Endüstriyel havuç suyu atıklarından enzim ve ultrason destekli ekstraksiyon ile gıda bileşeni üretilmesi
Production of food ingredients from industrial carrot juice waste using enzyme and ultrasound assisted extraction
Yazar: YASEMİN DEMİR
Danışman: DOÇ. DR. DERYA KAHVECİ KARINCAOĞLU
İstanbul Teknik Üniversitesi / Lisansüstü Eğitim Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı / Gıda Mühendisliği Bilim Dalı
Derece: Yüksek Lisans, 2024
Konu: Gıda Mühendisliği = Food Engineering

Özet:
Küresel olarak, insan tüketimi için üretilen gıdaların yaklaşık üçte birinin kaybolması veya israf edilmesi en önemli gıda sistemi sorunlarından biridir. Nüfus ve tüketim sürekli artarken, gıda talebinin en azından bir sonraki 40 yıl boyunca artması beklenmektedir, bu da doğal kaynaklar olan toprak, su ve enerji üzerinde baskı oluşturmaktadır. Gıda israfının önlenmesi bir önceliktir, ancak bazen bu ekonomik veya teknolojik olarak mümkün olmamaktadır. Bu nedenle, gıda atıklarının yem ve enerji üretimi gibi çeşitli süreçler için kaynak olarak kullanılması için çaba gösterilmektedir. Tarım ve gıda sektörlerinden gelen atıklar besin maddeleri bakımından zengin olmasına rağmen, bunların bertarafı çevresel ve sağlık sorunlarına yol açabilmektedir. Araştırmacılar, bu sorunları en aza indirmek ve değerli biyoaktif bileşikleri çıkarmak için ultrasonik ve enzimatik destekli ekstraksiyon gibi çevre dostu teknolojileri araştırmaktadır. Çevre dostu metotlar arasında yer alan enzim ve ultrason destekli ekstraksiyon; çözgen kullanımını azaltarak, daha az enerji sarfiyatı sağlayarak ve verimi artırarak fayda sağlamayı amaçlamaktadır. Havuç sebzesi meyve suyu fabrikalarında işlendikten sonra genellikle endüstriyel atık olarak atılır veya hayvan yemi olarak kullanılırken, ortaya çıkan bu yan ürün karotenoidler ve fenolik bileşikler gibi yüksek oranda yararlı bileşikler içermektedir. Dolayısıyla bu yan ürünün farklı endüstrilerde (gıda, sağlık, kozmetik vb.) kullanılmasının hem katma değerli ürün elde edilmesinde hem de çevresel konularda faydalı olabileceği düşünülmektedir. Bu çalışmanın amacı, endüstriyel havuç suyu atıklarına çevre dostu ön işlemler (enzimatik ve ultrasonik destekli ekstraksiyon) uygulanarak gıda endüstrisinde önemli kullanım alanı olan bileşenlerin elde edilmesi için Sürdürülebilir Kalkınma Hedefleri'ne uygun bir yaklaşım geliştirilmesi amaçlanmakla birlikte çalışmada bahsedilen yöntemler üzerinde optimizasyon koşullarının belirlenmesidir.
Öncelikle, geleneksel, enzimatik ve ultrasonik yöntemlerin her biri için solvent türü, katı-sıvı oranı, enzim türü, enzim miktarı, enzimatik ve ultrasonik destekli ön işlem süresi parametreleri beta karoten, likopen, toplam fenolik madde konsantrasyonlarına ve DPPH % inhibisyonuna göre optimize edilmiştir. Analizler spektrofotometre ile belirlenen dalga boylarında ölçümlenmiştir. Yapılan çalışmalar sonucunda solvent türü ve katı-sıvı oranı sırası ile etanol ve 1:40 (w/v) olmuştur. Enzimatik ön işlem parametreleri ise enzim türü için Pectinex® XXL, enzim miktarı için 300 μL ve ekstraksiyon süresi için 120 dk optimum koşulları vermiştir. Ultrasonik ön işlemin süresi 20 dk olarak belirlenmiştir. Sonuçlara göre, belirlenen bu optimum koşulların geleneksel, enzimatik ve ultrasonik ön işlem destekli ekstraksiyon yöntemlerine uygulanması ile istatistiksel olarak enzim ve ultrason ile ön işlem uygulamasının bu gıda bileşenlerinin geri kazanımını artırabileceğini göstermiştir (p<0.05).
Optimum koşullar belirlendikten sonra havuç posaları geleneksel, enzimatik destekli, ultrasonik destekli, enzimatik-ultrasonik destekli ve ultrasonik-enzimatik destekli 5 farklı ekstraksiyon yöntemi ve kombinasyonu ile ekstrakte edilmiştir. Elde edilen bütün sıvı ekstraktların önce dönel buharlaştırıcı ile içerdiği etanol uçurulmuş daha sonra ise dondurarak kurutulmuştur. Bütün analizler katı madde üzerine uygulanmıştır. Ekstraksiyon yöntemlerine göre elde edilen bileşenlerin konsantrasyon sonuçlarına baktığımızda enzimatik destekli ekstraksiyonun sonuçları diğer yöntemlere göre daha etkili bulunmuştur. Enzim destekli ekstraksiyonun sonuçları şu şekildedir: 26,37 ± 0,26 mg/g beta-karoten konsantrasyonu, 20,93 ± 0,005 mg/g likopen konsantrasyonu, 9,97 ± 1,38 mg GAE/g toplam fenolik madde konsantrasyonu, 209,44 ± 7,98 mg rutin/g toplam flavonoid madde konsantrasyonu ve 25,57 ± 4,15 DPPH % inhibisyonudur. Ayrıca hem E.coli hem de Salmonella Typhimurium için en etkili antimikrobiyal aktivite gösteren yöntemin enzim destekli ekstraksiyon yöntemi olduğu gözlemlenmiştir. Bulgulara bakıldığında enzim uygulamasının hücre duvarını parçalaması özelliğinden dolayı hedef bileşenlerin salınımını büyük ölçüde etkilediği söylenebilmektedir. Ek olarak Enzim - Ultrason Destekli Ekstraksiyon uygulamasının ise ikinci etkili yöntem olduğu tespit edilmiştir. Bu uygulamada ise ek olarak ultrason kullanımının kavitasyon etkisi ile hücre duvarını parçalayarak verimi artırmada etkili rol oynadığı gözlemlenmiştir.

Abstract:
It is estimated that globally, one-third of the edible parts of food produced for human consumption is lost or wasted. Wasted food is a major component of the world's food system problems. The global population is rapidly increasing, urbanizing and becoming richer; This is leading to diversification of dietary patterns and increased demand for land, resources and greenhouse gas-intensive foods such as meat and dairy. It is estimated that continuing population and consumption growth worldwide will lead to an increase in global food demand for at least another 40 years, which will lead to an intensified use of natural resources, especially land, water and energy. These challenges are further exacerbated by the world's changing environmental conditions, which cause food production to become unpredictable and increasingly difficult globally. Although preventing/reducing food waste is considered a top priority, it is sometimes not economically or technologically possible. In this sense, food waste should be evaluated as much as possible because it can be used as a resource for many other processes such as feed and energy production. Wastes from the agricultural and food sectors are often rich in proteins, carbohydrates and lipids, which serve as breeding grounds for numerous microorganisms. The presence of nutrients and high moisture content in food waste is the main cause of dioxin formation during the incineration of food waste, leading to various health and environmental problems. With the concept of sustainable development, researchers strive to transform waste into high-value products. Sustainable development of the circular bioeconomy concept is only possible by adopting potential advanced technologies for the utilization of food waste. Advanced technologies such as ultrasound-assisted extraction, microwave- assisted extraction, bioreactors, enzyme immobilization-assisted extraction and their combination can minimize the global concern arising from mismanagement of food waste. Although artificial bioactive compounds have been approved for food applications in many countries, their use is not well received by consumers. Therefore, there is increasing interest in replacing artificial bioactive compounds with natural bioactive compounds. These natural compounds can be used as food additives to maintain food quality, food safety and appearance, and as food supplements or nutraceuticals to correct nutritional deficiencies, maintain appropriate nutrient intake or support physiological functions, respectively. Recent research reveals that a large number of food wastes, especially fruit and vegetable by-products, are a good source of bioactive compounds that can be extracted and reintroduced into the food chain as natural food additives or in food matrices to obtain nutraceuticals and functional foods. Utilizing such food waste not only reduces the burden of bio-waste on industries and the ecosystem, but also positively affects the profitability of the industry by ensuring the production of high value-added products. Enzyme and ultrasound-assisted extraction, which are among the environmentally friendly methods; It aims to provide benefits by reducing solvent use, providing less energy consumption and increasing efficiency. Food waste contains different types of valuable substances depending on their type. Carotenoids and phenolic compounds are two important compounds that can be obtained from food waste through these environmentally friendly methods. Since the majority of food waste consists of fruits and vegetables, there are many studies on recycling. Citrus peel, apple peel, carrot pulp, grape peel, and pomegranate peel are common examples used in many studies. While the carrot vegetable is usually discarded as industrial waste or used as animal feed after being processed in juice factories, this resulting by-product contains highly beneficial compounds such as carotenoids and phenolic compounds. Therefore, it is thought that the use of this by-product in different industries (food, health, cosmetics, etc.) can be beneficial both in obtaining value-added products and in environmental issues. The aim of this study is to develop an approach in line with the Sustainable Development Goals to obtain components that have important uses in the food industry by applying environmentally friendly pre-treatments (enzymatic and ultrasonic assisted extraction) to industrial carrot juice waste, and to determine optimization conditions on the methods mentioned in the study. When we look at the literature, there is no study in which combinations of different environmentally friendly methods are used to extract all of these components.
First of all, carrot pulps, which were frozen at -40oC for traditional extraction, were thawed at room temperature. Then, 5 g of carrot pulp was weighed and 100 mL of ethanol was added. The samples were kept in a shaking water bath at 150 rpm for 60 minutes. At the end of 60 minutes, the liquid and solid phases were separated from the samples. While the liquid phases were taken into separate falcons, 100 mL of ethanol was added to the solid phases and kept in a shaking water bath under the same conditions for another 60 minutes. At the end of 60 minutes, the liquid and solid phases were separated again and added to the liquid phase taken at the end of the first hour. Finally, the obtained liquid part was subjected to phenolic substance amount, flavonoid substance amount, beta carotene and lycopene amount and DPPH % inhibition analysis. To select the most effective solvent and solid-liquid ratio for extraction, 3 different solvents (ethanol, hexane and 1:1 ethanol-hexane mixture) and 3 different ratios (1:20, 1:30 and 1:40) were tried. Then, before enzyme-assisted extraction, 5 g of carrot pulp was weighed and 50 mL of pure water was added. The pH of the samples was adjusted to 5 and 300 μL of Pectinex® XXL was added. It was kept in a shaking water bath previously heated to 50oC for 120 minutes. Then, the samples were taken into falcons and centrifuged at 25oC and 8000 rpm for 10 minutes to separate water and pulp. The shaking water bath was brought back to room temperature and the traditional extraction method was applied to the carrot pulps in the same way. Celluclast® 1.5L, Pectinex® XXL and Celluclast® 1.5L - Pectinex® XXL (1:1 v/v) Mixture enzymes were used to decide the enzyme type. To determine the amount of enzyme and pretreatment time required for enzymatic pretreatments, 3 different enzyme amounts (200, 300 and 500 μL) and 3 different times (30, 60 and 120 min) were used. In the ultrasound-assisted extraction method, 100 mL of ethanol was added to 5 g of carrot pulp and subjected to 35 kHz frequency for 20 minutes in an ultrasonic water bath. Then, the traditional extraction method was applied to the samples in the same way. Pretreatment times of 10, 20 and 30 min were used to determine the ultrasound pretreatment time. Additionally, in order to see the effect of sequential combinations of methods, enzymatic-ultrasonic sequential pretreatment and ultrasonic-enzymatic sequential pretreatment were applied according to the selected optimum conditions. The ethanol contained in all the liquid extracts obtained was first evaporated using a rotary evaporator and then freeze-dried. All analyzes were performed on solid matter. As a result of the studies, the solvent type and solid-liquid ratio were ethanol and 1:40 (w/v), respectively. Enzymatic pretreatment parameters gave the optimum conditions as Pectinex® XXL for enzyme type, 300 μL for enzyme amount and 120 min for extraction time. The duration of ultrasonic pretreatment was determined as 20 min. According to the results, the application of these optimum conditions to traditional, enzymatic and ultrasonic pretreatment assisted extraction methods statistically showed that enzyme and ultrasound pretreatment can increase the recovery of these food components (p<0.05). Total beta carotene, lycopene, phenolic substance amount, flavonoid substance amount and antioxidant activity of the extract obtained under the selected optimum conditions were determined using spectrophotometric methods. Antimicrobial activity analyzes were performed by the double-layer serial dilution method. When we look at the concentration results of the components obtained according to the extraction methods, the results of enzymatic assisted extraction were found to be more effective than other methods. The results of enzyme-assisted extraction are as follows: 26.37 ± 0.26 mg/g beta-carotene concentration, 20.93 ± 0.005 mg/g lycopene concentration, 9.97 ± 1.38 mg GAE/g total phenolic substance concentration, 209.44 ± 7.98 mg routin/g total flavonoid substance concentration and 25.57 ± 4.15 DPPH % inhibition. Additionally, it was observed that the method showing the most effective antimicrobial activity for both E.coli and Salmonella Typhimurium was the enzyme-assisted extraction method. Considering the findings, it can be said that enzyme application greatly affects the release of target components due to its ability to break down the cell wall. Additionally, Enzyme - Ultrasound Assisted Extraction has been found to be the second effective method. In this application, it has been observed that the use of ultrasound also plays an effective role in increasing the efficiency by breaking down the cell wall with the cavitation effect.
This system, which has been tested at laboratory scale, needs to be produced in industry-used sizes and its applicability tested. However, the sensitivity of the components to be recovered may make this application difficult on a large scale and may create an additional cost. On the other hand, due to increasing demand, sustainability and environmental concerns, the importance of recycling with environmentally friendly methods will increase even more. Based on the findings obtained in this study, studies can be carried out to obtain components with higher efficiency or more advanced quality properties.