Pediococcus acidilactici and application thereof in feed and food

Abstract

The invention discloses pediococcus acidilactici and application of the pediococcus acidilactici in feed and food, the pediococcus acidilactici HJ516 has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.31438, and the preservation date is July 24, 2024. The strain has the remarkable capability of resisting oxidation and regulating the immune system, so that the resistance of the organism is improved. The invention further discloses application of the pediococcus acidilactici HJ516 serving as an important functional component to preparation of feed for efficiently preventing and / or relieving and / or treating aquatic product death caused by the extreme environment. The invention also provides pediococcus acidilactici HJ516 as a functional probiotic, and the pediococcus acidilactici HJ516 has a remarkable effect of inhibiting amylase activity in preparation of wheat-based flour food. The application range of the pediococcus acidilactici is expanded, and the utilization value of the pediococcus acidilactici in the aspects of feed, food and the like is improved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Pediococcus acidilactici screened from yak milk cheese and application of the same in feed and food. Background Art

[0002] Lactic acid bacteria, as a type of microorganism widely distributed in animals, plants, fermented foods and dairy products, occupy an important position in biological research due to its unique sugar fermentation ability (producing lactic acid or other acids) and safety, stability and easy cultivation. In recent years, lactic acid bacteria have attracted widespread attention from the scientific community due to their many potential health benefits, such as regulating oxidative stress, enhancing immunity, and reducing inflammation. In particular, the colonization of lactic acid bacteria in the host intestine not only promotes the balance and homeostasis of the intestinal microecology, but also significantly improves intestinal health and effectively alleviates a variety of gastrointestinal diseases, becoming one of the important types of probiotics. In addition, probiotics can also respond to or affect the oxidative stress response in the human body by regulating the composition and function of the intestinal flora and the production of short-chain fatty acids.

[0003] As a major agricultural country, my country's rapid development of animal husbandry and fishery economy is accompanied by the continuous expansion of breeding scale, but it also brings about the problem of frequent animal diseases. Traditionally, the main way to prevent and control diseases is to add antibiotics to feed, which not only causes a certain degree of environmental pollution, but also leads to the accumulation of antibiotics in animals, posing a threat to the health of consumers at the top of the food chain. Therefore, there is a need for an additive that can replace antibiotics, which can achieve a green and pollution-free effect while preventing and controlling diseases more reasonably and effectively. Summary of the invention

[0004] The purpose of the present invention is to provide a Pediococcus acidilactici and its application in feed and food, wherein the strain has significant antioxidant and immune system regulation capabilities, thereby improving the body's resistance. It can replace antibiotics and be added to feed, achieving a green and pollution-free effect while more reasonably and effectively preventing and treating diseases.

[0005] To achieve the above-mentioned purpose, the solution of the present invention is: a Pediococcus acidilactici, wherein the Pediococcus acidilactici HJ516 is deposited in the China General Microbiological Culture Collection Center (CGMCC), with a deposit number of CGMCC No.31438 and a deposit date of July 24, 2024.

[0006] Furthermore, the Pediococcus acidilactici HJ516 is obtained by enriching, separating, purifying and extracting the traditional fermented yak milk cheese in Xining City, Qinghai Province.

[0007] Further, enrichment: In an ultra-clean workbench, 30 yak milk cheese samples were inoculated into sterile MRS broth at a 5% inoculum size, and cultured in a constant temperature incubator at 37°C for 18-24 hours until the MRS broth became turbid and white bacteria precipitated at the bottom, completing the enrichment.

[0008] Further, separation and purification: 1 mL of the enriched MRS broth suspension was taken and diluted to 10% with sterile saline under sterile conditions. -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 , take 10 respectively -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of the diluted bacterial suspension was spread on MRS plate medium, then cultured at 37°C for 24-48 h, and the colony morphology was observed and recorded; Pick colonies with different morphology on the plate and separate them by streaking. Repeat this process until pure single colonies with consistent morphology are obtained through separation and purification. Then perform Gram staining to eliminate non-target strains. Store the target strains that have not been eliminated in 50% glycerol at -80°C for future use.

[0009] The application of the above-mentioned Pediococcus acidilactici in feed comprises, by weight percentage, 55-65% corn flour, 25-35% soybean meal, 3-6% chicken bone meal, 0.5-2.5% Pediococcus acidilactici HJ516 and other substances.

[0010] Furthermore, the feed comprises, by weight percentage, 55-65% corn flour, 25-35% soybean meal, 3-6% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 0.5-2% Pediococcus acidilactici HJ516.

[0011] Furthermore, the feed comprises, by weight percentage, 60-63% corn flour, 28-31% soybean meal, 3-6% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 0.5-2% Pediococcus acidilactici HJ516.

[0012] Furthermore, the composition comprises, by weight percentage, 60.5% corn flour, 30% soybean meal, 5% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 2% Pediococcus acidilactici HJ516.

[0013] The use of the above-mentioned Pediococcus acidilactici in fermented food, wherein the fermented food comprises, by weight, 250g of high-gluten flour, 10g of sugar, 125g of water, 5g of yeast and 2.5g of Pediococcus acidilactici HJ516; The above raw materials are weighed, slowly stirred for 20 minutes until the dough is uniform, and fermented at 25° C. for 4 hours to obtain fermented dough; the fermented dough is divided, rolled into a ball, and placed in a proofing box at 38° C. and a humidity of 80% for 30 minutes after shaping, and finally placed in an oven and baked at 180° C. for 35 minutes to obtain bread.

[0014] After adopting the above scheme, the beneficial effects of the present invention are: 1. The present invention isolates a strain of Pediococcus acidilactici HJ516 from yak cheese for the first time. The strain has significant abilities of inhibiting amylase activity, resisting oxidation, regulating the immune system, and thus improving the body's resistance.

[0015] 2. The present invention also discloses that Pediococcus acidilactici HJ516 is used as an important functional ingredient in the preparation of feed for the efficient prevention and / or alleviation and / or treatment of aquatic deaths caused by extreme environments (high sugar, high salt, high cadmium). It has been verified that the strain has positive active functions on aquatic products, such as anti-oxidation, inhibition of enzyme activity, and improvement of body immunity, thereby improving the tolerance of animals to the environment. Moreover, as a biological agent, lactic acid bacteria have a relatively fast degradation rate in the natural environment and are not prone to drug resistance. Therefore, the risk of environmental pollution can be reduced.

[0016] 3. The present invention also provides the use of Pediococcus acidilactici HJ516 as a functional probiotic in the preparation of wheat-based noodle foods. It has been proven that it has a significant effect of inhibiting the activity of amylase and can improve the taste of noodle foods.

[0017] The invention expands the application scope of probiotics, especially Pediococcus acidilactici, and improves the utilization value thereof in functional foods, medicines, feeds and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figures are the growth conditions of Pediococcus acidilactici HJ516 of the present invention on MRS culture medium (A), blood plate culture medium (B) and the microscopic picture after Gram staining (C); Figure 2 Schematic diagram of the amylase inhibition ability of Pediococcus acidilactici HJ516 of the present invention; Figure 3 Schematic diagram of the antioxidant capacity of Pediococcus acidilactici HJ516 of the present invention; Figure 4is the average lymphocyte proliferation rate of Pediococcus acidilactici (1- represents its concentration of 104 CFU / g, 2- represents its concentration of 106 CFU / g).

[0019] Pediococcus acidilactici HJ516 is deposited in the China General Microbiological Culture Collection Center (CGMCC), the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit number is CGMCC No. 31438, and the deposit date is July 24, 2024. DETAILED DESCRIPTION

[0020] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0021] The present invention provides a Pediococcus acidilactici HJ516, which is deposited in the China General Microbiological Culture Collection Center (CGMCC), with a deposit number of CGMCC No.31438 and a deposit date of July 24, 2024.

[0022] Example 1: Preliminary screening of probiotics 1.1 Pretreatment of experimental materials Thirty samples containing lactic acid bacteria were collected from traditional fermented yak milk cheese, a local specialty food in Xining City, Qinghai Province.

[0023] The Qinghai-Tibet Plateau, as the world's third pole, is known as the "Roof of the World". Its special climatic and geographical environment, such as low oxygen, low temperature, low pressure, high altitude and high ultraviolet radiation, imposes unique biological restrictions on species. The organisms here have unique genetic characteristics through natural selection and genetic evolution. These conditions also have a great impact on the diversity and function of microorganisms including lactic acid bacteria. Although the Qinghai-Tibet Plateau is under extreme conditions all year round, it still has rich and unique biological resources and functional microorganisms. Therefore, the present invention selects yak cheese, a local specialty food from the Qinghai-Tibet Plateau, as a material, and screens out probiotics with multiple functional characteristics.

[0024] In the clean bench, the yak cheese sample was inoculated into sterile MRS (Lactobacilli MRS Broth) broth at a 5% inoculation rate and cultured at 37°C in a constant temperature incubator for 20 hours. When the MRS broth became turbid and white bacterial precipitates were present at the bottom, it indicated that good enrichment was achieved and the next step of separation and purification could be carried out.

[0025] 1.2 Isolation and purification of lactic acid bacteria Take 1 mL of the enriched MRS broth suspension and dilute it to 10% with sterile saline. -3 , 10-4 , 10 -5 , 10 -6 , 10 -7 , take 10 respectively -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of the diluted bacterial suspension was spread on MRS plate medium, then placed at a constant temperature of 37°C for 36 h, and the colony morphology was observed and recorded.

[0026] Pick colonies with different morphology on the plate and separate them by streaking. Repeat this process until pure single colonies with consistent morphology are obtained. Then perform Gram staining to eliminate non-target strains. Store the target strains that have not been eliminated in 50% glycerol at -80°C for later use.

[0027] Before starting the experiment, each bacterial culture was subcultured at least three times in MRS broth at 37°C with an interval of 24 h.

[0028] 1.3 DNA extraction of suspected lactic acid bacteria and 16S rDNA PCR amplification The purified suspected target strain was inoculated into MRS broth and cultured at 37°C for 20 h. DNA was then extracted using a bacterial genomic DNA extraction kit.

[0029] The extracted DNA was amplified by PCR, and the PCR reaction system was: Upstream primer 27F: (5'-AGAGTTTGATCCTGGCTCAG-3') 1 μL, Downstream primer 1492R: (5'-CTACGGCTACCTTGTTACGA-3') 1 μL, 2×Taqplus Buffer 12.5μL, Template DNA 1 μL, Sterile ddH2O was used to make up the system to 25 μL, and sterile ultrapure water was used instead of template DNA as a negative control.

[0030] Amplification conditions were as follows: 94°C for 5 min; 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, for a total of 29 cycles, with a final extension at 72°C for 5 min.

[0031] 1.4 Detection of PCR products by agarose gel electrophoresis 2.5 μL of PCR product was taken for agarose gel electrophoresis, and the amplified product with clear bands and no obvious tailing was selected for cleaning. The cleaned product was then connected to the pMD18-T vector and transformed into the competent Escherichia colitop10. The cloned sub-products with positive identification results were selected and sequenced by Beijing Qingke Biotechnology Co., Ltd. The successfully sequenced sequences were compared and analyzed using the BLAST (Basic Local Alignment Search Tool) program in NCBI.

[0032] Experimental results: After a series of screening and identification, a strain of bacteria selected from yak milk cheese was Pediococcus acidilactici, named Pediococcus acidilactici HJ516. The strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) with the deposit number CGMCC No.31438 and the deposit date of July 24, 2024.

[0033] Growth of Pediococcus acidilactici HJ516 on MRS medium Figure 1 (A) shows the growth of Figure 1 (B) Gram staining is shown in Figure 1 (C). The gene sequence of the lactic acid Pediococcus is shown in SEQ ID NO. 1. The blood plate culture medium results showed that the bacteria did not have hemolysis and had good safety.

[0034] Examples 2 to 6: Functional properties of lactic acid bacteria Lactic acid bacteria were cultured in MRS medium containing agar and MRS medium without agar, respectively, and the α-glucosidase and α-amylase inhibition tests of the Pediococcus acidilactici strain were further determined, and then its antioxidant capacity, improvement of body immunity, and its use in fermented food and feed were investigated.

[0035] Example 2: Determination of the ability of Pediococcus acidilactici to inhibit enzyme activity 2.1α-Glucosidase Inhibitory Activity 2.1.1 Preparation of CFS (cell-free supernatant) After the lactic acid Pediococcus HJ516 was cultured in MRS medium at 37°C for 18 hours, the cells were collected by centrifugation at 4°C and 8000 r / min for 10 minutes. The collected cells were washed twice with 0.1 mol / L sterile PBS (Phosphate Buffered Saline, pH 6.8), and then resuspended in sterile PBS to adjust the bacterial solution concentration to 1×10 9 CFU / mL, mix well. Then incubate at 37℃ for 12h, centrifuge at 4℃ and 8000r / min for 10min, collect the supernatant, filter with 0.22μm water microfiltration membrane to obtain CFS sample solution, and store at -80℃.

[0036] 2.1.2 Determination of α-glucosidase inhibitory activity The inhibitory ability of the samples on α-glucosidase activity was determined by referring to the method of Zhang Zhengkuan et al.

[0037] At 37°C, 100 μL of CFS sample solution was added to a 96-well plate, and the solution was diluted two-fold with PBS buffer (pH 7.3) and incubated for 5 min. Then add 5 μL of enzyme solution (reaction concentration is 0.05 U / mL), incubate at 37 °C for 30 min, and then add 50 μL of 4-nitrophenyl-aD-pyranoglucoside (reaction concentration is 500 μmol / L) to start the reaction. Immediately detect the absorbance value at 405nm as the data at 0 min. After incubation at 37 °C for 30 min, detect the absorbance value at 30 min of incubation. Take the difference between this value and the OD value at 0 min of incubation (AOD) as the final test result.

[0038] PBS buffer (pH 7.3) was used as blank sample, and the OD value of the blank sample was detected by the same method. 1 mg / mL acarbose was used as positive control.

[0039] Zhang Zhengkuan, Yang Feiyu, Liu Huaiqin, et al. Chemical components and α-glucosidase inhibitory activity of wax gourd seeds [J / OL]. Modern Food Science and Technology, 1-9 [2024-08-07]. 2.2 α-Amylase Inhibitory Activity The α-amylase inhibition rate was determined for the strains with better α-glucosidase inhibition rate.

[0040] 0.25 mL of CFS sample solution was mixed with 1 mg / mL α-amylase solution in a volume ratio of 1:1, incubated at 37 °C for 10 min, and then the reaction solution was added to 0.5 mL of 1.5% soluble starch solution at 37 °C and reacted at 37 °C for 5 min; Then add 1 mL of DNS solution (3,5-dinitrosalicylic acid solution), react in a boiling water bath for 5 minutes, and then quickly cool to room temperature; add 8 mL of PBS and let stand for 30 minutes, and measure the absorbance at 540 nm.

[0041] PBS solution (0.1 mol / L, pH 6.8) was used as a blank control for the α-amylase solution and the sample to be tested. Three parallels were set for each group. The inhibition rate is shown in the following formula:

[0042] Wherein, A is the sample group, containing CFS sample solution and α-amylase solution; B is the sample blank group, containing CFS sample solution but not α-amylase solution; C is the control group, containing α-amylase solution but not CFS sample solution; D is the blank group, containing neither CFS sample solution nor α-amylase solution, serving as a complete blank group.

[0043] Experimental results: The inhibitory effect of Pediococcus acidilactici HJ516 on the above two enzymes, such as Figure 2 shown.

[0044] Figure 2 The results showed that HJ516 has a certain inhibitory effect on the activity of α-glucosidase and α-amylase, so it has potential application value in low GI foods. HJ516 can help maintain the stability of post-meal energy release by slowing down the digestion and absorption of starch, thus providing consumers with a more balanced energy supply.

[0045] Example 3: Antioxidant effect of Pediococcus acidilactici The bacterial suspension cultured in MRS medium was centrifuged and collected, washed three times with sterile 0.01 M PBS buffer (pH = 7.2 ± 0.2), and then resuspended in sterile PBS solution to make the colony count about 1 × 10 8 CFU / mL. The cells were disrupted by ultrasonication in an ice bath (30 min, 800 W). The supernatant was collected as a cell-free extract at 12000 rpm and 4°C for 10 min.

[0046] 3.1 DPPH scavenging ability determination First, accurately weigh 0.008 g of DPPH powder and dissolve it in anhydrous ethanol to make a 0.2 mmol / L DPPH solution.

[0047] Experimental group: 0.5 mL of cell-free extract (1×10 8CFU / mL), add 0.5mL 0.2 mmol / L DPPH solution, react at room temperature in the dark for 30 min, then centrifuge and take the supernatant.

[0048] OD2 blank group: In order to eliminate the influence of the sample color on the absorbance, a control group was set up, in which 0.5 mL of sample solution was added with 0.5 mL of PBS solution and reacted under the same conditions.

[0049] OD3 standard set: add 0.5 mL of anhydrous ethanol to 0.5 mL of sample solution.

[0050] Take 200 μL of the supernatant and measure the absorbance of the experimental group at 517 nm, which is recorded as OD1. Each sample is repeated three times. The calculation formula is as follows: Clearance rate (%) = (1-(OD1-OD2) / OD3) × 100% Wherein, OD1: 0.5mL sample + 0.5mL DPPH; OD2: 0.5 mL sample + 0.5 mL PBS; OD3: 0.5 mL sample + 0.5 mL anhydrous ethanol. [1] Zhang Han, Chen Haijun, Long Xiaoyu, et al. Isolation and identification of a strain of Leuconostoc producing high-yield extracellular polysaccharide from beet molasses, optimization of fermentation process and study of its antioxidant activity[J]. Journal of Light Industry, 2024, 39(03): 9-20. 3.2 Determination of hydroxyl radical (OH·) scavenging ability Experimental group: 0.5 mL of cell-free extract and 1 mL of O-phenanthroline (diluted to a concentration of 0.1% with double distilled water), 1 mL of sterile PBS buffer (pH = 7.2±0.2), 0.5 mL of sterile ultrapure water, 1 mL of FeSO4 (2.5 mmol / L), and 1 mL of H2O2 (20 mmol / L) were mixed thoroughly and incubated at 37°C for 1.5 h.

[0051] Positive control group: LGG (Lactobacillus GG) was used as a positive control instead of the cell-free extract in the experimental group, and the method was the same as above.

[0052] OD2: Mix 1 mL H2O2 and 1 mL ultrapure distilled water, and the rest is added according to the experimental group, excluding cell-free extract or LGG.

[0053] OD3: Mix 0.5 mL of sample (cell-free extract or LGG) and 0.5 mL of ultrapure distilled water, and add the remaining reagents according to the experimental group, but do not add H2O2.

[0054] Absorbance measurement: The absorbance value of each reaction solution was measured at a wavelength of 536 nm using a spectrophotometer to reflect the degree of inhibition of the redox reaction by the cell-free extract. Three replicates were set for each sample. The calculation formula is as follows: Scavenging capacity = [(OD1-OD3) / (OD2-OD3)] × 100% Where: OD1: absorbance of the experimental group; OD2: absorbance caused by hydroxyl free radicals produced when H2O2 exists alone; OD3: the effect of cell-free extract or LGG itself on absorbance, used for correction; [2] He, ZS, Luo, H., Cao, CH, & Cui, ZW (2004). Photometric determination of hydroxyl free radical in Fenton system by brilliant green. Am. J. Chin. Clin. Med, 6, 236-243. Experimental results: The antioxidant capacity of Pediococcus acidilactici HJ516, such as Figure 3 shown.

[0055] As can be seen from the figure, P. acidilactici HJ516 exhibits strong scavenging ability against both free radicals, both reaching more than 80%. This indicates that the strain can effectively neutralize or inhibit the activity of free radicals, thereby reducing their oxidative damage to cells. Compared with commercially available P. acidilactici, P. acidilactici HJ516 exhibits better performance in antioxidant capacity. Compared with astaxanthin, an active substance recognized to have excellent antioxidant effects, the scavenging ability of P. acidilactici HJ516 against free radicals is second only to the former.

[0056] Example 4: Effect of Pediococcus acidilactici on Immunity Enhancement The method for determining the in vitro immune enhancement activity of Pediococcus acidilactici HJ516 in this example refers to the method of Yue Chanjuan with slight modifications.

[0057] Leukocyte separation and culture: Blood was collected from tilapia, diluted with an equal amount of Hank's solution, added to an equal amount of cell separation solution, centrifuged, and the leukocyte liquid was drawn with a sterile pipette, and then washed with Hank's solution. When the live cell count was greater than 90%, the cell concentration was adjusted to 2.5x10 6 mL -1 , add the cell solution to a 96-well cell culture plate, 100 μL / well.

[0058] Preparation of lactic acid bacteria Pediococcus HJ516 liquid: Prepared lactic acid Pediococcus liquid (viable bacteria count 108 CFU / g) were diluted with RMINI1640 culture medium to different concentrations.

[0059] Cell culture and stimulation: Add the diluted bacterial solution to the cell culture plate, 100 μL / well, and repeat 4 wells for each concentration. Set up a cell control well (BC) and add an equal volume of R / MINI1640 culture medium. No liquid is added to the blank zero well.

[0060] After culturing the cell plate at 37.5°C and 5% CO2 for 44 hours, 30 ml of MTT solution was added to each well and the culture was continued for 4 hours. The cell plate was taken out, 100 μL of DMSO lysis solution was added to each well, and the absorbance at 570 nm was measured after mixing to determine the safe concentration.

[0061] Lymphocyte proliferation experiment: The 96-well plate was divided into two parts, one of which was added with 20 μL of PHA solution as the stimulation group, and the other was added with 20 μL of cell growth medium as the control group.

[0062] Set up a cell control group (BC) and a blank zeroing well. Then add 100 μL of lactic acid Pediococcus solution of each concentration to each well, repeat 4 wells for each sample, and then add the prepared lymphocytes (80 mL / well) to each well. Cultivate under appropriate conditions, add 30L of MTT solution to each well after 48 hours, and then culture for another 4 hours. Add 100 μL of DMSO lysis solution to each well on the cell plate, mix well, and detect the absorbance value at 570 nm. Calculate the lymphocyte proliferation rate of polysaccharide according to the formula: Lymphocyte proliferation rate (%) = (OD 菌组 -OD 对照组 ) / (OD 对熙组 ) ×100% Lymphocyte proliferation refers to the use of stimulants to co-culture lymphocytes in vitro to cause cell differentiation and new DNA synthesis, thereby increasing the number of effector lymphocytes. It is one of the indicators for evaluating the body's immune capacity and is often used as an in vitro test method for the preliminary screening of immune-enhancing drugs.

[0063] Experimental results: Figure 4 As shown, Figure 4 The results of the average lymphocyte proliferation rate experiments of levamisole, Pediococcus acidilactici HJ516 and commercially available Pediococcus acidilactici are given in the table, where 1- represents a concentration of 10 4 CFU / g, 2- represents its concentration is 10 6 CFU / g.

[0064] Depend on Figure 4It can be seen that levamisole, as a common drug for improving immunity, shows the best ability to improve the proliferation of immune cells. The lactic acid Pediococcus HJ516 of the present invention shows a better lymphocyte proliferation ability at two different concentrations (104 CFU / g and 106 CFU / g), and its effect is lower than levamisole, but much higher than the commercially available lactic acid Pediococcus. Therefore, it can be concluded that the lactic acid Pediococcus HJ516 of the present invention can improve the proliferation and differentiation of immune cells, promote cytokine production and complement activation, regulate the immune system, and thus improve the body's resistance.

[0065] [3] Yue Chanjuan. Study on the immune enhancement and antioxidant activities of yam polysaccharides and schisandra polysaccharides and their selenized products [D]. Nanjing Agricultural University, 2017. Example 5: Application of Pediococcus acidilactici in feed Crucian carp (2-3 months old) were purchased from the market and cultured in different environments for 30 days. The mortality of tilapia was recorded: (1) Control group (normal water); (2) High cadmium group (water with a cadmium content of 450 mg / kg); (3) High sugar group (water with a glucose content of 20 g / kg); (4) High salt group (water with a NaCl content of 20 g / kg); (5) High arsenic group (water with an arsenic content of 450 mg / kg); (6) High mercury group (water with a mercury content of 450 mg / kg); (7) High lead group (water with a lead content of 450 mg / kg) The following feeds were fed during the rearing period, and the survival rate of tilapia during the rearing period was recorded.

[0066] Feed A: corn meal 62.5%, soybean meal 30%, chicken bone meal 5%, salt 0.5%, feed grade calcium carbonate 1%, feed grade calcium bicarbonate 1%.

[0067] Feed B: corn flour 62%, soybean meal 30%, chicken bone meal 5%, salt 0.5%, feed grade calcium carbonate 1%, feed grade calcium bicarbonate 1%, Pediococcus acidilactici HJ516 0.5%.

[0068] Feed C: corn flour 60.5%, soybean meal 30%, chicken bone meal 5%, salt 0.5%, feed grade calcium carbonate 1%, feed grade calcium bicarbonate 1%, Pediococcus acidilactici HJ516 2%.

[0069] Feed D: corn flour 60.5%, soybean meal 30%, chicken bone meal 5%, salt 0.5%, feed grade calcium carbonate 1%, feed grade calcium bicarbonate 1%, Pediococcus acidilactici (commercially available) 2%.

[0070] Feed E: corn meal 60.5%, soybean meal 30%, chicken bone meal 5%, salt 0.5%, feed grade calcium carbonate 1%, feed grade calcium bicarbonate 1%, enrofloxacin 2%.

[0071] Experimental results: Table 1: Survival rate of tilapia in different environments (%)

[0072] The breeding industry, especially the aquaculture industry, faces many water pollution problems.

[0073] As can be seen from Table 3, feeding feeds containing the lactic acid bacteria HJ516 of the present invention (feed B and feed C) can significantly improve the survival rate of aquatic products in harsh environments. In particular, it can improve the survival rate of aquatic products in high cadmium, high sugar, high salt (high sodium), and high mercury environments. Its effect is better than that of not adding probiotics (feed group A) and adding commercially available lactic acid bacteria (feed group D).

[0074] Enrofloxacin in feed E is a type of antibiotic, a quinolone antibacterial drug for livestock and aquatic products, and a broad-spectrum antibacterial agent that has antibacterial effects on Gram-positive bacteria, Gram-negative bacteria, and Mycoplasma. It has been used to control Vibrio and Escherichia coli diseases in farmed fish. Comparing feed group C with feed group E, it can be concluded that the lactic acid bacteria HJ516 screened in this experiment has a more significant ability to improve aquatic products' tolerance to heavy metal pollution than commonly used antibiotics.

[0075] In summary, the Pediococcus acidilactici HJ516 of the present invention has the ability to improve aquatic products' tolerance to various heavy metal pollutions, thereby improving the immunity of aquatic products to extreme growth environments.

[0076] Example 6: Application of Pediococcus acidilactici in fermented food Ratio 1: 250g high-gluten flour, 10g sugar, 125g water, 5g yeast, without adding Pediococcus acidilactici; Ratio 2: 250g high-gluten flour, 10g sugar, 125g water, 5g yeast, and 2.5g of the lactic acid bacteria HJ516 of the present invention; Ratio 3: 250g high-gluten flour, 10g sugar, 125g water, 5g yeast, and add 2.5g commercially available lactic acid bacteria.

[0077] The above raw materials are weighed by weight, slowly stirred for 20 minutes until the dough is uniform, and fermented at 25° C. for 4 hours to obtain fermented dough; then the fermented dough is divided, rolled into a ball, and after shaping, placed in a proofing box at 38° C. and a humidity of 80% for 30 minutes, and finally placed in an oven and baked at 180° C. for 35 minutes to obtain bread.

[0078] Sensory evaluation 20 food research and development personnel were selected as sensory evaluators to evaluate the sensory performance of the bread under the same environment, and the average value was selected as the test result. The sensory evaluation standard table and evaluation results are shown in Table 2 and Table 3 respectively.

[0079] Table 2 Sensory evaluation standards

[0080] Table 3 Sensory evaluation results

[0081] The results in Table 3 show that the bread with the addition of Pediococcus acidilactici HJ516 (ratio 2) has a better taste.

[0082] The above description is only a preferred embodiment of the present invention and is not a limitation on the design of this case. Any equivalent changes made based on the design key of this case shall fall within the protection scope of this case.

[0083] Sequence Listing Sequence Listing <110> Xiamen Aiyi Snacks Research Institute Co., Ltd. <120> A kind of Pediococcus acidilactici and its application in feed and food <160> 1 <170> WIPO Sequence 2.3.0 <210> 1 <211> 1446 <212> DNA <213> Pediococcus acidilactici HJ516 ( Pediococcus acidilactici ) <400> 1 atgccgtaaa cgatgattac taagtgttgg agggtttccg cccttcagtg ctgcagctaa 840 cgcattaagt aatccgcctg gggagtacga ccgcaaggtt gaaactcaaa agaattgacg 900 gggcccgca caagcggtgg agcatgtggt ttaattcgaa gctacgcgaa gaaccttacc 960 aggtcttgac atcttctgcc aacctaagag attaggcgtt cccttcgggg acagaatgac 1020 aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga 1080 gcgcaaccct tattactagt tgccagcatt cagttgggca ctctagtgag actgccggtg 1140 acaaaccgga ggaaggtggg gacgacgtca aatcatcatg ccccttatga cctgggctac 1200 acacgtgcta caatggatgg tacaacgagt cgcgaaaccg cgaggtttag ctaatctctt 1260 aaaaccattc tcagttcgga ctgtaggctg caactcgcct acacgaagtc ggaatcgcta 1320 gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg gccttgtaca caccgcccgt 1380 cacaccatga gagtttgtaa cacccaaagc cggtggggta accttttagg agctagccgt 1440 ctaagg 1446

Claims

1. A Pediococcus acidilactici, characterized in that: The Pediococcus acidilactici HJ516 is deposited in the China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC No.31438 and a deposit date of July 24, 2024.

2. The Pediococcus acidilactici according to claim 1, characterized in that: The Pediococcus acidilactici HJ516 is obtained by enriching, separating, purifying and extracting yak milk cheese traditionally fermented in Xining City, Qinghai Province.

3. The Pediococcus acidilactici according to claim 2, characterized in that The enrichment process is as follows: in an ultra-clean workbench, 30 yak cheese samples are inoculated into sterile MRS broth at a 5% inoculum size, and cultured at 37°C in a constant temperature incubator for 18-24 hours until the MRS broth becomes turbid and white bacteria are precipitated at the bottom, completing the enrichment.

4. The Pediococcus acidilactici according to claim 3, characterized in that: The separation and purification process is as follows: take 1 mL of the enriched MRS broth suspension, dilute it to 10% with sterile saline under sterile conditions, and -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 , take 10 respectively -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 100 μL of the diluted bacterial suspension was spread on MRS plate medium, then cultured at 37°C for 24-48 h, and the colony morphology was observed and recorded; Pick colonies with different morphology on the plate and separate them by streaking. Repeat this process until pure single colonies with consistent morphology are obtained through separation and purification. Then perform Gram staining to eliminate non-target strains. Store the target strains that have not been eliminated in 50% glycerol at -80°C for future use.

5. The use of Pediococcus acidilactici in feed according to claim 1, characterized in that: The feed comprises, by weight percentage, 55-65% corn flour, 25-35% soybean meal, 3-6% chicken bone meal, 0.5-2.5% Pediococcus acidilactici HJ516 and other substances.

6. The use according to claim 5, characterized in that: The feed comprises, by weight percentage, 55-65% corn flour, 25-35% soybean meal, 3-6% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 0.5-2% Pediococcus acidilactici HJ516.

7. The use according to claim 5, characterized in that: The feed comprises, by weight percentage, 60-63% corn flour, 28-31% soybean meal, 3-6% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 0.5-2% Pediococcus acidilactici HJ516.

8. The use according to claim 5, characterized in that: The feed comprises, by weight percentage, 60.5% corn flour, 30% soybean meal, 5% chicken bone meal, 0.5% salt, 1% feed-grade calcium carbonate, 1% feed-grade calcium bicarbonate and 2% Pediococcus acidilactici HJ516.

9. The use of Pediococcus acidilactici in fermented food according to claim 1, characterized in that: The fermented food comprises, by weight, 250 g of high-gluten flour, 10 g of sugar, 125 g of water, 5 g of yeast and 2.5 g of Pediococcus acidilactici HJ516; The above raw materials are weighed, slowly stirred for 20 minutes until the dough is uniform, and fermented at 25° C. for 4 hours to obtain fermented dough; the fermented dough is divided, rolled into a ball, and placed in a proofing box at 38° C. and a humidity of 80% for 30 minutes after shaping, and finally placed in an oven and baked at 180° C. for 35 minutes to obtain bread.

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