1. Introduction
Soybeans fermented in water for a short time (Shuidouchi) is a traditional Chinese fermented soybean product, whose process is similar to Chungjukjang from South Korea and Natto from Japan [
1]. It rich in nutrients, including proteins, vitamins and minerals, and its content of vitamin E is especially noteworthy [
2]. Fermented soybeans not only have high nutritive value, but are also used as a drug in Traditional Chinese Medicine [
3]. According to Traditional Chinese Medicine, fermented soybeans can clear heat and detoxify, which can treat headaches due to pathogenic wind-heat, chest distress and vomiting. Numerous soy oligosaccharides in fermented soybeans can improve the body’s immunity and reduce the intestinal levels of toxic substances, which can prevent intestinal tumors [
4]. Scholars in Japan and South Korea found that Chungjukjang and Natto have many health benefits, including anti-oxidation, anti-inflammatory and anti-cancer activities. As a soy product, the most active ingredient of soybeans fermented in water is soy isoflavones. Isoflavones in fermented soybean products are more active than that in raw soybeans, which have very strong anti-tumor and anti-aging effects and prevent embrittlement of blood capillaries [
5].
Shuidouchi is a soy product fermented for a short time [
6]. In addition to factories, it can also be homemade in many areas. Similarly, in Japan and South Korea, they often make Natto and Chungkukjang at home. Different kinds of containers are often used in fermentation of Natto and Chungkukjang both in the factories and at home. Japan has even developed an automatic Natto fermentation machine with a metal tank. South Korean scholars have studied the sensory, physical and chemical properties as well as antioxidant effects of Chungkukjang fermentation with jars and glass, they found that jars were much better for the production of Chungkukjang [
7]. In China, metal, glass, plastic and ceramic containers are often used in Shuidouchi production. This research aims to study the anti-gastric mucosal injury effects of Shuidouchi fermented in different containers and explain the mechanism of the anti-oxidation effects of Shuidouchi.
The stomach is in a protected anatomical position in abdominal cavity and can move within some limits, so it is not easy to injure it with outside violence [
8]. Gastric mucosal damage is very common and caused by many factors, including chemical factors such as smoking, drinking strong tea, coffee, and drugs stimulating the gastric mucosa such as aspirin and indomethacin, physical factors such as too much cold or heat, eating rough food, bacteria or their toxins [
9]. The alcohol in wine can greatly stimulate the gastric mucosa, and taking in too much alcohol can lead to gastric mucosa damage and congestion in the stomach [
10]. Most bean products can protect the stomach, as they take advantage of their alkaline characteristics to neutralize the amount of hydrochloric acid due to gastric damage and alleviate stomach injuries. In addition to their alkalinity, soy isoflavones in soybean products may play a key role in alleviating stomach damage [
2]. Soybean foods contain many isoflavones, such as daidzein, genistein, glycitein,
etc., but these isolflavones cannot be immediate absorbed in the human body, and they must be hydrolysed to absorbable aglycones by β-glucosidase from the intestinal microbiota. Shuidouchi is produced by microorganisms and these microorganism could make these isoflavones change into functional compounds which could be readily absorbed by humans.
A mice model of gastric mucosal injury induced by hydrochloric acid and alcohol can determine the health effects of functional food components. Alcohol is the main factor causing gastric mucosal injury, and a certain concentration of hydrochloric acid can promote and increase gastric mucosa lesions caused by alcohol. Based on this animal model, mice are gavaged with soybean isoflavones extracted from fermented soybeans to test biochemical indicators of serum levels (MTL, Gas, SS, VIP, IL-6, IL-12, TNF-α and IFN-γ), tissue levels (SOD, NO and MDA) and mRNA expression (NF-κB, IκB-α, EGF, EGFR, nNOS, eNOS, iNOS, COX-2, Mn-SOD, Gu/Zn-SOD and CAT) in gastric tissues. The experimental results prove the gastric mucosa damage prevention effects of isoflavones in fermented soybeans and help elucidate its possible mechanism. This study also aimed to know the physicochemical properties of Shuidouchi produced by fermentation in different vessels, and the relationship between the physicochemical properties discrepancies (isoflavones content, moisture content, fermentation temperature, acidity and total bacterial count) and anti-gastric mucosa damage effects.
3. Experimental Section
3.1. Shuidouchi Fermentation
Five kilograms of dry soybeans were soaked in 12.5 L distilled water for 12 h, and cooked under 120 °C for 1 h. then the water was removed and the cooked soybeans were equally divide into five parts, which were placed in ceramic, plastic, metal and glass vessels covered by gauze in a constant temperature incubator at 45 °C so natural fermentation could occur for 72 h. After fermentation, soybeans were cooled, dried and crushed to extract the soybean isoflavones.
3.2. Isoflavone Determination
Two kilograms of freeze-dried fermented soybean powder were Soxhlet extracted with 32 L 70% aqueous ethanol solution for 6 h, and then the distilled ethanol was added with 5 mol/L HCl and N-503. The mixture was kept in a water bath at 30 °C for hydrolysis and extraction of soybean isoflavones for 2 h. Stratification in the separating funnel also takes up the N-503 layer. NaOH (5 mol/L) solution was added for back extraction, then the water layer was added with hydrochloric acid after stratification to precipitate soy isoflavones. After centrifugation, the sediment was washed to a neutral state and freeze-dried to extract soybean isoflavones. Taking daidzein and genistein as standards, the absorbance of different concentrations of daidzein and genistein were determined at 260 nm, and a standard curve of soybean isoflavone concentration was plotted for the content of isoflavones in soybeans [
4].
3.3. Mice Experiment
Mice for this experiment were divided into seven groups, including normal group, control group, ceramic vessel fermented Shuidouchi (CVFS) group, plastic vessel fermented Shuidouchi (PVFS) group, metal vessel fermented Shuidouchi (MVFS) group, glass vessel fermented Shuidouchi (GVFS) group and ranitidine group, having 10 mice in each group. During the first 14 d, mice in the normal group and the control group were gavaged with 0.2 mL distilled water once a day, while mice in the other groups were gavaged 0.2 mL of Shuidouchi extract with the concentration of 500 mg/kg once. Mice in the drug treatment group were gavaged 0.2 mL ranitidine with a concentration of 50 mg/kg. From the 14th day, all mice were cut off food, but allowed to drink water freely. In addition to mice in the normal group, all mice in other group were gavaged a stomach injury inducer (0.1 mL HCl/ethanol/10 g body weight, 60% in 150 mM HCl) after 24 h and then killed after 1 h [
8]. Heart blood was taken for centrifugal separation (4000 r/min, 10 min), where the upper serum was kept and the stomach was anatomized for further use. The experiments were performed following a protocol approved by the Animal Ethics Committee of Chongqing Medical University (Chongqing, China).
3.4. Mice Gastric Mucosal Injury Evaluation
The gastric secretion volume of mice were determined with a 10 mL measuring cylinder, and the pH of gastric juice of mice were determined using a SevenEasy pH meter (Mettler Toledo, Schwerzenbach, Switzerland). The isolated stomachs were inflated by injecting 1% formalin solution (10 mL) for 10 min to fix the tissues, and opened along the greater curvature. The area (mm2) of hemorrhagic lesions that had developed in the stomach was measured under a Leica MZ7.5 dissecting microscope (Leica, Bensheim, Germany) with a square grid.
3.5. Mice Serum Levels Measurement
Serum MTL, Gas, SS and VIP levels were determined with radioimmunoassay kits (Beijing Puer Weiye Biotechnology Co., Ltd., Beijing, China) according to the manufacturer’s protocols.
3.6. Mice Cytokine IL-6, IL-12, TNF-α and IFN-γ Levels Measurement
Serum IL-6, IL-12, TNF-α and IFN-γ levels were measured with a commercial ELISA kit (ELISA MAX, Biolegend, San Diego, CA, USA) according to the manufacturer’s protocol.
3.7. Gastric Tissues SOD, NO and MDA Activities Measurement
Gastric tissues SOD, NO and MDA activities were determined with appropriate assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) according to the manufacturer’s protocols.
3.8. mRNA Expression Determination (RT-PCR Assay)
Stomach tissues in mice were shattered by an ultrasonic pulverizer and RNA was extracted using RNAzol reagent. RNA extract of stomach tissues was diluted to 1 μg/μL. oligodT18 (1 μL), RNase, dNTP with MLV enzymes and 10 μL 5 × buffer were added into 2 μL RNA extraction of stomach tissues to synthesize cDNA under the conditions of 37 °C for 120 min, 99 °C for 4 min, 4 °C for 3 min. Then the expressions were amplified by the reverse transcription-polymerase chain reaction method, while house-keeping gene GAPDH was taken as reference. Agarose gel (1%) with ethidium bromide was used for electrophoresis to check the PCR amplification products [
8].
3.9. Statistical Analysis
Experimental data were presented as mean ± standard deviation (SD). Differences between the mean values for individual groups were assessed by one-way analysis of variance (ANOVA) with Duncan’s multiple range test. p < 0.05 was considered to indicate a statistically significant difference. SAS version 9.2 (SAS Institute Inc., Cary, NC, USA) was used to conduct the statistical analyses.
4. Conclusions
Through molecular biology methods, this research built stomach injury animal models to study the effect on inhibition of stomach injuries when Shuidouchi fermented in different vessels was administered to mice. By analyzing the content of soybean isoflavones, the results showed that the glass vessel was more advantageous for fermentation, producing more soybean isoflavones. These isoflavones had functional effects, which could cause molecular changes in mice bodies, as inflammation and oxidation factors were changed by Shuidouchi. Shuidouchi could inhibit the inflammation factors and increase the antioxidation factors. By further analyzing animal serum and tissues, using glass vessel to ferment Shuidouchi could decrease the MTL, Gas serum levels and increase the SS, VIP serum levels compared with Shuidouchi fermented in other vessels and no Shuidouchi treatment for control mice. The Shuidouchi fermented in glass vessel could also better lower cytokine levels (IL-6, IL-12, TNF-α and IFN-γ) in stomach injury of mice, increase the content of SOD, NO and reduce the content of MDA in mice gastric tissues. By further analyzing mRNA in related genes in stomach tissues with RT-PCR experimental technology at the molecular level, it had been found out that Shuidouchi could improve the strength of expression of IκB-α, EGF, EGFR, nNOS, eNOS, Mn-SOD, Gu/Zn-SOD, CAT in stomach-injured mice tissues and reduce the expression strength of NF-κB, COX-2, iNOS. The effect of Shuidouchi fermentation in glass vessels was more intense, which was significantly different from that of Shuidouchi produced in other kinds of vessels.