Effect of cook temperature on starter and non-starter lactic acid bacteria viability, cheese composition and ripening indices of a semi-hard cheese manufactured using thermophilic cultures

ARTICLE IN PRESS

International Dairy Journal 17 (2007) 704–716 www.elsevier.com/locate/idairyj

Effect of cook temperature on starter and non-starter lactic acid bacteria viability, cheese composition and ripening indices of a semi-hard cheese manufactured using thermophilic cultures Jeremiah J. Sheehana,, Mark A. Fenelona, Martin G. Wilkinsonb, Paul L.H. McSweeneyc a

Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland b Department of Life Sciences, University of Limerick, Ireland c Department of Food and Nutritional Sciences, University College, Cork, Ireland Received 29 November 2005; accepted 25 August 2006

Abstract Semi-hard cheeses were manufactured using Streptococcus thermophilus and Lactobacillus helveticus cultures and their ripening was characterised. During cheese manufacture, curds were cooked to a maximum temperature of 47, 50 or 53 1C, pre-pressed under whey at pH 6.15, moulded, pressed and brined. Increased cook temperature resulted in increased manufacture time, a significantly reduced growth rate of S. thermophilus during manufacture in the order 47E50 1C453 1C and in significantly lower mean viable cell counts of S. thermophilus up to 56 d of ripening. Increasing cook temperature had no significant effect on mean viable cell numbers of L. helveticus or non-starter lactic acid bacteria (NSLAB). Cheeses produced from curds cooked to 47 1C had significantly higher levels of moisture in non-fat substances (MNFSs), salt-in-moisture and a significantly lower pH and levels of butyrate compared with cheeses produced from curds cooked to 50 or 53 1C. r 2006 Elsevier Ltd. All rights reserved. Keywords: Semi-hard cheese; Increased cook temperature; Starter viability; Ripening

1. Introduction Substantial growth in world cheese consumption is predicted to continue (Donnellan & Keane, 2000). However, there also exists an increased demand for diversity of sensory and textural attributes of cheese (Sheehan & Wilkinson, 2002; Wilkinson, Meehan, & Guinee, 2000). The ability to diversify the range of cheese produced in modern high-volume industrial plants may proceed via two routes: (1) production of soft, semi-soft, mould, smearripened and other speciality cheeses, but which requires major capital investment in specialised curd manufacture or ripening facilities; or (2) production of diverse, innovative and novel cheese types in existing Cheddar or Emmental-type cheese plants with a resulting broader product portfolio (Wilkinson, Sheehan, & Kelly, 1997). Corresponding author. Tel.: +353 25 42232; fax: +353 25 42340.

E-mail address: [email protected] (J.J. Sheehan). 0958-6946/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2006.08.011

The production of novel cheese types on existing equipment involves the manipulation of process and ripening variables, e.g., starter type, cook temperature and draining pH, to generate unique flavours and textures. Such cheeses can incorporate some of the characteristics of a diverse range of cheese types, e.g., Swiss and Cheddar. Novel or hybrid cheese types already produced in an existing plant include ‘‘Short method’’ Cheddar (Czulak, Hammond, & Meharry, 1954; Hammond, 1979; Radford & Hull, 1982), Egmont (Gillies, 1974) and Dubliner (Wilkinson et al., 1997). However, a high degree of technical knowledge as to the interactive effects of manipulation of process variables such as cook temperature on parameters such as starter cultures and ripening of resulting cheeses is required to achieve a successful novel cheese product. Thermophilic cultures (e.g., Streptococcus thermophilus and Lactobacillus helveticus) and elevated cook temperatures, typically 50–56 1C, are used in the manufacture of

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Swiss-type (e.g., Emmental, Gruye`re) and some Italiantype cheeses (e.g., Grana Padano and Parmigiano Reggiano). Production of acid in the early stages of manufacture of these cheese types depends substantially on the activity of the coccus, i.e., S. thermophilus, while lactobacilli, e.g., L. helveticus, are responsible for continuing the acidification process in the cheese press post drainage (Turner, Martley, Gilles, & Morris, 1983). Maximum growth rates for the above strains are in the range 42–45 1C (Martley, 1983), with acidifying activities being strain related (Chamba & Prost, 1989). The most rapid acid production by S. thermophilus and L. helveticus in milk requires a mean temperature of 42.7 and 44.0 1C, respectively (Martley, 1983). Heating inoculated milk to 53 1C has been shown to result in a variable slowing of the acidification of thermophilic lactobacilli due to decreased cellular viability, probably because of thermal stress (Neviani, Divizia, Abbiati, & Gatti, 1995). Similarly, cooking curds to a maximum scald of 53 1C delayed but did not arrest the growth of thermophilic lactobacilli in the core of Grana cheese (52 1C after 6 h; Giraffa, Rossetti, Mucchetti, Addeo, & Neviani, 1998). However, limited information exists regarding the effect of different cook temperatures on rates of acid production by thermophilic starter strains or on their ability to withstand the subsequent holding times experienced during cheese manufacture (Giraffa et al., 1998; Turner, Martley, et al., 1983). The degree of sensitivity of S. thermophilus and lactobacilli to cooking temperatures used in cheese manufacture would be expected to have a considerable effect on the rate of acid production (Martley, 1983) and in turn on cheese composition and ripening. This information is vital in order to enable a diversification strategy. Differences in the manufacture of Cheddar-type cheeses over Swiss and Italian-type cheeses include: the use of mesophilic cultures; longer vat residence times with separation of curd and whey at a lower pH; curd milling and dry salting, and not brine salting (Kosikowski & Mistry, 1997). In this study a novel semi-hard cheese was manufactured in which curds and whey were separated at pH 6.15 as in Cheddar type manufacture (Guinee, Auty, & Fenelon, 2000) but thermophilic cultures, elevated cook temperatures and brine salting were utilised as in Swissand Italian-type cheeses (Kosikowski & Mistry, 1997). The objectives of the study were to characterise the manufacture and ripening of a semi-hard cheese manufactured using S. thermophilus and L. helveticus as starter and to determine the effect of altered cook temperature on microbiological and biochemical parameters including starter viability and ripening indices. 2. Materials and methods 2.1. Starter strains Thermophilic starter cultures used in Swiss-type cheese manufacture (Scott, 1981), i.e., S. thermophilus TH3 (ST)

705

and L. helveticus LHB02 (LH) were purchased from Chr. Hansens Ltd. (Little Island, Co. Cork, Ireland) as individual frozen concentrates and stored at 45 1C until cheese manufacture. 2.2. Cheese manufacture Three replicate cheesemaking trials, each consisting of three vats of 500 L cheese milk, were undertaken over a 4week period. Raw milk was obtained from a local dairy company, standardised to a casein to fat ratio of 0.78:1.0, held overnight at o10 1C, pasteurised at 72 1C for 15 s, and pumped at 32 1C into cylindrical, jacketed, stainless steel vats (500 L) with automated variable-speed cutting and stirring equipment (APV Schweiz AG, Worb, Switzerland). Cheesemilk was inoculated with 0.1 g L1 ST (109 cfu L1) and 0.05 g L1 LH (5  108 cfu L1). After a 60 min ripening period, chymosin (Chymax plus, Chr. Hansens Ltd.), diluted 1:6 with de-ionised water, was added at a level of 18 mL kg1 milk. A coagulation period of 50 min was allowed prior to a cut programme of 5 min. After a 10 min healing period, the curd/whey mixture was stirred and cooked by steam injection into the jacket of the vat. Curds were cooked at a rate of 0.5 1C min1 from 32 to 45 1C and at 1 1C min1 from 45 1C to maximum scald. The process variable applied in each trial was that one vat was cooked to a maximum scald of 47 1C, the second vat to 50 1C and the third to 53 1C. At pH 6.15, curds were transferred to a pre-press, pressed at 5.4 kPa for 5 min under whey. The whey was then drained and the curds were moulded in 5 kg moulds. The moulded cheeses were pressed at 35.2 kPa until pH 5.25, de-moulded and brined for 24 h in a saturated brine solution (23%, w/w, NaCl, 0.2% Ca, pH 5.20) at 12 1C. Post brining, cheeses were dried at ambient temperature for 24 h, vacuum packaged and ripened at 9 1C. 2.3. Enumeration of starter and non-starter bacteria During cheese manufacture, curd samples were removed aseptically at 30 min post inoculation, prior to cook, at maximum scald, at transfer to pre-press and prior to brining. Further samples were removed at 2, 9, 31, 84, 140, 210 and 270 d of ripening. The samples were placed in a stomacher bag, diluted 1:10 with sterile trisodium citrate (2%, w/v) and homogenised in a stomacher (Stomacher, Lab-Blender 400, Seward, Thetford, Norfolk, UK) for 5 min. Further dilutions were prepared as required. Independent duplicate samples were taken at each sampling point. Viable S. thermophilus cells were enumerated in duplicate on lactose-M17 agar after aerobic incubation at 42 1C for 3 d (Terzaghi & Sandine, 1975), L. helveticus cells on MRS agar pH 5.4 after anaerobic incubation for 3 d at 42 1C (IDF, 1988b) and non-starter lactic acid bacteria (NSLAB) overlaid with LBS agar after aerobic incubation at 30 1C for 5 d (Rogosa, Mitchell, & Wiseman, 1951).

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2.4. Cheese composition At 14 d post-manufacture, two cheeses from each vat were sampled. Grated cheese samples were analysed for moisture (IDF, 1982), protein (IDF, 1993), salt (IDF, 1988a) and fat (IDF, 1996). Ash content was determined by the method described by Kindstedt and Kosikowski (1985) and the ash was further analysed for Ca (IDF, 1992). The pH was measured on cheese slurry prepared from 20 g of cheese and 12 g of H2O (British Standards Institution, 1976) throughout ripening. Samples for biochemical analyses were taken after 2, 9, 31, 84, 140, 210 and 270 d of ripening and held at 20 1C until analysed. On each sampling date, two cheeses from each vat were sampled. D- and L-lactic acid contents were determined in cheeses after 2, 31, 84, 140, 210 and 270 d of ripening using a Boehringer Mannheim test kit (Boehringer BCL, Blackrock, Dublin, Ireland). Samples were prepared for analysis according to the method of Bouzas, Knat, Bodyfelt, and Torres (1993). Free amino acid contents were determined in pH 4.6soluble extracts (pH4.6-SN) of cheeses after 9, 31, 84, 140, 210 and 270 d of ripening prepared by a modification of the method of Kuchroo and Fox (1982) as described by Fenelon, O’Connor, and Guinee (2000). Samples were deproteinised by mixing equal volumes of pH4.6-SN and trichloroacetic acid (240 g L1). Free amino acids (FAAs) were separated using ion-exchange chromatography with post-column ninhydrin derivatisation and visible colourimetric detection. Values reported were the means of three replicate trials. Acetate, propionate and n-butyrate (C2:0, C3:0, C4:0) contents were determined in cheeses after 2, 31, 81, 140, 210 and 270 d of ripening. Acetate, propionate and n-butyrate were recovered by steam distillation and quantified by ligand-exchange, ion-exclusion HPLC as described by Kilcawley, Wilkinson, and Fox (2001). 2.5. Statistical analysis A randomised complete block design, which incorporated the three treatments (cooking to maximum scalds of 47, 50 or 53 1C) and three blocks (replicate trials), was used for analysis of the response variables relating to cheese composition (Table 1). Analysis of variance (ANOVA) was performed using the general linear model (GLM) procedure (SAS, 1995) where the effects of treatment (cook temperature) and replicates were estimated for all response variables. Duncan’s multiple comparison test was used as a guide for paired comparisons of the treatment means. The level of significance was determined at Po0.05. A split plot design was used to determine the effects of cook temperature, ripening time and their interaction on the response variables measured at regular intervals during manufacture and/or ripening, i.e., counts of L. helveticus and S. thermophilus during manufacture and ripening, counts of NSLAB during ripening, pH, total and

Table 1 Mean squares (MS) and probabilities (P) for aggregated changes in mean viable counts of Streptococcus thermophilus and Lactobacillus helveticus during manufacture of semi-hard cheeses cooked to different max scalds during manufacturea,b Factor

S. thermophilus MS

P

L. helveticus MS

P

Main plot Temperature Error

0.168 0.008

0.0074**

0.025 0.047

0.3469

Sub-plot Time Interaction (temp.  time) Error

4.407 0.086 0.033

o0.0001*** 0.0376*

3.551 0.047 0.035

o0.0001*** 0.2847

a For the response variable mean viable cell counts of S. thermophilus during manufacture the degrees of freedom (df) for the main plot and subplot were 4 and 20, respectively; the corresponding values for L. helveticus were 4 and 21, respectively. b Significance levels: *Po0.05, **Po0.01 and ***Po0.001.

individual free amino acids, acetic and butyric acids, L-, and total lactate and lactate to protein ratio. ANOVA for the split plot design was carried out using a GLM procedure (SAS, 1995). Statistically significant differences (Po0.05) between means were determined by Fisher’s least significant difference. Application of a split plot design to determine the effects of cook temperature, ripening time and their interaction on starter and NSLAB counts was performed as reported by Fenelon, Beresford, and Guinee (2002).

D-

3. Results and discussion 3.1. Viability of S. thermophilus and L. helveticus during manufacture Mean viable cell numbers of S. thermophilus TH3 were significantly affected by cook temperature, time and their interaction (Table 1) during cheese manufacture. Mean viable cell counts increased in all cheeses during cheese manufacture from 107 cfu mL1 after inoculation to 107.9 at maximum scald (Fig. 1). However, increasing cook temperature from 47 1C or 50–53 1C significantly reduced the growth rate of S. thermophilus during the period from maximum scald to transfer to the pre-press and also to brining in the following order: 47E50 1Co53 1C. The respective viable cell counts observed for S. thermophilus were 108.5, 108.2 or 107.9 cfu g1 at transfer of the curds to the pre-press and 109.2, 108.9 and 108.6 cfu g1 at brining of the cheeses. Martley (1983) reported that the most rapid acid production in milk for S. thermophilus and for L. helveticus is at 42.7 and 44.0 1C, respectively. Increasing cook temperature from 47 to 53 1C had no significant effect on mean viable cell counts of Lb. helveticus LHBO2 during cheese manufacture (Table 1). Generally,

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mean viable cell counts of Lb. helveticus increased significantly during manufacture from 106.5 cfu g1 after inoculation and maximum scald to 107.1 cfu g1 at transfer to the pre-press and to 108 cfu g1 at brining (Fig. 1). Increasing cook temperature from 47 to 53 1C resulted in an increase in cheese manufacture time from 265 to 314 min from inoculation to transfer to the pre-press and from 340 to 460 min from inoculation to brining (Fig. 1). Overall, raising process temperatures away from the optimum growth temperatures for both S. thermophilus and L. helveticus led to a considerable slowing of the acidification process due to decreased cellular viability, probably as a result of thermal stress (Giraffa et al., 1998; Neviani et al., 1995). This would result in increased manufacturing times which would impact on vat residence times and on overall plant capacity during commercial manufacture of these cheese types.

9.5

Log10 cfu g-1 cheese

9 8.5 8 7.5 7 6.5

(A)

707

6 9 8.5

3.2. Starter and NSLAB viability during cheese ripening Log10 cfu g-1 cheese

8 7.5 7 6.5 6 5.5

(B)

5 6.8 6.6 6.4

pH

6.2 6 5.8 5.6 5.4 5.2 5 0

(C)

100

200

300

400

500

Manufacture time (min)

Fig. 1. The effect of varying cook temperature (47 1C (’), 50 1C (K), 53 1C (m)) on (A) viable cell counts of Streptococcus thermophilus during cheese manufacture enumerated on LM17 agar; (B) viable cell counts of Lactobacillus helveticus during cheese manufacture enumerated on MRS pH 5.4 agar; and (C) pH development during cheese manufacture. Samples were analysed after inoculation, prior to cook, at max scald, at transfer to prepress and at brining. Values presented are the means from three replicate trials.

There was a significant effect of cook temperature and ripening time on viable cell counts of S. thermophilus during ripening (Table 2). Counts in curds cooked to 47 1C were significantly higher than in those cooked to 53 1C; viable cell counts in curds cooked to 50 1C were intermediate between those cooked to 47 and 53 1C and did not differ significantly from either. Turner, Martley, et al. (1983) observed that increasing cook temperature from 48 to 54 1C in Swiss cheese manufacture reduced S. thermophilus counts at 1 d of ripening. In this study, mean viable cell counts of S. thermophilus decreased from 109 cfu g1 at 2 d to 108 cfu g1 at 56 d of ripening (Fig. 2) representing a reduction in cell viability of between 70% and 90%. Similar observations have been made in Swiss cheeses (Thierry, Salvat-Brunaud, Madec, Michel, & Maubois, 1998; Valence, Deutsch, Richoux, Gagnaire, & Lortal, 2000). No significant effect of cook temperature was observed on mean cell viability of L. helveticus during ripening (Table 2). Similarly, Turner, Martley, et al. (1983) found that increasing cook temperature from 48 to 54 1C did not have any effect on lactobacillus counts at 1 d of ripening. During ripening, mean viable cell counts of L. helveticus decreased from 108.3 cfu g1 at 2 d to 106 cfu g1 at 56 d (Fig. 2), representing a drop in viability of 499%, in agreement with the studies of Thierry et al. (1998) and Valence et al. (2000). Cook temperature (Table 2) did not significantly affect mean viable counts of NSLAB during ripening. However, there was a significant effect of ripening time on mean viable NSLAB cell populations which initially increased rapidly to 105.2 cfu g1 at 56 d of ripening and then more slowly to 107 cfu g1 at 210 d (Fig. 2). Similar trends were observed in Cheddar cheese by Fenelon et al. (2002) and in Swiss cheese by Valence et al. (2000). Thierry et al. (1998) reported NSLAB counts 4108 cfu g1 at 20 d of ripening in Emmental cheeses. However, unlike cheeses in this study,

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Table 2 Mean squares (MS) and probabilities (P) for aggregated changes in mean viable cell counts of Streptococcus thermophilus, Lactobacillus helveticus and NSLAB during ripening of novel semi-hard cheeses cooked to different max scalds during manufacturea,b Factor

Main plot Temperature Error

S. thermophilus

L. helveticus

MS P

MS

1.42 0.16

0.0343*

0.02 0.27

P

NSLAB MS

0.915

3.37 3.45

P

0.4516

Sub-plot Time 1.09 o0.0001*** 8.82 o0.0001*** 54.24 o0.0001*** Interaction (temp.  time) 0.10 0.415 0.09 0.938 0.59 0.9317 Error .09 0.258 1.24

10

9 Log10 cfu g-1 cheese

708

5 (A)

For the response variable mean viable count of S. thermophilus the degrees of freedom (df) for the main plot and sub-plot were 4 and 24, respectively; the corresponding values for L. helveticus were 4 and 22 and for NSLAB were 4 and 36, respectively. b Significance levels: *Po0.05, **Po0.01 and ***Po0.001.

0

10

0

10

20

30

40

50

60

9

Log10 cfu g-1 cheese

3.3. Cheese composition

7

6

a

Emmental- and Swiss-type cheeses undergo propionic acid fermentation.

8

8

7

6

5 (B)

20

30

50

40

60

8 7 6 Log10 cfu g-1 cheese

The gross composition of the cheeses is summarised in Table 3. Cheeses produced from curds cooked to 47 1C had significantly higher levels of moisture than cheeses cooked to 53 1C and significantly higher levels of moisture in nonfat substance (MNFS) and salt-in-moisture (S/M) values, and significantly lower levels of protein than in cheeses cooked to both 50 and 53 1C. Turner, Martley, et al. (1983) observed that increased synaeresis caused by increasing the cook temperature from 48 to 54 1C during the manufacture of a Swiss-type cheese outweighed the reduced synaeresis caused by a higher pH at transfer of curds to the pre-press. In this study the pH at transfer of curds to the pre-press was held constant, and it appears that a reduction in cheese moisture content resulted from increased synaeresis due to the direct effects of elevated cook temperatures, a significantly reduced growth rate of S. thermophilus and an increased manufacture time. Although not statistically different, there were numerically lower levels of calcium in the cheeses cooked to 47 1C in comparison to those cooked to 50 or 53 1C. As discussed below, the pH after brining was significantly lower in the cheeses cooked to 47 1C than in those cooked to 50 or 53 1C. This lower pH may have increased the degree of solubilisation of micellar calcium phosphate (Van Hooydonk, Hagedoorn, & Boerrigter, 1986) permitting increased soluble calcium to be lost from the curds during whey drainage (Czulak, Conochie, Sutherland, & van Leeuwen, 1969). In the context of varietal characteristics, the semi-hard cheeses in this study had similar levels of moisture, MNFS

5 4 3 2 1 0 0

(C)

60

120 Ripening (d)

180

240

Fig. 2. The effect of varying cook temperature (47 1C (’), 50 1C (K), 53 1C (m)) on (A) viable cell counts of Streptococcus thermophilus enumerated on LM17 agar; (B) viable cell counts of Lactobacillus helveticus enumerated on MRS pH 5.4 agar and (C) viable cell counts of nonstarter lactic acid bacteria enumerated on LBS agar during ripening. Values presented are the means from three replicate trials.

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709

Table 3 The effect of cooking temperature on the pH at 2 d of ripening and the composition at 14 d of ripening of semi-hard cheeses manufactured using Streptococcus thermophilus and Lactobacillus helveticusa Cook temp. (1C) 47 50 53 SED a

Moisture a

39.71 38.74ab 37.58b 0.49

MNFSb

Fat a

28.75 28.15a 29.54a 0.78

a

55.72 53.93b 53.33b 0.29

FDM (% wt/wt) a

47.69 45.95a 47.32a 0.95

S/M a

3.42 2.65b 2.71b 0.12

Protein b

26.22 27.98a 27.94a 0.46

Salt

Ca (mg%) a

1.36 1.03b 1.02b 0.05

a

718 778a 746a 23.75

pH day 2 5.14b 5.20a 5.22a 0.02

Values presented are the means of three replicates. Values within a column not sharing a common superscript letter differ (Po0.05). MNFS, moisture in the nonfat substance; FDM, fat in dry matter; S/M, salt in moisture; SED, standard error of difference; degrees of freedom ¼ 4.

b

and slightly higher levels of salt relative to Swiss type cheese (Lucey, Gorry, & Fox, 1993) and similar levels of protein, calcium and slightly lower levels of fat and FDM in comparison to a typical Cheddar cheese (Guinee et al., 2000). 3.4. Changes in lactate Unlike Cheddar where only L-lactate is produced (Thomas & Crow, 1983; Turner & Thomas, 1980) both D- and L-lactate were produced during manufacture (Fig. 3) due to S. thermophilus and L. helveticus which produce L-lactate and a mixture of both D- and L-lactate, respectively (Steffen, Flueckiger, Bosset, & Ruegg, 1987; Turner, Morris, & Martley, 1983). Cook temperature had no significant effect on D-, L- or total lactate levels found in curds. The levels of total lactic acid during ripening in the cheeses in this study (1300–1400 mg 100 g1) were slightly higher than in Cheddar cheese (1200 mg 100 g1) (Thomas & Crow, 1983; Turner & Thomas, 1980) and similar to levels in Swiss-type cheeses (1200–1500 mg 100 g1) (Steffen et al., 1987; Turner, Morris, et al., 1983). There was no significant change in total lactate levels during ripening; however, a significant increase in D-lactate levels (250–650 mg 100 g1 cheese) with a significant decrease in 1 L-lactate levels (1000–650 mg 100 g cheese) was observed. Similar trends were reported in Cheddar cheese with NSLAB counts of 107–108 cfu g1 by Turner and Thomas (1980) and Thomas and Crow (1983) which was attributed to the production of DL-lactate from lactose and to the racemisation of L- to D-lactate. Unlike in many Swiss-type cheeses, there was no propionic acid fermentation in the cheeses in this study, which would convert lactate to acetate and propionate.

53 1C. Decreased moisture, and thus lactose and lactate contents, resulted in lower lactate to protein ratios and thus a higher buffering capacity in the cheese (Fenelon & Guinee, 2000; Fox, Lucey, & Cogan, 1990; Huffman & Kristoffersen, 1984; Shakeel-Ur-Rehman, Waldron, & Fox, 2004). This may have resulted in higher cheese pH values at day 1. Similarly, the extent of acid production during manufacture determines the extent of solubilisation of colloidal calcium phosphate (Van Hooydonk et al., 1986) and therefore its contribution to buffering capacity (Lucey et al., 1993). Significantly lower moisture contents have been associated also with reduced levels of carbohydrate available for metabolism by the Lactobacillus during pressing of Swiss-type cheeses and thus may result also in higher cheese pH at 1 d (Turner, Morris, et al., 1983). Increasing cook temperature beyond 47 1C also resulted in a significantly higher mean pH throughout ripening (Table 4 and Fig. 4). Although there was no statistically significant difference in the mean total lactate to protein ratios between cheeses cooked to 47 1C and those cooked to 50 or 53 1C during ripening, the ratios of total lactate to protein in the cheeses cooked to 47 1C were notably higher throughout ripening (Fig. 5) and thus may have influenced the buffering capacity and cheese pH. Mean pH values also increased significantly during ripening, rising from 5.20 at 2 d to pH 5.90 at 286 d. Dolby (1941) reported greater buffering capacity at a curd pHo5.5, which may partly explain why the pH of acidic cheeses increases less than that of cheeses with a higher pH (Fox et al., 1990). This may also possibly explain the relatively low change in pH at o84 d of ripening in all cheeses in the current study and the more dramatic increase in pH thereafter. The lower pH in the cheeses will also result in lower enzyme activity during ripening. 3.6. Changes in levels of free amino acids

3.5. Age-related changes in pH Cheese pH at 2 d was significantly lower in the cheeses cooked to 47 1C (pH 5.14) than in those cooked to 50 or 53 1C (pH 5.20 and 5.22, respectively; Table 3). It is proposed that this significant difference in cheese pH between cook temperatures may possibly be attributed to the lower MNFS contents in the cheeses cooked to 50 and

Levels of total FAAs increased from 3000 to 27,000 mg kg1 during ripening. These levels were much greater than those reported, at comparable ages, in full fat Cheddar (Guinee et al., 2000) and Gouda (Fox & Wallace, 1997), but were similar to those observed by Lawlor, Delahunty, Wilkinson, and Sheehan (2002) in mature Swiss-type cheeses (Appenzeller, Emmental and Gruye`re).

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D lactate / protein ratio

710 0.03

Table 4 Mean squares (MS) and probabilities (P) for aggregated changes pH during ripening of semi-hard cheeses cooked to different max scalds during manufacturea,b

0.02

Factor

pH during ripening P

MS

0.01

(A) 0.00

Main plot Temperature Error

0.144 0.012

Sub-plot Time Interaction (temp.  time) Error

0.62 0.002 0.004

0.02*

o 0.0001*** 0.8671

a

For the response variable pH during ripening the degrees of freedom (df) for the main plot and sub-plot were 4 and 48, respectively. b Significance levels: *Po0.05, **Po0.01 and ***Po0.001.

0.04

6.2

L-lactate / protein ratio

0.03

6 0.02

5.8

pH

0.01

5.6

(B) 0.00 5.4 0.06

Total lactate / protein ratio

0.05

5.2

0.04

5 0 0.03

50

100

150

200

250

300

Ripening (d) Fig. 4. The effect of varying cook temperature during cheese manufacture (47 1C (’), 50 1C (K), 53 1C (m)) on pH development during ripening. Values presented are the means from three replicate trials.

0.02

0.01

0.00 0

(C)

50

100

150

200

250

300

Ripening time (d)

Fig. 3. The effect of varying cook temperature during cheese manufacture (47 1C (’), 50 1C (K), 53 1C (m)) on ratios of D-lactate, L-lactate and total lactate to protein during ripening. Values presented are the means from three replicate trials.

Both Swiss-type cheeses and the cheeses characterised in this study were produced using lactobacillus strains that are more proteolytic and can produce greater amounts of

amino acids than lactococci (Broome, Krause, & Hickey, 1990; Hickey, Hillier, & Jago, 1983), which are predominantly used in Cheddar- and Gouda-type cheese manufacture. Cook temperature had no significant effect on total free amino acid levels produced in the cheeses during ripening (data not shown). Other studies (Al-Otaibi & Wilbey, 2004; Ardo¨, Thage, & Madsen, 2002; Kelly, Fox, & McSweeney, 1996; Kristiansen, Deding, Jensen, Ardo¨, & Qvist, 1999) have shown differences in cheese composition to influence free amino acid levels. However, the magnitude of

ARTICLE IN PRESS J.J. Sheehan et al. / International Dairy Journal 17 (2007) 704–716

D lactate (mg 100g-1cheese)

800

600

400

200

0 (A)

0

50

0

50

0

50

100

150

200

250

300

L lactate (mg 100g-1 cheese)

1200

900

600

300

0 (B)

100

150

200

250

300

1600

Total lactate (mg 100g-1 cheese)

1400

1200

1000

800

600

400 (C)

100

150

200

250

300

Ripening time (d)

Fig. 5. The effect of varying cook temperature during cheese manufacture (47 1C (’), 50 1C (K), 53 1C (m)) on levels of (A) D-lactate, (B) L-lactate and (C) total-lactate in cheeses during ripening. Values presented are the means from three replicate trials.

711

compositional differences reported by those authors were greater than in this study. Increased levels of total FAAs are associated with the release of intracellular peptidases, particularly from lactobacilli as a result of cell lysis as reviewed by Khalid and Marth (1990). Cook temperature had no significant effect on mean viable L. helveticus counts up to 56 d of ripening and thus there was probably no effect on lysis of L. helveticus, however lysis of L. helveticus was not quantified. The concentrations of individual FAAs (mg kg1 of cheese) in the cheeses at 84 and 210 d are shown Fig. 6. Cook temperature significantly affected mean levels of certain individual FAAs (Table 5). Levels of threonine and glutamate were similar in cheeses cooked to 50 and 53 1C and significantly higher in those cooked to 47 1C. Levels of leucine, phenylalanine and histidine were similar in cheeses cooked to 47 and 50 1C and significantly higher than in those cooked to 53 1C. Significant differences in cheese compositional parameters may have influenced relative quantities of individual FAAs as differences in cheese composition affect aminopepetidase activities (Kristiansen et al., 1999; Laan, Eng Tan, Bruinenberg, Limsowtin, & Broome, 1998). Higher cook temperatures may have had a greater attenuating affect on the starter cells (particularly with significant differences in mean viable cell counts of S. thermophilus during ripening) resulting in a greater leakage of cytoplasmic enzymes due to lysis after death (Lowrie, Lawrence, & Pearce, 1972; Wilkinson, Guinee, O’Callaghan, & Fox, 1995). Catabolism of individual amino acids, e.g., by glutamate dehydrogenase activity in S. thermophilus, may also have been influenced by differences in mean viable cell counts of S. thermophilus during ripening (Helinck, Le Bars, Moreau, & Yvon, 2004). The principal FAAs observed in all cheeses at most ripening times were serine, glutamate, valine, leucine, lysine, proline and phenylalanine which, with the exception of proline, reflects trends observed in Cheddar cheese (Fenelon, O’Connor, & Guinee, 2000) and, with the exception of serine, reflects the relative proportions of FAAs observed in Swiss and Gouda cheese (Fox & Wallace, 1997). Glutamate is positively correlated with caramel (Lawlor et al., 2002) and sour (Fox & Wallace, 1997; McSweeney & Sousa, 2000) flavours. Histidine is positively correlated with caramel (Lawlor et al., 2002), sour (Fox & Wallace, 1997) and bitter (McSweeney & Sousa, 2000) flavours. Leucine and phenylalanine are positively correlated with caramel (Lawlor et al., 2002) and bitter flavours (Fox & Wallace, 1997; McSweeney & Sousa, 2000). Threonine is positively correlated with caramel (Lawlor et al., 2002) and sweet (Fox, Guinee, Cogan, & McSweeney, 2000; Lawlor et al., 2002; McSweeney & Sousa, 2000) flavours, as is proline (Hintz, Slatter, & Harper, 1956; Langsrud & Reinbold, 1973; McSweeney & Sousa, 2000).

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712

2000 Free amino acids (mg kg-1 cheese)

d84 1600

1200

800

400

0 ASP THR SER GLU GLY ALA VAL MET ILE LEU TYR PHE HIS LYS ARG PRO 3500 d 210 Free amino acids (mg kg-1 cheese)

3000

2500

2000

1500

1000

500

0 ASP THR SER GLU GLY ALA VAL MET ILE LEU TYR PHE HIS LYS ARG PRO Fig. 6. The effect of varying cook temperature during cheese manufacture (47 1C (’), 50 1C ( ), 53 1C (&)) on levels of individual free amino acids in pH 4.6-soluble N extracts from cheeses at 84 and 210 d of ripening. Values presented are the means from three replicate trials.

Table 5 Mean squares (MS) and probabilities (P) for aggregated changes in threonine, glutamate, leucine, phenylalanine and histidine during ripening of semi-hard cheeses cooked to different max scalds during manufacturea,b Factor

Main plot Temperature Error

Threonine

Glutamate

Leucine

Phenylalanine

Histidine

MS

P

MS

P

MS

P

MS

P

MS

P

32,889 3365

0.0289*

145,648 3782

0.0024**

390,071 22846

0.011*

79,815 3460

0.0064**

48,603 3696

0.0174*

Sub-plot Time 1,233,047 o0.0001*** 5,668,220 o0.0001*** 15,878,655 o0.0001*** 3,399,774 o0.0001*** 2,118,649 o0.0001*** Interaction (Temp.  time) 2457 0.9383 86,642 0.149 66,705 0.2455 15,248 0.0921 7001 0.3268 Error 6426 52,914 48,642 8078 5775 a

For the response variables the degrees of freedom (df) for the main plot and sub-plot were 4 and 27, respectively. Significance levels: *Po0.05, **Po0.01 and ***Po0.001.

b

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3.7. Determination of acetate, propionate and butyrate levels in cheese during ripening

3.7.2. n-Butyrate There was a significant effect of cook temperature on levels of n-butyrate with cheeses cooked to 50 and 53 1C having similar and significantly higher levels than cheeses cooked to 47 1C (Fig. 7 and Table 6). Butyrate is released by lipases present in cheese and/or is synthesised by cheese microflora (Bills & Day, 1964; McSweeney & Sousa, 2000). Esterolytic and lipolytic enzymes have been identified in a diverse range of lactic acid bacteria including L. helveticus (El-Soda, Abd El-Wahab, Ezzat, Desmazeaud, & Ismail, 1986; Khalid & Marth, 1990) and S. thermophilus (Liu, Holland, & Crow, 2001). Esterolytic activity in the latter has been shown to be more than double that of lactococcal strains (Crow, Holland, Pritchard, & Coolbear, 1994). A recent study (Collins, McSweeney, & Wilkinson, 2003a) has shown evidence for a relationship between cell lysis and lipolysis in cheese. In this study, mean viable cell counts of S. thermophilus were significantly lower in cheeses cooked to 50 and 53 1C than in cheeses cooked to 47 1C up to 56 d of ripening, probably due to a reduced growth rate during cooking at elevated temperatures. However, the effect of increased cooking temperature on cell lysis or permeability was not determined.

2500

Acetic acid (mg kg-1)

2000

1500

1000

500

0 0

50

100

150

0

50

100

150

200

250

300

100

80

Butyric acid (mg kg-1)

3.7.1. Acetate and propionate Acetate was detected in the cheeses at 2 d of ripening (250 mg kg1) and mean levels increased significantly during ripening to 2000 mg kg1 at day 270 (Fig. 7). There was no statistically significant effect of cook temperature on levels of acetate (Table 6). The concentration of acetate reported in Cheddar varies widely from 100 to 6560 mg kg1 (Kristoffersen, Gould, & Harper, 1959); levels of acetate in Swiss type cheeses range from 3000 to 7000 mg kg1 for Emmental (Lawlor et al., 2002; Steffen et al., 1987) and 100–5000 mg kg1 for Swiss-type cheeses without a propionic acid fermentation (Lawlor et al., 2002). Acetic acid contributes to the characteristic flavours of Swiss-type cheeses (Steffen et al., 1987), to a burnt aftertaste (Lawlor et al., 2002), to sourness (Grazier, Bodyfelt, McDaniel, & Torres, 1991) and, at concentrations X700 mg kg1, gives rise to off flavour in young Cheddar, though the threshold probably increases with overall flavour intensity (Thomas, 1987). The increase in acetate levels during ripening was substantial after 84 d of ripening coinciding with NSLAB attaining counts of 4106 cfu g1 (Fig. 2). NSLAB have been associated with production of acetate from citrate or lactate (Thomas, 1987), the most probable pathway in cheeses in this study, from amino acids (Nakae & Elliott, 1965), or from lactose (Bouzas et al., 1993; Thomas, Ellwood, & Longyear, 1979). Acetate is also a product of propionic acid fermentation; however, propionate was present in only minute quantities in the cheeses in this study (data not shown).

713

60

40

20

0 200

250

300

Ripening time (d) Fig. 7. The effect of varying cook temperature during cheese manufacture (47 1C (’), 50 1C (K), 53 1C (m)) on levels of (A) acetic acid and (B) butyric acid in cheeses during ripening. Values presented are the means from three replicate trials.

Butyrate may also be produced by amino acid catabolism, e.g., iso-butyrate from valine (Christensen., Dudley, Pederson, & Steele, 1999; McSweeney & Sousa, 2000; Yvon & Rijnen, 2001). Iso-butyrate levels were not measured in this study. In addition, butyrate may result from the activity of clostridia (Steffen et al., 1987), however, clostridia were not detected in the cheeses (data not shown).

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Table 6 Mean squares (MS) and probabilities (P) for aggregated changes in C2 and C4 concentrations during ripening of semi-hard cheeses cooked to different max scalds during manufacturea,b Factor

C2

C4

MS

P

MS

P

Main plot Temperature Error

1026 854

0.390

5.67 0.463

0.0197*

Sub-plot Time Interaction (temp.  time) Error

41052 290 500

o0.0001*** 0.817

83.17 2.755 2.06

o01*** 0.257

a

The degrees of freedom (df) for the main plot and sub-plot were 4 and

30. b

Significance levels: *Po0.05, **Po0.01 and ***Po0.001.

Mean levels of butyrate increased significantly during ripening (Fig. 7 and Table 6). Initial increases occurred up to 84 d before reaching a plateau with further increases at ripening times beyond 140 d. Overall, levels of butyrate were low (60–100 mg kg1 at 270 d). They were comparable to those for Cheddar but lower than those for Swiss cheeses as reviewed by Collins, McSweeney, and Wilkinson (2003b) and by Lawlor et al. (2002). Butyrate (along with propionic and acetic acid) has been correlated with Swiss cheese flavour notes (Vangtal & Hammond, 1986; Zerfiridis, Vafopoulou-Mastrogiannaik, & Litopoulou-Tzanetaki, 1984) and with Cheddar flavour notes at 45–50 mg kg1 (Barlow et al., 1989). Butyrate can also contribute to rancid and cheesy flavours (Collins et al., 2003b) and has been correlated with intensity of cheese flavour (Lawlor et al., 2002).

4. Conclusions This study characterised the manufacture of a novel semi-hard cheese capable of production on existing manufacturing plant and also characterised the affect of altered cook temperature on its composition and ripening. Increasing cook temperature from 47 to 50 or 53 1C during the manufacture of the semi-hard cheeses significantly reduced growth of S. thermophilus during manufacture in the order 47E50453 1C with a concomitant increase in manufacture time, but had no significant effect on mean viable cell numbers of L. helveticus or NSLAB. Increased cook temperature significantly reduced levels of moisture, MNFS, S/M, and also resulted in significant differences in mean pH, n-butyrate and individual amino acids during ripening. Overall, the study showed how the cook temperature process variable may be used to create novel cheeses with varied compositional and ripening characteristics on existing manufacturing plant.

Acknowledgements This work was funded by the Department of Agriculture and Food, under the Food Institutional Research Measure (National Development Plan). The authors kindly acknowledge the technical assistance of E.O. Mulholland, MFRC, Teagasc, Moorepark and the statistical advice of K. O’Sullivan, Department of Statistics, University College, Cork, Ireland.

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