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.     

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.     

Genetic manipulation system in propionibacteria

Members of the genus Propionibacterium are widely used in the production of vitamin B12, tetrapyrrole compounds, and propionic acid as well as in probiotic and cheese industries. Shuttle vectors were developed in propionibacteria using replicons from endogenous plasmids in Propionibacterium and Escherichia coli and an appropriate selection marker. The efficient transformation was achieved using the shuttle vector prepared from Propionibacterium freudenreichii to overcome the high restriction modification system in propionibacteria. Expression vectors with native promoters for use in propionibacteria were also developed. Using this system, cholesterol oxidase, which is used as a diagnostic enzyme, was produced in P. freudenreichii. Genes involved in 5-aminolevulinic acid (ALA) and vitamin B12 biosynthesis in propionibacteria were isolated. ALA in propionibacteria could be synthesized via both the C4 pathway (condensation of glycine and succinyl CoA) and the C5 pathway (from glutamate). The hemA gene encoding ALA synthase from Rhodobacter spheroides, was overexpressed and ALA accumulated in P. freudenreichii. Thus, the genetic manipulation systems in propionibacteria will facilitate genetic studies of probiotics and the vitamin B12 biosynthetic pathway.     

Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The enzyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline: bradykinin, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminus or other internal position were not hydrolyzed. The enzyme was most active at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme was inhibited strongly by the serine protease inhibitor 3,4-dichloroisocoumarin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and oligopeptides during the ripening of cheese.     

Autolysis of Lactobacillus sanfranciscensis

 Autolysis of Lactobacillus sanfranciscensis in phosphate buffer was strain-dependent and maximal in the exponential growth phase and at pH 5.6. It decreased to 15% of the maximum at pH 4.5. In culture medium, autolysis did not correspond to the cell death rate because the low pH reached after fermentation limited it markedly. Appreciable autolysis was only found when buffering citrate or fumarate were added to the culture medium. Leakage of peptidases from cells to the culture medium confirmed the autolysis. Renaturating SDS-PAGE showed an autolytic profile composed of four clean lytic bands of ca. 102, 87, 76.5 and 61.5 kDa. This profile was observed in all strains and did not vary with the growth phase or with the use of the crude cell-envelope or crude extracts. Sourdough fermentations lasting 24–48 h showed no differences in the leakage of peptidases when strains which differed greatly for autolysis were compared. However, if citrate was added to the dough it enhanced the release of intracellular peptidases by fast autolytic strains because of the less acidic conditions which favored microbial autolysis during sourdough fermentation. Received: 5 February 1999     

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.     

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.     

Characterization of lytic enzyme activities of Lactobacillus gasseri with special reference to autolysis

Lactobacillus gasseri JCM 1130 and JCM 1131(T) exhibited autolytic activity in agar containing autoclaved cells of each strain as substrate. By zymogram analysis of JCM 1131(T), two lytic bands with apparent molecular masses of 54.5 and 35 kDa, were detected. Similarly, JCM 1130 yielded two lytic bands with apparent molecular masses of 35 and 33.5 kDa. In simple buffers as well, JCM 1131(T) suffered a drastic decrease in cell turbidity, but JCM 1130 did not undergo the decrease. The optimal pH for autolysis of JCM 1131(T) was in the range of 6.0-7.0, and the lysis was completely inhibited at pH 4-5. The lysis of JCM 1131(T) was suppressed by NaCl, in a concentration-dependent way. When subjected to UV irradiation or mitomycin C (MMC) treatment, cultures of both strains elicited conspicuous turbidity decrease after 2-4 h of growth, suggesting the occurrence of prophage induction. The 35-kDa lytic band of JCM 1131(T) and the 33.5-kDa protein of JCM 1130 were considerably increased by UV irradiation.     

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.     

Characterization of thoeniicin 447, a bacteriocin isolated from Propionibacterium thoenii strain 447

Fifteen strains of propionibacteria, isolated from dairy products, were screened for the production of bacteriocins. Propionibacterium thoenii 447 produced an antimicrobial peptide, thoeniicin 447, which acted bactericidal against Lactobacillus delbrueckii subsp. bulgaricus and bacteriostatic against Propionibacterium acnes. Thoeniicin 447 remained active after 15 min at 100 degrees C and after 30 min of incubation at pH 1-10. The peptide was inactivated when treated with pepsin, pronase, alpha-chymotrypsin, trypsin and proteinase K. Optimal bacteriocin production was detected during late exponential growth. The peptide was partially purified by ammonium sulfate precipitation, followed by SP-Sepharose cation exchange chromatography. The estimated size of thoeniicin 447, according to tricine-SDS-PAGE, is 6 kDa. Based on DNA sequencing, the mature peptide is 7130.20 Da in size and homologous to propionicin T1, produced by P. thoenii strain 419 (=NCFB 568(T)). Strain 447 is phenotypically different from strain 419 and belongs to a separate ribotype cluster. To our knowledge, this is the first report of a bacteriocin from a Propionibacterium species active against P. acnes.     

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Production of extracellular bifidogenic growth stimulator by anaerobic and aerobic cultivations of several propionibacterial strains

Production of a bifidogenic growth stimulator (BGS) by propionic acid bacteria was investigated under anaerobic and aerobic culture conditions. To measure the concentration of extracellular BGS produced by propionic acid bacteria, we evaluated the effects of bioassay conditions using Bifidobacterium longum as a test microorganism on the formation of a growth-stimulation zone. The diameter of the growth-stimulation zone was significantly affected by both the component concentrations and the pH of a bioassay medium. The optimum component concentrations and pH of a bioassay medium were one-half of the normal values and 8.5, respectively. Using the bioassay method, we can measure the concentration of BGS produced by propionic acid bacteria ranging in concentrations from 0.1 microg/l to 1 mg/l using 1,4-dihydroxy-2-naphthoic acid (DHNA) and 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ) as standards. Of six dairy propionic acid bacterial strains tested, the four strains (Propionibacterium freudenreichii ET-3, P. shermanii PZ-3, P. acidipropionici JCM 6432, and P. jensenii JCM 6433) produced BGS at a concentration range of 4-23 mg/l under the anaerobic culture conditions. Analysis of high performance liquid chromatography (HPLC) showed that more than 70% of total BGS produced in supernatant samples was DHNA and no ACNQ was produced by the strains. The effect of oxygen supply on BGS production was investigated for the four BGS-producing strains. The aerobic conditions exerted in positive effects on BGS production by only P. acidipropionici JCM 6432. The concentration of BGS obtained in the aerobic cultivation of P. acidipropionici JCM 6432 was 1.3-fold than that in anaerobic cultivation. Different properties (BGS production as well as cell growth and glucose metabolism) occurring in response to the aerobic conditions were observed, depending on the propionic acid bacterial strain used. This paper is the first report on BGS production by propionibacterial strains except for P. freudenreichii.     

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.     

Autolysis of goatfish (Mulloidichthysmartinicus) mince: Characterisation and effect of washing and skin inclusion

Autolysis of goatfish mince and washed mince incubated at different temperatures (30–70 °C) was investigated. The highest autolytic activity was generally observed in mince and washed mince at 60 °C as evidenced by the highest trichloroacetic acid (TCA) soluble peptide content and the greatest disappearance of myosin heavy chains (MHCs). Autolysis of both mince and washed mince was maximised at pH 4, and lower autolytic activity was observed at pH 7. trans-epoxysuccinyl-l-leucyl-amido (4-guanidino) butane (E-64) showed the greatest inhibition of autolysis at pH 4, showing that at least one cysteine protease was active in goatfish muscle. Nevertheless, soybean trypsin inhibitor effectively inhibited the autolysis at neutral pH (pH 7), suggesting that goatfish muscle also contained at least one serine protease. Generally, autolysis of mince was more pronounced than that of washed mince, indicating that washing could lower the autolytic activity of mince. In the presence of skin, a higher autolysis was obtained with the goatfish mince. Therefore, both sarcoplasmic and myofibril-associated proteases in muscle as well as the contamination of skin likely contributed to the degradation of muscle proteins of goatfish.     

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology 180 5947-5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.     

Properties of Proteolytic Enzymes from Muscle of Octopus (Octopus vulgaris) and Effects of High Hydrostatic Pressure

ABSTRACT: The proteolytic activity of octopus arm muscle exhibited optimum activity at 40°C and 60°C, at optimum pH 2.5 and 4.0, respectively. The proteinases were inhibited strongly by cysteine- and aspartic-proteinase inhibitors and, to a lesser degree, by serine-proteinase inhibitors at 40°C, and by cysteine-proteinase inhibitors at 60°C. High pressure did not modify the temperature and pH autolytic activity profiles. The autolytic activity at 40°C was reduced by high pressure; however, it was increased at an incubation temperature of 60°C, mainly in muscle pressurized at 7°C. Aspartic-proteinase was the most sensitive to high pressure. The autolysis of myofibrillar proteins was reduced by high pressure, which was evident in MHC band.