A probiotic preparation and preparation method thereof

Abstract

The invention discloses a probiotic drop and a preparation method thereof, which comprises the following raw material components by weight: 5-8 parts of lactobacillus acidophilus powder, 3-6 parts of bifidobacterium breve powder, 4-6 parts of grape seed oil, 25-35 parts of olive oil, 10-20 parts of DHA algae oil, 25-35 parts of medium-chain triglycerides, and 0.5-1.5 parts of emulsifier; the lactobacillus acidophilus is lactobacillus acidophilus NCFM, and the bifidobacterium breve is bifidobacterium breve M-16V. The present invention forms a composite probiotic powder by compounding lactobacillus acidophilus NCFM and bifidobacterium breve M-16V, and introduces notoginseng saponin R1 copolymer drug-loaded microspheres for coordinated use therewith, which is beneficial to intestinal health.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotic preparations, and in particular to a probiotic preparation and a preparation method thereof. Background Art

[0002] As a kind of microorganisms that are beneficial to intestinal health, probiotics can play an important role by changing the intestinal microecology of the host. Their main functions include promoting the digestion and absorption of nutrients, enhancing the body's immunity, maintaining the balance of intestinal flora, inhibiting intestinal inflammation, and protecting the intestinal mucosal barrier. However, the activity of probiotics is not stable and is easily affected by environmental conditions such as temperature, humidity and pH, which may cause the death of probiotics and reduce their health benefits.

[0003] Probiotic drops have attracted the attention of the market and consumers due to their high probiotic content, small size, and ease of use and carrying. This type of product usually uses vegetable oil as the main carrier to disperse probiotic powder in it. However, vegetable oil contains a large amount of unsaturated fatty acids, which has poor stability and is easily oxidized during storage, causing product deterioration and odor, which in turn affects the activity of probiotics. In addition, due to the high water activity of the oil, the activity of probiotics decreases rapidly. In order to reduce the decay rate of probiotics, the storage conditions of probiotic drops products usually need to be set below 4°C, which limits the convenience of use and circulation range of the product.

[0004] More importantly, probiotic powder, as a water-soluble raw material, is incompatible with oil-based carriers, resulting in poor dispersibility and uneven distribution in oil-based carriers. This will cause the probiotic powder of probiotic drops to settle directly at the bottom of the bottle before use, and stick to the bottom of the bottle during the shelf life, so that the live probiotic content in the probiotic droplets of probiotic drops during actual use may be far lower than the labeled amount, which is insufficient to exert the expected health effects.

[0005] Therefore, it is particularly important to develop a probiotic drop product with high survival rate, good storage stability, good dispersibility and low bacterial powder sedimentation in an oil-based carrier. CN112494529B discloses a probiotic drop for increasing gastrointestinal motility and a preparation method thereof, and the probiotic drop provided by the invention includes coconut oil, DHA algae oil, olive oil, Lactobacillus reuteri, Lactobacillus rhamnosus, silicon dioxide, chamomile oil and lauric acid monoglyceride. The probiotic preparation preparation method provided by the invention is simple, and the drop has stable performance, good stability under freeze-thaw conditions and high temperature conditions, and low temperature does not solidify, and can significantly improve infantile colic and flatulence. The live bacteria in the probiotic drop are stored for a long time and stably, and the probiotic survival rate is high, but the probiotic drop has poor dispersibility, and sedimentation phenomenon is prone to occur, which affects use.

[0006] CN115531424A discloses a probiotic drop, which, based on the total weight of the drop, contains: 1-15wt% probiotics; 31-50wt% sunflower oil; 31-50wt% olive oil; and 17-30wt% triglycerides. Although the probiotic drop can effectively improve the stability of the probiotics and maintain the activity of the probiotics during long-term storage, the sunflower oil contains a lot of unsaturated fatty acids and is easily oxidized, which is not conducive to maintaining the activity of the probiotics. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a probiotic drops and a preparation method thereof. The provided probiotic drops have good storage stability, high probiotic survival rate, and good dispersibility of probiotic powder in oil-based carriers, and the sedimentation phenomenon is significantly improved.

[0008] One of the purposes of the present invention is to provide a method for preparing a probiotic drop, comprising the following steps:

[0009] (1) grinding Lactobacillus acidophilus powder and Bifidobacterium breve powder and passing them through a 50-100 mesh sieve respectively, and then mixing the sieved Lactobacillus acidophilus powder and the sieved Bifidobacterium breve powder to obtain a composite probiotic powder;

[0010] (2) Grape seed oil, olive oil, DHA algae oil, medium chain triglycerides and emulsifier are sterilized respectively; the sterilized grape seed oil, DHA algae oil, olive oil, medium chain triglycerides and emulsifier are stirred and mixed at 7500-8500 rpm for 4-8 minutes, and then homogenized by a homogenizer at a homogenization pressure of 18-25 MPa for 2-3 cycles to obtain an oil-based carrier; then the composite probiotic powder obtained in step (1) is added, and the mixture is stirred and mixed at a stirring speed of 450-550 rpm for 40-60 minutes to obtain a probiotic drop.

[0011] Preferably, the weight ratio of each raw material component is:

[0012] 5-8 parts of Lactobacillus acidophilus powder, 3-6 parts of Bifidobacterium breve powder, 4-6 parts of grape seed oil, 25-35 parts of olive oil, 10-20 parts of DHA algae oil, 25-35 parts of medium chain triglycerides, and 0.5-1.5 parts of emulsifier.

[0013] Preferably, the Lactobacillus acidophilus is Lactobacillus acidophilus NCFM; and the Bifidobacterium breve is Bifidobacterium breve M-16V.

[0014] Preferably, the total bacterial count of Lactobacillus acidophilus and Bifidobacterium breve in the probiotic drops is 5×10 8 -1×10 10 CFU / mL.

[0015] Preferably, the emulsifier is selected from one of polyglycerol polyricinoleate, polyethylene glycol fatty acid ester and polyglycerol fatty acid ester.

[0016] Preferably, in step (2), sterilization is performed at 118-125° C. for 15-30 min.

[0017] Preferably, the step (2) can also be: taking grape seed oil, olive oil, DHA algae oil, medium-chain triglycerides, emulsifier and Panax notoginseng saponin R1 copolymer drug-loaded microspheres for sterilization respectively; stirring and mixing the sterilized grape seed oil, DHA algae oil, olive oil, medium-chain triglycerides and emulsifier at 7500-8500rpm for 4-8min, and then using a homogenizer to perform 2-3 cycles of homogenization at a homogenization pressure of 18-25MPa to obtain an oil-based carrier; then adding the composite probiotic powder obtained in step (1) and the above-mentioned sterilized Panax notoginseng saponin R1 copolymer drug-loaded microspheres, stirring and mixing at a stirring speed of 450-550rpm for 40-60min, to obtain probiotic drops.

[0018] Further preferably, the method for preparing the notoginsenoside R1 copolymer drug-loaded microspheres comprises the following steps, calculated by weight:

[0019] S1. Dry polyethylene glycol monomethyl ether 2000 in vacuum for 45-50h, then mix 8-12 parts of the dried polyethylene glycol monomethyl ether 2000, 85-95 parts of L-lactide and 0.1-0.5 parts of stannous octoate, stir, condense and reflux at 135-145°C for 110-130h under nitrogen protection, and then cool to room temperature to obtain a product; dissolve the product in 8-12 parts of dichloromethane, then add 90-110 parts of methyl tert-butyl ether to precipitate to obtain a white solid, filter, dissolve the white solid in dichloromethane again, then precipitate with methyl tert-butyl ether, filter, repeat this operation 3-5 times to obtain a white solid product; wash the obtained white solid product with 50-60°C hot water 2-3 times and then vacuum dry for 45-50h to obtain polyethylene glycol monomethyl ether-polylactic acid copolymer;

[0020] S2, adding 0.02-0.03 parts of notoginseng saponin R1 to 1-2 parts of anhydrous ethanol, mixing and stirring to obtain notoginseng saponin R1 solution; adding 0.1-0.2 parts of polyethylene glycol monomethyl ether-polylactic acid copolymer obtained in the above step S1 to 4-6 parts of anhydrous dichloromethane, mixing and stirring to obtain a copolymer solution; then adding the notoginseng saponin R1 solution to the copolymer solution at a temperature of -1°C to 1°C and a stirring speed of 20000-22000rpm, and continuing to stir at 20000-30000rpm for 3-5min to obtain colostrum; at a temperature of -1-1°C and a stirring speed of 30000-32000rpm, Under the following conditions, colostrum is added dropwise at a rate of 5.5-6.5 mL / min into 15-25 parts of polyvinyl alcohol aqueous solution through an automatic syringe, and stirring is continued at 30000-40000 rpm for 3-5 min to obtain a double emulsion; the double emulsion is quickly transferred to 350-450 parts of isopropanol aqueous solution, and stirred at room temperature and 400-600 rpm for 23-26 h, and then the solution is centrifuged at 6500-8000 rpm for 7-10 min, the white solid is collected, washed with water 2-3 times, and freeze-dried at -55°C to -45°C for 23-26 h to obtain notoginseng saponin R1 copolymer drug-loaded microspheres.

[0021] Further preferably, the weight ratio of each raw material component is:

[0022] 5-8 parts of Lactobacillus acidophilus, 3-6 parts of Bifidobacterium breve, 4-6 parts of grape seed oil, 25-35 parts of olive oil, 10-20 parts of DHA algae oil, 25-35 parts of medium chain triglycerides, 0.5-1.5 parts of emulsifier, and 1-3 parts of notoginseng saponin R1 copolymer drug-loaded microspheres.

[0023] The second object of the present invention is to provide a probiotic drop prepared by the above method.

[0024] The grape seed oil in the present invention is rich in vitamin E and polyphenol compounds, which helps to prevent oil oxidation, thereby reducing oxidation and deterioration and odor generation of the probiotic drops during storage. As an oil-based carrier component in the probiotic drops, the grape seed oil can improve the stability of the oil-based carrier and extend the shelf life of the probiotic drops.

[0025] Olive oil in the present invention is a kind of vegetable oil, which is squeezed from the fruit of olive of the Oleaceae family and is a commonly used edible oil. Olive oil is known as "liquid gold". It contains a high proportion of monounsaturated fatty acids, especially oleic acid, which makes olive oil have good thermal stability and antioxidant capacity, and has multiple health benefits such as antioxidant, anti-inflammatory and reduced cardiovascular disease risk. Olive oil, as an oil-based carrier component in probiotic drops, can enhance the antioxidant properties of probiotic drops and reduce the decline in probiotic activity.

[0026] Medium-chain triglyceride of the present invention is also known as medium-chain fatty acid triglyceride, which is a triglyceride composed of saturated medium-chain fatty acids of 6-12 carbon chains, naturally present in foods such as palm kernel oil, coconut oil and breast milk, and is one of the sources of dietary fat. Medium-chain triglyceride is composed only of saturated fatty acids, has a smaller molecular structure, is easily digested and absorbed, and is rapidly metabolized in the body, and is not easy to cause fat accumulation. Medium-chain triglyceride has a low freezing point, is liquid at room temperature, has a small viscosity, has good oxidation stability and thermal stability, is not easy to oxidize and deteriorate, and medium-chain triglyceride is due to its molecular structure, is more difficult to combine with water than long-chain fatty acids, which helps to reduce the water activity of the oil-based carrier, thereby slowing down the rate of decline of probiotic activity. Using medium-chain triglyceride as the oil-based carrier component in probiotic drops can improve the stability of probiotic drops and reduce the activity loss of probiotics during storage and processing.

[0027] In the present invention, DHA algae oil is pure vegetable DHA, commonly known as brain gold, belongs to the Omega-3 unsaturated fatty acid family, is a main element of nervous system cell growth and maintenance, and is crucial to infant intelligence and vision development. The EPA content in DHA algae oil is extremely low, making it relatively more stable and safe, suitable for pregnant women and infants to eat. Because DHA itself has antioxidant properties, DHA algae oil is used as the oil-based carrier component in probiotic drops to help maintain the stability of probiotics.

[0028] Beneficial effects of the present invention:

[0029] 1. Compared with the prior art, the present invention mixes grape seed oil, olive oil, DHA algae oil, medium-chain triglycerides and emulsifiers in a specific proportion to form an oil-based carrier with antioxidant properties and stability. The antioxidant components in the oil-based carrier reduce oil oxidation, reduce the activity loss of probiotics, and improve the storage stability of probiotics, so that the probiotics can maintain their efficacy for a longer time, extend the shelf life of the probiotic drops, and through the appropriate ratio of the components in the oil-based carrier, the probiotics can be more evenly dispersed in the oil base, reducing the aggregation and precipitation of the probiotics.

[0030] 2. The present invention forms a composite probiotic powder by mixing Lactobacillus acidophilus NCFM and Bifidobacterium breve M-16V in a specific ratio, and introduces notoginseng saponin R1 copolymer drug-loaded microspheres. The composite probiotic powder is used in coordination, which can more effectively regulate the content of proinflammatory cytokines TNF-α, IFN-γ and IL-6 and anti-inflammatory cytokine IL-10 in the blood of mice with DSS-induced colitis in mice, and can more effectively improve the excessive inflammation in the body of mice with colitis, which is beneficial to intestinal health. In addition, the introduction of notoginseng saponin R1 copolymer drug-loaded microspheres can improve the dispersibility of probiotic powder in oil-based carriers, and effectively improve the situation that probiotic powder settles and adheres to the bottom of the bottle during the shelf life of probiotic drops. In addition, notoginseng saponin R1 copolymer drug-loaded microspheres can also cooperate with polyglycerol polyricinoleate to protect probiotics, which is conducive to maintaining the stability of probiotics during long-term storage, thereby maintaining the activity of probiotics. DETAILED DESCRIPTION

[0031] Parameters for specific chemical substances used, sources.

[0032] Polyvinyl alcohol, purity: 99%, purchased from Hubei Jiangming Taihua Chemical Co., Ltd., model: TH;

[0033] Polyethylene glycol monomethyl ether 2000 was purchased from Shanghai Myrel Biochemical Technology Co., Ltd., product number: TCl-P2186;

[0034] Notoginsenoside R1, purity: 98%, purchased from Chengdu Purifa Technology Development Co., Ltd., catalog number: BP1010;

[0035] Polyglycerol polyricinoleate, purity: 99%, purchased from Hubei Shiteng Chemical Technology Co., Ltd., model: 26520;

[0036] Medium chain triglycerides, purity: 99%, purchased from Shandong Haizhou Bioengineering Co., Ltd., product number: 538-24-9;

[0037] Polyglycerol fatty acid esters, purity: 99%, purchased from Leshengyuan Biotechnology (Nanjing) Co., Ltd., catalog number: 97536;

[0038] Polyethylene glycol fatty acid esters, purity: 99%, purchased from Chongqing Ruiya Biotechnology Co., Ltd., catalog number: 9004-74-4;

[0039] DHA algae oil, light yellow oily liquid, purity: 99%, purchased from Jishui County Hongxing Natural Medicinal Spice Oil Factory;

[0040] Grape seed oil, light yellow clear liquid, purity: 99%, purchased from Jiangxi Jingwang Natural Flavor Co., Ltd., item number: JW-1150;

[0041] Olive oil, light yellow liquid, purchased from Jiangxi Jingwang Natural Flavor Co., Ltd., item number: JW-J49;

[0042] Lactobacillus acidophilus NCFM, strain number: SHBCC D24720, specification: freeze-dried powder, purchased from Shanghai Ruichu Biotechnology Co., Ltd., product number: SHBCC D24720;

[0043] Bifidobacterium breve M-16V, strain number: SHBCC D24678, specification: lyophilized powder, purchased from Shanghai Ruichu Biotechnology Co., Ltd., product number: SHBCC D24678.

[0044] Example 1

[0045] A method for preparing probiotic drops comprises the following steps:

[0046] (1) grinding Lactobacillus acidophilus NCFM powder and Bifidobacterium breve M-16V powder and passing them through a 100-mesh sieve respectively, and then mixing 6 parts by weight of the sieved Lactobacillus acidophilus NCFM powder and 4 parts by weight of the sieved Bifidobacterium breve M-16V powder to obtain a composite probiotic powder;

[0047] (2) 5 parts by weight of grape seed oil, 30 parts by weight of olive oil, 15 parts by weight of DHA algae oil, 30 parts by weight of medium-chain triglycerides and 1 part by weight of polyglycerol polyricinoleate are sterilized at 120° C. for 20 min; the sterilized grape seed oil, DHA algae oil, olive oil, medium-chain triglycerides and polyglycerol polyricinoleate are stirred and mixed at 8000 rpm for 5 min, and then homogenized by a homogenizer at a homogenization pressure of 20 MPa for 3 cycles to obtain an oil-based carrier; and then the composite probiotic powder obtained in step (1) is added, and the mixture is stirred and mixed at a stirring speed of 500 rpm for 50 min to obtain a probiotic drop.

[0048] Example 2

[0049] A method for preparing probiotic drops, which differs from Example 1 in that 1 part by weight of polyglycerol polyricinoleate is replaced by 1 part by weight of polyglycerol fatty acid ester.

[0050] Example 3

[0051] A method for preparing probiotic drops, which differs from Example 1 in that 1 part by weight of polyglycerol polyricinoleate is replaced by 1 part by weight of polyethylene glycol fatty acid ester.

[0052] Example 4

[0053] A method for preparing probiotic drops comprises the following steps:

[0054] (1) grinding Lactobacillus acidophilus NCFM powder and Bifidobacterium breve M-16V powder and passing them through a 100-mesh sieve respectively, and then mixing 6 parts by weight of the sieved Lactobacillus acidophilus NCFM powder and 4 parts by weight of the sieved Bifidobacterium breve M-16V powder to obtain a composite probiotic powder;

[0055] (2) 5 parts by weight of grape seed oil, 30 parts by weight of olive oil, 15 parts by weight of DHA algae oil, 30 parts by weight of medium-chain triglycerides, 1 part by weight of polyglycerol polyricinoleate and 2 parts by weight of notoginseng saponin R1 copolymer drug-loaded microspheres are sterilized at 120° C. for 20 min; the sterilized grape seed oil, DHA algae oil, olive oil, medium-chain triglycerides and polyglycerol polyricinoleate are stirred and mixed at 8000 rpm for 5 min, and then homogenized by a homogenizer at a homogenization pressure of 20 MPa for 3 cycles to obtain an oil-based carrier; then the composite probiotic powder obtained in step (1) and the sterilized notoginseng saponin R1 copolymer drug-loaded microspheres are added, and the mixture is stirred and mixed at a stirring speed of 500 rpm for 50 min to obtain a probiotic drop.

[0056] The method for preparing notoginseng saponin R1 copolymer drug-loaded microspheres in step (2) comprises the following steps:

[0057] S1. The polyethylene glycol monomethyl ether 2000 was vacuum dried for 48 hours, and then 10 parts by weight of the dried polyethylene glycol monomethyl ether 2000, 90 parts by weight of L-lactide and 0.3 parts by weight of stannous octoate were mixed and stirred, and condensed and refluxed at 140° C. for 120 hours under nitrogen protection, and then cooled to room temperature to obtain a product; the product was dissolved in 10 parts by weight of dichloromethane, and then precipitated in 100 parts by weight of methyl tert-butyl ether to obtain a white solid, which was filtered, and the white solid was dissolved in dichloromethane again, and then precipitated with methyl tert-butyl ether, filtered, and the operation was repeated 4 times to obtain a white solid product; the obtained white solid product was washed twice with 55° C. hot water and then vacuum dried for 48 hours to obtain a polyethylene glycol monomethyl ether-polylactic acid copolymer;

[0058] S2, adding 0.025 weight parts of notoginseng saponin R1 to 1 weight part of anhydrous ethanol, mixing and stirring to obtain notoginseng saponin R1 solution; adding 0.15 weight parts of polyethylene glycol monomethyl ether-polylactic acid copolymer obtained in the above step S1 to 5 weight parts of anhydrous dichloromethane, mixing and stirring to obtain a copolymer solution; then adding the notoginseng saponin R1 solution to the copolymer solution at a temperature of 0°C and a stirring speed of 21000 rpm, and continuing to stir at 25000 rpm for 3 minutes to obtain colostrum; stirring at a temperature of 0°C and a stirring speed of 31 ... Under pm conditions, colostrum was added dropwise at a rate of 6 mL / min into 20 parts by weight of 2 wt% polyvinyl alcohol aqueous solution through an automatic syringe, and stirring was continued at 35000 rpm for 4 min to obtain a double emulsion; the double emulsion was quickly transferred to 400 parts by weight of 5 wt% isopropanol aqueous solution, and stirred at room temperature and 500 rpm for 24 h, then the solution was centrifuged at 7000 rpm for 8 min, the white solid was collected, washed with water 3 times, and freeze-dried at -50°C for 24 h to obtain notoginseng saponin R1 copolymer drug-loaded microspheres.

[0059] Example 5

[0060] A method for preparing probiotic drops, which differs from Example 4 in that 2 parts by weight of notoginseng saponin R1 copolymer drug-loaded microspheres are replaced with 1 part by weight of notoginseng saponin R1 copolymer drug-loaded microspheres.

[0061] Example 6

[0062] A method for preparing probiotic drops, which differs from Example 4 in that 2 parts by weight of notoginseng saponin R1 copolymer drug-loaded microspheres are replaced with 3 parts by weight of notoginseng saponin R1 copolymer drug-loaded microspheres.

[0063] Comparative Example 1

[0064] A method for preparing probiotic drops, which differs from Example 4 in that polyglycerol polyricinoleate is not added.

[0065] Comparative Example 2

[0066] A method for preparing probiotic drops, which differs from Example 4 in that medium-chain triglycerides are not added, and the missing parts by weight are replaced by 30 parts by weight of olive oil.

[0067] Comparative Example 3

[0068] A method for preparing probiotic drops, which differs from Example 4 in that grape seed oil is not added, and the missing parts by weight are replaced by 5 parts by weight of olive oil.

[0069] Test Example 1

[0070] Storage stability test

[0071] In order to verify the storage stability of the probiotic drops provided by the present invention during the period of being sold on the shelf, the probiotic drops of Examples 1-6 and Comparative Examples 1-3 were subjected to a storage stability test.

[0072] The probiotic drops prepared in Examples 1-6 of the present invention and Comparative Examples 1-3 were taken respectively, and then stored at 4° C., 25° C. and 37° C. for 24 months respectively, and samples were taken after 0, 6, 12, 18 and 24 months to detect the number of viable bacteria in the samples. The specific test results are shown in Tables 1-3;

[0073] The probiotic survival rate (%) of the probiotic drops after 24 months of shelf life was calculated according to the number of viable probiotic bacteria in the probiotic drops of Examples 1-6 and Comparative Examples 1-3 after storage at 4° C., 25° C. and 37° C. The probiotic survival rate (%) was calculated according to the following formula:

[0074] Probiotic survival rate (%) = number of viable probiotics at the 24th month / number of viable probiotics at the 0th month × 100%

[0075] The specific data of the probiotic survival rate (%) of the probiotic drops after a shelf life of 24 months are shown in Table 4.

[0076] Table 1 Number of viable probiotic bacteria in probiotic drops stored at 4°C

[0077]

[0078]

[0079] Table 2 Probiotic viable bacteria count of probiotic drops stored at 25°C

[0080]

[0081] Table 3 Probiotic viable counts of probiotic drops stored at 37°C

[0082]

[0083] Table 4 Probiotic survival rate after 24 months of probiotic drops (%)

[0084]

[0085] As shown in Table 1-3, the number of live probiotic bacteria in the probiotic drops provided by Examples 1-6 and Comparative Examples 1-3 exceeded 10×10 8 CFU / mL, which is much higher than the requirement for the number of viable probiotics in the "GB7101-2022 National Food Safety Standard Beverage" (required to be greater than 1×10 6CFU / mL). This indicates that the probiotic drops provided by Examples 1-6 and Comparative Examples 1-3 of the present invention have good storage stability.

[0086] As shown in Table 4, the survival rates of probiotics stored at 4°C, 25°C and 37°C for 24 months in Examples 1-6 and Comparative Examples 1-3 were compared, and it was found that the survival rate at 4°C was higher than that at 25°C and 37°C, which may be due to the fact that low temperature is more suitable for maintaining the activity of probiotics. Under the conditions of 4°C, 25°C and 37°C, Examples 1-6 and Comparative Example 3 have good probiotic activity, and the survival rate is still higher than 50% after storage at 25°C and 37°C for 24 months, which is significantly better than Comparative Examples 1-2, and maintains good probiotic activity, indicating that Examples 1-6 can maintain good stability without refrigeration and have excellent storage stability. By comparing Examples 1-6 and Comparative Example 3, it was found that at 4°C, 25°C and 37°C, after a shelf life of 24 months, the survival rates of the probiotics in Examples 4-6 were significantly higher than those in Examples 1-3 and Comparative Example 3, among which Example 4 was the best, and the survival rates of the probiotics were maintained at 83.04%, 58.20% and 55.85% at 4°C, 25°C and 37°C, respectively. The reason for this may be that the hydrophilic polyglycerol part of the polyglycerol polyricinoleate molecule can interact with the polar part of the probiotic cells, and form a protective barrier on the surface of the probiotics through its hydrophobic chain segment. This barrier can prevent moisture and oxygen in the external environment from directly contacting the probiotics, which helps to maintain the stability of the probiotics, and can slow down the evaporation of water in the probiotic cells, helping the probiotics to remain moist, thereby maintaining the activity of the probiotics. At the same time, the hydrophilic polyethylene glycol segments in the notoginseng saponin R1 copolymer drug-loaded microsphere structure can interact with the probiotic powder, which can not only further improve the stability of the surface protective barrier of the probiotic molecule, but also reduce the aggregation of probiotics in the oil-based carrier, improve its dispersibility, further improve the stability of probiotics, and help improve the activity of probiotics. In addition, the added grape seed oil contains vitamin E and polyphenol compounds with strong antioxidant effects, which can help scavenge free radicals and effectively reduce the damage of oxidative stress to probiotics. And the linoleic acid in grape seed oil can enhance the stability of cell membranes, prevent lipid peroxidation of cell membranes, and help maintain the survival rate of probiotics.

[0087] In summary, the addition of polyglycerol polyricinoleate, notoginseng saponin R1 copolymer drug-loaded microspheres and grape seed oil can improve the survival rate of probiotics stored at 4°C, 25°C and 37°C for 24 months, improve the storage stability of probiotics during the sales shelf, and extend the shelf life of probiotic drops.

[0088] Test Example 2

[0089] Anti-inflammatory effect of probiotic drops on mice with colorectal inflammation

[0090] 48 healthy C57BL / 6 adult mice weighing 20-22g were selected and fed adaptively for 1 week. The experimental period was 4 weeks, including 1 week of modeling and 3 weeks of normal feeding. The mice were randomly divided into 6 groups, 8 mice in each group, namely, the control group, the enteritis model group, the Example 1 group, and the Example 4-6 groups.

[0091] During the modeling phase, the enteritis model group, Example 1 group, and Example 4-6 groups were administered drugs, and 3% dextran sulfate sodium (DSS) (3g / 100mL) drinking water was prepared for free drinking by mice. The mice were monitored every day to ensure that approximately equal amounts of DSS water were ingested. The control group was given normal saline without DSS. After 7 days of DSS stimulation, the positive test phase was entered, and the Example 1 group and Example 4-6 groups were orally administered with the probiotic drops of Example 1 group and Example 4-6 groups, and the control group and the enteritis model group were given equal amounts of normal saline.

[0092] The Example 1 group and the Example 4-6 group were gavaged once a day with 0.5 mL of the probiotic drops prepared in Example 1 and Example 4-6, respectively, for 21 days. The control group and the enteritis model group were given 0.5 mL of normal saline per mouse every day during the entire test period. 0.5 h after gavage on the 21st day, the mice were anesthetized with 0.9% sodium pentobarbital, and the mice were bled from the heart with a 1 mL syringe. The blood was placed in an EP tube and allowed to stand for 2 h, centrifuged at 4000 rpm for 8 min, and the upper serum was collected for ELISA to detect cytokines in the serum.

[0093] The levels of TNF-α, IL-6, IFN-γ and IL-10 in mouse blood were determined according to the instructions of the ELISA detection kits. The ELISA kits for TNF-α (catalog number: KBR-hlk6685), IL-6 (catalog number: KBR-hlk6696), IFN-γ (catalog number: KBR-hlk6691) and IL-10 (catalog number: KBR-hlk6699) were purchased from Shanghai Keborui Biotechnology Co., Ltd.

[0094] The test results are averaged. The specific test results are shown in Table 5 below.

[0095] Table 5 TNF-α, IL-6, IFN-γ and IL-10 levels in the blood of mice in each group

[0096]

[0097] As shown in Table 5, compared with the control group, the pro-inflammatory cytokines TNF-α, IL-6, and IFN-γ in the enteritis model group increased, and the IL-10 content decreased significantly, indicating that the modeling was successful. Compared with the enteritis model group, the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-6 content in the blood of mice in Example 1 and Example 4-6 groups decreased significantly, and the anti-inflammatory cytokine IL-10 content increased significantly, indicating that the probiotic drops of Example 4-6 can effectively exert anti-inflammatory effects. In addition, compared with implementation 1, the decrease in TNF-α, IFN-γ and IL-6 content and the increase in IL-10 content in Example 4-6 are obvious, among which the change in Example 4 is the most significant, indicating that Example 4-6 can more effectively regulate the pro-inflammatory cytokines TNF-α, IFN-γ and IL-6 and the anti-inflammatory cytokine IL-10 in the blood of mice with DSS-induced colorectal inflammation in mice by combining Lactobacillus acidophilus NCFM and Bifidobacterium breve M-16V to form a composite probiotic powder and use it in conjunction with notoginseng saponin R1 copolymer drug-loaded microspheres, and can more effectively improve the excessive inflammation in mice with colorectal inflammation. The reason for the analysis may be that Lactobacillus acidophilus NCFM and Bifidobacterium breve BB536 can enhance the intestinal barrier function and reduce the penetration of inflammatory mediators by interacting with intestinal epithelial cells. And the composite probiotic powder formed by the two may stimulate the intestinal associated lymphoid tissue, regulate the host's immune response, increase the production of anti-inflammatory cytokines such as IL-10, and reduce the levels of pro-inflammatory cytokines such as TNF-α, IFN-γ and IL-6. At the same time, notoginseng saponin R1 has anti-inflammatory and immunomodulatory effects, can inhibit the production of proinflammatory cytokines, and may promote the release of anti-inflammatory cytokines, which may reduce inflammatory responses by affecting cell signaling pathways, such as inhibiting the activation of the NF-κB signaling pathway. In addition, the notoginseng saponin R1 copolymer drug-loaded microsphere structure can protect notoginseng saponin R1 from damage by adverse conditions such as light, high temperature and gastric acid, ensuring that its active ingredients reach the intestines, and has a sustained-release property, which can control the release rate of notoginseng saponin R1, allowing it to continue to play a role in the site of colorectal inflammation, further improving the improvement effect on excessive inflammation in mice with colorectal inflammation.

[0098] Test Example 3

[0099] Probiotic powder dispersibility test

[0100] In order to detect the dispersibility of the probiotic powder in the probiotic drops prepared in the examples of the present invention in the oil-based carrier, the probiotic drops in Examples 1 and 4 were subjected to weight sampling test and total viable bacteria count test of the drop samples. The uniform stability of the samples in terms of weight and activity was analyzed from the test results, which indirectly reflected the dispersibility of the probiotics in the oil-based carrier.

[0101] The probiotic drops prepared in Example 1 and Example 4 were filled separately, and the net weight was controlled at an average of 9.0 mL per bottle, and the difference in the amount per bottle was ±2.5%. The nominal value of the product was 50×10 8 CFU / mL of probiotic powder are recorded as Example 1 and Example 4. It should be noted that 50×10 8 The probiotic powder in CFU / mL is the nominal value of the supplier's probiotic drops product. Since probiotics are easily affected by environmental conditions such as temperature, humidity, and pH during the actual supply process, which may cause the death of probiotics, in order to avoid the nominal value not being reached due to the death of probiotics during the actual supply process, the actual detection values ​​of probiotics in the probiotic drops products provided by the supplier are greater than the nominal values, so the detection values ​​of the final supplied products will also be greater than the nominal values.

[0102] Weight sampling test: According to the actual use of consumers, 5 drops of liquid were dripped from the probiotic drops of Example 1 and Example 4 as a group of dripping samples, and the total weight of the 5 drops was 220 mg as the benchmark for the repeatability test of the weight of the dripped liquid. Five drops of liquid were dripped from the probiotic drops of Example 1 and Example 4 every day, and the total weight of the droplets and the dripping error with the benchmark droplet weight (220 mg) were recorded. The sampling test was repeated for 30 days, and the test results are shown in Tables 6-7.

[0103] Detection of total number of viable bacteria in drip samples: According to the detection method of GB4789.35-2016 National Food Safety Standard Food Microbiology Inspection Lactic Acid Bacteria Inspection, the drip samples of Example 1 and Example 4 in the above weight sampling test were proportionally detected for the total number of probiotics in the drip samples of each group every day, and the total number of probiotics in the drip samples of Example 1 and Example 4 every day for 30 days of weight sampling test was recorded, and the difference between the total number of probiotics in the drip samples of Example 1 and Example 4 and the nominal value (50×10 8 The test results are shown in Table 8-9.

[0104] Table 6 Total weight and error of dripping liquid in Example 1 (30d)

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[0106]

[0107] Table 7 Example 4 dripping liquid total weight and error (30d)

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[0109] Table 8 Example 1 Drop sample total number of live bacteria and error (30d)

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[0112] Table 9 Example 4 Total number of live bacteria in the sample dripped out and error (30d)

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[0114]

[0115] It can be seen from Tables 6-7 that the total weight error range of Example 1 is -22 mg-29 mg, the total weight error range of Example 1 is -11 mg-18 mg, and the total weight error range of Example 1 and Example 4 is within ±30 mg. The error range is small, which shows that the amount of liquid dripped out of the prepared probiotic drops each time is relatively stable, and has good uniformity and stability in weight, indicating that the probiotics are evenly dispersed in the oil-based carrier with less local aggregation.

[0116] As shown in Tables 8-9, the error range of the total number of live bacteria in the sample of Example 1 is -16%-16%; the error range of the total number of live bacteria in the sample of Example 4 is -7%-7%; it can be seen that the number of live probiotics in the liquid dripped out of the prepared probiotic drops is relatively stable, and there is no large fluctuation in the number of live bacteria, indicating that the probiotics are evenly dispersed in the oil-based carrier and have good dispersibility and stability. Comparing the total weight error range and the live bacteria error range of Example 1 and Example 4, it is found that the total weight and live bacteria value fluctuations of Example 4 are smaller, indicating that the probiotics in the probiotic drops prepared in Example 4 have better dispersibility than Example 1. The reason for the analysis may be that the notoginseng saponin R1 copolymer drug-loaded microspheres are added in Example 4, and the structure thereof contains a hydrophilic polyethylene glycol chain and a hydrophobic polylactic acid chain, which has an amphiphilic property, which may help to improve the dispersibility of the probiotic powder in the oil-based carrier and reduce the sedimentation of the probiotic powder. Due to its hydrophobicity, the polylactic acid chain can form good compatibility with the oil-based carrier, so that the microspheres can be evenly dispersed in the oil-based carrier, reducing sedimentation and aggregation. At the same time, the polyethylene glycol segment on the microspheres can interact with the probiotic powder due to its hydrophilicity, which may help to disperse the probiotic powder in the oil-based carrier, maintain the uniform distribution of probiotics in the drops, reduce the aggregation of probiotic powder, and help prevent the sedimentation of probiotic powder, effectively improving the situation where the probiotic powder settles and sticks to the bottom of the bottle during the shelf life of the probiotic drops.

Claims

1. A method for preparing probiotic drops, characterized in that: The following steps are involved: (1) grinding Lactobacillus acidophilus powder and Bifidobacterium breve powder and passing them through a 50-100 mesh sieve respectively, and then mixing the sieved Lactobacillus acidophilus powder and the sieved Bifidobacterium breve powder to obtain a composite probiotic powder; (2) Grape seed oil, olive oil, DHA algae oil, medium-chain triglycerides, emulsifier and notoginseng saponin R1 copolymer drug-loaded microspheres are sterilized respectively; the sterilized grape seed oil, DHA algae oil, olive oil, medium-chain triglycerides and emulsifier are stirred and mixed at 7500-8500 rpm for 4-8 minutes, and then homogenized by a homogenizer at a homogenization pressure of 18-25 MPa for 2-3 cycles to obtain an oil-based carrier; then the composite probiotic powder obtained in step (1) and the sterilized notoginseng saponin R1 copolymer drug-loaded microspheres are added, and the mixture is stirred and mixed at a stirring speed of 450-550 rpm for 40-60 minutes to obtain a probiotic drop; The weight proportion of each raw material component is: 5-8 parts of Lactobacillus acidophilus, 3-6 parts of Bifidobacterium breve, 4-6 parts of grape seed oil, 25-35 parts of olive oil, 10-20 parts of DHA algae oil, 25-35 parts of medium chain triglycerides, 0.5-1.5 parts of emulsifier, 1-3 parts of notoginseng saponin R1 copolymer drug-loaded microspheres; The Lactobacillus acidophilus is Lactobacillus acidophilus NCFM; the Bifidobacterium breve is Bifidobacterium breve M-16V; The emulsifier is selected from one of polyglycerol polyricinoleate, polyethylene glycol fatty acid ester and polyglycerol fatty acid ester.

2. The method for preparing the probiotic drops according to claim 1, characterized in that: The total bacterial count of Lactobacillus acidophilus and Bifidobacterium breve in the probiotic drops is 5×10 8 -1×10 10 CFU / mL.

3. The method for preparing the probiotic drops according to claim 1, characterized in that: In step (2), sterilization is performed at 118-125° C. for 15-30 min.

4. The method for preparing the probiotic drops according to claim 1, characterized in that: The preparation method of the notoginseng saponin R1 copolymer drug-loaded microspheres comprises the following steps, calculated by weight: S1. Dry polyethylene glycol monomethyl ether 2000 in vacuum for 45-50h, then mix 8-12 parts of the dried polyethylene glycol monomethyl ether 2000, 85-95 parts of L-lactide and 0.1-0.5 parts of stannous octoate, stir, condense and reflux at 135-145°C for 110-130h under nitrogen protection, and then cool to room temperature to obtain a product; dissolve the product in 8-12 parts of dichloromethane, then add 90-110 parts of methyl tert-butyl ether to precipitate to obtain a white solid, filter, dissolve the white solid in dichloromethane again, then precipitate with methyl tert-butyl ether, filter, repeat this operation 3-5 times to obtain a white solid product; wash the obtained white solid product with 50-60°C hot water 2-3 times and then vacuum dry for 45-50h to obtain polyethylene glycol monomethyl ether-polylactic acid copolymer; S2, adding 0.02-0.03 parts of notoginseng saponin R1 to 1-2 parts of anhydrous ethanol, mixing and stirring to obtain notoginseng saponin R1 solution; adding 0.1-0.2 parts of polyethylene glycol monomethyl ether-polylactic acid copolymer obtained in the above step S1 to 4-6 parts of anhydrous dichloromethane, mixing and stirring to obtain a copolymer solution; then adding the notoginseng saponin R1 solution to the copolymer solution at a temperature of 0°C and a stirring speed of 20000-22000rpm, and continuing to stir at 20000-30000rpm for 3-5min to obtain colostrum; stirring at a temperature of -1°C to 1°C and a stirring speed of 30000-32000rpm. Under the conditions, colostrum is added dropwise at a rate of 5.5-6.5 mL / min into 15-25 parts of polyvinyl alcohol aqueous solution through an automatic syringe, and stirring is continued at 30000-40000 rpm for 3-5 min to obtain a double emulsion; the double emulsion is quickly transferred to 350-450 parts of isopropanol aqueous solution, and stirred at room temperature and 400-600 rpm for 23-26 h, and then the solution is centrifuged at 6500-8000 rpm for 7-10 min, the white solid is collected, washed with water 2-3 times, and freeze-dried at -55°C to -45°C for 23-26 h to obtain notoginseng saponin R1 copolymer drug-loaded microspheres.

5. A probiotic drop, characterized in that: Prepared by the method according to any one of claims 1 to 4.

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