Small molecule peptide with antibacterial activity and application of selenium-rich hericium erinaceus in synergistically enhancing antibacterial effect of small molecule peptide

Abstract

This invention belongs to the field of medicinal chemistry and discloses a small molecule peptide with antibacterial activity and its application in synergistic enhancement of the antibacterial activity of selenium-enriched Hericium erinaceus. Specifically, it involves a small molecule peptide with antibacterial activity and provides a synergistic enhancement effect of selenium-enriched Hericium erinaceus powder on the antibacterial activity of the small molecule peptide. This invention identifies the genome of *Acidobacterium* from the NCBI microbial genome database and predicts the structure of non-ribosomal peptides synthesized by its non-ribosomal peptide synthase (NRPS) sequence. Based on the substrate selection of NRPS biosynthetic gene clusters (BGCs), peptides with antibacterial potential are selectively screened. A small molecule peptide, compound one, is obtained through chemical synthesis, and it is found to have an inhibitory effect on *Bacillus subtilis*. Simultaneously, the selenium-enriched Hericium erinaceus powder prepared by this invention through a specific method has a synergistic effect with compound one, enhancing the antibacterial activity of compound one, which has significant value.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry and relates to the application of a small molecule peptide with antibacterial activity and selenium-enriched Hericium erinaceus in synergistically enhancing the antibacterial effect of the small molecule peptide. Background Technology

[0002] Microorganisms, especially bacteria and fungi, play a crucial role in the discovery and development of human medicines and are an important source of many clinical drugs. In recent years, with the development of genomics and bioinformatics technologies, the potential of bacteria as a source of novel drug candidates has received increasing attention. Nonribosomal peptide synthases (NRPSs) are a class of biosynthetic systems mediated by specific nonribosomal polypeptide synthesis proteins, capable of assembling into structurally complex and functionally diverse peptide compounds. Many of these nonribosomal peptides possess significant biological activity and have been developed for clinical treatment. Currently, more than twenty related drugs have been successfully marketed, including antibacterial drugs such as penicillin and vancomycin, antitumor drugs such as bleomycin, and immunosuppressants such as cyclosporine. However, traditional natural product discovery processes typically involve multiple steps, including strain isolation, cultivation under specific conditions, induction of target compound expression, isolation and purification, structural identification, and activity testing—a complex and time-consuming process. Recent studies have shown that bioinformatics methods can effectively overcome these limitations. Software such as AntiSMASH and PRISM can be used to predict NRPS biosynthetic gene clusters in genomic data and infer the structural characteristics of their potential encoded products. Further integration with tools like PharmMapper allows for the screening and evaluation of the potential antibacterial activity and targets of candidate small peptides. This strategy enables the rapid discovery of potential biosynthetic gene clusters from large-scale genomic data, significantly improving the efficiency of novel bioactive molecule discovery and effectively reducing the time and economic costs of experimental screening. Selenium is an important trace mineral essential for maintaining normal bodily functions and effectively enhancing the immune system. Selenium has multiple functions, including cancer prevention, anti-oxidation, scavenging harmful substances, and promoting health. However, the human body cannot synthesize selenium endogenously and must obtain it from external sources. Long-term selenium deficiency can significantly lead to various metabolic diseases, making regular selenium supplementation crucial. Different forms of selenium possess different biological characteristics. Inorganic selenium is poorly absorbed by the body and has high cytotoxicity, making it unsuitable as a dietary supplement. Conversely, organic selenium not only has high absorption and utilization rates and low toxicity but also provides antioxidant protection for cells. Research on how to utilize plants to transform and enrich selenium into organic selenium has become a key research topic in recent years.

[0003] Hericium erinaceus, belonging to the Basidiomycetes family, has been a prized medicinal and edible fungus since ancient times. It is beneficial not only to the five internal organs but also to nourish the body, aid digestion, and possesses high medicinal and nutritional value. Modern medical research has discovered that Hericium erinaceus has antibacterial, anti-inflammatory, anticancer, antioxidant, immune-boosting, and nerve-regulating effects, and has good conditioning effects on duodenal ulcers, gastric ulcers, immune diseases, and neurasthenia. Currently, Hericium erinaceus products such as "Hericium erinaceus biscuits," "Hericium erinaceus rice porridge," and "compound Hericium erinaceus granules" are widely popular in the market.

[0004] Selenium and Hericium erinaceus have similar therapeutic effects in antibacterial and other aspects. There are many studies on selenium and Hericium erinaceus, but there are relatively few studies on selenium-enriched Hericium erinaceus that combines the two. Developing new selenium-enriched Hericium erinaceus production technology and its application research is not only of great significance for developing new organic selenium resources, but also improves the rational utilization of Hericium erinaceus and provides new scientific research directions for both.

[0005] This invention collects the genomes of *Acidobacterium* from the NCBI microbial genome database, uses bioinformatics software to predict the possible molecular structures of non-ribosomal peptides synthesized by NRPS in the genome, screens for potentially active small molecule peptides, and then obtains these small molecule peptides through chemical synthesis. Testing revealed that one of the small molecule peptides had an inhibitory effect on the growth of *Bacillus subtilis*. Subsequently, the small molecule peptide was combined with selenium-enriched *Hericium erinaceus* powder and applied to *Bacillus subtilis* cells, revealing a significant enhancement in the antibacterial activity of the small molecule peptide. Summary of the Invention

[0006] This invention uses software to predict the possible molecular structures of non-ribosomal peptides synthesized by Bacillus subtilis NRPS, and then chemically synthesizes these small peptides. Testing revealed that one of these small peptides (compound one) has an inhibitory effect on the growth of Bacillus subtilis, with the chemical formula C... 37 H 64 N8O 10 (Compound 1), with the following structural formula: The small molecule peptides with antibacterial activity can be used to prepare antibacterial drugs or biopesticides.

[0007] The antibacterial activity of the small molecule peptides includes inhibition of Bacillus subtilis.

[0008] Furthermore, this invention also provides the application of selenium-enriched Hericium erinaceus in the preparation of formulations that synergistically enhance the antibacterial activity of the small molecule peptides.

[0009] According to a specific embodiment of the present invention, a method for preparing selenium-enriched Hericium erinaceus powder that synergistically inhibits the growth of Bacillus subtilis with compound one is provided, comprising the following steps: (1) Inoculate the Hericium erinaceus test tube culture into a shake flask culture medium. The shake flask culture medium consists of: 2-4% glucose, 1-3% starch, 1-2% yeast extract, 0.1-0.2% potassium dihydrogen phosphate, 0.1-0.2% magnesium sulfate, and the remainder is water. The pH value is 5-6. Shake flask culture is carried out at a temperature of 26-28℃ and a rotation speed of 160-200 rpm for 5-7 days.

[0010] (2) Incubate in a fermenter at 26-28℃, with a liquid volume of 65-75% and an aeration rate of 1:0.4-0.6V / V·min for 2-4 days. The composition of the culture medium in the fermenter is the same as that in the shake flask.

[0011] (3) The bacterial culture after step (2) is kept in a homogenizer at a speed of 2000-2500 rpm for 0.5-1.5 minutes.

[0012] (4) Prepare solid cultivation material: 80-83% cottonseed hulls, 15-18% wheat bran, 1-2% urea, 1-2% superphosphate, adjust the pH value to 5-6 with citric acid, and add sodium selenite to form solid cultivation material. Then add water to form solid cultivation medium.

[0013] (5) Inoculate the bacterial solution obtained in step (3) into a solid culture medium, and culture it at a temperature of 24-27℃ and a humidity of 70%-90% for 18-25 days. After ventilation, the fruiting period is 16-21℃, the fruiting humidity is 90%-95%, and the fruiting period is 4-6 days.

[0014] (6) Dry and pulverize the selenium-enriched Hericium erinaceus obtained in step (5) to obtain selenium-enriched Hericium erinaceus powder.

[0015] Preferably, the shake flask culture medium in step (1) consists of: 3% glucose, 2% starch, 1% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and the remainder is water, with a pH of 5.5.

[0016] Preferably, the shaking flask culture temperature in step (1) is 27°C and the rotation speed is 180 rpm.

[0017] Preferably, the fermenter temperature in step (2) is 27°C, the liquid volume is 70%, and the aeration rate is 1:0.5V / V·min.

[0018] Preferably, the homogenizer speed in step (3) is 2200-2300 rpm, and is maintained for 1 minute.

[0019] Preferably, the solid cultivation material formula in step (4) is: 82% cottonseed hulls, 16% wheat bran, 1% urea, 1% superphosphate, and citric acid to adjust the pH value to 5.5.

[0020] Preferably, the sodium selenite content in step (4) is 240 mg / kg to 920 mg / kg.

[0021] Preferably, the cultivation temperature in step (5) is 25℃ and the humidity is 80%-85%. After 20 days of cultivation, the mushrooms are ventilated and the fruiting temperature is 18-20℃, the fruiting humidity is 92%-93%, and the fruiting cycle is 5 days.

[0022] The beneficial effects of the present invention are at least as follows: This invention identifies the genome of *Bacillus subtilis* from the NCBI microbial genome database and predicts the structure of non-ribosomal peptides synthesized by its non-ribosomal peptide synthase (NRPS) sequence. Based on the substrates of the NRPS biosynthetic gene clusters (BGCs), peptides with antibacterial potential are selectively screened. A small molecule peptide, compound one, is identified, which has an inhibitory effect on the growth of *Bacillus subtilis*. Furthermore, when this small molecule peptide is combined with selenium-enriched *Hericium erinaceus* powder and applied to *Bacillus subtilis* cells, the antibacterial activity of the small molecule peptide is significantly enhanced. Attached Figure Description

[0023] Figure 1 This is the positive ion mass spectrum of the antibacterial compound of this invention; Figure 2 This is an HPLC spectrum of the antibacterial compound of the present invention; Figure 3 This is the hydrogen spectrum of the antibacterial compound of this invention. Detailed Implementation

[0024] The present invention will be further described in conjunction with the accompanying drawings and embodiments. The liquid nutrient broth culture medium used in the embodiments comprises: 1% peptone, 1% beef extract, 0.5% NaCl, 1% glucose, and pH 7.0. All percentages are by weight.

[0025] (I) Analysis of compounds and preparation of selenium-enriched Hericium erinaceus The small molecule peptides of the present invention can be directly prepared by all-chemical peptide synthesis technology based on their structural formulas. This embodiment only provides one specific synthesis method for illustration and is not intended to limit it. This embodiment uses the fmoc solid-phase synthesis method, specifically: (1) Resin selection and swelling: Weigh Fmoc-His(Trt)-Wang resin with a degree of substitution of approximately 0.50 mmol / g. Weigh approximately 200 mg of resin according to a synthesis scale of 0.10 mmol and place it in a solid-phase synthesis tube. Add 5 mL of DCM and shake at room temperature for 30 min to swell. After drying, wash three times with DMF, 5 mL each time for 3 min, and then dry for later use.

[0026] (2) Deprotection of Fmoc on resin: Add 4 mL of 20% piperidine / DMF solution to the swollen Fmoc-His(Trt)-Wang resin, shake at room temperature for 10 min, and then dry under vacuum; add 4 mL of fresh 20% piperidine / DMF solution and continue shaking for 15 min to completely remove the Fmoc protecting group. Then wash with DMF 6 times, 4 mL each time for 2 min. Take a small amount of resin for ninhydrin detection; if it turns blue, it indicates that the free amino group is completely exposed.

[0027] (3) Coupling of Fmoc-NMe-Thr(tBu)-OH: Since NMe-Thr is an N-methyl amino acid, its steric hindrance is relatively large, and its coupling efficiency is lower than that of ordinary amino acids. It is advisable to use the HATU / DIPEA system and extend the reaction time. Weigh 0.30 mmol (3.0 eq) of Fmoc-NMe-Thr(tBu)-OH, add 0.30 mmol (3.0 eq) of HATU, dissolve in 3 mL of DMF, add 0.60 mmol (6.0 eq) of DIPEA, and pre-activate at room temperature for 4 min. Add the activated solution to the resin and shake at room temperature for 2 h. After the reaction is complete, dry the resin and wash it 4 times with DMF. Take a small amount of resin for testing. If the coupling is incomplete, the coupling can be repeated once under the above conditions, i.e., double coupling. After coupling is complete, add 4 mL of capping solution (acetic anhydride:DIPEA:DMF = 1:1:8), shake at room temperature for 10 min to block unreacted amino groups, and then wash 4 times with DMF.

[0028] (4) Removal of the Fmoc protecting group from NMe-Thr: Add 4 mL of 20% piperidine / DMF, shake at room temperature for 10 min, and then dry under vacuum; add 4 mL of fresh deprotection solution and shake for 15 min. After drying, wash 6 times with DMF. Since the deprotected product is an N-methyl secondary amine, the ninhydrin reaction may not be obvious. The chloroquinone / acetaldehyde detection method can be used to help determine whether the secondary amine is completely free.

[0029] (5) Coupling of Fmoc-Gly-OH: Gly needs to be coupled to the secondary amine of NMe-Thr. This step is a key difficult coupling step, and the feed equivalent should be increased and the reaction time extended. Weigh 0.50 mmol (5.0 eq) of Fmoc-Gly-OH, add 0.45 mmol (4.5 eq) of HATU, dissolve in 4 mL of DMF, add 1.0 mmol (10 eq) of DIPEA, and pre-activate at room temperature for 3 min. Add the activated solution to the resin and shake at room temperature for 3.5 h. After the reaction is complete, dry the solution and wash with DMF 4 times. Perform secondary coupling: Add the same ratio of Fmoc-Gly-OH / HATU / DIPEA activation solution again and continue the reaction at room temperature for 2 h. After coupling is complete, wash with DMF 4 times and treat with 4 mL of acetic anhydride end-capping solution (acetic anhydride:DIPEA:DMF = 1:1:8) for 10 min, and then wash with DMF 4 times.

[0030] (6) Removal of the Fmoc protecting group from Gly: Add 4 mL of 20% piperidine / DMF, shake at room temperature for 10 min, and then dry under vacuum; add 4 mL of fresh deprotection buffer and shake for 15 min. After drying, wash 6 times with DMF. The ninhydrin test should show a blue color, indicating that the Gly amino group is completely freed.

[0031] (7) Coupling of Fmoc-D-Lys(Boc)-OH: Weigh 0.30 mmol (3.0 eq) of Fmoc-D-Lys(Boc)-OH, 0.30 mmol (3.0 eq) of HBTU, and 0.30 mmol (3.0 eq) of HOBt, dissolve in 3 mL of DMF, add 0.60 mmol (6.0 eq) of DIPEA, and pre-activate at room temperature for 3 min. Add the activated solution to the resin and shake at room temperature for 90 min. After the reaction is complete, dry the resin and wash it 4 times with DMF. Take a small amount of resin for ninhydrin detection. If it still turns blue, the coupling is incomplete and can be repeated once. After coupling, the resin can be treated with acetic anhydride end-capping solution for 10 min, and then washed 4 times with DMF.

[0032] (8) Remove the Fmoc protecting group from D-Lys: Add 4 mL of 20% piperidine / DMF, shake at room temperature for 10 min, and then dry under vacuum; add 4 mL of fresh deprotection buffer and shake for 15 min. After drying, wash 6 times with DMF. Ninhydrin should show a blue color.

[0033] (9) Coupling of Fmoc-Thr(tBu)-OH: Weigh 0.30 mmol (3.0 eq) of Fmoc-Thr(tBu)-OH, 0.30 mmol (3.0 eq) of HBTU, and 0.30 mmol (3.0 eq) of HOBt, dissolve in 3 mL of DMF, add 0.60 mmol (6.0 eq) of DIPEA, and pre-activate at room temperature for 3 min. Add the activated solution to the resin and shake at room temperature for 90 min. After the reaction is complete, dry the solution under vacuum and wash 4 times with DMF. The coupling is confirmed to be complete by ninhydrin detection; if free amino groups are still detected, the coupling is repeated once. After coupling is complete, wash 4 times with DMF.

[0034] (10) Deprotection of the N-terminal Fmoc to obtain free N-terminal Thr: Add 4 mL of 20% piperidine / DMF, shake at room temperature for 10 min, and then dry under vacuum; add 4 mL of fresh deprotection buffer and shake for 15 min. After drying under vacuum, wash with DMF 5 times, DCM 3 times, methanol 2 times, and DCM 2 times in sequence. Finally, vacuum dry the resin to obtain resin loaded with the target peptide chain.

[0035] (11) Myr acylation of N-terminal Thr: After removing the Fmoc protecting group from the N-terminal Thr and washing it clean, a free N-terminal primary amine is obtained on the resin. At this time, myristication (Myr-acylation) is performed. Weigh 0.30 mmol (3.0 eq) of myristic acid (Myr-OH), add 0.30 mmol (3.0 eq) of HATU, dissolve in DMF / DCM = 1:1, 3 mL, then add 0.60 mmol (6.0 eq) of DIPEA, and pre-activate at room temperature for 4 min. Add the activated solution to the resin and shake at room temperature for 3 h. After the reaction is complete, dry the resin and wash it 4 times with DMF. Take a small amount of resin for ninhydrin detection. If it still turns blue, it indicates that the acylation is incomplete, and the coupling can be repeated once under the above conditions. After the reaction is complete, a capping solution (acetic anhydride:DIPEA:DMF = 1:1:8) can be added for 10 min to block unreacted amino groups. Then, wash with DMF 4 times, followed by washing with DMF and DCM in sequence and vacuum drying.

[0036] (12) Resin cleavage and removal of side chain protecting groups: Prepare cleavage buffer (TFA:TIS:H2O = 95:2.5:2.5, v / v / v), transfer the dried peptide resin to a reaction flask, add 10 mL of cleavage buffer, shake or stir in an ice bath for 5 min, then move to room temperature and stir in the dark for 2.5 h. This step can be completed simultaneously: the peptide chain is cleaved from the Wang resin to form a C-terminal carboxylic acid; the His side chain Trt protecting group is removed; the Lys side chain Boc protecting group is removed; and the Thr side chain tBu protecting group is removed. After cleavage, filter the TFA filtrate using a sintered glass funnel, and wash the resin twice with a small amount of TFA or DCM, then combine the filtrates.

[0037] (13) Precipitation and collection of crude peptides: The combined lysis filtrate was slowly added dropwise to 45 mL of pre-cooled anhydrous diethyl ether while stirring. After the addition was complete, the mixture was placed in an ice bath for 30 min to allow the crude peptides to precipitate fully. Then, it was centrifuged at 4000 r / min for 10 min and the supernatant diethyl ether was discarded. The precipitate was washed three times with pre-cooled anhydrous diethyl ether to remove TFA, TIS and detached side chain protecting groups. After washing, the precipitate was placed in a fume hood to evaporate the residual diethyl ether, yielding the crude peptide solid.

[0038] (14) Reversed-phase HPLC purification: Dissolve the crude peptide in a small amount of water / acetonitrile mixture, and add a small amount of 0.1% TFA to promote dissolution. Filter the solution through a 0.22 μm filter membrane and then perform preparative reversed-phase HPLC purification. The chromatographic conditions are as follows: Column: C18 preparative column; Mobile phase A: 0.1% TFA / water; Mobile phase B: 0.1% TFA / acetonitrile; Detection wavelength: 214 nm; Gradient: 5% B → 40% B, 30-40 min; Flow rate: set according to the preparative column specifications. Collect the target main peak fraction, confirm by analytical HPLC or LC-MS, combine the target fractions, remove acetonitrile by rotary evaporation, and freeze-dry the remaining aqueous phase to obtain a white or off-white powder target product: Myr-Thr-D-Lys-Gly-NMe-Thr-His-OH.

[0039] The characterization data of the obtained compounds are as follows: Figure 1 , Figure 2 , Figure 3 The antibacterial compound of this invention is a white solid, readily soluble in dimethyl sulfoxide, methanol, and slightly soluble in water.

[0040] The selenium-enriched Hericium erinaceus of this invention can be obtained through liquid spawn cultivation, homogenization of the spawn, solid-state fermentation cultivation, and drying and pulverization. In at least one of the liquid spawn cultivation and solid-state fermentation cultivation stages, selenium is added to the culture medium. The selenium content in the selenium-enriched Hericium erinaceus should meet national and industry standards.

[0041] According to a specific embodiment of the present invention, it can be prepared by the following method: Hericium erinaceus in vitro culture was inoculated into shake flask culture medium. The shake flask culture medium consisted of: 3% glucose, 2% starch, 1% yeast extract, 0.1% potassium dihydrogen phosphate, 0.1% magnesium sulfate, and the remainder was water, with a pH of 5.5. The culture was carried out at 27°C and 180 rpm for 5-7 days. Then, it was cultured in a fermenter at 27°C, 70% liquid volume, and an aeration rate of 1:0.5V / V·min for another 2-4 days. The fermenter culture medium had the same composition as the shake flask culture medium. The culture solution of the liquid bacterial culture was kept in a homogenizer at a speed of 2200-2300 rpm for 1 minute; The basic formula for solid cultivation substrate is: 82% cottonseed hulls, 16% wheat bran, 1% urea, 1% superphosphate, and citric acid to adjust the pH to 5.5. Sodium selenite is added to the basic formula to make the sodium selenite content 240mg / kg-920mg / kg to form solid cultivation substrate. Water is then added to form solid cultivation medium. The homogenized bacterial solution is then inoculated into the solid cultivation medium. After culturing at 25℃ and 80%-85% humidity for 20 days, the substrate is ventilated for fruiting. The fruiting temperature is 18-20℃, the fruiting humidity is 92%-93%, and the fruiting cycle is 5 days. The obtained selenium-enriched Hericium erinaceus was dried and pulverized to obtain selenium-enriched Hericium erinaceus powder.

[0042] The selenium-enriched Hericium erinaceus described in this invention can also be cultivated using any other feasible method.

[0043] (II) Antibacterial activity test (1) Test bacteria: Bacillus subtilis.

[0044] (2) Determination of the antibacterial activity of compound 1: The bacterial culture was obtained by shaking in liquid nutrient broth medium at 30 °C, and then the bacterial culture was diluted to a concentration of about 10. 6CFU / mL. Add 90 μL of diluted bacterial culture and 10 μL of compound-1 to a 96-well plate to achieve a concentration of 64 μg / mL. After incubation at 30 ℃ for 16 h, record the optical density (OD 600) at 600 nm using a multi-mode microplate reader and calculate the inhibition rate. Apramycin was used as a positive control, and DMSO as a negative control; each group was repeated three times. The inhibition rate was (negative control OD value - sample OD value) / negative control OD value × 100%.

[0045] (3) Determination of the antibacterial activity of selenium-enriched Hericium erinaceus powder and the synergistic antibacterial activity of compound 1 with selenium-enriched Hericium erinaceus powder: The bacterial solution was obtained by shaking in liquid culture medium at 30 °C, and then the bacterial solution was diluted to a concentration of about 10. 6 CFU / mL; Set up multiple sets of experiments as follows: Add 0.1% (w / v) and 0.5% (w / v) of selenium-enriched Hericium erinaceus powder to the diluted bacterial solution, respectively; Add 90 μL of diluted bacterial culture containing selenium-enriched Hericium erinaceus powder and 10 μL of compound 1 to a 96-well plate to make the concentration of compound 1 64 μg / mL, the concentration of selenium-enriched Hericium erinaceus powder 0.1% (w / v) and 0.5% (w / v). After incubating each experimental group at 30 ℃ for 16 h, the optical density (OD 600) at 600 nm was recorded using a multi-mode microplate reader, and the inhibition rate was calculated. Apramycin was used as a positive control, and DMSO was used as a negative control. Each group was repeated three times.

[0046] Table 1. Antibacterial activity of compound 1 Experimental conclusions: As shown in Table 1, the inhibition rate of 0.5% selenium-enriched Hericium erinaceus powder was 12.3%, and increasing the concentration of selenium-enriched Hericium erinaceus did not increase the inhibition rate by more than 50%; the inhibition rate of compound one at 64 μg / mL was 14.5%; the combined inhibition rate of the two reached 92.4%. It can be seen that the antibacterial compounds of this invention have good inhibitory activity against Bacillus subtilis and exhibit synergistic antibacterial effects with selenium-enriched Hericium erinaceus powder.

[0047] The content of this invention is not limited to the specific embodiments listed. Any equivalent modifications made by those skilled in the art to the technical solutions of this invention by reading this specification are covered by the claims of this invention.

Claims

1. A small molecule peptide with antibacterial activity, characterized in that, The small molecule peptide has the chemical formula C. 37 H 64 N8O 10 Compound one has the following structural formula: 。 2. The use of the small molecule peptide according to claim 1 in the preparation of antibacterial drugs or biopesticides.

3. The application according to claim 2, characterized in that, The antibacterial activity of the small molecule peptides includes inhibition of Bacillus subtilis.

4. The use of selenium-enriched Hericium erinaceus in the preparation of formulations that synergistically enhance the antibacterial activity of the small molecule peptides as described in claim 1.

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