Microbial culture medium containing peptone derived from hairtail by-product and preparation method thereof

Abstract

The present invention relates to a microbial culture medium containing a peptone derived from a hairtail by-product, and a preparation method thereof. More specifically, the present invention relates to technology for improving microbial growth and recombinant protein production by producing a microbial culture medium using a peptone extracted from hairtail head muscle protein, which is fish waste. In addition, the present invention recycles fish by-products that cause serious issues such as environmental pollution and economic loss, and thus is expected to have the effect of creating a large amount of added value commercially.

Description

Microbial culture medium containing peptone derived from hairtail by-product and method for producing the same

[0001] The present invention relates to a microbial culture medium containing peptone derived from hairtail by-products and a method for producing the same, and more particularly, to a technology for improving microbial growth and production of recombinant proteins by producing a microbial culture medium using peptone extracted from hairtail head muscle protein, which is a fish waste.

[0002]

[0003] As global fish production and processing continue to increase, the amount of fish waste has also increased. Approximately 70% of total aquatic animal production is used for further processing in the fish processing industry. Fish waste generated by the fish processing industry is estimated to account for 20-80% of the total weight, depending on the species, size, and type of processing. Despite the fact that fish waste still contains many reusable components, the vast majority of this waste is simply disposed of in oceans and landfills, posing environmental and aesthetic concerns as well as incurring significant economic losses.

[0004] Solid waste from fish processing waste, including flesh, head, bones, intestines, skin, fins, blood, and viscera, contains a large amount of nutrients such as proteins, essential amino acids, peptides, lipids, carotenoids, omega-3 polyunsaturated fatty acids (PUFAs), vitamins, and minerals. Therefore, some protein hydrolysates derived from fish waste have already been used in industrial applications such as functional foods, cosmetics, and pharmaceuticals, having their functional value verified.

[0005] Protein hydrolysates are defined as protein products obtained by chemical or enzymatic hydrolysis to produce free amino acids or peptides. The most commonly described methods for producing protein hydrolysates are chemical or biological hydrolysis, with biological methods involving enzyme-based processes that enhance the functional and nutritional value of the final product.

[0006] Protein hydrolysates produced through hydrolysis provide a nitrogen source suitable for microbial growth and protein production. Peptone, a water-soluble hydrolysate rich in amino acids, various polypeptides, vitamins, lipids, sugars, and mineral salts, is well-known as a nitrogen substrate. It serves as a nitrogen source for microbial cell culture in biological product production and is widely used in biotechnology fermentation industries. Nitrogen sources are one of the most expensive components in culture media, and the cost of peptone accounts for a significant portion of the production costs of microbial cells and bioproducts. Therefore, extracting high-quality peptone from inexpensive organic materials such as fish waste is a crucial aspect of fish waste management and nutrient recycling.

[0007] The present inventors have completed the present invention, which can improve recombinant protein productivity and microbial growth by manufacturing a medium using peptone extracted through hydrolytic enzyme treatment of muscle protein derived from hairtail by-products as an alternative to peptone used as a nitrogen source in a microbial culture medium.

[0008]

[0009] The present invention provides a microbial culture medium containing peptone derived from hairtail by-product.

[0010] In addition, the present invention provides a method for producing a microbial culture medium containing peptone derived from hairtail by-product.

[0011] The technical problems to be solved by the invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0012]

[0013] In order to solve the above problem, the present invention provides a microbial culture medium containing peptone derived from hairtail by-product.

[0014] The above peptone is characterized as a nitrogen source for a microbial culture medium.

[0015] The above microbial culture medium is characterized by increasing the production of recombinant proteins.

[0016] In addition, the present invention provides a method for producing a microbial culture medium, comprising the steps of boiling a hairtail by-product to powder muscle protein separated from bones; mixing the muscle protein powder with water and hydrolyzing it; freeze-drying the hydrolyzed muscle protein to obtain peptone powder; and adding the peptone powder to produce a microbial culture medium.

[0017] The above hydrolysis step is characterized in that it is performed by one or more hydrolytic enzymes selected from the group consisting of protamax, papain, bromelain, trypsin, and flavorzyme.

[0018] The above hydrolysis step is characterized by adjusting the muscle protein mixed with the water to a pH of 6 to 8, adding the hydrolytic enzyme, and treating the mixture at a temperature of 55 to 65°C for 23 to 25 hours.

[0019] The above hydrolyzing step is characterized by further including a step of inactivating the hydrolytic enzyme at 90 to 100°C for 5 to 15 minutes after hydrolyzing the muscle protein.

[0020]

[0021] The present invention relates to a microbial culture medium containing peptone derived from hairtail by-products and a method for producing the same, and has the effect of improving microbial growth and production of recombinant proteins by producing a microbial culture medium using peptone extracted from hairtail head muscle protein, which is a fish waste.

[0022] In addition, the present invention is expected to have the effect of creating high added value industrially by recycling fish by-products that cause serious problems such as environmental pollution and economic loss.

[0023]

[0024] Figure 1 is a diagram comparing the hydrolysis efficiency of muscle protein derived from hairtail by-products using various hydrolytic enzymes.

[0025] Figure 2 is a diagram comparing the hydrolysis effect of muscle protein derived from hairtail by-product according to the concentration of protamax hydrolase according to the concentration of muscle protein.

[0026] Figure 3 is a diagram showing the hydrolysis effect of muscle protein derived from hairtail by-product according to the concentration of protamax hydrolase, analyzed by SDS-PAGE.

[0027] Figure 4 is a schematic diagram showing the process of manufacturing a microbial culture medium containing peptone using dried pollack byproducts.

[0028] Figure 5 is a diagram comparing the expression of chitosan decomposing enzyme recombinant protein and hSOD recombinant protein in a microbial culture medium containing various types of peptone.

[0029] Figure 6 is a diagram showing the change in growth rate over time of E. coliBL21(DE3) in a microbial culture medium containing various types of peptone.

[0030]

[0031] The terms used in this specification will be briefly explained, and the present invention will be described in detail.

[0032] The terms used in this invention have been selected from widely used, common terms, taking into account their functions. However, these terms may vary depending on the intentions of those skilled in the art, precedents, the emergence of new technologies, etc. Therefore, the terms used in this invention should be defined based on their meaning and the overall content of the invention, rather than simply their names.

[0033] When a part of a specification is said to “include” a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0034] Below, with reference to the attached drawings, embodiments of the present invention are described in detail so that those skilled in the art can easily implement the invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[0035] Specific details, including the problems to be solved, means of solving them, and the effects of the invention, are included in the embodiments and drawings described below. The advantages and features of the present invention, and methods for achieving them, will become clearer with reference to the embodiments described below in detail, along with the accompanying drawings.

[0036] Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

[0037]

[0038] The present invention provides a microbial culture medium containing peptone derived from hairtail by-product.

[0039] As used herein, the term "microbial culture medium" refers to a medium that provides an environment for the growth of microorganisms, including nutrients necessary for their growth. Microorganism culture compositions typically include a carbon source, a nitrogen source, and various inorganic salts. Some microorganisms can utilize carbon dioxide as a carbon source and atmospheric nitrogen as a nitrogen source, but generally require sugars or organic acids as carbon sources, inorganic or organic nitrogen compounds as nitrogen sources, and various inorganic salts. Additionally, some microorganisms require vitamins or trace elements. Nitrogen sources are used for protein synthesis. Microorganisms can utilize inorganic nitrogen sources such as ammonium salts or nitrates, but some require organic nitrogen sources such as amino acids or peptones, depending on the type of microorganism being cultured. Depending on the type of microorganism being cultured and the purpose of the culture, a person skilled in the art to which the present invention pertains can select and use an appropriate microbial culture composition.

[0040] Additionally, suitable precursors may be used in the microbial culture medium. The above-mentioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acid compounds such as phosphoric acid or sulfuric acid, in an appropriate manner.

[0041] The above microbial culture medium may be a Luria-Bertani (LB) medium, or may be any medium commonly used for microbial culture.

[0042] The above peptone can be a nitrogen source for a microbial culture medium.

[0043] The above microorganism may be transformed with a genetically recombinant plasmid.

[0044] The above microbial culture medium may increase the production of recombinant proteins.

[0045] The above peptone is preferably separated and extracted from anchovy by-products, but more preferably separated and extracted from anchovy head.

[0046]

[0047] The present invention provides a method for producing a microbial culture medium, comprising the steps of boiling a hairtail by-product to powder muscle protein separated from bones; mixing the muscle protein powder with water and hydrolyzing it; freeze-drying the hydrolyzed muscle protein to obtain peptone powder; and adding the peptone powder to produce a microbial culture medium.

[0048] The above muscle protein may be a protein from the muscle part of the head of the hairtail.

[0049] It is preferable to separate the above muscle protein by washing the hairtail by-product with water and boiling it at 100℃ for 15 minutes.

[0050] The step of powdering the above muscle protein may be to powderize it by freeze-drying and grinding it with a mixer.

[0051] The above hydrolysis step is preferably performed by one or more hydrolases selected from the group consisting of protamax, papain, bromelain, trypsin, and flavorzyme, but is more preferably performed by the hydrolase of protamax.

[0052] The hydrolysis step is preferably performed by adjusting the muscle protein mixed with water to a pH of 6 to 8, adding the hydrolytic enzyme, and treating the muscle protein at a temperature of 55 to 65°C for 23 to 25 hours. It is more preferable to adjust the muscle protein mixed with water to a pH of 7, adding the hydrolytic enzyme, and treating the muscle protein at a temperature of 60°C for 24 hours.

[0053] It is preferable that the above hydrolyzing step further includes a step of inactivating the hydrolytic enzyme at 90 to 100°C for 5 to 15 minutes after hydrolyzing the muscle protein, but it is more preferable that the above hydrolyzing step further includes a step of inactivating the hydrolytic enzyme at 95°C for 10 minutes after hydrolyzing the muscle protein.

[0054] The above hydrolysis step can be performed by centrifugation to remove unhydrolyzed muscle proteins.

[0055] The step of preparing a microbial culture medium by adding the above peptone powder may be prepared by further including yeast extract and NaCl, but is not limited thereto.

[0056]

[0057] Hereinafter, to aid understanding of the present invention, examples will be given in detail. However, the following examples are intended only to illustrate the scope of the present invention and are not intended to limit its scope. These examples are provided to more fully explain the present invention to those of average skill in the art.

[0058]

[0059] <Example 1> Production of peptone derived from hairtail byproducts

[0060] 1-1. Isolation of muscle protein from the head of the hairtail

[0061] As shown in Fig. 4, 134 heads of hairtail, a by-product of hairtail, were washed with water, boiled at 100°C for 15 minutes, and muscle proteins were obtained by separating the muscle from the bones. The separated muscle proteins were freeze-dried, finely ground in a blender, and stored at -20°C.

[0062]

[0063] 1-2. Manufacturing of peptone from hairtail head

[0064] As shown in Fig. 4, 100 g of muscle protein powder separated according to Example 1 was dissolved in 2 L of water, the pH was adjusted to 7.0, and protamex hydrolase was added at a ratio of 0.5% (w / v). The mixture was reacted at 60°C for 24 hours, and then the mixture was heated at 95°C for 10 minutes to inactivate the hydrolase. The hydrolyzed mixture was centrifuged at 7,000 rpm at 4°C for 30 minutes, and the supernatant was recovered and freeze-dried to prepare powdered peptone.

[0065]

[0066] <Example 2> Manufacturing of microbial culture medium using hairtail head peptone

[0067] A microbial culture medium using peptone isolated from the hairtail head according to Example 1 above was prepared with the same composition as Luria-Bertani (LB) broth (BD Biosciences), which is commonly used for microbial culture. Yeast extract (10 g / liter), NaCl (10 g / liter), and the hairtail head peptone prepared according to Example 1 above (5 g / liter) were mixed in distilled water, and then subjected to high-pressure heat treatment at 121°C for 15 minutes in an autoclave (Fig. 4).

[0068]

[0069] <Comparison Example>

[0070] Each peptone (5 g / liter) of commercially available peptones, milk peptone (MP), animal peptone (AP), vegetable tryptone (VT), vegetable peptone (VP), potato peptone (PP), and wheat peptone (WP), was added instead of the peptone from dried pollack by-product in the same manner as in Example 2 to prepare a microbial culture medium.

[0071]

[0072] <Experimental Example 1> Measurement of the weight ratio (%) of muscle protein isolated from the head of a hairtail

[0073] As in Example 1 above, the weight ratio (%) of muscle protein and bone separated from the hairtail head was measured. As shown in [Table 1] below, the total weight of the hairtail head before muscle protein separation was 5,426 g including moisture, and the total weight of muscle protein containing moisture separated from the hairtail head was 934 g (dry weight 230 g), indicating that muscle protein accounts for 17.2% of the total weight of the fish head.

[0074]

[0075]

[0076] <Experimental Example 2> Analysis of hydrolysis efficiency of hairtail head muscle protein by type of hydrolytic enzyme

[0077] According to Example 1, the muscle protein isolated from the hairtail head was analyzed for hydrolysis efficiency using commercially available protein hydrolyzing enzymes, such as protamax, papain, bromelain, trypsin, and flavorzyme, and the results are shown in Fig. 1. The hydrolysis enzyme reaction was used so that the muscle protein powder of the hairtail head was 1%, and the substrate (muscle protein of the hairtail head) and enzyme were mixed at a ratio of 1:1 (v / v) and hydrolysis was performed for 12 hours. After hydrolysis, the hydrolytic enzyme was inactivated at 95°C for 10 minutes, centrifuged, and the supernatant was recovered. The sediment was dried in a dryer at 60°C and weighed. The recovery rate of the soluble fraction of the supernatant was calculated by comparing it with the initial weight of the sediment. The protein concentration of each sample supernatant (water-soluble portion) of the hydrolyzate was measured using a Pierce Bicinchoninic acid (BCA) protein assay kit, and SDS-PAGE electrophoresis analysis was performed to confirm the degree of protein hydrolysis, as shown in Fig. 1. As a result of hydrolysis using commercial enzymes for protein hydrolysis of muscle protein isolated from the hairtail head, the hydrolyzate of the hairtail head muscle protein hydrolyzed using Protamax showed a high protein content and a high hydrolysis yield. The hydrolysis efficiency produced by Protamax was over 90%, followed by Papain and Trypsin with over 80% (Fig. 1a).

[0078] In addition, the results of SDS-PAGE electrophoresis to detect the hydrolyzed protein fraction of hairtail head muscle protein by hydrolase showed that protamax hydrolyzed the hairtail head muscle protein the best, while the hydrolase head muscle protein by papain, bromelain, and trypsin was not completely hydrolyzed, as two protein bands in the hydrolyzate with a molecular size of around 9 kDa were confirmed (Fig. 1b). Therefore, it was found that among the protein hydrolases used in the above experiment, protamax was the most suitable for producing peptone.

[0079]

[0080] <Experimental Example 3> Effect of concentration-dependent hydrolysis of hairtail head muscle protein (substrate) according to the concentration of protamax hydrolase

[0081] In order to optimize the hydrolysis of hairtail head muscle protein using protamax hydrolase, the concentration of hairtail head muscle protein (substrate) separated according to Example 1 was changed to 1%, 5%, 10%, and 20%, and the protamax hydrolase concentration was added to 0.05%, 0.25%, 0.5%, and 1%, and the hydrolase reaction was performed under the conditions. The hydrolase reaction was performed at 60°C for 12 hours with stirring at 150 rpm, and the hydrolase was inactivated at 95°C for 10 minutes, after which the supernatant and the precipitate were collected, respectively, by centrifugation. The supernatant was used to measure the protein concentration, and the precipitate was weighed to determine the weight of the unhydrolyzed hairtail head muscle protein. Regardless of the concentration of hydrolase, a hydrolysis rate of over 90% was observed in the hydrolysis reaction using 1% of the hairtail head muscle protein (substrate), and it was confirmed that the overall hydrolysis rate decreased as the substrate concentration increased (Fig. 2a). However, when the hydrolysis effect of hairtail head muscle protein at various substrate concentrations according to the concentration of protamax hydrolase was analyzed by protein concentration measurement and SDS-PAGE, the protein hydrolysis rate according to the enzyme concentration at each substrate concentration did not have a significant effect (Fig. 2b, Fig. 3).

[0082]

[0083] <Experimental Example 4> Analysis of free amino acid and nitrogen content in hairtail head peptone

[0084] The peptone from the hairtail head prepared according to Example 1 was mixed with an extraction solution (0.1 M perchloric acid, 0.1% metaphosphoric acid) and extracted using an ultrasonic grinder for 1 hour. The sample was shaken and cultured at room temperature for 1 hour, filtered through a 0.2 μm filter, and analyzed for free amino acid composition, total nitrogen content, and amino nitrogen content using high-pressure liquid chromatography (HPLC) equipment (Dionex Ultimate 3000).

[0085] The total nitrogen and amino acid nitrogen contents of the hairtail head peptone manufactured according to Example 1 were confirmed to be 12.22% and 3.19%, respectively, and the amino acid profile results were confirmed as shown in [Table 2] below.

[0086]

[0087]

[0088]

[0089] According to the amino acid profile results above, the relative content of leucine was confirmed to be 17.26%, and the relative content of alanine was confirmed to be 12.45%. In addition, amino acids such as glutamic acid (12.24%), lysine (11.9%), and serine (9.55%) were confirmed to be present in high concentrations in the peptone of hairtail head, while the relative contents of citrulline and arginine were found to be less than 1%.

[0090]

[0091] <Experimental Example 5> Measurement of recombinant protein productivity efficiency using hairtail head peptone

[0092] The productivity of genetically recombinant proteins was analyzed using milk peptone (MP), animal peptone (AP), vegetable tryptone (VT), vegetable peptone (VP), potato peptone (PP), wheat peptone (WP) and the hairtail head peptone (FP) prepared according to the above Example 1, and seven media prepared according to the above Example 2 and Comparative Examples and commercially available LB medium (control group), and the results are shown in Fig. 5. As test strains, E. coli transformed with the chitosan degrading enzyme gene derived from Bacillus subtilis (GenBank accession no. GU001716) and the human antioxidant protein cu, zinc-superoxide dismutase (hSOD, GenBank accession no. GU001716) gene, which had the signal sequence removed and inserted into the pET11a vector, was used in E. coliBL21 (DE3). Each transformed strain was inoculated into the seven prepared media and LB medium (control) with ampicillin at a concentration of 100 μg / ml, and cultured with shaking at 37°C. When the strain concentration reached 0.7–0.8 at OD600nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to 0.01 mM. Protein expression was induced in E. coli transformed with the chitosan degrading gene at 15°C for 72 hours, and in E. coli transformed with the human antioxidant protein (hSOD) gene at 20°C for 24 hours. Protein expression was confirmed by collecting the cells, disrupting them with ultrasound, and analyzing them using SDS-PAGE. It was confirmed that the degree of protein expression varied depending on the type of peptone medium, and the expression of chitosan degrading enzyme and hSOD proteins was the highest in the medium containing hairtail head peptone (Fig. 5a, 5c). In addition, as shown in the quantitative results in Fig. 5b and d, the chitosan decomposition enzyme protein increased by 20% in the medium containing the hairtail head peptone compared to the LB medium (control group), and the hSOD protein showed a 32% increased expression.The relative expression level of chitosan degrading enzyme protein was expressed at more than 50% in all media except for the media containing milk peptone and potato peptone, where the expression level was less than 50%, and hSOD protein expression was expressed at more than 50% in all types of peptone media.

[0093]

[0094] <Experimental Example 6> Analysis of E. coli Growth Using Hairtail Head Peptone

[0095] The growth rate of E. coliBL21(DE3), a host strain for recombinant protein production, was confirmed using the media containing peptone (FP) derived from hairtail head, animal peptone (AP), and vegetable peptone (VP) among the media manufactured according to the above Examples 1 to 2 and Comparative Examples, and the LB medium as a control medium. The E. coliBL21(DE3) strain was inoculated into 20 ml of each of the above media and cultured with shaking at 200 rpm at 37°C. The growth of the strain was measured by absorbance at 850 nm (OD) using a Real Time Cell Growth Logger (Biosan), and monitored every 20 minutes for 48 hours. When comparing the 850 nm absorbance (OD) values ​​that indicate the highest growth values ​​of the strains in the above four media, the hairtail head peptone (FP) medium showed the highest growth at 2.84, followed by LB medium at 2.62, vegetable peptone (VP) medium at 2.58, and animal peptone (AP) medium at 1.79 (Fig. 6).

[0096]

[0097] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred implementation examples and are not intended to limit the scope of the present invention. Therefore, the actual scope of the present invention is defined by the appended claims and their equivalents. The scope of the present invention is set forth in the claims below, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

1. Microbial culture medium containing peptone derived from hairtail by-product.

2. In paragraph 1, A microbial culture medium containing peptone derived from hairtail by-products, characterized in that the above peptone is a nitrogen source for the microbial culture medium.

3. In paragraph 1, The above microbial culture medium is a microbial culture medium containing peptone derived from hairtail by-products, characterized in that it increases the production of recombinant proteins.

4. Step of boiling the dried pollack by-products and pulverizing the muscle protein separated from the bones; A step of mixing the above muscle protein powder with water and hydrolyzing it; A step of freeze-drying the hydrolyzed muscle protein to obtain peptone powder; and A method for producing a microbial culture medium, comprising: a step of producing a microbial culture medium by adding the above peptone powder.

5. In paragraph 4, A method for producing a microbial culture medium, characterized in that the hydrolyzing step is performed by one or more hydrolytic enzymes selected from the group consisting of protamax, papain, bromelain, trypsin, and flavourzyme.

6. In paragraph 4, A method for producing a microbial culture medium, characterized in that the hydrolyzing step comprises adjusting the muscle protein mixed with the water to a pH of 6 to 8, adding the hydrolytic enzyme, and treating the mixture at a temperature of 55 to 65°C for 23 to 25 hours.

7. In paragraph 4, A method for producing a microbial culture medium, characterized in that the hydrolyzing step further includes a step of inactivating the hydrolytic enzyme at 90 to 100° C. for 5 to 15 minutes after hydrolyzing the muscle protein.

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