Method for producing high-protein aquatic bait by heterotrophic fermentation of isochrysis sp. FQZ-2

Abstract

The invention relates to the technical field of microbial fermentation engineering, in particular to a method for producing high-protein aquatic bait by heterotrophic fermentation of isochrysis sp. FQZ-2. Comprising the following steps: inoculating isochrysis into a culture medium, culturing a seed culture solution until the dry cell weight is 4-10g / L, transferring the seed culture solution into a fermentation container, controlling the cell density to be 0.5-3g / L and the ventilation volume of the fermentation container to be 0.6-1vvm, and controlling the total sugar concentration to be 3-4.5% and the total nitrogen concentration to be 0.02-0.035% in the first 24 hours; at 24-96 hours, the total sugar concentration is controlled to be 2%-4%, and the total nitrogen concentration is controlled to be 0.006%-0.03%; the total sugar concentration is controlled to be 0.2-0.5% and the total nitrogen concentration is controlled to be 0.2-0.4.5% from 96h to 8h before putting into the tank. According to the application, the biomass, the protein content and the fucoxanthin content of the isochrysis sp. After fermentation for 120 hours are improved.

Description

Technical Field

[0001] This application relates to the technical field of microbial fermentation engineering, and particularly to a method for producing high-protein aquatic feed by heterotrophic fermentation of Isochrysis galbana FQZ-2. Background Art

[0002] Currently, protein-rich feeds are widely used in aquaculture feeds, mainly sourced from soybean meal and fish meal. With the continuous expansion of the aquaculture industry and the ever-changing aquaculture environment, the aquafeed industry is facing a series of challenges: such as the increasingly tight supply and high price of fish meal, and the frequent occurrence of nutritional and metabolic diseases. How to effectively improve the utilization rate of feeds, reduce the negative impact on the environment, and precisely meet the complex and variable nutritional needs of aquatic animals during their growth cycle has become the core problem that the current industry urgently needs to overcome.

[0003] Microalgae contain 40 - 70% protein. Compared with soybean meal (with a protein content of 45 - 57%), microalgae are considered a more environmentally sustainable and economical protein source. Notably, in July 2023, Isochrysis galbana was officially approved by the Ministry of Agriculture and Rural Affairs of China and successfully entered the new feed raw material catalog, becoming an ideal basic bait for various marine economic fish species and providing an important nutritional source for the aquaculture industry. Additionally, Isochrysis galbana is rich in DHA and EPA. As an initial feed for fry, it can improve the feed intake rate and growth performance of aquatic animals, enhance the immunity of aquatic animal bodies, and increase the nutritional value of aquatic animals. Currently, the production and development of Isochrysis galbana lack large-scale leading enterprises. The most prominent problem is that the yield of photoautotrophically cultured Isochrysis galbana is low (concentration is about 1 g / L), it is easily contaminated by other microorganisms, and the production cost is high, making it difficult to meet the large demand for algae by aquatic animal seedlings.

[0004] Chinese Patent with Publication No. CN110423692B discloses a method for culturing Isochrysis zhanjiangensis in a photobioreactor. This method uses a floating photobioreactor to culture Isochrysis zhanjiangensis on a large scale, obtaining a maximum cell density of 3.88×107 cells / mL and an average volumetric daily productivity of 0.048 g / L / d. Through the design of the floating reactor, Isochrysis galbana can fully receive light in a larger water body, thus achieving efficient cultivation. The floating design may also help control cultivation conditions such as temperature and pH value, improving the cultivation efficiency. However, the floating photobioreactor needs to consider the influence of factors such as the water environment and weather changes on cultivation.

[0005] The Chinese patent with publication number CN117946860A discloses a method and a culture device for promoting the growth of Isochrysis chrysophylla. The method sets an artificial light source to supplement light for Isochrysis chrysophylla, and the highest cell density can be obtained, which is about 2.4g / L. By supplementing light with an artificial light source, it is not limited by natural light conditions and can effectively increase the yield of Isochrysis chrysophylla. However, due to the external light source, the method requires a large amount of additional energy consumption. At the same time, the cell density of Isochrysis chrysophylla obtained by these two methods is also low, the culture cycle is long, and the later harvesting cost is high.

[0006] In recent years, scientific and technological workers have discovered that a small number of Isochrysis strains can use organic matter as a carbon source for heterotrophic growth. This discovery provides a new way for the large-scale cultivation of Isochrysis, which has important scientific research value and industrialization prospects. For example, Isochrysis 3011 can reach a maximum biomass of 37.17 g / L when cultured in the dark under suitable carbon, nitrogen and phosphorus sources (Ji Heng, Jiang Aili, Guo Xiaochen, et al. Heterotrophic culture of Isochrysis spherica [J]. Journal of Guangdong Ocean University, 2021, 41(02): 47-52.). The Chinese patent with publication number CN119120203A discloses Isochrysis strain FQZ-2 with heterotrophic growth ability, which can reach a maximum cell density of 55 g / L under heterotrophic conditions. E. Mohammad et al. heterotrophically cultivated marine Isochrysis, and the maximum biomass was 4.21 g / L, of which the protein content was 26.2%. It is worth noting that not only the Isochrysis obtained through heterotrophic culture has a generally low protein content; even the Isochrysis obtained through photoautotrophic culture has not been reported to have a protein content higher than 50%.

[0007] On the other hand, Isochrysis is rich in fucoxanthin, which has a wide range of biological activities when used in aquatic feed, including antioxidant, anti-inflammatory, health-promoting and immunity-enhancing effects, which helps to improve the disease resistance and growth performance of aquatic animals. With the application of heterotrophic culture technology, the culture process of Isochrysis is highly controllable, and the cell density is significantly improved. However, compared with the fucoxanthin content of Isochrysis cultured in photoautotrophic culture (about 20 mg / g), the fucoxanthin content of heterotrophic Isochrysis is generally lower (0.1 mg / g), and the low fucoxanthin algae powder also reduces the nutritional value of Isochrysis products.

[0008] Based on the above information, the main technical problems existing in the prior art can be summarized as follows: 1. The current cultivation method of Isochrysis mainly relies on the photoautotrophic mode, and its yield is low, which is difficult to meet the demand for aquatic bait; 2. Only a small number of Isochrysis strains can be cultured in a heterotrophic mode, and most algae species do not have a complete mechanism for absorbing and utilizing extracellular organic carbon sources; 3. The protein and fucoxanthin content of heterotrophically cultured Isochrysis is low, and the quality of algal powder is poor, and it cannot be developed as aquatic bait. Summary of the Invention

[0009] In view of this, the present application provides a method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis galbana FQZ-2, which can increase the biomass, protein content and fucoxanthin content of Isochrysis galbana after 120 h of fermentation.

[0010] To achieve the above object, the present application is realized through the following technical solutions:

[0011] A method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis galbana FQZ-2, characterized in that it comprises the following steps;

[0012] Step 1: Inoculate Isochrysis galbana FQZ-2 into a basal medium to form a seed culture solution. The pH of the seed culture solution is between 7 and 8. The seed culture solution is cultured until the dry weight of Isochrysis galbana FQZ-2 cells reaches 4-10 g / L, and the inoculation age is 48 h-96 h;

[0013] Step 2: Transfer the seed culture solution obtained in Step 1 into a fermentation container containing a basal medium. The cell density is controlled at 0.5-3 g / L, the aeration rate of the fermentation container is controlled at 0.6-1 vvm, the fermentation temperature is 25-30 °C, and the stirring speed is 50-450 r / min. Fermentation culture is carried out for 120-176 h; during the fermentation culture process, by supplementing the feeding substrate, within the first 24 h of the culture process, the mass percentage concentration of the total sugar is controlled between 3% and 4.5%, and the mass percentage concentration of the total nitrogen is controlled between 0.02% and 0.035%; between 24-96 h, the mass percentage concentration of the total sugar is controlled between 2% and 4%, and the mass percentage concentration of the total nitrogen is controlled between 0.006% and 0.03%; from 96 h to 8 h before discharging the tank, the mass percentage concentration of the total sugar is controlled between 0.2% and 0.5%, and the mass percentage concentration of the total nitrogen is controlled between 0.2% and 0.45%. No feeding operation is performed within 8 h before discharging the tank.

[0014] In some embodiments, the post-consumption concentration of the total sugar in the basal medium is between 3% and 5%, and the post-consumption concentration of the total nitrogen is between 0.02% and 0.04%. The meaning of the post-consumption concentration of the total sugar or total nitrogen is the mass percentage of the total sugar or total nitrogen in the medium after high-temperature sterilization.

[0015] In some embodiments, the post-consumption concentration of the total sugar in the feeding substrate is 60%, and the post-consumption concentration of the total nitrogen is 30%.

[0016] In some embodiments, the nitrogen source used in the basal medium is one or a combination of two or more of potassium nitrate, ammonium sulfate, urea, and ammonium chloride. The sugar source of the basal medium and the feeding substrate is glucose.

[0017] In some embodiments, the basal medium further comprises components with the following mass percentages: potassium dihydrogen phosphate: 0.005%-0.030%; disodium hydrogen phosphate dodecahydrate: 0.020%-0.050%; magnesium sulfate heptahydrate: 0.005%-0.020%; calcium chloride: 0.005-0.020%. By reasonably designing the ratio of these inorganic salts, a stable physical and chemical environment and essential nutrient support can be provided for the growth and product synthesis of Isochrysis galbana FQZ-2.

[0018] In some embodiments, the basal medium is sterilized under the conditions of 115°C and steam disinfection for 30 min.

[0019] As can be seen from the above technical solutions, the present application has at least the following advantages and positive effects:

[0020] 1. In the existing Isochrysis galbana culture technology, it is difficult to simultaneously achieve satisfactory results in both the cell density and protein content of the obtained Isochrysis galbana, and the large-scale production cost is high. In the present invention, by combining Isochrysis galbana FQZ-2 with heterotrophic ability and carefully designing the optimal carbon-nitrogen ratio of the initial fermentation medium by researchers, after Isochrysis galbana is inoculated into the medium, it can quickly absorb carbon and nitrogen sources to support the growth of microalgae, enabling the microalgae to accumulate a large amount of biological matrix (dry weight > 50 g / L) in a short period. When the culture system accumulates more biomass, by feeding operation to regulate the carbon source and nitrogen source, under the appropriate carbon-nitrogen ratio, a large number of metabolic activities of microalgae cells can be concentrated on protein synthesis without losing biomass, and finally a high-density and high-protein Chlorella product can be obtained. The main mechanism of this effect is that in the first 96 h of fermentation, Chlorella cells will accumulate a large amount of starch-rich biological matrix in a high-carbon and low-nitrogen environment. After 96 h, when the medium is rich in nitrogen elements, they can greedily absorb this element for storage, prompting the microalgae cell metabolism to tend to protein synthesis, converting the starch in the cells into protein, and without losing the original biomass under the appropriate feeding carbon-nitrogen ratio.

[0021] 2. In related technologies, based on this effect mechanism, the protein content of heterotrophic Chlorella vulgaris has been successfully increased. However, due to the improper selection of the culture medium and the complexity of the operation process, the biomass productivity of microalgae is not ideal (about 25 g / L). In addition, Isochrysis galbana is difficult to resist the shear force generated during the stirring process of traditional fermenters due to its lack of cell wall. Therefore, the present invention ingeniously designs a stirring speed control strategy that matches the cell density, which not only ensures sufficient dissolved oxygen supply but also minimizes the damage to algal cells. At the same time, the present invention carefully designs the carbon-nitrogen ratio of the initial basal medium, and performs feeding operations and feeding carbon-nitrogen ratios at reasonable time points, so that while ensuring a high-density and high-protein Isochrysis galbana biological matrix, the nutrient input and biological conversion rate can be maximized, reducing resource waste and not causing pollution to the environment.

[0022] 3. The cell density of the heterotrophic Isochrysis galbana obtained by the present invention can reach more than 60 g / L, and the cell protein content is more than 56%, which has the potential to be applied to aquaculture bait.

[0023] 4. The process of the present invention is simple, requires low equipment, has a high nutrient input and biological conversion rate, good benefits, does not cause waste of nutrients, can be completed in a single culture system, is extremely easy to scale up production, and has the advantages of short-time high efficiency, green safety and energy saving. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a process flow chart of an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0025] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings. The terms used in the embodiment part of the present application are only used to explain the specific embodiments of the present application, rather than to limit the present application.

[0026] The methods for protein detection and biomass detection involved in the embodiments are as follows:

[0027] (1) The detection of protein content includes the following steps:

[0028] a. Using an automatic Kjeldahl nitrogen analyzer, determine the protein content of the Isochrysis galbana biological matrix;

[0029] b. Calculate: The percentage of protein in the sample = M1 / M0 * 100%.

[0030] Wherein, M1 is the protein content of the sample in g;

[0031] M0 is the dry weight of the test sample.

[0032] (2) Take 1 mL of the algal solution and place it in a pre-weighed 2 mL centrifuge tube with a label. Centrifuge at 5000 rpm for 5 min, pour off the supernatant, wash the algal sludge with distilled water, centrifuge again and discard the supernatant. Repeat the operation three times, then place it in an oven at 80 °C and dry it to a constant weight, weigh it and record.

[0033] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions.

[0034] The Isochrysis galbana FQZ-2 used in this application was isolated from the mangroves in Quanzhou and is preserved in the China Center for Type Culture Collection. The preservation number is CCTCC NO: M 20241307; the preservation address is Wuhan University, Wuhan, China. Zip code: 430072.

[0035] Example 1:

[0036] A method for heterotrophic fermentation of Isochrysis galbana FQZ-2 to produce high-protein aquatic bait algae includes the following steps:

[0037] S1. Medium preparation: Prepare the basal medium and the feeding substrate. The volume of the feeding substrate accounts for 20% of the total volume of the fermentation broth. After preparing the basal medium, sterilize it in an autoclave at 115 °C for 30 minutes.

[0038] The basal medium includes the following components: (1) glucose, 50 g / L; (2) urea, 4 g / L; (3) KH2PO4, 0.1 g / L; (4) 12H2O·Na2HPO4, 0.2 g / L; (5) 7H2O·MgSO4, 0.2 g / L; (6) CaCl2, 0.05 g / L. The feeding substrate includes: (1) glucose, 600 g / L; (2) urea, 300 g / L.

[0039] S2. Preparation of Isochrysis galbana algal seeds: Scrape the algal cells on the agar slant and inoculate them into a 250 mL conical flask containing the basal medium to culture the Chlorella seeds. The liquid loading is 100 mL, the culture temperature is 30 °C, the culture is carried out in the dark, the shaking speed of the shaker is 200 r / min, the cell dry weight of the seed liquid is cultured to 5 g / L, the pH of the culture broth is 7, and the inoculation age is 48 h.

[0040] S3. Fermentation culture of Isochrysis galbana: Transfer the seed liquid into a 10 L fermenter containing 6 L of the basal medium. After inoculation, the cell density in the fermenter is 3 g / L. The culture temperature is 30 °C, the stirring speed is 450 r / min, and the aeration rate is 0.8 vvm.

[0041] S3. Feeding operation: In the first 24 hours of the culture process controlled by a peristaltic pump, the total sugar concentration is controlled at about 3%, and the total nitrogen concentration is about 0.02%; from 24h to 96h, the total sugar concentration is controlled at 3%, and the total nitrogen concentration is controlled at 0.01%; from 96h to 112h, the total sugar concentration is controlled at about 0.5%, and the total nitrogen concentration is controlled at about 0.3%. No feeding operation is performed from 112h to 120h, and the culture is terminated at 120h for discharging the fermenter.

[0042] S4. Discharging the fermenter and measuring the dry weight of algal cells to be 65 g / L.

[0043] S5. Collection of Isochrysis galbana: Take the cultured algal solution, centrifuge it at 5000 r / min for 5 min, remove the supernatant, and wash it with clear water. Repeat the above steps 3 times to obtain wet algal bodies.

[0044] S6. Drying of Isochrysis galbana: Dry the obtained wet algal bodies with a freeze dryer for 2 days to obtain Isochrysis galbana dry powder. The content of fucoxanthin detected is: 17.5 mg / g; the protein accounts for 56%. Its essential amino acid composition ratio is higher than the FAO / WHO feed standard and soy protein, and it is suitable for aquatic feed.

[0045] Example 2

[0046] A method for co-producing protein and fucoxanthin by heterotrophic fermentation of Isochrysis galbana FQZ-2, comprising the following steps:

[0047] S1. Medium preparation: Prepare a basal medium, prepare a feeding substrate, and the feeding volume accounts for 10% of the total fermentation broth volume. After the medium is prepared, sterilize it at 115 °C for 20 minutes;

[0048] The basal medium includes the following components: (1) glucose, 30 g / L; (2) urea, 2 g / L; (3) KH2PO4, 0.3 g / L; (4) 12H2O·Na2HPO4, 0.2 g / L; (5) 7H2O·MgSO4, 0.2 g / L; (6) CaCl2, 0.2 g / L. The feeding substrate includes: (1) glucose, 600 g / L; (2) urea, 300 g / L.

[0049] S2. Preparation of Isochrysis galbana seed: Scrape the algal cells on the above agar slant and inoculate them into a 1 L conical flask containing a basal medium with 30 g / L glucose to culture Isochrysis galbana seeds. The liquid loading volume is 500 mL, the culture temperature is 30 °C, and it is cultured in the dark with a shaker speed of 2000 r / min for 2 days as the primary seed liquid. Transfer the primary seed liquid into a 500 L fermenter containing 300 L of basal medium, culture the seed liquid to 10 g / L, the pH of the culture broth is 7, and the inoculation age is 48 h.

[0050] S3. Culturing Isochrysis galbana in a 2000L fermenter: Transfer the above seed liquid into a 2000L fermenter containing 1200L of basic medium. After inoculation, the cell density is 2g / L, the culture temperature is 25°C, the stirring speed is 300r / min, and the ventilation rate is 0.6vvm. Feed the 2000L fermenter through a feed tank. Control the total sugar concentration at about 3% and the total nitrogen concentration at about 0.03% in the first 24h of the culture process; control the total sugar concentration at 2% and the total nitrogen concentration at 0.01% from 24 - 96h; control the total sugar concentration at about 0.3% and the total nitrogen concentration at about 0.2% from 96 - 142h. Do not perform any feeding operation during 142 - 150h, and harvest the fermenter at 150h.

[0051] S4. Harvest the fermenter and measure the dry weight of algal cells to be 57g / L.

[0052] S5. Collection of Isochrysis galbana thalli: Take the cultured algal liquid, harvest it with a disc centrifuge, and wash it with clear water. Repeat the washing 3 times to obtain wet thalli.

[0053] S6. Drying of Isochrysis galbana thalli: Dry the obtained wet thalli into powder with a spray dryer. Detect that the content of fucoxanthin is 18.3mg / g; the protein proportion is 56%. Its essential amino acid composition ratio is higher than the FAO / WHO feed standard and soy protein, and it is suitable for aquatic feed.

[0054] Example 3

[0055] A method for producing high - protein aquatic feed algae by heterotrophic fermentation of Isochrysis galbana FQZ - 2, the steps are as follows:

[0056] S1. Medium preparation: The basic medium includes 50g / L of glucose, 4g / L of potassium nitrate, 0.05g / L of KH2PO4, 0.5g / L of 12H2O·Na2HPO4, 0.05g / L of 7H2O·MgSO4, 0.1g / L of CaCl2. The feeding substrate is 500g / L of glucose and 300g / L of potassium nitrate. The feeding volume accounts for 15% of the total fermentation broth volume. The medium is sterilized at 115°C for 30 minutes.

[0057] S2. Algal strain preparation: Inoculate from a slant seed into a 250mL conical flask containing basic medium, culture until the cell dry weight is 4g / L. The culture conditions are 30°C, dark, the shaker speed is 180r / min, the pH of the culture solution is 8, and the inoculation age is 96h.

[0058] S3. Fermentation culture: Transfer the seed liquid into a 10L fermenter containing 6L of basic medium. After inoculation, the cell density is 0.5g / L. The culture conditions are 28°C, the stirring speed is 400r / min, and the ventilation rate is 0.7vvm.

[0059] S4. Feeding operation: The total sugar concentration is controlled at 4% in the first 24 hours, and the total nitrogen concentration is 0.02%; the total sugar concentration is 2.5% from 24 to 96 hours, and the total nitrogen concentration is 0.008%; the total sugar concentration is 0.2% from 96 to 160 hours, and the total nitrogen concentration is 0.4%. The feeding is stopped at 160 hours, and the culture is continued until 170 hours when the fermentation broth is discharged. The dry cell weight obtained is 60 g / L.

[0060] S5. Harvesting and drying: The algal bodies are collected by centrifugation, washed three times with clear water, and freeze-dried to obtain algal powder. The content of fucoxanthin in the algal powder is detected to be 20.0 mg / g, and the protein content is 55%.

[0061] Example 4

[0062] A method for producing high-protein aquatic bait algae by heterotrophic fermentation of Isochrysis galbana FQZ-2, the steps are as follows:

[0063] S1. Medium preparation: The basal medium contains 50 g / L of glucose, 3 g / L of ammonium sulfate, appropriate amounts of other inorganic salts and antifoaming agent, and the feeding substrate is 600 g / L of glucose and 300 g / L of ammonium sulfate. The feeding volume accounts for 12% of the total fermentation broth volume, and the sterilization conditions of the medium are 115 °C for 30 minutes.

[0064] S2. Algal strain preparation: Inoculate the slant seeds into the seed culture medium, and culture until the dry cell weight reaches 6 g / L. The culture conditions are 32 °C, dark, the shaker speed is 200 r / min, the pH of the culture medium is 7.5, and the inoculation age is 72 h.

[0065] S3. Fermentation culture: Transfer the seed liquid into a 15-L fermenter containing 10 L of basal medium, and the cell density after inoculation is 3 g / L. The culture conditions are 30 °C, the stirring speed is 50 r / min, and the aeration rate is 0.6 vvm.

[0066] S4. Feeding operation: Adjust the feeding according to the fermentation process. The total sugar concentration is 4.5% in the first 24 hours, and the total nitrogen concentration is 0.035%; the total sugar concentration is 2% from 24 to 96 hours, and the total nitrogen concentration is 0.006%; the total sugar concentration is 0.4% from 96 to 168 hours, and the total nitrogen concentration is 0.45%. The feeding is stopped at 168 hours, and the culture is continued until 176 hours when it ends. The dry cell weight obtained is 45 g / L.

[0067] S5. Harvesting and drying: Collect the algal bodies by using a plate and frame filter press, wash them with clear water, and then spray-dry. The content of fucoxanthin in the algal powder is detected to be 15.1 mg / g, and the protein content is 45%.

[0068] Example 5

[0069] A method for producing high-protein aquatic bait algae by heterotrophic fermentation of Isochrysis galbana FQZ-2, the steps are as follows:

[0070] S1. Culture medium preparation: The basal medium includes 50 g / L of glucose, 2 g / L of urea, inorganic salts and antifoam agent added in a certain proportion. The feeding substrate is 600 g / L of glucose and 400 g / L of urea. The feeding volume accounts for 18% of the total fermentation broth volume. The medium is sterilized at 118 °C for 35 minutes.

[0071] S2. Algal strain preparation: Inoculate the slant seed into the seed liquid and culture until the dry cell weight reaches 5.5 g / L. The culture conditions are 31 °C, dark, shaker speed 200 r / min, the pH of the culture solution is 7.5, and the inoculation age is 48 h.

[0072] S3. Fermentation culture: Transfer the seed liquid into a 30 L fermenter containing 20 L of basal medium. The cell density after inoculation is 3 g / L. The culture conditions are 30 °C, stirring speed 450 r / min, and aeration rate 1 vvm.

[0073] S4. Feeding operation: Adjust the feeding strategy. The total sugar concentration is 4% and the total nitrogen concentration is 0.03% in the first 24 hours; the total sugar concentration is 2.8% and the total nitrogen concentration is 0.009% from 24 to 96 hours; the total sugar concentration is 0.35% and the total nitrogen concentration is 0.38% from 96 to 156 hours. Stop feeding at 156 hours and continue culturing until discharging the fermenter at 170 hours. The obtained dry cell weight is 60 g / L.

[0074] S5. Harvesting and drying: Centrifuge to collect the algal bodies, wash them with clear water and then dry them by vacuum freeze-drying method. The content of fucoxanthin in the algal powder is detected to be 20.2 mg / g, and the protein content is 58%.

[0075] Using the method of the present invention can maximize the conversion of the input nutrient substrate into microalgae high-protein biomass, with good benefits, without causing waste of nutrients, nor causing eutrophication pollution to the surrounding water quality. Moreover, the operation of the present invention is simple, the equipment requirements are low, it is easy to be applied to industrial production, and it has the advantages of short-time high efficiency, green safety and energy saving.

[0076] The above are only the embodiments of the present invention, and do not limit the patent scope of the present invention accordingly. All equivalent structural or equivalent process transformations made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, are equally included in the patent protection scope of the present invention.

Claims

1. A method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2, characterized in that: The steps include: Step 1: inoculate Isochrysis chrysophylla FQZ-2 into the basic culture medium to form a seed culture solution, the pH of the seed culture solution is between 7-8, and the seed culture solution is cultured until the dry weight of Isochrysis chrysophylla FQZ-2 cells is 4-10 g / L, and the seeding age is 48h-96h; Step 2: The seed culture solution obtained in step 1 is transferred into a fermentation container containing a basal culture medium, the cell density is controlled at 0.5-3 g / L, the ventilation volume of the fermentation container is controlled at 0.6-1 vvm, the fermentation temperature is 25-30°C, the stirring speed is 50-450 r / min, and the fermentation culture is carried out for 120-176 hours; during the fermentation culture process, the mass percentage of the total sugar concentration is controlled within 3%-4.5% within the first 24 hours of the culture process by supplementing the feed matrix. During the period from 1h to 2h, the mass percentage of total nitrogen concentration was controlled between 0.02% and 0.035%; from 24h to 96h, the mass percentage of total sugar concentration was controlled between 2% and 4%, and the mass percentage of total nitrogen concentration was controlled between 0.006% and 0.03%; from 96h to 8h before tanking, the mass percentage of total sugar concentration was controlled between 0.2% and 0.5%, and the mass percentage of total nitrogen concentration was controlled between 0.2% and 0.4.5%, and no feeding operation was performed within 8h before tanking.

2. The method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2 according to claim 1, characterized in that: The total sugar concentration of the basic culture medium after elimination is between 3% and 5%, and the total nitrogen concentration after elimination is between 0.02% and 0.04%.

3. The method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2 according to claim 1, characterized in that: The total sugar concentration after elimination of the feed matrix is ​​60%, and the total nitrogen concentration after elimination is 30%.

4. The method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2 according to claim 2, characterized in that: The nitrogen source used in the basic culture medium is one or a combination of two or more of potassium nitrate, ammonium sulfate, urea and ammonium chloride in any proportion.

5. The method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2 according to claim 2, characterized in that: The basic culture medium also includes the following components in percentage by mass: potassium dihydrogen phosphate: 0.005%-0.030%; disodium hydrogen phosphate dodecahydrate: 0.020%-0.050%; Magnesium sulfate heptahydrate: 0.005%-0.020%; Calcium chloride: 0.005-0.020%.

6. The method for producing high-protein aquatic bait by heterotrophic fermentation of Isochrysis chrysotrichum FQZ-2 according to claim 1, characterized in that: The basic culture medium is sterilized at 115° C. and steam sterilized for 30 minutes.

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