Isolation and phenotypic characterization of Lactobacillus casei and Lactobacillus paracasei bacteriophage-resistant mutants

Aims: To isolate and characterize bacterial strains derived from Lactobacillus casei and Lactobacillus paracasei strains and resistant to phage MLC‐A. Methods and Results: Two of nine assayed strains rendered resistant mutants with recovery efficiencies of 83% (Lact. paracasei ATCC 27092) and 100% (Lact. casei ATCC 27139). DNA similarity coefficients (RAPD–PCR) confirmed that no significant genetic changes occurred while obtaining resistant mutants. Neither parent nor mutant strains spontaneously released phages. Phage‐resistant mutants were tested against phages PL‐1, J‐1, A2 and MLC‐A8. Lactobacillus casei ATCC 27092 mutants showed, overall, lower phage resistance than Lact. paracasei ATCC 27092 ones, but still higher than that of the parent strain. Lactobacillus paracasei ATCC 27092 mutants moderately adsorbed phage MLC‐A only in calcium presence, although their parent strain successfully did it with or without calcium. Adsorption rates for Lact. casei ATCC 27139 and its mutants were highly influenced by calcium. Again, phage adsorption was higher on the original strain. Conclusions: Several isolates derived from two Lact. casei and Lact. paracasei strains showed resistance to phage MLC‐A but also to other Lact. casei and Lact. paracasei phages. Significance and Impact of the Study: This study highlights isolation of spontaneous bacteriophage‐resistant mutants from Lact. casei and Lact. paracasei as a good choice for use in industrial rotation schemes.     

Effect of carbohydrates on the survival of Lactobacillus helveticus during vacuum drying

AIMS: To assess four carbohydrates for the protective effect against Lactobacillus helveticus cells inactivation during vacuum drying, and to study the effect of selected carbohydrate on changes of inactivation kinetics. METHODS AND RESULTS: Early stationary phase L. helveticus cells grown in MRS media were recovered from fermentation broth, washed with PBS buffer (pH 7.0), and then mixed with different concentrations of four carbohydrates, namely lactose, sorbitol, inulin, and xanthan gum. Cells were dried in a vacuum drier at 100 mbar, 43 degrees C for 12 h. Only cells with 1% sorbitol addition showed higher survival (18%) over cells without added carbohydrate (8%). Using in situ microbalance technique whereby cell weight during vacuum drying was continuously monitored via precision balances integrated into the vacuum chamber, drying and inactivation kinetics of cells and cells mixed with sorbitol were established. CONCLUSION: Survival of L. helveticus during the vacuum drying could be improved by the addition of optimal concentration of 1% sorbitol. Addition of sorbitol did not cause drastic changes in drying rate, water content and water activity of samples. The protection mechanisms of sorbitol seemed not to be due to a direct physical effect, which could be related to drying rate. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in survival of cells after vacuum drying by the addition of a protective carbohydrate may provide an alternative mean to preserve starter cultures at a higher level of activity.     

Changes in membrane fatty acids of Lactobacillus helveticus during vacuum drying with sorbitol

Aims:  To examine changes in membrane fatty acid profile attributed to the physiological adaptation of Lactobacillus helveticus during vacuum drying.
Methods and Results:  The viability and membrane integrity of the cells after vacuum drying were measured by plate counts and DNA fluorescence dyes. The physiological adaptation of cells dried in the presence of sorbitol was observed by determining changes in membrane fatty acid composition using gas chromatography. Results showed that viability and membrane integrity of Lact. helveticus cells increased when drying in the presence of sorbitol. The occurrence of the very low melting point polyunsaturated fatty acids linoleic and arachidonic acid was observed in cells dried in the presence of sorbitol.
Conclusions:  The physiological adaptation of cells occurred with cell membrane of Lact. helveticus during vacuum drying of cells in the presence of sorbitol.
Significance and Impact of the Study:  The study showed that physiological adaptation with membrane of the cells occurred during the drying process. The insight implies that instead of viability improvement of dried cells by the conventional stress induction during cultivation, the induction may be exercised thereafter without compromising growth of the cells.     

Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms

Aim: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. Methods and Results: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. Conclusion: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. Significance and Impact of the Study: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.     

Survival of Lactobacillus paracasei subsp. paracasei LBC 81 in Fermented Milk Enriched with Green Banana Pulp Under Acid Stress and in the Presence of Bile Salts

This study evaluated the in vitro effect of 3.0, 6.0, and 9.0% of green banana pulp (GBP) incorporation in fermented milk on the survival of Lactobacillus paracasei subsp. paracasei LBC 81 subjected to acid stress conditions and in the presence of bile salts. Tolerance to acid stress in pH 2.0 and in the presence of 0.30% of bile salts was evaluated right after the incorporation of the fermented milk in each of these conditions, and also during 3 and 4 h of exposure, respectively. The addition of GBP (3.0%) gives a protective effect on L. paracasei LBC 81 when exposed to stress conditions evaluated, while of 9.0% there is a marked decrease of L. paracasei LBC 81. In the absence of GBP, a decrease of L. paracasei LBC 81 is observed, but lower in the presence of GBP (9.0%).

     

Stabilization of freeze-dried Lactobacillus paracasei subsp. paracasei JCM 8130T with the addition of disaccharides,polymers, and their mixtures

Although freeze-drying is a widely used dehydration technique for the stabilizing of unstable lactic acid bacteria, Lactobacillus paracasei subsp. paracasei JCM 8130^T (L. paracasei) is destabilized after freeze-drying and subsequent storage. In order to improve the stability of freeze-dried L. paracasei, effects of disaccharides (sucrose and trehalose), polymers (maltodextrin; MD and bovine serum albumin; BSA), and their mixtures on the survival rate of freeze-dried L. paracasei were investigated. The survival rate of non-additive sample decreased slightly after freeze-drying but decreased drastically after subsequent storage at 37 °C for 4 weeks. The reduction was diminished by the addition of disaccharides and polymers. The stabilizing effect of disaccharides was not affected by the co-addition of MD. In contrast, the disaccharide–BSA mixtures had a synergistic stabilizing effect, and the survival rates were largely maintained even after storage. It is suggested that the synergistic effect originates from the conformational stabilization of the dehydrated bacteria.     

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

     

Distinct immunomodulatory properties of Lactobacillus paracasei strains

Aims: This study was performed to ascertain the immunomodulatory effect of Lactobacillus paracasei strains. These strains were also genetically characterized. Methods and Results: The strains were genetically differentiated by using the fluorescent‐amplified fragment length polymorphism technique, which led to the identification of several molecular markers unique to each strain. To determine the immunomodulatory properties, we evaluated the effect of strains on dendritic cell maturation, dextran uptake, ability to induce proliferation of allogenic T cells and cytokine secretion. The results indicated that all the strains stimulated phenotypic maturation of dendritic cells (DCs), but they acted differently on DCs in relation to the other tested properties; notably, a different effect on cytokine secretion was detected. Conclusions: The results of this study revealed different immunomodulatory properties of strains of the species Lact. paracasei. Strain IMPC 4.1 showed an interesting anti‐inflammatory ability. Probiotic strains IMPC 2.1 and LMG P‐17806 were characterized by a similar and intermediate ability to induce cytokine secretion in contrast to the very low ability of strain LMG 23554. Significance and Impact of Study: Our results confirm that each single strain of a bacterial species appears to influence the immune system in a peculiar manner. The evaluation of the different types and/or levels of cytokines whose secretion is induced by each strain could be relevant to define its pro‐ or anti‐inflammatory properties and its more appropriate clinical use.     

A study of the effect of sorbitol on osmotic tolerance during partial desiccation of bovine sperm

The goal of the study was to improve the partial desiccation survival of bovine sperm by decreasing the dehydration induced osmotic injury. The protective role of sorbitol, a polyol, was investigated by (i) studying the osmotic behavior of sperm in hypertonic Tyrode’s buffer in the presence of sorbitol and trehalose, (ii) studying the effect of sorbitol and trehalose on sperm motility following partial dehydration. The osmotic behavior studies included the assessment of motility and volumetric responses in the presence of the additives. For the drying experiments, motility was assayed after drying the samples to different end water content followed by immediate rehydration. Compared to the effect of “intracellular + extracellular” trehalose alone, results showed a much improved motility in the presence of sorbitol and trehalose. While the drying results suggest an enhanced osmotolerance in the presence of sorbitol, the study of motility under hypertonic conditions combined with the sperm volume excursion experiments suggest that sorbitol imparts the enhancement by permeating into the cell cytoplasm.     

Metabolism of Klebsiella pneumoniae freeze‐dried cultures for the design of BOD bioassays

Aims: The survival rate of freeze‐dried cultures is not enough information for technological applications of micro‐organisms. There could be serious metabolic/structural damage in the survivors, leading to a delay time that can jeopardize the design of a rapid biochemical oxygen demand (BOD) metabolic‐based bioassay. Therefore, we will study the metabolic activity (as ferricyanide reduction activity) and the survival rate (as colony‐forming units, CFU) of different Klebsiella pneumoniae freeze‐dried cultures looking for stable metabolic conditions after 35 days of storage. Method and Results: Here, we tried several simple freeze‐drying processes of Kl. pneumoniae. Electrochemical measurements of ferrocyanide and survival rates obtained with the different freeze‐dried cultures were used to choose the best freeze‐drying process that leads to a rapid metabolic‐based bioassay. Conclusions: The use of milk plus monosodium glutamate was the best choice to obtain a Kl. pneumoniae freeze‐dried culture with metabolic stable conditions after storage at ?20°C without the need of vacuum storage and ready to use after 20 min of rehydration. We also demonstrate that the viability and the metabolic activity are not always directly correlated. Significance and Impact of the Study: This study shows that the use of this Kl. pneumoniae freeze‐dried culture is appropriate for the design of a rapid BOD bioassay.     

Antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20

Aims: The aim of this study was to determine the antimicrobial and antiadhesive properties of a biosurfactant isolated from Lactobacillus paracasei ssp. paracasei A20 against several micro‐organisms, including Gram‐positive and Gram‐negative bacteria, yeasts and filamentous fungi. Methods and Results: Antimicrobial and antiadhesive activities were determined using the microdilution method in 96‐well culture plates. The biosurfactant showed antimicrobial activity against all the micro‐organisms assayed, and for twelve of the eighteen micro‐organisms (including the pathogenic Candida albicans, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus agalactiae), the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were achieved for biosurfactant concentrations between 25 and 50 mg ml^?1. Furthermore, the biosurfactant showed antiadhesive activity against most of the micro‐organisms evaluated. Conclusions: As far as we know, this is the first compilation of data on antimicrobial and antiadhesive activities of biosurfactants obtained from lactobacilli against such a broad group of micro‐organisms. Although the antiadhesive activity of biosurfactants isolated from lactic acid bacteria has been widely reported, their antimicrobial activity is quite unusual and has been described only in a few strains. Significance and Impact of the Study: The results obtained in this study regarding the antimicrobial and antiadhesive properties of this biosurfactant opens future prospects for its use against micro‐organisms responsible for diseases and infections in the urinary, vaginal and gastrointestinal tracts, as well as in the skin, making it a suitable alternative to conventional antibiotics.     

Influence of drying methods over in vitro antitumoral effects of exopolysaccharides produced by Agaricus blazei LPB 03 on submerged fermentation

Agaricus blazei is a mushroom that belongs to the Brazilian biodiversity and is considered as an important producer of bioactive compounds beneficial to human health. Studies have demonstrated that these compounds present immuno-modulatory, antioxidant and antitumor properties. In order to compare the most used method for fungal polysaccharide drying, lyophilization with other industrial-scale methods, the aim of this work was to submit A. blazei LPB 03 polysaccharide extracts to vaucum, spray and freeze drying, and evaluate the maintenance of its antitumoral effects in vitro. Exopolysaccharides produced by A. blazei LPB 03 on submerged fermentation were extracted with ethanol and submitted to drying processes. The efficiency represents the water content that was removed during the drying process. The resultant dried products showed water content around 3% and water activity less than 0.380, preventing therefore the growth of microorganisms and reactions of chemical degradation. Exopolysaccharide extracts dried by vacuum and spray dryer did not showed any significant cytotoxic effect on cell viability of Wistar mice macrophages. Content of total sugars and protein decrease after drying, nevertheless, 20 mg/ml of exopolysaccharides dried by spray dryer reached 33% of inhibition rate over Ehrlich tumor cells in vitro.     

Suitability of buttermilk for fermentation with Lactobacillus helveticus and production of a functional peptide‐enriched powder by spray‐drying

Aim: To ferment buttermilk, a low‐cost by‐product of the manufacture of butter, with a proteolytic strain of Lactobacillus helveticus, to enhance its value by the production of a functional peptide‐enriched powder. Methods and Results: Buttermilk was fermented with Lact. helveticus 209, a strain chosen for its high proteolytic activity. To enhance the release of peptidic fractions, during fermentation pH was kept at 6 by using NaOH, Ca(CO)₃ or Ca(OH)₂. Cell‐free supernatant was recovered by centrifugation, supplemented or not with maltodextrin and spray‐dried. The profile of peptidic fractions released was studied by RP‐HPLC. The lactose, Na and Ca content was also determined. The powder obtained was administered to BALB/c mice for 5 or 7 consecutive days, resulting in the proliferation of IgA‐producing cells in the small intestine mucosa of the animals. Conclusions: Buttermilk is a suitable substrate for the fermentation with Lact. helveticus 209 and the release of peptide fractions able to be spray‐dried and to modulate the gut mucosa in vivo. Significance and Impact of the Study: A powder enriched with peptides released from buttermilk proteins, with potential applications as a functional food additive, was obtained by spray‐drying. A novel use of buttermilk as substrate for lactic fermentation is reported.     

Preservation of Bacteria by Circulating-Gas Freeze Drying

Water-washed Serratia marcescens and Escherichia coli were freeze dried in a circulating-gas system at atmospheric pressure. This convective procedure resulted in a substantially higher survival of organisms than could be obtained by the vacuum method of freeze drying. There was little or no decrease in cell viability during convective drying when the residual moisture content was 15% or higher. Below this level, survival declined with decreasing moisture content. A detailed comparison of the convective and vacuum methods indicated that the advantage gained by freeze drying bacteria in air accrues in the early period of sublimation, at which time cells were found to be sensitive to vacuum drying but insensitive to air drying. An explanation for this difference is proposed, based upon the kinetics of water removal in the two processes. In brief, it is suggested that the convective method permits samples to be dried more uniformly; and regional over-drying, which may be deleterious even if transient, is thus avoided in achieving the optimal level of moisture.     

Selection of Escherichia coli-inhibiting strains of Lactobacillus paracasei subsp. paracasei

The aim of this study was to select Escherichia coli-inhibiting strains among lactic acid bacteria. On the basis of phenotypical and technological characteristics, 20 strains of lactic acid bacteria were screened from a total of 225 isolates, obtained from nine samples of artisanal Caprino d'Aspromonte cheese, made from raw goats' milk. The antagonistic activity of these 20 strains was detected in plates against three different strains of E. coli. Two strains of Lactobacillus paracasei subsp. paracasei showed a marked anti-E. coli activity against all three strains tested; the other lactic acid bacteria did not exhibit inhibiting activity. The E. coli inhibition can be ascribed to production of bacteriocin-like compounds. The use of L. paracasei subsp. paracasei strains to increase the safety of the cheeses made from raw milk is recommended because these cultures strongly inhibit E. coli, without foreseeable adverse sensory changes. Received 27 December 2001/ Accepted in revised form 28 June 2002     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Reconstitution conditions for dried probiotic powders represent a critical step in determining cell viability

Aims: Resuscitation of dried cultures represents a critical control point in obtaining active and effective probiotic strains. This study examined the effects of various rehydration conditions on the viability of Bifidobacterium longum NCC3001 and Lactobacillus johnsonii La1. Methods and Results: Reconstitution conditions for these strains were optimized using a multivariate experimental design approach. Furthermore, using flow cytometry, the cell integrity was followed during reconstitution. By adjusting the pH, availability of a metabolizable sugar, reconstitution duration, powder matrix and ratio of powder to reconstitution solution, the recovery of Bif. longum NCC3001 and Lact. johnsonii La1 following reconstitution was increased eight‐ and two‐fold, respectively, over standard reconstitution in maximum recovery diluent. It was shown that pH had a significant effect on the recovery of Bif. longum NCC3001 and Lact. johnsonii La1. Conclusions: The recovery of dried probiotic cultures is greatly dependent on the reconstitution conditions. The maximum recovery of 11·7 ¹⁰log CFU g^?1Bif. longum NCC3001 was achieved at 30‐min reconstitution at pH 8, in the presence of 2%l ‐arabinose and a ratio of 1 : 100 of powder to diluent. Lact. johnsonii La1 showed highest recovery (9·3 ¹⁰log CFU g^?1) after reconstitution, when mixed with maltodextrin at pH 4. Significance and Impact of the Study: To achieve accurate viable probiotic numbers from dried probiotic cultures, the reconstitution conditions should be optimized for the strain used.     

Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying

The influence of low temperature vacuum drying process parameters on the survival, metabolic activity and residual water content of three different bacterial strains (Lactobacillus paracasei ssp. paracasei, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium lactis) was investigated. Shelf temperature and chamber pressure were varied and optimized by response surface methodology with regard to survival and residual water content. It is shown that the survival rate after low temperature vacuum drying is comparable to that of freeze drying. Based on the optimization experiments the combined influence of fermentation pH and drying process parameters was studied for the most detrimental and the best process condition, respectively. The results show that interactions between process and fermentation conditions have to be taken in account and that these influences are highly strain specific.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

Enhanced Survival of GroESL-Overproducing Lactobacillus paracasei NFBC 338 under Stressful Conditions Induced by Drying

GroESL-overproducing Lactobacillus paracasei NFBC 338 was dried, and its viability was compared with that of controls. Spray- and freeze-dried cultures overproducing GroESL exhibited ~10-fold and 2-fold better survival, respectively, demonstrating the importance of GroESL in stress tolerance, which can be exploited to enhance the technological performance of sensitive probiotic cultures.