Composite lactic acid bacteria metagen as well as preparation method and application thereof

Abstract

The invention discloses a compound lactic acid bacteria metagen as well as a preparation method and application thereof. Raw materials for preparing the compound lactic acid bacteria comprise lactic acid bacteria, glucose, palm meal, cellulase, alpha-galactosidase and xylanase. A culture process of lactobacillus acidophilus GBW1019 and lactobacillus plantarum GBW-LP001 is optimized through a technical means of composite solid fermentation, a co-culture with high bacterial quantity is obtained, a lactic acid bacteria metagen product with stable performance is further obtained through thermal inactivation, and when the lactic acid bacteria metagen product is applied to livestock and poultry animal feed addition, animal intake can be improved, the feed conversion ratio can be reduced, and the feed conversion rate can be increased. The preparation method of the product is simple, the period is short, the cost is low, and the product has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of microbial fermentation technology and livestock and poultry feed additives, and in particular to a composite lactic acid bacteria postbiotic and a preparation method and application thereof. Background Art

[0002] In recent years, probiotics and postbiotics have received increasing attention in the field of animal nutrition and health. Probiotics refer to beneficial microorganisms that can promote animal growth by maintaining the balance of intestinal flora, improving animal immunity and improving nutrient utilization. However, live probiotics are often adversely affected by environmental conditions (temperature, humidity, oxygen, acidity, etc.) during production, storage and transportation, resulting in reduced survival rate of live bacteria and unstable effects. To overcome these shortcomings, the concept of postbiotics came into being. Postbiotics refer to metabolites (such as organic acids, peptides, cell wall components, extracellular polysaccharides, etc.) produced by probiotics during their growth and metabolism, as well as the inactivated bacteria themselves. These components can exert beneficial effects on the host without the presence of live bacteria.

[0003] Traditional postbiotic preparation usually adopts liquid fermentation, followed by spray drying or freeze drying. However, in large-scale industrial production, liquid fermentation is costly, the process is difficult to control, energy consumption is high, and separation and purification operations are cumbersome.

[0004] Palm meal is a byproduct of palm oil extraction. It is rich in cellulose, hemicellulose, lignin and residual soluble sugars and is a potential fermentation substrate resource. By developing a composite solid fermentation technology to achieve efficient utilization of palm meal resources, it has a significant effect on promoting the production performance and health level of livestock and poultry. Summary of the invention

[0005] The object of the present invention is to provide a composite lactic acid bacteria postbiotic and its preparation method and application. The preparation method optimizes the lactic acid bacteria culture process to obtain a co-culture with a high bacterial count, and further obtains a lactic acid bacteria postbiotic product with stable performance through heat inactivation treatment. The composite lactic acid bacteria postbiotic can also improve the production performance and health level of animals.

[0006] In order to achieve the above-mentioned object of the invention, the present invention is implemented by the following technical solutions: The present invention provides a method for preparing a composite lactic acid bacteria postbiotic, which comprises the following steps: (1) Lactobacillus acidophilus and Lactobacillus plantarum are used as lactic acid bacteria raw materials, mixed evenly with glucose, palm meal, cellulase, α-galactosidase, xylanase and water for solid fermentation to obtain a lactic acid bacteria co-culture; (2) The lactic acid bacteria co-culture is subjected to heat treatment to inactivate the live bacteria and retain the beneficial components thereof, thereby obtaining a composite lactic acid bacteria postbiotic.

[0007] Furthermore, in step (1), the solid fermentation temperature is 25-35°C, the humidity is 60%-80%, and the time is 1-3 days.

[0008] Furthermore, in step (2), the heat treatment temperature is 70-90° C. and the time is 30-60 minutes.

[0009] Furthermore, the palm meal is used as a fermentation substrate, and its addition amount accounts for 50%-80% of the total weight of the raw materials.

[0010] Further, the added amount of the Lactobacillus acidophilus is 15 billion CFU-20 billion CFU, which is Lactobacillus acidophilus GBW1019 with a preservation number of CCTCC NO: M 20221403; the added amount of the Lactobacillus plantarum is 10 billion CFU-16 billion CFU, which is Lactobacillus plantarum GBW-LP001 with a preservation number of CGMCC No.22078.

[0011] Furthermore, per kilogram of fermentation substrate, the added amounts of the cellulase, α-galactosidase and xylanase are 60,000 U-100,000 U, 10,000 U-40,000 U and 20,000 U-50,000 U respectively.

[0012] Furthermore, the amount of glucose added is 40g-80g per kilogram of fermentation substrate.

[0013] The present invention also provides the composite lactic acid bacteria postbiotics prepared by the preparation method.

[0014] The present invention also provides the use of the composite lactic acid bacteria postbiotics in preparing livestock and poultry breeding feed.

[0015] Furthermore, the addition amount of the composite lactic acid bacteria postbiotics is 0.05%-0.2% of the total weight of livestock and poultry feed.

[0016] Furthermore, the poultry are broiler chickens and ducks.

[0017] Compared with the prior art, the present invention has the following advantages: The present invention optimizes the culture process of lactic acid bacteria through composite solid fermentation technology, obtains a co-culture with a high bacterial count, which contains rich metabolites and matrix nutrients in addition to bacteria, thereby comprehensively improving the utilization rate of lactic acid bacteria and the effect of the product.

[0018] The invention obtains a lactic acid bacteria postbiotic product with stable performance through heat inactivation treatment, thereby solving the problem of poor stability of traditional lactic acid bacteria preparations.

[0019] The composite lactic acid bacteria postbiotic product of the present invention is applied to livestock and poultry feed, which can significantly improve the production performance and health level of the animals and reduce breeding costs.

[0020] The preparation method of the invention is simple, has a short cycle, low cost, is easy for industrial production, and has broad application prospects. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with specific embodiments. However, the protection scope of the present invention is not limited to the following embodiments.

[0022] The depository of Lactobacillus acidophilus GBW1019 is China Center for Type Culture Collection (CCTCC); Address: Wuhan University, Wuhan, China; Deposit date: September 9, 2022; Lactobacillus acidophilus Lactobacillus acidophilus The deposit number of GBW1019 is CCTCC NO: M 20221403.

[0023] The depository of Lactobacillus plantarum GBW-LP001 is: China General Microbiology Center (CGMCC); Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit date: March 26, 2021; Lactobacillus plantarum Lactobacillus plantarum The deposit number is CGMCC No.22078.

[0024] Example 1: Preparation of compound lactic acid bacteria postbiotics 1. Raw material preparation Lactobacillus acidophilus GBW1019 and Lactobacillus plantarum GBW-LP001 were streaked on MRS solid medium (glucose 20 g / L, peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1 g / L, agar 15 g / L, pH 6.5) plates, and cultured anaerobically at 37 °C for 48 h. Single colonies were picked and transferred to liquid MRS medium (glucose 20 g / L, peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, dipotassium hydrogen phosphate 2 g / L, diammonium hydrogen citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1g / L, pH 6.5) for 24h, and the viable bacteria were counted by dilution plate method. 8 billion CFU of Lactobacillus acidophilus GBW1019 culture fluid and 6 billion CFU of Lactobacillus plantarum GBW-LP001 culture fluid were taken for use; Glucose: 50g; Palm meal: As fermentation substrate, the addition amount is 500g; Cellulase: added amount is 80,000 U; α-Galactosidase: added amount is 20,000 U; Xylanase: added amount is 30,000 U; Water: 250g.

[0025] 2. Solid fermentation After the above raw materials are fully mixed, they are placed in a sealed bag, the temperature is controlled at 32°C, and the ambient humidity is 70%, and static fermentation is carried out for 2 days.

[0026] After fermentation, a high-quality lactic acid bacteria co-culture was obtained, with a live bacterial count of 2×10 10 CFU / g and above.

[0027] 3. Heat inactivation The co-culture obtained by fermentation was dry-heat treated at 80°C for 40 minutes to inactivate the live bacteria and retain its beneficial components. After heat treatment, the product was crushed by a grinder through a 60-mesh sieve. The final product was in powder form, with no live bacteria detected, but retained beneficial substances such as metabolites and cell wall components of lactic acid bacteria.

[0028] Example 2: Application of compound lactic acid bacteria postbiotics in broiler breeding 1. Feed preparation 1000 kg of commercially available basic broiler feed was selected, and 1 kg of the composite lactic acid bacteria postbiotic product prepared in Example 1 was added thereto (ie, the added amount was 1% of the total weight of the feed).

[0029] After being fully mixed, broiler feed containing compound lactic acid bacteria postbiotics is obtained.

[0030] 2. Broiler breeding experiment: 320 one-day-old white-feathered broilers were pre-fed with mash for 7 days and then divided into groups as follows (Table 1), with 20 broilers in each pen.

[0031] Table 1: Evaluation of feeding scheme of compound lactic acid bacteria postbiotics in broiler feeding

[0032] The experimental phase was from the 8th day to the 29th day, during which the feed intake and weight gain of each group were recorded. On the morning of the 30th day, 4 birds were randomly selected from each pen, and venous blood was collected from under the wings. The upper serum was collected by centrifugation at 6000rpm for 10 minutes for the detection of serum antioxidant indicators and immunoglobulin content. The jejunal mucosa was dissected and collected for the detection of mucosal immunoglobulin content.

[0033] 3. Test results Growth performance: As shown in Table 2, compared with the control group, the daily weight gain of the group fed with compound lactic acid bacteria postbiotics increased by an average of 12%, and the daily feed intake increased by 6.5%. The feed-to-meat ratio was calculated on the 30th day. The feed-to-meat ratio of the experimental group was reduced by 0.08, or 6.5%, compared with the control group. The differences in various indicators were all at a significant level.

[0034] Table 2: Growth performance

[0035] Immune indicators: As shown in Table 3, compared with the control group, the serum IgG content of the group fed with compound lactic acid bacteria postbiotics increased by 33.3%, and the jejunal mucosal secretory immunoglobulin sIgA content increased by 35.2%, and the immune capacity was significantly enhanced.

[0036] Table 3: Immunity Levels

[0037] Antioxidant index: As shown in Table 4, compared with the control group, the serum The malondialdehyde (MDA) content decreased by 5.8%, the superoxide dismutase (SOD) activity increased by 1.9%, and the serum total antioxidant capacity (T-AOC) significantly increased by 14.7%, indicating that the overall antioxidant level of the experimental group was improved compared with the control group.

[0038] Table 4: Serum antioxidant indexes

[0039] Example 3: Application of compound lactic acid bacteria postbiotics in broiler breeding Palm meal was selected as the matrix, accounting for 70% of the total weight of the raw materials. 17 billion CFU of Lactobacillus acidophilus GBW1019 and 13 billion CFU of Lactobacillus plantarum GBW-LP001 were added to each kilogram of the matrix. At the same time, 80,000 U of cellulase, 20,000 U of α-galactosidase, 30,000 U of xylanase, 60 g of glucose were added, and water was added in proportion to about 70% of the humidity. The above mixture was solid fermented at 25°C for 2 days, and after the fermentation, the fermentation product was placed at 80°C for 40 minutes for heat treatment to obtain a composite lactic acid bacteria postbiotic.

[0040] The obtained postbiotic product was added to broiler feed at 0.5% of the total feed amount. After continuous feeding for 35 days, compared with the control group, the broiler feed intake increased by 5%, the feed-to-meat ratio decreased by 3%, the serum T-AOC increased by 10%, and the serum IgG and sIgA levels increased by more than 20%.

[0041] Example 4: Application of compound lactic acid bacteria postbiotics in meat duck breeding Similar to Example 3, the amount of palm meal added was 60% of the total weight of the raw material, the amount of Lactobacillus acidophilus GBW1019 added was 18 billion CFU / kg matrix, the amount of Lactobacillus plantarum GBW-LP001 added was 15 billion CFU / kg matrix, the amount of cellulase added was 90,000 U / kg matrix, α-galactosidase was 30,000 U / kg matrix, xylanase was 40,000 U / kg matrix, and glucose 80g / kg matrix was added, the humidity was controlled at 75%, solid fermentation was carried out at 30°C for 3 days, and then heat-treated at 90°C for 30 minutes to obtain a composite lactic acid bacteria postbiotic product. The composite lactic acid bacteria postbiotic product was mixed into the Cherry Valley duck feed at an addition amount of 0.15%. After continuous feeding for 28 days, the results showed that the granulation feed intake increased by about 6%, the feed-egg ratio decreased by 3%, and the serum T-AOC, IgG and sIgA levels were significantly improved.

[0042] Through the verification of the above examples, the preparation method of the composite lactic acid bacteria postbiotic provided by the present invention is not only simple and effective, but also the application effect of the product in livestock and poultry feed is significant and stable. The meat and poultry feeding experiment shows that the product of the present invention can significantly improve the production performance and health level of meat and poultry, especially the enhancement effect on immunity and antioxidant capacity is particularly prominent. Therefore, the present invention has broad application prospects and important practical value.

[0043] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for a person skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to replace some of the technical features therein by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions claimed to be protected by the present invention.

Claims

1. A method for preparing a composite lactic acid bacteria postbiotic, characterized in that: The following steps are involved: (1) Lactobacillus acidophilus and Lactobacillus plantarum are used as lactic acid bacteria raw materials, mixed evenly with glucose, palm meal, cellulase, α-galactosidase, xylanase and water for solid fermentation to obtain a lactic acid bacteria co-culture; (2) The lactic acid bacteria co-culture is subjected to heat treatment to inactivate the live bacteria and retain the beneficial components thereof, thereby obtaining a composite lactic acid bacteria postbiotic.

2. The preparation method according to claim 1, characterized in that: In the step (1), the solid fermentation temperature is 25-35°C, the humidity is 60%-80%, and the time is 1-3 days.

3. The preparation method according to claim 1, characterized in that: In the step (2), the heat treatment temperature is 70-90°C and the time is 30-60 minutes.

4. The preparation method according to claim 1, characterized in that: The palm meal is used as a fermentation substrate, and its addition amount accounts for 50%-80% of the total weight of the raw materials.

5. The preparation method according to claim 1, characterized in that: The added amount of the Lactobacillus acidophilus is 15 billion CFU-20 billion CFU, which is Lactobacillus acidophilus GBW1019 with a preservation number of CCTCC NO: M 20221403; the added amount of the Lactobacillus plantarum is 10 billion CFU-16 billion CFU, which is Lactobacillus plantarum GBW-LP001 with a preservation number of CGMCC No.22078.

6. The preparation method according to claim 4, characterized in that: The added amounts of the cellulase, α-galactosidase and xylanase are 60,000 U-100,000 U, 10,000 U-40,000 U and 20,000 U-50,000 U per kilogram of fermentation substrate, respectively.

7. The preparation method according to claim 4, characterized in that: The amount of glucose added is 40g-80g per kilogram of fermentation substrate.

8. The composite lactic acid bacteria postbiotic prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the composite lactic acid bacteria postbiotics according to claim 8 in preparing livestock and poultry breeding feed.

10. The use according to claim 9, characterized in that: The added amount of the compound lactic acid bacteria postbiotics is 0.05%-0.2% of the total weight of livestock and poultry feed.

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