Effects of low-pressure homogenisation pre-treatment of cheesemilk on the ripening of Cheddar cheese

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1 Effects of low-pressure homogenisation pre-treatment of cheesemilk on the ripening of Cheddar cheese Kevin Deegan, Paul Mcsweeney To cite this version: Kevin Deegan, Paul Mcsweeney. Effects of low-pressure homogenisation pre-treatment of cheesemilk on the ripening of Cheddar cheese. Dairy Science Technology, EDP sciences/springer, 2013, 93 (6), pp < /s >. <hal > HAL Id: hal Submitted on 17 Sep 2015 HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

2 Dairy Sci. & Technol. (2013) 93: DOI /s ORIGINAL PAPER Effects of low-pressure homogenisation pre-treatment of cheesemilk on the ripening of Cheddar cheese Kevin C. Deegan & Paul L. H. McSweeney Received: 21 December 2012 / Revised: 24 May 2013 / Accepted: 24 May 2013 / Published online: 14 June 2013 # INRA and Springer-Verlag France 2013 Abstract Homogenisation of milk breaks the protective fat globule membrane, allowing access of indigenous lipoprotein lipase and hydrolysis of free fatty acids (FFA) from triglycerides. The use of homogenisation in cheesemaking is limited due to the deleterious impact on cheese quality. The objective was to investigate the effects of a pre-processing routine (homogenisation of raw milk, incubation and pasteurisation) on the resultant cheese. Pilot- and laboratory-scale Cheddar cheeses were produced where raw bovine milk was homogenised at various pressures (0, 5 and 10 MPa in the pilot-scale and 0, 5, 10, 15, 20 and 25 MPa in the laboratory-scale trial), incubated at 37 C for 1 h and batch-pasteurised at 63 C for 30 min. A control milk was not homogenised. Acid degree value increased with increasing homogenisation pressure. Moisture and salt contents were higher in cheeses made with milk homogenised at 5 and 10 MPa, as was ph. The cheese made from milk homogenised at 10 MPa contained the highest level of FFA initially and throughout ripening. Breakdown of α s1 -casein was more extensive in cheese made from non-homogenised milks and breakdown of β-casein was more extensive in homogenised milk cheeses. The use of low-pressure homogenisation in a controlled pre-processing routine may be suitable for cheesemaking; however, further investigation into the effects of the pre-treatment and effects on cheese microstructure and sensory properties is warranted. Keywords Cheddar cheese. Lipolysis. Homogenisation. Lipoprotein lipase K. C. Deegan : P. L. H. McSweeney School of Food and Nutritional Sciences, University College, Cork, Ireland K. C. Deegan (*) Department of Food and Environmental Sciences, University of Helsinki, P.O. Box 66, Agnes Sjöbergin katu 2, Helsinki FI-00014, Finland kevin.deegan@helsinki.fi

3 642 K.C. Deegan, P.L.H. McSweeney 1 Introduction Homogenisation, developed in 1899 by Gaulin, is a widely used method to prevent undesirable creaming in milk and milk products during their shelf life (Jana and Upadhyay 1992). This is achieved by mechanical reduction of the size of milk fat globules, which retards the coalescence and subsequent separation of the fat layer (Hayes and Kelly 2003a; Huppertz and Kelly 2006). Disruption of the fat globules produces a five- to tenfold-larger surface area of fat which is stabilised by casein micelles and whey proteins (Michalski et al. 2002). Homogenisation is almost always followed by a heat treatment, e.g., pasteurisation, to destroy pathogenic bacteria and to inactivate indigenous lipoprotein lipase (LPL) which could access the fat and cause excessive lipolysis (Deeth 2006). Homogenisation of cheesemilk is performed only when high levels of lipolysis are necessary to the sensory qualities (e.g. in some blue cheeses) or for formation of correct texture (in some fresh cheeses). Homogenisation of milk has various effects on cheesemaking and cheese parameters, including decreased rennet coagulation time (Robson and Dalgleish 1984), decreased syneresis (Green et al. 1983), a weaker rennet coagulum and lower curd tension (Green et al. 1983), increased cheese yield (Jana and Upadhyay 1992), compositional changes in the cheese (Jana and Upadhyay 1992; Peters 1956) and excessive free fatty acids (FFA) production. Lipolysis is one of the key biochemical events during the ripening of cheese and is mainly caused by enzymes (hydrolytic) resulting in the hydrolysis of milk lipids to partial glycerides, FFA and further breakdown products. Such products contribute to the characteristic flavour of cheese varieties, especially in Blue and Italian varieties (Collins et al. 2003). Enzymes responsible for liberation of FFA in milk and cheese come from six main sources, namely; the milk itself, rennet paste, starter bacteria, secondary organisms, non-starter lactic acid bacteria (NSLAB) and exogenous lipases (Collins et al. 2003). Bovine milk contains one indigenous lipase, LPL, whose primary function is the catabolism of triacylglycerols in lipoproteins (Deeth 2006). LPL is present at a concentration of nmol.l 1 in bovine milk of which 80% is electrostatically associated with casein micelles. The lipolytic potential of LPL in raw bovine milk is quite high (Driessen 1983), but such action is normally prevented by the protection of the substrate by the milk fat globule membrane (MFGM). However, where the MFGM is damaged (e.g. by homogenisation, temperature abuse or foaming), lipolysis occurs rapidly. In cheesemaking, LPL is almost completely inactivated by pasteurisation (Driessen 1983; Andrews et al. 1987) and that which survives is immobilised in the casein gel system, where it is thought to make only a small contribution to overall lipolysis in cheese made from pasteurised milk (Fox and Stepaniak 1993). The aim of this study was to develop a pre-treatment routine consisting of lowpressure homogenisation, incubation and batch pasteurisation of cheesemilk, which could potentially be used to improve ripening and quality properties of Cheddar cheese. The effects of the pre-treatment routine on the properties of resultant cheeses, including microbiological parameters and subsequent chemical changes during ripening were studied to evaluate the possible advantageous use of such a routine in commercial cheesemaking.

4 Low-pressure homogenisation and cheese ripening Material and Methods 2.1 Experimental design Raw whole bovine milk was pre-treated as described below and used to produce miniature laboratory-scale cheese and pilot-scale cheeses. Laboratory- and pilot-scale cheeses were produced in triplicate. All cheesemaking and processing was carried out in the food processing facilities in the School of Food and Nutritional Sciences, University College, Cork, Ireland 2.2 Pre-treatment of milk For the preliminary laboratory-scale trial, raw whole milk, obtained from a local farm, was pre-heated to a temperature of 55 C and subjected to two-stage homogenisation at pressures of 0, 5, 10, 15, 20 or 25 MPa. Homogenisation was carried out in a two-stage homogeniser (model APV 1000, APV Homogenisers AS, Albertslund, Denmark). Output was passed through a coiled stainless steel pipe immersed in ice-water to bring the temperature to 37 C. Approximately 800 ml milk was collected for each homogenisation pressure and placed in glass beakers. A control milk sample was also heated to 55 C but not passed through the homogeniser. Glass beakers were placed in a waterbath at 37 C and incubated for 60 min once milk temperature reached 37 C. Following this incubation stage, milks were batch-pasteurised at 63 C for 30 min in a waterbath. Milks were cooled to 4 C and portions were taken for the production of laboratory-scale cheese ( 600 ml) and determination of acid degree value (ADV). For pilot-scale trials, batches of milk ( 6 L) were pre-heated to 55 C in a waterbath and immediately homogenised. Because of practical limitations, milk was homogenised in 18 L (3 6 L) batches. The first 18 L batch was homogenised in a two-stage homogeniser at a pressure of 0 MPa, followed by cooling to 37 C by passing through a stainless steel coil immersed in ice-water. Following this 18 L batches of pre-heated milk were homogenised at 5 or 10 MPa. A control milk sample, which was also heated to 55 C but not passed through the homogeniser, was also collected in the same manner. The homogenised milks ( 18 L was treated at each homogenisation pressure) were transferred (in 6 L batches in three stainless steel containers) to a 37 C waterbath. The milks were incubated at 37 C for 1 h and then transferred to a 63 C waterbath. The milks were then batch-pasteurised at 63 C for 30 min and cooled to refrigeration temperature. 2.3 Alkaline phosphatase assay Alkaline phosphatase activity in raw and batch-pasteurised milks was determined as described by Hayes and Kelly (2003b) using a Fluorophos (FLM200, Advanced Instruments Inc., Needham Heights, MA, USA). 2.4 Cheese production Laboratory-scale Cheddar-type cheeses were manufactured in triplicate for each of the milks homogenised at 0, 5, 10, 15, 20 or 25 MPa, including the control milk,

5 644 K.C. Deegan, P.L.H. McSweeney using the method described by Shakeel-Ur-Rehman et al. (1998). Direct vat set culture (DVS R604, Christian Hansen, Hørshom, Denmark) was used as a starter culture at a rate of 325 µl.l 1. Rennet (Maxiren-180, DSM Food Specialities, Delft, Netherlands) was added at 43.5 μl per 200 ml milk. Resultant cheeses were brine salted (200 g.l 1 NaCl, 0.50 g.l 1 CaCl 2 2H 2 O) for 30 min at room temperature, vacuum-packed and ripened at 8 C. Pilot-scale Cheddar cheeses were manufactured using milks homogenised at 0, 5 or 10 MPa and control milk, according to a standard protocol (Fox et al. 2000). Direct vat set culture (DVS R604) was added as a starter culture (3 g per 15 L vat) and allowed to stand for 30 min. Chymosin was added to the cheesemilk at a level of 0.3 ml.l 1. After pressing at 0.15 MPa, cheeses were vacuum-packed and ripened at 8 C for 180 days. Three replicate cheesemaking trials were performed for the pilotscale cheeses. The cheeses produced will be coded in this paper as C (control pretreatment cheese) and H0, H5 and H10 for the cheeses produced from milk homogenised at 0, 5 and 10 MPa, respectively. 2.5 Acid degree value ADVof the pre-treated milks were determined using the method described by Bradley et al. (1992). 2.6 Compositional analysis The fat content of cheeses was determined using the Gerber method according to Bradley et al. (1992). Salt content was calculated by the potentiometric method described by Fox (1963). ph measurements were determined by probing the cheeses directly with a pointed glass electrode connected to a ph meter (PHM210, Radiometer, Copenhagen, Denmark). Moisture levels were calculated using an oven-drying method (IDF 1982). Levels of fat in whey from the production of pilot-scale cheese were determined by the Rose Gottlieb method (Bradley et al. 1992). Each analysis was performed in triplicate. 2.7 Lipolysis Individual free fatty acids were extracted from the pilot-scale cheeses at 1, 90 and 180 days of ripening and quantified by gas chromatography using the method of de Jong and Badings (1990) as described by O Mahony (2005). All samples were extracted in triplicate with each extract analysed once. 2.8 Microbiological analysis Enumeration of starter lactococci and NSLAB was performed on pilot-scale cheeses at 1, 30, 60, 90, 150 and 180 days of ripening. Starter lactococci were enumerated on LM17 agar after incubation for 3 days at 30 C. NSLAB were enumerated on Rogosa agar incubated aerobically with an overlay for 5 day at 30 C. Enumeration of psychrotrophic bacteria in raw milk was carried out on total plate count agar incubated at 7 C for 10 days. All agars were purchased from Merck (Darmstadt, Germany).

6 Low-pressure homogenisation and cheese ripening Urea polyacrylamide gel electrophoresis Electrophoretic analysis was carried out directly on the pilot-scale cheeses at 180 days ripening. Electrophoresis in polyacrylamide gels (12.5% T, 4% C, ph 8.9) was performed using a Protean II xi vertical slab gel unit (Bio-rad Laboratories Ltd., Watford, Herts., UK) according to the method of Andrews (1983) with modifications described by Shalabi and Fox (1987). Gels were stained directly by the method of Blakesley and Boezi (1977) and digitized using a flat-bed scanner (Scanjet 6300 C, Hewlett Packard, Singapore) Statistical analysis Two-way analysis of variance (ANOVA) with repeated measures was carried out on composition and ADV data to evaluate differences between the cheeses. The main effects and interactions of sample (4) and trial (3) which were evaluated at a 5% significance level. Post hoc comparisons between samples for each attribute were performed with Sidak confidence interval adjustment. One-way ANOVA, followed by Tukey s HSD post hoc test, was used to investigate differences between attributes in samples which showed a significant sample trial effect. This was done to rule out the possibility that individual trials lead to conflicting conclusions about the chemical characteristics of homogenisation treatments. ANOVA tests were performed with PASW Statistics 18.0 (SPSS Inc). Principal component analysis (PCA) was conducted on the mean values of the individual free fatty acids, using a correlation matrix, with The Unscrambler 10.1 (Camo Software). 3 Results 3.1 Alkaline phosphatase activity In all trials, the batch pasteurisation routine inactivated alkaline phosphatase activity (results not shown). 3.2 Acid degree value The ADV of milks after incubation were higher as homogenisation pressure increased from 0 to 10 MPa in the laboratory-scale trials (not shown). The control milk had an ADVof 0.95 meq.100 g 1 fat, significantly lower (P 0.05) than the milk homogenised at 0 MPa (1.47 meq.100 g 1 fat). The ADV did not increase >10 MPa and no significant differences (P>0.05) were found between the ADVof the 10, 15, 20 and 25 MPa milks. ADV of the cheesemilks used in the production of pilot-scale cheeses are shown in Table 1. The raw milk sample was taken prior to the pre-treatment while 0, 5 and 10 MPa milks refer to samples of milk taken after homogenisation at 0, 5 and 10 MPa, incubation at 37 C for 1 h, and subsequent batch pasteurisation (63 C for 30 min). No significant sample trial effects were found (not shown). No significant differences (P>0.05) in ADV were found between the raw milk, the control milk and the milks homogenised at 0 and 5 MPa. The milk homogenised at 10 MPa had an ADVof 4.00±0.67 meq.100 g 1 fat, significantly higher than the other treatments and similar to the value (4.04± 0.06 meq.100 g 1 fat) obtained in the laboratory-scale trials.

7 646 K.C. Deegan, P.L.H. McSweeney Table 1 Average acid degree value (ADV) of raw milk, control milk and milks homogenised at 0, 5 or 10 MPa, incubated at 37 C 1 h and subsequently batch-pasteurised. Values are means of three trials Sample ADV (meq.100 g 1 fat) Raw milk 1.95 a (0.35) Control milk 1.60 a (0.26) 0 MPa milk 2.01 a (0.26) 5 MPa milk 2.21 a (0.57) 10 MPa milk 4.00 b (0.67) Numbers represent mean and standard deviation, with the latter in parenthesis (n=6). Means with different superscripts are significantly different (Tukey s HSD, P 0.05) 3.3 Composition The ph and moisture, fat and salt levels of the pilot-scale cheeses and the fat content of the whey removed during cheesemaking are shown for individual trials in Table 2. The ph of the H10 cheese was significantly higher than that of the C cheese in the individual trials. In the second and third trials, significantly higher (P 0.05) moisture contents were determined in the H10 cheeses (41.43% and 40.80%, for Trials 2 and 3, respectively) than in the C cheeses (36.99% and 39.72%, respectively). Significant differences were seen between the fat contents of the cheeses in Trials 2 and 3, where a general decrease in fat content was noted with increasing homogenisation pressure. Salt levels were significantly higher (P 0.05) in H10 cheeses than in C cheeses in each trial (1.57%, 1.47% and 1.57% for H10 cheeses in Trials 1, 2 and 3, respectively, versus, 1.25%, 1.17% and 1.30%, for C cheeses in Trials 1, 2 and 3, respectively). The fat-in-dry-matter of cheeses showed no significant increase or decrease with increasing homogenisation pressure in Trials 2 or 3, while a slight increase was seen in Trial 3. Similarly, the MNFS values showed no significant trends in Trials 1 or 2, but increased in Trial 3. A decrease in fat content of whey (Trials 1 and 3) was seen with increasing homogenisation pressure of milk in the pre-treatment. 3.4 Free fatty acids The levels of total FFA detected in each of the cheeses from the three pilot-scale trials are shown in Table 3. Significant sample trial effects were found with two-way (sample trial) ANOVA and hence each trial was subjected separately to a one-way ANOVA. In general, an increase in total FFA was noted with increasing ripening time. At 1 day, no significant differences (P>0.05) were found between total FFA levels in the C and H0 cheeses in Trials 1 and 3. The H5 cheeses contained significantly higher (P 0.05) total FFA concentrations than the C and H0 cheeses, while the 1-day H10 cheeses in Trials 1 and 2 contained higher FFA concentrations than cheeses made from milk subjected to all other treatments. Levels of total short-chain FFA increased with increasing homogenisation pressure of the cheesemilk, with H10 cheeses containing significantly higher (P 0.05) levels of total short-chain FFA at each ripening time in Trials 1 and 2, than the other treatments and the C cheese. The total short-chain FFA concentrations also increased with ripening

8 Low-pressure homogenisation and cheese ripening 647 Table 2 ph and composition of Cheddar cheeses made using control milk (C) and milk homogenised at 0, 5 or 10 MPa (H0, H5 and H10, respectively) and fat contents of whey removed during cheesemaking Sample ph Moisture (%) Fat (%) Salt (%) Fat in dry matter (%) Moisture in non-fat (%) Fat in whey (%) Trial 1 C 4.99 c ab a b (0.01) (0.34) (0.00) (0.01) (0.27) (0.48) (0.01) H b a a b (0.01) (0.37) (0.29) (0.01) (0.40) (0.45) (0.01) H a b b a (0.02) (0.90) (2.08) (0.03) (1.39) (1.49) (0.01) H d ab b a (0.01) (0.34) (0.58) (0.05) (3.81) (2.33) (0.00) Trial 2 C 4.96 bc a bc 1.17 b (0.06) (0.29) (0.00) (0.02) (0.27) (0.46) (0.00) H a a c 1.22 c (0.02) (2.66) (0.58) (0.13) (3.39) (4.70) (0.06) H ab a ab 1.06 a (0.01) (0.46) (1.15) (0.00) (2.24) (1.69) (0.02) H c b a 1.47 d (0.04) (0.30) (0.58) (0.01) (1.00) (0.68) (0.02) Trial 3 C 4.97 a ab b 1.30 a a ab 0.15 b (0.01) (0.09) (0.00) (0.02) (0.07) (0.10) (0.01) H b a ab 1.25 a b a 0.17 b (0.01) (0.08) (1.15) (0.06) (2.00) (1.28) (0.00) H a bc a 1.55 b ab b 0.14 ab (0.01) (0.36) (0.00) (0.02) (0.38) (0.55) (0.01) H b c a 1.57 b ab ab 0.11 a (0.03) (1.38) (0.58) (0.02) (0.85) (1.71) (0.01) Numbers represent means and standard deviations, with the latter in parenthesis (n=3 for ph, moisture, fat, salt, fat in dry matter and moisture in non-fat, n=2 for fat in whey). Means in a column with different superscripts are significantly different (Tukey s HSD, P 0.05) time, an effect which mirrored the total FFA content. Trends in medium-chain FFA were generally similar to those for short-chain fatty acids and an increase was seen with increasing homogenisation pressure of the cheesemilk and with ripening time. In general, there were no significant differences (P>0.05) between the C cheese and H0 cheese at the same ripening time, in any of the trials. The H10 cheeses had, in general, significantly higher levels of FFA (P 0.05) than the C and H0 cheeses at each ripening time. The H10 cheeses had significantly higher (P 0.05) levels of total long-chain FFA than the C and H0 cheeses, initially (1 day), and throughout ripening, however with increasing ripening time there appeared to be no general pattern of increase or decrease in levels of total short- and total medium-chain FFA.

9 648 K.C. Deegan, P.L.H. McSweeney Table 3 Concentration of short- (Σ (C 4:0 C 8:0 )), medium- (Σ (C 10:0 C 14:0 )), long- (Σ (C 16:0 C 18:3 )) chain and total (Σ (C 4:0 C 18:3 )) free fatty acids, determined by gas chromatography with flame ionization detection and expressed as mg.kg 1 fat for cheese made using control milk (C) and milk homogenised at 0, 5 or 10 MPa (H0, H5 and H10, respectively) at 1, 90 and 180 days of ripening Free fatty acid class Ripening time (d) Trial Concentration of free fatty acids (mg.kg 1 fat) C H0 H5 H10 Short chain Σ (C 4:0 C 8:0 ) Medium chain Σ (C 10:0 C 14:0 ) Long chain Σ (C 16:0 C 18:3 ) Total Σ (C 4:0 C 18:3 ) b (34) 206 a (7) 299 b (13) 353 c (24) a (1) 165 a (9) 504 c (45) 329 b (23) a (11) 126 a (19) 138 a (7) 143 a (7) a (48) 443 a (22) 613 a (117) 1,342 b (56) b (6) 521 c (30) 254 a (10) 1,004 d (68) a (14) 433 b (32) 334 a (15) 775 c (47) a (25) 949 b (29) 605 a (7) 1,812 c (56) a (21) 736 b (45) 517 a (23) 1,575 c (72) a (44) 579 a (43) 1,050 c (21) 756 b (23) 1 1 2,537 a (182) 2,134 a (37) 3,471 b (97) 4,364 c (302) 2 1,568 a (39) 1,886 ab (96) 2,879 b (157) 4,175 c (783) 3 1,224 a (269) 1,284 a (198) 2,037 b (201) 1,896 b (172) ,616 a (221) 2,531 a (192) 4,448 b (47) 6,575 c (509) 2 2,229 a (175) 2,555 a (210) 1,798 a (81) 6,277 b (635) 3 2,157 a (89) 2,353 a (90) 2,183 a (162) 4,932 b (227) ,813 a (197) 3,496 a (65) 2,961 a (148) 8,337 b (902) 2 2,387 a (210) 3,075 a (184) 2,611 a (220) 7,509 c (433) 3 1,004 a (72) 1,013 a (164) 1,378 ab (57) 1,216 b (16) 1 1 9,954 a (120) 8,808 a (214) 14,640 b (409) 18,392 c (1,329) 2 6,500 a (212.2) 7,506 ab (99.2) 9,283 b (1,399) 16,488 c (1,423) 3 5,865 a (1,369) 5,284 a (391) 8,958 b (322) 8,023 b (305) ,431 a (154) 8,808 a (214) 14,640 b (409) 18,392 c (1,329) 2 6,629 ab (1,228) 7,506 ab (99.2) 9,283 b (1,399) 16,488 c (1,423) 3 6,913 a (40) 5,284 a (391) 8,958 b (322) 8,023 b (305) ,198 a (525) 8,666 a (303) 14,741 b (565) 22,306 c (603) 2 7,060 a (471) 9,120 b (786) 5,917 a (560) 20,042 c (1,197) 3 8,092 a (582) 7,817 a (565) 7,207 a (483) 15,518 c (1,215) ,769 a (188) 11,148 a (233) 18,410 b (495) 23,109 c (1,620) 2 8,219 a (250) 9,556 ab (186) 12,665 b (1,511) 20,952 c (2,082) 3 7,229 a (1,100) 6,693 a (542) 11,133 b (513) 10,063 b (221) ,493 a (122) 11,631 a (516) 19,802 b (449) 30,223 c (983) 2 9,257 ab (1,371) 12,195 b (982) 7,968 a (630) 27,323 c (1,732) 3 9,374 a (55) 10,604 a (675) 9,724 a (647) 21,225 b (1,381) ,610 a (689) 14,448 b (612) 12,303 ab (778) 33,244 c (1,401) 2 9,980 a (694) 12,897 b (890) 10,603 ab (800) 30,275 c (1,167) 3 11,417 a (818) 11,413 a (1,050) 16,594 c (470) 14,347 b (385) Numbers represent mean and standard deviation, with the latter in parenthesis (n=3). Means in a row with different superscripts are significantly different (Tukey s HSD, P 0.05)

10 Low-pressure homogenisation and cheese ripening 649 PCA was performed on the individual FFA data (Fig. 1), where PC1 and PC2 explained 74% and 15% of the total variation, respectively, between FFA concentrations. C and H0 cheeses were generally on the negative side of PC1. PC2 separated the cheeses (with some exceptions) on the basis of ripening time (from positive to negative as ripening progressed). Figure 1 (bottom) shows that all FFA had positive loadings on PC1 which agreed with the levels of individual FFA (Table 3). Longchain saturated FFA C18:0 and unsaturated FFA (C18:1 and C18:2) had positive loadings on PC2, indicating that levels of these particular FFA decreased as values of PC2 decrease, or as shown in the analysis of data from individual trials, as ripening time increased. In general, levels of FFA in all homogenised milk cheeses increased with increasing ripening time; however, as ripening time progressed, a larger relative increase in short-chain FFA was noted (Table 3) compared with a decrease in longchain FFA, which compared favourably with loadings of C4:0 and C6:0 in Fig Bacterial numbers No discernible differences were found between numbers of starter bacteria or NSLAB throughout ripening (not shown). Starter numbers decreased in all cheeses from levels of cfu.g 1 to between approximately and cfu.g 1 after 180 days of ripening. The numbers of NSLAB increased from between 1 and cfu.g 1 at 1 day ripening to between and cfu.g 1 after 180 days of ripening. The number of NSLAB in both the C and H10 cheeses was cfu.g 1 at the end of ripening. Total and psychrotrophic plate counts were performed on a sample of milk obtained from the same farm as the milk used for the manufacture of pilot-scale cheeses. Total and psychrotrophic numbers were and cfu.ml 1, respectively. 3.6 Proteolysis Figure 2 shows the electrophoretograms of the C, H0, H5 and H10 cheeses at 180 days. The breakdown of α s1 -casein (α s1 -CN) was apparent as ripening progressed in cheeses made from milk subjected to each of the treatments. α s1 -CN was almost completely degraded by 30 days. Degradation of the α s1 -CN breakdown product α s1 -CN(f24-199) to α s1 -CN(f ) appeared to be more extensive in the C and H0 cheese. Extensive breakdown of β-cn did not occur in the C and H0 cheeses; however, in H5 and H10 cheeses, β-cn breakdown products; β-cn(29 209), β-cn(f ) and β-cn(f ) (γ 1 -, γ 2 - and γ 3 -CN) were produced. 4 Discussion Homogenisation resulted in higher levels of FFA as measured by ADV in both the laboratory- and pilot-scale cheeses. Homogenisation leads to increased levels of lipolysis (Deeth 2006) by causing damage to the protective MFGM. In this study, lipolysis was allowed to proceed unhindered during the incubation stage of 37 C for 1 h after homogenisation, where LPL could gain access to the fat made vulnerable by homogenisation and catalyse the release of FFA, resulting in higher ADV. It is most

11 650 K.C. Deegan, P.L.H. McSweeney C4:0 C6:0 C8:0 C10:0 C12:0 C14:0 C16:0 C18:0 C18:1 C18:2 C18:3 Fig. 1 top Biplot of scores and loadings obtained from principal component analysis (PCA) of free fatty acid data of cheeses made from milk homogenised at 0 (H0; open circle), 5 (H5; filled square) and 10 (H10; open diamond) MPa and a control milk (C; filled upright triangle) followed by the ripening time in days (1; green, 90; orange and 180; blue) and the trial (a, b or c). bottom Loadings of fatty acids from PCA on principal components 1 (black bars) and 2 (white bars) likely that indigenous LPL was the lipase activity responsible for increased ADV values as the milk used was fresh (Datta et al. 2005). In the laboratory-scale cheese trial, no significant differences in ADV values were observed for milks homogenised at pressures >10 MPa (results not shown), which could be due to strong feedback

12 Low-pressure homogenisation and cheese ripening 651 C H0 STD T1 T2 T3 STD T1 T2 T3 -CN s1-cn s1-cn (f24-199) H5 H10 STD T1 T2 T3 STD T1 T2 T3 2 -CN 1 -CN 3 -CN -CN X s1-cn s1-cn (f ) s1-cn (f24-199) Fig. 2 Urea polyacrylamide gel electrophoretograms of sodium caseinate standard (STD), and C and cheeses made from milk homogenised at 0 (H0), 5 (H5) and 10 (H10) MPa and a control milk (C) in Trials 1, 2 and 3 (T1, T2, T3) at 180 days ripening

13 652 K.C. Deegan, P.L.H. McSweeney inhibition by the FFA produced as a result of its hydrolysis of milk triacylglycerols (Olivecrona et al. 2003). At the start of ripening, H5 and H10 cheeses generally contained significantly higher levels of FFA than C and H0 cheeses. As mentioned, homogenisation resulted in a very strong activation of lipolysis in the incubation stage, which allowed access of LPL to a large surface area of milk fat made vulnerable by damage to the protective MFGM. In all treatments and trials, with the exception of H5 cheeses, C cheeses in Trial 1 and H10 cheeses, total FFA concentrations increased with increasing ripening time which can be attributed to lipolysis during cheese ripening caused by indigenous LPL and enzymes from the starter and NSLAB (Collins et al. 2003). LPL plays a very minor role in the production of FFA in cheese made from pasteurised milk due to the very extensive inactivation by pasteurisation (Deeth 2006) and to the sub-optimal conditions of the cheese curd for its activity. Increasing homogenisation pressure resulted in a higher rate of increase of total FFA. Homogenisation distributes fat more evenly through the cheese matrix (Jana and Upadhyay 1992), which increases the surface area to volume ratio of the globules and could allow greater access by lipolytic enzymes, resulting in a greater relative increase in FFA levels with ripening time. The lower levels of short-chain FFA relative to medium- and long-chain FFA at 1 day did not suggest LPL activity, which tends to release short-chain FFA preferentially. The content of long-chain FFA was much higher than that of short-chain FFA in all cheeses at 1 day. A possible explanation could be the loss of the volatile (e.g. butyric acid) or whey-soluble short-chain FFA during the cheesemaking process. The increase in short-chain FFA during ripening, relative to other FFA, was more pronounced. The preferential hydrolysis of short-chain FFA from triacylglycerols during ripening has been attributed to the lipolytic activities of starter bacteria and NSLAB. LAB present in starter cultures are the main lipolytic agents in Cheddar cheese made from pasteurised milk (Collins et al. 2003) while NSLAB can contribute to lipolysis in cheese, especially in cheese made from raw milk (McSweeney et al. 1993). Gobbetti et al. (1997) studied an intracellular lipase from L. plantarum, which was most active for the release of butyric acid (C4:0) from triacylglycerols, however, in general, NSLAB are weakly lipolytic (McSweeney and Sousa 2000). Also, as numbers of NSLAB did not significantly differ between samples in the present study, their significant contribution to lipolysis may be discounted. Another possible explanation for the larger relative increase in short-chain FFA relative to other FFA may be LPL activity, which exhibits positional specificity, preferentially on the sn-1 and sn-3 positions of triacylglycerols (Somerharju et al. 1978). As the short-chain fatty acids present in the triacylglycerols of milkfat are esterified mainly at the sn-3 position, LPL appears to release short-chain fatty acids preferentially (Deeth 2006). LPL is not regarded as having an important role in lipolysis during the ripening of cheese made from pasteurised milk (Fox and Stepaniak 1993). A possible explanation for the occurrence of lipolytic activity after pasteurisation may be the presence of heat-stable lipase enzymes produced by psychrotrophic bacteria. Total plate and psychrotrophic counts were similar to the results of a study by Smiddy et al. (2007) on raw whole milk. It is unlikely that psychrotrophs significantly contributed to the lipolysis in the incubation stage of this study, as lipases are generally produced by psychrotrophs during late log and early stationary phases, usually when numbers reach cfu.ml 1 (Deeth 2006).

14 Low-pressure homogenisation and cheese ripening 653 An increase of ph of Cheddar cheese with homogenisation of cheesemilk is thought to be as a result of a slower rate of acid production during cheese manufacture (Jana and Upadhyay 1992). O Mahony (2005) also noticed that the ph values of cheeses made from milks processed at increasing homogenisation pressures were higher and suggested that the slower acid production was related to the increased amount of FFA liberated with increasing homogenisation pressure, which could retard or inhibit the growth of starter lactic acid bacteria. Higher moisture levels found in the H10 pilot-scale cheeses in Trials 2 and 3 may be a result of reduced syneresis and therefore increased water retention in the curd. This was also found by Peters (1956). A decrease in fat levels with homogenisation pressure was not found in these previous studies, where fat levels in cheese increased with increasing homogenisation pressure of milk (Peters 1956; Nair et al. 2000). Salt content of cheese made from homogenised milk increases with homogenisation pressure due to lower syneresis and increased moisture which retains salt in the cheese matrix. The decrease in the fat content of whey in Trials 1 and 3 is in agreement with numerous other studies (Jana and Upadhyay 1992; Green et al. 1983; Peters 1956; Nair et al. 2000) and thought to be as a result of greater fat retention in the curd made from homogenised milk (Jana and Upadhyay 1992). However, as the levels of fat in whey of the cheeses agreed with the trends described in the literature, the fat contents of the cheeses did not and the reason for this is unclear. The almost complete degradation of α s1 -CN by 30 days of ripening is due to the action of residual chymosin at the Phe 23 Phe 24 bond, producing α s1 -CN(f1-23) and α s1 -CN(f24-199) (Sousa and McSweeney 2001). The relatively higher degradation of the fragment α s1 -CN(f24-199) to α s1 -CN(f ) in the C and H0 cheeses could also be due to residual chymosin activity. Homogenisation has been shown to retard the breakdown of α s1 -CN in Mozzarella cheese made from homogenised milk (Tunick et al. 1995). As the C and H0 cheeses had significantly lower ph values than the H10 cheese in Trials 1 and 3, chymosin activity may have increased. The β- CN breakdown products, β-cn(29 209), β-cn(f ) and β-cn(f ) (γ 1 -, γ 2 - and γ 3 -CN), were liberated from β-cn by the action of plasmin and were more pronounced in the H5 and H10 cheeses. Homogenisation may have caused more plasmin activity in the cheese due to their higher ph levels (plasmin ph optimum is 7.5). Hayes and Kelly (2003b) found an approximate 40% reduction in plasmin activity when milk underwent homogenisation (18 MPa) and attributed this to rearrangement of casein structure on homogenisation. As the total surface area of fat in milk fat globules is increased by approximately five- to tenfold by homogenisation, casein micelles are absorbed onto the newly formed MFGM (Michalski et al. 2002). The rearrangement of the casein micelle may promote plasmin activity which survives homogenisation. 5 Conclusion The pre-treatment routine resulted in pilot-scale cheeses which contained higher moisture and salt and increased ph. Cheese made from milk homogenised at 10 MPa (H10) contained generally higher levels of FFA initially and throughout ripening. The use of the pre-treatment routine described in this paper on a large

15 654 K.C. Deegan, P.L.H. McSweeney commercial scale could be warranted to produce a cheese with increased FFA levels. However, as FFA are strong contributors to the overall odour and flavour of cheese, further study needs to be undertaken to examine the consequences of such a pretreatment on the sensory characteristics of the resultant cheese. Investigation into the effects of such a pre-treatment on the microstructure of the resultant cheese would also be of interest in future research. Acknowledgements The assistance of Mr. Dave Waldron, MSc, in pre-treatment and cheesemaking is greatly appreciated. Ms. Elina Kokkonen, MSc, is thanked for her advice concerning statistical analysis. The authors appreciate the constructive comments made by Dr. Seamus O Mahony. Dr. Therese Uniacke-Lowe and Dr. Sinead Fitzsimons are thanked for their practical assistance. This work was funded by a grant awarded under the Food Institutional Research Measure (FIRM) administered by the Department of Agriculture, Food and the Marine of the Government of Ireland References Andrews AT (1983) Proteinases in normal bovine milk and their action on caseins. J Dairy Res 50:45 55 Andrews AT, Anderson M, Goodenough PW (1987) A study of the heat stabilities of a number of indigenous milk enzymes. J Dairy Res 54: Blakesley RW, Boezi JA (1977) A new staining technique for proteins in polyacrylamide gels using Coomassie brilliant blue G250. Anal Biochem 82: Bradley RL, Arnold E, Barbano DM, Semerard RG, Smith DE, Vines BK (1992) Chemical and physical methods, fat. In: Marshall RT (ed) Standard methods for the examination of dairy products. American Public Health Association, Washington, DC, pp Collins YF, McSweeney PLH, Wilkinson MG (2003) Lipolysis and free fatty acid catabolism in cheese: a review of current knowledge. Int Dairy J 13: Datta N, Hayes MG, Deeth HC, Kelly AL (2005) Significance of frictional heating for effects of high pressure homogenisation on milk. J Dairy Res 72: De Jong C, Badings HT (1990) Determination of free fatty acids in milk and cheese procedures for extraction, clean up, and capillary gas chromatographic analysis. J High Res Chromatog 13:94 98 Deeth HC (2006) Lipoprotein lipase and lipolysis in milk. Int Dairy J 16: Driessen FM (1983) Lipases and proteinases in milk. Occurrence, heat inactivation, and their importance for the keeping qualities of milk products. Neth Milk Dairy J 37: Fox PF (1963) Potentiometric determination of salt in cheese. J Dairy Sci 46: Fox PF, Stepaniak L (1993) Enzymes in cheese technology. Int Dairy J 3: Fox PF, Guinee TO, Cogan TM, McSweeney PLH (2000) Fundamentals of cheese science. Aspen Publishers Inc., Gaithersburg, MD Gobbetti M, Fox PF, Stepaniak L (1997) Isolation and characterization of a tributyrin esterase from Lactobacillus plantarum J Dairy Sci 80: Green ML, Marshall RJ, Glover FA (1983) Influence of homogenization of concentrated milks on the structure and properties of rennet curds. J Dairy Res 50: Hayes MG, Kelly AL (2003a) High pressure homogenisation of raw whole bovine milk. (a) Effects on fat globule size and other properties. J Dairy Res 70: Hayes MG, Kelly AL (2003b) High pressure homogenisation of raw whole bovine milk. (b) Effects on indigenous enzymatic activity. J Dairy Res 70: Huppertz T, Kelly A (2006) Physical chemistry of milk fat globules. In: Fox PF, McSweeney PLH (eds) Advanced dairy chemistry, vol 2, Lipids. Springer, NY, pp IDF (1982) Processed cheese. Determination of the total solids content. Standard 4A Jana A, Upadhyay K (1992) Homogenisation of milk for cheesemaking a review. Aust J Dairy Technol 47:72 79 McSweeney PLH, Sousa MJ (2000) Biochemical pathways for the production of flavour compounds in cheeses during ripening: a review. Lait 80: McSweeney P, Fox P, Lucey J, Jordan K, Cogan T (1993) Contribution of the indigenous microflora to the maturation of Cheddar cheese. Int Dairy J 3:

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