Candida tropicalis and method for preparing single-cell protein from vinegar residues

Abstract

The invention discloses Candida tropicalis, and the preservation number of the Candida tropicalis in the Guangdong Microbial Culture Collection Center is GDMCC No: 64869. The Candida tropicalis has the advantages that the preservation number of the Candida tropicalis is CGMCC No: 64869; the invention further provides application of the candida tropicalis in fermentation production of single-cell protein, a culture medium suitable for the candida tropicalis and a method for fermentation production of the single-cell protein. The candida tropicalis is a high-stress-resistance strain of vinegar residue hydrolysate, is suitable for large-scale production, can effectively promote recycling of vinegar residue waste biomass resources, and relieves the current situation of protein feed shortage in the breeding industry to a certain extent.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to tropical Candida and a method for preparing single-cell protein using vinegar residues. Background Art

[0002] Single-cell protein is biomass or protein extracted from microbial cultures such as yeast, bacteria, mold and algae. Microorganisms can convert low-value non-food biomass from forestry and agriculture into high-value protein feed, and they have a small footprint, high production efficiency and low dependence on environmental conditions. The protein they produce has the potential to be used as animal feed. Among them, yeast has become an important source of single-cell protein due to its rapid growth, rich nutrition and probiotic properties. At present, a large number of studies are focused on the production of single-cell protein using various biodegradable agricultural and industrial wastes. Cheap and suitable substrates for yeast growth are the key to reducing production costs.

[0003] Vinegar dregs are the residue left after making vinegar from rice, wheat, sorghum, etc., and are the main by-product of vinegar making. In ancient books, such as "Compendium of Materia Medica" and "Dietary Therapy Materia Medica", the medicinal value of "vinegar dregs" is mentioned. In the vinegar making process, vinegar dregs are also commonly referred to as "vinegar dregs" or "vinegar making residues", and more than 3 million tons are produced each year in the country. Vinegar dregs are mainly produced from raw materials such as sorghum, bran, and rice husks. They are rich in nutrients such as cellulose, hemicellulose, protein, and multiple amino acids. They are potential raw materials for feed production and single-cell protein production. However, there is a large amount of lignocellulose in vinegar dregs, which has the characteristics of stable structure and difficult to destroy, which limits its application as a biomass resource. In order to efficiently utilize dregs biomass resources, it is necessary to pre-treat its lignocellulose.

[0004] At present, the main method for pretreatment of vinegar grains lignocellulose is acid hydrolysis. Although acid hydrolysis can degrade cellulose into fermentable sugars that can be used by microorganisms, it has certain limitations. First, the acid hydrolysis process requires high-temperature and high-pressure equipment, and the processing cost is relatively high. Secondly, the acid hydrolysis process will produce harmful substances such as furfural, 5-hydroxymethylfurfural, and acetic acid, which inhibit the growth of microorganisms and require further detoxification processes, increasing production costs.

[0005] For example, Chinese patent application CN 117448181A discloses a method for producing single-cell protein by fermenting red yeast with vinegar lees, the method comprising: inoculating red yeast seed liquid into a fermentation medium, fermenting and culturing to obtain a fermentation product; collecting the single-cell protein in the fermentation product; wherein the fermentation medium contains a vinegar lees hydrolyzate. The method of the present invention can enable red yeast to utilize the byproduct vinegar lees hydrolyzate as a carbon source for growth metabolism and high-yield single-cell protein, and has the advantages of high single-cell protein yield, fast fermentation speed, high efficiency, low cost, etc., and can be used for large-scale production with high commercial value.

[0006] The vinegar lees hydrolyzate disclosed in the document is obtained by acid hydrolysis of the vinegar lees, specifically, by heating a sulfuric acid solution at 130°C for 1 hour. Therefore, there must be the aforementioned problems of "the acid hydrolysis process requires high temperature and high pressure equipment, and the processing cost is high" and the need for detoxification. The high hydrolysis temperature and harsh conditions will produce inhibitors that require detoxification. Detoxification will also adsorb the sugar after enzymatic hydrolysis and reduce the sugar content. At the same time, the culture medium disclosed in the document also needs to add exogenous nutrients such as yeast powder, which is expensive.

[0007] Therefore, breeding a yeast strain with high protein content and high biomass, which can directly use vinegar grains hydrolyzate as its main nutrient source and whose growth is not inhibited by the hydrolyzate, and using it to produce single-cell protein can not only promote the recovery of vinegar grains biomass resources, but also reduce the production cost of single-cell protein, and will have broad application prospects. Summary of the invention

[0008] In view of the above problems, the present invention aims to provide a highly stress-resistant bacterial strain capable of utilizing vinegar grains hydrolyzate, which is suitable for large-scale production, can effectively promote the recycling of vinegar grains waste biomass resources, and to a certain extent alleviate the current shortage of protein feed in the breeding industry.

[0009] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0010] The invention provides a tropical Candida (Candida tropicalis), whose preservation number in Guangdong Province Microbiological Culture Collection Center is GDMCC No: 64869.

[0011] The invention provides the use of the tropical Candida in fermentation production of single-cell protein.

[0012] The present invention also provides a culture medium suitable for culturing the tropical Candida. The culture medium contains vinegar lees hydrolyzate, which is obtained by pre-treating the vinegar lees with ammonia water and then enzymolyzing the vinegar lees.

[0013] Preferably, in the culture medium, the vinegar grains hydrolyzate treated with ammonia water provides a carbon source and a partial nitrogen source, and also includes a composite nitrogen source of an inorganic nitrogen source and an organic nitrogen source, wherein the inorganic nitrogen source is selected from one or more of ammonium sulfate, urea, ammonium chloride, ammonia water, ammonium carbonate, and ammonium bicarbonate, and the organic nitrogen source is selected from one or more of trypsin, animal peptone, soy peptone, yeast powder, corn dry pulp powder, and beef powder.

[0014] More preferably, the composite nitrogen source uses an inorganic nitrogen source provided by ammonia water used to treat the vinegar grains, and yeast powder is used as an organic nitrogen source, and the weight ratio of the two is 1:4-4:1.

[0015] Also preferably, the culture medium further comprises growth factors, and the growth factors are selected from one or more of magnesium sulfate, zinc sulfate, manganese sulfate, biotin, pantothenic acid, and thiamine.

[0016] The present invention also provides a yeast fermentation production method for single-cell protein, which comprises the following steps:

[0017] 1) treating the vinegar grains with ammonia water and performing enzymatic hydrolysis to obtain a vinegar grains hydrolyzate;

[0018] 2) adding organic nitrogen source yeast powder to the vinegar lees hydrolyzate obtained in step 1) to form a composite nitrogen source with the inorganic nitrogen source ammonia water in the vinegar lees hydrolyzate, wherein the weight ratio of the ammonia water to the yeast powder is 1:4-4:1, and then adding 0.2-5 mg / L of growth factor to prepare a yeast culture medium;

[0019] 3) inoculating the tropical Candida into the yeast culture medium obtained in step 2), fermenting to obtain yeast fermentation liquid, centrifuging and drying the precipitate to obtain single cell protein.

[0020] Preferably, the step 1) specifically includes the following steps:

[0021] S1. Pretreatment of vinegar residue: Pass the vinegar residue through an 80-mesh sieve for later use;

[0022] S2. Ammonia pretreatment: Add 16% ammonia water by mass to the vinegar residue obtained in step S1, control the moisture content to 40%, and then seal and ammoniaize at 70 ° C for 60h;

[0023] S3. Scattered ammonia treatment: After the ammonia treatment is completed, the excess ammonia is recovered by air stripping;

[0024] S4. Enzymatic hydrolysis: add an appropriate amount of distilled water to the ammoniated vinegar residue obtained in step S3 to adjust the material-liquid ratio to 1:10 (w / v), shake in a shaker at 150 rpm for 30 min to balance the ammonia nitrogen, then adjust the pH to 5.0 with 6 mol / L phosphoric acid solution, add 2% cellulase to the mixture, and place it at 50°C, 150 rpm in a shaker for enzymatic hydrolysis for 72 h;

[0025] S5. Centrifugal filtration: After the enzymatic hydrolysis is completed, centrifuge at 9000 rpm for 10 minutes to obtain the vinegar lees hydrolyzate.

[0026] Most preferably, the yeast fermentation production method of the single-cell protein specifically comprises the following steps: 1) weighing a certain amount of vinegar lees, adding 25% ammonia water of 16% of the mass of the vinegar lees, adjusting the water content to 40%, ammoniation at 70°C for 60h, after the ammoniation, adjusting the pH to 5.0 with 6mol / L phosphoric acid solution, adding an appropriate amount of distilled water to control the solid-liquid ratio to 1:10 (w / v), adding 2% (v / w dry matter) of cellulase, shaking enzymolysis at 50°C, 150r / min for 72h, and centrifuging at 9000rpm for 10min to obtain a vinegar lees hydrolyzate;

[0027] 2) taking an appropriate amount of the vinegar lees hydrolyzate in step 1), adding organic nitrogen source yeast powder, and combining it with inorganic nitrogen source ammonia water in the vinegar lees hydrolyzate to form a composite nitrogen source, wherein the mass ratio of ammonia water to yeast powder is 3:2, and adding 2 g / L of growth factor magnesium sulfate to prepare a yeast culture medium;

[0028] 3) The tropical Candida albicans described in claim 1 is inoculated into the yeast culture medium described in step 2), and fermented at 30° C. and 220 rpm for 48 h. After centrifugation at 8000 rpm for 10 min, the precipitate is dried to obtain single cell protein.

[0029] Beneficial effects:

[0030] 1. The present invention screened out a highly stress-resistant bacterial strain that can utilize vinegar lees hydrolyzate. The strain can convert inorganic nitrogen sources such as ammonia water and ammonium sulfate into its own bacterial protein, and the conversion rate of ammonia water is as high as 99.85%.

[0031] 2. The present invention proposes a method for preparing vinegar lees hydrolysate by combining ammonia pretreatment with enzymatic hydrolysis for fermentation and production of single-cell protein. The method does not require high-temperature and high-pressure special equipment for preparing vinegar lees hydrolysate, and does not produce any harmful substances that affect the growth of microorganisms. At the same time, the obtained hydrolysate contains the carbon source and nitrogen source required for yeast growth, and there is no need to use expensive peptone as a nitrogen source. The method has the characteristics of low cost, fast fermentation rate, and safe and reliable product quality, and can be used for large-scale production.

[0032] 3. The process of the present invention has the advantages of high single-cell protein yield, safety, environmental protection, low cost, etc. It is suitable for large-scale production, can effectively promote the recycling of vinegar lees waste biomass resources, and to a certain extent alleviate the current shortage of protein feed in the breeding industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The condition screening of ammoniated vinegar grains is shown. Figure 1 A shows the results of the ammoniation test after changing the amount of ammonia water added while other basic treatment conditions remain unchanged, using 4%, 8%, 12%, 16%, 20%, 24%, and 28% ammonia water addition amounts respectively. Figure 1 B shows the results of the ammoniation test after changing the ammonia moisture content and using a moisture content gradient of 40%, 45%, 50%, 55%, and 60% while other basic treatment conditions remain unchanged. Figure 1 C shows the results of ammoniation after changing the ammoniation temperature and using an ammoniation temperature gradient of 30°C, 40°C, 50°C, 60°C, and 70°C, respectively, while other basic treatment conditions remain unchanged. Figure 1 D shows the results of ammoniation after changing the ammoniation time to 1d, 2d, 3d and 4d respectively while other basic treatment conditions remain unchanged.

[0034] Figure 2 The present invention shows the utilization rate of reducing sugars in vinegar grains hydrolyzate by three yeast strains: Saccharomyces cerevisiae RA2, Candida tropicali GB3, and Meyerozyma caribbica GD1, which were activated and cultured in a medium containing the vinegar grains hydrolyzate of the present invention at 30°C for 24 hours.

[0035] Figure 3 The growth curve of the Candida tropicalis CQGB3 of the present invention is shown, and it can be seen that it is in the logarithmic growth phase from 3 to 18 hours and reaches a plateau phase at 18 hours.

[0036] Figure 4 The figure shows the utilization efficiency of Candida tropicalis CQGB3 of the present invention on different types of inorganic nitrogen sources. Different lowercase letters a, b, c and d above the columns in the figure represent significant differences, and the same lowercase letters represent insignificant differences.

[0037] Figure 5 The figure shows the utilization efficiency of Candida tropicalis CQGB3 of the present invention on different types of organic nitrogen sources. Different lowercase letters a, b, c, d and e above the columns in the figure represent significant differences, and the same lowercase letters represent insignificant differences.

[0038] Figure 6 The effect of composite nitrogen sources of different mass ratios on the growth of tropical Candida CQGB3 of the present invention is shown. In the figure, Y represents yeast powder, and A represents ammonia water. Different lowercase letters a, b, and c above the columns in the figure represent significant differences, and the same lowercase letters represent insignificant differences.

[0039] Figure 7The figure shows the effects of different growth factors on the dry weight of the Candida tropicalis CQGB3 of the present invention. Different lowercase letters a, b, c above the columns in the figure represent significant differences, and the same lowercase letters represent insignificant differences. DETAILED DESCRIPTION

[0040] The present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art. In addition, the specific embodiments of the present invention should be understood as illustrative rather than limiting the scope of protection of the present invention, and the operating essence and scope of protection of the present invention are defined by the claims. For those skilled in the art, various changes and modifications to the materials, addition amounts, and fermentation conditions in the embodiments of the present invention, without violating the essence and scope of the present invention, all belong to the scope of protection of the present invention.

[0041] It is clear to those skilled in the art that the vinegar lees involved in the present invention refers to the by-product of the vinegar brewing process, which is mainly the residue left after the vinegar liquid is extracted after fermentation and brewing of raw materials such as wheat bran, sorghum and a small amount of broken rice. These residues contain a certain amount of water, crude protein, crude fiber and rich trace elements, such as iron, zinc, selenium, manganese, etc. The water content of vinegar lees is usually between 65% and 70%, but the water content of vinegar lees can be reduced to about 10% after air drying. According to relevant data, air-dried vinegar lees contain 9.6-20.4% crude protein, 15.1-28% crude fiber, and a variety of trace elements. These ingredients make the vinegar lees have certain feeding value and nutritional value.

[0042] At present, vinegar dregs have a large number of applications in the fields of feed, medicine and gardening. In terms of feed, vinegar dregs are mainly used to feed pigs and cattle, and the feeding amount is usually about 5% of the daily diet; in addition, vinegar dregs can also be used to feed rabbits and other poultry, which helps to reduce feed costs. Because vinegar dregs have a sour aroma and can increase the appetite of livestock, they are widely welcomed in practical applications. Vinegar dregs have certain applications in Chinese medicinal materials: according to ancient records and modern research, vinegar dregs can be used in combination with other Chinese medicines to treat symptoms such as qi stagnation and wind congestion, arm, foot and knee pain. At the same time, vinegar dregs also have certain effects of reducing swelling and dispersing blood stasis, promoting blood circulation and removing blood stasis, and can be used for hot compresses to relieve pain. Vinegar dregs contain nitrogen, phosphorus, potassium and other elements needed for the growth of flowers, and have excellent characteristics such as light weight, clean, loose, and good air permeability. Therefore, vinegar dregs are also often used as a seedbed matrix, which helps the growth and development of flowers and trees.

[0043] It is clear to those skilled in the art that the single cell protein (SCP) referred to in the present invention, also known as microbial protein, refers to microbial cells artificially cultured using industrial and agricultural waste and petroleum waste. These microbial cells are mainly composed of a mixture of proteins, fats, carbohydrates, nucleic acids, and non-protein nitrogen-containing compounds, vitamins, and inorganic compounds. According to different production raw materials, single cell proteins can be divided into petroleum proteins, methanol proteins, methane proteins, etc.; according to different types of producing bacteria, they can be divided into bacterial proteins, fungal proteins (such as yeast proteins) and algae proteins, etc.

[0044] Due to the rapid growth and reproduction rate of microorganisms, the production efficiency of single-cell protein is thousands of times higher than that of animals and plants. The raw materials for the production of single-cell protein include agricultural waste (such as straw, bagasse, beet pulp, sawdust, etc.), industrial waste (such as sugar-containing organic wastewater, sulfite pulp waste liquid, etc.), petroleum and natural gas and its related products (such as crude oil, diesel, methane, ethanol, etc.), and waste gas (such as H2, CO2, etc.). The protein content of single-cell protein is as high as 40%-80%, which is 10%-20% higher than soybeans and more than 20% higher than meat, fish, and cheese. In addition, it also contains a variety of vitamins, carbohydrates, lipids, minerals, as well as rich enzymes and bioactive substances. The production process of single-cell protein is simple, requires less labor, is not restricted by region, season and climate, and has high yield and good quality.

[0045] Single-cell protein can not only be made into "artificial meat" for direct consumption, but is also often used as a food additive to supplement protein or vitamins, minerals, etc. Because some single-cell proteins have antioxidant properties, they prevent food from spoiling easily, so they are often used in baby powder, soups, and seasonings. As a feed additive, single-cell protein can save food, improve the nutritional value of feed, and promote the development of animal husbandry. Many useful things can be extracted from single-cell protein, such as pharmaceutical products such as coenzyme A, cytochrome C, and coenzyme I, as well as biological reagents such as yeast extract.

[0046] Yeast is widely used in the production of single-cell protein. Yeast types include Candida utilis, Candida lipolytica, Saccharomyces cerevisiae, etc. They can use a variety of organic substances as carbon sources to produce a large amount of protein through the fermentation process. Yeast can use a variety of raw materials for fermentation to produce single-cell protein, including agricultural waste (such as straw, bagasse, etc.), industrial waste (such as sugary organic wastewater, sulfite pulp waste liquid, etc.) and petroleum and natural gas and its related products (such as crude oil, diesel by-products, etc.).

[0047] However, there are still many unavoidable obstacles in using yeast to produce single-cell protein. First, the production cost is high: yeast production of single-cell protein requires the use of high-purity nutrients, and the cost of these raw materials is relatively high; at the same time, the temperature, pH value and oxygen conditions need to be strictly controlled during the fermentation process to ensure the growth and reproduction of yeast, which also increases the production cost; the most critical thing is that the utilization rate of carbon sources and nitrogen sources in vinegar dregs by existing microorganisms is still very low, and the inhibition of microbial activity by hydrolysis treatment liquid leads to greatly limited output, which greatly increases production costs. Second, it is difficult to digest: the yeast cell wall is thick, which makes it difficult to digest and absorb, which limits the application of yeast in single-cell protein production. Third, the taste is bad: because the taste and mouthfeel of single-cell protein are different from traditional proteins, it is not only unpopular with consumers as a food, but also may be difficult for livestock to accept as feed.

[0048] The strain used in the present invention is Candida tropicalis CQGB3 isolated from the Ronglong Hongyuan Vineyard in Chongqing Rongchang. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 15, 2024, with the address at No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the collection number is GDMCC No: 64869.

[0049] The strain isolated by the present invention is identified as short oval in shape under a microscope. The 16SrRNA gene of the strain is amplified by PCR, sequenced and analyzed by phylogeny, and identified as tropical Candida, named as tropical Candida (Candida tropicalis) CQGB3.

[0050] The formula of the culture medium for strain screening used in the present invention is: 40mL vinegar lees hydrolyzate (containing about 23.8g / L of soluble sugar to provide a carbon source, and about 10.4g / L of crude protein to provide a partial nitrogen source), 0.8g of peptone (providing an additional nitrogen source), 0.02g of magnesium sulfate heptahydrate, 0.004g of sodium chloride, and the pH is adjusted to 6.0.

[0051] The formula of the basic fermentation medium used in the present invention is: 20 g / L glucose, 20 g / L peptone, the pH value is adjusted to 6.0, 121°C, and sterilized for 20 minutes. Inorganic nitrogen sources such as ammonium sulfate, urea, ammonium chloride, ammonia water, ammonium carbonate, ammonium bicarbonate, etc. or organic nitrogen sources such as tryptone, animal peptone, soy peptone, yeast powder, corn dry pulp powder, beef powder, etc. are used to replace the peptone in the basic medium in the experiment; MgSO4, ZnSO4, MnSO4, biotin, pantothenic acid, thiamine, etc. are used as additional growth additives or regulators in the experiment.

[0052] The formula of the best fermentation medium for producing single-cell protein used in the present invention is: 1L of vinegar lees hydrolyzate (containing about 23.8g / L of soluble sugar to provide a carbon source, and about 10.4g / L of crude protein to provide a partial nitrogen source), 6.9g / L of yeast powder, 2g / L of magnesium sulfate heptahydrate (MgSO4·7H2O), the pH value is adjusted to 6.5, 121°C, and sterilized for 20min.

[0053] The formula of the conventional YPD medium (full name: yeast extract peptone glucose medium) mentioned in the present invention is: yeast extract 0.5 g / L; peptone 20 g / L; glucose 20 g / L.

[0054] The cellulase used in the present invention was purchased from Novozymes Biotech Co., Ltd., product model CTec3 HS, filter paper enzyme activity 7135 FPU / mL.

[0055] If solid culture medium is to be prepared, 2% agar powder (adjustable according to specific needs) needs to be added to form a solidified culture medium.

[0056] Except for the vinegar residue hydrolyzate of the present invention, the other culture medium components are all commercially available conventional chemical products. The ammonia water uses conventional industrial ammonia water, which is generally an aqueous solution containing 25%-28% ammonia.

[0057] The specific fermentation process steps and parameters involved in the present invention can be appropriately optimized and adjusted by those skilled in the art according to actual needs. As long as they do not deviate from the purpose of the present invention, these solutions are within the scope of protection of the present invention.

[0058] Example 1 Preparation of vinegar residue ammoniation hydrolyzate

[0059] Add 20g of vinegar residue that has passed through an 80-mesh sieve to an appropriate amount of ammonia water in a sealed bag and mix thoroughly, and then perform ammonia treatment under certain conditions. The specific steps of ammonia treatment are as follows:

[0060] S1. Pretreatment of vinegar residue: Pass the vinegar residue through an 80-mesh sieve for later use;

[0061] S2. Ammoniation pretreatment: adding a certain amount of aqueous ammonia to the vinegar residue obtained in step S1, controlling the moisture content, sealing and performing ammoniation at a certain ammoniation temperature for a certain period of time;

[0062] S3. Scattered ammonia treatment: After the ammonia treatment is completed, the excess ammonia is recovered by air stripping;

[0063] S4. Enzymatic hydrolysis: add a certain amount of distilled water to the ammoniated vinegar lees obtained in step S3, adjust the solid-liquid ratio to 1:10 (w / v), shake in a shaker at 150 rpm for 30 min to balance the ammonia nitrogen, then adjust the pH to 5.0 with 6 mol / L phosphoric acid solution, add 2% (v / w dry matter) of cellulase to the mixture, and place it at 50°C, 150 rpm in a shaker for enzymatic hydrolysis for 72 h;

[0064] S5. Centrifugal filtration: After the enzymatic hydrolysis is completed, centrifuge at 9000 rpm for 10 min to obtain the vinegar lees hydrolyzate of the present invention.

[0065] The basic treatment conditions of ammoniation treatment are: the amount of ammonia water added is 12% (weight percentage) of the vinegar lees, the moisture content is 50% (weight percentage), the ammoniation temperature is 60°C, the ammoniation time is 3 days, after the ammoniation is completed, the pH is adjusted to 5.0 with 6 mol / L phosphoric acid, a certain amount of distilled water is added, the solid-liquid ratio is controlled to be 1:10 (w / v), and then 2% (v / w dry matter) of cellulase is added CTec3 HS (converted to enzyme activity 142.72 FPU / g vinegar lees dry weight; 1 FPU is defined as the amount of enzyme used to produce 1 μmol of glucose in 1 min) was oscillated at 50°C and 150 r / min for 72 h. Different gradients were set, and the amount of ammonia water added, moisture content, ammoniating temperature, and ammoniating time were determined in turn through single factor experiments.

[0066] When other basic treatment conditions remain unchanged, the amount of ammonia water added is changed, and the ammonia water addition amount of 4%, 8%, 12%, 16%, 20%, 24%, and 28% is used to carry out the ammoniation test, and the optimal amount of ammonia water addition is determined by the soluble sugar content in the supernatant of the vinegar lees enzymatic hydrolysis. The soluble sugar content in the supernatant is determined by the anthrone colorimetric method, and the specific determination steps are: take 1mL of the enzymatic hydrolysis supernatant and add 4mL of anthrone reagent, determine its absorbance at 620nm, and calculate the soluble sugar content in the enzymatic hydrolysis supernatant according to the standard curve.

[0067] The results are as follows Figure 1 As shown in A, the soluble sugar content in the supernatant increased with the increase of the amount of ammonia water added, reaching the highest level of 271.1 mg / g dry matter when the addition amount was 16%, so 16% was selected as the optimal addition amount of ammonia water.

[0068] With other basic treatment conditions unchanged, the moisture content of ammonia was changed. Ammoniaization tests were carried out using a moisture content gradient of 40%, 45%, 50%, 55%, and 60%, and the optimal moisture content of ammonia was determined by the soluble sugar content in the supernatant of vinegar lees enzymatic hydrolysis.

[0069] The results are as follows Figure 1As shown in Figure B, previous studies reported that the suitable moisture content for ammoniation was between 40% and 60%. Experiments in this range found that the soluble sugar content in the supernatant decreased with the increase of moisture content during ammoniation treatment. In this experiment, when the moisture content was 40%, the soluble sugar content was the highest, at 206.28 mg / g dry matter, which was significantly higher than that of other treatment groups (P<0.05). Therefore, 40% was selected as the optimal moisture content for ammoniation.

[0070] With other basic treatment conditions unchanged, the amination temperature was changed, and ammoniating temperature gradients of 30°C, 40°C, 50°C, 60°C, and 70°C were used for ammoniating. The optimal ammoniating temperature was determined by the soluble sugar content in the supernatant of the vinegar lees enzymatic hydrolysis.

[0071] The results are as follows Figure 1 As shown in Figure C, the soluble sugar content in the supernatant increased with the increase of temperature during ammoniation treatment. When the ammoniation temperature was 70°C, the soluble sugar content was 366.12 mg / g dry matter, which was significantly higher than that of other treatment groups (P<0.05). Therefore, 70°C was selected as the optimal ammoniation temperature based on comprehensive cost considerations.

[0072] With other basic treatment conditions unchanged, the ammoniation time was changed to 12, 24, 36, 48, 60, and 72 h, respectively. The optimal ammoniation time was determined by the soluble sugar content in the supernatant of the vinegar lees enzymatic hydrolysis.

[0073] The results are as follows Figure 1 As shown in Figure D, with the increase of ammoniation time, the soluble sugar content showed an overall upward trend. At 60 h of ammoniation pretreatment, the soluble sugar content reached 513.23 mg / g dry matter, which was significantly higher than that of other treatment groups (P<0.05). Therefore, 60 h was selected as the optimal time for ammoniation treatment.

[0074] In summary, the conditions for ammoniation pretreatment of vinegar grains were optimized through single factor experiments, and the preparation conditions of the vinegar grains hydrolyzate used in the subsequent examples were determined: a certain amount of vinegar grains was weighed, 16% of the mass of vinegar grains was added with ammonia water, the moisture content was adjusted to 40%, and ammoniation was performed at 70°C for 60h. After the ammoniation was completed, the pH was adjusted to 5.0 with 6mol / L phosphoric acid solution, a certain amount of distilled water was added, the solid-liquid ratio was controlled to be 1:10 (w / v), and 2% (v / w dry matter) of cellulase was added, and the enzymolysis was oscillated at 50°C and 150r / min for 72h.

[0075] Example 2 Screening of strains and utilization of carbon sources by yeast CQGB3 strain

[0076] First, a culture medium for strain screening containing the vinegar grains hydrolyzate of the present invention was prepared: 1 L of the vinegar grains hydrolyzate prepared according to the optimized conditions in Example 1 (containing about 23.8 g / L of soluble sugar to provide a carbon source, and about 10.4 g / L of crude protein to provide a partial nitrogen source), 20 g of peptone, 0.5 g of magnesium sulfate heptahydrate, and 0.1 g of sodium chloride, and the pH was adjusted to 6.0.

[0077] Three yeast strains with high protein yields isolated from soil, leaves and fruits in the laboratory in the early stage: Saccharomyces cerevisiae RA2, Candida tropicali GB3, Meyerozyma caribbica GD1, were activated and cultured in the above-mentioned culture medium containing the vinegar lees hydrolyzate of the present invention at 30°C for 24 hours, and the best strain for producing single-cell protein was determined according to its utilization rate of reducing sugar in the vinegar lees hydrolyzate; the culture medium containing vinegar lees hydrolyzate without strains was used as the control group (CON). The utilization rate of reducing sugar was determined according to the following method: the reducing sugar content in the supernatant after fermentation of the control group, RA2, GB3, and GD1 was determined, and calculated according to the following formula: (reducing sugar content of the control group - reducing sugar content in the supernatant after fermentation of RA2 / GB3 / GD1) / reducing sugar content of the control group.

[0078] The results are as follows Figure 2 As shown, compared with the control group, the three strains utilized the reducing sugars in the vinegar lees hydrolyzate to different degrees. Among them, the utilization rate of reducing sugars in the vinegar lees hydrolyzate by tropical Candida GB3 reached 41.02%, which was significantly higher than that of the other two strains (P<0.05). Therefore, the tropical Candida CQGB3 of the present invention was selected as the preferred strain for producing single-cell protein for subsequent experiments.

[0079] After DNA was extracted from the screened yeast CQGB3 strain, ITS sequencing was performed to determine that this strain was Candida tropicali. This strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on July 15, 2024, with the deposit number GDMCC No: 64869.

[0080] The growth curve of the strain was determined, and its bacterial protein content and biomass were determined after 24 hours of culture. The specific method for determining the growth curve was: using a spectrophotometer to measure the OD of the culture solution at 0, 3, 6, 9, 12, 15, 18, 21, and 24 hours. 600nmThe specific method for determining the protein content is as follows: weigh 0.5 g of the sample, add 10 mL of sulfuric acid to digest it, and then determine it in an automatic Kjeldahl nitrogen analyzer. The specific method for determining the dry weight of the bacteria is as follows: centrifuge the fermentation liquid of the CQGB3 strain at 8000 rpm for 10 minutes to obtain a precipitate, wash the precipitate twice with deionized water, centrifuge it again to obtain a precipitate, dry the precipitate in a 65°C oven, weigh it, and obtain the dry weight of the bacteria. The protein dry weight is obtained by calculation, and the specific calculation method is: bacterial dry weight * protein content.

[0081] The results are as follows Figure 3 As shown, Candida tropicalis CQGB3 was in the logarithmic growth phase from 3 to 18 hours and reached the plateau phase at 18 hours. As shown in Table 1, after culturing in YPD medium for 24 hours, the bacterial protein reached 10.70±0.82 g / L and the protein content was 55.40±0.34%.

[0082] Table 1 The amount of bacteria and protein of Candida tropicalis GB3 after 24h culture in YPD medium

[0083] project Dry weight of bacteria (g / L) Protein content (%) Protein dry weight (g / L) GB3 10.70±0.08 55.40±0.34 5.93±0.01

[0084] Even when using the traditional YPD medium, the dry weight of the cells and the dry weight of protein obtained by the tropical Candida CQGB3 of the present invention are significantly higher than the data of other known yeast strains reported in the literature. The results are shown in Table 2 below.

[0085] Table 2 The amount of yeast cells and proteins after culture in YPD medium

[0086]

[0087]

[0088] We also compared the utilization efficiency of ammonia nitrogen by multiple yeast strains from different sources reported in the literature. The specific determination method was: the fermentation liquid after culturing the bacteria was added with indophenol blue reaction reagent for color development, the absorbance was measured at 637 nm, and the ammonia nitrogen content was obtained after substituting into the standard curve. The ammonia nitrogen conversion rate was calculated as follows: (ammonia nitrogen content in the blank control - ammonia nitrogen content in the supernatant after 48 hours of strain culture) / ammonia nitrogen content in the blank control.

[0089] Table 3 Ammonia nitrogen conversion rate of yeast in ammonia water medium

[0090]

[0091] The results are shown in Table 3. The results show that the yeast CQGB3 of the present invention has the highest utilization efficiency of ammonia nitrogen, exceeding 99%.

[0092] Example 3 Study on the utilization of different carbon sources by yeast CQGB3 of the present invention

[0093] Vinegar lees are rich in cellulose and hemicellulose. The hydrolyzate after hydrolysis of vinegar lees contains fermentable sugars such as glucose, xylose, and cellobiose. We compared the utilization of different types of common carbon sources by yeast CQGB3 and yeast reported in the literature. We measured the OD of the bacterial solution. 600nm The value of is used to reflect the biomass of yeast, and the results are shown in Table 4. It can be seen that the yeast CQGB3 of the present invention can not only make good use of glucose, but also make good use of xylose, cellobiose and molasses.

[0094] Table 4 Yeast utilization of different carbon sources (OD 600nm )

[0095]

[0096] Example 4 Study on the utilization rate of other nitrogen sources by yeast CQGB3 of the present invention

[0097] First, prepare the basic fermentation medium of CQGB3 strain: 20 g / L glucose, 20 g / L peptone, 0.5 g / L magnesium sulfate heptahydrate (MgSO4·7H2O), 0.1 g / L sodium chloride (NaCl), adjust the pH value to 6.0, 121°C, and sterilize for 20 min.

[0098] Under the conditions of the basic fermentation medium, inorganic nitrogen sources such as ammonium sulfate, urea, ammonium chloride, ammonia water, ammonium carbonate, and ammonium bicarbonate were used to replace the peptone in the basic fermentation medium, and the tropical Candida CQGB3 screened in Example 2 was inoculated therein, cultured at 30°C for 24 hours, and the OD 600 Screening the best inorganic nitrogen source for indicators.

[0099] The results are as follows Figure 4 As shown in Figure 2, Candida tropicalis CQGB3 has different utilization efficiencies for different types of inorganic nitrogen sources, among which ammonia is the best. 600 It reached 3.24, which was significantly higher than other types of inorganic nitrogen sources (P<0.05), which was consistent with the results of ammonia nitrogen conversion rate in the previous ammonia water medium (Table 3). Therefore, ammonia water is preferred as the best inorganic nitrogen source for tropical Candida CQGB3 fermentation. In addition, from this figure, we know that the growth effect of tropical Candida CQGB3 in inorganic nitrogen sources is not as good as the growth effect in the medium with peptone as the nitrogen source. Therefore, we further screened the organic nitrogen source suitable for CQGB3, and explored the optimal ratio of organic nitrogen source and inorganic nitrogen source considering the cost factor.

[0100] Under the conditions of basic fermentation medium, organic nitrogen sources such as tryptone, animal peptone, soy peptone, yeast powder, corn dry pulp powder, and beef powder were used to replace the peptone in the basic fermentation medium, and tropical Candida CQGB3 was inoculated into them and cultured at 30°C for 24 h. The OD 600nm Screening the best organic nitrogen source for indicators.

[0101] The results are as follows Figure 5 As shown in Figure 2, Candida tropicalis CQGB3 has different utilization efficiencies for different types of organic nitrogen sources, among which yeast powder has the best utilization effect. 600 It reached 5.95, which was significantly higher than other types of organic nitrogen sources (P<0.05). Therefore, yeast powder was preferred as the best organic nitrogen source for fermentation of Candida tropicalis CQGB3.

[0102] Under the conditions of the basic fermentation medium, the combination of the above-screened inorganic nitrogen source ammonia water (the addition ratios were 0, 20%, 40%, 60%, 80%, and 100%, respectively) and the above-screened organic nitrogen source yeast powder (the addition ratios were 100%, 80%, 60%, 40%, 20%, and 0), was used as a nitrogen source to replace the peptone in the basic fermentation medium, and tropical Candida CQGB3 was inoculated therein and cultured at 30°C for 24 h. The OD 600nm Screen the optimal ratio of complex nitrogen source for indicators.

[0103] The results are as follows Figure 6 As shown in the results of screening the combined ratio of inorganic nitrogen source ammonia water and organic nitrogen source yeast powder, we learned that the combined use of 40% yeast powder and 60% ammonia water had no significant difference in the growth effect of CQGB3 with that of using 100% yeast powder (P>0.05). Therefore, 60% ammonia water + 40% yeast powder (the mass ratio of inorganic nitrogen source and organic nitrogen source is 3:2) was selected as the optimal nitrogen source ratio for culturing CQGB3 for subsequent experiments.

[0104] Example 5 Study on Optimization of Culture Medium for Vinegar Grains Hydrolyzate

[0105] The crude protein of the vinegar lees hydrolyzate can provide part of the nitrogen source, and the residual ammonia water used for ammoniation treatment can provide an inorganic nitrogen source. Yeast powder is added according to the optimal ratio of the composite nitrogen source in Example 4, so that the weight ratio of the inorganic nitrogen source ammonia water to the yeast powder organic nitrogen source is 3:2, and the basic vinegar lees hydrolyzate fermentation medium is: 40mL vinegar lees hydrolyzate (containing about 23.8g / L of soluble sugar to provide a carbon source, and about 10.4g / L of crude protein to provide part of the nitrogen source), 6.9g / L of yeast powder, the pH value is adjusted to 6.5, 121°C, and sterilized for 20min.

[0106] Under the basic vinegar lees hydrolyzate fermentation medium conditions, 2g / L MgSO4, 2g / L ZnSO4, 2g / LMnSO4, 0.2mg / L biotin, 5mg / L pantothenic acid, and 2mg / L thiamine were added as growth factors, and the group without additional growth factors was used as the control (CON). Candida tropicalis CQGB3 was inoculated into it and cultured at 30℃ for 24h, and the dry weight of the bacteria was used as an indicator to screen the best growth factor.

[0107] The results are as follows Figure 7 As shown in the figure, different growth factors have different effects on the dry weight of CQGB3. The addition of magnesium sulfate and manganese sulfate significantly increased the dry weight of GB3 strain (P<0.05), while other growth factors had no significant effect on the dry weight of GB3 (P>0.05). During the experiment, it was found that the addition of manganese sulfate may form a small amount of black precipitate, affecting the accuracy of dry weight. Therefore, magnesium sulfate is the best growth factor to be added to GB3 medium for the production of single cell protein.

[0108] In summary, the medium components (inorganic nitrogen source, organic nitrogen source, ratio of inorganic to organic nitrogen source, growth factor) of CQGB3 yeast strain were optimized through single factor experiment, and the optimal composition of single cell protein production medium was determined as follows: the addition ratio of inorganic nitrogen source ammonia water and organic nitrogen source yeast powder was 3:2 (mass ratio), and the growth factor magnesium sulfate was added at 2g / L (final concentration of medium). The final formula was: 1L of vinegar lees hydrolyzate (containing about 23.8g / L soluble sugar to provide carbon source, and about 10.4g / L crude protein to provide part of the nitrogen source), 6.9g / L yeast powder, 2g / L magnesium sulfate heptahydrate (MgSO4·7H2O), pH value was adjusted to 6.0, 121℃, and sterilized for 20min.

[0109] Example 6 Effect of using vinegar lees hydrolyzate to produce single cell protein

[0110] According to the optimal fermentation medium conditions for single-cell protein production determined in Example 5, the yeast CQGB3 strain was expanded in a 2.5L fermenter in a shake flask according to the conventional yeast culture method, and fermented for 22 hours at 30°C, an initial speed of 220rpm, and a dissolved oxygen condition of 30%-80%. The precipitate was collected by centrifugation at 8000rpm for 10min, washed twice with sterile water, and then freeze-dried to obtain single-cell protein.

[0111] After the culture was completed, the dry weight of the bacteria was measured, and the nutritional components (crude protein, amino acids, nucleotides, and mannan) in the bacterial protein were measured. The specific determination method was as follows: crude protein was determined by the Kjeldahl method. Amino acids were analyzed by acid hydrolysis and then by a fully automatic amino acid analyzer. Nucleotides and mannan were determined by high performance liquid chromatography. The nutritional components of bacterial protein are shown in Table 5 below.

[0112] Table 5 Analysis of nutritional components of CQGB3 bacterial protein

[0113]

[0114] As can be seen from Table 5, the dry weight and yeast protein concentration of the CQGB3 yeast strain obtained by culturing the vinegar lees hydrolyzate medium of the present invention are higher than those of the YPD medium, wherein the dry weight of the yeast strain increases by 9.41 g / L and the protein concentration increases by 3.16 g / L. The contents of crude protein, total amino acids, nucleotides, etc. are only slightly different from those of the YPD medium commonly used in the prior art. These data prove that the tropical Candida CQGB3 screened by the present invention can make full use of the vinegar lees hydrolyzate treated with ammonia water as a carbon source and a partial nitrogen source, and obtain a substantially equivalent single-cell protein product in a low-cost culture medium.

[0115] Table 6 Culture medium cost accounting

[0116]

[0117]

[0118] As shown in Table 6, the cost of producing 1 kg of bacterial protein using conventional YPD medium and vinegar residue hydrolyzate medium of the present invention was analyzed, and it was found that the production cost of the vinegar residue hydrolyzate medium of the present invention was reduced by more than half compared with the YPD basic medium.

[0119] The above data prove that the tropical Candida CQGB3 screened by the present invention can utilize vinegar lees hydrolyzate, has high stress resistance, can convert inorganic nitrogen sources such as ammonia water and ammonium sulfate into its own bacterial protein, and has a very high conversion rate to ammonia water. The present invention uses ammonia water pretreatment combined with enzymolysis to prepare vinegar lees enzymolysis liquid and then ferments and produces a method for fermenting single-cell protein as a fermentation nutrient component. The process of preparing vinegar lees enzymolysis liquid by this method does not require high-temperature and high-pressure special equipment, and no additional harmful substances that affect microbial growth are produced. At the same time, the enzymolysis liquid obtained contains the carbon source and nitrogen source required for yeast growth, and has the characteristics of low cost, fast fermentation rate, and safe and reliable product quality, and can be used for large-scale production. The process of the present invention has the advantages of high single-cell protein yield, safety and environmental protection, low cost, etc., is suitable for large-scale production, can effectively promote the recycling of vinegar lees waste biomass resources, and to a certain extent alleviate the current situation of protein feed shortage in the breeding industry.

[0120] The above is only an embodiment of the present invention. The commonly known technical common sense in the scheme is not described in detail here. The ordinary technicians in the relevant field are aware of all the common technical knowledge in the technical field to which the invention belongs before the application date, can obtain all the existing technologies in the field, and have the ability to apply the conventional experimental means before that date. The ordinary technicians in the relevant field can improve and implement this scheme in combination with their own abilities under the enlightenment given by this application. Some typical commonly known technologies should not become obstacles for ordinary technicians in the relevant field to implement this application. It should be pointed out that for those skilled in the art, without departing from the structure of the present invention, they can also make some adjustments and improvements, which should also be regarded as the scope of protection of the present invention, which will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection claimed by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A tropical Candida species ( Candida tropicalis ), its deposit number in Guangdong Provincial Microbiological Culture Collection Center is GDMCC No:64869.

2. Use of the tropical yeast Candida albicans according to claim 1 in fermentation production of single-cell protein.

3. A culture medium suitable for culturing the tropical Candida according to claim 1, characterized in that: The culture medium component contains vinegar lees hydrolyzate, and the vinegar lees hydrolyzate is obtained by pre-treating the vinegar lees with ammonia water and then performing enzymatic hydrolysis.

4. The culture medium according to claim 3, characterized in that In the culture medium, the vinegar grains hydrolyzate treated with ammonia water provides a carbon source and a partial nitrogen source, and also includes a composite nitrogen source of an inorganic nitrogen source and an organic nitrogen source, wherein the inorganic nitrogen source is selected from one or more of ammonium sulfate, urea, ammonium chloride, ammonia water, ammonium carbonate, and ammonium bicarbonate, and the organic nitrogen source is selected from one or more of trypsin, animal peptone, soy peptone, yeast powder, corn dry pulp powder, and beef powder.

5. The culture medium according to claim 4, characterized in that The composite nitrogen source uses an inorganic nitrogen source provided by ammonia water used to treat the vinegar dregs, and yeast powder is used as an organic nitrogen source, and the weight ratio of the two is 1:4-4:

1.

6. The culture medium according to any one of claims 3 to 5, characterized in that The culture medium also includes growth factors, which are selected from one or more of magnesium sulfate, zinc sulfate, manganese sulfate, biotin, pantothenic acid, and thiamine.

7. A method for producing single-cell protein by yeast fermentation, characterized in that The following steps are involved: 1) treating the vinegar grains with ammonia water and performing enzymatic hydrolysis to obtain a vinegar grains hydrolyzate; 2) adding organic nitrogen source yeast powder to the vinegar lees hydrolyzate obtained in step 1) to form a composite nitrogen source with the inorganic nitrogen source ammonia water in the vinegar lees hydrolyzate, wherein the weight ratio of the ammonia water to the yeast powder is 1:4-4:1, and then adding 0.2-5 mg / L of growth factor to prepare a yeast culture medium; 3) Inoculating the tropical Candida albicans described in claim 1 into the yeast culture medium obtained in step 2), fermenting to obtain yeast fermentation liquid, centrifuging and drying the precipitate to obtain single cell protein.

8. The yeast fermentation production method of single-cell protein according to claim 7, characterized in that: The step 1) specifically includes the following steps: S1 vinegar lees pretreatment: the vinegar lees through an 80 mesh sieve standby; S2. Ammonia pretreatment: Add 16% ammonia water by mass to the vinegar residue obtained in step S1, control the moisture content to 40%, seal and ammoniaize at 70°C for 60h; S3. Scattered ammonia treatment: After the ammonia treatment is completed, the excess ammonia is recovered by air stripping; S4. Enzymatic hydrolysis: Add an appropriate amount of distilled water to the ammoniated vinegar residue obtained in step S3 to adjust the material-liquid ratio to 1:10 (w / v), shake in a shaker at 150 rpm for 30 min to balance the ammonia nitrogen, then adjust the pH to 5.0 with 6 mol / L phosphoric acid solution, add 2% cellulase to the mixture, and place it in a shaker at 50 ° C and 150 rpm for enzymatic hydrolysis for 72 h; S5. Centrifugal filtration: After the enzymatic hydrolysis is completed, centrifuge at 9000 rpm for 10 min to obtain the vinegar lees hydrolyzate.

9. The yeast fermentation production method of single-cell protein according to claim 8, characterized in that: The method specifically comprises the following steps: 1) Weigh a certain amount of vinegar lees, add 25% ammonia water (16% of the weight of the vinegar lees) to adjust the moisture content to 40%, and perform ammonia treatment at 70℃ for 60 h. After ammonia treatment, adjust the pH to 5.0 with 6 mol / L phosphoric acid solution, add appropriate amount of distilled water to control the solid-liquid ratio to 1:10 (w / v), add 2% (v / w dry matter) cellulase, perform enzymolysis at 50℃ and 150 r / min for 72 h, and centrifuge at 9000 rpm for 10 min to obtain the vinegar lees hydrolyzate; 2) taking an appropriate amount of the vinegar lees hydrolyzate in step 1), adding organic nitrogen source yeast powder, and combining it with inorganic nitrogen source ammonia water in the vinegar lees hydrolyzate to form a composite nitrogen source, wherein the mass ratio of ammonia water to yeast powder is 3:2, and adding 2 g / L of growth factor magnesium sulfate to prepare a yeast culture medium; 3) The tropical Candida albicans described in claim 1 is inoculated into the yeast culture medium described in step 2), and fermented at 30° C. and 220 rpm for 48 h. After centrifugation at 8000 rpm for 10 min, the precipitate is dried to obtain single cell protein.

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