بهینه‌سازی عوامل موثر بر ویژگی آنتی‌اکسیدانی و ضد میکروبی پروتئین هیدرولیز شده کنجاله کنجد با تخمیر توسط باسیلوس سابتیلیس

استفاده از پروتئین‌های هیدرولیز شده یا پپتیدهای حاصل از آنها با خاصیت ضد میکروبی و آنتی‌اکسیدانی یکی از روش‌های چایگزین ترکیبات شیمیایی ضد میکروب و آنتی‌اکسیدان می‌باشد. هیدرولیز میکروبی جهت رسیدن به این هدف نسبت به روش‌های هیدرولیز آنزیمی و شیمیایی روشی کارآمد و اقتصادی می‌باشد. در این پژوهش ایزوله پروتئین کنجاله کنجد از طریق فرآیند تخمیر توسط باسیلوس سابتبلیس با هدف دستیابی به بیشینه فعالیت ضد میکروبی و آنتی‌اکسیدانی هیدرولیز شد. بهینه‌سازی شرایط تخمیر (پارامترهای تخمیر شامل دما 30 تا 45 درجه سانتی‌گراد، زمان 12 تا 36 ساعت و غلظت سوبسترا 2 تا 6 درصد) به روش سطح پاسخ در قالب طرح مرکب مرکزی جهت دستیابی به بیشترین فعالیت ضد میکروبی و آنتی‌اکسیدانی انجام شد. دمای تخمیر (68/39 درجه سانتی‌گراد)، زمان (30/07 ساعت) و غلظت سوبسترا (4/85 درصد) به‌عنوان شرایط بهینه انتخاب شد. تحت این شرایط قدرت مهار کنندگی رادیکال  dpphو احیاء کنندگی یون آهن به‌ترتیب 5763 % و 0/9951 (جذب در 700 نانومتر) به‌دست آمد. برای تعیین اثر ضد میکروبی پروتئین هیدرولیز شده از روش کدورت‌سنجی استفاده شد و درصد مهار کنندگی استافیلوکوکوس اورئوس 59/58 %، اشرشیاکلای 46/55%، لیستریا مونوسیتوژنز 62/43 % و کلستریدیوم پرفرنجنس 50/97 % بدست آمد. آزمون پروتئازی با روش نقطه‌گذاری انجام شد. باسیلوس سابتیلیس در حضور پروتئین کنجد فعالیت پروتئازی به‌صورت معنی‌دار (0/05>p) در طی زمان نشان داد. به‌طوری‌که در مدت زمان 48 ساعت هاله شفاف به قطر 22 میلی متر ایجاد کرد. نتایج حاصل نشان داد که پروتئین هیدرولیز شده کنجاله کنجد توسط باسیلوس سابتیلیس دارای فعالیت زیست‌فعالی مناسبی است و از آن می‌توان به‌عنوان ترکیب ضد میکروب و آنتی‌اکسیدان طبیعی استفاده نمود.

optimization of effective factors on the antioxidant and antimicrobial activity of sesame meal protein hydrolysate with fermentation by bacillus subtilis

introductionnowadays, antibiotic resistance is increasing in all parts of the world and is emerging and expanding globally. due to their natural antimicrobial properties and low tendency to develop bacterial resistance, antimicrobial peptides can be a good candidate as an alternative to synthetic antibiotics. bioactive peptides are produced using enzymatic hydrolysis by enzymes extracted from microorganisms and plants, digestive enzymes, and fermentation by proteolytic starter cultures. enzymatic hydrolysis of proteins is performed by commercial proteases or a combination of several proteolytic enzymes. commercial proteases are expensive due to their specificity. among the strategies for protein hydrolysis with the aim of obtaining bioactive peptides is microbial fermentation, which is more environmentally friendly and has a high potential for use in industry due to its relatively low cost compared to commercial enzymes. it is a suitable method for the hydrolysis of sesame meal protein. bacillus species are bacteria that have high proteolytic activity and are able to produce different endopeptidases in the fermentation medium. the activity of endopeptidases in the environment containing proteins causes the production of peptides with small sizes and free amino acids in higher amounts compared to enzymatic hydrolysis, which is one of the advantages of using bacillus species with high proteolytic activity compared to pure enzymes. in general, the purpose of this research was to produce sesame meal protein hydrolysate by fermentation with bacillus subtilis and to investigate its antimicrobial and antioxidant activity. materials and methodsin this study, at the first step, sesame meal was defatted with hexane at a ratio of 1:5, then it was dried and sesame meal protein isolate was extracted, and the optimization of fermentation conditions was determined by the response surface methodology (rsm). independent variables, including temperature (30 to 45 ˚c), time (12 to 36 h), and substrate concentration (2 to 6%), were considered. the antioxidant properties of the treatments, including dpph radical scavenging activity, ferric ion reducing power, and antimicrobial activity, were investigated, and the optimum treatment was selected. then the protein hydrolysate was freez-dried and stored at -20 °c. results and discussionaccording to the results, temperature (39.68 °c), time (30.07 h), and substrate concentration (4.85%) were selected as optimum conditions. under these conditions, dpph radical scavenging activity and ferric ion reducing power of hydrolysate were 63.57% and 0.9951 (absorbance at 700 nm), respectively. the inhibition percentages of staphylococcus aureus (59.58%), escherichia coli (6.55%), listeria monocytogenes (62.43%), and clostridium perfringens (50.97%) were obtained in the optimized condition. bacillus subtilis, in the presence of sesame meal protein, showed significant (p<0.05) protease activity over time. after 48 hours, the clear zone diameter was determined to be 22 mm. the clear zone created by this strain showed that bacillus subtilis has high proteolytic activity and can be a suitable bacterium for hydrolyzing sesame meal protein with the aim of obtaining hydrolysates with the highest antimicrobial and antioxidant activities. the antimicrobial activity of the protein hydrolysate can be due to the higher degree of hydrolysis. by increasing the hydrolysis time, peptides with low molecular weight are produced, which cause better interaction with the microbial cell membrane, disrupt the membrane, and lead to the inhibition of the microorganism. according to the results, the sesame meal protein hydrolysate showed more inhibitory effect against gram-positive bacteria than gram-negative bacteria (escherichia coli). researchers reported that the difference in sensitivity to antimicrobial compounds between gram-positive and gram-negative bacteria can be attributed to the structure and composition of the cell envelope (cytoplasmic membrane or outer membrane, and cell wall). in general, the bioactivity properties of protein hydrolysate depend on the amino acid composition, sequence, and molecular weight of the amino acids. the antioxidant activity can be due to the high content of polar and aromatic amino acids. by further hydrolysis of proteins, peptides and polar free amino acids are produced that interact with free radicals and converted into safe and stable intermediate products. conclusionin this study, bacillus subtilis strain was used to ferment sesame meal protein, which is a rich source of protein, to produce protein hydrolysate with maximum antimicrobial and antioxidant activities. results showed that the protein hydrolysate obtained from sesame meal protein isolate had antimicrobial and antioxidant activities. it can be used as a natural antimicrobial or antioxidant agent in the formulation of food or pharmaceutical industry to improve the health of society.