Flavone-enriched citrus peel-derived bacillus and application thereof

Abstract

The application discloses a flavone-enriched pericarpium citri reticulatae source bacillus and application thereof. The pericarpium citri reticulatae source bacillus is Bacillus subtilis CP-J2440, which is collected in the China Center for Type Culture Collection on July 3, 2024, and the preservation number is CCTCC NO: M 20241459. The Bacillus subtilis CP-J2440 in the application is from dominant bacteria in pericarpium citri reticulatae aged for 3 years, can affect the generation of volatile and non-volatile metabolites, and can be applied to the preparation of functional food for resisting blood fat, functional food for resisting oxidation and probiotic functional food.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a flavonoid-rich Bacillus tangerine peel-derived strain and its application. Background Technology

[0002] In China, Japan, South Korea, India, and Southeast Asia, dried citrus peels aged for more than three years (known as Chenpi in China) are widely used in cooking and traditional medicine practices. China has several varieties of Chenpi, the most famous being Guangchenpi or Xinhuichenpi, made from peels grown in Xinhui District, Jiangmen City, Guangdong Province. Chenpi is rich in volatile oils, polyphenols, flavonoids, and alkaloids, possessing various physiological benefits such as antioxidant, anti-inflammatory, digestive system-improving, and anti-obesity effects. Due to its pleasant sensory characteristics, unique production methods, and potential health benefits, Chenpi has regained global attention in recent decades and is favored by consumers and researchers. Generally, the quality of Chenpi is closely related to its aging process; the longer the aging time, the higher the quality. However, the natural aging of citrus peel is a complex and slow process, susceptible to pests and mold, posing challenges to its processing and preparation. Microbial fermentation has been identified as a crucial factor influencing the quality and bioactivity characteristics of citrus peel during aging. Current research has identified several microbial genera in dried tangerine peel, including yeasts, aspergillus, ascomycetes, Bacillus, lactococci, pseudomonas, and enterococci, with Bacillus and lactococci being particularly prominent. However, direct evidence regarding the detailed dynamic mechanisms by which probiotics in dried tangerine peel can accelerate the accumulation of bioactive compounds during the aging process of citrus peel remains limited.

[0003] Probiotics produce hydrolytic enzymes that precisely degrade plant cell walls, releasing bioactive substances from raw materials. Therefore, probiotic fermentation has become crucial for enhancing the bioactivity of these materials. Bacillus possesses several advantageous traits, including the ability to form endospores. Some strains or species have received "Generally Recognized as Safe" (GRS) certification from the U.S. Food and Drug Administration (FDA) and "Qualified Presumption of Safety" (QPS) certification from the European Food Safety Authority (EFSA). Research has documented significant changes in metabolites such as polyphenols, flavonoids, organic acids, and other flavor compounds during Bacillus fermentation, potentially improving the sensory and functional properties of raw materials. Of particular note is the significant research interest in the biotransformation and enhanced functional activity of metabolites during Bacillus fermentation. A previous study highlighted that ginseng exhibited significantly enhanced antioxidant activity after fermentation with Bacillus, attributed to an increase in phenolic compounds and flavonoids. Another study showed that soy isoflavones only possess bioactivity after being hydrolyzed into free aglycones via β-glucosidase-mediated hydrolysis during Bacillus fermentation. Citrus peel is an important byproduct of the citrus industry, and its active ingredients and bioactivity can be enhanced through microbial fermentation. A study using *Aspergillus niger* to ferment citrus peel powder in a solid-state environment showed a significant increase in total phenolic and total flavonoid content by 70.0% and 26.8%, respectively. The contents of hesperidin, naringenin, and nobiletin increased from 19.36, 6.31, and 2.91 mg / g to 28.23, 7.78, and 3.49 mg / g, respectively. Another study using *Saccharomyces cerevisiae* similarly found that fermentation significantly increased the contents of limonene, naringenin, and 3'-methoxynaringenin in citrus peel, exhibiting a significant anti-obesity effect in rats fed a high-fat diet. Furthermore, recent research indicates that after 12 days of co-fermentation of citrus peel with *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae*, the free flavonoid content increased by 48.12% compared to the unfermented sample, indicating enhanced antioxidant activity. However, solid-state fermentation also presents challenges, such as long fermentation time, inconsistent quality, microbial contamination, and health and safety concerns. In contrast, liquid fermentation has advantages such as shorter fermentation time and suitability for large-scale automated production. To date, no systematic study has been conducted on the metabolic characteristics of citrus peel fermentation using Bacillus from dried tangerine peel under pure culture conditions. Summary of the Invention

[0004] The technical problem this invention aims to solve is to overcome the shortcomings of existing technologies and provide a Bacillus spp. from tangerine peel that enriches flavonoids and its applications. This invention employs various omics techniques, including volatile metabolomics, untargeted metabolomics, and targeted metabolomics, as well as in vitro lipid-lowering experiments, to elucidate the dynamic changes and assess the functional activity of incompletely aged citrus peel infusion (Pericarpium Citri Reticulatae infusion, PCRI) during fermentation using Bacillus spp. This research aims to explore in depth the high-value processes for developing citrus byproducts into functional foods, thereby accelerating the accumulation and biotransformation of bioactive compounds in citrus peel through endophytic bacteria. Furthermore, compared to yeasts and lactic acid bacteria, Bacillus spp., commonly used as a starter culture in solid-state fermentation food processes, has received far less systematic research. Therefore, this study contributes to broadening our understanding of the potential applications of Bacillus spp. and related application processes.

[0005] To address the aforementioned technical problems, this invention provides a flavonoid-enriched Bacillus subtilis CP-J2440, which was deposited at the China Center for Type Culture Collection on July 3, 2024, with accession number CCTCC NO:M 20241459.

[0006] Based on a general technical concept, the present invention also provides the application of Bacillus subtilis var. tangerine peel in the enrichment of flavonoids.

[0007] Based on a general technical concept, the present invention also provides the development and application of Bacillus subtilis var. tangerine peel in the preparation of functional foods with antioxidant and anti-lipid properties.

[0008] Based on a general technical concept, the present invention also provides an application of Bacillus subtilis derived from tangerine peel in the preparation of functional foods such as probiotics.

[0009] The above application, further, the method of the application includes:

[0010] S1. Fermentation broth was obtained by fermenting Bacillus CP-J2440 strain;

[0011] S2. Pulverize the dried citrus peel and extract it with purified water to obtain a tea branch citrus peel extract.

[0012] S3. After sterilizing the extract of citrus peel, inoculate it with the fermentation broth, and collect the fermentation supernatant after fermentation.

[0013] In the above application, further, in S1, the inoculation amount of the Bacillus CP-J2440 strain is 8% (volume fraction).

[0014] In the above application, further, in S1, the fermentation culture specifically refers to: culturing at 37°C for 5 hours.

[0015] In the above application, S2 specifically involves: pulverizing dried citrus peel, passing it through a 60-mesh sieve, adding ultrapure water at a solid-liquid ratio of 1:20, and sterilizing at 121°C for 20 minutes to obtain a tea branch citrus peel extract.

[0016] Further, in the above application, S3 specifically involves: cooling the tea branch and citrus peel extract to 20°C, adding 8% fermentation liquid, and fermenting it at 37°C on a shaker at 120g speed for 4 days; collecting the fermentation liquid every 24 hours, centrifuging the collected fermentation supernatant at 12,000g, and then storing it at -80°C.

[0017] Compared with the prior art, the advantages of the present invention are as follows:

[0018] This invention provides a flavonoid-rich Bacillus spore-forming bacterium derived from tangerine peel. Bacillus CP-J2440, a dominant bacterium from 3-year-aged tangerine peel, influences the formation of volatile and non-volatile metabolites. During the fermentation of citrus peel extract (Pericarpium Citri Reticulatae infusion, PCRI) by Bacillus CP-J2440, total phenols and flavonoids significantly increased, especially on the third day of fermentation. Soluble sugars and proteins initially decreased and then increased, while free amino acids continued to decrease. After the third day of fermentation with Bacillus CP-J2440, the antioxidant activity of the citrus peel extract was significantly enhanced, with DPPH, ABTS·+, and FRAP free radical scavenging abilities increasing by 62.84%, 19.27%, and 42.88%, respectively. Furthermore, in vitro experiments showed that the fermented PCRI had a stronger anti-lipidemia effect. Fermentation with Bacillus CP-J2440 significantly altered the flavor profile and metabolite composition of unfermented PCRI, resulting in a unique aroma reminiscent of fermented and ripe fruit with herbal notes. Furthermore, targeted quantification of flavonoids elucidated the interconversions between flavonoid glycosides and aglycones, as well as between different aglycones, during fermentation. Future research will integrate genomics, proteomics, and metabolomics to elucidate the specific pathways of flavonoid metabolism and enzymatic catalysis, which is crucial for advancing the development and application of probiotic products and processes.

[0019] Bacillus subtilis CP-J2440 was deposited at the China Center for Type Culture Collection on July 3, 2024, and was confirmed to be viable on July 10, 2024. The accession number is CCTCC NO:M 20241459, and the deposit address is Wuhan University, Wuhan, Hubei Province, China. Attached Figure Description

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0021] Figure 1 The colony morphology, scanning electron microscopy, and phylogenetic tree results of the Bacillus CP-J2440 isolated strain in Example 1 of this invention are shown.

[0022] Figure 2 This is a flowchart illustrating the application of Bacillus CP-J2440 in the enrichment of flavonoids in Example 2 of the present invention.

[0023] Figure 3 The results show the differences in chemical composition of PCRI fermented by Bacillus CP-J2440 in Experiment 2 of this invention.

[0024] Figure 4 The results show the differences in antioxidant activity of Bacillus CP-J2440 fermented with PCRI in Experiment 3 of this invention.

[0025] Figure 5 This is the result of the dynamic changes in soluble protein content (SPC), water-soluble sugar content (SSC), and total free amino acid content (FAAC) in Experiment 4 of this invention.

[0026] Figure 6 The result is the pH change in Experiment 4 of this invention.

[0027] Figure 7 These are photographs and trend graphs of the color changes in Experiment 4 of this invention.

[0028] Figure 8 This invention provides a comparison of volatile components in the fermentation process of Bacillus CP-J2440 in PCRI during Experiment 4.

[0029] Figure 9 This study analyzes the non-volatile metabolites produced during the fermentation of Bacillus CP-J2440 in PCRI during Experiment 5 of this invention.

[0030] Figure 10 This invention relates to the classification and analysis of differential metabolites during the fermentation process of Bacillus CP-J2440 in PCRI in Experiment 5 of this invention.

[0031] Figure 11 The results show the effects of PCRI on TC and TG in Experiment 7 of this invention.

[0032] Figure 12 The results show the effect of PCRI on lipid accumulation in HepG2 cells in Experiment 7 of this invention.

[0033] Figure 13The images are from Experiment 7 of this invention, stained with BODIPY 493 / 503.

[0034] Figure 14 This is the result of using the DCFH-DA probe to monitor the cellular response to oxidative stress in an environment rich in mixed acids in Experiment 7 of this invention.

[0035] Figure 15 This is a Spearman correlation heatmap showing the chemical composition of PCRI and its acidity, color, and biological activity in Experiment 8 of this invention. Detailed Implementation

[0036] The present invention will be further described below with reference to specific preferred embodiments, but this does not limit the scope of protection of the present invention.

[0037] The materials, reagents, and instruments used in the following examples are all commercially available. Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art.

[0038] The two-year and three-year-old Pericarpium Citri Reticulatae samples were purchased from Xinhui District, Jiangmen City, Guangdong Province, China. Folin-Ciocalteu, Trolox (GA, >98%), Rutin (GA, >98%), and Gallic acid (GA, >98%) were all purchased from Chengdu Master Biotechnology Co., Ltd. (Chengdu, China). Methanol and isopropanol were obtained from China National Pharmaceutical Group Chemical Reagent Co., Ltd. (Shanghai, China). The plant soluble protein and free amino acid ELISA kit was purchased from Shanghai Sinovac Biotech Co., Ltd. (Shanghai, China). Methylthiazolyl diphenyl-tetrazolium bromide (MTT), dimethyl sulfoxide (cell culture grade), and bovine serum albumin (BSA) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Oleic acid and palmitic acid were purchased from Sigma-Aldrich (St. Louis, USA). Fetal bovine serum (FBS), phosphate-buffered saline (PBS), trypsin, penicillin-streptomycin (PS), and high-glucose Duchenne modified chickpea medium (DMEM) were purchased from Gibco (Rockville, MD, USA). 4',6-Diamidino-2-phenylindole (DAPI) was purchased from Wuhan Saiweier Biotechnology Co., Ltd. (Wuhan, China). DCFH-DA was purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China). BODIPY 493 / 503 was purchased from Glpbio (California, USA).

[0039] Example 1

[0040] A Bacillus subtilis CP-J2440 of the present invention was deposited at the China Center for Type Culture Collection on July 3, 2024, and was found to be viable on July 10, 2024. The accession number is CCTCC NO:M20241459, and the deposit address is Wuhan University, Wuhan, Hubei Province, China.

[0041] The screening method is as follows:

[0042] (1) Cut the dried tangerine peel into small pieces of about 0.5cm×0.5cm, about 5g per bag.

[0043] (2) Prepare four 100ml Erlenmeyer flasks, each containing 100ml of LPDA, MRS, LB and modified Martin medium respectively. Transfer one bag of dried tangerine peel slices into each Erlenmeyer flask.

[0044] (3) Incubate at 37℃ and 150r / min on a shaker for 3 days. Every 24 hours, take 100μL and spread it on PDA, MRS, LB and modified Martin solid medium, and incubate in a constant temperature incubator at 37℃.

[0045] (4) After 24 hours, different colony morphologies were selected from the petri dishes and streaked onto the corresponding new solid culture medium.

[0046] Experiment 1: Examine the morphology of Bacillus CP-J2440 from Example 1.

[0047] Colony size and morphological characteristics were observed, and purity was confirmed using scanning electron microscopy. Finally, PCR sequencing and BLAST analysis were performed to identify closely related species, and a phylogenetic tree was constructed.

[0048] Figure 1 Colony morphology, scanning electron microscopy, and phylogenetic tree results for the isolated strain of Bacillus CP-J2440 (CP01 in the figure is CP-J2440).

[0049] Figure A shows the colony morphology of the isolated strain. As can be seen from the figure, on modified Martin solid medium, Bacillus CP-J2440 has a rough surface, irregular colonies, and a light pink center.

[0050] B in the figure is a scanning electron microscope image, which shows that the colonies have round, rod-shaped bacilli at both ends.

[0051] C in the figure represents the phylogenetic tree. The NCBI BLAST results show that Bacillus CP-J2440 is Bacillus subtilis and belongs to the same phylogenetic branch (NR043242.1) as Bacillus subtilis strain NBRC13719.

[0052] Example 2:

[0053] Application of Bacillus CP-J2440 in the enrichment of flavonoids (Example 1) Figure 2 The flowchart for its application method includes the following steps:

[0054] (1) Crush the dried tea branch mandarin orange peel (Pericarpium Citri Reticulatae, PCR), sieve it through a 60-mesh sieve, and then store it in a drying oven for later use.

[0055] (2) A single colony of the isolated and purified Bacillus CP-J2440 strain from Example 1 was inoculated into modified Martin liquid medium and cultured at 37°C for 5 h (OD = 0.5) to obtain the inoculum.

[0056] (3) Add the PCR powder from step (1) to water at a ratio of 1:20 (w / v). Pour the mixture into a 250 mL conical flask, seal the flask with a film, and sterilize at 121 °C for 20 min to obtain sterilized, unfermented tea branch and citrus peel extract (PCRI).

[0057] (4) After cooling the PCRI to 20°C, add 8% (v / v) of inoculum to the sterile PCRI and ferment at 37°C with stirring for 120g for 4 days. Collect the fermentation broth every 24 hours, centrifuge the collected fermentation supernatant at 12,000g, and then store at -80°C. Use unfermented PCRI as a control.

[0058] Experiment 2: Investigating the chemical composition of the fermentation broth.

[0059] The polyphenol content in PCRI was determined using the Folin-Ciocalteu colorimetric method with gallic acid as a standard. The total flavonoid content was determined using the aluminum chloride-sodium nitrite colorimetric method, with rutin as the quantitative standard. The soluble sugar content was analyzed using the phenol-sulfuric acid method, with glucose as the quantitative standard. Soluble protein content and free amino acids were assessed according to the protocols outlined in their respective kit manuals.

[0060] Figure 3 The results show the differences in chemical composition of PCRI fermented by Bacillus CP-J2440. In the figure, A represents the change in total flavonoid content, and B represents the change in total polyphenol content.

[0061] As can be seen from the figure, the total flavonoid and total polyphenol contents of PCRI fermented by Bacillus CP-J2440 first increased and then decreased with fermentation time.

[0062] Flavonoids are the most abundant polyphenolic compounds in citrus fruits, including flavones, flavonols, flavanones, isoflavones, anthocyanins, and flavanols. The flavonoid content of Bacillus CP-J2440 increased significantly in the early stages of fermentation, then gradually decreased. On the third day of fermentation, the PCRI content of Bacillus CP-J2440 was the highest (1329.13±27.59 μg RE / mL), and compared with the pre-fermentation level, the total flavonoid content of PCRI after fermentation was significantly increased by 38.36% compared with the unfermented PCRI. The increase in flavonoid content may be due to cellulase and pectinase secreted by Bacillus, which can disrupt the cell wall structure of citrus peel and promote the release of active ingredients from plant cells. Furthermore, these enzymes break the covalent bonds between flavonoids and cell wall components, thereby releasing the bound active ingredients into the plant matrix. Flavonoid glycosides are more water-soluble than aglycones. The reason why the flavonoid content decreases at the end of fermentation may be that Bacillus secretes β-glucosidase, which can hydrolyze the β-glucosidic bond between aglycone and sugar molecules, thereby increasing the aglycone content and reducing the content of soluble flavonoids in the aqueous phase.

[0063] The total polyphenol content of PCRI fermented by Bacillus subtilis CP-J2440 initially increased and then decreased with fermentation time, reaching its highest level (3406.78 ± 24.91 μg RE / mL) on the third day of fermentation, which was 142.524 μg RE / mL higher than that of unfermented sterile tangerine peel infusion (CPI). Notably, the total phenol content of Bacillus subtilis CP-J2440 was 4.45% higher than that of CPI on the third day. The increase in polyphenol content may be due to the activity of β-glucosidase in Bacillus subtilis, which can hydrolyze bound polyphenols into free polyphenols. The decrease in total phenol content from the third to the fourth day may be due to extracellular enzymes secreted by Bacillus, such as lyases, polyphenol oxidases, and peroxidases, which promote the oxidation, decomposition, and transformation of phenolic compounds. In addition, the use of polyphenols as an energy source by Bacillus mycelium during growth may reduce the total phenol content in PCRI. In summary, the findings regarding total phenol content highlight the important role of Bacillus in regulating polyphenol content in PCRI.

[0064] Experiment 3: Investigating the in vitro antioxidant activity of PCRI

[0065] The antioxidant capacity of PCRI was assessed using in vitro antioxidant assays, including DPPH radical scavenging, ABTS radical scavenging, and FRAP assays, following the guidelines provided by the manufacturer (Kemin Biotechnology Co., Ltd., Suzhou, Jiangsu Province, China). Known antioxidants such as Trolox were used as reference standards, and results are expressed as μmol Trolox equivalents per ml (TE μmol / mL).

[0066] Figure 4 The figure shows the differences in antioxidant activity of PCRI fermented by Bacillus CP-J2440; A in the figure represents the difference in ABTS activity; B in the figure represents the difference in FRAP activity; and C in the figure represents the difference in DPPH activity.

[0067] As shown in the figure, fermented foods associated with Bacillus exhibit higher antioxidant activity compared to non-fermented foods. This experiment used DPPH, ABTS·+, and FRAP methods to evaluate the antioxidant activity of PCRI. Fermentation with Bacillus CP-J2440 significantly affected the antioxidant capacity of PCRI, with the most pronounced effect observed after three days of fermentation. Specifically, compared to the unfermented control group, the free radical scavenging capacity of DPPH, ABTS·+, and FRAP increased by 62.84%, 19.27%, and 42.88%, respectively. Generally, there was a close relationship between total phenolic and total flavonoid content and antioxidant activity. The trend in antioxidant activity with increasing fermentation days was consistent with the changes in total phenolic and total flavonoid content. In conclusion, these results indicate that Bacillus CP-J2440-driven fermentation can significantly enhance the in vitro antioxidant bioactivity of PCRI.

[0068] Experiment 4: Investigate the changes in chemical composition, acidity, and color during the fermentation of Bacillus CP-J2440 in PCRI.

[0069] 4.1 Changes in chemical composition.

[0070] Based on the above experimental results, Bacillus CP-J2440 exhibited a strong ability to accumulate flavonoids in PCRI. Therefore, this study investigated in detail the dynamic changes in soluble protein content (SPC), water-soluble sugar content (SSC), and total free amino acid content (FAAC) during the fermentation of Bacillus CP-J2440.

[0071] Figure 5The figures show the dynamic changes in soluble protein content (SPC), water-soluble sugar content (SSC), and total free amino acid content (FAAC). In the figure, A represents free amino acid content, B represents free polysaccharide content, and C represents free protein content. As shown, SPC and SSC initially decreased and then increased during fermentation, while FAAC continuously decreased. During the first three days, Bacillus CP-J2440 produced proteases to meet its growth and metabolic needs, hydrolyzing soluble proteins into peptides and amino acids, leading to a decrease in SPC. Subsequently, by the fourth day, SPC increased significantly, likely due to Bacillus CP-J2440 breaking covalent bonds in biopolymers (such as insoluble proteins and polysaccharides), converting them into water-soluble small protein molecules. SSC decreased significantly on the first day of fermentation, possibly due to the rapid proliferation of Bacillus consuming water-soluble sugars in the PCRI. The subsequent increase in SSC is likely due to the release of cellulase, pectinase, and other hydrolytic enzymes by Bacillus, thereby degrading cellulose and pectin in the citrus peel. The reason for the continuous decrease in FAAC throughout the fermentation process may be that Bacillus absorbs free amino acids from PCRI to support its growth, reproduction and metabolic functions, which leads to the oxidation, degradation and transformation of these amino acids.

[0072] 4.2 Changes in pH:

[0073] Figure 6 The pH change results show that the pH value of fermented PCRI decreased by 4.1 ± 0.01 compared to unfermented PCRI. Specifically, the lowest pH value of PCRI was observed on day four of fermentation, at 3.28 ± 0.02. Previous studies have shown that Bacillus can ferment xylose into lactic acid via the phosphatase pathway, thus explaining the observed pH decrease during fermentation.

[0074] 4.3 Color Changes:

[0075] Color is a key sensory attribute in food, playing a decisive role in product marketing. Total color difference (ΔE) and chromaticity parameters (L*, a*, and b*) were used to evaluate how Bacillus CP-J2440 fermentation affects the color characteristics of PCRI. The evaluation results are shown in Table 1.

[0076] Table 1: Changes in color properties of PCRI after fermentation with Bacillus CP-J2440

[0077] L* a* b* DE*(ΔE) DC* DH* CP 24.72±0.04e 3.32±0.09e 3.73±0.01f 64.28±0.04a 4.07±0.08e -5.98±0.09a 0d 27.76±0.03a 7.06±0.05a 7.35±0.02a 62.06±0.04d 12.48±0.06a -8.45±0.04c 1d 27.33±0.15b 6.48±0.33b 6.92±0.21b 62.3±0.05c 11.37±0.58b -8.11±0.20b 2d 26.89±0.13c 6.14±0.14bc 6.46±0.06c 62.62±0.13b 10.42±0.15c -7.95±0.09b 3d 25.81±0.02d 4.28±0.13d 4.82±0.09e 63.22±0.04a 6.43±0.04d -6.70±0.13a 4d 26.84±0.03c 6.01±0.04c 6.22±0.09d 62.59±0.05b 9.93±0.13c -7.88±0.05b

[0078] In Table 1, different letters indicate significant differences between different samples (P<0.05). CP: Unfermented tangerine peel extract (CPI); 0d: Unfermented RCPI; 1d: Unfermented RCPI; 1d: PCRI fermented for 1 day; 2d: PCRI fermented for 2 days; 3d: PCRI fermented for 3 days; 4d: PCRI fermented for 4 days.

[0079] Figure 7 Photos and trend charts showing color changes. From Figure 7 As shown in Table 1, fermentation with Bacillus CP-J2440 significantly reduced ΔE, L*, a*, and b* values ​​compared to unfermented PCRI, especially on the third day. After three days of fermentation, the ΔE and color of PCRI were close to the values ​​observed in CPI. These results indicate that Bacillus fermentation darkens the color of PCRI, consistent with findings from studies on Bacillus fermentation in wine. Previous studies have highlighted the significant influence of flavonoids on the color and brightness of tea, suggesting that color changes during PCRI fermentation may be related to changes in flavonoid composition.

[0080] Experiment 4: Analysis of volatile compounds.

[0081] 1 mL of sample was transferred to a headspace vial, and saturated NaCl solution and 20 μL of 10 μg / mL internal standard solution were added. The sample was then subjected to automated headspace solid-phase microextraction (HS-SPME) for subsequent gas chromatography-mass spectrometry (GC-MS) analysis (Agilent 8890-7000D, Agilent, Santa Clara, CA, USA). The extraction process was performed with shaking at 60 °C for 5 min. A 120 μm DVB / CWR / PDMS extraction fiber (Agilent, Santa Clara, CA, USA) was inserted into the vial for headspace extraction for 15 min. GC-MS analysis was then performed at 250 °C for 5 min. Before sampling, the extraction fiber was conditioned at 250 °C for 5 min using a fiber conditioning station. Separation and identification were performed using a DB-5MS capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA) with high-purity helium as the carrier gas at a flow rate of 1.2 mL / min. The injection port temperature was set to 250 °C, with no splitting injection and a solvent delay of 3.5 min. The temperature program was as follows: initially held at 40 °C for 3.5 min, then increased at 10 °C / min to 100 °C, then at 7 °C / min to 180 °C, and finally at 25 °C / min to 280 °C for 5 min. Electron ionization (EI) was used as the ionization source at a source temperature of 230 °C, a quadrupole temperature of 150 °C, an interface temperature of 280 °C, and an electron energy of 70 eV, operating in the selected ion monitoring (SIM) scan mode.

[0082] The evaluation of high-quality health products has evolved from nutritional value to sensory flavor. This experiment identified 1388 volatile metabolites (VMs). Principal component analysis (PCA) was used to analyze the differences in volatile metabolites produced by different stages of PCRI fermentation. Figure 8 This figure compares the volatile components during the fermentation of Bacillus CP-J2440 in PCRI. Figure A shows the PCA scores of the mass spectrometry data for each sample group. Figure B shows the classification of volatile substances. The figures show that PC1 and PC2 account for 34.44% and 21.59% of the total variation, respectively, totaling 56.03%. There are significant differences in flavor characteristics between non-fermented and fermented PCRI. However, this difference gradually decreases with prolonged fermentation time, possibly due to the significant decrease in pH from day 0 to day 1 of fermentation, highlighting the important role of environmental factors in the metabolism of flavor compounds.

[0083] Experiment 5: Analysis of non-volatile metabolites during the fermentation of Bacillus CP-J2440 in PCRI.

[0084] The PCR I sample was freeze-dried under vacuum for 63 h, then ground into powder using a grinder at 30 Hz for 1.5 min. 50 mg of the powder sample was then weighed and added to 1.2 ml of pre-cooled 70% methanol-water extraction buffer. The internal standard solution was prepared by dissolving 1 mg of the standard in 1 ml of 70% methanol-water to a stock solution of 1000 μg / ml, which was then further diluted to 250 μg / ml. The sample was vortexed for 30 s every 30 min, repeated 6 times. Finally, the sample was centrifuged at 12000 rpm for 3 min, the supernatant was discarded, and the sample was filtered through a 0.22 μm microfiltration membrane and stored in a sample vial for UPLC-MS / MS analysis. An ACQUITY UPLC HSS T3 column (2.1 mm * 100 mm, 1.8 μm, Waters, Milford, MA, USA) was used in the experiment. Mobile phase A was a 0.1% formic acid aqueous solution, and mobile phase B was a 0.1% formic acid-acetonitrile aqueous solution. The column temperature was maintained at 40℃, the injection volume was 4 μL, and the flow rate was 0.4 mL / min. Mass spectrometry analysis was performed using electrospray ionization. The chromatographic gradient conditions and mass spectrometry parameters are detailed in Tables 2 and 3.

[0085] Table 2: Chromatographic gradient conditions for non-targeted metabolomics

[0086] Time (min) Mobile phase A (%) Mobile phase B (%) 0.0 95 5 2.0 80 20 5.0 40 60 6.0 1 99 7.5 1 99 7.6 95 5 10.0 95 5

[0087] Table 3: Mass Spectrometry Parameters for Non-Targeted Metabolomics

[0088] ESI+ ESI- Duration (min) 10 10 IonSpray Voltage (V) 5000 -4000 Temperature (°C) 550 450 Ion Source Gas 1 (psi) 50 50 Ion Source Gas 2 (psi) 60 60 Curtain Gas (psi) 35 35 Declustering Potential (V) 60 -60 MS1 Collision Energy (V) 10 -10 MS2 Collision Energy (V) 30 -30 Collision Energy Spread (V) 15 15

[0089] Figure 9 This figure shows the analysis of non-volatile metabolites during the fermentation of Bacillus CP-J2440 in PCRI. Figure A shows the PCA score of all metabolites in ESI+ mode; Figure B shows the PCA score of all metabolites in ESI- mode.

[0090] As shown in the figure, untargeted metabolomics was used to study the changes in metabolite profiles during PCRI. A total of 4041 metabolites were identified, including 2217 in the positive ion mode and 1824 in the negative ion mode. Principal component analysis (PCA) was performed to assess the differences in metabolite composition before and after fermentation. In ESI+ mode, PC1 and PC2 explained 39.10% and 24.30% of the total variance, respectively, while in ESI- mode, PC1 and PC2 accounted for 39.70% and 24.20% of the total variance, respectively. In both modes, there were significant differences in PC1 between samples before and after fermentation, but this difference decreased with prolonged fermentation time, which is consistent with the observations of flavor compounds.

[0091] Figure 10The figure shows the classification and analysis of differential metabolites; A in the figure represents the classification of differential metabolites, B in the figure represents the cluster analysis of the top 20 differential metabolites, and the bubble chart, annotations, and enrichment plot of the top 10 KEGG pathways of differential metabolites.

[0092] Identifying differentially expressed metabolites in untargeted metabolomics is a significant challenge. To further investigate how fermentation affects nonvolatile PCRI metabolites, we employed stringent screening criteria (|logFC|>1.0 and p<0.05) to analyze differences between different fermentation stages and day 0. Figure A lists a total of 1176 differential metabolites, divided into 19 major categories: alcohols and amines (2.13%), alkaloids (6.63%), amino acids and their derivatives (49.06%), benzene and its substituted derivatives (7.39%), fatty acyls (1.02%), flavonoids (1.62%), glycerides (0.17%), glycerophospholipids (2.81%), heterocyclic compounds (3.66%), lignans and coumarins (1.11%), lipids (2.13%), nucleotides and their derivatives (3.15%), organic acids (7.14%), others (8.50%), phenolic acids (2.55%), quinones (0.09%), sphingolipids (0.09%), steroids (0.77%), and triterpenoids (1.96%).

[0093] Figure B shows the top 20 metabolic pathways associated with different metabolites. Fermentation affects purine metabolism, biosynthesis of various alkaloids, biosynthesis of isoquinoline alkaloids, amino sugar and nucleotide sugar metabolism, nucleotide metabolism, zeatin biosynthesis, linoleic acid metabolism, and isoflavone biosynthesis. Purine nucleotides are essential molecules for DNA and RNA synthesis, energy metabolism, and signal transduction in organisms. The growth of Bacillus CP-J2440 requires a continuous supply of purine nucleotides. Therefore, purine metabolism is the most prominent differential metabolic pathway in fermented PCRI. Furthermore, isoflavone biosynthesis is an important enrichment pathway among the differential metabolites of fermented PCRI, further confirming the key role of Bacillus CP-J2440 in the metabolism and transformation of isoflavones during PCRI fermentation. In summary, this study highlights the significant impact of Bacillus CP-J2440 on PCRI metabolites, providing valuable insights into the role of bacteria in shaping the PCRI metabolite profile.

[0094] Experiment 6: Targeted quantitative analysis of flavonoids.

[0095] To investigate the transformation of flavonoids during the fermentation of Bacillus CP-J2440, researchers extensively employed LC-MS / MS for targeted quantitative analysis. To further determine the changes in flavonoid content during PCRI fermentation, a directional quantitative method for flavonoids was used. First, PCRI was freeze-dried under vacuum for 63 hours, then ground into powder at 30 Hz for 1.5 min. Subsequently, 20 mg of powder was weighed and mixed with 10 μL of a 4000 nmol / L internal standard working solution and 500 μL of 70% methanol solution. The mixture was sonicated at 4 °C for 30 min, then centrifuged at 12000 g for 5 min, and the supernatant was separated. The supernatant was filtered through a 0.22 μm membrane filter and stored in sample vials for subsequent LC-MS / MS analysis. An ACQUITY UPLC HSS T3 column (2.1 mm * 100 mm, 1.8 μm, Waters, Milford, MA, USA) was used in the experiment. Mobile phase A consisted of ultrapure water containing 0.05% formic acid, and mobile phase B consisted of acetonitrile containing 0.05% formic acid. The column temperature was maintained at 40℃, the injection volume was 2 μL, and the flow rate was 0.35 mL / min. Electrospray ionization (ESI) was used for mass spectrometry analysis. Quantitative evaluation of various flavonoid subclasses was performed, and the results are listed in Table 4.

[0096] Table 4: Results of changes in flavonoids during Bacillus CP-J2440 fermentation.

[0097]

[0098]

[0099] Different letters indicate significant differences between different samples (P<0.05). CP: Unfermented tangerine peel extract; 0d: Unfermented RCPI; 1d: Unfermented RCPI; 1d: PCRI fermented for 1 day; 2d: PCRI fermented for 2 days; 3d: PCRI fermented for 3 days; 4d: PCRI fermented for 4 days.

[0100] Table 4 provides a quantitative assessment of various flavonoid subclasses: Phlorizin, Trilobatin, Naringenin chalcone, (-)-Catechin, (-)-Catechin gallate, Hesperidin, Naringenin-7-glucoside, 7,4'-di-O-methylpersicogenin, Nariruti, Hesperetin, Isosakuranin, Eriocitrin, Isosakuranetin, Pinocembrin, Eriodictyol, Taxifolin, and Taxifolin 7-O-rhamnoside. 7-O-rhamnoside, dihydromyricetin, vitexin, spinosin, pudumetin (genkwanin), narcissin, 7,4'-di-O-methylapigenin, and apigenin 7-glucoside 7-glucoside, Diosmin, Homoplantaginin, Scutellarin, Diosmetin, Eupatorin, Chrysin, Apigenin, Acacetin, 5-O-Demethylnobiletin, Baicalin, Trimethylapigenin, Tangeretin, Wogonoside, 5,7,3',4'-Tetramethoxyflavone, Sinensetin, Jaceosidin, Nobiletin, Scutellarein tetramethyl ether), luteolin, isorhamnetin, quercetin, typhaneoside, 3,The fermentation process yielded the following compounds: 3,7-di-O-methylquercetin, hyperoside, isorhamnetin-3-O-neohespeidoside, tiliroside, quercetin, kaempferol 3-neohespeidoside, and kaempferitrin. Among these, 24 were flavonoids, 12 were flavonols, 10 were dihydroflavonoids, 5 were isoflavones, 3 were chalcones, 3 were dihydrochalcones, and 2 were flavanols. Notably, the content of flavonoid glycosides decreased significantly throughout the fermentation process. Specific compounds, such as naringin-7-glucoside, naringin, isonaringin, ergoside, taxane-7-O-rhamnoside, vitexin, narcissin, apigenin-7-glucoside, diosgenin, isopyrrolidone, tyroside, hyperoside, isorhamnoside-3-O-mannoside, isorhamnoside-3-O-mannoside, isorhamnoside-3-O-neohesperidin, kaempferol, isorhamnoside-3-O-glucoside, rutin, trifolin, sennain, and chlorophyll, were detected, with quercetin only detected at day 0 and becoming undetectable at the end of fermentation. Conversely, flavonoid acetal derivatives formed through hydrolysis and other reactions, such as isopyroxin, 7,4'-di-O-methylapigenin, genistein, trimethylapigenin, 5,7,3',4'-tetramethoxyflavonoids, baicalin tetramethyl ether, geraniol, and genistein, showed a significant increase, especially genistein, which was not detected until the fourth day. These findings suggest that the changes in flavonoid concentration and composition observed during fermentation may be caused by a biotransformation process mediated by Bacillus CP-J2440.

[0101] Flavonoids are the most abundant polyphenols in fruits, vegetables, and plants, primarily existing in glycosylated forms. However, their biological activity, especially their antioxidant properties, is usually inhibited in glycosides, requiring hydrolysis to aglycones to exert stronger physiological effects. Bacillus possesses a variety of enzymes, including β-glucosidase, β-cyclodextrin glycosyltransferase, O-methyltransferase, flavonol synthase, CYP450 BM3, flavonoid 3-hydroxylase, and flavonoid-3'-monooxidase. These microbial enzymes catalyze a series of reactions, such as hydrolysis, methoxylation, demethylation, hydroxymethylation, reduction, and demethylation. For example, under the action of O-methyltransferase, apigenin can undergo methylation to form trimethylapigenin, baicalin tetramethyl ether, or scrophularia glycoside. Notably, chrysanthin was only detectable after 4 days of fermentation, while lutein content decreased significantly after fermentation, possibly due to leucocyanidin reductase in Bacillus converting lutein to chrysanthin. Similarly, the increased concentration of (-)-catechin gallate observed on the fourth day of fermentation is likely due to the metabolic transformation of (-)-catechin. These results indicate that the fluctuations in flavonoid content in PCRI are mainly a result of Bacillus CP-J2440 fermentation, which promotes both the hydrolysis of flavonoid glycosides and the interconversion and accumulation of flavonoid glycosides.

[0102] During the fermentation of PCRI by Bacillus CP-J2440, total phenols and flavonoids increased significantly, especially on the third day of fermentation. Soluble sugars and proteins initially decreased and then increased, while free amino acids continued to decrease. After the third day of fermentation by Bacillus CP-J2440, the antioxidant activity of PCRI was significantly enhanced, with DPPH, ABTS·+, and FRAP free radical scavenging abilities increasing by 62.84%, 19.27%, and 42.88%, respectively. Furthermore, in vitro experiments showed enhanced anti-lipidemia effects of fermented PCRI. Fermentation by Bacillus CP-J2440 also significantly altered the flavor characteristics and metabolite composition of PCRI, resulting in a unique aroma reminiscent of fermented and ripe fruit with herbal notes. In addition, targeted quantification of flavonoids elucidated the interconversion of flavonoid glycosides and aglycones during fermentation, as well as the conversion between different aglycones. Future research will integrate genomics, proteomics, and metabolomics to elucidate the specific pathways of flavonoid metabolism and enzymatic catalysis, which is crucial for promoting the development and application of tangerine peel probiotics.

[0103] Experiment 7: Investigate the effect of Bacillus CP-J2440 fermentation on the anti-lipid activity of PCRI.

[0104] 7.1. Investigating the effects of PCRI on TC and TG: Treatment with trans fatty acids induces hepatic steatosis characterized by intracellular lipid accumulation. Figure 11The figures show the effects of PCRI on TC and TG. Figure A represents the effect of PCRI on TC, and Figure B represents the effect of PCRI on TG. As can be seen from the figures, compared with the control group, the TG and TC contents in HepG2 cells increased by 229.98% and 236.99%, respectively. However, intervention with PCRI significantly reduced TG content, with fermented PCRI showing a stronger effect. Specifically, after three days of fermentation, compared with the FFA group, PCRI reduced TC and TG levels by 31.57% and 60.36%, respectively, and compared with unfermented PCRI, reduced TC and TG levels by 19.38% and 52.46%, respectively, with effects comparable to CPI.

[0105] 7.2. Investigate the effect of PCRI on intracellular lipid accumulation in HepG2 cells:

[0106] Figure 12 The figure shows the effect of PCRI on intracellular lipid accumulation in HepG2 cells. The CON group represents untreated HepG2 cells that do not show lipid droplets, while the FFAs group represents cells treated with FFAs that exhibit morphological distortion and severe lipid accumulation, appearing as merged red lipid droplets. PCRI treatment reduces the number and size of intracellular lipid droplets, especially fermented PCRI, which shows smaller and more dispersed lipid droplets in HepG2 cells.

[0107] BODIPY493 / 503 is a lipophilic fluorescent dye that is used to label neutral lipids in live and fixed cells because of its superior specificity and consistency with lipid fluorescence compared to PCRI dyes. Figure 13 Images stained with BODIPY493 / 503 show that HepG2 cells treated with fermented PCRI exhibited significantly weaker fluorescence intensity compared to the model group and unfermented PCRI, indicating a marked reduction in intracellular lipid droplets. These findings highlight the potent lipid-lowering effect of fermented PCRI. Furthermore, evidence suggests that flavonoids in citrus fruits can effectively regulate lipid metabolism. Previous studies have shown that rutin can induce AMPK phosphorylation in oleic acid-treated HepG2 cells, activating PPARα to promote lipolysis while inhibiting SREBP-1 activation, thereby downregulating lipid synthesis-related genes such as FAS and ACC.

[0108] This study aimed to significantly enhance the nutritional value and bioactivity of plant-based foods. The effects of fermentation on PCRI were investigated in an in vitro lipid-lowering experiment. The MTT assay was used to assess the viability of HepG2 cells in order to optimize efficacy while maintaining cell viability. At all tested concentrations, PCRI did not affect the viability of HepG2 cells; therefore, a concentration of 0.64 mg / mL PCRI was selected for subsequent experiments.

[0109] 7.3 Monitoring cellular responses to oxidative stress in a mixed acid-rich environment using the DCFH-DA probe: Under normal physiological conditions, the human body maintains a balance between oxidation and antioxidation. However, disruption of this balance induces oxidative stress, leading to excessive production of free radicals and non-free radicals, resulting in cell damage. DCFH-DA is a non-fluorescent reactive probe that easily penetrates the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to form DCFH, which is non-fluorescent and therefore accumulates intracellularly. Intracellular reactive oxygen species (ROS) oxidize the non-fluorescent DCFH, generating fluorescent DCF; the intensity of the green fluorescence is directly related to the ROS level. Figure 14 To monitor cellular responses to oxidative stress in a mixed acid-rich environment using the DCFH-DA probe, the results showed a significant increase in fluorescence signal in HepG2 cells treated with fatty acids compared to the control group. In contrast, the fluorescence intensity of the PCRI-treated group decreased significantly, especially after three days of fermentation. Generally, increased oxidative stress in vivo is associated with aggravated abnormal lipid accumulation and significant changes in new lipid synthesis, suggesting that reducing oleic acid-induced oxidative stress may be one of the mechanisms by which PCRI exerts its lipid-lowering effect. Fermented PCRI has a significant protective effect against oxidative stress, which may be attributed to its high content of polyphenols and flavonoids that help maintain cellular redox balance. The unique structure of citrus flavonoids exhibits excellent antioxidant and chelating properties. Previous studies have shown that citrus flavonoids can effectively regulate oxidative stress induced by various substances.

[0110] Experiment 8: Investigate the relationship between the chemical composition of PCRI and its color and biological activity after 3 days.

[0111] To further observe the relationship between the chemical composition, color, and biological activity of PCRI after 3 days of fermentation, further analysis was conducted. The effect of PCRI fermented with Bacillus CP-J2440 on oxidative stress in HeppG2 cells was also investigated.

[0112] Figure 15 Spearman correlation heatmap showing the relationship between the chemical composition of PCRI and acidity, color, and bioactivity. Different letters indicate significant differences between different samples (P<0.05). CP: Unfermented tangerine peel infusion; FFAs: 200 μM oleic acid and 100 μM palmitic acid; 0d: Unfermented PCRI; 1d: PCRI fermented for 1 day; 2d: PCRI fermented for 2 days; 3d: PCRI fermented for 3 days; 4d: PCRI fermented for 4 days.

[0113] Blue indicates negative effects. The graph clearly shows that the increase in the antioxidant activity, antilipidemic activity, color properties, total phenols, and flavonoids of PCRI is significantly positively correlated with the noticeable changes in certain flavonoids after fermentation, such as hesperidin, isochoria flavonoids, echinacea flavonoids, 7,4'-di-O-methylapigenin, trimethylapigenin, flavonoid glycosides, and baicalin tetramethyl ether. In summary, driven by Bacillus CP-J2440, the bioactive substances of PCRI accumulate significantly, and its bioactivity is significantly enhanced.

[0114] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solutions of the present invention shall still fall within the protection scope of the technical solutions of the present invention.

Claims

1. A flavonoid-rich Bacillus tangerine peel-derived species, characterized in that, The Bacillus subtilis strain from which the tangerine peel is derived is Bacillus subtilis CP-J2440, which was deposited at the China Center for Type Culture Collection on July 3, 2024, with accession number CCTCC NO:M 20241459.

2. The application of the Bacillus tangerine peel source as described in claim 1 in the preparation of functional foods with anti-lipidemia properties.

3. The application of the Bacillus tangerine peel source as described in claim 1 in the preparation of antioxidant functional foods.

4. The application of the Bacillus tangerine peel source as described in claim 1 in the preparation of probiotic functional foods.

5. The application according to any one of claims 2 to 4, characterized in that, The application method includes: S1. Fermentation broth was obtained by fermenting Bacillus CP-J2440 strain; S2. The dried tangerine peel of the tea branch mandarin orange is crushed and extracted with ultrapure water to obtain a tea branch mandarin orange peel extract. S3. After sterilizing the tea branch and citrus peel extract, inoculate it with the fermentation broth and ferment at 37°C for 4 days. Collect the fermentation supernatant, centrifuge at 12,000g, and then store it at -80°C.

6. The application according to claim 5, characterized in that, In S1, the inoculation amount of the Bacillus CP-J2440 strain is 8 v / v.

7. The application according to claim 5, characterized in that, In S1, the fermentation culture specifically refers to: culturing at 37°C for 5 hours.

8. The application according to claim 5, characterized in that, S2 specifically involves: pulverizing the dried citrus peel of the tea branch mandarin orange, passing it through a 60-mesh sieve, adding water at a solid-liquid ratio of 1:20, and sterilizing at 121°C for 20 minutes to obtain a tea branch mandarin orange peel extract.

9. The application according to claim 5, characterized in that, S3 specifically involves: sterilizing the tea branch and citrus peel extract and cooling it to 20°C, adding 8 v / v% fermentation broth, and fermenting it at 37°C with a stirring speed of 120g for 4 days; collecting the fermentation broth every 24 hours, centrifuging the collected fermentation supernatant at 12000g, and then storing it at -80°C.

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