Preparation method and application of a composite preparation based on probiotics and prebiotics

Abstract

The present invention relates to the technical field of food nutrition, and in particular to a preparation method and application of a composite preparation based on probiotics and prebiotics. The present invention is prepared from a probiotic composition, gaussose, lotus leaf pectin-type polysaccharide LLP, morel polysaccharide MEP-1, puerarin, oligofructose, stachyose and oligogalactose. The probiotic composition is obtained by mixing probiotic composition A, probiotic composition B and probiotic composition C. Through the compounding of the three compositions, the effect of relieving constipation is maximized, the growth of probiotics can be promoted, and the probiotics can be protected from inactivation by low pH value in the gastrointestinal tract, thereby improving the survival rate of the probiotics. Moreover, the probiotic composition can quickly relieve constipation and prolong the duration of action of the composite preparation, thereby better conditioning gastrointestinal flora. Compared with the prior art, the probiotic composition has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of food nutrition, and in particular to a preparation method of a composite preparation based on probiotics and prebiotics and applications thereof. Background Art

[0002] Constipation during pregnancy is a common complication among pregnant women. The incidence of constipation varies across gestational ages, with studies reporting an incidence of 11%-38%. Pregnant women who are bedridden for fetal preservation are at a higher risk of constipation than the general population. Constipation can lead to a buildup of fecal matter in the intestines, causing abdominal discomfort such as bloating and pain. It can also impair a woman's ability to absorb nutrients, thereby impacting fetal growth and development.

[0003] Probiotics are live, non-pathogenic microorganisms that can also alter the intestinal microbiome, benefiting the host. Experiments have shown that probiotics can promote nutrient digestion and absorption, enhance the body's innate immunity and antioxidant capacity, maintain a balanced intestinal microbiome, inhibit the growth of harmful bacteria, protect the intestinal mucosal barrier from bacterial translocation, inhibit intestinal inflammation, and reduce the release of inflammatory cytokines by regulating gastrointestinal surface enzymes and microbial flora. Commonly used probiotics include Lactobacillus, Bifidobacterium, Enterococcus, Clostridium, and Saccharomyces.

[0004] Prebiotics are the food of probiotics and have multiple functional properties. In addition to maintaining the balance of intestinal flora, they also have multiple effects such as promoting mineral absorption, improving lipid metabolism, and moisturizing the intestines and promoting bowel movements. They are widely used in various foods by domestic and foreign food companies.

[0005] Prebiotics generally refer to an ideal dietary supplement that promotes the growth and metabolism of probiotics. These substances are organic substances that are not digested or absorbed by the host but selectively promote the metabolism and proliferation of beneficial bacteria in the body, thereby improving host health. They primarily fall into four categories: functional oligosaccharides, such as fructooligosaccharides, isomalto-oligosaccharides, xylooligosaccharides, and galacto-oligosaccharides; polysaccharides, such as those derived from algae like Spirulina and Arthrospira; extracts from natural plant resources, including everyday vegetables, traditional Chinese herbal remedies, and wild plants; and protein hydrolysates and polyols. Functional oligosaccharides are the most common type of prebiotic due to their widespread use and significant efficacy.

[0006] In the prior art, there are many probiotic and prebiotic compound preparations for relieving constipation. For example, patent document CN110693008A discloses a prebiotic polypeptide compound probiotic for treating constipation and indigestion, which includes: mannitol, tomato seed powder, 5-6 parts of tangerine peel, oligoxylose, baking soda, licorice, bitter melon peptide powder, Lactobacillus reuteri, sunflower oil, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus lactis, Streptococcus lactis, Lactobacillus bulgaricus, roasted malt, and Citrus aurantium. The invention contains a lot of dietary fiber and can supplement the nutrients needed by the human body, but the probiotics are easily affected by low gastric pH, small intestinal bile and different digestive enzymes and lose their activity, and thus cannot play the role of moisturizing the intestines and promoting bowel movements.

[0007] Therefore, according to the above-mentioned related technologies, there is an urgent need to develop a preparation method and application of a composite preparation based on probiotics and prebiotics. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to propose a preparation method and application of a composite preparation based on probiotics and prebiotics, so as to solve the problem in the prior art that the activity of probiotics is easily affected by the environment.

[0009] Based on the above objectives, the present invention provides a preparation method and application of a composite preparation based on probiotics and prebiotics.

[0010] A method for preparing a composite preparation based on probiotics and prebiotics, comprising the following raw materials in parts by weight:

[0011] 25-29 parts of probiotic composition, 4-8 parts of gosaccharide, 3.3-6 parts of lotus leaf pectin-type polysaccharide LLP, 14-7 parts of morel polysaccharide MEP, 0.5-0.9g of puerarin, 15-18 parts of oligofructose, 12-16 parts of stachyose, and 9-13 parts of galacto-oligosaccharide;

[0012] The probiotic composition is obtained by mixing probiotic composition A, probiotic composition B and probiotic composition C in a mass ratio of 24-33:10-12:2.4-3.8;

[0013] The probiotic composition A is prepared from composite probiotics, sodium alginate and wheat cellulose;

[0014] The probiotic composition B is prepared from inulin, glutamine, Lactobacillus acidophilus and anaerobic Corynebacterium faecalis;

[0015] The probiotic composition C is prepared from Bifidobacterium longum and Lactobacillus rhamnosus;

[0016] The composite probiotics are obtained by mixing Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus.

[0017] Preferably, the preparation method of the composite probiotics is as follows:

[0018] Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus are respectively activated in a sterile MRS culture medium at 37° C. for 22-24 hours, then centrifuged at 4000-5000 rpm for 15-20 minutes, the supernatant is discarded, the bacteria are collected, and sterile water is used to prepare a bacterial suspension to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension and Lactobacillus rhamnosus suspension, respectively; and the Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension and Lactobacillus rhamnosus suspension are then mixed to obtain a composite probiotic;

[0019] The MRS culture medium formula is: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 1mL Tween-80, 2g diammonium hydrogen citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate tetrahydrate, 1000mL distilled water, and pH 6.8.

[0020] Preferably, the mass ratio of the Lactobacillus plantarum suspension, the Lactobacillus acidophilus suspension, the Bifidobacterium longum suspension, and the Lactobacillus rhamnosus suspension is 4-7:2-5:8-12:10-15;

[0021] The number of viable Lactobacillus plantarum in the composite probiotics is 5×10 8 -5×10 10 CFU / mL, the number of viable Lactobacillus acidophilus is 3×10 7 -3×10 9 CFU / mL, the number of viable bacteria of Bifidobacterium longum was 2.2×10 7 -2.2×10 10 CFU / mL, the number of viable Lactobacillus rhamnosus was 2×10 7 -2×10 8 CFU / mL.

[0022] Preferably, the preparation method of the probiotic composition A is as follows:

[0023] Step A1. Add ethanol to the wheat cellulose, stir evenly, and distill under reduced pressure at 30-40°C for 22-30 minutes. Then, add the pomegranate peel ethanol extract, the composite probiotics, Tween-80, and a 3% by mass aqueous solution of sodium alginate and mix evenly to obtain a mixture A.

[0024] Step A2. Add the calcium chloride solution to the mixture A using a syringe pump, and centrifuge at 7200-7800 rpm for 20-24 min to obtain a probiotic composition A.

[0025] Preferably, the ratio of the wheat cellulose, ethanol, pomegranate peel ethanol extract, compound probiotics, Tween-80 and sodium alginate aqueous solution in step A1 is 5-9 g: 2-3 g: 1-2 g: 28-33 mL: 0.4-0.6 g: 90-100 mL;

[0026] The concentration of the calcium chloride solution in step A2 is 2-2.5 mol / L;

[0027] The volume ratio of the calcium chloride solution to the mixture A is 45-55:135-140.

[0028] Preferably, the preparation method of the probiotic composition B is as follows:

[0029] Step B1. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37°C for 22-24 hours, then centrifuged at 4000-5000 rpm for 15-20 minutes, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a Lactobacillus acidophilus suspension and an anaerobic Corynebacterium faecalis suspension, respectively;

[0030] Step B2. Dissolve inulin and glutamine in phosphate buffered saline and stir magnetically at 37°C for 15-20 minutes, then add the Lactobacillus acidophilus suspension and the anaerobic Corynebacterium faecalis suspension, mix well, and freeze-dry to obtain a probiotic composition B.

[0031] Preferably, the usage ratio of inulin, glutamine, phosphate buffered saline, Lactobacillus acidophilus suspension and anaerobic Corynebacterium faecalis suspension in step B2 is 16-19 g: 5-9 g: 6-8 mL: 5-8 mL: 3-6 mL;

[0032] The number of viable Lactobacillus acidophilus in the probiotic composition B is 1×10 8 -1×10 10 CFU / g, the number of viable bacteria of anaerobic Corynebacterium faecalis is 1x10 6 -10 8 CFU / g;

[0033] The temperature during the freeze drying is -82-76°C and the time is 23-25 ​​hours.

[0034] Preferably, the preparation method of the probiotic composition C is as follows:

[0035] Bifidobacterium longum and Lactobacillus rhamnosus were activated in sterile MRS culture medium at 37°C for 22-24 hours, centrifuged at 4000-5000 rpm for 15-20 minutes, washed three times with sterile water and suspended in phosphate buffered saline to a concentration of 1×10 10 -3.5×10 10CFU / mL, and then heat-inactivation treatment was performed to obtain probiotic composition C;

[0036] The temperature during the heat inactivation treatment is 90-100° C. and the time is 10 minutes.

[0037] Preferably, the method comprises the following steps:

[0038] Probiotic composition A, probiotic composition B and probiotic composition C are mixed to obtain a probiotic composition, which is then evenly mixed with gaussose, lotus leaf pectin-type polysaccharide LLP, morel polysaccharide MEP-1, puerarin, oligofructose, stachyose and oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

[0039] An application of a compound preparation based on probiotics and prebiotics, wherein the compound preparation based on probiotics and prebiotics is used for alleviating constipation during pregnancy.

[0040] Beneficial effects of the present invention:

[0041] The present invention provides a method for preparing a composite preparation based on probiotics and prebiotics and its application. The present invention prepares a composite preparation that can effectively relieve constipation by combining a probiotic composition with multiple prebiotics. The probiotic composition is obtained by mixing probiotic composition A, probiotic composition B, and probiotic composition C. By compounding the three compositions, the constipation-relieving effect is maximized.

[0042] Among them, sodium alginate and wheat cellulose in probiotic composition A, and inulin and glutamine in probiotic composition B can not only promote the growth of probiotics, but also protect probiotics from being inactivated by the low pH value of the gastrointestinal tract, thereby improving the survival rate of probiotics; and the probiotic composition can not only quickly relieve constipation, but also prolong the duration of action of the compound preparation and better regulate gastrointestinal flora. Compared with the existing technology, it has broad application prospects. DETAILED DESCRIPTION

[0043] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments.

[0044] The sources and properties of some of the raw materials used in the present invention are as follows:

[0045] Lotus leaf pectin-type polysaccharide LLP was purchased from Sichuan Weikeqi Biotechnology Co., Ltd.; Morchella polysaccharide MEP-1 was purchased from Ningxia Kaiyuan Biotechnology Co., Ltd.; puerarin was purchased from Jiangsu Xingyao Biotechnology Co., Ltd.; oligofructose was purchased from Guangzhou Tianjia Biotechnology Co., Ltd.; stachyose was purchased from Zhengzhou Dewang Chemical Industry Co., Ltd.; oligogalactose was purchased from Shandong Baolingbao Co., Ltd.; inulin was purchased from Sichuan Huatang Jurui Biotechnology Co., Ltd.; glutamine was purchased from Zhengzhou Haiweili Food Industry Co., Ltd.; pomegranate peel ethanol extract was purchased from Shaanxi Yijun Biotechnology Co., Ltd.

[0046] The Lactobacillus plantarum used in the present invention is Lactobacillus plantarum YS1, which is disclosed in the invention patent with the authorization publication number "CN106906165B" and the name "A Lactobacillus plantarum and its use in preparing food for preventing constipation", and the deposit number is CCTCC NO: M2016747;

[0047] The Lactobacillus acidophilus used in the present invention is disclosed in the invention patent with the authorization publication number "CN116286524A" and the title "A probiotic combination for improving constipation in pregnant women, its products and uses", and the deposit number is CGMCC No.1.2686;

[0048] The Bifidobacterium longum used in the present invention is disclosed in the invention patent with the authorization publication number "CN116286524A" and the name "A probiotic combination for improving constipation in pregnant women and its products and uses", and the deposit number is CGMCC No.1.2186;

[0049] The Lactobacillus rhamnosus used in the present invention is Lactobacillus rhamnosus VHProbi M15, which is disclosed in the invention patent with the authorization publication number "CN114717129B" and the name "A strain of Lactobacillus rhamnosus and its use in preventing and alleviating constipation symptoms", and the deposit number is CCTCC NO: M2021904;

[0050] The anaerobic Corynebacterium faecalis used in the present invention is Anaerostipes caccae AF04-45, which is disclosed in the invention patent with the authorization announcement number “CN110062806B” and the name “Anaerobic Corynebacterium faecalis (Anaerostipes caccae) and its application”, and the deposit number is GDMCC No.: 60088.

[0051] Example 1: A method for preparing a composite preparation based on probiotics and prebiotics, comprising the following steps:

[0052] S1. Weigh 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate heptahydrate, and 0.25 g of manganese sulfate tetrahydrate. Add 1000 mL of distilled water and 1 mL of Tween-80 and stir until completely dissolved. Adjust the pH to 6.8, filter, and autoclave at 121°C for 15 min to obtain MRS culture medium.

[0053] S2. Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus were placed in a sterile MRS culture medium at 37 ° C for 22h, then centrifuged at 4000rpm for 15min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension, Lactobacillus rhamnosus suspension, and then 4g of Lactobacillus plantarum suspension, 2g of Lactobacillus acidophilus suspension, 8g of Bifidobacterium longum suspension, 10g of Lactobacillus rhamnosus suspension were mixed to obtain a composite probiotic;

[0054] S3. Add 2 g of ethanol to 5 g of wheat cellulose, stir well, and distill under reduced pressure at 30°C for 22 min. Then, add 1 g of pomegranate peel ethanol extract, 28 mL of the composite probiotics, 0.4 g of Tween-80, and 90 mL of a 3% sodium alginate aqueous solution and mix well to obtain mixture A.

[0055] S4. Using a syringe pump, 45 mL of a 2 mol / L calcium chloride solution was added to 135 mL of the mixture A and centrifuged at 7200 rpm for 20 min to obtain a probiotic composition A;

[0056] S5. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37 ° C for 22 h, then centrifuged at 4000 rpm for 15 min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a suspension of Lactobacillus acidophilus and an anaerobic Corynebacterium faecalis, respectively;

[0057] S6. Weigh 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 8 g NaCl, add 1000 mL distilled water, stir until completely dissolved, and adjust the pH to 7.2 to obtain a phosphate buffered saline solution;

[0058] S7. 16 g of inulin and 5 g of glutamine were dissolved in 6 mL of phosphate buffered saline and magnetically stirred at 37 ° C for 15 min, and then 5 mL of Lactobacillus acidophilus suspension and 3 mL of anaerobic Corynebacterium faecalis suspension were added, mixed evenly, and freeze-dried at -82 ° C for 23 h to obtain a probiotic composition B;

[0059] S8. Activate Bifidobacterium longum and Lactobacillus rhamnosus in sterile MRS medium at 37°C for 22 h, centrifuge at 4000 rpm for 15 min, wash three times with sterile water, and resuspend in phosphate-buffered saline to a concentration of 1 × 10 10 CFU / mL, and then heat-inactivated at 90°C for 10 min to obtain probiotic composition C;

[0060] S9. Mix 24 g of probiotic composition A, 10 g of probiotic composition B and 2.4 g of probiotic composition C to obtain a probiotic composition, and then mix 25 g of the probiotic composition with 4 g of gosaccharide, 3.3 g of lotus leaf pectin-type polysaccharide LLP, 4 g of morel polysaccharide MEP-1, 0.5 g of puerarin, 15 g of oligofructose, 12 g of stachyose and 9 g of oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

[0061] Example 2: A method for preparing a composite preparation based on probiotics and prebiotics, comprising the following steps:

[0062] S1. Weigh 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate heptahydrate, and 0.25 g of manganese sulfate tetrahydrate. Add 1000 mL of distilled water and 1 mL of Tween-80 and stir until completely dissolved. Adjust the pH to 6.8, filter, and autoclave at 121°C for 15 min to obtain MRS culture medium.

[0063] S2. Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus were placed in a sterile MRS culture medium at 37 ° C for 23h, then centrifuged at 4300rpm for 17min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension, Lactobacillus rhamnosus suspension, and then 5g of Lactobacillus plantarum suspension, 3g of Lactobacillus acidophilus suspension, 9g of Bifidobacterium longum suspension, 12g of Lactobacillus rhamnosus suspension were mixed to obtain a composite probiotic;

[0064] S3. Add 2.5 g of ethanol to 6 g of wheat cellulose, stir well, and distill under reduced pressure at 33°C for 25 min. Then, add 1.5 g of pomegranate peel ethanol extract, 30 mL of the composite probiotics, 0.45 g of Tween-80, and 93 mL of a 3% sodium alginate aqueous solution and mix well to obtain a mixture A.

[0065] S4. Using a syringe pump, 46 mL of a 2 mol / L calcium chloride solution was added to 136 mL of the mixture A, and the mixture was centrifuged at 7500 rpm for 21 min to obtain a probiotic composition A;

[0066] S5. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37 ° C for 23 h, then centrifuged at 4300 rpm for 16 min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a Lactobacillus acidophilus suspension and an anaerobic Corynebacterium faecalis suspension, respectively;

[0067] S6. Weigh 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 8 g NaCl, add 1000 mL distilled water, stir until completely dissolved, and adjust the pH to 7.2 to obtain a phosphate buffered saline solution;

[0068] S7. 17 g of inulin and 6 g of glutamine were dissolved in 7 mL of phosphate buffered saline and magnetically stirred at 37 ° C for 16 min, and then 6 mL of Lactobacillus acidophilus suspension and 4 mL of anaerobic Corynebacterium faecalis suspension were added, mixed evenly, and freeze-dried at -80 ° C for 24 h to obtain a probiotic composition B;

[0069] S8. Bifidobacterium longum and Lactobacillus rhamnosus were activated in sterile MRS culture medium at 37°C for 23 h, centrifuged at 4300 rpm for 17 min, washed three times with sterile water, and suspended in phosphate-buffered saline to a concentration of 2 × 10 10 CFU / mL, and then heat-inactivated at 93°C for 10 min to obtain probiotic composition C;

[0070] S9. Mix 27g of probiotic composition A, 11g of probiotic composition B and 2.8g of probiotic composition C to obtain a probiotic composition, and then mix 26g of the probiotic composition with 5g of gosaccharide, 4.1g of lotus leaf pectin-type polysaccharide LLP, 5g of morel polysaccharide MEP-1, 0.6g of puerarin, 16g of oligofructose, 14g of stachyose and 10g of oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

[0071] Example 3: A method for preparing a composite preparation based on probiotics and prebiotics, comprising the following steps:

[0072] S1. Weigh 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate heptahydrate, and 0.25 g of manganese sulfate tetrahydrate. Add 1000 mL of distilled water and 1 mL of Tween-80 and stir until completely dissolved. Adjust the pH to 6.8, filter, and autoclave at 121°C for 15 min to obtain MRS culture medium.

[0073] S2. Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus were placed in a sterile MRS culture medium at 37 ° C for 23h, and then centrifuged at 4600rpm for 18min, the supernatant was discarded, the bacteria were collected, and the bacterial suspension was prepared with sterile water to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension, Lactobacillus rhamnosus suspension, and then 6g of Lactobacillus plantarum suspension, 4g of Lactobacillus acidophilus suspension, 10g of Bifidobacterium longum suspension, 13g of Lactobacillus rhamnosus suspension were mixed to obtain a composite probiotic;

[0074] S3. 2.5 g of ethanol was added to 8 g of wheat cellulose, stirred, and distilled under reduced pressure at 36°C for 27 min. Then, 1.5 g of pomegranate peel ethanol extract, 31 mL of the probiotic complex, 0.5 g of Tween-80, and 96 mL of a 3% sodium alginate aqueous solution were added and mixed to obtain a mixture A.

[0075] S4. Using a syringe pump, 52 mL of a 2.5 mol / L calcium chloride solution was added to 138 mL of the mixture A, and the mixture was centrifuged at 7600 rpm for 22 min to obtain a probiotic composition A;

[0076] S5. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37 ° C for 23 h, then centrifuged at 4800 rpm for 18 min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a Lactobacillus acidophilus suspension and an anaerobic Corynebacterium faecalis suspension;

[0077] S6. Weigh 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 8 g NaCl, add 1000 mL distilled water, stir until completely dissolved, and adjust the pH to 7.2 to obtain a phosphate buffered saline solution;

[0078] S7. 18 g of inulin and 8 g of glutamine were dissolved in 8 mL of phosphate buffered saline and magnetically stirred at 37 ° C for 18 min, and then 7 mL of Lactobacillus acidophilus suspension and 5 mL of anaerobic Corynebacterium faecalis suspension were added, mixed evenly, and freeze-dried at -78 ° C for 24 h to obtain a probiotic composition B;

[0079] S8. Bifidobacterium longum and Lactobacillus rhamnosus were activated in sterile MRS culture medium at 37°C for 23 h, centrifuged at 4800 rpm for 18 min, washed three times with sterile water, and suspended in phosphate buffered saline to a concentration of 3 × 10 10 CFU / mL, and then heat-inactivated at 97°C for 10 min to obtain probiotic composition C;

[0080] S9. Mix 30g of probiotic composition A, 11.5g of probiotic composition B and 3.2g of probiotic composition C to obtain a probiotic composition, and then mix 28g of the probiotic composition with 6g of gosaccharide, 5g of lotus leaf pectin-type polysaccharide LLP, 6g of morel polysaccharide MEP-1, 0.7g of puerarin, 17g of oligofructose, 15g of stachyose and 12g of oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

[0081] Example 4: A method for preparing a composite preparation based on probiotics and prebiotics, comprising the following steps:

[0082] S1. Weigh 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 20 g of glucose, 2 g of diammonium hydrogen citrate, 5 g of sodium acetate, 2 g of dipotassium hydrogen phosphate, 0.58 g of magnesium sulfate heptahydrate, and 0.25 g of manganese sulfate tetrahydrate. Add 1000 mL of distilled water and 1 mL of Tween-80 and stir until completely dissolved. Adjust the pH to 6.8, filter, and autoclave at 121°C for 15 min to obtain MRS culture medium.

[0083] S2. Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus were placed in a sterile MRS culture medium at 37 ° C for 24 h, and then centrifuged at 5000 rpm for 20 min. The supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension, Lactobacillus rhamnosus suspension, and then 7 g of Lactobacillus plantarum suspension, 5 g of Lactobacillus acidophilus suspension, 12 g of Bifidobacterium longum suspension, 15 g of Lactobacillus rhamnosus suspension were mixed to obtain a composite probiotic;

[0084] S3. 3 g of ethanol was added to 9 g of wheat cellulose, stirred, and distilled under reduced pressure at 40°C for 30 min. Then, 2 g of pomegranate peel ethanol extract, 33 mL of the composite probiotics, 0.6 g of Tween-80, and 100 mL of a 3% sodium alginate aqueous solution were added and mixed to obtain a mixture A.

[0085] S4. 55 mL of 2.5 mol / L calcium chloride solution was added to 140 mL of mixture A using a syringe pump and centrifuged at 7800 rpm for 24 min to obtain a probiotic composition A;

[0086] S5. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37 ° C for 24 h, then centrifuged at 5000 rpm for 20 min, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a suspension of Lactobacillus acidophilus and an anaerobic Corynebacterium faecalis, respectively;

[0087] S6. Weigh 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, and 8 g NaCl, add 1000 mL distilled water, stir until completely dissolved, and adjust the pH to 7.2 to obtain a phosphate buffered saline solution;

[0088] S7. 19 g of inulin and 9 g of glutamine were dissolved in 8 mL of phosphate buffered saline and magnetically stirred at 37 ° C for 20 min, and then 8 mL of Lactobacillus acidophilus suspension and 6 mL of anaerobic Corynebacterium faecalis suspension were added, mixed evenly, and freeze-dried at -76 ° C for 25 h to obtain a probiotic composition B;

[0089] S8. Activate Bifidobacterium longum and Lactobacillus rhamnosus in sterile MRS medium at 37°C for 24 h, centrifuge at 5000 rpm for 20 min, wash three times with sterile water, and resuspend in phosphate-buffered saline to a concentration of 3.5 × 10 10 CFU / mL, and then heat-inactivated at 100°C for 10 min to obtain probiotic composition C;

[0090] S9. Mix 33 g of probiotic composition A, 12 g of probiotic composition B and 3.8 g of probiotic composition C to obtain a probiotic composition, and then mix 29 g of the probiotic composition with 8 g of gaussose, 6 g of lotus leaf pectin-type polysaccharide LLP, 7 g of morel polysaccharide MEP-1, 0.9 g of puerarin, 18 g of oligofructose, 16 g of stachyose and 13 g of oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

[0091] Comparative Example 1:

[0092] Compared with Example 1, the probiotic composition A was not added during the preparation of the composite preparation based on probiotics and prebiotics in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite preparation based on probiotics and prebiotics was obtained.

[0093] Comparative Example 2:

[0094] Compared with Example 1, the probiotic composition B was not added during the preparation of the composite preparation based on probiotics and prebiotics in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite preparation based on probiotics and prebiotics was obtained.

[0095] Comparative Example 3:

[0096] Compared with Example 1, the probiotic composition C was not added during the preparation of the composite preparation based on probiotics and prebiotics in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite preparation based on probiotics and prebiotics was obtained.

[0097] Comparative Example 4:

[0098] Compared with Example 1, wheat cellulose was not added during the preparation of the probiotic composition A in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite preparation based on probiotics and prebiotics was obtained.

[0099] Comparative Example 5:

[0100] Compared with Example 1, glutamine was not added during the preparation of the probiotic composition B in this comparative example. The remaining steps and parameters were the same and will not be repeated in this comparative example. Finally, a composite preparation based on probiotics and prebiotics was obtained.

[0101] Comparative Example 6:

[0102] Compared with Example 1, this comparative example only replaces "heat inactivation treatment at 90°C for 10 min" with "heat inactivation treatment at 121°C for 10 min", and the remaining steps and parameters are the same. This comparative example will not be repeated, and finally a composite preparation based on probiotics and prebiotics is obtained.

[0103] Comparative Example 7:

[0104] Compared with Example 1, this comparative example only replaces the "probiotic composition" with "lyophilized powder of Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus rhamnosus and Anaerobic Corynebacterium faecalis", and the remaining steps and parameters are the same. This comparative example will not be repeated, and finally a composite preparation based on probiotics and prebiotics is obtained.

[0105] Performance testing:

[0106] 220 patients with constipation aged 22-42 years and 27-34 weeks of gestation were randomly divided into 11 groups. Each group was given the probiotic and prebiotic combination preparations prepared in Examples 1-4 and Comparative Examples 1-7. The constipation symptoms of the 220 patients who took the different preparations were scored according to the constipation symptom scoring table, and the total effectiveness of each preparation in relieving constipation symptoms was determined.

[0107] Inclusion criteria: individuals with less than 3 bowel movements per week and at least one of the following symptoms: painful defecation once a week; individuals with thick and large stools and obstruction of the toilet due to large stool volume; individuals with stopping bowel movements during defecation, individuals with hard stools found during abdominal or rectal examination; and the duration lasted for more than 2 weeks;

[0108] Exclusion criteria: ① history of inflammatory bowel disease or irritable bowel syndrome; ② combined with severe liver or kidney dysfunction; ③ combined with intestinal obstruction; ④ combined with heart or lung disease; ⑤ combined with neurological disease or mental illness;

[0109] Constipation symptom scoring: Stool consistency is graded according to the Bristol stool scale: Grade 1 is nut-like hard stool, small hard pieces that are difficult to pass; Grade 2 is dry and hard stool, which is hard but sticks together; Grade 3 is stool with cracks on the surface, like sausage; Grade 4 is soft, with a smooth surface, like a banana; Grade 5 is soft semi-solid, with clear edges and an uneven shape; Grade 6 is slightly shaped stool, which is mushy and has unclear edges; Grade 7 is watery stool, which is completely liquid and has no solid components.

[0110] Efficacy for constipation: Significantly effective refers to stool consistency grade 4-6, bowel movement frequency once a day, and constipation symptoms of 0 points; effective refers to stool consistency grade 2-3, bowel movement frequency >3 times a week, and constipation symptoms of 1 point; ineffective refers to stool consistency grade 1-2, bowel movement frequency <3 times a week, and constipation symptoms of 2-3 points. Significantly effective and effective are counted into the total effective.

[0111] Table 1 Constipation symptom score sheet

[0112]

[0113] Table 2

[0114]

[0115] Data Analysis:

[0116] As can be seen from Tables 1 and 2, the composite preparation based on probiotics and prebiotics prepared by the present invention is more conducive to relieving constipation. This may be because the probiotic composition A is prepared from composite probiotics, sodium alginate and wheat cellulose. Sodium alginate, wheat cellulose and pomegranate peel ethanol extract can not only promote the growth of probiotics, but also provide a good living environment for the composite probiotics, preventing the probiotics from losing their activity due to low gastric pH, small intestinal bile and different digestive enzymes, and wheat cellulose can improve the mechanical strength of sodium alginate, so that the probiotics in the composition are more effective. The release time of bacteria is prolonged, which can prolong the duration of action of the compound preparation, better regulate the gastrointestinal flora and achieve better relief effect; the probiotic composition B is prepared from inulin, glutamic acid, Lactobacillus acidophilus and anaerobic Corynebacterium faecalis. On the one hand, inulin, as a prebiotic, promotes the growth of probiotics while controlling the release of probiotics in the small intestine and improving the survival rate of probiotics. On the other hand, inulin and glutamine are combined to replace water molecules, protect proteins and lipid membranes, and promote the recovery of probiotic activity; Lactobacillus acidophilus enhances the activity of serotonin transporter and water channel protein -3 expression, thereby enhancing the intestinal barrier function, and can also degrade inulin to release fructose and convert it into lactic acid and acetic acid, while anaerobic Corynebacterium faecalis can convert lactic acid into butyric acid in the presence of acetic acid. Probiotic composition B synthesizes short-chain fatty acids, reduces the pH value of the small intestine, inhibits the growth of pathogens, increases colon smooth muscle contraction, improves intestinal peristalsis, and relieves constipation and indigestion symptoms; in probiotic composition C, Bifidobacterium longum and Lactobacillus rhamnosus are heated, and the heat-inactivated probiotics are in the form of undamaged inactive cells, cell walls and membranes, and intracellular The partial mixed form can also regulate intestinal inflammation and relieve constipation. At the same time, the probiotics after heat inactivation are less harmful to the human body and are safer when used. Prebiotics such as Morchella polysaccharide MEP-1 can prevent harmful substances from entering the systemic circulation by promoting the production of short-chain fatty acids and enhancing the intestinal barrier function, inhibiting inflammatory pathways, regulating intestinal health, and also improving lipid metabolism disorders caused by a high-fat diet in pregnant women. The present invention improves the activity of probiotics in the intestine by compounding multiple probiotic compositions, and when mixed with multiple prebiotics, it can effectively relieve constipation.

[0117] As can be seen from Tables 1 and 2, the total effective rate of Comparative Example 1 is worse than that of Example 1 because the probiotic composition A is not added. This may be because the probiotic composition A can improve the activity of the composite probiotics and protect them from the influence of low gastric pH, small intestinal bile and different digestive enzymes. As can be seen from Tables 1 and 2, the total effective rate of Comparative Example 2 is worse than that of Example 1 because the probiotic composition B is not added. This may be because the probiotic composition B can control the release of probiotics in the small intestine and play a faster role in relieving constipation, which is beneficial to the maintenance of probiotic activity and can also encourage probiotics to use prebiotics to synthesize short-chain fatty acids, improve intestinal peristalsis, and relieve constipation. and indigestion symptoms; Comparative Example 3 does not add probiotic composition C, as can be seen from Table 1 and Table 2, its total effective rate is worse than that of Example 1, which may be because the probiotics after heat inactivation appear in the form of undamaged inactive cells, cell walls and membranes, and intracellular parts, which can also regulate intestinal inflammation and relieve constipation; Comparative Example 4 does not add wheat cellulose, as can be seen from Table 1 and Table 2, its total effective rate is worse than that of Example 1, which may be because the addition of wheat cellulose can enhance the mechanical strength and stability of sodium alginate gel, and can also improve the acid resistance of probiotics, so that they can smoothly pass through gastric juice to reach the intestine to play a role, which helps The growth and colonization of probiotics in the intestine can better play the probiotic function and relieve constipation; Comparative Example 5 does not add glutamine. As can be seen from Table 1 and Table 2, its total effective rate is worse than that of Example 1. This may be because glutamine helps to improve constipation symptoms, enhance intestinal barrier function, and maintain the integrity of intestinal mucosa by regulating the composition of intestinal microflora, and glutamine and inulin are combined to replace water molecules, protect protein and lipid membranes, and promote the recovery of probiotic activity; Comparative Example 6 replaces "heat inactivation treatment at 90°C for 10 min" with "heat inactivation treatment at 121°C for 10 min". As can be seen from Table 1 and Table 2 It can be seen that its total effective rate is worse than that of Example 1. This may be because the properties of the probiotics are significantly changed after heat inactivation treatment at 121°C. Excessive heating will lead to the degradation of heat-sensitive active ingredients and the weakening of their biological efficacy, and thus they cannot play a good role. Comparative Example 7 replaces the "probiotic composition" with "Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus rhamnosus and anaerobic Corynebacterium faecalis freeze-dried powder". It can be seen from Tables 1 and 2 that its total effective rate is worse than that of Example 1. This may be because the activity of probiotics not encapsulated with sodium alginate and inulin is easily affected by the harsh environment of the gastrointestinal tract and inactivated.

[0118] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present invention is limited to these examples. Within the scope of the present invention, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the present invention as described above, which are not provided in detail for the sake of simplicity.

[0119] The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a composite preparation based on probiotics and prebiotics, characterized in that: Including the following raw materials by weight: 25-29 parts of probiotic composition, 4-8 parts of gosaccharide, 3.3-6 parts of lotus leaf pectin-type polysaccharide LLP, 4-7 parts of morel polysaccharide MEP-1, 0.5-0.9 parts of puerarin, 15-18 parts of fructooligosaccharide, 12-16 parts of stachyose, and 9-13 parts of galacto-oligosaccharide; The probiotic composition is obtained by mixing probiotic composition A, probiotic composition B and probiotic composition C in a mass ratio of 24-33:10-12:2.4-3.8; The probiotic composition A is prepared from wheat cellulose, ethanol, pomegranate peel ethanol extract, composite probiotics, Tween-80, sodium alginate aqueous solution and calcium chloride solution; The probiotic composition B is prepared from inulin, glutamine, Lactobacillus acidophilus and anaerobic Corynebacterium faecalis; The probiotic composition C is prepared from Bifidobacterium longum and Lactobacillus rhamnosus; The composite probiotics are obtained by mixing Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus; The Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus are respectively in the form of Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension and Lactobacillus rhamnosus suspension to form a composite probiotic; The mass ratio of the Lactobacillus plantarum suspension, the Lactobacillus acidophilus suspension, the Bifidobacterium longum suspension, and the Lactobacillus rhamnosus suspension is 4-7:2-5:8-12:10-15; The preparation method of the probiotic composition C is as follows: Bifidobacterium longum and Lactobacillus rhamnosus were activated in sterile MRS culture medium at 37°C for 22-24 hours, centrifuged at 4000-5000 rpm for 15-20 minutes, washed three times with sterile water and suspended in phosphate buffered saline to a concentration of 1×10 10 -3.5×10 10 CFU / mL, and then heat-inactivation treatment was performed to obtain probiotic composition C; The temperature during the heat inactivation treatment is 90-100° C. and the time is 10 minutes.

2. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 1, characterized in that: The preparation method of the compound probiotics is as follows: Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium longum and Lactobacillus rhamnosus are respectively activated in a sterile MRS culture medium at 37° C. for 22-24 hours, then centrifuged at 4000-5000 rpm for 15-20 minutes, the supernatant is discarded, the bacteria are collected, and sterile water is used to prepare a bacterial suspension to obtain Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension and Lactobacillus rhamnosus suspension, respectively; and the Lactobacillus plantarum suspension, Lactobacillus acidophilus suspension, Bifidobacterium longum suspension and Lactobacillus rhamnosus suspension are then mixed to obtain a composite probiotic; The MRS culture medium formula is: 10g peptone, 10g beef extract, 5g yeast extract, 20g glucose, 1mL Tween-80, 2g diammonium hydrogen citrate, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate heptahydrate, 0.25g manganese sulfate tetrahydrate, 1000mL distilled water, and pH 6.

8.

3. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 2, characterized in that: The number of viable Lactobacillus plantarum in the composite probiotics is 5×10 8 -5×10 10 CFU / mL, the number of viable Lactobacillus acidophilus is 3×10 7 -3×10 9 CFU / mL, the number of viable bacteria of Bifidobacterium longum was 2.2×10 7 -2.2×10 10 CFU / mL, the number of viable Lactobacillus rhamnosus was 2×10 7 -2×10 8 CFU / mL.

4. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 1, characterized in that: The preparation method of the probiotic composition A is as follows: Step A1. Add ethanol to the wheat cellulose, stir evenly, and distill under reduced pressure at 30-40°C for 22-30 minutes. Then, add the pomegranate peel ethanol extract, the composite probiotics, Tween-80, and a 3% by mass aqueous solution of sodium alginate and mix evenly to obtain a mixture A. Step A2. Add the calcium chloride solution to the mixture A using a syringe pump, and centrifuge at 7200-7800 rpm for 20-24 min to obtain a probiotic composition A.

5. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 4, characterized in that: The ratio of wheat cellulose, ethanol, pomegranate peel ethanol extract, composite probiotics, Tween-80 and sodium alginate aqueous solution in step A1 is 5-9 g: 2-3 g: 1-2 g: 28-33 mL: 0.4-0.6 g: 90-100 mL; The concentration of the calcium chloride solution in step A2 is 2-2.5 mol / L; The volume ratio of the calcium chloride solution to the mixture A is 45-55:135-140.

6. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 1, characterized in that: The preparation method of the probiotic composition B is as follows: Step B1. Lactobacillus acidophilus and anaerobic Corynebacterium faecalis were activated in sterile MRS culture medium at 37°C for 22-24 hours, then centrifuged at 4000-5000 rpm for 15-20 minutes, the supernatant was discarded, the cells were collected, and sterile water was used to prepare a bacterial suspension to obtain a Lactobacillus acidophilus suspension and an anaerobic Corynebacterium faecalis suspension, respectively; Step B2. Dissolve inulin and glutamine in phosphate buffered saline and stir magnetically at 37°C for 15-20 minutes, then add the Lactobacillus acidophilus suspension and the anaerobic Corynebacterium faecalis suspension, mix well, and freeze-dry to obtain a probiotic composition B.

7. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 6, characterized in that: The usage ratio of inulin, glutamine, phosphate buffered saline, Lactobacillus acidophilus suspension and anaerobic Corynebacterium faecalis suspension in step B2 is 16-19 g: 5-9 g: 6-8 mL: 5-8 mL: 3-6 mL; The number of viable Lactobacillus acidophilus in the probiotic composition B is 1×10 8 -1×10 10 CFU / g, the number of viable bacteria of anaerobic Corynebacterium faecalis is 1x10 6 -10 8 CFU / g; The temperature during the freeze drying is -82-76°C and the time is 23-25 ​​hours.

8. The method for preparing the composite preparation based on probiotics and prebiotics according to claim 1, characterized in that: The following steps are involved: Probiotic composition A, probiotic composition B and probiotic composition C are mixed to obtain a probiotic composition, which is then evenly mixed with gaussose, lotus leaf pectin-type polysaccharide LLP, morel polysaccharide MEP-1, puerarin, oligofructose, stachyose and oligogalactose to obtain a composite preparation based on probiotics and prebiotics.

9. Use of the composite preparation based on probiotics and prebiotics according to any one of claims 1 to 8 in the preparation of a health product for relieving constipation during pregnancy.

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