Kangping Bio Granted Invention Patent: A Bioactive Peptide PIGSENSGKTTMPL and Its Preparation Technology

A peptide preparation technology achievement of Kangping Bio has been granted an invention patent by the China National Intellectual Property Administration, with patent number ZL201711288374.8 and patent publication date of March 2, 2021. To date, Kangping Bio has accumulated a total of 17 authorized technical patents, and its core technology barriers are continuously consolidated.

I. Technical Field and Background

The present invention relates to the field of proteins, and in particular to a bioactive peptide PIGSENSGKTTMPL, its preparation method, and its applications.

During the fermentation of bovine milk by lactic acid bacteria, a portion of the milk proteins are metabolized and utilized by the lactic acid bacteria, and a series of physiological and biochemical reactions occur, converting the proteins into peptides or free amino acids. Searching for safe bioactive peptides from natural food sources is of particular importance.

Immunoactive peptides are the first class of bioactive peptides discovered from milk following the identification of opioid peptides, and their physiological activity has been demonstrated. Research has shown that immunoactive peptides can not only enhance the body’s immunity, stimulate the proliferation of lymphocytes, enhance the phagocytic function of macrophages, promote the release of cytokines, improve the body’s ability to resist infection by external pathogens, and reduce the incidence of disease, but also do not elicit immune rejection responses in the body.

With the development of modern biotechnology and the increasing public demand for health, the discovery of functional peptides with multiple bioactivities such as immunomodulatory and anti-aging properties from natural food proteins has become an important research direction in the fields of functional foods, health products, and biomedicine. Milk-derived bioactive peptides have received extensive attention from academia and industry due to their advantages of natural origin, high safety, and diverse biological activities.

II. Summary of the Invention

1. Amino Acid Sequence of Bioactive Peptide PIGSENSGKTTMPL

In a first aspect, the present invention provides a bioactive peptide PIGSENSGKTTMPL, whose amino acid sequence is Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu, as shown in SEQ ID NO: 1.

This bioactive peptide is milk-derived, specifically originating from alpha s1-casein, and comprises amino acid residues at positions 185 to 198 of the alpha s1-casein variant C.

The bioactive peptide possesses immunomodulatory and anti-aging functions.

2. Nucleotide Sequence Encoding the Peptide

In a second aspect, the present invention provides a nucleotide fragment encoding the bioactive peptide PIGSENSGKTTMPL, whose sequence is: 5’-cct att ggc tct gag aac agt ggc aag act act atg cca ctg-3’, as shown in SEQ ID NO: 2.

3. Method for Preparing the Peptide

In a third aspect, the present invention provides a method for preparing the bioactive peptide PIGSENSGKTTMPL, which can be artificially synthesized by genetic engineering methods, obtained directly from dairy products through isolation and purification, or prepared directly by chemical synthesis.

III. Beneficial Effects

The milk-derived bioactive peptide PIGSENSGKTTMPL of the present invention exhibits favorable activity in regulating the body’s immune function and anti-aging activity.

On the one hand, the bioactive peptide PIGSENSGKTTMPL of the present invention can enhance the in vitro proliferative capacity of lymphocytes and macrophages, improve the body’s ability to resist infection by external pathogens, and reduce the incidence of disease.

On the other hand, the bioactive peptide PIGSENSGKTTMPL of the present invention can increase the activity of the body’s anti-peroxidase system and enhance the body’s ability to resist exogenous stimuli, thereby reducing the probability of aging, senescence, and illness.

The peptide PIGSENSGKTTMPL of the present invention was subjected to in vitro immunomodulatory experiments and in vivo anti-aging experiments, which verified its favorable immunomodulatory and anti-aging activities. These findings are of great significance for the development of foods, health products, and pharmaceuticals with immunomodulatory and anti-aging functions.

IV. Specific Embodiments

1. Example 1: Artificial Synthesis of Bioactive Peptide PIGSENSGKTTMPL

This example employs the solid-phase peptide synthesis (SPPS) method for the artificial synthesis of bioactive peptide PIGSENSGKTTMPL. The specific synthetic steps are as follows:

Synthesis method: The solid-phase peptide synthesis (SPPS) method was employed using RINK resin (substitution degree 0.3 mmol/g) with an Fmoc protecting group strategy. Amino acids were coupled sequentially: Pro, Ile, Gly, Ser, Glu, Asn, Ser, Glu, Lys, Thr, Thr, Met, Pro, and Leu. The coupling system used HOBT/DIC, and deprotection was performed using 20% piperidine/DMF.

Product identification: Analysis by UPLC-ESI-Q-TOF-MS confirmed a mass-to-charge ratio (m/z) of 716.3632 Da and a retention time of 28.4 min. Sequence analysis using Mascot software confirmed the sequence as Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu (PIGSENSGKTTMPL).

2. Example 2: Immune Regulation Activity Assay of Bioactive Peptide

This example systematically validated the immunomodulatory activity of bioactive peptide PIGSENSGKTTMPL through two in vitro experiments.

(1) MTT Assay for Lymphocyte Proliferation Capacity in Vitro

The MTT assay is a classical method for detecting cell proliferation activity, which reflects the proliferative status of cells by measuring the activity of succinate dehydrogenase in the mitochondria of viable cells. In this experiment, bioactive peptide PIGSENSGKTTMPL was used as a stimulant to evaluate its effect on the in vitro proliferation capacity of mouse lymphocytes.

Experimental results: Under the condition of a mass concentration of 100 μg/mL of bioactive peptide PIGSENSGKTTMPL, the stimulation index (SI) reached 1.182, which showed a statistically significant difference compared with the negative control group (P < 0.05).

Experimental conclusion: Therefore, it can be concluded that this bioactive peptide PIGSENSGKTTMPL possesses a significant capacity to promote mouse lymphocyte proliferation, and can be consumed as a health product or additive to enhance the immunity of animals and humans.

This result indicates that peptide PIGSENSGKTTMPL can enhance the body’s cellular immune response by promoting lymphocyte proliferation, thereby improving the body’s ability to resist infection by external pathogens, and holds important application value in the development of foods, health products, and pharmaceuticals with immunomodulatory functions.

(2) Macrophage Phagocytosis of Neutral Red Assay

Macrophages are the core effector cells of the innate immune system, and their phagocytic capacity directly reflects the body’s non-specific immune function. This experiment evaluated the activity of bioactive peptide PIGSENSGKTTMPL in enhancing macrophage function by detecting its effect on macrophage phagocytosis of neutral red.

Experimental results: Compared with the cell blank, the phagocytic capacity of neutral red by macrophages in the inflammatory group supplemented with 1 mg/mL bioactive peptide PIGSENSGKTTMPL was significantly increased, with a statistically significant difference (P < 0.01).

Experimental conclusion: This indicates that bioactive peptide PIGSENSGKTTMPL exerts a significant promoting effect on the in vitro phagocytosis of neutral red by macrophages under inflammatory conditions.

The enhancement of macrophage phagocytic function means improved efficiency in clearing pathogenic microorganisms and foreign substances by the body, which is of great significance for enhancing the body’s anti-infection capability. In particular, the fact that peptide PIGSENSGKTTMPL can still significantly enhance macrophage phagocytic function under inflammatory conditions makes its application prospects in the field of immunomodulation even more promising.

3. Example 3: Anti-Aging Activity Assay of Bioactive Peptide

This example employed Caenorhabditis elegans as a model organism to comprehensively evaluate the anti-aging activity of bioactive peptide PIGSENSGKTTMPL from three dimensions: reproductive capacity, body length development, and oxidative stress survival rate.

(1) Effect of the Peptide on Reproductive Capacity of C. elegans

Reproductive capacity is one of the important indicators for evaluating the degree of aging in an organism. Changes in the egg-laying number of nematodes can reflect their overall physiological status and aging process.

Experimental results: When the feeding concentration of peptide PIGSENSGKTTMPL was 300 mg/L, the average egg-laying number of nematodes showed an extremely significant difference compared with the blank group (P < 0.01). At concentrations of 400 mg/L and 500 mg/L, only a statistically significant difference was observed compared with the blank group (P < 0.05).

Experimental conclusion: In summary, peptide PIGSENSGKTTMPL can significantly improve the reproductive capacity of nematodes at certain concentrations.

The improvement in reproductive capacity indicates that peptide PIGSENSGKTTMPL can ameliorate the overall physiological function of nematodes and delay reproductive system aging, providing direct experimental evidence for its anti-aging efficacy.

(2) Effect of the Peptide on Body Length of C. elegans

To address concerns that the peptide might adversely affect the growth and development of nematodes, this experiment examined changes in nematode body length under treatment with different concentrations of peptide PIGSENSGKTTMPL.

Experimental results: The experimental results showed that the concentration of peptide PIGSENSGKTTMPL did not affect the growth of nematodes.

This result demonstrates that peptide PIGSENSGKTTMPL exerts its anti-aging effects without negatively impacting the normal growth and development of nematodes, indicating favorable biological safety.

(3) Acute Oxidative Stress Survival Rate Assay

Oxidative stress is one of the important factors leading to organismal aging. This experiment evaluated the efficacy of peptide PIGSENSGKTTMPL in enhancing the body’s antioxidant capacity and resisting oxidative damage by detecting the survival rate of nematodes under oxidative stress conditions.

Experimental results: Under oxidative stress conditions, the survival rates of all experimental groups were significantly higher than that of the blank group.

Experimental conclusion and mechanistic analysis: This indicates that the survival rate of nematodes under oxidative stress conditions was significantly improved, possibly because peptide PIGSENSGKTTMPL can effectively help nematodes resist oxidative damage, scavenge free radicals generated in the body, and reduce the accumulation of peroxides.

The results of this experiment demonstrate that peptide PIGSENSGKTTMPL can significantly increase the stress resistance and oxidative stress capacity of nematodes, and improve their survival rate, indicating that peptide PIGSENSGKTTMPL at certain concentrations possesses anti-aging efficacy for nematodes.

The results of the oxidative stress survival rate assay further confirm the anti-aging activity of peptide PIGSENSGKTTMPL from the perspective of antioxidant effects. The peptide may enhance the activity of the body’s anti-peroxidase system and strengthen the body’s ability to resist exogenous oxidative stimuli, thereby reducing the harmful effects of oxidative damage on the body and slowing down the aging process.

V. Summary and Outlook

In summary, the present invention discloses a bioactive peptide PIGSENSGKTTMPL, its preparation method, and its applications. The amino acid sequence of this peptide is Pro-Ile-Gly-Ser-Glu-Asn-Ser-Glu-Lys-Thr-Thr-Met-Pro-Leu, derived from amino acid residues at positions 185 to 198 of the alpha s1-casein variant C, and it belongs to the class of milk-derived bioactive peptides.

Through systematic in vitro immunomodulatory experiments and in vivo anti-aging activity experiments, the present invention fully validated that peptide PIGSENSGKTTMPL possesses the following multiple biological activities:

Immunomodulatory function: (1) It can significantly enhance the in vitro proliferative capacity of lymphocytes. At a concentration of 100 μg/mL, the stimulation index reached 1.182, showing a statistically significant difference compared with the negative control group (P < 0.05), indicating its significant capacity to promote mouse lymphocyte proliferation. (2) Under inflammatory conditions, it can significantly promote the phagocytosis of neutral red by macrophages. At a concentration of 1 mg/mL, it showed an extremely significant difference compared with the cell blank (P < 0.01), indicating its effective enhancement of macrophage phagocytic function.

Anti-aging function: (1) It can significantly improve the reproductive capacity of C. elegans at certain concentrations. At a concentration of 300 mg/L, the average egg-laying number of nematodes showed an extremely significant difference compared with the blank group (P < 0.01); at concentrations of 400 mg/L and 500 mg/L, a statistically significant difference was observed compared with the blank group (P < 0.05). (2) It does not affect the normal growth and development of nematodes, demonstrating favorable biological safety. (3) It can significantly improve the survival rate of nematodes under oxidative stress conditions, effectively help nematodes resist oxidative damage, scavenge free radicals generated in the body, and reduce the accumulation of peroxides, thereby significantly increasing the stress resistance and oxidative stress capacity of nematodes.

The bioactive peptide PIGSENSGKTTMPL of the present invention, on the one hand, can enhance the in vitro proliferative capacity of lymphocytes and macrophages, improve the body’s ability to resist infection by external pathogens, and reduce the incidence of disease; on the other hand, it can increase the activity of the body’s anti-peroxidase system, enhance the body’s ability to resist exogenous stimuli, thereby reducing the probability of aging, senescence, and illness.

The present invention provides important technical support and a material foundation for the development of foods, health products, and pharmaceuticals with immunomodulatory and anti-aging functions, and is of great scientific significance and application value.

Disclaimer

This patent pertains solely to the technical solutions of an innovative food ingredient, encompassing its preparation methods, compositional characteristics, and physicochemical properties, and falls within the scope of a basic-level invention patent. The relevant experimental data, test results, and conclusions documented in the patent are all derived from validation studies conducted under laboratory conditions, intended solely to demonstrate the physicochemical performance and preliminary bioactive characteristics of this raw material within specific test systems, and do not involve actual product formulations, processing techniques, or human consumption scenarios.

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