Effect of glycation on the gastrointestinal digestibility and immunoreactivity of bovine β-lactoglobulin

Immunoreactivity of bovine β-lactoglobulin (β-Lg) hydrolysates obtained after a simulated gastrointestinal digestion and previously glycated via Maillard reaction with galactose, tagatose, and dextran of 10 or 20 kDa has been determined, with a view to study the effect of glycation and aggregation degree of β-Lg on its residual immunoreactivity. High levels of glycation impaired β-Lg proteolysis and, consequently, increased the IgG- and IgE-reactivities of hydrolysates, regardless of the carbohydrate used. Protein aggregation during the advanced stages of Maillard reaction had a masking effect on β-Lg epitopes, counteracting the negative effect of the lower digestibility of glycated protein on its allergenicity. Finally, the use of polysaccharides as glycation agents did not contribute to enhancement of the masking effect of the attached carbohydrate on β-Lg epitopes. These findings stress the importance of evaluating the impact of glycation on protein gastrointestinal digestibility prior to investigation of the immunoreactivity of protein Maillard complexes.     

Maillard-type glycoconjugates from dairy proteins inhibit adhesion of Escherichia coli to mucin

In this study, glycoconjugates of β-lactoglobulin (β-Lg) and sodium caseinate (SC) were obtained via Maillard reaction with galactose and lactose, and their ability to inhibit the adhesion of different Escherichia coli strains (CBL2, CBM1 and CBL8) to mucin was evaluated. The strains tested exhibited different interaction patterns with the glycoconjugates, suggesting the participation of different carbohydrate-recognition sites in adhesion. Galactosylation and lactosylation of both β-Lg and SC significantly decreased the adhesion values of E. coli CBL2 to mucin. Whereas the adhesion of E. coli CBM1 was preferably interfered by galactosylated glycoconjugates obtained under the harshest incubation conditions, the adhesion capacity of E. coli CBL8 was not affected. Competitive adhesion assays with lectins, which recognise different epitopes, supported the idea that galactose-reactive adhesins are partly responsible for the recognition of these glycoconjugates. The analysis of the presence of gene coding for several virulence factors in the E. coli strains by PCR revealed the absence of K88 gene in the CBL2 strain assayed. These findings suggest that the formation of Maillard-type neoglycoproteins under controlled conditions may be a simple and cost-effective method for producing new food ingredients with the potential ability to block pathogen adhesins involved in mucosal colonisation.     

Interfacial and foaming properties of bovine β-lactoglobulin: Galactose Maillard conjugates

In this paper, the effect of the initial and advanced steps of glycosylation by Maillard reaction (MR) (glycation) of β-lactoglobulin (β-Lg) with galactose on the interfacial and foaming (foamability and foam stability) properties of this protein has been studied at both pH 7 and pH 5. Hardly any effect of glycation was observed at pH 7. However, a pH 5, due to its increased solubility, β-Lg glycated at 50 °C during 48 h (advanced steps of MR) presented the best dynamic of adsorption which lead to an increase of the surface dilatational modulus of adsorbed film. This resulted in a better foaming capacity, as well as higher stability of foams of β-Lg glycoconjugates with respect to native and control heated protein. These results could extend the applicability of β-Lg as a foaming agent, particularly in acid foods.     

Preventive effect of fermented Maillard reaction products from milk proteins in cardiovascular health

The aim of this study was to determine the dual effect of Maillard reaction and fermentation on the preventive cardiovascular effects of milk proteins. Maillard reaction products (MRP) were prepared from the reaction between milk proteins, such as whey protein concentrates (WPC) and sodium caseinate (SC), and lactose. The hydrolysates of MRP were obtained from fermentation by lactic acid bacteria (LAB; i.e., Lactobacillus gasseri H10, L. gasseri H11, Lactobacillus fermentum H4, and L. fermentum H9, where human-isolated strains were designated H1 to H15), which had excellent proteolytic and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities (>20%). The antioxidant activity of MRP was greater than that of intact proteins in assays of the reaction with 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and trivalent ferric ions; moreover, the effect of MRP was synergistically improved by fermentation. The Maillard reaction dramatically increased the level of antithrombotic activity and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) inhibitory effect of milk proteins, but did not change the level of activity for micellar cholesterol solubility. Furthermore, specific biological properties were enhanced by fermentation. Lactobacillus gasseri H11 demonstrated the greatest activity for thrombin and HMGR inhibition in Maillard-reacted WPC, by 42 and 33%, respectively, whereas hydrolysates of Maillard-reacted SC fermented by L. fermentum H9 demonstrated the highest reduction rate for micellar cholesterol solubility, at 52%. In addition, the small compounds that were likely released by fermentation of MRP were identified by size-exclusion chromatography. Therefore, MRP and hydrolysates of fermented MRP could be used to reduce cardiovascular risks.     

Characterization and improvement of rheological properties of sodium caseinate glycated with galactose, lactose and dextran

The aim of this work was to investigate the effect of non-enzymatic glycosylation with galactose, lactose, and 10 kDa dextran on the rheological properties of sodium caseinate. To promote the formation of covalent complexes, the reaction was done in solid state (a_w = 0.67), pH 7.0 (0.1 M sodium phosphate buffer), and temperature set at 50 and 60 °C. The progress of Maillard reaction was indirectly traced by measuring the formation of the Amadori compound, through furosine (2-furoylmethyl-lysine) analysis, and brown polymers, and the resulting glycoconjugates were characterized by LC/ESI-MS and SEC. Results showed a higher reactivity of galactose than lactose and dextran to form the glycoconjugates, due to its smaller molecular weight. Glycation with galactose and lactose increased the viscosity of caseinate and also altered its flow characteristics from Newtonian to shear-thinning. Oscillatory testing showed a higher elastic modulus (G′) in glycoconjugates when compared to non-glycated caseinate, especially with galactose, where a gel-like behaviour was observed after long incubation times. Glycation with dextran did not produce substantial improvements in the rheological properties of caseinate, probably due to the limited extent of the reaction. Our results show that by controlling the rate and extent of the Maillard reaction is a technologically feasible operation to improve the viscosity and gelling properties of sodium caseinate-based ingredients.     

Anti-inflammatory activities of Maillard reaction products from whey protein isolate fermented by Lactobacillus gasseri 4M13 in lipopolysaccharide-stimulated RAW264.7 cells

Maillard reaction products formed from whey protein isolate (WPI) and sugar have been shown to have an anti-inflammatory effect in vitro. Here, we incubated WPI and galactose (GWA) in an aqueous solution at 65°C for 24 h to produce a glycated conjugate, which was then fermented using Lactobacillus gasseri 4M13 to obtain the fermented product (F-GWA). We demonstrated that F-GWA had an anti-inflammatory effect on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. It reduced both LPS-stimulated nitric oxide production and LPS-stimulated increases in the gene expression levels of tumor necrosis factor-α and cyclooxygenase-2 in a dose-dependent manner. Furthermore, F-GWA inhibited the LPS-induced phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase, members of the mitogen-activated protein kinase family. The glycation process was evaluated by measuring fluorescence intensity and the furosine concentration during the Maillard reaction to form GWA. The protein modifications of WPI were analyzed using MALDI-TOF tandem mass spectrometry. We found that the combination of the Maillard reaction and L. gasseri 4M13 fermentation increased the prebiotic properties of GWA as well as organic acid production, compared with the nonreacted WPI and galactose.     

Effect of combined treatment of hydrolysis and polymerization with transglutaminase on β-lactoglobulin antigenicity

The effect of combined treatments of hydrolysis with different proteases, and subsequent polymerization with transglutaminase on the antigenic activity of β-Lg was studied. For the hydrolysis of β-Lg using Alcalase, Neutrase or bromelain, the reaction conditions were 3?% β-Lg and enzyme:substrate 25?U?g^?1 of protein, as was defined using factorial study. Under these conditions, the degree of hydrolysis (DH) of the hydrolysates was 12.6?% when obtained with Alcalase and approximately 4?% with Neutrase or bromelain. Post-hydrolysis polymerization did not result in an increase in molecular mass of the protein, but these samples presented a lower DH, determined by trinitrobenzenosulfonic acid (TNBS) method, suggesting that polymerization had occurred. Hydrolysis with the three enzymes reduced the β-Lg antigenicity, as evaluated by ELISA and immunoblotting analyses. The IgE-binding responses were practically null (<9?μg?mL^?1), 22.82 and 55.73?μg?mL^?1 towards the hydrolysates obtained with Alcalase, bromelain, and Neutrase, respectively. The post-hydrolysis polymerization increased or had no significant effect (P?≥?0.05) on the antigenic response of the hydrolysates.     

The effect of lactose derivatives lactulose,lactitol and lactobionic acid on the functional and technological properties of potentially probiotic Lactobacillus strains

Lactulose, lactitol and lactobionic acid are unabsorbable lactose derivatives with prebiotic potential. They are utilised in varying extent by different Lactobacillus and Bifidobacterium species/strains. To explore the possibility of improving the properties of probiotic strains with a specific prebiotic, the effect of the lactose derivatives on the technological and functional properties of putative probiotic Lactobacillus strains was studied in vitro. The properties studied were growth in different conditions (aerobic versus anaerobic with varying substrate concentrations), acid and bile tolerance, antimicrobial activity, and stability during cold storage. In general lactose derivatives did not effect the technological or functional properties of the Lactobacillus strains. However, our results suggest that lactulose might improve the bile tolerance and cold-storage stability of Lactobacillus salivarius. Finding synbiotic pairs where the prebiotic would benefit the specific probiotic strain is not a simple task, but worth pursuing for, since this kind of capacity would give added value to the synbiotic product.     

Growth promotion of Bifidobacterium and Lactobacillus species by proteinaceous hydrolysates derived from poultry processing leftovers

Bacterial growth media represent a high cost in industrial applications, and for this reason, it is economically important to find less expensive supplements to replace the traditional ones. In the present work, peptide hydrolysates obtained from poultry meat and bone residues (functional animal protein [FAP]) and from feathers (functional feather protein [FFP]) were studied to determine their ability for the production of microbial biomass with improved viability. The results obtained were compared with those obtained with other supplement nutritive compounds used in fermentation growth media. The molecular composition of the hydrolysates in terms of total and soluble nitrogen, molecular weight distribution, total and free amino acids, was determined. The growth and cellular state of Bifidobacterium and Lactobacillus strains were studied by turbidimetric measurements and direct count by fluorescence microscopy. Overall, this study suggested that by‐products from poultry industry provide a good alternative to substitute expensive supplements for growth of Lactobacillus and Bifidobacterium with a high level of viability.     

Effect of drying methods on the reactivity of chitosan towards Maillard reaction

The effect of drying methods on chitosan reactivity towards Maillard reaction during storage of dried chitosan–lactose systems was investigated. Two different structural forms of chitosan, scaffold and microspheres, were prepared. Then they were dried by lyophilization or using supercritical fluid technology (SF-CO₂) and stored with lactose under controlled temperature conditions (60 °C) and water activity (a_w = 0.65). The drying method produced slight modifications on the chitosan structure especially in samples submitted to SF-CO₂ treatment. Differences were more evident in the reactivity of chitosan–lactose systems during storage. Maillard reaction development was assessed by quantification of a new compound which originated from the acid hydrolysis of Amadori compounds (NFMD) resulting from chitosan–lactose interactions, and not detected in the acid hydrolysates of freshly prepared chitosan-lactose systems. Maillard reaction was influenced by different factors such as the manner of lactose addition, structure of chitosan (microspheres and scaffolds) and drying method. Lyophilized chitosan in the form of microspheres with lactose, presented the highest values of NFMD (428.45 mg/100 g of sample). The determination of this compound could be used as an index of the progress of Maillard reaction.     

Structure and antioxidant activity of high molecular weight Maillard reaction products from casein–glucose

Maillard reaction products (MRPs) were prepared from a casein–glucose reaction and ultrafiltrated to provide six fractions. The high molecular weight glycated proteins (melanoprotein) were further purified with a Sephadex G-75 column. Two fractions were obtained and analysed for their reducing power and Fe²⁺ chelating activity. Results obtained from the first fraction, analysed by Fourier transform infrared spectroscopy (FT-IR) indicated that the amide I, II and III bands of casein were changed by the Maillard reaction. The obtained samples were also hydrolysed with pepsin and trypsin in vitro, and the proteolytic hydrolysates were evaluated for their antioxidant activities. Non-hydrolysed melanoproteins exhibited the highest reducing power, but peptic hydrolysates of different MRPs were more efficient in radical-scavenging activity.     

Conjugation of gluten hydrolysates with glucosamine at mild temperatures enhances antioxidant and antimicrobial properties

Gluten represents one of the principal by-products of the wheat starch industry. Peptides obtained by wheat hydrolysis can be used for specific functional and biological activities, albeit at relatively low yields. Although the Maillard reaction (glycation) is widely used to increase functionality of proteins, its main disadvantage is the production of undesirable compounds due to high processing temperature. In this research, functional and biologically active glycopeptides were obtained from gluten. Alcalase or Flavourzyme proteases were used to hydrolyse gluten protein, and the resulting peptides were conjugated with glucosamine by enzymatic glycosylation, using transglutaminase, or through glycation. Both reactions were performed at mild temperatures (25 or 37 °C). The formation of glycopeptides depended mostly on the glycation process, as demonstrated by MALDI-TOF-MS. The bioactivities of the conjugated hydrolysates were compared to the native hydrolysates. Although a reduction in the anti-ACE activity was detected, improved DPPH scavenging activity and enhanced antimicrobial activity against Escherichia coli were observed in the glycated Alcalase-derived hydrolysates and in the glycated Flavourzyme-derived hydrolysates, respectively. This study showed that mild conditions are an alternate approach to the traditional Maillard process conducted at elevated temperatures in creating conjugated gluten hydrolysates with enhanced bioactivities.     

Glycation sites and bioactivity of lactose-glycated caseinate hydrolysate in lipopolysaccharide-injured IEC-6 cells

During the thermal processing of milk, Maillard reactions occur between proteins and lactose to generate glycated proteins. In this study, a lactose-glycated caseinate was hydrolyzed by trypsin. The obtained glycated caseinate (GCN) hydrolysate had a lactose content of 10.8 g/kg of protein. We identified its glycation sites and then assessed it for its protective effect against lipopolysaccharide-induced barrier injury using a rat intestinal epithelial cell line (IEC-6 cells) as a cell model and unglycated caseinate (CN) hydrolysate as a reference. Results from our liquid chromatography–mass spectrometry analysis of the GCN hydrolysate verified that lactose glycation occurred at the Lys residues in 3 casein components (α_S1-casein, β-casein, and κ-casein), and this resulted in the formation of 5 peptides with the following amino acid sequences: EMPFPKYPKYPVEPF, HIQKEDVPSE, GSENSEKTTMPL, NQDKTEIPT, and EGIHAQQKEPM. The results from cell experiments showed that the 2 hydrolysates could promote cell growth and decrease lactate dehydrogenase release in the lipopolysaccharide-injured cells; more importantly, they could partially protect the damaged barrier function of the cells by increasing trans-epithelial electrical resistance, decreasing epithelial permeability, and upregulating the expression of the 3 tight junction proteins zonula occludens-1, occludin, and claudin-1. However, compared with CN hydrolysate, GCN hydrolysate showed lower efficacy in protecting against cellular barrier dysfunction. We propose that the different chemical characteristics of the CN hydrolysate and the GCN hydrolysate (i.e., amino acid loss and lactose conjugation) contributed to the lower barrier-protective efficacy of the GCN hydrolysate. During dairy processing, protein glycation of the Maillard type might have a non-negligible, unfavorable effect on dairy proteins, in view of the resulting protein glycation we found and the critical function of proteins for maintaining the integrity of the intestinal barrier.     

超高压诱导β-乳球蛋白-壳聚糖共价复合物及其稳定乳状液的制备与表征

目的：探究超高压处理对美拉德反应的影响，通过蛋白质-多糖共价复合物反应，开发更高效的油-水界面膜以提高乳液稳定性。方法：以不同质量比（1∶1、1∶2、1∶4）的β-乳球蛋白和壳聚糖为原料，经不同压力（200、400、600 MPa）处理后，进行干法美拉德反应，以β-乳球蛋白和壳聚糖的物理混合物为对照，研究产物的褐变程度、荧光光谱和圆二色光谱图，以及制备的乳液粒径、Zeta-电位、物理稳定性等。结果：蛋白与多糖质量比为1∶1和1∶2时，随压力增加，褐变程度先升高后降低；蛋白与多糖质量比为1∶4时，褐变程度随压力增加持续升高。超高压诱导的美拉德反应使蛋白质荧光发生猝灭，壳聚糖比例越高，猝灭程度越大。在蛋白与多糖不同质量比（1∶1、1∶2、1∶4）时，共价复合物的α-螺旋相对含量分别由19.5%、21.1%、22.5%（物理混合物）降至18.7%、16.9%、15.9%，共价复合物α-螺旋相对含量均在400 MPa达到最大值，分别为19.6%、19.0%、16.6%。蛋白与多糖共价复合物制备的乳液粒径（399.1、481.5、584.4 nm）以及4 h时稳定性动力学指数（1.60、2.31、0.49）均比其物理混合物要小，而Zeta-电位更高（46～48 mV），乳液更稳定。结论：超高压诱导美拉德反应对其产物乳液的乳化稳定性有积极影响。     

Effect of vegetal-oil emulsion and passion fruit peel-powder on sensory acceptance of functional yogurt

Vegetal oil emulsion and passion fruit peel powder were concomitantly incorporated to milk aiming to produce new functional yogurt with health benefits. Four milk bases with similar energetic content and macronutrient composition, enriched or not with vegetal oil emulsion and/or passion fruit peel powder, were fermented with Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Bifidobacterium lactis (Bifidobacterium species 420) to supply yogurts with similar appearance, flavor, and stability. Milk without supplementation was used as control. Applying hedonic scale and projective map approaches, respectively, 227 and 25 panelists analyzed the samples concerning flavor, texture-in-spoon, creaminess-in-mouth, and global impression. Instrumental firmness was also investigated. Considering the same fermentation temperature, the addition of vegetal-oil emulsion and passion fruit peel-powder did not influence fermentation time, but significantly affected instrumental firmness. Despite the observed differences in flavor and global impression there were no significant changes in consumers' overall liking between supplemented and conventional yogurts.     

Untargeted metabolic footprinting reveals key differences between fermented brown milk and fermented milk metabolomes

Fermented brown milk has gained popularity because of its unique taste and flavor. Lactobacillus bulgaricus ND02 is a starter culture that has good milk fermentation characteristics. This study aimed to profile the metabolites produced during Maillard browning and to identify metabolomic differences between fermented brown milk and fermented milk produced by the ND02 strain. This study used liquid chromatography–mass spectrometry to compare the metabolomes of milk, fermented milk, brown milk, and fermented brown milk. Significant differences were observed in the abundances of various groups of metabolites, including peptides, AA, aldehydes, ketones, organic acids, vitamins, and nucleosides. The Maillard browning reaction significantly increased the intensity of a wide spectrum of flavor compounds, including short peptides, organic acids, and compounds of aldehydes, ketones, sulfur, and furan, which might together contribute to the unique flavor of brown milk. However, Maillard browning led to an increase in Nε-(carboxymethyl)lysine, which might cause negative health effects such as diabetes, uremia, or Alzheimer's disease. On the other hand, fermenting brown milk with the ND02 strain effectively countered such an effect. Finally, 5 differentially abundant metabolites were identified between fermented brown milk and fermented milk, including l-lysine, methylglyoxal, glyoxal, 2,3-pentanedione, and 3-hydroxybutanoic acid, which might together contribute to the different nutritional qualities of fermented brown milk and fermented milk. This study has provided novel information about the Maillard reaction and compared the metabolomes of the 4 types of dairy products.     

Comparative study on the effects of d-psicose and d-fructose in the Maillard reaction with β-lactoglobulin

d-Psicose, recognized as a noncaloric sweetener, has shown a great potential in food industry. In the present study, d-psicose and d-fructose were used to modify bovine β-lactoglobulin (β-Lg) through Maillard reaction. The Maillard reaction process and the physicochemical and structural properties of the modified proteins were also investigated. The result showed that compared to d-fructose, d-psicose played a more effective role in the Maillard reaction, especially after the initial stage of the reaction. Moreover, the modified β-Lg with d-psicose had more polymeric compounds, higher antioxidant activity, but lower thermal stability than that with d-fructose. These findings, especially the structural changes of the modified proteins, supplied detail information on the Maillard reaction of d-psicose, and could provide some guidance to the practical applications of this rare sugar on food industry.     

Acid production by bifidobacteria and yoghurt bacteria during fermentation and storage of milk

Three species of bifidobacteria, namelyBifidobacterium bifidum, Bifidobacterium longumandBifidobacterium adolescentiswere used in pure culture and in combination with yoghurt bacteria (B3 and SBI cultures) for the production of fermented milks. The number of bacteria during fermentation and the level of acid produced during fermentation and storage were assessed using Rogosa's modified selective agar and high performance liquid chromatography (HPLC). It was found that during fermentation all bifidobacteria exhibited growth uncoupled from acid production. Two of the species examined produced only low levels of acids when grown individually and onlyB. adolescentisproduced appreciable amounts. In mixed cultures, the level of acid was a reflection of the combination of yoghurt culture and species ofBifidobacterium, and this, observation suggests that there is a degree of influence between the cultures. During storage, the acid concentration remained quite stable in most samples. The prevention of post-production acidification that normally occurs during storage of yoghurt can be attributed to the presence of bifidobacteria, and it could be that acetic acid has a marginally inhibitory effect on theLactobacillusandStreptococcusspp.     

Prebiotic effects of structurally identified galacto-oligosaccharides produced by β-galactosidase from Aspergillus oryzae

Novel galacto-oligosaccharides were produced by β-galactosidase from Aspergillus oryzae using lactose, and their structural characteristics and prebiotic effects were examined. Highly purified oligosaccharide fraction (HP) was prepared from a crude one (low purified, LP) by gel-filtration on Biogel P-2 column, which was further purified into S1 and S2 fractions by prep-HPLC. S1 and S2 were comprised of galactose (Gal) and glucose (Glc) in the ratio of 2 to 1. ESI-MS-MS and methylation analysis indicated that S1 and S2 were trisaccharides with structures of β-d-Galp-(1,6)-β-d-Galp-(1,4)-β-d-Glcp and β-d-Galp-(1,3)-β-d-Galp-(1,4)-β-d-Glcp, respectively. Herein, LP and HP were used as the carbon sources for determining the prebiotic activity score of probiotics including Lactobacillus and Bifidobacterium species. LP and HP at 1% and 2% (w/v) were observed at all positive scores on several probiotics, especially, B. infantis ATCC 15697 at the 2% level of HP (p<0.05). Consequently, structurally identified trisaccharides of HP can significantly enhance the growth of B. infantis.     

Bacterial community succession and metabolite changes during the fermentation of koumiss,a traditional Mongolian fermented beverage

The microbial population in koumiss, a traditional Mongolian fermented beverage, plays a vital role in its nutritional value, beneficial functions, and flavour. We used the Illumina MiSeq platform to investigate the successive bacterial communities produced during koumiss fermentation; dynamic changes in chemical composition of koumiss were also analysed. The correlation between bacterial species and major metabolites was then evaluated. The dominant bacterial species identified during the process of koumiss fermentation were Lactobacillus helveticus, Streptococcus parauberis (phylum Firmicutes), and Acetobacter pasteurianus (phylum Proteobacteria). Lactose content decreased during fermentation, whereas lactic acid, acetic acid, and butyric acid content increased, resulting in decreased pH. The changes in lactose, lactic acid, butyric acid, and pH were mainly correlated with the presence of the bacterial genera Acetobacter, Leuconostoc, Pediococcus, and Bacteroides. Information from these results could advance our understanding of koumiss fermentation, and also help improve the safety, flavour, and therapeutic applications of koumiss.