Microbial community dynamics during the Scamorza Altamurana cheese natural fermentation

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.     

Application of Different Molecular Techniques for Characterization of Catalase‐Positive Cocci Isolated from Sucuk

This study was carried out for the characterization and discrimination of the indigenous Gram positive, catalase‐positive cocci (GCC) population in sucuk, a traditional Turkish dry‐fermented sausage. Sucuk samples, produced by the traditional method without starter culture were collected from 8 local producers in Kayseri/Turkey and a total of 116 GCC isolates were identified by using different molecular techniques. Two different molecular fingerprinting methods; namely, randomly amplified polymorphic DNA‐PCR (RAPD‐PCR) and repetitive extragenic palindrome‐PCR (rep‐PCR), were used for the clustering of isolates and identification at species level was carried out by full length sequencing of 16S rDNA. Combining the results obtained from molecular fingerprinting and 16S rDNA sequencing showed that the dominant GCC species isolated from the sucuk samples was Staphylococcus saprophyticus followed by Staphylococcus succinus and Staphylococcus equorum belonging to the Staphylococcus genus. Real‐time PCR DNA melting curve analysis and high‐resolution melting (HRM) analysis targeting the V1 + V3 regions of 16S rDNA were also applied for the discrimination of isolates belonging to different species. It was observed statistically different Tm values and species‐specific HRM profiles for all except 2 species (S. saprophyticus and Staphylococcus xylosus) that have high 16S rDNA sequence similarity. The combination of rep‐PCR and/or PCR‐RAPD with 16S rRNA gene sequencing was an efficient approach for the characterization and identification of the GCC population in spontaneously fermented sucuk. On the other hand, intercalating dye assays were found to be a simple and very promising technique for the differentiation of the GCC population at species level.     

Lactobacillus casei, dominant species in naturally fermented Sicilian green olives

This study investigated the phenotypic and genotypic characteristics of lactic acid bacteria in naturally fermented green olives, collected from different areas of Sicily. Both classical biochemical tests and PCR/Restriction Fragments Length Polymorphism (RFLP) of 16S rDNA were used to characterize the isolates. The identity of the isolates was obtained by the partial sequencing analysis of the 16S rDNA. The BioMerieux software assigned the 13 heterofermentative strains to the Lactobacillus brevis species; 24 homofermentative strains were classified as Lactobacillus casei and the remaining 11 homofermentative lactobacilli were identified as Lactobacillus plantarum. The rapid ID 32 STREP test identified coccal-shaped strains as Enterococcus faecium species. The PCR/RFLP analysis showed a remarkable bacterial heterogeneity within the isolates. The 16S rDNA partial sequencing did not confirm biochemical identification, revealing a strong dominance of isolates belonging to the L. casei species. It is noteworthy that this species has never been reported as dominant species in fermented vegetables.A combination of molecular and biochemical analysis allowed the identification of species involved in natural food fermentations.     

信阳黑猪腊肉的微生物多样性分析及优势菌分离鉴定

为全面解析信阳光山自然发酵黑猪腊肉中的微生物菌落结构组成,采用Illumina MiSeq高通量测序和传统选择性培养基分离筛选相结合的方法,分析腊肉中的细菌多样性和真菌多样性并对分离优势细菌和真菌进行16SrDNA和26S rDNA鉴定。结果显示,信阳腊肉优势细菌中厚壁菌门( Firmicutes) 和变形菌门( Proteobacteria)相对丰度占比均值之和达到99.67%,为绝对优势细菌门;优势真菌为酵母,其菌落总数为4.65 lg(cfu/g)低于细菌2个数量级。在属水平上,相对丰度占比前5的优势细菌依次为乳球菌属(Lactococcus)、嗜冷杆菌属(Psychrobacter)、乳杆菌属(Lactobacillus)、肉食杆菌属(Carnobacterium)、葡萄球菌属(Staphylococcus)。并从腊肉中分离鉴定得到6株优势细菌和2株优势酵母分别为格式乳球菌,清酒乳杆菌,马胃葡萄球菌,广布肉毒杆菌,溶酪巨球菌,孤独四联球菌,胶红酵母和隐球酵母各1株,从而为后续进一步研究优势菌种在发酵过程中的具体作用和演替变化提供了可能,并为发酵肉行业的标准化生产提供理论依据和技术支撑。     

接种乳酸菌发酵剂对风干肠成熟过程中微生物群落动态变化及感官品质的影响

为研究乳酸菌发酵剂对风干肠成熟过程中微生物数量、微生物多样性及感官品质的影响,分别将SHI-59(木糖葡萄球菌+戊糖片球菌+植物乳杆菌)、WBL-45(木糖葡萄球菌+肉葡萄球菌+清酒乳杆菌)、PRO-MIX5(木糖葡萄球菌+清酒乳杆菌+类植物乳杆菌)复合型商业发酵剂接种到肉馅中,经过12?d的成熟过程生产发酵型风干肠,...     

米发糕储藏过程中微生物分析

米发糕是中国传统发酵食品,在我国尤其是南方地区颇受消费者欢迎。本实验利用16S rDNA 基因序列的PCR 扩增技术对米发糕在室温储藏过程分离出的菌株进行鉴定。结果表明：细菌是致使米发糕迅速腐败的主要因素,分离出的3 株菌经鉴定分别为发酵乳杆菌、巴氏葡萄球菌和蜡样芽胞杆菌。对米发糕腐败菌的菌相变化进行分析,确定乳杆菌和芽孢杆菌是米发糕储藏过程中的优势腐败菌,且都随着时间的延长呈明显上升趋势。     

Diversity of lactic acid bacteria in fermented brines used to make stinky tofu

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.     

传统发酵香肠中菌种的分子生物学鉴定

为探究发酵香肠的微生物菌群的作用机理,利用传统的微生物学平板稀释分离法,分离出发酵香肠中酵母、大肠杆菌和乳酸菌等微生物,并提取其DNA,PCR扩增16S rDNA、ITS后测序,将测序结果与NCBI基因库中序列进行比对,MegAlign软件进行序列分析并构建系统发育树,结果表明,发酵香肠中富含德氏酵母、YSY1-6腐生葡萄球菌、德式乳酸乳杆菌亚种。     

腌腊肉制品中乳酸菌的筛选鉴定及其在腊肠中的应用

为筛选适合传统腌腊肉制品的优良乳酸菌菌株,从多种农家自制传统腌腊肉制品中分离纯化出9株优势乳酸菌。通过发酵特性筛选,得到一株性状优良菌株10M-7,并制备该菌株的干粉发酵剂,以未接种发酵剂腊肠为对照,分析此发酵剂对腊肠感官品质和微生物变化的影响。结果表明,10M-7菌株具有良好的产酸特性和抑菌性能。根据形态学、生理生化特征和16S rRNA序列分析,鉴定其为植物乳杆菌,采用冷冻干燥法制备纯种发酵剂,并制作人工发酵腊肠。发酵剂组pH值在初期便迅速下降,且始终低于对照组;发酵剂组乳酸菌迅速生长繁殖,且葡萄球菌和大肠杆菌数量与对照组相比明显降低。感官评价表明,当添加量为10~4CFU/g原料肉时,能够很好地保持和改善产品风味,使产品整体感觉更好。     

鲊肉粉中乳酸菌和葡萄球菌的筛选及鉴定

从传统发酵鲊肉粉中分离、筛选发酵性能优良的乳酸菌和葡萄球菌,为鲊肉粉接种发酵提供理论依据。按照肉制品发酵菌株的筛选标准,利用形态学特征和16S rDNA序列分析鉴定菌株,筛选出2株乳酸菌A1、C7和2株葡萄球菌S6、S7。结果表明:乳酸菌菌株A1、C7均为植物乳杆菌(Lactobacillus plantarum),能快速产酸,具有较好抑菌特性;葡萄球菌菌株S6为沃氏葡萄球菌(Staphylococcus warneri),S7为巴氏葡萄球菌(Staphylococcus pasteuri),能产蛋白酶和脂肪酶,具有良好的耐盐性和耐亚硝酸盐性,对不同抗生素有不同的敏感性。筛选得到的4株菌株具有良好的发酵特性,乳酸菌和葡萄球菌之间无拮抗作用,复配后用可于鲊肉粉接种发酵。     

葡萄球菌和乳酸菌对广式腊肠风味的影响

本文研究了葡萄球菌、乳酸杆菌单独发酵和混合发酵对广式腊肠生产过程中风味变化的影响,并考察了不同发酵时间对成品腊肠风味及相关理化指标的影响,旨在研究以微生物发酵技术提高广式腊肠的风味。结果显示,葡萄球菌(M1、M14)和乳酸杆菌(PED、STR)单独或混合发酵均能在一定程度上提高广式腊肠的腊香味,混合菌种比单一菌种发酵效果好。随着生产时间的推移腊肠香味逐渐显现,烘烤至成品后腊肠风味的感官分值最高。腊肠生产过程的pH值随菌种或配比的不同而有不同的变化,pH过低对腊肠风味有不利的影响。添加菌种处理组在不同的发酵天数所对应的广式腊肠成品的挥发性盐基氮、氨基态氮、酸价都比空白对照组的高,而过氧化值比空白对照组的低,证明所选用菌种的发酵对腊肠的蛋白质和脂肪的水解有一定的促进作用。研究结果表明当菌种的混合配比为葡萄球菌(M1∶M14=1∶1):乳酸杆菌(PED:STR=1∶1)=1∶100时,发酵2d后广式腊肠的风味最好。     

Species-specific identification of commercial probiotic strains

Products containing probiotic bacteria are gaining popularity, increasing the importance of their accurate speciation. Unfortunately, studies have suggested that improper labeling of probiotic species is common in commercial products. Species identification of a bank of commercial probiotic strains was attempted using partial 16S rDNA sequencing, carbohydrate fermentation analysis, and cellular fatty acid methyl ester analysis. Results from partial 16S rDNA sequencing indicated discrepancies between species designations for 26 out of 58 strains tested, including two ATCC Lactobacillus strains. When considering only the commercial strains obtained directly from the manufacturers, 14 of 29 strains carried species designations different from those obtained by partial 16S rDNA sequencing. Strains from six commercial products were species not listed on the label. The discrepancies mainly occurred in Lactobacillus acidophilus and Lactobacillus casei groups. Carbohydrate fermentation analysis was not sensitive enough to identify species within the L. acidophilus group. Fatty acid methyl ester analysis was found to be variable and inaccurate and is not recommended to identify probiotic lactobacilli.     

盐浓度对自制香肠理化因子及原核微生物群落多样性的影响

为研究盐浓度对香肠发酵的影响，采用16S rRNA测序和理化分析技术，对不同盐度下发酵的自制香肠中微生物多样性和物理化学特性进行研究。结果表明，盐浓度可以显著影响pH、水分含量及过氧化值，并且也会显著影响微生物的多样性和组成。乳杆菌属（Lactobacillus）在香肠发酵中占优势，其相对丰度在30.29%～58.15%范围之间，明串珠菌属（Leuconostoc）、弧菌属（Vibrio）、不动杆菌属（Acinetobacter）、魏斯氏菌属（Weissella）、盐单胞菌属（Halomonas）、片球菌属（Pediococcus）、盐弧菌属（Salinivibrio）和四联球菌属（Tetragenococcus）受盐度的影响显著。冗余分析（RDA）结果表明，盐度对菌群的影响最大。综上所述，添加4%食盐的香肠具有更高的乳酸菌相对丰度（58.15%）。该结果有助于了解加盐对香肠发酵微生态的影响，为香肠的质量控制提供依据。     

16S rDNA-RFLP技术鉴定西藏地区乳制品中的乳杆菌

将16S rDNA PCR技术和RFLP技术相结合,对分离自乳制品中的乳杆菌进行分类和鉴定.从我国西藏地区传统发酵乳中分离出51株乳杆菌,采用通用引物扩增16S rDNA,利用限制性内切酶AluI,HaeIII和HinfI将16S rDNA扩增产物进行酶切后,经聚丙烯酰胺凝胶电泳图谱将菌种鉴定到种的水平.根据16S rDNA-RFLP的结果从中选出8株有代表性的菌株进行生理生化实验和16S rDNA序列的测定,实验结果与RFLP鉴定结果相吻合,表明此方法是一种快速、准确的可用于大量乳杆菌分类和鉴定的方法.     

一株来自河南的雪莲菌TK3优势菌相分析

对一株来自河南省的雪莲菌TK3进行初步菌相分析,扫描电镜结果表明：TK3是由乳酸菌及酵母菌构成的混菌共生体系,菌体附着在基质上,形成紧密不易分离的颗粒。对其菌相进行分离纯化得到6种优势菌,经镜检分析、生理生化分析及16S rDNA/26S rDNA鉴定,鉴定结果分别为：乳明串珠菌(Leuconostoc lactis)、乳酸乳球菌(Lactococcus lactis)、植物乳杆菌(Lactobacillus plantarum)、单孢酿酒酵母(Kazachstania unispora)、马克斯克鲁维酵母(Kluyveromyces marxianus)以及exigua酿酒酵母(Kazachstania exigua),在发酵乳中存在的数量级分别为107、107、107、106、106、107CFU/mL,并且在4℃存放7d后各个菌群数量仍保持稳定。     

Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk

In this study, the culture-dependent and culture-independent molecular methods were used for the identification of lactic acid bacteria (LAB) in sucuk a Turkish fermented dry sausage. On the one hand, the PCR-DGGE method targetting the V1 and V3 regions of 16S DNA was applied to DNA that was directly extracted from sucuk samples. On the other hand, rep-PCR fingerprinting was performed for the primary differentiation and grouping of the isolates, and the results were confirmed by sequencing of the 16S rDNA and 16S-23S rDNA intergenic spacer region. As a result of the PCR-DGGE analysis of all the samples, total 8 different lactic acid bacteria were identified, and Lactobacillus sakei, Lactobacillus curvatus and Weissella viridescens were the dominant microbiota among these bacteria. The culture-dependent approach indicated that the majority of the strains belonged to the Lactobacillus genera including Lb. sakei, Lactobacillus plantarum, Lb. curvatus, Lactobacillus brevis, Lactobacillus farciminis and Lactobacillus alimentarius. However, Leuconostoc and Weisella were also detected as minor genera. Again, Lactococcus piscium, Weissella halotolerans, Staphylococcus succinus and the comigrated Staphylococcus piscifermentans/Staphylococcus condimenti/Staphylococcus carnosus group were detected only with the culture-independent method while Lb. plantarum, Leuconostoc mesenteroides and Leuconostoc citreum were identified only by using the culture-dependent method. In the results, it was concluded that the combination of culture-dependent and culture-independent methods was necessary for reliable and detailed investigation of LAB communities in fermented food products.     

不同发酵肉中优良葡萄球菌的筛选鉴定

为获得适用于发酵香肠的优势葡萄球菌,本研究从国内外6种具有代表性的不同来源的发酵肉样品中分离到120株葡萄球菌.按照肉用发酵剂的基本原则进行初筛后共得到10株性能优良菌株.对这些菌株的蛋白酶、脂肪酶、硝酸还原酶和过氧化氢酶活性以及其在高盐和亚硝酸盐环境中的生长情况进行测定.发现菌株RG18和SL4有较高的蛋白酶、脂肪酶...     

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.     

Monitoring the lactic acid bacterial diversity during shochu fermentation by PCR-denaturing gradient gel electrophoresis

The presence of lactic acid bacteria (LAB) during shochu fermentation was monitored by PCR-denaturing gradient gel electrophoresis (DGGE) and by bacteriological culturing. No LAB were detected from fermented mashes by PCR-DGGE using a universal bacterial PCR primer set. However, PCR-DGGE using a new primer specific for the 16S rDNA of Lactococcus, Streptococcus, Tetragenococcus, Enterococcus, and Vagococcus and two primers specific for the 16S rDNA of Lactobacillus, Pediococcus, Leuconostoc, and Weissella revealed that Enterococcus faecium, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus nagelii, Lactobacillus plantarum, Lactococcus lactis, Leuconostoc citreum, Leuconostoc mesenteroides, and Weissella cibaria inhabited in shochu mashes. It was also found that the LAB community composition during shochu fermentation changed after the main ingredient and water were added during the fermentation process. Therefore, we confirmed that PCR-DGGE using all three primers specific for groups of LAB together was well suited to the study of the LAB diversity in shochu mashes. The results of DGGE profiles were similar to the results of bacteriological culturing. In conclusion, LAB are present during shochu fermentation but not dominant.