Preparation method and application of probiotics capable of improving intestinal colonization efficiency

Abstract

This invention relates to the technical field of food processing, specifically to a method for preparing and applying probiotics that can improve intestinal colonization efficiency. The method uses corydalis yanhusuo protein as one of the reaction substrates and employs a two-stage fermentation process to cultivate probiotics. In the first stage of fermentation, the probiotics rapidly proliferate and produce spores. In the second stage of fermentation, they form a biofilm. The resulting probiotic fermentation broth is then dried and pulverized to produce the probiotic powder that improves intestinal colonization efficiency. This invention's preparation method is the first to employ a high-density fermentation mode that enhances the formation of a probiotic biofilm. By adjusting the initial fermentation medium ratio and fermentation parameters, the probiotics rapidly proliferate and produce spores. In the second stage of fermentation, the medium is supplemented and parameters are changed to achieve biofilm formation. This method solves the technical difficulties of slow proliferation and low biomass in single-stage biofilm static culture of probiotics, as well as the problems of poor processing tolerance and intestinal membrane adhesion of planktonic probiotics.

Description

Technical Field

[0001] This invention relates to the technical field of food processing, specifically to a method for preparing and applying probiotics that can improve intestinal colonization efficiency. Background Technology

[0002] The human body possesses a highly complex innate immune system that can respond appropriately to the invasion of pathogens. This system helps the body resist the invasion of bacteria, viruses, and other pathogenic microorganisms, and promptly eliminates aging or diseased cells, providing a reliable guarantee for maintaining a balanced state in the complex and ever-changing internal and external environment. The various organs, tissues, cells, and molecules that perform immune functions form a vast and complex immune system, which, under the body's regulation, maintains a balanced state to sustain human health.

[0003] However, humanity's living environment and lifestyle have undergone tremendous changes. The pace of society has accelerated, and a large number of people are in a sub-healthy state. People generally suffer from insufficient sleep, are prone to mental tension, and experience health problems such as anxiety, insomnia, vivid dreams, poor concentration, and weakened immunity. An overly refined diet can also lead to nutritional imbalances. Excessive intake of fat and sugar, coupled with a lack of high-quality protein, vitamins, and minerals, can also cause a decline in immunity, leading to various chronic diseases and posing a significant challenge to the human immune system.

[0004] Selenium, as an essential trace element, plays a vital role in human health. Selenoproteins, in which selenium is a key component, possess excellent histological and enzymatic activities, including glutathione peroxidase (GPx), thioredoxin reductase (TRxR), and iodothyronine deiodinase (DIO). These are well-known antioxidants and catalysts for the secretion of active thyroid hormones. Selenium is also essential for maintaining a normal immune system, effectively resisting viral infections and the development of AIDS, while also improving sperm motility and reducing the risk of miscarriage. Furthermore, selenium has been shown to have significant effects in preventing cardiovascular disease and fighting cancer.

[0005] Probiotics are live microorganisms that provide health benefits to the body when given in sufficient quantities. Lactic acid bacteria and bifidobacteria are important multifunctional probiotics in food. Their inherent properties, such as antibacterial activity and the secretion of various vitamins and extracellular polysaccharides, make them highly distinctive functional foods or medicines. Most probiotics in the body reside in the gut, so the key to their beneficial effects is successful colonization within the gut. A crucial step in probiotic colonization is adhesion to intestinal mucus. Goblet cells in the intestinal mucosal epithelium secrete mucus to cover the intestine, forming a mucus layer containing mucin, nutrients, etc., which is an important site for bacterial colonization. Reports indicate that most probiotics are excreted from the colon in feces shortly after oral administration and cessation of consumption, often resulting in poor clinical outcomes, mainly due to their inability to effectively colonize the gut. However, colonization is not easy. Oral administration is a common method of probiotic intake, but due to the harsh gastrointestinal environment, its bioavailability is generally low.

[0006] Biofilms are closed substrates produced by bacterial populations to adhere to an interface, where bacteria attach to each other, forming a form of communal survival for the bacterial community. This substrate is composed of extracellular polymers secreted by the bacteria themselves, mainly exopolysaccharides (EPS), proteins, and extracellular deoxyribonucleic acid (eDNA). Biofilms can form at any interface.

[0007] Most probiotics have the characteristic of forming biofilms. Utilizing biofilms can make probiotics more likely to survive and colonize in the body, thereby improving the efficacy and ability to fight pathogens, showing good research and application prospects. At present, most research focuses on how to remove the biofilm of harmful microorganisms, while research on how to enhance the biofilm of beneficial microorganisms is still very scarce. Summary of the Invention

[0008] One of the objectives of this invention is to provide a method for preparing probiotics that can improve intestinal colonization efficiency. Through an innovative two-stage organic selenium-rich fermentation mode, the method increases the proliferation rate of probiotics and effectively forms a biofilm. The organic selenium-rich fermentation and biofilm protection enhance the resilience of probiotics, solving the problems of significant loss of effective live bacteria in product manufacturing and processing and the low colonization efficiency of probiotics on the intestinal mucosa. At the same time, it can quantitatively supplement the daily selenium requirement of the human body, and synergistically enhance the beneficial effects of selenium and probiotics on improving the human gut, boosting immunity, and preventing and fighting cancer.

[0009] The second objective of this invention is to provide an application of probiotics that can improve intestinal colonization efficiency, specifically for the preparation of organic selenium nutritional supplements.

[0010] One of the objectives of this invention is achieved through a method for preparing probiotics that can improve intestinal colonization efficiency. This method uses corydalis yanhusuo protein as one of the reaction substrates and employs a two-stage fermentation process to cultivate probiotics. In the first stage of fermentation, the probiotics are rapidly multiplied and spores are produced. In the second stage of fermentation, the probiotics are biofilm-forming. The resulting probiotic fermentation broth is then dried and pulverized to produce the probiotic powder that can improve intestinal colonization efficiency.

[0011] Preferably, the procedure specifically includes the following steps:

[0012] (1) Cultivation of probiotics: The freeze-dried probiotic powder was activated into a seed fermentation broth and inoculated into a modified nutrient broth liquid culture medium. The initial pH was adjusted to 5.7-6.5, and the broth was cultured at 32-40℃ and 80-150r / min for 12-24h to complete the initial fermentation. Then, the next fermentation stage was carried out by supplementing the modified nutrient broth culture medium, adjusting the pH to 6.8-7.2, and then incubating at 30-38℃ for 6-18h to obtain the probiotic fermentation broth.

[0013] (2) Preparation of probiotic powder: The probiotic fermentation liquid obtained in step (1) is centrifuged to obtain a mixture of selenium-enriched probiotic bodies, and a low-temperature protectant is added. The probiotic solids are obtained by freeze drying and then crushed and ground into powder to obtain the probiotics that can improve intestinal colonization efficiency.

[0014] In step (1), violet leaf broken rice protein is used as one of the reaction substrates in the modified nutrient broth liquid culture medium.

[0015] Preferably, in step (1), the probiotics used are at least one of Lactobacillus plantarum, Bifidobacterium, and Bacillus coagulans. When compound probiotics are used, the inoculation amount of each strain is the same.

[0016] Preferably, in step (1), the initial nutrient broth liquid culture medium consists of the following components: 1.5–4.0 g of beef extract, 5.0–10.0 g of peptone, 2.0–5.5 g of violet leaf and rice porridge protein powder, 3.0–8.0 g of glucose, 1.5–5.0 g of NaCl, 0.5–2 g of K2HPO4, and 0.04–0.06 g of MnSO4·H2O per 1 L of distilled water.

[0017] The components of the supplemental nutrient broth liquid culture medium are as follows: dissolve 1-3.0g of beef extract, 2.0-6.0g of peptone, 1.0-3.0g of violet leaf and rice porridge protein powder, and 2.0-5.0g of sucrose in 1L of distilled water.

[0018] Preferably, in step (1), the selenium content of the violet leaf crushed rice protein powder is 800-2200 ppm, the protein content is ≥40%, and the selenocysteine ​​content is ≥70%.

[0019] Preferably, in step (2), the cryoprotectant is one or more of sucrose, lactose, maltodextrin, and skim milk powder; the amount of cryoprotectant added is 15% to 25% of the mass of the selenium-enriched probiotic mixture.

[0020] The second objective of this invention is achieved by applying the probiotic prepared by the aforementioned method, which can improve intestinal colonization efficiency, to the preparation of an organic selenium nutritional supplement.

[0021] Preferably, the probiotic powder, prebiotics, selenium-enriched plant powder, and excipients are mixed in a certain proportion and then directly compressed into tablets or directly mixed and packaged to make the organic selenium nutritional supplement.

[0022] Preferably, the selenium-enriched plant powder is one or more of selenium-enriched corydalis leaf powder, selenium-enriched broccoli, selenium-enriched cabbage, and selenium-enriched soybean; the prebiotic is one or more of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, and inulin; and the excipient is one or more of sorbitol, mannitol, erythritol, and mannosaccharides.

[0023] Preferably, the organic selenium nutritional supplement, by weight percentage, comprises 5%–15% probiotic powder, 15%–35% selenium-enriched plant powder, 30%–50% prebiotics, and 10%–25% excipients; the organic selenium nutritional supplement contains 20–60 μg / g of selenium and has a live probiotic count of 1 × 10⁻⁶. 6 ~1×10 8 CFU / g.

[0024] The preparation method of this invention uses corydalis yanhusuo protein as one of the reaction substrates and employs a two-stage fermentation process to cultivate probiotics. In the first stage of fermentation, the probiotics rapidly proliferate and produce spores. In the second stage of fermentation, the probiotics are biofilm-formed. The resulting probiotic fermentation broth is then dried and pulverized to produce the probiotic powder that enhances intestinal colonization efficiency. This method increases the probiotic proliferation rate and effectively forms a biofilm. Simultaneously, the biofilm protects and enhances the probiotics' resilience, solving the problems of significant loss of viable bacteria in the production and processing of probiotics and their products, as well as low colonization efficiency on the intestinal mucosa.

[0025] The application of this invention, combined with a rational formulation, can simultaneously achieve quantitative supplementation of the daily selenium requirement of the human body, and synergistically enhance the beneficial effects of selenium and probiotics on improving the human gut, boosting immunity, and preventing and fighting cancer.

[0026] The present invention has the following advantages and beneficial effects:

[0027] (1) The preparation method of the present invention adopts a high-density fermentation mode that can improve the formation of biofilm of probiotics for the first time. By adjusting the ratio of the culture medium and fermentation parameters in the first stage of fermentation, the probiotics can rapidly multiply and produce spores. In the second stage of fermentation, the culture medium is supplemented and the parameters are changed to make the probiotics biofilm. This solves the technical difficulties of slow proliferation and low biomass of probiotics in the static culture of one-stage biofilm. It also solves the problems of poor processing tolerance and poor intestinal adhesion of traditional planktonic probiotics.

[0028] (2) The innovative preparation method of the present invention uses selenocysteine ​​as the main selenium form in the selenocyanin culture medium to replace part of the nitrogen source, which is beneficial to accelerate the proliferation of probiotics, improve the stress resistance of probiotics, and upregulate the expression of the luxS gene to stimulate the formation of probiotic biofilm in a targeted manner, thus solving the problems of poor processing tolerance and poor intestinal adhesion of traditional planktonic probiotics.

[0029] (3) In this invention, the loss of effective live bacteria in the drying and preparation processing of biofilm-enriched selenium probiotics is effectively improved, while the use of excipients such as cryoprotectants and carriers can be reduced.

[0030] (4) In this invention, the highly biofilm-bound probiotics and their products can be retained to the maximum extent and reach the intestinal mucosa and adhere efficiently during the digestion process. The selected selenoprotein and prebiotic components can promote the establishment of a dominant reproductive position of probiotics in the intestine, inhibit the growth of harmful microorganisms, regulate the intestinal flora and improve intestinal health.

[0031] (5) The application of this invention fully utilizes the synergistic effect of selenium and probiotics on human health from product preparation to efficacy, providing an effective probiotic-containing organic selenium nutritional supplement, filling the relevant market gap and with broad market prospects. Attached Figure Description

[0032] Figure 1 This is a graph showing the relative abundance of species at the phylum level in Examples 1-3 and Comparative Examples 1-3;

[0033] Figure 2 The bar chart shows the expression levels of the luxS gene in Examples 1-3 and Comparative Examples 1-3. Detailed Implementation

[0034] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0035] Implementation Example 1

[0036] A method for preparing probiotics that can improve intestinal colonization efficiency includes the following steps:

[0037] 1. Probiotic culture: Lactobacillus plantarum and Bacillus coagulans lyophilized powder were activated into seed fermentation broth and inoculated into modified nutrient broth liquid culture medium at a 1:1 ratio. The fermentation tank was placed at 38℃, the initial pH was adjusted to 6.2, and the culture was carried out for 20 hours at a stirring speed of 150 r / min to complete the first stage of fermentation. Then, the second stage of fermentation was carried out. Modified nutrient broth culture medium was added, the pH was adjusted to 7.0, and the culture was continued at 37℃ for 18 hours to obtain probiotic fermentation broth.

[0038] The initial nutrient broth liquid culture medium is as follows: 3.0g beef extract, 6.0g peptone, 3.0g corydalis yanhusuo protein powder, 5.0g glucose, 5.0g NaCl, 2.0g K2HPO4, and 0.04g MnSO4·H2O are dissolved in 1L of distilled water.

[0039] Supplemental nutrient broth liquid culture medium: Dissolve 3.0g beef extract, 2.0g peptone, 2.0g violet leaf and rice porridge protein powder, and 4.0g sucrose in 1L of distilled water.

[0040] The violet leaf and rice cereal protein powder contains 2000 ppm selenium, 50% protein, and 75% selenocysteine.

[0041] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. Maltodextrin (7.5% by mass) and skim milk powder (12% by mass) are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0042] The probiotics prepared in this embodiment are used to prepare a probiotic-containing organic selenium nutritional supplement, which, by weight percentage, comprises: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched corydalis leaf and rice straw powder; 25% fructooligosaccharides; 20% xylooligosaccharides; 5% inulin; 10% erythritol; and 5% mannose. These components are directly mixed and packaged to form the final product, which has a selenium content of 50 μg / g and a probiotic viable count of 1 × 10⁻⁶. 7 CFU / g.

[0043] Implementation Example 2

[0044] A method for preparing probiotics that can improve intestinal colonization efficiency includes the following steps:

[0045] 1. Probiotic culture: Lactobacillus plantarum, Bacillus coagulans, and Bifidobacterium lyophilized powder were activated into seed fermentation broths and inoculated into modified nutrient broth liquid culture medium at a ratio of 1:1:1. The fermentation tank was placed at 38℃, the initial pH was adjusted to 6.2, and the culture was carried out for 24 hours at a stirring speed of 150 r / min to complete the primary fermentation. Then, the next fermentation stage (secondary fermentation) was carried out. Modified nutrient broth culture medium was added, the pH was adjusted to 7.0, and the culture was continued at 37℃ for 16 hours to obtain the probiotic fermentation broth.

[0046] The initial nutrient broth liquid culture medium is as follows: 3.0g beef extract, 5.0g peptone, 4.0g corydalis yanhusuo protein powder, 5.0g glucose, 5.0g NaCl, 2.0g K2HPO4, and 0.04g MnSO4·H2O are dissolved in 1L of distilled water.

[0047] Supplemental nutrient broth liquid culture medium: Dissolve 3.0g beef extract, 2.0g peptone, 2.0g violet leaf and rice porridge protein powder, and 4.0g sucrose in 1L of distilled water.

[0048] The violet leaf and rice cereal protein powder contains 2000 ppm selenium, 50% protein, and 75% selenocysteine.

[0049] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. Maltodextrin (7.5% by mass) and skim milk powder (12% by mass) are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0050] The probiotics prepared in this embodiment are used to prepare a probiotic-containing organic selenium nutritional supplement, which, by weight percentage, comprises: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched corydalis leaf and rice straw powder; 25% fructooligosaccharides; 20% xylooligosaccharides; 5% inulin; 10% erythritol; and 5% mannose. These components are directly mixed and packaged to form the final product, which has a selenium content of 50 μg / g and a probiotic viable count of 1 × 10⁻⁶. 7 CFU / g.

[0051] Implementation Example 3

[0052] A method for preparing probiotics that can improve intestinal colonization efficiency includes the following steps:

[0053] 1. Probiotic culture: Lactobacillus plantarum and Bacillus coagulans lyophilized powder were activated into seed fermentation broth and inoculated into modified nutrient broth liquid culture medium at a ratio of 1:1. The fermentation tank was placed at 38℃, the initial pH was adjusted to 6.2, and the culture was carried out for 24 hours at a stirring speed of 150 r / min to complete the primary fermentation. Then, the next fermentation stage (second stage fermentation) was carried out. Modified nutrient broth culture medium was added, the pH was adjusted to 7.0, and the culture was continued at 37℃ for 16 hours to obtain probiotic fermentation broth.

[0054] The initial nutrient broth liquid culture medium is as follows: 3.0g beef extract, 5.0g peptone, 4.0g corydalis yanhusuo protein powder, 5.0g glucose, 5.0g NaCl, 2.0g K2HPO4, and 0.04g MnSO4·H2O are dissolved in 1L of distilled water.

[0055] Supplemental nutrient broth liquid culture medium: Dissolve 3.0g beef extract, 2.0g peptone, 2.0g violet leaf and rice porridge protein powder, and 4.0g sucrose in 1L of distilled water.

[0056] The violet leaf and rice cereal protein powder contains 2000 ppm selenium, 50% protein, and 75% selenocysteine.

[0057] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. 5% sucrose, 5% maltodextrin, and 10% skim milk powder (by mass percentage) of the selenium-enriched probiotic body mixture are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0058] The probiotics prepared in this embodiment were used to prepare a probiotic-containing organic selenium nutritional supplement, which, by weight percentage, comprises: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched corydalis leaf and rice straw powder; 25% fructooligosaccharides; 20% xylooligosaccharides; 5% inulin; 10% erythritol; and 5% mannose. These components were directly mixed and packaged to form the final product, which has a selenium content of 60 μg / g and a probiotic viable count of 1 × 10⁻⁶. 8 CFU / g.

[0059] Comparative Example 1

[0060] A method for preparing an organic selenium nutritional supplement includes the following steps:

[0061] 1. Probiotic culture: Lactobacillus plantarum and Bacillus coagulans lyophilized powder were activated into seed fermentation broth and inoculated into commercially available nutrient broth liquid culture medium at a ratio of 1:1. 0.027 g of Na2SeO3 was dissolved in each 1 L of culture medium. The mixture was placed in a fermenter at 38 °C, the initial pH was adjusted to 7.0, and the mixture was cultured at a stirring speed of 150 r / min for 38 h to obtain the probiotic fermentation broth.

[0062] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. Maltodextrin (7.5% by mass) and skim milk powder (12% by mass) are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0063] The probiotics prepared using this comparative example were used to prepare a probiotic-containing organic selenium nutritional supplement. By weight percentage, it comprises: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched corydalis leaf and water chestnut powder; 25% fructooligosaccharides; 20% xylooligosaccharides; 5% inulin; 10% erythritol; and 5% mannose. These components were directly mixed and packaged to form the final product. Its selenium content is 50 μg / g, and the live probiotic count is 1 × 10⁻⁶. 7 CFU / g.

[0064] Comparative Example 2

[0065] A method for preparing an organic selenium nutritional supplement includes the following steps:

[0066] 1. Probiotic culture: Lactobacillus plantarum and Bacillus coagulans lyophilized powder were activated into seed fermentation broth and inoculated into modified nutrient broth liquid culture medium at a ratio of 1:1. The fermentation tank was placed at 38℃, the initial pH was adjusted to 6.2, and the culture was carried out for 24 hours at a stirring speed of 150 r / min to complete the primary fermentation. Then, the next fermentation stage (second stage fermentation) was carried out. Modified nutrient broth culture medium was added, the pH was adjusted to 7.0, and the culture was continued at 37℃ for 16 hours to obtain probiotic fermentation broth.

[0067] The initial nutrient broth liquid culture medium is: 5.0g beef extract, 7.0g peptone, 5.0g glucose, 5.0g NaCl, 2.0g K2HPO4, and 0.04g MnSO4·H2O dissolved in 1L of distilled water.

[0068] Supplemental nutrient broth liquid culture medium: Dissolve 3.0g beef extract, 4.0g peptone, and 4.0g sucrose in 1L of distilled water.

[0069] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. Maltodextrin (7.5% by mass) and skim milk powder (12% by mass) are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0070] The probiotics prepared in this embodiment were used to prepare a probiotic-containing organic selenium nutritional supplement, which, by weight percentage, comprises: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched corydalis leaf and rice water chestnut powder; 25% fructooligosaccharides; 20% xylooligosaccharides; 5% inulin; 10% erythritol; and 5% mannose oligosaccharides. These components are directly mixed and packaged to form the final product. The selenium content is 50 μg / g, and the live probiotic count is 1 × 10⁻⁶. 7 CFU / g.

[0071] Comparative Example 3

[0072] A method for preparing an organic selenium nutritional supplement includes the following steps:

[0073] 1. Probiotic culture: Lactobacillus plantarum and Bacillus coagulans lyophilized powder were activated into seed fermentation broth and inoculated into modified nutrient broth liquid culture medium at a ratio of 1:1. The fermentation tank was placed at 38℃, the initial pH was adjusted to 6.2, and the culture was carried out for 24 hours at a stirring speed of 150 r / min to complete the primary fermentation. Then, the next fermentation stage (second stage fermentation) was carried out. Modified nutrient broth culture medium was added, the pH was adjusted to 7.0, and the culture was continued at 37℃ for 16 hours to obtain probiotic fermentation broth.

[0074] The initial nutrient broth liquid culture medium is: 3.0g beef extract, 6.0g peptone, 3.0g casein hydrolysate, 5.0g fructose, 5g NaCl, 2g K2HPO4, and 0.04g MnSO4·H2O dissolved in 1L of distilled water.

[0075] Supplemental nutrient broth liquid culture medium: Dissolve 3.0g beef extract, 2.0g peptone, 2.0g casein hydrolysate, and 4.0g fructose in 1L of distilled water.

[0076] 2. Preparation of probiotic powder: The probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies. Maltodextrin (7.5% by mass) and skim milk powder (12% by mass) are added as cryoprotectants. The probiotic solids are obtained by freeze-drying and then pulverized and ground into probiotic powder.

[0077] The probiotic prepared in this embodiment is used to prepare a probiotic organic selenium nutritional supplement, which, calculated by mass percentage, includes: 10% selenium-enriched probiotic powder; 10% selenium-enriched broccoli powder; 10% selenium-enriched cabbage powder; 5% selenium-enriched Cardamine violifolia powder; 25% fructo-oligosaccharide; 20% xylo-oligosaccharide; 5% inulin; 10% erythritol; 5% mannan oligosaccharide, which are directly mixed and bagged to form the final product. Its selenium content is 50 μg / g, and the viable count of probiotics is 1×10 7 CFU / g.

[0078] The viable counts of the selenium-enriched probiotic liquids fermented in Examples 1-3 and the probiotic liquids fermented in Comparative Examples 1-3 were measured according to "GB 4789.2-2016 National Food Safety Standard Microbiological Examination of Foods - Determination of Total Number of Colonies". The formation of biofilms was measured by the crystal violet staining method. The OD value reflects the firmness of the adhesion of the biofilm to the contact surface. According to the critical ODc value (the OD value obtained by adding the average value of the blank wells to three times its standard deviation), the biofilms can be classified: OD ≤ ODc is non-adhesion (0), ODc < OD ≤ 2ODc is weak adhesion (+), 2ODc < OD ≤ 4ODc is medium adhesion (++), OD > 4ODc is strong adhesion (+++).

[0079] The viable counts of the probiotic organic selenium nutritional supplements obtained in Examples 1-3 and Comparative Examples 1-3 were measured by in vitro simulated digestion in artificial gastric and intestinal juices.

[0080] The probiotic organic selenium nutritional supplements obtained in Examples 1-3 and Comparative Examples 1-3 were fully dissolved and then used for rat gavage experiments. One week later, rat fecal samples were taken, and the bacterial flora composition of the rat intestines was analyzed by high-throughput 16srDNA sequencing. The relative abundance map of species at the phylum level is as Figure 1 shown. It can be seen that after feeding with the probiotic organic selenium nutritional supplement of the embodiment of the present invention, the main probiotics in the rat intestines are Firmicutes, Bacteroidota, Proteobacteria, and Actinobacteria. The microbial diversity of the example group is higher than that of the control group, and the relative abundance of Bacteroidota, which is mostly pathogenic bacteria, is significantly reduced. This shows that the probiotic organic selenium nutritional supplement of the present invention can be efficiently colonized in the animal intestine.

[0081] The fermented bacterial liquids of each treatment were centrifuged at 4°C and 12,000 r / min for 2 min, and the supernatant was discarded to collect the bacterial cells. According to the instructions of the Tiangen Bacterial Total RNA Extraction Kit (DP430), the total RNA of the above-mentioned bacterial cells was extracted respectively. Before reverse transcription, genomic DNA was removed, and then on ice bath according to The RT Reagent Kit with gDNA Eraser was used to mix the reagents and synthesize cDNA, which was then stored at 4°C. Using the single-stranded cDNA obtained from reverse transcription as a template, relative semi-quantitative analysis was performed according to the instructions of the mRNA selective PCR kit (TAKARA, Japan). The relative expression level of the luxS gene in different treatments was determined and analyzed using GeneTools fromSynGene software. Figure 2 The bar charts shown are of the expression levels of the luxS gene in Examples 1-3 and Comparative Examples 1-3. As can be seen from the figures, the gene expression detection results of the fermentation broth show that, compared with the comparative examples, the expression of the probiotic LuxS gene in the fermentation broth of the examples was significantly upregulated. The luxS gene can encode the synthesis of LuxS protein, and LuxS protein is a key enzyme in the synthesis of AI-2. The amount of bacteriocin synthesis, tolerance, and adhesion characteristics of bacteria can be positively regulated by inducer-2 (autoinduction-2, AI-2). Therefore, the upregulation of the expression of the probiotic LuxS gene in the examples led to the formation of a highly biofilm-like state in the probiotics.

[0082] The results are shown in Table 1 below:

[0083] Table 1. Summary of indicators of probiotics prepared in Examples 1-3 and Comparative Examples 1-3

[0084]

[0085]

[0086] The data in Table 1 shows that:

[0087] Compared with conventional fermentation culture with the same concentration of sodium selenite (Ratio 1), comparative ratio 2 without corydalis yanhusuo protein fermentation nitrogen source, and comparative ratio 3 fermentation in conventional biofilm formation promoting medium (fructose + casein hydrolysate), the preferred fermentation mode of the present invention significantly increases the biomass of probiotics and forms a more adhesive biofilm. Simultaneously, in vitro gastrointestinal digestion simulation tests of the product formulations showed that the effective viable bacteria count was effectively retained after in vitro digestion, with a survival rate 5-10 times that of the comparative ratios. A one-week oral gavage test on rats with different formulations showed that the intestinal flora of the rats in the example group was effectively regulated, with the highest relative abundance of Firmicutes (a representative probiotic phylum) reaching 50%, generally more than twice that of the comparative treatment group. This indicates that the organic selenium-rich high-density fermentation mode of the present invention can significantly increase the biomass of probiotic fermentation and has good resistance to digestive juices, while effectively colonizing the intestinal mucosa of humans and animals.

[0088] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing a probiotic powder that can improve intestinal colonization efficiency, characterized in that: Specifically, the following steps are included: Cultivation of probiotics: The freeze-dried probiotic powder was activated into a seed fermentation broth and inoculated into an initial nutrient broth liquid culture medium. The initial pH was adjusted to 5.7-6.5 and cultured under stirring conditions to complete the initial fermentation. Then, the next fermentation stage was carried out by supplementing the nutrient broth liquid culture medium, adjusting the pH to 6.8-7.2, and allowing it to stand for culture to obtain the probiotic fermentation broth. Preparation of probiotic powder: The obtained probiotic fermentation liquid is dried and pulverized to prepare the probiotic powder that can improve intestinal colonization efficiency; The initial nutrient broth liquid culture medium and the supplementary nutrient broth liquid culture medium contain Viola yedoensis protein powder, which has a selenium content of 800~2200ppm, a protein content of ≥40%, and a selenocysteine ​​content of ≥70%. The probiotics are either two types: Lactobacillus plantarum and Bacillus coagulans, or three types: Lactobacillus plantarum, Bifidobacterium, and Bacillus coagulans.

2. The method for preparing probiotic powder that can improve intestinal colonization efficiency according to claim 1, characterized in that: Specifically, the following steps are included: Cultivation of probiotics: The freeze-dried probiotic powder was activated into a seed fermentation broth and inoculated into an initial nutrient broth liquid medium. The initial pH was adjusted to 5.7-6.5, and the broth was cultured at 32-40℃ and 80-150 r / min for 12-24 h to complete the initial fermentation. Then, the next fermentation stage was carried out by supplementing the nutrient broth liquid medium, adjusting the pH to 6.8-7.2, and incubating at 30-38℃ for 6-18 h to obtain the probiotic fermentation broth. Preparation of probiotic powder: The obtained probiotic fermentation broth is centrifuged to obtain a mixture of selenium-enriched probiotic bodies, and a low-temperature protectant is added. The probiotic solids are obtained by freeze drying, and then crushed and ground into powder to obtain the probiotic powder that can improve intestinal colonization efficiency.

3. The method for preparing probiotic powder that can improve intestinal colonization efficiency according to claim 1 or 2, characterized in that: The initial nutrient broth liquid culture medium consists of the following components: 1.5-4.0g beef extract, 5.0-10.0g peptone, 2.0-5.5g corydalis yanhusuo protein powder, 3.0-8.0g glucose, 1.5-5.0g NaCl, 0.5-2g K2HPO4, and 0.04g-0.06g MnSO4·H2O dissolved in each 1L of distilled water. The supplementary nutrient broth liquid culture medium consists of the following components: 1-3.0g of beef extract, 2.0-6.0g of peptone, 1.0-3.0g of violet leaf and rice porridge protein powder, and 2.0-5.0g of sucrose dissolved in 1L of distilled water.

4. The method for preparing probiotic powder that can improve intestinal colonization efficiency according to claim 2, characterized in that: The cryoprotectant is one or more of sucrose, lactose, maltodextrin, and skim milk powder; the amount of cryoprotectant added is 15% to 25% of the mass of the selenium-enriched probiotic mixture.

5. The application of a probiotic powder prepared by the method described in any one of claims 1 to 4, which can improve intestinal colonization efficiency, characterized in that: The probiotic powder was used to prepare an organic selenium nutritional supplement.

6. The application according to claim 5, characterized in that: The probiotic powder, prebiotics, selenium-enriched plant powder, and excipients are mixed in a certain proportion and then directly compressed into tablets or directly mixed and packaged to produce the organic selenium nutritional supplement.

7. The application according to claim 6, characterized in that: The selenium-enriched plant powder is one or more of the following: selenium-enriched corydalis leaf powder, selenium-enriched broccoli, selenium-enriched cabbage, and selenium-enriched soybean; the prebiotic is one or more of the following: fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, and inulin; the excipient is one or more of the following: sorbitol, mannitol, erythritol, and mannosaccharides.

8. The application according to claim 6, characterized in that: The organic selenium nutrient supplement is 5-15% probiotic powder, 15-35% selenium-rich plant powder, 30-50% prebiotic, and 10-25% auxiliary materials in terms of mass percentage; the selenium content in the organic selenium nutrient supplement is 20-60 μg / g, and the viable cell count of the probiotic is 1×10 6 ~1×10 8 CFU / g.

Images