WO2023010910A1 - 一种有机富硒酵母、其制备方法及制品和应用 - Google Patents

一种有机富硒酵母、其制备方法及制品和应用 Download PDF

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WO2023010910A1
WO2023010910A1 PCT/CN2022/089885 CN2022089885W WO2023010910A1 WO 2023010910 A1 WO2023010910 A1 WO 2023010910A1 CN 2022089885 W CN2022089885 W CN 2022089885W WO 2023010910 A1 WO2023010910 A1 WO 2023010910A1
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organic
selenium
enriched yeast
starch
preparation
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PCT/CN2022/089885
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English (en)
French (fr)
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陈晖�
肖明华
王茜
刘秀继
俞灿
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安琪酵母股份有限公司
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Publication of WO2023010910A1 publication Critical patent/WO2023010910A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/14—Yeasts or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention relates to the technical field of fermentation industry, in particular to an organic selenium-enriched yeast, its preparation method, product and application.
  • Selenium is an important trace element necessary for human and animal physiology. It mainly exists in organisms in the form of selenium enzymes and selenium-containing proteins. It has been reported in published literature that selenium has the functions of anti-oxidation, regulating immunity, scavenging free radicals, and regulating gene expression in organisms. Selenium can protect myocardium, prevent and treat Keshan disease, Kaschin-Beck disease, liver disease and various cancers, and activate pancreatic islets. Adjuvant treatment of diabetes, selenium deficiency and the occurrence and development of Keshan disease, coronary heart disease, atherosclerosis, hypertension, cardiomyopathy and other cardiovascular diseases have obvious correlations. In terms of animal breeding, the addition of organic selenium to sow diets can promote the development of embryos in the early stages of pregnancy and improve the reproductive performance of sows and boars.
  • selenium-enriched yeast has been approved for food nutrition fortification, feed additives and other purposes.
  • the first is the chelation method, in which a certain amount of selenate or selenite is chelated with yeast living cell emulsion at low temperature, and then dried.
  • the second is the fermentation method.
  • Yeast yeast strains are used as carbon source in sugar or starch or molasses, ammonia water, ammonium sulfate, ammonium carbonate or urea as nitrogen source, and sodium selenite or selenite are added.
  • the inorganic selenium is converted into yeast selenium through yeast growth, absorption and conversion.
  • Chinese patent CN107058208A discloses a production process of selenium-enriched yeast powder, which includes the following steps: preparation of medium ⁇ slant strain cultivation ⁇ primary liquid seed cultivation ⁇ secondary liquid seed cultivation ⁇ fermenter seed cultivation ⁇ fermenter fermentation cultivation ⁇ Separation ⁇ drying ⁇ crushing ⁇ packaging.
  • the selenium-enriched yeast powder prepared by the invention is a selenium-enriched yeast product with high selenium content, and its selenium content reaches 1200mg/kg, wherein the organic selenium content accounts for 92.8% of the total selenium, and the selenium in the form of selenomethionine accounts for 44.5% of the total selenium.
  • CN102181371B discloses a yeast and a method for producing selenium-enriched yeast by using the yeast.
  • Specific yeast strains are selected, and an organic nitrogen source containing amino nitrogen is added every 5 to 10 hours during the process of feeding the inorganic selenium salt solution;
  • the inorganic selenium salt solution is an aqueous solution of selenate, selenite or a mixture of selenate and selenite;
  • the organic nitrogen source containing amino nitrogen is peptone, yeast extract, vegetable protein, one or more of amino acids.
  • selenium in the form of selenomethionine in selenium-enriched yeast accounts for 55-70% of the total selenium,
  • the organic selenium active ingredient selenomethionine content in the selenium-enriched yeast prepared by the prior art fermentation method is relatively low, the selenium absorption and utilization rate is low, and the fermentation medium contains a large amount of inorganic selenium, inorganic nitrogen and Inorganic phosphorus does not meet the requirements of organic product certification.
  • one of the purposes of the present invention provides a kind of organic selenium-enriched yeast;
  • the second object of the present invention is to provide the preparation method of the above-mentioned organic selenium-enriched yeast;
  • the third object of the present invention is to provide a An organic selenium-enriched yeast product prepared from the above organic selenium-enriched yeast;
  • the fourth object of the present invention is to provide the application of the above-mentioned organic selenium-enriched yeast or organic selenium-enriched yeast product in feed additives and food nutrition enhancers.
  • the invention provides an organic selenium-enriched yeast, wherein the organic selenium accounts for more than 99% of the total selenium weight, and the selenium in the form of selenomethionine accounts for more than 80% of the total selenium weight.
  • the selenium in the form of selenomethionine accounts for more than 90% of the total selenium weight.
  • the organic selenium-enriched yeast contains 800-2100 mg of total selenium per kg, and preferably, the selenium absorption rate of the yeast is above 95%.
  • the above-mentioned organic selenium-enriched yeast is obtained by fermenting and culturing yeast strain Saccharomyces cerevisiaed.
  • the bacterial strain was preserved in the China Center for Type Culture Collection (CCTCC) on August 1, 2016, and the preservation number is CCTCC NO: M2016418. This bacterial strain has been recorded in the patent publication with the publication number CN108220175A.
  • the above-mentioned organic selenium-enriched yeast is obtained by fermenting and culturing the yeast strain Saccharomyces cerevisiaed FX-2 with organic starch hydrolyzed sugar, organic plant protein enzymatic hydrolyzate and selenium-enriched yeast extract.
  • the present invention provides a method for preparing the above-mentioned organic selenium-enriched yeast inoculant, which comprises expanding and culturing yeast strains step by step, fermenting and cultivating, collecting and drying yeast cells, wherein the fermenting and cultivating uses organic starch hydrolyzed sugar, organic plant Protein hydrolyzate and selenium-enriched yeast extract are used as culture medium.
  • the pH of the fermentation culture is 5.0-7.0, and preferably, the fermentation temperature is 25-35°C.
  • the organic starch hydrolyzed sugar is obtained from organic starch through enzymatic hydrolysis of amylase and glucoamylase in sequence.
  • the organic starch is organic corn starch, organic wheat starch, organic tapioca starch and organic rice starch.
  • the amylase is high-temperature amylase and/or pullulanase.
  • the amylase has a pH of 5.6-5.8 during enzymatic hydrolysis, and preferably, the amylase is used in an amount of 1-2 ⁇ based on the weight of organic starch.
  • the pH of the enzymatic hydrolysis of the glucoamylase is 4.2-4.4, preferably, based on the weight of organic starch, the amount of the glucoamylase is 1-2 ⁇ , further preferably, the enzymatic hydrolysis time of the glucoamylase is 15- 20 hours.
  • the total sugar content in the organic starch hydrolyzed sugar is 26-31%.
  • the organic plant protein enzymatic hydrolyzate is obtained by proteolysis of organic plant protein
  • the organic plant protein is one of organic soybean meal powder, organic corn protein, organic soybean protein and organic rice protein or two or more, further preferably, the protease is one or more of papain, bromelain, trypsin, alkaline protease, flavor protease, acid protease and neutral protease, still more preferably, the Protease activity is greater than 5000u/g.
  • the enzymatic hydrolysis with protease includes two enzymatic hydrolysis.
  • the pH of the first protease hydrolysis is 6.0-6.5, preferably, the enzymolysis temperature is 53-57°C, more preferably, the amount of protease is 1-3 ⁇ based on the weight of organic vegetable protein, Even more preferably, the enzymatic hydrolysis time is 2-5 hours.
  • the pH of the second protease hydrolysis is 6.0-6.5, preferably, the enzymolysis temperature is 50-54°C, more preferably, the amount of protease is 6-8 ⁇ based on the weight of organic vegetable protein, Even more preferably, the enzymatic hydrolysis time is 10-15 hours.
  • the amino acid nitrogen content in the organic plant protein hydrolyzate is >0.3%.
  • the selenium content in the selenium-enriched yeast extract is greater than 2000ppm based on 100% of the dry matter weight of the selenium-enriched yeast extract, preferably the selenium content in the selenium-enriched yeast extract is greater than 4000ppm, preferably, the selenium-enriched yeast extract
  • the total nitrogen content in the yeast extract is >6%, and further preferably, the amino acid nitrogen content in the selenium-enriched yeast extract is >3.5%.
  • the amount of organic starch in the medium raw material is 30-40%, the amount of organic vegetable protein is 55-65%, and the amount of selenium-enriched yeast extract is 2.4-4.2%, preferably Preferably, the sum of the amount of organic starch and organic vegetable protein is more than 95%.
  • the present invention also provides an organic selenium-enriched yeast product, which is prepared from the above-mentioned organic selenium-enriched yeast or the organic selenium-enriched yeast inoculum prepared by the above-mentioned preparation method.
  • the organic selenium-enriched yeast product can be organic selenium-enriched yeast hydrolyzate or organic selenium-enriched yeast extract, which is obtained by autolyzing, enzymatically hydrolyzing, separating and purifying organic selenium-enriched yeast.
  • the present invention also provides the application of the above-mentioned organic selenium-enriched yeast or the organic selenium-enriched yeast inoculum prepared by the above-mentioned preparation method or the above-mentioned organic selenium-enriched yeast product in feed additives or food nutrition enhancers.
  • the present invention uses organic starch hydrolysis sugar, organic plant protein enzymatic hydrolyzate and selenium-enriched yeast extract as culture medium, and the selenomethionine form selenium in the obtained organic selenium-enriched yeast accounts for more than 80% of the total selenium weight ratio, even More than 90%, organic selenium accounts for more than 99% of the total selenium weight, the total selenium content is 800-2100ppm, the absorption and utilization rate of yeast to selenium can reach more than 95%, and it has a higher absorption and utilization rate and Conversion rate.
  • the present invention adopts organic starch as the raw material of starch hydrolysis sugar, adopts organic vegetable protein as the raw material of proteolyzate, and the organic raw material consumption exceeds 95%, and does not add inorganic phosphorus source, inorganic nitrogen source and metal trace elements etc. from exogenous source, It can meet the requirements of organic product certification, meet the requirements of organic farming and organic food.
  • the invention provides an organic selenium-enriched yeast, wherein the organic selenium accounts for more than 99% of the total selenium weight, and the selenium in the form of selenomethionine accounts for more than 80% of the total selenium weight.
  • the selenium in the form of selenomethionine accounts for more than 90% of the total selenium weight.
  • the organic selenium-enriched yeast contains 800-2100 mg of total selenium per kg, preferably, the selenium absorption rate of the yeast is above 95%.
  • the above-mentioned organic selenium-enriched yeast is obtained by fermenting and culturing the yeast strain Saccharomyces cerevisiaed FX-2.
  • the above-mentioned organic selenium-enriched yeast is obtained by making the yeast strain into Saccharomyces cerevisiae FX-2 (Saccharomyces cerevisiaed) with organic starch hydrolysis sugar, organic plant protein enzymatic hydrolyzate and selenium-enriched yeast extract as culture medium Obtained by fermentation.
  • the present invention also provides a method for preparing an organic selenium-enriched yeast inoculant, which comprises expanding and culturing yeast strains step by step, fermenting and cultivating, collecting and drying yeast cells, wherein the fermenting and culturing uses organic starch hydrolyzed sugar, organic plant Protein hydrolyzate and selenium-enriched yeast extract are used as culture medium.
  • organic plant protein enzymatic hydrolyzate as nitrogen source and phosphorus source
  • selenium-enriched yeast extract as selenium source
  • vitamin and metal trace element source and also provides a part of nitrogen source and phosphorus source
  • organic plant protein enzymatic hydrolyzate and selenium-enriched yeast extract contain amino acids, which can promote the ability of yeast to absorb and utilize selenium salts.
  • Selenium-enriched yeast extract also contains vitamins and metal trace elements, which can improve yeast nutrition, so that organic selenium accounts for more than 99% of the total selenium weight in the prepared organic selenium-enriched yeast, and selenium in the form of selenomethionine accounts for more than 80% or even more than 90% of the total selenium weight. Containing 800-2100mg of total selenium, the absorption and utilization rate of selenium by yeast can reach more than 95%, the nutrition is balanced and rich, and it is easy to digest and absorb.
  • the pH of the fermentation culture is 5.0-7.0, preferably, the fermentation temperature is 25-35°C.
  • the main reason is to consider that the yeast in this pH range is in a relatively good acidic environment, which promotes the maturation of the yeast and facilitates the end of fermentation; controlling the fermentation temperature within the above range is mainly to consider the growth and reproduction of the yeast, and this temperature range is the best. growth temperature.
  • the organic starch hydrolyzed sugar is obtained by enzymatic hydrolysis of organic starch by amylase and glucoamylase in turn, preferably, the organic starch is organic corn starch, organic wheat starch, organic tapioca starch and one or more of organic rice starches, and more preferably, the amylase is one or more of high-temperature amylase and/or pullulanase.
  • organic starch hydrolyzed sugar is prepared as follows: take organic starch and add water to adjust the slurry, control the Baume degree to be about 15.5-17, adjust the pH to 5.6-5.8; add 1-2 ⁇ (with organic Starch weight) amylase is stirred and mixed, liquefied and sprayed at 105°C-115°C at high temperature; then kept at 95°C for 1 hour, cooled to 60°C, and adjusted to pH 4.2-4.4 to inactivate the enzyme; add 1-2 ⁇ (with organic Starch weight) and glucoamylase were mixed evenly, and after saccharification and heat preservation at 60°C for 15-20 hours, diatomaceous earth was added and the supernatant was collected by plate frame filtration to obtain organic starch hydrolyzed sugar.
  • the total sugar content in the organic starch hydrolyzed sugar is 26-31%.
  • the organic plant protein hydrolyzate is obtained from organic plant protein through proteolysis, preferably, the organic plant protein is organic soybean meal powder, organic corn protein, organic soybean protein and organic One or more than two of rice protein, more preferably, the protease is one or more of papain, bromelain, trypsin, alkaline protease, flavor protease, acid protease and neutral protease, more preferably Further preferably, the protease activity is greater than 5000u/g. During proteolysis, it can be hydrolyzed once, twice or more times. As long as the content of amino acid nitrogen in the final hydrolyzate is >0.3%, it can meet the requirements. The choice of enzymatic hydrolysis depends on the enzyme solution effects and production costs.
  • the organic plant protein enzymatic hydrolyzate is prepared as follows. Weigh the organic plant protein, add water according to the organic plant protein:water ratio of 1:5, stir and mix well, heat up to 53-57°C, and adjust the pH with sodium carbonate To 6.0-6.5, add 1-3 ⁇ (by organic plant protein weight) protease, enzymolysis for 2-5 hours, then add 6-8 ⁇ (by organic plant protein weight) protease, heat up to 50-54 ° C, The pH is 6.0-6.5.
  • Enzyme hydrolysis for 10-15 hours after the end of the enzymolysis, adjust the pH to 4.5-5.0, heat up to 90°C to inactivate the enzyme for 1.5 hours, then cool down to 50°C, add perlite and mix evenly, and collect through plate and frame filtration supernatant to obtain a liquid organic plant protein hydrolyzate.
  • the amino acid nitrogen content in the organic plant protein hydrolyzate is >0.3%.
  • the selenium content in the selenium-enriched yeast extract is greater than 2000ppm, preferably greater than 4000ppm in the selenium-enriched yeast extract based on 100% dry matter weight of the selenium-enriched yeast extract , preferably, the total nitrogen content in the selenium-enriched yeast extract is >6%, further preferably, the amino acid nitrogen content in the selenium-enriched yeast extract is >3.5%.
  • the amount of organic starch in the medium material is 30-40%, the amount of organic vegetable protein is 55-65%, and the amount of selenium-enriched yeast extract is 2.4-4.2%, preferably, the sum of the amount of organic starch and organic vegetable protein is more than 95%.
  • the weight of the medium raw material is the sum of the weight of organic starch, organic vegetable protein, selenium-enriched yeast extract, and protease, amylase and glucoamylase used in enzymatic hydrolysis.
  • the present invention also provides an organic selenium-enriched yeast product, which is prepared from the above-mentioned organic selenium-enriched yeast or the organic selenium-enriched yeast inoculum prepared by the above-mentioned preparation method.
  • the organic selenium-enriched yeast product is organic selenium-enriched yeast hydrolyzate or organic selenium-enriched yeast extract, which is obtained by autolyzing, enzymatically hydrolyzing, separating and purifying organic selenium-enriched yeast.
  • the present invention also provides the application of the above-mentioned organic selenium-enriched yeast or the organic selenium-enriched yeast inoculum prepared by the above-mentioned preparation method or the above-mentioned organic selenium-enriched yeast product in feed additives or food nutrition enhancers.
  • Each index detection method in the present invention is as follows:
  • X 2 organic selenium content, the unit is mg/kg;
  • TRIS buffer solution Accurately weigh 50mg (accurate to 0.1mg) of the sample in a glass container, add 2.5mL of TRIS buffer solution (accurately weigh 18.17g of tris, dissolve in 900mL of water, adjust the pH value to 7.60-7.70 with hydrochloric acid , constant volume 1L), put the glass container in a constant temperature water bath at 90°C and heat it for 10 minutes, take it out and add 2.5mL protease when it is cooled to room temperature, seal the glass container and put it in a water bath shaker, at 37°C, 150 rpm 40-48 hours of enzymatic hydrolysis per minute. Take out and transfer to a 100mL volumetric flask, and make to volume.
  • Liquid chromatography-tandem mass spectrometry detects and calculates the content of selenomethionine, and the weight percent content of selenomethionine form selenium accounting for total selenium is calculated according to the following formula:
  • X 3 the content of selenomethionine, the unit is mg/kg.
  • Table 1 embodiment of the present invention and comparative example use raw material and equipment source
  • the fermentation culture temperature was 25°C, and ventilation was cultivated for 35 hours. After the fermentation was over, 35.4m3 fermented liquid was obtained, which was centrifuged, concentrated and then spray-dried to obtain 0.88 tons of organic selenium-enriched yeast inoculum, with a total selenium content of 835ppm.
  • Selenium accounts for 99.8%
  • selenomethionine form selenium accounts for 90.6% of the total selenium
  • the selenium absorption rate of yeast reaches 96.8%.
  • the supernatant was collected by frame filtration to obtain 21 cubic meters of organic vegetable protein hydrolyzate, with an amino acid nitrogen content of 0.34%.
  • the fermentation culture temperature was 25°C, and ventilation was cultivated for 37 hours. After the fermentation, 33.9m 3 fermentation liquid was obtained, which was centrifuged, concentrated and then spray-dried to obtain 0.82 tons of organic selenium-enriched yeast inoculum. After testing, its total selenium content 1029ppm, organic selenium accounts for 99.7%, selenium in the form of selenomethionine accounts for 89.1% of the total selenium, and the selenium absorption rate of yeast reaches 96.4%.
  • the organic selenium-enriched yeast prepared in Example 2 was formulated into a 10% (yeast dry matter content) solution in the batching tank, the temperature was raised to 80° C., and heat shock was performed for 50 seconds, and the pH was adjusted with citric acid 5; lower the temperature to 50°C, keep warm for 12h, add 2 ⁇ papain for enzymolysis reaction for 6h; adjust pH to 6.8, temperature 60°C, add 3 ⁇ neutral protease for enzymolysis reaction for 6h; then adjust pH to 7, temperature 65°C, add 1 ⁇ alkaline protease for enzymolysis reaction for 10 hours, after the end of the enzymolysis reaction, heat up to 90°C for 1 hour to inactivate the enzyme, concentrate to 35% of dry matter, spray dry, and obtain organic selenium-enriched yeast hydrolyzate product .
  • the total selenium content in the organic selenium-enriched yeast hydrolyzate product is 1882ppm
  • the protein content is 40.2%
  • the acid-soluble protein content is 33%
  • the small peptide content is 22%
  • the cell wall content is 31%.
  • the organic selenium-enriched yeast extract in the batching tank, the organic selenium-enriched yeast prepared in Example 3 was formulated into a solution of 20% (yeast dry matter content), heated to 90°C for 30 minutes, cooled to 55°C, Adjust the pH to 8.0, add 2 ⁇ of alkaline protease, enzymatically hydrolyze for 20 hours, then heat up to 90°C for 1 hour to inactivate the enzyme, centrifuge at 5000rpm to remove the cell wall, collect the supernatant and concentrate it to a dry matter of 37%, then spray dry to obtain Organic selenium-enriched yeast extract product. After testing, the selenium content of the organic selenium-enriched yeast extract product reaches 1288ppm, the total nitrogen content is 10.8%, and the amino acid nitrogen content is 3.3%.
  • the applicant added the organic selenium-enriched yeast obtained in Examples 1-3 and the organic selenium-enriched yeast products obtained in Examples 4-5 to pig feed for animal experiments on fattening pigs.
  • the experiments showed that the feed has the ability to improve the growth of fattening pigs Speed, the trend of reducing the effect of feed-to-meat ratio, and at the same time, it can significantly improve the meat color and reduce drip loss during the storage period of fresh meat after slaughter.
  • the organic selenium-enriched yeast obtained in Examples 1-3 and the organic selenium-enriched yeast products obtained in Examples 4-5 are added to chicken feed respectively, which can improve the production performance of laying hens, increase egg weight and also increase selenium in eggs deposition.
  • the present invention uses organic starch hydrolysis sugar, organic plant protein enzymatic hydrolyzate and selenium-enriched yeast extract as the medium, and the selenium in the form of selenomethionine in the obtained organic selenium-enriched yeast accounts for more than 80% of the total selenium by weight , even more than 90%, organic selenium accounts for more than 99% of the total selenium weight ratio, the total selenium content is 800-2100ppm, and the absorption and utilization rate of yeast to selenium can reach more than 95%, which has a higher absorption and utilization rate and The conversion rate can increase the growth rate of fattening pigs, reduce the trend of feed-to-meat ratio, and can also improve the production performance of laying hens, increase egg weight and increase selenium deposition in eggs.
  • the present invention uses organic starch as raw material for starch hydrolysis sugar, and organic plant protein as raw material for proteolyzate.
  • the amount of organic raw material exceeds 95%, and there is no external source of inorganic phosphorus source, inorganic nitrogen source and metal trace elements, etc., which can meet the requirements of organic
  • the requirements of product certification meet the requirements of organic farming and organic food.

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Abstract

提供了一种有机富硒酵母、其制备方法及制品和应用。该有机富硒酵母通过包括将酵母菌株逐级扩大培养、发酵培养、收集酵母细胞和干燥制得，其中，该发酵培养以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基。该有机富硒酵母中硒代蛋氨酸形式硒占总硒重量比例大于80％，甚至大于90％，有机硒占总硒重量比例大于99％，总硒含量为800-2100ppm，酵母对硒的吸收利用率可达到95％以上，应用于饲养和食品中有较高的吸收利用率和转化率。

Description

一种有机富硒酵母、其制备方法及制品和应用 技术领域
本发明涉及发酵工业技术领域，具体涉及一种有机富硒酵母、其制备方法及制品和应用。
背景技术
硒是人和动物一种生理必须的重要微量元素，主要以硒酶和含硒蛋白质的形式存在于生物体内。已有公开文献报道，硒在生物体内具有抗氧化、调节机体免疫,清除自由基,调控基因表达等功能，硒可以保护心肌、防治克山病、大骨节病、肝病及多种癌症、激活胰岛辅助治疗糖尿病，缺硒与克山病、冠心病、动脉粥样硬化、高血压、心肌病等心血管疾病的发生、发展均有明显的相关性。在动物养殖方面，在母猪日粮中添加有机硒能促进胚胎在怀孕早期阶段的发育，提升母猪和公猪的繁殖性能。
鉴于富硒酵母的健康功能，富硒酵母以被批准用于食品营养强化剂、饲料添加剂等用途。目前富硒酵母生产技术，主要有两种：第一种是螯合法，将一定量的硒酸盐或亚硒酸盐与酵母活细胞乳液在低温螯合，然后干燥制成。第二种是发酵法，将酵母酵母菌种，在糖质或淀粉质或糖蜜为碳源，以氨水、硫酸铵、碳酸铵或尿素为氮源，在添加亚硒酸钠或亚硒酸盐的培养液中，通过酵母生长吸收转化，将无机硒转为酵母硒的方法。
中国专利CN107058208A公开一种富硒酵母粉的生产工艺，包括以下步骤：培养基的制备→斜面菌种培养→一级液体种子培养→二级液体种子培养→发酵罐种子培养→发酵罐发酵培养→分离→干燥→粉碎→包装。该发明制得的富硒酵母粉为高硒含量的富硒酵母产品，其硒含量达到1200mg/kg，其中有机硒含量占总硒的92.8％，硒蛋氨酸形式硒占总硒的比例44.5％。
CN102181371B公开了一种酵母及利用该酵母生产富硒酵母的方法，选用特定酵母菌种，在流加无机硒盐水溶液过程中，每隔5～10小时补加一次含氨基氮的有机氮源；所述无机硒盐水溶液为硒酸盐水溶液、亚硒酸盐水溶液或硒酸盐与亚硒酸盐的混合物的水溶液；所述含氨基氮的有机氮源为蛋白胨、酵母提 取物、植物蛋白、氨基酸中的一种或多种。该发明制得富硒酵母中硒蛋氨酸形式硒占总硒的55-70％，
发明内容
本发明要解决的技术问题：现有技术发酵法所制备富硒酵母中有机硒活性成分硒代蛋氨酸含量较低，硒吸收利用率低，且发酵培养基中含有大量的无机硒、无机氮和无机磷，不符合有机产品认证的要求。
针对现有技术存在的不足，本发明的目的之一提供一种有机富硒酵母；本发明的目的之二是提供上述有机富硒酵母的制备方法；本发明的目的之三是提供一种由上述有机富硒酵母制得的有机富硒酵母制品；本发明的目的之四是提供上述有机富硒酵母或有机富硒酵母制品在饲料添加剂和食品营养强化剂中的应用。
本发明的技术方案：
本发明提供一种有机富硒酵母，其有机硒占总硒重量的99％以上，硒代蛋氨酸形式硒占总硒重量的80％以上。
优选的是，其中，硒代蛋氨酸形式硒占总硒重量的90％以上。
优选的是，其中，每kg有机富硒酵母中含总硒800-2100mg，优选地，酵母对硒的吸收率为95％以上。
优选的是，上述有机富硒酵母通过酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)发酵培养得到。该菌株于2016年8月1日保藏在中国典型培养物保藏中心(CCTCC)，保藏编号为CCTCC NO:M2016418，该菌株在公开号为CN108220175A的专利公开文本中已有记载。
优选的是，上述有机富硒酵母通过将酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基进行发酵培养得到。
本发明提供一种上述有机富硒酵母菌剂的制备方法，包括将酵母菌株逐级扩大培养，发酵培养，收集酵母细胞和干燥制得，其中，所述发酵培养以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基。
优选的是，所述发酵培养时pH＝5.0-7.0，优选地，发酵温度为25-35℃。
优选的是，所述有机淀粉水解糖由有机淀粉依次经淀粉酶和糖化酶酶解制得，优选地，所述有机淀粉为有机玉米淀粉、有机小麦淀粉、有机木薯淀粉和有机大米淀粉中的一种或两种以上，进一步优选地，所述淀粉酶为高温淀粉酶和/或普鲁兰酶。
优选的是，所述淀粉酶酶解时pH为5.6-5.8，优选地，以有机淀粉重量计，所述淀粉酶的用量为1-2‰。
优选的是，所述糖化酶酶解时pH为4.2-4.4，优选地，以有机淀粉重量计，所述糖化酶的用量为1-2‰，进一步优选地，糖化酶酶解时间为15-20小时。
优选的是，以有机淀粉水解糖重量100％计，所述有机淀粉水解糖中总糖含量为26-31％。
优选的是，所述有机植物蛋白酶解液由有机植物蛋白经蛋白酶酶解制得，优选地，所述有机植物蛋白为有机豆粕粉、有机玉米蛋白、有机大豆蛋白和有机大米蛋白中的一种或两种以上，进一步优选地，所述蛋白酶为木瓜蛋白酶、菠萝蛋白酶、胰蛋白酶、碱性蛋白酶、风味蛋白酶、酸性蛋白酶和中性蛋白酶的一种或两种以上，更进一步优选地，所述蛋白酶酶活大于5000u/g。
优选的是，所述蛋白酶酶解包括两次酶解。
优选的是，所述第一次蛋白酶酶解pH为6.0-6.5，优选地，酶解温度为53-57℃，进一步优选地，以有机植物蛋白重量计，蛋白酶的用量为1-3‰，更进一步优选地，酶解时间为2-5小时。
优选的是，所述第二次蛋白酶酶解pH为6.0-6.5，优选地，酶解温度为50-54℃，进一步优选地，以有机植物蛋白重量计，蛋白酶的用量为6-8‰，更进一步优选地，酶解时间为10-15小时。
优选的是，以有机植物蛋白酶解液重量100％计，所述有机植物蛋白酶解液中氨基酸态氮含量>0.3％。
优选的是，以富硒酵母抽提物干物质重量100％计，所述富硒酵母抽提物中硒含量大于2000ppm，优选富硒酵母抽提物中硒含量大于4000ppm，优选地，富硒酵母抽提物中总氮含量>6％,进一步优选地，富硒酵母抽提物中氨基酸态氮含量>3.5％。
优选的是，以培养基原料重量计，所述培养基原料中有机淀粉用量为30-40％、有机植物蛋白用量为55-65％和富硒酵母抽提物用量为2.4-4.2％，优选地，所述有机淀粉和有机植物蛋白用量之和为95％以上。
本发明还提供一种有机富硒酵母制品，其以上述有机富硒酵母或上述制备方法制得有机富硒酵母菌剂为原料制得。其中，有机富硒酵母制品可以为有机富硒酵母水解物或有机富硒酵母抽提物，其通过将有机富硒酵母自溶、酶解、分离和纯化得到。
本发明还提供上述有机富硒酵母或上述制备方法制得有机富硒酵母菌剂或上述有机富硒酵母制品在饲料添加剂或食品营养强化剂中的应用。
本发明的有益效果：
(1)本发明使用有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物作为培养基，制得的有机富硒酵母中硒代蛋氨酸形式硒占总硒重量比例大于80％，甚至大于90％，有机硒占总硒重量比例大于99％，总硒含量为800-2100ppm，酵母对硒的吸收利用率可达到95％以上，应用于饲养和食品中有较高的吸收利用率和转化率。
(2)本发明采用有机淀粉作为淀粉水解糖原料，采用有机植物蛋白作为蛋白酶解物的原料，有机原料用量超过95％，且无外源添加无机磷源、无机氮源和金属微量元素等，可满足有机产品认证的要求，满足有机饲养和有机食品的要求。
具体实施方式
需要说明的是，在不冲突的情况下，本申请中的实施例及实施例中的特征可以相互结合。下面结合实施例来详细说明本发明。
本发明提供一种有机富硒酵母，其有机硒占总硒重量的99％以上，硒代蛋氨酸形式硒占总硒重量的80％以上。
在本发明的一个优选实施方式中，其中，硒代蛋氨酸形式硒占总硒重量的90％以上。
在本发明的一个优选实施方式中，其中，每kg有机富硒酵母中含总硒800-2100mg，优选地，酵母对硒的吸收率为95％以上。
在本发明的一个优选实施方式中，上述有机富硒酵母通过酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)发酵培养得到。
在本发明的一个优选实施方式中，上述有机富硒酵母通过将酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基进行发酵培养得到。
本发明还提供一种有机富硒酵母菌剂的制备方法，包括将酵母菌株逐级扩大培养，发酵培养，收集酵母细胞和干燥制得，其中，所述发酵培养以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基。
通过在发酵培养过程中采用有机淀粉水解糖为碳源，有机植物蛋白酶解液为氮源、磷源，富硒酵母抽提物作为硒源、维生素以及金属微量元素的来源，也提供一部分氮源和磷源，有机植物蛋白酶解液和富硒酵母抽提物中均含有氨基酸，可促进酵母对硒盐的吸收利用能力，富硒酵母抽提物中还含有维生素和金属微量元素，可提高酵母的营养性，使得制备得到的有机富硒酵母中有机硒占总硒重量的99％以上，硒代蛋氨酸形式硒占总硒重量的80％以上，甚至90％以上，每kg有机富硒酵母中含总硒800-2100mg，酵母对硒的吸收利用率可达到95％以上，营养均衡丰富，容易消化吸收。
在本发明的一个优选实施方式中，所述发酵培养时pH＝5.0-7.0，优选地，发酵温度为25-35℃。主要是考虑到此pH范围内酵母处于比较优良的偏酸性环境中，促进酵母成熟，便于发酵结束；将发酵温度控制在上述范围内主要是考虑酵母的生长和繁殖，此温度范围为较佳的生长温度。
在本发明的一个优选实施方式中，所述有机淀粉水解糖由有机淀粉依次经淀粉酶和糖化酶酶解制得，优选地，所述有机淀粉为有机玉米淀粉、有机小麦淀粉、有机木薯淀粉和有机大米淀粉中的一种或两种以上，进一步优选地，所述淀粉酶为高温淀粉酶和/或普鲁兰酶中的一种或两种以上。
具体来说，有机淀粉水解糖按如下方法制得，取有机淀粉加水调浆，控制波美度为15.5-17左右，调节pH为5.6-5.8；调浆完成后加入1-2‰(以有机淀粉重量计)淀粉酶搅拌混匀，105℃-115℃高温液化喷射；然后在95℃保温1小时后冷却降温至60℃，调节pH至4.2-4.4灭酶；加入1-2‰(以有 机淀粉重量计)糖化酶混合均匀，60℃糖化保温15-20小时后，加入硅藻土后通过板框过滤收集上清液，得到有机淀粉水解糖。
在本发明的一个优选实施方式中，以有机淀粉水解糖重量100％计，所述有机淀粉水解糖中总糖含量为26-31％。
在本发明的一个优选实施方式中，所述有机植物蛋白酶解液由有机植物蛋白经蛋白酶酶解制得，优选地，所述有机植物蛋白为有机豆粕粉、有机玉米蛋白、有机大豆蛋白和有机大米蛋白中的一种或两种以上，进一步优选地，所述蛋白酶为木瓜蛋白酶、菠萝蛋白酶、胰蛋白酶、碱性蛋白酶、风味蛋白酶、酸性蛋白酶和中性蛋白酶的一种或两种以上，更进一步优选地，所述蛋白酶酶活大于5000u/g。蛋白酶酶解时可以一次酶解、二次酶解或者更多次酶解，只要最终酶解液中氨基酸态氮含量>0.3％，就可以满足要求，酶解方式的选择取决于不同酶的酶解效果和生产成本。
具体来说，有机植物蛋白酶解液按如下方法制得，称取有机植物蛋白，按照有机植物蛋白：水为1:5加水，充分搅拌混匀，升温到53-57℃，用碳酸钠调节pH到6.0-6.5，加入1-3‰(以有机植物蛋白重量计)蛋白酶，酶解2-5小时，然后加入6-8‰(以有机植物蛋白重量计)蛋白酶，升温至50-54℃，pH在6.0-6.5.酶解10-15小时，酶解结束后将pH调至4.5-5.0，升温至90℃灭酶1.5小时后降温至50℃，加入珍珠岩混合均匀，通过板框过滤收集上清液，得到液体有机植物蛋白酶解液。
在本发明的一个优选实施方式中，以有机植物蛋白酶解液重量100％计，所述有机植物蛋白酶解液中氨基酸态氮含量>0.3％。
在本发明的一个优选实施方式中，以富硒酵母抽提物干物质重量100％计，所述富硒酵母抽提物中硒含量大于2000ppm，优选富硒酵母抽提物中硒含量大于4000ppm，优选地，富硒酵母抽提物中总氮含量>6％,进一步优选地，富硒酵母抽提物中氨基酸态氮含量>3.5％。
在本发明的一个优选实施方式中，以培养基原料重量计，所述培养基原料中有机淀粉用量为30-40％、有机植物蛋白用量为55-65％和富硒酵母抽提物用量为2.4-4.2％，优选地，所述有机淀粉和有机植物蛋白用量之和为95％以 上。具体计算重量时，培养基原料重量为有机淀粉、有机植物蛋白、富硒酵母抽提物以及酶解用到的蛋白酶、淀粉酶和糖化酶重量之和。
本发明还提供一种有机富硒酵母制品，其以上述有机富硒酵母或上述制备方法制得有机富硒酵母菌剂为原料制得。其中，有机富硒酵母制品为有机富硒酵母水解物或有机富硒酵母抽提物，其通过将有机富硒酵母自溶、酶解、分离和纯化得到。
本发明还提供上述有机富硒酵母或上述制备方法制得有机富硒酵母菌剂或上述有机富硒酵母制品在饲料添加剂或食品营养强化剂中的应用。
下面将通过具体的实施例进一步说明本发明的有益效果。
本发明中各项指标检测方法如下：
(1)总氮的测定
采用国家标准GB/T 23530-2009中6.4的凯式定氮法：取样品(相当于总氮30-440mg)，在混合催化剂a(硫酸钾与污水硫酸铜按97:3比例混合)5g和催化剂b(硒粉与硫酸钾按0.1:100比例混合)2.5g作用下，加入20mL浓硫酸进行消煮；再进行蒸馏，用硼酸吸收产物氨；再用0.1mol/L的盐酸滴定，读取数据，计算总氮含量。
(2)氨基酸态氮的测定
采用国家标准GB/T 23530-2009中6.5所述的氨基酸态氮的检测方法：取5g样品，稀释后用0.5mol/L的氢氧化钠溶液滴定至pH为8.2，并保持1min。缓慢加入36％的甲醛溶液10mL，与中性氨基酸中的非解离型氨基反应，生成单羟甲基和二羟甲基诱导体，此反应完全定量进行。此时放出的氢离子用上述氢氧化钠滴定，根据碱液的消耗量，计算出氨基酸态氮的含量。
(3)总硒含量的测定
本发明中总硒含量参见GB5009.93-2017中第一法氢化物原子荧光光谱法。
(4)有机硒含量的测定
称取约1g试样，精确值0.0001g，置于50mL容量瓶中，加入约25mL水，超声溶解10min，使试样充分溶解，加水定容至刻度，摇匀。将定容后的溶液置于5000r/min的离心机中，离心10min，吸取10.0mL离心上清液于15mL 离心管中，加盐酸2.0mL、铁氰化钾溶液1.0mL，混匀待测。同时做空白试验。按照GB5009.93-2017中第一法氢化物原子荧光光谱法测定无机硒含量，然后又(3)得到的总硒含量减去无机硒含量得到有机硒含量，按下式计算出有机硒的百分含量，
ω 1＝X 2/X*100％
ω 1——有机硒百分含量
X 2——有机硒含量，单位为mg/kg；
X——总硒含量，单位为mg/kg。
(5)硒代蛋氨酸含量的测定
精确称取50mg(精确至0.1mg)样品于玻璃容器内，加入2.5mLTRIS缓冲溶液(准确称取18.17g三羟甲基氨基甲烷，用900mL水溶解，用盐酸调节pH值至7.60～7.70之间，定容1L)，将玻璃容器放入90℃的恒温水浴锅中加热10min，取出待冷却至室温时加入2.5mL蛋白酶，将玻璃容器密闭后置于水浴振荡器中，在37℃，150转/分钟酶解40～48小时。取出转移至100mL容量瓶内，并定容。液相色谱-串联质谱法检测并计算硒代蛋氨酸含量，按下式计算硒代蛋氨酸形式硒占总硒的重量百分含量：
ω 2＝0.4026*X 3/X*100％
ω 2——硒代蛋氨酸形式硒占总硒的重量百分含量
X——总硒含量，单位为mg/kg；
X 3——硒代蛋氨酸含量，单位为mg/kg。
(6)酵母对硒的吸收率
酵母对硒的吸收率％＝有机富硒酵母产量*酵母总硒含量/(富硒酵母抽提物用量*富硒酵母抽提物总硒含量)*100％
本发明实施例和对比例使用原料和设备来源见表1。
表1本发明实施例和对比例使用原料和设备来源
使用原料或设备 型号\含量 出售厂家
富硒酵母抽提物 总硒≥2000ppm，食品级 安琪酵母股份有限公司
富硒酵母抽提物 总硒≥4000ppm，食品级 安琪酵母股份有限公司
有机豆粕粉 食品级 黑龙江富锦福慧食品科技有限
     公司
有机玉米淀粉 食品级 保龄宝生物股份有限公司
木瓜蛋白酶 食品级，酶活60万U/g 诺维信生物技术有限公司
风味蛋白酶 食品级，酶活60万U/g 诺维信生物技术有限公司
高温淀粉酶 食品级，酶活20万U/g 诺维信生物技术有限公司
糖化酶 食品级，酶活26万U/g 诺维信生物技术有限公司
普鲁兰酶 食品级，酶活2000U/g 诺维信生物技术有限公司
硅藻土 食品级 临江市亨泰助滤剂有限公司
珍珠岩 食品级 河南信阳市汇通实业有限公司
实施例1
(一)有机淀粉水解糖的制备
称取3000kg有机玉米淀粉，在溶配罐中溶解，加水调浆，控制波美度为17左右，调节pH为5.8；调浆完成后加入6kg高温淀粉酶搅拌混匀，105℃高温液化喷射；在95℃保温1小时后冷却降温至60℃，调节pH至4.2灭酶；然后加入6kg糖化酶混合均匀，60℃糖化保温15小时；加入硅藻土，混合均匀，通过板框过滤，得到有机淀粉水解糖10.2立方米，总糖含量为31.1％。
(二)有机植物蛋白酶解液的制备
称取5000kg有机豆粕粉，加水定容至25立方米，充分搅拌混匀；升温到55℃，用碳酸钠调节pH到6.2，加入15kg木瓜蛋白酶，酶解3小时；然后加入风味蛋白酶40kg，控制温度在52℃，pH在6.0-6.5.酶解12小时；酶解结束后将pH调至4.5，升温至90℃灭酶1.5小时后降温至50℃；加入珍珠岩混合均匀，通过板框过滤收集上清液，得到有机植物蛋白酶解液22立方米，其氨基酸态氮含量为0.38％。
(三)富硒酵母抽提物的制备
称取300kg富硒酵母抽提物(总硒含量为2530ppm)，按照质量比富硒酵母抽提物：水＝1:10溶配，得到富硒酵母抽提物溶液。
(三)有机富硒酵母菌剂的制备
取保存的酿酒酵母FX-2(保藏编号为CCTCC NO:M2016418)于YPD培养基上活化，30℃恒温培养48h，放4℃冰箱保藏备用；将得到的菌种用三角瓶和卡氏瓶扩培到10立方米的种子罐中，将制得的种子通过压差法接入50立方米发酵罐中进行发酵，用碳酸钠和柠檬酸调pH＝5.0，发酵过程中流加步骤(一)制得有机淀粉水解糖，每小时流加量为100-1000L，发酵过程中流加步骤(二)制得有机植物蛋白酶解液，每小时流加量为200-2000L，发酵过程中流加步骤(三)制备的富硒酵母抽提物溶液，每小时流加量为50-250L。在发酵培养温度25℃，通风培养35小时，发酵结束后，得到35.4m 3发酵液，经离心分离，浓缩后喷雾干燥，最终得到有机富硒酵母菌剂0.88吨，其总硒含量835ppm，有机硒占比99.8％，硒代蛋氨酸形式硒占总硒比例为90.6％，酵母对硒吸收率达到96.8％。
实施例2
(一)有机淀粉水解糖的制备
称取2600kg有机玉米淀粉，在溶配罐中溶解，加水调浆，控制波美度为15.5左右，调节pH为5.6；调浆完成后加入2.6kg普鲁兰酶搅拌混匀，115℃高温液化喷射；在95℃保温1小时后冷却降温至60℃，调节pH至4.4灭酶；然后加入2.6kg糖化酶混合均匀，60℃糖化保温20小时；加入硅藻土，混合均匀，通过板框过滤，得到液体有机淀粉水解糖9.7立方米，总糖含量为26.3％。
(二)有机植物蛋白酶解液的制备
称取5400kg有机豆粕粉，加水定容至25立方米，充分搅拌混匀；升温到55℃，用碳酸钠调节pH到6.5，加入5.4kg木瓜蛋白酶，酶解5小时；然后加入风味蛋白酶32.4kg，控制温度在52℃，pH在6.0，酶解15小时；酶解结束后将pH调至5.0，升温至90℃灭酶1.5小时后降温至50℃；加入珍珠岩混合均匀，通过板框过滤收集上清液，得到液体有机植物蛋白酶解液20立方米，其氨基酸态氮含量0.31％。
(三)富硒酵母抽提物的制备
称取350kg富硒酵母抽提物(总硒含量为4376ppm)，按照质量比富硒酵母抽提物：水＝1:10溶配，得到富硒酵母抽提物溶液。
(四)有机富硒酵母菌剂的制备
取保存的酿酒酵母FX-2(保藏编号为CCTCC NO:M2016418)于YPD培养基上活化，30℃恒温培养48h，放4℃冰箱保藏备用；将得到的菌种用三角瓶和卡氏瓶扩培到10立方米的种子罐中，将制得的种子通过压差法接入50立方米发酵罐中进行发酵，用碳酸钠和柠檬酸调pH＝5.0，发酵过程中流加步骤(一)制得有机淀粉水解糖，每小时流加量为100-1000L，发酵过程中流加步骤(二)制得有机植物蛋白酶解液，每小时流加量为200-2000L，发酵过程中流加步骤(三)制得富硒酵母抽提物溶液，每小时流加量为50-250L。在发酵培养温度25℃，通风培养40小时，发酵结束后，得到33.3m 3发酵液，经离心分离，浓缩后喷雾干燥，最终得到有机富硒酵母菌剂0.73吨，其总硒含量2011ppm，有机硒占比99.5％，硒代蛋氨酸形式硒占总硒比例为86.6％，酵母对硒吸收率达到95.8％。
实施例3
(一)有机淀粉水解糖的制备
称取3300kg有机玉米淀粉，在溶配罐中溶解，加水调浆，控制波美度为17左右，调节pH为5.8；调浆完成后加入6.6kg高温淀粉酶搅拌混匀，105℃高温液化喷射；在95℃保温1小时后冷却降温至60℃，调节pH至4.2灭酶；然后加入6.6kg糖化酶混合均匀，60℃糖化保温15小时；加入硅藻土，混合均匀，通过板框过滤，得到有机淀粉水解糖10.8立方米，总糖含量为30.7％。
(二)有机植物蛋白酶解液的制备
称取4700kg有机豆粕粉，加水定容至25立方米，充分搅拌混匀；升温到55℃，用碳酸钠调节pH到6.2，加入9.4kg木瓜蛋白酶，酶解3小时；然后加入风味蛋白酶23.5kg，控制温度在52℃，pH在6.0-6.5.酶解12小时；酶解结束后将pH调至4.5，升温至90℃灭酶1.5小时后降温至50℃；加入珍珠岩混合均匀，通过板框过滤收集上清液，得到有机植物蛋白酶解液21立方米，其氨基酸态氮含量0.34％。
(三)富硒酵母抽提物的制备
称取200kg富硒酵母抽提物(总硒含量为4376ppm)，按照质量比富硒酵母抽提物：水＝1:10溶配，得到富硒酵母抽提物溶液。
(四)有机富硒酵母菌剂的制备
取保存的酿酒酵母FX-2(保藏编号为CCTCC NO:M2016418)于YPD培养基上活化，30℃恒温培养48h，放4℃冰箱保藏备用；将得到的菌种用三角瓶和卡氏瓶扩培到10立方米的种子罐中，将制得的种子通过压差法接入50立方米发酵罐中进行发酵，用碳酸钠和柠檬酸调pH＝5.0，发酵过程中流加步骤(一)制得有机淀粉水解糖，每小时流加量为100-1000L，发酵过程中流加步骤(二)制得有机植物蛋白酶解液，每小时流加量为200-2000L，发酵过程中流加步骤(三)制得富硒酵母抽提物溶液，每小时流加量为50-250L。在发酵培养温度25℃，通风培养37小时，发酵结束后，得到33.9m 3发酵液，经离心分离，浓缩后喷雾干燥，最终得到有机富硒酵母菌剂0.82吨，经检测，其总硒含量1029ppm，有机硒占比99.7％，硒代蛋氨酸形式硒占总硒比例89.1％，酵母对硒吸收率达到96.4％。
实施例4
有机富硒酵母水解物制备：在配料罐中将实施例2制得的有机富硒酵母配制成10％(酵母干物质含量)的溶液，升温到80℃进行热击50s，用柠檬酸调节pH为5；降温至50℃，保温12h，加入2‰木瓜蛋白酶进行酶解反应6h；调节pH为6.8，温度60℃，加入3‰中性蛋白酶进行酶解反应6h；再调节pH为7，温度65℃，加入1‰碱性蛋白酶进行酶解反应10h，酶解反应结束后，升温到90℃保温1h灭酶，并浓缩到干物质35％后，喷雾干燥，获得有机富硒酵母水解物产品。经检测，有机富硒酵母水解物产品中总硒含量1882ppm，蛋白质含量40.2％，酸溶蛋白含量33％，小肽含量22％，细胞壁含量31％。
实施例5
有机富硒酵母抽提物制备：在配料罐中将实施例3制得的有机富硒酵母配制成20％(酵母干物质含量)的溶液，升温至90℃反应30分钟，降温至55℃，调节pH为8.0，加入2‰的碱性蛋白酶，酶解20小时，然后升温至90℃保温 1h灭酶，5000rpm离心分离去除细胞壁，收集上清液浓缩至干物质37％后进行喷雾干燥，得到有机富硒酵母抽提物产品。经检测，有机富硒酵母抽提物产品硒含量达到1288ppm，总氮含量10.8％，氨基酸态氮含量3.3％。
另外，申请人将实施例1-3得到的有机富硒酵母和实施例4-5得到的有机富硒酵母制品分别添加到猪饲料中进行育肥猪动物实验，实验表明该饲料具有提高育肥猪生长速度，降低料肉比的功效的趋势，同时可以明显改善屠宰后鲜肉放置期内的肉色和降低滴水损失。将实施例1-3得到的有机富硒酵母和实施例4-5得到的有机富硒酵母制品分别添加到鸡饲料中，可以提高蛋鸡的生产性能，增加蛋重的同时也增加鸡蛋中硒的沉积。
综上所述，本发明使用有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物作为培养基，制得的有机富硒酵母中硒代蛋氨酸形式硒占总硒重量比例大于80％，甚至大于90％，有机硒占总硒重量比例大于99％，总硒含量为800-2100ppm，酵母对硒的吸收利用率可达到95％以上，应用于饲养中有较高的吸收利用率和转化率，可提高育肥猪生长速度，降低料肉比功效的趋势，也可提高蛋鸡的生产性能，增加蛋重的同时也增加鸡蛋中硒的沉积。本发明采用有机淀粉作为淀粉水解糖原料，采用有机植物蛋白作为蛋白酶解物的原料，有机原料用量超过95％，且无外源添加无机磷源、无机氮源和金属微量元素等，可满足有机产品认证的要求，满足有机饲养和有机食品的要求。
以上所述，仅是本发明实施的较佳实施例，并非对本发明做任何形式上的限制，凡在本发明的精神和原则之内所做的修改、等同替换和改进等，均需要包含在本发明的保护范围之内。

Claims (20)

  1. 一种有机富硒酵母，其特征在于，有机硒占总硒重量的99％以上，硒代蛋氨酸形式硒占总硒重量的80％以上。
  2. 根据权利要求1所述的有机富硒酵母，其中，硒代蛋氨酸形式硒占总硒重量的90％以上。
  3. 根据权利要求1或2所述的有机富硒酵母，其中，每kg有机富硒酵母中含总硒800-2100mg，优选地，酵母对硒吸收率95％以上。
  4. 根据权利要求1-3任一项所述的有机富硒酵母，其通过酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)发酵培养得到。
  5. 根据权利要求1-4任一项所述的有机富硒酵母，其通过将酵母菌株为酿酒酵母FX-2(Saccharomyces cerevisiaed)以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基进行发酵培养得到。
  6. 权利要求1-5任一项所述的有机富硒酵母菌剂的制备方法，其特征在于，包括将酵母菌株逐级扩大培养，发酵培养，收集酵母细胞和干燥制得，其中，所述发酵培养以有机淀粉水解糖、有机植物蛋白酶解液和富硒酵母抽提物为培养基。
  7. 根据权利要求6所述制备方法，其特征在于，所述发酵培养时pH＝5.0-7.0，优选地，发酵温度为25-35℃。
  8. 根据权利要求6或7所述制备方法，其特征在于，所述有机淀粉水解糖由有机淀粉依次经淀粉酶和糖化酶酶解制得，优选地，所述有机淀粉为有机玉米淀粉、有机小麦淀粉、有机木薯淀粉和有机大米淀粉中的一种或两种以上，进一步优选地，所述淀粉酶为高温淀粉酶和/或普鲁兰酶。
  9. 根据权利要求8所述制备方法，其特征在于，所述淀粉酶酶解时pH为5.6-5.8，优选地，以有机淀粉重量计，所述淀粉酶的用量为1-2‰。
  10. 根据权利要求8或9所述制备方法，其特征在于，所述糖化酶酶解时pH为4.2-4.4，优选地，以有机淀粉重量计，所述糖化酶的用量为1-2‰，进一步优选地，糖化酶酶解时间为15-20小时。
  11. 根据权利要求6-10任一项所述制备方法，其特征在于，以有机淀粉水解糖重量100％计，所述有机淀粉水解糖中总糖含量为26-31％。
  12. 根据权利要求6-11任一项所述制备方法，其特征在于，所述有机植物蛋白酶解液由有机植物蛋白经蛋白酶酶解制得，优选地，所述有机植物蛋白为有机豆粕粉、有机玉米蛋白、有机大豆蛋白和有机大米蛋白中的一种或两种以上，进一步优选地，所述蛋白酶为木瓜蛋白酶、菠萝蛋白酶、胰蛋白酶、碱性蛋白酶、风味蛋白酶、酸性蛋白酶和中性蛋白酶的一种或两种以上，更进一步优选地，所述蛋白酶酶活大于5000u/g。
  13. 根据权利要求12所述制备方法，其特征在于，所述蛋白酶酶解包括两次酶解。
  14. 根据权利要求13所述制备方法，其特征在于，所述第一次蛋白酶酶解pH为6.0-6.5，优选地，酶解温度为53-57℃，进一步优选地，以有机植物蛋白重量计，蛋白酶的用量为1-3‰，更进一步优选地，酶解时间为2-5小时。
  15. 根据权利要求13或14所述制备方法，其特征在于，所述第二次蛋白酶酶解pH为6.0-6.5，优选地，酶解温度为50-54℃，进一步优选地，以有机植物蛋白重量计，蛋白酶的用量为6-8‰，更进一步优选地，酶解时间为10-15小时。
  16. 根据权利要求6-15任一项所述制备方法，其特征在于，以有机植物蛋白酶解液重量100％计，所述有机植物蛋白酶解液中氨基酸态氮含量>0.3％。
  17. 根据权利要求6-16任一项所述制备方法，其特征在于，以富硒酵母抽提物干物质重量100％计，所述富硒酵母抽提物中硒含量大于2000ppm，优选富硒酵母抽提物中硒含量大于4000ppm，优选地，富硒酵母抽提物中总氮含量>6％,进一步优选地，富硒酵母抽提物中氨基酸态氮含量>3.5％。
  18. 根据权利要求6-17任一项所述制备方法，其特征在于，以培养基原料重量计，所述培养基原料中有机淀粉用量为30-40％、有机植物蛋白用量为55-65％和富硒酵母抽提物用量为2.4-4.2％，优选地，所述有机淀粉和有机植物蛋白用量之和为95％以上。
  19. 一种有机富硒酵母制品，其特征在于，其以权利要求1-5任一项所述有机富硒酵母或权利要求6-18任一项所述制备方法制得有机富硒酵母菌剂为原料制得。
  20. 权利要求1-5任一项所述有机富硒酵母或权利要求6-18任一项所述制备方法制得有机富硒酵母菌剂或权利要求19所述有机富硒酵母制品在饲料添加剂或食品营养强化剂中的应用。
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