A bifidobacterium-yeast symbiont and its preparation method, preservation method and application

Abstract

The present invention provides a bifidobacterium-yeast symbiont and a preparation method, a preservation method and an application thereof, and belongs to the field of microbial technology. The present invention uses a specific method to prepare a bifidobacterium-yeast symbiont from Bifidobacterium breve i1088 (CGMCC NO.27281) and Saccharomyces cerevisiae ZB431 (GDMCC NO.61292), and uses a specific preservation method to maximize the survival rate of bacteria in the symbiotic system. In addition, the bifidobacterium-yeast symbiont prepared by the present invention has a significant therapeutic effect on diarrhea caused by flora imbalance.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a bifidobacterium-yeast symbiont and a preparation method and a preservation method thereof. Background Art

[0002] Bifidobacterium is one of the important beneficial microbial flora in the human intestine. It plays a key role in maintaining the balance of intestinal microecology. However, there are some limitations in the practical application of bifidobacteria. Bifidobacteria are sensitive to environmental factors, especially to changes in oxygen, temperature and humidity. In an aerobic environment, metabolic enzymes in bifidobacterial cells are easily inactivated, causing cell damage or even death. Moreover, under high temperature conditions, the cell membrane fluidity of bifidobacteria increases, and the stability of membrane proteins and membrane lipids is affected, affecting its normal physiological function. During the storage and processing process, these factors greatly limit the survival rate and activity retention of bifidobacteria, thereby affecting the full play of its probiotic effect.

[0003] Yeast is a type of unicellular fungus that is widely used in the fields of food and medicine. At the same time, yeast has a relatively strong ability to adapt to the environment, especially its tolerance to oxygen is much better than that of bifidobacteria. However, the cell structures and physiological characteristics of bifidobacteria and yeast are different, making it difficult to balance the storage conditions of the two. During the storage process, the symbiotic relationship between bifidobacteria and yeast may be affected. Due to the different growth rates and metabolic requirements of the two, a competitive relationship may occur in a storage environment with limited nutrients. For example, when nutrients such as carbon sources and nitrogen sources are in short supply, bifidobacteria and yeast may compete for these nutrients, thereby destroying the symbiotic balance. In addition, environmental changes during the storage process, such as temperature fluctuations, oxygen penetration, etc., may change the microenvironment of the symbiotic system. If the growth of yeast is inhibited due to environmental changes and oxygen cannot be effectively consumed, bifidobacteria will be exposed to a relatively aerobic environment, resulting in a decrease in its activity or even death. Therefore, developing an effective preparation method and preservation method for a bifidobacterium and yeast symbiont is a technical problem that needs to be solved at this stage. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a bifidobacterium-yeast symbiont and its preparation method, preservation method and application. The survival rate of the bifidobacterium-yeast symbiont system under this specific preservation method is the highest. At the same time, the bifidobacterium-yeast symbiont prepared by the method of the present invention has a significant therapeutic effect on diarrhea caused by dysbacteriosis.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides a bifidobacterium-yeast symbiont, wherein the bifidobacterium is Bifidobacterium breve i1088, with a preservation number of CGMCC NO.27281; and the yeast is Saccharomyces cerevisiae ZB431, with a preservation number of GDMCC NO.61292.

[0007] The present invention provides a method for preparing the bifidobacterium-yeast symbiosis, comprising the following steps: inoculating yeast into a liquid culture medium for cultivation, then adding nutrient solution, inoculating bifidobacteria for anaerobic cultivation, and obtaining a bifidobacterium-yeast symbiotic system.

[0008] Preferably, the liquid culture medium is MRS medium.

[0009] Preferably, the nutrient solution comprises: 10-20 g of peeled potato mash, 2-8 g of tea powder, 5-10 g of carrot stock solution, and 0.3-0.7 L of water.

[0010] Preferably, the method for preparing the tea powder comprises: crushing tea leaves to 180-220 meshes to obtain tea powder.

[0011] Preferably, the culture temperature is 28-35°C, the culture time is 23-50h, and the rotation speed is 100-150r / min.

[0012] The present invention provides a method for preserving the bifidobacterium-yeast symbiota or the bifidobacterium-yeast symbiota obtained by the preparation method, comprising the following steps: mixing the bifidobacterium-yeast symbiota with a protective agent, and freeze-drying, wherein the freeze-drying program is set as follows: first pre-freezing the sample at -50 to -70°C for 0.5 to 2.5h, then heating to -30 to -45°C at a rate of 0.5 to 2.5°C / min and drying for 4 to 7h, then raising the temperature to -15 to -20°C at a rate of 0.5 to 2.5°C / min for secondary drying for 9 to 13h, and then raising the temperature to 10 to 28°C at a rate of 0.5 to 2.5°C / min for tertiary drying for 0.5 to 3.5h.

[0013] Preferably, the protective agent includes one or more of glucose, glycerol, and carrageenan.

[0014] The present invention provides use of the bifidobacterium-yeast symbiont or the bifidobacterium-yeast symbiont obtained by the preparation method in preparing a product for treating diarrhea caused by intestinal flora imbalance.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention prepares a bifidobacterium-yeast symbiont by using Bifidobacterium breve i1088 (CGMCC NO. 27281) and Saccharomyces cerevisiae ZB431 (GDMCC NO. 61292) for the first time by adopting a specific method. A nutrient solution prepared by mashed potatoes, tea powder and carrot stock solution is added in the preparation method, which can provide better nutrients for yeast and bifidobacteria, so that the co-cultured strain has stronger activity and is easy to preserve.

[0017] Furthermore, the present invention adopts freeze-drying technology and selects protective agents for the two strains, so that the obtained symbiotic freeze-dried powder has a high survival rate and the activities of yeast and bifidobacteria are basically not affected.

[0018] Furthermore, the bifidobacterium-yeast symbiont prepared by the present invention is applied to rats with diarrhea caused by intestinal flora, which can adjust the beneficial bacteria and effective enzymes in the intestine to normal levels, and has a significant therapeutic effect on diarrhea caused by flora imbalance. DETAILED DESCRIPTION

[0019] The present invention provides a bifidobacterium-yeast symbiont, wherein the bifidobacterium is Bifidobacterium breve i1088, with a preservation number of CGMCC NO.27281; and the yeast is Saccharomyces cerevisiae ZB431, with a preservation number of GDMCC NO.61292. The Bifidobacterium breve i1088 (CGMCC NO.27281) of the present invention is disclosed in CN117143778A and is obtained through existing known channels. The Saccharomyces cerevisiae ZB431 (GDMCC NO.61292) of the present invention is disclosed in CN113493746A and is obtained through existing known channels.

[0020] The present invention provides a method for preparing the bifidobacterium-yeast symbiont, comprising the following steps: inoculating yeast into a liquid culture medium for cultivation, then adding nutrient solution, inoculating bifidobacteria for anaerobic cultivation, and obtaining a bifidobacterium-yeast symbiotic system. The present invention first inoculates yeast to consume oxygen in the culture environment and produces vitamins, amino acids, short peptides and other components suitable for bifidobacterium cultivation, which is conducive to cultivating a bifidobacterium-yeast symbiont with better live bacteria performance.

[0021] In the present invention, yeast was first inoculated onto MRS medium and cultured until OD 600 =0.2-0.4, to obtain an activated yeast culture; inoculate Bifidobacterium breve i1088 (CGMCC NO.27281) onto MRS medium and culture until OD 600=0.2-0.4, to obtain activated bifidobacterium liquid. The present invention prepares a symbiotic body by using activated yeast liquid and activated bifidobacterium liquid. The inoculation amount of the yeast liquid of the present invention is 2-4% (V / V), and the inoculation amount of the bifidobacterium liquid is 2-4% (V / V).

[0022] In the present invention, the liquid culture medium is MRS culture medium. The MRS culture medium of the present invention can be purchased through commercial channels.

[0023] In the present invention, the nutrient solution comprises: 10-20g peeled mashed potatoes, 2-8g tea powder, 5-10g carrot stock solution, and 0.3-0.7L of water; preferably 15g peeled mashed potatoes, 6g tea powder, 8g carrot stock solution, and 0.5L of water. The preparation of the peeled mashed potatoes of the present invention comprises: steaming yellow-heart potatoes and peeling them, and then mashing the peeled mashed potatoes into a paste to obtain peeled mashed potatoes. The preparation method of the tea powder of the present invention comprises: crushing Longjing tea to 180-220 meshes to obtain tea powder. The crushing mesh number of the present invention is preferably 200 meshes. The preparation of the carrot stock solution of the present invention comprises the following steps: crushing 100 parts of carrots and 250 parts of water with a wall-breaking machine, filtering, and obtaining carrot stock solution.

[0024] In the present invention, the culture temperature is 28-35°C, the time is 23-50h, and the rotation speed is 100-150r / min. The yeast of the present invention is inoculated into the liquid culture medium and the culture temperature is 30°C, the time is 24h, and the rotation speed is 120r / min. The culture temperature after inoculation of bifidobacterium of the present invention is 32°C, the time is 48h, and the rotation speed is 150r / min.

[0025] The present invention also provides a method for preserving the bifidobacterium-yeast symbiota, comprising the following steps: mixing the bifidobacterium-yeast symbiota with a protective agent, freeze drying, and the freeze drying program is set as follows: first pre-freeze the sample at -50 to -70°C for 0.5-2.5h, then heat it to -30 to -45°C at a rate of 0.5-2.5°C / min and dry it for 4-7h, then increase the temperature to -15 to -20°C at a rate of 0.5-2.5°C / min for secondary drying for 9-13h, and then increase the temperature to 10-28°C at a rate of 0.5-2.5°C / min for tertiary drying for 0.5-3.5h. The protective agent of the present invention includes one or more of glucose, glycerol, and carrageenan, preferably glucose and carrageenan.

[0026] The present invention also provides use of the bifidobacterium-yeast symbiont or the bifidobacterium-yeast symbiont obtained by the preparation method in preparing a product for treating diarrhea caused by intestinal flora imbalance.

[0027] In the present invention, unless otherwise specified, all components, reagents or culture media are commercially available products well known to those skilled in the art.

[0028] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] 1. Obtaining culture medium and nutrient solution

[0031] MRS culture medium (containing glucose), including liquid culture medium and solid culture medium, was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.

[0032] Preparation of nutrient solution: Steam yellow-heart potatoes and peel them, then mash the peeled potatoes into a paste to obtain peeled potato mash; crush Longjing tea to 200 mesh to obtain tea powder; crush 100 parts of carrots and 250 parts of water with a wall-breaking machine, filter, and obtain carrot stock solution. Evenly mix 15g of peeled potato mash, 6g of tea powder, 8g of carrot stock solution, and 0.5L of water to obtain a nutrient solution.

[0033] The above culture medium and nutrient solution were packaged, sterilized under high pressure at 121°C for 15 min, and set aside.

[0034] 2. Preparation of Bifidobacterium-Yeast Symbiont

[0035] (1) Preparation of bacterial solution: Saccharomyces cerevisiae ZB431 (GDMCC No. 61292) was inoculated into MRS medium and cultured at 30°C until OD 600 =0.3, and yeast culture was obtained; Bifidobacterium breve i1088 (CGMCC NO.27281) was inoculated into MRS medium and cultured at 32°C until OD 600 =0.25, and obtain bifidobacterium liquid.

[0036] (2) The yeast culture liquid was inoculated into 250 mL of MRS medium at an inoculum volume of 3% (V / V) and cultured at a rotation speed of 120 r / min and 30° C. for 24 h.

[0037] (3) Add 50 mL of nutrient solution and inoculate bifidobacterium culture solution at an inoculum rate of 3% (V / V), and culture anaerobically at a speed of 150 r / min and 32° C. for 48 h to obtain a bifidobacterium-yeast symbiont.

[0038] 3. Preservation of Bifidobacterium-Yeast Symbiont

[0039] 250mL of the prepared bifidobacterium-yeast symbiont was mixed with 5g of glucose (CAS 50-99-7, Ruichengkang Pharmaceutical Technology (Shaanxi) Co., Ltd.) and 5g of k-type carrageenan (CAS11114-20-8, Shanghai Hongshun Biotechnology Co., Ltd.), placed in a freezing tube, placed in a freeze dryer with a set program, and the drying program was started. Among them, the freeze drying program was set as follows: the sample was first pre-frozen at -60°C for 1.5h, then heated to -35°C at a rate of 1.5°C / min and dried for 6h, then the temperature was raised to -18°C at a rate of 0.5-2.5°C / min for secondary drying for 11h, and then the temperature was raised to 25°C at a rate of 0.5-2.5°C / min for tertiary drying for 1.5h.

[0040] Example 2

[0041] 1. The culture medium and nutrient solution were obtained in the same manner as in Example 1.

[0042] 2. Preparation of Bifidobacterium-Yeast Symbiont

[0043] (1) Preparation of bacterial solution: The purchased Saccharomyces cerevisiae ZB431 (GDMCC NO.61292) was inoculated into MRS medium and cultured until OD 600 =0.25, and yeast culture was obtained; the purchased Bifidobacterium breve i1088 (CGMCC NO.27281) was inoculated into MRS medium and cultured until OD 600 =0.3, and obtain bifidobacterium liquid.

[0044] (2) The purchased yeast culture liquid was inoculated into 250 mL of MRS medium at an inoculum volume of 4% (V / V) and cultured at a rotation speed of 120 r / min and 32° C. for 24 h.

[0045] (3) Add 50 mL of nutrient solution and inoculate bifidobacterium culture solution at an inoculum rate of 4% (V / V), and culture anaerobically at a speed of 150 r / min and 35° C. for 48 h to obtain a bifidobacterium-yeast symbiont.

[0046] 3. Preservation of Bifidobacterium-Yeast Symbiont

[0047] 250 mL of the prepared bifidobacterium-yeast symbiont was mixed with 6 g of glucose and 6 g of carrageenan, placed in a freezing tube, placed in a freeze dryer with a set program, and the drying program was started. The freeze drying program was set as follows: the sample was first pre-frozen at -70°C for 1 hour, then heated to -45°C at a rate of 2.5°C / min for 10 hours, then the temperature was raised to -20°C at a rate of 2.5°C / min for secondary drying for 9 hours, and then the temperature was raised to 28°C at a rate of 2.5°C / min for tertiary drying for 2 hours.

[0048] Comparative Example 1

[0049] The cerevisiae yeast ZB431 (GDMCC NO.61292) and the related steps were discarded, and the remaining steps were the same as those in Example 1.

[0050] Comparative Example 2

[0051] Bifidobacterium breve i1088 (CGMCC NO.27281) was replaced by Bifidobacterium lactis 80 (Bla80) (purchased from Weikang Probiotics (Suzhou) Co., Ltd.), and the remaining steps were the same as in Example 1.

[0052] Comparative Example 3

[0053] Mashed potatoes are replaced with mashed sweet potatoes, and the remaining steps are the same as in Example 1.

[0054] Comparative Example 4

[0055] Tea powder is replaced by tea polyphenols, which are purchased through commercial channels. The step of preparing tea powder is removed, and the remaining steps are the same as those in Example 1.

[0056] Comparative Example 5

[0057] The carrot stock solution was replaced with white radish stock solution, and the remaining steps were the same as in Example 1.

[0058] Comparative Example 6

[0059] The difference from Example 1 is that the steps of step (2) and step (3) in Example 1 are changed to: the yeast solution and the bifidobacterium solution are inoculated into 250 mL of MRS culture medium at an inoculation rate of 3% (V / V), respectively, and cultured at a speed of 120 r / min and 32° C. for 24 h, and then 50 mL of nutrient solution is added, and the speed is 150 r / min and 35° C. for anaerobically cultured for 48 h to obtain a bifidobacterium-yeast symbiont.

[0060] Comparative Example 7

[0061] The difference from Example 1 is that glucose is removed, carrageenan is 10 g, and the remaining steps are the same as Example 1.

[0062] Comparative Example 8

[0063] The difference from Example 1 is that glucose and carrageenan are replaced by sodium alginate, and the remaining steps are the same as those in Example 1.

[0064] Comparative Example 9

[0065] The difference from Example 1 is that carrageenan is removed, the amount of glucose is 10 g, and the remaining steps are the same as those in Example 1.

[0066] Comparative Example 10

[0067] The difference from Example 1 is that glucose and carrageenan are replaced by mannitol, and the remaining steps are the same as those in Example 1.

[0068] Test Example 1

[0069] The bacterial powders of Example 1 and Comparative Examples 1-10 after freeze drying were counted by gradient dilution counting method, 10 μL of dilution solution was taken for plate counting, and the plate was placed at 37°C for 36-48 hours. The freeze drying survival rate was: the number of viable bacteria after freeze drying / the number of viable bacteria before freeze drying. The results are shown in Table 1.

[0070] Table 1 Freeze-drying survival rate of each group

[0071]

[0072]

[0073] As can be seen from Table 1, the bifidobacterium-yeast symbiotic system obtained by the preparation method of the present invention has a higher survival rate after freeze-drying. Changes in the composite strains, changes in the ingredients in the nutrient solution or the preparation method during the preparation process, or changes in the protective agent during the freeze-drying process will affect the freeze-drying survival rate of the strains in the bifidobacterium-yeast symbiotic system.

[0074] Test Example 2

[0075] Healthy adult SD rats weighing 200-250g were selected. After the rats were adaptively fed for 3 days, they were randomly divided into groups and the experiment was started. They were divided into a control group, a model group, an embodiment 1-2 group, and a comparative example 1-6 group. Each of the control group was gavaged with 0.2mL of normal saline. The remaining groups were gavaged with 0.2mL of antibiotic mixture. Twice a day, for 5 consecutive days, the feces became thin and soft, that is, the model was successfully established. After the model was successfully established, the drug was administered by gavage (embodiment 1-2 group, comparative example 1-6 group), twice a day, for 3 consecutive days. The control group and the model group were given sterile normal saline, and the remaining groups were given 3.5g / kg by mice.

[0076] Immediately place the mice killed in each group on a clean bench, collect the contents of the cecum segment of each group aseptically, and collect the intestinal contents of the mice in the same group and mix them by aseptic operation for later use.

[0077] Determination of intestinal microorganism quantity: according to each group, a certain amount of intestinal contents were weighed aseptically and placed in a sterile water bottle containing glass beads. The shaker was shaken at 120r / min for 30min to fully disperse the microorganisms. The appropriate dilution was selected and the mixed bacteria method was used for counting. The colonies of bacteria and coliforms were counted after culturing in a 37℃ incubator for 24h, the colonies of fungi were counted after culturing in a 30℃ incubator for 96h, and the colonies of lactic acid bacteria and bifidobacteria were counted after culturing in a 37℃ anaerobic incubator for 48h. Each dilution was repeated 3 times, and the average value was calculated and the number of bacteria per gram of intestinal contents was calculated. The results are shown in Table 2.

[0078] Analysis of mouse intestinal enzyme activity: Take the intestinal contents, dilute the intestinal contents with sterile water, keep warm in a 40℃ water bath for 30 minutes, fully dissolve the enzyme protein, centrifuge at 2000r / min for 10 minutes, take the supernatant, and analyze the activities of cellulase, xylanase, protease, amylase and other enzymes. Cellulase activity, amylase activity and xylanase activity were all determined by DNS colorimetry. Cellulase activity was defined as 1μg of reducing sugar generated by 1g of intestinal contents at 46℃ for 30 minutes, amylase activity was defined as 1μg of reducing sugar generated by 1g of intestinal contents at 37℃ for 60 minutes, xylanase activity was defined as 1μg of reducing sugar generated by 1g of intestinal contents at 46℃ for 60 minutes, and protease activity was determined by Folin-phenol method, and 1μg of amino acid generated by 1g of intestinal contents at 37℃ for 40 minutes was defined as one enzyme activity unit U. The results are shown in Table 3.

[0079] Table 2 Effects of each group on intestinal microorganisms and specific microorganisms in mice with dysbacteriosis and diarrhea

[0080]

[0081] Table 3 Effects of each group on intestinal enzyme activities in mice with dysbacteriosis and diarrhea

[0082] Group Cellulase(U) Xylanase(U) Protease (U) Amylase (U) Example 1 1.65±0.07 5.68±0.24 4.05±0.47 130.25±6.59 Example 2 1.73±0.05 5.67±0.21 4.24±0.52 129.28±8.47 Comparative Example 1 0.78±0.25 3.98±0.57 1.36±0.29 50.36±9.23 Comparative Example 2 0.67±0.12 4.01±0.24 1.49±0.37 52.27±3.97 Comparative Example 3 0.74±0.35 3.79±0.51 2.25±0.21 55.39±2.76 Comparative Example 4 0.89±0.24 3.89±0.72 1.89±0.34 54.37±2.41 Comparative Example 5 0.95±0.35 4.09±0.54 2.04±0.24 56.45±1.97 Comparative Example 6 0.84±0.59 3.72±0.18 1.96±0.37 53.25±2.01 Control group 1.53±0.05 5.36±0.13 3.89±0.54 123.24±2.67 Model Group 0.58±0.18 3.42±0.16 1.25±0.24 45.36±0.38

[0083] It can be seen from Tables 2 and 3 that the symbiotic system freeze-dried powder prepared in Examples 1-2 and Comparative Examples 1-6 have a tendency to increase the intestinal microorganisms, the number of specific microorganisms and the related enzyme activities of mice with dysbacteriosis diarrhea compared with the model group, but the effect of the symbiotic system freeze-dried powder prepared in Examples 1-2 is significantly higher than that of Comparative Examples 1-6, and even better than the control group, which significantly balances the intestinal flora and enzyme activity of mice with dysbacteriosis diarrhea, and has a significant effect in treating dysbacteriosis diarrhea.

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A bifidobacterium-yeast symbiont, characterized in that: The bifidobacterium is Bifidobacterium breve i1088, with a preservation number of CGMCC NO.27281; the yeast is Saccharomyces cerevisiae ZB431, with a preservation number of GDMCC NO.61292.

2. The method for preparing a bifidobacterium-yeast symbiont according to claim 1, characterized in that: The method comprises the following steps: inoculating yeast into a liquid culture medium for cultivation, then adding nutrient solution, inoculating bifidobacteria for anaerobic cultivation, and obtaining a bifidobacterium-yeast symbiotic system.

3. The preparation method according to claim 2, characterized in that: The liquid culture medium is MRS culture medium.

4. The preparation method according to claim 2, characterized in that: The nutrient solution comprises: 10-20 g of peeled potato mash, 2-8 g of tea powder, 5-10 g of carrot stock solution, and 0.3-0.7 L of water.

5. The preparation method according to claim 4, characterized in that: The preparation method of the tea powder comprises: crushing tea leaves to 180-220 meshes to obtain tea powder.

6. The preparation method according to claim 2, characterized in that: The culture temperature is 28-35°C, the culture time is 23-50h, and the rotation speed is 100-150r / min.

7. A method for preserving the bifidobacterium-yeast symbiota according to claim 1 or the bifidobacterium-yeast symbiota obtained by the preparation method according to any one of claims 2 to 5, characterized in that: The steps include: The bifidobacterium-yeast symbiont is mixed with a protective agent and freeze-dried. The freeze-drying program is set as follows: the sample is first pre-frozen at -50 to -70°C for 0.5-2.5h, then the temperature is increased to -30 to -45°C at a rate of 0.5-2.5°C / min and dried for 4-7h, then the temperature is increased to -15 to -20°C at a rate of 0.5-2.5°C / min for secondary drying for 9-13h, and then the temperature is increased to 10-28°C at a rate of 0.5-2.5°C / min for tertiary drying for 0.5-3.5h.

8. The storage method according to claim 7, characterized in that: The protective agent includes one or more of glucose, glycerol and carrageenan.

9. Use of the bifidobacterium-yeast symbiont according to claim 1 or the bifidobacterium-yeast symbiont obtained by the preparation method according to any one of claims 2 to 5 in preparing a product for treating diarrhea caused by intestinal flora imbalance.

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