Fermented lactobacillus mucus J0273 and culture medium thereof

Abstract

The application discloses a fermented Lactobacillus mui J0273, which is preserved in the Guangdong Microbial Culture Collection Center on March 28, 2024, and has a preservation number of GDMCC No: 64463; the fermented Lactobacillus mui J0273 provided by the application has antioxidant activity, can produce indole-3-methanol, and has good DPPH free radical clearance rate, hydroxyl radical clearance rate, high reducing power, Fe 2+ Chelating capacity, can enhance the antioxidant capacity of the host, regulate the host immune, protect the intestinal barrier, and relieve inflammation.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a fermenting *Lactobacillus mucilaginosus* strain J0273 with antioxidant properties and indole-3-carboxaldehyde production, and the optimization of its culture medium. Background Technology

[0002] Oxidative stress is an abnormal state in which oxidation and antioxidation processes in the body are disrupted. It is a negative effect of free radicals produced in the body, playing a double-edged sword role in physiological adaptation and signal transduction. Under physiological conditions, cells maintain redox homeostasis by producing and eliminating reactive oxygen species (ROS) and reactive nitrogen species (RNS), enabling the body to maintain adaptive responses. However, if the redox balance is disrupted, an excessive increase in ROS and RN can lead to inflammatory neutrophil infiltration, the production of large amounts of oxidative intermediates, and increased protease secretion, inducing ischemia, inflammation, fibrosis, apoptosis, necrosis, and malignant transformation. These are considered important factors in aging and the development of various diseases. Furthermore, in the body, excessive ROS can be further converted into other, more toxic ROS. For example, when ferrous ions are present in cells, hydrogen peroxide can gain ferrous iron through the Fenton reaction, transforming an electron into a hydroxyl radical. Hydroxyl radicals are highly reactive molecules without any selectivity; once generated, they immediately react with other surrounding biomolecules, causing damage to fats, proteins, nucleic acids, and other substances. Studies have shown that *Lactobacillus fermentatus* has certain application potential in antioxidant applications. For example, patent CN202111501025.6 discloses a strain of *Lactobacillus fermentatus* GZSC-1 with antioxidant activity, exhibiting a DPPH scavenging rate of 95.45% and a hydroxyl radical scavenging rate of 70.21%. Patent CN201911107792.1 discloses a strain of *Lactobacillus fermentatus* JX306 with antioxidant function, where the bacterial concentration is 4 × 10⁻⁶. 8 At CFU / mL, Fe 2+ The ion chelation capacity is 54.38%.

[0003] Indole-3-carboxaldehyde is an indole derivative of tryptophan metabolites. In the intestines of humans and animals, approximately 4%–6% of tryptophan is metabolized and broken down into indole and its derivatives by intestinal microorganisms. Currently, indole and its derivatives have been shown to enhance host antioxidant capacity, regulate host immunity, protect the intestinal barrier, and alleviate inflammation. Studies by Liu Yufei et al. have found that indole-3-carboxaldehyde produced by *Bifidobacterium animalis* SHXXA4M1 can alleviate inflammation in mice with colitis-associated colorectal tumors by regulating immunity and enhancing intestinal barrier function. Other studies have shown that indole derivatives also exhibit certain therapeutic activities in tumor models. For example, studies have found that indole-3-carboxaldehyde can inhibit the progression of abdominal aortic tumors by regulating the phenotypic transformation of vascular smooth muscle cells. Currently, research and patent reports indicate that *Lactobacillus reuteri* and *Lactobacillus plantarum* are lactobacillus strains capable of producing indole derivatives. Studies by Shuhua Shan et al. have found that *Lactobacillus reuteri* SY523 has anti-obesity effects, and the intestinal metabolite indole-3-carboxaldehyde content in mice treated with this strain was significantly increased. Arong Wang et al. demonstrated that indole-3 lactic acid derived from *Lactobacillus plantarum* DPUL-S164 improves intestinal barrier integrity through the AhR / Nrf2 / NF-κB axis. Currently, there are no patent reports on indole derivatives produced by *Lactobacillus fermentans*.

[0004] *Lactobacillus fermentum* is a type of probiotic lactic acid bacteria that is generally recognized as safe (GRAS). It is one of the strains approved for use in food by authoritative agencies such as the National Health Commission of my country, the US Food and Drug Administration (FDA), and the European Food Safety Authority (EFSA). Zuo Mengnan et al., in their growth curve analysis of *Lactobacillus fermentum* BLHN3, found that this strain entered the stationary phase after 14 hours of culture in ordinary MRS. However, Liao Yixiao et al., under the same culture conditions, found that *Lactobacillus fermentum* NCO only entered the stationary phase after 16 hours of culture. This indicates that the nutrient requirements of *Lactobacillus fermentum* are strain-specific, and optimizing the culture medium for strains with superior characteristics is essential for large-scale cultivation. Summary of the Invention

[0005] The purpose of this invention is to provide a fermenting Lactobacillus mucilaginosus J0273 with antioxidant properties and indole-3-carboxaldehyde production, and to optimize its culture medium.

[0006] The objective of this invention is achieved as follows: a fermenting *Lactobacillus fermentum* J0273, characterized in that the fermenting *Lactobacillus fermentum* J0273 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 28, 2024, with the accession number GDMCCNo: 64463, and classified as *Limosilactobacillus fermentum*.

[0007] As a further preferred embodiment of the present invention, the 16S rRNA sequence of the fermenting Lactobacillus mucinus J0273 is shown in SEQ ID NO:1.

[0008] SEQ ID NO:1:

[0009] J0273

[0010]

[0011] The colony morphology of *Limosilactobacillus fermentum* J0273 is as follows: the colonies are round or spherical, milky white, opaque, raised, with smooth edges and a smooth surface. The colonies are medium-sized, with a diameter of about 2-3 mm, and are relatively moist.

[0012] As a further preferred embodiment of the present invention, the fermented Lactobacillus mucilaginosus J0273 produces indole-3-carboxaldehyde.

[0013] Application of fermented Lactobacillus mucinus J0273 in regulating immunity and enhancing the intestinal barrier.

[0014] The culture medium composition for fermenting Lactobacillus mucinus J0273 is characterized by comprising the following components: 20g glucose, 5g Angel yeast extract FM 802, 10g Angel peptone FP 401, 1g Tween 80, 10g beef extract, 2g anhydrous sodium acetate, 2g diamine citrate, 2.6g dipotassium hydrogen phosphate, pH 7.0, 0.25g MnSO4, and 0.25g MgSO4.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The fermented Lactobacillus mucinus J0273 of the present invention has antioxidant activity, produces indole-3-carboxaldehyde, can regulate immunity and enhance intestinal barrier function to relieve colitis, and inhibits the deterioration of abdominal aortic tumors by regulating the phenotypic transformation of vascular smooth muscle cells; it has high free radical scavenging ability, superoxide anion free radical scavenging ability, metal ion chelating ability, hydroxyl free radical scavenging ability, and linoleic acid oxidation inhibition ability. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the technical description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a morphological diagram of the fermented Lactobacillus mucinus J0273 of this invention.

[0018] Figure 2 A graph showing the DPPH free radical scavenging capacity of different fermented mucilage lactis.

[0019] Figure 3 A graph evaluating the superoxide anion free radical scavenging ability of different fermenting *Lactobacillus mucilaginosus*.

[0020] Figure 4A graph showing the metal ion chelating ability of different fermenting mucinous lactobacilli.

[0021] Figure 5 A graph evaluating the hydroxyl radical scavenging ability of different fermenting mucinous lactobacilli.

[0022] Figure 6 A graph showing the ability of different fermenting *Lactobacillus mucilaginosus* to inhibit linoleic acid oxidation.

[0023] Figure 7 A graph evaluating the ability of fermenting Lactobacillus mucilaginosus J0273 to produce indole-3-carboxaldehyde.

[0024] Figure 8 The optimal pH for the growth of fermenting Lactobacillus mucilaginosus J0273 is shown in the figure.

[0025] Figure 9 The optimal yeast extract for fermenting Lactobacillus mucilaginosus J0273 is shown in the figure.

[0026] Figure 10 The optimal peptone content for fermenting Lactobacillus mucilaginosus J0273 is shown in the figure.

[0027] Figure 11 The graph shows the determination of the limiting trace elements for the optimal growth of fermenting Lactobacillus mucilaginosus J0273.

[0028] Figure 12 The growth curve of Lactobacillus mucilaginosus J0273 fermentation medium was optimized. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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 protection scope of the present invention.

[0030] The culture medium formulation involved in this invention is as follows:

[0031] MRS agar medium: peptone 10g, beef extract 10g, yeast extract 5g, diammonium citrate 2g, glucose 20g, Tween 80 1g, sodium acetate 5g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.58g, manganese sulfate 0.25g.

[0032] 15g agar, 1L distilled water, pH 6.2-6.6.

[0033] MRS-optimized agar medium: Angel peptone FP 401 10g, beef extract 10g, Angel yeast extract FM80 25g, diammonium citrate 2g, glucose 20g, Tween 80 1g, sodium acetate 2g, dipotassium hydrogen phosphate 2g, magnesium sulfate 0.25g, manganese sulfate 0.25g, agar 15g, distilled water 1L, pH 7.

[0034] Experiment 1: Evaluation of DPPH free radical scavenging ability

[0035] Lactobacillus mucinus J0273 was activated three times with an inoculum of 1%, each time for 18 hours. 3 ml of the activated fermentation broth was centrifuged at 3500 rpm for 10 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm sterile membrane to obtain the fermentation supernatant FS. 1 mL of the fermentation supernatant FS was mixed with 1 mL of 0.2 mmol / L DPPH-anhydrous ethanol solution and reacted at room temperature in the dark for 30 minutes. The mixture was then centrifuged at 3500 rpm for 10 minutes, and the absorbance of the supernatant at 517 nm was measured in triplicate. Lactobacillus rhamnosus LGG was used as a positive control.

[0036]

[0037] Among them, A i DPPH solution + sample, A j A0: Anhydrous ethanol + sample, A0: DPPH solution + MRS.

[0038] The results are as follows Figure 2 :

[0039] Depend on Figure 2 It was found that *Lactobacillus fermentum* J0273, *Lactobacillus fermentum* JO275, and *Lactobacillus rhamnosus* LGG all showed highly significant differences. However, *Lactobacillus fermentum* J0273 had a higher DPPH free radical scavenging rate than *Lactobacillus fermentum* JO275, reaching 91.31%, while the DPPH free radical scavenging rate of *Lactobacillus fermentum* JO275 was 90.77%. Except for *Lactobacillus fermentum* J0273 and *Lactobacillus fermentum* JO275, the DPPH free radical scavenging rates of other strains showed little difference from LGG. Among them, *Lactobacillus fermentum* JO416 had a relatively high DPPH free radical scavenging rate of 88.09%, while *Lactobacillus fermentum* JO415 had the lowest DPPH free radical scavenging rate of 76.57%.

[0040] Experiment 2: Evaluation of superoxide anion free radical scavenging capacity

[0041] Lactobacillus mucinus J0273 was activated three times with an inoculum of 1%, each time for 18 hours. 3 ml of the activated fermentation broth was centrifuged at 3500 rpm for 10 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm sterile membrane; this was the fermentation supernatant (FS). The superoxide anion radical scavenging capacity experiment was conducted in four groups, with a total reaction volume of 3 mL. After reacting in a 25℃ water bath for 20 minutes, the absorbance was measured in triplicate at 325 nm. Lactobacillus rhamnosus LGG was used as a positive control.

[0042] (1)A 11 Group: Add 0.6 mL of sample to 2 mL of Tris-HCl buffer (150 mmol / L, pH 8.0), then add 0.4 mL of pyrogallol solution preheated to 25 °C to start the reaction;

[0043] (2)A 10 Group: Add 0.6 mL of sample to 2 mL of Tris-HCl buffer (150 mmol / L, pH 8.0), then add 0.4 mL of MRS liquid culture medium to start the reaction;

[0044] (3)A 01 Group: Add 0.6 mL of MRS liquid culture medium to 2 mL of Tris-HCl buffer (150 mmol / L, pH 8.0), then add 0.4 mL of pyrogallol solution preheated to 25 °C to start the reaction;

[0045] (4)A 00 Group: Add 1 mL of MRS liquid medium to 2 mL of Tris-HCl buffer (150 mmol / L, pH 8.0) to start the reaction;

[0046] Superoxide anion radical scavenging rate (%) = (A 11 -A 10 ) / (A 01 -A 00 )×100%

[0047] The results are as follows Figure 3 :

[0048] Depend on Figure 3It was found that all experimental *Lactobacillus fermentans* strains exhibited lower superoxide anion radical scavenging capabilities than *Lactobacillus rhamnosus* LGG, with *Lactobacillus rhamnosus* LGG achieving a superoxide anion radical scavenging rate of 54.73%. *Lactobacillus fermentans* JO416 showed the second-highest superoxide anion radical scavenging rate, reaching 53.09%. The superoxide anion radical scavenging rates of *Lactobacillus fermentans* JO275, JO283, and JO374 showed relatively small differences, at 49.16%, 48.76%, and 48.79%, respectively. *Lactobacillus fermentans* J0273 had a mid-range superoxide anion radical scavenging rate of 41.01%, while *Lactobacillus fermentans* JO344 had the lowest rate at 32.02%.

[0049] Experiment 3: Metal ions (Fe) 2+ Chelation capacity evaluation

[0050] Lactobacillus mucinus J0273 was activated three times with an inoculum of 1%, each time incubated for 18 hours. 3 ml of the activated fermentation broth was centrifuged at 3500 rpm for 10 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm sterile membrane; this was the fermentation supernatant (FS). Metal ions (Fe...) 2+ The chelation capacity experiment was divided into three groups. 0.1 mL of ascorbic acid solution, 0.1 mL of ferrous sulfate solution, and 1 mL of sodium hydroxide solution were mixed with 0.5 mL of FS and incubated in a 37°C water bath for 20 min. Then, 0.2 mL of trichloroacetic acid (TCA) was added, and the mixture was centrifuged at 3000 rpm for 10 min to obtain the supernatant. 0.2 mL of the supernatant was then added to 2 mL of o-phenanthroline solution and reacted for 10 min. The absorbance was measured in triplicate at 510 nm, with *Lactobacillus rhamnosus* LGG as a positive control. The specific groupings are as follows:

[0051] (1)A0: 0.1mL ascorbic acid + 0.1mL ferrous sulfate + 1mL sodium hydroxide + 0.5mL MRS + 0.2mL trichloroacetic acid + 2mL o-phenanthroline.

[0052] (2) A1: 0.1 mL ascorbic acid + 0.1 mL ferrous sulfate + 1 mL sodium hydroxide + 0.5 mL FS + 0.2 mL trichloroacetic acid + 2 mL o-phenanthroline.

[0053] (3) A2: 0.1 mL ascorbic acid + 0.1 mL MRS + 1 mL sodium hydroxide + 0.5 mL FS + 0.2 mL trichloroacetic acid + 2 mL o-phenanthroline

[0054] Metal ion chelating ability = [1 - (A1 - A2) / A0] × 100%

[0055] The results are as follows Figure 4 :

[0056] Depend on Figure 4 It was found that there was a significant difference in the metal ion chelating ability between the fermenting *Lactobacillus myxobolus* J0273 strain and the control group *Lactobacillus rhamnosus* LGG. The metal ion chelating ability of *Lactobacillus myxobolus* J0273 reached 97.85%, while that of *Lactobacillus rhamnosus* LGG was 87.75%. The metal ion chelating ability of *Lactobacillus myxobolus* JO373 was second only to *Lactobacillus myxobolus* J0273, reaching 95.91%. The metal ion chelating ability of the experimental group *Lactobacillus myxobolus* JO275 was the lowest at 82.35%.

[0057] Experiment 4: Evaluation of hydroxyl radical scavenging ability

[0058] Lactobacillus mucinus J0273 was activated three times with an inoculum of 1%, each time for 18 hours. 3 ml of the activated fermentation broth was centrifuged at 3500 rpm for 10 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm sterile membrane; this was the fermentation supernatant (FS). The hydroxyl radical scavenging capacity experiment was divided into three groups. The samples were incubated at 37℃ for 1 hour, and the absorbance was measured in triplicate at 536 nm. Lactobacillus rhamnosus LGG was used as a positive control. The specific groupings are as follows:

[0059] (1) Sample group A s Add 1 mL of 0.02 mol / L PBS (pH 7.4) solution to 0.5 mL of 2.5 mmol / L o-phenanthroline solution, 0.5 mL of 2.5 mmol / L FeSO4 solution, and 0.5 mL of 20 mmol / L H2O2 solution in sequence, mix thoroughly, and then add 0.5 mL of sample.

[0060] (2) Control group A c Add 1 mL of 0.02 mol / L PBS (pH 7.4) solution to 0.5 mL of 2.5 mmol / L o-phenanthroline solution, 0.5 mL of 2.5 mmol / L FeSO4 solution, and 0.5 mL of 20 mmol / L H2O2 solution in sequence, mix thoroughly, and then add 0.5 mL of distilled water.

[0061] (3) Blank group A b Add 1 mL of 0.02 mol / L PBS (pH 7.4) solution to 0.5 mL of 2.5 mmol / L o-phenanthroline solution, 0.5 mL of 2.5 mmol / L FeSO4 solution, and 1 mL of distilled water in sequence.

[0062] Hydroxyl radical scavenging rate = (A s -A b ) / (A c-A b )×100%

[0063] Depend on Figure 5 It was found that, compared with *Lactobacillus rhamnosus* LGG strain, most experimental strains of *Lactobacillus fermentum* exhibited superior hydroxyl radical scavenging ability. Among them, *Lactobacillus fermentum* J0273 had the highest hydroxyl radical scavenging rate at 76.19%, while *Lactobacillus rhamnosus* LGG strain had a hydroxyl radical scavenging rate of 73.63%. The hydroxyl radical scavenging rates of *Lactobacillus fermentum* JO275, JO283, and JO342 showed little difference and were all higher than those of *Lactobacillus rhamnosus* LGG, at 75.86%, 75.96%, and 75.84%, respectively. The lowest hydroxyl radical scavenging rate was 70.86% for *Lactobacillus fermentum* JO415.

[0064] Experiment 5: Evaluation of Linoleic Acid Oxidation Inhibition Capacity

[0065] Lactobacillus mucinus J0273 was activated three times with an inoculum of 1%, each time for 18 hours. 3 ml of the activated fermentation broth was centrifuged at 3500 rpm for 10 minutes, the precipitate was discarded, and the supernatant was filtered through a 0.22 μm sterile membrane to obtain the fermentation supernatant (FS). 1 mL of linoleic acid emulsion and 1 mL of ferrous sulfate (FeSO4, 1% by mass) were added to 0.5 mL of phosphate buffer (pH 7.4), followed by 0.5 mL of the fermentation supernatant. The mixture was incubated at 37°C for 1.5 hours. 0.2 mL of trichloroacetic acid (TAC, 4% by mass) and 2 mL of thiobarbituric acid (TBA, 0.8% by mass) were added to the mixture. After mixing, the mixture was incubated at 100°C for 30 minutes. After rapid cooling, the mixture was centrifuged at 5000 rpm for 10 minutes to collect the supernatant. The absorbance was measured at 532 nm, with phosphate buffered saline (PBS) as a blank control. Lactobacillus rhamnosus LGG was used as a positive control.

[0066] Total reducing power (%) = (1-A) 样品 ) / A blank × 100%

[0067] Depend on Figure 6It can be seen that the experimental strains of *Lactobacillus fermentatus* showed superior linoleic acid oxidation inhibition ability compared with the control strain *Lactobacillus rhamnosus* LGG. Significant differences were found between *Lactobacillus fermentatus* JO374, JO303, and J0273 and *Lactobacillus rhamnosus* LGG. The linoleic acid oxidation inhibition abilities of *Lactobacillus fermentatus* JO374, JO303, and J0273 were 65.33%, 61.55%, and 57.19%, respectively, while that of *Lactobacillus rhamnosus* LGG was 40.27%. *Lactobacillus fermentatus* JO344 had the lowest linoleic acid oxidation inhibition ability at 40.57%.

[0068] Among the five antioxidant capacity indicators—DPPH free radical scavenging capacity, superoxide anion free radical scavenging capacity, metal ion chelating capacity, hydroxyl free radical scavenging capacity, and linoleic acid oxidation inhibition capacity—Lactobacillus fermentum J0273 showed the best performance in three aspects: DPPH free radical scavenging capacity, metal ion chelating capacity, and hydroxyl free radical scavenging capacity. Therefore, Lactobacillus fermentum J0273 was selected as the best antioxidant strain.

[0069] Experiment 6: Evaluation of the ability of fermenting Lactobacillus mucilaginosus J0273 to produce indole-3-formaldehyde

[0070] The contents of indole-3-lactic acid and indole-3-carboxaldehyde in the fermentation broth of *Lactobacillus myxitis* JOU273 (with added tryptophan) were determined using an Ultimate 3000 HPLC system coupled with an ultra-high resolution QTOF mass spectrometer equipped with an ESI interface. A Waters ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 mm × 100 mm) was used as the liquid chromatography column. The mobile phase consisted of an aqueous solution containing 0.1% (v / v) formic acid (A) and acetonitrile (B), and the chromatography was performed at 30 °C with a flow rate of 0.3 mL / min. For gradient elution, the percentage of acetonitrile (B) was set as follows: 0.5% for 0-0.5 min; linearly increasing from 0.5% to 35% for 0.5-1 min; 35% for 1-4 min; linearly increasing from 35% to 70% for 4-5 min; maintaining 70% acetonitrile (B) for 0.5 min, then increasing to 100% within 0.5 min; and maintaining 100% for 6-8 min. Mass spectrometry was performed in negative ion mode with the following parameters: ESI nebulization voltage 2 kV, endplate offset -500 V, capillary voltage 2.5 kV, nebulizer pressure 2 bar, drying gas flow rate 4.0 L / min, vaporization temperature 200 °C, and scan range 50–1000 m / z.

[0071] Depend on Figure 7 It was found that the retention time of indole-3-carboxaldehyde was 2.95 min, and the concentration of tryptophan-indole-3-carboxaldehyde in the fermentation broth of *Lactobacillus plantarum* J0273 was 0.356 μg / mL, with no indole-3-lactic acid detected. Patent 202310503602.8 indicates that the concentration of indole-3-carboxaldehyde produced in vitro by *Lactobacillus plantarum* DPUL-S164 is close to 1 μg / mL.

[0072] Experiment 7: Optimization of culture medium for fermenting Lactobacillus mucinus J0273

[0073] Based on the traditional MRS culture medium components (10g peptone, 10g beef extract, 5g yeast extract, 2g diammonium citrate, 20g glucose, 1mL Tween 80, 5g sodium acetate, 2g dipotassium hydrogen phosphate, 0.58g magnesium sulfate, 0.25g manganese sulfate, 1000mL distilled water, pH 6.2–6.6), the yeast extract, peptone, trace elements, and pH of the culture medium were adjusted. The yeast extracts used in the experiment included: Angel Yeast Extract FM 985, Angel Yeast Extract FM 503, Angel Yeast Extract FM802, and Angel Yeast Extract FM 601; the peptones used included: Angel Peptone FP 401, Angel Peptone FP 103, and Angel Peptone FP 101. The specific steps are as follows:

[0074] Yeast preference analysis: Angel Yeast Peptone FP 401 was used as the fixed peptone (10 g / L); glucose 20 g / L, Tween 80 1 g / L, MnSO4 0.05 g / L, MgSO4 0.05 g / L, pH 7.0; Angel Yeast Extract FM 985, Angel Yeast Extract FM503, Angel Yeast Extract FM 802, and Angel Yeast Extract FM 601 (25 g / L) were used as variant yeasts for analysis.

[0075] Peptone preference analysis: Angel Yeast Extract FM 503 was used as the fixed yeast extract (25 g / L); glucose 20 g / L, Tween 80 1 g / L, MnSO4 0.05 g / L, MgSO4 0.05 g / L, pH 7.0; Angel Peptone FP 401, Angel Peptone FP 103, and Angel Peptone FP 101 (9 g / L) were used as variable peptones for analysis.

[0076] Trace elements: Angel peptone FP401 was used as the fixed nitrogen source (9 g / L); Angel yeast extract powder FM503 was used as the fixed yeast extract (25 g / L), glucose 20 g / L, Tween 80 1 g / L, pH 7.0; trace elements MnSO4 and MgSO4 were analyzed with concentration gradients of 0.05 g / L, 0.25 g / L, and 0.45 g / L, respectively.

[0077] pH: Angel peptone FP 401 was used as the nitrogen source (9 g / L), Angel yeast extract FM 503 was used as the carbon source (25 g / L), glucose 20 g / L, Tween 80 1 g / L, MnSO4 0.05 g / L, MgSO4 0.05 g / L; pH gradients of 5.5, 6, 6.5, and 7 were set for analysis.

[0078] The above culture medium was dispensed into 50ml Erlenmeyer flasks, with an inoculation volume of 1%-0.5ml. OD was measured at 2-hour intervals. 600 nm value.

[0079] Optimal growth pH determination

[0080] In the experiment to determine the optimal growth pH, ​​the initial pH of the culture medium was set at 5.5, 6.0, 6.5, and 7.0, while other culture conditions remained unchanged. The same culture medium (20 g / L glucose, 10 g / L Angel peptone FP 103, 5 g / L Angel yeast extract FM802, 10 g / L beef extract, 0.05 g / L MnSO4, 0.05 g / L MgSO4, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 2 g / L anhydrous sodium acetate, 1 g / L Tween 80) was placed in Erlenmeyer flasks. Two generations of activated *Lactobacillus fermentum* J0273 were inoculated at a 1% inoculum and cultured anaerobically at 37℃. The absorbance (OD) was measured every 2 hours. 600nm Value, result as Figure 8 .

[0081] Depend on Figure 8 It can be seen that during the 0-2h period, the growth rate of *Lactobacillus fermentatus* J0273 did not differ significantly at the four pH concentrations; during the 2-10h period, the growth rate of *Lactobacillus fermentatus* J0273 at pH 7 was slightly higher than that at pH 5.5, pH 6, and pH 6.5; during the 10-14h period, the growth rate slowed down, and the bacterial concentration of *Lactobacillus fermentatus* J0273 at pH 7 was slightly higher than that at pH 5.5, pH 6, and pH 6.5. Therefore, pH 7.0 was selected as the optimal pH for subsequent experiments.

[0082] Determination of optimal yeast extract

[0083] In the determination of optimal yeast extract, yeast extracts were prepared using Angel Yeast Extract FM 985, Angel Yeast Extract FM 503, Angel Yeast Extract FM 802, and Angel Yeast Extract FM 601, respectively, with all other culture conditions remaining the same. The same culture medium (20 g / L glucose, 10 g / L Angel peptone FP 103, 10 g / L beef extract, 0.05 g / L MnSO4, 0.05 g / L MgSO4, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 2 g / L anhydrous sodium acetate, 1 g / L Tween 80) and culture conditions (pH 7.0) were used in Erlenmeyer flasks. After inoculation, the cultures were incubated at 37°C, and the absorbance (OD) was measured every 2 hours. 600nm The optimal yeast extract was selected based on the value. The results are as follows: Figure 9 .

[0084] Depend on Figure 9 The results show that during the first 0-2 hours, the growth rates of *Lactobacillus myxoidus* J0273 fermented with four different Angel yeast powders were not significantly different. During the first 2-10 hours, the growth rate of *Lactobacillus myxoidus* J0273 fermented with Angel yeast powder FM 985 was slightly higher than that with Angel yeast powder FM 503, Angel yeast extract powder FM 802, and Angel yeast extract powder FM 601. During the first 10-14 hours, the growth rate slowed down, and the bacterial concentration of *Lactobacillus myxoidus* J0273 fermented with Angel yeast extract powder FM 802 was slightly higher than that with Angel yeast powder FM 985, Angel yeast powder FM 503, and Angel yeast extract powder FM 601. *Lactobacillus myxoidus* J0273 exhibited the highest growth rate. Therefore, Angel yeast powder FM 802 was ultimately selected as the most suitable yeast extract for use.

[0085] Optimal Peptone Assay

[0086] In the determination of the optimal peptone concentration, Angel peptone FP 401, FP 103, and FP 101 were used, with other culture conditions remaining the same. The same culture medium (20 g / L glucose, 10 g / L Angel peptone FP 103, 5 g / L Angel yeast extract FM 503, 10 g / L beef extract, 0.05 g / L MnSO4, 0.05 g / L MgSO4, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 2 g / L anhydrous sodium acetate, 1 g / L Tween 80) and culture conditions (pH 7.0) were used in Erlenmeyer flasks. After inoculation, the strains were cultured at 37°C, and the absorbance (OD) was measured every 2 hours. 600nm Value, and select the best peptone.

[0087] Depend on Figure 10The results show that, from 0 to 2 hours, the growth rate of *Lactobacillus mucinus* J0273 fermented under three different Angel peptone conditions was not significantly different; from 2 to 10 hours, the growth rate of *Lactobacillus mucinus* J0273 fermented under Angel peptone FP 401 conditions was significantly higher than that under Angel peptone FP 103 and Angel peptone FP 101 conditions; from 10 to 14 hours, the growth rate slowed down, and the bacterial concentration of *Lactobacillus mucinus* J0273 fermented under Angel peptone FP 401 conditions was slightly higher than that under Angel peptone FP 103 and Angel peptone FP 101 conditions; the growth amount of *Lactobacillus mucinus* J0273 fermented under Angel peptone FP 103 and Angel peptone FP 101 conditions was not significantly different, therefore, Angel peptone FP 401 was ultimately selected as the optimal peptone. Optimal growth limiting trace element analysis (MgSO4, MnSO4)

[0088] 0.05, 0.25, and 0.45 g / L of MnSO4 and MgSO4 were weighed into the culture medium, respectively, with m(MnSO4):m(MgSO4) = 1:1. The samples were placed in Erlenmeyer flasks using the same culture medium (20 g / L glucose, 10 g / L Angel peptone FP 103, 5 g / L Angel yeast extract FM 503, 10 g / L beef extract, 2 g / L diammonium citrate, 2.6 g / L K2HPO4·3H2O, 2 g / L anhydrous sodium acetate, 1 g / L Tween 80) and culture conditions (pH 7.0). After inoculation with the bacterial strain, the absorbance (OD) was measured every 2 hours at 37°C. 600nm The optimal concentration is selected based on the value.

[0089] Depend on Figure 11 It can be seen that, from 0 to 2 hours, the growth rate of *Lactobacillus mucinus* J0273 under the three different concentrations of trace elements was not significantly different; from 2 to 10 hours, the growth rate of *Lactobacillus mucinus* J0273 under the conditions of 0.45 g / L MnSO4 and MgSO4 was slightly higher than that under the conditions of 0.05 g / L and 0.25 g / L; from 10 to 14 hours, the growth rate slowed down, and the bacterial concentration of *Lactobacillus mucinus* J0273 under the conditions of 0.25 g / L MnSO4 and MgSO4 was slightly higher than that under the conditions of 0.05 g / L and 0.45 g / L MnSO4 and MgSO4; the growth of *Lactobacillus mucinus* J0273 was lowest under the conditions of 0.05 g / L MnSO4 and MgSO4. Therefore, the optimal conditions were selected with MgSO4 and MnSO4 contents of 0.25 g / L.

[0090] Optimal growth conditions for Lactobacillus fermentum J0273

[0091] Growth curves of fermented *Lactobacillus mucilaginosus* on optimized culture medium and MRS basal medium are shown below. Figure 12 .

[0092] Depend on Figure 12 It was found that under optimized culture conditions, the growth of *Lactobacillus mucinus* J0273 was significantly higher than that under ordinary MRS culture. In the optimized culture medium, with the extension of fermentation time, the number of viable *Lactobacillus mucinus* cells grew slowly from 0-4 hours, then increased exponentially from 4-8 hours, gradually slowed down from 8-14 hours, and stabilized from 14-18 hours, with no further increase in viable cell count. Therefore, the optimal fermentation time for *Lactobacillus mucinus* J0273 was 18 hours.

[0093] Table 1. Optimized culture medium formulation for fermenting Lactobacillus mucinus J0273

[0094]

[0095] The above description is merely a specific embodiment of the present invention, but the scope of protection of the invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A fermenting *Lactobacillus mucinus* ( Limosilactobacillus fermentum J0273, characterized in that, The fermenting Lactobacillus mucinus J0273 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 28, 2024, with accession number GDMCC No: 64463.

2. The fermented Lactobacillus mucinus J0273 according to claim 1, characterized in that: The 16S rRNA sequence of the fermenting Lactobacillus mucinus J0273 is shown in SEQ ID NO:

1.

3. The fermented Lactobacillus mucinus J0273 according to claim 1, characterized in that: The fermented Lactobacillus mucinus J0273 produces indole-3-carboxaldehyde.

Images