Optimizing the Fermentation Conditions of Cudrania tricuspidata Fruit Using Bacillus amyloliquefaciens for Anti-Inflammatory Activity and GC-MS-Based Volatile Component Characteristics

The aim of this study is to optimize the performance conditions used for maximum anti-inflammatory activity and to clarify in vitroanti-inflammatory properties of fermented C. tricuspidata fruit. Based on the single-factor experiment and Box–Behnken design, the optimized fermentation conditions of C. tricuspidata fruit for maximum anti-inflammatory activity were 3.8 d fermentation period, 8.4% (v/w) inoculation concentration, and 29.2°C fermentation temperature. Under optimal conditions, anti-inflammatory activity-based nitric oxide of fermented C. tricuspidata fruit reached 93.9%. Moreover, this study provides a theoretical basis and experimental data containing β-hexosaminidase and reactive oxygen species for the medical use and industrialization of C. tricuspidata fruit fermentation. Interestingly, the results of GC-MS analysis confirmed that fermented C. tricuspidata fruits detect volatile components different from unfermented C. tricuspidata fruits. We suggested that this volatile component may have been involved in the anti-inflammatory reaction, but scientific verification of this is needed later. Therefore, an in-depth study of volatile components detected from fermented C. tricuspidata fruits will need to be conducted later.


Introduction
Cudrania tricuspidata (C.tricuspidata), a small deciduous tree of the Moraceae family, is widely distributed throughout East Asia and is commonly referred to as cudrang or mandarin melon berry [1].Previous studies have reported that C. tricuspidata is one of the common folk remedies for preventing cancer over the past few decades [2].C. tricuspidata fruit has several health benefts [3]; however, most studies on its leaves and roots have been conducted because the raw fruits have unique favors that are difcult for consumers to accept [4][5][6].In addition, the fruits contain relatively fewer polyphenols and favonoids than other parts of the plant, such as leaves and roots [7].C. tricuspidata fruit has an extremely short shelf life and high moisture content, making it vulnerable to mechanical damage and microbial injury.Tis is similar to the characteristics of other berries [8,9].Terefore, there is an increasing trend in the postprocessing of C. tricuspidata fruits into vinegar, capsules, beverages, and powders [10,11].Previous studies have reported that the addition of appropriate processes to C. tricuspidata fruits signifcantly improves their functional properties and health characteristics [12,13].
C. tricuspidata fruit has been used in several studies for its various medicinal properties.C. tricuspidata extracts have been reported to have therapeutic efects on various diseases, including cancer [14].Park et al. [15] reported that favonoids isolated from chloroform extracts from C. tricuspidata showed a signifcant inhibitory efect on NO production and iNOS expression in RAW264.7 cells.In addition, they have been demonstrated to have various medicinal efects, including prostaglandin E2 production in macrophages [16].
Chronic infammatory diseases are the leading cause of mortality worldwide [17].Recently, considerable attention has been focused on exploring the potential role of natural substances in the prevention and treatment of chronic infammatory diseases [18][19][20].Natural fermentation products are popular worldwide [21].Tese products are rich sources of natural compounds, such as probiotics, with known biological properties [22].Terefore, these fermented products have signifcant potential as natural substances for the treatment of infammatory diseases.Fermentation has been used for a long time to preserve food.However, it has recently garnered considerable attention owing to an increase in the nutritional value of food and the production of health-promoting ingredients [23].Microorganisms used in fermentation metabolize fermentable carbohydrates to produce bioactive ingredients [24,25].Te active ingredients produced by fermentation can protect against diseases, including allergies [26].Kim et al. [27] reported that fermented Zizyphus jujuba played a protective role against infammation induced by nitric oxide (NO) and that the fermentation process improved anti-infammatory activity.Seo et al. [28] reported that the total polyphenol content improved by 47% by organic acid fermentation of C. tricuspidata fruits.Tis was regarded as immune activity in fermented C. tricuspidata fruits, as the levels of interleukin (IL)-2 and IL-4 secretion increased.Johnson et al. [29] reported that fermentation improves the antioxidant capacity and increases the polyphenol content, causing anti-infammatory activity in berries.
Although probiotic microorganisms guarantee the fermentation process, they can be destroyed during heating to produce fermented products without living microorganisms [30].However, the fermentation process before pasteurization enriches fermented foods by releasing active metabolites from existing plant nutrients or through the fermentation process [31].Terefore, it is important to determine the optimal fermentation conditions to preserve microorganisms in certain foods and enhance their medicinal efects.In fact, the fndings of Yusuf et al. [32] indicate that the optimal fermentation conditions of total mixed rations with cottonseed meal or rapeseed meal enhance the nutritional value, thereby making them viable and usable feedstufs for potential use in livestock industries.
Te frst goal of this study was to select Lactobacillus suitable for the fermentation of C. tricuspidata fruits based on OD600, pH, and solubility.Finally, the selected Lactobacillus strains were used to optimize the fermentation conditions to maximize the anti-infammatory efect, based on the NO inhibition rate of the fermented C. tricuspidata fruit.Tis study is expected to provide a fermentation technology to increase the anti-infammatory efects of C. tricuspidata fruits and provide evidence of their efcacy.

Fermentation of C. tricuspidata Fruit Using Lactobacillus.
Te mature C. tricuspidata fruit was cleaned and fermented in a 1-5 d fermentation period, 2-10% inoculation concentration, and 24-35 °C temperature.We used whole fruits during fermentation.It is understood that there is no universal strain or species that can provide a complete range of benefts.Terefore, the current work focused on screening anti-infammation activities of seven available Lactobacillus strains: Bacillus coagulans, Bacillus subtilis subsp.inaquosorum, Bacillus amyloliquefaciens, Enterococcus faecalis, Enterococcus faecium, Lactobacillus acidophilus, and Lactobacillus delbrueckii subsp.bulgaricus (Table 1).After fermentation, the juice produced during the fermentation process was freeze-dried for 48 h to remove moisture.Te dried juice was stored at −80 °C before use to maintain that Lactobacillus no longer grows and completely dissolved in a culture medium containing dimethyl sulfoxide (DMSO) before use in the cell experiments.

NO Expression Analysis.
Te nitrite accumulated in the fermented C. tricuspidata was measured as an indicator of nitric oxide production according to the Griess reaction described by Green et al. [35] with slight modifcation.In detail, 100 μL of each sample was mixed with 50 μL of 1% sulfanilamide (in 5% phosphoric acid) and 50 μL of 0.1% naphthalenediamine dihydrochloride and then incubated at room temperature for 10 min.Te absorbance of the soluble purple products was measured at 550 nm using an ELISA plate reader (SpectraMax 190, Molecular Devices, LLC).For the standard curve, absorbance at 550 nm was measured with a NaNO 2 serial dilution standard curve from 0 to 100 μM, and nitrite production was determined.

Box-Behnken Experimental Design of Response Surface
Methodology for Optimization of Variables.Te Box-Behnken design was used to determine the optimum levels of the most signifcant variables (fermentation 2 Evidence-Based Complementary and Alternative Medicine period, X 1 ; inoculation concentration, X 2 ; and fermentation temperature, X 3 ) and study their interactions.Te predicted response (Y) was the inhibition rate of NO (%) determined by measuring the survival rate of CCD-986sk cells.Each efective variable in the design was studied at three diferent levels (coded as −1, 0, and +1).A total of 17 experiments were conducted, and the entire experimental design considered three center points.Te ftness of the second-order polynomial model was expressed through the regression coefcient R 2 , and a detailed analysis of variance was conducted at the coded level to determine the efects of individual variables.Statistical Analysis System (SAS) software (version 11.0; SAS Institute Inc., Cary, NC, USA) was used for regression and graphical analyses of the experimental data.

Te β-Hexosaminidase
Assay.RBL-2H3 cells were dispensed in 24-well plates at a concentration of 5 × 10 5 cells/ well.Te medium was contained with penicillin, streptomycin, and anti-DNP IgE.Te cells were incubated overnight at 37 °C in a 5% CO 2 incubator.Te cells were washed twice with piperazine-N, N-bis-(2-ethanesulfonic acid) bufer.Tis medium was cultured at 37 °C for 10 min in a PIPES bufer.Cells were treated with optimized fermentation juice (250-1000 ppm) or 20 µM quercetin (positive control).After that, the cells were incubated at 37 °C for 20 min, and the cells were activated by treating them with 25 ng/mL of antigen DNP-BSA for 30 min at 37 °C.Te supernatant was cultured with a 1 mM substrate (p-nitrophenyl-N-acetyl-β-D-glucosaminide) at 37 °C for 1 h.Te reaction was stopped by addition of 2 ml of 200 mM glycine NaOH bufer pH 10.6.Te absorbance of the reaction solution was measured at 405 nm using a microplate reader (SpectraMax 190 Molecular Devices, LLC) [38].

ROS Analysis.
Intracellular ROS levels were measured using DCFH-DA according to a previously described method [39].RAW264.7 cells were seeded in 96-well plates at a density of 5 × 10 5 cells per well and incubated for 24 hours.Te cells were then treated with diferent concentrations of 250-1000 ppm fermented C. tricuspidata, or the 20 µM quercetin (positive control) for 72 hours.After treatment, DCFH-DA was added to the cells and incubated for 30 minutes.Te fuorescence intensities were measured using a microplate reader (SpectraMax 190, Molecular Devices LLC) at an excitation wavelength of 504 nm and an emission wavelength of 524 nm.

Gas Chromatography-Mass Spectrometry (GC-MS)
Analysis.Volatile compounds were extracted from C. tricuspidata fruits using headspace solid-phase microextraction (HS-SPME) and analyzed via gas chromatography-mass spectrometry (GC-MS).Te GC-MS analysis was performed using an Agilent 6890N GC DB-5 MS fused silica capillary column (30 mm × 0.25 mm i.d., flm thickness 0.25 μm).Te GC-MS was equipped with an electron ionization system with an ionization energy of 70 eV, and the carrier gas used was helium at a constant fow rate of 1 mL/min.Te injector and MS transfer line temperatures were set at 280 °C and 250 °C, respectively.Te initial oven temperature was 50 °C and was maintained for 2 min, then increased to 250 °C at a rate of 10 °C/min, and held at 250 °C for 10 min.Diluted samples (1/100, v/v, in methanol) of 1.0 μL were injected manually in the splitless mode.Te relative percentages of components were expressed as percentages by peak area normalization.Te identifcation of components was based on GC retention time on a DB-5 capillary column relative to computer matching of mass spectra using NIST libraries for the GC-MS system (software: NIST MS Search Program version 2.3).

Statistical Analysis.
Data are presented as mean-± standard deviation (n � 3).Statistical analyses of the results were performed at a 5% signifcance level.Diferences between the means of individual groups were assessed using Student's t-test (language R, R Development Core Team 2020, Vienna, Austria) and Duncan's multiple range test (SAS, SAS Institute, Inc., North Carolina, USA).

Efect of the Strains on the Fermentation of C. tricuspidata.
Te Lactobacillus density for fermentation was evaluated by measuring OD600 (Figure 1(a)).Te optical density (OD) using a spectrophotometer of increasing microbial mass of various Lactobacillus during incubation at 37 °C was measured in the presence of C. tricuspidata at diferent culture periods [40] Evidence-Based Complementary and Alternative Medicine fermentation (Figure 2).Visual changes in C. tricuspidata during fermentation for 5 days are shown in Figure 2. Tis study demonstrated the signifcant efects of Lactobacillus growth on the physicochemical parameters (pH and soluble solids; Figures 1(b) and 1(c)) of fermented C. tricuspidata, including nonvaccination controls.Te pH decreased by approximately 1-2 during lactobacillus treatment, probably because of the conversion of sugars to organic acids (e.g., malic and lactic acids) [41], and the initial pH value also decreased.Lactobacillus culture medium decreases the pH by producing acid-based substances.It has been reported that this increases the acidity of the medium, creating an environment unfavorable to pathogens [42].
C. tricuspidata juice fermented using Lactobacillus exhibited a signifcant diference from the nonvaccinated control (raw) in the graph as a result of the pH comparison according to the incubation period.
Figure 1(c) shows the efect of Lactobacillus on the total soluble solid content of C. tricuspidata.Te soluble solids content ( °Brix) of C. tricuspidata containing Lactobacillus was lower than that of C. tricuspidata without Lactobacillus.Te main sources of carbon and energy for Lactobacillus are glucose and fructose, respectively.Braga and Conti-Silva [43] reported a tendency of total soluble solids, similar to our results in papaya connectors with added Lactobacillus.Terefore, during fermentation, the solid content of the fruit

Efects of Fermented C. tricuspidata with Various Lactobacillus on NO in Macrophages.
NO is an infammationinducing mediator linked to various diseases [44].Overproduction of NO activates infammation-related tissue damage and infection [45].In this study, NO inhibition was measured after treatment with C. tricuspidata fermented with various Lactobacillus strains.A noticeable increase in NO metabolites (1.8-fold) was observed in LPS-treated RAW264.7 (Figure 3) compared to the untreated control group (see Control).However, fermented C. tricuspidata signifcantly (p < 0.01) reduced the expression of NO metabolites in the cell lines.Notably, a higher NO inhibition rate was observed in C. tricuspidata after fermentation than before fermentation.Fermented fruits exhibit high bioactivities, such as antioxidant and anti-infammatory properties [46,47].Previous studies have indicated that the anti-infammatory active ingredients of Trapa japonica fruit extract can be supplemented by fermentation with two microorganisms, Bacillus subtilis and Bacillus methylotrophicus [48].Tese results follow the same trend as that observed in our study.In particular, the fruit juice exhibited the lowest NO in C. tricuspidata fermented using B. amyloliquefaciens.C. tricuspidata fermented using B. amyloliquefaciens was a signifcant diference with the LPS treatment group (p < 0.001); however, it was similar to quercetin, a positive control.B. amyloliquefaciens belongs to the superfamily of bacteria belonging to the Bacillaceae family and genus Bacillus [49].Te previous study reported the investigated cytotoxic potential of B. amyloliquefaciens used in industrial production of enzyme products and discovered that none demonstrated any in vitro cytotoxic activity.[50].Te U.S. Food and Drug Administration (FDA) considers B. amyloliquefaciens generally safe for food and medical purposes [51].Moreover, some strains of B. amyloliquefaciens also suppress the synthesis of toxic substances [52].Te presence of B. amyloliquefaciens in some products has been reported to improve product quality, increase hydrophilicity, and increase the amount of bioactive compounds [53].We performed the subsequent analyses using B. amyloliquefaciens, which exhibited the lowest NO levels.

Cytotoxicity of the Fermentation Juice Obtained from B. amyloliquefaciens Fruits in RAW264.7 and RBL-2H3 Cells.
We evaluated the cytotoxicity of the fermentation juice obtained from fermented C. tricuspidata fruit with B. amyloliquefaciens in RAW264.7 and RBL-2H3 cells.As shown in Figure 4, 1000 ppm juice induced nonsignifcant cytotoxicity in RBL-2H3 (82.7%, p < 0.05).Moreover, the juice had no efect on RAW264.7 cells, even at a concentration of 1000 ppm.Terefore, in subsequent experiments, both RAW264.7 and RBL-2H3 cells were treated with nontoxic 1000 ppm concentrations of the juice.

Experimental Design.
As little information is available on NO control in fermented C. tricuspidata, a fractional factorial 17 design was initially designed to investigate the infuence of the fermentation period, Lactobacillus inoculation concentration, and fermentation temperature.Te experimental design, factors, levels (coded and decoded), and responses are summarized in Table 2.

Evidence-Based Complementary and Alternative Medicine
Te NO inhibition rate ranged from 19% to 85% of fermented C. tricuspidata.Te highest inhibition rate (93.92%) was obtained when C. tricuspidata fermentation conditions were fermentation period of 3.8 days, inoculation concentration 8.4%, and fermentation temperature 29.2 °C (Figure 5).Te quadratic efects of the inoculation concentration had the greatest efect on the NO inhibition rate, as summarized in Table 3 (regression coefcient: 2.79).
Terefore, the NO inhibition rate changed signifcantly with the controlled inoculation concentration (p < 0.05).Tis model was adopted as a model of the quadratic form, not as a linear one.Terefore, lack of ft can be signifcant, which can clarify the reliability of the model [54,55].Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine
where A is the fermentation period (days), B is the Lactobacillus inoculation concentration (%), and C is the fermentation temperature ( °C).
We show the reliability test results for the measured values and predicted values in Figure S1 (Supplementary data).Tis result shows that the measured and predicted values are very similar.

Efects of Fermented C. tricuspidata on the β-Hexosaminidase Activity in DNP-BSA-Stimulated
RBL-2H3 Cells.We performed β-hexosaminidase assay to prove the anti-infammatory efect of C. tricuspidata fermented under optimized conditions.We examined the effects of fermented C. tricuspidata on the DNP-BSA-induced release of β-hexosaminidase from RBL-2H3 cells [56].RBL-2H3 cells were treated with fermented C. tricuspidata and quercetin as a positive control to evaluate their inhibitory efects on the release of β-hexosaminidase.All test samples signifcantly (p < 0.001) inhibited the release of β-hexosaminidase from antigen-stimulated RBL-2H3 cells (Figure 6).
Among them, fermented C. tricuspidata exhibited excellent inhibitory efects on β-hexosaminidase activity at concentrations ranging from 500 to 1000 ppm.Tese results demonstrated that fermented C. tricuspidata is a potent antiinfammatory drug.

Efects of Fermented C. tricuspidata on ROS Level in LPS-
Stimulated RAW264.7.Te efects of fermented C. tricuspidata on intracellular free radical production in LPS-stimulated RAW264.7 cells were analyzed.Te level of ROS was similar to that of the positive control quercetin (Figure 7).LPS stimulation increased ROS production, which was signifcantly decreased upon treatment with diferent concentrations of fermented C. tricuspidata.Tese results suggest that fermented C. tricuspidata, which are considered to be involved in anti-infammatory events, may stimulate the secretion of ROS.

Volatile Compounds of Fermented C. tricuspidata.
According to the results of this paper, the fermented C. tricuspidata fruits contained various components including esters, alcohols, acids, ketones, volatile phenol, organic matter, and miscellaneous compounds.Te major components were diethyl phthalate (27.85%), aristolone (11.16%), furfuryl alcohol (8.69%), and scyllitol (8.16%), as presented in Table 4, Figure S2 (Supplementary data), and Figure S3 (Supplementary data).To the best of our knowledge, the composition of fermented C. tricuspidata fruit has not been previously reported.Terefore, our fndings represent the frst report on the composition of fermented C. tricuspidata fruit.

Discussion
Anti-infammatory response in the human body is crucially important for several reasons.Infammation is generally a defense mechanism of the immune system in response to external stimuli such as injury, infection, irritants, and toxins However, if chronic infammation occurs or the immune system continuously triggers infammation, it can be detrimental to health.Chronic infammation is associated with many diseases, such as cardiovascular disease, diabetes, cancer, atopic dermatitis, and rheumatoid arthritis.Tus, suppressing infammation through anti-infammatory drugs or food components is benefcial for overall health.
Te results of this study demonstrated that fermented C. tricuspidata also has the ability to inhibit the production of NO, as measured in the Griess assay.Ryu et al. [57] reported that Artemisia iwayomogi, Machilus thunbergii, Populus davidiana, and Populus maximowiczii exhibited potent NO inhibition above 70%.Jabit et al. [58] reported Most studies have reported that plant leaves or stems effectively inhibit NO; however, reports on inhibition owing to fruits are insufcient.In this study, an NO inhibitory activity of 90% or more could be reached by fermenting the fruits.Tese results are expected to provide potent antiinfammatory agents, which could pave the way for the discovery of novel clinical candidates.β-Hexosaminidase is also considered a degranulation marker of mast cells [59].Overproduction of free radicals during infammatory processes is involved in signal transduction and NF-κB activation.In this study, we suggest that fermented C. tricuspidata, which is considered to be involved in anti-infammatory events, may stimulate the secretion of β-hexosaminidase or ROS.
In previous paper, short-chain fatty acids, including butanoic acid, 3-methyl butanoic acid, 2-methyl propanoic acid, pentanoic acid, and hexanoic acid, are characterized by unpleasant, acidic, parmesan cheese, sour fruit, apple peel, rancid, sharp, or pungent aromas [60].3-Methylbutanoic acid has pungent, cheesy, sweaty, or old sock sensory characteristics, but its esters have a fruity character.In our study, these same components were detected in both fermented and nonfermented C. tricuspidata fruits.Interestingly, in fermented C. tricuspidata fruits, all acids except 3-methylbutanoic acid were reduced, while all ketones except Lanost-8-en-3-one were newly detected.Furfuryl alcohol, which was detected in fermented C. tricuspidata fruits, belongs to the furan compound family.Previous studies have demonstrated that furan compounds can be a source of novel tyrosinase inhibitors and can be developed as bioinsecticides and antimicrobials [61].Furfuryl alcohol, a sugar degradation product, inhibits aldehyde oxidation [62].Furanones, which are formed from the Maillard reactions between carbohydrates and proteins, have antioxidant activity [63,64].Furthermore, some furanones are found in fruits, and their antioxidant and antiinfammatory activities have been reported [65].2-Methoxy-4-vinylphenol, which is found in fermented C. tricuspidata fruits, possess antioxidant activity as well as antiinfammatory activity [66].Terefore, we assumed that Evidence-Based Complementary and Alternative Medicine 9 the volatile substances found in fermented C. tricuspidata fruits afected anti-infammatory activity.

Conclusion
To the best of our knowledge, this is the frst study to determine the optimal fermentation conditions under which C. tricuspidata fruits maximally inhibit NO production.B. amyloliquefaciens was used in the optimal fermentation process of C. tricuspidata to maximize NO inhibition.Tis strain was confrmed to exhibit the highest NO inhibition rate among the seven Lactobacillus species.Te obtained results verifed that fermented C. tricuspidata under optimal conditions signifcantly suppressed the production of proinfammatory markers β-hexosaminidase induced by DNP-BSA from RBL-2H3 cells.Terefore, we demonstrated that fermented C. tricuspidata could inhibit infammation from harmful factors such as NO or β-hexosaminidase.Te results of GC-MS analysis were interesting, as they confrmed the presence of volatile components in fermented C. tricuspidata fruits that were not detected in unfermented C. tricuspidata fruits.We suggested that these volatile components may be involved in the anti-infammatory reaction, but scientifc verifcation is required in future studies.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Figure 2 :
Figure 2: Visual variation of C. tricuspidata fruits fermented with various Lactobacillus.

5 F
ti c a c id b a c te r ia in o c u la ti o n c o n c e n tr a ti o n (% ) F e rm e n ta ti o n p e ri o d (d a y ) e n ta ti o n p e ri o d (d a y ) e r m e n ta ti o n te m p e r a tu re (°C ) ti c a c id b a c te r ia in o c u la ti o n c o n c e n tr a ti o n

Figure 5 :
Figure 5: Regression analysis of the Box-Behnken design experiments 3D plot.(a) Response surface graphs for nitric oxide inhibition rate as a function of fermentation period and inoculation concentration; (b) response surface graphs for nitric oxide inhibition rate as a function of fermentation period and fermentation temperature; (c) response surface graphs for nitric oxide inhibition rate as a function of inoculation concentration and fermentation temperature; (d) optimal fermentation conditions of C. tricuspidata and maximum nitric oxide inhibition rate.

Table 1 :
Lactobacillus used for the fermentation of C. tricuspidata fruits.
C. tricuspidata.A photograph of C. tricuspidata fermented using B. amyloliquefaciens also showed the occurrence of

Table 2 :
Results of three-factor Box-Behnken experimental design.

Table 3 :
ANOVA results of the ft model from Box-Behnken design.