Preliminary Purification and Partial Characterization of a Functional Bacteriocin of Lacticaseibacillus paracasei Zhang and Mining for its Gene Cluster.

Bacteriocins produced by lactic acid bacteria (LAB) have good potential for use as food biopreservatives. Lacticaseibacillus paracasei Zhang (L. paracasei Zhang) is both a food use and a probiotic bacterium. This study aimed to purify and preliminary characterize the active antibacterial metabolite of L. paracasei Zhang. The cell-free supernatant of L. paracasei Zhang was collected and purified by ultrafiltration and gel filtration chromatography. The 1-3 kDa active fraction could inhibit the growth of Staphylococcus aureus but not Escherichia coli. Further antibacterial activity assays revealed its capacity to suppress various foodborne and human opportunistic pathogens (including Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Listeria monocytogenes, and Bacillus cereus), but not fungi. The antibacterial activity showed good tolerance to heat (40 to 100 °C), acid-base (pH 2-3 and pH 6-10), and digestions by a number of industrial and animal/human enzymes (such as trypsin, pepsin, α-amylase, and protease K, except papain); these desired properties make it a suitable biopreservative to be used in harsh and complex industrial production processes. The high papain sensitivity suggested a proteinaceous/peptide nature of the bioactivity. Moreover, our genomic data mining for bacteriocin through BAGEL4 revealed an area of interest encoding a complete set of putative genes required for bacteriocin production. In conclusion, our study showed that L. paracasei Zhang can produce extracellular functional antibacterial metabolite, likely a class II bacteriocin. Our preliminary extraction and characterization of the active metabolite demonstrated that it has good potential to be used as a biopreservative or an agent for suppressing gastrointestinal infections.

Both viable Bifidobacterium longum subsp. infantis B8762 and heat-killed cells alleviate the intestinal inflammation of DSS-induced IBD rats.

In view of the safety concerns of probiotics, more and more attention is paid to the beneficial effects of dead probiotics cells. Herein, we investigated and compared the alleviation effects of viable Bifidobacterium longum subsp. infantis B8762 (B. infantis B8762) and its heat-killed cells on dextran sodium sulfate (DSS)-induced inflammatory bowel disease (IBD) rats. Four groups of rats (n = 12 per group) were included: normal control, DSS-induced colitis rats without bacterial administration (DSS), DSS-induced colitis rats with viable B. infantis B8762 administration (VB8762), and DSS-induced colitis rats with dead B. infantis B8762 administration (DB8762). Our results showed that both VB8762 and DB8762 administration exerted significant protective effects on DSS-induced IBD rats, as evidenced by a reduction in mortality, disease activity index score, body weight loss, as well as decreased histology score, which were companied by a significant decrease in serum pro-inflammatory factors compared with DSS group, and a stronger effect on modulating the fecal microbiota alpha-diversity and beta-diversity compared with DSS group. Additionally, the fecal metabolome results showed that both VB8762 and DB8762 interventions indeed altered the fecal metabolome profile and related metabolic pathways of DSS-induced IBD rats. Therefore, given the alleviation effects on colitis, the DB8762 can be confirmed to be a postbiotic. Overall, our findings suggested that VB8762 and DB8762 had similar ability to alleviate IBD although with some differences. Due to the minimal safety concern of postbiotics, we propose that the postbiotic DB8762 could be a promising alternative to probiotics to be applied in the prevention and treatment of IBDs.IMPORTANCEInflammatory bowel disease (IBD) has emerged as a global disease because of the worldwide spread of western diets and lifestyles during industrialization. Up to now, many probiotic strains are used as a modulator of gut microbiota or an enhancer of gut barrier to alleviate or cure IBD. However, there are still many issues of using probiotics, which were needed to be concerned about, for instance, safety issues in certain groups like neonates and vulnerable populations, and the functional differences between viable and dead microorganisms. Therefore, it is of interest to investigate the beneficial effects of dead probiotics cells. The present study proved that both viable Bifidobacterium longum subsp. infantis B8762 and heat-killed cells could alleviate dextran sodium sulfate-induced colitis in rats. The findings help to support that some heat-killed probiotics cells can also exert relevant biological functions and can be used as a postbiotic.

Protective effects of whey protein hydrolysate on Bifidobacterium animalis ssp. lactis Probio-M8 during freeze-drying and storage.

We evaluated the potential of whey protein hydrolysate as a lyoprotectant for maintaining the cell viability of Bifidobacterium animalis ssp. lactis Probio-M8 during freeze-drying and subsequent storage. The moisture content and water activity of the lyophilized samples treated by different concentrations of whey protein hydrolysate were ≤5.23 ± 0.33 g/100 g and ≤0.102 ± 0.003, respectively. During storage at 25°C and 30°C, whey protein hydrolysate had a stronger protective effect on B. lactis Probio-M8 than the same concentration of whey protein. Using the Excel tool GinaFit, we estimated the microbial inactivation kinetics during storage. Whey protein hydrolysate reduced cell damage caused by an increase in temperature. Whey protein hydrolysate could protect cells by increasing the osmotic pressure as a compatible solute. Whey protein hydrolysate improved cell membrane integrity and reduced the amounts of reactive oxygen species and malondialdehyde produced. The findings indicated that whey protein hydrolysate was a novel antioxidant lyoprotectant that could protect probiotics during freeze-drying and storage.

Design, synthesis, and biological evaluation of novel 2'-methyl-2'-fluoro-6-methyl-7-alkynyl-7-deazapurine nucleoside analogs as anti-Zika virus agents.

Zika virus (ZIKV) is a mosquito-borne flavivirus and outbreaks of ZIKV have been reported in Africa, Americas and other parts of the world lately. The ZIKV epidemic has received extensive attention due to its ability to cause serious medical consequences and complications such as microcephaly and Guillain-Barre syndrome in recent years. Up to now, there are no specific treatments or vaccines available for ZIKV infection, which highlights the urgent need for developing new therapies. In this work, we designed and synthesized a series of novel 6-methyl-7-acetylenenyl-7-deazapurine nucleoside analogs as potential inhibitors of ZIKV replication. The biological activities against ZIKV replication were evaluated and the structure-activity relationship (SAR) was also studied. Among the compounds evaluated, nucleoside analog 38 (EC50 = 2.8 ± 0.8 µM, EC90 = 6.8 ± 2.3 µM) showed the most potent anti-ZIKV activity with low cytotoxicity (CC50 = 54.1 ± 6.9 µM) in an A549 based cellular model. The inhibitory activity of 38 was about 5 times more potent than the positive control NITD008. Notably, 38 showed similar inhibition potency against different ZIKV strains (ZG-01 and MR766) in a variety of host cell types including SNB19, A549, Huh7, Vero. In addition, 38 (Kd = 1.87 µM) has a stronger affinity to ZIKV RNA-dependent RNA polymerase (RdRp) protein than NITD008 (Kd = 3.43 µM) in the non-phosphorylation assay. These results indicated that compound 38 may serve as a promising candidate in future anti-ZIKV drug discovery.

Comparative effects of the single and binary probiotics of Lacticaseibacillus casei Zhang and Bifidobacterium lactis V9 on the growth and metabolomic profiles in yogurts.

In the present study, comparative effects of the single and binary probiotics of Lacticaseibacillus casei Zhang (L. casei Zhang) and Bifidobacterium lactis V9 (B. lactis V9) on the growth and metabolomic profiles during milk fermentation and storage has been analyzed using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS). The growth of B. lactis V9 can be significantly increased (p < 0.001) by co-cultivation with L. casei Zhang at the end of fermentation and storage 10 days, and valine, leucine and isoleucine biosynthesis may be a major contributor to the growth promotion of B. lactis V9. However, the growth of L. casei Zhang was not affected by co-cultivation with B. lactis V9. There were notable distinctions in volatile and non-volatile metabolomic profiles and pathways between the single and binary probiotics cultures; binary probiotics L. casei Zhang and B. lactis V9 significantly affected the volatile, non-volatile metabolic profiles compared to the single probiotics. The levels of acetic acid, hexanoic acid, butanoic acid and pentanoic acid were significantly higher (p < 0.05) in binary probiotics cultures compared to the single probiotic cultures at the storage 10 days, which indicates that binary probiotics had additive effects on the production of short-chain fatty acids during storage. This work provides a detailed insight into metabolomic profiles and growth that differ between the single and binary probiotics cultures, and it can be helpful to develop probiotic yogurt with high probiotic viability and distinct metabolomic profiles.

Lactobacillus casei Zhang exerts probiotic effects to antibiotic-treated rats.

Probiotics administration can facilitate the restoration of host gut microbiota/metabolome after antibiotic treatment. Yet, the mechanism behind such beneficial effects remains unclear. This study constructed a rat model of antibiotic-induced gut dysbiosis to monitor the effects and mechanism of probiotic (Lactobacillus casei Zhang) treatment in maintaining gut homeostasis and restoring the gut microbiota/metabolome. Forty rats were randomly divided into four groups (n = 10 per group): control receiving only saline (Ctrl), antibiotic (AB-Ctrl), antibiotic followed by probiotic (AB-Prob), and antibiotic plus probiotic followed by probiotic (AB + Prob). Rat fecal microbiota and sera were collected at four time points from pre-treatment to post-treatment. The probiotic-treated group (AB + Prob) had significantly more Parabacteroides (P.) goldsteinii after one week of antibiotic and probiotic intervention but fewer antibiotic resistance genes (ARGs)-possessing bacteria (Clostridioides difficile and Burkholderiales bacterium). Consistently, metabolomics data revealed that both probiotic groups had more acetic acid, propionic acid, butyric acid, and valeric acid post treatment. Moreover, a potential probiotic species, P. goldsteinii, strongly correlated with L. casei, as well as propionic acid, butyric acid, and valeric acid. Furthermore, administering probiotic lowered the serum IL-1α level. In contrast, the antibiotic-recipients had a higher irreversible level of IL-1α, suggesting inflammation of the rats. Thus, antibiotic treatment not only led to host gut dysbiosis, but inflammatory responses and an increase in gut ARGs. Daily L. casei Zhang supplementation could alleviate the side effect of cefdinir intervention and facilitate the restoration of gut microbial homeostasis, and these probiotic effects might involve P. goldsteinii-mediated beneficial activities.

Comparative genomics of in vitro and in vivo evolution of probiotics reveals energy restriction not the main evolution driving force in short term.

Probiotics have attracted much attention because of their health-promoting effects, but little is known about the in vivo evolution of probiotics. This study analyzed the genome adaptation of the probiotic Lactiplantibacillus plantarum P-8 strain cultivated in ordinary and glucose restrictive growth media. Then, this study re-analyzed genomes of P-8 isolates recovered from the gut contents of subjects in two feeding trials (in rat and human). The sampling time points were similar to that of the in vitro evolution experiment, which might give parallel comparison of the in vitro and in vivo evolution processes. Our results showed that intra-individual specific microbial genomic variants of the original strain were detected in all human and some rat subjects. The divergent patterns of evolution within the host gastrointestinal tract suggested intra-individual-specific environmental adaptation. Based on comprehensive analysis of adapted-isolates recovered from these experiments, our results showed that the energy restriction was not the main driving force for evolution of probiotics. The individual-specific adaptation of probiotics might partially explain the varying extent of health effects seen between different individuals after probiotic consumption. In addition, the results suggest that probiotics should not only adapt to the environment of the birth canal, but also adapt to other species in the gut, revealing the Red Queen hypothesis in the process of intestinal flora.

Probiotics as Adjunctive Treatment for Patients Contracted COVID-19: Current Understanding and Future Needs.

The coronavirus disease 2019 (COVID-19) is caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which rages all over the world and seriously threatens human life and health. Currently, there is no optimal treatment for COVID-19, and emerging evidence found that COVID-19 infection results in gut microbiota dysbiosis. The intestinal microbial richness of patients of COVID-19 does not return to normal levels even six months after recovery, but probiotic adjunctive treatment has been found to restore gut homeostasis. An updated PubMed search returned four finished clinical trials that supported the use of probiotics as adjunctive treatment for COVID-19, while at least six clinical trials aiming to investigate beneficial effects of probiotic intake in managing COVID-19 are currently in progress worldwide. Here in we tentatively summarized the understanding of the actions and potential mechanisms of probiotics in the management of COVID-19. We also highlighted some future needs for probiotic researchers in the field. The success in using probiotics as adjunctive treatment for COVID-19 has expanded the scope of application of probiotics, meanwhile deepening our knowledge in the physiological function of probiotics in modulating the gut-lung axis.

pH-dependent and cathepsin B activable CaCO3 nanoprobe for targeted in vivo tumor imaging.

Background: The intraoperative visualization of tumor cells is a powerful modality for surgical treatment of solid tumors. Since the completeness of tumor excision is closely correlated with the survival of patients, probes that can assist in distinguishing tumor cells are highly demanded. Purpose: In the present study, a fluorescent probe JF1 was synthesized for imaging of tumor cells by conjugating a substrate of cathepsin B (quenching moiety) to Oregon Green derivative JF2 using a self-immolative linker. Methods: JF1 was then loaded into the folate-PEG modified CaCO3 nanoparticles. The folate receptor-targeted, pH-dependent, and cathepsin B activable CaCO3 nanoprobe was test in vitro and in vivo for tumor imaging. Results: CaCO3 nanoprobe demonstrated good stability and fast lighting ability in tumors under low pH conditions. It also showed lower fluorescence background than the single cathepsin B dependent fluorescent probe. The pH-dependent and cathepsin B controlled "turn-on" property enables precise and fast indication of tumor in vitro and in vivo. Conclusion: This strategy of controlled drug delivery enables in vivo imaging of tumor nodules with a high signal-to-noise ratio, which has great potential in surgical tumor treatment.

A Perspective Study of Koumiss Microbiome by Metagenomics Analysis Based on Single-Cell Amplification Technique.

Koumiss is a traditional fermented dairy product and a good source for isolating novel bacteria with biotechnology potential. In the present study, we applied the single-cell amplification technique in the metagenomics analysis of koumiss. This approach aimed at detecting the low-abundant bacteria in the koumiss. Briefly, each sample was first serially diluted until reaching the level of approximately 100 cells. Then, three diluted bacterial suspensions were randomly picked for further study. By analyzing 30 diluted koumiss suspensions, a total of 24 bacterial species were identified. In addition to the previously reported koumiss-associated species, such as Lactobacillus (L.) helveticus. Lactococcus lactis. L. buchneri, L. kefiranofaciens, and Acetobacter pasteurianus, we successfully detected three low-abundant taxa in the samples, namely L. otakiensis. Streptococcus macedonicus, and Ruminococcus torques. The functional koumiss metagenomes carried putative genes that relate to lactose metabolism and synthesis of typical flavor compounds. Our study would encourage the use of modern metagenomics to discover novel species of bacteria that could be useful in food industries.

Complete genome sequence of probiotic Bacillus coagulans HM-08: A potential lactic acid producer.

Bacillus coagulans HM-08 is a commercialized probiotic strain in China. Its genome contains a 3.62Mb circular chromosome with an average GC content of 46.3%. In silico analysis revealed the presence of one xyl operon as well as several other genes that are correlated to xylose utilization. The genetic information provided here may help to expand its future biotechnology potential in lactic acid production.

Complete genome sequence of Bacillus licheniformis BL-09, a probiotic strain isolated from naturally fermented congee.

Bacillus licheniformis BL-09 is a probiotic strain isolated from naturally fermented congee in Inner Mongolia of China. The genome of this strain contains a 4.39 Mb circular chromosome with an average GC content of 45.9%. The analysis of the genome revealed two gene clusters that are related to the peptide biosynthesis. The available genetic information of these peptides makes it possible to construct an improved strain for the industrial production of antimicrobial agents.