Aerobic culture of Propionibacterium freudenreichii ET-3 can increase production ratio of 1,4-dihydroxy-2-naphthoic acid to menaquinone

This is the first report on the production of both 1,4-dihydroxy-2-naphthoic acid (DHNA) and menaquinone by Propionibacterium freudenreichii ET-3. DHNA can be a stimulator of bifidogenic growth, and menaquinone has important roles in blood coagulation and bone metabolism. During anaerobic culture, DHNA and menaquinone concentrations reached 0.18 mM and 0.12 mM, respectively. The molar ratio between these products was approximately 3:2, which was not affected by culture pH and temperature over the ranges of 6.0-7.0 and 31-35 degrees C, respectively. As for organic acid, propionate and acetate accumulated at concentrations of 0.3 M and 0.15 M, respectively, and the propionate accumulation particularly inhibited further production of DHNA. To improve DHNA production, we switched from anaerobic condition to aerobic condition during the culture when lactose was depleted. DHNA concentration continued to increase even after lactose exhaustion, reaching 0.24 mM. In contrast to DHNA production, menaquinone production stopped after the switch to aerobic condition. The total molar production of DHNA and menaquinone was 0.3 mM irrespective of aerobic culture and anaerobic-aerobic switching culture. Therefore, the anaerobic-aerobic switching culture could increase the production ratio of DHNA to menaquinone. The DHNA concentration obtained from the anaerobic-aerobic switching culture was 1.3-fold higher than that in the anaerobic culture, because P. freudenreichii ET-3 utilized propionate accumulated in the medium via the reversed methylmalonyl CoA pathway under aerobic condition. The culture method proposed in this study could be applicable to industrial-scale fermentation using 1000 l of media, by which 0.23 mM DHNA was produced.     

Optimal aerobic cultivation method for 1,4-dihydroxy-2-naphthoic acid production by Propionibacterium freudenreichii ET-3

To investigate the effects of oxygen supply on Propionibacterium freudenreichii ET-3 metabolism and 1,4-dihydroxy-2-naphthoic acid (DHNA) production in detail, the strain was cultured by switching from anaerobic condition to aerobic condition at 72 h (termed anaerobic-aerobic switching culture hereafter) employing different oxygen transfer rates (OTRs) in the range of 0.08-0.90 mg/(l.h). It was found that a 0.08 mg/(l.h) OTR could not change the metabolism or improve the DHNA production of P. freudenreichii ET-3. When the OTR was in the range of 0.23-0.66 mg/(l.h), propionate, which inhibits DHNA production significantly, was consumed during the aerobic phase. Final DHNA concentration increased to 0.22 mM, irrespective of OTR. When the OTR was 0.90 mg/(l.h), a sudden increase in dissolved oxygen (DO) concentration during the aerobic phase resulted in a sudden decrease in DHNA concentration. To attenuate the stresses caused by propionate and oxygen exposure, we designed an optimal cultivation in which the anaerobic and aerobic phases were repeated three times alternately. As a result, propionate concentration was maintained below the level that inhibits DHNA production, and no DO concentration was detected throughout the culture. The final DHNA concentration in this culture was 0.33 mM, which is 2.7-fold that in the anaerobic culture and 1.5-fold that in the anaerobic-aerobic switching culture.     

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.     

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.     

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.     

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.     

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.     

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。     

Viability and beta-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids

An important criterion to consider in the selection of strains for dietary adjuncts is the ability of the microorganisms to survive the severe conditions of acidity and bile concentrations usually found in the gastrointestinal tract. In the present work, we report the effects of digestions by artificial gastric and intestinal fluids on beta-galactosidase activity and survival of four strains of dairy propionibacteria previously selected by their bile tolerance and beta-galactosidase activity. The strains were exposed to artificial gastric juice at pH values between 2 and 7 and then subjected to artificial intestinal digestion. Both viability and beta-galactosidase activity were seriously affected at pH 2. Skim milk and Emmental cheese juice exerted a protective effect on the parameters tested. The trypsin present in the intestinal fluid inactivated the enzyme beta-galactosidase in strains of Propionibacterium freudenreichii but not in Propionibacterium acidipropionici. Moreover, the presence of bile salts enhanced the beta-galactosidase activity of these strains by permeabilization of the cells during the first hour of exposure. The intestinal transit rate confirmed the permanence of the bacteria in the intestine for long enough to be permeabilized. These results suggest that P. acidipropionici would be a good source of beta-galactosidase activity in the intestine. We also propose a practical and effective in vitro method as a tool of screening and selection of potential probiotic bacteria.     

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。     

Chemical studies with silage microorganisms in artificial media and sterile herbages

The fermentation changes in artificial media and in sterile herbages inoculated with strains of yeast, hetero-and homo-fermentative lactic acid bacteria isolated from silage were studied in two experiments. In the first, using malt extract broth, four treatments were employed: aerobic, high level of glucose; aerobic, low level of glucose; anaerobic, high level of glucose; anaerobic, low level of glucose. The pH decreased in all cultures. There were no appreciable differences in pH among the cultures of the heterofermentative lactic acid bacterium, but in the cultures of the homofermentative organism there was a greater decrease in pH aerobically than anaerobically. The lowest pH values in the yeast cultures, occurred in the high glucose treatments both aerobically and anaerobically. In all the yeast cultures, acetic, propionic, butyric, isobutyric and isovaleric acids were present. The heterofermentative lactic acid bacterium produced only acetic acid, the greatest amount occurring under aerobic conditions. Ethanol was detected in the culture of the heterofermentative lactic acid bacterium but not in that of the homofermentative organism. Relatively large amounts of i-pentanol were found in the aerobic yeast cultures. In the second experiment, inoculated grass was ensiled with similar treatments to those used in the first experiment. Two incubation periods, 3d and 10d were used. The pH values in all treatments were high (> pH 5.0). In aerobic conditions, the lactic acid contents after the 10d incubation were higher than after 3d. The losses of water soluble carbohydrates during fermentation were greater in the aerobic than anaerobic treatments. Acetic acid was present in all cultures but the higher volatile fatty acids (C₃-iC₅) were only detected in appreciable amounts in the aerobically treated herbages after 10d. In addition to ethanol, n-propanol and i-butanol were also present in all the 10d anaerobic treatments. i-Pentanol occurred in both the aerobic and anaerobic treated herbages.     

GROWTH RESPONSE OF A SALMONELLA TYPHIMURIUM POULTRY ISOLATE TO PROPIONIC ACID UNDER AEROBIC AND ANAEROBIC CONDITIONS

Organic acids have long been used as additives in poultry feed to reduce microbial populations, including Salmonella spp. Propionic acid addition to poultry feed has a potential role in reducing Salmonella spp. in the chicken intestine. In this study , in vitro growth response of S. typhimurium isolated from poultry to propionic acid under aerobic and anaerobic conditions was determined. When grown in tryptic soy broth (TSB) containing buffered propionic acid (BPA), the growth rate of S. typhimurium gradually decreased as the level of BPA increased and broth pH decreased. No growth was detected with BPA concentrations greater than 3% (volume/volume) of the broth. When the growth rates of S. typhimurium in aerobic and anaerobic TSB were compared at two pH levels (pH 5.0 and 7.0), the growth inhibition of S. typhimurium by propionic acid was markedly suppressed by anaerobiosis at both pHs, as indicated by significantly (p<0.05) higher half-inhibition constants (K°). Also, the growth rates of S. typhimurium in the presence of propionic acid were dramatically reduced by the decrease in pH from 7.0 to 5.0. The results of this study indicated that the growth inhibitory effect of propionic acid against S. typhimurium strains was enhanced by pH decrease and suppressed by anaerobiosis, suggesting that the growth response of S. typhimurium to propionic acid in the chicken intestine might be affected by the environmental conditions such as pH and anaerobiosis.     

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.     

Influence of manufacturing conditions on the conjugated linoleic acid content and the isomer composition in ripened French Emmental cheese

In a study of the evolution of conjugated linoleic acid (CLA) during cheese production, the influence of Emmental cheese processing on the CLA content and the CLA isomer composition was evaluated. The use of raw and thermised milk, changes of processing temperature and the effect of propionic acid bacteria (PAB) were investigated. The content of CLA in raw milk was 8.6 +/- 1.9 mg/g fat and in the ripened cheese at 70 d was 8.6 +/- 1.6 mg/g fat, under normal processing conditions. No changes in the CLA content and CLA isomer composition were observed during Emmental cheese manufacturing process. Changes in cooking and moulding temperatures did not influence the CLA content. CLA content of cheese made from microfiltered milk with two different Propionibacterium freudenreichii strains was very close to cheeses made without PAB. CLA levels seem to be stable in this type of dairy product under the conditions examined.     

Industrial implementation of black ripe olive storage under acid conditions

Restrictions on the discharge of chloride in wastewater streams have recently increased, and processors of black ripe olives have to look for alternative storage systems to the traditional brines. Ripe olives of the Hojiblanca variety were stored under aerobic and anaerobic conditions in industrial underground tanks for one year. The aerobic systems assayed with or without NaCl produced a continuous consumption of sugars as they diffused from the fruits to the surrounding liquid. At the same time sugars accumulated in the liquid for months when anaerobic conditions were employed and a high concentration of acetic acid was used. In the end, glucose was consumed with time as well and, in addition to yeasts, acetic acid and lactic acid bacteria grew in the cover solutions. The assessment of olives stored under the different systems and processed as black ripe olives revealed that the traditional aerobic brines gave rise to darker and firmer fruits. However, olive industries must eliminate chlorides from their waste streams. Promising new storage systems were (i) anaerobic preservation of olives in water with an initially high concentration of acetic acid, and (ii) aerobic preservation in water with an initially low concentration of acetic acid. Both systems produced a good quality product, free of microbial spoilage and organoleptic defects.     

外源有机酸对于Propionibacterium freudenreichii CCTCC M207015发酵生产丙酸的影响

考察了外源有机酸(丙酸、乙酸、琥珀酸)对于游离细胞与纤维床反应器生产丙酸的影响。结果表明,以Propionibacterium freudenreichii CCTCC M207015为生产菌株时,纤维床反应器具有比游离细胞更强的丙酸、乙酸耐受性,其中丙酸的抑制现象比乙酸的更加严重。此外,外源琥珀酸可以作为丙酸合成的前体物质对P.freudenreichii CCTCC M207015发酵生产丙酸产生促进作用。     

Biochemical changes during the preservation stage of ripe olive processing

The influences of initial sodium chloride (6% and 0% w/v in tap water) and acetic acid concentrations (0.3%, and 0.6% v/v), use of starter culture, and aerobic versus anaerobic conditions on the biochemical changes that take place throughout the preservation stage of ripe olive processing were investigated. Glucose, fructose and sucrose were completely consumed during preservation. Mannitol and malic acid were metabolized only in the presence of lactic acid bacteria or oxidative yeast (aerobic treatment). The main metabolites produced were lactic and acetic acid in aerobic or anaerobic treatments inoculated with Lactobacillus plantarum. Methanol and ethanol were present in all the brines although in a lower concentration when conditions were aerobic. Thus, induced lactic fermentation led to the most efficient utilization of carbohydrates and yielded the most suitable physicochemical characteristics for ripe olive preservation.     

粗玉米抗性淀粉的模拟肠道发酵产酸与外源乳酸菌关联性

在模拟大肠环境(厌氧和37℃)下,利用健康成人粪便提取物发酵粗玉米抗性淀粉,研究短链脂肪酸的产生情况,并剖析外源乳酸菌对产酸模式的影响。实验结果表明,粪便提取物单独发酵抗性淀粉时,短链脂肪酸含量随发酵时间增加而呈增加趋势;粪便提取物和2株乳酸杆菌共同发酵粗玉米抗性淀粉时,发酵产物中乙酸含量增加,丙酸和丁酸含量没有明显变化;当粪便提取物和2株乳酸球菌共同发酵粗玉米抗性淀粉时,发酵产物中丙酸和丁酸含量明显增加,乙酸含量没有明显变化。研究结果表明,外源性乳酸菌影响粗玉米抗性淀粉的模拟肠道发酵产酸模式。