The Antimutagenic and Antioxidant Activity of Fermented Milk Supplemented with Cudrania tricuspidata Powder

In this study, Cudrania tricuspidata (CT) containing abundant phytochemicals, such as xanthones and flavonoids, was evaluated as an additive to fortify the functionality and organoleptic quality of fermented milk. The physicochemical, functional, and sensory properties of fermented milk supplemented with different concentrations of CT powder were investigated. Increasing amounts of CT powder elevated the malic acid concentration, increasing the total acidity and decreasing the pH of fermented milk supplemented with CT powder. The viable cell count and free sugar contents of fermented milk indicated that supplementing with CT powder improved lactic acid fermentation slightly. The color of fermented milk supplemented with CT powder was darker, redder, yellower, and more pleasing than the control fermented milk. The total phenolic and flavonoid contents of fermented milk supplemented with CT powder rose as the concentration of supplemented CT powder increased, resulting in enhanced antioxidant and antimutagenic activities. The CT powder improved the functionality of the fermented milk; still, at 2% or more, it had some unfavorable sensory properties, such as sourness, taste, and texture, which reduced the overall consumer preference. Therefore, a CT powder concentration of 0.5% or 1% may be acceptable to consumers.


Introduction
Fermented milk, one of the popular functional foods, is typically produced by thermophilic lactic acid bacteria (LAB), such as Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus; abundant and live LAB are still present in the final fermented milk product [1]. The LAB are probiotics that balance between beneficial and undesirable microorganisms in the gastrointestinal tract, affect the immune system, and enhance health [2]. Fermented milk is an excellent source of nutrients, including protein, calcium, phosphorus, riboflavin, thiamin, vitamin B12, folate, niacin, magnesium, zinc, and bioactive peptides [1,3,4]. Several studies have revealed that bioactive peptides have good potential for preventing cancer [5][6][7], lowering blood pressure [8,9], and managing type 2 diabetes [10,11]. Adding fruits, vegetables, or herbs into fermented milk has been recently considered a great a strategy to satisfy varying consumer preferences by boosting the functionality and organoleptic quality of fermented milk [12][13][14][15][16].
Cudrania tricuspidata, a member of the Moraceae family mainly cultivated in China and Korea [17], can be a good additive for fermented milk. Several studies have reported that two major groups of phytochemicals, such as xanthones and flavonoids, are present in all parts of C. tricuspidata. phytochemicals, such as xanthones and flavonoids, are present in all parts of C. tricuspidata. Prenylated xanthones and flavonoids, primarily present in leaves and root bark, showed remarkable antiinflammatory [18,19], antitumor [20,21], hepatoprotective [22,23], neuroprotective [24,25], and anticoagulant [26] activities. Hydroxybenzyl flavonoid glycosides, abundant in stem bark, were reported to be a natural antioxidant and potential antitumor agents [27,28]. Prenylated isoflavonoids and benzylated flavonoids in the fruits have anti-inflammatory and antioxidant activities [29,30]. Despite its numerous health benefits, C. tricuspidata's utilization has been focused on its leaf and root since its raw fruit has a distinctive flavor that can be unacceptable to consumers [31]. Furthermore, the fruit contains relatively less polyphenols and flavonoids than the other parts [32,33]. However, several recent studies indicate that adding an appropriate amount of C. tricuspidata can improve the organoleptic properties of various food product with significantly enhanced functional properties [31,34,35].
A mutagen is a chemical or physical agent that is responsible for a genetically critical damage in the DNA sequence of an organism [36]. Since a mutation is related to the initiation and progression phase of carcinogenesis, the development of an antimutagenic agent can be a promising way to prevent genetic disease in humans, including cancer [37]. Several studies have reported that various polyphenols and flavonoids in plants induce antimutagenic activity [38,39]. Moreover, a study by de Oliveria et al. revealed that xanthones and flavones of Syngonanthus could serve as a novel antimutagenic agents [40]. Since these compounds are also plentiful in C. tricuspidata, we speculated that C. tricuspidata could be a potential antimutagenic agent.
This study aims to study how C. tricuspidata improves the functional properties of fermented milk and optimize the amount of C. tricuspidata to be organoleptically acceptable to potential consumers. This study also examines the antioxidant and antimutagenic activities of fermented milk supplemented with different concentrations of C. tricuspidata powder. Moreover, C. tricuspidata's physicochemical and sensory properties were evaluated.

Preparation of Cudrania Tricuspidata Powder
Fully ripe Cudrania tricuspidata fruits (15.2 ± 0.1 °Brix, pH 5.3 ± 0.1, acidity 0.20% ± 0.03%) were obtained from Sangju, South Korea, during the 2017 harvest season ( Figure 1). C. tricuspidata fruits were selected from the uniformity of color and lack of decay or rot. The proximate compositions of C. tricuspidata fruit were analyzed by the means of Association of Official Analytical Chemists (AOAC) [41] (Table 1). Samples were washed five times with tap water to remove any black spot on the skin and then manually squeezed to obtain the juice. The juice was filtered with cheesecloth and centrifuged at 10,000× g for 10 min. The supernatant was collected and freeze-dried into powder.

Preparation of Fermented Milk Supplemented with Cudrania tricuspidata
First, the bulk starter was prepared using milk with 32 g/L protein, 32 g/L fat, and 1.05 g/L calcium (Seoul Dairy Co., Seoul, Korea). The milk was heat-treated at 95 • C for 15 min and cooled down to 30 • C. After that, the milk was inoculated with 0.3% (w/w) of a commercial yogurt starter powder containing Streptococcus thermophilus, Lactobacillus paracasei, and L. delbrueckii subsp. bulgaricus (Plain Foods 2020, 9, 1762 3 of 14 morning, Cellbiotech Co. Ltd., Gimpo, Korea). The mixture was incubated at 40 • C for 8 h and then stored at 4 • C. Four percent (w/w) skim milk was added to milk to create the fermentation substrate for yogurt production. The mixture was heat-treated at 95 • C for 15 min and cooled down to 30 • C. Subsequently, C. tricuspidata powder was added to the mixture at the concentration of 0%, 0.5%, 1%, 2%, or 3% (w/w) ( Table 2). The mixtures were mixed well and inoculated with 3% (w/w) of bulk starter and incubated at 40 • C for 12 h. All the fermented milk samples were prepared by centrifugation at 10,000× g for 10 min. Soluble solid contents were determined using a refractometer (RA250, ATAGO, Tokyo, Japan). The pH of fermented milk samples was measured with a pH meter (Mettler-Toledo, Schwerzenbach, Switzerland). Total acidity, expressed as % of lactic acid, was determined by titration with 0.1 N NaOH until neutralization to pH 8.2. Each sample was serially diluted with distilled water using an appropriate dilution factor to achieve around 30-300 CFU/plate to determine the viable cell count in fermented milk. Each sample was then spread onto MRS (Difco Laboratories, Detroit, MI, USA) agar media plates and incubated at 37 • C for 48 h.

Total Phenolic and Flavonoid Compounds
The total phenolic compounds in fermented milk samples were determined with the Folin-Ciocalteau method [42], with some modifications. Two milliliters of samples were mixed with 2 mL of 50% (v/v) Folin-Ciocalteu reagent and incubated at room temperature for 3 min. Each mixture was then mixed with 2 mL of 10% Na 2 CO 3 , vortexed, and allowed to stand at room temperature for 1 h. The absorbance was measured at 700 nm. The results were expressed as the equivalent of gallic acid mg/mL in fermented milk using a standard curve with gallic acid (0-0.5 mg/mL). The total flavonoid content in fermented milk samples was determined [43]. The fermented milk samples were examined spectrophotometrically at 510 nm against a blank solution containing all reagents in 200 µL of distilled water instead of fermented milk samples (UV-1601, Shimadzu Co., Kyoto, Japan). First, 430 µL of 50% ethanol, 70 µL of fermented milk sample, and 50 µL of 5% NaNO 2 were combined in a test tube. After 30 min of incubation, the samples were mixed with 50 µL of 10% Al(NO 3 ) 3 ·9H 2 O. Six minutes later, 500 µL of NaOH (1 N) was added, and the solutions vortexed. The results were expressed as the equivalents of quercetin mg/mL of fermented milk using a standard curve with quercetin (0-200 µg/mL).

Free Sugar and Organic Acid Contents
Next, free sugar and organic acid contents of fermented milk samples were examined. Fermented milk samples were centrifuged at 10,000× g for 15 min, and the supernatant was filtered (Millex-HV 0.45 µm, Millipore Co., Bedford, MA, USA). Filtered samples were determined by high-performance liquid chromatography (HPLC) (Model Prominence, Shimadzu, Kyoto, Japan) with a Sugar-Pak I column (diameter 6.5 × 300 mm; Waters, Milford, MA, USA) and PL Hi-Plex H column (diameter 7.7 × 300 mm; Agilent Technologies, Santa Clara, CA, USA) [44]. The chromatography for the free sugars was run at a flow rate of 0.5 mL/min at 90 • C in the mobile phase in 50 mg/L Ca-ethylenediaminetetraacetic acid (Ca-EDTA) buffer. The chromatography for the organic acids was run at a flow rate of 0.6 mL/min at 65 • C in the mobile phase in 0.005 M sulfuric acid. Free sugars and organic acids were detected with a refractive index detector (RID-10A, Shimadzu, Tokyo, Japan). The results were expressed as each compound's equivalents in mg/mL of fermented milk using a standard curve with each compound (0-10 mg/mL).

Hunter's Color Value
Changes in Hunter's color value of the fermented milk before and after fermentation were measured with a colorimeter (Konica Minolta CM-3600A, Osaka, Japan) calibrated with a standard calibration slide (50 × 12 mm). The results were expressed as L (brightness), a (redness), and b (yellowness). The L, a, and b values for standard tile were (L = 97.78, a = 0.39, b = 2.05).

Assessment of Antioxidant Activities in Fermented Milk
All antioxidant activities were analyzed according to Oszmianski et al. [45]. For diphenylpicrylhydrazyl (DPPH) radical scavenging activity analysis, 100 µL of DPPH was dissolved in pure ethanol (96%). The radical stock solution was prepared right before experimentation. Then, 1 mL of DPPH was added to 1 mL of fermented milk and 3 mL of 96% ethanol. The mixture was thoroughly shaken and placed at room temperature in the dark for 10 min. The resulting solution's change in absorbance was observed at 517 nm using a spectrophotometer (UV-1601, Shimadzu Co., Kyoto, Japan). The results were corrected for dilution and expressed in µM of Trolox/mL of fermented milk using a standard curve with Trolox (0-50 µM/mL).
2,2 -azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was dissolved in water to make a 7-µM stock solution for the ABTS radical scavenging activity analysis. The ABTS stock solution reacted with a final concentration of 2.45 µM potassium persulfate to produce ABTS radical cations (ABTS + ). The reaction was kept in the dark at room temperature for 12-16 h before use; the radical would remain stable in this condition for more than 2 days. The ABTS + -containing samples were diluted with double distilled water to an absorbance of 0.700 ± 0.02 at 734 nm and equilibrated at 30 • C. After adding 3.0 mL of diluted ABTS + solution (A 734 nm = 0.700 ± 0.02) to 30 µL of fermented milk sample, the absorbance was read exactly at 6 min after the initial mixing (UV-1601, Shimadzu Co., Kyoto, Japan). The results were corrected for dilution and expressed in µM Trolox/1 mL of fermented milk using a standard curve with Trolox (0-50 µM/mL).
The ferric ion reducing antioxidant power (FRAP) assay was based on an antioxidant's tendency to reduce. An antioxidant reduces ferric ions (Fe 3+ ) to ferrous ions (Fe 2+ ) to form a blue complex (Fe 2+ /TPTZ) with increased absorbance at 593 nm. Moreover, the FRAP reagent was prepared by mixing 300 µM acetate buffer at pH 3.6, 10 µM of TPTZ in 40 µM of HCl, and 20 µM of FeCl 3 at a ratio of 10:1:1 (v/v/v). An amount of 300 µL of FRAP reagent and 10 µL of fermented milk samples were mixed in each well. The absorbance was measured at 593 nm after 10 min (UV-1601, Shimadzu Co., Kyoto, Japan). A standard curve was plotted using different Trolox concentrations. All solutions were Foods 2020, 9, 1762 5 of 14 prepared on the same day of experimentation. The results were corrected for dilution and expressed in µM of Trolox/1 mL of fermented milk using a standard curve with Trolox (0-50 µM/mL).

Assessment of the Antimutagenic Activity of Fermented Milk
The antimutagenic activity of fermented milk supplemented with C. tricuspidata powder was investigated using the pre-incubation method [44]. Two mutagens, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 4-nitro-O-phenylenediamine (NPD) (Sigma Co., St. Louis, MO, USA), were dissolved in dimethyl sulfoxide (DMSO) to the final concentrations of 5 and 15 µg/plate, respectively. Salmonella typhimurium histidine operon mutant strains derived from LT-2, TA98 (hisD3052 rfa ∆uvrB) (TA98), and TA100 (hisG46 rfa ∆uvrB) (TA100), were also used. One hundred microliters of a fermented milk sample, 100 µL of MNNG or NPD solution, 100 µL of an overnight culture of TA98 or TA100, and 0.5 mL of 0.2 M sodium phosphate buffer (pH 7) were mixed in a capped glass tube. The mixture was then incubated at 37 • C for 30 min with agitation. Then, 3 mL of molten agar containing histidine and biotin was added to the mixture; the final mixtures were plated on a minimal glucose agar medium and incubated at 37 • C for 48 h in the dark. Subsequently, the number of His + revertants per plate was counted. The antimutagenic activity was expressed as follows: A was the number of mutagen-induced His + revertants in the absence of a sample, B was the number of mutagen-induced His + revertants in the presence of a sample, and C was the number of spontaneous His+ revertants.

Sensory Evaluation
Sensory evaluation of fermented milk samples was performed by a panel of twenty judges with sensitive taste discrimination from the Department of Food Science and Technology, Kyungpook National University, Korea. Each judge evaluated the color, odor, sourness, taste, texture, and overall preference of the fermented milk samples with intervals of at least 3 min between samples. Water was provided to cleanse the judges' palates. Sensory scores were assigned on a scale ranging from 1 (dislike extremely) to 7 (like extremely). Before sensory evaluation was conducted, the experimental plan was approved by Kyungpook National University Industry Foundation on 1 October 2018 (IRB approval number: 2018-0160). All panels were given explanation and agreed with the precaution related to the possible side effects of C. tricuspidata before performing the sensory evaluation.

Statistical Analysis
All experiments were conducted in triplicate or more; the data were analyzed using the SAS software (version 9.4, SAS Institute, Cary, NC, USA). Significance was determined at a threshold of p < 0.05 using ANOVA, followed by Duncan's multiple range test.

Physicochemical Properties and Viable Cell Count of Fermented Milk
The various physicochemical properties, such as soluble solids content, pH, total acidity, and viable cell count, of fermented milk supplemented with different concentrations of C. tricuspidata powder were investigated ( Table 3). The soluble solids content of all the fermented milk samples was 9.8 • Brix. The pH decreased, and total acidity increased in the fermented milk samples as the concentration of supplemented C. tricuspidata powder increased since various organic acids of C. tricuspidata were concentrated by freeze-drying. The viable cell counts of all fermented milk samples were slightly increased depending on the supplemented amount of C. tricuspidata powder, indicating some components in C. tricuspidata powder contribute minimally to promoting lactic acid fermentation. Total phenolic compounds and total flavonoid contents of fermented milk supplemented with C. tricuspidata increased from 3.7-to 12.8-fold and from 10.1-to 30.10-fold when compared with the control fermented milk, respectively. C. tricuspidata fruit has been reported to contain chlorogenic acid, rutin, and other phenolic compounds as well as prenylated and benzylated forms of flavonoids [29,46,47]. In addition, five parishin derivatives (gastrodin, parishin A, B, C, and E) were newly identified and detected at high amounts in C. tricuspidata fruit [48]. Many plant phytochemicals, including polyphenols and flavonoids, have been recently defined as prebiotics since they meet the criteria, and many studies have demonstrated that health benefits are more attributed to the metabolites produced by microorganisms than the parent polyphenolic compounds [49,50]. It has been reported that supplementing various fruit juices (aronia, blueberry, and grape) into yogurt enhances the viability of S. thermophilus, indicating that increased phenolics may act as prebiotics [51]. Nguyen and Hwang also reported that viable count of LAB in yogurt increased during lactic acid fermentation with increasing supplemented aronia juice [1]. C. tricuspidata and Morus alba L. leaf extracts have also been reported to shorten fermentation time and increase the viability of LAB, which should be related to the phenolic compounds present in the extracts [35]. In the present study, C. tricuspidata fruit powder containing high amounts of phenolics and flavonoids may increase the growth of LAB in fermented milk. Glucose and fructose have been reported to be the main components of C. tricuspidata [52]. In free sugar analysis, the glucose and fructose contents of the fermented milk samples rose with the increasing concentration of supplemented C. tricuspidata powder, except for 0.5% C. tricuspidata powder. LAB typically hydrolyzes lactose to glucose and galactose; however, several LAB, such as S. thermorphilus and L. delbrueckii subsp. bulgaricus, are galactose negative (Gal -) strains and can metabolize only glucose, thereby accumulating galactose in the milk [53]. This is because these strains do not have Tagtose-6P pathway and do not metabolize galactose [54]. The lactose content of fermented milk decreased, while galactose content increased as the concentration of the C. tricuspidata supplement increased. This data supported that C. tricuspidata powder affected lactic acid fermentation as viable cell count of fermented milk supplemented with C. tricuspidata powder increased. In the organic acid analysis, malic acid contents of the fermented milk were significantly increased as the concentration of C. tricuspidata supplement increased. Malic acid has been reported as one of the primary organic acids in C. tricuspidata [52] and the cause of sour and astringent tastes in food [55]. Lactic acid contents of fermented milk showed a slightly increasing trend, depending on the C. tricuspidata supplement concentration, which is due to lactic acid fermentation promoted by the addition of C. tricuspidata powder. The increases in malic and lactic acid contents caused a decrease in pH values and increased total acidities of fermented milk supplemented with C. tricuspidata powder.

Measuring Color Changes in Fermented Milk Supplemented with Cudrania tricuspidata
Color is one of the most critical parameters for consumer preference. Although C. tricuspidata's fruit offers less potential health benefits than its leaf or root bark, it can be an excellent additive for various food products due to its pleasant coloring. The color values of fermented milk supplemented with different concentrations of C. tricuspidata powder before and after fermentation were investigated ( Table 4). The L values, the brightness of the product, significantly decreased with increasing concentration of C. tricuspidata powder. Moreover, L values in all the fermented milk samples were slightly reduced after fermentation since pH decreased during fermentation. According to García-Pérez et al., lowering pH decreased the lightness in fermented milk due to gelation [56]. The a value, the indicator of redness, significantly increased with an increasing amount of C. tricuspidata powder. Additionally, the a values in most fermented milk samples, except for 0.5% of C. tricuspidata powder, were slightly increased after fermentation. Several studies have described that a values of yoghurt increased when various fruits such as grape, aronia, blueberry, and cherry, which contain a large amount of anthocyanin, were added [51,57]. Kim et al. also reported that the total carotenoid content of C. tricuspidata fruit increased during ripening, leading to the significantly higher a value compared to C. tricuspidata fruit in the lower maturity stages [48]. The b values, the yellowness indicator (if b > 0), increased significantly with an increasing amount of C. tricuspidata powder. Likewise, b values were higher in all the fermented milk samples after fermentation. Novruzov and Agamirov [58] reported that C. tricuspidata fruit contains various carotenoids, leading to an increase in b values of fermented milk supplemented with C. tricuspidata powder. Overall, the results of Hunter's color value indicated that the fermented milk supplemented with C. tricuspidata powder were redder, darker, and more yellow than the control fermented milk as the concentration of C. tricuspidata increased. Along with Hunter's color value, appearances of fermented milk supplemented with C. tricuspidata powder were also pleasant ( Figure 2).

Functional Properties of Fermented Milk Supplemented with Cudrania tricuspidata
The DPPH radical scavenging, ABTS radical scavenging, and FRAP activities of fermented milk samples were analyzed to determine the effect of C. tricuspidata supplement on the functional properties of fermented milk (Table 5). With the C. tricuspidata supplement, the DPPH radical scavenging activity increased from 0.63 to 1.46 µM TE/mL, the ABTS scavenging activity increased from 0.18 to 0.36 µM TE/mL, and the FRAP activity increased from 0.21 to 1.65 µM TE/mL compared to the control fermented milk. In other words, the antioxidant activities of fermented milk supplemented with C. tricuspidata increased in a concentration-dependent manner. C. tricuspidata leaf extract has been considered an excellent source for high-value-added food materials and functional foods [16,[59][60][61]; the leaves have a high level of polyphenols, especially quercetin, which have higher antioxidant activity than other parts of the plant [62,63]. On the other hand, C. tricuspidata fruit's antioxidant activity is strongly affected by maturation and associated with the changes of prenylflavonoids, such as artocarpesin, alpinumisoflavone, 6-isopentenylgenistein, 4 -O-methylalpinumisoflavone, and 6,8-diprenylgenistein; fully matured C. tricuspidata fruits reach its peak of antioxidant activity [64]. The present study revealed that the addition of C. tricuspidata powder improved the antioxidant activity of fermented milk in vitro, indicating that C. tricuspidata has promising potential as a functional food. In vivo testing should also be performed to verify its functionality. . From left to right, the fermented milk supplemented with 0%, 0.5%, 1%, 2%, or 3% of C. tricuspidata powder.

Functional Properties of Fermented Milk Supplemented with Cudrania tricuspidata
The DPPH radical scavenging, ABTS radical scavenging, and FRAP activities of fermented milk samples were analyzed to determine the effect of C. tricuspidata supplement on the functional properties of fermented milk (Table 5). With the C. tricuspidata supplement, the DPPH radical scavenging activity increased from 0.63 to 1.46 μM TE/mL, the ABTS scavenging activity increased from 0.18 to 0.36 μM TE/mL, and the FRAP activity increased from 0.21 to 1.65 μM TE/mL compared to the control fermented milk. In other words, the antioxidant activities of fermented milk supplemented with C. tricuspidata increased in a concentration-dependent manner. C. tricuspidata leaf extract has been considered an excellent source for high-value-added food materials and functional foods [16,[59][60][61]; the leaves have a high level of polyphenols, especially quercetin, which have higher antioxidant activity than other parts of the plant [62,63]. On the other hand, C. tricuspidata fruit's antioxidant activity is strongly affected by maturation and associated with the changes of prenylflavonoids, such as artocarpesin, alpinumisoflavone, 6-isopentenylgenistein, 4′-Omethylalpinumisoflavone, and 6,8-diprenylgenistein; fully matured C. tricuspidata fruits reach its peak of antioxidant activity [64]. The present study revealed that the addition of C. tricuspidata powder improved the antioxidant activity of fermented milk in vitro, indicating that C. tricuspidata has promising potential as a functional food. In vivo testing should also be performed to verify its functionality. The antimutagenic activity of fermented milk supplemented with different concentrations of C. tricuspidata powder was investigated (Table 6). MNNG and NPD were used as mutagens for Salmonella typhimurium TA 100 strain. The antimutagenic activities, or the inhibition rate, of fermented milk supplemented with different amounts of C. tricuspidata powder increased from 18.75% to 64.58% for MNNG and from 23.31% to 60.11% for NPD. As with S. typhimurium TA 98  The antimutagenic activity of fermented milk supplemented with different concentrations of C. tricuspidata powder was investigated (Table 6). MNNG and NPD were used as mutagens for Salmonella typhimurium TA 100 strain. The antimutagenic activities, or the inhibition rate, of fermented milk supplemented with different amounts of C. tricuspidata powder increased from 18.75% to 64.58% for MNNG and from 23.31% to 60.11% for NPD. As with S. typhimurium TA 98 strain, the inhibition rate of fermented milk supplemented with different amounts of C. tricuspidata powder increased from 9.01% to 54.05% for MNNG from 20.00% to 63.81% for NPD. Like antioxidant activity, antimutagenic activities of fermented milk supplemented with C. tricuspidata were also concentration-dependent. Fermented milk prepared with LAB has been known to be antimutagenic toward a broad spectrum of mutagens such as MNNG, 4-nitroquinoline N-oxide (NQNO), and 3,2-dimethyl-4-aminobiophenyl (DMAB) [65,66]. Milk proteins, such as casein, R-lactalbumin, and β-lactoglobulin, have been shown to bind mutagenic heterocyclic amines at a high percentage [67,68]. Another study reported a robust inhibitory activity by casein against NQNO, and casein's anti-NQNO activity increased when it was hydrolyzed by pepsin [69]. Matar reported that antimutagenic compounds are produced in milk during fermentation by L. helveticus, and the author suggested that the release of peptides is one possible contributing mechanism [70]. On the other hand, several studies have focused on antimutagenic activity of some phytochemical compounds, such as flavonoids (e.g., quercetin) and phenolic compounds (e.g., tannin) which are ubiquitous in plants [71,72]. The present study also identified C. tricuspidata's antimutagenic activity due to its abundant phenolic compounds and flavonoids; therefore, C. tricuspidata could be an antimutagenic functional food. Table 6.

Strain
Mutagen

Sensory Evaluation
The sensory aspects of fermented milk supplemented with different concentrations of C. tricuspidata powder were evaluated to determine the acceptable concentration of C. tricuspidata for consumer preference ( Table 7). The color scores of fermented milk became higher as the concentration of supplemented C. tricuspidata powder increased, possibly due to the various pigments, such as carotenoids and anthocyanins, in C. tricuspidata. The odor scores of all the fermented milk supplemented with C. tricuspidata were higher than the control. However, the sourness, taste, and texture scores of fermented milk with the C. tricuspidata supplement exhibited different patterns. The fermented milk sample supplemented with 0.5% C. tricuspidata powder obtained the highest score in sourness and taste; however, higher concentrations of C. tricuspidata negatively impacted these scores due to the increase in malic acid concentration ( Table 3). The supplementation C. tricuspidata powder also lowered the texture scores; only the fermented milk supplemented with 0.5% of C. tricuspidata powder was similar to the control. In overall preference, fermented milk supplemented with 0.5% of C. tricuspidata powder obtained the highest score. Of all the sensory properties, except for texture, the fermented milk supplemented with 1% of C. tricuspidata powder was not significantly different from the control even though it obtained lower sourness and taste scores. Therefore, the fermented milk supplemented with 0.5% of C. tricuspidata powder had the highest consumer preference. Moreover, fermented milk supplemented with 1% of C. tricuspidata powder can also be considered as a potential functional food for the health-conscious consumers since it contains 1.5-3 times higher antioxidant activities and 2-3 times higher antimutagenic activities than the control fermented milk. Table 7. Sensory evaluation of fermented milk supplemented with Cudrania tricuspidata powder.

Sensory Property
Cudrania tricuspidata Powder Content (%, w/w) a-c Different letters within the same column indicate a significant difference (p < 0.05). All data are expressed as the mean ± SD (n = 3).

Conclusions
In the present study, we evaluated various physicochemical and functional properties of fermented milk supplemented with C. tricuspidata and found that bioactive compounds, such as total phenolic and flavonoid contents of fermented milk, were increased by the addition of C. tricuspidata powder. Moreover, sensory evaluation was performed to determine the appropriate concentration of supplemented C. tricuspidata powder for optimal consumer preference. Various antioxidant activities, including DPPH radical scavenging, ABTS radical scavenging, and FRAP activities, and antimutagenic activity, were significantly enhanced as the concentration of supplemented C. tricuspidata powder increased. In sensory evaluation, color scores of fermented milk samples were raised with the increase in C. tricuspidata concentration. However, most sensory parameter scores such as sourness, taste, texture, and overall preference dramatically declined as the concentration of C. tricuspidata reached or exceeded 2%. Therefore, supplementing C. tricuspidata powder at 0.5% or 1% may be more acceptable to consumers while still providing good functional properties. Further in vivo study will prove the enhanced functionalities of fermented milk supplemented with C. tricuspidata powder.

Conflicts of Interest:
The authors declare no conflict of interest.