Preparation method and application of probiotic fermentation liquor for improving reproductive performance of sows

Abstract

The invention relates to a preparation method and application of probiotic fermentation broth capable of improving reproductive performance of sows, and the preparation method of the probiotic fermentation broth comprises the following steps: (1) inoculating a first probiotic into a seed culture medium for culture, transferring into a fermentation culture medium for culture, centrifuging the obtained fermentation broth, and collecting supernate; and (2) mixing the supernate obtained in the step (1), a carbon source and second probiotics, and carrying out secondary fermentation to obtain the probiotic fermentation liquor. According to the method, the supernate obtained after lactobacillus fermentation is recycled, the good preservation effect and the good use effect are achieved, resource recycling is achieved, the breeding benefit is improved, and the wide application prospect is achieved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a preparation method and application of a probiotic fermentation liquid for improving the reproductive performance of sows. Background Art

[0002] In intensive pig production, pregnant and lactating sows are under a certain degree of parturition stress, which leads to a decrease in their body resistance. The immune function of the sow's body directly affects the secretion of colostrum, milk composition and the immune function of colostrum. Lactating sows affect group productivity through milk production and their reproductive performance. In production practice, lactating sows often cause piglet deaths, high rates of milk-induced diarrhea and poor growth and development due to low feed intake, no milk and low milk production, which in turn leads to many problems such as short sow life, high culling rate, low piglet vitality and easy diarrhea. The health status, milk production and milk quality of lactating sows have a significant impact on piglet growth and development, weaning weight and piglet disease resistance. Improving the health level and lactation ability of sows is the key to improving the production performance of suckling piglets.

[0003] Lactic acid bacteria is a kind of probiotic, which is often used in the food industry, such as making yogurt. In recent years, studies have found that lactic acid bacteria and their metabolites (such as lactic acid bacteria supernatant) also have positive effects on animal health, including improving intestinal health and enhancing immunity. The main components of lactic acid bacteria fermentation supernatant are lactic acid content exceeding 1%, total acid exceeding 10%, more than 16 kinds of amino acids, total amino acids> 2%, pH value between 4.5-6, rich in nutrients, and can be used in livestock and poultry farming.

[0004] During the large-scale, high-density fermentation production process, lactic acid bacteria produce abundant metabolites. Often, the fermentation liquid after centrifugation becomes industrial waste and is discharged as wastewater. On the one hand, this increases the processing cost and pollutes the environment; on the other hand, resources are not effectively utilized, resulting in a waste of resources.

[0005] In summary, how to reuse the supernatant after lactic acid bacteria fermentation and apply it to the fields of livestock and poultry breeding has become one of the urgent problems to be solved in this field. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a preparation method and application of a probiotic fermentation liquid for improving the reproductive performance of sows. Through scientific formulation and fermentation process, the supernatant after lactic acid bacteria fermentation is reused, which has good preservation effect and use effect, thereby realizing resource recycling and improving breeding efficiency.

[0007] In a first aspect, the present invention provides the use of a probiotic secondary fermentation broth in the preparation of a probiotic agent for improving the reproductive performance of sows.

[0008] By reusing the supernatant after probiotic fermentation, the present invention obtains a secondary probiotic fermentation broth, which effectively increases the content of organic acids. The organic acids can lower the intracellular pH, disrupt the cell membrane structure, or act as promoters for other antibacterial compounds, thereby playing an antibacterial role, further balancing the intestinal flora, and maintaining intestinal health.

[0009] For sows, using the supernatant of lactic acid bacteria can improve their reproductive performance, which is mainly achieved through the following aspects: 1. Improving intestinal health: Lactic acid bacteria can regulate the balance of intestinal microorganisms and inhibit the growth of harmful bacteria, thereby reducing reproductive disorders in sows caused by intestinal diseases. 2. Enhancing immune function: Lactic acid bacteria can stimulate the body to produce an immune response, improve the resistance of sows, help prevent diseases, and ensure the smooth progress of the reproductive process. 3. Promoting nutrient absorption: Lactic acid bacteria help decompose macromolecular substances in feed, increase the availability of nutrients, provide better nutritional support for sows, and are beneficial to embryo development and piglet growth.

[0010] Preferably, the preparation method of the secondary probiotic fermentation broth includes the following steps:

[0011] (1) Inoculate the first probiotic into a seed medium for cultivation, transfer it to a fermentation medium for cultivation, and after centrifuging the obtained fermentation broth, collect the supernatant;

[0012] (2) Mix the supernatant, carbon source, and the second probiotic in step (1) and perform secondary fermentation to obtain the secondary probiotic fermentation broth.

[0013] Preferably, the first probiotic includes lactic acid bacteria.

[0014] The preparation method of the secondary probiotic fermentation broth provided by the present invention can be widely applied to the secondary utilization of the supernatant after fermentation of various lactic acid bacteria, which can be either the supernatant after fermentation of a single type of lactic acid bacteria or the supernatant after mixed fermentation of two or more types of lactic acid bacteria.

[0015] Preferably, by weight percentage, the seed medium contains 2% - 3% glucose, 1% - 2% yeast extract powder, 0.5% - 1.5% peptone, 0.02% - 0.04% MgSO4·7H2O, 0.005% - 0.015% MnSO4, 0.03% - 0.07% NaCl, 0.05% - 0.15% K2HPO4, 0.0005% - 0.0015% FeSO4·7H2O, and 0.03% - 0.07% CaCO3.

[0016] Specific point values within the range of 2% - 3% can be selected as 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8%, or 3%, etc.

[0017] The specific point values of the above 1% - 2% can be selected as 1%, 1.2%, 1.4%, 1.5%, 1.6%, 1.8% or 2%, etc.

[0018] The specific point values of the above 0.5% - 1.5% can be selected as 0.5%, 0.7%, 0.9%, 1%, 1.1%, 1.3% or 1.5%, etc.

[0019] The specific point values of the above 0.02% - 0.04% can be selected as 0.02%, 0.25%, 0.28%, 0.03%, 0.32%, 0.35% or 0.04%, etc.

[0020] The specific point values of the above 0.005% - 0.015% can be selected as 0.005%, 0.007%, 0.009%, 0.01%, 0.011%, 0.013% or 0.015%, etc.

[0021] The specific point values of the above 0.03% - 0.07% can be selected as 0.03%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06% or 0.07%, etc.

[0022] The specific point values of the above 0.05% - 0.15% can be selected as 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.13% or 0.15%, etc.

[0023] The specific point values of the above 0.0005% - 0.0015% can be selected as 0.0005%, 0.0007%, 0.0009%, 0.001%, 0.0011%, 0.0013% or 0.0015%, etc.

[0024] Preferably, the pH of the seed culture medium is 4 - 5 (for example, it can be 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5, etc.).

[0025] Preferably, by weight percentage, the fermentation culture medium contains 2% - 3% glucose, 3% - 5% yeast extract powder, 2% - 4% peptone, 0.04% - 0.08% MgSO4·7H2O, 0.005% - 0.015% MnSO4, 0.05% - 0.15% NaCl, 0.15% - 0.25% K2HPO4, 0.0015% - 0.0025% FeSO4·7H2O and 0.05% - 0.15% CaCO3.

[0026] The specific point values of the above 2% - 3% can be selected as 2%, 2.2%, 2.4%, 2.5%, 2.6%, 2.8% or 3%, etc.

[0027] The specific point values of the above 3% - 5% can be selected as 3%, 3.5%, 3.8%, 4%, 4.2%, 4.5% or 5%, etc.

[0028] The specific point values of the above 2% - 4% can be selected as 2%, 2.5%, 2.8%, 3%, 3.2%, 3.5% or 4%, etc.

[0029] The specific point values of the above 0.04% - 0.08% can be selected as 0.04, 0.05, 0.055, 0.06, 0.065, 0.07 or 0.08, etc.

[0030] The specific point values of the above 0.005% - 0.015% can be selected as 0.005%, 0.007%, 0.009%, 0.01%, 0.011%, 0.013% or 0.015%, etc.

[0031] The specific point values of the above 0.05% - 0.15% can be selected as 0.05%, 0.07%, 0.09%, 0.1%, 0.11%, 0.13% or 0.15%, etc.

[0032] The specific point values of the above 0.15% - 0.25% can be selected as 0.15%, 0.17%, 0.19%, 0.2%, 0.21%, 0.23% or 0.25%, etc.

[0033] The specific point values of the above 0.0015% - 0.0025% can be selected as 0.0015%, 0.0017%, 0.0019%, 0.002%, 0.0021%, 0.0023% or 0.0025%, etc.

[0034] Preferably, the pH of the fermentation medium is 4 - 5 (for example, it can be 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5, etc.).

[0035] Preferably, the fermentation end point of the culture in the fermentation medium in step (1) is an OD value of the fermentation broth of 3.8 - 4.5 (for example, it can be 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4 or 4.5, etc.).

[0036] Preferably, the rotation speed of the centrifugation in step (1) is 10000 - 15000 rpm (for example, it can be 10000, 11000, 12000, 13000, 14000 or 15000, etc.).

[0037] Preferably, the centrifugation time in step (1) is 3 - 4 h (for example, it can be 3 h, 3.2 h, 3.4 h, 3.5 h, 3.6 h, 3.8 h or 4 h, etc.).

[0038] Preferably, after the supernatant, carbon source and the second probiotic bacteria in step (2) are mixed, the carbon content in the mixed solution is 1-2 g / 100 mL (for example, it can be 1 g / 100 mL, 1.2 g / 100 mL, 1.4 g / 100 mL, 1.5 g / 100 mL, 1.6 g / 100 mL, 1.8 g / 100 mL or 2 g / 100 mL, etc.).

[0039] Preferably, after the supernatant, carbon source and the second probiotic bacteria in step (2) are mixed, the content of the second probiotic bacteria is 200 million - 800 million CFU / 100 mL (for example, it can be 200 million CFU / 100 mL, 300 million CFU / 100 mL, 400 million CFU / 100 mL, 500 million CFU / 100 mL, 600 million CFU / 100 mL, 700 million CFU / 100 mL or 800 million CFU / 100 mL, etc.).

[0040] Preferably, the carbon source includes any one or a combination of at least two of glucose, maltose, lactose, fructose, starch, syrup, glycerol, sodium acetate or sorbitol.

[0041] Preferably, the carbon source is glucose, maltose and fructose.

[0042] By adding three carbon sources, namely glucose, maltose and fructose, the present invention enriches the types of carbon sources in the probiotic medium, synergistically improves the energy utilization efficiency of microorganisms, promotes the complete utilization of carbon sources and the efficient synthesis of products. Too high or too low content of carbon sources may affect the metabolic pathways of microorganisms and product formation, resulting in a decrease in yield or a change in the synthesis direction of products.

[0043] Preferably, the mass ratio of glucose, maltose and fructose in the carbon source is 1:(0.5 - 2):(0.5 - 2).

[0044] The specific point values of (0.5 - 2) above are 0.5, 0.7, 0.9, 1, 1.2, 1.4, 1.6, 1.8 or 2, etc.

[0045] Preferably, the second probiotic bacteria include Lactobacillus plantarum and / or Bifidobacterium.

[0046] Preferably, the second probiotic bacteria is a combination of Lactobacillus plantarum and Bifidobacterium.

[0047] Through the mixed fermentation of Lactobacillus plantarum and Bifidobacterium, the present invention synergistically improves the content of organic acids and other substances in the secondary fermentation broth, and can be applied to fields such as improving the reproductive performance of sows.

[0048] Preferably, the bacterial count ratio of Lactobacillus plantarum and Bifidobacterium in the second probiotic bacteria is 1:(0.5 - 2).

[0049] The specific point values of the above (0.5 - 2) are 0.5, 0.7, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, etc.

[0050] Preferably, the temperature of the secondary fermentation in step (2) is 25 - 35°C (for example, it can be 25°C, 27°C, 29°C, 30°C, 31°C, 33°C, 35°C, etc.).

[0051] Preferably, the time of the secondary fermentation in step (2) is 20 - 30 h (for example, it can be 20 h, 22 h, 24 h, 25 h, 26 h, 28 h, 30 h, etc.).

[0052] Preferably, improving the reproductive performance of sows includes improving the intestinal health of sows, enhancing the immunity of sows, and promoting the absorption of nutrients by sows.

[0053] Preferably, specifically improving the reproductive performance of sows includes: reducing the constipation rate of sows, increasing the number of live piglets per litter of sows, increasing the average birth weight of piglets, increasing the average weaning weight of piglets, increasing the survival rate of piglets, reducing the diarrhea rate of piglets, increasing the estrus rate of sows after weaning, and increasing the content of milk protein, lactose, and milk fat in the colostrum of sows after parturition, either alone or in combination of at least two.

[0054] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] The probiotic secondary fermentation broth provided by the present invention can improve the reproductive performance of sows, specifically manifested as: significantly alleviating the occurrence of constipation in sows, effectively increasing the average birth weight of piglets, reducing the diarrhea rate of piglets, increasing the average weaning weight of piglets, and increasing the estrus rate of sows after weaning, increasing the content of milk protein and milk fat in the colostrum of sows at 0 h, 12 h, and 24 h, and the content of lactose at 24 h. By reusing the supernatant after lactic acid bacteria fermentation and through scientific formulation and fermentation process, a probiotic secondary fermentation broth for improving the reproductive performance of sows is provided, which has good preservation effect and use effect, realizes resource reuse and improves breeding efficiency, and has broad application prospects. Specific Embodiments

[0057] To further illustrate the technical means and effects adopted by the present invention, the present invention will be further described below in conjunction with embodiments. It can be understood that the specific embodiments described here are only used to explain the present invention, rather than limiting the present invention.

[0058] For those without specific technologies or conditions indicated in the examples, follow the technologies or conditions described in the literature in this field or the product specifications. For reagents or instruments without the manufacturer indicated, they are all conventional products that can be obtained through regular commercial channels.

[0059] The Lactobacillus acidophilus used in the following examples and test examples is Lactobacillus acidophilus LA71 strain, classified and named as Lactobacillus acidophilus. The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation number is CGMCC 21765, the preservation date is January 29, 2021, and the preservation address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0060] The Pediococcus acidilactici used in the following examples and test examples is Pediococcus acidilactici PA153 strain, classified and named as Pediococcus acidilactici PA153 Lactobacillus plantarum PA153. The preservation unit is the China Center for Type Culture Collection, the preservation time is March 2, 2023, the preservation number is CCTCC NO: M 2023230, and the preservation address is Wuhan University, Wuhan, China.

[0061] The Lentilactobacillus buchneri used in the following examples and test examples is Lentilactobacillus buchneri LBu101 strain, classified and named as Lentilactobacillus buchneri. The preservation unit is the Guangdong Provincial Microbiology Culture Collection Center, the preservation time is December 26, 2022, the preservation number is GDMCC No: 63094, and the preservation address is 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou.

[0062] The Lactobacillus plantarum used in the following examples and test examples is Lactobacillus plantarum LP194 strain, classified and named as Lactobacillus plantarum. The preservation unit is the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, the preservation time is January 29, 2021, the preservation number is CGMCC 21766, and the preservation address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0063] The Bifidobacterium used in the following examples and test examples is Bifidobacterium animalis strain BA20, taxonomically named Bifidobacterium animalis, deposited with the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on February 25, 2021, with the deposit number CGMCC No. 21830 and the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0064] Example 1

[0065] This example provides a secondary fermentation method for probiotics, which includes the following steps:

[0066] (1) Starting from a glycerol tube, Lactobacillus acidophilus is inoculated into a test tube slant for activation. After being cultured in a first-stage seed shake flask (450 mL / 500 mL), it is transferred to a seed tank (3 L / 5 L) for expansion culture at an inoculation amount of 5%, obtaining a secondary seed liquid; the secondary seed liquid is transferred to a 100 L fermenter (60 L / 100 L) for fermentation until the OD value of the fermentation broth is 4.2. The obtained fermentation broth is centrifuged at 12000 rpm for 3.5 h, and the supernatant is collected.

[0067] By weight percentage, the seed medium contains 2.5% glucose, 1.5% yeast extract powder, 1% peptone, 0.03% MgSO4·7H2O, 0.01% MnSO4, 0.05% NaCl, 0.1% K2HPO4, 0.001% FeSO4·7H2O, and 0.05% CaCO3, with the balance being water. The initial pH is adjusted to 4.6 and sterilized at 115°C for 30 min for standby.

[0068] By weight percentage, the fermentation medium contains 2.5% glucose, 4% yeast extract powder, 3% peptone, 0.06% MgSO4·7H2O, 0.01% MnSO4, 0.1% NaCl, 0.2% K2HPO4, 0.002% FeSO4·7H2O, and 0.1% CaCO3, with the balance being water. The initial pH is adjusted to 4.6 and sterilized at 115°C for 30 min for standby.

[0069] (2) The supernatant of the primary fermentation of Lactobacillus acidophilus obtained in step (1), glucose, maltose, fructose, Lactobacillus plantarum, and Bifidobacterium are mixed. After mixing, the carbon content in the mixed solution is 1.5 g / 100 mL, the mass ratio of glucose, maltose, and fructose is 1:1:1, the bacterial content in the mixed solution is 500 million CFU / 100 mL, and the ratio of the number of bacteria of Lactobacillus plantarum to Bifidobacterium is 1:1. Ferment at 30°C for 24 h to obtain a secondary fermentation broth of probiotics.

[0070] Example 2

[0071] This example provides a secondary fermentation method for probiotics, which includes the following steps:

[0072] (1) Pediococcus acidilactici starts from a glycerol tube, is inoculated into a test tube slant for activation, and after being cultured in a first-stage seed shaking flask (450 mL / 500 mL), it is transferred to a seed tank (3 L / 5 L) for expansion culture according to an inoculation amount of 5%, and a secondary seed liquid is obtained; the secondary seed liquid is transferred to a 100 L fermentation tank (60 L / 100 L) for fermentation until the OD value of the fermentation broth is 4.5, and the obtained fermentation broth is centrifuged at 15000 rpm for 3 h, and then the supernatant is collected.

[0073] By weight percentage, the seed culture medium contains 2% glucose, 2% yeast extract powder, 0.5% peptone, 0.04% MgSO4·7H2O, 0.005% MnSO4, 0.07% NaCl, 0.05% K2HPO4, 0.0015% FeSO4·7H2O, and 0.03% CaCO3, with the balance being water. The initial pH is adjusted to 4, and it is sterilized at 115 °C for 30 min for standby.

[0074] By weight percentage, the fermentation culture medium contains 3% glucose, 3% yeast extract powder, 4% peptone, 0.04% MgSO4·7H2O, 0.015% MnSO4, 0.05% NaCl, 0.25% K2HPO4, 0.0015% FeSO4·7H2O, and 0.15% CaCO3, with the balance being water. The initial pH is adjusted to 5, and it is sterilized at 115 °C for 30 min for standby.

[0075] (2) The supernatant of the primary fermentation of Pediococcus acidilactici obtained in step (1), glucose, maltose, fructose, Lactobacillus plantarum, and Bifidobacterium are mixed. After mixing, the carbon content in the mixed solution is 2 g / 100 mL, the mass ratio of glucose, maltose, and fructose is 1:0.5:2, the bacterial content in the mixed solution is 200 million CFU / 100 mL, and the bacterial count ratio of Lactobacillus plantarum and Bifidobacterium is 1:2. Fermentation is carried out at 25 °C for 30 h to obtain a secondary fermentation broth of probiotics.

[0076] Example 3

[0077] This example provides a secondary fermentation method for probiotics, which includes the following steps:

[0078] (1) Lactobacillus buchneri starts from a glycerol tube, is transferred to a slant test tube for activation, and after being cultured in a first-stage seed shake flask (450 mL / 500 mL), it is transferred to a seed tank (3 L / 5 L) for expansion culture at an inoculation amount of 5% to obtain a secondary seed liquid; the secondary seed liquid is transferred to a 100 L fermenter (60 L / 100 L) for fermentation until the OD value of the fermentation broth is 3.8. The obtained fermentation broth is centrifuged at 10000 rpm for 4 h, and the supernatant is collected.

[0079] By weight percentage, the seed culture medium contains 3% glucose, 1% yeast extract powder, 1.5% peptone, 0.02% MgSO4·7H2O, 0.015% MnSO4, 0.03% NaCl, 0.15% K2HPO4, 0.0005% FeSO4·7H2O, and 0.07% CaCO3, with the balance being water. The initial pH is adjusted to 5 and sterilized at 115 °C for 30 min for standby.

[0080] By weight percentage, the fermentation culture medium contains 2% glucose, 5% yeast extract powder, 2% peptone, 0.08% MgSO4·7H2O, 0.005% MnSO4, 0.15% NaCl, 0.15% K2HPO4, 0.0025% FeSO4·7H2O, and 0.05% CaCO3, with the balance being water. The initial pH is adjusted to 4 and sterilized at 115 °C for 30 min for standby.

[0081] (2) The primary fermentation supernatant of Lactobacillus buchneri obtained in step (1), glucose, maltose, fructose, Lactobacillus plantarum, and Bifidobacterium are mixed. After mixing, the carbon content in the mixed solution is 1 g / 100 mL, the mass ratio of glucose, maltose, and fructose is 1:2:0.5, the bacterial content in the mixed solution is 800 million CFU / 100 mL, and the ratio of the number of Lactobacillus plantarum to Bifidobacterium is 1:0.5. Fermentation is carried out at 35 °C for 20 h to obtain a probiotic secondary fermentation broth.

[0082] Example 4

[0083] This example provides a method for secondary fermentation of probiotics. The difference from Example 1 is only that in step (2), glucose is not added, and the proportion of glucose is distributed to maltose and fructose in proportion.

[0084] Example 5

[0085] This example provides a method for secondary fermentation of probiotics. The difference from Example 1 is only that in step (2), maltose is not added, and the proportion of maltose is distributed to glucose and fructose in proportion.

[0086] Example 6

[0087] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that fructose is not added in step (2), and the proportion of fructose is distributed to glucose and maltose proportionally.

[0088] Embodiment 7

[0089] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that glucose is replaced with an equal amount of galactose in step (2).

[0090] Embodiment 8

[0091] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that maltose is replaced with an equal amount of sucrose in step (2).

[0092] Embodiment 9

[0093] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that fructose is replaced with an equal amount of galactose in step (2).

[0094] Embodiment 10

[0095] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that only Lactobacillus plantarum is added in step (2), and the proportion of Bifidobacterium is replaced with Lactobacillus plantarum.

[0096] Embodiment 11

[0097] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that only Bifidobacterium is added in step (2), and the proportion of Lactobacillus plantarum is replaced with Bifidobacterium.

[0098] Embodiment 12

[0099] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that Lactobacillus plantarum is replaced with an equal amount of Lactobacillus acidophilus in step (2).

[0100] Embodiment 13

[0101] This embodiment provides a method for secondary fermentation of probiotics, which is only different from Embodiment 1 in that Bifidobacterium is replaced with an equal amount of Lactobacillus buchneri in step (2).

[0102] Comparative Example 1

[0103] This comparative example provides a method for fermenting probiotics, which is only different from Embodiment 1 in that the supernatant is not centrifugally collected in step (1) and step (2) is not carried out, that is, the primary fermentation broth of probiotics is obtained.

[0104] Test Example 1

[0105] This test example studied the effect of the secondary fermentation broth of probiotics on the reproductive performance of sows. The specific test method was as follows: 150 two - hybrid sows with similar parity and body weight were randomly divided into 15 groups, including 1 blank control group and 14 experimental groups. The test period was from 85 days of pregnancy until the piglets were weaned. The diet was formulated according to the nutritional requirements of sows in NRC (2012), and its composition and nutritional levels are shown in Table 1. During the test period, the control group drank normal water, and the experimental groups drank water added with the probiotic fermentation broth (diluted 1:25) obtained by the methods provided in the examples and comparative examples.

[0106] Table 1

[0107] Raw material name Late pregnancy (%) Lactation period (%) Nutritional level Late pregnancy Lactation period Corn 32.55 40.85 DM / % 86.45 86.2 Soybean meal 43 2.41 14.96 CP / % 13 17 Flour 10.00 12.00 EE / % 3.64 4.39 Wheat middlings 10.00 10.00 CF / % 7.67 3.76 Bran 14.55 0.00 Lys 0.73 1.12 Corn DDGS 5.00 0.00 Ca / % 0.65 0.85 Extruded soybean 0.00 5.00 P-total / % 0.61 0.59

[0108]

[0109] During the late pregnancy of sows, the constipation rate of sows was recorded. After parturition, the number of live piglets per litter, the average birth weight of piglets, the average weaning weight of piglets, the survival rate of piglets, the diarrhea rate of piglets, and the estrus rate of sows after weaning in different groups were recorded. The results are shown in Table 2.

[0110] Table 2

[0111]

[0112]

[0113] It can be seen from Table 2 that:

[0114] (1) By comparing the example with the control group, it can be seen that the secondary fermentation broth of probiotics can significantly relieve the occurrence of constipation, effectively increase the average birth weight of piglets, increase the average weaning weight of piglets, increase the survival rate of piglets, reduce the diarrhea rate of piglets, increase the estrus rate of sows after weaning, and improve the reproductive performance of sows.

[0115] (2) By comparing Example 1 with Examples 4 - 9, it can be seen that the type of carbon source will affect the improvement effect of the reproductive performance of sows, and glucose, maltose, and fructose have a synergistic effect.

[0116] (3) By comparing Example 1 with Examples 10 - 13, it can be seen that Lactobacillus plantarum and Bifidobacterium have a synergistic effect on improving the reproductive performance of sows, and the lack or replacement of any one of the bacteria will weaken the improvement ability.

[0117] After sows gave birth, milk samples were collected at 0 hour, 12 hours, and 24 hours after parturition, taken from the nipples at the front, middle, and rear positions of the sows, mixed evenly and placed in centrifuge tubes to measure the routine milk components. The milk protein, lactose, and milk fat contents of each group are shown in Table 3.

[0118] Table 3

[0119]

[0120]

[0121] It can be seen from Table 3 that:

[0122] (1) By comparing the examples with the control group, it can be seen that in the examples, the contents of milk protein and milk fat in colostrum at 0, 12, and 24 hours increased significantly, and the lactose content increased significantly at 24 hours. Milk protein, lactose, and milk fat are the main components of colostrum and also important nutritional sources for piglets. The protein in sow colostrum can provide essential amino acids and immune protection for neonatal growth and development. Lactose is the main energy source for neonatal piglets and can also regulate the intestinal flora of piglets.

[0123] (2) By comparing Example 1 with Examples 4-9, it can be seen that the type of carbon source will affect the composition of sow colostrum. Glucose, maltose, and fructose have a synergistic effect on increasing the contents of milk protein, lactose, and milk fat in sow colostrum.

[0124] (3) By comparing Example 1 with Examples 10-13, it can be seen that Lactobacillus plantarum and Bifidobacterium have a synergistic effect on improving the composition of sow colostrum.

[0125] In summary, the present invention provides a secondary fermentation liquid of probiotics. By reusing the supernatant after lactic acid bacteria fermentation, the secondary fermentation liquid has good preservation and use effects, realizes resource reuse, and is applied to improve breeding efficiency, having broad application prospects.

[0126] The applicant declares that the above description is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Application of the probiotic secondary fermentation broth in preparing a probiotic agent for improving the reproductive performance of sows.

2. The application according to claim 1, wherein The preparation method of the probiotic secondary fermentation broth comprises the following steps: (1) Inoculate the first probiotic into a seed medium for cultivation, transfer it to a fermentation medium for cultivation, and after centrifuging the obtained fermentation broth, collect the supernatant; (2) Mix the supernatant, carbon source and the second probiotic in step (1) and conduct secondary fermentation to obtain the probiotic secondary fermentation broth.

3. The application according to claim 2, wherein The first probiotic includes lactic acid bacteria; Preferably, by weight percentage, the seed medium contains 2% - 3% glucose, 1% - 2% yeast extract powder, 0.5% - 1.5% peptone, 0.02% - 0.04% MgSO4·7H2O, 0.005% - 0.015% MnSO4, 0.03% - 0.07% NaCl, 0.05% - 0.15% K2HPO4, 0.0005% - 0.0015% FeSO4·7H2O and 0.03% - 0.07% CaCO3; Preferably, the pH of the seed medium is 4 - 5.

4. The application according to claim 2 or 3, characterized in that, By weight percentage, the fermentation medium contains 2% - 3% glucose, 3% - 5% yeast extract powder, 2% - 4% peptone, 0.04% - 0.08% MgSO4·7H2O, 0.005% - 0.015% MnSO4, 0.05% - 0.15% NaCl, 0.15% - 0.25% K2HPO4, 0.0015% - 0.0025% FeSO4·7H2O and 0.05% - 0.15% CaCO3; Preferably, the pH of the fermentation medium is 4 - 5.

5. The application according to any one of claims 2 to 4, characterized in that, The fermentation end point of the cultivation in the fermentation medium in step (1) is that the OD value of the fermentation broth is 3.8 - 4.5; Preferably, the rotation speed of the centrifugation in step (1) is 10000 - 15000 rpm; Preferably, the centrifugation time in step (1) is 3 - 4 h.

6. The application according to any one of claims 2 to 5, characterized in that After the supernatant, carbon source and the second probiotic are mixed in step (2), the carbon content in the mixed solution is 1 - 2 g / 100 mL; Preferably, after the supernatant, carbon source and the second probiotic are mixed in step (2), the content of the second probiotic is 2 - 8 hundred million CFU / 100 mL.

7. The application according to any one of claims 2 to 6, characterized in that, The carbon source includes any one or a combination of at least two of glucose, maltose, lactose, fructose, starch, syrup, glycerol, sodium acetate or sorbitol; Preferably, the carbon source is glucose, maltose and fructose; Preferably, the mass ratio of glucose, maltose and fructose in the carbon source is 1:(0.5 - 2):(0.5 - 2).

8. The application according to any one of claims 2 to 7, characterized in that The second probiotic includes Lactobacillus plantarum and / or Bifidobacterium; Preferably, the second probiotic is a combination of Lactobacillus plantarum and Bifidobacterium; Preferably, the ratio of the number of Lactobacillus plantarum to Bifidobacterium in the second probiotic is 1:(0.5 - 2).

9. The application according to any one of claims 2 to 8, characterized in that, The temperature of the secondary fermentation in step (2) is 25 - 35 °C; Preferably, the time of the secondary fermentation in step (2) is 20 - 30 h.

10. The application according to any one of claims 1 to 9, characterized in that, The improvement of the reproductive performance of sows includes improving the intestinal health of sows, enhancing the immunity of sows and promoting the absorption of nutrients by sows.

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