A Bifidobacterium adolescentis - resistant starch complex with high stress resistance and its preparation method

Abstract

The present invention discloses a high stress-resistant Bifidobacterium adolescentis - resistant starch complex and its preparation method. Ammonium carboxylate salt is added to an aqueous solution containing 3 - 5 wt% glycerol, and high-temperature sterilization is carried out to obtain a glycerol - ammonium carboxylate salt aqueous solution, which is cooled and reserved. The Bifidobacterium adolescentis cells and resistant starch are added to the glycerol - ammonium carboxylate salt aqueous solution, stirred for 0.5 - 1 h, and ultrasonic treatment is used simultaneously to obtain a suspension. The suspension is allowed to stand for 10 - 15 min, and the precipitate is separated and recovered. The precipitate is allowed to stand for 2 - 3 h under sterile and anaerobic conditions at a temperature maintained at 38 - 40 °C, and then freeze-dried to obtain the high stress-resistant Bifidobacterium adolescentis - resistant starch complex. By constructing an environmental condition conducive to the easy adhesion of Bifidobacterium adolescentis to resistant starch, Bifidobacterium adolescentis adheres to the surface of resistant starch, improving its stress resistance. It has the advantages of remarkable effect and simple operation, and solves the problem of poor stress resistance of Bifidobacterium adolescentis.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a high stress-resistant Bifidobacterium adolescentis - resistant starch complex and a preparation method thereof. Background Art

[0002] Bifidobacterium adolescentis is an important probiotic in the human intestine. It is closely related to human health, and the number of it in the intestine is one of the indicators to measure human health. In addition to its physiological functions such as nutrition, immunity, tumor inhibition, and regulation of gastrointestinal function, Bifidobacterium adolescentis also has many health promotion effects. With the enhancement of people's health awareness, the market for probiotic preparations and live bacteria foods of Bifidobacterium adolescentis is very active, and the varieties are also increasing. The number and activity of Bifidobacterium adolescentis entering the human body directly become important indicators for testing product quality. During the process of passing through the human digestive tract, Bifidobacterium adolescentis is damaged by gastric acid and bile acid, and its activity and number are significantly reduced, and the number that can reach the large intestine intact is extremely small. Therefore, it is very important to improve the stress resistance of Bifidobacterium adolescentis.

[0003] To improve the tolerance of Bifidobacterium adolescentis, the main method is to screen strains with better tolerance. For example, Guo Dejun compared the acid and bile tolerance of different strains and their survival rates in simulated gastric juice and simulated intestinal fluid. Although this method can select strains with certain stress resistance, other properties of the strains are not considered, so this method has certain limitations. In addition, stimulating the enzyme properties of Bifidobacterium itself is also a method. For example, the published patent (CN202211153021.8) discloses a cultivation method for improving the stability of Bifidobacterium lactis, which mainly relies on changes in temperature conditions to stimulate Bifidobacterium lactis to metabolize more heat-resistant enzymes, thereby improving the tolerance and storage stability of Bifidobacterium lactis powder. This method is relatively convenient, but the improvement of the stability of the bacteria is limited, and the indicators for tolerance to gastric acid, bile, etc. are also disclosed. Continuing to find better methods to improve the resistance of Bifidobacterium strains, or reducing the influence of Bifidobacterium on harsh external environments, is a research hotspot of Bifidobacterium adolescentis. Summary of the Invention

[0004] Aiming at the problem that Bifidobacterium adolescentis is damaged by gastric acid and bile acid when passing through the human digestive tract, the present invention provides a high stress-resistant Bifidobacterium adolescentis - resistant starch complex and a preparation method thereof, solving the problem of poor stress resistance of Bifidobacterium adolescentis.

[0005] The present invention is achieved through the following technical solutions:

[0006] A preparation method of a high stress-resistant Bifidobacterium adolescentis - resistant starch complex, which comprises the following steps:

[0007] (1) Add ammonium carboxylate to an aqueous solution containing 3 - 5 wt% glycerol, sterilize at high temperature to obtain a glycerol-ammonium carboxylate aqueous solution, and cool for later use;

[0008] (2) Add Bifidobacterium adolescentis cells and resistant starch to the glycerol-ammonium carboxylate aqueous solution in step (1), stir for 0.5 - 1 h, and simultaneously use ultrasonic treatment to obtain a suspension; Use glycerol to adjust the activity and viscosity of the aqueous solution to provide a suitable macroscopic environment for the adhesion of Bifidobacterium adolescentis. Use ammonium carboxylate to change the pH environment of the solution and the state of the surface proteins of Bifidobacterium adolescentis, and reduce the surface tension of starch, which is beneficial for the proteins of Bifidobacterium adolescentis to approach the starch surface. In addition, glycerol has beneficial effects on subsequent sedimentation and freeze-drying protection;

[0009] (3) Let the suspension in step (2) stand for 10 - 15 min, separate and recover the precipitate. At this standing time, unadhered starch and the Bifidobacterium adolescentis-resistant starch complex can be effectively separated;

[0010] (4) Let the precipitate in step (3) stand under sterile and anaerobic conditions for 2 - 3 h at a temperature of 38 - 40 °C; Through the standing treatment for 2 - 3 h under sterile, anaerobic and 38 - 40 °C conditions, the adhered Bifidobacterium adolescentis can grow preliminarily and then be better fixed on the surface of resistant starch;

[0011] (5) Freeze-dry the precipitate after standing in step (4) to obtain a Bifidobacterium adolescentis-resistant starch complex with high stress resistance.

[0012] Furthermore, the addition amount of ammonium carboxylate in step (1) is 0.3 - 0.6% of the mass of the glycerol aqueous solution.

[0013] Furthermore, the ammonium carboxylate in step (1) is one of ammonium oxalate and ammonium citrate.

[0014] Furthermore, the number of Bifidobacterium adolescentis cells in the suspension in step (2) is 10 10 ~10 12 CFU; the addition amount of resistant starch is 2 - 3 g / mL of the glycerol-ammonium carboxylate aqueous solution.

[0015] Furthermore, the stirring speed in step (2) is 100 - 150 rpm, the ultrasonic frequency is 1 - 3 MHz, and the ultrasonic intensity is 0.5 - 1 W / cm 2 ; By stirring at a low speed, the possibility of collision between Bifidobacterium adolescentis and resistant starch is increased; Using low-frequency and low-intensity ultrasonic treatment to promote the adjustment of the positions of Bifidobacterium adolescentis and resistant starch, and Bifidobacterium adolescentis and resistant starch are more likely to adhere.

[0016] Further, the method for obtaining the Bifidobacterium adolescentis cells in step (2) is as follows: The Bifidobacterium adolescentis strain that has been passaged 2 to 3 times is cultured in CM0233 medium for 10 to 15 h, and the cells are collected by centrifugation to obtain the Bifidobacterium adolescentis cells.

[0017] Further, the resistant starch in step (2) is resistant starch prepared from legume starch.

[0018] In the present invention, a high stress-resistant Bifidobacterium adolescentis-resistant starch complex is prepared by the above preparation method.

[0019] The beneficial effects achieved by the present invention are as follows:

[0020] In view of the problem that the effective number of Bifidobacterium adolescentis reaching the large intestine is small due to the low resistance of Bifidobacterium adolescentis to gastric acid, intestinal juice, etc., by constructing an environmental condition conducive to the easy adhesion of Bifidobacterium adolescentis to resistant starch, Bifidobacterium adolescentis is made to adhere to the surface of resistant starch, improving its stress resistance. It can also provide energy for its fermentation and growth in the intestine, avoiding the use of complex means such as strain screening, having no specific requirements for the strain, and having the advantages of remarkable effect and simple operation. It solves the problem of poor stress resistance of Bifidobacterium adolescentis and provides a good solution for the development of high-activity Bifidobacterium adolescentis products. Specific embodiments

[0021] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications of the present invention by those skilled in the art fall within the scope defined by the appended claims of this application.

[0022] The resistant starch in the following examples and comparative examples is prepared by an enzymatic hydrolysis-pressure heat treatment method using pea starch.

[0023] Example 1

[0024] (1) The Bifidobacterium adolescentis strain that has been passaged 2 times is cultured in CM0233 medium for 15 h, and the cells are collected by centrifugation to obtain the Bifidobacterium adolescentis cells;

[0025] (2) Ammonium citrate accounting for 0.5% of the mass of the aqueous solution of glycerol is added to the aqueous solution containing 4 wt% glycerol, and sterilized at 100 °C for 10 min to obtain an aqueous solution of glycerol-ammonium carboxylate, and it is cooled for standby;

[0026] (3) The Bifidobacterium adolescentis cells and resistant starch are added to the aqueous solution of glycerol-ammonium carboxylate in step (2), stirred for 1 h, the stirring speed is 100 rpm, and at the same time, ultrasonic treatment is used, the ultrasonic frequency is 2 MHz, and the ultrasonic intensity is 0.8 W / cm 2, a suspension is obtained, and the cell count of Bifidobacterium adolescentis in the suspension is controlled to be 2×10 10 CFU, and the addition amount of resistant starch is 2.5 g / mL glycerol-ammonium citrate aqueous solution;

[0027] (4) The suspension in step (3) is allowed to stand at room temperature for 15 min for natural sedimentation separation, and the precipitate is recovered;

[0028] (5) The precipitate in step (4) is allowed to stand under sterile and anaerobic conditions for 2.5 h at a temperature maintained at 38°C;

[0029] (6) The precipitate after standing in step (5) is freeze-dried to obtain a highly stress-resistant Bifidobacterium adolescentis-resistant starch complex.

[0030] Example 2

[0031] (1) The Bifidobacterium adolescentis strain after 2 subcultures is cultured in CM0233 medium for 12 h, and the cells are collected by centrifugation to obtain Bifidobacterium adolescentis cells;

[0032] (2) Ammonium citrate at 0.6% of the mass of the aqueous solution of glycerol is added to the aqueous solution containing 3 wt% glycerol, and sterilized at 100°C for 15 min to obtain a glycerol-ammonium citrate aqueous solution, which is cooled for standby;

[0033] (3) The Bifidobacterium adolescentis cells and resistant starch are added to the glycerol-ammonium citrate aqueous solution in step (2), stirred for 1 h at a stirring speed of 120 rpm, and simultaneously treated with ultrasonic waves at an ultrasonic frequency of 3 MHz and an ultrasonic intensity of 0.6 W / cm 2 , a suspension is obtained, and the cell count of Bifidobacterium adolescentis in the suspension is controlled to be 5×10 10 CFU, and the addition amount of resistant starch is 3 g / mL glycerol-ammonium citrate aqueous solution;

[0034] (4) The suspension in step (3) is allowed to stand at room temperature for 15 min for natural sedimentation separation, and the precipitate is recovered;

[0035] (5) The precipitate in step (4) is allowed to stand under sterile and anaerobic conditions for 3 h at a temperature maintained at 38°C;

[0036] (6) The precipitate after standing in step (5) is freeze-dried to obtain a highly stress-resistant Bifidobacterium adolescentis-resistant starch complex.

[0037] Example 3

[0038] (1) The Bifidobacterium adolescentis strain after 2 subcultures is cultured in CM0233 medium for 15 h, and the cells are collected by centrifugation to obtain Bifidobacterium adolescentis cells;

[0039] (2) Add ammonium oxalate, which is 0.5% of the mass of the aqueous solution of glycerol, to the aqueous solution containing 5 wt% glycerol, and sterilize it at 100 °C for 15 min to obtain an aqueous solution of glycerol-ammonium carboxylate, and cool it for standby;

[0040] (3) Add Bifidobacterium adolescentis cells and resistant starch to the aqueous solution of glycerol-ammonium carboxylate obtained in step (2), stir for 0.8 h, and the stirring speed is 140 rpm. At the same time, use ultrasonic treatment, the ultrasonic frequency is 1 MHz, and the ultrasonic intensity is 1 W / cm 2 , to obtain a suspension, and control the number of Bifidobacterium adolescentis cells in the suspension to be 10 10 CFU, and the addition amount of resistant starch is 2 g / mL of the aqueous solution of glycerol-ammonium carboxylate;

[0041] (4) Let the suspension in step (3) stand at room temperature for 12 min, perform natural sedimentation separation, and recover the precipitate;

[0042] (5) Let the precipitate in step (4) stand under sterile and anaerobic conditions for 2 h, and keep the temperature at 40 °C;

[0043] (6) Freeze-dry the precipitate after standing in step (5) to obtain a Bifidobacterium adolescentis-resistant starch complex with high stress resistance.

[0044] Comparative Example 1

[0045] (1) For the Bifidobacterium adolescentis cells, culture the Bifidobacterium adolescentis strain that has been passaged twice in CM0233 medium for 15 h, and centrifuge to collect the cells, thus obtaining the Bifidobacterium adolescentis cells;

[0046] (2) Add Bifidobacterium adolescentis cells and resistant starch to water, stir for 1 h, and the stirring speed is 100 rpm. At the same time, use ultrasonic treatment, the ultrasonic frequency is 2 MHz, and the ultrasonic intensity is 0.8 W / cm 2 , to obtain a suspension, and control the number of Bifidobacterium adolescentis cells in the suspension to be 2×10 10 CFU, and the addition amount of resistant starch is 2.5 g / mL of water;

[0047] (3) Let the suspension in step (2) stand at room temperature for 15 min, perform natural sedimentation separation, and recover the precipitate;

[0048] (4) Freeze-dry the precipitate in step (3) to obtain a Bifidobacterium adolescentis-resistant starch complex.

[0049] Comparative Example 2

[0050] (1) The Bifidobacterium adolescentis strain after 2 subcultures was cultured in CM0233 medium for 15 h, and the cells were collected by centrifugation to obtain Bifidobacterium adolescentis cells.

[0051] (2) Bifidobacterium adolescentis cells and resistant starch were added to water, and stirred for 1 h at a stirring speed of 100 rpm. Meanwhile, ultrasonic treatment was used with an ultrasonic frequency of 2 MHz and an ultrasonic intensity of 0.8 W / cm 2 , to obtain a suspension. The cell count of Bifidobacterium adolescentis cells in the suspension was controlled to be 2×10 10 CFU, and the addition amount of resistant starch was 2.5 g / mL of water.

[0052] (3) The suspension in step (2) was allowed to stand at room temperature for 15 min for natural sedimentation separation, and the precipitate was recovered.

[0053] (4) The precipitate in step (3) was allowed to stand under sterile and anaerobic conditions for 2.5 h at a temperature maintained at 38°C.

[0054] (5) The precipitate after standing in step (4) was freeze-dried to obtain the Bifidobacterium adolescentis-resistant starch complex.

[0055] Comparative Example 3

[0056] (1) The Bifidobacterium adolescentis strain after 2 subcultures was cultured in CM0233 medium for 15 h, and the cells were collected by centrifugation to obtain Bifidobacterium adolescentis cells.

[0057] (2) Ammonium citrate at 0.5% of the mass of the aqueous solution containing 4 wt% glycerol was added to the aqueous solution of glycerol, and sterilized at 100°C for 10 min to obtain an aqueous solution of glycerol-ammonium carboxylate, which was cooled for standby.

[0058] (3) Bifidobacterium adolescentis cells and resistant starch were added to the aqueous solution of glycerol-ammonium carboxylate in step (2), and stirred for 1 h at a stirring speed of 100 rpm. Meanwhile, ultrasonic treatment was used with an ultrasonic frequency of 2 MHz and an ultrasonic intensity of 0.8 W / cm 2 , to obtain a suspension. The cell count of Bifidobacterium adolescentis cells in the suspension was controlled to be 2×10 10 CFU, and the addition amount of resistant starch was 2.5 g / mL of the aqueous solution of glycerol-ammonium carboxylate.

[0059] (4) The suspension in step (3) was allowed to stand at room temperature for 15 min for natural sedimentation separation, and the precipitate was recovered.

[0060] (5) The precipitate in step (4) was freeze-dried to obtain the Bifidobacterium adolescentis-resistant starch complex.

[0061] Performance Test:

[0062] The adhesion rates of the high stress-resistant Bifidobacterium adolescentis - resistant starch complex prepared in Example 1 and the Bifidobacterium adolescentis - resistant starch complexes prepared in Comparative Examples 1 to 3 were tested, and the survival rates of Bifidobacterium adolescentis after being placed in simulated gastric juice (10 mg·mL-1 pepsin at pH = 2) and simulated intestinal juice (0.3% sodium cholate) for two hours were tested. The results are shown in Table 1 below:

[0063] Table 1

[0064]

[0065] As can be seen from Table 1, glycerol was used to adjust the activity and viscosity of the aqueous solution to provide a suitable macroscopic environment for the adhesion of Bifidobacterium adolescentis. Ammonium carboxylate was used to change the pH environment of the solution and the state of the surface proteins of Bifidobacterium adolescentis, reduce the surface tension of starch, and facilitate the approach of Bifidobacterium adolescentis proteins to the starch surface. Through static treatment at 38 - 40 °C under sterile and anaerobic conditions for 2 - 3 h, the adhered Bifidobacterium adolescentis could grow initially, and then be better fixed on the surface of resistant starch. The high stress-resistant Bifidobacterium adolescentis - resistant starch complex prepared by the method of the present invention solves the problem of poor stress resistance of Bifidobacterium adolescentis and maintains a higher survival rate in the gastrointestinal tract.

Claims

1. A preparation method of a Bifidobacterium adolescentis - resistant starch complex with high stress resistance, characterized in that, It includes the following steps: (1) Add ammonium carboxylate to an aqueous solution containing 3-5 wt% glycerol, sterilize at high temperature to obtain an aqueous solution of glycerol-ammonium carboxylate, and cool for standby; (2)Add the Bifidobacterium adolescentis cells and the resistant starch prepared from bean starch to the glycerol-ammonium carboxylate aqueous solution in step (1), stir at a stirring speed of 100-150 rpm for 0.5-1 h, and at the same time, perform ultrasonic treatment with an ultrasonic frequency of 1-3 MHz and an ultrasonic intensity of 0.5-1 W / cm 2 to obtain a suspension; (3) Let the suspension in step (2) stand for 10-15 min, and separate and recover the precipitate; (4) Let the precipitate in step (3) stand for 2-3 h under sterile and anaerobic conditions, and keep the temperature at 38-40 °C; (5) Freeze-dry the precipitate after standing in step (4) to obtain a high stress-resistant Bifidobacterium adolescentis-resistant starch complex.

2. The preparation method of the Bifidobacterium adolescentis - resistant starch complex with high stress resistance according to claim 1, characterized in that, In step (1), the addition amount of ammonium carboxylate is 0.3-0.6% of the mass of the glycerol aqueous solution.

3. The preparation method of the Bifidobacterium adolescentis - resistant starch complex with high stress resistance according to claim 1, characterized in that The ammonium carboxylate described in step (1) is one of ammonium oxalate and ammonium citrate.

4. The preparation method of the Bifidobacterium adolescentis-resistant starch complex with high stress resistance according to claim 1, characterized in that, In step (2), the cell count of Bifidobacterium adolescentis in the suspension is 10 10 ~10 12 CFU; the addition amount of resistant starch is 2 - 3 g / mL of glycerol-ammonium carboxylate aqueous solution.

5. The preparation method of the Bifidobacterium adolescentis - resistant starch complex with high stress resistance according to claim 1, wherein, The method for obtaining Bifidobacterium adolescentis cells in step (2) is: culture the Bifidobacterium adolescentis strain that has been passaged 2-3 times in CM0233 medium for 10-15 h, and centrifuge to collect the cells, thus obtaining Bifidobacterium adolescentis cells.

6. A high stress-resistant Bifidobacterium adolescentis-resistant starch complex prepared by the preparation method according to any one of claims 1-5.

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