简并引物法克隆保加利亚乳杆菌β-N-乙酰氨基己糖苷酶基因

β-N-乙酰氨基己糖苷酶(β-Hexcase)在细菌、植物和动物中广泛存在,具有典型的外切酶活性并可以催化切除β-N-乙酰氨基己糖的非还原性氨基己糖残基,在细菌细胞分裂中具有重要作用.选取与保加利亚乳杆菌LJJ亲缘关系近的菌种的β-N-乙酰氨基己糖苷酶蛋白序列,利用ICO-DEHOP和CODEHOP在线简并引物设计软件设计β-Hexcase简并引物,选取Hex1-f和Hex1-r引物对,以LJJ基因组DNA为模板经PCR扩增得到614 bp产物.PCR产物连接pGEM-T质粒载体后克隆至大肠杆菌DH5α中,筛选阳性克隆提取质粒进行测序.测序结果显示产物长度为614 bp,该DNA序列经blastx比对发现与其他已知β-Hexcase基因具有相似性,表明所克隆的序列即为LJJβ-Hexcase的基因片段.β-N-乙酰氨基己糖苷酶基因的获得为进一步研究β-N-乙酰氨基己糖苷酶与保加利亚乳杆菌自溶的关系奠定了基础.

糯米乳酸饮料的研究

以糯米为发酵主原料,强化锌、钙等成分,应用保加利亚乳杆菌[Laclobacillus bulgoricus]和嗜热链球菌[Strepococcus thermophilus]发酵,生产新型保健饮料。发酵控制温度38℃～40℃,发酵时间22h～25h。所得产品不仅保留了糯米的原有营养成分和天然芳香,而且柔和适口。

Production of High Quality Functional Labneh Cheese Using Exopolysaccharides Producing Encapsulated Lactobacillus Bulgaricus

Production of polysaccharides by lactic acid bacteria is vital and its technology is very important, too in dairy study. The selection ofexpolysaccharides producing encapsulated Lactobacillus bulgaricus in alginate gel and the factors affecting the production of polysaccharides such as time, temperature, pH and the growth with Streptococcus thermophilus were studied, pH 5.5 and temperature 35℃/18hr were found to be optimal for exopolysaccharides production by encapsulated L. bulgaricus and free culture yielding 600 mg/L and 380 mg/L respectively. Co-culturing L. bulgaricus with S. thermophilus increased exopolysaccharides production at 35 ℃/18hr to 660 mg/L by encapsulated cells and 400 mg/L by free culture. Results of this study were applied in the manufacture of Labneh cheese vitally containing probiotic species ofL. acidophilus, L. gasseri, L. johnsonii and protection of these bacteria by polysaeeharide produced encapsulated L. bulgaricus. The use of exopolysaccharides producing encapsulated L. bulgaricus and probiotic bacteria can provide acceptable quality to functional Labneh. This product can be used as therapeutic and diabetic milk product with highly acceptable qualities. It is noted that all of the results are the means of triplicate experiments.

Fermented Goat Milk and Cow Milk Produced by Different Starters of Lactic Acid Bacteria： Quality Studies

Lactic acid bacteria （LAB） is widely used as culture starters in dairy fermentation. The aim of this study was to investigate the quality of fermented goat milk and cow milk, as well as the viability of LAB in the same products. Fermentations were performed with pasteurized goat milk or cow milk added with skim milk （18% of solids） using three separately different starters; yoghurt starter （a combination of Streptococcus thermophilus FNCC-0040 and Lactobacillus bulgaricus FNCC-0041）, single starter of Lactobacillus acidophilus FNCC-0029 and Lactobacillus casei FNCC-0051. The parameters observed were pH, acidity, nutritional quality including protein, fat and lactose content and product＇s viscosity. Acidity, pH and viability of LAB were also monitored during storage at refrigerated temperature （4 ℃） for 28 days. Results show that the different LAB starters did not affect the pH, acidity, lactose and protein content. Differences on LAB starters affected fat content and viscosity. The highest score of viscosity （30.00 Pa.s ± 7.02 Pa.s） was observed on products fermented by yoghurt starters, followed by products obtained using starter of L. acidophilus （17.7 ±11.4） and L. casei （8.62 ±0.35）. Protein content, acidity, pH and viscosity were not significantly different between products obtained from goat milk and cow milk. Fat content in fermented goat milk was higher （5.03% ±0.62%） than in fermented cow milk （3.52% ±0.37%）, however, lactose content was higher in fermented cow milk （5.16% ±0.40%） than in fermented goat milk （4.53% ±0.35%）. Total LAB concentration in fermented cow milk during storage was 8.03± 0.52 logt0 cfu/mL, while in fermented goat milk was 7.81 loglo cfu/mL ± 0.67 loglo cfu/mL. There was a 10.83% decrease in LAB viability in fermented cow milk and 11.40% in fermented goat milk after 28 days of storage. In conclusion, quality of fermented milk is affected by the starters applied, raw milk source and storage period.

Effect of Using Sourdough on the Rheological Properties, Sensory and Shelf Life Stability of Loaf Bread

Cereal-based foods play an important role as a source of dietary energy and nutrients in human nutrition. The use of microorganisms by humans has a long tradition. Bread making is one of the oldest arts known to man. Today we know that lactic acid bacteria （LAB） and yeasts are prevailing organisms in carbohydrate-rich environments. They break down carbohydrates to mainly lactic acid, ethanol and CO2. The present study is to determine the effect of Lactobacillus bulgaricus alone and as a combination with yeast （Saccharomyces cerevisiae） using yeast as a control. In this work, different percentage of Lactobacillus bulgaricus were used to investigate how sourdough affect on the bread sensory characteristics such as volume, specific loaf volume, crust, grain, color crumb, aroma, taste, texture, pH of bread and extended shelf life of bread. Also different storage times were used to know it is affected on the shelf life and sensory characteristics of bread. The results suggest that use of sourdough in bread production is useful in improving sensory properties, decreasing fungal spoilage.

The Antagonistic Activity of the Lactic Acid Bacteria （Streptococcus thermophilus, Bifidobacterium bifidum and Lactobacillus bulgaricus） against Helicobacter pylori Responsible for the Gastroduodenals Diseases

The lactic acid bacteria are used in fermentation and the bio conservation of food, because it produces organic acids and other antibacterial substances as the bacteriocin to inhibit certain pathogenic stocks. In order to demonstrate the inhibitory effect of these bacteria, we studied the antagonistic capacity of the two leavens of yoghourt （Streptococcus thermophilus and Lactobacillus bulgaricus） and Bifidobacterium bifidum with respect to Helicobacterpylori by the method of diffusion by discs. Their interactions led to the emergence of large zones of inhibition. Bifidobacterium bifidum led the greatest halo with a diameter of 0.8 cm by contribution to Lactobacillus bulgaricus （0.5 cm） and Streptococcus thermophilus （0.3 cm）. The additional tests were required to know the exact nature of the inhibitors. The results showed that the antibacterial peptides （bacteriocins） have a narrow spectrum of activity against the species pathogen compared to that caused by acidity.

Antimicrobial Activity of Probiotics on Swiss Mice

Salmonella enterica serovar typhimurium is the first cause identified of food poisoning in humans; new data indicate that the resistant strains of Salmonella sp. can be inhibited by a bacterial population present in foods have been called probiotics. Our study was conducted on Swiss albino mice in order to know the effect of probiotics or biotherapeutic agents, and suggest the different therapeutic possibilities of intestinal complications related to the presence of Salmonella enterica serovar typhimurium; Reference strains, Lactobacillus delbrueckii subsp, bulgaricus, Lactobacillus acidophilus and Lactobacillus paracasei in combination with Bifidobacterium sp. were tested for their health benefits. The number of Salmonella enterica serovar typhimurium decreases in intestine, in the stomach and in the liver for group 2, 3 in case of preventive treatment and group 4, 5 in case of a therapeutic treatment due to the use of a cuRure of probiotics, in comparison with group 1 where we recorded a high number. The bio-therapeutic treatment was finally confirmed by macroscopic and microscopic observations of different target organs before and after treatment for the experiment period.

Viability of Bifidobacteria in frozen yogurt made from camel milk

The purpose of this research was to incorporate Bifidobacterium angulatum and Bifidobacterium infantis in frozen fermented dairy desserts made from camel or cow milk and to determine their viability during freezing and storage at .20℃. To meet this objective, ice cream mixtures were formulated using camel or cow milk constituents, inoculated with regular yogurt starter （Lactobacillus delbruecldi ssp bulgaricus and streptococcus thermophilus） and incubated at 42℃ till a pH value of 5.0 was attained. The fermented mixes were heated to 80℃ for 5 min in water bath to inhibit yogurt organisms. Bifidobacteria were then added at 2 g/kg mix （1 gram from each Bifidobacterium strain）. The results showed that the initial counts of Bifidobacteria before freezing were 7.3 × 10^8 and 7.1 × 108 cfu/g for camel and cow mix respectively and decreased to 1.06× 10^8 and 1.1×10^8 in the same order （about 0.8 log reduction in the count of Bifidobacteria was observed） after freezing and storage for one day. No significant changes in counts of Bifidobaeteria were found during storage at -20℃ for 17 weeks. Changes in pH and titratable acidity were also studied. No significant changes in titratable acidities of frozen yogurt made from camel or cow milk constituents during storage period were observed.

Exopolysaccharides from Lactic acid Bacteria as Corrosion Inhibitors

Bacterial EPSs （exopolysaccharides） are believed to play an important role in the environment by promoting survival strategies such as bacterial attachment to surfaces and nutrient trapping, which facilitate processes of biofilm formation and development. These microbial biofilms have been implicated in corrosion of metals, bacterial attachment to prosthetic devices, fouling of heat exchange surfaces, toxicant immobilization, and fouling of ship hulls. In this paper, data on EPS production and the effect of EPS on corrosion of steel produced by Lactobacillus fermentum Ts are presented and discussed. The Lactobacillus fermentum Ts strain was isolated from types of Bulgarian rye flour. It was tested for its ability to produce exopolysaccharides when cultivated in a media containing 10% sucrose, 10% fructose and 10% maltose. The study of the corrosive stability of steel samples was conducted on the gravimetrique method. The rate of corrosion, the degree of protection, and coefficient of protection have been calculated. The structure of layer over steel plates was analysed by SEM （scanning electron microscopy） JSM 5510. It could be underlined that 10% sucrose and 10% maltose in the media stimulated the process of protection of corrosion.

Optimization of the quenching method for metabolomics analysis of Lactobacillus bulgaricus

This study proposed a quenching protocol for metabolite analysis of Lactobacillus delbrueckii subsp.bulgaricus.Microbial cells were quenched with 60% methanol/water,80% methanol/glycerol,or 80% methanol/water.The effect of the quenching process was assessed by the optical density（OD）-based method,flow cytometry,and gas chromatography-mass spectrometry（GC-MS）.The principal component analysis（PCA） and orthogonal partial least squares-discriminant analysis（OPLS-DA） were employed for metabolite identification.The results indicated that quenching with 80% methanol/water solution led to less damage to the L.bulgaricus cells,characterized by the lower relative fraction of prodium iodide（PI）-labeled cells and the higher OD recovery ratio.Through GC-MS analysis,higher levels of intracellular metabolites（including focal glutamic acid,aspartic acid,alanine,and AMP） and a lower leakage rate were detected in the sample quenched with 80% methanol/water compared with the others.In conclusion,we suggested a higher concentration of cold methanol quenching for L.bulgaricus metabolomics due to its decreasing metabolite leakage.

Effect of culturing conditions on the expression of key enzymes in the proteolytic system of Lactobacillus bulgaricus

The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions （different phases, initial pH values, temperatures, and nitrogen sources） using real-time polymerase chain reaction （RT-PCR）. The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains （P〈0.05）. All these results indicated that the culturing conditions affected the expression of the proteolytic system genes in Lactobacillus bulgaricus at the transcription level.