Iranian Scientific Fisheries Journal

fa مقاله علمی – پژوهشی:‌ افزودن نانوکپسول‌‌های حامل و فرم آزاد پپتیدهای زیست‌‌فعال حاصل از هیدرولیز آنزیمی فانوس‌ماهی (Benthosema pterotum) به فرمولاسیون دوغ و ارزیابی خصوصیات میکروبی، شیمیایی و حسی محصول Adding carrier nanocapsules and free form of bioactive peptides derived from enzymatic hydrolysis of lanternfish (Benthosema pterotum) to doogh formulation and evaluating the microbial, chemical and sensory properties of the product بيوتكنولوژي و فرآوري هاي شيلاتي بيوتكنولوژي و فرآوري هاي شيلاتي كاربردي Applicable عوارض خطرناک نگهدارنده‌‌های شیمیایی برای سلامت مصرف‌‌کنندگان، حدود دو دهه است که توجه محققین را به سمت استخراج و تولید ترکیبات طبیعی قابل جایگزین با نگهدارنده‌‌های شیمیایی معطوف کرده است. از مهم‌‌ترین ترکیبات با منشاء طبیعی که فعالیت ضد باکتریایی و آنتی‌‌اکسیدانی آنها در تحقیقات مختلف تائید شده است، می‌‌توان به پروتئین‌‌های هیدرولیزشده یا پپتیدهای زیست‌‌فعال اشاره کرد. در تحقیق حاضر، پپتیدهایی با وزن مولکولی کمتر از 3 کیلودالتون از فانوس ماهی (Benthosema pterotum) به روش آنزیمی جداسازی و سپس با کمک تکنیک خشک‌‌کردن انجمادی و پوشش ترکیبی مالتودکسترین-صمغ عربی (با نسبت برابر) نانوریزپوشانی شدند. سپس نانوکپسول‌‌های حامل (200 و 400 میکروگرم بر میلی‌‌لیتر) و پپتیدهای آزاد (400 و 500 میکروگرم بر میلی‌‌لیتر) به فرمولاسیون دوغ اضافه و خصوصیات کیفی محصول طی 60 روز نگهداری در دمای یخچال ارزیابی گردید. نتایج نشان داد که تیمارهای حاوی نانوکپسول‌‌های حامل نسبت به شاهد و تیمارهای حاوی پپتیدهای آزاد، در زمینه مهار رشد Staphylococcus aureus  و Escherichia coli و حذف رادیکال آزاد ABTS موفق‌‌تر عمل کردند (05/0p<). ضمن این‌که pH، اسیدیته و فعالیت حذف رادیکال این تیمارها برخلاف شاهد و تیمارهای حاوی پپتیدهای آزاد، طی دوره نگهداری فاقد تغییر معنی‌‌داری بودند (05/0<p). با افزایش غلظت پپتیدهای آزاد و نانوکپسول‌‌های حامل، شمارش باکتری‌‌ها و قدرت مهار رادیکال ABTS در تیمارها به‌ترتیب کاهش و افزایش یافتند (05/0p<). اما pH و اسیدیته تحت تأثیر قرار نگرفتند. ارزیابی رنگ، بو و مزه تیمارها نشان داد که فقط امتیازات تیمار حاوی 400 میکروگرم بر میلی‌‌لیتر نانوکپسول‌‌های حامل در روز اول و 60 برابر بود (05/0<p) اما در سایر تیمارها، امتیازات شاخص‌‌های حسی در طول دوره نگهداری به صورت معنی‌‌داری کاهش یافتند (05/0p<). یافته‌‌های این پژوهش نشان داد که نانوکپسولاسیون پپتیدهای زیست‌‌فعال با استفاده از روش خشک‌‌کردن انجمادی و پوشش ترکیبی مالتودکسترین-صمغ عربی، تکنیک موثری به منظور ارتقاء و پایداری خواص نگهدارندگی آنها برای استفاده در صنعت لبنیات (به‌ویژه دوغ) است. Introduction The chemical types of antioxidants and antimicrobial compounds used in food industries as preservatives causes some diseases in consumers due to their harmful effects (Reyhani et al., 2024). For this reason, over the last two decades, various researches have been conducted to produce and extract preservative compounds of natural origin, and have yielded acceptable results. Hydrolyzed proteins or bioactive peptides that one of the sources of their production are aquatic animals (with low marketability) and the wastes from their processing, are antioxidant (Ng and Wong, 2024) and antimicrobial (Rivera-Pérez et al., 2023) compounds of natural origin. The use of bioactive peptides in food formulations as preservatives always requires the use of a suitable protection system in harsh environmental conditions. Because the structure of peptides may be destroyed in thermal processes during food production. One of the appropriate protection systems for active compounds is encapsulation, which is usually done by spray drying and freeze drying methods (Sarabandi et al., 2020). During this process, a microcapsule or nanocapsule is created around the bioactive peptides, which protects these compounds from possible changes in adverse conditions. Meanwhile, the capsules formed around the peptide in this technique release their contents at a controlled rate and under special conditions (Rao et al., 2016). Doogh is one of the favorite drinks of Iranians, which is produced from the lactic fermentation of milk. Due to its low pH and rich in nutrients, this product suffers from bacterial contamination, especially at ambient temperature, which ultimately causes spoilage and changes in its aroma during storage (Karimi et al., 2022). This is an important challenge in the milk industry. On the other hand, due to the disadvantages of chemical preservatives, it is very important to use natural preservatives that can solve this problem. The aim of the present research in the first stage is the nanoencapsulation of bioactive peptides obtained from lanternfish (Benthosema pterotum) by freeze drying method and maltodextrin-gum Arabic combined coating. In the next step, carrier nanocapsules and free peptides are used in doogh formulation and the microbial, chemical and sensory properties of the product are evaluated during 60 days of storage at refrigerator temperature. Methodology Lanternfish (B. pterotum) were hydrolyzed using Protamex enzyme under optimal conditions (50ºC, Amount of enzyme: 30 Anson units, 90 min) and then by ultrafiltration, peptides with a molecular weight of less than 3 kilodaltons were isolated. Then these peptides were encapsulated using the freeze drying method and combined maltodextrin-gum Arabic coating. In the next step, carrier nanocapsules (200 and 400 µg/ml) and free peptides (400 and 500 µg/ml) were added to the doogh formulation. Finally, Microbial (Staphylococcus aureus and Escherichia coli count), antioxidant (ABTS radical scavenging activity), chemical (pH and acidity) and sensory (color, aroma and taste) properties of the product were evaluated during 60 days of storage at refrigerator temperature (4±1°C). The data were analyzed by one-way analysis of variance (One-Way ANOVA in SPSS22 software) and the difference between the means was evaluated by Duncan's test at 95% confidence level (p<0.05). Result The results showed that the treatments containing nanocapsules carrying bioactive peptides (200 and 400 µg/ml) were more successful in inhibiting the growth and proliferation of S. aureus and E. coli bacteria as well as removing the ABTS free radical during the storage period compared to the control and the treatments containing free peptides (400 and 500 µg/ml) (p<0.05). The number of S. aureus bacteria in treatments containing 400 and 500 µg/ml of bioactive peptides as well as treatments containing 200 and 400 µg/ml of carrier nanocapsules on the first day of storage was 3.26 ± 0.08, 2.25±0.12, 2.24±0.09 and 1.11±0.06 Log CFU/mL, respectively. These numbers for Escherichia coli were recorded as 4.42±0.09, 3.21±0.17, 3.23±0.11, and 1.94±0.08 Log CFU/mL, respectively. The ABTS free radical scavenging rate in these treatments was measured on the same day as 29.73±1.11, 35.52±1.72, 39.65±2.12, and 47.14±1.18 Unit/ml, respectively. By increasing the concentration of free peptides (from 400 to 500 µg/ml) and carrier nanocapsules (from 200 to 400 µg/ml), as well as the storage time (1, 20, 40 and 60 days), the number of bacteria under study in the treatments decreased significantly (p<0.05). pH, acidity and ABTS radical scavenging power of treatments containing 200 and 400 µg/ml carrier nanocapsules did not change significantly during the storage period (p>0.05) and had a constant trend. While in treatments containing free peptides and control, antioxidant activity and pH decreased (p<0.05), but acidity increased by increasing storage time (p<0.05). The pH values for the control, treatments containing 400 and 500 μg/mL of bioactive peptides, as well as 200 and 400 μg/mL of carrier nanocapsules on day 60 of storage were measured as 3.03±0.02, 3.55±0.01, 3.56±0.05, 4.74±0.04, and 4.75±0.09, respectively. These values for acidity were recorded as 0.85±0.03, 0.67±0.02, 0.68±0.01, 0.36±0.07, and 0.37±0.03, respectively. By increasing the concentration of free peptides (from 400 to 500 µg/ml) and carrier nanocapsules (from 200 to 400 µg/ml) in doogh, the antioxidant activity (ABTS free radical scavenging power) of the treatments increased significantly (p<0.05), but the pH and acidity values of them did not change (p>0.05). The evaluation of sensory indicators (color, smell and taste) of doogh samples showed that only the treatment containing 400 µg/ml of carrier nanocapsules on the first and 60 day earned equal points (p>0.05), but in other treatments, the sensory indicators during the storage period were significantly have decreased (p<0.05). Of course, the treatment containing 200µg/ml also had equal points in two color and smell indices on the first day and 60, but the score of the taste index in this treatment decreased on the 60th day compared to the first day. The result of examining the sensory indicators of different treatments compared to the control on the first day showed that adding bioactive peptides and carrier nanocapsules to the doogh formulation had no significant effect (positive or negative) on the color, smell and taste of the product. Discussion and conclusion The reduction in the number of S. aureus and E. coli bacteria in treatments with the addition of bioactive peptides and carrier nanocapsules indicates the ability of the aforementioned compounds to combat the growth and proliferation of the studied bacteria That in this regard and also ABTS free radica scavenging, nanocapsules carrying bioactive peptides were more successful and stronger. This finding has been recorded due to the higher active surface area of the nanocapsules, as well as the capsule's (maltodextrin-gum Arabic) ability to protect the core (peptides) structure in adverse environmental conditions and its gradual release. Two mechanisms of peptides antibacterial activity are: A- Peptides are attached to the membrane of bacterial cells and create irreversible pores on the surface of the membrane, as a result of which the cytoplasm flows out of the cell and the cell is completely destroyed. Bioactive Peptides pass through the cell membrane and bind to intracellular organelles such as ribosomes and genomes and disrupt the normal growth process of the cell (Amissah, 2012). In the present research prove that the sensitivity of S. aureus, which is a Gram-positive bacteria, to free peptides and carrier nanocapsules is higher than E. coli. This higher sensitivity is related to the absence of the lipopolysaccharide layer in the cell wall of gram-positive bacteria (Reyhani Poul and Yeganeh, 2023). The results of the evaluation of pH, acidity and sensory properties of treatments during 60 days of storage showed that only in two treatments containing carrier nanocapsules, this indexes remained constant. But in the control and two treatments containing free peptides, mentioned indexes changed in a negative direction (decrease in the quality of dough). This finding indicates that in regard of controlling chemical and microbial reactions that cause food spoilage, bioactive peptides carrier nanocapsules are more powerful than free peptides. According to the obtained results, bioactive peptides from lanternfish with a molecular weight of less than 3 kilodaltons can be used as a natural preservative in the formulation of doogh. Also, if these peptides are nanoencapsulated using a combined maltodextrin-gum Arabic coating and freeze-drying technique, the resulting nanocapsules more actively combat oxidative and bacterial spoilage in doogh during storage at refrigerated temperatures. Conflict of interest The authors declare no conflicts of interest. Acknowledgment The Authors would like to thank the Sari Agricultural Sciences and Natural Resources University (SANRU), for financial support of this research under contract number, 03-1404-03. پپتیدهای زیست‌‌فعال, صمغ عربی, دوغ, فعالیت آنتی‌‌اکسیدانی و ضد میکروبی Bioactive peptides, Doogh, Antioxidant and antimicrobial activity 13 31 http://isfj.ir/browse.php?a_code=A-10-2360-15&slc_lang=fa&sid=1 Sakineh Yeganeh سکینه یگانه skyeganeh@gmail.com 100319475328460041414 100319475328460041414 Yes Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran گروه شیلات، دانشکده علوم دامی و شیلات، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران Soheyl Reyhani Poul سهیل ریحانی پول soheylreyhani@gmail.com 100319475328460041415 100319475328460041415 No Department of Processing of Fishery Products, Faculty of Fisheries and Environment, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran گروه فرآوری محصولات شیلاتی، دانشکده شیلات و محیط زیست، دانشگاه علوم کشاورزی و منابع طبیعی گرگان، گرگان، ایران Faezeh Tork pahnabi فائزه ترک پهنابی pahnabifaezeh@gmail.com 100319475328460041416 100319475328460041416 No Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran گروه شیلات، دانشکده علوم دامی و شیلات، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران Farzaneh Shakerian فرزانه شاکریان farzaneshakerian64@gmail.com 100319475328460041417 100319475328460041417 No Department of Fisheries, Faculty of Animal Science and Fisheries, Sari Agricultural Sciences and Natural Resources University, Sari, Iran گروه شیلات، دانشکده علوم دامی و شیلات، دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری، ایران