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.     

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.     

Genomic Diversity of Phages Infecting Probiotic Strains of Lactobacillus paracasei

Strains of the Lactobacillus casei group have been extensively studied because some are used as probiotics in foods. Conversely, their phages have received much less attention. We analyzed the complete genome sequences of five L. paracasei temperate phages: C_L1, C_L2, iLp84, iLp1308, and iA2. Only phage iA2 could not replicate in an indicator strain. The genome lengths ranged from 34,155 bp (iA2) to 39,474 bp (C_L1). Phages iA2 and iLp1308 (34,176 bp) possess the smallest genomes reported, thus far, for phages of the L. casei group. The GC contents of the five phage genomes ranged from 44.8 to 45.6%. As observed with many other phages, their genomes were organized as follows: genes coding for DNA packaging, morphogenesis, lysis, lysogeny, and replication. Phages C_L1, C_L2, and iLp1308 are highly related to each other. Phage iLp84 was also related to these three phages, but the similarities were limited to gene products involved in DNA packaging and structural proteins. Genomic fragments of phages C_L1, C_L2, iLp1308, and iLp84 were found in several genomes of L. casei strains. Prophage iA2 is unrelated to these four phages, but almost all of its genome was found in at least four L. casei strains. Overall, these phages are distinct from previously characterized Lactobacillus phages. Our results highlight the diversity of L. casei phages and indicate frequent DNA exchanges between phages and their hosts.     

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.     

Comparative studies of the degradation of grass fructan and inulin by strains of Lactobacillus paracasei subsp. paracasei and Lactobacillus plantarum

Four strains of Lactobacillus paracasei subsp. paracasei and Lact. plantarum are investigated within 16 d in order to determine the formation of metabolites during the degradation of grass fructan and inulin as well as the subsequent fermentation to lactic acid. The decrease of the total content of fructans throughout the entire time of investigation shows differences specific for strains as for either fructan substrate. The strain Lact. plantarum V 54/6 completely degrades the grass fructan and inulin within no longer than 13 d. The utilization of fructan by the other strains is temporally delayed, and in a smaller degree of degradation, especially remarkable for inulin cleavage. The structural modifications of decomposed fructans are characterized by a noticeable shift of the mean DP from approximately 80 to the oligomeric range analysed by anion exchange chromatography. Additionally, a newly formed series of peaks of oligomeric saccharides was detected during the degradation of grass fructan and inulin. Part of the fructose that is derived from cleavage of fructans is fermented immediately by the LAB strains into differently high amounts of lactic acid. The abundance of formed fructose is enriched in the medium to a varying extent, depending on the strain as well as the substrate used. From these results a number of fructan degradative enzymes in lactobacilli have been concluded to possibly vary their modes of regulation: strain specific exo- and endohydrolases with different activities against β-2,1 and β-2,6 linked fructan.     

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.     

Rates of Drying and Survival of Rhizobium meliloti Strains During Storage at Different Relative Humidities

An investigation was made of the survival of six strains of Rhizobium meliloti filtered on membrane filters and held in atmospheres of controlled relative humidities (RH) of from 0 to 100% at 30°C in the presence of air. The rate of water loss in the desiccator was determined by the humidity-controlling solution used. Drying was accelerated by a mild evacuation of the desiccator during the drying step. Survival rates of R. meliloti strains were much higher after slow drying to 0% RH than immediately after rapid drying. Fast drying (drying period less than 3.4 h) was shown to adversely affect the tolerance to storage at all RH values tested (no survival after 2 to 5 days of storage). When survival during storage was measurable (after slow drying), the optimum RH values for storage were 43% for strains A145 and Wu498, 22 to 43% for strains RCR2011, Wu499, and Ar16, and 83% for strain RCR2004. The most favorable drying periods were 8, 9.2, 14.2, and 50.1 h for the subsequent storage of strain RCR2011 at RH values of 0, 22, 43, and 83%, respectively. The damaging effects of rapid drying on the tolerance of strain RCR2011 to storage at different RH values could be prevented either by rehydration and subsequent slow redrying or incomplete rapid drying followed by slow drying. It is suggested that R. meliloti strains are susceptible to desiccation stresses. However, the quantitative differences among strains appear to be large enough to permit selection with regard to tolerance to desiccation and storage in dried states.     

Probiotic Characterization of Lactobacillus paracasei subsp. paracasei KNI9 Inhibiting Adherence of Yersinia enterocolitica on Caco-2 Cells In Vitro

The study was aimed to characterize the probiotic properties of Lactobacillus paracasei subsp. paracasei strain KNI9 and its antagonistic activity against Yersinia enterocolitica subsp. enterocolitica. The strain KNI9 was susceptible to antibiotics such as chloramphenicol, tetracycline, erythromycin, and streptomycin recommended by European food safety authority (EFSA). Strain KNI9 exhibited tolerance to simulated oro-gastrointestinal (OGT) condition, adherence to Caco-2 cells, and antimicrobial activity against intestinal enteric pathogens such as Yersinia enterocolitica subsp. enterocolitica, Shigella boydii, and Listeria monocytogenes. Furthermore, the strain KNI9 inhibited the adherence and invasiveness of Y. enterocolitica subsp. enterocolitica to Caco-2 cell line. These results indicate that the L. paracasei subsp. paracasei KNI9 could be further developed into a potential probiotic strain after appropriate in vivo studies.

     

Lactobacillus salivarius and L. gasseri down-regulate Aggregatibacter actinomycetemcomitans exotoxins expression

Beneficial microbes, such as lactobacilli establish a symbiosis with the host and confer health-associated effects, by limiting the growth of indigenous pathogens and challenging microbes introduced by altered foods. Nevertheless, there is scarce information on the effects of beneficial microbes on the virulence properties of bacterial species associated with oral diseases, such as periodontitis. Aggregatibacter actinomycetemcomitans is a Gram-negative species highly implicated in the etiology of localized aggressive periodontitis. The objective of this study was to investigate the effect of lactobacilli on the expression of the two major virulence factors of A. actinomycetemcomitans. Lactobacillus salivarius and L. gasseri were selected as beneficial species. The gene expressions of leukotoxin (LtxA) and cytolethal distending toxin (CdtB) by A. actinomycetemcomitans were analyzed in response to challenge by lactobacilli cell-free supernatants. Neither lactobacilli affected the growth, but strongly attenuated the expressions of both CdtB and LtxA in the two A. actinomycetemcomitans strains tested. This reduction of the expression of these two exotoxins was time-dependent. These fundamental findings may indicate that lactobacilli can reduce the virulence of putative opportunistic oral pathogens, and may provide insights to future therapeutic approaches for the respective diseases.     

Comparative Analysis of Antigiardial Potential of Heat Inactivated and Probiotic Protein of Probiotic Lactobacillus rhamnosus GG in Murine Giardiasis

The present study was designed to envisage the antigiardial efficacy of killed probiotic and probiotic protein (PP) of Lactobacillus rhamnosus GG in murine giardiasis. Experimentally, it was observed that animal administered either with probiotic protein emulsified with adjuvant (PP_(E) + Giardia) or killed probiotic (killed probiotic (i/p) + Giardia) had significantly reduced Giardia cycle with respect to observed severity and duration of giardiasis compared with Giardia-infected mice. Further, it was found that animals belonging to PP_(E) + Giardia and killed probiotic (i/p) + Giardia had significantly high levels of antigiardial IgA antibody and nitric oxide both in serum and in intestinal fluid compared with Giardia-infected and counter control mice. Histopathologyically, also animals belonging to PP_(E) + Giardia and killed probiotic (i/p) + Giardia animals had intact mucosal epithelium lining, basal crypts, and normal villi along with increased goblet cells compared with severe microvillus atrophy, vacuolated epithelial cells, and ileitis in Giardia-infected mice. This is the first-ever study to demonstrate that prior administration of either killed probiotics or probiotic protein of effective probiotic reduced both the severity and the duration of giardiasis mainly by modulating the gut microbiome and morphology along with mucosal immunity, but animals belonging to PP_(E) + Giardia had better response than killed probiotic (i/p) + Giardia suggesting that probiotic components do have adjuvant potential and may be used as the vaccine candidate for gastrointestinal diseases.

     

Cell surface characteristics of Lactobacillus casei subsp. casei, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus strains.

Hydrophilic and electrostatic cell surface properties of eight Lactobacillus strains were characterized by using the microbial adhesion to solvents method and microelectrophoresis, respectively. All strains appeared relatively hydrophilic. The strong microbial adhesion to chloroform, an acidic solvent, in comparison with microbial adhesion to hexadecane, an apolar n-alkane, demonstrated the particularity of lactobacilli to have an important electron donor and basic character and consequently their potential ability to generate Lewis acid-base interactions with a support. Regardless of their electrophoretic mobility (EM), strains were in general slightly negatively charged at alkaline pH. A pH-dependent behavior concerning cell surface charges was observed. The EM decreased progressively with more acidic pHs for the L. casei subsp. casei and L. paracasei subsp. paracasei strains until the isoelectric point (IEP), i.e., the pH value for which the EM is zero. On the other hand, the EM for the L. rhamnosus strains was stable from pH 8 to pH 3 to 4, at which point there was a shift near the IEP. Both L. casei subsp. casei and L. paracasei subsp. paracasei strains were characterized by an IEP of around 4, whereas L. rhamnosus strains possessed a markedly lower IEP of 2. The present study showed that the cell surface physicochemical properties of lactobacilli seem to be, at least in part and under certain experimental conditions, particular to the bacterial species. Such differences detected between species are likely to be accompanied by some particular changes in cell wall chemical composition.     

Synergistic Antibacterial Efficiency of Bacteriocin and Silver Nanoparticles Produced by Probiotic Lactobacillus paracasei Against Multidrug Resistant Bacteria

Contamination of wounds with multidrug-resistant microorganisms is one of the greatest challenges in the curing of many diseases, and therefore, the search for alternative antimicrobial agents becomes urgent. In the present study, out of 280 bacterial isolates recovered from 57 wound swabs of patients, 199 isolates (71.1%) showed multiple drug resistance (MDR). Staphylococcus aureus was the most predominant (80.0%), followed by Pseudomonas aeuroginosa (78.87%). A number of 121 isolates (60.80%) of MDR was sensitive to bacteriocin produced by a probiotic strain of Lactobacillus paracasei. However, considering that bacteriocins may be degraded before reaching its target, silver nanoparticles (AgNPs) can be used as a suitable candidate for combinations with bacteriocin in order to be more effective. AgNPs were synthesized using the same strain and then characterized by UV–Vis spectroscopy, TEM and XRD analyses. Bacteriocin/AgNPs bioconjugate exhibited a higher antibacterial efficiency against MDR strains than that of bacteriocin or AgNPs alone. Interestingly, the bioconjugate has significantly increased the disruption of cell membrane permeability, leakages of protein and DNA, in addition to the formation of bactericidal reactive oxygen species than any other extracts. Therefore, the combination of bacteriocin with metal nanoparticles is recommended as a promising approach for the elimination of MDR organisms.

     

Safety and Stability of Two Potentially Probiotic Lactobacillus Strains After In Vitro Gastrointestinal Transit

According to FAO and WHO, probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Most probiotic bacteria used today belong to the genera Lactobacillus and Bifidobacterium and are of animal or human origin. The fundamental characteristic routinely evaluated in potential probiotics strains is their limited viability loss during gastrointestinal transit (GIT), but to date, no studies reported whether probiotics, besides viability, still also maintain their beneficial properties intact. To study this aspect, we considered two strains, Lactobacillus rhamnosus DTA 79 and L. paracasei DTA 83, previously characterised for the presence of some probiotic properties, isolated from faeces of 7- to 21-day-old babies. Here, we examined some additional properties, namely antibiotic resistance, resistance to lysozyme, presence of haemolytic activity and inhibition of pathogen biofilm formation. We then tested the effect of in vitro GIT on all these features and our results show evidence that this procedure had in some cases limited and in others no significant effects on them. Additionally, we examined the gastrointestinal resistance of the strains after skim milk fermentation and successive storage of the product for 20 and 40 days at refrigeration temperature, to see whether prolonged storage could weaken cell resistance to GIT. Our results demonstrate that a protracted refrigeration period before in vitro GIT did not affect or influenced very weakly this essential probiotic property.

     

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.     

A mixed culture of Propionibacterium jensenii and Lactobacillus paracasei subsp. paracasei inhibits food spoilage yeasts

Screening for antimicrobial features of 197 propionibacteria and tests with several antifungal lactobacilli led to the development of three protective cultures containing Propionibacterium jensenii SM11 and Lactobacillus paracasei subsp. paracasei strain SM20, SM29 or SM63. These cultures showed inhibitory activities (up to 5 orders of magnitude) against yeasts in dairy products such as yoghurt or cheese surface at refrigerator temperatures (6 degrees C) without an influence on the quality properties of the food. Initial cell numbers of 5 x 10(7) cells/g of propionibacteria and 1 x 10(8) cells/g of lactobacilli were the optimal concentrations to yield a total inhibition of the spoilage yeasts (Candida pulcherrima, Candida magnoliae, Candida parapsilosis and Zygosaccharomyces bailii).     

Purification and N-terminal amino acid sequence determination of the cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei.

The cell-wall-bound proteinase from Lactobacillus paracasei subsp. paracasei NCDO 151 was purified to homogeneity by anion-exchange and hydrophobic-interaction chromatography, chromatofocusing and gel-filtration. The purification resulted in a 600-700-fold increase in specific activity of the proteinase and the final yield was approximately 20%. Upon chromatofocusing, two proteolytically active components, termed pro135 and pro110, were detected. pro135 had an isoelectric point of 4.2. It had an Mr of about 300,000 as determined by gel-filtration and 135,000 as judged by SDS-PAGE, indicating that it may exist as a dimer in its native state. pro110 had an isoelectric point of 4.4, and an Mr of about 150,000 as determined by gel-filtration and 110,000 as judged by SDS-PAGE. pro110 appears to be a degradation product of pro135 as they have the same N-terminal amino acid sequence. The first N-terminal amino acid was ambiguous for both components, whereas the sequence from the second to the ninth amino acid was Ala-Lys-Ala-Asn-Ser-Met-Ala-Asn. This is identical to the corresponding sequence of the lactococcal cell-wall-bound proteinases. Although the Lactobacillus proteinase was a little smaller than the lactococcal proteinase, their purification characteristics were very similar, suggesting that these proteinases are related.     

Complete Genome Sequence of Lactobacillus salivarius CECT 5713, a Probiotic Strain Isolated from Human Milk and Infant Feces

Lactobacillus salivarius is a homofermentative lactic acid bacterium and is frequently isolated from mucosal surfaces of healthy humans. L. salivarius CECT 5713, a strain isolated simultaneously from breast milk and infant feces of a healthy mother-infant pair, has immunomodulatory, anti-inflammatory, and anti-infectious properties, as revealed by several in vitro and in vivo assays. Here, we report its complete and annotated genome sequence.In the last years, culture-dependent and -independent analyses of the bacterial diversity of human milk and colostrum have revealed that these biological fluids are a source of live staphylococci, streptococci, lactic acid bacteria, and bifidobacteria in the infant gut (5, 6, 8, 9, 11, 13), where they play a key role in the initiation and development of the gut microbiota (12). In a previous study, we isolated L. salivarius CECT 5713 from human milk and infant feces of a mother-child pair (10). Subsequent studies revealed that this strain was a good probiotic candidate since it achieved high survival rates when exposed to the gastrointestinal tract conditions, showed a strong adherence to intestinal cells, stimulated the expression of mucin-encoding genes, produced antimicrobial compounds (lactate, acetate, and hydrogen peroxide), and displayed in vivo and in vitro immunomodulatory, anti-inflammatory, and antibacterial properties against pathogenic bacteria (2, 10, 15). Moreover, oral administration of L. salivarius CECT 5713 appears to be an efficient alternative for the treatment of infectious mastitis in lactating women (7). Similarly, studies with other L. salivarius strains in animal models and clinical trials have demonstrated their probiotic function and, particularly, their anti-inflammatory effects (3, 14, 16).In order to interrogate the genome sequence of L. salivarius CECT 5713 with regard to its probiotic properties, the complete genome sequence was determined by a whole-genome shotgun strategy using pyrosequencing technology (454 Life Sciences, Banford, CT). The initial draft assembly provided by 454 Life Sciences was based on 444,604 high-quality pyrosequencing reads, which assembled into 59 contigs. The genome sequence of L. salivarius UCC118 (1), a well-characterized probiotic strain, was used to order these contigs into large scaffolds.The genome of L. salivarius CECT 5713 consists of a circular chromosome of 1,828,169 bp, two plasmids (pHN1, 44,581 bp; pHN2, 20,426 bp), and a megaplasmid (pHN3, 242,962 bp). The overall GC content of the chromosome is 32.93%, similar to that of the megaplasmid but lower than those of the plasmids (>38%). The entire genome of CECT 5713 contains 1,558 protein-, 87 tRNA-, and 51 rRNA-encoding genes. A comparison between the genomes of L. salivarius CECT 5713 and UCC118 revealed the presence of 52 protein-encoding genes that are exclusive for CECT 5713, including genes encoding a 6-phospho-β-glucosidase and three collagen-binding proteins, which may explain the high potential for competitive exclusion of pathogens displayed by this strain. The genes responsible for the bacteriocin activity of L. salivarius CECT 5713 are located in pHN3. This megaplasmid contains six open reading frames (ORFs) closely related, but not identical, to the genes responsible for the biosynthesis of salivaricin ABP-118, a two-component class II bacteriocin (4), in L. salivarius UCC118. Globally, several features of the L. salivarius CECT 5713 genome suggest a strong probiotic potential in humans.