Cell Envelope-associated Proteinase Research Articles

In Silico Comparative Genomic Analysis Revealed a Highly Conserved Proteolytic System in Lactobacillus delbrueckii.

Lactobacillus delbrueckii, the type species of the genus Lactobacillus, is widely recognized as the primary starter culture in the dairy industry due to its proteolytic activity, which enables it to growth in milk. In this study, a comprehensive genomic analysis of the proteolytic system was conducted on L. delbrueckii strains. The analysis included 27 genomes of L. delbrueckii, with a specific focus on the key enzyme involved in this system, the cell envelope-associated proteinase (CEP). The amino acid sequences, as well as the protein-structure prediction of the CEPs, were compared. Additionally, syntenic analysis of the genomic locus related to the CEPs revealed high conservation in L. delbrueckii subsp. bulgaricus strains, while L. delbrueckii subsp. lactis strains exhibited greater variability, including the presence of insertion sequences, deletions, and rearrangements. Finally, the CEP promoter region and putative regulatory elements responsible for controlling the expression of the proteolytic system in lactobacilli were investigated. Our genomic analysis and in silico characterization of the CEPs contribute to our understanding of proteolytic activity and the potential applications of these lactic acid bacteria in the dairy industry. Further research in this area will expand our knowledge and potential practical uses of these findings.

Distribution of Cell Envelope Proteinases Genes among Polish Strains of Lactobacillus helveticus.

Most of the lactic acid bacteria (LAB) are able to grow in milk mainly due to the activity of a complex and well-developed proteolytic system. Cell envelope-associated proteinases (CEPs) begin casein hydrolysis and allow for releasing the peptides, enclosed in the structure of native milk proteins that are essential for growth of Lactobacillus helveticus. The biodiversity of genes encoding CEPs among L. helveticus strains can have an effect on some technological parameters such as acid production, bacterial growth rate in milk as well as liberation of biologically active peptides. The study reveals significant differences in the presence of various variants of CEPs encoding genes among ten novel Polish strains and indicates the intraspecific diversity exhibited by L. helveticus. In terms of distribution of CEPs genes, four different genetic profiles were found among the microorganisms analyzed. Furthermore, the strains exhibited also various levels of proteolytic activity. Molecular analysis revealed that prtH3 is the most abundant CEPs-encoding gene among the strains investigated. The results indicate also that ecological niche and environmental conditions might affect proteolytic properties of L. helveticus strains. The greatest variety in terms of quantity of the detected CEP encoding genes was noticed in L. helveticus 141, T105 and T104 strains. In these strains, the combination of three nucleotide gene sequences (prtH/prtH2/prtH3) was identified. Interestingly, T104 and T105 exhibited the highest proteolytic activity and also the fastest dynamic of milk acidification among the tested strains of L. helveticus.

YebC, a putative transcriptional factor involved in the regulation of the proteolytic system of Lactobacillus

The proteolytic system of Lactobacillus plays an essential role in bacterial growth, contributes to the flavor development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. In this work, a genomic analysis of all genes involved in the proteolytic system of L. delbrueckii subsp. lactis CRL 581 was performed. Genes encoding the cell envelope-associated proteinase, two peptide transport systems, and sixteen peptidases were identified. The influence of the peptide supply on the transcription of 23 genes involved in the proteolytic system of L. delbrueckii subsp. lactis was examined after cell growth in a chemically defined medium (CDM) and CDM supplemented with Casitone. prtL, oppA1, optS, optA genes as well as oppDFBC and optBCDF operons were the most highly expressed genes in CDM; their expression being repressed 6- to 115-fold by the addition of peptides. The transcriptional analysis was confirmed by proteomics; the up-regulation of the PrtL, PepG, OppD and OptF proteins in the absence of peptides was observed while the DNA-binding protein YebC was up-regulated by peptides. Binding of YebC to the promoter region of prtL, oppA1, and optS, demonstrated by electrophoretic mobility shift assays, showed that YebC acts as a transcriptional repressor of key proteolytic genes.

Lactic Acid Bacteria as Cell Factories for the Generation of Bioactive Peptides.

There is a growing interest in the incorporation of functional foods in the daily diet to achieve health promotion and disease risk reduction. Numerous studies have focused on the production of biologically active peptides as nutraceuticals and functional food ingredients due to their health benefits. These short peptides, displaying antihypertensive, antioxidant, mineral binding, immunomodulatory and antimicrobial activities are hidden in a latent state within the primary sequences of food proteins requiring enzymatic proteolysis for their release. While microbial fermentation is one of the major and economically most convenient processes used to generate bioactive peptides, lactic acid bacteria (LAB) are widely used as starter cultures for the production of diverse fermented foods. This article reviews the current knowledge on LAB as cell factories for the production of bioactive peptides from a variety of food protein sources. These microorganisms depend on a complex proteolytic system to ensure successful fermentation processes. In the dairy industry, LAB containing cell envelope-associated proteinases (CEPs) are employed as biocatalysts for the first step of casein breakdown releasing bioactive peptides during milk fermentation. A better understanding of the functionality and regulation of the proteolytic system of LAB opens up future opportunities for the production of novel food-derived compounds with potential health-promoting properties.

Proteolytic Activity and Production of γ-Aminobutyric Acid by Streptococcus thermophilus Cultivated in Microfiltered Pasteurized Milk

A set of 191 strains of Streptococcus thermophilus were preliminarily screened for the presence of the genes codifying for cell envelope-associated proteinase (prtS) and for glutamate decarboxylase (gadB) responsible for γ-aminobutyric acid (GABA) production. The growth and proteolytic activity of the gadB-positive strains (9 presenting the prtS gene and 11 lacking it) were studied in microfiltered pasteurized milk. Degradation of both caseins (capillary electrophoresis) and soluble nitrogen fractions (HPLC) and changes in the profile of free amino acids (FAAs; ion-exchange chromatography) were evaluated at inoculation and after 6 and 24 h of incubation at 41 °C. None of the strains was capable of hydrolyzing caseins and β-lactoglobulin, and only two hydrolyzed part of α-lactalbumin, these proteins being present in their native states in pasteurized milk. Contrarily, most strains were able to hydrolyze peptones and peptides. For initial growth, most strains relied on the FAAs present in milk, whereas, after 6 h, prtS+ strains released variable amounts of FAA. One prtS+ strain expressed a PrtS- phenotype, and two prtS- strains showed a rather intense proteolytic activity. Only five strains (all prtS+) produced GABA, in variable quantities (up to 100 mg/L) and at different rates, depending on the acidification strength. Addition of glutamate did not induce production of GABA in nonproducing strains that, however, unexpectedly were shown to adopt the degradation of arginine into citrulline and ornithine as an alternative acid resistance system and likely as a source of ATP.

Characterization of the mature cell surface proteinase of Lactobacillus delbrueckii subsp. lactis CRL 581.

The cell envelope-associated proteinase (CEP) of Lactobacillus delbrueckii subsp. lactis CRL 581 (PrtL) has an essential role in bacterial growth, contributes to the flavor and texture development of fermented products, and can release bioactive health-beneficial peptides during milk fermentation. The genome of L. delbrueckii subsp. lactis CRL 581 possesses only one gene that encodes PrtL, which consists of 1924 amino acids and is a multidomain protein anchored to the cell via its W domain. PrtL was extracted from the cell under high ionic strength conditions using NaCl, suggesting an electrostatic interaction between the proteinase and the cell envelope. The released PrtL was purified and biochemically characterized; its activity was maximal at temperatures between 37 and 40°C and at pH between 7 and 8. Under optimal conditions, PrtL exhibited higher affinity for succinyl-alanyl-alanyl-prolyl-phenylalanine-p-nitroanilide than for succinyl-alanyl-glutamyl-prolyl-phenylalanine-p-nitroanilide, while methoxy-succinyl-arginyl-prolyl-tyrosyl-p-nitroanilide was not degraded. A similar α- and β-casein degradation pattern was observed with the purified and the cell envelope-bound proteinase. Finally, on the basis of its specificity towards caseins and the unique combination of amino acids at residues thought to be involved in substrate specificity, PrtL can be classified as a representative of a new group of CEP.

Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins

Ability of industrially relevant species of thermophilic lactobacilli strains to hydrolyze proteins from animal (caseins and β-lactoglobulin) and vegetable (soybean and wheat) sources, as well as influence of peptide content of growth medium on cell envelope-associated proteinase (CEP) activity, was evaluated. Lactobacillus delbrueckii subsp. lactis (CRL 581 and 654), L. delbrueckii subsp. bulgaricus (CRL 454 and 656), Lactobacillus acidophilus (CRL 636 and 1063), and Lactobacillus helveticus (CRL 1062 and 1177) were grown in a chemically defined medium supplemented or not with 1% Casitone. All strains hydrolyzed mainly β-casein, while degradation of αs-caseins was strain dependent. Contrariwise, κ-Casein was poorly degraded by the studied lactobacilli. β-Lactoglobulin was mainly hydrolyzed by CRL 656, CRL 636, and CRL 1062 strains. The L. delbrueckii subsp. lactis strains, L. delbrueckii subsp. bulgaricus CRL 656, and L. helveticus CRL 1177 degraded gliadins in high extent, while the L. acidophilus and L. helveticus strains highly hydrolyzed soy proteins. Proteinase production was inhibited by Casitone, the most affected being the L. delbrueckii subsp. lactis species. This study highlights the importance of proteolytic diversity of lactobacilli for rational strain selection when formulating hydrolyzed dairy or vegetable food products.

In-depth characterization of Lactobacillus delbrueckii subsp. lactis 313 for growth and cell-envelope-associated proteinase production

The effect of process conditions on the growth and production of cell-envelope-associated proteinase (PrtL) by Lactobacillus delbrueckii subsp. lactis ATCC® 7830™ (LDL 313) was studied. Cell growth was profuse under the temperature conditions of 37°C, 40°C and 45°C, with cell growth rates, μmax increasing with temperatures. Initial culture pH of 6 displayed the highest PrtL yield of 2.33U. For each growth temperature the cell growth rates under anaerobic conditions were markedly higher than microaerophilic conditions, attributable to efficient sugar utilization under anaerobic fermentation. Overall, the proteolytic activities of cell-bound PrtL (bPrtL) were found to be higher than that of released PrtL (rPrtL). Higher rPrtL activities were displayed by anaerobic cultures over the entire fermentation period whereas the converse was true for bPrtL with microaerophilic cultures in the mid-late exponential phase displaying higher bPrtL activities than anaerobic cultures. Further, the proteinases had caseinolytic specificity for β-casein and κ-casein placing them in the cell-envelope-associated proteinases (CEPs) class I (CEPI) of the lactococcal CEPs grouping. This study provides insights into conditions for profuse growth and proteinase production by LDL 313 for subsequent technological applications in fermented foods, the dairy industry and bioactive peptide production.

β-Casein hydrolysate generated by the cell envelope-associated proteinase of Lactobacillus delbrueckii ssp. lactis CRL 581 protects against trinitrobenzene sulfonic acid-induced colitis in mice

Lactobacillus delbrueckii ssp. lactis CRL 581, a thermophilic lactic acid bacterium used as a starter culture for the manufacture of several fermented dairy products, possesses an efficient proteolytic system that is able to release a series of potentially bioactive peptides (i.e., antihypertensive and phosphopeptides) from α- and β-caseins. Considering the potential beneficial health effects of the peptides released by L. delbrueckii ssp. lactis CRL 581 from milk proteins, the aim of this work was to analyze the anti-mutagenic and anti-inflammatory properties of the casein hydrolysates generated by the cell envelope-associated proteinase of this bacterium. The ability of α- and β-casein hydrolysates to suppress the mutagenesis of a direct-acting mutagen 4-nitroquinoline-N-oxide on Salmonella typhimurium TA 98 and TA 100 increased concomitantly with the time of casein hydrolysis. The anti-inflammatory effect of the β-casein hydrolysate was evaluated using a trinitrobenzene sulfonic acid (TNBS)-induced Crohn's disease murine model. The hydrolysate was administered to mice 10 d before the intrarectal inoculation of TNBS. The mice that received β-casein hydrolysate previously to TNBS showed decreased mortality rates, faster recovery of initial body weight loss, less microbial translocation to the liver, decreased β-glucuronidase and myeloperoxidase activities in the gut, and decreased colonic macroscopic and microscopic damage compared with the animals that did not receive this hydrolysate. In addition, β-casein hydrolysate exerted a beneficial effect on acute intestinal inflammation by increased interleukin 10 and decreased IFN-γ production in the gut. Our findings are consistent with the health-promoting attributes of the milk products fermented by L. delbrueckii ssp. lactis CRL 581 and open up new opportunities for developing novel functional foods.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

Potential immunomodulatory activity of bovine casein hydrolysates produced after digestion with proteinases of lactic acid bacteria

During hydrolysis of bovine milk caseins, cell envelope-associated proteinases (CEPs) of lactic acid bacteria (LAB) may be able to produce hydrolysates that possess immunomodulatory activity. In this study, the selection of LAB by screening for bacterial CEP genes as well as by testing the proteolytic activity on bovine milk caseins was performed. Diverse hydrolytic ability against α S1- and β-casein fractions was observed. Our experiments demonstrated that immunomodulatory activity of obtained casein hydrolysates (CHs) cannot be attributed to casein-derived bioactive peptides only and underlined the necessity to thoroughly remove bacterial components before assessing bioactivity of CHs. On this basis, this study established that 3 kDa-ultrafiltered CHs, produced after digestion with proteinases of Lactobacillus acidophilus ATCC 4356 and Lactococcus lactis subsp. lactis GR5, significantly decreased the basal nuclear factor (NF)-κB activity in Caco-2 cells, demonstrating immunomodulatory activity.

Variability and molecular typing of Streptococcus thermophilus strains displaying different proteolytic and acidifying properties

Proteolytic and acidifying properties of Streptococcus thermophilus strains isolated from yoghurt or cheeses were evaluated. Among 30 strains tested, 12 exhibited cell envelope-associated proteinase activity (PrtS +), three displayed a slight PrtS activity (PrtS +/−) and 15 were PrtS −, despite the presence of the corresponding gene ( prtS) in eight of them. Sequencing of the prtS gene in four PrtS − and one PrtS + strains revealed that the absence of PrtS activity in the PrtS − strain probably results from an alteration of the prtS regulation. The strains displaying the highest acidifying capacities were all PrtS +. All but one PrtS + strains were phylogenetically close, as shown by the sequencing of their rDNA internal transcribed spacer (ITS) 16S-23S. More specifically, the high proteolytic and acidifying capacities are associated with the presence of a type II-ITS.

Characterization of the Pattern of α s1 - and β-Casein Breakdown and Release of a Bioactive Peptide by a Cell Envelope Proteinase from Lactobacillus delbrueckii subsp. lactis CRL 581

The cell envelope-associated proteinases (CEPs) of the lactobacilli have key roles in bacterial nutrition and contribute to the development of the organoleptic properties of fermented milk products as well, as they can release bioactive health-beneficial peptides from milk proteins. The influence of the peptide supply, carbohydrate source, and osmolites on the CEP activity of the cheese starter Lactobacillus delbrueckii subsp. lactis CRL 581 was investigated. The CEP activity levels were controlled by the peptide content of the growth medium. The maximum activity was observed in a basal minimal defined medium, whereas in the presence of Casitone, Casamino Acids, or yeast extract, the synthesis of CEP was inhibited 99-, 70-, and 68-fold, respectively. The addition of specific di- or tripeptides containing branched-chain amino acids, such as leucylleucine, prolylleucine, leucylglycylglycine, or leucylproline, to the growth medium negatively affected CEP activity, whereas dipeptides without branched-chain amino acids had no effect on the enzyme's production. The carbon source and osmolites did not affect CEP activity. The CEP of L. delbrueckii subsp. lactis CRL 581 exhibited a mixed-type CEP(I/III) variant caseinolytic specificity. Mass-spectrometric screening of the main peptide peaks isolated by reverse-phase high-pressure liquid chromatography allowed the identification of 33 and 32 peptides in the alpha(s1)- and beta-casein hydrolysates, respectively. By characterizing the peptide sequence in these hydrolysates, a pattern of alpha(s1)- and beta-casein breakdown was defined and is reported herein, this being the first report for a CEP of L. delbrueckii subsp. lactis. In this pattern, a series of potentially bioactive peptides (antihypertensive and phosphopeptides) which are encrypted within the precursor protein could be visualized.

Potential of lactic acid bacteria isolated from specific natural niches in food production and preservation

Autochthonous strains of lactic acid bacteria (LAB) have been isolated from traditionally homemade cheeses collected from specific ecological localities across Serbia and Montenegro. Genetic and biochemical analysis of this LAB revealed that they produce bacteriocins, proteinases and exopolysaccharides. LAB produces a variety of antimicrobial substances with potential importance for food fermentation and preservation. Apart from the metabolic end products, some strains also secrete antimicrobial substances known as bacteriocins. Among the natural isolates of LAB from homemade cheeses, bacteriocin producers were found in both lactococci and lactobacilli. Lactococcus lactis subsp. lactis BGMN1–5 was found to produce three narrow spectrum class II heat-stable bacteriocins. In addition to bacteriocin production, BGMN1–5 synthesized a cell envelope-associated proteinase (CEP) and shows an aggregation phenotype. Another isolate, L. lactis subsp. lactis BGSM1–19 produces low molecular mass (7 kDa) bacteriocin SM19 that showed antimicrobial activity against Staphylococcus aureus, Micrococcus flavus and partially against Salmonella paratyphi. Production of bacteriocin reaches a plateau after 8 h of BGSM1–19 growth. Bacteriocin SM19 retained activity within the wide pH range from 1 to 12 and after the treatment at 100 °C for 15 min. Among collection of lactobacilli, the isolate Lactobacillus paracasei subsp. paracasei BGSJ2–8 produces heat-stable bacteriocin SJ (approx. 5 kDa) polypeptide. It retained activity after treatment for 1 h at 100 °C, and in the pH range from 2 to 11. In addition to isolates from cheeses, bacteriocin-producing human oral lactobacilli were detected. Most of them showed antimicrobial activity against streptococci, staphylococci and micrococci, but not against Candida. Isolate BGHO1 that showed the highest antimicrobial activity was determined as L. paracasei. Interestingly, Lactobacillus helveticus BGRA43, which was isolated from the human intestine showed strong activity against Clostridium sporogenes, but it was not possible to detect any bacteriocin production in this isolate by using standard procedures. Further analysis of antimicrobial activity revealed that BGRA43 has a relatively broad spectrum. Lactobacilli resistant to nisin were also detected among natural isolates. They produce bacteriocins, which have no activity against nisin producing lactococci.

Hydrolysis of bovine caseins by cathepsin B, a cysteine proteinase indigenous to milk

The presence of the lysosomal cysteine proteinase cathepsin B in milk has been demonstrated recently. The potential significance of this enzyme to proteolysis in milk and dairy products was evaluated by the study of its proteolytic specificity towards the caseins. Bovine cathepsin B (1.4 units mL−1) was added separately to β-casein or αs1-casein (5 mg mL–1, in 100 mm Na acetate buffer, pH 6.0, containing 1.5 mm dithiothreitol). Samples were taken over a 24 h period and analysed by urea polyacrylamide gel electrophoresis and RP-HPLC; peptides were identified by N-terminal sequencing and mass spectrometry. Cathepsin B cleaved β-casein at 32 sites and αs1-casein at 35 sites, extensively hydrolysing both proteins. Thus, cathepsin B has a very broad specificity against the caseins, with an apparent preference for bonds incorporating the amino acids Leu, Val, Gln, Pro and Ser. Cathepsin B cleaved some bonds in common with chymosin (including the Phe23–Phe24 bond of αs1-casein), plasmin and the cell envelope-associated proteinases of Lactococcus, suggesting that it could be involved in proteolysis in dairy products, particularly if manufactured from high somatic cell count milk.

Adaptation to high salt in Lactobacillus: role of peptides and proteolytic enzymes.

To study the influence of peptides and proteolytic enzymes in the osmotic adaptation of Lactobacillus casei. Di- and tri-peptides added individually increased the osmotolerance of Lact. casei when grown in a chemically defined medium (CDM) containing NaCl. Growth stimulation and the re-establishment in their presence of plasmid DNA supercoiling (recovery of the linking number) in hyperosmotic medium indicated that they are used as osmocompatible solutes as carnithine a known osmoprotector does. The investigation of the proteolytic system showed that in high osmolarity medium, the cell envelope-associated proteinase (PrtP), and PepX (X-prolyl-dipeptidyl aminopeptidase) increased activity and lost repression by peptides. PepI, an iminopeptidase was also derepressed. PepQ, a prolidase that specifically liberated proline from dipeptides, was almost unaffected. Derepression in the presence of peptides took place at the transcriptional level. However, the twofold activation of PrtP in CDM hyperosmotic medium was essentially through an increase of the apparent Vmax of the enzyme. These results strongly suggest a contribution of the proteolytic system peptide supply in the osmotic adaptation. Advances in understanding the role of peptides in the adaptation to high osmolarity particularly involved in dairy processes.

Proteolytic specificity of cathepsin G on bovine α s1 - and β-caseins

Abstract Mastitis is an inflammation of the mammary gland which results in an increase in numbers of somatic cells, particularly polymorphonuclear leucocytes (PMN), which contain very active proteinases. The objective of this study was to determine the cleavage specificity of cathepsin G, one of the principal PMN proteinases, on αs1- and β-casein. αs1- or β-casein (5 mg ml−1) were dissolved in 0.1 M HEPES buffer, pH 7.5, containing 0.05% NaN3. Cathepsin G was dissolved in 0.1% Brij 35 and 0.5 M NaCl and 0.25 units ml−1 of this stock solution was added to αs1- or β-casein in buffer. Samples were taken over a 24 h incubation at 37 °C and analysed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Isolated peptides were identified by N-terminal sequencing and mass spectrometry. Cathepsin G cleaved αs1-casein at at least 16 sites and β-casein at at least 21 sites, some of which were also cleavage sites of chymosin, plasmin, elastase, cathepsin B or the cell envelope-associated proteinase of Lactococcus. Thus, cathepsin G had a broad specificity on αs1- and β-casein and it is therefore possible that indigenous cathepsin G in milk may be of significance for the proteolysis of milk proteins. Of particular interest was the production of the small peptide αs1-casein (f1-23), which also results from cleavage of the Phe23-Phe24 bond by chymosin in cheese, and is hydrolysed rapidly during cheese ripening by the cell envelope-associated proteinase of Lactococcus.

Genetic characterization of a cell envelope-associated proteinase from Lactobacillus helveticus CNRZ32.

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.

Proteolytic specificity of elastase on bovine β-casein

The lysosomes of the principal somatic cell type recruited on mastitic infection, polymorphonuclear leucocytes (PMN), contain a wide range of hydrolytic enzymes which aid in the destruction of ingested bacteria. These enzymes are of increasing interest in terms of milk quality, but little is known about their activity on the caseins. The objective of this study was to determine the cleavage specificity of elastase, one of the principal PMN proteinases, on β-casein. β-Casein (5 mg ml −1) was dissolved in 0.1 M phosphate buffer, pH 7.5 and 1.76×10 −3 U ml −1 of elastase were added. Samples were taken over a 24 h period and analysed by urea polyacrylamide gel electrophoresis and high performance liquid chromatography. Peptides were identified by N-terminal sequencing and mass spectrometry. Elastase cleaved β-casein at several sites including Ile 26-Asn 27, Gln 40-Thr 41, Ile 49-His 50, Phe 52-Ala 53, Gln 56-Ser 57, Leu 58-Val 59, Asn 68-Ser 69, Val 82-Val 83, Val 95-Ser 96, Ser 96-Lys 97, Lys 97-Val 98, Ala 101-Met 102, Glu 108-Met 109, Phe 119-Thr 120, Glu 131-Asn 132, Leu 163-Ser 164, Ala 189-Phe 190, Phe 190-Leu 191 and Pro 204-Phe 205. Some of these sites are also cleaved by chymosin, plasmin or the cell envelope-associated proteinase of Lactococcus. The results show that elastase has a broad specificity on β-casein and it is therefore possible that indigenous elastase in milk may be of significance to the proteolysis of milk proteins.

Inhibition of Residual Coagulant in Cheese using Pepstatin

Abstract Pepstatin A, an inhibitor of acid proteases, was added (7.5, 15 or 30 μ mol L -1 ) to the curds/whey mixture at the start of cooking to inhibit residual coagulant in miniature (20 g) Cheddar-type cheeses. No degradation of α s1 -casein was observed by urea–polyacryl amide gel electrophoresis (PAGE) in the pepstatin-treated cheeses, indicating that all the concentrations of pepstatin used in this study effectively inhibited residual coagulant throughout ripening. The level of water-soluble N (WSN) as % of total N increased very slowly in the pepstatin-treated cheeses, while there was a steady increase in WSN in the control cheeses; after 4 months of ripening, the level of WSN in the control cheese was nearly three times as high as in the cheese treated with 30 μ mol L -1 pepstatin. Urea–PAGE of water-soluble fractions (WSF) showed marked differences between pepstatin-treated cheeses and their respective controls throughout ripening. Reverse-phase HPLC of the WSF of the cheeses showed that the peptides α s1 -CN f1-9/13, which are formed from the chymosin-produced peptide, α s1 -CN f1-23, by the action of the cell envelope-associated proteinase of Lactococcus , were not present in pepstatin-treated cheeses. Levels of total free amino acids (as determined by the Cd–ninhydrin method) were higher in controls than in pepstatin-treated cheeses throughout ripening. The results of this study demonstrated that pepstatin is a very effective inhibitor of residual coagulant in cheese.