Preparation method of yeast extract with antioxidant activity

Abstract

The invention discloses a preparation method of a yeast extract with antioxidant activity, which comprises the following steps: by taking malt and papaya as main raw materials, pretreating the raw materials, saccharifying to prepare wort, adjusting the concentration of the wort for proliferation and fermentation of saccharomycetes, and preparing the yeast extract with the antioxidant activity from the harvested saccharomycetes cells by taking pure water as a solvent and saccharomycetes autolysis. And finally, harvesting the yeast extract with antioxidant activity. According to the invention, the selected raw materials malt and pawpaw are both medicinal and edible substances, can be eaten and have a health-care function, and contain substance molecules beneficial to skin; the prepared yeast extract has scavenging activities of 59.4%, 50.1% and 37.6% on three free radicals of DPPH, ABTS and. OH respectively, and has excellent oxidation resistance and actual development and application potential; the extraction solvent is pure water, and the obtained yeast extract can avoid the irritation of organic solvents to the skin, is more mild and skin-friendly, and is small in actual popularization and application resistance.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering, and particularly relates to a process for producing yeast extract with antioxidant activity. Background Art

[0002] The clarified wort obtained from malt through steps such as crushing, gelatinization, saccharification, filtration, boiling, and precipitation removal is rich in nutrients such as carbohydrates, proteins, small molecule peptides, amino acids, various vitamins, and minerals, which meet the requirements of microorganisms for carbon sources, nitrogen sources, growth factors, and trace elements, and is widely used for the proliferation and fermentation of yeast.

[0003] Yeast is rich in nucleotides, proteins, amino acids, polysaccharides, and various trace elements. It has been widely recognized that the extract of yeast has reliable antioxidant activity. The yeast extract with antioxidant activity is an important active ingredient used in skin care products, which can achieve beneficial biological effects on the skin, such as moisturizing the skin, promoting cell renewal, slowing down skin aging, and accelerating wound healing.

[0004] A large number of yeast resources have not been developed and utilized. Developing yeast with high antioxidant activity is of great significance and can become a new source of high-efficiency antioxidant active ingredients for skin care products. In summary, developing a method for proliferating and fermenting yeast using wort as a culture medium and extracting the antioxidant active ingredients of yeast can provide a new raw material source for the vast skin care product market. Summary of the Invention

[0005] In view of the above background advantages, the object of the present invention is to use natural medicated and edible homologous substances, malt and papaya, as the main raw materials, prepare wort for the nutritional environment of yeast proliferation and fermentation, produce yeast extract with antioxidant activity for skin care products, and provide a preparation method of yeast extract with antioxidant activity.

[0006] A preparation method of yeast extract with antioxidant activity includes the following steps:

[0007] (1) Wort preparation: Malt is crushed by a pair-roll crusher, and papaya is made into pulp by a juicer. Then, malt powder∶papaya pulp∶water are added and mixed in a mass ratio of 10∶5∶40 for temperature-controlled saccharification. The temperature control program is: maintain at 42°C for 15 min, 55°C for 30 min, 65°C for 40 min, and finally 75°C for 15 min. Then, it is filtered through 8 layers of gauze, the filtrate is collected, the filtrate is boiled for 15 min, and then centrifuged at 6000 rpm for 15 min to obtain clear and transparent 100% original wort.

[0008] In some implementation details of the present invention, in step (1), papaya with good maturity and no pests and diseases is selected, and after peeling and seeding, it is used to prepare papaya pulp.

[0009] (2) Yeast proliferation and fermentation: Dilute the wort prepared in step (1) with pure water to a concentration of 60%, dispense it into 250 mL Erlenmeyer flasks, with 50 mL of liquid in each flask, sterilize at 121 °C for 20 min, and after cooling, inoculate the activated yeast seed solution at an inoculation amount of 2%, and culture and ferment in a shaker at 28 °C and 150 rpm for 72 h, then centrifuge to harvest yeast cells.

[0010] In some implementation details of the present invention, in step (2), the wort is diluted to a concentration of 60%, and the reducing sugar content is 80 g / L.

[0011] In some implementation details of the present invention, the yeast (Rhodotorula mangiferae Y62) used in step (2) is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China (No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province).

[0012] (3) Extraction of antioxidant active ingredients: Wash the yeast cells harvested in step (2) 3 times with an equal amount of pure water and then resuspend them, perform yeast cell autolysis treatment, maintain at 50 °C for 8 h, then continue to maintain at 52 °C for 4 h, centrifuge at 8000 rpm for 5 min to remove the precipitate, and harvest the supernatant, which is rich in yeast extract with antioxidant activity.

[0013] In some implementation details of the present invention, the solvent used for extraction in step (3) is pure water, and the harvested yeast extract is filled into small brown glass bottles and stored away from light.

[0014] The preparation method of the yeast extract according to the implementation mode of the present invention has at least the following advantages and beneficial effects:

[0015] (1) The raw materials malt and papaya selected in the present invention are both substances homologous to medicine and food, which can be eaten and have health care functions at the same time, and they also contain molecular substances beneficial to the skin.

[0016] (2) The yeast extract prepared in the present invention has scavenging activities against DPPH, ABTS, and ·OH free radicals reaching 59.4%, 50.1%, and 37.6% respectively, and has excellent antioxidant ability and actual development and application potential.

[0017] (3) The solvent used in the present invention is pure water, and the obtained yeast extract can avoid the irritation of organic solvents to the skin, has better mild skin-friendly properties, and has less resistance to actual promotion and application. Description of the Drawings

[0018] Figure 1 It is the analysis chart of reducing sugar of worts with different concentrations in the embodiment of the present invention.

[0019] Figure 2 In the embodiment of the present invention, it is the proliferation and fermentation change curve of yeast Y62 in worts with different concentrations.

[0020] Figure 3 In the embodiment of the present invention, it is the antioxidant activity curve of yeast extract against different kinds of free radicals.

[0021] Figure 4 In the embodiment of the present invention, the yeast extract is stored in a small brown glass bottle. Detailed implementation manners

[0022] Example 1: Wort preparation and reducing sugar content analysis

[0023] 1.1 Wort preparation

[0024] The malt needs to be wetted with water to ensure that the malt husk remains relatively intact during the crushing process of the malt, which plays a role as a filter layer during filtration. Select papayas with good maturity and no pests and diseases, peel and seed them, and then make them into papaya pulp with a juicer. Malt powder∶papaya pulp∶water are added and mixed in a mass ratio of 10∶5∶40, and the temperature is controlled by a water bath. During this period, continuous stirring is carried out to make the heating uniform for saccharification treatment. The temperature control process is as follows: maintain at 42°C for 15 min, and the amylase in the malt dissolves into the liquid; maintain at 55°C for 30 min, and the protease in the malt itself and papain act to hydrolyze proteins to form polypeptides and amino acids; maintain at 65°C for 40 min, and β-amylase hydrolyzes starch to provide easily utilizable reducing sugar carbon sources such as maltose and glucose for fermentation; maintain at 75°C for 15 min, mainly α-amylase acts to further hydrolyze starch to form low-molecular-weight dextrins. Use iodine solution to detect whether there is unhydrolyzed starch in the wort. If the iodine solution turns blue, extend the saccharification time until there is no starch. Use 8 layers of gauze to filter the saccharified mash to remove the spent grains to obtain wort, and boil the wort for 15 min to precipitate heat coagulants such as unhydrolyzed proteins, and then centrifuge at 6000 rpm for 15 min to obtain clear and transparent 100% original wort, which is frozen in a -20°C refrigerator for standby.

[0025] 1.2 Reducing sugar content analysis

[0026] The DNS method was used to measure the reducing sugar content. The DNS method is also known as the 3,5-dinitrosalicylic acid colorimetric method. Under alkaline conditions, DNS and reducing sugar will undergo an oxidation-reduction reaction, and the product will produce a color reaction after boiling. The darker the color, the higher the reducing sugar content. Take 0, 0.2, 0.4, 0.6, 0.8, 1.0 mL of 1 g / L glucose standard solution and add them to centrifuge tubes respectively. For the glucose standard solution with less than 1 mL, add pure water to make up to 1 mL. Then add 2.0 mL of DNS reagent, mix well and place in a boiling water bath for 5 min. After cooling, add 4 mL of pure water to each tube and mix thoroughly. Use the control group without glucose to zero the instrument, measure the absorbance at 550 nm, and draw the standard curve for measuring reducing sugar by the DNS method. Using the glucose standard solution with gradient concentrations as the abscissa and the absorbance of the reaction system at 550 nm as the ordinate, the fitted regression equation is y = 0.4757x - 0.0032, R 2 = 0.9993. Take an appropriate amount of the previously prepared original wort, adjust the wort concentration to 100%, 80%, 60%, 40%, 20% by adding pure water, and then after appropriate dilution, use the DNS method to measure the reducing sugar content at each wort concentration.

[0027] Example 2: Yeast proliferation and fermentation

[0028] Adjust the concentration of the wort to 100%, 80%, 60%, 40%, 20% with pure water. After dispensing different concentrations of wort medium into Erlenmeyer flasks, sterilize at 121 °C for 20 min and set aside. Inoculate the activated Y62 seed liquid into the wort medium at an inoculation amount of 2%, and culture it in a shaker at 28 °C and 150 rpm. Take samples every 8 h and draw the growth and proliferation change curve with time to compare and select the optimal wort concentration. 60% wort can achieve the accumulation of cell biomass, and at the same time can also take into account reducing production time and making full use of the nutritional components of the wort.

[0029] Example 3: Extraction and evaluation of antioxidant active components

[0030] 3.1 Extraction of antioxidant active components

[0031] Inoculate the activated Y62 seed liquid into the wort medium with an initial pH value of 6.5 and a wort concentration of 60% that has been sterilized at an inoculation amount of 2%, and culture it in a shaker at 28 °C and 150 rpm. Take 1 mL of samples every 8 h and after 72 h of fermentation culture, centrifuge to remove the fermentation supernatant, wash the cells 3 times with an equal volume of pure water and then resuspend the cells; according to the autolysis procedure: maintain at 50 °C for 8 h, and then continue to maintain at 52 °C for 4 h; centrifuge at 8000 rpm for 5 min to remove the precipitate, harvest the yeast extract with antioxidant activity, and store it in a small brown glass bottle at room temperature.

[0032] 3.2 Evaluation of the Antioxidant Activity of Yeast Extract

[0033] (1) Evaluation method for DPPH radical scavenging activity: Prepare a 1 mmol / L DPPH solution by dissolving 0.01 g of DPPH in 25 ml of methanol. Add the reaction mixture to a light-proof brown tube according to the following reaction system, mix well, let stand at room temperature for 30 min, measure the absorbance at 517 nm, and zero with methanol.

[0034] System A0: 900 μL of methanol + 100 μL of 1 mmol / L DPPH;

[0035] System A1: 800 μL of methanol + 100 μL of 1 mmol / L DPPH + 100 μL of yeast extract;

[0036] System A2: 900 μL of methanol + 100 μL of yeast extract;

[0037] DPPH radical scavenging activity = (A0 - A1 + A2) / A0 × 100%.

[0038] (2) Evaluation method for ·OH radical scavenging activity

[0039] Prepare a reaction solution of 0.1% H2O2, 6 mmol / L FeSO4, and 6 mol / L salicylic acid - ethanol. Add the reaction mixture to a light-proof brown tube according to the following reaction system, mix well, react in a water bath at 37°C for 30 min, measure the absorbance at 510 nm, and zero with pure water.

[0040] System A0: 300 μL of FeSO4 + 300 μL of H2O2 + 300 μL of salicylic acid - ethanol + 100 μL of pure water;

[0041] System A1: 300 μL of FeSO4 + 300 μL of H2O2 + 300 μL of salicylic acid - ethanol + 100 μL of yeast extract;

[0042] System A2: 300 μL of FeSO4 + 300 μL of pure water + 300 μL of salicylic acid - ethanol + 100 μL of yeast extract;

[0043] ·OH radical scavenging activity = (A0 - A1 + A2) / A0 × 100%.

[0044] (3) Evaluation method for ABTS radical scavenging activity

[0045] Prepare 7 mmol / L ABTS solution and 2.45 mmol / L potassium persulfate solution. Mix the ABTS and potassium persulfate solutions in a volume ratio of 1:1 and react overnight in the dark at room temperature to obtain free radicals. Dilute with 0.1 mol / L PBS to prepare the ABTS working solution, with an OD 734 = 0.75. Add the mixed reaction solution to a light-proof brown tube according to the following reaction system, mix well, let stand at room temperature for 6 min, measure the absorbance at 734 nm, and zero with 0.1 mol / L PBS.

[0046] A0 system: 900 μL ABTS working solution + 100 μL pure water;

[0047] A1 system: 900 μL ABTS working solution + 100 μL yeast extract;

[0048] ABTS free radical scavenging activity = (A0 - A1) / A0 × 100%

[0049] The yeast extract obtained by treating yeast showed scavenging activities against DPPH, ABTS, and ·OH free radicals of 59.4%, 50.1%, and 37.6% respectively, with excellent antioxidant capacity and potential for practical development and application.

Claims

1. A method for preparing a yeast extract having antioxidant activity, characterized in that: The method comprises the following steps: (1) Preparation of wort: malt was crushed by a roller mill, papaya was pulped by a juicer, and then malt powder: papaya pulp: water were added and mixed in a mass ratio of 10:5:40, and temperature-controlled saccharification was performed; the temperature control procedure was: 42°C for 15 min, 55°C for 30 min, 65°C for 40 min, and finally 75°C for 15 min; then filtered through 8 layers of gauze, the filtrate was collected, the filtrate was boiled for 15 min, and then centrifuged at 6000 rpm for 15 min to obtain clear and transparent 100% original wort. (2) Yeast proliferation and fermentation: Pure water was added to the wort prepared in step (1) to dilute the wort to a concentration of 60%, and the wort was divided into 250 mL Erlenmeyer flasks, with each flask containing 50 mL of liquid. The wort was sterilized at 121° C. for 20 min. After cooling, the activated yeast seed liquid was inoculated at an inoculum rate of 2%, and the wort was cultured and fermented in a shaker at 28° C. and 150 rpm for 72 h. The yeast cells were harvested by centrifugation. (3) Extraction of antioxidant active ingredients: The yeast cells harvested in step (2) were washed three times with an equal amount of pure water and then resuspended for yeast cell autolysis. The mixture was maintained at 50° C. for 8 h and then at 52° C. for 4 h. After centrifugation at 8000 rpm for 5 min, the precipitate was removed and the supernatant was harvested, which was rich in yeast extract with antioxidant activity.

2. The method according to claim 1 (1), characterized in that: Malt and papaya are used as raw materials, and the papaya is processed into papaya pulp and then added to prepare wort.

3. The method according to claim 1 (2), characterized in that: The wort is diluted to a concentration of 60% and a reducing sugar content of 80g / L before it is used for yeast proliferation and fermentation.

4. The method according to claim 1 (2), characterized in that: The yeast used was Rhodotorulamangiferae Y62.

5. The method according to claim 1 (3), characterized in that: The solvent used for extraction was pure water, and the harvested yeast extract was stored in brown glass vials away from light.

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