Antagonistic effect of enterocin CCM 4231 from Enterococcus faecium on “bryndza”, a traditional Slovak dairy product from sheep milk

“Bryndza” is a traditional Slovak dairy product (type of soft cheese) made from sheep cheese which was ripened for 14 days. Because its manufacture, transporting and/or storing represent conditions which facilitate contamination, the effect of enterocin CCM4231 in “bryndza” was investigated with the aim to reduce the contaminant agents. “Bryndza” was divided into equal portions (50 g). The experimental sample (ES) as well as the control sample one (C1) were inoculated with Listeria innocua Li1 strain. The other control samples C2 and C3 were without Li1 strain. C3 control was selected as a reference control. ES and C2 portions were treated with purified enterocin CCM4231 in a concentration of 6400 AU/ml. Before the experimental inoculation, “bryndza” was checked for the presence of contaminant agents. The experiment lasted 1 week and the samples were stored in the refrigerator at 4 °C. Sampling was performed on day 1, on day 4 and on day 7. The control samples C2 and C3 were checked only on day 1 and then after 1 week. The following contaminant agents were detected in “bryndza” before its experimental inoculation with L. innocua Li1 strain: Escherichia coli in the amount 10³ cfu/ml/g, Staphylococcus aureus (10² cfu/ml/g) and enterococci (10⁴ cfu/ml/g). In the control sample C2, the number of E. coli was reduced to 10² cfu/ml/g. Enterococci and staphylococci were totally eliminated there. Concerning C3 control, natural decrease of bacteria was found and/or their unchanged counts. The value of pH (5) was stable during the whole experiment. In the experimental sample inoculated with Li1 strain, its counts were decreased immediately after enterocin CCM4231 addition approximately by one order of magnitude. This inhibitory effect was also detectable on day 4 by the difference of one order of magnitude between ES and C1. On day 7, 10³ cfu/ml/g of Li1 strain were detected in both samples (ES, C1). The difference by one order of magnitude indicated, an inhibitory effect of enterocin CCM4231 in “bryndza”. However, bacteriocin activity was not determined by laboratory analyses.     

The use of enterocin CCM 4231 in soy milk to control the growth of Listeria monocytogenes and Staphylococcus aureus

The inhibitory effect of enterocin CCM 4231 (concentration 3200 AU ml-1) was used to control the growth of Listeria monocytogenes Ohio and Staphylococcus aureus in soy milk. The growth and bacteriocin (enterocin) production of producer strain CCM 4231 in soy milk was also checked. Bacteriocin production by CCM 4231 strain in soy milk was first detected after 2 h from the beginning of cultivation (100 AU ml-1). The stationary phase for CCM 4231 was reached after 6 h reaching 10.38 cfu ml-1 (log10) with a slight increase up to 24 h (10.43 cfu ml-1, log10), and the maximum bacteriocin production in soy milk (200 AU ml-1) was noted after 8 h of the beginning of cultivation with stability up to 24 h. The addition of enterocin CCM 4231 at 3200 AU ml-1 to a growing indicator strain, L. monocytogenes Ohio, in soy milk resulted in inhibition for 24 h. The high inhibitory effect of enterocin was found after 1 h and 2 h of its addition (in 5 h-6 h of cultivation), the difference between the experimental and the control samples (ES, CS) being 4.96 log cycles at 5 h and 5.15 log cycles at 6 h. Staphylococcus aureus was not fully inhibited, although a difference of 3.55 log cycles was found when ES and CS were compared at the end of cultivation (24 h). The pH was not influenced by enterocin addition. The inhibitory effect of enterocin CCM 4231 against L. monocytogenes Ohio in soy milk was probably bacteriocidal; while Staph. aureus was influenced bacteriostatically. In general, the observed inhibitory activity confirmed the possibility for further application of bacteriocins in food environments as the protective agents. Of course, legislation problems must be solved.     

Effect of enterocin CCM 4231 on Listeria monocytogenes in Saint-Paulin cheese

The bacteriocin production byEnterococcus faecium strain in cheese milk and cheese was demonstrated. Purified enterocin CCM 4231 exhibited an anti-listerial effect during Saint-Paulin cheese manufacture. During cheese production the strain grew to a final concentration of 10.1±0.01 log CFU per mL per g in cheese. Then only a slight decrease of the cell concentration was noticed during ripening and was almost stable for 8 weeks. No significant differences in pH were observed between the experimental and reference cheeses. Bacteriocin production during cheese manufacture was detected only in milk samples and curd, reaching a level of 100 AU/mL. After addition of purified enterocin CCM 4231 (concentration 3200 AU/mL) into the experimental cheese, the initial concentration of 6.7±0.06 log CFU per mL ofListeria monocytogenes Ohio was reduced up to 1.9±0.01 log CFU per mL per g. After 6 weeks and at the end of the experiment the difference of surviving cells ofL. monocytogenes Ohio in ECH was only one or 0.7 log cycle compared to the control cheese. Although enterocin CCM 4231 partially inhibitedL. monocytogenes in Saint-Paulin cheese manufacture, an inhibitory effect of enterocin added was shown in 1-week cheese; however, it was not possible to detect bacteriocin activity by the agar spot test. The traditional fermentation and ripening process was not disturbed, resulting in acceptable end-products, including sensory aspects.     

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.     

Anti-staphylococcal effect of enterocin in Sunar® and yogurt

Enterocin was used to control the growth of Staphylococcus aureus strains SA1 and Oxford 209P in Sunar (milk nourishment for suckling babies) and during the yogurt-making process. Reduction by three orders of magnitude was noted in the growth of SA1 strain in Sunar milk nourishment between the enterocin-containing (ES) and the control samples (CS) at 1-d cultivation. An inhibitory effect of enterocin was observed when surviving of SA1 cells were checked 6 h after the start of cultivation (2 h after enterocin application; enterocin was applied after 4 h). Decrease in the count of Oxford 209P strain in yogurt was detected in ES after 1 d of storage in comparison with CS (10(3) and 10(0) CFU mL-1 g-1). Thus a decrease by three orders of magnitude was found between ES and CS at the time mentioned. On the other hand, no bacteriocin activity was detected in ES after 1 d. Activity was detected only immediately after enterocin addition to ES (400 AU/mL) as well as after 1 and 3 1/2 h (200 AU/mL). Although the slight regrowth of the indicator was obtained up to 1 week of yogurt storage, the difference between ES and CS persisted. The lowest pH of the final yogurt product was noted in the reference yogurt sample but differences among the pH values of yogurt samples were not significant.     

Enterocin M-Producing Enterococcus faecium CCM 8558 Demonstrating Probiotic Properties in Horses

The effects of non-authochtonous Enterococcus faecium AL41 = CCM 8558, enterocin M-producing and probiotic strain were tested on the microbiota, phagocytic activity, hydrolytic enzymes, biochemical parameters and dry matter in horses based on its previous benefits demonstrated in other animals. E. faecium CCM 8558 sufficiently colonized the digestive tract of horses. At day 14, its counts reached 2.35 ± 0.70 CFU/g (log 10) on average. The identity of CCM 8558 was confirmed by means of PCR after its re-isolation from horse faeces. The inhibition activity of CCM 8558 was demonstrated against Gram-negative aeromonads, counts of which were significantly reduced (P < 0.001). After 14 days application of CCM 8558, a tendency towards increased phagocytic activity (PA) was measured; PA value was 73.13% ± 8.55 on average at day 0/1; at day 14, it was 75.11 ± 8.66%. Cellulolytic, xylanolytic and pectinolytic activity in horse faeces was significantly increased (P < 0.001) at day 14 (after CCM 8558 application) and amylolytic activity as well (P < 0.01) compared to day 0/1. Inulolytic activity increased with mathematical difference 1.378. Dry matter value reached 20.81 ± 2.29% on average at day 0/1; at day 14, it was 20.77 ± 2.59% (P = 0.9725). Biochemical parameters were influenced mostly in the physiological range. These results achieved after application of CCM 8558 in horses are original, giving us further opportunity to continue these studies, to measure additional parameters and to show the benefits of CCM 8558 application in horses.

     

The effect of salinomycin and lasalocid on laboratory cultures ofEnterococcus fœcium andStaphylococcus gallinarum strains

The growth ofEnterococus fœcium strains CCM 4231 and EF 26, andStaphylococcus gallinarum SG 31 was inhibited by salinomycin and lasalocid at concentrations of 25 and 50 mg/L.Staphylococcus gallinarum was more sensitive to the additives used than were enterococci. Maximum inhibition (90%) was measured after the growth with the SG 31 strain in the presence of both ionophores. Growth of organisms was more inhibited by salinomycin at 25 mg/L (67.5%) than at 50 mg/L (63%). The inhibitory effect in enterococcal strains reached after the addition of salinomycin and lasalocid (on average) 63 and 58%, respectively. The CCM 4231 strain was more inhibited by salinomycin as well as by lasalocid than was the EF 26 strain.     

Selected microbial consortium of raw and digested pig slurry and its susceptibility to enterocins

A consortium of bacterial genera from raw and digested pig slurry (pig farm at Figa, Slovakia; input and output samples) was counted from February to October 2000. The total counts of enterococci and staphylococci were well-balanced in input samples, with visible reduction of cells in May (3.22 and/or 4.21 log c.f.u./ml). Among organisms important from a sanitary perspective only a slight reduction after standard slurry treatment was found between input and output samples (2.0 log cycles), with no effect in April and May. However, their counts were high (8.1–9.01 log c.f.u./ml). Yersinia sp. were detected in rather high counts (6.47; 6.39 log c.f.u./ml). But these species, as well as pseudomonads and Aeromonas sp. were very effectively reduced by standard slurry treatment. Enterocins (CCM4231, V24 and EC24) produced by our own isolates of enterococci were used to determine the susceptibility of selected microbial strains from slurry to those enterocins. For quantifying the inhibitory activity of enterocins, the titre (expressed in activity-arbitrary units [AU/ml]), corresponding to the reciprocal of the highest dilution showing a distinct inhibition zone of the indicator, was determined. Under the conditions used, enterocins used were active against the selected microbial consortium by activity from 100 up to 800 AU/ml. Moreover, enterocin V24 reduced the growth of Enterobacter cloacae ECL751 as well as Pseudomonas sp. minimally with differences of 1. 54 and 2.2 log cycles.     

Enterococcus faecium AL 41: Its Enterocin M and Their Beneficial Use in Rabbits Husbandry

Enterococci are ubiquitous microbiota constituting a large proportion of autochthonous microflora in animals. Some produce bacteriocins mostly enterocins; some of bacteriocin-producing strains also possess probiotic properties. Enterococcus faecium AL 41, Ent M-producing strain was tested for beneficial effect in rabbits. Five-week-old animals (72, Hycole) were divided into experimental groups (E1, E2) and control (C); 24 animals in each. Rabbits in E1 were administered AL 41 (500???l per animal/day, 10⁹?cfu/ml) in water for 21?days; rabbits in E2 were administered Ent M (50???l/animal/day, activity 12,800?AU/ml) in water for 21?days. Rabbits in C fed a commercial diet. The experiment lasted 42?days. Sampling of faeces and blood was provided on day 0?C1 and 21, 42; 3 animals per group were slaughtered. Caecum and appendix were separated. AL 41 colonized rabbits intestines?<1.0 (log10) cfu/g, but stimulation of immunity was noted (P?<?0.01; P?<?0.001). Antimicrobial activity of both was noticed in faeces and/or caecum against pseudomonads. Significant decrease of coliform bacteria in faeces of E1 was noted on day 42 comparing with E2 (P?<?0.05). On day 21, S. aureus cells were not detected in E1, E2. On day 42, S. aureus was not found in E2; in E1 their counts were?<1.0?cfu/g, while in C it was in the count more than 1.0?cfu/g. In appendix, on day 21, significant decrease of not specified bacteria was found in E1, E2 comparing with C (P?<?0.01). Administration of additives has not evoked oxidative stress. Biochemical parameters were not influenced. Higher average daily weight gains were detected by both, AL 41 and Ent M.     

Long term and large-scale cultivation of human hepatoma Hep G2 cells in hollow fiber bioreactor

Long-term and large scale cultivation of an anchorage-dependent cell line using an industrial scale hollow fiber perfusion bioreactor is described. Hep G2 cells (a human hepatoma cell line) were cultivated in an Acysyst-P^® (Endotronic) with a total fiber surface area of 7.2 m² (6×1.2 m²) to produce Hep G2 crude conditioned medium (CCM). Pretreatment of the cellulose acetate hollow fibers with collagen enhances the attachment of the anchorage-dependent cells. We have succeeded in growing the Hep G2 cells in an antibiotics-and serum-free IMDM medium, supplemented with 50g/ml of Hep G2 CCM protein at inoculation. The Hep G2 cells replicate and secrete CCM protein in quantities comparable to those produced in DMEM containing 10% fetal calf serum (FCS). The highest CCM protein productivity during the 80-day cultivation was 1.1 g/day with a total of 30 g of protein accumulated. Hep G2 CCM (20–40 g protein/ml) was comparable to or even better than 10% FCS in supporting the growth of Molt-4 (a human T leukemia cell line) and FO (a mouse myeloma cell line) cells in vitro. The availability of this large amount of Hep G2 CCM will aid the further purification and characterization of growth factor(s) which could be used as serum substituents.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes' milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10(3) and 10(5) cfu/ml and stored at 4 degrees, 10 degrees and 15 degrees C and at room temperature (10 degrees-15 degrees C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C1 and C2 were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10(8) cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10(7) cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Inhibition of Bacillus licheniformis LMG 19409 from ropy cider by enterocin AS-48

AIMS: To determine the activity of enterocin AS-48 against ropy-forming Bacillus licheniformis from cider. METHODS AND RESULTS: Enterocin AS-48 was tested on B. licheniformis LMG 19409 from ropy cider in MRS-G broth, fresh-made apple juice and in two commercial apple ciders (A and B). Bacillus licheniformis was rapidly inactivated in MRS-G by 0.5 microg ml(-1)AS-48 and in fresh-made apple juice by 3 microg ml(-1). Concentration-dependent inactivation of this bacterium in two commercial apple ciders (A and B) stored at 4, 15 and 30 degrees C for 15 days was also demonstrated. Counts from heat-activated endospores in cider A plus AS-48 decreased very slowly. Application of combined treatments of heat (95 degrees C) and enterocin AS-48 reduced the time required to achieved complete inactivation of intact spores in cider A to 4 min for 6 microg ml(-1) and to 1 min for 12 microg ml(-1). D and z values also decreased as the bacteriocin concentration increased. CONCLUSION: Enterocin AS-48 can inhibit ropy-forming B. licheniformis in apple cider and increase the heat sensitivity of spores. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study support the potential use of enterocin AS-48 to control B. licheniformis in apple cider.     

Changes in prostaglandin production and ovarian function in gilts during endometritis induced by Escherichia coli infection

The aim of this study was to determine the prostaglandins (PGs) production and ovarian function in gilts after intrauterine infusions of 10(6) and 10(9) colony-forming units (cfu)/ml of Escherichia coli (E. coli). In Experiments 1 and 2, 30 ml of saline or 30 ml of E. coli suspension containing 10(6) or 10(9)cfu/ml, were infused once into each uterine horn in three groups of gilts on day 3 of the estrous cycle, respectively. In Experiment 1, 17 days after treatment it was revealed that inoculation of E. coli 10(9)cfu/ml induced severe acute or subacute endometritis while 10(6)cfu of E. coli evoked moderate acute endometritis or resulted in no inflammatory changes. In the gilts receiving 10(9)cfu/ml of E. coli, the concentration of 13,14-dihydro-15-keto-PGF(2)alpha in blood from the jugular vein was elevated (P<0.05-0.001) compared to concentration in the gilts inoculated with 10(6)cfu on days 8-17 after treatment. Both the E. coli-treated groups had a lower (P<0.05, P<0.01) progesterone plasma level from days 10 to 14 after administration than the control group. On day 17 of the study, infusion of E. coli 10(9)cfu/ml, in comparison to 10(6)cfu, resulted in the greater (P<0.001) content of PGE(2) in the myometrium. The content of both PGs in the endometrium as well as PGF(2alpha) in the myometrium of gilts-treated with 10(9)cfu/ml of E. coli was lower (P<0.001) than in gilts-treated with 10(6)cfu of bacteria. Newly formed corpora lutea were found in the gilts infused with 10(6), but not those infused with 10(9)cfu/ml of E. coli on day 17 after infusion. On day 8 of the study (Experiment 2), the blood from utero-ovarian vein of the gilts-treated with 10(9)cfu/ml of bacteria had a higher (P<0.05) PGF(2alpha) level and lower (P<0.001) PGE(2) level than following infusion of E. coli 10(6)cfu/ml. Also on day 8 of the study, the content of PGE(2) in the endometrium, both the PGs in the myometrium as well as cyclooxygenase-2 in the endometrium and myometrium was greater (P<0.01, P<0.001) after applying 10(9)cfu/ml than 10(6)cfu/ml of E. coli. These results indicate that intrauterine infusions of 10(6) or 10(9)cfu/ml of E. coli lead to the development of inflammatory states of different intensities which is connected with different PGF(2alpha) and PGE(2) production and function of ovaries.     

Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on inactivation of Escherichia coli O157:H7 in raw-milk cheese

The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of Escherichia coli O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with E. coli O157:H7 at approximately 10(5) CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 10(6) CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10 degrees C in both cases. After 60 days, E. coli O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of E. coli O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated E. coli O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced E. coli O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated E. coli O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.     

Effect of combined physico-chemical preservatives on enterocin AS-48 activity against the enterotoxigenic Staphylococcus aureus CECT 976 strain

AIMS: Control of the enterotoxigenic Staphylococcus aureus CECT 976 strain by enterocin AS-48 in laboratory cultures, and behaviour of the AS-48 activity in the presence of food preservatives. METHODS AND RESULTS: Enterocin AS-48 shows inhibitory activity on the majority of the Staphylococcus species tested. This enterocin has a bactericidal and bacteriolytic mode of action on S. aureus CECT 976, a strain selected for this study by its enterotoxigenic character (SEA production). The inhibitory effect of AS-48 was pH and temperature dependent, and enterocin activity was higher at pH 5. The minimum bactericidal concentration (MBC) of AS-48, decreased from 15 microg ml(-1) at 37 degrees C to 10 microg ml(-1) at 15 degrees C. Sublethally injured cells showed an increased sensitivity with a MBC of 5 microg ml(-1). In this way, the highest effectiveness of Ent AS-48 against S. aureus CECT 976 was obtained at 4 degrees C in combination with high concentrations of NaCl (6 and 7%). Interestingly, enterotoxin SEA production by strain CECT 976 was markedly inhibited by subinhibitory concentrations of Ent AS-48. These low concentrations also provoked a delay of bacterial growth. CONCLUSION: The results presented indicated that Ent AS-48 has a potential for application as a protective agent against S. aureus in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we have established the conditions for an efficient inhibition of growth and enterotoxin production by S. aureus CECT 976 in culture media by a combination of environmental factors and Ent AS-48.     

Characteristics of<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from rabbits

The occurrence of Staphylococcus aureus in rabbit feces, cecum and meat and its enterotoxin production, susceptibility to antibiotics and its sensitivity or resistance to bacteriocins produced by enterococci with probiotic properties were determined. Isolates were resistant to ampicillin, penicillin, phosphomycin and methicillin; a high percentage of susceptibility was also recorded to vancomycin, chloramphenicol, tetracycline and tobramycin. S. aureus isolates did not produce enterotoxins and were sensitive to partially purified enterocins (PPB) EK13, AL41 and EF2019 in the range of 100 to 12800 AU/mL; all S. aureus isolates, except the strain SA 2A/3, exhibited the highest sensitivity to PPB EK13. On the other hand, all strains were resistant to PPB CCM4231.     

Quantification of citrate lyase by enzyme-linked immunosorbent assay for determining the population of Lactococcus lactis subsp. Lactis biovar diacetylactis in pure and mixed cultures

Citrate lyase production by Lactococcus lactis subsp. lactis biovar diacetylactis DRC2 was quantified by an enzyme-linked immunosorbent assay (ELISA). The citrate lyase reached a concentration equivalent to 41 ± 4 g/ml purified citrate lyase in pure culture. When the strain DRC2, grown in mixed culture with L. lactis subsp. cremoris AM2, represented around 70% (DC culture) or 30% (CD culture) of the total initial population, the level of citrate lyase decreased to 21 ± 7 g/ml and 4.5 ± 1.5 g/ml respectively. The maximum bacterial concentration of strain DRC2 in pure culture reached 2.6 × 10⁹ cfu/ml and decreased to 1.5 (± 0.2) × 10⁹ cfu/ml and 0.5 (± 0.3) × 10⁹ cfu/ml in DC and CD mixed cultures respectively. In mixed cultures, the proportion of the strain DRC2 was 8.5 ± 5.0% lower at the end of the fermentation than immediately after inoculation, thus showing that this strain was clearly inhibited. However, the maximum rate of citrate consumption was the same during pure DRC2 culture and CD mixed culture (2.5 ± 0.3 mmol/h) and slightly highre in DC culture (3.07 mmol/h). The maximum rate of acidification was 0.37 ± 0.04 pH unit/h regardless of the culture. A good correlation was obtained between the population of the strain DRC2 and the citrate lyase concentration determined by ELISA but no relationship was found between citrate consumption and citrate lyase synthesis. Therefore an ELISA test of this kind can be used to monitor the growth of L. lactis subsp. lactis biovar diacetylactis in mixed cultures.     

Oral administration of Lactobacillus plantarum strain Lq80 to weaning piglets stimulates the growth of indigenous lactobacilli to modify the lactobacillal population

The composition and the number of fecal or intestinal lactobacilli were determined in weaned piglets before (pre-administration and day 0) and during (days 4, 7, and 14) the administration of Lactobacillus plantarum strain Lq80 (daily dose=10(10) cells). Fecal or intestinal lactobacilli were isolated, and the isolates were grouped by RAPD-PCR and further identified by 16S rDNA partial sequences. During administration, the number of lactobacilli in feces and intestinal contents (log cfu/g) increased from day 0 to day 4 (7.96 vs. 10.07, p<0.05), to day 7 (7.96 vs. 10.18, p<0.05), and to day 14 (7.96 vs. 9.02, p=0.07) in the administered group, although L. plantarum was isolated from the feces of piglets in the administered group at 5.0x10(6 cfu/g. The level of culturable lactobacilli was significantly higher on day 7 in the administered group than in the control group (8.93 vs. 10.18, p)=0.0002). Furthermore, the minor species in the lactobacillal population under the control condition become detectable with the administration of strain Lq80. The oral administration of L. plantarum strain Lq80 was effective to stimulate the development of the lactobacillal population in the intestine of post-weaning piglets.     

Characterization and enhanced production of enterocin HJ35 byEnterococcus faecium HJ35 isolated from human skin

A strain named as HJ35 was isolated from the skin of sixty-five men and fourteen women for acne therapy, in order to find an effective antimicrobial agent againstPropionibacterium acnes. Isolate HJ35 was identified asEnterococcus faecium based on 16 rDNA sequence and produced enterocin HJ35 having antimicrobial activities against most lactic acid bacteria,Enterococcus spp.,Staphylococcus aureus, S. epidermidis, Clostridium perfringens, some bacilli,Micrococcus flavus, Listeria monocytogenes, L. ivanovii, Escherichia coli, Pseudomonas fluorescens andPropionibacterium acnes, in the modified well diffusion method. Especially, enterocin HJ35 showed a bactericidal activity againstPropionibacterium acnes P1. The antimicrobial activity of enterocin HJ35 was disappeared completely with the use of protease XIV. But enterocin HJ35 activity is very stable at high temperature (up to 100°C for 30 min), in wide range of pH 3.0∼9.0), and by treatment with organic solvents. The apparent molecular mass of enterocin HJ35 was estimated to be approximately 4∼4.5 kDa on detection of its bactericidal activity after SDS-PAGE. In batch fermentation ofE. faecium HJ35, enterocin HJ35 was produced at the midlog growth phase, and its maximum production was obtained up to 2,300 AU/mL at the late stationary phase. By employing fed-batch fermentation, the enhanced production of enterocin HJ35 was achieved up to 12,800 AU/mL by feeding with 10 g/L glucose or 6 g/L lactate.     

Effects of medium containing heparin and theophylline on capacitation and metabolic enzyme activities of ejaculated spermatozoa from dogs with asthenozoospermia

The percentages of motile sperm (%MO), hyperactivated sperm (%HA), and acrosome-reacted sperm (%AR) of four beagle dogs with asthenozoospermia (AS) and five normal beagle dogs were determined during 7 h of incubation. The metabolic enzyme activities of the sperm was examined after 0 and 4 h of incubation. The sperm were incubated in canine capacitation medium (CCM) and CCM containing either 20 microg ml(-1) heparin (HE), 10 microg ml(-1) theophylline (TH) or 20 microg ml(-1) HE + 10 microg ml(-1) TH in glass tubes at 38 degrees C under 5% CO2 in air. The %HA and %AR were determined by counting the sperm exhibiting star-spin like movement and by the triple stain technique. The spermatozoa in HE + TH CCM were homogenized and centrifuged, and the metabolic activities of hexokinase, fructokinase, glucose-6-phosphodehydrogenase (G6PD), and pyruvate kinase in the sperm cytosol in the supernatant was measured with a spectrophotometer. The mean %MO and %HA values of both AS and normal dogs in the four types of CCM were highest in HE + TH CCM, with a mean %HA in HE + TH CCM of 78 +/- 5% (S.E.) after 7 h of incubation. However, there was little difference in %AR among the four types of CCM. The mean activities of the four enzymes in the sperm of AS dogs before incubation was significantly lower than in the sperm of normal dogs (P < 0.05, 0.01). However, after 4 h of incubation the activities of all enzymes in the sperm of both AS and normal dogs was clearly higher in HE + TH CCM than in the control CCM. These findings indicate that HE and TH in the medium are effective inactivating metabolic enzymes, maintaining longer sperm motility, and efficiently inducing HA even of the sperm of AS dogs.