Geotrichum candidum GZCTW-1 and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and relates to a yeast strain from a dairy product and the technical field of a high-density fermentation process of the yeast strain. The invention provides Geotrichum candidum GZCTW-1, and the preservation number of the Geotrichum candidum GZCTW-1 is CGMCC (China General Microbiological Culture Collection Center) No. 33018. The Geotrichum candidum GZCTW-1 is preserved in the China General Microbiological Culture Collection Center on December 10, 2024, the preservation number of the strain is CGMCC No.33018, the center is called CGMCC for short, and the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The Geotrichum candidum GZCTW-1 provided by the invention has the characteristics of acid resistance and high carbon source utilization efficiency, and can grow well at the temperature of 28-32 DEG C and the pH value of 3.0-4.0.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a yeast strain derived from dairy products and the technical field of high-density fermentation process thereof. Background Art

[0002] Geotrichum candidum is commonly found in moist substrates, including soil, plants, fruits, water bodies, the digestive tracts of humans and other animals, and dairy products such as milk, cream, and cheese. Geotrichum candidum has the characteristics of both filamentous fungi and yeasts, and grows and reproduces quickly. It is not restricted by substrates, climate, and regions, has stable production, and has strong environmental adaptability. The optimal growth temperature is 25-28°C, and the optimal pH is 5-7. Generally, the nutritional composition of well-cultivated dry thallus of Geotrichum candidum is as follows: crude protein 40%-60%, crude fat 3%-6%, nitrogen-free extract 3%-10%, crude ash 8%-10%, calcium 3%-11%, phosphorus 0.01%-0.1%, and some strains of Geotrichum candidum have the characteristics of producing fragrance.

[0003] Corn steep liquor, as one of the byproducts of corn deep processing, is the soaking water remaining after corn kernels are soaked and softened with a solution containing sulfurous acid or sulfite at about 50°C and the softened corn kernels are separated. This soaking water contains a large amount of sulfuric acid or sulfite, soluble substances in corn, such as starch, maltose, soluble protein, ammonium salt, phosphorus, various trace elements, and various vitamins, as well as lactic acid and ethanol produced by lactic acid bacteria and yeast fermentation during the soaking process. Corn steep liquor is cheap, nutritious, and is a good fermentation substrate. In the modern society where resources are increasingly limited, if corn steep liquor is directly treated as waste, not only is the environmental pressure huge, but it is also very wasteful.

[0004] During high-density fermentation, as the bacteria accumulate in large quantities, the viscosity of the fermentation liquid increases significantly and becomes a non-Newtonian fluid, which has a significant impact on the transfer of oxygen and the mass transfer of nutrients. Therefore, in the conventional fermentation process of yeast, a refined feeding strategy is required to achieve rapid growth of yeast and accumulation of biomass. Summary of the invention

[0005] In view of this, the present invention provides a Geotrichum candidum strain GZCTW-1, whose deposit number is CGMCC No.33018.

[0006] The Geotrichum candidum GZCTW-1 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration on December 10, 2024. The deposit number of the strain is CGMCC №.33018. The center is abbreviated as CGMCC. The address of the center is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0007] The Geotrichum candidum GZCTW-1 provided by the invention has the characteristics of acid resistance and high carbon source utilization efficiency, and can grow well at a temperature of 28 to 32° C. and a pH of 2.5 to 5.0.

[0008] The present invention optimizes high-density fermentation and batch-fed fermentation processes by improving the fermentation medium, so that the cell dry weight of Geotrichum candidum GZCTW-1 can reach 41.5 g / L within 7 hours. The cost of the optimized fermentation medium is greatly reduced, and low-cost and high-efficiency single-cell protein production is achieved.

[0009] The invention provides a strain GZCTW-1 which can grow well at a temperature of 28 to 32° C. and a pH of 3.0 to 4.0, can use lactic acid as a carbon source for growth, has high carbon source utilization efficiency and high protein content.

[0010] The D1 / D2 region sequence of Geotrichum candidum GZCTW-1 is shown as SEQ ID No. 1 in the sequence list, and the ITS region sequence is shown as SEQ ID No. 2 in the sequence list.

[0011] The present invention also provides a fermentation medium with 25% corn soaking liquid added, the medium comprises two parts: ① 10g / L glucose, 5g / L ammonium sulfate, 2.5g / L potassium dihydrogen phosphate sterilized at 115°C for 30 minutes; ② corn steep liquor sterilized at 60°C for 30 minutes. When in use, add 25% of ② to the sterilized ① and mix well.

[0012] The third object of the present invention is to provide a high-density fermentation culture method for the Geotrichum candidum CGMCC No.33018 strain, which uses the residual sugar content and dissolved oxygen changes to adjust the feeding rate, and does not feed initially. When the residual sugar decreases to below 5g / L, feeding is gradually started to maintain the residual sugar concentration at 5-15g / L. In the late fermentation stage, when the dissolved oxygen decreases to below 2%, the upper limit of the instrument's oxygen supply is reached, and the bacterial growth is limited by the dissolved oxygen, and the feeding is stopped. When the residual sugar is consumed, the fermentation ends.

[0013] The method is as follows: (1) the seed liquid of Geotrichum candidum is introduced into the fermentation tank, the initial stirring speed is 500 rpm, the initial ventilation volume is 2 L / min, and the stirring speed and ventilation volume are set to automatically adjust according to the dissolved oxygen (60%); (2) when the glucose drops below 5 g / L, feed is started, initially at 60 to 80 mL / h, and the feed rate is gradually increased, and sampling is performed every 1-2 hours. (3) when the dissolved oxygen drops below 2%, and the ventilation volume and stirring rate are increased to the maximum limit of the instrument, feed is stopped, and the fermentation is terminated after the residual sugar is consumed (less than 1 g / L).

[0014] The stepwise increase in the feeding rate means increasing the feeding rate by 40 to 60 mL / h every hour. The stepwise decrease in the feeding rate means decreasing the feeding rate by 40 to 60 mL / h every hour when the residual sugar is higher than 15 g / L.

[0015] In a specific embodiment of the present invention, the time when the dissolved oxygen drops below 2% is 6 hours.

[0016] The specific steps include: (1) After the fermentation medium is sterilized, keep it warm to 28-30°C, inoculate it with Geotrichum candidum seed liquid, inoculate it with an inoculation amount of 10%, and the initial OD 600 16, stirring speed 500rpm, maintain pH 3.8-4.2, dissolved oxygen control 58%-62%; (2) After 3h of fermentation, the glucose concentration was lower than 5g / L, and the feed was added at a rate of 60-80 mL / h, and the feeding rate was increased by 40-60 mL / h every hour. The SBA-40E biosensor analyzer was used to measure the changes in glucose and lactic acid concentrations (g / L) during the fermentation process. The stirring speed, ventilation volume and dissolved oxygen (60%) were linked and automatically controlled. (3) After 6h of fermentation, when the ventilation volume and stirring speed reached the maximum value of the fermentation equipment and the dissolved oxygen was lower than 2%, the feeding was stopped and the fermentation continued at the highest ventilation volume and stirring speed. After 7h, the residual sugar was lower than 1g / L and the fermentation was stopped.

[0017] The fermentation is terminated, in a specific embodiment of the present invention, when the fermentation is completed after 7 hours.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are:

[0019] (1) High carbon source utilization efficiency: The GZCTW-1 strain of Geotrichum candidum derived from dairy products of the present invention has strong carbon source utilization ability and acid resistance. Under specific environmental conditions (such as pH 3.0-4.0, temperature 28-32°C), it can effectively utilize corn steep liquor as a fermentation substrate, thereby reducing fermentation costs and increasing the yield of bacterial protein.

[0020] (2) Reducing production costs: The fermentation medium of the present invention not only solves the problem of waste disposal but also significantly reduces the cost of the medium by adding a portion of corn steep liquor, a cheap waste, to replace the nitrogen source yeast extract powder and peptone.

[0021] (3) High-density fermentation method for efficient production: The fermentation flow acceleration is controlled according to the changes in residual sugar and dissolved oxygen, which can not only understand the fermentation dynamics in real time, but also accurately control the materials used to avoid wasting carbon sources. The fermentation tank stirring speed and ventilation equipment are linked to control the dissolved oxygen to 60%, which enables rapid and stable growth of Geotrichum candidum. Conventional yeast high-density fermentation takes several days, but the present invention can complete the fermentation in only 7 hours, greatly shortening the fermentation cycle, which not only improves the utilization rate of the fermentation tank, but also saves time and cost. The fermentation control is timely and accurate, and the equipment requirements are relatively simple.

[0022] (4) Industrial prospects: The strain of the present invention has good prospects for industrial application and is suitable for the food, feed and other industries. In particular, in the context of the growing demand for protein, this technology provides a sustainable and economical solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The morphology of strain GZCTW-1 on YPD agar medium, where A is the front view and B is the microscopic view.

[0024] Figure 2 The morphology of strain GZCTW-1 on PDA agar medium, where A is the front view and B is the microscopic view.

[0025] Figure 3 OD after adding different concentrations of corn soaking liquid in shake flask experiment 600 Graph of changes over time.

[0026] Figure 4 During the first fermentation, the feed amount, ventilation volume, stirring speed and dissolved oxygen changed within 11 hours.

[0027] Figure 5 In the first fermentation, changes in bacterial protein content, glucose and lactic acid content and dry weight within 11 hours.

[0028] Figure 6 Liquid chromatography of various sugar alcohols in the culture medium at 0h and 11h in the first fermentation

[0029] Figure 7 Liquid chromatograms of various acids in the culture medium at 0h and 11h during the first fermentation.

[0030] Figure 8 During the second fermentation, the feed amount, ventilation volume, stirring speed and dissolved oxygen changed within 7 hours.

[0031] Fig. 9 In the second fermentation, the content of bacterial protein, glucose and lactic acid and the changes of dry weight within 7 hours.

[0032] Fig.10Liquid chromatograms of various sugar alcohols in the culture medium at 0h and 7h in the second fermentation

[0033] Fig.11 Liquid chromatograms of various acids in the culture medium at 0h and 7h in the second fermentation. DETAILED DESCRIPTION

[0034] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased from the market.

[0035] Example 1

[0036] Isolation, purification and identification of Geotrichum candidum

[0037] By observing the colony characteristics under solid plate culture, observing the cell morphology under a microscope, and identifying by 26S rDNA and ITS sequencing, the strain GZCTW-1 was identified as Geotrichum candidum. The strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on December 10, 2024, with the deposit number: CGMCC №.33018; the center is referred to as CGMCC, and the address of the center is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0038] Sample collection: In November 2020, samples were collected from cheese in Xinjiang Uyghur Autonomous Region. The samples were stored in 50mL centrifuge tubes and brought back to the laboratory at low temperature.

[0039] Sample separation: Using the dilution spreading method, take 1 mL of the dairy sample and place it simultaneously in a sterile 250 mL conical flask, each containing 99 mL of sterile distilled water, with a final dilution of (1:100). The flasks are then shaken on a mechanical orbital shaker for 30 minutes.

[0040] Aseptically transfer 100 μL of the diluted solution onto YPD and PDA agar media and spread evenly.

[0041] Incubate the plates (5 plates for each culture medium type) at 17°C, 25°C and 30°C for 3-5 days, pick a single colony with a morphology similar to that of the Geotrichum candidum strain, and transfer it to a new YPD plate using a sterile inoculation loop to ensure that a pure culture is obtained. Perform purification culture again to ensure that no foreign bacteria grow in the colony.

[0042] Colony morphology: on YPD plates Figure 1 and PDA tablets such as Figure 2In YPD agar medium, the colony diameter was 40-50 mm, and the front was white, rough, wrinkled, and filamentous. In PDA medium, the colony diameter was 40-45 mm, and the front was rough, and the strain was named GZCTW-1.

[0043] Molecular identification method: The fungal DNA was extracted using the alkaline lysis method, and PCR amplification was performed using universal primers ITS1 / ITS4 and NL1 / NL4 as primers. The specific implementation content is as follows.

[0044]

[0045] The sequencing results are as follows:

[0046] The D1 / D2 region sequence of strain GZCTW-1 is shown in SEQ ID No. 1 in the sequence listing, and the ITS region sequence of strain GZCTW-1 is shown in SEQ ID No. 2 in the sequence listing.

[0047] Example 2

[0048] Acid resistance test of Geotrichum candidum

[0049] 1. Culture medium preparation

[0050] The culture medium is YPD liquid medium, the composition content is: 2% glucose, 2% peptone, 1% yeast extract powder

[0051] 2. Acid resistance test

[0052] (1) Pick a single colony of Geotrichum candidum CGMCC No.33018 plate and inoculate it into YPD liquid medium sterilized at 115℃ for 30min. Fill the medium with 100mL / 250mL. Incubate at 30℃ for 18h, 200r / min to obtain seed solution.

[0053] (2) Prepare 250 mL of YPD and divide it into 5 bottles, with a filling volume of 50 mL / 100 mL. Use 36% hydrochloric acid to adjust the pH to make five pH gradients of 2.0, 2.3, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0. Divide each gradient into three portions, two of which are placed in test tubes, each with 5 mL; the remaining 40 mL is left in the bottle for pH measurement after sterilization. Sterilize at 115℃ for 30 minutes

[0054] (3) The seed solution in step 1 was inoculated into the test tube in step 2, with an inoculum volume of 0.5 mL. Two test tubes were inoculated for one pH gradient. The cells were cultured at 30°C for 18 h and 200 r / min. The bacterial growth was observed every day. The specific data are shown in Table 1. Geotrichum candidum CGMCC No. 33018 could still grow weakly at pH 2.61.

[0055] Table 1 Comparison of bacterial growth at different pH

[0056]

[0057] (+++ (strong growth); ++ (growth); + (weak growth); - (no growth))

[0058] Example 3

[0059] Experiment on the effect of corn soaking liquid addition on the growth rate of strains

[0060] (1) Pick a single colony of Geotrichum candidum CGMCC No.33018 plate and inoculate it into YPD liquid medium sterilized at 115℃ for 30min. Fill the medium with 100mL / 250mL, culture at 30℃ for 18h, 200r / min to obtain seed solution.

[0061] (2) Prepare the culture medium with different contents of corn steep liquor according to the following formula:

[0062]

[0063] (3) Inoculate the candidum seed solution cultured in step 1 into the 6 different culture media in step 2, with a bottling volume of 100 mL / 250 mL and an inoculum volume of 1 mL. Culture at 30°C for 18 h and 200 r / min for 80 h. Take 1 mL of samples at 4, 8, 12, 24, 48, and 80 h to record the OD values. 600 , observe the growth of bacteria, the results are as follows Figure 3 When the corn steep liquor was added at 25%, the growth rate was similar to that of YPD.

[0064]

[0065] Example 4

[0066] High density fermentation of Geotrichum candidum

[0067] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article.

[0068] In the present invention, the term "corn soaking liquid" has a well-known meaning in the art, and is a byproduct of the corn processing industry, which is the soaking water remaining after the corn kernels are soaked and softened in a solution containing sulfurous acid or sulfite and the softened corn kernels are separated. This soaking water contains a large amount of soluble substances in corn, such as soluble sugars, soluble proteins, inorganic nitrogen (various ammonium salts), minerals (calcium, phosphorus), various trace elements, and various vitamins, etc., and is rich in nutritional value.

[0069] 1. Culture medium preparation:

[0070] (1) The primary seed culture medium is YPD medium, and its composition and content are as follows: peptone 20 g / L, yeast extract powder 10 g / L, glucose 20 g / L.

[0071] (2) Secondary seed culture medium: YPD medium, composition and content are as follows: peptone 20 g / L, yeast extract powder 10 g / L, glucose 20 g / L.

[0072] (3) Fermentation medium: 10 g / L glucose, 5 g / L ammonium sulfate, 2.5 g / L potassium dihydrogen phosphate, 25% corn soaking liquid, 10% inoculation. The ammonia nitrogen content after inoculation is 1.5-2.0 g / L.

[0073] 2. High-density fermentation method of Saccharomyces cerevisiae

[0074] (1) Take a single colony of Geotrichum candidum CGMCC No. 33018 plate, inoculate it into a primary seed culture medium with a liquid volume of 50 mL / 250 mL, and culture it at 30° C. for 18 h at 200 r / min to obtain a primary seed solution.

[0075] (2) Secondary seed liquid culture of Geotrichum candidum

[0076] The first-level seed liquid of Geotrichum candidum cultured in step 1 was inoculated into two bottles of second-level seed culture medium with a liquid volume of 100 mL / 500 mL and an inoculation amount of 1%. The culture was carried out at 30° C. for 18 h and 200 r / min to obtain the second-level seed liquid.

[0077] (3) Geotrichum candidum high-density fermentation

[0078] First fermentation:

[0079] Fermentation medium part 1: glucose 10g / L, ammonium sulfate 5g / L, potassium dihydrogen phosphate 2.5g / L 1.3L115℃, after 30min sterilization, connect to the Xin Annuo 5L fermenter, control the temperature at 28-32℃, inoculate with 500mL of No. 2 corn soaking liquid sterilized at 65℃ for 30min, inoculate with 200mL of secondary seed liquid, inoculate with 10%, initial OD 6001, ammonia nitrogen content 1.58g / L, pH adjusted to 3.8, stirring speed 500rpm, fermentation started, set automatic pH adjustment, maintain pH 3.8-4.2, dissolved oxygen control 60%, linkage stirring rate and ventilation, start feeding feed medium after 3h of fermentation, monitor the changes of glucose concentration and lactic acid concentration in real time during fermentation by SBA-40E biosensor analyzer, and adjust the flow acceleration. The fermentation broth of the first fermentation for 11h.

[0080] After the first fermentation, the amount of stem cell obtained was 48.37 g / L, the residual ammonia nitrogen was 3.69 g / L, the residual glucose content was 0.14 g / L, and the residual lactose content was 0.12 g / L. The results are as follows Figure 4 As shown in Figure 5, the carbon source utilization efficiency is 0.64 grams of dry bacteria per gram of glucose, and the final production rate of dry bacteria is 4.64g / L / h, that is, 4.64g of dry bacteria can be produced per liter of fermentation medium per hour.

[0081] The fermentation broth obtained at 0h and 11h of fermentation was subjected to liquid phase determination of sugar and alcohol acids. The results were as follows: Figure 6 , as shown in 7. And the chemical oxygen demand COD was measured. The result dropped from 33.7g / L at 0h to 10.81g / L at 11h.

[0082] Second fermentation:

[0083] The fermentation liquid from the first fermentation was used as the seed liquid for the second fermentation. 200 mL of the fermentation liquid from the first fermentation was added to 1.8 L of the mixed fermentation medium. The initial OD 600 It is 16, and the ammonia nitrogen content is 1.58g / L.

[0084] Adjust pH to 3.8, stirring speed 500rpm, fermentation starts, set automatic pH adjustment, maintain pH 3.8-4.2, control dissolved oxygen at 60%, and link stirring speed and ventilation volume.

[0085] After 1 h of fermentation, the feed-batch fermentation medium was added. The changes in glucose concentration and lactic acid concentration during the fermentation process were monitored in real time by an SBA-40E biosensor analyzer to adjust the flow acceleration.

[0086] When the glucose concentration was lower than 5 g / L after 3 h of fermentation, feeding was carried out at a rate of 60-80 mL / h, and the feeding rate was increased by 40-60 mL / h every hour. The SBA-40E biosensor analyzer was used to measure the changes in glucose and lactic acid concentrations (g / L) during the fermentation process. The stirring speed, ventilation volume and dissolved oxygen (60%) were linked and automatically controlled.

[0087] After 6 hours of fermentation, when the ventilation volume and stirring speed reached the maximum value of the fermentation equipment and the dissolved oxygen was lower than 2%, the feeding was stopped and the fermentation was continued at the highest ventilation volume and stirring speed.

[0088] After 7 hours of fermentation, the residual sugar content was lower than 1g / L and the fermentation was stopped.

[0089] After the second fermentation, the dry cell volume was 41.48 g / L, the residual ammonia nitrogen was 0.23 g / L, the residual glucose content was 0.10 g / L, the residual lactic acid content was 0.10 g / L, and the carbon source utilization efficiency was 0.56 g of dry cell per gram of glucose. Figure 8 and 9 shown.

[0090] The second fermentation proved the feasibility of using the first fermentation liquid as seed liquid for cascade production.

[0091] The fermentation broth obtained at 0h and 7h was subjected to liquid phase determination of sugar alcohol acid. The results were as follows: Fig.10 , as shown in 11. And the chemical oxygen demand COD was measured. The result dropped from 30.8g / L at 0h to 10.83g / L at 7h.

Claims

1. Geotrichum candidum GZCTW-1, whose deposit number is CGMCC No.33018.

2. Use of Geotrichum candidum GZCTW-1 according to claim 1 in preparing an acid-resistant bacterial agent.

3. Use of Geotrichum candidum GZCTW-1 according to claim 1 in preparing an acid-resistant bacterial agent, characterized in that: The acid-resistant pH value is 2.5 to 5.

0.

4. Use of Geotrichum candidum GZCTW-1 in the preparation of an acid-resistant bacterial agent according to claim 3, characterized in that: The acid-resistant pH value is 3.8 to 4.

2.

5. The fermentation and culturing method of Geotrichum candidum GZCTW-1 according to claim 1, characterized in that: The steps include: The mixture of Geotrichum candidum GZCTW-1 seed solution and 9 times volume of fermentation medium was added to the fermentation tank, the pH was adjusted to 3.8-4.2, the dissolved oxygen was controlled to 60%, and the stirring speed was 500 rpm; When the glucose concentration is below 5 g / L, feed at a rate of 60-80 mL / h; When the ventilation volume and stirring speed reach the maximum value of the fermentation equipment and the dissolved oxygen is lower than 2%, stop feeding and continue fermentation at the highest ventilation volume and stirring speed; When the residual sugar level is lower than 1g / L, the fermentation is stopped.

6. The fermentation and culturing method of Geotrichum candidum GZCTW-1 according to claim 5, characterized in that: The initial OD of the seed solution 600 It is 16, and the ammonia nitrogen content is 1.58g / L.

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