Peptone derived from salmon by-product and medium composition for culturing microorganisms containing same

Abstract

The present invention relates to a method for preparing peptone derived from a salmon by-product, and a medium composition, for culturing microorganisms, comprising the peptone. The method for preparing peptone derived from a salmon by-product comprises the steps of: heating a salmon by-product and pulverizing muscle proteins separated from bones; mixing the muscle protein powder with water to prepare a mixture; adding hydrolase to the mixture to hydrolyze same; and drying the hydrolyzed mixture to obtain peptone powder. In addition, the present invention provides peptone derived according to the present invention from a salmon by-product, and a medium composition comprising the peptone for culturing microorganisms, the peptone having skin regeneration, skin barrier strengthening, anti-aging, reverse aging, and antioxidant effects, and thus the present invention can be used in the commercial development of health functional foods, cosmetics, and pharmaceuticals, and can help reduce environmental pollution since fish waste is utilized.

Description

Peptone derived from salmon by-products and a microbial culture medium composition containing the same

[0001] The present invention relates to peptone derived from salmon by-products and a microbial culture medium composition containing the same. More specifically, it relates to a microbial culture medium composition that utilizes peptone extracted from salmon head muscle protein, which is a fish waste, and has excellent skin regeneration, skin barrier strengthening, anti-aging, reverse aging, and antioxidant activities, and improves the production of recombinant proteins.

[0002]

[0003] As global fisheries production has increased significantly due to population growth and advancements in fishing technology, fish waste has also risen substantially. This global increase in fish waste is causing massive environmental problems and economic losses. Therefore, there is a need to develop technologies for the efficient processing and recycling of fish waste.

[0004] Maximizing the utilization of currently underutilized, low-value fish residues—such as skin, scales, fins, bones, flesh, heads, and internal organs—can produce high-demand compounds like collagen, gelatin, fish oil, omega-3 fatty acids, bioactive peptides, protein hydrolysates, and enzymes. These high-value compounds are utilized in various industries, including food processing, health functional foods, pharmaceuticals, cosmeceuticals, and biotechnology.

[0005] Therefore, converting nutrient-rich fish waste, which is often discarded, into high-value-added products through sustainable and economically viable technologies helps maximize the efficient utilization of fish resources.

[0006] Meanwhile, peptone serves as an essential nutrient source for microbial and cell culture, playing a significant role in various industries such as life science research, pharmaceuticals, the production of genetically modified drugs, and the fermentation industry. In the food industry, it enhances flavor and nutrition, while in cosmetics, it aids in skincare products by promoting cell regeneration and hydration. In the biopharmaceutical industry, peptone is used as a primary ingredient to increase the productivity of protein drugs and antibiotics.

[0007] In particular, the production of biopharmaceuticals is one of the most promising achievements in the field of biotechnology, and research to increase productivity plays an important role as demand rises due to advancements in biotechnology and human aging.

[0008] As part of efforts to increase such productivity, the inventors have completed the present invention regarding the production of peptone derived from salmon by-products, which has excellent skin regeneration, skin barrier strengthening, anti-aging, reverse aging, and antioxidant activities, by utilizing salmon heads, which are fish waste.

[0009]

[0010] To solve the above-mentioned problems, the present invention aims to provide a method for producing peptone derived from salmon by-products.

[0011] In addition, the present invention aims to provide a microbial culture medium composition comprising peptone produced by a method for producing peptone derived from salmon by-products.

[0012]

[0013] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0014]

[0015] To solve the above problems, the present invention aims to provide a method for producing peptone derived from salmon by-products, characterized by comprising the steps of: heating salmon by-products to pulverize muscle protein separated from bone; mixing water with the muscle protein powder to prepare a mixture; adding a hydrolytic enzyme to the mixture to hydrolyze it; and drying the hydrolyzed mixture to obtain peptone powder.

[0016] The above hydrolytic enzyme is characterized by being one or more selected from the group consisting of papain, protamax, trypsin, bromelain, alcalase, and pronase.

[0017] The hydrolysis step is characterized by adjusting the mixture to a pH of 6 to 8, adding the hydrolytic enzyme, and treating it at 45 to 60 ℃ for 5 to 7 hours.

[0018] The above hydrolysis step is characterized by further including a step of inactivating the hydrolysis enzyme at 90 to 100 ℃ for 5 to 15 minutes after hydrolysis.

[0019]

[0020] In addition, the present invention aims to provide a microbial culture medium composition comprising peptone produced by the above-described method for producing peptone derived from salmon by-products.

[0021] The above peptone is characterized as being a nitrogen source for the above microbial culture medium.

[0022] The above peptone is characterized by having anti-aging, reverse aging, and antioxidant activities.

[0023] The above peptone is characterized by exhibiting skin regeneration and skin barrier strengthening functions.

[0024] The above-mentioned culture medium composition is characterized by increasing the productivity of genetically modified proteins.

[0025]

[0026] By providing the peptone derived from salmon by-products of the present invention, which has skin regeneration, skin barrier strengthening, anti-aging, reverse aging, and antioxidant effects, and a microbial culture medium composition containing the same, it can be utilized in the development of health functional foods, cosmetics, and pharmaceuticals.

[0027] In addition, since the present invention recycles fish waste such as salmon by-products, environmental pollution can be reduced, and economic losses can be reduced by lowering the cost of processing said fish waste.

[0028]

[0029] The effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description in the claims.

[0030]

[0031] FIG. 1 is a protein electrophoresis diagram comparing the hydrolysis efficiency of salmon head-derived muscle protein prepared according to one embodiment of the present invention by various hydrolytic enzymes. ((a) bromelain, (b) papain, (c) pronase, (d) protamax, (e) trypsin, (f) alkalase)

[0032] Figure 2 is a diagram showing the recovery rate of water-soluble protein after protein hydrolysis of salmon head-derived protein prepared according to one embodiment of the present invention, categorized by type of hydrolytic enzyme.

[0033] Figure 3 is a diagram analyzing the active oxygen scavenging efficacy of salmon head peptone prepared according to one embodiment of the present invention.

[0034] Figure 4 is a diagram analyzing the efficacy of inhibiting HDF cell aging caused by LPS according to treatment with salmon head peptone prepared according to one embodiment of the present invention.

[0035] Figure 5 is a diagram analyzing the efficacy of inhibiting HDF cell aging caused by H2O2 according to treatment with salmon head peptone prepared according to one embodiment of the present invention.

[0036] Figure 6 is a diagram showing the change in HDF cell size after treating aged HDF cells with salmon head peptone, PBS control, plant peptone, and animal peptone prepared according to one embodiment of the present invention.

[0037] Figure 7 is a diagram showing the analysis of changes in CDKN1A mRNA, cell viability by the MTT method, and cell viability by the cell counting method after treating aged HDF cells with salmon head peptone, PBS control, plant peptone, and animal peptone prepared according to one embodiment of the present invention.

[0038] Figure 8 is a figure showing the changes in expression of IL1B, SAA1, SLIT2, KLF4, SOX2, and POU5F1 mRNA after treating aged HDF cells with salmon head peptone prepared according to one embodiment of the present invention, PBS control, plant-based peptone, and animal-based peptone.

[0039] Figure 9 is a diagram analyzing changes in the expression of p21 and IL-1β proteins by treating artificial skin tissue irradiated with ultraviolet rays with salmon head peptone prepared according to one embodiment of the present invention.

[0040] FIG. 10 is a diagram analyzing changes in the expression of Collagen I, Collagen IV, Filaggrin, and Elastin proteins by treating artificial skin tissue irradiated with ultraviolet rays with salmon head peptone prepared according to one embodiment of the present invention.

[0041] FIG. 11 is a diagram analyzing changes in the expression of Ki-67 and OCT4 proteins by treating artificial skin tissue irradiated with ultraviolet rays with salmon head peptone prepared according to one embodiment of the present invention.

[0042] FIG. 12 is a diagram comparing the recombinant protein productivity of microbial culture media containing salmon head peptone, a PBS control, plant peptone, and animal peptone, respectively, prepared according to one embodiment of the present invention.

[0043]

[0044] The terms used in this specification will be briefly explained, and the invention will be described in detail.

[0045] The terms used in this invention have been selected based on currently widely used general terms while considering their functions within the invention; however, these terms may vary depending on the intent of those skilled in the art, case law, the emergence of new technologies, etc. Therefore, the terms used in this invention should be defined not merely by their names, but based on their meanings and the overall context of the invention.

[0046] When a part of a specification is described as “comprising” a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0047] Embodiments of the present invention are described below with reference to the attached drawings so that those skilled in the art can easily implement them. However, the present invention may be embodied in various different forms and is not limited to the embodiments described herein.

[0048] Specific details regarding the problem to be solved by the present invention, the means for solving the problem, and the effects of the invention are included in the embodiments and drawings described below. The advantages and features of the present invention, and the methods for achieving them, will become clear by referring to the embodiments described in detail below in conjunction with the accompanying drawings.

[0049] Hereinafter, the present invention will be described in more detail with reference to the attached drawings.

[0050]

[0051] The present invention provides a method for producing peptone derived from salmon by-products, characterized by comprising the steps of: heating salmon by-products to pulverize muscle protein separated from bone; mixing water with the muscle protein powder to prepare a mixture; adding a hydrolytic enzyme to the mixture to hydrolyze it; and drying the hydrolyzed mixture to obtain peptone powder.

[0052] The above salmon by-product may be a salmon head, but is not limited to it.

[0053] The above muscle protein may be the muscle protein of the above salmon head.

[0054] Heating the above salmon by-product can be performed by washing the above salmon by-product with water and boiling it at 100°C for 30 minutes.

[0055] The step of pulverizing the muscle protein can be performed by freeze-drying the protein of the muscle part separated from the bone and finely grinding it with a mixer.

[0056] The above hydrolytic enzyme may be one or more selected from the group consisting of papain, protamax, trypsin, bromelain, alcalase, and pronase, but preferably may be protamax or alcalase.

[0057] The above hydrolysis step may be performed by adjusting the mixture to a pH of 6 to 8 and adding the hydrolytic enzyme, and treating it at 45 to 60 ℃ for 5 to 7 hours, but it is preferable to adjust the mixture to a pH of 7 and add the hydrolytic enzyme, and treat it at 55 ℃ for 6 hours.

[0058] In addition, the hydrolysis step preferably further includes a step of inactivating the hydrolytic enzyme at 90 to 100°C for 5 to 15 minutes after hydrolysis, but it is even more preferable to further include a step of inactivating the hydrolytic enzyme at 95°C for 10 minutes after hydrolysis.

[0059] The step of obtaining the peptone powder may further include the step of centrifuging the hydrolyzed mixture at 10,000 rpm for 20 minutes.

[0060] Centrifuging the above hydrolyzed mixture may remove the above unhydrolyzed muscle protein.

[0061]

[0062] In addition, the present invention provides a microbial culture medium composition comprising peptone produced by the above-described method for producing peptone derived from salmon by-products.

[0063] In this specification, the term "medium for microbial culture" refers to a medium that contains nutrients necessary for the growth of microorganisms and provides an environment for their growth. Generally, a composition for microbial culture includes a carbon source, a nitrogen source, and various inorganic salts. While some microorganisms may utilize carbon dioxide as a carbon source and nitrogen from the air as a nitrogen source, they generally require sugars or organic acids as carbon sources, inorganic nitrogen compounds or organic nitrogen compounds as nitrogen sources, and simultaneously require various inorganic salts. In addition, some microorganisms require vitamins or trace elements. Nitrogen sources are used for protein synthesis. Microorganisms may utilize inorganic nitrogen such as ammonium salts or nitrates, but depending on the type of microorganism, some may require organic nitrogen such as amino acids or peptones. Depending on the type of microorganism to be cultured and the purpose of culture, a person skilled in the art to which this invention belongs may select and use a suitable composition for microbial culture.

[0064] In addition, suitable precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner in a manner suitable for the culture process, but are not particularly limited thereto. The pH of the culture may be controlled by using basic compounds such as sodium hydroxide, potassium hydroxide, and ammonia, or acid compounds such as phosphoric acid or sulfuric acid in a suitable manner.

[0065] The above-mentioned microbial culture medium composition may be Luria-Bertani (LB) medium, and may also be a medium commonly used for microbial culture.

[0066] The above-mentioned microbial culture medium composition may include yeast extract and NaCl, but is not limited thereto.

[0067] The above peptone may be a nitrogen source for a microbial culture medium composition.

[0068] The above peptone is preferably isolated and extracted from salmon by-products, but more preferably isolated and extracted from salmon heads.

[0069] The above peptone may exhibit anti-aging, reverse aging, and antioxidant activities.

[0070] The above peptone may exhibit skin regeneration and skin barrier strengthening functions.

[0071] The above-mentioned culture medium composition may increase the productivity of genetically modified proteins.

[0072] The above microorganism may be transformed with a genetically recombinant plasmid, but is not limited thereto.

[0073]

[0074] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0075]

[0076] <Example 1> Preparation of Peptone Derived from Salmon By-products

[0077] 1-1. Isolation of Salmon Head Muscle Protein

[0078] 10.4 kg of salmon heads, which are salmon byproducts, were washed with water and boiled at 100°C for 30 minutes to separate the muscle portion from the bone and obtain muscle protein. The separated muscle protein was freeze-dried, finely ground with a blender, and stored at -20°C.

[0079]

[0080] 1-2. Preparation of Salmon Head Peptone

[0081] The muscle protein powder separated according to Example 1-1 above was dissolved in distilled water to a concentration of 5% and the pH was adjusted to 7.0. Then, a mixture was prepared by adding protamex hydrolase at a ratio of 0.6% (w / v), reacted at 55°C for 6 hours, and then heated at 95°C for 10 minutes to inactivate the hydrolase. The hydrolyzed mixture was centrifuged at 10,000 rpm for 20 minutes to recover the supernatant, and dried in a dryer to produce powdered peptone.

[0082]

[0083] <Example 2> Preparation of microbial culture medium using salmon head peptone

[0084] A microbial culture medium containing peptone isolated from salmon heads 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 (5 g / L), NaCl (10 g / L), and salmon head peptone (10 g / L) prepared according to Example 1 above were mixed in distilled water and subjected to high-pressure heat treatment at 121°C for 15 minutes in an autoclave.

[0085]

[0086] <Comparison Example 1>

[0087] A microbial culture medium was prepared in the same manner as in Example 2 above, but with the addition of commercially available animal peptone (10 g / L) instead of salmon head peptone.

[0088]

[0089] <Comparison Example 2>

[0090] A microbial culture medium was prepared in the same manner as in Example 2 above, but with the addition of commercially available plant-based peptone (10 g / L) instead of salmon head peptone.

[0091]

[0092] <Experimental Example 1> Measurement of muscle protein isolated from salmon head

[0093] As in Example 1 above, the weight ratio (%) of muscle protein and bone separated from the salmon head was measured. As shown in Table 1 below, the total weight of the salmon head before muscle protein separation was 10.4 kg with water content, and the total weight of the muscle protein with water content separated from the salmon head was 2.25 kg, which indicates that the muscle protein accounts for 21.6% of the total weight of the fish head.

[0094]

[0095] Salmon Head Wet Weight (kg) Wet Weight (%) Total Head Weight 10.4kg - Muscle Weight 2.25kg 21.6% Bone Weight 3.07kg 29.6% Residue 3.08kg 28.5% Loss 2kg 18.5%

[0096]

[0097] <Experiment Example 2> Hydrolysis of Salmon Head Muscle Protein by Type of Hydrolytic Enzyme

[0098] The hydrolysis efficiency of the muscle protein isolated from the salmon head in Example 1 above was analyzed using commercially available protein hydrolyzing enzymes, namely papain, protamax, trypsin, bromelain, alkalase, and pronase, and is shown in Figure 1. For the hydrolytic enzyme reaction, the salmon head muscle protein powder was used at a concentration of 1%. After mixing at a substrate (salmon head muscle protein) to enzyme ratio of 1:1 (v / v), hydrolysis was performed at 40°C and 50°C for 6, 12, 18, and 12 hours, respectively. The pH used for hydrolysis was the recommended pH for each enzyme and is shown in Table 2 below.

[0099]

[0100] Enzyme activity pH Papain 1.5-10 U / mg 7.0 Bromelain >3 U / mg 6.0 Trypsin >250000 USP Unit / g 8.0 Protamax >1.5 AU-N / g 7.0 Pronase 7 U / mg 8.0 Alcalase >5 U / g 8.0

[0101]

[0102] After hydrolysis, the hydrolytic enzyme was inactivated at 95°C for 10 minutes, and the supernatant was recovered by centrifugation. The precipitate 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 precipitate. The degree of hydrolysis of the supernatant (water-soluble portion) of each hydrolysate sample was confirmed through protein electrophoresis as shown in Fig. 1. As a result of hydrolysis of muscle protein isolated from salmon heads using commercially available protein hydrolytic enzymes, high hydrolysis efficiency was observed in the hydrolysates of salmon head muscle protein hydrolyzed using Protamax (Fig. 1d), bromelain (Fig. 1b), and alcalase (Fig. 1f).

[0103] In addition, to analyze the final recovery rate, the weight of the precipitate after hydrolysis was analyzed and presented as shown in Figure 2. The recovery rate indicates how much insoluble protein is converted into a water-soluble form and represents the final amount that can be obtained in industrial applications. After 6 hours of hydrolysis, the recovery rates of the hydrolysates using each enzyme were found to be 75% for papain, 69% for bromelain, 67% for trypsin, 82% for protamax, 87% for pronase, and 83.5% for alcalase. At 24 hours, papain was 79.5%, bromelain 78%, trypsin 69.5%, protamax 85%, pronase 87%, and alcalase 88.5%, with protamax, pronase, and alcalase showing similarly high recovery rates.

[0104] When considering the combined hydrolysis results and recovery rates via protein electrophoresis, it was confirmed that Protamax and Alcalase are suitable for hydrolysis as they cleave salmon head muscle protein most effectively and have the highest recovery rates.

[0105]

[0106] <Experiment Example 3> Analysis of Total Nitrogen and Amino Acid Nitrogen Content in Salmon Head Peptone

[0107] To measure the total nitrogen and amino acid nitrogen content of the salmon head peptone prepared according to Example 1 above, an analysis was commissioned to the Center for Life Science & Technology Innovation at Jeju National University, and the animal peptone of Comparative Example 1 and the plant peptone of Comparative Example 2 above were used as comparison groups. As shown in Table 3 below, the total nitrogen and amino acid nitrogen content of the salmon head peptone were confirmed to be 12.5% ​​and 4.9%, respectively.

[0108]

[0109] Types of Peptone Total Nitrogen (%) Amino Acid Nitrogen (%) Salmon Head Peptone 12.5 4.9 Animal Peptone 15.2 2.6 Plant Peptone 11.5 3.4

[0110]

[0111] <Experiment Example 4> Analysis of the Molecular Weight of Salmon Head Peptone

[0112] GPC analysis was performed to confirm the molecular weight distribution of the salmon head peptone prepared according to Example 1 above, and the results are shown in Table 4 below in comparison with animal peptone and plant peptone. The average molecular weight of the salmon head peptone was 557 daltons, and the highest peak molecular weight was 424 daltons, indicating that the protein was cleaved into lower molecular weights compared to animal peptone and plant peptone. The polydispersity index indicates the variety of peptides produced; a lower value is recorded as more degradation occurs, because the number of amino acid complexes decreases. The salmon head peptone showed a polydispersity index of 1.934, which was lower than that of animal peptone (2.507) and plant peptone (2.081).

[0113]

[0114] Types of Peptone Mn (Dalton) Mw (Dalton) Mp (Dalton) PDI Salmon Head Peptone 288 557 424 1.934 Animal Peptone 487 1222 136 12.507 Plant Peptone 309 64 2825 2.081

[0115] (Mn; number average molecular weight, Mw; weight average molecular weight, Mp; peak molecular weight, PDI; polydispersity index)

[0116]

[0117] <Experiment Example 5> Analysis of the Anti-aging Efficacy of Salmon Head Peptone

[0118] 5-1. Antioxidant Benefits of Salmon Head Peptone

[0119] To confirm the DPPH radical scavenging efficacy of salmon head peptone prepared as in Example 1 above, 0.1 mg / ml vitamin C (Ascorbic acid) was set as a positive control, and 100 µl of salmon head peptone samples at concentrations of 0 mg / ml, 2 mg / ml, 4 mg / ml, 6 mg / ml, 8 mg / ml, and 10 mg / ml were mixed with 100 µl of 0.2 mM DPPH solution, reacted at room temperature in a dark room for 30 minutes, and measured at 517 nm absorbance.

[0120] To confirm the ABTS radical scavenging efficacy of salmon head peptone prepared as in Example 1 above, 7.0 mM ABTS and 2.45 mM potassium persulfate solution were reacted in a dark room at room temperature for 12 hours, and then the radical solution was diluted with 10% ethanol to an absorbance of 0.7 at 732 nm. For analysis, 0.1 mg / ml vitamin C (ascorbic acid) was set as a positive control, and 180 μL of each sample of salmon head peptone at concentrations of 0 mg / ml, 2 mg / ml, 4 mg / ml, 6 mg / ml, 8 mg / ml, and 10 mg / ml was mixed with 20 μL of ABTS+ radical solution in a 96-well plate and reacted at room temperature in a dark room for 10 minutes. Afterward, the absorbance was measured at 734 nm, and the radical scavenging ability was expressed as a percentage (%) by comparing the absorbance between the sample solution addition group and the non-addition group.

[0121] As shown in Figure 3, the antioxidant efficacy of salmon head peptone could be confirmed as the group treated with salmon head peptone (salmon peptone) showed concentration-dependent DPPH and ABTS radical scavenging effects.

[0122]

[0123] 5-2. Anti-aging effects of salmon head peptone by inhibiting inflammatory responses

[0124] To confirm the antioxidant efficacy of salmon head peptone prepared as in Example 1 above at the cellular level, it was evaluated using a cellular aging model induced by an inflammatory response. Cellular aging was induced in Human dermal fibroblasts (HDF) through treatment with LPS (Lipopolysaccharide). HDF cells (normal cells) were seeded in a 6-well plate at a density of 0.8 × 10^5 cells, treated with 1x PBS or LPS (1 μg / mL) the next day, and after 24 hours, 1x PBS or 0.1% salmon head peptone was additionally added to confirm the efficacy of salmon head peptone in inhibiting LPS-induced cellular aging.

[0125] As shown in Fig. 4, it was confirmed that the cell size increased and the shape was deformed in the LPS-treated group compared to the PBS-treated control group (Fig. 4-1-1) or the salmon head peptone-treated group (salmon peptone, Fig. 4-1-3) (Fig. 4-1-2), and it was confirmed that the cell shape deformation caused by LPS treatment was restored to the control level by additional treatment with salmon head peptone (Fig. 4-1-4).

[0126] In addition, when the expression of IL1B mRNA, a major cytokine exhibiting an inflammatory response, and CDKN1A mRNA, an important molecular marker of cellular senescence, was confirmed by qPCR in LPS-treated HDF, it was observed that both genes were significantly increased, confirming that LPS induced an inflammatory response and cellular senescence (Figs. 4-2, 4-3).

[0127] In addition, as a result of additional treatment with salmon head peptone, a significant decrease in the mRNA expression of CDKN1A and IL1B was observed (Figs. 4-2, 4-3), which demonstrates that the inhibition of cellular aging by alleviating the inflammatory response of salmon head peptone works effectively. These results indicate that salmon head peptone can exert anti-aging efficacy by simultaneously regulating inflammation and aging pathways.

[0128]

[0129] 5-3. Anti-aging effects of salmon head peptone by inhibition of oxidative stress

[0130] To evaluate the anti-aging function of salmon head peptone prepared as in Example 1 above in a cell aging model induced by oxidative stress, HDF cells were seeded in a 96-well plate at a density of 0.5 × 10^4 cells, treated with 1x PBS, 0.1% animal peptone, and 0.1% salmon head peptone the next day, and after 24 hours of culture, treated with 1 mM H₂O₂ for an additional hour.

[0131] As shown in Figure 5-1, cell viability in the PBS control and animal peptone-treated groups (animal peptone, AP) decreased significantly upon H₂O₂ treatment, while cell viability was observed to be significantly restored in the group treated with salmon head peptone (salmon peptone, SP). This suggests that salmon head peptone is effective in alleviating or repairing cell damage caused by oxidative stress.

[0132] In addition, the ROS (reactive oxygen species) levels increased by H₂O₂ treatment were measured by fluorescence intensity using the DCF-DA (dichlorofluorescein diacetate) staining method. The experimental method involved seeding HDF cells in a 96-well plate at a density of 0.5 × 10^4 cells. The following day, the cells were treated with 1x PBS, 0.1% animal peptone, and 0.1% salmon head peptone, cultured for 24 hours, and then treated with 1 mM H₂O₂ for an additional hour. Subsequently, 20 μM DCFH-DA was applied to the cells, and after a 30-minute reaction, absorbance was measured at λex = 485 nm and λem = 528 nm.

[0133] As shown in Figure 5-2, ROS levels were significantly increased by H₂O₂ in the PBS control and animal peptone treatment groups (animal peptone, AP), and in the salmon head peptone treatment group (salmon peptone, SP), ROS levels were significantly reduced compared to the PBS control and animal peptone treatment groups.

[0134] Therefore, it is shown that the above salmon head peptone can effectively alleviate cell damage and aging caused by oxidative stress, increase cell viability, and effectively exert antioxidant and anti-aging effects by regulating ROS levels.

[0135]

[0136] <Experiment Example 6> Analysis of the Anti-aging Efficacy of Salmon Head Peptone

[0137] 6-1. Confirmation of Anti-aging Efficacy through Observation of Cellular Morphological Changes Following Peptone Treatment of Salmon Heads

[0138] Based on the excellent antioxidant and anti-aging efficacy of salmon head peptone prepared as in Example 1 above, the possibility of reverse aging, which can return cells undergoing the aging process to normal cells, was confirmed.

[0139] For reference, reprogramming aging-induced cells into normal cells is called reverse aging or rejuvenation. While conventional anti-aging focuses on inhibiting free radicals to delay the aging of normal cells, reverse aging is defined as a next-generation anti-aging technology that can fundamentally resolve various side effects of aging by restoring the function of aging cells to the level of normal cells through cell reprogramming, using advanced biotechnology that carries the meaning of rejuvenation.

[0140] To evaluate the anti-aging efficacy of cells, changes in the expression of stem cell transcription factors such as POU5F1 (OCT4), SOX2, and KLF4 are used as representative molecular markers, and changes in the expression of SLIT2, which is reported to be expressed more highly in normal cells than in senescent cells, as well as morphological changes in cells and restoration of cell activity, are used as major analytical markers.

[0141] Cells that had already aged through continuous subculture were seeded in a 6-well plate at a density of 1 x 10^5 cells, and the morphological anti-aging effects were compared and evaluated by treating the control group (PBS), animal peptone, plant peptone, and salmon head peptone at a concentration of 0.03% each.

[0142] As shown in Fig. 6, when observing the cell morphology 4 weeks after treatment, microscopic observation revealed that the cell morphology of the salmon head peptone treatment group (salmon peptone, SP) had returned to a form similar to normal cells compared to the control group, the animal peptone treatment group (animal peptone, AP), and the plant peptone treatment group (plant peptone, VP) (Fig. 6-1). Additionally, measuring the cell size using the Image J program confirmed that significant anti-aging had occurred (Fig. 6-2).

[0143]

[0144] 6-2. Confirmation of Anti-aging Efficacy through Observation of Cell Cycle and Survival Rate Following Peptone Treatment of Salmon Heads

[0145] Cell cycle arrest and the expression of p21, an important marker of aging, were analyzed in senescent cells following treatment with salmon head peptone prepared as in Example 1 above.

[0146] Cells were obtained after a total of 4 weeks of treatment, RNA was extracted, and cDNA was synthesized. CDKN1A mRNA expression was measured using real-time PCR. As shown in Figure 7-1, CDKN1A expression was significantly reduced in the salmon head peptone treatment group (salmon peptone, SP) compared to the control group, animal peptone treatment group (animal peptone, AP), and plant peptone treatment group (plant peptone, VP). This suggests that salmon head peptone plays an important role in inhibiting cell cycle arrest and the aging process.

[0147] In addition, to evaluate cell viability, 7 x 10^4 cells were seeded in a 24-well plate in each treatment group, and an MTT assay was performed after 4 days. As shown in Figures 7-2 and 7-3, it was confirmed that the cell viability of the salmon head peptone treatment group (salmon peptone, SP) was significantly increased compared to the control group, the animal peptone treatment group (animal peptone, AP), and the plant peptone treatment group (plant peptone, VP). This confirms that salmon head peptone is effective in preventing the decline in cell function due to aging by promoting cell survival and proliferation.

[0148]

[0149] 6-3. Confirmation of Anti-aging Efficacy through Observation of Stem Cell Molecular Markers Following Peptone Treatment of Salmon Head

[0150] To evaluate the anti-aging and reverse aging efficacy of the salmon head peptone prepared as in Example 1 above through cell reprogramming, molecular marker expression was analyzed. Cells treated for a total of 4 weeks were obtained, RNA was extracted and cDNA was synthesized, and real-time PCR was used to confirm the mRNA expression of IL1B, a major cytokine exhibiting an inflammatory response, SAA1, an important molecular indicator of cellular aging, and SLIT2, which has been reported to have higher expression in normal cells compared to senescent cells, using qPCR.

[0151] As shown in Fig. 8, a significant increase was observed specifically in the salmon head peptone treatment group (salmon peptone, SP) for all three genes IL1B, SAA1, and SLIT2 (Fig. 8-1).

[0152] In addition, compared to the control group, animal peptone treatment group (animal peptone, AP), and plant peptone treatment group (plant peptone, VP), it was confirmed that the expression of KLF4, SOX2, and POU5F1 factors, which are stem cell molecular markers involved in cell reprogramming, was significantly increased in the group treated with salmon head peptone (salmon peptone, SP) for a total of 4 weeks (Fig. 8-2). This suggests that salmon head peptone can enhance the reprogramming ability of cells and restore cell function, ultimately inducing cellular anti-aging.

[0153] These results show that salmon head peptone plays an important role in inhibiting cellular aging, reducing oxidative stress, alleviating inflammatory responses, and increasing the expression of reprogramming factors, demonstrating that it is a promising material for exhibiting anti-aging and reverse-aging effects.

[0154]

[0155] <Experiment Example 7> Analysis of the Anti-aging Efficacy of Artificial Skin by Salmon Head Peptone

[0156] 7-1. Confirmation of Anti-aging and Anti-inflammatory Efficacy of Salmon Head Peptone in Artificial Skin

[0157] As the salmon head peptone prepared as in Example 1 above showed excellent antioxidant, anti-aging, and reverse-aging effects as in Experiment 6 above, the anti-aging and anti-inflammatory efficacy of salmon head peptone was confirmed by inducing an aging response caused by UV irradiation on artificial skin made of human-derived skin cells and treating it with salmon head peptone to measure the expression levels of the aging marker p21 and the inflammation marker IL-1β.

[0158] For reference, the above p21 is a type of cyclin-dependent kinase inhibitor (CDKI) that plays an important role in regulating G1 / S switching and acts as an important factor in the progression of aging, and the above IL-1β is an inflammatory factor and is an immune cytokine formed in response to infection or toxins within microorganisms.

[0159] The anti-aging and anti-inflammatory effects of salmon head peptone on artificial skin were analyzed by irradiating the surface of artificial skin tissue with UVA 1J / ㎠ and UVB 30mJ / ㎠, treating it with salmon peptone, treating the tissue surface once for 2 days, culturing it for 5 days, fixing and staining the tissue, and acquiring images at 200x magnification using a fluorescence microscope (Leica THUNDER Imager Tissue microscope). The acquired images were objectively analyzed by quantifying the positive staining area using the Image J program.

[0160] As shown in Figures 9-1 and 9-2, it can be confirmed that p21 exhibited green fluorescence inside and around the nucleus of the upper epidermal layer, while the cell nucleus exhibited blue. Analysis of the changes in p21 expression revealed a significant change, with p21 expression increasing by 457.05% in the UV-treated group compared to the PBS-treated control group. This indicates that the expression of P21 increased to a high level due to UV radiation. Furthermore, the group treated with salmon head peptone after UV treatment (UV-treated + salmon peptone) showed a statistically significant change, with p21 expression decreasing by 63.31% compared to the UV-treated group (UV-treated only).

[0161] As shown in Figures 9-3 and 9-4, it can be observed that IL-1β exhibits green fluorescence in the epidermal layer, while the cell nucleus appears blue. Analysis of the changes in IL-1β expression revealed a significant change, with IL-1β expression increasing by 115.33% in the UV-VR treated group compared to the PBS-treated control group. This indicates that the expression of IL-1β increased to a high level due to UV radiation. Furthermore, the group treated with salmon head peptone after UV treatment (UV-VR + salmon peptone) showed a statistically significant change, with IL-1β expression decreasing by 24.12% compared to the group treated only with UV (UVR).

[0162] These results suggest that salmon head peptone can prevent skin cell aging by reducing p21 and IL-1β expression increased by ultraviolet rays.

[0163]

[0164] 7-2. Confirmation of Skin Barrier Improvement Effect of Salmon Head Peptone on Artificial Skin

[0165] To confirm the skin barrier improvement effect of salmon head peptone prepared as in Example 1 above, an aging reaction caused by UV irradiation was induced in artificial skin made of human-derived skin cells, and salmon head peptone was treated to confirm the skin barrier improvement effect of salmon head peptone by the expression levels of Collagen type I, Collagen type IV, Filaggrin, and Elastin.

[0166] For reference, the above Collagen type I accounts for 70% of the dermis and is an essential protein for preventing wrinkles and maintaining elastic skin by protecting against skin damage from ultraviolet rays and maintaining moisture balance; the above Collagen type IV is an epidermal-dermal junction protein that prevents skin aging by ensuring a smooth supply of nutrients so that the skin is not easily damaged; the above Filaggrin is a barrier-strengthening element that is attached to intermediate microfilaments of keratin to provide strong physical support and then forms natural moisturizing factors through a decomposition process to play an important role in skin moisturization; and the above Elastin is a protein that provides elasticity to the skin so that skin deformed by external factors returns to its original state.

[0167] The skin barrier-improving effect of salmon head peptone on artificial skin was analyzed by irradiating the surface of artificial skin tissue with UVA 1J / ㎠ and UVB 30mJ / ㎠, treating it with salmon peptone, treating the tissue surface once for 2 days, culturing it for 5 days, fixing and staining the tissue, and acquiring images at 200x magnification using a fluorescence microscope (Leica THUNDER Imager Tissue microscope). The acquired images were objectively analyzed by quantifying the positive staining area using the Image J program.

[0168] As shown in Figure 10-1, it can be observed that Collagen Type I (COLI) appears green in the dermal layer, while the cell nuclei appear blue. Analysis revealed a significant change in Collagen Type I (COLI), decreasing by 76.87% in the UV-treated group compared to the control group treated only with PBS. This indicates that the expression level of Collagen Type I was reduced by UV radiation. Furthermore, the group treated with salmon head peptone after UV treatment (UV-treated + salmon peptone) showed a statistically significant change, with Collagen Type I expression increasing by 182.34% compared to the UV-treated group (UV-treated only).

[0169] As shown in Figure 10-2, it can be observed that Collagen Type IV (COL IV) appears green at the epidermal-dermal junction, while the cell nucleus appears blue. Analysis revealed a significant change in Collagen Type IV (COL IV), decreasing by 61.24% in the UV-treated group compared to the control group treated only with PBS. This indicates that the expression level of Collagen Type IV was reduced by UV radiation. Furthermore, the group treated with salmon head peptone after UV treatment (UV-+Salmon Peptone) showed a statistically significant change, with Collagen Type IV expression increasing by 99.02% compared to the UV-treated group (UVR).

[0170] As shown in Figure 10-3, it can be observed that Filaggrin (FLG) appears green in the stratum corneum, while the cell nuclei appear blue. Analysis revealed a significant change in Filaggrin (FLG), decreasing by 60.50% in the UV-treated group compared to the control group treated only with PBS. This indicates that the expression level of Filaggrin was reduced by UV radiation. Additionally, the group treated with salmon head peptone after UV treatment (UV-treated + salmon peptone) showed a statistically significant change in Filaggrin expression, increasing by 82.54% compared to the UV-treated group (UV-treated only).

[0171] As shown in Figure 10-4, it can be observed that Elastin appears red around fibroblasts in the dermal layer, while the cell nuclei appear blue. Analysis revealed a significant change in Elastin expression, decreasing by 82.69% in the UV-VR treated group compared to the control group treated only with PBS. This indicates that the expression level of Elastin was reduced by UV radiation. Furthermore, the group treated with salmon head peptone after UV treatment (UV-VR + salmon peptone) showed a statistically significant change, with Elastin expression increasing by 169.15% compared to the UV-VR-only group.

[0172] These results suggest that salmon head peptone can help improve the skin barrier by increasing the expression of Collagen type I, Collagen type IV, Filaggrin, and Elastin, which are reduced by ultraviolet rays.

[0173]

[0174] 7-3. Confirmation of Skin Tissue Regeneration and Anti-Aging Effects of Salmon Head Peptone on Artificial Skin

[0175] To confirm the skin tissue regeneration and anti-aging effects of salmon head peptone prepared as in Example 1 above, an aging reaction caused by UV irradiation was induced in artificial skin made of human-derived skin cells, and salmon head peptone was treated to confirm the skin tissue regeneration and anti-aging effects of salmon head peptone based on the expression levels of Ki-67 and OCT4.

[0176] For reference, the above Ki-67 is expressed during the active phase of the cell cycle and is involved in cell proliferation, and the above OCT4 is a protein that regulates the regeneration of skin cells.

[0177] The skin tissue regeneration and anti-aging effects of salmon head peptone on artificial skin were analyzed by irradiating the surface of artificial skin tissue with UVA 1J / ㎠ and UVB 30mJ / ㎠, treating it with salmon peptone, treating the tissue surface once for 2 days, culturing it for 5 days, fixing and staining the tissue, and acquiring images at 200x magnification using a fluorescence microscope (Leica THUNDER Imager Tissue microscope). The acquired images were objectively analyzed by quantifying the positive staining area using the Image J program.

[0178] As shown in Figures 11-1 and 11-2, it can be observed that Ki-67 appears sky blue (light blue) in the basal layer of the epidermis, and the cell nuclei appear blue. Analysis revealed a significant change in Ki-67 expression, decreasing by 41.18% in the UV-VR treated group compared to the control group treated only with PBS. This indicates that the expression level of Ki-67 was reduced by UV radiation. Additionally, the group treated with salmon head peptone after UV treatment (UV-VR + salmon peptone) showed a statistically significant change, with Ki-67 expression increasing by 59.53% compared to the UV-VR-only group.

[0179] As shown in Figures 11-3 and 11-4, it can be observed that OCT4 appears green in the epidermal layer and the cell nuclei appear blue. Analysis revealed a significant change in OCT4 expression, decreasing by 65.14% in the UV-VR treated group compared to the control group treated only with PBS. This indicates that the expression level of OCT4 was reduced by UV radiation. Additionally, the group treated with salmon head peptone after UV treatment (UV-VR + salmon peptone) showed a statistically significant change, with OCT4 expression increasing by 91.54% compared to the UV-VR-only group.

[0180] These results suggest that salmon head peptone can help prevent skin aging by increasing the expression of Ki-67 and Oct-4, which are reduced by ultraviolet rays.

[0181]

[0182] <Experimental Example 8> Analysis of the Efficacy of Genetically Recombinant Protein Production Using Microbial Culture Medium Containing Salmon Head Peptone

[0183] The productivity of genetically recombinant proteins was analyzed using salmon head peptone medium prepared as in Example 2 above, animal peptone medium prepared as in Comparative Example 1 above, plant peptone medium prepared as in Comparative Example 2 above, and commercially available culture medium LB medium (control group). As test strains, Escherichia coli transformed with the human antioxidant protein (hSOD) gene inserted into the pET11a vector and Escherichia coli transformed with the human growth hormone (hGH) gene inserted into the pET11a vector were used. Each transformed strain was inoculated into four different media with ampicillin added at a concentration of 100 μg / ml and cultured with shaking at 37°C. When the strain concentration reached 0.6–0.7 at OD 600 nm, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a concentration of 0.1 mM, and protein expression was induced by shaking culture at 180 rpm for 24 hours at 20°C. After the induction of protein expression was complete, 1 ml was transferred to a 1.5 ml tube and centrifuged at 10,000 rpm for 1 minute to collect only the precipitated cells. 1 ml of distilled water was added to perform sonication, and intracellular proteins were identified via protein electrophoresis. For quantitative analysis, BSA was used as a standard, and the amount of each protein was measured using the Image J program.

[0184] As shown in Figure 12 and Table 5 below, the amount of the recombinant protein hSOD was produced at the highest concentration of 288.14 ug / ml in the salmon head peptone (SP) medium, followed by animal peptone (AP) medium (279.13 ug / ml), LB medium (249.17 ug / ml), and plant peptone (VP) medium (128.23 ug / ml). In addition, the recombinant protein hGH was produced at similarly high levels in the animal peptone medium (216.71 ug / ml) and salmon head peptone medium (193.76 ug / ml), and increased by more than 1.5 times compared to the LB medium (126.87 ug / ml) and plant peptone medium (119.02 ug / ml). This suggests that salmon head peptone medium and animal peptone medium exhibited similar recombinant protein productivity and can be considered the most suitable. However, considering the virus issue in biopharmaceutical production, the use of salmon head peptone is advantageous because there have been no cases of human infection by fish-derived viruses.

[0185]

[0186] Protein LB (μg / mL)AP (μg / mL)VP (μg / mL)SP (μg / mL)hSOD249.17 ± 1.09279.13 ± 4.92128.23 ± 1.89288.14 ± 13.84hGH126.87 ± 5.48216.71 ± 3.53119.02 ± 10.49193.76 ± 5.26

[0187] (AP; culture medium containing animal peptone, VP; culture medium containing plant peptone, SP; culture medium containing salmon head peptone)

[0188]

[0189] As specific parts of the present invention have been described in detail above, it is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention shall be defined by the appended claims and their equivalents. The scope of the invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and their equivalents shall be interpreted as being included within the scope of the invention.

Claims

1. A step of heating salmon by-products to powderize the muscle protein separated from the bones; A step of preparing a mixture by mixing water with the above muscle protein powder; A step of hydrolyzing the above mixture by adding a hydrolytic enzyme; and A method for producing peptone derived from salmon by-products, comprising the step of drying the hydrolyzed mixture to obtain peptone powder.

2. In Paragraph 1, The above hydrolytic enzyme is, A method for producing peptone derived from salmon by-products, characterized by being one or more selected from the group consisting of papain, protamax, trypsin, bromelain, alcalase, and pronase.

3. In Paragraph 1, The above hydrolysis step is, A method for producing peptone derived from salmon by-products, characterized by adjusting the above mixture to a pH of 6 to 8, adding the above hydrolytic enzyme, and treating at 45 to 60 ℃ for 5 to 7 hours.

4. In Paragraph 1, The above hydrolysis step is, A method for producing peptone derived from salmon by-products, further comprising the step of inactivating the hydrolytic enzyme at 90 to 100 ℃ for 5 to 15 minutes after the above hydrolysis.

5. A microbial culture medium composition comprising peptone produced by the method for producing peptone derived from salmon by-products of claim 1.

6. In Paragraph 5, The above peptone is, A microbial culture medium composition characterized by being a nitrogen source for the microbial culture medium.

7. In Paragraph 5, The above peptone is, A microbial culture medium composition characterized by exhibiting anti-aging, reverse aging, and antioxidant activities.

8. In Paragraph 5, The above peptone is, A culture medium composition for microorganisms characterized by exhibiting skin regeneration and skin barrier strengthening functions.

9. In Paragraph 5, The above culture medium composition is, A microbial culture medium composition characterized by increasing the productivity of genetically modified proteins.

Images