بهینه سازی شرایط تشکیل کونژوگه های حاصل از پروتئین های شیر با بخش محلول صمغ های بومی ایرانی (صمغ فارسی و کتیرا)

واکنش میلارد یک روش شناخته شده جهت ایجاد اتصال کوالانسی بین پروتئین ها و بسیارقندی ها می باشد که به منظور بهبود ویژگی های عملکردی پروتئین ها استفاده می شود. بنابراین، هدف از پژوهش حاضر بهینه سازی تولید کونژوگه های پروتئین های شیر–بخش محلول صمغ های بومی با استفاده از روش سطح پاسخ و کاربرد آن ها به‌منظور افزایش پایداری امولسیون های آب در روغن بود. برای تولید کونژوگه های پروتئین– بسیارقندی، حرارت دهی به روش خشک (دمای 60 درجه سانتی گراد و رطوبت نسبی 79 درصد) انجام شد و تاثیر هم زمان نسبت پروتئین:بسیارقندی و زمان حرارت‌دهی بر متغییرهای وابسته (میزان جایگزینی آمین آزاد و تغییر رنگ) بررسی گردید. به علاوه، جهت بررسی امکان بهبود پایداری امولسیون ها با استفاده از کونژوگه های حاصل، روغن آفتابگردان (4 درصد وزنی/ وزنی) به محلول کونژوگه پروتئین– بسیارقندی (4/0 درصد وزنی/ وزنی) افزوده شده و همگن سازی توسط امواج فراصوت انجام شد. طبق نتایج به‌دست آمده، شرایط بهینه از لحاظ زمان حرارت دهی و نسبت پروتئین: بسیارقندی در روش میلارد خشک به ترتیب برای ایزوله پروتئین های سرمی–بخش محلول صمغ فارسی 8 روز و 59:1/1، برای کازئینات سدیم–بخش محلول صمغ فارسی 7 ساعت و 2:1، برای ایزوله پروتئین های سرمی–بخش محلول صمغ کتیرا 64/13 روز و 3:1 و برای کازئینات سدیم–بخش محلول صمغ کتیرا 82/7 ساعت و 3:1 بود. نتایج این تحقیق نشان دادند که صمغ فارسی در مقایسه با صمغ کتیرا توانایی بیشتری در ایجاد اتصال کوالانسی با پروتئین های شیر داشت. هم چنین، اتصال پروتئین به بخش محلول صمغ های بومی نه تنها منجر به پایداری کامل امولسیون ها نشد، بلکه سبب کاهش کارایی پروتئین ها در پایداری امولسیون ها شد.

Optimization of milk proteins–soluble fraction of Iranian native gums conjugate formation via the Maillard reaction

Introduction: Maillard reaction is a wellknown technique for covalent coupling of protein–polysaccharide which is usually used to improve the functional properties of proteins. Conjugation by the dry heating method is occurred during Amadori rearrangement step in the Maillard reaction where amine groups of the proteins are linked to the reducing end of the polysaccharides. This glycation process leads to the improvement of solubility, foaming and emulsifying properties of proteins (Liu et al., 2012). Proteinbased emulsions are sensitive to pH and ionic strength alterations as well as heating and freezing–thawing processes. While, joining proteins to high molecular weight polysaccharides protects them against environmental stresses. A wide range of carbohydrates were already utilized to enhance the emulsifying properties of proteins–polysaccharides namely whey protein isolate–dextran (Akhtar et al., 2003), βlactoglobulin–dextran (Wooster et al., 2006), sodium caseinate–maltodextrin (O’Regan et al., 2009), deamidated wheat protein–maltodextrin or glucose (Wong et al., 2011), whey protein isolate–pectin (Xu et al., 2012), yolk phosvitin–dextran (Chen et al., 2014), βlactoglobulin–sixcarbon monosaccharides (Cheetangdee et al., 2014), soy protein isolate–soy soluble polysaccharides (Yang et al., 2015), lysozyme–tragacanth (Koshani et al., 2015), and βlactoglobulin–Persian gum (Golkar et al., 2015). However, based on the existing literature, it seems that conjugation of Iranian native gums and proteins needs to be more attended to show their potential applications. Therefore, in this study, conjugate formation between milk proteins (sodium caseinate and whey protein isolate) and soluble fraction of Iranian native gums (gum tragacanth and Persian gum) was optimized using response surface methodology (RSM) and the resulting conjugates were used in emulsion formulation in order to compare protein capability before and after being attached to the gums. Materials and methods: Iranian native gums were pulverized and sieved (mesh size < 60) after being prepared from local herbal stores. To obtain protein–polysaccharide conjugates, dry heat treatment was accomplished at 60oC and 79% relative humidity at different protein:polysaccharide ratios and heating times. Free amino group content was determined by the ninhydrin method described by Doi et al. (1981). To measure color changes before and after Maillard reaction, Hunter Lab was used. Besides, possibility of stabilizing oilinwater emulsions using the conjugates was studied. Emulsions were formed by addition of oil phase (4% w/w) into protein–polysaccharide conjugates solution (0.4% w/w) following by ultrasound treatment (amplitude of 100%, for 4 min). Samples were kept at 4 oC for 30 days after adjusting the pH at 3, 5 and 7 to compare the effect of protein–polysaccharide conjugates on their stability at different protein charges. For optimization of Maillard reaction using RSM (central composite design), protein:polysaccharide ratio and heating time were selected as independent variable and were studied at 5 levels. The dependent variables were the substitution degree of free amino groups and color change. Results & Discussion: Based on our findings, the decrease of free amino group indicated that NH2 group of amino acids in milk proteins was covalently linked to carbonyl group of the gums. Comparing free amino group reduction of the samples containing soluble fraction of gum tragacanth and Persian gum also showed that Persian gum was more capable of forming covalent linkage with milk proteins than gum tragacanth; probably, due to the lower side branches of Persian, its molecular weight as well as its structural flexibility. Moreover, we believe the higher side branches of attachedtragacanthin molecules might prevent further attachment of protein to the other polysaccharide molecules via steric repulsion. According to the results of color measurement before and after dry Maillard reaction, conjugation led to reduction of L* and increase of a* and b*. In addition, higher heating time and protein:polysaccharide ratio caused progress of Maillard reaction (especially in the case of milk proteins–soluble fraction of Persian gum). Based on our findings, in dry Maillard reaction, the optimum heating time and protein:polysaccharide ratio were 8 days and 1:1.59 for whey protein isolate–soluble fraction of Persian gum, 7 h and 1:2 for sodium caseinate–soluble fraction of Persian gum, 13.64 days and 1:3 for whey protein isolate–soluble fraction of gum tragacanth, and 7.82 h and 1:3 for sodium caseinate–soluble fraction of gum tragacanth, respectively. Besides, the obtained Maillard reaction products did not cause complete stability of emulsions at pHs 3, 5 and 7. Our findings also declared that attachment of polysaccharides to proteins might negatively affect the proteins functionality, as emulsifier, possibly by hindering adsorption of hydrophobic groups of proteins to oil droplets. Furthermore, homogenization process during emulsion preparation using ultrasound could break the formed covalent bonds and polysaccharide structure leading to lower steric repulsion and viscosity.