Post-combination prebiotic ferment powder and application thereof

Abstract

The invention discloses combined prebiotic ferment powder and application thereof. The combined prebiotics ferment powder is a dried product obtained after the compound lactic acid bacteria composition is fermented; wherein the cell number ratio of each fermentation strain is as follows: LRH10 (12 + / -0.5%), LPC12 (2 + / -0.5%), LF26 (26.3 + / -0.5%), ST30 (21.6 + / -0.5%) and LH43 (38.1 + / -0.5%). According to the present invention, the combined probiotic fermentation powder has significant promotion effect on the growth of Bifidobacterium longum BL986, such that the combined probiotic fermentation powder can be used for preparing the drug for relieving the senile intestinal flora disorder.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a combined postbiotic fermentation powder and a use thereof. Background Art

[0002] At present, probiotic preparations are widely used in the fields of food, health care products and medicine. Probiotics have many benefits for human health, such as improving the balance of intestinal flora and enhancing immunity. Among them, intestinal flora, especially bifidobacteria in the intestinal flora of the elderly, plays a pivotal role. As an important type of probiotics, bifidobacteria can not only promote intestinal peristalsis and improve nutrient absorption efficiency, but also inhibit the growth of harmful bacteria and maintain the balance of intestinal microecology. In the elderly population, the number of bifidobacteria usually decreases with age, but the long bifidobacterium has unique effects, such as enhancing immunity, promoting intestinal health and helping to prevent intestinal-related diseases.

[0003] However, probiotics are easily inactivated during storage and transportation, and their colonization rate in the intestine is also challenging. In addition, existing probiotic preparations usually contain only a single strain or a few strains, and their efficacy may be limited. Postbiotics, as metabolites of probiotics, also show potential prebiotic effects (i.e., some functions similar to prebiotics and some value-added functions for probiotics), but their preparation methods and applications still need further study.

[0004] The existing preparation methods of postbiotics are inefficient and difficult to ensure the stability of postbiotics. Directly adding probiotics also faces problems such as strain stability, low intestinal colonization rate and individual differences:

[0005] Low survival rate of probiotics: Probiotics are easily affected by environmental factors and become inactivated during production, storage and transportation, resulting in reduced efficacy of the final product.

[0006] Low intestinal colonization rate: Not all ingested probiotics can effectively colonize and exert their effects in the intestines, especially for the elderly, whose intestinal environment is complex and colonization is more difficult.

[0007] Strain homogeneity: Many existing probiotic preparations contain only a single strain, such as only one type of Bifidobacterium, while ignoring the synergistic effects between different strains, and their efficacy is limited.

[0008] Lack of synergy: The lack of in-depth research on the synergistic effects of probiotic combinations (such as the combination of multiple bifidobacteria with other probiotics) and their metabolites (postbiotics) makes it difficult to develop efficient and stable probiotic preparations.

[0009] How to explore and improve the probiotic effects of postbiotics, especially for the elderly, improve the stability and biological activity of postbiotics, and conduct in-depth research on the synergistic mechanism between different probiotics and their postbiotics to make up for some of the defects of probiotics are urgent issues to be solved by researchers. This will help develop probiotic preparations that are more suitable for the elderly and provide strong support for improving the intestinal health of the elderly and improving their quality of life. Summary of the invention

[0010] Purpose of the invention: The purpose of the present invention is to provide a combined postbiotic fermentation powder and its use in view of the many problems faced by directly adding probiotics. The combined postbiotic fermentation powder of the present invention has a significant promoting effect on the growth of Bifidobacterium longum BL986, and can therefore be used to prepare a drug for alleviating aging intestinal flora imbalance.

[0011] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0012] The present invention provides a combined postbiotic fermentation powder, which is a dry product after the fermentation of a composite lactic acid bacteria composition; the cell number proportions of each fermentation strain in the combined postbiotic fermentation powder are: LRH10 (12±0.5%), LPC12 (2±0.5%), LF26 (26.3±0.5%), ST30 (21.6±0.5%), and LH43 (38.1±0.5%).

[0013] Biomaterial deposit information:

[0014] Lactobacillus rhamnosus LRH10, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24513;

[0015] Lactobacillus paracasei LPC12, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.24512;

[0016] Limosilactobacillus fermentum LF26, which was deposited in the China General Microbiological Culture Collection Center on February 24, 2020, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.19431;

[0017] Streptococcus thermophilus ST30, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24514;

[0018] Lactobacillus helveticus LH43, this strain was deposited in the China General Microbiological Culture Collection on March 11, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24511.

[0019] The above strains have been recorded in patent application CN116179414A published in the patent gazette.

[0020] The cell number of the combined postbiotic fermentation powder is ≥1.0×10 9 cell / g.

[0021] The fermentation conditions of the composite lactic acid bacteria composition are as follows: first fermentation: fermentation at 40-44° C. for 5-8 h; second fermentation: fermentation at 37-39° C. for 10-16 h.

[0022] The dry product is a powder obtained by spray drying the composite lactic acid bacteria composition after fermentation and maltodextrin.

[0023] The added amount of the maltodextrin is 15-20%.

[0024] The spray drying conditions are as follows: an inlet air temperature of 180°C and an outlet air temperature of 110°C.

[0025] Specifically, the present invention provides a method for preparing a composite postbiotic fermentation powder using a mixture of skimmed milk powder, yeast powder, white sugar and water as a base, comprising the following steps:

[0026] (1) Prepare a base according to the ratio of 20-25% skim milk powder, 0.1-0.5% yeast powder, 1-5% white sugar, and the rest water by mass percentage, and stir to dissolve;

[0027] (2) sterilizing the substrate of step (1): 90-95° C., 10-15 min;

[0028] (3) Cooling: Cool to the inoculation temperature of 40-44°C;

[0029] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain in Table 1; the base viable bacterial count after inoculation is 1.0×10 5 CFU / ml;

[0030] Table 1 Proportion of inoculated cells of each strain

[0031] Lactobacillus rhamnosus LRH10 40％ Lactobacillus mucilaginosus fermentation LF26 17％ Lactobacillus paracasei LPC12 17％ Lactobacillus helveticus LH43 17％ Streptococcus salivarius subsp. thermophilus ST30 9％

[0032] (5) First fermentation: 40-44°C for 5-8 hours;

[0033] (6) Adjust the test pH to 4.8-5.0, add 0.5-1% yeast extract (based on the base before inoculation); 0-1% isomaltooligosaccharide (based on the base before inoculation);

[0034] (7) Secondary fermentation: fermentation at 37-39°C for 10-16 hours;

[0035] (8) Preparation: adding 15-20% maltodextrin to the fermentation product obtained in step (7);

[0036] (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C.

[0037] After spray drying, due to the instant inactivation of cells, the structure of the lactic acid bacteria strains was basically consistent with that before spraying, and a combined postbiotic fermentation powder was obtained, in which the cell number proportion of each strain was: LRH10 (12±0.5%), LPC12 (2±0.5%), LF26 (26.3±0.5%), ST30 (21.6±0.5%), and LH43 (38.1±0.5%).

[0038] The present invention also provides use of the combined postbiotic fermentation powder in preparing a medicine for alleviating aging intestinal flora imbalance.

[0039] The combined postbiotic fermented powder has a significant promoting effect on the growth of Bifidobacterium longum BL986.

[0040] The medicine comprises the above-mentioned postbiotic fermentation liquid or fermentation powder, and a pharmaceutically acceptable carrier or excipient.

[0041] The auxiliary materials include one or more of preservatives, antioxidants, emulsifiers, wetting agents, diluents, disintegrants or binders.

[0042] The drug of the present invention can be administered in various known ways, such as oral administration, injection, etc. The drug of the present invention can be administered alone or in combination with other drugs. The oral composition can be in any oral acceptable dosage form, including but not limited to tablets, granules, capsules, pills, suspensions and solutions.

[0043] Beneficial effects:

[0044] The combined postbiotic fermented powder of the present invention has stable bacterial counts of various lactic acid bacteria and has a significant promoting effect on the growth of Bifidobacterium longumBL986. Therefore, the combined postbiotic fermented powder of the present invention can be used to prepare a drug for alleviating aging intestinal flora imbalance. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The effects of different probiotics on the growth of BL986;

[0046] Figure 2 The effects of different probiotics on the growth of BL1015T;

[0047] Figure 3 The effects of different probiotics on the growth of BI17;

[0048] Figure 4 The effects of different probiotics on the growth of BI1024T;

[0049] Figure 5 The effects of different probiotics on the growth of BI1082;

[0050] Figure 6 This is the effect of postbiotic fermentation broth and fermentation powder on the growth of BL986. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0052] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0053] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0054] The lactic acid bacteria of the present invention, such as LRH10, LPC12, LF26, ST30 and LH43, are all from frozen vials in a syngeneic strain library. Before use, the frozen vials are placed in a 37° C. water bath for rapid thawing.

[0055] The method for detecting the number of LRH10, LPC12, LF26, ST30, and LH43 fermented lactic acid bacteria cells in the postbiotic fermented powder prepared in the embodiment of the present invention is:

[0056] 1. Preparation of MRS medium

[0057] Peptone 10.0g; beef powder 5.0g; yeast powder 4.0g; glucose 20.0g; Tween 80 1.0mL; triammonium citrate 2.0g; sodium acetate 5.0g; magnesium sulfate 0.2g; manganese sulfate 0.05g; dipotassium hydrogen phosphate 2.0g; agar powder 15.0g.

[0058] Add the above ingredients into 1000 mL of distilled water, heat to dissolve, adjust the pH to 6.2±0.2, and sterilize at 121°C for 20 min after packaging.

[0059] 2. Preparation of (MRS+Ciprofloxacin) medium

[0060] (1) Preparation of Ciprofloxacin (Sigma) Solution: Weigh 40 mg of ciprofloxacin and dissolve it in 10 mL of 0.1 M HCl. Mix well using an oscillator and filter the mixture using a 0.22 μm filter into a sterile 15 mL centrifuge tube for later use.

[0061] (2) Add the ingredients obtained in step (1) to 1000 mL of distilled water, heat to dissolve, adjust the pH to 6.4, and sterilize by autoclave at 121° C. for 20 min after packaging. Heat to melt agar before use, cool to 48° C. in a water bath, transfer 5 mL of ciprofloxacin solution to 1000 mL of MRS medium using a syringe with a 0.22 μm microporous filter membrane, mix well, pour 12 mL of the solution into a culture dish, and wait for solidification before use.

[0062] 3. Sample preparation and dilution

[0063] Accurately weigh 10.00g of the sample to be tested, put it into a 250mL Erlenmeyer flask filled with 90mL sterile water and small glass beads, and shake it thoroughly on a shaker to make a 1:10 dilution; then use a 1mL sterile pipette to draw 1mL of the 1:10 dilution, transfer it into a test tube filled with 9mL sterile water, and mix it thoroughly to make a 1:100 dilution. Take another 1mL sterile pipette and make 10-fold incremental dilutions according to the above operation sequence to make 10 according to the bacterial content of the sample. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 A series of dilutions of bacterial solution were taken, 10 -6 , 10 -7 , 10 -8 Three dilutions for inoculation of plates.

[0064] 4. Coating:

[0065] According to the estimation of the total number of viable bacteria in the sample to be tested, use a 0.1mL sterile pipette to take 0.1mL of three different dilutions respectively, repeat 3 times for each dilution, add them to the plates containing culture medium respectively, and use a sterile glass spreading rod to evenly spread the dilutions on the surface of the culture medium.

[0066] 5. Cultivation

[0067] Place the coated culture medium dish upside down in a constant temperature incubator for culture.

[0068] MRS medium was cultured anaerobically at 37°C for 48 hours.

[0069] (MRS+ciprofloxacin) medium was anaerobically cultured at 37°C for 48 hours.

[0070] 6. Colony morphology identification and counting

[0071] (1)LPC12

[0072] In the (MRS+Ciprofloxacin) culture medium, yellow colonies can be seen at the bottom of the plate. Obvious larger colonies are milky white, round, raised, with neat edges and a diameter of 1.8 mm±0.2 mm, which are LPC12.

[0073] (2)LRH10

[0074] In the (MRS+Ciprofloxacin) culture medium, yellow colonies can be seen on the bottom of the plate. Obviously smaller colonies are milky white, round, raised, with neat edges, and a diameter of 0.7mm±0.3mm, which is LRH10.

[0075] (3)LH43

[0076] In the (MRS+Ciprofloxacin) culture medium, yellow colonies can be seen at the bottom of the plate. Obvious smaller colonies are milky white, round, with raised colonies and neat edges. The diameter is 2.5 mm ± 0.2 mm, which is LH43.

[0077] (4) LF26

[0078] Lactobacillus fermentum LF26 does not grow in (MRS+ciprofloxacin) medium. The total number of bacteria on MRS medium minus the total number of bacteria on (MRS+ciprofloxacin) medium is the number of Lactobacillus fermentum colonies.

[0079] The calculation formula of the number of lactic acid bacteria in each fermentation is:

[0080]

[0081] The principle of this method is based on the fact that each bacteria has a different inhibitory effect on antibiotics, and the strains can be distinguished based on their colony size.

[0082] Example 1 Preparation of postbiotic fermentation powder

[0083] (1) Prepare a base in the following proportions by mass: 20% skim milk powder, 0.5% yeast powder, 5% white sugar, and the remainder water, and stir to dissolve;

[0084] (2) sterilizing the substrate of step (1): 95°C, 10 min;

[0085] (3) Cooling: Cool to the inoculation temperature of 42°C;

[0086] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain in Table 1; the base viable bacterial count after inoculation is 1.0×10 5 CFU / ml;

[0087] Table 1 Proportion of inoculated cells of each strain

[0088] Lactobacillus rhamnosus LRH10 40％ Lactobacillus mucilaginosus fermentation LF26 17％ Lactobacillus paracasei LPC12 17％ Lactobacillus helveticus LH43 17％ Streptococcus salivarius subsp. thermophilus ST30 9％

[0089] (5) First fermentation: 42°C for 8 h;

[0090] (6) Adjust the test pH to 4.8-5.0, add 1% yeast extract (based on the base before inoculation) and 1% isomaltooligosaccharide (based on the base before inoculation);

[0091] (7) Secondary fermentation: fermentation at 37°C for 16 h;

[0092] (8) Preparation: adding 15% maltodextrin to the fermentation product obtained in step (7);

[0093] (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C.

[0094] After spray drying, due to the instant inactivation of cells, the structure of the lactic acid bacteria strains was basically consistent with that before spraying, and a combined postbiotic fermentation powder was obtained, in which the cell number proportion of each fermentation strain was: LRH10 (12%), LPC12 (2%), LF26 (26.3%), ST30 (21.6%), and LH43 (38.1%).

[0095] Comparative Example 1 Preparation of postbiotic fermentation broth

[0096] (1) Prepare a base in the following proportions by mass: 22% skim milk powder, 0.25% yeast powder, 3% white sugar, and the remainder water, and stir to dissolve;

[0097] (2) Sterilize the substrate in step (1): 95°C, 15 min;

[0098] (3) Cooling: Cool to the inoculation temperature of 44°C;

[0099] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain in Table 2; the base viable bacterial count after inoculation is 1.0×10 5 CFU / ml;

[0100] Table 2 Proportion of inoculated cells of each strain

[0101] Lactobacillus rhamnosus LRH10 40％ Lactobacillus mucilaginosus fermentation LF26 17％ Lactobacillus paracasei LPC12 17％ Lactobacillus helveticus LH43 17％ Streptococcus salivarius subsp. thermophilus ST30 9％

[0102] (5) First fermentation: 44°C for 8 h;

[0103] (6) Adjust the test pH to 5.0, add 0.75% yeast extract (based on the base before inoculation) and 0.5% isomaltooligosaccharide (based on the base before inoculation);

[0104] (7) Secondary fermentation: fermentation at 37°C for 10 h;

[0105] The cell number percentage of each strain is: LRH10 (12.5%), LPC12 (2.3%), LF26 (26.1%), ST30 (21.9%), and LH43 (38.5%).

[0106] (8) Sterilization: 90°C, 10 min, to obtain the fermentation liquid.

[0107] Example 2 Intestinal bacteria growth effect test

[0108] Test bacteria: Bifidobacterium longum BL986, B. longum BL1015 T , B.infantisBI17, B.infantisBI1024 T , B.infantis BI1082 are both from Shenghe Biotechnology Co., Ltd.

[0109] Prebiotics to be tested:

[0110] 1. Postbiotic fermentation powder: prepared in Example 1, tested at 0%, 0.5%, 1%, and 2% (on a dry basis)

[0111] 2. Postbiotic fermentation broth: prepared according to comparative example 1, the test dosages were 0%, 0.5%, 1%, and 2% (on a dry basis)

[0112] 3. Maltodextrin: purchased from Roquette Frères, France; tested dosages were 0%, 0.5%, 1%, and 2% (on a dry basis)

[0113] 4. Galacto-oligosaccharide: purchased from Neo Cremar Co., Ltd., South Korea; the test dosage is 0%, 0.5%, 1%, 2% (on dry basis)

[0114] 5. Isomalto-oligosaccharide: purchased from Shouxiang Foods, Taiwan, China. The test dosages were 0%, 0.5%, 1%, and 2% (on a dry basis).

[0115] 6. Fructooligosaccharide: purchased from Taiwan Shenghe Biotechnology Co., Ltd., China. The tested dosages were 0%, 0.5%, 1%, and 2% (on a dry basis).

[0116] 7. Xylooligosaccharide: purchased from Shandong Longli Technology, the test dosage is 0%, 0.5%, 1%, 2% (on dry basis)

[0117] 8. Inulin: purchased from Beneo-orafti SA, Belgium. The tested dosages were 0%, 0.5%, 1%, and 2% (on dry basis).

[0118] Bacterial culture conditions:

[0119] The strain was cultured anaerobically at 37°C for 16 hours with MRS (BD Difco) + 0.05% cysteine, activated three times, and then inoculated into 10% skim milk powder SMP aqueous solution containing different doses of the probiotics to be tested, and the bacterial count was analyzed after anaerobic culture at 37°C for 16 hours. The above experiments were repeated three times. The bacterial count was performed by plate count method.

[0120] Statistical methods:

[0121] SPSS, One-way ANOVA, Tukey, p<0.05.

[0122] Experimental results:

[0123] 1. The effects of different probiotics on the growth of B. longum are shown in Table 3. Figure 1 , Figure 2 .

[0124] in, Figure 1 The effects of different probiotics on the growth of BL986; Figure 2 Effects of different probiotics on BL1015 T The impact of growth.

[0125] Table 3 Effects of different probiotics on the growth of B. longum

[0126]

[0127]

[0128] From Table 3, Figure 1 , Figure 2It can be seen that the combined postbiotic fermentation powder prepared by the present invention has a significant promoting effect on the growth of B. longum, especially at higher dosages (such as 1%, 2%), the growth promoting effect on the strain is more obvious.

[0129] 2. The effects of different probiotics on the growth of B. infantis are shown in Table 4. Figure 3 , Figure 4 , Figure 5 .

[0130] in, Figure 3 The effects of different probiotics on the growth of BI17; Figure 4 Effects of different prebiotics on BI1024 T The impact of growth, Figure 5 The effects of different probiotics on the growth of BI1082.

[0131] Table 4 Effects of different probiotics on the growth of B. infantis

[0132]

[0133]

[0134] From Table 4, Figure 3 , Figure 4 , Figure 5 It can be seen that the combined postbiotic fermentation powder prepared by the present invention has a significant promoting effect on the growth of B. infantis, especially at higher dosages (such as 1%, 2%), the growth promoting effect on the strain is more obvious.

[0135] Example 3 Experiment on the effect of postbiotic fermentation broth and fermentation powder on the growth of BL986

[0136] In order to further compare the differences between postbiotic fermentation broth and fermentation powder on strain growth, we conducted experiments on the effects of postbiotic fermentation broth and fermentation powder on the growth of BL986.

[0137] Postbiotic fermentation liquid and fermentation powder: the sources are the same as those in Example 2, and the test dosages are 0%, 0.2%, 0.5%, 1%, and 2% (on a dry basis)

[0138] The bacterial culture conditions and statistical methods are the same as those in Example 2.

[0139] Experimental results:

[0140] The effects of postbiotic fermentation broth and fermentation powder on the growth of BL986 are shown in Table 5. Figure 6 .in, Figure 6 This is the effect of postbiotic fermentation broth and fermentation powder on the growth of BL986.

[0141] Table 5 Effects of postbiotic fermentation broth and fermentation powder on the growth of BL986

[0142]

[0143] From Table 5, Figure 6 It can be seen that postbiotic fermentation powder has a more significant promoting effect on the growth of BL986 than fermentation broth.

[0144] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A combined postbiotic fermentation powder, characterized in that: It is a dry product after the composite lactic acid bacteria composition is fermented; the cell number proportion of each fermentation strain in the combined postbiotic fermentation powder is: LRH10 (12±0.5%), LPC12 (2±0.5%), LF26 (26.3±0.5%), ST30 (21.6±0.5%), LH43 (38.1±0.5%).

2. The combined postbiotic fermentation powder according to claim 1, characterized in that: The cell number of the combined postbiotic fermentation powder is ≥1.0×10 9 cell / g.

3. The combined postbiotic fermentation powder according to claim 1, characterized in that: The fermentation conditions of the composite lactic acid bacteria composition are as follows: first fermentation: fermentation at 40-44° C. for 5-8 h; second fermentation: fermentation at 37-39° C. for 10-16 h.

4. The combined postbiotic fermentation powder according to claim 1, characterized in that: The dry product is a powder obtained by spray drying the composite lactic acid bacteria composition after fermentation and maltodextrin.

5. The combined postbiotic fermentation powder according to claim 4, characterized in that: The added amount of the maltodextrin is 15-20%.

6. The combined postbiotic fermentation powder according to claim 4, characterized in that: The spray drying conditions are as follows: an inlet air temperature of 180°C and an outlet air temperature of 110°C.

7. Use of the combined postbiotic fermentation powder according to any one of claims 1 to 6 in the preparation of a medicament for alleviating aging intestinal flora imbalance.

8. The use according to claim 7, characterized in that The combined postbiotic fermented powder has a significant promoting effect on the growth of Bifidobacterium longum BL986.

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