Kangping Bio Granted Invention Patent: A Bioactive Peptide EPGIVNLD 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 ZL201810712475.1 and patent publication date of June 9, 2020. To date, Kangping Bio has cumulatively obtained 17 authorized technical patents, with its core technology barriers continuing to strengthen.

I. Technical Field and Scientific Background

1. Technical Field

The present invention relates to the field of proteins, specifically to a bioactive peptide EPGIVNLD and its preparation method and applications. This peptide is derived from Lactobacillus helveticus cellular protein and, upon isolation and identification, exhibits defined anti-inflammatory and anti-aging activities, representing a significant technical achievement in the field of bioactive peptide development.

2. Background Art

During the fermentation of bovine milk by lactic acid bacteria, a portion of the milk proteins is metabolized by the lactic acid bacteria, undergoing a series of physiological and biochemical reactions that convert proteins into peptides or free amino acids. Among these peptides, a subset possesses special physiological functions and is referred to as “bioactive peptides.”

Immunoactive peptides are the first class of bioactive peptides obtained from milk and proven to have physiological activity, following the discovery of opioid peptides. In 1981, Jolles et al. first discovered that tryptic hydrolysis of human milk protein yielded a hexapeptide with the amino acid sequence Val-Glu-Pro-Ile-Pro-Tyr, and in vitro experiments demonstrated that this peptide could enhance the phagocytic activity of mouse peritoneal macrophages against sheep red blood cells.

Aging is a natural phenomenon, and the process is often accompanied by changes in antioxidant levels, organ and tissue structures, and immune factors. As an emerging anti-aging agent, anti-aging peptides offer physiological advantages that amino acids cannot match.

Building upon the above scientific background, the R&D team of the present invention conducted in-depth exploration of the Lactobacillus helveticus fermented milk system, successfully isolating and identifying a novel bioactive peptide EPGIVNLD, and through systematic in vitro and in vivo experiments validated its significant anti-inflammatory and anti-aging activities, providing important technical support for the functional development and industrial application of bioactive peptides.

II. Summary of the Invention

1. Discovery and Identification of the Bioactive Peptide

The present invention provides a bioactive peptide EPGIVNLD, with the amino acid sequence Glu-Pro-Gly-Ile-Val-Asn-Leu-Asp (i.e., SEQ ID NO: 1). This peptide is derived from Lactobacillus helveticus cellular protein, specifically from the LBH_0034|m.31 protein, and constitutes the amino acid residues at positions 380–387 of this protein.

The nucleotide coding sequence of this peptide is: 5’-cga acc tgg cat agt caa cct cga-3’ (i.e., SEQ ID NO: 2). This nucleotide sequence provides the molecular basis for the genetic engineering preparation of the peptide.

2. Functional Characteristics of the Peptide

The bioactive peptide EPGIVNLD of the present invention possesses the following core functional characteristics:

(I) Anti-inflammatory function: The bioactive peptide EPGIVNLD of the present invention can promote cytokine secretion by macrophages, increase nitric oxide production by macrophages, enhance the body’s ability to resist exogenous pathogen infection, and reduce the incidence of disease.

(II) Anti-aging function: The bioactive peptide EPGIVNLD of the present invention can enhance the activity of the in vivo anti-peroxidation enzyme system, strengthen the body’s ability to resist exogenous stimuli, thereby reducing the probability of aging, senescence, and illness.

3. Preparation Methods

The present invention provides three approaches for the preparation of the bioactive peptide EPGIVNLD:

1. Artificial synthesis by genetic engineering methods: Utilizing genetic engineering technology, employing the above nucleotide sequence (SEQ ID NO: 2) for recombinant expression and purification;
2. Direct isolation and purification from Lactobacillus helveticus cellular biomass: Obtaining directly from Lactobacillus helveticus cellular protein through cell disruption, isolation, and purification methods;
3. Chemical synthesis: Direct preparation through chemical synthesis.

4. Application Scope

The bioactive peptide EPGIVNLD of the present invention can be widely applied in the following fields:

• Anti-inflammatory product development: Applications in the preparation of foods, health products, pharmaceuticals, or cosmetics with anti-inflammatory functions;
• Anti-aging product development: Applications in the preparation of foods, health products, or pharmaceuticals with anti-aging functions;
• Dual-function composite product development: Applications in the preparation of foods, health products, or pharmaceuticals possessing both anti-inflammatory and anti-aging functions.

III. Specific Embodiments and Experimental Validation

This patent establishes a systematic scientific validation system through three complete embodiments, spanning peptide synthesis, in vitro anti-inflammatory activity validation, and in vivo anti-aging activity evaluation.

1. Embodiment 1: Artificial Synthesis of the Active Peptide EPGIVNLD

This patent employed solid-phase peptide synthesis (SPPS) for the artificial synthesis of the target peptide, with the following specific process parameters:

(1) Synthesis Materials and Conditions

• Resin: RINK resin (substitution degree 0.3 mmol/g)
• Protection strategy: Fmoc protecting group strategy
• Amino acid coupling sequence: Glu, Pro, Gly, Ile, Val, Asn, Leu, and Asp coupled sequentially
• Coupling system: HOBT/DIC
• Deprotection reagent: 20% piperidine/DMF

(2) Cleavage and Purification

Following synthesis completion, the peptide was cleaved from the resin using 95% cleavage solution (TFA:ethanedithiol:triisopropylsilane:water = 95:2:2:1), and the crude peptide was obtained via centrifugal precipitation with ice-cold diethyl ether.

(3) Quality Confirmation

Analysis by UPLC-ESI-Q-TOF-MS confirmed that the synthetic product had a mass-to-charge ratio of 856.4322 Da and a retention time of 34.8 min. Sequence analysis using Mascot software confirmed the sequence as Glu-Pro-Gly-Ile-Val-Asn-Leu-Asp (EPGIVNLD), completely consistent with the target sequence.

The successful establishment of this synthesis method lays the material foundation for the large-scale preparation of peptide EPGIVNLD and subsequent functional studies.

2. Embodiment 2: Anti-inflammatory Activity Experiments of the Bioactive Peptide

This embodiment systematically validated the anti-inflammatory activity of peptide EPGIVNLD through two classical in vitro immunological experiments.

(1) Macrophage Cytokine Secretion Assay (ELISA Method)

Experimental Method and Objective: Enzyme-linked immunosorbent assay (ELISA) was used to detect the effects of different concentrations of peptide EPGIVNLD on cytokine secretion by mouse peritoneal macrophages, evaluating its immunomodulatory activity.

Key Experimental Results:

• TNF-alpha (tumor necrosis factor-alpha): At concentrations of 0.2 mg/mL and above, a statistically significant difference (P < 0.01) was observed compared with the control group, indicating that peptide EPGIVNLD could significantly promote macrophage secretion of TNF-alpha at these concentrations.
• IL-1beta (interleukin-1beta): At concentrations of 0.2 mg/mL and above, a statistically significant difference (P < 0.01) was observed compared with the control group, indicating that peptide EPGIVNLD could significantly promote macrophage secretion of IL-1beta at these concentrations.
• IL-6 (interleukin-6): At a concentration of 0.5 mg/mL, a statistically significant difference (P < 0.01) was observed compared with the control group, indicating that peptide EPGIVNLD could significantly promote macrophage secretion of IL-6 at this concentration.

Scientific Significance: The above results demonstrate that EPGIVNLD at certain concentrations can promote the activation of mouse peritoneal macrophages and the release of TNF-alpha, IL-1beta, and IL-6. These three cytokines are key signaling molecules in the innate immune response, and the elevation of their secretion levels indicates that peptide EPGIVNLD possesses biological activity in activating macrophages and enhancing the body’s immune defense capabilities.

(2) Determination of Macrophage Nitric Oxide Production (Griess Method)

Experimental Method and Objective: The Griess method was used to determine the effect of peptide EPGIVNLD on nitric oxide (NO) production by macrophages. Nitric oxide is an important effector molecule for macrophage bactericidal activity, and its production directly reflects the degree of macrophage activation and anti-infection capability.

Key Experimental Results:

At concentrations of 1 mg/mL and 0.5 mg/mL, nitric oxide production was promoted for macrophages growing under both normal conditions and under LPS (lipopolysaccharide)-induced inflammatory conditions, with statistically significant differences (P < 0.05).

Scientific Significance: Nitric oxide is a key mediator for macrophages to kill pathogenic microorganisms. The ability of peptide EPGIVNLD to significantly promote NO production by macrophages means it can enhance the body’s ability to resist exogenous pathogen infection, thereby reducing the incidence of disease. This discovery provides direct experimental evidence for the development of foods and health products with immune-enhancing functions.

3. Embodiment 3: Anti-aging Activity Experiments of the Bioactive Peptide

This embodiment comprehensively evaluated the anti-aging activity of peptide EPGIVNLD through three in vivo animal experiments. Experiments employed a D-galactose (D-gal)-induced aging mouse model, with peptide EPGIVNLD administered via intragastric gavage, systematically investigating its effects on spleen tissue structure, in vivo antioxidant levels, and serum immune cytokines.

(1) Experiment on the Effect on Spleen Tissue Structure

Experimental Method and Objective: The spleen is an important immune organ, and changes in its tissue structure are one of the important hallmarks of organismal aging. This experiment evaluated the protective effect of peptide EPGIVNLD on spleen tissue structure in aging mice through histopathological observation.

Key Experimental Results:

Compared with the animal model group, the spleen tissue of mice in the peptide gavage group showed less severe white pulp atrophy, and the boundary between red pulp and white pulp was relatively well-defined.

Scientific Significance: Spleen white pulp is the region where lymphocytes aggregate, and its atrophy is an important manifestation of declining immune function during aging. The ability of peptide EPGIVNLD to alleviate the degree of white pulp atrophy and maintain normal spleen tissue structure demonstrates that the bioactive peptide EPGIVNLD of the present invention exerts a certain protective effect against spleen aging and atrophy caused by adverse factor stimulation in animals. This result provides direct histological evidence for the anti-aging efficacy of the peptide.

(2) Experiment on the Effect on Organ Antioxidant Levels

Experimental Method and Objective: Oxidative stress is one of the core mechanisms of organismal aging. This experiment evaluated the regulatory effect of peptide EPGIVNLD on the antioxidant system by measuring three key antioxidant indicators—superoxide dismutase (SOD), malondialdehyde (MDA), and total antioxidant capacity (T-AOC)—in mouse liver and kidney.

Key Experimental Results:

• SOD (superoxide dismutase) content: The SOD content in the liver and kidney of mice in the peptide gavage group showed a significant increase (P < 0.01). SOD is the first line of defense against superoxide anion radicals in the body, and the elevation of its activity indicates that the body’s antioxidant defense capability has been significantly enhanced.
• MDA (malondialdehyde) content: The MDA content in the liver of mice in both peptide gavage groups showed a statistically significant difference (P < 0.01) compared with the model group. MDA is the end product of lipid peroxidation, and the decrease in its content indicates that the degree of oxidative damage in the body has been alleviated.
• T-AOC (total antioxidant capacity): Both the high-dose and low-dose peptide gavage groups showed a statistically significant difference (P < 0.05) compared with the model group. T-AOC reflects the comprehensive level of the body’s antioxidant system, and its elevation indicates that the overall antioxidant capacity of the body has been improved.

Scientific Significance: The significant improvement in the above three antioxidant indicators confirms at the molecular level that peptide EPGIVNLD can enhance the activity of the in vivo anti-peroxidation enzyme system, strengthen the body’s ability to resist exogenous stimuli, and has positive significance in delaying the aging process.

(3) Experiment on the Effect on Serum Immune Cytokines

Experimental Method and Objective: By detecting the levels of inflammatory cytokines in serum, this experiment evaluated the regulatory effect of peptide EPGIVNLD on the body’s inflammatory state, further elucidating the molecular mechanism of its anti-aging activity.

Key Experimental Results:

The serum inflammatory cytokine levels of IL-6 and TNF-alpha in the peptide gavage group were both lower than those in the animal model group.

Multi-dimensional Scientific Interpretation:

• From the oxidative damage perspective: The oxidative damage caused by free radical attack and accumulation of peroxidation products in mice may have been inhibited to a certain extent;
• From the inflammatory perspective: The inflammation caused by oxidation in mice has been effectively suppressed;
• From the aging perspective: The series of age-related diseases caused by long-term D-gal injection-induced aging in mice may potentially be controlled.

Scientific Significance: Chronic low-grade inflammation is an important hallmark of aging, referred to as “inflammaging.” IL-6 and TNF-alpha are core representatives of pro-inflammatory cytokines, and elevated levels of these factors are closely associated with various age-related diseases. The ability of peptide EPGIVNLD to significantly reduce the levels of these two pro-inflammatory factors suggests that it may exert anti-aging effects by suppressing chronic inflammatory responses. The discovery of this mechanism provides an important theoretical basis for the functional application of the peptide.

IV. Summary of Beneficial Effects

The bioactive peptide EPGIVNLD of the present invention possesses favorable anti-inflammatory and anti-aging activities, with its beneficial effects primarily manifested in the following two major aspects:

1. Anti-inflammatory Activity

On the one hand, the bioactive peptide EPGIVNLD of the present invention can promote cytokine secretion by macrophages, increase nitric oxide production by macrophages, enhance the body’s ability to resist exogenous pathogen infection, and reduce the incidence of disease.

In vitro experimental results showed that peptide EPGIVNLD, at concentrations in the range of 0.2–1 mg/mL, could significantly promote macrophage secretion of key cytokines including TNF-alpha, IL-1beta, and IL-6, and significantly increase nitric oxide production, with all effects achieving statistical significance.

2. Anti-aging Activity

On the other hand, the bioactive peptide EPGIVNLD of the present invention can enhance the activity of the in vivo anti-peroxidation enzyme system, strengthen the body’s ability to resist exogenous stimuli, thereby reducing the probability of aging, senescence, and illness.

In vivo experimental results showed that peptide EPGIVNLD could protect spleen tissue structure from atrophy damage caused by aging, significantly increase SOD content and decrease MDA content in the liver and kidney, elevate T-AOC levels, while simultaneously reducing the levels of pro-inflammatory factors IL-6 and TNF-alpha in serum, exerting anti-aging effects from multiple dimensions including antioxidation, anti-inflammation, and tissue protection.

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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