Influence of ripening container on the lactic acid bacteria population in Tulum cheese

The objective of the present study was to investigate the influence of container material (plastic or goat-skin bag) on the growth of lactic acid bacteria in Tulum cheese during 9 months of ripening. The lactic acid bacteria in Tulum cheeses were periodically counted on MRS and M17 agars throughout ripening. Results showed that the highest counts of lactic acid bacteria on MRS or M17 were observed at the beginning of ripening and their counts decreased during later stages of ripening. The cheese samples ripened in plastic bags exhibited higher numbers of LAB on MRS and M-17 agars than those ripened in goat-skin bags. A total of 112 strains of lactic acid bacteria were isolated from Tulum cheeses ripened in plastic or goat-skin bags during ripening. The lactic acid bacteria present in the cheese were classified by Microbial Identification System (MIS) based on a comparison of the fatty acid methyl ester profiles. Different species including Enteroccocus, Lactobacillus, Streptococcus, Lactococcus and Pediococcus genera were found in unripened cheese. As ripening proceeded, the species Streptococcus and Lactococcus disappeared and the percentages of the species Enterococcus was unchanged in both containers. There were slight differences between the cheeses ripened in plastic or goat-skin bags in terms of the profiles of lactic acid bacteria isolated. Some species including L. brevis, L. mesenteroides subsp. dextranicum, P. damnosus and E. mundtii were isolated only in the cheeses ripened in plastic bags; however, L. coryniformis and L. malafermentans were isolated only in the cheeses ripened in goat-skin bags at 6 or 9 months of ripening. Also the numbers of E. faecalis isolates were higher in the cheeses ripened in plastic containers than cheeses ripened goat-skin bags at the 6 or 9 months of ripening. The results showed that Lactobacillus and Enterococcus were the predominant species in matured Tulum cheeses in both ripening containers. It seemed possible to produce Tulum cheese with similar characteristics from both the containers used.     

Biodiversity of lactic acid bacteria in Romanian dairy products

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS-PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.     

Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio

A group of 85 isolates of haloalkaliphilic obligately chemolithoautotrophic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio were recently obtained from soda lakes in Mongolia, Kenya, California, Egypt and Siberia. They have been analyzed by repetitive extragenic palindromic (rep)-PCR genomic fingerprinting technique with BOX- and (GTG)5-primer set. Cluster analysis was performed using combined fingerprint profiles and a dendrogram similarity value (r) of 0.8 was used to define the same genotype. Fifty-six genotypes were found among the isolates, revealing a high genetic diversity. The strains can be divided into two major clusters, including isolates from the Asiatic (Siberia and Mongolia) and the African (Kenya and Egypt) continents, respectively. The majority (85.9%) of the genotypes were found in only one area, suggesting an endemic character of the Thioalkalivibrio strains. Furthermore, a correlation between fingerprint clustering, geographic origin and the characteristics of the lake of origin was found.     

DNA指纹图谱在乳酸菌分类鉴定中的应用

目的 探讨DNA指纹图谱在乳酸菌分类鉴定中的应用。方法 选用S23随机引物,对乳酸菌基因组DNA进行RAPD随机扩增,获得能够区分不同菌株的DNA指纹图谱,依据图谱DNA条带的多态性,对10株乳酸菌菌株进行分类与鉴定。结果 实验室保藏菌株LAP2、LAT、LAM、LAC和LAO之间的基因组DNA相似性达80%,亲缘关系最为相近,而LAB菌株与所有菌株的亲缘关系最远。结论 DNA指纹图谱技术与常规方法结合使用,将使乳酸菌的分类、鉴定更为准确、便捷。     

Identification of lactic acid bacteria in Moroccan raw milk and traditionally fermented skimmed milk 'lben'

Aims:  To identify lactic acid bacteria (LAB) present in Moroccan dairy products to establish and preserve their microbial species diversity.
Methods and Results:  Thirty-seven samples were collected from different farms. A total of 146 LAB were isolated and subjected to (GTG)₅-PCR analysis. Comparison of the profiles with data available at the Moroccan Coordinated Collections of Micro-organisms allowed identification of 85 isolates. The remaining 61 were subjected to SDS-PAGE analysis of whole cell proteins. Comparison of the profiles with data available at the Belgian Coordinated Collections of Micro-organisms allowed identification of 43 isolates. Several of the remaining 18 isolates exhibited identical protein electrophoretic fingerprints. Therefore, eight representatives of them were subjected to partial pheS gene sequencing which allowed identification of all remaining isolates. In raw milk, six genera were found while in 'lben', three were found. This is the first report of Leuconostoc kimchii in dairy products.
Conclusions:  LAB diversity was established using a stepwise polyphasic identification approach. It used the expertise of both research bodies involved in this study and proved to be cost-effective for the identification of all isolates.
Significance and Impact of the Study:  To establish LAB diversity in Moroccan dairy products which could be a source of strains with specific properties.     

酸马奶中乳酸菌的鉴定及生物学特性的研究

[目的]对从酸马奶中分离出来的10株乳酸菌进行鉴定和生理生化特性研究,为工业生产筛选特性优良的菌种.[方法]通过形态学观察、生理生化特性、分子生物学特性及其对致病菌抑制作用的研究对其进行鉴定,并筛选特性优良菌株.[结果]10株乳酸菌分别为2株Lactobacillus plantarum、2株Enterococcus villorum、2株Enterococcus dispar、3株Enterococcus durans和1株Enterococcus raffinosus;其对Staphylococcus aureus、Escherichia coli和Enteritidis bacillus有不同程度的抑制作用.[结论]菌株HZ24、HZ25具有良好的生物学特性和益生功能,可以应用到食品发酵工业生产中.     

Distribution of antimicrobial‐resistant lactic acid bacteria in natural cheese in Japan

To determine and compare the extent of contamination caused by antimicrobial‐resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P = 0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm‐made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm‐made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to > 512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial‐resistant LAB in imported and Japanese farm‐made cheeses on the Japanese market, but not in Japanese commercial cheeses.     

Progress and potential in the biotechnology of lactic acid bacteria

Abstract: Current activities and future prospects for the biotechnology of lactic acid bacteria are reviewed. Genetic engineering technology, including advances and limitations of plasmid vectors and chromosomal integration strategies are discussed together with the status of gene expression and the importance of in vivo gene transfer systems and transposition. Areas of biotechnological application considered include proteolysis and flavour generation, bacteriophage resistance, antimicrobials, metabolic engineering and the possible uses of lactic acid bacteria in relation to health.     

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.     

Chromosome mapping in lactic acid bacteria

The chromosome structure of lactic acid bacteria has been investigated only recently. The development of pulsed-field gel electrophoresis (PFGE) combined with other DNA-based techniques enables whole-genome analysis of any bacterium, and has allowed rapid progress to be made in the knowledge of the lactic acid bacteria genome. Lactic acid bacteria possess one of the smallest eubacterial chromosomes. Depending on the species, the genome sizes range from 1.1 to 2.6 Mb. Combined physical and genetic maps of several species are already available or close to being achieved. Knowledge of the genomic structure of these organisms will serve as a basis for future genetic studies. Macrorestriction fingerprinting by PFGE is already one of the major tools for strain differentiation, identification of individual strains, and the detection of strain lineages. The genome data resulting from these studies will be of general application strain improvement.     

新疆喀什地区母乳中乳酸菌的多样性分析

【目的】对新疆喀什地区母乳中乳酸菌多样性进行分析。【方法】采用菌落培养、显微镜观察、Repetitive genomic fingerprinting(Rep-PCR)指纹图谱和16S r RNA基因序列分析相结合的方法研究母乳中乳酸菌的菌群分布。【结果】从11份母乳中共分离出乳酸菌193株,利用16S r RNA基因序列同源分析和系统发育树对代表菌株进行了分子鉴定,193株乳酸菌隶属于4个属,分别为Lactobacillus(22株)、Streptococcus(42株)、Lactococcus(40株)、Enterococcus(89株)。其中,Enterococcus所占比例最大,达到46%。【结论】新疆喀什地区母乳中乳酸菌多样性丰富,极具开发潜力,将为开发母乳中的益生菌以及安全可靠的微生态制剂提供理论依据。     

应用特异PCR快速鉴定微生物肥料中4种乳酸菌

【目的】植物乳杆菌(Lactobacillus plantarum)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和德氏乳杆菌(L.delbrueckii)是微生物肥料生产中常用的乳酸菌,它们表型特征相似,若采用传统方法鉴定则费时费力,为准确、快速地鉴定这些种,建立种特异PCR方法。【方法】利用NCBI中Primer-BLAST(引物设计和特异性检验工具),以GenBank数据库中上述菌种的recA和gyrB为靶基因,设计和筛选种特异性引物从而建立相应特异PCR鉴定方法。【结果】经过乳杆菌属(Lactobacillus)、乳球菌属(Lactococcus)、片球菌属(Pediococcus)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、短芽孢杆菌属(Brevibacillus)和假单胞菌属(Pseudomonas)7个属24个种共40株标准菌株的实验验证,4个目标种分别扩增出唯一的目的产物,而其他种均无目的扩增产物。采用建立的4种特异PCR方法对产品中分离的16株乳杆菌进行鉴定,结果与16S rDNA序列分析、Biolog鉴定结果一致。【结论】建立的特异PCR鉴定方法均具有较高的种内通用性和种间特异性,可快速、准确的用于微生物制剂中植物乳杆菌、德氏乳杆菌、鼠李糖乳杆菌、嗜酸乳杆菌的检测和鉴定,具有较好的应用前景。     

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.     

乳酸菌低温菌株的复合诱变选育

【目的】采用复合诱导的方法,选育出能在低温条件下性状稳定、生长良好且具有工业生产价值的低温乳酸菌菌株。【方法】使用紫外照射和亚硝基胍处理的方法对分离的Q1菌株进行诱变处理,将选育得到的低温菌株Q1-4-6与出发菌株Q1在15°C、20°C、37°C下,比较其生长速率、产酸量和糖降解量。【结果】从玛曲牧民自制酸奶中分离得到一株乳酸菌Q1,根据其形态学特征、生理生化特性,同时结合16SrDNA序列分析结果,将其鉴定为肠球菌属棉籽糖肠球菌(Enterococcus raffinosus)。经过多轮诱变选育出一株能在低温下良好生长、遗传性状稳定的菌株Q1-4-6,将出发菌株与诱变菌株在15°C、20°C比较其生长、产酸和糖降解量发现,诱变菌株均好于出发菌株。在37°C的自然条件下,诱变菌株和出发菌株的生长速率差异不大,但均略高于出发菌株。在15°C培养发现,诱变菌株生长速率、产酸量均高于购买菌株干酪乳杆菌(CL04)和嗜热链球菌(CL05)。【结论】通过UV+NTG复合诱变,最终选育出一株在低温下生长良好、遗传性状稳定的菌株Q1-4-6。     

鄂尔多斯地区自然发酵苦菜中优良特性乳酸菌的筛选

【背景】鄂尔多斯地区传统自然发酵苦菜风味和作用独特,但目前尚无关于发酵苦菜中乳酸菌种类及其生物学特性研究的报道。【目的】获得适用于工业化生产特性优良的乳酸菌。【方法】通过测定总酸度筛选高产酸菌株,并进行形态学鉴定、生理生化特性研究,以及16S rRNA基因片段分析。【结果】经鉴定5株高产酸的菌株均为植物乳杆菌,生长温度范围宽,并且具有耐酸、耐碱、耐盐、耐热的特性。【结论】试验菌株具有优良的生理特性和潜在的益生特性,可用于食品工业的生产。     

乳酸菌对高脂小鼠降胆固醇作用的研究

目的探讨乳酸菌对高脂小鼠降胆固醇的作用。方法对昆明小鼠饲喂高脂饲料14 d建立高脂小鼠模型,确定模型建立成功后用1株经体外实验证实具有降胆固醇效果的乳酸菌对小鼠灌胃,测定小鼠经灌胃14、28 d的脏器指数、血清胆固醇含量和肝脏胆固醇含量。结果灌胃组小鼠脏器指数明显低于高脂组小鼠,且对于血清和肝脏胆固醇含量具有明显的降低作用。结论该菌株具有降低血清胆固醇,抑制肝脏胆固醇堆积的功能,为将来利用该菌株制作出降胆固醇功能的食品或药品提供实验基础。     

Detection of polygalacturonases and pectin esterases in lactic acid bacteria

Of 80 strains of lactic acid bacteria tested, only Lactobacillus casei strains HNK10 and L1–8, Lactobacillus plantarum Lc5 and Lactococcus lactis NN01 produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.1.). Crude extracellular extracts of strain L1–8 were able to clarify pectin.     

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.