Inorganic Additive Improves the Survival of the Probiotic Lacticaseibacillus rhamnosus CRL1505 During Spray Drying, Rehydration, and Storage.

In previous in vitro studies, an inorganic additive (MCM3) showed a thermo-protective effect on the cell viability of Lacticaseibacillus rhamnosus CRL1505 (Lr-CRL1505). In this work, cultures of this probiotic strain were spray dried at lab scale using two carriers: maltodextrin (powder MA) and maltodextrin plus MCM3 (powder MA/MCM3). The cell survival was higher in powder MA/MCM3 (72.8%) than in powder MA (42.8%). Different rehydration media, including the additive MCM3, and two temperatures (37 °C and 45 °C) were evaluated. The best results were obtained in cells rehydrated at 37 °C in MCM3. During the storage of the powders, the highest cell counts were observed in the MA/MCM3 powder. Our results demonstrated that the presence of MCM3 in the carrier and in reconstitution media benefits the spray drying process and the recovery of dehydrated cells. Thus, the use of this additive of inorganic nature and low cost represents a promising technological alternative.

Whey protein coating bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH.

AIMS: To evaluate the efficacy of a novel microencapsulation procedure using whey protein and pectin to improve the survival rate of Lactobacillus rhamnosus CRL 1505 to low pH and bile. METHODS AND RESULTS: Lactobacillus rhamnosus CRL 1505 was encapsulated by ionotropic gelation using pectin (PE) and pectin-whey protein (PE-WP). Both types of beads (MC(PE/WP) and MC(PE-WP/WP)) were covered with a layer of whey protein by complex coacervation. The noncapsulated lactobacilli were not sensitive to bile salts but to acid. Both microparticles protected Lact. rhamnosus CRL 1505 at pH 2.0, but only MC(PE/WP) was effective at pH 1.2. CONCLUSIONS: The combination of ionotropic gelation and complex coacervation techniques is efficient to obtain microcapsules of pectin covered with whey proteins. The MC(PE/WP) beads were more stable than the MC(PE-WP/WP) beads in simulated gastric conditions, thus offering better protection to Lact. rhamnosus CRL 1505 at low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: Pectin beads with a whey protein layer (MC(PE/WP)) could be used as probiotic carrier in functional foods of low pH (e.g. apple juice), thus protecting Lact. rhamnosus CRL 1505 against the stressful conditions of the gastric tract.

Effect of biosynthetic intermediates and citrate on the phenyllactic and hydroxyphenyllactic acids production by Lactobacillus plantarum CRL 778.

AIM: To evaluate the influence of biosynthetic precursors, intermediates and electron acceptors on the production of antifungal compounds [phenyllactic acid (PLA) and hydroxyphenyllactic acid (OH-PLA)] by Lactobacillus plantarum CRL 778, a strain isolated from home-made sourdough. METHODS AND RESULTS: Growth of fermentative activity and antifungal compounds production by Lact. plantarum CRL 778 were evaluated in a chemically defined medium (CDM) supplemented with biosynthetic precursors [phenylalanine (Phe), tyrosine (Tyr)], intermediates [glutamate (Glu), alpha-ketoglutarate (α-KG)] and electron acceptors [citrate (Cit)]. Results showed that the highest PLA production (0.26 mmol l(-1)), the main antifungal compound produced by Lact. plantarum CRL 778, occurred when greater concentrations of Phe than Tyr were present. Both PLA and OH-PLA yields were increased 2-folds when Cit was combined with α-KG instead of Glu at similar Tyr/Phe molar ratio. Similarly, glutamate dehydrogenase (GDH) activity was significantly (P < 0.01) stimulated by α-KG and Cit in Glu-free medium. CONCLUSION: Phe was the major stimulant for PLA formation; however, Cit could increase both PLA and OH-PLA synthesis by Lact. plantarum CRL 778 probably due to an increase in oxidized NAD(+). This effect, as well as the GDH activity, was enhanced by α-KG and down regulated by Glu. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where the role of Glu and GDH activity in the PLA and OH-PLA synthesis was evidenced in sourdough lactic acid bacteria (LAB) using a CDM. These results contribute to the knowledge on the antifungal compounds production by sourdough LAB with potential applications on the baked goods.

A ready-to-use antifungal starter culture improves the shelf life of packaged bread.

Fungal spoilage is the main cause of economic loss in the baking industry. In this study, we developed a ready-to-use biopreserver (slurry [SL]) for nonsliced packed bread by using selected antifungal lactic acid bacteria (LAB) and low-cost ingredients that are compatible with the food matrix. Four LAB strains (Lactobacillus brevis CRL 772, L. brevis CRL 796, L. plantarum CRL 778, and L. reuteri CRL 1100) tested in bread preservation were able to inhibit Penicillium sp. growth and lengthen shelf life twofold with respect to breads prepared using only Saccharomyces cerevisiae (2 days shelf life). The best biopreservation effect (5 days shelf life) was obtained with 40% antifungal slurry SL778 containing L. plantarum CRL 778; this was as effective as 0.2% calcium propionate (PCa). The antifungal effect of SL778 was related to the synthesis of acetic and phenyllactic acid as well as lactic acid, which was produced at a high concentration (31.2 mmol/kg) and lowered the pH of the dough, favoring the undissociated fraction of the organic acids. The combination of the starter SL778 with 0.4% PCa extended the shelf life of packaged bread to 24 days, 2.6-fold longer than breads prepared with only 0.4% PCa.

Functionality of exopolysaccharides produced by lactic acid bacteria in an in vitro gastric system.

AIMS: To evaluate whether slime-exopolysaccharides (EPS) or capsular-polysaccharide (CPS) production could protect the polymer-producing strains Streptococcus thermophilus CRL 1190 and Lactobacillus casei CRL 87 against the harsh conditions of an in vitro gastric system (GS). EPS stability on the GS was studied. METHODS AND RESULTS: An in vitro GS model containing human saliva and gastric juice was standardized. Polymer functionality on the cell viability and metabolic activity of the EPS-producing strains in the GS acidic conditions was evaluated. Two isogenic EPS/CPS deficient mutants were used for comparison. EPS or CPS conferred no significant protection on the cell viability of the studied strains after passage through the GS conditions. However, the phospho- and beta-galactosidase activities of the EPS(+) strains were higher than those of the EPS(-). Cytoplasmic alterations in the wild-type and mutant strains and partial degradation of both EPS were detected. CONCLUSIONS: The presence of EPS/CPS protected the metabolic activity of the assayed LAB strains, but had no effect on survival at low pH. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of EPS/CPS as well as polymer resistance to the harsh conditions of the human GS could impact positively in probiotic strains to exert their properties in the host.

Lactobacillus reuteri CRL 1098 prevents side effects produced by a nutritional vitamin B deficiency.

AIMS: To evaluate the efficiency of the vitamin B(12-)producing Lactobacillus reuteri CRL1098 strain in preventing the symptoms caused by a nutritional cobalamin-deficient diet in pregnant female mice and their weaned offspring. METHODS AND RESULTS: Pregnant female mice were divided into three groups: animals fed with a B(12)-deficient diet (DD), animals fed with DD plus L. reuteri CRL1098 and animals fed with a B(12)-sufficient diet. The animals received the different feedings from the end of gestation up to weaning. At the end of the trials, they and their corresponding offspring were bled to determine haematological, immunological and histological parameters. The administration of the pseudovitamin B(12)-producing strain prevented the symptoms observed in female and weaned young animals fed with a nutritional B(12)-deficient diet. CONCLUSIONS: Our data suggest that the pseudovitamin B(12) produced by L. reuteri CRL1098 is biologically active and effective in preventing the pathologies caused by the nutritional deficiency of B(12) both in pregnant mice and their offspring. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability of L. reuteri CRL1098 to prevent a nutritional vitamin deficiency was demonstrated for the first time. The addition of a GRAS micro-organism to complement the B(12) content in deficient foods is an interesting biotechnological alternative.

Prevention of chronic gastritis by fermented milks made with exopolysaccharide-producing Streptococcus thermophilus strains.

Acetyl-salicylic acid (ASA) is a nonsteroidal antiinflammatory/analgesic drug, which may cause gastritis or stomach ulcers if intensively employed. Exopolysaccharide (EPS)-producing lactic acid bacteria have been claimed to induce immunostimulatory/antiulcer effects in the host. This study investigated the potential preventive effect of fermented milks (FM) with EPS-producing Streptococcus thermophilus strains (CRL 1190 and CRL 804) on an in vivo model of chronic gastritis. Fermented milks (2 EPS(+) and 1 EPS(-), separately) were fed to BALB/c mice for 7 d before inducing gastritis with ASA (400 mg/kg of body weight per day for 10 d; gastritis group, n = 5). Appropriate control groups (ASA administered but not given FM, n = 5; and ASA not administered but given FM) were included in this study. Gastric inflammatory activity was evaluated through the stomach's histology and the number of IFNgamma(+) and IL-10(+) cytokine-producing cells in the gastric mucosa. Only mice preventively treated with the EPS-producing Strep. thermophilus CRL 1190 FM and later administered ASA did not develop gastritis, showing a conserved gastric mucosa structure similar to those of healthy mice. A marked decrease of IFNgamma(+)- and increase of IL-10(+)-producing cells compared with the gastritis group mice were observed. Purified EPS from Strep. thermophilus CRL 1190 resuspended in autoclaved milk was also effective for gastritis prevention. The EPS-protein interaction might be responsible for the observed gastroprotective effect; such interactions may be affected by industrial manufacturing conditions. The results indicate that the FM with Strep. thermophilus CRL 1190 or its EPS could be used in novel functional foods for preventing chronic gastritis.

Functionality of lactic acid bacteria peptidase activities in the hydrolysis of gliadin-like fragments.

AIMS: To evaluate the role of the peptidase activities from sourdough lactic acid bacteria (LAB) in the degradation of alpha-gliadin fragments. METHODS AND RESULTS: Different proline-containing substrates were hydrolysed by LAB indicating pro-specific peptidase activities. Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792 displayed the highest tri- and di-peptidase activities, respectively. Lactobacillus plantarum strains hydrolysed more than 60%alpha-gliadin fragments corresponding to the 31-43 and 62-75 amino acids in the protein after 2 h. None of the LAB strains alone could hydrolyse 57-89 alpha-gliadin peptide; however, the combination of L. plantarum CRL 775 and P. pentosaceus CRL 792 led to hydrolysis (57%) of this peptide in 8 h. CONCLUSIONS: The capacity of LAB strains to degrade alpha-gliadin fragments was not correlated to individual peptidase activities. Several strains separately degraded the 31-43 and 62-75 alpha-gliadin fragments, while the 57-89 peptide degradation was associated with the combination of peptidase profiles from pooled LAB strains. This is the first report on the peptide hydrolase system of sourdough pediococci and its ability to reduce alpha-gliadin fragments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to a better knowledge of sourdough LAB proteolytic system and its role in the degradation of proline-rich alpha-gliadin peptides involved in celiac disease.

Sensitivity of capsular-producing Streptococcus thermophilus strains to bacteriophage adsorption.

AIMS: To determine whether the presence and type of exopolysaccharides (EPS), slime-EPS or capsular, and the structural characteristics of the polymers produced by Streptococcus thermophilus strains could interfere with or be involved in phage adsorption. METHODS AND RESULTS: Phage-host interactions between eight EPS-producing Strep. thermophilus strains (CRL419, 638, 804, 810, 815, 817, 821, 1190) and five streptococcus specific phages (phiYsca, phi3, phi5, phi6, phi8) isolated from Argentinean faulty fermentation failed yoghurts were evaluated. No relationship was found between the EPS chemical composition and the phage sensitivity/resistance phenotype. In general, the capsular-producing strains were more sensitive to phage attacks than the noncapsular-producing strains. Streptococcus thermophilus CRL1190 (capsular-producing) was the only strain sensitive to all bacteriophages and showed the highest efficiency of plating. Phage adsorption to a capsular-negative, EPS low-producing mutant of strain CRL1190 was reduced, especially for phiYcsa and phi8. CONCLUSIONS: The presence of capsular polysaccharide surrounding the cells of Strep. thermophilus strains could play a role in the adsorption of specific phages to the cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Capsular-producing Strep. thermophilus strains should be evaluated for their bacteriophage sensitivity if they are included in starter cultures for the fermented food industry.

Lactobacillus reuteri CRL 1100 as starter culture for wheat dough fermentation.

The effect of sucrose on the fermentation balance of Lactobacillus reuteri CRL 1100 and the invertase activity of this strain in wheat dough and culture medium (MRSs) was evaluated. The enzyme activity was dependent on the environmental pH releasing glucose and fructose from sucrose hydrolysis. Glucose was used as carbon source, while fructose was mainly used as electron acceptor to produce mannitol up to 10h of fermentation. Thereafter, fructose seemed to be metabolized by the heterofermentative pathway, which determined an increase in the concentration of acetate (6 mmol l(-1)), lactate (2 mmol l(-1)) and ethanol (1 mmol l(-1)) and the lack of mannitol formation after glucose depletion. The fermentation balance of Lb. reuteri CRL 1100 during the dough fermentation resulted in lower (63%) ethanol, higher (75%) acetate production and soluble carbohydrates concentrations, like MRSs cultures. This fermentation profile would be important to obtain an optimal growth of yeast and the optimal bread flavor and taste.

Evidence of a glucose proton motive force-dependent permease and a fructose phosphoenolpyruvate:phosphotransferase transport system in Lactobacillus reuteri CRL 1098.

Sugar uptake and phosphoenolpyruvate phosphorylation assays have shown that the heterofermentative strain Lactobacillus reuteri CRL 1098, of likely probiotic value, can transport D-fructose through an inducible fructose-specific phosphotransferase system (K(m) 95 microM) and D-glucose mainly through a proton motive force-driven permease. These data open new perspectives for metabolic and regulatory studies in this bacterium.

The peptide hydrolase system of Lactobacillus reuteri.

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co2+ and inhibited by Cu2+, Hg2+, Cd2+ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn2+ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn2+ and Co2+, respectively.

Selection of bacteriocin producer strains of lactic acid bacteria from a dairy environment.

Two strains showing bacteriocin production were selected from a total of 206 lactic acid bacteria isolated from samples of milk, milk serum, whey and homemade cheeses in Southern Cordoba, Argentina. This property was detected by means of well diffusion assays. The strains were identified as Enterococcus hirae and Enterococcus durans. The protein nature of those substances was proved by showing their sensitivity to type IV and XXV proteases, papaine, trypsin, pepsin and K proteinase. The bacteriocins inhibited the growth of Listeria monocytogenes, Bacillus cereus, Clostridium perfringes and two strains of Staphylococcus aureus, an A-enterotoxin and a B-enterotoxin producers. All of these bacteria are common pathogens usually associated with food borne diseases (ETA). These lactic acid bacteria or their bacteriocins could be suitable candidates for food preservation and specially useful in the our regional dairy industry.

Isolation and identification of anaerobic contaminants from a machine for producing processed cheese.

Some bacteria of the genus Clostridium can contaminate milk. These bacteria can cause "the late gas" or "late blowing" defect in the cheese if this is made with milk containing such contaminants. In this study, six samples from a processed cheese contaminated in a manufacturing machine were analysed. Out of 60 strains studied, 30 were classified as Clostridium tyrobutyricum, 20 as Clostridium butyricum, and 10 as Desulfotomaculum ruminis.

Inhibition of citrus fungal pathogens by using lactic acid bacteria.

The effect of lactic acid bacteria (LAB) on pathogenic fungi was evaluated and the metabolites involved in the antifungal effect were characterized. Penicillium digitatum (INTA 1 to INTA 7) and Geotrichum citri-aurantii (INTA 8) isolated from decayed lemon from commercial packinghouses were treated with imazalil and guazatine to obtain strains resistant to these fungicides. The most resistant strains (4 fungal strains) were selected for evaluating the antifungal activity of 33 LAB strains, among which only 8 strains gave positive results. The antifungal activity of these LAB strains was related to the production of lactic acid, acetic acid, and phenyllactic acid (PLA). A central composite design and the response surface methodology were used to evaluate the inhibitory effect of the organic acids produced by the LAB cultures. The antifungal activity of lactic acid was directly related to its concentration; however, acetic acid and PLA showed a peak of activity at 52.5 and 0.8 mM, respectively, with inhibition rates similar to those obtained with Serenade((R)) (3.0 ppm) imazalil (50 ppm) and guazatine (50 ppm). Beyond the peak of activity, a reduction in effectiveness of both acetic acid and PLA was observed. Comparing the inhibition rate of the organic acids, PLA was about 66- and 600-fold more effective than acetic acid and lactic acid, respectively. This study presents evidences on the antifungal effect of selected LAB strains and their end products. Studies are currently being undertaken to evaluate the effectiveness in preventing postharvest diseases on citrus fruits.

Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807.

Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme alpha-phosphoglucomutase (alpha-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in alpha-PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both alpha-PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.

Citrate catabolism and production of acetate and succinate by Lactobacillus helveticus ATCC 15807.

The citrate metabolism of Lactobacillus helveticus ATCC 15807 was studied under controlled-pH fermentations at pH 4.5 and pH 6.2. The micro-organism was able to co-metabolize citrate and lactose at both pH from the beginning of growth, which enhanced the rate of lactose consumption and lactic acid production, compared with cultures without citrate. The effect of citrate on cell growth was dependent on the balance between the ratio of dissociated to non-dissociated forms of the acetic acid produced and the extra ATP gained by the cells, both facts related to the citrate metabolism. The citrate catabolism determined a change in the fermentation pattern of L. helveticus ATCC 15807 from homolactic to a mixed-acid profile, regardless of the external pH. Within this new fermentation pattern, acetate was the major product formed (13-20 mM), followed by succinate (2.4-3.7 mM), while acetoine, dyacetile or butanediol were not detected. The mixed-acid profile displayed by L. helveticus ATCC 15807 was linked to NADH(2) oxidase activity rather than the acetate kinase enzyme.

Growth and exopolysaccharide production by Lactobacillus helveticus ATCC 15807 in an adenine-supplemented chemically defined medium.

AIMS: To analyse the exopolysaccharide (EPS) production by Lactobacillus helveticus ATCC 15807 in a chemically defined medium (CDM) and the effect of nutrients and stress culture conditions on cell growth and EPS formation. METHODS AND RESULTS: Cultures were conducted in CDM: (i) containing essential and nonessential bases and vitamins; (ii) without nonessential bases and vitamins [Simplified CDM (SCDM)]; (iii) SCDM supplemented individually with vitamins and bases. The influence of carbohydrates, pH and osmotic culture conditions on growth and polymer formation was analysed. Adenine and lactose stimulated both growth and EPS production. Constant pH fermentations (4.5 and 6.2) did not improve EPS synthesis while NaCl and glycerol were detrimental for growth and polymer formation. In all media the EPS monomer composition was glucose and galactose (2.5 : 1). CONCLUSIONS: A SCDM containing adenine and lactose was optimal for cell growth and EPS formation by Lact. helveticus ATCC 15807. Controlled pH (6.2 and 4.5) and osmotic stress culture conditions did not improve polymer production. The EPS characteristics were identical in all media. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides a better knowledge on EPS synthesis by Lact. helveticus. A CDM to perform regulation studies on EPS production by Lact. helveticus species is now available.

UDP-galactose 4-epimerase: a key enzyme in exopolysaccharide formation by Lactobacillus casei CRL 87 in controlled pH batch cultures.

AIMS: To evaluate the relationship between exopolysaccharide (EPS) production and the sugar nucleotide biosynthetic enzymes in Lactobacillus casei CRL 87 under optimum growth conditions for polymer formation: controlled pH on galactose or glucose. Studies with an EPS mutant were carried out to determine the key enzymes in EPS synthesis under the above culture conditions. METHODS AND RESULTS: EPS concentration was estimated by the phenol/sulphuric acid method, while the activities of the biosynthetic enzymes were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. An environmental pH of 5.0, using galactose as carbon source, markedly improved not only polymer production and yield but also, cell growth and lactic acid production. Analysis of the activities of the EPS precursor-forming enzymes revealed that polysaccharide synthesis was correlated with uridine-diphosphate (UDP)-glucose pyrophosphorylase and UDP-galactose 4-epimerase under these growth conditions. CONCLUSIONS: EPS synthesis by Lact. casei CRL 87 was considerably improved at a controlled pH of 5.0 with galactose as carbon source, and was correlated with the activity of UDP-glucose pyrophosphorylase and UDP-galactose 4-epimerase. The results obtained with the wild-type and EPS- strains suggest that UDP-galactose 4-epimerase plays an essential role in EPS formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Unravelling the key enzymes involved in EPS biosynthesis under optimum culture conditions for polymer production provides important information for the design of strategies, via genetic engineering, to enhance polysaccharide formation.

Changes in the surface potential of Lactobacillus acidophilus under freeze-thawing stress.

The zeta potential of Lactobacillus acidophilus CRL 640, a measure of the net distribution of electrical charges on the bacterial surface, is a function of the glucose concentration in the growing media. With 2% glucose, cells in the stationary phase showed a zeta potential of -45 +/- 2 mV. With these cells, the zeta potential after freezing and thawing decreased to -32 +/- 2 mV and there was a decrease in viability. The changes in the surface potential correlated with damage to the cell surface as shown by electron microscopy. Freeze-thawed cells incubated in a rich medium recovered a zeta potential of -38 +/- 2 mV without cell growth. L. acidophilus CRL 640 showed the same value of surface potential as control cells when they were frozen and thawed in 2 M glycerol.