Copper ion sensitive lactobacillus bulgaricus and application thereof

Abstract

The invention discloses a copper ion sensitive type lactobacillus bulgaricus and an application of the copper ion sensitive type lactobacillus bulgaricus. According to the expression quantity of the acidification marker gene LDBRS05285, a strain of lactobacillus bulgaricus E9 with high sensitivity in CU1 is screened through high-throughput grading. The transcription factor CopR in the strain is found to be changed from lysine to arginine at the 71st amino acid, so that the binding force between the transcription factor CopR and the LDBRS05285 promoter region is enhanced, and the expression quantity of LDBRS05285 is further up-regulated. In the glycometabolism detection of the yoghourt, the lactose metabolism of the E9 fermented yoghourt is accelerated, the content of galactose and glucose is increased, and the content of lactic acid is reduced. Therefore, the acidifying degree of yogurt fermented by the CUhigh-sensitive strain E9 is lower after the yogurt is subjected to CUtreatment, and the yogurt has better texture and taste.

Description

Technical Field

[0001] This invention belongs to the field of post-fermentation technology of yogurt, specifically relating to a copper ion-sensitive Lactobacillus bulgaricus and its application. Background Technology

[0002] Yogurt is a popular fermented dairy product with a long history and is favored by consumers worldwide. Its unique flavor, nutritional value, and health benefits have driven continuous growth in global consumption, making it an important part of modern healthy diets and often recommended as part of a balanced diet. Yogurt is rich in probiotics, protein, calcium, and other nutrients, and is widely recognized for its multiple health benefits, including improving gut health, boosting immunity, and regulating metabolism. However, even after fermentation, some metabolically active lactic acid bacteria continue to metabolize lactose, producing acid, leading to a slow and sustained increase in acidity, known as post-acidification. Severe post-acidification produces an unacceptable taste and can cause whey separation, dehydration, and shrinkage, ultimately reducing the quality of the yogurt, shortening its shelf life, and consequently decreasing consumer satisfaction. This problem not only causes economic losses to the dairy industry but has also become a major factor restricting the development of fermented dairy products.

[0003] Currently, many scholars have proposed various measures to inhibit post-acidification in yogurt, mainly falling into three categories: physical, chemical, and biological methods. Physical methods include heat treatment, high hydrostatic pressure, and pulsed electric fields; chemical methods primarily involve the use of additives; and biological methods include genetic engineering. However, these methods inevitably have some impact on yogurt. For example, the number of viable bacteria in yogurt is significantly reduced after heat treatment; the use of additives not only affects the survival of viable bacteria but may also alter the natural flavor of the yogurt. Furthermore, these methods do not fundamentally solve the problem of post-acidification; they only delay the acidification process. Therefore, it is essential to understand the molecular mechanisms regulating post-acidification and to breed food-grade bacterial strains resistant to post-acidification as starter cultures.

[0004] Previous studies have found that the LDB_RS05285 gene in Lactobacillus bulgaricus ATCC11842 undergoes specific regulation and acidification, and its heterologous expression can inhibit acid production without affecting the viable cell count. Notably, screening using LDB_RS05285 as a biomarker revealed that Cu... ²⁺The significant upregulation of its expression suggests the existence of a potential transcriptional regulatory network, possibly mediating the association between copper signaling and gene expression through specific transcription factors. Numerous studies have shown that transcription factors, acting as molecular switches, play a central role in environmental signal transduction and metabolic regulation. For example, LacI family transcription factors precisely regulate the expression of lactose metabolism-related genes, CsoR family factors specifically sense copper signals and regulate resistance genes, while CodY acts as a global regulator coordinating multiple metabolic pathways. However, the transcriptional regulatory mechanism of the LDB_RS05285 gene in *Lactobacillus bulgaricus* remains poorly understood, particularly regarding its specific transcription factors and regulatory network. This knowledge gap significantly limits our comprehensive understanding of the acidification network following the regulation of this gene's expression. Summary of the Invention

[0005] The purpose of this invention is to provide a copper ion-sensitive Lactobacillus bulgaricus and its applications.

[0006] A type of Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus The strain, with preservation number CGMCC No.36610, is named E9.

[0007] The Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus It is sensitive to copper ions, and the degree of post-acidification of yogurt is reduced after Cu²⁺ treatment.

[0008] The Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus Application of yogurt post-acidification in inhibiting lactic acid production.

[0009] A fermentation agent comprising the Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus ).

[0010] A transcription factor mutant that inhibits the production of post-acidified lactic acid in yogurt, wherein the transcription factor mutant is a CopR mutant, in which the amino acid at position 71 is changed from lysine to arginine.

[0011] The amino acid sequence of the transcription factor mutant is shown in SEQ ID NO: 1.

[0012] CopR mutant and LDB_RS05285 The combination of the starter subregions leads to the encoding of CopB. LDB_RS05285 Increased gene expression levels allow CopB to promote lactose breakdown by enhancing the activity of β-galactosidase through protein-protein interactions, thereby increasing the galactose and glucose content in yogurt. It also inhibits lactic acid formation by reducing the activity of lactate dehydrogenase.

[0013] The taxonomic name of the *Lactobacillus bulgaricus* E9 strain of this invention is *Lactobacillus delbrueckii* subsp. bulgaricus; it was deposited on November 13, 2025, at the China General Microbiological Culture Collection Center (CGMCC), and has proof of viability. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 36610.

[0014] The beneficial effects of this invention: This invention uses neomycin sulfate to induce mutations and bind to acidification marker genes. LDB_ RS05285 High-throughput screening based on expression levels was used to identify copper-sensitive mutant strains with weak post-acidification properties. Simultaneously, high-performance liquid chromatography (HPLC) was used to monitor the dynamic changes of sugar metabolites during fermentation, and texture analysis and rheological testing systems were employed to evaluate the physical properties of the yogurt. Subsequently, whole-genome sequencing and comparative genomics analysis were used to identify transcription factors that could potentially regulate post-acidification marker genes. AlphaFold3 was used to predict the changes in the protein structure of transcription factors before and after mutation, as well as their binding affinity to target gene promoter regions. DNA pull-down and electrophoretic mobility shift assays (EMSA) were used to further validate the transcriptional regulatory relationships. Finally, the network regulatory relationships in the copper transport system and the intrinsic mechanism by which copper ions affect post-acidification were predicted and analyzed. This invention focuses on post-acidification genes... LDB_RS05285 This study provides new insights into the mechanism of copper ion inhibition of post-acidification at the upstream level, namely at the transcription factor level, and offers a new strategy for developing superior strains resistant to post-acidification. Attached Figure Description

[0015] Figure 1 Based on post-acidification marker genes LDB_RS05285 Screening for highly sensitive Cu²⁺ Lactobacillus bulgaricus; In the figure, A represents the establishment of a mutant library; B and C represent the stepwise screening of target strains, from the pool with the highest expression of marker genes to the subpool and then to the target strain.

[0016] Figure 2 To determine whether the mutant strain contains Cu 2+ Cluster analysis of the processed data; A shows the morphology of *Lactobacillus bulgaricus* strains under a microscope, scale bar = 10 μm; B shows... LDB_RS05285 C represents the gene expression level; D represents the growth curve of the strain, with OD600 measured every 2 hours; E represents the change in viable cell count of yogurt during storage; and E represents the change in titratable acidity of yogurt during storage.

[0017] Figure 3The content of reducing sugars (including lactose, galactose, and glucose) and organic acids (including pyruvic acid and lactic acid) in different groups of yogurt at different time periods was determined.

[0018] Figure 4 Sensory analysis of yogurt.

[0019] Figure 5 Results of DNA affinity chromatography and electrophoretic mobility shift assay (EMSA) experiments; A shows the protein SDS-PAGE analysis after DNA pull-down, with lanes 1, 2, and 3 representing the protein marker, experimental group, and control group without biotin probe, respectively; B shows the MEME-chip prediction of CopR and gene... LDB_RS05285 The binding site in the promoter region; C represents the electrophoretic mobility variation measurement, the results reflecting the direct binding of CopR and its mutants to the promoter region. LDB_RS05285 Promoter binding capability; unlabeled probes are used as competitors; the symbol "-" indicates that no protein or probe has been added. "+" indicates that an unlabeled or mutant probe has been added for competition and binding assays.

[0020] Figure 6 To assess the transcriptional association between LDB_RS06425 (encoding CopR) and copper homeostasis genes in wild-type (WT), E1, and E9 strains using Pearson correlation analysis; A is LDB_RS06425 and LDB_RS05285 Transcriptional relevance of (copB); B is LDB_RS06425 and LDB_RS02050 Transcriptional relevance of (copZ); C is LDB_RS06425 and LDB_RS02845 Transcriptional relevance of (copY); D is LDB_RS06425 and LDB_RS02855 Transcriptional correlation of (copA); values ​​represent Pearson correlation coefficient (R) and statistical significance (P), P<0.05 indicates significant correlation; E represents the EMSA experimental symbol for transcription factor CopR and other copper homeostatic genes in the Cop network. "-" indicates no protein or probe was added; "+" indicates the addition of unlabeled or mutated probes for competition and binding tests. Detailed Implementation

[0021] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention. Example

[0022] Experimental methods: 1. Bacterial strains and growth conditions Lactobacillus bulgaricus ATCC 11842 (purchased from the China General Microbiological Culture Collection Center) was used as the starting strain for the experiment. All Lactobacillus bulgaricus strains in the experiment were activated and passaged at 42°C in MRS broth medium (Beijing Luqiao Technology Co., Ltd.) with an initial pH of 6.5.

[0023] 2. Preparation of skim milk culture medium and yogurt The skim milk culture medium was prepared by mixing skim milk powder (Inner Mongolia Yili Industrial Group Co., Ltd.) and distilled water at a ratio of 1:7 (w / v), sterilized at 112℃ for 10 min, cooled, and stored at 4℃ for later use. The logarithmic growth phase strain was inoculated into the sterile skim milk culture medium at a 1% (v / v) inoculation rate and fermented at 42℃ until curdling. After curdling, Cu²⁺ (in the form of CuSO₄) was added to the treatment group to a final concentration of 2.5 mg / kg.

[0024] 3. RNA extraction, cDNA synthesis, and real-time quantitative PCR (qPCR) The bacterial strain was cultured in MRS medium at 42℃ to the logarithmic growth phase. Treatment groups were then treated with a final concentration of 2.5 mg / L CuSO4 (42℃ for 40 min). 1 mL of the sample was collected by centrifugation at 12,000 ×g for 10 min at 4℃. Total RNA was extracted using Trizol reagent (Promega, Germany), and RNA purity was assessed by detecting the A260 / A280 ratio using a NanoDrop ND-1000 (Thermo Scientific, USA).

[0025] 2 μL of RNA was reverse transcribed using EasyScript® One-Step gDNA Removal cDNA Synthesis SuperMix (TransGen, China). qPCR experiments were performed on a LightCycler 96 system (Roche, Switzerland) using PerfectStart® Green qPCR SuperMix (TransGen Biotech, Beijing, China), with rpob selected as the internal control gene.

[0026] The program parameters were set as follows: 94℃ for 5 min; 45 cycles (94℃ for 5 s, 51℃ for 15 s, 72℃ for 10 s). Each sample was tested three times, using 2... −ΔΔCt The relative expression level of genes can be calculated.

[0027] 4. Construction of mutant libraries and screening of copper-sensitive strains Wild-type strain L. delbrueckii subsp. bulgaricus ATCC 11842 was plated on MRS solid medium containing (0, 40, 80, 120, 160 μg / mL) neomycin sulfate (Solarbio, China) at mid-log growth. Plates with colony counts of 30-300 were selected, and 864 single colonies were picked and inoculated into 9 wells of 96-well plates (numbered Pool 1-Pool 9). The plates were incubated at 42°C until mid-log growth.

[0028] High-throughput hierarchical screening was used to screen for copper ion-sensitive mutants, and... LDB_RS05285 As a screening marker gene, specifically, 96 bacterial strains from each pool were mixed and incubated at a final concentration of 2.5 mg / kg CuSO4 for 40 min at 42°C. RNA was then extracted and reverse transcribed into cDNA, and wild-type ATCC 11842 was used as a control for screening by qPCR analysis. LDB_RS05285 The pool with the highest expression level was further divided into 8 subpools (Subpool AH), and finally, individual... LDB_RS05285 The mutant with the highest expression level. To clarify... LDB_RS05285 To investigate the negative correlation between expression and post-acidification of yogurt, a mutant strain with moderate expression was also included in the experiment. Together with the wild-type strain, an LDB_RS05285 expression gradient (low → high) was constructed to verify the phenotypic differences.

[0029] 5. Determination of yogurt acidity The acidity of the yogurt sample during fermentation and storage was determined according to the People's Republic of China National Standard GB 5009.239-2016 "Determination of Acidity in Food". The specific steps were as follows: Accurately weigh 10g of the yogurt sample and mix it with 20mL of distilled water that had been pre-boiled to remove carbon dioxide. Using 0.5% (w / v) phenolphthalein ethanol solution as an indicator, titrate the mixture with 0.1 mol / L sodium hydroxide standard solution until a persistent pale pink endpoint is reached (remaining for 5 seconds without fading). Record the volume of sodium hydroxide consumed. The acidity (expressed as °T) was calculated using the following formula: °T = (c × V × 100) / (m × 0.1); Where: c - concentration of NaOH standard solution (mol / L); V - volume of NaOH consumed (mL); m - sample mass (g).

[0030] 6. Determination of sugars and organic acids in yogurt Ultra-high performance liquid chromatography (UHPLC) was used to quantitatively analyze the reducing sugar and organic acid contents in yogurt samples stored at 4℃ for 0, 7, 14, and 21 days (Leaps UHPLC system, Chromai, China). Sample pretreatment followed the method of Yue et al. with slight modifications: 2g of yogurt sample was accurately weighed, dissolved in 10 mL of ultrapure water, and 1.00 mL of 10... 6 Protein precipitation was performed using 1.00 mL of 220 g / L potassium ferrocyanide solution (Solepro, China) and 1.00 mL of 220 g / L zinc acetate solution (Solepro, China). The mixture was brought to a final volume of 25.00 mL, magnetically stirred for 30 min, centrifuged at 6000×g for 30 min at 4 °C, filtered through a 0.22 μm filter membrane, and stored at 4 °C.

[0031] For the analysis of reducing sugars (lactose, galactose, glucose), the 1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization method described by Wu et al. was used: 1.00 mL of supernatant was taken, 200 μL of 0.3 M NaOH and 150 μL of 0.5 MPMP-methanol reagent were added, vortexed, and derivatized at 70 °C for 30 min. After cooling in an ice bath, the solution was neutralized with 200 μL of 0.3 M HCl, and then extracted three times with 3.00 mL of chloroform to remove excess reagent. The aqueous phase was filtered through a 0.22 μm filter before loading. Chromatographic separation was performed using an Agilent 1200-C18 column (4.6 mm × 250 mm, 5 μm), with an acetonitrile-ammonium acetate buffer (100 mM, pH 5.5; volume ratio 22:78, Thermo Fisher Scientific) as the mobile phase, a flow rate of 1.0 mL / min, a column temperature of 25 °C, a detection wavelength of 245 nm, and an injection volume of 10.0 μL.

[0032] Organic acid (lactic acid, pyruvic acid) analysis was performed using a Rezex™ ROA-Organic Acid H+ (8%) column (300 × 7.8 mm, 8 μm), with a mobile phase of 2.5 mM H2SO4 solution, a flow rate of 0.6 mL / min, a column temperature of 60 °C, a detection wavelength of 210 nm, and an injection volume of 10.0 μL.

[0033] Quantitative analysis was performed using the external standard method. Reducing sugars: a mixed standard (containing 1.44 g / L lactose, 0.72 g / L galactose, and 0.72 g / L glucose); organic acids: lactic acid (1.00 g / L) and pyruvic acid (1.00 g / L) as single standards. Standard curves were prepared by serial dilutions (1 / 2, 1 / 4, 1 / 8, and 1 / 16 of the original concentration) in the corresponding mobile phases. All standards were purchased from Shanghai Yuanye Biotechnology Co., Ltd., and each sample was measured in triplicate.

[0034] 7. Enzyme activity assay of β-galactosidase and lactate dehydrogenase Enzyme activities of β-galactosidase and lactate dehydrogenase were determined according to the manufacturer's instructions using the β-Galactosidase (β-GAL) Activity Assay Kit (BC2580) and the Lacate Dehydrogenase (LDH) Activity Assay Kit (BC0680) (Solarbio, Beijing, China). Protein concentrations of the bacterial extracts were determined using the Protein Content Assay Kit (Biuret Method) (BC3185) (Solarbio, Beijing, China). Enzyme activities are presented as U / mg protein.

[0035] 8. Determination of the texture and water-holding capacity of yogurt The texture and water-holding capacity (WHC) of yogurt samples stored at 4°C for 0, 7, 14, and 21 days were determined according to the methods reported by Sun et al. and Meng et al. Texture analysis was performed using a TA-XT Plus texture analyzer (Stable Micro Systems, UK), equipped with a 35 mm cylindrical probe (Type A / BE). The test parameters were set as follows: pre-test speed 1 mm / s, test speed 1 mm / s, compression distance 15 mm, post-test speed 10 mm / s, and trigger force 5 g. The determined texture parameters included consistency (g·s), viscosity index (g·s), hardness (g), and cohesiveness (g), with each sample measured in triplicate.

[0036] For the water-holding capacity determination, 25.0 ± 0.1 g of yogurt sample was weighed into a 50 mL centrifuge tube and centrifuged at 6000 × g for 10 min (Hettich Universal 320 R centrifuge, Germany). The supernatant was discarded, and the mass of the remaining sample was weighed. The water-holding capacity was calculated using the following formula: ; In the formula: W1: sample mass before centrifugation (g); W2: sample mass after centrifugation (g).

[0037] 9. Sensory evaluation of yogurt A panel of 10 experts (5 men and 5 women, aged 22-28) was formed to conduct sensory analysis. Panel members received two weeks of professional training following the methods of Utz et al. Quantitative descriptive analysis (QDA) was used to evaluate yogurt samples stored at 4°C on days 0 and 21, using a 0-10 scale (0: imperceptible; 10: extremely strong). The 10 key sensory attributes evaluated included: sweetness, sourness, astringency, milkiness, fermentation flavor, graininess, stringiness, whey separation, thickness, and fattiness. Samples were randomly coded with three-letter codes and placed in disposable plastic cups. Evaluators rinsed their mouths with purified water between samples to eliminate any aftertaste. Each sample was evaluated three times to ensure data reliability.

[0038] 10. Comparative genomic variation analysis Genomic DNA was extracted from *Lactobacillus delbrueckii* subsp. bulgaricus using a commercial genomic DNA extraction kit (Ruisen Biotechnology, China). After quantification using NanoDrop 2000 (10-50 ng / μL), the DNA was fragmented using an ultrasonic homogenizer (Covaris M220). Sequencing libraries were constructed using the NEXTFLEX Rapid DNA-Seq kit, and paired-end sequencing (2 × 150 bp) was performed on an Illumina NovaSeq 6000 platform. Raw data were quality controlled using FastQC (v0.11.9), retaining high-quality reads (Q30 > 90%). Genome assembly was performed using SPAdes (v3.15.4) software. The whole genome was aligned with the reference strain ATCC11842 (NCBI accession number: NC_008054.1) using MUMmer (v4.0.0), and single nucleotide polymorphisms (SNPs) and small insertions / deletions (<50 bp) were identified using the show-snps function. Mutation annotation is done using snpEff (v5.0).

[0039] 11. DNA pull-down assay Will LDB_RS05285The promoter region was biotin-labeled at the 5' end. The whole protein of the starting strain ATCC11842 was then prepared using a protein extraction kit R00185 (Beyotime, Shanghai, China) and stored at -80°C. Finally, DNA pull-down assays were performed using the Pull-down Kits for Using Biotin-Probes (Viagene, Changzhou, China). Streptavidin magnetic beads (Beyotime, Shanghai, China) were incubated with the biotin-labeled probe on a shaker at room temperature for 1 h, according to the manufacturer's instructions. The extracted sample protein was then mixed with the DNA probe-bound magnetic beads and incubated overnight on a shaker at 4°C. The mixture was then washed and separated onto a 10% acrylamide SDS-PAGE gel. The DNA-bound protein bands were then excised for proteomic analysis. The probe and primer sequences are shown in Table 1.

[0040] 12. Electrophoretic mobility shift assay (EMSA) The coding sequences of transcription factor CopR and its mutants were cloned into the pET-28a-His vector, and the recombinant vector was then transformed into E. coli BL21(DE3) competent cells for protein expression. After induction with 0.5 mM IPTG (37℃, 200 rpm, 4 h), the target protein was obtained and purified using a His-tagged purification kit. Based on MEME-ChIP bioinformatics analysis, the protein was predicted... LDB_RS05285 The optimal motif for CopR binding to the promoter region was determined, and a 5' biotin-labeled probe (BGI) was designed around this binding site. The probe sequences are shown in Table 1. EMSA experiments were performed using a chemiluminescence detection kit (GS009, Beyotime). First, the purified protein and labeled probe were incubated at room temperature (25°C) for 30 min. Then, after separation by 6% non-denaturing PAGE electrophoresis, the protein was transferred to a positively charged nylon membrane (0.5×TBE transfer buffer, Servicebio). Finally, after UV cross-linking, protein-DNA interactions were detected using a chemiluminescence imaging system (Agilent, USA).

[0041] Table 1. Upstream and downstream primers for biomarker genes and internal reference genes

[0042] Experimental results: 1. Mutagenesis screening of copper ion-sensitive strains The screening process is as follows Figure 1 As shown, it can be seen that, in the wild-type strain ATCC11842 LDB_RS05285The gene expression level of Pool 3 was used as a control. LDB_RS05285 The highest expression level ( Figure 1 B); later it was located in subpool E ( Figure 1 C), finally E9 was obtained as a highly sensitive Cu²⁺ Lactobacillus bulgaricus (C), Figure 1 C).

[0043] Lactobacillus bulgaricus has received widespread attention as a core starter culture for yogurt. Transcriptome analysis revealed that... LDB_ RS05285 Gene expression levels showed a significant negative correlation with acid production kinetics, suggesting that it may play a key role in post-acidification metabolic regulation. Based on this finding, we constructed a mutant library of *Lactobacillus bulgaricus* and screened strains based on the expression level of LDB_RS05285.

[0044] 2. Physicochemical characteristics analysis of the strain To investigate the differences between mutant and wild-type strains, this invention compared the physiological characteristics and fermentation performance of *Lactobacillus delbrueckii* subsp. bulgaricus before and after Cu²⁺ treatment. Two groups were set up in the experiment: the wild-type strain ATCC11842 (WT) and the highly sensitive strain E9+ Cu²⁺ (E9+). Morphological observation showed that all strains maintained a typical rod-shaped morphology, and could be solitary or arranged in chains. Figure 2 A). Growth kinetics analysis showed that all strains entered the mid-logarithmic growth phase at approximately 11 hours of culture. Figure 2 C). Meanwhile, the number of live bacteria in yogurt decreased continuously during the 21-day storage period. Figure 2 D), but all met the viable cell count requirement (≥10⁷ CFU / g) during storage. Gene expression analysis showed that Cu²⁺ treatment significantly upregulated the expression level of LDB_RS05285, with the E9+ group showing a 2.87-fold increase compared to the WT group ( Figure 2 B). Although there was no significant difference in acidity among the groups at the fermentation endpoint, after 21 days of storage, the E9+ group had the lowest acidity (80.8°T), significantly lower than the WT group (94.4°T). Figure 2 E).

[0045] Liquid chromatography results showed that there were no significant differences in the changes in reducing sugar and organic acid content during storage between yogurt fermented by the WT and E9 strains without Cu²⁺ treatment. However, after Cu²⁺ treatment, the WT+ and E9+ groups exhibited significant metabolic changes. Specifically, in the WT group, lactose consumption was 18.7 mg / mL, galactose accumulation was 2.26 mg / mL, and the final glucose concentration was 0.21 mg / mL during the 21-day storage period. Pyruvic acid and lactic acid increased by 1.34 mg / mL and 12.57 mg / mL, respectively. Compared with the WT group, the E9+ group showed a 1.4-fold increase in lactose consumption, a 4-fold increase in galactose accumulation, a 6.57-fold increase in final glucose concentration, a 41.49% decrease in pyruvic acid increase, and a 34.29% decrease in lactic acid increase. Figure 3 ).

[0046] The acidity and LDB_RS05285 expression levels of yogurt samples showed opposite trends during 21-day storage, indicating a negative correlation between LDB_RS05285 expression and acidity. Furthermore, Cu²⁺ treatment inhibited post-acidification of yogurt but had no significant effect on the viable cell morphology and count of fermented milk. In *Lactobacillus bulgaricus* ATCC11842, lactose is transported into the cell via lactose permease and then hydrolyzed into galactose and glucose by β-galactosidase. Glucose is metabolized into pyruvate via glycolysis and ultimately converted into lactic acid under the catalysis of lactate dehydrogenase (LDH). This process is the main cause of post-acidification in yogurt. Notably, this strain cannot metabolize galactose and can only excrete it extracellularly. Based on the direct correlation between sugar metabolism and post-acidification, we quantitatively analyzed the effect of Cu²⁺ treatment on the dynamic changes of sugars and organic acids during storage. It can be seen that the three groups of yogurt samples treated with Cu²⁺ showed the same trend of change. This means that lactose metabolism in yogurt is accelerated, leading to an increase in galactose and glucose content; however, the content of pyruvate and lactic acid decreases.

[0047] 3. Texture analysis of yogurt at different storage periods Table 2 shows that as the storage time was extended to 21 days, the water-holding capacity (WHC) of all sample groups decreased significantly (P<0.05), with the WT group showing the lowest decrease (38.53%). Notably, Cu²⁺ treatment significantly improved WHC (P<0.05), especially the E9+ group (45.73%). This result is highly correlated with changes in acidity, as excessive acidification disrupts the gel structure of yogurt, causing water molecules bound to casein molecules to leak out, ultimately leading to a decrease in WHC. The hardness of yogurt in all groups increased significantly on day 21 of storage (P<0.05). However, the E9+ group had the lowest hardness on day 21 (43.27 g), a significant difference compared to the WT group (P<0.05). The viscosity of the E9+ group was significantly higher than other groups on day 21 (P<0.05). Throughout the measurement process, cohesion was consistently lower than viscosity, and the cohesion of the yogurt continuously decreased during storage, consistent with the results reported by Harbinder Kaur et al. At the end of the storage period, the cohesiveness of the yogurt treated with Cu²⁺ was significantly higher than that of the WT group (P<0.05). However, the cohesiveness of the E9+ group was significantly higher than that of the other Cu²⁺ treatment groups (P<0.05), indicating that the yogurt in the E9+ group had a denser structure. The consistency of the yogurt decreased continuously during storage. At the end of the storage period, the consistency of the yogurt in the E9+ group decreased significantly (P<0.05), but was still significantly higher than that of the other groups (P<0.05).

[0048] During storage, lactose in yogurt continuously metabolizes and breaks down, producing acid. Severe post-acidification can cause dehydration, shrinkage, and whey separation, leading to a decline in yogurt quality. Texture is one of the most critical parameters for evaluating fermented milk, directly impacting consumer acceptance. Texture analysis showed that Cu²⁺ treatment significantly affected the texture characteristics of yogurt during storage (Table 2). The water-holding capacity of yogurt showed an inverse trend to the dehydration shrinkage rate, representing the level of whey separation. Therefore, improving water-holding capacity can enhance yogurt stability, thereby improving its quality. The acidity of yogurt increased with prolonged storage time, and its hardness also increased. Yogurt with lower hardness was considered to have better lubrication properties. Mutagenized strains E1 and E9 showed varying degrees of sensitivity to Cu²⁺. E9, after Cu²⁺ treatment, significantly improved the texture and quality of yogurt. This may be because its fermented yogurt exhibited lower post-acidification, milder dehydration, shrinkage, and whey separation, and had less impact on the overall yogurt gel network, resulting in higher texture parameters.

[0049] Table 2. Changes in the texture characteristics of yogurt during 21 days of refrigeration.

[0050] 4. Sensory analysis of yogurt Sensory evaluation results showed that after 21 days of storage, all samples exhibited increased acidity, whey precipitation (pale yellow color), and deterioration in texture (increased astringency and decreased viscosity). Figure 4 However, the E9+ group performed exceptionally well across several key metrics: lowest acidity score, least whey separation, and optimal viscosity retention. Figure 4 ).

[0051] Furthermore, Cu²⁺ treatment significantly enhanced the fermentation flavor, which may be related to the enhanced synthesis of aromatic substances under the regulation of metal ions. These findings confirm that Cu²⁺ treatment of the E9 strain not only improves the rheological properties of yogurt but also significantly enhances its sensory quality during storage. This is closely related to the acidity of fermented milk during storage, and is associated with the lowest expression level of LDB_RS05285 in E9+ during storage. There is limited research on transcription factors in Lactobacillus bulgaricus that can bind to and regulate the expression of the LDB_RS05285 promoter region. Therefore, identifying this core switch—the transcription factor—is crucial for achieving stable acidity control and maintaining good yogurt quality during storage.

[0052] 5. Analysis of CopR mutation sites through comparative genomic and AlphaFold3 studies Using the reference sequence as a benchmark, a genome-wide alignment map constructed with Circos (v0.64) software visually illustrated the distribution patterns of SNPs and InDels. Therefore, we conducted comparative genomic variation analysis on WT and E9 strains. Comparison with WT revealed that the E9 strain had 38 SNP sites (including 7 synonymous mutations, 9 nonsense mutations, and 22 missense mutations) and 10 InDels <50 bp in length (9 located in the coding region and 1 in the non-coding region). Notably, non-3-fold InDels in the coding region may lead to frameshift mutations, thus affecting gene function. Under 2.5 mg / kg Cu²⁺ treatment, the expression of the LDB_RS05285 gene in the E9 strain was significantly upregulated, and the fermented yogurt showed a significant effect in inhibiting post-acidification and improving texture properties. Since changes in gene expression are often regulated by transcription factors, we hypothesize that some changes have occurred in the upstream transcription factors regulating LDB_RS05285 in the highly sensitive CU²⁺ strain E9. Analysis of comparative genomic variation detection results revealed a c.212A>G mutation in the gene CopR, causing the amino acid at position 71 of the encoded transcription factor CopR to change from lysine (K) to arginine (R). AlphaFold3 structure prediction showed that the tertiary structure of the protein before and after the mutation of the transcription factor CopR is mainly composed of α-helices and random coils. Furthermore, the change in amino acid position 71 of CopR makes the protein tertiary structure more compact and stable. In addition, MEME-chip prediction identified the binding site with the highest score for transcription factor CopR in the promoter region of the target gene LDB_RS05285. Figure 5 (B) This suggests that CopR may be a transcription factor regulating the gene LDB_RS05285. Furthermore, the predicted results show increased binding affinity between the mutant CopR and the promoter region, and more stable conformational binding kinetics. Structural analysis based on AlphaFold3 suggests that this increased interaction force, resulting in stronger binding affinity and a more stable complex conformation, is likely due to changes in the hydrogen bond distance when the mutant CopR binds to the LDB_RS05285 gene promoter region.

[0053] 6. CopR mutation enhances its interaction with... LDB_RS05285 Binding affinity of gene promoters Through DNA pull-down experiments, we successfully captured [a specific substance] from the whole protein extract of wild-type strain WT. LDB_RS05285 The protein complex bound to the promoter region probe. SDS-PAGE analysis showed significantly different protein bands between the experimental and control groups. Figure 5A), mass spectrometry confirmed that the transcription factor CopR specifically binds to this region, indicating that CopR is a transcription factor regulating the LDB_RS05285 gene. MEME-chip prediction analysis further revealed an 8-base conserved binding motif ( Figure 5 B). To verify the effect of the mutated CopR on binding affinity, we purified wild-type and mutant (p.Lys71Arg) CopR proteins for EMSA experiments. The results showed that both the unmutated and mutant CopR proteins could bind to biotin-labeled probes, producing migration bands, and this binding could be inhibited by unlabeled competing probes, while the mutant probe had no such effect. Figure 5 C).

[0054] Notably, the mutant protein exhibited a stronger binding signal (wider band and deeper staining), which may be due to the fact that the substitution of lysine (K) with arginine (R) at position 71 allows the guanidine group to form more hydrogen bonds, significantly enhancing its interaction with DNA. Lysine, on the other hand, primarily forms hydrogen bonds with DNA through its amino group, and its ability and number of hydrogen bonds are generally lower than that of arginine. Based on the structural differences predicted by AlphaFold3, it is speculated that after the K71R mutation, the arginine at position 71 of CopR can form more hydrogen bonds than the wild-type lysine. This is the structural basis for the stronger binding signal between the mutant CopR and the LDB_RS05285 promoter in the EMSA experiment, directly leading to the upregulation of LDB_RS05285 expression. The CopR protein was identified through a DNA pull-down experiment, further validating that CopR is a regulator of DNA activity. LDB_RS05285 CopR is a transcription factor. EMSA in vitro experiments further demonstrated that CopR directly interacts with… LDB_RS05285 Promoter region binding, and the p.Lys71Arg mutation upregulates target genes by enhancing protein-DNA binding. LDB_RS05285 Express.

[0055] 7. LDB_RS06425 Transcriptional correlation analysis of (encoding CopR) with key copper homeostasis genes Experimental verification shows that CopR directly binds LDB_RS05285 The promoter was activated and its expression was upregulated. These findings extend the hypothetical regulatory scope of CopR to other key copper homeostasis genes. Pearson correlation analysis of wild-type (WT), E1, and E9 strains revealed the existence of a tightly coordinated transcriptional network regulating copper homeostasis. LDB_RS06425 (CopR encoding) expression and LDB_RS05285 (copB; R=0.944, P<0.0001; Figure 6 A), LDB_RS02050 (copZ; R=0.839, P<0.0001; Figure 6B) and the copper-responsive repressor protein gene LDB_RS02845 (copY; R=0.931, P<0.0001; Figure 6 C) Shows a strong positive correlation. Conversely, LDB_RS06425 The expression and LDB_RS02855 (copA; R=−0.852, P<0.0001; Figure 6 D) shows a significant negative correlation.

[0056] Gene function annotation analysis showed that, LDB_RS05285 The gene encodes copper-to-P-type ATPase B (CopB), belonging to the CPx-ATPase family. Its function is to expel Cu²⁺ ions extracellularly via ATP hydrolysis. In addition, the entire copper ion transport system also includes: LDB_RS02855 Encoded CopA (copper transporter P-type ATPase A), LDB_RS02050 Encoded CopZ (copper molecular chaperone) LDB_RS02845 The encoded CopY (copper-responsive repressor protein), and LDB_RS06425 The encoded transcriptional regulator CopR has been reported to play a crucial role in copper homeostasis regulation in *Lactobacillus plantarum*. Cop homeostasis is primarily regulated by the activation of other Cop proteins by CopR in response to high copper stress to maintain intracellular copper homeostasis. This study discovered a novel regulatory function of CopR: the K71R mutation enhances the interaction between CopR and... LDB_RS05285 The mechanism of CopR's binding ability to the promoter and its upregulation is consistent with the "feedback inhibition" model of copper homeostasis. CopB, as a potential copper chaperone, has its expression reduced, limiting intracellular free copper concentration and thus inhibiting copper-induced post-acidification. This finding expands the regulatory scope of CopR in *Lactobacillus bulgaricus*, revealing its multifunctional role from "copper efflux regulation" to "metabolic adaptation regulation," and enriching our understanding of the *Lactobacillus bulgaricus* copper homeostasis regulatory network.

[0057] Furthermore, these correlations suggest that CopR may have cross-regulatory interactions with copper homeostasis genes under different copper loads, making it a potential transcriptional activator in this network. EMSA experiments demonstrated that CopR binds not only to the LDB_RS05285 promoter region but also to the LDB_RS02855 and LDB_RS02845 promoter regions, indicating that CopR may play a co-regulatory role as a core transcription factor in the Cop regulatory network. The synergistic upregulation of copB (efflux), copZ (molecular chaperone), and copY (repressor protein), coupled with the inhibition of copA (influx), establishes a metabolic state biased towards copper export. The coordinated expression profile further suggests that CopR may act as a core regulator of the Cu²⁺ sensing network, forming a complex regulatory network to inhibit the post-acidification process of yogurt by coordinating the expression of multiple key genes in the copper homeostasis network (including the upregulation of LDB_RS05285 and synergistic changes in other related genes).

[0058] In this application, we used the expression level of the post-acidification marker gene LDB_RS05285 as a basis to screen a strain highly sensitive to CU²⁺ using high-throughput fractionation. Changes in gene expression levels are usually controlled and regulated by the binding of upstream transcription factors to the promoter regions of target genes. Through comparative genomic variation detection analysis, we found that the transcription factor CopR changed from lysine (K) to arginine (R) at amino acid position 71, a mutation that attracted our attention. Subsequently, DNA pulldown and EMSA in vitro experiments demonstrated that CopR is a transcription factor regulating LDB_RS05285, which can directly bind to the promoter region of LDB_RS05285, and the mutated CopR has a stronger binding affinity. The enhanced binding affinity leads to an increase in the expression level of the LDB_RS05285 gene encoding CopB. CopB may further promote lactose breakdown by increasing the activity of β-galactosidase through protein-protein interactions, thereby increasing the galactose and glucose content in yogurt; and inhibit lactate formation by decreasing the activity of lactate dehydrogenase. This results in lower post-acidification levels in yogurt fermented by the CU²⁺-sensitive strain E9 after CU²⁺ treatment, leading to improved texture and taste. This study provides new insights into the molecular mechanism by which CU²⁺ regulates post-acidification in yogurt.

[0059] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A type of Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus The strain, with preservation number CGMCC No.36610, is named E9.

2. The Lactobacillus bulgaricus (Lactobacillus bulgaricus) according to claim 1 Lactobacillus delbrueckii subsp. bulgaricus ) strain, characterized in that, The Lactobacillus bulgaricus ( Lactobacillus delbrueckii subsp. bulgaricus It is sensitive to copper ions, and the degree of post-acidification of yogurt is reduced after Cu²⁺ treatment.

3. The Lactobacillus bulgaricus of claim 1 ( Lactobacillus delbrueckii subsp. bulgaricus Application of yogurt post-acidification in inhibiting lactic acid production.

4. A fermentation agent, characterized in that, Contains the Lactobacillus bulgaricus of claim 1 ( Lactobacillus delbrueckii subsp. bulgaricus ).

5. A transcription factor mutant that inhibits the production of post-acidified lactic acid in yogurt, characterized in that, The transcription factor mutant is the CopR mutant, in which the amino acid at position 71 is changed from lysine to arginine.

6. The transcription factor mutant according to claim 5, characterized in that, The amino acid sequence of the transcription factor mutant is shown in SEQ ID NO:

1.

7. The transcription factor mutant according to claim 5, characterized in that, CopR mutant and LDB_RS05285 The combination of the starter subregions leads to the encoding of CopB. LDB_RS05285 Increased gene expression levels allow CopB to promote lactose breakdown by enhancing the activity of β-galactosidase through protein-protein interactions, thereby increasing the galactose and glucose content in yogurt. It also inhibits lactic acid formation by reducing the activity of lactate dehydrogenase.

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