In vitro study of the antioxidative and antiproliferative capabilities of Lactobacillus casei 16‐fermented soymilk

Abstract In this study, soymilk was fermented with Lactobacillus casei 16. The contents of aglycone isoflavones, saponins, total phenolic, and free amino acid in the fermented soymilk, and the protection for the HepG2 cells against 2,2'‐azobis(2‐amidinopropane) dihydrochloride (ABAP) damage and the antiproliferative effects of the fermented soymilk on the HT‐29 cells and Caco‐2 cells were studied. The results showed that the levels of total phenolic, aglycone isoflavones, and free amino acids in the L. casei 16‐fermented soymilk were enhanced. The ethanol extract and the water extract of the L. casei 16‐fermented soymilk showed protection for HepG2 cells against ABAP damage and inhibited the proliferation of the HT‐29 cells and Caco‐2 cells, which may be attributed to the enhanced level of total phenolic, aglycone isoflavones, and free amino acids in the L. casei 16‐fermented soymilk.


| INTRODUC TI ON
Soybean is rich in polyphenol, protein, and fatty acids and consumed in the forms of soymilk, tofu, soy sauce, tempeh, and so on (Dajanta & Chukeatirote, 2012;Erdman & Committee, 2000). Soymilk is manufactured by grounding soybean with water in some ratio. Soymilk was greeting by some consumers due to its protein, fatty acid, and isoflavones. On the other hand, soymilk was disgusting due to the undesirable bean odor and the flatulence.
The nutritional content changes of the fermented soybean and their products could account for some enhanced physiological functions of the fermented soybean and their products. In general, antioxidative activities of the fermented soybean products are significantly
The food with antioxidant capacities was reported to exert anticancer activities. Wen-Huei, Jun-Jen, Ching-Hsein, Tien-Shang, and Fung-Jou. (2002) demonstrated that fermented soymilk product (FSP) has a growth-inhibitory effect on various human breast carcinoma cell lines, especially on MCF-7 cells. The soymilk fermented with Bifidobacterium breve Yakult could inhibit female Sprague Dawley rats mammary carcinogenesis due to the increased content of isoflavone aglycone (Ohta et al., 2000).
In our previous work, Lactobacillus casei 16 showed higher β-glucosidase activity, and the L. casei 16-fermented soymilk exhibited the DPPH free radical scavenging capacity and oxygen radical absorbance capacity (ORAC) (Tang et al., 2018). In this study, we further investigated the effect of fermentation by L. casei 16 on the aglycone isoflavone, total phenolic, free amino acid, and saponin content in the fermented soymilk. In addition, the ethanol and water extracts of the fermented soymilk were studied for the protection for HepG 2 cell against ABAP damage and the antiproliferative effect on human colon cancer cell lines.

| Preparation of soymilk and soymilk fermentation
The soymilk preparation and fermentation by L. casei 16 was carried out according to our previous work (Tang et al., 2018). Soybean seeds were obtained from a local supermarket and stored at 4•C until use.
Whole soybeans were cleaned by washing and soaked overnight in 2 times their weight of distilled water. The soaked soybeans were blended with 4 times their weight of distilled water and ground in a blender for 3 min. And the ground mixture was filtered through a double-layered cloth to obtain soymilk. The soymilk was dispensed into sterile bottle and sterilized by autoclaving for 15 min at 105°C.
After cooling, the sterile soymilk was inoculated with L. casei 16 by 2% (v/v) and incubated at 37 •C for 24 hr.

| Preparation of solvent extracts
The method of the extraction was according to our previous work (Tang et al., 2018). To prepare solvent extracts, nonfermented or fermented soymilk was freeze-dried using freeze dryer (FD-IC-5D, Bo Medical Experimental Instrument Co., Ltd.), and the freeze-dried soymilk or fermented soymilk was extracted by ultrasound (100 W) with solvent (water or 80% ethanol) (1:10, w/v) and held at 25°C for 6 hr. The extracts were centrifuged at 10,000 g for 10 min at 4°C before the supernatants were collected. The supernatants were freeze-dried again. The freeze-dried extracts were stored at −80°C.

| Determination of total phenolic content
The total phenolic contents of samples were examined as described by Chen et al. (2013) with minor modifications. The freeze-dried samples were dissolved in 0.1 ml DMSO (Merck).The 1.9 ml of deionized water and 1.0 ml of Folin-Ciocalteu phenol reagent (Sigma-Aldrich Co.) were added to the solution. Then, 5.0 ml of 20% Na 2 CO 3 was added to the mixture at room temperature in darkness and allowed to react for 20 min. Absorbance of samples was then measured at 735 nm. The total phenolic content of the sample was compared with a standard curve of prepared gallic acid solution, and the results were expressed as milligrams of gallic acid per milligram of extract.

| The free amino acid content measurement
The free amino acid contents of the fermented and unfermented soymilk were measured as described by Xing et al. (2017) with minor modifications. The pH of unfermented and fermented soymilk was adjusted to 4.6 before determination. The samples were then centrifuged at 3,000 g for 30 min at 4°C to obtain supernatants. Fifty microliters of the supernatants was mixed with 2 ml O-phthalaldehyde and incubated for 2 min at room temperature. The absorbance of samples was read at 340 nm. The free amino acid content was determined from a standard curve constructed with Leucine standard, and the results were expressed as milligrams of Leucine extract.

| Quantification of total saponin
The saponin quantification in the soymilk was determined as described by Helaly method (Dini, Schettino, Simioli, & Dini, 2001) with slight modification. Briefly, the freeze-dried samples were dissolved in MeOH 80%. Aliquots of the samples (0.1 ml) were mixed with fresh made vanillin solution (8% in ethanol, 0.1 ml) and added to sulfuric acid (72%, 1 ml). The mixtures were allowed to bath at 60°C water for 20 min and then stand for 5 min at ice-cold water. The absorbance of the mixture at 544 nm was measured, and saponin content was calculated from a standard curve constructed with purified soyasaponin standard.

| HPLC analysis of the soy isoflavone
The soy isoflavone contents in the soymilk were determined according to our previous work (Tang et al., 2018). The ethanol extracts of soymilk and fermented soymilk were redissolved in 80% methanol. The samples were filtered through a 0.22 μm-pore-size polyvinylidene fluoride filter (PVDF) (Teknokroma, Barcelona, Spain) prior to analyzed by HPLC. The analytical HPLC was consisted with Waters 2,695 Alliance, a Waters 2,998 PDA detector, and C18 column (Optimapak, 4.5 × 250 mm, 5 μm). The autoinjector was used to inject 20 μl of the samples, and isoflavone was detected by monitoring the elution at 260 nm. The identification of isoflavones was calculated from retention time, and PDA spectrums were compared with those of the standards (genistein, genistin, daidzin, and daidzein, purchased from Sigma-Aldrich chemical company). The mobile phase was composed of 0.1% acetic acid in distilled water (solution A) and acetonitrile (solution B), and the solvent flow rate was maintained at 1 ml/min. The gradient conditions were as follows: 0-28 min, 85% A, 1.0 ml/min; 28-42 min, 65% A, 1.0 ml/min; 42-47 min, 55% A, 1.0 ml/min; and 47-59 min, 85% A, 1.0 ml/min.

| Cell culture
The human colon cancer cell lines HT-29 and Caco-2, and liver cancer cell line HepG 2 used in this study were purchased from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences, in Shanghai, China. HT-29, Caco-2, and HepG 2 cells were routinely cultured at 37°C in a 5% CO 2 and 95% air atmosphere. HT-29 cells were grown in Roswell Park Memorial Institute (RPMI) 1,640 medium (Gibco Life Technologies) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco Life Technologies), penicillin (100 U/ ml), and streptomycin (100 U/ml; Sigma-Aldrich). HepG 2 and Caco-2 cells were grown in DMEM medium (Gibco Life Technologies) supple- Technologies), penicillin, and streptomycin (100 U/ml; Sigma-Aldrich).

| Cytotoxicity
Cytotoxicity was measured using the method of Wolfe and Hai (2008) with modifications. 100 μl of HepG 2 , Caco-2, and HT-29 cells at a concentration of 4 × 10 5 Cell/ml was seeded in the wells of 96well plate. After incubation for 24 hr at 37°C, the grown medium was taken out, and the wells were washed with PBS twice. Then, the wells of experimental group were added with soymilk or fermented soymilk at the concentration of 50 μg/ml, 125 μg/ml, 250 μg/ml, and 500 μg/ml, respectively, and the plates were incubated at 37°C for 24 hr. After the medium was removed, the wells were washed with PBS twice, and the plate was added with fifty microliter methylene blue staining solution (98% HBSS, 0.67% glutaraldehyde and 0.6% methylene blue). After incubation at 37°C for 1 hr, the methylene blue was removed; the plate was washed with fresh distilled water several times and then the plate dried in room temperature. 100 μl of elution solution (49% PBS, 50% ethanol and 1% acetic acid) was added to the dried plate and incubated on a bench-top shaker for 20 min at room temperature. The absorbance of the wells in the plate was determined at 570 nm using Multiskan GO microplate reader (Thermo Fisher Scientific). The absorbance decreased by >10%, compared with the control, was considered to be cytotoxic.

| Effects of soymilk extracts on Survival of HepG 2 cells by ABAP oxidative damage
The ABAP oxidative damage was measured using the method of Song et al. (2010) with some modifications. HepG 2 cells were seeded in the wells in a 96-well plate at a density of 1 × 10 5 cell/ml using 100 μl of growth medium and incubated for 24 hr at 37°C to allow for cell attachment. After 24 hr incubation, the growth medium was removed and washed with PBS twice. The cells were treated with 100 µl ABAP (final concentration of 50 mmol/L, 60 mmol/L, 70 mmol/L, 75 mmol/L, 80 mmol/L, 85 mmol/L, 90 mmol/L, and 100 mmol/L dissolved in DEME medium, respectively, Sigma-Aldrich)or DEME medium of 0 mmol/L ABAP as control for up to 2 hr at 37°C. To assess cell viability, the modified methylene blue assay was to be used. The viability of HepG 2 cells was measured at 570 nm using Multiskan GO microplate reader (Thermo Fisher Scientific). The semilethal concentration (IC50) was determined. The HepG 2 cell survival rate was calculated using the following equation: where A control is the absorbance at 570 nm of the control, A sample is the absorbance at 570 nm of the sample, and A blank is the optical absorbance at 570 nm of the blank group. All samples were analyzed in triplicate.
To evaluate the protective effect of dried samples on HepG 2 cells against ABAP injury, HepG 2 cells were seeded at a density of 1 × 10 5 Cell/ml on a 96-well plate in 100 μl of growth medium and incubated for 24 hr at 37°C. After cells adhere to the wall, the experimental group was fed with soymilk and fermented soymilk at the final concentration of 125 μg/ml, 250 μg/ml, and 500 μg/ml for one hour. Cells were treated with ABAP at the concentration of IC50 to induce oxidative stress for 2 hr and fresh medium as control. The HepG 2 cell viability was calculated as described above.

| Effects of soymilk extracts on Superoxide Dismutase (SOD) in HepG 2 cells by ABAP oxidative damage
To evaluate the effects of fermented soymilk extracts on the production of SOD in HepG 2 cells by ABAP oxidative damage, HepG 2 cells were cultured in 96-well plate as described above.
Then, the HepG 2 cells were treated with different extracts at the final concentration of 125 μg/ml, 250 μg/ml, and 500 μg/ ml for one hour. Cells were treated with ABAP at the concentration of IC50 to induce oxidative stress for 2 hr and fresh medium as control. Cultured medium was removed, and cells were then lysed in a suitable volume of buffer (50 mM Tris-HCl pH 8.0, 50 mM EDTANa2, 0.2 M NaCl, 1% Triton X-100). Cell homogenate was then centrifuged at 10,000 × g, 4°C for 10 min at 4°C.
The afforded supernatant was stored at −20°C prior to the assays. Superoxide dismutase (SOD) activity was determined using the detection kit provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

| Effects of fermented soymilk extracts on human colon cancer cells proliferation
After the assessing of the fermented and unfermented soymilk extracts cytotoxicity, the antiproliferative activity of the crude extracts was measured using methylene blue assay as described above. HT-29 cells and Caco-2 cells were seeded in the wells of 96-well plates at a density of 5 × 10 4 cells/ml in 200 μl of fresh medium for 24 hr. Cells were then treated with the extracts at different concentration (50μg/ml, 125 μg/ml, 250 μg/ ml, 500 μg/ml) and incubated for 72 hr at 37°C. The inhibition rates of HT-29 cells and Caco-2 cells were calculated using the following equation:

| Western Blot Assay
The Western blot analysis was carried out as Mu et al. (2018)

| Statistics
All the experiments were repeated 3 times or more, the results were expressed as mean standard deviation, and the results were statistically analyzed by SPSS 20.0 software. The multiple comparisons were tested by Duncan's test, and the difference was significant ( p < .05).

| RE SULTS AND D ISCUSS I ON
The strain L. casei 16 was screened in our previous work (Tang et al., 2018). L. casei 16 had higher β-glucosidase activity, and the L. casei 16-fermented soymilk showed antioxidant activities evidenced by the DPPH free radical scavenging capacity and ORAC value (Tang et al., 2018). In this study, the contents of total phenolic, aglycone isoflavones, and free amino acid in the L. casei 16-fermented soymilk were determined, and the cellular antioxidant activity and the antiproliferative effects on human colon cancer cell lines of the fermented soymilk were studied.

| The total phenolic, aglycone isoflavones, free amino acids, and saponin levels in the soymilk fermented by L. casei 16
In this study, the total phenolic content of the soymilk fermented by L. casei 16 significantly increased (p < .05) with the prolonging of fermentation time, as shown in Figure 1 Our previous work found that the β-glucosidase activities of the L. casei 16-fermented soymilk increased significantly during the fermentation time ranging from 6 to 24 hr (Tang et al., 2018). Catalyzing the release of phenolics by β-glucosidase of L. casei 16 during fermentation could account for the increase in the total phenolic content and aglycone isoflavones contents. Rekha and Vijayalakshmi (2011) reported that the isoflavone glucosides in soymilk could be converted to aglycone isoflavone catalyzed by β-glucosidase produced by some lactobacilli strains. The β-glucosidase produced by lactic acid bacteria is responsible for the breakdown of β-1-6 glucosidic bond, which conjugates the pran ring of isoflavone and the sugar moieties (Donkor & Shah, 2010). The breakdown of isoflavone glycosides into sugar moieties and bioactive isoflavone aglycones during fermentation could improve the biological activity of soymilk. (2018) found that the β-glucosidase BIBG3 from B. longum catalyzed the hydrolysis of saponin at higher efficiency. Chen et al. (2013) reported that the peptide and amino acid contents of black soybeans fermented by Aspergillus awamori were enhanced. Su, Cheng, Hsiao, Han and Yu (2018) reported that the solid-state fermentation of soybean by Lactobacillus species and Clostridium butyricum could increase the degradation of soybean protein. Watanabe, Fujimoto, and Aoki (2007) suggested that antioxidant activity in the water-soluble fraction of Rhizopus-fermented tempeh may be due to the amino acids and peptides formed during fermentation. In this study, the fermentation of soymilk by L. casei 16 also increased the free amino acid contents, which may be ascribed to the proteases produced by L. casei 16.

| Effects of fermented soymilk extracts on the survival of HepG 2 cells treated by ABAP
In this study, whether the fermented soymilk ethanol extracts or water extract protect HepG 2 cells against ABAP damage was stud- The polyphenols (isoflavones, phenolic acids, and flavanols), free amino acids, and peptides in fermented soybean are responsible for antioxidant activity of the fermented soybean (Sanjukta & Rai, 2016). Yang et al. (2017) reported that the increase of aglycone isoflavones in fermented soymilk explained the antioxidant activity of the fermented soymilk by L. acidophilus MF204. Suo et al. (2016) reported that the soymilk fermented by L. fermentum Zhao had antioxidant activity due to the higher levels of amino type nitrogen, genistein, and daidzein. In this study, the L. casei 16-fermented soymilk contains higher levels of amino type nitrogen, genistein, and daidzein compared with the unfermented soymilk. Both the water extract and the ethanol extract of the L. casei 16-fermented soymilk could enhance the survival rates and the SOD activities of the ABAP-damaged HepG 2 cells. As described above, aglycone isoflavones and total phenolics wer mainly in the ethanol extract, while free amino acids were mainly in the water extract. It can be concluded that the aglycone isoflavones, total phenolics, and free amino acids in the L. casei  and HT-29 cells, and the ethanol extract exhibited the higher antiproliferative activity. Chen et al. (2013) found that, compared with unfermented soymilk, all the water, 80% methanol or 80% ethanol extract of fermented black soybeans showed significantly higher (p < .05) antiproliferative effects on HT-29 and Caco-2 cells. Shafiee, Saidijam, Tavilani, Ghasemkhani, and Khodadadi (2016) reported that soybean genistein may exhibit its anticancer properties on HT29 colon cancer cells by modulating caspase-3 and p38 MAPK pathway at different transcriptional and protein levels. Ye, Li, and Wei (2017) determined that genistein exerted its tumor suppressor effect at least partially via inhibition of S-phase kinase-associated protein 2 and promotion of its downstream targets p21 and p27 in breast cancer cells. Khan and Kang (2017)  showed antiproliferative effect on human colon cancer HCT-116 cells through ROS-JNK signaling pathway. PCNA is often used as a proliferation marker due to its specifically expressed in proliferating cell nuclei. In our study, the ethanol extract and the water extract of the L. casei 16-fermented soymilk showed the suppression effect on the proliferation of both HT-29 and Caco-2 cells in a dose manner and inhibited the PCNA expression in HT-29 cells. In the ethanol extract, the isoflavone aglycone maybe accounts for the antiproliferative effect. In the water extract, the antiproliferative effects may be attributed to the increase in the free amino acid and peptide contents in the fermented soymilk.

| CON CLUS ION
This work showed the ethanol and water extracts of the L. casei 16fermented soymilk could protect HepG 2 cells against ABAP damage and inhibit the proliferation of human colon cancer cell lines, HT-29 cells, and Caco-2 cells. The fermentation of soymilk by L. casei 16 enhanced the level of total phenolic, aglycone isoflavones, and free amino acids in the fermented soymilk, which maybe accounts for the protection for the HepG 2 cells, and the antiproliferative effects on the HT-29 cells and Caco-2 cells.

ACK N OWLED G M ENTS
This research project was supported by the National Natural Science Foundation of China (No.31571813, No.31671828).

CO N FLI C T O F I NTE R E S T
The authors do not have any conflicting interests.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

Yanfeng Tuo
https://orcid.org/0000-0001-7255-3753 F I G U R E 9 Effects of the extracts of fermented soymilk and nonfermented expression of PCNA in HT-29 cells. Data are represented as mean ± SD (n = 3). Mean values in the same group with different letters (A-B) are significantly different by Duncan's multiple range test (p < .05)