ABSTRACT
Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 µg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 µg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 µg/mL, respectively. We observed that at least 256 µg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16-128 µg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 µg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 µg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 µg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 µg/mL. Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.
ABSTRACT
Background and Aim: Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice (Oryza sativa L.) is exclusively indigenous to Thailand's Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides. Materials and Methods: Antifungal activity in four Sangyod rice seed extracts (SYPs) - namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 - was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin-Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs - SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs]). Results: The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract - SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoformans, Talaromyces marneffei yeast phase, Trichophyton mentagrophytes, and Trichophyton rubrum. A partial inhibitory action on Candida albicans and Microsporum gypseum was possessed while showing the least activity to C. neoformans. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications. Conclusion: These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods.
ABSTRACT
This experiment was conducted to investigate the effects of dietary supplementation with coated granules (CG) on performance, in vitro digestibility and the gastrointestinal functionality in laying hens. A total of 40 Hisex Brown laying hens (36 weeks of age) were randomly divided into five equal groups; one served as a control and the other four were actual experimental groups supplemented with 0.75, 1.5, 3 or 6 g CG per day. All the hens were fed restrictively with target feed intake of 100 g basal diet/hen per day. There were no differences in egg production or egg weight among the groups, but feed conversion ratio was significantly improved (linearly, p < 0.05) with the dose level of CG in diet. Dietary CG increased the jejunal weight (linear and quadratic terms, p < 0.05) but decreased the ileal weight (linearly, p < 0.05). There was mostly statistical interaction between dietary CG and specific activity of intestinal digestive enzymes with similar patterns for dietary CG treatments (p < 0.05). A positive linear correlation was observed with in vitro protein digestibility by using the crude enzyme extract from dietary CG supplementation. Dietary CG decreased the caecal Escherichia coli population while the Lactobacillus spp.: E. coli ratio increased (quadratic fit, p < 0.05). CG supplementation, on the other hand, significantly altered intestinal morphology by increasing the height of duodenal and ileal villi (linearly, p < 0.05). Also, duodenal antioxidant capacity observed via remaining reducing power improved linearly (p < 0.05). This suggests that CG, unlike garlic oils without encapsulation, may be a good candidate for feed supplementation in commercial egg production. It could be included up to 6 g CG per day without any adverse effects on performance, which may relate to improving nutrient digestibility and better utilization of limited feed intake when using a low amount of diet or other observations in this study.
