Effect of nitrogen gas flushing treatments on total antioxidant capacity and ascorbic acid content in raw bovine milk during cold storage

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1 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) 155 Originl scientific pper - Izvorni znnstveni rd UDK: Effect of nitrogen gs flushing tretments on totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk during cold storge doi: /mljekrstvo Oguz Gursoy 1 *, Ptrici Munsch-Altossv 2, Kubr Ertn 1, Yusuf Yilmz 1, Tpni Altossv 2 1 Mehmet Akif Ersoy University, Fculty of Engineering nd Architecture, Deprtment of Food Engineering, TR Burdur, Turkey 2 University of Helsinki, Fculty of Agriculture nd Forestry, Deprtment of Food nd Environmentl Sciences, FI Helsinki, Finlnd Abstrct Received - Prispjelo: Accepted - Prihvćeno: Continuous nitrogen gs ( ) flushing extends the shelf life of rw milk (RM) during cold storge. The effect of tretment on the totl ntioxidnt cpcity (TAC) nd scorbic cid (AA) content of RM ws determined during cold storge. TAC of RM or deproteinized RM ws determined by ABTS nd DPPH methods, while L(+)-AA content of RM ws determined chromtogrphiclly on dys 0, 4 nd 7 during storge t 6±1 C. With the ABTS method, the TAC of RM decresed from ±16.70 to ±62.06 µm TEAC while it reduced from 13.30±0.84 to 8.20±0.66 µm TEAC with DPPH method during cold storge. TAC of RM determined with ABTS method decresed fter 4 dy-storge; however, they remined sttisticlly similr for -treted smples during 7 dy-storge. The AA content of RM rnged from to mg/l during storge but -tretment did not influence AA content significntly. Deproteiniztion reduced TAC vlues of milk smples significntly, nd the reduction with the ABTS method ws bout % for control smples cold-stored for four dys, while it ws % for -treted deproteinized RM. In conclusion, -flushing through the hedspce of milk contining vessels showed significnt protective effect on the ntioxidnt components of RM during cold storge. Key words: rw milk, nitrogen gs ( ), shelf-life, ntioxidnt, scorbic cid Introduction Cellulr formtion of free rdicls nd other rective oxygen species such s superoxide, hydroxyl, peroxyl nd lkoxy rdicls tkes plce continuously by severl mechnisms s prt of norml cellulr functions (Young nd Woodside, 2001). There is sensitive blnce between oxidizing gents, which re produced in living systems in excessive mounts for vrious resons (i.e. chemicl oxidnts, UV nd ir pollution), nd ntioxidnts from endogenous nd exogenous sources. Disruption of this blnce towrds oxidnts (oxidtive stress) cn ply n importnt role in etiopthogenesis of severl diseses (Buyuktuncel, 2013). Antioxidnts interct with free rdicls nd cese the chin rection before cellulr components re dmged (Oroin nd Escriche, 2015). Antioxidnts cn demonstrte their function by different mechnisms such s (i) scvenging free rdicls, (ii) binding of metl ions, (iii) scvenging oxygen, (iv) converting hydroperoxides to non-rdicl species, (v) bsorbing UV rdition nd (vi) dectivting singlet oxygen (Gordon, 2010). The intke of ntioxidnts cn prevent oxidtive stress formtion in living orgnisms. Severl epidemiologicl studies hve shown tht dietry intke of foods contining nturl ntioxidnts is highly ssocited with the decresed risk of coronry hert disese (Young nd Woodside, 2001; Virgili et l., 2010), tumor development (Johnson, 2010), *Corresponding uthor/dopisni utor: Phone/Tel: , E-mil: ogursoy@yhoo.com, ogursoy@mehmetkif.edu.tr

2 156 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) nd lowering incidence of different diseses nd/or complictions such s dibetes (Bynes, 1991) nd ctrct (Spector, 1995). Thus, the prevention of the losses of individul ntioxidnt compounds together with the protection of the totl ntioxidnt cpcities of foods throughout food production chins become criticlly importnt in providing high dietry intke of ntioxidnts in humn nutrition. Milk nd diry products contin severl compounds hving ntioxidnt properties. Min ntioxidnt compounds in milk re proteins (α-, β- nd k-cseins nd lctoferrin), enzymes (superoxide dismutse, ctlse nd glutthione peroxidse) (Cervto et l., 1999; Lindmrk-Mnsson nd Akesson, 2000; Prvst et l., 2000), vitmins (vitmin E, C nd A), coenzyme Q10, crotenoids (minly β-crotene) (Lindmrk-Mnsson nd Akesson, 2000), phenolic compounds (Vzquez et l., 2015) nd orgnic cids (uric cid) (Zuluet et l., 2009). The concentrtion of ntioxidnt compounds in milk is ffected by both feeding rtions of nimls nd milk storge conditions (Lindmrk- Mnsson nd Akesson, 2000). Since there re numerous ntioxidnt components in milk nd diry products, the totl ntioxidnt cpcity mesurement my be useful method for detecting the sum of the ntioxidnt role of ech component in milk (Gjorgievski et l., 2014). Numerous bcteril gener successfully survive nd grow despite the cold chin conditions during rw milk storge nd trnsporttion. More precisely cold storge does not prevent the growth of psychrotrophic bcteri which cn constitute more thn 90 % of the totl bcteril popultion in cooled rw milk (Cousin, 1981; Smržij et l., 2012). Psychrotrophs re usully ble to form extrcellulr, het-stble enzymes (proteses nd lipses), which re minly responsible for spoilge of milk nd diry products (Cousin, 1981). Some psychrotrophic bcteril species ( mjority re Grm-negtive representtives) hve lso shown to exhibit multiple ntibiotic resistnce fetures (Munsch-Altossv nd Altossv, 2007). Thus, controlling psychrotrophic bcteril growth during cold storge of rw milk is importnt for both - technologicl nd humn helth spects. Severl studies hve indicted tht the combintion of modified tmosphere tretments (bsed on the use of CO 2, or mixtures of both) combined with cold storge my hve significnt potentil to extend shelf life of rw milk (Mrtin et l., 2003; Dechemi et l., 2005; Rjgopl et l., 2005). Previously, our reserch group showed tht continuous gs flushing when pplied in so-clled open system improved the microbiologicl qulity nd shelf life of rw nd psteurized milk smples during cold storge (Munsch-Altossv et l., 2010,b; Munsch-Altossv et l., 2011; Munsch-Altossv et l., 2013). Although, there re some evidences regrding improved microbiologicl qulity of rw milk by btch or continuously treted modified tmospheres, to the best of our knowledge, no study tht considered the effect of tretment on the totl ntioxidnt cpcity of rw milk hs been published so fr. Therefore, the objective of this reserch ws to determine the effect of continuous flushing on the totl ntioxidnt cpcity nd scorbic cid content of rw milk smples during cold storge, by considering the sme experimentl setting s for previous investigtions. Mterils nd methods Chemicls 2,2 -zinobis(3-ethylbenzothizoline-6- sulfonic cid) (ABTS), ethnol ( 99.9 %), potssium persulfte nd trichlorocetic cid (TCA) were purchsed from Merck (Drmstdt, Germny). Methnol ws obtined from Sigm Chemicl Co. (St. Louis, MO, USA). 6-Hydroxy-2,5,7,8-tetrmethylchromn- 2-crboxylic cid (Trolox, wter-soluble vitmin E nlogue) nd 2,2-diphenyl-1-picrylhydrzyl (DPPH) were purchsed from Fluk (St. Louis, MO, USA). All other chemicls used were of nlyticl or HPLC grde. L(+)-scorbic cid ws purchsed from PnRec AppliChem (Drmstdt, Germny). Nitrogen gs tretment nd microbiologicl nlyses of rw milk smples Three rw milk smples, representing lorry tnk milk from June nd August 2014, provided by the Helsingin Meijeriliike Ltd. (Helsinki, Finlnd) were considered. Experimentl setup previously described by Munsch-Altossv et l. (2010) ws used for the continuous flushing tretments of rw milk (Figure 1). Briefly, experiments were conducted in cold room t constnt temperture of 6 C. For nitrogen tretments, glss bottle ws

3 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) 157 connected to flowmeter (Brooks Instruments B.V., Veenendl, the Netherlnds), vi 0.2-μm sterile filters (Schleicher & Schuell GmbH, Dssel, Germny). The purity of nitrogen gs (AGA Ltd, Riihimäki, Finlnd) ws %. For nitrogen tretments, the hedspce of bottle ws continuously purged by fresh nitrogen gs t flow rte of 120 ml/min. Rw milk smples (100 ml) were continuously mixed with mgnetic stirrer (Vriomg, Oberschleißheim, Germny) for both the control (C) nd the -treted milk, while cold stored t 6 C in refrigerted wter bth (MGW Lud MS/2, Lud-Königshofen, Germny) during the course of the experiments. At the given smpling times, the gs flow ws shortly interrupted nd 0.5 ml of rw milk smples ws serilly diluted nd cultured on PCA gr for 3 d t 30 C in erobic conditions. Simultneously, milk frctions were collected nd frozen (-20 C) until subjected to chemicl nlyses. Milk deproteiniztion The procedure described by Zuluet et l. (2009) ws used for the deproteiniztion of milk smples. Ech of the rw milk smple ( flushed or untreted) (5 ml) ws deproteinized by the ddition of trichlorocetic cid solution (5 ml, 20 % w/v). Subsequently, the milk smples were incubted for 10 min t 42 C to remove ll milk proteins, then cooled to room temperture nd centrifuged for 10 min t 9500 g t room temperture. The obtined superntnts were collected from protein precipittes, nd then promptly nlyzed. ABTS Rdicl Scvenging Assy Totl ntioxidnt cpcity of rw nd deproteinized rw milk smples corresponding to TCA superntnt frctions obtined from the corresponding rw milk ws determined by the ABTS method described by Re et l. (1999), with slight modifictions. Stock solution of ABTS rdicls were prepred by the ddition of potssium persulfte (2.6 mm) into queous solution of ABTS (7 mm), nd the mixture ws stored t room temperture for h in drk plce. The working solution ws prepred by diluting the stock solution with methnol (finl bsorbnce ws bout 1.1±0.02 t 734 nm). Then, 0.3 ml rw milk or TCA superntnts (deproteinized rw milk) were trnsferred into the working solution (2.7 ml). After incubting the mixture t room temperture for 30 min, the smples were centrifuged t 12,000 g for 2 min t room temperture. Decrese in bsorbnce vlues ws mesured t 734 nm ginst methnol s reference. Results were expressed s Trolox equivlent ntioxidnt cpcity (TEAC). Figure 1. Experimentl setup used for nitrogen tretments of rw milk in glss bottles t flow rte of 120 ml/min

4 158 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) DPPH Rdicl Scvenging Activity The DPPH rdicl scvenging ctivity of smples ws mesured by the modified method of McCue nd Shetty (2005). DPPH stock solution ws prepred by dissolving DPPH (24 mg) in ethnol (100 ml). Working solution ws prepred by diluting the stock solution with ethnol (finl bsorbnce ws bout 1.1±0.02 t 517 nm). Ethnolic DPPH solution (2.7 ml) ws mixed with 0.3 ml of rw milk or TCA superntnt (deproteinized rw milk) nd the mixture ws vortexed well. After incubting the smples t room temperture for 30 min, they were centrifuged t 12,000 g for 2 min t room temperture. Decrese in bsorbnce vlues ws mesured t 517 nm ginst ethnol, nd results were expressed s TEAC. Determintion of scorbic cid content Smple preprtion for scorbic cid determintion ws performed ccording to Kondyli et l. (2007). Metphosphoric cid (1.12 %) ws mixed with rw milk t rtio of 1:1 (v/v). After centrifugtion t 12,000 g for 30 min, the superntnt ws filtered nd nlyzed immeditely by the HPLC system. HPLC conditions described by Akts et l. (2005) were used to determine the scorbic cid content of rw milk smples. HPLC nlyses were crried out by Shimdzu Prominence HPLC System (Shimdzu Corportion, Kyoto, Jpn) equipped with diode-rry detector (SPD-M20A), pump (LC20 AT), n uto smpler (SIL 20ACHT) 10 nd column oven (CTO-10ASVp). The column used ws ODS-4 (250 mm x 4.6 mm I.D., 5 μm) (GP Sciences, Intersil ODS-4, Jpn). The mobile phse ws ultrpure wter djusted to ph 3 with orthophosphoric cid. A flow rte of 1 ml/min ws used for chromtogrphic seprtions. Sttisticl nlyses Anlysis of vrince (ANOVA) ws used to determine sttisticlly significnt differences by mens of the SAS softwre progrm (The SAS System for Windows 9.0, Chicgo, USA). Seprtion of mens for significnt differences ws conducted using the Duncn s multiple-rnge test t α=0.01 level. Dt were presented s mens of three replictes (±stndrd devition). Results nd discussion Microbiologicl results Totl bcteril counts t initil conditions (dy 0) rnged from 3.6 to 4.2 log-units, indictive of good bcteriologicl qulity (Figure 2). After 4 dys of cold storge, counts from the controls (C1, C2, C3) incresed by over 10 times for C3 nd up to 1000 times for C1 during the 4 dys cold storge, nd exceeded the threshold vlue of cfu/ml (5.5 log-units) of bcteriologicl cceptnce; counts still incresed between dys 4 nd 7 for the controls until log units. 9 log cfu/ml Dy 0 Dy 4 Dy C1 N1 C2 N2 C3 N3 Figure 2. Totl bcteril counts of control nd -treted rw milk smples during 7 dys of storge t 6 C (C1, C2 nd C3: Control rw milk smples, N1, N2 nd N3: -treted rw milk smples). Error brs indicte stndrd devitions

5 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) 159 Under the flushing, in contrry, the bcteril growth could be hlted for 4 dys (Figure 2); in following, fter 7 dys, counts hd incresed by bout 10 times for N1 nd N2, but remined unchnged for N3; however, the flushing tretment kept counts in ll three experiments below the limit of cfu/ml, which is in greement with previous results (Munsch-Altossv et l., 2010). Totl ntioxidnt cpcity of rw milk smples Vlues of the totl ntioxidnt cpcity (TAC) of rw milk smples determined by both ABTS nd DPPH methods re presented in Figures 3 nd 4, respectively. TEAC vlues, for the controls, obtined by the ABTS method during storge rnged from ±62.06 to ±16.70 µm while those determined by the DPPH method rnged from 8.09±0.49 to 13.30±0.84 µm TEAC. In this present study, the ntioxidnt cpcity vlues of rw milk smples determined by the ABTS method were considerbly higher thn those determined by the DPPH method. Similr results were previously reported by Mrtysik-Zurowsk nd Went (2012). The ABTS rdicl is soluble in both queous nd orgnic solvents, nd the ABTS method cn be used to determine both hydrophilic nd lipophilic ntioxidnt cpcities of different smples like food extrcts nd body fluids. DPPH, long-lived nitrogen rdicl, hs been widely used to determine ntioxidnt potentil of different compounds (Villňo et l., 2007). However, this rdicl does not hve ny similrity with the highly rective nd trnsient peroxyl rdicls involved in lipid peroxidtion (Hung et l., 2005). DPPH rdicl dissolves in polr orgnic mtrices like ethnol nd methnol, nd the DPPH ssy is bsed minly on the electron trnsfer rection, while hydrogen-tom trnsfer pthwy is mrginl in this ssy (Hung et l., 2005; Prior et l. 2005). Unlike -tocopherol, BHA nd BHT, crotenes nd xnthophylls hve no DPPH rdicl scvenging ctivity (Müller et l., 2011). Conformtionl inccessibility of DPPH rdicls with ntioxidnts nd reduced rectivity of some compounds with DPPH rdicls re other disdvntges of this method (Hung et l., 2005; Prior et l. 2005). Moreover, prticulr components (i.e. crotenoids) present in rection medium my interfere with the DPPH ssy becuse their spectr my overlp with the spectr of DPPH rdicl itself (Prior et l., 2005). The dvntges nd disdvntges of these two methods hve been reviewed extensively in literture (Prior et l., 2005; Cloetens et l., 2013). Results from both methods tested to determine the ntioxidnt cpcity indicted tht -treted rw milk smples exhibited higher TAC vlues thn untreted smples (Figures 3 nd 4). While the TAC vlues of the control smples decresed fter 4 dys of storge (p<0.01), the vlues for -treted smples were found sttisticlly similr during 7 dys of storge (p>0.01) (Figure 3). According to the results of the ABTS ssy, the ntioxidnt ctivity of control rw milk smples decresed bout 22 % during 7 dys of storge. Results of the DPPH method indicted tht t the end of the storge period, the b 400 TAC (µm TEAC) C(4) N(4) C(7) N(7) Tretment & Storge Time (dy) Figure 3. Totl ntioxidnt cpcity of rw milk smples ssyed by the ABTS method during storge t 6 C (C: Control, N: -treted; different letters bove the columns indicte sttisticl significnt differences t the p<0.01 level)

6 160 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) men TAC vlues of -treted smples decresed bout 20 %, wheres this decrese ws bout 38 % in untreted smples (Figure 4). Therefore, tretment could be effective in preventing loss of ntioxidnt components in rw milk. Moreover, in previous reserch we observed tht 30-minute flushing of the hedspce of rw milk contining flsks, t flow rte of 120 ml/min, is effective in decresing dissolved oxygen content of rw milk smples from 10.5 mg/l to 0.1 mg/l (Munsch-Altossv et l., 2010). The retined ntioxidnt cpcity of -treted rw milk smples ws most likely due to the replcement of dissolved O 2 with in milk. Totl ntioxidnt cpcity of deproteinized milk smples The totl ntioxidnt cpcity (TAC) vlues of deproteinized rw milk smples determined by the ABTS method were noticebly lower thn those obtined for the originl rw milk smples (Figure 5). At the beginning of the storge, the TAC vlue of control rw milk smple ws ±16.70 µm TEAC (Figure 3) nd decresed to 5.74±0.27 µm TEAC fter milk protein removl (Figure 5). Deproteiniztion of milk smples with trichlorocetic cid my be responsible for the reduced TAC vlues since the deproteiniztion step could exclude b c TAC (µm TEAC) d d C(4) N(4) C(7) N(7) Tretment & Storge Time (dy) Figure 4. Totl ntioxidnt cpcity of rw milk smples ssyed by the DPPH method during storge t 6 C (C: Control, N: -treted, different letters bove the columns indicte sttisticl significnt differences t the p<0.01 level) Figure 5. Totl ntioxidnt cpcity of deproteinized rw milk smples ssyed by the ABTS nd DPPH methods during storge t 6 C (C: Control, N: -treted, different letters bove columns indicte sttisticl significnt differences t the p<0.01 level)

7 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) 161 some ntioxidnt components like scorbic nd uric cids (Zuluet et l., 2009) s well s proteins with ntioxidnt properties. Interestingly, both -treted rw milk smples nd their deproteinized counterprts hd higher TAC vlues compred to untreted deproteinized smples (Figures 3-5). While TAC vlues of control smples decresed by bout % fter four dys of storge (p<0.01), the reduction for -treted deproteinized rw milk smples ws of only % (p<0.01) (Figure 5). After three dditionl dys storge, the TAC vlues of both groups (either controls or treted milk) did not chnge (p>0.01) (Figure 5). The totl ntioxidnt cpcity (TAC) vlues of deproteinized rw milk smples, determined by the DPPH method, re presented in Figure 5. The TAC vlue of control smples decresed from 12.26±0.22 to 9.40±0.27 µm TEAC during seven dys of storge. Results with the DPPH method indicted tht the TAC vlue of the control smples ws 9.83±0.04 µm TEAC while the TAC vlue of -treted smples ws 11.04±0.25 µm TEAC fter four dys of storge. With respect to the initil TAC vlues, the TAC vlues of control nd -treted smples t the end of four dy storge reduced bout 20 nd 10 %, respectively. While the TAC vlues of -treted smples remined similr for three dditionl dys of storge (p>0.01), the TAC vlues of untreted smples still decresed significntly (p<0.01) (Figure 5). During the deproteiniztion step, trichlorocetic cid ddition increses the cidity of the medium, which my lso hve n influence on the ntioxidnt ctivity vlues of deproteinized milk smples. He et l. (2015) reported tht ph djustment to either 3.7 or 6.8 in fruit juice-milk beverge models did not hve ny significnt effect on the ntioxidnt cpcity determined with the ABTS nd FRAP (ferric reducing ntioxidnt power) ssys. Chen et l. (2003) determined TAC vlues of milk smples with the ABTS nd FRAP ssys nd found tht cidity of medium hd significnt effect on the ntioxidnt ctivity vlues. In our study, lthough possible effect of cidity in the rection medium of the ABTS nd DPPH ssys on the TAC vlues my be present, this effect remins similr for ll smples. Therefore, differences in the TAC vlues of deproteinized milk smples (either control or -treted) most likely reflected the effect of -tretment on the ntioxidnt cpcity of smples. The TAC vlues of rw nd deproteinized milk smples obtined by both ABTS nd DPPH methods showed tht milk proteins were the mjor components responsible for the ntioxidnt cpcity of rw milk. Similr results were lso reported by Cervto et l. (1999) nd Zuluet et l. (2009), who found tht csein frctions of milk were responsible for the mjority of the ntioxidnt cpcity. Free rdicl quenching ctivity of cseins is due to the oxi dtion of minocid residues of cseins themselves (Zuluet et l., 2009). Hydrolyzed csein frctions formed by the proteolytic ctivity of enzymes (Rivl et l., 2001) or different microbil cultures (Gjorgievski et l., 2014) my lso hve ntioxidtive properties cting by different mechnisms. Ascorbic cid contents of milk smples Typicl chromtogrms of stndrd scorbic cid () nd scorbic cid in -treted rw milk smple (b) re presented in Figure 6. Retention time, limit of detection, limit of quntifiction nd coefficient of correltion were 6.7 min, 0.02 mg/l, 0.06 mg/l nd 1.00, respectively. Recovery for scorbic cid determintion rnged from 91 to 98 %. Ascorbic cid contents of ll rw milk smples rnged from to mg/l during cold storge. In previous studies, scorbic cid contents between nd mg/l were reported for rw milk (Woessner et l., 1939; Andersson nd Ostle, 1994; Lindmrk-Mnsson nd Akesson, 2000). Our Figure 6. Chromtogrm of stndrd scorbic cid () nd chromtogrphic seprtion of scorbic cid of -treted rw milk smple by the ODS 4 column () (b)

8 162 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) results were in good greement with the results reported in the literture. The chnge in scorbic cid contents of ll smples ws insignificnt in the first four dys of storge while scorbic cid contents decresed significntly fterwrds (p<0.01) (Figure 7). Ascorbic cid contents of both -treted nd control smples were sttisticlly similr t the 4 th nd 7 th dy of storge (p>0.01). Some studies indicted tht scorbic cid content of milk nd humn milk cn be influenced by severl fctors including storge time nd conditions (Lindmrk-Mnsson nd Akesson, 2000; Buss et l., 2001). In our study, scorbic cid content of rw milk smples decresed bout 23.5 % fter seven dys of storge. Buss et l. (2001) reported tht refrigertion for 24 h decresed the totl biovilble vitmin C (scorbic cid nd dehydroscorbic cid) content of humn milk smples by bout 35 %. Possible mechnism for vitmin C losses ws lso indicted to be cused by lctoperoxidse ctivity in milk during cold storge (Buss et l., 2001). Although the dehydroscorbic cid form of vitmin C ws not determined in this study, results of this present study showed tht -tretment did not hve ny protective effect on scorbic cid contents of rw milk smples. Conclusions In this study, rw milk smples possessed higher ntioxidnt cpcity when determined by the ABTS method compred to the DPPH method. Nitrogen gs treted rw milk smples hd higher totl ntioxidnt cpcity (DPPH nd ABTS) vlues thn Ascorbic Acid Content (mg/l) untreted smples when cold stored t 6 C. With the ABTS method, deproteinized rw milk smples hd prticulrly lower TAC vlues compred to their non-deproteinized counterprts. Reduced TAC vlues my hve risen from deproteiniztion which results in reduction of ntioxidnt components. TAC vlues of rw nd deproteinized milk smples determined by both, ABTS nd DPPH methods suggest tht the min components responsible for the ntioxidnt cpcity of rw milk were proteins. Unlike the totl ntioxidnt cpcity vlues, -tretment did not show ny protective effect on L(+)-scorbic cid contents of rw milk smples under the studied conditions. In conclusion, results of this study clerly indicted tht -flushing through the hedspce of milk contining vessels to control microbiologicl growth nd retrd spoilge of rw milk during cold storge hs lso significnt protective effect on the ntioxidnt components of rw milk. Acknowledgements This study ws prtilly supported by the Commission of Mehmet Akif Ersoy University Scientific Reserch Projects (Project No: 0158-KAYDEP-13) nd the Support Progrm of Mehmet Akif Ersoy University for Acdemic Activities with Foreign Countries. The uthors re most grteful to Dml Byn for her technicl ssistnce in lbortory studies. We grtefully cknowledge Antti Alvuotunki /Helsingin Meijeriliike Ltd. for the gift of the rw milk smples. Tpni Altossv thnks the support of the Mtti Sundberg qulity reserch foundtion, nd the Finnish Culturl Foundtion. b b C(4) N(4) C(7) N(7) Tretment & Storge Time (dy) Figure 7. Ascorbic cid content of rw milk smples during storge t 6 C (C: Control, N: -treted, different letters bove columns indicte sttisticl significnt differences t the p<0.01 level).

9 O. GURSOY et l.: Totl ntioxidnt cpcity nd scorbic cid content in rw bovine milk, Mljekrstvo 67 (2), (2017) 163 Utjecj tretmn protočnim plinovitim dušikom n ukupni ntioksidtivni kpcitet i sdržj skorbinske kiseline u sirovom krvljem mlijeku tijekom hldnog skldištenj Sžetk Tretmn kontinuirnim protokom plinovitog dušik ( ) produžuje trjnost sirovog mlijek tijekom hldnog skldištenj. Utjecj tretmn dušikom n ukupni ntioksidtivni kpcitet (TAC) i sdržj skorbinske kiseline (AA) u sirovom mlijeku određivn je tijekom hldnog skldištenj. TAC sirovog ili deproteinizirnog mlijek određivn je pomoću ABTS i DPPH metode, dok je sdržj L (+) - AA u sirovom mlijeku određen kromtogrfski i to nkon 0., 4. i 7. dn skldištenj n 6±1 C. TAC utvrđen pomoću ABTS metode snizio se tijekom hldnog skldištenj s 472,33±16,70 n 369,47±62,06 µm TEAC, dok je TEAC izmjeren pomoću DPPH metode po s 13,30±0,84 n 8,20±0,66 µm. TAC sirovog mlijek određen ABTS metodom smnjio se nkon 4-dnevnog skldištenj, međutim, ostle izmjerene vrijednosti bile su sttistički jednke z -tretirne uzorke tijekom 7-dnevnog skldištenj. Sdržj skorbinske kiseline (AA) sirovog mlijek kreto se u rsponu od 14,06 do 10,76 mg/l tijekom skldištenj, te tretmn nije znčjno utjeco n sdržj AA. Deproteinizirnjem je znčjno smnjen TAC vrijednost uzork mlijek, te u slučju mjerenj pomoću ABTS metode iznosilo je oko 47,50 % z kontrolne uzorke hldno skldištenih tijekom četiri dn, 11,67 % z deproteinizirno sirovo mlijeko tretirno. Zključno, tretmn protočnim kroz gornji dio posude u kojoj se skldišti mlijeko tkođer je pokzo znčjn zštitni učink n ntioksidtivne sstojke sirovog mlijek tijekom hldnog skldištenj. Ključne riječi: sirovo mlijeko, plinoviti dušik (N2), rok trjnj, ntioksidns, skorbinsk kiselin References 1. Akts, A.H., Sen, S., Yilmzer, M., Cubuk, E. (2005): Determintion of crboxylic cids in pple juice by RP HPLC. Irnin Journl of Chemistry nd Chemicl Engineering 24, Andersson, I., Oste, R. (1994): Nutritionl qulity of psteurized milk. Vitmin B12, folcin nd scorbic cid content during storge. Interntionl Diry Journl 4, Bynes, J.W. (1991): Role of oxidtive stress in development of complictions in dibetes. Dibetes 40, Buss, I.H., McGill, F., Drlow, B.A., Winterbourn, C.C. (2001): Vitmin C is reduced in humn milk fter storge. Act Peditric 90, Buyuktuncel, E. (2013): Toplm fenolik içerik ve ntioksidn kpsite tyininde kullnıln bşlıc spektrofotometrik yöntemler, Mrmr Phrmceuticl Journl 17, Cervto, G., Czzol, R., Cestro, B. (1999): Studies on the ntioxidnt ctivity of cseins. Interntionl Journl of Food Science nd Nutrition 50, Chen, J., Lindmrk-Mnsson, H., Gorton, L., Akesson, B. (2003): Antioxidnt cpcity of bovine milk s ssyed by spectrophotometric nd mperometric methods. Interntionl Diry Journl 13, Cloetens, L., Pnee, J., Akesson, B. (2013): The ntioxidnt cpcity of milk - the ppliction of different methods in vitro nd in vivo. Journl of Cell nd Moleculr Biology 59, Cousin, M.A. (1981): Proteolytic ctivity of psychrotrophic microorgnisms in milk nd diry products. In: Psychrotrophic microorgnisms in spoilge nd pthogenicity, Acdemic Press, London, pp Dechemi, S., Benjelloun, H., Lebeult, J.M. (2005): Effect of modified tmospheres on the growth nd extrcellulr enzymes of psychrotrophs in rw milk. Engineering in Life Sciences 5, Gjorgievski, N., Tomovsk, J., Dimitrovsk, G., Mkrijoski, B., Shriti, M.A. (2014): Determintion of the ntioxidnt ctivity in yogurt. Journl of Hygienic Engineering nd Design 8, Gordon, M.H. (2010): Antioxidnts nd food stbility. In: Antioxidnts in food: prcticl pplictions, Woodhed Publishing Ltd., Cmbridge, UK, pp He, Z., Yun, B., Zeng, M., To, G., Chen, J. (2015): Effect of simulted processing on the ntioxidnt cpcity nd in vitro protein digestion of fruit juice-milk beverge model systems. Food Chemistry 175, Hung, D., Ou, B., Prior, R.L. (2005): The chemistry behind ntioxidnt cpcity ssys. Journl of Agriculturl nd Food Chemistry 53,

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