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.     

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.     

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.     

Molecular Fingerprinting of the Fecal Microbiota of Children Raised According to Different Lifestyles

In this population-based study, 90 children from three European countries were examined to determine the impact of lifestyle on the fecal microbiota. The study was designed to assess the impact of two extreme lifestyles that we hypothesized could impact the microbial composition in the gut: i.e., an anthroposophic lifestyle (restricted use of antibiotics, greater consumption of fermented vegetables, etc.) versus living on a farm (greater consumption of farm milk, contact with animals, etc.). In previous studies, these lifestyles correlated with lower prevalence of allergies. Terminal restriction fragment length polymorphism (T-RFLP) was used to assess the bacterial composition in fecal samples since recent studies have shown that the majority of this community cannot be cultivated. The T-RFLP data were used to calculate richness and evenness of the fecal microbiota. Children that were attending Steiner schools (anthroposophic children) had a significantly higher diversity of microbes in their feces than farm children, who in turn also had lower diversity than the control groups. Specific primers were also used to focus on the Lactobacillus-like community (lactic acid bacteria [LAB]). Large differences were found in the LAB subpopulations in the sampled groups. In some children, the LAB subpopulation was dominated by a species that has not yet been cultivated.     

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。     

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.     

新疆伊犁地区原牛乳中乳酸菌的多样性分析

【目的】对新疆伊犁地区原牛奶中乳酸细菌的遗传多样性进行分析。【方法】采用菌落培养、Rep-PCR(Repetitive genomic fingerprinting)指纹图谱和16S r RNA基因序列分析相结合的方法研究牛乳内乳酸菌的遗传多样性。【结果】从5份原牛乳中分离出乳酸菌29株,基因序列分析和系统进化分析显示29株乳酸菌隶属于5个属,分别为:Lactococcus、Lactobacillus、Leuconostoc、Pediococcus和Enterococcus。优势属为Leuconostoc(27.6%),其次为Lactococcus(24.0%)。【结论】新疆伊犁地区原牛乳中乳酸菌多样性丰富,为开发新疆地区益生乳酸菌提供了丰富的活性资源。     

Phenotypic diversity of lactic acid bacteria isolated from fermented sausages produced in Basilicata (Southern Italy)

AIMS: to evaluate the evolution of lactic acid bacteria (LAB) populations in traditional fermented sausages (salsiccia and soppressata) produced in artisanal and industrial plants in Basilicata (Southern Italy). METHODS AND RESULTS: Four hundred and fourteen lactic acid bacteria (LAB) cultures were isolated from samples of sausages at different stages of ripening. A phenotypic characterization of the isolates was carried out using a set of 28 tests, and 34 clusters were identified at the 80% similarity level using hierarchical cluster analysis. Of the isolates 50% were identified as Lactobacillus sakei (with several biotypes), 22% as Pediococcus spp. (mainly Ped. pentosaceus), 7% as Leuconostoc (Leuc. carnosum, Leuc. gelidum, Leuc. pseudomesenteroides), 6% as Lact. plantarum, 1% as Lact. curvatus. Other lactobacilli, including unidentified species, were present in lower numbers. CONCLUSION: The phenotypic diversity and composition of the LAB flora varied as a function of the production plant, product type and ripening time. SIGNIFICANCE AND IMPACT OT THE STUDY: A new procedure based on bootstrapping and Multidimensional Scaling was successfully used to obtain a graphical representation of the evolution of the LAB populations.     

Inventory of non starter lactic acid bacteria from ripened Parmigiano Reggiano cheese as assessed by a culture dependent multiphasic approach

The objective of this study was to investigate microbial species diversity and strain complexity of the cultivable non starter lactic acid bacteria (NSLAB) occurring in 31 ripened Parmigiano Reggiano (PR) cheeses. Dereplication of 127 lactobacilli isolates by (GTG)(5)-PCR fingerprinting yielded a total of 51 genotypes. Phylogenetic relatedness of all the genotypes with known Lactobacillus species was determined by a novel combined amplified 16S rDNA restriction analysis (16S-ARDRA), species-specific PCR assays and 16S rRNA gene sequencing. The species Lactobacillus rhamnosus and Lactobacillus paracasei comprise the largest portions of the cultivable NSLAB community in PR cheese, with an inter-individual diversity ranging from one to four dominant genotypes per sample. Lactobacillus casei, Lactobacillus harbinensis and Lactobacillus fermentum species were also detected at low frequency. The data showed differences in cultivable NSLAB population, with an overall decrease in diversity and complexity from early to advanced stages of ripening. Finally the de-replicated collection of genotypes resulting from this work is the bases for further functional screening.     

Influence of geographical origin and flour type on diversity of lactic acid bacteria in traditional Belgian sourdoughs

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.     

Cow teat skin, a potential source of diverse microbial populations for cheese production

The diversity of the microbial community on cow teat skin was evaluated using a culture-dependent method based on the use of different dairy-specific media, followed by the identification of isolates by 16S rRNA gene sequencing. This was combined with a direct molecular approach by cloning and 16S rRNA gene sequencing. This study highlighted the large diversity of the bacterial community that may be found on teat skin, where 79.8% of clones corresponded to various unidentified species as well as 66 identified species, mainly belonging to those commonly found in raw milk (Enterococcus, Pediococcus, Enterobacter, Pantoea, Aerococcus, and Staphylococcus). Several of them, such as nonstarter lactic acid bacteria (NSLAB), Staphylococcus, and Actinobacteria, may contribute to the development of the sensory characteristics of cheese during ripening. Therefore, teat skin could be an interesting source or vector of biodiversity for milk. Variations of microbial counts and diversity between the farms studied have been observed. Moreover, Staphylococcus auricularis, Staphylococcus devriesei, Staphylococcus arlettae, Streptococcus bovis, Streptococcus equinus, Clavibacter michiganensis, Coprococcus catus, or Arthrobacter gandavensis commensal bacteria of teat skin and teat canal, as well as human skin, are not common in milk, suggesting that there is a breakdown of microbial flow from animal to milk. It would then be interesting to thoroughly study this microbial flow from teat to milk.     

青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性及低温发酵菌株的筛选

【目的】探究青藏高原垂穗披碱草青贮饲料中乳酸菌的多样性,筛选在低温条件下(10、15和25°C)生长性能较好的优良菌株。【方法】将垂穗披碱草青贮饲料中分离纯化的乳酸菌进行形态特征观察及16S r RNA基因测序鉴定;用MRS液体培养基在10、15和25°C条件下分离、初筛乳酸菌,选取高吸光度值的菌株作为优势菌株。用绿汁发酵液在10、15和25°C条件下培养测定其p H值,选取低p H值菌株作为优势菌株,并综合MRS培养基筛选结果确定优良菌株。【结果】从不同温度和发酵阶段的垂穗披碱草青贮饲料中共分离得到108个乳酸菌菌株,它们分属于6个属、18个种。其中,清酒乳杆菌LS-24在15°C条件下发酵液p H值显著降低(P0.05),戊糖片球菌PP-63在发酵初期生长速度较快,植物乳杆菌LP-21在15°C条件下发酵液p H值降至3.9且有最大活菌数。【结论】在青藏高原垂穗披碱草青贮饲料中发现的乳酸菌属基本涵盖了前人在常温青贮饲料中发现的所有属,但种数略少;在108株菌中,清酒乳杆菌LS-24、戊糖片球菌PP-63和植物乳杆菌LP-21在低温条件下均表现出较好的繁殖和发酵特性,可作为青贮饲料低温发酵的备选菌株。     

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.     

Inhibition of psychrotrophic bacteria in raw milk by immobilized lactic acid bacteria

Summary Cells ofLactococcus lactis orLactobacillus helveticus were immobilized in calcium-alginate beads, added to raw milk, and incubated 48 h at 7°C. The addition of 2.7×10⁷ immobilizedLc.lactis or 13×10⁷ immobilizedLb. helveticus cells per mL reduced the development of the psychrotrophic bacteria of raw milk by approximately 50%. The pH of the raw milk dropped 0.10 to 0.22 units under these conditions. Periodic agitation of the seeded raw milk increased the inhibitory activity of the immobilized lactic acid bacteria (LAB). Free LAB cells in the system were only of 0.5% of total LAB. The use of immobilized LAB to inhibit psychrotrophic bacteria might be extended to raw milks destined to the manufacture of non-fermented dairy products.     

Influence of Geographical Origin and Flour Type on Diversity of Lactic Acid Bacteria in Traditional Belgian Sourdoughs

A culture-based approach was used to investigate the diversity of lactic acid bacteria (LAB) in Belgian traditional sourdoughs and to assess the influence of flour type, bakery environment, geographical origin, and technological characteristics on the taxonomic composition of these LAB communities. For this purpose, a total of 714 LAB from 21 sourdoughs sampled at 11 artisan bakeries throughout Belgium were subjected to a polyphasic identification approach. The microbial composition of the traditional sourdoughs was characterized by bacteriological culture in combination with genotypic identification methods, including repetitive element sequence-based PCR fingerprinting and phenylalanyl-tRNA synthase (pheS) gene sequence analysis. LAB from Belgian sourdoughs belonged to the genera Lactobacillus, Pediococcus, Leuconostoc, Weissella, and Enterococcus, with the heterofermentative species Lactobacillus paralimentarius, Lactobacillus sanfranciscensis, Lactobacillus plantarum, and Lactobacillus pontis as the most frequently isolated taxa. Statistical analysis of the identification data indicated that the microbial composition of the sourdoughs is mainly affected by the bakery environment rather than the flour type (wheat, rye, spelt, or a mixture of these) used. In conclusion, the polyphasic approach, based on rapid genotypic screening and high-resolution, sequence-dependent identification, proved to be a powerful tool for studying the LAB diversity in traditional fermented foods such as sourdough.     

Isolation and identification of cultivable lactic acid bacteria in traditional yak milk products of Gansu Province in China

Various traditional fermented yak milk and raw milk foods could be considered as an abundant resource for obtaining novel lactic acid bacteria (LAB) with unique properties. Eighty-eight samples of yak milk products were collected from Gansu Province in China. Three hundred and nineteen strains of LAB isolated from these samples were identified by phenotypic methods, 16S rRNA gene sequence analysis and PCR-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technology. Among the isolates, one hundred and sixty-four isolates (51.41% of the total) were classified under Lactobacilli, and one hundred and fifty-five (48.59%) belonged to cocci. All the isolates were classified to six genera (Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus and Weissella) and twenty-one species. Lactobacillus helveticus (87 strains), Leuconostoc mesenteroides subsp. mesenteroides (49 strains), Streptococcus thermophilus (39 strains), Lactobacillus casei (31 strains) and Lactococcus lactis subsp. lactis (19 strains) were considered as the predominant populations in the yak milk products. The results showed that there were abundant genus and species LAB existing in yak milk products in Gansu Province in China. The obtained LAB pure cultures may be a valuable source for further starter selection.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

ICR小鼠肠道中乳酸菌组成的克隆文库研究

目的对健康雄性ICR小鼠肠道内乳酸菌的组成结构进行研究。方法收集10只健康雄性ICR小鼠的新鲜粪便样品,提取粪便样品中微生物的总DNA,采用乳酸菌类群特异性引物（Lac1）和细菌通用性引物（1391r）的组合扩增16SrRNA基因并构建乳酸菌特异性克隆文库,研究小鼠肠道内各种乳酸菌的组成和比例。结果克隆文库的分析结果表明罗伊乳杆菌（Lactobacillus reuteri）和约氏乳杆菌（Lactobacillus johnsonii）为ICR小鼠肠道内的优势种,其他还包括鼠乳杆菌（Lactobacillus murinus）、阴道乳杆菌（Lactobacillus vaginalis）、肠乳杆菌（Lacto-bacillus intestinalis）等乳酸菌以及一个潜在的乳酸菌新种。结论健康ICR小鼠肠道内乳酸菌的多样性较高;L.re-uteri种可能具有较高的菌株水平多样性。     

The effect of Lactobacillus buchneri on the fermentation, aerobic stability and ruminal degradability of maize silage

AIMS: To evaluate the effect of Lactobacillus buchneri, heterofermentative lactic acid bacteria (LAB), on the fermentation, aerobic stability and ruminal degradability of whole-crop maize silages under laboratory conditions. Two homofermentative LAB were tested for the purpose of comparison. METHODS AND RESULTS: Maize was harvested at early dent [290 g kg(-1) dry matter (DM)] and one-half milk line (355 g kg(-1) DM) stages. Both homofermentative LAB were applied at 1 x 10(5) CFU g(-1) of fresh forage. Lactobacillus buchneri was applied at 1 x 10(5), 5 x 10(5) and 1 x 10(6) CFU g(-1) of fresh forage. Silages with no additives served as control. After treatment, the chopped forages were ensiled in 1.5-l anaerobic jars. Three jars per treatment were sampled on day 60. After 60 days of storage, silages were subjected to an aerobic stability test lasting for 5 days, in which CO(2) production, as well as chemical and microbiological parameters, was measured to determine the extent of aerobic deterioration. Both homofermentative LAB increased the concentration of lactic acid and the numbers of yeasts, and decreased the concentration of acetic acid and impaired the aerobic stability of silages. In contrast, applying L. buchneri decreased the concentration of lactic acid and increased the concentration of acetic acid of the silages. Under aerobic conditions, silages treated with 5 x 10(5) and 1 x 10(6) CFU g(-1) of L. buchneri, had lower pH, CO(2) production and the numbers of yeasts than the silages treated with 1 x 10(5) CFU g(-1) of L. buchneri (P < 0.05). However, all doses of L. buchneri and both homofermentative LAB did not affect in situ rumen DM, organic matter and neutral detergent fibre degradability of the silages. CONCLUSIONS: Lactobacillus buchneri was very effective in protecting maize silages exposed to air under laboratory conditions. All doses of L. buchneri, especially 5 x 10(5) CFU g(-1) or more, markedly decreased the numbers of yeasts and improved the aerobic stability of silages. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of L. buchneri, as a silage inoculant, can improve the aerobic stability of maize silages by inhibition of yeast activity.     

A gene network engineering platform for lactic acid bacteria

Recent developments in synthetic biology have positioned lactic acid bacteria (LAB) as a major class of cellular chassis for applications. To achieve the full potential of LAB, one fundamental prerequisite is the capacity for rapid engineering of complex gene networks, such as natural biosynthetic pathways and multicomponent synthetic circuits, into which cellular functions are encoded. Here, we present a synthetic biology platform for rapid construction and optimization of large-scale gene networks in LAB. The platform involves a copy-controlled shuttle for hosting target networks and two associated strategies that enable efficient genetic editing and phenotypic validation. By using a nisin biosynthesis pathway and its variants as examples, we demonstrated multiplex, continuous editing of small DNA parts, such as ribosome-binding sites, as well as efficient manipulation of large building blocks such as genes and operons. To showcase the platform, we applied it to expand the phenotypic diversity of the nisin pathway by quickly generating a library of 63 pathway variants. We further demonstrated its utility by altering the regulatory topology of the nisin pathway for constitutive bacteriocin biosynthesis. This work demonstrates the feasibility of rapid and advanced engineering of gene networks in LAB, fostering their applications in biomedicine and other areas.