Pediococcus acidilactici GOLDGUT-EZMind and application thereof in preparation of product for improving mental health

Abstract

The invention discloses a strain of pediococcus acidilactici GOLDGUT-EZMind and an application of the strain of pediococcus acidilactici GOLDGUT-EZMind in preparation of a product for improving mental health. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35671, the preservation date is August 18, 2025, and the strain is named as pediococcus acidilactici in taxonomy. The strain has the capacity of producing ILA at high yield, also has excellent acid resistance and cholate resistance, can be used as an efficient ILA producer, and is used for preparing medicines, foods, nutritional supplements and the like to improve the ILA level in the body so as to improve mental health.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and more specifically to a strain of Pediococcus lactis GOLDGUT-EZMind and its application in the preparation of products that improve mental health. Background Technology

[0002] Studies have shown that only about 5% of dietary tryptophan is absorbed and utilized by the human body from the small intestine. Most of the unabsorbed tryptophan enters the colon and becomes an important substrate for gut microbiota. Under the transformation of microorganisms, tryptophan is metabolized along multiple pathways. Among them, indole-3-lactic acid (ILA), produced through the indolepyruvate pathway, is one of the core bioactive molecules that has attracted much attention in recent years.

[0003] As a representative product of tryptophan microbial metabolism, indole-3-lactic acid (ILA) is an important endogenous signaling molecule in the human body. It can efficiently activate the aryl hydrocarbon receptor (AhR) on the cell surface, a signaling pathway crucial for maintaining intestinal homeostasis. After AhR activation, ILA not only significantly enhances the integrity of the intestinal epithelial barrier and reduces the entry of harmful substances into the bloodstream, but also precisely regulates the host's immune response, such as promoting immune cell differentiation and inhibiting excessive inflammation.

[0004] Compared to other major indole derivatives (such as indole-3-acetic acid), indole-3-lactic acid exhibits unique functional characteristics. Studies have confirmed that it can effectively inhibit the growth of various pathogenic intestinal bacteria, thereby optimizing the intestinal microecological environment. More notably, indole-3-lactic acid can also cross the intestinal wall and enter the circulatory system, playing a powerful antioxidant role in the human body, helping to eliminate free radicals that damage cells; its activity is even superior to other similar derivatives in some aspects. Furthermore, in patients with depression, studies have consistently found that ILA levels are significantly lower than in healthy individuals, and its concentration is negatively correlated with the severity of depressive symptoms—that is, the lower the ILA level, the more severe the depressive symptoms may be. Therefore, the medical community currently mainly explores its association with depression from the perspective of its "deficiency" or "lack," and regards it as a potential therapeutic target.

[0005] Although the physiological functions of indole-3-lactic acid are well-defined and promising, its development and application still face the challenge of targeted delivery. The key obstacle to realizing its health value is how to accurately and efficiently deliver exogenously supplemented indole-3-lactic acid to specific sites in the intestine and ensure its stable release in the target area to continuously exert its core functions of regulating immunity and strengthening the barrier.

[0006] Probiotics, as a class of live microorganisms that are beneficial to the host, have received much attention in recent years for their potential to regulate the function of the gut-brain axis and improve mood.

[0007] Therefore, how to develop a probiotic strain that can efficiently produce specific beneficial metabolites (such as indole-3-lactic acid) and tolerate the complex environment of the human gastrointestinal tract is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide a strain of Pediococcus lactis GOLDGUT-EZMind and its application in the preparation of products that improve mental health, so as to overcome the shortcomings of the prior art.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A strain of *Pediococcus acidilactici*, obtained by screening, isolation, and purification from sweet fermented mash, a specialty fermented food from the Qinghai-Tibet Plateau region, was deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.35671 and deposit date of August 18, 2025. Its taxonomic name is *Pediococcus acidilactici*.

[0011] This invention also claims protection for the use of the above-mentioned *Pediococcus lactis* GOLDGUT-EZMind in the preparation of products with high ILA yield.

[0012] Furthermore, the aforementioned Pediococcus lactis GOLDGUT-EZMind includes at least one of Pediococcus lactis GOLDGUT-EZMind cells, metabolites, and fermentation broth.

[0013] Furthermore, the aforementioned products include pharmaceuticals, foods, and nutritional supplements, which are used to increase ILA levels in the body.

[0014] Furthermore, the aforementioned drugs are administered orally, and the dosage forms include capsules, tablets, granules, oral liquids, and lyophilized powders.

[0015] Furthermore, the aforementioned foods include ordinary foods, foods for special medical purposes, functional foods, and health foods.

[0016] Furthermore, the aforementioned foods are consumed orally, and their dosage forms include yogurt and dairy beverages.

[0017] This invention also claims protection for the use of the above-mentioned Pediococcus lactis GOLDGUT-EZMind in the preparation of products that improve mental health.

[0018] Furthermore, the aforementioned Pediococcus lactis GOLDGUT-EZMind includes at least one of Pediococcus lactis GOLDGUT-EZMind cells, metabolites, and fermentation broth.

[0019] Furthermore, the aforementioned products include pharmaceuticals, food, and nutritional supplements, used to improve mental health.

[0020] Furthermore, the aforementioned drugs are administered orally, and the dosage forms include capsules, tablets, granules, oral liquids, and lyophilized powders.

[0021] Furthermore, the aforementioned foods include ordinary foods, foods for special medical purposes, functional foods, and health foods.

[0022] Furthermore, the aforementioned foods are consumed orally, and their dosage forms include yogurt and dairy beverages.

[0023] As can be seen from the above technical solution, compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] 1. The present invention, Pediococcus lactis GOLDGUT-EZMind, has a high ILA production capacity and also possesses excellent acid and bile salt resistance. It can be used as a highly efficient ILA producer to prepare pharmaceuticals, food, and nutritional supplements to increase ILA levels in the body, thereby improving mental health.

[0025] 2. In vitro fermentation experiments showed that the *Pediococcus lactis* GOLDGUT-EZMind strain of the present invention has an outstanding ability to metabolize tryptophan and produce ILA, with a concentration of up to 17.34 μg / mL.

[0026] 3. Animal experiments have confirmed that oral administration of the present invention, *Pediococcus lactis* GOLDGUT-EZMind (at a dose of 1×10⁻⁶), is effective. 9 ~1×10 11 (CFU / mL) can significantly increase the level of ILA in the host serum.

[0027] 4. The dosage and administration method of the Lactococcus lactis GOLDGUT-EZMind of this invention are reasonable and effective, effectively avoiding the adverse effects that may result from unclear dosage in existing probiotic preparations, such as no effect after administration or excessive administration. Attached Figure Description

[0028] Figure 1 A photograph showing the colony morphology of Pediococcus lactis GOLDGUT-EZMind.

[0029] Figure 2 Morphological image of Pediococcus lactis GOLDGUT-EZMind observed under a microscope;

[0030] Figure 3 A phylogenetic tree was constructed by homology comparison of the sequences of Pediococcus lactis GOLDGUT-EZMind and similar strains included in GenBank;

[0031] Figure 4 The ability of *Pediococcus lactis* GOLDGUT-EZMind to produce ILA in vitro was evaluated. The results are expressed as mean ± standard error (n = 3).

[0032] Figure 5 The experiment showed that Pyocortisone lactis GOLDGUT-EZMind significantly increased serum ILA levels in depressed mice in vivo. The results are expressed as mean ± standard error (n = 3). Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] Isolation, identification and preservation of Pediococcus lactis GOLDGUT-EZMind

[0036] 1. Sample collection

[0037] Samples of sweet fermented food, a specialty fermented food from the Qinghai-Tibet Plateau region, were collected in 50-100g portions in sterile self-sealing bags. Two to three portions of each sample were collected separately, sealed, transported at low temperature, and temporarily stored in a 4°C refrigerator (for isolation and culture within one week).

[0038] 2. Preparation of culture medium

[0039] ① Preparation of MRS solid plates: Accurately weigh 48g of MRS broth medium, 1mL of resazurin, 1000mL of distilled water, and 15.00g of agar powder into a beaker. After fully dissolving, dispense the solution into anaerobic flasks. Pour nitrogen gas for 3 minutes, then sterilize in an autoclave at 115℃ for 15 minutes. After sterilization, allow the solution to cool slightly, then quickly transfer it to an anaerobic chamber. Pour approximately 20mL of the solution into disposable petri dishes. After complete cooling and solidification, flip the dishes over and transfer them to an incubator. The plates are ready for use after one day if there is no contamination.

[0040] ② Preparation of MRS liquid culture medium: Accurately weigh 48g of MRS broth medium, 1mL of resazurin, and 1000mL of distilled water into a beaker. After thorough dissolution, aliquot the solution into anaerobic flasks. Purge with nitrogen for 3 minutes, then sterilize in an autoclave at 115℃ for 15 minutes. After sterilization, quickly transfer the solution to an anaerobic chamber. After cooling, aliquot 1.4mL of MRS liquid culture medium into 2.0mL round-bottom centrifuge tubes and transfer to an incubator. The solution is ready for use after one day if uncontaminated.

[0041] 3. Isolation and culture of bacterial strains

[0042] ① Sample dilution: Transfer fresh samples to an anaerobic chamber, remove the sample packaging bag, and carefully wipe the bag opening or bottle opening twice with an alcohol swab. Add approximately 0.2g to 1.0mL of PBS solution and thoroughly pipette until a suspension forms. Take 100μL of the suspension and add it to 900μL of PBS solution, mix thoroughly, and then gradually dilute in 10-fold serial steps to a final concentration of 10. -8 .

[0043] ② Sample coating and incubation: Take the diluted sample from ① and select 10 -5 10 -6 10 -7 and 10 -8 The bacterial suspensions were plated using a 100 μL dilution gradient. 100 μL of the dilution was placed on a plate and spread using a spreader. Two plates were prepared for each sample and each dilution gradient. The evenly spread plates were incubated in a 37°C anaerobic incubator for 48 h. A blank plate was also placed as a control.

[0044] ③ Colony purification and subculturing: Take out the culture dishes prepared in ② and carefully observe the morphological characteristics of the colonies (colony diameter, shape, color, water content, edge, transparency, and ridges) in an anaerobic chamber. Observe with a magnifying glass and distinguish different colonies according to their morphology. After roughly distinguishing them, use an inoculation loop to select colonies of different morphologies (select 3-5 strains of the same morphology repeatedly). Streak the colonies on plates for 24-48 hours and repeat 2-3 times to obtain pure single colonies. Then, use a pipette tip to pick them and place them in 1.5 mL of MRS liquid medium. After culturing for 16-24 hours, aspirate 100 μL of the bacterial solution into a new 1.5 mL of MRS liquid medium. Repeat this subculturing process 2-3 times.

[0045] 4. Preservation of bacterial strains

[0046] After subculturing the purified bacterial strain, it was preserved at the end of the logarithmic growth phase. 200 μL of bacterial culture was mixed with 200 μL of 40% glycerol in a 2.0 mL cryovial, and the strain was numbered and labeled. At least three cryovials of each strain were preserved. The labeled strains were placed in pre-chilled cryovials, and the strain information and growth status were recorded on the lid. The cryovials were then stored at -80°C. One backup cryovial was used for subsequent DNA sequencing, probiotic function evaluation, and metabolite analysis.

[0047] 5. 16S rDNA identification

[0048] ① Performed PCR amplification of the 16S rRNA gene of the selected single bacterial strain: Centrifuged 1 mL of bacterial culture after 24 h of culture (10000 r / min, 5 min, 4℃), discarded the supernatant, and resuspended in 200 μL PBS. Universal primers for the full-length bacterial 16S rRNA gene were used: the upstream primer was 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', and the downstream primer was 1492R: 5'-CTACGGCTACCTTGTTACGA-3'. The PCR amplification system (25 μL) included 1 μL each of universal primers 27F and 1492R, 1 μL of bacterial template, 12 μL of 2×TaqPCRMasterMix, and finally, ddH2O was added to bring the total volume to 25 μL. PCR reaction conditions: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 1 min, for a total of 35 cycles; 72℃ extension for 10 min.

[0049] ② Electrophoresis detection: Prepare 100 mL of 1.5% agarose gel, heat in a microwave oven on medium heat until completely dissolved, cool slightly, add 6 μL of GelRed nucleic acid gel dye, mix well, pour into a gel tray with a gel holder and comb, cool until solidified, remove the comb, and remove the gel along with the gel holder into the electrophoresis tank. Load 5 μL of the amplified PCR product and 5 μL of DL 2000 DNA Marker onto the gel, and perform electrophoresis at 200V for 15 min. After electrophoresis, perform gel imaging to check the banding in the 1500 bp region. In the absence of a band in the blank control, select PCR products with positive amplification results for purification and Sanger sequencing.

[0050]

[0051] This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35671 and deposit date of August 18, 2025. Its taxonomic name is Pediococcus acidilactici.

[0052] 6. Test Results

[0053] like Figure 1 As shown, the morphology of the plate colonies is as follows: approximately 0.5-2 mm in diameter, round, with neat edges, opaque, milky white or grayish white in color, with a raised center, smooth surface, moist texture, and easy to pick up.

[0054] like Figure 2 As shown, under a microscope, this strain is observed to be Gram-positive, spherical cells, usually arranged in pairs, chains, or clusters, and does not form spores or move.

[0055] like Figure 3 As shown, the 16S identification results indicate that, according to the phylogenetic tree of the model bacteria, this bacterium is a strain of Pediococcus acidilactici.

[0056] Example 2

[0057] Evaluation of acid and bile salt tolerance of Pediococcus lactis GOLDGUT-EZMind

[0058] 1. Draw the growth curve

[0059] The preserved bacterial strain was inoculated into MRS liquid medium and subcultured. After incubation at 37°C for 16 hours, the bacterial culture was inoculated at 10% into honeycomb plates dispensed with fresh MRS medium and transferred to an automated microbial growth curve analyzer. Absorbance at 600 nm was measured every 2 hours, and the culture was incubated at 37°C for 24 hours. Data were then exported and growth curves were plotted.

[0060] 2. Bile salt tolerance test

[0061] The method is the same as shown in "1. Drawing Growth Curves". Replace the MRS medium in the honeycomb plate with MRS medium containing 0.2% and 0.3% bile salts, and compare the growth curve containing bile salts with the growth curve in "1. Drawing Growth Curves". Select strains that can grow in bile salt medium for subsequent experiments.

[0062] 3. Acid resistance test

[0063] The bile salt-tolerant strains selected in "2. Bile Salt Tolerance Assay" were inoculated into MRS liquid medium and subcultured. After incubation at 37°C for 16 hours, the bacterial suspension was inoculated at 10% with a pH 3 PBS solution. A 10-fold serial dilution was performed at 0 hours and 2 hours (the specific dilution was determined based on the bacterial growth concentration). 100 μL of each dilution was spread onto plates using a spreader. For each sample, 3-4 appropriate dilution gradients were used to spread onto two plates. The evenly spread plates were incubated in a 37°C anaerobic incubator for 24 hours. The acid tolerance of the strain was determined by the ratio of viable bacteria counts at 2 hours and 3 hours to viable bacteria counts at 0 hours.

[0064] 4. Test Results

[0065] Table 1 Evaluation of the strain's acid and choline salt tolerance

[0066] growth medium pH=3 0.2% bile salts 0.3% bile salts time 2h 3h 1h Survival rate 101.89％ 100％ 90.63％

[0067] As shown in Table 1, the survival rate of *Pediococcus lactis* GOLDGUT-EZMind was 101.89% after incubation at pH 3 for 2 hours, 100% after incubation at 0.2% bile salt concentration for 3 hours, and 90.63% after incubation at 0.3% bile salt concentration for 1 hour.

[0068] Example 3

[0069] Evaluation of in vitro ILA production capacity of Pediococcus lactis GOLDGUT-EZMind

[0070] 1. Take 200 μL of the fermentation broth after 24 h of microbial culture into a 1.5 mL centrifuge tube, add 800 μL of chromatographic grade methanol to precipitate the protein, and place the tube in an ultrasonicator to sonicate and disrupt the cells for 10 min to fully extract the target metabolites. Then, place the tube in a -20℃ freezer for 20 min to accelerate protein precipitation. After centrifugation (12000 r / min, 10 min, 4℃), collect the supernatant. Filter the supernatant through an organic phase filter membrane, add it to an inner liner tube, place the inner liner tube in a sample vial, and store it in a 4℃ freezer until analysis.

[0071] 2. After sample preparation, the filtered supernatant was analyzed using High-Performance Liquid Chromatography (HPLC). For ILA quantification, 10 μL of sample was injected into the chromatograph, which was equipped with a C18 column (4.6 × 250 mm, 5 μm) and an RF-20A fluorescence detector. The excitation wavelength was 282 nm, and the emission wavelength was 352 nm. Chromatographic separation was performed at a column temperature of 30 °C and a flow rate of 1.0 mL / min, using chromatographic grade methanol (mobile phase A) and 15 mmol / L sodium dihydrogen phosphate (pH 2.8, mobile phase B). The gradient program was as follows: 0–12 min: 42% mobile phase A; 12–28 min: 50% mobile phase A; 28–35 min: 85% mobile phase A.

[0072] 3. Data and Statistical Analysis

[0073] Data and statistical analyses were performed using GraphPad Prism 8.0. Results are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used to determine significant differences between groups, and Tukey's multiple comparison test was used for post-hoc analysis. p < 0.05 was considered statistically significant, where * indicates p < 0.05 (significant difference) and ** indicates p < 0.01 (highly significant difference).

[0074] 4. Test Results

[0075] pass Figure 4 It can be seen that *Pediococcus lactis* GOLDGUT-EZMind, *Lactobacillus plantarum* LP550, and *Bifidobacterium breve* AF71-02 all have the ability to metabolize tryptophan and produce ILA. Among them, *Pediococcus lactis* GOLDGUT-EZMind has the most outstanding ILA production ability, with an ILA concentration of 17.34 μg / mL in its fermentation broth.

[0076] Example 4

[0077] Pediococcus lactis GOLDGUT-EZMind increases ILA levels in the body.

[0078] 1. Grouping of experimental animals

[0079] Eight-week-old male C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All animal procedures were approved by the Animal Ethics Committee of Northwest A&F University (Ethics No.: IACUC2024-1110).

[0080] After one week of acclimatization, mice were randomly divided into three groups: control group (CON), model group (CUMS), and GOLDGUT-EZMind intervention group (CUMS+GOLDGUT-EZMind), with eight mice in each group. Mice in the model and intervention groups received a 6-week chronic, unpredictable, mild stress regimen. Stressors included 5 minutes of swimming in 4°C cold water, 3 hours of restraint, 24 hours of fasting or water deprivation, damp bedding, cage tilting, and 5 minutes of tail clamping, with the sequence randomly assigned weekly to reduce predictability. The CON group received no stress treatment.

[0081] 2. Processing methods for each group

[0082] Control group: Normal drinking water, supplemented with 100μL PBS by gavage daily.

[0083] Model group: received a 6-week chronic unpredictable mild stress regimen, with daily gavage supplementation of 100 μL of PBS.

[0084] GOLDGUT-EZMind intervention group: Received a 6-week chronic unpredictable mild stress regimen, with cultured Pediococcus lactis GOLDGUT-EZMind suspended in PBS until the viable count reached 1×10⁻⁶. 10 CFU / mL, prepare fresh before use. Administer 100 μL of bacterial suspension via gavage daily.

[0085] 3. Experimental Methods

[0086] After a 6-week probiotic intervention, mice were sacrificed and their serum was collected. ILA levels in the serum were detected using high-performance liquid chromatography (HPLC). 30 μL of serum was diluted to 300 μL with methanol, then incubated at -20°C for 20 min. The sample was then centrifuged at 4°C and 13000 rpm for 10 min. The supernatant was filtered through a 0.22 μm organic filter membrane and added to a brown bottle, stored at 4°C for analysis. The HPLC detection method was the same as in Example 4.

[0087] 4. Data and Statistical Analysis

[0088] Data and statistical analyses were performed using GraphPad Prism 8.0. Results are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used to determine significant differences between groups, and Tukey's multiple comparison test was used for post-hoc analysis. p < 0.05 was considered statistically significant, where * indicates p < 0.05 (significant difference) and ** indicates p < 0.01 (highly significant difference).

[0089] 5. Test Results

[0090] Depend on Figure 5It was found that after 6 weeks of probiotic intervention, compared with the CUMS group, the Lactococcus GOLDGUT-EZMind intervention group significantly increased the serum ILA level in mice (p<0.05).

[0091] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of *Pediococcus lactis* GOLDGUT-EZMind, characterized in that, This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35671 and deposit date of August 18, 2025. Its taxonomic name is Pediococcus acidilactici.

2. The application of the *Pediococcus lactis* GOLDGUT-EZMind as described in claim 1 in the preparation of a product with high ILA production.

3. The application according to claim 2, characterized in that, The *Pediococcus lactis* GOLDGUT-EZMind includes at least one of the *Pediococcus lactis* GOLDGUT-EZMind cells, metabolites, and fermentation broth.

4. The application according to claim 2, characterized in that, The products include pharmaceuticals, food, and nutritional supplements.

5. The application according to claim 4, characterized in that, The drug is administered orally, and its dosage forms include capsules, tablets, granules, oral liquids, and lyophilized powders.

6. The application according to claim 4, characterized in that, The food products include general food, food for special medical purposes, functional food, and health food, which are consumed orally and include yogurt and dairy beverages in dosage forms.

7. The use of the lactic acid porphyria GOLDGUT-EZMind as described in claim 1 in the preparation of products that improve mental health.

8. The application according to claim 7, characterized in that, The *Pediococcus lactis* GOLDGUT-EZMind includes at least one of the *Pediococcus lactis* GOLDGUT-EZMind cells, metabolites, and fermentation broth.

9. The application according to claim 7, characterized in that, The products include pharmaceuticals, food, and nutritional supplements.

10. The application according to claim 7, characterized in that, The drug is administered orally, and its dosage forms include capsules, tablets, granules, oral liquids, and lyophilized powders. The food products include general food, food for special medical purposes, functional food, and health food, which are consumed orally and include yogurt and dairy beverages in dosage forms.

Images