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

Kangping Bio’s peptide preparation technology achievement has been granted an invention patent by the China National Intellectual Property Administration, with patent number ZL201810715273.2 and patent publication date of February 7, 2020. To date, Kangping Bio has accumulated a total of 17 authorized technical patents, continuing to strengthen core technology barriers.

I. Technical Field and Research Background

1. Technical Field

This invention belongs to the field of proteins, particularly relating to a bioactive peptide SNLIEVT and its preparation method and application. This field represents a frontier direction at the intersection of life sciences and food science, aiming to discover physiologically active functional peptide segments from natural protein resources to provide safe and effective functional ingredients for human health.

2. Background Art

The search for safe bioactive peptides from natural food sources is of paramount importance. In particular, during the fermentation of bovine milk by lactic acid bacteria, a portion of the milk proteins is metabolized and utilized by the lactic acid bacteria, undergoing a series of physiological and biochemical reactions that convert proteins into polypeptides or free amino acids. This natural transformation process provides a rich resource foundation for the isolation and identification of biologically active functional peptides from fermented dairy products.

Immunologically active peptides are the first class of bioactive peptides discovered from milk following the discovery of opioid peptides, and their physiological activity has been demonstrated. As early as 1981, Jolles et al. first discovered that trypsin hydrolysis of human milk protein yields a hexapeptide with the amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr. In vitro experiments demonstrated that this peptide could enhance the phagocytic activity of mouse peritoneal macrophages against sheep red blood cells.

A large body of subsequent research has shown that immunologically active peptides 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 external pathogen infections, and reduce the incidence of disease, but also do not elicit immune rejection responses in the body. These unique biological characteristics make immunologically active peptides ideal candidate molecules for the development of functional foods and health products.

Against this research background, the scientific research team behind this invention, through in-depth research and systematic screening, successfully discovered and identified a novel bioactive peptide SNLIEVT from Lactobacillus helveticus cellular protein, and conducted comprehensive scientific validation of its immunomodulatory activity and anti-aging activity.

II. Overview of the Invention Content

1. Peptide Sequence and Source

The bioactive peptide SNLIEVT provided by this invention has the amino acid sequence Ser-Asn-Leu-Ile-Glu-Val-Thr, as shown in SEQ ID NO: 1. This heptapeptide consists of seven amino acid residues: serine (Ser), asparagine (Asn), leucine (Leu), isoleucine (Ile), glutamic acid (Glu), valine (Val), and threonine (Thr), with a well-defined molecular structure and clear sequence characteristics.

According to the patent disclosure, this bioactive peptide is derived from Lactobacillus helveticus cellular protein, specifically from the LBH_0240|m.224 protein, and constitutes amino acid residues 71–77 of this protein. This origin confers upon the peptide natural and safe characteristics, providing a solid safety foundation for its application in food and health product sectors.

2. Encoding Nucleotide Sequence

This invention also provides a nucleotide fragment encoding the aforementioned bioactive peptide SNLIEVT, with the sequence: 5’-caa act taa tcg aag tta ctg-3’, as shown in SEQ ID NO: 2. This nucleotide sequence provides the molecular basis for the large-scale production of this peptide through genetic engineering technology.

3. Core Functional Properties

The bioactive peptide SNLIEVT of this invention possesses two core functional properties: immunomodulatory function and anti-aging function. Detailed experimental data and scientific evidence demonstrating the experimental validation process and results for these two functions are presented below.

III. Preparation Method

This invention provides multiple preparation methods for the bioactive peptide SNLIEVT, offering flexibility and operability:

Method 1: Chemical Synthesis. Artificial synthesis using solid-phase peptide synthesis (SPPS). Specifically, RINK resin (substitution degree 0.3 mmol/g) is employed, with amino acids Ser, Asn, Leu, Ile, Glu, Val, and Thr sequentially coupled via the Fmoc protecting group strategy. The condensation system uses HOBT/DIC. The synthetic product is analyzed and confirmed by UPLC-ESI-Q-TOF-MS.

Method 2: Genetic Engineering. Artificial synthesis through genetic engineering methods, utilizing the aforementioned encoding nucleotide sequence for recombinant expression and purification in an appropriate expression system.

Method 3: Isolation and Purification. Direct isolation and purification from Lactobacillus helveticus cellular material through cell disruption.

IV. Specific Embodiments and Experimental Verification

This patent comprehensively validated the physicochemical properties and biological activity of the bioactive peptide SNLIEVT through the following three embodiments.

1. Example 1: Artificial Synthesis and Identification of Active Peptide SNLIEVT

To ensure the purity and sequence accuracy of the peptide used in experiments, the research team artificially synthesized the target peptide using solid-phase peptide synthesis (SPPS). The synthesis was performed using RINK resin (substitution degree 0.3 mmol/g), with amino acids Ser, Asn, Leu, Ile, Glu, Val, and Thr sequentially coupled via the Fmoc protecting group strategy, and HOBT/DIC as the condensation system.

The synthetic product was analyzed and confirmed by UPLC-ESI-Q-TOF-MS: mass-to-charge ratio of 775.4212 Da, retention time 37.7 min. Subsequent analysis using Mascot software confirmed that the synthetic product sequence was Ser-Asn-Leu-Ile-Glu-Val-Thr (SNLIEVT), with accurate sequence and purity meeting the requirements for subsequent experiments.

2. Example 2: Bioactive Peptide Immune Regulation Activity Experiments

To comprehensively evaluate the immunomodulatory function of peptide SNLIEVT, the research team designed and conducted in vitro lymphocyte proliferation experiments and macrophage phagocytosis experiments.

(1) MTT Assay for In Vitro Lymphocyte Proliferation

The MTT assay is a classical method for evaluating the ability of compounds to promote lymphocyte proliferation. The experimental results showed: under the condition of bioactive peptide SNLIEVT at a mass concentration of 100 μg/mL, the milk-derived bioactive peptide SNLIEVT reached a stimulation index of 1.213, with a statistically significant difference from the negative control group (P<0.05).

Based on the above experimental data, the research team concluded that this bioactive peptide SNLIEVT has the ability to significantly promote mouse lymphocyte proliferation and can be used as a health product or dietary additive to enhance the immunity of animals and humans.

(2) Macrophage Neutral Red Phagocytosis Assay

Macrophages are an important component of the body’s non-specific immune system, and their phagocytic capacity directly affects the efficiency of pathogen clearance. The experiment evaluated the effect of peptide SNLIEVT on macrophage function by measuring the phagocytosis of neutral red by macrophages.

The experimental results showed: compared with the blank cell control, macrophages in the inflammation group treated with 1 mg/mL bioactive peptide SNLIEVT exhibited significantly enhanced neutral red phagocytic capacity, with a statistically significant difference (P<0.01).

This result demonstrates that bioactive peptide SNLIEVT significantly promotes the neutral red phagocytic capacity of macrophages in vitro under inflammatory conditions.

Integrating the results of the two immune function experiments, peptide SNLIEVT can significantly enhance the in vitro proliferative capacity of lymphocytes and the phagocytic function of macrophages at the cellular level, demonstrating its favorable immunomodulatory activity.

3. Example 3: Bioactive Peptide Anti-Aging Activity Experiments

To evaluate the anti-aging efficacy of peptide SNLIEVT, the research team used Caenorhabditis elegans as a model organism and conducted a systematic evaluation from three dimensions: reproductive capacity, body length development, and oxidative stress resistance.

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

Reproductive capacity is one of the important indicators for evaluating the aging state of an organism. The experimental results showed: when the feeding concentration of peptide SNLIEVT was 300 mg/L, the average egg-laying count of the worms showed an extremely statistically significant difference compared with the blank control group (P<0.01).

Based on the above experimental data, the research team concluded that peptide SNLIEVT can significantly enhance the reproductive capacity of C. elegans at certain concentrations.

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

To ensure the safety of peptide SNLIEVT, the research team simultaneously evaluated the effect of different peptide concentrations on the growth and development of C. elegans. The experimental results showed that the concentration of peptide SNLIEVT did not affect the growth of the worms. This result demonstrates that peptide SNLIEVT has favorable safety while exerting anti-aging effects, without producing adverse effects on normal growth and development.

(3) Acute Oxidative Stress Survival Rate Experiment

Oxidative stress is one of the important factors leading to organismal aging. To evaluate the effect of peptide SNLIEVT in enhancing the body’s antioxidant capacity, the research team conducted an acute oxidative stress survival rate experiment.

The experimental results showed: under oxidative stress conditions, the survival rate of the experimental groups was significantly higher than that of the blank control group. This indicates that under oxidative stress conditions, the survival rate of C. elegans was significantly improved, possibly because peptide SNLIEVT can effectively help C. elegans resist oxidative damage, scavenge free radicals generated in the body, and reduce the accumulation of peroxides.

These experimental results demonstrate: peptide SNLIEVT can significantly increase the stress resistance and oxidative stress capacity of C. elegans, improve the survival rate of C. elegans, indicating that peptide SNLIEVT at certain concentrations has anti-aging effects on C. elegans.

V. Summary of Beneficial Effects

Integrating all the above experimental results, the bioactive peptide SNLIEVT of this invention has the following two significant beneficial effects:

First, in terms of immunomodulatory function: The bioactive peptide SNLIEVT of this invention can enhance the in vitro proliferative capacity of lymphocytes and the phagocytic function of macrophages, improve the body’s ability to resist external pathogen infections, and reduce the incidence of disease. Specifically, at a mass concentration of 100 μg/mL, SNLIEVT reached a stimulation index of 1.213 on lymphocytes (P<0.05); at a concentration of 1 mg/mL, it significantly promoted the phagocytic capacity of macrophages under inflammatory conditions (P<0.01).

Second, in terms of anti-aging function: The bioactive peptide SNLIEVT of this invention can increase the activity of the body’s anti-peroxidation enzyme system, enhance the body’s ability to resist exogenous stimuli, thereby reducing the probability of aging, senescence, and illness. Specifically, at a concentration of 300 mg/L, it significantly enhanced the reproductive capacity of C. elegans (P<0.01); under oxidative stress conditions, it significantly improved the survival rate of C. elegans, without affecting the normal growth and development of the worms.

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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