Guillermo Oliver

Viability and β-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.

Protective effect of Enterococcus faecium J96, a potential probiotic strain, on chicks infected with Salmonella Pullorum.

Enterococcus faecium J96 was isolated from a healthy free-range chicken and it inhibited Salmonella Pullorum, in vitro, due to its lactic acid and bacteriocin production. In vivo assays were carried out with 30-h-old broiler chicks. The lactic acid bacteria (approximately 1 x 10(9) cells per chick) were orally administered as preventive and as therapeutic treatments. In the first case they were given to the chicks twice a day for 3 consecutive days. In the second case the lactic bacteria were administered in the same way after a 24-h challenge by Salmonella Pullorum (in both instances the salmonella dose was 1 x 10(5) cells per chick). Cecal contents, liver, and spleens were analyzed and liver and spleen fragments were also fixed in formaldehyde (pH 7.00) in order to determine salmonella translocation. The chickens that were preventively treated with E. faecium J96 survived the Salmonella Pullorum challenge. Those that were infected on the first day and then inoculated with lactic bacteria died 4 days later. Salmonellae were isolated from their livers and spleens. From these results we may conclude that E. faecium J96 can protect newly hatched chicks from Salmonella Pullorum infection but cannot act as a good therapeutic agent.

Combined Effect of Bacteriocins on the Survival of Various Listeria Species in Broth and Meat System

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population.

Hydrolysis of muscle myofibrillar proteins by Lactobacillus curvatus and Lactobacillus sake.

Proteolytic enzyme activities of whole cells and cell free extracts (CFE) of Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were characterised using synthetic chromogenic compounds and myofibrillar proteins as substrates. The hydrolytic action was monitored by SDS-PAGE and reverse phase-HPLC analyses. The CFE of L. sake partially contributed, together with muscle enzymes, to the initial hydrolysis of myofibrillar proteins. Whole-cells of both L. curvatus and L. sake generated peptides considered important for cured-meat taste. The peptide mapping, resulting from the action on the substrates assayed, revealed a profile of extra and intracellular enzymes. Both strains expressed strong amino acid metabolism.

Comparative study of the efficiency of some additives in protecting lactic acid bacteria against freeze-drying.

Cultures of 14 lactic acid bacteria species were freeze-dried in 10 or 20% non-fat skim milk and in distilled water containing one of the following additives: bovine albumin, glycogen, dextran, polyethylene glycol (PEG) 1000, PEG 4000, PEG 6000, glycerol, beta-glycerophosphate, sodium glutamate, asparagine, or cysteine. Each of the potential protective agents tested exhibited marked variations in the protection afforded to different species, none of them was effective for the preservation of viability of thermophilic lactobacilli. However, glycerol provided effective protection for L. leichmannii ATCC 4797 (90% survival), while L. bulgaricus ATCC 11842 reached a viability of 78% with 0.04 M cysteine.

Physical Adsorption of Aflatoxin B1 by Lactic Acid Bacteria and Saccharomyces cerevisiae: A Theoretical Model

The ability of lactic acid bacteria (LAB) and Saccharomyces cerevisiae to remove aflatoxin B1 (AFB1) from liquid medium was tested. The experimental results indicated that (i) AFB1 binding to microorganisms was a rapid process (no more than 1 min); (ii) this binding involved the formation of a reversible complex between the toxin and microorganism surface, without chemical modification of the toxin; (iii) the amount of AFB1 removed was both toxin- and bacteria concentration-dependent; and (iv) quantitatively similar results were obtained with viable and nonviable (heat-treated) bacteria. According to these details, a physical adsorption model is proposed for the binding of AFB1 to LAB and S. cerevisiae, considering that the binding (adsorption) and release (desorption) of AFB1 to and from the site on the surface of the microorganism took place (AFB1 + S <--> S - AFB1). The model permits the estimation of two parameters: the number of binding sites per microorganism (M) and the reaction equilibrium constant (K(eq)) involved, both of which are useful for estimating the adsorption efficiency (M x K(eq)) of a particular microorganism. Application of the model to experimental data suggests that different microorganisms have similar K(eq) values and that the differences in toxin removal efficiency are mainly due to differences in M values. The most important application of the proposed model is the capacity to select the most efficient microorganism to remove AFB1. Furthermore, it allows us to know if a modification of the adsorption efficiency obtained by physical, chemical, or genetic treatments on the microorganism is a consequence of changes in M, K(eq), or both.

Characterization of the Lactic Acid Bacteria in Ewe's Milk and Cheese from Northwest Argentina

Indigenous lactic acid bacteria in ewes milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Tyramine degradation and tyramine/histamine production by lactic acid bacteria and Kocuria strains

Of 53 strains of lactic acid bacteria and Kocuria, screened for production or degradation of biogenic amines, 29 Kocuria varians and four strains of Enterococcusfaecalisproduced tyramine and, at lower concentrations, histamine. In contrast, Lactobacillus strains that did not possess amino acid decarboxylase activity degraded tyramine. The greatest tyramine oxidase activity was present in the strains L. casei CRL705 (98% degradation) and CRL678 (93%) as well as in L. plantarum CRL681 (69%) and CRL682 (60%).

Rehydration conditions and viability of freeze-dried lactic acid bacteria

Abstract The influence of rehydration conditions on the recovery of 16 species of freeze-dried lactic acid bacteria was investigated. The survival of dried cultures during reconstitution to the wet state was increased by rehydration with small volumes of the medium used for that purpose (0.3 and 0.5 ml). Comparison of rehydration at several times of exposure showed best survival at 10 min for the majority of the species analyzed.

Proteolytic activity of Lactobacillus strains isolated from dryfermented sausages on muscle sarcoplasmic proteins.

The proteolytic activity of seven strains of Lactobacillus from two species isolated from dry cured sausages was assayed using a soluble muscle extract as a source of proteins, at a temperature of 30 °C. The results indicated that the strains of Lactobacillus plantarum tested had the more active proteolytic system, showing the highest amino acid release in the medium after 72 hr of incubation (L. plantarum CRL 681) when the microorganism was in the stationary phase of growth. The strains of L. casei showed a continued hydrolytic activity with a lower amino acids concentration along the studied period. The SDS-PAGE profiles showed that the major changes in sarcoplasmic proteins were produced in the 13 kDa and 36-40 kDa molecular weights region.