补加氨水对薛氏丙酸杆菌分批发酵及代谢物抑菌活性的影响

研究了在分批发酵过程中补加氨水对1株薛氏丙酸杆菌(Propionibacterium shermanii)发酵及其代谢物对恶臭假单胞菌(Pseudomonas pudia)抑菌活性的影响。结果表明,薛氏丙酸杆菌代谢物在初始pH7.0时抑菌活性最高,为7.59AU/mL,显著高于其他实验组(p0.05);每隔24h补加10%氨水溶液调节发酵液pH6.0时,薛氏丙酸杆菌的生物量和代谢物的抑菌活性都最大,分别为9.62mg/mL和16.42AU/mL;研究分别从第2、3或4d补加10%氨水开始调节发酵液pH6.0时发现,从发酵第2d开始调节pH效果最好,生物量和代谢物的抑菌活性分别达到9.55mg/mL和16.47AU/mL。这些结果表明,在分批发酵过程中补加氨水调节发酵液的pH可以显著提高丙酸杆菌代谢物的抑菌活性。     

丙酸杆菌代谢物与山梨酸钾抑菌作用比较研究

以一株费氏丙酸杆菌突变株为出发菌株,通过改变发酵培养基、接种量和初始培养pH值3方面,研究其代谢产物对大肠杆菌和沙门氏菌的抑菌作用,并与山梨酸钾的抑菌作用进行对比。结果表明:3种发酵培养基均对大肠杆菌和沙门氏菌均存在一定的抑制作用,其中以乳酸钠(sodium lactate broth,SLB)培养基发酵时,该菌株代谢产物的抑菌活性分别为25.3 AU/mL和18.3 AU/mL,结果要优于0.1%的山梨酸钾的抑菌活性(21.1 AU/mL和14.1 AU/mL);对于接种量而言,对大肠杆菌和沙门氏菌的抑菌活性最佳的接种量分别为6%和5%,其抑菌活性分别为28.1 AU/mL和18.0 AU/mL,抑菌效果均优于0.1%的山梨酸钾;对于不同培养pH值而言,对大肠杆菌和沙门氏菌的抑菌活性最适初始pH值分别为6.5和6,抑菌活性分别为27.6 AU/mL和14.5 AU/mL,其抑菌效果也优于0.1%的山梨酸钾。所以,选择在适宜条件下发酵所得的丙酸杆菌代谢物,对大肠杆菌和沙门氏菌的抑制效果要优于山梨酸钾。对丙酸杆菌代谢物抑菌作用的研究将为新型天然防腐剂的开发提供依据与试验数据支撑。     

分批补料高密度发酵生产丙酸杆菌素及其结构分析

采用分批补料高密度发酵的方法提高丙酸杆菌素的抑菌活性。从发酵96 h开始,每隔24 h补加2.5 g/L复合碳源（乳酸钠-葡萄糖质量比3∶1）和复合氮源（酵母溶液-玉米酒糟清液体积比1∶30）各100 mL,连续补加5 次后,丙酸杆菌素的抑菌活性从17.6 AU/mL提高到31.2 AU/mL。本研究创新性地将玉米酒糟清液作为发酵氮源代替部分酵母溶液,大大降低了丙酸杆菌素的生产成本。另外,通过红外光谱及蛋白酶敏感实验发现丙酸杆菌素是一种具有抑菌活性的蛋白类物质。本研究结果为丙酸杆菌素的工业化生产及其应用提供一定理论支持。     

嗜酸乳杆菌细菌素Lactobacilin XH1发酵条件优化

以细菌素相对抑菌效价为考察指标,通过单因素和正交试验对嗜酸乳杆菌细菌素Lactobacillin XH1发酵条件进行优化,研究了培养基初始pH、培养温度、培养时间及接种量4个因素对细菌素产生的影响。结果表明,优化后最佳发酵条件为培养基初始pH 6.5、培养温度35℃、接种量为2.5%和培养时间28 h,在此条件下细菌素Lactobacillin XH1相对抑菌效价值达到247.49±1.05 AU/mL,比优化前提高了2倍。     

pH对费氏丙酸杆菌细菌素抑菌性的影响

以实验室保存的一株费氏丙酸杆菌CS1420(Propionibacterium freudenreichii CS1420)为试验菌株。首先考察了不同初始pH下对其发酵产细菌素的影响。结果表明:以大肠杆菌ATCC25922为指示菌,当培养基初始pH为6.0时,细菌素抑菌效果最好;以Saccharomyces cerevisiae 2-10515为指示菌,当培养基初始pH为5.5时,细菌素抑菌效果最好。然后用丙酸调节细菌素粗提物的pH进行抑菌试验,结果表明,pH对粗提得到的细菌素抑菌性影响很大,当用丙酸将细菌素溶液的pH调至5.5时,细菌素的抑菌活性有显著提高。     

响应面法优化植物乳杆菌LPL-1产细菌素发酵条件及细菌素理化性质分析

为提高新型植物乳杆菌LPL-1所产细菌素（植物乳杆菌素LPL-1）的产量,以单核细胞性李斯特菌为指示菌,相对抑菌效价为响应值,通过响应面法对发酵条件进行优化,确定了最优发酵条件。利用单因素试验与Plackett-Burman试验,确定主要影响因素为温度、发酵时间与初始pH值,通过最陡爬坡试验与Box-Behnken响应面试验,确定最优发酵条件为发酵温度31?℃、培养基初始pH?6.40、发酵时间32?h、接种量0.5%、装液量60%,在此条件下细菌素效价（674.29?AU/mL）比优化前（292.02?AU/mL）提高了1.31倍。通过对细菌素理化性质的分析,证明了细菌素具有热稳定性（100?℃,30?min）、酸碱稳定性（pH?2～10）、蛋白酶敏感性与抑菌性,同时利用二硫键变性剂对细菌素结构中的二硫键进行变性处理,证明二硫键对其抑菌特性的重要性。因此,通过细菌素发酵条件的优化与理化性质的分析,为菌种与细菌素的产业化生产与应用提供了理论支持。     

响应面法优化双歧杆菌B04代谢产细菌素的发酵条件

以分离自中国广西巴马长寿老人肠道的产细菌素动物双歧杆菌(Bifidobacterium animal)B04为研究对象,以单核细胞增生李斯特菌为细菌素抑菌活性测试指示菌,以相对抑菌效价为考察指标,对双歧杆菌菌株B04所产细菌素的发酵条件进行优化。首先考察不同接种量、培养时间、培养温度及起始pH值4个因素对细菌素合成的影响,确定培养时间、培养温度及起始pH值3个因素对细菌素的相对抑菌效价影响较为显著,故再选取这3个因素进行响应面试验优化。获得优化后的最佳培养条件确定为:接种量1%、起始pH 6.3、培养温度36℃、培养时间29h。在此优化条件下,细菌素相对抑菌效价高达3479.25AU/mL,比优化前(1032.66AU/mL)提高了2.37倍。同时,最优发酵条件下获得的实验结果与模型预测值相吻合,说明所建立的回归模型是切实可行的。     

发酵条件对费氏丙酸杆菌代谢物抑菌活性的影响

费氏丙酸杆菌代谢物是一种新型的天然防腐剂。试验研究氮源、碳源表面活性剂和温度对费氏丙酸杆菌代谢物抑菌活性的影响。结果表明,葡萄糖是其最适碳源,抑菌活性最高,为19AU/mL,玉米浆为最适氮源,抑菌活性为17.4AU/mL。0.1%的Tween80能提高代谢物的抑菌活性,最高为23.6AU/ mL。产生丙酸杆菌代谢物的最适培养温度为30℃。     

1株产细菌素乳酸菌的鉴定及所产细菌素的诱导合成现象

从发酵大蒜中筛选到一株具有抑菌作用的菌株Br26,通过用不同蛋白酶处理判断该菌株是否为细菌素产生菌。随后,通过分析自身发酵上清液与其他乳酸菌对细菌素合成的影响,探讨该细菌素的诱导合成现象。结果表明：菌株Br26的发酵上清液经胃蛋白酶和木瓜蛋白酶处理后抑菌活性降低,确定该菌为细菌素产生菌,经16S rDNA鉴定为Weissella sp. Br26。Weissella sp. Br26在42 ℃、1/4 MRS培养时,细菌素抑菌活性消失,而当添加不同生长时期的发酵上清液时,抑菌活性显著增加,表明该细菌素可进行自我诱导。另一方面,Weissella sp. Br26与卷曲乳杆菌、瑞士乳杆菌、副干酪乳杆菌和肠膜明串珠菌的活菌共同培养后,发酵液pH值未有明显变化,但细菌素抑菌活性显著增加,表明细菌素的合成亦可受其他乳酸菌的诱导。综上,Weissella sp. Br26细菌素的合成是受自身发酵液及其他菌诱导调控的。     

丙酸杆菌素高产菌株的诱变选育

丙酸杆菌素是乳品丙酸杆菌代谢合成的一种重要的多肽或蛋白质,能抑制革兰氏阴性菌、部分革兰氏阳性菌、霉菌和酵母菌的生长。以丙酸杆菌Propionibacterium feudenreichiiCS01为出发菌株,采用紫外(UV)和亚硝基胍(NTG)复合诱变的方法,获得高产突变株N11和N42,其抑菌活性达到28.63、28.83AU/mL,分别比原始出发菌株(21.14AU/mL)提高了35.4%和36.4%。     

Enhancement of trehalose production in dairy propionibacteria through manipulation of environmental conditions

We have shown that the ability to produce trehalose is widespread within the genus Propionibacterium. Eighteen strains isolated from dairy sources were screened for trehalose synthesis; the effect of environmental conditions on trehalose production was evaluated in Propionibacterium freudenreichii ssp. shermanii NIZO B365, a strain that accumulated high amounts of this disaccharide. Lactose was the best carbohydrate source for trehalose production, whereas lactate, the substrate that led to the highest specific growth rate, was a poor precursor. Trehalose was consumed after exhaustion of the carbon source in the medium, suggesting its role as a reserve compound. The production of trehalose was not affected by lowering the growth temperature from 30 to 20 degrees C. On the other hand, the maximum trehalose accumulation increased from about 200 to 400 mg of trehalose/g of cell protein upon decreasing the pH from 7.0 to 4.7, by increasing the concentration of NaCl to 2% (w/v), or during growth under aerobic conditions (50% air saturation, 24 microM O(2), pH 7.0). In the absence of NaCl, trehalose accumulated concomitantly with growth, but an increase in salinity triggered a high trehalose production already in the early exponential growth phase. The data provide evidence for a dual function of trehalose as a reserve compound and as a stress-response metabolite. Moreover, P. freudenreichii ssp. shermanii NIZO B365 was able to produce high levels of trehalose in skim milk, which is promising for the implementation of fermented dairy products.     

Research on some factors influencing acid and exopolysaccharide produced by dairy propionibacterium strains isolated from traditional homemade Turkish cheeses

In this study, a total of 32 isolated strains and 5 reference strains of dairy propionibacteria were analyzed for acid and exopolysaccharide (EPS) production in skim milk and yeast extract-lactate broth (YEL) media in order to investigate the physiological background and preservative role of acid and EPS. The effects of final culture pH and optical density on acid and EPS production were also determined. On average, all strains produced more acid and reached lower final pH values in skim milk than in YEL medium. While the correlations obtained between the acid produced by propionibacterium strains and their final culture pH in skim milk medium were significant (P < 0.01), no correlations were found between optical density, final pH, and produced acid in YEL medium. Sixteen isolated and five reference strains of propionibacteria were tested further for the ability to produce propionic and acetic acids. On average, Propionibacterium freudenreichii subsp. shermanii and P. freudenreichii subsp. freudenreichii strains produced higher amounts of propionic and acetic acids than did Propionibacterium jensenii in YEL medium. The acid produced by these strains may be used as a preservative in the food industry for replacement or reduction of the increasing use of chemical additives. The EPS production by propionibacterium strains during growth in YEL medium was 72 to 168 mg/liter, while in skim milk it was 94 to 359 mg/liter. The monomer compositions of the EPSs formed by the six selected dairy propionibacteria strains were analyzed. The EPSs may have applications as food grade additives and viscosity-stabilizing agents.     

Capsular exopolysaccharide biosynthesis gene of Propionibacterium freudenreichii subsp. shermanii

In the dairy industry, exopolysaccharides (EPS) contribute to improving the texture and viscosity of cheese and yoghurt and also receive increasing attention because of their beneficial properties for health. For lactic acid bacteria, the production of EPS is well studied. However, for dairy propionibacteria the biosynthesis of EPS is poorly documented. A polysaccharide synthase-encoding gene was identified in the genome of Propionibacterium freudenreichii subsp. shermanii TL 34 (CIP 103027). This gene best aligns with Tts, the polysaccharide synthase gene of Streptococcus pneumoniae type 37 that is responsible for the production of a beta-glucan capsular polysaccharide. PCR amplification showed the presence of an internal fragment of this gene in twelve strains of P. freudenreichii subsp. shermanii with a ropy phenotype in YEL+ medium. The gene sequence is highly conserved, as less than 1% of nucleotides differed among the 10 strains containing the complete gtf gene. The same primers failed to detect the gene in Propionibacterium acidipropionici strain TL 47, which is known to excrete exopolysaccharides in milk. The presence of (1-->3, 1-->2)-beta-d-glucan capsule was demonstrated for 7 out of 12 strains by agglutination with a S. pneumoniae-type 37-specific antiserum. The presence of mRNA corresponding to the gene was detected by RT-PCR in three strains at both exponential and stationary growth phases. This work represents the first identification of a polysaccharide synthase gene of P. freudenreichii, and further studies will be undertaken to elucidate the role of capsular EPS.     

Inhibition of Clostridium tyrobutyricum by bacteriocin-like substances produced by lactic acid bacteria

Lactic acid bacteria were selected for their inhibitory activity against Clostridium tyrobutyricum under conditions that eliminate the effects of lactic acid and hydrogen peroxide. Four strains were isolated belonging to the species Lactococcus lactis ssp. lactis. The sensitivity of the inhibitory substances to pronase and trypsine indicates that they are proteins or peptides different from nisin. Their resistance to phospholipase D indicates that they are also different from lactostrepcin. The inhibitory substances are produced during the exponential phase of growth. Their activity is bactericidal and directed toward some strains of Clostridium tyrobutyricum, Lactobacillus helveticus, and Streptococcus thermophilus, but strains used as dairy starters, Lactobacillus lactis, Streptococcus thermophilus, and Propionibacterium shermanii, are not all affected by the inhibition.     

Characterization of the 16S-23S and 23S-5S rRNA intergenic spacer regions of dairy propionibacteria and their identification with species-specific primers by PCR.

In this study, the 16S-23S and 23S-5S rRNA intergenic spacer region sequences of Propionibacterium acidipropionici, P. freudenreichii ssp. freudenreichii and ssp. shermanii, P. jensenii and P. thoenii were determined. The sequences were shown to vary greatly between the species. Specific primer pairs were derived from the 16S-23S rRNA spacer sequences and used for the identification of the species by PCR.     

Production of acetic and propionic acids from labneh whey by fermentation with propionibacteria

Whey produced during the manufacture of labneh was supplemented with yeast extract (10 g/1), and then fortified with lactose, treated with β-galactosidase or fermented with Lactobacillus helveticus, prior to inoculation with free living cells of Propionibacterium freudenreichii ssp shermanii or Propionibacterium acidipropionici or cells immobilized in aliginate beads. Under anaerobic batch conditions, fermentation of the whey with Lb helveticus followed by P acidipropionici (free cell system) for 2.5 days at 32°C gave a broth with 5.9 g/l of propionic acid and 2.4 gll of acetic acid, while immobilized cells of the same organisms gave a broth with 11.0 gll propionic acid and 3.2 g/l acetic acid over 4 days. These latter values were the maximum levels recorded with any of the treatments, and it is suggested that such yields might make recovery economically feasible in certain countries.     

Assessment of root uptake and systemic vine-transport of Salmonella enterica sv. Typhimurium by melon (Cucumis melo) during field production

Bifidobacterium adolescentis Int57 (Int57) and Propionibacterium freudenreichii subsp. shermanii ATCC 13673 (ATCC 13673) were grown either in coculture or as pure cultures in different media, such as cow's milk, soybean milk, and modified MRS medium. The viable cell counts of bacteria, changes in pH, concentrations of organic acids, and contents of various sugars were analyzed during incubation up to 7days. In soy milk, the survival of cocultured Int57 was six times higher than the monocultured cells, and ATCC 13673 cocultured with Int57 consumed 69.4% of lactic acid produced by Int57 at the end of fermentation. In cow's milk, coculture with ATCC 13673 increased the growth of Int57 from 24h until 120h by approximately tenfold and did not affect the survival of Int57 cells. After 96h of fermentation of modified MRS, the survival of ATCC 13673 cells cocultured with Int57 increased by 3.2- to 7.4-folds as compared with ATCC 13673 monoculture, whereas the growth of Int57 cells was unaffected. The growth and metabolic patterns of two strains during coculture showed noticeable differences between food grade media and laboratory media. The consumption of stachyose in soy milk during coculture of Int57 with ATCC 13673 was increased by more than twice compared with Int57 monoculture, and completed within 24h. The combinational use of Bifidobacterium and Propionibacterium could be applied to the development of fermented milk or soy milk products.     

Ability of Lactobacillus and Propionibacterium strains to remove aflatoxin B, from the chicken duodenum

The ability of Lactobacillus rhamnosus strains GG and LC-705 to remove AFB1 from the intestinal luminal liquid medium has been tested in vivo using a chicken intestinal loop technique. In this study, the GG strain of L. rhamnosus decreased AFB1 concentration by 54% in the soluble fraction of the luminal fluid within 1 min. This strain was more efficient in binding AFB1 compared with L. rhamnosus strain LC-705 (P < 0.05) that removed 44% of AFBl under similar conditions. Accumulation of AFB1 into the intestinal tissue was also determined. There was a 74% reduction in the uptake of AFB1 by the intestinal tissue, in the presence of L. rhamnosus strain GG compared with 63% and 37% in the case of Propionibacterium freudenreichii ssp. shermanii JS and L. rhamnosus strain LC-705, respectively. The complexes formed in vitro between either L. rhamnosus strain GG or L. rhamnosus strain LC-705 and AFB1 were stable under the luminal conditions for a period of 1 h.     

Production of extracellular bifidogenic growth stimulator (BGS) from Propionibacterium shermanii using a bioreactor system with a microfiltration module and an on-line controller for lactic acid concentration

Production of a bifidogenic growth stimulator (BGS) by Propionibacterium freudenreichii subsp. shermanii (Propionibacterium shermanii) using lactic acid as a carbon source was investigated using different cultivation methods. When a continuous bioreactor system with a filtration device was used at a dilution rate of 0.075 h(-1), the average BGS concentration was 2.4 mg/l, which corresponds to a BGS productivity per cultivation time of 1.8 x 10(-1) mg x l(-1) x h(-1). The BGS productivity per cultivation time in continuous cultivation with filtration was 1.9-fold that (9.4 x 10(-2) mg x l(-1).h(-1)) in a conventional batch cultivation. In fed-batch cultivation with feed-back control using an on-line lactic acid controller with a lactic acid biosensor, it was possible to prevent substrate inhibition by maintaining the lactic acid concentration in culture broth low at 3.3 g/l, and an enhanced BGS production (31 mg/l) was successfully attained. The BGS productivity per cultivation time (2.1x10(-1) mg x l(-1) x h(-1)) in the fed-batch cultivation with feed-back control was 2.2-fold that in the conventional batch cultivation. A new bioreactor system was developed by coupling a continuous bioreactor system with a filtration device to an on-line lactic acid controller. Using the new bioreactor system, we produced BGS continuously at a high level of 47 mg/l. The BGS productivities per cultivation time (3.5 mg.l(-1) x h(-1)) and the total volume of medium used (1.7 x 10(-1) mg x l(-1) x h(-1)) obtained in the new bioreactor system were 37-fold and 2.1-fold those in the conventional batch cultivation, respectively. These results described above clearly demonstrate the positive effects of both the continuous filtration for removal of metabolites (propionic and acetic acids) inhibitory to cell growth and feed-back control of lactic acid concentration in the culture broth on BGS production by P. shermanii. This paper is the first report on BGS production by the propionic acid bacterium using lactic acid as a carbon source.     

丙酸杆菌发酵条件的优化

讨论pH值和不同碳源对丙酸杆菌的影响,同时利用正交实验优化了此菌发酵的培养基。