CN117941785A

CN117941785A - Preparation method of Tibetan concave parsley and highland barley compound fermented beverage with high antioxidation

Info

Publication number: CN117941785A
Application number: CN202311854374.5A
Authority: CN
Inventors: 南木加; 刘双平; 罗布顿珠; 多吉卓玛; 米久; 次白拉
Original assignee: Tibet University Of Tibetan Medicine; Jiangnan University
Current assignee: Tibet University Of Tibetan Medicine; Jiangnan University
Priority date: 2023-12-29
Filing date: 2023-12-29
Publication date: 2024-04-30

Abstract

The invention discloses a preparation method of a Tibetan concave parsley highland barley compound fermented beverage with high antioxidation, which comprises the following steps: step S1, germination treatment of highland barley; s2, heating germinated highland barley in a water bath to inactivate enzyme, drying, crushing by a crusher, and sieving; step S3, uniformly mixing the crushed highland barley with pure water, adding saccharifying enzyme and liquefying enzyme, uniformly mixing, and saccharifying and liquefying to obtain highland barley saccharifying liquid; s4, cooling the highland barley saccharification liquid to room temperature, and inoculating saccharomycetes and lactobacillus for fermentation; s5, centrifuging the fermented highland barley saccharification liquid to obtain highland barley stock solution; s6, adding the Tibetan parsley polysaccharide extract into the highland barley stock solution, and continuously stirring and uniformly mixing; and S7, packaging the uniformly mixed feed liquid, and sterilizing to obtain the Tibetan concave parsley highland barley compound fermented beverage. The invention adopts Tibetan concave parsley polysaccharide to compound highland barley fermentation stock solution, and obtains the compound fermented beverage with good antioxidant activity.

Description

Preparation method of Tibetan concave parsley and highland barley compound fermented beverage with high antioxidation

Technical Field

The invention belongs to the field of beverage processing, and particularly relates to a preparation method of a Tibetan concave parsley highland barley compound fermented beverage with high antioxidation.

Background

The Tibetan celery is the root of the plant Apium graveolens of Apium of Umbelliferae, and in recent years, research at home and abroad finds that the Tibetan celery polysaccharide is used as a main active ingredient of the Tibetan celery, has anti-fatigue and in-vivo and in-vitro anti-oxidation activities, and has the effects of resisting fatigue, resisting oxidative stress, regulating immunity and the like. Highland barley is rich in various functional components such as beta-glucan, linoleic acid, linolenic acid, gamma-aminobutyric acid (GABA) and the like. The highland barley has larger change of various nutritional ingredients in the fermentation process, and the flavor ingredients are obviously increased compared with the unfermented highland barley, so that the color, the aroma and the taste of the composite beverage are formed, and the composite beverage has unique flavor and deep nutritional value.

The cereal fermented beverage is a liquid beverage prepared by using cereal as a main raw material, converting starch in the raw material into sugar usable by microorganisms by using enzymes and the like, and fermenting the sugar by probiotics. The cereal fermented beverage can not only completely retain the nutritional ingredients of the raw materials, but also ensure that the raw materials have better taste and are more convenient to drink, and the nutritional ingredients in the cereal fermented beverage are easier to be absorbed and utilized by human bodies.

In recent years, as plant polysaccharide has various health care effects, more and more functional beverages related to plant polysaccharide are researched by people; therefore, a specific preparation process is explored, proper plant polysaccharide is selected and compounded with highland barley beverage, and the functional composite cereal beverage with high antioxidant activity and aromatic flavor is developed, so that the method has wide market prospect.

At present, as the Tibetan celery polysaccharide has anti-fatigue and in-vivo and in-vitro anti-oxidation activities, no beverage which takes Tibetan celery polysaccharide compounded highland barley as a main raw material appears in the market, the proposal of a Tibetan celery and highland barley compound fermentation beverage is necessary.

Disclosure of Invention

The embodiment of the invention aims to provide a preparation method of a highland barley compound fermented beverage with high antioxidation, which adopts Tibetan concave parsley polysaccharide to compound highland barley fermentation stock solution to obtain the compound fermented beverage with good antioxidation activity.

In order to solve the technical problems, the invention adopts the technical scheme that the preparation method of the Tibetan concave parsley highland barley compound fermented beverage with high antioxidation comprises the following steps:

Step S1, selecting highland barley with complete and full particles, cleaning, draining off water on the surface, soaking with saline water, draining off water, placing into a constant temperature and humidity incubator for culture, flushing once every certain time, and germinating for a period of time;

S2, heating germinated highland barley in a water bath to inactivate enzyme, then placing the highland barley in an oven for drying, crushing the highland barley by a crusher, sieving the highland barley, and placing the highland barley in a dryer for preservation;

step S3, uniformly mixing crushed highland barley with pure water, adding saccharifying enzyme and liquefying enzyme, uniformly mixing, and saccharifying and liquefying for a period of time at a certain temperature to obtain highland barley saccharifying liquid;

S4, cooling the highland barley saccharification liquid to room temperature, and inoculating saccharomycetes and lactobacillus for fermentation;

s5, centrifuging the fermented highland barley saccharification liquid to obtain highland barley stock solution;

s6, adding the Tibetan parsley polysaccharide extract into the highland barley stock solution, and continuously stirring and uniformly mixing;

and S7, sub-packaging the uniformly mixed feed liquid into cleaned and sterilized beverage bottles, sealing, sterilizing, and rapidly cooling to room temperature by cold water to obtain the Tibetan-concave-parsley highland barley compound fermented beverage.

Further, the step S1 specifically includes:

Selecting highland barley with complete and full grains, cleaning, draining off water on the surface, soaking the highland barley in warm water at 28-30 ℃ for 12-13 h, draining off water, placing the highland barley in a constant temperature and constant humidity incubator at 28-30 ℃ for culture, flushing once every 5-6 h, and carrying out germination treatment for 24-48 h.

Further, the step S2 specifically includes:

heating germinated highland barley in water bath at 85-90 deg.c for 10-12 min to deactivate enzyme, stoving in 50-52 deg.c oven, crushing in crusher, sieving with 50-60 mesh sieve, and storing in drier.

Further, the step S3 specifically includes: mixing crushed highland barley with pure water according to the following ratio of 1: mixing uniformly in a mass ratio of 10-30, adding saccharifying enzyme and liquefying enzyme, mixing uniformly, saccharifying and liquefying at 50-70 ℃ for 3.5-4.5 h, and obtaining highland barley saccharifying liquid; stirring once every 25-35 min in the saccharification and liquefaction process;

The mass concentration of the saccharifying enzyme in the highland barley aqueous solution is 1-3 per mill, and the mass concentration of the liquefying enzyme in the highland barley aqueous solution is 0.5-1.5 per mill.

Further, in the step S4, the mass concentration of the saccharomycete in the highland barley saccharification liquid is 0.05% -0.3%, the mass concentration of the lactobacillus in the highland barley saccharification liquid is 1% -5%, and the fermentation time is 24-48 h.

Further, in the step S4, the yeast with the mass concentration of 0.25% is inoculated for fermentation for 16 hours, and then the lactobacillus with the mass concentration of 4% is inoculated for fermentation for 18 hours, and the total fermentation is 34 hours.

Further, the step S5 specifically includes:

centrifuging the highland barley saccharification liquid after fermentation at 8000-8500 rpm for 5-6 min to obtain highland barley stock solution.

Further, in the step S6, the volume ratio of the Tibetan concave parsley polysaccharide extract to the highland barley stock solution is 10% -40%.

Further, in the step S7, the temperature during sterilization is 80-85 ℃ and the sterilization time is 15-25 min.

Further, the preparation method of the Tibetan parsley polysaccharide extract in the step S6 comprises the following steps:

Weighing Tibetan rue, adding 10-50 times of purified water, adding complex enzyme, stirring uniformly, performing ultrasonic treatment at the ultrasonic power of 350-380W and the ultrasonic temperature of 55-60 ℃ for 55-60 min, performing water bath enzyme deactivation at the ultrasonic temperature of 90-95 ℃ for 10-12 min, cooling, centrifuging at 4500-5000 rpm for 15-18 min, taking supernatant, performing rotary concentration at the temperature of 50-60 ℃ to 1/8-1/10 of the original volume, and standing for 12-15 h to obtain Tibetan rue polysaccharide extract;

The mass concentration of the complex enzyme in the Tibetan parsley aqueous solution is 1-3%;

the compound enzyme is cellulase and papain, and the mass ratio of the cellulase to the papain is 3-5: 5 to 7.

The beneficial effects of the invention are as follows:

The compound fermented beverage with good oxidation resistance is obtained by extracting the Tibetan concave parsley polysaccharide and then compounding the Tibetan concave parsley polysaccharide with the fermented highland barley stock solution, the flavor component content of the highland barley stock solution is improved by adding the Tibetan concave parsley polysaccharide, the mouthfeel and the aroma are better, and the Tibetan concave parsley has light Tibetan concave parsley fragrance.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a flow chart of a method for preparing the Tibetan kefir highland barley compound fermented beverage with high antioxidant activity.

FIG. 2 is a graph showing the effect of a yeast strain on a composite fermented beverage according to an embodiment of the present invention.

FIG. 3 is a graph showing the effect of lactobacillus strains on a composite fermented beverage according to an embodiment of the present invention, wherein (a) is a histogram of total acid content and pH value of each lactobacillus strain; (b) a line graph of sensory scores for each lactic acid bacterial species.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

As shown in fig. 1, the embodiment of the invention provides a preparation method of a Tibetan-concave-parsley highland barley compound fermented beverage with high antioxidation, which comprises the following steps:

Firstly, selecting highland barley with complete and full grains, cleaning, draining the water on the surface of the highland barley, soaking the highland barley in warm water at 28-30 ℃ for 12-13 hours, draining the highland barley, placing the highland barley in a constant temperature and humidity incubator at 28-30 ℃ for culture, flushing the highland barley every 5-6 hours, and carrying out germination treatment for 24-48 hours;

Heating germinated highland barley in water bath at 85-90 deg.c for 10-12 min to deactivate enzyme, stoving in 50-52 deg.c oven, crushing in crusher, sieving with 50-60 mesh sieve, and storing in drier;

Step three, crushed highland barley and pure water are mixed according to the following ratio of 1: uniformly mixing (10-30) by mass ratio, adding 1-3%o of saccharifying enzyme (i.e. the mass concentration of saccharifying enzyme in highland barley water solution is 1-3%o) and 0.5-1.5%o of liquefying enzyme (i.e. the mass concentration of liquefying enzyme in highland barley water solution is 0.5-1.5%o), uniformly mixing, saccharifying and liquefying at 50-70 ℃ for 3.5-4.5 h, and obtaining highland barley saccharifying liquid. Stirring every 25-35 min in the saccharification and liquefaction process to prevent the slurry from being heated unevenly;

Step four, cooling highland barley saccharification liquid to room temperature, and then inoculating saccharomycetes with the mass concentration of 0.05-0.3% and lactobacillus with the mass concentration of 1-5% for fermentation for 24-48 h;

Step five, centrifuging the highland barley saccharification liquid after fermentation at 8000-8500 rpm for 5-6 min to obtain highland barley stock solution;

Step six, adding 10% -40% (volume ratio v/v) of Tibetan concave parsley polysaccharide extract to highland barley stock solution into the highland barley stock solution, and continuously stirring and mixing uniformly;

And seventhly, subpackaging the prepared uniformly mixed feed liquid into cleaned and sterilized beverage bottles, sealing, placing the beverage bottles in a sterilizing pot at 80-85 ℃ for sterilization for 15-25 min, and rapidly cooling the beverage with cold water to room temperature to obtain the composite fermented beverage.

Further, the preparation method of the Tibetan rue polysaccharide extract comprises the steps of weighing Tibetan rue, adding 10-50 times of purified water, adding 1-3% of compound enzyme (cellulase and papain with the mass concentration W/v of the compound enzyme in the Tibetan rue water solution), stirring uniformly, carrying out ultrasonic sterilization for 10-12 min at the ultrasonic power of 350-380W and the ultrasonic temperature of 55-60 ℃ under the conditions of carrying out ultrasonic sterilization for 55-60 min at the temperature of 90-95 ℃, cooling, centrifuging for 15-18 min at 4500-5000 rpm, taking supernatant, carrying out rotary concentration at the temperature of 50-60 ℃ to 1/8-1/10 of the original volume, and standing for 12-15 h to obtain the Tibetan rue polysaccharide extract.

Highland barley is a typical high protein, low fat food. The main minerals include calcium, magnesium, potassium, phosphorus, sodium and sulfur; major trace minerals include iron, zinc, copper and manganese. In addition, highland barley also contains a large amount of vitamins, in particular vitamin B, wherein the content of vitamins B1, B2 and nicotinic acid is 30.4 mg.kg ^-1～1327.4mg·kg^-1 which is higher than that of corn (3.9 mg.kg ^-1～36.3mg·kg^-1) and wheat (0.16 mg.kg ^-1～13.55mg·kg^-1). Highland barley contains 18 amino acids, and human essential amino acids are complete, wherein gamma-aminobutyric acid (gamma-aminobutric acid, GABA) is a four-carbon non-protein amino acid, which can inhibit sympathetic excitation, and exert hypnotic, sedative and anxiety relieving effects. The Tibetan celery is also called Tibetan celery. Is the most distinctive plant for both medicine and food in Tibetan medicine, is one of five root drugs of Tibetan medicine, and the pharmacological effect research is mainly focused on the aspects of anti-fatigue, anti-dysmenorrhea, anti-oxidation, intelligence promotion and the like at present.

According to the invention, the fermentation process of the feed liquid ratio, the yeast strain, the lactic acid strain and the mixed bacteria is optimized according to the process flow. The lactobacillus can produce acid in large quantity, so that the pH value in the solution is stable, and the lactobacillus can be produced in the fermentation process, thereby delaying the spoilage speed of the beverage. In addition, the Angel active dry yeast can bring good flavor to the product during fermentation, and can enrich the flavor components of the beverage after the composite fermentation with lactobacillus, so that the research adopts the yeast and lactobacillus composite fermentation beverage, and the inoculation process of the yeast and lactobacillus composite fermentation beverage is determined.

Step one: optimization of composite fermented beverage-to-liquid ratio

(1) Optimization of feed-liquid ratio

Setting initial fermentation conditions, liquefying and saccharifying highland barley powder for 4 hours, inoculating 0.2% of saccharomycetes and 2% of lactic acid bacteria after cooling, fermenting at 30 ℃ for 36 hours, adding 10% of Tibetan concave celery polysaccharide into the highland barley powder after fermentation, exploring the influence of different feed liquid ratios (highland barley: water=1:10, 1:15, 1:20, 1:25 and 1:30) on reducing sugar, acidity and ethanol content, and carrying out sensory evaluation, wherein when the feed liquid ratio is 1:5 and 1:10, the highland barley fermented beverage has thick flavor, deep color, but has the problems of thick mouthfeel, heavy bitter taste and the like, and the beverage has bad sensory. When the feed liquid ratio is more than 1:15, the fermented beverage has light highland barley fragrance, light color and proper sour and sweet taste but insufficient highland barley special flavor. When the feed-liquid ratio is 1:15, the acidity of the fermented beverage is proper, the highland barley has a thicker taste and a better taste, so that the final feed-liquid ratio is 1:15. And comprehensively comparing to determine that the optimal feed-liquid ratio is 1:15.

Step two: optimization of individual fermentation conditions for yeasts

The microzyme is facultative anaerobic microorganism, in order to make the taste of the composite fermented beverage good, the microzyme is utilized for fermentation, but the microzyme can produce ethanol by fermentation, and the microzyme is used for preparing the composite fermented beverage with more comfortable taste and low alcoholic strength, so that the ethanol content and sensory evaluation are used as evaluation indexes of the single fermentation of the microzyme.

(1) Determination of fermentation species

Six active dry yeasts of H21, HL, BH, YL, GH and BTH are selected for fermentation, the information of yeast starter is shown in table 1, the fixed bottling amount is 150mL (total capacity is 250 mL), 0.2% of yeast is inoculated, 160 r.min ^-1 is used for fermentation for 24 hours at 30 ℃, 10% of Tibetan celery polysaccharide is added after the fermentation is finished, as shown in figure 2, the ethanol production capacity of 6 Angel active dry yeasts is not significantly different after the fermentation is finished, but the ethanol content of GH is the lowest, and the content of GH is 0.51 g.L ^-1. Yeast fermentation produces a number of metabolites that together constitute the characteristic aroma and taste of the beverage, with a wide variation in sensory scores among 6 active dry yeasts, with the highest sensory score for GH. This may be due to the different volatile substances produced by the different yeasts, which makes the beverage different in flavor. Based on the sensory evaluation and the results of ethanol content, GH is selected as yeast for subsequent fermentation.

TABLE 1 Yeast starter information

(2) Determination of fermentation time

Fixing 150mL (total volume of 250 mL), 0.2% of yeast inoculum size, 30 ℃ of fermentation temperature and the yeast strain determined in (1), fermenting highland barley saccharification liquid at 160 r.min ^-1 for 0h, 4h, 8h, 12h, 16h, 20h and 24h respectively, adding 10% of Tibetan celery polysaccharide after fermentation, measuring ethanol content, and performing sensory evaluation on a sample to determine the optimal fermentation time of the yeast. Along with the extension of fermentation time, the ethanol yield of the saccharomycete is obviously increased within 0-16 h, probably because the growth and propagation speed of the saccharomycete is obviously accelerated, a large amount of oxygen is required to be consumed, the oxygen content of the solution is obviously reduced, and thus anaerobic respiration is carried out, and the ethanol yield is also increased. In addition, the beverage sensory tended to rise first and then slightly drop with increasing yeast fermentation time, reaching a maximum at 20 h. The fermentation time of the saccharomycetes plays an important role in the quality of the beverage, the fermentation time is short, the alcohol production is insufficient, and the taste of the beverage is poor; the fermentation time is too long, the alcohol yield is too high, and the alcoholic smell of the beverage is too heavy. The sensory evaluation of the beverage for 16 hours is similar to that for 20 hours, and no significant difference exists. Therefore, the fermentation time of the selected yeasts is 16 hours from the viewpoint of integrating ethanol content, sensory evaluation and fermentation industrial cost.

(3) Determination of inoculum size

150ML (total volume 250 mL) of fixed bottle volume, 0.2% of yeast inoculation amount, fermentation temperature of 30 ℃, the yeast strain determined in (1) and the fermentation time determined in (2), 0.05%, 0.1%, 0.15%, 0.2%, 0.25% and 0.3% of yeast are respectively inoculated to the saccharified highland barley saccharification liquid, and 10% of Tibetan concave parsley polysaccharide is added after fermentation is finished. The ethanol content of the saccharomycetes tends to decrease along with the increase of the inoculation amount, which is probably due to the fact that the saccharomycetes grow vigorously in the fermented beverage with low inoculation amount when the fermentation time is the same, so that the time of the anaerobic respiration stage is prolonged, and the ethanol content is greatly increased; with the increase of inoculation amount, the quantity of saccharomycetes in the fermentation liquor is increased, and nutrient substances are rapidly consumed and utilized, so that the saccharomycetes grow poorly, the aerobic respiration time is prolonged, and the ethanol content in the fermentation liquor is reduced. The inoculation amount of the beverage is 0.25% and the ethanol content of the beverage is 0.30%, the sensory score of the inoculation amount of the beverage is 0.25% and is relatively higher, the taste is sweet and sour, the mellow and suitable, and the inoculation amount of the saccharomycetes is 0.25% in order to reduce the process cost.

Step three: optimization of individual fermentation conditions for lactic acid bacteria

(1) Determination of fermentation species

Five lactic acid bacteria including BF-ZR, MYT, GM, WM, L1901 are selected for experiments (Table 2, L1901-lactobacillus plantarum, MYT-lactobacillus casei, BF-ZR-lactobacillus plantarum, GM-lactobacillus fermentum and WM-pediococcus acidilactici), 3% of lactic acid bacteria are inoculated in a constant temperature incubator for fermentation for 30 hours under the conditions of 150mL (total capacity of 250 mL) of bottling volume and fermentation temperature of 37 ℃, 10% of concave celery polysaccharide is added after fermentation is finished, total acid and pH value of the beverage are measured after uniform mixing, and sensory evaluation is carried out, so that the optimal fermentation strain is determined.

The lactic acid production capacities of the 5 lactic acid bacteria are obviously different, wherein the total acid content of MYT, GM and BF-ZR groups is more than 4.95 g.L ^-1, which is higher than the total acid content of L1901 and WM groups, and the total acid content of the WM groups is the lowest and is 3.78 g.L ^-1. Meanwhile, the pH value of the beverage can be obviously reduced by fermentation according to the figure, the final pH value of fermentation in the commercial fermented beverage is approximately between 3.9 and 4.2, wherein the pH values of lactobacillus plantarum L1901 group and Pediococcus acidilactici WM are respectively 3.91 and 3.98, and the pH value of the commercial fermented beverage is within the pH range of the commercial beverage.

TABLE 2 lactic acid bacteria Strain information

The sensory evaluation of the composite fermented beverage after being fermented by 5 lactic acid bacteria is different, and the flavor and the taste of the beverage are different due to the great differences of metabolic pathways and metabolites of different lactic acid bacteria; and the growth rate of different lactic acid bacteria is different, and the sense of the beverage is also different. The sensory evaluation of the beverage fermented by the WM group is highest, the score is 73.6, the taste and acidity are proper, no bitter and astringent taste exists, and the color of the product is yellow and no layering phenomenon exists; the sensory score of the beverage after the fermentation in the L1901 group is 66.4, and the taste of the beverage is slightly sour and slightly bitter; the fermentation effects of MYT, GM and BF-ZR groups are basically similar, the taste is too sour, the bitter taste is obvious, the color of the product is pale yellow, no obvious layering phenomenon exists, and the sensory scores of the three are lower than those of the former two bacteria.

Therefore, the fermentation effect of WM group is obviously better than other 4 bacteria by combining the measurement results of the sensory evaluation, total acid content and pH value of the beverage, so the invention selects Pediococcus acidilactici WM for subsequent fermentation experiments.

(2) Determination of fermentation time

Fixing lactobacillus strains with the inoculation amount of 3%, the bottling amount of 150mL (total capacity of 250 mL) and the lactobacillus strain determined in the step (1), fermenting highland barley saccharification liquid in a constant temperature incubator at 37 ℃ for 0h, 3h, 6h, 9h, 12h, 15h, 18h, 24h and 30h respectively, adding 10% Tibetan concave parsley polysaccharide after fermentation, uniformly mixing, measuring the total acid and pH value of the beverage, and performing sensory evaluation to determine the optimal fermentation time. As shown in FIG. 3 (a), the total acid content of the composite fermented beverage gradually increases along with the extension of the fermentation time, the total acid content of the beverage increases fastest during 18h of fermentation, the content is 3.42 g.L < -1 >, and the total acid content increases by 21.39%; the pH value of the composite fermented beverage in 0-18 h is obviously reduced from 5.07 to 4.17. When the fermentation is carried out for 30 hours, the total acid content of the composite fermentation beverage is 3.50g.L < -1 >, and the pH value is 4.10, which is not greatly different from the total acid content and the pH value of 18 hours. In addition, as shown in fig. 3 (b), the sensory scores of the composite fermented beverage tended to be a rising and falling trend with the increase of fermentation time. The beverage fermented for 18 hours has proper taste and acidity and is rich in fermentation smell; the beverage after 30 hours of fermentation has slightly sour taste, obvious bitter and astringent taste and reduced sensory score. Thus, the fermentation time of lactic acid bacteria was set to 18 hours by combining the measurement results of the total acid content, pH value and sensory score.

(3) Determination of inoculum size

Fixing lactobacillus inoculation amount of 3%, bottling amount of 150mL (total capacity of 250 mL), inoculating 1%, 2%, 3%, 4% and 5% lactobacillus respectively to saccharified highland barley saccharifying liquid at determined lactobacillus strain WM and determined fermentation time of 18h, and adding 10% Tibetan concave parsley polysaccharide after fermentation, thereby exploring influence of inoculation amount on acidity, pH value and sense organ, and determining optimal inoculation amount. The total acid content of the composite fermented beverage is correspondingly increased along with the increase of the inoculation amount, and the pH value is correspondingly reduced, which is probably due to the increase of the lactic acid bacteria amount of the fermentation liquor caused by the increase of the inoculation amount, so that the lactic acid production capacity is enhanced, the rising speed of the total acid content of the fermentation liquor is accelerated, and the pH value reducing trend is obvious. However, when the inoculation amount of lactic acid bacteria in the fermentation broth is too high, the lactic acid bacteria grow poorly, the lactic acid production capacity of the lactic acid bacteria is reduced, and the total acid content is slowly increased. In addition, the total acid content of the beverage with the inoculation amount of 4 percent is increased by 0.28 g.L ^-1 and the pH value is reduced by 0.13 compared with the beverage with the inoculation amount of 3 percent; while the total acid content, pH and the inoculum size of the beverage at 5% were not significantly different compared to the beverage at 4%. As the inoculation amount increases, the sensory scores of the composite fermented beverage rise first and then tend to be smooth. The composite fermented beverage with the inoculation amount of 4% has proper taste and acidity, no bitter taste and no obvious difference with the fermented beverage with the inoculation amount of 5%. Thus, the lactobacillus inoculation amount was set to 4% from the viewpoint of industrial cost by combining the total acid content, pH value and the result of sensory scoring.

Step four: determination of composite fermentation process

(1) Inoculation mode determination

The lactobacillus can produce acid in large quantity, so that the pH value in the solution is stable, and the lactobacillus can be produced in the fermentation process, thereby delaying the spoilage speed of the beverage. In addition, the Angel active dry yeast has poor ethanol production capability, but can bring good flavor to the product during fermentation, and can enrich the flavor components of the beverage after being subjected to compound fermentation with lactobacillus, so that the research adopts the yeast and lactobacillus compound fermentation beverage, and the inoculation sequence of the yeast and the lactobacillus compound fermentation beverage is determined.

Under the condition of 150mL (250 mL) of bottling amount, the inoculation amount of the immobilized saccharomycetes is 0.25 percent, the fermentation time is 16 hours, and the fermentation temperature is 30 ℃; the inoculation amount of the lactobacillus is 4%, the fermentation time is 18h, the fermentation temperature is 37 ℃, the total fermentation time is 34h, 10% of Tibetan celery polysaccharide is added after the fermentation is finished, the fermentation is carried out according to 3 fermentation modes of inoculating the lactobacillus firstly and then inoculating the saccharomycete (A), inoculating the saccharomycete firstly and then inoculating the lactobacillus (B), inoculating the saccharomycete and the lactobacillus simultaneously, determining the acidity and the ethanol content, and finally carrying out sensory evaluation, thereby determining the inoculation modes of the saccharomycete and the lactobacillus.

After the fermentation conditions of the saccharomycetes and the lactic acid bacteria are optimized respectively, in order to take the fermentation advantages of the saccharomycetes and the lactic acid bacteria into consideration, the drink is fermented jointly by the saccharomycetes and the lactic acid bacteria, so that the drink has the flavor of the saccharomycetes and the lactic acid bacteria, the total acid, the ethanol content, the pH value and the sensory evaluation are used as indexes, and the simultaneous inoculation and the sequential inoculation fermentation are compared, wherein the sequential inoculation is divided into two cases: inoculating lactobacillus for fermenting for 18 hours, and inoculating saccharomycete for continuous fermenting for 16 hours at 18 hours; inoculating yeast for 16h, and inoculating lactobacillus for continuous fermentation for 18h at 16h; inoculating for 0h simultaneously, inoculating lactobacillus and saccharomycete, fermenting for 34h simultaneously, and finally determining the process flow suitable for highland barley saccharification liquid fermentation.

TABLE 3 physicochemical index detection results of different fermentation modes

Properties of (C)	A	B	C
				Total acid content (g.L-1)	3.90±0.06b	3.71±0.06c	4.11±0.08a
Ethanol content (g.L-1)	0.79±0.02	0.80±0.03	0.73±0.05
				pH	4.00±0.03a	3.92±0.02b	3.80±0.03c

Note that: the different letters of the same row of data represent significant differences (P < 0.05)

(A is mixed bacteria co-fermentation, B is inoculation of yeast and then lactic acid bacteria, C is inoculation of lactic acid bacteria and then yeast)

As shown in Table 3, the ethanol content of the beverage A obtained by fermenting the highland barley saccharification liquid is similar to that of the beverage B obtained by fermenting sequentially, and the ethanol content of the beverage C obtained by fermenting sequentially is the lowest; at the same time, beverage C has the highest total acid content, followed by beverage A and finally beverage B. This is probably due to the fact that the preferential growth of lactic acid bacteria causes a decrease in the pH of the fermentation system, limiting the growth of a large amount of yeasts and thus the rapid accumulation of ethanol.

From a combination of the main volatile component content in the fermented beverage table 4 and the sensory evaluation table 5, it can be seen that the sensory evaluation of beverage B was the highest, probably due to the higher kinds and contents of alcohol, acid, ester, aldehyde compounds. Beverage A has the lowest sensory acceptability, is bitter and astringent to taste and has heavy killing taste. The sensory evaluation of beverage C was also poor and the mouthfeel was too sour. The indexes of total acid content, ethanol content, volatile flavor substances and sensory evaluation are combined, wherein the sensory evaluation is the most important index, so that the fermentation is carried out for 16 hours by inoculating yeast and then 18 hours by inoculating lactobacillus.

TABLE 4 content of essential volatile components of composite fermented beverage

TABLE 5 sensory evaluation of different fermentation modes

Experimental group	Sensory evaluation description	Sensory scoring
			A	The fragrance is coordinated; the taste is sour and sweet, the lactic acid is more irritant, and the bitter taste is heavy	64.20±7.05
B	The flavor is coordinated, the taste is sour and sweet, the lactic acid taste is obvious, the sour taste is soft, and the fluidity is good	80.10±4.79
			C	The taste is slightly bitter and astringent, the sour taste is too heavy, and the sweetness is too low	72.70±8.50

(2) Determination of the amount of added Apii Graveolentis polysaccharide

After the inoculation mode is determined, the highland barley beverage after fermentation is centrifuged, polysaccharides are added according to the proportion of 10%, 15%, 20%, 25%, 30%, 35% and 40% of the volume of the beverage respectively, and then sensory evaluation is carried out, and the evaluation is carried out on the highland barley beverage from the aspects of color, smell, tissue state and taste respectively, so that the proper polysaccharide addition amount is selected. The sensory score of the Tibetan concave parsley and highland barley compound fermented beverage has the trend of increasing and then decreasing with the increase of the addition amount of the polysaccharide. When the addition amount of the polysaccharide is low, the taste of the beverage is mainly sour, and the sweetness is insufficient; and too high an amount of polysaccharide can lead to too sweet beverages with poor flowability and precipitation, and reduced sensory scores. Researches show that when the addition amount of the polysaccharide is 20%, the beverage is sour and sweet in taste, good in fluidity, free of precipitation and highest in sensory score. Therefore, the subsequent experiments were performed with a polysaccharide addition of 20%.

Example 1

Firstly, selecting complete and full highland barley, cleaning, draining water on the surface of the highland barley, soaking the highland barley in warm water at 28 ℃ for 12 hours, draining the highland barley, placing the highland barley in a constant temperature and humidity incubator at 28 ℃ for culture, flushing the highland barley every 5 hours, and carrying out germination treatment for 24 hours;

Heating germinated highland barley in water bath at 85deg.C for 10min to deactivate enzyme, oven drying at 50deg.C, pulverizing with pulverizer, sieving with 50 mesh sieve, and storing in dryer;

Step three, the crushed highland barley powder and pure water are mixed according to the following ratio of 1:10, adding 1 per mill of saccharifying enzyme and 0.5 per mill of liquefying enzyme, mixing, saccharifying and liquefying at 50deg.C for 3.5 hr to obtain highland barley saccharifying liquid. Stirring every 25min in the saccharification and liquefaction process to prevent the slurry from being heated unevenly;

Step four, cooling highland barley saccharification liquid to room temperature, inoculating saccharomycetes with mass concentration of 0.05% for fermentation for 12 hours, inoculating lactobacillus with mass concentration of 1% for fermentation for 12 hours, and fermenting for 24 hours;

step five, centrifuging the highland barley saccharification liquid after fermentation at 8000rpm for 5min to obtain highland barley stock solution;

Step six, adding 40% (volume ratio of Tibetan concave parsley polysaccharide extract to highland barley stock solution) of Tibetan concave parsley polysaccharide extract into highland barley stock solution, and continuously stirring and mixing uniformly;

And seventhly, subpackaging the prepared feed liquid into cleaned and sterilized beverage bottles, sealing, placing the beverage bottles in a sterilizing pot at 80 ℃ for sterilizing for 15min, and rapidly cooling the beverage with cold water to room temperature to obtain the composite fermented beverage.

The preparation method of the Tibetan concave parsley polysaccharide extract comprises the steps of weighing Tibetan concave parsley, adding 10 times of purified water, adding 1% (the mass concentration W/v of compound enzyme in a Tibetan concave parsley aqueous solution) of compound enzyme (cellulase and papain, the mass ratio is 3:7), uniformly stirring, performing ultrasonic treatment at an ultrasonic power of 350W and an ultrasonic temperature of 55 ℃ for 55min, performing water bath enzyme deactivation at 90 ℃ for 10min, cooling, centrifuging at 4500rpm for 15min, taking a supernatant, performing rotary concentration at 50 ℃ to 1/8 of the original volume, and standing for 12h to obtain the Tibetan concave parsley polysaccharide extract.

Example 2

Firstly, selecting complete and full highland barley, cleaning, draining water on the surface of the highland barley, soaking the highland barley in warm water at 30 ℃ for 12 hours, draining the highland barley, placing the highland barley in a constant temperature and constant humidity incubator at 30 ℃ for culture, flushing the highland barley every 6 hours, and carrying out germination treatment for 48 hours;

Heating germinated highland barley in water bath at 90deg.C for 12min to deactivate enzyme, oven drying at 52deg.C, pulverizing with pulverizer, sieving with 60 mesh sieve, and storing in dryer;

step three, the crushed highland barley powder and pure water are mixed according to the following ratio of 1:30 mass ratio, adding saccharifying enzyme with mass concentration of 3%and liquefying enzyme with mass concentration of 1.5%, mixing, saccharifying and liquefying at 70deg.C for 4.5 hr to obtain highland barley saccharifying liquid. Stirring every 35min in the saccharification and liquefaction process to prevent the slurry from being heated unevenly;

Step four, cooling highland barley saccharification liquid to room temperature, inoculating saccharomycetes with the mass concentration of 0.3% for fermentation for 24 hours, inoculating lactobacillus with the mass concentration of 5% for fermentation for 24 hours, and performing total fermentation for 48 hours;

step five, centrifuging the highland barley saccharification liquid after fermentation at 8500rpm for 6min to obtain highland barley stock solution;

Step six, adding 25% (volume ratio v/v) of Tibetan concave parsley polysaccharide extract to highland barley stock solution into the highland barley stock solution, and continuously stirring and mixing uniformly;

and seventhly, subpackaging the prepared feed liquid into cleaned and sterilized beverage bottles, sealing, placing the beverage bottles in a sterilizing pot at 85 ℃ for sterilization for 25min, and rapidly cooling the beverage with cold water to room temperature to obtain the composite fermented beverage.

Further, the preparation method of the Tibetan rue polysaccharide extract comprises the steps of weighing Tibetan rue, adding 50 times of purified water, adding 3% (the mass concentration W/v of compound enzyme in the Tibetan rue water solution) of compound enzyme (cellulase and papain, the mass ratio is 5:5), stirring uniformly, performing ultrasonic treatment at the ultrasonic power of 380W and the ultrasonic temperature of 60 ℃ for 60min, performing water bath enzyme deactivation at the temperature of 95 ℃ for 12min, cooling, centrifuging at 5000rpm for 18min, taking supernatant, performing rotary concentration at the temperature of 60 ℃ to 1/10 of the original volume, and standing for 15h to obtain the Tibetan rue polysaccharide extract.

Example 3

Firstly, selecting complete and full highland barley, cleaning, draining water on the surface of the highland barley, soaking the highland barley in warm water at 29 ℃ for 12.5 hours, draining the highland barley, placing the highland barley in a constant temperature and constant humidity incubator at 29 ℃ for culture, flushing every 5.5 hours, and carrying out germination treatment for 36 hours;

Heating germinated highland barley in 87 ℃ water bath for 11min to inactivate enzyme, drying in a 51 ℃ oven, crushing by a crusher, sieving with a 55-mesh sieve, and storing in a dryer;

step three, the crushed highland barley powder and pure water are mixed according to the following ratio of 1:15 mass ratio, adding saccharifying enzyme with mass concentration of 2 permillage and liquefying enzyme with mass concentration of 1 permillage, mixing, saccharifying and liquefying at 60 ℃ for 4 hours, and obtaining highland barley saccharifying liquid. Stirring every 30min in the saccharification and liquefaction process to prevent the slurry from being heated unevenly;

fourthly, cooling the highland barley saccharification liquid to room temperature, inoculating 0.25 mass percent of saccharomycetes for fermentation for 16 hours, inoculating 4 mass percent of lactic acid bacteria for fermentation for 18 hours, and performing total fermentation for 34 hours;

Step five, centrifuging the highland barley saccharification liquid after fermentation at 8200rpm for 5.5min to obtain highland barley stock solution;

Step six, adding 10% (volume ratio v/v) of Tibetan concave parsley polysaccharide extract to highland barley stock solution into the highland barley stock solution, and continuously stirring and mixing uniformly;

And seventhly, subpackaging the prepared feed liquid into cleaned and sterilized beverage bottles, sealing, placing the beverage bottles in a sterilizing pot at 82 ℃ for 20min for sterilizing, and rapidly cooling the beverage with cold water to room temperature to obtain the composite fermented beverage.

Further, the preparation method of the Tibetan rue polysaccharide extract comprises the steps of weighing Tibetan rue, adding 35 times of purified water, adding (2% of compound enzyme (the mass concentration W/v of the compound enzyme in the Tibetan rue water solution is 4:6) into the Tibetan rue, stirring uniformly, carrying out ultrasonic 58min at the ultrasonic temperature of 58 ℃ under the ultrasonic power of 360W, carrying out water bath enzyme deactivation at 92 ℃ for 11min, cooling, centrifuging at 4750rpm for 16min, taking supernatant, carrying out rotary concentration at 55 ℃ to 1/9 of the original volume, and standing for 13.5h to obtain the Tibetan rue polysaccharide extract.

Example 4

The manufacturing process disclosed in this example is substantially identical to that of example 3, except that: in the sixth step, the volume ratio of the highland barley stock solution added with the concave parsley polysaccharide is 20 percent.

Example 5

The manufacturing process disclosed in this example is substantially identical to that of example 3, except that: in the sixth step, the volume ratio of the highland barley stock solution added with the concave parsley polysaccharide is 30 percent.

Example 6

The manufacturing process disclosed in this example is substantially identical to that of example 3, except that: in the sixth step, the volume ratio of the highland barley stock solution added with the concave parsley polysaccharide is 40 percent.

Example 7

The manufacturing process disclosed in this example is substantially identical to that of example 3, except that: after the highland barley saccharification liquid is cooled to room temperature, 0.05% of saccharomycetes is inoculated for fermentation for 16 hours, then 5% of lactic acid bacteria is inoculated for fermentation for 18 hours, and the total fermentation is carried out for 34 hours.

Example 8

The manufacturing process disclosed in this example is substantially identical to that of example 3, except that: after the highland barley saccharification liquid is cooled to room temperature, lactobacillus with the mass concentration of 4% is firstly inoculated for fermentation for 18 hours, then saccharomycetes with the mass concentration of 0.25% is inoculated for fermentation for 16 hours, and the total fermentation is carried out for 34 hours.

Comparative example 1

The preparation of the common highland barley fermented beverage, the preparation process disclosed in the comparative example is basically the same as that of the example 1, except that: the highland barley stock solution obtained after the highland barley beverage fermentation is finished is not added with Tibetan concave parsley polysaccharide.

Sample detection

Samples 1 to 9 were obtained according to the preparation methods of examples 1 to 8 and comparative example 1, respectively, and sensory test and antioxidant activity test were performed, respectively.

(1) Sensory evaluation

Referring to GB/T7101-2022 requirements of national Standard beverage for food safety, 10 sensory evaluators trained in profession are invited and establish a sensory evaluation group, sensory evaluation is carried out on the products from 4 aspects of color, smell, taste and organization state, the comprehensive score is 100 points, and finally, the average value of the sensory scores of 10 people is taken, and the specific scoring standard is shown in Table 6.

Table 6 sensory evaluation table

(2) Antioxidant Activity assay

And respectively detecting the DPPH free radical clearance, the hydroxyl free clearance, the ABTS+ free radical clearance and the iron ion reducing capability of the sample to be detected.

The results are shown in Table 7 and Table 8.

Table 7 sensory test evaluation table

Group of	Highest score	Least score	Average score
				Example 1	75	52	64.20
Example 2	84	73	80.60
				Example 3	86	75	76.30
Example 4	96	84	89.6
				Example 5	81	67	72.20
Example 6	70	52	59.5
				Example 7	88	73	80.10
Example 8	79	54	72.70
				Comparative example 1	68	50	58.30

TABLE 8 statistical table of antioxidant Activity assays

Group of	DPPH radical scavenging rate	ABTS ⁺ radical scavenging rate	Hydroxyl radical scavenging rate	Iron ion reducing ability
					Example 1	97.37％	84.16％	85.17％	0.91
Example 2	95.26％	82.45％	83.27％	0.89
					Example 3	93.94％	76.45％	79.50％	0.89
Example 4	95.11％	86.25％	90.60％	0.93
					Example 5	95.75％	88.41％	90.85％	0.96
Example 6	96.25％	89.01％	92.57％	1.04
					Example 7	95.41％	87.77％	89.77％	0.92
Example 8	95.26％	85.39％	88.75％	0.91
					Comparative example 1	86.39％	81.21％	80.59％	0.87

From the results in tables 7 and 8, it is clear that the sensory test of example 4 is optimal, the antioxidant activity is higher, the overall performance is excellent, and the present best solution is obtained. Meanwhile, tables 7 and 8 show that the fermented beverage prepared by the preparation method can meet the taste requirements of the masses, has better market reaction and has higher oxidation resistance.

In this specification, each embodiment is described in a related manner, and identical and similar parts of each embodiment are all referred to each other, and each embodiment mainly describes differences from other embodiments. In particular, for system embodiments, since they are substantially similar to method embodiments, the description is relatively simple, as relevant to see a section of the description of method embodiments.

The foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention are included in the protection scope of the present invention.

Claims

1. The preparation method of the Tibetan concave parsley highland barley compound fermented beverage with high antioxidation is characterized by comprising the following steps of:

2. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein the step S1 is specifically:

3. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein the step S2 is specifically:

4. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein the step S3 is specifically: mixing crushed highland barley with pure water according to the following ratio of 1: mixing uniformly in a mass ratio of 10-30, adding saccharifying enzyme and liquefying enzyme, mixing uniformly, saccharifying and liquefying at 50-70 ℃ for 3.5-4.5 h, and obtaining highland barley saccharifying liquid; stirring once every 25-35 min in the saccharification and liquefaction process;

5. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein in the step S4, the mass concentration of the saccharomycete in the highland barley saccharification liquid is 0.05-0.3%, the mass concentration of the lactobacillus in the highland barley saccharification liquid is 1-5%, and the fermentation time is 24-48 h.

6. The method for preparing the highland barley compound fermented beverage with high oxidation resistance according to claim 5, wherein in the step S4, saccharomycetes with the mass concentration of 0.25% are firstly inoculated for fermentation for 16 hours, then lactobacillus with the mass concentration of 4% is inoculated for fermentation for 18 hours, and the total fermentation is 34 hours.

7. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein the step S5 is specifically:

8. The method for preparing the highland barley compound fermented beverage with high oxidation resistance according to claim 1, wherein in the step S6, the volume ratio of the Tibetan concave parsley polysaccharide extract to the highland barley stock solution is 10% -40%.

9. The method for preparing the highland barley compound fermented beverage with high antioxidation according to claim 1, wherein in the step S7, the sterilization temperature is 80-85 ℃ and the sterilization time is 15-25 min.

10. The method for preparing the highly antioxidant compound fermented beverage of Tibetan parsley and highland barley according to any one of claims 1 to 9, wherein the preparation method of the Tibetan parsley polysaccharide extract in the step S6 is as follows: