Method for optimizing formula process of protogenetic lactobacillus fermented tsamba and determining polyphenol oxidation resistance of protogenetic lactobacillus fermented tsamba

Abstract

The invention belongs to the technical field of fermentation, and discloses a method for optimizing a formula technology of protogenetic lactic acid bacteria fermented tsamba and determining polyphenol oxidation resistance of the protogenetic lactic acid bacteria fermented tsamba, the optimized fermented tsamba not only has relatively high nutritional value and relatively good taste, but also has relatively good antioxidant activity, and is beneficial to improving the functional activity of the tsamba. The optimal level of the tsamba fermented by lactic acid bacteria is 9.1 mL of buttered tea, 2.7 g of butter, 6.0 g of white granulated sugar and 6.0 g of Qula. The tsamba made according to the formula is neat in structure and shape, fine and uniform in tissue, moderate in hardness, good in taste and best in sensory evaluation. The polyphenol content of the zanba fermented by the lactic acid bacteria is 109.8500 + / -1.070 [mu] mol FAE / g DW, the flavone content of the zanba fermented by the lactic acid bacteria is 0.5568 + / -0.009 mmol CE / g DW, the polyphenol, flavone and antioxidant activity indexes of the zanba fermented by the lactic acid bacteria are obviously higher than those of unfermented zanba, and it is indicated that the polyphenol and flavone content and the polyphenol antioxidant activity of the zanba fermented by the lactic acid bacteria can be improved.

Description

Technical Field

[0001] The invention belongs to the technical field of fermentation, and in particular relates to a method for optimizing the formula process of tsampa fermented by native lactic acid bacteria and a method for determining the antioxidant effect of polyphenols. Background Art

[0002] Highland barley (Hordeum vulgare L.var.nudum Hook.f) is an annual herbaceous plant of the Poaceae family and the genus Hordeum. It is also called hullless barley or naked barley and mainly grows in high altitude areas of my country. Highland barley grows in extreme environments such as high cold, hypoxia and strong ultraviolet rays. It not only has the nutritional characteristics of "three highs and two lows" (high protein, high fiber, high vitamins, low fat and low sugar), but also accumulates a variety of active ingredients such as rich polyphenol compounds, dietary fiber, β-glucan, arabinoxylan and γ-aminobutyric acid. Studies have found that highland barley has antioxidant, antibacterial, anti-inflammatory, anti-fatigue and anti-aging effects, and can prevent chronic diseases caused by metabolic syndrome such as cancer, diabetes, obesity and cardiovascular disease. It has a good health-promoting effect on the human body, which may be mainly attributed to the phenolic compounds therein. However, since highland barley polyphenols can be covalently bonded with macromolecular substances such as dietary fiber to form insoluble bonded polyphenols, it is difficult to extract and release, resulting in low bioavailability of highland barley polyphenols. At the same time, the high content of dietary fiber in highland barley also makes the taste of highland barley rough. Therefore, highland barley is mainly used to produce feed, brew highland barley wine and process tsampa. The lack of deep processing has limited the further development and utilization of highland barley.

[0003] Tsampa is a special food of the Tibetan people. It is made from highland barley, which is processed through a series of processes such as selection, cleaning, water control, drying, frying, and grinding. Then, butter tea, qula, and white sugar are added to the tsampa powder and stirred and mixed evenly. As the staple food of the Tibetan people, tsampa has greatly retained the nutrients and active ingredients in highland barley. Traditionally, tsampa is mainly processed by families or small workshops. It is usually eaten immediately after it is made and cannot be stored for a long time. In addition, the processing technology lacks unified standards, which restricts its promotion and consumption.

[0004] Lactic acid bacteria (LAB) is a general term for bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. Lactic acid bacteria is a Gram-negative bacterium that has the functions of promoting the growth of intestinal probiotics, inhibiting the proliferation of intestinal spoilage bacteria, and maintaining the balance of intestinal flora. At present, many traditional fermented foods are produced by lactic acid fermentation. Solid-state fermentation refers to the process in which microorganisms grow on the surface of solid particles that lack free water, but the substrate contains enough water to allow the growth and metabolism of microorganisms. Solid-state fermentation has the advantages of low production cost, simple operation and environmental friendliness. During the fermentation process, many hydrolases can be produced, which can promote the release of insoluble bonded polyphenols, thereby increasing the content and bioavailability of polyphenols, and having a better promoting effect on human health. At the same time, the fermentation process can promote the decomposition of nutrients in tsampa into more easily absorbed small molecular nutrients, which is conducive to the absorption of nutrients, improves its nutritional value, and improves the rough taste.

[0005] In the prior art, for example, the patent with application number CN118383475A prepares a fermented highland barley biscuit with plant lactobacillus and saccharomyces cerevisiae composite fermented highland barley powder, solves the problem of highland barley rough taste, and increases the flavor, texture and health benefits of highland barley biscuits. The patent with application number CN109674051A discloses a method for enriching polyphenols of lactobacillus fermented wheat and its application in antioxidation, and finds that lactobacillus fermented wheat increases its phenolic content and antioxidant activity. However, the current tsampa processing technology lacks a unified standard, and the optimization of the original lactobacillus fermented tsampa formula process and the determination method of polyphenol antioxidation have not been reported. The invention provides a method for optimizing the formula process of native lactic acid bacteria fermented tsampa and determining the antioxidant activity of polyphenols thereof. Firstly, native lactic acid bacteria separated from highland barley are used to perform solid-state fermentation on tsampa powder. Secondly, the process of tsampa is optimized by using response surface Box-Behnken experimental design. Finally, DPPH, ABTS·+, FRAP and antioxidant activity determination of hydroxyl radical scavenging ability of polyphenols in tsampa are performed to evaluate the antioxidant performance of the fermentation product.

[0006] In summary, the problems existing in the prior art are: highland barley has a relatively rough taste and insufficient deep processing, which limits the development and utilization of highland barley; 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] The difficulty of solving the above technical problems: During the fermentation process, fermentation time, humidity, and strain inoculation amount have a great influence on the taste, color and nutritional content of tsampa; secondly, the polyphenols in tsampa are less stable and easily oxidized and decomposed, and the extraction and determination process requires strict low-temperature and light-proof operations.

[0008] The significance of solving the above technical problems: By using native lactic acid bacteria to carry out solid-state fermentation of tsampa and optimizing the process formula of native lactic acid bacteria fermentation of tsampa, a scientific basis can be provided for the formulation of lactic acid bacteria fermentation tsampa process standards; at the same time, this technology 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. On the other hand, it can promote the release of insoluble bonded polyphenols, improve the bioavailability of polyphenols, and have a better promoting effect on human health. It can provide new ideas for the deep processing and development and utilization 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 optimizing the process of native lactic acid bacteria fermented tsampa formula and a method for determining the antioxidant activity of polyphenols.

[0010] The invention is achieved in that a process formula of tsampa fermented by native lactic acid bacteria is used, and process optimization is performed through single factor experiment and response surface Box-Behnken experimental design, and the optimal process formula of tsampa fermented by native lactic acid bacteria is determined as follows: 30g tsampa powder is used as a benchmark, 9.1mL of butter tea, 2.7g of butter, 6.0g of white sugar and 6.0g of qula are added.

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

[0012] Step 1, using native lactic acid bacteria separated 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 lactic acid bacteria.

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

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

[0017] S1, raw material pretreatment: select highland barley with full grains, no impurities, mildew, insect pests, or deterioration, weigh an appropriate amount of highland barley and rinse it with water for 3 to 4 times until the water is relatively clear;

[0018] S2, moistening the barley: placing the water-controlled highland barley on a tray and adding an appropriate amount of water to moisten the barley, so that the surface of the raw material is fully moistened, placing the highland barley on a tray for drying after moistening, and measuring the moisture content of the highland barley after moistening with a moisture meter;

[0019] S3, frying barley: weigh the barley with a moisture content of 15.0% to 17.0% after drying, and fry the barley in a frying pan with controlled temperature until the barley burst rate (burst rate) reaches more than 85% and the ripening rate reaches more than 95%; spread the ripened barley for natural cooling;

[0020] S4, grinding: grinding the roasted highland barley grains into powder using a multifunctional pulverizer, and passing through a 60-mesh sieve to obtain highland barley tsampa powder;

[0021] S5, fermentation: using native lactic acid bacteria isolated from highland barley in the laboratory to ferment tsampa, using MRS medium for lactic acid bacteria culture, and fermenting under the optimal fermentation process conditions of lactic acid bacteria solid-state fermentation of tsampa optimized in the early laboratory, controlling temperature, time and other conditions during the fermentation process, and the fermentation time is 4 days;

[0022] S6, tsampa preparation: taking 30 g highland barley tsampa powder as a basis, first add 9.1 mL butter tea to melt 6 g qula and 6 g white sugar, then add 30 g highland barley tsampa powder, stir evenly and knead the dough to prepare dough;

[0023] S7, pressing and molding: putting the prepared dough into a mold and pressing it to shape the tsampa fermented by native lactic acid bacteria;

[0024] Furthermore, the tsampa is stored:

[0025] Refrigerated storage is an effective way to maintain its flavor and quality. The product should be stored in an ambient temperature of 0℃-15℃, relative humidity <50%, in ventilated and dry conditions, with no pollutants in the storage area. Tsampa that has been mixed with raw materials should be refrigerated.

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

[0027] Step 1, determination of polyphenol content

[0028] Step 2, flavonoid content determination

[0029] Step 3, DPPH free radical scavenging ability determination;

[0030] Step 4, determination of iron ion reduction and antioxidant capacity;

[0031] Step 5, ·OH scavenging capacity determination;

[0032] Step 6: Determination of total antioxidant capacity.

[0033] Further, the step 1 specifically includes:

[0034] The colorimetric determination was performed using the Folin phenol method. Folin phenol solution was added to the polyphenol extract, followed by saturated sodium carbonate and distilled water. Afterwards, dark reaction and centrifugation were performed. 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 =0.998). Calculate the polyphenol content (μmol FAE / g DW).

[0035] Further, the step 2 specifically includes

[0036] According to the AlCl3 method, an appropriate amount of polyphenol extract was placed in a 50 mL centrifuge tube, and after dark reaction, distilled water, NaNO2, and AlCl3 were added in sequence. After the reaction, the supernatant was extracted 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).

[0037] Further, the step 3 specifically includes

[0038] The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. and slightly modified. Experimental and control groups were set up, and appropriate amounts of polyphenol extracts were measured and placed in three test tubes. The corresponding experimental groups added appropriate amounts of polyphenol solution and DPPH methanol solution, and the control group added polyphenol extract and methanol solution. After preparation, they were placed in the dark for 10 minutes, and the absorbance was measured at 517nm. The DPPH radical scavenging rate was calculated using the standard curve, and three parallel experiments were performed. The DPPH radical scavenging rate was calculated according to the following formula:

[0039] DPPH free radical scavenging ability (%) = [(c-cb)-(s-sb)] / (c-cb)×100%

[0040] Where s: absorbance value after the sample reacts with DPPH methanol solution;

[0041] sb: absorbance value after the sample reacts with methanol solution;

[0042] c: absorbance of DPPH methanol solution;

[0043] cb: absorbance of methanol solution;

[0044] A standard curve was drawn using ferulic acid as the standard, clearance rate (%) as the ordinate, and ferulic acid concentration (mmol) as the abscissa. The obtained standard equation was y=237.11x+43.681(R 2 =0.9948). The polyphenol DRSA in each gram of dry sample is expressed as ferulic acid equivalent (mmol FAE / g DW).

[0045] Further, the step 4 specifically includes:

[0046] The test sample was added with an appropriate amount of FRAP (Ferric reducing antioxidant power, FRAP) solution and mixed thoroughly, then placed at 37°C for 4 minutes and the absorbance was measured at 593 nm. 2+ It is expressed as equivalent μmol FE / g sample. A standard curve is drawn using FeSO4 standard stock solution.

[0047] The standard equation is: y = 6.24x + 0.0005 (R 2 =0.9984), where y is the absorbance, x is the concentration of ferrous sulfate in μmol / L, and the FRAP value of polyphenols in each g of dry sample is expressed as Fe 2+ Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmolFE / g DW).

[0048] Further, the step 5 specifically includes:

[0049] This project intends to use the Fenton method to generate hydroxyl free radicals. Salicylic acid is added to the system to generate hydroxyl free radicals, which react with salicylic acid, and its absorption peak at 510nm is measured, and compared with a blank to verify its ability to eliminate hydroxyl free radicals. FeSO4, H2O2 and salicylic acid are added to the sample solution in sequence, stirred evenly at a certain temperature, and then cooled in an ice water bath, and the absorbance at 510nm is recorded. Vitamin C (VC) is used to draw a standard curve, and the standard equation is: y = 47.562x + 2.9794 (R 2 =0.9959), and the polyphenol HPSA in each gram of dry sample is expressed as VC equivalent (VE) (μmolVE / gDW). The OH scavenging rate is calculated as follows:

[0050] Hydroxyl radical scavenging rate % = [1-(A x -A x0) / A0]×100%

[0051] In the formula: Ax: absorbance value of the sample group; Ax0: absorbance value of the blank group using distilled water instead of the sample; A0: absorbance value of the control group using distilled water instead of hydrogen peroxide.

[0052] Further, the step 6 specifically includes:

[0053] The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications. An appropriate amount of polyphenol extract was mixed with ABTS·+ working solution and reacted in the dark. After completion, the absorbance was measured at 734 nm.

[0054] Antioxidant capacity (%) calculation formula = [(c-cb)-(s-sb)] / (c-cb)*100%

[0055] In the calculation formula:

[0056] c represents the absorbance value of ABTS·+ working solution;

[0057] cb is expressed as the absorbance value of ethanol solution;

[0058] s represents the absorbance of ABTS·+ working solution and sample;

[0059] sb is the absorbance of ethanol solution and sample.

[0060] The standard curve was drawn using Trolox, and the standard equation was y = 103.95x + 1.5663 (R 2 =0.9984), and the polyphenol TEAC in each gram of dry sample was expressed as Trolox equivalents (TE) (μmol TE / g DW).

[0061] 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:

[0062] First, the problems of the prior art are: the taste of highland barley is relatively rough, and the deep processing is insufficient, which limits the development and utilization of highland barley; the processing technology of tsampa 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. Based on the single factor experiment, the present invention obtains the optimal process formula of native lactic acid bacteria fermented tsampa after optimization analysis by response surface method: 9.1mL of butter tea addition, 2.7g of butter, 6.0g of white sugar, and 6.0g of qula. At this time, the sensory score of tsampa is the highest, with a strong fragrance and the best taste. The results of the experiment showed that the polyphenol content of tsampa fermented by lactic acid bacteria was 109.85±1.07μmol FAE / g DW, and the flavonoid content was 0.5568±0.01mmol CE / g DW. The results showed that the polyphenol and flavonoid content of tsampa fermented by native lactic acid bacteria increased significantly, indicating that native lactic acid bacteria can increase the content of polyphenols and flavonoids in tsampa and improve its antioxidant capacity. According to the analysis of the antioxidant activity of polyphenols in fermented tsampa and unfermented tsampa, all antioxidant activity indicators increased after fermentation, indicating that the fermentation of lactic acid bacteria promoted the release of insoluble bonded polyphenols. The present invention uses native lactic acid bacteria to perform solid-state fermentation on tsampa, optimizes the process formula of native lactic acid bacteria fermentation of tsampa, and can provide a scientific basis for formulating lactic acid bacteria fermentation tsampa process standards; at the same time, the technology uses native lactic acid bacteria to perform solid-state fermentation on tsampa, which can promote the decomposition of nutrients in tsampa into more easily absorbed small molecule nutrients, which is beneficial to the absorption of nutrients, improves its nutritional value, and improves the rough taste, and can promote the release of insoluble bonded polyphenols, improve the content of phenolic substances and the bioavailability of antioxidant polyphenols, and has a better promoting effect on human health. It can provide new ideas for the deep processing and development and utilization of highland barley.

[0063] Second, the expected benefits and commercial value of the technical solution of the present invention after transformation are as follows: Based on the preliminary laboratory research, the present invention uses native lactic acid bacteria to ferment tsampa powder to make tsampa, optimizes the process formula of fermented tsampa on the basis of single factor test, and performs in vitro antioxidant activity test on polyphenols therein. It can promote the decomposition of nutrients into more easily absorbed small molecule nutrients, and can promote the release of insoluble bonded polyphenols, can improve the nutritional value and functional activity of tsampa, and promote the deep processing and development and utilization of highland barley.

[0064] The technical solution of the present invention fills the technical gap in the industry at home and abroad: the optimization of the process of fermenting tsampa with native lactic acid bacteria and the determination method of its polyphenol antioxidant have not been reported. The present invention uses native lactic acid bacteria as the fermentation strain and uses an optimized process formula to prepare fermented tsampa, which can promote the decomposition of nutrients into more easily absorbed small molecule nutrients, and can promote the release of insoluble bonded polyphenols, improve the nutritional value and functional activity of tsampa, promote the deep processing and development and utilization of highland barley, and promote the development of the highland barley industry in Yunnan Province.

[0065] The technical solution of the present invention solves the technical problem that people have been eager to solve but have never succeeded in solving: through the technology of the present invention, solid-state fermentation of tsampa is carried out by native lactic acid bacteria, and the process formula of native lactic acid bacteria fermentation of tsampa is optimized, which can provide a scientific basis for formulating the process standard of lactic acid bacteria fermentation of tsampa; at the same time, the technology can promote the decomposition of nutrients in tsampa into more easily absorbed small molecule nutrients, which is conducive to the absorption of nutrients, improves its nutritional value, and improves the rough taste; on the other hand, it can promote the release of insoluble bonded polyphenols, increase the content of phenolic substances and the bioavailability of antioxidant polyphenols, and has a better promoting effect on human health. It can provide new ideas for the deep processing and development and utilization of highland barley.

[0066] Third, the present invention provides a process formula for fermenting tsampa based on native lactic acid bacteria, and optimizes the process through single factor experiment and response surface Box-Behnken test design to determine the best formula. The specific process is to add 9.1mL butter tea, 2.7g butter, 6.0g white sugar and 6.0g Qula to 30g highland barley tsampa powder. The process optimizes the flavor of tsampa by adding butter, butter tea, white sugar and Qula, ensures the optimal activity of lactic acid bacteria during the fermentation process, and makes the product unique in flavor and excellent in texture.

[0067] The process optimization method includes the steps of fermenting tsampa powder with native lactic acid bacteria isolated from highland barley in the laboratory, and conducting a single factor experiment with sensory evaluation as the main indicator. Then, the optimal fermentation conditions of tsampa were determined by Box-Behnken experimental design response surface. At the same time, the activity of polyphenol antioxidant substances in the fermented tsampa was determined to improve the nutritional value and health benefits of tsampa. The systematic and scientific nature of the process improves the quality stability and flavor consistency of the product.

[0068] In the preparation process of fermented tsampa, high-quality highland barley is selected as the raw material. Through pretreatment, tempering, frying, grinding and other steps, the cracking rate and maturity rate of highland barley are ensured, thereby improving the nutrition and flavor of tsampa. The temperature and time are strictly controlled during the fermentation process to ensure the activity of lactic acid bacteria. The fermentation time is 4 days. In the modulation step, the texture and taste of the tsampa dough are guaranteed by accurately controlling the proportion of ingredients and the stirring method.

[0069] In addition, the polyphenol in vitro antioxidant activity determination method provided by the present invention verifies the antioxidant properties of fermented tsampa by determining the polyphenol and flavonoid contents, DPPH free radical scavenging ability, iron ion reduction antioxidant ability, etc. This determination method further demonstrates the technological progress of the process formula, which not only improves the nutritional value of tsampa, but also has certain health functions, meeting the needs of modern consumers for healthy food. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 It is a flow chart of the process optimization of the original lactic acid bacteria fermented tsampa formula and the determination method of its polyphenol antioxidant provided by the embodiment of the present invention;

[0071] Figure 2 is a phylogenetic tree of some of the isolated and purified lactic acid bacteria provided in the embodiments of the present invention;

[0072] Figure 3 This is a growth curve of isolated and purified lactic acid bacteria provided by an embodiment of the present invention;

[0073] Figure 4 This is a screening diagram of lactic acid bacteria with the best fermentation performance provided by an embodiment of the present invention;

[0074] Figure 5 is a graph showing the effect of the butter tea powder ratio on the quality of fermented tsampa provided by an embodiment of the present invention;

[0075] Figure 6 Graph showing the effect of butter provided by an embodiment of the present invention on the quality of fermented tsampa;

[0076] Figure 7 is a graph showing the effect of white sugar on the quality of fermented tsampa provided by an embodiment of the present invention;

[0077] Figure 8 is a graph showing the effect of Qula on the quality of fermented tsampa provided by an embodiment of the present invention;

[0078] Fig. 9 : The response surface two-way factor interaction diagram provided by the embodiment of the present invention is (a) the interaction between butter tea and butter (b) the interaction between butter tea and white sugar (c) the interaction between butter tea and qula (d) the interaction between butter and white sugar (e) the interaction between butter and qula (f) the interaction diagram between white sugar and qula;

[0079] Fig.10 is a moisture content diagram provided by an embodiment of the present invention;

[0080] Fig.11 is a comparison chart of polyphenol content provided by an embodiment of the present invention;

[0081] Fig.12 is a comparison chart of flavonoid content provided by an embodiment of the present invention;

[0082] Fig.13 is a graph of DPPH free radical scavenging ability provided by an embodiment of the present invention;

[0083] Fig.14 is an ABTS·+ removal capability diagram provided by an embodiment of the present invention;

[0084] Fig.15 is a graph of the iron ion reducing capacity provided by an embodiment of the present invention;

[0085] Fig.16 is a graph of hydroxyl radical scavenging capability provided by an embodiment of the present invention;

[0086] Fig.17 This is a picture of the finished product of tsampa fermented by native lactic acid bacteria provided by an embodiment of the present invention. DETAILED DESCRIPTION

[0087] 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.

[0088] The process formula of tsampa fermented by native lactic acid bacteria provided in an embodiment of the present invention is optimized by single factor experiment and response surface Box-Behnken experimental design, and the optimal process formula of tsampa fermented by native lactic acid bacteria is determined as follows: 30g tsampa powder is used as a benchmark, 9.1mL of butter tea, 2.7g of butter, 6.0g of white sugar, and 6.0g of qula are added.

[0089] S101, fermenting tsampa using native lactic acid bacteria isolated from highland barley in the laboratory;

[0090] S102, determine the best process by studying single factor experiments and response surface formulation process optimization;

[0091] S103, determining the antioxidant activity of polyphenols in the optimized fermented tsampa.

[0092] like Figure 1 As shown, the process optimization method of the process formula of native lactic acid bacteria fermented tsampa provided in the embodiment of the present invention comprises the following steps:

[0093] Step 1: fermenting tsampa powder using native lactic acid bacteria isolated from highland barley in the laboratory;

[0094] 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.

[0095] Step three, the Box-Behnken experiment was used to conduct response surface design to determine the optimal process formula for fermenting tsampa with native lactic acid bacteria.

[0096] (1) Process flow

[0097] Raw material cleaning → tempering → maturation → grinding → lactic acid bacteria fermentation → preparation → molding → finished product

[0098] (2) Key points of experimental operation

[0099] ① Raw material pretreatment

[0100] Select highland barley with full grains and no impurities, mildew, insect pests, or deterioration. Weigh an appropriate amount of highland barley and rinse it with water for 3 to 4 times until the water is clear.

[0101] ② Moistening wheat

[0102] Put the highland barley after water control in a tray and add appropriate amount of water to moisten the barley, so that the surface of the raw material is fully moistened, put it in the tray to dry after moistening, and use a moisture meter to measure the moisture content of the highland barley after moistening;

[0103] ③Fried wheat

[0104] Weigh the highland barley with a moisture content of 15.0% to 17.0% after drying, and fry it in a frying pan at a controlled temperature until the highland barley burst rate (burst rate) reaches more than 85% and the ripening rate reaches more than 95%; spread the ripened highland barley for natural cooling;

[0105] ④ Grinding

[0106] The roasted highland barley grains are ground into powder using a multifunctional grinder and passed through a 60-mesh sieve to obtain highland barley tsampa powder;

[0107] ⑤ Fermentation

[0108] The native lactic acid bacteria isolated from highland barley in the laboratory were used to ferment tsampa. The lactic acid bacteria were cultured using MRS medium. The fermentation was carried out under the optimal fermentation process conditions for solid-state fermentation of tsampa by lactic acid bacteria optimized in the early laboratory. The temperature, time and other conditions were controlled during the fermentation process. The fermentation time was 4 days.

[0109] ⑥ Preparation of tsampa

[0110] Taking 30g of highland barley tsampa powder as a basis, 9.1mL of butter tea was first added to melt 6g of qula and 6g of white sugar, and then 30g of highland barley tsampa powder was added, stirred evenly, and then kneaded to prepare dough;

[0111] ⑦Compression molding

[0112] Put the prepared dough into a mold and press it to shape the tsampa fermented by native lactic acid bacteria;

[0113] ⑧ Storage of Tsampa:

[0114] Refrigerated storage is an effective way to maintain its flavor and quality. The product should be stored in an ambient temperature of 0℃-15℃, relative humidity <50%, in ventilated and dry conditions, with no pollutants in the storage area. Tsampa that has been mixed with raw materials should be refrigerated.

[0115] (3) Single factor test

[0116] ① Determination of the amount of butter tea added

[0117] The addition amount of tsampa powder was fixed at 30g (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 20%, 30%, 40%, 50%, and 60% in turn, and the sensory evaluation and polyphenol content were determined.

[0118] ②Determination of the amount of ghee added

[0119] The addition amount of tsampa powder was fixed at 30g (mass 100%), and when the addition amount of butter tea was determined, 20% white sugar and 20% qula were added to conduct a single factor experiment on the selection of butter addition amount. The sensory evaluation and polyphenol content were determined when the butter percentage was 6%, 8%, 10%, 12%, and 14%.

[0120] ③Determination of the amount of white sugar added

[0121] The addition amount of tsampa powder was fixed at 30g (mass 100%), and when the addition amount of butter tea and butter was determined, the addition amount of qula was set at 20%, and a single factor experiment was conducted to select the addition amount of white sugar. The sensory evaluation and polyphenol content were determined when the percentage of white sugar was 16%, 18%, 20%, 22%, and 24%.

[0122] ④ Determination of the amount of Qula added

[0123] The addition amount of tsampa powder was fixed at 30g (mass 100%), and the addition amount of white sugar, butter, and butter tea was determined. The sensory evaluation and polyphenol content were determined when the percentage of qula was 16%, 18%, 20%, 22%, and 24%.

[0124] (4) Response surface optimization experiment

[0125] According to the results of the single-factor experiment, butter tea (A), butter (B), sugar (C), and qula (milk residue) (D) were used as variable factors, and the Box-Behnken test in Design Expert 8.0.6 was used to perform a four-factor three-level response surface design. The response surface design factors and levels are shown in Table 1.

[0126] Table 1 Factor level table

[0127]

[0128] The sensory evaluation was conducted by a sensory evaluation team composed of 10 professionals. They evaluated five factors, including the color, feel, texture, taste, and mouthfeel of tsampa. They were required to rinse their mouths with water after evaluating each sample, and score the next sample every ten minutes, and finally take the average value. The sensory indicators and scoring standards of tsampa are shown in Table 2.

[0129] Table 2 Rating standards for sensory quality of tsampa

[0130]

[0131] (5) Determination of various indicators

[0132] ① Determination of sample moisture

[0133] Unfermented tsampa and lactic acid bacteria fermented tsampa were measured using a rapid moisture meter according to the instructions, and three parallel experiments were conducted.

[0134] ②Determination of polyphenol content in tsampa extract

[0135] The colorimetric determination was performed using the Folin phenol method. Folin phenol solution was added to the polyphenol extract, followed by saturated sodium carbonate and distilled water. Afterwards, dark reaction and centrifugation were performed. 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 =0.998). Calculate the polyphenol content (μmol FAE / g DW).

[0136] ③ Determination of flavonoids content in tsampa extract

[0137] According to the determination method using AlCl3, an appropriate amount of polyphenol extract was taken and placed in a 50mL centrifuge tube. After dark reaction, distilled water, NaNO2, and AlCl3 were added in sequence. After the reaction, centrifugation was performed, the supernatant was extracted, and the absorbance was measured at 510nm. The results were expressed as catechin equivalents (CE) per g dry weight, and the flavonoid content (μmol CE / g DW) was calculated based on the standard curve y=2.5459x-0.01143 (R2=0.999).

[0138] ④Determination of antioxidant active substances

[0139] The antioxidant activity of tsampa polyphenol extracts was determined by DPPH, ABTS·+, FRAP and hydroxyl radical scavenging methods.

[0140] DPPH free radical scavenging ability determination:

[0141] The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. and slightly modified. Experimental and control groups were set up, and appropriate amounts of polyphenol extracts were measured and placed in three test tubes. The corresponding experimental groups added appropriate amounts of polyphenol solution and DPPH methanol solution, and the control group added polyphenol extract and methanol solution. After preparation, they were placed in the dark for 10 minutes, and the absorbance was measured at 517nm. The DPPH radical scavenging rate was calculated using the standard curve, and three parallel experiments were performed. The DPPH radical scavenging rate was calculated according to the following formula:

[0142] DPPH free radical scavenging ability (%) = [(c-cb)-(s-sb)] / (c-cb)×100%

[0143] Where s: absorbance value after the sample reacts with DPPH methanol solution;

[0144] sb: absorbance value after the sample reacts with methanol solution;

[0145] c: absorbance of DPPH methanol solution;

[0146] cb: absorbance of methanol solution;

[0147] A standard curve was drawn using ferulic acid as the standard, clearance rate (%) as the ordinate, and ferulic acid concentration (mmol) as the abscissa. The obtained standard equation was y=237.11x+43.681(R 2 =0.9948). The polyphenol DRSA in each gram of dry sample is expressed as ferulic acid equivalent (mmol FAE / g DW).

[0148] Determination of iron ion reducing ability:

[0149] The test sample was added with an appropriate amount of FRAP (Ferric reducing antioxidant power, FRAP) solution and mixed thoroughly, then placed at 37°C for 4 minutes and the absorbance was measured at 593 nm. 2+ It is expressed as equivalent μmol FE / g sample. A standard curve is drawn using FeSO4 standard stock solution.

[0150] The standard equation is: y = 6.24x + 0.0005 (R 2 =0.9984), where y is the absorbance, x is the concentration of ferrous sulfate in μmol / L, and the FRAP value of polyphenols in each g of dry sample is expressed as Fe 2+ Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmolFE / g DW).

[0151] OH scavenging capacity determination:

[0152] This project intends to use the Fenton method to generate hydroxyl free radicals. Salicylic acid is added to the system to generate hydroxyl free radicals, which react with salicylic acid, and its absorption peak at 510nm is measured, and compared with a blank to verify its ability to eliminate hydroxyl free radicals. FeSO4, H2O2 and salicylic acid are added to the sample solution in sequence, stirred evenly at a certain temperature, and then cooled in an ice water bath, and the absorbance at 510nm is recorded. Vitamin C (VC) is used to draw a standard curve, and the standard equation is: y = 47.562x + 2.9794 (R 2 =0.9959), and the polyphenol HPSA in each gram of dry sample is expressed as VC equivalent (VE) (μmolVE / gDW). The OH scavenging rate is calculated as follows:

[0153] Hydroxyl radical scavenging rate % = [1-(A x -A x0) / A0]×100%

[0154] 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.

[0155] Total antioxidant capacity determination:

[0156] The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications. An appropriate amount of polyphenol extract was mixed with ABTS·+ working solution and reacted in the dark. After completion, the absorbance was measured at 734 nm.

[0157] Antioxidant capacity (%) calculation formula = [(c-cb)-(s-sb)] / (c-cb)*100%

[0158] In the calculation formula: c represents the absorbance value of ABTS·+ working solution;

[0159] cb is expressed as the absorbance value of ethanol solution;

[0160] s represents the absorbance of ABTS·+ working solution and sample;

[0161] sb is the absorbance of ethanol solution and sample.

[0162] The standard curve was drawn using Trolox, and the standard equation was y = 103.95x + 1.5663 (R 2 =0.9984), and the polyphenol TEAC in each gram of dry sample was expressed as Trolox equivalents (TE) (μmol TE / g DW).

[0163] (6) Data analysis

[0164] All experiments were repeated three times, and the results were expressed as mean ± standard deviation. Origin 2021, GrahPad Prism, SPSS20 and Excel 2019 software were used for data analysis and drawing.

[0165] The present invention provides a method for optimizing the formula process of native lactic acid bacteria fermented tsampa and determining the antioxidant activity of polyphenols, which can provide a scientific basis for formulating the process standard of lactic acid bacteria fermented tsampa; it can improve the nutritional value of fermented tsampa, improve the rough taste, and promote the release of insoluble bonded polyphenols therein, improve the bioavailability of polyphenols, and have a better promoting effect on human health. It 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.

[0166] Relevant evidence of the technical effects achieved by the embodiments of the present invention.

[0167] 1. Single factor test results

[0168] Through single factor experiments, the optimal addition amount of each factor was determined. The results showed that when the addition amount of tsampa powder was fixed at 30g, the addition amount of butter tea was 30%, the addition amount of butter was 10%, the addition amount of white sugar was 20%, and the addition amount of qula was 20%, the quality of fermented tsampa was best.

[0169] (1) Effect of butter tea powder ratio on the quality of tsampa fermented by native lactic acid bacteria

[0170] like Figure 5 As shown in the figure, when the butter tea addition amount is 30%, the filled area of ​​the radar chart reaches the maximum, and the filled areas of the radar charts of other proportions are all smaller than the area of ​​this proportion, and show a trend of first increasing and then decreasing. When the butter tea addition amount is 0.3%, its sensory score is the highest and the fermented tsampa quality is the best.

[0171] (2) Effect of ghee on the quality of tsampa fermented by native lactic acid bacteria

[0172] like Figure 6 As shown in the figure, when the amount of ghee added reaches 10%, the filling area of ​​the radar chart reaches the maximum, and the filling areas of the radar charts of other proportions are all smaller than this proportion, showing a trend of first increasing and then decreasing. This shows that when the ghee content is 10%, the sensory score of fermented tsampa is the highest and the quality of fermented tsampa is the best.

[0173] (3) Effect of white sugar on the quality of tsampa fermented by native lactic acid bacteria

[0174] like Figure 7 As shown in the figure, when the white sugar reaches 20%, the filled area of ​​the radar chart reaches the maximum, and the filled areas of the radar charts of other proportions are all smaller than this proportion, showing a trend of first increasing and then decreasing. This shows that when the white sugar is 20%, the sensory score of fermented tsampa is the highest and the quality of fermented tsampa is the best.

[0175] (4) Effect of Qula on the quality of tsampa fermented by native lactic acid bacteria

[0176] like Figure 8 As shown in the figure, when the qula reaches 20%, the filling area of ​​the radar chart reaches the maximum, and the filling areas of the radar charts of other proportions are all smaller than this proportion, showing a trend of first increasing and then decreasing. This indicates that when the qula is 20%, the sensory score of fermented tsampa is the highest and the quality of fermented tsampa is the best.

[0177] 2. Response surface results and analysis

[0178] (1) Response surface analysis results

[0179] By using the software Design-Expert 13 to perform variance analysis on the experimental results, we get the quadratic polynomial regression equation:

[0180] Y=41.2+0.58A-0.42B+0.33C+0.17D+0.5AB+0.75AC-0.5AD-0.5BC+0.75BD-0.75CD-4.85A 2 -4.60B 2 -4.22C 2 -4.47D 2

[0181] The results of variance analysis are shown in Table 3. From Table 3, it can be seen that the model P < 0.0001, indicating that the model is extremely significant, and the lack of fit term is not significant (0.9815 > 0.05), indicating that the equation has a good fit, and the determination coefficient R 2 =0.9884, indicating that the actual value and the predicted value have a high correlation. Therefore, the model can better reflect the optimal process conditions for fermenting tsampa with lactic acid bacteria. From the F value, it can be seen that the order of influence of the above four factors on the sensory score of lactic acid bacteria fermented tsampa is A>B>C>D, that is, butter tea>butter>white sugar>qula.

[0182] Table 3 Analysis of variance table

[0183]

[0184] Note: *: P<0.05 significant; **: P<0.01 extremely significant

[0185] (2) Optimal formula and verification experiment

[0186] After comprehensive analysis by Design-Expert 13 software, the optimal process formula for fermenting highland barley tsampa with native lactic acid bacteria is 30.61% butter tea, 9.91% butter, 20.09% white sugar, and 20.02% qula, that is, the addition amount of tsampa powder is fixed at 30g (mass is 100%), the addition amount of butter tea is 9.09mL, the addition amount of butter is 2.73g, the addition amount of white sugar is 6.03g, and the addition amount of qula is 6.01g, and the theoretical sensory evaluation score is 41.24. Considering the operability in actual production, the optimal formula is adjusted to 9.1mL butter tea, 2.7g butter, 6.0g white sugar, and 6.0g qula. Under this condition, the verification experiment is carried out, and the sensory score of lactic acid bacteria fermented tsampa is 40, which is not much different from the theoretical calculation result, indicating that the model is reliable. Fig. 9 It is the response surface two-way factor interaction diagram.

[0187] 3. Determination results of tsampa extract content

[0188] (1) Analysis of moisture measurement results

[0189] Depend on Fig.10It can be seen that the moisture content of tsampa fermented by native lactic acid bacteria is 0.6343±0.0019g, and the moisture content of unfermented tsampa is 0.4219±0.0004g. From the numerical value, the moisture content of tsampa fermented by lactic acid bacteria is greater than that of unfermented tsampa, indicating that the fermentation process increases the moisture content of tsampa, that is, lactic acid bacteria fermentation can increase the moisture content of tsampa. Figure 8 .

[0190] (2) Analysis of polyphenol content results

[0191] The polyphenol content of unfermented tsampa was 32.13±0.57μmol FAE / g DW, and the polyphenol content of tsampa fermented by native lactic acid bacteria was 109.85±1.07μmol FAE / g DW. Fig.11 As shown in the figure, the extraction amount of polyphenols from tsampa fermented by native lactic acid bacteria is significantly higher than that from unfermented tsampa, indicating that the antioxidant activity of tsampa fermented by native lactic acid bacteria is higher than that of unfermented tsampa.

[0192] According to the research of Wang Xin and others, the extraction rate of polyphenols in tsampa fermented by native lactic acid bacteria increased significantly. Therefore, this project proposes that native lactic acid bacteria may improve the antioxidant properties of highland barley by enhancing the release of polyphenols in highland barley. The difference in polyphenol content between fermented tsampa and unfermented tsampa further confirms the value of the fermentation process. During the fermentation process, it is conducive to the growth of microorganisms and can improve their nutritional value and efficacy. This natural fermentation method is extremely important for enriching the nutritional content of tsampa, enhancing the market competitiveness of products and consumer acceptance.

[0193] (3) Analysis of flavonoid content results

[0194] The flavonoid content of unfermented tsampa was 0.2325±0.020mmol CE / g DW, and the flavonoid content of tsampa fermented by native lactic acid bacteria was 0.5568±0.009mmol CE / g DW. Fig.12 It can be seen that the flavonoid content is similar to the polyphenol content. The flavonoid content of tsampa fermented by native lactic acid bacteria is higher than the extraction amount of unfermented tsampa. Studies have shown that the flavonoid content in food can be increased by native lactic acid bacteria fermentation. This is because the metabolic activity of microorganisms during the fermentation process may cause the combination of flavonoids with other compounds, or the solubility of flavonoids in the food matrix may change, thereby increasing the leaching rate of flavonoids. This phenomenon is Fig.10 It was clearly shown in the results that the flavonoid content of tsampa samples fermented by native lactic acid bacteria was significantly higher than that of unfermented samples.

[0195] The antioxidant effect of flavonoids is due to the phenolic hydroxyl groups in their structure, which can stabilize free radicals and terminate free radical chain reactions, thereby protecting cells from damage caused by oxidative stress. In addition, some theoretical studies have also found that flavonoids have the potential to interact with specific cancer cells and may control cancer occurrence by affecting the cell's signal transduction pathways.

[0196] (4) Evaluation of antioxidant activity

[0197] Depend on Figure 13-16 As shown in the data, the four antioxidant activities of tsampa extracts in this study were positively correlated, that is, the antioxidant activity of tsampa with high polyphenol flavonoid content was higher than that of unfermented tsampa, indicating that the content of polyphenol flavonoids determines the scavenging capacity of DPPH, ABTS·+, FRAP, and hydroxyl radicals to a certain extent. It further shows that tsampa fermented by native lactic acid bacteria has strong antioxidant capacity, and its antioxidant performance is better than that of unfermented tsampa.

[0198] ① Analysis of DPPH free radical scavenging ability results

[0199] DPPH free radical scavenging ability Fig.13 As shown in the figure, the DPPH radical scavenging ability value of tsampa fermented by native lactic acid bacteria is 8.3911±1.17mmol FAE / g DW, and the DPPH radical scavenging ability value of unfermented tsampa is 3.6783±0.08mmol FAE / g DW. Compared with the unfermented tsampa, its ability to scavenge DPPH radicals is significantly stronger than that of unfermented tsampa, indicating that the fermentation process increases the DPPH radical scavenging ability of tsampa, that is, the DPPH radical scavenging ability of tsampa can be improved after fermentation by native lactic acid bacteria.

[0200] ②Analysis of ABTS·+ removal ability results

[0201] ABTS·+ removal ability Fig.14 As shown in the data, the ABTS·+ scavenging ability value of tsampa fermented by native lactic acid bacteria was 77.1347±10.94μmol TE / g DW, and the ABTS·+ scavenging ability value of unfermented tsampa was 19.0757±1.43μmol TE / g DW. The study found that after adding native lactic acid bacteria to tsampa, its ABTS·+ scavenging ability was significantly stronger than that of unfermented tsampa, indicating that under fermentation conditions, the ABTS·+ scavenging ability in tsampa was significantly improved, and after fermentation by native lactic acid bacteria, its ABTS·+ scavenging ability was significantly stronger than that of unfermented tsampa, indicating that native lactic acid bacteria fermentation can enhance the ABTS·+ scavenging rate in tsampa.

[0202] ③Analysis of iron ion reducing ability results

[0203] The results of the iron ion reducing ability test are as follows Fig.15 As shown in the results, the iron ion reducing capacity of tsampa fermented by native lactic acid bacteria is 38.2500±0.443μmol FE / g DW, and the iron ion reducing capacity of unfermented tsampa is 12.4261±0.179μmol FE / g DW. The study found that the iron ion reducing capacity of tsampa fermented by native lactic acid bacteria is significantly stronger than that of unfermented tsampa, indicating that fermentation can improve the iron ion reducing capacity of tsampa, and tsampa fermented by native lactic acid bacteria has stronger iron ion reducing capacity. Therefore, native lactic acid bacteria fermentation has a promoting effect on the degradation of iron ions in tsampa.

[0204] ④Analysis of hydroxyl radical scavenging ability

[0205] The results of hydroxyl radical scavenging ability are as follows Fig.16 As shown in the data, the hydroxyl radical scavenging ability value of tsampa fermented by native lactic acid bacteria is 38.2500±0.443μmol TE / g DW, and the hydroxyl radical scavenging ability value of unfermented tsampa is 12.4261±0.179μmol TE / g DW. From the numerical value, tsampa fermented by native lactic acid bacteria has a stronger hydroxyl radical scavenging ability, indicating that fermentation can improve the hydroxyl radical scavenging ability in tsampa, and tsampa fermented by native lactic acid bacteria has a stronger hydroxyl radical scavenging ability, indicating that native lactic acid bacteria fermentation can enhance the hydroxyl radical scavenging ability in tsampa.

[0206] 4 Discussion

[0207] ① In the raw material pretreatment stage, in order to improve the processing quality of highland barley and ensure the production effect of tsampa, it is usually necessary to temper the barley. The main purpose is to make the moisture inside and on the surface of the highland barley grains uniform and achieve a better cracking rate. Pay attention to controlling the moisture and tempering time. Sufficient tempering is enough to make the moisture inside the wheat grains reach a balanced state, which can ensure that the highland barley can be evenly heated and fully cracked when it is quickly fried at high temperature to form an ideal tsampa raw material. At the same time, too long or too short tempering time may have an adverse effect on the texture and nutritional content of tsampa. This process has an important impact on the taste, color and subsequent storage of tsampa.

[0208] ② Ripening

[0209] The maturation process of highland barley is a crucial link in the production of highland barley tsampa. This process causes physical and chemical changes in the starch and other components of highland barley through high-temperature heat treatment. The maturation temperature is set between 220℃ and 240℃, in order to make the highland barley reach a high degree of maturation and to achieve a high cracking rate. The combination of the two determines the texture and taste of tsampa. The control of this link has a decisive influence on the quality of the final product.

[0210] ③ Reasons for the increase of antioxidant activity of fermented tsampa

[0211] Through DPPH free radical scavenging test and ABTS·+ free radical scavenging test, it was found that fermented tsampa has strong antioxidant activity, and this enhancement is related to the content of its polyphenols and flavonoids.

[0212] Research on the content of polyphenols and flavonoids and antioxidant capacity of tsampa fermented by native lactic acid bacteria shows that this process can not only improve the nutritional value of tsampa, but also effectively improve the antioxidant properties of tsampa. By fermenting tsampa with lactic acid bacteria, the content of polyphenols and flavonoids in tsampa can be increased, and the antioxidant activity can be enhanced, thereby improving the total antioxidant capacity of tsampa.

[0213] ④The significance of fermenting tsampa with native lactic acid bacteria

[0214] Highland barley is the main ingredient of tsampa, a traditional Tibetan food. It contains physiologically active ingredients such as polyphenols and has natural antioxidant effects. Applying lactic acid bacteria fermentation technology to tsampa can not only give tsampa a new taste, but also increase its health value. It is a functional food that integrates traditional materials with modern health concepts. When native lactic acid bacteria ferment tsampa, it can not only improve the nutritional value of tsampa itself, but also generate new antioxidant active substances. In addition, the fermentation process may also promote the release of original antioxidants or improve their stability and bioavailability, thereby further enhancing the overall antioxidant capacity. Based on previous research, this experiment intends to further study the antioxidant mechanism under different fermentation conditions and reveal its mechanism of action.

[0215] Polyphenols and flavonoids are important natural antioxidants in plants. They have many benefits for human health, including reducing the risk of chronic diseases and anti-inflammatory effects. Flavonoids are a class of compounds containing phenolic hydroxyl groups (A, B) composed of two benzene rings (A, B). They contain three carbon atoms in their structure and have good antioxidant and anti-tumor effects. Lactic acid bacteria can regulate the biochemical substances in tsampa through their own metabolic activity, thereby promoting the release and synthesis of phenolic substances in tsampa. This process not only depends on the enzymes produced by lactic acid bacteria, but also is affected by fermentation conditions such as temperature, pH value and fermentation time. On this basis, a new lactic acid bacteria fermentation method was proposed and preliminarily studied. The phenomenon that native lactic acid bacteria fermentation significantly increases the flavonoid content in tsampa not only provides a new technical path for food industry production, but also brings new opportunities for the development of functional foods. In modern society where health awareness is constantly improving, lactic acid bacteria fermentation can not only enhance the health benefits of food and enhance the market competitiveness of products, but also meet people's demand for food with high nutritional and health value. Tsampa fermented by native lactic acid bacteria is not only more advantageous in nutrition, but also reflects the deepening of traditional food innovation and health attributes in food technology. This method of combining traditional food with modern technology can activate the potential functional factors in the original ingredients and bring more value-added possibilities to traditional grains such as highland barley. Therefore, the research and application of this project will greatly promote the rapid development of my country's cereal food industry.

[0216] 5 Conclusion

[0217] Based on the single factor experiment, this experiment obtained the optimal process formula of native lactic acid bacteria fermented highland barley tsampa through response surface method optimization analysis, which was 9.1mL butter tea addition, 2.7g butter, 6.0g white sugar, and 6.0g qula. The experiment showed that the polyphenol content of native lactic acid bacteria fermented tsampa was 109.85±1.07μmol FAE / g DW, and the flavonoid content was 0.5568±0.01mmol CE / g DW. From the comparison of the results, it can be seen that the polyphenol and flavonoid content in tsampa after fermentation by native lactic acid bacteria is significantly increased, indicating that native lactic acid bacteria can increase the content of polyphenols and flavonoids in tsampa. According to the analysis of the antioxidant activity comparison between fermented tsampa and unfermented tsampa, all antioxidant activity indicators increased in value after fermentation, indicating that the fermentation of lactic acid bacteria promoted the generation and accumulation of these antioxidant substances.

[0218] 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 lactic acid bacteria, 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 follows: 9.1 mL of butter tea, 2.7 g of butter, 6.0 g of white sugar, and 6.0 g of qula.

2. The process optimization method for the process formula of native lactic acid bacteria fermented tsampa as claimed in claim 1, characterized in that: The following steps are involved: Step 1: fermenting tsampa powder using native lactic acid bacteria isolated from highland barley in the laboratory; Step 2, using sensory evaluation as an indicator, selecting the addition amount of butter, butter tea, sugar, and qula to conduct a single factor experiment; Step 3, using Box-Behnken experiment to conduct response surface design to determine the optimal process formula of tsampa fermented by native lactic acid bacteria; Step 4: Determine the activity of polyphenol antioxidant substances in the optimized fermented tsampa.

3. The process optimization method for the process formula of native lactic acid bacteria fermented tsampa as claimed in claim 2, characterized in that: The preparation method of the tsampa comprises: S1, raw material pretreatment: select highland barley with full grains, no impurities, mildew, insect pests, or deterioration, weigh an appropriate amount of highland barley and rinse it with water for 3 to 4 times until the water is relatively clear; S2, moistening the barley: placing the water-controlled highland barley on a tray and adding an appropriate amount of water to moisten the barley, so that the surface of the raw material is fully moistened, placing the highland barley on a tray for drying after moistening, and measuring the moisture content of the highland barley after moistening with a moisture meter; S3, frying barley: weigh the barley with a moisture content of 15.0% to 17.0% after drying, and fry the barley in a frying pan with controlled temperature until the barley burst rate (burst rate) reaches more than 85% and the ripening rate reaches more than 95%; spread the ripened barley for natural cooling; S4, grinding: grinding the roasted highland barley grains into powder using a multifunctional pulverizer, and passing through a 60-mesh sieve to obtain highland barley tsampa powder; S5, fermentation: using native lactic acid bacteria isolated from highland barley in the laboratory to ferment tsampa, using MRS medium for lactic acid bacteria culture, and fermenting under the optimal fermentation process conditions of lactic acid bacteria solid-state fermentation of tsampa optimized in the early laboratory, controlling temperature, time and other conditions during the fermentation process, and the fermentation time is 4 days; S6, tsampa preparation: taking 30 g highland barley tsampa powder as a basis, first add 9.1 mL butter tea to melt 6 g qula and 6 g white sugar, then add 30 g highland barley tsampa powder, stir evenly and knead the dough to prepare dough; S7, pressing and molding: putting the prepared dough into a mold and pressing it to shape the tsampa fermented by native lactic acid bacteria; Storage of the tsampa: Refrigerated storage is an effective way to maintain its flavor and quality. The product is stored at an ambient temperature of 0°C-15°C, relative humidity <50%, in a ventilated and dry environment, and the storage area is free of any pollutants. The tsampa prepared with the addition of raw materials should be placed in a refrigerator for refrigeration.

4. The process optimization method for the process formula of native lactic acid bacteria fermented tsampa 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 tsamba fermented by native lactic acid bacteria as claimed in claim 4, characterized in that: The following steps are involved: Step 1, determination of polyphenol content Step 2, flavonoid content 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; Step 6: Determination of total antioxidant capacity.

6. The method for determining the in vitro antioxidant activity of polyphenols according to claim 4, characterized in that: The step 1 specifically includes: The colorimetric determination was performed using the Folin phenol method. Folin phenol solution was added to the polyphenol extract, followed by saturated sodium carbonate and distilled water. Afterwards, dark reaction and centrifugation were performed. 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 =0.998). Calculate the polyphenol content (μmol FAE / g DW).

7. The method for determining the in vitro antioxidant activity of polyphenols according to claim 4, characterized in that: The step 2 specifically includes: According to the determination method using AlCl3, an appropriate amount of polyphenol extract was taken and placed in a 50mL centrifuge tube. After dark reaction, distilled water, NaNO2, and AlCl3 were added in sequence. After the reaction, centrifugation was performed, the supernatant was extracted, and the absorbance was measured at 510nm. The results were expressed as catechin equivalents (CE) per g dry weight, and the flavonoid content (μmol CE / g DW) was calculated based on the standard curve y=2.5459x-0.01143 (R2=0.999).

8. The method for determining the in vitro antioxidant activity of polyphenols according to claim 4, characterized in that: The step 3 specifically includes: The DPPH radical scavenging activity (DRSA) was determined by referring to the method of Zou Qingfei et al. and slightly modified. Experimental and control groups were set up, and appropriate amounts of polyphenol extracts were measured and placed in three test tubes. The corresponding experimental groups added appropriate amounts of polyphenol solution and DPPH methanol solution, and the control group added polyphenol extract and methanol solution. After preparation, they were placed in the dark for 10 minutes, and the absorbance was measured at 517nm. The DPPH radical scavenging rate was calculated using the standard curve, and three parallel experiments were performed. The DPPH radical scavenging rate was calculated according to the following formula: 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; A standard curve was drawn using ferulic acid as the standard, clearance rate (%) as the ordinate, and ferulic acid concentration (mmol) as the abscissa. The obtained standard equation was y=237.11x+43.681(R 2 =0.9948). The polyphenol DRSA in each gram of dry sample is expressed as ferulic acid equivalent (mmol FAE / g DW).

9. The method for determining the in vitro antioxidant activity of polyphenols according to claim 4, characterized in that: The step 4 specifically includes: The test sample was added with an appropriate amount of FRAP (Ferric reducing antioxidant power, FRAP) solution and mixed thoroughly, then placed at 37°C for 4 minutes and the absorbance was measured at 593 nm. 2+ It is expressed as equivalent μmol FE / g sample. A standard curve is drawn using FeSO4 standard stock solution. The standard equation is: y = 6.24x + 0.0005 (R 2 =0.9984), where y is the absorbance, x is the concentration of ferrous sulfate in μmol / L, and the FRAP value of polyphenols in each g of dry sample is expressed as Fe 2+ Equivalents (Ferrous sulfate equivalents, FE) are expressed as (μmol FE / gDW); The Fenton method was used to generate hydroxyl radicals. Salicylic acid was added to the system to generate hydroxyl radicals, which reacted with salicylic acid. The absorption peak at 510 nm was measured and compared with a blank to verify its ability to eliminate hydroxyl radicals. FeSO4, H2O2 and salicylic acid were added to the sample solution in sequence, stirred evenly at a certain temperature, and then cooled in an ice water bath. The absorbance at 510 nm was recorded. Vitamin C (VC) was used to draw a standard curve. The standard equation was: 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% In the formula: Ax: absorbance value of the sample group; Ax0: absorbance value of the blank group using distilled water instead of the sample; A0: absorbance value of the control group using distilled water instead of hydrogen peroxide.

10. The method for determining the in vitro antioxidant activity of polyphenols according to claim 4, characterized in that: The step 6 specifically includes: The total antioxidant capacity (TEAC) was determined by referring to the method of Zhu Yulin et al. with slight modifications; an appropriate amount of polyphenol extract was mixed with ABTS·+ working solution and reacted in the dark. After completion, the absorbance was measured at 734 nm. Antioxidant capacity (%) calculation formula = [(c-cb)-(s-sb)] / (c-cb)*100% In the calculation formula: c represents the absorbance value of ABTS·+ working solution; cb is expressed as the absorbance value of ethanol solution; s represents the absorbance of ABTS·+ working solution and sample; sb represents the absorbance of ethanol solution and sample; The standard curve was drawn using Trolox, and the standard equation was y = 103.95x + 1.5663 (R 2 =0.9984), and the polyphenol TEAC in each gram of dry sample was expressed as Trolox equivalents (TE) (μmol TE / g DW).

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