CN104630099A - Bifidobacterium longum strain and application thereof in preparing active Bifidobacterium fermented beverage - Google Patents

Abstract

The invention discloses a bifidobacterium longum and its application in preparing active bifidobacterium fermented beverages. The Bifidobacterium longum (Bifidobacterium longum) BF-10 has a preservation number of CGMCC No.7789. Bifidobacterium longum (Bifidobacterium longum) BF-10 CGMCC №.7789 can be used to prepare active bifidobacterium fermented beverages. The bifidobacterium-fermented seabuckthorn and wolfberry compound live bacteria beverage prepared by the invention has mild color, good taste, good flavor, high number of viable bacteria and long shelf life, which can not only exert the nutritional and physiological effects of seabuckthorn and wolfberry, but also strengthen bifidobacteria It is a health drink with more physiological functions.

Description

A strain of bifidobacterium longum and its application in the preparation of active bifidobacterium fermented beverage

technical field

The invention relates to a bifidobacterium strain and its application in preparing active bifidobacterium fermented beverages, in particular to a bifidobacterium longum strain and a process for fermenting seabuckthorn and wolfberry compound juice live bacteria beverage.

Background technique

Lactic acid bacteria drinks are very popular among consumers due to their rich nutrition and health benefits. Commercially available lactic acid bacteria beverages are mainly divided into two categories: living bacteria and non-living bacteria. The live bacteria type is fermented and contains a large amount of live bacteria, which has been scientifically proven to have beneficial physiological effects on the human body; the non-viable type has almost no health benefits. Therefore, the development of live bacteria beverage is bound to become a research hotspot of lactic acid bacteria beverage.

At present, in the lactic acid bacteria drinks that have been developed at home and abroad, the fermentation strains used are mostly Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei (Lactobacillus paracasei) etc., have a few to also adopt bifidobacterium, as animal bifidobacterium (Bifidobacterium animal) etc., also do not see the patent and literature report of bifidobacterium longum active fermented beverage. Bifidobacterium spp. is a typical beneficial lactic acid bacteria parasitic in the intestinal tract of humans and animals. It can play a biological barrier, nutrition, immunity, anti-aging, anti-tumor and other physiological effects on the host. Fermenting bifidobacterium to produce live bacteria beverage will greatly enhance its health function and increase the added value of the product.

In the lactic acid bacteria drinks that have been developed, the fermentation raw materials used mainly include: 1) dairy products, such as milk, soybean milk, coconut milk, etc.; 2) fruit and vegetable products, such as carrots, apples, corn, pumpkin, red dates, tomatoes, small Berries, etc.; 3) Nuts, such as peanuts, walnuts, etc. At present, there is no bifidobacterium drink that uses seabuckthorn and wolfberry compound juice as a fermented raw material on the market.

Hippophae rhamnoides Linn (Hippophae rhamnoides Linn), also known as vinegar willow and ink thorn, is the mature fruit of Hippophae rhamnoides Linn, a plant of the genus Hippophae rhamnoides. It is a medicinal and edible plant, and the fruit contains rich nutrients and biologically active substances. In particular, vitamin C, which is rich in fruit, can promote the proliferation of bifidobacteria.

Lycium barbarum (Gycium barbarum), as a traditional Chinese herbal medicine and an important economic crop in my country, has a variety of physiological activities and health functions. Clinical medicine shows that wolfberry has a significant effect on diabetes, hypertension, central retinitis, optic atrophy, nephritis and hepatitis, and the polysaccharides in wolfberry have a growth-promoting effect on bifidobacteria.

The live bacteria drink made from bifidobacterium-fermented seabuckthorn and wolfberry compound juice can not only exert the nutritional and physiological effects of seabuckthorn and wolfberry, but also enhance the health care effect of bifidobacteria. It will definitely become a high-value-added live bacteria drink.

Contents of the invention

The purpose of the present invention is to provide a strain of Bifidobacterium longum with excellent probiotic properties and its application in the preparation of active Bifidobacterium fermented beverages.

Bifidobacterium longum (Bifidobacterium longum) BF-10 has been preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short) on June 21, 2013. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing , Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is: CGMCC №.7789.

The Bifidobacterium longum CGMCC №.7789 provided by the present invention has health care functions such as resistance to digestive tract adverse environment, biological antagonism and anti-oxidation, and can be used to prepare Bifidobacterium live bacteria beverage.

The present invention also provides a preparation method of live bacteria beverage of seabuckthorn and wolfberry compound juice fermented by Bifidobacterium longum, which is to activate Bifidobacterium longum (Bifidobacterium longum) BF-10 and inoculate it in compound juice containing seabuckthorn and wolfberry juice, milk powder and sucrose After anaerobic cultivation at a certain temperature for a certain period of time in the compound fruit juice culture medium of the solution, the fermentation is completed to obtain a bifidobacterium-fermented seabuckthorn and wolfberry compound juice living bacteria beverage.

In the method, the fermentation medium of seabuckthorn and wolfberry compound juice is prepared according to the following method: fresh seabuckthorn and water with a mass ratio of 1:1 are mixed for beating, and filtered with four layers of gauze to obtain seabuckthorn juice with a mass percentage of 50%; Weigh the fruit of Lycium barbarum, add it to the triangular flask according to the mass ratio of material and water at 1:10, soak it in a water bath at 85°C for two hours, beat it, filter it with four layers of gauze, and obtain the Lycium barbarum extract with an active ingredient mass percentage of 10%. The above-mentioned seabuckthorn juice and Chinese wolfberry extract were mixed in a volume ratio of 2:1 to obtain a mixed fruit juice; 75 g of sucrose was dissolved in 100 mL of water to obtain a sucrose solution; 10 g of skim milk powder was added to 1 L of water, and fully stirred to dissolve to obtain skim reconstituted milk; Fruit juice 50%, sucrose solution 12%, skimmed reconstituted milk 10%, water 28% prepare compound fruit juice culture medium; Carry out homogenization for the first time under 8MPa condition with homogenizer, carry out second homogenization under 12MPa condition, and use Baking soda (N _a HCO ₃ ) adjusts its pH to about 6.0; finally, fill the compound fruit juice fermentation medium with nitrogen to reduce the redox potential to below -0.03eV, and sterilize at 115°C for 10 minutes.

In the method, the fermentation temperature of the bifidobacterium-fermented live bacteria beverage of seabuckthorn and wolfberry compound juice is 38-42° C., the fermentation time is 24-26 hours, the initial pH value is 6.0, and the inoculation amount is 1×10 ⁶ cfu/mL.

The present invention uses Bifidobacterium longum BF-10CGMCC №.7789 as the fermentation strain, and at the same time selects sea buckthorn juice and Chinese wolfberry extract which are rich in nutrition and contain physiologically active substances that can promote the proliferation of bifidobacteria as fermentation materials, and add a certain amount of sucrose and skimmed milk to prepare a bifidobacterium-fermented seabuckthorn and wolfberry compound juice live bacteria beverage. After the compound fruit juice is fermented by the Bifidobacterium strain, the number of viable bacteria can reach 8.7×10 ¹⁰ CFU/mL, and at the same time, it can also produce acetic acid, lactic acid, B vitamins and other metabolites with important physiological activities, making the product form a good sensory and nutritional qualities. The finished product is aseptically filled and can be stored for 19 days at 4°C and 8 days at room temperature (22°C). The bifidobacterium-fermented seabuckthorn and wolfberry compound juice living bacteria beverage prepared by the invention has mild color, good taste, good flavor, high number of viable bacteria, and long shelf life. The health function of mycobacteria is a health drink with more physiological functions.

Description of drawings

Fig. 1 is the bifidobacterium-fermented seabuckthorn and wolfberry compound juice living bacteria beverage sample (one of the packaging forms) of the present invention.

Fig. 2 is a graph showing the changes in the number of viable bifidobacteria in fermented beverages over time under different storage temperatures.

Detailed ways

The experimental methods described in the following examples are conventional methods unless otherwise specified.

Example 1. Isolation and identification of Bifidobacterium longum (Bifidobacterium longum) BF-10CGMCC №.7789

1. Screening of excellent bifidobacteria strains

In order to obtain excellent Bifidobacterium strains resistant to the adverse environment of the digestive tract, 5 strains of Bifidobacteria derived from the feces of healthy breast-fed infants and the intestines of long-lived elderly people (Layisu Village, Yutian County, Hetian, Xinjiang) were used as the starting strains, and the improved MRS was used. Liquid culture medium simulates the environment of the human gastrointestinal tract to study its resistance to adverse environmental characteristics of the digestive tract. The starting strain numbers and sources are shown in Table 1.

(1) Acid tolerance test: Based on the PBS buffer solution with pH 7.0, adjust it to pH 3.0 with 37% hydrochloric acid, sterilize at 121°C for 20min, and insert the inoculum with 5% inoculum. The liquid culture of Bifidobacterium BF-10 activated for 3 generations was cultured anaerobically at 37°C, and samples were taken at 0 and 2 hours respectively. The number of viable bacteria was determined by the Hengate anaerobic rolling tube method, and the survival rate was calculated.

(2) Bile salt tolerance test: the liquid culture after 3 generations of strain activation was inserted into the improved MRS liquid medium containing 0.5% sodium taurocholate at a 2% inoculum size, and anaerobically cultured at 37°C After 12 hours, samples were taken to determine the number of viable bacteria; at the same time, the improved MRS liquid medium without bile salt was used as a control to determine the number of viable bacteria. Calculate the survival rate.

The composition of the modified MRS medium used is as follows (calculated in 1000 mL): 20 g of glucose, 10 g of beef extract powder, 10 g of peptone, 5 g of yeast extract powder, 3 g of corn extract, 5 g of anhydrous sodium acetate, 2 g of triamine citrate, and dipotassium hydrogen phosphate 2g, manganese sulfate 0.25g, magnesium sulfate 0.58g, L-cysteine hydrochloride 0.4g, Tween-80 1mL.

(3) Pepsin resistance test: Based on the phosphate buffer solution of pH 7.0, adjust it to pH 3.0 with 37% hydrochloric acid by volume fraction, then add 0.5% pepsin as simulated gastric juice, and use 5% inoculum Insert the liquid culture after 3 generations of activation of the strains, take samples at 0 and 2 hours to determine the number of viable bacteria and calculate the survival rate.

(4) Trypsin resistance test: Based on 1/15mol/mL, pH7.0 phosphate buffer, add 10mg/mL trypsin as simulated intestinal fluid, inoculate the bacterial solution aseptically at 5% of the inoculated amount, and keep at 37°C Under anaerobic conditions, samples were taken at 0h and 12h to determine the number of viable bacteria, and the survival rate was calculated.

Table 1 Comparison of the tolerance of the five tested strains to the adverse environment of the digestive tract

As can be seen from the screening results in Table 1, the survival rate of bacterial strain BF-10 after acid treatment was 96.1%, the survival rate after bile salt and pepsin treatment were all above 86%, and the strain increased after 12 hours of trypsin treatment, indicating that Bifidobacterium BF-10 has strong resistance to acid, bile salt, pepsin and trypsin, and the survival rate after the four treatments is significantly higher than that of other tested strains. It can be seen from the results that the strain BF-10 has great potential as a probiotic preparation, so Bifidobacterium BF-10 is selected as the target excellent strain.

2. Identification of excellent strains

The target strain BF-10 was taxonomically identified by morphological observation, API 20A physiological and biochemical reactions and 16S rDNA sequence analysis. Strain BF-10 grows well in the improved solid MRS anaerobic tube, the colony is white, round, with smooth and tidy edges, no obvious colonies under aerobic conditions; the picked colonies are diluted and then Gram stained, observed under a microscope It was found that BF-10 cells were G ⁺ , rod-shaped, and partly branched in a V or Y shape. They had no spores, did not move, and were arranged irregularly. According to the API bacterial identification standard, 20 kinds of sugar alcohol fermentation tests were carried out on the strain BF-10 using the API 20A anaerobic identification system. The reaction results of the reagent strips were analyzed by looking up the table, and it was preliminarily determined that BF-10 belonged to the genus Bifidobacterium spp. The DNA genome of strain BF-10 was extracted, and a 1349bp DNA sequence fragment (see sequence listing) was obtained by PCR amplification, which was recovered and purified, and submitted to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. The sequencing results were compared in Genbank on NCBI, and the phylogenetic tree was constructed using Mega3.1 software. It was found that the strain BF-10 and Bifidobacterium longum subsp.infantis belonged to the same small branch, with the closest relationship and high similarity 99.2%; on this basis, combined with the results of morphological observation and API 20A biochemical identification, it was determined that the strain BF-10 was Bifidobacterium longum.

Bifidobacterium longum (Bifidobacterium longum) BF-10 has been preserved in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short) on June 21, 2013. The address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing , Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is: CGMCC №.7789.

Example 2, Efficacy Evaluation of Bifidobacterium longum (Bifidobacterium longum) BF-10CGMCC №.7789

1. Evaluation of biological antagonistic ability

The bacteriostatic effect of the fermentation supernatant of strain BF-10 was detected by Oxford cup double-layer agar diffusion method. The biological antagonistic ability of strain BF-10 bacterial cells was detected by mixed culture test.

(1) Analysis of the antibacterial effect of the fermentation supernatant of the strain: Bifidobacterium longum BF-10 was anaerobically cultured at 37°C for 24 hours, the fermentation liquid was centrifuged at 5000rpm/min for 15min, and the supernatant was collected and divided into two groups, one group As the original fermentation supernatant sample, one group was neutralized to about pH 7.0 with 1M NaOH, and used as the post-neutralization fermentation supernatant sample. The final concentration of the indicator bacteria was 10 ⁷ cfu/mL. After adding 100 μL sample into the Oxford cup, the indicator bacteria were cultured under the optimum conditions, and the diameter of the inhibition zone (mm) was measured.

The results in Table 2 show that the original fermentation supernatant has a certain degree of antibacterial effect on 8 bacteria tested indicator bacteria, among which Escherichia coli 1.90, Staphylococcus aureus 1.128, Staphylococcus aureus 1.169, Listeria monocytogenes Bacteria 35152(4b) and Bacillus coagulans had strong antagonistic effects, and the diameter of the inhibition zone was above 20mm. After the fermentation supernatant is treated with neutralizing acid, it can only maintain a certain antibacterial effect on Staphylococcus aureus 1.128 and Listeria monocytogenes 35152, which shows that the strain BF-10 can not only secrete organic acids extracellularly, but also metabolize Produce some other antibacterial substances, such as bacteriocins. Furthermore, the biological antagonistic ability of strain BF-10 is excellent.

Table 2 Bacteriostatic effect of fermentation supernatant of bacterial strain BF-10

(2) Analysis of biological antagonism of thalline cells: Inoculate test indicator bacteria (totally 8 kinds, see Table 2) and Bifidobacterium longum BF-10 respectively by 1% inoculation amount in the improved MRS liquid medium, mix evenly , placed in anaerobic culture at 37°C for 24 hours, then took samples, and used the corresponding selective medium for each indicator bacteria to count the live bacteria of each indicator bacteria by plate counting method to investigate the survival of each indicator bacteria. And the pure culture of the test indicator bacteria in the MRS liquid medium was used as the control, and the culture conditions were the same. Among them, the selective medium used by Staphylococcus aureus is Baird-Parker medium, the selective medium used by Listeria is Listeria chromogenic medium, and the selective medium used by Escherichia coli is eosin-methylene blue medium . The results showed that each control group of indicator bacteria showed a good growth trend in the MRS liquid medium, and the logarithmic value of the number of viable bacteria was above 8 after 24 hours of culture. After mixed culture with Bifidobacterium BF-10, the growth of each indicator bacteria was significantly inhibited. Among them, the number of viable bacteria of Escherichia coli 1.90, Bacillus coagulans, Staphylococcus aureus 1.169 and 1.128 decreased significantly (2.1-2.5log). The above results show that Bifidobacterium longum BF-10 has obvious antagonistic effects on the tested 8 strains, which may be related to the nutrient competition or space competition among them, and the organic acid and other substances produced by BF-10 during the growth process. Bacteriostatic substances such as bacteriocins are involved.

In summary, the bacterial cells and fermentation supernatant of the strain BF-10 exhibit strong biological antagonistic ability, and their use in the preparation of live fermented beverages will greatly improve the safety of the product.

2. Antioxidant efficacy evaluation

Through four tests including superoxide anion radical scavenging, DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) radical scavenging, hydroxyl radical scavenging and reducing ability determination, the bacterial strain BF-10 The antioxidant capacity of somatic cells, cell-free extracts of bacteria and cell-free fermentation supernatant was studied.

(1) Preparation of samples: mainly including three types of samples, namely bacterial cells, bacterial cell-free extracts and cell-free fermentation supernatant. Culture Bifidobacterium longum BF-10 anaerobically at 37°C for 24 hours, take the fermentation liquid and centrifuge it at 5000rpm/min for 15 minutes, collect the supernatant, which is the cell-free fermentation supernatant sample, and collect the bacterial cells at the same time, and use double steaming Wash with water for 3 times, and finally adjust the number of bacteria to 10 ⁹ cells/mL with double distilled water. The resulting bacterial suspension was divided into two groups, one group was used as bacterial cell samples, and the other group was placed in an ice bath to sonicate the cells. The crushed solution was centrifuged at 6000r/min for 10min, and the supernatant was collected, which was the cell-free extract sample of the bacteria.

(2) Superoxide anion scavenging test: Pyrogallol autoxidation method was used to measure its antioxidant capacity. Take 4.5mL, 0.1mol/L Tris-HCl buffer solution (pH8.2) in a test tube, add 1mL, 1.0mmol/L ethylenediaminetetraacetic acid (dissolved in hot water), 1.0mL of sample, 2.4mL distilled water, and react at 25°C After 10 min, 2 mi of pyrogallol was added. After reacting at 25°C for 60 min, 100 μL of 12 mol/L HCl was added to terminate the reaction. Absorbance (As) was measured at a wavelength of 325 nm. Replace the sample with 1.0mL distilled water in the blank tube, the operation method is the same as that of the sample tube, and measure the absorbance (Ac). All the above were measured in parallel 3 times to get the average value, and the 3 times were measured to get the average value. Among them, the preparation of 0.1mol/L, pH 8.2 Tris-HCl: 0.346g Tris base, set the volume to 1000ml, intermodulate with concentrated hydrochloric acid to pH8.2. The clearance rate is calculated by the following formula: clearance rate/%=[(Ac-As)/Ac]×100

(3) DPPH free radical removal test: Take 4.0mL0.1mmol/LDPPH solution in a test tube, add 2.0mL sample and 1.0mL50% ethanol, react in the dark for 60min, centrifuge at 12000r/min for 20min, take the supernatant at a wavelength of 517nm Measure the absorbance (Aj). Replace the sample with 2.0mL distilled water in the blank tube, the operation method is the same as that of the sample tube, and measure the absorbance (Ai). All the above were measured in parallel 3 times to get the average value, and the 3 times were measured to get the average value. The clearance rate is calculated by the following formula: clearance rate/%=[(Ai-Aj)/Ai]×100

(4) Hydroxyl free radical scavenging test: Add 1 mL of 0.05 mol/L, pH7.4 phosphate buffer, 0.5 mL, 6 mmol/L of phenanthroline into the test tube, mix well, then add 0.5 mL, 6mmol/L FeSO4 solution, mix immediately after adding. Then add 0.5mL of sample solution to it, mix well, then add 0.5mL of 0.1% H ₂ O ₂ , and finally replenish the volume to 4mL with distilled water, and do a blank experiment at the same time. In addition, a damaged tube and an undamaged tube were made again, wherein 0.5 mL of 0.1% H ₂ O ₂ was added to the damaged tube, and no sample was added. Undamaged tubes received neither H ₂ O ₂ nor sample. Finally, the volume was added to 4 mL. Incubate at 37°C for 1h, and measure its absorbance at a wavelength of 536nm. The clearance rate is calculated by the following formula: clearance rate/%=[(Ai-A)/(A ₀ -A)]×100, A is the absorbance of the solution without adding the sample; A ₀ is the solution without adding the sample and H ₂ O ₂ When is the absorbance of the solution; Ai is the absorbance of the solution when the sample is added.

(5) Determination of reducing ability: take 0.5mL sample, add 0.5mL of phosphate buffer solution with a concentration of 0.2mol/L, pH6.6 and 0.5mL of 1g/100mL potassium ferricyanide, put it in a water bath at 50°C for 20min, and cool it rapidly. Then add 0.5 mL of 10 g/100 mL trichloroacetic acid, centrifuge at 3000 rpm/min for 5 min, take 1 mL of supernatant, add 1 mL of distilled water and 1 mL, 0.1 g/100 mL of ferric chloride, and mix well. After 10 min, the absorbance was measured at 700 nm. The greater the absorbance, the stronger the reducing ability of the sample to be tested.

Table 3 Antioxidant activity analysis of strain BF-10

The results in Table 3 show that the bacterium cells have a scavenging rate of up to 94.53% for hydroxyl radicals, indicating that the active substance for scavenging hydroxyl radicals mainly exists on the surface of the thalline cells of Bifidobacterium longum BF-10; the cell-free extract of the thalline The superoxide anion scavenging rate is higher (97.67%), indicating that the active substance for scavenging superoxide anion free radicals is mainly the intracellular substance of the infant bacterial cells; the cell-free fermentation supernatant has the highest DPPH scavenging rate, which can reach nearly 93 %; This shows that the active substances for scavenging DPPH free radicals may be metabolic secretions. In addition, the reducing ability of the cell-free fermentation supernatant was the strongest, indicating that there were active substances with reducing ability outside the cells of the bacteria. The results of this study show that the bacterial cells and extracellular secretions of the strain BF-10 have strong antioxidant capacity. In further product development, this strain can be considered as a starter strain for the production of live bacterial beverages, so as to give full play to its Antioxidant effect.

In summary, Bifidobacterium longum BF-10 not only has a strong tolerance to the adverse environment of the digestive tract, but also exerts biological antagonism against a variety of harmful microorganisms, and the cells and extracellular secretions of the bacteria have strong It has strong anti-oxidation and obvious physiological effects, and has the potential to be used as a probiotic strain and to develop a series of products.

Example 3, the preparation method of Bifidobacterium longum (Bifidobacterium longum) BF-10CGMCC №.7789 fermented seabuckthorn and wolfberry compound juice live bacteria beverage

The present invention utilizes the obtained Bifidobacterium longum (Bifidobacterium longum) BF-10CGMCC №.7789 fermented seabuckthorn and wolfberry compound juice obtained in Examples 1 and 2 to prepare Bifidobacterium live bacteria beverage, and the preparation method is as follows:

1. Preparation of Bifidobacterium Seeds

(1) Preparation of first-generation seeds: Bifidobacterium longum (Bifidobacterium longum) BF-10 was inoculated into the improved MRS liquid medium, and anaerobically cultured at 37° C. for 24h-48h to obtain first-generation seeds.

(2) Preparation of the second-generation seeds: inoculate the first-generation seeds in the improved MRS liquid medium and culture them anaerobically at 37°C for 14-16h (to the end of the logarithmic phase) Turning time period. By measuring the growth curve of the actual production situation and determining the time range of the logarithmic phase), the second generation of seeds is obtained.

(3) Preparation of the third-generation seeds: the second-generation seeds were inoculated in the improved MRS medium and cultured anaerobically for 14 hours (to the end of logarithmic phase) to obtain the third-generation seeds. The third generation seeds are used as cultivation seeds.

2. Preparation of seabuckthorn and wolfberry compound juice fermentation medium

Take fresh seabuckthorn and drinking water with a mass ratio of 1:1, mix and beat, filter with four layers of gauze, and obtain seabuckthorn juice with a mass percentage of 50%; accurately weigh wolfberry, and add it to a triangular flask according to the mass ratio of material and water at 1:10 , after soaking in a water bath at 85°C for two hours, beating, and filtering through four layers of gauze to obtain a wolfberry extract with an active ingredient mass percentage of 10%; the above-mentioned sea buckthorn juice and wolfberry extract were mixed in a volume ratio of 2:1, and mixed with a small Adjust the pH to about 6.0 with soda (N _a HCO ₃ ) to obtain mixed fruit juice; dissolve 75g of sucrose in 100mL of water to obtain a sucrose solution; add 10g of skim milk powder to 1L of water, stir well to dissolve, and obtain reconstituted skim milk. According to 50% of mixed fruit juice, 12% of sucrose solution, 10% of skimmed reconstituted milk, and 28% of water, the first homogenization is carried out with a homogenizer under the condition of 8MPa, and the second homogenization is carried out under the condition of 12MPa to obtain the compound fruit juice Fermentation medium; finally, fill the compound fruit juice fermentation medium with nitrogen to reduce the redox potential to below -0.03eV, and sterilize at 115°C for 10 minutes with high-pressure steam.

3. Preparation of bifidobacterium-fermented seabuckthorn and wolfberry compound juice live bacteria beverage

The seeds obtained in step 1 were inoculated into the compound fruit juice medium containing seabuckthorn and wolfberry at 2% inoculation amount (1×10 ⁶ cfu/mL), and cultured anaerobically at 38-42° C. for about 24-26 hours until the pH was 4.2-4.5. After the fermentation is finished, the seabuckthorn and wolfberry bifidobacterium fermented beverage is obtained. Divide the fermented beverage, aseptically fill as shown in Figure 1, and store at 4°C. The number of live bacteria counted by Hungate anaerobic rolling tube method can reach 8.7×10 ¹⁰ CFU/mL.

Example 4. Quality and shelf life evaluation of Bifidobacterium longum (Bifidobacterium longum) BF-10CGMCC №.7789 fermented seabuckthorn and wolfberry compound juice live bacteria beverage

1. Quality evaluation

The quality of the prepared active bifidobacteria fermented beverage was evaluated based on the determination of physical and chemical indicators and sensory evaluation.

(1) Determination of physical and chemical indicators: the bifidobacterium seabuckthorn and wolfberry compound juice fermented active beverage prepared in Example 3 is measured for the following indicators, 1) protein content: with reference to GB 50095-2010 "Measurement of Protein in Food"; 2) soluble solids content: refer to GB 12143.1-88 "Determination of Soluble Solids in Soft Drinks"; 3) Reducing sugar content: refer to GB/T 5009.7-2003 "Determination of Reducing Sugar in Food"; 4) Total sugar content: phenol sulfuric acid method 5) Total acid content: With reference to GB/T 12456-2008 "Determination of Total Acid in Food" (calculated as lactic acid), 6) pH value, measured by a pH electrode. The results are shown in Table 5. It can be seen that the physical and chemical indicators of the live bacteria beverage prepared in Example 3 all meet the requirements of the national standard GB16321-2003 "Hygienic Standards for Lactic Acid Bacteria Beverages".

Table 5 Analysis of the physical and chemical indicators of the live bacteria beverage prepared in Example 3

(2) Sensory evaluation: Sensory analysis was carried out on the bifidobacterium seabuckthorn and wolfberry compound juice fermented active beverage prepared in Example 3, using fuzzy mathematics seven-degree scale method, and a scoring team composed of 10 trained people. The comment domain is V=(1, 2, 3, 4, 5, 6, 7), 1: most difficult to accept; 2: relatively difficult to accept; 3: slightly difficult to accept; 4: barely acceptable; 5: relatively easy to accept ; 6: easy to accept; 7: most acceptable. The evaluation field U=(color, aroma, acidity, sweetness, bitterness, texture state) of fermented fruit juice, and its corresponding weight value is X=(0.15, 0.20, 0.20, 0.15, 0.20, 0.10). The results are shown in Table 6. It can be seen that the beverage has the fruity aroma of seabuckthorn and wolfberry and the unique aroma after fermentation of bifidobacteria, and its color, texture, and sweetness scores are all above 6.0. Its comprehensive sensory score is 6.28, which is a drink that is easily accepted by the public.

Table 6 The sensory evaluation score table of the live bacteria beverage prepared in Example 3

2. Shelf life evaluation

The live bacteria beverage was stored at 4°C and room temperature (22°C) respectively, and samples were taken every other day, and bifidobacteria were counted by the Hengate anaerobic rolling tube method. Refer to GB 16321-2003 "Hygienic Standards for Lactic Acid Bacteria Beverages" on the minimum limit standard for the number of viable lactic acid bacteria in lactic acid bacteria beverages, and the end of shelf life is judged when the number of viable bacteria is less than 1.0×10 ⁶ CFU/mL. The results are shown in accompanying drawing 2. It can be seen that the number of viable bifidobacteria in the beverage prepared in Example 3 declines slowly during storage at 4°C, and it can ensure that the number of viable bifidobacteria in 19 days of storage is at 1.0× 10 ⁶ CFU/mL or more; while the number of viable bifidobacteria decreased rapidly during storage at room temperature and could only be stored for 8 days. The above shows that the storage temperature has a significant impact on the shelf life of the live bacteria drink. As a live bacteria drink with a short shelf life without secondary sterilization, the fermented drink is more suitable for cold chain sales.

Claims (7)

1. bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789.

2. producing a method for bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage, is fermentation bifidus longum bb according to claim 1 (Bifidobacterium longum) BF-10CGMCC № .7789, the viable bacteria beverage obtained.

3. method according to claim 2, is characterized in that: described fermention medium is prepared as follows: get the water mixing making beating that fresh sea-buckthorn and mass ratio are 1: 1, by four layers of filtered through gauze, obtain the juice of Fructus Hippophae that mass percent is 50%; Precise wolfberry fruit, adds in triangular flask by material quality than 1: 10, and in 85 DEG C of water-baths, soak making beating after two hours, four layers of filtered through gauze, obtain the wolfberry fruit extract that effective constituent mass percent is 10%; Juice of Fructus Hippophae is mixed according to a certain volume with wolfberry fruit extract, obtains fruit juice blends; Sucrose 75g is dissolved in 100mL water and obtains sucrose solution; In 1L water, add 10g skimmed milk powder, abundant stirring and dissolving, obtain degreasing reconstituted milk; By fruit juice blends, sucrose solution, degreasing reconstituted milk and water mix by a certain percentage; Under 8MPa condition, carry out first time homogeneous with clarifixator, under 12MPa condition, carry out second time homogeneous, and with sodium bicarbonate (N- _ahCO ₃) regulate about 6.0 of its pH, obtain composite fruit juice fermention medium; Finally, in composite fruit juice fermention medium, fill nitrogen, make Redox potential be down to below-0.03eV, and at 115 DEG C, high pressure steam sterilization 10min.

4. method according to claim 3, is characterized in that: fermention medium consists of fruit juice blends 50%, sucrose solution 12%, degreasing reconstituted milk 10%, water 28%; Described percentage composition is percent by volume.

5. method according to claim 3 or 4, is characterized in that: in described fruit juice blends, the composite ratio of juice of Fructus Hippophae and wolfberry fruit extract is 2: 1.

6. method according to Claims 2 or 3 or 4 or 5, is characterized in that: described leavening temperature is 38-42 DEG C, and fermentation time is 24-26h, and initial ph value is 6.0, and inoculum size is 1 × 10 ⁶cfu/mL.

7. bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage prepared by the method in claim 2-6 described in any one.