Autolysis Of Lactic Acid Bacteria Research Articles

Peptidoglycan Hydrolases of Probiotic Pediococcus acidilactici NCDC 252: Isolation, Physicochemical and In Silico Characterization

Pediococcus acidilactici NCDC 252 is a Gram-positive lactic acid bacteria (LAB) that is being studied to develop it as probiotic and starter culture for dairy and meat industry. Peptidoglycan hydrolases (PGHs) are also gaining interest because of their antibacterial activity. NCDC 252 genome was searched for PGH encoded genes and product of these genes were in silico characterized for different parameters. Extracellular PGHs of NCDC 252 were precipitated and characterized. PGHs were screened turbidometrically with Bacillus cereus and Staphylococcus albus as substrates. Spectrum of PGH(s) was studied in renaturing SDS-PAGE by varying substrates and renaturing conditions. PGH(s) band intensity decreased in the presence of NaCl whereas some additional secondary lytic bands appeared in the presence of EDTA. Autolysis of LAB in cheese and other fermented food products is associated with release of oligopeptides by proteolysis and other enzyme activities. Autolytic phenotype of P. acidilactici was evaluated and characterized. Lytic activity studies revealed 45–60% activity against Micrococcus lysodeikticus and B. cereus greater than for P. acidilactici. PGH of P. acidilactici exhibited broad growth inhibiting spectrum of antibacterial activity against studied Gram positive and Gram negative bacteria and thus can be promising in controlling pathogenic bacteria. This also confers probiotic attribute to the studied strain.

Determination of the enzyme destruction rational mode of biomass autolysate of lactic acid bacteria

It is shown that the degradation products of peptidoglycans of lactic acid bacteria cell walls that are related to the muramyl peptide series compounds are promising components of food ingredients and dietary supplements for the nutritional support of the population suffering from disorders of the immune system. The expediency of autolysis of lactic acid bacteria biomass to increase the enzymatic degradation efficiency of peptidoglycans of their cell walls has been proven. The accumulation of low molecular weight peptides in the enzymatic hydrolysis of biomass under rational parameters, which was not subjected to autolysis, is 0.260 mg/cm 3 , in the enzymatic hydrolysis of biomass that was subjected to autolysis is 0.569 mg/cm 3 . Rational regimes of enzymatic hydrolysis of the composition of lactic acid bacteria (enzyme concentration of 12.5 mg/cm 3 , substrate concentration of 70.0 mg/cm 3 , duration of enzymatic hydrolysis of 245.6 min) were determined using mathematical planning methods of multifactorial experiments, which made it possible to significantly optimize and improve the work efficiency. The affiliation of low molecular weight peptides obtained under the rational conditions of enzymatic hydrolysis to immunological compounds of the muramyl peptide series has been proven by gel chromatography and IR spectroscopy methods. It is determined that the molecular weight of the obtained low molecular weight peptides is in the range of 294 – 650 Da, which, in fact, corresponds to the molecular weight of muramyl dipeptide and glucosaminylmuramyl dipeptide. In the IR spectrum of low molecular weight peptides, the absorption bands are noted, which correspond to fluctuations of free amino groups, peptide bonds, which, in fact, occur in the structure of peptides, pyranose glucose forms that are part of muramic acid and N-acetylglucosamine of peptidoglycan, muramyl dipeptide and glucosaminylmuramyl dipeptide. Also, fluctuations of β-glycoside bonds, which binds the remains of muramic acid and N-acetylglucosamine in peptidoglycan and glucosaminylmuramyl dipeptide are marked in the IR spectrum of low molecular weight peptides

Identification of Quorum Sensing Signal Molecule of Lactobacillus delbrueckii subsp. bulgaricus.

Many bacteria in nature use quorum sensing (QS) to regulate gene expression. The quorum sensing system plays critical roles in the adaptation of bacteria to the surrounding environment. Previous studies have shown that during high-density fermentation, the autolysis of lactic acid bacteria was regulated by the QS system, and the two-component system (TCS, LBUL_RS00115/LBUL_RS00110) is involved in the autolysis of Lactobacillus delbrueckii subsp. bulgaricus. However, the QS signal molecule, which regulates this pathway, has not been identified. In this study, we compared the genome of Lactobacillus bulgaricus ATCC BAA-365 with the locus of seven lactobacillus QS systems; the position of the QS signal molecule of Lactobacillus bulgaricus ATCC BAA-365 was predicted by bioinformatics tool. Its function was identified by in vitro experiments. Construction of TCS mutant by gene knockout of LBUL_RS00115 confirmed that the signal molecule regulates the density of the flora by the TCS (LBUL_RS00115/LBUL_RS00110). This study indicated that quorum quenching and inhibition based on the signal molecule might serve as an approach to reduce the rate of autolysis of LAB and increase the number of live bacteria in fermentation.

Can the development and autolysis of lactic acid bacteria influence the cheese volatile fraction? The case of Grana Padano

In this study, the relationship between the dynamics of the growth and lysis of lactic acid bacteria in Grana Padano cheese and the formation of the volatile flavor compounds during cheese ripening was investigated. The microbial dynamics of Grana Padano cheeses that were produced in two different dairies were followed during ripening. The total and cultivable lactic microflora, community composition as determined by length heterogeneity-PCR (LH-PCR), and extent of bacterial lysis using an intracellular enzymatic activity assay were compared among cheeses after 2, 6 and 13months of ripening in two dairies.The evolution of whole and lysed microbiota was different between the two dairies. In dairy 2, the number of total cells was higher than that in dairy 1 in all samples, and the number of cells that lysed during ripening was lower. In addition, at the beginning of ripening (2months), the community structure of the cheese from dairy 2 was more complex and was composed of starter lactic acid bacteria (Lactobacillus helveticus and Lactobacillus delbrueckii) and NSLAB, possibly arising from raw milk, including Lactobacillus rhamnosus/Lactobacillus casei and Pediococcus acidilactici. On the other hand, the cheese from dairy 1 that ripened for 2months was mainly composed of the SLAB L. helveticus and L. delbrueckii. An evaluation of the free-DNA fraction through LH-PCR identified those species that had a high degree of lysis. Data on the dynamics of bacterial growth and lysis were evaluated with respect to the volatile profile and the organic acid content of the two cheeses after 13months of ripening, producing very different results. Cheese from dairy 1 showed a higher content of free fatty acids, particularly those deriving from milk fat lipolysis, benzaldehyde and organic acids, such as pGlu and citric. In contrast, cheese from dairy 2 had a greater amount of ketones, alcohols, hydrocarbons, acetic acid and propionic acid. Based on these results, we can conclude that in the first cheese, the intracellular enzymes that were released from lysis were mainly involved in aroma formation, whereas in the second cheese, the greater complexity of volatile compounds may be associated with its more complex microbial composition caused from SLAB lysis and NSLAB (mainly L. rhamnosus/L. casei) growth during ripening.

Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.

Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.

Flavour precursor development in Cheddar cheese due to lactococcal starters and the presence and lysis of Lactobacillus helveticus.

The rapid release of intracellular enzymes due to autolysis of lactic acid bacteria in the cheese matrix has been shown to accelerate cheese ripening. The objective of this work was to investigate the evolution of the flavour precursors, individual free amino acids (FAAs), free fatty acids (FFAs) and volatile compounds that contribute to the sensory profiles of cheeses at 2, 6 and 8 months of ripening in Cheddar cheese manufactured using starter systems which varied with respect to their autolytic properties. Starter system A contained a blend of two commercial Lactococcus lactis strains (223 and 227) which had a low level of autolysis. System B was identical to A but also included a highly autolytic strain of Lactobacillus helveticus (DPC4571). System C contained only strain DPC4571. Levels of all individual FAAs were elevated in cheeses B and C relative to A after 2 months of ripening. By 8 months of ripening the main FAA were glutamate, leucine, lysine, serine, proline and valine. Levels of C 6:0, C 8:0, C 12:0 and C 18:0 fatty acids did not vary greatly over ripening, while levels of C 4:0, C 10:0, C 14:0, C 16:0 and C 18:1 were elevated in cheeses B and C. Principal component analysis of the headspace volatiles separated cheese A from cheeses B and C. Cheeses B and C had highest levels of dimethyl disulphide, carbon sulphide, heptanal, dimethyl sulphide, ethyl butanoate, 2-butanone, and 2-methyl butanal and were described as having a ‘caramel’ odour and ‘sweet’, ‘acidic’ and ‘musty’ flavour. Cheese A had highest levels of 2-butanol, 2-pentanone, 2-heptanone, 1-hexanol and heptanal and was described as having a ‘sweaty/ sour’ odour and ‘soapy’, ‘bitter’ and ‘mouldy’ flavour. The results highlight the impact of starter lactococci on flavour precursor development and the positive effect of Lb. helveticus and the lysis of this strain on enhancing levels of substrate and flavour precursors early during ripening resulting in early flavour development.

Use of autolytic starter systems to accelerate the ripening of Cheddar cheese

Abstract The rapid release of intracellular enzymes due to autolysis of lactic acid bacteria in the cheese matrix post-manufacture is thought to play a role in the acceleration of cheese ripening. To investigate this hypothesis Cheddar cheese was manufactured using three related starter systems which varied with respect to their autolytic properties. Starter system A contained a blend of two Lactococcus lactis strains (223 and 227) which had a low level of autolysis. System B was identical to A but included an adjunct of a highly autolytic strain of Lactobacillus helveticus (DPC4571). System C consisted only of strain DPC4571 as starter. The cheeses were evaluated during ripening for key ripening indices including autolysis of starter cells by release of intracellular marker enzyme lactate dehydrogenase (LDH), composition, proteolysis and flavour development by descriptive sensory analysis. Populations of Lb. helveticus DPC4571 decreased rapidly in cheeses B and C and were not detected by 8 weeks. The level of starter culture autolysis proceeded in the order C≫B>A. Levels of proteolysis were elevated in cheeses B and C relative to A. Principal component analysis of the sensory data separated the character of cheese A from that of cheeses B and C. Cheeses B and C developed a unique ‘balanced’ ‘strong’ flavour early in ripening with a ‘caramel’ and ‘musty’ odour and ‘sweet’ ‘astringent’ flavour compared to cheese A. Hierarchical cluster analysis grouped C at 2 months with B at 6 and 8 months reflecting accelerated flavour development. Proteolytic and sensory data support the hypothesis that autolysis accelerates the rate of cheese ripening.

Some Factors Influencing the Autolysis of Lactobacillus bulgaricus and Lactobacillus casei

The autolysis of lactic acid bacteria is important for cheese ripening. However, little information is currently available on the characteristics of autolytic enzymes in Lactobacillus bulgaricus and Lactobacillus casei spp. The current study showed that temperature, pH, NaCl concentration, and growth phase affect the rate and extent of autolysis of Lb. bulgaricus and Lb. casei when autolysis was assessed in 0.2 M NaCl at 40°C; the rate and extent of autolysis appeared to be strain dependent. Under our experimental conditions, extensive cell lysis was observed in 0.2 M NaCl, and maximum autolysis was 78% for Lb. bulgaricus UL12; Lb. casei ssp. casei L2A and Lb. casei ssp. pseudoplantarum 137 showed 56% and 57% lysis, respectively. As observed by electron microscopy, the highest autolytic activity occurred in cells during the early exponential growth phase. Ultrastructural observations revealed that cell-wall degradation occurred concomitantly with cell autolysis and was well correlated to optical density measurements during cell autolysis. This paper is the first report of a quantitative estimation of cell autolysis based on observations with electron microscopy.

The role of autolysis of lactic acid bacteria in the ripening of cheese

Abstract The importance of autolysis of lactic acid bacteria in cheese ripening is evident from the literature. However, the mechanisms and the consequences still require investigation. The consequences of autolysis of mesophilic starters in Cheddar cheese are discussed and highlights from current physiological and genetic studies on starter autolysis are presented. The relative merits of measuring starter autolysis in cheese by viable starter cell densities, electron microscopic observations and assay of cell-free cytoplasmic enzymes are discussed for cheese studies using different starter strains and added phage to achieve different levels of autolysis. The balance of both the intact and autolysed starter cells in young curd appear to be important in cheese ripening. The intact cells are necessary for physiological reactions such as lactose fermentation and oxygen removal and possibly for a number of flavour reactions. In contrast, the main consequence of autolysed cells in cheese is to accelerate the peptidolytic reactions. The possible influences of autolysis of adventitious lactic acid bacteria during cheese ripening are discussed.