OVER-EXPRESSION OF FOOD-GRADE LACTOBACILLAL Β-GALACTOSIDASES IN LACTOCOCCUS LACTIC USING THE NISIN-CONTROLLED EXPRESSION SYSTEM..7. FRUCTO-OLIGOSACCHARIDE PRODUCTION BY PSEUDOMONAS SYRINGAE DC3000 LSC3 PROTEIN: USE OF RANDOM MUTAGENESIS TO MODIFY THE POLYMERIZING PROPERTIES OF. THE SAT V 1 SCAFFOLD IS AGAIN UNSTABLE TO LOW PH AND PROTEOLYSIS..40 A.I.SANCHO,A.WANGORSCH,B.JENSEN,P.JOHNSON,A.WATSON,Y.ALEXEEV,G.REESE,B.BALLMER-WEBER, P . SKOV,K.HOFFMANN-SOMMERGRUBER,S.VIETHS,A.R.MACKIE &E.N.C.MILLS.
The lacL gene, which encodes the large subunit of the β-galactosidase, is translationally fused downstream of the nisin-inducible promoter nisA. Comparison of the standard and the food-grade expression system, which differ only in their selection marker, gave significant differences in volumetric β-galactosidase activity, depending on both the origin of the lacLM genes and the selection marker used. Quercetin and -tocopherol both acted synergistically in prolonging the lag phase with lettuce extract independent of initiator with quercetin showing the greatest effect.
The combination of stored lettuce extract with antioxidants (except ascorbic acid) showed a similar tendency to decrease the lag phase for both storage temperatures, while the combination of antioxidants with heated lettuce showed an increase in the lag phase. In the present work it was found that PPO is not inactivated by PEF treatments at moderate temperatures.
Enzymatic cross‐linking of β‐casein –impact on digestibility and allergenic properties
S ESSION 2: F UNCTIONAL FOOD INGREDIENTS
Fructo‐oligosaccharide production by the Pseudomonas syringae DC3000 Lsc3 protein: use of random mutagenesis to modify polymerizing properties of the enzyme
Gluconacetobacter diazotrophicus synthesizes a remarkable amount of fructo-oligosaccharides (FOS) from sucrose, while levansucrases of most bacteria produce long-chain levan as the main polymerization product. The TLC migration spectrum of FOS produced by Lsc3 is similar to that of commercial prebiotic preparations FOS P95 and Synergy1 from Orafti. In addition to long-chain levan, potentially prebiotic FOS with degrees of polymerization of up to seven were detected by TLC and up to five using mass spectrometry (1).
We used random chemical mutagenesis of Escherichia coli transformants containing lsc3 to mutate the Lcs3 protein. Screening of 1500 recombinant mutant clones on growth media containing sucrose and raffinose resulted in clones possibly expressing mutant Lsc3 proteins with altered polymerizing properties. Both mutants synthesized FOS and levan, but levan had a lower degree of polymerization than that produced by wild-type Lsc3.
Total catalytic activity of mutant levansucrases measured according to sucrose cleavage was approximately 20-fold reduced compared to wild-type Lsc3, while the ability to use raffinose as substrate was preserved in the mutants.
O2.4. Targeted oxidation of polysaccharides by galactose oxidase
O2.5. Identification of a novel bacteriocin from Lactobacillus rhamnosus strain 68
With regard to xylose release, Abf A.niger - as expected - showed a better synergistic effect than Abf Bifido with β-xylosidase. Thus, at equimolar addition levels of the four enzymes, endo-1,4-β-xylanase activity was rate-limiting for the β-xylosidase-catalyzed depolymerization to release xylose from arabinoxylan. Furthermore, the hydrolysates were tested as an alternative to commonly used complex nitrogen sources (peptones) for the type strains of the probiotic lactic acid bacteria Lactobacillus rhamnosus GG (Valio Ltd., Finland), Lactobacillus acidophilus NCDO 1748 and Lactobacillus gasseri 33323T.
Comparative studies using MRS-like media containing different nitrogen sources showed that all salmon hydrolysates performed as well as or better than the selected commercial peptones for the lactic acid bacteria included in this study. In particular, peptones from intestines and the soluble fraction after defatting supported excellent growth of the lactobacilli. Okara, a byproduct of soy milk production, typically contains 66% carbohydrates, 23% protein, 8% lipids and 3% ash (on a dry matter basis).
The dietary fiber component was enriched by hydrolysis of the protein component with serine peptidases. After extraction of the pectin fraction, a number of oligosaccharides (OS) together with poly- and monosaccharides were enzymatically generated. Ferulic acid (FA) released from plant cell walls by the action of feruloyl esterase (FAE) is an effective natural antioxidant with potential applications in the pharmaceutical and food industries.
3.1.1.73] represent a subclass of carboxylic ester hydrolases, which release phenolic acids (ferulic acid and p-coumaric acid) and their dimers from natural hemicelluloses and pectins. In this study, the synergistic action of recombinant FAE from Fusarium oxysporum (FoFaeC-12213) and xylanase M3 from Trichoderma longibrachiatum as monoenzymes was studied for FA release from spent beer grains (BSG). Spent brewer's grains (BSG), the barley malt residue resulting from the production of brewing salt, is a major co-product of the brewing industry.
The purpose of this work was to provide a scientific basis for the valorization of one of the main components of BSG, i.e. of enzymatic hydrolysis of BSG protein concentrate (BPC) prepared by alkaline extraction of BSG, followed by acid precipitation of the extracted substances, improved solubility and emulsion and foaming properties. The physicochemical characterization of the hydrolyzates showed the importance of the presence of protein fragments with a relatively high molecular weight (MW) (more than 14.5 k) and high surface hydrophobicity for favorable techno-functional properties.
O2.11. Transglutaminase enzyme as reticulating agent for protein component of hydrocolloid edible films
S ESSION 3: FOOD PROCESSING
O3.1. Targeted pectin modification to improve cell wall saccharification for biofuel production
The effect of carbohydrases on the intensification of tradional Lithuanian rye bread processing
In the Baltic region - especially in Lithuania - a special traditional process of rye and mixed rye bread is used. Compared to the rye bread preparation processes used in Europe, the main difference is the steaming step in the process, which contributes to the excellent quality and long shelf life of the bread. The aim of this study was to intensify and simplify the traditional Lithuanian bread-making process using different carbohydrases (A.oryzae amylase, A.niger amyloglucosidase and A.niger xylanase) and extruded rye products for sourdough preparation.
When using extruded products for burns, it is recommended to use a composition of amylase, amyloglucosidase and xylanase. The maximum amount of saccharides such as maltose, sucrose and glucose was observed in the scald after 20 minutes. We also improved the quality of bread by using new technological means for the preparation of sourdough.
O3.4. Application of selected microorganisms and enzyme preparations in reduction of allergic proteins during wheat dough fermentation
O3.5. New possibilities on noodle and cake productions based on non traditional raw materials
O3.6. Enzymes in production of gluten free breads
S ESSION 4: E NZYMOLOGY
O4.1. A novel expression system for enhanced laccase expression in Aspergillus niger
O4.2. Production and physicochemical properties of Lactobacillus plantarum tannase
The enzyme pectin methylesterase (PME) is believed to be involved in the destabilization and loss of cloudiness of vegetable juices through de-esterification of pectin followed by successive co-precipitation of pectate with insoluble materials present in the juices. Cloud destabilization is often observed even when plant products have been subjected to heat treatment. Therefore, it is possible that destabilization is due to thermostable PME isoforms that survive heat treatments.
Industrial fruit products were investigated for their content of PME residual activity by a new procedure that allows selective concentration of the active form of the enzyme by affinity chromatography. PMEI binds with high affinity the native active form of PME which can later be recovered by dissociating the complex at alkaline pH with high ionic strength. We investigated fruit products from various industrial preparations of peach, pear, apple, apricot and pineapple.
A dependence of the intensity of cloud loss on the levels of PME residual activity was found.
Pseudomonas aeruginosa san-ai, a strain that grows naturally under highly alkaline conditions (pH 10), extracellularly produces a lipase (EC 3.1.1.3) and a negatively charged exopolysaccharide (EPS) that is considered the alginate type of bacterial EPS. Based on that natural interaction between lipase and EPS, it was hypothesized that lipase could be immobilized on its autogenous EPS. Indeed, in these investigations, lipase from Pseudomonas aeruginosa san-ai was successfully immobilized from autogenous exopolysaccharide as a matrix, using the Ca-EPS bead entrapment method.
The lipase immobilized in autogenous EPS even without further stabilization maintained stability and can be reused several times for hydrolysis in stirred tank batch reactor, suggesting that the lipase is highly suitable as a biotechnological tool in a variety of applications.
O4.6. Arabinanases from Chrysosporium lucknowense and their potential in the complete degradation of sugar beet arabinan
P OSTERS
P1. Application of Raman microspectroscopy in the study of action of polygalacturonase treatment on apple cubes convectively dried
P2. The Bet v 1 scaffold is inherently unstable to low pH and proteolysis
P3. Influence of honey processing on diastase activity
P4. Application of amylolytic enzymes in baking of gluten‐free rolls
Enzymatic regeneration systems for the largest group of enzymes - oxidoreductases - are well known due to the expensive cofactor or coenzyme requirements of these attractive biocatalysts. Various procedures for regeneration are known, e.g. the electrochemical regeneration of coenzymes or the use of auxiliary enzymes for the regeneration of the coenzyme. Especially for the regeneration of the oxidized coenzymes NAD(P)+, no easily applicable procedure is known, and this thereby limits the application of several useful dehydrogenases for e.g. oxidative deracemization.
To provide an alternative, we are investigating a novel system for the regeneration of NAD(P)+ from NAD(P)H for use in dehydrogenase-catalyzed reactions using the multicopper oxidase laccase in combination with an intermediate electron carrier (redox mediator) to oxidize nicotinamide adenine dinucleotides with simultaneous reduction of oxygen to water. The laccase/mediator system was tested with meldolin blue as a redox mediator in the conversion of glucose to gluconate catalyzed by NAD(P)-dependent glucose dehydrogenase. Easy removal of the redox mediator can be achieved by absorption on a cation exchange resin. The results obtained show that this method may prove useful for other applications, such as the production of the raspberry ketone frambinone, which is a key component of the raspberry aroma, by ensuring a high oxidation rate of NAD(P)H and the stability of the regeneration system.
P7. Comparison of enzymatic hydrolyzability of proteins and polysaccharides in brewers` spent grain from different European breweries
P8. Kinetic study of ferulic acid cross linking in pectic polysaccharides
P9. Presence of residual pectin methylesterase activity in thermally stabilized industrial fruit preparations
P10. Oxidation of phenols from fruits juice using immobilized laccase
P11 . The application of enzymes in the process of enzymatic hydrolysis of starches from different wheat varieties
P12. Effect of prebiotics on quality parameters of wheat‐rye bread
P13. Traditional meat goods in Lithuania: creation the sensory profiles and description specificity
P14. Enzymatic hydrolysis of okara proteins and techno‐functional properties of the obtained hydrolysates
P15. Upscaling the biocatalytic conversion of lactose to lactobionic acid with the dynamic membrane aeration reactor
In this study, the lacZ gene (3027 bp) encoding β-galactosidase from Lactobacillus bulgaricus DSM 20081 was cloned into the lactobacillal-inducible expression vectors pSIP403 and pSIP409, which are based on the sakacin P expression level of L. bulgaricus cultures with yield of approx. 43 kU β-galactosidase activity per liter of fermentation, which is 5 times higher than wild type. As judged by SDS-PAGE and native-PAGE, β-galactosidase from L. bulgaricus DSM 20081 is a homodimer consisting of two identical subunits of approx. 110 kDa.
The optimum temperature and pH for o-nitrophenyl β-D-galactopyranoside and lactose were 45oC, pH 6.5 to 8 and 65oC, pH 7.5 to 8, respectively.
P17. Reduction vicinal diketones by used enzyme alfa –acetolactate decarboxylase in aim achieve characteristic beer
P18. Experimental optimization of mashing process in production of beer’s wort
P19. Evaluation of the oxidative enzymes activity during the processing of white grapes
P20. The properties of soft cheeses produced from milk cross‐linked by microbial transglutaminase
