Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59 degrees C. An air outlet temperature of 80 to 85 degrees C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 x 10(9) CFU/g for NFBC 338 and 5.2 x 10(7) CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at approximately 1 x 10(9) CFU/g during 2 months of powder storage at 4 degrees C, while a decline in the level of survival of approximately 1 log (from 7.2 x 10(7) to 9.5 x 10(6) CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.     

Comparative Survival Rates of Human-Derived Probiotic Lactobacillus paracasei and L. salivarius Strains during Heat Treatment and Spray Drying

Spray drying of skim milk was evaluated as a means of preserving Lactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 10⁹ CFU/g for NFBC 338 and 5.2 × 10⁷ CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ~1 × 10⁹ CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 10⁷ to 9.5 × 10⁶ CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Improved stress tolerance of GroESL-overproducing Lactococcus lactis and probiotic Lactobacillus paracasei NFBC 338

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52 degrees C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54 degrees C) and lactobacilli (at 60 degrees C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.     

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 approximately 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.     

Sequence analysis of the plasmid genome of the probiotic strain Lactobacillus paracasei NFBC338 which includes the plasmids pCD01 and pCD02

Lactobacillus paracasei NFBC338 is a probiotic strain that was isolated from the human gastrointestinal tract (GIT) and contains a plasmid genome of 80kb. Using a shotgun sequencing approach, two of the plasmids, pCD01 (19,882bp) and pCD02 (8554bp) have been completely sequenced, and four contiguous sequences (Contigs) have been assembled. Bioinformatic analysis of pCD01 revealed that it contains 23 putative open reading frames (ORFs) and that it contains regions characterised by potential replication functions and multidrug resistance (MDR). In contrast, the content of pCD02 is mainly cryptic, although, it does contain two insertion sequence (IS) elements. Indeed, up to 17% of the entire plasmid genome encodes putative transposable elements. In addition, there are a number of interesting ORFs distributed over the four Contigs that show significant homology to genes such as those involved in adherence and biotin metabolism, which may prove beneficial to Lb. paracasei NFBC338 under certain environmental conditions. This study provides a novel insight into the rich plasmid complement of this probiotic Lactobacillus strain, which may potentially be exploited as the basis for development of improved genetic tools for probiotic lactobacilli.     

Functional and safety characterization of autochthonous Lactobacillus paracasei FS103 isolated from sheep cheese and its survival in sheep and cow fermented milks during cold storage

The main objective of this study was to evaluate some probiotic characteristics of Lactobacillus spp. isolated from traditional sheep cheese, and to investigate the fermentative ability and viability in sheep and cow milks of a selected potential probiotic Lactobacillus (L.) strain, i.e., L. paracasei FS103. A total of 54 autochthonous Lactobacillus isolates were characterized for (i) acidity and bile salt resistance, (ii) tolerance to gastric and intestinal juice models, and (iii) antagonistic activity against pathogens and antibiotic resistance. Potential probiotic Lactobacillus has been used in sheep and cow milks for the manufacturing of experimental fermented milks. In these latter, pH value, microbial count, and sensory analysis were carried out. Lactobacillus FS103 classified as L. paracasei subsp. paracasei had a good survival in gastric and intestinal juice models, inhibited the growth of undesirable bacteria, and was susceptible to chloramphenicol, clindamycin, penicillin, amoxicillin, erythromycin, tetracycline, and ampicillin. Moreover, when used to produce experimental sheep and cow fermented milks, L. paracasei FS103 was able to acidify both milk types leading to a continuous pH decrease during all fermentation time (24 h). FS103 population remains viable at a level > 108 CFU mL−1 after 21 days of cold (4 °C) storage. The results of sensory analysis showed that scores related to consistency, taste, and astringent were significantly higher in sheep fermented milk while animal-like was less acceptable compared to cow fermented milk. Lactobacillus paracasei FS103 isolated from sheep cheese exhibited potential probiotic properties and suitable features for sheep and cow fermented milks maintaining high vitality during cold storage.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.     

Probiotic properties of Lactobacillus strains isolated from the feces of breast-fed infants and Taiwanese pickled cabbage

This study assessed potential probiotic Lactobacillus strains isolated from the feces of breast-fed infants and from Taiwanese pickled cabbage for their possible use in probiotic fermented foods by evaluating their (i) in vitro adhesive ability, resistance to biotic stress, resistance to pathogenic bacteria, and production of β-galactosidase; (ii) milk technological properties; and (iii) in vivo adhesive ability, intestinal survival and microbial changes during and after treatment. Five Lactobacillus isolates identified as Lactobacillus reuteri F03, Lactobacillus paracasei F08, Lactobacillus rhamnosus F14, Lactobacillus plantarum C06, and Lactobacillus acidophilus C11 that showed resistance to gastric juice and bile salts were selected for further evaluation of their probiotic properties. All the strains demonstrated the ability to adhere to Caco-2 cells, particularly, strain L. plantarum C06 and L. reuteri F03 showed satisfactory abilities, which were similar to that of the reference strain L. rhamnosus GG. The strains L. paracasei F08 and L. acidophilus C11 had the highest β-galactosidase activity. Most of the strains were resistant to aminoglycosides and vancomycin but sensitive to ampicillin, erythromycin, and penicillin. All the 5 strains elicited antibacterial activity against both Gram-positive (Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus) and -negative (Escherichia coli and Salmonella enterica) pathogens. Moreover, the strains L. reuteri F03, L. paracasei F08, and L. plantarum C06 could grow rapidly in milk without nutrient supplementation and reached 10? cfu/mL after 24 h of fermentation at 37 °C. The viable cell counts of the 3 strains remained above 10? cfu/mL after 21 d of storage at 4 °C. In the animal feeding trial, the number of intestinal lactobacilli increased significantly after administration of milk fermented with the 3 strains, and the counts of fecal coliforms and Clostridium perfringens were markedly reduced. Lactobacillus strains could also survive in the ileal intestinal tissue of the treated rats. Technologically interesting Lactobacillus isolates may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

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.     

Optimization of spray-drying conditions for the large-scale preparation of Bacillus thuringiensis var. israelensis after downstream processing

Reduction of water activity in the formulations of mosquito biocontrol agent, Bacillus thuringiensis var. israelensis is very important for long term and successful storage. A protocol for spray drying of B. thuringiensis var. israelensis was developed through optimizing parameters such as inlet temperature and atomization type. A indigenous isolate of B. thuringiensis var. israelensis (VCRC B-17) was dried by freeze and spray drying methods and the moisture content and mosquito larvicidal activity of materials produced by the two methods were compared. The larvicidal activity was checked against early fourth instars Aedes aegypti larvae. Results showed that the freeze-dried powders retained the larvicidal activity fairly well. The spray-dried powder moderately lost its larvicidal activity at different inlet temperatures. Between the two types of atomization, centrifugal atomization retained more activity than the nozzle type atomization. Optimum inlet temperature for both centrifugal and nozzle atomization was 160 degrees C. Keeping the outlet temperature constant at 70 degrees C the moisture contents for the spray-dried powders through centrifugal atomization and freeze-dried powders were 10.23% and 11.80%, respectively. The LC(50) values for the spray-dried and freeze-dried powders were 17.42 and 16.18 ng/mL, respectively. Spore count of materials before drying was 3 x 10(10) cfu/mL and after spray drying through nozzle and centrifugal atomization at inlet and outlet temperature of 160 degrees C/70 degrees C were 2.6 x 10(9) and 5.0 x 10(9) cfu/mL, respectively.     

Improved Stress Tolerance of GroESL-Overproducing Lactococcus lactis and Probiotic Lactobacillus paracasei NFBC 338

The bacterial heat shock response is characterized by the elevated expression of a number of chaperone complexes. Two-dimensional polyacrylamide gel electrophoresis revealed that GroEL expression in probiotic Lactobacillus paracasei NFBC 338 was increased under heat adaptation conditions (52°C for 15 min). Subsequently, the groESL operon of L. paracasei NFBC 338 was PCR amplified, and by using the nisin-inducible expression system, two plasmids, pGRO1 and pGRO2, were constructed on the basis of vectors pNZ8048 and pMSP3535, respectively. These vectors were transferred into Lactococcus lactis(pGRO1) and L. paracasei(pGRO2), and after induction with nisin, overexpressed GroEL represented 15 and 20% of the total cellular protein in each strain, respectively. Following heat shock treatment of lactococci (at 54°C) and lactobacilli (at 60°C), the heat-adapted cultures maintained the highest level of viability (5-log-unit increase, approximately) in each case, while it was found that the GroESL-overproducing strains performed only moderately better (1-log-unit increase) than the controls. On the other hand, the salt tolerance of both GroESL-overproducing strains (in 5 M NaCl) was similar to that of the parent cultures. Interestingly, both strains overproducing GroESL exhibited increased solvent tolerance, most notably, the ability to grow in the presence of butanol (0.5% [vol/vol]) for 5 h, while the viability of the parent strain declined. These results confirm the integral role of GroESL in solvent tolerance, and to a lesser extent, thermotolerance of lactic acid bacteria. Furthermore, this study demonstrates that technologically sensitive cultures, including certain probiotic lactobacilli, can potentially be manipulated to become more robust for survival under harsh conditions, such as food product development and gastrointestinal transit.     

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Bacteriocin production by spray-dried lactic acid bacteria

AIMS: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei. METHODS AND RESULTS: Bacterial cell concentrates were spray-dried and stored at 4 degrees C and 18 degrees C and 0.3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage. CONCLUSIONS: A higher survival rate was obtained when survival was performed at 4 degrees C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes. SIGNIFICANCE AND IMPACT OF THE STUDY: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.     

Fermentation of pomegranate juice by probiotic lactic acid bacteria

In this research, production of probiotic pomegranate juice through its fermentation by four strains of lactic acid bacteria: Lactobacillus plantarum, L. delbruekii, L. paracasei, L. acidophilus was examined. Fermentation was carried out at 30°C for 72 h under microaerophilic conditions. Microbial population, pH, titrable acidity, sugar and organic acid metabolism were measured during the fermentation period and the viability of all strains was also determined during the storage time at 4°C within 4 weeks. The results indicated that L. plantarum and L. delbruekii increased the pH sharply at the initial stages of fermentation and the sugar consumption was also higher in comparison with other strains, better microbial growth was also observed for these two strains during fermentation. Citric acid, as a major organic acid in pomegranate juice was significantly consumed by all probiotic lactic acid bacteria. L. plantarum and L. delbruekii showed higher viability during the storage time. Viable cells remained at their maximum level within 2 weeks but decreased dramatically after 4 weeks. Pomegranate juice was proved to be a suitable media for production of a fermented probiotic drink.     

Viability of vaginal probiotic lactobacilli during refrigerated and frozen storage

The viability of six different strains of probiotic vaginal Lactobacillus was examined in two different cryoprotective media, during refrigerated versus frozen storage, and using two traditional types of stock cultures for starting the biomass production. Freezing at -20 degrees C and -70 degrees C had much less adverse effect on viability than did storage at 7 degrees C, and the reduction in viability was greater at -20 degrees C than at -70 degrees C. The strains showed variation in the extent of the viability losses during both types of storage. Milk-yeast extract (MYE) was shown to be the more suitable protective medium to maintain viability of the strains during the storage. The vaginal Lactobacillus strains are most stable in MYE at -70 degrees C with only a small decrease of the viability observed under these conditions. The viable cell counts of Lactobacillus paracasei CRL 1251 and CRL 1289, L. crispatus CRL 1266 and L. salivarius CRL 1328 remained around 1 x 10(8) CFU/mL after 24 months of storage at -70 degrees C, or up to 18 months for L. acidophilus CRL 1259.     

Survival of Lactobacillus plantarum 44a after spraying and drying in feed and during exposure to gastrointestinal tract fluids in vitro

A good probiotic strain should be able to survive the conditions of handling and storage to be delivered in high concentration to the host. That is especially important when stressful conditions are prevalent in the carrier, for instance in low water content foods like animal feed. The aim of this research was to study the survival of the probiotic candidate Lactobacillus plantarum 44a after spraying and drying in feed, and during storage and exposure to gastrointestinal tract fluids in vitro. In addition, the viability of the strain during exposure to distilled water and 2% NaCl was studied. Feed was sprayed with a suspension of asymptotically equal to 2 x 10(10) CFU of L. plantarum 44a in 10, 15, 20, 25 and 30% v/w of the feed and dried to constant weight (6% moisture) in a convective oven at 25 degrees C. L. plantarum 44a survived 14.67, 36, 51.86, 78.9 and 105.3% respectively in relation to the original % v/w of the feed. After 3 weeks of storage at 25 degrees C, survival was similarly low in all the treatments. L. plantarum 44a stored in feed containing 13% moisture, vacuum-packaged and stored in refrigeration, maintained high viability (approximately 100%) after 1 year of storage. Survival was not affected after feed-containing lactobacilli was exposed to gastrointestinal fluids in a simulation model. Viability of L. plantarum 44a as a cell suspension in PBS added directly to distilled water or distilled water with 2% NaCl was maintained up to 48 h; after 72 h, viability started to decline. It is concluded that L. plantarum 44a maintained high viability after being dried and stored in feed even after exposure to gastric and intestinal fluids in vitro.     

Effect of some pharmaceutical excipients on the survival of probiotic vaginal lactobacilli

Lactobacilli are the predominant microorganisms of the vaginal bacterial microbiota, and they play a major role in the maintenance of a healthy urogenital tract. In consequence, the interest in their potential use as probiotics has significantly increased during the last decade. In the present study we assessed the influence of different excipients on the survival of 4 probiotic vaginal lactobacilli incorporated into glycerinated gelatin ovules and stored at 5 degrees C for 60 d. Results showed that viability after storage was a strain-dependent characteristic, but inclusion of ascorbic acid significantly increased survival in 3 of the 4 strains tested. The best survival was observed for Lactobacillus salivarius CRL 1328 in ovules containing skimmed milk. No significant differences in viability were observed between control ovules (glycerogelatin base without excipients) and those containing lactose or Tween 80 for any of the strains tested. Lactobacillus acidophilus CRL 1259 and Lactobacillus crispatus CRL 1266 were, respectively, the most resistant and sensitive strains to the storage with the different substances. In conclusion, these results provide a basis for selecting excipients to improve the survival of lactobacilli in a probiotic product, in an attempt to ensure the delivery of an adequate number of viable cells to the urogenital tract.     

Production of probiotic cabbage juice by lactic acid bacteria

Research was undertaken to determine the suitability of cabbage as a raw material for production of probiotic cabbage juice by lactic acid bacteria (Lactobacillus plantarum C3, Lactobacillus casei A4, and Lactobacillus delbrueckii D7). Cabbage juice was inoculated with a 24-h-old lactic culture and incubated at 30 degrees C. Changes in pH, acidity, sugar content, and viable cell counts during fermentation under controlled conditions were monitored. L. casei, L. delbrueckii, and L. plantarum grew well on cabbage juice and reached nearly 10x10(8) CFU/mL after 48 h of fermentation at 30 degrees C. L. casei, however, produced a smaller amount of titratable acidity expressed as lactic acid than L. delbrueckii or L. plantarum. After 4 weeks of cold storage at 4 degrees C, the viable cell counts of L. plantarum and L. delbrueckii were still 4.1x10(7) and 4.5x10(5) mL(-1), respectively. L. casei did not survive the low pH and high acidity conditions in fermented cabbage juice and lost cell viability completely after 2 weeks of cold storage at 4 degrees C. Fermented cabbage juice could serve as a healthy beverage for vegetarians and lactose-allergic consumers.     

Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze-drying survival, storage stability, and acid and bile exposure of Bifidobacterium animalis ssp. lactis cells produced without milk-based ingredients

AIMS: To investigate the stability of Bifidobacterium animalis ssp. lactis VTT E-012010 (=Bb-12) during freeze-drying, storage and acid and bile exposure. The effect of harvesting time and composition and pH of the cryoprotectant on the survival was evaluated. The procedure was performed by using a milk-free culture medium and cryoprotectants to produce cells for nonmilk-based applications. METHODS AND RESULTS: Bifidobacterial cells were grown in fermenters in general edible medium for 15 or 22 h. The cell mass was freeze-dried either as non-neutralized or neutralized using sucrose, betaine or reconstituted skim milk (control) as cryoprotectants. For stability studies freeze-dried powders were stored at 37, 5 and -20 degrees C for 2-6 months. In addition, acid and bile tolerance of the powders was tested. Sucrose-formulated B. animalis ssp. lactis preparations had an excellent stability during storage at refrigerated and frozen temperatures for 5-6 months. They also had a good survival during storage at 37 degrees C for 2 months as well as during exposure to pH 3 and 1% bile acids. No difference was observed between 15 and 22 h grown cells or between non-neutralized and neutralized cells. Betaine proved to be a poor cryoprotectant compared with sucrose. CONCLUSIONS: Fermentation time and neutralization of cell concentrate before freeze-drying had no impact on the storage stability and bile and acid tolerance of freeze-dried bifidobacterial cells. The nonmilk-based production protocol using sucrose as a cryoprotectant yielded powdery preparations with excellent stability in adverse conditions (storage at elevated temperatures and during acid and bile exposure). SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that it is feasible to develop nonmilk-based production technologies for probiotic cultures. This provides new possibilities for the development of nondairy-based probiotic products.