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

A peptide preparation technology of Kangping Bio has been granted an invention patent by the China National Intellectual Property Administration, with patent number ZL201810716089.X and patent publication date of May 12, 2020. To date, Kangping Bio has cumulatively obtained 17 authorized technical patents, with its core technology barriers continuing to strengthen.

I. Technical Field and Background

1. Technical Field

This invention relates to the field of proteins, and more particularly to a bioactive peptide PLLPQSL, as well as its preparation method and applications. This invention identifies a bioactive peptide with significant antioxidant activity and anti-aging activity from Lactobacillus helveticus cellular proteins, and systematically validates its biological efficacy through in vitro and in vivo experiments.

2. Background Art

Oxidation reactions and oxidative metabolism are of vital importance to both food and the human body. Free radicals and reactive oxygen species trigger a series of oxidative reactions. When excessive free radicals are formed, they exceed the protective capacity of protective enzymes such as superoxide dismutase and catalase, thereby leading to a series of side effects including lipid oxidation and apoptosis.

In the field of antioxidant research, some early synthetic antioxidants such as butylated hydroxyanisole (BHA) and 2,6-di-tert-butyl-4-methylphenol (BHT) were applied in food as lipid antioxidants. However, these synthetic additives pose potential risks to the human body, and their long-term safety has drawn considerable attention and scrutiny. It is against this backdrop that, in the course of natural antioxidant research, antioxidant peptides derived from food proteins have emerged as a major research focus. The discovery of bioactive peptides with antioxidant activity from natural protein resources has become an important research direction for replacing synthetic antioxidants, holding significant scientific importance and application prospects.

II. Overview of the Invention Content

1. Discovery and Identification of the Bioactive Peptide

This invention provides a bioactive peptide PLLPQSL, whose amino acid sequence is Pro-Leu-Leu-Pro-Gln-Ser-Leu, as shown in SEQ ID NO: 1. This peptide is derived from Lactobacillus helveticus cellular proteins, specifically from the LBH_0727|m.681 protein, and constitutes amino acid residues 27 to 33 of this protein.

Research has revealed that the said bioactive peptide possesses antioxidant function and anti-aging function, laying a theoretical foundation for the application of this peptide in the fields of functional foods and biomedicine.

2. Encoding Nucleotide Fragment

This invention also provides a nucleotide fragment encoding the said bioactive peptide PLLPQSL, with the sequence: 5’-cac ttt tgc cac aaa gct tga-3’, as shown in SEQ ID NO: 2. This nucleotide fragment provides a critical technical basis for the subsequent preparation of this peptide through genetic engineering methods.

3. Preparation Methods

This invention provides multiple preparation methods for the said bioactive peptide PLLPQSL, including: artificial synthesis through genetic engineering methods; direct obtainment from Lactobacillus helveticus cells through cell disruption followed by isolation and purification; and direct preparation through chemical synthesis. The availability of multiple preparation pathways provides flexible technical options for the large-scale production of this peptide.

III. Specific Embodiments

1. Embodiment 1: Artificial Synthesis of Bioactive Peptide PLLPQSL

This patent employs solid-phase peptide synthesis (SPPS) for the artificial synthesis of the bioactive peptide. The specific synthesis process is as follows:

The synthesis system uses RINK resin (substitution degree: 0.3 mmol/g), with amino acids Pro, Leu, Leu, Pro, Gln, Ser, and Leu sequentially coupled using the Fmoc protecting group strategy. The condensation system uses HOBT/DIC.

Upon completion of synthesis, quality confirmation was performed by UPLC-ESI-Q-TOF-MS analysis. The analytical results showed: mass-to-charge ratio of 767.4723 Da, and retention time 66.5 min. Sequence confirmation by Mascot software analysis identified the sequence as Pro-Leu-Leu-Pro-Gln-Ser-Leu（PLLPQSL）. The mass spectrometry and chromatography data of the synthetic product were in complete agreement with the theoretical values of the target peptide, demonstrating the success of the synthesis process.

2. Embodiment 2: Antioxidant Activity Assay of Bioactive Peptide

This patent systematically evaluated the antioxidant activity of peptide PLLPQSL through two classic in vitro antioxidant evaluation methods — the DPPH free radical scavenging assay and the FRAP total antioxidant capacity assay.

(1) DPPH Radical Scavenging Assay for In Vitro Antioxidant Activity

The DPPH (1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay is one of the classic methods for evaluating the antioxidant activity of compounds. DPPH free radicals appear purple in solution; when scavenged by antioxidants, the solution color lightens and the absorbance at a specific wavelength decreases. The free radical scavenging rate can be calculated by measuring the change in absorbance.

Experimental Results: The peptide PLLPQSL exhibited a [DPPH·] free radical scavenging rate of 31.44%, and as the concentration of PLLPQSL decreased, the free radical scavenging capacity diminished. This result indicates that peptide PLLPQSL possesses a certain scavenging capacity against DPPH free radicals, and its scavenging effect demonstrates a concentration-dependent characteristic — i.e., the higher the concentration, the stronger the scavenging capacity.

(2) FRAP Assay for In Vitro Antioxidant Capacity

FRAP (Ferric Reducing Antioxidant Power) is another widely applied method for evaluating antioxidant activity. It assesses the total antioxidant level by measuring the ability of a sample to reduce ferric iron to ferrous iron.

Experimental Results: At a concentration of 4 mg/mL, the total antioxidant level of peptide PLLPQSL reached 0.0246 mmol/g. This experimental result demonstrates that the total antioxidant capacity of bioactive peptide PLLPQSL was higher than that of phytic acid, which has weak antioxidant activity, at the same concentration, with a significant (p > 0.05) difference. Therefore, the bioactive peptide PLLPQSL of this invention can be determined to have significant antioxidant capacity.

The results from the above two in vitro antioxidant experiments indicate that peptide PLLPQSL demonstrated favorable biological activity in both free radical scavenging and total antioxidant level enhancement, providing solid experimental evidence for its application as an antioxidant functional ingredient.

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

To further validate the biological efficacy of peptide PLLPQSL, this patent designed and conducted a series of in vivo anti-aging activity experiments, systematically evaluating its effects from three dimensions: spleen tissue structure protection, organ antioxidant level enhancement, and serum immune cytokine regulation.

(1) Effect on Spleen Tissue Structure In Vivo

The spleen is an important immune organ of the body, and changes in its tissue structure can reflect the organism’s aging state. During the aging process, the spleen commonly exhibits pathological changes such as white pulp atrophy and blurred boundaries between red and white pulp.

Experimental Results: Compared with the animal model group, mice in the peptide gavage group showed milder white pulp atrophy and more distinct boundaries between red pulp and white pulp. This result demonstrates that the bioactive peptide PLLPQSL of this invention exerts a certain protective effect against spleen aging and atrophy caused by adverse factor stimulation. Peptide PLLPQSL can effectively alleviate aging-related pathological changes in spleen tissue, maintain the normal tissue structure of the spleen, and thereby sustain the body’s immune function to a certain extent.

(2) Effect on Organ Antioxidant Levels In Vivo

Superoxide dismutase (SOD) and malondialdehyde (MDA) are two key indicators for evaluating the body’s antioxidant status. SOD is an important antioxidant enzyme in the body that catalyzes the dismutation reaction of superoxide anion radicals, protecting cells from oxidative damage. MDA is the end product of lipid peroxidation; its content directly reflects the degree of lipid peroxidation and the severity of oxidative damage in the body.

SOD Content: Compared with the animal model group, SOD levels in the liver and kidney of mice in the peptide gavage group showed a significant increase (P < 0.01). The significant elevation in SOD content indicates that peptide PLLPQSL can effectively enhance the antioxidant enzyme activity of the body’s major organs and strengthen the function of the endogenous antioxidant defense system.

MDA Content: Compared with the animal model group, MDA content in the liver of mice in both peptide gavage groups showed a significant difference (P < 0.01). The significant reduction in MDA content indicates that peptide PLLPQSL can effectively inhibit lipid peroxidation reactions in the body and alleviate the degree of oxidative damage.

The above organ antioxidant level experimental results demonstrate that peptide PLLPQSL exerts anti-aging effects at two levels — by enhancing antioxidant enzyme activity and reducing peroxidation products — and exhibits significant in vivo antioxidant and anti-aging biological effects.

(3) Effect on Serum Immune Cytokines

The aging process is often accompanied by a state of chronic low-grade inflammation. Elevated levels of inflammatory cytokines in serum are an important hallmark of aging-related inflammation. Interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are two important pro-inflammatory cytokines, and changes in their levels can reflect the body’s inflammatory and aging status.

Experimental Results: The serum secretion levels of inflammatory cytokines IL-6 and TNF-alpha in mice of the peptide gavage group were lower than those of the animal model group.

This result carries multiple important biological implications. From the perspective of oxidative damage, oxidative damage in mice caused by free radical attack and peroxidation product accumulation may be inhibited to a certain degree. From the perspective of inflammation, inflammation induced by oxidation in mice was effectively suppressed. From the perspective of aging, age-related diseases caused by chronic D-gal injection-induced senescence in mice may be brought under control.

Taking together the results of the three in vivo experiments in Embodiment 3, it can be seen that peptide PLLPQSL demonstrates favorable anti-aging activity in terms of spleen tissue structure protection, organ antioxidant level enhancement, and serum immune cytokine regulation, fully confirming its potential and value as an anti-aging functional ingredient.

IV. Beneficial Effects

The bioactive peptide PLLPQSL of this invention exhibits favorable antioxidant activity and anti-aging activity, which are mainly manifested in the following two aspects:

On one hand, the bioactive peptide PLLPQSL of this invention possesses favorable antioxidant activity, capable of scavenging free radicals in the body and improving the quality of life. Through DPPH free radical scavenging experiments and FRAP total antioxidant capacity assays, it has been confirmed that peptide PLLPQSL can effectively scavenge free radicals and enhance total antioxidant levels; its antioxidant capacity is higher than that of phytic acid at the same concentration.

On the other hand, the bioactive peptide PLLPQSL of this invention can enhance the activity of anti-peroxidation enzyme systems in the body, strengthen the body’s ability to resist exogenous stimuli, and thereby reduce the probability of aging, senescence, and illness. Through in vivo anti-aging experiments, it has been confirmed that peptide PLLPQSL can effectively protect spleen tissue structure, increase SOD content in the liver and kidney, decrease MDA content in the liver, and effectively inhibit the secretion levels of pro-inflammatory cytokines IL-6 and TNF-alpha in serum.

In summary, through systematic validation by in vitro antioxidant experiments and in vivo anti-aging experiments, peptide PLLPQSL exhibits favorable antioxidant biological activity and anti-aging activity. The bioactive peptide PLLPQSL of this invention is derived from amino acid residues 27 to 33 of the Lactobacillus helveticus cellular protein LBH_0727|m.681, with the amino acid sequence Pro-Leu-Leu-Pro-Gln-Ser-Leu, and can be prepared through chemical synthesis or genetic engineering methods. This peptide holds broad application prospects in the preparation of foods, health products, pharmaceuticals, or cosmetics with antioxidant and anti-aging functions, and is of great significance for the development of antioxidant and anti-aging foods, health products, and pharmaceuticals.

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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