for lipid extraction. The FAMEs were prepared according to the standard EVS–EN ISO ⁵⁵0⁹:²000. The prepared methyl esters samples were injected into the gas chromatograph (Agilent ⁷⁸⁹0A GC System) equipped with a flame ionization detector (FID) at a split ratio of ¹:¹0. Helium served as the carrier gas (flow ¹ ml per min). Agilent J&W GC Column HP–⁸⁸ (⁶0 m x 0.²⁵ mm x 0.² μm) was used for the separation of FAMEs. The analytical conditions were: injector port temperature −²⁵0°C and detector temperature −²⁸0°C. The oven was programmed from ¹²⁵–²³0°C. Retention times of FAMEs of the standard mixture were used to identify chromatographic peaks of the sample. Supelco ³⁷ Component FAME mix was used as standard FAME mixture. Fatty acid content in the samples was calculated, based on the peak area ratio and expressed as g fatty acid per ¹00 g lipid.All necessary reagents were purchased from Sigma-Aldrich, Germany.Statistical AnalysisXLSTAT (²0¹0, AddInsoft, France) was used for lipid, moisture, protein, and FA(P = 0.0⁵) Analysis of Variance (ANOVA) between samples. Principal Component Analysis (PCA) was used to visualize relations between samples. Statistically significant correlations (Pearson, P = 0.0⁵) are given in this paper. Samples were clustered using K-means clustering according to the lipid content.RESULTS AND DISCUSSIONMoisture, lipid and protein composition of Estonian farmed and importedRainbow trout are shown in Table ¹. The moisture content was in the range of ⁶³.⁸-⁷³.⁴%, the lipid content was in the range of ².¹-¹¹.⁶%, and the protein ranged from ¹⁹.⁷-²³.¹%. There was a strong negative correlation between water and lipid content of the Rainbow trout (R = −0.⁹², P = 0.0⁵). According to the lipid content clustering analysis (k–means) was performed, which indicated that there were three Rainbow trout groups: group ¹ (G¹)–lipid content ².¹–³.⁹%; group ² (G²)–lipid content ⁴.⁶–⁷.¹%; and group ³ (G³)–lipid content ⁹.³–¹¹.⁶%.The PCA plot (Figure ¹) shows the location of the Rainbow trout in multivariatespace according to the first (PC¹) and second (PC²) principal component. The first and second principal components explained ⁹⁸% of the total variance between the samples. The PCA plot confirms Rainbow trout grouping into three groups as the first principal component divides the samples according to their lipid contents. Sample T³ was higher in protein content than the rest of the samples.The FA contents of Estonian farmed and imported Rainbow trout are shown inTable ². In all samples, C¹⁶:0, C¹⁸:¹, and docosahexaenoic acid (DHA, C²²:⁶n–³) were dominant, which has also been observed by other researchers (Blanchet et al., ²00⁵; Suzuki et al., ¹⁹⁸⁶). The linoleic acid (¹⁸:²n–⁶) content in all analyzed Rainbow

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Table 1. Moisture, lipid and protein composition (g per100 g wet meat) in Rainbow trout.SampleMoistureProteinLipidE170.3 ± 0.421.3 ± 0.03.0 ± 0.2E271.1 ± 0.320.7 ± 0.34.6 ± 0.1E371.1 ± 0.320.5 ± 0.24.9 ± 0.0E473.4 ± 0.720.4 ± 0.32.1 ± 0.2E567.6 ± 1.219.8 ± 0.05.3 ± 0.4E672.4 ± 0.220.2 ± 0.53.5 ± 0.1E770.3 ± 0.420.7 ± 0.25.3 ± 0.2E871.8 ± 1.519.8 ± 0.23.9 ± 0.1E970.0 ± 1.419.6 ± 0.95.4 ± 0.4E1067.6 ± 0.920.8 ± 0.97.1 ± 0.6T163.8 ± 0.619.9 ± 0.211.6 ± 0.3T265.2 ± 1.019.7 ± 0.19.3 ± 0.5T368.2 ± 0.223.1 ± 0.35.2 ± 0.1

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501-507 Key words: chlorophyll, growth stages, leaf nitrogen, protein, Rubisco, tissues, tomatoINTRODUCTIONThe rate of photosynthesis and biomass accumulation depends largely on thequantity and activity of Rubisco (Lorimer, ). Rubisco is the first and key enzyme in the Calvin cycle of photosynthetic assimilation of CO in C plants. It catalyzes the fixation of atmospheric CO to ribulosa-,-bisphosphate (RuBP) to form two molecules of -phosphoglycerate (PGA), which is subsequently used to build organic molecules. The enzyme is extremely inefficient and its carboxylation activity is compromised by competing side–reactions, the most notable being with another atmospheric gas, O. Both CO and O are mutually competitive at the same large-subunit active site. Whereas carboxylation accounts for net CO fixation, oxygenation leads to the loss of CO in the photorespiratory pathway. In order to catalyse photosynthetic CO fixation at high rates, large amounts of Rubisco are needed to compensate the slow catalytic rate of the enzyme. It has been estimated that Rubisco
Differences in Rubisco and Chlorophyll Content among Tissues and Growth Stages in Two tomato (Lycopersicon esculentum Mill.) Varieties R. Vicente1,2, R. Morcuende1 and J. Babiano2 1Institute of Natural Resources and Agrobiology of Salamanca, IRNASA–CSIC, Apartado 257, 37071 Salamanca, Spain; e-mails: ruben.vicente@irnasa.csic.es; rosa.morcuende@irnasa.csic.es 2University of Salamanca, Department of Plant Physiology, Campus Miguel de Unamuno, 37008 Salamanca, Spain; e-mail: babiano@usal.es Abstract. Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in the photosynthetic assimilation of CO2 and the most abundant leaf protein. The amounts ofchlorophyll (chl) and Rubisco have often been considered, respectively, as indices of light harvesting and Calvin cycle capacities of leaves. The purpose of this study was to analyze the changes in chlorophyll content and the level of Rubisco protein in various plant tissues at different growth stages in two tomato (Lycopersicon esculentum Mill.) varieties. The results show an increase of the amount of both chlorophyll and Rubisco protein at vegetative growth stages (leaf expansion), which was followed by a gradual decline during anthesis, probably as a consequence of changes in the balance of their synthesis and degradation reported previously –Rubisco could be remobilized and reused in the production of reproductive structures. However, the increase in the amount of Rubisco and chlorophyll at ripening stage (more in Tres Cantos variety) contrasts with the decrease reported in other studies when degradation is becoming predominant during senescence.
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Differences in Rubisco and Chlorophyll Content among Tissues and Growth Stages in Two tomato (Lycopersicon esculentum Mill.) Varieties R. Vicente1,2, R. Morcuende1 and J. Babiano2 1Institute of Natural Resources and Agrobiology of Salamanca, IRNASA–CSIC, Apartado 257, 37071 Salamanca, Spain; e-mails: ruben.vicente@irnasa.csic.es; rosa.morcuende@irnasa.csic.es 2University of Salamanca, Department of Plant Physiology, Campus Miguel de Unamuno, 37008 Salamanca, Spain; e-mail: babiano@usal.es Abstract. Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) is a key enzyme in the photosynthetic assimilation of CO2 and the most abundant leaf protein. The amounts ofchlorophyll (chl) and Rubisco have often been considered, respectively, as indices of light harvesting and Calvin cycle capacities of leaves. The purpose of this study was to analyze the changes in chlorophyll content and the level of Rubisco protein in various plant tissues at different growth stages in two tomato (Lycopersicon esculentum Mill.) varieties. The results show an increase of the amount of both chlorophyll and Rubisco protein at vegetative growth stages (leaf expansion), which was followed by a gradual decline during anthesis, probably as a consequence of changes in the balance of their synthesis and degradation reported previously –Rubisco could be remobilized and reused in the production of reproductive structures. However, the increase in the amount of Rubisco and chlorophyll at ripening stage (more in Tres Cantos variety) contrasts with the decrease reported in other studies when degradation is becoming predominant during senescence.

Key words: chlorophyll, growth stages, leaf nitrogen, protein, Rubisco, tissues, tomatoINTRODUCTIONThe rate of photosynthesis and biomass accumulation depends largely on thequantity and activity of Rubisco (Lorimer, ¹⁹⁸¹). Rubisco is the first and key enzyme in the Calvin cycle of photosynthetic assimilation of CO² in C³ plants. It catalyzes the fixation of atmospheric CO² to ribulosa-¹,⁵-bisphosphate (RuBP) to form two molecules of ³-phosphoglycerate (³PGA), which is subsequently used to build organic molecules. The enzyme is extremely inefficient and its carboxylation activity is compromised by competing side–reactions, the most notable being with another atmospheric gas, O². Both CO² and O² are mutually competitive at the same large-subunit active site. Whereas carboxylation accounts for net CO² fixation, oxygenation leads to the loss of CO² in the photorespiratory pathway. In order to catalyse photosynthetic CO² fixation at high rates, large amounts of Rubisco are needed to compensate the slow catalytic rate of the enzyme. It has been estimated that Rubisco

501

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accounts for a quarter of leaf nitrogen and up to half of the soluble protein in leaves of C3 plants (Ellis, 1979), and it is probably the most abundant protein of the world (Portis & Parry, 2007). In higher plants Rubisco is composed of large and small subunits in a hexadecameric structure. The catalytic large subunit is encoded by a single gene in the chloroplast genome and the small subunit is coded by a family of closely related nuclear genes (Portis & Parry, 2007; Andersson & Backlund, 2008).The amount of Rubisco in a leaf is determined by the balance between itssynthesis and degradation (Imai et al., 2008). Rubisco synthesis is most active during leaf expansion or during the greening of etiolated leaf tissue (Ishizuka et al., 2004; Imai et al., 2005; 2008; Suzuki et al., 2010) but very limited after full leaf expansion (Miller & Huffaker, 1982), and it is actively degraded during leaf senescence (Miller & Huffaker, 1982; Suzuki et al., 2010). Thus, in many higher plants Rubisco also appears to serve as a leaf storage protein that can be hydrolyzed and the nitrogen derived from the degraded Rubisco is remobilized and reused in developing new tissues (Evans, 1989; Imai et al., 2005) at the expense of leaf photosynthetic activity, which declines in parallel with Rubisco content (Makino et al., 1992). Changes in Rubisco synthesis have been primarily explained by changes in transcript abundance of the encoding genes of the enzyme. The levels of these mRNAs are high in expanding leaves and decline in senescing leaves. Different studies suggest a highly positive correlation between Rubisco content and mRNA levels in the leaves of plants grown under different nitrogen supplies (Evans, 1989; Makino et al., 1992; Imai et al., 2005; 2008; Suzuki et al., 2010). In addition, nitrogen influx into the leaf seems to be closely related to Rubisco synthesis, and N influx declines in the senescent leaf.Chlorophylls are the most important group of photosynthetic pigmentsresponsible for light absorption and are found in the thylakoids of the chloroplasts. They contain a porphyrin ring with a magnesium ion in the center of the molecule and a long hydrophobic tail that anchors them in the membrane. It has also been shown that the level of chlorophyll is increased in young expanding leaves and decreased substantially during senescence (Imai et al., 2005; 2008).Tomato is one of the most important vegetable crops cultivated for its fleshy fruit,which is a rich source of minerals, vitamins, organic acids and dietary fibers and is considered as a protective food. The dual role of Rubisco as a key photosynthetic enzyme and a major nitrogen-containing compound in leaves predetermines its importance for plant productivity and its use as a selection criterion for high yield can be hypothesized. With this objective the aim of the present study was to compare the developmental changes in the chlorophyll content and the level of Rubisco protein in different plant tissues in two tomato varieties –Tres Cantos and Cherry, which were grown hydroponically under controlled environmental conditions.MATERIALS AND METHODSPlant material and growth conditionsTomato seeds (Lycopersicon esculentum Mill.) of two varieties, Tres Cantos andCherry, were sown in rockwool in darkness at 24°C for germination. Afterwards, seedlings were grown hydroponically in pots containing vermiculite. The pots were placed in a growth chamber with 140 ± 14 μmol m−2 s−1 photon flux density, 19–502

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509-514 sodium chlorophylline, provitamin concentrate, etc. A small number of investigations were carried out on chlorophyll – carotene paste utilization in poultry feeding (Vitina et al., ).The investigative data testified that the inclusion of pine needles’ chlorophyll –carotene paste and oils in poultry feed increased live weight by .0–.0% and improved immunity, increasing the functional activity of blood immunocytes, the quality of circulating immune complexes, protein, albumen, beta – gamma globulins and lysozyme (Remeze & Аndersons, ). The investigations of improved poultry meat and egg quality by using pine needle products have not been carried out.According to our data, spruce needle biomass contains higher amounts ofbiologically active substances (β–carotenoids, fatty acids, vitamins. etc.) in comparison with pine needle biomass (Ievins et al., ). Therefore the aim of our investigations was to evaluate the influence of spruce needles’ total extractives and, separately, neutral extract group substances on broiler chicken productivity and meat quality by specifying the amount of ω– fatty acids and carotenoids in broiler chicken meat and to point to its possible use to obtain broiler chicken meat with innovative composition.MATERIALS AND METHODSThe investigation was carried out with cross ROSS 0 broiler chickens from 0 to
Applying Spruce Needle Extractives in Broiler Chicken Feeding I. Vitina1, V. Krastina1, M. Daugavietis2, J. Miculis1 and S. Cerina1 1Research Institute of Biotechnology and Veterinary Medicine “Sigra” of Latvia University of Agriculture, Institūta 1, Sigulda, Latvia, LV-2150; e-mail: sigra@lis.lv 2Latvian State Forest Research Institute “Silava”, Rigas 111, Salaspils, Latvia, LV 2169; e-mail: inst@silava.lv Abstract. Spruce needle extractive substances were produced from a forestry by-product –green biomass of spruce needles. Spruce needle extracts contain a significant amount of natural biologically active substances. During our investigations the evaluation of biologically active substances from spruce needle total extract and of neutral extracts was carried out to assess their effects on innovative composition broiler chicken meat. The feeding trial was conducted with cross Ross 308 broiler chickens by adding the spruce needle total extract and, separately, neutral extract substances to the composition of broiler chickens’ diet. Using spruce needle extractive substances increased live weight on average by 4.31–7.58% (P < 0.05) and decreased feed conversion by 6.28–7.33% in comparison with the control group. The use of neutral extract substances in the poultry diet improved the composition of fatty acids, increased the amount of total carotenoids by 0.45–0.57 mg kg−1 and decreased the cholesterol level by 11.16–23.66 mg 100g−1 in meat. Key words: broiler chickens, meat quality, spruce needle extractives INTRODUCTION Complexes of natural biologically active substances can be extracted by nonpolar organic solvent from spruce needle biomass and could be utilized to substitute for synthetic preparations in animal diets. The major active ingredients in the biologically active substance of pine and spruce needle total extract are chlorophyll and its derivates: carotenoids, vitamin E, vitamin K, phytosterols, polyprenols, squalene, sodium salts of resin acids (balsamic compounds) and essential oils (Andersons et al., 1983; Ievins et al., 1976). The previously mentioned biologically active substances have a broad therapeutic and prophylactic influence on poultry and on the human organism. By including the determined biologically active substances contained by spruce needle extracts in the poultry diet, fatty acids, antioxidants, and vitamins desirable for human organisms are transferred from the feed to poultry meat. Other researchers (Leskanich & Noble, 1997) have confirmed that the transfer of biologically active substances from feed to production takes place, resulting in products of innovative composition enriched with biologically active spruce needle extract natural substances. In the 1970’s, only the following biologically active complexes were extracted from pine needles and pine needle biomass extracts: chlorophyll – carotene paste, 509
Abstract |

Applying Spruce Needle Extractives in Broiler Chicken Feeding I. Vitina1, V. Krastina1, M. Daugavietis2, J. Miculis1 and S. Cerina1 1Research Institute of Biotechnology and Veterinary Medicine “Sigra” of Latvia University of Agriculture, Institūta 1, Sigulda, Latvia, LV-2150; e-mail: sigra@lis.lv 2Latvian State Forest Research Institute “Silava”, Rigas 111, Salaspils, Latvia, LV 2169; e-mail: inst@silava.lv Abstract. Spruce needle extractive substances were produced from a forestry by-product –green biomass of spruce needles. Spruce needle extracts contain a significant amount of natural biologically active substances. During our investigations the evaluation of biologically active substances from spruce needle total extract and of neutral extracts was carried out to assess their effects on innovative composition broiler chicken meat. The feeding trial was conducted with cross Ross 308 broiler chickens by adding the spruce needle total extract and, separately, neutral extract substances to the composition of broiler chickens’ diet. Using spruce needle extractive substances increased live weight on average by 4.31–7.58% (P < 0.05) and decreased feed conversion by 6.28–7.33% in comparison with the control group. The use of neutral extract substances in the poultry diet improved the composition of fatty acids, increased the amount of total carotenoids by 0.45–0.57 mg kg−1 and decreased the cholesterol level by 11.16–23.66 mg 100g−1 in meat. Key words: broiler chickens, meat quality, spruce needle extractives INTRODUCTION Complexes of natural biologically active substances can be extracted by nonpolar organic solvent from spruce needle biomass and could be utilized to substitute for synthetic preparations in animal diets. The major active ingredients in the biologically active substance of pine and spruce needle total extract are chlorophyll and its derivates: carotenoids, vitamin E, vitamin K, phytosterols, polyprenols, squalene, sodium salts of resin acids (balsamic compounds) and essential oils (Andersons et al., 1983; Ievins et al., 1976). The previously mentioned biologically active substances have a broad therapeutic and prophylactic influence on poultry and on the human organism. By including the determined biologically active substances contained by spruce needle extracts in the poultry diet, fatty acids, antioxidants, and vitamins desirable for human organisms are transferred from the feed to poultry meat. Other researchers (Leskanich & Noble, 1997) have confirmed that the transfer of biologically active substances from feed to production takes place, resulting in products of innovative composition enriched with biologically active spruce needle extract natural substances. In the 1970’s, only the following biologically active complexes were extracted from pine needles and pine needle biomass extracts: chlorophyll – carotene paste, 509

sodium chlorophylline, provitamin concentrate, etc. A small number of investigations were carried out on chlorophyll – carotene paste utilization in poultry feeding (Vitina et al., ¹⁹⁸⁹).The investigative data testified that the inclusion of pine needles’ chlorophyll –carotene paste and oils in poultry feed increased live weight by ¹².0–¹⁶.0% and improved immunity, increasing the functional activity of blood immunocytes, the quality of circulating immune complexes, protein, albumen, beta – gamma globulins and lysozyme (Remeze & Аndersons, ¹⁹⁹⁹). The investigations of improved poultry meat and egg quality by using pine needle products have not been carried out.According to our data, spruce needle biomass contains higher amounts ofbiologically active substances (β–carotenoids, fatty acids, vitamins. etc.) in comparison with pine needle biomass (Ievins et al., ¹⁹⁸⁶). Therefore the aim of our investigations was to evaluate the influence of spruce needles’ total extractives and, separately, neutral extract group substances on broiler chicken productivity and meat quality by specifying the amount of ω–³ fatty acids and carotenoids in broiler chicken meat and to point to its possible use to obtain broiler chicken meat with innovative composition.MATERIALS AND METHODSThe investigation was carried out with cross ROSS ³0⁸ broiler chickens from 0 to

42 days age (n = 300; Table 1).Table 1. Experimental design.GroupFeeding programmeBasic dietAdditives of biologically active substances complex1st group – controlBasic diet *–2nd group – trialBasic dietTotal extractive substances from spruce needle biomass3rd group – trialBasic dietNeutral extractives substances from spruce needle biomass
*the content complies with the standard requirements (Management Essentials, 1999).The basic diet composition was the same for all broiler chicken groups. In thediet, the content of crude protein and metabolizable energy at the different ages was on average 19.0–24.0% and 11.90–12.80 MJ kg−1. From the age of 7 days, the spruce needle total extractives-containing additive was added to the second group of broiler chickens in doses of 0.04–0.05%. To the diet of the 3rd group, the spruce needle neutral extractives substances additive that was obtained by extraction from the total extractives mass was added in doses of 0.04–0.05%. Additives used were in the form of paste, and were dark green in colour.The main indices of poultry productivity and meat quality were recorded andevaluated during the investigation period. The gas chromatography method was used for fatty acids and carotenoids analyses, the Blur colorimetric method, for cholesterol analysis. The statistical analysis was performed using SPSS17. Statistical significance was declared at P < 0.05. The data were presented as means and standard errors.510

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RESULTS AND DISCUSSIONDuring the trial cross ROSS 308 the broiler chickens’ productivity was high. Onaverage, the broiler chicken live weight at age 42 days ranged from 3.12–3.36 kg, live weight gain per day was from 73.32–78.97 g and feed consumption for obtaining 1 kg of live weight (feed conversion) was within limits from 1.77–1.91 kg kg−1 (Table 2).Table 2. Productivity of broiler chickens.Parameters1st group2nd group3rd groupLive weight at the age of 423,123 ± 513,360 ± 573,258 ± 54days, g % to control–7.58*4.31*Live weight gain per day, g73.3278.9776.48% to control–7.714.31Feed conversion, kg kg−1 1.911.79 1.77% to control–6.287.33*P < 0.05By including a composition of spruce needle total extractives and neutralextractives substance additives in their diet, the broiler chickens’ live weight for sale increased by 4.31–7.58%; correspondingly, the live weight daily gain increased by 4.31–7.71% and feed conversion positively decreased by 6.28–7.33% in comparison with the control group (P < 0.05). A similar stimulating effect on the chickens’ live weight gain was ascertained by feeding them the pine needle total extractives substance chlorophyll – carotene paste (Fishers, 1971).By using the spruce needle total extractives-containing additive in the broilerchickens’ diet, their live weight had a tendency to increase in comparison with the live weight of the 2nd and 3rd groups of chickens. The results can be connected with the comparative higher biologically active substances profile and amount in the total extractives additive.From the consumer’s point of view, the essential significance was the effect ofspruce needle extractives on fatty acids, cholesterol and antioxidant (carotenoids) content in muscle tissue.Muscle tissue of the broiler chickens contained on average 22.51–24.06%saturated, 48.90–45.80% monounsaturated, and 26.58–27.73% polyunsaturated (Table 3).Supplementing the broiler chickens’ diet with only a little (by 1.46–1.55%) spruceneedle extractive decreased the undesirable amount of saturated fat in broiler chicken meat, and also promoted myristic fatty acid decrease. The amount of monounsaturated and polyunsaturated fat, desirable for the human organism, was increased correspondingly by 3.10–2.59% and 1.13–1.15% in broiler chicken muscle tissue (P < 0.05).At the same time the ω–6 fatty acids level in muscle tissue was not influenced bythe spruce needle extractives additives to the broiler chicken diet. The addition of spruce needle extractives increased the ω–3 fatty acids total amount by 0.53–0.57%, including an increase in eicosapentaenoic fatty acid by 0.11–0.13% and511

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515-520 R. Vokk, T. Lõugas, K. Mets and M. Kravets
Dill (Anethum graveolens L.) and Parsley (Petroselinum crispum (Mill.) Fuss) from Estonia: Seasonal Differences in Essential Oil Composition
Abstract |

Dill (Anethum graveolens L.) and Parsley (Petroselinum crispum (Mill.) Fuss) from Estonia: Seasonal Differences in Essential Oil Composition

R. Vokk, T. Lõugas, K. Mets and M. Kravets

Tallinn University of Technology, Ehitajate tee ⁵, EE¹⁹0⁸⁶, Tallinn, Estonia;e-mail: raivov@hotmail.com

Abstract:

The essential oil content and composition of dill and parsley growing in summer and wintertime in Estonia were studied using the Clevenger distillation method for oil isolation and gas chromatography for identifying the extracts. Antimicrobial activity against several test microorganisms (Escherichia coli, Staphylococcus albus, Bacillus mesentericus and Aspergillus flavus) was studied using the zone-of-inhibition method. The essential oil yield of dried aromatic plants grown in wintertime was 0.²⁴% of dry weight for parsley, and 0.⁵⁶% for dill, and 0.²⁹% and 0.⁶⁵% for plants, grown in summer, respectively. Twenty-five (²⁵) compounds were identified representing over ⁹⁸% of the oil components of dill and dill seeds. The principal components of dill leaf oil were α-Phellandrene (⁴⁷.⁷–⁶².⁵%), myristicin (¹.⁷–²⁸.²%), dill ether (0.⁹–¹⁴.⁸%), β-phellandrene (⁷.⁴–⁷.⁵%), and limonene (³.⁷–³.⁸%). Thirty-four (³⁴) essential oil components were identified in parsley leaves (≥ ⁹⁶%) with the major constituents myristicin (³0.⁷–⁴².⁷%), β-phellandrene (²¹.⁸–³⁵.⁹%), p-¹,³,⁸-menthatriene (⁵.⁴–¹0.0%), andβ-myrcene (⁴.⁵–⁸.⁷%). Essential oils from summer grown plants possessed higher antimicrobial activity against all studied microorganisms.

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, , , ,




521-526 was probably due primarily to T- toxin (Sarkisov, ; Joffe, ; Yli-Mattila et al., 0). In the 0’s high levels of T- toxin were also found in maize in USA (Mirocha & Patre, ).In Nordic countries oats are more severely affected by mycotoxins than wheat andbarley, which may result high DON and T-/HT- levels (Yli-Mattila, 00). In the 000’s, T- toxin levels have been rising in oats in northern Europe (Yli-Mattila et al., 00, 00a; Edwards et al.; 00, Fredlund et al., 00). In 00 Norway had to import oat seed due to high Fusarium infection levels in Norwegian seed which inhibited germination. The increase of Fusarium toxin levels may be connected to climate change. The greatest oat producers in the world are in the Nordic countries. For example, in Finland about % of oats are used for human food (including baby food), thus the quality of the raw material for oat products is important.Until now, only a few sources of resistance to FHB in oats have been demonstrated(Björnstad, et al., 00; Tekauz et al., 00; Gagkaeva et al., 00). One of the problems in breeding resistant cultivars in oats and barley is the lack of clear visual symptoms, which could be scored to assess the level of FHB. That is why other parameters connected to mycotoxins, such as Fusarium DNA, should be evaluated. In the present review paper I will concentrate on trichothecene-producing Fusarium species.Type A and B trichothecene-producing fusarium speciesDON is the most important trichothecene in Europe and Asia. It is produced bytype B trichothecene-producing species of the F. graminearum species complex and F. culmorum. In northern Europe the highest DON levels have been found in oats (Yli-Mattila, 00). Nivalenol, which is also a type B trichothecene, is produced by F. poae, F. cerealis and NIV genotypes of F. graminearum species complex and F. culmorum.T- toxin is the main type A trichothecene. The main Fusarium species producingT- toxin have been difficult to identify. The researchers have agreed that they belong to the Sporotrichiella section of the Fusarium species. In the Soviet Union the Fusarium species causing ATA was identified as F. sporotrichioides (Sarkisov, ), but for a long time the researchers occasionally thought that both F. sporotrichioides and F. poae isolates of Sporotrichiella section or even F. tricinctum might be effective in producing T- toxin (Joffe, ; Burkin et al., 00) due to identification problems. Since then it has been found that T- producing F. poae isolates (Torp & Nirenberg, 00; Yli-Mattila et al., 0) differ from typical F. poae isolates, which do not produce high levels of T- toxin. Based on these investigations it seems that in Europe a new species F. langsethiae is the main T--producer, while in the Russian Far East another new species F. sibiricum is also involved in T- production together with F. sporotrichioides. There are two subgroups or populations of F. langsethiae, which are morphologically similar to F. poae, but phylogenetically are closer to F. sporotrichioides (Konstantinova & Yli-Mattila, 00; Yli-Mattila et al., 00; 0). T- toxin production is affected by water activity and temperature and the toxin is rapidly metabolized to HT- toxin (Jestoi et al., 00; 00; Medina & Magan, 0).DNA extraction, detection and quantificationDifferent commercial kits are available for DNA extraction and should beoptimised for the fungal DNA from plant material. The quality and quantity of DNA can be estimated by gel electrophoresis and spectrophotometer or by different
Detection of Trichothecene-producing Fusarium Species in Cereals in Northern Europe and Asia T. Yli-Mattila Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland.; e-mail: tymat@utu.fi Abstract. Several toxigenic trichothecene-producing and nonproducing Fusarium species are involved in Fusarium head blight, which reduces both crop yield and quality in cereals. Climate change has altered crop production in many countries, and this in turn influences the pathogen populations. E.g. in northern areas a risk will be new toxigenic Fusarium species spreading to the north due to higher temperatures and the increased use of alternative hosts, such as maize, winter barley and winter oats. Traditional identifications and classifications of Fusarium species have been used for grouping isolates to species and grouping species according to shared morphological and cultural characteristics. During the last years researchers have started to use alternative ways for species identification and classification based on molecular data and phylogenetic analyses. The best way to identify and classify Fusarium isolates is the polyphasic approach by using all available characters. Key words: deoxynivalenol, Fusarium head blight, F. graminearum, F. langsethiae, F. sibiricum, F. sporotrichioides, nivalenol, T-2 toxin, trichothecene INTRODUCTION Fusarium species are the most important phytopathogenic and toxigenic fungi in Nordic countries and globally. Several Fusarium species are involved in Fusariumhead blight (FHB), which reduces both yield and quality in cereal crops. FHB was first described in England and the Russian Far East in the 1880’s, but Fusarium outbreaks had already started much earlier, e.g. in the Far East. Since then FHB has increased worldwide (McMullen et al., 1997; Bottalico & Perrone, 2002; Goswami & Kistler, 2004; Desjardins, 2006; Yli-Mattila, 2010). Fusarium toxins can be divided into those which already are under international regulation, i.e. deoxynivalenol (DON), zearalenone and fumonisins and to new emerging varieties, such as beauvericin, enniatins and culmorins. European regulations for nivalenol (NIV), T-2 toxin and HT-2 toxin, also produced by Fusarium species, are under evaluation. Chemically, trichothecenes can be divided into type A (e.g. T-2 and HT-2) and type B (e.g. NIV and DON) and their mono- and di-acetylated derivatives (Ueno, 1983). The greatest tragedy due to the Fusarium toxins took place in former Soviet Union before and during World War II, when harvesting was delayed and overwintered mouldy grains were consumed. In the Orenburg region near the Ural River more than 10% of the population were affected by the disease called alimentary toxic aleukia and mortality was high. The ATA (Alimentary Toxic Aleukia) syndrome in Soviet Union 521
Abstract |

Detection of Trichothecene-producing Fusarium Species in Cereals in Northern Europe and Asia T. Yli-Mattila Molecular Plant Biology, Department of Biochemistry and Food Chemistry, University of Turku, FI-20014 Turku, Finland.; e-mail: tymat@utu.fi Abstract. Several toxigenic trichothecene-producing and nonproducing Fusarium species are involved in Fusarium head blight, which reduces both crop yield and quality in cereals. Climate change has altered crop production in many countries, and this in turn influences the pathogen populations. E.g. in northern areas a risk will be new toxigenic Fusarium species spreading to the north due to higher temperatures and the increased use of alternative hosts, such as maize, winter barley and winter oats. Traditional identifications and classifications of Fusarium species have been used for grouping isolates to species and grouping species according to shared morphological and cultural characteristics. During the last years researchers have started to use alternative ways for species identification and classification based on molecular data and phylogenetic analyses. The best way to identify and classify Fusarium isolates is the polyphasic approach by using all available characters. Key words: deoxynivalenol, Fusarium head blight, F. graminearum, F. langsethiae, F. sibiricum, F. sporotrichioides, nivalenol, T-2 toxin, trichothecene INTRODUCTION Fusarium species are the most important phytopathogenic and toxigenic fungi in Nordic countries and globally. Several Fusarium species are involved in Fusariumhead blight (FHB), which reduces both yield and quality in cereal crops. FHB was first described in England and the Russian Far East in the 1880’s, but Fusarium outbreaks had already started much earlier, e.g. in the Far East. Since then FHB has increased worldwide (McMullen et al., 1997; Bottalico & Perrone, 2002; Goswami & Kistler, 2004; Desjardins, 2006; Yli-Mattila, 2010). Fusarium toxins can be divided into those which already are under international regulation, i.e. deoxynivalenol (DON), zearalenone and fumonisins and to new emerging varieties, such as beauvericin, enniatins and culmorins. European regulations for nivalenol (NIV), T-2 toxin and HT-2 toxin, also produced by Fusarium species, are under evaluation. Chemically, trichothecenes can be divided into type A (e.g. T-2 and HT-2) and type B (e.g. NIV and DON) and their mono- and di-acetylated derivatives (Ueno, 1983). The greatest tragedy due to the Fusarium toxins took place in former Soviet Union before and during World War II, when harvesting was delayed and overwintered mouldy grains were consumed. In the Orenburg region near the Ural River more than 10% of the population were affected by the disease called alimentary toxic aleukia and mortality was high. The ATA (Alimentary Toxic Aleukia) syndrome in Soviet Union 521

was probably due primarily to T-² toxin (Sarkisov, ¹⁹⁵⁴; Joffe, ¹⁹⁸⁶; Yli-Mattila et al., ²0¹¹). In the ¹⁹⁶0’s high levels of T-² toxin were also found in maize in USA (Mirocha & Patre, ¹⁹⁷³).In Nordic countries oats are more severely affected by mycotoxins than wheat andbarley, which may result high DON and T-²/HT-² levels (Yli-Mattila, ²0¹0). In the ²000’s, T-² toxin levels have been rising in oats in northern Europe (Yli-Mattila et al., ²00⁸, ²00⁹a; Edwards et al.; ²00⁹, Fredlund et al., ²0¹0). In ²0¹0 Norway had to import oat seed due to high Fusarium infection levels in Norwegian seed which inhibited germination. The increase of Fusarium toxin levels may be connected to climate change. The greatest oat producers in the world are in the Nordic countries. For example, in Finland about ⁸% of oats are used for human food (including baby food), thus the quality of the raw material for oat products is important.Until now, only a few sources of resistance to FHB in oats have been demonstrated(Björnstad, et al., ²00⁶; Tekauz et al., ²00⁸; Gagkaeva et al., ²0¹0). One of the problems in breeding resistant cultivars in oats and barley is the lack of clear visual symptoms, which could be scored to assess the level of FHB. That is why other parameters connected to mycotoxins, such as Fusarium DNA, should be evaluated. In the present review paper I will concentrate on trichothecene-producing Fusarium species.Type A and B trichothecene-producing fusarium speciesDON is the most important trichothecene in Europe and Asia. It is produced bytype B trichothecene-producing species of the F. graminearum species complex and F. culmorum. In northern Europe the highest DON levels have been found in oats (Yli-Mattila, ²0¹0). Nivalenol, which is also a type B trichothecene, is produced by F. poae, F. cerealis and NIV genotypes of F. graminearum species complex and F. culmorum.T-² toxin is the main type A trichothecene. The main Fusarium species producingT-² toxin have been difficult to identify. The researchers have agreed that they belong to the Sporotrichiella section of the Fusarium species. In the Soviet Union the Fusarium species causing ATA was identified as F. sporotrichioides (Sarkisov, ¹⁹⁵⁴), but for a long time the researchers occasionally thought that both F. sporotrichioides and F. poae isolates of Sporotrichiella section or even F. tricinctum might be effective in producing T-² toxin (Joffe, ¹⁹⁸⁶; Burkin et al., ²00⁸) due to identification problems. Since then it has been found that T-² producing F. poae isolates (Torp & Nirenberg, ²00⁴; Yli-Mattila et al., ²0¹¹) differ from typical F. poae isolates, which do not produce high levels of T-² toxin. Based on these investigations it seems that in Europe a new species F. langsethiae is the main T-²-producer, while in the Russian Far East another new species F. sibiricum is also involved in T-² production together with F. sporotrichioides. There are two subgroups or populations of F. langsethiae, which are morphologically similar to F. poae, but phylogenetically are closer to F. sporotrichioides (Konstantinova & Yli-Mattila, ²00⁴; Yli-Mattila et al., ²00⁴; ²0¹¹). T-² toxin production is affected by water activity and temperature and the toxin is rapidly metabolized to HT-² toxin (Jestoi et al., ²00⁴; ²00⁸; Medina & Magan, ²0¹¹).DNA extraction, detection and quantificationDifferent commercial kits are available for DNA extraction and should beoptimised for the fungal DNA from plant material. The quality and quantity of DNA can be estimated by gel electrophoresis and spectrophotometer or by different

522

Abstract:

fluorescence methods. Fluorescence and gel electrophoresis are more specific than spectrophotometric analyses for quantification of DNA from grains. The amount of fungal DNA should be compared to the amount of total DNA obtained from plants (Yli-Mattila et al., 2011). Internal controls (Waalwijk, 2004; Kulik, 2011) should also be used in order to compensate for the effect of PCR inhibition.TaqMan real-time PCR can be used for quantifying DNA of Fusarium species inplants and the correlation between Fusarium graminearum DNA and DON levels have been good (e.g. Waalwijk et al., 2004; Yli-Mattila et al., 2008). The advantage of TaqMan primers based on ribosomal DNA is that there are numerous ribosomal DNA copies in the genome, making the reaction more sensitive. Each Fusarium species has a species-specific mycotoxin profile, which means that in several cases there is a good correlation between Fusarium DNA and mycotoxins produced by them (Yli-Mattila et al., 2008).Correlation between fusarium toxins and DNA levelsA highly significant correlation has been found between F. graminearum DNAand DON in Finnish oat, barley and spring wheat (Yli-Mattila et al., 2008, 2009a). F. culmorum seems to have a significant role in DON production only in barley. This is probably due to the fact that Finnish F. graminearum isolates are usually more effective in producing DON than F. culmorum isolates (Jestoi et al., 2008). The highly significant correlation between F. graminearum/F. culmorum DNA and DON is in agreement with previous results of Waalwijk et al. (2004) and Nicholson et al. (2003) in Europe and Sarlin et al., (2006) in North America. Highly significant correlations were also found between F. langsethiae/F. sporotrichioides DNA and HT-2/T-2 toxins and F. avenaceum DNA and enniatins/moniliformin (Yli-Mattila et al., 2008, 2009a). A slighter correlation has been found in Finland between NIV and F. poae DNA levels in barley and oats, while in Luxembourg F. culmorum seems to be the main NIV producer in winter wheat (Pasquali et al., 2010).Fusarium DNA levels during growing seasonF. langsethiae is one of the pioneer Fusarium fungi (together with F. poae) incereals during flowering (Wilson et al., 2004; Jestoi et al., 2008), especially in oats. It is a weak competitor, easily overgrown and replaced by other Fusarium species based on qPCR results. F. poae and F. langsethiae may be competitors in cereal grains, because it has been found that tillage with ploughing increases the amount of F. poae, while the amount of F. langsethiae decreases as compared to direct drilling (Yli-Mattila, 2010). After flowering, F. graminearum, F. culmorum, F. sporotrichioides and F. avenaceum cause higher infection than F. langsethiae (Yli-Mattila et al., 2009a).F. graminearum species complex and multilocus genotypingMultilocus genotyping and molecular phylogenetics are very useful inidentification of Fusarium isolates (O’Donnell et al., 2000, 2008). F. graminearum(sexual state Gibberella zeae) is the most important Fusarium species in central Europe and in large areas in North America and Asia (O’Donnell et al., 2000; 2008; Gagkaeva and Yli-Mattila, 2004; Goswami & Kistler, 2004; Yli-Mattila et al, 2009a,b). In recent years F. graminearum has been spreading to the north in Europe in the Netherlands and England (e.g. Waalwijk et al., 2003; Nicholson et al., 2003), Norway (Elen et al., 2007, personal communication), Poland (Stepien et al., 2008) and north-western Russia523

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