CN107815429B - Granulation-resistant lactobacillus preparation and preparation method thereof - Google Patents
Granulation-resistant lactobacillus preparation and preparation method thereof Download PDFInfo
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Abstract
The invention provides a pelletization-resistant lactobacillus preparation and a preparation method thereof, wherein the pelletization-resistant lactobacillus preparation comprises lactobacillus; the Lactobacillus is one or a combination of several of Enterococcus faecalis, Enterococcus faecium , Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus Lactis, Lactobacillus buchneri, Lactobacillus casei and Bifidobacterium animalis; the viable count of the lactobacillus reaches 1.0 multiplied by 1010‑1.0×1011CFU/g. The preparation method of the lactobacillus preparation provided by the invention improves the tolerance of the thallus and avoids the loss of the activity of the thallus in the processing process.
Description
Technical Field
The invention relates to a probiotic preparation, in particular to a pelletization-resistant lactobacillus preparation and a preparation method thereof.
Background
The lactobacillus preparation is a very effective live bacterium preparation for livestock and poultry breeding, and can improve the digestion and health level of livestock and poultry. Lactic acid bacteria are generally sensitive to both heat and oxygen. The culture temperature of the lactic acid bacteria is generally 35-40 ℃, and many lactic acid bacteria die in a high-temperature environment above 40 ℃. Therefore, when the temperature of the high-temperature solid medium in a steam-sterilized state or the like is not lowered to less than 40 ℃ by cooling, it is impossible to add lactic acid bacteria, and since it takes a certain time to cool the solid medium (the time required for cooling varies depending on the amount of the solid medium), it takes a long time to produce a feed.
The solid medium to which lactic acid bacteria are added is generally stored in silage, and it takes 2 to 3 months to ferment lactic acid, and the time taken to sterilize the solid medium, so that the production of lactic acid fermented feed is a long-term process.
In addition, when lactic acid is fermented in a temperature environment suitable for lactic acid fermentation such as a fermentation chamber, it takes at least more than 24 hours to complete the lactic acid fermentation and the time taken to sterilize the solid medium, and therefore, it takes at least 2 days or more to produce the lactic acid-fermented feed, resulting in a reduction in the production efficiency of the feed.
As described above, there are problems that the lactic acid fermentation feed cannot be produced by a routine procedure every day (1 unit per 1 day), and that the management is very complicated. Since the feed is expensive due to the above reasons, it is practically difficult to popularize the lactic acid fermented feed even when the lactic acid fermented feed is used as a food for feeding livestock such as cattle and pigs, and the feed is well fattened and has a high-grade meat quality.
Patent document 1 discloses a method for producing bean curd refuse containing lactic acid bacteria by lactic acid fermentation, in which a culture medium containing lactic acid bacteria is sterilized at a temperature of 100 ℃ or higher, thereby causing a large amount of lactic acid bacteria in a culture area to die, and the lactic acid bacteria that have survived are not sufficiently effective. Patent document 2 discloses a method for preparing heat-resistant lactic acid bacteria.
Patent document 1: japanese patent laid-open No. 2005-13206
Patent document 2: japanese patent laid-open No. 2001-292763
Disclosure of Invention
In view of the above, the present invention aims to provide an efficient lactobacillus preparation and a preparation method thereof, so as to solve the problems of poor heat stability and low granulation survival rate of lactobacillus.
A lactobacillus preparation comprises lactobacillus; the Lactobacillus is one or a combination of several of Enterococcus faecalis (Enterococcus faecalis), Enterococcus faecium (Enterococcus faecalis), Lactobacillus plantarum (Lactobacillus plantarum), Lactobacillus acidophilus (Lactobacillus acidophilus), Lactobacillus Lactis (Lactobacillus Lactis), Lactobacillus buchneri (Lactobacillus buchneri), Lactobacillus casei (Lactobacillus casei) and Bifidobacterium animalis (Bifidobacterium animalis); the viable count of the lactobacillus reaches 1.0 multiplied by 1010-1.0×1011CFU/g。
The invention also aims to provide a preparation method of the lactobacillus preparation, which comprises the following steps:
(1) strain activation
Thawing enterococcus faecalis, enterococcus faecium, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus lactis, lactobacillus buchneri, lactobacillus casei and bifidobacterium animalis stored in a glycerol tube, performing streak separation on an MRS plate under an aseptic condition, culturing for 48h at 35 ℃, selecting a single colony from the MRS plate, performing slant streak, culturing for 16h at 35 ℃, and preserving for later use;
(2) first stage culture
Respectively taking the vigorously growing inclined planes, scraping 1-ring bacteria in 100mL of primary culture medium by using a sterilized inoculating ring, and culturing at 35 ℃ for 10-20h to obtain primary culture solution of each lactic acid bacteria;
(3) second stage culture
Respectively inoculating the primary culture solution of each lactobacillus into a secondary culture medium of a 50L fermentation tank, wherein the inoculation amount is 1-5%, the liquid loading amount is 25-35L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and the secondary seed solution is obtained after culturing for 8-15 h;
(4) three-stage culture
Inoculating two or more secondary seed liquids of enterococcus faecalis, enterococcus faecium, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus lactis, lactobacillus buchneri, lactobacillus casei and bifidobacterium animalis into a three-stage culture medium of a 20-ton fermentation tank according to the inoculation amount of 1% (v/v), wherein the total inoculation amount is 4-12%, the liquid loading amount is 50-70L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and the three-stage fermentation liquid is obtained after culturing for 48-72 h;
(5) preparation of wet lactobacillus powder
Centrifuging the tertiary fermentation liquor at 8000r/min for 30min to obtain lactobacillus active bacteria mud, mixing the lactobacillus active bacteria mud and a protective agent according to the proportion of (0.5-2) to (1.5-3) for 30min to obtain high-concentration lactobacillus suspension, adding 20-50 parts of soluble starch in a mixer, and mixing for 5min to obtain wet lactobacillus powder;
(6) preparation of wet lactic acid bacteria granule
Adding the wet lactobacillus powder into a granulator, adjusting the granularity to 20-40 meshes to obtain microcapsule master batches of 20-40 meshes, adding the microcapsule master batches into a QZL-1300 type spherical shot blasting machine for granulation, wherein the rotating speed is 500r/min, atomizing in the granulation process, spraying a coating agent at the speed of 65mL/min according to the ratio of the lactobacillus powder to the coating agent of 1:5, and finally obtaining wet lactobacillus granules;
(7) preparation of lactic acid bacteria preparation
Drying wet lactobacillus granules in a fluidized bed for 15min, wherein the air inlet temperature is 65 ℃ and the air outlet temperature is 35 ℃; starting a side spraying coating device, spraying the coating solution to the lactobacillus wet granules at the speed of 30mL/min to form a coating, and preparing the lactobacillus preparation.
Further, the primary culture medium and the secondary culture medium comprise the following specific components in percentage by weight: 1-2.5% of glucose, 0.5-1.5% of trisodium citrate, 0.5-2% of corn flour, 1-2% of tryptone, 0.2-1% of anhydrous sodium acetate, 0.01-0.05% of dipotassium phosphate, 0.01-0.05% of monopotassium phosphate, 0.01-0.1% of magnesium sulfate, 0.01-0.05% of manganese sulfate, 0.02-0.08% of ferrous sulfate, 0.02-0.2% of calcium carbonate, 800.8-1.2% of Tween and the balance of water, wherein the pH value is 6.2-7.2; preferably: 1.25% of glucose, 0.8% of trisodium citrate, 1.5% of corn flour, 1.2% of tryptone, 0.4% of anhydrous sodium acetate, 0.03% of dipotassium phosphate, 0.01% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.02% of manganese sulfate, 0.02% of ferrous sulfate, 0.45% of calcium carbonate, 801.1% of tween and the balance of water, wherein the pH value is 6.5.
Further, the three-stage fermentation medium comprises the following specific components in percentage by weight: molasses 0.5-2.5%, cane sugar 0.5-1.5%, peptone 1-2%, corn steep liquor dry powder 0.5-1%, yeast extract powder 0.5-1%, sodium acetate 0.2-1%, ammonium chloride 0.01-0.05%, vitamin C0.01-0.1%, sodium chloride 0.1-0.5%, magnesium chloride 0.01-0.1%, dipotassium hydrogen phosphate 0.01-0.05%, potassium dihydrogen phosphate 0.01-0.05%, Tween 801-1.5%, and water in balance, and pH 6.2-7.2; preferably: molasses 2.25%, cane sugar 0.75%, peptone 1.25%, corn steep liquor dry powder 0.5%, yeast extract powder 0.8%, sodium acetate 0.45%, ammonium chloride 0.04%, vitamin C0.02%, sodium chloride 0.3%, magnesium chloride 0.05%, dipotassium hydrogen phosphate 0.02%, potassium dihydrogen phosphate 0.01%, Tween 801.1%, and water in balance, and the pH value is 6.5.
Further, the protective agent comprises the following specific components in parts by weight: 2-10 parts of trehalose, 1-5 parts of sorbitol, 0.5-2.5 parts of bovine serum albumin, 1-4 parts of gelatin, 5-15 parts of skim milk, 2-8 parts of sodium glutamate, 800.8-2 parts of tween, 1-2.5 parts of glycerol, 0.1-1.0 part of vitamin D, 1-8 parts of malto-oligosaccharide, 1-8 parts of xylo-oligosaccharide, 1-8 parts of glucan, 0.5-5 parts of polyethylene glycol, 0.1-1.0 part of vitamin C, 0.1-1.0 part of ethylene diamine tetraacetic acid and 20-40 parts of water; sequentially adding the substances into water, stirring while adding, and stirring for 60min to dissolve completely to obtain protectant; preferably: 7.5 parts of trehalose, 2 parts of sorbitol, 1.25 parts of bovine serum albumin, 2.4 parts of gelatin, 8 parts of skim milk, 6.8 parts of sodium glutamate, 801.2 parts of tween, 1.5 parts of glycerol, 0.5 part of vitamin D, 4.5 parts of malto-oligosaccharide, 6 parts of xylo-oligosaccharide, 8 parts of glucan, 2.6 parts of polyethylene glycol, 0.35 part of vitamin C, 0.6 part of ethylene diamine tetraacetic acid and 35 parts of water.
Further, the coating agent comprises the following specific components in parts by weight: 1-10 parts of hydroxypropyl methyl cellulose, 2-5 parts of polyethylene glycol, 4-8 parts of gelatin, 1-3 parts of polyvinylpyrrolidone, 1-5 parts of enteric acrylic resin, 2-6 parts of cellulose acetate, 2-10 parts of beta-cyclodextrin and 5-30 parts of water; preferably: 6 parts of hydroxypropyl methyl cellulose, 2.5 parts of polyethylene glycol, 5.8 parts of gelatin, 1.25 parts of polyvinylpyrrolidone, 2.2 parts of enteric acrylic resin, 3.5 parts of cellulose acetate, 4.8 parts of beta-cyclodextrin and 12.5 parts of water; sequentially adding the above components into water, stirring for 15min, and mixing completely.
Further, the coating solution comprises the following specific components in parts by weight: 2-10 parts of corn starch, 1-5 parts of mannitol, 2-10 parts of sodium carboxymethylcellulose, 2-10 parts of skimmed milk powder, 1-15 parts of fructo-oligosaccharide and 8-40 parts of water; preferably: 8 parts of corn starch, 3.2 parts of mannitol, 2 parts of sodium carboxymethylcellulose, 5.6 parts of skimmed milk powder, 8.8 parts of fructo-oligosaccharide and 15 parts of water; sequentially adding the above components into water, stirring for 20min, and mixing completely.
The invention further provides application of the pelletization-resistant lactic acid bacteria preparation in preparation of animal feed.
The invention has the beneficial effects that:
the preparation method of the lactobacillus preparation provided by the invention has the advantages that the tolerance of thalli is improved, and the loss of viable bacteria in the processing process is avoided;
the invention coats the lactobacillus by various protective agents and coating agents, thereby greatly improving the temperature resistance of the lactobacillus.
Detailed Description
The present invention is explained below with reference to examples, which are merely illustrative of the present invention. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention.
EXAMPLE 1 preparation of lactic acid bacteria preparation
(1) Strain activation
Thawing lactic acid bacteria (enterococcus faecalis, enterococcus faecium, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus buchneri, Lactobacillus casei, and Bifidobacterium animalis) stored in glycerin tube, performing streak separation on MRS plate under aseptic condition, culturing at 35 deg.C for 48 hr, selecting single colony from MRS plate, performing slant streak, culturing at 35 deg.C for 16 hr, and preserving.
(2) First stage culture
Respectively taking the vigorously growing inclined planes, scraping 1-ring bacteria by using a sterilized inoculating ring, and culturing in 100mL of primary culture medium at 35 ℃ for 10-20h to obtain primary culture solution of each lactic acid bacteria.
(3) Second stage culture
Respectively inoculating the primary culture solution of each lactobacillus into a secondary culture medium of a 50L fermentation tank, wherein the inoculation amount is 1-5%, the liquid loading amount is 25-35L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and culturing for 8-15h to obtain a secondary seed solution.
(4) Three-stage culture
Inoculating the secondary seed liquid in enterococcus faecalis, enterococcus faecium, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus lactis, lactobacillus buchneri, lactobacillus casei and bifidobacterium animalis into a 20-ton tertiary fermentation culture medium according to the inoculation amount of 1% (v/v) (each bacterium is in the same proportion), wherein the total inoculation amount is 4-12%, the liquid loading amount is 50-70L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and the tertiary fermentation liquid is obtained after culturing for 48-72 h.
(5) Preparation of wet lactobacillus powder
Centrifuging the tertiary fermentation liquid to obtain lactobacillus active bacteria mud, mixing with protective agent at a ratio of 1:1.8 for 30min to obtain high-concentration lactobacillus suspension, adding soluble starch (purchased from Nanjing Sen Bega Biotech limited) 20-50 parts in a mixer, and mixing for 5min to obtain lactobacillus wet bacteria powder.
(6) Preparation of wet lactic acid bacteria granule
Adding the wet lactobacillus powder into a granulator, adjusting the granularity to 20-40 meshes to obtain microcapsule master batches with the granularity of 20-40 meshes, adding the microcapsule master batches into a QZL-1300 type spherical shot blasting machine for granulation, wherein the rotating speed is 500r/min, atomizing in the granulation process, spraying a coating agent at the speed of 65mL/min according to the ratio of the lactobacillus powder to the coating agent of 1:5, and finally obtaining the wet lactobacillus granules.
(7) Preparation of lactic acid bacteria preparation
Drying wet lactobacillus granules in a fluidized bed for 15min, wherein the air inlet temperature is 65 ℃ and the air outlet temperature is 35 ℃; starting the side-spraying coating device, spraying the coating solution to the material at a speed of 30mL/min to form a coating, and making into lactobacillus preparation.
The first-stage culture medium and the second-stage culture medium comprise the following specific components in percentage by weight: 1-2.5% of glucose, 0.5-1.5% of trisodium citrate, 0.5-2% of corn flour, 1-2% of tryptone, 0.2-1% of anhydrous sodium acetate, 0.01-0.05% of dipotassium phosphate, 0.01-0.05% of monopotassium phosphate, 0.01-0.1% of magnesium sulfate, 0.01-0.05% of manganese sulfate, 0.02-0.08% of ferrous sulfate, 0.02-0.2% of calcium carbonate, 800.8-1.2% of Tween, and the balance of water, wherein the pH value is 6.2-7.2. Preferably: 1.25% of glucose, 0.8% of trisodium citrate, 1.5% of corn flour, 1.2% of tryptone, 0.4% of anhydrous sodium acetate, 0.03% of dipotassium phosphate, 0.01% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.02% of manganese sulfate, 0.02% of ferrous sulfate, 0.45% of calcium carbonate, 801.1% of tween and the balance of water, wherein the pH value is 6.5.
The three-stage fermentation medium comprises the following specific components in percentage by weight: molasses 0.5-2.5%, cane sugar 0.5-1.5%, peptone 1-2%, corn steep liquor dry powder 0.5-1%, yeast extract powder 0.5-1%, sodium acetate 0.2-1%, ammonium chloride 0.01-0.05%, vitamin C0.01-0.1%, sodium chloride 0.1-0.5%, magnesium chloride 0.01-0.1%, dipotassium hydrogen phosphate 0.01-0.05%, potassium dihydrogen phosphate 0.01-0.05%, Tween 801-1.5%, and water in balance, and pH 6.2-7.2. Preferably: molasses 2.25%, cane sugar 0.75%, peptone 1.25%, corn steep liquor dry powder 0.5%, yeast extract powder 0.8%, sodium acetate 0.45%, ammonium chloride 0.04%, vitamin C0.02%, sodium chloride 0.3%, magnesium chloride 0.05%, dipotassium hydrogen phosphate 0.02%, potassium dihydrogen phosphate 0.01%, Tween 801.1%, and water in balance, and the pH value is 6.5.
The protective agent comprises the following specific components in parts by weight: 2-10 parts of trehalose, 1-5 parts of sorbitol, 0.5-2.5 parts of bovine serum albumin, 1-4 parts of gelatin, 5-15 parts of skim milk, 2-8 parts of sodium glutamate, 800.8-2 parts of tween, 1-2.5 parts of glycerol, 0.1-1.0 part of vitamin D, 1-8 parts of malto-oligosaccharide, 1-8 parts of xylo-oligosaccharide, 1-8 parts of glucan, 0.5-5 parts of polyethylene glycol, 0.1-1.0 part of vitamin C, 0.1-1.0 part of ethylene diamine tetraacetic acid and 20-40 parts of water. Adding the above materials into water in sequence, adding under stirring, and stirring for 60min to dissolve completely to obtain the protectant. Preferably: 7.5 parts of trehalose, 2 parts of sorbitol, 1.25 parts of bovine serum albumin, 2.4 parts of gelatin, 8 parts of skim milk, 6.8 parts of sodium glutamate, 801.2 parts of tween, 1.5 parts of glycerol, 0.5 part of vitamin D, 4.5 parts of malto-oligosaccharide, 6 parts of xylo-oligosaccharide, 8 parts of glucan, 2.6 parts of polyethylene glycol, 0.35 part of vitamin C, 0.6 part of ethylene diamine tetraacetic acid and 35 parts of water.
The coating agent comprises the following specific components in parts by weight: 1-10 parts of hydroxypropyl methyl cellulose, 2-5 parts of polyethylene glycol, 4-8 parts of gelatin, 1-3 parts of polyvinylpyrrolidone, 1-5 parts of enteric acrylic resin, 2-6 parts of cellulose acetate, 2-10 parts of beta-cyclodextrin and 5-30 parts of water. Preferably: 6 parts of hydroxypropyl methyl cellulose, 2.5 parts of polyethylene glycol, 5.8 parts of gelatin, 1.25 parts of polyvinylpyrrolidone, 2.2 parts of enteric acrylic resin, 3.5 parts of cellulose acetate, 4.8 parts of beta-cyclodextrin and 12.5 parts of water; sequentially adding the above components into water, stirring for 15min, and mixing completely.
The coating solution comprises the following specific components in parts by weight: 2-10 parts of corn starch, 1-5 parts of mannitol, 2-10 parts of sodium carboxymethylcellulose, 2-10 parts of skimmed milk powder, 1-15 parts of fructo-oligosaccharide and 8-40 parts of water. Preferably: 8 parts of corn starch, 3.2 parts of mannitol, 2 parts of sodium carboxymethylcellulose, 5.6 parts of skimmed milk powder, 8.8 parts of fructo-oligosaccharide and 15 parts of water; sequentially adding the above components into water, stirring for 20min, and mixing completely.
EXAMPLE 2 Lactobacillus preparation resistance to Artificial gastric juice and Artificial intestinal juice
The food stays in the animal stomach for 1-2 hours, the test places the lactobacillus preparation in simulated artificial gastric juice (pH1.5, pH2.5, pH3.5, pH4.5), treats for 3 hours, and detects the activity once per hour, and the influence of the artificial gastric juice on the lactobacillus activity of the lactobacillus preparation is shown in Table 1. As can be seen from Table 1, when the pH of the artificial gastric juice is 4.5, the survival rate of the lactic acid bacteria reaches 95.05% after 3 hours of treatment; when the pH value of the artificial gastric juice is 3.5, the survival rate of the lactobacillus reaches 93.75 percent after the artificial gastric juice is treated for 3 hours; when the pH value of the artificial gastric juice is 2.5, the survival rate of the lactobacillus reaches 91.11 percent after the artificial gastric juice is treated for 3 hours; when the pH value of the artificial gastric juice is 1.5, the survival rate of the lactobacillus reaches 90.65 percent after the artificial gastric juice is treated for 3 hours.
TABLE 1 Effect of artificial gastric juice on Lactobacillus Activity of Lactobacillus preparation
The lactobacillus preparation was treated in a simulated artificial intestinal juice (ph6.8) for 3 hours, and the activity was measured every hour, and the effect of the artificial intestinal juice on the lactobacillus activity of the lactobacillus preparation is shown in table 2. As can be seen from Table 2, the survival rate of lactic acid bacteria reached 99.5% after the artificial intestinal juice was treated for 3 hours.
TABLE 2 Effect of artificial intestinal juice on Lactobacillus Activity of Lactobacillus preparation
EXAMPLE 3 measurement of bile salt resistance of lactic acid bacterium preparation
The lactobacillus preparation was treated in bile salt solutions of different concentrations (0.03%, 0.15%, 0.3%, 0.5%) for 3h, and the activity was measured every hour, and the effect of bile salts on the lactobacillus activity of the lactobacillus preparation is shown in table 3. As can be seen from Table 3, when the concentration of bile salts is 0.03%, the survival rate of the lactic acid bacteria reaches 100% after 3 hours of treatment; when the concentration of the bile salt is 0.15%, the survival rate of the lactobacillus reaches 99.5% after the treatment for 3 hours; when the concentration of the bile salt is 0.3%, the survival rate of the lactobacillus reaches 95.26% after the treatment for 3 hours; when the concentration of the bile salt is 0.5%, the survival rate of the lactobacillus reaches 93.14% after the treatment for 3 hours.
TABLE 3 Effect of bile salts on Lactobacillus Activity of Lactobacillus preparation
EXAMPLE 4 measurement of temperature resistance of lactic acid bacterium preparation
In the experiment, the survival rate of the lactobacillus is detected by artificially simulating a high-temperature and high-humidity environment to treat the lactobacillus preparation, and the results are shown in table 4. As can be seen from Table 4, the survival rate of the lactic acid bacteria reaches 100% after the high-temperature treatment at 85 ℃ and 95 ℃ for 6 min; the high temperature treatment is carried out for 6min at 105 ℃, and the survival rate of the lactic acid bacteria reaches 98.60%; the survival rate of the lactobacillus reaches 97.21 percent after being treated for 6min at the high temperature of 115 ℃. The survival rate of the lactobacillus reaches 90.25 percent after being treated for 1min at high temperature and high humidity of 85 ℃; the survival rate of the lactobacillus reaches 88.37 percent after the high-temperature and high-humidity treatment for 1min at the temperature of 95 ℃; the survival rate of the lactobacillus reaches 86.51 percent after being treated for 1min at high temperature and high humidity of 105 ℃; the survival rate of the lactic acid bacteria reaches 83.72 percent after being treated for 1min at the high temperature and the high humidity of 115 ℃.
TABLE 4 influence of high temperature and high humidity on the lactic acid bacteria Activity of the lactic acid bacteria preparation
Example 5 lactic acid bacteria preparation tolerance feed processing test
The feed processing temperature is generally 60-90 ℃, and the lactobacillus used as the feed additive can endure high temperature or high temperature and high humidity environment, so that the lactobacillus can still keep high activity through feed processing. In this test, the lactic acid bacteria preparation prepared in example 1 was mixed with a feed material (the ratio between the two was 100g/t), and after granulation, the activity of lactic acid bacteria in the granulated material was measured, and the results are shown in Table 5. As can be seen from Table 5, after the lactobacillus preparation is granulated under the conditions that the steam pressure is 0.5-0.6 MPa, the tempering temperature is 60-70 ℃, the tempering time is 60s, the compression ratio of a ring membrane is 1:4 and the aperture of the ring membrane is 2.0mm, the survival rate of the lactobacillus reaches 99.07%; after the lactobacillus preparation is granulated under the conditions that the steam pressure is 0.5-0.6 MPa, the tempering temperature is 80-85 ℃, the tempering time is 40s, the compression ratio of a ring membrane is 1:6, and the aperture of the ring membrane is 3.0mm, the survival rate of the lactobacillus reaches 97.67%; the survival rate of the lactobacillus reaches 98.14% after the lactobacillus preparation is granulated under the conditions that the steam pressure is 0.5-0.6 MPa, the tempering temperature is 75-80 ℃, the tempering time is 90-120 s, the compression ratio of the circular membrane is 1:8 and the aperture of the circular membrane is 3.2 mm.
TABLE 5 Effect of granulation conditions on the Activity of lactic acid bacteria formulations
EXAMPLE 6 shelf-Life measurement of lactic acid bacteria preparation
In this test, the lactobacillus preparation prepared in example 1 was left at room temperature for 12 months, and the activity of the lactobacillus was measured every month, and the results are shown in table 6, in which the lactobacillus preparation was stored at room temperature for 12 months, with no loss of the activity of the lactobacillus in the 1 st to 3 rd months, the survival rate reached 100%, the activity of the lactobacillus preparation started to decrease from the 4 th month, and the survival rate reached 98.10% when the lactobacillus preparation was stored up to the 12 th month.
TABLE 6 Effect of shelf life on Lactobacillus granulation Activity
| Time/month | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| Survival rate% | 100 | 100 | 100 | 99.67 | 99.24 | 99.01 | 98.95 | 98.57 | 98.43 | 98.21 | 98.18 | 98.10 |
Example 7 Effect of lactic acid bacteria preparation on weaned pig Productivity
160 healthy weaned piglets (24 +/-1 days old) with similar weight and sex are selected for test, and are randomly divided into 2 groups, each group is 8 in number of repetitions, each repetition is 10 in number, one group is a control group, and basic daily ration (shown in table 7) is fed; the other group is a test group, and the test group is fed with basic ration and lactobacillus preparation (the addition amount is 100 g/t). The test period is 4 weeks, all piglets to be tested freely eat and drink water during the test period, the health, diarrhea and death conditions of the piglets are observed every day, the daily food consumption and daily weight gain of the piglets are recorded every day, the diarrhea rate, the death rate, the average daily food consumption, the average daily weight gain and the feed-meat ratio are calculated, and fresh excrement samples are randomly collected from each group at the end of the test, and the viable count of lactic acid bacteria and escherichia coli in the fresh excrement samples is respectively measured (table 8 and table 9).
TABLE 7 basic ration ratio and Nutrition level
| Ration ratio | Content% | Nutritional levels | Content (wt.) |
| Corn% | 58.0 | Digestive energy MJ/kg | 13.85 |
| Bean pulp% | 25.0 | Metabolizable energy MJ/kg | 13.31 |
| Fish meal% | 5.0 | Crude protein% | 19.00 |
| Whey powder% | 5.0 | Crude fiber% | 2.5 |
| Soybean oil% | 1.0 | Crude fat% | 5.0 |
| Calcium hydrogen phosphate% | 0.6 | Lysine% | 0.78 |
| Stone powder% | 1.0 | Threonine% | 0.51 |
| Table salt% | 0.4 | Isoleucine% | 0.55 |
| Premix compound% | 4.0 | Methionine + cystine% | 0.51 |
| Phosphorus% | 0.64 | ||
| Calcium content% | 0.54 |
TABLE 8 Effect of lactic acid bacteria formulations on weaned piglet Productivity
| Item | Control group | Test group |
| Initial weight (kg) | 8.49±0.15 | 8.67±0.21 |
| Terminal weight (kg) | 21.83±2.56a | 23.78±2.29b |
| Average daily gain (g) | 333.5±3.72a | 377.75±4.68b |
| Average daily food intake (g) | 386.26±15.32 | 394.11±20.05 |
| Meat ratio of materials | 1.16±0.04a | 1.04±0.03b |
| Rate of diarrhea | 10.24％a | 1.04％b |
| Mortality rate | 6.25％a | 1.25％b |
Note: the difference of the same-person shoulder-marked letters is obvious (P < 0.05)
As can be seen from Table 8, the piglets in the test group had bright fur, strong and beautiful body, and lively and vigorous movements during the test period. Compared with a control group, the piglet daily gain of a test group can be obviously improved after the preparation is added, the feed conversion ratio is obviously reduced by 0.12, and the diarrhea rate and the death rate are obviously reduced.
TABLE 9 Effect of lactic acid bacteria formulations on the fecal microbiota of weaned piglets
| Item | Control group | Test group |
| Lactic acid bacteria (logCFU/g) | 6.78±0.25a | 8.46±0.09b |
| Escherichia coli (logCFU/g) | 6.15±0.11a | 4.67±0.15b |
| Lactobacillus/Escherichia coli | 1.10a | 1.81b |
Note: the difference of the capital letters of the shoulder marks of the same person is obvious (P < 0.05)
The quantity ratio of the lactic acid bacteria to the escherichia coli is considered as an important mark for evaluating the microbial balance of the animal digestive tract, and the organic acid generated by the lactic acid bacteria can effectively reduce the pH value of the intestinal tract and can also inhibit the growth of pathogenic bacteria of the intestinal tract such as the escherichia coli. As can be seen from Table 9, the lactobacillus preparation can significantly improve the content of lactobacillus in the feces of weaned piglets and significantly reduce the content of escherichia coli, and the ratio of lactobacillus to escherichia coli in the feces of the piglets in the test group is significantly higher than that in the control group.
Example 8 Effect of lactic acid bacteria preparation on broiler productivity
1000 healthy broiler chickens of 1 day old 45g are selected, weighed after 40 days, randomly divided into 2 groups, each experimental group is provided with 5 replicates, and each replicate is provided with 100 chickens. One group was the test group: basic ration (shown in table 10) + lactobacillus preparation (added in an amount of 100 g/t); the other group was a control group: and (4) common feed. The feed formula and the feeding amount of the test group and the control group are the same, and the test group and the control group are managed in a conventional daily way, cultivated in cages and fed with free food and water. The illumination, temperature and humidity in the house are strictly carried out according to the conventional feeding management requirements. During the test period, the health, diarrhea and death conditions of the broilers are observed every day, the daily feed intake and daily weight gain of the broilers are recorded, and the feed conversion ratio and the death and culling rate of the broilers are calculated (table 11).
TABLE 10 basal diet composition and nutritional level
TABLE 11 Effect of lactic acid bacteria preparation on broiler growth Performance
Note: the difference of the capital letters of the shoulder marks of the same person is obvious (P < 0.05)
As can be seen from table 11, the lactobacillus preparation added to the broiler feed can significantly improve the productivity and immunity of broilers, and compared with the control group, the feed-meat ratio is reduced by 18.86%, and the death and culling rate is reduced by 97.22%.
Example 9 Effect of lactic acid bacteria preparation on the Productivity of grass carp
Selecting 600 grass carp fries with the weight of about 8.0 +/-0.2 g, randomly dividing the grass carp fries into two groups, wherein each group is divided into 3 times, each group is divided into 100 times, a control group is fed with common feed, a test group is fed with the common feed (shown in table 12) and lactobacillus preparation (the addition amount is 100g/t), the grass carp fries are raised in a circulating water filtering aquarium, feeding is carried out regularly and quantitatively during the test period, feeding is carried out on the grass carp fries at 8:00, 12:00 and 16:00 every day, the test period is 50 days, the health condition and the death condition of the grass carp fries are observed and recorded every day, weighing is carried out once every 10 days, the water temperature is 25 +/-5 ℃, the pH value is 7.0, dissolved oxygen is higher than 5. The specific growth rate, survival rate, feed efficiency of the grass carp were calculated (table 13).
Specific growth rate (end ln weight-initial ln weight)/day of feeding × 100%
Survival rate ═ total number-number of deaths/total number of bars × 100%
The feed efficiency is (final weight-initial weight)/the total feed amount is multiplied by 100 percent
TABLE 12 complete feed (common feed) formula (in weight percent) for grass carp
| Feed raw material | Small Cao fish feed | Feed for big fish |
| Wheat middling | 8 | 8 |
| Rapeseed meal | 28 | 21 |
| Cotton seed dregs | 26 | 30 |
| Rice bran | 10 | 12 |
| Enzyme protein | 5 | 5 |
| Soybean meal | 2 | 10 |
| Root of barley malt | 10 | 5 |
| Soybean lecithin powder | 3 | 3 |
| Zeolite powder | 5 | 5 |
| Calcium dihydrogen phosphate | 1.5 | 1.5 |
| Salt | 0.3 | 0.3 |
| Premix compound | 1 | 1 |
| Choline chloride | 0.2 | 0.2 |
| Dried substance | 86 | 86.67 |
| Crude protein | 28.25 | 30 |
| Lysine | 1.229 | 1.375 |
| Methionine | 0.435 | 0.471 |
| Methionine + cystine | 0.962 | 1.039 |
| Calcium carbonate | 0.57 | 0.67 |
| Total phosphorus | 1.13 | 1.17 |
| Available phosphorus | 0.58 | 0.63 |
| Coarse fiber | 7.03 | 6.99 |
| Coarse ash content | 5.19 | 5.43 |
| Fat | 4.72 | 4.58 |
TABLE 13 influence of lactic acid bacteria preparation on the Productivity of grass carp
| Group of | Initial weight (g) | Terminal weight (g) | Survival rate (%) | Specific growthLength (%/d) | Feed efficiency (%) |
| Control group | 8.14±0.05 | 17.69±0.21 | 85.78±1.34a | 2.26±0.05a | 46.21±2.24a |
| Test group | 8.09±0.07 | 21.43±0.18 | 99.67±0.92b | 2.75±0.06b | 56.87±2.53b |
Note: the difference of the same row shoulder mark letters is obvious (P < 0.05)
As can be seen from table 13, the addition of the lactobacillus preparation prepared by the present invention can significantly improve the survival rate, specific growth rate and feed efficiency of grass carp, thereby indicating that the lactobacillus preparation prepared by the present invention can promote the growth and development of grass carp, improve the growth performance of grass carp, and has significant effect.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (10)
1. The preparation method of the pelletization-resistant lactic acid bacteria preparation is characterized by comprising the following steps of:
(1) strain activation
Thawing enterococcus faecalis, enterococcus faecium, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus lactis, lactobacillus buchneri, lactobacillus casei and bifidobacterium animalis stored in a glycerol tube, performing streak separation on an MRS plate under an aseptic condition, culturing for 48h at 35 ℃, selecting a single colony from the MRS plate, performing slant streak, culturing for 16h at 35 ℃, and preserving for later use;
(2) first stage culture
Respectively taking the vigorously growing inclined planes, scraping 1-ring bacteria in 100mL of primary culture medium by using a sterilized inoculating ring, and culturing at 35 ℃ for 10-20h to obtain primary culture solution of each lactic acid bacteria;
(3) second stage culture
Respectively inoculating the primary culture solution of each lactobacillus into a secondary culture medium of a 50L fermentation tank, wherein the inoculation amount is 1-5%, the liquid loading amount is 25-35L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and the secondary seed solution is obtained after culturing for 8-15 h;
(4) three-stage culture
Inoculating secondary seed liquid of enterococcus faecalis, enterococcus faecium, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus lactis, lactobacillus buchneri, lactobacillus casei and bifidobacterium animalis into a tertiary culture medium of a 20-ton fermentation tank according to the inoculation amount of 1% (v/v), wherein the total inoculation amount is 4-12%, the liquid loading amount is 50-70L, the fermentation temperature is 25-40 ℃, the stirring speed is 80-120r/min, and the tertiary fermentation liquid is obtained after culturing for 48-72 h;
(5) preparation of wet lactobacillus powder
Centrifuging the tertiary fermentation liquor at 8000r/min for 30min to obtain lactobacillus active bacteria mud, mixing the lactobacillus active bacteria mud and a protective agent according to the proportion of (0.5-2) to (1.5-3) for 30min to obtain high-concentration lactobacillus suspension, adding 20-50 parts of soluble starch in a mixer, and mixing for 5min to obtain wet lactobacillus powder;
(6) preparation of wet lactic acid bacteria granule
Adding the wet lactobacillus powder into a granulator, adjusting the granularity to 20-40 meshes to obtain microcapsule master batches of 20-40 meshes, adding the microcapsule master batches into a QZL-1300 type spherical shot blasting machine for granulation, wherein the rotating speed is 500r/min, atomizing in the granulation process, spraying a coating agent at the speed of 65mL/min according to the ratio of the lactobacillus powder to the coating agent of 1:5, and finally obtaining wet lactobacillus granules;
(7) preparation of lactic acid bacteria preparation
Drying wet lactobacillus granules in a fluidized bed for 15min, wherein the air inlet temperature is 65 ℃ and the air outlet temperature is 35 ℃; starting a side spraying coating device, spraying the coating solution to the lactobacillus wet granules at the speed of 30mL/min to form a coating, and preparing a lactobacillus preparation;
the protective agent comprises the following specific components in parts by weight: 2-10 parts of trehalose, 1-5 parts of sorbitol, 0.5-2.5 parts of bovine serum albumin, 1-4 parts of gelatin, 5-15 parts of skim milk, 2-8 parts of sodium glutamate, 800.8-2 parts of tween, 1-2.5 parts of glycerol, 0.1-1.0 part of vitamin D, 1-8 parts of malto-oligosaccharide, 1-8 parts of xylo-oligosaccharide, 1-8 parts of glucan, 0.5-5 parts of polyethylene glycol, 0.1-1.0 part of vitamin C, 0.1-1.0 part of ethylene diamine tetraacetic acid and 20-40 parts of water; sequentially adding the substances into water, stirring while adding, and stirring for 60min to dissolve completely to obtain protectant;
the coating agent comprises the following specific components in parts by weight: 1-10 parts of hydroxypropyl methyl cellulose, 2-5 parts of polyethylene glycol, 4-8 parts of gelatin, 1-3 parts of polyvinylpyrrolidone, 1-5 parts of enteric acrylic resin, 2-6 parts of cellulose acetate, 2-10 parts of beta-cyclodextrin and 5-30 parts of water; sequentially adding the above components into water, stirring for 15min, and mixing completely;
the coating solution comprises the following specific components in parts by weight: 2-10 parts of corn starch, 1-5 parts of mannitol, 2-10 parts of sodium carboxymethylcellulose, 2-10 parts of skimmed milk powder, 1-15 parts of fructo-oligosaccharide and 8-40 parts of water; sequentially adding the above components into water, stirring for 20min, and mixing completely.
2. The method according to claim 1, wherein the primary and secondary culture media comprise, in weight percent: 1-2.5% of glucose, 0.5-1.5% of trisodium citrate, 0.5-2% of corn flour, 1-2% of tryptone, 0.2-1% of anhydrous sodium acetate, 0.01-0.05% of dipotassium phosphate, 0.01-0.05% of monopotassium phosphate, 0.01-0.1% of magnesium sulfate, 0.01-0.05% of manganese sulfate, 0.02-0.08% of ferrous sulfate, 0.02-0.2% of calcium carbonate, 800.8-1.2% of Tween, and the balance of water, wherein the pH value is 6.2-7.2.
3. The method according to claim 2, wherein the primary and secondary culture media comprise, in weight percent: 1.25% of glucose, 0.8% of trisodium citrate, 1.5% of corn flour, 1.2% of tryptone, 0.4% of anhydrous sodium acetate, 0.03% of dipotassium phosphate, 0.01% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.02% of manganese sulfate, 0.02% of ferrous sulfate, 0.45% of calcium carbonate, 801.1% of tween and the balance of water, wherein the pH value is 6.5.
4. The method according to claim 1, wherein the tertiary fermentation medium comprises, in weight percent: molasses 0.5-2.5%, cane sugar 0.5-1.5%, peptone 1-2%, corn steep liquor dry powder 0.5-1%, yeast extract powder 0.5-1%, sodium acetate 0.2-1%, ammonium chloride 0.01-0.05%, vitamin C0.01-0.1%, sodium chloride 0.1-0.5%, magnesium chloride 0.01-0.1%, dipotassium hydrogen phosphate 0.01-0.05%, potassium dihydrogen phosphate 0.01-0.05%, Tween 801-1.5%, and water in balance, and pH 6.2-7.2.
5. The method according to claim 4, wherein the tertiary fermentation medium comprises, in weight percent: molasses 2.25%, cane sugar 0.75%, peptone 1.25%, corn steep liquor dry powder 0.5%, yeast extract powder 0.8%, sodium acetate 0.45%, ammonium chloride 0.04%, vitamin C0.02%, sodium chloride 0.3%, magnesium chloride 0.05%, dipotassium hydrogen phosphate 0.02%, potassium dihydrogen phosphate 0.01%, Tween 801.1%, and water in balance, and the pH value is 6.5.
6. The preparation method according to claim 1, wherein the protective agent comprises the following specific components in parts by weight: 7.5 parts of trehalose, 2 parts of sorbitol, 1.25 parts of bovine serum albumin, 2.4 parts of gelatin, 8 parts of skim milk, 6.8 parts of sodium glutamate, 801.2 parts of tween, 1.5 parts of glycerol, 0.5 part of vitamin D, 4.5 parts of malto-oligosaccharide, 6 parts of xylo-oligosaccharide, 8 parts of glucan, 2.6 parts of polyethylene glycol, 0.35 part of vitamin C, 0.6 part of ethylene diamine tetraacetic acid and 35 parts of water.
7. The preparation method according to claim 1, wherein the coating agent comprises the following specific components in parts by weight: 6 parts of hydroxypropyl methyl cellulose, 2.5 parts of polyethylene glycol, 5.8 parts of gelatin, 1.25 parts of polyvinylpyrrolidone, 2.2 parts of enteric acrylic resin, 3.5 parts of cellulose acetate, 4.8 parts of beta-cyclodextrin and 12.5 parts of water.
8. The preparation method according to claim 1, wherein the coating solution comprises the following specific components in parts by weight: 8 parts of corn starch, 3.2 parts of mannitol, 2 parts of sodium carboxymethylcellulose, 5.6 parts of skimmed milk powder, 8.8 parts of fructo-oligosaccharide and 15 parts of water.
9. A pelletization-resistant lactic acid bacterium preparation prepared by the method according to any one of claims 1 to 8, wherein the viable count of the lactic acid bacterium is 1.0 x 1010-1.0 × 1011CFU/g。
10. Use of a pelletization-resistant lactic acid bacteria preparation according to claim 9 for the preparation of an animal feed.
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