ORIGINAL ARTICLE/ORİJİNAL ÇALIŞMA FULL PAPER TAM MAKALE COMPARISON OF DIFFERENT METHODS FOR BETA LACTOGLOBULIN ISOLATION Ezgi Demir Özer1 , Zübeyde Öner2 1 Niğde Ömer Halisdemir University, Faculty of Engineering, Department of Food Engineering, Niğde, Turkey 2 Süleyman Demirel University, Faculty of Engineering, Department of Food Engineering, Isparta, Turkey Received: 09.02.2017 Corresponding author: Accepted: 02.07.2017 Ezgi Demir Özer, Niğde Ömer Halisdemir University, Fac- ulty of Engineering, Department of Food Engineering, Published online: 06.11.2017 51240, Niğde, Turkey E-mail: [email protected] Abstract: The aim of this study was to introduce a simple, high Keywords: Beta lactoglobulin, Whey, Ultrafiltration, efficient and less expensive method for isolation of β- Anion exchange chromatography, Pepsin Lg from whey. Anion exchange chromatography, pep- enzyme sin enzyme treatment and ultrafiltration tecniques were preferred for isolation process to compare differences. In addition to, centrifuge ultrafiltration techniques were using for the first time for isolation of β-Lg from whey. Physicochemical analysis of whey samples indicated that protein and β-Lg content in whey samples changed from 0.07 to 0.8% and 0.24 to 0.29 g/L, respectively. Treatment with the use of pepsin enzyme, anion ex- changes chromatography and ultrafiltration techniques, resulted to β-Lg of 1.43, 6.56 and 43.59 folds respec- tively. Our results showed that ultrafiltration tech- niques are rapid and efficient that allows high protein yield and has advantages over other methods since it preserves the native structure of β-Lg. Additionally, when the enzymatic hydrolysis was used together with ultrafiltration technique, it was found efficient and pure than the enzymatic hydrolysis together with dialyse membrane. Also this study concluded that pepsin en- zyme treatment and anion exchange chromatography are economic methods but they are not efficient enough and very time consuming. However isolation efficiency can be increased the use of isolation methods together. FOOD and HEALTH E-ISSN: 2602-2834 4(1), 1-8 (2018) doi: 10.3153/JFHS18001 1 © 2015-2018 ScientificWebJournals (SWJ) Food and Health, 4(1), 1-8 (2018) Journal abbreviation: Food Health Introduction Whey is obtained as a by-product during cheese been shown to have inhibitory activity against an- making and it has recognized as a valuable food giotensin converting enzyme (ACE) when deriv- ingredient with important nutritional and func- ing various peptides of β-Lg derived from proteo- tional properties in the last decades. However, be- lytic digestion. β-lg can be used as an ingredient in cause it’s low concentration of milk constituents the formulation of modern foods and beverages (5-6% dry matter), whey has commonly consid- because of its high nutritional and functional value ered waste or animal feed by providing amino ac- (Chatterton, Smithers, Roupas, & Brodkorb, ids required by the young animal (Aich et al., 2006). Recently, researchers have shown interests 2015). It consists of lactose, protein, minerals and in the bioactivities of β-Lg peptides. These pep- organic acids (Morr & Ha, 1993). Whey proteins tides are inactive within the sequence of parent which are a diverse mixture of true proteins, pep- protein, and become activated once released dur- tides and non-protein (NPN) components, is a de- ing gastrointestinal digestion or during food pro- fined as the components that are soluble at pH 4.6 cessing. Bioactive peptides may act as regulatory in their native form (Fox, 2003). Furthermore, compounds with hormone-like activity when they whey is an important source of beta lactoglobulin are released in the body (Hernández-Ledesma et (β-Lg), alfa lactoalbumin (α-La), bovine serum al- al., 2008). As well as known its high value as a bumin (BSA) and immunoglobulins (Ig) food ingredient and its technofunctional proper- (Yerlikaya, Kınık, & Akbulut, 2010). The most ties, it can be a significant health risk in patients abundant whey protein is β-lactoglobulin (β-Lg), allergic to milk (Stojadinovic et al., 2012). Also, which consists of approximately 50-60% of whey there has been an increased interest in recent years proteins and 12% of the total proteins in milk in ways to isolation and purification of β-lg at la- (Outinen, 2010). boratory and industrial-scale. β-Lg is a small, soluble and globular protein that Some studies have been made to isolate this pro- is a dimer at the normal pH of bovine milk with a tein because of its superior nutritional and func- molecular weight of 36 kDa and is a single chain tional properties. Some methods have been used polypeptide of 18 kDa comprising of 162 amino for isolation, such as the salting-out procedure, se- acid residues (Aich et al.,2015).Essential amino lective solubility in the presence of 3% w/w tri- acids such as threonine, valine, isoleucine, leu- chloroacetic acid (TCA), separation by ion-ex- cine, tryptophan and lysine are composed % 45.68 change chromatography, utilizing the differences of total amino acid composition of β-Lg (Young, in thermal stability in acidic conditions. 1994). β-Lg is a rich source of cysteine which is (Bhattacharjee, Bhattacharjee, & Datta, 2006; an essential amino acid for the synthesis of gluta- Cheang & Zydney, 2003; Kinekawa & Kitabatake, thione (Karagözlü & Bayarer, 2004). Five genetic 1996). variants of bovine and four genetic variants ovine The aim of the present study was to investigate the β-Lg of which the phenotypes A and B are most isolation of β-lg from whey by using ultrafiltration predominant have been discovered. β-Lg exists in process, pepsin enzyme treatment and anion ex- the solution as a dimer, with an effective molecu- change chromatography. In addition, the centrifu- lar mass of about 36.6 kDa at the normal pH of gal ultrafiltration technique was used for the first milk (6.5 - 6.7) (Hernández-Ledesma, Recio, & time for β-lg isolation from whey in this study. Amigo, 2008). Furthermore, β-Lg is an important These techniques compare the isolated β-lg re- source of biologically active peptides. These pep- tained purity degree, yield of isolate and native tides are inactive with the sequence of the precur- properties in terms of the different isolation tech- sor protein. But they can be released through ‘in niques. vivo’ or ‘in vitro’ enzymatic proteolysis. These peptides also play important roles in the human Materials and Methods health such as antihypertensive, antioxidant and in Fresh whey which was obtained from white antimicrobial activities. Its opioid-like features cheese manufacturing process using pasteurized gives it the ability to decrease body-cholesterol bovine milk was provide from the local dairy pro- levels (Hernández-Ledesma et al., 2008). ducer for each of three replications on separate β-Lg is involved with the transfer of passive im- production days. Three isolation methods which munity and the binding of retinol and fatty acids are ultrafiltration techniques, pepsin enzyme hy- (De Wit, 1998; Yerlikaya et al., 2010). They have 2 Food and Health, 4(1), 1-8 (2018) Journal abbreviation: Food Health drolysis and anion exchange column chromatog- (Sigma 3K30). The amount and purity of β-Lg iso- raphy were used for isolation of β-Lg. Ultrafiltra- lates were analyzed at 214 nm by HPLC. Samples tion techniques were carried out on flat membrane were taken from the last concentrate isolates for ultrafiltration (Vivaflow 200 PES, Sartorius, Ger- the determination of protein profiles by sodium many) and centrifugal ultrafiltration units (Vi- dodecyl sulfate polyacrylamide gel electrophore- vaspin 15R, Sartorius, Germany). Anion exchange sis (SDS-Page) (Laemmli, 1970). chromatography technique was performed by an- Isolation of β-Lg by Anion Exchange Chromatog- ion-exchange spin column (Vivapure Q mini H raphy column, Sartorius, Germany) and 2-(Diethyla- mino)ethyl-Sephadex (DEAE) A-50 (Sigma Al- The method used by Lozano et al. (2008) were drich, USA). Porcine pepsin enzyme (0.8 IU/mg; modified and used for the isolation of β-Lg by an- Merck, USA) was used for pepsin enzyme treat- ion exchange chromatography. The pH of free par- ment. ticulates was adjusted to 3.0 using concentrated phosphoric acid (85%, H PO Sigma, Germany) Physicochemical Analysis 3 4, . Then, precipitation was performed with 50% am- Physicochemical properties of whey which are monium sulfate at 4°C to obtain highly enriched acidity (SH, pH), fat, protein, dry matter contents fraction of β-Lg and precipate was obtained by and β-Lg contents were determined (Lieske, centrifugation at 15330xg, 4ºC for 15 min. The ob- Konrad, & Faber, 1997). Before the isolation pro- tained precipitate was dissolved in phosphate buff- cess of β-Lg from whey, all whey samples centri- ered saline (PBS) solution at the pH of 3.0. Precip- fuged at 15333 x g, 4ºC for 15 min as a pretreat- itations were obtained from 50% salt. Then, sam- ment procedure to remove fat and particulates ples were dialyzed using a 12-14 kDa cut off Spec- (Lozano, Giraldo, & Romero, 2008). β-Lg content tra/Pors membrane against PBS solution pH 7.2. of whey in the beginning of isolation process and After dialysis, salt was added to 70%. The result- differences between protein content and effective- ant precipitate was centrifuged at same centrifuga- ness of isolation techniques were measured by us- tion conditions and obtained supernatant which ing high-performance liquid chromatography contained high β-lactoglobulin was dialyzed under (HPLC) (Shimadzu LC-20AT, Japan) which was the same conditions. Finally, obtained product was performed on a Zorbax 300SB-C18 4.6 x 250mm lyophilized and stored for anion-exchange chro- (Agilent, USA) with a diode array detector. matography. Isolation of β-Lg by Ultrafiltration Techniques Anion-exchange chromatography was performed on a column packed with DEAE Sephadex A-50. Ultrafiltration methods were performed with flat A 50 milliliters sample, which was reconstituted membrane ultrafiltration and centrifugal ultrafil- in distilled water to a final concentration of 1 g/ tration units.