A compound powder containing Akk probiotics and its preparation process

Abstract

The present invention provides a composite powder containing AKK probiotics and its preparation process, belonging to the technical field of solid beverage foods; the preparation process includes the following steps: preparing perilla leaf extract; preparing soybean hypocotyl extract; preparing cranberry extract; preparing a composite coating material; activating AKK bacteria and performing encapsulation; and mixing to prepare the composite powder. The present invention first enzymatically hydrolyzes whey protein using neutral protease and papain to prepare hydrolyzed whey protein, and then grafts the hydrolyzed whey protein with carboxymethyl glucan to obtain a composite coating material. After encapsulating AKK probiotics with this composite coating material, the encapsulation 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 AKK probiotics, reducing their mortality rate in gastric juice, and thus increasing their survival rate in the gastrointestinal tract.

Description

Technical Field

[0001] The present invention relates to the technical field of solid beverage foods, and particularly relates 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 undertakes the important task of digesting and absorbing nutrients, but also plays an indispensable role in maintaining the body's immune balance and resisting the invasion of pathogens. 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 lifestyle, such as unreasonable diet structure (increased intake of high-sugar, high-fat, and high-salt foods, insufficient intake of dietary fiber), long-term excessive mental stress, improper use of antibiotics, and environmental pollution, have had a significant negative impact on the balance of the intestinal microbial community. Clinical studies have shown that intestinal flora dysbiosis is closely related to the occurrence and development of various diseases, such as inflammatory bowel disease (including ulcerative colitis and Crohn's disease), irritable bowel syndrome, obesity, diabetes, cardiovascular diseases, and even some neurological diseases (such as autism and depression) and cancers. These diseases 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 a type of 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 enhancing the tight junctions between intestinal epithelial cells, thus forming a strong defense line to prevent the invasion of harmful bacteria, toxins, and other pathogens, reducing the intestinal permeability, and reducing the risk of intestinal inflammation. On the other hand, Akk probiotics play a key role in regulating the balance of the 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 at the same time produces beneficial metabolites such as short-chain fatty acids, creating favorable conditions for the growth of beneficial bacteria such as Bifidobacterium and Lactobacillus, and promoting the development of the intestinal flora towards a healthy and balanced direction. At present, various oral nutritional supplement products have been made from Akk probiotics to maintain intestinal health. However, the strong acidic environment in gastric juice can denature the enzyme system and biological macromolecules in Akk probiotic cells by destroying the spatial structure of proteins, losing their activity, and thus 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 that can improve the survival rate of Akk probiotics. Summary of the Invention

[0005] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a composite powder containing Akk probiotics and its preparation process.

[0006] A preparation process of a composite powder containing Akk probiotics comprises the following steps:

[0007] S1: Prepare perilla leaf extract

[0008] After pretreating the perilla leaves, add them to the leaching solution, and carry out ultrasonic-assisted heating reflux extraction to prepare perilla leaf extract;

[0009] S2: Prepare soybean hypocotyl extract

[0010] Pretreat the soybeans and peel the hypocotyls, then grind the soybean hypocotyls into powder, and then add them to the ethanol solution for heating reflux extraction to obtain soybean hypocotyl extract;

[0011] S3: Prepare cranberry extract

[0012] After pretreating the cranberries, add them to the ethanol solution, and carry out ultrasonic-assisted heating reflux extraction to obtain cranberry extract;

[0013] S4: Prepare composite coating material

[0014] Dissolve whey protein, add composite enzyme for hydrolysis to prepare hydrolyzed whey protein powder, then dissolve the hydrolyzed whey protein powder and carboxymethyl glucan in PBS buffer solution, freeze-dry, and then heat for reaction to obtain composite coating material;

[0015] S5: Activate Akk bacteria and carry out embedding

[0016] Activate the Akk probiotics, then dissolve the above composite coating material in PBS buffer solution, add the activated Akk probiotics, stir well and freeze-dry to obtain coated Akk probiotics;

[0017] S6: Mix to prepare composite powder

[0018] Mix 6-10 parts by mass of porphyra polysaccharide, 8-10 parts by mass of fructooligosaccharide, 8-10 parts by mass of inulin, 18-20 parts by mass of the above perilla leaf extract, 13-15 parts by mass of the above soybean hypocotyl extract, 18-20 parts by mass of the above cranberry extract and 4-6 parts by mass of the above coated Akk probiotics and stir to obtain composite powder.

[0019] Furthermore, S1 specifically comprises the following steps:

[0020] S1.1: After drying, pulverizing, and sieving the dry perilla leaves, add them to the extraction solution at a solid-liquid ratio of 1 g : (15 - 25) mL, stir well to disperse, and perform ultrasonic-assisted heating reflux extraction at 50 - 60 °C and 300 - 350 W for 40 - 50 min to obtain the perilla leaf extract;

[0021] S1.2: Centrifuge the above perilla leaf extract at a rate of 6000 - 7000 r / min for 10 - 20 min, separate the supernatant, and perform vacuum distillation and vacuum drying on the supernatant to obtain the perilla leaf extract.

[0022] Further, S2 specifically includes the following steps:

[0023] S2.1: Wash, freeze-dry the soybeans, and then perform hypocotyl peeling to obtain soybean hypocotyls;

[0024] S2.2: After grinding the above soybean hypocotyls into powder, add them to 65% ethanol solution at a solid-liquid ratio of 1 g : (20 - 30) mL, heat under reflux at 60 - 70 °C for 1 - 2 h, perform centrifugal separation, vacuum distillation, and vacuum drying to obtain the soybean hypocotyl extract.

[0025] Further, S3 specifically includes the following steps:

[0026] S3.1: Wash, dry, pulverize, and sieve the cranberries, and then defat them with petroleum ether to obtain cranberry powder;

[0027] S3.2: Add the above cranberry powder to 50% ethanol solution at a solid-liquid ratio of 1 g : (18 - 22) mL, perform ultrasonic-assisted heating reflux extraction at 200 - 300 W and 60 - 70 °C for 30 - 40 min to obtain the cranberry extract;

[0028] S3.3: Filter the above cranberry extract by suction filtration, and then perform vacuum concentration and freeze drying on the filtrate to obtain the cranberry extract.

[0029] Further, S4 specifically includes the following steps:

[0030] S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g : (10 - 20) mL, stir well to dissolve to obtain a whey protein solution;

[0031] S4.2: Add a complex enzyme to the above whey protein solution, stir and mix, incubate at 40 - 60 °C for enzymatic hydrolysis for 2 - 3 h, then heat to 90 - 95 °C to inactivate the enzyme for 3 - 5 min, cool, and perform centrifugal filtration, concentration, and drying to obtain hydrolyzed whey protein powder;

[0032] S4.3: Add hydrolyzed whey protein powder and carboxymethyl dextran of equal mass to PBS buffer solution at a solid-liquid ratio of 1 g : (90 - 100) mL. After stirring until completely dissolved, conduct freeze-drying, and then place it in a sealed environment at 55 - 65 °C and a humidity of 70% - 80% for reaction for 20 - 24 h to obtain the composite coating material.

[0033] Further, S5 specifically includes the following steps:

[0034] S5.1: Inoculate AKK probiotics into Mucin medium, anaerobically culture at 35 - 37 °C for 18 - 24 h, then centrifuge at 4000 - 6000 rpm for 10 - 15 min, collect the bacterial cells, and wash them 2 - 3 times with PBS buffer solution to obtain activated AKK probiotics;

[0035] S5.2: Add the composite coating material prepared in step S4.3 to PBS buffer solution at a solid-liquid ratio of 1 g : (10 - 20) mL, and stir well to dissolve to obtain the coating material solution;

[0036] S5.3: While stirring, add the above-mentioned activated AKK probiotics to the above-mentioned coating material solution, and continue to stir and mix. After freeze-drying and grinding, obtain coated AKK probiotics.

[0037] Further, 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.

[0038] Further, the mass ratio of the composite enzyme to the whey protein powder is 1 : (90 - 100), and the composite enzyme is prepared by mixing neutral protease and papain at a mass ratio of 1 : (1 - 3).

[0039] Further, the AKK probiotics are AKK AM06 strain derived from healthy breast milk, and the addition amount of the activated AKK probiotics is (1 - 3) × 10 6 CFU / mL of the coating material solution.

[0040] Further, 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.

[0041] Compared with the prior art, the present invention has at least the following beneficial effects:

[0042] In the present invention, whey protein is first enzymatically hydrolyzed using neutral protease and papain to prepare hydrolyzed whey protein, and then the hydrolyzed whey protein is mixed with carboxymethyl dextran for grafting reaction to obtain a composite coating material. After coating AKK probiotics with the composite coating material, 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 AKK probiotics, reduce the erosion of hydrochloric acid, pepsin, etc. in gastric juice on the cells of AKK probiotics, and lower its mortality rate in gastric juice, thereby increasing its survival rate in the gastrointestinal tract.

[0043] In the present invention, perilla leaf and soybean hypocotyl are first extracted with ethanol solution respectively to prepare perilla leaf extract and soybean hypocotyl extract, and then the two are mixed with other components to form a composite powder. On the one hand, since the active ingredients in perilla leaf extract can promote intestinal peristalsis, and the dietary fiber and other components in soybean hypocotyl extract can absorb water and expand in the intestine. When the two are combined, the perilla leaf extract stimulates the contraction of intestinal smooth muscle, while the soybean hypocotyl extract increases the intestinal contents, more powerfully promoting the forward movement of intestinal contents, thereby achieving a better effect of moistening the intestines and relieving constipation. On the other hand, since both perilla leaf extract and soybean hypocotyl extract can delay the digestion and absorption of sugar in the small intestine by inhibiting the activities of α-glucosidase and α-amylase, achieving the effect of lowering blood sugar, so the two have a synergistic hypoglycemic effect.

[0044] In the present invention, cranberry extract is first extracted with ethanol solution to prepare cranberry extract, and then cranberry extract, porphyra polysaccharide and other components are mixed to prepare a composite powder. Since porphyra polysaccharide can provide a carbon source for AKK probiotics, and the active ingredients in cranberry extract can act as growth factors to activate the growth and metabolic genes of AKK probiotics, promoting its uptake and utilization of porphyra polysaccharide, thus synergistically promoting the growth of AKK probiotics. In addition, in the intestinal environment, porphyra polysaccharide has a certain water absorption capacity, and it can regulate the water balance in the intestine. When it acts synergistically with cranberry extract, on the one hand, the polysaccharide absorbs water and expands, making the intestinal contents more moist, creating a moist growth environment for AKK probiotics and being conducive to its reproduction. On the other hand, the organic acids in cranberry extract can regulate the pH value of the intestine, making the growth environment of AKK probiotics more suitable. Therefore, when porphyra polysaccharide and cranberry extract act together, they can synergistically promote the growth of AKK probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The drawings incorporated herein and constituting a part of the specification illustrate embodiments of the present disclosure, and together with the specification are further used to explain the principles of the present disclosure and enable those skilled in the relevant art to implement and use the present disclosure.

[0046] Figure 1 Process flow chart of the preparation of the composite powder containing Akk probiotics adopted in the embodiments of the present invention. Detailed implementation manners

[0047] The following describes in detail a composite powder containing Akk probiotics and its preparation process provided by the present invention in conjunction with the accompanying drawings and specific embodiments.

[0048] Preparation example

[0049] The preparation steps of the Mucin medium are as follows:

[0050] 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 well until dissolved, sterilize with a high-pressure steam sterilizer at 121 °C for 15 min, and then add 0.5 g of L-cysteine hydrochloride by filtration sterilization. After stirring and mixing evenly, the Mucin medium is obtained.

[0051] Example 1: The preparation process of a composite powder containing Akk probiotics is as Figure 1 shown, and includes the following steps:

[0052] S1: Prepare perilla leaf extract

[0053] S1.1: After drying, crushing, and sieving the dry perilla leaves, add them to the extraction solution according to the solid-liquid ratio of 1 g: 15 mL, stir well and disperse, and perform ultrasonic-assisted heating reflux extraction at 50 °C and 300 W for 40 min to obtain the perilla leaf extract. Among them, the extraction solution is prepared by mixing 50% ethanol solution and 0.1 mol / L hydrochloric acid, and the pH of the extraction solution is 2;

[0054] S1.2: Centrifuge the above perilla leaf extract at a rate of 6000 r / min for 10 min, separate the supernatant, and perform vacuum distillation and vacuum drying on the supernatant to obtain the perilla leaf extract;

[0055] S2: Prepare soybean hypocotyl extract

[0056] S2.1: Wash, freeze-dry the soybean, and then perform hypocotyl stripping to obtain the soybean hypocotyl;

[0057] S2.2: After grinding the above soybean hypocotyls into powder, add them to 65% ethanol solution according to the solid-liquid ratio of 1 g: 20 mL, heat and reflux extract at 60 °C for 1 h, and after centrifugal separation, vacuum distillation and vacuum drying, obtain the soybean hypocotyl extract;

[0058] S3: Prepare cranberry extract

[0059] S3.1: After cleaning, drying, pulverizing and sieving cranberries, degrease them with petroleum ether to obtain cranberry powder;

[0060] S3.2: Add the above-mentioned cranberry powder to a 50% ethanol solution at a solid-liquid ratio of 1 g: 18 mL, and perform ultrasonic-assisted heating and reflux extraction at 200 W and 60 °C for 30 min to obtain a cranberry extract;

[0061] S3.3: Filter the above-mentioned cranberry extract by suction, then concentrate the filtrate under reduced pressure and freeze-dry it to obtain a cranberry extract;

[0062] S4: Prepare a composite coating material

[0063] S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g: 10 mL, and stir well until dissolved to obtain a whey protein solution;

[0064] S4.2: Add a composite enzyme to the above-mentioned whey protein solution, stir and mix, incubate at 40 °C for 2 h, then heat to 90 °C to inactivate the enzyme for 3 min. After cooling, centrifuge, filter, concentrate and dry to obtain hydrolyzed whey protein powder. Among them, the mass ratio of the composite enzyme to the whey protein powder is 1:90, and the composite enzyme is prepared by mixing neutral protease and papain at a mass ratio of 1:1;

[0065] S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl dextran to PBS buffer at a solid-liquid ratio of 1 g: 90 mL, stir until completely dissolved, then perform freeze-drying, and then react in a sealed environment at 55 °C and 70% humidity for 20 h to obtain a composite coating material;

[0066] S5: Activate AKK bacteria and perform embedding

[0067] S5.1: Inoculate AKK probiotics into Mucin medium, anaerobically culture at 35 °C for 18 h, then centrifuge at 4000 rpm for 10 min, collect the bacterial cells, and wash them twice with PBS buffer to obtain activated AKK probiotics. Among them, the AKK probiotics are AKK AM06 strains derived from healthy breast milk;

[0068] S5.2: Add the composite coating material prepared in step S4.3 to PBS buffer at a solid-liquid ratio of 1 g: 10 mL, stir well until dissolved to obtain a coating material solution;

[0069] S5.3: While stirring, add the above-mentioned activated AKK probiotics to the above-mentioned coating material solution, and continue to stir and mix. After freeze-drying and grinding, obtain coated AKK probiotics. Among them, the addition amount of the activated AKK probiotics is 1×10 6 CFU / mL coating material solution;

[0070] S6: Mixing to prepare composite powder

[0071] 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.

[0072] Example 2: A preparation process of a composite powder containing AKK probiotics, such as Figure 1 As shown, the following steps are included:

[0073] S1: Preparation of Perilla Leaf Extract

[0074] 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;

[0075] 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;

[0076] S2: Preparation of soybean hypocotyl extract

[0077] S2.1: washing the soybeans, freeze-drying them, and then peeling off the hypocotyls to obtain soybean hypocotyls;

[0078] 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;

[0079] S3: Preparation of cranberry extract

[0080] S3.1: washing, drying, crushing and sieving cranberries, and then defatting with petroleum ether to obtain cranberry powder;

[0081] 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;

[0082] S3.3: filtering the cranberry extract, and then concentrating the filtrate under reduced pressure and freeze-drying it to obtain a cranberry extract;

[0083] S4: Preparation of composite coating material

[0084] S4.1: Add whey protein powder to deionized water at a solid-liquid ratio of 1 g: 15 mL. After fully stirring and dissolving, a whey protein solution is obtained;

[0085] S4.2: Add a composite enzyme to the above-mentioned whey protein solution. After stirring and mixing, incubate at 50 °C for 2.5 h for enzymatic hydrolysis, then heat to 92 °C to inactivate the enzyme for 4 min. After cooling, centrifugally filter, concentrate and dry to obtain hydrolyzed whey protein powder. Among them, the mass ratio of the composite enzyme to the whey protein powder is 1:95, and the composite enzyme is prepared by mixing neutral protease and papain at a mass ratio of 1:2;

[0086] S4.3: Add equal masses of hydrolyzed whey protein powder and carboxymethyl glucan to PBS buffer at a solid-liquid ratio of 1 g: 95 mL. After stirring until completely dissolved, perform freeze-drying, and then react in a closed environment at 60 °C and 75% humidity for 22 h to obtain a composite coating material;

[0087] S5: Activate AKK bacteria and perform embedding

[0088] S5.1: Inoculate AKK probiotics into Mucin medium, anaerobically culture at 36 °C for 21 h, then centrifuge at 5000 rpm for 12 min, collect the bacterial cells, and wash them twice with PBS buffer to obtain activated AKK probiotics. Among them, the AKK probiotics are AKK AM06 strains derived from healthy breast milk;

[0089] S5.2: Add the composite coating material prepared in step S4.3 to PBS buffer at a solid-liquid ratio of 1 g: 15 mL, and fully stir and dissolve to obtain a coating material solution;

[0090] S5.3: While stirring, add the above-mentioned activated AKK probiotics to the above-mentioned coating material solution, and continue to stir and mix. After freeze-drying and grinding, coated AKK probiotics are obtained. Among them, the addition amount of the activated AKK probiotics is 2×10 6 CFU / mL coating material solution;

[0091] S6: Mix and prepare a composite powder

[0092] Stir and mix 8 parts by mass of Gracilaria lemaneiformis polysaccharide, 9 parts by mass of fructooligosaccharide, 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 to obtain a composite powder.

[0093] Example 3: A preparation process of a composite powder containing AKK probiotics, as Figure 1As shown, it includes the following steps:

[0094] S1: Prepare perilla leaf extract

[0095] S1.1: After drying, crushing and sieving dry perilla leaves, add them to the extraction solution according to the solid-liquid ratio of 1 g: 25 mL, stir well to disperse, and perform ultrasonic-assisted heating reflux extraction at 60 °C and 350 W for 50 min to obtain perilla leaf extract. Among them, the extraction solution is prepared by mixing 50% ethanol solution and 0.1 mol / L hydrochloric acid, and the pH of the extraction solution is 3;

[0096] S1.2: Centrifuge the above perilla leaf extract at a rate of 7000 r / min for 20 min, separate the supernatant, and perform vacuum distillation and vacuum drying on the supernatant to obtain perilla leaf extract;

[0097] S2: Prepare soybean hypocotyl extract

[0098] S2.1: Wash, freeze-dry soybeans, and then perform hypocotyl peeling to obtain soybean hypocotyls;

[0099] S2.2: After grinding the above soybean hypocotyls into powder, add them to 65% ethanol solution according to the solid-liquid ratio of 1 g: 30 mL, heat under reflux at 70 °C for 2 h, and after centrifugal separation, vacuum distillation and vacuum drying, obtain soybean hypocotyl extract;

[0100] S3: Prepare cranberry extract

[0101] S3.1: After washing, drying, crushing and sieving cranberries, degrease them with petroleum ether to obtain cranberry powder;

[0102] S3.2: Add the above cranberry powder to 50% ethanol solution according to the solid-liquid ratio of 1 g: 22 mL, perform ultrasonic-assisted heating reflux extraction at 300 W and 70 °C for 40 min to obtain cranberry extract;

[0103] S3.3: Filter the above cranberry extract by suction, and then perform vacuum concentration and freeze-drying on the filtrate to obtain cranberry extract;

[0104] S4: Prepare composite coating material

[0105] S4.1: Add whey protein powder to deionized water according to the solid-liquid ratio of 1 g: 20 mL, stir well to dissolve, and obtain whey protein solution;

[0106] S4.2: Add a complex enzyme to the above whey protein solution. After stirring and mixing, incubate at 60 °C for 3 h for enzymatic hydrolysis, then heat to 95 °C to inactivate the enzyme for 5 min. After cooling, carry out centrifugal filtration, concentration and drying to obtain hydrolyzed whey protein powder. Among them, the mass ratio of the complex enzyme to the whey protein powder is 1:100, and the complex enzyme is prepared by mixing neutral protease and papain at a mass ratio of 1:3;

[0107] S4.3: Add the hydrolyzed whey protein powder and carboxymethyl glucan of equal mass to PBS buffer according to the solid-liquid ratio of 1 g:100 mL. After stirring until completely dissolved, carry out freeze-drying, and then react in a sealed environment at 65 °C and 80% humidity for 24 h to obtain a composite coating material;

[0108] S5: Activate AKK bacteria and carry out embedding

[0109] S5.1: Inoculate AKK probiotics into Mucin medium, anaerobically culture at 37 °C for 24 h, then centrifuge at 6000 rpm for 15 min to collect the bacteria, and wash with PBS buffer 3 times to obtain activated AKK probiotics. Among them, the AKK probiotics are AKK AM06 strain derived from healthy breast milk;

[0110] S5.2: Add the composite coating material prepared in step S4.3 to PBS buffer according to the solid-liquid ratio of 1 g:20 mL, and stir well to dissolve to obtain a coating material solution;

[0111] S5.3: While stirring, add the above activated AKK probiotics to the above coating material solution, and continue to stir and mix. After freeze-drying and grinding, obtain coated AKK probiotics. Among them, the addition amount of the activated AKK probiotics is 3×10 6 CFU / mL coating material solution;

[0112] S6: Mix and prepare a composite powder

[0113] Stir and mix 10 parts by mass of Gracilaria lemaneiformis polysaccharide, 10 parts by mass of fructooligosaccharide, 10 parts by mass of inulin, 20 parts by mass of the above perilla leaf extract, 15 parts by mass of the above soybean hypocotyl extract, 20 parts by mass of the above cranberry extract and 6 parts by mass of the above coated AKK probiotics to obtain a composite powder.

[0114] Comparative Example 1: The difference between this Comparative Example 1 and Example 1 is that steps S4 and S5.2 - S5.3 are removed, that is, the activated AKK probiotics are not embedded and directly added to the composite powder.

[0115] Comparative Example 2: The difference between this Comparative Example 2 and Example 1 is that the perilla leaf extract in step S6 is replaced with an equal amount of soybean hypocotyl extract.

[0116] Comparative Example 3: The difference between this Comparative Example 3 and Example 1 is that the soybean hypocotyl extract in step S6 is replaced with an equal amount of perilla leaf extract.

[0117] Comparative Example 4: The difference between this Comparative Example 4 and Example 1 is that the polysaccharide from Gelidium amansii in step S6 is replaced with an equal amount of cranberry extract.

[0118] Comparative Example 5: The difference between this Comparative Example 5 and Example 1 is that the cranberry extract in step S6 is replaced with an equal amount of polysaccharide from Gelidium amansii.

[0119] Test Example: Test Example 1: The encapsulated AKK probiotics prepared in Example 1 and the unencapsulated activated AKK probiotics in Comparative Example 1 were respectively suspended in sterilized simulated gastric juice, incubated in the dark for 2 h in a constant temperature incubator at 37 °C, then 0.5 mL was taken and transferred to a sterile centrifuge tube, centrifuged at 3000 rpm for 10 min, the supernatant was discarded, the precipitate was resuspended with sterile PBS buffer, and then spread on Mucin medium, anaerobically cultured at 37 °C for 48 h, and then the number of colonies on each plate was counted, and the number of viable bacteria in the sample after incubation in simulated gastric juice was calculated according to the dilution factor (CFU / mL). The calculation formula for the survival rate is as follows:

[0120] Survival rate = (number of viable AKK probiotics / initial number of AKK probiotics) × 100%, and the results are shown in Table 1.

[0121] Table 1: Survival rate of AKK probiotics

[0122] Survival rate (%) Example 1 87.45 Example 2 86.96 Example 3 87.32 Control Example 1 48.63

[0123] As can be seen from Table 1, when the AKK probiotics are not encapsulated with the composite encapsulating material in Comparative Example 1, the survival rate after 2 h in gastric juice is only about 48.63%, which is much lower than that in Example 1. Thus, 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 glucan for grafting reaction to obtain a composite encapsulating material, after encapsulating the AKK probiotics with this composite encapsulating material, the embedding structure formed by the composite encapsulating material can serve as a physical barrier to reduce the erosion of hydrochloric acid, pepsin, etc. in gastric juice on the AKK probiotic cells, thereby increasing its survival rate in the gastrointestinal tract.

[0124] Test Example 2: 70 male BALB / c mice at 7 weeks of age were randomly divided into a blank control group, a model control group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 2 group, and 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 h to establish a dry constipation model due to water loss. After modeling, the blank control group and the model control group were respectively intragastrically administered with 2% ink prepared with distilled water. For the remaining groups, after dissolving the corresponding compound powder prepared in each group in distilled water to prepare a 5% sample solution, 3 mL of the sample solution was used to prepare 2% ink, and the mice were intragastrically administered. Then, the time (min) for the first black stool excretion of each group of mice was observed and recorded, the total number of black stools excreted by the mice within 4 h was counted, and the water content of the feces was measured, and the average value was taken. The results are shown in Table 2.

[0125] Table 2: Time for the first black stool excretion of mice, total number of black stools excreted, and water content of feces

[0126] Time for the first appearance of black stools (min) Total number of black stools excreted (pieces) Moisture content of feces (%) Example 1 157.2 7.1 56.84 Example 2 155.9 7.2 57.21 Example 3 158.5 6.9 57.36 Control Example 2 219.3 2.7 43.22 Control 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

[0127] As can be seen from Table 2, when the compound powder prepared with a single soybean hypocotyl extract and a single perilla leaf extract was intragastrically administered in Comparative Example 2 and Comparative Example 3, the time for the first black stool excretion of the mice was longer than that in Example 1, the total number of black stools excreted was less than that in Example 1, and the water content of the feces was lower than that in Example 1. Thus, it can be seen that by first extracting perilla leaf and soybean hypocotyl with ethanol solution respectively to prepare perilla leaf extract and soybean hypocotyl extract, and then mixing the two with other components to prepare a compound powder, the perilla leaf extract and soybean hypocotyl extract can synergistically promote the laxative effect of the compound powder.

[0128] Test Example 3: Using 4-nitrophenyl-α-D-glucopyranoside as a substrate, a total of 12 groups were set up in the experiment, with 3 replicate wells in each group, namely 3 blank groups (a, buffer + enzyme + substrate), 3 blank control groups (b, buffer + substrate), 3 sample groups (c, 5% compound powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 + enzyme + substrate), and 3 sample control groups (d, 5% compound powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 + buffer + substrate). Using 25 μL of phosphate buffer (PBS, 20 mM, pH = 7.0) as the buffer system, 25 μL of α-glucosidase (0.6 U / mL) was added, 25 μL of the test sample solution (5% compound powder solution prepared in Example 1 / Comparative Example 2 / Comparative Example 3 by mass fraction) was added, and they were mixed by shaking. Incubate in a biochemical incubator at 37 °C for 15 min. After taking out, 50 μL of p-nitrophenyl-β-D-galactopyranoside (10 mM) solution was added, and they were mixed evenly. Incubate in a biochemical incubator at 37 °C for 15 min. After taking out, 100 μL of sodium carbonate solution was added to terminate the reaction. The absorbance (A) was measured at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The calculation formula for the α-glucosidase inhibition rate is as follows:

[0129] 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 absorbance of the sample group, A d is the absorbance of the sample control group. The results are shown in Table 3.

[0130] Table 3: α-Glucosidase inhibition rate

[0131] Example 1 Control Example 2 Control Example 3 Inhibition rate (%) 83.2 72.9 74.4

[0132] As can be seen from Table 3, the inhibition rates of the composite powders prepared with single soybean hypocotyl extract and single perilla leaf extract in Comparative Example 2 and Comparative Example 3 on α-glucosidase are both lower than those of the composite powder prepared with soybean hypocotyl extract and perilla leaf extract in Example 1. Thus, it can be seen that perilla leaf extract and soybean hypocotyl extract have a synergistic hypoglycemic effect.

[0133] Test Example 4: Add 5 mg of the composite powders prepared in Example 1, Comparative Example 4, and Comparative Example 5 into Mucin medium respectively. After incubating for 72 h under anaerobic conditions at 37°C, take 0.5 mL and transfer it to a sterile centrifuge tube. Centrifuge at 3000 rpm for 10 min, discard the supernatant, then resuspend the precipitate with sterile PBS buffer and coat it on Mucin medium. Culture it for 72 h under anaerobic conditions at 37°C, calculate the number of colony-forming units on the plate, then calculate the number of viable bacteria in the sample after incubating for 72 h according to the dilution factor, and calculate the growth rate of AKK probiotics. The calculation formula for the growth rate is as follows:

[0134] Growth rate = number of viable bacteria after incubating for 72 h / number of viable bacteria before incubation. The results are shown in Table 4.

[0135] Table 4: Growth rate of AKK probiotics

[0136] Growth rate Example 1 <![CDATA[3.26×10 3 > Control Example 4 <![CDATA[6.72×10 2 > Control Example 5 <![CDATA[4.37×10 2 >

[0137] As can be seen from Table 4, when single cranberry extract or single porphyra polysaccharide is added to the composite powder in Comparative Example 4 and Comparative Example 5, the growth rate of AKK probiotics is lower than that of the composite powder prepared by adding cranberry extract and porphyra polysaccharide simultaneously in Example 1. Thus, it can be seen that when porphyra polysaccharide and cranberry extract act together, they can synergistically promote the growth of AKK probiotics.

[0138] The above embodiments are only illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should 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.

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