ANGIOTENSIN I-CONVERTING ENZYME (ACE) INHIBITORY PEPTIDES FROM WHEY FERMENTED BY LACTOBACILLUS SPECIES

From 2% (w/w) whey powder in growth media, inhibitory peptides against angiotensin I-converting enzyme (ACE) were studied with nine Lactobacillus species. Lb. brevis, Lb. helveticus and Lb. paracasei were proved to be the most effective strains in liberating ACE inhibitory peptides from whey protein. The inhibition rates of these peptides against ACE ranging from 93.3 to 100%. Several distinct peaks were eluted when the whey proteins were fractionated on a Delta Pak C₁₈ column by reversed phase-high performance liquid chromatography (RP-HPLC). Among ACE inhibitory activities of 14 peptides purified by dialysis and by fractionation using RP-HPLC, two peptide fractions (H5 and H7) of Lb. helveticus showing IC₅₀ values of 5.3 and 7.8 were the most potent ACE inhibitors.
All of these peptides including some other peptides (H1 and B1), having strong inhibitory activities against ACE were pentapeptides positioning with Ala at their N-terminal and these petapeptides had mostly hydrophobic (Pro, Val and Leu) or aromatic (Phe) amino acids at the C-terminal.

PRACTICAL APPLICATIONS

There is a significant amount of research and interest in developing and charactering the peptides that inhibit angiotensin I-converting enzyme (ACE) activity as these natural products may have a role in blood pressure control in man. This study revealed that the identification of peptides, mostly composed of pentapeptides following fermentation of whey protein in growth medium with different strains have the ACE inhibitory activities. These peptides may have antihypertensive effect as natural and safe nutraceutical/functional ingredients, though the exact potency of the pentapeptides isolated in this experiment has not been determined.     

Aroma development in semi-hard reduced-fat cheese inoculated with Lactobacillus paracasei strains with different aminotransferase profiles

Reduced-fat, semi-hard round-eyed cheese was made from bovine milk with a mesophilic DL-starter and three Lactobacillus paracasei subsp. paracasei single-strain adjuncts with different aminotransferase (AT) activity profiles. The pilot-plant procedure was not influenced by the adjuncts, and similar cheese was made from all vats. The growth of each adjunct to dominate the cheese was confirmed by using DNA fingerprinting. Flavour profiles were different for cheeses made with the different Lactobacillus strains. Use of the adjunct CHCC 4256 significantly increased the content of flavour compounds that were produced from the branched-chain amino acids (BcAAs: Leu, Ile and Val) and Asp. These cheeses also had superior sensory characteristics as they tasted aromatic and sweet without bitterness. The adjunct CHCC 4256 did not have glutamate dehydrogenase activity, or the highest activity on BcAA, but showed typical AT activity, with a similar activity on Leu, Phe and Asp substrates.     

西藏地区发酵牛乳中产细菌素乳酸菌的筛选及鉴定

从西藏地区藏族传统发酵乳中分离乳酸菌,采用生理生化特性和16S基因序列同源性分析对其进行鉴定,通过双层琼脂平板扩散法筛选具有抑菌活性的菌株。结果表明,共分离37株乳酸菌,其中,乳杆菌属(Lactobacillus)35株、明串珠菌属(Leuconostoc)2株;35株乳酸杆菌为Lactobacillus casei 16株、Lactobacillus paracasei 7株、Lactobacillus plantarum 4株、Lactobacillus fermentum 2株、L.delbrueckii subsp.bulgaricus 2株、Lactobacillus helveti-cus 1株、Lactobacillus diolivorans 3株;7株L.casei和L.paracasei的发酵上清液对3株细菌指示菌表现出明显抑制作用,所有菌株对真菌无抑菌活性;在排除有机酸、H2O2等的干扰和经蛋白酶K处理后,初步确定7株乳酸菌发酵上清液中的抑菌物质为细菌素。     

Identification of lactic acid bacteria isolated from South African brandy base wines

In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates. The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.     

The potential of dairy lactic acid bacteria to metabolise amino acids via non-transaminating reactions and endogenous transamination

The metabolism of amino acids by 22 starter and 49 non-starter lactic acid bacteria (LAB) was studied in a system consisting of amino acids and non-growing cells without added amino acceptors such as alpha-ketoglutarate. There were significant inter- and intra-species differences in the metabolism of amino acids. Some amino acids such as alanine, arginine, aspartate, serine and branched-chain amino acids (leucine, isoleucine and valine) were utilised, whereas other amino acids such as glycine, ornithine and citrulline were produced. Alanine and aspartate were utilised by some LAB and accumulated during the incubation of other LAB. Arginine was degraded not only by Lactococcus lactis subsp. lactis (the lactococcal subspecies known to catabolise arginine), but also by pediococci, heterofermentative lactobacilli (Lactobacillus brevis and Lb. fermentum) and some unidentified homofermentative lactobacilli. Serine was utilised predominantly by homofermentative Lb. paracasei subsp. paracasei, Lb. rhamnosus and Lb. plantarum. Of the LAB studied, Lb. brevis and Lb. fermentum were the most metabolically active, utilising alanine, arginine, aspartate, glutamate and branched-chain amino acids. Leuconostocs were the least metabolically active, showing little potential to metabolise amino acids. The formation of ammonia and acetate from amino acid metabolism varied both between species and between strains within species. These findings suggest that the potential of LAB for amino acid metabolism via non-transaminating reactions and endogenous transamination will impact both on the physiology of LAB and on cheese ripening, especially when transamination is rate-limiting in the absence of an exogenous amino acceptor such as alpha-ketoglutarate.     

Synthetic peptides as substrate for assaying the proteolytic activity of Lactobacillus helveticus

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including alpha(s)1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro alpha(s)1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.     

Ripening of cheddar cheese with added attenuated adjunct cultures of lactobacilli

We made Milled curd Cheddar cheese with Lactococcus starter and an adjunct culture of Lactobacillus helveticus I or Lactobacillus casei T subjected to different attenuation treatments: freeze shocking (FS), heat shocking (HS), or spray drying (SD). Proteolysis during cheese ripening (0 to 6 mo), measured by urea-PAGE and water-soluble nitrogen, indicated only minor differences between control and most adjunct-treated cheeses. However, there were significant differences in the effect of Lactobacillus adjuncts on the level of free amino nitrogen in cheese. Cheeses made with FS or HS Lb. helveticus adjunct exhibited significantly greatest rates of free amino group formation. Lipolysis as measured by total free fatty acids was consistently highest in adjunct-treated cheeses, and FS Lb. casei-treated cheeses showed the highest rate of free fatty acid formation followed by FS Lb. helveticus treated cheeses. Mean flavor and aroma scores were significantly higher for cheeses made with Lb. helveticus strain. Freeze-shocked Lb. helveticus-treated cheeses obtained the highest flavor and aroma scores. Sensory evaluation indicated that most of the adjunct-treated cheeses promoted better texture and body quality.     

Lactic Microflora Present in Liqvan Ewes' Milk Cheese

This study aimed to isolate and characterize Lactic Acid Bacteria (LAB) in Liqvan Ewes' milk cheese. A total of 117 Lactic Acid Bacteria were isolated and identified phenotypically. They belonged to 4 genera and 17 species. The dominant LAB found in Liqvan cheese were from the genus Lactobacillus (75.21%) consisted of 70.08% facultatively heterofermentative and 5.12% obligately heterofermentative lactobacillus species. Other isolates were classified as Pediococci (5.12%), Enterococci (5.98%), and Leuconostocs (13.67%). Lb. paracasei subsp. paracasei was the predominant species accounted for 36.75%. Likewise, predominant species of each genus were Lb. paracasei subsp. paracasei, P. pentosaceus, E. faecalis, and Leu. lactis. The preponderance of isolates (86.32%) was referred to be as members of Non Starter Lactic Acid Bacteria (NSLAB).     

The growth and interaction of yeasts and lactic acid bacteria isolated from Zimbabwean naturally fermented milk in UHT milk.

Nine yeast and four lactic acid bacterial strains, previously isolated from Zimbabwean traditionally fermented milk, were inoculated into ultra-high temperature treated (UHT) milk in both single and yeast-lactic acid bacteria co-culture. The lactic acid bacteria (LAB) strains consisted of Lactococcus lactis subsp. lactis biovar. diacetylactis C1, L. lactis subsp. lactis Lc39, L. lactis subsp. lactis Lc261 and Lactobacillus paracasei subsp. paracasei Lb11. The yeast strains used were Candida kefyr 23, C. lipolytica 57, C. lusitaniae 63, C. lusitaniae 68, C. tropicalis 78, Saccharomyces cerevisiae 71, S. dairenensis 32, C. colliculosa 41 and Dekkera bruxellensis 43. After 48-h fermentation at 25 degrees C, the samples were analysed for pH, viable yeast and bacterial counts, organic acids, volatile organic compounds (VOC) and carbon dioxide. The Lactococcus strains reduced the pH from about 6.6 to between 4.0 and 4.2, while Lb. paracasei subsp. paracasei Lb11 reduced the pH to about 5.4. Most of the yeasts, however, did not affect the final pH of the milk except for C. kefyr 23, which reduced the pH from 6.6 to 5.8. All the Lactococcus strains grew two log cycles during the 48-h fermentation period, while Lb. paracasei subsp. paracasei Lb11 grew about one log cycle. S. cerevisiae 71, C. colliculosa 41 and D. bruxellensis 43 showed poor growth in the milk in both single and co-culture. The other species of yeast grew about two log cycles. Candida colliculosa 41, S. dairenensis 32 and D. bruxellensis 43 showed reduced viability when in co-culture with Lb. paracasei subsp. paracasei Lb11. The samples in which C. kefyr 23 was used were distinct and characterised by large amounts of acetaldehyde, carbon dioxide and ethanol. However, in the samples where S. dairenensis, C. colliculosa, D. bruxellensis, C. lusitaniae, C. tropicalis, C. lipolytica and S. cerevisiae were used in co-culture, the final pH and metabolite content were mainly determined by the correspondin     

瑞士乳杆菌H11发酵乳饮料的ACE抑制活性及代谢组学研究

目的:研究瑞士乳杆菌H11与副干酪乳杆菌Lc-01两种发酵乳饮料在贮藏期间的代谢差异变化。方法:使用气相色谱-质谱（SPME-GC-MS）联用、高效液相色谱（HPLC）和超高效液相色谱串联四级杆飞行时间质谱（UPLC/Q-TOF MS）技术对4 ℃、贮藏28 d期间发酵乳饮料中的挥发性风味物质、代谢物以及ACE抑制活性之间的差异进行分析。结果:在4 ℃贮藏28 d后,瑞士乳杆菌H11发酵乳饮料的体外ACE抑制活性比副干酪乳杆菌Lc-01高60%以上,ACE抑制肽VPP和IPP含量也显著高于Lc-01（P<0.05）。采用SPME-GC-MS发现瑞士乳杆菌H11发酵乳饮料中香气成分丰富,特征风味物质2-庚酮和2-壬酮相对含量较高,分别为43.84%和12.39%。基于UPLC/Q-TOF MS的结果表明,贮藏期间两种发酵乳饮料的主要代谢差异物为肽、氨基酸和有机酸。结论:瑞士乳杆菌H11在制备发酵乳饮料方面存在巨大潜力。     

Fatty acid composition and freeze-thaw resistance in lactobacilli

The fatty acid composition and freeze-thaw resistance of eight strains of thermophilic lactobacilli were studied. Seven of these contained the same polar and neutral lipids, the five major components making up 90% of the cellular fatty acid pool being 14:0, 16:0, 16:1, 18:1 and C19 cyclopropane (cyc19:0). Strain comparison by means of cluster analysis based on the fatty acid ratios using the overlap coefficient revealed two well defined clusters. One was formed by three strains of species Lactobacillus delbrueckii subsp. lactis and Lb. delbrueckii subsp. delbrueckii, the other included five strains of the species Lb. delbrueckii subsp. bulgaricus, Lb. acidophilus and Lb. helveticus. Resistance of strains with a high content of unsaturated fatty acids (66-70%) decreased with increasing cyc19:0 concentrations. In contrast, in strains with a low concentration of unsaturated fatty acids (42-49%), increasing cyc19:0 levels were associated with increased freeze-thaw resistance.     

具有ACE抑制活性乳酸菌菌株的筛选与鉴定

以ACE抑制活性和蛋白水解活性为检测指标,选择138株乳酸菌为出发菌株,筛选出具有强ACE抑制活性的乳酸菌菌株.结果表明,筛选出具有强ACE抑制活性的4株乳酸菌,其中3株菌为瑞士乳杆菌,1株菌为干酪乳杆菌.瑞士乳杆菌KLDS1.0485和干酪乳杆菌KLDS1.0486比例为1:1,制成的发酵乳ACE抑制活性可达到61.55%.因此,组合瑞士乳杆菌KLDS1.0485和干酪乳杆菌KLDS1.0486可作为制备乳源ACE抑制肽的优良菌株.     

Potential impact on cheese flavour of heterofermentative bacteria from starter cultures

Leuconostoc mesenteroides subsp. cremoris, Leuconostoc pseudomesenteroides and Lactobacillus danicus were grown to early stationary phase in MRS and a cheese based medium (CBM). Lb. danicus had lower aminopeptidase and aminotransferase activity after growth in CBM compared with growth in MRS. Lb. danicus showed aminotransferase activity on all 20 amino acids investigated after growth in CBM; growth in MRS gave higher activities but on fewer amino acids. Highest activity was on aromatic amino acids. Lb. danicus had generally higher and broader aminopeptidase and aminotransferase activity than the Leuconostoc species. When grown in CBM the aminotransferase activity was more similar between Lb. danicus and the leuconostocs. Lb. danicus and Leu. pseudomesenteorides strains had activity on substrates containing short chain fatty acids; activity on C8 and C12 was only seen for the leuconostocs. The results show that the potential role of heterofermentative bacteria in cheese flavour formation involves specific esterase and aminotransferase activities.     

Biodiversity of the microbial community in a Spanish farmhouse cheese as revealed by culture-dependent and culture-independent methods

The microbial diversity within Alberquilla cheese, made from a spontaneously fermented mixture of raw goats' and sheep's milk in the Alpujarra mountains (Granada, south-east Spain), has been studied by the classical culturing method and also by molecular analysis of community DNA. A collection of 206 isolates was obtained from the cheese on different selective/differential media, which were then re-grouped to 52 after randomly amplified polymorphic DNA (RAPD)-PCR analyses. Isolates on Man-Rogosa and Sharpe-agar (MRS), M17-glucose agar and Kenner Fecal (KF)-agar medium were identified by specific PCR or 16S rRNA gene sequencing and belonged mainly to the lactic-acid bacteria group. The predominant genus was Lactobacillus, which accounted for more than 50% of the isolates, the most abundant species being Lactobacillus paracasei, followed by considerably less quantities of Lb. plantarum and Lb. brevis. Other lactic-acid bacteria identified were Pediococcus urinaequi, Leuconostoc pseudomesenteroides, Leuc. mesenteroides, Lactococcus lactis and even the enterococci Enterococcus faecium and E. devriesei. Cluster analyses of RAPD-PCR patterns revealed a high degree of diversity among the lactobacilli. The Gram-negative bacterial strains belonged mainly to Hafnia alvei species. The microbes occurring in Alberquilla cheese were also studied by PCR temporal temperature-gradient gel electrophoresis (TTGE) of the 16S rRNA V3 region and partial 16S rRNA sequencing of the TTGE bands. The results showed a major presence of lactic-acid bacteria closely related to Lc. lactis, Lb. paracasei, Lb. plantarum, Lb. brevis, Lb. acidophilus and Enterococcus sp. The non-lactic-acid bacterium detected was identified as Escherichia coli. All the Enterococcus strains showed great susceptibility to the most clinically relevant antibiotics, harbouring only the virulence gene efaAfm. On the basis of their antimicrobial activity against Listeria monocytogenes we chose two strains of Ln. mesenteroides that produced mesenterocin B105 and mesenterocin Y105, as revealed by PCR techniques.     

Two strains of nonstarter lactobacilli increased the production of flavor compounds in soft cheeses

The contribution to flavor generation and secondary proteolysis of 2 strains of mesophilic lactobacilli isolated from cheese was studied. Miniature soft cheeses (200 g) were produced with or without the inclusion of a culture of Lactobacillus plantarum I91 or Lactobacillus casei I90 in the starter composed of Streptococcus thermophilus. During ripening, cheeses containing the added lactobacilli showed an increased content of total free amino acids, but this increase was only significant in cheeses with Lb. plantarum I91. In addition, free amino acid profiles were modified by selective increases of some amino acids, such as Asp, Ser, Arg, Leu, and Phe. Cheeses inoculated with Lb. plantarum I91 or Lb. casei I90 were also characterized by a significantly higher concentration of diacetyl, a key flavor compound, and an increased content of acetoin. Results suggest an increase in the catabolism of either citrate or aspartate, with the production of the derived aroma compounds. Overall, aspartate content increased in both lactobacilli-added cheeses, whereas citrate was more or less constant, suggesting that aspartate could be the source of increased diacetyl and acetoin. A triangle aroma test showed that the addition of the lactobacilli strains significantly changed the sensory attributes of cheeses. At least 11 of 12 panelists commented that the aroma of cheeses with adjuncts was more buttery than that of control cheeses, which is desirable in most soft cheeses. Both Lb. plantarum I91 and Lb. casei I90 performed well as adjunct cultures by influencing cheese aroma development and cheese proteolysis.     

Screening and selection of Lactobacillus strains for use as adjunct cultures in production of semi-hard cheese

Thirty-three Lactobacillus strains were tested as adjuncts in a cheese model system. Eighteen strains originated from cheese (nine Lactobacillus spp. and nine Lb. paracasei/casei) and 15 from human intestinal mucosa (11 Lb. rhamnosus; three Lb. paracasei; one Lb. plantarum). Model cheeses weighing 120 g were made of cheese grains from full-scale production of washed curd semi-hard cheese (Herrg?rd). The model system was reproducible and similar to full-scale production with respect to moisture, salt content, pH and microbial flora. The model cheeses were sampled for aerobic and anaerobic plate count and viable counts of Lactobacillus and Lactococcus. The presence of adjuncts in the model cheeses was confirmed by typing isolates with Randomly Amplified Polymorphic DNA (RAPD). The sensory properties of model cheeses were described. In a first trial 23 of the 33 adjuncts were re-isolated from the corresponding model cheeses after 9 or 13 weeks. Adjuncts of Lb. paracasei were re-isolated more frequently than adjuncts of Lb. rhamnosus. Nine strains were selected, on the basis of their ability to grow and be a dominating part of the microflora of model cheese with interesting sensory properties. These strains were further studied together with two commercial cultures. The sensory influences on model cheeses of six of the adjuncts were confirmed, and flavour scores were in the range of 2.9-7.1 for model cheeses with different adjuncts while the control had a flavour score of 5.6 (0-10 scale). Survival and growth of seven out of the nine strains correlated with the results of the first trial. Growth and influence on flavour of four adjunct cultures were confirmed in experimental cheese manufactured in a 400-1 open vat.     

Characterization of lactic acid bacteria isolated from a Thai low-salt fermented fish product and the role of garlic as substrate for fermentation

Lactic acid bacteria (LAB) isolated from raw materials (fish, rice, garlic and banana leaves) and processed som-fak (a Thai low-salt fermented fish product) were characterized by API 50-CH and other phenotypic criteria. Lactococcus lactis subsp. lactis and Leuconostoc citreum were specifically associated with fish fillet and minced fish, Lactobacillus paracasei subsp. paracasei with boiled rice and Weisella confusa with garlic mix and banana leaves. In addition, Lactobacillus plantarum, Lactobacillus pentosus and Pediococcus pentosaceus were isolated from raw materials. A succession of aciduric, homofermentative lactobacillus species, dominated by Lb. plantarum/pentosus, was found during fermentation. In total, 9% of the strains fermented starch and 19% fermented garlic, the two main carbohydrate components in som-fak. The ability to ferment garlic was paralleled by a capacity to ferment inulin. An increased percentage of garlic fermenting strains was found during fermentation of som-fak, from 8% at day 1 to 40% at day 5. No starch fermenting strains were isolated during fermentation. Three mixed LAB cultures, composed of either starch fermenting Lc. lactis subsp. lactis and Lb. paracasei subsp. paracasei, or garlic fermenting Lb. plantarum and Pd. pentosaceus, or a combination of these strains were inoculated into laboratory prepared som-fak with or without garlic. In som-fak without garlic, pH was above 4.8 after three days, irrespective of addition of mixed LAB cultures. The starch fermenting LAB were unable to ferment som-fak and sensory spoilage occurred after three days. Fermentation with the combined mix of starch and garlic fermenting strains led to production of 2.5% acid and a decrease in pH to 4.5 in two days. The fermentation was slightly slower with the garlic fermenting strains alone. This is the first report describing the role of garlic as carbohydrate source for LAB in fermented fish products.     

A polyphasic approach to highlight genotypic and phenotypic diversities of Lactobacillus helveticus strains isolated from dairy starter cultures and cheeses

In the present work, 67 strains of Lactobacillus helveticus isolated from whey starter cultures and cheeses were identified and grouped by genotypic and phenotypic methods. Strains were identified by sugar fermentation pattern, by cell-wall protein profile, and by probe hybridisation. Phenotypic diversity was evaluated by a chemometric model taking into account biochemical characteristics (i.e. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by RAPD-PCR, which provided stran-specific patterns and revealed the occurrence of different strains. The RAPD-PCR profiles were clustered according to their similarities: the groups obtained, together with the cell-wall protein profiling and the chemometric information, could be sometimes correlated with the type of cheese and/or dairy niches used as sources of strains. A computerised analysis of genotypic and phenotypic information could be successfully applied for rapid and reliable differentiation and characterisation of Lb. helveticus isolates occurring in different dairy products.     

Autolysis and related proteolysis in Swiss cheese for two Lactobacillus helveticus strains

Intracellular peptidases of Lactobacillus helveticus may play a major role in the proteolysis of Swiss cheeses, provided that they are released through bacterial lysis. Experimental Swiss cheeses were manufactured on a small scale from thermized and microfiltered milk using as starters (in addition to Streptococcus thermophilus and Propionibacterium freudenreichii) one of two Lb. helveticus strains, ITGLH1 and ITGLH77, which undergo lysis to different extents in vitro. All the cheeses were biochemically identical after pressing. The viability of Lb. helveticus ITGLH1 and ITGLH77 decreased to a similar extent (96-98%) while in the cold room, but the concomitant release of intracellular lactate dehydrogenase in cheeses made with strain ITGLH1 was 5-7-fold that in cheeses made with ITGLH77. Protein profiles and immunoblot detection of the dipeptidase PepD confirmed a greater degree of lysis of the ITGLH1 strain. Free active peptidases were detected in aqueous extracts of cheese for both strains, and proteolysis occurred principally in the warm room. Reversed-phase HPLC revealed a more extensive peptide hydrolysis for ITGLH1, which was confirmed by the greater release of free NH2 groups (+33%) and free amino acids (+75%) compared with ITGLH77. As the intracellular peptidase activities of ITGLH1 and ITGLH77 have previously been shown to be similar, our results indicated that the extent of lysis of Lb. helveticus could have a direct impact on the degree of proteolysis in Swiss cheeses.     

The aminopeptidase C (PepC) from Lactobacillus helveticus CNRZ32. A comparative study of PepC from lactic acid bacteria

 The aminopeptidase C (PepC) of Lactobacillus helveticus CNRZ32 was purified by anion exchange chromatography from cell free extracts of an E. coli DH5α clone overexpressing the Lactobacillus aminopeptidase. PepC was found to have a tetrameric structure in its native form with subunits of 50 kDa each, a pH optimum of 6.5 and maximum activity at 45  °C. Sulfhydryl-blocking reagents inhibited the enzyme activity whereas reducing or metal chelating reagents had an activating effect on the PepC activity. The PepC hydrolyzed a wide range of p-nitroaniline derivatives, dipeptides and several tripeptides which contained basic amino acids (Arg, Lys), Pro residues, or cheese flavour precursor amino acids (Met, Leu, Phe) at the N-terminal position. The substrate specificity and residual activity of PepC from several lactic acid bacteria, including the PepC described above, were compared at conditions of pH and NaCl present in cheese. Received: 25 February 2000