抑黄曲霉乳酸菌的筛选及菌种鉴定

目的筛选具有抑制黄曲霉(Aspergillus flavus)生长的乳酸菌。方法以各地泡菜、实验室自制泡菜、豆浆渣以及新鲜猪肠、鸡肠道内容物为材料,采用牛津杯法筛选所需菌株。对筛选出的菌株进行生理生化及16S rRNA基因序列同源性分析。结果分离得到756株乳酸菌,其中有6株菌株对黄曲霉的生长有明显的抑制作用。结论实验获得的6株产酸菌,3株为植物乳杆菌(Lactobacillus plantarum),2株为消化乳杆菌(L.alimentari-us),1株为亚利桑那乳杆菌(L.arizonenensis)。     

动物微生态制剂猪肠源乳球菌的分离与鉴定

试验的菌种是从宁夏平罗县边远农村基本自然生长的健康肉猪的小肠、大肠和盲肠中分离获得的,共分离出64株菌株。通过对这64株菌株的菌落形态观察和革兰氏染色镜检。筛选出9株进行了乳球菌属的生理生化鉴定,初步确定这9株属于乳球菌属(Lactococcus)。再通过糖醇类发酵产酸鉴定,确定2株为乳酸乳球菌乳酸亚种(L.lactissubsplactis),3株为植物乳球菌(L.plantarum),4株为棉籽糖乳球菌(L.raffi-nolactis)。各项鉴定结果均符合乳球菌属和相应种鉴定标准。     

内蒙古地区传统制作奶豆腐和乌日莫中乳酸菌多样性及分布特征

【背景】传统制作奶豆腐和酸性奶油(乌日莫)是内蒙古农牧地区最喜爱的食品,蕴含着十分丰富的乳酸菌资源,亟待开发利用。【目的】通过解析内蒙古农牧地区传统自制奶豆腐和乌日莫样品中乳酸菌多样性及分布特征,为优良菌株选育与利用提供资源和理论基础。【方法】采用稀释涂布法分离纯化乳酸菌,测定菌株16S rRNA基因序列鉴定种属关系,阐明乳酸菌系统发育、遗传分化及菌群结构。【结果】传统自制样品中共分离得到乳酸菌81株,主要归属于乳酸片球菌(Pediococcus acidilactici)、戊糖片球菌(Pediococcus pentosaceus)、短乳杆菌(Lactobacillus brevis)、瑞士乳杆菌(Lactobacillus helveticus)、副干酪乳杆菌(Lactobacillus paracasei)、食二酸乳杆菌(Lactobacillus diolivorans)、奥塔基乳杆菌(Lactobacillus otakiensis)、植物乳杆菌(Lactobacillus plantarum)、开菲尔乳杆菌(Lactobacillus kefir)、乳酸乳球菌(Lactoc...     

新疆发酵乳品中乳酸菌的分离鉴定及其耐药性

目的对新疆传统发酵乳品中乳酸菌进行分离鉴定并检测其耐药性。方法利用传统形态学鉴定法和生化鉴定等方法对新疆发酵乳中乳酸菌进行鉴定,采用纸片扩散法对分离鉴定的菌进行耐药性分析。结果从新疆发酵乳品中共分离出8株乳酸菌,经鉴定分别为瑞士乳杆菌(Lactobacillus helveticus)、植物乳杆菌(Lactobacillus plantarum)、马乳酒样乳杆菌(Lactobacillus kefianofaciens)、乳酸乳球菌(Lactococcus lactis)、副干酪乳杆菌(Lactobacillus paracasei)、副干酪乳杆菌类坚韧亚种(Lactobacillus paracasei subsp.tolerans)、哈尔滨乳杆菌(Lactobacillus harbinensis)、希氏乳杆菌(Lactobacillus hilgardii),并且发现8株乳酸菌对万古霉素、庆大霉素、阿莫西林、多西环素、环丙沙星、阿奇霉素、头孢他啶、头孢孟多具有一定敏感性。结论新疆发酵乳品中以乳杆菌居多,对常见抗生素具有一定的敏感性。     

产广谱细菌素乳酸菌的筛选和鉴定

以大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌和枯草芽孢杆菌为指示菌,从分离自四川传统发酵食品中的267株乳酸菌中,采用平板打孔法初筛、牛津杯法复筛(排除酸、过氧化氢干扰以及胰蛋白酶和木瓜蛋白酶处理),筛选出1株分离自醪糟的具有较强抑菌作用的产广谱细菌素的乳杆菌菌株P158,结合形态学、生理生化特性和16S rDNA序列同源性分析,该菌株被鉴定为植物乳杆菌(Lactobacillus plantarum)。     

山西高平市农家酸菜汁中乳酸菌的分离及鉴定

对山西高平市农家酸菜汁中乳酸菌含量和菌种进行分析研究,经分离筛选得到3株产酸能力较强的菌株,对其进行鉴定并对其生理特性进行研究。通过对其形态特征、菌落特征及生化特征进行分析可知,其中2株为植物乳杆菌(Lactobacillus plantarum),1株为短乳杆菌(Lactobacillus breris)。对筛选到的乳酸菌进行酸菜发酵性能研究发现,其中菌株L2在发酵24 h后,发酵液pH值达到3.47、酸度达到1.67 g乳酸/100 mL、菌落数达到1.37×107cfu/mL的高密度富集状态,且发酵无异味,口味自然,效果较好。     

30周龄笼养海蓝褐壳蛋鸡盲肠中优良乳酸菌的筛选

目的对笼养蛋鸡盲肠中的优良乳酸菌进行筛选与鉴定。方法以30周龄笼养海蓝褐壳蛋鸡盲肠分离纯化得到10株乳酸菌为试验菌,通过发酵产酸力、耐酸性和耐胆盐的筛选试验,获得产酸能力高、对酸和胆盐有高耐受力的乳酸菌,进行生长曲线和产酸曲线及对抗生素的耐受力的测定,并采用API CHL50试剂条对选出的乳酸菌进行鉴定。结果 10株乳酸菌在发酵起始时间点时p H在6.10~6.40,经过48 h的发酵阶段,发酵液的p H明显降低;酸性环境和胆盐对各菌株存活率均有抑制。CL1、CL12和CL54生长较快,最佳生长和产酸发酵时间是24 h,CL26和CL31生长相对较慢,到32 h时生长曲线达到最高点;对抗生素的耐受力检测结果显示,CL1、CL12和CL54对抗生素的耐受力很高,而CL26和CL31对抗生素的耐受力较弱。结论对CL1、CL12和CL54这三株菌进行种属鉴定。根据API CHL50生化鉴定条说明进行接菌发酵试验,经对照鉴定,CL1为Lactococcus Lactis ssp Lactis1(乳酸乳球菌亚种1),CL12为Pediococcus Acidilactici(乳酸片球菌),CL54为Lactobacillus Brevis1(短乳杆菌1)。     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

酸菜中高产γ-氨基丁酸乳酸菌的筛选和鉴定

从酸菜中分离筛选出产γ-氨基丁酸(GABA)乳酸菌18株,采用改良纸层析结合高效液相色谱法测定,获得1株高产乳酸菌菌株Lb-2,该Lb-2菌株在含10g/L谷氨酸钠的TYG培养基中静置培养48h,发酵液中GABA含量可达13.4g/L。根据乳酸菌的形态特征、生理生化特征和16S rDNA基因序列分析鉴定Lb-2菌株为乳杆菌属(Lactobacillus)的短乳杆菌(Lactobacillus brevis)。     

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。     

Usefulness of length heterogeneity-PCR for monitoring lactic acid bacteria succession during maize ensiling

The use of length-heterogeneity PCR was explored to monitor lactic acid bacteria succession during ensiling of maize. Bacterial diversity was studied during the fermentation of 30-day-old maize in optimal and spoilage-simulating conditions. A length heterogeneity PCR profile database of lactic acid bacteria isolated from the silage and identified by 16S rRNA gene sequencing was established. Although interoperonic 16S rRNA gene length polymorphisms were detected in some isolates, strain analysis showed that most of the lactic acid bacteria species thriving in silage could be discriminated by this method. The length heterogeneity PCR profiles of bacterial communities during maize fermentation were compared with those on a database. Under optimal fermentation conditions all the ecological indices of bacterial diversity, richness and evenness, deduced from community profiles, increased until day thirteen of fermentation and then decreased to the initial values. Pediococcus and Weissella dominated, especially in the first days of fermentation. Lactococcus lactis ssp. lactis and Lactobacillus brevis were mainly found after six days of fermentation. A peak corresponding to Lactobacillus plantarum was present in all the fermentation phases, but was only a minor fraction of the population. Unsuitable fermentation conditions and withered maize leaves in the presence of oxygen and water excess caused an enrichment of Enterococcus sp. and Enterobacter sp.     

Comparative analysis of amylolytic lactobacilli and Lactobacillus plantarum as potential silage inoculants

Abstract Two starch-degrading Lactobacillus strains, Lactobacillus amylovorus NRRLB4540 and Lactobacillus amylophilus NCIB11546, were assessed for their potential as silage inoculants. Lactobacillus amylovorus was considered suitable as a silage inoculant for crops which are low in water-soluble carbohydrate, but contain starch, which is unavailable to most conventional silage inoculants. This was on the basis of it exhibiting similar growth characteristics to an inoculant strain of Lactobacillus plantarum and secreting an amylase which was optimally active in the silage pH range. Lactobacillus amylophilus was found to be intolerant of low acid conditions but displayed considerable potential for the industrial production of lactic acid from inexpensive starch-based substrates as it fermented sugars to the desirable L(+) isomer of lactic acid only.     

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei , a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.     

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains

A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.     

Use of DNA probes in the study of silage colonization by Lactobacillus and Pediococcus strains

P.S. COCCONCELLI, E. TRIBAN, M. BASSO AND V. BOTTAZZI. 1991. A technique to monitor lactic acid bacteria inoculants in silage, based on specific DNA probes, was developed and used to evaluate the colonization properties of two strains of Lactobacillus plantarum and one strain of Pediococcus pentosaceus which were used as maize silage inoculants in farm conditions. The results indicated that these three strains were able to dominate the natural microflora of the silage, representing more than the 95% of the bacterial biomass of the maize silage. These studies indicate that the colony hybridization with specific DNA probes may be an effective method for monitoring bacteria and evaluating the colonization properties of inoculants in maize silage.     

Biological ensiling of sugarcane tops

The following lactic acid bacteria were isolated from sugarcane tops silage:Lactobacillus plantarum, Lactobacillus buchneri, Lactobacillus brevis, Lactobacillus delbrueckii, Pediococcus cerevisiae, Leuconostoc mesenteroides andStreptococcus lactis. The isolates were grown in a synthetic medium and the final pH, sugar uptake and the effect of adding CaCO₃ and L1, L2 and L3 factors was observed. The importance of the buffering capacity of the ensiled material for the silage process with a view to the occurrence of lactic acid bacteria is discussed.     

云南泡梨中乳酸菌的分离鉴定及其应用

【背景】泡梨是云南省常见的一种腌渍水果,在云南加工食用已经有一百多年的历史,因其味道酸甜可口、风味独特而深受人们喜爱,而目前对泡梨中微生物种群的系统分析和发酵原理的研究尚未见报道。【目的】研究乳酸菌在云南泡梨中的分布及应用,阐明乳酸菌种类对泡梨发酵中风味物质的影响。【方法】从云南省4个不同地区采集12份泡梨样品,经菌落菌体形态、生理生化特性和16SrRNA基因序列分析进行菌种分离与鉴定。利用分离的乳酸菌为菌种进行泡梨的制备,采用GC-MS技术对人工接种的复合乳酸菌发酵与自然发酵泡梨进行风味物质的分析与感官评价。【结果】分离鉴定出79株植物乳杆菌(Lactobacillus plantarum)、 3株类植物乳杆菌(Lactobacillus paraplantarum)、1株戊糖乳杆菌(Lactobacillus pentosus)、1株干酪乳杆菌(Lactobacillus casei)、2株副干酪乳杆菌(Lactobacillus paracasei)和1株短乳杆菌(Lactobacillus brevis),植物乳杆菌为泡梨发酵中的优势菌。将分离所得乳酸菌用于泡梨制备的结果表明,乳酸菌使泡梨的发酵时间缩短5d且品质更优,分析其中的风味物质发现接种乳酸菌发酵泡梨风味物质更丰富,其中酯类和醇类远多于自然发酵泡梨。【结论】云南泡梨中含有丰富的乳酸菌,选用分离出的优势乳酸菌作为复合乳酸菌用于泡梨发酵获得色泽、口感更好的泡梨,且发酵周期更短,风味物质更丰富。该研究对泡梨制备工艺和进一步标准化生产均具有重要意义。     

Diversity of a stable enrichment culture which is useful for silage inoculant and its succession in alfalfa silage

Alfalfa is a kind of forage that is difficult to ensile for good quality. Therefore, inoculants are always used to enhance the preservation of alfalfa silage. Through continuous restricted subcultivation, a lactic acid bacteria community (Al2) was selected from well-fermented alfalfa silage, which sharply decreased the pH level and produced a large amount of lactic acid. The adding of Al2 to alfalfa at ensiling resulted in a more rapid drop in pH and higher levels of lactic acid, and it also reduced the ammonia-nitrogen content significantly (P < 0.01). Plate isolation, denaturing gradient gel electrophoresis (DGGE) and the construction of a 16S rRNA gene clone library were used to identify the composition diversity of the Al2 community; seven strains were detected in the community, the predominant strain belonging to Lactobacillus plantarum. Samples of alfalfa silages of duration 0, 2, 5, 10, 20 and 30 days were studied with DGGE analysis. The DGGE band patterns of Al2-treated and non inoculated were rather different, and the components of Al2 were the dominant bacteria in Al2-treated silages, especially L. plantarum, while Pediococcus pentosaceaus was predominant in naturally fermented alfalfa silage.     

Growth and survival of lactic acid bacteria in lucerne silage

A rifampicin-resistant variant of two strains of Lactobacillus plantarum, one strain of Pediococcus acidilactici, and one strain of Enterococcus faecium were used for the experimental production of lucerne silage. Laboratory silage without inoculants served as a control. Counts of total anaerobes, total lactic acid bacteria (LAB), lactobacilli, pediococci, and enterococci were determined on days 14, 21, 30, 49, and 60 of lucerne fermentation. LAB dominated in silage microflora, reaching a percentage between 59 and 95 % of total anaerobes. Lactobacilli were found as a predominant group of LAB during the whole study. Lactobacilli reached numbers 8.74 log CFU/g in treated silage and 8.89 log CFU/g in the control at the first observation. Their counts decreased to 4.23 and 4.92 log CFU/g in treated silage and the control, respectively, on day 63 of fermentation. Similar decreases were observed in all bacterial groups. The treated silage samples possessed lower pH (4.2 vs. 4.5 in control samples) and contained more lactic acid compared to control silage. The identity of re-isolated rifampicin-resistant bacteria with those inoculated to the lucerne was evaluated by fingerprinting techniques. The fingerprint profiles of re-isolated bacteria corresponded to the profiles of strains used for the treatment. It could be concluded that supplemented LAB dominated in laboratory silage and overgrew naturally occurring LAB.