Documents

Determination of lipase activity in cheese using trivalerin as substrate

Description
Determination of lipase activity in cheese using trivalerin as substrate
Categories
Published
of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  International Dairy Journal 16 (2006) 423–430 Determination of lipase activity in cheese using trivalerin as substrate Ingemar Svensson, Igor Herna ´ndez 1 , Mailo Virto, Mertxe de Renobales  Biochemistry and Molecular Biology Department, Facultad de Farmacia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea,Aptdo. 450, E-01080 Vitoria-Gasteiz, Spain Received 24 June 2004; accepted 12 June 2005 Abstract Lipolysis during cheese ripening is usually assessed by the accumulation of free fatty acids (FFA). An assay to determine totallipolytic activity present in a cheese during ripening was established. Finely grated cheese (1g) was directly incubated with trivalerin(204mg) as a substrate at 35 1 C for 4h. Free valeric acid was extracted and quantified by gas chromatography. The assay was linearwith time up to 24h and up to 0.11LipaseUnitsg  1 cheese. The total amount of lipolytic activity determined with this assay wasconsistent with the total amount of FFA present in 5 types of cheese. In Idiazabal cheese samples manufactured with or withoutlipase added, the total lipolytic activity determined during ripening remained constant up to 6 months of ripening. In addition totrivalerin, other possible substrates investigated were triolein, triundecanoin and the endogenous butyric acid-containingtriacylglycerols present in the cheese sample. Activities measured with these substrates were considerably lower than values obtainedwith trivalerin due to the high levels of oleic acid present in cheese, or to difficulties in mixing triundecanoin (solid at 35 1 C) with thegrated cheese sample. Endogenous triacylglycerols gave increasingly lower activity values as ripening time progressed due tosubstrate depletion. r 2005 Elsevier Ltd. All rights reserved. Keywords:  Lipase activity assay; Cheese; Trivalerin; Lipolysis; Cheese ripening 1. Introduction Lipolysis of milk triacylglycerols during cheese ripen-ing is an important biochemical process that contributesdirectly to its sensory characteristics (for a review onlipolysis see Collins, McSweeney, & Wilkinson, 2003). Free fatty acids (FFA) are important sensory com-pounds by themselves (Molimard & Spinnler, 1996), and as precursors of other volatiles such as methyl ketones,and esters (for a review see McSweeney & Sousa, 2000). The amount of FFA accumulated during ripening isconsidered to be an overall measure of lipolysis, and it isquite variable, depending on the type of cheese, ripeningtime, and manufacturing technology. In cheeses likeCheddar or Gouda, lipolysis is rather low, with amountsbetween 7000 and 10,000 m mol total FFA kg  1 cheeseafter 7–10 months of ripening (Bills & Day, 1964). The highest lipolysis rates have been reported in blue cheesesdue to the presence of very active lipases from Penicillium  spp. (Gripon, 1993). In these cheeseslipolysis can reach up to 10% and 20%, which wouldcorrespond to over 300,000 m mol total FFA kg  1 cheese,with oleic acid (C18:1) being the major FFA (Gripon,Monnet, Lamberet, & Desmazeaud, 1991; Gonza  ´lez-Llano, Ramos, Rodrı ´guez, Montilla, & Jua ´rez, 1992). Incheeses from Mediterranean countries manufacturedwith lamb rennet pastes, the total amount of FFAranges between 30,000 and 80,000 m mol total FFA kg  1 cheese, with short-chain FFA (C4 to C10) representingover 60% of the total FFA, approximately 40,000 m moltotal FFA kg  1 cheese after 6 months of ripening (Woo& Lindsay, 1984; Fox & Guinee, 1987; Larra  ´yoz,Martinez, Barro ´n, Torre, & Barcina, 1999; Virto et al., ARTICLE IN PRESS www.elsevier.com/locate/idairyj0958-6946/$-see front matter r 2005 Elsevier Ltd. All rights reserved.doi:10.1016/j.idairyj.2005.06.004  Corresponding author. Tel.: +34945013097; fax: +34945013014. E-mail address:  mertxe.derenobales@ehu.es (M. de Renobales). 1 Present address: NIZO Food Research, B.V., Kernhemseweg 2,6718 ZB, Ede, The Netherlands.  2003). These FFA are responsible for imparting a sharp,characteristic flavour to the cheese which is oftendescribed as ‘piquant’ or ‘pungent’ (Nelson, Jensen, &Pitas, 1977; Woo & Lindsay, 1984; Fox & Guinee, 1987; Larra ´yoz et al., 1999; Virto et al., 2003). The accumulation of FFA during cheese ripeningoccurs by the combined action of lipases present in thecheese (Collins et al., 2003). Lipase activity duringcheese ripening can be of different srcins: (a) microbiallipases produced by the starter culture and/or the biotathat develops during ripening, which could includeendogenous species present in the milk, particularly incheeses made from raw milk; (b) milk lipoprotein lipasein cheeses made with raw milk; (c) added lipase activityduring cheese manufacture, either as part of lipolyticrennet paste (Bustamante et al. 2000), or as acommercial enzyme to accelerate ripening (Law, 2001);(d) lipolytic secondary cultures added to certain types of cheese, such as Roquefort (Gripon, 1993).Lipolysis during cheese ripening has been studiedmostly by the determination of the amounts of FFA atvarious ripening times. Results are often reported as mg, m mol, or milliequivalents of FFA kg  1 cheese. Someauthors have attempted to study the variation of theseFFA during ripening, observing that the FFA composi-tion of the cheese, and the proportions of short-,medium- and long-chain FFA, change during ripeningunder certain conditions (Cha ´varri et al., 1999, 2000;Virto et al., 2003). There are very few reports on thedetermination of partial glycerides resulting from thehydrolysis of triacylglycerols by lipases during cheeseripening (Barron et al., 2004; Precht & Abd El Salam, 1985; Contarini & Topino, 1995; Koprivnjak, Conte, & Boschelle, 1997). Attempts to directly measure lipolyticactivities in cheese samples are scarce. To the best of ourknowledge, very few reports have been published onlevels of lipase–esterase activity during ripening (Pitotti& Dal Bo, 1996; Humbert, Guingamp, & Linden, 1997; Weimer et al., 1997; Albenzio et al., 2001). In all these cases  p -nitrophenyl, or  b -naphthol esters of fatty acidsare used as substrates. Weimer et al. (1997) measuredactivity on aqueous extracts of cheeses and Humbertet al. (1997) measured it on cheese slurries. Theseauthors did not attempt to relate the levels of measuredactivity in cheese with the amounts of total FFA, orother lipolysis-related products (such as acyl–ester, oreven partial glycerides) present in the same sample.Esters of fatty acids and  p -nitrophenol are substrates forlipolytic enzymes of the esterase type (Desnuelle, 1976;Martinelle & Hult, 1994). These substrates allow for a convenient spectrophotometric assay, and are frequentlyused for this reason. However, these substrates may notbe the most suitable ones to determine the activity of lipases that hydrolyse the fatty acids from milktriacylglycerols, such as those that may play animportant role on the transformation of the milk fatduring cheese ripening. The study of lipolysis duringripening would considerably benefit from a reliablemethod to determine lipolytic activity directly in cheeseslurries.Previous work in our laboratory (Cha ´varri, Santiste-ban, Virto, & de Renobales, 1998) indicated that, although some enzyme activities could be determinedin water extracts of Idiazabal cheeses during ripening,no lipolytic activity could be measured in water extractswith emulsified triolein or  p -nitrophenyl esters assubstrates. This was most likely due to the fact thatthe lipolytic activity, which was extremely low in allcases (cheeses had been manufactured with no addedlipase, nor with any rennet paste), remained in theinsoluble precipitate, presumably adsorbed to the fatand/or insoluble protein. The objective of the presentstudy was to devise a method to measure the lipaseactivity present in the cheese matrix without the need forprior extraction into an aqueous phase. The methoddescribed herein is based on the quantitative determina-tion by gas chromatography of the valeric acid liberatedfrom the triacylglycerol substrate (trivalerin) addeddirectly to the finely ground cheese. 2. Materials and methods  2.1. Cheese samples Idiazabal cheese samples were from cheeses manu-factured, from the same batch of milk, with lipase-containing lamb rennet paste, or with commercialbovine rennet, or with a mixture of commercial bovinerennet and added commercial pregastric lipase, asdescribed by Hernandez et al. (2005). Lamb rennetpaste was prepared as described by Bustamante et al.(2000) from abomasa from 1-month-old lambs. Com-mercial bovine rennet (devoid of any lipase activity) was‘Standard Plus’, and commercial pregastric lipase was‘Animal Lipase’, both from Chr. Hansen (Madrid,Spain). Commercial lipase was added to the milk withthe rennet. The amounts of added lipase in each case arestated in table and figure legends. Samples fromManchego, Provolone, Roquefort and blue cheese, allof unknown ripening periods, were purchased in localmarkets.  2.2. Assay of lipase activity 2.2.1. Cheese samples The assay is based on the quantitative determinationof free valeric acid released after a given incubationperiod with a known amount of trivalerin added to 1gof grated cheese. One gram of finely grated cheese(particles were smaller than 3mm) was placed in a10mL glass vial with teflon-lined screw cap. The ARTICLE IN PRESS I. Svensson et al. / International Dairy Journal 16 (2006) 423–430 424  reaction was started by addition of 200 m L trivalerin(Sigma, Madrid, Spain), vortexing the mixture for 10s,and subsequently sonicating it for 10min at 35 1 C (MSESoniprep 150; Integrated Services TCP Inc., PalisadesPark, NJ, USA) with a medium size probe at 20kHz.The samples were immediately placed in a water bath at35 1 C. Shaking of the reaction mixture did not appearto be necessary. After an incubation period of 4h forroutine assays, reactions were stopped by placing thevessels on ice. The reaction blank was carried out byboiling the grated cheese for 10min to inactivatelipolytic activity.To determine the amount of free valeric acidproduced, the FFA were extracted immediately follow-ing the method of  de Jong and Badings (1990), asdescribed by Cha ´varri et al. (1997). Briefly, to the reaction vessel, 100 m L of an internal standard solution(1.0mgmL  1 heptanoic acid in heptane:diethyl ether,1:1 by volume), 0.3mL 2.5 M  H 2 SO 4 , 0.3mL absoluteethanol (99%) and 3.0g anhydrous Na 2 SO 4  were added.The mixture was vortexed for 10s (at room tempera-ture). The FFA were extracted 3 times with 3mLheptane:diethyl ether each time. After solvent addition,the mixture was vortexed for 20s and centrifuged at500g for 2min. All the organic phases were collectedand pooled. The reaction blank was prepared by placingthe sample vessel immediately on ice and subsequentlyextracting the free fatty acids.Cheese samples used to determine lipase activity wererefrigerated at 5 7 1 1 C for up to 48h until analysed.Values reported are the means 7 standard deviation of 4 replicates. When lipase activity in Idiazabal cheeseswas determined on various ripening days, 2 cheeses weretaken for analysis on each ripening day and lipase assayswere run in duplicate for each sample.  2.2.2. Commercial lipase preparations and extracts of lamb rennet pastes Lipase activity in the commercial pregastric lipasefrom Chr. Hansen (0.1g lipase powder in 1mLdeionised water), or in aqueous extracts of lamb rennetpastes, was assayed by titrating the free butyric acidreleased from a tributyrin emulsion, according to theprocedure described by Barton, O’Connor, and Turner(1996), with the following modifications. To prepare thesubstrate emulsion, 0.6g casein (Sigma, catalogue no.C-5890, Madrid, Spain), 120mg Na 2 HPO 4  and 50mglecithin (Merck, Madrid, Spain) were mixed in 95mLdeionised water. After stirring for 30min at 42 1 C allvisible particles were dissolved and the pH was around6.8. Tributyrin (5mL; Sigma) was added and stirredwith a magnetic stirring bar for 30min at 42 1 C. Thissolution was sonicated with a medium size probe at20kHz for a total of 6 cycles of 20s on and 10s pauseeach. The emulsion was kept at ambient temperaturewith stirring and it was used within 1h. The assaymixture contained 10.0mL emulsion and 1.0mL of enzyme-containing solution. The reaction was carriedout at 42 1 C. The pH was adjusted to 6.2 and the releaseof butyric acid was calculated from the amount of 0.05 M NaOH consumed to keep the pH constant at 6.2. Thereaction blank was prepared by adding to the substrateemulsion an aliquot of enzyme extract boiled for 10min.One Lipase Unit (LU) was defined as the amount of enzyme that liberated 1 m mol of the corresponding fattyacid (butyric acid from tributyrin; valeric acid fromtrivalerin) per minute.  2.3. Determination of FFA by gas chromatography The FFA extracted from cheese incubation mixtureswere purified on aminopropyl-bonded phase columns(0.5g; Waters, Madrid, Spain), previously equilibratedwith 10mL heptane, as described by Cha ´varri et al.(1997). The pooled organic phase was applied to thecolumn, and glycerides were washed with 10mL chloro-form:2-propanol (2:1, by volume). Elution of the boundFFA was accomplished with 5mL diethylether contain-ing 10mLL  1 formic acid. FFA were quantified under-ivatized by gas–liquid chromatography on a HewlettPackard gas chromatograph, model 5890 series II, on afused-silica capillary column (25m  0.32mm) coatedwith FFA phase (cross-linked polyethylene glycol,0.52 m m layer thickness), essentially as described pre-viously (Cha ´varri et al., 1997). Quantification wascompleted with heptanoic acid as internal standardadded to the cheese sample at the time of extraction, asdescribed above.  2.4. Statistical analysis The SPSS package (SPSS, Chicago, IL60611, USA)was used for statistical analysis of the results. Simplelinear regression analysis was applied to fit the amountof valeric acid released with the incubation time and thetotal amount of lipase added. 3. Results and discussion A preliminary experiment was carried out to deter-mine the best substrate to assay lipase activity in cheesesamples. Several triacylglycerides could be used as asubstrate, at least in principle. Triolein is a frequentlyused substrate for most lipases (Beisson, Tiss, Rivie `re, & Verger, 2000; Deeth & Touch, 2000), but free oleic acid is produced in a cheese in very large amounts (Woo &Lindsay, 1984; Cha ´varri et al., 1999), so the backgroundlevel of this acid in cheese samples would be very high,making the quantification of enzyme-released oleic aciddifficult. Triacylglycerides of odd-numbered carbonatom fatty acids, such as trivalerin or triundecanoin, ARTICLE IN PRESS I. Svensson et al. / International Dairy Journal 16 (2006) 423–430  425  offer good alternatives because endogenous levels of freevaleric or undecanoic acid present in cheese are almostnegligible. Two cheese samples were selected on accountof their presumed lipase content: a commercial sampleof blue cheese that should contain a very active lipaseproduced by  Penicillium  spp. (which has a broadspecificity for fatty acids of various chain lengths;Gripon, 1993), and an Idiazabal cheese manufacturedwith commercial pregastric lipase with a markedspecificity for short-chain fatty acids (Lai, Mackenzie,O’Connor, & Turner, 1997). As it can be seen in Table 1, trivalerin gave the best results of the 3 substrates withboth types of cheese. The low activity value obtainedwith triolein in blue cheese was probably due to thedifficulties in the quantification of the enzyme releasedoleic acid against the high content of oleic acid presentin the cheese. Triundecanoin was tested only withIdiazabal cheese and it gave an extremely low activityvalue, most likely due to the fact that it did not mix wellwith the cheese slurry because its melting point is around35 1 C. Trivalerin was used as a substrate in allsubsequent experiments.To optimise substrate concentration, incubationswere carried out with different amounts of trivalerin.Increasing the amount of trivalerin up to 400 m Lincreased the rate of release of valeric acid (Table 2).The optimum amount of substrate in the assay mixturewas 200 m L, which is the amount used in all subsequentassays. It was found that to ensure complete extractionof the free valeric acid produced at the end of theincubation period, a small amount of ethanol had to beadded to the cheese slurry to break up lipid-proteincomplexes and thus improve free fatty acid extraction,as reported also by de Jong and Badings (1990). In thisrange of substrate concentrations no substrate inhibitionwas observed. The release of valeric acid from samplesof cheese manufactured with commercial lipase(5LUL  1 milk) was linear up to 24h of incubationtime (Fig. 1;  R 2 : 0.99). However, routine assays werecarried out for 4h. No increase with time in the amountof released valeric acid was detected in boiled cheesesamples.The total amount of FFA in cheeses, which can varywidely with the type of cheese, can be taken as anindication of the total amount of lipolytic activitypresent during cheese ripening. To determine whetheror not this assay can be used in cheeses with verydifferent levels of lipolytic activity, the linearity of theassay as increased amounts of lipase were added wasstudied. To avoid having an excessively large amount of aqueous phase in the cheese slurry, which would resultin an emulsion of different characteristics, very smallvolumes of a commercial pregastric lipase solution wereadded to 1g of grated cheese, boiled first for 10min toinactivate the indigenous activity present in it. The assaywas linear up to 0.11LUg  1 cheese of added lipase(Fig. 2;  R 2 : 0.99). This range of lipolytic activity includesthe values present in highly lipolytic cheeses, such asblue cheeses (Table 1) and cheeses made with lipolyticrennet pastes (Table 4), described herein.To determine the usefulness of the assay for othercheeses, we purchased in the local market 4 different ARTICLE IN PRESS Table 1Lipase activity determined with 3 different substrates in blue cheeseand Idiazabal cheeseCheese Substrate Lipase activity a (mLUg  1 cheese)Blue cheese b Trivalerin 15.51 7 0.07Triolein 3.15 7 0.32Idiazabal c Trivalerin 6.47 7 0.55Triundecanoin 0.10 7 0.01 a Determinations were accomplished as described in Materials andmethods using 100 m L of each substrate. Values given are means andstandard deviations of 4 replicates. One unit of lipase activity (LU) isthe amount of lipase that releases 1 m mol FFA per minute. The FFAquantified were valeric, oleic and undecanoic acids. b A sample of blue cheese was purchased at a local market. c Idiazabal cheese was manufactured with added commercial lipase(5LUL  1 milk) and had been ripened for 60 days.Table 2Lipase activity with increasing concentration of trivalerinAmount of trivalerin ( m L) mLUg  1 cheese a 100 6.95 7 0.07200 8.55 7 0.20400 8.45 7 0.13 a Determinations were accomplished as described in Materials andmethods using 1-day-old Idiazabal cheese manufactured with addedcommercial lipase (5LUL  1 milk). One unit of lipase activity is theamount of lipase that releases 1 m mol valeric acid per minute.Fig. 1. Amount of valeric acid released during different incubationtimes up to 24h. Assay mixtures contained 1g boiled for 10min ( ’ ) ornot boiled ( ~ ) grated Idiazabal cheese manufactured with commercialbovine rennet and added commercial lipase (5LUL  1 milk), and200 m L trivalerin. Values given are means and standard deviations of 4replicates. I. Svensson et al. / International Dairy Journal 16 (2006) 423–430 426  types of cheese, of unknown ripening time, which wereexpected to have different levels of lipolysis. The amountof lipase present in these samples was determined withtrivalerin as a substrate, and compared with that in asample of an Idiazabal cheese manufactured with nolipase added and ripened for 60 days (Table 3).Considering the total amount of FFA, Idiazabal,Prolovone and Manchego cheeses would not beconsidered as ‘lipolytic’ cheeses, in contrast to Roque-fort and blue cheeses that could be considered ‘lipolytic’.In the 3 cheeses with low amounts of total FFA(Manchego, Provolone and 60-day-old Idiazabal), thelevel of lipase was low and of the same order of magnitude. Manchego cheese is manufactured withcommercial bovine, or ovine, rennet, with no lipaseadded. Lipolytic enzymes present in rennet pastes usedin the manufacture of Provolone cheese are inactivatedduring the heat treatment prior to the stretching of thecurd (Fox & Guinee, 1987). The low amount of lipolytic activity detected reflects, most likely, the lipase activityfrom the starter cultures (in which lipases are intracel-lular and of low activity) and other microbial speciesthat develop in the cheese during ripening (Collins et al.,2003). The differences observed among the 3 cheesescould be due, presumably, to differences in theirmicrobial ecosystems. In contrast, during the manufac-ture of Roquefort, or Blue-type cheeses, a secondaryinnoculum of highly lipolytic  Penicillium  spp. is added(Gripon, 1993). Lipases from these organisms hydrolysetriacylglycerols both with long- and short-chain fattyacids (Gripon et al., 1991). Accordingly, the totalamount of FFA in these cheeses was very high. In thetwo different types of cheeses analysed (not verylipolytic or highly lipolytic), the levels of totalFFA correlated with the level of measured lipaseactivity, although a quantitative correlation cannot beestablished.Idiazabal cheese, and some Italian (Fox & Guinee, 1987) and Greek cheeses (Anifantakis, 1976; Anifanta- kis & Green, 1980; Moatsu et al., 2004), can be manufactured with lamb rennet paste containing lipo-lytic activity (Bustamante et al., 2000), or with addedpregastric lipase (Hernandez et al., 2005). The lipaseactivity present in cheeses made with a commercialpregastric lipase added during manufacturing (50LUadded L  1 milk) was assayed after 1, 30, 60 and 180days of ripening (Fig. 3). Lipase activities are reportedas units per gram of dry matter (DM) to account for thegradual loss of water during ripening. The activitymeasured with trivalerin as a substrate was constant(10.94 7 0.41mLUg  1 DM) throughout the 180 days. Inthese cheeses the concentration of total FFA duringripening increased linearly up to 45,000 m molkg  1 cheese after 60 days of ripening, and then up to60,000 m molkg  1 cheese after 180 days of ripening(Herna ´ndez et al., 2005). In contrast, Weimer et al.(1997), who determined lipase activity with  b -naphtholesters of short-chain fatty acids during 180 days of ripening of Cheddar cheese, found a small increase inlipase activity on day 120 of ripening, but provided nodata regarding the amounts of FFA during ripening.The correlation between the total amount of FFApresent in the cheese with the lipolytic activity deter-mined should be interpreted carefully, particularly incheeses of short ripening time, or at the early ripeningstages, because the concentration of FFA at any time isthe result of two types of opposite processes, production(such as triacylglycerol hydrolysis, or de novo synthesis)and utilization (for example, degradation or esterformation). Thus, whereas high levels of total FFAcorrelate with high levels of lipase activity (Roquefortand blue cheese in Table 3, and Idiazabal in Fig. 3), it ARTICLE IN PRESS Table 3Lipase activity and total FFA ( m molkg  1 cheese) determined in5 different types of cheeseCheese sample a Lipase activity b (mLUg  1 cheese)Total free fatty acids( m molFFAkg  1 cheese)Idiazabal 0.22 7 0.02 6427 7 169Provolone 0.66 7 0.04 9218 7 178Manchego 0.30 7 0.02 11,161 7 180Blue 16.58 7 0.07 42,660 7 476Roquefort 13.81 7 0.03 50,204 7 2236 a Samples of Provolone, Manchego, Roquefort and Blue cheese werepurchased in a local market. Manufacturing conditions and ripeningage were unknown. Idiazabal cheese was manufactured with commer-cial bovine rennet and no lipase added and ripened for 60 days. b Values given are means of 4 determinations and standarddeviations. One unit of lipase activity is the amount of lipase thatreleases 1 m mol valeric acid per minute.Fig. 2. Relationship between the lipase activity of a commercial lipasesolution (measured with tributyrin) added to a boiled Idiazabal cheesesample manufactured with no lipase added ( X  -axis), and the activity inthe cheese sample (measured with trivalerin;  Y  -axis). One unit of lipaseactivity is the amount of enzyme that releases 1 m mol of fatty acid(butyric or valeric) per minute. Values given are means and standarddeviations of 4 replicates. I. Svensson et al. / International Dairy Journal 16 (2006) 423–430  427
Search
Similar documents
View more...
Tags
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks