Anti-bacterial and anti-inflammatory activities of lactic acid bacteria-bioconversioned indica rice (Oryza sativa L.) extract

In this study, indica rice (Oryza sativa L.) was bioconversioned using Lactobacillus plantarum ATCC14917 and Lactobacillus rhamnosus GG KCTC5033. Compared to the control treatment (RE), rice extract fermented with Lactobacillus plantarum ATCC14917 (LP-RE) and Lactobacillus rhamnosus GG KCTC5033 (LR-RE) inhibited the growth of pathogenic bacteria significantly except for E. coli O157:H7 (p < 0.05). The LR-RE treatment had the strongest inhibitory effect on Staphylococcus aureus biofilm formation (10.81 ± 1.98%). Additionally, real-time PCR analyses confirmed that LP-RE inhibited S. aureus-induced IL-8 expression in Caco-2 cells by 60%. HPLC-orbitrap analysis indicated that this treatment increased the levels of key metabolites with therapeutic properties such as acetophenone, 9-oxo-10(E), 12(E)-octadecadienoic acid, nicotinic acid, and indol-3-lactic acid. This study demonstrated that the secondary metabolites were linked to antibacterial and anti-inflammatory effects, suggesting that bioconversion is a promising means to produce dietary supplements with therapeutic properties.


Introduction
Nutrient intake not only prevents or treats diseases, but also improves overall health [1]. However, contrary to consumer expectations, obtaining enough nutrients through dietary intake alone is often not possible in our busy modern society. Therefore, there is a rise in the consumer demand for novel nutrient-dense foods and supplements, which has led to the recent expansion of the health functional food industry [2]. Health functional food is defined as food manufactured and processed using ingredients that benefit the human body [3], and increasingly more people are consuming these products to prevent diseases rather than treating them with medicine.
Bioconversion is also often referred to as bioprocessing, biotransformation, biosynthesis, and biocatalysis, and these terms are generally used to describe the technology used for manufacturing products using microorganisms [4]. The bioconversion process differs from existing fermentation processes in that the fermentation process starts from relatively simple raw materials, whereas the bioconversion process produces products from materials by taking advantage of the substrate selectivity of microorganisms or enzymes. In this regard, the bioconversion process is known to be an energy-saving and high-tech sector that has advanced fermentation technology. Particularly, this process can be used in various ways to improve the usefulness and effectiveness of medicines and cosmetics [5].
Biotechnology techniques that employ several lactic acid bacteria are promising alternatives for the establishment of cost-effective, renewable, and virtually inexhaustible sources of high-value aromatic and bioactive compounds. Bioactive compounds possess a variety of therapeutic properties such as antibacterial, anti-inflammatory, and antioxidant activities, as well as anti-wrinkle and anti-whitening properties. These properties can potentially improve various aspects of the food, pharmaceutical, chemical, and agrochemical industries. As mentioned earlier, the bioconversion method has a wide variety of applications [6].
Indica rice (Oryza sativa L.), also called Annammi in Korea, is a representative rice variety that accounts for 90% of the world's rice trade and is mainly grown in Southeast Asia. The name "indica" refers to the fact that this rice variety is widely consumed in India. Indica rice is an important cereal and is known to be highly adaptable to environmental stress [7]. Although indica rice contains several bioactive substances, there is a lack of knowledge regarding the health-promoting properties of bioconversioned indica rice. A previous study reported the bioconversion of indica rice via inoculation with Agrobacterium tumefaciens [8]. Furthermore, although lactic acid bacteria-bioconversioned mulberries (Morus alba L.) have been reported to exert antimicrobial and anti-inflammatory activities [9], very few studies have focused on characterising lactic acid bacteria-bioconversioned indica rice. In this study, the antimicrobial, anti-inflammatory, antioxidant, anti-wrinkle, and anti-whitening effects of indica rice bioconversioned using Lactobacillus plantarum and L. rhamnosus GG were carried out.

Rice extract bioconversion process by L. plantarum and L. rhamnosus GG
Indica rice (Oryza sativa L.) was purchased at a local market (Seoul, Korea) and ground using a food grinder (SMKANB-4000, Poongnyun Co., Ltd., Korea). The rice was then immersed in distilled water at a ratio of 1:2.5 and boiled at 55 °C for 12 h in a water bath. Afterwards, the solid content was filtered through a sterilised cotton cloth and the pH of the supernatant was adjusted to 6.5-6.8. The rice extracts (RE) were then autoclaved prior to inoculation. Next, the extracts were inoculated with the bacterial strains (L. plantarum and L. rhamnosus GG; 1%, w/w) and incubated at 37 °C for 72 h. After incubation, the supernatants were collected by centrifugation at 10,170×g for 10 min and their pH was adjusted to 6.5-6.8. The L. plantarum (LP-RE) and L. rhamnosus GG (LR-RE) bioconversioned RE was recovered by filtering the fermented samples through a syringe filter (0.25 μm, Advantec Dismic, ToyoRochi, Tokyo, Japan). Non-bioconversioned RE was obtained by incubating the RE without L. plantarum and L. rhamnosus GG inoculation under the same conditions.

Anti-bacterial effects Bactericidal activity of bioconversioned RE
Yersinia enterocolitica, S. Typhimurium, E. coli O157:H7, or S. aureus were incubated with or without LP-RE or LR-RE at 50% in each appropriate broth media at 37 °C for 1, 3, 6, 9, 12, 24, and 48 h. These pathogenic bacteria were also incubated with RE under the same conditions. At the indicated incubation times, the growth of the bacteria was determined by measuring the optical density of the samples at 595 nm using a microtiter plate reader (Allsheng, Hangzhou, China).

Effect of bioconversioned RE on pathogenic bacteria biofilm formation
Pathogenic bacteria (Y. enterocolitica, S. Typhimurium, E. coli O157:H7, and S. aureus) were cultured at 37 °C for 24 h and diluted with fresh broth to 1 × 10 7 CFU/mL. Next, 100 µL of the bacterial suspension was incubated in 96-well culture plates with or without LP-RE or LR-RE (0, 3.125, 6.25, 12.5, 25, and 50%, v/v) at 37 °C for 24 h. After incubation, the culture supernatants were removed and the plates were gently washed with phosphate-buffered saline (PBS). The biofilms formed by pathogenic bacteria were stained with crystal violet (0.1%) at room temperature for 30 min and the excess stain was removed by washing with PBS. The adherent stain was dissolved by adding 95% ethanol and 0.1% acetic acid, after which the optical density of the samples was measured at 595 nm.

Cell viability of Caco-2 cells in the presence of bioconversioned RE
The cytotoxicity of the bioconversioned RE was evaluated via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Caco-2 cells, a human intestinal epithelial cell line, were purchased from the ATCC and cultured in complete Dulbecco's modified Eagle's medium (DMEM, HyClone, Logan, UT, USA) with 10% foetal bovine serum (Gibco, Burlington, ON, Canada), 100 U/mL penicillin, and 100 μg/mL streptomycin (HyClone, Logan, UT, USA) at 37 °C in a humidified 5% CO 2 and 95% air atmosphere. Caco-2 cells (5 × 10 5 cells/ mL) were plated in 96-well culture plates and grown until fully confluent. The cells were then incubated in LP-RE, LR-RE, or RE (10%, v/v) at 37 °C for 24 h. After incubation, 10 μL of MTT (5 mg/mL) was added to 90 μL of cell suspensions and incubated at 37 °C for 4 h. The culture supernatants were removed and 100 μL of DMSO was added to each well. The plates were then shaken to dissolve the reduced intracellular formazan. The absorbance was measured at 570 nm.

Statistical analysis
All experimental results were reported as the mean ± standard deviation of three independent replicate   experiments. Statistical differences between the treatment groups and the appropriate controls were determined via the Student's t-test using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) or one-way analysis of variance [12] using IBM SPSS Statistics 23 (IBM, Armonk, NY, USA).

Anti-bacterial activity of bioconversioned RE
Our study determined the inhibitory effects of LP-RE and LR-RE on the growth of Y. enterocolitica, S. Typhimurium, E. coli O157:H7, and S. aureus. As illustrated in Fig. 1A, the growth of Y. enterocolitica was effectively suppressed when the bacteria were incubated with both LP-RE and LR-RE. The bioconversioned RE inhibited the growth of Y. enterocolitica more effectively than RE. LP-RE and LR-RE did not significantly inhibit the growth of Y. enterocolitica from 0 to 24 h of incubation; however, significant growth suppression effects were observed after 48 h of incubation. Furthermore, LP-RE and LR-RE suppressed the growth of S. Typhimurium at 24 and 48 h, whereas the RE treatment increased S. Typhimurium growth (Fig. 1B). In contrast, neither LP-RE nor LR-RE inhibited the growth of E. coli O157:H7 (Fig. 1C). RE significantly inhibited S. aureus growth from 6 to 24 h but did not inhibit bacterial growth at 48 h. Furthermore, only LR-RE (not RE nor LP-RE) suppressed S. aureus growth at 48 h (Fig. 1D).
Several studies have shown that antimicrobial activity can be enhanced via biotransformation. Enhancement of antimicrobial activity via biotransformation has been constantly reported such as biotransformed Chinese herbal medicine residue with Aspergillus oryzae showed increased antibacterial activity to pathogenic bacteria [13], and bioconversion of fruit juice using LAB has an anti-listerial effect [14]. Therefore, the increase in antimicrobial activity by bioconversion of rice extract supports that the research result increases the antimicrobial activity of food by fermentation.

Bioconversioned RE inhibits bacterial biofilm formation
To examine whether bioconversioned RE inhibits pathogenic bacteria biofilm formation, Y. enterocolitica, S. Typhimurium, E. coli O157:H7, and S. aureus were incubated with or without bioconversioned RE (0%, 3.125%, 6.25%, 12.5%, 25%, and 50%) for 24 h. As shown in  [15]. In our growth inhibition result, except for E. coli, growth inhibition was the effect. Therefore, the anti-biofilm activity of bioconversion rice extract could be deemed to suppress of growth of the pathogen.

Anti-inflammatory effects Effect of bioconversioned RE on the viability of Caco-2 cells
Cytotoxicity must be thoroughly tested to determine the suitability, applicability, and safety of food extracts. Therefore, the MTT assay was conducted to confirm the viability of Caco-2 cells in the presence or absence of bioconversioned RE (10%, v/v). Figure 2 illustrates the cell viability results of the bioconversioned RE after 24 h of incubation. Notably, cell viability was not affected by RE, LP-RE, and LR-RE treatment regardless of concentration, suggesting that bioconversioned RE is not cytotoxic toward Caco-2 cells.

Bioconversioned RE inhibits S. aureus-induced IL-8 expression
Consistent with our antibacterial and anti-biofilm results, bioconversion RE was affected on S aureus. Therefore we demonstrated to investigate whether bioconversioned RE could inhibit S. aureus-induced IL-8 expression, Caco-2 cells were treated with 0.1% LP-RE, LR-RE, or RE in the presence or absence of S. aureus (1 × 10 7 CFU/mL) for 3 h. As illustrated in Fig. 3, LP-RE, LR-RE, and RE did not increase IL-8 mRNA expression, whereas S. aureus significantly increased IL-8 mRNA expression in Caco-2 cells. When the cells were co-incubated with bioconversioned RE and S. aureus, IL-8 mRNA expression was significantly decreased in the presence of LP-RE and LR-RE. Although RE moderately inhibited S. aureus-induced IL-8 mRNA expression, both LP-RE and LR-RE more effectively inhibited S. aureus-induced IL-8 mRNA expression, suggesting that the bioconversioned RE has anti-inflammatory effects in human intestinal epithelial cells. In previous studies, the anti-inflammatory effect was caused by the increment of metabolites and enhanced bioaccessibility or bioavailability of phenolic compounds [16][17][18]. Table 2 summarises the results of secondary metabolite analysis in RE using HPLC-MS/MS (Additional file 1: Fig. S1). Compared with the control, the concentration of 13 compounds increased significantly after bioconversion. Among these compounds, four (acetophenone, 9-oxo-10(E), 12(E)-octadecadienoic acid, nicotinic acid, and indol-3-lactic acid) were found to enhance the therapeutic effects of the extract. Particularly, acetophenone and 9-oxo-10(E), 12(E)-octadecadienoic acid have been previously reported to increase anti-bacterial and anti-inflammatory activity [10,11,19]. Furthermore, nicotinic acid and l-indole-3-lactic acid exhibit anti-inflammatory effects and radical scavenging activity, respectively [20,21]. After the bioconversion process, the levels of many types of amino acids decreased and lactic acid bacteria likely used these free amino acids to synthesise the abovementioned compounds.

Conclusions
Collectively, our findings suggest that lactic acid bacteriabioconversioned RE can suppress S. Typhimurium, Y. enterocolitica, and S. aureus growth and biofilm formation more effectively than RE; however, E. coli O157:H7 remained largely unaffected. Additionally, this study demonstrated that LP-RE inhibited interleukin-8 secretion by S. aureus in the human intestinal epithelial cell line Caco-2. Moreover, our study demonstrated that LC/MS-MS can be used to monitor the production of a variety of secondary metabolites produced during bioconversion. More importantly, these secondary metabolites were linked to antibacterial and anti-inflammatory effects, suggesting that bioconversion is a promising means to produce dietary supplements with therapeutic properties. Nevertheless, additional studies are required to confirm the efficacy of bioconversioned RE as a health functional food, particularly when consumed daily.