CN118812643A - Yellow croaker maw oligopeptide with immunity-enhancing effect and application thereof - Google Patents

Abstract

The present invention discloses a yellow croaker maw oligopeptide with an immunity-enhancing effect and its use. The yellow croaker maw is used as a raw material, and the immune-enhancing oligopeptide FLVAPRH is prepared by defatting, chymotrypsin and neutral protease composite enzymolysis, ultrafiltration membrane classification and chromatography, and its molecular weight is 839.0 Da as determined by mass spectrometry. The yellow croaker maw oligopeptide FLVAPRH prepared by the present invention increases the growth rate of macrophages RAW 264.7 in a dose-dependent manner, enhances the phagocytic ability of RAW 264.7 cells, increases the NO synthesis ability of macrophages RAW 264.7, and promotes the secretion levels of cytokines TNF-α, IL-6 and IL-1β in macrophages RAW 264.7. Therefore, the yellow croaker maw oligopeptide FLVAPRH can significantly enhance the immune ability of macrophages, and is safe and has no toxic side effects, and can be used to prepare medicines and functional foods with immune-enhancing functions.

Description

Yellow croaker maw oligopeptide with immunity-enhancing effect and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and in particular relates to a yellow croaker maw oligopeptide having the function of enhancing immunity and an application thereof.

Background Art

The immune system has the functions of immune surveillance, defense, and regulation. This system is composed of immune organs (bone marrow, spleen, lymph nodes, tonsils, small intestinal lymph nodes, appendix, thymus, etc.), immune cells (lymphocytes, mononuclear phagocytes, neutrophils, basophils, eosinophils, mast cells, platelets (because platelets contain IgG), etc.), and immune active substances (antibodies, lysozyme, complement, immunoglobulins, interferons, interleukins, tumor necrosis factors and other cytokines). The immune system maintains homeostasis under normal physiological conditions. Immunity is the body's own defense mechanism, which is the body's ability to identify and eliminate foreign bodies that invade, deal with aging, dead, and degenerated self cells, and identify and deal with mutant cells and virus-infected cells in the body. However, excessive fatigue, abnormal emotions, lack of exercise, abuse of antibiotics, environmental factors, and genetic factors can lead to immune system disorders, which are manifested as easy to get sick, weak constitution, malnutrition, mental depression, fatigue, decreased appetite, sleep disorders, etc.

Immunity enhancement refers to strengthening one's own immunity through various methods. Modern immunology believes that improving immunity is a physiological reaction of the human body to identify and exclude "foreigners". The immune system performs this function in the human body. There are many ways to enhance immunity, such as supplementing foods high in zinc, selenium and protein, eating more fruits and vegetables, exercising moderately and maintaining good sleep quality.

Immunoenhancing peptides are peptide substances that can enhance the body's immunity. They can improve immunity by activating immune cells, enhancing antibody production, promoting the proliferation and differentiation of immune cells, promoting macrophage proliferation and phagocytosis, and improving the ability of macrophages to synthesize NO. Immunoenhancing peptides include thymosin, thymokinin, thymostimulating peptide, etc. In addition, immune-enhancing peptides can also be prepared from food-derived proteins through bioengineering technology, such as corn peptides, soybean peptides, whey peptides, etc. Therefore, immune-enhancing peptides have become a research focus and have also shown great application value in the field of peptide immune-enhancing drugs and functional foods.

Yellow croaker has high nutritional and medicinal value, and has the effects of nourishing the kidney, replenishing yang, strengthening yang, enhancing memory, calming the nerves, and helping development. Yellow croaker meat is sweet and salty, and has a neutral nature. It has the effects of nourishing the kidney, promoting diuresis, and reducing swelling. It is suitable for treating nephritis edema and other diseases; Yellow croaker otoliths are sweet and salty, and have a cold nature. They have the effects of clearing heat and removing blood stasis, promoting diuresis, and are suitable for treating urinary system stones, gallstones, suppurative otitis media, sinusitis, wild mushroom poisoning, arsenic poisoning and other diseases; and the processed product of the fish maw of yellow croaker - white fish maw, is rich in collagen, vitamins, amino acids, and a variety of trace elements and minerals and other nutrients. It has the effects of nourishing the kidney and essence, beautifying the skin, and enhancing immunity. It has very good nourishing, anti-aging, and physical fitness effects.

Based on this, the applicant uses yellow croaker maw as raw material and utilizes modern bioengineering technology and chromatography technology to prepare oligopeptides with the ability to promote macrophage proliferation and NO synthesis. The oligopeptides can be used in medicines and functional foods with immune enhancement function.

Summary of the invention

In view of the problems existing in the prior art, the purpose of the present invention is to provide a yellow croaker maw oligopeptide with the effect of enhancing immunity and its application. The oligopeptide is a heptapeptide compound with an amino acid sequence of Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH) and a molecular weight of 839.0 Da. The specific preparation method of the oligopeptide comprises the following steps:

(1) Pretreatment of yellow croaker maw: the yellow croaker maw is thawed, impurities are removed, and the maw is crushed with a tissue crusher. An isopropanol solution is added at a solid-liquid ratio of 1 g:16-20 mL. The maw is defatted at room temperature and 250-280 W ultrasonically for 35-40 min. The process is repeated three times. The solution is centrifuged at room temperature and 6000 g for 20-25 min. The solid precipitate is dried, ground, and passed through a 200-mesh sieve to obtain defatted yellow croaker maw powder.

(2) Enzymatic hydrolysis of defatted yellow croaker maw powder: the defatted yellow croaker maw powder is added to Na ₂ HPO ₄ -NaH ₂ PO ₄ buffer (0.2 M) at a solid-liquid ratio of 1 g:10-15 mL, stirred evenly, the solution is adjusted to a pH value of 7.2-7.6, 2.5-3.0% bromelain by weight of the defatted yellow croaker maw powder is added, enzymatic hydrolysis is performed for 4.5-5.0 h, and the enzyme activity is inactivated in a boiling water bath for 10 min; then the temperature is adjusted to 35-40° C., the pH value is adjusted to 6.0-7.0, 1.0-1.5% chymotrypsin by weight of the maw powder is added, and after enzymatic hydrolysis for 3.5-4.0 h, the solution is inactivated in a boiling water bath for 10 min; finally, the solution is centrifuged at 6000 rpm for 25-30 min, and the supernatant is collected to obtain yellow croaker maw enzymatic hydrolyzate;

(3) Fractional preparation of ultrafiltration hydrolysate of yellow croaker maw: The yellow croaker maw enzymatic hydrolysate was fractionated through ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 3.5 kDa, and three fractionated fractions (MW < 1 kDa, 1 kDa < MW < 3.5 kDa, and MW > 3.5 kDa) were collected. The effects of the three ultrafiltration fractions on the proliferation capacity of macrophages were measured, and the fraction with the best activity was selected, i.e., the ultrafiltration hydrolysate of yellow croaker maw;

(4) Preparation of yellow croaker maw immunoenhancing peptides: The yellow croaker maw ultrafiltration hydrolysate is purified by macroporous adsorption resin, gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC) to obtain yellow croaker maw immunoenhancing oligopeptides.

Preferably, the specific process of macroporous adsorption resin, gel chromatography and RP-HPLC purification in step 4) is:

Macroporous adsorption resin purification: the pretreated DA201-C macroporous resin is wet-packed into a column (2.5×100 cm), and the chromatographic column is balanced with ultrapure water; the ultrafiltration hydrolysate of yellow croaker maw is dissolved in ultrapure water to form a solution with a concentration of 25-30 mg/mL, and added to the DA201-C macroporous resin column, and allowed to stand for adsorption for 1.5-2.0 h, and then eluted with 1200-1400 mL of ultrapure water to remove the non-adsorbed substances, and then eluted with 1500-1600 mL of 95 ethanol solution at an elution flow rate of 1.5-2.0 mL/min, and the eluted solution is collected, and the effects of the four ultrafiltration components on the proliferation ability of macrophages are determined, and vacuum freeze-dried to obtain the yellow croaker maw macroporous resin chromatography hydrolysate.

Gel chromatography purification: The above-mentioned yellow croaker maw macroporous resin chromatography hydrolysate is prepared into a solution with a concentration of 20-25 mg/mL, separated by Sephadex LH-20 column chromatography (2.0×120 cm), eluted with ultrapure water at a flow rate of 0.6-0.8 mL/min, and each chromatographic peak is collected according to the chromatogram at 225 nm. The effect of each chromatographic peak on the proliferation ability of macrophages is determined, and the sample with the highest activity chromatographic peak is the yellow croaker maw gel chromatography hydrolysate.

RP-HPLC purification: The above-mentioned yellow croaker maw gel chromatography hydrolysate was prepared into a 20-25 μg/mL solution with double distilled water, and purified by RP-HPLC. According to the ability of the prepared oligopeptide to promote the proliferation of macrophages, an immune-enhancing oligopeptide was obtained.

Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH), the molecular weight was 839.0 Da as determined by ESI-MS.

More preferably, the RP-HPLC conditions are: injection volume 8-10 μL; chromatographic column Kromasil C18 (250 mm×4.6 mm, 5 μm); mobile phase: 45% acetonitrile; elution rate 1.0-1.2 mL/min; ultraviolet detection wavelength 225 nm.

Yellow croaker maw oligopeptide prepared by the above preparation method

Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH) can significantly promote the proliferation of macrophages RAW 264.7; at the same time, Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH) can significantly improve the phagocytic ability of macrophages RAW 264.7, enhance the secretion of NO in macrophages RAW 264.7, and promote the secretion of cytokines IL-1β and TNF-α in macrophages RAW 264.7. Therefore, FLVAPRH has the advantages of safety, no toxic side effects, and significant immune enhancement activity, and can be used in drugs and functional foods with immune enhancement function.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a Sephadex G-25 column chromatography chromatogram of MB-HA, an ultrafiltration fraction of yellow croaker maw protein hydrolysate of the present invention;

FIG2 is a RP-HPLC chromatogram of the HA-3 component separated by Sephadex G-25 column chromatography of the present invention;

FIG3 is a structure of the yellow croaker maw oligopeptide FLVAPRH of the present invention;

FIG4 is a mass spectrum of the yellow croaker maw oligopeptide FLVAPRH of the present invention;

FIG5 shows the effect of the yellow croaker maw oligopeptide FLVAPRH of the present invention on the proliferation rate of macrophages RAW264.7 at a concentration of 0 to 0.5 mg/mL;

FIG6 shows the effect of the yellow croaker swim bladder oligopeptide FLVAPRH of the invention on the phagocytic ability of macrophages RAW 264.7 at a concentration of 0 to 0.5 mg/mL;

FIG7 shows the effect of the yellow croaker maw oligopeptide FLVAPRH of the present invention on the NO synthesis ability in macrophages RAW264.7 at a concentration of 0 to 0.5 mg/mL;

FIG8 shows the effect of the yellow croaker maw oligopeptide FLVAPRH of the present invention on the secretion levels of TNF-α (A), IL-6 (B) and IL-1β (C) in macrophages RAW264.7 at a concentration of 0 to 0.5 mg/mL.

DETAILED DESCRIPTION

The present invention is further described below in conjunction with the accompanying drawings in order to better understand the technical solution.

A yellow croaker maw oligopeptide with immunity-enhancing effect, the preparation process of which is as follows: yellow croaker maw→defatting→composite enzymatic hydrolysis→ultrafiltration membrane classification→chromatographic preparation→immunity-enhancing oligopeptide→functional evaluation.

The specific steps are:

(1) Pretreatment of yellow croaker maw: The yellow croaker maw was thawed, impurities were removed, and the maw was crushed with a tissue crusher. Isopropanol solution was added at a solid-liquid ratio of 1 g:16-20 mL. The maw was defatted at room temperature and 260 W ultrasonic for 40 min. The process was repeated three times. The solution was centrifuged at room temperature and 6000 g for 25 min. The solid precipitate was dried, ground, and passed through a 200-mesh sieve to obtain defatted yellow croaker maw powder.

(2) Enzymatic hydrolysis of defatted yellow croaker maw powder: The defatted yellow croaker maw powder was added to _{Na2HPO4 - NaH2PO4} buffer (0.2M ₎ at a solid-liquid ratio of 1 g:11 mL, stirred evenly, the solution was adjusted to a pH value of 7.3, 3.0% bromelain by weight of the defatted yellow croaker maw powder was added, enzymatic hydrolysis was performed for 5.0 h, and the enzyme activity was inactivated in a boiling water bath for 10 min; then the temperature was adjusted to 37°C, the pH value was adjusted to 6.7, 1.3% chymotrypsin by weight of the maw powder was added, and after enzymatic hydrolysis for 4.0 h, the solution was inactivated in a boiling water bath for 10 min; finally, the solution was centrifuged at 6000 rpm for 30 min, and the supernatant was collected to obtain yellow croaker maw enzymatic hydrolysate (HGH).

(3) Fractional preparation of ultrafiltration hydrolysate of yellow croaker maw: The yellow croaker maw enzymatic hydrolysate (HGH) was fractionated through ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 3.5 kDa, and three fractionated fractions, HGH-I (MW < 1 kDa), HGH-II (1 kDa < MW < 3.5 kDa) and HGH-III (MW > 3.5 kDa), were collected. The effects of the three ultrafiltration fractions on the proliferation ability of macrophages were measured (see Table 1). HGH-I showed the strongest ability to promote the proliferation of macrophages. HGH-I was dried to obtain the ultrafiltration hydrolysate of yellow croaker maw.

(4) Preparation of yellow croaker maw immune enhancing peptide: The yellow croaker maw ultrafiltration hydrolysate (HGH-I) was purified by macroporous adsorption resin, gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC) to obtain yellow croaker maw immune enhancing oligopeptide, and its molecular weight was determined by mass spectrometry, and its amino acid sequence was determined by amino acid sequence analyzer.

The specific process of purification by macroporous adsorption resin, gel chromatography and reverse phase high performance liquid chromatography (RP-HPLC) is as follows:

① Purification by macroporous adsorption resin: The pretreated DA201-C macroporous resin was wet-packed on a column (2.5×100 cm), and the column was balanced with ultrapure water; the ultrafiltered hydrolysate of yellow croaker maw was dissolved in ultrapure water to prepare a solution with a concentration of 25 mg/mL, and added to the DA201-C macroporous resin column, and allowed to stand for adsorption for 2.0 h, and then eluted with 1400 mL of ultrapure water to remove the non-adsorbed substances, and then eluted with 1500 of 95 ethanol solution at an elution flow rate of 1.5 mL/min, and the eluted solution was collected and dried, and its effect on the proliferation ability of macrophages was determined (see Table 1), which is the yellow croaker maw macroporous resin chromatography hydrolysate (HGH-IA).

② Gel chromatography purification: The above-mentioned yellow croaker maw macroporous resin chromatography hydrolysate (HGH-IA) was prepared into a solution with a concentration of 25 mg/mL, separated by Sephadex LH-20 column chromatography (2.0×120 cm), eluted with ultrapure water at a flow rate of 0.7 mL/min, and chromatographic peaks (IA-1, IA-2, IA-3 and IA-4) were collected according to the chromatogram at 225 nm (see Figure 1). The effects of the four chromatographic peaks on the proliferation ability of macrophages were determined (see Table 1). The sample with the highest activity chromatographic peak was the yellow croaker maw gel chromatography hydrolysate (IA-3).

③ RP-HPLC purification: The above-mentioned yellow croaker swim bladder gel chromatography hydrolysate (IA-3) was prepared into a 20 μg/mL solution with double distilled water, and purified by RP-HPLC (injection volume 10 μL; chromatographic column Kromasil C18 (250 mm×4.6 mm, 5 μm); mobile phase: 45% acetonitrile; elution rate 1.0 mL/min; ultraviolet detection wavelength 225 nm). According to the RP-HPLC spectrum (see Figure 2), 8 peptide components (HGP-1 to HGP-8) were obtained, and the ability of the 8 peptide components to promote the proliferation of macrophages was determined (see Table 2); among them, HGP-6 showed the highest activity and was freeze-dried.

④ Structural detection: The peptide HGP-6 with the strongest ability to promote macrophage proliferation was collected and tested by RP-HPLC to meet the sequencing requirements. The amino acid sequence was determined by a protein/peptide sequence analyzer to be Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH) (see Figure 3), and the molecular weight was determined by ESI-MS to be 839.0 Da (see Figure 4).

⑤ Functional evaluation: Referring to the experimental method of the literature [Zhen Wang, Yue Fang, Yu Zeng, Xu Yang, Fang-Miao Yu, Bin Wang. Immunomodulatory peptides from thick-shelled mussel (Mytilus coruscus): Isolation, identification, molecular docking and immunomodulatory effects on RAW264.7 cells. Food Bioscience, 2024, 103874], macrophages RAW264.7 were used to evaluate the immune-enhancing function of yellow croaker swim bladder oligopeptide FLVAPRH.

The experimental results show that FLVAPRH at a concentration of 0-0.5 mg/mL can significantly enhance the proliferation of macrophages RAW 264.7 (see Figure 5), enhance the phagocytic ability of macrophages RAW 264.7 (see Figure 6), improve the NO synthesis ability of macrophages RAW 264.7 (see Figure 7), and promote the secretion of cytokines TNF-α, IL-6 and IL-1β in macrophages RAW 264.7 (see Figure 8).

The yellow croaker swim bladder oligopeptide Phe-Leu-Val-Ala-Pro-Arg-His (FLVAPRH) provided by the present invention can significantly enhance the immune ability of macrophages, is safe and has no toxic side effects, and can be used to prepare medicines and functional foods with immune enhancement function.

Table 1 Effects of yellow croaker swim bladder hydrolysate (HGH), ultrafiltration fractions (HGH-I, HGH-II and HGH-III), macroporous resin chromatography hydrolysate (HGH-IA) and gel chromatography separation fractions (IA-1, IA-2, IA-3 and IA-4) on the proliferation rate (%) of macrophages RAW 264.7 at a concentration of 1.0 mg/mL.

Table 2 Effects of 0.5 mg/mL enhanced oligopeptide from yellow croaker maw on the proliferation rate of macrophage RAW 264.7

Claims (8)

1. A yellow croaker maw oligopeptide with immunity enhancement, characterized in that the oligopeptide is a heptapeptide compound with an amino acid sequence of Phe-Leu-Val-Ala-Pro-Arg-His and a molecular weight of 839.0 Da. 2. Use of the yellow croaker maw oligopeptide with immunity enhancement as claimed in claim 1 in the preparation of medicines or functional foods with immunity enhancement function. 3. The use according to claim 2, characterized in that the medicine or functional food containing the oligopeptide can significantly promote the proliferation of macrophage RAW 264.7. 4. The use according to claim 2, characterized in that the medicine or functional food containing the oligopeptide can significantly improve the phagocytic ability of macrophages RAW 264.7 and enhance the ability to synthesize NO in macrophages RAW 264.7. 5. The use according to claim 2, characterized in that the medicine or functional food containing the oligopeptide can promote the secretion of cytokines TNF-α, IL-6 and IL-1β in macrophage RAW 264.7. 6. The yellow croaker maw oligopeptide with enhanced immunity as claimed in claim 1, characterized in that the oligopeptide is prepared by the following method: 1) Pretreatment of yellow croaker maw: thawing the yellow croaker maw, removing impurities, crushing it with a tissue crusher, adding isopropanol solution at a solid-liquid ratio of 1 g:16-20 mL, defatting at room temperature, 250-280 W ultrasonic for 35-40 min, repeating three times, centrifuging the solution at room temperature, 6000 g for 20-25 min, drying the solid precipitate, grinding it, and passing it through a 200-mesh sieve to obtain defatted yellow croaker maw powder; 2) Enzymatic hydrolysis of defatted yellow croaker maw powder: adding the defatted yellow croaker maw powder to 0.2M _{Na2HPO4 - NaH2PO4} buffer solution at a solid-liquid ratio of _1g :10-15mL, stirring evenly, adjusting the pH value of the solution to 7.2-7.6, adding 2.5-3.0% bromelain by weight of the defatted yellow croaker maw powder, enzymolysis for 4.5-5.0h, and inactivating the enzyme in a boiling water bath for 10min; then adjusting the temperature to 35-40°C, adjusting the pH value to 6.0-7.0, adding 1.0-1.5% chymotrypsin by weight of the maw powder, enzymolysis for 3.5-4.0h, and inactivating the enzyme in a boiling water bath for 10min; finally, centrifuging the solution at 6000rmp for 25-30min, collecting the supernatant, and obtaining yellow croaker maw enzymatic hydrolyzate; 3) Fractional preparation of ultrafiltration hydrolysate of yellow croaker maw: The yellow croaker maw enzymatic hydrolysate was fractionated through ultrafiltration membranes with molecular weight cutoffs of 1 kDa and 3.5 kDa, and three fractionated fractions (MW < 1 kDa, 1 kDa < MW < 3.5 kDa, and MW > 3.5 kDa) were collected. The effects of the three ultrafiltration fractions on the proliferation capacity of macrophages were measured, and the fraction with the best activity was selected, i.e., the ultrafiltration hydrolysate of yellow croaker maw; 4) Preparation of yellow croaker maw immune enhancing peptides: The yellow croaker maw ultrafiltration hydrolysate is purified by macroporous adsorption resin, gel chromatography and reversed-phase high performance liquid chromatography in sequence to obtain yellow croaker maw immune enhancing oligopeptides. 7. The yellow croaker maw oligopeptide with enhanced immunity as claimed in claim 6, characterized in that the specific process of purification by macroporous adsorption resin, gel chromatography and reversed-phase high performance liquid chromatography is: Macroporous adsorption resin purification: pre-treated DA201-C macroporous resin is wet loaded into a column, and the chromatographic column is balanced with ultrapure water; the ultrafiltration hydrolysate of yellow croaker maw is dissolved in ultrapure water to prepare a solution with a concentration of 25-30 mg/mL, and added to the DA201-C macroporous resin column, and adsorbed for 1.5-2.0 hours, and then eluted with 1200-1400 mL of ultrapure water to remove non-adsorbed substances, and then eluted with 1500-1600 mL of 95 ethanol solution, with an elution flow rate of 1.5-2.0 mL/min, and the eluted solution is collected, and the effects of four ultrafiltration components on the proliferation ability of macrophages are determined, and vacuum freeze-dried to obtain the yellow croaker maw macroporous resin chromatography hydrolysate; Gel chromatography purification: the above-mentioned yellow croaker maw macroporous resin chromatography hydrolysate is prepared into a solution with a concentration of 20-25 mg/mL, separated by Sephadex LH-20 column chromatography, eluted with ultrapure water at a flow rate of 0.6-0.8 mL/min, and each chromatographic peak is collected according to the chromatogram at 225 nm, and the effect of each chromatographic peak on the proliferation ability of macrophages is determined. The sample with the highest activity chromatographic peak is the yellow croaker maw gel chromatography hydrolysate; Reverse-phase high-performance liquid chromatography purification: The above-mentioned yellow croaker maw gel chromatography hydrolysate was prepared into a 20-25 μg/mL solution with double distilled water, and purified by RP-HPLC. Based on the ability of the prepared oligopeptide to promote the proliferation of macrophages, an immune-enhancing oligopeptide Phe-Leu-Val-Ala-Pro-Arg-His was obtained, and its molecular weight was determined to be 839.0 Da by ESI-MS. 8. The yellow croaker maw oligopeptide with enhanced immunity as claimed in claim 6, characterized in that the reversed-phase high performance liquid chromatography purification conditions are: injection volume 8-10 μL; chromatographic column Kromasil C18 (250 mm×4.6 mm, 5 μm); mobile phase: 45% acetonitrile; elution rate 1.0-1.2 mL/min; ultraviolet detection wavelength 225 nm.