Hovenia acerba fermented functional beverage with biological activity

Abstract

The invention provides a hovenia acerba fermented functional beverage with biological activity. The hovenia acerba fermented functional beverage is a beverage prepared by adding polypeptide with immunocompetence into prepared hovenia acerba fermented liquid with anti-fatigue activity. The hovenia acerba fermentation liquor is prepared by taking hovenia acerba and honey as main raw materials, adding pectinase for enzymolysis and then adding saccharomycetes for fermentation; the amino acid sequence of the polypeptide capable of improving the immunocompetence is SEQ ID NO: 1. The functional fermentation liquor provided by the invention is clear and transparent, is golden yellow in color, harmonious in wine body style, mellow and rich in fragrance, and palatable in sourness and sweetness, and has a remarkable anti-fatigue effect. Moreover, the functional polypeptide is added, so that the functional health-care wine has the function of improving the immunity of a body and can be developed and prepared into functional health-care wine.

Description

Technical Field

[0001] The invention belongs to the technical field of health wine, and in particular relates to a fermented jujube-shaped functional drink with biological activity. Background Art

[0002] Hovenia dulcis, scientifically known as Hovenia dulcis, belongs to the genus Hovenia of the Rhamnaceae family and is widely distributed in Guizhou, Sichuan, Yunnan, Gansu, Shaanxi and other places. Its fruit is rich in potassium malate and glucose, especially after the frost, the sweetness doubles, and the sugar content of mature fruit exceeds 30%. It can be tasted directly and is also an excellent raw material for winemaking. Hovenia dulcis not only has sufficient sugar (24.9 grams of total sugar per 100 grams of fresh fruit, including 8.9 grams of glucose), but is also rich in vitamins B1, B2, VC, as well as dihydromyricetin, organic acids and other nutrients. It is a plant resource with great potential.

[0003] Fermented fruit wine is an alcoholic beverage made by crushing and squeezing fruits, extracting their juice, filtering, adjusting the ingredients, adding microorganisms for fermentation and aging. Although wine dominates the market, in recent years, with the advancement of technology and the diversification of consumer preferences, more and more non-grape fruit wines have begun to emerge, adding more choices to the market.

[0004] The reason why fermented fruit wine is so popular is mainly due to its four major advantages: first, it is nutritionally comprehensive, rich in organic acids, aromatic esters, vitamins, amino acids and minerals, and drinking in moderation is good for health; second, fruit wine has a moderate alcohol content and low irritation, which can both refresh and relieve fatigue without burdening the body; third, fruit wine has a unique flavor, attractive color, fragrant fruity aroma, and mellow taste, which meets the taste buds of different consumers; finally, fruit wine uses fruit as raw material and does not require the consumption of food resources. It not only promotes the sustainable development of the fruit industry, but also helps solve the problem of fruit surplus in some areas. At the same time, the saved food can be used to support food-scarce areas and help solve the problem of food and clothing.

[0005] At present, although wine is still the mainstream of the fermented fruit wine market, with the improvement of technological automation and people's pursuit of healthy diet, other high-nutrition fruits such as jujube are gradually gaining attention. The development of jujube wine not only conforms to the market trend, but also provides consumers with a new choice that is both delicious and healthy, especially suitable for modern people who pursue quality of life and health and wellness. Summary of the invention

[0006] The object of the present invention is to provide a fermented jujube functional beverage with biological activity, that is, a beverage prepared by adding a polypeptide with immune activity to a fermented jujube liquid with anti-fatigue activity.

[0007] The invention first provides a fermented jujube liquor, which is prepared by taking jujube and honey as main raw materials, adding pectinase for enzymolysis, and then adding yeast for fermentation;

[0008] The fermentation process conditions of the jujube provided by the present invention are as follows: using 20% ​​honey, adding 0.05% pectinase at 30°C, enzymolysis for 2h, then adding 0.2% yeast, and fermenting for 6d;

[0009] The invention also provides that the jujube fermented liquid can be used to prepare anti-fatigue products.

[0010] In another aspect, the present invention provides a functional polypeptide having the function of improving immunity;

[0011] Furthermore, the polypeptide has an amino acid sequence of PQEGNG (SEQ ID NO: 1);

[0012] The present invention also provides an application of the functional polypeptide, which can be added to the jujube fermentation liquid to prepare a functional fermented beverage.

[0013] The functional fermented liquor provided by the present invention is clear and transparent, golden in color, harmonious in style, mellow and rich in flavor, sweet and sour in taste, and has a significant anti-fatigue effect. Moreover, the functional polypeptide is added to improve the body's immune ability, and can be developed into a functional health wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :The effect of fermentation time on the fermented liquid of jujube;

[0015] Figure 2 :The effect of the amount of koji on the fermented jujube liquor;

[0016] Figure 3 :The effect of honey addition on the fermented liquid of jujube;

[0017] Figure 4 :The influence of fermentation temperature on the fermentation liquid of jujube;

[0018] Figure 5 : The levels of BUN and LA in the serum of mice in each group

[0019] Figure 6 :The liver glycogen and muscle glycogen contents of mice in each group

[0020] Figure 7 : Polypeptide G-25 dextran gel chromatography chromatogram;

[0021] Figure 8 : Liquid phase distribution diagram of F2 component after reversed phase high performance liquid chromatography;

[0022] Fig. 9: Peptide mass spectrometry identification results. DETAILED DESCRIPTION

[0023] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0024] Example 1: Single factor experiment affecting the fermentation liquid of jujube

[0025] By referring to relevant literature and conducting preliminary experiments, we preliminarily determined the factors that affect the fermentation liquid of jujube and the data range of each factor for single factor experiments. After adding twice the amount of water to the jujube to squeeze the juice, the sugar content was measured to be about 10%. By consulting the data, we learned that the more suitable sugar content for fermenting fruit wine is about 15%-25%. Through preliminary experiments, we know that after adding 12% honey, the sugar content of jujube juice can reach 20%, so we selected two addition amounts on both sides of this value for single factor experiments. By consulting the data and referring to the instructions of the yeast used, we learned that the suitable survival temperature of yeast is 25-30℃, so we selected 22-28℃ for single factor experiments. By referring to the fermentation process of other fruit wines and the instructions of brewing yeast, we learned that the amount of yeast used is more suitable at 0.15%, so we selected 0.05%-0.25% of the koji amount for single factor experiments. Through preliminary experiments, it was found that the fermentation time of jujube syrup is preferably 4-6 days. If the fermentation time is too short, most of the sugars are not decomposed and the alcohol content is too low. If the fermentation time is too long, the wine is slightly sour and ethanol begins to convert into acetic acid. Therefore, a fermentation time of 3-7 days was selected for single factor experiments. Based on the above considerations, the fermentation days, the amount of honey used, the amount of koji used, the fermentation temperature and their factor gradients were selected for single factor experiments (Table 1).

[0026] Table 1: Single factor level table

[0027]

[0028] 1. Effect of fermentation time on the quality of jujube fermentation liquid

[0029] After enzymatic hydrolysis of the jujube juice with a ratio of 1:2 and water, 0.1% activated yeast and 12% honey were added, and fermented at 28°C. The fermentation days were designed to be 3 days, 4 days, 5 days, 6 days, and 7 days. The effect of fermentation time on the alcohol content and sensory score of the jujube fermented liquid was analyzed experimentally. The results are as follows Figure 1As shown. The chart shows that the alcohol content increases with the increase of fermentation days. When it reaches the 5th day, it starts to rise slowly, reaches the highest on the 6th day, and decreases slightly on the 7th day, indicating that after the 7th day, the alcohol in the wine begins to ferment into acetic acid. The amount of residual sugar in the wine decreases with the increase of time, until it starts to decrease slowly after the 5th day, indicating that after the 5th day, the fermentation in the wine begins to slow down, the sugar in it stops decomposing, and gradually reaches the end of fermentation. The sensory score of the fermented jujube liquid increases with the increase of time, and becomes flat on the fifth day. The score begins to decrease on the 7th day. It may be that the acid produced in the wine on the 7th day makes people feel uncomfortable, so the score is reduced. Comprehensive analysis shows that the best fermentation days for the fermented jujube liquid are 6 days.

[0030] 2. Effect of the amount of koji on the quality of jujube fermentation liquid

[0031] After enzymatic hydrolysis of jujube juice and water in a ratio of 1:2, 12% honey was added and fermented at 28°C for 6 days. Then, the amount of koji was divided into five levels of 0.05%, 0.1%, 0.15%, 0.2%, and 0.25% to conduct a single factor experiment on the effect of the amount of koji on the fermentation results. The experimental results are as follows: Figure 2 As shown. From the figure, it can be analyzed that the alcohol content increases slowly when the amount of koji increases to 0.15%. The residual sugar content in the wine decreases with the increase of the amount of koji, but it also decreases slowly when it reaches 0.15%. The sensory score of jujube wine increases with the increase of the amount of koji, but there is a significant decrease in the score when it reaches 0.2%. At this time, a clear yeast smell can be smelled in the wine, indicating that a large amount of yeast in the wine has not been fully used and the amount of yeast added is too much. Comprehensive analysis shows that the optimal amount of yeast added to jujube fermentation liquid is 0.15%.

[0032] 3. Effect of honey addition on the quality of jujube fermentation liquid

[0033] After enzymatic hydrolysis of the jujube juice with water in a ratio of 1:2, 0.15% activated yeast was added and fermented at 28°C for 6 days. The effect of different honey addition amounts on the quality of the jujube fermented liquid was observed by changing the honey addition amount to 4%, 8%, 12%, 16%, and 20%. The results are shown in the figure. Figure 3 As shown. It can be analyzed from the figure that the alcohol content increases with the increase of honey addition, and when the honey addition reaches 16%, the alcohol content almost stops rising. The residual sugar content in the wine increases with the increase of honey addition. When the addition amount is greater than 16%, the residual sugar content in the wine increases sharply, indicating that the honey addition amount is too high and a large amount of sugar can no longer be decomposed. The sensory score of the fermented jujube liquid increases with the increase of honey addition, but the score decreases after the addition amount is greater than 16%. At this time, the sugar content in the wine is too high, which seriously affects the taste of the wine. Therefore, a comprehensive analysis shows that the best honey addition amount for the fermented jujube liquid is 16%.

[0034] 4. Effect of fermentation temperature on the quality of jujube fermentation liquid

[0035] After enzymatic hydrolysis of the jujube juice with a ratio of 1:2 and water, 0.15% activated yeast and 16% honey were added and fermented at 28°C. The effect of different fermentation temperatures on the alcohol content and sensory score of the jujube fermentation liquid was observed by adjusting the fermentation temperature as a single factor. The results are as follows Figure 4 As shown. It can be analyzed from the figure that the alcohol content in the fermentation liquid increases significantly with the increase of fermentation temperature, and the alcohol content tends to decrease slightly when the fermentation temperature is greater than 28°C. The amount of residual sugar in the wine decreases with the increase of temperature. When the temperature is greater than 28°C, the amount of residual sugar no longer changes, indicating that 28-30°C has little effect on the decomposition of sugar in the wine. The sensory score of the fermented jujube liquid increases with the increase of fermentation temperature, but also has a slight downward trend when it reaches 28°C. Therefore, comprehensive analysis shows that the optimal fermentation time for the fermented jujube liquid is 28°C.

[0036] Example 2: Process Optimization Experiment of Jujube Fermentation Liquid

[0037] According to the results of single factor experiment, the optimal level of each single factor was determined, and then on the basis of the optimal level, the orthogonal experiment was carried out to optimize the process of jujube fermented liquid beverage. Three levels were selected for fermentation days, honey addition, koji amount, and fermentation temperature, respectively, to conduct an orthogonal experiment with four factors and three levels (see the table for details) to select the best brewing method for jujube fermented liquid.

[0038] Table 2: Orthogonal experiment level table

[0039]

[0040]

[0041] During the fermentation process of jujube wine, there are many factors that affect fermentation, and even different factors will have mutual influence. Single factor experiments show that the above factors have a great influence on the quality of jujube fermented liquid. Therefore, in order to optimize the process of jujube fermented liquid, four factors, namely fermentation time, fermentation temperature, amount of koji, and amount of honey added, were selected for orthogonal experiment L9 (34). The alcohol content and sensory score were comprehensively analyzed as the results, and then the optimal fermentation process of jujube fermented liquid was selected. The experimental results and analysis are shown in Table 3.

[0042] Table 3: Orthogonal experiment results

[0043]

[0044] According to the sensory analysis method in GB / T15038-2006 General Analytical Methods for Grape and Fruit Wine, the fermented jujube liquor was sensory evaluated and scored in terms of color, aroma, taste, typicality, etc. A group of 10 food professionals was selected to form an evaluation team, and the evaluation method in the sensory evaluation table was used to score, and the average value was taken as the result. The scoring table is shown in Table 4.

[0045] Table 4: Sensory scoring index table

[0046]

[0047] From the results in Table 3, it can be seen that the order of influence of each factor on the alcohol content of the fermented jujube is: B>D>A>C, that is, the factor that has the greatest influence on the alcohol content of the fermented broth is the fermentation temperature, followed by the amount of koji, then the number of fermentation days, and finally the amount of honey added. The best fermentation combination is: A3B3C3D3. The order of influence of each factor on the sensory score results is: A>C>B>D, that is, the factor that has the greatest influence on the sensory score results is the fermentation time, followed by the amount of honey added, then the fermentation temperature, and finally the amount of koji. The best fermentation combination is A3B3C3D1. The fermented jujube fermented broth fermented according to this process has the highest sensory score of 87.9 points, which is more popular with the public. Because the influence of factor D (the amount of koji) on sensory evaluation ranks fourth, which is the factor with the least influence, and the influence of factor D (the amount of koji) on alcohol content ranks second, which is a more important influencing factor, so the D factor (the amount of koji) should choose the third level, that is, 0.2%. Therefore, the best fermentation combination was finally analyzed to be A3B3C3D3, that is, the fermentation results were best when the fermentation days were 6 days, the fermentation temperature was 30°C, the honey addition was 20%, and the amount of koji was 0.2%. Therefore, the additional verification experiment A3B3C3D3 was conducted, and the experimental results showed that the alcohol content was 12.2%vol, and the average sensory score was 88.5, as shown in Table 5. The best fermentation combination of the jujube fermentation liquid was finally determined to be A3B3C3D3. The jujube wine brewed with this formula has a clear and transparent body, a golden color, a prominent fruity aroma, a rich and elegant wine aroma, a coordinated wine style, a rich mellow aroma, and a sweet and sour taste.

[0048] Table 5: Verification experiment scoring results

[0049]

[0050] Example 3: Determination of the physicochemical properties of the fermented jujube broth

[0051] 1) Determination of residual sugar content: Use a handheld saccharimeter to measure the initial sugar content and residual sugar content.

[0052] 2) Determination of alcohol content: After distilling the sample, use an alcohol meter and a thermometer to measure the value, and then convert it into alcohol content at 20°C.

[0053] 3) Determination of pH value and total acid: The pH value is directly determined using a pH meter, and the total acid is determined using the direct titration method in the national standard.

[0054] 4) Determination of methanol: gas chromatography in the general analysis method of fruit wine is adopted.

[0055] The test results of physical and chemical indicators are shown in Table 6.

[0056] Table 6: Test results

[0057]

[0058]

[0059] Microbial detection indicators: Total bacteria count: less than 50cfu / ml.

[0060] Sensory indicators: The wine is clear and transparent, golden in color, with prominent fruity aroma, rich and elegant wine aroma, harmonious style, mellow and rich aroma, and a sweet and sour taste.

[0061] Example 4: Functional determination of fermentation broth

[0062] The fermented liquid was added to the diet, and the mice were fed according to the above principles. American ginseng was set as a positive control, and the normal saline group was set as a blank control. The swimming exhaustion time of each group of mice was tested.

[0063] Table 7: Effects of different amounts of fermentation broth added on the exhaustion time of mice

[0064]

[0065] The results showed (Table 7) that the fermented jujube juice significantly increased the swimming exhaustion time of mice, and its effect was similar to that of American ginseng.

[0066] After the weight-bearing swimming experiment, 2 ml of blood was collected from the eyeball immediately and treated with heparin anticoagulation. The blood sample was centrifuged at 3000 r / min for 10 min to obtain the supernatant. The BUN and LA contents in the supernatant were determined by colorimetry.

[0067] BUN is a key indicator of protein metabolism and reflects exercise metabolic adaptation. During high-intensity exercise, the increase in BUN levels is more significant in those with weak adaptability, highlighting the importance of monitoring it. LA, as a product of glycolysis, surges during intense exercise, leading to increased muscle acidity, interfering with metabolism, and promoting fatigue. Therefore, testing serum LA after exercise can evaluate anaerobic energy supply, fatigue, and muscle damage. Figure 5It can be seen that the serum BUN and LA levels of mice in the blank control group were significantly higher than those in the positive control group, while after treatment with the fermentation liquid, these two indicators of mice in the experimental group decreased significantly (P<0.05), and the effect was similar to that of American ginseng, indicating that the fermentation liquid enhanced the mice's resistance to exercise fatigue and lactic acid tolerance, restored the homeostasis of the internal environment, promoted the normalization of metabolism, and had a significant anti-fatigue effect.

[0068] Dissect the mouse, take about 200 mg of liver and muscle tissue, and place them in 0.9% sodium chloride solution. High-speed homogenization to 10% homogenate, and then centrifuge at 12,000 rpm for 15 minutes to obtain the supernatant. Use a kit to measure liver glycogen and muscle glycogen levels. Glycogen reserves are related to endurance exercise performance. Sufficient endurance means strong endurance, while depletion means easy fatigue. Experiment ( Figure 6 ) showed that the glycogen reserves of mice in the positive control group were lower than those in the blank group, while the fermented liquid (experimental group) group significantly increased the glycogen content (P<0.05), prolonging the mice's exercise endurance, and the effect was similar to that of American ginseng.

[0069] Example 5: Preparation of functional polypeptides

[0070] The salmon meat tissue was washed and crushed into a homogenate, deionized water was added according to a liquid-to-solid ratio of 10:1, and papain was added, the pH was adjusted to 7.0, and the mixture was hydrolyzed at 60°C for 4 hours. The enzymatic hydrolyzate was boiled for 15 minutes to inactivate the enzyme and then freeze-dried to obtain salmon polypeptide dry powder.

[0071] The salmon peptide mixture was deeply purified using Sephadex G-25 column chromatography technology. First, 1g of salmon peptide freeze-dried powder was dissolved in sterile water to prepare a concentration solution of 100mg / mL. After filtering through a 0.45μm filter membrane, 3mL was loaded into a chromatography column filled with Superdex G-25 gel. Subsequently, sterile water was passed through at a flow rate of 0.5mL / min for elution, and the eluate was collected by an automatic collector. The absorbance of the eluate was monitored at a wavelength of 215nm using an ultraviolet spectrophotometer, and the eluate in a single peak area was selected and collected according to the elution curve. Figure 7 It can be seen that two components F1 and F2 were separated by dextran gel G-25 chromatography, among which F2 had a high peak and a large area, so F2 component was selected for subsequent experiments.

[0072] The F2 component was purified by reversed phase high performance liquid chromatography, PE C18 column (150 mm × 4.6 mm), mobile phase A (pure water) and B (acetonitrile containing 0.1% v / v trifluoroacetic acid), injection volume 30 μL, column temperature 25 ± 5 ° C, flow rate 1.0 mL / min, detection wavelength 215 nm. 0-8 min (99% A ~ 97% A, 1% B ~ 3% B); 8-12 min (97% A ~ 96% A, 3% B ~ 4% B); 12-16 min (96% A ~ 80% A, 4% B ~ 20% B); 16-20 min (80% A ~ 99% A, 20% B ~ 1% B), the results are shown in Figure 8 Nine elution peaks appeared within 8-30 min, among which the FG peak area accounted for a larger proportion, so it was inferred that the FG component had a higher content and response value, and might be the main effector substance. Therefore, mass spectrometry was used to sequence the FG component. Fig. 9 A total of 36 polypeptides were isolated from the FG component, of which 6 polypeptides had a molecular weight of <1000Da, as shown in Table 8. The antioxidant activity of polypeptide c and polypeptide e was higher than that of the others, and their sequences were PQEGNG and HFDYILA. The sequences were sent to a biological company for synthesis, and the immunity of the three polypeptides was determined. IgG is mainly a humoral immune antibody produced by the spleen and plasma cells, and plays a key role in enhancing immunity. Therefore, IgG was selected as the judgment indicator. The results showed that the IgG content in polypeptide c group was the highest, and its sequence was PQEGNG (SEQ ID NO: 1).

[0073] Table 8: Sequences and activities of isolated peptides

[0074]

[0075] Example 6: Animal experiments on polypeptides

[0076] A total of 200 clean-grade male BALB / c mice were randomly divided into four groups: low dose (100 mg / kg), medium dose (200 mg / kg), and high dose (300 mg / kg) were added to the fermentation broth, and a control group was set up. Each group of mice was gavaged daily at a volume of 0.2 ml / 10 g bw, and the corresponding dose of drug or an equal amount of normal saline (control group) was given.

[0077] 1. Thymus / body mass ratio

[0078] Before and after the experiment, the mice were fasted for 12 hours to measure their body weight. Subsequently, the mice were killed by cervical dislocation, dissected and the thymus was removed. After carefully removing the blood on the surface of the organ with filter paper, its mass was weighed. The ratio of organ mass to body mass was calculated to obtain the organ index.

[0079] Table 9: Ratio of fermentation broth of different doses of peptides to mouse thymus / body weight

[0080]

[0081] The ratio of thymus to body mass, known as the thymus index, is an important indicator for measuring the functional state of an individual's immune system. As shown in Table 9, there are significant differences between the groups with added polypeptide fermentation broth and the control group, indicating that the addition of polypeptides enhances the immune performance of the thymus.

[0082] 2. Delayed hypersensitivity (DTH)

[0083] After continuous gavage of mice for 30 days, the abdominal skin of each mouse was treated with a depilatory agent in an area of ​​about 3 cm × 3 cm, and then 50 μL of DNFB solution was evenly applied for sensitization. Five days after sensitization, 10 μL of DNFB solution was evenly applied to both sides of the right ear of the mouse to stimulate the reaction. After 24 hours, the mouse was killed by cervical dislocation, and the left and right ears were cut off. A circular ear piece with a diameter of 5 mm was removed from each ear using a punch and weighed separately.

[0084] Table 10: Effects of different doses of peptides on delayed-type hypersensitivity (DTH) in mice

[0085]

[0086] The delayed hypersensitivity reaction (DTH) of mice reflects the cellular immunity of mice. As shown in the results of Table 10, the ear swelling degree of each polypeptide dosage group was significantly higher than that of the control group, indicating that the addition of polypeptide improved the cellular immunity function of mice.

[0087] 3. Half hemolysis value (HC 50 )

[0088] After continuous gavage for 30 days, mice were intraperitoneally injected with 0.2 mL of 2% SRBC cell suspension to stimulate immune response. After 5 days, blood was collected by removing the eyeballs into a centrifuge tube, and the serum was precipitated by standing for 1 hour, and then centrifuged at 5000r / min for 8min to collect the serum. After the serum was diluted 300 times with normal saline, 1 mL of diluted serum was mixed with 0.5 mL of 10% SRBC by volume and 1 mL of diluted complement (1:10 dilution of normal saline) in a test tube, and a serum-free control tube (normal saline) was set up at the same time. This mixture was placed in a 37°C constant temperature water bath for 15 to 30 minutes, and then the reaction was terminated by ice bath. Centrifuge again at 5000r / min for 8min, take 1 mL of supernatant and mix with 3 mL of Du's reagent, and take another test tube to add 0.25 mL of 10% SRBC and an appropriate amount of Du's reagent to a total volume of 4 mL as a standard control. After mixing evenly, let it stand for 10 minutes, adjust the wavelength to zero with the control tube, and measure the optical density value of each tube at 540nm.

[0089] Table 11: Effects of different doses of polypeptide fermentation broth on mouse HC 50 Impact table

[0090]

[0091] Mouse HC 50 Used to evaluate the humoral immune response of mice, the results in Table 11 show that the addition of polypeptides enhanced the humoral immune ability of mice, and the medium and high dose groups had the strongest effect, which was significantly higher than the low dose group and the control group.

[0092] 4. Mouse NK cell activity assay

[0093] After continuous gavage for 30 days, the mice were killed by cervical dislocation, and the spleen was removed under sterile conditions and placed in a culture dish containing sterile Hanks solution. The spleen was gently torn and filtered through a 200-mesh sieve to obtain a single-cell suspension. The suspension was washed three times with Hanks solution, each time centrifuged for 10 minutes (2000r / min), and the cells were finally resuspended in 2ml of complete culture medium. Trypan blue staining was used to confirm that the proportion of live cells was higher than 95%, and then the cell concentration was adjusted to 5x10 using RPMI1640 complete culture medium. 6 CFU / mL.

[0094] 24 hours before the experiment, the target cells were subcultured and washed three times with Hanks solution before use. The final concentration was adjusted to 1x10 6 CFU / mL. During the experiment, 100 μl of target cells and effector cells were mixed at a ratio of 50:1 and added to a 96-well culture plate. At the same time, the target cell natural release well (containing only target cells and culture medium) and the target cell maximum release well (containing target cells and 1% NP40) were set up, and three replicates were set up for each condition. The culture plate was placed in a 37°C, 5% CO2 incubator for 4 hours.

[0095] After the incubation, the culture plate was centrifuged at 2000r / min for 5min, 100μl of supernatant was aspirated from each well and transferred to a new 96-well plate, followed by the addition of 100μl of LDH matrix solution for 3 minutes. Afterwards, 30μl of 1mol HCl was added to each well to terminate the reaction. Finally, the absorbance (OD) value of each well was measured at a wavelength of 490nm on an ELISA reader.

[0096] Table 12: Effects of different doses of peptide fermentation broth on NK cell activity

[0097]

[0098] NK cell activity is one of the important indicators for evaluating the state of individual immune function. Table 12 shows the NK cell activity under the influence of various polypeptide doses. Compared with the control group, there was no significant change in the NK cell activity in the low and medium dose groups, while under the influence of high doses, the NK cell activity was significantly increased, indicating that the addition of high-dose polypeptides significantly improved the body's immunity.

[0099] The prepared polypeptides were added to the fermentation broth, and the color, aroma, taste, typicality and other aspects of the wine were evaluated by sensory evaluation according to the standards in Table 4, and the physicochemical properties were determined. The alcohol content of each group of fermentation broth was 12.2% vol, the sugar content was 12.7 g / L, the wine was clear and transparent, the color was golden yellow, the fruity aroma was prominent, the wine aroma was rich and elegant, the wine style was coordinated, the mellow aroma was rich, the sweet and sour taste was suitable, and the bacterial content was less than 50 cfu / ml, which confirmed that the addition of polypeptides did not affect the quality of the wine, and it could be added as a functional substance. Considering the improvement of the immune performance of each addition amount, 300 mg / kg was determined to be the best addition amount.

[0100] In summary, the fermented jujube liquor used in the present invention has a significant anti-fatigue effect, and the functional polypeptide added therein can improve the body's immunity, and can be used as a health-care active product.

Claims

1. A fermented jujube liquor, characterized in that: The jujube fermented liquid is prepared by taking jujube and honey as raw materials, adding pectinase for enzymolysis, and then adding yeast for fermentation.

2. The fermented jujube juice according to claim 1, characterized in that The jujube fermented liquid is obtained by using 20% ​​honey, adding 0.05% pectinase, enzymolysis at 30°C for 2h, and then adding 0.2% yeast and fermenting for 6d.

3. Use of the fermented jujube liquor according to claim 1 in the preparation of anti-fatigue products.

4. A product with anti-fatigue effect, wherein the product contains the fermented jujube liquid of claim 1 at a pharmacologically effective concentration.

5. The preparation with anti-fatigue effect as claimed in claim 4, further comprising a polypeptide with immune effect.

6. The anti-fatigue product according to claim 5, wherein the amino acid sequence of the polypeptide is SEQ ID NO:

1.

7. A polypeptide having an immune effect, characterized in that: The amino acid sequence of the polypeptide is SEQ ID NO:

1.

8. Use of the polypeptide according to claim 7 in preparing functional fermented beverages.

9. A functional fermented beverage, characterized in that: The functional fermented beverage is prepared by adding the polypeptide described in claim 7 to the jujube fermentation liquid described in claim 1.

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