Method for improving functional activity of annona squamosa juice fermentation liquid

Abstract

This invention discloses a method for improving the functional activity of custard apple juice fermentation broth, which involves using Bacillus subtilis (… Bacillus subtilis ) or Lactobacillus reuteri ( Lactobacillus reuteri This invention involves fermenting custard apple juice to enhance its antioxidant, anti-inflammatory, uric acid-lowering, and antibacterial bioactivities. The fermented juice effectively exerts its antioxidant, anti-inflammatory, uric acid-lowering, and pathogen-inhibiting effects, providing a scientific basis for its application in the adjunctive treatment of metabolic syndrome. Furthermore, its inhibitory effect on various pathogens further expands the product's comprehensive application potential in functional foods. The preparation method of this invention is simple, controllable, and low-cost, demonstrating promising prospects for industrial application in the fields of functional and health foods.

Description

Technical Field

[0001] This invention belongs to the field of food biotechnology and microbial fermentation engineering, specifically relating to a method utilizing Bacillus subtilis (… Bacillus subtilis ) or Lactobacillus reuteri ( Lactobacillus reuteri Methods to improve the functional activity of custard apple fermentation broth. Background Technology

[0002] In recent years, with the increasing health awareness of residents, functional beverages with nutritional regulation and health benefits have received widespread attention. Fruit and vegetable juices, as an important dietary supplement, are rich in various vitamins, polyphenols, and minerals, playing a vital role in antioxidation, metabolism regulation, and immune enhancement. At the same time, the problem of foodborne pathogen contamination is receiving increasing attention, and the development of natural fermented fruit and vegetable products that combine nutritional and health benefits with antibacterial functions has become a research hotspot in the food processing field.

[0003] Custard apple (Annona squamosa L.) is a specialty fruit widely cultivated in tropical and subtropical regions. Rich in sugars, organic acids, vitamins, polyphenols, and various bioactive substances, it possesses high nutritional value and development potential. However, custard apples are prone to spoilage after ripening, exhibit poor processing stability, and traditional juice processing methods easily lead to the loss of nutrient-active substances, limiting its deep processing and high-value-added utilization. Furthermore, custard apple juice is susceptible to contamination by pathogenic microorganisms during storage and distribution, further hindering its industrial development.

[0004] Microbial fermentation technology can improve the flavor structure of fruit juice, increase the content of active ingredients and bioavailability through microbial metabolism, and has been widely used in the field of deep processing of fruits and vegetables in recent years.

[0005] Currently, there are few reports on the synergistic effects of various functional strains on custard apple juice, including its ability to improve nutritional quality, physicochemical properties, antioxidant and anti-inflammatory effects, uric acid reduction, and inhibition of pathogens. Summary of the Invention

[0006] This invention provides a method for improving the functional activity of custard apple fermentation broth, the method being the use of Bacillus subtilis (… Bacillus subtilis ) or Lactobacillus reuteri ( Lactobacillus reuteri Fermenting custard apple juice enhances its antioxidant, anti-inflammatory, uric acid-lowering, and antibacterial bioactivity, thereby comprehensively improving its functional properties and nutritional quality.

[0007] The custard apples were purchased from the Kunming market in China; selected fruits were free from mechanical damage and pests, and were fresh and ripe before the experiment. The bacterial strain used in this invention was purchased from the China Center for Type Culture Collection (CCTCC): Bacillus subtilis (… Bacillus subtilis The strain preservation number is CCTCC AB 2025223; Lactobacillus reuteri ( Lactobacillus reuteri The strain preservation number is CCTCC AB 2026004.

[0008] This invention utilizes Bacillus subtilis or Lactobacillus reuteri to treat custard apple ( Annona squamosa Fermenting L. fruit juice yields fermented fruit juice with the following significant advantages: (1) Significantly improves in vitro antioxidant capacity, enhances the scavenging effect on various free radicals, and effectively reduces oxidative stress damage; (2) It exhibits good anti-inflammatory activity and helps regulate the body's inflammatory response; (3) It has the effect of lowering uric acid and can play a positive role in regulating uric acid metabolism; (4) It exhibits significant inhibitory effects on common pathogens and has good broad-spectrum antibacterial properties; (5) Effectively improves the nutritional quality of fermentation broth and promotes the release and biotransformation of active ingredients such as polyphenols and free amino acids.

[0009] Furthermore, the preparation method of this invention is simple, the process is stable, the operation is controllable, and the cost is low, showing good prospects for industrial application in the field of functional foods and health foods. Attached Figure Description

[0010] Figure 1 The results of the hemolysis test were used to evaluate the safety of the strain. Figure 2 The pH value of the fermentation broth for fermenting custard apple juice using functional bacteria was measured. Figure 3 The results of Brix content determination in the fermentation broth of custard apple juice fermented with functional bacteria; Figure 4 Results of total phenol content detection in fermentation broth of custard apple juice fermented with functional bacteria; Figure 5 The results of amino acid content detection in the fermentation broth of custard apple juice fermented with functional bacteria; Figure 6 The results of the total antioxidant capacity test in the fermentation broth of custard apple juice fermented with functional bacteria; Figure 7 The results of hydroxyl radical scavenging rate detection in the fermentation broth of custard apple juice fermented with functional bacteria; Figure 8 To utilize the fermentation broth of custard apple juice fermented with functional bacteria for the treatment of ABTS + Results of free radical scavenging ability; Figure 9The results of the detection of the DPPH free radical scavenging ability of fermentation broth from custard apple juice fermented with functional bacteria were presented. Figure 10 The results of the inhibition rate of pancreatic lipase by fermentation broth of custard apple juice fermented with functional bacteria; Figure 11 The results of the inhibition rate of xanthine oxidase by fermentation broth of custard apple juice fermented with functional bacteria; Figure 12 To utilize the fermentation broth of custard apple juice fermented with functional bacteria to counteract protein denaturation rate results; Figure 13 To utilize the fermentation broth of custard apple juice fermented with functional bacteria to treat Fe 2+ Chelation rate results; Figure 14 The results of the taste determination in the fermentation broth of custard apple juice fermented with functional bacteria; Detailed Implementation

[0011] The methods described in this invention are further described below through examples, but the scope of protection of this invention is not limited by the examples. Unless otherwise specified, the reagents used in this embodiment are all commercially available reagents or reagents prepared by conventional methods, and the methods used are all conventional methods unless otherwise specified. Example 1: Preparation of strain seed liquid 1. Take out the strain stored at -80℃, spread it evenly on the surface of MRS solid medium with a sterile spreader, incubate in a constant temperature incubator at 37℃ for 24h, and then inoculate it into MRS broth medium and shake it at 37℃ for 24h. 2. After centrifuging the liquid bacterial culture and discarding the supernatant, rinse with sterile water, centrifuge again, and discard the supernatant. Repeat twice. Add sterile water, measure the absorbance under a UV spectrophotometer, and adjust the concentration to the OD value. 600 =1, used as seed solution.

[0012] Example 2: Preparation of Custard Apple Juice Select ripe and plump custard apples, rinse the surface of the fruit with running tap water to remove dust and impurities, remove the peel and seeds by hand, collect the pulp, mix the pulp with distilled water at a ratio of 3:7 (w / v), blend in a juicer for 2 minutes, filter through double-layer gauze to remove coarse fibers, sterilize in a 65℃ water bath for 30 minutes, quickly cool to room temperature, and then add 0.05% (w / v) L-ascorbic acid to prevent enzymatic browning, so as to avoid the browning of custard apples during fermentation and affect bacterial growth. The prepared juice is dispensed into sterile Erlenmeyer flasks (100 mL per flask) and stored at 4℃ for later use.

[0013] Example 3: Preparation and performance verification of custard apple fermentation broth Bacillus subtilis 2025223 and Lactobacillus reuteri 2026004 were inoculated into custard apple juice at an inoculation rate of 5% (v / v) (initial bacterial count approximately 10). 7 The samples were fermented at 37℃ and 150 rpm for 48 h at different fermentation stages (0 h, 4 h, 8 h, 16 h, 24 h, 36 h and 48 h) for various index determinations. At the same time, uninoculated custard apple juice was set as the control group (Nfa), Bacillus subtilis group (Bac), and Lactobacillus reuteri group (Lac). All fermentation experiments were performed in triplicate.

[0014] 1. Safety evaluation of the strain The strain was inoculated onto Columbia blood agar plates containing 5% (w / v) fresh sheep blood and incubated at 37°C for 48 hours. Hemolysis was then observed. If a grass-green hemolysis ring appeared around the colony, it was determined to be α-hemolysis; if a clear hemolysis ring appeared, it was determined to be β-hemolysis.

[0015] Hemolysis is the rupture and dissolution of red blood cells, which can be caused by a variety of physical and chemical factors, as well as toxins. To determine the pathogenicity of functional bacteria, their hemolytic properties were evaluated. Results are shown below. Figure 1 Bacillus subtilis (A) and Lactobacillus reuteri (B) did not form obvious hemolytic rings, indicating that they have no hemolytic effect, are relatively safe, and will not cause hemolysis-related toxic reactions.

[0016] 2. Determination of pH and Brix content of fermentation broth Fermentation broth was collected at 0h, 4h, 8h, 16h, 24h, 36h and 48h respectively. 5mL of fermentation broth was placed in a centrifuge tube and centrifuged at 4500r / min for 10 minutes. The pH value of the supernatant was measured with a pH meter and the Brix concentration (°Bx) was measured with a saccharimeter.

[0017] pH changes during custard apple juice fermentation, such as Figure 2 As shown, the pH values ​​of the fermentation broths of both strains showed a continuous decreasing trend with increasing fermentation time. Brix content was as follows... Figure 3 All groups showed a trend of initial decrease followed by stabilization or slight increase, but significant differences existed among different strains. After 16-24 h of fermentation, the Brix content in the Bacillus subtilis group continued to decrease, stabilizing at a low level after 36-48 h, indicating that it continuously consumed soluble sugars and had a strong metabolic capacity; while the Lactobacillus reuteri group showed a significant increase after 24-36 h.

[0018] 3. Determination of total phenol and amino acid content The total phenol content in sample solutions was determined using the Folin-Ciocalteu method. Under alkaline conditions, phenolic substances can reduce phosphotungstic acid-phosphomolybdic acid to form a blue complex, which has a characteristic absorption peak at 760 nm. The total phenol content in the sample was calculated by measuring its absorbance and referring to a standard curve. Under acidic conditions, amino acids react with ninhydrin upon heating to form a blue-violet compound (diketonine), which has a characteristic absorption peak at 570 nm. The free amino acid content in the sample was calculated by measuring its absorbance and referring to a standard curve. Both content determinations were performed using a kit-ELISA method according to the manufacturer's instructions.

[0019] like Figure 4 As shown, the total phenol content of different strains generally showed an upward trend during fermentation, but the magnitude of the change varied significantly. The Bacillus subtilis group showed a slow increase in the early stage of fermentation, a significant increase from 16 to 36 h (P < 0.05), and reached a high level at 48 h; the Lactobacillus reuteri group showed a similar trend, maintaining a high content level in the middle and late stages (24-48 h).

[0020] like Figure 5 As shown, the content of free amino acids generally showed a significant upward trend during fermentation (P < 0.05), but the change patterns differed among different strains. The content of Bacillus subtilis significantly increased from 24 to 36 hours; the content of Lactobacillus reuteri fluctuated relatively slowly, gradually increasing in the middle and late stages.

[0021] Example 4: Determination of the in vitro antioxidant capacity of fermentation broth The in vitro antioxidant activity of the fermentation broth was assessed through FRAP iron ion reducing capacity, hydroxyl groups, DPPH, and ABTS. + Free radical scavenging capacity was assessed. All four antioxidant capacities were determined using a kit-ELISA method according to the manufacturer's instructions. Iron ion reducing capacity was assessed using the FRAP method, utilizing Fe... 3+ - Tripyridine triazine (Fe 3+ -TPTZ) can be reduced to Fe by antioxidants. 2 + -TPTZ produces a blue color to assess the antioxidant capacity of a sample. The antioxidant capacity can be determined and calculated by measuring the absorbance at 593 nm. In the Fenton reaction system, Fe... 2+ABTS reacts with H₂O₂ to form -OH, which can then react with chromogenic substrates to generate colored products. When antioxidants are present in the system, they can competitively scavenge -OH, thereby reducing the intensity of the chromogenic reaction. The hydroxyl radical scavenging rate of the sample was calculated by measuring the absorbance change at 510 nm. Based on the characteristic that DPPH radicals have a single electron, a strong absorption peak at 517 nm, and a purple color in alcoholic solutions, the supernatant was collected after centrifugation of the fermentation broth at different time points, and the DPPH radical scavenging ability of the biological sample was quantitatively determined using a 96-well plate. ABTS is oxidized to green ABTS+ under the action of appropriate oxidants. When antioxidants are present, ABTS... + The production of ABTS will be suppressed, and ABTS was measured at 734 nm. + The antioxidant capacity of a sample can be determined and calculated by measuring its absorbance.

[0022] Free radical scavenging rate (%) = [(1-(A)] 测定 -A 对照 )÷A 空白 )×100% like Figure 6 As shown, the total antioxidant capacity of different strains during fermentation generally showed a trend of first increasing and then fluctuating. The Bacillus subtilis group showed a significant increase (P < 0.05) from 8 to 16 h of fermentation, followed by a slight decrease; the Lactobacillus reuteri group showed a significant increase from 12 to 24 h, reaching a peak value around 24 h.

[0023] Figure 7 The scavenging rate of hydroxyl radicals showed a decreasing and then increasing trend in the Bacillus subtilis group during fermentation, while the Lactobacillus reuteri group showed an increasing trend. The hydroxyl radical scavenging rate of the control group (Nfa) did not change significantly throughout the fermentation process.

[0024] Figure 8 ABTS was displayed + Trends in free radical scavenging capacity: All treatment groups showed varying degrees of enhancement during fermentation, with the Bacillus subtilis group reaching its peak at 24 h; the Lactobacillus reuteri group exhibited the strongest scavenging capacity at 24 h. like Figure 9 As shown, the DPPH free radical scavenging capacity also showed a significant increasing trend during fermentation (P < 0.05). The Bacillus subtilis group initially decreased and then increased, reaching a peak at 36 h; the Lactobacillus reuteri group exhibited the strongest scavenging capacity, which decreased somewhat in the later stages. Considering the changes in all indicators, the optimal fermentation termination time was determined to be 25–30 h, at which point lactic acid production was high and cell activity was good. The trends of the four antioxidant indicators were basically consistent, all indicating that fermentation could significantly enhance the antioxidant activity of the samples.

[0025] Example 5: Determination of in vitro functional activity of fermentation liquid The fermentation supernatant was used to determine enzyme inhibitory activity. The inhibitory activities of pancreatic lipase and xanthine oxidase were measured according to the method of Lankatillake et al., with appropriate modifications. Each experiment included a control group and a blank group, and the measurements were performed in triplicate. For pancreatic lipase inhibition, p-nitrophenyl palmitate (pNPP) was used as the substrate and porcine pancreatic lipase as the enzyme source. The absorbance change was measured at 405 nm, and the inhibition rate was calculated using Formula 2. The inhibitory effect of the samples on xanthine oxidase activity was assessed using uric acid production. For xanthine oxidase inhibition, xanthine was used as the substrate and xanthine oxidase as the enzyme source, and the absorbance of uric acid production was monitored at 295 nm. Sodium phosphate buffer (200 mM, pH 7.4) was used as a negative control, and allopurinol as a positive control. The inhibition rate was calculated using Formula 1.

[0026] Protein denaturation inhibitory activity assay: The reaction system contained 500 μL of sample and 500 μL of 0.2% ovalbumin. After incubation at room temperature for 10 min, the mixture was heated in a 75℃ water bath for 10 min, cooled in an ice bath, and centrifuged. The supernatant was measured at 660 nm. Acetylsalicylic acid (200 μg / mL) was used as a positive control, and PBS as a negative control. The assay was performed in triplicate. The inhibition rate was calculated using Formula 2. 2+ Chelating capacity determination (Ferrozine colorimetric method): Add 50 μL of sample, 50 μL of deionized water, and 50 μL of 2 mM FeCl2 to a 96-well plate, mix well, and then add 50 μL of 5 mM Ferrozine to start the reaction. After reacting at room temperature for 10 min, the result is measured at 562 nm. EDTA was used as a positive control, and the sample-free system was used as a negative control. A background group was set up to subtract color interference. The determination was performed in triplicate, and the inhibition rate was calculated using Formula 2.

[0027] Formula 1: Inhibition rate (%) = 1 - [(Sample A - Blank A) / Control A] × 100% Formula 2: Inhibition rate (%) = [1 - (Sample A - Sample A blank) / (Control B - Blank B)] × 100% Pancreatic lipase inhibitory activity such as Figure 10 Pancreatic lipase plays a crucial role in lipid metabolism. Both *Lactobacillus reuteri* and *Bacillus subtilis* groups showed strong inhibition rates. Studies have shown that phenolic compounds can combat obesity and metabolic syndrome by regulating key cellular signaling pathways, improving insulin sensitivity, and modulating lipid and glucose metabolism.

[0028] Xanthine oxidase inhibitory activity such as Figure 11In both groups, the XOD inhibitory activity continuously increased with prolonged fermentation time. The Bacillus subtilis group showed a significant increasing trend in its inhibitory effect on xanthine oxidase activity, reaching its peak at 24 hours (P<0.001). While the inhibitory activity gradually decreased with prolonged fermentation time, it remained at a high level throughout the entire fermentation cycle. This suggests that functional metabolites with antioxidant or antiuric acid effects may be produced during fermentation.

[0029] The inhibitory effect of the sample on protein denaturation and Fe were measured. 2+ Chelating ability can effectively assess the strength of its in vitro anti-inflammatory activity. The fermentation broth's inhibitory activity against protein denaturation, such as... Figure 12 Both groups showed varying degrees of inhibitory effects, and the inhibitory activity dynamically changed with fermentation time. Compared with the control group, the inhibition rate of both groups reached its highest anti-denaturing effect at 16 h of fermentation (P < 0.001), indicating that the active substances formed in the later stages of fermentation have strong anti-inflammatory potential. The fermentation broth also showed an effect on Fe... 2+ Chelating ability such as Figure 13 As shown, overall, the chelating capacity of the fermentation broths in both groups was significantly higher than that in the control group. The *Lactobacillus reuteri* group reached its maximum value at 4 h (P<0.001), and then decreased slightly but remained higher than the control. The *Bacillus subtilis* group showed less fluctuation. These results indicate that the fermentation process helps generate antioxidants with metal ion chelating capabilities and exhibits strong in vitro anti-inflammatory activity.

[0030] Example 6: Determination of the antibacterial effect of fermentation broth on pathogens The fermentation broths from the three groups, after 24 hours of fermentation, were centrifuged and filtered through a 0.22 μm microporous membrane to obtain the fermentation supernatant. Six pathogenic bacteria were selected as indicator strains (all purchased from Guangdong Huankai Microbial Technology Co., Ltd.), including: Staphylococcus aureus (ATCC25923), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (GDMCC1.2476), Salmonella enteritidis (GDMCC1.3028), Shigella flexneri (CMCC(B)51572), and Proteus vulgaris (CMCC(B)49027). Antimicrobial inhibition tests were performed using the agar diffusion method. Under aseptic conditions, the suspensions of each indicator bacteria were adjusted to 0.5 McFarland turbidity, approximately 1.5 × 10⁻⁶. 8CFU / mL was evenly spread onto the surface of agar plates. Then, sterile Oxford cups (6 mm in diameter) were placed at equal intervals on each plate, and 200 μL of the prepared red yeast rice fermentation supernatant was added to each cup. An equal volume of sterile water was used as a negative control. The plates were incubated at 37°C for 24 hours, after which the diameter of the inhibition zone on each plate was measured and recorded. All experiments were independently repeated three times, and results are expressed as mean ± standard deviation.

[0031] The inhibitory activity of three fermentation broths against six common pathogens was evaluated using the agar diffusion method, and the results are shown in Table 1. The inhibitory activity of different fermentation broths against pathogens showed strain-dependent differences. The *Lactobacillus reuteri* group showed the most significant antibacterial effect, with the strongest inhibitory activity against *Staphylococcus aureus* and *Salmonella enterica*. The *Bacillus subtilis* group showed the highest inhibitory activity against *Escherichia coli* and *Salmonella enterica*.

[0032] Table 1. Determination of pathogen sensitivity in fermentation broth ( (n=3)

[0033] Note: "-" indicates no inhibitory activity for diameters ≤ 8 mm; different lowercase letters in the same line indicate significant differences between treatment groups (P < 0.05); the same letters indicate no significant difference.

[0034] Example 7: Determination of the flavor of fermentation broth After 24 hours of fermentation, the custard apple juice was centrifuged, and 90 mL of the supernatant was divided into three portions. Each portion was placed in a dedicated detection cup for the electronic tongue, and nine biological replicates were performed. Taste data were acquired at room temperature following a procedure of washing → equilibration → measuring the initial taste value → washing → measuring the aftertaste value. The acquisition time was 120 seconds, the acquisition period was 1 second, and the acquisition delay was 0 seconds. The sensor was cleaned with pure water for 10 seconds.

[0035] Based on the electronic tongue radar images of custard apples before and after fermentation by functional bacteria ( Figure 14 Analysis revealed that the response trends of different fermentation broths to each sensor were consistent, with similar response intensities, indicating that custard apples fermented with different functional bacteria possessed similar taste characteristics, with sour, bitter, and salty flavors being the most prominent. Among the various sensors, the umami (AAE sensor) had the lowest response value (<0.3), while the sour taste sensor had the highest. Compared to before fermentation, after the addition of functional bacteria, the response values ​​of the sour, astringent, umami, sweet, and umami richness sensors all increased, while the response values ​​of the bitter and salty taste sensors decreased.

Claims

1. A method for improving the functional activity of custard apple juice fermentation broth, characterized in that: Bacillus subtilis ( Bacillus subtilis ) or Lactobacillus reuteri ( Lactobacillus reuteri Fermenting custard apple juice can enhance the biological activity of the fermentation liquid.

2. The method for improving the functional activity of custard apple juice fermentation liquid according to claim 1, characterized in that: Its biological activities include antioxidant, anti-inflammatory, uric acid-lowering, and antibacterial activities.

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