Green tea catechins of food supplements facilitate pepsin digestion of major food allergens,but hampers their digestion if oxidized by phenol oxidase

The in vitro gastric digestion of several food allergens (beta-lactoglobulin (BLG), alpha-lactalbumin (LA) and peanut allergens (PE)) in the presence of a catechin-enriched polyphenol extract of green tea (GTC), oxidized polyphenols and phenol oxidase processed food allergens and GTC was investigated. Pepsin-resistant proteins, such as BLG, major peanut allergens, Ara h 1 and Ara h 2, degrade faster in the presence of catechin-enriched green tea polyphenols. Phenol oxidase polymerized GTC affected adversely protein digestion of BLG and LA, but not digestion of PE proteins. Protecting effect of polyphenols correlated well with the ability of proteins to form insoluble complexes with oxidized catechins. Cross-linking of proteins and polyphenols further extended the half-lives of BLG and LA in the in vitro digestion by pepsin. Catechin-enriched green tea polyphenols of food supplements facilitate pepsin digestion of major food allergens, but hamper their digestion if oxidized and polymerized by phenol oxidase.     

Glycation of the Major Milk Allergen β‐Lactoglobulin Changes Its Allergenicity by Alterations in Cellular Uptake and Degradation

1 Scope

During food processing, the Maillard reaction (МR) may occur, resulting in the formation of glycated proteins. Glycated proteins are of particular importance in food allergies because glycation may influence interactions with the immune system. This study compared native and extensively glycated milk allergen β‐lactoglobulin (BLG), in their interactions with cells crucially involved in allergy.

2 Methods and results

BLG was glycated in MR and characterized. Native and glycated BLG were tested in experiments of epithelial transport, uptake and degradation by DCs, T‐cell cytokine responses, and basophil cell degranulation using ELISA and flow cytometry. Glycation of BLG induced partial unfolding and reduced its intestinal epithelial transfer over a Caco‐2 monolayer. Uptake of glycated BLG by bone marrow–derived dendritic cells (BMDC) was increased, although both BLG forms entered BMDC via the same mechanism, receptor‐mediated endocytosis. Once inside the BMDC, glycated BLG was degraded faster, which might have led to observed lower cytokine production in BMDC/CD4⁺ T‐cells coculture. Finally, glycated BLG was less efficient in induction of degranulation of BLG‐specific IgE sensitized basophil cells.

3 Conclusions

This study suggests that glycation of BLG by MR significantly alters its fate in processes involved in immunogenicity and allergenicity, pointing out the importance of food processing in food allergy.     

Tyrosinase, Laccase, and Peroxidase in Mushrooms (Agaricus, Crimini, Oyster, and Shiitake)

Enzyme assays and electrophoresis were used to monitor the activity of tyrosinase, laccase, and peroxidase in Agaricus bisporus (common cultivated button mushrooms and Crimini mushrooms), Oyster, and Shiitake mushrooms. The three enzymes could be differentiated using specific substrates and inhibitors. Tyrosinase seemed to be the major phenol oxidase in the Agaricus strains, while Oyster and Shiitake mushrooms had much lower levels. Peroxidase activity was low or undetectable in all types examined. Control of enzymatic browning in different mushroom types may depend upon the distribution of different oxidases within any given type.     

Enzymatic cross-linking of β-lactoglobulin in solution and at air-water interface: Structural constraints

Effective and controlled use of cross-linking enzymes in structure engineering of food systems depends on characterization of the favorable conditions for enzyme-substrate complex and the limiting factors for the desired modification. In this respect, we analyzed the susceptibility of bovine β-lactoglobulin (BLG) to enzymatic cross-linking by Trichoderma reesei tyrosinase (TrTyr) and transglutaminase (TG). Changes in BLG molecular structure were determined at pH 6.8, 7.5 and 9.0 before and after high-temperature heat treatment. The conformational change was linked to efficiency of protein cross-linking. BLG was not susceptible to TrTyr without heat treatment. TG, however, induced inter-molecular cross-links at pH 7.5 and 9.0. After the heat treatments, BLG molecules adopted a molten-globule-like conformation. Both of the enzymes were able to form inter-molecular cross-links between heat-denatured BLG molecules. Electrophoretic mobility and broadness of the oligomer bands created by both enzymes on SDS-PAGE gels showed differences which were linked to the availability and number of target amino acid residues. Evidence for intra-molecular cross-linking was obtained. Once adsorbed to air/water interface, BLG formed a viscoelastic surface film which was characterized by surface shear rheology. Application of cross-linking enzymes under a dense layer of BLG molecules at the interface led to decreasing G′ with time. Intra-molecular links were most probably favored against inter-molecular on packed BLG layer leading to constrained molecules. Results in general emphasize the importance of structural and colloidal aspects of protein molecules in controlling inter/intra-molecular bond formation by cross-linking enzymes.     

Changes in Grape Seed Phenols as Affected By Enzymic and Chemical Oxidation in vitro

An in vitro oxidation system containing all the phenolic compounds of an extract of grape seeds and phenol oxidases (catechol oxidase from grapes and laccase from grey mold of grape Botrytis cinerea) was used to determine the effects of enzymic oxidation on seven phenolic compounds: gallic acid, (+)-catechin; (-)-epicatechin and four procyanidins, dimers of (+)-catechin and (-)-epicatechin units. HPLC determinations showed that there was considerable depletion of phenolic compounds in the extract by both enzymes since the quantities of all the compounds fell during enzymic oxidation. When a pure chemical oxidation was compared, the decrease was very much slower.     

MARINE FISH DIGESTIVE PROTEASES—RELEVANCE TO FOOD INDUSTRY AND THE SOUTH-WEST ATLANTIC REGION—A REVIEW

The biological diversity of marine species provides a wide array of enzymes with unique properties. For this reason, there is great potential for the recovery and use of digestive proteases from fish provessing wastes. Fish digestive proteolytic enzymes most commonly studied include pepsin, trypsin, chymotrypsin, gastricsin and elastase. Enzymes from cold adapted ectothermic organisms have application in the food industry because their temperature and other characteristics differ from homologous proteases from warm-blooded animals. The South-West Atlantic ocean is an enormous reservoir of different fish species. The digestive proteases from these organisms can be isolated as a by-product and used for processing aids in the food industry.     

Enzymatic preparation of immunomodulatory hydrolysates from defatted wheat germ (Triticum Vulgare) globulin

Defatted wheat germ globulin (DWGG) was hydrolysed using the enzymes alcalase, neutrase, papain, pepsin or trypsin to improve the immunomodulatory effects of the protein. The degree of hydrolysis (DH), molecular weight distribution, hydrophobicity, secondary structures and immunomodulatory activities were determined. The DH of hydrolysate using alcalase or neutrase was higher (P < 0.05) than that of hydrolysate prepared by other proteases. Alcalase hydrolysate (AH) had the strongest (P < 0.05) immunomodulatory activity with respect to lymphocyte proliferation, phagocytosis of neutral red and secretion of pro‐inflammatory cytokines. The molecular weight of AH ranged between 300 and 1450 Da and was composed of α‐helix and β‐turns. AH also showed the highest hydrophobic value (P < 0.05). The DH and hydrophobicity appear to be related to immunomodulatory activities. The hydrolysate prepared by the alcalase treatment on DWGG may be used as an immune supplement by the food industry.     

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.     

Peptic hydrolysis of bovine beta‐lactoglobulin under microwave treatment reduces its allergenicity in an ex vivo murine allergy model

In this study, the in vivo allergenicity of bovine beta‐lactoglobulin (BLG) in peptic whey protein hydrolysates generated during microwave and conventional heating treatments was assessed. The allergenicity of the hydrolysates was explored by studying the reaction of the murine jejunum from previously immunised Balb/c mice to treated BLG in an Ussing chamber. Intestinal anaphylactic reactions after stimulation of the gut‐associated immune system are a good indicator of potential in vivo allergenicity of whey hydrolysates. Fifty‐two per cent of BLG was hydrolysed by pepsin after only 3 min of microwave irradiation at 200 watts (W), yet it remained intact under conventional heating. Far‐ and near‐UV circular dichroism spectra indicated significant changes in BLG secondary and tertiary structures with microwave irradiation at 200 W. Pepsin whey protein hydrolysates obtained with microwave irradiation at 200 W for 3 min did not stimulate secretion of chloride in the Ussing chamber, as shown by the intensity of the short current values recorded (27.86 μA cm^?2), compared to the conventional pepsin hydrolysates (68.21 μA cm^?2). This demonstrates the low allergenicity of whey protein hydrolysates generated in this manner. These results confirm that microwave treatment combined with peptic hydrolysis could be applied to produce low allergenicity milk peptides.     

Effects of soaking, germination and fermentation on phytic acid, total and in vitro soluble zinc in brown rice

Rice is an important staple food in Asian countries. In rural areas it is also a major source of micronutrients. Unfortunately, the bioavailability of minerals, e.g. zinc from rice, is low because it is present as an insoluble complex with food components such as phytic acid. We investigated the effects of soaking, germination and fermentation with an aim to reduce the content of phytic acid, while maintaining sufficient levels of zinc, in the expectation of increasing its bioavailability. Fermentation treatments were most effective in decreasing phytic acid (56–96% removal), followed by soaking at 10 °C after preheating (42–59%). Steeping of intact kernels for 24 h at 25 °C had the least effect on phytic acid removal (<20%). With increased germination periods at 30 °C, phytic acid removal progressed from 4% to 60%. Most wet processing procedures, except soaking after wet preheating, caused a loss of dry mass and zinc (1–20%). In vitro solubility, as a percentage of total zinc in soaked rice, was significantly higher than in untreated brown rice while, in steeped brown rice, it was lower (p < 0.05). Fermentation and germination did not have significant effects on the solubility of zinc. The expected improvement due to lower phytic acid levels was not confirmed by increasing levels of in vitro soluble zinc. This may result from zinc complexation to other food components.     

Proteolytic activities and safety of use of <Emphasis Type="Italic">Enterococci</Emphasis> strains isolated from traditional Azerbaijani dairy products

A collection of 147 isolates obtained from 23 samples of traditional Azerbaijani dairy products was screened for the presence of proteolytic enzymes. Six Enterococcus faecalis strains obtained from three cheese samples have been identified as proteinase-producing strains, according to their ability to hydrolyze caseins. RAPD–PCR profiles of their total DNA showed different patterns for strains isolated from different cheese samples. The proteolytic activities of these strains were studied during their growth in milk and in non-proliferative cells system. Isolated strains were able to hydrolyze α_S1-, α_S2-, β-caseins and BLG albeit to different extents, at optimal pH in the range 6.0–7.2 and optimal temperature in the range 37–45 °C, depending on the strain. Proteolysis was strongly inhibited in the presence of EDTA—specific inhibitor of metalloproteases—but the presence of other types of proteases cannot be excluded. The potential pathogenicity of the strains was evaluated investigating the presence of the genes coding different virulence factors and their resistance to antibiotics. The obtained results yield new information about technological characteristics and safety of studied Enterococci strains from Azerbaijani artisanal dairy products. Many from the isolated strains contribute certainly to the differences in flavor, texture, and taste of Azerbaijani traditional cheeses and could represent new adjunct cultures for the dairy industry.     

Detection and impact of protease and lipase activities in milk and milk powders

Freshly drawn milk contains indigenous enzymes, including proteases and lipases. During handling and processing, milk acquires contaminating bacteria that produce further proteases and lipases, adding to the enzyme loading in the milk. Even when several heat treatment steps are used to prepare milk products, these will not be sufficient to inactivate all of the enzymes. Adverse effects of heat on the product limit the extent of heat treatments that can be used. The activities of proteinases and lipases that survive the heat treatments may cause changes in functionality and flavour of milk products, including milk powders, during storage. This review focuses on the characteristics of proteases (specifically proteinases) and lipases in milk and milk powders, detection methods for such enzymes and the effects of these enzymes on milk and milk powders during storage.     

Assessment of Thymus vulgaris L. essential oil as a safe botanical preservative against post harvest fungal infestation of food commodities

A total of 14 odoriferous angiospermic essential oils were tested against the toxigenic strain of Aspergillus flavus. The essential oil of Thymus vulgaris L. showed highest antifungal efficacy. The thyme oil absolutely inhibited the mycelial growth of A. flavus at 0.7 μl ml^− 1 and exhibited a broad fungitoxic spectrum against eight different food contaminating fungi viz. Fusarium oxysporum, Cladosporium herbarum, Curvularia lunata, Aspergillus terreus, Aspergillus niger, Aspergillus fumigatus, Alternaria alternata and Botryodiploidia theobromae. The oil also showed significant antiaflatoxigenic efficacy as it completely arrested the aflatoxin B₁ production at 0.6 μl ml^− 1. Thyme oil as fungitoxicant was also found superior over most of the prevalent synthetic fungicides. The LC₅₀ of thyme oil against mice was recorded as 7142.85 μl kg^− 1 body weight indicating its non-mammalian toxicity and strengthening its safe exploitation as preservative for stored food commodities. The findings recommend the thyme oil as potential botanical preservative in eco-friendly control of biodeterioration of food commodities during storage.

Industrial relevance

The thyme essential oil may be recommended for large scale application as a plant based preservative for stored food items because of its strong antifungal as well as antiaflatoxigenic efficacy. Because of broad antimicrobial spectrum, more efficacy over prevalent synthetic preservatives as well as non-mammalian toxicity, the thyme essential oil may be formulated as a safe and economical plant based preservative against post harvest fungal infestation and aflatoxin contamination of food commodities.     

Xyloglucan and β-glucans: origins and destinations

In ultrastructural studies, cellulose microfibrils can be seen to form an integral part of the primary wall framework along with xyloglucan, which is firmly bound to the wall matrix. Callose (1, 3-β-glucan) is not part of the wall proper but is deposited after wounding between wall and plasmamembrane in the periplasmic space. Of these extracellular end products, only xyloglucan has been detected intracellularly. The four enzymes required for xyloglucan assembly have all been found in Golgi and pulse-chase experiments in vivo show that xyloglucan molecules are completed in Golgi before being secreted via dictyosome vesicles. Cellulose and 1,3-β-glucan, in contrast, both appear to be formed at the cell surface. Plant membrane preparations assayed immediately after isolation can form a small amount of 1,4-β-linked product from UDP-glucose, which has a relatively low mol. wt equivalent to dextran 70 000. Only a few glucose units appear to be added to an endogenous acceptor. However, plasmamembrane, isolated from protoplasts after a period of digestion in a mixture of hydrolases, shows no capacity to form 1,4-β-glucan, but instead forms 1,3-β-glucan with a high mol. wt. The available data are consistent with the possibility that 1,4-β-glucan synthase is converted to l,3-β-glucan synthase in isolated (perturbed) membranes and in wounded tissues. The question of particular current interest is whether such a conversion takes place in a reversible manner, in which event conditions may be found to isolate 1,3-β-glucan synthase and reconstitute it into a complex that regains the capacity to form 1,4-β-glucan. This may never be possible, however, if 1,4-β-glucan linkages are differentially susceptible to decay in the presence of proteases while the capacity to form 1, 3-β-linkages is stimulated. It may be, therefore, that if membranes were isolated under conditions that completely inhibited endogenous proteases, the yield of 1,4-β-glucan synthase would be maximized and the development of 1,3-β-glucan synthase activity minimized.     

Allergenicity of acidic peptides from bovine β-lactoglobulin is reduced by hydrolysis with Bifidobacterium lactis NCC362 enzymes

Cow milk proteins, particularly β-lactoglobulin (BLG) are a leading cause of food allergy in children. In vivo, gastrointestinal hydrolysis of milk proteins decreases their allergenicity, but the intestinal microbiota also contributes to their breakdown. This study investigated the effect of hydrolysis of BLG-derived tryptic-chymotryptic (TC) peptides by Bifidobacterium lactis NCC362 enzymes, on their allergenicity. B. lactis enzymes have been shown to hydrolyze both acidic and basic peptide fractions isolated from tryptic-chymotryptic hydrolysate, but not whole proteins. The IgE binding capacity of acidic TC peptides was reduced by B. lactis peptidases-driven hydrolysis. This is partly explained by the breakdown of known allergenic peptides. Moreover, the resulting peptide fragments significantly up-regulated IFN-γ and IL-10 production and down-regulated IL-4 secretion by murine splenocytes. These results indicate that B. lactis NCC362 could be a potential probiotic for preventing cow's milk allergy through hydrolysis of the allergenic portion of BLG and the release of peptides which down-regulate the allergic immune response, but further studies using living bacteria are needed to confirm these data.     

Novel touchdown-PCR method for the detection of putrescine producing Gram-negative bacteria in food products

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99–91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.     

β-Lactoglobulin conformation and mixed sugar beet pectin gel matrix is changed by laccase

Sugar beet pectin (SBP) and β-lactoglobulin (BLG) contain ferulic acid and tyrosine, respectively, potential substrates for laccase. Dispersions of BLG were treated with laccase and/or heat and SBP to assess potential for ferulic acid in SBP to influence laccase conjugation of BLG. Changes were investigated by size exclusion chromatography combined with multi angle laser light scattering (HPSEC-MALLS), refractive index (RI) and UV detector, particle size and ζ-potential analysis. Molecular weight (MW) of BLG decreased from 3.7 × 10⁴ to 2.9 × 10⁴ and root mean square (RMS) decreased from 41 to 36 after laccase treatment. The slope of a conformation plot increased from 0.35 to 0.72, indicating a change in shape in laccase treated BLG to more random coil. While laccase did not affect MW of BLG, MW of BLG increased after sequential heat and laccase treatment to 2.9 × 10⁶ and 4.4 × 10⁶, respectively. Free thiol (SH) increased by heat and laccase treatment of BLG; tyrosine in BLG increased only by heat. An increased MW and decreased ferulic acid in SBP was observed in the presence of BLG and laccase. The increase in MW was attributed to entrapment of BLG in SBP cross-linked matrix catalyzed by laccase.     

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.     

Immunogold labelling to localize polyphenol oxidase (PPO) during wilting of red clover leaf tissue and the effect of removing cellular matrices on PPO protection of glycerol‐based lipid in the rumen

BACKGROUND: The enzyme polyphenol oxidase (PPO) reduces the extent of proteolysis and lipolysis within red clover fed to ruminants. PPO catalyses the conversion of phenols to quinones, which can react with nucleophilic cellular constituents (e.g. proteins) forming protein–phenol complexes that may reduce protein solubility, bioavailability to rumen microbes and deactivate plant enzymes. In this study, we localized PPO in red clover leaf tissue by immunogold labelling and investigated whether red clover lipid was protected in the absence of PPO‐induced protein–phenol complexes and plant enzymes (lipases). RESULTS: PPO protein was detected to a greater extent (P < 0.001) within the chloroplasts of mesophyll cells in stressed (cut/crushed and wilted for 1 h) than freshly cut leaves for both palisade (61.6 and 25.6 Au label per chloroplast, respectively) and spongy mesophyll cells (94.5 and 40.6 Au label per chloroplast, respectively). Hydrolysis of lipid and C18 polyunsaturated fatty acid biohydrogenation during in vitro batch culture was lower (P < 0.05) for wild‐type red clover than for red clover with PPO expression reduced to undetectable levels but only when cellular matrices containing protein–phenol complexes were present. CONCLUSION: Damaging of the leaves resulted in over a doubling of PPO detected within mesophyll cells, potentially as a consequence of conversion of the enzyme from latent to active form. PPO reduction of microbial lipolysis was apparent in macerated red clover tissue but not in the absence of the proteinaceous cellular matrix, suggesting that the PPO mechanism for reducing lipolysis may be primarily through the entrapment of lipid within protein–phenol complexes. Copyright © 2009 Society of Chemical Industry     

A Simplified Isolation of High‐Amylose Maize Starch Using Neutral Proteases

Breeding projects aimed at increasing starch amylose would benefit by having a rapid starch extraction method requiring only non‐hazardous reagents and leaving the native granule intact for functional analyses. A study was, therefore, designed to investigate the use of a neutral protease for the removal of protein during the starch extraction process from the grain of high‐amylose corn. Sets of F2 ears presumed to be homozygous for the recessive amylose‐extender (ae) allele and segregating high amylose modifier gene(s) were used in the study and ranged in amylose content from 55% to around 70%, although several non‐mutant genotypes (˜25%) occurred because of visual misclassification of the ae kernels. Starches from samples were all initially extracted by grinding and filtering, then further treated in three ways including either 1) no protein removal, 2) toluene and saline washes or 3) use of neutral proteases. In general, amylose values corresponded among samples extracted using the three methods. Samples purified using proteases had higher mean amylose values (62.5%) attributed to the lower presence of contaminating protein compared to samples prepared with toluene (61%) and grinding/filtration only (57.5%). Little change occurred among the starches as a result of the protease treatments according to thermal properties obtained using differential scanning calorimetry. In addition, gel permeation chromatography profiles (GPC) were unaffected by this treatment. A low level of amylase activity from the protease was found which degraded less than 1% of the starch sample. The results demonstrated that this protease method gave an increased yield of starch with a quality similar to that of starch prepared with toluene.