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Manufacture of Cheddar cheese with and without adjunct lactobacilli under controlled microbiological conditions
Int. Dairy Journal 6 (1996) 85 l-867 Copyright
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PIl:SO958-6946(96)00019-Z
1996 Published by Elwier Science Limited Printed in Ireland. All rights reserved 0958-6946/96/U
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ELSEVIER
Manufacture of Cheddar Cheese with and without Adjunct Lactobacilli under Controlled Microbiological Conditions
C. M. Lynch,“* P. L. H. McSweeney,” P. F. Fox,~ T. M. Cogad & F. D. Drinad ‘National
QDepartment of Food Chemistry, University College, Cork, Ireland Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland (Received 26 October 1995; accepted 7 April 1996)
ABSTRACT Cheddar cheeses were manufactured under controlled microbiological conditions to study the influence of selected strains of mesophilic lactobacilli on proteolysis andflavour development. In each of two trials, a control cheese (containing only a Lactococcus starter) and four experimental cheeses (containing the Lactococcus starter and adjunct lactobacilli) were manujactured. The Lactobacillus inocula were Lb. casei ssp. casei (4 strains), Lb. casei ssp. pseudoplantarum (4 strains), Lb. curvatus (4 strains) or Lb. plantarum ( 2 strains). In the experimental cheeses, counts of lactobacilli ranged from IO4 to IO’ cjii gg’ at milling and increased to -5x IO7 cfu g-t after 4 weeks. Control cheeses remained free of lactobacilli for up to 97 days and thereafter the counts did not exceed NIX 10’
*Author to whom correspondence
should be addressed. 851
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INTRODUCTION Non-starter lactic acid bacteria (NSLAB) in Cheddar cheese have been defined (Franklin & Sharpe, 1963) as the microbial population that grows in the cheese during ripening and is comprised of organisms that are able to grow under the highly selective conditions of the cheese environment (typically < 39% moisture, 46% salt-in-moisture, pH 4.9-5.3 and 513°C ripening temperature). The principal NSLAB in Cheddar cheese are species of mesophilic lactobacilli, although pediococci and micrococci may be also present (Peterson & Marshall, 1990; Bhowmik & Marth, 1990). A recent study of commercial Irish Cheddar showed that the non-starter flora was almost completely mesophilic lactobacilli (Jordan & Cogan, 1993). During cheese manufacture, the starter lactococci multiply rapidly, reaching -lo9 cfu g-i of curd at pressing, but numbers decline after salting, at a rate dependent on the strain of starter used, due to the presence of salt, the low curd pH and the absence of a fermentable carbohydrate (lactose). Concomitant with this decrease in starter numbers, NSLAB multiply, typically starting from lo’-lo4 cfu g-l of curd on the day of manufacture to become the dominant viable microflora (106-10’ cfu g-l) in the mature cheese (Chapman & Sharpe, 1981). While there is still much uncertainty as to the energy sources used by these organisms during growth in cheese, it is thought that residual lactose, lactate, citrate, peptides, amino acids, casein-derived sugars, glycerol (formed via lipolysis) and material released from autolysing starter bacteria may serve as potential growth substrates (Martley & Crow, 1993). The role of NSLAB in proteolysis and flavour development in Cheddar cheese remains unclear. However, it is widely accepted that the relatively recent introduction of pasteurisation of milk for cheesemaking, as well as improvements to the sanitary condition in the cheesemaking environment, has led to an overall reduction in NSLAB numbers and a more bland flavour in the mature cheese. Cheese made from raw milk generally ripens faster and develops a stronger, more intense flavour than cheese made from pasteurised milk. McSweeney et al. (1993) concluded that the differences observed between Cheddar cheeses made from microfiltered or pasteurised milk (which were similar in most respects) or from raw milk were due principally to the presence of indigenous NSLAB in the rawmilk cheese. The apparent importance of NSLAB in flavour development has prompted much interest in the deliberate addition of selected lactobacilli to cheesemilk with the objective of accelerating ripening and/or improving cheese quality, either by their own metabolic activities or by inhibiting the growth of undesirable adventitious bacteria (McSweeney et al., 1995). Some authors, e.g. Puchades et al. (1989), Lee et al. (1990), Broome et al. (1990) and Trepanier et al. (1991), have used mesophilic lactobacilli as adjuncts to the starter with more or less satisfactory results; increased levels of proteolysis (particularly free amino acids) and improved flavour characteristics of the adjunct-containing cheeses were generally observed. In some instances, however, adjunct lactobacilli were responsible for flavour defects (Puchades et al., 1989; Lee et al., 1990). Interpretation of the results of these studies for possible selection of strains of Lactobacillus for use as adjuncts is complicated by the fact that adventitious NSLAB almost invariably gain access to the ‘control’ cheese and eventually reach similar - and in some cases higher (e.g., Trepanier et al., 1991) - numbers in comparison to those in
Injluence of adjunct lactobacillion Cheddar cheese ripening
853
the experimental cheese. Differences in experimental design and level of inoculum of adjunct lactobacilli create further complications in interpretation. In this study, it was decided to adopt cheesemaking procedures whereby a number of strains of 4 species of mesophilic lactobacilli isolated from a good quality, strongly-flavoured raw-milk cheese, could be screened for their cheesemaking properties. Similar levels of inocula and manufacture of cheese under strictly controlled microbiological conditions (so that the influence of ‘wild’ lactobacilli could be minimised) facilitated this comparison.
MATERIALS
AND METHODS
Microbial strains and cultures Lactococcus lactis subsp. cremoris 223 (Chr. Hansen’s Laboratories, Little Island, Co. Cork, Ireland), grown overnight at 21°C in sterile (1 10°C 10 min) 10% (w/v) reconstituted low-heat skim milk powder, was used as the starter. The following strains of Lactobacillus were added to the experimental cheese vats: Lb. plantarum DPC 2739 and 2741 (Vat A); Lb. casei ssp. pseudoplantarum DPC 2742, 2745, 2749, 2750 (Vat B); Lb. casei ssp. casei DPC 2766, 2763, 2756, 2752 (Vat C) and Lb. curvatus DPC 2768, 2770, 2771, 2775 (Vat D). All Lactobacillus strains, obtained from the culture collection of the National Dairy Products Research Centre, Moorepark, Fermoy, had been isolated from a good quality, stronglyflavoured raw-milk cheese (McSweeney et al., 1993). Strains were grown individually in MRS broth (deMan et al., 1960) modified by reducing the glucose concentration to 1.0% (w/v) and omitting acetate and Tween 80, except for strains of Lb. curvatus, which were grown in unmodified MRS broth since they grew better in this medium. Equal volumes of stationary-phase cultures of strains of the same species were mixed together and used as adjuncts. Manufacture
of controlled microflora cheese
Cheeses were made on two occasions using small-scale cheese vats (25 L of cheesemilk) and cheesemaking equipment, all of which were steam-sterilised at 137°C for 5 min prior to use. Milk for cheesemaking was pasteurised at 78°C for 15 s and stored in sterile containers. The higher than normal pasteurisation conditions were used to ensure the destruction of the indigenous microflora in the milk. The cheese vats were placed in thermostatically-controlled water baths placed in a modified laminar air-flow unit. Apart from inoculation of the experimental cheesemilk with the various Lactobacillus adjuncts, cheesemaking was according to a standard protocol (Kosikowski, 1977). The aseptic precautions taken during cheesemaking were as described by McSweeney et al. (1994). The cheeses were ripened at 7°C for 6 months. Bacteriological
analysis
Lactobacilli and coliform bacteria in cheesemilk and cheeses were enumerated on Lactobacillus Selection Agar (LBS; Baltimore Biological Laboratories, Rockville, MD, USA) at 30°C for 5 days, or Violet Red Bile Agar (VRBA; Oxoid, Basingstoke, UK) at 30°C for 24 h, respectively.
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Starter bacteria in the cheeses were enumerated on LM17 agar (Oxoid, Basingstoke, UK), incubated at 30°C for 3 days (Terzaghi & Sandine, 1975). Total bacterial count (TBC) in pasteurised cheesemilk was determined on tryptone-glucose-yeast extract agar (Oxoid, Basingstoke, UK), incubated at 30°C for 2 days. Sensory analysis Cheeses were graded when 4 and 6 months old by an experienced taste panel (eight members, including a commercial grader) at the National Dairy Products Research Centre, for flavour intensity and flavour acceptability on a O-8 point scale (&l unacceptable; 2-3 poor; 45 acceptable; 6-7 good; 8 excellent). Samples were held for 1 h at room temperature before being presented to panel members in semi-enclosed booths designed for sensory analysis. Samples were coded l-5 (Icontrol, 2-Vat A, 3-Vat B, 4-Vat C and 5-Vat D). The five samples from each trial were presented at separate sessions and the results recorded individually. Compositional analysis All cheeses were analysed for fat (Gerber method; IS, 1955), protein (macroKjeldahl; IDF, 1964), moisture (oven drying at 102°C; IDF, 1982) and salt (Fox, 1963). The pH of a 1:l cheese:water slurry was determined using a standard pH meter (Radiometer, Copenhagen, Denmark). Assessment of proteolysis Water-soluble extracts (WSE) of the cheeses were prepared according to Kuchroo and Fox (1982). The nitrogen content of the WSEs was determined in triplicate by a modification of the Lowry method, as described by Dunn (1989). Total free amino acids in the cheeses were determined in triplicate by the Cd-ninhydrin method (Folkertsma & Fox, 1992). Freeze-dried aliquots of the WSEs were analysed by reverse phase high performance liquid chromatography (RP-HPLC) and urea-polyacrylamide gel electrophoresis (urea-PAGE). RP-HPLC was performed using a Waters 626 solvent delivery system with a Waters 600s controller, a Waters 717 plus autosampler (Waters Corp., Milford, MA, USA), nucleosil Cs guard (4.6 mm x 1,O cm) and analytical (4.6 mm x 25 cm) columns (5 pm particle size and 300A pore size; Macherey-Nagel GmbH, Duren, Germany). Column eluates were monitored at 214 nm using a Waters 486 detector interfaced with an IBM-compatible PC running on Millennium software (Waters). Chromatographic conditions were as follows: solvent A: 0.1% (v/v) trifluoroacetic acid (TFA, sequential grade, Sigma, St Louis, MO, USA) in deionised, HPLC-grade water; Solvent B: 0.1% (v/v) TFA in CHsCN (HPLC-grade acetonitrile, Labscan Ltd, Dublin, Ireland). Samples (4 mg mL-‘) were dissolved in solvent A and filtered through 0.45 pm cellulose acetate filters (Sartorius GmbH, Gottingen, Germany). Forty PL of filtrate were applied to the column and eluted at a flow-rate of 0.75 mL min-’ with 100% A for 5 min. A gradient of 0 to 50% B (0.91% B min-‘) was then commenced, followed by elution with 50% B for 6 min. A further gradient from 50 to 60% B (2.5% B min-‘) was then applied,
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Influence of adjunct lactobacilli on Cheddar cheese ripening
followed by 60% B for 3 min. The column was washed with 95% B for 5 min, followed by equilibration with 100% A for 5 min before the next injection. Cheeses and their WSEs were analysed by urea-PAGE (Shalabi & Fox, 1987), using a Protean IIxi vertical slab gel unit (Bio-Rad Laboratories Ltd, Watford, Herts, UK) with the stacking gel system described by Andrews (1983). The gels were stained using a modification of the method of Blakesley and Boezi (1977) with Coomassie Brilliant Blue G250. Amino acid analysis The concentration of free amino acids (FAA) in freeze-dried WSEs was determined per single injection on to a Beckman model 6300 amino acid analyser (Beckman Instruments Ltd, High Wycombe, UK) using a Beckman P-N 338052 Na cation exchange column (12 x 0.4 cm, i.d). A standard amino acid mixture (Beckman) was used to calibrate the column and norleucine (Sigma) was added to all samples before injection as an internal standard. Each freeze-dried sample (2.5-5.0 mg) was dissolved in sample buffer (0.2 M sodium citrate, pH 2.2) filtered through Whatman 0.22 pm filters and 50 PL of filtrate loaded onto the column. Amino acids were post-column derivatised with ninhydrin and detected by absorbance at 440 nm (proline and hydroxyproline) or 570 nm (all other amino acids). Results were analysed using a VG Minichrom computer system with a chromatography data handling software package.
RESULTS AND DISCUSSION Microbiological analyses Milk
The total bacterial, coliform and Lactobacillus counts in the milk for both cheesemaking trials are summarised in Table 1. Before addition of the cultures, the milk in all cases was free of coliforms, indigenous lactobacilli and had very low total bacterial counts. Numbers of lactobacilli in the experimental cheesemilk after adjunct addition were ~10~ cfu mL_’ in each vat. Pasteurisation of the cheesemilk at 78°C for 15 s was quite effective in providing milk of good microbiological quality for the manufacture of controlled microflora cheese. Elimination of indigenous NSLAB in the raw milk is particularly important TABLE 1 Bacterial Counts (cfu mL_‘) in Milk Used for the Manufacture of Cheddar Cheese Under Controlled Microbiological Conditions Uninoculated milk
Coliforms Lactobacilli TBC”
“Total bacterial count
Trial 1
Trial 2
Inoculated milk Trial 1
Trial 2
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200
Time (days)
Time (days)
Fig. 1. Growth of lactobacilli in Cheddar cheeses (a, trial 1; b, trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. pluntarum (0), Lb. casei subsp. pseudopluntarum (O), Lb. cusei subsp. cusei (0) or Lb. curvatus (m) as adjunct.
even very low numbers in the cheesemilk grow rapidly during cheese ripening (McSweeney et al., 1993). Turner et al. (1986) recommended pasteurisation of cheesemilk > 72°C for 15 s if NSLAB-free milk is required for cheesemaking under controlled microbiological conditions. since
Cheese
The numbers of lactobacilli in the control and experimental cheeses during ripening for both cheesemaking trials are shown in Fig. 1. The control cheese (to which no adjunct lactobacilli were added) remained free of ‘wild’ lactobacilli for 34 (trial 1) and 97 (trial 2) days. Numbers of lactobacilli in the experimental cheeses ex-press were between lo4 and 10’ cfu g-’ in both trials, but they grew rapidly during the first month of ripening, reaching a maximum in all cases of 10’ to lo8 cfu gg’ after -3 months. Broome et al. (1990) also noted rapid initial growth of strains of Lb. casei added (lo6 cfu mL_’ cheesemilk) to Cheddar cheese. Although ‘wild’ NSLAB grew in our control cheeses, a l-2 log difference in NSLAB numbers was maintained between the control and experimental cheeses in both trials, even towards the end of ripening. This
Influence of adjunct lactobacilli on Cheddar cheese ripening
857
demonstrates the effectiveness of the aseptic vat technique used in minimising the growth of NSLAB in Cheddar cheese during ripening. While numbers of Lb. casei ssp. casei (vat C) and Lb. curvatus (vat D) showed no decline towards the end of ripening, numbers of Lb. plantarum (vat A) and Lb. casei ssp. pseudoplantarum (vat B) decreased -10 fold between 3 and 6 months. This decrease may have been accompanied by autolysis of the adjunct lactobacilli. Lee et al. (1990) also observed decreases in the numbers of Lb. casei ssp. pseudoplantarum and Lb. plantarum strains added as adjuncts to Cheddar cheese, between 3 and 9 months of ripening. Starter numbers in both sets of cheeses during the first month of ripening are shown in Fig. 2. Starter bacteria grew to the same extent in the control and experimental cheeses, reaching between 10’ and lo9 cfu g-’ cheese after overnight pressing, indicating that the adjuncts did not interfere with starter growth (or acid production) during manufacture. Starter numbers showed a typical decline during the first month of ripening, after which it was not possible to monitor starter numbers due to the growth of lactobacilli on the non-selective medium used to enumerate the starter organisms.
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Fig. 2. Growth of starter bacteria in Cheddar cheeses (a, trial 1; b trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. plantarum (0), Lb. casei subsp. pseudoplantarum (a), Lb. casei subsp. casei (U) or Lb. curvatus (m) as adjunct.
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Compositional analysis The mean composition of the cheeses from trials 1 and 2 is shown in Table 2. The values obtained were within the range typical for Cheddar. Cheeses in trial 1 generally had similar compositions (as did those in trial 2), but cheeses in trial 2 generally had slightly higher fat and salt-in-moisture, and slightly lower moisture and protein contents than those in trial 1. The pH of the experimental cheeses was also slightly lower than that of the controls in both trials, presumably due to the metabolism of residual lactose by the adjunct lactobacilli in the experimental cheeses which were absent from the controls at 1 month, when the cheeses were analysed. Sensory analysis The mean grades received by the cheeses after 4 and 6 months ripening are shown in Table 3. After 4 months, scores received by the control cheeses for both flavour intensity and flavour acceptability were similar to those received by the experimental cheeses. However, after 6 months the control cheeses received the highest scores for flavour intensity and the lowest scores for flavour acceptability. Slight bitterness detected in the control cheeses by some members of the taste panel may explain why they received higher scores for flavour intensity in comparison to the experimental cheeses. Adjunct lactobacilli have been associated with a debittering effect in Cheddar through increased peptidolytic activity (Broadbent, 1994) which may have occurred in the experimental cheeses in this study. Improvements in flavour acceptability over the controls were noted for all of the experimental cheeses. Cheeses A (Lb. plantarum) and B (Lb. casei ssp. pseudopfantarum) generally scored better than cheeses C (Lb. casei ssp casei) and D (Lb. curvatus) in this regard. General improvement in Cheddar cheese quality by the use of adjunct mesophilic lactobacilli has been reported by other authors. TABLE 2 Composition” of Cheddar Cheeses Manufactured Under Controlled Microbiological Conditions Without (control) or With Adjunct Cultures of Mesophilic Lactobacilli (Vat A, Lb. plantarum; Vat B, Lb. casei ssp. pseudoplantarum; Vat C, Lb. casei ssp. casei and Vat D, Lb. curvatus) Fat A
Protein B
A
B
Moisture A
NaCl
B
PH
A
B
A
B
1.61 1.58 1.60 1.57 1.58
1.57 1.57 1.53 1.54 1.57
4.1 4.2 4.2 4.1 4.1
4.5 4.5 4.2 4.3 4.2
A
B
5.12 5.01 5.06 5.02 5.06
5.17 5.08 5.01 5.08 5.03
5%
Control Vat A Vat B Vat C Vat D
29.3 29.3 29.0 29.0 29.5
33.5 34.3 33.8 34.3 33.3
25.8 25.9 25.6 25.6 26.2
A, trial 1; B, trial 2. ‘Means of duplicate analyses. bSalt-in-moisture.
23.6 23.6 23.1 23.1 22.9
39.1 37.5 38.6 38.3 38.3
35.0 34.9 36.4 35.8 37.2
Injluence of adjunct lactobacilli on Cheddar cheese ripening
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TABLE 3
Gradesa (mean f SD) of Cheddar Cheeses (O-l Unacceptable; 2-3 Poor; 45 Acceptable; 67 Good; 8 Excellent) Manufactured Under Controlled Microbiological Conditions Without (Control) or With Adjunct Cultures of Mesophilic Lactobacilli (Vat A, Lb. plantarum; Vat B, Lb. casei ssp. pseudoplantarum; Vat C, Lb. casei ssp. casei and Vat D, Lb. curvatus) Flavour intensity
Control Vat A Vat B Vat C Vat D
Flavour acceptability
16 weeks
26 weeks
16 weeks
26 weeks
4.38f1.30 4.56f1.05 4.13f0.84 3.631tO.95 3.81f1.19
6.41f0.99 6.00f0.97 5.25f1.00 5.06f 1.24 4.94f1.61
3.38hO.79 3.31f1.03 3.38fl.28 3.13f1.09 3.50f1.41
4.00*1.59 5.22zt1.40 5.00f1.37 4.69f1.45 4.69f1.54
“Data from trials 1 and 2 combined.
Broome et al. (1990) observed enhancement of flavour development in Cheddar cheeses inoculated with Lb. casei strains in comparison to controls, while Puchades et al. (1989) found improvements in the taste quality of Cheddar cheeses inoculated with Lb. plantarum, Lb. casei ssp. casei and Lb. casei ssp. pseudoplantarum after 9 months of ripening. However, comparison of the present results with published work is complicated by differences in experimental design (e.g. level of inoculum, aseptic precautions, etc.) and differing effects on cheese quality by different strains of the same species (as demonstrated by Lee et al., 1990). Assessment
of proteolysis
Since the results for both sets of cheeses were similar, the results for only trial 2 are shown. Urea-PAGE electrophoretograms of whole cheese samples after ripening for 3 or 6 months and their corresponding WSEs are shown in Fig. 3. No differences in primary proteolysis (which were typical for Cheddar cheese) were apparent between the control and experimental cheeses during ripening. This was expected since proteolysis in Cheddar cheese detectable by urea-PAGE is mediated mainly by the action of rennet and plasmin (O’Keeffe et al., 1976). The proteolytic activity of the adjunct lactobacilli may have been responsible for the small quantitative differences in peptides in the electrophoretograms of the WSEs, apparent at 3 and 6 months. Levels of water-soluble nitrogen (WSN), expressed as a % of total nitrogen (TN), were more or less identical for all cheeses of the same age (Fig. 4). Visser (1977) concluded that rennet and, to a lesser extent, starter bacteria, were responsible for the formation of WSN in Gouda cheese. The only area where differences emerged between the control and experimental cheeses was in the formation of free amino acids (Fig. 5). After 6 months, cheeses A (Lb. plantarum), B (Lb. casei ssp. pseudoplantarum) and D (Lb. curvatus) had elevated levels of FAA compared to the controls and
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3 months
6 months
3 months
6 months
Fig., 3. Urea-polyacrylamide gel electrophoretograms of Na caseinate (CN) and samples at 3 and .6 months of ripening from trial 2 Cheddar cheeses made under controlled microbiological con ditions with and without (control) adjunct lactobacilli. (A) Electrophoretograms of cheese and (B) water-soluble extracts therefrom. Lane 1 - control cheese, lane 2 - cheese made usir lg an adjunct consisting of strains of Lb. plantarum, lane 3 - using Lb. casei subsp. pseudoplantarum, lane 4 - using Lb. cmei subsp. casei and lane 5 -using Lb. curvatus.
Influence of adjunct lactobacilli on Cheddar cheese ripening
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100
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Fig. 4. Development of water-soluble N in Cheddar cheeses (trial 2) made under control-
led microbiological conditions without (A) or with strains of Lb. plantarum (0), Lb. casei subsp. pseudoplantarum (a), Lb. casei subsp. casei (0) or Lb. curvatus ( n) as adjunct. experimental cheese C (Lb.casei ssp. casei), which were comparable throughout the 6 month ripening period. Higher concentrations of FAA in cheeses containing adjunct lactobacilli has been noted by other authors, e.g. Broome et al. (1990) found higher levels of PTA-soluble amino N and individual amino acids in Cheddar cheeses made with an adjunct of Lb. casei, and attributed this increase to higher peptidase activity in cheeses containing the adjunct. M&Sweeney et al. (1994) also observed elevated levels of FAA in Cheddar cheeses inoculated with a ‘cocktail’ of six strains of mesophilic lactobacilli, particularly in the later stages of ripening. At 3 months, the concentration of most individual FAA in the control and experimental cheeses were more or less similar, with the exception of glutamic acid and leucine, which were present in higher concentrations in the experimental cheeses, particularly cheeses B and D, than in the control (Fig. 6 A). At 6 months, concentrations of aspartic acid, glutamic acid, serine, leucine, tyrosine, phenylalanine and arginine were higher in the experimental cheeses than in the control, while all other individual FAA were present at similar concentrations in all cheeses (Fig. 6 B). Puchades et al. (1989), who also found elevated levels of aspartic acid, glutamic acid, phenylalanine and arginine in 7 month-old Cheddar cheeses to which strains of Lb. casei, Lb. plantarum and Lb. casei ssp. pseudoplantarum were added. The individual amino acid profiles at both time points reflect the results obtained using the Cd-ninhydrin method for total FAA determination, i.e. higher levels of individual FAA in experimental cheeses A, B and D than in the control and experimental cheese C. RP-HPLC of the WSEs from 3 or 6 month-old cheeses (Fig. 7 and Fig. 8,
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1.0 -
F
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0.5 -
1
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Fig. 5. Development of total free amino acids in Cheddar cheeses (trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. pluntarum (0), Lb. casei subsp. pseudoplantarum (o), Lb. casei subsp. casei (0) or Lb. curvatus (m) as adjunct.
respectively) showed essentially no quantitative or qualitative differences between the control and experimental cheeses during ripening, although at 3 months, some peptides (eluting between 60 and 70 min) were detected in the hydrophobic region of the chromatogram of the control in trial 1 (not shown) which were not present in the chromatograms of any of the experimental cheeses. This feature was observed only in trial 1 and not in trial 2 at this time. Broome et al. (1990) also found only minor differences in the HPLC elution profiles of WSEs from control cheeses and cheeses containing an adjunct of Lb. cusei, even after 48 weeks of ripening.
CONCLUSIONS Controlled conditions with respect to the growth of non-starter lactobacilli during ripening were maintained so that the influence of selected species of mesophilic lactobacilli on Cheddar cheese ripening could be assessed. In agreement with other workers, proteolysis during cheese ripening was affected by the adjuncts mainly at the level of FAA formation. Mesophilic lactobacilli have been shown to have an extensive peptidase system (e.g. El Soda et al., 1978). Elevated levels of FAA in the experimental cheeses probably arose from peptidase activity by the adjunct lactobacilli. Cell death and autolysis during ripening (accompanied by the release of intracellular peptidases) would obviously increase peptidase activity and this may have occurred to some
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Influence of adjunct lactobacilli on Cheddar cheese ripening
n Control Ei 0
q 0
Cys
Asp Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
VatA Vat B VatC Vat D
His
Lys
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Arg
Csy
n Control El 0 E 0
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Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
Vat Vat Vat Vat
His
A B C D
Lys
Fig. 6. Concentrations of individual free amino acids in 3 month-old (a) and 6 month-old (b) Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (Vat A), Lb. casei subsp. pseudoplantarum (Vat B), Lb. casei subsp. casei (Vat C) and Lb. curvatus (Vat D) as adjunct.
extent in cheeses containing pseudoplantarum (Vat B).
strains of Lb. plantarum
(Vat A) and Lb. casei ssp.
The importance of FAA in relation to flavour development in Cheddar cheese is well recognised. FAA contribute to cheese flavour either directly or by acting as precursors for the formation of cheese flavour compounds by enzymatic and non-enzymatic reactions (Engels & Visser, 1994). Results shown here demonstrate that adjunct cultures of mesophilic lactobacilli increase the concentration
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et al.
0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02
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10
20
30
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50
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30
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Minutes (cj)
0.08’ 0.06
10
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70
0
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10
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40
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Fig. 7. Reverse-phase HPLC chromatograms of water-soluble extracts from 3 month-old Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (a), Lb. casei subsp. pseudoplantarum (b), Lb. casei subsp. casei (c) or Lb. curvatus (d) as adjunct. AU, absorbance units.
of FAAs in Cheddar cheese during ripening. Sensory data also suggest a general improvement in cheese quality by the use of these adjuncts, in particular, Lb. plantarum and Lb. casei ssp. pseudoplantarum. These findings warrant further investigation of the effect of these two species on the sensory properties of Cheddar cheese.
ACKNOWLEDGEMENTS
The technical assistance of Mr Eugene O’Connor and the financial support of FORBAIRT are gratefully acknowledged. This research project was part funded from EU structural funds under the food sub-programme of the industry programme.
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Influence of adjunct lactobacilli on Cheddar cheese ripening
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Fig. 8. Reverse-phase HPLC chromatograms of water-soluble extracts from 6 month-old Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (a), Lb. casei subsp. pseudoplantarum (b), Lb. casei subsp. casei (c) or Lb. curvatus (d) as adjunct. AU, absorbance units.
REFERENCES Andrews, A.T. (1983). Proteinases in normal bovine milk and their action on the caseins. J. Dairy Res., 50, 45-55. Bhowmik, T. & Marth, E.H. (1990). Role of Micrococcus and Pediococcus species in cheese ripening: a review. J. Dairy Sci., 73, 859-866. Blakesley, R.W. & Boezi, J.A. (1977). A new staining technique for proteins in polyacrylamide gels using Coomassie Brilliant Blue G250. Anal. Biochem., 82, 580-58 1. Broadbent, J. (1994). Lactobacillus adjunct cultures for low-fat cheese. Proc. Eleventh Biennial Cheese Conference, Logan, UT, August 1618. Broome, M.C., Krause, D.A. & Hickey, M.W. (1990). The use of non-starter iactobacilli in Cheddar cheese manufacture. Aust. J. Dairy Technol., 45, 67-73. Chapman, H.R. & Sharpe, M.E. (1981). Microbiology of cheese. In Dairy Microbiology, Vol. 2. ed. R.K. Robinson. Applied Science Publishers, London, UK, pp. 1577243.
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deMan, J.C., Rogosa, M. & Sharpe, M.E. (1960). A medium for the cultivation of lactobacilli. J. Appl. Bacterial., 23, 130-135. Dunn, M.J., 1989. Determination of total protein concentration. In Protein PuriJication Methods, eds E.L.V. Harris & S. Angal. Oxford University Press, pp. 10-16. El Soda, M., Bergere, J.L. & Desmazeaud, M.J. (1978). Detection and localisation of peptide hydrolyases in Lactobacillus casei. J. Dairy Res., 45, 519-524. Engels, W.J.M. & Visser, S. (1994). Isolation and comparative characterisation of components that contribute to the flavour of different types of cheese. Neth. Milk Dairy J., 48, 127-140.
Folkertsma, B. & Fox, P.F. (1992). Use of the Cd-ninhydrin reagent to assess proteolysis in cheese during ripening. J. Dairy Res., 59, 217-224. Fox, P.F. (1963). Potentiometric determination of salt in cheese. J. Dairy Sci., 46,744-745. Franklin, J.G. & Sharpe, M.E. (1963). The incidence of bacteria in cheesemilk and Cheddar cheese and their association with flavour. J. Dairy Res., 30, 87-99. IDF (1964). Determination of the protein content of processed cheese products. Standard 25, International Dairy Federation, Brussels. IDF (1982). Cheese and processed cheese. Determination of the total solids content, reference method 4A, International Dairy Federation, Brussels. IS (1955), Irish Standards 69. Determination of the percentage of fat in cheese. Institute for Industrial Research and Standards, Dublin. Jordan, K.N. & Cogan, T.M. (1993). Identification and growth of non-starter lactic acid bacteria in Irish Cheddar cheese. Irish J. Agric. Food Res., 32, 47-55. Kosikowski, F. (1977). Cheddar cheese and related types. In Cheese and Fermented Milk Foods. Edwards Brothers Inc., Ann Arbor, MI, pp. 228-260. Kuchroo, C.N. & Fox, P.F. (1982). Soluble nitrogen in Cheddar cheese: comparison of extraction procedures. Milchwissenschaft, 37, 331-335. Lee, B.H., Laleye, L.C., Simard, R.E., Munsch, M.H. & Holley, R.A. (1990). Influence of homofermentative lactobacilli on the microflora and soluble nitrogen components in Cheddar cheese. J. Food Sci., 55, 391-397. Martley, F.G. & Crow, V.L. (1993). Interactions between non-starter microorganisms during cheese manufacture and ripening. Znt. Dairy J., 3, 461484. McSweeney, P.L.H., Fox, P.F., Lucey, J.A., Jordan, K.N. & Cogan, T.M. (1993). Contribution of the indigenous microflora to the maturation of Cheddar cheese. Znt. Dairy J., 3, 613634.
McSweeney, P.L.H., Lynch, C. M., Walsh, E.M., Fox, P.F., Jordan, K.N., Cogan, T.M. & Drinan, F.D. (1995). Role of non-starter lactic acid bacteria in Cheddar cheese ripening. In Proc. 4th Cheese Symposium, eds T.M. Cogan, P.F. Fox & R.P. Ross. Teagsc, Fermoy, Co. Cork, Ireland, pp. 3245. McSweeney, P.L.H., Walsh, E.M., Fox, P.F., Cogan, T.M., Drinan, F.D. & CasteloGonzalez, M. (1994). A procedure for the manufacture of Cheddar cheese under controlled bacteriological conditions and the effect of adjunct lactobacilli on cheese quality. Irish J. Agric. Food Res., 33, 183-192. O’Keeffe, R.B., Fox, P.F. & Daly, C. (1976). Contribution of rennet and starter proteases to proteolysis in Cheddar cheese. J. Dairy Res., 43, 97-107. Peterson, SD. & Marshall, R.T. (1990). Non-starter lactobacilli in Cheddar cheese: a review. J. Dairy Sci., 73, 1395-1410. Puchades, R., Lemieux, L. & Simard, R.D. (1989). Evolution of free amino acids during the ripening of Cheddar cheese containing added lactobacilli strains. J. Food Sci., 54, 885888.
Shalabi, S.I. & Fox, P.F. (1987). Electrophoretic analysis of cheese: Comparison of methods. Irish J. Food Sci. Technol., 11, 135-151. Terzaghi, B.E. & Sandine, W.E. (1975). Improved media for lactic streptococci and their bacteriophages. Appl. Microbial., 29, 807-8 13. Trepanier, G., Simard, R.E. & Lee, B.H. (1991). Lactic acid bacteria relation to accelerated maturation of Cheddar cheese. J. Food Sci., 56, 1238-1240.
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Turner, K.W., Lawrence, R.C. & Lelievre, J. (1986). A microbiological specification for milk for aseptic cheesemaking. N.Z. J. Dairy Sci. Technol., 21, 249-254. Visser, F.M.W. (1977). Contribution of enzymes from rennet, starter bacteria and milk to proteolysis and flavour development in Gouda cheese. 3. Protein breakdown: analysis of the soluble nitrogen and amino acid nitrogen fractions. Neth. Milk Dairy J., 31, 210239.
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ELSEVIER
Manufacture of Cheddar Cheese with and without Adjunct Lactobacilli under Controlled Microbiological Conditions
C. M. Lynch,“* P. L. H. McSweeney,” P. F. Fox,~ T. M. Cogad & F. D. Drinad ‘National
QDepartment of Food Chemistry, University College, Cork, Ireland Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland (Received 26 October 1995; accepted 7 April 1996)
ABSTRACT Cheddar cheeses were manufactured under controlled microbiological conditions to study the influence of selected strains of mesophilic lactobacilli on proteolysis andflavour development. In each of two trials, a control cheese (containing only a Lactococcus starter) and four experimental cheeses (containing the Lactococcus starter and adjunct lactobacilli) were manujactured. The Lactobacillus inocula were Lb. casei ssp. casei (4 strains), Lb. casei ssp. pseudoplantarum (4 strains), Lb. curvatus (4 strains) or Lb. plantarum ( 2 strains). In the experimental cheeses, counts of lactobacilli ranged from IO4 to IO’ cjii gg’ at milling and increased to -5x IO7 cfu g-t after 4 weeks. Control cheeses remained free of lactobacilli for up to 97 days and thereafter the counts did not exceed NIX 10’
*Author to whom correspondence
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INTRODUCTION Non-starter lactic acid bacteria (NSLAB) in Cheddar cheese have been defined (Franklin & Sharpe, 1963) as the microbial population that grows in the cheese during ripening and is comprised of organisms that are able to grow under the highly selective conditions of the cheese environment (typically < 39% moisture, 46% salt-in-moisture, pH 4.9-5.3 and 513°C ripening temperature). The principal NSLAB in Cheddar cheese are species of mesophilic lactobacilli, although pediococci and micrococci may be also present (Peterson & Marshall, 1990; Bhowmik & Marth, 1990). A recent study of commercial Irish Cheddar showed that the non-starter flora was almost completely mesophilic lactobacilli (Jordan & Cogan, 1993). During cheese manufacture, the starter lactococci multiply rapidly, reaching -lo9 cfu g-i of curd at pressing, but numbers decline after salting, at a rate dependent on the strain of starter used, due to the presence of salt, the low curd pH and the absence of a fermentable carbohydrate (lactose). Concomitant with this decrease in starter numbers, NSLAB multiply, typically starting from lo’-lo4 cfu g-l of curd on the day of manufacture to become the dominant viable microflora (106-10’ cfu g-l) in the mature cheese (Chapman & Sharpe, 1981). While there is still much uncertainty as to the energy sources used by these organisms during growth in cheese, it is thought that residual lactose, lactate, citrate, peptides, amino acids, casein-derived sugars, glycerol (formed via lipolysis) and material released from autolysing starter bacteria may serve as potential growth substrates (Martley & Crow, 1993). The role of NSLAB in proteolysis and flavour development in Cheddar cheese remains unclear. However, it is widely accepted that the relatively recent introduction of pasteurisation of milk for cheesemaking, as well as improvements to the sanitary condition in the cheesemaking environment, has led to an overall reduction in NSLAB numbers and a more bland flavour in the mature cheese. Cheese made from raw milk generally ripens faster and develops a stronger, more intense flavour than cheese made from pasteurised milk. McSweeney et al. (1993) concluded that the differences observed between Cheddar cheeses made from microfiltered or pasteurised milk (which were similar in most respects) or from raw milk were due principally to the presence of indigenous NSLAB in the rawmilk cheese. The apparent importance of NSLAB in flavour development has prompted much interest in the deliberate addition of selected lactobacilli to cheesemilk with the objective of accelerating ripening and/or improving cheese quality, either by their own metabolic activities or by inhibiting the growth of undesirable adventitious bacteria (McSweeney et al., 1995). Some authors, e.g. Puchades et al. (1989), Lee et al. (1990), Broome et al. (1990) and Trepanier et al. (1991), have used mesophilic lactobacilli as adjuncts to the starter with more or less satisfactory results; increased levels of proteolysis (particularly free amino acids) and improved flavour characteristics of the adjunct-containing cheeses were generally observed. In some instances, however, adjunct lactobacilli were responsible for flavour defects (Puchades et al., 1989; Lee et al., 1990). Interpretation of the results of these studies for possible selection of strains of Lactobacillus for use as adjuncts is complicated by the fact that adventitious NSLAB almost invariably gain access to the ‘control’ cheese and eventually reach similar - and in some cases higher (e.g., Trepanier et al., 1991) - numbers in comparison to those in
Injluence of adjunct lactobacillion Cheddar cheese ripening
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the experimental cheese. Differences in experimental design and level of inoculum of adjunct lactobacilli create further complications in interpretation. In this study, it was decided to adopt cheesemaking procedures whereby a number of strains of 4 species of mesophilic lactobacilli isolated from a good quality, strongly-flavoured raw-milk cheese, could be screened for their cheesemaking properties. Similar levels of inocula and manufacture of cheese under strictly controlled microbiological conditions (so that the influence of ‘wild’ lactobacilli could be minimised) facilitated this comparison.
MATERIALS
AND METHODS
Microbial strains and cultures Lactococcus lactis subsp. cremoris 223 (Chr. Hansen’s Laboratories, Little Island, Co. Cork, Ireland), grown overnight at 21°C in sterile (1 10°C 10 min) 10% (w/v) reconstituted low-heat skim milk powder, was used as the starter. The following strains of Lactobacillus were added to the experimental cheese vats: Lb. plantarum DPC 2739 and 2741 (Vat A); Lb. casei ssp. pseudoplantarum DPC 2742, 2745, 2749, 2750 (Vat B); Lb. casei ssp. casei DPC 2766, 2763, 2756, 2752 (Vat C) and Lb. curvatus DPC 2768, 2770, 2771, 2775 (Vat D). All Lactobacillus strains, obtained from the culture collection of the National Dairy Products Research Centre, Moorepark, Fermoy, had been isolated from a good quality, stronglyflavoured raw-milk cheese (McSweeney et al., 1993). Strains were grown individually in MRS broth (deMan et al., 1960) modified by reducing the glucose concentration to 1.0% (w/v) and omitting acetate and Tween 80, except for strains of Lb. curvatus, which were grown in unmodified MRS broth since they grew better in this medium. Equal volumes of stationary-phase cultures of strains of the same species were mixed together and used as adjuncts. Manufacture
of controlled microflora cheese
Cheeses were made on two occasions using small-scale cheese vats (25 L of cheesemilk) and cheesemaking equipment, all of which were steam-sterilised at 137°C for 5 min prior to use. Milk for cheesemaking was pasteurised at 78°C for 15 s and stored in sterile containers. The higher than normal pasteurisation conditions were used to ensure the destruction of the indigenous microflora in the milk. The cheese vats were placed in thermostatically-controlled water baths placed in a modified laminar air-flow unit. Apart from inoculation of the experimental cheesemilk with the various Lactobacillus adjuncts, cheesemaking was according to a standard protocol (Kosikowski, 1977). The aseptic precautions taken during cheesemaking were as described by McSweeney et al. (1994). The cheeses were ripened at 7°C for 6 months. Bacteriological
analysis
Lactobacilli and coliform bacteria in cheesemilk and cheeses were enumerated on Lactobacillus Selection Agar (LBS; Baltimore Biological Laboratories, Rockville, MD, USA) at 30°C for 5 days, or Violet Red Bile Agar (VRBA; Oxoid, Basingstoke, UK) at 30°C for 24 h, respectively.
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Starter bacteria in the cheeses were enumerated on LM17 agar (Oxoid, Basingstoke, UK), incubated at 30°C for 3 days (Terzaghi & Sandine, 1975). Total bacterial count (TBC) in pasteurised cheesemilk was determined on tryptone-glucose-yeast extract agar (Oxoid, Basingstoke, UK), incubated at 30°C for 2 days. Sensory analysis Cheeses were graded when 4 and 6 months old by an experienced taste panel (eight members, including a commercial grader) at the National Dairy Products Research Centre, for flavour intensity and flavour acceptability on a O-8 point scale (&l unacceptable; 2-3 poor; 45 acceptable; 6-7 good; 8 excellent). Samples were held for 1 h at room temperature before being presented to panel members in semi-enclosed booths designed for sensory analysis. Samples were coded l-5 (Icontrol, 2-Vat A, 3-Vat B, 4-Vat C and 5-Vat D). The five samples from each trial were presented at separate sessions and the results recorded individually. Compositional analysis All cheeses were analysed for fat (Gerber method; IS, 1955), protein (macroKjeldahl; IDF, 1964), moisture (oven drying at 102°C; IDF, 1982) and salt (Fox, 1963). The pH of a 1:l cheese:water slurry was determined using a standard pH meter (Radiometer, Copenhagen, Denmark). Assessment of proteolysis Water-soluble extracts (WSE) of the cheeses were prepared according to Kuchroo and Fox (1982). The nitrogen content of the WSEs was determined in triplicate by a modification of the Lowry method, as described by Dunn (1989). Total free amino acids in the cheeses were determined in triplicate by the Cd-ninhydrin method (Folkertsma & Fox, 1992). Freeze-dried aliquots of the WSEs were analysed by reverse phase high performance liquid chromatography (RP-HPLC) and urea-polyacrylamide gel electrophoresis (urea-PAGE). RP-HPLC was performed using a Waters 626 solvent delivery system with a Waters 600s controller, a Waters 717 plus autosampler (Waters Corp., Milford, MA, USA), nucleosil Cs guard (4.6 mm x 1,O cm) and analytical (4.6 mm x 25 cm) columns (5 pm particle size and 300A pore size; Macherey-Nagel GmbH, Duren, Germany). Column eluates were monitored at 214 nm using a Waters 486 detector interfaced with an IBM-compatible PC running on Millennium software (Waters). Chromatographic conditions were as follows: solvent A: 0.1% (v/v) trifluoroacetic acid (TFA, sequential grade, Sigma, St Louis, MO, USA) in deionised, HPLC-grade water; Solvent B: 0.1% (v/v) TFA in CHsCN (HPLC-grade acetonitrile, Labscan Ltd, Dublin, Ireland). Samples (4 mg mL-‘) were dissolved in solvent A and filtered through 0.45 pm cellulose acetate filters (Sartorius GmbH, Gottingen, Germany). Forty PL of filtrate were applied to the column and eluted at a flow-rate of 0.75 mL min-’ with 100% A for 5 min. A gradient of 0 to 50% B (0.91% B min-‘) was then commenced, followed by elution with 50% B for 6 min. A further gradient from 50 to 60% B (2.5% B min-‘) was then applied,
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Influence of adjunct lactobacilli on Cheddar cheese ripening
followed by 60% B for 3 min. The column was washed with 95% B for 5 min, followed by equilibration with 100% A for 5 min before the next injection. Cheeses and their WSEs were analysed by urea-PAGE (Shalabi & Fox, 1987), using a Protean IIxi vertical slab gel unit (Bio-Rad Laboratories Ltd, Watford, Herts, UK) with the stacking gel system described by Andrews (1983). The gels were stained using a modification of the method of Blakesley and Boezi (1977) with Coomassie Brilliant Blue G250. Amino acid analysis The concentration of free amino acids (FAA) in freeze-dried WSEs was determined per single injection on to a Beckman model 6300 amino acid analyser (Beckman Instruments Ltd, High Wycombe, UK) using a Beckman P-N 338052 Na cation exchange column (12 x 0.4 cm, i.d). A standard amino acid mixture (Beckman) was used to calibrate the column and norleucine (Sigma) was added to all samples before injection as an internal standard. Each freeze-dried sample (2.5-5.0 mg) was dissolved in sample buffer (0.2 M sodium citrate, pH 2.2) filtered through Whatman 0.22 pm filters and 50 PL of filtrate loaded onto the column. Amino acids were post-column derivatised with ninhydrin and detected by absorbance at 440 nm (proline and hydroxyproline) or 570 nm (all other amino acids). Results were analysed using a VG Minichrom computer system with a chromatography data handling software package.
RESULTS AND DISCUSSION Microbiological analyses Milk
The total bacterial, coliform and Lactobacillus counts in the milk for both cheesemaking trials are summarised in Table 1. Before addition of the cultures, the milk in all cases was free of coliforms, indigenous lactobacilli and had very low total bacterial counts. Numbers of lactobacilli in the experimental cheesemilk after adjunct addition were ~10~ cfu mL_’ in each vat. Pasteurisation of the cheesemilk at 78°C for 15 s was quite effective in providing milk of good microbiological quality for the manufacture of controlled microflora cheese. Elimination of indigenous NSLAB in the raw milk is particularly important TABLE 1 Bacterial Counts (cfu mL_‘) in Milk Used for the Manufacture of Cheddar Cheese Under Controlled Microbiological Conditions Uninoculated milk
Coliforms Lactobacilli TBC”
“Total bacterial count
Trial 1
Trial 2
Inoculated milk Trial 1
Trial 2
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Fig. 1. Growth of lactobacilli in Cheddar cheeses (a, trial 1; b, trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. pluntarum (0), Lb. casei subsp. pseudopluntarum (O), Lb. cusei subsp. cusei (0) or Lb. curvatus (m) as adjunct.
even very low numbers in the cheesemilk grow rapidly during cheese ripening (McSweeney et al., 1993). Turner et al. (1986) recommended pasteurisation of cheesemilk > 72°C for 15 s if NSLAB-free milk is required for cheesemaking under controlled microbiological conditions. since
Cheese
The numbers of lactobacilli in the control and experimental cheeses during ripening for both cheesemaking trials are shown in Fig. 1. The control cheese (to which no adjunct lactobacilli were added) remained free of ‘wild’ lactobacilli for 34 (trial 1) and 97 (trial 2) days. Numbers of lactobacilli in the experimental cheeses ex-press were between lo4 and 10’ cfu g-’ in both trials, but they grew rapidly during the first month of ripening, reaching a maximum in all cases of 10’ to lo8 cfu gg’ after -3 months. Broome et al. (1990) also noted rapid initial growth of strains of Lb. casei added (lo6 cfu mL_’ cheesemilk) to Cheddar cheese. Although ‘wild’ NSLAB grew in our control cheeses, a l-2 log difference in NSLAB numbers was maintained between the control and experimental cheeses in both trials, even towards the end of ripening. This
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demonstrates the effectiveness of the aseptic vat technique used in minimising the growth of NSLAB in Cheddar cheese during ripening. While numbers of Lb. casei ssp. casei (vat C) and Lb. curvatus (vat D) showed no decline towards the end of ripening, numbers of Lb. plantarum (vat A) and Lb. casei ssp. pseudoplantarum (vat B) decreased -10 fold between 3 and 6 months. This decrease may have been accompanied by autolysis of the adjunct lactobacilli. Lee et al. (1990) also observed decreases in the numbers of Lb. casei ssp. pseudoplantarum and Lb. plantarum strains added as adjuncts to Cheddar cheese, between 3 and 9 months of ripening. Starter numbers in both sets of cheeses during the first month of ripening are shown in Fig. 2. Starter bacteria grew to the same extent in the control and experimental cheeses, reaching between 10’ and lo9 cfu g-’ cheese after overnight pressing, indicating that the adjuncts did not interfere with starter growth (or acid production) during manufacture. Starter numbers showed a typical decline during the first month of ripening, after which it was not possible to monitor starter numbers due to the growth of lactobacilli on the non-selective medium used to enumerate the starter organisms.
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Fig. 2. Growth of starter bacteria in Cheddar cheeses (a, trial 1; b trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. plantarum (0), Lb. casei subsp. pseudoplantarum (a), Lb. casei subsp. casei (U) or Lb. curvatus (m) as adjunct.
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Compositional analysis The mean composition of the cheeses from trials 1 and 2 is shown in Table 2. The values obtained were within the range typical for Cheddar. Cheeses in trial 1 generally had similar compositions (as did those in trial 2), but cheeses in trial 2 generally had slightly higher fat and salt-in-moisture, and slightly lower moisture and protein contents than those in trial 1. The pH of the experimental cheeses was also slightly lower than that of the controls in both trials, presumably due to the metabolism of residual lactose by the adjunct lactobacilli in the experimental cheeses which were absent from the controls at 1 month, when the cheeses were analysed. Sensory analysis The mean grades received by the cheeses after 4 and 6 months ripening are shown in Table 3. After 4 months, scores received by the control cheeses for both flavour intensity and flavour acceptability were similar to those received by the experimental cheeses. However, after 6 months the control cheeses received the highest scores for flavour intensity and the lowest scores for flavour acceptability. Slight bitterness detected in the control cheeses by some members of the taste panel may explain why they received higher scores for flavour intensity in comparison to the experimental cheeses. Adjunct lactobacilli have been associated with a debittering effect in Cheddar through increased peptidolytic activity (Broadbent, 1994) which may have occurred in the experimental cheeses in this study. Improvements in flavour acceptability over the controls were noted for all of the experimental cheeses. Cheeses A (Lb. plantarum) and B (Lb. casei ssp. pseudopfantarum) generally scored better than cheeses C (Lb. casei ssp casei) and D (Lb. curvatus) in this regard. General improvement in Cheddar cheese quality by the use of adjunct mesophilic lactobacilli has been reported by other authors. TABLE 2 Composition” of Cheddar Cheeses Manufactured Under Controlled Microbiological Conditions Without (control) or With Adjunct Cultures of Mesophilic Lactobacilli (Vat A, Lb. plantarum; Vat B, Lb. casei ssp. pseudoplantarum; Vat C, Lb. casei ssp. casei and Vat D, Lb. curvatus) Fat A
Protein B
A
B
Moisture A
NaCl
B
PH
A
B
A
B
1.61 1.58 1.60 1.57 1.58
1.57 1.57 1.53 1.54 1.57
4.1 4.2 4.2 4.1 4.1
4.5 4.5 4.2 4.3 4.2
A
B
5.12 5.01 5.06 5.02 5.06
5.17 5.08 5.01 5.08 5.03
5%
Control Vat A Vat B Vat C Vat D
29.3 29.3 29.0 29.0 29.5
33.5 34.3 33.8 34.3 33.3
25.8 25.9 25.6 25.6 26.2
A, trial 1; B, trial 2. ‘Means of duplicate analyses. bSalt-in-moisture.
23.6 23.6 23.1 23.1 22.9
39.1 37.5 38.6 38.3 38.3
35.0 34.9 36.4 35.8 37.2
Injluence of adjunct lactobacilli on Cheddar cheese ripening
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TABLE 3
Gradesa (mean f SD) of Cheddar Cheeses (O-l Unacceptable; 2-3 Poor; 45 Acceptable; 67 Good; 8 Excellent) Manufactured Under Controlled Microbiological Conditions Without (Control) or With Adjunct Cultures of Mesophilic Lactobacilli (Vat A, Lb. plantarum; Vat B, Lb. casei ssp. pseudoplantarum; Vat C, Lb. casei ssp. casei and Vat D, Lb. curvatus) Flavour intensity
Control Vat A Vat B Vat C Vat D
Flavour acceptability
16 weeks
26 weeks
16 weeks
26 weeks
4.38f1.30 4.56f1.05 4.13f0.84 3.631tO.95 3.81f1.19
6.41f0.99 6.00f0.97 5.25f1.00 5.06f 1.24 4.94f1.61
3.38hO.79 3.31f1.03 3.38fl.28 3.13f1.09 3.50f1.41
4.00*1.59 5.22zt1.40 5.00f1.37 4.69f1.45 4.69f1.54
“Data from trials 1 and 2 combined.
Broome et al. (1990) observed enhancement of flavour development in Cheddar cheeses inoculated with Lb. casei strains in comparison to controls, while Puchades et al. (1989) found improvements in the taste quality of Cheddar cheeses inoculated with Lb. plantarum, Lb. casei ssp. casei and Lb. casei ssp. pseudoplantarum after 9 months of ripening. However, comparison of the present results with published work is complicated by differences in experimental design (e.g. level of inoculum, aseptic precautions, etc.) and differing effects on cheese quality by different strains of the same species (as demonstrated by Lee et al., 1990). Assessment
of proteolysis
Since the results for both sets of cheeses were similar, the results for only trial 2 are shown. Urea-PAGE electrophoretograms of whole cheese samples after ripening for 3 or 6 months and their corresponding WSEs are shown in Fig. 3. No differences in primary proteolysis (which were typical for Cheddar cheese) were apparent between the control and experimental cheeses during ripening. This was expected since proteolysis in Cheddar cheese detectable by urea-PAGE is mediated mainly by the action of rennet and plasmin (O’Keeffe et al., 1976). The proteolytic activity of the adjunct lactobacilli may have been responsible for the small quantitative differences in peptides in the electrophoretograms of the WSEs, apparent at 3 and 6 months. Levels of water-soluble nitrogen (WSN), expressed as a % of total nitrogen (TN), were more or less identical for all cheeses of the same age (Fig. 4). Visser (1977) concluded that rennet and, to a lesser extent, starter bacteria, were responsible for the formation of WSN in Gouda cheese. The only area where differences emerged between the control and experimental cheeses was in the formation of free amino acids (Fig. 5). After 6 months, cheeses A (Lb. plantarum), B (Lb. casei ssp. pseudoplantarum) and D (Lb. curvatus) had elevated levels of FAA compared to the controls and
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3 months
6 months
3 months
6 months
Fig., 3. Urea-polyacrylamide gel electrophoretograms of Na caseinate (CN) and samples at 3 and .6 months of ripening from trial 2 Cheddar cheeses made under controlled microbiological con ditions with and without (control) adjunct lactobacilli. (A) Electrophoretograms of cheese and (B) water-soluble extracts therefrom. Lane 1 - control cheese, lane 2 - cheese made usir lg an adjunct consisting of strains of Lb. plantarum, lane 3 - using Lb. casei subsp. pseudoplantarum, lane 4 - using Lb. cmei subsp. casei and lane 5 -using Lb. curvatus.
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Time (days)
Fig. 4. Development of water-soluble N in Cheddar cheeses (trial 2) made under control-
led microbiological conditions without (A) or with strains of Lb. plantarum (0), Lb. casei subsp. pseudoplantarum (a), Lb. casei subsp. casei (0) or Lb. curvatus ( n) as adjunct. experimental cheese C (Lb.casei ssp. casei), which were comparable throughout the 6 month ripening period. Higher concentrations of FAA in cheeses containing adjunct lactobacilli has been noted by other authors, e.g. Broome et al. (1990) found higher levels of PTA-soluble amino N and individual amino acids in Cheddar cheeses made with an adjunct of Lb. casei, and attributed this increase to higher peptidase activity in cheeses containing the adjunct. M&Sweeney et al. (1994) also observed elevated levels of FAA in Cheddar cheeses inoculated with a ‘cocktail’ of six strains of mesophilic lactobacilli, particularly in the later stages of ripening. At 3 months, the concentration of most individual FAA in the control and experimental cheeses were more or less similar, with the exception of glutamic acid and leucine, which were present in higher concentrations in the experimental cheeses, particularly cheeses B and D, than in the control (Fig. 6 A). At 6 months, concentrations of aspartic acid, glutamic acid, serine, leucine, tyrosine, phenylalanine and arginine were higher in the experimental cheeses than in the control, while all other individual FAA were present at similar concentrations in all cheeses (Fig. 6 B). Puchades et al. (1989), who also found elevated levels of aspartic acid, glutamic acid, phenylalanine and arginine in 7 month-old Cheddar cheeses to which strains of Lb. casei, Lb. plantarum and Lb. casei ssp. pseudoplantarum were added. The individual amino acid profiles at both time points reflect the results obtained using the Cd-ninhydrin method for total FAA determination, i.e. higher levels of individual FAA in experimental cheeses A, B and D than in the control and experimental cheese C. RP-HPLC of the WSEs from 3 or 6 month-old cheeses (Fig. 7 and Fig. 8,
C. M. Lynch et al.
862 1..5-
1.0 -
F
2
0.5 -
1
0
200
Time (days)
Fig. 5. Development of total free amino acids in Cheddar cheeses (trial 2) made under controlled microbiological conditions without (A) or with strains of Lb. pluntarum (0), Lb. casei subsp. pseudoplantarum (o), Lb. casei subsp. casei (0) or Lb. curvatus (m) as adjunct.
respectively) showed essentially no quantitative or qualitative differences between the control and experimental cheeses during ripening, although at 3 months, some peptides (eluting between 60 and 70 min) were detected in the hydrophobic region of the chromatogram of the control in trial 1 (not shown) which were not present in the chromatograms of any of the experimental cheeses. This feature was observed only in trial 1 and not in trial 2 at this time. Broome et al. (1990) also found only minor differences in the HPLC elution profiles of WSEs from control cheeses and cheeses containing an adjunct of Lb. cusei, even after 48 weeks of ripening.
CONCLUSIONS Controlled conditions with respect to the growth of non-starter lactobacilli during ripening were maintained so that the influence of selected species of mesophilic lactobacilli on Cheddar cheese ripening could be assessed. In agreement with other workers, proteolysis during cheese ripening was affected by the adjuncts mainly at the level of FAA formation. Mesophilic lactobacilli have been shown to have an extensive peptidase system (e.g. El Soda et al., 1978). Elevated levels of FAA in the experimental cheeses probably arose from peptidase activity by the adjunct lactobacilli. Cell death and autolysis during ripening (accompanied by the release of intracellular peptidases) would obviously increase peptidase activity and this may have occurred to some
863
Influence of adjunct lactobacilli on Cheddar cheese ripening
n Control Ei 0
q 0
Cys
Asp Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
VatA Vat B VatC Vat D
His
Lys
Arg
Csy
Arg
Csy
n Control El 0 E 0
Cys Asp
Thr
Ser
Glu
Pro
Gly
Ala
Val
Met
Ile
Leu
Tyr
Phe
Vat Vat Vat Vat
His
A B C D
Lys
Fig. 6. Concentrations of individual free amino acids in 3 month-old (a) and 6 month-old (b) Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (Vat A), Lb. casei subsp. pseudoplantarum (Vat B), Lb. casei subsp. casei (Vat C) and Lb. curvatus (Vat D) as adjunct.
extent in cheeses containing pseudoplantarum (Vat B).
strains of Lb. plantarum
(Vat A) and Lb. casei ssp.
The importance of FAA in relation to flavour development in Cheddar cheese is well recognised. FAA contribute to cheese flavour either directly or by acting as precursors for the formation of cheese flavour compounds by enzymatic and non-enzymatic reactions (Engels & Visser, 1994). Results shown here demonstrate that adjunct cultures of mesophilic lactobacilli increase the concentration
864
C. M. Lynch
et al.
0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02
Minutes
3 (
0.22 0.20 0.18 0.16 0.14 0.12 0.10 0.08 0.06 0.04 0.02 0
10
20
30
40
50
60
70
0
10
20
0.220.20: 0.18 0.16. 0.14
0.22 0.20 0.18 0.16 0.14 3 0.12 < 0.10 0.08 0.06 0.04 0.02 0
30
40
50
60
7;
50
60
7(
Minutes
Minutes (cj)
0.08’ 0.06
10
20
30
40
50
60
70
0
Minutes
10
20
30
40
Minutes
Fig. 7. Reverse-phase HPLC chromatograms of water-soluble extracts from 3 month-old Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (a), Lb. casei subsp. pseudoplantarum (b), Lb. casei subsp. casei (c) or Lb. curvatus (d) as adjunct. AU, absorbance units.
of FAAs in Cheddar cheese during ripening. Sensory data also suggest a general improvement in cheese quality by the use of these adjuncts, in particular, Lb. plantarum and Lb. casei ssp. pseudoplantarum. These findings warrant further investigation of the effect of these two species on the sensory properties of Cheddar cheese.
ACKNOWLEDGEMENTS
The technical assistance of Mr Eugene O’Connor and the financial support of FORBAIRT are gratefully acknowledged. This research project was part funded from EU structural funds under the food sub-programme of the industry programme.
865
Influence of adjunct lactobacilli on Cheddar cheese ripening
lo
0
20
30
40
50
60
70
Minutes 0.22.
(a)
;:;; 0.16
I
0.14. 3 <
0.12. 0.10. 0.08: 0.06. 0.04. 0.02 0
IO
20
30
40
50
60
70
Minutes
Minutes
J
0
IO
20
30
40
50
60
70
0
IO
20
30
40
50
60
7(
Minutes
Minutes
Fig. 8. Reverse-phase HPLC chromatograms of water-soluble extracts from 6 month-old Cheddar cheeses (trial 2) made under controlled microbiological conditions without (control) or with strains of Lb. plantarum (a), Lb. casei subsp. pseudoplantarum (b), Lb. casei subsp. casei (c) or Lb. curvatus (d) as adjunct. AU, absorbance units.
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