Phenotypic and genotypic characterization of non-starter lactic acid bacteria in mature cheddar cheese.

Non-starter lactic acid bacteria were isolated from 14 premium-quality and 3 sensorially defective mature Irish Cheddar cheeses, obtained from six manufacturers. From countable plates of Lactobacillus-selective agar, 20 single isolated colonies were randomly picked per cheese. All 331 viable isolates were biochemically characterized as mesophilic (i.e., group II) Lactobacillus spp. Phenotypically, the isolates comprised 96.4% L. paracasei, 2.1% L. plantarum, 0.3% L. curvatus, 0.3% L. brevis, and 0.9% unidentified species. Randomly amplified polymorphic DNA (RAPD) analysis was used to rapidly identify the dominant strain groups in nine cheeses from three of the factories, and through clustering by the unweighted pair group method with arithmetic averages, an average of seven strains were found per cheese. In general, strains isolated from cheese produced at the same factory clustered together. The majority of isolates associated with premium-quality cheese grouped together and apart from clusters of strains from defective-quality cheese. No correlation was found between the isomer of lactate produced and RAPD profiles, although isolates which did not ferment ribose clustered together. The phenotypic and genotypic methods employed were validated with a selection of 31 type and reference strains of mesophilic Lactobacillus spp. commonly found in Cheddar cheese. RAPD analysis was found to be a useful and rapid method for identifying isolates to the species level. The low homology exhibited between RAPD banding profiles for cheese isolates and collection strains demonstrated the heterogeneity of the L. paracasei complex.     

Use of autochthonous lactic acid bacteria starters to ferment mango juice for promoting its probiotic roles

Strains of Leuconostoc mesenteroides, Pediococcus pentosaceus, and Lactobacillus brevis were identified from mango fruits by partial 16S rDNA gene sequence. Based on the ability of producing mannitol and diacetyl, Leuconostoc mesenteroides MPL18 and MPL39 were selected within the lactic acid bacteria isolates, and used as mixed starters to ferment mango juice (MJ). Both the autochthonous strains grew well in fermented mango juice (FMJ) and remained viable at 9.81 log cfu mL^?1 during 30 days of storage at 4°C. The content of total sugar of FMJ was lower than that of MJ, while the concentration of mannitol was higher than that of MJ, and the concentration of diacetyl was 3.29 ± 0.12 mg L^?1. Among detected organic acids including citric acid, gallic acid, lactic acid, and acetic acid, only citric acid and gallic acid were found in MJ, while all detected organic acids were found in FMJ. The concentration of lactic acid of FMJ was the highest (78.62 ± 13.66 mM) among all detected organic acids. The DPPH radical scavenging capacity of FMJ was higher than that of MJ. Total phenolic compounds were better preserved in FMJ. The acidity and sweetness had a noticeable impact on the overall acceptance of the treated sample.     

Spatial and temporal distribution of non-starter lactic acid bacteria in Cheddar cheese

AIMS: The aim of this work was to investigate the spatial and temporal distribution of species and strains of non-starter lactic acid bacteria (NSLAB) within Cheddar cheese. METHODS AND RESULTS: Randomly amplified polymorphic DNA was used to identify and track the principle species and strain groups of NSLAB present. The same strains dominated each location examined within a cheese at any particular time point. Temporal change in species and strains of NSLAB during ripening was observed. A mixture of Lactobacillus paracasei, Lact. plantarum, Lact. rhamnosus and unidentified strains was found up to 6 weeks of maturation, thereafter only Lact. paracasei strains were isolated. CONCLUSION: Little variation in the spatial distribution of NSLAB strains occurs within Cheddar cheese; however, temporal changes in the species and strains were observed during ripening. SIGNIFICANCE AND IMPACT OF THE STUDY: The complex changes in the composition of the NSLAB community of Cheddar cheese may be the source of the variation in flavour that is seen in commercial practice.     

Response of lactic acid bacteria to amino acid derivatives. II. Glycine

The response of five lactobacilli (for which glycine is an essential nutrient) to two tripeptides, five dipeptides, and seventeen other glycine derivatives has been tested over a range of concentrations and at four or five incubation times from 18 to 229 hours. In general the activities decreased with increasing concentrations and incubation times although in some cases they remained nearly constant, increased, or increased and then decreased. Hippuric acid, all of the dipeptides, and the tripeptide, l-leucylglycylglycine, exhibited greater activity than glycine for one or more of the organisms. These results may be interpreted to signify that (a) the apparent decrease in apparent glycine in hydrolyzed urines may be accounted for in part by the higher activity of hippuric acid in unhydrolyzed urines, and (b) some peptides may be utilized directly by lactic acid bacteria under some conditions.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Manganese, superoxide dismutase, and oxygen tolerance in some lactic acid bacteria

A previous study of the aerotolerant bacterium Lactobacillus plantarum, which lacks superoxide dismutase (SOD), demonstrated that it possesses a novel substitute for this defensive enzyme. Thus, L. plantarum contains 20 to 25 mM Mn(II),m in a dialyzable form, which is able to scavenge O2- apparently as effectively as do the micromolar levels of SOD present in most other organisms. This report describes a survey of the lactic acid bacteria. The substitution of millimolar levels of Mn(II) for micromolar levels of SOD is a common occurrence in this group of microorganisms, which contained either SOD or high levels of Mn(II), but not both. Two strains were found which had neither high levels of Mn(II) nor SOD, and they were, as was expected, very oxygen intolerant. Lactic acid bacteria containing SOD grew better aerobically than anaerobically, whereas the organisms containing Mn(II) in place of SOD showed aerobic growth which was best, at best, equal to anaerobic growth. Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone) increases the rate of O2- production in these organisms. Lactobacillus strains containing high intracellular Mn(II) were more resistant to the oxygen-dependent toxicity of plumbagin than were strains containing lower levels of Mn(II). The results support the conclusion that a high internal level of Mn(II) provides these organisms with an important defence against endogenous O2-.     

Flavour formation by lactic acid bacteria and biochemical flavour profiling of cheese products

Flavour development in dairy fermentations, most notably cheeses, results from a series of (bio)chemical processes in which the starter cultures provide the enzymes. Particularly the enzymatic degradation of proteins (caseins) leads to the formation of key-flavour components, which contribute to the sensory perception of dairy products. More specifically, caseins are degraded into peptides and amino acids and the latter are major precursors for volatile aroma compounds. In particular, the conversion of methionine, the aromatic and the branched-chain amino acids are crucial. A lot of research has focused on the degradation of caseins into peptides and free amino acids, and more recently, enzymes involved in the conversion of amino acids were identified. Most data are generated on Lactococcus lactis, which is the predominant organism in starter cultures used for cheese-making, but also Lactobacillus, Streptococcus, Propionibacterium and species used for surface ripening of cheeses are characterised in their flavour-forming capacity. In this paper, various enzymes and pathways involved in flavour formation will be highlighted and the impact of these findings for the development of industrial starter cultures will be discussed.     

Relationship between lactose utilization of lactic acid bacteria and browning of cheese during storage

ABSTRACT

To produce processed cheese turning hardly brown during transportation and storage at room temperature, natural cheese showing less discoloration should be used as a raw material. The purpose of this study was to clarify the relationship between the lactose utilization of lactic acid bacteria and the browning of cheese during storage. Three type-cultures (Lactobacillus plantarum and Streptococcus thermophilus) and five isolates from Japanese pickles (Lactobacillus spp.) were used. Cheese curds inoculated with these bacteria were prepared and stored. The L. plantarum-inoculated curds showed smaller ΔE-values after storage, an indicator for the browning, compared to the others. Accumulation of galactose was observed in the curd to which S. thermophilus was inoculated. The sample showed larger ΔE-value after storage. These results showed the lactose utilization of bacteria affected galactose concentration in cheese and its browning during storage. L. plantarum might be a good starter for preparing cheese turning hardly brown.     

Survey on the community and dynamics of lactic acid bacteria in Grana Padano cheese

Grana Padano (GP) is a Protected Designation of Origin cheese made with raw milk and natural whey culture (NWC) that is characterised by a long ripening period. In this study, six GP productions were considered in order to evaluate the trend of microbial dynamics and compare lactic acid bacteria (LAB) population levels in cheeses during the entire cheese-making process. To reach this goal, for each GP production, samples of vat raw milk, NWC and cheeses at 48 h, 2, 6, 9 and 13 months were subjected to plate counts and direct counts by fluorescence microscopy, as well as amplicon length heterogeneity-PCR (LH-PCR). Statistical analysis was applied to the results and ecological indices were estimated. It was demonstrated that the LAB able to grow in the cheese-environment conditions could arise from both raw milk and NWC. Starter lactobacilli (SLAB) from NWC were the main species present during acidification, and non-starter LAB (NSLAB), mainly from milk but also from NWC, were able to grow after brining and they dominated during ripening. The peak areas of LH-PCR profiles were used to determine ecological indices during manufacture and ripening. Among cheese ecosystems with different ageing times, diversity, Evenness and Richness were different, with highest bacterial growth and diversity occurring in cheese ripening at 2 months. At this time point, which seemed to be a crucial moment for GP microbial evolution, cell lysis of both SLAB and NSLAB was also observed.     

Spelt and emmer flours: characterization of the lactic acid bacteria microbiota and selection of mixed starters for bread making

Aims: This study aimed at characterizing the lactic acid bacteria microbiota and selecting mixed endogenous starters to be used for sourdough fermentation of spelt or emmer flours. Methods and Results: Identification of lactic acid bacteria was carried out by partial sequencing of the 16S rRNA, recA, 16S/23S rRNA spacer region and pheS genes. Spelt flour showed the largest biodiversity, while Lactobacillus plantarum dominated in emmer flour. Isolates were subjected to RAPD‐PCR analysis and screened based on the kinetics of growth and acidification, quotient of fermentation and liberation of free amino acids (FAA) during sourdough fermentation. After selection, mixed starters were used according to a two‐step fermentation process. Wheat flour was fermented by the same starters. Spelt and emmer sourdoughs had slightly higher pH than wheat sourdoughs but titratable acidity, concentration of FAA and phytase activity were higher. Specific volume and crumb grain of emmer and, especially, spelt breads approached those of wheat breads. Sensory analysis confirmed the suitability of spelt and emmer for bread making. Conclusions: The sourdough biotechnology was indispensable to completely exploit the potential of spelt and emmer flours. Significance and Impact of the Study: Results filled up the lack of knowledge on the lactic acid bacteria microbiota and technological performances of spelt and emmer flours.     

Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy

AIMS: Identification and biotyping of lactic acid bacteria (LAB) isolated from raw-milk Pecorino cheese manufactured in the Marche region (central Italy) for selection of suitable starter cultures or adjuncts. METHODS AND RESULTS: Preliminary characterization with morphological and biochemical assays were undertaken for 112 Gram-positive and catalase-negative isolates. Unequivocal identification of the isolates was obtained through restriction analysis of the amplified 16S rRNA gene and sequencing of 360-380 bp amplicons. Fifty-nine isolates belonging to LAB species generally recognized as safe and potentially utilized as starters or flavour-producing adjuncts were preselected and tested for their acidifying, proteolitic and autolytic activities. Fifty-five of these isolates were also subject to RAPD (randomly amplified polymorphic DNA) fingerprinting and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis for the estimation of genotypic intra-species variation. As a result, in Pecorino cheese, a heterogeneous lactic acid bacteria population, which includes strains with metabolic characteristics of technological interest, was characterized. CONCLUSIONS: The polyphasic approach proposed allows the bacterial ecology of Pecorino cheese to be investigated and allows to assess the potential role of autochthonous LAB strains for the dairy industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The great economic importance of Pecorino cheese encouraged a deeper knowledge of its microbiota, which is known to influence the peculiar sensory properties of this cheese, also in view of its exploitation.     

Comparison of pattern recognition techniques for the identification of lactic acid bacteria

AIMS: The goal of this study was to evaluate three pattern recognition methods for use in the identification of lactic acid bacteria. METHODS AND RESULTS: Lactic acid bacteria (21 unknown isolates and 30 well-characterized strains), including the Lactobacillus, Lactococcus, Streptococcus, Pediococcus and Oenococcus genera, were tested for 49 phenotypic responses (acid production on carbon sources). The results were scored in several ways. Three procedures, k-nearest neighbour analysis (KNN), k-means clustering and fuzzy c-means clustering (FCM), were applied to the data. CONCLUSION: k-Nearest neighbour analysis performed better with five-point-scaled than with binary data, indicating that intermediate values are helpful to classification. k-Means clustering performed slightly better than KNN and was best with fuzzified data. The best overall results were obtained with FCM. Genus level classification was best with FCM using an exponent of 1.25. SIGNIFICANCE AND IMPACT OF THE STUDY: The three pattern recognition methods offer some advantages over other approaches to organism classification.