Development of lactic acid production from coffee grounds hydrolysate by fermentation with Lacticaseibacillus rhamnosus.

Spent coffee grounds (SCG) are commercial waste that are still rich in numerous valuable ingredients and can be further processed into useful products such as coffee oil, antioxidant extract, lactic acid, and lignin. The challenge and innovation is to develop the SCG processing technology, maximizing the use of raw material and minimizing the use of other resources within the sequential process. The presented research is focused on the aspect of biotechnological production of lactic acid from SCG by using the Lacticaseibacillus rhamnosus strain isolated from the environment. Thanks to the optimization of the processes of acid hydrolysis, neutralization, enzymatic hydrolysis of SCG, and fermentation, the obtained concentration of lactic acid was increased after 72 hr of culture from the initial 4.60 g/l to 48.6 g/l. In addition, the whole process has been improved, taking into account the dependence on other processes within the complete SCG biorefinery, economy, energy, and waste aspects. Costly enzymatic hydrolysis was completely eliminated, and it was proven that supplementation of SCG hydrolysate with expensive yeast extract can be replaced by cheap waste from the agri-food industry. ONE-SENTENCE SUMMARY: A process for efficient lactic acid production from spent coffee grounds using the Lacticaseibacillus rhamnosus strain was developed and optimized, including nutrient solution preparation, supplementation and fermentation.

Integrating fruit by-products and whey for the design of folate-bioenriched innovative fermented beverages safe for human consumption.

Global concerns over folate deficiency, the risks of excessive synthetic folic acid consumption, and food loss implications for environmental sustainability and food security drive needs of innovative approaches that align food by-product valorisation with folate bio-enrichment. This study explored the use of three fruit by-products extracts (grape, passion fruit, and pitaya) and whey to develop a folate bio-enriched fermented whey-based beverage. Three strains (Lacticaseibacillus rhamnosus LGG, Bifidobacterium infantis BB-02, and Streptococcus thermophilus TH-4) were tested for folate production in different fermentation conditions in modified MRS medium and in a whey-based matrix prepared with water extracts of these fruit by-products. B. infantis BB-02 and S. thermophilus TH-4, alone and in co-culture, were the best folate producers. The selection of cultivation conditions, including the presence of different substrates and pH, with grape by-product water extract demonstrating the most substantial effect on folate production among the tested extracts, was crucial for successfully producing a biofortified fermented whey-based beverage (FWBB). The resulting FWBB provided 40.7 µg of folate per 100 mL after 24 h of fermentation at 37 °C, effectively leveraging food by-products. Moreover, the beverage showed no cytotoxicity in mouse fibroblast cells tests. This study highlights the potential for valorising fruit by-products and whey for the design of novel bioenriched foods, promoting health benefits and contributing to reduced environmental impact from improper disposal.

Eugenia uniflora (pitanga) juice as a new alternative vehicle for Limosilactobacillus fermentum ATCC 23271: evaluation of antioxidant and anti-infective effects.

Probiotic-containing foods are among the most appreciated functional foods; however, probiotic-based dairy products cannot be consumed by people who are lactose intolerant, allergic to milk, or vegetarian or vegan individuals. Thus, new non-dairy matrices have been tested for probiotics delivery. This study evaluated the growth and viability of Limosilactobacillus fermentum ATCC 23271 and Lacticaseibacillus rhamnosus ATCC 9595 in Pitanga juice (Eugenia uniflora L.). The effects of the fermentation on the antioxidant and anti-infective properties of the juice were also analyzed. The E. uniflora juice allowed lactobacilli growth without supplementation, reaching rates around 8.4 Log CFU/mL and producing organic acids (pH values < 4) after 72 h of fermentation. The strain remained viable after 35 days of refrigerated storage. Fermentation by these bacteria increases the antioxidant capacity of the juice. The central composite rotational design was employed to evaluate the effects of bacterial inoculum and pulp concentration on growth and organic acids production by L. fermentum ATCC 23271. The strain was viable and produced organic acids in all tested combinations. L. fermentum-fermented juice and its cell-free supernatant significantly increased the survival of Tenebrio molitor larvae infected by enteroaggregative Escherichia coli 042. The results obtained in this study provide more insights into the potential of Pitanga juice to develop a functional non-dairy probiotic beverage with antioxidant and anti-infective properties.

Exoproteome Analysis of Antagonistic Interactions between the Probiotic Bacteria Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F and Multidrug Resistant Strain of Klebsiella pneumonia.

The expansion of multiple drug resistant (MDR) strains of Klebsiella pneumoniae presents an immense threat for public health. Annually, this microorganism causes thousands of lethal nosocomial infections worldwide. Currently, it has been shown that certain strains of lactic acid bacteria (LAB) can efficiently inhibit growth of K. pneumoniae and the formation of its biofilms; however, the active principle of such action remains unknown. In the current article, the growth inhibition of MDR K. pneumoniae by two LAB-Limosilactobacillus reuteri LR1 and Lacticaseibacillus rhamnosus F-is demonstrated, and the nature of this inhibition studied at the level of exoproteome. This article shows that the exoproteomes of studied LAB contains both classically and non-classically secreted proteins. While for L. reuteri LR1 the substantial portion of classically secreted proteins was presented by cell-wall-degrading enzymes, for L. rhamnosus F only one out of four classically secreted proteins was presented by cell-wall hydrolase. Non-classically secreted proteins of both LAB were primarily metabolic enzymes, for some of which a possible moonlighting functioning was proposed. These results contribute to knowledge regarding antagonistic interaction between LAB and pathogenic and opportunistic microorganisms and set new perspectives for the use of LAB to control the spread of these microorganisms.

Kinetic analysis of microcalorimetric data derived from microbial growth: Basic theoretical, practical and industrial considerations.

We report here a mathematical framework for the quantitative interpretation of exponential bacterial growth measured with isothermal microcalorimetry. The method allows determination of many parameters that define the exponential growth phase. To automate the analysis, we also wrote a coding program, so that the approach could be embedded in a commercial setting. As an exemplar, we apply the method to a commercial probiotic product. The outcome was that we could identify characteristic parameters of growth (including rate constant and doubling time), and hence authenticate product quality, within 15 h. This compares favourably with the current 7-10 days required for conventional microbiological assessment (to allow release of product for bottling and marketing) via plating methods. The method would lend itself to growth analysis of single and mixed bacterial cultures.

Maillard conjugates of whey protein isolate-xylooligosaccharides for the microencapsulation of Lactobacillus rhamnosus: protective effects and stability during spray drying, storage and gastrointestinal digestion.

The Maillard reaction products (MRPs) of whey protein isolate (WPI) and xylooligosaccharides (XOS) were prepared by a moist heat method for use as protectants to encapsulate Lactobacillus rhamnosus via spray drying. The protective effects of MRPs on bacterial cells during drying, storage, and in vitro digestion were explored. FTIR results indicated that MRPs were successfully prepared. All MRPs showed good thermo-protective effect on the bacteria, and the survival ratio achieved with 1 : 2 XOS-WPI as a wall material reached 99.83 ± 8.44%, which was around 2 times as high as that of the WPI wall material and 1.5 times as high as that of the 1 : 2 XOS-WPI mixture. The dried lactobacilli showed similar growth curves to the fresh culture. After 10 weeks of storage at 4 °C, the decrease in the bacterial activity was less than 1 log CFU g-1 for all types of microcapsules, while the microcapsules composed of all MRPs had better storage stability. MRPs improved the stability of microcapsules during in vitro digestion. The number of viable bacteria in 1 : 2 XOS-WPI MRPs microcapsules was maintained at 4.09 ± 0.59 × 109 CFU g-1 after simulated gastrointestinal digestion for 4 hours, which only decreased by 0.20 log CFU g-1.

Growth, survival, and metabolic activities of probiotics Lactobacillus rhamnosus GG and Saccharomyces cerevisiae var. boulardii CNCM-I745 in fermented coffee brews.

Amidst rising demand for non-dairy probiotic foods, and growing interest in coffees with added functionalities, it would be opportune to ferment coffee brews with probiotics. However, challenges exist in maintaining probiotic viability in high-moisture food products. Here, we aimed to enhance the viability of the probiotic bacteria, Lactobacillus rhamnosus GG, in coffee brews by co-culturing with the probiotic yeast, Saccharomyces cerevisiae var. boulardii CNCM-I745. The yeast significantly enhanced the viability of L. rhamnosus GG, as bacterial populations beyond 7 Log CFU/mL were maintained throughout 14 weeks of storage at 4 and 25 °C. In contrast, the single culture of L. rhamnosus GG suffered viability losses below 6 Log CFU/mL within 10 weeks at 4 °C, and 3 weeks at 25 °C. Growth and survival of S. boulardii CNCM-I745 remained unaffected by the presence of L. rhamnosus GG. Volatile profiles of coffee brews were altered by probiotic metabolic activities, but co-culturing led to suppressed generation of diacetyl and ethanol compared to single cultures. Probiotic fermentation did not alter principal coffee bioactive compounds and antioxidant capacities; however, declines in peroxyl radical scavenging capacities were observed after ambient storage. Overall, we illustrate that yeasts are effective in enhancing probiotic bacterial viability in coffee brews, which may be useful in developing shelf stable probiotic food products.

Woodfordia fruticosa extract supplementation stimulates the growth of Lacticaseibacillus casei and Lacticaseibacillus rhamnosus with adapted intracellular and extracellular metabolite pool.

AIM: To investigate the effect of Woodfordia fruticosa extract (WfE) on two probiotic bacteria: Lacticaseibacillus casei and Lacticaseibacillus rhamnosus. METHODS AND RESULTS: WfE supplementation at 0·5 and 1 mg ml-1 stimulated probiotic growth (P < 0·05), enhanced adhesion to CaCO2 cells (P < 0·05) while inhibiting foodborne pathogens Escherichia coli and Staphylococcus aureus (P < 0·05). 1 H-NMR based metabolomic studies indicated higher glucose : lactate and glucose : acetate in the extracellular matrix with significant variation (P < 0·05) in intracellular concentrations of lactate, acetate, glutamate, dimethylamine, phenylalanine, branched-chain amino acids and total cellular lipid composition. Fatty acid methyl ester analysis showed a chemical shift from saturated to unsaturated lipids with WfE supplementation. PCA plots indicated clear discrimination between test groups, highlighting variation in metabolite pool in response to WfE supplementation. CONCLUSION: Phytonutrient-rich WfE exhibited prebiotic-like attributes, and probiotic bacteria showed altered metabolite pools as an adaptive mechanism. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report providing insights into the prebiotic-like activity of WfE on gut representative probiotics. The extended metabolomic studies shed light on the positive interaction between phytonutrients and beneficial bacteria that possibly help them to adapt to a phytonutrient-rich WfE environment. WfE with potential prebiotic attributes can be used in the development of novel synbiotic functional products targeting gut microbial modulation to improve health.

High-efficient L-lactic acid production from inedible starchy biomass by one-step open fermentation using thermotolerant Lactobacillus rhamnosus DUT1908.

The purpose of this study was to establish a simplified operational process for lactic acid (LA) production from inedible starchy biomass by open fermentation using thermotolerant Lactobacillus rhamnosus DUT1908. One step simultaneous liquefaction, saccharification and fermentation (SLSF) was proposed to produce LA using aging paddy rice with hull (APRH) as feedstock. First, a robust microbial strain was obtained by adaptive laboratory evolution under high temperature stress. As a result, L. rhamnosus DUT1908 showed high thermotolerance up to 50 °C and high efficiency of substrate utilization. Then, the performance of this thermotolerant L-lactic acid producing strain was demonstrated. Finally, various fermentation strategies were compared for LA production from APRH, including simultaneous saccharification and fermentation (SSF) and SLSF. In one-step open SLSF process, 107.8 g/L lactic acid was obtained with a productivity of 3.4 g/(L.h) and a yield to theoretical glucose of 0.89 g/g. This is the highest yield and productivity of lactic acid reported on starchy residues, and provides an efficient route for the development of high value-added products.

Microencapsulation of Lactobacillus casei and Lactobacillus rhamnosus in pectin and pectin-inulin microgel particles: Effect on bacterial survival under storage conditions.

The main objective of the research was to evaluate the performance of synbiotic delivery systems using pectin microgels on the protection of two probiotic strains (Lactobacillus casei ATCC 393 and Lactobacillus rhamnosus strain GG [ATCC 53103]) to simulated gastrointestinal digestion (GD) and storage conditions (4 ± 1 °C) in a 42 days trial. Microgel particles were prepared by ionotropic gelation method and three variables were evaluated: incubation time (24 and 48 h), free vs encapsulated cells, and presence or absence of prebiotic (commercial and Jerusalem artichoke inulin). Results demonstrated an encapsulation efficiency of 96 ± 4% into particles with a mean diameter between 56 and 118 µm. The viability of encapsulated cells after 42 days storage stayed above 7 log units, being encapsulated cells in pectin-inulin microgels more resistant to GD compared to non-encapsulated cells or without prebiotics. In all cases incubation time influenced the strains' survival.

Lactobacillus rhamnosus AD3 as a Promising Alternative for Probiotic Products.

Lactobacillus strains dominate the vaginal habitat and they are associated with a lower risk of genital infections. In addition, they contribute to the conservation of the vaginal microbiota by producing postbiotic agents. Previous studies have shown that their predominance involves antimicrobial activity against urogenital pathologies. In this context, probiotics may improve treatment outcomes. The aim of this study was to evaluate the probiotic properties of lactobacilli strains of vaginal origin using a multidisciplinary approach. For this purpose, safety criteria, ability to resist at low pH and bile salts, antimicrobial activity, ability to produce biofilm, capacity to produce hydrogen peroxide and more importantly, auto-aggregation, co-aggregation (with Candida spp.) and adhesion to human cells were evaluated. The strains belonged to the species of L. crispatus, L. gasseri, L. rhamnosus and L. delbruckii. Among these, a strain of L. rhamnosus named AD3 showed the best probiotic properties. As probiotics are already in use in many clinical practice and there are no major safety concerns, L. rhamnosus AD3 showed promise in becoming a prevention and complementary treatment option for urogenital diseases. Indeed, these results suggest that strain L. rhamnosus AD3 is non-pathogenic and likely to be safe for human consumption. This study revealed the great amensalistic properties of a new L. rhamnosus strain which can aim to be used as probiotic in pharmaceutical applications.

Generation of Lactose- and Protease-Positive Probiotic Lacticaseibacillus rhamnosus GG by Conjugation with Lactococcus lactis NCDO 712.

Lacticaseibacillus rhamnosus GG (LGG) is the most studied probiotic bacterium in the world. It is used as a probiotic supplement in many foods, including various dairy products. However, LGG grows poorly in milk, as it neither metabolizes the main milk carbohydrate lactose nor degrades the major milk protein casein effectively. In this study, we made L. rhamnosus GG lactose and protease positive by conjugation with the dairy Lactococcus lactis strain NCDO 712 carrying the lactose-protease plasmid pLP712. A lactose-hydrolyzing transconjugant colony was obtained on agar containing lactose as the sole source of carbohydrates. By microscopic analysis and PCR with LGG- and pLP712-specific primers, the transconjugant was confirmed to have originated from LGG and to carry the plasmid pLP712. The transconjugant was named L. rhamnosus LAB49. The isolation of plasmids revealed that not only pLP712 but also other plasmids had been transferred from L. lactis into LGG during conjugation. With plasmid-specific PCR primers, four additional lactococcal plasmids were detected in LAB49. Proteolytic activity assay and SDS-PAGE analysis verified that L. rhamnosus LAB49 effectively degraded ß-casein. In contrast to its parental strain, LGG, the ability of LAB49 to metabolize lactose and degrade casein enabled strong and fast growth in milk. As strains with new properties made by conjugation are not regarded as genetically modified organisms (GMOs), L. rhamnosus LAB49 could be beneficial in dairy fermentations as a probiotic starter culture.IMPORTANCE Probiotic strain Lacticaseibacillus rhamnosus GG (LGG) is widely sold on the market as a probiotic or added as a supplement in dairy foods because of its benefits in human health. However, due to the deficiency of lactose and casein utilization, LGG does not grow well in milk. On the other hand, lactose intolerance and cow's milk protein allergy are the two major problems related to milk consumption. One option to help with these two conditions is the use of probiotic or lactose- and casein-hydrolyzing bacteria in dairy products. The purpose of this study was to equip LGG with lactose/casein-hydrolyzing ability by bacterial conjugation. As a result, we generated a non-GMO LGG derivative with improved properties and better growth in milk.

Intestinal Dysbiosis Correlates With Sirolimus-induced Metabolic Disorders in Mice.

BACKGROUND: Long-time use of pharmacological immunosuppressive agents frequently leads to metabolic disorders. Most studies have focused on islet toxicity leading to posttransplantation diabetes mellitus. In contrast, the link between intestinal dysbiosis and immunosuppressive drug-induced metabolic disorders remains unclear. METHODS: We established a mouse model of metabolic abnormality via sirolimus treatment. Fecal microbiota was examined using 16S rRNA gene MiSeq sequencing. Intestinal barrier function was assessed using fluorescein isothiocyanate-dextran assay and mucus immunostaining. Systemic inflammation was determined using a multiplexed fluorescent bead-based immunoassay. RESULTS: Sirolimus induced dyslipidemia and glucose intolerance in mice in a dose-dependent manner. Interestingly, the clinical-mimicking dose of sirolimus altered the intestinal microbiota community, which was characterized by the enrichment of Proteobacteria, depletion of Akkermansia, and potential function shifts to those involved in lipid metabolism and the immune system. In addition, the clinical-mimicking dose of sirolimus reduced the thickness of the intestinal mucosal layer, increased the intestinal permeability, and enriched the circulating pro-inflammatory factors, including interleukin (IL)-12, IL-6, monocyte chemotactic protein 1, granulocyte-macrophage colony stimulating factor, and IL-1ß. Our results showed a close association between intestinal dysbiosis, intestinal barrier failure, systemic inflammation, and metabolic disorders. Furthermore, we demonstrated that oral intervention in the gut microbiota by Lactobacillus rhamnosus HN001 protected against intestinal dysbiosis, especially by depleting the lipopolysaccharide-producing Proteobacteria, and attenuated the sirolimus-induced systemic inflammation, dyslipidemia, and insulin resistance. CONCLUSIONS: Our study demonstrated a potentially causative role of intestinal dysbiosis in sirolimus-induced metabolic disorders, which will provide a novel therapeutic target for transplant recipients.

Restraining the TiO2 nanoparticles-induced intestinal inflammation mediated by gut microbiota in juvenile rats via ingestion of Lactobacillus rhamnosus GG.

Human were given a lot of opportunities to ingest TiO2 NPs in the environment. Children have low, sensitive intestinal tolerance, and they could be exposed to higher levels of TiO2 NPs than adults. Few studies have been conducted on the interaction between TiO2 NPs and juvenile intestine phase models. Thus, in this work, weaning rats were orally exposed to TiO2 NPs for 7 and 14 days. Results indicate that Ti accumulated in the intestine, liver, and feces. Inflammatory infiltration damage was observed in the colonic epithelial tissue, and gut microbiota fluctuated with a decreased abundance of Lactobacilli in feces. Oral supplementation with Lactobacillus rhamnosus GG (LGG) lessened TiO2 NPs-induced colonic inflammatory injury, which might due to downregulation of nuclear factor kappa-B (NF-κB). Meanwhile, LGG maintained normal intestinal microbiome homeostasis, thereby improving TiO2 NPs-induced colon injury in juvenile rats. Moreover, fecal microbiota transplant (FMT) experiment indicated possible TiO2 NPs-induced intestinal microbiota disorder led to colonic inflammation. Our works suggested the urgent need for additional studies on the risk safety assessment, mechanism, and prevention of juvenile health damage from exposure to TiO2 NPs.

Isolation and characterization of lactic acid bacteria from human milk.

Human milk is the main source of nutrition for infants and the transmission of various microorganisms. The lactic acid bacteria (LAB) in breast milk allow for the establishment of the gut microflora of infants. In this study, we aimed to assess the probiotic potential of LAB strains isolated from breast milk of healthy Chinese women. Two strains, Lacticaseibacillus rhamnosus (formerly Lactobacillus rhamnosus) LHL6 and LHL7, were selected and identified through morphology observation, Gram staining, and 16S rDNA phylogenetic analysis. Using Limosilactobacillus fermentum (formerly Lactobacillus fermentum) CECT5716 as the standard reference strain, the screened strains were characterized for aspects of growth, production of lactic acid and H2O2, antibiotic susceptibility, survival under simulated gastrointestinal conditions, and tolerance to cadmium (Cd). In de Man, Rogosa, and Sharpe (MRS) broth, LHL6 and LHL7 showed longer lag phases than CECT5716 but higher specific growth rates. For the production of lactic acid and H2O2, LHL7 performed better than LHL6 and CECT5716, indicating better antimicrobial ability. Strain LHL7 generated 9.99 mg/L H2O2, considerably higher than 1.25 mg/L for LHL6 and 2.33 mg/L for CECT5716. According to European Food Safety Authority minimum inhibitory concentrations, all of the investigated strains were resistant to chloramphenicol, streptomycin, and kanamycin. However, unlike LHL6 and CECT5716, LHL7 was susceptible to ampicillin and resistant to tetracycline. Resistance to azithromycin, cephalexin, and penicillin G were similar for all 3 strains, whereas CECT5716 was resistant to a higher concentration of roxithromycin. All 3 strains were able to survive in a simulated gastric-like solution, but a low percentage survived in the presence of 0.4% bile salt and 7% pancreatin. Encapsulation with protectants may enhance the survival rate. All 3 strains were tolerant to 500 mg/L Cd in MRS broth and to 1,000 mg/L Cd on MRS agar medium. In summary, 2 novel strains of LAB were obtained that have similar characteristics to the reference strain CECT5716. This work identified potential probiotic candidates for application in the food and pharmaceutical industries and facilitated identification of further probiotics.

Light at night affects gut microbial community and negatively impacts host physiology in diurnal animals: Evidence from captive zebra finches.

The gastrointestinal tract (GIT) hosts a large number of diverse microorganisms, with mutualistic interactions with the host. Here, in two separate experiments, we investigated whether light at night (LAN) would affect GIT microbiota and, in turn, the host physiology in diurnal zebra finches (Taeniopygia guttata). Experiment I assessed the effects of no-night (LL) and dimly illuminated night (dim light at night, dLAN) on fecal microbiota diversity and host physiology of birds born and raised under 12 h photoperiod (LD; 12 h light: 12 h darkness). Under LL and dLAN, compared to LD, we found a significant increase in the body mass, subcutaneous fat deposition and hepatic accumulation of lipids. Although we found no difference in total 24 h food consumption, LL/ dLAN birds ate also at night, suggesting LAN-induced alteration in daily feeding times. Concurrently, there were marked differences in amplicon sequence and bacterial species richness between LD and LAN, with notable decline in Lactobacillus richness in birds under LL and dLAN. We attributed declined Lactobacillus population as causal (at least partially) to negative effects on the host metabolism. Therefore, in experiment II with similar protocol, birds under LL and dLAN were fed on diet with or without Lactobacillus rhamnosus GG (LGG) supplement. Clearly, LGG supplement ameliorated LL- and dLAN-induced negative effects in zebra finches. These results demonstrate adverse effects of unnatural lighting on GIT bacterial diversity and host physiology, and suggest the role of GIT microbiota in the maintenance of metabolic homeostasis in response to LAN environment in diurnal animals.

Growth conditions and survival kinetics during storage of Lactobacillus rhamnosus GG for the design of a sustainable probiotic whey-based beverage containing Costa Rican guava fruit pulp.

The finding of economical and practical applications for milk whey is still a challenge for dairy industries. This paper presents information about the development of a probiotic-prebiotic beverage based on Lactobacillus rhamnosus GG (LGG) and Costa Rican guava (CRG) fruit pulp with industrial potential. First, a supplemented whey media was developed for LGG growth, and the whey-supplemented media was used for fermentation in bioreactors. LGG reached a maximum growth rate of 0.32 hr-1 after 48 hr of fermentation. The whey-grown probiotics were then mixed with CRG pulp to produce the probiotic-prebiotic beverage. The survival kinetics of LGG in the formulated drink was not affected by the addition of CRG pulp (P > 0.05), and the shelf-life of the inoculated beverage surpassed 40 days with a minimum population of 106 colony forming units (CFU)/mL. Properties as pH, fructose, glucose, sucrose, and proanthocyanidins (PACs) content exhibited a significant difference after storage time (P < 0.05). Finally, three different formulas of the beverage with different whey content were compared through sensory evaluation. The prototype with 50% whey content was one of the most valuable beverage formulas according to the organoleptic parameters, which remarks about the possibility of developing a probiotic whey-based beverage containing CRG pulp. Furthermore, this is the first report about CRG beverages as a probiotic vector. PRACTICAL APPLICATION: This research focuses on the evaluation of the properties of a probiotic beverage, with a promissory industrial application using whey, as a dairy industry byproduct, combined with the pulp of the highly nutritious and subutilized Costa Rican guava (CRG) fruit.

Short-Chain Cello-oligosaccharides: Intensification and Scale-up of Their Enzymatic Production and Selective Growth Promotion among Probiotic Bacteria.

Short-chain cello-oligosaccharides (COS; degree of polymerization, DP ≤ 6) are promising water-soluble dietary fibers. An efficient approach to their bottom-up synthesis is from sucrose and glucose using glycoside phosphorylases. Here, we show the intensification and scale up (20 mL; gram scale) of COS production to 93 g/L product and in 82 mol % yield from sucrose (0.5 M). The COS were comprised of DP 3 (33 wt %), DP 4 (34 wt %), DP 5 (24 wt %), and DP 6 (9 wt %) and involved minimal loss (≤10 mol %) to insoluble fractions. After isolation (≥95% purity; ≥90% yield), the COS were examined for growth promotion of probiotic strains. Benchmarked against inulin, trans-galacto-oligosaccharides, and cellobiose, COS showed up to 4.1-fold stimulation of cell density for Clostridium butyricum, Lactococcus lactis subsp. lactis, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus but were less efficient with Bifidobacterium sp. This study shows the COS as selectively functional carbohydrates with prebiotic potential and demonstrates their efficient enzymatic production.

New insights in enumeration methodologies of probiotic cells in finished products.

The number of bacterial cells is currently recognized as the most important parameter for the efficacy and quality of finished probiotic or live biotherapeutic products (LBP). Cell enumeration is generally performed by culture-dependent methodologies like plate count (PC). These techniques are able to reveal the number of viable cells able to replicate and generate a colony. However, they are limited by their dependence on the combination of culture conditions (e.g. nutrients, temperature) selected for cell recovery. Additionally, they do not provide information on the heterogeneity of a bacterial culture, namely they do not detect the cells in a viable but not cultivable (VBNC) status. Flow-cytometry (FC) is a culture-independent methodology having the potential to enumerate selectively live and damaged or dead cells. FC relies on the use of specific probes for different cell targets (e.g. membrane, enzymes) to unveil information on the cell structure and physiological statuses within a bacterial population. In this context, we monitored three batches of freeze-dried Lactobacillus rhamnosus GG (ATCC 53103) during a 3 year of storage at different conditions of temperature and relative humidity, according to ICH guidelines, by means of PC and FC. The Arrhenius model was applied to assess the suitability of the model to predict the mortality of probiotic cells in finished products. The higher destruction rate (k) obtained by PC data compared to FC data suggests a faster reduction of cultivability compared to membrane integrity, probably representing a dynamic shift of the bacterial population into a VBNC/dormant status during storage time. Interestingly, this mechanistic approach works both for PC and FC methodologies increasing the chances to monitor biological phenomenon within a mathematical modelling. The combined use of PC and FC shed lights on the true bacterial potency within a closed system like a finished product and the complexity of its heterogeneity.

The effect of selected Lactobacillus strains on dextran sulfate sodium-induced mouse colitis model.

Inflammatory bowel disease (IBD) comprises two major illnesses: Crohn's disease (CD) and ulcerative colitis (UC). Dextran sulfate sodium (DSS) mouse colitis model has been used in understanding the mechanism of IBD. This study was conducted to examine selected Lactobacillus spp. as potential IBD treatment in the DSS-induced animal model. Balb/c mice were used and colitis was induced by adding 5% dextran sodium sulfate into the drinking water for 8 days. Colon length, disease activity index (DAI) and histological analysis were measured as markers of inflammation in DSS colitis mice. The majority of the Lactobacillus species significantly prevented the shortening of the colon length compared with the DSS group. The DAI scores of mice were significantly reduced following usage of four Lactobacillus strains included: Lactobacillus plantarum 03 and 06, Lactobacillus brevis 02 and Lactobacillus rhamnosus 01. The histological analysis exhibited that oral administration of Lactobacillus strains had therapeutic effects on mice colitis. L. plantarum and L. brevis showed better therapeutic effect against DSS-induced acute colitis mice. The probiotic activities of these three isolates indicated that the probiotic effects were strain specific and none of these useful bacteria could exhibit all of the valued probiotic properties simultaneously.