Bakladan ultrases destekli alkali ekstraksiyon ile protein eldesi: Teknofonksiyonel, ın vıtro biyoerişebilirlik ve ade inhibisyon özelliklerinin incelenmesi
Protein extraction by ultrasound assisted alkaline extraction from faba beans: Investigation of technofunctional, in vitro bioavailability and ace inhibition properties
Yazar: ZEHRA MERTDİNÇ
Danışman: PROF. DR. BERAAT ÖZÇELİK
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı / Gıda Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2022
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Alkaliler = Alkalies ; Proteinler = Proteins ; Ultrases dalgaları = Ultrasound waves ; Özütleme = Extraction

Özet:
Son yıllarda nüfus artışı, gıda kaynaklarının tükenmesi, iklim değişikliği ve hayvan hakları gibi birçok sebepten dolayı hayvansal gıda kaynaklarına alternatifler aranmaya başlamıştır. Bitkisel diyetlerin çevresel ve ekonomik katkılarının yanı sıra, kardiyovasküler hastalıklar, Tip-2 diyabet, obezite ve hipertansiyon gibi hastalıkların önlenmesinde de etkili olduğu bilinmektedir. Bakla, ülkemizde genellikle Ege, Marmara ve Akdeniz bölgelerinde üretimi yapılan, besleyici değeri yüksek ve biyoaktif bileşenler açısından zengin bir bitkisel protein kaynağıdır. Gıdaların besleyici ve fonksiyonel özelliklerinin geliştirilmesi için bakliyatlardan protein konsantresi/izolatı elde edilmektedir. Bu amaçla, özellikle alkali ekstraksiyon ve izoelektrik noktada çöktürme metodu sıklıkla kullanılan yöntemlerden biridir. Aynı zamanda bu yöntemler, ultrases, mikrodalga ve enzim destekli uygulamalar ile daha verimli hale getirilmektedir. Elde edilen proteinlerin, gıda matrisinde uygulamaları, depolanması ve işlenmesi noktasında teknofonksiyonel özelliklerinin (su/yağ tutma, köpürme kapasitesi/stabilitesi, çözünürlük gibi) incelenmesi gerekmektedir. Bu çalışmada da İzmir'de yetiştirilmiş bir bakla türünden alkali ekstraksiyon ve alkali ekstraksiyon öncesi ultrases uygulaması ile elde edilen protein konsantrelerinin teknofonksiyonel özellikleri incelenmiştir. 
Gıdalardan biyoaktif peptilerin eldesinde pepsin, alkalaz, tripsin gibi enzimler kullanılarak hidrolizasyonu gerçekleştirilebilir. Elde edilen hidrolizatlar, aminoasit dizilimleri, büyüklükleri ve bazı fizikokimyasal özelliklerine bağlı olarak anti-hipertansif, anti-oksidan ve anti-mikrobiyal etki gösterebilirler. Aynı zamanda hidrolize uğrayan peptitlerin insan vücudunda in vitro gastrointestinal sindirimin ardından biyoerişilebilirlikleri de etkilenmektedir. Bu çalışmada da alkali ve ultrases destekli alkali ekstraksiyonla elde edilen protein konsantrelerine pepsin ile enzimatik hidrolizasyon uygulanmış, ardından elde edilen hidrolizatlarda da in vitro protein biyoerişilebilirliği ve anti-hipertansif etkisinin belirlenmesi amacıyla ADE inhibisyon analizi uygulanmıştır. Bu çalışmada elde edilen sonuçlara göre, %27,32 protein miktarına sahip olan bakla unundan, alkali ve ultrases destekli alkali ekstraksiyonla sırasıyla %65,93 ve %75,04 protein içeriğine sahip konsantratlar elde edilmiştir. Ekstraktların teknofonksiyonel içerikleri de incelendiğinde, alkali ve ultrases destekli uygulamalar sonucunda ekstraktlarda protein çözünürlüğü, su ve yağ tutma kapasiteleri, köpürme stabiliteleri gibi özellikleri geliştirilmiş olup, çoğu uygulamada da ultrases uygulamasının alkali konsantrelerden istatistiksel olarak anlamlı bir farklılık gösterdiği tespit edilmiştir. Ayrıca hammadde, ekstraktlar ve enzimatik yöntemlerle elde edilen hidrolizatların, in vitro gastrointestinal sindirim sonrasındaki protein biyoerişilebilirlikleri incelenmiştir. Bu çalışmada da en yüksek protein biyoerişilebilirliği %46,39 ile ultrases destekli hidrolizatlarda tespit edilmiştir. Öte yandan, hidrolizatlarda ADE inhibisyon değerleri hesaplanarak baklanın potansiyel anti-hipertansif etkisi incelenmiştir. Analizler sonunda sırasıyla alkali ve ultrases yöntemlerinden elde edilen hidrolizatların IC50 değerleri 504,42 ve 222,89 μg/ml olarak hesaplanmıştır. Elde edilen sonuçlar göstermiştir ki, ultrases uygulaması ile ADE inhibisyon aktivitesinin arttığı gözlemlenmiştir. Özetle, bu çalışma ile Türkiye'ye özgü bir bakla türünden iki farklı yöntemle ekstrakte edilen proteinlerin teknofonksiyonel özellikleri tanımlanmış olup, enzimatik hidrolizasyonu ile de in vitro biyoerişilebilirlik ve ADE inhibisyon potansiyelleri incelenmiştir. Sonuç olarak, ultrases teknolojisinin, proteinlerin bazı teknofonksiyonel özellikleri, protein biyoerişilebilirliği ve anti-hipertansif etkileri üzerindeki olumlu etkileri incelenmiş ve gıda uygulamalarındaki potansiyeline dair bir bakış açısı geliştirilmesi hedeflenmiştir.

Abstract:
In recent years, people have tended toward consuming alternative plant protein sources rather than animal-based diets for many reasons, such as increasing population growth, depletion of food resources, climate change and animal rights. In addition to the environmental and economic contributions of plant-based diets, it is known that they are effective for preventing diseases such as cardiovascular diseases, type 2 diabetes, obesity and hypertension. One of the most important food groups used in plant-based diets is legumes. The vegetarian/vegan diet and the increasing trend toward gluten-free products, especially legume-based flour, pasta, and snack products, have increased interest in this market in developed countries. It is thought that the orientation toward legumes in particular will play an important role in the fight against hunger, which is one of the 2030 Sustainable Development Goals. In this context, international programs are also implemented in the sector that encourages the consumption of legumes. Faba beans are an excellent source of protein, energy, soluble fiber, and micronutrients (such as vitamin B, iron, zinc, calcium, magnesium, copper, etc.). In addition to being a protein source, it is a food that stands out for its low-fat content and high fiber content, especially its vitamin B and iron contents. Faba bean has a higher protein content than common legumes such as beans, lentils, and chickpeas except soy bean. However, as in other legumes, there are some anti-nutrients, such as tannin, phytic acid, oxalate, and lectins, in faba bean that cause a decrease in the absorption of protein and minerals in the body. Faba bean is largely produced in the Aegean, Marmara and Mediterranean regions, and Mersin, İzmir, Antalya, Aydın and Bursa are the provinces with the highest production rates in Turkey. In this study, the chemical composition of faba bean flour consisted of 9.76% moisture, 3.20% ash, 1.17% fat, and 27.32% protein. Additionally, the fatty acid profile of the selected faba bean flour was investigated in this study. According to the results, the fatty acid profile included palmitic acid (13.78%), oleic acid (32.50%), stearic acid (3.35%), cis-linoleic acid (45.59%), arachidonic acid (1.15%) and gamma linolenic acid (3.62%).
Protein concentrates/isolates are obtained from legumes to improve the nutritional and functional properties of foods. For this purpose, dry and wet extraction techniques can be used to obtain protein extracts. Alkaline extraction, which is one of the wet extraction methods, and isoelectric point precipitation are generally preferred in plant-based protein extraction. These conventional methods become more efficient with ultrasound-, microwave- and enzyme-assisted applications.
The basic principle in the ultrasonic extraction method is based on the collapse of microscopic bubbles formed by sound waves by releasing high energy in a short time, in other words, cavitation. It is known that the intracellular components/extract pass faster through the cell wall, which is mechanically destroyed by the effect of acoustic cavitation, to the solvent, and thus mass transfer increases. It is also known that the energy generated by cavitation is affected by many parameters, such as the amplitude and frequency of ultrasound application, the ambient temperature and pH, the type of solvent, the application time, and the characteristic properties of the proteins in the food matrix. Since ultrasound technology is an easily applicable, fast and environmentally friendly technology, it is involved in many applications, such as enzyme inhibition, microbial inactivation, sterilization, freezing, drying and extraction. The techno functional properties (such as water/oil binding capacity, foaming capacity/stability, solubility) of the obtained proteins should be analyzed to examine their storage and functionality during food processing. In this study, the techno functional properties (water/oil binding capacity, foaming capacity/stability, solubility, zeta potential, emulsion capacity/stability) of protein concentrates obtained from a faba bean species grown in İzmir by alkaline extraction and ultrasound-assisted alkaline extraction were investigated. Hydrolysis of bioactive peptides from foods can be performed using enzymes such as pepsin, alkalase, and trypsin. Protein hydrolysates may show antihypertensive, antioxidant, and antimicrobial effects depending on their amino acid sequence and some physicochemical properties. The degree of hydrolysis of proteins is a parameter that measures the degradation rate of peptide bonds and is used as an indicator of protein recovery. The hydrolysis process was carried out with pepsin in both extracts for 90 minutes. Then, at the end of the hydrolysis period, the hydrolysis degrees of alkaline and ultrasound-assisted extraction were calculated as 8.58% and 8.14%, respectively, and there was no statistically significant difference. At the same time, the bioaccessibility of hydrolysed peptides in the human body after in vitro gastrointestinal digestion is also important to reveal their bioactive properties. In addition, due to increasing heart diseases and negative side effects of medical drugs, there has been a tendency toward more reliable and economical food sources with antihypertensive effects in recent years. The role of angiotensin-converting enzyme (ACE) is important in the study of antihypertensive agents. There are many in vivo and in vitro studies on ACE inhibition in foods, especially meat and dairy products, aquaculture, cereals and legumes. In this study, enzymatic hydrolysis with pepsin was applied to protein concentrates obtained by alkaline and ultrasound-assisted alkaline extraction. Then, in vitro protein bioaccessibility and ACE inhibition analyses were applied to determine the antihypertensive effect of the obtained hydrolysates. According to the results of this study, faba bean flour had 27.32% protein content, and alkaline and ultrasound-assisted extraction protein concentrates with 65.93% and 75.04% protein content, respectively, were obtained. Ultrasound-assisted alkaline extraction revealed improved techno-functional properties, such as protein solubility, water and oil holding capacities, and foaming stability. In this study, the solubility of alkaline and ultrasound-assisted alkaline extracts of faba bean was investigated between pH 2 and 11. As a result, it is observed that the solubility of the protein concentrates is low between pH 3-5. Therefore, in this range, the isoelectric point of this raw material can be selected based on these values. Additionally, the solubility values and zeta potential of the products are convenient. In other experiments, the foaming stability of the extracts increased with the alkaline and ultrasound-assisted extraction methods, whereas there was no significant difference between the foaming capacity of the raw material and the extracts. In general, it was determined that ultrasound application showed a statistically significant difference from alkaline concentrates in most of the technofunctional properties applications. Legumes are very rich foods in terms of protein content and have biological activity. How much of these beneficial compounds are absorbed and used in the human body can be explained by the concepts of bioavailability and bioaccessibility. While "bioaccesibility" refers to the fractions that are digested and ready to be absorbed from the intestinal epithelial cells, leaving the food matrix and reaching the gastrointestinal tract. The bioaccessibility of food components is affected by many factors, such as the way the food is processed and prepared, the nutritional and health status of the individual, the physical and chemical properties of the components in the diet, the interactions between the components, the pH and microbiota of the digestive system. In this study, the protein bioaccessibility of raw materials, extracts, and hydrolysates obtained by enzymatic methods after in vitro gastrointestinal digestion was investigated. Protein bioaccessibility or digestibility is the ratio between the amount of protein consumed and the amount of protein absorbed by the human body and depends on the protein structure, preprocessing and food matrix. At the same time, protein digestibility is an important criterion in determining protein quality. In this study, the highest protein bioaccessibility was detected in ultrasound-assisted hydrolysates at 46.39%.
In addition, the potential antihypertensive effect of faba beans was investigated by analyzing ACE inhibition of hydrolysates. At the end of the analysis, the IC50 values of the hydrolysates obtained from alkaline and ultrasound-assisted extraction were calculated as 504.42 and 222.89 μg/ml, respectively. Differences in ACE inhibition values depend on hydrolysis conditions (time, pH, etc.), used enzymes, process effects (such as heat treatment, microwave, ultrasound) and the chemical structure of the raw material. Literature studies and the results obtained from this study showed convenience, and it was observed that ACE inhibition activity of the protein hydrolysates was increased by ultrasound application. At the same time, all these results support the use of ACE inhibitors obtained from plant-derived proteins as functional foods.
The food industry is interested in the manufacture of plant protein concentrates because of the increased demand for protein and the increasing use of plant-derived proteins in foods. This study will help to integrate faba bean protein concentrates into the food industry. Further studies are needed to improve the quality of the protein by assisting alkaline extraction with different novel extraction methods, such as microwave, irradiation, and high pressure.In summary, in this study, the techno-functional properties of proteins extracted by alkaline and ultrasound-assisted extraction from a faba bean species native to Turkey were investigated, along with enzymatic hydrolysis, ACE inhibition potentials, and in vitro protein digestibility. As a result, the positive effects of ultrasound technology on some techno-functional properties, protein bioaccessibility, and anti-hypertensive effects were examined to develop a perspective on its potential in food applications.