Neuroprotective Effects Exerted by a Combination of Selected Lactic Acid Bacteria in a Mouse Parkinsonism Model under Levodopa-Benserazide Treatment.

Alterations of the microbiota-gut-brain axis has been associated with intestinal and neuronal inflammation in Parkinson's disease (PD). The aim of this work was to study some mechanisms associated with the neuroprotective effect of a combination (MIX) of lactic acid bacteria (LAB) composed by Lactiplantibacillus plantarum CRL2130 (riboflavin overproducing strain), Streptococcus thermophilus CRL808 (folate producer strain), and CRL807 (immunomodulatory strain) in cell cultures and in a chronic model of parkinsonism induced with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in aged mice, and under levodopa-benserazide treatment. In vitro, N2a differentiated neurons were exposed to the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and treated with intracellular bacterial extracts or with conditioned media from BV-2 cells exposed to the bacterial extracts. In vivo, motor skills, tyrosine hydrolase (TH) in brain and cytokine concentrations in serum and in brain were evaluated. The study of the faecal microbiota and the histology of the small intestine was also performed. The results showed that the neuroprotective effect associated with LAB MIX administration did not interfere with levodopa-benserazide treatment. This effect could be associated with the antioxidant and immunomodulatory potential of the LAB selected in the MIX, and was associated with the significant improvement in the motor tests and a higher number of TH + cells in the brain. In addition, LAB MIX administration was associated with modulation of the immune response. LAB administration decreased intestinal damage with an increase in the villus length /crypt depth ratio. Finally, the administration of the LAB MIX in combination with levodopa-benserazide treatment was able to partially revert the intestinal dysbiosis observed in the model, showing greater similarity to the profiles of healthy controls, and highlighting the increase in the Lactobacillaceae family. Different mechanisms of action would be related to the protective effect of the selected LAB combination which has the potential to be evaluated as an adjuvant for conventional PD therapies.

Multi-omics Approach Reveals How Yeast Extract Peptides Shape Streptococcus thermophilus Metabolism.

Peptides present in growth media are essential for nitrogen nutrition and optimal growth of lactic acid bacteria. In addition, according to their amino acid composition, they can also directly or indirectly play regulatory roles and influence global metabolism. This is especially relevant during the propagation phase to produce high cell counts of active lactic acid bacteria used as starters in the dairy industry. In the present work, we aimed at investigating how the respective compositions of two different yeast extracts, with a specific focus on peptide content, influenced Streptococcus thermophilus metabolism during growth under pH-controlled conditions. In addition to free amino acid quantification, we used a multi-omics approach (peptidomics, proteomics, and transcriptomics) to identify peptides initially present in the two culture media and to follow S. thermophilus gene expression and bacterial protein production during growth. The free amino acid and peptide compositions of the two yeast extracts differed qualitatively and quantitatively. Nevertheless, the two yeast extracts sustained similar levels of growth of S. thermophilus and led to equivalent final biomasses. However, transcriptomics and proteomics showed differential gene expression and protein production in several S. thermophilus metabolic pathways, especially amino acid, citrate, urease, purine, and pyrimidine metabolisms. The probable role of the regulator CodY is discussed in this context. Moreover, we observed significant differences in the production of regulators and of a quorum sensing regulatory system. The possible roles of yeast extract peptides on the modulation of the quorum sensing system expression are evaluated.IMPORTANCE Improving the performance and industrial robustness of bacteria used in fermentations and food industry remains a challenge. We showed here that two Streptococcus thermophilus fermentations, performed with the same strain in media that differ only by their yeast extract compositions and, more especially, their peptide contents, led to similar growth kinetics and final biomasses, but several genes and proteins were differentially expressed/produced. In other words, subtle variations in peptide composition of the growth medium can finely tune the metabolism status of the starter. Our work, therefore, suggests that acting on growth medium components and especially on their peptide content, we could modulate bacterial metabolism and produce bacteria differently programmed for further purposes. This might have applications for preparing active starter cultures.

Suppressive effects of Streptococcus thermophilus KLDS 3.1003 on some foodborne pathogens revealed through in vitro, in vivo and genomic insights.

Foodborne diseases (FBDs) remain a persistent global challenge and recent research efforts suggest that lactic acid bacteria (LAB) strains can contribute towards their prevention and treatment. This study investigates the genetic properties of Streptococcus thermophilus KLDS 3.1003 as a potential probiotic and health-promoting LAB strain as well as its in vitro and in vivo activities against two foodborne pathogens. In vitro, its antimicrobial activities and tolerance levels in simulated bile salts and acids were determined. The cytotoxic effects of the LAB strain in RAW264.7 cells were also evaluated. For in vivo evaluation, 24 BALB/c mice were orally administered control and trial diets for 14 days. Genomic analyses of this strain's bacteriocin configuration, stress response system and multidrug resistance genes were annotated to validate in vitro and in vivo results. In vitro antimicrobial results show that the cells and CFS of S. thermophilus KLDS 3.1003 could inhibit both pathogens with the former being more effective (P < 0.05). In addition, its cell-free supernatant (CFS) could inhibit the growth of both pathogens, with catalase treatment having the highest effect against it. More so, after 3 h of incubation, survivability levels of S. thermophilus KLDS 3.1003 were significantly high (P < 0.05). LPS-induced RAW264.7 cell activities were also significantly reduced by 108-109 CFU mL-1 of S. thermophilus KLDS. In vivo, significant weight losses were inhibited in the TSTEC group compared to the TSTSA group (P < 0.05). Moreover, pathogen-disrupted blood biochemical parameters like HDL, LDL, TP, TG, AST, ALT and some minerals were restored in the respective prevention groups (TSTEC and TSTSA). Genomic analyses showed that S. thermophilus KLDS 3.1003 has bacteriocin-coding peptides, which accounts for its antimicrobial abilities in vitro and in vivo. S. thermophilus KLDS 3.1003 is also endowed with intact genes for acid tolerance, salt-resistance, cold and heat shock responses and antioxidant activities, which are required to promote activities against the selected foodborne pathogens. This study showed that S. thermophilus KLDS 3.1003 has the genomic capacity to inhibit foodborne pathogens' growth in vitro and in vivo, thus qualifying it as a potential probiotic, antimicrobial and bio-therapeutic candidate.

No more cleaning up - Efficient lactic acid bacteria cell catalysts as a cost-efficient alternative to purified lactase enzymes.

ß-galactosidases, commonly referred to as lactases, are used for producing lactose-free dairy products. Lactases are usually purified from microbial sources, which is a costly process. Here, we explored the potential that lies in using whole cells of a food-grade dairy lactic acid bacterium, Streptococcus thermophilus, as a substitute for purified lactase. We found that S. thermophilus cells, when treated with the antimicrobial peptide nisin, were able to hydrolyze lactose efficiently. The rate of hydrolysis increased with temperature; however, above 50 °C, stability was compromised. Different S. thermophilus strains were tested, and the best candidate was able to hydrolyze 80% of the lactose in a 50 g/L solution in 4 h at 50 °C, using only 0.1 g/L cells (dry weight basis). We demonstrated that it was possible to grow the cell catalyst on dairy waste, and furthermore, that a cell-free supernatant of a culture of a nisin-producing Lactococcus lactis strain could be used instead of purified nisin, which reduced cost of use significantly. Finally, we tested the cell catalysts in milk, where lactose also was efficiently hydrolyzed. The method presented is natural and low-cost, and allows for production of clean-label and lactose-free dairy products without using commercial enzymes from recombinant microorganisms. KEY POINTS: • Nisin-permeabilized Streptococcus thermophilus cells can hydrolyze lactose efficiently. • A low-cost and more sustainable alternative to purified lactase enzymes. • Reduction of overall sugar content. • Clean-label production of lactose-free dairy products.

Jamelão capsules containing bioactive compounds and its aplication in yoghurt.

BACKGROUND: Jamelão fruit (Syzygium cumini), has recently attracted interest as a functional food for being rich in anthocyanins, which has antioxidant power, attractive color and stability in high acid foods. METHODS: Samples of yoghurt with jamelão capsules obtained through the gelling process with sodium alginate solution and bioactive yoghurt (control + capsules) were evaluated. The samples were evaluated for composition, microbial viability, the stability on the anthocyanic pigments and its antioxidant activity. RESULTS: With the addition of jamelão pulp capsules there was a reduction in the Streptococcus thermophilus count. The addition of capsules in yoghurts were able to increase the amount of phenols and anthocyanins, as well as antioxidant potential at 28 days. The chromatographic analysis showed that process was efficient, being capable of encapsulating a large part of the bioactive compounds present in the pulp. CONCLUSIONS: The addition of jamelão pulp capsules to yoghurts was shown to be a promising and viable alternative, since the bioactive compounds present in the fruit were present in the yoghurt.

Effects of gelatin-based antifreeze peptides on cell viability and oxidant stress of Streptococcus thermophilus during cold stage.

Cold stage adversely affects cell proliferation and cell viability of probiotics such as Streptococcus thermophilus in food industry, new type of cryoprotectants continues to be needed. Gelatin-based antifreeze peptide becomes a popular topic because of its cryoprotective effects on cold-stressed probiotics. In this study the effects of tilapia scales antifreeze peptides (TSAPP) on cell viability and oxidant stress of S. thermophilus during cold stage were investigated. The results showed that the percentage of viable cells was increased 10.85 folds compared with control groups. Addition of TSAPP activated the activities of ATPases, relieved the hyperpolarization of cell membrane potential and regulated the intracellular Ca2+ concentration. Furthermore, TSAPP significantly inhibited reactive oxygen species level and malonaldehyde content in cells. Under cryopreservation with TSAPP, cells of S. thermophilus maintained higher activities of antioxidant enzymes including catalase, peroxidase and total antioxidant capacity. These findings indicate that TSAPP likely offered its cellular protection by maintaining membrane integrity and alleviation of oxidative stress.

Laxative effect of probiotic chocolate on loperamide-induced constipation in rats.

The fecal morphology, defecation frequency, bowel function, intestinal motility, and fecal bacterial composition were evaluated to investigate the laxative effect of probiotic chocolate containing Streptococcus thermophilus MG510 and Lactobacillus plantarum LRCC5193 (LYC) on loperamide-induced constipated rats. Daily oral administration of LYC in constipated rats for two weeks was shown to significantly increase (n = 14) the defecation frequency, fecal moisture content, and relative abundance of fecal Lactobacillus and Faecalibacterium prausnitzii. Moreover, histological analysis of the distal colon of constipated rats revealed that LYC treatment can also increase the thickness of the colonic mucosa and muscle layers, and crypt of Lieberkühn. LYC also significantly increased (n = 5) the intestinal motility and modulated (n = 9) mRNA expression levels of colonic ZO-1 and Cldn-1 in the constipated rats. Altogether, these results demonstrate that probiotic chocolate has potential as a dietary adjunct for the treatment of constipation.

Short communication: Influence of an aqueous myrrh suspension on yogurt culture bacteria over yogurt shelf life.

Myrrh is an essential oil and natural flavoring approved by the US Food and Drug Administration, and it has antibacterial and antifungal activity against pathogens. Our objective was to determine the effect of an aqueous myrrh suspension on Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus counts in peptone solution and yogurt, as well as pH and titratable acidity of yogurt during 5 wk of storage at 1 to 4°C. The myrrh suspension (10% wt/vol) was prepared and incorporated into a pure culture dilution in peptone and into yogurt mix at a 1% (vol/vol) level. A control with no myrrh was also prepared, and 3 replications were conducted. Streptococcus thermophilus were enumerated using Streptococcus thermophilus agar with aerobic incubation at 37°C for 24 h, and Lactobacillus delbrueckii ssp. bulgaricus were enumerated using de Man, Rogosa, and Sharpe agar adjusted to pH 5.2, with anaerobic incubation at 43°C for 72 h. During the 8-h period after inoculation, S. thermophilus and L. delbrueckii ssp. bulgaricus counts in peptone solution at 37°C and 43°C, respectively, were not significantly different in the presence or absence of the aqueous myrrh suspension. Counts of S. thermophilus in yogurt containing myrrh (mean ± SD; 4.96 ± 0.58 log cfu/mL) were not significantly different from those in the control yogurt (4.87 ± 0.39 log cfu/mL). The log counts for L. delbrueckii ssp. bulgaricus in yogurt containing myrrh (5.04 ± 1.44 log cfu/mL) and those of the control (5.52 ± 1.81 log cfu/mL) did not differ, and the counts remained within 1 log of each other throughout 5 wk of storage. The pH of the yogurts containing the aqueous myrrh suspension was not significantly different from that of the control yogurts, and their pH values were within 0.1 pH unit of each other in any given week. Titratable acidity values remained steady around 1.1 to 1.2% lactic acid for both yogurt types throughout the storage period, with no significant differences between them. Yogurt culture bacteria can survive in the presence of a myrrh suspension in yogurt with no significant change in pH or titratable acidity. Therefore, it may be beneficial to add an aqueous myrrh suspension to yogurt.

Influence of passion fruit by-product and fructooligosaccharides on the viability of Streptococcus thermophilus TH-4 and Lactobacillus rhamnosus LGG in folate bio-enriched fermented soy products and their effect on probiotic survival and folate bio-accessibility under in vitro simulated gastrointestinal conditions.

This study aimed to evaluate the influence of passion fruit by-product (PFBP) and fructooligosaccharides (FOS) on the viability of Streptococcus thermophilus TH-4 and Lactobacillus rhamnosus LGG in folate bio-enriched fermented soy products and their effect on probiotic survival and folate bio-accessibility under in vitro simulated gastrointestinal conditions during storage of the products at 4 °C for up to 28 days (at days 1, 14, and 28). Kinetic parameters and folate contents before and after fermentation were also evaluated. Four different bio-enriched soy products in which the two microorganisms were used in co-cultures were studied and PFBP and/or FOS were added at 1 g/100 g, except for the control product. No differences (p < 0.05) between the fermented soy products (FSP) were observed for the maximum acidification rate (Vmax) and the time to reach the Vmax (Tmax) or pH 5.5 (Tf), indicating that the use of PFBP and/or FOS did not affect the fermentation kinetic parameters. Only Lb. rhamnosus LGG retained the desired viability (>8 log CFU/mL) during storage, whereas St. thermophilus TH-4 populations decreased by day 14 reaching counts between 6.4 and 5.5 log CFU/mL by day 28. The folate content of all FSP increased after fermentation and the simultaneous presence of PFBP and FOS stimulated the co-culture to increase folate production. Folate content in all FSP decreased during storage. Lb. rhamnosus LGG was recovered at the end of the simulated digestion, but PFBP and/or FOS did not affect recovery. The folate content increased during the gastrointestinal assay for all FSP, especially for FSP without supplementation, suggesting an in vitro increase of folate bio-accessibility. Therefore, the bio-enriched probiotic FSP presented a great potential as an innovative functional food by delivering probiotic microorganisms and providing 14% of the recommended daily folate intake. The folate content of the FSP might be increased during gastrointestinal stress conditions, which could contribute to increase the folate bio-accessibility in the gut.

Cryoprotective Activity and Action Mechanism of Antifreeze Peptides Obtained from Tilapia Scales on Streptococcus thermophilus during Cold Stress.

Cold stress adversely affects cell viability and acidification, and new cryoprotective methods continue to be needed in cold-chain food industry. Given this, we investigated the cryoprotective effects and action mechanism of antifreeze peptides obtained from tilapia scales (TSAPP) on Streptococcus thermophilus during cold stress. Our results showed that the molecular weight of TSAPP ranged from 180 to 2000 Da and its thermal hysteresis activity was 0.29 °C. Growth of S. thermophilus was improved after treatment with TSAPP (1 mg/mL) under cold stress. This growth was notable when compared with the effects of other cryoprotectants. Furthermore, TSAPP improved the metabolic activity of S. thermophilus during cold stress. TSAPP likely offered its cellular protection by maintaining cell membrane fluidity through hydrogen bonding of the phospholipid bilayer. These results indicate that TSAPP has potential as a novel biological peptide material with cryoprotective activity for future use in probiotic or other processed food applications.

Antibiotic Resistance of Lactobacillus spp. and Streptococcus thermophilus Isolated from Chinese Fermented Milk Products.

The aim of the present study was to investigate the phenotypic and genotypic antimicrobial resistance and the transferability of resistance markers in 87 lactic acid bacterial strains recovered from fermented milk products obtained from different areas of China. The isolates were identified as 21 Lactobacillus bulgaricus, 8 Lactobacillus casei, 6 Lactobacillus rhamnosus, 3 Lactobacillus paracasei, 2 Lactobacillus acidophilus, and 47 Streptococcus thermophilus strains. High levels of intrinsic resistance were revealed among the tested species. The following resistance genes were detected in strains isolated from fermented milk products: tet(M) in two L. bulgaricus and two S. thermophilus isolates, strA and strB in nine and seven S. thermophilus isolates, respectively; sul1 in six L. bulgaricus and seven S. thermophilus isolates, sul2 in one S. thermophilus isolate, aac(6')-aph(2″) in two L. bulgaricus isolates, and aph(3″)-II and aph(3″)-III in one S. thermophilus and two L. bulgaricus isolates, respectively. Transfer of the monitored antibiotic resistance genes was not observed in the filter mating assays of this study. To our knowledge, the strA, strB, sul1, sul2, and aph(3″)-II genes in S. thermophilus, and the sul1 and aac(6')-aph(2″) genes in L. bulgaricus were identified for the first time. These results indicate the potential risks posed by lactic acid bacteria (LAB) in fermented milk products in expanding the antibiotic resistance gene reservoir and transferring antibiotic resistance genes among bacteria. Further investigations are required to identify the potential sources of contamination and the dissemination routes of antibiotic resistance genes among LAB in fermented milk products.

Characterization and transfer of antimicrobial resistance in lactic acid bacteria from fermented dairy products in China.

INTRODUCTION: Lactic acid bacteria (LAB) are commonly found in foods and are also natural intestinal inhabitants in humans and most animals. However, information regarding antimicrobial resistance and the transfer of resistance genes of LAB from fermented dairy products in China is limited. METHODOLOGY: In this study, LAB isolates (n = 82) of Lactobacillus (n = 43) and Streptococcus thermophilus (n = 39) were isolated from 51 commercial fermented food samples in China. All isolates were subjected to pulsed-field gel electrophoresis (PFGE), antimicrobial susceptibility, detecting resistance genes, as well as investigating the transferability of resistance genes. RESULTS: The 43 Lactobacillus isolates yielded 24 PFGE patterns and the 34 isolates of S. thermophilus generated 32 different PFGE patterns. Among the 43 Lactobacillus strains, the most commonly observed resistance was that to streptomycin (83.7%) and gentamycin (83.7%). Among the 39 S. thermophilus strains, the most frequently observed resistance was that to streptomycin (92.3%), gentamycin (87.2%), ciprofloxacin (79.5%), and chloramphenicol (71.8%), whereas the lowest level of resistance was that against erythromycin (7.7%). Antimicrobial resistance genes for erythromycin (emrB), gentamycin (aac(6')-aph(2")), streptomycin (ant(6)), sulfamethoxazole (sulI and sulII), tetracycline (tetM and tetS) were detected in the 18 resistance LAB strains. Conjugation experiments showed that tetM from L. delbrueckii subsp. bulgaricus R6 and tetS from L. plantarum R41 were successfully transferred to L. monocytogenes by filter mating. CONCLUSIONS: LAB strains could potentially act as reservoirs of resistance genes and play an active role in the transfer of resistance to humans via the food chain.

A Cryptic Non-Inducible Prophage Confers Phage-Immunity on the Streptococcus thermophilus M17PTZA496.

Streptococcus thermophilus is considered one of the most important species for the dairy industry. Due to their diffusion in dairy environments, bacteriophages can represent a threat to this widely used bacterial species. Despite the presence of a CRISPR-Cas system in the S. thermophilus genome, some lysogenic strains harbor cryptic prophages that can increase the phage-host resistance defense. This characteristic was identified in the dairy strain S. thermophilus M17PTZA496, which contains two integrated prophages 51.8 and 28.3 Kb long, respectively. In the present study, defense mechanisms, such as a lipoprotein-encoding gene and Siphovirus Gp157, the last associated to the presence of a noncoding viral DNA element, were identified in the prophage M17PTZA496 genome. The ability to overexpress genes involved in these defense mechanisms under specific stressful conditions, such as phage attack, has been demonstrated. Despite the addition of increasing amounts of Mitomycin C, M17PTZA496 was found to be non-inducible. However, the transcriptional activity of the phage terminase large subunit was detected in the presence of the antagonist phage vB_SthS-VA460 and of Mitomycin C. The discovery of an additional immune mechanism, associated with bacteriophage-insensitive strains, is of utmost importance, for technological applications and industrial processes. To our knowledge, this is the first study reporting the capability of a prophage integrated into the S. thermophilus genome expressing different phage defense mechanisms. Bacteriophages are widespread entities that constantly threaten starter cultures in the dairy industry. In cheese and yogurt manufacturing, the lysis of Streptococcus thermophilus cultures by viral attacks can lead to huge economic losses. Nowadays S. thermophilus is considered a well-stablished model organism for the study of natural adaptive immunity (CRISPR-Cas) against phage and plasmids, however, the identification of novel bacteriophage-resistance mechanisms, in this species, is strongly desirable. Here, we demonstrated that the presence of a non-inducible prophage confers phage-immunity to an S. thermophilus strain, by the presence of ltp and a viral noncoding region. S. thermophilus M17PTZA496 arises as an unconventional model to study phage resistance and potentially represents an alternative starter strain for dairy productions.

Differences in Carbohydrates Utilization and Antibiotic Resistance Between Streptococcus macedonicus and Streptococcus thermophilus Strains Isolated from Dairy Products in Italy.

Streptococcus thermophilus and S. macedonicus are the only two species of the genus related to food productions so far known. In the present study, eight S. thermophilus and seven S. macedonicus strains isolated from dairy environments in Italy were compared in order to evidence possible species-specific technological characteristics. Their capability to use lactose, galactose, fructose, and glucose, sugars commonly present in foods and two carbohydrates considered as prebiotics, xylose and inulin, along with the respective growth kinetics were studied. Results showed a luxuriant growth on lactose and different behaviors on galactose, glucose, and fructose. No growth on inulin and xylose was recorded, which is a positive feature for strains intended to be used as starter cultures. Growth parameters, namely, λ, µmax, and Nmax, were estimated by using the Gompertz model. Antibiotic resistance to 14 drugs revealed an overall similar behavior between the two species with only a marked difference regarding gentamycin. Antimicrobial activity was also tested against six deleterious bacterial strains, but none of the strains evidenced inhibitory capabilities. The results presented here could be helpful to compare technological potentialities of the two species and to choose strains of the most suitable species for selected microbiological food transformations.

Preparation of Acid-Resistant Microcapsules with Shell-Matrix Structure to Enhance Stability of Streptococcus Thermophilus IFFI 6038.

Microencapsulation is an effective technology used to protect probiotics against harsh conditions. Extrusion is a commonly used microencapsulation method utilized to prepare probiotics microcapsules that is regarded as economical and simple to operate. This research aims to prepare acid-resistant probiotic microcapsules with high viability after freeze-drying and optimized storage stability. Streptococcus thermophilus IFFI 6038 (IFFI 6038) cells were mixed with trehalose and alginate to fabricate microcapsules using extrusion. These capsules were subsequently coated with chitosan to obtain chitosan-trehalose-alginate microcapsules with shell-matrix structure. Chitosan-alginate microcapsules (without trehalose) were also prepared using the same method. The characteristics of the microcapsules were observed by measuring the freeze-dried viability, acid resistance, and long-term storage stability of the cells. The viable count of IFFI 6038 in the chitosan-trehalose-alginate microcapsules was 8.34 ± 0.30 log CFU g-1 after freeze-drying (lyophilization), which was nearly 1 log units g-1 greater than the chitosan-alginate microcapsules. The viability of IFFI 6038 in the chitosan-trehalose-alginate microcapsules was 6.45 ± 0.09 log CFU g-1 after 120 min of treatment in simulated gastric juices, while the chitosan-alginate microcapsules only measured 4.82 ± 0.22 log CFU g-1 . The results of the long-term storage stability assay indicated that the viability of IFFI 6038 in chitosan-trehalose-alginate microcapsules was higher than in chitosan-alginate microcapsules after storage at 25 °C. Trehalose played an important role in the stability of IFFI 6038 during storage. The novel shell-matrix chitosan-trehalose-alginate microcapsules showed optimal stability and acid resistance, demonstrating their potential as a delivery vehicle to transport probiotics.

Production of reuterin in a fermented milk product by Lactobacillus reuteri: Inhibition of pathogens, spoilage microorganisms, and lactic acid bacteria.

We assessed the antimicrobial activity of reuterin produced in vitro in glycerol aqueous solutions in situ by Lactobacillus reuteri ATCC 53608 as part of a fermented milk product against starter (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus), spoilage (Penicillium expansum), pathogenic (Staphylococcus aureus Salmonella enterica ssp. enterica, and Listeria monocytogenes), and pathogen surrogate (Escherichia coli DH5α) microorganisms. We also assayed the influence of cold storage (28 d at 4°C) and reuterin on the color and rheology of the fermented milk product. We obtained maximum reuterin concentrations of 107.5 and 33.97 mM in glycerol aqueous solution and fermented milk product, respectively. Reuterin was stable throughout its refrigerated shelf life. Gram-positive microorganisms were more resistant to reuterin than gram-negative microorganisms. Penicillium expansum and Lactobacillus reuteri ATCC 53608 survived at concentrations up to 10 and 8.5 mM, respectively. Escherichia coli DH5α was the most sensitive to reuterin (0.9 mM). The presence of reuterin did not cause relevant changes in the quality parameters of the fermented milk product, including pH, acidity, soluble solids, color, and rheological aspects (storage and loss moduli and viscosity). This study demonstrated the viability of using Lactobacillus reuteri ATCC 53608 as a biopreservative in a fermented milk product through reuterin synthesis, without drastically modifying its quality parameters.

In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria.

Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200-350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85-200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60-280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100-500 nm), but higher relative to those isolated from Streptococcus thermopilus (50-100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics.

Salt Reduction in a Model High-Salt Akawi Cheese: Effects on Bacterial Activity, pH, Moisture, Potential Bioactive Peptides, Amino Acids, and Growth of Human Colon Cells.

This study evaluated the effects of sodium chloride reduction and its substitution with potassium chloride on Akawi cheese during storage for 30 d at 4 °C. Survival of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum) and starter bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), angiotensin-converting enzyme-inhibitory and antioxidant activities, and concentrations of standard amino acids as affected by storage in different brine solutions (10% NaCl, 7.5% NaCl, 7.5% NaCl+KCl [1:1], 5% NaCl, and 5% NaCl+KCl [1:1]) were investigated. Furthermore, viability of human colon cells and human colon cancer cells as affected by the extract showing improved peptide profiles, highest release of amino acids and antioxidant activity (that is, from cheese brined in 7.5% NaCl+KCl) was evaluated. Significant increase was observed in survival of probiotic bacteria in cheeses with low salt after 30 d. Calcium content decreased slightly during storage in all cheeses brined in various solutions. Further, no significant changes were observed in ACE-inhibitory activity and antioxidant activity of cheeses during storage. Interestingly, concentrations of 4 essential amino acids (phenylalanine, tryptophan, valine, and leucine) increased significantly during storage in brine solutions containing 7.5% total salt. Low concentration of cheese extract (100 µg/mL) significantly improved the growth of normal human colon cells, and reduced the growth of human colon cancer cells. Overall, the study revealed that cheese extracts from reduced-NaCl brine improved the growth of human colon cells, and the release of essential amino acids, but did not affect the activities of potential bioactive peptides.

Implication of sortase-dependent proteins of Streptococcus thermophilus in adhesion to human intestinal epithelial cell lines and bile salt tolerance.

Streptococcus thermophilus (ST) is a lactic acid bacterium widely used in dairy industry and displays several properties which could be beneficial for host. The objective of this study was to investigate, in vitro, the implication of sortase A (SrtA) and sortase-dependent proteins (SDPs) in the adhesion of ST LMD-9 strain to intestinal epithelial cells (IECs) and resistance to bile salt mixture (BSM; taurocholoate, deoxycholate, and cholate). The effect of mutations in prtS (protease), mucBP (MUCin-Binding Protein), and srtA genes in ST LMD-9 in these mechanisms were examined. The HT29-MTX, HT29-CL.16E, and Caco-2 TC7 cell lines were used. HT29-MTX and HT29-CL.16E cells express different mucins found in the gastro intestinal tract; whereas, Caco-2 TC7 express cell surface proteins found in the small intestine. All mutants showed different adhesion profiles depending on cell lines. The mutation in genes srtA and mucBP leads to a significant decrease in LMD-9 adhesion capacity to Caco-2 TC7 cells. A mutation in mucBP gene has also shown a significant decrease in LMD-9 adhesion capacity to HT29-CL.16E cells. However, no difference was observed using HT29-MTX cells. Furthermore, ST LMD-9 and srtA mutant were resistant to BSM up to 3 mM. Contrariwise, no viable bacteria were detected for prtS and mucBP mutants at this concentration. Two conclusions could be drawn. First, SDPs could be involved in the LMD-9 adhesion depending on the cell lines indicating the importance of eukaryotic-cell surface components in adherence. Second, SDPs could contribute to resistance to bile salts probably by maintaining the cell membrane integrity.

Antimicrobial potential of commercial silver nanoparticles and the characterization of their physical properties toward probiotic bacteria isolated from fermented milk products.

The application of nanotechnology in the agriculture and food sector is relatively recent compared to its usage in drug delivery or pharmaceuticals. Therefore, this paper presents a study of the effect of silver nanoparticles on probiotic bacteria based on the example of Lactobacillus acidophilus LA-5, Bifidobacterium animalis subsp. lactis BB-12 and Streptococcus thermophilus ST-Y31 isolated from fermented milk products. Probiotic bacteria are one of the most crucial groups of bacteria for the food industry, because of their claimed health-promoting properties. Studies have shown that the type and concentration of silver nanoparticle solutions have a significant impact on the tested probiotic bacteria which are profitable for the digestive system. In the presence of all tested silver nanoparticles, St. thermophilus ST-Y31 growth was inhibited significantly by the dilution method as opposed to the disk-diffusion method. Both the disk-diffusion and the dilution methods showed no significant differences between L. acidophilus LA-5 and B. animalis subsp. lactis BB-12. The concentrations 2 µg mL(-1) and 0.25 µg mL(-1) had the highest antibacterial activity and statistically significant impacts on the tested probiotic strains. To our knowledge, this is the first report on potential antimicrobial effect of nanosilver against the health-promoting probiotic bacteria L. acidophilus LA-5, B. animalis subsp. lactis BB-12 and St. thermophilus ST-Y31 isolated from fermented milk products.