Characterization of microflora in homemade semi-hard white Zlatar cheese

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.     

Identification of Lactic Acid Bacteria and Gram‐Positive Catalase‐Positive Cocci Isolated from Naturally Fermented Sausage (Sucuk)

ABSTRACT: The aim of the study was to identify lactic acid bacteria and Gram‐positive catalase‐positive cocci isolated from Turkish dry fermented sausage (sucuk) produced by 7 different manufacturers without using starter culture. A total of 129 isolates of lactic acid bacteria were identified phenotypically. Lactobacillus plantarum was the dominant species (45.7%) followed by L. curvatus (10.9%) and L. fermentum (9.3%). Pediococcus isolates were identified as P. pentosaceus and P. acidilactici. All the isolates of gram‐positive and catalase‐positive cocci (123 isolates) were classified as Staphylococcus except for 1 isolate assigned to Kocuria rosea. The species isolated most often were S. xylosus (41.5%) and S. saprophyticus (28.5%). Four isolates were identified as S. equorum (3.3%), 1 isolate was assigned to S. carnosus (0.8%).     

Evolution of microbial populations during traditional Feta cheese manufacture and ripening

In three different dairies (A, B and C) located in Peloponess region (Southern Greece), traditional Feta cheese trials took place February to March using mixtures of sheep's and goat's milk. Only small variations in the evolution of microbial groups were observed during the whole ripening period. The main groups, such as thermophilic cocci, mesophilic lactococci, thermophilic lactobacilli, nonstarter lactic acid bacteria (NSLAB), presumptive Leuconostoc, enterococci and micrococci, reached their highest levels during the first 16 days, and then declined approximately 1-2 log units until the end of ripening. The remaining groups investigated, comprising yeasts, coliforms and Escherichia coli, were highest at day 4. The yeasts remained constant, while coliforms and E. coli decreased sharply and were not detectable after 120 days of ripening. A number of 146 isolates (dairy A) taken from all stages of the manufacturing and ripening process were purified and studied. Lactobacillus plantarum (58/146) and isolates of related species Lactobacillus pentosus and Lactobacillus paraplantarum (16/146) were the most common microorganisms found during cheese ripening. Streptococcus thermophilus (23/146) and Lactobacillus delbrueckii subsp. bulgaricus (20/146) were detected in high levels up to 20 days, and then gradually reduced. Enterococcus faecium (29/146) was found in all manufacturing and ripening stages.     

Characterization of natural starter cultures used in the manufacture of Pasta Filata Cheese in Basilicata (Southern Italy)

Microbiological, chemical and technological analyses were used to characterize nine natural starter cultures used for the mnnufacture of Pasta Filata cheese in Basilicata (Southern Italy). The cultures were either dominated by thermophilic rods or mesophilic and/or thermophilic cocci. Principal component and cluster analysis discriminated between rods and coccus cultures and helped to estabilish relationships between the cultures and to evaluate the variability of cultures obtained from the same plant. A total of 156 isolates of lactic acid bacteria were obtained from the cultures and tentatively classified using biochemical and physiological tests and cluster analysis. Most of the lactobacilli were identified as Lactobacillus helveticus. Most cocci were classified in the genera Lactococcus or Enterococcus but identification at the species level was often impossible. The acid production and proteolyti activity of the isolates in skim milk were evaluated. Cluster analysis was used to group the isolates according to technological properties. Isolates belonging to some phenotypic clusters were consistently associated with some technological clusters.     

Indigenous raw milk microbiota influences the bacterial development in traditional cheese from an alpine natural park

Nostrano di Primiero is a 6-month ripened cheese produced from raw milk collected in the Paneveggio-Pale di San Martino Natural Park area in the Italian Dolomites. In summer, this cheese is made using milk collected from two different areas, Passo Rolle and Vanoi, in the Paneveggio Natural Park. During the experiment, the milk from the two areas was separately processed, and cheeses were made in the same cheese factory using the same technological process. The microbiota of raw milk and cheeses of the two areas was isolated and the dominant population was monitored by RAPD analysis and identified by 16S rRNA sequence. The milk of the Passo Rolle area was mainly composed of mesophilic strains, thermophilic Streptococcus thermophilus, and low amounts of enterococci were also found; the milk of the Vanoi area was dominated by mesophilic microbiota mostly Lactococcus lactis ssp. cremoris and ssp. lactis and Lactobacillus paracasei ssp. paracasei. The plating of the natural starter culture revealed the presence of a relevant community of thermophilic cocci and lower amounts of enterococci. The dynamic population analysis showed the importance of the natural starter culture in the first 2 days of cheese ripening in both cheeses. Moreover, the large biodiversity observed in the raw milks was also detected in the cheeses during ripening. The Vanoi cheese was dominated by Enterococcus faecium and Streptococcus macedonicus in the first two days and mesophilic 21 Lb. paracasei ssp. paracasei became the most represented population after 15 days of ripening. In the first few days, the Rolle cheese was characterized by being mainly composed of thermophilic S. macedonicus and S. thermophilus and secondarily by mesophilic cocci. During ripening, the microbiota composition changed, and at 15 days, mesophilic lactobacilli were the dominant population, but later, this was mainly composed of mesophilic cocci and lactobacilli. The taxonomical identification by 16S rRNA sequence confirmed a large biodiversity related to raw milk microbiota and only five strains of S. macedonicus, Lactobacillus plantarum, 21 Lb. paracasei ssp. paracasei, Lactobacillus fermentum and E. faecium were detected in both cheeses.     

Development of fermented instant Chinese noodle using Lactobacillus plantarum

A new-type of instant Chinese noodle was developed with the application of lactic acid fermentation by lactobacilli. Since the pH value of the noodle sheets is alkaline with kansui (around 8.5), alkaline tolerance is required for the lactobacilli to ferment noodle sheets. The screening of the lactobacilli strains suitable for the fermentation was conducted using 46 strains from 12 species (including subspecies) of lactobacilli. Several strains of Lactobacillus pentosus and Lactobacillus plantarum were found to be fermenters. Among these, L. plantarum NRIC 0380, that showed the highest fermentation rate and favorable modification of noodle, was selected as the best strain, and was employed for the pilot scale manufacture of instant Chinese noodle. During fermentation, L. plantarum NRIC 0380 produced lactic acid to about 11 g/kg noodle sheet after 24 h with a concomitant pH decrease from an initial of about 7.9 down to 3.9. Sensory test after rehydration with boiled water revealed that the fermented instant Chinese noodle sheets at pH 7.5 had increased hardness, elasticity and light sour taste.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

Bacterial diversity of Darfiyeh, a Lebanese artisanal raw goat's milk cheese

In order to contribute to the preservation of the Lebanese dairy heritage, the aim of this study was to characterize the Darfiyeh cheese, a traditional variety made from raw goat's milk and ripened in goat's skin. Three independent batches of Darfiyeh production were analyzed after 20, 40 and 60 days of ripening. Mesophilic lactobacilli, thermophilic coccal-shaped lactic acid bacteria (LAB) and thermophilic lactobacilli were enumerated. In order to explore the Darfiyeh natural ecosystem, a combination of phenotypical and molecular approaches was applied. The latter included Polymerase Chain Reaction-temporal temperature gel electrophoresis (PCR-TTGE), classical PCR and quantitative PCR. These methods revealed the presence of Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus malodoratus, group D Streptococcus sp., Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus haemolyticus, Escherichia coli, Clostridium sp./Eubacterium tenue. Real-time PCR enabled quantification of E. faecium, with a detection of 10⁷–10⁹ cfu g⁻¹ of product. The present molecular approaches combined with phenotypic method allowed describing the complex natural ecosystem of Darfiyeh, giving useful information for the preservation of Lebanese artisanal dairy products.     

Isolation and identification of cultivable lactic acid bacteria in traditional fermented milk of Tibet in China

One hundred and seventy‐one strains of lactic acid bacteria were isolated from 44 traditional fermented milk samples from different region of Tibet. Among these samples, the concentrations of lactic acid bacteria varied from 10 ⁵ to 10 ⁹ colony forming unit/mL. The ratio of rod bacteria (71.3%) was higher than that of cocci (28.7%), and was considered to be the major population. Lactobacillus fermentum (31%) and Lactobacillus casei (28%) were the most predominant species among all these isolates. This study systematically analysed the microbial composition of traditional fermented milk in China, which may become a valuable source for further starter selection.     

Evaluation of microbial diversity during the manufacture of Fior di Latte di Agerola, a traditional raw milk pasta-filata cheese of the Naples area

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.     

Isolation and applied potential of lactic acid bacteria from Chinese traditional fermented food in specific ecological localities

The isolation and screening of lactic acid bacteria (LAB) from natural sources have been one of the powerful means to obtain strains for the food industry. A total of 275 indigenous isolates were obtained from 43 samples of traditional fermented foods in specific ecological niches throughout the northwestern China, and among which 13 strains of LAB were selected for their potential in food preservation and production. Among the 13 isolates, Lactobacillus (10) was dominant over Lactococcus (3). The distribution of the isolates was as follows: Lactobacillus paracasei ssp. paracasei (J23, M10, M20, M22), Lactobacillus rhamnosus (J20, M18), Lactococcus lactis ssp. lactis (X20, Q7), Lactobacillus casei (Q1, Q12), Lactobacillus plantarum (J11), Lactococcus lactis ssp. cremoris (X8), Lactobacillus delbrueckii ssp. bulgaricus (Q5). All 13 isolates produced bacteriocin with a broad inhibitory spectrum against selected Gram-positive as well as Gram-negative pathogenic and spoilage species. Biochemical analysis revealed that they possessed high acidification and coagulation activity. Several strains possessed the high activity of 2 or 3 technological characteristics, related to novel starters and food preservatives.     

Characterisation of Lactobacillus helveticus strains isolated from home‐made dairy products in Iran

The aims of this study were the isolation and characterisation of a number of lactobacilli strains from traditional dairy products. Fifteen home‐made samples were pour‐plated onto MRS and predominant colonies were randomly picked up. Nine isolated lactobacilli were grouped using rep‐PCR fingerprinting, and partial sequencing of 16S‐rRNA of group's representatives confirmed them as Lactobacillus helveticus. Detection of two CEP (prtH and prtH2) genes and examination of acidification and growth in milk revealed intradiversity among isolates. The findings indicate the possibility of isolating novel wild strains of L. helveticus from home‐made products and emphasises on the necessity of both genetic and technological characterisation for deeper differentiation of strains.     

Effects of Elaidic Acid,a Predominant Industrial Trans Fatty Acid,on Bacterial Growth and Cell Surface Hydrophobicity of Lactobacilli

The consumption of trans fatty acids (TFAs) increases the risk of cardiovascular diseases and coronary heart disease in human, and there are no effective ways to remove TFAs after consumption. The aim of this study was to investigate the effects of elaidic acid on bacterial growth, cell surface hydrophobicity of lactobacilli, and metabolism of elaidic acid by lactobacilli. Lactobacilli were inoculated in MRS broth containing 0, 100, 200, and 500 mg/L of elaidic acid. Viable cell counts of lactobacilli were enumerated, concentrations of elaidic acid were determined, and cell surface hydrophobicity of lactobacilli was measured. The results showed that the growth of lactobacilli was significantly inhibited by 500 mg/L of elaidic acid, however, a cell count of 8.50 log₁₀ CFU/mL was still reached for tested lactobacilli after 24‐h incubation. In particular, a reduction of elaidic acid was found for tested lactobacilli after 24‐h incubation as compared to its initial concentration of 200 mg/L. However, cell surface hydrophobicity showed no correlations with the metabolism of elaidic acid by lactobacilli. Moreover, elaidic acid was able to influence cell surface hydrophobicity, and the decrease in hydrophobicity was more obvious in Lactobacillus paracasei and Lactobacillus casei compared with that in other tested lactobacilli. This study suggests that elaidic acid could change physiochemical surface properties of lactobacilli and the lactobacilli have the potential to reduce TFAs.     

Identification and Characterization of Lactic Acid Bacteria Involved in Natural and Lactic Acid Bacterial Fermentations of Pastes of Soybeans and Soybean-Maize Blends Using Culture-Dependent Techniques and Denaturing Gradient Gel Electrophoresis

Pastes of soybeans and soybean-maize blends were fermented with inoculum through back-slopping using lactic acid bacteria (LAB) fermented cereal gruel, thobwa and without inoculum (naturally). LAB involved in the fermentations were characterized using culture-dependent and culture-independent analyses. Decreases in pH from 6.4 to 3.9–4.2 and from 6.9 to 5.4–5.8 after 72 h were observed in LAB fermented pastes (LFP) and in naturally fermented pastes (NFP), respectively. LAB increased from 5.0 to 8.7–9.6 log₁₀ cfu/g in NFP and from 8.1 to 9.3 log₁₀ cfu/g in LFP. LAB in both fermentations were heterofermentative lactobacilli (82.4%) and homofermentative cocci (17.6%), of which 44.7% and 42.9% were exopolysaccharide producers, respectively. Principal component analysis based on carbohydrate fermentation, CO₂ production and arginine hydrolysis showed four clusters dominated by Lactobacillus fermentum, Weissella confusa, Lactobacillus brevis 1 and Pediococcus pentosaceus, respectively. Sequencing of 16S rDNA gene confirmed Lb. fermentum, W. confusa/W. cibaria, and P. pentosaceus as identities of species in three clusters. Denaturing gradient gel electrophoresis (DGGE) confirmed these species as the dominant microbiota. DGGE showed higher similarity in microbial profiles of LFP throughout fermentation and low similarity in NFP during early and late stages of fermentation.     

Metabolic activity and symbiotic interactions of lactic acid bacteria and yeasts isolated from water kefir

Water kefir is a mildly sour and alcoholic drink fermented by a stable microbial multispecies community. With its high sugar content and low amino acid concentration water kefir medium represents a demanding habitat. In this ecological niche only well adapted microorganisms which are fit to the consortium are able to grow and mutually provide essential nutrients. The synergism between main representatives of water kefir yeasts and lactobacilli was studied in a co-culture model system. Co-cultivation of yeasts and lactobacilli in water kefir medium significantly increased cell yield of all interaction partners, delineating the interaction of these water kefir isolates as mutualism. The support of Zygotorulaspora (Z.) florentina was due to the acidification of the medium by the lactobacilli, whereas lactobacilli are improved in growth by the disposal of essential nutrients produced by yeasts. The trophic interaction between Lactobacillus (Lb.) hordei and yeasts is constituted by the release of amino acids and Vitamin B₆ from yeasts, whereas Lb. nagelii is supported in growth by their production of amino acids. The interaction of Z. florentina and Lb. nagelii was further examined to reveal that co-cultivation induced the yeast to release arginine, which was essential for Lb. nagelii.     

Genotypic diversity of bacteria and yeasts isolated from Tibetan kefir

In this study, we have investigated the intraspecies genotypic diversity of bacteria and yeasts isolated from Tibetan kefir via the (GTG)₅‐REP‐PCR technology. According to the cluster analysis of generated fingerprints, forty‐nine bacterial isolates were grouped into nine clusters and formed thirteen genotypes, and twenty‐two yeast isolates were grouped into two clusters and formed two genotypes. It's worth noting that isolates of Lactobacillus kefiranofaciens, Streptococcus thermophilus, Lactobacillus kefiri and Lactobacillus paracasei exhibited the intraspecies genotypic diversity, whereas isolates of Saccharomyces cerevisiae and Kazachstania unispora did not exhibit. These results confirmed that bacterial isolates possessed higher level of intraspecies genotypic diversity than yeast isolates studied in this research. These findings revealed that (GTG)₅‐REP‐PCR was an efficient and sensitive molecular typing tool for revealing the intraspecies genotypic diversity among the bacterial isolates originated from Tibetan kefir.     

Microbiological and chemical properties of kefir manufactured by entrapped microorganisms isolated from kefir grains

In this study, various yeasts (Kluyveromyces marxianus, Saccharomyces turicensis, Pichia fermentans) and lactic acid bacteria (Lactobacillus kefiranofaciens, Lactobacillus kefiri, Leuconostoc mesenteroides) were entrapped in 2 different microspheres using an entrapment ratio for the strains that was based on the distribution ratio of these organisms in kefir grains. The purpose of this study was to develop a new technique to produce kefir using immobilized starter cultures isolated from kefir grains. An increase in cell counts with fermentation cycles was observed for both the lactic acid bacteria (LAB) and yeasts, whereas the cell counts of kefir grains were very stable during cultivation. Scanning electron microscopy showed that the short-chain lactobacilli and lactococci occupied the surface of the LAB microspheres, whereas the long-chain lactobacilli were inside the microspheres. When the yeasts were analyzed, cells at a high density were entrapped in cracks on the surface and within the microspheres, where they were surrounded by the short-chain lactobacilli. The distribution of the LAB and yeast species in kefir produced from grains and microspheres showed that there was no significant difference between the kefirs produced by the 2 methods; moreover, Leu. mesenteroides and K. marxianus were the predominating microflora in both types of kefir. There was no significant difference in the ethanol and exopolysaccharide contents between the 2 kefirs, although the acidity was different.     

Amino acid-decarboxylase activity of bacteria isolated from ice-preserved anchovies

The amino acid-decarboxylase activity of bacteria isolated from Engraulis encrasicholus preserved in ice was investigated throughout 23 days of storage. A total of 140 bacterial isolates were studied, including 37 enterobacteria, 23 pseudomonads, 49 Gram+ catalase+ cocci, 27 lactic acid bacteria and 4 enterococci. The percentage of strains that decarboxylated amino acids was low, 12% on average. None of the Gram+ catalase+ cocci showed aminogenic activity, but all enterococci isolates did. The enterobacteria with aminogenic capacity were identified as Enterobacter cloacae and they simultaneously produced putrescine and cadaverine (up to 500 mg/l), but not histamine. Among pseudomonads, two species decarboxylated ornithine (producing 350–650 mg/l putrescine): Pseudomonas cepacia and Pseudomonas fluorescens. Lactic acid bacteria decarboxylated tyrosine, yielding up to 2,000 mg/l tyramine, and they were identified as Lactobacillus brevis, Lactococcus lactis, Pediococcus pentosaceous and Enterococcus spp. As a general rule, the positive Gram-negative isolates (enterobacteria and pseudomonads) were diamine producers and they were found only during the first 2 weeks of storage. In contrast, lactic acid bacteria and enterococci were mainly tyramine producers and they were isolated only at the end of storage.Part of this study was presented at the XIII Congreso de Microbiología de los Alimentos held in Bilbao (Spain), 17–19 September 2002.     

Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Microbial characterization of Iranian traditional Lighvan cheese over manufacturing and ripening via culturing and PCR-DGGE analysis: identification and typing of dominant lactobacilli

This paper reports on the diversity and dynamics of the dominant microbial populations during manufacturing and ripening of Lighvan, a traditional, starter-free Iranian cheese made from raw ewe and goat’s milk as determined by culturing and PCR-DGGE. Similar dominant populations, composed of Lactococcus lactis and Lactobacillus spp. strains, were found by both techniques. However, discrepancies regarding the identity of the Lactobacillus species were encountered. Lactobacillus curvatus and Lactobacillus sakei proved to be dominant by PCR-DGGE; in contrast, Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis and Lactobacillus plantarum were the majority cultivable organisms. RAPD typing of lactobacilli isolates showed wide genetic diversity among the species. Moreover, strain compositions change over time; L. brevis and L. paraplantarum were dominant in milk and were replaced by L. plantarum and L. paracasei strains as ripening progressed.