发酵乳中双岐杆菌种类快速识别

建立发酵乳制品中双歧杆菌快速识别方法。采用酶解前处理法获取样品中菌体细胞,基于PCR-DGGE技术确定发酵乳中双歧杆菌种属。该方法能准确、快速鉴别双歧杆菌,检出限为105cfu/mL。该方法可用于发酵乳中双歧杆菌的准确识别。     

实时荧光定量PCR法测定发酵乳中双歧杆菌

对实时荧光定量聚合酶链式反应(PCR)技术检测发酵乳中双歧杆菌的DNA提取方法、PCR扩增效率、标准曲线绘制进行探讨,通过比较分析碱式提取法、玻璃珠破碎法和酶解法3种提取方法对发酵乳中总DNA提取效率、4种不同参考菌株的PCR扩增效率以及单一菌株标准曲线与混合菌株标准曲线的计数结果,建立实时荧光定量PCR快速测定发酵乳制品中双歧杆菌数量方法。结果表明：酶解法提取发酵乳中总DNA效果最好,OD260/280比值基本接近1.80,且提取的质量浓度含量最高;不同菌株的PCR扩增效率不同,其中参考菌株1.2213的Ct值与其他3菌株的Ct值存在显著性差异;根据单一菌株绘制标准曲线与混合菌株绘制标准曲线计数结果无显著性差异,后者计数结果更客观、准确。采用酶解法获取样品中的菌体细胞,基于混合菌液绘制标准曲线,采用实时荧光定量PCR技术确定发酵乳中双歧杆菌种属数量,可快速、准确地测定发酵乳中双歧杆菌的数量。     

海南传统发酵蔬菜中微生物多样性分析

为研究海南发酵蔬菜中微生物的多样性,以采自海南白沙、昌江、临高、琼中、文昌、万宁和五指山7个地区的30份不同种类的发酵竹笋、小西瓜、白菜和豆角样品为研究对象,通过传统分离培养方法、16S rRNA基因序列分析和荧光定量PCR技术对样品中的微生物进行多样性分析。共获得了172株乳酸菌,分属于3个属,16个种,其中乳杆菌属数量最多,占菌株总数的84.30%,肠球菌属和片球菌属分别占15.12%和0.58%,测得每份样品中乳杆菌的平均含量为5.22×10^7 CFU/g,片球菌的平均含量为9.42×10^5 CFU/g,肠球菌的平均含量为1.07×10^4 CFU/g。乳杆菌属是海南发酵蔬菜中的优势菌属,植物乳杆菌是海南发酵蔬菜中的优势菌种。研究表明不同地区、不同种类的发酵蔬菜中微生物多样性存在差异。本研究初步揭示了海南发酵蔬菜中微生物的多样性,为海南微生物资源的深入研究和开发利用打下基础。     

采用实时荧光定量PCR法同时定性定量检测发酵乳中多种益生菌

为快速、准确地对益生菌发酵乳中的干酪乳杆菌及罗伊氏乳杆菌进行定性、定量检测,通过设计干酪乳杆菌及罗伊氏乳杆菌种属特异性引物,建立了双重实时荧光定量聚合酶链式反应(polymerase chain reaction,PCR)检测方法。结果表明,所建标准曲线方程线性关系良好,相关系数R²均达到0.99以上。对市售产品随机抽样检测发现:样品1、样品2未添加干酪乳杆菌及罗伊氏乳杆菌。样品3中检出干酪乳杆菌(1.39±0.25)×10⁹copies/mL,未检出罗伊氏乳杆菌。自制益生菌发酵乳检出干酪乳杆菌(3.7±0.35)×10⁹copies/mL、罗伊氏乳杆菌(4.8±0.26)×10⁹copies/mL,检测结果均与所知的益生菌种类及数量信息相符。结果表明,上述建立的实时荧光定量PCR方法能够同时对益生菌发酵乳中的干酪乳杆菌及罗伊氏乳杆菌进行快速、准确地定性、定量检测,克服了平板培养法和流式细胞术的局限,具有一定的优势和应用前景。     

一种发酵乳中双歧杆菌鉴别培养基计数评估

目的:建立发酵乳中双歧杆菌数量计数测定方法。方法:采用果糖为碳源的鉴别培养基,基于乳酸菌同型发酵和异型菌落形态的差异,借助pH指示剂确定样品中双歧杆菌数量。结果:该培养基所计双歧杆菌数量与对照培养基所计数量在同一数量级且差异不显著(P>0.05)。结论:该培养基可用于发酵乳中双歧杆菌的准确计数。     

基于微滴数字PCR技术的婴幼儿配方乳粉中双歧杆菌定量检测及应用

应用微滴式数字聚合酶链式反应（droplet digital polymerase chain reaction，ddPCR）技术，建立婴幼儿配方乳粉中双歧杆菌定量检测方法。以双歧杆菌的单拷贝特异性基因rpsL为目标基因设计引物探针，对ddPCR条件进行优化，考察方法的特异性、灵敏度和重复性，并与平板计数方法进行对照验证。结果表明，建立的方法具有良好的特异性、灵敏性和重复性。细菌纯培养液的检出限为296 CFU/mL，模拟样品检出限为7 300 CFU/g，不与10 种近缘乳酸菌发生交叉反应，且重复性较好，采用已建立的ddPCR方法和平板计数方法对市售婴幼儿配方乳粉样品进行检测，2 种方法测定值结果偏差小于10%，结果一致性较好。本研究建立的ddPCR方法对婴幼儿配方乳粉中的双歧杆菌定量检测能够更快速、灵敏、准确，具有一定的应用前景。     

类食品乳杆菌412对酸面团发酵的影响

从酸面团中分离得到1株类食品乳杆菌(Lactobacillus parali mentarius)412,添加该菌株的面团在28℃发酵4h后的pH为5.2,发酵24h后pH为3.5;而添加商业化安琪酵母发酵的面团在28℃发酵4h后的pH为5.9,发酵24h后pH为5.2。类食品乳杆菌412与安琪酵母(接种量为107CFU/g)联合发酵测定表明,若菌株412起始接种量为109CFU/g,则面团在28(C发酵24h后的滴定酸度为0.76%(滴定酸度以乳酸计,%为质量分数);若菌株412的起始接种量是108CFU/g或107CFU/g时,则面团在28℃发酵24h后的滴定酸度为0.57%。当向面团添加6.75g/kg的葡萄糖或蔗糖可以促进类食品乳杆菌412的生长。面团在28℃发酵12h后,添加葡萄糖可以使菌株412的细胞数量从起始的2×108CFU/g提高到2.5×1010CFU/g,添加蔗糖则可以使其细胞数量提高到2.6×1010CFU/g;而添加果糖的面团在28℃发酵12h,菌株412的细胞生物量仅3.8×108CFU/g。与25(C或32℃发酵条件相比,28℃下发酵的酸面团中类食品乳杆菌412和安琪酵母菌的活菌数都比较高,且面团酸化速率高于单独采用安琪酵母菌发酵的面团。结果证明,类食品乳杆菌412能够单独或与商业化的安琪酵母联合应用到酸面团发酵中,并对面团的酸化起到积极促进作用。     

乳制品中活双歧杆菌检测试剂盒的研制及应用

在EMAReal-Time PCR检测方法的基础上,组装检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。建立了EMAReal-Time PCR检测乳制品中活双歧杆菌的标准曲线。组装出检测乳制品中活双歧杆菌的荧光定量PCR试剂盒。该试剂盒批内及批间变异系数(CV)均小于5%;对乳制品中常见乳酸菌无交叉反应;最低检测限为2×104CFU/mL。该试剂盒在-20℃下,至少可保存6个月,4℃可保存3个月。分别采用研究所获得的试剂盒和平板菌落计数法检测12份市售酸乳中的活双歧杆菌的数量,2种检测方法计数结果差异不显著(P>0.05)。该方法所建立的标准曲线相关系数大于98%。所研制的试剂盒重复性较好、特异性较强、灵敏度较高,可以准确地定量检测乳制品中的活双歧杆菌。     

鼠李糖乳杆菌HN001菌株水平快速定量方法的建立及应用

目的 应用实时荧光定量聚合酶链式反应(real-timefluorescencequantitativepolymerasechainreaction,qPCR),建立鼠李糖乳杆菌HN001菌株水平的快速定量检测方法。方法 通过比对鼠李糖乳杆菌HN001的近缘菌株的基因组,筛选出鼠李糖乳杆菌HN001菌株水平的特异基因,以菌株水平的特异基因为靶标设计引物及探针,优化反应体系和条件,建立鼠李糖乳杆菌HN001株水平的q PCR检测方法。对方法的特异性、灵敏度、检出限进行验证,并采用已建立的q PCR方法进行模拟添加样品和实际样品的检测。结果 所建立的方法特异性强,与近缘菌株无交叉反应,鼠李糖乳杆菌HN001纯培养液检出限为102 CFU/mL,灵敏度可达到650 copies/μL,可在2h内完成样品检测。对模拟添加样品以及实际样品检测中q PCR和平板计数法结果的对数值进行显著性分析检验,两种方法的测定值结果均无显著性差异(P>0.05)。结论 本研究建立的q PCR检测方法可有效应用于益生菌产品中鼠李糖乳杆菌HN001菌株水平定量检测,具有快速、灵敏、准确的特点。     

益生菌切达干酪成熟过程中细菌群落的多样性分析

采用选择性培养基和聚合酶链式反应和变性梯度凝胶电泳（polymerase chain reaction-denaturing gradient gelelectrophoresis,PCR-DGGE）技术,研究益生菌切达干酪成熟过程中（6 ℃,180 d）细菌群落构成及益生菌（干酪乳杆菌LC2W）的存活情况。结果表明：SBM和MSE等选择性培养基存在选择专一性不强的缺点,不能客观反映干酪内各种微生物的动态变化;随着切达干酪成熟时间的增加,发酵剂嗜热链球菌和乳酸乳球菌的数量明显下降,而非发酵剂菌群乳杆菌的数量和主要种类呈上升趋势;干酪成熟180 d后,干酪乳杆菌LC2W的存活量仍高于1×108CFU/g。切达干酪能作为干酪乳杆菌LC2W存活的良好载体;PCR-DGGE技术和选择计数法联用更加适合干酪细菌群落结构的分析。     

Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis

The use of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) technique in identifying the microorganisms present in commercial probiotic yoghurts and lyophilised products was evaluated. Two reference ladders were assembled constituted by PCR-amplified V2-V3 regions of 16S rDNA from bacterial species generally used as probiotics. Identification was achieved comparing the PCR-DGGE patterns obtained from the analysed products with the ladder bands. Bands from members of the same species showed the same migration distance in denaturing gel, hence supporting the identificative value of the method. The validity of the technique was also proven confirming the PCR-DGGE identification results by sequence data analysis and by species-specific PCR. General congruence between microorganisms declared on the label and those revealed by PCR-DGGE was found for probiotic yoghurts. Conversely, some discrepancies were observed for probiotic lyophilised preparations, i.e. the incorrect identification of some Bifidobacterium and Bacillus species and the presence of not declared microorganisms. PCR-DGGE turned out to be an appropriate culture-independent approach for a rapid detection of the predominant species in mixed probiotic cultures.     

Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR

Real-time quantitative PCR assays were developed for the absolute quantification of lactic acid bacteria (LAB) (Streptococcus thermophilus, Lactobacillus delbrueckii, L. casei, L. paracasei, L. rhamnosus, L. acidophilus and L. johnsonii) in fermented milk products. The results of molecular quantification and classic bacterial enumeration did not differ significantly with respect to S. thermophilus and the species of the L. casei group which were detected in the six commercial fermented products tested, thus showing that DNA extraction was efficient and that genomic DNA solutions were free of PCR inhibitors. For L. delbrueckii, the results of bacterial enumeration were generally lower by a factor 10 to 100 than those of PCR quantification, suggesting a loss of viability during storage of the dairy products at 1-8 degrees C for most of the strains in this species. Real-time quantitative assays enabled identification of the species of lactic acid bacterial strains initially present in commercial fermented milk products and their accurate quantification with a detection threshold of 10(3) cells per ml of product.     

Identification of probiotic microorganisms in South African products using PCR-based DGGE analysis

Probiotic microorganisms in commercial yoghurts and other food products are currently identified by traditional methods such as growth on selective media, morphological and biochemical characteristics. In this study, PCR-based DGGE analysis was used for the rapid and accurate identification of probiotic microorganisms from South African yoghurts and lyophilized preparations in capsule and tablet form. To identify the microorganisms present in these products, the DGGE profiles obtained were compared to two reference markers (A and B) composed of five lactobacilli and seven Bifidobacterium species, respectively. The results obtained were confirmed by species-specific PCR, as well as sequence analyses of unknown bands not present in the reference markers. It was found that only 54.5% of the probiotic yoghurts contained the microorganisms stated on the label compared to only a third (33.3%) of the lyophilized probiotic products. Some Bifidobacterium species were incorrectly identified and various microorganisms were detected that were not listed on the label. Sequence analyses confirmed the presence of Streptococcus spp. other than the yoghurt starter, Streptococcus thermophilus, in some of these products and in some instances label information was vague and non-scientific. PCR-based DGGE analyses proved to be a valuable culture-independent approach for the rapid and specific identification of the microbial species present in South African probiotic products.     

PCR method for detection and identification of Lactobacillus casei/paracasei bacteriophages in dairy products

Bacteriophage infections of starter lactic acid bacteria (LAB) pose a serious risk to the dairy industry. Nowadays, the expanding use of valuable Lactobacillus strains as probiotic starters determines an increase in the frequency of specific bacteriophage infections in dairy plants. This work describes a simple and rapid Polymerase Chain Reaction (PCR) method that detects and identifies bacteriophages infecting Lactobacillus casei/paracasei, the main bacterial species used as probiotic. Based on a highly conserved region of the NTP-binding genes belonging to the replication module of L. casei phages phiA2 and phiAT3 (the only two whose genomes are completely sequenced), a pair of primers was designed to generate a specific fragment. Furthermore, this PCR detection method proved to be a useful tool for monitoring and identifying L. casei/paracasei phages in industrial samples since specific PCR signals were obtained from phage contaminated milk (detection limit: 10(4) PFU/mL milk) and other commercial samples (fermented milks and cheese whey) that include L. casei/paracasei as probiotic starter (detection limit: 10(6) PFU/mL fermented milk). Since this method can detect the above phages in industrial samples and can be easily incorporated into dairy industry routines, it might be readily used to earmark contaminated milk for use in processes that do not involve susceptible starter organisms, or processes which involve phage-deactivating conditions.     

Encapsulation in an alginate–goats’ milk–inulin matrix improves survival of probiotic Bifidobacterium in simulated gastrointestinal conditions and goats’ milk yoghurt

In this work, a new encapsulating matrix, alginate–goats’ milk–inulin, was used to encapsulate Bifidobacterium animalis subsp. lactis BB‐12. The addition of inulin resulted in capsules with a compact structure, and a higher probiotic cell count under simulated gastrointestinal conditions and in probiotic goats’ milk yoghurt during refrigerated storage. Encapsulation of the probiotic bacteria led to slower post‐acidification yoghurts. The results of this study showed that the alginate–goats’ milk–inulin matrix has potential to be used as a new encapsulation material to encapsulate probiotics for use in goats’ milk‐based probiotic fermented dairy products, avoiding the cross‐contamination caused by using capsules based on cows’ milk.     

Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry

Probiotic food is manufactured by adding probiotic strains simultaneously with starter cultures in fermentation tanks. Here, we investigate the accuracy and feasibility of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for bacterial identification at the species level in probiotic food and yoghurts. Probiotic food and yoghurts were cultured in Columbia and Lactobacillus specific agar and tested by quantitative real-time PCR (qPCR) for the detection and quantification of Lactobacillus sp. Bacterial identification was performed by MALDI-TOF analysis and by amplification and sequencing of tuf and 16S rDNA genes. We tested 13 probiotic food and yoghurts and we identified by qPCR that they presented 10(6) to 10(7) copies of Lactobacillus spp. DNA/g. All products contained very large numbers of living bacteria varying from 10(6) to 10(9) colony forming units/g. These bacteria were identified as Lactobacillus casei, Lactococcus lactis, Bifidobacterium animalis, Lactobacillus delbrueckii, and Streptococcus thermophilus. MALDI-TOF MS presented 92% specificity compared to the molecular assays. In one product we found L. lactis, instead of Bifidus spp. which was mentioned on the label and for another L. delbrueckii and S. thermophilus instead of Bifidus spp. MALDI-TOF MS allows a rapid and accurate bacterial identification at the species level in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified. Practical Application: MALDI-TOF MS is rapid and specific for the identification of bacteria in probiotic food and yoghurts. Although the safety and functionality of probiotics are species and strain dependent, we found a discrepancy between the bacterial strain announced on the label and the strain identified.     

Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts

This study evaluated the effect of the supplementation of total dietary fiber from apple, banana or passion fruit processing by-products on the post-acidification, total titratable acidity, bacteria counts and fatty acid profiles in skim milk yoghurts co-fermented by four different probiotics strains: Lactobacillus acidophilus L10 and Bifidobacterium animalis subsp. lactis BL04, HN019 and B94. Apple and banana fibers increased the probiotic viability during shelf-life. All the fibers were able to increase the short chain and polyunsaturated fatty acid contents of yoghurts compared to their respective controls. A synergistic effect between the type of fiber and the probiotic strain on the conjugated linoleic acid content was observed, and the amount of α-linolenic acid was increased by banana fiber. The results of this study demonstrate, for the first time, that fruit fibers can improve the fatty acid profile of probiotic yoghurts and point out the suitability of using fibers from fruit processing the by-products to develop new high value-added fermented dairy products.     

Performance of different microbial cultures in potentially probiotic and prebiotic yoghurts from cow and goat milks

Lactobacillus acidophilus or Bifidobacterium animalis subsp. lactis Bb‐12 and green banana pulp were used in order to obtain potentially probiotic and prebiotic yoghurts, which were compared over a 45‐day storage period. Goat milk yoghurts demonstrated probiotic effects up to 45 days of storage. Cow milk yoghurts produced with B. animalis subsp. lactis Bb‐12 showed a probiotic effect reduction during the storage period (1.74 log CFU/g). The type of milk affected the yoghurts’ chemical and physicochemical properties. Sensory acceptance was also affected, where cow milk yoghurts were better accepted than goat milk ones.