BAMLET proteini üretimi ve koaservasyon yöntemiyle stabilitesinin arttırılması
Production of BAMLET protein and increase stability with coacervation
Yazar: REYHAN KOYUNCU
Danışman: PROF. DR. BERAAT ÖZÇELİK
İstanbul Teknik Üniversitesi / Fen Bilimleri Enstitüsü / Gıda Mühendisliği Ana Bilim Dalı
Derece: Yüksek Lisans, 2017
Konu: Gıda Mühendisliği = Food Engineering
Dizin: Mikroenkapsülasyon = Microencapsulation ; Serum proteinleri = Serum proteins ; Süt proteinleri = Milk proteins ; İlaç stabilitesi = Drug stability

Özet:
Enkapsülayon; gıda bileşenlerinin korunması, stabilizasyonu ve yavaş salınımı için kullanılan nispeten yeni bir teknolojidir. Bununla birlikte enkapsülasyon gıda dışında ilaç ve kozmetik gibi geniş bir kapsamda kullanılmaktadır. Bahsedilen alanlarda birçok çalışma mevcuttur. Ancak önemli terapötik etkiye sahip protein ve peptit kaynaklı ilaç etkin maddelerinin enkapsülasyonu ile ilgili çalışma sayısı fazla değildir. Bu tip ilaçların klinik kullanımı için üretim ve depolama koşulları gibi in vitro şartların kararlılığa etkisi yanında; in vivo koşulların (örn: enzimatik hidroliz) terapötik etki üzerine olumsuz sonuçları bulunmaktadır. Bu stabilite sorunlarını enkapsülasyon ile elimine etmek mümkündür.
Bu tezde, terapötik etkiye sahip BAMLET proteini koaservatı oluşturularak stabilitesi arttırılmıştır (O7 w/w). Koaservasyonda pektin kullanılmış ve püskürtmeli kurutulmuştur. Proteinin stabilitesini etkileyen faktörler ve koaservasyon verimi incelenmiştir. Oral biyoyararlanımı arttırmak için yapılan çalışmada, mide asitliğine yakın bir pH seçilmek istenmiştir. Bu nedenle pH 3 koaservasyon için hedef pH değerini oluşturmuştur. Bağırsaklara gelindiğinde ilacın salınımı istenmektedir. Bu nedenle pH 7 ve sonrasında koaservasyonun yapısının bozulması ilacın salınması beklenmektedir.
BAMLET (Bovine Alpha lactalbumin Made LEthal to Tumor cells) proteini tümorisidal etkiye sahip etkin bir proteindir. Memeli sütlerinden elde edilen ve α-laktalbuminin-oleik asit ile birleşiminden oluşan bu proteinin, ilaç etkin maddesi haline getirilme sürecindeki olumsuz koşullardan etkilenmesi önlenmeye çalışılmış hatta bu sayede ilaç haline getirilmesi olası hale gelmiştir.
BAMLET proteinini üretmek için öncelikle inek sütünden α -laktalbumin elde edilmiştir. Literatüre uygun olarak 1 litre sütten 1.2 g α-laktalbumin elde edilmiştir. Proteinin oleik asit ile birleşimi sıcaklık uygulaması yöntemiyle gerçekleştirilmiştir. Alfa-laktalbumin ve BAMLET proteini, Ultra Performans Sıvı Kromatografisi (UPLC) ile tanımlanmıştır. Kromatogramda tek pik çıkmış ve literatürde tanımlandığı gibi 280 nm dalga boyunda sonuç elde edilmiştir. Elde edilen BAMLET için en uygun kaplama materyali pektin olarak belirlenmiştir.
BAMLET proteini validasyonu için HPLC (yüksek performanslı sıvı kromatografisi) kullanılmıştır. Bunun için öncelikle doğrusal aralık ve kalibrasyon eğrisi oluşturulmuştur. Yöntemin doğruluğu ve kesinliği, standart sapma ve bağıl hata değerleri yaklaşık olarak % 4.3'den küçük olduğu verisi ile ispatlanmıştır. Ayrıca geri kazanım değeri BAMLET proteinini süt ve elma suyuna ilave edilerek hesaplanmıştır. Gıda preparatlarından elde edilen geri kazanım değerleri % 98.0 ile % 100.16 arasında olduğu belirlenmiş ve proteinin validasyonu sağlanmıştır.
BAMLET proteininin  pektin ile farklı oranlarda (O1-O7; protein:pektin) koaservatı oluşturulmuştur. Koaservasyon veriminin belirlenmesi için analitik yöntemlere başvurulmuş ve Flaş kromatografide absorbans ölçümleri yapılmıştır. En iyi verim, bulanıklık gözlemlerine paralel olarak O7 w/w Protein:pektin oranı olarak belirlenmiştir. Bu konsantrasyonda partikül büyüklüğü 335,6 nm ve ζ-potansiyeli -25,4 mV olarak ölçülmüştür ve koaservasyon veriminin moleküller arasında elektrostatik etkileşiminden büyük oranda etkilendiği belirlenmiştir.  Daha sonra koaservat püskürterek kurutulmuş bu sayede kararlılığı arttırılmıştır.
Fourier Transform Infrared (Kızılötesi) Spektroskopisi(FTIR) ile BAMLET, pektin ve koaservat (O7 protein:pektin w/w) üzerine yapılan analizde; BAMLET-pektin koaservatının 1653 cm ̄ ˡ ve 1572 cm ̄ ˡ 'aralığında oluştuğu gözlenmiştir. BAMLET (-COO ̄ ) karboksil grupları ile pektin amin grupları (-NH₃⁺) arasındaki elektrostatik etkileşimi bu aralıkta belirlenmiştir. FTIR koaservasyonun oluşum şeklini bağ yapıları ile ifade etmektedir.
Püskürtülerek kurutulmuş BAMLET-pektin koaservatınn termal özelliklerinin belirlenmesi için diferansiyel taramalı kalorimetri (DSC) kullanılmıştır. Bu sayede püskürtülerek kurutulmuş koaservatın denatürasyon pik sıcaklığı ve denatürasyon entalpisinin (Td=163.57°C, ΔHd=261.6 j/g), hem BAMLET proteininin denatürasyon pik sıcaklığı ve denatürasyon entalpisinden (Td=136.33°C, ΔHd=35.95 j/g), hem de sıvı formadaki koaservatın denatürasyon pik sıcaklığı ve denatürasyon entalpisinden (Td=157.6°C, ΔHd=104.3 j/g) daha yüksek olduğu gözlenmiştir. 
BAMLET proteini dielektrik sabiti incelendiğinde (10.05 ±0.05) sıvı koaservatın polaritesinden (78.063 ±1.3) çok daha düşük olduğu görülmüştür. Solventin poleritesinin bir ölçütü olan dielektrik sabiti, BAMLET proteinin koaservatına göre daha az polariteye sahip olduğunu göstermiştir. Çözeltide daha büyük bir dielektrik sabiti olan solventin daha yüksek bir net yük yoğunluğuna sahip olduğu düşünülmektedir.

Abstract:
Encapsulation is a relatively new technology, which can be used for protection of food items, stabilization of food and slow release. In addition, encapsulation able to use other disciplines such as medicines and cosmetics widely. There is a lot of work in the areas mentioned. However, the therapeutic effect of protein and peptide-induced significant drug encapsulation of active ingredients is not more about running count. The clinical use of this type of drugs can be affected by production and storage conditions namely invitro conditions determination. Invivo conditions (e.g.: enzymatic hydrolysis) has negative consequences on the therapeutic impact. These stability problems are possible to eliminated with encapsulation method. From the formulation perspective, proteins are encapsulated to provide efficient protection for active protein moiety against a physiological environment and targeting to the site of action allowing optimal therapeutic efficiency. Some proteins are influenced by gastric medium. In order to prevent this instability, we suggest to encapsulate model protein with pectin.
Protein pharmaceuticals are the fastest growing class of new therapeutic agents and are an important research topic in the (bio) pharmaceutical industry. For this purpose, the methods of encapsulation in the field are studied. The therapeutic effect is based on obtaining a low dose of the pharmacotherapeutic effect on the beneficial component. In addition to this, the use of carrier systems provides controlled release of the drug and preservation of the stability of the active substance.
The active ingredients namely protein and peptides which are the most widely used in the pharmaceutical field. Proteins that are very small proportions in the human body may be transported by diffusion or active transport to the site of action. It is desirable that the proteins in the protein structure should be transported to these effect areas in small doses in the appropriate dosage forms. Targeting these drugs brings many difficulties.
Protein and peptide structured proteins are cleaved by enzymes in the gastrointestinal tract. We do not want the a-lactalbumin-oleic acid composition (BAMLET) to break down in the digestive tract, which we want to make the drug active substance. If the half-life is considered to be short, controlled release and increased gastric stability are essential.
HAMLET (Human Alpha Lactalbumin Made LEthal to Tumor cells) protein, which we use because of its anticarcinogenic and therapeutic effect, is the molecular complex of human α-lactalbumin derived from human oil and oleic acid, a naturally occurring unsaturated fatty acid. As in some tumor cells, such as skin, breast and lung cancer, they induce apoptosis through autophagy in cells, but do not damage normally distributed cells. However, the mechanism of action is not fully understood. After proving the presence of the protein's tumoricidal effect, other mammalian milks were investigated and then the same effect was found in the complexes of the a-lactalbumin derived from all mammalian milk with oleic acid. The protein name varies according to the mammal species obtained. (E.g. BAMLET: Bovine Alpha lactalbumin Made LEthal to Tumor cells).
In this thesis, coacervation with pectin (O7 w / w) is proposed and investigated to increase the gastric stability and to provide controlled release of the proteinaceous BAMLET to be administered orally. Cellulose derivatives are used as coating material because they have a beneficial effect on coacervation. However, there are few studies in the literature about encapsulation of proteins. Apart from that, BAMLET / HAMLET etc. There is no current study of the encapsulation of proteins. For this reason, the coacervates of BAMLET selected as model protein using the coacervation method have been prepared and characterized in our study. Pectin had been used for coacervation and also it had been done with spray drying method. Factors, which are affecting the stability of the protein and the coacervation efficiency, have been investigated.
BAMLET (Bovine Alpha lactalbumin Made LEthal to Tumor cells) is an effective protein with a tumoricidal effect. It has been tried to prevent the adverse conditions in the process of making this protein, which is obtained from mammalian milk and which is composed of a-lactalbumin-oleic acid, to become a drug active substance.
In order to produce BAMLET protein, firstly alpha-lactalbumin was obtained from the cow's milk. In accordance with the literature, 1.2 grams of alpha lactalbumin was obtained from 1-liter milk. The association of the protein with oleic acid has been carried out by means of temperature application. Alpha-lactalbumin, hydrolyzed alpha-lactalbumin and BAMLET protein were identified by Ultra Performance Liquid Chromatography (UPLC). A single peak was given in the chromatogram and a result of 280 nm wavelength was obtained as described in the literature. The most suitable coating material for the obtained BAMLET was identified as pectin.
HPLC (high performance liquid chromatography) was used for BAMLET protein validation. For this, firstly linear interval and calibration curve are created. The accuracy and accuracy of the method has been proved by the data that the standard deviation and relative error values are less than approximately 4.3%. In addition, recovery value of BAMLET protein can be calculated by adding milk and apple juice. The recovery values obtained from the food preparations were determined to be between 98.0% and 100.16% and protein validation was achieved.
The BAMLET protein was coacervated with pectin at different ratios (O1-O8, protein: pectin). Analytical methods were used to determine the coherence efficiency and absorbance measurements were performed on the flash chromatograph. The best efficiency was determined as O7 w / w Protein: pectin ratio parallel to turbidity observations. At this concentration, the particle size was measured as 335.6 nm and ζ-potential -25.4 mV and it was determined that the efficiency of coacervation was greatly affected by the electrostatic interaction between the molecules. It was then dried by spraying coacervate and its stability was increased. 
Analysis by Fourier Transform Infrared Spectroscopy (FTIR) on BAMLET, pectin and coacervate (O7 protein: pectin w / w); BAMLET-pectin coacervate was observed at 1653 cm ̄ ˡ and 1572 cm ̄ ˡ '. The FTIR spectrum of BAMLET  showed absorption bands related to C=O stretching at 1629 cm ̄ ˡ (free carboxyl groups) and N–H bending at 1536 cm ̄ ˡ (amide II). The absorption band at 1387 cm ̄ ˡ was attributable to the C–N stretching and N–H bending (amide III) vibrations. 1598 cm ̄ ˡ a strong band associated to the amide III (-NH₃⁺ groups). The BAMLET–pectin coacervate displayed a band at 1653 cm ̄ ˡ and 1572 cm ̄ ˡ attributed to –COO‾ and -NH₃⁺ Groups stretching vibration.  The coacervation between the two biopolymers changed the FTIR in the carbonyl-amide region. The -NH₃⁺ groups (band at 1598 cm ̄ ˡ) and asymmetric and -COO ̄ stretching vibration at 1629 and 1536 cm ̄ ˡ, disappeared, respectively, indicating the electrostatic interaction between the carboxyl groups of SPI (-COO ̄ ) and amine groups of chitosan (-NH₃⁺). Besides, the spectrum of BAMLET–pectin coacervate showed a broad band at around 3000-3600 cm ̄ ˡ, indicating enhanced hydrogen bonding compared to that of BAMLET and pectin. This implied that hydrogen bonding was also involved in the interaction between BAMLET and pectin.
Differential scanning calorimetry (DSC) was used to determine the thermal properties of the spray-dried BAMLET-pectin coacervate. Thus spray-dried coacervate denaturation peak temperature and denaturation enthalpy (Td=163.57°C, ΔHd=261.6 j/g) and peak temperature denaturation of BAMLET protein and denaturing the enthalpy (Td=136.33°C, ΔHd=35.95 j/g) As well as higher than the denaturation peak temperature and denaturation enthalpy (Td = 157.6 ° C, ΔHd = 104.3 j / g) of coacervate in liquid form. This shows us that pectin is a good coating material and is more stable against temperature changes. In addition, the BAMLET protein has become more resistant to denaturation at this point. Because denaturation peak temperature and denaturation enthalpy are observed to be extremely low. In a coherent form, the resistance is increased by spray drying.
When the dielectric constant of BAMLET protein was examined (10.05 ± 0.05), it was found to be much lower than that of liquid coacervate (78.063 ± 1.3). The dielectric constant, a measure of the solvent's polarity, has shown that the BAMLET protein has less polarity than coacervate. The solvent, which is a larger dielectric constant in the solution, is thought to have a higher net charge density. The dielectric holder has given us information about the polarity of the coacervate formed. Thus, food and food varieties consumption compliance with this drug and also provided with the idea of the content that will be presented in the form of drugs. It has been observed that BAMLET has a less polar form. In UPLC analyzes, BAMLET proves the accuracy of our dielectric measurement, which may be later than the system, relative to alpha-lactalbumin. Because in the reversed-phase UPLC, the analyte with high polarity will first come out of the column. For this reason, the retention time is longer than that of our long BAMLET protein coacervate. However, the alpha-lactalbumin polarity was found to be low in accordance with the literature.