ENRICHMENT OF BIOACTIVE MATERIAL BY ENZYMATIC DEGRADATION AND MEMBRANE SEPARATION
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1 1. Szilárd SZÉLPÁL, 2. Kitti FEJES, 3. Józse SANÁDI, 4. Dragana ŠOONJA-SIMOVIĆ, 5. Zsuzsanna LÁSZLÓ, 6. Gábor KESZTHELYI-SZABÓ, 7. ecília HODÚ ENIHMENT OF BIOATIVE MATEIAL BY ENZYMATI DEGADATION AND MEMBANE SEPAATION 1-2, 4-7. UNIVESITY OF SZEGED, FAULTY OF ENGINEEING, DEPATMENT OF POESS ENGINEEING, SZEGED, HUNGAY 3. UNIVESITY OF SZEGED, FAULTY OF ENGINEEING, DEPATMENT OF FOOD ENGINEEING, SZEGED, HUNGAY ABSTAT: The aim o the present investigation was to examine the applicability o ultrailtration and enzyme degradation process in enhancing o bioactive components. Skimmed milk was treated with hy-max TM Plus (which is produced by ermentation o a 100% chymosin rennet enzym) and the eect o the enzyme concentration, the time o the enzymatic degradation, and the cut o value o the membrane on the protein retention, lux, membrane ouling and gel ormation were measured. KEYWODS: ultrailtration, skimmed milk, enzymatic degradation, whey, bioactive components INTODUTION The bioactive materials have more eective biological properties than the well known nutrients, and thereore they have health-protective and health care eects. The bioactive materials are special macro-and micronutrients; they protect the human body, and the human health better than the other nutrients. These materials contribute to the harmonious unctioning o the human body, promote health and disease prevention, and also they have an important role in the treatment o disease, to speed up the healing processes o diseases (Szakaly et al., 2001). The biologically active materials can be ound in large quantities in the skimmed milk. The α- casein and β-casein makes up the largest component in it. A high proportion o trace elements such as calcium and phosphorus can be ound in these proteins. The biologically active materials have poor solubility in water, and thereore only in the orm o suspensions can be ound in the milk. The whey proteins and casein proteins have dierent physiological eects because they have dierent physical property. The biggest dierences between the two types o proteins are in the rate o digestion and in the speed o the absorption. Nowadays a lot o dierent techniques are known to extracting the whey proteins rom the valuable components o the milk. Among them, the membrane technology is a current solution (ATA et al., 2005; Yorgun et al., 2008; Fachin et al., 2005). The natural properties o milk proteins - solubility, oaming and emulsiying capacity, and gelling capacity - are retained ater the application o membrane technology, and the separation process is protected the product (Saxena, 2009; Poulin, 2008). The ultrailtration is a prevalent separation process among the pressure driven membrane separation techniques to separate proteins o the milk. Several research groups have investigated the eectiveness o ultrailtration o skimmed milk (inaldoni et al., 2009, Salvatore et al., 2011; Hodúr et al., 2009) however, the separation o the proteins ollowed ultrailtration is not possible, since the sizes o the protein molecules are very close to each other (or example, β-lactoglobulin and α- lactalbumin) (Metsamuuronen et al., 2011). The protein because o their molecular size may be rented in the concentrate phase meanwhile the lactose and the other mineral substances can separated through the membrane (Yee et al., 2007; heang & Zydney, 2004; ektor &Vatai, 2004). The milk and whey protein clogs relatively quick the membrane thereore a pre-treatment or other method would be useul beore the ultrailtration o whey or milk (ektor & Vatai, 2004). hymosin is an enzyme which is recovered in rennet and is used in the cheese manuacturing. The most o the used chymosin enzyme is produced recombinant in dierent bacteria and ungi or example E. coli, or Aspergillus niger (awlings & Barret, 1995). The chymosin enzyme is produced by gastric chie cells in the new born animals. The human body is used another type o enzymes to digest milk such as pepsin or lipase. The chymosin enzyme is important in the cheese production because i this aspartic peptidase enzyme can cleave eiciently the κ-cazein in their sensitive region, at the copyright FAULTY o ENGINEEING HUNEDOAA, OMANIA 221
2 ANNALS OF FAULTY ENGINEEING HUNEDOAA International Journal O Engineering Phenil-alanin 105 Methionin 106 bond, the cheese production can become more economical (Kappeler et al., 2006). The chymosin releasing the κ-cazein s negatively charged -terminus, which is the irst step in the separation process o whey and curd (Langholm Jensen et al., 2013). This enzyme has ound in three dierent orms in the animals, the A, B and the orm. The activity measurements were demonstrated that the chymosin A has increased the milk clotting ability than the other orms (Williams et al., 1997). The extent o membrane ouling is highly depend on parameters such as: share orce on the surace (Konrad et al., 2012; Karlsson et al., 2007), thereore the use o vibrated membrane modules appropriate may be increased the membranes lie (Al-Akoum et al., 2005; Jarin, 2012; Kertész et al., 2010). The aim o the present investigation was to examine the applicability o ultrailtration and enzyme degradation process in enhancing o bioactive components and it was also examined what kind o correlation is between membrane characteristics and the success o enzymatic degradation, or the amount o potential bioactive components.. Skimmed milk was treated with hy-max TM Plus (which is produced by ermentation o a 100% chymosin rennet enzym) and the eect o the enzyme concentration, the time o the enzymatic degradation, and the cut o value o the membrane on the protein retention, lux, membrane ouling and gel ormation were measured. MATEIALS AND METHODS The mass transer and the luid dynamics the lux is deined as the rate o volumetric low across a unit area. In this case the eectiveness o a membrane separation can be determined also by the lux. (Bélainé, 2002): dv J = [ Lm h ] (1.) dt A where J is the lux, A is the active membrane area [m 2 ], V is the iltrate volume[m 3 ] and t is the time [s]. The retention, (retention), was calculated using the ollowing equation: p p = = 1 [%] (2.) where F is the solute concentration in the eed [mg/l], and p is the solute concentration in the permeate [mg/l]. Determination o the degree o ouling is proportional to the power low exponent rom an analysis o the lux-time unctions (Hodúr et al, 2007.): k 2 1 J = J 0 t [ Lm h ] (3.) where J 0 is the initial lux [Lm -2 h -1 ], t is the membrane iltration time [h], and the k is the ouling index. The membrane resistance ( M ) was calculated rom the ollowing equation: Δp 1 M = [m ] (4.) J w ηw where J W is the water lux o [m 3 m -2 h -1 ] o the clean membrane, and η w is the water viscosity at 25 [Pas]. The ouling resistance ( ) was determined ollowed the washing o the gel layer rom the membrane. The ouling resistance (6) and the resistance o the polarization layer ( g ) were calculated as ollowing (7): Δ p = [m -1 ] (5.) M J WA η WA Δp g = M [m -1 ] (6.) J ηww where Δp is the pressure dierence between the two sides o the membrane (Pa), η WA [Pas] is the viscosity o the iltered solution and J WA is the water lux ater concentration tests. The gel layer resistance s mathematical ormula the J is the constant lux and the η ww is the wastewater viscosity. The well known membrane iltration parameters were measured in both cases. The volume reduction ratio (V) was calculated using the ollowing ormula: Veed V = (7.) Veed Vperm where V eed is the volume o the eed [L] and V perm is the volume o the permeate [L]. The analytical values o skimmed milk and separated samples were determined by Bentley B-150 type Milk Analyzer. Skimmed mild was used as eed in our work. It has 2.79g/100g protein raction and 2.97g/100g total N protein, 0.02 g/100g milk at, 4.5 g/100g o lactose and 8.09 g/100 g o dry material were measured in the skimmed milk samples. Skimmed milk samples were stored deep rozen until used. 222 Tome XI (Year 2013). Fascicule 4. ISSN
3 ANNALS OF FAULTY ENGINEEING HUNEDOAA International Journal O Engineering Two dierent equipments were used or membrane separation; the VSEP Vibratory Shear Enhanced Processing laboratory module and the MEUF micellar enhanced ultrailtration device. Torsional vibration reduced ouling in both dead-end and cross low modes. Increasing vibration amplitude decreased membrane ouling, increased rejection o most solutes and changed the morphology o the scales rom a tightly packed layer to a more scattered distribution o particles. The VSEP system consists o disk-shaped lat-sheet membranes. This laboratory module attached to a central shat. The shat was rotated a short distance at a requency o 54 Hz. Under examination 503 cm 2 active iltering surace, regenerated cellulose,, lat-sheet ultrailtration membranes were used at controlled temperature and ph. The cut-o values o applied membranes were 100 kda and 10kDa. The trans-membrane pressure dierence was 0.5 MPa. The MEUF equipment has 15 cm 2 active iltering surace. A regenerated cellulose based latsheet UF membranes were used with a cut-o value at 10 kda. During the measurements 0.5 MPa pressure was applied and the eed side was stirred with a magnetic stirrer at 200 rpm to keep the cross-low on the membrane surace. The permeate o skimmed milk s 100 kda separation (F10) was treated by hy-max TM Plus enzyme (which is produced by ermentation o a 100% chymosin rennet enzyme). The enzimatic degradation were made on 43 and 1cm 3 /L enzyme concentration was used. The untreated samples, i.e. permeat o 100 kda separation added enyme () and samples taken every third hours o degradation procedure (P10E3, ) were iltered on a 10 kda cut-o value ultrailtration regenerated cellulose membrane in the MEUF equipment. ESULTS AND DISUSSION The lux time unctions o the skimmed milk membrane separation are presented in Figure 1. The lux was increasing at the irst 20 min o the separation and it was decreasing slowly ollows. This interesting and unusual behaviour could explain with the eect o vibration P100_ 4 P100_ 3 P100_ 2 P100_ 1 J [dm 3 m 2 h 1 ] Time [min] Fig. 1: Flux o the skimmed milk as a unction o time ( membrane with cut o 100 kda, vibration requency: 54 Hz, TMP:0.5 MPa) 6.10E+13 The values o the resistances [1/m] 5.10E E E E E E+12 t g m Dierent resistances Fig. 2: esistances o the skimmed milk ultrailtration ( membrane with cut o 100 kda, vibration requency: 54 Hz, TMP: 0.5 MPa) The dierent resistance values were calculated: the membrane resistance (m), the resistance o the gel layer on the surace o the membrane (g), the ouling resistance () and the total resistance (t) (Fig. 2). The resistances values showed there is no signiicant dierences between the measured values o the samples demonstrated that the skimmed milk samples weren t changed during Tome XI (Year 2013). Fascicule 4. ISSN
4 ANNALS OF FAULTY ENGINEEING HUNEDOAA International Journal O Engineering the deep rozen storage. The gel layer resistance is smaller than the ouling resistance similarly as it was published by Shi & Benjamin (2009) and as it is known rom the other publication about the vibrated membrane separation. The proportion the resistances enhanced the well known data: vibrated systems reduce the gel layer resistance. g/100g F Protein Total N F10 Fig. 3: Analytical values o the 100 kda permeate as a Feed (F10) and the enzyme treated () and untreated () The analitical values o the eed (F10) and the untreated () and treated () permeate ractions were demonstrated in the Fig. 3. The protein and total nitrogen contents were higher in the treated samples than in the untreated samples which means that the enzymatic degradation o protein was unctioned (Fig. 3). The retention was calculated on the base o protein and the total N content and it was ound that the permeate o enzyme treated samples had more protein and total nitrogen, which also proos the enzyme degradation was successul. The resistance values o the ultrailtration in the case o the enzyme treated and untreated samples are shown in the Fig.4. The total resistance and the gel layer resistance are smaller and the ouling resistance permeate samples is higher in the case o enzyme-degraded samples, it means that the smaller protein ractions increased during the 6 hour long treatment. Table 1: The retention values and the dierent protein and total Nitrogen values Protein Samples [%] [g/100g] F P10E Total N [g/100g] 6.00E E E E E E E+00 t g m t g m 5.46E E E E E E E E+13 Fig. 4.: esistances on the treated() and untreated () samples This tendency could ollow in the Fig.5 also where the percentage o gel layer resistance and the ouling resistance are shown % g 20 0 P10E3 Fig. 5.: The percentage o gel layer (g) and ouling resistance () at untreated (), the 3 hour long treated (P10E3) and 6 hour long treated () samples. 224 Tome XI (Year 2013). Fascicule 4. ISSN
5 ANNALS OF FAULTY ENGINEEING HUNEDOAA International Journal O Engineering Presumably the lower lux values (Fig. 6) detected at the treated samples was caused by the higher ouling resistance. J [dm 3 m 2 h 1 ] y = 70.33x = y = x = y = x = Time [min] P10E3 Fig. 6: The enzyme-treated (P10E3, ) and untreated () samples lux values as a unction o time k g/ Fig. 7: Fouling index values as a unction o t the gel and the ouling resistance ratio ONLUSIONS This research is an initial step o the project to enhance the milk originated bioactive components in the membrane separated ractions. In our experiment were perormed the skimmed milk s iltration on regenerated cellulose membranes, and chymosin-treated ultrailtered permeate raction. The dierent resistance values obtained chymosin enzyme treated samples showed that the enzymatic degradation o protein was unctioned; thereore the protein content o the permeate phase increased and it was bigger in the 6 h degradeted samples than the 3 h degradeted ones. Since the ouling index has a linear relationship with the ratio o the gel resistance and ouling resistance, it could be relevant parameter or expression o the degradation in adequate conditions. The next step should be the investigation o the correlation o the g/ ratio and the bioactive component content o the samples. AKNOWLEDGEMENT We would like to thank or the OTKA Hungarian Scientiic Foundation No: the TÁMOP /B-09/1/KONV identiication number, "Establish the Excellence esearch entre in the University o Szeged" in the TÁMOP-4.2.2/B-10/ "the Department o esearch University entre o Excellence knowledge base expansion and long-term technical sustainability basis o scientiic excellence in recruitment by providing" entitled projects, which are assistanced by the EU, co-inanced by the European Social Fund, and which made it possible to perorm our work. EFEENES [1.] Al-Akoum O., Jarin M. Y., Ding L. H. oncentration o total milk proteins by high shear ultrailtration in a vibrating membrane module, Journal o Membrane Science, Vol. 247., Issues 1-2, pp (2005) [2.] Atra., Vatai Gy., Bekassy-Molnar E., Balint A. Investigation o ultra- and nanoiltration or utilization o whey protein and lactose, Journal o Food Engineering, Vol. 67. pp (2005) [3.] Bouzid H., abiller-baudry M., Paugam L., ousseau F., Derriche Z., Bettahar N. E. Impact o zeta potential and size o caseins as precursors o ouling deposit on limiting and critical luxes in spiral ultrailtration o modiied skim milks, Journal o Membrane Science, Vol pp (2008) [4.] heang B., Zydney A. L. A two-stage ultrailtration process or ractionation o whey protein isolate, Journal o Membrane Science, Vol pp (2004) [5.] Fachin L., Viotto W. H. Eect o ph and heat treatment o cheese whey on solubility and emulsiying properties o whey protein concentrate produced by ultrailtration, International Dairy Journal, Vol. 15. pp (2005) [6.] Hodúr., Kertész Sz., sanádi J., Szabó G., László Zs. Investigation o Vibratory Shear-enhanced Processing System, Progress in Agricultural Engineering Sciences, Vol. 5. pp (2009) [7.] Jarin Y. Michel Dynamic iltration with rotating disks, and rotating and vibrating membranes: an update, urrent Opinion in hemical Engineering, Vol. 1., Issue 2, pp (2012) [8.] Kappeler S.., Van Den Brink J. M., ahbek-nielsen H., Farah Z., Puhan Z., Bech Hansen E., Johansen E. haracterization o recombinant camel chymosin reveals superior properties or the coagulation o bovine and camel milk, Biochemical and Biophysical esearch ommunications, Vol. 342., Issue 2, pp (2006) [9.] Karlsson A. O., Ipsen., Ardo Y. heological properties and microstructure during rennet induced coagulation o UF concentrated skim milk, International Dairy Journal, Vol. 17. pp (2007) [10.] Kertész Sz., Szép A., sanádi J., Szabó G., Hodúr. omparison between stirred and vibrated UF modules, Desalination and Water Treatment, Vol. 14., Issue 1-3, pp (2010) Tome XI (Year 2013). Fascicule 4. ISSN
6 ANNALS OF FAULTY ENGINEEING HUNEDOAA International Journal O Engineering [11.] Konrad G., Kleinschmidt T., Faber W. Ultrailtration lux o acid whey obtained by lactic acid ermentation, International Dairy Journal, Vol. 22. pp (2012) [12.] Langholm Jensen J., Mølgaard A., Navarro Poulsen J-., Harboe M. K., Simonsen J. B., Lorentzen A. M., Hjernø K., Van Der Brink J. M., Qvist K. B., Larsen S. amel and bovine chymosin: The relationship between their structures and cheese-making properties, Acta rystallographica Section D: Biological rystallography, Vol. 69., Issue 5, pp (2013) [13.] Metsämuuronen S., Mänttäri M., Nyström M. omparison o analysis methods or protein concentration and its use in UF ractionation o whey, Desalination, Vol pp (2011) [14.] Popovic S., N. Tekic M. N. Twisted tapes as turbulence promoters in the microiltration o milk, Journal o Membrane Science, Vol. 384., pp (2011) [15.] Paugam L., abiller-baudry M., Delaunay D. Physico-chemical eect o simple alkaline and acid solutions in cleaning sequences o spiral ultrailtration membranes ouled by skim milk, Desalination, Vol pp (2006) [16.] Pouliot Y. Membrane processes in dairy technology From a simple idea to worldwide panacea, International Dairy Journal, Vol. 18. pp (2008) [17.] awlings N. D., Barrett A. J. Families o aspartic proteases, and those o unknown catalytic mechanisms, Methods in Enzymology, Vol. 248., pp (1995) [18.] ektor A., Vatai Gy., Membrane iltration o Mozzarella whey, Desalination, Vol pp (2004) [19.] inaldoni A. N., Tarazaga.., ampderrós M. E., Padilla A. P. Assessing perormance o skim milk ultrailtration by using technical parameters, Journal o Food Engineering, Vol. 92. pp (2009) [20.] Salvatore E., Pirisi A., orredig M. Gelation properties o casein micelles during combined renneting and bacterial ermentation: Eect o concentration by ultrailtration, International Dairy Journal, Vol. 21. pp (2011) [21.] Saxena A., Tripathi B.P., Kumar M., Shahi V.K. Membrane-based techniques or the separation and puriication o proteins. An overview, Advances in olloid and Interace Science, Vol pp (2009) [22.] Shi W., Benjamin M. M. Fouling o O membranes in a vibratory shear enhanced iltration process (VSEP) system, Journal o Membrane Science, Vol. 331., Issues 1-2, pp (2009) [23.] Szakály S.(szerk.) Tejgazdaságtan. Dinasztia Kiadó, Budapest, Vol pp (2001) [24.] Yee K. W.K., Wiley D. E., Bao J. Whey protein concentrate production by continuous ultrailtration: Operability under constant operating conditions, Journal o Membrane Science, Vol pp (2007) [25.] Yorgun M.S., Balcioglu I. A., Saygin O. Perormance comparison o ultrailtration, nanoiltration and reverse osmosis on whey treatment, Desalination, Vol pp (2008) [26.] Williams M. G., Wilsher J., Nugent P., Mills A., Dhanaraj V., Fabry M., Sedlacek J., Uusitalo J. M., Penttila M. E., Pitts J. E., Blundell T. L. Mutagenesis, biochemical characterization and X-ray structural analysis o point mutants o bovine chymosin, Protein Engineering, Vol. 10., pp (1997) ANNALS o Faculty Engineering Hunedoara International Journal o Engineering copyright UNIVESITY POLITEHNIA TIMISOAA, FAULTY OF ENGINEEING HUNEDOAA, 5, EVOLUTIEI, , HUNEDOAA, OMANIA Tome XI (Year 2013). Fascicule 4. ISSN
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