CN114868914A - A prebiotics and its preparation method - Google Patents

A prebiotics and its preparation method Download PDF

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CN114868914A
CN114868914A CN202210366346.8A CN202210366346A CN114868914A CN 114868914 A CN114868914 A CN 114868914A CN 202210366346 A CN202210366346 A CN 202210366346A CN 114868914 A CN114868914 A CN 114868914A
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metazoan
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CN114868914B (en
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彭孝雄
尹修权
徐彩虹
旷文丰
陈晓霞
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Guangzhou Zhengming Houshengyuan Technology Co ltd
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    • A23V2400/00—Lactic or propionic acid bacteria
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    • A23V2400/113—Acidophilus
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Abstract

The invention discloses a metazoan and a preparation method thereof, belonging to the technical field of biology. The preparation method of the anagen comprises the following steps: s1, inoculating Lactobacillus acidophilus into a lactic acid bacteria culture solution for culturing, culturing at 35-38 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 25-30 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35-38 ℃ to obtain a third fermentation solution; s2, filtering the third fermentation liquor to obtain supernatant, and concentrating the supernatant to obtain concentrated liquor; s3, adding gelatin into the concentrated solution, and then refrigerating at 5-10 ℃ to obtain a fourth fermentation liquid; s4, inactivating the fourth fermentation liquid. The invention also comprises the anagen prepared by the preparation method. The obtained Lactobacillus acidophilus has high content and the metazoan has high content of antibacterial peptide.

Description

A prebiotics and its preparation method
Technical Field
The invention relates to the technical field of biology, in particular to a metazoan and a preparation method thereof.
Background
The metazoan is a general name of the metabolite components of the probiotics after the probiotics are processed and treated, and comprises thalli and metabolites. Recent studies have found that inactivated probiotics (non-viable probiotics) and their lysates (lysates), extracts or isolates (known as postbiotics) have similar efficacy to probiotics and are more acid tolerant and easier to preserve than probiotics.
Lactobacillus acidophilus, one of lactic acid bacteria, produces many antibacterial substances in the metabolic process, mainly including acidic substances, antibacterial peptides, etc., and can effectively resist food deterioration and food-borne pathogenic microorganisms. The antibacterial peptide has broad-spectrum antibacterial activity and strong killing effect on bacteria, and particularly the killing effect on certain drug-resistant pathogenic bacteria draws more attention from people; the method is a technical problem which needs to be solved urgently in the prior art, the prior art promotes the production of the antibacterial peptide by adding salmonella into the lactic acid bacteria as a competitive relationship, but the survival rate of the lactic acid bacteria can be influenced in the competitive process, and further the yield of the antibacterial peptide can be influenced, and how to improve the yield of the antibacterial peptide while ensuring the number of lactobacillus acidophilus is a technical problem which needs to be solved urgently in the prior art.
Disclosure of Invention
The invention aims to overcome the technical defects, provides a metazoan and a preparation method thereof, and solves the technical problem of how to ensure the number of lactobacillus acidophilus and improve the yield of antibacterial peptide in the prior art.
In order to achieve the technical purpose, the technical scheme of the invention provides a preparation method of a metazoan, which comprises the following steps:
s1, inoculating lactobacillus acidophilus into a lactic acid bacteria culture solution for culturing, culturing at 35-38 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 25-30 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35-38 ℃ to obtain a third fermentation solution;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5-10 ℃ to obtain a fourth fermentation liquid;
s4, inactivating the fourth fermentation liquid.
Further, in step S1, the addition amount of citric acid is 0.1-0.5% of the volume of the first fermentation solution, and the concentration of citric acid is 0.3-0.5 mol/L.
Further, in step S1, the amount of the hydrolyzed protein added is 0.1-0.5% by mass of the first fermentation solution.
Further, in step S4, the gelatin is added in an amount of 0.5 to 1% by mass of the concentrated solution.
Further, after step S4, the method further includes: and step S5, adding chitosan into the inactivated fourth fermentation liquid, and then carrying out spray drying to obtain the secondary prebiotics.
Further, in step S5, the chitosan is added in an amount of 1-3% by mass of the fourth fermentation broth after inactivation.
Further, in step S3, the volume of the concentrated solution is 0.5 to 0.6 times the volume of the supernatant.
Further, in step S3, the refrigerating time is 30-60 min.
Further, in step S1, culturing at 35-38 deg.C for 2-3 days to obtain a first fermentation liquid; culturing at 25-30 deg.C for 3-5h to obtain second fermentation broth, and culturing at 35-38 deg.C for 1-2 days to obtain third fermentation broth.
In addition, the invention also provides a metazoan prepared by the preparation method.
Compared with the prior art, the invention has the beneficial effects that: inoculating Lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing at a higher temperature of 35-38 ℃ to be beneficial to the growth of the Lactobacillus acidophilus, adding citric acid and hydrolyzed protein into a first fermentation solution, continuously culturing at a lower temperature of 25-30 ℃ to obtain a second fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at a lower temperature of 35-38 ℃ to obtain a third fermentation solution, providing amino acid required for the generation of antibacterial peptide by the hydrolyzed protein, improving the stability of the Lactobacillus acidophilus by the citric acid, culturing at a low temperature and a high temperature to be beneficial to the growth of the Lactobacillus acidophilus again and promoting the generation of the antibacterial peptide, filtering and concentrating to obtain a concentrated solution, adding gelatin into the concentrated solution, refrigerating at a temperature of 5-10 ℃ to obtain a fourth fermentation solution, wherein the gelatin has an adhesive effect, the lactobacillus acidophilus inactivation method has the advantages that the position of lactobacillus acidophilus can be stabilized at a lower temperature, the inactivation accuracy can be improved by further performing inactivation treatment, the inactivation efficiency is further improved, and the lactobacillus acidophilus can be completely inactivated only by inactivating at 80-85 ℃ for 30-60min, so that the lactobacillus acidophilus with a large number of bacteria is obtained, and the content of antibacterial peptide in metazoan is high.
Detailed Description
The specific embodiment provides a preparation method of a metazoan, which comprises the following steps:
s1, inoculating Lactobacillus acidophilus into a lactic acid bacteria culture solution for culturing, culturing at 35-38 ℃ for 1-2 days to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 25-30 ℃ for 3-5h to obtain a second fermentation solution, and culturing the second fermentation solution at 35-38 ℃ for 1-2 days to obtain a third fermentation solution; the hydrolyzed protein is preferably wheat hydrolyzed protein; the addition amount of the citric acid is 0.1-0.5% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.3-0.5 mol/L; the addition amount of the hydrolyzed protein is 0.1-0.5% of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5-0.6 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5-10 ℃ for 30-60min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.5-1% of the mass of the third fermentation liquor;
s4, inactivating the fourth fermentation liquid at 80-85 ℃ for 30-60 min;
s5, adding chitosan into the inactivated fourth fermentation liquid, and then carrying out spray drying to obtain a secondary cell; the addition amount of the chitosan is 1-3% of the mass of the inactivated fourth fermentation broth. Chitosan enables the anagen to form microcapsules, the microcapsules can start to release the anagen when entering the intestinal tract, and the adhesion force with the intestinal tract is improved under the action of gelatin in the anagen, so that the action time of the anagen is prolonged, and the treatment effect on the intestinal inflammation is favorably improved.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the following examples, the lactic acid bacteria culture medium was composed of 1000ml of water, 10g of beef extract, 20g of peptone, 10g of yeast extract, 200g of tomato juice, 10g of glucose, 0.5ml of tween, 17g of calcium carbonate, 0.1g of bromocresol green, and 15g of agar. The Lactobacillus acidophilus in the following examples is a known strain from the prior art collected by itself.
Example 1
This example presents a prebiotic made by the following steps:
s1, mixing the powder with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing for 2 days at 35 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing for 3 hours at 25 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35 ℃ for 1 day to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.5 × 10 10 CFU/mL; the addition amount of the citric acid is 0.3% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.3 mol/L; the addition amount of the hydrolyzed protein is 0.4 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5 ℃ for 30min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.8 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 60 min;
s5, adding chitosan into the inactivated fourth fermentation broth, then carrying out spray drying to obtain 162g of metazoan, and repeating the method of the embodiment by adopting a dialysis concentration method in the prior art to obtain 0.923g of antibacterial peptide; the addition amount of the chitosan is 1% of the mass of the inactivated fourth fermentation broth.
The yield of the antimicrobial peptide was calculated to be 0.923g/(162g +0.923g) 100% ═ 0.567%.
Example 2
This example presents a metazoan prepared by the following steps:
s1, mixing the powder with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing at 37 ℃ for 1 day to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 30 ℃ for 5 hours to obtain a second fermentation solution, and culturing the second fermentation solution at 37 ℃ for 2 days to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.2 × 10 10 CFU/mL; the addition amount of the citric acid is 0.1% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.5 mol/L; the addition amount of the hydrolyzed protein is 0.2 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.6 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 10 ℃ for 60min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.5 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 85 ℃ for 45 min;
s5, adding chitosan into the inactivated fourth fermentation liquid, and then performing spray drying to obtain 158g of secondary prebiotics; the mass of the obtained antibacterial peptide obtained by repeating the method of the embodiment by adopting a dialysis concentration method of the prior art is 0.884 g; the addition amount of the chitosan is 2% of the mass of the inactivated fourth fermentation liquid.
The yield of antimicrobial peptide was calculated to be 0.884g/(158g +0.884g) × 100% — 0.556%.
Example 3
This example presents a prebiotic made by the following steps:
s1, mixing the powder with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing at 38 ℃ for 1.5 days to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 28 ℃ for 4 hours to obtain a second fermentation solution, and culturing the second fermentation solution at 38 ℃ for 1 day to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 2.9 × 10 10 CFU/mL; the addition amount of the citric acid is 0.5% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.4 mol/L; the addition amount of the hydrolyzed protein is 0.1 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.6 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 8 ℃ for 50min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 1% of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 85 ℃ for 40 min;
s5, adding chitosan into the inactivated fourth fermentation liquid, and then performing spray drying to obtain 162g of secondary prebiotics; the mass of the antibacterial peptide prepared by repeating the method of the embodiment by adopting a dialysis concentration method of the prior art is 0.824 g; the addition amount of the chitosan is 3% of the mass of the inactivated fourth fermentation liquid.
The yield of antimicrobial peptide was calculated to be 0.828g/(162g +0.824g) 100% — 0.506%.
Example 4
This example presents a prebiotic made by the following steps:
s1, mixing the powder with 10 4 Individual acidophilus milkInoculating bacillus into a lactic acid bacteria culture solution for culturing, culturing for 1 day at 36 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing for 5 hours at 27 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 36 ℃ for 2 days to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.1 × 10 10 CFU/mL; the addition amount of the citric acid is 0.4% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.4 mol/L; the addition amount of the hydrolyzed protein is 0.3 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 7 ℃ for 40min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.8 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 30 min;
s5, adding chitosan into the inactivated fourth fermentation liquid, and then carrying out spray drying to obtain 159g of secondary prebiotics; the mass of the antibacterial peptide prepared by repeating the method of the embodiment by adopting a dialysis concentration method in the prior art is 0.817 g; the addition amount of the chitosan is 3% of the mass of the inactivated fourth fermentation liquid.
The yield of antimicrobial peptide was calculated to be 0.817g/(159g +0.817g) 100% ═ 0.511%.
Example 5
This example presents a prebiotic made by the following steps:
s1, mixing the raw materials with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing at 37 ℃ for 1 day to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 25 ℃ for 4 hours to obtain a second fermentation solution, and culturing the second fermentation solution at 37 ℃ for 2 days to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.2 × 10 10 CFU/mL; the addition amount of the citric acid is the volume of the first fermentation liquid0.5% of (A), wherein the concentration of the citric acid is 0.3 mol/L; the addition amount of the hydrolyzed protein is 0.1 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5 ℃ for 30min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.6 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 50 min;
s5, adding chitosan into the inactivated fourth fermentation liquid, then carrying out spray drying to obtain 155g of secondary prebiotics, and repeating the method of the embodiment by adopting a dialysis concentration method of the prior art to obtain 0.851g of antibacterial peptide; the addition amount of the chitosan is 2% of the mass of the inactivated fourth fermentation liquid.
The yield of antimicrobial peptide was calculated to be 0.851g/(155g +0.851g) × 100% ═ 0.546%.
Comparative example 1
This comparative example differs from example 1 only in that: lactobacillus acidophilus was always cultured at 35 ℃ as follows:
this example presents a prebiotic made by the following steps:
s1, mixing the powder with 10 4 Inoculating Lactobacillus acidophilus into the lactobacillus culture solution, culturing at 35 deg.C for 2 days to obtain a third fermentation liquid; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.5 × 10 7 CFU/mL; the addition amount of the citric acid is 0.3% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.3 mol/L; the addition amount of the hydrolyzed protein is 0.4 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5 ℃ for 30min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.8 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 60 min;
s5, adding chitosan into the inactivated fourth fermentation broth, then performing spray drying to obtain 112g of secondary prebiotics, and repeating the method of the embodiment by adopting a dialysis concentration method in the prior art to obtain 0.512g of antibacterial peptide; the addition amount of the chitosan is 1% of the mass of the inactivated fourth fermentation broth.
The yield of antimicrobial peptide was calculated to be 0.512g/(112g +0.512g) × 100% — 0.457%.
Comparative example 2
This comparative example differs from example 1 only in that: citric acid and hydrolyzed protein are not added in the fermentation process, and the specific steps are as follows:
this example presents a prebiotic made by the following steps:
s1, mixing the raw materials with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing at 35 ℃ for 2 days to obtain a first fermentation solution, continuously culturing the first fermentation solution at 25 ℃ for 3 hours to obtain a second fermentation solution, and then culturing the second fermentation solution at 35 ℃ for 1 day to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.2 × 10 9 CFU/mL；
S2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5 ℃ for 30min to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.8 percent of the mass of the third fermentation liquid;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 60 min;
s5, adding chitosan into the inactivated fourth fermentation broth, then performing spray drying to obtain 145g of metagens, and repeating the method of the embodiment by adopting a dialysis concentration method of the prior art to obtain 0.312g of antibacterial peptide; the adding amount of the chitosan is 1% of the mass of the inactivated fourth fermentation liquor.
The yield of antimicrobial peptide was calculated to be 0.312g/(145g +0.312g) × 100% — 0.215%.
As can be seen from comparative example 1, the fermentation broth obtained without the preparation method of high and low temperature treatment has a small number of Lactobacillus acidophilus and a low yield of antibacterial peptide; it can also be seen from comparative example 2 that fermentation without the addition of citric acid and hydrolyzed protein had a smaller effect on the number of L.acidophilus but a larger effect on the yield of antimicrobial peptides.
Comparative example 3
This comparative example differs from example 1 in that no gelatin was added, as follows:
the comparative example proposes a prebiotic prepared by the steps of:
s1, mixing the powder with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing for 2 days at 35 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing for 3 hours at 25 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35 ℃ for 1 day to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.5 × 10 10 CFU/mL; the addition amount of the citric acid is 0.3 percent of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.3 mol/L; the addition amount of the hydrolyzed protein is 0.4 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, refrigerating the concentrated solution at 5 ℃ for 30min to obtain a fourth fermentation solution;
s4, inactivating the concentrated solution at 80 ℃ for 60 min;
s5, adding chitosan into the inactivated concentrated solution, then carrying out spray drying to obtain 163g of metazoan, and repeating the method of the embodiment by adopting a dialysis concentration method of the prior art to obtain 0.911g of antibacterial peptide; the addition amount of the chitosan is 1% of the mass of the inactivated fourth fermentation broth.
The yield of antimicrobial peptide was calculated to be 0.921g/(163g +0.921g) × 100% — 0.562%.
Comparative example 4
This comparative example differs from example 1 in that no refrigeration is performed, as follows:
the comparative example proposes a prebiotic prepared by the steps of:
s1, mixing the powder with 10 4 Inoculating lactobacillus acidophilus into a lactobacillus culture solution for culturing, culturing for 2 days at 35 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing for 3 hours at 25 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35 ℃ for 1 day to obtain a third fermentation solution; detecting the third fermentation liquid, the number of Lactobacillus acidophilus is 3.5 × 10 10 CFU/mL; the addition amount of the citric acid is 0.3% of the volume of the first fermentation liquid, and the concentration of the citric acid is 0.3 mol/L; the addition amount of the hydrolyzed protein is 0.4 percent of the mass of the first fermentation liquid;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution; the volume of the concentrated solution is 0.5 times of the volume of the supernatant;
s3, adding gelatin into the concentrated solution to obtain a fourth fermentation liquid; the adding amount of the gelatin is 0.8 percent of the mass of the third fermentation liquor;
s4, inactivating the fourth fermentation liquid at 80 ℃ for 60 min;
s5, adding chitosan into the inactivated fourth fermentation broth, then carrying out spray drying to obtain 163g of metagens, and repeating the method of the embodiment by adopting a dialysis concentration method in the prior art to obtain 0.923g of antibacterial peptide; the addition amount of the chitosan is 1% of the mass of the inactivated fourth fermentation broth.
The yield of the antimicrobial peptide was calculated to be 0.923g/(163g +0.923g) 100% ═ 0.563%.
The survival of Lactobacillus acidophilus in the fourth fermentation broth or concentrate after fire extinguishing in examples 1-5 and comparative examples 3 and 4 was examined and the results are shown in Table 1.
TABLE 1 survival of Lactobacillus acidophilus in fourth fermentation broth or concentrate after extinguishing fire in examples 1-5 and comparative example 3
Figure BDA0003587277680000121
As can be seen from Table 1, some of the Lactobacillus acidophilus bacteria were not completely inactivated after the inactivation of comparative example 3 without addition of gelatin, some of the Lactobacillus acidophilus bacteria were not completely inactivated after the inactivation of comparative example 4 without refrigeration, and all of the Lactobacillus acidophilus bacteria of examples 1 to 5 were inactivated.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. A preparation method of anagen is characterized by comprising the following steps:
s1, inoculating lactobacillus acidophilus into a lactic acid bacteria culture solution for culturing, culturing at 35-38 ℃ to obtain a first fermentation solution, adding citric acid and hydrolyzed protein into the first fermentation solution, continuously culturing at 25-30 ℃ to obtain a second fermentation solution, and culturing the second fermentation solution at 35-38 ℃ to obtain a third fermentation solution;
s2, filtering the third fermentation liquid to obtain a supernatant, and concentrating the supernatant to obtain a concentrated solution;
s3, adding gelatin into the concentrated solution, and then refrigerating at 5-10 ℃ to obtain a fourth fermentation liquid;
s4, inactivating the fourth fermentation liquid.
2. The method for preparing metazoan according to claim 1, wherein in step S1, the citric acid is added in an amount of 0.1-0.5% by volume of the first fermentation solution, and the concentration of the citric acid is 0.3-0.5 mol/L.
3. The method for producing metazoan according to claim 1, wherein the hydrolyzed protein is added in an amount of 0.1 to 0.5% by mass of the first fermentation solution in step S1.
4. The method for preparing metazoan according to claim 1, wherein the gelatin is added in an amount of 0.5-1% by mass of the concentrate in step S4.
5. The method for preparing an metagenin according to claim 1, further comprising, after step S4: and step S5, adding chitosan into the inactivated fourth fermentation liquid, and then carrying out spray drying to obtain the secondary prebiotics.
6. The method for producing metazoan according to claim 5, wherein the chitosan is added in an amount of 1-3% by mass of the inactivated fourth fermentation broth in step S5.
7. The method for producing metazoan according to claim 1, wherein the volume of the concentrated solution is 0.5 to 0.6 times the volume of the supernatant in step S3.
8. The method for preparing metazoan according to claim 1, wherein the time for cold storage is 30-60min in step S3.
9. The method for producing metazoan according to claim 1, wherein in step S1, the first fermentation solution is obtained by culturing at 35-38 ℃ for 1-2 days; culturing at 25-30 deg.C for 3-5h to obtain second fermentation broth, and culturing at 35-38 deg.C for 1-2 days to obtain third fermentation broth.
10. An anagen produced by the production method according to any one of claims 1 to 9.
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