A wolfberry wine rich in organic selenium and its preparation method

Abstract

The present invention discloses a wolfberry wine rich in organic selenium. This wolfberry wine is prepared by fermenting with selenium-rich yeast and additionally adding organic selenium of selenium-rich yeast. Its preparation method includes: breaking the cell walls of the separately fermented selenium-rich yeast, enzymatically hydrolyzing selenium proteins, and adding the released selenium-containing amino acids to the wine to prepare an organic selenium-type selenium-rich wolfberry wine. The wolfberry wine provided by the present invention contains abundant selenium-containing amino acids, which are beneficial for direct absorption by the human body. After drinking this product, it can achieve the effects of antioxidation and inhibiting residual free radicals in the human body. In addition, the wine-making technology of the present invention is simple, the organic selenium content meets the standards of selenium-rich beverages, meets the needs of consumers for selenium-rich products, and expands the channels for scientific selenium supplementation for the human body.

Description

Technical Field

[0001] The present invention belongs to the technical field of health care wine, and relates to a wolfberry wine rich in organic selenium with antioxidant activity and a preparation method thereof. Background Art

[0002] Fruit wine has rich nutritional and health care functions. With the gradual enhancement of people's health and health care awareness, health care fruit wine as a daily drink for the public has great potential and development space in the consumer market.

[0003] Selenium plays an important role in maintaining human health and preventing and treating diseases. For human health, the intake of selenium can improve the body's antioxidant function, inhibit oxidative damage of cells, and enhance the body's ability to resist inflammatory responses. Selenium can be used as an auxiliary antioxidant to inhibit diseases such as thyroid cancer and female HPV infection. The Chinese Nutrition Society has listed selenium as one of the daily dietary nutrients. Among them, the activity and utilization rate of organic selenium have been proven to be significantly higher than those of inorganic selenium (selenate and selenite).

[0004] At present, the selenium content in natural crops is relatively low and cannot meet the human body's needs. The ability of wolfberries to absorb inorganic selenium from the soil and convert it into organic selenium is limited, resulting in relatively low total selenium content and the proportion of organic selenium in the brewed wine, so that the selenium content in wolfberry wine often fails to meet the standards of selenium-rich foods. Summary of the Invention

[0005] To solve this problem, the present invention aims to provide a wolfberry wine rich in organic selenium and having high antioxidant capacity.

[0006] To achieve the above object, by mass percentage, the raw materials required for the selenium-rich wolfberry wine provided by the present invention include: 76-79% of wolfberry juice, 2% of selenium-rich yeast, 0.01-3.01% of selenium-rich yeast extract, 0.98% of citric acid, 1% of pectinase, 0.01% of sulfur dioxide, and 17% of sucrose.

[0007] In particular, the wolfberry juice of the present invention is obtained by crushing and pressing wolfberries that are both medicine and food, and the organic selenium content in the selenium-rich yeast used in the present invention is not less than 80% of the total selenium content.

[0008] Furthermore, the selenium-rich yeast extract of the present invention is obtained by alkaline extraction of yeast crude protein, enzymatic hydrolysis, and then ultrafiltration to release selenium amino acids.

[0009] Furthermore, the preparation steps of the selenium-rich wolfberry wine of the present invention include: mixing wolfberry juice, sucrose, and sulfur dioxide to make a liquid fermentation medium, activating selenium-rich yeast and inoculating it into the liquid fermentation medium for fermentation to obtain selenium-rich wine; extracting selenium amino acids from the selenium-rich yeast to obtain the selenium-rich yeast extract, and adding the selenium-rich yeast extract to the selenium-rich wine after the brewing of the selenium-rich wine is completed to obtain selenium-rich wolfberry wine; sterilizing and filling the brewed selenium-rich wolfberry wine, and storing it under cool conditions.

[0010] Furthermore, the present invention measures the antioxidant capacity of the selenium-rich wolfberry wine by FRAP (ferric ion reducing antioxidant power method) and DPPH (1,1-diphenyl-2-picrylhydrazyl) method. After measurement, the antioxidant capacity of the selenium-rich wolfberry wine of the present invention is significantly superior to that of ordinary wolfberry wine.

[0011] Furthermore, the present invention also uses HG-AFS (hydride generation-atomic fluorescence spectrometer) to measure the selenium content in the selenium-rich wolfberry wine. After measurement, the content of organic selenium in the selenium-rich wolfberry wine prepared by the present invention is not less than 80% of the total selenium content.

[0012] Through the above technical solutions, combined with the embodiments, the wolfberry wine rich in organic selenium provided by the present invention has at least the following beneficial effects or advantages:

[0013] 1. The selenium in the wolfberry wine of the present invention mainly exists in the form of organic selenium, and the content of organic selenium is not less than 80% of the total selenium content, which will not cause problems of acute toxicity and is safe and efficient.

[0014] 2. The wolfberry wine of the present invention fully exerts and enhances the antioxidant capacity of wolfberry wine. After being detected by FRAP and DPPH methods, its antioxidant capacity is significantly higher than that of ordinary wolfberry wine. Among them, the highest antioxidant capacity detected by the FRAP method can reach 67.47 μmol TE / g; the highest antioxidant capacity detected by the DPPH method can reach 60.91 μmol TE / g.

[0015] 3. The wolfberry wine of the present invention is significantly superior to ordinary wolfberry wine in taste through sensory evaluation. Specific Embodiments

[0016] Next, the technical solutions of the present invention will be described in conjunction with the embodiments. However, the present invention is not limited to the following embodiments. Those not specified in the embodiments are carried out according to conventional conditions. Those reagents or instruments not specified in the manufacturer are all conventional products that can be obtained through the market.

[0017] Example 1

[0018] This example describes a method for self-making selenium-rich yeast and an extract of selenium-rich yeast based on Saccharomyces cerevisiae.

[0019] In this example, selenium-enriched yeast strains HYJ1 and YN6 with different selenium enrichment amounts were obtained by culturing Saccharomyces cerevisiae CICC 31869 and CICC 32883 (purchased from China Center of Industrial Culture Collection) in selenium-enriched media. The specific preparation steps of selenium-enriched yeast and selenium-enriched yeast extract are as follows:

[0020] 1. Preparation of self-made selenium-enriched yeast

[0021] 1.1 Activate Saccharomyces cerevisiae CICC 31869 and CICC 32883 strains, and add selenite to the medium at a final concentration of 20 μg / L during the culture stage. After culturing for 48 h, selenium-enriched yeast strains HYJ1 and YN6 are obtained.

[0022] 1.2 Wash the obtained selenium-enriched yeast with sterile saline 5 times to remove the medium and inorganic selenium attached to the outer wall of yeast cells. Among them, the selenium-enriched yeast strain HYJ1 can be directly used for brewing wine.

[0023] 1.3 Lyophilize the YN6 selenium-enriched yeast strain obtained in the previous step to obtain selenium-enriched yeast powder, which is used for the subsequent extraction of organic selenium.

[0024] The activation conditions in step 1.1 are as follows: Use the experimental YPD medium (Yeast Extract Peptone Dextrose Medium) as the medium for yeast inoculation, activation and culture. The YPD liquid medium contains 1% yeast extract powder, 2% peptone and 2% glucose. Its solid medium needs to add 2% agar powder again. Take the Saccharomyces cerevisiae stored in an ultra-low temperature refrigerator at -80 °C, immediately place it in a water bath at 38 - 40 °C to resuscitate and shake quickly until all the internal ice is dissolved (50 - 100 s). Open the plastic cryopreservation tube, streak the strain on the YPD solid medium, and culture it at 28 °C for 48 h. Then pick well-dispersed single colonies from the YPD solid medium and inoculate them into a conical flask containing YPD liquid medium (100 mL / 250 mL), and culture at 28 °C for 48 h, with a rotation speed of 160 r / min, and the activated seed liquid can be obtained.

[0025] 2. Preparation of selenium-enriched yeast YN6 extract

[0026] 2.1 Mix NaOH with a concentration of 1.8%, NaCl with a concentration of 0.1%, and SDS (sodium dodecyl sulfate) with a concentration of 1% to obtain an alkaline solution.

[0027] 2.2 Dissolve the freeze-dried selenium-enriched yeast powder obtained in step 1.3 in the above alkaline solution at a volume ratio of 1:40. At the same time, use a high-speed oscillating homogenizer (IKA T25 high-speed homogenizer from Germany) for cell wall breaking treatment. The treatment time is 15 min (intermittent treatment, totaling 15 min), the power is 700 W, the frequency is 50 / 60 Hz, and the shear rotation speed is 10,000 rpm.

[0028] 2.3 After the cell wall breaking treatment, perform ammonium sulfate gradient precipitation (concentration of 55 - 85%) of proteins. The precipitation conditions are 4°C and 12 h.

[0029] 2.4 Centrifuge the precipitated proteins (using a high-speed refrigerated centrifuge HC-3018R, 6000 rpm, 15 min). After centrifugation, perform dialysis for 72 h to remove salts, and then freeze-dry to obtain selenium-enriched yeast protein powder.

[0030] 2.5 Enzymatically hydrolyze the above selenium-enriched yeast protein. The enzymatic hydrolysis conditions are shown in Table 1.

[0031] 2.6 After enzymatic hydrolysis, centrifuge and take the supernatant, and perform ultrafiltration using a 10KD ultrafiltration tube to obtain the selenium-enriched yeast YN6 extract.

[0032] Table 1 Enzymatic hydrolysis protein parameter conditions

[0033]

[0034] In particular, due to the limitation of enzymatic hydrolysis ability, not all of the above selenium-enriched yeast protein can be enzymatically hydrolyzed and converted into selenium amino acids. The uncompletely enzymatically hydrolyzed part exists in other selenium forms, while selenium amino acids are still the main components of the selenium-enriched yeast YN6 extract.

[0035] Example 2

[0036] Based on the selenium-enriched yeast and selenium-enriched yeast extract obtained in Example 1, this example prepared selenium-enriched wolfberry wine with different raw material ratios, and also prepared reference example wolfberry wine.

[0037] 1. Preparation of selenium-enriched wolfberry wine

[0038] The raw materials for the preparation of selenium-enriched wolfberry wine in this example include wolfberry juice, selenium-enriched yeast, selenium-enriched yeast extract, citric acid, pectinase, sulfur dioxide, and sucrose. Among them, the wolfberry juice is obtained by crushing Ningxia red medicinal and edible organic wolfberries (using a Philips blender, model HR2096, crushing time 3 min (intermittent crushing for 30 s, rated frequency 50 Hz, output power 800 W)).

[0039] Table 2 Raw material ratios of each experimental group

[0040]

[0041] In this embodiment, the mass ratio of each group of raw materials is shown in Table 2, and the specific preparation steps are as follows:

[0042] S1. Material selection: Select high-quality organic goji berries from Ningxia.

[0043] S2. Ingredient preparation: Add goji berry juice, sucrose, sulfur dioxide, citric acid, and pectinase according to the mass ratio of each experimental group in Table 2.

[0044] S3. Inoculation and fermentation: Cultivate selenium-enriched yeast and activate it, then inoculate it into goji berry juice for fermentation.

[0045] S4. Extraction: Extract selenium amino acids from the self-made selenium-enriched yeast powder.

[0046] S5. Blending: After the brewing of selenium-enriched wine is completed, add the extracted organic selenium to the selenium-enriched wine to obtain selenium-enriched goji berry wine of groups I, II, and III respectively.

[0047] 2. Preparation of reference example goji berry wine

[0048] In this embodiment, the reference example goji berry wine is a self-fermented ordinary goji berry wine, fermented with Saccharomyces cerevisiae CICC31869. The raw materials include 79% goji berry juice, 2% Saccharomyces cerevisiae CICC31869 strain, 0.98% citric acid, 1% pectinase, 0.01% sulfur dioxide, and 17% sucrose. The specific steps are as follows:

[0049] S1. Material selection: Select high-quality organic goji berries from Ningxia.

[0050] S2. Ingredient preparation: Add goji berry juice, sucrose, sulfur dioxide, citric acid, and pectinase according to the above mass ratio.

[0051] S3. Inoculation and fermentation: Cultivate yeast and activate it, then inoculate it into goji berry juice for fermentation.

[0052] S4. Storage: Sterilize and fill the brewed goji berry wine, and store it under cool conditions.

[0053] Example 3

[0054] In this embodiment, the antioxidant capacities of the selenium-enriched goji berry wine of groups I, II, and III prepared in Example 2 and the reference example goji berry wine were compared and tested.

[0055] In this embodiment, the antioxidant capacities of each experimental group and the reference example goji berry wine were determined by FRAP (ferric ion reducing antioxidant power method) and DPPH method (1,1-diphenyl-2-picrylhydrazyl).

[0056] 1. Determination of antioxidant capacity by DPPH method

[0057] 1.1 Add 2 mL of diluted samples from each group to 4 mL of methanol containing 0.18 mg of 1,1-diphenyl-2-picrylhydrazyl (DPPH).

[0058] 1.2 After incubating for 30 minutes in the dark, measure the absorbance of the mixtures from each group at 517 nm, where the antioxidant capacity is calculated in terms of trolox (μmol TE / g).

[0059] 2. FRAP determination of antioxidant capacity

[0060] 2.1 Mix 0.1 M acetate buffer solution (pH 3.6), 10 mM TPTZ reagent (prepared with 40 mM HCl solution), and 20 mM ferric chloride in a volume ratio of 10:1:1 to prepare the FRAP reagent.

[0061] 2.2 Take 0.5 mL of the sample and mix it with 9.5 mL of the FRAP reagent, then incubate in the dark for 30 minutes for absorbance detection, where the antioxidant capacity is calculated in terms of trolox (μmol TE / g).

[0062] After measurement, the antioxidant capacities of each experimental group and the wolfberry wine in the reference example are shown in Table 3.

[0063] Table 3 Antioxidant capacities of four kinds of wines

[0064]

[0065] Combined with the above data, the antioxidant effects of the wolfberry wines in Groups I, II, and III prepared in Example 2, with FRAP as the antioxidant index, are all better than those in the reference example. When detected with DPPH as the antioxidant index, there is no significant difference between Group I and the reference example, but the antioxidant effects of Groups II and III are both better than those in the reference example; in summary, Groups II and III have the characteristic of stronger antioxidant capacity. Due to different detection principles, there are also differences in the comparison of the results between the experimental groups and the reference example for different antioxidant detection methods. However, based on the above detection principles, the presence of selenium amino acids is efficient and safe for human health and can directly participate in the synthesis of proteins in the body.

[0066] Example 4

[0067] In this example, the total selenium and organic selenium contents in the wolfberry wines in Groups I, II, and III prepared in Example 2 and the wolfberry wine in the reference example were compared and tested.

[0068] (1) Total selenium determination method

[0069] In this example, a hydride generation-atomic fluorescence spectrometer (HG-AFS) was used to detect the selenium content (the detailed parameters are shown in Table 4). The specific detection method is as follows: After the sample is pre-digested and microwave digested, in a 6 mol / L hydrochloric acid medium, the hexavalent selenium in the sample is reduced to tetravalent selenium, and potassium borohydride and sodium hydroxide are used as reducing agents to reduce the tetravalent selenium to hydrogen selenide. It is carried into the atomizer by the carrier gas (argon) for atomization. Through the irradiation of the selenium hollow cathode lamp, the ground-state selenium atoms are excited to the high-energy state and emit fluorescence with a characteristic wavelength. The fluorescence intensity is proportional to the selenium content, and quantitative detection and analysis are carried out by comparing with the standard series to obtain the selenium content of the sample.

[0070] Table 4 HG-AFS Instrument Parameter Table

[0071]

[0072] (2) Selenium Species Determination Method

[0073] A liquid chromatography coupled with hydride generation-atomic fluorescence spectrometer was used to measure the selenium species. Before sample determination, a series of standard products of Sec 2 , SeMet, SeMeCys, Se(IV), and Se(VI) with different concentrations were prepared for determination and the drawing of the standard curve. After the wine sample was treated through a 0.22 μm filter (SYRINGE FILTER 13mm Luer) and a 10KD ultrafiltration tube (MILLIPORE), it was directly injected, and the LC-HG-AFS was used to determine the sample content. The specific determination conditions are shown in Table 5.

[0074] Table 5 LC-HG-AFS Instrument Parameter Table

[0075]

[0076] After detection, the total selenium and organic selenium contents of the above-mentioned wolfberry wines in groups I, II, and III and the wolfberry wine in the reference example are shown in Table 6.

[0077] Table 6 Total Selenium and Selenium Amino Acid Contents of Each Group of Wolfberry Wines

[0078]

[0079]

[0080] Note: In the table, "--" represents not detected. "Other selenium forms" in the table are calculated by subtracting organic and inorganic selenium from the total selenium content, and the possibility of other selenium forms formed by enzymatic hydrolysis ability and yeast metabolism is not excluded.

[0081] Example 5

[0082] In this example, sensory evaluations were conducted on the wolfberry wines of Groups I, II, and III prepared in Example 2 and the wolfberry wine of the reference example.

[0083] The jury consisted of 8 postgraduate students (five females and three males, aged 20 - 25) from the College of Food Science and Engineering, Northwest A&F University. Before the formal sample evaluation, the subjects were trained with the minismell aroma toolkit for 4 consecutive weeks, so that the aroma recognition deviation was less than 5%. The evaluation members evaluated the samples in triplicate. During the evaluation, it was carried out in a sensory analysis laboratory, and pure water was provided to clean the mouth. The sensory scoring criteria are shown in Table 7, and the evaluation results are shown in Table 8.

[0084] Table 7 Sensory Scoring Criteria

[0085]

[0086]

[0087] Table 8 Sensory Evaluation Results

[0088]

[0089] As can be seen from Table 8, there are no significant differences among the four wines in terms of color and appearance, and aroma. They are all clear, transparent, and have a pure wolfberry wine fragrance. In terms of taste, the experimental groups are significantly better than the reference example. The wine bodies of the experimental groups are more plump, with a moderate sweetness and sourness, while the balance of the reference example is relatively poor. In terms of typicality, Groups I and II have a more obvious wolfberry style, and the typicality is better than that of the reference example and Group III.

[0090] In summary, the wolfberry wine rich in organic selenium provided by the present invention has strong antioxidant ability, the proportion of organic selenium content is not less than 80%, and it is significantly superior to ordinary wolfberry wine in terms of taste.

[0091] As described above, the present invention can be preferably implemented. The above embodiments are only descriptions of the preferred implementation modes of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A wolfberry wine rich in total selenium and organic selenium with strong antioxidant capacity, characterized in that, by mass percentage, the preparation raw materials include: 76% - 79% of Ningxia red wolfberry juice, 2% of selenium-enriched yeast, 0.01 - 3.01% of selenium-enriched yeast extract, 17% of sucrose, 0.01% of sulfur dioxide, 0.98% of citric acid and 1% of pectinase; the content of organic selenium in the selenium-enriched yeast is not less than 80% of the total selenium content; the selenium-enriched yeast extract includes selenium-containing amino acids; the preparation method of the wolfberry wine includes: mixing the Ningxia red wolfberry juice, sucrose, sulfur dioxide, citric acid and pectinase to make a liquid fermentation medium; activating the selenium-enriched yeast and inoculating it into the liquid fermentation medium for fermentation to obtain selenium-enriched wine; adding the selenium-enriched yeast extract to the selenium-enriched wine to obtain selenium-enriched wolfberry wine; sterilizing and filling the brewed selenium-enriched wolfberry wine, and storing it under cool conditions; the organic selenium includes selenocysteine (Sec), methylselenocysteine (SeMeCys) and selenomethionine (SeMet); the preparation method of the selenium-enriched yeast includes: after activating the Saccharomyces cerevisiae CICC31869 and CICC32883 strains purchased from the China Center for Industrial Culture Collection, adding selenite to the medium at a final concentration of 20 μg / L during the cultivation stage, and after culturing for 48 h, selenium-enriched yeasts HYJI and YN6 can be obtained respectively; washing the obtained selenium-enriched yeasts with sterile normal saline 5 times to remove the medium and inorganic selenium attached to the outer wall of the yeast cells; freezing and drying the washed YN6 selenium-enriched yeast to obtain selenium-enriched yeast powder; the preparation method of the selenium-enriched yeast extract includes: dissolving the selenium-enriched yeast powder in an alkaline solution prepared by mixing NaOH, NaCl and SDS at a volume ratio of 1:40, and at the same time using a high-speed oscillating homogenizer for cell wall breaking treatment. The treatment time of the homogenizer is 15 min, the power is 700 W, the frequency is 50 / 60 Hz, and the shear rotation speed is 10000 rpm; after the cell wall breaking is completed, ammonium sulfate gradient precipitation of proteins is carried out, the precipitation temperature is 4°C, and the time is 12 h; the precipitated proteins are centrifuged and then dialyzed for 72 h to remove salts, and then freeze-dried at low temperature to obtain selenium-enriched yeast protein powder; enzymatically hydrolyzing the above selenium-enriched yeast protein powder, centrifuging after enzymatic hydrolysis to take the supernatant, and ultrafiltering with a 10KD ultrafiltration tube to obtain the selenium-enriched yeast extract; the enzyme in the enzymatic hydrolysis is trypsin, streptomyces protease E and protease K, under the enzymatic hydrolysis conditions of pH 7.2, enzymatic hydrolysis time of 6 h, and enzymatic hydrolysis temperature of 37°C, the concentration ratio of trypsin to the substrate is 1:10; under the enzymatic hydrolysis conditions of pH 7.2, enzymatic hydrolysis time of 6 h, and enzymatic hydrolysis temperature of 37°C, the concentration ratio of streptomyces protease E to the substrate is 1:50; under the enzymatic hydrolysis conditions of pH 7.2, enzymatic hydrolysis time of 12 h, and enzymatic hydrolysis temperature of 55°C, the concentration ratio of protease K to the substrate is 1.88:100.

Images