Prebiotic composition for proliferating bacillus subtilis and application thereof

Abstract

The invention discloses a prebiotic composition for proliferating bacillus subtilis and application of the prebiotic composition. The prebiotic composition comprises resistant dextrin and stachyose, wherein the content of the resistant dextrin is 6.2 to 80.9 parts, and the content of the stachyose is 19.1 to 93.8 parts. The bacillus subtilis is used as a research object, an in-vitro simulation fermentation method is adopted, a growth curve of microorganisms is detected in real time, the prebiotic composition capable of proliferating the bacillus subtilis in a synergistic interaction mode is screened out, the bacillus subtilis is efficiently proliferated through combination of resistant dextrin and stachyose, the bacillus subtilis can be specifically proliferated, and the bacillus subtilis can be efficiently proliferated. The effects of regulating the intestinal health, inhibiting harmful bacteria in the intestinal tract and the like are achieved.

Description

Technical Field

[0001] The invention belongs to the field of functional foods, and in particular relates to a prebiotic composition for proliferating Bacillus subtilis and application thereof. Background Art

[0002] Prebiotics refer to carbohydrates that cannot be digested and utilized by the human body but can selectively promote the proliferation and metabolism of intestinal probiotics. Their essence is oligosaccharides and bifidobacteria, such as stachyose, oligofructose, oligogalactose, oligomannosaccharide, oligoxylose, and oligomaltolsaccharide. They provide an effective carbon source for endogenous probiotics, stimulate their growth and control them, and indirectly protect intestinal health and promote the absorption of nutrients. Generally, they will not produce immune reactions such as allergies. Prebiotics are non-organic living things that reach the intestines in an undigested form, so there is no problem with their viability.

[0003] Probiotics are microorganisms that are beneficial to humans and animals. The commonly known ones include Bacillus subtilis, Lactobacillus and Bifidobacterium. They are organic living organisms that need to pass the test of strong acids and bile salts. Only some of them can survive and reach the intestines, replenish probiotics, colonize the human intestines, inhibit the growth of harmful bacteria, and improve the intestinal microenvironment.

[0004] Therefore, how to promote its proliferation and industrialization has become the focus of current R&D workers.

[0005] At present, there is no report on the specific proliferation of Bacillus subtilis using resistant dextrin combined with stachyose. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the object of the present invention is to overcome the deficiencies in the prior art and provide a prebiotic composition for efficiently proliferating Bacillus subtilis.

[0009] In order to solve the above technical problems, the present invention provides the following technical solution: comprising resistant dextrin and stachyose; wherein, in parts by weight, the resistant dextrin is 6.2 to 80.9 parts, and the stachyose is 19.1 to 93.8 parts.

[0010] As a preferred embodiment of the composition of the present invention, the resistant dextrin is 13.7 to 73.4 parts, and the stachyose is 26.6 to 86.3 parts by weight.

[0011] As a preferred embodiment of the composition of the present invention, the resistant dextrin is 23.7 to 63.5 parts, and the stachyose is 36.5 to 76.3 parts by weight.

[0012] As a preferred embodiment of the composition of the present invention, the composition further comprises Bacillus subtilis.

[0013] As a preferred embodiment of the composition of the present invention, the number of viable Bacillus subtilis in the composition is not less than 1×10 9 CFU / g.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a prebiotic composition for proliferating Bacillus subtilis for use in preparing food and health food having the function of promoting the proliferation of Bacillus subtilis.

[0015] As a preferred embodiment of the present invention, the content of Bacillus subtilis in the food or health food is 1×10 9 CFU / 100g~1×10 12 CFU / 100g. As a preferred solution of the application of the present invention, it includes: adding the composition to food raw materials to prepare the food and health food finished products.

[0016] As a preferred embodiment of the application of the present invention, the food includes solid beverages, tablets, soft candies and capsules.

[0017] Another object of the present invention is to overcome the deficiencies in the prior art and provide a prebiotic composition for proliferating Bacillus subtilis for use in the preparation of a medicine for promoting the proliferation of Bacillus subtilis.

[0018] Beneficial effects of the present invention:

[0019] (1) The present invention provides a prebiotic composition for efficiently proliferating Bacillus subtilis. It is found that resistant dextrin combined with stachyose has a synergistic effect on the proliferation of Bacillus subtilis, which is better than any single material.

[0020] (2) The present invention uses Bacillus subtilis as the research object, adopts the method of in vitro simulated fermentation, detects the growth curve of microorganisms in real time, and screens out a prebiotic composition that can synergistically enhance the proliferation of Bacillus subtilis. By combining resistant dextrin with stachyose to efficiently proliferate Bacillus subtilis, Bacillus subtilis can be specifically proliferated to play a role in regulating intestinal health and inhibiting harmful intestinal bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0022] Figure 1 This is a comparison chart of the effects of different carbon sources on the proliferation of Bacillus subtilis in the examples of the present invention.

[0023] Figure 2 This is a diagram showing the effect of different carbon sources or carbon source combinations on the proliferation of Bacillus subtilis in the examples of the present invention. DETAILED DESCRIPTION

[0024] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0025] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0026] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0027] The probiotics used in the embodiments of the present invention are powders composed of Bacillus subtilis, wherein the Bacillus subtilis adopts a strain with a strain collection number of NRRLB-67989, which is commonly available on the market.

[0028] The prebiotics used in the embodiments of the present invention: isomaltooligosaccharide, mannooligosaccharide, galacto-oligosaccharide, stachyose, polydextrose, oligofructose, resistant dextrin, and other materials: lactitol, L-arabinose are all common commercial products.

[0029] The culture medium used in the embodiments of the present invention is prepared as follows:

[0030] (1) 0.1% peptone water: accurately weigh 1.0 g of peptone and dissolve it in 1000 mL of deionized water. Adjust the pH to 7.0 with hydrochloric acid / NaOH. Aliquot 10 mL into each test tube and sterilize it by high pressure steam at 121°C for 15 min for later use.

[0031] (2) Trace mineral solution: Accurately weigh 0.5 g sodium chloride, 0.9 g ferrous sulfate heptahydrate, 0.8 g manganese sulfate monohydrate, 0.08 g zinc sulfate heptahydrate, 0.08 g copper sulfate pentahydrate, and 0.08 g cobalt sulfate heptahydrate and add to 50 mL of deionized water.

[0032] (3) Proliferation liquid medium: Accurately weigh 5 g of yeast extract, 5 g of peptone, 20 g of D-glucose, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate, and 1 mL of trace mineral solution and dissolve them in 1000 mL of deionized water. Adjust the pH to 7.2 with hydrochloric acid / NaOH. Aliquot 10 mL into each test tube and sterilize by high-pressure steam at 121°C for 20 min for later use.

[0033] (4) Carbon-free fermentation liquid medium: Accurately weigh 0.5 g of peptone, 0.5 g of potassium dihydrogen phosphate, 0.5 g of potassium dihydrogen phosphate, 0.3 g of magnesium sulfate, and 1 mL of trace mineral solution and dissolve them in 1000 mL of deionized water. Adjust the pH to 7.2 with hydrochloric acid / NaOH and dispense 10 mL into each test tube. Add 200 mg of the corresponding different carbon sources to each test tube. After sufficient dissolution, adjust the pH to 7.2 and sterilize by high-pressure steam at 121°C for 20 min for use.

[0034] Example 1

[0035] Effects of lactitol, L-arabinose and common prebiotics on the proliferation of Bacillus subtilis:

[0036] (1) Methods of adding lactitol, L-arabinose and prebiotics:

[0037] Each test tube was filled with 10 mL of carbon-free fermentation liquid medium, and 200 mg of the corresponding different carbon sources (lactitol, L-arabinose and prebiotics, see Table 1) were added to each test tube. After being fully dissolved, the pH was adjusted to 7.2, and then sterilized by high-pressure steam at 121°C for 20 min for use;

[0038] The Bacillus subtilis strains stored in a freeze-drying tube at -80°C were inoculated into a proliferation liquid culture medium at a rate of 3.33% (v / v), and cultured in a shaking incubator at 37°C for 24 hours to obtain a strain stock solution;

[0039] Centrifuge the bacterial stock solution at 1000 rpm for 10 min at 4°C, discard the supernatant, re-dissolve in sterile water, shake thoroughly and mix evenly, repeat twice, wash away the proliferation medium, and add the same volume of sterile water;

[0040] The obtained bacterial liquid was inoculated into the fermentation liquid medium (with different carbon sources, see Table 1) at an inoculum volume of 1% (v / v), and cultured at 37°C for 24 h. Each group of samples was measured in triplicate.

[0041] (2) Proliferation-promoting effect detection:

[0042] The OD600nm value of the fermentation liquid was used as the evaluation index: 200 μL of the fermentation liquid was added to a 96-well plate, and then its absorbance at 600 nm was measured using an ELISA reader. The temperature was set to 37°C, the time was set to 24 h, and the OD value was measured every 30 min.

[0043] Draw the change curve of OD600nm under different fermentation times.

[0044] The test results are shown in Table 1 below.

[0045] Table 1 Effect of different carbon sources on the proliferation of Bacillus subtilis

[0046]

[0047]

[0048] The results are shown in Table 1 and Figure 1 It can be seen that the proliferation effect of the three materials of commonly used prebiotics, resistant dextrin, stachyose and oligofructose, was significantly better than that of the positive control group (glucose group) and other test groups when cultured for 24 hours.

[0049] Example 2

[0050] Promoting effects of different carbon sources or carbon source combinations on the proliferation of Bacillus subtilis:

[0051] The experimental method is the same as that in Example 1, and the specific experimental group settings are as follows (Table 2):

[0052] Experimental group 1 was 1% resistant dextrin + 1% stachyose;

[0053] Experimental group 2 was 1% resistant dextrin: 1% oligofructose;

[0054] Experimental group 3 was 1% stachyose: 1% oligofructose;

[0055] Test group 4 was 2% resistant dextrin;

[0056] Experimental group 5 was 2% stachyose;

[0057] Test group 6 was 2% fructo-oligosaccharide.

[0058] Table 2 Proliferation promoting effect of different carbon sources or carbon source combinations on Bacillus subtilis

[0059] Experimental materials 0h 4h 8h 12h 16h 20h 24h 1% resistant dextrin + 1% stachyose 0.0034 0.0061 0.1185 0.1572 0.1867 0.1998 0.2073 1% resistant dextrin + 1% oligofructose 0.0089 0.0021 0.1057 0.1489 0.1520 0.1644 0.1734 1% Stachyose + 1% Fructooligosaccharide 0.0025 0.0050 0.1141 0.1492 0.1766 0.1888 0.1944 2% resistant dextrin 0.0039 0.0083 0.0819 0.1326 0.1510 0.1583 0.1598 2% Stachyose 0.0033 0.0055 0.0833 0.1329 0.1514 0.1716 0.1747 2% Fructo-oligosaccharides 0.0064 0.0087 0.1322 0.1548 0.1698 0.1812 0.1831

[0060] See the figure for the effect of different carbon sources or carbon source combinations on the proliferation of Bacillus subtilis. Figure 2From the above results, it can be seen that the proliferation effect of 1% resistant dextrin combined with 1% stachyose on Bacillus subtilis is better than that of any of the two materials alone, which shows that the two have a synergistic effect on the proliferation of Bacillus subtilis. However, the proliferation effect of 1% resistant dextrin combined with 1% oligofructose on Bacillus subtilis is lower than that of any of the two materials alone, indicating that there is no synergistic effect between the two.

[0061] Note: The experiment of Example 1 and the experiment of Example 2 were done separately. Table 1 was done first, and then Table 2. The two experiments were relatively independent. The bacterial strains selected for the experiment were stored in freeze-dried tubes at -80°C. In the first experiment, the bacterial strain activity was the highest. After the experiment in Table 1 was completed, the freeze-dried tube was put back into the refrigerator. After a period of time, the same freeze-dried tube was taken out again to do the experiment in Table 2. The freeze-dried tube experienced freezing - room temperature - freezing again. The bacterial strain activity was affected by the first experiment and the bacterial strain activity decreased to a certain extent. Therefore, when conducting the experiment in Table 2, the OD value decreased due to the decrease in bacterial strain activity.

[0062] Example 3

[0063] Optimal point fitting:

[0064] 3.1 Obtaining the Optimal Ratio Experimental Group Setting: Tests were conducted according to the five groups of composition ratios shown in Table 3, with three replicates in each group.

[0065] According to Minitab software, a two-material mixing experiment design was performed, as shown in Table 3. Corresponding experiments were performed according to the corresponding proportions to obtain the OD600 values ​​corresponding to the five groups of experiments. The experimental results were brought into the software to perform mixture analysis, including analysis of mixture design, response tracking diagram and response optimizer, so as to obtain the optimal proportion. Finally, the proportion was confirmed to be the optimal addition proportion through experimental verification. The specific example is shown in Experimental Example 5 in Table 4.

[0066] Experimental group setting: The test was conducted according to the composition ratios of the three groups shown in the following table, with three replicates in each group.

[0067] Table 3

[0068] Experimental Group Resistant dextrin addition ratio Stachyose addition ratio 1 100％ 0 2 0 100％ 3 50％ 50％ 4 75％ 25％ 5 25％ 75％

[0069] Training system:

[0070] (1) Anaerobic culture in small bottles, fermentation liquid medium without carbon source + 2% mixture of resistant dextrin and stachyose;

[0071] (Fitting point + optimal boundary conditions) The optimal boundary conditions are the five groups of mixture ratios corresponding to Table 3. The optimal ratios are obtained by fitting the corresponding points of the experimental results based on the analysis of the mixture design, response tracking diagram and response optimizer analysis of the five groups of experimental results. (5 ratios, see Table 3)

[0072] (2) Cultivation with automatic growth curve instrument, liquid medium without carbon source + mixture of 2% resistant dextrin and stachyose. (Fitting point + optimal boundary conditions) (5 ratios, see Table 3).

[0073] Detection indicators: real-time growth curve (automatic growth curve instrument, uninoculated system is required as control);

[0074] According to the results in Table 3, the best combination at a carbon source concentration of 2% was fitted to a ratio of 43.43%:56.57%.

[0075] 3.2 Experiment on the ratio of stachyose and resistant dextrin in the composition

[0076] Experimental steps:

[0077] (1) Addition method of resistant dextrin and stachyose:

[0078] Each test tube was filled with 10 mL of carbon-free fermentation liquid medium, and 200 mg of the corresponding different carbon sources (resistant dextrin and stachyose, see Table 4) were added to each test tube. After being fully dissolved, the pH was adjusted to 7.2, and then sterilized by high-pressure steam at 121°C for 20 min for later use;

[0079] The Bacillus subtilis strains stored in a freeze-drying tube (the same tube as the freeze-drying tube used in Example 1 and Example 2) in a -80°C refrigerator were inoculated into a proliferation liquid culture medium at an inoculum rate of 3.33% (v / v), and cultured in a shaking incubator at 37°C for 24 hours to obtain a strain mother solution;

[0080] Centrifuge the bacterial stock solution at 1000 rpm for 10 min at 4°C, discard the supernatant, re-dissolve in sterile water, shake thoroughly and mix evenly, repeat twice, wash away the proliferation medium, and add the same volume of sterile water;

[0081] The obtained bacterial liquid was inoculated into the fermentation liquid medium (with different carbon sources, see Table 4) at an inoculum volume of 1% (v / v) and cultured at 37°C for 24 h. Each group of samples was measured in triplicate.

[0082] (2) Proliferation-promoting effect detection:

[0083] The OD600nm value of the fermentation liquid was used as the evaluation index: 200 μL of the fermentation liquid was added to a 96-well plate, and then its absorbance at 600 nm was measured using an ELISA reader. The temperature was set to 37°C, the time was set to 24 h, and the OD value was measured every 30 min.

[0084] Draw the change curve of OD600nm under different fermentation times.

[0085] The test results are shown in Table 4 below.

[0086] Table 4

[0087]

[0088]

[0089] The total amount of resistant dextrin and stachyose is 200 mg (in a 10 ml system).

[0090] It can be seen from Table 4 that the OD value corresponding to Experimental Example 5 is the highest, which is the optimal addition ratio, and is consistent with the results of software analysis.

[0091] Preferably, the mass ratio of resistant dextrin and stachyose in the composition is (6.2-80.9): (19.1-93.8), at which time, the corresponding OD value of the composition is increased by 5% compared with that of the single material of stachyose. More preferably, the mass ratio of resistant dextrin and stachyose in the composition is (13.7-73.4): (26.6-86.3), at which time, the corresponding OD value of the composition is increased by 10% compared with that of the single material of stachyose. Further optimized, the mass ratio of resistant dextrin and stachyose in the composition is (23.7-63.5): (36.5-76.3), at which time, the corresponding OD value of the composition is increased by 15% compared with that of the single material of stachyose.

[0092] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A composition, characterized in that: including, resistant dextrin and stachyose; Wherein, in parts by weight, the resistant dextrin is 6.2 to 80.9 parts, and the stachyose is 19.1 to 93.8 parts.

2. The composition according to claim 1, characterized in that: In parts by weight, the resistant dextrin is 13.7 to 73.4 parts, and the stachyose is 26.6 to 86.3 parts.

3. The composition according to claim 2, characterized in that: In parts by weight, the resistant dextrin is 23.7 to 63.5 parts, and the stachyose is 36.5 to 76.3 parts.

4. The composition according to any one of claims 1 to 3, characterized in that: The composition also includes Bacillus subtilis.

5. The composition according to claim 4, characterized in that: The viable count of the Bacillus subtilis is not less than 1×10 9 CFU / g.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of food or health food having the function of promoting the proliferation of Bacillus subtilis.

7. The use according to claim 6, characterized in that: The content of Bacillus subtilis in the food or health food is 1×10 9 CFU / 100g~1×10 12 CFU / 100g.

8. The use according to claim 6, characterized in that: include, The composition is added into food raw materials to prepare the food and health food finished products.

9. The use according to claim 6, characterized in that: The food includes solid beverages, tablets, soft candies and capsules.

10. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicine for promoting the proliferation of Bacillus subtilis.

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