Thermal and Chemical Inactivation of Bacillus Phage BM-P1.

Bacillus spp. are often used as probiotics; however, they can be infected by phages, leading to significant economic losses. Biocidal and thermal treatments are considered rapid and effective methods for controlling microbial contamination. To prevent viral contamination in industrial dairy production, the impact of temperature and biocides on the viability of Bacillus methylotrophic phage BM-P1 was assessed. The results demonstrated that reconstituted skim milk (RSM) as a medium showed the most effective protective effect on phage BM-P1. Treatment at 90°C for 5 min or 72°C for 10 min inactivated it to nondetectable levels from the initial titer of 7.19 ± 0.11 log, regardless of the culture medium. Sodium hypochlorite exhibited the best inactivating effect, which could reduce the phage titer below the detection level in 4 min at 50 ppm. Additionally, treatment with 75% ethanol for 20 min or 50% isopropanol for 30 min could achieve inactivation to nondetectable levels. The inactivating effect of peracetic acid was limited; even when treated at the highest concentration (0.45%) for 60 min, only a 2.47 ± 0.17 log reduction was observed. This study may provide some theoretical basis and data support for establishing measures against Bacillus spp. phages.

A large-scale comparative genomics study reveals niche-driven and within-sample intra-species functional diversification in Lacticaseibacillus rhamnosus.

Lacticaseibacillus rhamnosus (L. rhamnosus) is widely recognized as a probiotic species, and it exists in a variety of environments including host gut and dairy products. This work aimed at conducting a large-scale comparative genomics analysis of 384 L. rhamnosus genomes (257 whole-sequence or metagenomic-assembled genomes from gut-associated isolates [122 and 135 retrieved from the UHGG and NCBI databases, respectively] and 127 genomes from dairy isolates [34 from the NCBI database; 93 isolated from a cheese sample and sequenced here]). Our results showed that L. rhamnosus had a large and open pan-genome (15,253 pan-genes identified from all 384 genomes; 15,028 pan-genes if the 93 cheese-originated isolates were excluded). The core-gene phylogenetic tree constructed from the 384 L. rhamnosus genomes comprised five phylogenetic branches, with a random distribution of dairy and gut-associated isolates/genomes across the tree. No significant difference was identified in the overall profile of metabolism-related genes between dairy and gut-associated genomes; however, notably, the gut-associated strains/isolates contained more genes coding for specific metabolic pathways and carbohydrate-active enzymes, e.g., lacto-N-biosidase (EC 3.2.1.140; GT20) and lacto-N-biose phosphorylase/galacto-N-biose phosphorylase (EC 2.4.1.211; GH112). Further, we found that there was obvious intra-species diversification of the 93 cheese-originated L. rhamnosus isolates, forming three clades (Clades A, B, and C) in the reconstructed core-gene phylogenetic tree. There were numerous single nucleotide variations (over 10,000) across the three clades. Moreover, significant differences were observed in the content of metabolism-related genes across clades (p < 0.05, Adonis test), characterized by the enrichment in glycoside hydrolases in Clade C and the possession of unique metabolic pathways in each clade. These results implicated genomics/functional diversification of L. rhamnosus in a single food matrix and niche-driven adaptive evolution of isolates from dairy and host gut-associated origins. Our study shed insights into the selection of candidate strains for food industry applications.

Intraspecific microdiversity and ecological drivers of lactic acid bacteria in naturally fermented milk ecosystem.

Traditional fermented milks are produced by inoculating technique, which selects well-adapted microorganisms that have been passed on through generations. Few reports have used naturally fermented milks as model ecosystems to investigate the mechanism of formation of intra-species microbial diversity. Here, we isolated and whole-genome-sequenced a total of 717 lactic acid bacterial isolates obtained from 12 independent naturally fermented milks collect from 12 regions across five countries. We further analyzed the within-sample intra-species phylogenies of 214 Lactobacillus helveticus isolates, 97 Lactococcus lactis subsp. lactis isolates, and 325 Lactobacillus delbrueckii subsp. bulgaricus isolates. We observed a high degree of intra-species genomic and functional gene diversity within-/between-sample(s). Single nucleotide polymorphism-based phylogenetic reconstruction revealed great within-sample intra-species heterogeneity, evolving from multiple lineages. Further phylogenetic reconstruction (presence-absence gene profile) revealed within-sample inter-clade functional diversity (based on carbohydrate-active enzyme- and peptidase-encoding genes) in all three investigated species/subspecies. By identifying and mapping clade-specific genes of intra-sample clades of the three species/subspecies to the respective fermented milk metagenome, we found extensive potential inter-/intra-species horizontal gene transfer events. Finally, the microbial composition of the samples is closely linked to the nucleotide diversity of the respective species/subspecies. Overall, our results contribute to the conservation of lactic acid bacteria resources, providing ecological insights into the microbial ecosystem of naturally fermented dairy products.

Characteristics and Whole-Genome Analysis of Limosilactobacillus fermentum Phage LFP02.

Limosilactobacillus fermentum is a bacterium widely used in food production, medicine, and industrial fermentation. However, fermentation could fail due to phage contamination. L. fermentum bacteriophage LFP02 can be induced from L. fermentum IMAU 32579 using mitomycin C. To better understand the characteristics of this phage, its physiological and genomic characteristics were evaluated. The results showed that its optimal multiplicity of infection was 0.01, and the burst size was 148.03 ± 2.65 pfu/infective center. Compared to temperature, pH had a more obvious influence on phage viability, although its adsorption capacity was not affected by the divalent cations (Ca2+ and Mg2+) or chloramphenicol. Its genome size was 43,789 bp and the GC content was 46.06%, including 53 functional proteins. Compared to other L. fermentum phages, phage LFP02 had chromosome deletion, insertion, and inversion, which demonstrated that it was a novel phage. This study could expand the knowledge of the biological characteristics of L. fermentum bacteriophages and provide some theoretical basis for bacteriophage prevention during fermentation.

Inactivation of Lactobacillus Bacteriophages by Dual Chemical Treatments.

Phage contamination is one of the significant problems in the food fermentation industry, which eventually causes economic losses to the industry. Here, we investigated the viability of Lactobacillus plantarum phage P1 and P2 using various biocides treatments (ethanol, isopropanol, sodium hypochlorite and peracetic acid). Results indicated that phage P1 and P2 could be completely inactivated by treatment with 75% ethanol for 5 min, followed by 400 ppm of sodium hypochlorite treatment for 5 min. Phage P2 could be completely inactivated in the reverse sequence, while 800 ppm of sodium hypochlorite was required to achieve a similar effect for phage P1. Moreover, 100% isopropanol could increase the inactivating effect of 75% ethanol. This study may provide basic information on using multiple antimicrobials for phage control in laboratories and food plants.

Whole Genome Sequence Analysis of Lactiplantibacillus plantarum Bacteriophage P2.

Phage P2 was isolated from failed fermentation broth carried out by Lactiplantibacillus plantarum IMAU10120. A previous study in our laboratory showed that this phage belonged to the Siphoviridae family. In this study, this phage's genomic characteristics were analyzed using whole-genome sequencing. It was revealed that phage P2 was 77.9 kb in length and had 39.28% G + C content. Its genome included 96 coding sequences (CDS) and two tRNA genes involved in the function of the structure, DNA replication, packaging, and regulation. Phage P2 had higher host specificity; many tested strains were not infected. Cell wall adsorption experiments showed that the adsorption receptor component of phage P2 might be a part of the cell wall peptidoglycan. This research might enrich the knowledge about genomic information of lactobacillus phages and provide some primary data to establish phage control measures.

PacBio sequencing revealed variation in the microbiota diversity, species richness and composition between milk collected from healthy and mastitis cows.

Mastitis is the economically most important disease of dairy cows. This study used PacBio single-molecule real-time sequencing technology to sequence the full-length 16S rRNAs from 27 milk samples (18 from mastitis and nine from healthy cows; the cows were at different stages of lactation). We observed that healthy or late stage milk microbiota had significantly higher microbial diversity and richness. The community composition of the microbiota of different groups also varied greatly. The healthy cow milk microbiota was predominantly comprised of Lactococcus lactis, Acinetobacter johnsonii, and Bacteroides dorei, while the milk from mastitis cows was predominantly comprised of Bacillus cereus. The prevalence of L. lactis and B. cereus in the milk samples was confirmed by digital droplets PCR. Differences in the milk microbiota diversity and composition could suggest an important role for some these microbes in protecting the host from mastitis while others associated with mastitis. The results of our research serve as useful references for designing strategies to prevent and treat mastitis.

Differential proteomics reveals main determinants for the improved pectinase activity in UV-mutagenized Aspergillus niger strain.

OBJECTIVES: To reveal the potential mechanism and key determinants that contributed to the improved pectinase activity in Aspergillus niger mutant EIMU2, which was previously obtained by UV-mutagenesis from the wild-type A. niger EIM-6. RESULTS: Proteomic analysis for Aspergillus niger EIMU2 by two-dimensional electrophoresis demonstrated that mutant EIMU2 harbored a multiple enzyme system for the degradation of pectin, mainly constituting by main-chain-cleaving enzymes polygalacturonase, pectate lyase, pectinesterase, and some accessory enzymes rhamnogalacturonan lyase and arabinofuranosidase. Further quantitatively differential proteomic analysis revealed that the quantities of four proteins, pectinesterase, rhamnogalacturonan lyase A, DNA-directed RNA polymerase A, and a hypothetical protein in strain EIMU2 were much higher than those in EIM-6. PCR amplification, sequencing and alignment analysis of genes for the two main members of pectin-degrading enzymes, pectate lyase and polygalacturonase showed that their sequences were completely consistent in A. niger EIM-6 and mutant EIMU2. CONCLUSIONS: The result demonstrated that the improved pectinase activity by UV-mutagenesis in A. niger EIMU2 was probably contributed to the up-regulated expression of rhamnogalacturonan lyase, or pectinesterase, which resulted in the optimization of synergy amongst different components of pectin-degrading enzymes.

Comparison of Bacterial Microbiota in Raw Mare's Milk and Koumiss Using PacBio Single Molecule Real-Time Sequencing Technology.

Koumiss is a traditional fermented raw mare's milk product. It contains high nutritional value and is well-known for its health-promoting effect as an alimentary supplement. This study aimed to investigate the bacterial diversity, especially lactic acid bacteria (LAB), in koumiss and raw mare's milk. Forty-two samples, including koumiss and raw mare's milk, were collected from the pastoral area in Yili, Kazakh Autonomous Prefecture, Xinjiang Uygur Autonomous Region in China. This work applied PacBio single-molecule real-time (SMRT) sequencing to profile full-length 16S rRNA genes, which was a powerful technology enabling bacterial taxonomic assignment to the species precision. The SMRT sequencing identified 12 phyla, 124 genera, and 227 species across 29 koumiss samples. Eighteen phyla, 286 genera, and 491 species were found across 13 raw mare's milk samples. The bacterial microbiota diversity of the raw mare's milk was more complex and diverse than the koumiss. Raw mare's milk was rich in LAB, such as Lactobacillus (L.) helveticus, L. plantarum, Lactococcus (Lc.) lactis, and L. kefiranofaciens. In addition, raw mare's milk also contained sequences representing pathogenic bacteria, such as Staphylococcus succinus, Acinetobacter lwoffii, Klebsiella (K.) oxytoca, and K. pneumoniae. The koumiss microbiota mainly comprised LAB, and sequences representing pathogenic bacteria were not detected. Meanwhile, the koumiss was enriched with secondary metabolic pathways that were potentially beneficial for health. Using a Random Forest model, the two kinds of samples could be distinguished with a high accuracy 95.2% [area under the curve (AUC) = 0.98] based on 42 species and functions. Comprehensive depiction of the microbiota in raw mare's milk and koumiss might help elucidate evolutionary and functional relationships among the bacterial communities in these dairy products. The current work suffered from the limitation of a low sample size, so further work would be required to verify our findings.

Probiotic-directed modulation of gut microbiota is basal microbiome dependent.

As an effective means to improve quality of life and prevent diseases, the demand for probiotics and related products has increased in recent years. However, it is still unclear whether a particular probiotic strain will have similar beneficial effects on healthy adults from different regions. In this study, the probiotic Lactobacillus casei Zhang (LCZ) was consumed by healthy adults from six different Asian regions and the changes in gut microbiota were compared using PacBio single molecule, real-time (SMRT) sequencing technology based on samples collected before, during and after consumption of LCZ. Our results reveal that the effect of LCZ consumption on individuals was closely related to the composition of that individual's basal gut microbiota. A Gut Microbiota Variability Index (GMVI) was proposed to quantitatively compare the effects of LCZ on human gut microecology. Subjects from Xinjiang and Singapore regions had the highest and lowest GMVI, respectively. In general, consumption of LCZ increased the relative abundance of certain beneficial bacteria such as Lactobacillus, Roseburia, Coprococcus and Eubacterium rectale, while it inhibited growth of certain harmful bacteria such as Blautia and Ralstonia pickettii. In addition, consumption of LCZ was responsible for the conversion of some participants from Prevotella copri/Faecalibacterium prausnitzii (PF) enterotype to Faecalibacterium prausnitzii/Bacteroides dorei (FB) enterotype and consistently increased the abundance of lactic acid bacteria in the gut. It also increased/enhanced phosphate metabolic modules, amino acid transport systems, and isoleucine biosynthesis, but conversely decreased lipopolysaccharide biosynthesis. These changes could have health benefits for healthy adults.

Probiotics maintain the intestinal microbiome homeostasis of the sailors during a long sea voyage.

The challenging conditions encountered during long sea voyages increase the risk of health-threatening physiological and psychological stress for sailors compared with land-based workers. However, how the intestinal microbiota responds to a long sea voyage and whether there is a feasible approach for protecting gut health during sea voyage are still unexplored. Here, we designed a 30-d longitudinal study including a placebo group (n = 42) and a probiotic group (n = 40) and used shotgun metagenomic sequencing to explore the impacts of sea voyage on the intestinal microbiome of sailors. By comparing the intestinal microbiome of subjects in the placebo group at baseline (d 0) and at the end of the sea voyage (d 30), we observed an alteration in the intestinal microbiome during the long sea voyage based on the microbial structure; the results revealed an increase in the species Streptococcus gordonii and Klebsiella pneumoniae as well as a decrease in some functional features. However, the change in the microbial structure of sailors in the probiotic group between d 0 and d 30 was limited, which indicated a maintenance effect of probiotics on intestinal microbiome homeostasis. At the metagenomic strain level, a generally positive correlation was observed between probiotics and the strains belonging to Bifidobacterium longum and Bifidobacterium animalis, whereas a common negative correlation was observed between probiotics and Clostridium leptum; this result revealed the potential mechanism of maintaining intestinal microbiome homeostasis by probiotics. The present study provided a feasible approach for protecting gut health during a long sea voyage.

Community structure and succession regulation of fungal consortia in the lignocellulose-degrading process on natural biomass.

The study aims to investigate fungal community structures and dynamic changes in forest soil lignocellulose-degrading process. rRNA gene clone libraries for the samples collected in different stages of lignocellulose degradation process were constructed and analyzed. A total of 26 representative RFLP types were obtained from original soil clone library, including Mucoromycotina (29.5%), unclassified Zygomycetes (33.5%), Ascomycota (32.4%), and Basidiomycota (4.6%). When soil accumulated with natural lignocellulose, 16 RFLP types were identified from 8-day clone library, including Basidiomycota (62.5%), Ascomycota (36.1%), and Fungi incertae sedis (1.4%). After enrichment for 15 days, identified 11 RFLP types were placed in 3 fungal groups: Basidiomycota (86.9%), Ascomycota (11.5%), and Fungi incertae sedis (1.6%). The results showed richer, more diversity and abundance fungal groups in original forest soil. With the degradation of lignocellulose, fungal groups Mucoromycotina and Ascomycota decreased gradually, and wood-rotting fungi Basidiomycota increased and replaced the opportunist fungi to become predominant group. Most of the fungal clones identified in sample were related to the reported lignocellulose-decomposing strains. Understanding of the microbial community structure and dynamic change during natural lignocellulose-degrading process will provide us with an idea and a basis to construct available commercial lignocellulosic enzymes or microbial complex.