PaPer Ital. J. Food Sci., vol. 27 - 2015 437 - Keywords: lipolysis, ovine blue cheese, Penicillium roqueforti, volatile compounds - Evaluation of lipolysis and volatilE compounds producEd by thrEE Penicillium roqueforti commErcial culturEs in a bluE-typE chEEsE madE from ovinE milk E. salvatorE*, m. addis, m. pEs, m. fiori and a. pirisi Agris Sardegna, Department of Animal Science, Loc. Bonassai, 07040 Olmedo, SS, Italy *Corresponding author: Tel. +39 079 2842391, Fax +39 079 389450, email: esalvatore@agrisricerca.it AbstrAct the aim of this work was to compare the effect of three different Penicillium roqueforti commer- cial cultures (named Ps1, Ps2 and Ps3) on proteolysis, lipolysis and volatile flavour profile of a blue cheese made from ovine milk and lamb paste rennet. Proteolytic parameters were not signif- icantly affected by the Penicillium roqueforti culture, while cheeses manufactured using Ps2 and Ps3 cultures showed the higher amount of free fatty acids (FFA) and volatile FFA when compared with Ps1 culture after 30 days of ripening. this study can provide important information for ob- taining the desired extent of lipolysis in this type of blue cheese. mailto:esalvatore%40agrisricerca.it?subject= 438 Ital. J. Food Sci., vol. 27 - 2015 INtrODUctION blue cheeses represent a cheese variety char- acterised by the presence of blue or blue-green veins, caused by induced growth of the mould Penicillium roqueforti within the cheese matrix. this category includes, among others, PDO (pro- tected designation of Origin) or PGI (protect- ed geographical indication) cheeses made from bovine (Gorgonzola, Italy; Danablu, Denmark; stilton, United Kingdom), and ovine (roquefort, France) milk. the manufacture process of blue mould cheeses has been well described previ- ously (ArDö, 2011), but it can vary depending on country or region where cheese is produced. In particular in sardinia island (Italy), a small production of ovine blue cheese is manufactured on industrial scale. this cheese is character- ized by the use of lamb paste rennet for its pro- duction, differently from most of blue cheeses, where the milk coagulation is usually induced by the action of liquid rennet. cheeses produced with paste rennet are characterized, at late rip- ening stages, by high amounts of free fatty ac- ids due to the presence of lipolytic enzymes (li- pases) in the rennet extract (ADDIs et al., 2005; VIrtO et al., 2003). the ovine blue cheese is made following the process described herein. thermised whole ovine milk is inoculated with a Penicillium roqueforti culture and a mesophilic starter at 36°c. Milk is coagulated using a water solution of lamb paste rennet, and the coagulum is cut into small gran- ules (about 4 mm in size), drained, moved into moulds, dry salted and ripened for 30 days at 10°c and 85% of relative humidity. cheeses are pierced using a stainless steel needle 7 days af- ter production. At the end of ripening (30 days) cheeses are cylindrical in shape (height and di- ameter around 100 and 200 mm, respectively) and weigh between 2.5 and 3.0 kg. the growth of Penicillium roqueforti within the cheese matrix results in a high production of its extracellular enzymes, and consequently in an extensive secondary proteolysis and lipolysis of blue cheeses during ripening (cAlzADA et al., 2013; cONtArINI and tOPPINO, 1995; PrIetO et al., 1999; 2000). Furthermore, blue cheeses are characterised by an high level of flavour compounds produced by lipid, lactose and protein catabolism (ArDö, 2011); in particular a large amount of methyl ke- tones is produced by the β-oxidation of free fat- ty acids followed by a decarboxylation reaction (QIAN et al., 2002; VOIGt et al., 2010). the aim of this work was to compare the effect of three different Penicillium roqueforti commer- cial cultures on proteolysis, lipolysis and volatile flavour profile of sardinian ovine blue cheese af- ter 30 days of ripening, in order to provide use- ful information to cheese makers about the bio- chemical effects produced by each culture dur- ing ripening of this cheese. MAterIAls AND MetHODs Mould cultures three commercially available P. roqueforti cultures, named Ps1 (Prb 6 HYP 5 D, Danis- co Deutschland GmbH, Niebull, Germany), Ps2 (Pr4, chr. Hansen, Hørsholm, Denmark) and Ps3 (PV lYO 10 D, Danisco Deutschland GmbH, Niebull, Germany), were separately used to pro- duce blue-type cheeses. More details about the specific properties of each mould culture can be found in the respective product description doc- uments provided by the supplier. Mould cultures were dissolved in water and added to 50 l of milk before renneting at a fi- nal concentration of 5.0e+6 cFU per l of milk. Small-scale cheese-making cheese production was performed at the dairy technology laboratories of Agris sardeg- na (Olmedo, Italy). Whole ovine milk was placed in a staining steel cheese vat, batch-heated at 65°c in 10 min, and quickly cooled down to 36°c (in 5 min). After cooling, a mould cul- ture and a milk starter culture, prepared us- ing a freeze-dried mixed culture (bulk set HM M4 lYO, Danisco, Deutschland GmbH, Nie- bull, Germany; 1 l·100 l-1 of milk), were add- ed to milk. the composition of the milk start- er culture was: Lactococcus lactis subsp. lac- tis, Lactococcus lactis subsp. cremoris, Lacto- coccus lactis subsp. lactis biovar. diacetylac- tis, Leuconostoc mesenteroides subsp. cremo- ris. Milk was coagulated using a water solu- tion (30 g·100 l-1 of milk) of lamb paste rennet (caglificio Manca, thiesi, Italy). About 45 min after the addition of rennet, the coagulum was cut into small granules (about 4 mm in size), and the drained curd was moved into moulds and kept at 20-25°c under saturated humidi- ty conditions for 18 h. cheeses were then kept at 10°c for 24 h and 90-95% of relative humid- ity, dry salted and finally ripened for 30 days at 10°c and 85% of relative humidity. chees- es were pierced using a stainless steel needle 7 days after production. three replicates were carried out for each treatment level, for a total of nine cheese-making trials. Cheese composition and nitrogen fractions samples were taken for analysis 1 day af- ter production and after 30 days of ripen- ing. cheeses were analysed for pH (pH meter 420 A, Orion, boston, UsA), total solids (ts) (IsO, 2004), fat (soxhlet method), total nitro- gen (tN) (IDF, 1993), soluble nitrogen at pH 4.6 (sN), soluble nitrogen in 12% trichloro- acetic acid (tcA-sN), and soluble nitrogen in 10% phosphotungstic acid (PtA-sN) (GrIPON et al., 1975). Ital. J. Food Sci., vol. 27 - 2015 439 Free fatty acids analysis the free fatty acids content was determined as previously described by ADDIs et al. (2005) on cheeses 1 day after production and after 30 days of ripening. Volatile flavour profile analysis Volatile compounds were determined by sPMe (solid-phase microextraction) -Gc-FID/Ms on cheeses after 30 days of ripening. 0.5 g of fresh- ly grated cheese was placed in a 10 ml vial, her- metically sealed with a seal and thin Viton sep- ta. the vials were held at 37°c in a thermostat- ed autosampler (8200 cX Varian, Walnut creek, cA, UsA) for 5 min to reach equilibrium between sample and above headspace prior to sPMe head- space sampling. A divinylbenzene (DVb)/carbox- en (cAr)/polydimethylsiloxane (PDMs) 50/30 µm fiber (supelco Inc., bellefonte, PA, UsA) was ex- posed to headspace under constant stirring for 7 min in samples after 30 days of ripening. During headspace sampling, samples were maintained at 37°c, and volatile compounds adsorbed on the fiber were immediately thermally desorbed in the injector port of a 3800cX Varian Gc (Wal- nut creek, cA, UsA) equipped with a 1077 split/ splitless injector (250°c), coupled with a flame ionization detector (FID; 250°c), and a saturn 2000 ion trap mass spectrometer system (Ms detector) (Walnut creek, Varian, cA, UsA). Vol- atile compounds were injected in splitless mode in two identical capillary columns (Db-WAX 30 m, 0.32 mm i.d., 0.25 µm film thickness; J. & W. scientific, Folsom, cA, UsA) connected one to FID and the other to mass spectrometer. the column was operated with Helium (1 ml·min-1, constant flow), and the column temperature was held at 40°c for 3 min, then increased to 200°c at a rate of 4°c·min-1, and finally held at 200°c for 5 min. Ms detector was programmed in electron Ioniza- tion (eI) mode at an ionization voltage of 70 eV in the acquisition range between 20-300 m/z, and at a scan rate of 1 scan/sec. the trap, manifold and transfer line temperature were set to 200°, 80° and 200°c respectively. Volatile compounds were identified by comparison of their mass spec- tral data with those of the NIst 98 library (NIst, UsA), by their linear retention indexes (VAN DeN DOOl and KrAtz, 1963) and by comparison with authentic standard compounds (when available). Statistical analysis statistical treatment of data was performed using the sPss statistical package, release 11.5 (sPss, chicago, Il, UsA). Data of chemical com- position, nitrogen fractions and free fatty acids were examined using a bifactorial ANOVA model with “P. roqueforti culture factor” (Pc) and “rip- ening stage factor” (r) as fixed effects, while lsD test (least significant difference test, P < 0.05) for multiple comparisons was used to separate treatment means. the results of volatile com- pounds were examined using a monofactorial ANOVA model with “P. roqueforti culture factor” (Pc) as fixed effect. resUlts AND DIscUssION Chemical composition and nitrogen fractions the chemical composition of cheeses 1 day after production and after 30 days of ripening is reported in table 1. Gross composition was not significantly affected by the Penicillium roque- forti culture at 1 day and after 30 days, where- as it changed significantly (P < 0.05) during the ripening (with the exception of protein content). the values of moisture, fat and protein to to- tal solids ratio after 30 days of ripening were in agreement with data reported by lAWlOr et al. (2003) for other blue-type cheeses. pH values increased of around 1.4 units for all treatments from day 1 to day 30, probably as consequence of the consumption of lactate and the oxidative formation of NH 3 from amino acids operated by moulds during ripening (cANtOr et al., 2004). It has been seen, for example, that the pH may increase of around 2 units in Danablu during the first 5 weeks of ripening (ArDö, 2011). the data reported in table 1 indicate that all proteolytic parameters increased significantly throughout ripening (P < 0.05). the level of pH 4.6-sN (expressed as a percentage of total nitro- gen) ranged from 31.03 to 33.97% after 30 days of ripening (table 1), in some agreement with re- sults published for a number of different blue- type cheeses with longer ripening times (from 34% up to 72% of pH 4.6-sN; FerNáNDez-sAlGUerO et al., 1989; lAWlOr et al., 2003; VOIGt et al., 2010). the level of secondary proteolysis is high- er in blue cheeses compared to other cheese vari- eties (cANtOr et al., 2004); therefore, the raised values at 30 days reported here for tcA-sN and PtA-sN (both expressed as a percentage of total nitrogen; table 1), compared with the values of pH 4.6-sN, highlighted that the most of the sol- uble fraction included non-protein substances (FerNANDez-sAlGUerO et al., 1989). Free fatty acids analysis table 2 summarises the extent of lipolysis (expressed as mmol of free fatty acids per kg of cheese) of cheeses made with each of the three Penicillium roqueforti cultures both 1 day and 30 days after production. the type of Penicillium roqueforti culture sig- nificantly influenced (P < 0.05) the amount of short-chain (c4:0-c8:0), medium-chain (c10:0- c14:0), and long-chain (c16:0-c18:3) FFA. Over- all, all individual FFA increased significantly (P < 440 Ital. J. Food Sci., vol. 27 - 2015 table 1 - composition and nitrogen fractions of cheeses (Ps1, Ps2, Ps3) 1 day after production and after 30 days of ripening. 1 day 30 days SEMe F testf PS1 PS2 PS3 PS1 PS2 PS3 PCg Rh pH 4.85 4.82 4.83 6.35 6.38 6.05 0.18 NS * Moisture (g.100g-1) 49.26 49.63 50.04 42.28 42.97 42.30 0.89 NS * Fata 55.02 54.93 54.88 56.15 56.63 55.60 0.26 NS * Proteina 38.25 38.02 38.19 37.50 37.67 37.42 0.16 NS NS pH 4.6-SNb 8.92 8.84 9.25 31.03 33.97 32.10 2.89 NS * TCA-SNc 5.40 5.60 5.65 28.28 30.86 29.42 2.93 NS * PTA-SNd 1.11 1.23 1.49 12.92 13.05 11.85 1.42 NS * aExpressed as a percentage of total solids (% w/w). b,c,dExpressed as a percentage of total nitrogen (% w/w); bSoluble nitrogen at pH 4.6; cSoluble nitrogen in 12% trichloroacetic acid (TCA); dsoluble nitrogen in 10% phosphotungstic acid (PTA). eStandard error mean. fSignificant differences: * P < 0.05; NS, no significant differences. gP. roqueforti culture factor. hRipening stage factor. 0.05) during ripening, and cheeses manufactured with the cultures named Ps2 and Ps3 showed the higher amount of each FFA after 30 days of ripening when compared with Ps1 culture. three free fatty acids (c4:0, c16:0, c18:1) represented around 60% of total free fatty acids for all treat- ments both 1 day and 30 days after production. After 30 days of ripening, the content of c4:0, c16:0, and c18:1 ranged on average (depending on the culture tested) from 16.07 to 31.65 mmol. kg-1, from 11.88 to 22.90 mmol.kg-1, and from 17.09 to 32.14 mmol.kg-1 of cheese, respective- ly. In general, the action of Penicillium roquefor- ti lipases releases higher concentrations of long- chain FFA than short- and medium-chain FFA (ArDö, 2011). A recent study reported that pal- table 2 - Free fatty acids content (mmol.kg-1 of cheese) in cheeses (Ps1, Ps2, Ps3) 1 day after production and after 30 days of ripening. 1 day 30 days SEMb F testc PS1 PS2 PS3 PS1 PS2 PS3 PCd Re C4:0a 0.47 0.57 0.74 16.07 28.28 31.65 3.37 * * C6:0 0.11 0.14 0.17 3.89 9.46 11.55 1.19 * * C8:0 0.20 0.20 0.20 2.74 5.50 6.85 0.68 * * C10:0 0.15 0.17 0.21 5.83 10.93 13.81 1.39 * * C12:0 0.11 0.11 0.13 2.56 4.64 5.96 0.59 * * C14:0 0.21 0.22 0.23 6.53 9.11 12.68 1.25 * * C16:0 0.46 0.45 0.47 11.88 15.20 22.90 2.24 * * C18:0 0.12 0.11 0.11 2.71 3.51 4.98 0.49 * * C18:1 0.34 0.32 0.31 17.09 27.38 32.14 3.31 * * C18:2 0.07 0.05 0.02 2.62 3.97 4.51 0.47 * * C18:3 0.02 0.02 0.02 2.08 2.85 3.88 0.39 * * TFFAs 2.26 2.41 2.61 74.00 120.83 150.90 15.16 * * aC4:0, butyric acid; C6:0, caproic acid; C8:0, caprylic acid; C10:0, capric acid; C12:0, lauric acid; C14:0, myristic acid; C16:0, palmitic acid; C18:0, stearic acid; C18:1, oleic acid; C18:2, linoleic acid; C18:3, linolenic acid; TFFAs, total free fatty acids. bStandard error mean. cSignificant differences: * P < 0.05. dP. roqueforti culture factor. eRipening stage factor. mitic and oleic acids reached the highest levels of long-chain FFA in a blue cheese (cAlzADA et al., 2013). On the contrary, the values of butyric acid presented in the present study were high- er than those found by other authors in differ- ent blue cheese varieties (cAlzADA et al., 2013; WOO et al., 1984). In particular, Ps1, Ps2 and Ps3 cheeses after 30 days of ripening showed about 1.8-3.6 times higher values of c4:0 when compared with results of cAlzADA et al. (2013), who reported that the content of butyric acid in a blue cheese after 90 days of ripening reached a value of 1.32 mg.g-1 (14.98 mmol.kg-1) of cheese dry matter. the raised values of c4:0 and, in gen- eral, of short-chain FFA observed in the present study can be ascribed to the use of lamb paste Ital. J. Food Sci., vol. 27 - 2015 441 rennet for milk coagulation. lamb paste rennet contains a pregastric lipase, which preferential- ly hydrolyzes short chain fatty acids (KIM HA and lINDsAY, 1993). Furthermore, it is important to point out that even the lipolytic system of Peni- cillium roqueforti can exhibit a selectivity similar to that of the pregastric lipase (KIM HA and lIND- sAY, 1993). SPME analysis cheeses (Ps1, Ps2 and Ps3) after 30 days of ripening were subjected to volatile flavour pro- file analysis by sPMe-Gc-FID/Ms (table 3). A total of 34 volatile compounds were identified in cheese samples, and among them only some vol- atile FFA (butyric, pentanoic and hexanoic acids) and some alcohols (2-pentanol, 1-pentanol and phenyl ethyl alcohol) were significantly affected by Penicillium roqueforti culture (P < 0.05). Ketones and acids represented almost the totality of vola- tile fraction and resulted as more abundant in all samples (about 70 and 27%, respectively). Among ketones, 2-heptanone and 2-nonanone present- ed the highest values of FID Peak Area (table 3). these results were in agreement with those re- ported in literature relating to concentration of ketones in this category of cheeses (ArDö, 2011; cANtOr et al., 2004). the presence of ketones is correlated to the typical flavour of blue cheeses, and they are produced by the β-oxidation of free fatty acids followed by a decarboxylation reaction. table 3 - Volatile compounds (FID Peak Area) in cheeses (Ps1, Ps2, Ps3) after 30 days of ripening. LRIa PS1 PS2 PS3 SEMb PCc 839 2-propanone 275,452 196,359 179,488 70,535 NS 926 2-butanone 4,681 3,307 3,295 671 NS 1,004 2-pentanone 949,455 706,739 742,756 108,431 NS 1,107 2-hexanone 22,208 12,493 17,289 2,347 NS 1,211 2-heptanone 1,791,884 1,299,484 1,624,590 146,423 NS 1,315 2-octanone 28,394 19,954 30,148 4,275 NS 1,333 3-hydroxy 2-butanone 7,969 8,587 5,481 1,753 NS 1,422 2-nonanone 1,047,296 678,913 1,204,917 187,792 NS 1,481 8-nonen 2-one 78,175 55,547 87,753 12,625 NS 1,631 2-undecanone 13,624 3,499 11,418 3,510 NS 1,690 acetophenone 362 183 295 95 NS Ketones 4,219,501 2,985,068 3,907,431 493,937 NS 1,581 propanoic acid 1,805 2,052 2,763 435 NS 1,609 2-methyl propanoic acid 951 1,141 1,400 139 NS 1,671 butyric acid 407,334 1,137,832 957,216 136,292 * 1,712 3-methyl butyric acid 6,539 3,390 4,240 912 NS 1,787 pentanoic acid 2,476 8,945 6,831 1,142 * 1,889 hexanoic acid 314,878 673,581 540,975 63,090 * 1,990 heptanoic acid 1,313 2,965 2,445 363 NS 2,103 octanoic acid 62,326 102,518 81,564 8,519 NS 2,316 decanoic acid 8,299 17,473 11,960 1,935 NS Acids 805,921 1,949,897 1,609,395 208,018 * 910 ethyl acetate 293 176 125 36 NS 1,057 ethyl butyrate 5,024 6,800 3,500 1,703 NS 1,256 ethyl hexanoate 9,744 15,343 10,222 2,315 NS 1,459 ethyl octanoate 11,645 28,596 26,947 4,415 NS Esters 26,511 50,914 40,794 7,354 NS 945 2-propanol 10,390 2,791 7,434 2,096 NS 1,137 2-pentanol 75,497 24,527 45,705 9,397 * 1,270 1-pentanol 2,115 281 0 368 * 1,344 2-heptanol 90,554 34,925 52,055 14,579 NS 1,538 2-nonanol 8,074 2,898 8,414 2,048 NS 1,575 2,3-butanediol 1,402 2,296 2,664 264 NS 1,950 phenyl ethyl alcohol 236 0 0 30 * 2,047 phenol 323 322 341 31 NS Alcohols 188,591 68,368 117,699 26,439 NS 1,301 styrene 2,537 5,351 2,784 940 NS Hydrocarbons 2,537 5,351 2,784 940 NS 940 3-methylbutanal 229 403 245 66 NS Aldehydes 229 403 245 66 NS Totals 5,243,291 5,060,001 5,678,349 398,884 NS aLinear Retention Indexes using a DB-WAX column. bStandard error mean. cP. roqueforti culture factor, significant differences: * P < 0.05; NS, no significant differences. 442 Ital. J. Food Sci., vol. 27 - 2015 the reaction is catalysed by enzymes contained both in spores and mycelium of Penicillium spp. (QIAN et al., 2002; VOIGt et al., 2010). table 3 also highlights that Ps1 present- ed significantly lower values of acids than oth- er cheeses. this was consistent with what dis- cussed above concerning the higher lipolytic ac- tivity of Ps2 and Ps3 compared to Ps1 (table 2), and was also probably due to the greater apti- tude of Ps1 in converting FFA to 2-alkanones. Among volatile FFA, butyric acid was the most abundant (50, 58 and 59% of total FFA in Ps1, Ps2 and Ps3, respectively) followed by hexano- ic (39, 35 and 34% of total FFA in Ps1, Ps2 and Ps3, respectively) and octanoic (8, 5 and 5% of total FFA in Ps1, Ps2 and Ps3, respectively) ac- ids. the origin of raised values of butyric acid has been discussed in the previous section, while hexanoic and octanoic acids are important fla- vor compounds of blue cheeses (ArDö, 2011). esters are produced by free fatty acids esterifi- cation with primary alcohols, and may attenuate the typical pungent flavour of blue cheeses due to the methyl ketones (MOIO et al., 2000). they represented only 1% on average of totals vola- tile compounds (table 3); Ps2 and Ps3 showed higher FID Peak Area (but without any statis- tical significance) of esters as a consequence of their higher content of FFA compared to Ps1. the strong reducing environment present in ripened cheese favoured the production of 2-al- kanols from corresponding 2-alkanones. Ps1 showed significantly higher values of 1- and 2-pentanol when compared with other samples (P < 0.05), and tended to have the highest levels both of 2-alkanones and 2-alkanols. the parallel evolution of these volatile compounds was previ- ously observed in other blue cheeses (GONzáles De llANO et al., 1990). cONclUsIONs the results indicated that the ovine blue cheese made in sardinia was more subjected to lipolysis and presented higher amounts of short chain fatty acids when compared to the most known blue cheese varieties. this evolu- tion of lipolysis in the product was also due to the use of lamb paste rennet. two cultures (Ps2 and Ps3) were characterised by the highest val- ues of total free fatty acids. In contrast, prote- olytic parameters and the most volatile com- pounds did not vary significantly depending on the culture tested. 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Paper Received July 7, 2014 Accepted January 19, 2015