Anti-helicobacter pylori composite nutritional composition and preparation method thereof

Abstract

The invention belongs to the field of nutritional food preparation, and particularly relates to a helicobacter pylori-resistant composite nutritional composition and a preparation method thereof. The composite nutritional composition contains the functional plant protein hydrogel, the probiotic preparation and the like, the functional plant protein hydrogel has the effects of various plant-derived active substances at the same time, and the probiotic preparation is used for encapsulating the lactobacillus rhamnosus D8A, so that the survival rate of the lactobacillus rhamnosus D8A in gastrointestinal tracts is increased. The functional vegetable protein hydrogel and the probiotic preparation can synergistically improve the intestinal flora disorder condition. After egg yolk globulin powder, maltodextrin and Arabic gum are mixed, a coating material is prepared and covers the outside of a pressed tablet, and finally, the composite nutritional composition obtained by air drying is safe, non-toxic and edible through an acute oral toxicity test and a 30-day feeding test, and meanwhile, the composite nutritional composition is beneficial to regulating intestinal flora and improving the immunity of a human body. The field planting of stomach helicobacter pylori can be inhibited, and the application range is wide.

Description

Technical Field

[0001] The invention belongs to the field of nutritious food preparation, and in particular relates to an anti-Helicobacter pylori composite nutritious composition and a preparation method thereof. Background Art

[0002] The gastrointestinal tract is a complex ecosystem that achieves functions such as digestion, absorption, secretion and immunity through the different functions of a series of organs from the mouth to the anus. In addition, the enteric nervous system can independently control most of the functions of the intestine and is often referred to as the "second brain". The gastrointestinal system of humans and animals contains about trillions of microorganisms, which are a collection of bacteria, archaea, viruses and eukaryotes, known as "intestinal flora". After thousands of years of co-evolution, it has formed an intricate symbiotic relationship with the host. The intestinal flora of a normal human body includes Firmicutes, Bacteroidetes, Proteobacteria, Clostridium, Actinobacteria, Verrucomicrobia, etc., which together maintain the homeostasis of the intestinal environment. When the intestinal environment is disordered, the increase in pathogenic bacteria and the endotoxins they release will damage the biological barrier of the intestinal mucosa. The pathogenic bacteria and their antigens release a variety of active substances that can easily cause patients to have gastrointestinal diseases such as diarrhea, constipation and irritable bowel syndrome. In addition, intestinal flora also plays an important role in preventing gastrointestinal diseases and inhibiting pathogens. Currently, common treatments for intestinal flora imbalance include fecal microbiota transplantation, probiotics, prebiotics, postbiotics and dietary intervention.

[0003] Among the intestinal flora, Helicobacter pylori is a well-known microorganism. It was first reported by Australian scholars in 1982 that it was isolated from the gastric mucosa of patients with chronic gastritis and gastric ulcers. It is a Gram-negative bacterium with a typical morphology of S-type, C-type or seagull-shaped curves. It can survive in the strong acidic environment of the stomach and pass through the mucus layer to reach the epithelial surface and firmly connect to avoid being emptying by the stomach. The strong motility, adhesin and urease produced by Helicobacter pylori enable it to firmly connect to the surface of the gastric mucosa. Helicobacter pylori can be divided into type I and type II. Type I strains are highly virulent strains that contain cytotoxin-associated protein (CagA) genes and express CagA proteins. They can cause gastroduodenal ulcers, lymphomas, gastric cancer, etc. in the stomach, causing relatively large damage to the stomach. Type II is a low-virulence strain that does not contain the CagA gene and can cause chronic superficial gastritis in the stomach, which is relatively easy to treat. The infection routes of Helicobacter pylori mainly involve oral-oral transmission, fecal-oral transmission, and medical-induced transmission. Therefore, disinfecting tableware and implementing a separate dining system can prevent the spread of Helicobacter pylori to a certain extent. Summary of the invention

[0004] Based on the above problems, the present invention provides a composite nutritional composition against Helicobacter pylori, containing functional plant protein hydrogel, probiotic preparations, etc. The functional plant protein hydrogel has the efficacy of multiple plant-derived active substances at the same time. The probiotic preparation is to encapsulate Lactobacillus rhamnosus D8A, and the biochar, sodium alginate and xanthan gum from oyster shells are processed and cultured, which improves the survival rate of Lactobacillus rhamnosus D8A in the gastrointestinal tract. Functional plant protein hydrogel and probiotic preparations can synergistically improve the disorder of intestinal flora. After mixing egg yolk globulin powder, maltodextrin and gum arabic, a coating material is prepared, which is covered on the outside of the material compressed by functional plant protein hydrogel, probiotic preparations, etc., and finally the composite nutritional composition obtained by air drying is safe and non-toxic under oral acute toxicity test and 30-day feeding test, edible, and at the same time helps to regulate intestinal flora, can inhibit the colonization of Helicobacter pylori in the stomach, and is widely applicable to the population.

[0005] The invention provides an anti-Helicobacter pylori composite nutritional composition, comprising the following raw materials in parts by weight: 2-3 parts of functional plant protein hydrogel, 0.3-1 parts of probiotic preparation, 0.1-0.25 parts of black pepper extract, and 1-3 parts of turmeric.

[0006] The preparation method of the functional plant protein hydrogel of the present invention is as follows:

[0007] L1. Wash and slice Momordica grosvenori and Hericium erinaceus, dry and grind into powder, sieve and weigh according to the mass ratio of 8-12:3-5:1 to obtain a mixed powder, add 10-12 times the mass of water, adjust the pH to 8.0, stir at a speed of 180-240rpm for 3-4h, filter through a nylon mesh, centrifuge the filtrate at a centrifugal force of 8000-10000g for 30-45min, adjust the pH to 4.5, let stand at room temperature for 8-10h, centrifuge at a centrifugal force of 10000-12000g for 15-20min, wash the precipitate with deionized water, add deionized water to dissolve and dialyze for 8-10h, and freeze-dry to obtain substance 1;

[0008] L2. The substance 1 obtained in step L1 was dispersed in deionized water, stirred at 150-200 rpm for 2-4 hours at room temperature, and then placed in a 4°C environment for 10-15 hours, heated in a water bath to 90-95°C and maintained for 30-45 minutes, cooled naturally to room temperature, and the pH was adjusted to 7.0 to obtain solution 1, and glutamine transaminase was added, and ultrasonic treatment was performed at a power of 400-450 W for 1-2 minutes to obtain solution 2;

[0009] L3. Weigh konjac flour, psyllium husk powder and water in a mass ratio of 1:5-8:15-20, stir until evenly dispersed, and then stand at room temperature for 15-18 hours. After completion, freeze-dry, grind and sieve to obtain substance 2, weigh substance 2 and solution 2 obtained in step L2 in a mass volume ratio of 1g:8-12mL, stir until evenly dispersed, heat to 30-40℃, ultrasonically treat at a power of 100-150W for 30-45min, and naturally cool to room temperature to obtain functional plant protein hydrogel.

[0010] Preferably, in step L1, the sieve is preferably a 10-mesh sieve, the nylon mesh is preferably a 180-mesh sieve, and the dialysis bag with a molecular weight cutoff of 4.5 kDa is selected for dialysis.

[0011] Preferably, the mass percentage concentration of substance 1 in deionized water in step L2 is 3-6%, the amount of transglutaminase added is 1-5% of the mass of solution 1, and the enzyme activity of transglutaminase is preferably 12 U / g.

[0012] Preferably, the sieving in step L3 is preferably a 2-mesh sieve.

[0013] The preparation method of the probiotic preparation of the present invention is as follows:

[0014] S1. Cultivate Lactobacillus rhamnosus D8A in lactic acid bacteria culture medium until the concentration is 1×10 6 -1×10 9 cfu / mL bacterial suspension, centrifuge the bacterial suspension at 2000-2500g for 10-20min, discard the supernatant, wash the precipitate with PBS buffer solution 2-3 times, and centrifuge again at 3000-3200g for 10-20min to obtain the precipitate;

[0015] S2. The oyster shell was calcined at 1000-1200°C for 2-3h, and then naturally cooled to room temperature, washed with 0.1mM hydrochloric acid solution for 2-3 times, and then washed with deionized water for 2-3 times, and then frozen at -80°C for 24-30h after drying, and ground at 300-360rpm for 2-3h, and then substance 3 was obtained;

[0016] S3. Weigh the substance 3 obtained in step S2, sodium alginate, xanthan gum, the precipitate obtained in step S1 and distilled water according to the mass volume ratio of 0-1g:0.5-1g:1-2g:0.2-0.5g:30-40mL, stir at a speed of 120-160rpm for 15-20min to obtain solution 3, drop solution 3 into 0.1mM calcium chloride solution at an electrode frequency of 90-100Hz, a voltage of 450-500mV and a flow rate of 40-50mL / min, collect the insoluble matter and wash it with deionized water for 3-5 times, and obtain the probiotic preparation after air drying.

[0017] Preferably, the rhamnosus Lactobacillus D8A in step S1 has a Latin name of Lacticaseibacilusrhamnosus, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration on March 7, 2024, with a deposit number of CGMCC No.29961. The lactic acid bacteria culture medium is prepared as follows: 7.5 g yeast extract, 7 g beef extract, 7.5 g peptone, 1.5 g diammonium hydrogen citrate, 2 g potassium dihydrogen phosphate, 0.2 g anhydrous magnesium sulfate, 0.5 mL Tween-80, 7.5 g glucose, 0.03 g manganese sulfate, and 1000 mL distilled water, and the pH is adjusted to 7.0. The pH of the PBS buffer solution used in step S1 is 6.5.

[0018] Preferably, the grinding in step S2 is preferably performed in a planetary grinder, the grinding ball particle size is preferably 5 mm, and the ball-to-material ratio is preferably 10-15:1.

[0019] The present invention also provides a method for preparing an anti-Helicobacter pylori composite nutritional composition, the specific steps of which are as follows:

[0020] V1. Dissolve egg yolk globulin powder in water to obtain a solution 4 with a mass percentage concentration of 5-8%, heat to 70-80°C and maintain for 12-18 minutes, adjust the pH to 6.5, add papain, and bathe at a speed of 80-120 rpm and a temperature of 58-64°C for 3-4 hours, heat to 95°C and maintain for 8-10 minutes to terminate the enzyme activity, and centrifuge at a centrifugal force of 6000-8000g for 20-25 minutes, collect the supernatant and freeze-dry to obtain substance 4;

[0021] V2. Dissolve maltodextrin in water to obtain a solution 5 with a mass percentage concentration of 10-12%, keep it at room temperature for 12-15 hours, dissolve gum arabic in water to obtain a solution 6 with a mass percentage concentration of 4-6%, weigh the substance 4 obtained in step V1, solution 5 and solution 6 according to the mass volume ratio of 1g:3-6mL:2-5mL, stir until uniformly dispersed, and obtain solution 7;

[0022] V3. The functional plant protein hydrogel, probiotic preparation, black pepper extract and turmeric are mixed, dispersed evenly and then dried and granulated to obtain substance 5. The solution 7 obtained in step V2 is evenly covered on the outside of substance 5, and after air drying, a composite nutritional composition against Helicobacter pylori is obtained.

[0023] Preferably, the amount of papain added in step V1 is 2-5% of the mass of solution 4, the enzyme activity of papain is preferably 6000 U / mg, and the pH needs to be controlled to 6.5 using sodium hydroxide solution during the water bath.

[0024] The beneficial effects of the present invention are as follows:

[0025] The composite nutritional composition of the present invention contains functional plant protein hydrogel, probiotic preparations and the like. The functional plant protein hydrogel is prepared by extracting proteins from monk fruit and hericium erinaceus, and obtaining an active part of 4.5 kDa in size. The protein is treated with glutamine transaminase so that the protein presents a cross-linked network, which is convenient for loading other active ingredients. The other active ingredients specifically come from konjac flour and psyllium husk powder. The addition of konjac flour realizes the modification of psyllium husk powder, thereby reducing the viscosity of psyllium husk powder, making it more suitable for food processing. After the substance 2 obtained by combining konjac flour and psyllium husk powder is combined with solution 2, the obtained functional plant protein hydrogel has the efficacy of multiple plant-derived active substances at the same time, and has the effect of inhibiting bacteria and resisting Helicobacter pylori. The probiotic preparation specifically encapsulates Lactobacillus rhamnosus D8A, which has been previously shown to help with sleep. The present invention further develops its food use. It is known that Lactobacillus rhamnosus D8A is mostly present in the intestines of humans and animals, and has the functions of regulating microecological balance, enhancing host intestinal resistance, preventing and treating diarrhea, etc. Lactobacillus rhamnosus D8A is isolated from the feces of healthy infants. Compared with other isolation routes, the infant intestine is not affected by antibiotics, etc., and the isolated strains have greater potential. Lactobacillus rhamnosus D8A has a strong adhesion ability to the intestinal mucosa, can improve the distribution of intestinal flora, and antagonize the colonization of harmful bacteria, but its inhibitory effect on Helicobacter pylori is not strong. On this basis, the present invention uses biochar, sodium alginate and xanthan gum from oyster shells for processing and cultivation, and calcium chloride solution encapsulation to obtain micro-nano particles. Experiments verify that Lactobacillus rhamnosus D8A forms a biofilm therein, and has a high survival rate under gastrointestinal fluid simulation. The functional plant protein hydrogel and probiotic preparation of the present invention can synergistically improve the disorder of intestinal flora.

[0026] In the preparation of the composite nutritional composition against Helicobacter pylori, the present invention processes egg yolk globulin powder to obtain protein substances of small molecule fragments, and then mixes them with maltodextrin and gum arabic to prepare a coating material, which is covered on the outside of a substance 5 compressed with functional plant protein hydrogel, probiotic preparations, etc., and finally air-dried to obtain the composite nutritional composition, which is safe and non-toxic in oral acute toxicity tests and 30-day feeding tests and is edible.

[0027] The anti-Helicobacter pylori composite nutritional composition obtained by the present invention is helpful to regulate intestinal flora, can inhibit the colonization of Helicobacter pylori in the stomach, and is suitable for a wide range of people. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The illustrative embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0029] Figure 1 This is the test result of blood biochemical indexes TC, ALT and Alb in the 30-day feeding test;

[0030] Figure 2 This is the test result chart of blood biochemical indexes UREA, Glu and TG in 30-day feeding test;

[0031] Figure 3 This is the test result chart of blood biochemical indexes Cr, TP, and AST in the 30-day feeding test;

[0032] Figure 4 This is a comparison chart of the concentration of fecal microorganism Lactobacillus before and after the experiment;

[0033] Figure 5 This is a comparison chart of fecal microbial Bifidobacterium concentrations before and after the experiment;

[0034] Figure 6 This is a comparison chart of the concentration of fecal microorganism Enterobacter before and after the experiment;

[0035] Figure 7 This is a comparison chart of the concentration of fecal microorganism Enterococcus before and after the experiment;

[0036] Figure 8 This is a comparison chart of the concentration of fecal microorganism Clostridium perfringens before and after the experiment;

[0037] Fig. 9 This is a graph showing the results of urease activity measurement;

[0038] Fig.10 This is a SEM image of the morphological changes of Helicobacter pylori before and after cultivation with the composite nutritional composition. DETAILED DESCRIPTION

[0039] In order to more clearly explain the overall concept of the present application, the following is described in detail in conjunction with the accompanying drawings of the specification by way of embodiment. In the following description, a large number of specific details are provided to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some technical features well known in the art are not described.

[0040] Example 1: This example provides a method for preparing a functional plant protein hydrogel, and the specific steps are as follows:

[0041] L1. Wash and slice Momordica grosvenori and Hericium erinaceus, dry and grind into powder, pass through a 10-mesh sieve and weigh in a mass ratio of 8:3:1 to obtain a mixed powder, add 10 times the mass of water of the mixed powder, adjust the pH to 8.0, stir at 180 rpm for 3 hours, filter through a 180-mesh nylon mesh, centrifuge the filtrate at 8000 g for 30 minutes, adjust the pH to 4.5, let stand at room temperature for 8 hours, centrifuge at 10000 g for 15 minutes, wash the precipitate with deionized water, dissolve it in deionized water, dialyze it with a dialysis bag with a molecular weight cutoff of 4.5 kDa for 8 hours, and freeze-dry to obtain substance 1;

[0042] L2. The substance 1 obtained in step L1 was dispersed in deionized water, the mass percentage concentration of the substance 1 in the deionized water was 3%, and the mixture was stirred at 150 rpm for 2 h at room temperature. After the mixture was stirred, the mixture was placed at 4°C for 10 h, heated in a water bath to 90°C for 30 min, cooled naturally to room temperature, and the pH was adjusted to 7.0 to obtain solution 1. Glutamine transaminase (12 U / g) was added, and the amount of glutamine transaminase added was 1% of the mass of solution 1. The mixture was ultrasonically treated at 400 W for 1 min to obtain solution 2;

[0043] L3. Weigh konjac flour, psyllium husk powder and water in a mass ratio of 1:5:15, stir until evenly dispersed and let stand at room temperature for 15 hours, freeze-dry after completion, grind and pass through a 2-mesh sieve to obtain substance 2, weigh substance 2 and solution 2 obtained in step L2 in a mass-to-volume ratio of 1g:8mL, stir until evenly dispersed, heat to 30°C, ultrasonically treat at 100W power for 30 minutes, and naturally cool to room temperature to obtain functional plant protein hydrogel.

[0044] Example 2: This example provides a method for preparing a functional plant protein hydrogel, and the specific steps are as follows:

[0045] L1. Wash and slice Momordica grosvenori and Hericium erinaceus, dry and grind into powder, pass through a 10-mesh sieve and weigh in a mass ratio of 10:4:1 to obtain a mixed powder, add 11 times the mass of water of the mixed powder, adjust the pH to 8.0, stir at 210 rpm for 3.5 hours, filter through a 180-mesh nylon mesh, centrifuge the filtrate at 9000 g for 38 minutes, adjust the pH to 4.5, let stand at room temperature for 9 hours, centrifuge at 11000 g for 18 minutes, wash the precipitate with deionized water, dissolve it in deionized water, dialyze it with a dialysis bag with a molecular weight cutoff of 4.5 kDa for 9 hours, and freeze-dry to obtain substance 1;

[0046] L2. The substance 1 obtained in step L1 was dispersed in deionized water, the mass percentage concentration of the substance 1 in the deionized water was 4%, and the mixture was stirred at a speed of 180 rpm for 3 h at room temperature. After the mixture was stirred, it was placed in a 4°C environment for 12 h, heated to 93°C in a water bath and maintained for 38 min, cooled naturally to room temperature, and the pH was adjusted to 7.0 to obtain solution 1. Glutamine transaminase (12 U / g) was added, and the amount of glutamine transaminase added was 3% of the mass of solution 1. The mixture was ultrasonically treated at a power of 420 W for 1.5 min to obtain solution 2;

[0047] L3. Weigh konjac flour, psyllium husk powder and water in a mass ratio of 1:6:18, stir until evenly dispersed and let stand at room temperature for 16 hours, freeze-dry after completion, grind and pass through a 2-mesh sieve to obtain substance 2, weigh substance 2 and solution 2 obtained in step L2 in a mass-to-volume ratio of 1g:10mL, stir until evenly dispersed, heat to 36°C, ultrasonically treat at 120W power for 39 minutes, and naturally cool to room temperature to obtain functional plant protein hydrogel.

[0048] Example 3: This example provides a method for preparing a functional plant protein hydrogel, and the specific steps are as follows:

[0049] L1. Wash and slice Momordica grosvenori and Hericium erinaceus, dry and grind into powder, pass through a 10-mesh sieve and weigh in a mass ratio of 12:5:1 to obtain a mixed powder, add 12 times the mass of water of the mixed powder, adjust the pH to 8.0, stir at 240 rpm for 4 hours, filter through a 180-mesh nylon mesh, centrifuge the filtrate at 10,000 g for 45 minutes, adjust the pH to 4.5, let stand at room temperature for 10 hours, centrifuge at 12,000 g for 20 minutes, wash the precipitate with deionized water, dissolve it in deionized water, dialyze it with a dialysis bag with a molecular weight cutoff of 4.5 kDa for 10 hours, and freeze-dry to obtain substance 1;

[0050] L2. The substance 1 obtained in step L1 was dispersed in deionized water, the mass percentage concentration of the substance 1 in the deionized water was 6%, and the mixture was stirred at 200 rpm for 4 h at room temperature. After the mixture was stirred, it was placed in a 4°C environment for 15 h, heated in a water bath to 95°C and maintained for 45 min, cooled naturally to room temperature, and the pH was adjusted to 7.0 to obtain solution 1. Glutamine transaminase (12 U / g) was added, and the amount of glutamine transaminase added was 5% of the mass of solution 1. The mixture was ultrasonically treated at a power of 450 W for 2 min to obtain solution 2;

[0051] L3. Weigh konjac flour, psyllium husk powder and water in a mass ratio of 1:8:20, stir until evenly dispersed and let stand at room temperature for 18 hours, freeze-dry after completion, grind and pass through a 2-mesh sieve to obtain substance 2, weigh substance 2 and solution 2 obtained in step L2 in a mass-to-volume ratio of 1g:12mL, stir until evenly dispersed, heat to 40°C, ultrasonically treat at 150W power for 45 minutes, and naturally cool to room temperature to obtain functional plant protein hydrogel.

[0052] Embodiment 4: This embodiment provides a method for preparing a probiotic preparation, and the specific steps are as follows:

[0053] S1. Lactobacillus rhamnosus D8A (Latin name: Lacticaseibacilus rhamnosus, the strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 7, 2024, with the deposit number CGMCC No. 29961) was cultured in lactic acid bacteria medium until a concentration of 1×10 6 cfu / mL bacterial suspension, centrifuge the suspension at 2000g for 10 min, discard the supernatant, wash the precipitate twice with PBS buffer solution with a pH of 6.5, and centrifuge again at 3000g for 10 min to obtain the precipitate;

[0054] S2. Sodium alginate, xanthan gum, the precipitate obtained in step S1 and distilled water were weighed according to a mass volume ratio of 0.5 g: 1 g: 0.2 g: 30 mL, and solution 3 was obtained by stirring at a speed of 120 rpm for 15 min. Solution 3 was dripped into a 0.1 mM calcium chloride solution (involving a B-395Pro granulator) at an electrode frequency of 90 Hz, a voltage of 450 mV, and a flow rate of 40 mL / min. The insoluble matter was collected and washed three times with deionized water, and the probiotic preparation was obtained after air drying;

[0055] Preparation of lactic acid bacteria culture medium in step S1: 7.5 g yeast extract, 7 g beef extract, 7.5 g peptone, 1.5 g diammonium hydrogen citrate, 2 g potassium dihydrogen phosphate, 0.2 g anhydrous magnesium sulfate, 0.5 mL Tween-80, 7.5 g glucose, 0.03 g manganese sulfate, 1000 mL distilled water, and adjust the pH to 7.0.

[0056] Embodiment 5: This embodiment provides a method for preparing a probiotic preparation, and the specific steps are as follows:

[0057] S1. Lactobacillus rhamnosus D8A (the same as that used in Example 4) was cultured in a lactic acid bacteria culture medium (the same as that used in Example 4) until a concentration of 1×10 8cfu / mL bacterial suspension, the bacterial suspension was centrifuged at 2300g for 15 min, the supernatant was discarded, the precipitate was washed three times with PBS buffer solution with a pH of 6.5, and centrifuged again at 3100g for 15 min to obtain the precipitate;

[0058] S2. The oyster shell was calcined at 1000°C for 2h, then naturally cooled to room temperature, washed 3 times with 0.1mM hydrochloric acid solution, and then washed 3 times with deionized water. After drying, it was frozen at -80°C for 24h, and ground in a planetary grinder at 300rpm for 2h. The grinding ball size was 5mm and the ball-to-material ratio was 10:1. After the end, substance 3 was obtained;

[0059] S3. Weigh the substance 3 obtained in step S2, sodium alginate, xanthan gum, the precipitate obtained in step S1 and distilled water according to the mass volume ratio of 0.5g:0.8g:1.5g:0.3g:35mL, stir at a speed of 140rpm for 18min to obtain solution 3, drop solution 3 into 0.1mM calcium chloride solution (involving B-395Pro granulator) at an electrode frequency of 95Hz, a voltage of 480mV and a flow rate of 45mL / min, collect the insoluble matter and wash it 4 times with deionized water, and obtain the probiotic preparation after air drying.

[0060] Embodiment 6: This embodiment provides a method for preparing a probiotic preparation, and the specific steps are as follows:

[0061] S1. Lactobacillus rhamnosus D8A (the same as that used in Example 4) was cultured in a lactic acid bacteria culture medium (the same as that used in Example 4) until a concentration of 1×10 9 cfu / mL bacterial suspension, the bacterial suspension was centrifuged at 2500g for 20 min, the supernatant was discarded, the precipitate was washed three times with PBS buffer solution with a pH of 6.5, and centrifuged again at 3200g for 20 min to obtain the precipitate;

[0062] S2. The oyster shell was calcined at 1200°C for 3 h, then naturally cooled to room temperature, washed 3 times with 0.1 mM hydrochloric acid solution, then washed twice with deionized water, dried and frozen at -80°C for 30 h, and ground in a planetary grinder at 360 rpm for 3 h. The grinding ball size was 5 mm, and the ball-to-material ratio was 15:1. After the grinding, substance 3 was obtained.

[0063] S3. Weigh the substance 3 obtained in step S2, sodium alginate, xanthan gum, the precipitate obtained in step S1 and distilled water according to the mass volume ratio of 1g:1g:2g:0.5g:40mL, stir at a speed of 160rpm for 20min to obtain solution 3, drop solution 3 into 0.1mM calcium chloride solution (involving B-395Pro granulator) at an electrode frequency of 100Hz, a voltage of 500mV and a flow rate of 50mL / min, collect the insoluble matter and wash it with deionized water 5 times, and obtain the probiotic preparation after air drying.

[0064] Embodiment 7: This embodiment provides a method for preparing a composite nutritional composition for resisting Helicobacter pylori, and the specific steps are as follows:

[0065] The anti-Helicobacter pylori composite nutritional composition of this embodiment comprises the following raw materials in parts by weight: 2 parts of functional plant protein hydrogel, 0.3 parts of probiotic preparation, 0.1 parts of black pepper extract, and 1 part of turmeric, wherein the functional plant protein hydrogel is prepared by Example 1, and the probiotic preparation is prepared by Example 4;

[0066] V1. Dissolve egg yolk globulin powder in water to obtain a solution 4 with a mass percentage concentration of 5%, heat to 70°C and maintain for 12 minutes, adjust the pH to 6.5 after the solution is dissolved, add papain (6000U / mg), the amount of papain added is 2% of the mass of solution 4, and place in a water bath at a speed of 80rpm and a temperature of 58°C for 3 hours, heat to 95°C and maintain for 8 minutes to terminate the enzyme activity, and centrifuge at a centrifugal force of 6000g for 20 minutes after the solution is dissolved. Collect the supernatant and freeze-dry it to obtain substance 4;

[0067] V2. Dissolve maltodextrin in water to obtain a solution 5 with a mass percentage concentration of 10%, keep it at room temperature for 12 hours, dissolve gum arabic in water to obtain a solution 6 with a mass percentage concentration of 4%, weigh the substance 4 obtained in step V1, solution 5 and solution 6 according to a mass volume ratio of 1g:3mL:2mL, stir until uniformly dispersed, and obtain solution 7;

[0068] V3. The functional plant protein hydrogel, probiotic preparation, black pepper extract and turmeric are mixed, dispersed evenly and then dried and granulated to obtain substance 5. The solution 7 obtained in step V2 is evenly covered on the outside of substance 5, and after air drying, a composite nutritional composition against Helicobacter pylori is obtained.

[0069] Embodiment 8: This embodiment provides a method for preparing a composite nutritional composition for resisting Helicobacter pylori, and the specific steps are as follows:

[0070] The anti-Helicobacter pylori composite nutritional composition of this embodiment comprises the following raw materials in parts by weight: 2.5 parts of functional plant protein hydrogel, 0.5 parts of probiotic preparation, 0.2 parts of black pepper extract, and 2 parts of turmeric, wherein the functional plant protein hydrogel is prepared by Example 2, and the probiotic preparation is prepared by Example 5;

[0071] V1. Dissolve egg yolk globulin powder in water to obtain a solution 4 with a mass percentage concentration of 6%, heat to 75°C and maintain for 15 minutes, adjust the pH to 6.5 after the solution is dissolved, add papain (6000U / mg), the amount of papain added is 3% of the mass of solution 4, and bathe at a speed of 95rpm and a temperature of 60°C for 3.5 hours, heat to 95°C and maintain for 9 minutes to terminate the enzyme activity, and centrifuge at a centrifugal force of 7000g for 24 minutes after the solution is dissolved. Collect the supernatant and freeze-dry it to obtain substance 4;

[0072] V2. Dissolve maltodextrin in water to obtain a solution 5 with a mass percentage concentration of 11%, keep it at room temperature for 14 hours, dissolve gum arabic in water to obtain a solution 6 with a mass percentage concentration of 5%, weigh the substance 4 obtained in step V1, solution 5 and solution 6 according to a mass volume ratio of 1g:4mL:3mL, stir until uniformly dispersed, and obtain solution 7;

[0073] V3. The functional plant protein hydrogel, probiotic preparation, black pepper extract and turmeric are mixed, dispersed evenly and then dried and granulated to obtain substance 5. The solution 7 obtained in step V2 is evenly covered on the outside of substance 5, and after air drying, a composite nutritional composition against Helicobacter pylori is obtained.

[0074] Embodiment 9: This embodiment provides a method for preparing a composite nutritional composition for resisting Helicobacter pylori, and the specific steps are as follows:

[0075] The anti-Helicobacter pylori composite nutritional composition of this embodiment comprises the following raw materials in parts by weight: 3 parts of functional plant protein hydrogel, 1 part of probiotic preparation, 0.25 parts of black pepper extract, and 3 parts of turmeric, wherein the functional plant protein hydrogel is prepared by Example 3, and the probiotic preparation is prepared by Example 6;

[0076] V1. Dissolve egg yolk globulin powder in water to obtain a solution 4 with a mass percentage concentration of 8%, heat to 80°C and maintain for 18 minutes, adjust the pH to 6.5 after the solution is dissolved, add papain (6000U / mg), the amount of papain added is 5% of the mass of solution 4, and water bath is performed at a speed of 120rpm and a temperature of 64°C for 4 hours, heat to 95°C and maintain for 10 minutes to terminate the enzyme activity, and centrifuge at a centrifugal force of 8000g for 25 minutes after the solution is dissolved. Collect the supernatant and freeze-dry it to obtain substance 4;

[0077] V2. Dissolve maltodextrin in water to obtain a solution 5 with a mass percentage concentration of 12%, keep it at room temperature for 15 hours, dissolve gum arabic in water to obtain a solution 6 with a mass percentage concentration of 6%, weigh the substance 4 obtained in step V1, solution 5 and solution 6 according to the mass volume ratio of 1g:6mL:5mL, stir until uniformly dispersed, and obtain solution 7;

[0078] V3. The functional plant protein hydrogel, probiotic preparation, black pepper extract and turmeric are mixed, dispersed evenly and then dried and granulated to obtain substance 5. The solution 7 obtained in step V2 is evenly covered on the outside of substance 5, and after air drying, a composite nutritional composition against Helicobacter pylori is obtained.

[0079] Comparative Example 1: This comparative example provides a method for preparing a probiotic preparation, and the specific steps are as follows:

[0080] The difference between this comparative example and Example 5 is that Lactobacillus rhamnosus D8A is replaced by Lactobacillus rhamnosus D8A purchased from China Center for Type Culture Collection (strain collection number: CCTCC AB 2015377, model strain), and the other steps are consistent with Example 5 to obtain the probiotic preparation of this comparative example.

[0081] Comparative Example 2: This comparative example provides a method for preparing a composite nutritional composition, and the specific steps are as follows:

[0082] The difference between this comparative example and Example 8 is that the probiotic preparation obtained in Comparative Example 1 is used to replace the probiotic preparation obtained in Example 5, and the other steps are consistent with Example 8, and finally the composite nutritional composition of this comparative example is obtained.

[0083] Experimental test:

[0084] 1. Toxicological safety evaluation:

[0085] 1) Oral acute toxicity test:

[0086] SPF grade Kunming mice, weighing 18-20g, half male and half female, were raised in an SPF grade experimental environment, and the temperature and relative humidity were controlled at 20-25°C and 40%-70%, respectively. During the feeding process, the experimental animals were given 12 hours of light, and they were free to eat and drink. With a gavage dose of 20mL / kg BW, gavage was performed twice, with an interval of 6h, and continuous observation for 14d, weighing, observing and recording the general state of the animals, signs of poisoning, and death, etc. After the experiment, all rats were blood sampled from the abdominal aorta, and their hematological indicators were further detected, and the rats were grossly dissected, and the main organs such as the heart, liver, spleen, lungs, and kidneys were observed with the naked eye to see if they were abnormal, and their organ indexes were calculated. The experiment involves the liquid preparations obtained by Examples 1-9 and Comparative Examples 1-3, wherein the liquid preparations of Examples 1-6 match the mass fractions in Examples 7-9 to be limited, the liquid preparations of Comparative Example 1 are obtained in accordance with Example 5, and the liquid preparations of Comparative Example 2 are obtained in accordance with Example 8. The following shows the weight changes and mortality of mice during the oral toxicity test:

[0087] Table 1 Results of oral acute toxicity test in mice (n=10, )

[0088]

[0089]

[0090] During the oral toxicity test, the mice in each group involved in Examples 1-9 and Comparative Examples 1 and 2 had shiny fur, active spirits, normal activities, normal mucous membranes, and no symptoms of poisoning. As shown in Table 1, the number of deaths was 0 and the mortality rate was 0%.

[0091] 2) 30-day feeding trial:

[0092] Wistar rats were fed for 3 days, and males and females were divided into groups, and randomly divided into a control group and three test groups, high, medium and low, with 10 males and females in each group. The high-dose group was 3.30 / kg BW, the medium-dose group was 2.30g / kg BW, and the low-dose group was 1.65g / kg BW. The test substances of the three test groups were evenly mixed into the basic feed at the content of 3.07%, 2.30%, and 1.53%, respectively. The method of gradual expansion, full mixing, and repeated sieving was adopted. The control group was fed with normal basic feed. The feed intake of rats was calculated as 10% of body weight. Single feeding, free diet, and the general performance, behavior, poisoning performance and death of the animals were observed and recorded every day. The body weight and food intake were weighed once a week and twice a week, and the weekly and total food utilization was calculated. Continuous observation for 30 days. After 30 days, fasting for 16 hours, the animals were weighed on the 31st day of the experiment, anesthetized with 1% sodium pentobarbital saline solution at 5.0 mL / kg BW by intraperitoneal injection, and blood was collected from the inferior vena cava for hematological index detection and blood biochemical index detection. The hematological indexes included hemoglobin (HGB), red blood cell (RBC), white blood cell (WBC) counts and white blood cell classification, and the blood biochemical indexes included alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), creatinine (Cr), blood glucose (Glu), total protein (TP), albumin (Alb), cholesterol (TC), and triglycerides (TG). All animals were subjected to gross examination, and the organ weights were weighed and histopathological changes were examined. The organs included liver, kidney, spleen, stomach, duodenum, testis or ovary.

[0093] Figures 1 to 3 The results of blood biochemical indexes were shown. Figure 1 , Figure 2 , Figure 3 It can be seen that there is no significant difference in alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea (UREA), creatinine (Cr), blood glucose (Glu), total protein (TP), albumin (Alb), cholesterol (TC), and triglyceride (TG) of the Wistar rats in Examples 7-9 and Comparative Example 2 compared with those in the control group, that is, the composite nutritional compositions obtained in Examples 7-9 and Comparative Example 2 are safe and non-toxic to rats.

[0094] 2. Evaluation of the effect of improving bacterial flora:

[0095] A total of 100 Babl / C female mice weighing 18-22g were used and randomly divided into 10 groups. The test was started after adaptive feeding for one week. Different groups were treated differently during the experiment. The gavage time was fixed at 9 o'clock in the morning. The mice were gavaged once a day and weighed before gavage. Each group ensured sufficient feed and drinking water. The control group was gavaged with 200 μL of sterile saline, and the other nine experimental groups were gavaged with the obtained materials of embodiment 1-9, respectively, and physiological saline solution was used to prepare the results. Before the experiment started, 0.1g of feces in the anus of mice was taken aseptically, diluted 10 times, and suitable dilution was selected to be inoculated in different selective culture media respectively. According to colony morphology or Gram staining microscopy, the colony count was counted and the colony count in each gram of feces was calculated. The index detected was body weight, lactobacillus, bifidobacterium, enterobacter, enterococcus and clostridium perfringens.

[0096] Table 2 Culture medium and conditions used for fecal microbial culture

[0097] Fecal microbes Culture medium Culture conditions Lactobacillus LBS medium Culture at 36±1℃ for 2 days Bifidobacterium TPY medium Anaerobic, 36±1℃ for 2 days Enterobacteriaceae Eosin-methylene blue medium Incubate at 36±1℃ for 1 day Enterococci Sodium azide-crystal violet-esculin agar medium Culture at 36±1℃ for 2 days Clostridium perfringens TSC medium Anaerobic, 36±1℃ for 1 day

[0098] The colony concentration of fecal microorganisms before and after the experiment is as follows Figures 4 to 8 As shown by Figure 4 , Figure 5 It can be seen that compared with the control group, the number of lactobacilli and bifidobacteria increased significantly after the experiment. Figure 6 , Figure 7 It can be seen that the changes in Enterobacteriaceae and Enterococci were not obvious. Figure 8 It can be seen that Clostridium perfringens has different degrees of reduction, which shows that the functional plant protein hydrogel obtained in Examples 1-3, the probiotic preparations obtained in Examples 4-6, and the composite nutritional compositions obtained in Examples 7-9 are all helpful in regulating the intestinal flora of mice, and the functional plant protein hydrogel can synergistically improve the disorder of intestinal flora after being mixed with the probiotic preparation. The addition of oyster shells can enhance the encapsulation effect. If strictly considered, not adding oyster shells may be more suitable for the production process, but it does not affect the use effect of the composite nutritional composition. In addition, it can be seen that the rhamnosus lactobacillus obtained from other channels does not have the effect of rhamnosus lactobacillus D8A on improving intestinal flora, and Comparative Example 2 once again verifies the advantage that the functional plant protein hydrogel can promote the effect of the probiotic preparation.

[0099] 3. Evaluation of anti-Helicobacter pylori effect:

[0100] 1) Construction of mouse model infected with Helicobacter pylori:

[0101] SPF grade C57 / BL male mice, 8 weeks old, weighing 20-25 g, were used for infection with the standard strain of Helicobacter pylori NCTC11637. NCTC11637 was prepared with PBS buffer solution to 1×10 9cfu / mL bacterial suspension, 0.6 mL was gavaged into each mouse every other day for three times. After two weeks, five mice were randomly killed, and the stomach was removed by laparotomy. The stomach was cut into three parts along the longitudinal axis, and the rapid urease test, 40g / L formaldehyde fixation for histological examination, and bacterial culture were performed to check whether Helicobacter pylori was infected;

[0102] 2) Experimental design:

[0103] The mice with successful modeling were randomly divided into 5 groups, 10 in each group, respectively, the control group, Example 7, Example 8, Example 9, and Comparative Example 2. The control group was fed with normal saline, and the other experimental groups were given the corresponding formulated liquid preparations, and the feeding dose of any mouse was 0.5 mL. It was given once a day, and the experiment was carried out for 21 days, and fasted for 24 hours after the end. On the second day, the eyeballs were removed and the animals were bled to death, and the stomach was cut into 3 parts along the longitudinal axis. The Helicobacter pylori culture was cultured by semi-quantitative bacterial culture method, and the gastric tissue was weighed and made into a homogenate. After diluting it in multiple ratios with Brucella broth, 200 μL was taken and applied to the solid culture medium, and the colony count was used to calculate the colonization bacterial density per unit gastric tissue.

[0104] After 21 days of experiment, the average density of Helicobacter pylori in the control group was 1.3×10 6 cfu / g gastric tissue, the average density of Helicobacter pylori in Example 7 was 2.1×10 2 cfu / g gastric tissue, the average density of Helicobacter pylori in Example 8 was 2.3×10 2 cfu / g gastric tissue, the average density of Helicobacter pylori in Example 9 was 1.9×10 2 cfu / g gastric tissue, the average density of Helicobacter pylori in comparative example 2 was 8.6×10 4 cfu / g. After statistical analysis, the colonization density of Helicobacter pylori in the stomach of mice after administration of the composite nutritional composition of Examples 7-9 was significantly different compared with that of the control group (P<0.05).

[0105] 4. Urease activity measurement:

[0106] The urease activity detection kit (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number MAK120) was used. The detection principle is that urease catalyzes the hydrolysis of urea, resulting in the production of ammonia. Ammonia is determined by the Berthelot method, resulting in a colorimetric product measured at 670 nm, which is proportional to the urease activity present in the sample. The experiment was carried out with reference to the kit instructions.

[0107] The higher the absorbance value, the more urease there is, which may come from more or more active Helicobacter pylori. The urease activity measurement results are as follows: Fig. 9 As shown by Fig. 9It can be seen that compared with the control group, the absorbance values ​​corresponding to Examples 7-9 are all lower, that is, their urease activity is lower, which proves that the composite nutritional composition obtained in Examples 7-9 has the effect of inhibiting urease activity. At the same time, the composite nutritional composition obtained in Comparative Example 2 also has the effect of inhibiting urease activity, but the effect is poor.

[0108] In addition, the composite nutritional composition obtained in Example 9 was used to culture Helicobacter pylori. The SEM image after 24 hours of culture was as follows: Fig.10 As shown by Fig.10 It can be seen that the complex nutritional composition will cause certain changes in the morphology of Helicobacter pylori, such as the disappearance of the spiral. Subsequent experiments verified that the virulence of the overall culture was weakened (compared with the overall culture not cultured with the complex nutritional composition). This shows that the complex nutritional composition may achieve the effect of inhibiting the reproduction and colonization of Helicobacter pylori by affecting the growth and morphology of Helicobacter pylori.

[0109] The above are only several embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention is disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, using the technical content disclosed above to make slight changes or modifications are equivalent to equivalent implementation cases and fall within the protection scope of the technical solution of the present invention.

Claims

1. A composite nutritional composition for resisting Helicobacter pylori, characterized in that: The invention comprises the following raw materials in proportions by weight: 2-3 parts of functional plant protein hydrogel, 0.3-1 parts of probiotic preparation, 0.1-0.25 parts of black pepper extract and 1-3 parts of turmeric.

2. The anti-Helicobacter pylori composite nutritional composition according to claim 1, characterized in that: The preparation method of the functional plant protein hydrogel is as follows: L1. Wash and slice Momordica grosvenori and Hericium erinaceus, dry and grind into powder, sieve and weigh according to the mass ratio of 8-12:3-5:1 to obtain a mixed powder, add 10-12 times the mass of water of the mixed powder, adjust the pH to 8.0, stir, filter through nylon mesh, centrifuge the filtrate, adjust the pH to 4.5, let stand at room temperature, centrifuge after completion, wash the precipitate with deionized water, add deionized water to dissolve and dialyze, and freeze-dry to obtain substance 1; L2. The substance 1 obtained in step L1 was dispersed in deionized water, stirred at room temperature, and placed at 4 ° C for 10-15 hours, heated in a water bath and maintained, cooled naturally to room temperature, and the pH was adjusted to 7.0 to obtain solution 1, and glutamine transaminase was added, and ultrasonic treatment was performed to obtain solution 2; L3. Weigh konjac flour, psyllium husk powder and water in a mass ratio of 1:5-8:15-20, stir until evenly dispersed and let stand at room temperature, freeze-dry after completion, grind and sieve to obtain substance 2, weigh substance 2 and solution 2 obtained in step L2 in a mass volume ratio of 1g:8-12mL, stir until evenly dispersed, heat and ultrasonically treat, and naturally cool to room temperature to obtain functional plant protein hydrogel.

3. The anti-Helicobacter pylori composite nutritional composition according to claim 2, characterized in that: In step L2, the mass percentage concentration of substance 1 in deionized water is 3-6%, and the amount of transglutaminase added is 1-5% of the mass of solution 1.

4. The anti-Helicobacter pylori composite nutritional composition according to claim 3, characterized in that: The preparation method of the probiotic preparation is as follows: S1. Lactobacillus rhamnosus D8A was cultured in lactic acid bacteria culture medium until the concentration was 1×10 6 -1×10 9 cfu / mL bacterial suspension, centrifuge the bacterial suspension, discard the supernatant, wash the precipitate with PBS buffer solution, centrifuge again, and obtain the precipitate; S2. The oyster shells are calcined at 1000-1200°C, cooled naturally to room temperature, washed with hydrochloric acid solution, washed with deionized water, dried, frozen at -80°C, and ground to obtain substance 3; S3. Weigh the substance 3 obtained in step S2, sodium alginate, xanthan gum, the precipitate obtained in step S1 and distilled water according to the mass volume ratio of 0-1g:0.5-1g:1-2g:0.2-0.5g:30-40mL, stir to obtain solution 3, drop solution 3 into the calcium chloride solution at an electrode frequency of 90-100Hz, a voltage of 450-500mV and a flow rate of 40-50mL / min, collect the insoluble matter and wash it with deionized water, and obtain the probiotic preparation after air drying.

5. A method for preparing the anti-Helicobacter pylori composite nutritional composition according to any one of claims 1 to 4, characterized in that: The specific steps are as follows: V1. Dissolve egg yolk globulin powder in water to obtain a solution 4 having a mass percentage concentration of 5-8%, heat and maintain, adjust the pH to 6.5 after completion, add papain, treat in a water bath, heat to terminate the enzyme activity, centrifuge after completion, collect the supernatant and freeze-dry to obtain substance 4; V2. Dissolve maltodextrin in water to obtain a solution 5 with a mass percentage concentration of 10-12%, keep it at room temperature for 12-15 hours, dissolve gum arabic in water to obtain a solution 6 with a mass percentage concentration of 4-6%, weigh the substance 4 obtained in step V1, solution 5 and solution 6 according to the mass volume ratio of 1g:3-6mL:2-5mL, stir until uniformly dispersed, and obtain solution 7; V3. The functional plant protein hydrogel, probiotic preparation, black pepper extract and turmeric are mixed, dispersed evenly and then dried and granulated to obtain substance 5. The solution 7 obtained in step V2 is evenly covered on the outside of substance 5, and after air drying, a composite nutritional composition against Helicobacter pylori is obtained.

6. The method for preparing the anti-Helicobacter pylori composite nutritional composition according to claim 5, characterized in that: The amount of papain added in step V1 is 2-5% of the mass of solution 4.

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