Preparation method and application of gamma-PGA-rich feed additive for broiler chickens

Abstract

The invention relates to a preparation method and application of a gamma-PGA-rich feed additive for broiler chickens, and the technical scheme for solving the problems is as follows: screening bacillus licheniformis DY-0089 for high yield of gamma-PGA from fermented foods such as fermented soya beans, fermented bean curds and natto, inoculating the bacillus licheniformis DY-0089 on a solid fermentation culture medium, fermenting for 24-96 hours at 28-45 DEG C, drying in a drying oven at 55-70 DEG C, and crushing to obtain the gamma-PGA-rich feed additive for broiler chickens. And preparing the feed additive rich in gamma-PGA. Research finds that when 1% of the gamma-PGA-rich feed additive is fed to broilers, the immune function of the body can be enhanced, cecum microbiota can be improved, cecum metabonomics can be adjusted, then the growth performance and feed conversion rate of the broilers are improved, serum biochemical indexes, oxidation resistance, slaughter performance, pectoral muscle nutritional ingredients and the like of the broilers are basically not affected, and the feed additive is suitable for large-scale production. And a theoretical basis is provided for development and application of the gamma-PGA-rich feed additive in animal breeding, especially broiler breeding.

Description

1. Technical Field

[0001] The present invention relates to the technical field of animal feed additives, in particular to a preparation method and application of a gamma-PGA-rich feed additive for broilers. 2. Background Technology

[0002] my country has completely banned the use of antibiotics in feed since January 1, 2020. This measure ensures the safety of farmed animal food and public health, but it also impacts animal health and growth performance, reduces feed conversion efficiency, and compounds the already significant shortage of feed resources. Therefore, identifying new, green, alternative antibiotic feed additives that promote animal health, improve growth performance, and feed conversion efficiency, while reducing feed costs, is a pressing bottleneck in the livestock and poultry industry.

[0003] γ-Polyglutamic acid (γ-PGA) is a polymer composed of L-glutamic acid and / or D-glutamic acid linked by γ-amide bonds. Produced primarily by Bacillus sp., it is a novel multifunctional biomaterial with properties such as metal chelation, water absorption, and moisturizing. Furthermore, its non-toxicity, biodegradability, and edibility make it widely used in various fields, including medicine, environmental protection, agriculture, and food. Studies have shown that adding γ-PGA to animal diets can improve the absorption of minerals such as calcium and phosphorus, thereby increasing eggshell strength and reducing fat deposition. Furthermore, the Bacillus sp. that produces γ-PGA rapidly develops into nutrient cells after entering the intestine, modulating intestinal microecological balance, enhancing immune function, and improving animal growth performance. Therefore, it is a widely used microecological agent in the livestock and poultry industry. Therefore, fermentation products rich in γ-PGA have broad application prospects as new feed additives in livestock and poultry farming. However, the dosage and efficacy of γ-PGA in broilers remain unclear, and no reports have been published to date. 3. Summary of the Invention

[0004] To address this bottleneck, the present invention aims to provide a method for preparing a γ-PGA-rich feed additive for broiler chickens and its application. This method offers the advantages of simple operation, low cost, and technical ease of implementation. It can promote broiler health, improve growth performance and feed conversion rate, contribute to cost reduction and efficiency improvement, and provide new insights into the development and application of γ-PGA.

[0005] One of the technical solutions provided by the present invention is a method for preparing a γ-PGA-rich feed additive for broilers, specifically comprising: picking Bacillus licheniformis DY-0089 from the inclined surface of a test tube, inoculating it into a seed culture medium, culturing it in a shaking incubator at 28-45°C and 150-220 r / min overnight, inoculating it into a γ-PGA solid fermentation medium at an inoculum size of 2-8%, fermenting it at 28-45°C for 24-96 hours, drying it in a drying oven at 55-70°C, and crushing it to obtain the γ-PGA-rich feed additive.

[0006] The γ-PGA-rich feed additive has a pH of 6.2-7.5, a γ-PGA content of 30-50 g / kg, and a viable bacterial count of 1.2×10 9 -6.5×10 10 cfu / g.

[0007] The Bacillus licheniformis DY-0089, which is classified as Bacillus licheniformis, was deposited with the General Microbiology Center of the China Culture Collection Administration on February 14, 2023, with the deposit number: CGMCC NO.26553, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The 16S rDNA gene sequence of this strain is shown in SEQ ID No. 1.

[0008] The seed culture medium is LB liquid culture medium, and the formula is 10 g / L of tryptone (Tryptone), 5 g / L of yeast extract (Yeast Extract) and 10 g / L of sodium chloride (NaCl).

[0009] The formula of the γ-PGA solid fermentation medium is as follows: 200-300g corn flour, 150-250g soybean meal, 25-70g bran, 20-50g sodium glutamate, and 700-1200mL water.

[0010] The second technical solution provided by the present invention is the use of the feed additive rich in γ-PGA in the preparation of broiler feed.

[0011] The third technical solution provided by the present invention is the use of the feed additive rich in γ-PGA in the preparation of animal feed.

[0012] Preferably, the amount of the γ-PGA-rich feed additive added to the broiler feed is 1%.

[0013] The beneficial technical effects of the present invention are:

[0014] 1) Adding 1% γ-PGA-rich feed additive to the diet can improve the cecal microbiome of broiler chickens, enhance the immune function of broiler chickens, regulate the cecal metabolic characteristics, and significantly improve the growth performance and feed conversion rate of broiler chickens, but has little effect on the serum biochemical indicators and antioxidant capacity, slaughter performance, and breast muscle nutritional components of broiler chickens.

[0015] 2) By studying the effects of adding γ-PGA-rich feed additives to feed on the growth performance, immune function, cecal microbial diversity and metabolomics of broiler chickens, a theoretical basis is provided for the development and application of γ-PGA-rich feed additives in animal husbandry, and new green alternative antibiotic feed additives are explored to promote health, improve growth performance and feed conversion rate, and reduce feed costs. IV. Description of the Figures

[0016] Figure 1 This figure shows the effect of the γ-PGA-rich feed additive of the present invention on the β diversity of the cecal microbiome of broiler chickens. In the figure, A is the PCoA analysis and B is the NMDS analysis.

[0017] Figure 2 The figure shows the effect of the γ-PGA-rich feed additive of the present invention on the composition of the cecal microbiome at the phylum level (A) and genus level (B) of broiler chickens.

[0018] Figure 3 This is a Venn diagram of the effect of the γ-PGA-rich feed additive of the present invention on the distribution of cecal microbial flora in broiler chickens (A phylum level, B family level).

[0019] Figure 4 This is a pie chart of the microbial composition at the genus level in the cecum of broiler chickens in different treatment groups of the present invention (A: CK; B: PGA1; C: PGA2; D: PGA3).

[0020] Figure 5 This is a heat map of the association between broiler chicken cecal microorganisms and ADG and F / D of the present invention. In the figure, FG in the horizontal axis represents F / G.

[0021] Figure 6 This is a scatter plot of the PCA model scores of cecal metabolites in broiler chickens in the PGA2 group and CK group of the present invention.

[0022] Figure 7 Figure 2 is the OPLS-DA score graph (A) and permutation test graph (B) of cecal metabolites of broiler chickens in the PGA2 group and CK group of the present invention.

[0023] Figure 8 This is the volcano plot of differential metabolites in the cecum of broiler chickens in the PGA2 group and CK group of the present invention.

[0024] Figure 9This is a KEGG differential metabolic pathway bubble diagram of the present invention. In the figure, A is the differential metabolic pathway between the PGA2 group and the CK group, B is the metabolic pathway upregulated in the PGA2 group compared with the CK group, and C is the metabolic pathway downregulated in the PGA2 group compared with the CK group. The horizontal axis is the enrichment rate, and the vertical axis is the KEGG metabolic pathway. The size of the bubble indicates the amount of enriched metabolites in the metabolic pathway, and the color of the bubble indicates the size of the significant P value of different enrichment. V. Specific Implementation Methods

[0025] The specific implementation of the present invention is described in detail below with reference to the embodiments and drawings.

[0026] Example 1

[0027] A method for preparing a γ-PGA-rich feed additive for broilers comprises the following steps: picking Bacillus licheniformis DY-0089 from the inclined surface of a test tube, inoculating it into a seed culture medium, culturing it in a shaking incubator at 28°C and 150 rpm overnight, inoculating it into a γ-PGA solid fermentation medium at a 2% inoculum amount, fermenting it at 28°C for 24 hours, drying it at 55°C, and crushing it to obtain the γ-PGA-rich feed additive.

[0028] The seed culture medium is LB liquid culture medium, and the formula is 10 g / L of tryptone (Tryptone), 5 g / L of yeast extract (Yeast Extract) and 10 g / L of sodium chloride (NaCl).

[0029] The γ-PGA solid fermentation medium formula is as follows: 200g corn flour, 150g soybean meal, 25g bran, 20g sodium glutamate, and 700mL water.

[0030] Example 2

[0031] A method for preparing a feed additive rich in γ-PGA for broiler feed comprises the following steps: picking Bacillus licheniformis DY-0089 from the inclined surface of a test tube, inoculating it into a seed culture medium, culturing it in a shaking incubator at 37°C and 180 rpm overnight, inoculating it into a γ-PGA solid fermentation medium at a 4% inoculum amount, fermenting it at 35°C for 36 hours, drying it at 65°C, and crushing it to obtain the feed additive rich in γ-PGA.

[0032] The seed culture medium is LB liquid culture medium, and the formula is 10 g / L of tryptone (Tryptone), 5 g / L of yeast extract (Yeast Extract) and 10 g / L of sodium chloride (NaCl).

[0033] The γ-PGA solid fermentation medium formula is as follows: 250g corn flour, 200g soybean meal, 47.5g bran, 35g sodium glutamate, and 950mL water.

[0034] Example 3

[0035] In a specific implementation of the present invention, a preparation method of a feed additive rich in γ-PGA for broiler feed comprises the following steps: picking Bacillus licheniformis DY-0089 from the inclined surface of a test tube, inoculating it into a seed culture medium, culturing it in a shaking incubator at 45°C and 220 rpm overnight, inoculating it into a γ-PGA solid fermentation medium at an inoculum rate of 8%, fermenting it at 45°C for 96 hours, drying it in a drying oven at 70°C, and crushing it to obtain the feed additive rich in γ-PGA.

[0036] The seed culture medium is LB liquid culture medium, and the formula is 10 g / L of tryptone (Tryptone), 5 g / L of yeast extract (Yeast Extract) and 10 g / L of sodium chloride (NaCl).

[0037] The γ-PGA solid fermentation medium formula is as follows: 300g corn flour, 250g soybean meal, 70g bran, 50g sodium glutamate, and 1200mL water.

[0038] The DY-0089 of the present invention is a γ-PGA-producing strain obtained by screening in the laboratory in October 2022. The specific screening method is as follows: 1.0 g of fermented foods such as fermented black beans, fermented bean curd, and natto sold on the market were selected, and 1.0 g of each was added to 100 mL of sterilized water, shaken, and boiled in a boiling water bath for 5 minutes. After standing for a while, 100 μL of the upper turbid liquid was taken and 900 μL of sterilized water was added for 10 minutes. -1 , diluted to 10 -5 ; Take 100uL of the diluted solution with different dilutions and add it to the LB separation plate, spread it evenly with a spreading rod, invert and culture at 37℃ for 24 hours, pick out the single colony with stringing, inoculate it into a triangular flask (10mL / 50mL) containing γ-PGA liquid screening medium, ferment it at 37℃ for 48h, measure the viscosity of the fermentation liquid with a digital viscometer, and screen the strain with high viscosity of the fermentation liquid; transfer the screened strain to a triangular flask (100mL / 500mL) containing γ-PGA liquid re-screening medium and ferment it at 37℃ for 48h The γ-PGA production was determined by the CTAB method and rescreened, and finally a strain DY-0089 with the highest γ-PGA production was obtained. After fermentation process optimization, the formula of the γ-PGA solid fermentation medium was obtained: 200-300g corn flour, 150-250g soybean meal, 25-70g bran, 20-50g sodium glutamate, 700-1200mL water, inoculation amount 2-8%, fermentation temperature 28-45℃, fermentation time 24-96h, and after drying and crushing at 55-70℃, the feed product had a pH of 6.2-7.5, a γ-PGA content of 30-50g / kg, and a viable bacterial count of 1.2×10 9 -6.5×10 10cfu / g.

[0039] The genome of strain DY-0089 was extracted, and 16S rDNA was amplified using bacterial universal primers 27F and 1492R. The amplified product was sent to Shenzhen BGI Genomics Co., Ltd. for sequencing. The measured 16S rDNA full gene sequence of strain DY-0089 was compared with the sequence in GenBank. It was found that the 16S rDNA full gene sequence of strain DY-0089 was 99.6% identical to the 16S rDNA sequence of Bacillus licheniformis published online, and 99% identical to Bacillus subtilis and Bacillus amyloliquefaciens. Combined with the results of physiological and biochemical identification, strain DY-0089 was confirmed to be Bacillus licheniformis. The strain was deposited with the China General Microbiological Culture Collection on February 14, 2023, with the deposit number CGMCC NO.26553.

[0040] The results of 16S rDNA full gene sequencing of strain DY-0089 are as follows:

[0041]

[0042] In this study, 360 healthy, 50-day-old "Liangfenghua" broiler chickens (half male and half female) of uniform weight were randomly divided into four groups, each containing three replicates and 30 chickens per replicate. The CK group was fed a basal diet, while the treated groups supplemented with 0.5%, 1%, and 2% γ-PGA-rich feed additives (PGA1, PGA2, and PGA3, respectively) to partially replace the basal diet. The trial lasted 49 days. The results showed that: 1) the average daily gain (ADG) of broilers in PGA1, PGA2 and PGA3 groups increased by 7.50%, 11.88% and 6.04% respectively compared with the CK group, and the feed consumption / gain ratio (F / G) decreased by 7.27%, 11.21% and 5.76% respectively compared with the CK group, among which the PGA1 and PGA2 groups showed significant differences from the CK group; 2) compared with the CK group, the relative abundance of probiotics such as unclassified_o__Ruminococcaceae, Olsenella, unclassified_o__Bacteroidales, NK4A214_group, Phascolarctobacterium, unclassified_f__Tannerellaceae, Christensenellaceae_R-7_group, Lachnoclostridium, norank_f__Oscillospiraceae and unclassified_f__Lachnospiraceae in the cecum of broilers in PGA2 group increased significantly. The relative abundance of norank_f__Oscillospiraceae and unclassified_f__Lachnospiraceae were significantly positively correlated with the growth performance of broiler chickens (P<0.05), while Bacteroides, norank_o__Clostridia_UCG-014, Shuttleworthia, Lactobacillus and Barnesiella were negatively correlated with the growth performance of broiler chickens. The relative abundance of bacterial genera decreased significantly; 3) Serum immunoglobulins in the three treatment groups The concentrations of white IgA and IgM and the levels of cytokines IL-6 and LCN2 were significantly higher than those in the CK group (P<0.05), and the level of cytokine IL-10 was significantly lower than that in the CK group (P<0.05); 4) Compared with the CK group, the metabolic pathways of amino sugar and nucleotide sugar metabolism, nucleotide sugar biosynthesis, arachidonic acid metabolism, O-antigen nucleotide sugar biosynthesis, and bile secretion in the cecum of broiler chickens in the PGA2 group were upregulated, while the metabolic pathways of steroid biosynthesis, isoflavone biosynthesis, antibiotic (neomycin, kanamycin and gentamicin) biosynthesis, and steroid hormone biosynthesis were downregulated.It can be seen that adding 1% γ-PGA-rich feed additives to the diet can improve the cecal microbiome of broiler chickens, regulate the cecal metabolic characteristics, enhance the immune function of broiler chickens, and significantly improve the growth performance and feed conversion rate of broiler chickens, but basically has no effect on the serum biochemical indicators and antioxidant capacity, slaughter performance, and breast muscle nutritional components of broiler chickens. The specific experimental data are as follows:

[0043] 1 Materials and Methods

[0044] 1.1 Preparation of γ-PGA-rich feed additive

[0045] 1.1.1 Strain The γ-PGA producing strain is Bacillus licheniformis DY-0089 screened in the laboratory.

[0046] 1.1.2 Culture medium

[0047] The seed culture medium was LB liquid medium.

[0048] γ-PGA liquid rescreening medium: glucose 30 g / L, yeast powder 8 g / L, sodium glutamate 30 g / L, magnesium sulfate 0.25 g / L, potassium hydrogen phosphate 2 g / L, pH 7.2.

[0049] The solid fermentation of γ-PGA adopts the tray fermentation method. The loading amount and culture medium formula of each tray are: corn flour 200-300g, soybean meal 150-250g, bran 25-70g, sodium glutamate 20-50g, and water 700-1200mL.

[0050] 1.1.3 Preparation method of feed additive rich in γ-PGA

[0051] Bacillus licheniformis DY-0089 was picked from the slant of the test tube, inoculated into a seed culture medium, and cultured overnight in a shaking incubator at 28-45°C and 150-220 r / min. The inoculum size was then inoculated onto a γ-PGA solid fermentation medium in a tray at a rate of 2-8%. The culture was carried out at 28-45°C for 24-96 hours, dried at 55-70°C, and crushed to obtain a feed additive rich in γ-PGA. The results showed that the pH was 6.2-7.5, the γ-PGA content was 30-50 g / kg, and the number of viable Bacillus licheniformis cells was 1.2×10 9 -6.5×10 10 cfu / g.

[0052] 1.2 Experimental animals and experimental design

[0053] The experimental broilers were "Liangfenghua" broilers provided by Wugu Farm in Weishi County. A total of 360 healthy, healthy, and uniformly weighted "Liangfenghua" broilers (half male and half female) were randomly divided into four groups, each containing three replicates of 30 birds. The CK group was fed a basal diet, while the treatment groups, PGA1, PGA2, and PGA3, had their diets supplemented with 0.5%, 1%, and 2% of a γ-PGA-rich feed additive, respectively. The experimental period lasted 49 days. During the experimental period, the diets were formulated according to the nutrient requirements of yellow-feathered broilers (NY / T3645-2020). The nutritional levels of the diets were essentially the same across all groups. The composition and nutritional levels of the experimental diets are shown in Table 1.

[0054] Table 1 Composition and nutritional level of experimental diet (air-dried basis) %

[0055]

[0056] Note: ① Each kg of premix contains: Vitamin A 8 000IU, Vitamin B1 4mg, Vitamin B2 3.6mg, Vitamin B5 40mg, Vitamin B6 3.5mg, Vitamin B 12 0.02mg, Vitamin D3 3,000IU, Vitamin E 20IU, Vitamin K3 2mg, Biotin 0.16mg, Folic Acid 0.8mg, D-Pantothenic Acid 10mg, Niacin 15mg, Copper 8.0mg, Iron 80mg, Manganese 80mg, Zinc 70mg, Iodine 0.50mg, Selenium 0.35mg. ② Metabolizable energy is calculated; the remaining values ​​are measured.

[0057] 1.3 Experimental Animal Husbandry and Management

[0058] The experimental broiler chickens were housed in a semi-open, three-tiered cage system, with 10 birds per cage (198 cm × 38 cm × 38 cm). Natural ventilation combined with negative pressure ventilation was used. During the experimental period, the broiler houses were naturally lit, with a temperature of 20-25°C and humidity consistent with ambient conditions. During the experimental period, the birds had free access to feed and water. House disinfection and routine immunizations were performed according to standard farm practices.

[0059] 1.4 Detection indicators and methods

[0060] 1.4.1 Nutritional level of feed

[0061] A certain amount of basal feed was mixed with 0.5%, 1% and 2% of γ-PGA-rich feed additive to prepare broiler diets for PGA1, PGA2 and PGA3 groups, respectively. The basal feed (CK group diet), PGA1, PGA2 and PGA3 group diets were taken and their nutritional levels were determined. The crude protein (CP) content was determined according to the "Determination of crude protein in feeds - Kjeldahl method" (GB / T 6432-2018), the crude fiber (CF) content was determined according to the "Determination of crude fiber content in feeds" (GBT 6434-2022), the total phosphorus (TP) content was determined according to the "Determination of phosphorus in feeds" (GB / T 6437-2018), and the calcium (Ca) content was determined according to the "Determination of calcium in feeds" (GB / T 6436-2018).

[0062] 1.4.2 Growth performance

[0063] Each replicate group was weighed on an empty stomach at the beginning and end of the experiment. During the experiment, the feeding situation, initial weight, final weight, health status, and number of deaths of the broilers were recorded on a replicate basis. The average daily feed intake (ADFI), ADG, and F / G of the broilers in each group were calculated using the following formula:

[0064] ADFI (g / d) = total feed consumption / (number of pieces × number of days)

[0065] ADG (g / d) = total weight gain / (number of birds × number of days)

[0066] F / G = total feed consumption / total weight gain.

[0067] 1.4.3 Slaughter performance

[0068] After the end of the experiment, the chickens were fasted for 12 hours. One male and one female broiler were randomly selected from each replicate. 5 mL of blood was collected from the subwing vein and centrifuged at 3000 r / min for 15 minutes to separate the serum. The serum was collected and packaged and stored at -20°C for later use. The chickens were then sacrificed by bleeding from the jugular vein. The carcass weight, half-eviscerated weight, full-eviscerated weight, breast muscle weight, and leg muscle weight were weighed. The slaughter rate, half-eviscerated rate, full-eviscerated rate, breast muscle rate, and leg muscle rate were calculated using the following formula:

[0069] Slaughter rate (%) = carcass weight / live weight × 100

[0070] Eviscerated rate (%) = Eviscerated weight / live weight × 100

[0071] Eviscerated rate (%) = eviscerated weight / live weight × 100

[0072] Chest muscle ratio (%) = weight of chest muscles on both sides / total eviscerated weight × 100

[0073] Leg muscle rate (%) = weight of both leg muscles / total eviscerated weight × 100

[0074] 1.4.4 Serum index determination

[0075] Serum antioxidant indices (glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA)) and serum biochemical indices (blood glucose (GLU), total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), triglycerides (TG), creatinine (CRE), albumin (ALB), total protein (TP), urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT)) were all measured using kits produced by Nanjing Jiancheng Bioengineering Research Institute. Serum immune indicators: immunoglobulin M (IgM), immunoglobulin G (IgG), immunoglobulin A (IgA), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor (TNF-α), and total lipocalin-2 (LCN2) were determined by Qingdao Stand Standard Testing Co., Ltd.

[0076] 1.4.5 Determination of nutritional components of breast muscle

[0077] After the breast muscles were weighed, 50 g of each sample was taken and sent to the Institute of Agricultural Product Quality and Safety of Henan Academy of Agricultural Sciences to determine the crude protein, crude fat and amino acid contents.

[0078] 1.4.6 Diversity of cecal microbiota and non-targeted metabolomics analysis

[0079] About 3 g of cecal contents were collected in cryovials, rapidly frozen in liquid nitrogen, and sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. for microbial diversity and non-targeted metabolomics analysis.

[0080] 1.5 Data Processing

[0081] Data Statistics and Analysis: Experimental data were organized using Excel 2003, and the results were analyzed and compared using SPSS 20.0 and Duncan's method. Microbial diversity was analyzed using Uparse software for OTU clustering and bioinformatics analysis based on valid sequences at 97% similarity. Bacterial diversity indices based on OTU classification were calculated using Mothur and Qiime software. Metabolomics data were analyzed using orthogonal partial least squares discriminant analysis (OPLS-DA) to identify differential metabolites between groups. Kyoto Encyclopedia of Genomics (KEGG) IDs were then enriched to identify enriched metabolic pathways.

[0082] 2 Results and Analysis

[0083] 2.1 Effects of γ-PGA-rich feed additives on broiler growth performance

[0084] As shown in Table 2, the γ-PGA-rich feed additive had little effect on the ADFI of broiler chickens. Compared with the CK group, the ADG of broiler chickens in the PGA1 and PGA2 treatment groups increased significantly (P < 0.05), and the F / G ratio decreased significantly (P < 0.05). The ADG and F / G ratio of broiler chickens in the PGA3 group also increased and decreased, respectively, but the differences from the CK group were not significant (P > 0.05). Compared with the CK group, the ADG of the three treatment groups increased by 7.50%, 11.88%, and 6.04%, respectively, and the F / G ratio decreased by 7.27%, 11.21%, and 5.76%, respectively. This indicates that the addition of a γ-PGA-rich feed additive to the diet can improve the growth performance and feed conversion rate of broiler chickens.

[0085] Table 2 Effects of γ-PGA-rich feed additives on broiler growth performance

[0086]

[0087] Note: Data in the same row without letters or with the same letters indicate no significant difference (P>0.05), while data with different lowercase letters indicate significant difference (P<0.05).

[0088] 2.2 Effects of γ-PGA-rich feed additives on broiler slaughter performance and breast muscle nutritional composition

[0089] As shown in Table 3, there were no significant differences in the slaughter rate, semi-eviscerated rate, fully eviscerated rate, leg muscle rate and breast muscle rate between the three treatment groups and the CK group (P>0.05), indicating that feeding feed additives rich in γ-PGA did not affect the slaughter performance of broilers.

[0090] Table 3 Effects of γ-PGA-rich feed additives on broiler slaughter performance (%)

[0091]

[0092] As shown in Table 4, there were no significant differences in crude protein, crude fat and amino acid contents between the broiler breast muscles in the three treatment groups and the CK group (P>0.05), indicating that feeding feed additives rich in γ-PGA did not affect the nutritional composition of broiler breast muscles.

[0093] Table 4 Effects of γ-PGA-rich feed additives on nutrient composition of broiler breast muscle %

[0094]

[0095] 2.3 Effects of γ-PGA-rich feed additives on broiler serum parameters

[0096] As shown in Table 5, after feeding the γ-PGA-rich feed additive, there were no significant differences in serum GSH-Px, SOD, T-AOC, and MDA between the three treatment groups and the CK group. Therefore, in this experiment, feeding the γ-PGA-rich feed additive had no significant effect (P>0.05) on the antioxidant capacity of broilers.

[0097] Table 5 Effects of γ-PGA-rich feed additives on antioxidant indicators in broiler chickens

[0098]

[0099] The effects of γ-PGA-rich feed additives on broiler serum biochemical indices are shown in Table 6. There were no significant differences (P>0.05) between the three treatment groups and the CK group in serum GLU, T-CHO, LDL-C, HDL-C, TG, CRE, ALB, BUN, AST, and ALT. Compared with the CK group, serum TP levels in the three treatment groups increased, with the PGA3 group showing an extremely significant (P<0.01) higher serum TP level than the CK group. This suggests that γ-PGA-rich feed additives have little effect on broiler serum biochemical indices.

[0100] Table 6 Effects of γ-PGA-rich feed additives on serum biochemical parameters of broiler chickens

[0101]

[0102] The effects of γ-PGA-enriched feed additives on broiler serum immune indices are shown in Table 7. Compared with the CK group, serum IgA, IgM, and LCN2 levels in the three treatment groups were significantly increased (P<0.05), and IL-10 levels were significantly decreased (P<0.05). The IgG content in the PGA1 group was extremely significantly higher than that in the CK group (P<0.01), but there was no significant difference between the PGA2 and PGA3 groups and the CK group (P>0.05). TNF-α levels in broilers in the PGA2 treatment group were significantly higher than those in the CK group (P<0.05), but there was no significant difference between the PGA1 and PGA3 groups and the CK group (P>0.05). IL-6 levels in broilers in the PGA2 and PGA3 groups were significantly higher than those in the CK group (P<0.05), and IL-6 levels in broilers in the PGA1 group were also higher than those in the CK group, but the differences were not significant (P>0.05). This suggests that γ-PGA-enriched feed additives can enhance the immune function of broilers.

[0103] Table 7 Effects of γ-PGA-rich feed additives on immune performance of broiler chickens

[0104]

[0105] 2.4 Effects of γ-PGA-enriched feed additives on cecal microbiota in broiler chickens

[0106] A total of 622 922 valid sequencing reads were obtained from 12 samples. Based on 97% species similarity, 5 363 OTUs were obtained from broiler samples with an average length of 416 bp, belonging to 16 phyla, 26 classes, 54 orders, 92 families, 212 genera, and 420 species.

[0107] 2.4.1 Effects of γ-PGA-enriched feed additives on the α-diversity of cecal microbiota in broiler chickens

[0108] Table 8 shows that the coverage rate of all four sample groups in this experiment was 99.6%, indicating that the sequencing depth was reasonable and that all species in the samples were essentially covered, reflecting the true situation. There were no significant differences in the Sobs, Shannon, Simpson, Ace, and Chao indices between the treatment groups (P>0.05). However, the OUTs, Sobs, Shannon, Ace, and Chao indices in the PAG2 group were higher than those in the CK group, while the Simpson index was lower than that in the CK group. This indicates that the richness, diversity, and evenness of the cecal microbiota in broiler chickens in the PAG2 group were higher than those in the CK group. Compared with the CK group, the richness and diversity of the cecal microbiota in the PAG1 group increased, but the evenness decreased. The richness, diversity, and evenness of the cecal microbiota in broiler chickens in the PGA3 group were all lower than those in the CK group. This suggests that the addition of a 1% γ-PGA-enriched feed additive to the diet can improve the diversity and evenness of the cecal microbiota in broiler chickens.

[0109] Table 8 Effects of γ-PGA-rich feed additives on α-diversity of cecal microbiota in broiler chickens

[0110]

[0111] 2.4.2 Effects of γ-PGA-enriched additives on β-diversity in cecum of broiler chickens

[0112] Depend on Figure 1 A shows that the contribution rate of the first principal component of broiler β diversity is 24.38%, and the contribution rate of the second principal component is 15.18%. Figure 1 A and Figure 1 B, The samples between PGA2 and CK and PGA3 groups were dispersed far apart, indicating that the microbial community structure was quite different. However, the samples between PGA2 and PGA1 groups were dispersed relatively closely, and the community structure difference was relatively small. The samples between CK and PGA1 and PGA3 groups were dispersed closely, and the community structure difference was small.

[0113] 2.4.3 Effects of γ-PGA-enriched feed additives on the composition of cecal microbiota in broiler chickens

[0114] Depend on Figure 2 A showed that the top four phyla with high abundance in the cecum of broiler chickens in each group were Bacteroidetes, Firmicutes, Verrucomicrobia and Actinobacteria; compared with the CK group, the abundance of Bacteroidetes and Verrucomicrobia in the cecum of broiler chickens in the PGA1 group increased significantly, and the abundance of Firmicutes decreased significantly; the abundance of Bacteroidetes in the cecum of broiler chickens in the PGA2 group decreased significantly, and the abundance of Firmicutes and Actinobacteria increased significantly; there was no significant difference between the top four phyla of the cecal microbiota of broiler chickens in the PGA3 group and the CK group.

[0115] Depend on Figure 3 A shows that the bacteria detected in the cecum of each group of broiler chickens belong to 16 phyla, namely Bacteroidetes, Firmicutes, Verrucomicrobia, Actinobacteria, Campylobacterota, Cyanobacteria, Deferribacterota, Desulfobacterota, Elusimicrobiota, Patescibacteria, Proteobacteria, Synergistot, unclassified_k__norank_d__Bacteria, Fusobacteriota, Spirochaetota and WPS-2. Figure 3A and 3B show that the microorganisms in the cecum of the treatment groups PGA1 and PGA2 contained all 16 detected bacterial phyla; the cecum of the PGA1 and PGA2 groups contained one more Fusobacterium and 7 more families than the CK group, while the PGA3 group had two fewer phyla (Spiral bacteria and WPS-2) and 4 fewer families than the CK group. Therefore, the microbial diversity in the cecum of broiler chickens in the PGA1 and PGA2 treatment groups increased, but the diversity of the microbial community in the PGA3 treatment group decreased, which is consistent with the results of α diversity.

[0116] Figure 4 The pie chart shows the composition of bacteria with abundance >1% in the cecum of broiler chickens at the genus level in each treatment group. There were 19, 16, 28 and 8 bacterial genera with abundance >1% in the cecum of broiler chickens in the CK, PGA1, PGA2 and PGA3 groups, respectively, indicating that the diversity and uniformity of the cecal microbiota in the PGA2 group were significantly increased compared with the CK group, the diversity and uniformity in the PGA3 group were significantly decreased compared with the CK group, and the richness and diversity differences between the PGA1 group and the CK group were small, which was consistent with the results of α-diversity determination.

[0117] Table 9 Composition and function of cecal microbial communities at genus level in broiler chickens in different treatment groups %

[0118]

[0119]

[0120]

[0121] 2.4.4 Correlation analysis between cecal microbiota and growth performance of broiler chickens

[0122] Spearman correlation analysis between cecal microbiota and growth performance indicators (ADG and F / G) of broiler chickens is shown in Figure 5The abundance of norank_f__Oscillospiraceae and unclassified_f__Lachnospiraceae of the Firmicutes phylum was significantly positively correlated with ADG and significantly negatively correlated with F / G, that is, the abundance of the two genera was significantly positively correlated with the growth performance of broiler chickens; the relative abundance of strains such as unclassified_o__Ruminococcaceaes, Olsenella, NK4A214_group, Phascolarctobacterium, Lachnoclostridium, Christensenellaceae_R-7_group, unclassified_f__Tannerellaceae, and unclassified_o__Bacteroidales also had positive and negative correlation trends with ADG and F / G, respectively; the relative abundance of genera such as Bacteroides, norank_o__Clostridia_UCG-014, Shuttleworthia, Lactobacillus, and Barnesiella had negative and positive correlation trends with ADG and F / G, respectively.

[0123] 2.5 Effects of γ-PGA-enriched feed additives on cecal metabolomics in broiler chickens

[0124] 2.5.1 Principal Component Analysis

[0125] The principal component analysis results are as follows Figure 6 As shown, there is a clear separation between the PGA2 and CK group samples. The sample contribution rate on PC1 is 35.40%, and the contribution rate on PC2 is 23.60%, with all samples within the 95% confidence interval. Based on these results, further analysis of the metabolomics data can be performed.

[0126] In the present invention, the OPLS-DA model score ( Figure 7 A) The corresponding results are R2Y=0.994, Q2=0.835, indicating that 99.4% of the samples meet the established model. The permutation test of OPLS-DA ( Figure 7 B) It can be seen that the regression line of Q2Y fitting has a positive slope, and the blue points are mainly located above the red points, indicating that the OPLS-DA model has good stability and predictive ability, and can truly reflect objective facts. Therefore, it can be used for screening and analysis of differential metabolites.

[0127] 2.5.2 Differential marker screening

[0128] A total of 2779 metabolites were detected by non-targeted metabolomics (LC-MS / MS) of cecal contents of broiler chickens in the PGA2 and CK groups, of which 262 were significantly differentially expressed, of which 173 were significantly up-regulated and 89 were significantly down-regulated ( Figure 8 ).

[0129] VIP and P values ​​were obtained according to OPLS-DA, and differential metabolite screening was performed according to the criteria of P < 0.01 and VIP > 1.5. Eleven metabolites were screened out in the PGA2 group (7-benzylidenenalone, N-oleoylhistidine, 2-[3-(methoxymethyl)-1,2,4-oxadiazol-5-yl]-N-propan-2-ylazacyclopentane-1-carboxamide, prolyl-lysine, silamadol, (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H -indol-7-yl] amine, (r)-lisofrine, 4-(5-chloro-3-isopropyl-1H-pyrazol-4-yl)-N-(5-(4-(dimethylamino)piperidin-1-yl)pyridin-2-yl)pyrimidin-2-amine, N-cyclohexylidene-2-carbamoylcyclohexyl-1-aniline, N,N,O-tridesmethylvenlafaxine, 2-acetylamino-5-(4-cyclohexylpiperazine-1 -yl)benzoic acid) (Table 8), among which the expression levels of 7 metabolites (7-benzylidenalone, N-oleoylhistidine, 2-[3-(methoxymethyl)-1,2,4-oxadiazol-5-yl]-N-propan-2-ylazacyclopentane-1-carboxamide, prolyl-lysine, siramadol, (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine and (r)-lisofrin) in broilers of the three treatment groups (PGA1, PGA2 and PGA3) compared with those in the CK group all met the criteria of P < 0.01 and VIP > 1.5 (Tables 10-12), indicating that these 7 metabolites can be used as differential markers, and the expression levels of the 7 differential markers in the three treatment groups were all upregulated compared with those in the CK group.

[0130] Table 10 Significantly different metabolites in the cecum of broiler chickens in the PGA2 group and the CK group

[0131]

[0132]

[0133] Table 11 Significantly different metabolites in the cecum of broiler chickens in the PGA1 group and the CK group

[0134]

[0135] Table 12 Significantly different metabolites in the cecum of broiler chickens in the PGA3 group and the CK group

[0136]

[0137]

[0138] 2.5.3 KEGG metabolic pathway enrichment analysis

[0139] KEGG metabolic pathway enrichment analysis showed that ( Figure 9 ), the differential metabolites in the cecum of broiler chickens in the PGA2 group and the CK group were mainly enriched in pathways such as steroid hormone biosynthesis, amino sugar and nucleotide sugar metabolism, steroid biosynthesis, isoflavone biosynthesis, plant hormone signal transduction, and arachidonic acid metabolism ( Figure 9 A), among which the metabolic pathways significantly upregulated mainly include amino sugar and nucleotide sugar metabolism, nucleotide sugar biosynthesis, arachidonic acid metabolism, O-antigen nucleotide sugar biosynthesis, bile secretion, etc. ( Figure 9 B), the metabolic pathways that were significantly downregulated mainly included steroid biosynthesis, isoflavone biosynthesis, antibiotic (neomycin, kanamycin and gentamicin) biosynthesis, steroid hormone biosynthesis, etc. ( Figure 9 C).

[0140] 3 Conclusion

[0141] 3.1 Effects of γ-PGA-rich feed additives on broiler growth performance

[0142] Adding 1% of γ-PGA-rich feed additives to the feed can significantly increase the ADG of broilers and reduce F / G, indicating that adding 1% of γ-PGA-rich feed additives to broiler feed can improve the growth performance and feed conversion efficiency of broilers and reduce feed costs.

[0143] 3.2 Effects of γ-PGA-rich feed additives on broiler slaughter performance

[0144] In this experiment, there was no significant difference in the slaughter rate, semi-eviscerated rate, fully eviscerated rate, leg muscle rate, and breast muscle rate of broiler chickens among each group, and all met the standards for good meat performance, indicating that feed additives rich in γ-PGA have no adverse effects on the slaughter performance of broiler chickens.

[0145] 3.3 Effects of γ-PGA-rich feed additives on broiler serum parameters

[0146] Feed additives rich in γ-PGA can regulate the immune function of broilers and are potential immunomodulators, but they basically have no effect on the serum biochemical indicators and antioxidant capacity of broilers.

[0147] 3.4 Effects of γ-PGA-enriched feed additives on cecal microbiota in broiler chickens

[0148] Low doses (0.5%) of γ-PGA-rich feed additives have little effect on the diversity of cecal microbiota in broiler chickens, but the growth performance of broiler chickens is significantly improved. As the dose of feed additives increases (up to 1%), the diversity and uniformity of cecal microbiota increase, and the growth performance of broiler chickens further improves. However, as the dose of γ-PGA feed additives continues to increase (to 2%), the diversity and uniformity of cecal microbiota decrease rapidly, and the growth performance of broiler chickens also decreases compared to 1%. This shows that there may be a threshold for the dose of γ-PGA-rich feed additives fed to broiler chickens. When this threshold is exceeded, excessive γ-PGA may inhibit the diversity of microbiota by changing the osmotic pressure of the intestinal environment, and may affect intestinal absorption by chelating too many minerals (such as calcium, zinc, etc.), thereby causing a decrease in broiler growth performance. In the present invention (Table 9 and Figure 5 ), compared with the CK group, the levels of unclassified_o__Ruminococcaceae, Olsenella, unclassified_o__Bacteroidales, NK4A214_group, Phascolarctobacterium, unclassified_f__Tannerellaceae, Christensenellaceae_R-7_group, Lachnoclostridium, norank_f__Oscillospiraceae, unclassified_f__Lachnospiraceae in the cecum of broiler chickens in the PGA2 group were significantly associated with the growth performance of broiler chickens. The relative abundance of probiotics that were positively correlated with broiler growth performance increased significantly, among which the abundance of norank_f__Oscillospiraceae and unclassified_f__Lachnospiraceae were significantly positively correlated with broiler growth performance; the relative abundance of bacterial genera such as Bacteroides, norank_o__Clostridia_UCG-014, Shuttleworthia, Lactobacillus and Barnesiella that were negatively correlated with broiler growth performance decreased significantly, indicating that feeding 1% γ-PGA-rich feed additives can increase the relative abundance of bacterial genera in the cecum that are positively correlated with growth performance, and reduce the relative abundance of bacterial genera that are negatively correlated with growth performance, thereby improving the cecal microbiome.

[0149] 3.5 Effects of γ-PGA-enriched feed additives on cecal metabolomics in broiler chickens

[0150] The experimental results showed that feed additives rich in γ-PGA can regulate the metabolic characteristics of broiler cecum. Compared with the CK group, the main metabolic pathways of the body, such as amino sugar and nucleotide sugar metabolism, nucleotide sugar biosynthesis, arachidonic acid metabolism, O-antigen nucleotide sugar biosynthesis, and bile secretion, were upregulated in the cecum of broiler chickens in the γ-PGA2 group. The secondary metabolic pathways of the body, such as steroid biosynthesis, isoflavone biosynthesis, antibiotic (neomycin, kanamycin and gentamicin) biosynthesis, and steroid hormone biosynthesis metabolism, were downregulated in the cecum of broiler chickens in the γ-PGA2 group compared with the CK group.

[0151] In the present invention, compared with the CK group, 7 differential markers with important biological functions (Table 10) in the γ-PGA2 group (7-benzylidenenalone, N-oleoylhistidine, 2-[3-(methoxymethyl)-1,2,4-oxadiazol-5-yl]-N-propyl-2-ylazacyclopentane-1-carboxamide, prolyl-lysine, silamadol, (tetrahydropyran-4-yl)-[2-phenyl-5-(1,1-dioxo-thiomorpholin-4-yl)methyl-1H-indol-7-yl]amine and (r)-lisofrin) were all upregulated, thus being beneficial to the growth of broilers and improving feed conversion rate. In summary, adding 1% γ-PGA-rich feed additive to the feed of the present invention can improve the cecal microbiome of broiler chickens, enhance immune function, regulate cecal metabolic characteristics, significantly increase the ADG of broiler chickens, reduce F / G, promote broiler growth, improve feed conversion rate, and help reduce costs and increase efficiency, but basically does not affect the slaughter performance, breast muscle nutritional quality, serum biochemical and antioxidant indicators of broiler chickens.

[0152] It should be pointed out that the above is only a preferred embodiment of the present invention and does not impose any form of limitation on the present invention. Any technician familiar with this profession can make changes or modify the technical content disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention, and all of them fall within the scope of protection of the present invention.

Claims

1. A method for preparing a feed additive rich in γ-PGA for broiler chickens, characterized in that: Bacillus licheniformis DY-0089 was picked from the slant of the test tube, inoculated into a seed culture medium, cultured overnight at 28-45°C and 150-220 r / min in a shaking incubator, inoculated into a γ-PGA solid fermentation medium at an inoculum size of 2-8%, fermented at 28-45°C for 24-96 hours, dried at 55-70°C, and crushed to obtain a feed additive rich in γ-PGA.

2. The method for preparing the γ-PGA-rich feed additive for broiler chickens according to claim 1, wherein: The γ-PGA-rich feed additive has a pH of 6.2-7.5, a γ-PGA content of 30-50 g / kg, and a viable bacterial count of 1.2×10 9 -6.5×10 10 cfu / g.

3. The method for preparing the γ-PGA-rich feed additive for broiler chickens according to claim 1, wherein: The Bacillus licheniformis DY-0089 is classified and named as Bacillus licheniformis ( Bacillus licheniformis ), which has been deposited in the General Microbiology Center of China Culture Collection Administration Committee with the deposit number: CGMCC NO. 26553. The 16S rDNA gene sequence of the strain is shown in SEQ ID No.

1.

4. The method for preparing the γ-PGA-rich feed additive for broilers according to claim 1, characterized in that: The seed culture medium is LB liquid culture medium, and the formula is 10 g / L tryptone, 5 g / L yeast extract and 10 g / L sodium chloride.

5. The method for preparing the γ-PGA-rich feed additive for broilers according to claim 1, characterized in that: The formula of the γ-PGA solid fermentation medium is as follows: 200-300g corn flour, 150-250g soybean meal, 25-70g bran, 20-50g sodium glutamate, and 700-1200mL water.

6. Use of the feed additive rich in γ-PGA prepared by the preparation method according to any one of claims 1 to 5 in preparing broiler feed.

7. Use of the γ-PGA-rich feed additive prepared by the preparation method according to any one of claims 1 to 5 in preparing animal feed.

8. The use of the γ-PGA-rich feed additive according to claim 6 in preparing broiler feed, characterized in that: The addition amount of the gamma-PGA-rich feed additive in the broiler feed is 1%.

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