Autolysis of propionibacteria: detection of autolytic enzymes by renaturing SDS-PAGE and additional buffer studies

Five strains of propionibacteria with 70-90% autolysis in sodium lactate broth (SLB) were studied by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several lytic bands ranging in size between 25 and 143 kDa were detected by using propionibacteria cells or cell walls as substrate in the gel. Four Propionibacterium freudenreichii strains showed similar autolytic-enzyme profiles, consisting of two autolytic bands, one with molecular mass 162 kDa and one in the range 123-143 kDa. However, the Propionibacterium acidipropionici strain showed a completely different profile, consisting of 8 autolytic bands with molecular masses of 122, 97, 71, 55, 43, 39, 31, and 25 kDa. Lytic enzymes from P. freudenreichii INF-alpha, P. freudenreichii ISU P-59, P. freudenreichii ISU P-24, and P. freudenreichii ISU P-50 showed lytic activity against cells from all these four strains, but not against P. acidipropionici ATCC 4965. However, P. acidipropionici ATCC 4965 autolysed only its own cells. Effects of pH, temperature, and ions on autolytic activity were tested by renaturing SDS-PAGE and in buffer systems. Results from the SDS-PAGE electrophoresis showed optimal autolytic activity of P. acidipropionici ATCC 4965 at 37 degrees C and in the pH range 7 to 8.5 and of P. freudenreichii ISU P-59 at 20 degrees C and in the pH range 5 to 7. The autolytic activity of P. acidipropionici ATCC 4965 was extremely heat stable (100 degrees C, 2 h), in contrast to the lytic activity of P. freudenreichii ISU P-59, which was heat labile. The autolytic activities of P. acidipropionici ATCC 4965 were inhibited by divalent cations, however, the lytic activities of P. freudenreichii ISU P-59 were activated by Mn(2+), Ca(2+), and Co(2+). In buffer, optimum autolysis of P. acidipropionici ATCC 4965 was observed at pH 8.5 and at 40 degrees C. P. freudenreichii ISU P-59 showed optimum autolysis in buffer at pH 7.5 and at 30 degrees C.     

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.     

Production of vitamin B12 in genetically engineered Propionibacterium freudenreichii

Since the chemical synthesis of vitamin B12 requires more than 70 steps, the production of vitamin B12 has been achieved by microorganism fermentation with additional brief chemical modifications. In an effort to increase the productivity of vitamin B12, we tried to express 10 genes belonging to the hem, cob and cbi gene families involved in the synthesis of vitamin B12 in Propionibacterium freudenreichii, which is a known producer of vitamin B12. In a recombinant P. freudenreichii clone that harbored the expression vector containing a cobA, cbiLF, or cbiEGH, we obtained an increase in vitamin B12 production of 1.7-, 1.9-, and 1.5-fold higher, respectively, than that in the microorganism without any cloned genes in the expression vector pPK705. The cobU and cobS genes caused a slight increase in the production of vitamin B12. Furthermore, we achieved multigene expression in P. freudenreichii. In a recombinant P. freudenreichii clone that harbored an exogenous gene, hemA, from Rhodobacter sphaeroides and endogenous hemB and cobA genes, we successfully achieved the production of about 1.7 mg/l vitamin B12, 2.2-fold higher than that produced by P. freudenreichii harboring pPK705.     

Antifungal effect of dairy propionibacteria--contribution of organic acids

Large amounts of food and feed are lost every year due to spoilage by moulds and yeasts. Biopreservation, i.e. the use of microorganisms as preservatives instead of chemicals, has gained increased interest. Lactic acid bacteria and propionibacteria might be particularly useful due to their important role in many food fermentations. Knowledge of the antifungal effects of the organic acids produced by these bacteria is necessary to understand their inhibitory activity. We evaluated the antifungal activity of the type strains of five dairy propionibacteria, Propionibacterium acidipropionici, P. jensenii, P. thoenii, P. freudenreichii subsp. freudenreichii and P. freudenreichii subsp. shermanii against eight food- and feedborne moulds and yeasts. A dual culture system assayed the inhibitory activity on three different agar media, sodium lactate (SL), de Man Rogosa Sharp (MRS) and MRS without acetate (MRS-ac). The amounts of organic acids produced during growth of propionibacteria in liquid SL, MRS and MRS-ac were also determined. The minimal inhibitory concentration (MIC) values of propionic, acetic and lactic acid were established for all fungi at pH 3, 5 and 7. Propionic acid, followed by acetic acid, was the most potent antifungal acid. Inhibition at pH 7 generally required concentrations above 500 mM for all three acids, at pH 5 the MIC values for propionic and acetic acids were 20-120 mM and above 500 mM for lactic acid. At pH 3, the MIC values were, with one exception, below 10 mM for both propionic and acetic acid and above 160 mM for lactic acid. The yeast Pichia anomala was the fungus most resistant to organic acids. The propionibacteria exhibited a pronounced species variation in antifungal activity on MRS (+/-acetate) agar, with P. thoenii being the most potent. Four of the five propionibacteria species produced more propionic and acetic acid in liquid SL medium than in MRS (+/-acetate) broth. However, when SL agar was used as the growth medium, none of the propionibacteria inhibited fungal growth.     

丙酸对费氏丙酸杆菌生长及脱氧腺苷钴胺素合成的影响

通过添加控制丙酸浓度考查了费氏丙酸杆菌发酵生产VB12的过程中丙酸抑制细胞生长的浓度范围,在此基础上利用树脂对发酵过程中丙酸进行了选择性吸附后细胞的生长和脱氧腺苷钴胺素合成的变化。研究结果表明,丙酸的浓度在10.0g/L时,对菌体细胞生长产生了明显的抑制。在丙酸浓度积累到10.0g/L之前,利用树脂对其吸附分离出2.5g/L的丙酸后,生物量提高了37.5%,脱氧腺苷钴胺素产量提高了50.0%。该实验为实现丙酸的在线分离和丙酸/VB12高效联产的新型耦合发酵工艺提供了基础。     

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.     

The influence of Lactobacillus rhamnosus LC705 together with Propionibacterium freudenreichii ssp. shermanii JS on potentially carcinogenic bacterial activity in human colon

The bacterial enzymes beta-glucosidase, beta-glucuronidase, and urease may contribute to the development of colon cancer by generating carcinogens. A reduction in the activity of these enzymes by certain lactic acid bacteria is considered to be beneficial. This study examined fecal beta-glucosidase, beta-glucuronidase, and urease activities during administration of Lactobacillus rhamnosus LC705 (LC705) together with Propionibacterium freudenreichii ssp shermanii JS (PJS). Thirty-eight healthy men participated in this randomized, double-blind, placebo-controlled, two-period crossover study with treatment periods of 4 weeks. Subjects consumed daily bacterial or placebo capsules. Bacterial capsules contained viable LC705 and PJS (2x10(10) CFU of each strain daily). The activities of beta-glucosidase, beta-glucuronidase and urease, recovery of LC705 and PJS, and counts of total lactobacilli and propionibacteria were determined from feces. The mean fecal counts of total lactobacilli and propionibacteria as well as strains LC705 and PJS were significantly increased during the administration of bacteria (3.5-, 13-, 80- and 11-fold, respectively). beta-glucosidase activity decreased by 10% (P=0.18) and urease activity by 13% (P=0.16) during bacterial supplementation versus placebo. The change in beta-glucosidase activity was negatively correlated with the change in propionibacteria counts (R=-0.350, P=0.039), being -2.68 versus 0.94 nmol/min/mg protein in subjects with increased and unchanged/decreased propionibacteria, respectively (P=0.003). To conclude, the administration of LC705 and PJS was followed by an increase in the fecal counts of lactobacilli and propionibacteria and a decrease in the activity of beta-glucosidase with increasing counts of propionibacteria.     

Heterologous expression of a gene encoding cholesterol oxidase in probiotic strains of Lactobacillus plantarum and Propionibacterium freudenreichii under the control of native promoters

To develop systems for the expression of heterologous genes in probiotic strains of Lactobacillus and Propionibacterium, we used Lactobacillus plantarum and Propionibacterium freudenreichii and a modified gene encoding cholesterol oxidase (choA) from Streptomyces sp. to generate working models. The acetyl coenzyme A carboxylase (acc) promoter derived from the acc operon of L. plantarum L137 and a previously constructed shuttle vector, pRN14, were used to construct vectors for the expression of heterologous genes in lactic acid bacteria. The concentration of cholesterol oxidase in recombinant L. plantarum carrying choA fused to the NH2-terminal region of the first open reading frame of the acc operon was 3.6 mU/mg of protein. Using the promoters from Propionibacterium, namely, P4, P8, and P138, which enabled high-level expression of choA in Escherichia coli, and a previously constructed shuttle vector pPK705, we constructed expression vectors for Propionibacterium. In recombinant P. freudenreichii subsp. shermanii IFO12426, the activities of cholesterol oxidase generated under the control of promoters P4, P8, and P138 were 1.6, 4.3, and 7.2 U/mg of protein, respectively. The expression of heterologous genes may facilitate the production of useful proteins in these economically important bacteria.     

费氏丙酸杆菌发酵生产V_B_(12)

VB12广泛应用于医药业和食品工业,费氏丙酸杆菌是其主要的工业生产菌种。费氏丙酸杆菌发酵过程中会产生大量丙酸和乙酸,有机酸的积累会抑制菌体生长,延长发酵周期。通过改进发酵工艺来解除有机酸抑制,提高发酵产量是费氏丙酸杆菌发酵工艺的主要研究内容。VB12是费氏丙酸杆菌胞内产物,提取工艺对生产总收率具有重要作用。耦合发酵工艺改造和膜分离技术目前是费氏丙酸杆菌发酵VB12的研究热点。     

费氏丙酸杆菌生物学特性及与5株益生菌拮抗性试验研究

为了评价费氏丙酸杆菌的益生特性,费氏丙酸杆菌活化后,划线和镜检观察其形态特征,然后利用体外共培养法测定费氏丙酸杆菌对3种致病菌的抑制效果,同时利用平板计数法研究费氏丙酸杆菌的耐酸性、耐胆盐和耐高温等生物学特性,以及5株益生菌拮抗性试验。结果表明,费氏丙酸杆菌对大肠杆菌和沙门氏菌没有显著的抑制作用,而对金黄色葡萄球菌的抑制效果明显（P＜0.01）;其在pH值为2.0条件下维持3 h,活菌数降低一个数量级;在胆盐含量为0.30%条件下维持6 h,存活率为18.67%;在60 ℃下处理20 min,存活率为12%,因此费氏丙酸杆菌具有一定的耐酸、耐高温能力以及很强的耐胆盐活性。除枯草芽孢杆菌与丁酸梭菌有拮抗作用外,其余菌株间均没有拮抗作用。     

Substances with biological activity of vitamin B12 formed during cultivation of Propionibacterium freudenreichii in the presence of precursors (short communications)

The papers of Kolhouse et al. and Cooper et al. described the occurrence of vitamin B12 analogues of unknown origin in blood serum. Some of these analogues may be derived from slaughter cattle raised on feed supplemented with vitamins and minerals, as was observed by Allen. Herbert et al. found vitamin B12 analogues in multivitamin preparations produced in U.S.A., and Kanazawa et al. in human liver, red cells and brain. It is not clear so far, if and how do vitamin B12 analogues interfere with vitamin B12 metabolism. When P. freudenreichii was cultivated in the presence of o-phenylenediamine and 5,6-dimethylbenzimidazole, which may be considered precursors as well as antimetabolites of vitamin B12, the stimulation of biosynthesis of substances with biological activity of vitamin B12 took place. Various signs show that these substances are probably vitamin B12 analogues. During stimulated and nonstimulated production of vitamin B12 by P. freudenreichii, two substances with vitamin B12 biological activity have always been obtained. Their relation was not stable and differed according to the conditions of cultivation. Every attempt to stimulate the biosynthesis of vitamin B12 resulted in the suppression of production of the substance with higher molecular weight, even if the biosynthesis of cobalamin (lower molecular weight) was increased. In our note we want to pay attention to the character of substances arising in the stimulated biosynthesis.     

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.     

Changes in protein synthesis during thermal adaptation of Propionibacterium freudenreichii subsp. shermanii

Dairy propionibacteria are present in Graviera Kritis, a traditional Gruyère-type cheese made without added propionic starter. Ten isolated strains were identified by a combination of SDS-PAGE, species-specific PCR and according to their ability to ferment lactose. They were all found to belong to the Propionibacterium freudenreichii subsp. shermanii species. Because of the stressing Gruyère technology, which includes cooking at 52 to 53 degrees C their thermotolerance was investigated at 55 degrees C. Thermotolerant and thermosensitive strains were clearly discriminated. Interestingly, the reference strain CIP 103027 belongs to the sensitive subset. One sensitive strain, ACA-DC 1305 and one tolerant, ACA-DC 1451, were selected for further study and compared to CIP 103027. For the sensitive strains ACA-DC 1305 and CIP 103027, heat pre-treatment at 42 degrees C conferred thermoprotection of cells at the lethal temperature of 55 degrees C, while there was less effect on the tolerant ACA-DC 1451. No cross-protection of salt-adapted cells against heat stress was observed for none of the strains. Differential proteomic analysis revealed distinct but overlapping cell responses to heat stress between sensitive and tolerant strains. Thermal adaptation upregulated typical HSPs involved in protein repair or turnover in the sensitive one. In the tolerant one, a distinct subset of proteins was overexpressed, whatever the temperature used, in addition to HSPs. This included enzymes involved in propionic fermentation, amino acid metabolism, oxidative stress remediation and nucleotide phosphorylation. These results bring new insights into thermoprotection in propionibacteria and the occurrence of divergent phenotypes within a same subspecies.     

鲜牛奶中丙酸杆菌的筛选、鉴定及特性分析

从新鲜生牛奶中分离筛选产丙酸较高的菌株,经形态学特征、生理生化及糖发酵试验、16S rDNA基因序列同源性分析以及多位点序列分型(multilocus sequence typing)等实验鉴定该菌株,分析了其对不同碳源的利用率及产酸情况,并从溶血性试验及抗生素抗性试验方面来评估该菌株的安全性。结果表明,从4批次样品中共分离获得54株能产丙酸的菌株,其中一株菌的丙酸产量达到7.38 g/L,为费氏丙酸杆菌(Propionibacterium freudenreichii)。比较了葡萄糖和甘油对其生长的影响,发现其对葡萄糖的利用率大于对甘油的利用率,在含葡萄糖的培养基中于30 ℃厌氧培养120 h后丙酸产量达到7.38 g/L。该菌株无溶血性,对卡那霉素等氨基糖苷类抗生素有耐药性,对氨苄西林、万古霉素、氯霉素、克林霉素、四环素敏感,对红霉素中介。综上,费氏丙酸杆菌B1具有一定的潜在应用价值。     

The first dairy product exclusively fermented by Propionibacterium freudenreichii: A new vector to study probiotic potentialities in vivo

Dairy propionibacteria display probiotic properties which require high populations of live and metabolically active propionibacteria in the colon. In this context, the probiotic vector determines probiotic efficiency. Fermented dairy products protect propionibacteria against digestive stresses and generally contain a complex mixture of lactic and propionic acid bacteria. This does not allow the identification of dairy propionibacteria specific beneficial effects. The aim of this study was to develop a dairy product exclusively fermented by dairy propionibacteria. As they grow poorly in milk, we determined their nutritional requirements concerning carbon and nitrogen by supplementing milk ultrafiltrate (UF) with different concentrations of lactate and casein hydrolysate. Milk or UF supplemented with 50 mM lactate and 5 g L⁻¹ casein hydrolysate allowed growth of all dairy propionibacteria studied. In these new fermented dairy products, dairy propionibacteria remained viable and stress-tolerant in vitro during minimum 15 days at 4 °C. The efficiency of milk fermented by the most tolerant Propionibacterium freudenreichii strain was evaluated in piglets. Viability and SCFA content in the colon evidenced survival and metabolic activity of P. freudenreichii. This work results in the design of a new food grade vector, which will allow preclinical and clinical trials.     

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.     

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.     

Production of 5-aminolevulinic acid by Propionibacterium acidipropionici TISTR442

Propionibacterium acidipropionici TISTR442 produced the highest amount of 5-aminolevulinic acid (ALA) when cultivated in medium supplemented with glycine at 18g/l. ALA production correlated with ALA synthase activity, whereas ALA dehydratase activity was maintained at a low level. ALA yield reached 405mg/l after prolonged cultivation for 1 month.