KR102581772B1 - A method of optimal cultivation for preparing cultured meat using plant and insect protein hydrolysate and application of the same - Google Patents

Abstract

The present invention relates to a medium composition for animal cell culture, which contains plant protein hydrolyzate or insect protein hydrolyzate as an active ingredient as a substitute for serum among additives, and includes plant and insect-derived protein hydrolyzate (10 %) improves in vitro viability and myoblast protein marker expression compared to the crude isolate, which allows protein hydrolysates to be used as serum substitutes in culture compositions and as an ideal dietary source for alleviating oxidative stress. It acts as a naturally derived protein supplement and offers potential antioxidant properties.

Description

Optimal culture method and application for producing cultured meat using plant and insect protein hydrolysates {A METHOD OF OPTIMAL CULTIVATION FOR PREPARING CULTURED MEAT USING PLANT AND INSECT PROTEIN HYDROLYSATE AND APPLICATION OF THE SAME}

The present invention relates to an optimal culture method and application for producing cultured meat using plant and insect protein hydrolysates.

It is estimated that by 2050, the world's population will reach 9 billion, and human meat consumption will also reach 465 million tons. Accordingly, the Food and Agriculture Organization of the United Nations (FAO) plans to produce 200 million tons of meat every year to meet the increasing demand for meat. It has been announced that additional production is necessary.

In addition, in recent years, problems with livestock diseases such as mad cow disease, foot-and-mouth disease, and cases where pathogens contained in livestock intestines are not killed during the processing process, causing various diseases, are occurring one after another, causing major social problems.

Meanwhile, the cytoprotective effects of functional foods have been studied for a long time because they play an important role in eliminating cellular oxidative stress. Oxidative stress plays an important role in disease development due to the accumulation of reactive oxygen species (ROS) such as superoxide anion (O-), hydroxyl anion (OH-), and hydrogen peroxide (H2O2) inside cells.

In general, active oxygen is generated inside cells during respiration or metabolism, which performs various biological functions such as cell homeostasis, signal transduction, pathogen killing, autophagy, and cell division. In fact, excessive ROS accumulation causes abnormal oxidative and antioxidant effects, leading to cell death due to oxidative stress.

Elevated levels of intracellular ROS also cause severe DNA damage, protein damage, and lipid peroxidation. As a result, various fatal diseases such as cardiovascular disease (CVD), neurodegenerative disease (NDD), chronic inflammatory disease (CID), and even cancer are occurring.

Therefore, the use of antioxidants to improve oxidative stress is of major interest. Compared to naturally derived antioxidants, synthetic antioxidants (butylated hydroxyanisole and butylated hydroxytolune) have potential risks, including liver damage.

Therefore, the development of naturally derived antioxidants is important to reduce the body's stress response. Naturally derived non-protein components such as polyphenols, alkaloids, etc. are often used as sources of antioxidants.

Naturally derived protein hydrolysates (PH) have received great attention due to their high antioxidant properties. PHs contain several bioactive peptides that primarily act as stress reducers.

Meanwhile, skeletal muscles are complex multinucleated structures responsible for contractile movements in living organisms.

During the embryonic stage, myogenic progenitor cells increase in number by a process known as proliferation. Later, the progenitor cells align and fuse to form multinucleated cells known as myotubes, which further develop into contractile muscle fibers.

In adults, satellite cells (SCs) are located between the basal layer and the plasmalemma of muscle fibers. The formation of new myotubes is greatly influenced by the activation, proliferation and fusion of SCs in muscle tissue. When muscle tissue receives various external stimuli, such as injury or stretching, SCs become activated and fuse to form myoblasts. Therefore, the isolation and in vitro culture of muscle cells in a controlled environment provides an opportunity to study muscle physiology and differentiation processes.

[Prior patent literature]

Republic of Korea Patent Publication No. 10-2018-0132325 (December 12, 2018)

The present invention was created in response to the above-mentioned need, and the purpose of the present invention is to provide a composition for animal cell culture containing a novel serum replacement or reduced serum addition amount.

Another object of the present invention is to provide a novel bovine muscle satellite cell (bMSC) culture method.

In order to achieve the above object, the present invention provides a medium composition for animal cell culture, which includes plant protein hydrolyzate or insect protein hydrolyzate as an active ingredient instead of serum as an additive.

In one embodiment of the present invention,

The plant protein hydrolyzate is preferably, but not limited to, a protein hydrolyzate selected from the group consisting of soy protein hydrolyzate, pea protein hydrolyzate, and gluten protein hydrolyzate.

In another embodiment of the present invention,

When the soy protein hydrolyzate is hydrolyzed with Alcalase, the soy protein hydrolyzate contains the highest content of peptides with a molecular weight of 12.7 kilodaltons compared to other peptides,

When soy protein is hydrolyzed with Neutrase, it is preferable that the protein hydrolyzate contain the highest content of peptides with a molecular weight of 35 kilodaltons relative to other peptides, but it is not limited to this.

In another embodiment of the present invention,

When the pea protein hydrolyzate is hydrolyzed with Neutrase, the pea protein hydrolyzate preferably contains the highest content of peptide with a molecular weight of 36 kilodaltons relative to other peptides, but is not limited to this.

In another embodiment of the present invention,

When the gluten protein hydrolyzate is hydrolyzed with a flavourzyme, the protein hydrolyzate preferably contains the highest content of peptides with a molecular weight of 160 kilodaltons relative to other peptides, but is not limited to this. .

In one embodiment of the present invention,

The insect protein hydrolyzate is preferably, but not limited to, a protein hydrolyzate selected from the group consisting of beetle protein hydrolyzate, cricket protein hydrolyzate, and meal-worm protein hydrolyzate.

In another embodiment of the present invention,

When the beetle protein hydrolyzate is hydrolyzed with Alcalase, the beetle protein hydrolyzate contains the highest content of peptides with a molecular weight of 12 kilodaltons compared to other peptides,

When beetle protein is hydrolyzed with Neutrase, it is preferable that the protein hydrolyzate contains a peptide with a molecular weight of 26 kilodaltons that is relatively high compared to other peptides, but is not limited to this.

In another embodiment of the present invention,

The cricket protein hydrolyzate, when the cricket protein is hydrolyzed with Alcalase, contains the highest content of peptides with a molecular weight of 12 kilodaltons in the protein hydrolyzate relative to other peptides,

When cricket protein is hydrolyzed with Neutrase, it is preferable that the protein hydrolyzate contain the highest content of peptides with a molecular weight of 12 kilodaltons relative to other peptides, but it is not limited to this.

In another embodiment of the present invention,

When the mealworm protein hydrolyzate is hydrolyzed with Neutrase, the protein hydrolyzate preferably contains the highest content of peptides with a molecular weight of 12 kilodaltons relative to other peptides, but is not limited to this.

In another embodiment of the present invention,

The composition preferably contains 10% by weight of plant protein hydrolyzate or insect protein hydrolyzate and 5% by weight or less of fetal bovine serum based on the total composition, but is not limited thereto.

In a preferred embodiment of the present invention,

The animal cells are bovine myosatellite cells. It is preferable, but is not limited to this.

In addition, the present invention involves adding 10% of a protein hydrolyzate obtained by hydrolyzing beetle protein with Neutrase to the culture composition during bovine myosatellite cell culture in vitro to remove desmin and myogenin from the cells. A method of increasing expression is provided.

In another embodiment of the present invention,

The glucose concentration of the high glucose medium is preferably 4500.0 mg/L, but is not limited thereto.

In another embodiment of the present invention,

The composition of the growth medium preferably includes, but is not limited to, 10% fetal bovine serum containing L-alanyl-L-glutamine dipeptide, 1% penicillin-streptomycin, and Amphotericin-B.

[0036] The food composition containing the protein hydrolyzate of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), and pects. It may further contain acids and their salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages, as well as natural fruit juices and synthetic beverages. It may further contain pulp for the production of fruit juice and vegetable drinks. These ingredients can be used independently or in combination.

In addition, food compositions may additionally contain food additives, and unless otherwise specified, the suitability as a “food additive” is determined by the specifications for the item in accordance with the general provisions and general test methods of the food additive code approved by the Korea Food and Drug Administration. and standards.

Items listed in the "Food Additives Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, cold pigment, and guar gum, L -Mixed preparations such as sodium glutamate preparations, noodle-added alkaline preparations, preservative preparations, and tar color preparations are included.

The food composition may be in the form of any one of meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, gum, ice cream, soup, beverage, tea, functional water, drink, alcohol, and vitamin complex. , but is not limited to this.

At this time, the content of the protein hydrolyzate according to the present invention added to food in the process of manufacturing the food composition can be appropriately adjusted as needed.

The present invention will be described below.

In general, protein hydrolysates (PHs) are a rich source of several bioactive components, including antioxidant peptides.

In the present invention, the effect and antioxidant properties of PH derived from some plants and edible insects were investigated.

For this purpose, various commercially available proteases (alcalase, neutrase and flavourzyme) are used and prepared from Glycine max (soybean), Cajanus cajan (pigeon pea), Triticum vulgare (gluten extract), Protaetia bervitarsis (beetle), Gryllus bimaculatus (cricket) and Tenebrio molitor ( Mealworm) protein isolate was prepared.

PHs were ultimately tested to measure in vitro biocompatibility of bovine myosatellite cells. Cytoprotective properties were also investigated through DCF-DA staining method.

The resulting hydrolyzate exhibited lower molecular weight bands compared to the crude isolate, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Moreover, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay showed improvement in free radical reduction of alcalase- and neutrase-treated PH. Additionally, both plant-derived PH and insect-derived PH showed excellent cytoprotective activity against H ₂ O ₂ -induced oxidative stress in myosatellite cells. Expression of myoblast protein markers such as desmin and myogenin were significantly increased in PHs-treated cells. Taken together, our results suggest that soybean- and beetle-derived PH can be used as ideal food ingredients due to their excellent cytoprotective effects.

As can be seen from the present invention, the present invention has demonstrated that protein hydrolysates of plant and insect origin are antioxidant and helpful in eliminating intracellular ROS in myogenic cells extracted from meat samples, with excellent antioxidant activity. The hydrolyzate (10%) improves in vitro viability and myoblast protein marker expression compared to the crude isolate, making this protein hydrolyzate useful as a serum substitute in culture compositions and an ideal diet for alleviating oxidative stress. It serves as a naturally derived protein supplement that can be used as a source and offers potential antioxidant properties.

1 is a schematic diagram related to the production of plant protein hydrolyzate and insect protein hydrolyzate;
Figure 2 is a diagram showing the characterization of protein hydrolyzate.
(ab) Degree of hydrolysis after enzymatic digestion of PPH and IPH.
(c) Digital photo of the hydrolyzate. and (d) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein isolates and corresponding hydrolysates.
In the figure, AH, Alcalase hydrolyzate; NH, Neutrase enzyme hydrolyzate; FH, flabozyme hydrolyzate; M, marker. Data are mean ± SD of three replicate experiments (n = 3), *statistical significance at p < 0.05.
Figure 3 is a diagram showing the antioxidant properties of protein hydrolyzate.
Antioxidant activity over time of (a) SPI(AH) and SPI(NH), (b) PPI(AH), PPI(NH) and GI(FH), (c) BPI(AH) and BPI(NH), (d) CPI (AH) and CPI (NH), (e) WPI (AH) and WPI (NH). Data are mean ± SD of three replicate experiments (n = 3), *statistical significance at p < 0.05.
Figure 4 is a diagram showing the isolation and characteristics of bovine myosatellite cells.
(a) Morphological features of myogenic cells showing initial growth within the first 7-12 days. Afterwards, the cells were cultured in differentiation medium. Differentiated cells display typical myoblast morphology. Scale bar: Scale bar: 100 μm.
(b) Representative immunocytochemical staining of myosatellite cells in growth and differentiation medium for 3 days. Scale bar: Scale bar: 100 μm.
Figure 5 depicts the in vitro cytotoxicity evaluation of bovine myosatellite cells in the presence of (a) PPH and (b) IPH at the indicated time intervals. Data are the mean ± SD of three replicate experiments (n = 3), * Statistical significance at p <0.05.
Figure 6 shows immunocytochemical staining of bovine myosatellite cells in the presence of PPH and IPH after 3 days of incubation.
(a) Representative fluorescence microscopy images of cells treated with 10% SPI (NH) and BPI (NH) (b) positive for desmin and myogenin with corresponding quantification values.
Data are mean ± SD of three replicate experiments (n = 3), *statistical significance at p < 0.05. Scale bar: 100 μm.
Figure 7 is a diagram showing the cytoprotective effect of PH against oxidative cell damage caused by H ₂ O ₂ .
(a) Fluorescence-activated cell sorting (FACS) analysis of PHs-treated cells. Cells were stained with DCF-DA and approximately 5,000 events were counted in the M1 gate.
(b) Representative fluorescence microscopy of a cell showing ROS inside the cell. Scale bar: 100 μm.
(c) Measurement of fluorescence intensity of DCF. Data are means ± SD of three replicate experiments (n = 3), *statistical significance at p < 0.05.
Figures 8 and 9 show quantification values of band intensities for plant protein hydrolyzate (PPH) and insect protein hydrolyzate (IPH) of the present invention by SDS-PAGE, respectively;
10 is a digital photograph of DPPH reduction of PPH and IPH of the present invention, and
Figure 11 is a graph showing the quantitative values of the fluorescence intensity of Desmin and Myogenin.

The present invention will be described in more detail below through non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example 1: Materials

Plant-derived protein isolates (soybean, pea and gluten) were purchased from Pingdingshan Tianjin Plant Albumen Co. Ltd. (Pingdingshan, China), The Green Labs LLC (Clifton, New Jersey, USA) and ADM Chamtor Ltd. (Bazancourt, France).

Protease enzymes, including Alcalase (2.4 L), Neutrase (0.8 L) and Flavourzyme (1000 L), were procured from Novozymes Ltd., Bagsvaerd, Denmark.

Insect samples (Protaetia bervitarsis, Gryllus bimaculatus and Tenebrio molitor) were obtained from a local farm (Korea Farm, Wonju, South Korea).

Example 2: Preparation of plant protein hydrolyzate (PPH)

Plant-derived protein hydrolysates include Yoon, S.; Wong, N.A.; Chae, M.; Auh, J.-H. It was manufactured with slight modifications to the one reported in Foods 2019 , 8 , 563.

Briefly, three commercial protein isolates were hydrated in 20-fold sterile distilled water and pasteurized at 90 °C for 15 min with vigorous agitation.

Next, the temperature was set to 50 °C, and the enzyme solution was slowly added to the sterilized solution. The resulting mixture was reacted at 50 °C for 12 hours. After enzymatic digestion, the reaction was stopped by heating at 100 °C for 20 min.

Next, the mixture was cooled to room temperature, purified by centrifugation at 3,220 g for 15 minutes, and then filtered. The manufacturing process is briefly described in Figure 1.

Example 3: Preparation of insect protein hydrolyzate (IPH)

Ganguly, K.; Jeong, M.-S.; Dutta, SD; Patel, D.K.; Cho, S.-J.; Lim, K.-T. Insect protein isolates were prepared as reported in Molecules 2020 , 25 , 6056.

The IPH manufacturing process is similar to PPH, as mentioned above.

A list and names of plant- and insect-derived protein isolates are listed in Table 1.

Isolate name Enzyme treatment Sample ID Soy Protein Isolate (SPI) Alcalase/Neutrase SPI(AH)/SPI(NH) Pea protein isolate (PPI) Alcalase/Neutrase PPI(AH)/PPI(NH) Gluten Isolate (GI) Flavorzyme GI(FH) Protaetia bervitarsis (beetle) protein isolate (BPI) Alcalase/Neutrase BPI(AH)/BPI(NH) Gryllus bimaculatus (cricket) protein isolate (CPI) Alcalase/Neutrase CPI(AH)/CPI(NH) Tenebrio molitor (meal-worm) protein isolate (WPI) Alcalase/Neutrase WPI(AH)/WPI(NH)

Table 1 lists the names of protein isolates and their corresponding hydrolysates: AH; Alcalase hydrolysates, NH; Neutrase hydrolysates, FH; Flavorzyme hydrolysates.

Example 4: Degree of hydrolysis

Hall, F.; Johnson, P.E.; Liceaga, A. Effect of enzymatic hydrolysis on bioactive properties and allergenicity of cricket (Gryllodes sigillatus) protein. The degree of hydrolysis was determined using the trinitrobenzene sulfonic acid (TNBS) test, as reported in Food chemistry 2018 , 262 , 39-47.

Briefly, PPH and IPH were mixed with an equal volume of trichloroacetic acid (TCA, Sigma-Aldrich, USA), vortexed for 10 s, and then centrifuged at 12,000 g for 5 min. Next, the supernatant (0.2 mL) was added to 0.2 M sodium borate buffer (pH 9.2) followed by 4mM TNBS solution (1 mL). The mixture was incubated at room temperature for 30 min, then 1 mL of 2M monosodium phosphate (Sigma-Aldrich, USA) and 18mM sodium sulfite (Sigma-Aldrich, USA) were added. Absorbance was recorded at 420 nm.

The degree of hydrolysis (%) is given by Eq. It was calculated as follows: degree of hydrolysis (%) = m/n × 100 (1)

where m is the peptide content of the hydrolyzate and n is the total protein content of the sample before enzymatic hydrolysis.

Example 5: Molecular weight estimation

The molecular weight of PPH and IPH was measured by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) method.

Briefly, electrophoresis was performed using 5% stacking gels at 60 V current and 10% separating gels at 100 V current, respectively.

Next, the gel was stained with 0.05% Coomassie Brilliant Blue (Sigma-Aldrich, USA) and destained using the target solution (acetic acid:methanol:deionized water; 2:5:5, v/v/v). Images were captured using Chemi-Dox XRS+ (Bio-Rad Laboratories, Hercules, CA, USA) and band intensities were quantified using Image Lab software (v6.0, Bio-Rad, USA).

Example 6: DPPH radical scavenging activity

Antioxidant properties of hydrolysates Liao, X.; Zhu, Z.; Wu, S.; Chen, M.; Huang, R.; Wang, J.; Wu, Q.; Ding, Y. Preparation of Antioxidant Protein Hydrolysates from Pleurotus geesteranus and Their Protective Effects on H2O2 Oxidative Damaged PC12 Cells. It was determined using the DPPH analysis reported in Molecules 2020 , 25 , 5408.

Briefly, 10% protein hydrolysates were prepared by vortexing in DPPH solution (0.4mM) for 10 s and incubated for 30 min in the dark at room temperature. The spectrophotometer measured absorbance at 517 nm at 10 min intervals for 60 min. A 1% ascorbate (Sigma-Aldrich, USA) solution was prepared as a control and compared to the test samples.

Example 7: Isolation and culture of myosatellite cells

Beef (mature female cattle) was obtained from Hongcheon Meat Plant, Gangwon-do, South Korea.

Meat samples were collected from the psoas muscle and cell culture experiments were performed within 30 min of slaughter. The Animal Experiment Ethics Committee (Kangwon National University Animal Care and Use Committee, permit number KW-210317-1) approved the animal tissue culture and handling procedures.

Myosatellite cell isolation Baquero-Perez, B.; Kuchipudi, S.V.; Nelli, R.K.; Chang, K.-C. A simplified but robust method for the isolation of avian and mammalian muscle satellite cells. It was performed with slight modifications as reported in BMC cell biology 2012 , 13 , 1-12.

Briefly, meat samples were washed several times with phosphate-buffered saline (PBS; Welgene, South Korea) to remove red blood cells (RBCs) and then incubated with 20 mL of washing medium. Approximately 3–4 g of minced samples were incubated with 30 mL of enzymatic digestion solution containing 1.4 mg/mL pronase enzyme (Sigma-Aldrich, USA) for 1.5 h at 37 °C in a shaking water bath. Afterwards, the enzyme-digested tissue samples were centrifuged at 300 g for 3-5 min and the supernatant was discarded. The obtained tissue was resuspended in 15 mL collection medium and subjected to vigorous pipetting (10-15 times) to release the cells of the tissue. The supernatant was centrifuged at 300 g for 3 min and the broth containing cells was collected. Next, the supernatant was passed through a 40 μm cell strainer (BD Falcon, Corning, U.S.A.), resuspended in collection medium, and centrifuged at 800 g for 10 min to pellet the cells. A list of media compositions used for cell isolation and culture is provided in Table 2.

procedure MediaㅣComposition Muscle tissue harvest DMEM, 1% P/S solution, Amphotericin-B Stock solution for enzymatic digestion DMEM/Ham's F12 (1:1), pronase (1.4 mg/mL), 1% P/S solution, Amphotericin-B Collection medium DMEM-Glutamax-1, 1% P/S, Amphotericin-B Growth medium DMEM-Glutamax-1, 10% FBS, 1% P/S, Amphotericin-B Differentiation medium DMEM-Glutamax-1, 20% FBS, 10% HS, 1% P/S, Amphotericin-B

Table 2 is a list of medium compositions used for bovine cell isolation and culture : DMEM; Dulbecco's Modified Eagle Media, P/S; Penicillin/streptomycin, FBS; fetal bovine serum, HS; Means horse serum.

For cell plating, the pellet-containing cells were resuspended in growth medium and seeded into T75 flasks coated with 0.5% gelatin (Sigma-Aldrich, USA) at 37 °C in a 5% CO environment. During the first 48 hours, the medium was changed frequently to remove contamination. Thereafter, the medium was changed daily. Passage 1 cells were used for each cell culture experiment.

Example 8: Immunocytochemical (ICC) staining

Myogenic cell populations were identified by staining with myoblast-specific protein markers. For this purpose, 4 × ¹⁰ cells were grown in 24 well plates for 24 h. Afterwards, cells were fixed with 3.7% paraformaldehyde (PFA; Sigma-Aldrich, USA) for 15 minutes at room temperature and then permeabilized with 100% methanol for 5 minutes.

Next, cells were washed twice with PBS and blocked with 1% bovine serum albumin (BSA; Sigma-Aldrich, USA) overnight at 4 °C. Blocked cells were then stained for 1 h with specific primary antibodies against Desmin (1:100) and Myogenin (1:200) followed by AF-488 (green emission) and AF-647 (red emission) conjugated secondary antibodies. Incubated with antibodies for 1 hour. All antibodies were purchased from Santa Cruz Biotechnology (SCBT), USA.

Nuclei were counterstained by DAPI (Sigma-Aldrich, USA) and cells were visualized under an inverted light microscope (DMi8, Leica Microsystems, Wetzlar, Germany). Images were captured at 40× magnification and processed with ImageJ software (ImageJ, v1.8, NIH, Bethesda, MD, USA).

Example 9: Cell viability assay

Cell viability of myosatellite cells was assessed by WST-8 assay.

For this, passage-1 myosatellite cells (1 × 10 ^cells /100 μL medium) were cultured in 96-well plates in the presence of growth medium. Afterwards, cells were supplemented with 100 μL of medium containing 10% PPH and IPH + 5% FBS and cultured for 24 and 48 h. After desired incubation, cells were washed with PBS and replenished with fresh medium. Culture plates containing only 5% FBS were considered controls. Finally, cells were incubated with 100 μL of WST-8 dye (Celix Viability Assay Kit, Korea) and incubated for 2 h. Reacted formazan was quantified using a microplate reader at 450 nm (625 nm as reference value). All experiments were performed in triplicate (n = 3) and data are expressed as mean ± standard deviation (SD).

Example 10: Cell morphology and intracellular ROS detection

The effects of PPH and IPH on the expression of myoblastic-protein markers were assessed using immunocytochemical techniques.

For this purpose, cells (2.5 × ¹⁰ cells/500 μL medium) were cultured in confocal dishes and treated with 10% PPH and IPH for 3 days. Medium containing only 5% FBS was used as a control.

The staining procedure was described in Ganguly, K.; Jeong, M.-S.; Dutta, SD; Patel, D.K.; Cho, S.-J.; Lim, K.-T. Protaetia brevitarsis seulensis Derived Protein Isolate with Enhanced Osteomodulatory and Antioxidative Property. Described in Molecules 2020 , 25 , 6056.

Briefly, cells were fixed with 3.7% paraformaldehyde (PFA) for 15 min at room temperature and then permeabilized with Triton-X 100 (0.1%) for 10 min. Afterwards, cells were blocked by 1% BSA for 1 h and incubated with specific primary and secondary antibodies against Desmin and Myogenin. Nuclei were counterstained with DAPI solution for 1–2 min in the dark and visualized under an inverted fluorescence microscope (DMi8, Leica Microsystems, Wetzlar, Germany). Images were captured at 40× magnification and processed with Cell Suite-X software (Leica Microsystems, Wetzlar, Germany).

For intracellular ROS detection, myosatellite cells (1.5 × ¹⁰ cells/well) were seeded in 96-well plates and incubated with growth medium.

Next, the cells were supplemented with 100 μL of medium containing 10% PPH and IPH + 5% FBS, while the control group was supplemented with 5% FBS only. Cells were grown for an additional 24 h and then treated with 200 μM of 30% (v/v) H ₂ O ₂ for 20 min in dark conditions.

Next, cells were washed with PBS and incubated with 20 μM DCF-DA (Sigma-Aldrich, USA) for 30 min. Afterwards, cells were harvested and 50 μL of cell suspension was taken onto a slide and visualized under an inverted fluorescence microscope. Fluorescence intensity was quantified using ImageJ software (ImageJ v1.8, NIH Lab., Bethesda, MD, USA).

All experiments were performed in triplicate (n = 3) and data are expressed as mean ± standard deviation (SD).

Statistical analysis performed in the present invention was performed by one-way analysis of variance using Origin Pro 9.0 software. All experiments were performed in triplicate (n = 3) and results are expressed as mean ± SD, *statistical significance at p < 0.05.

The results of the above examples are described below.

Enzymatic hydrolysis of protein isolates

To investigate the effect of enzymatic hydrolysis, the degree of hydrolysis efficiency was evaluated. As shown in Figure 2a, the extent of hydrolysis increased after 12 h of protease digestion. TCA soluble peptides were significantly increased in alcalase, neutrase and flavourzyme treated isolates. Additionally, SPI, PPI and GI showed the highest level of hydrolysis (*p <0.05) in the presence of alkalase compared to other proteases. A similar trend was observed when BPI, CPI and WPI were incubated with alcalase enzyme.

The hydrolysis degrees of SPI, PPI, GI, BPI, CPI, and WPI were 34.7%, 31%, 26.8%, 48.9, 60.25, and 65.5%, respectively.

A digital photograph of the hydrolyzate is shown in Figure 2b. Moreover, the molecular weight of the peptide fragments was analyzed by SDS-PAGE.

Quantification values of band intensity for PPH and IPH are shown in Figures 8 and 9. Undigested PPH samples showed characteristic bands ranging from 12 to 160 kDa. Interestingly, the major band disappeared after hydrolysis using alcalase and neutrase. Flabozyme-treated samples also showed a similar behavior. The different cleavage patterns of the isolated proteins indicate the hydrolytic effect and are closely related to the enzyme used.

The enhancement of TCA-soluble peptide content and the absence of major bands in the presence of alcalase, neutrase and flavourzyme suggest that PPI and IPI were sufficiently hydrolyzed.

Antioxidant properties of hydrolyzate

Reactive oxygen species (ROS) are metabolic by-products and are actively involved in the pathogenesis of several diseases such as cancer and cardiovascular disease (CVD).

In the present invention, the antioxidant activity of PPH and IPH was evaluated through DPPH radical scavenging analysis, and the results are shown in Figure 3.

Ascorbic acid (1%) solution was used as a control. The antioxidant properties of 10% PPH and IPH changed over time and an increasing trend was observed in alcalase-treated plant hydrolysates (SPI and PPI), as shown in Figure 3a. In particular, flavourzyme-treated samples (GI) showed enhanced antioxidant activity compared to SPI or PPI samples, indicating the radical scavenging potential of the gluten isolate (Figure 3b).

However, IPH demonstrated an alternative mode of action. The absorbance at 517 nm changed dramatically in neutrase-treated samples compared to alcalase-treated samples, indicating that neutrase hydrolysis produced more antioxidant peptides than alcalase hydrolysis (Figure 3c–e). The antioxidant activity is probably due to the presence of specific amino acids (tryptophan, cysteine, methionine, histidine) or peptides (cryptides) present in PPH and IPH.

Pictures of DPPH reduction in PPH and IPH are shown in Supplementary Figure 3.

Characterization of myosatellite cells

Myosatellite cells are the main progenitor cells of skeletal tissue. To study muscle tissue development and physiology, myosatellite cells can be isolated, cultured, and differentiated to a stage suitable for experiments.

In the present invention, bovine myosatellite cells were isolated from adult female dairy cows and then cultured for 2 weeks. As shown in Figure 4a, the cells were initially round in shape and grew adequately in gelatin-coated T75 flasks within 7 days of primary culture. Growing cells are relatively small in size and exhibit fibroblast-like growth when cultured in growth medium. In particular, the cells display typical myoblast morphology when cultured in differentiation medium (high-glucose DMEM + 20% FBS + 10% HS + 1% P/S).

Myosatellite cells expressed various myogenic genes and protein markers, such as desmin (Des), myogenin (MyoA, MyoD), paxillin-7, etc. Cells were cultured in growth and differentiation medium until passage 1 to assess protein marker expression. Cells were stained with myoblast-specific markers to determine desmin- and myogenin-positive cells, and the results are shown in Figure 4b.

In fact, cells were shown to be positive for both desmin and myogenin markers when cultured in growth medium. Interestingly, the expression of both markers was higher when cultured in differentiation medium for 3 days. Quantitative values of fluorescence intensity are shown in Supplementary Figure 4.

PHs improve myosatellite cell viability

The effect of PH on the viability of myosatellite cells was assessed by WST-8 assay after 48 h of culture and the results are shown in Figure 5. Cells were cultured in medium supplemented with 10% PPH and IPH with 5% FBS. Plates containing only 5% FBS were used as controls. In particular, PPH was confirmed to be biocompatible, except for PPI(NH), which showed a slight decrease in viability. The viability of myosatellite cells significantly increased in the presence of SPI(NH) supplements compared to the control and other groups (*p < 0.05) (Figure 5a).

A similar kind of observation was observed in the presence of IPH, where BPI (NH) supplementation resulted in maximum viability of myogenic cells after 48 h of culture (Figure 5b). None of the IPHs were found to be toxic to cultured cells, suggesting biocompatibility.

cell shape

The morphology of PPH- and IPH-treated cells was examined by fluorescence microscopy. Cells were treated with 10% SPI (NH) and BPI (NH) supplements and stained with myoblast-specific protein markers. Plates without PH were considered controls. Fluorescence images with corresponding intensity profiles are shown in Figure 6. The expression of markers in SPI and BPI supplemented media indicates that PHs sufficiently enhanced the proliferation and expression of desmin and myogenin. No abnormalities in cell morphology were observed in the presence of PH.

H ₂ O ₂ Effect of PH on oxidative damage caused by

We investigated the effects of PPH and IPH on _{H2O2 -} induced oxidative damage in bovine myosatellite cells by DCF-DA staining. For this purpose, PHs-treated cells were stained with DCF-DA and analyzed by FACS and fluorescence microscopy.

As shown in Figure 7a, intracellular ROS levels increased rapidly in HO-treated cells, whereas 10% SPI (NH) and BPI (NH) supplemented cells showed very low levels of intracellular ROS, suggesting ROS scavenging properties. This was further confirmed by fluorescence microscopy. Interestingly, PHs-treated myosatellite cells showed lower levels of DCF fluorescence than H2O2-treated cells, which is characterized by the intense color of DCF.

The average fluorescence intensity of DCF is shown in Figure 7c. Control cells maintain very low physiological ROS levels due to partial reduction of molecular oxygen.

Claims (12)

delete delete delete delete delete delete delete delete delete delete delete During bovine myosatellite cell culture in vitro, 10% of a protein hydrolyzate obtained by hydrolyzing beetle protein with Neutrase was added to the culture composition to increase the expression of desmin and myogenin in the cells. method.