Composite powder containing AKK probiotics and preparation process thereof

Abstract

The invention provides compound powder containing AKK probiotics and a preparation technology of the compound powder, and belongs to the technical field of solid beverage food. The preparation process comprises the following steps: preparing the perilla leaf extract; preparing a soybean hypocotyl extract; preparing a cranberry extract; preparing a composite coating material; activating the AKK bacteria and embedding the AKK bacteria; and mixing to prepare composite powder. The preparation method comprises the following steps: performing enzymolysis on whey protein by using neutral protease and papain to prepare hydrolyzed whey protein, mixing the hydrolyzed whey protein with carboxymethyl dextran, performing grafting reaction to obtain a composite coating material, coating AKK probiotics by using the composite coating material, and performing freeze-drying to obtain the AKK probiotics coated with the composite coating material. An embedding structure formed by the composite coating material can be used as a physical barrier to prevent gastric acid and digestive enzymes in gastric juice from directly contacting with AKK probiotics, so that the death rate of the AKK probiotics in the gastric juice is reduced, and the survival rate of the AKK probiotics in gastrointestinal tracts is improved.

Description

Technical Field

[0001] The invention relates to the technical field of solid beverage food, and in particular to a composite powder containing AKK probiotics and a preparation process thereof. Background Art

[0002] As a key part of the human digestive system, the intestine not only bears the heavy responsibility of digesting and absorbing nutrients, but also plays an indispensable role in maintaining the body's immune balance and resisting pathogen invasion. According to statistics, about 70% of the human immune system is concentrated in the intestine, and intestinal health is directly related to the overall health level. However, changes in modern lifestyles, such as unreasonable dietary structure (increased intake of high-sugar, high-fat, and high-salt foods, insufficient dietary fiber intake), long-term mental stress, improper use of antibiotics, and environmental pollution, have had a significant negative impact on the balance of intestinal microbial communities. Clinical studies have shown that intestinal flora imbalance is closely related to the occurrence and development of a variety of diseases, such as inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome, obesity, diabetes, cardiovascular disease, and even some neurological diseases (such as autism, depression) and cancer, which not only bring physical pain to patients, but also have a serious impact on their quality of life and economic burden.

[0003] AKK probiotics are an obligate anaerobic Gram-negative bacterium that mainly colonizes the intestinal mucus layer of humans and animals. A large number of studies have shown that AKK probiotics have a variety of important physiological functions. On the one hand, it can effectively improve the intestinal barrier function by promoting the thickening of the intestinal mucus layer and strengthening the tight connection between intestinal epithelial cells, thereby forming a solid line of defense to prevent the invasion of harmful bacteria, toxins and other pathogens, reduce intestinal permeability, and reduce the risk of intestinal inflammation. On the other hand, AKK probiotics play a key role in regulating the balance of intestinal flora. It competes with harmful intestinal bacteria for nutrients and adhesion sites, inhibits the growth and reproduction of harmful bacteria (such as Escherichia coli, Salmonella, etc.), and produces beneficial metabolites such as short-chain fatty acids, creating favorable conditions for the growth of beneficial bacteria such as bifidobacteria and lactic acid bacteria, and promoting the development of intestinal flora in a healthy and balanced direction. Currently, AKK probiotics are made into various oral nutritional supplements to maintain intestinal health. However, the strong acid environment in gastric juice will denature the enzyme system and biological macromolecules in the AKK probiotic cells by destroying the spatial structure of the protein, causing them to lose their activity, thereby affecting their normal metabolism and survival ability.

[0004] Therefore, it is necessary to propose a composite powder containing AKK probiotics and a preparation process thereof which can improve the survival rate of AKK probiotics. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a composite powder containing AKK probiotics and a preparation process thereof.

[0006] A preparation process of a composite powder containing AKK probiotics comprises the following steps: S1: Preparation of Perilla Leaf Extract The perilla leaves are pretreated and then added into the leaching solution to perform ultrasonic-assisted heating reflux extraction to prepare the perilla leaf extract; S2: Preparation of soybean hypocotyl extract The soybeans are pretreated and the hypocotyls are peeled off, and then the soybean hypocotyls are ground into powder, and then added into an ethanol solution for heating and refluxing to obtain a soybean hypocotyl extract; S3: Preparation of cranberry extract After pretreatment, cranberries are added to an ethanol solution and subjected to ultrasonic-assisted heating reflux extraction to obtain a cranberry extract; S4: Preparation of composite coating materials After the whey protein is dissolved, a composite enzyme is added to hydrolyze it to prepare a hydrolyzed whey protein powder, and then the hydrolyzed whey protein powder and carboxymethyl dextran are dissolved in a PBS buffer solution, freeze-dried, and then heated to react to obtain a composite coating material; S5: Activation of AKK bacteria and encapsulation Activate the AKK probiotics, then dissolve the composite coating material in PBS buffer, add the activated AKK probiotics, fully stir and mix, and freeze-dry to obtain coated AKK probiotics; S6: Mixing to prepare composite powder 6-10 parts by mass of red algae polysaccharide, 8-10 parts by mass of oligofructose, 8-10 parts by mass of inulin, 18-20 parts by mass of the above-mentioned perilla leaf extract, 13-15 parts by mass of the above-mentioned soybean hypocotyl extract, 18-20 parts by mass of the above-mentioned cranberry extract and 4-6 parts by mass of the above-mentioned coated AKK probiotics are stirred and mixed to obtain a composite powder.

[0007] Furthermore, S1 specifically includes the following steps: S1.1: Dry, crush and sieve the dried perilla leaves, add them into the extracting solution at a solid-liquid ratio of 1 g: (15-25) mL, stir and disperse them thoroughly, and perform reflux extraction at 50-60°C and 300-350W with ultrasonic assisted heating for 40-50 min to obtain a perilla leaf extract; S1.2: After centrifuging the perilla leaf extract at a rate of 6000-7000 r / min for 10-20 min, separating the supernatant, and performing reduced pressure distillation and vacuum drying on the supernatant to obtain the perilla leaf extract.

[0008] Furthermore, S2 specifically includes the following steps: S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls; S2.2: Grind the above soybean hypocotyls into powder, add them into 65% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, heat and reflux at 60-70℃ for 1-2h, and obtain soybean hypocotyl extract by centrifugation, reduced pressure distillation and vacuum drying.

[0009] Furthermore, S3 specifically includes the following steps: S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder; S3.2: Add the cranberry powder to a 50% ethanol solution at a solid-liquid ratio of 1 g: (18-22) mL, and perform reflux extraction at 200-300 W and 60-70 ° C with ultrasonic assisted heating for 30-40 min to obtain a cranberry extract; S3.3: The cranberry extract is filtered, and the filtrate is concentrated under reduced pressure and freeze-dried to obtain a cranberry extract.

[0010] Furthermore, S4 specifically includes the following steps: S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g: (10-20) mL, stir thoroughly to dissolve, and obtain a whey protein solution; S4.2: Adding compound enzyme to the above whey protein solution, stirring and mixing, keeping the temperature at 40-60°C for 2-3h for enzymolysis, then heating to 90-95°C for 3-5min to inactivate the enzyme, cooling, centrifugally filtering, concentrating and drying to obtain hydrolyzed whey protein powder; S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran into PBS buffer at a solid-liquid ratio of 1g:(90-100)mL, stir until completely dissolved, freeze-dry, and then place in a closed environment at 55-65℃ and 70%-80% humidity to react for 20-24h to obtain a composite coating material.

[0011] Furthermore, S5 specifically includes the following steps: S5.1: Inoculate AKK probiotics into Mucin medium, culture anaerobically at 35-37°C for 18-24h, then centrifuge at 4000-6000rpm for 10-15min, collect the bacteria, and wash them with PBS buffer 2-3 times to obtain activated AKK probiotics; S5.2: Add the composite coating material prepared in step S4.3 into PBS buffer at a solid-liquid ratio of 1 g: (10-20) mL, stir thoroughly to dissolve, and obtain a coating material solution; S5.3: Add the activated AKK probiotics to the coating material solution while stirring, and continue stirring and mixing. After freeze drying and grinding, coated AKK probiotics are obtained.

[0012] Furthermore, the leaching solution is prepared by mixing 50% ethanol solution and 0.1 mol / L hydrochloric acid, and the pH of the leaching solution is 2-3.

[0013] Furthermore, the mass ratio of the complex enzyme to the whey protein powder is 1:(90-100), and the complex enzyme is prepared by mixing neutral protease and papain in a mass ratio of 1:(1-3).

[0014] Furthermore, the AKK probiotics are AKK AM06 strains derived from healthy breast milk, and the amount of activated AKK probiotics added is (1-3)×10 6 CFU / mL coating material solution.

[0015] Furthermore, a composite powder containing AKK probiotics is prepared by the preparation process of a composite powder containing AKK probiotics described in any one of the above items.

[0016] Compared with the prior art, the present invention has at least the following beneficial effects: The present invention firstly utilizes neutral protease and papain to enzymolyze whey protein to prepare hydrolyzed whey protein, and then mixes the hydrolyzed whey protein with carboxymethyl dextran for grafting reaction to obtain a composite coating material. After the composite coating material is used to coat AKK probiotics, the embedding structure formed by the composite coating material can serve as a physical barrier to prevent gastric acid and digestive enzymes in gastric juice from directly contacting the AKK probiotics, reduce the erosion of hydrochloric acid, pepsin and the like in gastric juice on AKK probiotic cells, reduce the mortality rate of AKK probiotic cells in gastric juice, and thus improve the survival rate of AKK probiotic cells in the gastrointestinal tract.

[0017] The invention extracts perilla leaf and soybean hypocotyl with ethanol solution respectively to prepare perilla leaf extract and soybean hypocotyl extract, and then mixes the two with other components to prepare composite powder. On the one hand, since the effective ingredients in the perilla leaf extract can promote intestinal peristalsis, and the dietary fiber and other ingredients in the soybean hypocotyl extract can absorb water and swell in the intestine, when the two are combined, the perilla leaf extract stimulates the contraction of intestinal smooth muscle, and the soybean hypocotyl extract increases the intestinal contents and more effectively pushes the intestinal contents forward, thereby achieving a better effect of moisturizing the intestine and relieving constipation; on the other hand, since the perilla leaf extract and the soybean hypocotyl extract can delay the digestion and absorption of sugar by the small intestine by inhibiting the activities of alpha-glucosidase and alpha-amylase, thereby achieving the effect of lowering blood sugar, the two have a synergistic blood sugar-lowering effect.

[0018] The present invention extracts cranberry extract with an ethanol solution to prepare the cranberry extract, and then mixes the cranberry extract and red hair algae polysaccharide with other components to prepare the composite powder. Since the red hair algae polysaccharide can provide a carbon source for AKK probiotics, and the active ingredients in the cranberry extract can be used as growth factors to activate the growth and metabolic genes of the AKK probiotics, promote the uptake and utilization of the red hair algae polysaccharide, and thus synergistically promote the growth of the AKK probiotics. In addition, in the intestinal environment, the red hair algae polysaccharide has a certain water absorption, which can regulate the water balance in the intestine. When it synergistically acts with the cranberry extract, on the one hand, the polysaccharide absorbs water and expands to make the intestinal contents more moist, creating a moist growth environment for the AKK probiotics, which is conducive to its reproduction. On the other hand, the organic acid in the cranberry extract can regulate the pH value of the intestine, so that the growth environment of the AKK probiotics is more suitable. Therefore, when the red hair algae polysaccharide and the cranberry extract act together, they can synergistically promote the growth of the AKK probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which are incorporated herein and constitute a part of the specification, illustrate embodiments of the present disclosure and, together with the description, further serve to explain the principles of the present disclosure and to enable those skilled in the relevant art to make and use the present disclosure.

[0020] Figure 1 This is a flow chart of the preparation process of the composite powder containing AKK probiotics used in the embodiments of the present invention. DETAILED DESCRIPTION

[0021] The composite powder containing AKK probiotics and its preparation process provided by the present invention are described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Preparation Example The steps for preparing Mucin medium are as follows: Weigh 10 g of porcine gastric mucin, 5 g of yeast extract, 5 g of tryptone, and 5 g of sodium chloride, add them to 1 L of deionized water, stir thoroughly to dissolve, sterilize in a high-pressure steam autoclave at 121°C for 15 min, then add 0.5 g of L-cysteine ​​hydrochloride by filtration sterilization, stir and mix evenly to obtain Mucin medium.

[0023] Example 1: A preparation process of a composite powder containing AKK probiotics, such as Figure 1 As shown, the following steps are included: S1: Preparation of Perilla Leaf Extract S1.1: After drying, crushing and sieving the dried perilla leaves, add them into the leaching solution at a solid-liquid ratio of 1g:15mL, fully stir and disperse, and reflux extract at 50℃ and 300W ultrasonic assisted heating for 40min to obtain the perilla leaf extract, wherein the leaching solution is prepared by mixing 50% ethanol solution and 0.1mol / L hydrochloric acid, and the pH of the leaching solution is 2; S1.2: centrifuging the perilla leaf extract at a rate of 6000 r / min for 10 min, separating the supernatant, and performing reduced pressure distillation and vacuum drying on the supernatant to obtain a perilla leaf extract; S2: Preparation of soybean hypocotyl extract S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls; S2.2: After the soybean hypocotyls are ground into powder, they are added into a 65% ethanol solution at a solid-liquid ratio of 1 g:20 mL, and the mixture is heated under reflux at 60°C for 1 h. The soybean hypocotyl extract is obtained by centrifugation, vacuum distillation and vacuum drying. S3: Preparation of cranberry extract S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder; S3.2: Add the cranberry powder to 50% ethanol solution at a solid-liquid ratio of 1 g:18 mL, and perform reflux extraction at 200 W and 60 °C for 30 min to obtain a cranberry extract; S3.3: filtering the cranberry extract, and then concentrating the filtrate under reduced pressure and freeze-drying it to obtain a cranberry extract; S4: Preparation of composite coating materials S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g:10 mL, stir thoroughly to dissolve, and obtain a whey protein solution; S4.2: Add compound enzyme to the above whey protein solution, stir and mix, keep it at 40°C for 2h for enzymolysis, then heat it to 90°C for 3min to inactivate the enzyme, cool it, centrifuge, concentrate and dry it to obtain hydrolyzed whey protein powder, wherein the mass ratio of compound enzyme to whey protein powder is 1:90, and the compound enzyme is prepared by mixing neutral protease and papain in a mass ratio of 1:1; S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran into PBS buffer at a solid-liquid ratio of 1 g:90 mL, stir until completely dissolved, freeze-dry, and then place in a closed environment at 55°C and 70% humidity for reaction for 20 hours to obtain a composite coating material; S5: Activation of AKK bacteria and encapsulation S5.1: inoculating AKK probiotics into Mucin medium, culturing anaerobically at 35°C for 18 hours, then centrifuging at 4000 rpm for 10 minutes, collecting the bacteria, and washing twice with PBS buffer to obtain activated AKK probiotics, wherein the AKK probiotics are AKK AM06 strains derived from healthy breast milk; S5.2: Add the composite coating material obtained in step S4.3 into PBS buffer at a solid-liquid ratio of 1 g:10 mL, stir and dissolve thoroughly to obtain a coating material solution; S5.3: Add the activated AKK probiotics to the coating material solution while stirring, and continue stirring and mixing, freeze-drying and grinding to obtain coated AKK probiotics, wherein the amount of activated AKK probiotics added is 1×10 6 CFU / mL coating material solution; S6: Mixing to prepare composite powder 6 parts by mass of red hair algae polysaccharide, 8 parts by mass of oligofructose, 8 parts by mass of inulin, 18 parts by mass of the above-mentioned perilla leaf extract, 13 parts by mass of the above-mentioned soybean hypocotyl extract, 18 parts by mass of the above-mentioned cranberry extract and 4 parts by mass of the above-mentioned coated AKK probiotics were stirred and mixed to obtain a composite powder.

[0024] Example 2: A preparation process of a composite powder containing AKK probiotics, such as Figure 1 As shown, the following steps are included: S1: Preparation of Perilla Leaf Extract S1.1: After drying, crushing and sieving the dried perilla leaves, add them into the leaching solution at a solid-liquid ratio of 1g:20mL, fully stir and disperse, and reflux extract with ultrasonic assisted heating at 55℃ and 325W for 45min to obtain a perilla leaf extract, wherein the leaching solution is prepared by mixing 50% ethanol solution and 0.1mol / L hydrochloric acid, and the pH of the leaching solution is 2.5; S1.2: centrifuging the perilla leaf extract at a rate of 6500 r / min for 15 min, separating the supernatant, and performing reduced pressure distillation and vacuum drying on the supernatant to obtain a perilla leaf extract; S2: Preparation of soybean hypocotyl extract S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls; S2.2: After grinding the above soybean hypocotyl into powder, add it into 65% ethanol solution at a solid-liquid ratio of 1g:25mL, heat and reflux at 65°C for 1.5h, centrifuge, distill under reduced pressure and dry in vacuum to obtain soybean hypocotyl extract; S3: Preparation of cranberry extract S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder; S3.2: Add the cranberry powder to 50% ethanol solution at a solid-liquid ratio of 1 g:20 mL, and perform reflux extraction at 250 W and 65 °C for 35 min to obtain a cranberry extract; S3.3: filtering the cranberry extract, and then concentrating the filtrate under reduced pressure and freeze-drying it to obtain a cranberry extract; S4: Preparation of composite coating materials S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g:15 mL, stir thoroughly to dissolve, and obtain a whey protein solution; S4.2: Add compound enzyme to the whey protein solution, stir and mix, heat at 50°C for 2.5 hours, heat to 92°C for 4 minutes to inactivate the enzyme, cool, centrifuge, concentrate and dry to obtain hydrolyzed whey protein powder, wherein the mass ratio of compound enzyme to whey protein powder is 1:95, and the compound enzyme is prepared by mixing neutral protease and papain in a mass ratio of 1:2; S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran into PBS buffer at a solid-liquid ratio of 1 g:95 mL, stir until completely dissolved, freeze-dry, and then place in a closed environment at 60°C and 75% humidity for reaction for 22 hours to obtain a composite coating material; S5: Activation of AKK bacteria and encapsulation S5.1: inoculating AKK probiotics into Mucin medium, culturing anaerobically at 36°C for 21 hours, then centrifuging at 5000 rpm for 12 minutes, collecting the bacteria, and washing them twice with PBS buffer to obtain activated AKK probiotics, wherein the AKK probiotics are AKK AM06 strains derived from healthy breast milk; S5.2: Add the composite coating material obtained in step S4.3 into PBS buffer at a solid-liquid ratio of 1 g:15 mL, stir and dissolve thoroughly to obtain a coating material solution; S5.3: Add the activated AKK probiotics to the coating material solution while stirring, and continue stirring and mixing, freeze-drying and grinding to obtain coated AKK probiotics, wherein the amount of activated AKK probiotics added is 2×10 6 CFU / mL coating material solution; S6: Mixing to prepare composite powder 8 parts by mass of red hair algae polysaccharide, 9 parts by mass of oligofructose, 9 parts by mass of inulin, 19 parts by mass of the above-mentioned perilla leaf extract, 14 parts by mass of the above-mentioned soybean hypocotyl extract, 19 parts by mass of the above-mentioned cranberry extract and 5 parts by mass of the above-mentioned coated AKK probiotics were stirred and mixed to obtain a composite powder.

[0025] Example 3: A preparation process of a composite powder containing AKK probiotics, such as Figure 1 As shown, the following steps are included: S1: Preparation of Perilla Leaf Extract S1.1: After drying, crushing and sieving the dried perilla leaves, add them into the leaching solution at a solid-liquid ratio of 1g:25mL, fully stir and disperse, and reflux extract with ultrasonic assisted heating at 60℃ and 350W for 50min to obtain a perilla leaf extract, wherein the leaching solution is prepared by mixing 50% ethanol solution and 0.1mol / L hydrochloric acid, and the pH of the leaching solution is 3; S1.2: centrifuging the perilla leaf extract at a rate of 7000 r / min for 20 min, separating the supernatant, and performing reduced pressure distillation and vacuum drying on the supernatant to obtain a perilla leaf extract; S2: Preparation of soybean hypocotyl extract S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls; S2.2: After the soybean hypocotyls are ground into powder, they are added into a 65% ethanol solution at a solid-liquid ratio of 1 g:30 mL, and the mixture is heated under reflux at 70°C for 2 h. The soybean hypocotyl extract is obtained by centrifugation, reduced pressure distillation and vacuum drying. S3: Preparation of cranberry extract S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder; S3.2: Add the cranberry powder to 50% ethanol solution at a solid-liquid ratio of 1 g:22 mL, and perform reflux extraction at 300 W and 70 °C for 40 min to obtain a cranberry extract; S3.3: filtering the cranberry extract, and then concentrating the filtrate under reduced pressure and freeze-drying it to obtain a cranberry extract; S4: Preparation of composite coating materials S4.1: Add whey protein powder into deionized water at a solid-liquid ratio of 1 g:20 mL, stir thoroughly to dissolve, and obtain a whey protein solution; S4.2: Add compound enzyme to the whey protein solution, stir and mix, heat at 60°C for 3h, heat to 95°C for 5min to inactivate the enzyme, cool, centrifuge, concentrate and dry to obtain hydrolyzed whey protein powder, wherein the mass ratio of compound enzyme to whey protein powder is 1:100, and the compound enzyme is prepared by mixing neutral protease and papain in a mass ratio of 1:3; S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran into PBS buffer at a solid-liquid ratio of 1 g:100 mL, stir until completely dissolved, freeze-dry, and then place in a closed environment at 65°C and 80% humidity for reaction for 24 hours to obtain a composite coating material; S5: Activation of AKK bacteria and encapsulation S5.1: inoculating AKK probiotics into Mucin medium, culturing anaerobically at 37°C for 24 hours, then centrifuging at 6000 rpm for 15 minutes, collecting the bacteria, and washing them three times with PBS buffer to obtain activated AKK probiotics, wherein the AKK probiotics are AKK AM06 strains derived from healthy breast milk; S5.2: Add the composite coating material obtained in step S4.3 into PBS buffer at a solid-liquid ratio of 1 g:20 mL, stir and dissolve thoroughly to obtain a coating material solution; S5.3: Add the activated AKK probiotics to the coating material solution while stirring, and continue stirring and mixing, freeze-drying and grinding to obtain coated AKK probiotics, wherein the amount of activated AKK probiotics added is 3×10 6 CFU / mL coating material solution; S6: Mixing to prepare composite powder 10 parts by mass of red algae polysaccharide, 10 parts by mass of oligofructose, 10 parts by mass of inulin, 20 parts by mass of the above-mentioned perilla leaf extract, 15 parts by mass of the above-mentioned soybean hypocotyl extract, 20 parts by mass of the above-mentioned cranberry extract and 6 parts by mass of the above-mentioned coated AKK probiotics were stirred and mixed to obtain a composite powder.

[0026] Comparative Example 1: The difference between Comparative Example 1 and Example 1 is that step S4 and steps S5.2-S5.3 are removed, that is, the activated AKK probiotics are not embedded and are directly added to the composite powder.

[0027] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that the perilla leaf extract in step S6 is replaced by an equal amount of soybean hypocotyl extract.

[0028] Comparative Example 3: The difference between Comparative Example 3 and Example 1 is that the soybean hypocotyl extract in step S6 is replaced by an equal amount of perilla leaf extract.

[0029] Comparative Example 4: The difference between Comparative Example 4 and Example 1 is that the red hair algae polysaccharide in step S6 is replaced by an equal amount of cranberry extract.

[0030] Comparative Example 5: The difference between Comparative Example 5 and Example 1 is that the cranberry extract in step S6 is replaced by an equal amount of red hair algae polysaccharide.

[0031] Test Example: Test Example 1: The coated AKK probiotics prepared in Example 1 and the uncoated activated AKK probiotics in Comparative Example 1 were suspended in the sterilized simulated gastric fluid, incubated in a 37°C constant temperature incubator in the dark for 2 hours, and then 0.5 mL was transferred to a sterile centrifuge tube, centrifuged at 3000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended with sterile PBS buffer, and then spread on Mucin medium, anaerobically cultured at 37°C for 48 hours, and then the number of colonies on each plate was calculated, and the number of surviving bacteria (CFU / mL) in the sample after incubation in simulated gastric fluid was calculated according to the dilution multiple. The calculation formula of the survival rate is as follows: Survival rate = (number of surviving AKK probiotics / initial number of AKK probiotics) × 100%. The results are shown in Table 1.

[0032] Table 1: Survival rate of AKK probiotics Survival rate (%) Example 1 87.45 Example 2 86.96 Example 3 87.32 Comparative Example 1 48.63 As can be seen from Table 1, when the composite coating material is not used to coat the AKK probiotics in Comparative Example 1, its survival rate in gastric juice after 2 hours is only about 48.63%, which is much lower than that in Example 1. It can be seen that by first enzymatically hydrolyzing whey protein with neutral protease and papain to prepare hydrolyzed whey protein, and then mixing the hydrolyzed whey protein with carboxymethyl dextran for grafting reaction to obtain a composite coating material, after the AKK probiotics are coated with the composite coating material, the embedding structure formed by the composite coating material can serve as a physical barrier to reduce the erosion of AKK probiotic cells by hydrochloric acid, pepsin, etc. in gastric juice, thereby improving its survival rate in the gastrointestinal tract.

[0033] Test Example 2: 70 7-week-old BALB / c male mice were randomly divided into a blank control group, a model control group, an embodiment 1 group, an embodiment 2 group, an embodiment 3 group, a comparative example 2 group, and a comparative example 3 group. Except for the blank control group, the mice in the other groups were deprived of water but not food for 72 hours to create a dryness and dehydration constipation model. After modeling, the blank control group and the model control group were gavaged with 2% ink made of distilled water, and the other groups were respectively dissolved in distilled water to prepare a 5% sample solution, and then 3mL of the sample solution was prepared into 2% ink, and the mice were gavaged, and then the time (min) for the first discharge of black stools in each group of mice and the total number of black stools discharged by mice within 4 hours were observed and recorded, and the feces water content was measured, and the average value was taken. The results are shown in Table 2.

[0034] Table 2: Time of first black stool excretion, total number of black stool excretion and feces water content of mice Time of first black stool discharge (min) Total number of black stools discharged Fecal water content (%) Example 1 157.2 7.1 56.84 Example 2 155.9 7.2 57.21 Example 3 158.5 6.9 57.36 Comparative Example 2 219.3 2.7 43.22 Comparative Example 3 203.7 3.6 44.63 Blank control group 126.8 8.5 61.32 Model control group 265.4 2.1 40.16 As can be seen from Table 2, when the composite powders prepared by single soybean hypocotyl extract and single perilla leaf extract in Comparative Examples 2 and 3 were gavaged, the time for the first black stool excretion of mice was greater than that in Example 1, the total number of black stools excreted was less than that in Example 1, and the water content of feces was lower than that in Example 1. It can be seen that by first extracting perilla leaves and soybean hypocotyls with ethanol solution respectively to prepare perilla leaf extract and soybean hypocotyl extract, and then mixing the two with other components to form a composite powder, the perilla leaf extract and the soybean hypocotyl extract can synergistically promote the intestinal moisturizing and laxative effect of the composite powder.

[0035] Test Example 3: Using 4-nitrobenzene-α-D-pyranoglucoside as the substrate, the experiment set up 12 groups, each with 3 replicates, including 3 blank groups (a, buffer + enzyme + substrate), 3 blank control groups (b, buffer + substrate), 3 sample groups (c, 5% composite powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 + enzyme + substrate), and 3 sample control groups (d, 5% composite powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 + buffer + substrate). 25 μL of phosphate buffer (PBS, 20 mM, pH = 7.0) was used as the buffer system, 25 μL of α-glucosidase (0.6 U / mL), 25 μL of the sample solution to be tested (the composite powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 with a mass fraction of 5%) was added, and the mixture was shaken and mixed, and incubated in a 37 ° C biochemical incubator for 15 min. After taking out, 50 μL of p-nitrophenyl-β-D-pyranogalactoside (10 mM) solution was added, mixed evenly, and incubated in a 37 ° C biochemical incubator for 15 min. After taking out, 100 μL of sodium carbonate solution was added to terminate the reaction, and the absorbance (A) was measured at 405 nm using a microplate reader. The α-glucosidase inhibition rate was calculated as follows: Inhibition rate = [1-(A c -A d ) / (A a -A b )]×100%, where A a is the absorbance of the blank group, A b is the absorbance of the blank control group, A c is the sample group absorbance, A d is the absorbance of the sample control group. The results are shown in Table 3.

[0036] Table 3: α-glucosidase inhibition rate Example 1 Comparative Example 2 Comparative Example 3 Inhibition rate (%) 83.2 72.9 74.4 As can be seen from Table 3, the inhibition rates of α-glucosidase by the composite powders prepared using single soybean hypocotyl extract and single perilla leaf extract in Comparative Examples 2 and 3 were lower than those of the composite powders prepared using both soybean hypocotyl extract and perilla leaf extract in Example 1. This shows that perilla leaf extract and soybean hypocotyl extract have synergistic hypoglycemic effects.

[0037] Test Example 4: 5 mg of the composite powder prepared in Example 1, Comparative Example 4 and Comparative Example 5 were added to Mucin medium, incubated under anaerobic conditions at 37 ° C for 72 hours, 0.5 mL was transferred to a sterile centrifuge tube, centrifuged at 3000 rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended with sterile PBS buffer and spread on Mucin medium. Incubated under anaerobic conditions at 37 ° C for 72 hours, the number of colonies on the plate was calculated, and the number of live bacteria in the sample after incubation for 72 hours was calculated according to the dilution multiple, and the growth rate of AKK probiotics was calculated. The growth rate calculation formula is as follows: Growth rate = number of viable bacteria after 72 h of incubation / number of viable bacteria before incubation. The results are shown in Table 4.

[0038] Table 4: Growth rate of AKK probiotics Growth rate Example 1 <![CDATA[3.26×10 3 ]]> Comparative Example 4 <![CDATA[6.72×10 2 ]]> Comparative Example 5 <![CDATA[4.37×10 2 ]]> As can be seen from Table 4, when a single cranberry extract or a single red hair algae polysaccharide was added to the composite powder in Comparative Examples 4 and 5, the growth rate of AKK probiotics was lower than that of the composite powder prepared by adding cranberry extract and red hair algae polysaccharide at the same time in Example 1. It can be seen that when red hair algae polysaccharide and cranberry extract act together, they can synergistically promote the growth of AKK probiotics.

[0039] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A preparation process of a composite powder containing AKK probiotics, characterized in that: The steps include: S1: Preparation of Perilla Leaf Extract The perilla leaves are pretreated and then added into the leaching solution to perform ultrasonic-assisted heating reflux extraction to prepare the perilla leaf extract; S2: Preparation of soybean hypocotyl extract The soybeans are pretreated and the hypocotyls are peeled off, and then the soybean hypocotyls are ground into powder, and then added into an ethanol solution for heating and refluxing to obtain a soybean hypocotyl extract; S3: Preparation of cranberry extract After pretreatment, cranberries are added to an ethanol solution and subjected to ultrasonic-assisted heating reflux extraction to obtain a cranberry extract; S4: Preparation of composite coating materials After the whey protein is dissolved, a composite enzyme is added to hydrolyze it to prepare a hydrolyzed whey protein powder, and then the hydrolyzed whey protein powder and carboxymethyl dextran are dissolved in a PBS buffer solution, freeze-dried, and then heated to react to obtain a composite coating material; S5: Activation of AKK bacteria and encapsulation Activate the AKK probiotics, then dissolve the composite coating material in PBS buffer, add the activated AKK probiotics, fully stir and mix, and freeze-dry to obtain coated AKK probiotics; S6: Mixing to prepare composite powder 6-10 parts by mass of red algae polysaccharide, 8-10 parts by mass of oligofructose, 8-10 parts by mass of inulin, 18-20 parts by mass of the above-mentioned perilla leaf extract, 13-15 parts by mass of the above-mentioned soybean hypocotyl extract, 18-20 parts by mass of the above-mentioned cranberry extract and 4-6 parts by mass of the above-mentioned coated AKK probiotics are stirred and mixed to obtain a composite powder.

2. The preparation process of a composite powder containing AKK probiotics according to claim 1, characterized in that: S1 includes the following steps: S1.1: Dry, crush and sieve the dried perilla leaves, add them into the extracting solution at a solid-liquid ratio of 1 g: (15-25) mL, stir and disperse them thoroughly, and perform reflux extraction at 50-60°C and 300-350W with ultrasonic assisted heating for 40-50 min to obtain a perilla leaf extract; S1.2: After centrifuging the perilla leaf extract at a rate of 6000-7000 r / min for 10-20 min, separating the supernatant, and performing reduced pressure distillation and vacuum drying on the supernatant to obtain the perilla leaf extract.

3. The preparation process of a composite powder containing AKK probiotics according to claim 2, characterized in that: S2 includes the following steps: S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls; S2.2: Grind the above soybean hypocotyls into powder, add them into 65% ethanol solution at a solid-liquid ratio of 1g:(20-30)mL, heat and reflux at 60-70℃ for 1-2h, and obtain soybean hypocotyl extract by centrifugation, reduced pressure distillation and vacuum drying.

4. The preparation process of a composite powder containing AKK probiotics according to claim 3, characterized in that: S3 includes the following steps: S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder; S3.2: Add the cranberry powder to a 50% ethanol solution at a solid-liquid ratio of 1 g: (18-22) mL, and perform reflux extraction at 200-300 W and 60-70 ° C with ultrasonic assisted heating for 30-40 min to obtain a cranberry extract; S3.3: The cranberry extract is filtered, and the filtrate is concentrated under reduced pressure and freeze-dried to obtain a cranberry extract.

5. The preparation process of a composite powder containing AKK probiotics according to claim 4, characterized in that: S4 specifically includes the following steps: S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g: (10-20) mL, stir thoroughly to dissolve, and obtain a whey protein solution; S4.2: Adding compound enzyme to the above whey protein solution, stirring and mixing, keeping the temperature at 40-60°C for 2-3h for enzymolysis, then heating to 90-95°C for 3-5min to inactivate the enzyme, cooling, centrifugally filtering, concentrating and drying to obtain hydrolyzed whey protein powder; S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran into PBS buffer at a solid-liquid ratio of 1g:(90-100)mL, stir until completely dissolved, freeze-dry, and then place in a closed environment at 55-65℃ and 70-80% humidity to react for 20-24h to obtain a composite coating material.

6. The preparation process of a composite powder containing AKK probiotics according to claim 5, characterized in that: S5 specifically includes the following steps: S5.1: Inoculate AKK probiotics into Mucin medium, culture anaerobically at 35-37°C for 18-24h, then centrifuge at 4000-6000rpm for 10-15min, collect the bacteria, and wash them with PBS buffer 2-3 times to obtain activated AKK probiotics; S5.2: Add the composite coating material prepared in step S4.3 into PBS buffer at a solid-liquid ratio of 1 g: (10-20) mL, stir thoroughly to dissolve, and obtain a coating material solution; S5.3: Add the activated AKK probiotics to the coating material solution while stirring, and continue stirring and mixing. After freeze drying and grinding, coated AKK probiotics are obtained.

7. The preparation process of a composite powder containing AKK probiotics according to claim 2, characterized in that: The leaching solution is prepared by mixing 50% ethanol solution and 0.1 mol / L hydrochloric acid, and the pH of the leaching solution is 2-3.

8. The preparation process of a composite powder containing AKK probiotics according to claim 5, characterized in that: The mass ratio of the complex enzyme to the whey protein powder is 1:(90-100), and the complex enzyme is prepared by mixing neutral protease and papain in a mass ratio of 1:(1-3).

9. The preparation process of a composite powder containing AKK probiotics according to claim 6, characterized in that: AKK probiotics are AKK AM06 strains derived from healthy breast milk, and the amount of activated AKK probiotics added is (1-3)×10 6 CFU / mL coating material solution.

10. A composite powder containing AKK probiotics, characterized in that: The composite powder containing AKK probiotics is prepared by the preparation process of the composite powder containing AKK probiotics according to any one of claims 1 to 9.

Images