Microbiological quality of artisanal Sepet cheese

Microbial diversity in milk and in cheese itself affects the biochemical and sensory characteristics of artisanal cheeses. In this study, the microflora of Sepet cheese, which is a traditional artisanal cheese in Turkey, was investigated. Average lactococci, lactobacilli, enterococci, yeast, mould, coliform, psychrotrophic and total aerobic bacteria, presumptive Staphylococcus aureus counts were; 7.31 ± 1.08, 7.19 ± 1.02, 6.84 ± 0.92, 3.19 ± 1.40, 0.84 ± 0.89, 2.18 ± 1.81, 4.92 ± 1.15, 7.53 ± 1.13 and 1.25 ± 1.70 log cfu/g, respectively. Staphylococci, coliform and mould counts were less than 1.00 log cfu/g at the end of ripening, which was at around 6–8 °C for 3 months. According to phenotypic and genotypic identifications, isolates were closely related to Lactobacillus plantarum, Weisella confusa, Weisella paramesenteroides, Pediococcus pentasaceous, Enterococcus casseliflavus, Enterococcus durans and Enterococcus faceium. This study provides baseline data on the microflora of traditional artisanal Sepet cheese, which is a prerequisite for a successful scale up to industrial production.     

Polyphasic characterization of indigenous lactobacilli and lactococci from PDO Canestrato Pugliese cheese

A polyphasic approach, involving both genotypic and phenotypic analyses was used to characterize 33 isolates of lactic acid bacteria from the raw milk Protected Designation of Origin (PDO) Canestrato Pugliese cheese, in order to select candidate strains that can be used as autochthonous starter cultures in the dairy industry. Randomly amplified polymorphic DNA (RAPD) analysis and clustering by the unweighted pair-group method with arithmetic averages (UPGMA) were used to evaluate the genotypic diversity, while phenotypic characterization was performed through miniaturized assays and traditional biochemical tests. Technological properties of major interest for cheese-making (acidification, tendency to lysis, proteolytic and peptidase activity) were also evaluated, and selected subset of data was statistically examined by the one-way ANOVA and the Tukey-Kramer HSD test, to highlight strain-specific and species-specific differences among the isolates. A high degree of diversity appeared in the phenotypic and technological traits in opposition to a relatively low genotypic diversity. Although none of the isolates showed the best performances in all the activities, an appropriate mixture of strains could be selected for providing an efficacious autochthonous starter culture.     

新疆特色干酪中乳酸菌的分离鉴定

从新疆不同牧区采集工艺不同的干酪制品,对其中的乳酸菌进行分离纯化、生理生化性质试验和16S rRNA分析.结果表明,分离、纯化出的104株乳酸菌种,有82株为乳杆菌属(Lactobacillus),12株为肠球菌属(Enterococcus),10株为魏斯氏菌属(Weissella).利用16S rRNA序列同源分析和系统发育树分析对具有不同生理生化特性的代表菌株进行了分子鉴定,鉴定结果为TNM-2与干酪乳杆菌(Lactobacillus casei)、Y5-4与食窦魏斯氏菌(Weissella cibaria)、NS2-2与植物乳杆菌(Lactobacillus plantarum)的同源性达到100％,NM-2与瑞士乳杆菌(Lactobacillus helveticus)、Y1-1与马乳酒乳杆菌(Lactobacillus kefiranofaciens)、WG与耐久肠球菌(Enterococcus durans)的同源性达到99％.     

Identification and first characterization of lactic acid bacteria isolated from the artisanal ovine cheese Pecorino del Poro

A total of 225 lactic acid bacteria, isolated from the artisanal raw ovine milk cheese Pecorino del Poro, were identified and given an initial characterization; the best 18 strains were tested for their antagonistic activity against autochthonous Escherichia coli . An intense antagonistic activity against E. coli growth, induced by strains of Lactobacillus paracasei ssp. paracasei or L. curvatus , was observed; the E. coli inhibition can be ascribed to the production of bacteriocin-like compounds. The lactobacilli seem able to improve the hygienic quality of other artisanal raw milk cheeses.     

Technological characterization of the natural lactic acid bacteria of artisanal Turkish White Pickled cheese

The aim of this study was to characterize the lactic acid bacteria (LAB) isolated from White Pickled cheeses produced with traditional methods; and to improve the quality of cheesemaking with a selection of bacterial cultures from artisanal White cheeses. LAB were isolated and identified from 30 White Pickled cheese samples collected from various cities in Turkey. Also, the numbers of several microbial groups (total aerobic mesophilic bacteria, LAB, enterococci, coliforms, moulds and yeasts) of cheese samples were enumerated. Lactobacilli, lactococci and enterococci were the most abundant microbial groups. The numbers of Enterococcus and Lactobacillus isolates were higher than those of the other LAB. Enterococcus faecalis (24.43%), Enterococcus faecium (17.61%) and Lactobacillus fermentum (19.88%) isolates were the most frequently isolated species. Lactococcus strains showed the highest acidifying activity, followed by Enterococcus and Lactobacillus strains. Proteolytic activity of Enterococcus faecalis strains was higher than that of the other enterococci species, except Enterococcus avium strains. Within lactobacilli strains, the highest mean proteolytic activity was that of Lactobacillus bifermentans, Lactobacillus brevis and Lactobacillus casei strains.     

A polyphasic approach in order to identify dominant lactic acid bacteria during pasta manufacturing

Using a polyphasic approach, we have isolated and identified, lactic acid bacteria (LAB) in samples directly collected from an artisanal pasta-making manufactory located in Puglia (South Italy). Samples were collected during several steps of pasta processing and LAB were isolated on MRS and M17 plates. Furthermore, strains were grouped in a total of eight species by means of randomly amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) typing and 16S rDNA sequencing. The majority of strains were identified as belonging to Pediococcus pentosaceus and Enterococcus faecium species. The remaining strains were characterized and assigned to Weissella confusa, Pediococcus acidilactici, Leuconostoc mesenteroides, Leuconostoc citreum, Lactobacillus fermentum and Lactobacillus plantarum species. Strains were identified from all the analysed samples, and growth of LAB was monitored from extrusion to pre-heating steps.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

The effect of wild lactic acid bacteria on the production of goat’s milk soft cheese

A blend of four wild strains of lactic acid bacteria, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei subsp. paracasei and Enterococcus faecalis, was used as starter to produce a goat’s milk soft cheese. The cheese was analysed microbiologically, physicochemically and organoleptically through a 30‐day period of ripening. Counts of the starter cultures increased in the first 24 h and then remained stable during ripening. Yeasts and coliforms were found at low numbers initially and gradually decreased to undetectable levels. Micrococcal counts were high throughout ripening. The end product was a soft cheese characterised by a mild, aromatic taste as well as a smooth structure.     

Diversity of lactic acid bacteria population in ripened Parmigiano Reggiano cheese

The diversity of dominant lactic acid bacteria population in 12 months ripened Parmigiano Reggiano cheeses was investigated by a polyphasic approach including culture-dependent and independent methods. Traditional plating, isolation of LAB and identification by 16S rDNA analysis showed that strains belonging to Lactobacillus casei group were the most frequently isolated. Lactobacillus helveticus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus parabuchneri, and Lactobacillus buchneri species were detected with lower frequency. PCR-denaturing gradient gel electrophoresis (DGGE) applied to DNA extracted directly from cheese samples and sequencing of rDNA amplicons confirmed the complex microbiological pattern of LAB in ripened Parmigiano Reggiano cheeses, with the significant exception of the Lactobacillus fermentum species, which dominated in several samples, but was not detected by cultivation. The present combination of different approaches can effectively describe the lactic acid bacteria population of Parmigiano Reggiano cheese in advanced stages of ripening, giving useful information for elucidating the role of LAB in determining the final cheese quality.     

Application of indigenous strains of lactic acid bacteria for semi‐industrial production of autochthonous Montenegrin Njeguši cheese

Most of the traditional dairy products of south‐eastern Europe are produced from unpasteurised milk without addition of selected starter cultures. These artisanal products have an interesting biodiversity of indigenous microbiota. In Montenegro, one of the most popular traditional cheeses is Njegu?i cheese, belonging to the group of hard cheeses. Most of the cheeses are produced from ewes' milk, but cows' milk is also widely used. In Montenegro, there are no existing legal regulations defining the specificities of the production or the raw material used for the production of autochthonous food products. Therefore, the producers make the decision about what type of milk to use for cheese production. One of the priorities of the food producers in Montenegro is to obtain a larger scale, standardised production of traditional food products, which are of good quality and considered safe for the consumers. Therefore, in this study, indigenous strains of lactic acid bacteria isolated from various Montenegrin artisanal dairy products were tested as starter cultures for the production of Njegu?i cheese. Three isolates were selected and used as starter cultures in pilot plant experiments for the production of Njegu?i cheese. When applying an adequate combination of added bacterial strains as starters, the flavour of Njegu?i cheese, produced under pilot plant conditions, was comparable to that of the artisanal origin.     

干酪中非发酵剂乳酸菌

非发酵剂乳酸菌是干酪中主要的次生菌群，不属于发酵微生物，通常不能很好的在牛奶中生长，不能产酸，但对干酪的风味形成有很重要作用。本文概述了非发酵剂乳酸菌的一些特征，包括不同原料制成的干酪中非发酵剂乳酸菌的来源、生长能源的利用、自身存在的蛋白分解系统对干酪风味形成影响及作为一种提高干酪品质的附属发酵剂的应用展望。     

Evaluation of isolated starter lactic acid bacteria in Ras cheese ripening and flavour development

Eighteen cultures of starter lactic acid bacteria with or without added adjunct cultures, isolated from Egyptian dairy products, were evaluated in experimental Ras cheese for flavour development. Chemical composition of experimental cheeses was within the legal limit for Ras cheese in Egypt. All cultures used in this study had no effect on chemical composition of Ras cheese. Very significant variations in free amino acids, free fatty acids and sensory evaluation have been found among the cultures used in Ras cheesemaking. The levels of free amino acids and free fatty acids were correlated well with flavour development in Ras cheese. Seven of the tested cultures produced acceptable flavour and texture of Ras cheese. The highest overall score of flavour intensity, flavour and texture acceptability were in cheese made using YY47 lactic culture in addition to adjunct culture of Lactobacillus helveticus, Lactobacillus paracasei subsp. paracasei, Lactobacillus delbrueckii subsp. lactis and Enterococcus faecium. This culture can be recommended for Ras cheese manufacture using pasteurized milk.     

Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese

Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains).     

Molecular and technological characterization of lactic acid bacteria from traditional fermented sausages of Basilicata region (Southern Italy)

Lactic acid bacteria (LAB) from traditional fermented sausages of the Basilicata region were investigated by ARDRA-PCR and RAPD-PCR for taxonomic identification at species and strain level and characterized on the basis of the growth and acidification at different temperatures, incubation times, levels of NaCl and KNO₂, hydrolysis of sarcoplasmatic and myofibrillar proteins and antimicrobial, peptide/amino acid release and nitrate reductase activities.Lactobacillus sakei was the predominant species (67%) followed by Pediococcus pentosaceus (16%), Leuconostoc carnosum (8%), Lactobacillus plantarum (4%), Lactobacillus brevis (2%) and Leuconostoc pseudomesenteroides (2%). The technological characterization revealed that most of the isolates had good acidifying and proteolytic properties. Moreover, Lb. sakei strains showed antimicrobial ability, while Leuconostoc strains the highest reduction of nitrates.This work was a preliminary study in the formulation of autochthonous starter cultures in order to standardize the production process of sausages, to preserve their typical organoleptic and sensory characteristics and to improve the quality of final product.     

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.     

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.     

新疆哈族奶酪中产蛋白酶非发酵剂乳酸菌的筛选及其系统发育分析

以采集10份新疆塔城地区哈萨克族牧民自制的奶酪为原料,用改良的ROGOSA和MRS两种培养基对其分离纯化,共筛选出43株菌种,其中球菌32株,杆菌11株。对筛选出的菌株进行产蛋白酶和菌株自溶能力测定,结合生理生化特性和16S rDNA序列同源分析和建立系统发育树分析,对挑选出的10株优势菌进行了菌株分子生物学鉴定,鉴定结果表明：菌株R6-6自溶度最高,为42.32%;而自溶度最低的菌株为R4-4,达到6.21%;菌株R2-2、R4-2、R4-7为表皮葡萄球菌（Staphylococcus epidermidis）;菌株R3-5、R10-6为乳酸片球菌（Pediococcus acidilactici）;菌株R3-2为戊糖片球菌（Pediococcus pentosaceus）;菌株R6-6为干酪乳杆菌（Lactobacillus casei）;菌株R9-5、R9-6为鼠李糖乳杆菌（Lactobacillus rhamnosus）。其中产蛋白酶的最优势非发酵剂乳酸菌（NSLAB）菌株为乳酸片球菌（P. acidilactici）。     

德宏水牛乳饼中乳酸菌的分离鉴定及发酵性能研究

德宏水牛奶乳饼是云南特有的乳制品,微生物资源丰富,是乳酸菌的重要来源,也是分离筛选优良发酵剂的天然基础。本文采用16S rRNA基因序列分析法和纯培养法对15份水牛奶乳饼中乳酸菌种属进行分离鉴定,通过对菌株产酸、产香、发酵乳活菌计数等比较,以筛选出具有优良发酵特性的乳酸菌。结果表明:15份水牛奶乳饼样品中共分离鉴定出57株乳酸菌（3个属,6个种和1个亚种）,其中Lactobacillus fermentum和Lactobacillus oris为优势菌属,占总菌属的36.84%和24.56%。57株乳酸菌发酵特性比较后得到两株优势菌株MGR3-1和MBR1-1。此研究为后续开发和应用优良发酵剂提供理论基础。      

Esterase activities of indigenous lactic acid bacteria from Argentinean goats’ milk and cheeses

Esterase activities of indigenous lactic acid bacteria isolated from goats’ milk and cheese were investigated. All of the strains exhibited esterase activity on α-naphthyl derivative of fatty acids of 2–6 carbon atoms. Lactobacillus fermentum ETC1 and L. bulgaricus ETC2 showed the highest specific activity on α-naphthyl acetate; L. rhamnosus ETC14 presented the highest specific activity on α-naphthyl butyrate and caproate. All enterococci strains presented the highest specific activities on α-naphthyl propionate, butyrate and caproate, and the lowest specific activities on α-naphthyl acetate. Pedicoccus pentocaseus ETC5 only had esterase activity on α-naphthyl acetate. The electrophoretic zymogram showed for each strain an individual enzyme profile on α-naphthyl acetate and revealed the presence of more than one esterase. L. plantarum ETC11 and Enterococcus faecium ETC9 showed four and five bands of esterase activity, respectively. The strains evaluated in this work showed different esterase activities, which were species and strain specific.     

Microbial diversity of Livanjski cheese with the emphasis on lactic acid bacteria based on culture‐dependent and sequencing method

The microbial diversity of artisanal Livanjski cheese from traditional farms and dairies was analysed. The polyphasic approach included the isolation of microorganisms by using culture‐dependent methods and the identification of viable isolates by sequencing methods. In total, 159 isolates were identified, including five strains of yeast and 15 strains of fungi. Twenty‐two species of homofermentative and heterofermentative lactic acid bacteria were classified. The Shannon–Wiener index of diversity ranged from 0.462 to 2.51 within the cheese samples from the farms and dairies. The hygiene conditions during cheese production and the age of the cheese significantly influenced the counts and diversity of microorganisms.