L-theanine-producing bifidobacterium longum subsp. longum hc2904, fermentation product and application thereof

Abstract

This invention relates to the field of probiotic screening and application technology, specifically to a *Bifidobacterium longum* subsp. *longum* HC2904 that produces L-theanine, its fermentation products, and its applications. *Bifidobacterium longum* subsp. *longum* ( Bifidobacterium longum subsp. longum HC2904 was deposited with the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, with accession number CGMCC No. 26586. This strain can produce high levels of L-theanine during the fermentation of specific raw materials, with a yield of up to 9.66 mg / mL, overcoming the technical limitations of existing probiotic technologies for improving sleep, which mainly rely on the γ-aminobutyric acid (GABA) metabolic pathway. L-theanine fermented beverages prepared from this strain can significantly improve sleep quality in people with insomnia, significantly reduce PSQI scores, and synergistically improve emotional health problems such as gastrointestinal discomfort, stress, anxiety, and depression caused by long-term poor sleep quality. It can also significantly reduce cortisol levels in the body, demonstrating a clear physiological regulatory mechanism and promising application prospects.

Description

Technical Field

[0001] This invention relates to the field of probiotic screening and application technology, specifically to a Bifidobacterium longum subsp. HC2904 that produces L-theanine, its fermentation products, and applications. Background Technology

[0002] Insomnia is a common disorder characterized by difficulty maintaining a stable sleep state or entering a sleep stage, resulting in insufficient sleep duration and depth. Its clinical manifestations are diverse, ranging from mild cases such as prolonged sleep onset (more than 30 minutes), sleep interruptions, or difficulty falling back asleep after waking; to severe cases where it may be impossible to fall asleep at all. Specific manifestations include: ① delayed sleep onset and restlessness; ② difficulty maintaining sleep, frequent awakenings at night, and insufficient deep sleep time; ③ poor subjective sleep quality, with no relief of drowsiness after waking; ④ total sleep time often less than 6.5 hours; ⑤ a series of daytime functional impairments, such as dizziness, fatigue, poor concentration, lethargy, drowsiness, and decreased memory.

[0003] An international study revealed that 24% of respondents reported unsatisfactory sleep quality, with the prevalence of insomnia even higher at 45.2% in the Chinese population. The situation is similarly concerning among university students, with 16.9% reporting insomnia symptoms and 6.3% potentially meeting the clinical diagnostic criteria for insomnia. Chronic sleep deprivation not only causes daytime drowsiness, fatigue, and memory loss, interfering with normal life and work, but is also a significant contributing factor to emotional problems such as anxiety and depression. Furthermore, it significantly increases the risk of gastrointestinal diseases, obesity, diabetes, cardiovascular disease, atherosclerosis, and even cancer, posing a serious threat to an individual's physical and mental health.

[0004] Insomnia is primarily treated with psychological intervention and medication. Psychotherapy aims to correct patients' negative cognitions and behavioral habits regarding sleep, thereby improving their self-efficacy in coping with insomnia. Commonly used non-pharmacological interventions include cognitive behavioral therapy, sleep restriction techniques, stimulus control training, analytic intention therapy, multimodal therapy, music therapy, and hypnotherapy. However, in practice, due to relatively limited resources and high implementation costs of cognitive therapy, individual differences in patient responses, and the widespread impact of insomnia itself, the dependence on medication is still on the rise. It is important to emphasize that any medication used for insomnia must have both short-term and long-term safety and effectiveness. Many medications can impair daytime functioning due to sedative residual effects upon waking, thus negatively impacting work performance, family life, and social interactions.

[0005] L-Theanine is a non-protein amino acid derived from tea. It can be transported to the small intestine via Na+-dependent transporters, absorbed into the bloodstream, and then cross the blood-brain barrier into the nervous system. It has physiological functions such as combating exercise fatigue, improving sleep, combating depression, improving cognition, and protecting the nervous system. The concentration of L-theanine in the blood reaches its maximum 5 hours after intestinal absorption. Ultimately, part of it is broken down into glutamate and ethylamine, while the other part is transported to the kidneys and excreted in the urine.

[0006] Patent CN202410295182 discloses *Bifidobacterium longum* subsp. *kS*, which secretes γ-aminobutyric acid (GABA) and has sleep-aiding effects; patent CN202310494077 discloses *Lactobacillus plantarum* FPHC0500, which has the effect of fermenting and producing high levels of GABA. Currently, the probiotic strains in published patents that improve sleep are all focused on GABA production; no probiotic strains that metabolize and produce L-theanine to improve sleep have been reported. Therefore, screening a strain of *Bifidobacterium longum* subsp. *kS* with high L-theanine production for improving insomnia and thus enhancing sleep quality is of significant value. Summary of the Invention

[0007] To address the issues that existing probiotics for improving sleep have a single mechanism of action, are prone to causing excessive sedation and affecting daytime function, and have poor efficacy stability in different individuals, making it difficult to cover multiple types of sleep disorders, this invention provides a Bifidobacterium longum subsp. HC2904 that produces L-theanine, its fermentation products, and their applications.

[0008] In a first aspect, the present invention provides a *Bifidobacterium longum* subsp. *longum* HC2904 that produces L-theanine. Bifidobacteriumlongum subsp. longum HC2904 was deposited on February 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26586. The deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0009] Furthermore, the 16S rDNA sequence of Bifidobacterium longum subspecies HC2904 is shown in SEQ ID NO:3.

[0010] Secondly, the present invention provides an L-theanine ferment, which is prepared by inoculating the above-mentioned Bifidobacterium longum subsp. HC2904 into a grain fermentation medium for fermentation. The grain fermentation medium contains the following components in weight percentage: 1%~3% ground sprouted millet, 0.1%~0.5% grape seed powder, 2%~5% jujube powder, and the remainder is distilled water.

[0011] Furthermore, the preparation method of the grain fermentation culture medium is as follows: After washing the millet with distilled water, let it stand and skim off the millet floating on the water surface; spread the remaining millet evenly in the culture cell of the germination box plate, add water to the bottom of the germination box to the designated water level line, start the germination box for germination culture, and stop the culture after germination for 48~72 hours; put the germinated millet into a food processor to grind it, mix the ground germinated millet, grape seed powder, red date powder and distilled water, and sterilize it under high temperature and high pressure to obtain the grain fermentation culture medium.

[0012] Furthermore, the preparation method of L-theanine fermentation product includes the following steps:

[0013] (1) Preparation of primary seed culture: The activated Bifidobacterium longum subsp. HC2904 monoclonal strain was inoculated into a shake flask containing seed culture medium and glucose monohydrate solution was added. The culture was carried out at 30-40℃ for 10-12h to obtain primary seed culture.

[0014] (2) Preparation of secondary seed liquid: Inoculate the primary seed liquid into a shake flask containing grain fermentation medium, add glucose monohydrate solution, and culture at 30~40℃ for 3~4 days to obtain secondary seed liquid;

[0015] (3) Preparation of L-theanine fermentation product: The secondary seed liquid was transferred to a shake flask containing grain fermentation medium and glucose monohydrate solution was added. The mixture was cultured at 30-40℃ for 5-10 days, and the pH was maintained between 6.8 and 7.5 to obtain L-theanine fermentation product.

[0016] Furthermore, the seed culture medium comprises the following components in the following amounts: glucose 25 g / L, peptone 2 g / L, yeast extract 15 g / L, L-cysteine ​​hydrochloride 1.0 g / L, KH₂PO₄ 1.5 g / L, MgSO₄ 0.3 g / L, FeSO₄·7H₂O 8 mg / L, MnSO₄·7H₂O 8 mg / L, VB₁ 1.5 mg / L, VB₃ 1.5 mg / L, VB₅ 1.5 mg / L, VB₂ 12 1.5 mg / L, VH 2 mg / L.

[0017] Thirdly, the present invention provides an application of the above-mentioned Bifidobacterium longum subsp. HC2904 in the production of L-theanine.

[0018] Fourthly, the present invention also provides the application of the above-mentioned L-theanine fermentation product in the preparation of medicines that improve sleep quality and relieve anxiety.

[0019] Furthermore, the medicine is a medicine for improving gastrointestinal discomfort.

[0020] Furthermore, the drug is one that lowers cortisol levels.

[0021] The beneficial effects of this invention are as follows:

[0022] 1. The *Bifidobacterium longum* subsp. *longum* HC2904 provided in this invention can produce high levels of L-theanine during the fermentation of specific raw materials, with a yield of up to 9.66 mg / mL. This overcomes the technical limitations of existing probiotic technologies for improving sleep, which mainly rely on the γ-aminobutyric acid (GABA) metabolic pathway. L-theanine-fermented beverages prepared from this strain can significantly improve sleep quality in people with insomnia, significantly reduce PSQI scores, and synergistically improve emotional health problems such as gastrointestinal discomfort, stress, anxiety, and depression caused by long-term poor sleep quality. Simultaneously, it can significantly reduce cortisol levels in the body, demonstrating a clear physiological regulatory mechanism and promising application prospects.

[0023] 2. The *Bifidobacterium longum* subspecies HC2904 provided by this invention possesses excellent biological characteristics and environmental adaptability. This strain can ferment glucose to produce acid without producing gas, and has a well-defined carbon source metabolic profile. It can effectively utilize multiple carbon sources such as fructose, D-ribose, D-galactose, D-mannose, L-rhamnose, and D-mannitol, which is beneficial for its stable growth and continuous metabolism in the complex intestinal nutrient environment. Simultaneously, this strain has a highly hydrophobic cell surface, exhibits significant adhesion to Caco-2 intestinal epithelial cells, and demonstrates excellent tolerance to simulated gastric and intestinal fluids. It can cross the gastrointestinal barrier with a high survival rate, reach the intestine alive, and stably colonize, thereby continuously exerting its probiotic function.

[0024] 3. The *Bifidobacterium longum* subsp. *longum* HC2904 provided by this invention exhibits significantly superior antioxidant capacity compared to the control strain. Its fermentation supernatant and intracellular extract show high scavenging rates against both DPPH and HRS free radicals and effectively inhibit lipid peroxidation, indicating that this strain can significantly reduce oxidative stress levels in vivo. These antioxidant properties help improve sleep disorders and related metabolic imbalances caused by increased oxidative stress, providing an important biological basis for its application in sleep health and overall bodily regulation. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a colony morphology diagram of Bifidobacterium longum subspecies HC2904.

[0027] Figure 2 This is an optical microscope image of Bifidobacterium longum subspecies HC2904.

[0028] Figure 3 The RAPD fingerprint of Bifidobacterium longum subspecies HC2904.

[0029] Figure 4 The rep-PCR fingerprint of Bifidobacterium longum subspecies HC2904.

[0030] Figure 5 The graph shows the changes in L-theanine levels during the fermentation of Bifidobacterium longum subsp. HC2904.

[0031] Figure 6 This is a graph showing the gastrointestinal discomfort symptom scores of the subjects in Example 9.

[0032] Figure 7 This is a graph showing the emotional problem scoring results of the subjects in Example 9.

[0033] Figure 8 The graph shows the results of the salivary cortisol level test in the subjects of Example 9. Detailed Implementation

[0034] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0035] Example 1: Isolation and Screening of Strains

[0036] 1. Initial screening of lactic acid bacteria

[0037] Fresh fecal samples were collected from elite track and field athletes who had not consumed probiotic preparations within the past six months. The fecal samples were serially diluted using sterile PBS buffer, and 10⁻⁶ samples were collected. -5 10 -6 10 -7 100 μL of each of the three dilution gradients was spread onto MRS selective medium and anaerobic incubated at 37°C for 48 h. After single bacteria grew on the plates, 35 strains of lactic acid bacteria with the shape of bacilli were selected by microscopic examination.

[0038] 2. Secondary screening of acid-resistant strains

[0039] Prepare 1L of MRS liquid culture medium and autoclave at 115℃ for 30min. After the medium cools, add 3.2g of porcine mucosal pepsin, shake well to dissolve, and incubate in a 37℃ water bath for 1h to prepare an acid-resistant culture medium. Inoculate the 10 selected lactobacillus strains into the above acid-resistant culture medium at a 5% inoculum and incubate at 37℃ for 72h. Take the fermentation broth and count the bacterial count according to GB4789.35-2023 "Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0040] The results showed that the fermentation broth of strain HC2904 had the highest viable cell count, reaching 8.71 × 10⁻⁶. 9 CFU / mL. Therefore, strain HC2904 was selected for further research.

[0041] Example 2 Identification of strain HC2904

[0042] 1. Colony morphology identification

[0043] The HC2904 strain was inoculated onto MRS solid medium and anaerobically cultured at 37°C for 24 hours. Colony photos are shown below. Figure 1 As shown in the image, single colonies of HC2904 are milky white, with a diameter of approximately 1.0–3.5 mm. They are round, convex or lenticular in shape, opaque in texture, and range in surface from smooth to mucous-like and soft. After crystal violet staining, under an optical microscope, the HC2904 strain appears as rod-shaped cells with smooth ends and does not form spores. The optical microscope image is shown below. Figure 2 As shown.

[0044] 2. Identification of physiological and biochemical characteristics

[0045] The inoculum in this embodiment was prepared as follows: the activated HC2904 strain was added at an inoculum volume of 2% to a modified MRS liquid medium containing 0.5 g / L L-cysteine ​​hydrochloride, and then anaerobically cultured at 37°C for 36 h.

[0046] 2.1 Temperature growth range experiment

[0047] The HC2904 strain inoculum was inoculated into 10 ml LMR liquid medium at a 10% inoculation rate. The uninoculated 10 ml LMR liquid medium served as a control. The medium was placed in constant temperature shaking incubators at 0℃, 20℃, 37℃, 45℃ and 60℃ for anaerobic incubation for 48 h, and the turbidity of the culture medium was observed.

[0048] The results showed that the culture medium for strain HC2904 remained clear after 48 hours of incubation in constant temperature shaking incubators at 0℃, 20℃, 45℃, and 60℃. However, the culture medium became turbid after 48 hours of incubation at 37℃. This indicates that the optimal growth temperature for strain HC2904 is 37℃.

[0049] 2.2 Salinity tolerance test

[0050] Under aseptic conditions, the activated HC2904 bacterial suspension was inoculated at a rate of 10% into 5 mL of MRS liquid culture medium with salt concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 8%, respectively. 5 mL of uninoculated MRS liquid culture medium was used as a control. The culture was placed at 37°C with shaking for 48 h, and the culture medium was observed to see if it became turbid.

[0051] The results showed that strain HC2904 grew in culture media with salt concentrations of 1% to 3%, but did not grow in culture media with salt concentrations above 4%. The highest salt concentration that strain HC2904 could tolerate was 3%.

[0052] 2.3 Carbon source metabolism experiment

[0053] The phenol red basal medium formula is as follows: 1.5g peptone, 0.6g yeast extract, 1g Tween 800, 0.5mL salt solution, 18mg phenol red, 100mL distilled water, pH 7.4.

[0054] The salt solution consists of the following components: 11.5 g MgSO4·7H2O, 8 g MnSO4·4H2O, and 100 mL distilled water.

[0055] Prepare 10 g / mL solutions of sugars, alcohols, and glycosides, and filter them through a 0.22 μm sterile filter. Under aseptic conditions, add 20 μL of the sterilized carbohydrate solution to each well of a 96-well plate, with three replicates for each carbohydrate. Then add 170 μL of sterilized basal medium containing phenol red, followed by 10 μL of inoculum. Wells without inoculation serve as controls. Add 50 μL of liquid paraffin to each well to prevent moisture evaporation during culture. Incubate at 37°C, using phenol red as an indicator, and observe the color changes of the medium. The results are shown in Table 1.

[0056] Table 1. Results of carbon source metabolism

[0057]

[0058] 2.4 Glucose Acid and Gas Production Test

[0059] The culture medium formulation used in this embodiment is as follows:

[0060] Weigh out 0.5g of peptone, 0.3g of yeast extract, 0.1mL of Tween 80, 0.5mL of salt solution A, 0.5mL of salt solution B, 0.5g of sodium acetate, 2.5g of glucose, and 0.05mL of 2% bromocresol green (w / v). Dissolve these in distilled water to a final volume of 100mL, adjusting the pH to 6.8-7.0. Dispense the prepared culture medium into large test tubes containing inverted small test tubes, 3mL per tube, and autoclave at 121℃ for 15min.

[0061] Preparation method of salt solution A: Dissolve 10g of KH2PO4 and 1.0g of K2HPO4 in distilled water and make up to 100mL.

[0062] Preparation method of salt solution B: Dissolve 11.5g of MgSO4·7H2O, 2.4g of MnSO4·2H2O and 0.68g of FeSO4·7H2O in distilled water and make up to 100mL.

[0063] Under aseptic conditions, the inoculum was inoculated into the culture medium at a rate of 10%, with an uninoculated medium serving as a control. The top was then sealed with 2 mL of sterile liquid paraffin and incubated at 37°C. The medium was incubated for 6 consecutive days, with daily observation of any changes in color. Results showed that after 6 days of incubation, the medium changed from green to yellow, and no gas was detected in the small inverted tubes, indicating that strain HC2904 ferments glucose to produce acid but not gas.

[0064] 2.5 MALDI-TOF-MS Qualification Test

[0065] The procedure according to the kit instructions is as follows: A single clone of the activated HC2904 strain was evenly coated onto a target plate in the form of a thin film. 1 μL of lysis buffer was added to cover the sample, and after drying, 1 μL of matrix solution was added to cover the sample again. After drying, the sample target was placed in a mass spectrometer for identification. The co-crystallized film formed by the sample and matrix was irradiated with a laser to ionize the proteins in the sample. The ions were accelerated through the flight tube under an electric field of 10–20 kV. The molecular weight of the protein was determined based on the different flight times to the detector. Protein fingerprints were obtained using Autofms 1000 Analyzer v1.0 software. The identification results showed that the HC2904 strain was *Bifidobacterium longum* subsp. *longum* (…). Bifidobacteriumlongum subsp. longum ).

[0066] 2.6 Molecular biological identification

[0067] Single colonies of strain HC2904 were picked from the plate and placed in MRS liquid medium. After incubation at 37°C for 24 hours, 800 μL of fermentation broth was taken and processed according to TIANGEN. ®The bacterial genomic DNA extraction kit (catalog number: DP302) was used to obtain the genome of this strain for subsequent molecular biological identification.

[0068] (1) Identification of 16S rDNA gene sequence

[0069] The 16S rDNA gene of strain HC2904 was amplified using the TIANGEN® 2×TaqPCR premix kit. The reaction system and reaction cycle settings were performed in accordance with the kit instructions.

[0070] The primer sequences are as follows:

[0071] 27F: AGAGTTTGATCCTGGCTCA (SEQ ID NO: 1);

[0072] 1492R: GGTTACCTTGTTACGACTT (SEQ ID NO: 2).

[0073] Electrophoresis confirmed that the PCR amplification product size was approximately 1500 bp, which meets the requirements. The 16S rDNA sequencing results are as follows:

[0074]

[0075] The sequence was BLAST-aligned on the EzBioCloud website, and it showed the highest similarity to *Bifidobacterium longum* subsp. *longum*. Therefore, strain HC2904 was identified as *Bifidobacterium longum* subsp. *longum*. Bifidobacteriumlongum subsp. longum ).

[0076] (2) RAPD fingerprint identification

[0077] The HC2904 strain was amplified using the TIANGEN® 2×TaqPCR premix kit. The reaction system and reaction cycle settings were performed according to the kit instructions. The primer sequence was M1: 5'-GAGGGTGGCGGTTCT-3' (SEQ ID NO:4).

[0078] A 1.5% agarose gel plate was prepared, with a DL2000 DNA Marker used as a result control. Electrophoresis was performed at a constant voltage of 100V for 80 minutes. Finally, the electrophoresis pattern was detected using a gel imaging system. The RAPD fingerprint of strain HC2904 is shown below. Figure 4 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 3 The matching RAPD fingerprint pattern indicates that strain HC2904 is a novel subspecies of Bifidobacterium longum.

[0079] (3) Rep-PCR fingerprint identification

[0080] Using TIANGEN ® The 2×TaqPCR premix kit was used to amplify strain HC2904. The reaction system and reaction cycle settings were performed according to the kit instructions. The rep-PCR primer was 5'-GTGGTGGTGGTGGTG-3' (SEQ ID NO:5).

[0081] Prepare 1.5% agarose gel plates, using a DL2000 DNA Marker as a result control. Electrophoresis was performed at 100V for 80 minutes to detect the amplification results. The rep-PCR fingerprint of strain HC2904 is shown below. Figure 5 As shown. A comparison revealed no similarity in existing publicly available reports. Figure 4 The matching rep-PCR fingerprint pattern indicates that the HC2904 strain screened in this invention is a new subspecies of Bifidobacterium longum.

[0082] Based on the combined results of MALDI-TOF-MS identification, molecular biology experiments, carbon source metabolism experiments, and glucose acid and gas production experiments, strain HC2904 is identified as *Bifidobacterium longum* subsp. *longum*. Bifidobacteriumlongum subsp. longum The new strain was named Bifidobacterium longum subsp. HC2904.

[0083] Bifidobacterium longum subsp. longum HC2904 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 20, 2023, with accession number CGMCC No. 26586, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and classified as Bifidobacterium longum subsp. longum. Bifidobacteriumlongum subsp. longum .

[0084] Example 3: Determination of the antioxidant function of Bifidobacterium longum subsp. HC2904

[0085] 1. Determination of DPPH and hydroxyl radical scavenging capacity

[0086] 1.1 Determination of DPPH scavenging ability

[0087] Take 1 mL of PBS fermentation supernatant or intracellular extract of the test strain, add 1 mL of 0.4 mM freshly prepared DPPH radical solution, mix well, and incubate at room temperature in the dark for 30 min. Then measure the absorbance A of the fermentation supernatant and intracellular extract samples at a wavelength of 517 nm. 样品 The test was performed in triplicate. The control group sample was prepared with an equal volume of PBS solution and DPPH-ethanol mixture, and a blank was zeroed with an equal volume of fermentation supernatant or intracellular extract sample mixed with ethanol. Clearance rate = [1 - (A...] 样品 -A 空白 ) / A 对照 ×100%. The commercially available *Bifidobacterium longum* subsp. *longum* NCC3001 was used as a control strain.

[0088] Table 2 DPPH free radical scavenging rate of strain HC2904

[0089]

[0090] As shown in Table 2, the DPPH scavenging rate of fermentation supernatant and intracellular extract of Bifidobacterium longum subsp. HC2904 was significantly better than that of the control strain NCC3001.

[0091] 1.2 Determination of hydroxyl radical (HRS) scavenging ability

[0092] Mix 100 μL of 5 mM sodium salicylate-ethanol solution, 100 μL of 5 mM ferrous sulfate, 500 μL of deionized water, and 200 μL of fermentation supernatant or intracellular extract of *Bifidobacterium longum* subsp. *longum* HC2904. Add 100 μL of 3 mM hydrogen peroxide solution. Incubate at 37°C for 15 min. Measure the absorbance of sample A at 510 nm. Calculate the HRS clearance rate using the following formula: HRS clearance rate (%) = (A...) / (A...) 样品 -A 控制 ) / (A 空白 -A 控制 ) × 100%, where: A 控制 A represents the absorbance of the sample reaction, where deionized water is used instead. 空白 The absorbance is calculated by substituting deionized water for the sample and reacting with H2O2. The commercially available *Bifidobacterium longum* subsp. *longum* NCC3001 was used as a control strain. The results are shown in Table 3.

[0093] Table 3 HRS free radical scavenging rate of strain HC2904

[0094]

[0095] As shown in Table 3, the fermentation supernatant and intracellular extract of Bifidobacterium longum subsp. HC2904 showed significantly better HRS clearance rates than the control strain NCC3001.

[0096] 2. Determination of anti-lipid peroxidation capacity

[0097] Preparation of linoleic acid emulsion: Mix 0.1 mL linoleic acid, 0.2 mL Tween 20, and 19.7 mL deionized water thoroughly. Add 1 mL of the linoleic acid emulsion and 1 mL of 1% FeSO4 solution to 0.5 mL of PBS solution, then add 0.5 mL of fermentation supernatant or intracellular extract of the test strain. Incubate at 37°C for 1.5 h. Add 0.2 mL of 4% TCA solution and 2 mL of 0.8% TBA solution to the mixture, incubate at 100°C for 30 min, cool rapidly, centrifuge at 4000 r / min for 15 min, and collect the supernatant. 532nm The absorbance measured is A. A blank control was set up, and 0.5 mL of distilled water was used to replace the sample reaction, and the absorbance A0 was measured. The inhibition rate was calculated according to the following formula: Inhibition rate (%) = (A0 - A) / A0 × 100%. The commercially available Bifidobacterium longum subsp. NCC3001 was used as the control strain. The results are shown in Table 4.

[0098] Table 4. Lipid peroxidation resistance rate of HC2904 strain

[0099]

[0100] As shown in Table 4, the fermentation supernatant and intracellular extract of Bifidobacterium longum subsp. HC2904 provided by this invention have strong anti-lipid peroxidation ability, and the anti-lipid peroxidation effect is better than that of the control strain NCC3001.

[0101] Based on the combined performance of Bifidobacterium longum subsp. longum HC2904 in DPPH scavenging rate, HRS scavenging rate and anti-lipid peroxidation, it can be concluded that the Bifidobacterium longum subsp. longum HC2904 provided by the present invention has excellent antioxidant capacity.

[0102] Example 4: Gastrointestinal Fluid Tolerance Test

[0103] Simulated gastric juice (pH 3) with 3 g / L pepsin and simulated intestinal juice (pH 8) with 1 g / L ox bile salts and 1 g / L trypsin were prepared. The HC2904 strain was activated twice consecutively, and the absorbance (OD) of the activated bacterial solutions was measured. 600 Adjust to 1.5. Centrifuge 1 mL of bacterial culture at 6000 × g for 10 min at 4℃, discard the supernatant, resuspend the bacterial culture in 1 mL of simulated gastric fluid, and incubate in an anaerobic environment at 37℃ for 3 h. Perform plate colony counting on the sample at the beginning (0 h) and end (3 h) of the incubation. Subsequently, centrifuge the bacterial culture in the simulated gastric fluid for 3 h again at 6000 × g for 10 min at 4℃, discard the supernatant, and resuspend the bacterial culture in an equal volume of simulated intestinal fluid. Continue incubation at 37℃ for 2 h, followed by plate colony counting. Use commercially available Bifidobacterium longum subsp. longum NCC3001 as the control strain. The survival rate of the strains was calculated according to the following formula, and the results are shown in Table 5.

[0104]

[0105] In the formula, N0 and N1 are the number of surviving colonies (CFU / mL) of the strain before and after treatment with simulated gastrointestinal fluid, respectively.

[0106] Table 5. Tolerance of HC2904 to simulated gastrointestinal fluid

[0107]

[0108] Table 5 shows that *Bifidobacterium longum* subsp. *longum* HC2904 exhibits high tolerance to simulated gastric and intestinal fluids, superior to the control strain NCC3001. This indicates that *Bifidobacterium longum* subsp. *longum* HC2904 can tolerate the harsh acid-base environment of the human gastrointestinal tract.

[0109] Example 5: Cell Surface Hydrophobicity Test

[0110] Activated colonies of Bifidobacterium longum subsp. HC2904 were picked and inoculated into freshly prepared MRS liquid medium. The culture was incubated at 37°C with shaking for 24 h. Then, 1% of the culture was added to the MRS liquid medium and cultured at 37°C with shaking for another 24 h. After centrifugation at 6000×g for 10 min, the bacterial cells were collected and washed twice with sterile physiological saline. The bacterial cells were then resuspended in 1 mL of sterile 0.1M KNO3 solution to obtain the bacterial suspension for testing.

[0111] Add 50 μL of the above bacterial suspension to 2450 μL of 0.1 M KNO3 solution and record the OD. 600 The result is A0. 1.5 mL of bacterial suspension was mixed with 500 μL of xylene and allowed to stand at room temperature for 10 min. The two-phase system was vortexed for 2 min and then allowed to stand for 20 min to reform the aqueous and organic phases. The absorbance A1 of the aqueous phase was carefully measured at 600 nm. Cell hydrophobicity was calculated using the following formula: Hydrophobicity (%) = (A0 - A1) / A1 × 100%, with three parallel measurements taken as the average. The commercially available *Bifidobacterium longum* subsp. *longum* NCC3001 was used as a control strain. The results are shown in Table 6.

[0112] Table 6 Results of cell surface hydrophobicity test

[0113]

[0114] As shown in Table 6, the hydrophobicity of the cell surface of *Bifidobacterium longum* subsp. HC2904 provided by this invention is superior to that of the control strain NCC3001. Previous studies have shown a positive correlation between the hydrophobicity of bacterial cell surface and its non-specific adhesion ability to host intestinal epithelial cells or mucus layer. Therefore, the above results indicate that *Bifidobacterium longum* subsp. HC2904 has greater adhesion potential.

[0115] Example 6: Intestinal Epithelial Cell Adhesion Test

[0116] Intestinal epithelial cells (Caco-2) were revived and cultured to the required volume. When the cell density reached approximately 80%, they were digested with trypsin into a single-cell suspension and counted using a hemocytometer to obtain a cell count of 5 × 10⁶ cells. 5 Cells / mL. Then, 500 μL of cell suspension was seeded into 24-well plates with cell spreaders at a seeding density of 2.5 × 10⁻⁶ cells / mL. 5 Cells per well. After overnight culture until fully adhered, discard the culture medium, rinse twice with fresh culture medium, and set aside.

[0117] Fresh culture of *Bifidobacterium longum* subsp. *longum* HC2904 was washed twice with phosphate-buffered saline (pH 7.0), then resuspended in an equal volume of RPMI-1640 medium containing 10% fetal bovine serum. The absorbance was adjusted to achieve the desired OD value. 600The concentration was between 0.8 and 1.0. The control strain, *Bifidobacterium longum* subsp. *longum* NCC3001, was cultured using the same method to obtain a bacterial suspension.

[0118] Add 500 μL of bacterial suspension to a prepared 24-well plate containing Caco-2 intestinal epithelial cells and co-culture in a CO2 incubator for 2 h. Wash three times with phosphate-buffered saline (pH 7.0) to remove unadhered bacteria. Fix the cell smears with methanol for 15 min, then stain with Giemsa stain for 5 min, and rinse with phosphate-buffered saline (pH 7.0). After rinsing, remove the cell smears, place them on a glass slide, and observe them under a microscope. Calculate the adhesion ability of the tested strains using the following formula; the results are shown in Table 7.

[0119]

[0120] Table 7. Results of cell adhesion ability test (CFU / cell)

[0121]

[0122] The results show that the adhesion ability of *Bifidobacterium longum* subsp. *longum* HC2904 to intestinal epithelial cells (Caco-2) is 4.4 times that of strain NCC3001. Specific proteins produced on the surface of probiotic cells, such as fibronectin-binding proteins (FnBPs), S-layer proteins (SLPs), extracellular polysaccharides (EPS), and mucus-binding proteins (MUBs), facilitate colonization of the strain cells in the mucus layer. The adhesion ability of *Bifidobacterium longum* subsp. *longum* HC2904 reaches 93.45 (CFU / cell), indicating that this strain can effectively adhere to intestinal epithelial cells, exerting antioxidant effects and producing beneficial metabolic substances.

[0123] Example 7: Determination of L-theanine production capacity

[0124] 1. Preparation of L-theanine fermentation product

[0125] (1) Preparation of primary seed culture: The activated Bifidobacterium longum subsp. HC2904 monoclonal strain was inoculated into a shake flask containing 30 mL of seed culture medium. 1 mL of sterile glucose monohydrate solution (60%, v / v) was added to the shake flask. The shake flask was placed in a 37℃ incubator and incubated for 10 h.

[0126] The seed culture medium formula is as follows: glucose 25 g / L, peptone 2 g / L, yeast extract 15 g / L, L-cysteine ​​hydrochloride 1.0 g / L, KH₂PO₄ 1.5 g / L, MgSO₄ 0.3 g / L, FeSO₄·7H₂O 8 mg / L, MnSO₄·7H₂O 8 mg / L, VB₁ 1.5 mg / L, VB₃ 1.5 mg / L, VB₅ 1.5 mg / L, VB₂ 12 1.5 mg / L, VH 2 mg / L.

[0127] (2) Preparation of grain fermentation culture medium: After washing the millet with distilled water, let it stand for 10 minutes and skim off the millet floating on the surface. Spread the remaining millet evenly in the culture compartment of the germination chamber plate, add water to the bottom of the germination chamber to the designated water level line, and start the germination chamber for germination culture. Stop the culture after germination for 72 hours, and change the water once a day during the period. Put the germinated millet into a food processor and grind it thoroughly until it is homogeneous. Mix the ground germinated millet, grape seed powder, red date powder and distilled water at a mass ratio of 15:2:25:900, and sterilize the mixture at 121℃ for 15 minutes under high temperature and high pressure to obtain the grain fermentation culture medium.

[0128] (3) Preparation of secondary seed culture: The primary seed culture was inoculated into a shake flask containing 30 mL of grain fermentation medium. 1 mL of sterile glucose monohydrate (60%, v / v) was added to the shake flask. The shake flask was placed in a 37℃ incubator and incubated for 3 days to obtain the secondary seed culture of Bifidobacterium longum subsp. HC2904.

[0129] (4) Preparation of L-theanine fermentation product: 3 mL of secondary seed culture was transferred to a shake flask containing 100 mL of grain fermentation medium. 1 mL of sterile glucose monohydrate (60%, v / v) and 2 mL of phenol red solution were added to the shake flask. The shake flask was then placed in a 37°C incubator and incubated for 7 days. Phenol red solution was used as a pH indicator. During the incubation period, the pH was adjusted by observing the color change in the shake flask and adding 25% ammonia solution as needed to maintain the pH between 6.8 and 7.5. After the incubation was completed, L-theanine fermentation product was obtained.

[0130] 2. Determination of L-theanine content by high performance liquid chromatography

[0131] (1) Fermentation broth treatment: Dilute the supernatant of the sample obtained from fermentation by 10 times so that the concentration of theanine in the diluted sample is within the range of the standard used.

[0132] (2) Pre-column derivatization: Use a pipette to add 10 μL of diluted sample, 200 μL of derivatization buffer and 300 μL of derivatizing agent solution to a 1.5 mL EP tube. After mixing, incubate in a 65 °C water bath for 1 h (this process should be protected from light). After the reaction is complete, allow it to cool to room temperature naturally. Add 690 μL of volume buffer (mix well and filter through a 0.22 μm organic membrane into a 2 mL brown liquid chromatography vial).

[0133] (3) High-performance liquid chromatography (HPLC) detection: An Agilent ZORBAX Eclipse AAA column (4.6 mm × 150 mm, 5 mm) was used for detection at a wavelength of 360 nm. The column operating temperature was 33 °C. Mobile phase A was 50% (v / v) acetonitrile solution, and mobile phase B was 50 mM sodium acetate and 130 mM N,N-dimethylformamide aqueous solution (pH 5.0). The retention time of theanine was approximately 12.2 min. Analysis was performed using a binary gradient elution method with a mobile phase flow rate of 1 mL / min. The gradient changes are shown in Table 8.

[0134] Table 8. Changes in the mobile phase gradient

[0135]

[0136] During the static culture process, starting from day 0, 1.5 mL of bacterial culture was collected daily, centrifuged at 1300 rpm for 2 min to obtain the supernatant of the fermentation broth, and the L-theanine content was detected. Three samples were taken at each point, and the test results are as follows. Figure 5 As shown. (Through) Figure 5 It can be seen that Bifidobacterium longum subsp. HC2904 can produce high concentrations of L-theanine using the fermentation medium formula, and the L-theanine yield reaches 9.66 mg / mL after fermentation on the 7th day, indicating that Bifidobacterium longum subsp. HC2904 has excellent L-theanine production capacity.

[0137] Example 8: Preparation of fermented beverage made from Bifidobacterium longum subsp. HC2904

[0138] 1. Preparation of L-theanine fermentation product

[0139] The preparation of primary seed culture, secondary seed culture, and grain fermentation medium is described in Example 7.

[0140] The secondary seed culture was inoculated into a fermenter containing 15L of grain fermentation medium at an inoculation rate of 10%. The fermentation temperature was 37℃, the pressure in the fermenter was not lower than 0.03MPa, the pH was maintained at 6.5, the rotation speed was 200rpm, and the culture was carried out for 7 days until the end of fermentation. After fermentation was completed, the L-theanine content in the fermentation supernatant was detected according to the method in Example 7, and the L-theanine content was 7.48g / L. The fermentation supernatant was then subjected to ultra-high pressure treatment at 450MPa and 15℃ for 10min to kill the bacteria and obtain an inactivated fermentation supernatant.

[0141] 2. Preparation of Fermented Beverages

[0142] After adding the inactivated supernatant of the fermentation material to the preparation tank, 0.5% xanthan gum, 0.05% galactooligosaccharides, 0.05% fructooligosaccharides, 0.01% nicotinamide, and 0.01% cyclamate are added by weight / volume ratio. The mixture is then sterilized at 135℃ for 8 seconds using an instantaneous high-temperature sterilizer. After the material temperature cools to room temperature, it is aseptically filled, packaged, and stored to obtain L-theanine fermented beverage.

[0143] Example 9: Observation on the therapeutic effect of Bifidobacterium longum subsp. longum HC2904 fermentation product on sleep quality in community insomnia patients.

[0144] 1. General Information

[0145] Fifty insomnia patients who visited a traditional Chinese medicine clinic were selected. The diagnostic criteria were based on the "Guidelines for the Diagnosis and Treatment of Insomnia in Chinese Adults (2017)". All participants signed informed consent forms and had an undisturbed and constant sleep environment. The inclusion and exclusion criteria for the participants are shown in Table 9.

[0146] Table 9. Intake and Excretion Standards for Studies on the Sleep-Improving Effects of Fermented Drinks

[0147]

[0148] 2. Intervention Plan

[0149] All subjects orally took the L-theanine fermented beverage prepared in Example 8, 30 minutes before bedtime, once a day, 50 mL each time, for 28 days, followed up for one month.

[0150] 3. Test Plan and Result Analysis

[0151] 3.1 Sleep quality test

[0152] The Pittsburgh Sleep Quality Index (PSQI) was used to assess the sleep status of the subjects at day 0, day 28, and 58 days after discontinuation of medication. The PSQI includes seven factors: sleep quality, sleep onset time, sleep duration, sleep efficiency, sleep disturbances, hypnotics, and daytime dysfunction. Each factor is scored from 0 to 3, and the total score of all factors is the PSQI score. The results are shown in Table 10.

[0153] Table 10. Total PSQI Score and Factor Scores for Each Visit Site

[0154]

[0155] Note: * indicates a significant difference compared to day 0 (P<0.05); ** indicates an extremely significant difference compared to day 0 (P<0.01).

[0156] The subjects showed significant improvement in overall sleep quality after using L-theanine fermented beverage prepared from *Bifidobacterium longum* subsp. *longum* HC2904 for 28 days. As shown in Table 11, on day 0, the subjects' total PSQI score was 16.89, indicating very poor sleep quality. After 28 days of using the L-theanine fermented beverage, the total PSQI score decreased by 48.68% to 8.67, showing a significant improvement in sleep quality compared to day 0 (P<0.01). One month later (day 58), the improvement in sleep quality continued, with the total PSQI score at 7.89, also showing a significant difference compared to day 0 (P<0.01). Looking at the factors of the PSQI scoring scale, on day 28, the subjects' sleep quality, sleep onset time, and sleep duration all showed significant improvement compared to day 0 (P<0.05). Furthermore, after one month of follow-up (day 58), the subjects' sleep quality, sleep onset time, and sleep duration still showed sustained significant improvement compared to day 0 (P<0.05). The subjects' sleep efficiency, sleep disturbance, hypnotic drug use, and daytime dysfunction all decreased on day 28 and after one month of follow-up (day 58) compared to day 0, but the differences were not statistically significant (P>0.05).

[0157] 3.2 Methods for assessing gastrointestinal symptoms

[0158] The Gastrointestinal Symptom Scale (GSRS) was used to assess the improvement of gastrointestinal symptoms in subjects, including six indicators: abdominal pain, bloating, borborygmus, heartburn, constipation, and diarrhea. A score of 1 represents none; 2 represents slight symptoms; 3 represents minor symptoms; 4 represents moderate symptoms; 5 represents significant discomfort; 6 represents severe symptoms; and 7 represents very severe symptoms. GSRS scores were observed on days 0, 14, and 28.

[0159] After the test subjects consumed L-theanine fermented beverage prepared from *Bifidobacterium longum* subsp. *longum* HC2904, their gastrointestinal discomfort symptoms showed significant improvement. Figure 6 As shown, after 14 days of using L-theanine fermented beverage, the subjects' gastrointestinal discomfort symptom scores decreased from an initial 29.30 to 17.20, a decrease of 41.3%, which was significantly different from day 0 (P<0.01). By day 28, the gastrointestinal discomfort symptom scores had decreased by 54.95% to 13.20%, which was also significantly different from day 0 (P<0.01). This indicates that after taking L-theanine fermented beverage prepared from Bifidobacterium longum subsp. HC2904, the subjects' sleep indicators improved, and their gastrointestinal health also improved, with a significant reduction in discomfort such as abdominal pain, diarrhea, constipation, and bloating.

[0160] 3.3 Emotion Assessment Methods

[0161] The DASS-21 self-rating emotion scale was used to assess the improvement of depression, anxiety and stress in the subjects. The observation indicators are shown in Table 11. The subjects' emotion scores were observed on day 0 and day 28.

[0162] Table 11 Emotional Self-Rating Scale

[0163]

[0164] Note: Scoring criteria: 0 - No; 1 - Sometimes; 2 - Often; 3 - Always; Items 1-7 assess stress; Items 8-14 assess anxiety; Items 15-21 assess depression.

[0165] After the test subjects consumed an L-theanine fermented beverage prepared from *Bifidobacterium longum* subsp. *longum* HC2904, their long-term poor sleep quality and related emotional health problems showed significant improvement. Figure 7 As shown, after 28 days of using the L-theanine fermented beverage, the subjects' stress scores decreased from 11.75 to 7.50, a decrease of 36.17%, which was significantly different from day 0 (P<0.05); the subjects' anxiety scores decreased from 9.63 to 6.50, a decrease of 32.47%, which was also significantly different from day 0 (P<0.05); the subjects' depression scores decreased from 3.13 to 3.00, with no significant difference in depression compared to day 0 (P>0.05). This indicates that after consuming the L-theanine fermented beverage prepared from Bifidobacterium longum subsp. HC2904, the subjects' stress and anxiety levels were significantly improved.

[0166] 3.4 Detection of salivary cortisol levels

[0167] Saliva samples were collected from subjects at each of the following visit times: day 0, day 14, day 28, and day 58 (one month follow-up). Subjects were prohibited from brushing their teeth or eating before saliva sample collection. Saliva cortisol levels were measured using a human cortisol ELISA kit.

[0168] like Figure 8 As shown, after 14 days of using L-theanine fermented beverage, the subjects' cortisol levels decreased from 24.04 nmol / L to 20.53 nmol / L, a decrease of 14.61%, which was significantly different from day 0 (P<0.01). By day 28, the subjects' cortisol levels had decreased by 27.81% to 17.36 nmol / L, which was also significantly different from day 0 (P<0.05). After one month of follow-up, the cortisol level maintained its decreased level at 18.61 nmol / L. Cortisol is a glucocorticoid produced by the adrenal glands and regulated by the hypothalamic-pituitary-adrenal axis. High cortisol levels reduce the duration of deep sleep, leading to increased fatigue the next day, and can also overactivate mood centers such as the amygdala, thereby increasing anxiety. The decrease in cortisol levels in the subjects was consistent with the results of improved sleep quality and reduced anxiety symptoms.

[0169] In summary, after using the L-theanine fermented beverage prepared from *Bifidobacterium longum* subsp. *longum* HC2904, the participants experienced a 48.68% decrease in their total PSQI score and a significant improvement in sleep quality. The participants also showed significant improvement in gastrointestinal discomfort, stress, anxiety, and depression caused by long-term poor sleep quality. Furthermore, cortisol levels significantly decreased after using the L-theanine fermented beverage. These findings indicate that the L-theanine fermented beverage prepared from *Bifidobacterium longum* subsp. *longum* HC2904 can improve sleep quality and alleviate gastrointestinal and emotional health problems caused by poor sleep quality.

[0170] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. 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 also be covered within the protection scope of the present invention.

Claims

1. A long subspecies of Bifidobacterium longum HC2904 that produces L-theanine, characterized in that, Bifidobacterium longum subsp. ( Bifidobacteriumlongum subsp. longum HC2904 was deposited on February 20, 2023, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 26586.

2. An L-theanine ferment, characterized in that, The product is prepared by inoculating Bifidobacterium longum subsp. HC2904 as described in claim 1 into a grain fermentation medium for fermentation. The grain fermentation medium contains the following components in weight percentage: 1%~3% milled sprouted millet, 0.1%~0.5% grape seed powder, 2%~5% jujube powder, and the remainder is distilled water.

3. The L-theanine ferment as described in claim 2, characterized in that, The preparation method of grain fermentation culture medium is as follows: After washing the millet with distilled water, let it stand and skim off the millet floating on the water surface; spread the remaining millet evenly in the culture cell of the germination chamber plate, add water to the bottom of the germination chamber to the specified water level line, start the germination chamber for germination culture, and stop the culture after germination for 48~72 hours; put the germinated millet into a food processor to grind it, mix the ground germinated millet, grape seed powder, red date powder and distilled water, and sterilize it under high temperature and high pressure to obtain the grain fermentation culture medium.

4. The L-theanine ferment as described in claim 2, characterized in that, Its preparation method includes the following steps: (1) Preparation of primary seed culture: The activated Bifidobacterium longum subsp. HC2904 monoclonal strain was inoculated into a shake flask containing seed culture medium and glucose monohydrate solution was added. The culture was carried out at 30-40℃ for 10-12h to obtain primary seed culture. (2) Preparation of secondary seed liquid: The primary seed liquid is inoculated into a shake flask containing grain fermentation medium, and glucose monohydrate solution is added. The mixture is cultured at 30-40℃ for 3-4 days to obtain the secondary seed liquid. (3) Preparation of L-theanine fermentation product: The secondary seed liquid was transferred to a shake flask containing grain fermentation medium and glucose monohydrate solution was added. The mixture was cultured at 30-40℃ for 5-10 days, and the pH was maintained between 6.8 and 7.5 to obtain L-theanine fermentation product.

5. The L-theanine ferment as described in claim 4, characterized in that, The seed culture medium comprises the following components in the following proportions: glucose 25 g / L, peptone 2 g / L, yeast extract 15 g / L, L-cysteine ​​hydrochloride 1.0 g / L, KH₂PO₄ 1.5 g / L, MgSO₄ 0.3 g / L, FeSO₄·7H₂O 8 mg / L, MnSO₄·7H₂O 8 mg / L, VB₁ 1.5 mg / L, VB₃ 1.5 mg / L, VB₅ 1.5 mg / L, VB₂ 12 1.5 mg / L, VH 2 mg / L.

6. The use of Bifidobacterium longum subsp. HC2904 as described in claim 1 in the production of L-theanine.

7. The use of the L-theanine ferment as described in any one of claims 2-5 in the preparation of a medicine for improving sleep quality and relieving anxiety.

8. The application as described in claim 7, characterized in that, The medicine is for improving gastrointestinal discomfort.

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