A high-density culture method for Interneuron phagocytosis and its application

Abstract

The present invention discloses a high-density culture method of Interneuron pulex and its application, and belongs to the field of microbial fermentation technology. By adopting a strategy of timed or continuous feeding, the fermentation efficiency of Interneuron pulex is significantly improved, and the high-density growth of the strain is achieved. After systematic optimization of the culture medium formula and fermentation parameters, the dry weight of the bacteria in the shake flask reached more than 80g / L, and the highest reached 104g / L in the fermenter, which was more than 12 times higher than before feeding. The present invention takes industrial production as the goal, optimizes the nutrient supply and environmental control during the fermentation process, greatly improves the dry weight yield of the bacteria, and successfully realizes the efficient and high-density culture of the strain. The wide application of the present invention in the microbial fermentation industry, especially in food, feed and other protein demand fields, has huge market potential.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial fermentation, and in particular to a high-density fermentation culture method of Interneuron CGMCC No. 40496 and an application thereof in the efficient production of alternative proteins. Background Art

[0002] Fungal protein is a safe, sustainable, nutritious and environmentally friendly source of protein. As an alternative protein source, fungal protein can effectively reduce dependence on traditional meat protein and provide a stable protein supply, thereby solving food safety issues. The production process of fungal protein is environmentally friendly, requiring significantly less land, water and energy than traditional animal husbandry, and significantly reducing greenhouse gas emissions. In addition, fungi have the advantages of fast growth and short production cycle.

[0003] Neurocystis is a naturally occurring fungus with a long history of use in the food industry. In Chinese patent CN 116162554 A, a strain of Neurocystis CGMCC NO. 40496 is disclosed, which is derived from Pu'er tea, a traditional fermented food in my country. This strain can grow rapidly using cheap organic nitrogen sources and produce a large amount of nutrient-rich mycelium. In this patent, the highest yield of fungal protein is 4.96g / L. However, in the process of industrial production, in order to improve economic benefits, further increase the yield of fungal protein per unit volume, help reduce production costs, improve production efficiency, reduce environmental pollution and save energy, thereby improving the market competitiveness of fungal protein products.

[0004] High-density fermentation is a key technology to improve the volumetric productivity of fermentation. It has been successfully applied to the production of strains such as Escherichia coli, Bacillus, algae, yeast and their metabolites. However, there are relatively few reports on high-density fermentation of filamentous fungi. For example, Chen Gong et al. optimized the medium composition and fermentation conditions to achieve a mycelium yield of 39.77 g / L of Monascus after 16 days of fermentation; Graziella Chuppa-Tostain et al. used winery wastewater to ferment Aspergillus niger, and the biomass reached 35.29 g / L after 10 days. However, high-density fermentation of filamentous fungi faces many technical challenges, including low substrate solubility, substrate restriction on growth, inhibition of metabolites, and high oxygen demand. As the concentration of filamentous fungi increases, the increase in medium viscosity is also a major limiting factor for high-density fermentation.

[0005] Therefore, the present invention proposes innovative solutions from aspects such as culture medium raw material selection and fermentation process control, develops a high-density fermentation medium and fermentation method for Interneuron sp., provides important technical support for the industrial production of Interneuron sp., and has significant practical application value. Summary of the invention

[0006] The purpose of the present invention is to provide a high-density fermentation method for Interneuron CGMCC NO.40496 to solve the problems existing in the above-mentioned prior art. Through this method, the bacterial concentration can reach more than 100g / L, realizing the high-density culture of Interneuron, and laying a solid theoretical and technical foundation for the subsequent industrial production of Interneuron protein.

[0007] To achieve the above object, the present invention provides the following scheme:

[0008] The present invention provides a high-density fermentation method of Interneuron sp., which comprises the following steps:

[0009] The seed liquid of Interneuron (filamentous fungus FF171) with a deposit number of CGMCC NO. 40496 is inoculated into the initial fermentation medium for fermentation, and the feed medium is added regularly during the fermentation process until the fermentation is completed;

[0010] The composition and proportion of the initial fermentation medium are: 25 mL / L carbon source, 4.72 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.75 g / L MgSO4·7H2O, 0.03 g / L EDTA, 4.57 g / L organic nitrogen source, 100 mL / L each of 7 trace metal mother solutions, pH 6.2, and 1 mL / L mixed vitamin mother solution is added after sterilization;

[0011] The composition and proportion of the feed medium are: carbon source 570 mL / L, (NH4)2SO4 54.3-108.5 g / L, KH2PO4 40.3-80.5 g / L, MgSO4·7H2O 8.6-17.2 g / L, EDTA 0.35-0.7 g / L, organic nitrogen source 52.6-105.1 g / L, 7 trace metal mother solutions 2.3 mL / L each, and after sterilization, 23 mL / L of mixed vitamin mother solution is added;

[0012] The seven trace element mother solutions are: CaCl2·2H2O 0.09 g / mL, ZnSO4·7H2O 0.09 g / mL, FeSO4·7H2O 0.06 g / mL, MnCl2·2H2O 0.016 g / mL, Na2MO4·2H2O 0.008 g / mL, CuSO4·5H2O 0.006 g / mL, KI 0.002 g / mL;

[0013] The mixed vitamin mother solution comprises: aminobenzoic acid 0.2 g / L, niacin 1 g / L, calcium pantothenate 1 g / L, pyridoxine hydrochloride-vitamin B6 1 g / L, thiamine hydrochloride-vitamin B1 1 g / L, biotin 0.05 g / L, and inositol 25 g / L.

[0014] The carbon source is selected from one or more of glucose, cane molasses, maltose, starch hydrolyzate, bran, and bean dregs hydrolyzate; the organic nitrogen source is selected from one or more of yeast powder, corn syrup, bean cake, soybean meal, and corn protein.

[0015] Preferably, the carbon source sugarcane molasses in the initial fermentation medium is added in an amount until the total sugar content of the medium is 8-12 g / L; the carbon source sugarcane molasses in the feed medium is added in an amount until the total sugar content of the medium is 200-260 g / L.

[0016] Specifically, the seed liquid is inoculated into the initial fermentation medium at a ratio of 5 to 15%.

[0017] More specifically, the preparation method of the seed solution is as follows: inoculate the Neurocystis spp. stored at -80°C onto a PDA solid plate and culture at 35°C for 2-3 days until the mycelium grows all over the plate, cut the activated Neurocystis spp. plate into a seed culture medium, and culture at 35°C and 220 rpm for 24 hours to obtain a seed solution;

[0018] The seed culture medium comprises: 10 g / L glucose, 5 g / L yeast extract, 7.08 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.75 g / L MgSO4·7H2O, 100 mL / L of each of 7 trace metal elements, 1 mL / L vitamins, and a pH of 6.2.

[0019] In a specific embodiment, the high-density fermentation is a shake flask high-density fermentation: the shaker speed is 200-300 rpm, the temperature is 30-38°C, the defoaming agent is soybean oil; the feeding interval is 6-12h, and the total sugar content in the shake flask is maintained at 10-20g / L after feeding.

[0020] In another specific embodiment, the high-density fermentation is a high-density fermentation in a fermenter: the rotation speed is adjusted according to the mycelium morphology to maintain the mycelium length between 100 and 500 mm; the ventilation volume is 1 to 3 VVM, and the dissolved oxygen is maintained at not less than 15% in the early stage of fermentation, not less than 10% in the middle stage, and not less than 5% in the late stage.

[0021] Preferably, the fermentation tank rotation speed is 400-800 rpm; feeding starts at the 4th to 7th hour of fermentation, and the feeding speed is adjusted according to the total sugar consumption rate to maintain the total sugar content in the fermentation tank at 10-15 g / L; the fermentation temperature is 30-38°C.

[0022] More preferably, the fermentation tank rotation speed is 500-700 rpm; feeding starts at the 5th to 6th hour of fermentation, and the feeding speed is adjusted according to the total sugar consumption rate to maintain the total sugar content in the fermentation tank at 12-14 g / L; the fermentation temperature is 30-38°C.

[0023] The present invention provides a method for producing a substitute protein by using the Interneuron sp. After the fermentation is completed, the fermentation broth is filtered through a 500-mesh gauze to collect a sample, and washed with sterile water to obtain wet biomass, or further freeze-dried or conventionally dried to obtain biomass powder, namely, the substitute protein.

[0024] The present invention provides a protein product that partially or completely replaces animal-derived protein, characterized in that part or all of the animal-derived protein in the product is replaced by the replacement protein obtained by the method.

[0025] Specifically, the product can be used in food, beverage, health care product or feed.

[0026] The present invention discloses the following technical effects:

[0027] From the perspective of industrial production, the present invention systematically optimizes the formula and culture process of the high-density fermentation medium of Interneuron, and clarifies the composition and ratio of the initial medium and feed medium for high-density fermentation. The optimized medium uses agricultural byproduct sugarcane molasses as a carbon source and corn syrup as a nitrogen source, thereby significantly reducing the production cost of fungal protein. In the shake flask high-density fermentation using the optimized medium and feeding method, the fermentation cycle was 76h, the bacterial concentration reached more than 80g / L, which was 12.9 times that before feeding, the carbon conversion rate was 67.5% (g dry weight / g total sugar), the nitrogen conversion rate was 35.7%, and the bacterial growth rate was 1.07g / L / h. Furthermore, the fermentation tank was used for high-density fermentation using the optimized medium and feeding method, the fermentation cycle was 96h, and the bacterial concentration after fermentation reached 104g / L. During this process, the carbon conversion rate was 70.7% (g dry weight / g total sugar), the bacterial growth rate was 1.08g / L / h, the total protein content was 36.9g / L, and the nitrogen conversion rate was 30.9%. Compared with shake flask fermentation, the bacterial concentration in the fermenter increased by 30%, the carbon conversion rate increased by 3.2%, and the upper limit of high-density fermentation was further increased. In particular, compared with before optimization, the method of the present invention increased the bacterial concentration by more than 12 times and the growth rate by more than 5 times, achieving high-density culture of Interneuron sp. This provides an important theoretical basis and technical support for the industrial production of Interneuron sp. protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the change of mycelial morphology during the fermentation of Intercellular Sporangium.

[0029] Biomaterial deposit information: filamentous fungus FF171, classified as Interstitial Neurocystis Neurospora intermedia It was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on February 10, 2023. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is CGMCCNO.40496. DETAILED DESCRIPTION

[0030] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0031] Example 1: Effects of different carbon sources on high-density culture of Neurocystis

[0032] Carbon sources that can be used for fermentation include glucose, maltose, sugarcane molasses, and hydrolyzates of bran, palm meal, and soybean dregs as agricultural waste. Among them, sugarcane molasses is valued for its wide range of sources, low price, and rich nutrients, but it contains 5-10% colloidal substances and 10-12% ash, so it needs to be pretreated before being used for microbial fermentation. Starch hydrolyzate is easy to obtain and has low cost, but it may require more energy consumption during the production process. Maltose has good fermentation performance and is easily utilized by microorganisms, but its price is relatively high. Bran, palm meal, and soybean dregs hydrolyzate are agricultural wastes with low cost and abundant sources, but they may contain substances that inhibit fermentation. The above six organic carbon sources were used for fermentation of Intersporus after appropriate treatment to explore the effects of different carbon sources on the yield and cost of high-density fermentation.

[0033] The Intertype Neurocystis CGMCC NO.40496 (also disclosed in CN116162554A) cultured on the PDA plate was cut into pieces and inoculated into the shaking flask seed liquid medium for fermentation culture. The culture temperature was 30-38°C, preferably 35°C, the shaking speed was 200-250rpm / min, preferably 220rpm / min, and the culture time was 18-30h to obtain seed liquid. The seeds were inoculated into the fermentation medium of different carbon sources at an inoculation rate of 1%, and fermented at 35°C and 220rpm for 24h. After the fermentation was completed, the bacteria were collected by filtering through 500 mesh gauze, and the dry weight and total protein content after fermentation were determined. The results are shown in Table 1.

[0034] The remaining components of the fermentation medium include: 5 g / L yeast powder, 7.08 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.75 g / L MgSO4·7H2O, 0.03 g / L EDTA, 100 mL / L each of 7 trace metal mother solutions, 1 mL / L mixed vitamin mother solution, pH 6.2.

[0035] Table 1. Effects of different carbon sources on the fermentation of Neurospora intermedia

[0036]

[0037] In the control group, glucose was used as the carbon source for fermentation. After fermentation, the dry weight of the bacteria was 4.77 g / L, the total protein content was 2.74 g / L, and the conversion rate was 0.48 g dry weight / g glucose. The fermentation yield of bran, palm meal and dregs hydrolyzate was only 20-41% of that of the control group, which may be affected by factors such as fermentation inhibitors and nutrients. Therefore, these raw materials need to further optimize the processing process to be more effectively used in the fermentation of Intersporus. In addition, the yield after fermentation using starch hydrolyzate increased by 4.2% compared with the control group, while the fermentation yield using maltose decreased by 8.5%, which was equivalent to the yield of the control group. When using sugarcane molasses for fermentation, the dry weight of the bacteria reached 6.01 g / L, an increase of 26% compared with the control group; the total protein content was 3.19 g / L, an increase of 16.4% compared with the control group; and the conversion rate was 0.6 g dry weight / g sugar. In view of the low cost, simple pretreatment and high yield of sugarcane molasses, sugarcane molasses is preferred as the fermentation carbon source.

[0038] Example 2: Effects of different nitrogen sources on high-density culture of Neurocystis

[0039] In the fermentation industry, commonly used nitrogen sources include corn syrup, soybean cake, soybean meal, corn protein and yeast powder. Corn syrup, as a nitrogen source for fermentation, has the advantages of low cost and rich nutrition. Bean cake, soybean meal and corn protein are usually hydrolyzed for fermentation, but the hydrolysis process is difficult and costly, and they may also contain ingredients that inhibit fermentation. Yeast powder is rich in nutrients, easily absorbed by microorganisms and promotes fermentation, but its cost is relatively high. The above five nitrogen sources were selected for Interneuron fermentation to explore the effects of different nitrogen sources on the yield and cost of high-density fermentation.

[0040] The remaining components of the fermentation medium include: glucose 10 g / L, (NH4)2SO4 7.08 g / L, KH2PO4 3.5 g / L, MgSO4·7H2O 0.75 g / L, EDTA 0.03 g / L, 7 trace metal stock solutions 100 mL / L each, mixed vitamin stock solution 1 mL / L, pH 6.2.

[0041] The Intertype Neurocystis CGMCC NO.40496 cultured on the PDA plate was cut into pieces and inoculated into the shake flask seed liquid medium for fermentation culture. The culture temperature was 30-38°C, preferably 35°C, the shaker speed was 200-250rpm / min, preferably 220rpm / min, and the culture time was 18-30h, preferably 24h, to obtain seed liquid. The seeds were inoculated into the fermentation medium with different nitrogen sources at an inoculation rate of 1%, and fermented at 35°C and 220rpm for 24h. After the fermentation was completed, the bacteria were collected by filtering through 500 mesh gauze, and the dry weight and total protein content after fermentation were determined. The results are shown in Table 2.

[0042] Table 2. Effects of different nitrogen sources on the fermentation of Neurospora intermedia

[0043]

[0044] In the control group, yeast powder was used as the nitrogen source for fermentation. After fermentation, the dry weight of the bacteria was 4.77 g / L, the total protein content was 2.74 g / L, and the conversion rate was 0.48 g dry weight / g glucose. When corn protein was used as the nitrogen source, the dry weight after fermentation was only 80% of that in the control group, and the total protein content was 85% of that in the control group. In contrast, the dry weight obtained after fermentation of corn slurry, soybean cake, and soybean meal was 6.5-15.1% higher than that of the control group, and the total protein content was 4.4-8.4% higher. However, due to the relatively high prices of soybean cake and soybean meal and the complicated hydrolysis steps, corn slurry was selected as the fermentation nitrogen source.

[0045] Example 3: Effects of different nitrogen-phosphorus ratios on high-density culture of Neurocystis

[0046] Sugarcane molasses was selected as the carbon source and corn steep liquor was selected as the nitrogen source according to Example 1 and Example 2. The experiment further investigated the effects of the phosphorus source content and the ratio of the organic nitrogen source to the inorganic nitrogen source in the initial culture medium on the fermentation of Interneuron fusobacteria.

[0047] The main differences in fermentation media are:

[0048] Comparative Example 1 Fermentation conditions: corn steep liquor 1g / L, potassium dihydrogen phosphate 2g / L, ammonium sulfate 7.14g / L, pH 5.6;

[0049] Comparative Example 2 Fermentation conditions: corn steep liquor 1g / L, potassium dihydrogen phosphate 3.5g / L, ammonium sulfate 9.52g / L, pH6.2;

[0050] Comparative Example 3 Fermentation conditions: corn steep liquor 3g / L, potassium dihydrogen phosphate 1g / L, ammonium sulfate 7.14g / L, pH6.2;

[0051] Comparative Example 4 Fermentation conditions: corn steep liquor 6.25 g / L, potassium dihydrogen phosphate 1 g / L, ammonium sulfate 9.52 g / L, pH 5.6;

[0052] Comparative Example 5 Fermentation conditions: corn steep liquor 6.25 g / L, potassium dihydrogen phosphate 2 g / L, ammonium sulfate 4.72 g / L, pH 6.2;

[0053] Comparative Example 6 Fermentation conditions: corn steep liquor 4.57 g / L, potassium dihydrogen phosphate 4.72 g / L, ammonium sulfate 3.5 g / L, pH 6.2;

[0054] The remaining components of the fermentation medium include: MgSO4·7H2O 0.75 g / L, EDTA 0.03 g / L, 7 trace metal stock solutions 100 mL / L each, mixed vitamin stock solution 1 mL / L, pH 6.2.

[0055] The Intertype Neurocystis CGMCC NO.40496 cultured on the PDA plate was cut into pieces and inoculated into the shaking flask seed liquid medium for fermentation culture, the culture temperature was 30-38°C, preferably 35°C, the shaking speed was 200-250rpm / min, preferably 220rpm / min, the culture time was 18-30h, preferably 24h, to obtain seed liquid; the seeds were inoculated into the above fermentation medium at an inoculation rate of 1%, and fermented at 35°C and 220rpm for 24h. After the fermentation was completed, the bacteria were collected by filtering through 500 mesh gauze, and the dry weight and total protein content after fermentation were determined. The results are shown in Table 3.

[0056] Table 3. Effects of different nitrogen-phosphorus ratios on the fermentation of Neurocystis

[0057]

[0058] The experiment investigated the effects of different nitrogen-phosphorus ratios in the initial fermentation medium on the fermentation products and production costs of Intersporus. In comparative examples 1 to 6, the bacterial concentration after fermentation showed a gradual upward trend, the bacterial dry weight increased from 3.81g / L to 6.24g / L, and the total protein content increased from 2.05g / L to 3.48g / L. The results showed that the increase in bacterial concentration did not show a linear correlation trend with the single variable of phosphorus source, organic nitrogen source or inorganic nitrogen source, indicating that there was a significant interaction between different phosphorus sources and nitrogen sources. In comparative example 6, the ratio of corn steep liquor, potassium dihydrogen phosphate and ammonium sulfate was 4.57:4.72:3.5, and the dry weight of the bacterial body after fermentation was significantly higher than that of other comparative examples, reaching 6.24g / L, and the total protein content reached 3.48g / L. At this time, both the bacterial dry weight and the total protein content showed obvious advantages, which were determined as the final composition of the initial culture medium.

[0059] Example 4: Effect of batch feeding on high-density culture of Neurocystis in shake flasks

[0060] The experiment further evaluated the effects of different feed medium compositions on high-density fermentation of Neurocystis in shake flasks:

[0061] Feed medium 1: cane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 105.1 g / L, (NH4)2SO4 108.5 g / L, KH2PO4 80.5 g / L, MgSO4·7H2O 17.2 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, mixed vitamin stock solution 23 mL / L, pH 4.3.

[0062] Feed medium 2: cane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 52.6 g / L, (NH4)2SO4 54.3 g / L, KH2PO4 80.5 g / L, MgSO4·7H2O 17.2 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, mixed vitamin stock solution 23 mL / L, pH 4.3.

[0063] Feed medium 3: cane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 105.1 g / L, (NH4)2SO4 108.5 g / L, KH2PO4 40.3 g / L, MgSO4·7H2O 17.2 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, mixed vitamin stock solution 23 mL / L, pH 4.3.

[0064] Feed medium 4: cane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 105.1 g / L, (NH4)2SO4 108.5 g / L, KH2PO4 80.5 g / L, MgSO4·7H2O 8.6 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, vitamin stock solution 23 mL / L, pH 4.3.

[0065] Feed medium 5: cane molasses 570 mL / L (total sugar 230 g / L), corn syrup 52.6 g / L, (NH4)2SO4 54.3 g / L, KH2PO4 40.3 g / L, MgSO4·7H2O 8.6 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, vitamin stock solution 23 mL / L, pH 4.3.

[0066] Use an inoculation loop to pick a 1cm×1cm square block of bacteria from the middle and upper part of the Intertype Neurospora plate, transfer it to the seed culture medium, and shake culture for about 24 hours at 35℃ and 220rpm. Fermentation was carried out in a 1L baffled conical flask, with an initial fermentation medium of 150mL and an inoculation amount of 10%. After 6 hours of culture, feed to 20g / L of total sugar; then feed every 12 hours, and increase the total sugar concentration by 20g / L each time until the final concentration is 120g / L. The specific feed addition amounts are: 7.14mL, 16.54mL, 20.43mL, 25.88mL, 33.85mL, 46.15mL. The total fermentation time is 76h. Soybean oil is added as a defoamer according to the foaming condition during the fermentation process.

[0067] Table 4 Effects of different feed media on the fermentation of Neurocystis

[0068]

[0069] Feed medium 1 is 23 times the concentration of the initial medium. Feed medium 1 was used for feeding. The highest bacterial concentration after fermentation reached 81g / L, which was 12.9 times that before feeding. The carbon conversion rate was 67.5% (g dry weight / g total sugar), the nitrogen conversion rate was 35.7%, and the bacterial growth rate was 1.07g / L / h. The composition of feed medium 2 to 5 was tested for the reduction of total nitrogen, total phosphorus, total magnesium, nitrogen, phosphorus and magnesium in feed medium 1. After fermentation, the bacterial dry weight was 61.1-66.1g / L, which was more than 10 times that before feeding. The carbon conversion rate was 50.9%-55.1%, and the bacterial growth rate was 0.8-0.87g / L / h.

[0070] Example 5: High-density fermentation of Neurocystis in a fermenter

[0071] Seed culture medium: glucose 10g / L, yeast extract 5g / L, (NH4)2SO47.08g / L, KH2PO43.5g / L, MgSO4·7H2O 0.75g / L, EDTA 0.03g / L, 7 trace metal stock solutions 100mL / L each, vitamin stock solution 1mL / L, pH 6.2.

[0072] Initial fermentation medium: sugarcane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 4.57 g / L, (NH4)2SO4 4.72 g / L, KH2PO4 3.5 g / L, MgSO4·7H2O 0.75 g / L, EDTA 0.03 g / L, 7 trace metal mother solutions 100 mL / L each, vitamin mother solution 1 mL / L, pH 6.2.

[0073] Fermentation feed medium: sugarcane molasses 570 mL / L (total sugar 230 g / L), corn steep liquor 105.1 g / L, (NH4)2SO4 108.5 g / L, KH2PO4 80.5 g / L, MgSO4·7H2O 17.2 g / L, EDTA 0.7 g / L, 7 trace metal stock solutions 2.3 mL / L each, vitamin stock solution 23 mL / L.

[0074] Use an inoculation loop to pick a 1cm×1cm square cake from the middle of the Interneuron plate, transfer it to the seed culture medium, and culture it at 35℃ and 220rpm for about 24 hours to obtain vigorous seeds. Inoculate it into the initial fermentation medium at a ratio of 10%, and maintain the fermentation temperature at 35℃.

[0075] At the beginning of fermentation, the stirring speed was set to 400 rpm, and the stirring was linked to the dissolved oxygen. At the same time, the speed was adjusted according to the mycelium morphology to maintain the mycelium morphology between 100 and 500 mm. Figure 1 shown.

[0076] After 5 hours of fermentation, the mycelium is clumped and long. At this time, set the stirring and dissolved oxygen to maintain 25% dissolved oxygen, and the speed range is 400-700rpm; after 21 hours of fermentation, the mycelium is still not dispersed, and there are mycelium <100mm, and the speed is increased by 25rpm to 725rpm. After 21 hours of fermentation, until the end of fermentation, the mycelium length is in the range of 100-500mm, and the speed is maintained at 725 rpm until the end of fermentation.

[0077] At the beginning of fermentation, the ventilation volume is 1.5L / min (1VVM). During the fermentation process, the ventilation volume is increased according to the feed volume and dissolved oxygen measurement value, and the ventilation ratio does not exceed 3VVM.

[0078] Batch fermentation was carried out during the 1st to 6th hour of fermentation without feeding; the feeding rate was then adjusted according to the sugar consumption, and the average sugar consumption rate during the fermentation process was 1.5 g / L / h.

[0079] When the fermentation lasted for 96 hours, the cells were collected by filtration through 500 mesh gauze after the fermentation was completed, and the dry weight was determined by drying at 70°C. The cell concentration was 104 g / L, the carbon conversion rate was 70.7% (g dry weight / g total sugar), the cell growth rate was 1.08 g / L / h, the protein content was 35.47%, and the nitrogen conversion rate was 30.9%.

[0080] Example 6: Separation and application of fermentation products

[0081] Fermentation is carried out according to the method described in Example 5 to obtain a fungal protein product. After fermentation, heat treatment is performed at 65°C for 30 minutes to degrade intracellular RNA. The fermented biomass is then further heated to 90°C for 30 minutes to ensure that the fungal cells are killed. After that, it is filtered through a 500-mesh gauze and washed with 5 times the volume of sterile water to completely remove the culture medium. The aqueous biomass obtained after filtration can be directly used in the development of meat substitutes, such as meat patties, meatballs and other products. In addition, the filtered biomass can be dried or freeze-dried and ground into powder, which can be used in the development of products such as dairy products, health products or feed.

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A high-density fermentation method for Interneuron sp., characterized in that: The following steps are involved: The seed liquid of Interstitial Neurocystis with the deposit number of CGMCC NO. 40496 is inoculated into the initial fermentation medium for fermentation, and the feed medium is added regularly during the fermentation process until the fermentation is completed; The composition and proportion of the initial fermentation medium are: 25 mL / L carbon source, 4.72 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.75 g / L MgSO4·7H2O, 0.03 g / L EDTA, 4.57 g / L organic nitrogen source, 100 mL / L each of 7 trace metal mother solutions, pH 6.2, and 1 mL / L mixed vitamin mother solution is added after sterilization; The composition and proportion of the feed medium are: carbon source 570 mL / L, (NH4)2SO4 54.3-108.5 g / L, KH2PO4 40.3-80.5 g / L, MgSO4·7H2O 8.6-17.2 g / L, EDTA 0.35-0.7 g / L, organic nitrogen source 52.6-105.1 g / L, 7 trace metal mother solutions 2.3 mL / L each, and after sterilization, 23 mL / L of mixed vitamin mother solution is added; The seven trace metal mother solutions are: CaCl2·2H2O 0.09 g / mL, ZnSO4·7H2O 0.09 g / mL, FeSO4·7H2O 0.06 g / mL, MnCl2·2H2O 0.016 g / mL, Na2MO4·2H2O 0.008 g / mL, CuSO4·5H2O 0.006 g / mL, KI 0.002 g / mL; The mixed vitamin mother solution comprises: aminobenzoic acid 0.2 g / L, niacin 1 g / L, calcium pantothenate 1 g / L, pyridoxine hydrochloride-vitamin B6 1 g / L, thiamine hydrochloride-vitamin B1 1 g / L, biotin 0.05 g / L, and inositol 25 g / L; The organic nitrogen source is selected from one or more of yeast powder, corn steep liquor, soybean cake and soybean meal; Wherein, the carbon source in the initial fermentation medium is sugar cane molasses, and the amount added to the total sugar content of the medium is 8 to 12 g / L; the carbon source in the feed medium is sugar cane molasses, and the amount added to the total sugar content of the medium is 200 to 260 g / L; The high-density fermentation is a shake flask high-density fermentation: the shaker speed is 200-300 rpm, the temperature is 30-38° C., the defoaming agent is soybean oil; the feeding interval is 6-12 hours, and the total sugar content in the shake flask is maintained at 10-20 g / L after feeding; Or the high-density fermentation is a high-density fermentation in a fermenter: the rotation speed is adjusted according to the mycelium morphology to maintain the mycelium length between 100 and 500 mm; the ventilation volume is 1 to 3 VVM, the dissolved oxygen is maintained at not less than 15% in the early stage of fermentation, not less than 10% in the middle stage, and not less than 5% in the late stage, and the fermenter rotation speed is 400 to 800 rpm; feeding starts at the 4th to 7th hour of fermentation, and the feeding speed is adjusted according to the total sugar consumption rate to maintain the total sugar content in the fermenter at 10 to 15 g / L; the fermentation temperature is 30 to 38°C; The seed liquid is inoculated into the initial fermentation medium at a ratio of 5 to 20%.

2. The high-density fermentation method according to claim 1, characterized in that: The preparation method of the seed solution is as follows: inoculate the Neurocystis spp. stored at -80°C onto a PDA solid plate and culture at 35°C for 2-3 days until the mycelium grows all over the plate, cut the activated Neurocystis spp. plate into a seed culture medium, and culture at 35°C and 220 rpm for 24 hours to obtain the seed solution; The seed culture medium comprises: 10 g / L glucose, 5 g / L yeast extract, 7.0 8 g / L (NH4)2SO4, 3.5 g / L KH2PO4, 0.75 g / L MgSO4·7H2O, 100 mL / L each of 7 trace metal mother solutions, 1 mL / L mixed vitamin mother solution, and pH 6.

2.

3. The high-density fermentation method according to claim 1, characterized in that The rotation speed of the fermentation tank is 500-700rpm; feeding starts at the 5th to 6th hour of fermentation, and the feeding speed is adjusted according to the total sugar consumption rate to maintain the total sugar content in the fermentation tank at 12-14g / L; the fermentation temperature is 30-38°C.

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