Phytobacterium plantarum for producing lipase and application of phytobacterium plantarum in preparation of fermented single cream

Abstract

The invention relates to a lactobacillus plantarum for producing lipase and an application of the lactobacillus plantarum in preparation of fermented single cream, the lactobacillus plantarum strain is separated from a goat milk sample from Diqing of Yunnan, is named as lactobacillus plantarum B22, has a preservation number of CCTCC M 2025779, is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC M 2025779. The preservation address is Wuhan University Campus, Wuchang District, Wuchang District, Hubei Province, No. 299. The strain can produce lipase at high yield, and has good oxidation resistance, bacteriostasis and a plurality of other probiotic functions; in addition, the strain and a commercial leavening agent are combined to prepare the fermented single cream, and the obtained fermented single cream is fine and smooth in texture and has a strong flavor.

Description

Technical Field

[0001] The invention relates to the field of lactic acid bacteria, in particular to a lipase-producing Lactobacillus plantarum and application thereof in preparing fermented cream. Background Art

[0002] Lipases, also known as glycerol ester hydrolases or triacylglycerol acyl hydrolases, primarily catalyze the hydrolysis of triglycerides into glycerol and fatty acids, thereby enhancing the nutritional value and flavor of the product. Cream is a high-fat dairy product with a soft texture and rich nutrition. Adding cream to other foods can enhance their nutritional value, leading to its widespread use in dairy products, baked goods, confectionery, and beverages. However, traditional cream has a monotonous flavor and lacks functional fatty acids, which significantly limits its market potential and consumer appeal.

[0003] Therefore, using microbial fermentation to improve the flavor and nutritional content of cream to expand its market reach and consumer base has become a research focus. The present invention provides a lactic acid bacterium that produces high levels of lipase and its use in the preparation of fermented cream. The present invention isolates a strain of Lactobacillus plantarum from goat milk samples collected from Diqing, Yunnan. This strain produces high levels of lipase and can significantly improve the flavor of fermented cream. Summary of the Invention

[0004] The present invention screened a high-lipase-producing lactic acid bacteria B22 from goat milk samples in Diqing, Yunnan. The lactic acid bacteria B22 was identified by morphological identification and whole genome sequencing. Lactiplantibacillus plantarum and named it Lactobacillus plantarum ( Lactiplantibacillus plantarum The lipase content of the Lactobacillus plantarum B22 provided by the present invention is higher than that of the existing Lactobacillus plantarum.

[0005] The technical solution of the present invention is as follows: a lipase-producing Lactobacillus plantarum ( Lactiplantibacillus plantarum ), the plant lactobacillus is named plant lactobacillus ( Lactiplantibacillus plantarum ) B22, the deposit number is CCTCC M 2025779, the depositor is: China Center for Type Culture Collection, the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province; the deposit time is: April 14, 2025.

[0006] The colony of the plant lactobacillus B22 is milky white, translucent, relatively moist, smooth, with neat edges and obvious protrusions. Microscopic examination results of the plant lactobacillus B22 show that it does not produce spores, has a dispersed rod-shaped cell morphology, and is a purple-positive bacterium in Gram staining.

[0007] The present invention also provides the use of the Lactobacillus plantarum in the preparation or production of lipase; the yield of lipase produced by fermentation of the Lactobacillus plantarum is as high as 3.45±0.13 U / mL.

[0008] The present invention also provides application of the Lactobacillus plantarum in preparing fermented food.

[0009] Preferably, the fermented food is cheese, sausage or cream.

[0010] As a preferred method, when the plant lactobacillus is used to prepare fermented cream, the inoculation amount of the strain is 1.5×10 9 -4.5×10 9 CFU / mL; the fermented cream is rich in fatty acid content and flavor, which can reduce the use of food additives.

[0011] As a further preference, the Lactobacillus plantarum is used in combination with a commercial yogurt starter; the usage amount of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 8 h-12 h, and the fermentation temperature is 35°C-37°C.

[0012] Compared with the prior art, the present invention has the following beneficial effects: the Lactobacillus plantarum provided by the present invention can produce a high amount of lipase, and can be used alone or in combination with a commercial starter to prepare fermented cream, and the obtained fermented cream has a delicate texture and a very rich flavor.

[0013] In addition, the Lactobacillus plantarum provided by the present invention has good antioxidant, antibacterial, high production of biofilm, high production of extracellular polysaccharides and multiple other beneficial functions, and is suitable for the fermentation of various dairy products. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 (A) Lactobacillus plantarum Lactiplantibacillus plantarum Colony morphology of B22 on MRS plate; (B) Lactobacillus plantarum Lactiplantibacillus plantarum Colony morphology of B22 under light microscope; Figure 2 These are the transparent circles on the tributyrin plate; (A) the transparent circle of Lactobacillus plantarum B22 on the tributyrin plate; (B) the transparent circle of Lactobacillus plantarum E97 on the tributyrin plate; (C) the transparent circle of Lactobacillus plantarum A74 on the tributyrin plate; (D) the transparent circle of Lactobacillus plantarum D73 on the tributyrin plate; (E) the transparent circle of Lactobacillus plantarum A70 on the tributyrin plate; Figure 3 This is the result of the lipase activity test of Lactobacillus plantarum; Figure 4is the phylogenetic tree of Lactobacillus plantarum B22 lipase; Figure 5 Molecular docking of lipase; (AD) shows the molecular docking of gene3060, gene3059, gene2553, gene0817 with tributyrin; Figure 6 It is the acid resistance test of the strain; Figure 7 Bile salt tolerance test for strains; Figure 8 To simulate the artificial gastric juice tolerance test; Figure 9 To simulate the tolerance test of artificial intestinal fluid; Figure 10 The results of biofilm content testing of multiple lactic acid bacteria strains are shown below; Figure 11 The results are for the detection of exopolysaccharide content of multiple lactic acid bacteria; Figure 12 Results of antibacterial activity tests: (A) Antibacterial effect of Lactobacillus plantarum against Salmonella CICC2148; (B) Antibacterial effect of Lactobacillus plantarum against Staphylococcus aureus ATCC25923; (C) Antibacterial effect of Lactobacillus plantarum against Escherichia coli CICC10389; (D) Antibacterial effect of Lactobacillus plantarum against Pichia pastoris BNCC380011; (E) Antibacterial effect of Lactobacillus plantarum against Geotrichum candidum BNCC33599. Figure 13 For the determination of AI-2 content of multiple lactic acid bacteria; Figure 14 is the DPPH free radical scavenging rate of multiple lactic acid bacteria; Figure 15 is the ABTS+ free radical scavenging rate; Figure 16 For hemolytic test, (A) is Lactobacillus plantarum B22, (B) is Staphylococcus aureus ATCC25923; Figure 17 It is the amino acid decarboxylase test; Figure 18 Radar chart for sensory evaluation of fermented cream; Figure 19 It is composed of fermented cream flavor substances; Figure 20 Fermented cream flavor content; Figure 21 The contents of the top eight lipid subclasses in fermented cream are given below; Figure 22 for the lipid differences of fermented cream; Figure 23It is the key lipid in fermented cream. DETAILED DESCRIPTION

[0015] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0016] The plant lactobacillus B133 that the present invention uses derives from Baoshan pickled bean curd, and plant lactobacillus A152 derives from Dehong soil, and plant lactobacillus D138 derives from Yuxi pickled cabbage, and fermentation mucus lactobacillus A261 derives from Dehong water pickled cabbage, and above 4 strains of lactic acid bacteria are all sequenced by 16srDNA after separation, and sequencing result and GenBank database are compared, and result shows that it is 100% with fermentation lactobacillus homology.Commercial leavening agent lactobacillus bulgaricus and thermophilus streptococcus are enough to be purchased from DSM Food Specialties (DSM).Staphylococcus aureus ATCC25923, Escherichia coli CICC10389, Salmonella CICC21482 and candidum BNCC33599 are purchased from Guangdong Huankai Microbiology Technology Co., Ltd., standard strain.Pichia pastoris BNCC380011 is purchased from Guangzhou Yongjin Biotechnology Co., Ltd.

[0017] Example 1 Isolation and purification of bacterial strains

[0018] A 10g sample of goat milk collected from Diqing, Yunnan, was added to 100mL of sterile saline and shaken thoroughly. After a gradient dilution of the sample, 100μL was spread onto MRS solid medium containing 1% calcium carbonate and incubated at 37°C for 48 hours. Colonies that formed a calcium-dissolving ring on the solid medium were selected and repeatedly streaked onto MRS solid medium until the strains had consistent colony morphology. The purified lactic acid bacteria were inoculated onto MRS slant medium and stored at 4°C. The strains were then mixed with 20% glycerol and placed into cryovials. After mixing thoroughly, they were stored at -80°C for subsequent use.

[0019] (1) Identification of bacterial species ①Morphological characteristics of the strain like Figure 1 As shown, after culturing Lactobacillus plantarum B22 on MRS agar medium for 48 hours, the colony morphology was milky white, translucent, moist, smooth, with neat edges and obvious protrusions. Figure 1 AB. Microscopic examination of Lactobacillus plantarum B22 showed that it did not produce spores, had a dispersed rod-shaped cell morphology, and was a purple-positive Gram-stained bacterium.

[0020] ②Molecular genetic identification of bacterial species Whole-genome sequencing Lactobacillus plantarum B22 was cultured in 150 mL of MRS medium for 14 hours and then centrifuged at 8000 g at 4°C for 5 minutes. After centrifugation, the pellet was ground with liquid nitrogen and lysed with SDS lysis buffer containing proteinase K and mercaptoethanol. After lysis was complete and the mixture cooled to room temperature, the supernatant was centrifuged and extracted twice with chloroform / isoamyl alcohol (24:1). DNA was then precipitated with isopropanol, gently agitated, and centrifuged. DNA was initially purified using OMEGA purification columns and further purified using AmpureXP beads. Finally, DNA quality was assessed using a NanoDrop spectrophotometer and a Qubit fluorometer, and final purification was performed by electrophoresis. 2.5 μg of quality-controlled DNA was extracted and purified using magnetic beads, and 1 μL of the sample was quantified using a Qubit fluorometer. DNA fragments were mechanically fragmented by sonication, followed by purification, end-repair, 3' tailing, and ligation for sequencing. Fragments were size-selected using agarose gel electrophoresis, and sequencing libraries were generated by PCR amplification using the NEBNext® Ultra™ DNA Library Preparation Kit. The constructed libraries were then quality-controlled. The libraries were loaded onto an R9.4 sequencing chip and sequenced using a PromethION sequencer within 48–72 hours.

[0021] Sequence similarity analysis was performed using NCBI BLAST (http: / / blast.ncbi.nlm.nih.gov / ). The sequence homology between the test strain and the corresponding sequences of known strains was 100% identical to that of Lactobacillus plantarum, confirming that the selected lactic acid bacteria was Lactobacillus plantarum. The results in Table 1 show that the total length of the Lactobacillus plantarum B22 gene is 3,290,520 bp, with an average G+C content of 44.57%.

[0022] Table 1 Characteristics of the Lactobacillus plantarum B22 genome

[0023] Finally, the strain screened was identified as Lactobacillus plantarum through morphology, 16S rDNA sequencing, and whole genome sequencing, and was named Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B22, deposited in China Center for Type Culture Collection, with the accession number: CCTCC M 2025779.

[0024] Example 2 Determination of lipase activity of Lactobacillus plantarum B22 (1) Initial screening of lipase-producing lactic acid bacteria Punch a 0.8 cm diameter well on a tributyrin plate. Accurately inoculate 100 μL of a culture solution of various lactic acid bacteria strains into the well. Incubate the plate in a 37°C incubator for 48 hours and observe whether a clear zone appears on the plate. Measuring the diameter of the clear zone formed by different strains provides a preliminary assessment of the strains' lipase-producing capacity.

[0025] like Figure 2 As shown in Table 2, Lactobacillus plantarum B22 formed a clear hydrolysis zone (24.88 ± 1.04 mm) on tributyrin agar plates, demonstrating its ability to hydrolyze tributyrin, thus demonstrating its potent lipase activity. Tributyrin is a substrate widely used to screen for lipase- and esterase-producing microorganisms. By observing the hydrolysis zone surrounding colonies grown on nutrient agar containing tributyrin, larger hydrolysis zones indicate higher lipase activity, effectively assessing lipase activity.

[0026] Table 2 Diameters of transparent circles on tributyrin plates

[0027] (2) Rescreening of high-lipase-producing lactic acid bacteria Lipase activity was determined by referring to existing literature. Add 2 mL of 0.4 mM p-NPP standard substrate solution, preheat in a 40°C water bath for 5 minutes, then add 50 μL of enzyme solution. Mix thoroughly and incubate for 10 minutes. Terminate the reaction with 1 mL of 10% trichloroacetic acid, remove from the container, and allow to stand at room temperature for 5 minutes. Add 1 mL of 10% Na₂CO₃, develop color for 5 minutes, and measure absorbance at 405 nm. Enzyme activity is defined as the amount of enzyme required to release 1 μmol of p-NPP per minute at pH 7.5 and 40°C.

[0028] Enzyme activity (U / mL) = cV / tV1 Where: c: p-NP concentration (μmol / mL); V: volume of reaction solution after termination (mL); V1: amount of enzyme solution used; t: action time (min) like Figure 3 As shown, Lactobacillus plantarum B22 exhibited the highest lipase activity (3.45 ± 0.13 U / mL), which has important application potential in biotechnology and industry.

[0029] Example 3 Lipase Production and Gene Annotation of Lactobacillus plantarum B22 As shown in Table 3, functional gene annotation revealed 13 genes in Lactobacillus plantarum B22 that were annotated as esterases / lipases. A comprehensive phylogenetic analysis was subsequently performed to determine the evolutionary relationships among these esterases / lipases, revealing distinct clustering patterns. Furthermore, the basic physicochemical properties of these esterases / lipases were predicted and analyzed using the ProtParam tool in the ExPASy database (Table 3).

[0030] Table 3 Bioinformatics analysis of potential lipases in the genome of Lactobacillus plantarum B22

[0031] Subsequently, these 13 lipases were compared with known lipases (TAT54020.1, AZU39759.1, AQY70037.1) through phylogenetic analysis to reveal their evolutionary relationships. Figure 4 As shown, the lipases encoded by four genes in Lactobacillus plantarum, gene3060 (sequence shown in SEQ ID NO. 1), gene3059 (sequence shown in SEQ ID NO. 2), gene2553 (sequence shown in SEQ ID NO. 3), and gene0817 (sequence shown in SEQ ID NO. 4), have high sequence similarity with the known lipases TAT54020.1 and AZU39759.1. Therefore, the present application selected the above four lipases for further molecular docking.

[0032] Molecular docking analysis was further performed using MOE 2019 software to study the interaction between these four lipases and tributyrin. Generally, a docking energy value less than -4.25 kcal / mol indicates a certain binding activity, less than -5.0 kcal / mol indicates good binding activity, and less than -7.0 kcal / mol indicates strong binding activity. Figure 5 All four lipases showed good binding activity with tributyrin, with docking energies ranging from -5.4811 to -5.6039 kcal / mol. This suggests that the annotated lipases can bind to tributyrin. The enzyme encoded by gene 3060 may have stronger binding activity with tributyrin, forming hydrogen bonds with tributyrin through Ser114 and Ser41, and residue Ser41 also forming a carbon-hydrogen bond with tributyrin. In addition, Ile199 and Pro228 in the protein form hydrophobic interactions with the compound. These lipase genes highlight the potential of Lactobacillus plantarum B22 in lipase-substrate binding. Combined with the gene level, the present invention demonstrates that Lactobacillus plantarum B22 has the ability to produce high levels of lipase.

[0033] Example 4 Evaluation of the probiotic properties of Lactobacillus plantarum B22

[0034] (1) Acid tolerance The test bacterial liquid was inoculated into MRS liquid medium at a volume ratio of 2%, cultured at 37℃ for 18-24 h, and then transferred to MRS medium with pH 2.0 and 3.0 at the same inoculum volume. The culture was continued at 37℃ for 16 h, and the acid resistance of the strain was evaluated by measuring the absorbance at OD600nm.

[0035] If probiotics are to work stably in the body, they must first be in a viable state in the intestines. The human stomach secretes gastric acid with a very low pH, so the isolated strains must be tested for acid resistance by simulating the acidic environment of the stomach. Figure 6 As shown, the OD value of Lactobacillus plantarum B22 was higher than that of other strains, indicating that it had good acid tolerance and could survive in the gastric environment.

[0036] (2) Bile salt tolerance Based on the MRS medium formula, add 0.3% (w / v) ox bile salts by volume and sterilize for later use. Inoculate the test bacteria into the modified MRS liquid medium at a 2% (v / v) inoculum and culture at 37°C for 18–24 h. Then, inoculate the test bacteria into MRS liquid medium supplemented with 0.3% ox bile salts at a 2% (v / v) inoculum and culture anaerobically at 37°C for 16 h. Measure the absorbance at OD600nm to assess the strain's tolerance to bile salts.

[0037] Probiotic fermented dairy products are digested and absorbed in the stomach and eventually colonize in the intestines. They need to have good tolerance to the substances in the small intestine. The bile salt content in the human body is generally 0.3%. Figure 7 As shown, Lactobacillus plantarum B22 has a good tolerance to 0.3% (w / v) bile salts. Genome analysis annotated that Lactobacillus plantarum B22 has two of four bile salt tolerance genes.

[0038] (3) Gastrointestinal tolerance To prepare artificial gastric fluid at pH 2.5, first take 20 mL of 1 mol / L hydrochloric acid, adjust the pH to 2.5 by adding distilled water and 1 mol / L sodium hydroxide, and add pepsin to a concentration of 1 g / 100 mL. Then, filter through a 0.22 μm filter to complete the preparation. Inoculate 100 μL of the test bacterial solution into 5 mL of artificial gastric fluid and incubate anaerobically at 37°C. Samples are taken at 0 h and 3 h after the start of the experiment, and the viable bacterial count (CFU / mL) is determined. The survival rate is then calculated according to the formula:

[0039] Nt: viable bacteria count at 3 h; No: viable bacteria count at 0 h.

[0040] like Figure 8 As shown, Lactobacillus plantarum B22 had good tolerance in artificial gastric fluid, with a survival rate of 81.28±3.08%.

[0041] First, dissolve 0.27 g of potassium dihydrogen phosphate in 20 mL of distilled water. Then, adjust the solution's pH to 6.8 with 1 mol / L sodium hydroxide. Add trypsin to a final concentration of 1 g / 100 mL and allow it to completely dissolve. Then, filter through a 0.22 μm sterile filter to obtain ready-to-use artificial intestinal fluid. Inoculate 100 μL of bacterial solution into 5 mL of simulated artificial intestinal fluid and incubate anaerobically at 37°C. Samples were taken at 0 h and 3 h, and the viable bacterial count (CFU / mL) was determined. The survival rate was calculated using the formula:

[0042] Nt: viable bacteria count at 3 h; No: viable bacteria count at 0 h.

[0043] like Figure 9 As shown, the survival rate of Lactobacillus plantarum B22 in artificial intestinal fluid was higher than that of other strains, with a survival rate of 69.18±3.11%.

[0044] (4) Determination of biofilm production capacity Multiple lactic acid bacteria strains from experimental collections were activated for three generations in MRS liquid medium and cultured at 37°C for 24 hours. The activated strains were inoculated into MRS liquid medium at a 2% volume fraction, shaken, and then added to each well of a 96-well microtiter plate (200 μL). The plates were then incubated at 37°C for 24 hours. After incubation, the supernatant was discarded and the plates were washed twice with sterile PBS (pH 7.2) to remove non-adherent bacteria. The plates were fixed with methanol for 10 minutes, the methanol was removed, and the plates were stained with 0.1% (v / v) crystal violet for 20 minutes. The crystal violet was removed, and the plates were washed with sterile PBS (pH 7.2). The plates were dried at room temperature and decolorized with 33% acetic acid for 10 minutes. The biofilm formation of the different strains was measured at OD 595 nm.

[0045] OD595nm can indicate biofilm content; higher values ​​indicate greater biofilm content. Biofilms allow strains to tolerate extreme environments such as the gastrointestinal tract, thereby promoting the adhesion and colonization of lactic acid bacteria in the intestines, which is beneficial for regulating intestinal flora and exerting probiotic functions.

[0046] like Figure 10 As shown, the amount of biofilm produced by Lactobacillus plantarum B22 (OD595 was 1.51) was significantly higher than that of other lactic acid bacteria, indicating that it had a higher survival rate in adverse environments and the gastrointestinal tract.

[0047] (5) Ability to produce extracellular polysaccharides The activated lactic acid bacteria strains were inoculated into MRS liquid culture medium and cultured at 37 °C for 24 h. The fermentation broth was centrifuged at 12000 r / min for 10 min, and three volumes of 95% ethanol were added to the supernatant. The exopolysaccharides were precipitated in a 4 °C refrigerator overnight. Glucose standard solutions of different concentrations were prepared separately. Subsequently, 2 mL of glucose standard solutions of different concentrations were added to the test tubes, followed by 2 mL of 6% (v / v) phenol solution, and finally 10 mL of concentrated sulfuric acid. The mixture was mixed and allowed to stand. After cooling, the absorbance at a wavelength of 490 nm was measured. Three parallels were made for each group. A standard curve was drawn with glucose content (mg / L) as the horizontal axis and OD490nm absorbance as the vertical axis. The absorbance of the exopolysaccharide solutions of different strains precipitated in a 4 °C refrigerator overnight at 490 nm was determined by the same method, and the regression equation (y = 0.0061x + 0.0417, R 2 =0.995) to calculate the extracellular polysaccharide content of the strain.

[0048] Lactic acid bacteria exopolysaccharides have good rheological properties, can improve the taste and texture of dairy products and other foods, extend the shelf life of food, and also have anti-cancer, immune regulation, blood sugar lowering, antioxidant and other properties. Figure 11 As shown, the exopolysaccharide content of Lactobacillus plantarum B22 (344.21 mg / L) was much higher than that of other strains.

[0049] (6) Antibacterial ability Staphylococcus aureus, Escherichia coli, and Salmonella were used as indicator strains for the antibacterial activity assay of Lactobacillus plantarum B22. A 3% (v / v) suspension of the activated indicator strains was inoculated into unsolidified agar medium, mixed thoroughly, and then poured onto plates. After the plates were placed in a clean bench for 30 minutes, 200 μL of fermentation supernatant (CFS) was added to the wells of each well. For the control group, 200 μL of sterile MRS broth was added. The plates were then incubated at 37°C for 24 hours. The size of the inhibition zone was observed and measured. Three replicates were performed.

[0050] like Figure 12As shown, compared to the control sterile MRS broth, Lactobacillus plantarum B22 produced distinct zones of inhibition on Salmonella, Staphylococcus aureus, and Escherichia coli plates, with zone diameters of 16.04±1.08 mm, 17.70±0.42 mm, and 14.86±0.75 mm, respectively. This indicates that Lactobacillus plantarum B22 has a high antibacterial activity, inhibiting the growth of Staphylococcus aureus, Escherichia coli, and Salmonella strains. Lactobacillus plantarum B22 also inhibited the growth of yeast (Pichia pastoris), with a zone diameter of 18.37±2.02 mm. Furthermore, Lactobacillus plantarum B22 significantly inhibited the growth of mold (Geotrichum candidum).

[0051] Genomic analysis of Lactobacillus plantarum B22 revealed the presence of four genes encoding dipeptide bacteriocins, which may contribute to its antimicrobial activity. Genes associated with bacteriocin biosynthesis were identified. In addition to bacteriocins, organic acids such as lactic acid (ldh) and phenyllactic acid (ldhA) are key antimicrobial metabolites produced by lactic acid bacteria, and related genes have also been annotated in the genome.

[0052] (7) AI-2 activity assay Using Vibrio harveyi ( V. harveyi )BB170 (ATCC BAA-1117) was used as the indicator bacteria, which was purchased from Guangdong Provincial Microbial Culture Collection Center (GDMCC). V. harveyi BB170 was streaked onto Marine 17 agar medium and incubated at 30°C for 24 h. It was inoculated into the autogenous bioassay (AB) liquid medium and incubated at 30°C for 12 h. V. harveyi The fermentation broth of BB170 was centrifuged at 4000 rpm for 30 min, and the supernatant was filtered through a 0.2 μm filter. V. harveyi BB170 was then mixed with culture medium (sterile MRS broth), different culture time groups, and AB liquid culture medium (negative control) at a ratio of 1:50 (v:v). 200 μL of each mixed sample was then placed into a 96-well cell culture plate, with each sample placed in a separate well. The fluorescence intensity of each sample was measured using a full-wavelength multifunctional microscope in bioluminescence mode. The AI-2 content of each experimental group was calculated when the fluorescence value of the negative control reached its lowest value within 1-7 hours. The AI-2 content is the ratio of the fluorescence value of the experimental group to that of the negative control, reaching its lowest value within 1-7 hours. The ratio of the fluorescence value of the experimental group to that of the culture medium was also calculated.

[0053] AI-2 is a signal molecule for strain information exchange, such as Figure 13 The results showed that Lactobacillus plantarum B22 had a high AI-2 (1.06), which was beneficial to the exchange of strains and promoted the performance of prebiotic functions.

[0054] (8) Determination of antioxidant capacity The lactic acid bacteria from the experimental collection were activated for three generations in MRS liquid medium and cultured at 37°C for 24 hours. The activated three-generation strain was inoculated into MRS liquid medium at a volume fraction of 2% and cultured at 37°C for 24 hours. After culture, the bacterial solution was mixed and centrifuged at 6000 rpm for 5 minutes. The supernatant and bacterial cells were collected and stored at 4°C for later use.

[0055] ①DPPH free radical scavenging ability Mix 2 mL of sample with 2 mL of 0.2 mol / L DPPH anhydrous ethanol solution and allow to react at room temperature in the dark for 30 minutes. Centrifuge (4°C, 6000 rpm, 10 minutes), and collect the supernatant. Measure the absorbance at 517 nm using a spectrophotometer. For the blank group, replace the DPPH anhydrous ethanol solution with an equal volume of anhydrous ethanol, and for the control group, replace the sample solution with an equal volume of blank solvent. The DPPH radical scavenging efficiency is calculated as follows:

[0056]

[0057] Where: Ai: absorbance value of supernatant; A0: absorbance value of blank group; Aj: absorbance value of control group; like Figure 14 As shown in the figure, the DPPH radical scavenging rate of the fermentation supernatant of Lactobacillus plantarum B22 reached 98.41%, which was significantly higher than that of other strains.

[0058] ②ABTS + Free radical scavenging ability ABTS + Preparation of diluted working solution (λ=734 nm, absorbance 0.7-0.8): Accurately weigh 0.192 g of ABTS + Dissolve the standard sample in distilled water and accurately dilute to the mark on a 50 mL volumetric flask. Also, accurately weigh 0.067 g of potassium persulfate and dissolve it in the same manner to 50 mL. Mix the mixture in a 1:1 ratio and store in a dark environment for 12 h. Dilute 30-35 times with PBS buffer (5 mmol / L, pH 7.4) before use. Transfer 25 μL of the lactic acid bacteria sample solution to a 96-well plate, then add 190 μL of the ABTS working solution. Incubate at room temperature for 6 min. After the reaction is complete, shake the plate at 37°C for 20 s and measure the absorbance at 734 nm using a microplate reader. Pipette 25 μL of ultrapure water as a blank control.

[0059] Calculation formula: ABTS + Free radical scavenging rate (%) = [(A0-A1) / A0] × 100 Where: A0 is the absorbance of blank control; A1 is the absorbance of ABTS + The absorbance of the working solution + PBS.

[0060] like Figure 15 As shown, the fermentation supernatant and ABTS of Lactobacillus plantarum B22 + The free radical scavenging rate (79.56%) was significantly higher than that of other strains ( p <0.05).

[0061] The genome of Lactobacillus plantarum B22 contains genes encoding key antioxidant enzymes, including catalase, glutathione peroxidase, thioredoxin, thioredoxin reductase, and thiol peroxidase. In addition, the genome also contains multiple genes related to manganese (Mn) transport, including manganese transporter, Mn ABC transporter, and manganese-dependent inorganic pyrophosphatase. Murgas et al. showed that the manganese accumulation system regulates the transport and accumulation of manganese, enhancing the strain's tolerance to oxidative stress. Manganese ions (Mn 2+ ) protects the body from ROS by acting as a cofactor for superoxide dismutase and other enzymes involved in redox regulation. These findings indicate that Lactobacillus plantarum B22 is a probiotic with strong antioxidant potential.

[0062] Example 5 Safety Evaluation of the Probiotic Properties of Lactobacillus plantarum B22

[0063] (1) Hemolytic test Lactobacillus plantarum B22, a third-generation activated strain from an experimental collection, was cultured in MRS liquid medium at 37°C for 24 hours. The third-generation activated strain was streaked onto Columbia blood agar and incubated at 37°C for 48 hours. After incubation, the colonies were observed for the formation of hemolytic zones. Staphylococcus aureus served as a positive control to facilitate comparison and evaluation of the hemolytic activity of the test strains.

[0064] In the present invention, the hemolytic activity of Lactobacillus plantarum B22 was tested on blood agar plates, with Staphylococcus aureus (a known β-hemolytic strain) as a positive control. Figure 16 As shown, compared with Staphylococcus aureus, no obvious clear zone was observed around Lactobacillus plantarum B22, indicating that this strain is γ-hemolytic and does not produce β-hemolysin.

[0065] (2) Amino acid decarboxylase test The Lactobacillus plantarum B22 seed liquid activated three times was inoculated into four groups of amino acid (tyrosine, arginine, histidine and lysine) biochemical tube culture media respectively. The biochemical tube mouth was sealed with liquid paraffin and cultured anaerobically at 37°C for 48 hours. The color change of the amino acid biochemical tube culture media was observed. If the color of the control group without inoculation of the test bacteria liquid remained yellow, the result was negative; if the color of the culture medium turned purple after inoculation, it indicated a positive reaction.

[0066] After being decarboxylated by amino acid decarboxylase, amino acids are converted into biogenic amines. Excessive biogenic amines can cause headaches, high blood pressure, and breathing difficulties. Amines in food can also form strong carcinogens, such as nitrosamines. Therefore, it is very necessary to test the amino acid decarboxylase activity of the target strain. Figure 17 As can be seen, the indicators for tyrosine, arginine, histidine, and lysine all display the same yellow (negative) color as the blank control. Furthermore, the genome of Lactobacillus plantarum B22 does not contain genes for tyrosine, arginine, histidine, or lysine decarboxylases. It lacks the complete gene clusters responsible for the biosynthesis of tyramine, putrescine, histamine, or cadaverine. This indicates that Lactobacillus plantarum B22 does not produce amino acid decarboxylases and, therefore, does not break down amino acids to form biogenic amines. Therefore, consumption of Lactobacillus plantarum B22 does not pose a food safety concern due to biogenic amines.

[0067] (3) Antibiotic sensitivity test Antibiotic susceptibility testing was performed using the disk diffusion method. 8 Lactobacillus plantarum B22 (200 μL) was evenly plated onto MRS agar. Flame-sterilized forceps were then used to place the antibiotic susceptibility test discs onto the plates. The plates were incubated overnight at 37°C, and the diameter of the zone of inhibition (mm) was measured. The diameter of the zone of inhibition surrounding the discs was measured and categorized as sensitive (>20.5 mm), intermediate (10.5-20.5 mm), and resistant (<10.5 mm) according to the method described by Zareie et al. (2023).

[0068] Antibiotic resistance has become a key criterion for evaluating the suitability of bacterial strains as feed additives for humans and animals. To evaluate the potential application of Lactobacillus plantarum B22 in food, its resistance to 20 common antibiotics was assessed (Table 4). As shown in Table 4, Lactobacillus plantarum B22 exhibited susceptibility to a variety of antibiotics, including penicillin, piperacillin, ampicillin, minocycline, cefuroxime, ceftazidime, ceftriaxone, cefoperazone, and erythromycin, with clear inhibition zone diameters of 27.60±2.84 mm, 35.27±0.33 mm, 31.89±3.16 mm, 40.09±3.46 mm, 41.55±0.72 mm, 25.51±2.80 mm, 39.43±1.31 mm, 24.61±0.99 mm, and 27.42±1.46 mm, respectively. It was moderately sensitive to kanamycin, gentamicin, tetracycline, and lincomycin. The number of antibiotics it was sensitive to accounted for 65% of the total number of antibiotics tested. Therefore, Lactobacillus plantarum B22 was shown to be sensitive to most antibiotics and safe and reliable.

[0069] Table 4 Antibiotic sensitivity analysis of Lactobacillus plantarum B22

[0070] Note: R stands for resistant; I stands for intermediate; S stands for sensitive.

[0071] Example 6 Preparation of Fermented Cream with Lactobacillus plantarum B22 (1) Preparation of fermented cream A certain amount of cream (fat content 35.00%, protein content 1.2%) was weighed and homogenized at a pressure of 15-18 MPa. After sterilization in a 95°C water bath for 10 minutes, the mixture was allowed to cool. After the temperature dropped to 30-40°C, a 4% (v / v) inoculum of Lactobacillus plantarum B22 seed solution and a 0.01% inoculum of a commercial direct-inoculation starter (Lactobacillus bulgaricus and Streptococcus thermophilus, 1:1) were inoculated into the cream. The viable cell count of Lactobacillus plantarum B22 was 3.68×10 9 CFU / mL, and fermented at 37°C for 8 h, followed by sensory evaluation. The starter cultures were mixed evenly and fermented at a fixed temperature and time. Fermented cream was prepared using the same procedure as controls (Lactobacillus plantarum D138 and B133).

[0072] like Figure 18As shown, fermented cream prepared with Lactobacillus plantarum B22 has a similar color to other strains. Furthermore, Lactobacillus plantarum B22 outperforms other strains in terms of odor, flavor, texture, and overall acceptability. This is attributed to the high lipase activity and high exopolysaccharide content of Lactobacillus plantarum B22. Lipase can break down fats and produce flavor compounds, while exopolysaccharides can enhance the texture of the product.

[0073] (2) Flavoromics of fermented cream ① Flavor composition of fermented cream Lactic acid bacteria (LAB) produce a variety of volatile compounds during cream fermentation. To comprehensively evaluate the impact of the lipase-producing strain Lactobacillus plantarum B22 on the flavor of fermented cream, headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to analyze the volatile flavor compounds of fermented creams prepared from Lactobacillus plantarum B22 and Lactobacillus plantarum D138, respectively. A total of 77 volatile compounds were identified. These compounds were classified into eight chemical classes: 18 esters (23.38%), 10 ketones (12.99%), 10 acids (12.99%), 24 alcohols (31.17%), 6 heterocyclic compounds (7.79%), 4 nitrogen compounds (5.19%), 3 halogens (3.9%), and 2 aldehydes (2.6%).

[0074] Based on the semi-quantitative analysis of 77 aroma compounds, principal component analysis (PCA) was performed to differentiate between cream samples fermented with Lactobacillus plantarum B22 and Lactobacillus plantarum D138. The first principal component (PC1) and the second principal component (PC2) explained 79.59% and 9.42% of the total variance in the original dataset, respectively. Consistent with the findings of Zhou et al. (2024), a clear separation was observed between the two sample groups. This distinct separation clearly distinguished the flavor profiles of the two fermented creams, demonstrating that the addition of Lactobacillus plantarum B22 significantly altered the flavor of the fermented cream.

[0075] ② Identification of key flavor compounds in LSB and LS fermented creams using GC-O and OAV GC-O analysis identified 23 aroma-active compounds in cream fermented by Lactobacillus plantarum B22 and Lactobacillus plantarum D138 ( Figure 19), including two aldehydes, seven ketones, four alcohols, six acids, and three esters. Key sensory characteristics include cheese, rancidity, sourness, cream, bread, almonds, sweetness, boiled milk, soap, and green. For example, acetoin (buttery, creamy), 2,3-butanedione (buttery, sour), 2-nonanone (hot milk, soapy, grassy), butyric acid (rancidity, cheese, sweat), tetrahydro-6-propyl-2H-pyran-2-one (peach), caprylic acid (sweat, cheese), and tetrahydro-6-pentyl-2H-pyran-2-one (coconut) exhibited high aroma intensity in creams fermented with Lactobacillus plantarum B22 and Lactobacillus plantarum D138. It is noteworthy that the aroma intensity of 2-heptanone, acetoin, 2-nonanone, 2-undecanone, butyric acid, 2-tridecanone, tetrahydro-6-propyl-2H-pyran-2-one, octanoic acid, tetrahydro-6-pentyl-2H-pyran-2-one, 4-methyl-3-hexanol, phenylethyl alcohol, benzoic acid and 2,3-butanedione in cream fermented with Lactobacillus plantarum B22 was higher than that in cream fermented with Lactobacillus plantarum D138.

[0076] like Figure 20 As shown, the contents of acetoin, δ-dodecalactone, 4-methyl-3-hexanol, 2-hydroxy-3-pentanone, 2-(2-hydroxypropyloxy)-1-propanol, phenylethyl alcohol, benzoic acid, and 2,3-butanedione in cream fermented with Lactobacillus plantarum B22 were significantly higher than those in cream fermented with Lactobacillus plantarum D138. This is likely due to the lipase produced by Lactobacillus plantarum B22, which promotes the breakdown of fats into aldehydes, ketones, and esters. Acetoin, 2,3-butanedione, lactones, 2-nonanone, and butyric acid are key aroma components in fermented milk.

[0077] The impact of volatile compounds on aroma perception depends not only on their concentration but also on their odor threshold. Odor activity value (OAV) analysis provides a more intuitive understanding of the contribution of each compound to the overall aroma profile. Volatile compounds with an OAV ≥ 1 are generally considered to be important components of the aroma profile. Therefore, OAV analysis was used to further explore different flavor compounds. Seven key volatile flavor compounds (OAV > 1) were identified in lactic acid bacteria-fermented cream: acetoin (OAV = 3.28), 2-nonanone (OAV = 1.25), tetrahydro-6-pentyl-2H-pyran-2-one (OAV = 1.29), δ-dodecalactone (OAV = 28.08), 2-hydroxy-3-pentanone (OAV = 2.20), benzoic acid (OAV = 2.44), and 2,3-butanedione (OAV = 3.24). Lactobacillus plantarum B22 was added to lactic acid bacteria-fermented cream. Studies have shown that B22 promotes acetoin production in the cream, significantly contributing to the fruity and creamy flavor of the cream. 2,3-Butanedione imparts a delicate, rich, buttery flavor to the cream, indicating that the addition of the independently screened strain B22 can promote 2,3-butanedione production. 2-Hydroxy-3-pentanone imparts a sweet aroma, mouthfeel, and buttery flavor. 2-Nonanone, a unique flavor component, has been reported to contribute to the flavor of cheese. Furthermore, lactones significantly enhance the woody, sweet, and coconutty notes of cheese. Overall, the presence of these flavor compounds contributes to a better palatability of cream fermented with B22 than with D138. Analysis suggests that the lipase produced by B22 enhances flavor, likely by hydrolyzing and oxidizing lipids.

[0078] (3) Lipidomics of Fermented Cream Sensory ① Lipid composition of fermented cream Absolute lipidomics was used to identify the lipid species and relative abundances in cream during fermentation. A total of 2,728 lipid species were detected in both positive and negative ion modes, and these lipids were classified into eight major categories based on the classification criteria of Dahdah et al. (2021). The identified lipids included glycerophospholipids (GPs; 464 species), glycolipids (SLs; 83 species), sphingolipids (SPs; 510 species), glycerides (GLs; 1,530 species), fatty acyl groups (FAs; 42 species), sterol lipids (STs; 25 species), polyketides (PKs; 1 species), and pro-alcohol lipids (PRs; 90 species). These lipids are further subdivided into 38 subclasses, showcasing the diversity of lipid molecules, such as hexosylceramide (HexCer), ceramide (Cer), cardiolipin (CL), diglyceride (DG), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), N-acetylethanolamine (NAE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol ceramide (PI-Cer), sphingomyelin (SM), sterols (ST), phosphatidylglycerol (PG), and triglyceride (TG). The four major lipid groups in fermented butter are triglycerides (TGs; 1036 species), diglycerides (DGs; 445 species), hexosylceramide (Hex1Cer; 157 species), phosphatidylethanolamine (PE; 145 species), and ceramide (Cer; 114 species).

[0079] In terms of lipid content percentage, GL (55.28%) was the most abundant, followed by SP (18.77%) and GP (17.08%). TGs and DGs were two important lipids in GL, accounting for 38.12% and 16.37%, respectively. Among the identified GPs, PE was the most predominant lipid, accounting for 5.33% of the total lipid content, followed by PC at 3.13%. These results are consistent with previous studies showing that GL, SP, and GP are the major lipid classes in dairy products (Song, 2024). In PR, the contents of ZyE and ZE were 1.32% and 1.99%, respectively. ST had the highest content of St, reaching 2.24%. Furthermore, SP had the highest contents of Hex1Cer and Cer, at 5.78% and 4.19%, respectively. Furthermore, FA and OAHFA accounted for 0.48% and 1.07% of the FA, respectively.

[0080] ② Lipid changes in fermented cream Changes in lipid subclasses reflect altered lipid functions. Therefore, by evaluating the differential expression of lipid subclasses in creams fermented with Lactobacillus plantarum B22 and Lactobacillus plantarum D138, we can identify important lipid subclasses that may be involved in specific biological activities. Figure 21The top eight lipid subclasses during fermentation were shown to include triglycerides (TG), diacylglycerol (DG), phosphatidylglycerol (PG), fatty acids (FA), sphingomyelin (SPH), hexadecylmuramerol (Hex1Cer), phosphatidylethanolamine (PE), and phosphatidylcholine (PC). Lactobacillus plantarum B22 had the lowest levels of TG, DG, PG, SPH, Hex1Cer, PE, and PC, while the highest level of FA. Previous studies have shown that fatty acids are primarily derived from the breakdown of triglycerides and diacylglycerol. Therefore, Lactobacillus plantarum B22 promotes the production of fatty acids and enhances the production of flavor compounds.

[0081] ③Key lipids of fermented cream like Figure 22 As shown in the figure, a total of 42 significantly different lipids were identified between the samples of Lactobacillus plantarum B22 fermented cream and Lactobacillus plantarum D138 fermented cream, including 5 up-regulated and 37 down-regulated lipids. The target lipids that changed significantly between the different groups were further selected by the selection criteria of VIP value greater than 5 ( Figure 23 Seven characteristic lipids were observed in cream fermented with Lactobacillus plantarum B22, including FA (18:1), TG (10:0_12:0_14:0), TG (16:0_14:0_18:1), TG (6:0_18:1_18:1), TG (6:0_14:0_18:1), DG (24:2e), and PC (18:2_18:2). FA (18:1) is a monounsaturated fatty acid that serves as a precursor for flavor compounds. Therefore, cream fermented with Lactobacillus plantarum B22 may have a stronger flavor.

[0082] It should be understood that the above-described specific embodiments of the present invention are merely illustrative or illustrative of the principles of the present invention and do not constitute limitations of the present invention. Therefore, any modifications, equivalent substitutions, improvements, etc. made without departing from the spirit and scope of the present invention should be included within the scope of protection of the present invention. In addition, the appended claims are intended to cover all variations and modifications that fall within the scope and metes and bounds of the appended claims, or equivalents thereof.

Claims

1. A lipase-producing Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, The plant lactobacillus is named plant lactobacillus ( Lactiplantibacillus plantarum ) B22, the deposit number is CCTCC M 2025779, the depository is China Center for Type Culture Collection, and the deposit address is: Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. Use of the plant lactobacillus according to claim 1 in the preparation or production of lipase.

3. Use of the plant lactobacillus according to claim 1 in preparing fermented foods.

4. The use of plant lactobacillus in preparing fermented food according to claim 3, characterized in that The fermented food is milk cake, cheese, sausage or cream.

5. The use of plant lactobacillus in preparing fermented food according to claim 4, characterized in that When the plant lactobacillus is used to prepare fermented cream, the inoculation amount of the strain is 1.5×10 9 -4.5×10 9 CFU / mL.

6. The use of plant lactobacillus according to claim 4 in preparing fermented food, characterized in that The Lactobacillus plantarum is combined with a commercial yogurt starter to ferment the cream; the usage amount of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 8 h-12 h, and the fermentation temperature is 35° C.-37° C.

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