Preparation and application of nitrogen source in fungus culture medium

Abstract

The invention discloses preparation and application of a nitrogen source in a fungus culture medium, and belongs to the technical field of microbial culture mediums. The nitrogen source in the fungus culture medium is a powdery solid which is finally obtained by taking seriola quinqueradiata processing leftovers as a raw material, carrying out treatments such as mincing and the like to obtain minced fillet slurry, and carrying out treatments such as protease enzymolysis, centrifugation, filtration, drying and the like on the minced fillet slurry. According to the method, the seriola quinqueradiata leftovers are efficiently utilized, the high-quality peptone is prepared, the seriola quinqueradiata leftovers serve as a nitrogen source to be applied to a fungus culture medium component so as to improve the nutrient value of a culture medium and the growth effect of fungi, and the obtained nitrogen source shows a good growth promoting effect in a microorganism culture experiment.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial culture medium, and particularly relates to the preparation and application of a nitrogen source in a fungal culture medium. Background Art

[0002] Fungal culture is an important research topic in the fields of biology, bioengineering and medicine. Fungi require suitable nutrients to maintain their growth and metabolic activities during the culture process. Fungal culture media are usually composed of multiple components such as carbon sources, nitrogen sources, mineral salts, trace elements and vitamins. In fungal culture media, nitrogen sources play a key role and are mainly used to support the synthesis of proteins and nucleic acids, as well as energy metabolism processes. Traditional culture media usually use peptone as the main nitrogen source and nutrient source. Among the peptone products prepared from different raw materials, the enzymatic hydrolysate of fish protein resources has the characteristics of high protein content, good water solubility, rich peptides and free amino acids, etc. Therefore, fish peptone can be used as the best nitrogen source for microbial culture in fermentation industry and laboratories.

[0003] Yellowtail amberjack is widely distributed in the Yellow Sea, East China Sea and Bohai Sea in my country. It is known for its fast growth rate, delicious meat and high economic value. This makes it an ideal fish species for deep-sea aquaculture in my country. At present, the processing of yellowtail amberjack is mainly concentrated in food fields such as sashimi, while the waste is often ignored or discarded, resulting in a huge waste of resources. The main raw materials of the by-products of yellowtail amberjack processing include fish bones, fish heads, fish gills and other wastes. These wastes are rich in valuable nutrients such as protein and polyunsaturated fatty acids. The development and utilization of yellowtail amberjack waste to prepare peptone can be used as a nitrogen source for culture medium with more nutritional value and biological activity, thereby improving the culture conditions of fungi. Summary of the invention

[0004] The present invention aims to provide a preparation and application of a nitrogen source in a fungal culture medium.

[0005] To achieve the above purpose, the present invention adopts the following technical solution:

[0006] A nitrogen source in a fungus culture medium. The nitrogen source in the fungus culture medium is an enzymatic hydrolysis product obtained by enzymatically hydrolyzing yellowtail fish waste as a raw material.

[0007] A method for preparing a nitrogen source in a fungal culture medium comprises the following steps: using yellowtail fish scraps as raw materials, adding protease to the pulverized fish paste for enzymolysis, obtaining peptone by centrifugation and filtration, and freeze-drying the peptone to obtain the final powdered fish peptone, which is the nitrogen source in the fungal culture medium.

[0008] The yellowtail scraps are derived from byproducts or wastes generated by aquaculture and / or food processing.

[0009] The yellowtail fish comprises one or more of fish head, fish milt, fish liver, fish intestine, fish roe, fish fillet and fish tail.

[0010] For the enzymolysis, the enzyme addition amount accounts for 1.0-2.0% of the weight ratio of the surimi slurry, the material-liquid ratio is 1:2-1:4 g / mL, the reaction temperature is 50-55° C., and the enzymolysis time is 4-5 hours.

[0011] Furthermore, in the enzymatic hydrolysis, the amount of enzyme added is 1.0-2.0% by weight of the fish paste, deionized water is added to the fish paste according to the solid-liquid ratio of 1:2-1:4 g / mL, the reaction temperature is 50-55°C, the enzymatic hydrolysis time is 4-5 hours, and then the enzyme is inactivated at 90°C for 30-40 minutes.

[0012] The protease enzymolysis adopts flavor protease, papain and double enzyme enzymolysis in different mass ratios.

[0013] The double enzymes are papain and flavor protease, and the mass ratio of the two enzymes is 1:1, 1:2 or 2:1.

[0014] The above-mentioned middle clear liquid after centrifugation is placed in a vacuum freeze dryer, the freeze drying temperature is -50 to -40°C, the vacuum degree is 13 to 26 Pa, and the peptone powder is collected.

[0015] An application of the nitrogen source in the fungal culture medium, wherein the enzymatic hydrolysis product obtained by enzymatic hydrolysis of yellowtail fish waste as a raw material is used as the nitrogen source in the fungal culture medium.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The peptone prepared by the invention with yellowtail fish scraps has high amino acid content, provides a high-quality peptone source for the microbial culture field, and is suitable for the culture needs of various fungi; the obtained method can effectively convert yellowtail fish scraps into high-quality peptone, thereby realizing efficient utilization of waste resources, and has the advantages of low preparation cost and simple process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The present invention provides the variation of mycelial growth diameter of Trichoderma on solid culture medium containing different nitrogen sources.

[0019] Figure 2 The present invention provides the spore conditions of Trichoderma on solid culture media with different nitrogen sources.

[0020] Figure 3 The present invention provides a crude extract of fermentation broth obtained by growing Trichoderma in liquid culture medium containing different nitrogen sources for 14 days.

[0021] Figure 4The present invention provides the dry weight of biomass of Trichoderma grown in liquid culture medium containing different nitrogen sources for 14 days. Specific implementation method:

[0022] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0023] The structures, proportions, sizes, etc. drawn in the drawings of the present invention are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any modification of the structure, change in the proportion relationship, or adjustment of the size should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0024] The peptone obtained by the invention is applied to the fungal culture medium component to improve the nutritional value of the culture medium and the growth effect of the fungi. The fish peptone obtained by the invention shows a good growth-promoting effect in a microbial culture experiment.

[0025] Solid culture medium: different nitrogen sources 2g / L, glucose 20g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, agar 20g / L, pH 6-7.

[0026] Liquid culture medium: different nitrogen sources 2g / L, glucose 20g / L, dipotassium hydrogen phosphate 1g / L, magnesium sulfate 0.5g / L, pH 6-7.

[0027] The nitrogen sources are: fish protein obtained by enzymatic hydrolysis of the protease in the following examples of the present invention, commercially available peptone, yeast powder, beef extract, potato extract powder, and no nitrogen source added.

[0028] The experimental methods in the following examples without specific experimental conditions are usually carried out according to conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are all obtained from commercial sources.

[0029] Unless otherwise specified, the devices used in the present invention are all conventional devices in the art. The present invention is further described below in conjunction with specific embodiments:

[0030] Example 1: Preparation method of fish peptone

[0031] Specifically:

[0032] Clean the yellowtail fish scraps (including fish head, milt, liver, intestine, roe, fillet, and tail), chop them into small pieces, and put them into a tissue grinder to grind them into a muddy state, which is fish paste. Take 100g of fish paste and place it in a 500mL beaker, add 300mL of water and stir evenly, and the solid-liquid ratio is 1:3. The enzymes used for enzymatic hydrolysis are flavor protease and papain, the amount of enzyme added is 1.2%, and the mass ratio of the two enzymes is 2:1, that is, take 0.8g of flavor protease and 0.4g of papain. Add the two enzymes to the beaker, the enzymatic hydrolysis reaction temperature is 53℃, and the enzymatic hydrolysis time is 4h. After the reaction is completed, inactivate at 90℃ for 30min. After cooling to room temperature, take the lower layer of filtrate and place it in a 50mL centrifuge tube, centrifuge it at 6500r / min for 15min, and take the middle layer of clear liquid for freeze drying to obtain fish protein powder. The collected fish peptone powder was analyzed, and the amino nitrogen content was 8.78% and the total nitrogen content was 11.00%.

[0033] Example 2: Fungal growth on fish peptone solid medium

[0034] Specifically:

[0035] The fish peptone powder obtained by the above embodiment is configured according to the configuration method of the above solid culture medium. Different nitrogen sources are commercially available peptone, yeast powder, beef extract and potato extract powder as the control group, and the culture medium without nitrogen source is used as the blank control group for fungal culture. The fungus used is dark green Trichoderma. A circular fungus block with a diameter of 5 mm is punched out from the edge of the pure cultured dark green Trichoderma colony with a hole puncher, and it is inverted and inoculated in the center of the plate containing different nitrogen sources. 3 repetitions are set, and the culture temperature is set to 25 ° C. The growth rate is monitored by measuring the diameter of the colony every half a day. When Trichoderma grows on solid culture medium for 7 days, 10 ml of sterile water is taken to rinse the solid plate and collect spores. Take 1 mL of sterile water containing spores and dilute 10 times, and observe the spore production of Trichoderma under different culture media with a blood cell counting plate and count. Figure 1 The diameter of mycelium of Trichoderma grown on solid culture medium containing different nitrogen sources varies. Figure 2 The spores of Trichoderma on solid culture medium with different nitrogen sources.

[0036] The results showed that under the conditions of culture medium with fish peptone as nitrogen source, the growth rate of Trichoderma was significantly better than that of other culture medium. It was observed that Trichoderma showed stronger growth momentum in fish peptone medium, which was manifested by entering the logarithmic growth phase earlier and the mycelium expanding faster. In addition, fish peptone medium also prompted Trichoderma to produce more spores, and the number of spores increased significantly. This result suggests that fish peptone has a good ability to promote fungal growth as a nitrogen source.

[0037] Example 3: Fungal growth in fish peptone liquid culture medium

[0038] Specifically:

[0039] The fish peptone powder obtained in Example 1 was prepared according to the above-mentioned liquid culture medium preparation method. Different nitrogen sources were used as control groups with commercially available peptone, yeast powder, beef extract and potato extract powder, and a blank control group with no nitrogen source added as culture medium. The liquid culture medium was respectively filled into 250 mL conical flasks, each conical flask was filled with 100 mL of culture medium, and 3 parallels were set for each group. The pure cultured dark green Trichoderma was rinsed with sterile water to collect spores, 50 μL of spore liquid was added to the sterilized culture medium, and cultured at 25 ° C for 14 days.

[0040] After the culture is completed, add an appropriate amount of ethyl acetate to the conical flask and soak for 48 hours, then filter and dry the mycelium after filtration naturally and weigh it, which is the dry weight of biomass. The organic phase of the fermentation broth is extracted with a separatory funnel, and the crude extract of the fermentation broth is obtained by vacuum distillation at 40°C. The quality comparison of the crude extract of the fermentation broth grown for 14 days in the culture medium containing different nitrogen sources is shown in Figure 3 . Figure 4 It is the dry weight of biomass of Trichoderma grown in liquid culture medium containing different nitrogen sources for 14 days.

[0041] The results showed that Trichoderma showed significant advantages in liquid culture medium with fish peptone as nitrogen source compared with other culture media. After 14 days of culture, the growth rate of Trichoderma in fish peptone medium increased and the biomass increased significantly. This shows that the nitrogen source provided by fish peptone is more conducive to the growth of Trichoderma and the accumulation of biovolume. The liquid culture medium with fish peptone as nitrogen source provides a good environment for the biomass accumulation and secondary metabolite production of Trichoderma.

[0042] The above embodiments further illustrate the purpose and technical solutions of the present invention. It should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

[0043] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present disclosure will not further describe various possible combinations.

[0044] In addition, the various embodiments disclosed in the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the content invented by the present invention.

Claims

1. A nitrogen source in a fungal culture medium, characterized in that: The nitrogen source in the fungal culture medium is the enzymatic hydrolysis product obtained by enzymatic hydrolysis of yellowtail fish waste as raw material.

2. A method for preparing a nitrogen source in a fungal culture medium according to claim 1, characterized in that: Yellowtail fish waste is used as raw material, protease is added to the pulverized fish paste for enzymatic hydrolysis, and peptone is obtained by centrifugation and filtration. The peptone is freeze-dried to obtain the final powdered fish peptone, which is the nitrogen source in the fungal culture medium.

3. The method for preparing a nitrogen source in a fungal culture medium according to claim 2, characterized in that: The yellowtail scraps are derived from byproducts or wastes generated by aquaculture and / or food processing.

4. The method for preparing a nitrogen source in a fungal culture medium according to claim 2, characterized in that: The yellowtail fish comprises one or more of fish head, fish milt, fish liver, fish intestine, fish roe, fish fillet and fish tail.

5. The method for preparing a nitrogen source in a fungal culture medium according to claim 2, characterized in that: For the enzymolysis, the enzyme addition amount accounts for 1.0-2.0% of the weight ratio of the surimi slurry, the material-liquid ratio is 1:2-1:4 g / mL, the reaction temperature is 50-55° C., and the enzymolysis time is 4-5 hours.

6. The method for preparing a nitrogen source in a fungal culture medium according to claim 5, characterized in that: The protease enzymolysis adopts flavor protease, papain and double enzyme enzymolysis in different mass ratios.

7. Use of a nitrogen source in a fungal culture medium according to claim 1, characterized in that: In claim 1, yellowtail fish scraps are used as raw materials, and the obtained enzymatic hydrolysate is used as a nitrogen source in fungal culture medium.

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