Lactococcus lactis expression system based on bglx mutant and its application in development of health care and skin care products

Abstract

The application discloses a lactic acid streptococcus expression system based on a BglX mutant and application thereof in development of health products and skin care products, and belongs to the technical field of bioengineering and food health. The application provides a glycosidase mutant, which can efficiently convert ginsenosides and improve the content of rare saponins in a fermentation product. The application also uses a product of fermentation of ginseng extract by a recombinant bacterium expressing the glycosidase mutant to prepare a child skin care product, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a BglX mutant-based Lactococcus lactis expression system and its application in the development of health products and skin care products, belonging to the fields of bioengineering and food health technology. Background Technology

[0002] Ginseng ( Panax ginseng As a traditional medicinal plant with a long history, ginseng occupies an important position in the medical and health care fields due to its rich bioactive components. Its main active substance—ginsenosides—has been proven to have various physiological functions beneficial to human health, such as enhancing immune defense, anti-fatigue, regulating the cardiovascular system, and delaying aging. Among them, ginsenoside CK is a class of secondary metabolites with a unique structure, exhibiting significant pharmacological potential, including anti-tumor, anti-inflammatory, and neuroprotective effects. However, due to the extremely low content of ginsenoside CK in natural ginseng roots and extracts, its industrial preparation and pharmaceutical development have been severely limited, urgently requiring efficient and safe biotransformation technologies to increase its yield.

[0003] Traditional methods for extracting active ingredients from ginseng, such as decoction and alcohol extraction, while simple and easy to operate, often suffer from low extraction efficiency and limited content of target components, making them unsuitable for industrial production. With the development of biotechnology, methods to increase the content of target components in plant extracts through microbial fermentation have attracted widespread interest from researchers. In particular, the use of specific microbial strains or engineered mutants of transcriptional regulatory proteins can specifically increase the content of one or more active ingredients in plant extracts, demonstrating unique advantages. BglX is a type of glycosidic bond hydrolase that catalyzes the deglycosylation of various glycosides. Natural BglX has low substrate specificity and limited hydrolysis efficiency for ginsenosides, limiting its application in the preparation of rare saponins.

[0004] In recent years, *Lactococcus lactis* has been widely used in various fields due to its high safety, edibility, and excellent fermentation performance. As a probiotic, *Lactobacillus* is not only widely used in the food industry but has also shown unique potential in the field of bioengineering. Especially in research on increasing the content of active ingredients in plant extracts, *Lactococcus lactis* fermentation technology has significant advantages. Through its own metabolic activities, *Lactococcus lactis* can efficiently convert common saponins in ginseng into rare saponins. This bioconversion is not only highly efficient but also ensures the safety and environmental friendliness of the conversion process. However, the conversion rate of saponins from ginseng by *Lactococcus lactis* in current technologies is less than 30%, and there is still room for improvement. If a highly efficient BglX mutant can be obtained through molecular modification and a stable expression system can be constructed in *Lactococcus lactis*, efficient bioconversion of ginseng saponins can be achieved, providing a new technological path for the development of novel ginseng functional foods and health products. Therefore, it is urgent to develop a new technical means to enhance the content of rare saponins in ginseng extracts, with the aim of also extending to the extraction and optimization of other traditional Chinese medicine components. Summary of the Invention

[0005] This invention provides a Lactococcus lactis expression system based on the BglX mutant. By performing site-directed mutagenesis on key catalytic residues of BglX, its desugaring efficiency for substrates such as ginsenoside Rb1 is improved, thereby promoting the generation of rare ginsenoside CK.

[0006] This invention also provides the application of this expression system in fermented ginseng extract, realizing a green, edible, solvent-free rare saponin biotransformation technology for the development of novel ginseng health products and children's skin care products.

[0007] The technical solution of the present invention includes: The present invention provides a glycosidase mutant BglX-M, whose amino acid sequence has undergone directed mutations at multiple key sites, including positions 88, 157, 163, 178 and 215, compared with the parent glycosidase. The amino acid sequence of the parent is shown in SEQ ID NO: 3.

[0008] In one embodiment, the mutant is one in which proline at position 88 is mutated to leucine, glycine at position 157 is mutated to isoleucine, alanine at position 163 is mutated to isoleucine, histidine at position 178 is mutated to proline, and histidine at position 215 is mutated to glycine.

[0009] In one embodiment, the amino acid sequence of the mutant is shown in SEQ ID NO.1.

[0010] The present invention also provides a gene encoding the mutant.

[0011] The present invention also provides an expression vector carrying the gene.

[0012] The present invention also provides genetically engineered bacteria that express the mutant.

[0013] In one embodiment, the genetically engineered bacteria use Lactococcus lactis NZ3900 as the host and pNZ8048 as the vector to express the mutant.

[0014] The present invention also provides a method for preparing CK, wherein the genetically engineered bacteria are inoculated into a fermentation system containing ginseng extract for fermentation to prepare ginsenoside CK.

[0015] In one embodiment, the ginseng extract is prepared by mixing dried and ground ginseng powder with sterile water at a ratio of 1:5 to 1:20, applying 50-80Hz ultrasonic treatment for 30 minutes, followed by filtration, evaporation and drying to obtain the ginseng extract.

[0016] In one embodiment, the genetically engineered bacteria are inoculated into the fermentation system to achieve an OD ≥ 0.2 after inoculation, and fermentation is carried out at 37°C for 48 hours.

[0017] The present invention also provides a method for increasing the CK content in ginseng extract, characterized in that the genetically engineered bacteria are used as a fermentation agent to ferment and transform the ginseng extract, thereby increasing the CK content.

[0018] The present invention also provides the application of the mutant, the gene, the expression vector, the genetically engineered bacteria, or the method in the preparation of ginseng health products or children's skin care products.

[0019] Beneficial effects: (1) Improved conversion efficiency: The Proteus glycosidase mutant obtained by screening in this invention showed a high efficiency of 80% in the conversion of CK in the extract, which significantly increased the content of CK.

[0020] (2) Safety and environmental friendliness: This invention uses a biological fermentation method, which is safer and more environmentally friendly than traditional methods such as chemical synthesis. Using genetically engineered Lactococcus lactis for fermentation not only ensures the green and environmentally friendly nature of the fermentation process, but also avoids chemical residues and ensures the safety of the final product.

[0021] (3) Broad application prospects: By significantly increasing the content of rare saponin CK in ginseng water extract, a new way is provided for the high-value utilization of ginseng and other Chinese herbal medicine ingredients. It can be used not only to develop new ginseng health care products, but also to develop new children's skin care products, and has broad market prospects. Attached Figure Description

[0022] Figure 1Map of the pNZ8048-BglX-M expression plasmid.

[0023] Figure 2 The expression of the BglX-M mutant protein in Lactococcus lactis.

[0024] Figure 3 The ability of wild-type BglX and mutant BglX-M to catalyze the generation of CK from Rb1.

[0025] Figure 4 The study investigated the changes in Rb1 and CK levels in ginseng extract before and after fermentation using Lactococcus lactis expressing the BglX-M mutant protein. Detailed Implementation

[0026] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0027] Test method: Detection of active ingredients (Rb1 and CK): The sample was centrifuged, and the supernatant was filtered through a 0.22 μM filter membrane. Detection conditions: Shimadzu LC-20AT high-performance liquid chromatography system, SPD-M20A PAD detector, Waters C18 column (250 x 4.6 mm, 5 μM), column temperature 35℃. Mobile phase A was water, and mobile phase B was acetonitrile. The linear gradient of mobile phase B (0...) 20 minutes, 30 40%; 20 35 minutes, 40 100%; 35 40 minutes, 100%; 40 Separation was performed at a flow rate of 1 mL / min for 45 minutes (30%). The injection volume was 10 μL, and the detection wavelength was 203 nm.

[0028] Enzyme activity is defined as the amount of enzyme that produces 1 μmol of Compound K (CK) per minute under standard conditions (37℃, pH 6.5).

[0029] The raw materials used in the embodiments of this invention are as follows: 1. The glycosidase gene used was derived from the transcriptome of Proteus mirabilis. After reverse transcription, cDNA was obtained and used as a template for gene cloning. The expression vector was the nisin-inducible expression plasmid pNZ8048, which was self-constructed.

[0030] 2. The culture media used for microbial culture are as follows: MRS medium: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g sodium citrate, 2.0 g K₂HPO₄, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 1.0 mL Tween 80, final pH adjusted to 6.5; used for the growth and fermentation of Lactococcus lactis. Nisin was used as an expression inducer, with a final concentration controlled at 10 ng / mL.

[0031] 3. The ginseng extract, the fermentation substrate, was obtained in-house. Ginseng was ground into powder and sieved to obtain a homogeneous and fine powder. 200 grams of this powder was mixed with 2 liters of sterile water and treated using ultrasonic disruption technology for 30 minutes. The mixture was then filtered and subjected to evaporation and drying processes to obtain a pure ginseng extract. The obtained ginseng extract was dissolved in 1 liter of distilled water and mixed thoroughly to prepare a culture medium. This culture medium was then pasteurized at 80°C for 30 minutes to ensure complete sterilization.

[0032] 4. Rb1 and CK used in the in vitro catalytic reaction and activity verification were purchased from Sigma-Aldrich and had a purity of ≥99%.

[0033] 5. The glycerin, petrolatum, cetyl alcohol, propylene glycol, glyceryl stearate, sodium lauryl sulfate, stearyl alcohol, methyl paraben, propyl paraben, butyl paraben, phenoxyethanol and other ingredients used in the children's skin care product formula are all purchased from Sinopharm Group and have a purity of ≥98%.

[0034] 6. All water used in the experiments was deionized water, all reagents were of analytical grade or higher, and all consumables were treated to be free of DNase and RNase contamination to ensure the accuracy and repeatability of the experiments.

[0035] Example 1: Construction of Lactococcus lactis expressing the BglX-M mutant Using the pNZ8048 vector as a template, PCR amplification was performed using 5'-AAGCTTTCTTTGAACCAAAA-3' (upstream primer) and 5'-GGTGAGTGCCTCCTTATAAT-3' (downstream primer). Using the BglX-M gene fragment as a template, the BglX-M gene (SEQ ID NO: 2) was amplified using 5'-ATTATAAGGAGGCACTCACCATGAGCCAGGCGTTCGAATT-3' (upstream primer) and 5'-TTTTGGTTCAAAGAAAGCTTCTACCCGGCGAGGACGCGAC-3' (downstream primer). PCR system: 0.2 μM primers each, 0.2 mM dNTPs, 10 ng template, 5 μL 2× Buffer, 0.5 μL Hieff, and deionized water to bring the volume to 100 μL. The PCR reaction conditions were: 94 ℃ for 20 s, 94 ℃ for 30 s, 55 ℃ for 30 s, 72 ℃ for 2 min, and 4 ℃ for 5 min, with 30 cycles of steps 2-5 above. The obtained PCR products were ligated using a Gibson Assembly kit (Anhui General Biotechnology). The ligation products were transformed into *Lactococcus lactis* NZ3900, and the correct clones were selected through sequencing and named... L. plantarum -BglX-M. The plasmid pNZ8048-BglX-M was obtained by extracting the plasmid from the recombinant bacteria. The plasmid map is shown below. Figure 1 .

[0036] Example 2: Wild-type BglX and mutant BglX-M catalyze the generation of CK from Rb1 right L. plantarum -BglX-M was cultured and induced to express, and NZ3900 was inoculated into GM17 medium containing 10 μg / mL chloramphenicol and cultured at 30°C until OD. 600 Add 5 ng / mL nisin to a temperature of 0.4–0.6 and continue culturing for 3–5 h. Expression vector construction was performed as described in Example 1. Plasmids pNZ8048-BglX-WT and pNZ8048-BglX-M, containing the wild-type BglX and mutant BglX-M genes respectively, were transformed into *Lactococcus lactis* NZ3900 to construct recombinant strains NZ3900-BglX-WT and NZ3900-BglX-M, respectively.

[0037] The above-mentioned bacterial strains were inoculated into MRS liquid medium containing chloramphenicol (5 μg / mL) and incubated statically at 37°C for 12 hours. Once the cell density reached OD600 = 0.6, nisin (final concentration 10 ng / mL) was added to induce expression of the target enzyme, and the culture was continued for another 24 hours. After expression, the bacterial cells were collected, sonicated with PBS buffer (pH 6.8), and cell debris was removed by centrifugation. The supernatant was the crude enzyme solution. The supernatant was purified using a Ni-NTA affinity chromatography column. Purification conditions: Equilibration buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl; Elution buffer: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 250 mM imidazole; The eluted fraction was analyzed by SDS-PAGE, yielding the target protein band. The expression of the BglX-M mutant protein is shown below. Figure 2 As shown.

[0038] Example 3: Wild-type BglX and mutant BglX-M catalyze the generation of CK from Rb1 Glycosidase BglX and mutant BglX-M were prepared according to the method in Example 2. The following reaction system was prepared: total volume 1.0 mL; final Rb1 concentration: 10 mM; enzyme solution prepared in Example 2: 50 μL (concentration 0.5 U / mL); PBS buffer was added to make up the volume, pH 6.5. The reaction system was reacted at 37°C for 60 minutes. After termination of the reaction, the pH was adjusted to 3.0 with acetic acid. The precipitate was removed by centrifugation, and the CK content in the supernatant was detected by HPLC. The results are as follows: Figure 3 As shown, the CK content of the mutant catalytic reaction reached 0.90 mM, which is significantly higher than that of the wild type (0.25 mM).

[0039] Example 4: Lactococcus lactis fermenting ginseng extract expressing the BglX-M mutant increases CK content. Ginseng was ground into powder and sieved to obtain a homogeneous and fine powder. 200g of this powder was mixed with 2L of sterile water and treated using ultrasonic disruption technology for 30 minutes. The mixture was then filtered and subjected to evaporation and drying processes to obtain a pure ginseng extract. The obtained ginseng extract was dissolved in 1L of distilled water and mixed thoroughly to prepare GM17 medium containing 10 g / L (final concentration). This medium was pasteurized at 80°C for 30 minutes to ensure complete sterilization. The *Lactococcus lactis* expressing the BglX-M mutant constructed in Example 2 was inoculated at 10% of the medium volume, ensuring the OD600 of the inoculated *Lactococcus lactis* reached 0.2. Fermentation was carried out at 37°C for 48 hours. After fermentation, the filtrate was separated by filtration. This filtrate contained ginseng extract transformed by *Lactobacillus* fermentation and was rich in CK. The contents of Rb1 and CK before and after fermentation of the ginseng extract by *Lactococcus lactis* are as follows: Figure 4 As shown, the Rb1 content after fermentation was 0.5 mM, and the CK content was 4.5 mM.

[0040] Example 5: Preparation of children's skin care products from the obtained CK-rich fermentation filtrate In this embodiment, the preparation method of the children's skin care product is as follows: Petrolatum, cetyl alcohol, stearyl alcohol, and glyceryl stearate are heated to 70-75°C; deionized water, glycerin, propylene glycol, and soluble components are heated to 70-75°C; the oil phase is slowly added to the aqueous phase, and vigorous stirring / emulsification (using a stirrer or high-speed disperser) is performed until the emulsion is uniform; when cooled to <40°C, heat-sensitive active ingredients (ginseng extract, fragrance, preservatives, stabilizers, etc.) are added, stirred evenly, and allowed to cool to room temperature. The content of each component is as follows: water 60-70%, petrolatum 5-10%, glycerin 4-6%, glyceryl stearate 3-4%, cetyl alcohol 1-3%, stearyl alcohol 1-2%, propylene glycol 1-3%, ginseng fermentation extract 0.5-1.0%, preservatives (by system) 0.7-1.0%, and the remainder being pH adjusters and antioxidants.

[0041] Example 6: Application effect of children's skin care products prepared from fermentation filtrate rich in CK The children's skincare product prepared in Example 5 was compared with the "Cetaphil White Jar" product in an efficacy comparison test. The specific steps were as follows: Ten healthy volunteers, aged 18–27 years, with balanced gender, were recruited. Those with known allergies to any ingredient in the formula, recent use of immunosuppressive / anti-allergy medications, skin diseases, or who were pregnant or breastfeeding were excluded. For comparative evaluation, a parallel group design was used: Group A (experimental product): 5 participants; Group B (Cetaphil white jar, control product): 5 participants. The testing apparatus used was a breathable patch with a diameter of 8 mm → 10 mm. 20–50 μL (or 0.02–0.05 mL) of product was applied to filter paper or the inner ring of the patch on each patch (ensuring the amount was consistent with the reference). The product was used directly in its original packaging (undiluted), and the control product was treated in the same way.

[0042] Induction period: Apply the patches to different sites on the same side of the back or upper arm. Remove the patches 48 hours after each application, observe for 30 minutes after removal, and record the immediate stimulus response. Patch frequency: 3 times a week (e.g., Monday / Wednesday / Friday), for a total of 9 induction applications over 3 consecutive weeks.

[0043] Rest period: No application for 2 weeks after induction (to observe for latent sensitization).

[0044] Challenge period: Apply the test sample once to another unexposed site on the back for 48 hours. After removal, evaluate the sample and take readings at 24, 48, and 72 hours (e.g., 48h and 72h scores).

[0045] Assessment time points and records: Record the instantaneous stimulus score after each patch removal; each time during the induction period; during the challenge period, focus on the delayed response (sensitive response) at 48 and 72 hours.

[0046] Scoring System: The score for the test sample is calculated as follows: (Number of subjects) × (Number of patches) × (Frequency) = Number of scoring data; The total score is the sum of all the scores.

[0047] Average score = Total score ÷ Number of ratings. The average score is used to indicate the overall intensity of the stimulus.

[0048] The results are shown in Tables 1 and 2. Table 1 divides the stimuli into four levels according to the average score: an average score between 0 and 0.5 is considered no stimulation; 0.5–1.5 is mild stimulation; 1.5–2.5 is moderate stimulation; and 2.5–4.0 is severe stimulation.

[0049] According to the test results in Table 2, the ginseng children's skincare product (Group A) underwent 90 tests, with a total score of 18 points and an average score of 0.2, falling within the "0–0.5" range, and was therefore judged as non-irritating. The Cetaphil large white jar (Group B) was also tested 90 times, with a total score of 54 points and an average score of 0.6, falling within the "0.5–1.5" range, and was classified as slightly irritating. Overall, the ginseng children's skincare product was significantly less irritating than the Cetaphil large white jar.

[0050] Table 1 Stimulus Level Classification

[0051] Table 2 Comparison of the effects of ginseng children's skincare products and Cetaphil's large white jar product.

[0052] Comparative Example 1: Following the method of Example 4, children's skin care products containing corresponding concentrations of Rb1 and CK were prepared. The effects were verified following the methods of Examples 5 and 6. The results showed that the skin care products prepared were slightly less effective than those using fermentation broth, as shown in Table 3.

[0053] Table 3 Comparison of the effects of RB1 and CK skincare products with fermented liquid skincare products

[0054] Comparative Example 2: Following the method of Example 1, mutants as shown in Table 4 were constructed. Following the methods of Examples 2 and 3, different mutants were expressed, and the transformation rate was verified. The results are shown in Table 4.

[0055] Table 4 Mutants and Transformation Rate

[0056] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A glycosidase mutant BglX-M, characterized in that, The amino acid sequence was mutated at one or more sites at positions 88, 157, 163, 178 and 215 compared to the parent shown in SEQ ID NO:

3.

2. The glycosidase mutant BglX-M according to claim 1, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

3. The gene encoding the mutant of claim 1 or 2.

4. An expression vector carrying the gene of claim 3.

5. Genetically engineered bacteria expressing the mutant of claim 1 or 2.

6. Recombinant Lactococcus lactis, characterized in that, Using Lactococcus lactis NZ3900 as the host and pNZ8048 as the vector, the mutant described in claim 1 or 2 is expressed.

7. A method for preparing CK, characterized in that, The recombinant lactococcus as described in claim 6 is fermented in a fermentation system containing ginseng extract to prepare ginsenoside CK.

8. The method according to claim 7, characterized in that, The preparation method of the ginseng extract is as follows: dry and ground ginseng powder is mixed with sterile water at a ratio of 1:5 to 1:20, treated with 50-80Hz ultrasound, and then filtered, evaporated and dried to obtain the ginseng extract.

9. A method for increasing the CK content in ginseng extract, characterized in that, Using the recombinant lactococcus as described in claim 6 as a fermenting agent, ginseng extract is fermented and transformed to increase CK content.

10. The use of the mutant of claim 1 or 2, or the gene of claim 3, or the expression vector of claim 4, or the genetically engineered bacteria of claim 5, or the method of any one of claims 7 to 9 in the preparation of ginseng health products or children's skin care products.

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