Facilitating Production of Rice Dreg Peptides via Lactic Acid Bacterial Fermentation on Anti-Hypertension Efficacy

Facilitating Production of Rice Dreg Peptides via Lactic Acid Bacterial Fermentation on Anti-Hypertension Efficacy Chia Chi Chen1#, Yin Shen Lin2#, Chien Chao Chiu1, Yi Tsz Liu2, Ruei Chen Hung1, Cho Lin Li1, Ying Ching Hung1, Chia Yu Lin1, Tzu Yun Chi1, Ya Peng Wang2, Hung Chiao Lai2, Meng Ting Wang2, Wen Lin Lai3,4, Wei Huang Tsai6, Ching Feng Chiu7, Jyh Shiun Lin1, Meng Hwan Lee2* and Shao Wen Hung1,5* 1Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Xiangshan, Hsinchu 300, Taiwan 2Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Xiangshan, Hsinchu 300, Taiwan 3Department of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan 4Clinical Laboratory, Chung Shan Medical University Hospital, Taichung, Taiwan 5Nursing Department of Yuanpei University, Xiangshan, Hsinchu 300, Taiwan 6Department of Science and Technology, Council of Agriculture, Executive Yuan, Taipei 100, Taiwan 7Graduate Institute of Metabolism and Obesity Sciences, College of Nutrition, Taipei Medical University, Taiwan #Contributed equally to this study *Corresponding author: Meng Hwan Lee, Division of Animal Technology, Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan Shao Wen Hung, Division of Animal Industry, Animal Technology Laboratories, Agricultural Technology Research Institute, Taiwan DOI: 10.26717/BJSTR.2019.18.003121


Introduction
Rice (Oryza sativa L.) represents one of the leading food crops in the world. A global annual production was about 480 million metric tons (milled rice basis) in 2015 [1]. It is cultivated today in more than 100 countries except Antarctica. It is the staple food for over half the world's population, mainly in Asian countries, where it provides a considerable proportion of the protein intake for millions of people [2][3]. The total food protein production of rice per hectare is second only to that of wheat, although the yield of utilisable protein is actually higher for rice than for wheat, due to the superior quality of rice proteins [3][4]. Rice is known to have nutritional, hypoallergenic and healthy properties, which are retained by Rice Dreg Peptides (RDP) derived from rice starch by-products that contains more than 50% protein content and is used as a protein source [5]. With the massive of rice starch syrups industry in China, RDP is available in large amounts and at minimal costs. RDP are currently used as animal feed with low economic benefits in China. In China, the development of efficient methods to recovering RDP for human consumption that could significantly increase economic and social benefits [6].
High blood pressure has now become a major global health concern. The disease affected approximately 73 million people in USA [7]. There will be about 1.56 billion people suffered with hypertension worldwide in 2025 [7][8]. At present, hundreds of peptides with antihypertensive activity have already been reported and novel peptides are also discovered every day [7]. These biofunctional peptides were derived from 35 major sources that include animal matrix as milk, egg, fish, pork, and chicken and plant as soybean etc. [7]. In general, plant-derived functional peptides are considered more safe and healthy compounds. They are low molecular weight, possess different biological activities, and are easily absorbed by the small intestine in human [9][10]. Usually, plant-derived functional peptides may show not only antioxidant properties but also a wide range of other physiologic and biological activities as anti-hypertension, anti-hypercholesterolemia, immunomodulation, and anti-microbial actions [5,[9][10].
Angiotensin Converting Enzyme (ACE) increases blood pressure by converting the inactive angiotensin I (decapeptide) to the active angiotensin II (octapeptide). Several chemicals have been verified to act as anti-hypertensive drugs. These clinical anti-hypertensive drugs included as nifedipine, captopril, fosinopril, lisinopril etc., which act either by direct inhibition of ACE or block of the angiotensin II receptors. Although these chemicals are effective in the blood pressure control, however, there are many side effects found [11][12]. For the example, in the nifedipine intake, there are many side effects found as mild dizziness, flushing (warmth, redness, or tingly feeling), weakness, headache, mood changes, heartburn, nausea, tremors, muscle cramps, cough, wheezing, sore throat, stuffy nose, taste disturbance, skin rashes, kidney failure etc [7].
Besides chemical drugs, diet and lifestyle also play a significant role in the prevention of hypertension. Lots of small molecular peptides with antihypertensive activity have been discovered and many of these peptides are part of proteins present in our daily food. They are not toxic in general and may represent a new therapeutic strategy for the prevention and treatment of hypertension [7]. Hence, RDP were obtained from the rice dreg fermentation with Pediococcus pentosauceus L11 in this study. This novel RDP with anti-hypertensive activity was verified via in vitro ACE inhibition assay and in vivo SHRs animal experiment. There is a substantial interest in discovering RDP with antihypertensive activity in this study.

Food by Products, Chemicals and Reagents
Rice dreg (moisture: 80%) was provided from rice winery in

Fermentation of Rice Dreg
Rice dreg was adjusted to pH 7.0 using sodium hydroxide solution and fermented with 5% sucrose and 10% of L11 (final concentration of approximately 10 6 CFU/mL) and inoculated at

Measurement of RDP content
Measurement of the peptide content of the fermented rice dregs was followed as the method of Tsai and Wu [13] with some modification. Twenty-five milliliter of working solution was

Determination of In Vitro ACE Inhibitory Activity
The ACE inhibitory activity was assayed by Cushman and Cheung The same volume and frequency of physiological saline was administrated to WKY rats by p.o. using disposable feeding needles.
The blood pressure values were measured for each group once a week. The efficacy of RDP on the blood pressure was compared with each group. Following p.o. of sample, the blood pressure of rats was measured by a tail-cuff method (Model MK-2000ST; Muromachi Kikai, Tokyo, Japan) without warming rats in a chamber maintained at 38°C for 5 min. Five times at a time and take the average for the detection of blood pressure.

Statistical Analysis
Values of ACE inhibitor activity and production concentration of RDP are reported as mean ± Standard Deviation (S.D.). Values of blood pressure, body weight, and liver and kidney function indexes are represented as mean ± Standard Error of the Mean (SEM). Statistical evaluation was performed using Student's t-test, one-way analysis of variance (ANOVA), and SAS 8.0 software. Differences between groups were considered statistically significant at *p < 0.05.

The Most Ideal Fermentation Conditions of Rice Dregs
The fermentation conditions as fermentation time (h

Effect of RDP on ACE Inhibitory Activity In Vitro
The ACE inhibitory activity (%) and IC 50   Abbreviation: angiotensin I-converting enzyme (ACE); half maximal inhibitory concentration (IC 50 ); rice dreg peptides (RDP); standard deviation (SD). Data were presented as mean ± SD. Different latters (a, b, and c) was presented significant difference intergroups.   The mean blood pressure in two RDP groups was significant lower than negative control group (p < 0.01-p < 0.001) ( Figure 1).

Effect of RDP on BW, Anti-hypertension, and Liver and Kidney Functions In Vivo
Moreover, 4 indexes (GOT, GPT, BUN, and CRE) of the liver and kid-ney functions of RDP-administrated SHRs were located at the normal levels (Table 4).

Discussion
Functional peptides can be isolated from the natural food or their by-products. These natural food sources include mainly animal matrix as milk, egg, meat, fish, etc. and plants as cereal, wheat, rice, garlic, spinach, grapes, etc. [15][16][17][18][19][20][21][22][23]. These functional peptides have also been reported from various biological processes such as enzymatic hydrolysis, fermentation, and chemical synthesis properties compared to other plant-sourced protein [27][28] and is suitable as a hypoallergenic protein source to replace milk and soy infant formulas [6,[28][29]. Rice manufacturing industry produces large volumes of co-and by-products, which are generally undervalued and under-utilised. Although rice-sourced by-products were cheap. However, it can be renewable to producing the abundant antioxidant and bioactive compounds/peptides [5]. Currently, ricesourced by-products are mostly used as animal feed. These proteins of rice-sourced by-products were as a valuable source for the recovery of antioxidant peptides following whole cell or proteolytic enzyme treatments [5].
Many ACE inhibitory peptides have been characterized from food proteins such as gelatin, maize, fish, eggs, pea, and whey protein [30]. In this study, the source of RDP was rice dregs via lactic acid bacterial fermentation. This RDP is a valuable source for anti-hypertension according to the data of in vitro ACE inhibitory activity and in vivo animal experiment with SHRs. We have successfully R&D to evaluate the rice sourced by-products became the renewable bio-matrix and produced the anti-ACE and anti-hypertensive peptides (RDP). RDP with ACE inhibitory activity was mainly produced by many factories such as monosodium glutamate factory, glucose factory, fermentation industry etc. Commonly, rice dregs are severed as animal feed and they can cause environmental pollution problem. Moreover, rice dregs contain at least 65% protein content, which is higher than soybean protein content.
Therefore, rice dregs are good materials for producing the anti-hypertensive peptides [30]. In this study, ACE inhibitory peptides sourced from rice dregs has some effect on blood pressure regulation in vivo with SHRs. Additionally, Increase BW and normal liver and kidney functions of RDP-administrated SHRs were found in this study. We have found a safer, effective, and economical ACE inhibitor (RDP) for the remedy of hypertension. So, rice dregs-derived ACE inhibitory peptides via the lactic acid bacterial fermentation are safer and more efficient substitution for human health.

Conclusion
Our study mainly focused on the ACE inhibitory activity and anti-hypertension efficacy of RDP. The present study indicated the RDP had significant antihypertensive efficacy and no side effects were found in vitro and/or in vivo. Pediococcus pentosauceus L11-fermentated products (RDP) from rice dregs might serve as alternative sources of dietary health care products with good nutritional quality, safety, and protection against hypertension.
Our data might contribute to further research into food derived antihypertensive compounds, meanwhile it also provides some reference for the clinical drug use of RDP in traditional Chinese medicine.