Preparation method and application of copper-enriched yeast

Abstract

The invention provides a preparation method and application of copper-enriched yeast. The preparation method comprises the following steps: carrying out copper-rich fermentation by utilizing saccharomyces cerevisiae with a known preservation number of CCTCC NO: M 2017781, so as to obtain the copper-rich yeast. The saccharomyces cerevisiae (the preservation number is CCTCC NO: M 2017781) in the application is used for absorbing inorganic copper in the copper-containing solution and converting the inorganic copper into organic copper. By utilizing the method, a large amount of yeast with higher copper content can be obtained under the condition of shorter preparation time, the cost is low, and the method is more beneficial to industrial production.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular, to a method for preparing copper-rich yeast and its application. Background Art

[0002] Copper is an essential trace element for animals and plants. On the one hand, as a cofactor of various enzymes, copper participates in hematopoiesis by affecting the absorption and metabolism of iron ions, enhancing the body's hematopoietic function; promotes the development of blood vessels and bones by activating lysyl oxidase and monoamine oxidase, etc., and maintains normal blood vessel elasticity and bone strength; promotes the formation of melanin by activating tyrosinase and maintains the health and brightness of hair. On the other hand, as an antioxidant factor, copper has an anti-stress function, can effectively scavenge free radicals, protect cells from oxidative stress damage, and plays an important role in the prevention and treatment of oxidative stress damage. In addition, copper also participates in regulating the body's cellular immunity and humoral immunity, enhancing the immune function.

[0003] Since copper is widely present in foods, there are few cases of copper deficiency in normal diet populations, but copper deficiency still exists in some special regions or some special populations. In animal feeding, the lack of copper element in the diet often affects the slaughter speed of animals. Currently, the commonly used copper element supplements are inorganic copper salts such as copper sulfate and / or copper chloride, which have large toxic and side effects and are prone to cause diarrhea. Moreover, the absorption and utilization rate of such inorganic copper by animals is low, and large doses are taken to achieve the physiological effect, causing serious pollution to the environment.

[0004] Yeast can convert inorganic copper into biogenic copper with higher biocompatibility and absorption utilization rate through biological absorption and biological transformation, thus breaking through the limitations of inorganic copper in practical applications, and can achieve low-dose, high-absorption and low-emission on the basis of meeting physiological needs; at the same time, combined with the rich and balanced nutrition of yeast itself, the synergism and compatibility with other nutritional components are improved. However, the existing conversion efficiency of organic copper using yeast is low, and the copper content in unit yeast is low, which may be related to the yeast fermentation process and the types of yeast used. Therefore, providing a yeast fermentation method that can efficiently convert organic copper is very important for the production of copper supplement products. Summary of the Invention

[0005] The main object of the present invention is to provide a method for preparing copper-rich yeast and its application, so as to solve the problem of low content of organic copper converted by yeast in the prior art.

[0006] To achieve the above object, according to the first aspect of the present invention, a method for preparing copper-rich yeast is provided, and the preparation method includes: performing copper-rich fermentation using the known Saccharomyces cerevisiae with the preservation number of CCTCC NO: M 2017781 to obtain copper-rich yeast.

[0007] Furthermore, the preparation method includes: performing batch seed culture on the activated Saccharomyces cerevisiae to obtain a seed culture milk; and performing copper-rich fermentation culture on the seed culture milk to obtain copper-rich yeast.

[0008] Furthermore, the batch seed culture includes: performing primary seed culture on the activated Saccharomyces cerevisiae to obtain a primary seed culture solution; inoculating the primary seed culture solution into a fermentation nutrient solution to perform secondary seed culture to obtain a secondary seed culture solution; and inoculating the secondary seed culture solution into the fermentation nutrient solution to perform multiple tertiary seed cultures to obtain a seed culture milk.

[0009] Furthermore, the primary seed culture includes: inoculating a single colony of the activated Saccharomyces cerevisiae into a seed medium to perform first-stage shake flask culture to obtain a seed solution; inoculating the seed solution into the seed medium to perform second-stage shake flask culture to obtain a primary seed culture solution; preferably, by mass fraction, the seed medium includes: 10-13% glucose, 2-4% yeast extract, 0.1-0.2% KH2PO4, and 0.1-0.2% MgSO4; preferably, the pH value of the seed medium is 5-6; preferably, the volume of the seed medium accounts for 40-60% of the container volume of the first-stage shake flask culture and the second-stage shake flask culture; preferably, the container volume of the first-stage shake flask culture is 500-750 mL; preferably, the container volume of the second-stage shake flask culture is 5-10 L; preferably, by volume fraction, the inoculation amount of the seed solution is 1-5%; preferably, the temperature of the first-stage shake flask culture and the second-stage shake flask culture is 30-32 °C; preferably, the rotation speed of the first-stage shake flask culture and the second-stage shake flask culture is 200-220 rpm; preferably, the time of the first-stage shake flask culture and the second-stage shake flask culture is 17-20 h.

[0010] Further, the secondary seed culture includes: the fermentation nutrient solution is mixed with the primary seed culture solution in a way of feeding while culturing to conduct the secondary seed culture, and a secondary seed culture solution is obtained; preferably, the fermentation nutrient solution includes: a carbon source; when the carbon source is molasses, the fermentation nutrient solution further includes: a nitrogen source and phosphate; when the carbon source is one or more of glucose, fructose or sucrose, the fermentation nutrient solution further includes: a nitrogen source, phosphate, magnesium salt, potassium salt, calcium salt, sodium salt, trace elements and vitamins; preferably, the nitrogen source is selected from any one or more of the following substances: yeast extract, peptone, corn steep liquor, soy protein, ammonia water, ammonium sulfate, ammonium phosphate and ammonium dihydrogen phosphate; preferably, the trace elements are selected from any one or more of the following: zinc salt, copper salt, nickel salt, iron salt, manganese salt, borate, iodide and nickel salt; preferably, the trace elements are copper, iron and zinc; preferably, the volume of the fermentation nutrient solution accounts for 40-60% of the volume of the container for the secondary seed culture; preferably, the volume of the container for the secondary seed culture is 10-15L; preferably, the inoculation amount of the primary seed culture solution, based on the wet weight of the initial yeast in the culture, is 10-90g / L, preferably 40-70g / L; preferably, the temperature of the secondary seed culture is 30-32°C; preferably, the rotation speed of the secondary seed culture is 200-220rpm; preferably, when the wet weight of the yeast in the secondary seed culture solution is greater than 150g / L, the secondary seed culture is stopped and centrifugal concentration is carried out to obtain the secondary seed culture solution.

[0011] Further, the tertiary seed culture includes: the fermentation nutrient solution is mixed with the secondary seed culture solution in a way of feeding while culturing to conduct the tertiary seed culture, and a seed culture milk is obtained; preferably, multiple tertiary seed cultures are carried out, specifically two tertiary seed cultures; preferably, the volume of the fermentation nutrient solution accounts for 60-70% of the volume of the container for the tertiary seed culture; preferably, the volume of the container for the tertiary seed culture is 200-360L; preferably, the inoculation amount of the tertiary seed culture solution, based on the wet weight of the initial yeast in the culture, is 10-90g / L, preferably 40-70g / L; preferably, the temperature of the tertiary seed culture is 30-32°C; preferably, the rotation speed of the tertiary seed culture is 200-700rpm; preferably, when the wet weight of the yeast in the tertiary seed culture solution is greater than 150g / L, the tertiary seed culture is stopped and centrifugal concentration is carried out.

[0012] Further, the copper-rich fermentation culture includes: the fermentation nutrient solution is mixed with the seed culture milk in a way of feeding while culturing or batch supplementing. At 600 min - 960 min of the copper-rich fermentation culture, ventilation and the addition of the fermentation nutrient solution are stopped, and a copper-containing solution is added to the solution of the copper-rich fermentation culture; continue the copper-rich fermentation culture to obtain copper-rich yeast; preferably, the ventilation volume is 150-200m 3 / h.

[0013] Furthermore, the inoculation amount of the seed culture milk is 40-70 g / L based on the wet weight of the initial yeast culture; preferably, the volume of the copper-rich fermentation nutrient solution accounts for 60-70% of the volume of the fermentation culture container; preferably, the volume of the copper-rich fermentation culture container is 10-15 m3; preferably, the copper-containing solution is added until the copper concentration in the copper-rich fermentation culture solution is 500-600 mg / kg; preferably, before adding the copper-containing solution, the pH value of the copper-rich fermentation culture solution is adjusted to 4.5-5.5; preferably, the temperature of the copper-rich fermentation culture is 28-35°C; preferably, the rotation speed of the copper-rich fermentation culture is 200-700 rpm; preferably, continuing the copper-rich fermentation culture to obtain the copper-rich yeast comprises: when the volume of the culture solution for the copper-rich fermentation culture is 7.0-7.2 m 3 When the copper-rich fermentation culture is stopped, centrifugal concentration is performed to obtain yeast milk; the yeast milk is dried to obtain copper-rich yeast.

[0014] In order to achieve the above object, according to a second aspect of the present invention, a copper-rich yeast is provided. The copper-rich yeast is obtained by the above preparation method, and the copper content in the copper-rich yeast is 10000-15000 mg / kg.

[0015] In order to achieve the above object, according to the third aspect of the present invention, there is provided a copper-rich yeast obtained by the above preparation method or the use of the above copper-rich yeast in the fields of food, health products, medicine, environmental protection and feed.

[0016] By applying the technical solution of the present invention, the inorganic copper in the copper-containing solution can be absorbed and converted into organic copper by using Saccharomyces cerevisiae (the deposit number is CCTCC NO: M 2017781). By using the above method, a large amount of yeast with high copper content can be prepared in a short time, which is low in cost and more conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0018] Figure 1 A schematic diagram of the method for preparing copper-rich yeast of the present invention is shown. DETAILED DESCRIPTION

[0019] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0020] As mentioned in the background art, currently, inorganic copper salts such as copper sulfate and / or copper chloride are often used as copper element supplements. They have large toxic and side effects and are prone to cause diarrhea. Moreover, animals have low absorption and utilization rates of such inorganic copper. To achieve the physiological effect, large doses are taken, which causes serious environmental pollution. Yeasts can convert inorganic copper into biogenic copper with higher biocompatibility and absorption and utilization rates. However, due to certain defects in the yeast species and production technologies used in the prior art, the copper content of the copper-rich yeast obtained is low, and the production time is long and the cost is high, which is not conducive to industrial production. To improve this situation, the inventors of the present application discovered a yeast strain capable of achieving high-efficiency copper enrichment from numerous existing fermentation strains, and then used this yeast strain for copper enrichment fermentation to obtain copper-rich yeast and its derivative copper-rich products, which can be used as copper element supplements. Based on this research, the applicant proposed a series of technical solutions of the present application.

[0021] In the first typical embodiment of the present application, a method for preparing copper-rich yeast is provided. The preparation method includes: performing copper enrichment fermentation using the known Saccharomyces cerevisiae with the preservation number CCTCC NO: M 2017781 to obtain copper-rich yeast. Among them, Saccharomyces cerevisiae is classified and named as Saccharomyces cerevisiae, and it was preserved in the China Center for Type Culture Collection on December 11, 2017. The preservation address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the preservation number is CCTCC NO: M 2017781.

[0022] In order to obtain a large amount of yeast with a high copper content under the condition of a relatively short preparation time, in a preferred embodiment, the preparation method includes: performing batch seed culture on the activated Saccharomyces cerevisiae to obtain a seed culture milk; performing copper enrichment fermentation culture on the seed culture milk to obtain copper-rich yeast.

[0023] Using the above-mentioned Saccharomyces cerevisiae for batch seed culture to gradually expand the culture system. First, the Saccharomyces cerevisiae is multiplied and activated in large quantities, and then the Saccharomyces cerevisiae is used to absorb and convert the inorganic copper in the copper-containing solution into organic copper. This preparation process has a low cost and is more conducive to industrial production.

[0024] Gradually increasing the dosage of the culture system can produce a large amount of highly active Saccharomyces cerevisiae for subsequent conversion of inorganic copper within an appropriate raw material consumption. In a preferred embodiment, the batch seed culture includes: performing primary seed culture on the activated Saccharomyces cerevisiae to obtain a primary seed culture solution; inoculating the primary seed culture solution into a fermentation nutrient solution for secondary seed culture to obtain a secondary seed culture solution; inoculating the secondary seed culture solution into a fermentation nutrient solution for multiple tertiary seed cultures to obtain a seed culture milk. Performing tertiary seed culture can efficiently obtain a large amount of highly active Saccharomyces cerevisiae for subsequent production of copper-rich yeast.

[0025] In this application, the activated strain is first subjected to primary seed culture to obtain a bacterial solution with a certain quantity and strong growth to efficiently complete subsequent scale-up culture. In a preferred embodiment, the primary seed culture includes: inoculating a single colony of the activated Saccharomyces cerevisiae into a seed medium for first-stage shake flask culture to obtain a seed solution; inoculating the seed solution into a seed medium for second-stage shake flask culture to obtain a primary seed culture solution.

[0026] Any seed culture that can provide nutrients for the growth and reproduction of yeast is applicable to this application. In a preferred embodiment, by mass fraction, the seed medium includes: 10 - 13% glucose, 2 - 4% yeast extract, 0.1 - 0.2% KH2PO4, and 0.1 - 0.2% MgSO4; the remaining component is water.

[0027] During the seed culture process, to provide a sufficient nutrient environment for the strain without causing waste of materials, in a preferred embodiment, the volume of the seed medium accounts for 40 - 60% of the container volume of the first-stage shake flask culture and the second-stage shake flask culture; preferably, the container volume of the first-stage shake flask culture is 500 - 750 mL; preferably, the container volume of the second-stage shake flask culture is 5 - 10 L. Any seed culture method that can obtain a bacterial solution with strong growth is applicable to this application. In a preferred embodiment, by volume fraction, the inoculation amount of the seed solution is 1 - 5%; preferably, the temperature of the first-stage shake flask culture and the second-stage shake flask culture is 30 - 32 °C; preferably, the rotation speed of the first-stage shake flask culture and the second-stage shake flask culture is 200 - 220 rpm; preferably, the time of the first-stage shake flask culture and the second-stage shake flask culture is 17 - 20 h.

[0028] Any method capable of completing the expansion culture and reproduction of yeast is applicable to this application, and it can activate yeast in a larger culture system (10 - 15 L). In a preferred embodiment, the secondary seed culture includes: mixing the fermentation nutrient solution with the primary seed culture solution in a way of feeding while culturing to conduct the secondary seed culture and obtain the secondary seed culture solution. Providing growth substances for yeast in a batch or continuous feeding manner with the fermentation nutrient solution can timely and sufficiently provide the required nutrients for yeast and improve the growth efficiency of yeast.

[0029] Any component of the fermentation nutrient solution capable of providing nutrients for the growth and reproduction of yeast is applicable to this application. In a preferred embodiment, the fermentation nutrient solution includes: a carbon source; when the carbon source is molasses, the fermentation nutrient solution further includes: a nitrogen source and phosphate. Among them, molasses is the solution after sugarcane or beet is used for sugar refining in a sugar refinery, rich in sucrose, various metal ions and vitamins, including various metal ions such as magnesium salts, potassium salts, calcium salts, sodium salts and trace elements (such as zinc salts, copper salts, nickel salts, iron salts, manganese salts, boron salts, iodine salts, etc.). When the carbon source is one or more of glucose, fructose or sucrose, the fermentation nutrient solution further includes: a nitrogen source, phosphate, magnesium salts, potassium salts, calcium salts, sodium salts, trace elements and vitamins; preferably, the nitrogen source is selected from any one or more of the following substances: yeast extract, peptone, corn steep liquor, soy protein, ammonia water, ammonium sulfate, ammonium phosphate and ammonium dihydrogen phosphate.

[0030] Any vitamins, phosphates, magnesium salts, potassium salts, calcium salts, sodium salts, and trace elements that can meet the growth requirements provided by yeast are applicable to this application. In a preferred embodiment, preferably, the vitamins are selected from any one or more of the following substances: biotin, VB1 and / or its salts, VB2 and / or its salts, VB3 and / or its salts, VB4 and / or its salts, VB5 and / or its salts, VB6 and / or its salts, VC and / or its salts, nicotinic acid and / or its salts; preferably, the trace elements are selected from any one or more of the following: zinc salts, copper salts, nickel salts, iron salts, manganese salts, boron salts, iodine salts, and nickel salts; preferably, the trace elements are copper, iron, and zinc; preferably, the phosphates are selected from any one or more of the following substances: KH2PO4, NH4H2PO4, NaH2PO4, NaH2PO4*2H2O, K2HPO4, (NH4)2HPO4, Na2HPO4, Na2HPO4*12H2O, H3PO4, and more preferably NH4H2PO4; preferably, the magnesium salts are selected from any one or more of the following substances: MgSO4, MgCl2, magnesium glycinate, magnesium threonate, magnesium citrate, magnesium glycinate, magnesium orotate, and more preferably MgSO4, MgCl2; preferably, the zinc salts are selected from any one or more of the following substances: ZnSO4, ZnCl2, zinc gluconate, zinc citrate, zinc acetate, zinc orotate, and more preferably ZnSO4; preferably, the copper salts are selected from any one or more of the following substances: CuSO4, CuCl2, copper acetate, copper nitrate; preferably, the potassium salts are selected from any one or more of the following substances: K2HPO4, KH2PO4, K2SO4, KHSO4, KNO3, KNO2, KCl, and more preferably KCl; preferably, the calcium salts are selected from any one or more of the following substances: CaCl2, calcium gluconate, calcium hydrogen phosphate, and calcium lactate, and more preferably CaCl2; preferably, the sodium salts are selected from any one or more of the following substances: NaCl, NaNO3, Na2SO4, and more preferably NaCl. The above substances have low costs and can be better absorbed and utilized by yeast, improving the growth efficiency of yeast.

[0031] During the seed culture process, to provide sufficient nutritional environment for the strains without causing waste of materials, in a preferred embodiment, the volume of the fermentation nutrient solution accounts for 40-60% of the volume of the container for secondary seed culture; preferably, the volume of the container for secondary seed culture is 10-15L. Preferably, the inoculation amount of the primary seed culture solution, based on the wet weight of the initial yeast in the culture, is 10-90g / L, preferably 40-70g / L; preferably, the temperature for secondary seed culture is 30-32°C; preferably, the rotation speed for secondary seed culture is 200-220rpm; preferably, when the wet weight of the yeast in the secondary seed culture solution is greater than 150g / L, stop the secondary seed culture and perform centrifugal concentration to obtain the secondary seed culture solution. The wet weight of the yeast in the seed solution after centrifugal concentration increases, which is convenient for adjusting the addition amount in the subsequent fermentation culture.

[0032] Any method capable of completing the expansion culture and reproduction of yeast is applicable to this application and can activate yeast in a larger culture system (200-360L). In a preferred embodiment, the tertiary seed culture includes: the fermentation nutrient solution is mixed with the secondary seed culture solution in a way of feeding while culturing to perform tertiary seed culture to obtain the seed culture milk. In a preferred embodiment, performing multiple tertiary seed cultures means performing two tertiary seed cultures. The number of tertiary seed cultures can be adjusted according to the initial growth activity of the yeast required for fermentation culture. Providing growth substances for yeast in the form of batch feeding or continuous feeding of the fermentation nutrient solution can timely and sufficiently provide the required nutrients for yeast and improve the growth efficiency of yeast.

[0033] Any seed culture method capable of obtaining a vigorously growing bacterial solution is applicable to this application, and during the seed culture process, to provide sufficient nutritional environment for the strains without causing waste of materials, in a preferred embodiment, the volume of the fermentation nutrient solution accounts for 60-70% of the volume of the container for tertiary seed culture; preferably, the volume of the container for tertiary seed culture is 200-360L; preferably, the inoculation amount of the secondary seed culture solution, based on the wet weight of the initial yeast in the culture, is 10-90g / L, preferably 40-70g / L; preferably, the temperature for tertiary seed culture is 30-32°C; preferably, the rotation speed for tertiary seed culture is 200-700rpm; preferably, when the wet weight of the yeast in the tertiary seed culture solution is greater than 150g / L, stop the tertiary seed culture and perform centrifugal concentration. The wet weight of the yeast in the seed solution after centrifugal concentration increases, which is convenient for adjusting the addition amount in the subsequent fermentation culture.

[0034] The yeast-containing seed culture solution after the expanded activation culture is fermented and cultured, so that the yeast therein converts inorganic copper into organic copper using the copper-containing raw material. In order to increase the growth rate of yeast and the copper content of yeast per unit volume, shorten the production time for preparing copper-rich yeast, and improve production efficiency, in a preferred embodiment, the copper-rich fermentation culture includes: the fermentation nutrient solution is mixed with the seed culture milk in a manner of simultaneous addition or batch supplementation during the culture, and at 600min-960min of the copper-rich fermentation culture, the ventilation and the addition of the fermentation nutrient solution are stopped, and the copper-containing solution is added to the copper-rich fermentation culture solution; the copper-rich fermentation culture is continued to obtain copper-rich yeast; preferably, the ventilation volume is 150-200m 3 / h.

[0035] Among them, in order to continue to promote yeast growth and reproduction in the early stage of copper-rich fermentation, the copper-containing solution is added to the reaction system in the late stage of fermentation to avoid the toxicity of copper ions in the fermentation system to yeast caused by long-term presence, and the fermentation culture solution is added in batches to avoid the toxicity to yeast caused by the conversion of part of the carbon source into alcohol and unnecessary waste of raw materials after the yeast cannot use all the culture solution at one time. In addition, ventilation is stopped while the copper-containing solution is added to avoid ventilation blowing away the copper ions in the copper-containing solution, which can make the copper ions in the copper-containing solution better mixed with the culture solution and improve the absorption and conversion of copper by yeast.

[0036] In order to avoid overflowing due to excessive volume of culture medium during fermentation and to provide sufficient nutrients for the fermentation process, in a preferred embodiment, the volume of the copper-rich fermentation nutrient solution accounts for 60-70% of the volume of the fermentation culture container; preferably, the volume of the copper-rich fermentation culture container is 10-15m 3 .

[0037] In consideration of efficient yeast culture and production cost, in a preferred embodiment, the inoculation amount of the seed culture milk is 40-70 g / L based on the wet weight of the initial yeast culture; preferably, the copper-containing solution is added until the copper concentration in the copper-rich fermentation culture solution is 500-600 mg / kg; preferably, before adding the copper-containing solution, the pH value of the copper-rich fermentation culture solution is adjusted to 4.5-5.5; preferably, the temperature of the copper-rich fermentation culture is 28-35°C; preferably, the rotation speed of the copper-rich fermentation culture is 200-700 rpm. Providing appropriate ventilation volume, temperature environment, and rotation speed for the fermentation reaction can enable the yeast in the seed culture solution to proceed at a higher activity, and utilize the copper-containing solution as much as possible to convert it into organic copper. Any raw material containing inorganic copper that can be used by yeast is suitable for this application. In a preferred embodiment, the copper-containing solution includes: copper sulfate, CuCl2, and copper nitrate.

[0038] To avoid the adverse effects on the activity of yeast caused by too long cultivation time and cost considerations, stopping the fermentation reaction at an appropriate fermentation time and drying the yeast can facilitate subsequent utilization and storage. In a preferred embodiment, continuous copper-rich fermentation culture is carried out to obtain copper-rich yeast, including: when the volume of the culture solution for copper-rich fermentation culture is 7.0 - 7.2m 3 When it reaches this volume, stop the copper-rich fermentation culture, perform centrifugal concentration to obtain yeast milk, and dry the yeast milk to obtain copper-rich yeast. Stopping the reaction when the culture solution for fermentation culture reaches this volume can avoid overflow of the fermentation broth due to excessive fermentation broth and can meet the production requirements.

[0039] In the third typical embodiment of the present application, a copper-rich yeast is provided. The copper-rich yeast is obtained by the above preparation method, and the copper content in the copper-rich yeast is 10,000 - 15,000 mg / kg. Using the above preparation process can obtain copper-rich yeast with a copper content higher than the prior art level, and can obtain copper-rich yeast with good quality and high copper content at a relatively low cost, which has certain advantages in industrial production and subsequent utilization.

[0040] In the fourth typical embodiment of the present application, an application of the copper-rich yeast obtained by the above preparation method or the above copper-rich yeast in the fields of food, health products, medicine, environmental protection, and feed is provided. The copper-rich yeast of the present application has a high copper content. Therefore, when preparing products such as food, health products, and feed that require copper supplementation, since the copper in the yeast is organic copper, it has high biocompatibility and utilization rate, can be efficiently absorbed by humans and animals at a relatively low intake, and has less biological toxicity and side effects compared with inorganic copper, and is more environmentally friendly.

[0041] The following further describes the present application in detail with specific embodiments, and these embodiments should not be construed as limiting the scope claimed by the present application.

[0042] The Saccharomyces cerevisiae used in the following examples was all deposited at the China Center for Type Culture Collection on December 11, 2017. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2017781.

[0043] Example 1 Verification of the copper-rich ability of Saccharomyces cerevisiae strains

[0044] Fermentation broth (g / L): Glucose 100 g, yeast extract 20 g, KH2PO4 1 g, MgSO4 0.5 g, adjust pH to 5.0.

[0045] Seed culture: Add 200 mL of fermentation broth into a 500 mL conical flask. Use an inoculation loop to scrape the well-grown Saccharomyces cerevisiae strain on the solid medium twice and inoculate it into the fermentation broth. Incubate at 30 °C and 200 rpm for 24 h to obtain the seed solution.

[0046] Fermentation culture: Add 200 mL of fermentation broth into a 500 mL conical flask, and add copper-containing solutions with different concentrations to the fermentation broth, with concentrations of 100 mg / L, 200 mg / L, and 300 mg / L respectively. Inoculate 4% of the seed solution into each experimental group and ferment at 30 °C and 200 rpm for 72 h to obtain the copper-rich yeast fermentation broth.

[0047] Collect yeast mud: Centrifuge the copper-rich yeast fermentation broth at 5000 rpm for 3 min to discard the supernatant. Dissolve the yeast mud in an appropriate amount of tap water and then centrifuge at 5000 rpm for 3 min. Centrifuge a total of three times to collect the yeast mud.

[0048] Prepare yeast filter cake: Dissolve the yeast mud in an appropriate amount of tap water, then perform suction filtration to obtain the filter cake. Dry it in an oven at 105 °C until constant weight, and determine the copper content in the yeast by atomic absorption spectrometry. The results are shown in Table 1.

[0049] Table 1:

[0050] Copper content in the fermentation broth (mg / L) Copper content in yeast (mg / kg) 100 7186 200 10395 300 14472

[0051] Example 2 (the preparation process as Figure 1 shown)

[0052] (1) Primary shake flask culture: Add 20 g of glucose, 6 g of yeast extract, 0.2 g of KH2PO4, and 0.2 g of MgSO4 into a 500 mL conical flask. Add purified water to make up the volume to 200 mL, adjust the pH to 5.5, and sterilize at 121 °C for 20 min. Use an inoculation loop to scrape the well-grown Saccharomyces cerevisiae on the solid medium and inoculate it into the seed medium. Incubate at 30 °C and 200 rpm for 18 h to obtain the seed solution;

[0053] (2) Secondary shake flask culture: Add 500 g of glucose, 200 g of yeast extract, 5 g of KH2PO4, and 5 g of MgSO4, make up the volume to 5 L, adjust the pH to 5.5, and sterilize at 121 °C for 20 min. Inoculate 1% of the seed solution in (1) and incubate at 30 °C and 200 rpm for 18 h to obtain the primary seed culture solution;

[0054] (3) Secondary seed fermentation: Conduct seed fermentation in a 10 m 3 fermenter. Inoculate the primary seed culture solution in (2) into the fermenter, make the initial wet weight of yeast 10 g / L, and add the fermentation nutrient solution during the cultivation period for fermentation. Incubate at 30 °C and 200 rpm until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate to obtain the secondary seed culture solution;

[0055] (4) Primary three-stage seed fermentation: Conduct seed fermentation in a 200 - 360 m 3 fermentation tank. Transfer the secondary seed culture solution in (3) to the fermentation tank, with the initial wet weight of yeast being 40 g / L. During the cultivation period, add fermentation nutrient solution continuously for fermentation. Cultivate at 30 °C until the wet weight of yeast reaches 150 g / L, then stop fermentation, centrifuge, and concentrate;

[0056] (5) Secondary three-stage seed fermentation: Conduct seed fermentation in a 200 - 360 m 3 fermentation tank. Inoculate the primary three-stage seed fermentation broth in (4) into the fermentation tank, with the initial wet weight of yeast being 40 g / L. During the cultivation period, add fermentation nutrient solution continuously for fermentation. Cultivate at 30 °C until the wet weight of yeast is above 150 g / L, then stop fermentation, centrifuge, and concentrate to obtain the seed culture solution;

[0057] (6) Copper-rich fermentation culture: Inoculate the seed culture solution in (5) into a 10 m 3 fermentation tank, with the initial wet weight of yeast being 40 g / L. During the cultivation period, add fermentation nutrient solution continuously for fermentation; Stop adding fermentation nutrient solution and ventilation at the 600th minute, with the ventilation volume being 150 m 3 / h. Adjust the pH to 5.5 and add copper sulfate solution until the copper concentration in the fermentation broth reaches 500 mg / kg, then stop adding copper sulfate solution. During the whole fermentation culture process, the fermentation temperature is 32 °C, cultivate at 200 - 700 rpm until the total fermentation volume reaches 7.2 m 3 at this time, stop fermentation, centrifuge, and dry to obtain copper-rich yeast.

[0058] When the fermentation volume is 7.2 m 3 at this time, the fermentation nutrient solution includes: 2.3 m 3 of glucose solution with a mass fraction of 30% (760 kg of glucose), 190 L of 16% ammonia water, and add the following: 15 kg of NH4H2PO4, 5 kg of MgSO4, 1 kg of ZnSO4, 8 kg of KCl, 4 kg of CaCl2, 20 kg of NaCl, 0.17 kg of FeSO4·7H2O, 90 g of MnSO4·H2O, 30 g of Na2B4O7·10H2O, 40 g of KI, 40 g of (NH4)6Mo7O 24 ·4H2O, 460 g of CuSO4·5H2O, 44 g of NiSO4·6H2O, 46 g of VB1, 7 g of VB2, 170 g of VB6, 23 g of biotin, 172 g of nicotinic acid, 46 g of D-calcium pantothenate.

[0059] Among them, the addition amount of glucose is adjusted according to the growth situation of yeast, fluctuating around 760 kg. The system can be enlarged proportionally as needed.

[0060] Detection: Measure 100 mL of the culture broth after the fermentation culture is completed, centrifuge it at 5000 rpm for 3 min. Dissolve the yeast mud with an appropriate amount of purified water, centrifuge again, and then extract it with an appropriate amount of purified water to obtain fresh yeast. After drying the fresh yeast at 105 °C to constant weight, the yeast content in the fermentation broth is 42 g / L.

[0061] The fermentation broth is centrifuged twice to obtain yeast cream, and then dried to obtain yeast powder. The copper content in the yeast powder is detected by atomic absorption method to be 10535 mg / kg.

[0062] Example 3

[0063] (1) Primary shake flask culture: Add 26 g of glucose, 4 g of yeast extract, 0.3 g of KH2PO4, and 0.3 g of MgSO4 to a 500 mL conical flask, make up the volume to 200 mL with purified water, adjust the pH to 5.0, and sterilize at 121 °C for 20 min. Use an inoculation loop to scrape the Saccharomyces cerevisiae with good growth state on the solid medium and inoculate it into the seed medium. Culture at 30 °C and 220 rpm for 18 h to obtain the seed liquid;

[0064] (2) Secondary shake flask culture: 550 g of glucose, 100 g of yeast extract, 10 g of KH2PO4, 10 g of MgSO4, make up the volume to 5 L, adjust the pH to 5.5, and sterilize at 121 °C for 20 min. Add 2% of the seed liquid in (1) and culture at 30 °C and 220 rpm for 18 h to obtain the primary seed culture broth;

[0065] (3) Secondary seed fermentation: Conduct seed fermentation in a 10 m 3 fermentation tank. Add the primary seed culture broth in (2) to the PC tank, make the initial wet weight of yeast 40 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C and 220 rpm until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate to obtain the secondary seed culture broth;

[0066] (4) First tertiary seed fermentation: Conduct seed fermentation in a 200 - 360 m 3 fermentation tank. Inoculate the secondary seed culture broth in (3) into the fermentation tank, make the initial wet weight of yeast 90 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate;

[0067] (5) Second tertiary seed fermentation: Conduct seed fermentation in a 200 - 360 m 3 fermentation tank. Inoculate the first tertiary seed fermentation broth in (4) into the fermentation tank, make the initial wet weight of yeast 90 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C until the wet weight of yeast is above 150 g / L, stop fermentation, centrifuge and concentrate to obtain the seed culture broth;

[0068] (6) Copper-rich fermentation culture: Inoculate the seed culture solution in (5) into a 10 m 3 fermenter, with the initial wet weight of yeast being 70 g / L. During the culture period, add the fermentation nutrient solution continuously for fermentation; stop adding the fermentation nutrient solution and ventilation at the 960th minute, and the ventilation volume is 200 m 3 / h. Adjust the pH to 4.5 and add copper sulfate solution. Stop adding the copper sulfate solution when the copper concentration in the fermentation broth reaches 550 mg / kg. During the whole fermentation culture process, the fermentation temperature is 28 °C, and culture at 200 - 700 rpm until the total fermentation volume reaches 7 m 3 at this time, stop fermentation, centrifuge and dry to obtain copper-rich yeast.

[0069] Among them, the fermentation nutrient solution includes: 2.3 m of molasses with a mass fraction of 30% 3 (2600 kg of molasses), 140 L of 16% ammonia water, 15 kg of NH4H2PO4, and the system can be expanded proportionally as needed.

[0070] Detection: Measure 100 mL of the culture solution at the end of fermentation culture, centrifuge at 5000 rpm for 3 min. Dissolve the yeast mud with an appropriate amount of purified water and centrifuge again. Then, dissolve it with an appropriate amount of purified water and extract to obtain fresh yeast. After drying the fresh yeast at 105 °C to a constant weight, the amount of yeast in the fermentation broth is 41 g / L.

[0071] Centrifuge the yeast mother twice with a centrifuge to obtain yeast powder. Detect the copper content in the yeast powder by atomic absorption method, which is 11092 mg / kg.

[0072] Example 4

[0073] (1) Primary shake flask culture: Add 22 g of glucose, 8 g of yeast extract, 0.4 g of KH2PO4, and 0.4 g of MgSO4 into a 500 mL conical flask, add purified water to make the volume up to 200 mL, adjust the pH to 6.0, and sterilize at 121 °C for 20 min. Use an inoculation loop to scrape the Saccharomyces cerevisiae with good growth state on the solid medium and inoculate it into the seed medium, and culture at 32 °C and 200 rpm for 20 h to obtain the seed solution;

[0074] (2) Secondary shake flask culture: 650 g of glucose, 150 g of yeast extract, 7.5 g of KH2PO4, 7.5 g of MgSO4, make the volume up to 5 L, adjust the pH to 5.0, and sterilize at 121 °C for 20 min. Inoculate 5% of the seed solution in (1) and culture at 32 °C and 200 rpm for 20 h to obtain the primary seed culture solution;

[0075] (3) Secondary seed fermentation: In a 10 m 3Seed fermentation is carried out in a fermenter. The primary seed culture solution in (2) is inoculated into a PC tank, with the initial wet weight of yeast being 90 g / L. During the cultivation period, a fermentation nutrient solution is added dropwise for fermentation. It is cultivated at 32 °C and 200 rpm until the wet weight of yeast reaches 160 g / L, then fermentation is stopped, centrifuged and concentrated to obtain a secondary seed culture solution; the fermentation nutrient solution used in this example is the same as that in Example 2.

[0076] (4) Primary tertiary seed fermentation: In a 200 - 360 m 3 Seed fermentation is carried out in a fermenter. The secondary seed culture solution in (3) is inoculated into the fermenter, with the initial wet weight of yeast being 10 g / L. During the cultivation period, a fermentation nutrient solution is added dropwise for fermentation. It is cultivated at 32 °C until the wet weight of yeast reaches 150 g / L, then fermentation is stopped, centrifuged and concentrated;

[0077] (5) Secondary seed fermentation: In a 200 - 360 m 3 Seed fermentation is carried out in a fermenter. The primary tertiary seed fermentation broth in (4) is inoculated into the fermenter, with the initial wet weight of yeast being 10 g / L. During the cultivation period, a fermentation nutrient solution is added dropwise for fermentation. It is cultivated at 32 °C until the wet weight of yeast is above 150 g / L, then fermentation is stopped, centrifuged and concentrated to obtain a seed culture solution;

[0078] (6) Copper - rich fermentation culture: The seed culture solution in (5) is inoculated into a 10 m 3 fermenter, with the initial wet weight of yeast being 50 g / L. During the cultivation period, a fermentation nutrient solution is added dropwise for fermentation; at the 720th minute, the addition of the fermentation nutrient solution is stopped and ventilation is stopped, with the ventilation volume being 150 m 3 / h. The pH is adjusted to 5.0 and a copper sulfate solution is added dropwise until the copper concentration in the fermentation broth reaches 600 mg / kg, then the addition of the copper sulfate solution is stopped. During the whole fermentation culture process, the fermentation temperature is 35 °C, and it is cultivated at 200 - 700 rpm until the total fermentation volume reaches 7 m 3 at which time fermentation is stopped, centrifuged and dried to obtain copper - rich yeast.

[0079] Detection: Measure 100 mL of the culture broth at the end of fermentation culture, centrifuge at 5000 rpm for 3 min. The yeast mud is dissolved with an appropriate amount of purified water and centrifuged again, and then dissolved with an appropriate amount of purified water and extracted to obtain fresh yeast. The fresh yeast is dried at 105 °C to a constant weight, and the amount of yeast in the fermentation broth is 41 g / L.

[0080] The fermentation broth is centrifuged twice to obtain yeast cream, which is dried to obtain yeast powder. The copper content in the yeast powder is detected by atomic absorption method to be 10681 mg / kg.

[0081] Example 5

[0082] Steps (1), (2), (3), (4), and (5) have the same composition of the fermentation nutrient solution as in Example 2. However, during the addition of copper sulfate in step (6), ventilation is not stopped. The specific operation is as follows:

[0083] (6) Copper-rich fermentation culture: Inoculate the seed culture solution in (5) into a 10 m 3 fermentation tank, with the initial wet weight of yeast being 50 g / L. During the culture, add the fermentation nutrient solution for fermentation; stop adding the fermentation nutrient solution at the 720th minute, adjust the pH to 5.0 and add copper sulfate solution, and stop adding the copper sulfate solution when the copper concentration in the fermentation broth reaches 600 mg / kg. During the entire fermentation culture process, the fermentation temperature is 35 °C, and culture at 220 rpm until the total fermentation volume reaches 7 L, then stop fermentation, centrifuge, and dry to obtain copper-rich yeast.

[0084] Detection: Measure 100 mL of the culture solution at the end of fermentation culture, centrifuge at 5000 rpm for 3 minutes, dissolve the yeast mud with an appropriate amount of purified water and centrifuge again, and then dissolve with an appropriate amount of purified water and extract to obtain fresh yeast. After drying the fresh yeast at 105 °C to a constant weight, the amount of yeast in the fermentation broth is 37 g / L.

[0085] Centrifuge the fermentation broth twice to obtain yeast cream, and dry to obtain yeast powder. The copper content in the yeast powder is detected by atomic absorption method to be 5847 mg / kg.

[0086] Example 6

[0087] Steps (1), (2), (3), (4), and (5) have the same composition of the fermentation nutrient solution as in Example 2. However, during the addition of copper sulfate in step (6), the ventilation volume is reduced to about half, and the ventilation volume is 70 m 3 / h. The specific operation is as follows:

[0088] (6) Copper-rich fermentation culture: Inoculate the seed culture solution in (5) into a 10 m 3 fermentation tank, with the initial wet weight of yeast being 50 g / L. During the culture, add the fermentation nutrient solution for fermentation; stop adding the fermentation nutrient solution at the 720th minute, adjust the pH to 5.0 and add copper sulfate solution, and stop adding the copper sulfate solution when the copper concentration in the fermentation broth reaches 600 mg / kg. During the entire fermentation culture process, the fermentation temperature is 35 °C, and culture at 220 rpm until the total fermentation volume reaches 7 L, then stop fermentation, centrifuge, and dry to obtain copper-rich yeast.

[0089] Detection: Measure 100 mL of the culture solution at the end of fermentation culture, centrifuge at 5000 rpm for 3 minutes, dissolve the yeast mud with an appropriate amount of purified water and centrifuge again, and then dissolve with an appropriate amount of purified water and extract to obtain fresh yeast. After drying the fresh yeast at 105 °C to a constant weight, the amount of yeast in the fermentation broth is 45 g / L.

[0090] The fermentation broth was centrifuged twice to obtain yeast cream, which was then dried to obtain yeast powder. The copper content in the yeast powder was detected by atomic absorption method to be 7209 mg / kg.

[0091] Example 7

[0092] Steps (1), (2), (3), (4), and (5) had the same composition of the fermentation nutrient solution as in Example 2. However, during step (6) when adding copper sulfate, the ventilation rate was reduced to about 1 / 5 of the original, and the ventilation rate was 30 m 3 / h. The specific operation is as follows:

[0093] (6) Copper-rich fermentation culture: The seed culture solution in step (5) was inoculated into a 10 m 3 fermentation tank, with the initial wet weight of yeast being 50 g / L. During the culture period, the fermentation nutrient solution was added dropwise for fermentation; at the 720th minute, the addition of the fermentation nutrient solution was stopped, the pH was adjusted to 5.0, and a copper sulfate solution was added dropwise until the copper concentration in the fermentation broth reached 600 mg / kg, at which point the addition of the copper sulfate solution was stopped. During the entire fermentation culture process, the fermentation temperature was 35 °C, and the culture was carried out at 220 rpm until the total fermentation volume reached 7 m 3 at which point fermentation was stopped, and the copper-rich yeast was obtained by centrifugation and drying.

[0094] Detection: 100 mL of the culture broth at the end of the fermentation culture was measured, centrifuged at 5000 rpm for 3 minutes, the yeast mud was dissolved in an appropriate amount of purified water and centrifuged again, and then dissolved in an appropriate amount of purified water and extracted to obtain fresh yeast. The fresh yeast was dried at 105 °C to a constant weight, and the amount of yeast in the fermentation broth was 46 g / L.

[0095] The fermentation broth was centrifuged twice to obtain yeast cream, which was then dried to obtain yeast powder. The copper content in the yeast powder was detected by atomic absorption method to be 7793 mg / kg.

[0096] Comparative Example 1 (implemented according to Example 4 of Patent CN111434767B)

[0097] (1) Slant culture: The Saccharomyces cerevisiae screened in this patent was inoculated on a YEPD solid slant medium containing 50 μg / mL of copper ions, cultured at 30 °C for 48 hours, and then stored in a 4 °C refrigerator.

[0098] (2) Liquid culture: After activating the Saccharomyces cerevisiae stored in the above step (1), one full loop of cells was inoculated into a triangular flask containing 20 mL of YEPD medium and cultured with shaking at 30 °C for 18 hours to obtain a liquid culture.

[0099] (3) Primary liquid seed culture: The liquid culture in the above step (2) was inoculated into a triangular flask containing 0.2 L of YEPD medium at an inoculation amount of 10%, and cultured with shaking at 30 °C for 18 hours to obtain a primary liquid seed culture.

[0100] (4) Secondary liquid seed culture: Inoculate the primary seed culture from the above step (3) into a small fermenter containing 2 L of YEPD medium at an inoculation amount of 10%, and stir and culture at 25 - 35 °C for 18 hours to obtain a secondary liquid seed culture.

[0101] (5) Tertiary liquid seed culture: Inoculate the secondary seed culture from the above step (4) into a fermenter containing 20 L of YEPD medium at an inoculation amount of 10%, stir and culture at 30 °C for 18 hours to obtain a tertiary liquid seed culture.

[0102] (6) Fermentation culture in the fermenter: Inoculate the tertiary seed culture from the above step (5) into a fermenter containing 75 L of basal sugar fermentation medium at an inoculation amount of 10% for fermentation. Start feeding 25 L of fermentation medium A at a feeding rate of 5 L / hr at the 4th hour of fermentation. At the 4th hour of fermentation, add 250 ml of copper chloride aqueous solution with a copper ion concentration of 0.236 mol / L at one time. The fermentation conditions are: 30 °C, stirring speed: the speed is 200 rpm from the start of fermentation to the 4th hour of fermentation, and the speed is 200 - 800 rpm from the 4th hour of fermentation to the end of fermentation (control the dissolved oxygen to be greater than 20%). When the dissolved oxygen rises to greater than 60%, end the fermentation.

[0103] Among them, the basal sugar fermentation medium consists of a solute and a solvent. The solvent is water, and the solute and its concentration in the basal sugar fermentation medium are respectively molasses reducing sugar 20 g / L (that is, the concentration of reducing sugar in molasses in the fermentation medium), copper ion (copper chloride) 2.36 mmol / L, (NH4)2SO4 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 1 g / L, and the pH value of the basal sugar fermentation medium is 6.0.

[0104] Fermentation medium A consists of a solute and a solvent. The solvent is water, and the solute and its concentration in fermentation medium A are respectively molasses reducing sugar 180 g / L (that is, the concentration of reducing sugar in molasses in the fermentation medium), (NH4)2SO4 5 g / L, KH2PO4 1 g / L, MgSO4·7H2O 1 g / L, and the pH is 6.0.

[0105] (7) Detection: Measure 100 mL of the culture solution after the fermentation culture ends, centrifuge at 5000 rpm for 3 min. Dissolve the yeast mud with an appropriate amount of purified water and centrifuge again. Then, dissolve it with an appropriate amount of purified water and extract to obtain fresh yeast. After drying the fresh yeast at 105 °C to constant weight, the yeast amount in the fermentation broth is 16 g / L.

[0106] Centrifuge the fermentation broth twice to obtain yeast cream, and dry it to obtain yeast powder. The copper content in the yeast powder is detected by atomic absorption method to be 8593 mg / kg.

[0107] Comparative Example 2 (Candida, prepared by the method of this patent)

[0108] (1) Primary shake flask culture: Add 26 g of glucose, 4 g of yeast extract, 0.3 g of KH2PO4, and 0.3 g of MgSO4 into a 500 mL conical flask, make up the volume to 200 mL with purified water, adjust the pH to 5.0, and sterilize at 121 °C for 20 min. Use an inoculation loop to scrape the well-grown Candida (CCTCC No: 2017782) on the solid medium and inoculate it into the seed medium, and culture at 30 °C and 220 rpm for 18 h to obtain the seed liquid;

[0109] (2) Secondary shake flask culture: 550 g of glucose, 100 g of yeast extract, 10 g of KH2PO4, and 10 g of MgSO4, make up the volume to 5 L, adjust the pH to 5.5, and sterilize at 121 °C for 20 min. Inoculate 1% of the seed liquid in (1) into it and culture at 30 °C and 220 rpm for 18 h to obtain the primary seed culture solution;

[0110] (3) Secondary seed fermentation: Carry out seed fermentation in a 10 m3 fermenter. Transfer the primary seed culture solution in (2) into a PC tank, make the initial wet weight of yeast 40 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C and 220 rpm until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate to obtain the secondary seed culture solution;

[0111] (4) First tertiary seed fermentation: Carry out seed fermentation in a 200 - 360 m3 fermenter. Inoculate the secondary seed culture solution in (3) into the fermenter, make the initial wet weight of yeast 90 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C and 220 rpm until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate;

[0112] (5) Second tertiary seed fermentation: Carry out seed fermentation in a 200 - 360 m3 fermenter. Inoculate the first tertiary seed fermentation liquid in (4) into the fermenter, make the initial wet weight of yeast 90 g / L, and add the fermentation nutrient solution during the culture period for fermentation. Culture at 30 °C and 220 rpm until the wet weight of yeast reaches 150 g / L, stop fermentation, centrifuge and concentrate to obtain the seed culture solution;

[0113] (6) Copper-rich fermentation culture: Inoculate the seed culture solution in (5) into a 10 m3 fermenter, make the initial wet weight of yeast 50 g / L. Add the fermentation nutrient solution during the culture period for fermentation; stop adding the fermentation nutrient solution and stop ventilation at the 720th minute, adjust the pH to 4.5 and add the copper sulfate solution, and stop adding the copper sulfate solution until the copper concentration in the fermentation broth reaches 550 mg / kg. During the whole fermentation culture process, the fermentation temperature is 28 °C and culture until the total fermentation volume reaches 7 m 3 When it reaches, stop fermentation, centrifuge and dry to obtain copper-rich yeast.

[0114] Detection: Measure 100 mL of the culture broth after the fermentation culture ends, centrifuge it at 5000 rpm for 3 min. Dissolve the yeast mud with an appropriate amount of purified water and centrifuge again. Then, dissolve it with an appropriate amount of purified water and extract to obtain fresh yeast. After drying the fresh yeast at 105 °C to a constant weight, the amount of yeast in the fermentation broth is 34 g / L.

[0115] The fermentation broth is centrifuged twice to obtain yeast milk, which is then dried to obtain yeast powder. The copper content in the yeast powder is detected by atomic absorption spectrometry to be 6346 mg / kg.

[0116] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: Using Saccharomyces cerevisiae (preservation number: CCTCC NO: M 2017781) for batch seed culture, gradually expanding the culture system. First, Saccharomyces cerevisiae is propagated and activated in large quantities, and then Saccharomyces cerevisiae is used to absorb and convert the inorganic copper in the copper-containing solution into organic copper. And at the initial stage of fermentation culture, the fermentation nutrient solution is supplemented in a batch or fed-batch form to promote the efficient growth of yeast. When the fermentation time is 600 - 960 min, the addition of the fermentation nutrient solution is stopped, and ventilation is stopped. The copper-containing solution is added to make the yeast subsequently convert the inorganic copper into organic copper. Using the above method, a large amount of yeast with a high copper content can be obtained under the condition of a relatively short preparation time, with low cost and being more conducive to industrial production.

[0117] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing copper-rich yeast, characterized in that, The preparation method includes: Performing copper-rich fermentation using the known Saccharomyces cerevisiae with the preservation number CCTCC NO: M 2017781 to obtain the copper-rich yeast.

2. The preparation method according to claim 1, wherein The preparation method includes: Performing batch seed culture on the activated Saccharomyces cerevisiae to obtain a seed culture broth; Performing copper-rich fermentation culture on the seed culture broth to obtain the copper-rich yeast.

3. The preparation method according to claim 2, characterized in that, The batch seed culture includes: Performing primary seed culture on the activated Saccharomyces cerevisiae to obtain a primary seed culture solution; Inoculating the primary seed culture solution into a fermentation nutrient solution for secondary seed culture to obtain a secondary seed culture solution; Inoculating the secondary seed culture solution into the fermentation nutrient solution for multiple tertiary seed cultures to obtain the seed culture broth.

4. The preparation method according to claim 3, characterized in that, The primary seed culture includes: Inoculating a single colony of the activated Saccharomyces cerevisiae into a seed medium for first-stage shake flask culture to obtain a seed solution; Inoculating the seed solution into the seed medium for second-stage shake flask culture to obtain the primary seed culture solution; Preferably, by mass fraction, the seed medium includes: 10 - 13% glucose, 2 - 4% yeast extract, 0.1 - 0.2% KH2PO4, and 0.1 - 0.2% MgSO4; Preferably, the pH value of the seed medium is 5 - 6; Preferably, the volume of the seed medium accounts for 40 - 60% of the container volume of the first-stage shake flask culture and the second-stage shake flask culture; Preferably, the container volume of the first-stage shake flask culture is 500 - 750 mL; Preferably, the container volume of the second-stage shake flask culture is 5 - 10 L; Preferably, by volume fraction, the inoculation amount of the seed solution is 1 - 5%; Preferably, the temperature of the first-stage shake flask culture and the second-stage shake flask culture is 30 - 32 °C; Preferably, the rotation speed of the first-stage shake flask culture and the second-stage shake flask culture is 200 - 220 rpm; Preferably, the time of the first-stage shake flask culture and the second-stage shake flask culture is 17 - 20 h.

5. The preparation method according to claim 3, characterized in that, The secondary seed culture includes: Mixing the fermentation nutrient solution with the primary seed culture solution in a manner of feeding while culturing to perform the secondary seed culture to obtain the secondary seed culture solution; Preferably, the fermentation nutrient solution includes: a carbon source; When the carbon source is molasses, the fermentation nutrient solution further includes: a nitrogen source and phosphate; When the carbon source is one or more of glucose, fructose, or sucrose, the fermentation nutrient solution further includes: a nitrogen source, phosphate, magnesium salt, potassium salt, calcium salt, sodium salt, trace elements, and vitamins; Preferably, the nitrogen source is selected from any one or more of the following substances: yeast extract, peptone, corn steep liquor, soy protein, ammonia water, ammonium sulfate, ammonium phosphate, and ammonium dihydrogen phosphate; Preferably, the trace elements are selected from any one or more of the following: zinc salt, copper salt, nickel salt, iron salt, manganese salt, boron salt, iodine salt, and nickel salt; Preferably, the trace elements are copper, iron, and zinc; Preferably, the volume of the fermentation nutrient solution accounts for 40 - 60% of the container volume of the secondary seed culture; Preferably, the volume of the container for the secondary seed culture is 10 - 15 L; Preferably, the inoculation amount of the primary seed culture medium, based on the wet weight of the initial yeast in the culture, is 10 - 90 g / L, preferably 40 - 70 g / L; Preferably, the temperature of the secondary seed culture is 30 - 32 °C; Preferably, the rotation speed of the secondary seed culture is 200 - 220 rpm; Preferably, when the wet weight of the yeast in the secondary seed culture medium is greater than 150 g / L, stop the secondary seed culture and perform centrifugal concentration to obtain the secondary seed culture medium.

6. The preparation method according to claim 3, characterized in that, The tertiary seed culture includes: The fermentation nutrient solution is mixed with the secondary seed culture medium in a manner of feeding while culturing to perform the tertiary seed culture to obtain the seed culture milk; Preferably, performing the tertiary seed culture multiple times means performing the tertiary seed culture twice; Preferably, the volume of the fermentation nutrient solution accounts for 60 - 70% of the volume of the container for the tertiary seed culture; Preferably, the volume of the container for the tertiary seed culture is 200 - 360 L; Preferably, the inoculation amount of the tertiary seed culture medium, based on the wet weight of the initial yeast in the culture, is 10 - 90 g / L, preferably 40 - 70 g / L; Preferably, the temperature of the tertiary seed culture is 30 - 32 °C; Preferably, the rotation speed of the tertiary seed culture is 200 - 700 rpm; Preferably, when the wet weight of the yeast in the tertiary seed culture medium is greater than 150 g / L, stop the tertiary seed culture and perform centrifugal concentration.

7. The preparation method according to claim 2, characterized in that, The copper-rich fermentation culture includes: The fermentation nutrient solution is mixed with the seed culture milk in a manner of feeding while culturing or batch supplementing; At 600 min - 960 min of the copper-rich fermentation culture, stop ventilation and the addition of the fermentation nutrient solution, and add a copper-containing solution to the solution of the copper-rich fermentation culture; Continue the copper-rich fermentation culture to obtain the copper-rich yeast; Preferably, the ventilation volume is 150 - 200 m 3 / h.

8. The preparation method according to claim 7, characterized in that, The inoculation amount of the seed culture milk, based on the wet weight of the initial yeast in the culture, is 40 - 70 g / L; Preferably, the volume of the copper-rich fermentation nutrient solution accounts for 60 - 70% of the volume of the container for the fermentation culture; Preferably, the volume of the container for the copper-rich fermentation culture is 10-15 m 3 ; Preferably, add the copper-containing solution until the copper concentration in the solution of the copper-rich fermentation culture reaches 500 - 600 mg / kg; Preferably, before adding the copper-containing solution, adjust the pH value of the solution of the copper-rich fermentation culture to 4.5 - 5.5; Preferably, the temperature of the copper-rich fermentation culture is 28 - 35 °C; Preferably, the rotation speed of the copper-rich fermentation culture is 200 - 700 rpm; Preferably, the step of continuing the copper-rich fermentation culture to obtain the copper-rich yeast includes: When the volume of the culture medium for the copper-rich fermentation culture is 7.0 - 7.2 m 3 stop the copper-rich fermentation culture, perform centrifugal concentration to obtain fermented milk mother liquor; Dry the yeast milk to obtain the copper-rich yeast.

9. A copper-rich yeast, characterized in that, The copper-rich yeast is obtained by the preparation method described in any one of claims 1 - 8, and the copper content in the copper-rich yeast is 10000 - 15000 mg / kg.

10. Application of the copper-rich yeast obtained by the preparation method described in any one of claims 1 - 8 or the copper-rich yeast described in claim 9 in the fields of food, health products, medicine, environmental protection, and feed.

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