Improving Broiler Health through Cecal Microbiota Transplantation: A Comprehensive Study on Growth, Immunity, and Microbial Diversity.

Cecal microbiota has emerged as a prominent intervention target for improving the production and welfare of poultry. This is essential for the overall health and performance of broiler chickens. The current study focused on investigating the effect of cecal microbiota transplantation (CMT) from healthy donor chickens on the growth performance, immunity, and microbial composition of newly hatched chicks and evaluated the effect of sample storage on the microbial diversity of the cecal samples. A healthy "Wannan Yellow Chicken line" was selected as the donor, and 180 1-day-old chicks from the same line were used as recipients for a 60-day feed trial. The chicks were randomly allocated to three groups (60 birds per group) with three replicates in each group. The three treatment groups were CMT-0 (control, normal saline solution), CMT-I (1:12 cecal content, normal saline supplemented with 10% glycerol), and CMT-II (1:6 cecal content, normal saline supplemented with 10% glycerol). The results of weight gain and absolute organ weight showed significant improvements in the CMT-II group compared with the CMT-0 group. Serum IgG level was significantly improved (p < 0.05) in CMT-I compared with that in the CMT-0. However, IL-6 levels increased in CMT-I and then significantly decreased in CMT-II. The cecal microbial diversity of CMT treatment was compared between two groups, fresh samples (FS) and stored samples at -80°C (SS). The results showed that beneficial taxa, such as Firmicutes and Verrucomicrobiota, were substantially more abundant in both CMT-I and CMT-II than in CMT-0 in both FS and SS. Microbial function analysis at levels 1, 2, and 3 showed improved metabolism, genetic information processing, cellular processes, environmental information processing, and organismal systems in CMT-I and CMT-II for both FS and SS groups. However, the SS group showed decreased microbial diversity and function. To conclude, cecal microbiota transplantation is a promising strategy for enhancing the productivity and health of broiler chickens.

Site-Directed Mutagenesis: Improving the Acid Resistance and Thermostability of Bacillus velezensis α-Amylase and Its Preliminary Feed Application.

The current study aimed to improve the acid resistance and thermostability of Bacillus velezensis α-amylase through site-directed mutagenesis, with a specific focus on its applicability to the feed industry. Four mutation sites, P546E, H572D, A614E, and K622E, were designed in the C domain of α-amylase, and three mutants, Mut1 (E), Mut2 (ED), and Mut3 (EDEE), were produced. The results showed that the specific activity of Mut3 was 50 U/mg higher than the original α-amylase (Ori) after incubation at 40 °C for 4 h. Compared to Ori, the acid resistance of Mut3 showed a twofold increase in specific activity at pH 2.0. Moreover, the results of preliminary feed hydrolysis were compared between Ori and Mut3 by designing three factors, three levels of orthogonal experiment for enzymatic hydrolysis time, feed quantity, and amount of amylase. It was observed that the enzymatic hydrolysis time and feed quantity showed an extremely significant difference (p < 0.01) in Mut3 compared to Ori. However, the amount of enzyme showed significant (p < 0.05) improvement in the enzymatic hydrolysis in Mut3 as compared to Ori. The study identified Mut3 as a promising candidate for the application of α-amylase in the feed industry.

Effect of Bacillus subtilis Supplemented Diet on Broiler's Intestinal Microbiota and TLRs Gene Expression.

This study aimed to investigate the effects of dietary Bacillus subtilis supplementation on gut microbiota diversity, digestive enzyme activity, and Toll-like receptor (TLR) expression in broiler chickens. A total of 240 "817" crossbred broiler chickens were randomly assigned to four groups: control (basal diet, BD), group I (BD + 300 g/d B. subtilis at 1.08 × 107 CFU/kg), group II (BD + 600 g/d B. subtilis at 2.16 × 107 CFU/kg), and group III (BD + 900 g/d B. subtilis at 3.24 × 107 CFU/kg). Gut microbiota analysis revealed significant improvements in the abundance of specific microorganisms in the treatment groups, with distinct variations in the core microorganisms between the groups. Notably, protease activity in the ileum was significantly increased in groups II (22.59%; p < 0.01) and III (14.49%; p < 0.05) compared to that in the control group. Moreover, significant up-regulation of TLR1A and TLR7 expression was observed in jejunum and cecum of the treated groups. Additionally, the TLR1B expression in the ileum was significantly increased. Furthermore, TLR2A and MyD88 transcription levels were significantly elevated in the jejunum, liver, spleen, and kidneys of experimental groups. Modulations in the expression of various TLR's (TLR2B, TLR3, TLR4, TLR15, and TLR21) were also observed in different organs. The spleen and kidney of B. subtilis-supplemented chickens exhibited upregulated expression of the proinflammatory cytokine IL-1ß. Dietary supplementation with B. subtilis in broiler chickens improved the gut microbiota diversity and significantly upregulated TLR's expression in various organs. B. subtilis could be a valuable feed additive, contributing to improved disease management and overall health in broiler chickens.

Improving the anti-tumor effect of EGCG in colorectal cancer cells by blocking EGCG-induced YAP activation.

(-)-Epigallocatechin-3-gallate (EGCG) is the primary active ingredient in green tea and has been used for cancer prevention in clinical trials. The anti-tumor effects of EGCG stem from its ability to inhibit the activities of many oncoproteins, such as AKT, VEGFR, STAT3, and mutant p53. However, the clinical efficacy of EGCG is unsatisfactory. How to improve the anti-tumor effects of EGCG is an open question. Here we report that EGCG inhibits the tumor suppressive Hippo signaling pathway and activates downstream YAP in colorectal cancer (CRC) cells. Activation of YAP impedes the anti-tumor effects of EGCG. YAP blockade increases the sensitivity of CRC cells to EGCG treatment.

Microbial fermentation technology for degradation of saponins from peony seed meal.

Peony seed meal is a very important feed protein raw material with a high potential for development; however, the presence of some anti-nutritional factors, such as saponins, reduces its reusability. This study aimed to establish ideal microbial fermentation conditions for the degradation of saponins in peony seed meal for its subsequent use in poultry feed. First, saponins were extracted via two methods: ethanol extraction and reflux. Then, response surface methodology and orthogonal array testing were used to establish the optimal conditions for the degradation of saponins by (a) liquid fermentation of single bacteria, (b) liquid fermentation of compound bacteria, and (c) solid-state fermentation. The degradation efficiencies were 40.21% (±1.62), 59.82% (±1.54), and 69.31% (±2.95), respectively. The maximum degradation was obtained via solid-state fermentation, and the soluble protein content for this fermentation product was found to be 14% higher than that of unfermented peony seed meal.

Effect of Spore-Forming Probiotics on the Poultry Production: A Review.

Due to the bad aspects associated with the use of antibiotics, the pressure on poultry production prompted the efforts to find out suitable growth-promoting and disease-preventing alternatives. Although many cost-effective alternatives have been developed, currently, one of the most auspicious alternatives for poultry feed is spore-forming probiotics, which can exert more beneficial effects as compared to normal probiotics, because of their ability to withstand the harsh external and internal conditions which result in increased viability. Many studies have already used spore-forming probiotics to improve different parameters of poultry production. Our laboratory has recently isolated a spore-forming bacterial strain, which has the potential to be used as a probiotic. So, to provide a detailed understanding, the current review aimed to collect valuable references to describe the mechanism of action of spore-forming probiotics and their effect on all the key aspects of poultry production.

Effect of fermented corn by-products on production performance, blood biochemistry, and egg quality indices of laying hens.

Residual wastes can be fermented by using probiotics to formulate a well-balanced diet for poultry. The current study was conducted to investigate the effect of fermented feed (FF) formulated by the supplementation of probiotics into corn by-products, on the production performance of laying hens. A total of 468 49-wk-old Hy-Line Brown chickens were randomly allocated into four treatments with 3 replicates, control group (basal diet), group I (4% FF, w/w), group II (8% FF, w/w), and antibiotic group (flavomycin, 50 mg/kg). Laying hens were housed in 3-tier cages (28 × 48 cm × 48 cm) and the temperature was maintained at 21 °C. The birds were fed 4.56 kg of feed to each group twice a day. Compared with the control group, the egg production and feed intake in the FF groups increased significantly. Egg weight in group I was higher than that of group II and antibiotic (P < 0.01) while that in group II was lower than control (P < 0.05). The feed-to-egg ratio of FF groups compared with control was decreased (P < 0.05). Eggshell strength and egg shape index in group II were decreased significantly when compared with the control group (P < 0.05). Yolk color, albumen height (AH), and Haugh unit (HU) in FF groups were improved compared with the control group and antibiotic group (P < 0.01). Yolk triglyceride level was significantly reduced in group II, compared with group I. These results indicated that the FF has no adverse effects and has beneficial effects on production performance and egg quality parameters of laying hens.

Feed costs the major portion of poultry industry which is estimated to be 60% to 70%. The current study focused to add the fermented agro-industrial by product most of which were waste residues to the laying hen's diet. The result showed beneficial effects, which proved that fermentation is a good method to improve the nutritional quality of feed raw materials, which is beneficial to the gastrointestinal health of the poultry. The study elaborated the potential of waste residues to be used as a kind of functional feed ingredient, which can enhanced the production parameters and egg quality indices of poultry.

Fermentation optimization and amylase activity of endophytic Bacillus velezensis D1 isolated from corn seeds.

AIMS: To acquire quality amylase adopted in practical applications, endophytic bacteria were identified as Bacillus velezensis strain D1 which was isolated from corn seeds. The fermentation conditions and amylase properties of the strain were investigated. METHODS AND RESULTS: The strain D1 was identified via morphological, physiological and 16S rDNA phylogenetic analysis. The fermentation conditions of secreting amylase were optimized by single-factor and orthogonal experiments. The α-amylase gene was expressed in E. coli and purified by means of immobilized metal ion affinity chromatography (IMAC), upon which the enzyme activity of purified recombinant α-amylase was determined. The results outlined that (1) The strain D1 was identified as Bacillus velezensis. (2) The optimized fermentation conditions for maximum amylase yields included 44°C for 48 h at pH 7.5. (3) The enzyme had an optimal reaction temperature of 60°C with the highest activity at 50°C and tolerance to 4-h incubation at 70°C. (4) The enzyme was strong acid resistant and tolerated at pH 5.0-6.0 while the optimal pH was 8.0. (5) Besides, the amylase activity was elevated by the presence of Ca2+ and Cu2+ . (6) The activity of purified recombinant amylase was 20.59 U/ml under optimal conditions, nearly seven times that of crude amylase preparations. CONCLUSIONS: The amylase produced by Bacillus velezensis D1 is strongly tolerant towards acid and high temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Amylases with thermophilic and acid-resistant characteristics are useful for a wide range of applications in food, brewing, textile, starch, paper and deterrent industries. The enzyme from Bacillus velezensis D1 can be effectively used in different areas of industries.

Effects of dietary fermented peony seed dreg on the laying performance, albumen quality, antioxidant capacity, and n-3 PUFA-enriching property of laying hens.

Introduction: It is of great importance to seek agro-industrial byproducts that can serve as unconventional or alternative feedstuffs for poultry, especially those that are substitutive sources of n-3 polyunsaturated fatty acids (PUFAs), as this will contribute to alleviating feed shortage pressure and improving poultry performance and product quality. In this study, the effects of dietary fermented peony seed dreg (FPSD) on the production performance of hens and fatty acid composition of meat and egg yolk were evaluated. Methods: A total of 480 54-week-old Xinyang chickens were divided into 5 groups: control (basal diet); 5% peony seed dreg (PSD); and 5%, 7% and 9% FPSD, and each group consisted of 6 replicates with 16 birds per replicate. Production performances were recorded daily, and egg quality, serum parameters, antibody titers and fatty acid profile in the muscle and yolk were measured at 64 weeks of age. Results and discussion: Egg production, feed conversion ratio and egg albumen quality were improved (p < 0.05) by 5% and 7% FPSD groups compared to 5% PSD and the control diet. Immune status was not influenced by PSD, but antibody titres against H7N9 and ND were improved by FPSD diets during most of the experimental periods. Dietary 5% and 7% FPSD increased (p < 0.05) serum high density lipoprotein concentrations and glutathione peroxidase actives. Total n-3 polyunsaturated fatty acids (PUFA) in meat and yolk increased gradually, while the total saturated fatty acids (SFA) and the n-6/n-3 PUFA ratio decreased gradually with increasing dietary FPSD levels. In conclusion, up to 7% dietary FPSD has the potential use to be utilized as a supplement in Xinyang laying hen diets to produce n-3 PUFA-enriched meat and eggs and positively affect production performance and health status.

Potential of Bacillus velezensis as a probiotic in animal feed: a review.

Bacillus velezensis is a plant growth-promoting bacterium that can also inhibit plant pathogens. However, based on its properties, it is emerging as a probiotic in animal feed. This review focuses on the potential characteristics of B. velezensis for use as a probiotic in the animal feed industry. The review was conducted by collecting recently published articles from peer-reviewed journals. Google Scholar and PubMed were used as search engines to access published literature. Based on the information obtained, the data were divided into three groups to discuss the (i) probiotic characteristics of B. velezensis, (ii) probiotic potential for fish, and (iii) the future potential of this species to be developed as a probiotic for the animal feed industry. Different strains of B. velezensis isolated from different sources were found to have the ability to produce antimicrobial compounds and have a beneficial effect on the gut microbiota, with the potential to be a candidate probiotic in the animal feed industry. This review provides valuable information about the characteristics of B. velezensis, which can provide researchers with a better understanding of the use of this species in the animal feed industry.

Production of ß-glucanase and protease from Bacillus velezensis strain isolated from the manure of piglets.

In this study, a strain producing ß-glucanase and protease, identified as Bacillus velezensis Y1, was isolated from the manure of piglet. We attempted to produce ß-glucanase and protease after optimization of various process parameters with the submerged fermentation. The effects of each factor on producing ß-glucanase and protease were as follows: temperature > time > pH > loaded liquid volume. The properties of the ß-glucanase showed that the most suitable reaction temperature was 65 °C and pH was 6.0. However for protease optimum reaction temperature was 50 °C, and pH was 6.0. The amplified PCR fragments of ß-glucanase and protease were 1434 bp containing an open reading frame of 1413 bp encoding a protein with 444 amino acids and 1752 bp containing an open reading frame of 1521 bp encoding a protein with 506 amino acids, respectively. So, the study demonstrated a viable approach of using newly identified B. velezensis Y1 strain for the maximum yield of two industrially important enzymes.

Effect of Bacillus velezensis to substitute in-feed antibiotics on the production, blood biochemistry and egg quality indices of laying hens.

BACKGROUND: The excessive use of antibiotics in the livestock feed industry caused inevitable side effects of microbial resistance. Besides this residual antibiotics in animal-derived foodstuff imposed serious health problems for humans. So this study aimed to investigate the potential use of Bacillus velezensis to substitute antibiotics for poultry production. A total of 468, 49-week-old Hy-Line Brown chickens, were randomly divided into four groups the control group (regular diet), experiment group I (0.1% B. veleznesis), experiment group II (0.2% B. veleznesis), and antibiotic group (50 mg/kg flavomycin), with three replicates per group and trial period consisted on 42 days. RESULTS: The results showed that, compared with the control group, the average egg production rate and daily feed intake of experimental groups I and II increased significantly (P < 0.05), while the average egg weight was increased in experimental group II as compared to (I) (P < 0.01). The feed conversion ratio was decreased (P > 0.05) in group (II) Egg quality parameters such as yolk weight of the experimental group II was increased, but that of the antibiotic group and experiment group I was decreased, neither significant (P > 0.05). Moreover, the eggshell strength, yolk color, albumen height, and Haugh unit were significantly increased (P < 0.05). Compared with the control group, probiotic groups can increase the progesterone and motilin (P > 0.05) but decrease the secretin and cholecystokinin in the blood plasma (P > 0.05). CONCLUSIONS: This study suggested that B. velezensis can substitute in-feed-antibiotics and improved most of the study parameters significantly. Which suggested that B. velezensis has potential future application value to replace the feed antibiotics.

Characteristics and Application of a Novel Species of Bacillus: Bacillus velezensis.

Bacillus velezensis has been investigated and applied more and more widely recently because it can inhibit fungi and bacteria and become a potential biocontrol agent. In order to provide more clear and comprehensive understanding of B. velezensis for researchers, we collected the recent relevant articles systematically and reviewed the discovery and taxonomy, secondary metabolites, characteristics and application, gene function, and molecular research of B. velezensis. This review will give some direction to the research and application of this strain for the future.

The optimization of fermentation conditions for producing cellulase of Bacillus amyloliquefaciens and its application to goose feed.

The proper culture conditions for producing cellulase of Bacillus amyloliquefaciens S1, isolated from the cecum of goose was optimized by single-factor experiment combined with orthogonal test. The properties of the cellulase were investigated by DNS method. The appropriate doses of B. amyloliquefaciens S1 were obtained by adding them to goose feed. It indicated that the suitable culture conditions of producing cellulase were the culture temperature of 37°C, the initial pH of 7.0, the incubation time of 72 h and the loaded liquid volume of 75 ml per 250 ml. The effects of each factor on producing cellulase by B. amyloliquefaciens S1 were as follows: initial pH > incubation time = culture temperature > loaded liquid volume. The optimum reaction temperature and pH were 50°C and 7.0, respectively. This enzyme is a kind of neutral cellulase that possesses resistance to heat and acidity. It showed high activity to absorbent cotton, soya bean meal and filter paper. By adding different doses of B. amyloliquefaciens S1 to the goose feed, it was found that the egg production, average egg weight, fertilization rate and the hatching rate were promoted both in experiment 1 (1.5 g kg-1) and experiment 2 (3 g kg-1). Also the difference of egg production, fertilization rate and hatching rate between experiment 1 and control group was obvious (p < 0.05), and the average egg weight was significantly increased in experiment 2 (p < 0.05).

Gene Cloning and Expression of Cellulase of Bacillus amyloliquefaciens Isolated from the Cecum of Goose.

A kind of bacteria secreting cellulase and showing probiotic attributes was isolated from the cecum of goose and identified as Bacillus amyloliquefaciens by analysis of 16S rRNA gene sequence and named as B. amyloliquefaciens S1. In vitro assays, the enzymatic activity of the strain was determined by the reducing-sugar method, and the proper culture conditions of producing cellulase and some properties of the cellulase were investigated. The cultural mixture of the bacteria had a high cellulase activity of 1.25 U/mL. In order to improve the utilization rate of the cellulase, some properties of the cellulase were studied. The best reaction pH of the enzymes was 7.0 and the optimum reaction temperature was 60°C. The enzyme was a kind of neutral cellulase that possessing strong resistance against heat and acidity. It showed high activity to absorbent cotton, soybean meal, and filter paper. Meanwhile, a gene encoding a kind of cellulase was cloned and prokaryotic expressed in Escherichia coli. The gene had 1500 bp in length, encoding a protein of 55 kDa, which was confirmed by SDS-PAGE and Western blotting. This study explored the possibility of degrading ability of bacteria with its probiotic attributes to enhance digestibility of the feed and gut health of animal. It also provided some basis for its further functional analysis and practical application as a microbial preparation for the breeding.

The optimization of fermentation conditions and enzyme properties of Stenotrophomonas maltophilia for protease production.

Intestinal bacteria play a significant physiological role in silkworms. Proteases secreted by intestinal microbes can promote the digestion of the nutrient by Bombyx mori and the absorption of mulberry leaves. Intestinal bacteria from Jingsong × Haoyue in the fourth larvae were isolated and purified to obtain high activity protease-producing bacteria. The morphology of the identified bacterial colony was examined by microscopy combined with the 16S rDNA method. The results showed that this bacterium was Gram negative and that it belonged to Stenotrophomonas maltophilia, which produces the proteases. To improve the utilization rate of these proteases, we studied the proper culture conditions for producing proteases, and we further studied the properties of the proteases that were produced. The results showed that the optimal enzyme-producing conditions were as follows: pH of 7.0, culture temperature of 35 °C, incubation time of 36 H, and outfit fluid amount of 60 mL per 100 mL. Meanwhile, the properties of the preliminary enzyme purification indicated that the best pH of the enzymes was 9.0 and the optimal reaction temperature was 50 °C. The enzymes are alkaline proteases that show satisfactory stability at 30 °C and pH 9.0. Consequently, it is suitable for the proteases secreted by S. maltophilia to play a bioactive role in the silkworm gut.

Molecular characterization of swine leukocyte antigen class I genes by sequence-based and PCR-SSP method in Guizhou minipigs.

The highly polymorphic swine leucocyte antigen (SLA) genes play an important role in swine immune responses to infectious diseases, vaccines and production performance. The pig resource with well defined SLA genes is useful for xenotransplantation and immunological studies. In this study, we have characterized three SLA class I genes (SLA-1, SLA-3, SLA-2) of 22 founder Guizhou minipigs using sequence-based typing method. Thirteen alleles were detected in this population, compared with the SLA allele sequences in GenBank, 11 of 13 SLA class I alleles were novel in Guizhou minipigs. There are four SLA I haplotypes, none of them previously reported in other pigs. Based on these alleles sequences information, we developed a simple method implemented to SLA-typing for unknown offsprings of Guizhou minipigs, relying on designed 13 sequence specific primers that could discriminate each one among which located in each locus using PCR in a SLA typing assay. According the combination methods of sequence-based typing and PCR-SSP, we were able to rapidly conduct SLA typing for Guizhou breeding stock and identify four SLA haplotypes present in the herd. This resource population of SLA-defined Guizhou minipigs will be useful as animal models for xenotransplantation and further immunological research.