Ripening and quality of Swiss-type cheese made from raw, pasteurized or microfiltered milk

hr. Dairy Journal 7 (1997) 311-323 CC“1997 Elsevier Science Ltd PII:

ELSEVIER

All rights reserved. Printed in Great Britain 09X6946/97 $17.00+0.00

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Ripening and Quality of Swiss-type Cheese Made from Raw, Pasteurized or Microfiltered Milk Eric Beuvier a*, Karine Berthaud ‘, Sophie Cegarra ‘, Andri Dasen a, Sylvie Pochet II, Solange Buchin” and Gabriel Duboz” uInstitut National de la Recherche Agronomique, Station de Recherches en Technologie et Analyses Laititfres. BP 89, 39801 Poligny Cedex, France hI.S.A. R. A., 3 1 pl. Bellecow, 69002 Lyon, France (Received

13 January

1997; accepted

7 April 1997)

ABSTRACT Experimental mini-cheeses were made from raw (Ra), microfiltered (MF), pasteurized (Pa) (72°C 30 s) or pasteurized mixed with microfiltration retentate (PR) milk to study the influence of the indigenous microflora and pasteurization on the quality of Swisstype cheese. To estimate biochemical transformations during cheese ripening, several methods were used: nitrogen fractionation (water-soluble fraction and phosphotungstic acid (PTA)-soluble fraction), urea-polyacrylamide gel electrophoresis of caseins, reverse phase liquid chromatography of the water-soluble fraction, lactate and volatile fatty acids. Microbial populations were also enumerated. At the end of ripening, in comparison with MF and Pa milk cheeses, Ra and PR milk cheeses exhibited higher overall aroma intensit and pungency, characteristics which correlated with higher populations of facultatively heterofermentative sv -I (lO*cfu gg’), and enterococci ( lo6 cfu g ‘). These cheeses had high levels of PTA-soluble lactobacilli (10 cfu g ), propionibacteria N and acetic, propionic and isovaleric acids. MF and Pa milk cheeses, although somewhat different from one another, were very different from the two other types of cheese. Pa milk cheese had a lower pH than MF milk cheese, and contained a higher proportion of y-caseins due to the activation of plasmin. Moreover, Pa milk cheese was more acidic, but demonstrated a higher overall aroma intensity. The addition of raw milk flora (retentate) to Pa milk restored almost all the biochemical and sensory characteristics of Ra milk cheese measured in this study. @> 1997 Elsevier Science Ltd. All rights reserved

Keywords:

raw milk microflora;

pasteurization;

microliltration;

cheese ripening

Swiss-type cheeses are made either from raw, thermized or pasteurized milk, depending on the type of manufacture and the country. Generally, raw milk cheese develops a more intense flavour than pasteurized milk cheese due, in part, to higher concentrations of amino acids, fatty acids or volatile compounds (Scarpellino and Kosikowski, 1962; Price and Call, 1969; McSweeney et al., 1993). Little information is available concerning the nature of the raw milk flora responsible for the differences between raw and pasteurized milk cheeses. Nevertheless, McSweeney et al. (1993) observed that non-starter lactic acid bacteria (NSLAB), such as mesophilic lactobacilli, were more important and diversified in raw milk Cheddar cheese than in pasteurized milk Cheddar. Several authors have described the influence of pasteurization on the quality of cheese, but mainly in the case of Cheddar. However, they did not agree upon the cause of changes produced by pasteurization. Indeed, Sherwood (1936) and Lau et al. (1991) attributed the differences in proteolysis observed between raw and pasteurized milk cheese to the chemical alteration of the composition of milk by heat treatment; heat-induced interactions of whey proteins with the casein micelles were primarly involved. However, the microflora of the cheeses was not investigated in either of these studies. On the other

INTRODUCTION Different Swiss-type cheeses are manufactured in many countries, including Comte, Emmental, Gruylre, Appenzeller, Maasdamer and Jarbergost. Generally, they have ‘eyes’, varying in size, which result mainly from the propionic acid fermentation caused by propionic acid bacteria (Steffen et al., 1987). Typical Swiss-type cheese has a characteristic nut-like, sweet flavour, due to either free fatty acids, peptides, amino acids, interactions between these carbonyls or compounds (Paulsen et al., 1980; Griffith and Hammond, 1989). High scalding temperatures used in the manufacture of Swiss-type cheeses are necessary to adjust, particularly, the moisture content at the start of ripening, but they also have a selective effect on the microflora of the cheese (Mocquot, 1979). The starters used for Swiss-type cheeses contain thermophilic lactic acid bacteria such as Streptococcus thermophilus, Lactohacillus helveticus and Lb delbrueckii subsp. lactis (Mocquot, 1979). The propionic acid bacteria present in Swiss-type cheese are often added to the milk, but sometimes they are indigenous to the raw milk, as in Comte, Emmental ‘Grand cru’ and Swiss Gruyere cheeses. *Corresponding

author. 311

312

E. Beuvier et al.

hand, Kristoffersen (1985) claimed that the lack of flavour in cheese made from heat-treated milk in comparison to that made from raw milk was due only to heat-induced interactions of sulphur groups of milk proteins. This phenomenon, in relation to redox potential, would reduce the ability of the sulphur groups to accept hydrogen during the cheese fermentation process and adversely affect cheese flavour. In contrast, Gallmann (1982), Gaya et al. (1990) and McSweeney et al. (1993) underlined the essential role of the natural microflora, eliminated by pasteurization of milk, in cheese quality. To our knowledge, no comparison has been made between Swiss-type cheeses made from raw or pasteurized milk. Recently, Bouton and Grappin (1995) studied the role of the indigenous microflora of milk on the physico-chemical and sensory characteristics of Comte-type cheese by comparing mini-cheeses made from raw or microfiltered milk. Using different thermophilic starters, they observed that proteolysis was enhanced in raw milk cheese, demonstrating possible interactions between the raw milk flora and thermophilic starters. However, no microbiological analyses, except thermophilic lactic acid bacteria, were carried out on ripened cheeses. The originality of this work is the simultaneous application of two different milk treatments, pasteurization and microfiltration, in order to determine whether the biochemical and sensory differences observed between raw and pasteurized milk cheeses result mainly from the diminution in the natural milk flora, or from chemical, biochemical and enzymatic modifications induced by heat treatment of the milk.

MATERIALS AND METHODS Cheese manufacture Raw cows’ milk was totally skimmed and microfiltered using an ALFA-LAVAL MFSl crossflow microfiltration unit (pore size of ceramic membrane 1.4pm; membrane area 0.2 m2; flow rate 600 L hh’ rnp2, temperature 35°C). Cheeses were manufactured in four 12-L vats (Cardenas et al., 1991) according to a Swiss-type cheese technology (Bouton et al., 1993) using the following experimental design: -

-

-

raw milk (Ra milk): mixture of microfiltered skim milk, retentate and unpasteurized cream; microfiltered milk (MF milk): mixture of microfiltered skim milk and pasteurized cream (72°C 30 s); pasteurized milk (Pa milk): mixture of microfiltered skim milk and retentate. This mixture was then pasteurized (72°C 30s) and recombined with pasteurized cream (72°C 30 s); ‘pasteurized + retentate’ milk (PR milk): microtiltered skim milk was pasteurized (72°C 30s) and recombined with pasteurized cream (72°C 30s) and retentate.

Cheesemaking was performed in quadruplicate within a two-week period. The milk for each day (stored for 24 h at + 4°C) came from different cheese

plants close to the experimental plant, but the four treatments were applied to the same initial milk. All milks were microliltered in order to obtain the same physico-chemical composition for each treatment and to get the same level of total flora in Ra and PR milks. The total flora of retentate was rapidly estimated by the DEFT method (Direct Epifluorescent Filter Technique) (Dasen et al., 1987) and total flora in Ra and PR milks was adjusted to approximately 50,000 cfu mL_’ with an appropriate volume of retentate. Milk fat was standardized to 29-32 gL_’ with a fat/protein ratio of 1. Two strains of Streptococcus thermophilus and three strains of Lactobacillus helveticus were used as thermophilic starters. No propionibacteria were added to the milk. After waxing, cheeses were ripened for 3 weeks at 14°C and 9 weeks at 18°C.

Microbiological analyses The microbiological was assessed by microorganisms:

-

quality of the different milks enumerating the following

total bacteria on Plate Count Agar (PCA) medium (Difco, [Osi], Maurepas, France), incubated for 72 h at 30°C; thermoduric bacteria after heat treatment of milk, 63°C 30min, on PCA medium (Difco), incubated for 72 h at 30°C; psychrotrophic bacteria on PCA medium (Difco), incubated for 10days at 7°C; coliforms on Violet Red Bile Agar medium (Difco), incubated for 24 h at 30°C; Gram-negative bacteria on Nutrient Agar medium (Diagnostics Pasteur, Lyon, France) with crystalviolet (2mgL-‘), incubated for 72 h at 30°C (Piton, 1988); enterococci on Kanamycin Aesculin Azide agar medium (Fernandez de1 Pozo et al., 1988; Paleari et al., 1993; Rodriguez Medina et al., 1995) incubated for 48 h at 37°C; Micrococcaceae (micrococci and staphylococci) on Mannitol Salt Agar medium (Difco), incubated for 72 h at 30°C; facultatively heterofermentative lactobacilli on Facultative Heterofermentativen agar medium, incubated for 72 h at 38°C (Isolini et al., 1990; Demarigny et al., 1996). This medium is characterized by the use of vancomycin (50mg L-i), and mannitol as the only source of carbohydrate; thermophilic lactobacilli on de Man-RogosaSharpe medium (Difco); incubated for 72 h at 42°C (De Man et al., 1960); thermophilic streptococci on M 17 medium; incubated for 48 h at 42°C (Terzaghi and Sandine, 1975); propionic acid bacteria on Sodium Lactate Agar Cloxacillin medium, incubated for 4 and 7 days at 30°C (Drinan and Cogan, 1992); butyric acid bacteria on Bryant and Burkey modified Berg&e broth (Biokar, Beauvais,

Ripening and quality of Swiss-type cheese

~

France), incubated for 7 days at 37°C; results were obtained using the most probable number technique with five tubes for each dilution (CERNA, 1986); yeast on Yeast Extract Glucose Agar medium with chloramphenicol (0.1 g L-l), incubated for 3 days at 25°C.

The above-mentioned microorganisms, except total flora, thermoduric and psychrotrophic bacteria, were counted throughout the ripening period at 1 day, 3, 6 and 12 weeks. Ten grams of a cheese sample were dissolved in 50mL of a trisodium citrate solution (4% w/v) and adjusted to a final concentration of l/l0 (w/ w) by adding sterile deionized water. All microbiological analyses were performed in duplicate using a SPIRAL plater (Interscience, St Nom la Breteche, France), except for the determination of butyric acid bacteria which was carried out in tubes. The Spiral system allowed a detection limit of 10 cfu mL-’ in milk and 100 cfu gg ’ in cheese. Chemical analyses of cheese Samples of cheese were collected at three periods of ripening: 1 day, 3 and 12 weeks. They were subsequently wrapped in strong sulphurized aluminium foil and stored at -20°C before being analysed. The pH of the cheese was determined by placing an electrode within a cylinder packed with grated cheese; the pH value was recorded within 304.5 s. Dry matter (DM) content (g lOOg_’ cheese) was determined according to a simplified procedure of the FIL-IDF (FIL-IDF, 1982) and moisture (M) content was calculated as lOO-DM (g lOOg-’ cheese). Fat (F) content (g lOOg-’ cheese) was determined by the method of Heiss (1961) as modified by Pien (1976). Non-Fat Dry Matter (NFDM) was calculated as DM-F (g lOOg-’ cheese). The ratio M/NFDM was then calculated. NaCl content was determined using a chloride analyser (Model 926, Corning), and expressed as g 100 gg ’ moisture. Assessment of proteolysis Total nitrogen was determined on grated cheese by the Kjeldhal method according to the semi-micro procedure (FIL-IDF, 1993). The water-soluble fraction (WSF) was prepared according to the modified method of Kuchroo and Fox (1982) as described by Bouton et al. (1994). The phosphotungstic acid-soluble fraction was prepared from WSF, according to Gripon et al. (1975). The nitrogen content of these fractions was determined as previously described, and water-soluble nitrogen (WSN) and phosphotungstic acid-soluble nitrogen (PTASN) were expressed as a percentage of total N. The water-insoluble fraction, representing intact caseins and large casein peptides produced by proteolysis, was recovered during the water-soluble extraction and freeze-dried. It was then analysed by urea-polyacrylamide gel electrophoresis (PAGE) (Protean II xi, Biorad, Ivry sur Seine, France) according to the method of Andrews (1983). Staining was for 16 h in 0.2% Coomassie blue G250 in 50%

313

sulphuric acid 2N containing 12% TCA (Blakesley and Boezi, 1977). This was followed by three washes in deionized water for 30 min, 6 h and 18 h, respectively. Optical density of the bands was recorded as a profile using a densitometer (Hoefer Scientific Inst., San Francisco, USA); the area of each peak was integrated an HSI programme and expressed as a using percentage of the total area. Peptides in the WSF were separated by reverse phase-high performance liquid chromatography (RPHPLC) (Varian LC5000, Les Ulis, France; automatic sample injector: Kontron 456, St Quentin en Yvelines, France). Peptides were eluted at 37°C and at a flow rate of 0.8 mLmin-’ from a 4.6x225mm column packed with Cl 8-bonded silica gel (NucleosilTM, porosity 3000nm, particle diameter 5pm). A gradient of two solvents (solvent A: 0.105% (v/v) trifluoroacetic acid (TFA) in ultra-high quality water (uhqw); solvent B: 0.1% TFA in acetonitrile/uhqw (60/40, v/v) was used, as follows: 100% A for 5min, linearly to 100% B over lOOmin, then 100% B for 5 min and finally back to the initial conditions. All solvents and samples were filtered through 0.45pm filters. Absorbance of the effluate was monitored at 214 nm using a UV detector (Varian UVlOO, Les Ulis, France) interfaced with COCONUT software (Almanza and Mielle, INRA Dijon, France). Peptides maps were visually compared to detect differences in the profiles. WSN and PTASN analyses were carried out at three stages of ripening (1 day, 3 and 12 weeks) and PAGE and RP-HPLC analyses at the end of ripening. Plasmin activity and plasminogen content were measured in cheeses at the beginning of ripening (1 day) according to the method of Song et al. (1993). Results were expressed in pmol p-nitroaniline released hh’ gg ’ of cheese using a standard curve for pnitroaniline. Analyses of lactates and volatile fatty acids L( +) and D(-) Lactate (mg 100 gg’ cheese) were determined on the WSF at three stages of ripening (1 day, 3 and 12 weeks) by an enzymatic method using the Boehringer Mannheim kit (Meylan, France). Volatile fatty acids, C2 to C6 (mg 100 gg’ cheese), were determined at the end of ripening by gas chromatography according to the method of Bouton et al. (1994).

Sensory assessment At the end of ripening, cheeses were evaluated by a trained panel of 12 assessors, who were members of the Institut National de la Recherche Agronomique staff. A &lo scale was used, considering nine criteria including taste (saltiness, acidity, bitterness), aroma (overall aroma intensity), pungency, the occurrence of off-flavours, and texture (elasticity, firmness and granular character). Statistical

analysis

Statistical analyses were carried out using Stat-ITCF software (version 5, 1991) from the Institut Technique des Cereales et des Fourrages (Paris, France). The

314

E. Beuvier et al.

effect of each milk treatment was assessed by an analysis of variance of the results (averages of four replicates). When a significant difference (p < 0.05) was observed between the treatments, the Newman-Keuls test was applied to allow constitution of homogeneous groups. A principal component analysis showed the relationship between microbiological and physicochemical characteristics, and organoleptic characteristics of the cheeses.

of ripening (about 6x 108cfug-‘), and no significant difference was observed between the cheeses. During the first period of cheese ripening (14°C) thermophilic streptococci counts decreased by 0.6 to 1.61og, and thermophilic lactobacilli by 2.2 to 2.91og, depending on the treatment applied to the milk. The decrease of thermophilic lactobacilli was greater for MF and Pa milk cheeses than for Ra and PR milk cheeses (p < 0.05). After 6 weeks of ripening, the number of thermophilic streptococci was significantly lower (p < 0.001) in MF and Pa milk cheeses than in Ra and PR milk cheeses. No significant difference was detected in thermophilic lactic acid bacteria counts at the end of cheese ripening. Facultatively heterofermentative lactobacilli were lower than lo4 cfu gg’ in Ra and PR milk cheeses before ripening, and reached more than 107cfug-1 after the first eriod of ripening at 14°C and increased further to 10Pcfug -1 during the warm room period. Approximately 100 cfu g-’ of facultatively heterofermentative lactobacilli were detected at the beginning of ripening of MF milk cheese. They grew rapidly at 14°C reaching about lo’cfug-’ and then 107cfug-’ at the end of ripening. On the other hand, they grew more slowly in Pa milk cheese. Propionibacteria were determined only during the warm room period. They reached maximum numbers at the end of ripening, 10’ cfu g-’ in Ra and PR milk cheeses, 1O7cfu g-i in MF milk cheese (difference nonsignificant). However, after 6 weeks of ripening, the population of propionibacteria was lower (p < 0.001) in MF milk cheese (2.3x 105cfug-1) than in Ra and PR milk cheeses (on average, 3.7x 106cfug-‘). In Pa milk cheese, their number remained at a low level (less than 104cfu g-l) throughout the warm room period. At the beginning of ripening, the number of enterococci was higher than facultatively heterofermentative lactobacilli in Ra and PR milk cheeses ( > lo4 cfu g-l), but enterococci increased more slowly at 14°C reaching only 105cfug-‘. In MF and Pa milk cheeses, enterococci remained at a level 10 to lOOO-fold lower than in Ra and PR milk cheeses throughout ripening. Again, as for FH lactobacilli,

RESULTS Microbiological

analysis

Milk

The bacterial population of different cheese milks is shown in Table 1. The mean total flora of Ra and PR milks were, respectively, 46,000 and 42,000 cfu mL-‘, close to the desired value (50,000 cfu mL_‘), and varied from 26,000 to 53,000 cfumL_‘. In addition, the composition of the microflora was found to be similar; psychrotrophic and thermoduric bacteria, the most important groups, represented 50 and 20% of the total flora, respectively. Microfiltration was more efficient than pasteurization in reducing the bacterial population (99 and 88%, respectively). The main difference between the microflora of these two milks was that Pa milk had a more homogeneous bacterial population and was composed primarly of thermoduric bacteria (90%). Coliforms, facultatively heterofermentative lactobacilli, enterococci and yeast counts were less than 1Ocfu mL_’ in both MF and Pa milks. None or only a few propionic acid bacteria were found in MF and Pa milks. Finally, no butyric acid bacteria were counted in any milk. Cheese The quantitative evolution of the dominant microbial groups throughout cheese ripening is described in Table 2. Thermophilic lactic acid bacteria, enumerated on M 17 and MRS media, represented the major flora at the beginning

Table 1.

Bacterial

Counts

(cfumL_‘)

in Ra, MF, Pa and PR Cheese Milk? Treatmentb

Ra Total flora Psychrotrophic bacteria Thermoduric bacteria Coliforms Gram-negative bacteria Enterococci Micrococcaceae FH lactobacilliC Thermophilic lactobacilli Thermophilic streptococci Propionic acid bacteria Butyric acid bacteria Yeast

MF

PR

Pa

46,000 21,000 9800 320 1700 390 3300 280 3000

440 630 240 < 10 30 < 10 30 < 10 30

5700 < 10 5000 < 10 20 < 10 < 10 < 10 < 10

42,000 21,000 8100 420 1600 290 3000 230 2900

8500

40

60

7600

4300 co.18 1200

10 CO.18 < 10

< 10 <0.18 < 10

3000 CO.18 1000

a Arithmetic mean of four replicates. b Ra: raw milk cheese; MF: microfiltered milk cheese; Pa: pasteurized ’ Facultatively heterofermentative lactobacilli.

milk cheese; PR: ‘pasteurized

+ retentate’

milk cheese.

315

Ripening and quality of Swiss-type cheese Table 2.

Counts”

(log cfug-I)

Microflora

Ripening

Thermo. streptococci

of Microflora

FH lactobacilli

Propionibacteria

Enterococci

Micrococcaceae

Ripening of Cheeses Made from Ra, MF, Pa or PR Milk (3 Weeks at 14°C and 9 Weeks at 18°C) Treatmentb

stages

Significance

Ra

MF

Pa

PR

8.38

8.36

8.45

8.52

NS

3 weeks 6 weeks 12 weeks 1 day

1.45 6.92A 6.87 8.74

7.09 6.12’ 5.68 8.40

6.68 5.72B 6.26 8.54

7.09 6.70A 6.68 8.57

NS ***

3 weeks 6 weeks 12 weeks 1 day 3 weeks 6 weeks 12 weeks 1 day 3 weeks 6 weeks 12 weeks I day 3 weeks 6 weeks 12 weeks 1 day 3 weeks 6 weeks 12 weeks

5.77AB 7.15 7.19 3&IA 7.02A 7.64A 8.09A ND ND 6.37A 7.85A 4.10A 4.80A 6.70A 6.20A 3.04 3.49 4.10c 4.85

4.87’ 6.50 6.12 2.00B 5.02’ 6.72’ 7.27A ND ND 5.36’ 7.03B 1.98” 2.15’ 5.40B 5.1gA 1.50 3.77 5.19A 5.27

4.84” 6.51 6.36 < 1.40B 2.83’ 5.63’ 6.46’ ND ND < 4.00c < 4.00c 2.10B < 1.40B 2.74’ 3.60B 1.22 2.67 4.77AB 5.15

6.06A 7.08 7.07 3.6gA 7.56A 8.15A 8.09A ND ND 6.76A 8.15A 4.30A 5.00A 7.01A 6.1gA 3.59 4.01 4.62’ 4.72

1 day

Thermo. lactopbacilli

During

level

NS NS

* NS NS *** *** *** ** I ***

*** *** *** *** ** NS

NS ** NS

‘Mean of four replicates. b Ra: raw milk cheese; MF: microtiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. ND: not determined. Significance level: non-significant (NS); significant p < 0.001 ( ***); significant p i 0.01 (**); significant p < 0.05 (*). Cheese classification (decreasing log cfu g-‘) in different groups noted as A, B and C according to Newman-Keuls test.

lower counts of enterococci were observed in Pa milk cheese than in MF milk cheese. Generally, Micrococcaceae were present at lower numbers than the other major microbial groups and no difference was observed between cheeses at the end of ripening. Gram-negative microorganisms and yeasts were present in low numbers (results not shown), whereas no coliform and butyric acid bacteria were detected throughout ripening.

difference was observed between measured variables except for the NaCl/moisture ratio, which was slightly higher in Pa and PR milk cheeses (p < 0.05). However, changes in pH during ripening differed between cheeses (Fig. I). At the end of ripening, Ra and PR milk cheeses had a significantly higher pH, 5.74, than MF milk cheese, 5.63, or Pa cheese, 5.57 (p < 0.001).

Physico-chemical

Significant differences between cheeses in the composition of the water-insoluble fraction were evident at the end of ripening (Table 4). Raw milk cheese contained higher amounts of B-caseins than the

analysis

The composition of the cheeses at the beginning of ripening is summarized in Table 3. No significant

Table 3.

Composition

of Swiss-type

Assessment of proteolysis

Cheese (1 Day Old) Made from Ra, MF, Pa or PR Milk” Treatmentb

Dry matter % Fat/dry matter % M/NFDM Ca/NFDM % PH S/M %

Significance

Ra

MF

Pa

63.60 (0.93) 48.82 (0.78) 1.12 (0.05) 3.12 (0.09) 5.31 (0.05) 1.73’ (0.08)

63.78 (0.59) 49.50 (0.90) 1.12 (0.06) 3.13 (0.06) 5.3 1 (0.07) 1.74B (0.06)

63.10 (1.13) 48.92 (0.48) I. 15 (0.04) 2.97 (0.06) 5.26 (0.03) 1.82A (0.07)

level

PR 63.64 (0.68) 49.40 (0.85) 1.13 (0.04) 3.05 (0.07) 5.29 (0.03) 1.87A (0.09)

NS NS NS NS NS *

‘Mean of four replicates (standard deviation). b Ra: raw milk cheese; MF: microfiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. M/NFDM: Moisture/Non-Fat Dry Matter; S/M: Salt/Moisture. Significance level: non-significant (NS); significant p < 0.05 (*). Cheese classification (decreasing %) in different groups noted as A and B according to Newman-Keuls test.

E. Beuvier et al.

316 5.8,

5.21 0

peptide profiles of cheeses at the end of ripening (Fig. 2). Cheeses made from Ra and PR milks contained lower concentrations of peptides, which eluted between 3000 and 3600s (region of medium hydrophobicity), than MF and Pa milk cheeses. Moreover, these latter cheeses were characterized by the occurrence of a larger peak with an elution time of 3200 s (indicated by an arrow). These results were obtained with all four replicates. Since the PTASN content, composed of very small peptides (ten residues maximum) and amino acids, was higher in Ra and PR milk cheeses, it may be supposed that the peptides of medium hydrophobicity were further degraded by enzymes of raw milk microflora.

I

:

: 20

:

I

:

40

:

:

80

: 80

:

I 100

Days

Fig. 1. Changes in pH throughout ripening of cheeses made from raw milk cheese (o), microtiltered milk cheese (o), pasteurized milk cheese (V) or ‘pasteurized + retentate’ milk cheese (m) (mean of four replicates).

Lactate metabolism and production of volatile fatty acids

Because other cheeses, whereas y-caseins, resulting from the degradation of /I-caseins by plasmin, were found at higher concentrations in heated milk cheeses, reflecting higher plasmin activity in these cheeses (Table 5). The greater degradation of /3-caseins in MF milk cheese did not lead to a higher concentration of y-caseins and the activity of plasmin was the same as in Ra milk cheese. Breakdown of a,i-casein was slower in cheeses made from heat-treated milk (Pa and PR). However, CQ_~casein (cI,, f24-199) was not statistically different between cheeses. WSN in cheeses increased throughout ripening, but most rapidly in the warm room (Table 4). No difference was observed except for a slightly, but significantly, higher content (pcO.05) in PR milk cheese at the end of ripening (19.5% versus a mean of 18.3% for the other cheeses). The PTASN content was 30% higher (p ~0.01) in lZweek-old cheeses with a raw milk flora (Ra and PR). Marked variations were observed between the Table 4.

fl-CN

Total y-CN

WSN/TN

PTAN/TN

nature

of

the

starters

(Str.

lactate was found at a higher concentration lactate at the beginning of ripening (Fig. 3). After 12 weeks of ripening, the concentration of L( +) lactate was significantly lower (p
Treatmentb

Ripening stages Ra

cc,,.t-CN

the

Evolution of Principal Caseins (Expressed as Percent of Total Area of the Peaks) and Soluble Nitrogen (Expressed as a Percent of Total N) at Three Stages of Ripening in Ra, MF, Pa or PR Milk Cheesea

N fractions

c(,t-CN

of

thermophilus and Lb helveticus strains),

1 day 3 weeks 12 weeks 1 day 3 weeks 12 weeks 1 day 3 weeks 12 weeks 1 day 3 weeks 12 weeks 1 day 3 weeks 12 weeks 1 day 3 weeks 12 weeks

MF

27.1 (2.8) 25.5 (3.5) 17.0A (0.5) 8.6 (1.4) 9.9 (0.9) 19.4B (0.9) 37.9 (4.8) 34.0 (3.6) 15.4B (2.4)

25.5 (4.4) 24.9 (3.1) 15.0B (0.8) 9.3 (1.7) 10.2 (0.7) 19.4B (1.0) 36.2 (4.6) 35.8 (2.1) 16.0’ (1.3)

4.Yg.5) 14.3 (0.8) 3.1 (0.2) 6.3 (0.4) 18.3B (0.5) 0.71 (0.1) 1.19 (0.1) 4.gA (0.2)

4.:; .2) 16.6 (0.7) 3.2 (0.1) 6.4 (0.2) 18.1’ (0.2) 0.54 (0.2) 0.91 (0.2) 3.9B (0.4)

Significance level Pa 27.1 (3.1) 24.0 (3.9) 14.0B (0.4) 7.3 (0.8) 11.5 (1.6) 21.gA (1.5) 36.5 (4.6) 34.7 (2.9) 19.0A (0.5) ND 4.0 (1.9) 13.3 (2.0) 3.3 (0.2) 6.5 (0.3) 18.6B (0.4) 0.54 (0.2) 0.97 (0.3) 3.9B (0.5)

PR 25.6 (5.1) 23.5 (4.8) 14.2B (1.1) 10.6 (3.5) 11.6 (3.2) 22.5* (1.3) 36.2 (4.7) 35.1 (4.4) 18.1A (1.3) ND 4.3 (1.5) 12.6 (0.9) 3.1 (0.1) 6.5 (0.4) 19sA (0.9) 0.58 (0.1) 1.12 (0.1) 5.2A (0.6)

NS

NS ** NS NS * NS NS *

ND NS NS NS NS * NS NS **

aMean of four replicates (standard deviation). b Ra: raw milk cheese; MF: microtiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. ND, not determined. Significance level: non-significant (NS); significant p < 0.01 (**); significant p < 0.05 (*). Cheese classification (decreasing % N content and % caseins) in different groups noted as A and B according to Newman-Keuls test.

Ripening and qualit?: c~f Swiss-type Table 5.

Plasmin

Activity

and Plasminogen Content P-Nitroanilide

317

chersc>

in Cheeses at the Beginning of Ripening Released h-’ g-’ of Cheese)”

(Results Expressed in /Lmoi Significance level

Treatmentb Ra Plasmin activity Plasminogen

MF

501B (43) X37 (31)

495’ (42) X69 (42)

Pa

PR

592A (26) x53 (43)

575A (65) 886 (101)

**

NS

content ‘Mean of four replicates (standard deviation). ’ Ra: raw milk cheese; MF: microfiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. Significance level: non-significant (NS); significant p < 0.01 (**). Cheese classification (decreasing plasmin activity) in different groups

noted as A and B according

Table 6.

to Newman--Keuls

Free Fatty Acids Composition

test.

(Expressed

in mg lOOg_’ Cheese) of Cheeses at the end of Ripening”

Treatmentb Ra Acetic acid Propionic acid Butyric acid lsovaleric acid Caproic acid Total volatile fatty acids

173.3A (39.8) 197.5* (89.7) 8.5 (1.2) 1.4” (0.2) 3.1 (0.8) 383.5* (125.7)

MF 61.6’ (50.7) 22.7’ (53.4) 9.7 (2.5) o.4B (0.1) 4.0 (1.3) 98.8B (92.9)

Significance Pa

PR

37.3B (26.4) 1.1” (72.5) 9.9 (3.1) o.2” (0.4) 3.4 (1.1) 54.0’ (95.2)

187.9A (35.6) 247.0A (62.9) 7.2 (0.7) l.5A (0.3) 2.3 (1.1) 446.0A (83.7)

** ** NS *** NS **

‘Mean of four replicates (standard deviation). ’ Ra: raw milk cheese; MF: microfiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. Significance level: non-significant (NS); significant p < 0.01 (**); significant p < 0.001 ( ***). Cheese classification (decreasing fatty acids concentrations) in different groups noted as A and B according to Newman-Keuls test.

of propionic acid bacteria (r = + 0.73 and Y= + 0.71). In these cheeses, the proportion of isovaleric acid was higher @ < 0.001). When the indigenous microflora was removed (MF and Pa milk cheeses), the quantity, as well as the composition, of volatile fatty acids was affected greatly. In these cheeses, acetic acid tended to predominate ( > 60%) whereas propionic acid tended to be at the same level as butyric acid. Concentrations of butyric and caproic acids were not affected by any of the treatments. Sensory analysis Sensory characteristics of cheeses were evaluated at the end of ripening (Table 7). In general, no offflavours were found in any of the cheeses and the sensory profile was different for each treatment. Cheeses made with a raw milk flora (Ra and PR) showed a higher overall aroma intensity and pungency. Acidity and bitterness were predominant in cheeses made from pasteurized milks (Pa and PR). MF milk cheese had the lowest scores for saltiness, overall aroma intensity and pungency. Raw milk cheese received the highest scores for firmness and granular character. The values for elasticity were not affected by any of the treatments. Relationships between sensory characteristics and biochemical and microbiological composition of cheeses The relationships between organoleptic and physico-chemical and microbiological characteristics of cheeses were analysed via a principal component analysis. Physico-chemical and microbiological

level

free

characteristics, which were found to be the most discriminating between treatments, were used as active variables (i.e. pH, NaCl, acetic, propionic and isovaleric acids, PTASN, propionibacteria, facultatively heterofermentative lactobacilli and enterococci) with sensory criteria as additional variables (Fig. 4). Most of the variables were well correlated to axis 1 (66% of the variance) which seems to describe the intensity of microbial activity, except NaCl content, acid and elasticity characteristics to axis 2 (16% of the variance). The propionic acid fermentation (production of acetic and propionic acids), and proteolysis variables (PTASN and isovaleric acid) were positively correlated to the overall aroma intensity and pungency of cheeses (+ 0.79~~~ +0.52). On the other hand, PTASN and isovaleric acid were positively correlated to propionibacteria, facultatively heterofermentative lactobacilli and enterococci (+ 0.73>r> + 0.62). A negative correlation was observed between isovaleric acid and the elasticity of cheeses (r = -0.54, p < 0.05). Microbial flora (propionibacteria, facultatively heterofermentative lactobacilli and enterococci) were positively correlated to the pungent characteristic (Y= + 0.51, p < 0.05), and negatively to the acidity of the cheeses (r= -0.52, p
318

E.Baker et al.

TIME

s; signal expressed in UVZI~~~ Fig. 2. RP-HPLC profiles ofthe water-soluble fraction ofcheeses at the end of ripening. Timeexpressedin arbitrary units. (a) Raw milk cheese; (b) ‘pasteurized + retentate’ milk cheese; (c) microfiltered milk cheese; (d) pasteurized milk cheese.

319

Ripening and quality of Swiss-type cheese

Fig. 2.

with raw milk flora added (Ra and PR milk cheeses) differed from the other cheeses (MF and Pa milk cheeses) in terms of higher propionic acid fermentation and more extensive proteolysis, and stronger aromas and less pronounced acidity and bitterness. In addition, it is interesting to note that Ra and PR milk cheeses

(a)[

600 --

T :

4004 0

:

:

20

:

:

:

40

:

60

\I :

:

80

Con timed.

were somewhat different was added to Pa milk.

DISCUSSION At the beginning of ripening, the microflora of Swisstype cheese is composed mainly of thermophilic lactic acid bacteria, log-109cfugp’ (Accolas et al., 1978). In our study, we observed the same level of thermophilic lactic acid bacteria regardless of the treatment applied to the milk. Surprisingly, following a decrease, the number of thermophilic lactobacilli increased again after 3 weeks of ripening. Bouton et al. (1996) also found that mesophilic lactobacilli isolated from Comtk cheese, such as Lactobacillus paracasei and L. rhamnosus, are able to grow on MRS incubated at

I 100

Axis 2

Days

N&l

,id

250 2001 0

: 20

:

: 40

:

I 60

:

: 80

:

I 100

Days

Fig. 3. Changes in L( +) lactate (a) and D(-) lactate (bj during ripening of cheeses made from raw (o), microfiltered (oj,pasteur.ifed (V) or ‘pasteurized + retentate’ (W) milk (mean of tour rephcates).

....

1

ris

Principal component analysis of physico-chemical, microbiological and sensory measurements of cheeses made from raw, microfiltered, pasteurized or ‘pasteurized + retentate’ milk. C2: acetic acid; C3: propionic acid; iC5: isovaleric acid; PTA: PTA-soluble N; propio: propionibacteria; FhLb: facultatively heterofermentative lactobacilli; Entero: enterococci; aroma: overall aroma intensity; pung: pungent; gran: granular; elas: elastic. Only active variables are underlined. Additional variables are in italic and indicated by a dotted Fig. 4.

:

..

bil

(b)]

Z

even when the raw milk flora

line

E. Beuvier et al.

320 Table 7.

Sensory Analysis of Cheeses at the End of Ripening” Treatmentb

Ra Saltiness Acidity Bitterness Overall aroma intensity Pungency Elasticity Firmness Granular character

MF

Significance level Pa

PR

3sB (0.09) l.@ (0.01) 1.2B (0.21) 3.9B (0.23)

3.0c (0.14) 2.0BC (0.18) 1.6AB (0.29) 3.0c (0.18)

3.sB (0.21) 2.4A (0.08) 1.9A (0.19) 3.6B (0.24)

4.0A (0.19) 2.2AB (0.20) 1.7A (0.09) 4.5A (0.23)

*** ** ** ***

1.7B (0.37) 4.4 (0.13) 5.5* (0.32) 4.5A (0.16)

0.8’ (0.14) 4.6 (0.15) 5.2B (0.17) 3.9B (0.25)

l.lc (0.28) 4.2 (0.21) 4.4’ (0.42) 3.6’ (0.44)

2.0A (0.15) 4.3 (0.15) 4.9c (0.15) 3.7B (0.28)

*** NS *** ***

LMean of four replicates (standard deviation); scale: 0 to 10. Ra: raw milk cheese; MF: microtiltered milk cheese; Pa: pasteurized milk cheese; PR: ‘pasteurized + retentate’ milk cheese. Significance level: non-significant (NS); significant p < 0.01 (**); significant p < 0.001 (***). Cheese classification (decreasing sensory notes) in different groups noted as A, B and C according to Newman-Keuls test.

42°C. Generally, throughout ripening, high numbers ( 108-lo9 cfu g-i) of propionibacteria and adventitious facultatively heterofermentative lactobacilli, composed of Lactobaciilus paracasei, L. plantarum and L. rhamnosus species (Demarigny et al., 1996), have been reported in Swiss-type cheese (Veaux et al., 1974; Gilles et al., 1983). Enterococci have also been enumerated in Swiss-type cheese but at lower numbers, 106-lo* cfu gg’ (Veaux et al., 1974; Demarigny et al., 1996). The level of populations we observed for these groups of bacteria in Ra and PR milk cheeses agree with these results. As previously found by McSweeney et al. (1993) and Demarigny et al. (1996) pasteurization and microfiltration delayed the growth of facultatively heterofermentative lactobacilli, propionibacteria and enterococci in cheeses. The counts of these three bacterial populations were low or not detected in MF and Pa milks. In agreement with Martley and Crow (1993) their presence in MF and Pa milk cheeses may have been the result of contamination during cheese fabrication. In contrast, the origin of facultatively heterofermentative lactobacilli, propionibacteria and enterococci in Ra and PR milk cheeses was most probably the raw milk. Furthermore, it is worth noting that in this study, MF and Pa milk cheeses differed with respect to microbial counts. The main conditions that regulate the rate and extent of growth of microorganisms, i.e. moisture content, pH and ripening temperature (Martley and Crow, 1993), were similar at the start of ripening for both MF and Pa milk cheeses. It is unlikely that the difference in salt concentrations, 0.08% (S/M), between MF and Pa milk cheeses (within a total salt concentration slightly lower than 2%) affected the growth of bacteria. Moreover, no difference in bacterial counts was observed between Ra and PR milk cheeses with a similar difference in S/M (0.14%). Therefore, it would seem that pasteurization of milk, in contrast to microfiltration, delays the growth of microorganisms during cheese ripening. Another possible explanation could be that microorganisms remaining in Pa milk, despite the fact that they were more numerous than in MF milk, 5000 cfu mL_’ 500 cfu mL-r and respectively, developed poorly in cheese. The important role of the indigenous microflora of

milk in proteolysis has been emphasized in Manchego cheese (Gaya et al., 1990) in Cheddar cheese (McSweeney et al., 1993) and in a Swiss-type mini-cheese (Bouton and Grappin, 1995). In this study, Ra and PR milk cheeses containing higher levels of facultatively heterofermentative lactobacilli, propionibacteria and enterococci throughout ripening had higher amounts of PTASN, showing a more extensive proteolysis. Even if similar counts of facultatively heterofermentative lactobacilli and enterococci were observed in MF milk cheese, and Ra and PR milk cheeses at the end of ripening, the late growth of these bacteria in MF milk cheese most likely delayed biochemical transformations. According to Peterson and Marshall (1990) facultatively heterofermentative lactobacilli, as well as enterococci (Hokoma and Salle, 1958), have peptidase activities and could contribute to the increased proteolysis noted in Ra and PR milk cheeses. Moreover, the smaller RP-HPLC peaks observed in Ra and PR milk cheeses, in agreement with the results of Bouton and Grappin (1995) suggest increased aminopeptidase activity in cheeses made with a raw milk microflora. This is also supported by McSweeney et al. (1993) who found marked differences in peptide profiles between raw milk Cheddar cheese, and pasteurized and microfiltered milk Cheddar cheese. These authors attributed these differences to non-starter lactic acid bacteria composed mainly of facultatively heterofermentative lactobacilli (Lactabacillus casei, L. plantarum and L. curvatus species). On the other hand, the peptidase activities of dairy propionibacteria have been described by several authors: prolyl dipeptidyl aminopeptidase (El-Soda et al., 199 l), phenylalanine aminopeptidase (Dupuis, 1994) and dipeptidase activities (Ostlie et al., 1995).

Therefore, in our study, propionibacteria may also have contributed to the higher levels of PTASN measured in cheeses made with a raw milk flora. In addition, high levels of isovaleric acid were found in Ra and PR milk cheeses. It is known that this compound is formed by the catabolism of leucine (Baraud et al., 1970) and in this case, could be attributed to propionibacteria (Biede et al., 1979). MF and Pa milk cheeses were almost identical in terms of WSN and PTASN, peptide profiles, but their

Ripening and quality of Swiss-type cheese

urea-PAGE profiles were different. These results differ somewhat from those obtained in Cheddar cheese by McSweeney et al. (1993) who found no difference in breakdown between MF and primary casein pasteurized milk cheeses. It is known that b-casein is hydrolysed to y-caseins by indigenous plasmin and that pasteurization of milk increases plasmin activity due to inactivation of plasmin inhibitors and/or increases in the activity of plasminogen activators (Fox and Stepaniak, 1993; Farkye and Imalidon, 1995). The higher proportion of y-caseins and higher levels of plasmin found in pasteurized milk cheese confirm these results. Since similar levels of plasminogen were found at the beginning of ripening, in all cheeses pasteurization probably inactivated plasmin inhibitor rather than the inhibitors of plasminogen activators. Moreover, it is likely that the high % salt in moisture (4.555%) and low pH (5.10-5.20) in Cheddar cheese reduce the activity of plasmin (Delacroix-Buchet and Trossat, 1991; Fox and Stepaniak, 1993). Therefore, no effect of pasteurization on plasmin activity was observed in Cheddar cheese. These results suggest that the breakdown of rsland fl-caseins is highly dependent on cheese type. A low content of fl-casein in MF milk cheese was observed by Bouton and Grappin (1995) who suggested that plasmin could be activated due to the addition of pasteurized cream to MF skim milk. However, the results of our study, regarding the y-casein and plasmin content in MF milk cheese, suggest that it is unlikely that plasmin was activated. Another reason for the low [I-casein content could be that some of the [i’-casein micelles were retained by the microfiltration membrane, as suggested by Bouton and Grappin (1995). It is known that chymosin is denatured during Swisstype cheese processing (Delacroix-Buchet and Fournier. 1992; Igoshi and Arima, 1993). Therefore, the lower degradation of r,i-casein noted in Pa and PR milk cheeses in comparison to Ra and MF milk cheeses. could be attributed to the denaturation of the indigenous acid proteinase of milk by pasteurization. Indeed, according to Kaminogawa et al. (1980), this protease is able to degrade x,r-casein like chymosin. Moreover, we noted an increase in WSN in PR milk cheese due probably to the combined effect of the raw milk flora and plasmin. Moir (1930) found that Cheddar cheese made from pasteurized milk (74°C. 20s) but still containing a total bacterial count of 100,000 cfu mL -I, developed more soluble nitrogen than raw milk cheese. The role of propionibacteria in the ripening of Swiss cheese is known (Langsrud and Reinbold, 1973). They produce CO2 and propionic and acetic acids from L( + ) lactate in preference to D(-) lactate (Crow, 1986). Microfiltration and pasteurization, which dramatically reduced the number of propionibacteria in milk, led to a large reduction of the propionic acid fermentation in the cheeses. Bouton and Grappin (1995) found that Swiss-type cheese (4-month-old) made from MF milk had a lower concentration of propionic acid than cheese made with raw milk, 47mg and 252mg lOOg_’ of cheese, respectively. The increase of D(-) lactate in all cheeses after 3 weeks of ripening probably resulted from the presence in high numbers of facultatively heterofermentative lactobacilli, because these bacteria

321

possess a system for lactate racemization (Thomas and Crow, 1983). The concentrations of lactate, acetate and propionate expressed in mmollO0 gg ’ (results not shown) illustrated that a complex metabolism occurred in Ra and PR milk cheeses. However, the measurement of citrate, formate, succinate and aspartate involved in the metabolism of acetate and propionate (Crow and Turner, 1986; Davies et al., 1994) was not carried out in this study. Therefore, conclusions concerning the metabolic pathways involved in acetate and propionate production could not be reached. By removal of the natural flora of raw milk by pasteurization or microfiltration, this study shows its essential role on the flavour of Swiss-type cheese. Cheeses with the raw milk microflora showed a higher overall aroma intensity and pungency due, in part, to higher amounts of PTASN, and acetic and propionic acids. This work confirmed the results obtained by several authors in the study of other types of cheese: Scarpellino and Kosikowski (1962) McSweeney et al. (1993) in Cheddar cheese, Gallmann (1982) and Gaya et al. (1990) in semi-hard cheeses such as Raclette and Manchego. The addition of raw milk flora by means of microfiltration retentate allowed Pa milk cheese to recover certain sensory characteristics of Ra milk cheese. This is supported by Law et al. (1976) who found that flavour intensity score was higher in pasteurized milk Cheddar cheese supplemented with certain groups of microflora (lactobacilli, leuconostocs, micrococci, Gram-negative rods) in comparison to control Cheddar cheese made without these microorganisms. Nevertheless, in our study. some differences still existed between PR and Ra milk cheeses such as higher acidity and bitterness, as well as higher overall aroma intensity for the former cheese. In future studies, it could be of interest to extend the ripening period of cheese to see if sensory differences would decrease or increase with the age of cheese.

CONCLUSION This work has demonstrated the role of the raw milk microflora in the biochemical and sensory characteristics of Swiss-type cheese. Cheeses with the raw milk microflora showed a more extensive proteolysis and a stronger propionic acid fermentation leading to more pronounced flavour. The biochemical and sensory differences observed between cheeses were probably due to facultatively heterofermentative lactobacilli, propionibacteria and enterococci observed in higher counts in Ra and PR milk cheeses. MF and Pa milk cheeses, which were very different from the two other types, also differed from one another in terms of microbiological, biochemical or sensory evaluation. Pasteurization, which was more efficient in reducing the subsequent growth of microorganisms during cheese ripening, gave a higher proportion of y-caseins and slightly higher acidity than microfiltration. Addition of raw milk flora to pasteurized milk restored for the most part the characteristics of Ra milk cheese measured in this study.

322

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ACKNOWLEDGEMENTS The authors thank R. Grappin for reviewing the manuscript and F. Dufrene for technical assistance. The authors also thank Sister N. Mascellino for reading the manuscript.

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