Arnous* & Meyer*

Center for BioProcess Engineering, Technical University of Denmark Lyngby, Denmark e-mail: am@kt.dtu.dk

Keywords: Plant cell wall polymers, polysaccharides, pectin.

Knowledge about the structural makeup of fruit skin polysaccharides is a prerequisite for tailoring of enzymatic treatments for efficient upgrading of press residues or for improving the release of colour and bioactive phenolics from the fruit skins into juice and wine. Based on monosaccharide analysis after acid hydrolysis of skin samples of three wine grape cultivars (Vitis vinifera L.: Cabernet Sauvignon, Merlot, Shiraz) and of two types of apple skin samples (Malus domestica: Red Delicious, Golden Delicious) we here report an iterative calculation method for the quantitative allocation of plant cell wall monomers into relevant structural polysaccharide elements from monosaccharide analysis. By this method we estimated that the polysaccharides, which made up 48-54 % by weight of the skins (the rest mainly being lignin) of red wine grapes (Vitis vinifera L.) were made up of 57-62%

homogalacturonan, 6.0-14% cellulose, 10-11% xyloglucan, 7% arabinan, 4.5-5.0%

rhamnogalacturonan I, 3.5-4.0% rhamnogalacturonan II, 3% arabinogalactan, and 0.5-1.0% mannans – the ranges indicating minor variations among the skin compositions of the different cultivars. The calculated relative levels of polysaccharides in the apple skins appeared to be similar to those of the grape skins, but in agreement with the available knowledge on apple cell wall pectin the apple skins were estimated to be relatively richer in arabinan (10-13%) and having relatively lower mannan content than grape skins. The data also demonstrated the superiority of trifluoroacetic acid (TFA) hydrolysis versus hydrochloric acid (HCl) hydrolysis, notably with respect to the levels of galacturonic acid, and in turn concerning the predicted relative homogalacturonate contents.

P9: The influence of rye and triticale inhibitors on the functionality of commercial endoxylanases.

G. Juodeikiene & D. Vidmantiene

Department of Food Technology, Kaunas University of Technology, Lithuania

Keywords: rye, triticale, endoxylanase, inhibition, functionality.

In recent years, the interest in carbohydrate-active enzymes such as xylanases has increased due to their potential application in the food and feed industry. However, efficiency of added commercial endoxylanases can vary depending on the variety and the growing location making the optimal dosage of the enzyme difficult to determine. It may be due to the levels of endoxylanase and endoxylanase inhibitors in cereals.

The present work has the objective to measure the inhibition activity of partial purified rye and triticale albumins against commercial endoxylanases. Winter rye cultivars were grown in different parts of Lithuania after the growing season of 2003-2004 as well as winter triticale varieties grown in 2006.

The study includes a partial purification of proteinaceous cereal inhibitors by Flash chromatography using gradient (pH 3.0-8.6) elution. The inhibition activity of rye and triticale albumins against fungal endoxylanases from Trichoderma reesei and Aspergillus oryzae was analyzed. The endoxylanase activity was quantified with the dinitrosalicylic acid assay based on the measurement of reducing sugars released from the wheat arabinoxylan and birchwood xylan. The levels of the endoxylanase inhibition activity were essential influenced by genetic factors. The microbial endoxylanase from A.

oryzae was found to be more active than T. reesei endoxylanase. However, the A. oryzae endoxylanase was much more sensitive to inhibition by rye and triticale inhibitors. The results obtained may be useful in explaining the differences in functionality of different endoxylanases in biotechnological applications.

P10: Variability in the levels of arabinoxylan, xylanase and xylanase inhibitors in different wheat types and varieties

K. Gebruers, E. Dornez, Z. Bedo, C. Courtin & J. Delcour LFoRCe, Katholieke Universiteit Leuven, Leuven, Belgium

The use of high-quality raw materials and knowledge of their health-promoting properties becomes increasingly important in cereal processing. After all, variation in the (nutritional) quality of cereal- based products is caused to a large extent by differences in the quality of raw materials. The functionality of the arabinoxylan, xylanase and xylanase inhibitor system has already been studied in- depth during the past decade. Today, xylanases with tailor-made catalytic properties and improved stability, and with different degrees of inhibition by cereal xylanase inhibitors are (becoming) available. We here report on the variability of arabinoxylan, xylanase and xylanase inhibitor levels in flour and bran of different varieties of spring, winter, durum, spelt, emmer and einkorn wheats. The contribution of genotype, environment and their covariation to the total variance was determined. To this end, selected wheat varieties were cultivated during three successive years on one location and in one year these varieties were grown on four different locations in Europe. Based on the results obtained, the potential impact of the observed variations on biotechnological processes in which xylanases are often used and on the (nutritional) quality of cereal-based products will be discussed.

This study was carried out within HEALTHGRAIN, a 6th Framework EU project (2005-2010).

P11: Effects of cooperation of phytase B with phytase A on phytate dephosphorylation routes of sodium phytate and in enzymatically modified fermented rye dough

K. Zyla & R. Dulinski

Department of Food Biotechnology, Agricultural University in Krakow, Poland

Commercial preparations of phytase A were used alone and in combination with phytase B to dephosphorylate 5 mM sodium phytate solution at pH 4,0 and 40^oC. Phytic acid and myo-inositol phosphates generated in the course of the reaction were separated by high-performance anion-exchange chromatography on CarboPac PA-100 (250x4) column with postcolumn derivatization using 0.1%

Fe(NO₃)₃ x 9H₂O in 0,33M H₂SO₄ and spectrophotometric detection at 280 nm. The same procedure was applied to separate myo-inositol phosphates from enzymatically modified rye doughs fermented for 150 minutes. 3-Phytase A generated DL-I(1,2,4,5,6)P5, DL-I(1,2,5,6)P4 and DL-I(1,2,6)P3 and IP2 as the main intermediates, whereas 6-phytase A produced DL-I(1,2,3,4,5)P5 along with minor amounts of DL-I(1,2,4,5,6), DL-I(1,2,4,5)P4 as well as D-I(1,4,5)P3 and IP2. The addition of phytase B along with either of phytases A did not produced any new intermediates but only speeded up the conversion process. Similar isomers of lower myo-inositol phosphates were isolated from enzymatically modified rye dough supplemented with 6-phytase A and phytase B and fermented for 150 minutes. It may be postulated that each of the phytase A generate its own family of lower myo-inositol phosphates and phytase B when applied along with phytase A does not influence dephosphorylation routes. Phytase B along with phytase A may serve therefore as convenient tools for generating inositol phosphates with possible functional properties

P12: Acoustic method for bread staling assessment: Applied to enzyme- supplemented wheat bread

L. Basinskiene, G. Juodeikiene & S. Garmuviene

Department of Food Technology, Kaunas University of Technology, Kaunas, Lithuania

Keywords: amylolytic enzymes, β-xylanase, bread staling, acoustic method.

Wheat bread is regarded as one of the essential food products in most countries. Therefore, great attention is paid to its quality improvement. Freshness is one of the characteristics that consumers most appreciate in bread. To prolong bread freshness qualities or inhibit staling various technological means have been applied, such as specific packaging, freezing, use of enzymes.

In this study, an acoustic method developed at Kaunas University of Technology was used to evaluate the effects of amylolytic and non-amylolytic enzymes and their combination on the textural properties of wheat bread over a storage time of 5 days. An acoustic spectrometer working in a range of frequencies of 4.95-35.70 kHz and measuring the amplitude of the acoustic signal penetrated through the tested sample were used in the experiment. A strong correlation between the acoustic signal amplitude and the breadcrumb hardness measured by the Texture Analyzer was found. This means possible application of the acoustic method to determine the changes that took place in bread during storage. The non-destructive character of the acoustic method enables the testing of breadcrumb hardening in the same sample during storage and enhances the effectiveness of the analysis. The results showed that tested fungal β-xylanase, α-amylase and glucoamylase preparations could be used as wheat bread staling inhibitors. Bread samples with the combined enzyme supplements showed the smallest signs of ageing. Tested multienzyme composition of β-xylanase, α-amylase and glucoamylase could therefore be successfully used in bread making to produce bread with a good consumer quality.

P13: Talaromyces Emersonii enzymes and their application in cereal based functional food

D. Waters*, P. G. Murray & M. G. Tuohy

Dept. Biochemistry, National University of Ireland, Galway, Ireland

The fungus Talaromyces emersonii (T.E.) can produce an assortment of thermostable xylanolytic and cellulolytic enzymes which can improve or create texture using enzymes, particularly in the cereal food processing. The isolation and characterisation of novel enzymes can be used to improve food quality and nutrition. Certain T.E. enzymes with potential functions in this area have been identified including two acetyl esterase genes (AEs), two catalase genes and two glutaminase genes.

AEs aid the degradation of acetylated polysaccharides like pectins and xylans, and are used in baking.

Catalase can reduce lipid oxidation in foods, promoting a longer shelf life. It also acts as a stabilizer and when added to dough in conjunction with glucose oxidase and cystine it is useful for bread whitening. Glutaminase catalyzes the hydrolysis of glutamine to glutamic acid, imparting a savory flavour and increasing nutrition. Glutaminase/asparaginase can combat acrylamide produced during the Maillard reaction contributing to the color, smell and taste of bread.

A range of assays were performed on various T.E. cocktails, xylanases, α-amylase, β-amylase, glucoamylase and tripeptidyl peptidase were found. Temperature and pH optimums of these activities have been established.

P14: Tyrosinase-induced cross-linking of milk proteins: Effects on the texture of chemically acidified sodium caseinate and milk gels

D. Ercili Cura*, M. Lille, J. Buchert & R. Lantto

VTT Technical Research Centre of Finland, Espoo, Finland e-mail: dilek.ercili@vtt.fi

Enzyme-aided structure engineering of dairy products has taken much interest since the introduction of transglutaminase (EC 2.3.2.13, TG), a transferase capable of forming inter- or intramolecular isopeptide bonds in protein systems. TG-aided cross-linking of caseins has been reported to lead to milk protein gels with increased firmness and less syneresis. Tyrosinase (EC 1.14.18.1) is an interesting alternative to TG, due to its different mode of action. In the presence of oxygen, tyrosinase catalyses the hydroxylation of a monophenol to an o-diphenol, which is further oxidised to the corresponding o- quinone. These quinones react non-enzymatically resulting in covalent bonds in proteinaceous and other systems. Tyrosinase is capable of oxidizing tyrosine residues in protein molecules, resulting in the formation of covalent tyrosine-tyrosine, tyrosine-cysteine or tyrosine-lysine cross-links. In this work the effects of a novel tyrosinase from Trichoderma reesei in structure modification of chemically acidified sodium caseinate and skim milk (raw and heat treated) gels is reported. The action of T. reesei tyrosinase (TrTYR) is compared with the action of the commercially available mushroom tyrosinase and TG. Protein modification was analyzed by SDS-PAGE and the firmness of the resulting gels was determined by penetration tests with a texture analyser.

T. reesei tyrosinase induced cross-linking of caseins in raw milk, while mushroom tyrosinase and TG were ineffective. TrTYR treatment of raw milk prior to acidification resulted in increased gel firmness.

Even though cross-linking was achieved in heated milk as well, no effect on gel firmness was observed with TrTYR. TG-induced cross-linking led to significantly firmer gels in heated milk. In the case of sodium caseinate, both TrTYR and TG resulted in significant increase in gel firmness as a result of protein cross-linking. It was observed that TrTYR caused formation of a brownish color in both sodium caseinate and milk gels.

H. Ma¹*, P. Forsell¹, R. Partanen¹, R. Seppänen², J. Buchert¹ & H. Boer¹

1VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Finland,

2YKI, Institute for Surface Chemistry, Box 5607, SE-114 86 Stockholm, Sweden e-mail: hairan.ma@vtt.fi

Proteins, such as casein and whey protein, are widely used as emulsifiers in various industrial application areas such as in food products, cosmetics and pharmaceuticals. A drawback with proteins as emulsifiers is their low solubility and low net charge at pH values close to their isoelectric points (pI) and as a result of this, their emulsifying activity in that pH range is considerably reduced. In the case of caseins with an average isoelectric point around 4.5, reduction of its emulsifying activity at pH values 4-5 has been a severe limitation in its applications in the acidic pH range. In this work, sodium caseinate was chemically modified in order to alter its isoelectric point (pI). Negatively charged carboxylic groups were introduced to lower the pI, and positively charged amino groups to achieve the opposite. Different chemical amino acid modification approaches were studied and the modified proteins were characterized using free amino group assays, SDS-PAGE, MALDI-TOF mass spectrometry and zeta potential measurements. Oil in water emulsions were prepared using these modified caseinates. The pH stability behavior of the emulsions was monitored and interestingly the stability of the emulsion could be modulated through steering the pI of caseinate. Using different modified caseinates it was possible to create emulsions that were stable in the acid, neutral and alkaline regions of the pH spectrum. The stability behavior of the emulsions correlated well with the theoretical and experimentally determined pI values of the caseinates. Storage stability of emulsions was also studies at pH around 7, and emulsions made of modified caseinates showed storage similar stability to the unmodified caseinate emulsions.

P16: Cross-linking of β-casein with Trichoderma reesei tyrosinase and Streptoverticillium mobaraense transglutaminase

E. Monogioudi¹*, N. Creusot², K. Kruus¹, H. Gruppen², M-L. Mattinen¹, & J. Buchert¹

1VTT Technical research Centre of Finland, Espoo, Finland

2Laboratory of Food Chemistry, Wageningen University, Wageningen, The Netherlands e-mail: evanthia.monogioudi@vtt.fi

Transglutaminase is an acyltransferase being able to crosslink proteins via lysine and glutamine residues. Transglutaminase has been extensively studied on different food matrices including milk products. Tyrosinase is an oxidative enzyme, known to catalyze the oxidation of the phenolic ring of tyrosine residues to the corresponding quinones. About 25% of the milk proteins are β-casein. β-casein contains 11 lysine, 19 glutamic acid and 4 tyrosine amino acids and is thus a potential substrate for these two different types of enzymes.

In this work cross-linking of β-casein by fungal Trichoderma reesei tyrosinase (TrTyr) and microbial transglutaminase (TGase) was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-Page) and size exclusion chromatography (SEC) equipped with UV/Vis and multi-angle light scattering (MALLS) detectors to determinate the molecular masses of the cross-linked products. Three different enzyme dosages (10, 100 and 1000 nkat g -1) and reaction times (2, 6 and 24 h) were used to optimize the enzymatic reaction conditions at 40 °C. Oxygen consumption measurement was used to verify the reactivity of TrTyr on β-casein. Cross-linking with TrTyr resulted in the formation of coloured products and hence the progress of the enzymatic reaction was also visually followed as a function of time. The molecular masses of the cross-linked products ranged from 500 to 1700 kDa for TrTyr and from 500 to 1500 kDa for Tgase.

P17: Effect of prebiotic and enzyme supplementation on chicken‘s meat quality characteristics

A. Mieželienė^1*, G. Alenčikienė¹, R.Gružauskas², A. Semaškaitė², V. Šašytė² & A. Stupelienė²

1Food institute of Kaunas University of Technology, Kaunas, Lithuania,

2Lithuanian Veterinary Academy, Kaunas, Lithuania

Keywords: broiler chickens, meat quality, feeds, xylanase, prebiotic, sensory profiles.

The aim of this research was to determine the effect of endo-1,4-β-xylanase and its combination with prebiotics on sensory attributes of meat quality of broiler chickens. Prebiotic preparation.

Raftifeed^®OPS is powder for animal feed purposes containing mainly oligofructose produced by partial hydrolysis of chicory inulin. Six hundred chicks of the strain Cobb 500 were reared from 1 to 35 days old. The birds were assigned to three dietary treatments. First group was control (control diet), second group diet was supplemented with 0.01 % endo-1,4-β-xylanase and third group diet was supplemented with 2 % prebiotic and 0,01 % enzyme preparation.

By using descriptive sensory analysis method recommended list of descriptors was presented for panelists and it was explained to use it or free choiced descriptors. Sensory profiles from 15 attributes were created. It was determined that the addition of xylanase and its combination with Raftifeed®OPS had no significant effect (p>0,05) on the majority of the chicken breast and thigh meat sensory attributes. The addition of xylanase and prebiotic preparation had no significant influence on overall acceptability of tested samples also.

P18: A new strategy to prebiotic arabinoxylooligosaccharides

V. Puchart & P. Biely

Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia

Strains of the thermophilic fungus Thermomyces lanuginosus showed limited growth on wheat arabinoxylan as a carbon source despite a high production of extracellular endo-β-1,4-xylanase. An essential part of the polysaccharide was not utilized, and it was converted to a series of arabinoxylooligosaccharides differing in the degree of polymerization. The structure of the shorter arabinoxylooligosaccharides remaining in the wheat arabinoxylan-spent medium was established using mass spectrometry and digestion with three glycosidases, one β-xylosidase two distinct α-L- arabinofuranosidases. The oligosaccharides contained singly and doubly substituted xylopyranosyl residues. Xylose and linear β-1,4-xylooligosaccharides generated extracellularly during growth on the cereal xylan were efficiently taken up by the cells and metabolized intracellularly. The data suggest that due to a lack of extracellular β-xylosidase and α-L-arabinofuranosidases, the widely used Thermomyces lanuginosus strains might become efficient producers of branched xylooligosaccharides from cereal arabinoxylans. Arabinoxylooligosaccharides have a potential to be used as prebiotics.

P19: Production of food grade carotenoids from microorganisms