Panda-derived tryptophan-producing streptococcus non-lactolyticus C49 and application thereof

Abstract

The invention discloses panda-derived tryptophan-producing streptococcus non-lactolyticus C49 and application thereof, and belongs to the field of microorganisms, the strain is preserved in Guangdong Microbial Culture Collection Center, and the preservation number is GDMCC No: 67209. The strain has good acid-resistant and cholate-resistant characteristics, and can ensure that enough biomass passes through stress of a digestive tract environment; whole genome sequencing finds that C49 is significantly related to the pathway and module of tryptophan biosynthesis. Whole genome sequencing and in-vitro fermentation confirm that C49 has tryptophan biosynthesis capability, and in-vivo animal tests confirm that C49 has synthetic nerve conditioning capability. Therefore, as a probiotic feed additive, the streptococcus non-lactolyticus C49 has a good application prospect in the aspects of improving the feed value, improving the intestinal health of animals, enhancing the immunity of organisms and the like.

Description

Technical Field

[0001] This invention relates to the field of microbial development and utilization technology, specifically to a tryptophan-producing non-lactolytic streptococcus C49 from giant pandas and its applications. Background Technology

[0002] Giant pandas consume highly specialized bamboo rich in lignocellulose, resulting in a high abundance of key microbial species and genes involved in lignocellulose degradation within their gut microbiota. Lignocellulose is the most abundant renewable biomass resource on Earth, widely found in agricultural and forestry waste. However, due to its complex structure—primarily composed of cellulose, hemicellulose, and lignin, interconnected by covalent and hydrogen bonds to form a robust cell wall—lignocellulose is difficult for microorganisms to utilize directly. Currently, the main methods of lignocellulose disposal are incineration or landfill, which not only result in significant resource waste but also cause severe environmental pollution.

[0003] Although some microorganisms are capable of degrading lignocellulose, most are fungal strains, such as Trichoderma reesei. These fungal strains face numerous challenges in practical applications, such as slow growth rates, demanding environmental requirements, and difficulty in colonizing complex environments like the gut, thus limiting their further development in the utilization and biotransformation of lignocellulose resources.

[0004] Streptococci are Gram-positive bacteria that provide a wide range of benefits to their hosts through their metabolic activities and various biological functions. Streptococci can directly provide essential amino acids and vitamins (such as B vitamins and vitamin K) to their hosts, and through fermentation, they produce organic acids that promote the absorption of calcium, iron, phosphorus, and vitamin D in animals, while also enhancing gastrointestinal motility. Streptococci can also produce various digestive enzymes such as cellulase, protease, and lipase, regulating the absorption and utilization of nutrients in the digestive tract and benefiting animal growth and development. Among them, *Streptococcus non-lactolyticus* is a type of streptococcus associated with carbohydrate fermentation. Streptococci form a favorable symbiotic relationship with other lactic acid-producing bacteria, potentially promoting the growth and reproduction of these bacteria. Studies have shown that *Streptococcus non-lactolyticus* can colonize the intestine and exert various biological effects, including inhibiting the growth of pathogenic bacteria, regulating the intestinal microecological balance, and promoting the absorption and conversion of nutrients. Currently, research and application of *Streptococcus non-lactolyticus* are limited, but related studies indicate that it is a potential probiotic with significant research potential. Therefore, screening out a strain of non-lactolytic streptococcus that can efficiently degrade lignocellulose and convert it into high-value-added products, promote the synthesis of tryptophan and neuromodulators, and maintain the balance of gut microbiota in giant pandas and livestock is of great practical significance. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a non-lactolytic streptococcus that can degrade lignocellulose and convert it into tryptophan, and its application.

[0006] The technical solution of the present invention is: a strain of non-lactolytic streptococcus C49, which is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67209.

[0007] The above-mentioned application of non-lactolytic streptococcus alactolyticus C49 in the degradation of lignocellulose.

[0008] The above-mentioned application of non-lactolytic streptococcus (Streptococcus alactolyticus) C49 in the synthesis of tryptophan.

[0009] Furthermore, the synthetic tryptophan refers to the conversion of lignocellulose into tryptophan.

[0010] Biological agents containing the aforementioned Streptococcus alactolyticus C49.

[0011] Furthermore, the biological agent is a feed additive, health food, or bio-fertilizer.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The giant panda-derived tryptophan-producing non-lactose-degrading Streptococcus C49 of the present invention has good in vivo tolerance and can be used as a health food or feed additive.

[0014] 2. The non-lactose-degrading streptococcus C49 of the present invention has a variety of lignocellulose-degrading enzyme activities, and has the ability to degrade lignocellulose, and can be used as a bio-fertilizer.

[0015] 3. The non-lactolytic streptococcus C49 of the present invention has the ability to biosynthesize tryptophan and can be used for tryptophan production.

[0016] 4. The non-lactose-degrading Streptococcus C49 of the present invention has the ability to convert lignocellulose into tryptophan and can be used as a feed additive.

[0017] 5. Animal experiments have shown that C49 can enhance tryptophan synthesis, significantly increase the concentration of neurotransmitters such as 5-hydroxyindoleacetic acid and kynurenine in serum or feces, and promote intestinal peristalsis. Transcriptomics and anatomy have further revealed that C49 can regulate colon gene expression, enhance intestinal mucosal function, and promote intestinal development. Therefore, it can be used as a health food or feed additive. Attached Figure Description

[0018] Figure 1 A culture image of the giant panda-derived non-lactolytic streptococcus C49 isolated in Example 1 of this invention.

[0019] Figure 2 The effect of non-Lactobacillus fermentation on the concentrations of total amino acids (a) and tryptophan (b) in Example 4 of this invention.

[0020] Figure 3 The potential tryptophan biosynthesis pathway in *Streptococcus non-lactamase* in Example 5 of this invention; Note: Shikimicacid pathway, Erythrose 4-phosphate, Phosphoenolpyruvate, 3-Deoxy-arabino-heptulonate 7-phosphate, 3-Dehydroquinate, 3-Dehydroshikimate, Shikimate, Shikimate 3-phosphate, 5-O-(1-Carboxyvinyl)-3-phosphoshikimate; Chorimate pathway, Tryptophan. Indoleglycerolphosphate, 1-(2-Carboxyphenylamino)1-deoxy-D-ribulosephosphate, N-(5-Phospho-D-ribosyl)-anthranilate, Anthranilate, and Chorismate.

[0021] EC 2.5.1.54: 3-Deoxy-7-phosphate heptanulose synthase, EC 4.2.3.4: 3-Dehydroquinic acid synthase, EC 4.2.1.10: 3-Dehydroquinic acid dehydratase, EC 1.1.1.25: Shikimate dehydrogenase, EC 2.7.1.71: Shikimate kinase, EC 2.5.1.19: 3-phosphate shikimate 1-carboxyvinyltransferase, EC 4.2.3.5: branched acid synthase, EC 4.1.3.27: an-aminobenzoic acid synthase, EC 2.4.2.18: an-aminobenzoic acid phosphoribosyltransferase, EC 5.3.1.24: phosphoribosylan-aminobenzoic acid isomerase, EC 4.1.1.48: indole-3-glycerol phosphate synthase, EC 4.2.1.20: tryptophan synthase.

[0022] Figure 4 Principal coordinate analysis plots of Bray-Curtis distance (a) based on gut microbial species level, Jaccard distance (b) based on gut microbial species level, and Jaccard distance (c) of KO distribution in gut microbial community in Example 6; Note: PCoA1 (first principal component), PCoA2 (second principal component).

[0023] Figure 5 The composition (a) and differences (b) of the intestinal MAGs (Metagenome Assembled Genomes) in mice in Example 6 of this invention; Note: BFS is the experimental group fed with streptococci, and BF is the control group fed with physiological saline; Note: Actinomycetia, Bacilli, Bacteroldia, Campylobacteria, Clostridia, Coriobacteriia, Deferribacteres, Dehalobacteriia, Desulfovibrionia, Jeotgalicoccus nanhaiengis, Corynebacterium slationls, Odoribacter, Ventrimonas, Mucispirilum, Bifidobacterium globosum.

[0024] Figure 6Comparison of the differences in amino acid metabolism pathways (a) and KEGG modules (b) between the mouse experimental group and the control group in Example 6 of this invention; Note: Lysine degradation, Valine, leucine and isoleucine degradation, Phenylalanine metabolism, Tyrosine, Tryptophan, Histidine, Lysine biosynthesis, Glycine, serine and threonine, Cysteine ​​and methionine, Alanine, aspartate and glutamate, Betaine biosynthesis, choline => betaine, Lysine degradation, bacteria, L-lysine => succinate / acetyl CoA (lysine degradation, bacteria, L-lysine => succinate / acetyl-CoA), Lysine degradation, lysine => saccharopine => acetyl-CoA, Lysine degradation, bacteria, L-lysine => succinate, Staphylopine biosynthesis, L-lysine => staphylopine, Glycine cleavage system, Arginine biosynthesis, glutamate => N-acetylornithine => arginine, Arginine biosynthesis, ornithine => arginine, Tyrosine biosynthesis...The biosynthesis pathways chorismate, prephenate, and tyrosine (chorismate biosynthesis, chorismate => prephenate => tyrosine), threonine biosynthesis, aspartate => homoserine => threonine (threonine biosynthesis, aspartate => homoserine => threonine), shikimate pathway, phosphoenolpyruvate + erythrose-4-phosphate => shikimate (shikimate pathway, phosphoenolpyruvate + erythrose-4-phosphate => shikimate), cysteine ​​biosynthesis, serine => cysteine ​​(cysteine ​​biosynthesis, serine => cysteine), methionine biosynthesis, aspartate => homoserine (methionine biosynthesis, aspartate => homoserine), and lysine biosynthesis, DAP dehydrogenase pathway, aspartate => lysine (lysine biosynthesis, diaminopimelic acid dehydrogenase pathway, aspartate => lysine). DAPaminotransferase pathway, aspartate => lysine (lysine biosynthesis, diaminopimelic acid transaminase pathway, aspartate => lysine), Tryptophan biosynthesis, chorismate => tryptophan (tryptophan biosynthesis, chorismate => tryptophan), Histidine biosynthesis, PRPP => histidine (histidine biosynthesis, phosphoribosyl pyrophosphate => histidine), Valine / isoleucine biosynthesis, pyruvate => valine / isoleucine (valine / isoleucine biosynthesis, pyruvate => valine / isoleucine), Isoleucine biosynthesis, threonine => 2-oxobutanoate => isoleucine (isoleucine biosynthesis, threonine => 2-oxobutanoate => isoleucine), Leucine biosynthesis...2-oxoisocaproate => 2-oxoisocaprate => leucine (leucine biosynthesis, 2-oxoisocaproate => 2-oxoisooctanoic acid => leucine), Proline biosynthesis, glutamate => proline (proline biosynthesis, glutamate => proline).

[0025] Figure 7 In Example 6 of this invention, the lengths of the colon and small intestine, as well as the villus / crypt (V / C) ratio in the ileum, were measured in the mouse experimental group and the control group.

[0026] Figure 8 The results of hematoxylin-eosin staining of mouse ileum tissue in Example 6 of this invention.

[0027] Figure 9 Comparison of metabolic differences between the mouse experimental group and the control group in Example 6 of this invention; Note: Concentration in faces (fecal concentration), Concentration in serum (serum concentration), Trp (tryptophan), TrpA (tryptophan), X5.HT (5-hydroxytryptamine), X5.HIAA (5-hydroxyindoleacetic acid), Kyn (kynurenine).

[0028] Figure 10 The enrichment of differentially expressed genes in the colon of the mouse experimental group and the control group in Example 6 of this invention.

[0029] Figure 11The differential gene functional enrichment in the colon of the mouse experimental group and control group in Example 6 of this invention; Note: Normalized Enrichment Score, Regulation, Downregulated, Upregulated; spindle checkpoint signaling, mitotic spindle checkpoint signaling, metaphase chromosome alignment, chromosome separation, mitotic sister chromatid segregation, regulation of chromosome separation, regulation of sister chromatid segregation, sister chromatid segregation, chromosome segregation, axon development, regulation of membrane potential, regulation of synapse structure or activity, regulation of synapse organization, postsynaptic density (postsynaptic density), postsynaptic specialization, asymmetric synapse, neuron-to-neuron synapse, synaptic membrane, postsynaptic membrane.

[0030] Preservation information:

[0031] Streptococcus alactolyticus C49 was deposited on November 4, 2025, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No. 67209), located at Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Detailed Implementation

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial sources.

[0033] Example 1: Isolation, identification, and activation of tryptophan-producing non-lactose-degrading Streptococcus C49 from giant pandas

[0034] 1. Isolation and identification of tryptophan-producing non-lactose-degrading Streptococcus C49 from giant pandas

[0035] Dilute fresh panda feces 10% with sterile phosphate-buffered saline (PBS). 6 Take 200 μL of the suspension and spread it evenly on the surface of Gifu anaerobic medium (GAM) agar plates. Incubate at 37°C for 48 hours. Select single colonies and incubate them further on GAM agar plates at 37°C for 48 hours. Select single colonies with distinct morphological characteristics (such as color, size, and shape) and incubate them further on GAM agar plates at 37°C for 48 hours. Select single colonies and preserve them in GAM broth containing glycerol at -80°C.

[0036] Total DNA was extracted from the isolates using a bacterial DNA extraction kit (QIAGEN). The taxonomic position of the isolates was identified by PCR amplification of their 16S rRNA gene. The classification of the isolates was determined by the V1-V9 region of the 16S rRNA gene, amplified by PCR using primers 27F and 1492R. Primers were provided by Sangon Biotech (Chengdu) Co., Ltd., and Sanger sequencing was performed at the same company. The identified strain was *Streptococcus alactolyticus*.

[0037] 2. Activation of tryptophan-producing non-lactolytic streptococci C49 from giant pandas

[0038] The bacterial culture tubes were removed from the -80°C freezer, and strain C49 was cultured in GAM broth in a 37°C anaerobic incubator for 48 hours. The cells were then centrifuged at 6000 g for 10 minutes to obtain the bacterial cells. The cells were diluted with sterile physiological saline to an optical density (OD600) of 0.6 for further investigation.

[0039] Example 2: Safety and tolerability test of tryptophan-producing non-lactolytic streptococcus C49 from giant pandas.

[0040] Hemolytic activity test: After streaking the preservative solution of the C49 strain to be tested onto a modified GAM agar plate, single colonies were selected and streaked onto a blood agar plate. After 18 h of incubation, hemolysis was observed. Each strain was tested in triplicate. Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were used as positive controls.

[0041] Acid tolerance test: After streaking the preservative solution of the C49 strain to be tested onto a modified GAM agar plate, single colonies were selected and incubated in GAM liquid medium at 37°C for 18 h of anaerobic culture. 2 mL of the bacterial suspension was then centrifuged and resuspended to prepare a bacterial suspension. The bacterial suspension was inoculated into GAM liquid medium at pH 2 and 3, and counted on modified GAM agar plates after 0 h and 4 h of anaerobic culture.

[0042] Bile salt tolerance test: The C49 strain to be tested was prepared into a bacterial suspension and inoculated into 2 mL of sterile PBS solution with a porcine bile salt concentration of 0.3% at an inoculation rate of 2%. After anaerobic incubation for 0 h and 4 h, the strains were counted on modified GAM agar plates.

[0043] Tolerance to artificial gastrointestinal fluid: After preparing a bacterial suspension of the C49 strain to be tested, it was added to artificial gastric fluid and artificial intestinal fluid at an inoculum volume of 2%, respectively. The suspensions were incubated at 37°C for 0 h and 4 h in artificial intestinal fluid, and 0 h and 3 h in artificial gastric fluid. The gastrointestinal fluid-treated liquids were then counted on modified GAM agar plates.

[0044] In the above experiment, the survival rate of the bacterial strain = (number of viable bacteria after culture / number of viable bacteria before culture) * 100%

[0045] Table 1. Tolerance analysis of *Streptococcus non-lactolyticus* C49 strain from giant pandas.

[0046]

[0047] Note: Percentages represent survival rates.

[0048] The results showed that the strain was non-hemolytic and exhibited strong tolerance to hydrochloric acid, bile salts, gastric acid, and intestinal fluid, demonstrating its potential as a probiotic.

[0049] Example 3: Test of Lignocellulose Degradation Ability of Giant Panda-Derived Tryptophan-Producing Non-Lactosolytic Streptococcus C49

[0050] 1. Experimental Materials

[0051] Bamboo powder was added to the culture medium as a carbon source. Bamboo (Pleioblastus amarus), the staple food of giant pandas, was collected from the China Conservation and Research Center for the Giant Panda. After drying at 40℃, it was pulverized and sieved (35 mesh) to obtain bamboo powder. To minimize the influence of microorganisms in the bamboo powder on the experimental results, the bamboo powder was sent to China Gold Irradiation Chengdu Co., Ltd. for sterilization by cobalt-60 gamma irradiation (8.00 kGy). The sterilized bamboo powder was then cultured aerobically on TSA medium and anaerobically on GAM medium for microbial testing; no colonies were detected. The sterilized bamboo powder was stored at 4℃ for later use.

[0052] β-glucosidase (β-GC) activity assay kit, neutral xylanase activity assay kit, and manganese peroxidase (Mnp) activity assay kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. Omega genomic DNA extraction kit. TEA buffer was purchased from Solarbio Science & Technology Co., Ltd. DNA Marker D2000 was purchased from Beijing Tiangen Biotech Co., Ltd., and agarose was purchased from Shanghai Bioengineering Co., Ltd.

[0053] 2. Determination of Lignocellulose Degrading Enzyme Activity

[0054] To quantitatively verify the degradation activity of C49 strain on bamboo lignocellulose, the activities of xylanase, β-glucosidase, and manganese peroxidase (MnP) were measured to quantitatively verify the degradation activity of the strain on bamboo lignocellulose, the staple food of giant pandas. Single cells of strain C49 were picked and inoculated into GAM liquid medium, placed in an anaerobic jar at 37℃ for 24 h, centrifuged at 6000 g for 10 min, the supernatant was discarded, and the precipitated cells were diluted with physiological saline to a bacterial suspension with an OD600 value of 0.6. 1 mL of the bacterial suspension was inoculated into 9 mL of bamboo powder degradation medium with bamboo powder as the sole carbon source. After anaerobic incubation for 24 hours, the fermentation broth was centrifuged at 4℃ (8000 g, 10 min), and the supernatant was collected for enzyme activity analysis. Enzyme activity was measured in triplicate, and the final enzyme activity value was the average of the three assays.

[0055] The hemicellulose degradation capacity was determined using a neutral xylanase activity assay kit. The principle is that xylanase catalyzes the degradation of xylan into reducing oligosaccharides and monosaccharides in a neutral environment. Under boiling water bath conditions, it further undergoes a colorimetric reaction with 3,5-dinitrosalicylic acid, exhibiting a characteristic absorption peak at 540 nm. The intensity of the reaction color is directly proportional to the amount of reducing sugar produced by enzymatic hydrolysis. The xylanase activity can be calculated by measuring the rate of increase in absorbance at 540 nm of the reaction solution.

[0056] The results showed that the xylanase activity was 17.50 U / mL, the β-glucosidase activity was 4.72 U / mL, and the MnP enzyme activity was 4.74 U / mL.

[0057] Example 4: In vitro tryptophan biosynthesis capacity test of *Streptococcus lactis* C49 (giant panda-derived)

[0058] 1. Determination of total amino acid content

[0059] One mL of bacterial suspension was added to a culture tube containing 9 mL of sterile physiological saline and 0.5 g of sterile bamboo fiber powder, serving as the treatment group. Separately, 10 mL of sterile physiological saline and 0.5 g of sterile bamboo fiber powder were mixed in a culture tube, serving as the control group. The mixture was incubated in an anaerobic incubator at 37°C for 12 hours. The fermentation mixture was centrifuged at 4°C and 8000 rpm for 15 minutes, and the supernatant was collected for targeted metabolomics analysis using ultra-high performance liquid chromatography-mass spectrometry. Four replicates were set for each group.

[0060] Amino acid (AA) content detection kit (Solepro) was used to detect amino acid content. The principle is that α-amino acids react with ninhydrin in an aqueous solution upon heating, producing a blue-violet compound with an absorption peak at 570 nm. The amino acid content is calculated by measuring the absorbance of the reaction solution at 570 nm. The specific operating steps are as follows, referring to the instruction manual:

[0061] Add 0.5 mL of the test solution to 0.5 mL of reagent one, place in a metal bath and heat at 100°C for 15 minutes. After cooling with running water, centrifuge at 8000g for 15 minutes at room temperature. Collect the supernatant for testing.

[0062] Before the assay, reagents 2, 3, and 4 were prepared into a working solution in a ratio of 10:10:1 according to the experimental dosage. 10 μL of supernatant sample, 10 μL of standard (1.25 μmol / mL glutamic acid), and 10 μL of distilled water were added to an EP tube containing 210 μL of working solution. After mixing, the tube was placed in a metal bath and heated at 100℃ for 15 minutes. After cooling, the EP tube was inverted several times. After centrifugation at 10,000 rpm for 10 minutes, the supernatant was collected, and the absorbance was measured at 570 nm. The total amino acid content was then calculated.

[0063] 2. Determination of chromoic acid content

[0064] Strains with higher total amino acid content than the control group were selected for further determination of tryptophan content. Simultaneously, supernatant samples from the strains in Example 3.1 and the control group were taken, flash-frozen in liquid nitrogen for 15 min, and transported to Beijing Novogene Technology Co., Ltd. on dry ice for tryptophan concentration determination. The concentration of tryptophan in feces and serum was determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS). Standards were accurately weighed to prepare a 5 mg / mL linear standard stock solution. The linear stock solution was diluted with methanol to obtain a series of concentrations. L-Alanine-d4 and Phenylanine-d2 solutions of specific concentrations were prepared and mixed to obtain the internal standard solution (IS). The linear, internal standard, and quality control stock solutions and working solutions were stored at -20℃ for later use. Samples were added to the mass spectrometer in water and vortexed to dilute the sample. Take 50 μL of diluted sample, add 200 μL of precipitant containing mixed internal standard (acetonitrile:methanol=1:1), vortex to mix, stand on ice for 30 min, centrifuge at 12000 rpm at 4℃ for 10 min, and take all supernatant for analysis.

[0065] The chromatographic conditions were as follows: column: ACQUITY UPLC BEH Amide (2.1 × 100 mm, 1.7 μm); mobile phase: phase A: 5 mM ammonium acetate aqueous solution containing 0.1% formic acid, phase B: acetonitrile containing 0.1% formic acid; column temperature: 50℃; injection volume: 1 μL; flow rate: 0.3 mL / min.

[0066] Mass spectrometry conditions were as follows: electrospray ionization (ESI) source, negative ionization mode. Ion source temperature 550°C, ion source voltage -4500 V, sheath gas 35 psi, auxiliary gas 50 psi, collision gas 55 psi. Multiple reaction monitoring was used for scanning.

[0067] The concentration series of standard solutions were analyzed by LC-MS. The linearity of the standard solutions was assessed by plotting the ratio of the standard concentration to the internal standard concentration on the x-axis and the ratio of the peak area of ​​the standard to the internal standard on the y-axis. QC tests were performed on each batch of samples at several intervals. The RSD of all QC tests was calculated using the peak area ratio of the standard to the internal standard to evaluate the stability of the method (RSD ≤ 15% indicates that the method is stable and reliable). Quantitative analysis was performed on all samples according to the established sample pretreatment and instrumental analysis methods.

[0068] The results show that ( Figure 2 In strain C49 (a), the total amino acid content was higher than that in the control group. Tryptophan levels showed (…). Figure 2 (b) Compared with the uninoculated control group, the C49 strain showed a significant increase in tryptophan concentration after fermentation (Wilcoxon rank-sum test, p < 0.05).

[0069] Example 5: Whole genome sequencing of tryptophan-producing non-lactolytic streptococcus C49 from giant pandas.

[0070] Genomic DNA was randomly fragmented into approximately 350 bp fragments using a Covaris fragmenter. After constructing a DNA library, the strain was sequenced using a second-generation whole-genome sequencing system on an Illumina platform (PE150). Sequencing was performed at Beijing Novogene Bioinformatics Technology Co., Ltd. After the raw sequencing data was processed, low-quality reads were removed using Trimmomatic, and the data were assembled using SPAdes v3.15.5. Subsequently, the reads were evaluated using Quast to determine total contig length, N50, N90, and GC content. Genome species identification was performed using GTDB-Tk v2.4.1, and genome draft annotation was completed using bakta v1.11.0. Finally, functional annotation of genes was performed using eggNOG-mapper v2.1.12 based on the KEGG database.

[0071] The results of gene annotation using Bokta and KEGG Mapper indicate that *Streptococcus non-lactolyticus* C49 from giant pandas possesses a complete gene / enzyme system in the tryptophan biosynthesis pathway, including the shikimic acid pathway for the generation of cladonic acid and the complete pathway for the synthesis of tryptophan from cladonic acid. Figure 3 ).

[0072] Table 2. Next-generation sequencing assembly information of non-lactolytic streptococcus cinerea C49 from giant pandas.

[0073]

[0074] Experimental Example 6: The ability of non-lactolytic streptococcus C49 to convert tryptophan into neuromodulators

[0075] 1. Experimental Materials

[0076] Experimental strain: The non-lactolytic streptococcus C49 strain that underwent whole-genome sequencing in Example 4 was selected.

[0077] Experimental animals: Ten 6-8 week old female balb / c mice were purchased from Chengdu Dashuo Biotechnology Co., Ltd. Experiments began after the mice had acclimatized for 7 days.

[0078] Animal feed: The high-fiber feed for mice is customized by Nantong Trofi Feed Technology Co., Ltd. It is made by adding 10% bamboo powder to the ordinary feed (feed code: LAD 0011). The ingredients of the ordinary feed are: wheat flour, corn flour, wheat middlings, soybean meal, corn gluten meal, vegetable oil, brewer's yeast, salt, lysine, methionine, minerals, and vitamins.

[0079] Preparation of bacterial suspension for feeding: daily, streak the test strains on modified GAM agar plates, anaerobically incubate at 37°C for 24 h, wash the colonies on the plates with sterile physiological saline, collect the bacterial cells by centrifugation, and resuspend them in sterile physiological saline. Adjust the concentration of the bacterial suspension to an OD600 value of 0.6 for later use.

[0080] 2. Gavage-feeding experiment on mice

[0081] After a week of environmental acclimatization, mice were randomly divided into two groups of five each. The experimental group (BFS) mice were gavaged daily at a dose of 0.2 mL per mouse, with a bacterial load of 10⁸ CFU per mouse. The control group (BF) mice were gavaged with physiological saline at a dose of 0.2 mL per mouse. The mice's mental state and behavioral characteristics were observed and recorded. Weight, food intake, and water intake were recorded every two days. Experimental materials were collected after 21 days of gavage. This animal experiment followed the guidelines for the husbandry and management of laboratory animals and was approved by the Animal Welfare and Ethics Committee of Chengdu Medical College (Approval No.: 2024-100).

[0082] Fecal samples were collected from each mouse for metagenomic sequencing and metagenomic binning. Sequencing was performed at Novogene Technology Co., Ltd. in Beijing.

[0083] Feces and serum were collected from each mouse for targeted metabolomics testing. Blood from the mouse's orbital rim was centrifuged at 4°C for 10 min to obtain serum. Feces and serum were flash-frozen in liquid nitrogen for 15 min and transported on dry ice to Beijing Novogene Technology Co., Ltd. to determine the concentrations of tryptophan, tryptophan, tryptophan, 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, and kynurenine. The method was the same as in Example 3.

[0084] 3. Transcriptome sequencing of mouse colon

[0085] Mice gavaged with non-lactolytic streptococcal C49 were dissected, and the lengths of the small intestine and colon were measured. Ileal tissue was collected for hematoxylin-eosin staining to determine villus height and crypt depth. Colon tissue from three mice in each group was randomly selected, flash-frozen in liquid nitrogen, and sent to Beijing Novogene Technology Co., Ltd. for transcriptome sequencing. Total RNA pairs from the quality-tested mouse colon tissue were sequenced using the Illumina sequencing platform. Low-quality reads were removed from the original sequences using NGS QC Toolkit v2.343 to obtain high-quality reads. High-quality reads were mapped to the mouse reference genome using HISAT2 v2.2.144. Gene counts and expression values ​​were calculated using StringTie v2.2.345. Differentially expressed genes (DEGs) were screened using the DESeq2 package in R with a p-value < 0.05 and |log2 fold-change| ≥ 1 as thresholds. Functional enrichment analysis was performed using the clusterProfiler package in R based on the GO and KEGG databases.

[0086] 4. Conclusion

[0087] 4.1. Analysis of the composition, structure, and function of non-lactolytic streptococcal citrate C49 from giant panda in the mouse gut microbiota

[0088] PCoA analysis based on species-level Bray-Curtis distance showed no significant difference in gut microbiome β-diversity between the BFS and BF groups (PERMANOVA, R² = 0.09, p = 0.627). Figure 4 (a) PCoA analysis based on Jaccard distance showed a clear separation in the gut microbiota composition between the two groups (PERMANOVA, R² = 0.18, p = 0.009). Figure 4 (b) indicates that the addition of non-lactolytic streptococcus C49 affects the structure of the mouse gut microbiota. β-diversity analysis based on the Jaccard distance of the KO functional spectrum showed that different groups of samples clustered together (PERMANOVA, R² = 0.39, p = 0.006), indicating that the addition of C49 affects the function of the mouse gut microbiota. Figure 4 (c)

[0089] Based on the threshold of ANI < 99%, after removing redundant bins, 97 non-redundant MAGs with integrity > 80% and contamination < 10% were further obtained. Figure 5 (a) All MAGs belong to 8 phyla, 9 classes, 16 orders, 24 families, 66 genera, and 91 species, and can be identified down to the family level. The abundance of MAGs in different groups was compared ( Figure 5In b), seven MAGs, including J. nanhaiensis, C. stationis, and Odoribacter sp 910578105, were found to be significantly enriched in the intestines of BFS group mice, and four MAGs, including COE1 sp 003513705, RGIG4057 sp 910577165, and B. globosum, were found to be significantly enriched in the intestines of BF group mice.

[0090] Subsequently, functional enrichment analysis was performed on KEGG pathways and modules related to amino acid metabolism in the gut microbiota of the two groups of mice. The results showed that multiple KEGG pathways, including lysine degradation, valine, leucine, and isoleucine degradation, phenylalanine metabolism, tyrosine metabolism, and tryptophan metabolism, were significantly enriched in the gut microbiota of the BFS group mice. Figure 6 (a). The BFS group mice showed significant enrichment in seven KEGG pathways, including valine, leucine, and isoleucine biosynthesis; alanine, aspartic acid, and glutamate metabolism; cysteine ​​and methionine metabolism; and phenylalanine, tyrosine, and tryptophan biosynthesis. At the KEGG module level, the BFS group showed significant enrichment in the betaine biosynthesis and lysine degradation modules (a). Figure 6 In group b), the BF group was significantly enriched with more modules, such as the shikimic acid pathway, tryptophan biosynthesis, and tyrosine biosynthesis.

[0091] This indicates that C49 can enhance amino acid synthesis, thereby promoting the growth of other gut microbes that can utilize these amino acids.

[0092] 4.2. Effects of giant panda-derived non-lactolytic streptococcus C49 on mouse development

[0093] Both the experimental and control groups showed good growth and mental condition during the experiment, with normal feeding, drinking, respiration, and excretion; no cases of diarrhea or disease were observed. Regarding physiological indicators, the colon length of mice in the BFS group was significantly longer than that in the BF group (Wilcoxon rank-sum test, p < 0.05). Figure 7 However, there was no significant difference in small intestinal length between the two groups of mice. Furthermore, the villus height / crypt depth (V / C) ratio in the ileum of the BFS group was significantly higher than that of the BF group (Wilcoxon rank-sum test, p < 0.01). Figure 7 , Figure 8 This indicates that C49 can enhance the intestinal mucosal barrier function.

[0094] 4.3. Effects of giant panda-derived non-lactolytic streptococcus C49 on amino acid metabolism in mice

[0095] The concentrations of tryptophan and its metabolites in mouse feces and serum were measured, revealing that ( Figure 9 The mean concentration of 5-hydroxyindoleacetic acid (5-HA) in the feces of mice in the BFS group (432.4 ± 113.3 ng / g) was significantly higher than that in the BF group (222.5 ± 67.3 ng / g) (p < 0.05). Simultaneously, the mean concentrations of 5-HA (54 ± 16.8 ng / mL) and kynurenine (353.3 ± 61.9 ng / mL) in the serum of the BFS group were also significantly higher than those in the BF group (30 ± 6.3 ng / mL) (226.2 ± 37.6 ng / mL) (p < 0.05). The mean concentration of tryptamine in the feces of the BFS group (9.2 ± 3.8 ng / g) was higher than that in the BF group (5.4 ± 2.4 ng / g), with the difference approaching statistical significance (p = 0.056). The mean concentrations of tryptophan, tryptophan, and serotonin in the serum of the BFS group (29200.1±12202.6 ng / mL, 0.4±0.3 ng / mL, and 54.1±32.3 ng / mL, respectively) were all higher than those in the BFS group (16761±4666.5 ng / mL, 0.3±0.1 ng / mL, and 39.3±32.2 ng / mL, respectively).

[0096] In the mammalian gut, tryptophan is primarily metabolized via three pathways: the serotonin pathway, the kynurenine pathway, and the indole derivative pathway. Over 95% of tryptophan is metabolized via the kynurenine pathway, which plays a crucial role in regulating tryptophan utilization by metabolizing excess tryptophan. Despite dietary variations, plasma tryptophan concentrations remain stable through two key rate-limiting enzymes in the kynurenine pathway: indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO). When plasma tryptophan levels are sufficient, TDO stability is enhanced, accelerating tryptophan degradation; conversely, when tryptophan is deficient, TDO is degraded to prevent over-metabolism. This study observed significantly elevated kynurenine concentrations in the serum of mice gavaged with the strain, indicating enhanced tryptophan metabolism along the kynurenine pathway, thus demonstrating that C49 can influence tryptophan metabolism in mice.

[0097] Furthermore, the concentrations of 5-hydroxyindoleacetic acid (5-HICA) in the feces and serum of mice in the BFS group were significantly increased. In mammals, 5-HICA is the end product of serotonin metabolism via monoamine oxidase (MAO). Given the rapid conversion of serotonin to 5-HICA, the latter is often used as a surrogate indicator for assessing serotonin concentration. Most serotonin is produced by enterochromaffin cells and is present in the mammalian gut, playing a crucial role in nerve signal transduction, nervous system development, intestinal epithelial barrier maintenance, and intestinal motility promotion. Studies have shown that Bacillus subtilis can accelerate intestinal motility by increasing tryptophan and 5-HICA concentrations in the mouse colon. Considering the poorly digestible bamboo diet of giant pandas, serotonin may accelerate chyme emptying by enhancing intestinal peristalsis, thereby reducing the risk of intestinal obstruction and driving giant pandas to compensate for inefficient energy intake through frequent feeding.

[0098] 4.4. Effects of Panda-derived non-lactolytic streptococcal C49 on gene expression in mouse colon

[0099] To investigate the effects of C49 on gene expression in the mouse colon, we performed transcriptome sequencing on colon tissue and identified differentially expressed genes (DEGs) between the BFS and BF groups. Based on a corrected p-value < 0.05 and a |log2 fold change| ≥ 1 threshold, a total of 56 colonic DEGs were identified. Figure 10 Of these, 14 genes were upregulated and 42 genes were downregulated in the BFS group.

[0100] Since these DEGs did not show significant enrichment in Gene Ontology (GO) functional entries, we used the GSEA method for functional enrichment analysis. The results showed that nine GO entries in the BFS group had normalized enrichment scores (NES) > 1 and corrected p < 0.05. The most significantly upregulated GO entries involved spindle checkpoint signaling, mitotic spindle checkpoint, metaphase chromosome alignment, chromosome separation, sister chromatid separation in mitosis, and related regulatory processes. Figure 11 Meanwhile, in the BFS group, 97 GO items had NES < -1 and corrected p < 0.05. The top 10 GO items that were most significantly downregulated included postsynaptic membrane, synaptic membrane, interneuronal synapse, asymmetric synapse, postsynaptic specialized structures, postsynaptic density, synaptic tissue and structural function regulation, membrane potential regulation, and axonal development.

[0101] The above studies indicate that C49 can significantly increase the concentrations of 5-hydroxyindoleacetic acid and kynurenine in serum and feces, synthesize neuromodulators, regulate colon gene expression, and promote intestinal development.

Claims

1. A strain of non-lactolytic streptococcus C49 is deposited at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 67209.

2. The application of the non-lactolytic streptococcus alactolyticus C49 as described in claim 1 in the degradation of lignocellulose.

3. The application of the non-lactolytic streptococcus alactolyticus C49 as described in claim 1 in the synthesis of tryptophan.

4. The application according to claim 3, characterized in that, The synthetic tryptophan refers to the conversion of lignocellulose into tryptophan.

5. A biological agent containing Streptococcus alactolyticus C49 as described in claim 1.

6. The biological agent according to claim 5, characterized in that, The biological agent is a feed additive, health food, or bio-fertilizer.

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