Microbial Communities, Metabolites, Fermentation Quality and Aerobic Stability of Whole-Plant Corn Silage Collected from Family Farms in Desert Steppe of North China

Whole-plant corn silages on family farms were sampled in Erdos (S1), Baotou (S2), Ulanqab (S3), and Hohhot (S4) in North China, after 300 d of ensiling. The microbial communities, metabolites, and aerobic stability were assessed. Lactobacillusbuchneri, Acinetobacter johnsonii, and unclassified Novosphingobium were present at greater abundances than others in S2 with greater bacterial diversity and metabolites. Lactobacillus buchneri, Lactobacillus parafarraginis, Lactobacillus kefiri, and unclassified Lactobacillus accounted for 84.5%, and 88.2%, and 98.3% of bacteria in S1, S3, and S4, respectively. The aerobic stability and fungal diversity were greater in S1 and S4 with greater abundances of unclassified Kazachstania, Kazachstania bulderi, Candida xylopsoci, unclassified Cladosporium, Rhizopus microspores, and Candida glabrata than other fungi. The abundances of unclassified Kazachstania in S2 and K. bulderi in S3 were 96.2% and 93.6%, respectively. The main bacterial species in S2 were L. buchneri, A. johnsonii, and unclassified Novosphingobium; Lactobacillus sp. dominated bacterial communities in S1, S3, and S4. The main fungal species in S1 and S4 were unclassified Kazachstania, K. bulderi, C. xylopsoci, unclassified Cladosporium, R. microspores, and C. glabrata; Kazachstania sp. dominated fungal communities in S2 and S3. The high bacterial diversity aided the accumulation of metabolites, and the broad fungal diversity improved the aerobic stability.

Probiotic Lactobacilli Isolated from Kefir Promote Down-Regulation of Inflammatory Lamina Propria T Cells from Patients with Active IBD

Ulcerative colitis and Crohn’s disease, the two main forms of inflammatory bowel disease (IBD), are immunologically mediated disorders. Several therapies are focused on activated T cells as key targets. Although Lactobacillus kefiri has shown anti-inflammatory effects in animal models, few studies were done using human mucosal T cells. The aim of this work was to investigate the immunomodulatory effects of this bacterium on intestinal T cells from patients with active IBD. Mucosal biopsies and surgical samples from IBD adult patients (n = 19) or healthy donors (HC; n = 5) were used. Lamina propria mononuclear cells were isolated by enzymatic tissue digestion, and entero-adhesive Escherichia coli-specific lamina propria T cells (LPTC) were expanded. The immunomodulatory properties of L. kefiri CIDCA 8348 strain were evaluated on biopsies and on anti-CD3/CD28-activated LPTC. Secreted cytokines were quantified by ELISA, and cell proliferation and viability were assessed by flow cytometry. We found that L. kefiri reduced spontaneous release of IL-6 and IL-8 from inflamed biopsies ex vivo. Activated LPTC from IBD patients showed low proliferative rates and reduced secretion of TNF-α, IL-6, IFN-γ and IL-13 in the presence of L. kefiri. In addition, L. kefiri induced an increased frequency of CD4+FOXP3+ LPTC along with high levels of IL-10. This is the first report showing an immunomodulatory effect of L. kefiri CIDCA 8348 on human intestinal cells from IBD patients. Understanding the mechanisms of interaction between probiotics and immune mucosal cells may open new avenues for treatment and prevention of IBD.

Probiotic Lactobacillus kefiri prevents endotoxin-induced preterm birth and stillbirth in mice

Preterm birth (PTB), defined as birth occurring before 37 weeks of pregnancy, affects 5-18% of pregnancies and is the leading cause of neonatal morbidity and mortality worldwide. Although PTB is considered a syndrome, infection-induced inflammation accounts up to 50% of all cases. Despite the effort to reduce the incidence of PTB, it continues rising worldwide and current approaches for preventing or treating PTB are largely unsatisfactory. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. It is well known that probiotics can modulate the host immune system exerting a potent anti-inflammatory activity. The main aim of this work was to evaluate the capacity of the probiotic Lactobacillus kefiri (Lk48) to prevent preterm birth in mice. C57BL/6 female mice were treated with Lk48 or vehicle a week before and during pregnancy and were challenged with LPS (10 µg), a dose known to induce 100% of PTB, on gestational day 16. Percentages of PTB as well as stillbirth were evaluated. We observed that oral administration of Lk48 significantly reduced the occurrence of LPS-induced PTB and stillbirth as well as improved post-natal development. This protective effect was associated with a reduction in leucocyte infiltration and reduced inflammation-induced damage in reproductive tissue. Besides, Lk48 treatment also modulated the diversity of vaginal microbiota. Our results demonstrated that prophylactic consumption of probiotic Lactobacillus kefiri prevented LPS-induced PTB and still birth in mice and open new avenues for exploring novel and promising strategies for preventing PTB in humans.

Lactobacillus Kefiri LKF01 (Kefibios®) for Prevention of Diarrhoea in Cancer Patients Treated with Chemotherapy: A Prospective Study

Diarrhoea is one of the main side effects that cancer patients face. The literature showsthat the incidence of chemotherapy (CT)-induced diarrhoea (grade 3–4) in treated patients is in the range of 10–20%, particularly after 5-fluorouracil (5-FU) bolus or some combination therapies of irinotecan and fluoropyrimidines. The aim of the present study was to evaluate the clinical effectiveness of Lactobacillus kefiri LKF01 (Kefibios®) in the prevention or treatment of CT-related diarrhoea in the cancer population. We conducted a prospective observational study. Patients enrolled were adults treated for at least four months with 5-FU-based CT. Kefibios® was administered to patients every day. The primary outcome was the evaluation of the incidence of grade 3–4 CT-induced diarrhoea. We included 76 patients in the final analysis. A 6.6% incidence of high-grade diarrhoea was found in the evaluated population (4.7% of patients treated with 5-FU-based therapy and 8.5% of patients treated with capecitabine-based CT). The overall incidence of high-grade diarrhoea observed was higher in the 1st and 2nd cycles (3.9%), with a subsequent sharp reduction from the 3rd cycle (1.3%) and negativisation from the 5th cycle. Lactobacillus kefiri LKF01 (Kefibios®) is safe and effective in preventing severe diarrhoea in cancer patients receiving 5-FU or capecitabine-based treatment.

Synthesis and Catalytic Application of Silver Nanoparticles Supported on Lactobacillus kefiri S-Layer Proteins

Research on nanoparticles obtained on biological supports is a topic of growing interest in nanoscience, especially regarding catalytic applications. Silver nanoparticles (AgNPs) have been studied due to their low toxicity, but they tend to aggregation, oxidation, and low stability. In this work, we synthesized and characterized AgNPs supported on S-layer proteins (SLPs) as bidimensional regularly arranged biotemplates. By different reduction strategies, six AgNPs of variable sizes were obtained on two different SLPs. Transmission electron microscopy (TEM) images showed that SLPs are mostly decorated by evenly distributed AgNPs; however, a drastic reduction by NaBH4 led to large AgNPs whereas a smooth reduction with H2 or H2/NaBH4 at low concentration leads to smaller AgNPs, regardless of the SLP used as support. All the nanosystems showed conversion values between 75–80% of p-nitrophenol to p-aminophenol, however, the increment in the AgNPs size led to a great decrease in Kapp showing the influence of reduction strategy in the performance of the catalysts. Density functional theory (DFT) calculations indicated that the adsorption of p-nitrophenolate species through the nitro group is the most favored mechanism, leading to p-aminophenol as the only feasible product of the reaction, which was corroborated experimentally.

Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

S-layer (glyco)-proteins (SLPs) form a nanostructured envelope that covers the surface of different prokaryotes and show immunomodulatory activity. Previously, we have demonstrated that the S-layer glycoprotein from probiotic Lactobacillus kefiri CIDCA 8348 (SLP-8348) is recognized by Mincle (macrophage inducible C-type lectin receptor), and its adjuvanticity depends on the integrity of its glycans. However, the glycan's structure has not been described so far. Herein, we analyze the glycosylation pattern of three SLPs, SLP-8348, SLP-8321, and SLP-5818, and explore how these patterns impact their recognition by C-type lectin receptors and the immunomodulatory effect of the L. kefiri SLPs on antigen-presenting cells. High-performance anion-exchange chromatography–pulse amperometric detector performed after β-elimination showed glucose as the major component in the O-glycans of the three SLPs; however, some differences in the length of hexose chains were observed. No N-glycosylation signals were detected in SLP-8348 and SLP-8321, but SLP-5818 was observed to have two sites carrying complex N-glycans based on a site-specific analysis and a glycomic workflow of the permethylated glycans. SLP-8348 was previously shown to enhance LPS-induced activation on both RAW264.7 macrophages and murine bone marrow–derived dendritic cells; we now show that SLP-8321 and SLP-5818 have a similar effect regardless of the differences in their glycosylation patterns. Studies performed with bone marrow–derived dendritic cells from C-type lectin receptor–deficient mice revealed that the immunostimulatory activity of SLP-8321 depends on its recognition by Mincle, whereas SLP-5818's effects are dependent on SignR3 (murine ortholog of human DC-SIGN). These findings encourage further investigation of both the potential application of these SLPs as new adjuvants and the protein glycosylation mechanisms in these bacteria.

The Microbiota of Edam Cheeses Determined by Cultivation and High-Throughput Sequencing of the 16S rRNA Amplicon

The aim of this study was to evaluate the microbiome of industrially produced ripened Edam cheeses by next-generation sequencing. The samples for analyses were collected in spring and autumn. Spring samples were characterized by significantly higher Lactococcus and Bacillus counts and lower counts of Enterobacteriaceae, Enterococcus, and yeasts than autumn samples. The predominant microorganisms identified by the Illumina high-throughput sequencing technology belonged to four phyla: Firmicutes, Actinobacteria, Proteobacteria and Bacteroidetes. The dominant species were starter culture bacteria. Lactobacillus rhamnosus, Lactobacillus kefiri, Lactobacillus kefiranofaciens, Lactobacillus casei, Streptococcus thermophilus, and Bifidobacterium had the highest share of microbial cheese communities. The number of γ-Proteobacteria reads was higher in autumn cheese samples. A high number of reads was also noted in the genus Clostridium. The counts of spore-forming bacteria of the genus Bacillus were higher in cheeses produced in spring. The study revealed highly similar relationships between the analyzed production periods. The present results contribute to the existing knowledge of cheese microbiota, and they can be used to improve and modify production processes based on the composition of microbial communities, as well as to improve the quality of the final product.