Formula process optimization and polyphenol oxidation resistance determination method for zanba fermented by native saccharomycetes

Abstract

The invention belongs to but is not limited to the technical field of fermentation, and particularly relates to a method for determining the antioxidant activity of polyphenol of a yeast-fermented tsamba, which obtains an optimal yeast-fermented highland barley tsamba technological formula through optimization on the basis of a single factor in combination with response surface design. Under the optimal formula process condition, the influence of yeast fermentation on polyphenol and antioxidant activity of the tsamba is measured, so that the nutritional value and functional activity of the tsamba are improved. Therefore, a certain theoretical basis is provided for development of highland barley products, the potential market competitiveness of the tsamba is improved, and the tsamba has great significance in protection and inheritance of the traditional national product, namely tsamba.

Description

Technical Field

[0001] The invention belongs to but is not limited to the field of fermentation technology, and in particular relates to a method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast. Background Art

[0002] Highland barley (Hordeum vulgare L. var. nudum Hook f.) belongs to the genus Hordeum of the Poaceae family. It is also called naked barley because its pericarp is separated from the caryopsis and the grains are exposed. It is a cultivated barley variety. Highland barley has the characteristics of cold resistance, drought resistance, and strong stress resistance. It is mainly distributed in five provinces of my country, namely Tibet, Qinghai, Sichuan, Yunnan, and Gansu, and is the main food crop of the Tibetan people. Highland barley is rich in protein, high dietary fiber, and polyphenols, and is an important raw material for the development of healthy foods. Recent studies have shown that highland barley is rich in a variety of bioactive substances, such as γ-aminobutyric acid, β-glucan, and phenolic compounds. Among them, phenolic compounds have attracted widespread attention due to their potential health benefits such as anti-oxidation, hypoglycemic, hypolipidemic, cardiovascular disease prevention, and anti-cancer. However, highland barley polyphenols are easily covalently bonded to dietary fiber to form insoluble bonded polyphenols, making them difficult to be released and absorbed by the body, thereby affecting their bioavailability. At the same time, the high dietary fiber content in highland barley makes its taste rough. At present, highland barley is mainly used for feed, wine making, tsampa, etc., and the degree of deep processing is not high, which restricts the nutritional potential of highland barley and restricts its further utilization and development.

[0003] Tsampa is a food made from highland barley as the main raw material. After the whole grain of highland barley is fried and crushed at high temperature, ghee, qula and white sugar are added to it for processing and preparation. It is also called "highland barley fried noodles". Tsampa has the characteristics of high calories, easy to store, easy to carry and rich in nutrition. It is widely loved by consumers. People in high-altitude and cold areas where fruits and vegetables are scarce can eat tsampa to satisfy their hunger and keep warm, which can solve the problem of human nutritional deficiencies. Traditionally, the production of tsampa is mostly done by families or small workshops. It is usually eaten immediately after being made and difficult to preserve for a long time. In addition, the lack of unified processing standards also limits its wider promotion and consumption.

[0004] Solid-state fermentation is a safe and efficient processing method with the advantages of low production cost, simple operation and environmental friendliness. During the fermentation process, a large number of hydrolases produced by microbial metabolism can promote the release of insoluble bonded polyphenols, enhance the content of soluble polyphenols and their biological activity, and has been widely used in the processing of grains. At the same time, the fermentation process can promote the decomposition of nutrients in tsampa into more easily absorbed small molecular nutrients, which is beneficial to the absorption of nutrients, improves its nutritional value, and improves the rough taste. Compared with bacteria with low tolerance to water activity, fungi are usually more able to grow in solid matrices lacking water, so that they can make better use of nutrients in the matrix. Yeast is a type of fungus that can ferment sugar into alcohol and carbon dioxide. It is a single-cell organism. The operation of yeast solid-state fermentation is relatively simple, with the advantages of strong selectivity, environmental protection, economy and high product yield, and is often used in the fermentation of traditional foods.

[0005] A patent with application number CN102258071B, which uses snow lotus fungus (a naturally occurring symbiotic system composed of lactic acid bacteria and yeast) to ferment highland barley flour to prepare a fermented highland barley biscuit, the product has good flavor and taste, and rich nutritional value. For example, the patent with application number CN116138392A discloses a method for increasing the content of highland barley nutrients and functional components based on solid-state fermentation, which uses the mycelium of the Chinese delicious mushroom Qaidam variant ZJU-TP-08 as a fermentation strain to solid-state ferment cereals to enrich its nutrients and improve antioxidant capacity, and has a very wide range of application prospects. At present, the processing technology standards of tsampa are not unified, and the optimization of the formula process of tsampa fermented by native yeast and the determination method of its polyphenol antioxidant have not been reported. The present invention proposes a formula process for optimizing the fermentation of tsampa by native yeast and a method for determining the polyphenol antioxidant. Firstly, tsampa powder was solid-state fermented by native yeast isolated from highland barley. Secondly, the processing technology of tsampa was optimized by response surface Box-Behnken experimental design. Finally, the antioxidant activity of fermented tsampa was evaluated by antioxidant activity assays such as DPPH, ABTS·+, FRAP and hydroxyl radical scavenging ability.

[0006] In summary, the problems existing in the prior art are: highland barley has a relatively rough taste, insufficient deep processing, which limits the development and utilization of highland barley, and the tsampa processing technology lacks a unified standard; the insoluble bonded polyphenols in highland barley and tsampa are difficult to extract and release, resulting in low bioavailability of polyphenols.

[0007] Difficulty in solving the above technical problems: During the fermentation process, fermentation time, humidity, and strain inoculation amount have a great impact on the taste, color and nutritional content of tsampa. Secondly, the polyphenols in tsampa are unstable and easily oxidized and decomposed during the extraction process. The extraction process requires strict low-temperature and light-proof operation.

[0008] The significance of solving the above technical problems: By using native yeast to carry out solid-state fermentation of tsampa and optimizing the process formula of native yeast fermentation of tsampa, a scientific basis can be provided for the formulation of yeast fermentation tsampa process standards; this technology can not only effectively decompose the nutrients in tsampa into small molecular nutrients that are easier to absorb, thereby improving its nutritional value and taste, but also promote the release of insoluble polyphenols and improve their bioavailability, thus having a more positive impact on human health. This provides new ideas for the deep processing and further development of highland barley. Summary of the invention

[0009] In view of the problems existing in the prior art, the present invention provides a method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast.

[0010] The invention is realized in this way: a process formula of fermenting tsampa with native yeast is optimized by single factor experiment and response surface Box-Behnken test design, and the best process formula of tsampa is determined as follows: 100g (100% by mass) of highland barley tsampa powder is used as a basis, 41mL of butter tea, 19.9g of qula, 20.0g of white sugar and 7.7g of butter.

[0011] Furthermore, the process optimization method of the process formula of tsampa comprises the following steps:

[0012] Step 1, using native yeast isolated and screened from highland barley in the laboratory to carry out solid-state fermentation of tsampa powder;

[0013] Step 2: Using sensory evaluation as an indicator, select the addition amount of butter, butter tea, sugar, and qula (milk residue) for single factor experiment.

[0014] Step three, the Box-Behnken experiment was used to conduct response surface design to determine the optimal process formula for fermenting tsampa with native yeast.

[0015] Step 4: Determine the activity of polyphenol antioxidant substances in the optimized native yeast fermented tsampa.

[0016] Further, the method for fermenting tsampa is as follows:

[0017] (1) Raw material pretreatment: Select highland barley grains with full grains, no insect infestation, no impurities, no mildew or other deterioration phenomena.

[0018] (2) Moistening: First, measure the moisture content of the raw barley. After cleaning and controlling the water content, weigh it and then moisten it. Use sealing film to seal the surface of the raw material to make it fully moist. After drying, use a moisture meter to measure the moisture content of the barley after moistening to 15.0% to 17.0% before grinding. Moistening is to ensure that the barley kernels have a better burst rate when frying.

[0019] (3) Stir-frying: Stir-fry the barley using an induction cooker until the barley is more than 85% crispy and more than 95% cooked, and then cool.

[0020] (4) Grinding: Grind the roasted highland barley into powder using a grinder, and then sieve the ground powder to obtain tsampa flour.

[0021] (5) Fermentation: This study used native yeast isolated from highland barley in the laboratory to ferment tsampa. Weigh tsampa powder into a conical flask, add an appropriate amount of sterile distilled water, and mix thoroughly. After sterilization, place it on a sterile operating table to inoculate yeast mother liquid; the tsampa inoculated with yeast was cultured and fermented in a shaking incubator at 28°C for 4 days.

[0022] (6) Preparation of tsampa: Taking 100 g (100% by mass) highland barley tsampa powder as a base, first add 41 mL of butter tea to melt 19.9 g of qula, 20.0 g of white sugar, and 7.7 g of butter, then add 100 g of highland barley tsampa powder, stir evenly, and knead the dough to prepare dough;

[0023] (7) Pressing and molding: placing the prepared dough into a mold and pressing it to shape the tsampa fermented by native yeast;

[0024] (8) Determination of biological activity, extraction and content determination of polyphenols and flavonoid content determination;

[0025] (9) Determination of in vitro antioxidant activity, including total antioxidant capacity, DPPH free radical scavenging capacity, iron ion reducing capacity, and hydroxyl free radical scavenging capacity.

[0026] Another object of the present invention is to provide a method for determining the in vitro antioxidant activity of polyphenols in tsampa fermented by native yeast prepared by the process formula of tsampa fermented by native yeast, comprising the following steps:

[0027] Step 1, extraction and content determination of polyphenols;

[0028] Step 2, extraction and content determination of flavonoids;

[0029] Step 3, total antioxidant capacity determination;

[0030] Step 4, DPPH free radical scavenging ability determination;

[0031] Step 5, determination of iron ion reduction and antioxidant capacity;

[0032] Step 6, ·OH scavenging capacity determination;

[0033] Further, the extraction and content determination of polyphenols specifically include:

[0034] Add the extractant to the weighed sample, perform ultrasonic extraction at low temperature and centrifuge, extract the supernatant and extract the residue again, combine the two supernatants and add an equal volume of trichloroacetic acid (TCA) solution to the supernatant, centrifuge again and collect the supernatant overnight, and finally use the extractant to make up the volume to obtain soluble polyphenols. Store at 0℃~4℃ away from light for future use;

[0035] The Folin-Ciocalateau method was used for colorimetric determination. The polyphenol extract was placed in a centrifuge tube, and Folin phenol solution was added. After shaking, saturated sodium carbonate was added, and then an appropriate amount of distilled water was added. After dark reaction, the supernatant was placed in a colorimetric dish and colorimetric determination was performed at 725 nm. The results were expressed as ferulic acid equivalents (FAE) per g dry weight (DW). According to the standard curve: y = 1.289x-0.003 (R 2 =0.9988) to calculate the polyphenol content (μmol FAE / g DW).

[0036] Further, the flavonoid content determination specifically includes:

[0037] The AlCl3 method was used for determination. An appropriate amount of polyphenol extract was added to a centrifuge tube, followed by NaNO2 solution and distilled water. After reaction in the dark, AlCl3 solution was added and mixed thoroughly. Then NaOH solution was added to make up the volume. The reaction was carried out at room temperature in a light-blocked state. Finally, after centrifugation, the supernatant was taken and the absorbance was measured at 510 nm. The results were expressed as catechin equivalents (CE) per g dry weight. According to the standard curve y = 2.5459x-0.01143 (R 2 =0.999) to calculate the flavonoid content (μmol CE / g DW).

[0038] Furthermore, the total antioxidant capacity determination specifically includes:

[0039] The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications. After adding an appropriate amount of polyphenol solution to the centrifuge tube, add ABTS·+ working solution, place it at room temperature for reaction, and measure the absorbance at 734nm for the tsampa samples after yeast fermentation and those without yeast fermentation;

[0040] The standard curve was drawn using Trolox, and the standard equation was: y = 103.95x + 1.563 (R 2 =0.9984)

[0041] Wherein: y is the ABTS·+ clearance rate, %; x is the Trolox concentration, mmol / L; polyphenol TEAC per gram of dry sample is expressed as Trolox equivalents (TE) (mmol TE / g DW).

[0042] Antioxidant capacity (%) = [(c-cb)-(s-sb)] / (c-cb)*100

[0043] Where:

[0044] c: absorbance of ABTS·+ working solution (measure A0);

[0045] cb: absorbance of ethanol;

[0046] sb: absorbance value after the sample reacts with ethanol;

[0047] s: absorbance value after the sample reacts with ABTS·+ working solution (measure A1).

[0048] Further, the DPPH free radical scavenging ability determination specifically includes:

[0049] The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. with slight modifications. An appropriate amount of polyphenol solution was added to the DPPH methanol solution, and the absorbance was measured at 517 nm after the reaction in the dark; and the DPPH radical scavenging activity was calculated based on the standard curve. The standard curve was drawn with ferulic acid, and the standard equation was:

[0050] y=451.75x+32.31(R 2 =0.9987)

[0051] Where:

[0052] y——DPPH free radical scavenging rate, %;

[0053] x——is the ferulic acid concentration (μmol / mL), and the polyphenol DRSA in each g of dry sample is expressed as ferulic acid equivalent (μmol FAE / g DW).

[0054] DPPH free radical scavenging ability (%) = [(c-cb)-(s-sb)] / (c-cb)*100;

[0055] Where:

[0056] s: absorbance value after the sample reacts with DPPH methanol solution;

[0057] sb: absorbance value after the sample reacts with methanol solution;

[0058] c: absorbance of DPPH methanol solution;

[0059] cb; absorbance of methanol solution.

[0060] Further, the determination of iron ion reducing ability specifically includes:

[0061] Take an appropriate amount of polyphenol solution, add FRAP (Ferric reducing antioxidant power, FRAP) solution and mix thoroughly. After a certain period of reaction at room temperature (37°C), measure the absorbance at 593nm. The iron ion reducing antioxidant power obtained is expressed as Fe 2+ It is expressed as mmol FE / g sample equivalent; acetic acid is used as blank; the standard curve is drawn using FeSO4 standard stock solution, and the standard equation is:

[0062] y=6.24x+0.0005(R 2 =0.9984) where: y is the absorbance; x is the concentration of ferrous sulfate, mmol / mL; the FRAP value of polyphenols per gram of dry sample is expressed as Fe 2+ Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmol FE / g DW).

[0063] Further, the hydroxyl radical scavenging ability determination specifically includes:

[0064] Take an appropriate amount of polyphenol solution in a centrifuge tube, add FeSO4 solution, H2O2 solution and salicylic acid in turn, incubate the mixture at 37°C for half an hour, cool it in an ice water bath, and plot a standard curve with vitamin C (VC) at 510 nm. The standard equation is: y = 47.562x + 2.9794 (R 2 =0.9959), and the polyphenol HPSA in each gram of dry sample was expressed as VC equivalent (VE) (μmol VE / g DW).

[0065] The calculation formula of OH removal rate is as follows:

[0066] Hydroxyl radical scavenging rate (%) = [1-(A x -A x0 ) / A0]*100;

[0067] Where: A x : Absorbance value of sample group; A x0 : The absorbance value of the blank group was obtained by replacing the sample with distilled water; A0: The absorbance value of the control group was obtained by replacing hydrogen peroxide with distilled water.

[0068] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0069] First, highland barley tastes relatively rough, and deep processing is insufficient, which limits the development and utilization of highland barley; the processing technology of tsampa lacks a unified standard; insoluble bonded polyphenols in highland barley and tsampa are difficult to extract and release, resulting in low bioavailability of polyphenols. Based on a single factor experiment, the present invention obtains the best process formula of highland barley tsampa fermented by native yeast after optimization analysis by response surface method: the addition amount of butter tea is 41mL, the addition amount of butter is 7.7g, the addition amount of qula is 19.9g, and the addition amount of white granulated sugar is 20.0g, and the sensory evaluation score of the finished product is 40.92, at which time the sensory score of tsampa is the highest, the fragrance is strong, and the taste is the best. On the basis of traditional tsampa production, tsampa powder is inoculated with yeast for fermentation. The polyphenol content and flavonoid content of fermented tsampa were 117.16μmol FAE / g DW and 0.59mmol CE / g DW, respectively. The DPPH scavenging ability, iron ion reducing ability, hydroxyl radical scavenging ability, and ABTS·+ scavenging ability of polyphenols in fermented tsampa were 10.09mmol FAE / g DW, 39.77mmol FE / g DW, 242.28mmol TE / g DW, and 81.27mmol TE / g DW, respectively, which were significantly improved compared with the highland barley tsampa before fermentation. This shows that the fermentation of yeast promoted the release of insoluble bonded polyphenols. The present invention uses native yeast to perform solid-state fermentation on tsampa, optimizes the process formula of native yeast fermentation of tsampa, and can provide a scientific basis for formulating yeast fermentation tsampa process standards; this technology can not only effectively decompose the nutrients in tsampa into small molecular nutrients that are easier to absorb, thereby improving its nutritional value and taste, but also promote the release of insoluble polyphenols and improve their bioavailability, thereby having a more positive impact on human health. It can provide new ideas for the deep processing and further development of highland barley.

[0070] In summary, the optimized formula process can make highland barley tsampa have better flavor, taste and active ingredients, and has good application prospects and market potential.

[0071] Second, (1) the expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0072] The present invention, based on laboratory preliminary research, uses native yeast isolated from highland barley to ferment highland barley, optimizes the process formula of fermented tsampa on the basis of single factor test, and performs in vitro antioxidant activity determination on polyphenols therein. The nutrient components in tsampa can be effectively decomposed into small molecule nutrients that are easier to absorb, thereby improving its nutritional value and taste, and can also promote the release of insoluble polyphenols and improve their bioavailability, thereby having a more positive impact on human health and promoting the deep processing and development and utilization of highland barley.

[0073] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0074] At present, there is no report on the optimization of the process of fermenting tsampa with native yeast and the determination method of its polyphenol antioxidant. The present invention uses native yeast as the fermentation strain and uses an optimized process formula to prepare fermented tsampa, which can effectively decompose the nutrients in tsampa into small molecule nutrients that are easier to absorb, thereby improving its nutritional value and taste, and can also promote the release of insoluble polyphenols and improve their bioavailability, thereby having a more positive impact on human health, promoting the deep processing and development and utilization of highland barley, and promoting the development of the highland barley industry in Yunnan Province.

[0075] (3) The technical solution of the present invention solves the technical problems that people have been eager to solve but have never been able to solve successfully:

[0076] Through the technology of the present invention, native yeast is used to carry out solid-state fermentation of tsampa, and the process formula of native yeast fermentation of tsampa is optimized, which can provide a scientific basis for the formulation of yeast fermentation tsampa process standards; at the same time, this technology can not only effectively decompose the nutrients in tsampa into small molecular nutrients that are easier to absorb, thereby improving its nutritional value and taste, but also promote the release of insoluble polyphenols and improve their bioavailability, thereby having a more positive impact on human health. This provides a new idea for the deep processing and further development of highland barley. BRIEF DESCRIPTION OF THE DRAWINGS

[0077] Figure 1 Schematic diagram of a method for determining the antioxidant activity of tsampa polyphenols fermented by yeast provided in an embodiment of the present invention;

[0078] Figure 2 is a phylogenetic tree of the isolated and purified yeast provided in an embodiment of the present invention;

[0079] Figure 3 It is a growth curve diagram of the separated and purified yeast provided in an embodiment of the present invention;

[0080] Figure 4 This is a screening diagram of yeast with the best fermentation performance provided by an embodiment of the present invention;

[0081] Figure 5 Schematic diagram of the effect of the amount of butter tea added on sensory evaluation provided by an embodiment of the present invention;

[0082] Figure 6 Schematic diagram of the effect of the amount of ghee added on sensory evaluation provided by an embodiment of the present invention;

[0083] Figure 7 Schematic diagram of the effect of the amount of added white sugar on sensory evaluation provided by an embodiment of the present invention;

[0084] Figure 8 Schematic diagram of the effect of the amount of Qula added on sensory evaluation provided by an embodiment of the present invention;

[0085] Fig. 9 It is a schematic diagram of a response surface diagram of the interaction of various factors provided in an embodiment of the present invention;

[0086] Fig.10 1 is a schematic diagram comparing the polyphenol content of highland barley tsampa before and after fermentation by yeast provided in an embodiment of the present invention;

[0087] Fig.11 is a schematic diagram comparing the flavonoid content before and after fermentation provided by an embodiment of the present invention;

[0088] Fig.12 Schematic diagram showing the comparison of ABTS·+ removal capacity before and after fermentation provided by an embodiment of the present invention;

[0089] Fig.13 Schematic diagram comparing the DPPH scavenging ability before and after fermentation provided by an embodiment of the present invention;

[0090] Fig.14 is a schematic diagram comparing the iron atomic capacity of yeast before and after fermentation provided by an embodiment of the present invention;

[0091] Fig.15 3 is a schematic diagram comparing the hydroxyl radical scavenging capabilities before and after fermentation provided by an embodiment of the present invention.

[0092] Fig.16 A finished product of highland barley tsampa fermented by native yeast provided in an embodiment of the present invention; DETAILED DESCRIPTION

[0093] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0094] The embodiment of the present invention provides a process formula for fermenting tsampa with native yeast. The process is optimized by single factor experiment and response surface Box-Behnken experimental design, and the optimal process formula of tsampa is determined as follows: 100 g (mass 100%) of highland barley tsampa powder is used as a benchmark, 41 mL of butter tea, 19.9 g of qula, 20.0 g of white sugar, and 7.7 g of butter.

[0095] like Figure 1 As shown, the method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast provided in the embodiment of the present invention comprises the following steps:

[0096] S101, using native yeast isolated and screened from highland barley in the laboratory to ferment tsampa flour;

[0097] S102, using sensory evaluation as an indicator, the addition amounts of butter, butter tea, sugar, and qula (milk residue) were selected for single factor experiment.

[0098] S103, Box-Behnken experiment was used for response surface design to determine the optimal process formula for tsampa fermentation with native yeast.

[0099] S104, determining the antioxidant activity of polyphenols in the optimized fermented tsampa.

[0100] (1) Process flow:

[0101] Highland barley selection → tempering → frying → grinding and sieving → tsampa powder → fermentation → preparation → molding → finished product.

[0102] (2) Key points of experimental operation

[0103] ① Raw material pretreatment: It is required to select highland barley grains with full grains, no insect infestation, no impurities, no mildew and other deterioration phenomena.

[0104] ② Moistening: First, measure the moisture content of the raw barley. It is required to clean it, control the water and weigh it before moistening. When moistening, seal it with a sealing film to make the surface of the raw material fully moist. After moistening and drying, use a moisture meter to measure the moisture content of the moistened barley to 15.0%-17.0% before grinding.

[0105] ③ Stir-frying: Stir-fry in an induction cooker until the highland barley is over 85% crispy and over 95% cooked, then cool.

[0106] ④ Grinding: Grind the roasted highland barley into powder using a grinder, and then sieve the ground powder to get tsampa flour.

[0107] ⑤ Fermentation: This study used native yeast isolated from highland barley in the laboratory to ferment tsampa. Weigh tsampa powder into a conical flask, add an appropriate amount of sterile distilled water, and mix thoroughly. After sterilization, place it on a sterile operating table to inoculate yeast mother liquid; the tsampa inoculated with yeast was cultured and fermented in a shaking incubator at 28°C for 4 days.

[0108] ⑥ Tsampa preparation: Taking 100 g (100% by mass) highland barley tsampa powder as the basis, first add 41 mL butter tea to melt 19.9 g qula, 20.0 g white sugar, and 7.7 g butter, then add 100 g highland barley tsampa powder, stir evenly, and knead the dough to prepare the dough;

[0109] ⑦Molding: put the prepared dough into the mold and press it to shape the tsampa fermented by native yeast;

[0110] (3) Single factor test

[0111] ① Determination of the amount of butter tea added

[0112] The addition amount of tsampa powder was fixed at 100g (mass was 100%), and the addition amount of butter 10%, white sugar 20%, and qula 20% was proposed to conduct a single factor experiment on the selection of the addition amount of butter tea. The addition amount of butter tea was 30%, 40%, 50%, 60%, and 70% in turn to determine the optimal addition amount of butter tea.

[0113] ②Determination of the amount of ghee added

[0114] The addition amount of tsampa powder was fixed at 100g (mass was 100%), and when the addition amount of butter tea was determined, the addition amount of white sugar was 20%, and the addition amount of qula was 20% to conduct a single factor experiment on the selection of the addition amount of butter. The addition amount of butter was 6%, 8%, 10%, 12%, and 14% in turn to determine the optimal addition amount of butter.

[0115] ③Determination of the amount of white sugar added

[0116] The addition amount of tsampa powder was fixed at 100g (mass was 100%). Through the above single factor test, when the addition amount of butter tea and butter was determined, the addition amount of qula was set at 20%, and the single factor test of the addition amount of white sugar was carried out. The addition amount was 16%, 18%, 20%, 22%, and 24% respectively, so as to determine the optimal addition amount of white sugar.

[0117] ④ Determination of the amount of Qula added

[0118] The addition amount of tsampa powder was fixed at 100g (mass was 100%). Through the above single factor test, when the addition amount of white sugar and butter tea was determined, a single factor test was conducted to select the addition amount of qula. The addition amount of qula was 16%, 18%, 20%, 22%, and 24% in turn to determine the optimal addition amount of qula.

[0119] (4) Response surface experiment

[0120] After completing the single factor experiment, with the help of Design-Expert software, four indicators including the amount of butter tea added (A), the amount of butter added (B), the amount of white sugar added (C), and the amount of qula added (D) were selected as experimental factors, and a 4-factor 3-level response surface optimization experiment was designed and carried out. The sensory evaluation score was used as the measurement indicator to determine the optimal formula of yeast fermented highland barley tsampa.

[0121] Table 1 Factor level table

[0122]

[0123] (5) Determination of biological activity

[0124] ① Extraction and content determination of polyphenols

[0125] The extraction of polyphenols was based on the method of Zhang Sha et al., and some modifications were made on this basis; the extractant was added to the weighed sample, ultrasonically extracted at low temperature and centrifuged, the supernatant was extracted and the residue was extracted again, the two supernatants were combined and collected and added with a TCA solution of equal volume to the supernatant, overnight, centrifuged again and the supernatant was collected, and finally the volume was fixed with the help of the extractant to obtain soluble polyphenols. Store at 0℃~4℃ away from light for future use.

[0126] The Folin-Ciocalateau method was used for colorimetric determination. The polyphenol extract was placed in a centrifuge tube, and Folin phenol solution was added. After shaking, saturated sodium carbonate was added, and then an appropriate amount of distilled water was added. After dark reaction, the supernatant was placed in a colorimetric dish and colorimetric determination was performed at 725 nm. According to the standard curve: y = 1.289x - 0.003 (R 2 =0.9988) to calculate the polyphenol content (μmol FAE / g DW).

[0127] ② Determination of flavonoid content

[0128] The AlCl3 method was used for determination. An appropriate amount of polyphenol extract was added to a centrifuge tube, followed by NaNO2 solution and distilled water. After reaction in the dark, AlCl3 solution was added and mixed thoroughly. Then NaOH solution was added to make up the volume. The reaction was carried out at room temperature in a light-blocked state. Finally, after centrifugation, the supernatant was taken and the absorbance was measured at 510 nm. The results were expressed as catechin equivalents (CE) per g dry weight. According to the standard curve y = 2.5459x-0.01143 (R 2 =0.999) to calculate the flavonoid content (μmol CE / g DW).

[0129] (7) Determination of in vitro antioxidant activity

[0130] ①Total antioxidant capacity assay (TEAC)

[0131] The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications. After adding an appropriate amount of polyphenol solution to the centrifuge tube, add ABTS·+ working solution, place it at room temperature for reaction, and measure the absorbance at 734nm for the tsampa samples after yeast fermentation and those without yeast fermentation;

[0132] The standard curve was drawn using Trolox, and the standard equation was: y = 103.95x + 1.563 (R 2 =0.9984)

[0133] Wherein: y is the ABTS·+ clearance rate, %; x is the Trolox concentration, mmol / L; polyphenol TEAC per gram of dry sample is expressed as Trolox equivalents (TE) (mmol TE / g DW).

[0134] Antioxidant capacity (%) = [(c-cb)-(s-sb)] / (c-cb)*100

[0135] Where:

[0136] c: absorbance of ABTS·+ working solution (measure A0);

[0137] cb: absorbance of ethanol;

[0138] sb: absorbance value after the sample reacts with ethanol;

[0139] s: absorbance value after the sample reacts with ABTS·+ working solution (measure A1).

[0140] ②Determination of DPPH free radical scavenging ability

[0141] The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. with slight modifications. An appropriate amount of polyphenol solution was added to the DPPH methanol solution, and the absorbance was measured at 517 nm after the reaction in the dark; and the DPPH radical scavenging activity was calculated based on the standard curve. The standard curve was drawn with ferulic acid, and the standard equation was:

[0142] y=451.75x+32.31(R 2 =0.9987)

[0143] Where:

[0144] y——DPPH free radical scavenging rate, %;

[0145] x——is the ferulic acid concentration (μmol), and the polyphenol DRSA in each g of dry sample is expressed as ferulic acid equivalent (μmol FAE / gDW).

[0146] DPPH free radical scavenging ability (%) = [(c-cb)-(s-sb)] / (c-cb)*100;

[0147] Where:

[0148] s: absorbance value after the sample reacts with DPPH methanol solution;

[0149] sb: absorbance value after the sample reacts with methanol solution;

[0150] c: absorbance of DPPH methanol solution;

[0151] cb; absorbance of methanol solution.

[0152] ③ Ferric reducing ability assay (FRAP)

[0153] Take an appropriate amount of polyphenol solution, add FRAP (Ferric reducing antioxidant power, FRAP) solution and mix thoroughly. After a certain period of reaction at room temperature (37°C), measure the absorbance at 593nm. The iron ion reducing antioxidant power obtained is expressed as Fe 2+ It is expressed as mmol FE / g sample equivalent; acetic acid is used as blank; the standard curve is drawn using FeSO4 standard stock solution, and the standard equation is:

[0154] y=6.24x+0.0005(R 2 =0.9984) where: y is absorbance; x is ferrous sulfate concentration, mmol / L; FRAP of polyphenols per g dry sample is expressed as Fe 2+Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmol FE / g DW).

[0155] ④ Determination of hydroxyl radical scavenging ability

[0156] Take an appropriate amount of polyphenol solution in a centrifuge tube, add FeSO4 solution, H2O2 solution and salicylic acid in turn, incubate the mixture at 37°C for half an hour, cool it in an ice water bath, and plot a standard curve with vitamin C (VC) at 510 nm. The standard equation is: y = 47.562x + 2.9794 (R 2 =0.9959), and the polyphenol HPSA in each gram of dry sample was expressed as VC equivalent (VE) (μmol VE / g DW).

[0157] The calculation formula of OH removal rate is as follows:

[0158] Hydroxyl radical scavenging rate (%) = [1-(A x -A x0 ) / A0]*100;

[0159] Where: A x : Absorbance value of sample group; A x0 A0: The absorbance value of the blank group was obtained by replacing the sample with distilled water; A1: The absorbance value of the control group was obtained by replacing hydrogen peroxide with distilled water.

[0160] (6) Data analysis

[0161] This experiment used Origin, Design-Expert8.0.6, and Excel2019 for data processing and analysis.

[0162] The invention provides a method for optimizing the formula process of native yeast fermented tsampa and determining the antioxidant activity of its polyphenols, which can provide a scientific basis for formulating the process standard of yeast fermented tsampa; can improve the nutritional value of fermented tsampa, improve the rough taste, and can promote the release of insoluble bonded polyphenols therein, improve the bioavailability of polyphenols, and have a more positive impact on human health; can cooperate with highland barley processing enterprises to promote the development of highland barley functional foods, and provide new ideas for the deep processing and development and utilization of highland barley.

[0163] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[0164] 1. Single factor test results

[0165] (1) Effect of butter tea addition on yeast fermentation of highland barley tsampa

[0166] Depend on Figure 5It can be seen that as the amount of butter tea added gradually increases, the sensory score of fermented tsampa will show a trend of first rising and then falling. When the amount of butter tea added is between 30% and 50%, the sensory score is the highest. Through observation, it is found that as the proportion of butter tea added increases, the increase in moisture content will cause the moldability of tsampa to gradually decrease, and the overall feel and touch will gradually deteriorate. When butter tea is added in excess, it will also lead to the deterioration of flavor and taste, so the sensory score shows a downward trend.

[0167] (2) Effect of butter addition on yeast fermentation of highland barley tsampa

[0168] Depend on Figure 6 It can be concluded that with the increase of the amount of ghee added, the sensory evaluation score of highland barley tsampa first increases and then decreases. When the amount of ghee added is in the range of 6% to 10% (the amount of tsampa powder added is fixed at 100g, and the mass is 100%), the sensory score reaches the maximum value.

[0169] The effect of ghee on the sensory evaluation of fermented tsampa is mainly reflected in the taste and mouthfeel. The appropriate amount of ghee added can make the fat aroma blend into the highland barley tsampa without affecting other flavor components and causing taste imbalance. In addition, it also has the effect of increasing appetite. When ghee is added in excess, a strong milky flavor will appear after tasting, which will greatly mask the smell of other ingredients and make it difficult for people to accept. When the amount of ghee added is too low, the product flavor will be too light and lack the delicate layering given by ghee, and the sensory score will be low.

[0170] (3) Effect of white sugar addition on yeast fermentation of highland barley tsampa

[0171] Depend on Figure 7 It can be concluded that the sensory evaluation score of highland barley tsampa first increases and then decreases with the increase of white sugar content. When the addition amount of white sugar is in the range of 18% to 22% (the addition amount of tsampa powder is fixed to 100g, and the mass is 100%), the sensory evaluation score of highland barley tsampa is the highest.

[0172] The influence of white sugar on the sensory score of tsampa is mainly on the taste. When the white sugar content is too low, the tsampa will not be sweet enough and the taste of tsampa will be too bland. When the white sugar content is too high, the tsampa will be too sweet and people will find it difficult to accept.

[0173] (4) Effect of Qula addition on yeast fermentation of highland barley tsampa

[0174] Depend on Figure 8 It can be seen that with the increase of the amount of qula added, the sensory evaluation score of highland barley tsampa first increases and then decreases. When the amount of qula added is in the range of 18% to 22% (the amount of tsampa powder added is fixed at 100g, and the mass is 100%), the sensory score reaches the maximum value.

[0175] The main effect of qula on sensory scores is the taste. When the amount of qula added is appropriate, it will give tsampa a sweet flavor. At the same time, qula is rich in nutrients such as protein and fat, which can promote the water absorption of tsampa powder to a certain extent and reduce the occurrence of caking. The fragrance of qula can cleverly neutralize the more shocking taste of ghee to people's senses and further enhance the taste of ghee. However, if too much qula is added, tsampa will have a certain milky smell, causing sensory discomfort.

[0176] 2. Response surface results and analysis

[0177] Based on the single factor experiment, response surface experiment was carried out to analyze the addition of butter tea at a ratio of 30%, 40% and 50%; butter was added at a content of 6%, 8% and 10%; white sugar and qula were added at a content of 18%, 20% and 22% respectively.

[0178] (1) Response surface experiment results

[0179] Table 2 Response surface experimental design and results

[0180]

[0181]

[0182] Table 3 Response surface variance analysis results

[0183]

[0184]

[0185] Note: * indicates significant, p < 0.05; ** indicates extremely significant, p < 0.01; p > 0.05 indicates not significant.

[0186] From the results presented in Table 3, it can be seen that the P value is extremely significant (P value <0.0001), indicating that the four factors of this model have a very significant effect on the sensory evaluation of highland barley tsampa. The P value of the lack of fit term below is equal to 0.2425, showing a non-significant difference, that is, the results presented by this response surface model are relatively ideal and reliable.

[0187] Comparing the F values ​​of the four factors, it can be seen that in terms of the degree of influence on the sensory evaluation score of yeast fermented tsampa, the four factors are ranked in the following order: C (amount of white sugar added) = D (amount of qula added) > B (amount of butter added) > A (amount of butter tea added); among them, A (amount of butter tea added) and B (amount of butter added) have no significant effect on the sensory evaluation of yeast fermented tsampa (P value > 0.05), while C (amount of white sugar added) and D (amount of qula added) significantly affect the sensory evaluation results of highland barley tsampa (P value < 0.05).

[0188] The fit of the model can be measured by the regression coefficient R 2 It can be determined from the above table that R 2 =0.9321, which proves that the response surface model has a relatively reliable fit and can accurately predict the regression value corresponding to the regression equation; and the adjusted R 2 adj =0.8642, proving that the regression equation has high reliability. According to the results presented in the table, CV = 0.6540%, indicating that the response surface optimization experiment has a small error and the reliability of the test results is high. In summary, we can use this response surface model to more accurately analyze and predict the sensory evaluation results of yeast fermented tsampa.

[0189] The quadratic regression fitting equation obtained from the above response surface model is:

[0190] Y=83.86+0.39A-0.062B-0.90C+1.97AB-1.00AC-0.75BC-5.02A 2 -2.47B 2 -6.54C 2 .

[0192] (2) Response surface analysis

[0193] The response surface diagram and contour diagram that can present the interaction between the experimental factors obtained through the response surface experiment were drawn by using Design-Expert 8.0.6 software. Through these two sets of diagrams, the pairwise interaction between the independent variables in the fermented tsampa process formula and their influence on the sensory score can be intuitively reflected and analyzed. If the contour diagram is elliptical and the contour lines are densely distributed, it means that the interaction between the two factors can have a significant effect on the response value; when the interaction between the two independent variables has no significant effect on the response value, the contour diagram is circular and the distribution of the contour lines is increasingly sparse. For the response surface diagram, the steeper the surface and the larger the inclination angle, the more obvious the effect of the interaction between the two independent variables on the sensory evaluation of fermented tsampa.

[0194] like Fig. 9 As shown, the obtained two-dimensional curve graph and three-dimensional surface graph are shown.

[0195] Depend on Fig. 9 It can be seen that all response surfaces are convex and open downward, indicating that the sensory scores have a maximum value. It can be seen that each interaction term has a significant impact on the response value, that is, the interaction between the four independent variables significantly affects the sensory quality of yeast-fermented tsampa, which is consistent with the results of variance analysis.

[0196] (3) Determine the optimal formula and conduct verification tests

[0197] After comprehensive analysis by Design-Expert8.0 software, the optimal process formula for yeast fermentation of highland barley tsampa is 41.27% butter tea, 7.71% butter, 19.85% white sugar, and 20.02% qula, that is, the addition amount of tsampa powder is fixed to 100g (100% by mass), the addition amount of butter tea is 41.27mL, the addition amount of butter is 7.71g, the addition amount of white sugar is 19.85g, the addition amount of qula is 20.02g, and the theoretical sensory evaluation score is 40.64. Combined with the actual production situation, the parameters are adjusted: the addition amount of tsampa powder is fixed to 100g (100% by mass), the addition amount of butter tea is 41mL, the addition amount of butter is 7.7g, the addition amount of qula is 19.9g, and the addition amount of white sugar is 20.0g. Under this condition, the average value of the results of three verification tests was taken, and the sensory evaluation score of the finished product was 40.92, which did not show significant difference compared with the theoretical result, and was significantly higher than the single factor test result, indicating that the results of using response surface analysis to optimize the process formula of yeast fermentation tsampa are more reliable.

[0198] 3. Determination of polyphenols and antioxidant activity

[0199] (1) Moisture content test results

[0200] Table 4 Moisture determination results

[0201]

[0202] According to the test results, fermentation increased the moisture content in tsampa.

[0203] (2) Results of polyphenol content determination test

[0204] Depend on Fig.10 It can be concluded that the polyphenol content (mmol CE / g DW) in highland barley tsampa fermented by yeast is: after fermentation > before fermentation.

[0205] Studies have shown that the total phenol content of tsampa made from fermented barley flour increases after adding yeast. This is because the bound phenols are released from the insoluble matrix during the yeast fermentation process through the action of proteases, cellulases and pectinases in grains and microorganisms.

[0206] (3) Flavonoid content determination test results

[0207] Depend on Fig.11It can be concluded that the flavonoid content (mmol CE / g DW) in highland barley tsampa fermented by yeast is: after fermentation> before fermentation. The average flavonoid content in the fermented tsampa can reach 0.59mmol CE / g DW, which is twice the flavonoid content in the tsampa that has not been fermented by yeast.

[0208] (4) Results of in vitro antioxidant activity test

[0209] ① Total antioxidant test results

[0210] Depend on Fig.12 It can be concluded that the ABTS·+ scavenging capacity (mmol TE / gDW) of highland barley tsampa fermented by yeast is: after fermentation> before fermentation. The ABTS·+ scavenging capacity of highland barley tsampa fermented by yeast is significantly higher than that of highland barley tsampa not fermented by yeast.

[0211] ②DPPH free radical scavenging rate test results

[0212] Depend on Fig.13 It can be concluded that the DPPH scavenging capacity (mmol FAE / g DW) in yeast fermented tsampa is: after fermentation> before fermentation. The DPPH free radical scavenging capacity of highland barley tsampa after yeast fermentation reaches 10.09mmol FAE / g DW, which is significantly greater than that of unfermented highland barley tsampa.

[0213] ③ Test results of iron ion reducing ability

[0214] Depend on Fig.14 It can be concluded that the iron ion reducing capacity (mmol FE / g DW) in yeast fermented tsampa is: after fermentation> before fermentation. That is, the iron ion reducing capacity of highland barley tsampa after yeast fermentation is much greater than that of unfermented highland barley tsampa.

[0215] ④ Hydroxyl free radical scavenging ability test results

[0216] Depend on Fig.15 It can be concluded that the hydroxyl radical scavenging capacity (mmol TE / gDW) of highland barley tsampa fermented by yeast is: after fermentation> before fermentation. The hydroxyl radical scavenging capacity of fermented highland barley tsampa reaches 242.28mmol TE / g DW, which is much higher than that of unfermented tsampa.

[0217] The invention optimizes the process by single factor test and response surface Box-Behnken test design, and determines that the optimal process formula of fermenting tsampa with native yeast is as follows: the addition amount of tsampa powder is fixed to 100g (the mass is 100%), the addition amount of butter tea is 41.27mL, the addition amount of butter is 7.71g, the addition amount of white sugar is 19.85g, the addition amount of qula is 20.02g, the sensory score of tsampa is the highest, the taste is good, the sweetness is moderate, the color is uniform, it is yellow-brown, the texture is good, and the whole presents the unique aroma of highland barley; the content of polyphenols and flavonoids in tsampa is high, and it has good in vitro antioxidant activity. The invention can provide a scientific basis for determining the process standard of fermenting tsampa with native yeast, and provide a new idea for the deep processing and development and utilization of highland barley.

[0218] The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast provided in an embodiment of the present invention first ferments tsampa powder by using native yeast separated and screened from highland barley (step S101). The yeast screened in the laboratory has been preliminarily identified to be able to effectively promote the fermentation reaction in tsampa powder and increase the functional components produced during the fermentation process, especially polyphenols. The use of native yeast helps to retain and activate the natural antioxidant components with biological activity in tsampa to the maximum extent, ensuring the accuracy of the experimental results.

[0219] In step S102, with sensory evaluation as the key indicator, the experiment uses a single factor test to study the effects of different additives on the quality of fermented tsampa. The experiment selects four traditional Tibetan foods, namely butter, butter tea, sugar, and qula (milk residue), as variables, and evaluates the color, smell, taste, etc. of fermented tsampa through a sensory scoring system. The purpose of this step is to screen out the most suitable formula additives for fermentation with native yeast to enhance the overall sensory experience of the fermented product.

[0220] Next, in step S103, a Box-Behnken experimental design response surface model is used to further optimize the process formula of yeast fermented tsampa. Response surface design is a multi-factor experimental optimization method that can comprehensively consider the interaction between multiple factors and determine the optimal combination of conditions in the fermentation process, including the amount of yeast added, fermentation time, fermentation temperature and the optimal ratio of additives. Through mathematical modeling and experimental verification, the optimal fermentation process formula is finally determined to maximize the polyphenol antioxidant activity of fermented tsampa.

[0221] Based on the optimized process, step S104 measures the polyphenol antioxidants in the fermented tsampa. Polyphenols are the main antioxidant components in tsampa, and their content may be significantly increased through the optimized fermentation process. This step uses standard antioxidant activity determination methods, such as DPPH free radical scavenging ability, ABTS free radical scavenging ability, etc., and these test methods are used to quantitatively determine the polyphenol content and antioxidant activity level in the fermentation product.

[0222] During the fermentation process, yeast not only promotes the fermentation of tsampa powder, but also converts polyphenols in the raw materials into more active antioxidant components through metabolic activities. This conversion helps to improve the antioxidant activity of fermented tsampa, giving it better health benefits. The yeast fermentation process and the optimized combination of additives have also greatly improved the nutritional value and market competitiveness of fermented tsampa.

[0223] The core of the whole method is to use native yeast for fermentation. Through scientific experimental design and optimization process, it not only effectively increases the content of polyphenols in fermented tsampa, but also greatly enhances its antioxidant activity. This method provides a scientific basis for the development and application of subsequent fermentation products. At the same time, through experimental verification and measurement, it ensures the functionality and quality controllability of the product.

[0224] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A process formula for fermenting tsampa with native yeast, characterized in that: The process was optimized through single factor experiment and response surface Box-Behnken experimental design, and the optimal process formula of tsampa was determined as: 41 mL of butter tea, 19.9 g of qula, 20.0 g of white sugar, and 7.7 g of butter.

2. The process optimization method for the process formula of fermenting tsampa with native yeast as claimed in claim 1, characterized in that: The following steps are involved: S101, using native yeast isolated and screened from highland barley in the laboratory to ferment tsampa flour; S102, using sensory evaluation as an indicator, selecting the addition amount of butter, butter tea, sugar, and qula (milk residue) for single factor experiment; S103, using Box-Behnken experiment for response surface design to determine the optimal process formula for fermenting tsampa with native yeast; S104, determining the antioxidant activity of polyphenols in the optimized fermented tsampa.

3. The process optimization method for the process formula of fermenting tsampa with native yeast as claimed in claim 2, characterized in that: The preparation method of the tsampa comprises: (1) Raw material pretreatment: Select highland barley grains with full grains, no insect infestation, no impurities, no mildew or other deterioration phenomena; (2) Moistening the wheat: First, measure the moisture content of the raw barley. After cleaning and controlling the water, weigh it and then moisten the wheat. Use sealing film to seal the wheat so that the surface of the raw material is fully moistened. After moistening and drying, use a moisture meter to measure the moisture content of the moistened barley to be 15.0% to 17.0% before grinding. Moistening the wheat is to ensure that the barley grains have a better burst rate when frying. (3) Stir-frying: stir-frying with an induction cooker until the highland barley has a crackling rate of more than 85% and a ripening rate of more than 95%, followed by cooling; (4) Grinding: Grinding the roasted highland barley into powder using a grinding machine, and then passing the ground powder through a sieve to obtain tsampa powder; (5) Fermentation: This study used native yeast isolated from highland barley in the laboratory to ferment tsampa. Weigh tsampa powder into a conical flask, add an appropriate amount of sterile distilled water, and mix thoroughly. After sterilization, place it on a sterile operating table to inoculate yeast mother liquid. The tsampa inoculated with yeast was cultured and fermented in a shaking incubator at 28°C for 4 days. (6) Preparation of tsampa: Taking 100 g (100% by mass) highland barley tsampa powder as a base, first add 41 mL of butter tea to melt 19.9 g of qula, 20.0 g of white sugar, and 7.7 g of butter, then add 100 g of highland barley tsampa powder, stir evenly, and knead the dough to prepare dough; (7) Pressing and molding: placing the prepared dough into a mold and pressing it to shape the tsampa fermented by native yeast; (8) Determination of biological activity, extraction and content determination of polyphenols and flavonoid content determination; (9) Determination of in vitro antioxidant activity, including total antioxidant capacity, DPPH free radical scavenging capacity, iron ion reducing capacity, and hydroxyl free radical scavenging capacity.

4. The process optimization method for the process formula of fermenting tsampa with native yeast as claimed in claim 3, characterized in that: In step 3, based on the single factor experiment, the sensory score is used as the response value, and the amount of butter, butter tea, sugar, and qula (milk residue) added is used as the variable factor, and the Box-Behnken test in Design Expert 8.0.6 is used to perform a four-factor three-level response surface design.

5. A method for determining the in vitro antioxidant activity of polyphenols in tsamba prepared by the process formula of fermenting tsamba with native yeast as claimed in claim 1, characterized in that: The following steps are involved: Step 1, extraction and content determination of polyphenols and flavonoids; Step 2, total antioxidant capacity determination; Step 3, DPPH free radical scavenging ability determination; Step 4, determination of iron ion reduction and antioxidant capacity; Step 5, ·OH scavenging capacity determination; The method for determining the antioxidant activity of tsampa polyphenols fermented by yeast as claimed in claim 5, characterized in that the extraction and content determination of polyphenols specifically include: The extractant is added to the weighed sample, and the sample is extracted by ultrasonication at low temperature and then centrifuged. The supernatant is extracted and the residue is extracted again. The two supernatants are combined and collected and a TCA solution of equal volume to the supernatant is added. The sample is centrifuged again overnight and the supernatant is collected. Finally, the volume is fixed with the extractant to obtain soluble polyphenols. The sample is stored at 0°C to 4°C away from light for future use. The Folin-Ciocalateau method was used for colorimetric determination. The polyphenol extract was placed in a centrifuge tube, and Folin phenol solution was added. After shaking, saturated sodium carbonate was added, and then an appropriate amount of distilled water was added. After dark reaction, the supernatant was placed in a colorimetric dish and colorimetric determination was performed at 725 nm. The results were expressed as ferulic acid equivalents (FAE) per g dry weight (DW). According to the standard curve: y = 1.289x-0.003 (R 2 =0.9988) to calculate the polyphenol content (μmol FAE / g DW).

6. The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast as claimed in claim 5, characterized in that: The flavonoid content determination specifically includes: The AlCl3 method was used for determination. An appropriate amount of polyphenol extract was added to a centrifuge tube, followed by NaNO2 solution and distilled water. After reaction in the dark, AlCl3 solution was added and mixed thoroughly. Then, NaOH solution was added to make up the volume. The reaction was carried out at room temperature in a light-blocked state. Finally, after centrifugation, the supernatant was taken and the absorbance was determined at 510 nm. The results were expressed as catechin equivalents (CE) per g dry weight. According to the standard curve y = 2.5459x-0.01143 (R 2 =0.999) to calculate the flavonoid content (μmol CE / g DW).

7. The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast as claimed in claim 5, characterized in that: The total antioxidant capacity determination specifically includes: The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications. After adding an appropriate amount of polyphenol solution to a centrifuge tube, ABTS·+ working solution was added and the tube was allowed to react at room temperature. The absorbance was measured at 734 nm for the tsampa samples that had been fermented with yeast and those that had not been fermented with yeast. The standard curve was drawn using Trolox, and the standard equation was: y = 103.95x + 1.563 (R 2 =0.9984) Where: y is the ABTS·+ clearance rate, %; x is the Trolox concentration, mmol / L; polyphenol TEAC per g dry sample is expressed as Trolox equivalents (TE) (mmol TE / g DW); Antioxidant capacity (%) = [(c-cb)-(s-sb)] / (c-cb)*100 Where: c: absorbance of ABTS·+ working solution (measure A0); cb: absorbance of ethanol; sb: absorbance value after the sample reacts with ethanol; s: absorbance value after the sample reacts with ABTS·+ working solution (measure A1).

8. The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast as claimed in claim 5, characterized in that: The DPPH free radical scavenging ability determination specifically includes: The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. with slight modifications; an appropriate amount of polyphenol solution was added to the DPPH methanol solution, and the absorbance was measured at 517 nm after the reaction in the dark; and the DPPH radical scavenging activity was calculated based on the standard curve; ferulic acid was used to draw the standard curve, and the standard equation was: y=451.75x+32.31(R 2 =0.9987) Where: y——DPPH free radical scavenging rate, %; x——Ferulic acid concentration (μmol), polyphenol DRSA per g dry sample is expressed as ferulic acid equivalent (μmol FAE / g DW); DPPH free radical scavenging ability (%) = [(c-cb)-(s-sb)] / (c-cb)*100; Where: s: absorbance value after the sample reacts with DPPH methanol solution; sb: absorbance value after the sample reacts with methanol solution; c: absorbance of DPPH methanol solution; cb; absorbance of methanol solution.

9. The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast according to claim 5, characterized in that: The determination of iron ion reducing capacity specifically includes: Take an appropriate amount of polyphenol solution, add FRAP (Ferric reducing antioxidant power, FRAP) solution and mix thoroughly. After a certain period of reaction at room temperature (37°C), measure the absorbance at 593nm. The iron ion reducing antioxidant power obtained is expressed as Fe 2+ It is expressed as mmol FE / g sample equivalent; acetic acid is used as blank; the standard curve is drawn using FeSO4 standard stock solution, and the standard equation is: y=6.24x+0.0005(R 2 =0.9984) where: y is absorbance; x is ferrous sulfate concentration, mmol; polyphenol FRAP per g dry sample is expressed as Fe 2+ Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmol FE / g DW).

10. The method for determining the antioxidant activity of tsampa polyphenols fermented by native yeast according to claim 5, characterized in that: The hydroxyl radical scavenging ability test specifically includes: Take an appropriate amount of polyphenol solution in a centrifuge tube, add FeSO4 solution, H2O2 solution and salicylic acid in turn, incubate the mixture at 37°C for half an hour, cool it in an ice water bath, and plot a standard curve with vitamin C (VC) at 510 nm. The standard equation is: y = 47.562x + 2.9794 (R 2 =0.9959), the polyphenol HPSA in each gram of dry sample is expressed as VC equivalent (VE) (μmol VE / g DW); the OH scavenging rate is calculated as follows: Hydroxyl radical scavenging rate (%) = [1-(A x -A x0 ) / A0]*100; Where: A x : Absorbance value of sample group; A x0 : The absorbance value of the blank group was obtained by replacing the sample with distilled water; A0: The absorbance value of the control group was obtained by replacing hydrogen peroxide with distilled water.

Images