Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 and development and application thereof in functional fermented food

Abstract

The invention discloses lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 and development and application of the lactobacillus delbrueckii SWU-DLB2 and the SWU-DLB3 in functional fermented food, and belongs to the technical field of microbial The lactobacillus delbrueckii SWU-DLB2 and the lactobacillus delbrueckii SWU-DLB3 are preserved in Guangdong Microbial Culture Collection Center on October 28, 2025, and the preservation numbers of the lactobacillus delbrueckii SWU-DLB2 and the lactobacillus delbrueckii SWU-DLB3 are respectively GDMCC No: 67173 and GDMCC No: 67174. Experiments show that in the dairy product containing the spina date seed powder, the content of bioactive substances can be remarkably increased through fermentation of the two strains, and the total phenol content, the flavone content and the antioxidant activity are remarkably improved. The lactobacillus delbrueckii strain provided by the invention is combined with a plant-based supplement to be applied to a food model, a functional food with the potential of enhancing neuroprotection is developed, and the important effect of microbial metabolism in food functionalization is highlighted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and particularly relates to Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 and development and application thereof in functional fermented food. BACKGROUND

[0002] Currently, neurodegenerative diseases (such as dementia, Alzheimer's disease, Parkinson's disease, etc.) are growing globally, and have become a serious social and economic and medical challenge. The pathogenesis of such diseases is complex, involving multiple key links such as oxidative stress, neural inflammation, acetylcholinesterase activity disorder, and intestinal-brain axis dysfunction, and the aging of the population exacerbates the severity of the problem. At present, effective treatment methods are scarce, and traditional pharmacological inhibitors also have hepatotoxicity and potential fatal risks, so it is of great significance to develop natural and safe neuroprotective functional foods.

[0003] In the prior art, probiotics have been proven to have the potential to resist neurodegenerative diseases, and some strains of lactic acid bacteria exhibit antioxidant, antibacterial, anti-inflammatory and neuroprotective properties, but the screening and application of specific strains with high acetylcholinesterase inhibitory activity and GABA synthesis capacity still need to be broken through. At the same time, as an agricultural byproduct with an annual output of more than 3 million tons, Zizyphus jujuba Mill. is rich in bioactive compounds such as flavonoids and alkaloids, and has antioxidant, neuroprotective and other effects, but has not been fully developed and applied in functional fermented food. In addition, existing fermented milk products have deficiencies in neuroprotective function enhancement, and lack mature technical solutions combining specific high-efficiency probiotics with plant-based bioactive ingredients, and the synergistic effect of functional ingredients in fermented milk and genomics mechanism research is not deep enough.

[0004] Traditional acetylcholinesterase pharmacological inhibitors for neurodegenerative diseases (such as Alzheimer's disease) have hepatotoxicity and potential fatal risks, and it is urgent to develop natural and safe neuroprotective functional foods as an alternative or complementary strategy. SUMMARY

[0005] To solve the above technical problems, the present application provides a Lactobacillus delbrueckii (Lactobacillus delbrueckii) and a preparation method thereof. Lactobacillus delbrueckii subsp. The preservation number of the Lactobacillus delbrueckii is GDMCC No: 67173 or GDMCC No: 67174.

[0006] The present application also provides an application of the above-mentioned Lactobacillus delbrueckii (Lactobacillus delbrueckii) in the preparation of a neuroprotective functional fermented food. Lactobacillus delbrueckii subsp.

[0007] Preferably, the food is fermented milk.

[0008] ​The application also provides a method for preparing fermented milk by using the Lactobacillus delbrueckii, comprising the following steps: adding 1.5-2.5% of the Semen Ziziphi Spinosae powder into liquid milk, adding the Lactobacillus delbrueckii with the preservation number of GDMCC No: 67173 and / or GDMCC No: 67174 after sterilization treatment, and then fermenting for 0.8-1.2 hours at 41-43 DEG C, and then adding yogurt starter to ferment.

[0009] Preferably, the liquid milk is cow milk.

[0010] More preferably, the addition amount of the Semen Ziziphi Spinosae powder is 2%.

[0011] More preferably, when only one kind of Lactobacillus delbrueckii is added into the sterilized liquid milk, the inoculation amount of the Lactobacillus delbrueckii is 1.5-2.5% by volume ratio; when two kinds of Lactobacillus delbrueckii are added into the sterilized liquid milk, the quantity ratio of the two kinds of Lactobacillus delbrueckii is 0.8-1.2:0.8-1.2, and the total inoculation amount of the two kinds of Lactobacillus delbrueckii is 1.5-2.5% by volume ratio.

[0012] More preferably, after inoculating the Lactobacillus delbrueckii, the fermentation is carried out at 42 DEG C for 1 hour.

[0013] The application also provides fermented milk prepared by the above method.

[0014] Compared with the prior art, the application has the following beneficial effects: (1) precise strain screening: the screened Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 strains have high acetylcholinesterase inhibitory activity, GABA synthesis capacity and excellent probiotic characteristics, and no drug resistance genes and virulence factors are verified by whole genome sequencing, and the safety is high; (2) synergistic enhancement of functions: the specific probiotics are combined with the Semen Ziziphi Spinosae powder to realize the synergistic effect of "probiotics + plant-based bioactive ingredients", and the total phenol content, flavonoid content and antioxidant activity are significantly improved, the total phenol content of the Semen Ziziphi Spinosae powder fortified group fermented by the SWU-DLB2 strain is 235.75±1.92 mg GAE / 100g, the flavonoid content is 114.07±4.54 mg RE / 100g, and the acetylcholinesterase inhibition rate is 30.66%, which is significantly higher than that of the control group (p<0.05); (3) reasonable process optimization: through the pre-fermentation process and the addition of the Semen Ziziphi Spinosae powder, the viscosity and sensory characteristics of the fermented milk are improved, and the survival rate of the probiotics during cold storage is improved, and the functional stability of the product during the shelf life is ensured; (4) efficient use of resources: the Semen Ziziphi Spinosae, an underutilized agricultural byproduct, is converted into high-value functional ingredients, expanding its application field and reducing the production cost of functional foods.

[0015] Biological preservation instruction of Lactobacillus delbrueckii SWU-DLB2: Preservation agency: Guangdong Microbial Culture Collection Center; Preservation number: GDMCC No: 67173; Preservation date: October 28, 2025; Preservation address: 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard; Taxonomic name: Lactobacillus delbrueckii subsp . bulgaricus .

[0016] Biological preservation instruction of Lactobacillus delbrueckii SWU-DLB3: Preservation agency: Guangdong Microbial Culture Collection Center; Preservation number: GDMCC No: 67174; Preservation date: October 28, 2025; Preservation address: 5th Floor, Building 59, Guangzhou Xianlie Middle Road 100 Courtyard; Taxonomic name: Lactobacillus delbrueckii subsp . allosunkii . BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 For the inhibitory ability of lactic acid bacteria isolates in Example 1 on acetylcholinesterase, wherein a: inhibitory ability of lactobacillus; b: inhibitory ability of coccus; c: acetylcholinesterase inhibition rate of lactic acid bacteria and enterococcus strains; GRS: galantamine control standard; DRS: donepezil control standard; DLB2: Lactobacillus delbrueckii SWU-DLB2; DLB3: Lactobacillus delbrueckii SWU-DLB3.

[0018] Figure 2 For the antibacterial activity of the screened strains in Example 1, wherein (a) inhibitory effect on Escherichia coli (10 9 CFU / mL); (b) inhibitory effect on Salmonella typhimurium (10 9 CFU / mL). C+: ampicillin (10 mg / mL), C-: culture medium control; DLB2: Lactobacillus delbrueckii SWU-DLB2; DLB3: Lactobacillus delbrueckii SWU-DLB3.

[0019] Figure 3 For the representative gel electrophoresis of gad gene detection of all tested strains in Example 1, wherein C+: Lactobacillus plantarum SWU-ZFT6 (GDMCC No: 66290, positive control); C-: Raffinose Lactococcus DSM 20021 (negative control); DLB2: Lactobacillus delbrueckii SWU-DLB2; DLB3: Lactobacillus delbrueckii SWU-DLB3.

[0020] Figure 4 Changes in acidity (%) and pH of functional fermented milk during cold storage in Example 1, wherein (a) acidity of functional fermented milk without addition of Zizyphus jujuba powder; (b) acidity of fermented milk with addition of 2% Zizyphus jujuba powder; (c) pH of functional fermented milk without addition of Zizyphus jujuba powder; (d) pH of fermented milk with addition of 2% Zizyphus jujuba powder.

[0021] Figure 5 Changes in (a) viscosity and (b) survival rate of lactic acid bacteria of fermented milk during cold storage in Example 1, wherein C2: control fermented milk prepared using yogurt starter; T4: fermented milk prepared using SWU-DLB2 as a secondary starter; T5: fermented milk prepared using SWU-DLB3 as a secondary starter; T6: fermented milk prepared using a mixture of SWU-DLB2 and SWU-DLB3 as a secondary starter; C1: control fermented milk prepared using yogurt starter (supplemented with 2% Zizyphus jujuba powder); T1: fermented milk prepared using SWU-DLB2 as a secondary starter and supplemented with 2% Zizyphus jujuba powder; T2: fermented milk prepared using SWU-DLB3 as a secondary starter and supplemented with 2% Zizyphus jujuba powder; T3: fermented milk prepared using a mixture of SWU-DLB2 and SWU-DLB3 as a secondary starter and supplemented with 2% Zizyphus jujuba powder.

[0022] Figure 6 Phylogenetic tree of SWU-DLB2 in Example 1.

[0023] Figure 7 Phylogenetic tree of SWU-DLB3 in Example 1. DETAILED DESCRIPTION

[0024] Example 1 1. Isolation of lactic acid bacteria and screening for acetylcholinesterase inhibitory potential Samples were taken from 25 kinds of traditional fermented dairy products and stored at 4°C. For isolation, the samples were cultured in skim milk at 30°C, 37°C, and 42°C until coagulation. Then, the samples were streaked onto M17 and MRS agar plates and anaerobically incubated at the same temperatures. The purified isolates were phenotypically evaluated and stored at -80°C for further studies.

[0025] The acetylcholinesterase inhibitory activity of the sterile cell-free filtrate obtained from 48-hour cultures was determined using a microplate method (Pandey et al., 2014). Donepezil hydrochloride and galantamine hydrobromide were used as positive controls. All experiments were performed in triplicate, and independently repeated three times. The acetylcholinesterase inhibition rate was calculated according to the following formula: AChEI (%) = [(blank OD value - sample OD value) / blank OD value] x 100; Where, blank control is phosphate buffered saline (PBS; 50 mM) for calculation of standard inhibition rate; and culture medium (MRS or M17) for calculation of cell-free filtrate inhibition rate.

[0026] 2. Probiotic potential and safety profile The 15 selected lactic acid bacterial isolates with acetylcholinesterase inhibitory potential were subjected to functional probiotic potential evaluation.

[0027] 2.1 Acid, pepsin and trypsin tolerance assessment The tolerance of the strains to gastrointestinal conditions was assessed following the method of Madian et al. (2025). Briefly, the procedure was as follows: the bacterial cells from overnight culture were collected by centrifugation, washed twice with PBS and resuspended in simulated gastric fluid to a final concentration of about 10 9 CFU / mL. Simulated gastric fluid was PBS at pH 3.0 with the addition of pepsin at a concentration of 1 :10,000. In the gastric phase, the bacterial suspension was incubated at 37°C for 0 and 3 hours, respectively. Subsequently, in the intestinal phase, 1 mL of the bacterial suspension from the gastric phase was added to 9 mL of simulated intestinal fluid and incubated at 37°C for 0 and 4 hours, respectively. The survival rate was calculated using the following formula: Survival rate (%) = (log of final viable count / log of initial viable count) x 100; According to the survival rate results, the acid tolerance ability of the strains was classified into four categories: sensitive (<10%), moderately resistant (10-60%), well resistant (60-80%) and highly resistant (>80%) according to the standard of (Narimani et al., 2015).

[0028] 2.2 Bile salt tolerance assessment The bile tolerance of the strains was assessed following the method of Guo et al. 2009. Overnight cultures were inoculated into MRS broth with and without the addition of 0.3% (w / v) bile salts and incubated at 37°C. The OD 600 absorbance was measured every hour using a spectrophotometer to record their growth for 24 hours, and the un-inoculated medium was used as a blank control. According to the survival rate of the strains in the presence of bile salts, they were classified into four categories according to the standard of Chateau et al. (1994).

[0029] 2.3 Antimicrobial activity against gastrointestinal pathogenic bacteria Agar diffusion method was used to evaluate the antibacterial activity of lactic acid bacteria against Salmonella typhimurium ATCC33110 and Escherichia coli NCTC12900 in vitro. The pathogenic bacteria (10 9 CFU / mL) were inoculated in specific medium to prepare holes with a diameter of 6 mm. 100 μL of sterile neutralized cell-free supernatant (nCFS) was added to each well, and incubated at 37°C for 24 hours. Ampicillin (10 mg / mL) was used as a positive control, and un-inoculated MRS broth as a negative control. The average diameter of the inhibition zone was measured in millimeters (mm).

[0030] 2.4 Gamma-aminobutyric acid (GABA) synthesis gene detection Total DNA was extracted from the bacterial broth in the logarithmic growth phase using the TIANamp Bacterial DNA Extraction Kit. The presence of the gad gene was detected by PCR reaction using specific primers (gad-F / gad-R) and the Direct PCR Kit. The reaction was performed in a Bio-Rad PCR instrument, and the amplification products were analyzed by 1% agarose gel electrophoresis. Lactiplantibacillus plantarum SWU-ZFT6 (positive control) and Lactobacillus rhamnosus DSM 20021 (negative control) were used as controls. The PCR reaction conditions were based on the method of Langa et al., 2024.

[0031] 2.5 Hemolytic activity detection The hemolytic activity of lactic acid bacteria isolates was evaluated to determine their application safety, following the method of Abedi et al. (2018). Each strain was streak-inoculated on blood agar plates containing 7% (v / v) defibrinated sheep blood and incubated at 37°C under microaerophilic conditions for 48 hours. Hemolytic phenomena were observed after incubation.

[0032] 2.6 Antibiotic susceptibility test (AST) The antibiotic susceptibility of lactic acid bacteria strains was determined using the disc diffusion method, using ten antibiotic discs: ampicillin (10 μg), ceftriaxone (30 μg), tetracycline (30 μg), gentamicin (10 μg), penicillin (10 U), erythromycin (15 μg), ciprofloxacin (5 μg), chloramphenicol (30 μg), lincomycin (2 μg), and sulfamethoxazole (25 μg). Plates were incubated under appropriate conditions for 24 hours, the diameter of the inhibition zone (mm) was measured, and the results were classified as sensitive (S), intermediate (I), or resistant (R) according to the CLSI guidelines (CLSI, 2017).

[0033] 3. Molecular identification The 16S rRNA gene sequencing was used to identify the lactic acid bacteria isolates. The purified genomic DNA was used as a template to amplify the 16S rRNA gene using specific primers 27F (SEQ ID NO. 1: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO. 2: 5'-CGGTTACCTTGTTACGACTT-3'). The amplified products were sequenced, and the obtained sequences were subjected to similarity analysis by the BLAST tool of the National Center for Biotechnology Information (NCBI) database. Based on the 16S rRNA sequence, 20 strains most similar at the species level were selected to construct a phylogenetic tree, see Figure 6 and Figure 7 SWU-DLB2 and SWU-DLB3 belong to lactic acid bacteria in terms of phylogeny, and are particularly closely related to Lactobacillus species.

[0034] 4. Genotype characteristics analysis of SWU-DLB2 and SWU-DLB3 strains 4.1 Genomic DNA extraction SWU-DLB2 and SWU-DLB3 strains were inoculated into MRS broth and cultured at 37°C with 150 rpm shaking for about 12 hours. After centrifugal collection of the bacterial cells, the genomic DNA was extracted according to the steps of the bacterial / fungal DNA extraction kit instructions.

[0035] 4.2 Whole genome sequencing Genome sequencing was commissioned to a professional agency, combining PacBio and Illumina dual-platform sequencing technology. In terms of Illumina sequencing, the genomic DNA was used to construct a sequencing library, the library was prepared using the Nextera XT library preparation kit, and double-end sequencing was performed on the Illumina NovaSeq 6000 platform. In terms of PacBio sequencing, the genomic DNA was fragmented, purified, and end-repaired, then connected to the SMRT bell sequencing adapter, followed by preparation of the PacBio library and completion of sequencing on the SMRT cell.

[0036] 4.3 Bioinformatics analysis Bioinformatics analysis was performed based on the data generated by PacBio and Illumina platforms, and the analysis work was completed on the Majorbio cloud platform of Shanghai Maiyao Information Technology Co., Ltd. Unicycler and Pilon software were used to assemble short reads and HiFi reads to construct a complete genome. The coding sequences of chromosomes and plasmids were predicted by Glimmer or Prodigal and GeneMarkS software, respectively.

[0037] 5. Functional fermented milk prepared with screening strains as adjunct starter cultures 5.1 Starter cultures and fermented milk preparation After three times of continuous subculture, the two adjunct strains SWU-DLB2 and SWU-DLB3 were inoculated into raw milk and incubated at 42°C for 18 hours to reach a final concentration of about 2 x 10 8 CFU / mL.

[0038] Using whole milk powder as base material, reconstituted milk was prepared with 12.5% (w / v g / mL). The reconstituted milk was divided into two groups: one group was added with 2% (w / v g / mL) Jujube powder (JP) (jujube powder was obtained by directly powdering jujube), and the other group was not added with JP as control. Both groups of reconstituted milk were heat treated at 90°C for 5 minutes (Tamime and Robinson, 2007) and then cooled to 42°C for fermentation. Each group (with and without JP) was further divided into four treatment groups, totaling eight experimental groups: C1 : inoculated with 1 g / L of direct vat set (DVS) starter culture (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus subsp. salivarius, with a ratio of 1:1), and then incubated at 42°C until coagulation at pH 4.70±0.1.

[0039] T1 : inoculated with SWU-DLB2 strain for pre-fermentation (1 hour, 42°C), and then added with the same DVS starter culture as in C1 group.

[0040] T2: inoculated with SWU-DLB3 strain for pre-fermentation (1 hour, 42°C), and then added with the same DVS starter culture as in C1 group.

[0041] T3: inoculated with SWU-DLB2 and SWU-DLB3 strains for pre-fermentation (1 hour, 42°C) at a ratio of 1:1, and then added with the same DVS starter culture as in C1 group.

[0042] The total inoculation amount of SWU-DLB2 in T1 group, SWU-DLB3 in T2 group, and SWU-DLB2 and SWU-DLB3 in T3 group was the same, and the concentration of Lactobacillus delbrueckii in T1-T3 groups was 2 x 10 8 CFU / mL, and the total inoculation ratio was 2%.

[0043] The sample C2, T4, T5, and T6 were treated as above, except that no JP was added. The fermentation was terminated when the end point pH reached 4.70±0.1, and then 5% sucrose was added after gentle stirring, and the samples were cooled and stored at 4.0±0.5°C for 14 days for analysis.

[0044] 5.2 Analysis of physicochemical indicators The physicochemical indexes of fermented milk were determined periodically during cold storage: pH value was determined by a pH meter; titratable acidity (expressed as lactic acid %), total solid content (%) were determined according to AOAC (2019) method, and total nitrogen content was determined by Kjeldahl method; fat content was determined by filter bag method (ANKOM XT15 fat determination instrument, USA).

[0045] 5.3 Viscosity and sensory evaluation Viscosity was determined by a rotary viscometer according to ISO 7884-2 standard during cold storage, and the test conditions were 10℃, No.3 rotor, and 60 rpm rotation speed. Sensory evaluation was performed by 10 trained evaluators (6 males and 4 females) from the College of Food Science of Southwest University, and the appearance, texture, aroma, taste and overall acceptability of the samples were scored (ethical review approval number: HF20251002). The samples were served in uniform colorless cups at room temperature, and the evaluation was performed in the afternoon using a 9-point hedonic scale (Meilgaard, 1999).

[0046] 5.4 Determination of viable lactic acid bacteria count According to ISO 19344:2015 standard, the viable lactic acid bacteria count was determined by anaerobic culture on MRS medium at 37℃ for 72 hours during cold storage.

[0047] 5.5 Bioactive components and antioxidant activity 20 g of fermented milk sample was accurately weighed, and n-hexane was added at a ratio of 1:3 (w / v) for defatting (Sellami et al., 2009). The sample was treated in an ultrasonic water bath (30℃, 15 minutes) and kept constant. The defatted residue was extracted with a methanol-water solution (80% methanol, v / v) at a ratio of 1:10 (w / v) to extract antioxidant components such as polyphenols (Duda-Chodak and Tarko, 2007), and the extraction was continued for 24 hours at 4℃ with continuous shaking. After centrifugation (10,000 ×g, 15 minutes, 4℃), the supernatant was filtered to obtain clear crude extract.

[0048] Total phenol content: determined by modified Folin-Ciocalteu method (Abirami et al., 2014).

[0049] Total flavonoid content: determined by aluminum chloride colorimetric method (Barros et al., 2011).

[0050] Antioxidant activity: the extract was dissolved in 10 mL of 80% methanol for DPPH method (Brand-Williams et al., 1995) and FRAP method (Benzie and Strain, 1996).

[0051] Acetylcholinesterase inhibitory activity: The extract was dissolved in 2 mL of 50 mM phosphate buffer containing 0.1% dimethyl sulfoxide (pH suitable for enzyme activity assay) and the assay was performed (Ellman et al., 1961; Vinutha et al., 2007).

[0052] 2.6 Statistical Analysis Except for sensory evaluation, which was repeated 10 times, all other data were expressed as mean ± standard deviation of 3 replicates. One-way ANOVA was performed using IBM SPSS Statistics 24.0 software (p<0.05), and graphs were generated using GraphPad Prism 9.5.0 software.

[0053] Results and Discussion 1. Acetylcholinesterase inhibitory activity of lactic acid bacteria strains Of the 95 lactic acid bacteria strains isolated from traditional dairy products, 48 ​​strains (50.5%) were identified as Lactobacillus and 47 strains (49.5%) as cocci. Preliminary screening for acetylcholinesterase inhibitory activity showed that 14 Lactobacillus strains exhibited high inhibitory activity. Figure 1 a), and among cocci, only one Enterococcus strain, EKA11, exhibited high inhibitory activity ( Figure 1 (b) The inhibitory activity of the remaining cocci was at a low to moderate level. Among them, strain SWU-DLB2 showed the strongest inhibitory activity, followed by Lactobacillus AY19 and Enterococcus EKA11 (Table 1). The inhibitory effects of these strains were all superior to the standard drug donepezil (0.1 mM), with an inhibition rate of 14.4% ± 0.05%. Figure 1 c). Furthermore, its effect was significantly higher than that of galantamine, and also significantly exceeded the inhibitory effect of *Lactobacillus brevis* P109 in previous studies (Qadah et al. (2023)). Previous studies have indicated that increasing cholinergic neurotransmitter levels has a positive effect on improving cognitive function and alleviating Alzheimer's disease symptoms (Smyrska-Wieleba and Mroczek, 2023). Considering the hepatotoxicity and potential fatal risks of traditional pharmacological inhibitors of this enzyme (Marucci et al., 2021), developing natural inhibitors has become an innovative therapeutic and preventative strategy.

[0054] 2. Potential characteristics of probiotics Tolerance assessments were conducted on 15 lactic acid bacteria isolates with potential probiotic functions, revealing significant differences in tolerance among the strains. After cultivation at pH 2.0, strains SWU-DLB3 and AY16 exhibited superior acid tolerance. Statistical analysis confirmed that the survival rates of each strain under these conditions ranged from 47.66% to 99.62%, with statistically significant differences (p<0.05). Furthermore, all strains demonstrated high tolerance to intestinal stressors: in trypsin-containing environments, survival rates remained at a high level of 46.66% to 100%; under 0.3% bile salt conditions, the survival rates of 5 strains exceeded 60%, while strains SWU-DLB2 and EMA9 showed moderate tolerance (Table 1). Based on the excellent and stable acid tolerance, high trypsin survival rate, and strong bile salt tolerance exhibited by strains SWU-DLB3 and AY16 in various key tests, [further details are needed]. Existing research evidence suggests that probiotics play an important regulatory role in neurological health and cognitive function primarily through the gut-brain axis mechanism (Akbarie et al., 2016; Athari et al., 2018).

[0055] Table 1. Acetylcholinesterase inhibition rate, antibacterial activity, and survival rate of lactic acid bacteria strains in 0.3% bile salts, low pH 2.0 and 4.0, and simulated gastric juice.

[0056] Data are the mean ± standard deviation of three repeated experiments. No significant difference exists between data points marked with the same lowercase letter in the same column (p>0.05).

[0057] Probiotics exert neuroprotective effects through a variety of key mechanisms. These mechanisms include inhibiting pro-inflammatory cytokines to reduce neuroinflammation, enhancing blood-brain barrier integrity, and regulating neurotransmitter balance. Crucially, probiotics can effectively alleviate oxidative stress—a core trigger for neurodegenerative diseases. Preclinical studies have demonstrated that probiotics can improve cognitive function in Alzheimer's disease model mice and reduce β-amyloid plaque deposition in the brain (Abraham et al., 2019). These benefits have also been validated in human studies: multiple clinical trials have shown that probiotic supplementation can significantly improve cognitive function in Alzheimer's patients and effectively delay the progression of neurological decline (Pistollato et al., 2018).

[0058] 3. Antibacterial activity The inhibitory effects of lactic acid bacteria isolates on *Escherichia coli* and *Salmonella typhimurium* are highly correlated with the gut-brain axis mechanism, as these two pathogens can induce neuroinflammation and neurodegenerative processes (Hu et al., 2016). Neutralized cell-free supernatant showed significant antibacterial activity against the tested strains (Table 1;).Figure 2 The inhibition zone diameter for *Escherichia coli* ranged from 11.25 ± 0.95 to 18.66 ± 0.57 mm, and for *Salmonella typhimurium*, it ranged from 11.33 ± 0.57 to 19.33 ± 1.52 mm (Chen et al., 2016). It is known that some *E. coli* species can produce amyloid-coil protein, while *Salmonella* infection can trigger a systemic inflammatory cascade (Yang et al., 2022). Both are associated with the misfolding and aggregation of neuronal proteins such as α-synuclein and β-amyloid, which are typical pathological features of Parkinson's disease and Alzheimer's disease (Sun et al., 2022). Therefore, lactic acid bacteria can help maintain intestinal homeostasis by effectively inhibiting the proliferation of Escherichia coli and Salmonella typhimurium, thereby preventing pathogen-induced intestinal flora imbalance, reducing pro-inflammatory factor load, and ultimately alleviating key triggers of brain dysfunction. This suggests that they may exert neuroprotective effects through the gut-brain axis pathway (Zhang et al., 2024).

[0059] 4. Glutamate decarboxylase ( Glutamate Decarboxylase , gad Gene testing The gad gene was detected in all screened lactic acid bacteria and enterococcal strains using PCR technology, confirming their inherent GABA synthesis ability. Figure 3 This finding, combined with the strain's confirmed acetylcholinesterase inhibitory activity, suggests that it exerts its neuroprotective effect through a synergistic dual mechanism: while inhibiting acetylcholinesterase to enhance cholinergic signaling and support cognitive function, the accompanying GABA can effectively alleviate the key excitotoxicity problem in neurological diseases (Rivera et al., 2023).

[0060] It is important to note that while existing research indicates the dysfunction of endogenous GABA in the progression of Alzheimer's disease (Lauterborn et al., 2021), this dysfunction is more likely due to weakened GABA inhibitory tone rather than the harmful effects of GABA itself. Therefore, bacterial-derived GABA may effectively antagonize glutamate-mediated excitotoxicity by restoring impaired inhibitory function and help restore the excitation-inhibition balance necessary for maintaining neuronal integrity (Lam et al., 2023). This multi-target mechanism represents a promising therapeutic strategy that goes beyond symptom relief, aiming to fundamentally regulate the disease by intervening in the core pathological processes of neurodegenerative diseases.

[0061] 5. Hemolytic activity and antibiotic susceptibility testing All tested lactic acid bacteria isolates exhibited γ-hemolytic activity, confirming their lack of hemolytic activity. Antibiotic susceptibility testing results showed differences in resistance and susceptibility to the tested antibiotics among the strains (Table 2). Overall, the strains showed high susceptibility to ampicillin, ceftriaxone, erythromycin, chloramphenicol, and ciprofloxacin; resistance to gentamicin and penicillin was observed in multiple strains; tetracycline resistance was strain-dependent, ranging from sensitive to moderate resistance. Notably, resistance to lincomycin and sulfamethoxazole was relatively rare, but some strains showed moderate resistance. Oh and Jung (2015) pointed out that the absence of antibiotic resistance and the lack of hemolytic activity are fundamental requirements for screening novel safe probiotic isolates.

[0062] Table 2. Identification results of 16S rRNA gene of lactic acid bacteria isolates and their safety characteristics.

[0063] 6. Molecular identification Strain-level identification was performed by amplifying and sequencing the 16S rRNA gene of lactic acid bacteria isolates (Table 2). All strains isolated from yak milk were identified as *Lactobacillus delbrueckii*, with differences in subspecies classification.

[0064] 7. Genomic characteristics of SWU-DLB2 and SWU-DLB3 strains Based on previous results, whole-genome sequencing was performed on two strains, SWU-DLB2 and SWU-DLB3. The genomes were circular chromosomes, with lengths of 1,897,414 bp and 1,890,519 bp, respectively, and a GC content of approximately 49.60%. The SWU-DLB2 chromosome contained 1,913 coding sequences, 27 rRNA genes, and 95 tRNA genes; the SWU-DLB3 chromosome contained 1,907 coding sequences, 24 rRNA genes, and 88 tRNA genes. Pathogenicity analysis showed that neither strain contained clinically relevant drug resistance genes, key virulence factors, or toxin-related genes, indicating their potential as probiotic supplements (El-Hosseny et al., 2025).

[0065] 7.1 Identification of probiotic-related genes Based on Lactobacillus delbrueckii subsp. bulgaricus SWU-DLB2 and Lactobacillus delbrueckii allosunkii Genome annotation of subspecies SWU-DLB3 identified several probiotic-related genes (Table 3). Genome analysis revealed the presence of adhesion and biofilm-related genes, including LPxTG motif protein and sortase A (…). srtA ) and enolase ( eno), which are essential for the specific interaction of bacterial surface components with host epithelial cell surface receptors (Xu et al., 2022). Furthermore, genes responsible for bile and acid tolerance ( cfa, nagB, ppa, nhaC, napA ), a property that supports their viability within the gastrointestinal tract, ensuring a sufficient number of viable bacteria reach specific host sites of action to exert probiotic efficacy (De Jesus et al., 2022). Stress adaptation mechanisms are also well-defined, with both sets of genomes containing complete heat shock protein gene clusters ( htpX, hrcA, grpE, dnaK, dnaJ, groES, groEL ), which not only function under high-temperature conditions but also constitute a defense mechanism against sudden heat shock stress (Jakaria Al-Mujahidy et al., 2024). Simultaneously, cold stress-regulating genes ( cspA ) present in the genomes aid in the survival of the strains in environments below their optimal growth temperature. The study also confirmed the antibacterial activity of the Type III RamC lantibiotic, a heat-stable bacteriocin that catalyzes bacterial cell wall hydrolysis, leading to cell lysis and death (Angelescu et al., 2022).

[0066] Table 3. Probiotic-related genes predicted in the genomes of Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 strains based on KEGG annotation

[0067] (+) : present; (-) : absent 7.2 Identification of acetylcholinesterase inhibition-related genes Multiple genes involved in acetylcholinesterase inhibition, either directly or indirectly, were identified in the genomes of SWU-DLB2 and SWU-DLB3 strains, which could have a protective effect on neurodegenerative diseases. Analysis revealed that both strains contain genes involved in the synthesis of neuroactive molecules, particularly the gadC gene encoding glutamate-GABA transporter, which is responsible for GABA efflux and glutamate uptake (Ma et al., 2012). GABA, a byproduct of glutamate decarboxylation, acts as an inhibitory neurotransmitter in the human central nervous system (Gong et al., 2019). Additionally, pyroglutamyl peptidase encoded by the pcp gene is a highly specific membrane-bound thyrotropin-releasing hormone-degrading enzyme (Charli et al., 1989) that functions as a tripeptide in the brain, directly affecting neuronal excitability and potentially participating in the regulation of emotional balance (Rodríguez-Molina et al., 2009).

[0068] In addition, riboflavin synthesis-related genes are present in the SWU-DLB2 strain ( ribE, ribD This indicates that they possess the potential to synthesize riboflavin (vitamin B2), which explains why SWU-DLB2 exhibits higher acetylcholinesterase inhibitory activity in in vitro experiments compared to the SWU-DLB3 strain, which lacks this gene. Riboflavin is essential for myelin synthesis, possesses important antioxidant properties, and exerts neuroprotective effects by maintaining cellular energy balance and reducing oxidative stress (Plantone et al., 2021). Furthermore, both strains contain enzymes encoding dihydrofolate synthase. folC The gene, containing the enzyme, is crucial for the synthesis of folic acid (vitamin B9). Previous studies have reported that adequate folic acid intake can reduce the risk of Alzheimer's disease (Zhang et al., 2021). The presence of thioredoxin in both strains further highlights their neuroprotective properties. This protein plays a key role in maintaining cellular redox homeostasis and has been considered a potential therapeutic agent for the prevention and treatment of various neurodegenerative, neuroinflammatory, and neurooxidative stress-related diseases (Bjorklund et al., 2022). Furthermore, both strains contain abundant extracellular polysaccharide gene clusters. Multiple studies have explored the potential protective role of lactic acid bacteria against Alzheimer's disease, and recent research further attributes this to the extracellular polysaccharides produced by lactic acid bacteria, which exert their effects through mechanisms such as scavenging free radicals, regulating peroxidation products, and influencing apoptosis (Sirin and Aslim, 2021).

[0069] 8. Effects of the selected bacterial strains and jujube seed powder on the properties of fermented milk 8.1 Physical and chemical properties Compared with the control group, the addition of 2% jujube seed powder (JP) did not significantly change the pH value of the fermented milk (p>0.05). This result can be attributed to the buffering capacity of the protein components in JP (Zhang, 2021), a property that can mitigate the typical pH decline trend during fermentation (Salaün et al., 2004). Nevertheless, the acidity of all samples increased significantly during storage (p<0.05), indicating that lactic acid bacteria maintained metabolic activity during storage. The acid production was more pronounced in the JP-enhanced samples, suggesting that the prebiotic components in the powder may have promoted bacterial activity and the acidification process. Figure 4 Nutritional analysis confirmed that JP fortification only slightly increased the fat content of fermented milk (Table 4), which is consistent with the residual fat content of the JP used. This treatment method is advantageous in that it can enrich the target bioactive components while minimizing potential problems caused by wax ester digestion. In addition, JP supplementation did significantly increase the protein content of fermented milk.

[0070] Table 4 Chemical composition analysis and sensory evaluation of functional fermented milk

[0071] Data are presented as mean ± standard deviation of three replicates. Means marked with the same lowercase letter in the same row indicate no significant difference (p>0.05).

[0072] C2: Control fermented milk prepared using yogurt starter; T4: Fermented milk prepared using SWU-DLB2 as an auxiliary starter; T5: Fermented milk prepared using SWU-DLB3 as an auxiliary starter; T6: Fermented milk prepared using a mixed strain of SWU-DLB2 and SWU-DLB3 as an auxiliary starter; C1: Control fermented milk prepared using yogurt starter (supplemented with 2% jujube seed powder); T1: Fermented milk prepared using SWU-DLB2 as an auxiliary starter and supplemented with 2% jujube seed powder; T2: Fermented milk prepared using SWU-DLB3 as an auxiliary starter and supplemented with 2% jujube seed powder; T3: Fermented milk prepared using a mixed strain of SWU-DLB2 and SWU-DLB3 as an auxiliary starter and supplemented with 2% jujube seed powder.

[0073] 8.2 Viscosity and Sensory Evaluation Significant differences in viscosity were observed among the treatment groups (p<0.05). For example... Figure 5 As shown in Figure a, the sample fermented with the exopolysaccharide-producing strain SWU-DLB3 and enhanced with jujube seed powder exhibited the highest viscosity. Whole-genome sequencing confirmed that the SWU-DLB3 genome contains a key gene cluster for exopolysaccharide biosynthesis (Table 3), a genetic characteristic directly related to its function as a natural biothickener (Jurášková et al., 2023). The addition of jujube seed powder itself also increased viscosity, attributed to its high protein content (mainly gluten and albumin) promoting gel network formation (Ravindran et al., 2025). The highest viscosity of the T2 sample indicates a significant synergistic effect: jujube seed powder may act as a prebiotic substrate to stimulate SWU-DLB3 to produce more exopolysaccharides, while its own proteins simultaneously enhance the gel matrix structure.

[0074] The improved rheological properties directly enhanced sensory acceptance. Table 4 shows that the treatment group using strain SWU-DLB3 had significantly higher texture scores, confirming the positive impact of in-situ extracellular polysaccharide synthesis on taste optimization. The addition of jujube seed powder further improved the texture scores of all strains, demonstrating its role as a functional texture modifier.

[0075] Notably, none of the treatment groups showed significant differences in flavor scores (p > 0.05), indicating that the addition of the adjunct starter culture and the Chinese date kernel powder did not introduce undesirable flavors. This result is consistent with previous research that Chinese date kernel powder can enhance the flavor profile of products without negative effects (Zhang et al., 2024). In terms of taste, all groups showed a positive trend, indicating that the slight sweetness and other desirable sensory characteristics brought by Chinese date kernel powder helped to improve overall taste acceptance, which is consistent with the improvement effect of plant-based fortifying ingredients in other dairy products (Azeem et al., 2024).

[0076] 9. Analysis of the prebiotic effect of Chinese date kernel powder From Figure 5 As can be seen from Figure b, the number of lactic acid bacteria showed a downward trend during cold storage, but except for the control group C1, the viable bacterial count of all samples remained above 10 6 CFU / mL, which is the basic requirement to ensure the efficacy of probiotics (Sohrabvandi et al., 2010). Notably, the number of lactic acid bacteria in the treatment groups with Chinese date kernel powder did not decrease significantly (p > 0.05), indicating that it had a significant protective effect on bacterial survival. The ability of Chinese date kernel powder to maintain the survival of lactic acid bacteria is mainly due to its unique biochemical composition. Chinese date is rich in polysaccharides, flavonoids, saponins, and other bioactive components (Ruan et al., 2022; Mao et al., 2018). These components mainly play a role through two mechanisms: one is the prebiotic effect, the polysaccharides in Chinese date kernel powder can act as a substrate for lactic acid bacteria fermentation, exerting a prebiotic effect (Guo et al., 2021); the other is antioxidant protection, the bioactive components such as flavonoids in it have strong antioxidant activity (Borisov et al., 2021). During storage, lactic acid bacteria will suffer oxidative stress, leading to damage to cell membranes and DNA, and thus causing cell death. The antioxidant substances in Chinese date kernel powder can alleviate this stress damage, thereby maintaining cell membrane integrity and improving bacterial survival rate. Maintaining the number of lactic acid bacteria above 10 6 CFU / mL is crucial, not only to ensure the biochemical stability of fermented products, but also to help maintain their potential probiotic function for intestinal health. Therefore, the addition of Chinese date kernel powder is a feasible natural strategy to improve the shelf life and functional quality of probiotic-containing products.

[0077] 10. Evaluation of the potential neuroprotective efficacy of fermented milk The results in Table 5 showed significant differences (p < 0.05) in total phenolic and flavonoid contents among the samples. The functional Zizyphus jujuba Mill. fermented milk sample T1 exhibited the best performance in key bioactive components and functional activities, with the highest total phenolic content (235.75 ± 1.92 mg GAE / 100 g) and flavonoid content (114.07 ± 4.54 mg RE / 100 g), which endowed it with superior antioxidant capacity, as evidenced by the strongest DPPH scavenging activity (110.24 ± 6.11 mg Ascorbic / 100 g) and the highest FRAP assay value (99.07%). Moreover, even without the addition of Zizyphus jujuba Mill. powder, the samples T4 and T5 fermented using the selected strains SWU-DLB2 and SWU-DLB3 exhibited significantly improved bioactivities compared to the control group C2. This improvement can be attributed to the metabolic activities of the strains used, which can biotransform complex phenolic compounds into simpler, more bioavailable flavonoid compounds or synthesize new flavonoid molecules during fermentation (Gaur and Gänzle, 2023). The results clearly showed that the treatment groups using SWU-DLB2 or SWU-DLB3 strains alone (T4, T5) were superior to the treatment groups using the two strains in combination (T3, T6) in promoting the accumulation of bioactive components.

[0078] Most importantly, the T1 sample exhibited the highest acetylcholinesterase inhibition rate of 30.66%, which is a key activity indicator for assessing its neuroprotective potential. The results showed that the bioactive component profile of T1 (fermented using the SWU-DLB2 strain and supplemented with Zizyphus jujuba Mill. powder) was most significantly improved compared to the control group, followed by T2 (fermented using the SWU-DLB3 strain and supplemented with Zizyphus jujuba Mill. powder). At the same time, samples T4 and T5 also exhibited high acetylcholinesterase inhibition rates, indicating a strong correlation between the increase in flavonoid content and the enhancement of inhibitory activity during fermentation. It has been well studied that flavonoid compounds can effectively inhibit acetylcholinesterase activity through mechanisms such as binding to the enzyme active site (Cichon et al., 2025). The excellent performance of the selected strains (SWU-DLB2 and SWU-DLB3) indicates their specific ability to produce potent inhibitory compounds or enhance the inhibitory effect by modifying substrates. Ultimately, the increase in these specific phytochemicals directly reflects the improvement in enzyme inhibition rates. Fermentation using specific strains, especially in a substrate fortified with Zizyphus jujuba Mill. powder, can significantly increase flavonoid and polyphenol levels, thereby greatly enhancing the bioactivity of the product through the enhancement of enzyme inhibition activity. These findings highlight the great potential of using specific fermentation strategies combined with Zizyphus jujuba Mill. powder to enhance the functional characteristics of foods.

[0079] Table 5 Total phenolic, total flavonoid content, antioxidant activity, and acetylcholinesterase inhibition rate of functional Zizyphus jujuba Mill. fermented milk

[0080] Data are means ± standard deviation of triplicate. Means with the same lower case letter in the same column are not significantly different (p > 0.05). C2: control fermented milk prepared using yogurt starter; T4: fermented milk prepared using SWU-DLB2 as a helper starter; T5: fermented milk prepared using SWU-DLB3 as a helper starter; T6: fermented milk prepared using SWU-DLB2 and SWU-DLB3 mixed strains as a helper starter; C1: control fermented milk prepared using yogurt starter (supplemented with 2% amomum seed powder); T1: fermented milk prepared using SWU-DLB2 as a helper starter and supplemented with 2% amomum seed powder; T2: fermented milk prepared using SWU-DLB3 as a helper starter and supplemented with 2% amomum seed powder; T3: fermented milk prepared using SWU-DLB2 and SWU-DLB3 mixed strains as a helper starter and supplemented with 2% amomum seed powder.

[0081] Conclusion This study successfully isolated a new probiotic candidate strain with significant in vitro neuroprotective potential, confirming its acetylcholinesterase inhibitory activity and GABA production capacity. The selected strain is suitable as a helper starter for fermented milk production. The addition of amomum seed powder significantly improves the survival rate and functional properties of probiotics in the product. Future research should focus on identifying the specific bioactive metabolites responsible for acetylcholinesterase inhibition, elucidating its molecular mechanism, and verifying its neuroprotective efficacy through in vivo experiments.

[0082] The above-described embodiments are merely preferred modes of the present application, and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solutions of the present application without departing from the design spirit of the present application shall fall within the scope of protection of the present application as defined by the claims.

Claims

1. A Lactobacillus delbrueckii (L. delbrueckii) strain, characterized in that, Lactobacillus delbrueckii subsp. The preservation number of the Lactobacillus delbrueckii is GDMCC No: 67173 or GDMCC No: 67174. ​ 2. Use of Lactobacillus delbrueckii (Lactobacillus delbrueckii ssp. bulgaricus) of claim 1 for the preparation of a neuroprotective functional fermented food. Lactobacillus delbrueckii subsp. ) 3. Use according to claim 2, characterized in that, The food is fermented milk.

4. A method of preparing a fermented milk using the Lactobacillus debruekii according to claim 1, characterized by, The method comprises the following steps: After adding 1.5-2.5% of the Semen Ziziphi Spinosae powder in the liquid milk and sterilizing, adding the Lactobacillus delbrueckii with the preservation number of GDMCC No: 67173 and / or GDMCC No: 67174, fermenting at 41-43℃ for 0.8-1.2h, and then adding the yogurt starter to ferment.

5. The method of claim 4, wherein, The liquid milk is cow milk.

6. The method of claim 5, wherein, The adding amount of the Semen Ziziphi Spinosae powder is 2%.

7. The method of claim 6, wherein, When only one kind of Lactobacillus delbrueckii is added in the sterilized liquid milk, the inoculation amount of the Lactobacillus delbrueckii is 1.5-2.5% by volume ratio; when two kinds of Lactobacillus delbrueckii are added in the sterilized liquid milk, the number ratio of the two kinds of Lactobacillus delbrueckii is 0.8-1.2:0.8-1.2, and the total inoculation amount of the two kinds of Lactobacillus delbrueckii is 1.5-2.5% by volume ratio.

8. The method of claim 7, wherein, After inoculating the Lactobacillus delbrueckii, fermenting at 42℃ for 1h.

9. Fermented milk prepared by the method of any one of claims 4-8.

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