# Kinetics of Hyaluronidazoleae Inhibition by Rice (_Oryza sativa_ L.) Protein Hydrolysate
## 3 Results
### Hyaluronidase Inhibitory Activity Kinetics  
Our rice protein hydrolysate has the ability to inhibit hyaluronase activity and degradation of HA. As illustrated in Figure 4, under the condition of 5 mg/mL HA as the substrate, the hyaluronidase activity increased with the hyaluronidase concentration. The relationship of hyaluronase activity with its concentration was linear, and the hyaluronidase activity decreased with the addition of the rice protein hydrolysate solution.  
Next, we investigated the mechanism of hyaluronase activity inhibition with HA as the substrate by constructing a Lineweaver-Burk double reciprocal plot for the relationship of the inverse of the reaction rate with that of HA concentration (Figure 5), where the concentration of the rice protein hydrolysate solution is a parameter. The results revealed that the five curves with different concentrations of the rice protein hydrolysate shared the same \(y\) intercept \(1/V_{max}\), indicating \(V_{max}=0.096\) mM for all cases. Moreover, \(-1/K_{m}\), the \(x\) intercept, increased as the rice protein hydrolysate concentration was increased, revealing that \(K_{m}\) increased with the inhibitors of  
Figure 4: Effects of rice protein hydrolysate on the hyaluronase activity with 5 mg/mL HA as a substrate.  
Our rice protein hydrolysate solution thus can inhibit hyaluronise activity effectively. Under this condition, the rice protein hydrolysate bound with hyaluronidase had an effect on the HA binding. Therefore, HA and rice protein hydrolysate both bound to the hyaluronidase. According to Figure 5, our rice protein hydrolysate's inhibition mechanism was competitive. A previous study [25] reported tannic acid to have a similar effect; however, chia seed peptides were noted to have a mixed inhibition pattern, where seven peptides (namely KKKRVYV, GFEWITE, GDAHWVY, GDAHWY, GDAHWAY, DQNPRSF, and APHWYTN) were identified in the fraction [26]. [PERSON] et al. [27] reported that rice protein hydrolysates' unique Val-Asn-Pro (VNP) and Val-Trp-Pro (VWP) characteristics enable their application in antihypertensive functional foods and drugs. In the rice tripeptides, the presence of proline may contribute to high angiotensin-converting enzyme (ACE) inhibitory activity. VNP and VWP are competitive inhibitors, and therefore their powerful ACE inhibition may be caused by reduction in ACE's capacity for substrate binding.  
In the past 10 years, the research on peptides and protein from cereals and with multiple biological activities has rapidly increased. In 2018, [PERSON] et al. [28] indicated proso millet to be a potential tyrosinase inhibitor and antioxidant; the authors also indicated its possible cosmetic applications. Industrial rice byproducts can also be hydrolyzed by selected enzymes and microbial systems to produce functional supplements and additives that are antioxidant-peptide rich and applicable in new food formulations for humans [29; 30]. Empirical and bioinformatics studies thus far have mainly provided in vitro data and limited clinical evidence to prove these biological activities. Peptides and hydrolysates are therefore reasonable for use as health and functional foods [31].