Coconut milk base freeze-drying protective agent capable of improving acid resistance of bifidobacterium and application of coconut milk base freeze-drying protective agent

Abstract

The invention provides a coconut milk base freeze-drying protective agent capable of improving acid resistance of bifidobacterium and application of the coconut milk base freeze-drying protective agent, and belongs to the technical field of microorganisms. The coconut milk base freeze-drying protective agent comprises a component 1, wherein the component 1 is one of coconut milk powder or coconut milk. The bifidobacterium freeze-dried product protected by the freeze-drying protective agent can also improve the survival rate of bifidobacterium treated by simulated gastric juice with the pH value of 2.0. When the freeze-drying protective agent is used for preparing freeze-dried bifidobacterium powder, the freeze-drying survival rate of the bifidobacterium reaches 94.21 + / -3.8%, and the freeze-drying survival rate of the bifidobacterium reaches 83.89 + / -9.64% after the freeze-dried bifidobacterium powder is treated in simulated gastric juice with the pH value of 2.0 for 2 hours.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a coconut milk-based freeze-drying protective agent capable of improving the acid resistance of bifidobacteria and application thereof. Background Art

[0002] Probiotics are a type of active microorganisms that play a beneficial role in the health or physiological functions of the host by promoting the ecological balance of various microbial flora in the host's intestines. As a type of probiotic, Bifidobacterium has many physiological functions such as enhancing immune function, anti-tumor, and anti-aging. It is used in the development and research of fermented dairy products, probiotic beer, fermented fruit and vegetable juice, probiotic solid beverages and other foods. However, Bifidobacterium is a strict anaerobic bacterium and is very sensitive to oxygen, gastric acid and other environments. This causes a large loss of Bifidobacterium in the production and processing of probiotic foods, increasing the difficulty of its application; in addition, Bifidobacterium will also die in large numbers during gastrointestinal digestion, thereby reducing the number of live bacteria that finally reach the intestines and reducing its health intervention function.

[0003] In order to improve the survival rate of bifidobacteria during the production, storage, transportation and digestion of bacterial powder, microencapsulation technology has gradually been used and developed. The basic principle of microencapsulation technology is to use natural or synthetic polymer materials to encapsulate probiotics in one or more layers of protective barriers through physical or chemical methods, thereby effectively isolating oxygen, acidic media, bile and other external adverse factors, ensuring that probiotics can maintain high activity during production, storage and passage through the gastrointestinal tract. At present, there have been many studies on probiotic microencapsulation technology, but most of the technologies have complex processing, high economic costs, safety risks (such as some encapsulation materials or auxiliary reagents used in the preparation process may have residual problems, posing a potential threat to human safety), and large loss of viable bacteria after encapsulation, and unsatisfactory anti-adversity effects, which limit its promotion and application.

[0004] Therefore, it is very necessary to develop probiotic encapsulation materials and methods that are natural, safe, simple in production process, and have high protection capabilities. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a coconut milk-based freeze-drying protective agent and application thereof which can improve the acid resistance of bifidobacteria. The agent takes natural coconut milk as a matrix, has a simple preparation process, and the prepared bacterial powder product has a high survival rate, is resistant to gastric acid, and is resistant to storage.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] The invention provides a coconut milk-based freeze-drying protective agent, comprising a component 1, wherein the component 1 is one of coconut milk powder or coconut milk.

[0008] Preferably, the mass volume concentration of the coconut milk powder in the coconut milk-based freeze-drying protective agent is 30 g / L to 300 g / L.

[0009] More preferably, the coconut milk powder is the product of vacuum freeze-drying of coconut milk.

[0010] More preferably, the dry matter yield of the coconut milk after vacuum freeze-drying is 33.34±0.15%.

[0011] More preferably, the coconut milk powder has composition characteristics of 1.66±0.02% moisture, 13.05±0.04% protein, 59.35±0.03% fat and 3.90±0.01% ash.

[0012] Preferably, component 2 is also included, and component 2 is one or more of fructooligosaccharides, galacto-oligosaccharides, trehalose, stachyose, sucrose, sorbitol, resistant starch, maltodextrin, sodium caseinate, skimmed milk powder, sodium glutamate, glycine or glycerol.

[0013] More preferably, the mass volume concentration of the component 2 in the coconut milk-based freeze-drying protectant is 4 g / L to 200 g / L.

[0014] Preferably, the coconut milk-based freeze-drying protective agent comprises 30 g / L to 150 g / L coconut milk powder, 20 g / L to 100 g / L fructooligosaccharides, 8 g / L to 40 g / L sodium glutamate, and 6 g / L to 30 g / L glycerol.

[0015] More preferably, the coconut milk-based freeze-drying protective agent comprises 120 g / L coconut milk powder, 60 g / L fructooligosaccharides, 24 g / L sodium glutamate, and 12 g / L glycerol.

[0016] The present invention also provides a method for preparing coconut milk-based bifidobacterium freeze-dried powder, which is achieved by using the coconut milk-based freeze-drying protective agent.

[0017] Preferably, the method comprises: centrifuging the bifidobacterium culture solution to collect the bacteria; resuspending the bacteria in physiological saline to obtain a bacterial suspension; mixing the bacterial suspension with the coconut milk-based freeze-drying protective agent to obtain a mixed solution; freeze-drying the mixed solution to obtain coconut milk-based bifidobacterium freeze-dried powder.

[0018] More preferably, the viable bacterial count of the bacterial suspension is 10^9 CFU / mL.

[0019] More preferably, the volume ratio of the bacterial suspension to the coconut milk-based freeze-drying protective agent is 4:1.

[0020] More preferably, the freeze-drying is completed in a vacuum freeze dryer, including pre-freezing, primary drying, gradient heating and secondary drying; the pre-freezing is to control the temperature of the layer plate to be cooled to -35°C and maintained for 4 to 6 hours; the primary drying is to control the temperature of the layer plate to be heated to -26°C for 1 hour and maintained for 20 to 30 hours; the gradient heating is to control the temperature of the layer plate to be heated to 0°C for 3 hours and maintained for 2 hours; the secondary drying is to control the temperature of the layer plate to be heated to 25°C for 2 hours and maintained for 5 to 10 hours.

[0021] More preferably, the bifidobacterium is Bifidobacterium animalis H22B-648.

[0022] The present invention also provides a coconut milk-based bifidobacterium freeze-dried powder, which is prepared by the method.

[0023] The present invention also provides application of the coconut milk-based freeze-drying protective agent or the method in freeze-drying of bifidobacteria.

[0024] Beneficial Effects

[0025] The invention provides a coconut milk-based freeze-drying protective agent capable of significantly improving the freeze-drying survival rate and acid resistance of bifidobacteria. The invention takes natural coconut milk as a matrix and has a simple preparation process. When bifidobacterium freeze-dried powder is prepared by using the coconut milk-based freeze-drying protective agent, the freeze-drying survival rate of the bifidobacteria reaches 94.21±3.8%, and the freeze-drying survival rate of the bifidobacteria reaches 83.89±9.64% after the bifidobacterium freeze-dried powder is treated with simulated gastric juice at pH 2.0 for 2 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Table 5 shows the freeze-dried survival rates of the freeze-dried coconut milk-based Bifidobacterium powders 1 to 20. DETAILED DESCRIPTION

[0027] The present invention is described in detail below in conjunction with specific implementation methods. The following specific embodiments are helpful for those skilled in the art to further understand the present invention, but do not limit the present invention in any form.

[0028] Example

[0029] 1. Experimental equipment and materials

[0030] The freeze dryer was purchased from Jinan Junde Instrument Co., Ltd., model FD-604.

[0031] Coconut milk raw materials were purchased from Hainan Taifengyuan Industrial Co., Ltd.; oligofructose was purchased from Shandong Bailong Chuangyuan Biotechnology Co., Ltd.; glycine was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; resistant starch, galacto-oligosaccharide, sodium glutamate, and pepsin were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; agar powder, cysteine ​​hydrochloride, and skim milk were purchased from Shanghai Bevanta Biotechnology Co., Ltd.; trehalose, stachyose, sucrose, sorbitol, maltodextrin, and sodium caseinate were purchased from Henan Gaobao Industrial Co., Ltd.; glycerol and sodium chloride were purchased from Xilong Science Co., Ltd.; PBS was purchased from Beijing Solebow Technology Co., Ltd.

[0032] The bifidobacterium used was Bifidobacterium animalis, which was deposited in the “Hainan Tropical Fungus Resource Bank” of Hainan University with the deposit number H22B-648 and the deposit address is Building 503, National Key Laboratory of Tropical Crops, Hainan University, No. 58 Renmin Avenue, Meilan District, Haikou City, Hainan Province.

[0033] c-MRS liquid culture medium: casein digest 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L, dimethyl phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1.0 g / L, cysteine ​​hydrochloride 1.0 g / L.

[0034] c-MRS solid culture medium: casein digest 10.0 g / L, beef extract powder 10.0 g / L, yeast extract powder 4.0 g / L, triammonium citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate heptahydrate 0.2 g / L, manganese sulfate tetrahydrate 0.05 g / L, dimethyl phosphate 2.0 g / L, glucose 20.0 g / L, Tween-80 1.0 g / L, cysteine ​​hydrochloride 1.0 g / L, agar 20.0 g / L.

[0035] Simulated gastric fluid: 3.5 g of pepsin was fully dissolved in 1 L of PBS buffer, adjusted to pH 2.0 with 2 M HCl solution, and sterilized by filtration through a 0.22 μm filter membrane.

[0036] 2. Experimental Methods

[0037] 2.1 Strain activation

[0038] The Bifidobacterium animalis H22B648 glycerol storage tube frozen and stored in the "Hainan Tropical Bacteria Resource Bank" was taken out and thawed at 24°C. The storage solution was dipped into an inoculation loop that was cooled to room temperature after burning and then streaked on a c-MRS plate culture medium. The plate was transferred to an anaerobic workstation and cultured in an anaerobic environment at 37°C for 48 hours. A single colony on the plate was picked and inoculated into a 5 mL c-MRS liquid culture medium test tube, and anaerobically cultured at 37°C for 24 hours. The plate was evenly shaken and transferred to a c-MRS liquid culture medium at a 4% V / V inoculation amount. After culturing for 24 hours, the second-generation bacterial solution was obtained.

[0039] 2.2 Preparation of bacterial suspension

[0040] The second-generation bacterial liquid obtained by continuous subculture was inoculated into fresh c-MRS liquid culture medium at an inoculum size of 4% V / V for enrichment culture. After static culture in an anaerobic workstation at 37°C for 24 hours, the bacteria were collected by centrifugation at 3050×g, 4°C for 15 minutes, resuspended in physiological saline and centrifuged. After repeating twice, the bacteria were resuspended in an appropriate amount of physiological saline to obtain a bacterial suspension, counted by the pouring method, and the volume of physiological saline used for resuspending the bacteria was adjusted so that the concentration of the bacterial suspension was about 10^9 CFU / mL.

[0041] 2.3 Preparation and characterization of freeze-dried coconut milk powder

[0042] Thaw the coconut milk stored at -20℃ in a 37℃ water bath, shear and homogenize, centrifuge at 40℃, discard the upper coconut oil, homogenize and freeze-dry to obtain coconut milk powder, store at 4℃ away from light for later use, and record and calculate the water loss rate (WL) of freeze-dried coconut milk:

[0043]

[0044] The moisture, ash, fat and crude protein of the freeze-dried coconut milk powder were determined respectively according to the first method of "GB5009.3-2016 National Food Safety Standard Determination of Water in Food", the first method of "GB 5009.4-2016 National Food Safety Standard Determination of Ash in Food", the second method of "GB 5009.6-2016 National Food Safety Standard Determination of Fat in Food" acid hydrolysis method, and the first method of "GB 5009.5-2016 National Food Safety Standard Determination of Protein in Food" Kjeldahl nitrogen method.

[0045] In this embodiment, the dry matter yield of coconut milk after vacuum freeze drying is 33.34±0.15%, the water content in coconut milk powder is 1.66±0.02%, the protein content is 13.05±0.04%, the fat content is 59.35±0.03%, and the ash content is 3.90±0.01%.

[0046] 2.4 Bifidobacterium viable count detection method

[0047] The method for detecting the number of live bifidobacteria adopts "GB 4789.34-2016 National Food Safety Standard Food Microbiology Inspection Bifidobacterium Inspection".

[0048] 2.5 Evaluation of freeze-drying protective effect of protective agent

[0049] The freeze-drying protection effect of the protective agent is evaluated by the freeze-drying survival rate of Bifidobacterium, and the specific method is as follows:

[0050] Before freeze-drying, take 1 mL of the sample to be tested, weigh it and record the weight m 1 , count the live bacteria, and the total number of live bacteria is recorded as F 1 ; 1mL sample is weighed after freeze-drying and recorded as m 2 , add m 3 Sterile deionized water of weight, mix thoroughly and count, the total number of viable bacteria is recorded as F 2 .

[0051] The formula is as follows:

[0052] m 3 =m 1 -m 2

[0053]

[0054] 2.6 Evaluation of gastric acid resistance of freeze-dried Bifidobacterium powder

[0055] Weigh 0.20g of the freeze-dried bacterial powder to be tested, add 5mL of simulated gastric juice to disperse, and culture for 2h in a constant temperature shaker (37℃, 180rpm) to simulate the digestive environment. Take 1mL of the gastric juice suspension of the above bacterial powder for counting; 0.2mL of physiological saline bacterial suspension is used as the control group. The gastric acid resistance survival rate of the freeze-dried bacterial powder is calculated according to the following formula:

[0056]

[0057] Where: N is the number of viable bacteria per unit (CFU / g) after the sample is treated with simulated gastric juice, N 0 It is the initial viable bacterial count (CFU / g) of the sample before being treated with simulated gastric fluid.

[0058] 3. Preparation of Coconut Milk-Based Freeze-Drying Protectant and Freeze-Dried Powder Containing Only Component 1

[0059] Coconut milk-based freeze-drying protective agents 1 to 4 were prepared according to the formula in Table 1 using deionized water as solvent.

[0060] Table 1 Coconut milk base freeze-drying protective agent 1 to 4 formula

[0061] Group formula Coconut milk based freeze-dried protective agent 1 Coconut milk powder 32.5g / L Coconut milk based freeze-dried protective agent 2 Coconut milk powder 75g / L Coconut milk based freeze-dried protective agent 3 Coconut milk powder 150g / L Coconut milk based freeze-dried protective agent 4 Coconut milk powder 300g / L

[0062] In the present invention, "mass volume concentration (unit: g / L)" refers to the proportion of the mass of each solute in the total volume of the coconut milk-based freeze-drying protective agent aqueous solution.

[0063] The coconut milk-based freeze-dried protective agent volume: bacterial suspension volume = 4:1 was fully mixed, and the control group was sterile deionized water volume: bacterial suspension volume = 4:1; after freeze-drying, coconut milk-based bifidobacterium freeze-dried powders 1 to 4 were obtained. The number of viable bifidobacteria in coconut milk-based bifidobacterium freeze-dried powders 1 to 4 was detected and the survival rate was calculated. The test results are shown in Table 2.

[0064] Table 2 Freeze-dried survival rate of bifidobacterium lyophilized powder 1 to 4 based on coconut milk

[0065]

[0066]

[0067] 4. Preparation of Coconut Milk-Based Freeze-Drying Protectant and Freeze-Dried Powder Containing Component 1 and Component 2

[0068] Use deionized water as solvent to prepare 1-70 coconut milk-based freeze-drying protective agents according to the formula in Table 3.

[0069] Table 3 Coconut milk base freeze-drying protective agent 1-70 formula

[0070]

[0071]

[0072] The coconut milk-based freeze-dried protective agent volume: bacterial suspension volume = 4:1 was fully mixed, and the control group was sterile deionized water volume: bacterial suspension volume = 4:1; after freeze-drying, coconut milk-based bifidobacterium freeze-dried powder 1-70 was obtained. The number of viable bifidobacteria in the bifidobacterium freeze-dried powder 1-70 was detected and the survival rate was calculated (the test results are shown in Table 4).

[0073] Table 4 Freeze-dried survival rate of bifidobacterium freeze-dried powder 1-70 based on coconut milk

[0074]

[0075]

[0076] 5. Preparation of Coconut Milk-Based Bifidobacterium Freeze-Dried Powder

[0077] On the basis of “IV. Preparation of coconut milk-based freeze-dried protective agent and freeze-dried powder containing component 1 and component 2”, the concentration of coconut milk powder was adjusted to 30g / L~150g / L; component 2 was set as a combination of oligofructose, sodium glutamate and glycerol, among which oligofructose was 20~100g / L, sodium glutamate was 8~40g / L, glycerol was 6~30g / L, and deionized water was also used as the solvent. Each group controlled a single variable. The specific grouping and configuration method are shown in Table 5.

[0078] Table 5 Coconut milk base freeze-drying protective agent 1-20 formula

[0079]

[0080] The coconut milk-based freeze-dried protective agent volume: bacterial suspension volume = 4:1 was fully mixed, and the control group was sterile deionized water volume: bacterial suspension volume = 4:1; after freeze drying, 1-20 coconut milk-based bifidobacterium freeze-dried powders were obtained; the number of viable bifidobacteria in the coconut milk-based bifidobacterium freeze-dried powder was detected and the survival rate was calculated (the results are shown in Figure 1 ). Although the average value of this factor was different after compounding, there was no statistically significant difference (P>0.05).

[0081] VI. Preparation of more preferably coconut milk-based bifidobacterium freeze-dried powder

[0082] On the basis of “V. Preparation of Bifidobacterium freeze-dried powder based on coconut milk”, the concentration of coconut milk powder was adjusted to 120 g / L; component 2 was set to be a combination of oligofructose, sodium glutamate and glycerol, including 60 g / L of oligofructose, 24 g / L of sodium glutamate, and 12 g / L of glycerol, and the solvent was deionized water.

[0083] The coconut milk-based freeze-dried protective agent volume: bacterial suspension volume was fully mixed in a ratio of 4:1, and the control group was a mixture of sterile deionized water volume: bacterial suspension volume = 4:1; the coconut milk-based bifidobacterium freeze-dried powder was obtained after freeze drying; the number of live bifidobacteria in the coconut milk-based bifidobacterium freeze-dried powder was detected and the survival rate was calculated (the results are shown in Table 6); the number of live bifidobacteria in the coconut milk-based bifidobacterium freeze-dried powder after treatment with simulated gastric juice at pH 2.0 for 2 hours was detected and the survival rate was calculated. The results are shown in Table 6.

[0084] Table 6 Freeze-dried survival rate and gastric juice-resistant survival rate of bifidobacterium lyophilized powder based on coconut milk

[0085] Group Freeze-dried survival rate Survival rate after 2 h treatment with simulated gastric fluid at pH 2.0 Control group 1.59±0.24％ 0.21±0.06％ Coconut milk based Bifidobacterium freeze-dried powder 94.21±3.8％ 83.89±9.64％

[0086] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A coconut milk-based freeze-drying protective agent, comprising component 1, wherein component 1 is one of coconut milk powder or coconut milk.

2. The coconut milk-based freeze-drying protective agent according to claim 1, characterized in that: The mass volume concentration of the coconut milk powder in the coconut milk-based freeze-drying protective agent is 30 g / L to 300 g / L.

3. A coconut milk-based freeze-drying protective agent according to claim 1, characterized in that: The invention also includes component 2, which is one or more of fructooligosaccharides, galacto-oligosaccharides, trehalose, stachyose, sucrose, sorbitol, resistant starch, maltodextrin, sodium caseinate, skimmed milk powder, sodium glutamate, glycine, cysteine ​​or glycerol.

4. A coconut milk-based freeze-drying protective agent according to claim 3, characterized in that: The mass volume concentration of the component 2 in the coconut milk-based freeze-drying protective agent is 4 g / L to 200 g / L.

5. The coconut milk-based freeze-drying protective agent according to claim 1, characterized in that: The coconut milk-based freeze-drying protective agent comprises 30 g / L to 150 g / L of coconut milk powder, 20 g / L to 100 g / L of oligofructose, 8 g / L to 40 g / L of sodium glutamate, and 6 g / L to 30 g / L of glycerol.

6. A method for preparing coconut milk-based bifidobacterium freeze-dried powder, which is achieved by using a coconut milk-based freeze-drying protective agent according to any one of claims 1 to 5.

7. The method according to claim 6, characterized in that The method comprises: centrifuging bifidobacterium culture fluid to collect bacterial cells; resuspending the bacterial cells in physiological saline to obtain bacterial suspension; mixing the bacterial suspension with the coconut milk-based freeze-drying protective agent to obtain a mixed solution; freeze-drying the mixed solution to obtain coconut milk-based bifidobacterium freeze-dried powder.

8. The method according to claim 6, characterized in that The viable bacteria count of the bacterial suspension is 10^9 CFU / mL; the volume ratio of the bacterial suspension to the coconut milk-based freeze-drying protective agent is 4:1; the freeze-drying is completed in a vacuum freeze dryer, including pre-freezing, primary drying, gradient heating and secondary drying; the pre-freezing is to control the temperature of the layer plate to cool to -35°C and keep it for 4 to 6 hours; the primary drying is to control the temperature of the layer plate to -26°C for 1 hour and keep it for 20 to 30 hours; the gradient heating is to control the temperature of the layer plate to 0°C for 3 hours and keep it for 2 hours; the secondary drying is to control the temperature of the layer plate to 25°C for 2 hours and keep it for 5 to 10 hours.

9. A coconut milk-based bifidobacterium freeze-dried powder, prepared by the method according to claim 6.

10. Use of a coconut milk-based freeze-drying protective agent according to any one of claims 1 to 5 or the method according to claim 6 in freeze-drying of bifidobacteria.

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