Lactic Microflora Research Articles

Discrimination of commercial Caciocavallo cheeses on the basis of the diversity of lactic microflora and primary proteolysis

A wide variety of semi-hard pasta-filata cheese fall under the common name “Caciocavallo”, including Caciocavallo Silano, a Protected Denomination of Origin (PDO) cheese produced in Southern Italy. Multivariate analysis techniques were used to investigate the specificity of Caciocavallo Silano PDO cheeses within the area of production and to compare them with cheeses of similar denomination or cheeses produced with similar technology. The composition of lactic acid bacteria (LAB) communities at the species level and the primary proteolysis of commercial cheeses were assessed. A high variability was observed in the composition of LAB communities, and no clear relationship was found with either the region of origin or the cheese denomination. However, the microflora of PDO cheeses was less heterogeneous than that of non-PDO cheeses and composed mainly of non-starter lactic acid bacteria (NSLAB) such as Lactobacillus paracasei , L. fermentum , and L. plantarum . A high diversity in the proteolytic pattern of the cheeses was also found. This diversity was associated with the trends observed in the community of LAB populating each of the cheeses. The main problems concerning the protection of the denomination are discussed.

Evidence for Formation of Heterooligosaccharides by Lactobacillus sanfranciscensis during Growth in Wheat Sourdough

Lactobacillus sanfranciscensis is a key organism of the lactic microflora in traditional and industrial sourdough fermentations. In this paper we provide evidence for the formation of heterooligosaccharides (HeOS) by L. sanfranciscensis during growth in sourdough. To identify the HeOS based on HPAEC-PAD analysis, HeOS standards were synthesized by enzymatic reactions with L. sanfranciscensis levansucrase in a chemically defined system in the presence of raffinose, maltotriose, maltose, xylose, or arabinose as acceptor carbohydrates. The oligosaccharides known to originate from the corresponding acceptor reactions, 1(F)-beta-fructosylraffinose, 1(F)-beta-fructofuranosylmaltotriose, erlose (1(F)-beta-fructofuranosylmaltose), xylsucrose, 1(F)-beta-fructosylxylsucrose, and arabsucrose, were identified by HPAEC-PAD. Evidence for the formation of further tri-, tetra-, and pentasaccharides was provided. Wheat doughs with sucrose were fermented with L. sanfranciscesis TMW 1.392 or the isogenic, levansucrase-negative strain TMW 1.392Deltalev, and the analysis of dough extracts or invertase-treated dough extracts provided evidence for the formation of arabsucrose and erlose in sourdough in addition to 1-kestose and nystose.

Phenotypic characterization of lactic acid bacteria from sourdoughs for Altamura bread produced in Apulia (Southern Italy)

In order to study the composition of the lactic acid bacteria (LAB) community of sourdoughs used for the manufacture of Altamura bread, a traditional durum wheat bread produced in Apulia (Southern Italy), 111 strains of LAB were isolated and characterized. The phenotypic characterization of the isolates, carried out using a set of 29 tests, allowed the identification of 15 clusters at the 80% similarity level by hierarchical cluster analysis. Of the isolates, 88% were identified as facultatively heterofermentative LAB ( Lactobacillus plantarum, Lb. paracasei, Lb. casei) and 12% as heterofermentative LAB ( Lb. brevis, Leuconostoc mesenteroides). SDS-PAGE profiles of whole cell proteins of 68 strains confirmed the identification. Both the diversity and structure of the lactic microflora for sourdoughs for Altamura bread varied among samples.

Microbiological characteristics of Batzos, a traditional Greek cheese from raw goat's milk

The changes of microbial flora in Batzos, raw goat's milk cheese, were studied during ripening, throughout the whole lactation season in nine cheese batches manufactured three in each, winter, spring and summer. High counts of Enterobacteriaceae and coliforms were recorded early in ripening, but their levels decreased significantly ( P<0.05) on ripening and storage. Lactic acid bacteria predominated over the other microbial groups throughout ripening, during the whole lactation season. It seems possible, that high NaCl content of the cheese was the main agent that regulated the microbial survival. No significant differences were observed in the counts related to the season when the cheeses were ripened and stored. However, the season affected the composition of the lactic microflora. Thus, in winter enterococci were abundant, while in spring and summer lactobacilli isolates were found more frequently. Lactococcus lactis subsp lactis was the most frequently isolated species in the cheese and its use as starter seems promising.

Characterization of a cell-associated inulosucrase from a novel source: a Leuconostoc citreum strain isolated from Pozol, a fermented corn beverage of Mayan origin.

A cell-associated fructosyltransferase was extracted from a novel source, a strain of Leuconostoc citreum isolated from Pozol, a Mexican traditional fermented corn beverage, where lactic microflora are partially responsible for the transformation process. The enzyme is associated with the cell wall. It was characterized both in its cell-associated insoluble form and after separation by urea treatment. The fructosyltransferase has a molecular mass of 170 kDa, the highest reported for this type of enzyme, and in its insoluble form is highly specific for polymer synthesis, with low fructose transferred to maltose and lactose added to the reaction medium (acceptor reactions). The synthesized polymer has an inulin-like structure with beta2-1 glycosidic linkages, as demonstrated by 13C nuclear magnetic resonance (NMR). Bacterial inulosucrases have only been reported in Streptococcus mutans.

Microbiological, compositional, biochemical and textural characterisation of Caciocavallo Pugliese cheese during ripening

A microbiological, compositional, biochemical and textural characterisation of the pasta filata Caciocavallo Pugliese cheese during ripening is reported. Fully ripened cheese contained a total of ca. log 8.0 cfu g −1 mesophilic bacteria and ca. log 6.0 cfu g −1 presumptive staphylococci, while the number of thermophilic and mesophilic rod and coccus lactic acid bacteria varied during ripening. A two-step RAPD-PCR protocol was used to differentiate biotypes. The natural whey starter was composed mainly of Lactobacillus delbrueckii, Lb. fermentum , Lb. gasseri , Lb. helveticus and Streptococcus thermophilus strains. After day 1 of ripening, Lb. delbrueckii became dominant and some strains of Enterococcus durans and E. faecalis appeared. Non-starter lactic acid bacteria, such as Lb. parabuchneri and Lb. paracasei subsp. paracasei formed a large part of the lactic microflora at 42 and 60 d of ripening. The level of pH 4.6-soluble nitrogen increased from the outer to the inner of the cheese and also increased in each section as ripening progressed, attaining values of 18–15%. Urea-PAGE electrophoresis showed that degradation of α s1 -casein was more rapid than that of β -casein throughout ripening and the rates at which both caseins were degraded greatly increased from the outside to the inside of the cheese. Based on the primary proteolysis products, both chymosin and plasmin appeared to be active. RP-HPLC profiles of the 70% ethanol-soluble, pH 4.6-soluble nitrogen, showed a large number of peaks, indicating a heterogeneous mixture of proteolytic products. There were both age- and section-related changes in the area of the different peptide peaks. Butyric (C4:0), caproic (C6:0), palmitic (C16:0) and oleic (C18:1) acids were the free fatty acids found at the highest concentrations. The level of short chain fatty acids (e.g., butyric and caproic) decreased from the middle and inner to outer sections of the cheese. Peptidase activity in the curd was pronounced, increased during ripening and varied with the cheese section. The greatest increase of the peptidase activity coincided with a change in the lactic microflora and with the prevalence of non-starter lactic acid bacteria. Microbial esterases were supposed to be active together with rennet paste. Little change in the firmness and fractures stress during maturation were found by textural analyses of the raw cheese. The flowability was similar to that of typical low-moisture Mozzarella cheese, while stretchability was lower. The heat-induced changes in phase angle of Caciocavallo Pugliese cheese indicated a phase transition from largely elastic rheological characteristics in unheated cheese to a more viscous and fluid character in melted cheese.

A preliminary study of lactic acid bacteria in whey starter culture and industrial Pecorino Sardo ewes’ milk cheese: PCR-identification and evolution during ripening

Lactic acid bacteria isolated from industrial Pecorino Sardo cheese, manufactured from thermized ewe's milk, and from its natural starter culture were characterized taxonomically. Cheese samples were collected from three dairies at different stages of production and 543 colonies were isolated. PCR-based taxonomic identification revealed that thermophilic lactic acid bacteria belonging to Lactobacillus delbrueckii , Lactobacillus helveticus and Streptococcus thermophilus species constituted the dominant lactic microflora isolated. Mesophilic facultatively heterofermentative lactobacilli of the L. casei group and enterococci were also isolated from the cheeses, together with starter lactic acid bacteria (SLAB). The counts of mesophilic lactobacilli and enterococci increase during ripening. The three batches of cheese differed in the range of the species isolated in the whey starter culture and their evolution in cheese during ripening. Differences in the microflora composition of industrial and homemade Pecorino Sardo cheese were also examined.

Nisin-producing organisms during traditional ‘Fior di latte’ cheese-making monitored by multiplex-PCR and PFGE analyses

In this work we studied using different molecular methods the population dynamics of nisin-producing organisms and the persistence of such organisms within a complex ecosystem, ‘Fior di latte’ cheese, a traditional high-moisture pasta filata cheese. Using the primers targeting the eubacterial 16S–23S rRNA spacer region, together with those amplifying the nisA or nisZ gene, we were able to provide a rapid species identification of the isolates. Inhibitors of Lactococcus lactis subsp. lactis DSM 20481 T used as indicator occurred during the whole process of cheese manufacture as a significant part of lactic microflora; however, only 12 among 109 isolates of bacteriocin producers were nisin producers. Amplification of the nisA or nisZ gene, using DNA extracted directly from dairy samples as templates, showed that the nisin structural gene was detected during cheese-making from milk samples up to the end of curd ripening but not in the final cheese. In order to monitor nisin-producing strains during cheese manufacturing, the 12 Lactococcus lactis nis + strains were analysed by low frequency restriction fragment and PFGE. Nine isolates among the 12 nisin-producers exhibited an unique and distinct DNA banding pattern and are considered to be genetically diverse. The other three isolates from curd after ripening showed the same restriction pattern and could be the same strain. In fact, it was also isolated 2 months after the first analysis of cheese-making of ‘Fior di latte’.

Viability of lactic acid microflora in different types of yoghurt

The suitability of three culture media (Skim milk agar, M17 agar and MRS agar, incubated under aerobic and anaerobic conditions) to obtain total and differential counts of yoghurt bacteria, was assayed. A total of 25 samples of different yoghurt types (set, skimmed set, drinking, and set with “dulce de leche”) obtained with the culture IA, were used. The lactic microflora viability was studied at 6°C and 12°C. Skim milk agar (aerobiosis) was the most useful medium for total and differential counts of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus. Cell viability depended on the yoghurt type and the storage temperature. On the basis of a minimum value of 10 7 CFU g −1, the shelf life of yoghurts at 6°C was longer than 60 days, with the exception of the drinking type (45 days). At 12°C, shelf life was identical for the skimmed drinking yoghurt but lower for the other yoghurt types. Both the storage temperature and the yoghurt type should be taken into account when shelf life is specified on the basis of the lactic microflora content.

Presence of peptidase activities in different varieties of cheese.

Lactic acid bacteria (LAB) counts, PepX activity towards H-Phe-Pro-beta NA, and aminopeptidase activity towards H-Arg-beta NA. HCl, H-Lys-beta NA, H-Leu-beta NA, H-Pro-beta NA, H-Glu-beta NA derivatives have been evaluated in 32 commercial samples of cheese, one processed cheese, and one yoghurt. The presence of intracellular exo-peptidase activities in cheese extracts free from bacterial cells was detected, even after 1 year of ripening. An inverse ratio between the presence of viable lactic microflora and peptidase activity in the cheese extracts was observed. The importance of LAB starter exo-peptidases in the degradation of casein oligopeptides, and the key role of autolysis in the release of peptidases in the cheese, are discussed.

Microbiological and Technological Characteristics of Natural Whey Cultures for Argentinian Hard-Cheese Production

Samples (29) of natural whey starters used in the Santa Fe (Argentina) area for hard cheese production were examined. The microbial composition (including lactic microflora identification) and technological characteristics (acidifying and proteolytic activities) were determined.The cultures consisted mainly of thermophilic lactic acid bacteria belonging to the genus Lactobacillus. L. helveticus (66% the total strains) and L. delbrueckii subsp. lactis (33%) were the dominant species. In every sample, yeasts appeared as the only natural contaminants, at variable contents.The samples showed high acidity levels (1.2 to 1.4% lactic acid) with a mean pH value of 3.3. The acidifying and proteolytic activities were high at temperatures ranging from 37 to 50°C and negligible at 55°C.

Microbiological, Chemical, and Sensory Changes in Irradiated Pica De Gallo

ABSTRACTThe effects of gamma processing (1 kGy) and refrigerated storage (2°C) on microbiological, sensory, and chemical quality of pico de gullo was studied. Color, flavor, texture, odor, and heat sensory attributes were not affected by radiation treatment. The treatment decreased populations of aerobic mesophilic, heterofermentative, and total lactic microflora during storage. L‐ascorbic acid content declined 50% in response to gamma processing, but levels were similar in irradiated and nonirradiated samples after 6 wk. Pectin solubility was affected by radiation treatment. Gamma processing caused a reduction in pectin degree of esterification, and conversion of chelator soluble to dilute alkali soluble and nonextractable pectins.

Quality Maintenance of “Ready‐to‐eat” Shredded Carrots by Gamma Irradiation

ABSTRACTShredded carrots were either chlorinated, rinsed, and spin‐dried as in industrial processes, or irradiated (2 kGy), replacing the three steps. Several factors defining the quality of minimally processed vegetables were monitored during storage at 10°. Atmospheres inside micro‐porous plastic bags stabilized at 7–15% O2 and 10–15% CO2. Sugar levels in tissues were twice as high in irradiated samples as in chlorinated ones. Irradiation also prevented losses of orange color and carotenes. Growth of aerobic mesophilic and lactic microflora was strongly inhibited by irradiation. Sensory analysis demonstrated preferences for irradiated vegetables. Irradiation, avoiding three potential denaturing steps preserved “ready‐to‐eat” shredded carrots with better quality than those prepared by conventional industrial processes.

A SURVEY OF MICROBIOLOGICAL ASPECTS OF CIDER MAKING

Among the lactic microflora of ciders it was found that the species Leuconostoc oenos was predominant at the end of the alcoholic fermentation as well as at the end of the malo-lactic conversion. Among yeasts, the genera Saccharomyces was predominant at the end of fermentation. However, in the case of one juice characterised by a low initial level of bacteria, a high amount of phenolic compounds and a small quantity of total nitrogen, the whole lactic flora was no longer detected after a fortnight.

Genetic heterogeneity among Lactobacillus acidophilus strains.

Physiological characteristics, DNA base composition (% GC) and DNA-DNA reassociation values were determined for 138 Lactobacillus acidophilus strains. Twenty seven strains were received from various culture collections and 111 strains were freshly isolated during a study on the composition of the intestinal lactic microflora of piglets and suckling calves. All strains had physiological characteristics which were substantially similar. The strains isolated from pigs were unable to ferment trehalose. The % GC ranged from 35.8 to 43.4. On the basis of the results of DNA-DNA hybridization the strains were divided into four genetic groups.

Inhibition of Listeria monocytogenes by defined lactic microflora in gnotobiotic silages of lucerne, fescue, ryegrass and maize — Influence of dry matter and temperature

The development of L. monocytogenes was studied in gnotobiotic silages of four plant species (ryegrass, fescue, lucerne, maize) with various dry matter concentrations and incubation temperatures. The microflora of these silages was perfectly defined and included Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Pediococcus spp., Enterobacter liquefaciens and Clostridium tyrobutyricum. The results show that in silages of good quality not only is there no development of L. monocytogenes, but the cells initially present are eliminated. It is therefore most unlikely that a silage of good quality with a pH lower than 5 plays a contaminating role. The hypothesis according to which silages, especially those of bad quality, contain factors favouring contamination seems to be more plausible.

LEBANON BOLOGNA

A process devised in our pilot plant to manufacture Lebanon bologna consists of three steps: (a) aging salted beef at 5 C for 10 days; (b) smoking the stuffed bolognas at 35 c and high relative humidity for 4 days; and (c) mellowing the smoked bolognas at 5 C for 3 days. Aging the salted beef serves to enrich for a lactic microflora which will carry out the fermentation and for a micrococcal flora which will reduce nitrate to nitrite. Development of a firm cohesive structure which is characteristic of Lebanon bologna is related principally to acid production and only slightly to smoking. Fermentation occurs during the smoking period with the pH of the bolognas falling at least one pH unit during the first 2 to 3 days. Nitrate reduction and subsequent formation of nitrosylmyoglobin occur within the first 24 h. Flavor of Lebanon bologna is described as both acid and smoky. Both components develop during the lengthy incubation in the smoke house.

ESTIMATION OF THE BIOPRESERVATIVE OBTAINING METHOD MODIFICATION FOR PERENNIAL GRASSES SILAGE MAKING BY FERMENTATIVE ACTIVITY

For a long time, scientific research has been carried out at the VNIIMZ on the basis of the Department of Biotechnology in the field of bioconversion of organic resources into biological means intended for use in agriculture as fertilizers, plant growth stimulants, and bioconservants for silage forage crops. One of the promising basic developments of the department is the patented way of obtaining a biokonservant by peat fermentation with excrement of agricultural animals, including extraction of bioactive substances, which, in fact, constitute the basis of the product being formed. The material of the publication presented contains a comparative analysis of a patented method for producing a biokonservant and a modified method in which new biostimulant and extractant are used. The investigated processes were compared in the dynamics of enzyme activity. The results of the studies showed that the introduction of a new multicomponent biostimulator activated the enzymatic activity in the bioconversion process of the initial substrate. Two bursts of oxidation-reduction reaction activity were recorded at the key stages of the process (the oxidation-reduction ratio of the initial mixture was 1.3 and the fermentation product was 1.23 in the new technology versus 0.14 and 0.06 in the base technology, respectively). In this case, the high activity of invertase in the initial substrate provided an intensification of the process of cleavage of complex carbohydrates to monomers. The change in the extractant had no significant effect on the enzymatic activity. The extraction period was characterized by a decrease in the activity of all the enzymes studied, regardless of the technology used. Additional studies of a complex biostimulator are required to increase and grow lactic acid microflora, as in the finished biokonservant by the new technology, the lactic acid microflora titer is nx107 cfu / ml, which is lower by an order of magnitude than in the finished product according to the basic technology.