The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 2. The levels and subcellular distributions of peptidase and esterase activities

The levels and subcellular distributions of various peptidase and esterase activities in a range of lactococcal and Streptococcus thermophilus strains were investigated. There was no correlation between the levels of the enzymes in the different strains and the ability of the strains to produce acid when grown in milk. While considerable differences between individual strains were apparent, average levels of X-prolyldipeptidyl aminopeptidase, dipeptidase and tripeptidase were similar in the Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris strains studied, while that of lysylaminopeptidase (i.e. activity assayed using lysine p-nitroanilide as substrate) in the L. lactis subsp. cremoris strains was approximately double that in the L. lactis subsp. lactis strains. The average levels of lysylaminopeptidase and X-prolyldipeptidyl aminopeptidase in the S. thermophilus strains studied were similar to those in the L. lactis subsp. cremoris strains, while the average levels of dipeptidase and tripeptidase were considerably lower. All peptidases studied were recovered predominantly in the cytoplasmic fraction, although in a few strains there was some evidence to suggest that a part of the tripeptidase activity may be associated with cell structures comprising the particulate fraction. The levels of esterase activity in the strains were considerably different between strains. However, the average level of esterase activity detected in the two lactococcal subspecies was similar, while that in the S. thermophilus strains was more than double the lactococcal average. The subcellular distribution of the esterase in all strains studied showed that a significant proportion of the activity is located on the cell surface.     

The diversity of potential cheese ripening characteristics of lactic acid starter bacteria: 1. Resistance to cell lysis and levels and cellular distribution of proteinase activities

By reference to subcellular fraction markers, the resistance to lysis of 23 strains of Lactococcus lactis subsp. cremoris, 30 strains of L. lactis subsp. lactis and five strains of Streptococcus thermophilus and the levels and distribution of proteinase activity in the strains were determined. Strains of L. lactis subsp. cremoris were readily lysed by transfer to hypotonic buffer after treatment with lysozyme alone, whilst strains of L. lactis subsp. lactis and S. thermophilus could be efficiently lysed in this way only after treatment with a combination of lysozyme and mutanolysin. With a few notable exceptions, those strains which gave the fastest rates of acid production also generally presented higher levels of cell surface proteinase, as determined by activity on fluorescein isothiocyanate-labelled β-casein. The highest levels of cell surface proteinase detected were found for strains of L. lactis subsp. cremoris. However, the levels of total proteinase activity in the lactococcal strains did not correlate with the rate of acid production in milk, some slow acid-producers yielding similar or greater total proteinase levels than fast acid-producers. Homology to DNA probes for the lactococcal cell surface proteinase gene and to the conserved region encoding the serine proteinase active site was shown by the fast acid-producing lactococcal strains, but not by most of the slow acid-producing lactococcal strains or by the strains of S. thermophilus. A significant proportion of the total proteinase activity was recovered in the subcellular fractions in which high levels of cytoplasmic marker enzyme activity were found. The total proteinase levels detected in strains ofL. lactis subsp. lactis showed a greater range of variation than in the strains of L. lactis subsp. cremoris. High levels of total proteinase activity were found in the slow acid-producers despite the strains having been grown in the presence of yeast extract. For many of the strains, the levels of proteinase released from the cell surface during cell wall degradation with lytic enzyme treatment were higher than those found using whole cells, suggesting that a significant amount of proteolytic activity was either inaccessible to substrate or present in an inactive form.     

Isolation,characterisation and identification of lactic acid bacteria from bushera: a Ugandan traditional fermented beverage

One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined.

The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml⁻¹. The coliform counts varied between <1 and 5.2 log cfu ml⁻¹. The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera.

The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml⁻¹ during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml⁻¹ at 24 h, but after 48 h, counts were less 4 log cfu ml⁻¹. Yeasts increased from 4.3 to 7.7 log cfu ml⁻¹ at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively.

Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain).     

Fermentation of salmon fillets with a variety of lactic acid bacteria

The influence of five strains of lactic acid bacteria (four Lactobacillus and one Carnobacterium) on the quality of fermented salmon fillets was studied. Best starter growth (increase of more than 1 log in 3 days) and acidification of muscle (e.g. pH reduction of approximately 0.7 units in 5 days) were achieved with the two commercial strains L. sake LAD and L. alimentarius BJ33. pH reduction was consistently lower (e.g. reduction of 0.2 units in 5 days) with C. piscicola 85. Protein breakdown as observed on SDS-PAGE gels was similar for all strains. In contrast, the starter strain did influence texture and colour changes. Fast acidifying strains L. sake LAD and L. alimentarius BJ33 brought about a firmer overall texture and a lighter colour, while softening of flesh occurred in samples processed with C. piscicola 85. Sensory evaluations indicated that samples processed with fast acidifying strains were preferred. L. sake LAD and L. alimentarius BJ33 are regarded as suitable starters for fermentation of salmon fillets.     

Probiotic white cheese with Lactobacillus acidophilus

The objective of the present study was to determine the effects of Lactobacillus acidophilus on the sensory attributes, ripening time, and composition of Turkish white cheese and to investigate the survival of L. acidophilus during ripening of the cheese stored in vacuum or in brine. Two types of white cheeses, traditional cheese (control, made with Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris) and probiotic cheese (made with Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris and L. acidophilus 593 N), were produced and ripened in vacuum pack or in brine at 4°C for 90 days. Cheese samples were assessed for microbiological and compositional properties, proteolysis, and sensory evaluation at different ripening stages. On ripening in vacuum pack, L. acidophilus survived to numbers >10⁷ cfu g⁻¹, which is necessary for positive effects on health. Protein, dry matter, salt content, and percentage of lactic acid in the vacuum-packed and brine-salted probiotic cheeses were significantly different. Also, the lactic acid content of probiotic cheeses was slightly higher than that of the controls for both vacuum- and brine-packed cheeses. Vacuum-packed probiotic cheese had the highest levels of proteolysis and the highest sensory scores of all cheeses. Consequently, L. acidophilus could be used for the manufacturing of probiotic white cheese to shorten ripening time and vacuum packaging is the preferred storage format.     

Inoculation of lactic acid bacteria on meat surfaces as a means of decontamination in semitropical conditions

Reduction of microbial populations on carcasses has been achieved and reported by some authors by spraying solutions of organic acids, mainly lactic acid, on the meat surface. However, in practice, lactic acid is very expensive. Production of lactic acid in situ by a controlled lactic fermentation seemed to be a feasible answer. The objective of the present study was to explore the viability of this concept at semitropical conditions, i.e. temperatures around 25°C. In a first experiment, seven starters were tested for their ability to produce lactic acid and reducing the growth rate of pseudomonads, taking these microorganisms as indicators of contamination by spoilage microorganisms. The lactic acid bacteria strains were isolated from native Mexican maize-based beverages, and inoculated on the meat surface. Addition of sucrose and wrapping the samples in PVC film in order to induce a microaerophilic environment encouraged the over-growth of L. bulgaricus and P. pentosaceous over pseudomonads. A second experiment was designed to test a mixture of these two strains and a commercial starter with respect to lactic acid production, overgrowth of lactic acid bacteria over pseudomonads, decolouration and oxidation of the meat samples. It was concluded that a commercial starter (L. plantarum + M. kristinae-varians) resulted in a reduction of pseudomonas growth rate, without notably affecting meat colour and degree of oxidation.     

Growth energetics of Lactococcus lactis subsp. lactis biovar diacetylactis in cometabolism of citrate and glucose

Certain strains of lactic acid bacteria present in commercial cheese starters, characterized by faint transparent colonies on an agar plate containing 1 mg kg ⁻¹ crystal violet (CVT), were identified as Lactococcus lactis subsp. (ssp) lactis biovar diacetylactis. The effect of citrate on the growth of these strains (CVT strains) in the presence of glucose was studied, in comparison with L. lactis strains. Molar growth yield from glucose (Y_G, g dry weight/mole of glucose consumed) for CVT strains grown on glucose plus citrate was significantly higher than the control (i.e. without citrate), but not for other L. lactis strains tested. Enhanced Y_G was also observed at a pH-controlled experiment, indicating that enhanced Y_G did not result from a buffering effect of citrate. CVT strains, in contrast to other strains of the same species, were shown to obtain enough energy to enhance Y_G on glucose–citrate mixtures.     

Genetic manipulation of the peptidolytic system in lactic acid bacteria

Due to their presumed involvement in product flavour the peptidases of lactic acid bacteria have been subject to extensive research. A major breakthrough was the isolation and purification of the various enzymes to homogeniety. This allowed a reevaluation of the number of different enzymes in one species by careful investigation of their specificities. Moreover, the purified proteins offered novel ways to the genetic analysis of the peptidolytic system. N-terminal amino acid sequences of, and antibodies against, the purified peptidases were used to clone the majority of peptidases described for Lactococcus lactis. Some peptidase genes, especially those from a number of lactobacilli, were obtained by complementation of peptidase-negative cloning hosts. Thus, by deduction, the amino acid sequences of these peptidases is known and all belong to known families of peptidases. The recently developed chromosomal integration systems for LAB have been used to construct (multiple) peptidase-negative mutants in L. lactis. The growth characteristics of these strains are now under study. As a number of the mutants were made in a food grade way their performance in cheesemaking can be evaluated. The review presented will focus on the latest developments in the genetic analysis of peptidases in LAB and will evaluate our current understanding of peptidolysis by LAB and its importance in dairy fermentations.     

Antagonistic effect on Listeria monocytogenes and L. innocua of a bacteriocin-like metabolite produced by lactic acid bacteria isolated from sucuk

Two Lactobacilli and four Pediococci strains producing bacteriocin-like metabolities isolated from sucuk were tested with agar spot tests and well diffusion assays for their inhibitory activity against 16 Listeria strains, also isolated from sucuk. The production of organic acids and hydrogen peroxide limited, L. sake Lb 706 (used as a bacteriocin producer strain) and the isolated lactic acid bacteria (LAB) showed inhibitory activity against all of the Listeria strains, while L. sake Lb 706-A (used as a bacteriocin non-producer mutant) had the same effects against only two Listeria monocytogenes strains (51, 52) in agar spot tests. In the well diffusion assays, while L sake Lb 706 and four Pediococci isolates (413, 416, 419, 446) exhibited inhibitory activity against all of the Listeria strains tested, L. sake Lb 706-A and two of the Lactobacilli isolates (77, 116) showed no effect on the Listeria strains tested.     

Antilisterial activity of lactic acid bacteria inoculated on cooked ham

This study was conducted to evaluate the ability of Lactobacillus sakei 1, a bacteriocin-producing (bac⁺) lactic acid bacterium (LAB), isolated from Brazilian fresh pork sausage to inhibit two Listeria monocytogenes strains (serotypes 4b and 1/2a) on cooked, sliced vacuum-packaged ham. L. sakei ATCC 15521 was used as a non-bacteriocin producer (bac⁻). L. monocytogenes (ca. 2 log CFU/mL) and LAB (ca. 6 log CFU/ml) were inoculated on the sterilized ham, vacuum-sealed and incubated at 8 °C for 10 days. A treatment with the bacteriocin Chrisin (UI/ml) was included. Both L. monocytogenes strains were significantly inhibited in the presence of either bac⁺ and bac⁻ LAB in comparison to the control (L. monocytogenes alone). Using a bacteriocinogenic strain of LAB did not offer an additional barrier to listerial growth in the studied meat system. The application of Chrisin did not affect at all the growth of L. monocytogenes.     

Antimicrobial activity of neutralized extracellular culture filtrates of lactic acid bacteria isolated from a cultured Indian milk product (‘dahi’)

Neutralized extracellular culture filtrate obtained from isolates of Lactobacillus acidophilus, Lactobacillus delbruecki ssp. bulgaricus, Lactobacillus salivarius and Lactococcus lactis ssp. lactis from ‘dahi’ showed weal to moderate inhibition of Staphylococcus aureus, Bacillus cereus, Escherichia coli, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus laterosporus, Bacillus subtilis and Pseudomonas aeruginosa when tested by the diffusion agar well assay method. The effective minimum quantity of lactic culture filtrates required to obtain complete inhibition of an inoculum of 10³ cfu/ml of the bacteria tested was between 20 and 26% (vol/vol), as determined by the agar incorporation method. Neutralized extracellular culture filtrate of these lactic cultures added at a level of 10% in sterile, 10% reconstituted non-fat dry milk was able to either suppress or retard growth of selected bacterial cultures when incubated at 37°C for 24 h. This study indicated the antimicrobial activity of dahi and the potential of using neutralized extracellular culture filtrate of lactic acid bacteria in the biopreservation of foods.     

乳酸菌的分类鉴定及在食品工业中的应用

乳酸菌是一类革兰氏阳性杆菌或球菌、可収酵乳糖或葡萄糖产生大量乳酸的细菌的通称,将乳酸菌应用到食品中具有提高其营养价值,改善食品风味,抑制食品中腐败菌的生长,延长食品保质期等作用。因其独特的生理特性,近年来国内外学者对乳酸菌的研究逐渐增多。本文主要论述了乳酸菌的分类和鉴定技术,介绍了传统鉴定斱法和分子生物学鉴定斱法的优缺点以及乳酸菌在食品工业(乳制品、肉制品、果蔬制品、食品保鲜)中的应用,为提高乳酸菌的鉴定效率以及乳酸菌在食品工业、饲料生产和临床医疗上的迚一步应用提供参考。     

Some characteristics of lactic acid bacteria present in commercial sucuk samples

A total of 10 sucuk samples, obtained from Denizli, Turkey were analysed for some physical, chemical and microbiological characteristics. In addition, lactic acid bacteria (LAB) strains producing bacteriocin-like metabolites were isolated and identified. The production of some typical metabolites of the cultures isolated was investigated. At the end of the research, the average values of the pH, water and fat content were 5.1, and 37.2% and 30.5%, respectively. Microbiological analyses results were determined: average 8.34 log CFU/g TAMB, 8.91 log CFU/g LAB (at the MRS agar) and average 8.25 log CFU/g LAB (at the Elliker's lactic agar). The average counts of yeast-mould, coliform and Enterobacteriaceae were found to be 5.0 log CFU/g, 3.28 log CFU/g and 3.27 log CFU/g, respectively. In this study, counts of yeast-mould in the two samples, coliform counts in the five samples, and Enterobacteriaceae counts in the three samples were < 1.0 log CFU/g. A total of 6 of 100 LAB isolates obtained from the sucuk samples were found as a strain producing bacteriocin-like metabolites. These 6 strains were identified as follows; 3 strains Lactobacillus plantarum and 3 strains Pediococcus pentosaceus. According to the findings, these strains have the potential to be used as a sucuk starter culture. Additionally, acid and flavour compounds, other undesirable metabolite-producing activities of the strains, were determined in the model system. From these results it was concluded, after the determination of the toxicological properties, that the 4 strains of LAB identified (L. plantarum 13 P. pentosaceus 15 P. pentosaceus 74 and P. pentosaceus 75) would be useful as the starter and protective culture in the processing of the sucuk and similar fermented products.     

PHYSIOLOGICAL AND MORPHOLOGICAL CHANGES IN IMMOBILIZED L. PLANTARUM NCIM 2084 CELLS DURING REPEATED BATCH FERMENTATION FOR PRODUCTION OF LACTIC ACID

L. Plantarum NCIM 2084 cells are homofermentative, essentially producing lactic acid as the main end product of glucose fermentation. Prolonged use through repeated batch fermentation with the cells of L. plantarum immobilized on chitosan treated polypropylene matrix showed a shift in the metabolic pathway from the homofermentative to heterofermentative, accompanied by morphological changes in immobilized cells from the normal rod shape to the coccoid shape. These changes appear to be related to a shift in the bacterial cell metabolism, resulting in a decrease in lactic acid yields.     

浅谈乳酸菌在黄酒生产中的作用

为了全面地了解乳酸茵兼在黄酒生产全过程中的作用，介绍了黄酒米浆水中乳酸茵的应用、黄酒深层酿造兼氧浸米中乳酸菌的作用和乳酸菌在黄酒发酵过程中的作用，同时也介绍了乳酸茵代谢产物乳酸和乳酸乙酯在黄酒中的含量和作用。     

Effect of fermented milks on humoral immune response in mice

Eight groups of 20 CD-1 mice were fed for eight days with UHT milk fermented by one of the following bacteria: Bifidobacterium longum, Lactobacillus acidophilus, L. delbrueckii spp. bulgaricus, L. casei spp.rhamnous, L. helveticus, Lactococcus lactis spp. cremoris, Lactococcus lactis spp. lactis, Streptococcus salivarius spp. cremoris. No significant differences were observed in the serum IgG and IgA level, nor in the bronchoalveolar IgG level. Only mice fed milk fermented with L. delbrueckii spp. bulgaricus showed a significant increase (P < 0·05) in their bronchol-alveolar IgA level after eight days. The extent of proteolysis of the fermented milks was not correlated with the bronchoalveolar IgA level.     

Carnocin U149, a potential biopreservative produced by Carnobacterium piscicola: large scale purification and activity against various Gram-positive bacteria including Listeria sp.

This paper describes a simple purification method for the purification of carnocin U149, a potential biopreservative produced by Carnobacterium piscicola U149. The protocol was also applicable for the isolation of nisin Z, which is a biopreservative produced by Lactococcus lactis SIK-83. The protocol consists of only two purification steps, XAD chromatography and cation exhange chromatography. It is quick, easy, and can be used for large scale purification of these lantibiotics. The bactericidal activity of carnocin U149 against carnobacteria, lactococci and Listeria was compared with that of nisin Z. The carnobacteria showed similar sensitivity towards carnocin U149 and nisin. The nisin producing L. lactis strains were very sensitive towards carnocin U149, while the non-producing L. lactis strains were more sensitive to nisin. The Listeria strains were weakly sensitive to carnocin U149, lower concentrations of nisin were needed to inhibit growth.     

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 activity of ethanol, glycerol monolaurate or lactic acid against Listeria monocytogenes

Minimal inhibitory concentrations (MIC) and antimicrobial effects of glycerol monolaurate (monolaurin), ethanol and lactic acid, either alone or in combination, against Listeria monocytogenes in tryptic soy broth were determined. Ethanol at concentrations up to 1.25% did not inhibit growth, but growth was strongly inhibited in the presence of 5% ethanol. MIC values of monolaurin and ethanol alone were 10 μg/ml (0.001%) and 50 000 μg/ml (5%), respectively. However, MIC values were not changed when monolaurin was combined with ethanol. When 5 μg/ml monolaurin was combined with 5% ethanol, the inhibitory effect of the combination was similar to the most active compound alone after 24 h incubation. These data indicate little interaction between monolaurin and ethanol against L. monocytogenes. MIC value of lactic acid alone was 5000 μg/ml (0.5%), but was lower when 1.25% ethanol was combined with 0.25% lactic acid. When 2.5% ethanol was combined with 0.25% lactic acid, the combination did not increase the inhibitory effect of the most active single compound alone. This result also indicates that there was little interaction between ethanol and lactic acid.     

Simultaneous saccharification and fermentation of furfural residues by mixed cultures of lactic acid bacteria and yeast to produce lactic acid and ethanol

In a cellulosic ethanol production system, yeasts cannot be reused, and it is difficult to avoid the formation of lactic acid. A novel mixed fermentation system based on water-rinsed furfural residue was designed to produce lactic acid and ethanol simultaneously by yeast and lactic acid bacteria. The fermentation broth can be used for the production of lactic acid, ethanol, or ethyl lactate, which is a very suitable alternative green solvent. Simultaneous saccharification and fermentation by mixed cultures of lactic acid bacteria and yeast (MSSF) in different conditions were carried out. Acid/alcohol molar ratios (molar ratio of lactic acid to ethanol) were investigated to determine the effects of temperature, substrate concentration, and mass rate of yeast to lactic acid bacterial cells on MSSF. Cellulose conversion rate was also calculated to evaluate the effectiveness of MSSF. The cellulose conversion rate of MSSF was higher than those of ethanol simultaneous saccharification and fermentation and lactic acid simultaneous saccharification and fermentation. The cellulose conversion rate increased with increasing substrate concentration, while the acid/alcohol molar ratio decreased with increasing substrate concentration. These results indicate that yeast cells could provide nutrients for lactic acid bacteria. MSSF at 38 °C is also apt to obtain a low acid/alcohol molar ratio. A 1:1 lactic acid/alcohol molar ratio will be obtained at a fluctuating temperature (38 °C between 0 and 54 h, 42 °C after 54 h) and a substrate concentration of 9%, while keeping a high cellulose conversion rate and final lactic acid concentration. A probable bottleneck for MSSF and its potential solution are also proposed in this paper.