Impact of Milk Derived β-Casomorphins on Physiological Functions and Trends in Research: A Review

β-Casomorphins are a group of opioid peptides released during gastrointestinal digestion or food processing from the β-casein of milk protein. Consequently, milk can be divided into A1 and A2 “like” groups depending upon the presence or absence of proline or histidine at the 67th position of β-casein. A1 “like” milk is postulated to be a source of BCM-7 as histidine allows the cleavage at this position, while A2 “like” milk has proline that resists the hydrolysis. On one hand, BCM-7 has been implicated as a risk factor for cardiovascular diseases, type I diabetes, and neurological disorders. On the other hand, various physiological effects of these peptides have also been documented, i.e., secretion of mucus, increased activity of superoxide dismutase and catalase, increased levels of prolactin, and analgesic role. In addition, many evidences correlate these peptides with various immunological functions, such as development of innate immunity, lymphocyte proliferation and cellular immunity, role in autoimmune diseases, histamine release, and allergy. In conclusion, the role of β-casomorphins in physiological functions remains controversial and more research with improved diagnostic techniques is needed to unravel the mechanism and study physiological functions of β-casomorphins. Thus, health-related aspects of β-casomorphins (positive, negative, and immunological impacts) have been comprehensively reviewed in this article.     

A2β-酪蛋白的功能性及其在乳制品中应用的研究进展

牛奶中富含多种营养成分,包括蛋白质、脂质、碳水化合物等,每100 g牛奶中就含有3 g蛋白质,其中主要包括乳清蛋白和酪蛋白。β-酪蛋白占牛奶蛋白总量的24%～28%,其主要包括两种基因型：A1型与A2型。A1β酪蛋白经消化后产生的β-酪啡肽-7会导致人体消化功能紊乱、心血管疾病等不良反应。A2β-酪蛋白则产生较少甚至并不产生β-酪啡肽-7,在胃肠道消化、提高抗氧化功能、降低胆固醇浓度等方面具有益生作用。本文综述了β-酪蛋白基因型在人体内消化后所产生的影响,并阐述了A2β-酪蛋白的益生功能,讨论其对人体健康的作用。并从β-酪蛋白基因型在乳制品中的应用入手,阐述A2β-酪蛋白乳制品的研究进展,为今后乳制品中A2β-酪蛋白的功能研究提供指导意义。     

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92–100, 125–135, 125–138, and 149–162) in β-LG, 2 peptides in α-LA (AA 80–93 and 63–79), 2 peptides in α_S1-casein (AA 84–90 and 125–132), and 1 peptide (AA 25–32) in α_S2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.     

Formation of soluble protein complexes and yoghurt properties influenced by the addition of whey protein concentrate

The effects of whey protein concentrate (WPC) on the formation of soluble protein complexes and yoghurt texture were evaluated. Skim milk (SM) and skim milk enriched with 1% WPC (SM + 1%WPC) or 2% WPC (SM + 2%WPC) were left unheated or heated and then made into yoghurt gels. Yoghurt prepared from heated SM + 2%WPC had significantly higher storage modulus, water holding capacity and firmness values and a denser microstructure than those prepared only from skim milk. Electrophoretic analysis of the milk showed that the level of β-lactoglobulin and κ-casein in the serum phase increased with increasing WPC concentration, indicating that the content of disulfide-linked β-lactoglobulin and κ-casein was higher in SM + 2%WPC than in SM, suggesting that more soluble protein complexes had been formed. Consequently, yoghurt prepared from heated SM enriched with WPC may have more bonds and more protein complexes in the protein network than yoghurt prepared only from SM, thus resulting in firmer gels.Practical applicationsYoghurt, one of the most popular fermented milk products, is of high economic importance to the dairy industry worldwide. In particular, high-protein yoghurt, such as Greek-style or set-type yoghurt, has been driving its ongoing popularity over recent years. In current industrial production of high-protein yoghurt, protein fortification and heat treatment of milk are two of the most important processing parameters affecting yoghurt texture. Whey protein concentrate has been added to milk to reduce whey separation and to increase the firmness of the yoghurt. From a technological point of view, the interaction of the denatured whey proteins with casein micelles or with κ-casein in the serum phases is regarded as responsible for obtaining a good yoghurt structure. The present research has shown that it is possible to produce yoghurt with a range of textural properties by precisely controlling the rate of whey protein fortification during its manufacture. Therefore, this study provides a better understanding of the effect of WPC fortification and aims to extend this insight for the production of good-quality yoghurt.     

Evaluation of the antihypertensive angiotensin-converting enzyme inhibitory (ACE-I) activity and other probiotic properties of lactic acid bacteria isolated from traditional Greek dairy products

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of α_S1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.     

Antioxidant activity of yoghurt peptides: Part 2 – Characterisation of peptide fractions

The aim of the present study was to elucidate previous findings showing that peptide fractions isolated from yoghurt had antioxidant effects. Therefore, peptides and free amino acids released during fermentation of milk were characterised. Yoghurt samples were stripped from sugars and lactic acid and subsequently fractionated by ultra filtration using membranes with cut off sizes of 30, 10 and 3 kDa. The peptides in these fractions were identified by LC–MS/MS. The identified peptides comprised a few N-terminal fragments of αs₁-, αs₂-, and κ-casein, and several fragments from β-casein. Almost all the peptides identified contained at least one proline residue. Some of the identified peptides included the hydrophobic amino acid residues Val or Leu at the N-terminus and Pro, His or Tyr in the amino acid sequence, which is characteristic of antioxidant peptides. In addition, the yoghurt contained a considerable amount of free amino acids such as His, Tyr, Thr and Lys, which have been reported to have antioxidant properties. Thus, our findings confirm that the antioxidant effects of the peptide fractions from yoghurt are due to the presence of certain peptides and free amino acids with recognised antioxidant activity in these fractions.     

Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas

The release of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated during simulated gastro-intestinal digestion (SGID) of bovine β-casein variants (n = 3), commercial milk-based infant formulas (n = 6) and experimental infant formulas (n = 3). SGID included pepsin digestion at pH 2.0, 3.0 and 4.0 and further hydrolysis with Corolase PP™. β-Casein (β-CN) variants were extracted from raw milks coming from cows of Holstein-Friesian and Jersey breeds. Genomic DNA was isolated from milk and the β-CN genotype was determined by a PCR-based method. Phenotype at protein level was determined by capillary zone electrophoresis in order to ascertain the level of gene expression. Recognition and quantification of BCMs involved HPLC coupled to tandem MS. Regardless of the pH, BCM7 generated from variants A1 and B of β-CN (5–176 mmol/mol casein) the highest amount being released during SGID of form B. As expected, the peptide was not released from variant A2 at any steps of SGID. BCM5 was not formed in hydrolysates irrespective of either the genetic variant or the pH value during SGID. Variants A1, A2 and B of β-CN were present in all the commercial infant formulae (IFs) submitted to SGID. Accordingly, 16–297 nmol BCM7 were released from 800 ml IF, i.e. the daily recommended intake for infant. Industrial indirect-UHT treatments (156 °C × 6–9 s) did not modify release of BCM7 and, during SGID, comparable peptide amounts formed in raw formulation and final heat-treated IFs.     

Identification of bioactive peptides and quantification of β-casomorphin-7 from bovine β-casein A1, A2 and I after ex vivo gastrointestinal digestion

This study investigated whether different genetic variants of β-casein (β-CN) give rise to different bioactive peptides during digestion. β-CN was purified from bovine milk of genetic variants A1, A2 and I, and digested with human gastrointestinal juices in a static ex vivo model. Mass spectrometry analyses revealed that the peptide ⁶⁰YPFPGPIPN⁶⁸ was exclusively identified from variants containing proline at position 67. Most strikingly, the opioid peptide β-casomorphin-7, ⁶⁰YPFPGPI⁶⁶, was identified from both variants A1 and A2 after simulated digestion, though with concentration being somewhat higher after digestion of the variant A1, compared with variants A2 and I. The peptides ¹³⁴HLPLP¹³⁸ and ¹³³LHLPLP¹³⁸ were both identified after initial 5 min of duodenal digestion. In conclusion, genetic variation of β-CN may affect proteolysis during digestion; however, the release of β-casomorphin-7 (BCM7) does not seem to be linked solely to variant A1, as earlier suggested by relevant published literature on in vitro digestion.     

Occurrence of β-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion

The occurrence of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated in commercial dairy products and in their digests, following in vitro simulated gastro-intestinal digestion (SGID), by means of HPLC–MS. The analysed dairy products were as follows: 10 cheeses (Gorgonzola, Caprino, Brie, Taleggio, Gouda, Fontina, Cheddar and Grana Padano 10-, 15- or 25-m ripened); 4 samples of drinking milk (unprocessed, pasteurised, UHT and in bottle-sterilised); 2 yoghurts and 4 fermented milks containing probiotics; 7 infant formulas; and 4 dried milk-derivatives (skim milk powder, calcium caseinate and milk protein concentrates). β-Casomorphin-5 was not detected in dairy products, either prior to or after SGID. β-Casomorphin-7 was detected only in cheeses with the exception of Taleggio, Caprino and Grana Padano samples. Peptide amount was in the range 0.01–0.15 mg kg⁻¹ the highest level being recovered in Brie sample. Following SGID, BCM7 formed in all dairy samples or increased up to 21.77 mg kg⁻¹ in digests of cheeses. The peptide level ranged from 0.29 to 1.23 mg kg⁻¹in fermented milks and from 3.46 to 22.18 mg kg⁻¹ in dried milk-derivatives. Digests of commercial infant formulas contained BCM7 at concentrations of 0.04–0.21 mg l⁻¹. For the first time, this work reports quantitative values for BCM5 and BCM7 in a range of dairy products providing evidence that, during processing, only proteolytic systems involved in manufacturing and ripening of cheese can potentially hydrolyse β-CN to BCM7. Nevertheless, formation or further release of BCM7 is mainly promoted by the action of gastrointestinal proteinases during in vitro digestion irrespective of the type of dairy product.     

Short communication: Tryptic β-casein hydrolysate modulates enteric nervous system development in primary culture

The intestinal tract of the newborn is particularly sensitive to gastrointestinal disorders, such as infantile diarrhea or necrotizing colitis. Perinatal development of the gut also encompasses the maturation of the enteric nervous system (ENS), a main regulator of intestinal motility and barrier functions. It was recently shown that ENS maturation can be enhanced by nutritional factors to improve intestinal maturation. Bioactivity of milk proteins is often latent, requiring the release of bioactive peptides from inactive native proteins. Several casein-derived hydrolysates presenting immunomodulatory properties have been described recently. Furthermore, accumulating data indicate that milk-derived hydrolysate can enhance gut maturation and enrichment of milk formula with such hydrolysates has recently been proposed. However, the capability of milk-derived bioactive hydrolysate to target ENS maturation has not been analyzed so far. We, therefore, investigated the potential of a recently described tryptic β-casein hydrolysate to modulate ENS growth parameters in an in vitro model of rat primary culture of ENS. Rat primary cultures of ENS were incubated with a bioactive tryptic β-casein hydrolysate and compared with untreated controls or to cultures treated with native β-casein or a Prolyve β-casein hydrolysate (Lyven, Colombelles, France). Differentiation of enteric neurons and enteric glial cells, and establishment of enteric neural network were analyzed using immunohistochemistry and quantitative PCR. Effect of tryptic β-casein hydrolysate on bone morphogenetic proteins (BMP)/Smad pathway, an essential regulator of ENS development, was further assessed using quantitative PCR and immunochemistry. Tryptic β-casein hydrolysate stimulated neurite outgrowth and simultaneously modulated the formation of enteric ganglia-like structures, whereas native β-casein or Prolyve β-casein hydrolysate did not. Additionally, treatment with tryptic bioactive β-casein hydrolysate increased the expression of the glial marker glial fibrillary acidic protein and induced profound modifications of enteric glial cells morphology. Finally, expression of BMP2 and BMP4 and activation of Smad1/5 was altered after treatment with tryptic bioactive β-casein hydrolysate. Our data suggests that this milk-derived bioactive hydrolysate modulates ENS maturation through the regulation of BMP/Smad-signaling pathway. This study supports the need for further investigation on the influence of milk-derived bioactive peptides on ENS and intestinal maturation in vivo.     

基于酪蛋白免疫快速检测的羊奶鉴伪方法建立

为了能够快速、灵敏、低成本和方便地对市场上的羊奶进行鉴伪,本文基于竞争法原理开发了牛源酪蛋白胶体金免疫层析试纸条并对其进行了评价。通过在纯羊奶中添加不同比例的牛奶,获得含有不同浓度牛源蛋白的羊奶溶液并进行免疫层析试纸条测定,评价了该方法的检测范围和灵敏度,分析了奶制品经蒸煮和酶解处理对试纸条检测结果的影响,评价了该方法对完整的和酶解的纯品牛源α-酪蛋白和β-酪蛋白鉴别能力,制备了酪蛋白抗体亲和柱结合UPLC-Q-Exactive Orbitrap MS液质联用仪器及Peaks软件测定亲和柱结合分离的多肽序列,合成了鉴定到的人工多肽并通过试纸条方法进行检测。结果表明,基于肉眼判断羊奶掺伪定性检出限为1.2 mg/100 g,试纸条消线值为6 mg/100 g,羊奶与牛奶进行煮沸和酶解处理后对检测结果没有影响。将纯牛源α-酪蛋白和β-酪蛋白分别添加到羊奶中后测定发现,该方法无法区分并检验出酶解后的α-酪蛋白和β-酪蛋白。亲和柱结合液质结果表明,酪蛋白经过多种酶联合酶解后得到的大部分多肽并不能与酪蛋白抗体结合,胶体金试纸条方法也无法检测出人工合成多肽。以上研究结果表明,该试纸条方法可以测定羊奶中完整空间结构的牛源酪蛋白,并可以作为有效的工具用于市场上羊奶产品的真实性鉴定。     

Plasmin digestion of photooxidized milk proteins

Plasmin-mediated hydrolysis of 6 different milk protein preparations [α_S-casein (α_S1 + α_S2), β-casein, κ-casein, α-lactalbumin, β-lactoglobulin, and lactoferrin] was found to be very dependent on photooxidation of the said proteins. Changes in plasmin proteolysis were investigated in a peptide-mapping study applying liquid chromatography-mass spectrometry. The changes were seen in the formation of peptides formed by plasmin-mediated hydrolysis after photooxidation, which was initiated with the naturally occurring photosensitizer riboflavin in all the milk protein preparations studied. The changes in the plasmin-mediated hydrolysis of photooxidized proteins are discussed in relation to changes introduced in the protein structure upon photooxidation. Plasmin-mediated hydrolysis of α_S-casein, consisting of a mixture of α_S1- and α_S2-casein and a preparation of β-casein, was most highly affected by photooxidation, which is in agreement with the fact that those 2 proteins have been found to be most labile toward photooxidation. Changes in the formation of potential angiotensin-I-converting enzyme-inhibitory peptides as well as peptides proposed to have antibactericidal activities by plasmin were observed by oxidation of milk proteins before plasmin-mediated hydrolysis.     

β-CN-5P and β-CN-4P components of bovine milk proteose–peptone: Large scale preparation and influence on the growth of cariogenic microorganisms

Indigenous proteolytic activity in milk, mostly due to plasmin, gives rise to many casein-derived peptides that subsequently are found in the proteose–peptone fraction of milk where they comprise 10% or more of the total whey protein. Prominent amongst proteose–peptone components are β-CN-5P (β-casein residues, 1–105/107) and β-CN-4P (β-casein residues 1–28). Many peptides have potentially valuable functional or biological properties that differ from those of the parent proteins, and this paper describes simple, rapid and cost-effective preparation of these two milk peptide components in a high degree of purity, and in gramme quantities, for evaluation of such properties. The purification process was more efficient if β-casein was used as starting material. In this work, we prepared 46 g of β-casein from sodium caseinate in a simple rapid DEAE-cellulose ion-exchange chromatography stage. This was followed by in vitro hydrolysis with plasmin and precipitation and gel filtration steps to yield 4.8 g of highly purified β-CN-5P and 1.2 g of β-CN-4P. Utilising either unfractionated sodium caseinate, or milk itself, as starting material was satisfactory but gave less purified material containing other peptide impurities. Peptides similar to these proteose–peptone components have been implicated in the protective effects of milk and dairy products against dental caries in teeth. The mechanism(s) by which this protection occurs is unclear, but some antibiotics are peptides. However, we have found that, even at peptide concentrations as high as 0.5 mg/ml, neither β-CN-5P nor β-CN-4P had any effect on the in vitro growth of cariogenic Streptococcus mutans bacteria, ruling out a simple antibiotic mechanism.     

Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity

Two sets of traditional Greek sheep milk yoghurt were produced: the first one (YC) using normal yoghurt culture (Lactobacillus delbrueckii subsp. bulgaricus ?10.13 and Streptococcus thermophilus ?10.7) and the second (PR) with the same normal culture mixed with Lactobacillus paracasei subsp. paracasei DC412. YC and PR had similar physicochemical properties and proteolysis patterns throughout storage. Both products showed similar peptide profiles by RP-HPLC but quantitative differences were observed in respect to storage time. Single-strain cultures of the microorganisms used showed similar peptide profiles for both lactobacilli, yet L. delbrueckii subsp. bulgaricus was the most proteolytic of all three microorganisms. The peptide content and the ACE-inhibitory activity of the water-soluble extracts of yoghurts, YC and PR, increased throughout storage. Major peptides were identified from yoghurt PR and from the separate cultures of L. delbrueckii subsp. bulgaricus and L. paracasei subsp. paracasei. Most of these peptides were derived from β-casein. A peptide, β-CN f114-121, with well-established ACE-inhibitory and opiate-like activity was identified in yoghurt PR. Further identified peptides were regarded as potential ACE-inhibitors according to their sequence.     

Milk-derived bioactive peptides: From science to applications

Milk proteins have received increasing attention as potential ingredients of health-promoting functional foods targeted at diet-related chronic diseases, such as cardiovascular disease, diabetes type two and obesity. To this end, growing interest has been focused on physiologically active peptides derived from milk proteins. These peptides are inactive within the sequence of the parent protein molecule and can be liberated by gastrointestinal digestion of milk, fermentation of milk with proteolytic starter cultures or hydrolysis by proteolytic enzymes. Milk protein-derived peptides have been shown under in vitro and in vivo conditions to exert a number of activities affecting the digestive, endocrine, cardiovascular, immune and nervous systems. A great variety of naturally formed bioactive peptides have been found in fermented dairy products, such as yoghurt, sour milk and cheese. Recently, industrial-scale technologies suitable for the industrial production of bioactive milk peptides have been developed. In addition, a few commercial food products supplemented with milk protein-derived bioactive peptides have been launched on limited markets. Some of these products carry clinically documented benefits, in particular for reduction of mild hypertension. The multifunctional properties of milk peptides appear to offer considerable potential for the development of many similar products in the near future.     

Production of angiotensin I converting enzyme inhibitory (ACE-I) peptides during milk fermentation and their role in reducing hypertension

Fermented milk is a potential source of various biologically active peptides with specific health benefits. Angiotensin converting enzyme inhibitory (ACE-I) peptides are one of the most studied bioactive peptides produced during milk fermentation. The presence of these peptides is reported in various fermented milk products such as, yoghurt, cheese, sour milk, etc., which are also available as commercial products. Many of the ACE-I peptides formed during milk fermentation are resistant to gastrointestinal digestion and inhibit angiotensin converting enzyme (ACE) in the rennin angiotension system (RAS). There are various factors, which affect the formation ACE-I peptides and their ability to reach the target tissue in active form, which includes type of starters (lactic acid bacteria (LAB), yeast, etc.), substrate composition (casein type, whey protein, etc.), composition of ACE-I peptide, pre and post-fermentation treatments, and its stability during gastrointestinal digestion. The antihypertensive effect of fermented milk products has also been proved by various in vitro and in vivo (animal and human trials) experiments. This paper reviews the literature on fermented milk products as a source of ACE-I peptides and various factors affecting the production and activity of ACE-I peptides.     

Impact of milk processing on the generation of peptides during digestion

Milk processing may induce changes in dairy product composition and influence digestibility and nutrient bioavailability. Differences in protein degradation and peptide generation were studied for β-lactoglobulin and α_S1-casein from commercially available dairy products before, during, and after in vitro digestion. All major milk proteins, except β-lactoglobulin, were degraded to smaller peptides during the gastric phase in all investigated products. After the gastric phase, a shortened fragment of β-lactoglobulin was identified in the non-fermented dairy products, underlining differences in protein conformation due to the fermentation process. During the gastric phase, greater numbers of small peptides were generated from α_S1-casein than from β-lactoglobulin. The monitoring of generation of specific β-lactoglobulin and α_S1-casein peptide profiles by liquid chromatography–mass spectrometry allowed the identification of potential bioactive peptides. Peptides with satiety-influencing DPP-4 inhibiting properties were monitored and quantities were compared between products to identify promising targets for the development of new health promoting products.     

Variation of six bioactive milk proteins from milk from Chinese commercial dairy farms: Effect of season,farm, breed and udder health status

The study investigated potential changes in bioactive milk proteins in relation to the season, breed, farm and udder health status. Nine hundred and sixty bulk milk samples from 12 commercial farms in northwest China were collected during 2020–2021. Six bioactive proteins including fraction of β-casein proteins, α-lactalbumin, β-lactoglobulin, lactoferrin (Lf) and osteopontin were evaluated. It was found that most proteins (with exception of β-casein A1) of Jersey cows are significantly higher than Holstein cows. Most bioactive proteins demonstrated seasonal variation, and some exhibited geographical variation (with exception of whey albumins). The milk somatic cell count showed a positive correlation with immune proteins such as Lf.     

Effect of β-casein concentration in cheese milk on rennet coagulation properties,cheese composition and cheese ripening

Effects of the use of a β-casein powder to enrich cheese milk on rennet coagulation properties of milk, cheese composition and cheese ripening were investigated. Casein content of control milk was 2.5%, whereas that for the three enriched milks was adjusted with β-casein powder at 2.7%, 2.9% and 3.1%. The β-casein to α-casein ratio of these cheese milks was, respectively, 0.70, 0.79, 0.89 and 0.99. Rennet coagulation properties were related not only to casein concentration but also to the proportion of β-casein and α_s-casein presents in milks. Milk with higher concentration of β-casein had poorer coagulation properties. Cheeses could be produced by using a miniature cheese making process. Moisture, ash and calcium contents decreased, while protein content and β-casein increased in cheese as casein and β-casein concentration increased in milk. As a result, hardness was higher in enriched cheeses than in control cheese. During cheese ripening, α-casein was hydrolyzed, but the rate of degradation of α-casein decreased as protein and β-casein concentration increased in cheese. β-Casein seemed to be not hydrolyzed. The rate of decrease of hardness was also slower for enriched cheeses.     

Variation in caseinolytic properties of six cheese related Lactobacillus helveticus strains

A large degree of strain variation was observed in caseinolytic properties of six cheese related Lactobacillus helveticus strains. Activity on intact α_s1- and β-casein was observed only after growth in milk and not in MRS. Totally 27 peptides from α_s1- and 22 from β-casein were identified from MS/MS fragmentation patterns. All six strains released peptides from the amino end of α_s1-casein, and the bonds Ile₆-Lys₇ and Gln₉-Gly₁₀ were identified as primary cleavage sites. Strain variation in the activity on intact β-casein was observed and five of the six strains released peptides from the C-terminal region. The strains had very different activities and some strains had only trace activities. L. helveticus CNRZ 32 had the highest activity towards α_s1-casein while L. helveticus LHC2 had the highest activity towards β-casein, and these two strains also produced unique peptides from both α_s1- and β-casein.