1、foodsArticleEffects of Proteolytic and Lipolytic EnzymeSupplementations on Lipolysis and ProteolysisCharacteristics of White CheesesOya Berkay Karaca1,*and Mehmet Gven21Karata s School of Tourism and Hotel Management,ukurova University,Adana 01903,Turkey2Department of Food Engineering,Agricultural F
2、aculty,ukurova University,Adana 01330,Turkey;mguvencu.edu.tr*Correspondence:obkaracacu.edu.tr;Tel.:+90-322-696-8401(ext.130)Received:8 June 2018;Accepted:7 August 2018;Published:8 August 2018 Abstract:Effects of proteolytic(Neutrase,Bacillus subtilis-originate,0.20(P1)and 0.40 g 100 L 1(P2)and lipol
3、ytic(Piccantase A,Mucor miehei-originated,0.05(L1)and 0.10 g 100 L 1(L2)enzymesupplementations to cheese milk on lipolysis and proteolysis characteristics of 90-day ripened cheesesamples were investigated in this study.While enzyme supplementation did not have signicanteffects on titratable acidity,
4、fat and protease-peptone nitrogen ratios of cheese samples,dry matter,salt,protein,water soluble nitrogen,12%trichloroacetic acid soluble nitrogen ratio(TCA-SN),5%phosphotungstic acid soluble nitrogen(PTA-SN),casein nitrogen ratios,penetrometer value,total free fatty acids(TFFA)and total free amino
5、acids(TFAA)were signicantly inuenced by enzymesupplementations.Individual free amino acids(15 of them)were also determined.Free amino acidcontents of enzyme-supplemented cheeses were higher than the control cheese and the valuesincreased in all cheese samples with the progress of ripening(p 0.05).Th
6、e highest amino acids inall periods of ripening were identied as glutamic acid,lysine,proline and aspartic acid.The major(Ca,P,Na,K,Mg)and minor(Zn,Fe,Cu,Mn)mineral levels of cheeses decreased with the progressof ripening and the effects of enzyme supplementations on these attributes(except for magn
7、esiumand manganese)were found to be signicant(p 0.01).As to conclude,enzyme supplementationsincreased proteolysis and lipolysis and accelerated ripening and thus reduced ripening durations.Especially the enzyme ratios in P1 and L1 cheeses were found to be suitable for reducing the ripeningperiod in
8、White cheese without any adverse effects.Keywords:white cheese;accelerate;lipase;protease;mineral;free amino acid;ripening1.IntroductionTextural,functional and sensory attributes of cheese usually develop with the progress of ripening.Ripening is a costly process and ripening durations largely vary
9、based on the type of cheese 1.It isa quite complex and slow biochemical process 2 and involves three basic reactions as of glycolysis,lipolysis and proteolysis 3.Proteolysis is the primary reaction takes place throughout the ripeningcheese 4,5 and it is the key factor to promote the diversity in tex
10、ture and avor 6.During theprimary proteolysis,residual rennet enzymes together with milk proteases hydrolyze casein and thusproduce large or medium-size peptides.Then in the secondary proteolysis,proteins and large peptidesare gradually hydrolyzed into smaller peptides and amino acids with the aid o
11、f intracellular andextracellular enzymes of starter cultures or other cheese microorganisms 7.Such a breakdown ofprotein networks plays a signicant role in textural attributes and in the release of free amino acidswith a key role in production of sapid compounds 8.Cheese avor is directly related to
12、release of fattyFoods 2018,7,0125;doi:10.3390/foods7080125 Foods 2018,7,0125 2 of 14acids during the lipolysis 9.Since ripening is a quite costly process,various economic and technicalprocesses were practiced accelerating the process and to reduce process costs 10.Cheese should bestored for certain
13、durations for proper ripening.However,such a longer storage duration requires highinvestment costs.Shortened storage durations may increase utilization capacities of the ripening rooms,increase production capacities,reduce production costs and provide signicant economic contributionsto producers 11,
14、12.Elevated ripening temperatures,addition of enzymes(recombinant enzymes,exogenous enzymesand microencapsulation of enzymes)and starters(attenuated,adjunct and genetically modiedcultures)addition of cheese slurry and high-pressure treatment are methods used to accelerate cheeseripening.Among these
15、practices,enzyme supplementation to the milk or curd and elevated ripeningtemperatures are the most common methods 1315.While elevated ripening temperatures areable to be applied to limited number of cheese types,the other methods may have legal(geneticallymodied starters)and technical problems(exce
16、ssive ripening with the use of some peptidase andproteinases)and such methods can also be quite costly(use of plasmin,attenuated starter cultureand free amino acids)16.Commercial lipase and protease enzyme are also used separately or incombination to accelerate ripening process of cheese 1723.In thi
17、s study,two previously determineddifferent doses of Bacillus subtilis-originated neutral protease(Neutrase)and Mucor miehei-originatedfungal esterase lipase(Piccantase A)enzymes were used in cheese production and control cheesewas produced without any microbial enzyme supplementation.Then produced c
18、heeses were ripenedfor 3 months.Experiments were conducted in 3 replications and physico-chemical characteristics,nitrogen fractions,free amino acid and mineral compositions of white cheeses were determined atdifferent stages of the ripening process and the effects of enzymes and ripening period on
19、theseattributes were determined.2.Materials and Methods2.1.MaterialsRaw milk supplied from morning milking of Livestock Facility of Agricultural Research andImplementation Center of Agricultural Faculty was used in cheese production.The Ezal MA014(Ezal;Texel,Dange Saint Romain,France)starter supplie
20、d from Ezal Company,commercial ECOREN200 rennet produced by Maysa Food Co.(mraniye,Istanbul,Turkey)were used.As lipolytic enzyme,Mucor miehei-originated Piccantase A GR(fungal esterase-lipase)enzyme supplied from DSM FoodSpecialties Co.(Izmir,Trkiye)(Turkish Distributor of French Gist-Brocades Co.,I
21、zmir,Turkey)and asproteolytic enzyme,Bacillus subtilis-originated Neutrase 1.5 MG enzymes supplied from NovozymesEnzyme Export Co.(Etiler,Istanbul)(Turkish Distributor of Novozymes A/SBagguaerd,Denmark)were used.Calcium chloride(Merck,Darmstandt,Germany)and NaCl(Merck)were also suppliedfrom commerci
22、al suppliers.2.2.Methods2.2.1.Manufacture of Experimental Turkish White CheesesMilk was checked for acidity and fats,passed through heat treatment at 68 1 C for 10 min,cooled down to 34 1 C and divided into 5 aliquots.These 5 sample aliquots were supplementedwith 1%starter culture and 0.002%CaCl2and
23、 subjected to pre-ripening for 30 min.Milk acidity levelswere continuously monitored throughout the pre-ripening period and enzyme supplementation andfermentation was performed when the pH level reached to 6.4.Following pre-ripening,two aliquotswere supplemented respectively with 0.05 g 100 L 1(P1 c
24、heese)and 0.10 g 100 L 1(P2 cheese)Bacillussubtilis-originated neutral protease,the other two aliquots were supplemented with 0.20 g 100 L 1(L1 cheese)and 0.40 g 100 L 1(L2 cheese)Mucor miehei-originated fungal esterase lipase enzymes.The remaining last aliquot was used as control(C cheese)without a
25、ny enzyme supplementation.Foods 2018,7,0125 3 of 14Milk was coagulated with rennet at 32 1 C for 90 min.The coagulum was cut into 23 cm3pieces,left to drain for 30 min.The gradually increasing weights(0.010.03 kg cm 2)were placed over thesamples to press and separate whey.Then,cheese curds were cut
26、into 7.58 cm3pieces and salted at12%brine and placed into 1 kg cheese containers.The containers were fully lled with 12%fresh brine,tightly closed and left for ripening in a cold storage at 7 1 C for 90 days.2.2.2.Methods of AnalysisGross Composition of Milk,Whey and Cheese SamplesDry matter(DM)rati
27、o of the milk and whey was determined by gravimetric method 24,fat ratiowith Gerber method 25,protein ratios with Micro-Kjeldahl method 26,pH values with Inolop WTW(Weilheim,Germany)digital pH meter,titration acidity with alkaline titration 25,lactose ratios withthe LayneEynon method 27.Cheese yield
28、 performance was calculated as the amount of cheese(kg)produced from 100 kg milk.Cheese dry matter ratios were determined with gravimetric method 24,fat ratios with Van-Gulik butyrometer 28,protein ratios with Micro-Kjeldahl method 26,salt ratioswith Mohr titration method and titration acidity with
29、alkaline titration method 29.Penetrometervalue was determined at 4 1 C with Sur-Berlin PNR 6-brand penetrometer and the results wereachieved as the penetration depth of 95.5 g penetrating tip in 5 s(1/10 mm).Water soluble nitrogen(WSN)ratio 30,5%phosphotungstic acid soluble nitrogen(PTA-SN)ratio 31
30、and 12%trichloroacetic acid soluble nitrogen ratio(TCA-SN)32 were determined withthe MicroKjeldahl method 26.The degree of ripening in terms of 12%TCA soluble nitrogen wascalculated as the ratio of 12%TCA soluble nitrogen to total nitrogen(TN).Casein nitrogen ratio wasdetermined by subtracting water
31、 soluble nitrogen ratio from total nitrogenous substance ratio andresults were expressed in%nitrogen.Protease-peptone nitrogen ratio was calculated by subtracting12%TCA soluble nitrogen ratio from water soluble nitrogen ratio 33.Total Free Amino Acid RatiosTotal free amino acid ratios of proteolytic
32、 enzyme-supplemented cheeses were determinedspectrophotometrically in accordance with Cd-ninhidrine method 34.Calculations were madebased on created standard curve and results were expressed in mg Leu g 1cheese.Total Free Fatty Acid RatiosFat extractions from lipolytic enzyme-supplemented cheeses we
33、re performed in accordance withNunez et al.35 with slight modications and results were expressed in terms of%oleic acid.Free Amino Acid CompositionPhenomenex EZ Faast GC-FID Hydrolyzed Amino Acid Analysis by GC-FID kit(Phenomenex,Lane Cove West,NSW,Australia)was used to determine individual amino ac
34、ids 36.The procedureis briey composed of three phases as of solid-phase extraction clean-up,derivation of the amino acidsand liquid/liquid extraction phases.Zebron ZB-AAA chromatography column(10 m 0.25 mm)tted to a Varian CP-3800 gas chromatograph was used to analyze resultant derivative amino acid
35、swith an organic layer.The gas chromatograph was equipped with an auto-sampler and a ameionization detector(FID 320 C,California,CA,USA)and operated at constant pressure ow mode(helium,60 kPa,1.5 mL/min)with a split injection ratio of 1:15.The injector temperature was 250 Cand detector temperatures
36、was 300 C.The column oven temperature was gradually increased from110 C to 310 C with an increment ratio of 30 C min 1.Then the temperature was held constant at310 C for 18 s.The overall run time was 7.56 min.The chromatographic data were managed by VarianStar Chromatography software(version 5.52;Va
37、rian Inc.,Palo Alto,CA,USA).Amino acid quantitiesof proteolytic enzyme-supplemented cheese samples were calculated from the GC peaks and resultsFoods 2018,7,0125 4 of 14were expressed in mg 100 g 1.These analyses were performed at the TUBITAK(The Scientic andTechnical Research Council of Turkey)Food
38、 Institute of Marmara Research Centre.Mineral Analyses with Atomic Absorption SpectrophotometerDry-ashing method was employed in mineral analyses 37.Phosphorus analyses of cheesesamples were performed colorimetrically in a spectrophotometer(Hitachi U-2000,Minato-ku,Tokyo,Japan).Readings were perform
39、ed with the standard samples prepared identical with thesamples prepared in accordance with Barton(0100 mg P mL 1)at 430 nm.K,Na,Ca,Mg,Fe,Cu,Znand Mn contents were determined with the aid of an Atomic Absorption Spectrophotometer(AAS)(Varian SpectrAA220,Victoria,Australia)provided that required dilu
40、tions were performed.Resultantvalues were multiplied by dilution factor and expressed in mg 100 g 1DM.Statistical AnalysesStatistical analyses were performed in accordance with one way ANOVA“Coincidental ParsellityTrial Plan”to determine the effects of different enzymes and ripening period and signi
41、cant differenceswere compared with Duncans multiple range test.3.Results and Discussion3.1.Raw Milk and Whey Characteristics and Cheese Yield PerformanceAs can be seen in Table 1,average dry matter content of the raw milk was 11.23%,fat contentwas 3.2%,protein content was 3.13%,lactose content was 4
42、.37%,pH was 6.61 and acidity was 6.69 SH.Whey pH values varied between 5.625.69%,titration acidity values varied between 8.268.75 SH,dry matter ratios between 6.526.64%,protein contents between 0.900.91%,lactose contents between4.254.49%and the values were close to each other(p 0.05).Fat contents of
43、 whey samples variedbetween 0.250.30%with the lowest values from the control and L1 cheese(p 0.01).Table 1.Composition of the raw cow milk and whey of White cheeses(n=3).PropertiesRaw Cow MilkWheyC*P1 P2 L1 L2pH 6.61 0.01 5.63 0.02a5.62 0.03a5.62 0.02a5.67 0.10a5.69 0.10aTA*6.69 0.02 8.74 0.12a8.71
44、0.08a8.75 0.09a8.32 0.81a8.26 0.67aDM*(%)11.23 0.10 6.52 0.08a6.55 0.09a6.64 0.08a6.56 0.10a6.64 0.08aFat(%)3.20 0.00 0.25 0.03b0.30 0.00a0.30 0.00a0.26 0.05b0.30 0.00aProtein(%)3.13 0.02 0.91 0.01a0.91 0.02a0.90 0.02a0.91 0.01a0.91 0.01aLactose(%)4.37 0.06 4.27 0.21a4.34 0.28a4.49 0.14a4.25 0.13a4.
45、32 0.10aa,bMeans in the same row followed by different letters were signicantly different(p 0.05).*Cheeses:C:Control without enzymes;P1;0.05 g 100 L 1protease,Bacillus subtilis;P2:0.10 g 100 L 1protease,Bacillus subtilis;L1:0.20 g 100 L 1lipase,Mucor miehei;L2:0.40 g 100 L 1lipase,Mucor miehei.*DM:D
46、ry Matter;TA:Titratableacidity as SH,Soxhelet Henkel Unit.Cheese yield performances varied between 12.9714.02%(Table 2)and enzyme-supplementedcheeses had signicantly different and lower values than the control cheese(p 0.05).The actualperformance is indicated as transition ratio of milk dry matter c
47、omponents into the cheese.In caseof performance calculations as to have 40%dry matter ratio in white cheese,it was observed thatprotease and lipase enzyme-supplemented cheeses had close performance values to each other but hadsignicantly lower values than the control cheese and decreasing performanc
48、e values were observedwith increasing enzyme supplementation rates(p 0.05).Previous researchers also indicated thatenzyme supplementation to cheese to accelerate ripening reduced performance values and such a caseprimarily resulted from increasing nitrogen losses in whey 14,19,38,39.Foods 2018,7,012
49、5 5 of 14Table 2.Yield rates of White cheeses(n=3).C*P1 P2 L1 L2Yield(%)14.02 0.31a13.26 0.32b12.97 0.22b13.24 0.63b12.99 0.42bYield(%40)15.53 0.36a14.79 0.14ab14.20 0.21b14.49 0.68b14.33 0.45ba,bMeans in the same row followed by different letters were signicantly different(p 0.05),but the effects o
50、fripening period were found to be signicant(p 0.01).It was also reported in previous studies thatenzyme-supplemented cheeses did not have signicantly different acidity values from the controlcheese 38,40,41.Table 3.Titratable acidity,penetrometer values and mean composition of experimental White che
