CN103932198A - Preparation method for selenium-enriched glutathione beer yeast biological product by utilizing waste beer yeast - Google Patents

Abstract

A selenium-enriched glutathione brewer's yeast biological product is characterized in that: using beer waste yeast to simultaneously transform inorganic selenium into organic selenium and glutathione precursor amino acid to synthesize glutathione, and obtain selenium-enriched glutathione yeast . It not only realizes the reuse of beer industry waste, but also glutathione can promote the activity of yeast selenium, which makes up for the deficiency of existing products that are purely enriched in selenium. At the same time, it also provides a variety of amino acids and vitamins. It is an efficient nutritional enhancer and supplement for the human body. The preparation method of selenium-enriched glutathione brewer's yeast biological product is characterized in that: after biotransformation of organic selenium and glutathione, the obtained yeast liquid is subjected to centrifugation, washing, resuspension, high-pressure homogenization, colloid mill, low-temperature drying, Granulated and tableted to obtain selenium-enriched glutathione yeast tablets that can be swallowed or chewed, and have the characteristics of convenient consumption, stable ingredients, and easy preservation.

Description

A method for preparing selenium-enriched glutathione brewer's yeast biological products by using beer waste yeast

technical field

The invention relates to a method for preparing selenium-enriched glutathione yeast biological products by utilizing fresh beer waste yeast to carry out biotransformation of sodium selenite to synthesize organic selenium, simultaneously transform glutathione precursor amino acid to synthesize glutathione.

Background technique

Selenium is one of the essential trace elements for humans and livestock. It is generally believed that the biological function of selenium mainly comes from selenoprotein (selenoprotein), and the biosynthesis of selenoprotein is encoded by the codon UGA on the mRNA selenocysteine (Selenocysteine, Sec) combines selenium into proteins by covalent bonds, so the biosynthesis and metabolic mechanism of selenoproteins have important theoretical value for molecular biology and biochemistry. Numerous studies have shown that selenoproteins play important roles in antioxidant activity, regulation of redox signaling, regulation of thyroid hormone metabolism, and immune response.

Studies have shown that the minimum and optimum daily safe intake of selenium for the human body are 22 μg and 50 μg respectively, and the maximum safe intake is 400-550 μg. Data show that the selenium intake of residents in many countries around the world is lower than the optimal selenium intake, while the selenium intake and blood selenium content of residents in surveyed areas in my country are lower than those in the United States and Japan. In addition, studies have shown that some special disease groups also need selenium supplementation, such as diabetes, cardiovascular disease, gastrointestinal disease, respiratory disease and other diseases and cancer patients, the blood selenium content is significantly lower than that of healthy people. Therefore, supplementing selenium, whether as a feed additive or as a dietary or drug supplement, has important significance and broad prospects.

Compared with inorganic selenium, organic selenium has the characteristics of high bioabsorption rate, high utilization rate, low toxicity, and less environmental pollution, so it has become the main type of selenium supplementation. At present, most organic selenium products use biological methods to transform inorganic selenium, including animal transformation, plant transformation and microbial transformation. Among them, microbial organic selenium, especially selenium-enriched yeast, has been widely valued and applied as an ideal organic selenium preparation.

Selenium-enriched products have been industrialized in many countries and entered the practical stage. Japan, the U.S. had yeast organic selenium commercial sales in the 1980s, and European countries such as France, Finland and Germany all had selenium-enriched yeast for sale as food additives and medicines. my country's selenium-enriched products are in the stage of extensive research and development and production. For example, Anhui Tiger Biotechnology Co., Ltd. provides a method of fermenting and culturing beer yeast strains ( Saccharomyces sp. , ATCC 21135) to produce selenium-enriched yeast, the selenium content of which can reach more than 2000 μg/g (CN 102277306 B); Guangzhou Gram Biotechnology Co., Ltd. used Rhodopseudomonas palustris as a selenium-enriched material to provide a preparation method for selenium-enriched Rhodopseudomonas palustris (CN 103284029 A), and the conversion rate of selenium reached 90.2 %, the selenium-enriched amount of the bacteria reached 75.3 mg/g dry bacteria.

One of the main problems with selenium-enriched products is high cost. At present, in the research on the biosynthesis of microbial selenium enrichment, almost all of them use the pure fermentation method. Pure breed fermentation requires large-scale initial investment, takes a long time to cultivate, consumes a lot of energy, and requires strict operating conditions, which makes the production cost higher, so the price of selenium-enriched biological products is generally higher.

Glutathione is divided into reduced glutathione (GSH) and oxidized glutathione (GSSG), commonly referred to as glutathione is reduced glutathione. Glutathione is an important active substance in the body. It has various physiological functions such as scavenging free radicals, detoxifying and maintaining DNA biosynthesis, normal cell growth and cellular immunity. It has important application value in the fields of medicine and health care. Glutathione is also an intrinsic natural component in brewer's yeast. Adding glutamic acid, cysteine and glycine to the yeast fermentation medium can effectively increase the content of glutathione in brewer's yeast cells. Glutathione peroxidase (GSH-Px) is an important peroxide-decomposing enzyme widely present in the body. Selenium is a component of the GSH-Px enzyme system. It can catalyze GSH into GSSG, reduce toxic peroxides to non-toxic hydroxyl compounds, and promote the decomposition of _H2O2 , thereby protecting the structure and function of cell membranes _. Interference and damage by peroxide, so glutathione is an important medium for selenium to exert physiological activity in the human body. Selenium and glutathione (GSH) system play a key role in the oxidative defense reaction. The preparation of selenium-enriched glutathione yeast can not only provide the health function of glutathione, but also better promote the production of selenium. Physiological functions, so as to exert a more comprehensive biological activity. For example, adding biological selenium and glutathione to fish feed can greatly enhance the fish's resistance to external poisons and their ability to decompose and excrete (CN 101438768A). Soochow University used Candida utilis as the starting strain and provided a method to increase the intracellular glutathione content of selenium-enriched Candida utilis (China CN 101875958 A).

Beer waste yeast is the main by-product in the production of beer industry. It is estimated that for every 100 tons of beer produced, about 1.5-2.0 tons of waste beer yeast (water content 75%-80%) can be obtained. With the rapid development of my country's beer industry, the annual beer output is increasing at a rate of 5% to 7%. According to the data released by the National Bureau of Statistics, my country's beer output in 2012 was 49.02 million kiloliters, and waste yeast accounted for about 1.5% of beer output , only 40%-50% of the waste yeast is recycled, and the rest is discarded, seriously polluting the environment (both the BOD and COD of the yeast sludge are as high as 100,000 mg/L or more). At present, most of the comprehensive utilization of waste beer yeast is concentrated on the protein, nucleic acid, vitamins, polysaccharides and enzymes, mainly the preparation of flavor food additive yeast extract, and the simultaneous enrichment and transformation of inorganic selenium and gluten The research on the development and application of stathione biosynthesis is still blank.

Most of the strains used in the production of selenium-enriched yeast are Saccharomyces cerevisiae, which is the same as that used in beer production. Therefore, it is theoretically feasible to use waste beer yeast as raw material to transform inorganic selenium and synthesize glutathione through re-fermentation; , studies have shown that the main period for Saccharomyces cerevisiae to synthesize organic selenium and glutathione is the stable period after 48 hours of yeast culture, and most of the fresh spent yeast cells are also in the period of 48-72 hours of anaerobic fermentation, so fresh spent yeast cells Yeast has the ability to enrich organic selenium and glutathione synthesis in terms of timing, and has the feasibility of turning waste into treasure.

Yeast products usually need to be broken or autolyzed to facilitate the digestion and absorption of animals. The selenium-enriched glutathione yeast prepared by this method has an autolysis rate of only about 16% in 2 hours in a simulated gastric environment, and the autolysis process of the yeast can be promoted by adding an appropriate amount of sodium chloride as an autolysis catalyst, which is beneficial to the animal body. digestion and absorption.

Contents of the invention

Aiming at the problem of high production cost of preparing selenium-enriched products by fermenting existing purebred microorganisms, the object of the present invention is to provide a method for preparing selenium-enriched glutathione yeast by using beer waste yeast.

Another object of the present invention is to provide a selenium-enriched glutathione yeast product and a preparation method thereof.

The technical scheme adopted in the present invention is:

Beer waste yeast ( Saccharomycescerevisiae ), fresh untreated beer sludge from Tsingtao Brewery (Jinan) Co., Ltd.

Resuspend the above waste beer yeast slime in medium, add sodium selenite and glutathione to synthesize precursor amino acids (cysteine, glutamic acid and glycine), and culture in shake flask or fermenter for 16-20 hours Selenium-enriched glutathione yeast was prepared by transforming inorganic selenium and precursor amino acids. Each gram of dry selenium-enriched glutathione yeast contained 1000-1500 μg of organic selenium, accounting for 82-92% of the total selenium weight, and selenoprotein content accounted for 75~80% of the total selenium weight; at the same time, each gram of dry selenium-enriched glutathione yeast contains about 6 mg of glutathione.

In this method, by adding 3-6 g/L NaCl, the autolysis rate of selenium-enriched yeast can reach 36% in 2 hours under simulated gastric environment, which can effectively improve the bioavailability of yeast products.

A method for producing the above-mentioned selenium-enriched yeast, comprising the steps of:

1) Wash the fresh waste beer yeast with water, centrifuge at 4000 r/min for 5-10 minutes to obtain yeast sludge (moisture content is about 58%), add the medium containing sodium selenite and precursor amino acids, and incubate at 24° Ferment at C~28°C for 12~16 hr;

2) The medium components are: phosphoric acid 8.0~10.0 g/L, sodium selenite 18~20 mg/L, ammonium sulfate 8.0~10.0 g/L, magnesium sulfate 3.0~5.0 g/L, calcium chloride 0.1 ~0.4 g/L, glucose 60.0~80.0 g/L, pH5.0, 1~2 g/L glutamic acid, 0.5~1.5 g/L glycine, 0.8~1.5 g/L cysteine (amino acids need to be separated Sterilization), add 150~250 g yeast mud (wet weight) per 1L of culture medium;

3) After the obtained yeast liquid is centrifuged, the bacterial cells are obtained. After washing with deionized water for 3 times, the yeast cells are resuspended with 2-5% lactic acid solution to obtain yeast milk. The yeast milk is repeatedly homogenized by high pressure homogenization 2~4 times, then add 1~2% hypromellose and 10%~20% 65% fructose syrup, and then pass through a colloid mill to obtain a uniform system. The system is dried at low temperature for 30 to 60 hours to reduce the water content to 1 to 5%, and the selenium-enriched glutathione yeast product is obtained through granulation and tableting.

In the present invention, the obtained selenium-enriched glutathione yeast can also be added to promote the autolysis of yeast cells by adding 3 to 6 g/L sodium chloride, thereby increasing its digestion and absorption in the stomach, which is beneficial to its digestion and absorption in the human body. Bioavailability.

Reduced glutathione (GSH) plays an important role in the conversion of inorganic selenium to organic selenium in organisms. As a reducing agent of selenite, GSH completes the synthesis process from sodium selenite to selenocysteine by generating GSSeSG and GSSeH. In the present invention, the conversion of waste beer yeast to inorganic selenium and the synthesis of organic selenium are improved by adding the precursor amino acid of glutathione, and yeast glutathione with higher yield can be obtained.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

Example 1

Dissolve 5.0 g of phosphoric acid, 4.0 g of ammonium sulfate, 1.8 g of magnesium sulfate, 0.15 g of calcium chloride, 9.0 mg of sodium selenite and 30 g of glucose into 400 mL of tap water, adjust the pH to 5.0, and place in a 2000 mL Erlenmeyer flask 115 °C, high-pressure damp heat sterilization for 30 min; dissolve 0.1 g of glutamic acid, 0.05 g of glycine, and 0.075 g of cysteine in 100 mL of distilled water, filter and sterilize with a 0.22 μm filter membrane, and merge into a container In a 2000 mL Erlenmeyer flask of sterilized culture medium. Add 100 g of fresh yeast sludge that has been washed and centrifuged, and incubate at 28±1°C and 150 rpm for 18 h.

The results showed that the selenium content in yeast was determined by conventional methods to be 1101 μg/g yeast, of which the organic selenium content accounted for 90%, the selenoprotein content accounted for 78.6%, and the glutathione content in each gram of selenium-enriched glutathione dry yeast was about 6.8 mg.

After the obtained yeast liquid is centrifuged, the bacterial cells are obtained. After washing 3 times with deionized water, the yeast cells are resuspended with 3% lactic acid solution to obtain yeast milk. Add 6 g/L NaCl to the yeast milk and stir Uniformity, the yeast milk was homogenized twice under high pressure (80 MPa), then added 1.5% hypromellose and 12% 65% fructose syrup, and then passed through a colloid mill twice to obtain a uniform system. The system was dried at low temperature for 45 hours to reduce the water content to 2%, and the obtained solid was granulated and tableted to obtain selenium-enriched glutathione yeast product.

Example 2

Dissolve 50.0 g of phosphoric acid, 13.4 g of ammonium sulfate, 20 g of magnesium sulfate, 2 g of calcium chloride, 33.4 mg of sodium selenite and 300.0 g of glucose into 4900 mL of tap water, adjust the pH to 5.0, and place in a 10 L fermenter 115 °C, high-pressure damp heat sterilization for 20 min; dissolve 10 g of glutamic acid, 7.5 g of cysteine, and 5 g of glycine in 100 mL of distilled water, filter and sterilize with a 0.22 μm filter membrane, and put them into a container in sterilized culture medium. Add 1250 g of fresh yeast sludge that has been washed and centrifuged, the temperature is 28±1°C, the stirring speed is 200-500 rpm, and the dissolved oxygen is kept greater than 25%, and the transformation is carried out for 19 hours.

The results showed that the selenium content in yeast was determined by conventional methods to be 1223 μg/g yeast, of which organic selenium content accounted for 91.0%, selenoprotein content accounted for 77.8%, and glutathione content was 8.03 mg/g dry yeast.

After the obtained yeast transformation liquid was centrifuged, the bacterial cells were obtained. After washing twice with deionized water, the yeast cells were resuspended with 5% citric acid solution to obtain yeast milk, and 5 g NaCl was added to the yeast milk, and stirred evenly , the yeast milk was subjected to high-pressure homogenization (75 MPa) for 3 times, then 1.2% hypromellose and 15% 65% fructose syrup were added, and then passed through a colloid mill for 3 times to obtain a uniform system. The system was dried at low temperature for 55 hours to reduce the water content to 1.5%, and the obtained solid was granulated and tableted to obtain selenium-enriched glutathione yeast product.

Example 3

Dissolve 150.0 g of phosphoric acid, 40 g of ammonium sulfate, 50 g of magnesium sulfate, 5.5 g of calcium chloride, 100 mg of sodium selenite, and 900.0 g of glucose into 14 L of tap water, adjust the pH to 5.0, and place in a 30 L fermentation tank 115 °C, high-pressure damp heat sterilization for 25 min; dissolve 35 g of glutamic acid, 25 g of cysteine, and 15 g of glycine in 1L of distilled water, filter and sterilize with a 0.22 μm filter membrane, and combine them into a well-preserved in sterile culture medium. Add 4000 g of fresh yeast sludge that has been washed and centrifuged, the temperature is 29±1°C, the stirring speed is 200-500 rpm, and the dissolved oxygen is kept greater than 30%, and the transformation is carried out for 17 hours.

The results showed that the selenium content in yeast was determined by conventional methods to be 1424 μg/g yeast, of which organic selenium content accounted for 90%, selenoprotein content accounted for 80.1%, and glutathione content was 7.93 mg/g dry yeast.

After the obtained yeast transformation liquid was centrifuged, the bacterial cells were obtained. After washing with deionized water for 3 times, the yeast cells were resuspended with 4% malic acid solution to obtain yeast milk, and 5 g/L NaCl was added to the yeast milk. And stir evenly, the yeast milk was homogenized twice under high pressure (90 MPa), then added 1% hypromellose and 18% 65% fructose syrup, and then passed through a colloid mill twice to obtain a uniform system. The system was dried at low temperature for 52 hours to reduce the water content to 1.2%, and the obtained solid was granulated and tableted to obtain selenium-enriched glutathione yeast product.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and modifications can also be made. These improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (4)

1. A selenium-enriched glutathione brewer's yeast biological product, which is obtained by bioconverting inorganic selenium and glutathione precursor amino group by spent beer yeast. 2. the described saccharomyces cerevisiae of claim 1 is beer industry waste fresh yeast slime, and the step of its conversion synthetic organic selenium and glutathione is as follows: (1) Fresh waste beer yeast was washed 3 times with tap water, and centrifuged at 4000 r/min for 5-10 minutes to obtain debittered and impurity-removed yeast sludge; (2) Prepare organic selenium and glutathione conversion solution, its composition is: phosphoric acid 8.0~10.0 g/L, sodium selenite 18~20 mg/L, ammonium sulfate 8.0~10.0 g/L, magnesium sulfate 3.0~ 5.0 g/L, calcium chloride 0.1~0.4 g/L, glucose 60.0~80.0 g/L, pH5.0, 1~2 g/L glutamic acid, 0.5~1.5 g/L glycine, 0.8~1.5g/L L-cysteine; (3) Add 150-250 g of wet yeast mud to each liter of yeast transformation liquid in (2), and disperse evenly; (4) Cultivate and biotransform the system in (3) for 18-22 hours in a constant temperature shaker or bioreactor at 28±1°C, 150-500 rpm. 3. according to the described selenium-enriched glutathione Saccharomyces cerevisiae biological product according to claim 1, its preparation step is: (1) After the cultivation and biotransformation are completed, the culture medium is centrifuged to obtain bacterial cells, washed with deionized water, and the yeast cells are resuspended with 2-5% food-grade organic acid solution, and yeast autolysis inducer is added to obtain acidification of yeast milk; (2) The acidified yeast milk is homogenized by high pressure for 2~4 times, then add 1~2% binder and 10%~20% flavoring agent, and stir evenly; (3) Pass the system in (2) through the colloid mill for 2 to 3 times to obtain a uniform yeast product system; (4) Dry the system in (3) at low temperature for 40-60 hours to make it a solid with a water content of 1-3%; (5) Granulate and tablet the solid obtained in (4) to obtain selenium-enriched glutathione yeast product. 4. according to claim 3, during the preparation of selenium-enriched glutathione Saccharomyces cerevisiae biological products, the yeast autolysis inducer used is 3 ~ 6 g/L NaCl; the binder used is 1 ~ 2% hydroxypropyl Methyl cellulose; The flavoring agent used is 10-20% 65% fructose syrup; The selected organic acids are lactic acid, citric acid, and malic acid.