Application of saccharomyces cerevisiae BYBC2.21100 in preparation of hypoxia-resistant food products

Abstract

The invention provides an application of saccharomyces cerevisiae BYBC2.21100 in preparation of an anti-hypoxia food product, and the preservation number of the saccharomyces cerevisiae BYBC2.21100 is CGMCC (China General Microbiological Culture Collection Center) No.28727. The invention also provides an application of the saccharomyces cerevisiae According to the method, the leaching rate of active substances of the highland barley powder is increased, fermentation conditions are optimized, and the content of beta-glucan in fermentation liquor after fermentation reaches 2.39 mg / mL and is increased by 64.83% compared with that before fermentation. A rich enzyme system generated by fermentation of the wine yeast BYBC2.21100 can further release hypoxia-tolerant substances in the highland barley powder, so that the hypoxia tolerance is improved, the movement distance and movement speed of the hypoxia zebra fish are remarkably increased by the highland barley fermentation liquor obtained by the method, the LDH activity of the zebra fish is remarkably reduced, the SOD activity and the tissue total antioxidant capacity are remarkably improved, and the hypoxia tolerance of the zebra fish is remarkably improved. Movement behaviors of zebrafish in an anoxic environment are improved, and tissues and cells are protected from being damaged to a certain extent.

Description

Technical Field

[0001] The invention belongs to the field of food processing, and particularly relates to a method for preparing anoxic-resistant highland barley fermentation liquid and application thereof. Background Art

[0002] Hypoxia refers to a pathological process in which tissue metabolism, function and morphological structure change abnormally due to insufficient oxygen supply or impaired oxygen use in tissues. Hypoxia is a very common pathological process in various clinical diseases. Hypoxia of important organs such as the brain and heart is also an important cause of death. Since high altitude areas are in a low-pressure and low-oxygen environment, the oxygen content in the local air is about 40% lower than that in plain areas. Local residents are in a low-oxygen environment for a long time, which leads to increased red blood cell compensation and increased blood viscosity.

[0003] Tibetan traditional food highland barley is rich in β-glucan and phenolic substances such as flavonoids, tocols, anthocyanins, etc., which can be used as natural antioxidants. Eating highland barley products every day can relieve oxidative stress and inflammation caused by low pressure and low oxygen environment in the plateau, and at the same time has a certain ability to lower blood sugar and blood lipids. Although highland barley has long been used as a food crop and feed crop, the problem of single use and low added value is becoming increasingly prominent.

[0004] Chen Xiaonan gavaged mice with highland barley β-glucan and conducted a hypoxia test. He found that in the hypoxia experiment, the hypoxia survival time and wheezing time of mice were 21.97 minutes and 20.17 minutes, respectively, which were significantly different from the blank control group, proving that highland barley β-glucan has the function of improving the hypoxia tolerance of mice (Chen Xiaonan, Li Xiaoli, Zhang Heng, et al. Research on the hypoxia tolerance effect of highland barley β-glucan [J]. Grain and Oil, 2023, 36(08): 42-45.).

[0005] Chinese patent CN 116998652 A discloses a highland barley solid beverage that helps improve hypoxia tolerance (Bao Haining, Yan Wenjie, Duan Hao, et al. A highland barley solid beverage that helps improve hypoxia tolerance [P]. Qinghai Province: CN202310988561.6, 2023-11-07.), which includes 80-120 parts of highland barley, 20-30 parts of ginseng, and 10-20 parts of Cordyceps militaris to make a solid beverage. Through mouse experiments, it was found that the highland barley solid beverage made by this patented method can prolong the normal pressure hypoxia tolerance time of mice.

[0006] Chinese patent CN 118000373 A discloses a process for preparing a highland barley lactic acid fermentation oral liquid (Jin Weiyun, Xue Jie, Yu Jiajun, et al. A process for preparing a highland barley lactic acid fermentation oral liquid [P]. Beijing: CN202311817604.0, 2024-05-10.). The process uses highland barley as raw material, and obtains highland barley juice through crushing, gelatinization, saccharification, and filtration. The highland barley lactic acid fermentation liquid with high lactic acid content is prepared by lactic acid liquid fermentation using plant lactobacillus seed liquid. Through mouse experiments, it was found that the oral liquid has the effects of lowering blood sugar, protecting the liver, and improving intestinal flora.

[0007] Chinese patent CN 115191551 A discloses an oxygen-resistant fermentation liquid, a preparation method and its application (Gao Ying, Fan Caifeng, Huang Jiali, et al. An oxygen-resistant fermentation liquid, a preparation method and its application [P]. Sichuan Province: CN202210853014.2, 2022-10-18.). The highland barley slurry is fermented for the first time by yeast, the fermentation liquid is distilled after fermentation, the residue is mixed with honey and then fermented for the second time by lactic acid bacteria to obtain the fermentation liquid. It was verified by mouse experiments that the fermentation liquid prepared by this patented method can prolong the normal pressure oxygen tolerance time of mice.

[0008] At present, there are very few studies on fermentation based on highland barley. As for how to increase the extraction rate of hypoxia-resistant substances in highland barley, such as β-glucan, the existing technology generally rarely involves. Therefore, how to use brewer's yeast to ferment highland barley flour, give full play to its maximum value, and enable it to enter industrial production has become the top priority of current research.

[0009] The above patent discloses the application of several types of highland barley in oxygen-deficient foods, but no patent publication documents related to the patent application of the present invention have been found. Summary of the invention

[0010] The object of the present invention is to provide the use of Saccharomyces cerevisiae BYBC2.21100 in preparing hypoxia-tolerant food products.

[0011] The brewer's yeast Saccharomyces cerevisiae BYBC2.21100 is deposited in the China General Microbiological Culture Collection Center, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is October 24, 2023, and the deposit number is CGMCC No.28727.

[0012] The prepared hypoxia-resistant food product of the present invention comprises solid food and / or liquid food, and the hypoxia-resistant food product of the present invention also comprises hypoxia-resistant health care products.

[0013] A preferred embodiment of the present invention provides a highland barley fermented liquor beverage with hypoxia resistance function. The highland barley fermented liquor is obtained by fermenting highland barley powder with brewer's yeast BYBC2.21100 as the highland barley fermented puree beverage.

[0014] A preferred embodiment of the present invention provides a highland barley fermentation concentrate with hypoxia resistance, wherein the highland barley fermentation liquid is obtained by fermenting saccharomyces cerevisiae BYBC2.21100 with highland barley powder, and a portion of water in the highland barley fermentation liquid is evaporated to obtain the highland barley fermentation concentrate. The highland barley fermentation concentrate is concentrated by a conventional method in the prior art, including but not limited to low-temperature vacuum concentration, vacuum concentration, freeze concentration, and membrane concentration.

[0015] Preferably, vacuum concentration utilizes vacuum concentration equipment to boil the juice under reduced pressure and rapidly evaporate the water in the juice. The recovery of aromatic substances is an indispensable process in vacuum concentration production.

[0016] Preferably, the membrane concentration method mainly uses reverse osmosis and ultrafiltration concentration to concentrate the juice. Its advantages are that it does not require heating, can achieve concentration at room temperature, has little quality change, and has low energy consumption. It operates in a closed environment and is not affected by oxygen; the loss of volatile components is small; and the energy consumption is low. The disadvantage is that the juice cannot be concentrated to a high concentration state, so it is mainly used as a pre-concentration process for juice.

[0017] Preferably, freeze concentration utilizes the water in the juice to freeze first at low temperature, and the freezing point of the remaining solution gradually decreases due to the continuous increase in solute concentration, thereby obtaining concentrated juice.

[0018] In order to achieve the above object, the present invention provides a highland barley fermentation liquid prepared by using brewer's yeast BYBC2.21100 and a method for preparing the highland barley fermentation liquid. The highland barley fermentation liquid prepared by the method of the present invention has a good effect on hypoxia tolerance, and is widely applicable and developmental value when applied to hypoxia-tolerant foods and health products.

[0019] The preparation method comprises the following steps: Step 1: Activate and culture the strains: take the frozen Saccharomyces cerevisiae BYBC2.21100 strains, activate them, and inoculate them into the seed culture medium; Step 2: Preparation of highland barley fermentation medium: mixing highland barley powder with deionized water, adjusting the initial pH to 3.5-7.5, sterilizing, and preparing highland barley fermentation medium; Step 3: Inoculation and fermentation: The brewer's yeast BYBC2.21100 seed solution cultured in step 1 is inoculated into the highland barley fermentation medium mixed in step 2, and the highland barley fermentation liquid is obtained by constant temperature shaking fermentation at 26-34° C. and 160 rpm-200 rpm for 12-60 h; Preferably, the method further comprises step 4: obtaining anoxic-resistant fermentation broth: after terminating the fermentation, taking out the fermentation broth, sterilizing it, centrifuging it, and collecting the supernatant.

[0020] In a preferred embodiment of the present invention, a method for preparing anoxic-resistant highland barley fermentation broth is provided, comprising the following steps: Step 1: Activation and cultivation of bacteria Take out the glycerol tube stored in the -80 ℃ refrigerator and place it in a 30 ℃ constant temperature incubator to activate for 1~2 hours, then streak on the plate and culture until a single colony grows, pick a single colony and transfer it to the slant culture medium, and culture it at 30 ℃ for 24 hours. Refrigerate the slant in a 4 ℃ refrigerator. Take out the slant from the 4 ℃ refrigerator, activate it in a 30 ℃ constant temperature incubator for 1~2 hours, take a loop of bacteria from the slant and inoculate it into the seed culture medium, and culture it in a shaking incubator at 30 ℃ and 200 r / min for 18~24 hours.

[0021] Step 2: Preparation of highland barley fermentation medium The highland barley powder was mixed with deionized water, the initial pH was adjusted to 3.5-7.5, and the mixture was sterilized at 121°C for 20 min to prepare the highland barley fermentation medium.

[0022] Step 3: Inoculation and Fermentation The brewer's yeast seed liquid cultured in step 1 is inoculated into the highland barley fermentation medium mixed in step (2), and then placed in a constant temperature shaking incubator at 26-34° C., and fermented at a rotation speed of 160 rpm-200 rpm for 12-60 hours to obtain highland barley fermentation liquid.

[0023] Through single-factor experiments, the effects of brewer's yeast inoculation amount, fermentation time, fermentation temperature, and initial pH of fermentation on the β-glucan content and hypoxia tolerance of the fermentation liquid were investigated.

[0024] Any of the above is preferably that in step 2, highland barley flour is mixed with deionized water to prepare a highland barley aqueous solution, wherein the volume percentage of highland barley flour to deionized water is 1:15-1:35. More preferably, 1:15, 1:20, 1:25, 1:30, 1:35 and the range therebetween.

[0025] Any one of the above is preferably that in step 2, the method for preparing highland barley powder is to wash highland barley, drain the water, dry it, and grind it to obtain highland barley powder.

[0026] Preferably, in any of the above items, in step 2, the highland barley raw material is washed with clean water and the water is drained. Preferably, it is placed in a drying oven at 50° C. for drying. Preferably, after being crushed, it is sieved through a 60-mesh sieve to obtain highland barley powder.

[0027] Any of the above is preferably that in step 3, the seed solution of Saccharomyces cerevisiae BYBC2.21100 is inoculated into the highland barley fermentation medium at an inoculation rate of 1-9% by volume. A further preferred inoculation ratio is 1, 2, 3, 4, 5, 6, 7, 8, 9% and ranges therebetween.

[0028] Any of the above is preferably that the initial pH of the highland barley flour aqueous solution is adjusted to 3.5-7.5. The preferred pH is 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and the range therebetween.

[0029] Preferably, the culture is carried out at 26-34° C. at 200 r / min. The preferred temperature is 26, 30, 34° C. and the range therebetween.

[0030] Preferably, any of the above items is cultured in a shaking incubator at 200 r / min for 18-24 h.

[0031] Any one of the above is preferably that step 4: obtaining anoxic-tolerant fermentation broth.

[0032] After the fermentation was terminated, the fermentation broth was taken out, sterilized, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The β-glucan content in the fermentation broth was detected using a β-glucan content determination kit.

[0033] Any of the above is preferably that the preferred slant culture medium of the present invention is YEPD slant culture medium: 10 g yeast powder, 20 g peptone, 20 g glucose, 20 g agar, natural pH, made up to 1 L with deionized water, and sterilized at 121°C for 20 min.

[0034] Any of the above is preferably the preferred seed culture medium of the present invention: 10 g yeast powder, 20 g peptone, 20 g glucose, natural pH, made up to 1 L with deionized water, and sterilized at 121° C. for 20 min.

[0035] Any of the above is preferably that the preferred fermentation medium of the present invention is: highland barley flour and deionized water are mixed at a solid-liquid ratio of 1:15, pH 3.5, and sterilized at 121°C for 20 min.

[0036] The present invention also provides the use of highland barley fermentation liquid prepared by using brewer's yeast BYBC2.21100 in the preparation of hypoxia-resistant food products.

[0037] Any of the above items is preferably that the highland barley fermentation broth prepared by using brewer's yeast BYBC2.21100 includes: at least one of the highland barley fermentation broth containing solids obtained in step 3, the supernatant of the highland barley fermentation broth obtained in step 4, the concentrated solution of the highland barley fermentation broth obtained in step 3, and the concentrated solution of the supernatant of the highland barley fermentation broth obtained in step 4.

[0038] A preferred embodiment of the present invention provides a highland barley fermented liquor biscuit with a function of tolerance to hypoxia.

[0039] Preferably, in step 3, brewer's yeast BYBC2.21100 is fermented with highland barley flour to obtain highland barley fermentation liquid, and then the highland barley fermentation liquid is used to prepare highland barley fermentation liquid biscuits.

[0040] The advantages and positive effects of the present invention are: 1. The present invention aims at the problems of insufficient extraction of hypoxia-resistant components in highland barley extract, single utilization of highland barley and low added value. The highland barley powder raw material is processed by liquid fermentation method, and highland barley powder is utilized to a large extent to prepare hypoxia-resistant fermentation liquid, which is directly used as hypoxia-resistant food or provides raw materials for the subsequent development of hypoxia-resistant food.

[0041] 2. The present invention utilizes yeast to ferment highland barley flour, thereby increasing the leaching rate of active substances in highland barley flour, especially increasing the leaching rate of β-glucan. The fermentation conditions are optimized from the aspects of solid-liquid ratio, inoculation amount, fermentation time, fermentation temperature and initial pH of fermentation. In the present invention, the β-glucan content in the highland barley fermentation liquid reaches 2.39 mg / mL, which is 64.83% higher than that before fermentation.

[0042] 3. Based on LC-MS metabolomics analysis, it was found that the rich enzyme system produced by yeast fermentation can further release hypoxia-resistant substances in highland barley flour, such as pseudonepetalactone glycosides, neurotensin 11-13, enalaprilat, flavan-3-ol, N-[4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]-2-[methyl-[2-(methylamino)ethyl]amino]phenyl]prop-2-enamide, lotosterol, 10,11-dihydrocarbamazepine, fluazifop, phenylbutyrylglutamine, L-β-aspartate-L-leucine, thereby improving hypoxia resistance. The hypoxia-resistant fermentation liquid prepared after fermentation was verified by a zebrafish sodium sulfite poisoning survival experiment. It was found that compared with the blank group, the hypoxia-resistant fermentation liquid can significantly increase the movement distance and movement speed of hypoxic zebrafish, and the zebrafish LDH activity is significantly reduced, and the SOD activity and total antioxidant capacity of tissues are significantly increased. This shows that the fermentation liquid can improve the movement behavior of zebrafish in hypoxic environment and protect tissues and cells from damage to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 It is the β-glucan content in the preferred embodiment 7 of the present invention.

[0044] Figure 2 This is the survival status of each group of zebrafish in the survival rate statistics of zebrafish with hypoxia injury in the preferred embodiment 8 of the present invention.

[0045] Figure 3 This is the behavioral test result of zebrafish with hypoxia injury in the preferred embodiment 8 of the present invention.

[0046] Figure 4 This is the test result of the behavioral swimming time of zebrafish injured by hypoxia in the preferred embodiment 8 of the present invention.

[0047] Figure 5 This is the behavioral swimming speed test result of the hypoxia-injured zebrafish in the preferred embodiment 8 of the present invention.

[0048] Figure 6 This is the result of the zebrafish lactate dehydrogenase activity test in the behavior of zebrafish with hypoxia injury in the preferred embodiment 8 of the present invention.

[0049] Figure 7 This is the result of detecting SOD activity in zebrafish tissues in the behavior study of zebrafish damaged by hypoxia in the preferred embodiment 8 of the present invention. DETAILED DESCRIPTION

[0050] The present invention is further described in detail below through the accompanying drawings and specific embodiments.

[0051] Example 1 1. Preparation of highland barley flour and culture medium (1) Highland barley flour: Wash the highland barley raw material with clean water, drain the water, dry it in a drying oven at 50°C, grind it, and pass it through a 60-mesh sieve to obtain highland barley flour.

[0052] (2) YEPD slant medium: 10 g yeast powder, 20 g peptone, 20 g glucose, 20 g agar, natural pH, dilute to 1 L with deionized water, and sterilize at 121 °C for 20 min.

[0053] (3) Seed culture medium: 10 g yeast powder, 20 g peptone, 20 g glucose, natural pH, dilute to 1 L with deionized water, sterilize at 121 °C for 20 min.

[0054] (4) Fermentation medium: Highland barley flour and deionized water were mixed at a solid-liquid ratio of 1:15, pH 3.5, and sterilized at 121 °C for 20 min.

[0055] (II) The production process of preparing hypoxia-resistant highland barley fermentation liquid by liquid culture method may specifically include the following steps: (1) Preservation slope Take out the Saccharomyces cerevisiae BYBC2.21100 strain stored in the glycerol tube from the -80 ℃ refrigerator and place it in a 30 ℃ constant temperature incubator for activation for 1~2 hours, then streak it on the plate and culture it until a single colony grows, pick a single colony and transfer it to the slant culture medium, and culture it at 30 ℃ for 24 hours. Store the slant in a 4 ℃ refrigerator.

[0056] (2) Seed solution preparation Take out the slant from the 4℃ refrigerator and activate it in a 30℃ constant temperature incubator for 1~2 h. Take a loopful of bacteria from the slant and inoculate it into the seed culture medium. Incubate it in a shaking incubator at 30℃ and 200r / min for 18 h.

[0057] (3) 250 mL shake flask fermentation The cultured liquid seeds were inoculated into the fermentation medium at an inoculation rate of 1%, and cultured at 26°C and 160 rpm for 12 h.

[0058] (4) Obtaining anoxic fermentation broth After the fermentation was terminated, the fermentation broth was taken out, sterilized, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. The β-glucan content in the fermentation broth was detected using a β-glucan content determination kit.

[0059] Example 2

[0060] According to the preparation method of Example 1, the fermentation medium was adjusted to highland barley: deionized water = 1:20, the initial pH was adjusted to 4.5, sterilized at 121°C for 20 min, the cultured liquid seeds were inoculated into the fermentation medium at a 3% inoculation rate, and cultured at 28°C, 160rpm for 24h.

[0061] Example 3

[0062] According to the preparation method of Example 1, the fermentation medium was adjusted to highland barley: deionized water = 1:25, the initial pH was adjusted to 5.5, sterilized at 121°C for 20 min, the cultured liquid seeds were inoculated into the fermentation medium at a 5% inoculation rate, and cultured at 30°C, 160rpm for 36h.

[0063] Example 4

[0064] According to the preparation method of Example 1, the fermentation medium was adjusted to highland barley: deionized water = 1:30, the initial pH was adjusted to 6.5, sterilized at 121°C for 20 min, the cultured liquid seeds were inoculated into the fermentation medium at a 7% inoculum amount, and cultured at 32°C, 160rpm for 48h.

[0065] Example 5

[0066] According to the preparation method of Example 1, the fermentation medium was adjusted to highland barley: deionized water = 1:35, the initial pH was adjusted to 7.5, sterilized at 121°C for 20 min, the cultured liquid seeds were inoculated into the fermentation medium at a 9% inoculation rate, and cultured at 34°C, 160rpm for 60h.

[0067] Example 6

[0068] Highland barley and deionized water were mixed at a solid-liquid ratio of 1:20 (mass ratio), sterilized at 121 °C for 20 min, and prepared into the control group sample solution.

[0069] Example 7

[0070] Example 7 The β-glucan content of the highland barley fermentation liquid obtained from the highland barley fermentation medium at pH 3.5-7.5 was determined.

[0071] The β-glucan content in the fermentation broth was detected using a β-glucan content determination kit purchased from Suzhou Grace Biotechnology Co., Ltd. The β-glucan content in the fermentation broth after yeast fermentation was 2.39 mg / mL, which was 64.83% higher than that before fermentation. The specific effects are as follows: Figure 1 .

[0072] Example 8

[0073] Example 8 conducted a zebrafish experiment.

[0074] (1) Zebrafish breeding and reagent preparation: AB strain zebrafish purchased from the National Zebrafish Resource Center were raised in a zebrafish culture system at a water temperature of 28.5°C, pH 6.9-7.2, and a photoperiod of 14 h light: 10 h dark. They were fed with newly hatched brine shrimp once a day at 9 am and 3 pm. The juveniles used in the experiment were obtained by natural mating of healthy male and female adult zebrafish in a 1:1 ratio.

[0075] Related reagent configuration: 5 mg / mL sodium sulfite solution: Accurately weigh 0.5 g of sodium sulfite, dissolve it in 100 mL of deionized water, and adjust the pH to 7.

[0076] Zebrafish culture medium stock solution I: Accurately weigh 8g NaCl and 0.4g KCl, add deionized water to dissolve and make up to 100mL.

[0077] Zebrafish culture medium storage solution II: Accurately weigh 0.06g KH2PO4 and 0.358g Na2HPO4, add deionized water to dissolve and make up to 100mL.

[0078] Zebrafish culture medium storage solution III: Accurately weigh 0.72 g of CaCl2, add deionized water to dissolve and make up to 50 mL.

[0079] Zebrafish culture medium stock solution IV: Accurately weigh 1.23 g of MgSO4, add deionized water to dissolve and make up to 50 mL.

[0080] Conventional zebrafish culture medium: Take 1 mL each of zebrafish culture medium storage solutions I, II, III, and IV, mix well, and adjust the volume to 1 L.

[0081] (2) Fermentation broth for zebrafish protection 3dpf wild-type AB strain zebrafish were randomly selected and placed in a 12-well plate, with 10 zebrafish in each well (two replicate wells were set). The zebrafish were placed in the following experimental group culture solution and cultured at 27°C for 24 hours.

[0082] The pre-protection methods and culture solutions used in each experimental group were: Blank control group (CON): the zebrafish culture medium was the conventional zebrafish culture medium, that is, no drug was added to the conventional zebrafish culture medium, cultured at 27°C for 24 hours; Model blank group (MOD): the zebrafish culture medium was the conventional zebrafish culture medium, that is, no drug was added to the conventional zebrafish culture medium, cultured at 27°C for 24 hours; Rhodiola rosea positive control group (RL): conventional zebrafish culture fluid and Rhodiola rosea oral liquid were mixed in proportion to obtain 8-fold diluted Rhodiola rosea oral liquid, the 8-fold diluted Rhodiola rosea oral liquid was used as zebrafish culture fluid, and zebrafish were placed in the 8-fold diluted Rhodiola rosea oral liquid, cultured at 27°C for 24 hours; preferably, the Rhodiola rosea oral liquid was purchased from Tibet Tibetan Medicine Group Co., Ltd., with a national medicine standard of B20070002; Control sample group (GN): a conventional zebrafish culture solution and the control sample solution obtained in Example 6 were mixed in proportion to obtain an 8-fold diluted control sample solution, the 8-fold diluted control sample solution was used as the culture solution, and zebrafish were placed in the 8-fold diluted control sample solution and cultured at 27° C. for 24 hours; Fermentation sample group (FL): The conventional zebrafish culture liquid and the highland barley fermentation liquid of the present invention are mixed in proportion to obtain an 8-fold diluted highland barley fermentation liquid, the 8-fold diluted highland barley fermentation liquid is used as the culture liquid, the zebrafish is placed in the 8-fold diluted highland barley fermentation liquid, and cultured at 27° C. for 24 hours. Preferably, the highland barley fermentation liquid obtained in Example 1 is diluted 8 times as the zebrafish culture liquid (the same below).

[0083] (3) Establishment of hypoxia zebrafish model The method of hypoxia treatment in this embodiment is: the zebrafish after the above pre-protection is placed in a 5 mg / mL sodium sulfite solution. Sodium sulfite can reduce the dissolved oxygen content in water, thereby creating an hypoxic environment. Each experimental group is subjected to hypoxia treatment according to the above method, wherein: Blank control group (CON): after the pre-protection, no hypoxia treatment was performed; Model blank group (MOD): After the pre-protection, the above hypoxia treatment was performed; Rhodiola rosea positive control group (RL): after the pre-protection, the above hypoxia treatment was performed; Control sample group (GN): after the pre-protection, the above hypoxia treatment was performed; Fermentation sample group (FL): After the pre-protection, the above-mentioned anoxic treatment was performed.

[0084] (4) Statistics on survival rate of zebrafish subjected to hypoxia injury After the pre-protection, randomly select zebrafish from each group and place them in a 12-well plate, with 10 fish in each well (two duplicate wells are set). Perform hypoxia treatment according to method (3), and record the asphyxiation time when the experimental fish loses balance and flips over. Count the number of zebrafish deaths in each group every 5 minutes.

[0085] like Figure 2 The figure shows the survival status of zebrafish in each group. It can be seen from the figure that all the CON group survived during the experiment, all the MOD group died at 35 minutes, all the GN group died at 45 minutes, all the FL group died at 60 minutes, and all the RL group died at 70 minutes. The survival time of zebrafish pre-protected by fermentation liquid was longer than that of zebrafish not treated with medication, and the FL group samples could prolong the survival time of zebrafish under hypoxic conditions more than the GN group samples.

[0086] (5) Behavioral testing of zebrafish for hypoxia injury The zebrafish were treated with hypoxia according to the method (3). After 10 min of hypoxia, the zebrafish were removed and placed in a 12-well plate, with one fish in each well. Zebrafish culture water was given and a 20-30 min video of the zebrafish movement was recorded to ensure that there was only one fish in the shooting range and there were no blind spots.

[0087] The behavioral tests were evaluated using DeepLabCut. DeepLabCut was trained using a total of 100,000 iterations. After training, a training error of 3.78 pixels and a test error of 4.32 pixels were obtained with a cutoff value of 0.6. After the successful training of the network, it was applied to analyze all the videos in the dataset. Subsequently, the behavior of each animal was analyzed using a Python script based on the results of the DeepLabCut model.

[0088] Depend on Figure 3 , Figure 4 and Figure 5It can be seen that compared with the CON group, the zebrafish in the MOD group had decreased locomotion, significantly shortened swimming distance, and significantly slowed swimming speed. From the movement trajectory, it can be seen that the movement trajectory of the CON group was concentrated on the edge of the hole plate, while the zebrafish in the MOD group swam around irregularly, indicating that hypoxic conditions would significantly affect the movement behavior of zebrafish. Compared with the MOD group, the swimming distance and swimming speed of each drug-treated group were significantly improved, and the movement trajectory was more regular, closer to the blank group, indicating that the samples of each drug-treated group could significantly improve the movement behavior of hypoxic zebrafish. There was no significant difference in swimming distance and swimming speed between the GN group and the FL group, but from the movement trajectory, the movement of the FL group was more regular.

[0089] (6) Behavioral testing of zebrafish for hypoxia injury The experimental fish used for asphyxiation in the sodium sulfite poisoning experiment were collected in centrifuge tubes after the experiment and tested using a lactate dehydrogenase (LDH) kit and a total superoxide dismutase (T-SOD) assay kit purchased from Nanjing Jiancheng Bioengineering Research Institute.

[0090] Depend on Figure 6 and Figure 7 It can be seen that the LDH activity of zebrafish tissues increased significantly after hypoxia, indicating that hypoxia caused damage to zebrafish organs and caused a large amount of LDH to be released. The LDH activity of zebrafish tissues in each drug-treated group decreased significantly compared with the MOD group, indicating that the pre-protection of highland barley hypoxia-resistant fermentation broth can alleviate the damage to zebrafish organs, inhibit the release of LDH under cellular hypoxia, and maintain the body's metabolic balance. The SOD activity of zebrafish tissues decreased significantly after hypoxia, and the decrease in SOD activity can cause cell damage. The SOD activity of the tissues in the fermentation broth intervention group was significantly higher than that in the MOD group, indicating that the fermentation broth can slow down the oxidative stress response caused by the hypoxic environment and reduce cell oxidative damage. Compared with the GN group, the SOD activity of the tissues in the FL group was significantly improved, indicating that the FL group samples can better protect tissue cells and enhance the antioxidant capacity of cells.

[0091] Example 9

[0092] Example 9 was detected and analyzed based on LC-MS metabolomics, and it was detected that the rich enzyme system produced by Saccharomyces cerevisiae BYBC2.21100 after fermentation further released hypoxia-resistant substances in highland barley flour, including pseudonepetalactone glycosides, neurotensin 11-13, enalaprilat, flavan-3-ol, N-[4-methoxy-5-[[4-(1-methylindol-3-yl)pyrimidin-2-yl]amino]-2-[methyl-[2-(methylamino)ethyl]amino]phenyl]prop-2-enamide, lotosterol, 10,11-dihydrocarbamazepine, fluazifop, phenylbutyrylglutamine, and L-β-aspartic acid-L-leucine, thereby improving the ability to tolerate hypoxia.

[0093] Example 10 Preparation of fermented biscuits (1) The fermentation broth was sterilized at 121°C for 20 min. After sterilization, the supernatant of the highland barley fermentation broth was collected by centrifugation, and then the supernatant of the highland barley fermentation broth was concentrated to a solid content of 60%.

[0094] (2) Weigh 50-70 parts of low-gluten flour, 30-40 parts of milk, 5-10 parts of vegetable oil, and 1-3 parts of baking soda.

[0095] (3) Mix the milk and flour, add 10% of the flour mass of fermented highland barley puree and stir evenly.

[0096] (4) Knead the dough until it is smooth, wrap it with plastic wrap, and refrigerate for about 20 minutes.

[0097] (5) After the refrigeration treatment, the dough is divided into small pieces, squeezed into round shapes, and baked at 180°C until golden brown. The pieces are then taken out of the oven, cooled to room temperature, and packaged to obtain fermented highland barley biscuits. The above embodiments are merely examples for the purpose of clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. Application of Saccharomyces cerevisiae BYBC2.21100 in the preparation of hypoxia-tolerant food products, characterized in that: The deposit number of the Saccharomyces cerevisiae BYBC2.21100 is CGMCC No.28727.

2. The use according to claim 1, characterized in that The hypoxia-resistant food product is highland barley fermentation liquid prepared by using the brewer's yeast BYBC2.21100.

3. An oxygen-deficient highland barley fermentation liquid obtained by the application according to claim 1, characterized in that: The highland barley fermentation liquid is prepared by fermenting highland barley with brewer's yeast BYBC2.21100.

4. The method for preparing anoxic-resistant highland barley fermentation liquid according to claim 3, characterized in that: The following steps are involved: Step 1: Activate and culture the strains: take the frozen Saccharomyces cerevisiae BYBC2.21100 strains, activate them, and inoculate them into the seed culture medium; Step 2: Preparation of highland barley fermentation medium: mixing highland barley powder with deionized water, adjusting the initial pH to 3.5-7.5, sterilizing, and preparing highland barley fermentation medium; Step 3: Inoculation and fermentation: The brewer's yeast BYBC2.21100 seed solution cultured in step 1 is inoculated into the highland barley fermentation medium mixed in step 2, and the highland barley fermentation liquid is obtained by constant temperature shaking fermentation at 26-34° C. and 160 rpm-200 rpm for 12-60 h.

5. The preparation method according to claim 4, characterized in that: In step 2, highland barley flour and deionized water are mixed to prepare a highland barley aqueous solution, wherein the volume percentage of highland barley flour to deionized water is 1:15-1:

35.

6. The preparation method according to claim 4, characterized in that: In step 3, the seed solution of Saccharomyces cerevisiae BYBC2.21100 is inoculated into highland barley fermentation medium at an inoculation rate of 1-9% by volume.

7. The preparation method according to claim 4, characterized in that: In step 3, the fermentation culture conditions are 200 rpm, 26-34° C., and 18-24 h.

8. The preparation method according to claim 4, characterized in that: Also includes step 4: Obtaining anoxic-resistant fermentation broth: After terminating the fermentation, take out the fermentation broth, sterilize it, centrifuge it, and collect the supernatant.

9. The preparation method according to claim 8, characterized in that: In step 4, the centrifugation condition is 12000 rpm, 5 min.

10. Application of highland barley fermentation liquid prepared by using brewer's yeast BYBC2.21100 in the preparation of hypoxia-tolerant food products, characterized in that: The deposit number of the Saccharomyces cerevisiae BYBC2.21100 is CGMCC No.28727.

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