Intestinal probiotic preparation

Abstract

The invention provides an intestinal probiotic preparation, relates to the technical field of biological medicines, in particular to a probiotic preparation designed for intestinal health and a preparation method of the probiotic preparation, and aims to realize effective regulation of intestinal microecology, promotion of intestinal health and prevention and treatment of intestinal related diseases by optimizing a formula and a release mechanism of probiotics.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and specifically to a probiotic preparation designed for intestinal health and a preparation method thereof, which aims to achieve effective regulation of intestinal microecology, promote intestinal health, and prevent and treat intestinal-related diseases by optimizing the formula and release mechanism of probiotics. Background Art

[0002] The balance of intestinal microecology is crucial to human health. As important microorganisms in the intestine, probiotics play an irreplaceable role in maintaining intestinal health. However, the existing probiotic products on the market have problems such as single strain, insufficient activity, and difficulty in effective release in specific parts of the intestine, resulting in limited effects. Therefore, it is particularly important to develop an intestinal probiotic preparation that can accurately locate and efficiently release. Summary of the invention

[0003] The present invention aims to solve the problems of existing probiotic products, such as single strain, insufficient activity, poor release effect, etc., and provide an intestinal probiotic preparation containing multiple high-effective probiotics. The preparation can be accurately released in specific parts of the intestine (such as the colon), thereby effectively regulating the intestinal microecology and promoting intestinal health.

[0004] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0005] An intestinal probiotic preparation, characterized in that it contains the following strains in parts by weight:

[0006] Lactobacillus plantarum CD fermentation precursor powder 1000 billion units;

[0007] Bifidobacterium bifidum ferment precursor powder 500 billion units;

[0008] Bifidobacterium animalis subsp. lactis fermentation precursor powder 400 billion units;

[0009] Lactobacillus acidophilus ferment precursor powder 100 billion units;

[0010] 100 parts by weight of oligofructose D-fructose and oligogalactose;

[0011] (Lactobacillus plantarum CD is patented with the number ZL200710014488.6 and the name is Lactobacillus plantarum CD and its uses)

[0012] The fermentation precursor powders of various strains are obtained by concentrating the content of live bacteria of the strains into the form of fermentation precursors through fermentation.

[0013] As a preferred method, the process of extracting live Lactobacillus plantarum CD is as follows: ① activation: select a Lactobacillus plantarum CD bacterial solution cultured to the end of the logarithmic phase to a concentration of 1 to 9×108 CFU / mL, inoculate it on a slant of a solid culture medium of Lactobacillus plantarum CD for activation, and culture it at 30°C for 1 to 3 days before taking out the activated bacterial lawn for culture; ② purification: take the activated bacterial lawn of Lactobacillus plantarum CD and inoculate it into MRS broth, culture it on a shaking table at 30°C 80 to 120 rpm for 20 hours, take 100 μL of the culture solution and transfer it to 9 mL MRS broth, and then placed at 30 ℃ 80 ~ 120rpm shaking incubator for 18 ~ 24h, and this was transferred continuously 3 to 6 times, and then 100μL of the last activation solution was inoculated into a centrifuge tube containing 5mL MRS broth and cultured at 30 ℃ 80 ~ 120rpm shaking incubator for 18 ~ 24h; finally, 100μL of the culture solution was mixed with a centrifuge tube containing 9mL 10% skim milk powder, shaken at 37 ℃ for 45min, centrifuged at 3000rpm / min for 10min, and then the supernatant was removed, the precipitate was washed and The bacteria were suspended in saline solution, and this step was repeated three times, and the cells were shaken at 37°C for 45 minutes to precipitate the bacteria and elute with saline solution, and finally a resuspension was prepared, which was the bacterial solution to be centrifuged; the number of viable bacteria in the bacterial solution was calculated to be 1 to 9×108 CFU / mL; ③ Concentration: The centrifuged bacterial solution was concentrated by a concentrator to prepare a concentrated bacterial solution with a bacterial count of 1 to 9×1011 CFU / mL; the number of viable Lactobacillus plantarum CD in the concentrated bacterial solution was checked by gradient dilution method to be equal to that of the bacterial solution before purification; ④ Filtration: Use 0.22μm A microporous filter membrane with a pH of 7.0 is prepared. Precooled sterilized physiological saline is added to the hollow part of the filter membrane. A concentrated bacterial solution with a bacterial count of 1 to 9×1011 CFU / mL is placed on top of the microporous filter membrane for filtration. A liquid culture medium is inserted into one end of the filter membrane. An appropriate amount of precooled pure water is placed on top of the filter membrane. The bacterial solution flows out of the filter membrane, and the effluent is the filtered bacterial solution; ⑤ Drying: The filtered bacterial solution is placed in an incubator for 24 hours at 48°C and dried; ⑥ Crushing: After drying, a powdered concentrated bacterial product is obtained, which is crushed by a crusher in an environment of -20°C; ⑦ Packaging: The powder is stored at -4°C for use; ⑧ Calculation of live bacteria in powder form: Weigh 0.1g of bacterial powder and 9.99g of sterile skimmed milk powder and mix them thoroughly. Take 0.1g of the mixed solution in a sterile centrifuge tube and add 9mL Mix with 10% sodium carbonate solution, incubate in a 70°C water bath for 10 min, and shake at 90-120 rpm for 30 min. After dilution, perform gradient dilution on the bacterial solution, spread 0.1 mL of the bacterial solution on a bromocresol purple solid plate, and culture at 37°C for 12-18 h. Count and calculate the number of live bacteria in the sample. The dilution degree is 1-100×101×107 CFU / g.

[0014] Preferably, the fermentation precursor powder of Bifidobacterium bifidum is activated by Bifidobacterium bifidum in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of Bifidobacterium bifidum and 45-50 parts of culture medium by weight, and the activated seed culture solution is inoculated into a primary seed tank with an inoculation amount of 6% by weight. After inoculation, the mixture is stirred and fermented for 10 hours under anaerobic conditions at a temperature of 14-20°C, and the mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2-5):(5-3), and the number of live bacteria and spores in the seed solution are both 1010-1011 / m L, the seed liquid of the primary fermentation tank is inoculated into the secondary fermentation tank, the inoculation amount is 4% by weight, and the fermentation culture is carried out in the tank under anaerobic conditions at a temperature of 14-20°C, and the fermentation is stirred for 5 hours. The mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2-5):(5-3), and the number of live bacteria and spores in the seed liquid are both 1010-1011 / mL. The culture liquid of Bifidobacterium bifidum in the secondary fermentation tank is inoculated into the tertiary concentration tank at an inoculation amount of 1:5 of the solid-liquid mass, and the tank is sealed and maintained in a sterile environment for concentrated fermentation. The tank is kept at a constant temperature of 42°C and a stirring rate of 20r pm / min stirring fermentation, culture time 4h, Bifidobacterium bifidum spore formation rate and bacterial number according to the mass ratio of (2-5): (5-3), the number of live bacteria and spores in the concentration tank is (1010-1011) / mL, the fermented bacterial liquid is diluted 100 times and counted using a hemocytometer, the residual liquid in the tertiary concentration tank is injected into the fourth secondary concentration tank at an inoculation amount of 1:5 solid-liquid mass ratio, the fermentation conditions in the concentration tank are repeated, the Bifidobacterium bifidum spores collected in the secondary concentration tank, the spore formation rate and bacterial number according to the mass ratio The fermentation liquid collected in the concentration tank of this stage is stored in a refrigerator at 4°C and kept sterile throughout the process; the bacterial count and spore count of the concentrated culture liquid are (1010-1011) / mL, and the fermentation liquid after secondary concentration is vibrated on an oscillator for 10 minutes, and the bacterial liquid is inoculated into a vacuum drying oven. After vibrating on the vibrator for 3 seconds, the valve of the vacuum drying oven is closed, and the vacuum is drawn, and the pressure is maintained at 0.02MPa and the temperature is maintained at 48°C for 1 hour. The dried fermentation crystals are taken out and stored in a -20°C environment.

[0015] Preferably, the animal Bifidobacterium lactis subspecies fermentation precursor powder is activated by animal Bifidobacterium lactis subspecies in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of animal Bifidobacterium lactis subspecies and 45 to 50 parts of culture medium by mass, the fermentation process of animal Bifidobacterium lactobacillus is the same as that of Bifidobacterium bifidum, except for the concentration and fermentation time, and the number of live bacteria of animal Bifidobacterium lactobacillus in the secondary fermentation tank is 1011 to 1012 cells / mL; the mass ratio of the number of animal Bifidobacterium lactobacillus spores to the number of bacteria is 3:7, and the number of live bacteria in the concentration tank is 1011 to 1012 cells / mL.

[0016] Preferably, the Lactobacillus acidophilus fermentation precursor powder is activated by Lactobacillus acidophilus in a pure culture medium.

[0017] Preferably, the fermentation precursor powders of each strain are subjected to multi-layer embedding treatment, comprising the following steps: 1) mixing the fermentation precursor powders of each strain with a protective agent, and obtaining a probiotic suspension after shaking treatment, wherein the protective agent comprises the following components: skim milk 20-30 g / L, trehalose 20-30 g / L, mannitol 10-20 g / L, sorbitol 20-30 g / L, sodium L-glutamate 1.2-3.8 g / L, vitamin C 0.1-0.2 g / L, resistant dextrin 10-22 g / L, taurine 0.1-0.5 g / L; 2) the probiotic suspension is loaded into soft capsules, and the probiotic powder is obtained by freeze drying; 3) a slurry is prepared, the slurry includes plant gum, honey, probiotic powder, konjac fiber, maltodextrin, hawthorn paste and deionized water, and the slurry is sterilized, stirred and mixed to obtain a sterilized slurry; the concentration of the plant gum is 1%-3%, the mass of the honey accounts for 3%-7% of the total mass of the slurry, the mass of the probiotic powder accounts for 5-10% of the total mass of the slurry, and the mass of the konjac fiber accounts for 3% of the total mass of the slurry. ~8%, the mass of the maltodextrin accounts for 0.1~0.3% of the total mass of the slurry; the mass of the hawthorn paste accounts for 0.3~0.7% of the total mass of the slurry; 4) adding the sterilized slurry to a cold isostatic pressing machine for pressing to form a probiotic preparation with a multi-layer structure; 5) packaging the probiotic preparation with a multi-layer structure through a vacuum bag filling machine, and the packaged intestinal probiotic preparation is irradiated with cobalt 60 for irradiation sterilization, the number of probiotic colonies in the intestinal probiotic preparation is ≥0.5×105CFU / g, and the total survival rate is above 90%; the irradiation dose of the irradiation sterilization is 10~20kGy.

[0018] Preferably, in step 1), in the probiotic suspension, the ratio of the fermentation precursor powder of each strain to the protective agent is 1:10 to 1:20; the number of viable bacteria is not less than 5×1010 cfu per gram;

[0019] The parameters of the shaking treatment include: a temperature of 40-50° C. and a shaking treatment time of 0.5-3 hours; then cooling at a rate of 0.4-0.7° C. / min and storing at 3-10° C. for more than 12 hours.

[0020] Preferably, in step 2), before freeze drying, the soft capsules are firstly quick-frozen at -10 to -78°C for 8 to 12 hours; then after the temperature is restored to 4°C, the soft capsules are compressed at a rate of 25 to 50 kg per minute on a cold isostatic press until the volume is reduced by 1 / 6 to 1 / 10 of its original volume; the freeze drying pressure is 0.2 to 0.4 MPa, the temperature is -50 to -10°C, the vacuum degree is 0.1 to 0.25 Pa, and the freeze drying time is 24 to 48 hours.

[0021] Preferably, in step 3), maltodextrin and plant gum are first formulated into a starch mixture, and placed in a water bath at 80°C for stirring for 20 to 60 minutes, and when the temperature drops to 75°C, hawthorn paste and honey are added, and the temperature is adjusted to 65 to 70°C, and the mixture is then allowed to stand for cooling, and allowed to stand at 4°C for 12 to 16 hours to obtain a starch gel for standby use; the starch gel is placed in a homogenizer, and the probiotic powder is added to the pre-prepared starch gel, mixed evenly, to obtain a crude emulsion slurry; the crude emulsion slurry is rapidly cooled and stored at -10 to -78°C for 10 to 12 hours and then added to a storage tank; the sterilization is performed by high-temperature sterilization at 121°C for 20 minutes; the pH of the sterilized slurry is 4.2 to 4.6, and the pH regulator is a mixture of citric acid and sodium citrate; the konjac fiber is obtained by ultrafine grinding for 10 to 30 minutes and passing through a 200-mesh sieve.

[0022] Preferably, in step 3), the plant gum is guar gum, which is extracted with water and then concentrated, diluted, centrifuged and freeze-dried to obtain a concentrated product; in the concentrated product, the plant gum extraction method comprises the following steps: S1: extracting the plant gum extract at a mass ratio of 1:3 to 1:10 at 90°C to 95°C for 2 to 4 hours; S2: centrifuging the obtained plant gum extract at a speed of 6000 to 10000 r / min for 10 to 15 minutes, and then taking the supernatant; S3: concentrating the supernatant by membrane separation to obtain a crude plant gum extract; S4: the dilution parameters are as follows: the concentration of the plant gum after dilution is 2% to 3%, and then the diluted plant gum crude extract is subjected to ultrasonic extraction, the ultrasonic extraction time is 2 to 4 hours, the extraction temperature is 35 to 55°C, and the ultrasonic power is 150 to 200W; S5: the feed liquid after ultrasonic extraction in step S4 is placed in a centrifuge and centrifuged at 3000 to 4000r / m to obtain a secondary extract, and the time of each centrifugation is 15 to 20min; the supernatant is collected for later use; S6: the residue after centrifugation in step S5 is added with deionized water, and ultrasonically treated at an ultrasonic power of 100 to 150W for 5 to 8min, and then centrifuged to obtain the upper clear liquid after two centrifugations;

[0023] S7: repeating step S6, and taking the upper supernatant after three centrifugations, and combining it with the supernatant obtained in step S5; S8: freeze-drying the combined secondary supernatants to obtain a plant gum concentrate;

[0024] Preferably, in step 3), the preparation method of the konjac fiber comprises the following steps: taking konjac powder and water, adding 2-3 mL of water per gram of konjac powder, mixing to form konjac water, adjusting the pH to 7.0, stirring for 5 min at a speed of 500 rm on a magnetic stirrer to obtain a konjac solution; subjecting the obtained konjac solution to shearing treatment, high pressure homogenization treatment, and ultrasonic treatment, and placing it in a freezer to treat at -10°C for more than 12 hours; drying the treated konjac solution to obtain a konjac extract; the shearing treatment The parameters are: shear rate of 18000rm, shear time of 6 minutes; the parameters of the high-pressure homogenization treatment are: feed pressure of 200Pa, homogenization times of 4 times, homogenization time of 20 minutes, and homogenization temperature of 50°C; the parameters of the ultrasonic treatment are: frequency of 1800Hz, ultrasonic time of 15 minutes; the drying comprises: taking the treated konjac solution and drying it at a freezing temperature of -10 to -78°C for 10 hours; the moisture content of the solid obtained after the drying treatment is less than 5%.

[0025] The beneficial effects of the present invention are:

[0026] The intestinal probiotic preparation of the present invention contains a variety of high-efficiency probiotics, which can more comprehensively regulate the intestinal microecology. Through the multi-layer embedding design, the probiotics are accurately released in specific parts of the intestine, which improves the utilization rate and effect of the probiotics. The preparation has a stable structure, is easy to prepare and store, and is convenient for patients to use. In addition, it also has:

[0027] (1) The number of live bacteria is high and can effectively regulate intestinal flora.

[0028] (2) The survival rate is high and it can maintain good activity during storage and use.

[0029] (3) It has significant efficacy and can improve intestinal health and relieve symptoms such as diarrhea and constipation.

[0030] (4) The preparation method is simple, low-cost, and suitable for large-scale production. DETAILED DESCRIPTION

[0031] In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments. The experimental methods in the following embodiments are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments are all commercially available unless otherwise specified.

[0032] Example 1

[0033] An intestinal probiotic preparation, comprising the following strains in parts by weight:

[0034] Lactobacillus plantarum CD fermentation precursor powder 1000 billion units;

[0035] Bifidobacterium bifidum ferment precursor powder 500 billion units;

[0036] Bifidobacterium animalis subsp. lactis fermentation precursor powder 400 billion units;

[0037] Lactobacillus acidophilus ferment precursor powder 100 billion units;

[0038] 100 parts by weight of oligofructose D-fructose and oligogalactose;

[0039] Lactobacillus plantarum CD is the Lactobacillus plantarum CD disclosed in Patent No. ZL200710014488.6 and its name: Lactobacillus plantarum CD and its use. The fermentation precursor powders of various strains are obtained by concentrating the content of live bacteria of the strains into the form of fermentation precursors through fermentation.

[0040] Extraction process of live Lactobacillus plantarum CD: ① Activation: Select the Lactobacillus plantarum CD bacterial solution cultured to the end of the logarithmic phase to a concentration of 1-9×108 CFU / mL, inoculate it on the slope of the Lactobacillus plantarum CD solid culture medium for activation, and culture it at 30°C for 1-3 days before taking the activated bacterial lawn for culture; ② Purification: Take the activated bacterial lawn of Lactobacillus plantarum CD and inoculate it into MRS broth, culture it on a shaking table at 30°C and 80-120rpm for 20h, take 100μL of the culture solution and transfer it to 9mL MRS broth, and then culture it on a shaking table at 30°C and 80-120rpm for 18-24h, and do this transfer 3-6 times in a row, and then take 100μL of the last activation solution and inoculate it into a centrifuge tube containing 5mL MRS broth for 30°C and 80-120rpm shaking culture for 18-24h; finally take 100μL of the culture solution and mix it with 9mL 10% skim milk powder in a centrifuge tube, shake at 37 ° C for 45 minutes, centrifuge at 3000 rpm / min for 10 minutes, remove the supernatant, wash the precipitate and suspend the bacteria in physiological saline, repeat this step three times, shake at 37 ° C for 45 minutes to elute the bacterial precipitate and physiological saline, and finally make a resuspension, which is the bacterial solution to be centrifuged; the number of viable bacteria in the bacterial solution is calculated to be 1 to 9×108 CFU / mL; ③ concentration: the centrifuged bacterial solution is concentrated by a concentrator to make a concentrated bacterial solution with a bacterial count of 1 to 9×1011 CFU / mL; the number of viable Lactobacillus plantarum CD in the concentrated bacterial solution is equal to the number of viable bacteria in the bacterial solution before purification by gradient dilution method; ④ filtration: use 0.22μm A microporous filter membrane with a pH of 7.0 is prepared. Precooled sterilized physiological saline is added to the hollow part of the filter membrane. A concentrated bacterial solution with a bacterial count of 1 to 9×1011 CFU / mL is placed on top of the microporous filter membrane for filtration. A liquid culture medium is inserted into one end of the filter membrane. An appropriate amount of precooled pure water is placed on top of the filter membrane. The bacterial solution flows out of the filter membrane, and the effluent is the filtered bacterial solution; ⑤ Drying: The filtered bacterial solution is placed in an incubator for 24 hours at 48°C and dried; ⑥ Crushing: After drying, a powdered concentrated bacterial product is obtained, which is crushed by a crusher in an environment of -20°C; ⑦ Packaging: The powder is stored at -4°C for use; ⑧ Calculation of live bacteria in powder form: Weigh 0.1g of bacterial powder and 9.99g of sterile skimmed milk powder and mix them thoroughly. Take 0.1g of the mixed solution in a sterile centrifuge tube and add 9mL Mix with 10% sodium carbonate solution, incubate in a 70°C water bath for 10 min, and shake at 90-120 rpm for 30 min. After dilution, perform gradient dilution on the bacterial solution, spread 0.1 mL of the bacterial solution on a bromocresol purple solid plate, and culture at 37°C for 12-18 h. Count and calculate the number of live bacteria in the sample. The dilution degree is 1-100×101×107 CFU / g.

[0041] The fermentation precursor powder of Bifidobacterium bifidum is activated in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of Bifidobacterium bifidum and 45-50 parts of culture medium by weight, the activated seed culture solution is inoculated into a primary seed tank, the inoculation amount is 6% by weight, after inoculation, it is stirred and fermented for 10 hours under anaerobic conditions at a temperature of 14-20°C, the mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2-5):(5-3), the number of live bacteria and the number of spores in the seed solution are both 1010-1011 / mL, and a The seed liquid of the first fermentation tank is inoculated into the second fermentation tank, the inoculation amount is 4% by weight, and the fermentation culture is carried out in the tank under anaerobic conditions at a temperature of 14 to 20° C., and the fermentation is stirred for 5 hours. The mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2 to 5): (5 to 3), and the number of live bacteria and spores in the seed liquid are both 1010 to 1011 cells / mL. The culture liquid of Bifidobacterium bifidum in the second fermentation tank is inoculated into the third concentration tank according to the inoculation amount of 1:5 of the solid-liquid mass, and the tank is sealed and maintained in a sterile environment for concentrated fermentation. The concentration is carried out at a constant temperature of 42° C. and a stirring rate of 20 rpm. / min stirring fermentation, culture time 4h, Bifidobacterium bifidum spore formation rate and bacterial number according to the mass ratio of (2-5): (5-3), the number of live bacteria and spores in the concentration tank is (1010-1011) / mL, the fermented bacterial liquid is diluted 100 times and counted using a hemocytometer, the residual liquid in the tertiary concentration tank is injected into the fourth secondary concentration tank at an inoculation amount of 1:5 solid-liquid mass ratio, the fermentation conditions in the concentration tank are repeated, the spore formation rate and bacterial number of Bifidobacterium bifidum spores collected in the secondary concentration tank, the mass ratio of (1~3):(7~2), the fermentation liquid collected in the concentration tank of this level is stored in a refrigerator at 4℃ and kept sterile throughout the process; the bacterial count and spore count of the concentrated culture liquid are (1010~1011) / mL, the fermentation liquid after secondary concentration is vibrated on an oscillator for 10 minutes, the bacterial liquid is inoculated into a vacuum drying oven, and after vibrating on the vibrator for 3s, the valve of the vacuum drying oven is closed, and the vacuum is drawn, and the pressure is maintained at 0.02MPa and the temperature is maintained at 48℃ for 1 hour of vibration drying, and the dried fermentation crystals are taken out and stored in a -20℃ environment.

[0042] The animal Bifidobacterium lactis subspecies fermentation precursor powder is activated by animal Bifidobacterium lactis subspecies in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of animal Bifidobacterium lactis subspecies and 45 to 50 parts of culture medium in parts by mass; the fermentation process of the animal Bifidobacterium lactobacillus is the same as that of Bifidobacterium bifidum, except for the concentration and fermentation time; the number of live bacteria of animal Bifidobacterium lactobacillus in the secondary fermentation tank is 1011 to 1012 cells / mL; the mass ratio of the number of animal Bifidobacterium lactobacillus spores to the number of bacteria is 3:7, and the number of live bacteria in the concentration tank is 1011 to 1012 cells / mL.

[0043] The Lactobacillus acidophilus fermentation precursor powder is activated by Lactobacillus acidophilus in a pure culture medium.

[0044] The fermentation precursor powders of each strain are subjected to multi-layer embedding treatment, comprising the following steps: 1) mixing the fermentation precursor powders of each strain with a protective agent, and obtaining a probiotic suspension after shaking treatment, wherein the protective agent comprises the following components: 20-30 g / L skim milk, 20-30 g / L trehalose, 10-20 g / L mannitol, 20-30 g / L sorbitol, 1.2-3.8 g / L sodium L-glutamate, and vitamin C. 0.1-0.2 g / L, resistant dextrin 10-22 g / L, taurine 0.1-0.5 g / L; 2) the probiotic suspension is loaded into soft capsules, and the probiotic powder is obtained by freeze drying; 3) a slurry is prepared, the slurry includes plant gum, honey, probiotic powder, konjac fiber, maltodextrin, hawthorn paste and deionized water, and the slurry is sterilized, stirred and mixed to obtain a sterilized slurry; the concentration of the plant gum is 1%-3%, the mass of the honey accounts for 3%-7% of the total mass of the slurry, the mass of the probiotic powder accounts for 5-10% of the total mass of the slurry, and the mass of the konjac fiber accounts for 3% of the total mass of the slurry. ~8%, the mass of the maltodextrin accounts for 0.1~0.3% of the total mass of the slurry; the mass of the hawthorn paste accounts for 0.3~0.7% of the total mass of the slurry; 4) adding the sterilized slurry to a cold isostatic pressing machine for pressing to form a probiotic preparation with a multi-layer structure; 5) packaging the probiotic preparation with a multi-layer structure through a vacuum bag filling machine, and the packaged intestinal probiotic preparation is irradiated with cobalt 60 for irradiation sterilization, the number of probiotic colonies in the intestinal probiotic preparation is ≥0.5×105CFU / g, and the total survival rate is above 90%; the irradiation dose of the irradiation sterilization is 10~20kGy.

[0045] The following is the comparative test data of the intestinal probiotic preparation of the present invention and the existing intestinal probiotic preparation:

[0046]

[0047] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only preferred examples of the present invention and are not intended to limit the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. An intestinal probiotic preparation, characterized in that: Contains the following strains in parts by weight: Lactobacillus plantarum CD fermentation precursor powder 1000 billion units; Bifidobacterium bifidum ferment precursor powder 500 billion units; Bifidobacterium animalis subsp. lactis fermentation precursor powder 400 billion units; Lactobacillus acidophilus ferment precursor powder 100 billion units; 100 parts by weight of oligofructose D-fructose and oligogalactose; The fermentation precursor powders of various strains are obtained by concentrating the content of live bacteria of the strains into the form of fermentation precursors through fermentation.

2. The intestinal probiotic preparation according to claim 1, characterized in that: Extraction process of live Lactobacillus plantarum CD: ① Activation: Select the Lactobacillus plantarum CD bacterial solution cultured to the end of the logarithmic phase to a concentration of 1-9×108 CFU / mL, inoculate it on the slope of the Lactobacillus plantarum CD solid culture medium for activation, and culture it at 30°C for 1-3 days before taking the activated bacterial lawn for culture; ② Purification: Take the activated bacterial lawn of Lactobacillus plantarum CD and inoculate it into MRS broth, culture it on a shaking table at 30°C and 80-120rpm for 20h, take 100μL of the culture solution and transfer it to 9mL MRS broth, and then culture it on a shaking table at 30°C and 80-120rpm for 18-24h, and do this transfer 3-6 times in a row, and then take 100μL of the last activation solution and inoculate it into a centrifuge tube containing 5mL MRS broth for 30°C and 80-120rpm shaking culture for 18-24h; finally take 100μL of the culture solution and mix it with 9mL 10% skim milk powder in a centrifuge tube, shake at 37°C for 45 minutes, centrifuge at 3000rpm / min for 10 minutes, remove the supernatant, wash the precipitate and suspend the bacteria in physiological saline, repeat this step three times, shake at 37°C for 45 minutes to elute the bacterial precipitate and physiological saline, and finally make a heavy suspension, which is the bacterial solution to be centrifuged; the number of viable bacteria in the bacterial solution is calculated to be 1 to 9×108CFU / mL; ③ Concentration: After the centrifuged bacterial solution is concentrated by a concentrator, a concentrated bacterial solution with a bacterial count of 1 to 9×1011CFU / mL is prepared; The number of live bacteria of Lactobacillus plantarum CD in the concentrated bacterial solution was checked by gradient dilution method to be equal to that of the bacterial solution before purification; ④ Filtration: Use a 0.22μm pH=7.0 microporous filter membrane, add pre-cooled sterilized physiological saline to the hollow part of the filter membrane, and the bacterial amount is 1~9×1011CFU / mL. The concentrated bacterial solution is placed on the microporous filter membrane for filtration, and one end of the filter membrane is inserted into the liquid culture medium. An appropriate amount of pre-cooled pure water is placed on the filter membrane. The bacterial solution flows out of the filter membrane, and the effluent is the filtered bacterial solution; ⑤ Drying: The filtered bacterial solution is placed in an incubator for culturing at 48℃ for 24h and drying; ⑥ Crushing: After drying, a powdered concentrated bacterial product is obtained, which is crushed by a crusher in an environment of -20℃; ⑦ Packaging: The powder is stored at -4℃ for use; ⑧ Calculation of live bacteria in powder: Weigh 0.1g of bacterial powder and 9.99g of sterile skimmed milk powder and mix them thoroughly. Take 0.1g of the mixed solution in a sterile centrifuge tube and add 9mL Mix with 10% sodium carbonate solution, incubate in a 70°C water bath for 10 min, and shake at 90-120 rpm for 30 min. After dilution, perform gradient dilution on the bacterial solution, spread 0.1 mL of the bacterial solution on a bromocresol purple solid plate, and culture at 37°C for 12-18 h. Count and calculate the number of live bacteria in the sample. The dilution degree is 1-100×101×107 CFU / g.

3. The intestinal probiotic preparation according to claim 1, characterized in that: The fermentation precursor powder of Bifidobacterium bifidum is activated in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of Bifidobacterium bifidum and 45-50 parts of culture medium by weight, the activated seed culture solution is inoculated into a primary seed tank, the inoculation amount is 6% by weight, after inoculation, it is stirred and fermented for 10 hours under anaerobic conditions at a temperature of 14-20°C, the mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2-5):(5-3), the number of live bacteria and the number of spores in the seed solution are both 1010-1011 / mL, and a The seed liquid of the first fermentation tank is inoculated into the second fermentation tank, the inoculation amount is 4% by weight, and the fermentation culture is carried out in the tank under anaerobic conditions at a temperature of 14 to 20° C., and the fermentation is stirred for 5 hours. The mass ratio of the spore formation rate of Bifidobacterium bifidum to the number of bacteria is (2 to 5): (5 to 3), and the number of live bacteria and spores in the seed liquid are both 1010 to 1011 cells / mL. The culture liquid of Bifidobacterium bifidum in the second fermentation tank is inoculated into the third concentration tank according to the inoculation amount of 1:5 of the solid-liquid mass, and the tank is sealed and maintained in a sterile environment for concentrated fermentation. The concentration is carried out at a constant temperature of 42° C. and a stirring rate of 20 rpm. / min stirring fermentation, culture time 4h, Bifidobacterium bifidum spore formation rate and bacterial number according to the mass ratio of (2-5): (5-3), the number of live bacteria and spores in the concentration tank is (1010-1011) / mL, the fermented bacterial liquid is diluted 100 times and counted using a hemocytometer, the residual liquid in the tertiary concentration tank is injected into the fourth secondary concentration tank at an inoculation amount of 1:5 solid-liquid mass ratio, the fermentation conditions in the concentration tank are repeated, the spore formation rate and bacterial number of Bifidobacterium bifidum spores collected in the secondary concentration tank, the mass ratio of (1~3):(7~2), the fermentation liquid collected in the concentration tank of this level is stored in a refrigerator at 4℃ and kept sterile throughout the process; the bacterial count and spore count of the concentrated culture liquid are (1010~1011) / mL, the fermentation liquid after secondary concentration is vibrated on an oscillator for 10 minutes, the bacterial liquid is inoculated into a vacuum drying oven, and after vibrating on the vibrator for 3s, the valve of the vacuum drying oven is closed, and the vacuum is drawn, and the pressure is maintained at 0.02MPa and the temperature is maintained at 48℃ for 1 hour of vibration drying, and the dried fermentation crystals are taken out and stored in a -20℃ environment.

4. The intestinal probiotic preparation according to claim 1, characterized in that: The animal Bifidobacterium lactis fermentation precursor powder is activated by animal Bifidobacterium lactis in a pure culture medium; wherein the pure culture medium contains 5 parts by weight of animal Bifidobacterium lactis and 45-50 parts of culture medium by weight; the fermentation process of animal Bifidobacterium lactobacillus is the same as that of Bifidobacterium bifidum, except for the concentration and fermentation time; the number of live bacteria of animal Bifidobacterium lactobacillus in the secondary fermentation tank is 1011-1012 cells / mL; the mass ratio of the number of animal Bifidobacterium lactobacillus spores to the number of bacteria is 3:7, and the number of live bacteria in the concentration tank is 1011-1012 cells / mL.

5. The intestinal probiotic preparation according to claim 1, characterized in that: The Lactobacillus acidophilus fermentation precursor powder is obtained by activating Lactobacillus acidophilus in a pure culture medium.

6. The intestinal probiotic preparation according to claim 1, characterized in that: The fermentation precursor powders of each strain are subjected to multi-layer embedding treatment, comprising the following steps: 1) mixing the fermentation precursor powders of each strain with a protective agent, and obtaining a probiotic suspension after shaking treatment, wherein the protective agent comprises the following components: 20-30 g / L skim milk, 20-30 g / L trehalose, 10-20 g / L mannitol, 20-30 g / L sorbitol, 1.2-3.8 g / L sodium L-glutamate, and vitamin C. 0.1-0.2 g / L, resistant dextrin 10-22 g / L, taurine 0.1-0.5 g / L; 2) the probiotic suspension is loaded into soft capsules, and the probiotic powder is obtained by freeze drying; 3) a slurry is prepared, the slurry includes plant gum, honey, probiotic powder, konjac fiber, maltodextrin, hawthorn paste and deionized water, and the slurry is sterilized, stirred and mixed to obtain a sterilized slurry; the concentration of the plant gum is 1%-3%, the mass of the honey accounts for 3%-7% of the total mass of the slurry, the mass of the probiotic powder accounts for 5-10% of the total mass of the slurry, and the mass of the konjac fiber accounts for 3% of the total mass of the slurry. ~8%, the mass of the maltodextrin accounts for 0.1~0.3% of the total mass of the slurry; the mass of the hawthorn paste accounts for 0.3~0.7% of the total mass of the slurry; 4) adding the sterilized slurry to a cold isostatic pressing machine for pressing to form a probiotic preparation with a multi-layer structure; 5) packaging the probiotic preparation with a multi-layer structure through a vacuum bag filling machine, and the packaged intestinal probiotic preparation is irradiated with cobalt 60 for irradiation sterilization, the number of probiotic colonies in the intestinal probiotic preparation is ≥0.5×105CFU / g, and the total survival rate is above 90%; the irradiation dose of the irradiation sterilization is 10~20kGy.

7. The intestinal probiotic preparation according to claim 6, characterized in that: In step 1), in the probiotic suspension, the ratio of the fermentation precursor powder of each strain to the protective agent is 1:10 to 1:20; the number of viable bacteria is not less than 5×1010 cfu per gram; The parameters of the shaking treatment include: a temperature of 40-50° C. and a shaking treatment time of 0.5-3 hours; then cooling at a rate of 0.4-0.7° C. / min and storing at 3-10° C. for more than 12 hours.

8. The intestinal probiotic preparation according to claim 6, characterized in that: In step 2), before freeze drying, the soft capsules are firstly quick-frozen at -10 to -78°C for 8 to 12 hours; then after the temperature is restored to 4°C, the soft capsules are compressed at a rate of 25 to 50 kg per minute in a cold isostatic press until the volume is reduced by 1 / 6 to 1 / 10 of the original volume; the freeze drying pressure is 0.2 to 0.4 MPa, the temperature is -50 to -10°C, the vacuum degree is 0.1 to 0.25 Pa, and the freeze drying time is 24 to 48 hours.

9. The intestinal probiotic preparation according to claim 6, characterized in that: In step 3), maltodextrin and plant gum are first prepared into a starch mixture, and placed in a water bath at 80° C. and stirred for 20 to 60 minutes. When the temperature drops to 75° C., hawthorn paste and honey are added, and the temperature is adjusted to 65 to 70° C., and the mixture is then allowed to stand for cooling, and allowed to stand at 4° C. for 12 to 16 hours to obtain a starch gel for standby use; the starch gel is placed in a homogenizer, and the probiotic powder is added to the pre-prepared starch gel, mixed evenly, to obtain a coarse emulsion slurry; the coarse emulsion slurry is rapidly cooled and stored at -10 to -78° C. for 10 to 12 hours and then added to a storage tank; the sterilization is performed by high-temperature sterilization at 121° C. for 20 minutes; the pH of the sterilized slurry is 4.2 to 4.6, and the pH regulator is a mixture of citric acid and sodium citrate; the konjac fiber is obtained by ultrafine grinding for 10 to 30 minutes and passing through a 200-mesh sieve.

10. The intestinal probiotic preparation according to claim 6, characterized in that: In step 3), the plant gum is guar gum, which is extracted with water and then concentrated, diluted, centrifuged and freeze-dried to obtain a concentrated product; the plant gum extraction method in the concentrated product comprises the following steps: S1: extracting the plant gum extract at a mass ratio of 1:3 to 1:10 at 90°C to 95°C for 2 to 4 hours; S2: centrifuging the obtained plant gum extract at a speed of 6000 to 10000 r / min for 10 to 15 minutes, and then taking the supernatant; S3: The supernatant is concentrated by membrane separation to obtain a crude plant gum extract; S4: the dilution parameters are: the concentration of the plant gum after dilution is 2% to 3%, and then the diluted crude plant gum extract is subjected to ultrasonic extraction, the ultrasonic extraction time is 2 to 4 hours, the extraction temperature is 35 to 55°C, and the ultrasonic power is 150 to 200W; S5: taking the liquid after ultrasonication in step S4, placing it in a centrifuge and centrifuging it at 3000 to 4000 r / m to obtain a secondary extract, and the centrifugation time for each time is 15 to 20 minutes; collecting the supernatant for later use; S6: adding deionized water to the residue after centrifugation in step S5, ultrasonically treating the residue at an ultrasonic power of 100 to 150 W for 5 to 8 minutes, and then centrifuging the residue to obtain a supernatant after two centrifugations; S7: repeating step S6, and taking the upper supernatant after three centrifugations, and combining it with the supernatant obtained in step S5; S8: freeze-drying the combined secondary supernatants to obtain a plant gum concentrate; Step 3), the preparation method of the konjac fiber comprises the following steps: taking konjac powder and water, dispensing 2-3 mL of water per gram of konjac powder, mixing to form konjac water, adjusting the pH to 7.0, stirring for 5 min at a speed of 500 rm on a magnetic stirrer to obtain a konjac solution; subjecting the obtained konjac solution to shearing treatment, high pressure homogenization treatment, and ultrasonic treatment, and placing the solution in a freezer to treat at -10 ° C for a treatment time greater than 12 hours; drying the treated konjac solution to obtain a konjac extract; the shearing treatment of the konjac solution is performed. The parameters of the high-pressure homogenization treatment are: a feed pressure of 200 Pa, a homogenization number of 4 times, a homogenization time of 20 minutes, and a homogenization temperature of 50° C.; the parameters of the ultrasonic treatment are: a frequency of 1800 Hz, and an ultrasonic time of 15 minutes; the drying comprises: taking the treated konjac solution and drying it at a freezing temperature of -10 to -78° C. for 10 hours; and the moisture content of the solid obtained after the drying treatment is less than 5%.

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