A composite microbial iron-enriched bacterial agent and its preparation and application

Abstract

The present invention discloses a composite microbial iron-rich agent and its preparation and application. The composite agent is prepared by co-culturing and fermenting saccharomyces cerevisiae (CGMCC No. 22727) and Lactobacillus plantarum (CCTCC No. M 2022915). The composite microbial iron-rich agent combines the probiotic functions of trace elements, yeast and lactic acid bacteria, and can be used as a feed additive for piglet breeding. The composite microbial iron-rich agent of the present invention and its use method have the effects of promoting piglet growth, preventing piglet anemia, improving immunity and antioxidant performance, reducing diarrhea rate, and reducing odor and greenhouse gas concentration in pig houses.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial preparations, and specifically relates to a composite microbial iron-enriched bacterial agent, a preparation method thereof, and an application thereof in piglet breeding. Background Art

[0002] Iron is a trace element necessary for animals to maintain normal growth and development. It is an important raw material for synthesizing hemoglobin and has a certain promoting effect on the body's immune function. If there is not enough iron in the process of pig farming, it will cause iron deficiency anemia, decreased immunity, growth retardation and other symptoms. In the breeding process, iron is generally supplemented in the form of inorganic salts, but this iron supplementation method has great irritation to the intestinal mucosa of animals and has a low absorption rate. In recent years, the use of microorganisms as carriers to enrich iron as feed additives has attracted people's attention. This can not only improve the bioavailability of iron, but also reduce the stress response caused by inorganic iron stimulation. At the same time, with the large-scale development of pig farming, a large amount of ammonia (NH3), hydrogen sulfide (H2S), nitrous oxide (N2O), methane (CH4) and other gases are produced in pig houses. If relevant improvement measures are not taken, it will not only affect the health of pigs, but also cause serious odor pollution and greenhouse effect, and will also cause certain harm to the health of breeders and residents around the farms. A large number of studies have shown that microecological preparations have good effects on the prevention and control of pig farming diseases and the improvement of production performance, and have broad application value and market prospects. Summary of the invention

[0003] In view of the shortcomings of the existing microecological preparations with single functions and the market demand, the primary purpose of the present invention is to provide a composite microbial iron-rich agent that integrates the probiotic functions of trace elements, yeast and lactic acid bacteria. The composite microbial iron-rich agent is used as a feed additive in piglet feeding to prevent anemia, improve piglet immunity and antioxidant properties, promote growth, and reduce the emission of odor and greenhouse gases during breeding.

[0004] In order to achieve the above object, the technical solution adopted by the present invention is:

[0005] The brewer's yeast of the present invention is the brewer's yeast (Saccharomyces cerevisiae) ZYJM-15 strain, which has been deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC No. 22727, a deposit date of June 17, 2021, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] The plant lactobacillus of the present invention is the plant lactobacillus (Lactobacillus plantarum) ZZ2022-615 strain, which has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC No.M 2022915, a deposit date of June 17, 2022, and a deposit address of Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] A composite microbial iron-enriching agent is prepared by co-culturing and fermenting Saccharomyces cerevisiae ZYJM-15 with a preservation number of CGMCC No. 22727 and Lactobacillus plantarum ZZ2022-615 with a preservation number of CCTCC No. M 2022915.

[0008] The composite microbial iron-rich bacterial agent has a live yeast count of ≥8×10 8 CFU / ml, Lactobacillus plantarum ≥2.0×10 9 CFU / ml.Fe 2+ The enrichment rate reached over 87.17%.

[0009] The second object of the present invention is to provide a method for preparing the composite microbial iron-rich agent, comprising the following steps:

[0010] 1) Bacteria activation

[0011] The solid cultures of Saccharomyces cerevisiae ZYJM-15 and Lactobacillus plantarum ZZ2022-615 were inoculated into the liquid culture medium to obtain their respective seed liquids;

[0012] 2) Seed cultivation

[0013] The seed liquids of the two bacteria are respectively inoculated into the seed culture medium to obtain their respective fermentation seed liquids;

[0014] 3) Fermentation tank co-culture

[0015] Firstly, Lactobacillus plantarum fermentation seed liquid is introduced into a fermentation tank and statically cultured; then, Saccharomyces cerevisiae fermentation seed liquid is introduced into the fermentation tank and shaken cultured to obtain a composite microbial iron-rich bacterial agent fermentation liquid.

[0016] Furthermore, the preparation method of the composite microbial iron-rich agent specifically comprises:

[0017] 1) Bacteria activation

[0018] The solid culture of Saccharomyces cerevisiae ZYJM-15 was inoculated into the malt juice liquid medium, and the culture was activated at 28-32°C and 180-220 rpm. The activation was performed at least three times to serve as the seed liquid.

[0019] The solid culture of Lactobacillus plantarum ZZ2022-615 was inoculated into MRS broth medium for activation culture, and then statically cultured at 35-37° C. The activation was performed at least three times to serve as seed liquid;

[0020] 2) Seed cultivation

[0021] The saccharomyces cerevisiae seed liquid obtained in step 1) is inoculated into a seed bottle at an inoculation rate of 3-5%, the filling coefficient is 40-50 / 250 volume ratio, the formula of the seed culture medium is: 3% glucose, 2.4% yeast extract, 0.6% urea, 0.25% K2PO4, 0.05% MgSO4·7H2O, the fermentation conditions are: fermentation temperature 28-32°C, rotation speed 180-220rpm, pH value 4.5-5.5, culture time 36-48h, to obtain saccharomyces cerevisiae fermentation seed liquid;

[0022] The Lactobacillus plantarum seed liquid obtained in step 1) is inoculated into a seed bottle at an inoculum amount of 3-6%, the filling coefficient is 40-60 / 250 volume ratio, the formula of the seed culture medium is: 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, and the fermentation conditions are: static culture at 35-37° C. for 24-40 hours to obtain Lactobacillus plantarum fermentation seed liquid;

[0023] 3) Fermentation tank co-culture

[0024] In a fermenter, 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, 0.6% urea and 1600 μg / ml of FeSO4·7H2O are added in a volume ratio of 25-35 / 50, and the mixture is sterilized at 121° C. for 30 minutes in the fermenter. After cooling, the plant lactobacillus fermentation seed liquid obtained in step 2) is added to the fermenter in an inoculation amount of 3-6%, and the mixture is statically cultured at 35-37° C. for 24-40 hours. Under sterile conditions, the brewer's yeast fermentation seed liquid obtained in step 2) is added in an inoculation amount of 3-5%, the fermentation temperature is 28-32° C., the rotation speed is 180-220 rpm, and the culture time is 36-48 hours to obtain a composite microbial iron-enriched bacterial agent fermentation liquid.

[0025] The third object of the present invention is to provide an application of the composite microbial iron-rich agent for preparing a preparation having at least one of the functions of promoting growth of weaned piglets, preventing anemia in piglets, improving immunity, improving antioxidant capacity, reducing diarrhea rate, reducing odor in pig houses, and reducing greenhouse gas concentrations in pig houses. Specifically, the composite microbial iron-rich agent is added to the daily feed of weaned piglets.

[0026] The present invention finds that adding the composite microbial iron-enriching agent of the present invention into the daily diet of piglets can significantly increase the average daily weight gain of piglets, and significantly reduce the feed-to-weight ratio and diarrhea rate.

[0027] The number of red blood cells, hemoglobin, serum iron and total iron binding capacity in the blood are important indicators reflecting the iron metabolism and nutrition of piglets. The present invention has found that adding the composite microbial iron-enriching agent of the present invention to the piglet diet can significantly increase the number of red blood cells, hemoglobin, serum iron and total iron binding capacity of piglets, and has a good effect on preventing anemia in piglets and ensuring their normal growth and development.

[0028] Serum biochemical indicators not only reflect the digestion, absorption and metabolic function of nutrients by the animal body, but also indirectly reflect the health status of the animal body. The total protein, albumin and urea nitrogen contents are highly correlated with the body's protein absorption and metabolic status. When the body has good protein metabolism, the total protein and albumin contents will increase, and the urea nitrogen content in the blood will decrease. In the embodiment of the present invention, the addition of the composite microbial iron-rich agent of the present invention increases the albumin and total protein contents, and serum urea nitrogen is significantly reduced, indicating that the addition of the composite microbial iron-rich agent increases the utilization rate of protein by piglets, promotes the synthesis of its own protein, and improves its growth performance. Serum ceruloplasmin not only has antioxidant activity, but also directly affects the yeast's uptake of iron. The present invention finds that the addition of the composite microbial iron-rich agent of the present invention has a serum ceruloplasmin activity that is better than the addition of brewer's yeast iron agent and inorganic iron in the diet.

[0029] When the body is under oxidative stress, the content of free radicals in the body increases, and the free radicals react with lipids to produce cytotoxic malondialdehyde. Important antioxidant enzymes such as glutathione peroxidase and superoxide dismutase can remove free radicals in the body and restore the body to a normal physiological state. The results in the embodiments of the present invention show that the addition of the composite microbial iron-rich bacterial agent of the present invention to the diet can significantly improve the total antioxidant capacity of piglets, as well as the activity levels of glutathione peroxidase and superoxide dismutase in serum, while the content of malondialdehyde is significantly reduced. The results show that the addition of the composite microbial bacterial agent of the present invention is beneficial to improving the antioxidant capacity of piglets.

[0030] Interleukin plays a role in information transmission between leukocytes or immune cells, and can regulate immune cells to produce immunoglobulins. Immunoglobulins are proteins with antibody activity in the body, which can bind to antigens, reduce damage to the body, and play an important role in inflammatory reactions. The embodiments of the present invention show that the addition of the composite microbial iron-rich bacteria agent or the saccharomyces cerevisiae iron-rich bacteria agent of the present invention significantly increases the content of IgG and IL-6 in serum, and significantly reduces the content of TNF-α. The composite microbial iron-rich bacteria agent is better than the saccharomyces cerevisiae iron-rich bacteria agent, indicating that the addition of the composite microbial iron-rich bacteria agent of the present invention to the diet can regulate the immune function of piglets and improve the body's disease resistance.

[0031] The odor and greenhouse gases in the pig house of the present invention include: NH3, H2S, CH4, N2O. The embodiments of the present invention show that by adding the composite microbial iron-enriching agent of the present invention to the diet, the concentrations of harmful gases NH3, H2S, CH4, and N2O in the pig house are reduced, achieving the coordinated reduction of odor and greenhouse gases, and greatly improving the breeding environment.

[0032] The present invention also provides the use of the two strains mentioned above in preparing the composite microbial iron-rich bacterial agent.

[0033] The present invention also provides the use of the above-mentioned strain in preparing a preparation having at least one of the functions of promoting the growth of weaned piglets, preventing anemia in piglets, improving immunity, improving antioxidant capacity, reducing diarrhea rate, reducing odor in pig houses, and reducing greenhouse gas concentration in pig houses.

[0034] The present invention has the following advantages:

[0035] 1. The composite microbial iron-rich agent adopts a co-culture fermentation process and optimizes the fermentation medium. Compared with the conventional fermentation technology of separate fermentation and then mixing, it shortens the fermentation cycle and reduces the production cost of the agent. The proportion of live bacteria of each strain in the product is better, which is conducive to the stable preservation of the product and improves the application effect.

[0036] 2. The composite microbial iron-enriching agent of the present invention adopts brewer's yeast and plant lactobacillus with strong iron-enriching ability as feed additives, which integrates the probiotic functions of trace elements, yeast and lactic acid bacteria. It not only uses yeast to provide a large amount of protein, but also uses plant lactobacillus to inhibit the growth of harmful bacteria. While supplementing iron, it promotes the growth performance of piglets, enhances the immunity and antioxidant capacity of piglets, reduces the feed-to-weight ratio and diarrhea rate, and improves the economic benefits of breeding.

[0037] 3. The composite microbial iron-rich bacteria agent of the present invention can be used as a feed additive to reduce the concentration of harmful gases NH3, H2S, CH4, and N2O in pig houses, eliminate the stench of pig houses, reduce the impact of greenhouse gases on the environment, and effectively improve the pig breeding environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Growth and microscopic examination of Saccharomyces cerevisiae ZYJM-15 on iron-rich plate medium;

[0039] Figure 2 :Growth and microscopic examination images of Lactobacillus plantarum ZZ2022-615 in iron-rich plate culture medium. DETAILED DESCRIPTION

[0040] The specific implementation methods will be described in detail below in conjunction with the embodiments of the present invention, but these implementation methods are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0041] Example 1: Breeding of functional strains

[0042] The 28 yeast strains and 15 lactobacilli strains isolated and identified by the research team from the natural environment were selected for iron resistance test. The strains to be screened were suspended in sterile water, and the concentration of the bacterial suspension was uniformly adjusted to 10 6 CFU / ml.

[0043] Apply yeast suspension on Fe 2+ Malt extract agar plates with concentrations of 0, 50, 100, 150, 200, 250, 300, 350, 400, 450, and 500 μg / ml were cultured at 28°C for 48 h, and the iron-resistant strains with good growth were selected to further determine their iron-enriching capacity. 2+ Malt extract liquid culture medium with concentrations of 600, 700, 800, 900, 1000, 1500 and 2000 μg / ml, respectively, was placed in a shaker at 28°C and 180 rpm for 48 h, the biomass and intracellular iron content of each strain were determined, and the iron enrichment rate (intracellular iron content / iron addition amount) was calculated.

[0044] Apply the Lactobacillus suspension on the Fe 2+ The iron-tolerant strains were cultured on MRS agar plates with concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg / ml at 37°C for 48 h, and the iron-tolerant strains with good growth were selected for further determination of their iron-rich capacity. 2+ MRS liquid culture medium with concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg / ml, respectively, was placed at 37°C and cultured for 40 h. The biomass and intracellular iron content of each strain were determined, and the iron enrichment rate (intracellular iron content / iron addition amount) was calculated.

[0045] The test results show that yeast ZYJM-15 2+ In the malt extract liquid medium with a concentration of 900 μg / ml, the bacterial biomass was (4.81±0.05) g / L and the iron enrichment rate was (32.23±1.14)%. 2+ In MRS liquid culture with a concentration of 700μg / ml, the bacterial biomass was (2.14±0.31)g / L and the iron enrichment rate was (28.95±1.82)%. These were the two strains with the best iron-enriching effect.

[0046] The yeast ZYJM-15 was confirmed to be Saccharomyces cerevisiae by 26S rDNA sequencing and physiological and biochemical reaction verification, and the homology comparison with the gene sequence in the gene bank reached 99%; the lactobacillus ZZ2022-615 was confirmed to be Lactobacillus plantarum by 16S rRNA sequencing and physiological and biochemical reaction verification, and the homology comparison with the gene sequence in the gene bank reached 100%. The growth and microscopic examination pictures of Saccharomyces cerevisiae ZYJM-15 and Lactobacillus plantarum ZZ2022-615 on iron-rich plate culture medium are shown in Figure 1 and Figure 2 .

[0047] Example 2: Optimization of fermentation parameters of functional strains

[0048] Different carbon sources, nitrogen sources and their concentration gradients, different concentration gradients of inorganic salts and FeSO4·7H2O, and different fermentation conditions were set to investigate the bacterial biomass, viable count and iron enrichment rate. The optimization parameters of the two bacteria fermentation alone are as follows:

[0049] Optimization of the iron-enriched fermentation parameters of Saccharomyces cerevisiae ZYJM-15: glucose 3%, yeast extract 2.4%, urea 0.6%, K2PO4 0.25%, MgSO4·7H2O 0.05%, FeSO4·7H2O 900μg / ml; inoculation volume 3-5%, loading coefficient 40-50 / 250 volume ratio, temperature 28-32℃, rotation speed 180-220rpm, pH 4.5-5.5, culture time 36-48h. The biomass of the bacteria was (14.57±0.02)g / L, and the number of live bacteria of Saccharomyces cerevisiae was ≥1×10 8 CFU / ml, and the iron enrichment rate reached (95.48±0.71)%. After optimization, the bacterial biomass increased by up to 202.91%, and the iron enrichment rate increased by up to 196.25%.

[0050] Optimization of iron-enriched fermentation parameters of Lactobacillus plantarum ZZ2022-615:

[0051] Brown sugar 10%, peptone 0.5%, yeast extract powder 0.5%, urea 0.6%, FeSO4·7H2O 700μg / ml; inoculation volume 3-6%, loading coefficient 40-60 / 250 volume ratio, temperature 35-37℃, static culture 24-40h. Bacterial biomass is (5.23±0.16)g / L, and the number of viable Lactobacillus plantarum is ≥4×10 8 CFU / ml. The iron enrichment rate reached (57.21±1.82)%. After optimization, the bacterial biomass increased by up to 144.39%, and the iron enrichment rate increased by up to 97.62%.

[0052] Example 3: Composite microbial agent iron-enriched co-fermentation process

[0053] The growth of viable bacteria and iron-enriching effect of composite microbial iron-enriching agents under different co-fermentation modes in different culture media were investigated.

[0054] A: Glucose 3%, yeast extract 2.4%, urea 0.6%, K2PO4 0.25%, MgSO4·7H2O 0.05%, FeSO4·7H2O 1600μg / ml

[0055] In the above culture medium, the fermentation seed liquid of Lactobacillus plantarum ZZ2022-615 was inoculated at 3% and cultured at 35°C for 24 hours; the fermentation seed liquid of Saccharomyces cerevisiae ZYJM-15 was inoculated at 4% under sterile conditions and cultured at 28°C, 180rpm, pH 4.5, and shaken for 48 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0056] In the above medium, 4% inoculum of Saccharomyces cerevisiae ZYJM-15 fermentation seed liquid was inoculated, and the culture was shaken at 28°C, 180rpm, pH 4.5 for 48 hours. Under sterile conditions, 3% inoculum of Lactobacillus plantarum ZZ2022-615 fermentation seed liquid was inoculated, and the culture was statically cultured at 35°C for 24 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0057] The fermentation seed liquid of Saccharomyces cerevisiae ZYJM-15 was inoculated with 4% inoculation amount, and the fermentation seed liquid of Lactobacillus plantarum ZZ2022-615 was inoculated with 3% inoculation amount into the above culture medium. The culture was shaken at 28°C, 180rpm, pH 4.5 for 72 hours, and the live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0058] The above culture medium was inoculated with 4% inoculum of fermented seed liquid of Saccharomyces cerevisiae ZYJM-15 and 3% inoculum of fermented seed liquid of Lactobacillus plantarum ZZ2022-615, and cultured at 35°C for 72 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0059] B: medium composed of 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, 0.6% urea, and 1600μg / ml FeSO4·7H2O

[0060] In the above culture medium, 3% inoculum was added to the fermentation seed liquid of Lactobacillus plantarum ZZ2022-615, and the culture was statically cultured at 35°C for 24 hours; under sterile conditions, 4% inoculum was added to the fermentation seed liquid of Saccharomyces cerevisiae ZYJM-15, and the culture was shaken at 28°C, 180rpm, pH 4.5 for 48 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0061] In the above medium, 4% inoculum of Saccharomyces cerevisiae ZYJM-15 fermentation seed liquid was inoculated, and the culture was shaken at 28°C, 180rpm, pH 4.5 for 48 hours. Under sterile conditions, 3% inoculum of Lactobacillus plantarum ZZ2022-615 fermentation seed liquid was inoculated, and the culture was statically cultured at 35°C for 24 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0062] The fermentation seed liquid of Saccharomyces cerevisiae ZYJM-15 was inoculated with 4% inoculation amount, and the fermentation seed liquid of Lactobacillus plantarum ZZ2022-615 was inoculated with 3% inoculation amount into the above culture medium. The culture was shaken at 28°C, 180rpm, pH 4.5 for 72 hours, and the live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0063] The above culture medium was inoculated with 4% inoculum of fermented seed liquid of Saccharomyces cerevisiae ZYJM-15 and 3% inoculum of fermented seed liquid of Lactobacillus plantarum ZZ2022-615, and cultured at 35°C for 72 hours. The live bacteria of Saccharomyces cerevisiae and Lactobacillus plantarum were counted, and the total biomass and iron enrichment rate of the bacteria were determined.

[0064] Comparing the above 8 co-fermentation methods, the following co-fermentation method obtained the highest total biomass, viable count and iron enrichment rate: in a medium composed of 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, 0.6% urea, and 1600μg / ml FeSO4·7H2O, the fermentation seed liquid of Lactobacillus plantarum ZZ2022-615 was inoculated at a 3% inoculation rate and cultured at 35℃ for 24h; in a sterile environment, the fermentation seed liquid of Saccharomyces cerevisiae ZYJM-15 was inoculated at a 4% inoculation rate and cultured at 28℃, 180rpm, pH 4.5, and shaking for 48h. The total biomass of the bacteria was (20.44±1.55)g / L, and the viable count of Lactobacillus plantarum was ≥9×10 8 CFU / ml, the number of live yeast cells ≥ 6×10 8 CFU / ml, and the iron enrichment rate reached (87.19±1.82)%.

[0065] Example 4: Preparation of composite microbial iron-enriched bacterial agent

[0066] Step 1: Bacteria Activation

[0067] The solid culture of Saccharomyces cerevisiae ZYJM-15 was inoculated into the malt juice liquid medium, and the culture was activated at 28°C and 180 rpm. This activation was repeated three times to obtain the seed liquid.

[0068] The solid culture of Lactobacillus plantarum ZZ2022-615 was inoculated into MRS broth medium for activation culture, and then cultured at 35°C for three times to serve as seed liquid.

[0069] Step 2: Seed cultivation

[0070] The saccharomyces cerevisiae seed liquid obtained in step 1) is inoculated into a seed bottle at an inoculation rate of 5%, the filling coefficient is 40 / 250 by volume, the formula of the seed culture medium is: 3% glucose, 2.4% yeast extract, 0.6% urea, 0.25% K2PO4, 0.05% MgSO4·7H2O, the fermentation conditions are: fermentation temperature 28°C, rotation speed 180rpm, pH value 4.5, culture time 44h, to obtain saccharomyces cerevisiae fermentation seed liquid;

[0071] The Lactobacillus plantarum seed liquid obtained in step 1) is inoculated into a seed bottle at a 3% inoculation rate, the filling coefficient is 60 / 250 volume ratio, the formula of the seed culture medium is: 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, and the fermentation conditions are: static culture at 35° C. for 24 hours to obtain Lactobacillus plantarum fermentation seed liquid;

[0072] Step 3: Co-cultivation in commercial fermenter

[0073] In a fermenter, 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, 0.6% urea and 1800 μg / ml of FeSO4·7H2O are added in a volume ratio of 30 / 50, and the mixture is sterilized at 121° C. for 30 min in the fermenter. After cooling, the plant lactobacillus fermentation seed liquid obtained in step 2) is inoculated in a 3% inoculation amount into the fermenter, and the mixture is statically cultured at 35° C. for 24 h; under sterile conditions, the brewer's yeast fermentation seed liquid is inoculated in a 5% inoculation amount into the fermenter, the fermentation temperature is 28° C., the rotation speed is 180 rpm, and the culture time is 44 h to obtain a composite microbial iron-enriched bacterial agent fermentation liquid.

[0074] The number of live yeast cells in the obtained high-efficiency iron-rich composite microbial agent was ≥8×10 8 CFU / ml, Lactobacillus plantarum ≥2.0×10 9 CFU / ml, Fe 2+ The enrichment rate reached over 87.17%.

[0075] Example 5: Effects of composite microbial iron-enriched agents on growth and health of weaned piglets

[0076] 1. Experimental design

[0077] 28-day-old weaned piglets were selected as the subjects and were assigned to the following three groups using a single-factor randomized experimental design: 1. Blank control group: basic diet + ferrous sulfate (containing 104 mg / kg of iron in feed); 2. Experimental group 1: basic diet + composite microbial iron-rich fermentation broth prepared in Example 4 of the present invention (containing 104 mg / kg of iron in feed); 3. Experimental group 2: basic diet + saccharomyces cerevisiae iron-rich fermentation broth (containing 104 mg / kg of iron in feed) (preparation method: saccharomyces cerevisiae fermentation seed liquid was obtained according to the method of Example 4, and then 3% glucose, 2.4% yeast extract, 0.6% urea, 0.25% K2PO4, 0.05% MgSO4·7H2O, and 0.05% FeSO4·7H2O were added to the fermentation tank. 900μg / ml, the loading coefficient is 30 / 50 volume ratio, add the culture medium, sterilize at 121℃ in the fermenter for 30min, and after cooling, add the fermentation seed liquid of brewer's yeast at a 5% inoculation amount, the fermentation temperature is 28℃, the rotation speed is 180rpm, the pH value is 4.5, and the culture time is 44h to obtain the brewer's yeast iron-rich fermentation liquid). Each group has 1 pig house, 3 replicates per group, and single-pen feeding is adopted, with 10 pigs in each replicate. The basic diet formula is described in Table 1 and Table 2. The piglet house was cleaned and disinfected before the start of the experiment. Feed once a day, free feeding, free drinking water from the automatic waterer, and natural light. The pigs were immunized using conventional immunization procedures. During the experiment, a dedicated person was responsible for feeding and managing the experimental piglets, and the health status of the piglets was carefully observed and recorded. The experimental period was 28d.

[0078] Table 1 Diet composition and nutritional level

[0079]

[0080] Table 2. Premix formulation

[0081]

[0082]

[0083] 2. Testing indicators and methods

[0084] 2.1 Growth-promoting effect

[0085] The mice were weighed on an empty stomach at the beginning of the experiment and before feeding in the morning of 28 days, and the average daily weight gain, average daily feed intake and feed-to-weight ratio were calculated.

[0086] Average daily weight gain = (final weight - initial weight) / number of days; average daily feed intake = total feed intake / number of days / number of heads;

[0087] Feed-to-weight ratio = average daily feed intake / average daily weight gain;

[0088] The diarrhea incidence of each group of piglets during the experiment was recorded to calculate the diarrhea rate, diarrhea rate = number of piglets with diarrhea / total number of piglets × 100%.

[0089] 2.2 Effects on improving immunity, antioxidant performance, and blood biochemical indicators

[0090] On the 28th day of feeding, 5 heads were randomly selected from each group, and 20 ml of blood was collected from the anterior vena cava. Part of the blood sample was immediately tested for red blood cells and hemoglobin. The other part was separated into serum, and the serum iron, total iron binding capacity, total protein, albumin, serum urea nitrogen, and ceruloplasmin activity were measured using a kit.

[0091] Total antioxidant capacity, malondialdehyde content, and glutathione peroxidase and total superoxide dismutase activities were determined using kits.

[0092] The kit was used to measure the contents of interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), immunoglobulin A (IgA), immunoglobulin G (IgG), and immunoglobulin M (IgM). The determination methods were carried out according to the instructions.

[0093] 3. Test results

[0094] 3.1 Effects of piglet growth performance and diarrhea rate

[0095] The results in Table 3 show that compared with the control group, the experimental groups can significantly increase the average daily weight gain of piglets, and the feed-to-weight ratio and diarrhea rate are significantly reduced. Compared with the control group, the addition of composite microbial iron-rich bacteria agent and brewer's yeast iron bacteria agent increased the average daily weight gain by 32.6% and 21.5%, respectively, the feed-to-weight ratio decreased by 22.15% and 15.19%, respectively, and the diarrhea rate decreased by 59.63% and 42.2%, respectively. The addition of composite microbial iron-rich bacteria agent is better than brewer's yeast iron bacteria agent.

[0096] Table 3 Effects of compound microbial agents on growth performance and diarrhea rate of piglets

[0097]

[0098]

[0099] Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (P<0.05), the same below.

[0100] 3.2 Effect of serum biochemical indicators

[0101] The number of red blood cells, hemoglobin and serum iron content in the blood are important indicators reflecting the iron metabolism and nutrition of piglets. The results in Table 4 show that compared with the control group, the addition of composite microbial iron-enriching bacteria and brewer's yeast iron-enriching bacteria increased the number of red blood cells, hemoglobin, serum iron and total iron binding capacity of piglets, which has a good effect on preventing anemia in piglets and ensuring their normal growth and development. The composite microbial iron-enriching bacteria is better than the brewer's yeast iron-enriching bacteria.

[0102] Serum biochemical indicators not only reflect the digestion, absorption and metabolism of nutrients by the animal body, but also indirectly reflect the health status of the animal body. The total protein, albumin and urea nitrogen contents are highly correlated with the body's protein absorption and metabolism. When the body has good protein metabolism, the total protein and albumin contents will increase, and the urea nitrogen content in the blood will decrease. The results in Table 4 show that the albumin and total protein contents of the test group 1 were higher than those of the control group and the test group 2, and the serum urea nitrogen contents of the test groups 1 and 2 were significantly lower than those of the control group, indicating that the addition of the composite microbial iron-rich bacteria agent increased the utilization rate of protein by piglets, promoted the synthesis of their own protein, and improved their growth performance.

[0103] Ceruloplasmin not only has antioxidant activity, but also directly affects the iron uptake of yeast. The results in Table 4 show that the activity of ceruloplasmin in Experimental Groups 1 and 2 was significantly higher than that in the control group, and the composite microbial iron-enriched bacteria agent was superior to the Saccharomyces cerevisiae iron bacteria agent.

[0104] Table 4 Effects of composite microbial iron-enriched agents on serum biochemical parameters of piglets

[0105]

[0106]

[0107] Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (P<0.05), the same below.

[0108] 3.3 Effect of antioxidant indicators

[0109] When the body is under oxidative stress, the content of free radicals in the body increases, and the free radicals react with lipids to produce cytotoxic malondialdehyde. Important antioxidant enzymes such as glutathione peroxidase and superoxide dismutase can remove free radicals in the body and restore the body to a normal physiological state. The results in Table 5 show that the addition of the composite microbial iron-rich bacterial agent of the present invention to the diet can significantly improve the antioxidant capacity of piglets, as well as the activity levels of glutathione peroxidase and superoxide dismutase in serum, compared with the other test groups, while the content of malondialdehyde is significantly reduced. The results show that the addition of the composite microbial iron-rich bacterial agent is beneficial to improving the antioxidant capacity of piglets.

[0110] Table 5 Effects of composite microbial iron-enriched bacteria on serum antioxidant indexes in piglets

[0111]

[0112] Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (P<0.05), the same below.

[0113] 3.4 Impact of immune indicators

[0114] Interleukin plays a role in information transmission between leukocytes or immune cells, and can regulate immune cells to produce immunoglobulins. Immunoglobulins are proteins with antibody activity in the body, which can bind to antigens, reduce damage to the body, and play an important role in inflammatory reactions. The results in Table 6 show that compared with the control group, the levels of IgG and IL-6 in the serum of test group 1 and test group 2 were significantly increased, and the content of TNF-α was significantly reduced, indicating that the addition of the composite microbial iron-rich bacterial agent of the present invention to the diet can regulate the immune function of piglets and improve the body's resistance to disease.

[0115] Table 6 Effects of compound microbial iron-enriched bacteria on immune indicators of piglets

[0116]

[0117]

[0118] Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (P<0.05), the same below.

[0119] Example 6: Effect of composite microbial iron-enriching agent on pig house air quality

[0120] On the central axis of each pig house, three points were selected at equal intervals, located in the east, middle (middle of the aisle), and west (near the pig house door), and two points in the vertical direction, close to the ground and 1.0m from the ground. The NH3, H2S, CH4, and N2O contents were measured three times a day, i.e. once at 8:00, 12:00, and 19:00. Before the measurement, the doors and windows of the pig house were closed, and the monitor was placed in the air. After the data stabilized, it was recorded and averaged. The average concentration of each group during the entire test period was calculated based on the daily concentration of each gas.

[0121] Compared with the other groups, the concentrations of harmful gases NH3, H2S, CH4, and N2O in the pig house were significantly reduced by adding the composite microbial iron-rich agent prepared in Example 4 of the present invention to the diet, achieving a coordinated reduction in odor and greenhouse gas emissions and greatly improving the breeding environment.

[0122] Table 7 Effects of composite microbial iron-enriched agents on air quality in pig houses

[0123]

[0124] Note: Different lowercase letters after the data in the same column indicate significant differences among treatments (P<0.05), the same below.

Claims

1. A composite microbial iron-rich bacterial agent, characterized in that: The saccharomyces cerevisiae ( Saccharomyces cerevisiae )ZYJM-15 and Lactobacillus plantarum with a deposit number of CCTCC No.M 2022915 ( Lactobacillus plantarum ) ZZ2022-615 co-cultured and fermented.

2. The composite microbial iron-rich bacterial agent according to claim 1, characterized in that: The number of live yeast cells is ≥8×10 8 CFU / ml, Lactobacillus plantarum ≥2.0×10 9 CFU / ml.

3. The method for preparing the composite microbial iron-rich agent according to claim 1 or 2, characterized in that: The following steps are involved: 1) Bacteria activation The solid cultures of Saccharomyces cerevisiae ZYJM-15 and Lactobacillus plantarum ZZ2022-615 were inoculated into the liquid culture medium respectively, and activated and cultured to obtain the respective seed liquids; 2) Seed cultivation The seed liquids of the two bacteria are respectively inoculated into the seed culture medium to obtain their respective fermentation seed liquids; 3) Fermentation tank co-culture Firstly, Lactobacillus plantarum fermentation seed liquid is introduced into a fermentation tank and statically cultured; then, Saccharomyces cerevisiae fermentation seed liquid is introduced into the fermentation tank and shaken cultured to obtain a composite microbial iron-rich bacterial agent fermentation liquid.

4. The method for preparing the composite microbial iron-rich agent according to claim 3, characterized in that: 1) Bacteria activation The solid culture of Saccharomyces cerevisiae ZYJM-15 was inoculated into the malt juice liquid medium, and the culture was activated by shaking at 28-32°C and 180-220 rpm. The activation was performed at least three times to serve as the seed liquid. The solid culture of Lactobacillus plantarum ZZ2022-615 was inoculated into MRS broth medium for activation culture, and then statically cultured at 35-37° C. The activation was performed at least three times to serve as seed liquid; 2) Seed cultivation The brewer's yeast seed liquid obtained in step 1) is inoculated into a seed bottle at an inoculation rate of 3-5%, and the filling coefficient is 40-50 / 250 by volume. The formula of the seed culture medium is: 3% glucose, 2.4% yeast extract, 0.6% urea, 0.25% K2PO4, 0.05% MgSO4·7H2O, and the fermentation conditions are: fermentation temperature 28-32°C, rotation speed 180-220 rpm, pH value 4.5-5.5, and culture time 36-48 h to obtain brewer's yeast fermentation seed liquid; The Lactobacillus plantarum seed liquid obtained in step 1) is inoculated into a seed bottle at an inoculation rate of 3-6%, and the filling coefficient is 40-60 / 250 by volume. The formula of the seed culture medium is: 10% brown sugar, 0.5% peptone, and 0.5% yeast extract powder. The fermentation conditions are: static culture at 35-37° C. for 24-40 h to obtain Lactobacillus plantarum fermentation seed liquid; 3) Fermentation tank co-culture In a fermenter, 10% brown sugar, 0.5% peptone, 0.5% yeast extract powder, 0.6% urea, and 1600 µg / ml FeSO4·7H2O are added in a volume ratio of 25-35 / 50, and the culture medium is sterilized at 121°C for 30 minutes in the fermenter. After cooling, the plant lactobacillus fermentation seed liquid obtained in step 2) is inoculated into the fermenter at an inoculation rate of 3-6%, and statically cultured at 35-37°C for 24-40 hours; under sterile conditions, the cerevisiae fermentation seed liquid obtained in step 2) is inoculated at an inoculation rate of 3-5%, the fermentation temperature is 28-32°C, the rotation speed is 180-220 rpm, and the culture time is 36-48 hours to obtain a composite microbial iron-rich bacterial agent fermentation liquid.

5. Use of the composite microbial iron-rich agent according to claim 1 or 2 in the preparation of a preparation having at least one function of promoting growth of weaned piglets, preventing anemia in piglets, improving immunity of piglets, improving antioxidant capacity of piglets, reducing diarrhea rate of piglets, reducing odor in pig houses and reducing greenhouse gas concentration in pig houses.

6. The use according to claim 5, characterized in that: The preparation was added into the daily feed of weaned piglets.

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