There are some limitations of traditional influenza vaccines concerning novel mutant strains. Therefore, it is particularly important to develop preventive means for antigen-unrelated types of influenza viruses. Recent studies have shown that probiotics can modulate the immune system and reduce the severity of viral infections. In this study, we investigated the potential of Lactiplantibacillus plantarum 0111 against influenza virus H9N2. Challenge experiments showed that L. plantarum 0111 pretreatments could effectively improve mice's survival rate and weight loss and reduce the inflammatory cytokines IL-6 and TNF-α in the lungs and bronchoalveolar lavage fluid (BALF) along with the degree of lung and intestinal injury. FMT experiment demonstrates that the protective effect produced by L. plantarum 0111 is associated with gut microorganisms. In addition, 16S high-throughput sequencing of the mouse intestinal microbiota showed that L. plantarum 0111 remodeled the intestinal microbiota after H9N2 infection and maintained the gut microbiota balance. In a mouse model, the oral administration of L. plantarum 0111 increased IFN-β expression in the serum and BALF. At the same time, the transcript levels of IFN-β and related ISGs in the intestine and lungs of mice were also increased. In addition, the activation and polarization of T cells in mesenteric lymph nodes (MLNs) and the spleen were detected by flow cytometry, and the results showed that L. plantarum 0111 modulated cytokines in T cells and increased IgA expression in B cells in the MLNs and spleen. Thus, L. plantarum 0111 may improve gut microbiota-mediated immune responses and thus, resist infection by the influenza virus, and it could be used as an effective preventive measure against the influenza virus.

Intelectin (ITLN) is a type of glycan-binding lectin involved in many physiological processes and some human diseases. Here we report a common carp intelectin (cITLN). Like other orthologs, cITLN also contains a conserved fibrinogen-related domain (FReD) and a unique intelectin domain, expresses in all the tissues tested with the highest level in the hindgut, and responds to bacterial challenge in the acute phase. We also expressed cITLN in Escherichia coli (E. coli) system, and the purified recombinant cITLN could neither affect the surface of bacteria nor inhibit the growth of bacteria, but it can agglutinate both gram-positive and gram-negative bacteria in a calcium-dependent manner. The cITLN's ability of agglutination of gram-positive bacteria is stronger than that of gram-negative bacteria. This is probably because recombinant cITLN could binding peptidoglycan (PGN) with a higher degree to lipopolysaccharide (LPS). Our results of cITLN provided new insight into the function of intelectin in the intestinal mucosal immunity.

Bacillus velezensis is widely used for agricultural biocontrol, due to its ability to enhance plant growth while suppressing the growth of microbial pathogens. However, there are few reports on its application in fermented feed. Here, a two-stage solid-state fermentation process using Bacillus velezensis followed by Lactobacillus plantarum was developed to degrade antinutritional factors (ANFs) and improve soybean meal (SBM) nutrition for animal feed. The process was evaluated for performance in degrading SBM antinutritional factors, dynamic changes in physicochemical characteristics, microorganisms and metabolites. After two-stage fermentation, degradation rates of glycinin and β-conglycinin contents reached 78.60% and 72.89%, respectively. The pH of fermented SBM (FSBM) decreased to 4.78 ± 0.04 and lactic acid content reached 183.38 ± 4.86 mmol/kg. NSP-degrading enzymes (Non-starch polysaccharide, NSPases) and protease were detected from the fermented product, which caused the changed microstructure of SBM. Compared to uninoculated SBM, FSBM exhibited increased proportions of crude protein (51.97 ± 0.44% vs. 47.28 ± 0.34%), Ca, total phosphorus (P), and trichloroacetic acid-soluble protein (11.79 ± 0.13% vs. 5.07 ± 0.06%). Additionally, cellulose and hemicellulose proportions declined by 22.10% and 39.15%, respectively. Total amino acid content increased by 5.05%, while the difference of AA content between the 24 h, 48 h and 72 h of fermentation was not significant (P > 0.05). Furthermore, FSBM also showed antibacterial activity against Staphylococcus aureus and Escherichia coli. These results demonstrated that two-stage SBM fermentation process based on Bacillus velezensis 157 and Lactobacillus plantarum BLCC2-0015 is an effective approach to reduce ANFs content and improve the quality of SBM feed.

The aim of this study was to investigate a novel means of treating piglet diarrhoea caused by enterotoxigenic Escherichia coli (ETEC) strain K88 using traditional Chinese medicine (TCM) fermented by selected probiotic strains. Six types of TCMs were determined to be efficient at inhibiting ETEC growth. In addition, two types of probiotics, Lactobacillus 0111 and 0115, were shown to be the most efficient at inhibiting ETEC. The results of studies with mice indicated that the administration of individual TCMs or probiotic strains increased the growth of mice to a different extent. Specifically, the fermentation of Fructus mume, Galla chinensis and Herba taraxaci by the Lactobacillus 0111 strain enhanced their antibacterial ability compared with traditional Jade screen, possibly due to the presence of a compound producing an absorption peak with a retention time of 19.5 min by HPLC. The fermentation of TCMs by probiotic strain provides a novel approach to relieve the effects of the ETEC strain K88.

试验旨在对一株杜仲树皮内生菌贝莱斯芽孢杆菌(Bacillus velezensis)的内切纤维素酶(CMCase)进行产酶条件优化及酶学性质分析,为探究贝莱斯芽孢杆菌CMCase的相关特性提供参考依据.采用单因子试验设计,分别优化pH、接种量、温度、时间、碳源和氮源等发酵条件,并探讨温度、pH、底物专一性、金属离子及表面活性剂对CMCase的影响.结果 表明,以玉米秸秆为碳源(30 g/L)、豆粕为氮源(30 g/L)、3％接菌量、pH7.0的条件下37℃培养48 h,贝莱斯芽孢杆菌157的CMCase最高可达(5.14±0.18) U/mL.经CMCase酶学性质分析发现,贝莱斯芽孢杆菌157的最适酶反应温度为60℃,最适pH为5.0;温度稳定性试验发现,在40和50℃时,相对剩余酶活力均高于80％,随着温度的升高,酶活力逐渐降低,当温度高于55℃,相对剩余酶活力约为50％;pH稳定性试验发现,在pH 5.0～10.0之间耐受性良好,相对剩余酶活力均高于90％,随作用时间的延长,相对剩余酶活力变化不大.贝莱斯芽孢杆菌157 CMCase属于典型内切型纤维素酶.Na+、Mg2+、Ca2+可促进贝莱斯芽孢杆菌157CMCase酶活力,而Co2+、Hg2+、Fe2、Cu2+和SDS抑制CMCase酶活力;表面活性剂Tween-80、Tween-20、TritonX-100对贝莱斯芽孢杆菌157 CMCase无影响.本试验通过对贝莱斯芽孢杆菌157进行产酶条件优化及CMCase酶学性质分析发现,贝莱斯芽孢杆菌157 CMCase具有产酶量高,耐受pH范围广的特点,在饲料添加剂、洗涤剂和造纸领域中具有一定的应用价值.

Bacillus velezensis 157 was isolated from the bark of Eucommia ulmoides, and exhibited antagonistic activity against a broad spectrum of pathogenic bacteria and fungi. Moreover, B. velezensis 157 also showed various lignocellulolytic activities including cellulase, xylanase, α-amylase, and pectinase, which had the ability of using the agro-industrial waste (soybean meal, wheat bran, sugarcane bagasse, wheat straw, rice husk, maize flour and maize straw) under solid-state fermentation and obtained several industrially valuable enzymes. Soybean meal appeared to be the most efficient substrate for the single fermentation of B. velezensis 157. Highest yield of pectinase (19.15 ± 2.66 U g−1), cellulase (46.69 ± 1.19 U g−1) and amylase (2097.18 ± 15.28 U g−1) was achieved on untreated soybean meal. Highest yield of xylanase (22.35 ± 2.24 U g−1) was obtained on untreated wheat bran. Here, we report the complete genome sequence of the B. velezensis 157, composed of a circular 4,013,317 bp chromosome with 3789 coding genes and a G + C content of 46.41%, one circular 8439 bp plasmid and a G + C content of 40.32%. The genome contained a total of 8 candidate gene clusters (bacillaene, difficidin, macrolactin, butirosin, bacillibactin, bacilysin, fengycin and surfactin), and dedicates over 15.8% of the whole genome to synthesize secondary metabolite biosynthesis. In addition, the genes encoding enzymes involved in degradation of cellulose, xylan, lignin, starch, mannan, galactoside and arabinan were found in the B. velezensis 157 genome. Thus, the study of B. velezensis 157 broadened that B. velezensis can not only be used as biocontrol agents, but also has potentially a wide range of applications in lignocellulosic biomass conversion.

Genomes of 24 sequenced Bacillus velezensis strains were characterized to identity shared and unique genes of lignocellulolytic enzymes and predict potential to degrade lignocellulose. All 24 strains had genes that encoded lignocellulolytic enzymes, with potential to degrade cellulose and hemicelluloses. Several lignocellulosic genes related to cellulose degradation were universally present, including one GH5 (endo-1,4-β-glucanase), one GH30 (glucan endo-1,6-β-glucosidase), two GH4 (6-phospho-β-glucosidase, 6-phospho-α-glucosidase), one GH1 (6-phospho-β-galactosidase), one GH16 (β-glucanase) and three GH32 (two sucrose-6-phosphate hydrolase and levanase). However, in the absence of gene(s) for cellobiohydrolase, it was predicted that none of the 24 strains would be able to directly hydrolyse cellulose. Regarding genes for hemicellulose degradation, four GH43 (1,4-β-xylosidase; except strain 9912D), one GH11 (endo-1,4-β-xylanase), three GH43 (two arabinan endo-1,5-α-L-arabinosidase and one arabinoxylan arabinofuranohydrolase), two GH51 (α-N-arabinofuranosidase), one GH30 (glucuronoxylanase), one GH26 (β-mannosidase) and one GH53 (arabinogalactan endo-1,4-β-galactosidase) were present. In addition, two PL1 (pectate lyase) and one PL9 (pectate lyase) with potential for pectin degradation were conserved among all 24 strains. In addition, all 24 Bacillus velezensis had limited representation of the auxiliary activities super-family, consistent with a limited ability to degrade lignin. Therefore, it was predicted that for these bacteria to degrade lignin, pretreatment of lignocellulosic substrates may be required. Finally, based on in silico studies, we inferred that Bacillus velezensis strains may degrade a range of polysaccharides in lignocellulosic biomasses.

Goose parvovirus (GPV) continues to be a threat to goose farms and has significant economic effects on the production of geese. Current commercially available vaccines only rarely prevent GPV infection. In our study, Lactobacillus (L.) plantarum NC8 was selected as a vector to express the VP2 gene of GPV, and recombinant L. plantarum pSIP409-VP2/NC8 was successfully constructed. The molecular weight of the expressed recombinant protein was approximately 70 kDa. Mice were immunized with a 2 × 109 colony-forming unit/200 μL dose of the recombinant L. plantarum strain, and the ratios and numbers of CD11c+, CD3+CD4+, CD3+CD8+, and interferon gamma- and tumor necrosis factor alpha-expressing spleen lymphocytes in the pSIP409-VP2/NC8 group were higher than those in the control groups. In addition, we assessed the capacity of L. plantarum SIP409-VP2/NC8 to induce secretory IgA production. We conclude that administered pSIP409-VP2/NC8 leads to relatively extensive cellular responses. This study provides information on GPV infection and offers a clear framework of options available for GPV control strategies.

It has been considered that the Avian influenza virus (AIV) causes severe threats to poultry industry. In this study, we constructed a series of recombinant Lactobacillus plantarum (L. plantarum) with surface displayed hemagglutinin subunit 2 (HA2) alone or together with heat-labile toxin B subunit (LTB) from enterotoxigenic Escherichia coli. Balb/c mice were used as model to evaluate the protective effects of recombinant L. plantarum strains against H9N2 subtype challenge. The results showed that the presence of LTB significantly increased the percentages of CD3+CD4+IL-4+, CD3+CD4+IFN-γ+ and CD3+CD4+IL-17+ T cells, as well as CD3+CD8+IFN-γ+ T cells in spleen and MLNs determined by Fluorescence-Activated Cell Sorting assay. Similar increased production of serum IFN-γ was also confirmed by enzyme linked immunosorbent assay (ELISA). The L. plantarum with surface displayed HA2-LTB also dramatically increased the percentages of B220+ IgA+ B cells in peyer patch, in consistent with elevated production of mucosal SIgA antibody determined by ELISA. Finally, the orally administrated HA2-LTB expressing strain efficiently protected mice against H9N2 subtype AIV challenge shown by increased survival percentages, body weight gains and decreased lung lesions in histopathologic analysis. In conclusion, this study provides more detail mechanisms underlying the adjuvant effects of LTB on heterologous antigen produced in recombinant lactic acid bacteria.

The coccidiosis caused by Eimeria tenella (coccidian) and other species is a serious parasitic disease that affects the global poultry breeding industry. Lactobacillus strains exhibit a number of properties that make them attractive candidates as delivery vehicles for presentation to the mucosa of compounds with pharmaceutical interest, particularly vaccines. Here, the recombinant Lactobacillus plantarum (co-expressing SO7 and DCpep gene) was constructed, and its efficacy against E. tenella challenge was evaluated in this study. Broiler chickens were orally immunized with live recombinant L. plantarum NC8-pSIP409-SO7-DCpep for two weeks and were then challenged with 5×104E.tenella sporulated oocysts per chicken. During the experiment, body weight gains, cecum lesion scores, fecal oocyst shedding and antibody responses in serum and intestinal washes were assessed as measures of protective immunity. The results indicated that chickens immunized with live recombinant L. plantarum can increase body weight gains and serum antibody responses compared to the control groups. Meanwhile, fecal oocyst shedding in the immunized group was significantly reduced (p<0.01). Moreover, recombinant L. plantarum can significantly relieve pathological damage in cecum, according to lesion scores and histopathologic cecum sections (p<0.01). Therefore, these results indicate that recombinant L. plantarum NC8-pSIP409-SO7-DCpep could become a promising oral vaccine candidate against E. tenella infection.

Recently, poly-γ-glutamic acid synthetase A (pgsA) has been applied to display exogenous proteins on the surface of Lactobacillus casei or Lactococcus lactis, which results in a surfacedisplayed component of bacteria. However, the ability of carrying genes encoded by plasmids and the expression efficiency of recombinant bacteria can be somewhat affected by the longer gene length of pgsA (1,143 bp); therefore, a truncated gene, pgsA, was generated based on the characteristics of pgsA by computational analysis. Using murine IL-10 as an exogenous gene, recombinant Lactobacillus plantarum was constructed and the capacity of the surface-displayed protein and functional differences between exogenous proteins expressed by these strains were evaluated. Surface expression of IL-10 on both recombinant bacteria with anchorins and the higher expression levels in L. plantarum-pgsA'-IL-10 were confirmed by western blot assay. Most importantly, up-regulation of IL-1β, IL-6, TNF-α, IFN-γ, and the nuclear transcription factor NF-κB p65 in RAW264.7 cells after stimulation with Poly(I:C) or LPS was exacerbated after co-culture with L. plantarum-pgsA. By contrast, IL-10 expressed by these recombinant strains could reduce these factors, and the expression of these factors was associated with recombinant strains that expressed anchorin (especially in L. plantarum-pgsA'-IL-10) and was significantly lower compared with the anchorin-free strains. These findings indicated that exogenous proteins could be successfully displayed on the surface of L. plantarum by pgsA or pgsA', and the expression of recombinant bacteria with pgsA' was superior compared with bacteria with pgsA.