Lactobacillus delbrueckii swu-dlb2 and swu-dlb3 and their development and application in functional fermented food

Abstract

The application discloses Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 and development and application thereof in functional fermented food, and belongs to the technical field of microbial fermentation. The Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 have been preserved in the Guangdong Microbial Culture Collection Center on October 28, 2025, and the preservation numbers are GDMCC No: 67173 and GDMCC No: 67174 respectively. It is found through experiments that the fermentation of the two strains mentioned above in dairy products containing Zizyphus jujuba Mill. powder can significantly improve the content of bioactive substances, and the total phenol content, flavonoid content and antioxidant activity are all significantly improved. The Lactobacillus delbrueckii strain provided by the application is combined with a plant-based supplement and applied to a food model, a functional food with enhanced neuroprotective potential is developed, and the important role of microbial metabolism in the functionalization of food is highlighted.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and particularly relates to Lactobacillus delbrueckii SWU-DLB2 and SWU-DLB3 and their development and application in functional fermented foods. Background Technology

[0002] Currently, neurodegenerative diseases (such as dementia, Alzheimer's disease, and Parkinson's disease) are on a global rise, posing a serious socioeconomic and medical challenge. The pathogenesis of these diseases is complex, involving multiple key aspects such as oxidative stress, neuroinflammation, dysregulation of acetylcholinesterase activity, and gut-brain axis dysfunction. Furthermore, population aging exacerbates the severity of this problem. At present, effective treatments are scarce, and traditional pharmacological inhibitors also pose hepatotoxicity and potential fatal risks.

[0003] In existing technologies, probiotics have been proven to have the potential to combat neurodegenerative diseases, and some lactic acid bacteria strains have exhibited antioxidant, antibacterial, anti-inflammatory, and neuroprotective properties. However, the screening and application of specific strains with both high acetylcholinesterase inhibitory activity and γ-aminobutyric acid (GABA) synthesis capabilities still require breakthroughs. Meanwhile, jujube seed, an agricultural by-product with an annual output exceeding 3 million tons, is rich in flavonoids, alkaloids, and other bioactive compounds, possessing antioxidant and neuroprotective effects, but its application in fermented foods has not yet been fully developed. Currently, there is a lack of mature technical solutions for combining specific high-efficiency probiotics with plant-based bioactive components, and research on the synergistic effects and genomic mechanisms of functional components in fermented milk is insufficient.

[0004] Traditional acetylcholinesterase inhibitors for neurodegenerative diseases (such as Alzheimer's disease) have hepatotoxicity and potential fatal risks, necessitating the development of natural and safe foods as alternative or supplementary strategies. Summary of the Invention

[0005] To solve the above-mentioned technical problems, the present invention provides a *Lactobacillus delbrueckii* (… Lactobacillus delbrueckii subsp. The Lactobacillus delbrueckii preservation number is GDMCC No: 67173 or GDMCC No: 67174.

[0006] The present invention also provides the above-mentioned Lactobacillus delbrueckii ( Lactobacillus delbrueckii subsp. Application of ) in the preparation of fermented foods.

[0007] Preferably, the food is fermented milk.

[0008] The present invention also provides a method for preparing fermented milk using the above-mentioned Lactobacillus delbrueckii, comprising the following steps: adding 1.5-2.5% of jujube seed powder to liquid milk, sterilizing it, adding an appropriate amount of Lactobacillus delbrueckii with preservation numbers GDMCC No: 67173 and / or GDMCC No: 67174, fermenting at 41-43℃ for 0.8-1.2h, and then adding yogurt starter for fermentation.

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

[0010] More preferably, the amount of jujube seed powder added is 2%.

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

[0012] More preferably, after inoculation with Lactobacillus delbrueckii, fermentation is carried out at 42°C for 1 hour.

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

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] (1) Precise strain screening: The selected Lactobacillus delbrueckii strains SWU-DLB2 and SWU-DLB3 have high acetylcholinesterase inhibitory activity, GABA synthesis ability and excellent probiotic characteristics. The whole genome sequencing verified that there are no drug resistance genes and virulence factors, and the safety is high.

[0016] (2) Enhanced Functional Synergy: Combining specific probiotics with jujube seed powder achieves the synergistic effect of "probiotics + plant-based bioactive components", significantly increasing the content of total phenols, flavonoids and antioxidant activity. The total phenol content of the jujube seed powder enhanced group fermented by SWU-DLB2 strain reached 235.75±1.92mg GAE / 100g, the flavonoid content reached 114.07±4.54 mg RE / 100g, and the acetylcholinesterase inhibition rate reached 30.66%, which were significantly higher than those of the control group (p<0.05).

[0017] (3) Optimized and reasonable process: By pre-fermentation process and addition of jujube seed powder, the viscosity and sensory characteristics of fermented milk are improved, while the survival rate of probiotics during refrigeration is increased, ensuring the functional stability of the product during its shelf life;

[0018] (4) Efficient utilization of resources: The unused agricultural by-product of jujube seed is transformed into a high-value functional component, expanding its application field.

[0019] Biological preservation instructions for Lactobacillus delbrueckii SWU-DLB2:

[0020] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0021] Accession number: GDMCC No: 67173;

[0022] Deposit date: October 28, 2025;

[0023] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0024] Taxonomic nomenclature: Lactobacillus delbrueckii subsp bulgaricus .

[0025] Biological preservation instructions for Lactobacillus delbrueckii SWU-DLB3:

[0026] Preservation institution: Guangdong Provincial Center for Microbial Culture Collection;

[0027] Accession number: GDMCC No: 67174;

[0028] Deposit date: October 28, 2025;

[0029] Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou;

[0030] Taxonomic nomenclature: Lactobacillus delbrueckii subsp allosunkii . Attached Figure Description

[0031] Figure 1 The inhibitory capacity of the lactic acid bacteria isolates in Example 1 against acetylcholinesterase is given by: a: inhibitory capacity of Lactobacillus; b: inhibitory capacity of cocci; c: acetylcholinesterase inhibition rate of Lactobacillus and Enterococcus strains; GRS: galantamine control standard; DRS: donepezil control standard; DLB2: Lactobacillus delbrueckii SWU-DLB2; DLB3: Lactobacillus delbrueckii SWU-DLB3.

[0032] Figure 2 The antibacterial activity of the strains screened in Example 1, wherein (a) against Escherichia coli (10 9 (a) Inhibition of Salmonella Typhimurium (CFU / mL); (b) Inhibition of Salmonella Typhimurium (10 CFU / mL); 9Inhibitory effect of CFU / mL. C+: ampicillin (10 mg / mL), C-: culture medium control; DLB2: Lactobacillus delbrueckii SWU-DLB2; DLB3: Lactobacillus delbrueckii SWU-DLB3.

[0033] Figure 3 This is a representative gel electrophoresis image of the gad gene detection of all tested strains in Example 1, where C+: *Lactobacillus plantarum* SWU-ZFT6 (GDMCC No: 66290, positive control); C-: *Lactobacillus rhamnosus* DSM 20021 (negative control); DLB2: *Lactobacillus delbrueckii* SWU-DLB2; DLB3: *Lactobacillus delbrueckii* SWU-DLB3.

[0034] Figure 4 The following figures show the changes in acidity percentage (%) and pH value of the functional fermented milk during refrigeration in Example 1, where (a) is the acidity percentage of the functional fermented milk without added jujube seed powder; (b) is the acidity percentage of the fermented milk with 2% added jujube seed powder; (c) is the pH value of the functional fermented milk without added jujube seed powder; and (d) is the pH value of the fermented milk with 2% added jujube seed powder.

[0035] Figure 5 The following figures illustrate the changes in (a) viscosity and (b) lactic acid bacteria survival rate of fermented milk during refrigeration in Example 1. 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.

[0036] Figure 6 This is the phylogenetic tree of SWU-DLB2 in Example 1.

[0037] Figure 7 This is the phylogenetic tree of SWU-DLB3 in Example 1. Detailed Implementation

[0038] Example 1

[0039] 1. Isolation of lactic acid bacteria and screening for their acetylcholinesterase inhibitory potential

[0040] Samples were taken from 25 conventionally fermented dairy products and stored at 4°C. For isolation, samples were incubated in skim milk at 30°C, 37°C, and 42°C until coagulation. The samples were then streaked onto M17 and MRS agar plates and anaerobically cultured at the same temperature. The purified isolates were phenotypically evaluated and stored at -80°C for further studies.

[0041] The inhibitory activity of acetylcholinesterase in sterile, cell-free filtrate obtained from 48-hour cultures was determined using a microplate assay (Pandey et al., 2014). Donepezil hydrochloride and galantamine hydrobromide were used as positive controls. All experiments were performed in triplicate and independently three times. The acetylcholinesterase inhibition rate was calculated using the following formula:

[0042] AChEI (%) = [(blank OD value - sample OD value) / blank OD value] × 100;

[0043] When calculating the inhibition rate of the standard, the blank control was phosphate-buffered saline (PBS; 50 mM); when calculating the inhibition rate of the cell-free filtrate, the blank control was culture medium (MRS or M17).

[0044] 2. Probiotic potential and safety characteristics

[0045] The functional probiotic potential of 15 selected lactic acid bacteria isolates with acetylcholinesterase inhibition potential was evaluated.

[0046] 2.1 Assessment of tolerance to acid, pepsin, and trypsin

[0047] The strain's tolerance to gastrointestinal conditions was assessed according to the method described by Madian et al. (2025). The simplified procedure was as follows: overnight culture was centrifuged to collect the bacterial cells, washed twice with PBS, and then resuspended in simulated gastric fluid to a final concentration of approximately 10⁻⁶. 9 CFU / mL. The simulated gastric fluid was PBS at pH 3.0, with pepsin added at a concentration of 1:10,000. During the gastric phase, the bacterial suspension was incubated at 37°C for 0 hours and 3 hours, respectively. Subsequently, during the intestinal phase, 1 mL of the gastric-treated bacterial suspension was added to 9 mL of simulated intestinal fluid, and incubated at 37°C for 0 hours and 4 hours, respectively. The survival rate was calculated using the following formula:

[0048] Survival rate (%) = (logarithm of final viable count / logarithm of initial viable count) × 100;

[0049] Based on the survival rate results, and referring to the criteria of (Narimani et al., 2015), the acid tolerance of the strains was divided into four categories: sensitive (<10%), moderately tolerant (10%–60%), well tolerant (60%–80%), and highly tolerant (>80%).

[0050] 2.2 Assessment of bile salt tolerance

[0051] Bile tolerance of the strain was assessed according to the method of Guo et al. 2009. Overnight cultures were inoculated into MRS broth with and without 0.3% (w / v) bile salts, and incubated at 37°C. OD was measured hourly using a spectrophotometer. 600 Absorbance was used to record growth over 24 hours, with uninoculated medium serving as a blank control. Based on the survival rate of the strains in the presence of bile salts, they were classified into four groups according to the criteria of Chateau et al. (1994).

[0052] 2.3 Antibacterial activity against gastrointestinal pathogens

[0053] The in vitro antibacterial activity of lactic acid bacteria against Salmonella Typhimurium ATCC33110 and Escherichia coli NCTC12900 was evaluated using the agar diffusion method. The pathogens (10... 9 Cell-free supernatant (nCFS) containing CFU / mL was inoculated into specific culture media to prepare wells with a diameter of 6 mm. 100 μL of sterile, neutralized, cell-free supernatant (nCFS) was added to each well, and the mixture was incubated at 37°C for 24 hours. Ampicillin (10 mg / mL) was used as a positive control, and uninoculated MRS broth was used as a negative control. The average diameter of the inhibition zone was measured in millimeters (mm).

[0054] 2.4 Detection of γ-aminobutyric acid (GABA) synthesis gene

[0055] Total DNA was extracted from bacterial cultures in the logarithmic growth phase using the TIANamp bacterial DNA extraction kit. The presence of the gad gene was detected by PCR using specific primers (gad-F / gad-R) and a Direct PCR kit. Reactions were performed in a Bio-Rad PCR instrument, and the amplified products were analyzed by 1% agarose gel electrophoresis. *Lactobacillus plantarum* SWU-ZFT6 (positive control) and *Lactobacillus rhamnosus* DSM 20021 (negative control) were used as controls. PCR reaction conditions followed the method described by Langa et al., 2024.

[0056] 2.5 Hemolytic activity assay

[0057] The hemolytic activity of lactic acid bacteria isolates was assessed according to the method of Abedi et al. (2018) to determine their application safety. Each strain was streaked onto blood agar plates containing 7% (v / v) defibrinated sheep blood and incubated at 37°C under microaerophilic conditions for 48 hours. Hemolysis was observed after the incubation period.

[0058] 2.6 Antibiotic susceptibility testing (AST)

[0059] Antibiotic susceptibility of lactic acid bacteria strains was determined using the disk diffusion method. Ten antibiotic disks were used: 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 suitable conditions for 24 hours, and the diameter of the inhibition zone (mm) was measured. Results were categorized as susceptible (S), intermediate (I), or resistant (R) according to the CLSI guidelines (CLSI, 2017).

[0060] 3. Molecular identification

[0061] Molecular identification of lactic acid bacteria isolates was performed using 16S rRNA gene sequencing. Purified genomic DNA was used as a template, and the 16S rRNA gene was amplified 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 sequences were analyzed for similarity using the BLAST tool in the National Center for Biotechnology Information (NCBI) database. Based on the 16S rRNA sequences, a phylogenetic tree was constructed using the 20 most species-related strains. (See [link to phylogenetic tree]). Figure 6 and Figure 7 Both SWU-DLB2 and SWU-DLB3 are phylogenetically classified as lactic acid bacteria, and are particularly closely related to species of the genus Lactobacillus.

[0062] 4. Genotypic characteristics analysis of SWU-DLB2 and SWU-DLB3 strains

[0063] 4.1 Genomic DNA Extraction

[0064] SWU-DLB2 and SWU-DLB3 strains were inoculated into MRS broth and cultured at 37°C with shaking at 150 rpm for approximately 12 hours. After centrifugation to collect the bacterial cells, genomic DNA was extracted according to the instructions of the bacterial / fungal DNA extraction kit.

[0065] 4.2 Whole genome sequencing

[0066] Genome sequencing was outsourced to a specialized institution, utilizing a dual-platform sequencing approach combining PacBio and Illumina technologies. For Illumina sequencing, sequencing libraries were constructed from genomic DNA using the Nextera XT library preparation kit, and paired-end sequencing was performed on the Illumina NovaSeq 6000 platform. For PacBio sequencing, genomic DNA was fragmented, purified, and end-repaired, then ligated with SMRT bell sequencing adapters. PacBio libraries were subsequently prepared and sequenced on SMRT cells.

[0067] 4.3 Bioinformatics Analysis

[0068] Bioinformatics analysis was performed on data generated from PacBio and Illumina platforms, and the analysis was completed on the Majorbio cloud platform of Shanghai Maiyue Information Technology Co., Ltd. Short reads and HiFi reads were assembled using Unicycler and Pilon software to construct complete genomes. Chromosome and plasmid coding sequences were predicted using Glimmer or Prodigal and GeneMarkS software, respectively.

[0069] 5. Preparation of functional fermented milk using screened strains as auxiliary starter.

[0070] 5.1 Preparation of starter culture and fermented milk

[0071] After three consecutive subcultures and activations of the two helper strains SWU-DLB2 and SWU-DLB3, they were inoculated into raw milk and cultured at 42°C for 18 hours to achieve a final starter culture concentration of approximately 2 × 10⁻⁶. 8 CFU / mL.

[0072] Whole milk powder was used as a base and rehydrated to prepare a 12.5% ​​(w / vg / mL) reconstituted milk. The reconstituted milk was divided into two groups: one group was supplemented with 2% (w / vg / mL) jujube seed powder (JP) (jujube seed powder was obtained by directly grinding jujube seeds), and the other group served as a control without JP. Both groups of reconstituted milk were heat-treated at 90℃ for 5 minutes (Tamime and Robinson, 2007) and then cooled to a fermentation temperature of 42℃. Each group (with and without JP) was further divided into four treatment groups, for a total of eight experimental groups:

[0073] C1: Inoculate directly with yogurt starter culture (two strains, Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, in a ratio of 1:1) at 1 g / L, and ferment at 42°C until coagulation occurs at pH 4.70±0.1.

[0074] T1: First, inoculate with SWU-DLB2 strain for pre-fermentation (1 hour, 42℃), then add the same yogurt starter as group C1.

[0075] T2: First, inoculate with SWU-DLB3 strain for pre-fermentation (1 hour, 42℃), then add the same yogurt starter as group C1.

[0076] T3: Simultaneously inoculate with SWU-DLB2 and SWU-DLB3 strains for pre-fermentation (1 hour, 42°C), with an inoculation ratio of 1:1 for the two strains, and then add the same yogurt starter as group C1.

[0077] The total inoculation amounts of SWU-DLB2 in group T1, SWU-DLB3 in group T2, and SWU-DLB2 and SWU-DLB3 in group T3 were the same, and the concentration of Lactobacillus delbrueckii in groups T1-T3 was 2×10⁻⁶. 8 CFU / mL, with a total inoculation rate of 2%.

[0078] Samples C2, T4, T5, and T6 were treated in the same manner as above, except that JP was not added. Fermentation was terminated when the endpoint pH reached 4.70±0.1. After gentle stirring, 5% sucrose was added, and the mixture was cooled and refrigerated at 4.0±0.5℃ for 14 days before analysis.

[0079] 5.2 Physicochemical Index Analysis

[0080] During refrigeration, the physicochemical properties of the fermented milk were measured periodically: pH was measured using a pH meter; titratable acidity (expressed as lactic acid%) and total solids content (%) were determined according to the AOAC (2019) method, and total nitrogen content was determined by the Kjeldahl method; fat content was determined by the filter bag method (ANKOM XT15 fat analyzer, USA).

[0081] 5.3 Viscosity and Sensory Evaluation

[0082] Apparent viscosity was determined using a rotational viscometer during refrigeration, according to ISO 7884-2 standard, under the following conditions: 10°C, rotor #3, and 60 rpm. Sensory evaluation was conducted by 10 trained evaluators (6 men and 4 women) from the College of Food Science and Technology, Southwest University, who scored the samples on appearance, texture, aroma, taste, and overall acceptability (ethics approval number: HF20251002). Samples were placed in uniform colorless cups at room temperature and evaluated in the afternoon using a 9-point preference scale (Meilgaard, 1999).

[0083] 5.4 Determination of the number of viable lactic acid bacteria

[0084] According to ISO 19344:2015, the number of viable lactic acid bacteria was counted after anaerobic incubation at 37°C for 72 hours on MRS medium during refrigeration.

[0085] 5.5 Bioactive components and antioxidant activity

[0086] Accurately weigh 20 g of fermented milk sample and defatt it with n-hexane at a ratio of 1:3 (w / v) (Sellami et al., 2009). Sonicate in a water bath (30℃, 15 min) while maintaining a constant temperature. The defatted residue is then extracted with a methanol-water solution (80% methanol, v / v) at a ratio of 1:10 (w / v) to extract polyphenols and other antioxidant components (Duda-Chodak and Tarko, 2007), with continuous shaking at 4℃ for 24 hours. The extract is centrifuged (10,000 × g, 15 min, 4℃), and the supernatant is collected and filtered to obtain a clear crude extract.

[0087] Total phenol content: determined by the modified Folin-Ciocalteu method (Abirami et al., 2014).

[0088] Total flavonoid content: determined by the aluminum chloride colorimetric method (Barros et al., 2011).

[0089] Antioxidant activity: The extract was dissolved in 10 mL of 80% methanol and determined by the DPPH method (Brand-Williams et al., 1995) and the FRAP method (Benzie and Strain, 1996).

[0090] 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 measured (Ellman et al., 1961; Vinutha et al., 2007).

[0091] 2.6 Statistical Analysis

[0092] 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.

[0093] Results and Discussion

[0094] 1. Acetylcholinesterase inhibitory activity of lactic acid bacteria strains

[0095] 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.

[0096] 2. Potential characteristics of probiotics

[0097] 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).

[0098] 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.

[0099]

[0100] 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).

[0101] 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).

[0102] 3. Antibacterial activity

[0103] 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).

[0104] 4. Glutamate decarboxylase ( Glutamate Decarboxylase , gad Gene testing

[0105] 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).

[0106] 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.

[0107] 5. Hemolytic activity and antibiotic susceptibility testing

[0108] 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.

[0109] Table 2. Identification results of 16S rRNA gene of lactic acid bacteria isolates and their safety characteristics.

[0110]

[0111] 6. Molecular identification

[0112] 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.

[0113] 7. Genomic characteristics of SWU-DLB2 and SWU-DLB3 strains

[0114] 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).

[0115] 7.1 Identification of probiotic-related genes

[0116] 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 These genes are crucial for the specific interaction between bacterial surface components and host epithelial cell surface receptors (Xu et al., 2022). Furthermore, genes responsible for bile and acid tolerance were detected in both strains. cfa, nagB, ppa, nhaC, napA This characteristic supports its viability in the gastrointestinal tract, ensuring a sufficient number of live bacteria reach specific host sites to exert probiotic effects (De Jesus et al., 2022). The stress adaptation mechanism is also well-defined; both genomes contain complete heat shock protein gene clusters. htpX, hrcA, grpE, dnaK, dnaJ, groES, groEL These genes not only function under high-temperature conditions but also constitute a defense mechanism against sudden heat stress (Jakaria Al-Mujahidy et al., 2024). Simultaneously, cold stress regulatory genes exist in the genome ( cspAThis helps the strain survive in environments below its optimal growth temperature. The study also confirmed its antibacterial activity by detecting type III RamC lanthanide, a heat-stable bacteriocin that catalyzes the hydrolysis of bacterial cell walls, leading to cell lysis and death (Angelescu et al., 2022).

[0117] Table 3. Probiotic-related genes in the genomes of Lactobacillus delbrueckii strains SWU-DLB2 and SWU-DLB3 predicted based on KEGG annotation.

[0118]

[0119]

[0120] (+): Present; (-): Missing

[0121] 7.2 Identification of genes related to acetylcholinesterase inhibition

[0122] Several genes directly or indirectly involved in acetylcholinesterase inhibition were identified in the genomes of SWU-DLB2 and SWU-DLB3 strains, suggesting a potential protective effect against neurodegenerative diseases. Analysis revealed that both strains contain genes involved in the synthesis of neuroactive molecules, particularly those encoding a glutamate-GABA transporter. gadC The pcp gene is responsible for GABA efflux and glutamate uptake (Ma et al., 2012). GABA, a byproduct of glutamate decarboxylation, functions as an inhibitory neurotransmitter in the human central nervous system (Gong et al., 2019). Furthermore, pyroglutamyl peptidase, encoded by the pcp gene, is a highly specific membrane-bound thyrotropin-releasing hormone degrading enzyme (Charli et al., 1989). As a tripeptide, it plays multiple homeostatic regulatory roles in the brain, directly affecting neuronal excitability and potentially participating in mood balance regulation (Rodríguez-Molina et al., 2009).

[0123] 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. folCThe 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).

[0124] 8. Effects of the selected bacterial strains and jujube seed powder on the properties of fermented milk

[0125] 8.1 Physical and chemical properties

[0126] 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.

[0127] Table 4 Chemical composition analysis and sensory evaluation of functional fermented milk

[0128]

[0129] 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).

[0130] 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.

[0131] 8.2 Viscosity and Sensory Evaluation

[0132] 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.

[0133] 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.

[0134] Notably, there were no significant differences in flavor scores among the treatment groups (p>0.05), indicating that the addition of the fermentation aid and jujube seed powder did not introduce undesirable flavors. This result is consistent with previous studies that jujube seed powder can enhance the flavor profile of products without negatively impacting flavor (Zhang et al., 2024). In terms of taste, all groups showed a positive trend, indicating that the slight sweetness and other desirable sensory characteristics of jujube seed powder contribute to improved overall taste acceptability, which is also consistent with the improving effects of other plant-based fortification ingredients in dairy products (Azeem et al., 2024).

[0135] 9. Analysis of the prebiotic effect of jujube seed powder

[0136] from Figure 5 b shows that the number of lactic acid bacteria decreased during refrigeration, but except for the control group C1, the viable bacteria count of all samples remained at 10. 6 A CFU / mL level or higher is a basic requirement for ensuring the efficacy of probiotics (Sohrabvandi et al., 2010). Notably, the decrease in lactic acid bacteria count in the treatment group with added jujube seed powder was not significant (p>0.05), indicating a clear protective effect on bacterial survival. The ability of jujube seed powder to maintain lactic acid bacteria survival is mainly attributed to its unique biochemical composition. Jujube seeds are rich in polysaccharides and various bioactive components such as flavonoids and saponins (Ruan et al., 2022; Mao et al., 2018). These components mainly exert their effects through two mechanisms: firstly, the prebiotic effect, where the polysaccharides in jujube seed powder can serve as substrates for lactic acid bacteria fermentation, exerting a prebiotic effect (Guo et al., 2021); secondly, antioxidant protection, where the flavonoids and other bioactive components have strong antioxidant activity (Borisov et al., 2021). During storage, lactic acid bacteria suffer from oxidative stress, leading to cell membrane and DNA damage, which in turn causes bacterial death. The antioxidants in jujube seed powder can alleviate this stress damage, thereby maintaining cell membrane integrity and improving bacterial survival rate. Maintaining the lactic acid bacteria count at 10... 6 A concentration of CFU / mL or higher is crucial, as it not only ensures the biochemical stability of fermented products but also helps maintain their potential beneficial functions for gut health. Therefore, adding jujube seed powder is a feasible natural strategy to improve the shelf life and functional quality of probiotic-containing products.

[0137] 10. Evaluation of the potential neuroprotective efficacy of fermented milk

[0138] Table 5 shows that there were significant differences in the total phenol and total flavonoid contents among the samples (p<0.05). The functional jujube seed fermented milk sample T1 showed the best performance in key bioactive components and functional activities, with the highest total phenol content (235.75±1.92 mg GAE / 100g) and the highest flavonoid content (114.07±4.54 mg RE / 100g). This gave it excellent antioxidant capacity, exhibiting the strongest DPPH scavenging activity (110.24±6.11 mg Ascorbic / 100g) and the highest FRAP value (99.07%). Furthermore, even without the addition of jujube seed powder, samples T4 and T5, fermented using the screened strains SWU-DLB2 and SWU-DLB3, showed significantly higher bioactivity than the control group C2. This improvement can be attributed to the metabolic activity of the strains used, which can bioconvert complex phenolic substances into simpler, more bioavailable flavonoids, or synthesize new flavonoid molecules during fermentation (Gaur and Gänzle, 2023). The results clearly show that the treatment groups using either SWU-DLB2 or SWU-DLB3 strains alone (T4, T5) were superior to the treatment groups using a combination of both strains (T3, T6) in promoting the accumulation of bioactive components.

[0139] Of particular importance, sample T1 exhibited the highest inhibition rate against acetylcholinesterase, reaching 30.66%, a key activity indicator for assessing its neuroprotective potential. The results showed that, compared to the control group, T1 (fermented using SWU-DLB2 strain with added jujube seed powder) showed the most significant improvement in its bioactive component profile, followed by T2 (fermented using SWU-DLB3 strain with added jujube seed powder). Simultaneously, samples T4 and T5 also demonstrated high acetylcholinesterase inhibition rates, indicating a strong correlation between increased flavonoid content and enhanced inhibitory activity during fermentation. Extensive research has confirmed that flavonoids can effectively inhibit acetylcholinesterase activity through mechanisms such as binding to enzyme active sites (Cichon et al., 2025). The superior performance of the selected strains (SWU-DLB2 and SWU-DLB3) suggests their specific ability to produce potent inhibitory compounds or enhance inhibitory effects through substrate modification. Ultimately, the increase in these specific phytochemicals directly translates into an improved enzyme inhibition rate. Fermentation using specific strains, especially in a matrix fortified with jujube seed powder, can significantly increase the levels of flavonoids and polyphenols, thereby greatly improving the product's bioactivity by enhancing enzyme inhibitory activity.

[0140] Table 5. Total phenolic and total flavonoid content, antioxidant activity, and acetylcholinesterase inhibition rate of functional jujube seed fermented milk.

[0141]

[0142] 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). C2: Control fermented milk prepared using yogurt starter; T4: Fermented milk prepared using SWU-DLB2 as co-starter; T5: Fermented milk prepared using SWU-DLB3 as co-starter; T6: Fermented milk prepared using a mixed strain of SWU-DLB2 and SWU-DLB3 as co-starter; C1: Control fermented milk prepared using yogurt starter (supplemented with 2% jujube seed powder); T1: Fermented milk prepared using SWU-DLB2 as co-starter and supplemented with 2% jujube seed powder; T2: Fermented milk prepared using SWU-DLB3 as co-starter and supplemented with 2% jujube seed powder; T3: Fermented milk prepared using a mixed strain of SWU-DLB2 and SWU-DLB3 as co-starter and supplemented with 2% jujube seed powder.

[0143] in conclusion

[0144] This study successfully isolated a novel probiotic candidate strain with significant in vitro neuroprotective potential, confirming its acetylcholinesterase inhibitory activity and γ-aminobutyric acid (GABA) production capacity. The selected strain is suitable as a co-fermentation agent in fermented milk production. The addition of jujube seed powder significantly improved the survival rate and functional properties of the probiotics in the product. Future research should focus on identifying the specific bioactive metabolites responsible for acetylcholinesterase inhibition, elucidating their molecular mechanism of action, and verifying their neuroprotective efficacy through in vivo experiments.

[0145] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of Lactobacillus delbrueckii ( Lactobacillus delbrueckii ), characterized in that, The Lactobacillus delbrueckii preservation number is GDMCC No: 67173 or GDMCC No: 67174.

2. The *Lactobacillus delbrueckii* as described in claim 1 (… Lactobacillus delbrueckii Application of ) in the preparation of fermented foods.

3. The application according to claim 2, characterized in that, The food product is fermented milk.

4. A method for preparing fermented milk using Lactobacillus delbrueckii, characterized in that, Includes the following steps: Add 1.5-2.5% of jujube seed powder to liquid milk, sterilize it, add an appropriate amount of Lactobacillus delbrueckii with preservation numbers GDMCC No: 67173 and / or GDMCC No: 67174, ferment at 41-43℃ for 0.8-1.2 hours, and then add yogurt starter for further fermentation.

5. The method according to claim 4, characterized in that, The liquid milk is cow's milk.

6. The method according to claim 5, characterized in that, The amount of jujube seed powder added is 2%.

7. The method according to claim 6, characterized in that, When only one type of Lactobacillus delbrueckii is added to sterilized liquid milk, the inoculum amount of Lactobacillus delbrueckii is 1.5-2.5% by volume; when two types of Lactobacillus delbrueckii are added to sterilized liquid milk at the same time, the ratio of the number of the two types of Lactobacillus delbrueckii is 0.8-1.2:0.8-1.2, and the total inoculum amount of the two types of Lactobacillus delbrueckii is 1.5-2.5% by volume.

8. The method according to claim 7, characterized in that, After inoculation with Lactobacillus delbrueckii, fermentation was carried out at 42°C for 1 hour.

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

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