Non-starter Lactic Acid Bacteria Populations Research Articles

Dynamics of Starter and Non-Starter Lactic Acid Bacteria Populations in Long-Ripened Cheddar Cheese Using Propidium Monoazide (PMA) Treatment.

The microbial community of industrially produced Canadian Cheddar cheese was examined from curd to ripened cheese at 30–32 months using a combination of viable plate counts of SLAB (GM17) and NSLAB (MRSv), qPCR and 16S rRNA gene amplicon sequencing. Cell treatment with propidium monoazide excluded DNA of permeable cells from amplification. The proportion of permeable cells of both Lactococcus spp. and Lacticaseibacillus spp. was highest at 3–6 months. While most remaining Lacticaseibacillus spp. cells were intact during later ripening stages, a consistent population of permeable Lactococcus spp. cells was maintained over the 32-month period. While Lactococcus sequence variants were significant biomarkers for viable cheese curd communities at 0–1 m, Lacticaseibacillus was identified as a distinctive biomarker for cheeses from 7 to 20 months. From 24 to 32 months, Lacticaseibacillus was replaced in significance by four genera (Pediococcus and Latilactobacillus at 24 m and at 30–32 m, Secundilactobacillus and Paucilactobacillus). These results underscore the importance of monitoring potential defects in cheeses aged over 24 months, which could be diagnosed early through microbial DNA profiling to minimize potential waste of product. Future perspectives include correlating volatile flavor compounds with microbial community composition as well as the investigation of intra-species diversity.

The potential of non-starter lactic acid bacteria from Cheddar cheese to colonise the gut

• NSLAB populations in Cheddar cheese can survive simulated gastric transit. • Many NSLAB strains capable of gastric transit exhibit probiotic traits. • Many strains bound to mucin suggesting potential for prolonged gut residence time. • No genes for antibiotic resistance, virulence or pathogenicity were identified. This study was undertaken to assess the potential of Non-Starter Lactic Acid Bacteria (NSLAB) from Cheddar cheese to survive gastric transit and display probiotic-related traits including bile salt hydrolase activity, the ability to adhere to the gut epithelium and inhibition of enteropathogen binding. Populations of NSLAB, up to 10 7 CFU/g per cheese were recovered following exposure of cheese to Simulated Stomach Duodenum Passage (SSDP) conditions. A total of 240 isolates were randomly selected from twelve Cheddar cheeses and assessed for probiotic traits. Two strains Lactobacillus paracasei DPC 7150 and Lactobacillus rhamnosus DPC 7102 showed the most probiotic potential. The Lb. paracasei and Lb. rhamnosus strains displayed adhesion rates of 64% and 79%, respectively and inhibited binding of pathogenic Escherichia coli by >20%. This research demonstrates that Cheddar cheese harbours potentially beneficial bacteria, a large portion of which can survive simulated digestion and potentially exhibit health beneficial effects once ingested.

The Microfloras and Sensory Profiles of Selected Protected Designation of Origin Italian Cheeses.

Approximately 39 Italian cheeses carry protected designation of origin (PDO) status. These cheeses differ in their manufacturing technology and the microbial flora which comprise the finished products. The evolution of lactic microflora in cheeses with PDO status is of particular interest because the biochemical activities of these organisms participate in cheesemaking and may play an acknowledged role in the development of organoleptic characteristics during ripening. Nonstarter lactic acid bacteria (NSLAB) constitute complex microbial associations that are characterized by the occurrence of various species and many biotypes as a result of a number of selective conditions persisting during the manufacturing process and different ecological niches. The evolution of different species during ripening of Fiore Sardo showed that, when present, Lactobacillus paracasei persists and dominates the microflora of the cheese in the last period of ripening, suggesting that this species, more resistant to the constraints of the mature cheese, could be involved in proteolysis and in other enzymatic processes occurring during cheese ripening. In contrast, the stretching step typical of pasta filata cheese, such as Ragusano, induced a simplification of the raw milk profiles, allowing the persistence only of some predominant species, such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. lactis, Lactococcus lactis, and Streptococcus macedonicus, after the stretching step. Lactobacillus plantarum and L. paracasei were isolated from ripened Castelmagno PDO cheese samples with the highest frequencies. These species, generally absent in the milk, occur in dairy ecosystems and dominate the bacterial flora of many ripened semihard cheeses. In PDO long-ripened Italian cheese such as Parmigiano Reggiano, the NSLAB population is mainly formed by L. paracasei, Lactobacillus rhamnosus, and Pediococcus acidilactici. Lactobacillus helveticus, L. delbrueckii subsp. lactis, and L. delbrueckii subsp. bulgaricus were also detected. Continued insight into the microbial populations of traditional Italian cheeses will allow continued production of characteristic, high-quality cheeses which have been enjoyed for many centuries.

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)5-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.

Optimal growth of Lactobacillus casei in a Cheddar cheese ripening model system requires exogenous fatty acids

Flavor development in ripening Cheddar cheese depends on complex microbial and biochemical processes that are difficult to study in natural cheese. Thus, our group has developed Cheddar cheese extract (CCE) as a model system to study these processes. In previous work, we found that CCE supported growth of Lactobacillus casei, one of the most prominent nonstarter lactic acid bacteria (NSLAB) species found in ripening Cheddar cheese, to a final cell density of 108 cfu/mL at 37°C. However, when similar growth experiments were performed at 8°C in CCE derived from 4-mo-old cheese (4mCCE), the final cell densities obtained were only about 106 cfu/mL, which is at the lower end of the range of the NSLAB population expected in ripening Cheddar cheese. Here, we report that addition of Tween 80 to CCE resulted in a significant increase in the final cell density of L. casei during growth at 8°C and produced concomitant changes in cytoplasmic membrane fatty acid (CMFA) composition. Although the effect was not as dramatic, addition of milk fat or a monoacylglycerol (MAG) mixture based on the MAG profile of milk fat to 4mCCE also led to an increased final cell density of L. casei in CCE at 8°C and changes in CMFA composition. These observations suggest that optimal growth of L. casei in CCE at low temperature requires supplementation with a source of fatty acids (FA). We hypothesize that L. casei incorporates environmental FA into its CMFA, thereby reducing its energy requirement for growth. The exogenous FA may then be modified or supplemented with FA from de novo synthesis to arrive at a CMFA composition that yields the functionality (i.e., viscosity) required for growth in specific conditions. Additional studies utilizing the CCE model to investigate microbial contributions to cheese ripening should be conducted in CCE supplemented with 1% milk fat.

Parmigiano Reggiano cheese: evolution of cultivable and total lactic microflora and peptidase activities during manufacture and ripening

Parmigiano Reggiano is a Protected Designation of Origin, long-ripened cheese, made from cow’s milk supplemented with natural whey starter, which thus contains a large microbial biodiversity. The aim of this study was to understand the population dynamics of the total lactic microflora throughout the manufacture and ripening of this cheese. Several approaches were combined to determine the quantitative changes in the different bacterial populations during 20 months of ripening of Parmigiano Reggiano cheeses from the same cheesemaking. Total and viable cells were enumerated after fluorescent labeling. Culturable bacteria were enumerated on different plate count agar media, including original media prepared from curd and ripened cheese. Six peptidase activities were quantified in curd and cheese samples free from cells. While the total bacterial cultivable population remained high and similar for the first six months, a decrease in viable starter lactic acid bacteria was observed during the first 48 h. The non-starter lactic acid bacteria populations, initially present in low numbers, began to grow after the brining and remained at high levels (about 107 CFU·g−1) for at least 10 months. During ripening, a strong decrease in the total bacterial population and a marked increase in 4 out of 6 peptidase activities were observed. In the external and internal zones of Parmigiano Reggiano cheese different trends in microbial growth, cell autolysis and peptidase activity were observed. This study gives for the first time a global view of the possible contribution of total, viable, cultivable and lysed bacterial cells throughout the ripening of Parmigiano Reggiano cheese.

Effect of yeast and bacterial adjuncts on the CLA content and flavour of a washed-curd, dry-salted cheese

Three strains of Propionibacterium freudenreichii ssp. shermanii that converted free linoleic acid to conjugated linoleic acid (CLA) in laboratory media were used as adjunct strains, together with strains of Geotrichum candidum and Yarrowia lipolytica, to make a dry-salted, washed-curd cheese. Lactobacillus fermentum was included to produce ethanol (from lactose), a potential substrate for ethyl ester synthesis, while Lactobacillus rhamnosus was used to control the adventitious non-starter lactic acid bacteria population. The total (esterified plus free) level of CLA was similar in the control and experimental cheeses and remained unchanged over 4 months of ripening. Addition of linoleic acid-rich safflower oil to the cheese curd increased the concentration of free linoleic acid generated in the cheese but the CLA content did not change. Free linoleic acid was released by the yeast lipase(s) but there was no conversion to CLA. High concentrations of ethyl esters were produced in the cheeses made with added yeast, giving a fruity flavour.

Influence of Adjunct Use and Cheese Microenvironment on Nonstarter Bacteria in Reduced-Fat Cheddar-Type Cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7°C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 104 cfu/ml did not completely suppress “wild” NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.

Variability of the species and strain phenotype composition of the non-starter lactic acid bacterial population of cheddar cheese manufactured in a commercial creamery

The non-starter lactic acid bacteria (NSLAB) present in cheddar cheese manufactured in a commercial creamery was monitored phenotypically to the strain level over a period of 12 months to examine the effects of maturity status and manufacturing practices on the composition of the population. Five Lactobacillus spp. and Leuconostoc lactis were identified among the 459 isolates selected. The predominant NSLAB, Lactobacillus paracasei and Lactobacillus brevis, were present in 59 and 31% of the cheeses examined and represented 52.7 and 25.8%, respectively, of the isolates identified. Among the NSLAB screened 71 different phenotypic profiles were identified and these included 26 biotypes of Lb. paracasei, 14 Lb. brevis, 11 Lactobacillus plantarum, 10 Lactobacillus curvatus and 7 Leuc. lactis. The average number of strains recovered from a cheese was 3.9±2.1 and ranged from 1 to 11. Although approximately 70% of the cheese samples were dominated by three or less strains the NSLAB populations were heterogeneous and the majority (61.5%) were comprised of four or more strains of one or more species. Only 30 of the biotypes were recovered from more than one population. There was no evidence for the repeated recurrence of any of the strains isolated although some of the Lb. paracasei strains were present intermittently in cheeses throughout the 12-month manufacturing period. Six Lb. brevis strains also recurred in some of the cheeses produced in a limited period during the autumn. Pronounced shifts in the species complement and strain profile occurred during maturation, while the average number of strains present in the cheese decreased with increasing maturity. Microbiological examination of the NSLAB population of cheese either produced in different vats during the same production run or manufactured in the same vat but in different production runs (vat fills) indicated that the number of strains common to paired samples from two vats or a single vat in successive production runs was only 1.7±1.4 and 1.5±1.2, respectively, and confirmed the inherent variability that exists, both within and between production runs, in the non-starter population of cheese manufactured in a commercial creamery.

The ecology of non-starter lactic acid bacteria (NSLAB) and their use as adjuncts in New Zealand Cheddar

This paper reviews the recent New Zealand studies on non-starter lactic acid bacteria (NSLAB) in Cheddar cheese. In New Zealand, cheese adventitious NSLAB were usually strains of homofermentative lactobacilli and occasionally pediococci and heterofermentative lactobacilli. In Cheddar cheese, manufactured in six factories at different times and ripened over 24 months, over 140 NSLAB strains were identified using pulse field gel electrophoresis profiles and fermentation patterns on 22 carbohydrates. The majority of NSLAB isolates identified belong to two species: Lactobacillus paracasei subsp. paracasei and Lactobacillus rhamnosus. The composition of the NSLAB strains in a cheese was variable and dependent on the manufacturing factory, the date of manufacture and the age of the cheese. In most cheeses, no single NSLAB strain predominated during ripening. Up to six strains formed the majority (about 90%) of the NSLAB population, the remainder was represented by up to 15 other strains. In only about 10% of the cheeses investigated was one strain predominant throughout the 24 month ripening period. The acid and salt tolerance, effect of temperature, ability to produce biogenic amines and the lipolytic and proteolytic activities of the NSLAB strains were studied to screen strains for use as adjuncts. Sixty suitable strains were selected for further screening in a cheese model system. The NSLAB were incubated at 30°C for 10 days in the model system and the flavour and biochemical changes were assessed. Twenty-three strains were eliminated as potential adjuncts because they produced defective flavours and a further 13 strains were eliminated on the basis of undesirable biochemical properties (e.g. excess fermentation of glutamate, lactate racemisation). The 24 remaining NSLAB strains that had acceptable microbiological biochemical and flavour properties were used as adjuncts in Cheddar trials. Seven of the 24 adjunct combinations studied (made up from 2–4 strains of NSLAB) provided consistent flavour benefits. Analysis of the cheeses during ripening in these trials provided significant understanding of the ripening mechanisms of the NSLAB adjuncts.

Methods used to study non‐starter microorganisms in cheese: a review

The significance of non‐starter lactic acid bacteria (NSLAB), which become the dominant organ‐isms in hard cheese varieties during ripening, is still controversial, but they have been shown to contribute to the release of amino acids and to influence flavour development. Since the NSLAB population is essentially uncontrolled, it is likely that at least some of the variability in cheese quality is due to the NSLAB. Several approaches used to assess the significance of NSLAB in cheese ripening are reviewed in this paper.

Proteolytic and other hydrolytic enzyme activities in non-starter lactic acid bacteria (NSLAB) isolated from cheddar cheese manufactured in the United Kingdom

The populations of non-starter lactic acid bacteria (NSLAB) in a selection of 15 good-quality UK-manufactured Cheddar cheeses that had been matured for 6–9 months ranged from 105 to 107 bacteria g-1. Fifteen different species of lactic acid bacteria were identified using commercially-available identification systems. The species isolated most frequently were Lactobacillus paracasei subsp. paracasei and Lb. plantarum; 10 other species were isolated from two or more cheeses and three species were recovered from only a single cheese. There were marked differences in the NSLAB populations of the cheeses produced by different UK manufacturers, and differences were also apparent in the populations of two cheeses produced on different occasions at the same creamery. Forty-one isolates, selected to include all the species identified and the dominant strains present in cheeses produced at several different creameries, were screened for activities of 34 proteolytic, five glycoside hydrolase and five esterolytic enzymes. All the NSLAB possessed a wide range of hydrolytic enzymes and therefore had the potential to contribute at some stage to the development of cheese flavour during the maturation and ripening period. Inter-species and strain differences in enzyme profiles and levels of activity were apparent and were determinants for the non-random selection of NSLAB for use as adjunct cultures in subsequent cheese-making trials. The breakdown of diagnostic substrates was indicative of the presence of multiple proteinase, tripeptidase, dipeptidase (including prolinase-and prolidase-like), dipeptidyl peptidase, prolyl, proline, aspartyl, pyroglutamyl (pyrrolidonecarboxyl) and general aminopeptidase activities.

Use of antibiotics to inhibit non-starter lactic acid bacteria in Cheddar cheese

The efficacy of Nisin, Penicillin G and Streptomycin at inhibiting the growth of the non-starter lactic acid bacteria (NSLAB) in Cheddar cheese was studied in cheeses treated with an antibiotic-salt mixture after milling. Nisin (500 U g −1 curd) and particularly Streptomycin (12 μg g −1 curd) did not effectively inhibit NSLAB. Penicillin G (12 U g −1 curd) reduced NSLAB numbers but mixtures of antibiotics (250 U Nisin, 6 U Penicillin G, 6 μg Streptomycin g −1 curd or 500 U Nisin, 12 U Penicillin G, 12 βg Streptomycin g −1 curd) were more effective than any individual antibiotic. The higher level antibiotic mixture reduced NSLAB populations by 4–5 log cycles compared with the control after 100–160 days. Viable starter numbers (after 35 days) were not affected by the antibiotic treatments. Antibiotic-treated cheeses contained higher levels of free amino acids but no differences between the cheeses were indicated by urea—polyacrylamide gel electrophoresis or reverse-phase HPLC.