Production of low antigenic cheese whey protein hydrolysates using mixed proteolytic enzymes

This study examined the effect of different proteolytic enzymes on the production of cheese whey protein (CWP) hydrolysates with low antigenicity. Four enzyme combinations (1:1) trypsin + papain W‐40 (TP), trypsin + neutrase 1.5 (TN), papain W‐40 + protease S (PP) and papain W‐40 + neutrase 1.5 (PN) were added at the rate of 1% of the CWP and it was incubated for 15, 30, 60, 90, 120 and 180 min at 50 °C. CWP hydrolysis and its non‐protein nitrogen concentrations were higher with TP and TN compared with PP and PN at all incubation times. The SDS‐PAGE revealed complete removal of α‐lactalbumin (α‐LA) and β‐lactoglobulin (β‐LG) from hydrolysates produced by trypsin‐containing enzyme mixtures. Reverse‐phase HPLC analysis ascertained the CWP hydrolysis and SDS‐PAGE results. The lowest antigenicity in CWP hydrolysates was observed with the use of trypsin‐containing enzyme mixtures compared with other enzyme combinations. Present results suggested that TP and TN combinations were the most effective for CWP hydrolysis for the removal of β‐LG from CWP. Further research is warranted to identify the peptides in CWP hydrolysates produced with these enzyme combinations that may help enhance the utilisation of whey protein in human food. Copyright © 2007 Society of Chemical Industry     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

优良“老酸奶”发酵剂组合的筛选

为了筛选出优良的酸奶发酵剂,根据菌株的稳定性、发酵活力、凝乳时间等指标对23菌株(其中有13株球菌与10株杆菌)进行初步筛选,获得13株具有优良发酵特性的菌株(其中有7株球菌与6株杆菌)。根据单菌株发酵实验结果,按菌种性能互补原则将各球菌与杆菌复配,进行28个组合发酵实验,测定发酵速度、质构、乳清析出以及后酸化等指标。最终筛选出球菌S1、S4与杆菌L2,此组合与目前商业化直投发酵剂比具有发酵前期产酸速度快、发酵时间短的优点,而且具有天然的发酵香气,乳清析出量很少,适用于直投式发酵剂的生产。     

Effect of milk supplementation and culture composition on acidification, textural properties and microbiological stability of fermented milks containing probiotic bacteria

In the present work, the combined effect of milk supplementation and culture composition on acidification, textural properties, and microbiological stability of fermented milks containing probiotic bacteria, was studied. Three powders (whey, casein hydrolysate, and milk proteins) were tested as supplementation. Two strains of probiotic bacteria, Lactobacillus acidophilus (LA5) and Lactobacillus rhamnosus (LC35), were used in pure culture, and in mixed culture with Streptococcus thermophilus (ST7). Acidifying activity was enhanced with mixed cultures, compared to pure cultures resulting in a shorter time to reach pH 4.5. Acidifying activity was greatly improved with casein hydrolysate, with a reduction of the fermentation time by about 55% by comparison with the other supplementations. The stability of probiotic bacteria was weakly affected by milk supplementation and culture composition. However, pure cultures were more stable than mixed cultures. The texture of the fermented products was not dependent on culture composition, but strongly dependent on milk supplementation. Sweet whey supplementation gave products with lower firmness and viscoelasticity than products supplemented with casein hydrolysate or milk proteins (decrease by 70%). It was observed that all products containing probiotic counts over 2.2×10⁷ CFU mL⁻¹ are suitable for the development of a lactic beverage containing probiotics.     

Purification and physicochemical characterization of ovine β‐lactoglobulin and α‐lactalbumin

Ovine whey proteins were fractionated and studied by using different analytical techniques. Anion‐exchange chromatography and reversed‐phase high‐performance liquid chromatography (HPLC) showed the presence of two fractions of β‐lactoglobulin but only one of α‐lactalbumin. Gel permeation and sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis allowed the calculation of the apparent molecular mass of each component, while HPLC coupled to electrospray ionisation‐mass spectrometry (ESI‐MS) technique, giving the exact molecular masses, demonstrated the presence of two variants A and B of ovine β‐lactoglobulin. Amino acid compositions of the two variants of β‐lactoglobulin differed only in their His and Tyr contents. Circular dichroism spectroscopy profiles showed pH conformation changes of each component. The thermograms of the different whey protein components showed a higher heat resistance of β‐lactoglobulin A compared to β‐lactoglobulin B at pH 2, and indicated high instability of ovine α‐lactalbumin at this pH.     

Relationship between proteolytic changes and tenderness in prerigor lactic acid marinated beef

A meat tenderising procedure involving injection of a lactic acid solution into prerigor muscle was investigated using beef M pectoralis profundus. The distribution of lysosomal enzymes in subcellular fractions, densities of myofibrillar protein bands after SDS‐PAGE and shear force were measured in non‐injected, 0.5 M and 1.0 M lactic‐acid‐injected samples during a 21 days ageing period. The activities of cathepsin B + L and β‐glucuronidase in the soluble fraction increased with level of lactic acid and with time post‐mortem (P < 0.001). Lactic acid and storage decreased densities of SDS‐PAGE bands migrating at the position of myosin heavy chain (MHC) and α‐actinin and increased densities of a 150 kDa band (P < 0.01). SDS‐PAGE of isolated perimysium cleaved with CNBr showed proteolytic cleavage of collagen after prolonged storage. Lactic acid injection significantly reduced shear force (P < 0.001). The cathepsin B + L activity in the soluble fraction correlated to shear force (r = −0.8), the degradation of MHC and α‐actinin (r = −0.88 and −0.90) and the generation of the 150 kDa fragment (r = 0.90) but not to the generation of a 31 kDa fragment (r = 0.05). A major part of the tenderness improvement after lactic acid injection was complete at 24 h post‐mortem, and was therefore due to a rapid process, eg pH‐induced swelling of the muscle structure. The data on enzyme activities and protein degradation, however, suggested that the action of lysosomal cathepsins also contributed to textural changes. © 1999 Society of Chemical Industry     

Influence of Maillard reaction conditions on the antigenicity of bovine α‐lactalbumin using response surface methodology

BACKGROUND: Maillard reaction can modify functional properties of proteins. Bovine α‐lactalbumin (α‐LA) is often supplemented to the new generation of infant formulae, but it is considered to be a main allergen. However, there is little information on the effect of Maillard reaction on α‐LA antigenicity. The objective of this study was to investigate the influence of Maillard reaction on the antigenicity of α‐LA in conjugates of whey protein isolate (WPI) with glucose under different conditions of protein/sugar weight ratio (0.17–7.83), temperature (40–60 °C) and time (24–120 h) using response surface methodology. RESULTS: Conjugation of WPI with glucose markedly reduced the antigenicity of α‐LA. This reduction in antigenicity could be controlled by regulating the three independent variables weight ratio, temperature and time. A model of optimal reaction conditions for lower antigenicity of α‐LA was established. According to the model, the minimum antigenicity of α‐LA was achieved at 52.8 °C, 78 h and 5.96:1 WPI/glucose weight ratio. WPI/glucose weight ratio had the greatest effect on the antigenicity of α‐LA, while reaction temperature influenced α‐LA antigenicity to a lesser extent. CONCLUSION: Well‐controlled Maillard reaction between WPI and glucose is an efficient method to reduce α‐LA antigencity. Copyright © 2009 Society of Chemical Industry     

Bioutilisation of whey for lactic acid production

The disposal of whey, the liquid remaining after the separation of milk fat and casein from whole milk, is a major problem for the dairy industry, which demands simple and economical solutions. The bioconversion of lactose present in whey to valuable products has been actively explored. Since whey and whey permeates contain significant quantities of lactose, an interesting way to upgrade this effluent could be as a substrate for fermentation. Production of lactic acid through lactic acid bacteria could be a processing route for whey lactose and various attempts have been made in this direction. Immobilised cell technology has also been applied to whey fermentation processes, to improve the economics of the process. A fermentative means of lactic acid production has advantages over chemical synthesis, as desirable optically pure lactic acid could be produced, and the demand for optically pure lactic acid has increased considerably because of its use in the production of poly(lactic acid), a biodegradable polymer, and other industrial applications. This review focuses on the various biotechnological techniques that have used whey for the production of lactic acid.     

Antimicrobial peptides generated from milk proteins: a survey and prospects for application in the food industry. A review

Milk proteins constitute a natural reservoir of bioactive peptides with physiological and/or antimicrobial properties, the release of which requires hydrolysis of the precursor molecules by digestive proteases or by fermentation with proteolytic micro‐organisms. Depending on the digestive or microbial proteases used, an array of bioactive peptides would be released either from caseins or whey proteins, but only a small part of these peptides has so far been identified and characterised with respect to their antimicrobial activity. The antimicrobial peptides known thus far have proven to be potent inhibitors to the growth of a wide range of undesirable micro‐organisms of health or spoilage significance. Nevertheless, previous research work has largely been oriented towards their possible application in medicine, which has hindered their high potential as food‐grade biopreservatives and/or as supplements in functional foods. This review attempts to study the literature pertaining to antimicrobial peptides derived from major milk proteins (caseins, α‐lactalbumin and β‐lactoglobulin) upon hydrolysis either by digestive proteases or by fermentation with proteolytic lactic acid bacteria. Their possible application in the food industry and their mechanism of action will also be discussed. Reference antimicrobial peptides produced by living micro‐organisms as innate immune defence components against microbial infections will occasionally be invoked for comparison purposes.     

Selection of Dominant NSLAB from a Mature Traditional Cheese According to their Technological Properties and in vitro Intestinal Challenges

Abstract: Isolates (47) of lactobacilli from 5 different productions of Melichloro cheese were examined for potential use as adjunct cultures. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) of whole‐cell proteins classified 29 isolates as L. paraplantarum and 18 as L. paracasei subsp. paracasei. Randomly amplified polymorphic DNA‐polymerase chain reaction (RAPD‐PCR) analysis differentiated the L. paraplantarum and L. paracasei subsp. paracasei isolates at strain level and both, RAPD analysis and whole‐cell protein profiling provided useful information about the diversity of nonstarter lactic acid bacteria (NSLAB) in the different cheese productions. The isolates were slow acidifiers and about 70% of them degraded, preferentially α_s‐casein. The amounts of amino acids accumulated in the milk increased with the incubation time. A similar enzyme profile was exhibited by strains of both species, except for α‐mannosidase and α‐fucosidase, which were not detected in the L. paracasei subsp. paracasei strains. All strains grew in the presence of bile at 0.3% and the majority was able to withstand pH 2.5 and pancreatin at 0.1%. Moreover, all strains reduced cholesterol in vitro, with higher removal ability recorded for strains of L. paraplantarum. A narrow spectrum of antibacterial activity was recorded for 88% of the strains. Selected isolates with appropriate technological and interesting in vitro intestinal challenges could be used as adjuncts and deserve further studies. Practical Application: Strains selected by this study could be used as adjuncts to make the Melichloro cheese. Their contribution to cheese flavor is then going to be studied to select the most appropriate. Of course these strains have to be also studied for their probiotic potential, to say that we have a probiotic food.     

蒙古族奶嚼口与下层凝乳中乳酸菌的筛选和比较研究

该研究以蒙古族奶嚼口和下层凝乳为原料,对乳酸菌进行计数和分离,通过产酸凝乳、耐胆盐实验及基因分析筛选并鉴定优良发酵菌株。结果表明,奶嚼口和下层凝乳中乳酸菌活菌数为（12.60±0.78）lg（CFU/mL）和（12.31±0.35）lg（CFU/mL）;分离获得的200株乳酸菌中有19株产酸凝乳能力较好,其中12株来源于奶嚼口,7株来自下层凝乳;在0.3 g/L和0.6 g/L胆盐环境中,奶嚼口中筛选出的乳酸菌具有更强的胆盐耐受性;奶嚼口筛选出11株乳酸乳球菌（Lactococcus lactis）和1株屎肠球菌（Enterococcus faecium）;下层凝乳筛选出的7株均为乳酸乳球菌。奶嚼口和下层凝乳中乳酸菌活菌数及种属并无明显差异,奶嚼口中分离的具有产酸凝乳能力的乳酸菌多于下层凝乳中所筛选的菌株,并有较强的胆盐耐受性。     

酸浆标准化生产工艺的研究

利用从豆腐酸浆老汤中筛选到的五株产酸菌,以大豆黄浆水为培养基,以酸浆的p H为考察指标,探讨了单菌发酵、双菌发酵、发酵温度、菌种接种量及菌种的混合比例对酸浆p H的影响。在单因素实验的基础上设计了正交实验,以确定酸浆纯种发酵的最佳生产工艺参数。结果表明:酸浆纯种发酵的最佳工艺参数为:双菌混合发酵,混合比例为1∶9(1号菌∶3号菌),接种量5%,发酵时间24h,发酵温度42℃。对正交实验的结果进行验证得到酸浆的p H为3.56。      

甘肃牧区传统发酵乳制品中优良乳酸菌的分离筛选

从甘肃牧区传统发酵乳制品中分离筛选适合发酵乳生产的乳酸菌,对分离纯化的乳酸菌进行了发酵性能检测,筛选出产酸快、发酵活力高、凝乳时间短、后酸化能力弱、遗传性状稳定的菌株。筛选的6株菌株单菌发酵牛乳,均在6.5 h内凝乳,发酵乳酸度＞70 °T,乳酸菌活菌数＞1×108 CFU/mL,发酵乳组织状态、滋味、气味等感官指标良好,可作为生产发酵乳的优良乳酸菌菌种进行开发利用。     

新疆传统发酵乳品中乳酸菌与酵母菌生长的相互影响

对新疆传统发酵乳酪乳清中的优势菌株:马乳酒样乳杆菌、乳酸乳球菌、瑞士乳杆菌、植物乳杆菌、东方伊莎酵母菌在驼乳中的生长特性进行了研究。探讨了4株乳酸菌与东方伊莎酵母菌之间的相互作用。结果表明,在发酵过程中东方伊莎酵母菌显著促进马乳酒样乳杆菌和乳酸乳球菌的生长(p<0.05),对瑞士乳杆菌的生长促进作用不明显(p>0.05)。同时,4株乳酸菌抑制东方伊莎酵母菌的生长(p<0.05)。此外,乳酸菌与东方伊莎酵母菌共同接种发酵有利于保持发酵乳冷藏期间活菌数的稳定及缓解乳酸菌的过度产酸。综上所述:乳酸菌与酵母菌之间可能存在相互作用的关系。      

Milk protein gene expression in the rat mammary gland

For the studies of the expression of milk proteins during the functional development of the rat mammary gland and in mammary tumor MTW9, milk proteins were purified, their mRNAs isolated, and the cDNA sequences complementary to these mRNAs cloned in E. coli cells. Results of such studies show: (1) rat α‐LA is unique in that it is larger than any known α‐LAs and is glycosylated; (2) rat milk contains unique whey phosphoproteins not found in other milks; (3) more than one plasmid DNA with differences in the restriction maps have been identified for several of these milk proteins, suggesting eithera presence of a family of genes or allelic differences for these proteins; (4) the expression of individual milk proteins is dependent on the functional stage of the gland; (5) there is an inverse relationship between the expression of milk proteins and the methylation of their gene sequences; (6) mammotrophic hormones required for synthesis and stability of milk proteins and their mRNAs, when withdrawn arrest the synthesis of α‐LA in the mammary tumor MTW9 at 6 hr or earlier of withdrawal but without any measurable effect on other proteins of the tumor.     

Changes in the soluble nitrogen fraction of milk throughout PDO Grana Padano cheese-making

The behaviour of soluble nitrogen compounds during Grana Padano cheese-making was studied at eight dairies. Raw milk, skimmed milk, sweet whey and the derived natural whey culture, collected from 24 processes, were analysed for soluble whey proteins (α-lactalbumin and β-lactoglobulin), proteose-peptones (PP), small peptides (SP), caseinomacropeptides (CMPs), and free amino acids (FAAs). The PP fraction increased during milk natural creaming, then part of it was selectively retained in the curd and the rest degraded in the first few hours of whey fermentation, together with α-lactalbumin, CMPs and part of SP. Features outlined for the whey culture were confirmed on 30 samples collected at six different dairies. A time course study of the whey fermentation showed that degradation of α-lactalbumin began when the pH dropped below 4, whereas β-lactoglobulin content did not change. Uptake of specific FAAs was shown to support the initial growth of lactic acid bacteria in whey.     

Molecular Characterization and Identification of Bioactive Peptides Producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing

In the present study, 3 bacterial cultures were isolated from faecal samples of human infant. The biochemical traits showed similarity with Lactobacillus sps and 16S rRNA sequence analyses, confirmed as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. The cultures were screened for their proteolytic activity and good ability to release peptides from milk proteins was found. Hence, these bacteria were used as a proteolytic starter culture for the fermentation of skim milk and whey for the liberation of small peptides. Bioactive nature of the peptides released from whey and skim milk was tested, and results demonstrated that peptides obtained after fermentation of whey and skim milk by Lactobacillus strains showed antimicrobial activity against all the pathogens causing food borne infections in humans. These peptides also indicated antioxidant as well as ACE (angiotensin-converting enzymes) inhibitory activity.     

Beta-lactoglobulin is a thermal marker in processed milk as studied by electrophoresis and circular dichroic spectra

As much of the sterilization process involves heat treatment during the preparation of milk on an industrial scale, the unpredictable measures of the process are an essential issue in determining the quality of the milk. The purpose of the present study was to investigate the major protein change(s) of whey proteins in processed milk and extend the knowledge for future reference in the dairy industry. Using a native polyacrylamide gel electrophoresis, we showed almost a 90% loss and denaturation of beta-lactoglobulin (LG), but not alpha-lactalbumin (LA), in some brands of the processed and dry milks. Immunochemical analysis using Western blotting revealed that part of the loss was attributed to the formation of large multiple forms of LG in the processed product. Such denaturation was presumably associated with the heating procedure used in the process. Essentially, LG was the only major fraction converted to aggregates in milk heated at 95 degrees C for 30 min on 2-dimensional PAGE. The detailed thermal denaturation of purified LG and LA at various temperatures (50 to 95 degrees C) and time (5 to 960 s) were investigated using a circular dichroic analysis. The maximal changes of ellipticity at 205 nm (converting beta-structure to disordered structure) were correlated to heating temperature and time. There were no significant conformational changes of LG at temperatures below 70 degrees C for as long as 480 s. Pronounced and rapid changes occurred between 80 to 95 degrees C in a time-dependent manner. Fifty percent of the maximal changes could be reached within 15 s. In conclusion, the unique chemical and immunochemical loss and conformational changes made LG a superior marker for evaluating the thermal processing of milk. The detailed thermal denaturation curves of LG constructed with its time and temperature in this study provide a valuable reference for the dairy industry. We postulate that heat treatment over 80 degrees C in 15 s may induce a significant denaturation of milk LG.     

核桃粕发酵乳菌种筛选及发酵条件优化研究

该试验对乳酸菌发酵核桃粕乳的不同菌株进行筛选,通过pH值、酸度及感官评定分析,从9株乳酸菌中筛选出嗜酸乳杆菌(Lactobacillus acidophilus)和植物乳杆菌(Lactobacillus plantarum)两株优良菌种。研究两株乳酸菌的复配比例,并与传统发酵剂发酵的核桃粕乳进行品质对比。结果表明,以嗜酸乳杆菌:植物乳杆菌(1∶1)接种发酵后得到的发酵核桃粕发酵乳综合品质最佳,其感官评分90分,氨基酸态氮含量57.0 mg/L,活菌总数7.35×107 CFU/mL,经发酵后的营养价值明显优于传统发酵剂发酵的核桃粕乳。     

乳酸菌降低过敏食物致敏性的研究进展

食物过敏的患病率在近几十年内急剧上升,越来越多的证据表明机体内微生物的生态失调是引起食物过敏的主要原因,因此乳酸菌等益生菌对过敏症状的潜在预防和治疗效果引起了人们的广泛关注。大量研究表明乳酸菌不仅能够通过发酵降低食物过敏原的致敏性,而且有可能通过调节肠道菌群的平衡在宿主免疫系统的发育和调节中发挥作用,从而改变过敏性疾病的发病风险。本文综述了乳酸菌的分类和生物特性,乳酸菌发酵在降低牛奶、大豆、花生、小麦等主要食物过敏原中发挥的作用,乳酸菌发挥免疫调节作用的主要机制,包括改变抗原递呈细胞的表型、调节Th1/Th2平衡、诱导调节性T细胞、增强肠道屏障功能。以期通过乳酸菌及其发酵作用达到预防和/或治疗食物过敏的效果,为乳酸菌的研究及应用提供参考。