A kind of blueberry juice fermentation method

Abstract

The present invention relates to a method for fermenting blueberry juice, and belongs to the field of food processing. A method for fermenting blueberry juice, characterized in that: the following three strains are used in sequence to ferment blueberry juice, specifically: Bifidobacterium lactis JYBR‑190CGMCC No.18092, Lactobacillus plantarum JYLP‑326CGMCC No.18038, and Lactococcus lactis JYLL‑60CGMCC No.2335. The present invention adopts different commercial bacteria to add sequence to ferment blueberry juice for the first time, effectively combining the probiotic function of lactic acid bacteria with the nutritional function of blueberry, and the CAA cell antioxidant capacity of blueberry juice is significantly enhanced after lactic acid bacteria fermentation, which is better than the traditional model of composite bacteria simultaneous fermentation. After the blueberry juice is fermented according to the strain addition sequence of the present invention, the content of its nutritional functional components such as total phenols and total flavonoids is significantly increased, which is better than the traditional model of composite bacteria simultaneous fermentation.

Description

Technical Field

[0001] The invention relates to a blueberry juice fermentation method and belongs to the field of food processing. Background Art

[0002] Blueberry is a good source of fiber, vitamins, folic acid, fatty acids, polyphenols and minerals. As an edible berry, it is characterized by high nutritional compounds and a wide variety of bioactive compounds. Every step of blueberry processing affects the increase and degradation of its bioactive compounds. Since blueberry is a seasonal fruit, its supply and storage time and other factors have led to the production of many products derived from it in the food industry, such as blueberry juice.

[0003] As the economy develops, people's pursuit of nutritious food has led to more products. Probiotic fermented blueberry juice meets the needs of more consumers, such as lactose intolerance, milk allergy, vegetarians, etc. Probiotics themselves have many benefits and have various health-promoting effects, such as preventing intestinal infections, improving lactose metabolism, lowering serum cholesterol levels, enhancing immunity, stimulating calcium absorption, improving protein digestion, synthesizing vitamins (vitamin B, including niacin and folic acid), and counteracting foodborne pathogens. In addition to its own characteristics, it also contributes to the increase in the content of bioactive compounds, citric acid and ascorbic acid, anthocyanins, polyphenols, flavonoids and health properties, such as antioxidant activity and inhibition of angiotensin converting enzyme, α-amylase and α-glucosidase. Probiotic fermentation can increase the content of different nutrients in the matrix or metabolic functional compounds.

[0004] At present, the common fermentation methods at home and abroad are single-strain fermentation or fermentation with compound probiotics. The fermentation method is single and it is impossible to confirm whether it is the best fermentation result. Sequential fermentation can be seen in wine fermentation. Since fermentation requires the selection of single-strain probiotics and yeast for fermentation, it is significantly different from this fermentation method. Due to the differences in the selection of fermentation matrix and fermentation strain, it is impossible to determine the optimal fermentation process, which cannot meet the development of the juice industry. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a process method for preparing probiotic fermented blueberry juice with better nutrient indexes in view of the current single probiotic blueberry juice fermentation process.

[0006] The invention uses blueberry juice, three lactic acid bacteria, namely, lactobacillus JYLP-326, lactococcus lactis JYLL-60 and bifidobacterium lactis JYBR-190, as research objects. The fermentation time and sequence of each strain are determined by measuring the growth curve of the strains, and the content of phenolic substances in the fermented blueberry juice between each group, the composition of phenolic substances, enzyme activity, CAA cell antioxidant activity, and the changes of various indicators of color difference value are determined to determine the optimal strain addition sequence and the optimal process for fermenting blueberry juice.

[0007] A blueberry juice fermentation method comprises the following three strains, namely: Bifidobacterium lactis JYBR-190 CGMCC No.18092, Lactobacillus plantarum JYLP-326 CGMCC No.18038, and Lactococcus lactis JYLL-60 CGMCC No.2335, for blueberry juice fermentation.

[0008] The Bifidobacterium lactis JYBR-190CGMCC No. 18092 described in the method of the present invention can be obtained from China General Microbiological Culture Collection Center (CGMCC).

[0009] The Lactobacillus plantarum JYLP-326 CGMCC No. 18038 described in the method of the present invention can be obtained from China General Microbiological Culture Collection Center (CGMCC).

[0010] The Lactococcus lactis JYLL-60 CGMCC No. 2335 described in the method of the present invention can be obtained from China General Microbiological Culture Collection Center (CGMCC).

[0011] Preferably, the mass ratio of Bifidobacterium lactis, Lactobacillus plantarum and Lactococcus lactis is 1:1:1.

[0012] Preferably, the accession amount of Bifidobacterium lactis is 1% of the blueberry juice, the accession amount of Lactobacillus plantarum is 1% of the blueberry juice, and the accession amount of Lactococcus lactis is 1% of the blueberry juice.

[0013] The blueberry juice fermentation method of the present invention is specifically as follows: at a constant temperature of 37°C, first, using Bifidobacterium lactis JYBR-190 to ferment the blueberry juice for 3 hours, then adding Lactobacillus plantarum JYLP-326 to ferment for 3 hours, and then adding Lactococcus lactis JYLL-60 for cooperative fermentation for 48 hours to end the fermentation.

[0014] Preferably, each bacterial strain is activated, selected and cultured on a solid culture medium and then inoculated into a liquid culture medium for later use.

[0015] Furthermore, the three strains were spread on the surface of solid culture medium respectively, cultured at 37°C for two days, single colonies on the plate were picked, transferred to solid culture medium and streaked for more than two times to obtain pure colonies, which were then picked into liquid culture medium for culture.

[0016] Preferably, the blueberry juice raw material is prepared by the following method: adding water to blueberry pulp to squeeze the juice, filtering, the ratio of blueberry pulp to water is 1g:2-3mL; adding sodium carbonate to the blueberry juice, adjusting its pH value to 5.0, adding 0.4% pectinase, and bathing in 45°C water for 90 minutes; centrifuging at 4500r / min for 20min for homogenization; bathing in 60°C water for 10min to inactivate the enzyme; and sterilizing at 75°C for 15min.

[0017] The beneficial effects of the present invention are as follows: the present invention uses different commercial bacteria to add in sequence to ferment blueberry juice for the first time, effectively combining the probiotic function of lactic acid bacteria with the nutritional function of blueberries. After lactic acid bacteria fermentation, the CAA cell antioxidant capacity of blueberry juice is significantly enhanced, which is better than the traditional model of composite bacteria simultaneous fermentation. After the blueberry juice is fermented according to the order of adding strains of the present invention, the content of nutritional functional components such as total phenols and total flavonoids is significantly increased, which is better than the traditional model of composite bacteria simultaneous fermentation. The color difference value of blueberry juice fermented according to the order of adding strains of the present invention is not significantly different, a * The value is significantly increased compared with the unfermented blueberry juice, showing bright red color and having good appearance. The blueberry juice fermented according to the order of adding strains of the present invention has high acidity value and good acid production ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 (A) (B) shows the biomass of lactic acid bacteria in MRS medium. It shows that Lactobacillus plantarum JYLP-326 becomes the dominant strain at the third hour. It indicates that after inoculation for the third hour, it is allowed to fully utilize nutrients to grow into the dominant strain, and then the next strain is inoculated, and the synergistic fermentation ends.

[0019] Figure 1 (C) (D) shows the biomass of lactic acid bacteria in MRS medium. It shows that Lactococcus lactis JYLL-60 becomes the dominant strain at the third hour. It means that after inoculation for the third hour, it is allowed to fully utilize nutrients to grow into the dominant strain, and then the next strain is inoculated, and the synergistic fermentation ends.

[0020] Figure 1 (E) (F) shows the biomass of lactic acid bacteria in MRS medium. It indicates at which hour Bifidobacterium lactis JYBR-190 becomes the dominant strain. It indicates that after inoculation for 3 hours, it is allowed to fully utilize nutrients to grow into the dominant strain, and then the next strain is inoculated, and the synergistic fermentation ends.

[0021] Figure 2 The antioxidant activity of CAA cells was optimized by adding different strains in the order of fermenting blueberry juice in Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0022] Figure 3 The total phenol content of the blueberry juice fermentation process was optimized by adding different strains in the order of Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0023] Figure 4 The total flavonoid content of blueberry juice fermented by adding different strains in the order of Example 1 was optimized. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0024] Figure 5 The total anthocyanin content of blueberry juice fermented by adding different strains in the order of Example 1 was optimized. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0025] Figure 6 The number of live bacteria in the blueberry juice fermentation process optimized by adding different strains in the order in Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0026] Figure 7 The total acidity of the blueberry juice fermentation process was optimized by adding different strains in the order of Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0027] Figure 8 The optimized pH value of the blueberry juice fermentation process was obtained by adding different strains in the order described in Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05).

[0028] Fig. 9 The color difference values ​​of the optimized blueberry juice fermentation process by adding different strains in the order in Example 1. The lowercase letters indicate that different letters in the same row indicate significant differences (p>0.05). DETAILED DESCRIPTION

[0029] The following non-limiting embodiments may enable a person skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0030] The test methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0031] A process for fermenting blueberry juice comprises the following steps:

[0032] (1) Preparation of blueberry juice: Select and wash 300g of blueberry pulp, pour it into a juicer and add water, the ratio of blueberry to water is 1g:2-3mL, and filter it with 300 mesh gauze for 3 times. Add sodium carbonate to the blueberry juice, adjust its pH value to 5.0, add 0.4% pectinase, bathe in 45℃ water for 90 minutes; centrifuge at 4500r / min for 20min for homogenization; bathe in 60℃ water for 10min to inactivate enzymes; sterilize at 75℃ for 15min, which can inactivate enzymes / harmful bacteria in the blueberry juice and avoid changes in nutrients caused by excessive temperature.

[0033] (2) Activation of bacterial strains: Use a sterile pipette to draw 1.0 mL of sterile water to completely dissolve the powders of Lactobacillus plantarum JYLP-326, Lactococcus lactis JYLL-60, and Bifidobacterium lactis JYBR-190. Take 100 μl of the bacterial solution and dilute it 10 times in a gradient manner. Spread it on the surface of a solid culture medium (solarbio cat#M8330 MRS agar) and culture it at 37°C for two days. Pick a single colony on the plate, transfer it to a solid culture medium, and streak it for more than two times to obtain a pure colony. Pick it into a liquid culture medium (solarbio cat#M8540 MRS broth) for culture.

[0034] (3) Determination of bacterial biomass: The OD values ​​of the bacterial species Lactobacillus plantarum JYLP-326, Lactococcus lactis JYLL-60, and Bifidobacterium lactis JYBR-190 samples obtained in step (2) were measured at a wavelength of 600 nm and the pH value of the fermentation liquid to obtain the growth curve and acid production performance corresponding to each bacterial species, and the fermentation time for adding a single bacterial species was determined based on the two.

[0035] (4) Fermentation time: at a constant temperature of 37° C., the blueberry juice obtained in step (1) was placed in a conical flask, and the bacterial strains were added in order and then placed in a constant temperature incubator for closed culture at 37° C. for 48 hours until the fermentation was completed, wherein the inoculation amount of Bifidobacterium lactis was 1% of the blueberry juice, the inoculation amount of Lactobacillus plantarum was 1% of the blueberry juice, the inoculation amount of Lactococcus lactis was 1% of the blueberry juice.

[0036] The order of adding strains is: Bifidobacterium lactis JYBR-190 (C) is added to blueberry juice and fermented for 3 hours, then Lactobacillus plantarum JYLP-326 (A) is added and fermented for 3 hours, and then Lactococcus lactis JYLL-60 (B) is added and cultured until the fermentation ends after 48 hours. The order of adding strains is (C, A, B)

[0037] The present invention establishes a process for preparing probiotic fermented blueberry juice with better nutrient index. The antioxidant capacity of CAA cells of blueberry juice after fermentation by adding probiotics in the optimal strain order is significantly enhanced, and the main material functions and color difference values ​​of sequential addition of strains are analyzed. The phenolic content in blueberry juice before and after fermentation is compared by ultraviolet spectrophotometer colorimetry. The phenolic content in the optimal fermentation method increases by 55.269 (mg / 100ml), the total flavonoid content also increases significantly by 97.76 (mg / 100ml), and the degradation degree of phenolic substances and anthocyanins is significantly improved, which proves the biological conversion function of sequential addition of strains on functional substances in blueberry juice. After the fermentation is completed, the pH of blueberry juice decreases, and the total acidity continues to accumulate and increase. The total acidity of the CAB group is the highest, which can reach 4.36g / L, which is significantly higher than other groups. The number of live bacteria is detected after 48 hours of fermentation. The number of live bacteria in the CAB group is higher than the national standard for probiotic fermented beverages, reaching 6.93lg (cfu) / mL.

[0038] The method established by the present invention can be applied to the commercial production of probiotic fermented blueberry juice, and provides an important reference for the optimal fermentation process of probiotic blueberry juice in the future.

[0039] Example 1: Preparation of blueberry juice by fermentation with different strains added in sequence

[0040] S1: Preparation method of blueberry juice:

[0041] After washing and selecting blueberries, pour 300g of blueberry pulp into a juicer and add water. Filter with 300-mesh gauze. Prepare, add deionized water to the filtrate to 600ml, and filter three times with a ratio of blueberries to water of 1:2; add 0.4% pectinase, bathe in 45℃ water for 90min, centrifuge at 4500r / min for 20min for homogenization, and bathe in 60℃ water for 10min to inactivate the enzyme. Other methods: sterilize in a water bath before inoculation (75℃, 15min) to inactivate the enzymes / harmful bacteria in the blueberry juice, and avoid changes in nutrients caused by excessively high temperatures.

[0042] S2: Methods for separation, purification, preservation and activation of bacterial strains:

[0043] Use a sterile pipette to absorb 1.0mL of sterile water to completely dissolve the powder of Lactobacillus plantarum JYLP-326, Lactococcus lactis JYLL-60, and Bifidobacterium lactis JYBR-190, take 100μl of bacterial solution and dilute it 10 times, respectively, spread it on the surface of solid culture medium, culture it at 37℃ for two days, pick single colonies on the plate, transfer them to solid culture medium and separate them by streaking for more than 10 times to obtain pure colonies. Strain preservation: pick a single colony and transfer it to 100mL liquid culture medium, culture it at 37℃ for 20h, take 1mL of bacterial solution and mix it with 60% glycerol in a ratio of 1:1 to make a glycerol tube, and freeze it at -20℃. Strain activation: inoculate the strain taken out of the glycerol tube into MRS broth culture medium, and culture it at 37℃ for 20h.

[0044] S3: Determination of lactic acid bacteria fermentation performance

[0045] The OD values ​​of the samples of the treated bacteria species Lactobacillus plantarum JYLP-326, Lactococcus lactis JYLL-60, and Bifidobacterium lactis JYBR-190 in S2 at a wavelength of 600 nm were measured to obtain the corresponding growth curves, and the fermentation time of a single bacteria species was determined according to the growth curve values.

[0046] S4: Preparation of blueberry juice fermented by adding different strains

[0047] The S1 sample was placed in a conical flask, and the S3 strains were added in sequence, and then placed in a constant temperature incubator for closed culture at 37°C for 48 hours until the fermentation was completed.

[0048] The order of adding strains is: Bifidobacterium lactis JYBR-190 (C) is added to blueberry juice and fermented for 3 hours, then Lactobacillus plantarum JYLP-326 (A) is added and fermented for 3 hours, and then Lactococcus lactis JYLL-60 (B) is added and cultured until the fermentation ends after 48 hours. The order of adding strains is (C, A, B)

[0049] Analysis of antioxidant activity of CAA cells based on the optimized blueberry juice fermentation process with different strains added sequentially according to Example 1

[0050] HepG2 cells were cultured in DEME medium containing 10% fetal bovine serum, 1% penicillin and streptomycin at 37°C and 5% CO. 2. Add cells in the logarithmic growth phase to a 96-well black board (60,000 / well), incubate for 48 hours, remove the culture medium, and wash the cells with PBS buffer solution. Treat 6 replicate wells with 50uL fermented blueberry juice, and add 50 microliters of culture medium to the blank group. Then add 50 microliters of fluorescent probe DCFH-DA and incubate for 1 hour. After the incubation, the sample solution is aspirated and discarded. Three replicate wells are washed with phosphate buffer (PBS wash), and three are not washed (No PBS wash). Use a spray gun to draw 100 microliters of ABAP working solution and add it to the sample well. Subsequently, place the 96-well plate in a fluorescence microplate reader and record the fluorescence every 5 minutes for 1 hour at 37°C at 535nm (emission) and 485nm (excitation). Calculate according to the following formula.

[0051] CAA value=100-(∫SA / ∫CA)x100

[0052] Wherein, ∫sA is the area under the sample fluorescence-time curve, and ∫CA is the area under the control curve.

[0053] CAA determination is a cell-based method for evaluating the antioxidant capacity of bioactive substances. Considering bioavailability and cellular uptake, it is a relatively representative detection method. In this experiment, the CAA values ​​of the seven blueberry juices were greater than those after PBS washing when not washed with PBS, indicating that the antioxidant activity of fermented blueberry juice on the cell membrane surface is better than that inside the cell. Three groups of cells in the washed group had significantly higher antioxidant activity than the traditional composite yABC fermentation method. Four groups in the non-PBS washed group had higher antioxidant activity than the yABC group. The antioxidant activity of the washed group CAB group was higher than that of the other groups.

[0054] Analysis of total phenol content in blueberry juice fermentation process optimization by adding different strains in sequence according to Example 1

[0055] Add 0.1 mL of fermented blueberry juice to the test tube. Then add 0.1 mL of Folin phenol reagent and 0.4 mL of distilled water to the test tube and vortex to mix. After 6 minutes, add 1 mL of 7% Na 2 CO 3 solution and 0.8 mL of distilled water. After vortexing, the mixture was developed at room temperature in the dark for 90 minutes. 200 μL of the reaction solution was transferred to a 96-well plate. The absorbance was measured at a wavelength of 760 nm using an ELISA reader. The absorbance was inserted into the standard curve to calculate the sample concentration. The total phenol content in the sample was expressed as gallic acid equivalents. Standard curve: y = 0.0022x + 0.0698R 2=0.999Different strain addition order fermentation has different effects on the changes of phenolic substances in blueberry juice, which will increase the content of phenolic substances. It may be that plant lactobacillus can produce enzymes such as phenolic acid esterase and β-galactosidase during metabolism, which can release phenolic substances in juice. In addition, the acidic environment created by lactic acid bacteria fermentation is conducive to the stability of polyphenols. The total phenol content of the groups with different strain addition order was significantly higher than that of the unfermented blueberry juice, among which the CAB group had the highest total phenol content.

[0056] Analysis of total flavonoid content in blueberry juice fermentation process optimization by adding different strains in sequence according to Example 1

[0057] Take 1mL of sample solution and put it in a 10mL volumetric flask, add 0.3mL of 5% sodium nitrite solution, mix well and let stand for 6min, then add 0.3mL of 10% aluminum nitrate solution, mix well and let stand for 6min, add 1mL of 4% sodium hydroxide solution and mix well, dilute to 10mL with 70% ethanol, water bath at 25℃ for 15min, measure the absorbance at 370nm with an enzyme marker, and make a standard curve with quercetin as the standard. Standard curve: y=0.4136x+0.0256R 2 =0.993The total flavonoid content is expressed as quercetin equivalent per gram of dry weight. The different addition order combinations of lactic acid bacteria metabolize or biotransform during the fermentation process to produce flavonoids, breaking down complex flavonoids into other compounds. In this process, lactic acid bacteria secrete glycosidases. On the one hand, flavonoid glycosides are hydrolyzed into water-insoluble flavonoid aglycones, and on the other hand, flavonoids bound to dietary fiber are easily hydrolyzed into water-soluble flavonoids, which may have a positive effect on the bioavailability of flavonoids in fermented juice. The total flavonoid content of the seven fermentation groups was also higher than that of the unfermented blueberry juice.

[0058] Analysis of total anthocyanin content in blueberry juice fermentation process optimization by adding different strains in order according to Example 1

[0059] Thaw the sample in an ice water bath at 37℃, add the sample to a centrifuge tube, centrifuge at 800rpm for 15min, take 0.2mL of 2 processed samples and place them in test tubes, add pH1.0 and pH4.5 respectively. The preparation process of pH1.0 buffer is: use distilled water to make the solution 3gKCI to a volume of 200mL, then mix it with a hydrochloric acid solution with a concentration of 0.2mol / L in a ratio of 1:3 to obtain the required solution: The preparation process of pH4.5 buffer is: use distilled water to make the solution 8.2g KCI to a volume of 100mL, then continue to add distilled water and 1mol, the volumes of the two are 90mL and 60mL respectively, to obtain the required solution) 4.8mL of buffer, place it in the dark for 1h, and measure the absorbance at 520nm and 700nm wavelengths, do 3 parallels, and take the average value. After the fermentation of blueberry juice in all groups, the total anthocyanin content of the seven fermentation sequences showed a downward trend. The decrease in total anthocyanin content may be due to the vigorous metabolism of lactic acid bacteria during the fermentation stage. Among them, the yABC group has the strongest ability to retain anthocyanins during the fermentation of blueberry juice. The fermentation order of adding different strains has different effects on the changes of phenolic substances in blueberry juice, which will increase the content of phenolic substances. It may be that Lactobacillus plantarum can produce enzymes such as phenolic acid esterase and β-galactosidase during metabolism, which can release phenolic substances in juice. In addition, the acidic environment created by lactic acid bacteria fermentation is conducive to the stability of polyphenols.

[0060] Analysis on the optimization of the viable bacterial count of blueberry juice fermentation process by adding different strains in the order of Example 1

[0061] Gradient dilution. Pipette 1 mL of bacterial suspension into a test tube containing 9 mL of sterile water, shake and mix, take out 1 mL and add it to the next test tube containing 9 mL of sterile water, and so on, and make a 10-fold gradient dilution of the bacterial suspension. According to the actual situation, the dilution multiple is generally suitable when the number of bacteria in the microscope field is 30-300. Pipette the bacterial suspension of appropriate dilution automatically into the counting chamber along the edge of the coverslip, and select the four corners of the counting chamber and a middle grid in the center for microscopic counting.

[0062] Analysis of total acid content in blueberry juice fermentation process optimization by adding different strains in order according to Example 1

[0063] The total acid content of the samples was determined with reference to GB / T 12456-2008 "Determination of total acid in food", and the results were expressed as lactic acid (g / L). The acidity value is also an important indicator for measuring the acid production capacity of the strain. After the fermentation of the seven groups, the acidity values ​​were significantly different, and the acidity of the CAB group was the highest.

[0064] Analysis on the optimization of pH value of blueberry juice fermentation process by adding different strains in the order of Example 1

[0065] The pH value was directly measured by pH meter and repeated three times. The variation of pH value in the fermented juice reflects the quality of the fermentation performance of the strain. The pH value of the seven fermentation groups decreased roughly the same after 48 hours of fermentation. The pH value of the BAC sequential group reached 3.53±0.04, and the growth was good.

[0066] Analysis of color difference determination for the optimization of blueberry juice fermentation process by adding different strains in sequence according to Example 1

[0067] The color of the fermented blueberry juice samples was measured by a fully automatic colorimeter (perspective mode), with unfermented blueberry juice as the reference solution, and the results were measured as total color difference ΔE*. Color change is one of the important evaluation indicators of fermented juice. After 48 hours of fermentation, the color difference values ​​of the seven groups of samples were not much different, but the color change was greater than that of the unfermented blueberry juice. It was an attractive red. The main coloring substance in blueberry juice is anthocyanin, and anthocyanin is mainly red and blue, which is consistent with the color difference value changes in the seven groups corresponding to the previous detection of anthocyanin content.

[0068] Example 2

[0069] Preparation of blueberry juice fermented by adding different strains in order:

[0070] Steps S1 to S3 are exactly the same as those in Example 1.

[0071] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2. After fermentation of strain A for 3 hours, strain B was added for fermentation for 3 hours, and strain C was added for synergistic fermentation for 48 hours. (A, B, C)

[0072] Table 1 Effects of the addition sequence of strains (A, B, C) on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0073]

[0074] Note: Values ​​are expressed as mean ± SD (n = 3)

[0075] Example 3

[0076] Preparation of blueberry juice fermented by adding different strains in order:

[0077] Steps S1 to S3 are exactly the same as those in Example 1.

[0078] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2. After fermentation of strain A for 3 hours, strain C is added and fermented for 3 hours, and strain B is added for synergistic fermentation for 48 hours. (A, C, B)

[0079] Table 2 Effects of the order of adding strains (A, C, B) on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0080]

[0081] Note: Values ​​are expressed as mean ± SD (n = 3)

[0082] Example 4

[0083] Preparation of blueberry juice fermented by adding different strains in order:

[0084] Steps S1 to S3 are exactly the same as those in Example 1.

[0085] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2, B strain is fermented for 3 hours, A strain is added for fermentation for 3 hours, and C is added for collaborative fermentation for 48 hours. (B, A, C)

[0086] Table 3 Effects of the addition sequence of strains (B, A, C) on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0087]

[0088] Note: Values ​​are expressed as mean ± SD (n = 3)

[0089] Example 5

[0090] Preparation of blueberry juice fermented by adding different strains in order:

[0091] Steps S1 to S3 are exactly the same as those in Example 1.

[0092] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2, B strain is fermented for 3 hours, C strain is added for fermentation for 3 hours, and A is added for collaborative fermentation for 48 hours. (B, C, A)

[0093] Table 4 Effects of (B, C, A) strain addition sequence on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0094]

[0095] Note: Values ​​are expressed as mean ± SD (n = 3)

[0096] Example 6

[0097] Preparation of blueberry juice fermented by adding different strains in order:

[0098] Steps S1 to S3 are exactly the same as those in Example 1.

[0099] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2, C strain is fermented for 3 hours, A strain is added for fermentation for 3 hours, and B strain is added for cooperative fermentation for 48 hours. (C, A, B)

[0100] Table 5 Effects of (C, A, B) strain addition sequence on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0101]

[0102] Note: Values ​​are expressed as mean ± SD (n = 3)

[0103] Example 7

[0104] Preparation of blueberry juice fermented by adding different strains in order:

[0105] Steps S1 to S3 are exactly the same as those in Example 1.

[0106] S4: The order of adding strains is: Lactobacillus plantarum JYLP-326 (A), Lactococcus lactis JYLL-60 (B), Bifidobacterium lactis JYBR-190 (C) after being treated according to S2, C strain is fermented for 3 hours, B strain is added for fermentation for 3 hours, and A is added for collaborative fermentation for 48 hours. (C, B, A)

[0107] Table 7 Effects of (C, B, A) strain addition sequence on the nutritional quality and color difference of blueberry juice after 48 hours of fermentation

[0108]

[0109] Note: Values ​​are expressed as mean ± SD (n = 3)

[0110] The present invention relates to a method for preparing an optimal strain addition sequence of probiotic fermented blueberry juice, and belongs to the field of food probiotic juice processing. The present invention uses blueberry juice as a substrate matrix for the fermented sample, selects three strains of lactic acid bacteria suitable for the matrix to develop a fermentation process, and determines the optimal strain addition sequence for fermentation. The changes of multiple indicators after the fermentation are analyzed, including the content of functional substances, antioxidant capacity, color difference value, etc., in order to provide new ideas and certain technical guidance for the blueberry deep processing industry, and provide a new theoretical data basis for the regular research of lactic acid bacteria fermented blueberry products.

Claims

1. A method for fermenting blueberry juice, Features: At a constant temperature of 37°C, first, the blueberry juice was fermented with Bifidobacterium lactis JYBR-190CGMCC No.18092 for 3 to 3.5 hours, then Lactobacillus plantarum JYLP-326 CGMCC No.18038 was added for 3 to 3.5 hours, and then Lactococcus lactis JYLL-60 CGMCC No.2335 was added for cooperative fermentation for 48 to 48.5 hours to end the fermentation. After the fermentation was completed, the pH of the blueberry juice decreased. Among them, the accession amount of Bifidobacterium lactis was 1% of the blueberry juice, the accession amount of Lactobacillus plantarum was 1% of the blueberry juice, and the accession amount of Lactococcus lactis was 1% of the blueberry juice.

2. The method according to claim 1, Features: The various bacterial strains are activated, selected and cultured on solid culture medium and then introduced into liquid culture medium for inoculation and standby use.

3. The method according to claim 2, Features: The three strains were spread on the surface of solid culture medium respectively, and cultured at 37°C for two days. Single colonies on the plate were picked, transferred to solid culture medium and streaked for more than two times to obtain pure colonies, which were then picked and cultured in liquid culture medium.

4. The method according to claim 2, Features: The blueberry juice raw material is prepared according to the following method: adding water to blueberry pulp to squeeze the juice, filtering, and the ratio of blueberry pulp to water is 1g:2-3mL; adding sodium carbonate to the blueberry juice to adjust its pH value to 5.0, adding 0.4% pectinase, and bathing it in a water bath at 45°C for 90 minutes; centrifuging at 4500r / min for 20min for homogenization; bathing it in a water bath at 60°C for 10min to inactivate the enzyme; and sterilizing it at 75°C for 15min.

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