A probiotic formulation having high activity and high stability and a method for preparing the same

Abstract

The application discloses a method for preparing probiotic preparation by fermenting pineapple peel and soapberry, and belongs to the field of food science and biotechnology. The operation steps of the method comprise the following steps: firstly, preparing pineapple peel fermentation liquor and soapberry fermentation liquor; soaking the pineapple peel and the soapberry in distilled water, adding yeast for fermentation and filtering to obtain the pineapple peel fermentation liquor and the soapberry fermentation liquor; then mixing the two kinds of fermentation liquors and adding black sugar; after filtering, adding yeast, and standing for 36h-63h to complete secondary fermentation, the probiotic preparation is prepared. The method can improve the survival rate and the colonization ability of the probiotic. The method is simple and easy to implement, can fully utilize waste resources such as pineapple peel and soapberry, and the prepared probiotic preparation has high activity and stability.

Description

Technical Field

[0001] This invention relates to a probiotic preparation with high activity and stability and its preparation method, belonging to the fields of food science and biotechnology. Background Technology

[0002] Modern lifestyles, including changes in dietary habits, environmental pollution, and increased stress, have led to gut microbiota imbalance, negatively impacting immunity, digestive function, and nutrient absorption. To regulate gut microbiota balance, enhance immunity, and improve gut health, probiotic preparations are often necessary. The gut is the largest microbial ecosystem in the human body, containing a vast array of bacteria, including both beneficial and harmful ones. Probiotics are beneficial microorganisms that help maintain gut microbiota balance, promote digestion and absorption, enhance immunity, and prevent and improve various digestive system-related diseases.

[0003] The journal article "Research Status of Probiotic Classification and Multi-field Applications" mentions that while the application fields of probiotics are constantly expanding with the continuous deepening and development of research, problems still exist in practical applications, such as poor strain stability, difficulty in colonization in the digestive tract, and unclear mechanisms of action for certain effects. For example, live bacteria preparations are easily inactivated during feed processing, transportation, and storage, resulting in a loss of survival rate, reduced biological activity, and impact on product quality. Most probiotics entering the animal digestive tract are affected by stomach acid and bile salts, resulting in insufficient numbers of live bacteria reaching the intestines and colonizing to exert their effects, leading to insignificant probiotic effects. Furthermore, probiotics also have certain toxicity issues while exerting their effects. Most lactobacilli and bifidobacteria produce various bacteriocins during metabolism. These bacteriocins, in addition to inhibiting or killing putrefactive bacteria and pathogens in the intestines, also act on other microorganisms in the intestines that are beneficial to maintaining normal physiological functions. Finally, the preparation process of probiotic preparations is relatively complex, resulting in high production costs, unstable product prices, and limited market penetration. Therefore, a new solution is needed to improve the aforementioned problems of traditional probiotic preparations in order to enhance product quality, efficacy, safety, and cost-effectiveness.

[0004] Patent CN 102150746A discloses a method for packaging and fermenting pineapple pomace feed and its energy-saving processing. Using pineapple pomace as the main raw material, the process involves crushing and juicing, adding meal and fermenting agent, rapid initial drying, pressing and bagging, sealing packaging, and solid-state fermentation to produce a special fermented feed for ruminants such as dairy cows, deer, and pigs. This invention solves the key processing technology for the high-value, large-scale utilization of pineapple pomace feed. The juicing, pressing, and bagging processes and equipment are advanced. By adding soybean meal and rapeseed meal to adjust the moisture content of the juiced pineapple pomace without drying, it not only saves investment and energy consumption in drying equipment, but also allows the soybean meal and rapeseed meal to undergo enzymatic fermentation, achieving multiple benefits. Rapid initial drying significantly reduces drying energy consumption, increases yield, and avoids nutrient loss caused by drying. This invention features a simple process, low equipment investment, low cost, and turns waste into treasure. The fermented feed product is small in volume, high in density, and has a neat and attractive shape, allowing for fresh storage and long-distance transportation, thus realizing the preservation, storage, and commercialization of pineapple pomace. Patent CN102150746A discloses a method for the deep processing of pineapple peel and pulp. After pineapple juice or preserves are produced, the peel and pulp are typically used as supplementary feed for livestock without any treatment. This extensive and inefficient method results in a huge waste of resources and puts significant pressure on the environment. This invention utilizes Rhizopus, Candida, and Lactobacillus, active enzymes, and EM stock solution. Through specific technical steps including primary fermentation, secondary fermentation, pressing, fermented juice treatment, and probiotic preparation, fermented feed and probiotic preparations are produced. Testing showed that the fermented feed had a crude protein content of 23%, and the crude protein content after washing reached 16%. The probiotic preparation can purify water and promote animal growth, enhance immunity and disease resistance, making it a potential alternative to antibiotics in animal husbandry. It has shown good results in cattle, sheep, and aquaculture. Although both patents disclose methods for preparing probiotic preparations by fermenting pineapple peels and residue, the probiotic preparations made by fermenting pineapple peels or residue have low probiotic activity, poor stability, and potential long-term safety risks. Summary of the Invention

[0005] This invention aims to solve the problems of existing probiotic preparations, such as easy inactivation, poor stability, certain toxicity, complex preparation process, and high production cost. It provides a method for preparing probiotic preparations by mixing pineapple peel fermentation liquid and soapberry fermentation liquid, then adding brown sugar for secondary fermentation. By controlling the fermentation temperature, pH value, and time, the active ingredients in the fermentation products are effectively extracted and new substances are generated, thus preparing a probiotic preparation with high activity and stability.

[0006] To achieve the above objectives, the present invention first provides a method for preparing a probiotic preparation with high activity and high stability, comprising the following steps:

[0007] (1) Mix the pineapple peel fermentation liquid and soapberry fermentation liquid together, add brown sugar, and stir to obtain a mixed solution;

[0008] (2) Filter the mixed solution prepared in step (1) to obtain the filtrate;

[0009] (3) Pour the filtrate obtained in step (2) into a fermentation tank, add yeast, stir until the yeast is completely dissolved, and let it ferment for 36h to 63h to obtain a probiotic preparation.

[0010] In one embodiment of the present invention, in step (1), the volume ratio of pineapple peel fermentation liquid to soapberry fermentation liquid is 1:3 to 3:1.

[0011] In one embodiment of the present invention, in step (1), the mass ratio of the mixture of brown sugar, pineapple peel fermentation liquid and soapberry fermentation liquid is 1:3 to 1:5.

[0012] In one embodiment of the present invention, step (1) of the method for preparing the pineapple peel fermentation liquid includes the following steps:

[0013] a1. Wash and peel the fresh pineapple skin, then cut it into small pieces;

[0014] b1. Place the small pieces of pineapple peel from step a1 into a fermentation tank, add distilled water, and soak them to obtain a fermentation solution.

[0015] c1. Add yeast to the solution to be fermented, let it ferment for 36h to 63h, and filter to obtain pineapple peel fermentation liquid.

[0016] In one embodiment of the present invention, the size range of the small piece in step a1 is 7mm×7mm to 11mm×11mm.

[0017] In one embodiment of the present invention, in step b1, the ratio of pineapple peel pieces to distilled water is 1:4 to 1:9.

[0018] In one embodiment of the present invention, in step b1, the soaking time is 35 min to 55 min.

[0019] In one embodiment of the present invention, in step c1, the yeast used is a cultured yeast strain. The culture method is to add yeast powder to HS medium for culture. The components of the HS medium include bacterial peptone 4g / L-6g / L, glucose 19g / L-21g / L, yeast extract 4g / L-6g / L, sodium phosphate dibasic 2.6g / L-2.8g / L, citric acid 1g / L-1.3g / L, and magnesium sulfate 0.5g / L-1.5g / L.

[0020] In one embodiment of the present invention, in step c1, the yeast culture conditions are: pH value of 2.3-5.2, temperature of 20℃-29℃, and culture time of 36h-80h, preferably 36h-63h.

[0021] In one embodiment of the present invention, in step c1, when culturing yeast, the amount of yeast powder added is 0.8%-2% of the mass of HS culture medium.

[0022] In one embodiment of the present invention, in step c1, the amount of yeast added is 26%-37% of the volume of the solution to be fermented.

[0023] In one embodiment of the present invention, in step c1, the temperature during static fermentation is 20℃-29℃, and the pH value is 2.3-5.2.

[0024] In one embodiment of the present invention, in step c1, the mesh size of the filter screen is 50 mesh during filtration.

[0025] In one embodiment of the present invention, step (1) of the method for preparing the Sapindus mukorossi fermentation broth includes the following steps:

[0026] a2. Wash the soapberry and grind it into a fine powder. Put the obtained soapberry powder into a fermentation tank, add distilled water, and let it soak to obtain a fermentation solution.

[0027] b2. Add yeast to the solution to be fermented in step a2, let it stand for fermentation, and filter to obtain the Sapindus mukorossi fermentation broth.

[0028] In one embodiment of the present invention, the particle size range of the Sapindus mukorossi powder in step a2 is 95 mesh to 137 mesh.

[0029] In one embodiment of the present invention, in step a2, the ratio of Sapindus mukorossi powder to distilled water is 1:4 to 1:9.

[0030] In one embodiment of the present invention, in step a2, the soaking time is 35 min to 55 min.

[0031] In one embodiment of the present invention, in step b2, the yeast used is a cultured yeast strain. The culture method is to add yeast powder to HS medium for culture. The components of the HS medium include bacterial peptone 4g / L-6g / L, glucose 19g / L-21g / L, yeast extract 4g / L-6g / L, sodium phosphate dibasic 2.6g / L-2.8g / L, citric acid 1g / L-1.3g / L and magnesium sulfate 0.5g / L-1.5g / L.

[0032] In one embodiment of the present invention, in step b2, the yeast culture conditions are: pH value of 2.3-5.2, temperature of 20℃-29℃, and culture time of 36h-80h, preferably 36h-63h.

[0033] In one embodiment of the present invention, in step b2, when culturing yeast, the amount of yeast powder added is 0.8%-2% of the mass of HS culture medium.

[0034] In one embodiment of the present invention, in step b2, the amount of yeast added is 26%-37% of the volume of the solution to be fermented.

[0035] In one embodiment of the present invention, in step b2, the temperature during static fermentation is 20℃-29℃, and the pH value is 2.3-5.2.

[0036] In one embodiment of the present invention, in step b2, the filter screen mesh size is 50 mesh during filtration.

[0037] In one embodiment of the present invention, in step (2), the mesh size of the filter screen is 70 mesh during filtration.

[0038] In one embodiment of the present invention, in step (3), the yeast used is a cultured yeast strain. The culture method is to add yeast powder to HS medium for culture. The components of the HS medium include bacterial peptone 4g / L-6g / L, glucose 19g / L-21g / L, yeast extract 4g / L-6g / L, sodium phosphate dibasic 2.6g / L-2.8g / L, citric acid 1g / L-1.3g / L and magnesium sulfate 0.5g / L-1.5g / L.

[0039] In one embodiment of the present invention, in step (3), the culture conditions of yeast are: pH value of 2.3-5.2, temperature of 20℃-29℃, and culture time of 36h-80h, preferably 36h-63h.

[0040] In one embodiment of the present invention, in step (3), when culturing yeast, the amount of yeast powder added is 0.8%-2% of the mass of HS culture medium.

[0041] In one embodiment of the present invention, in step (3), the amount of yeast added is 26%-37% of the filtrate volume.

[0042] In one embodiment of the present invention, in step (3), the temperature during static fermentation is 20℃-29℃ and the pH value is 2.3-5.2.

[0043] The present invention also provides a probiotic preparation prepared according to the above method.

[0044] The present invention also provides an application of the above-mentioned probiotic preparation in the preparation of food, pharmaceuticals, and health products.

[0045] In one embodiment of the present invention, the health product includes health products that enhance immunity, regulate intestinal flora, promote digestion, and relieve constipation.

[0046] The beneficial effects of this invention are:

[0047] (1) Enhance the activity of probiotic preparations: The enzymes, strains and active ingredients in pineapple peel and soapberry can promote the growth and metabolism of probiotics, thereby enhancing the activity of the preparations.

[0048] (2) Improve the stability of probiotic preparations: Through mixed fermentation, extraction and other steps, solid residues and impurities are removed to obtain pure probiotic preparations, thereby improving their stability and shelf life.

[0049] (3) Reduced toxicity: The present invention uses natural pineapple peel and soapberry as raw materials, which have lower toxicity and side effects compared with chemically synthesized components, and are safer and more reliable.

[0050] (4) Simplified preparation process: The preparation method of the present invention is relatively simple and does not require complex equipment and operation, which reduces the complexity of the preparation process and production cost. Detailed Implementation

[0051] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0052] To evaluate the effectiveness of different embodiments and comparative examples, the present invention conducted the following tests:

[0053] 1. Validity test

[0054] The effectiveness was tested in accordance with the testing methods in GB 4789.34 and GB 4789.35 of the China Health Care Association group standard "General Rules for Live Probiotic Count", including probiotic count test and probiotic activity test.

[0055] 2. Stability Test

[0056] Stability testing shall be conducted in accordance with the provisions of the former State Food and Drug Administration's document No. 500 of 2013, "Notice of the General Office of the State Food and Drug Administration on Issuing the Guiding Principles for Stability Testing of Health Foods." This includes temperature stability testing, humidity stability testing, and acid and alkali resistance stability testing.

[0057] 3. Security Testing

[0058] Safety tests were conducted in accordance with the "National Food Safety Standard for Safety Evaluation of Food-Use Microbial Strains" developed by the National Health Commission, including acute toxicity tests, long-term toxicity tests, immunogenicity tests, and drug resistance tests.

[0059] Example 1:

[0060] A method for preparing a probiotic preparation with high activity and high stability includes the following steps:

[0061] 1. Preparation of pineapple peel fermentation liquid

[0062] (1) Wash and peel the fresh pineapple skin, and cut it into small pieces with a size of 7mm×7mm.

[0063] (2) Place small pieces of pineapple peel into a fermentation tank, add distilled water, and soak for 50 minutes. The ratio of small pieces of pineapple peel to distilled water is 1:7 to obtain the fermentation solution.

[0064] (3) Add yeast to the fermentation tank. The amount of yeast added accounts for 37% of the volume of the solution to be fermented in step (2). Let it stand and let the pineapple peel ferment to obtain the fermentation product. The fermentation temperature is 26℃, the pH value is 4.8, and the fermentation time is 63h.

[0065] (4) Filter the fermentation product obtained in step (3) to remove the pineapple skin and leave the fermentation liquid. The mesh size of the filter screen is 50 mesh.

[0066] 2. Preparation of Sapindus mukorossi fermentation broth

[0067] (1) Wash the soapberry and grind it into a fine powder for later use. The particle size of the fine powder is 137 mesh.

[0068] (2) Put the soapberry powder into a fermentation tank, add distilled water, and soak for 50 minutes; the ratio of soapberry powder to distilled water is 1:7 to obtain the fermentation solution.

[0069] (3) Add yeast to the fermentation tank. The content of this yeast is 37% of the volume of the solution to be fermented in step (2). Let it stand and let the soapberry ferment to obtain the fermentation product. The fermentation temperature is 26℃, the pH value is 4.8, and the fermentation time is 63h.

[0070] (4) Filter the fermentation product obtained in step (3) to remove Sapindus mukorossi and leave the fermentation liquid. The mesh size of the filter screen is 50 mesh.

[0071] 3. Preparation of probiotic formulations

[0072] (1) Mix the pineapple peel fermentation liquid and soapberry fermentation liquid together at a volume ratio of 3:2, and add brown sugar; the mass ratio between brown sugar and the mixture is 1:4.

[0073] (2) Use a stirrer to completely dissolve the brown sugar in the mixture, then pour it into a filter and filter it using a 70-mesh filter.

[0074] (3) Pour the filtered juice into a fermentation tank and add yeast; the yeast content should be 37% of the volume of the filtered juice. Stir well, let stand, and allow the juice to ferment to obtain a probiotic preparation. The fermentation temperature is 26℃, the pH value is 4.8, and the fermentation time is 63h.

[0075] In Example 1, the yeast strain used was a cultured yeast culture. The yeast culture process was as follows: yeast powder was added to HS medium for culture. The components of the HS medium included bacterial peptone 5.2 g / L, glucose 20.2 g / L, yeast extract 5.2 g / L, sodium diphosphate 2.75 g / L, citric acid 1.2 g / L, and magnesium sulfate 1.1 g / L. The amount of yeast powder added during culture was 1.2% of the mass of the HS medium. The culture conditions for the yeast were: pH 4.8, temperature 26℃, and culture time 63 h. The yeast powder was sourced from Angel Yeast Co., Ltd., with a specification of 5 g / bag; the HS medium was sourced from Jiangsu Yizhe Teaching Instrument Co., Ltd., with a specification of 100 mm.

[0076] Example 2:

[0077] The difference between Example 2 and Example 1 is that the yeast was cultured for 80 hours.

[0078] Comparative Example 1:

[0079] The difference between Comparative Example 1 and Example 1 is that the mixing ratio of pineapple peel fermentation liquid and soapberry fermentation liquid was changed to 7:1.

[0080] Comparative Example 2:

[0081] The difference between Comparative Example 2 and Example 1 is that the pH value in the fermentation conditions for preparing the probiotic preparation was adjusted to 7.0.

[0082] Comparative Example 3:

[0083] The difference between Comparative Example 3 and Example 1 is that no brown sugar is added in step 3.

[0084] Comparative Example 4:

[0085] The difference between Comparative Example 4 and Example 1 is that the temperature in the fermentation conditions for preparing the probiotic preparation was adjusted to 50°C.

[0086] Comparative Example 5:

[0087] The difference between Comparative Example 5 and Example 1 is that step 2 is omitted and step 3 does not contain Sapindus mukorossi fermentation liquid.

[0088] Comparative Example 6:

[0089] The difference between Comparative Example 6 and Example 1 is that step 1 is omitted, and step 3 does not contain pineapple peel fermentation liquid.

[0090] Comparative Example 7:

[0091] The difference between Comparative Example 7 and Example 1 is that step 3 is omitted, and the pineapple peel fermentation liquid obtained in step 1 and the soapberry fermentation liquid obtained in step 2 are directly mixed without secondary fermentation.

[0092] Table 1. Results of efficacy tests on the probiotic preparations prepared in Examples 1-2 and Comparative Examples 1-7.

[0093]

[0094] Table 2 shows the stability test results of the probiotic preparations prepared in Examples 1-2 and Comparative Examples 1-7.

[0095]

[0096]

[0097] Table 3 shows the safety test results of the probiotic preparations prepared in Examples 1-2 and Comparative Examples 1-7.

[0098]

[0099] As can be seen from Tables 1-3, this invention successfully extracted the active ingredients from the fermentation products and produced new substances by mixing pineapple peel fermentation liquid and soapberry fermentation liquid, adding brown sugar for secondary fermentation, and controlling fermentation temperature, pH value and time. This resulted in the preparation of a probiotic preparation.

[0100] By comparing the examples and comparative examples, we can see that Example 1 performed best in terms of probiotic count and activity, exhibiting high stability and tolerability. In the temperature stability test, Example 1 showed the smallest decrease in both activity and count; in the humidity stability test, Example 1 showed relatively small decreases in both activity and count; and in the acid and alkali stability test, Example 1 showed smaller decreases in both activity and count compared to the other comparative groups. Furthermore, Example 1 exhibited low immunogenicity, and no drug resistance genes were detected.

[0101] In contrast, the other comparative samples showed lower probiotic counts and activity, and exhibited poor stability under varying temperatures, humidity, and pH conditions. Furthermore, these comparative samples showed slightly increased immunogenicity and detected drug resistance genes.

[0102] In summary, Example 1 is the optimal solution, exhibiting a high number and activity of probiotics, as well as excellent stability and tolerability. Therefore, Example 1 shows promising application prospects in the preparation of probiotic formulations. This invention, through the fermentation method of pineapple peel and Sapindus mukorossi, provides a more stable and effective probiotic formulation, offering benefits such as improved probiotic activity and stability, a simple and efficient preparation method, and the provision of higher quality and more reliable products, thus providing consumers with better intestinal health protection.

[0103] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined in the claims of this invention.

Claims

1. A method for preparing a probiotic preparation with high activity and high stability, characterized in that, Includes the following steps: (1) Mix the pineapple peel fermentation liquid and the soapberry fermentation liquid together, add brown sugar, and stir to obtain a mixed solution. The volume ratio of the pineapple peel fermentation liquid to the soapberry fermentation liquid is 1:3~3:

1. (2) Filter the mixed solution prepared in step (1) to obtain filtrate. The mesh size of the filter screen is 70 mesh. (3) Pour the filtrate obtained in step (2) into a fermenter, add yeast, stir until the yeast is completely dissolved, and let it ferment for 36h~63h to obtain a probiotic preparation; the yeast is a cultured yeast strain, and the culture method is to add yeast powder to HS medium for culture. The components of the HS medium include bacterial peptone 4g / L-6g / L, glucose 19g / L-21g / L, yeast extract 4g / L-6g / L, sodium diphosphate 2.6g / L-2.8g / L, citric acid 1g / L-1.3g / L and magnesium sulfate 0.5g / L-1.5g / L; the culture conditions of the yeast are: pH value 2.3-5.2, temperature 20℃-29℃, and culture time 36h-63h; The preparation method of the pineapple peel fermentation liquid includes the following steps: a1. Wash and peel the fresh pineapple peel, then cut it into small pieces of 7mm×7mm~11mm×11mm; b1. Place the small pieces of pineapple peel from step a1 into a fermentation tank, add distilled water at a material-to-liquid ratio of 1:4 to 1:9, and soak for 35 to 55 minutes to obtain the fermentation solution. c1. Add yeast to the fermentation solution in step b1, let it ferment for 36h~63h, and filter to obtain pineapple peel fermentation liquid; The preparation method of the Sapindus mukorossi fermentation broth includes the following steps: a2. Wash the soapberry and grind it into a fine powder of 95-137 mesh. Put the obtained soapberry powder into a fermentation tank, add distilled water, and soak it for 35-55 minutes to obtain the fermentation solution. b2. Add yeast to the fermentation solution from step a2, let it ferment for 36-63 hours, and then filter to obtain the Sapindus mukorossi fermentation broth.

2. The preparation method according to claim 1, characterized in that, In step (1), the mass ratio of the mixture of brown sugar, pineapple peel fermentation liquid, and soapberry fermentation liquid is 1:3 to 1:

5.

3. The preparation method according to claim 1, characterized in that, In step c1, the amount of yeast added is 26%-37% of the volume of the solution to be fermented, the temperature during static fermentation is 20℃-29℃, the pH value is 2.3-5.2, and the mesh size of the filter screen is 50 mesh.

4. The preparation method according to claim 1, characterized in that, In step a2, the ratio of Sapindus mukorossi powder to distilled water is 1:4 to 1:

9.

5. The preparation method according to claim 1, characterized in that, In step b2, the amount of yeast added is 26%-37% of the volume of the solution to be fermented, the temperature during static fermentation is 20℃-29℃, the pH value is 2.3-5.2, and the filtration is 50 mesh.

6. The preparation method according to claim 1, characterized in that, In step (3), the amount of yeast added is 28%-42% of the filtrate volume, the temperature during static fermentation is 20℃-29℃, and the pH value is 2.3-5.

2.

7. The probiotic preparation obtained by the method according to any one of claims 1 to 6.

8. The application of the probiotic preparation according to claim 7 in the preparation of food, medicine, and health products, wherein the health products include those with functions of enhancing immunity, regulating intestinal flora, promoting digestion, and relieving constipation.

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