Lactobacillus salivarius and its application for improving sleep and anxiety

Abstract

This invention discloses a *Ligilactobacillus salivarius* YS-SA09, with accession number CGMCC NO.37763 and Latin name *Ligilactobacillus salivarius*. This invention also discloses a *Citrus aurantium* ferment, obtained by fermenting *Citrus aurantium* with the aforementioned *Ligilactobacillus salivarius* YS-SA09. This invention further discloses the applications of *Ligilactobacillus salivarius* YS-SA09 and the *Citrus aurantium* ferment, which can effectively improve sleep in mice with insomnia models, effectively shorten sleep latency, and prolong sleep onset time; effectively improve depressive-like behavior in mice with CUMS models; significantly reverse the decrease in neurotransmitter levels caused by CUMS in mice, effectively increase the levels of GABA, 5-HT, DA, and NE in the serum of depressed mice; reduce serum TNF-α and IL-6 levels, and improve inflammatory factor levels.

Description

Technical Field

[0001] This invention belongs to the field of probiotic fermentation, specifically relating to a strain of Lactobacillus saliva and its application in improving sleep and anxiety. Background Technology

[0002] Insomnia is a sleep disorder characterized by persistent difficulty falling asleep, difficulty maintaining sleep, or early awakening, accompanied by daytime dysfunctions such as fatigue, decreased attention, and mood disturbances. In recent years, the prevalence of insomnia has shown a significant upward trend globally. China's prevalence rate of 15.00% is higher than the global average, and it is showing a trend towards affecting younger people, with an incidence rate of 18.20% in adolescents and as high as 67.20% in the elderly.

[0003] Insomnia is often accompanied by anxiety, depression, and other disorders. 40% of insomnia patients have one or more mental disorders, with anxiety disorders accounting for 24%. There is a moderate correlation between insomnia and anxiety. Among patients with comorbid insomnia and anxiety, anxiety disorder precedes insomnia in 73% of cases, while insomnia precedes anxiety in 69%. Therefore, insomnia is a common symptom of anxiety attacks and a major contributing factor to anxiety; conversely, anxiety can also be a risk factor for chronic insomnia. Therefore, alleviating anxiety is an important means of treating insomnia. Because drug treatment for insomnia and anxiety is fast-acting and effective, it remains the most widely used clinical treatment. However, currently used sleeping pills and anti-anxiety medications all have varying degrees of side effects. Long-term use can lead to dependence and tolerance, and withdrawal symptoms may occur upon discontinuation.

[0004] Gamma-aminobutyric acid (GABA) is a naturally occurring inhibitory neurotransmitter widely distributed in vertebrates, plants, and microorganisms, possessing various physiological functions such as anti-anxiety, blood pressure reduction, sleep promotion, anti-epileptic, and anti-depressant effects. Currently, many microorganisms, including lactic acid bacteria, yeasts, and certain Gram-negative bacteria, have been confirmed to possess the ability to synthesize GABA. Research on GABA production through microbial fermentation mainly focuses on screening dominant strains, optimizing fermentation processes, and verifying its anti-inflammatory, sleep-aiding, and anti-fatigue effects, and some progress has been made. However, existing technologies still have the following problems: in actual fermentation processes, the growth, metabolic pathways, and GABA yield of strains are easily affected by fermentation conditions, culture medium composition, and other factors, leading to unstable production processes and restricting its large-scale application.

[0005] Citrus aurantium L. var. amara Engl. (CAVA) is a resource used for both food and medicine. It is rich in various active ingredients such as flavonoids, volatile oils, and alkaloids. Traditional Chinese medicine believes it has the effects of regulating qi and soothing the liver, relieving chest tightness, and calming the mind. The use of microbial fermentation technology to enhance the functional properties of food-medicine homologous substances has been widely verified.

[0006] However, the existing applications of fermentation of bitter orange are mainly concentrated in the cosmetics field. For example, patent CN117965644B discloses a fermented product of bitter orange fruit, its preparation method and application. It improves the content of effective substances in bitter orange fruit and reduces toxic side effects through lactobacillus fermentation. It also verifies that the fermented product has a strong ability to protect against oxidative damage. However, it is mainly used as a cosmetic raw material.

[0007] It is worth noting that although some studies have reported probiotics capable of producing high levels of GABA, these strains often do not simultaneously possess excellent fermentation performance on the bitter orange substrate, making it difficult to achieve efficient conversion and enrichment of active ingredients. For bitter orange, a resource with both medicinal and edible properties, how to screen probiotics that possess both high GABA production capacity and good fermentation adaptability, and establish stable and efficient fermentation processes to improve the bioavailability and functional characteristics of its active ingredients, remains a pressing technical problem that needs to be solved.

[0008] Therefore, targeted screening of probiotics that combine high GABA production capacity with excellent bitter orange fermentation performance, and the development of functional fermented products that can effectively improve sleep and anxiety based on these probiotics, is of great significance for making up for the shortcomings of existing technologies and expanding the application of bitter orange in the field of functional foods. Summary of the Invention

[0009] The first objective of this invention is to provide a *Lactobacillus salivarius* YS-SA09 that can efficiently ferment *Citrus aurantium* and specifically release GABA.

[0010] The second objective of this invention is to provide a fermented product of bitter orange blossom.

[0011] A third objective of this invention is to provide a method for preparing the fermented product of *Citrus aurantium*.

[0012] A fourth objective of this invention is to provide an application of the aforementioned *Lactobacillus saliva-associated* YS-SA09.

[0013] A fifth object of the present invention is to provide an application of the said bitter orange blossom ferment.

[0014] This invention is achieved through the following technical solution: A strain of *Ligilactobacillus salivarius* YS-SA09, with accession number CGMCC NO.37763, was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China (Institute of Microbiology, Chinese Academy of Sciences). The strain was viable at the time of deposit.

[0015] A fermented product of bitter orange, obtained by fermenting bitter orange with Lactobacillus saliva-associated YS-SA09.

[0016] The method for preparing the fermented product of bitter orange includes the step of inoculating the seed liquid obtained by activating Lactobacillus saliva-associated with YS-SA09 into the fermentation medium of bitter orange to obtain the fermented product of bitter orange.

[0017] The fermentation temperature was 37°C; The fermentation time is 52 hours; The inoculation amount of the seed solution is 3% (v / v); The viable bacteria count in the seed liquid is 1~9×10⁻⁶. 9 CFU / mL.

[0018] The preparation method of the fermentation medium of Citrus aurantium includes the following steps: weighing Citrus aurantium powder, adding water at a ratio of 10% (w / v) and mixing evenly to a fixed volume, and sterilizing at 121°C for 15 min.

[0019] The method for preparing the fermented product of bitter orange blossom further includes a post-processing step; The post-processing steps include inactivating the fermented product of *Citrus aurantium* at 105°C for 30 min, cooling it to 4°C, centrifuging it at 8000 rpm for 5 min, and collecting the supernatant.

[0020] The method for preparing the seed culture includes streaking activation of *Lactobacillus saliva-associated* YS-SA09, which was frozen in glycerol at -80℃, on MRS solid medium and cultured at 37℃ for 48 h. Then, a single colony is picked and inoculated into MRS liquid medium, and cultured statically at 37℃ for 24 h. This is followed by two passages to obtain the activated bacterial culture. The activated bacterial culture is then centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant is discarded, and the culture is washed and resuspended with physiological saline. The bacterial concentration is adjusted to 1~9×10⁻⁶. 9 The steps for CFU / mL.

[0021] The application of the aforementioned Lactobacillus saliva-associated with salivary glands YS-SA09 is used in the preparation of health products and pharmaceuticals that help improve sleep.

[0022] The application of the aforementioned Lactobacillus saliva-associated with YS-SA09 is used in the preparation of antidepressant drugs.

[0023] The aforementioned bitter orange blossom ferment is used in the preparation of health products and medicines that improve sleep; or It is used in the preparation of antidepressant drugs.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a strain of *Lactobacillus salivarius* YS-SA09 obtained through targeted screening based on GABA release levels. This bacterium not only produces high levels of GABA but also effectively ferments *Citrus aurantium* and effectively enriches the fermentation product with active substances such as GABA and flavonoids.

[0025] This invention provides a method for preparing bitter orange blossom ferment. This method is simple and can effectively enrich GABA and increase the levels of various synergistic flavonoid aglycones without the addition of monosodium glutamate (MSG). It can be applied to the preparation of products that improve sleep and anxiety.

[0026] The salivary-associated Lactobacillus YS-SA09 and Citrus aurantium ferment broth provided by this invention can effectively improve the sleep status of mice with insomnia model, effectively shorten the sleep latency time of mice, and prolong the sleep onset time of mice; can effectively improve the depressive-like behavior of mice with CUMS model; can significantly reverse the decrease in neurotransmitter levels in mice caused by CUMS, effectively increase the content of GABA, 5-HT, DA, and NE in the serum of depressed mice; reduce the serum TNF-α and IL-6 levels, and improve the level of inflammatory factors. Attached Figure Description

[0027] Figure 1 The results of the acid resistance test of the lactic acid bacteria in Example 1; Figure 2 The relative expression levels of relevant genes before and after fermentation in Example 4; Figure 3 The sleep latency of mice in each group in Example 5; Figure 4 The sleep duration of each group of mice in Example 5; Figure 5 The sucrose preference index of each group of mice in Example 6; Figure 6 This refers to the resting time of the mice in each group in Example 6; Figure 7 The swimming and floating times of the mice in each group in Example 6; Figure 8 The activity distance of the mice in each group in Example 6; Figure 9 The NMR spectrum of high-senna aminobutyrate is shown. Detailed Implementation

[0028] Example 1 Targeted screening and identification of strains Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter that plays an important physiological role in the human body. It has beneficial effects on improving sleep, calming the nerves, and reducing anxiety, and can be applied in food, health products, and pharmaceuticals. Therefore, this invention aims to selectively screen a lactic acid bacterium that can produce high levels of GABA and has excellent fermentation performance.

[0029] The specific screening process is as follows: 1. Strains Isolation and Purification Saliva samples, infant feces, and breast milk were collected and serially diluted 10-fold with sterile physiological saline. 100 μL of each dilution was spread onto MRS solid medium and incubated at 37°C for 48 h. After colonies grew, typical lactic acid bacteria colonies (round, milky white, smooth and moist surface, regular edges, and a raised center) were selected and repeatedly streaked on MRS solid medium for purification until pure cultures were obtained. Gram staining and microscopic examination were performed on the pure cultures, retaining Gram-positive, cocci or short rod-shaped strains arranged in pairs or short chains.

[0030] A total of 15 strains were obtained in this experiment through isolation, purification, and microscopic examination. They were frozen at -80℃ with glycerol for later use.

[0031] 2. Initial screening of strains with high GABA production and acid tolerance To screen for target strains, given that the fermentation environment is slightly acidic, we designed a primary screening method using GABA release and acid tolerance as the core indicators.

[0032] Strain activation and inoculation: The 15 preserved bacterial strains were activated to prepare seed culture, and then inoculated into MRS liquid medium containing 0.3% L-glutamate at a 3% inoculum rate and cultured at 37°C for 24 hours. The fermentation broth was centrifuged at 8000 rpm for 10 minutes at 4°C, and the supernatant was collected.

[0033] Detection of GABA and glutamate levels: GABA and glutamic acid content determination: HPLC method was used. Chromatographic conditions: ZORBAX Eclipse Plus C18 column (4.6×250mm, 5μm), mobile phase: acetonitrile: 0.1% (v / v) phosphoric acid aqueous solution = 5:95 (v / v), flow rate: 1.0 mL / min, column temperature: 30℃, detection wavelength: 210 nm (after derivatization), injection volume: 10 μL. Pre-column derivatization: the sample was reacted with dansyl chloride solution at 60℃ in the dark for 30 min.

[0034] Initial screening results: The initial screening results of 15 strains are shown in Table 1.

[0035] Table 1 Initial screening results of strains Screening criteria were set for the above indicators: ① GABA content in group A ≥ 5.0 g / L; ② Glutamic acid conversion rate > 60%, indicating that the strain prefers to utilize L-glutamate and release GABA.

[0036] The initial screening results of 15 strains are shown in the table above. The strains that meet both criteria are M40S3, M40S17, M41S5, M41S13 and M42S14. Further acid resistance tests were conducted on these 5 strains.

[0037] Screening for acid-resistant strains: The bacterial suspensions of the above 5 strains were inoculated into MRS liquid medium at an inoculation rate of 3%. The pH values ​​were adjusted to 3, 3.5, 4, 4.5 and 5 with citric acid. The experiment was repeated in triplicate. The culture was carried out at 37℃ for 24 h. Uninoculated MRS liquid medium was used as a blank control. The OD600 of each sample was measured.

[0038] For details on the acid resistance results of the 5 strains, please refer to [link / reference]. Figure 1 Five lactic acid bacteria strains showed different growth states under different acidity conditions. M41S13 had the largest OD at pH 3, 4, and 5, while M40S17 had the largest OD at pH 3.5 and 4.5. Based on the GABA content, strains M41S13 and M40S17 were selected for the fermentation experiment of Citrus aurantium.

[0039] 3. Secondary screening of strains Preparation of fermentation medium for Citrus aurantium: Weigh Citrus aurantium powder, add purified water at a ratio of 10% (w / v) and mix well to a fixed volume. Sterilize at 121℃ for 15 min and cool before use.

[0040] M41S13 and M40S17 were inoculated at a 3% inoculum and then inoculated into the fermentation medium of *Citrus aurantium*. After anaerobic fermentation at 37°C for 48 hours, the fermentation broth was centrifuged at 4°C and 8000 r / min for 10 minutes, and the supernatant was collected.

[0041] The viable count of strains (logCFU / mL) was calculated using the plate count method, the content of GABA (g / L) was detected by HPLC, and the increase in total flavonoids (mg / L) was detected by colorimetry.

[0042] The results of the secondary screening are shown in Table 2: Table 2 Comparison of fermentation performance of dominant strains after 48 hours Note: Δ represents the difference between the 48h value and the 0h value.

[0043] As shown in Table 2, strain M40S17 exhibited the most comprehensive fermentation performance during the fermentation of multiflora. Therefore, M40S17 was selected as the core strain of this invention.

[0044] 4. Molecular biological identification of strain YS-SA09 (M40S17) The culture medium of the isolated strain was centrifuged at 5000 rpm for 10 min. After centrifugation, the supernatant was discarded, and the pure culture cells to be tested were collected. Genomic DNA of the strain was extracted using a DNA extraction kit according to the manufacturer's instructions. The extracted genomic DNA was used as a template for PCR amplification. Based on morphological and physiological biochemical characteristics, the strain was identified as *Lactobacillus salivarius* and named YS-SA09. It was deposited on February 9, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 37763. Its 16S rRNA gene sequence is shown in SEQ ID NO:1.

[0045] SEQ ID NO. 1: Example 2 Fermentation of strain YS-SA09 To further verify the fermentation capacity of *Lactobacillus salivarius* YS-SA09, this example uses *Lactobacillus salivarius* YS-SA09 to ferment *Citrus aurantium*, *St. John's wort*, *Albizia julibrissin*, *Bupleurum chinense*, *Acorus tatarinowii*, and *Morinda officinalis*. The specific fermentation process is as follows: 1. Preparation of fermentation products (1) Preparation of highly active seed liquid Lactobacillus saliva-associated strain YS-SA09, frozen in glycerol at -80℃, was streaked onto MRS solid medium (purchased from Wuhan Pronosai) for activation and cultured at 37℃ for 48 h. Single colonies were picked and inoculated into MRS liquid medium, incubated statically at 37℃ for 24 h, and passaged twice to restore good viability. The activated bacterial suspension was centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline and resuspended. The bacterial concentration was adjusted to 1~9×10⁻⁹. 9 The CFU / mL concentration yielded a highly active seed solution, which was then stored at 4°C for later use.

[0046] (2) Preparation of fermentation products The following were separately prepared as fermented seed liquids: *Citrus aurantium*, *St. John's wort*, *Albizia julibrissin*, *Bupleurum chinense*, *Acorus tatarinowii*, and *Morinda officinalis*. Purified water was added at a ratio of 10% (w / v) to the liquid, and the mixture was stirred until homogeneous and brought to a final volume. The mixture was then sterilized at 121℃ for 15 minutes. After cooling to room temperature, the above-mentioned highly active seed liquid was inoculated at a rate of 3% (v / v), and the mixture was stirred at low speed until homogeneous. The mixture was then allowed to ferment at 37℃ for 48 hours. After fermentation, the fermentation broth was immediately transferred to 4℃ to terminate the fermentation process, yielding fermentation broths of *Citrus aurantium*, *St. John's wort*, *Albizia julibrissin*, *Bupleurum chinense*, *Acorus tatarinowii*, and *Morinda officinalis*.

[0047] (3) Post-fermentation treatment The above fermentation broth was inactivated at 105℃ for 30 min, cooled to 4℃, centrifuged at 8000 rpm for 5 min, and the supernatant was collected to obtain the fermentation product.

[0048] The test data for GABA production and total flavonoid content in the fermentation product are shown in Table 3 below.

[0049] Table 3. Results of detection of major components after fermentation in different groups As shown in Table 3, glutamic acid in bitter orange flowers was efficiently converted into GABA under the fermentation of Lactobacillus salivarius YS-SA09. This indicates that the glutamic acid decarboxylase in Lactobacillus salivarius YS-SA09 may have expression specificity and can efficiently utilize the glutamic acid in bitter orange flowers. Therefore, we subsequently chose bitter orange flowers as the fermentation substrate.

[0050] Meanwhile, liquid chromatography-mass spectrometry (LC-MS) analysis of the fermentation broth of *Citrus aurantium* revealed the presence of high levels of senna-based aminobutyrate (GABA). The structure of the high-senna-based GABA is as follows: The NMR data for high-scented gentamicin gamma-aminobutyrate are as follows: 1H NMR (300MHz, DMSO, δ, ppm): 12.06 (1H in alcohol), 10.29 (1H inalcohol), 7.18 (1H in 1-benzene), 6.97 (1H in 1-benzene), 6.96 (1H in 1-benzene), 6.06 (1H in 1-benzene), 5.95 (1H in 1-benzene) 5.51 (1H inmethine), 3.83 (3H in methyl), 3.38 (1H in methylene), 3.135 (1H inmethylene), 2.68 (2H in methylene), 2.52 (2H in methylene), 2.04 (2H inmethylene).1.5 (2H in amine). This substance can serve as a prodrug for both succinate and GABA, thus extending their half-lives. Furthermore, the content of succinate-containing GABA in the fermentation product of *Citrus aurantium* is 2.11 ± 0.10 g / L. The presence of succinate-containing GABA also indicates that *Lactobacillus salivarius* YS-SA09 possesses highly efficient esterification enzymes.

[0051] To increase the GABA and total flavonoid content in the fermentation products of bitter orange blossom and thus enhance its potential efficacy in improving sleep and regulating mood, this embodiment further optimized key fermentation parameters. The evaluation indicators were defined as "GABA yield," which is directly related to sleep improvement and antidepressant activity, and "total flavonoid aglycone conversion rate," which reflects the synergistic potential of the active ingredients in bitter orange blossom.

[0052] 1. Single-factor experimental design: The pH was fixed at 4.5, and the concentration of bitter orange blossom was 10%.

[0053] (1) Effect of different inoculum amounts on fermentation: The inoculum amounts of YS-SA09 were 1%, 2%, 3%, 4% and 5%, respectively, while other processes remained unchanged. Samples were taken to determine the contents of GABA and total flavonoids.

[0054] (2) Effect of different fermentation times on fermentation: Fermentation times were set to 24h, 36h, 48h, 60h and 72h respectively, with other processes unchanged, and samples were taken to determine the contents of GABA and total flavonoids.

[0055] (3) The effect of different fermentation temperatures on fermentation: Fermentation temperatures were set at 31℃, 34℃, 37℃, 40℃, and 43℃, with other processes remaining unchanged. Samples were taken and the contents of GABA and total flavonoids were determined.

[0056] (4) Results Analysis: The effect of vaccination volume: The fermentation temperature was kept constant at 37℃ and the fermentation time was 48 h. The results are shown in Table 4. The results indicate that when the inoculum size was 3%, both the GABA yield and the total flavonoid content reached the highest levels.

[0057] Table 4. Effects of different inoculum sizes on fermentation: The effect of fermentation temperature: A fixed inoculum size of 3% and a fermentation time of 48 hours were used. The results are shown in Table 5. Strain YS-SA09 exhibited the best fermentation performance at 37℃, indicating that 37℃ is more conducive to its hydrolysis.

[0058] Table 5. Effects of different fermentation temperatures on fermentation: The effect of fermentation time: The inoculum size was fixed at 3%, and the fermentation temperature was 37℃. The results are shown in Table 6. The contents of GABA and total flavonoids reached their highest levels after 48 hours of fermentation; after 60 hours, the contents of GABA and total flavonoids began to decline, possibly because the cells began to enter the death phase or over-fermentation occurred.

[0059] Table 6. Effects of different fermentation times on fermentation: 2. Orthogonal optimization experimental design To obtain the optimal combination, based on the results of single-factor experiments, L9(3) was adopted. 4 Orthogonal optimization.

[0060] Factor A (vaccination volume): 2%, 3%, 4%; Factor B (fermentation temperature): 35℃, 37℃, 39℃; Factor C (fermentation time): 44h, 48h, 52h.

[0061] Results analysis: The results of the orthogonal experiment are shown in Table 7 below.

[0062] Table 7 Orthogonal experimental design and results Table 7 shows that group 5 (A2B2C3) is the optimal combination, namely, 3% inoculum size, 37℃ fermentation temperature, and 52h fermentation time. Under these conditions, the GABA yield reaches 3.68 g / L, and the total flavonoid content reaches 658.81 mg / L. Interaction analysis indicates a significant interaction between fermentation temperature, inoculum size, and fermentation time. The combination of 37℃, 3% inoculum size, and 52h is the key combination for achieving the best fermentation effect. Therefore, these conditions are determined to be the optimal fermentation process parameters.

[0063] Example 3 1. Preparation of fermentation products (1) Preparation of highly active seed liquid Lactobacillus saliva-associated strain YS-SA09, frozen in glycerol at -80℃, was streaked onto MRS solid medium for activation and cultured at 37℃ for 48 h. Single colonies were picked and inoculated into MRS liquid medium, incubated statically at 37℃ for 24 h, and passaged twice to restore good viability. The activated bacterial suspension was centrifuged at 8000 rpm for 10 min at 4℃, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline and resuspended. The bacterial concentration was adjusted to 1~9×10⁻⁹. 9 The CFU / mL concentration yielded a highly active seed solution, which was then stored at 4°C for later use.

[0064] (2) Preparation of fermented bitter orange flowers The substitute peanut powder was weighed and added to purified water at a ratio of 10% (w / v) to the liquid, mixed thoroughly, and brought to a final volume. The mixture was then sterilized at 121°C for 15 minutes. After cooling to room temperature, the highly active seed liquid was inoculated at a rate of 3% (v / v), stirred at low speed until homogeneous, and allowed to ferment at 37°C for 52 hours. After fermentation, the fermentation broth was immediately transferred to a 4°C environment to cool and terminate the fermentation, yielding the fermentation broth.

[0065] (4) Post-fermentation treatment The above fermentation broth was inactivated at 105℃ for 30 min, cooled to 4℃, centrifuged at 8000 rpm for 5 min, and the supernatant was collected to obtain the fermented product of Citrus aurantium.

[0066] Comparative Example 1 The difference from the preparation method in Example 3 is that commercially available Lactobacillus salivarius AP-32 was used for fermentation.

[0067] Comparison of main components before and after fermentation To further investigate the changes in fermentation products, the active components of the fermentation products were determined by HPLC, including GABA and flavonoids. The detection results are detailed in Table 8 below: Table 8. Results of detection of major components before and after fermentation As shown in Table 8, the strain YS-SA09 screened in this invention exhibited significantly superior metabolic activity compared to commercially available strains in a pure *Citrus aurantium* fermentation system. Its GABA production was 2.88 times that of commercially available strains. During fermentation, *Citrus aurantium*-rich components such as xylan, hemicellulose, and pectin were degraded by glycosidases contained in *Lactobacillus salivarius*, releasing free xylose, glucose, flavonoid aglycone precursors, glutamic acid, and other substances. The released xylose activated the expression of the xylose operon in strain YS-SA09. Simultaneously, xylose and glucose in the fermentation environment serve as suitable carbon sources, effectively enhancing intracellular energy and reducing power supply. This regulation promotes cell proliferation and optimizes the fermentation environment, while potentially providing an optimal catalytic environment for glutamate decarboxylase, β-glucosidase, and ferulic acid esterase. This results in the release and hydrolysis of more flavonoids, organic acids, and free amino acids, generating more GABA synthesis substrates and ultimately achieving efficient conversion of flavonoid glycosides (neohesperidin, naringin) into more bioavailable aglycones (naringenin, hesperidin, succinate) while simultaneously achieving efficient GABA enrichment. Furthermore, the formation of succinate GABA ester prolongs the half-life of succinate and GABA in vivo. Studies have shown that succinate is a weak inhibitor of GABA transaminase, increasing brain GABA levels and possessing certain anti-inflammatory and antioxidant activities; it also exhibits sedative and anti-anxiety activities. Naringenin has been reported to exert antidepressant and anti-anxiety effects by activating the BDNF signaling pathway; hesperidin has been reported to exert sedative and sleep-inducing effects by activating adenosine receptors. Therefore, the sedative and anti-anxiety effects of bitter orange blossom ferment are more lasting.

[0068] Example 4 Analysis of the efficient transformation mechanism of Lactobacillus saliva-associated with YS-SA09 To elucidate the efficient transformation mechanism of *Lactobacillus saliva-associated* YS-SA09, transcriptome sequencing results in this embodiment showed that xylose transporter (XylT), xylose isomerase (XylA), xylulokinase (XyIB), β-glucosidase (Bgl), ferulic acid esterase (faeR), and glutamate decarboxylase gene (Gad) were all actively expressed. The xyl operon is not only responsible for xylose metabolism, but its upstream regulators can also induce the expression of various glycoside hydrolases in the presence of xylose or xylose analogs (such as glucuronic acid and the glycosyl moiety of certain glycosides), such as upregulating the expression of β-glucosidase and glutamate decarboxylase. β-glucosidase, encoded by Bgl, is an important component of the cellulolytic enzyme system, capable of hydrolyzing the terminal non-reducing β-D-glucose bond, releasing β-D-glucose and the corresponding ligand. Ferulic acid esterase can release bound flavonoid precursors by hydrolyzing the flavonoid-phenolic acid ester bond. Glutamate decarboxylase is a key enzyme catalyzing the conversion of glutamate to GABA.

[0069] Table 9 Primer sequence listing qPCR validation showed that the relative expression levels of the above-mentioned genes were significantly upregulated after fermentation. Specifically, XylT peaked 1.8 times higher than the initial level, XylA increased 2.1 times, XylB increased 1.9 times, Bgl increased 2.5 times, Gad increased 2.1 times, and faeR increased 1.7 times. Analysis of the detection results revealed that the efficient induction of the xylose operon during fermentation synergistically upregulated the expression of β-glucosidase and ferulic acid esterase, promoting flavonoid glycoside hydrolysis and ester bond cleavage, thus releasing naringenin, hesperidin, and succinate. Simultaneously, by activating glutamate decarboxylase, it effectively promoted the efficient synthesis of GABA, ultimately forming a synergistic mechanism of "substrate induction—enzyme upregulation—metabolic linkage," resulting in the simultaneous enrichment of multiple active ingredients.

[0070] Example 5 Validation of mouse models for improving insomnia 1. Mouse feeding and grouping Fifty SPF-grade, 18-month-old male C57BL / 6J mice, weighing 18±2g, were selected. The rearing environment was controlled at 22±2℃, 55±10% humidity, and a 12-hour light-dark cycle, with free access to water and food. After one week of acclimatization, the mice were randomly divided into two groups: a control group (n=10) and a model group (n=40). Mice in the model group received intraperitoneal injections of PCPA (chlorophenylalanine) suspension (6mg PCPA / 20g mouse) at 8:00 AM for three consecutive days. On the third day after modeling, the mice were randomly divided into four groups. The mice were then administered intervention via gavage according to the following grouping and dosage guidelines: Control group (NC group): Administered an equal volume of normal saline by gavage daily; Model group (MG group): Daily gavage with an equal volume of physiological saline; Positive control group (PC group): daily oral administration of melatonin (0.4 mg / kg); Probiotic group (YS-AG group): Daily gavage administration of YS-SA09 probiotic solution (dose 2×10⁻⁶). 9 CFU / kg); Fermented bitter orange blossom group (CAYS group): The fermented broth prepared in Example 3 was administered by gavage daily (dose was 100 mg / Kg).

[0071] 2. Experimental Methods Evaluation of direct sleep effect: A direct sleep experiment was designed according to the "Methods for Functional Testing and Evaluation of Health Foods". The number of mice that fell asleep was observed 30 minutes after the last administration to evaluate the direct sleep effect of each drug on mice. The standard for entering a sleep state was the disappearance of the righting reflex for 60 seconds.

[0072] Synergistic sleep experiment: Mice were injected intraperitoneally with chloral hydrate (2.6%; 0.1 mL / 10 g), and the sleep was recorded using a video device. After the experiment, the sleep latency and sleep duration were recorded. The disappearance of the righting reflex (side-lying position, back-lying position) for 60 seconds was defined as entering a sleep state.

[0073] 3. Analysis of Experimental Results (1) Direct sleep effect As shown in Table 10, the direct sleep rate was zero in each group 30 minutes after the last administration, indicating that the samples in each group were safe and had no direct sleep effect.

[0074] Table 10 Direct Sleep Rate (2) Co-sleep experiment The synergistic sleep experiment is an experimental method used to evaluate the effects of sedative-sleep-aiding health foods on the sleep of mice. It assesses sleep quality, sleep latency, and sleep duration by observing the sleep behavior of mice under specific conditions. For example... Figure 3-4 As shown, in the synergistic sleep experiment, compared with the control group (NC group), the MG group had a 25.08% longer sleep latency and a 43.11% shorter sleep duration. Among the two intervention groups, both the YS-AG and CAYS groups shortened sleep latency and prolonged sleep duration, with the CAYS group showing the best effect, approaching the effect of the positive control group. This may be related to the increased GABA and flavonoid content in the fermented bitter orange blossom broth. Therefore, it can be concluded that the probiotics and bitter orange blossom ferment provided by this invention can improve sleep in mice, with the bitter orange blossom ferment showing a better effect.

[0075] Example 6 Validation of mouse models that improve anxiety 1. Mouse feeding and grouping Fifty SPF-grade, 18-month-old male C57BL / 6J mice, weighing 18±2g, were selected. The rearing environment was controlled at 22±2℃, 55±10% humidity, and a 12-hour light-dark cycle, with free access to water and food. After one week of acclimatization, the mice were randomly divided into 5 groups: a normal control group (CON: no intervention, conventional feeding), a model control group (CUMS), a positive control group (fluoxetine group, CUMS + 5.2mg / kg / d fluoxetine), and a probiotic group (YS-AG: CUMS + 2×10⁻⁶ mg / kg / day fluoxetine). 9The mice were divided into two groups: a CFU / kg / d YS-SA09 probiotic solution group and a fermentation group (CAYS: CUMS + 100mg / kg / d Citrus aurantium fermentation broth), with 10 mice in each group. Except for the normal control group, the other mice were subjected to CUMS modeling treatment. During this period, the mice were exposed to different stressors, including white noise, stroboscopic lighting, fasting and water restriction, day-night reversal, restraint, cage tilting, and wet cages.

[0076] Twenty-eight days after modeling, mice were administered the drug via gavage. The normal control and model control groups were given distilled water daily via gavage, while the other groups were given the corresponding drug solutions. Drug administration continued for 35 days, during which time the mice underwent continuous CUMS treatment. Behavioral tests were performed at 60-63 days. Mice were immediately euthanized to avoid further suffering, and relevant tissues were cryopreserved.

[0077] 3. Experimental Methods (1) Sugar water preference experiment The sucrose preference experiment consisted of two phases: a training period and a testing period. The first two days were the training period, during which the animals were fully acclimatized to sucrose-containing water. During the first 24 hours, the animals were provided with two bottles of 1% sucrose solution (w / v); during the second 24 hours, they were given one bottle of 1% sucrose solution and one bottle of pure water. Before the test, the animals ate normally but were deprived of water for 8 hours to ensure their thirst desire during the test. During the test, the mice were simultaneously provided with one bottle of 1% sucrose solution and one bottle of pure water, and the amount consumed by each was recorded over 15 hours. To eliminate the influence of positional preference on the results, the positions of the two bottles were periodically swapped. The formula for calculating the sucrose preference index (%) is: Sucrose solution consumption / (Water consumption + Sucrose solution consumption) × 100 (2) Tail suspension test During the experiment, mice were sequentially secured to a tail suspension device with a baffle to separate their line of sight and prevent interference with other mice. The mice's heads were approximately 5 cm from the platform, thus preventing them from climbing or grasping. The mice's activity and rest times were recorded over 5 minutes.

[0078] (3) Forced swimming experiment Mice were placed in glass cylinders (20 cm high × 14 cm inner diameter) filled with clean water (10 cm high, 25 ± 2 ℃). Rest time was quantified as the floating time of the mice, and the struggling time, swimming time, and floating time of the mice within 5 minutes were recorded.

[0079] (4) Mine field experiment During the experiment, the mice were first placed in the testing chamber and allowed to acclimatize for about 3 minutes to ensure they were in a stable state. Then, the testing program was started, and the mice's spontaneous activities were continuously observed and recorded over the next 10 minutes.

[0080] 4. Sample Detection Methods (1) Determination of monoamine neurotransmitter content The levels of monoamine neurotransmitters (GABA / 5-HT / DA / NE) were determined using an enzyme-linked immunosorbent assay (ELISA) kit. Whole blood samples were incubated overnight at 4°C and then centrifuged at 1000g for 15 minutes at 2-8°C. The supernatant was collected to obtain serum samples. The experiment was performed according to the manufacturer's instructions. Finally, the absorbance of each well was read at 450 nm using an ELISA reader. A standard curve was plotted based on the absorbance values ​​of the standards, and the concentration of each sample was calculated.

[0081] (2) Measurement of inflammatory factor levels To detect the effects of each group on pro-inflammatory cytokines in mouse serum, the levels of IL-6 and TNF-α were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit.

[0082] 5. Results Analysis (1) The sucrose preference test is a classic method for assessing responsiveness to positive stimuli. The sucrose preference index is often used to assess the degree of anhedonia symptoms. Experimental results are as follows: Figure 5 As shown, compared with the blank control group, the mice in the model control group exhibited a significantly reduced sucrose preference. Compared with the model control group, the positive control fluoxetine group, probiotic group, and fermentation group all increased the sucrose preference index of depressed mice, indicating that the administration reversed the depressive behavior of the mice. Among them, the fermentation group had a similar effect to fluoxetine.

[0083] (2) The tail suspension test is commonly used to assess depression in mice. The experimental results are as follows: Figure 6 As shown in the figure. Compared with the blank control group, CUMS significantly increased the resting time of mice in the tail suspension experiment; compared with the model control group, the fermentation group significantly reduced the resting time of mice in the tail suspension experiment, showing a better improvement effect, while the probiotic group had no significant effect.

[0084] (3) The forced swimming experiment provides an unavoidable, oppressive environment, reflecting the despair state of the experimental animals' behavior. The experimental results are as follows: Figure 7 As shown, compared with the blank control group, CUMS significantly reduced the swimming time and significantly increased the floating time of mice. Compared with the model control group, the fluoxetine group, probiotic group and fermentation group all increased the swimming time and reduced the floating time of mice. Among them, the effect of the fermentation group was comparable to that of the positive control fluoxetine group, which reflects its effect on improving the depressive-like behavior of mice.

[0085] (4) The open field test is a classic test widely used in research on emotion-related behaviors. Its basic principle is based on the natural fear of open spaces in mice, leading to a certain degree of avoidance behavior, and is used to assess the animal's curiosity and self-exploration behavior. Emotional responses are judged by observing the mouse's spontaneous activities and exploratory behavior; activity level is inversely proportional to the degree of depression. Experimental results are as follows: Figure 8 As shown in the figure. Compared with the blank control group, CUMS reduced the total distance, edge distance, and central distance of mice in the open field. Compared with the model control group, the positive control fluoxetine group, probiotic group, and fermentation group all increased the distance of mice in the open field. The central distance of mice in each group shows that the activity of mice in the fermentation group was effectively restored, reflecting its good antidepressant effect. The edge distance of mice in each group shows that administration of bitter orange fermentation significantly increased the distance of mice in the open field, reflecting the active exploration state of the mice.

[0086] (5) Results of the determination of monoamine neurotransmitter content in mouse serum The main biochemical cause of depression is the metabolic disorder of monoamine neurotransmitters (including GABA, 5-HT (5-hydroxytryptamine), DA (dopamine), and NE (norepinephrine)). These neurotransmitters are the main neural basis for regulating human mood. The experimental results of measuring the 5-HT content in mouse serum are shown in Table 11.

[0087] Table 11 Results of monoamine neurotransmitter content determination in mouse serum Compared with the blank control group, the serum GABA content of mice in the CUMS model control group was significantly decreased. PCPA affected serum GABA levels during modeling, leading to a decrease in serum GABA content. After intervention, the FLU, YS-AG, and CAYS groups all showed varying degrees of recovery. Compared with the model control group, the probiotic group and the bitter orange fermentation group increased the serum GABA content of experimental mice, especially the bitter orange fermentation group, where the GABA content increased more than that of the positive control group.

[0088] As shown in Table 11, compared with the blank control group, the serum 5-HT content of mice in the CUMS model control group was significantly decreased, reflecting the disorder of 5-HT synthesis, release, transport, and reuptake in mice, indicating a state of depression. Compared with the model control group, the probiotic group and the bitter orange fermentation group increased the serum 5-HT content of experimental mice, and the bitter orange fermentation group showed an effect comparable to the positive control group.

[0089] The results of the experiment measuring DA content in mouse serum are shown in Table 11. Compared with the blank control group, CUMS significantly reduced the DA content in the serum of mice in the model control group. This abnormality may affect the patients' perception of pleasure and reward, causing them to feel unable to experience normal pleasure or lose interest in life, reflecting a decreased sense of pleasure and a state of depression in the mice. Compared with the model control group, the probiotic group and the bitter orange fermentation group increased the DA content in the serum of experimental mice, and the effect of the bitter orange fermentation group was slightly better than that of the positive control group.

[0090] The results of the NE content experiment in mouse serum are shown in Table 11. As can be seen from the table, compared with the blank control group, CUMS significantly decreased the NE content in the serum of the model control group mice, reflecting a decreased sense of pleasure and an anxious state in the mice. Compared with the model control group, the fermented bitter orange group significantly increased the NE content in the serum of depressed mice, with an effect comparable to the positive control group, while the probiotic group showed a relatively less significant effect.

[0091] (6) Results of the determination of inflammatory factor content in mouse serum Overproduction of inflammatory markers is associated with cognitive changes in patients with depression. Among them, TNF-α and IL-6 are known as pro-inflammatory cytokines and are key cytokines in triggering inflammatory responses. The results of measuring the levels of inflammatory factors in mouse serum are shown in Table 12. Table 12 Results of the determination of inflammatory factor content in mouse serum As shown in the table, compared with the blank control group, the serum levels of TNF-α and IL-6 in CUMS-induced mice were higher. Compared with the model group, both the probiotic group and the bitter orange fermentation group effectively reduced the serum levels of inflammatory factors, indicating that the expression of inflammatory factors in mice was reduced. This demonstrates that probiotic saliva combined with Lactobacillus YS-SA09 and bitter orange fermentation can effectively improve inflammation in depressed mice and has a good anti-inflammatory effect, but the improvement effect of bitter orange fermentation is better.

[0092] In summary, the fermented bitter orange extract provided by this invention can effectively improve sleep and anxiety. Its mechanism of action is mainly manifested in: ① effectively shortening sleep latency and prolonging sleep onset time in mice; ② effectively improving depressive-like behavior in mice; ③ significantly reversing the decrease in neurotransmitter levels in mice caused by CUMS, and effectively increasing the levels of GABA, 5-HT, DA, and NE in the serum of depressed mice; ④ alleviating inflammation: significantly reducing serum TNF-α and IL-6 levels and improving inflammatory factor levels. Intergroup comparisons showed that its overall effect was not only superior to that of saliva combined with Lactobacillus YS-SA09 probiotics alone, but even superior to the positive control under conditions of GABA and DA. This fully demonstrates that the fermentation process not only preserves the nutrients of the raw materials, but also achieves a significant improvement in efficacy by enriching GABA and increasing various synergistic flavonoid aglycones.

Claims

1. A type of Lactobacillus salivarius YS-SA09, characterized in that: Its accession number is CGMCC NO.37763, and its Latin name is Ligilactobacillus salivarius.

2. A fermented product of bitter orange blossom, characterized in that: Obtained by fermenting *Lactobacillus salivarius* YS-SA09 as described in claim 1.

3. The method for preparing the fermented product of *Citrus aurantium* as described in claim 2, characterized in that: The method includes the step of inoculating the seed liquid obtained by activating Lactobacillus saliva-associated with YS-SA09 into the fermentation medium of Citrus aurantium to obtain the fermented Citrus aurantium product.

4. The method for preparing the fermented product of *Citrus aurantium* as described in claim 3, characterized in that: The fermentation temperature was 37°C; The fermentation time is 52 hours; The inoculation amount of the seed solution is 3% (v / v); The viable bacteria count in the seed liquid is 1~9×10⁻⁶. 9 CFU / mL.

5. The method for preparing the fermented product of *Citrus aurantium* as described in claim 3, characterized in that: The preparation method of the fermentation medium of Citrus aurantium includes the following steps: weighing Citrus aurantium powder, adding water at a ratio of 10% (w / v) and mixing evenly to a fixed volume, and sterilizing at 121°C for 15 min.

6. The method for preparing the fermented product of *Citrus aurantium* as described in claim 3, characterized in that: It also includes post-processing steps; The post-processing steps include inactivating the fermented product of *Citrus aurantium* at 105°C for 30 min, cooling it to 4°C, centrifuging it at 8000 rpm for 5 min, and collecting the supernatant.

7. The method for preparing the fermented product of *Citrus aurantium* as described in claim 3, characterized in that: The method for preparing the seed culture includes streaking activation of *Lactobacillus saliva-associated* YS-SA09, which was frozen in glycerol at -80℃, on MRS solid medium and cultured at 37℃ for 48 h. Then, a single colony is picked and inoculated into MRS liquid medium, and cultured statically at 37℃ for 24 h. This is followed by two passages to obtain the activated bacterial culture. The activated bacterial culture is then centrifuged at 4℃ and 8000 rpm for 10 min, the supernatant is discarded, and the culture is washed and resuspended with physiological saline. The bacterial concentration is adjusted to 1~9×10⁻⁶. 9 The steps for CFU / mL.

8. The application of the salivary lactobacillus YS-SA09 as described in claim 1, characterized in that: It is used in the preparation of health products and medicines that help improve sleep.

9. The application of the *Lactobacillus saliva-associated* YS-SA09 as described in claim 1, characterized in that: It is used in the preparation of antidepressant drugs.

10. The application of the bitter orange blossom ferment as described in claim 2, characterized in that: Used in the preparation of health products and medicines to improve sleep; or It is used in the preparation of antidepressant drugs.

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