J. Dairy Sci. 86:3038-3047
© American Dairy Science Association, 2003.
Proteolysis, Volatile Compounds, and Sensory Evaluation in Hispánico Cheese Manufactured with the Addition of a Thermophilic Adjunct Culture, Nisin, and Calcium Alginate-Nisin Microparticles
S. Garde,
P. Gaya,
E. Fernández-García,
M. Medina and
M. Nuñez
Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña Km 7, Madrid, 28040 Spain
Corresponding author: M. Nuñez; e-mail: nunez{at}inia.es.
 |
ABSTRACT
|
|---|
Nisin, free or incorporated in calcium alginate microparticles, was added to pasteurized milk (80% cows’ and 20% ewes’ milk) used for the manufacture of Hispánico cheese with a mesophilic starter and a thermophilic adjunct culture of high aminopeptidase activity. Addition of nisin incorporated in microparticles promoted early lysis of thermophilic adjunct culture bacteria. Extracellular aminopeptidase activity in 1-d-old cheese made with both thermophilic adjunct culture and nisin incorporated in microparticles was 1.8-fold higher than in cheese made with thermophilic adjunct culture and free nisin and 2.0-fold higher than in cheese made only with thermophilic adjunct culture without any addition of nisin. Addition of nisin, free or incorporated in microparticles, did not influence cheese proteolysis measured using hydrophilic or hydrophobic peptides, o-phthaldialdehyde ripening index, or free amino acids. Moreover, a total of 37 volatile compounds were identified in the volatile fraction of Hispánico cheese, using a dynamic headspace technique with a purge and trap system followed by a gas chromatography mass spectrometry analysis. The volatile compound profile was not influenced by nisin addition, either free or incorporated in microparticles, but addition of thermophilic adjunct culture enhanced the formation of 2-butanone, diacetyl, 2,3-pentanedione and acetoin and improved the flavor quality (sensory panel) of cheese.
Key Words: proteolysis • volatile compound • nisin microparticle • Hispánico cheese
Abbreviation key: OPA = o-phthaldialdehyde.
|
INTRODUCTION
|
|---|
Lactic acid bacteria are the main source of enzymes in a wide variety of cheeses. Their proteinases and peptidases transform caseins into small peptides and free amino acids (Kunji et al., 1996; Lane and Fox, 1997), which contribute to cheese flavor and serve as aroma precursors (Engels and Visser, 1996; Fox and Wallace, 1997). Peptidases and other enzymes such as esterases and amino acid catabolic enzymes are located within the cell. Therefore, the lysis of starter bacteria should favor the access of those enzymes to their substrates and may accelerate the development of cheese flavor and hence cheese ripening (Morgan et al., 1997; Garde et al., 1997). Bacteriocin-producing adjunct cultures have already been used to enhance the lysis of starter bacteria during ripening (Morgan et al., 1997; Garde et al., 1997; Martínez-Cuesta et al., 1998; Oumer et al., 2001b; Martínez-Cuesta et al., 2001; Garde et al., 2002a) to accelerate the maturation of cheese.
Addition of a bacteriocin-producing adjunct culture may retard starter growth and consequently lactic acid production and affect cheese sensory characteristics (Morgan et al., 1997; Garde et al., 1997; Oumer et al., 2001b). The delay in lactic acid production would increase when bacteriocins in free form are added to milk, compared with the gradual production of bacteriocins in the curd by adjunct cultures.
An alternative to the addition of free bacteriocins is the use of microencapsulated bacteriocins (Degnan et al., 1993; Wan et al., 1997; Benech et al., 2002). Encapsulation of pediocin in liposomes limited the degree of bacteriocin inactivation in model food systems, but the encapsulation efficiency was relatively low (Degnan et al., 1993). A considerably higher encapsulation efficiency in liposomes, up to 47%, was achieved for nisin Z (Benech et al., 2002). Cheeses made with encapsulated nisin contained after 6 mo less than 10 cfu/g of Listeria innocua and 90% of the initial nisin activity, compared with 104 cfu/g and only 12% of initial nisin activity in cheeses made with a nisinogenic starter. Incorporation of nisin in calcium alginate microparticles increased the incorporation efficiency up to 87 to 93% (Wan et al., 1997). Nisin incorporated in microparticles was 100% active against an indicator culture in both MRS broth and reconstituted skim milk.
In previous works carried out at our laboratory (Oumer et al., 2001a; Garde et al., 2002a), Streptococcus thermophilus INIA 463 and INIA 468 showed a high aminopeptidase activity in milk compared with other strains and higher sensitivity to nisin than to other bacteriocins. On that basis, both strains were selected as a source of aminopeptidases to accelerate cheese ripening. In the present work, the addition of nisin, both free and incorporated in calcium alginate microparticles, to pasteurized milk used for the manufacture of Hispánico cheese was tested. Its effects on the viability of mesophilic starter and thermophilic adjunct culture, release of intracellular enzymes, proteolysis, formation of volatile compounds, and sensory characteristics of the cheese are investigated.
|
MATERIALS AND METHODS
|
|---|
Nisin Incorporation into Calcium Alginate Microparticles
Nisaplin (Aplin and Barrett Ltd, Beaminster, UK) and sodium alginate at 1:4 ratio (wt/wt) were dry-mixed. Sterile 50 g of CaCl2/L solution in distilled water was sprayed onto the dry mixture while stirring, until calcium alginate macroparticles were formed. Preparation of microparticles and determination of incorporation efficiency were carried out as described by Wan et al. (1997). Incorporation efficiency was expressed as the percentage of nisin activity remaining in the microparticles compared with the total nisin activity of the original suspension in sterile distilled water. Control calcium alginate microparticles were prepared by the same procedure but without the addition of nisin. Nisin activity was determined in triplicate by the critical dilution method by an agar diffusion assay using Lactobacillus buchneri St2A as indicator strain and expressed as IU/ml (Nuñez et al., 1996).
Lactic Cultures and Cheese Manufacture
Lactococcus lactis subsp. lactis INIA 437 and L. lactis subsp. cremoris INIA 450, both from the culture collection of INIA (Madrid, Spain), were used as mesophilic starter. They were stored at -80°C in MRS broth (Biolife, Milano, Italy) and subcultured twice in reconstituted skim milk at 25°C for 16 h before use in cheese manufacture. Two Streptococcus thermophilus strains, INIA 463 and INIA 468, were used as thermophilic adjunct culture with a high aminopeptidase activity. They were stored at -80°C in M17 broth (Biolife) and subcultured twice in reconstituted skim milk at 37°C for 4 h before use in cheese manufacture.
Hispánico cheese was manufactured in duplicate experiments on different days from a mixture of pasteurized cows’ (80%) and ewes’ (20%) milk as previously described (Garde et al., 2002a). Each experiment consisted of four 45-L vats. Lactococcus lactis subsp. lactis INIA 437 and L. lactis subsp. cremoris INIA 450 cultures were added each at 5 ml/L to all vats. Moreover, S. thermophilus INIA 463 and INIA 468 cultures in milk were added each at 5 ml/L to vats 2, 3 and 4 (vat 1 was used as control). Nisin in the form of Nisaplin was added to vat 3 (4 IU/ml) and nisin incorporated in calcium alginate microparticles to vat 4 (8 IU/ml). Rennet was added 20 min after lactic culture inoculation. Cheeses were ripened at 12°C for 75 d.
Microbiological Analysis and Cheese pH Value
Viable counts of lactic acid bacteria were determined in duplicate on M17 agar (Biolife) using a Spiral plater (Interscience, Saint-Nom-La-Bretèche, France), after incubation at 37°C for 48 h aerobically. Previous trials had shown that small size colonies were thermophilic lactic acid bacteria and large size colonies were mesophilic lactic acid bacteria (Garde et al., 2002a).
Cheese pH value was measured in duplicate after homogenizing 10 g of cheese with 20 ml of distilled water at 70°C by means of a Stomacher 400 (Seward Laboratory, London, UK).
Determination of Aminopeptidase Activity and Cheese Proteolysis
Aminopeptidase activity released into the cheese was measured on duplicate samples with lysine p-nitroanilide and leucine p-nitroanilide as substrates (Garde et al., 1997). One activity unit corresponds to the activity of enzymes producing 1 nmol of p-nitroaniline per minute per gram of cheese.
Overall cheese proteolysis (cheese ripening index) was determined on duplicate samples by the o-phthaldialdehyde (OPA) method, based on the reaction of released 
-amino groups with this compound and with ß-mercaptoethanol to form a derivative that absorbs at 340 nm (Church et al., 1983).
Hydrophilic and hydrophobic peptides in the water-soluble fraction of cheese were determined on duplicate samples by reversed-phase-HPLC using a Beckman System Gold chromatograph (Beckman Instruments, Madrid, Spain) equipped with a diode array detector module 168, with detection wavelength at 280 nm, as previously described (Gómez et al., 1997). Results were expressed as units of chromatogram area per milligrams of cheese DM.
Individual free amino acids were extracted from duplicate samples of cheese using the method of Krause et al. (1995) and determined by reversed-phase HPLC using a Beckman System Gold chromatograph, after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate as described by Liu et al. (1995). Results were expressed as grams per kilogram of cheese DM.
Analysis of Volatile Compounds
Cheese pieces wrapped in aluminium foil were vacuum packed and frozen at -40°C until analysis. Prior to volatile extraction, frozen pieces were kept overnight at 4°C, and then left stabilized at room temperature. An automatic dynamic headspace apparatus (Purge and Trap, HP 7695, Hewlett-Packard, Palo Alto, CA) connected to a gas chromatograph-mass spectrometer (HP 6890, Hewlett-Packard) was used for volatile compounds analysis. Duplicate 15-g cheese samples were homogenized in an analytical grinder (IKA, Labortechnik, Staufen, Germany), with 20 g of Na2SO4 and 75 µl of an aqueous solution containing 0.5 mg/ml cyclohexanone (IS1) and camphor (IS2) as internal standards. The analytical procedure and the identification and relative quantification of volatile compounds were carried out as described by Oumer et al. (2000).
Sensory Evaluation
Flavor intensity and flavor quality (preference test) of 25-, 50- and 75-d-old cheeses from duplicate experiments were evaluated by 14 trained panelists on a 10-point scale as previously described (Fernández del Pozo et al., 1988).
Statistical Analysis
Analyses of variance with 1) nisin addition, 2) thermophilic adjunct culture addition, and 3) cheese age as independent variables were performed by means of SPSS Win 8.0 program. Comparison of means between cheeses of the same age was carried out using Tukey’s test (Steel and Torrie, 1980).
Two-tailed Pearson correlations between proteolysis (OPA test), peptides, total free amino acids, and flavor intensity or flavor quality were performed by means of SPSS Win 8.0 program. Principal component analysis (PCA) with Varimax rotation was carried out on selected volatile compounds and sensory characteristics by means of SPSS Win 8.0 program.
|
RESULTS AND DISCUSSION
|
|---|
Efficiency of Nisin Incorporation
Calcium alginate-nisin microparticles (60 mg) suspended in 10 ml of sterile distilled water presented a calculated concentration of 1200 IU/ml. After centrifugation of the suspension, only 75 IU/ml of nisin activity was detected in the supernatant and apparently 100% of nisin activity was retained in the resuspended microparticles. In the supernatant of the free nisin preparation, 100% of nisin activity was detected. Calcium alginate microparticles without addition of nisin did not produce any zone of growth inhibition in the indicator lawn culture. From the results obtained for supernatants, efficiency of nisin incorporation in calcium alginate microparticles was calculated to be 94%. A similar result was obtained by Wan et al. (1997).
Cheese pH Value and Lactic Acid Bacteria
Addition of nisin, either free or incorporated in microparticles, did not significantly affect the pH value of cheese (Table 1
). In some previous works (Morgan et al., 1997; Garde et al., 1997; Oumer et al., 2001b), the addition of bacteriocin-producing adjunct culture retarded lactic acid production in cheese. However, Garde et al. (2002a) did not find significant differences between the pH values of cheeses made from milk inoculated with a bacteriocin producer and those of cheeses made without bacteriocin producer. The pH value of cheese was significantly influenced (P < 0.001) by ripening (Table 1).
View this table:
[in this window]
[in a new window]
|
Table 1. Changes in pH value during the ripening of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
Growth and viability of mesophilic starter lactic acid bacteria were scarcely influenced by the addition of nisin, either free or incorporated in microparticles (Table 2). Inversely, during the first 15 d, counts of thermophilic lactic acid bacteria were significantly (P < 0.05) lower in cheeses made with nisin, free or incorporated in microparticles, than in cheese made without nisin (Table 2). Cheese made with nisin incorporated in microparticles exhibited the lowest counts of thermophilic lactic acid bacteria. Garde et al. (2002a) also found lower concentrations of thermophilic lactic acid bacteria in cheese made with L. lactis subsp. lactis INIA 415, a strain harboring the structural genes of nisin Z and lacticin 481, than in control cheese.
View this table:
[in this window]
[in a new window]
|
Table 2. Levels of mesophilic and thermophilic lactic acid bacteria (LAB) during ripening of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
Release of Intracellular Enzymes
Addition of nisin, free or incorporated in microparticles, addition of thermophilic adjunct culture, as well as cheese age significantly (P < 0.001) influenced aminopeptidase activity in cheese (Table 3). Cheeses made with thermophilic adjunct culture showed considerably higher values of aminopeptidase activity than cheese made without thermophilic adjunct culture. In the absence of nisin, aminopeptidase activity values on lysine-p-nitroanilide and leucine-p-nitroanilide in cheese made with thermophilic adjunct culture on d 1 were 14-fold and 21-fold, respectively, those found in cheese made without thermophilic adjunct culture. Similar results were reported by Garde et al. (2002a) for cheese made with a nisin-producing adjunct culture. The presence of two additional peptidases in S. thermophilus with respect to L. lactis (Rul and Monnet, 1997) should explain the higher aminopeptidase activity. The latter increased during the first 15 d of ripening in cheeses made with nisin and during the first 50 d in cheese made with thermophilic adjunct culture but without nisin. In cheeses made with thermophilic adjunct culture, aminopeptidase activity was higher during the first 15 d of ripening when nisin was added, both free and incorporated in microparticles. Aminopeptidase activity in 1-d-old cheese made from milk with nisin incorporated in microparticles almost doubled that found in the respective cheese made from milk with free nisin (Table 3). Nisin incorporated in microparticles probably was retained in the curd in a higher proportion than free nisin but, due to its reduced availability compared with free nisin, had a retarded effect on the thermophilic adjunct culture. This might have allowed the thermophilic adjunct culture to reach a high population in the curd that, when lysed, would have resulted in the higher aminopeptidase activity of the respective 1-d-old cheese. In fact, cheese made with thermophilic adjunct culture and nisin incorporated in microparticles reached on d 1 the same aminopeptidase activity on lysine-p-nitroanilide that cheese made with thermophilic adjunct culture but without nisin did on d 15. The increase in aminopeptidase activity due to bacteriocin-mediated lysis of lactic acid bacteria is in agreement with previous reports (Morgan et al., 1997; Garde et al., 1997; Oumer et al., 2001b; Garde et al., 2002a).
View this table:
[in this window]
[in a new window]
|
Table 3. Aminopeptidase activity during ripening of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
Proteolysis
Cheese proteolysis as determined by the OPA test (Table 4) increased significantly (P < 0.001) with ripening. Addition of nisin, free or incorporated in microparticles, and addition of thermophilic adjunct culture scarcely influenced cheese proteolysis.
View this table:
[in this window]
[in a new window]
|
Table 4. Overall proteolysis (OPA index)1 during ripening of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA) and nisin, in free form or incorporated in microparticles (MP).
|
|
Levels of hydrophobic and hydrophilic peptides in the water-soluble fraction of cheese increased significantly (P < 0.001) with cheese age (Table 5), but addition of nisin or addition of thermophilic adjunct culture did not influence hydrophobic or hydrophilic peptides. The level of hydrophobic peptides determined at 280 nm correlates well with mean panel bitterness scores in Hispánico cheese made from pasteurized milk (Gómez et al., 1997). In the present work, the levels of hydrophobic peptides were (mean values from all vats) 0.71 after 25 d, 1.05 after 50 d, and 1.84 after 75 d, whereas the respective values for hydrophilic peptides were 6.63, 7.84, and 10.97 (Table 5). In a previous work on the same cheese variety (Garde et al., 2002a), mean levels of hydrophobic peptides were higher (2.80, 3.05, and 2.49 after 25, 50, and 75 d, respectively), whereas levels of hydrophilic peptides were similar (6.16, 9.83, and 11.90 after 25, 50, and 75 d, respectively). Aminopeptidase activity was higher on average in the present work (Table 3
) than in the work by Garde et al. (2002a), a fact that would account for the low level of hydrophobic peptides (Figure 1) found in the present work.
View this table:
[in this window]
[in a new window]
|
Table 5. Hydrophilic and hydrophobic peptides determined at 280 nm and the ratio of hydrophobic peptides to hydrophilic peptides in the water-soluble fraction of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
View larger version (13K):
[in this window]
[in a new window]
|
Figure 1. Reversed-phase HPLC chromatogram of the water-soluble fraction of a 75-d-old Hispánico cheese manufactured with a mesophilic starter and a thermophilic adjunct culture, without any addition of nisin, at a detection wavelength of 280 nm.
|
|
Ripening significantly (P < 0.001) increased cheese content in total free amino acids, independently on the addition of nisin or the addition of thermophilic adjunct culture (Table 6). Concentrations of total free amino acids were (mean values from all vats) 5.40, 8.75, and 12.79 g/kg cheese DM after 25, 50, and 75 d, respectively; values comparable with those recorded for Hispánico cheese (Garde et al., 2002a), which were on average 5.91, 9.48, and 13.52 g/kg cheese DM after the same ripening time. Individual free amino acid concentrations were not influenced by addition of nisin, free or incorporated in microparticles (data not shown). Alanine was the single amino acid influenced by addition of thermophilic adjunct culture, with a concentration of 0.25 g/kg in 50-d-old cheese made without thermophilic adjunct culture and 0.33 g/kg as the average value for 50-d-old cheeses made with thermophilic adjunct culture.
View this table:
[in this window]
[in a new window]
|
Table 6. Total free amino acids during ripening of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
Volatile Compounds
A total of 37 volatile compounds were identified using the dynamic headspace analysis of Hispánico cheese, including mainly hydrocarbons, alcohols, ketones, aldehydes, acids, and esters. Eighteen peaks, two of which corresponded to a pair of volatile compounds chromatographically not separated, were not significantly (P < 0.05) influenced by the addition of nisin, the addition of thermophilic adjunct culture, or cheese age. Overall mean values of the relative abundances of those compounds (calculated from all ages and all vats) were 0.45 for heptane, 2.13 for cyclohexane, 1.49 for octane, 15.61 for acetone, 2.74 for ethyl acetate, 1.37 for 2-hexanone + 2-hexanal (chromatographically not separated), 0.34 for ethylbenzene, 0.25 for p-xylene, 1.01 for m-xylene + 1-butanol (chromatographically not separated), 3.14 for 2-heptanone, 0.30 for o-xylene, 0.72 for 3-methyl-2-buten-1-ol, 0.45 for 2-nonanone, 0.26 for nonanal, 0.10 for 1-heptanol, 0.10 for acetic acid, 0.11 for 1-octanol, and 0.19 for butanoic acid.
Ten further volatile compounds were significantly (P < 0.05) influenced by cheese age, but not by the addition of thermophilic adjunct culture or nisin. Their relative abundances (mean values from all vats) at 25 and 75 d, respectively, were 0.95 and 0.59 for acetaldehyde, 4.48 and 7.23 for 2-propanol, 306 and 362 for ethanol, 3.14 and 8.29 for 2-pentanone, 0.13 and 0.41 for ethyl butanoate, not detected and 0.40 for 1-propanol, 8.27 and 2.41 for toluene, 0.35 and 0.54 for 2-methyl-1-propanol, 0.14 and 0.41 for 2-pentanol, and 0.22 and 0.76 for ethyl hexanoate.
As expected from previous results (no influence of nisin on proteolysis and concentration of free amino acids), nisin addition did not significantly change the volatile compound profile of cheese. However, the addition of thermophilic adjunct culture had a significant (P < 0.05) effect on the level of seven volatile compounds (Figure 2). Their relative abundances are shown in Table 7. The level of 3-methylbutanal was slightly higher in cheese made without termophilic starter. This aldehyde originates from Leu by transamination or Strecker degradation (Christensen et al., 1999) and is responsible for unclean and harsh flavors in Cheddar cheese (Dunn and Lindsay, 1985) and for a spicy chocolate-like flavor in Gouda-type cheese (Ayad et al., 2000), in Proosdij cheese (Engels et al., 1997), in Saint-Paulin cheese (Saboya et al., 2001), in Emmental cheese (Rychlik et al., 1997) as well as in Gruyère cheese (Rychlik and Bosset, 2001a, b). On the contrary, cheeses made with thermophilic adjunct culture showed slightly higher levels of 3-methyl-1-butanol. Formation of this alcohol from 3-methylbutanal through the activity of lactic acid bacteria dehydrogenases should be favored by the strong reducing conditions present in cheese (Molimard and Spinnler, 1996; Engels et al., 1997). 3-Methyl-1-butanol has a fruity and alcohol flavor note (Molimard and Spinnler, 1996). This alcohol has also been associated with unclean and harsh flavor in Cheddar cheese (Dunn and Lindsay, 1985). The level of 3-methyl-3-buten-1-ol was slightly higher in cheese made without termophilic starter. The relative abundances of 2-butanone, 2,3-butanedione, 2,3-pentanedione and 3-hydroxy-2-butanone were higher in cheeses made with thermophilic adjunct culture. Diacetyl (2,3-butanedione) originates from the unstable precursor -acetolactate during citrate metabolism and is one of the major aromatic compounds in fermented milk and fresh cheese (Molimard and Spinnler, 1996). The higher levels of acetoin (3-hydroxy-2-butanone) and 2-butanone in cheeses made with thermophilic adjunct culture were to be expected, since diacetyl reduction generates acetoin, and acetoin reduction generates 2-butanone. 2,3-Pentanedione might be formed from -aceto--hydroxybutyrate, an intermediate of the isoleucine metabolism (Imhof et al., 1995). Some S. thermophilus strains have been reported to produce high levels of diacetyl and 2,3-pentanedione (Imhof et al., 1995). Garde et al. (2002b) also recorded higher levels of diacetyl and 2,3-pentanedione in cheeses made with thermophilic adjunct culture than in cheeses made without thermophilic adjunct culture.
View larger version (16K):
[in this window]
[in a new window]
|
Figure 2. Gas chromatogram corresponding to the headspace of a 75-d-old Hispánico cheese made with a mesophilic starter and a thermophilic adjunct culture, without any addition of nisin. Peaks were: 7, 2-butanone; 8, 3-methylbutanal; 12, 2,3-butanedione; 16, 2,3-pentanedione; 25, 3-methyl-1-butanol; 27, 3-methyl-3-buten-1-ol; 28, 3-hydroxy-2-butanone; CY, cyclohexanone; CA, camphor. See Materials and Methods for chromatographic conditions.
|
|
View this table:
[in this window]
[in a new window]
|
Table 7. Volatile compounds in cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP), influenced by TA.
|
|
Sensory Evaluation
Flavor intensity and flavor quality were not significantly influenced by the addition of nisin, free or incorporated in microparticles (Table 8). Addition of thermophilic adjunct culture significantly enhanced flavor quality (P < 0.001), but did not influence flavor intensity. Cheese age influenced flavor intensity (P < 0.001), which tended to increase during ripening of cheeses. The current results do not completely agree with those of Garde et al. (2002a), who recorded a significant enhancement of the flavor quality (P < 0.001) and the flavor intensity (P < 0.05) of Hispánico cheese by the addition of thermophilic adjunct culture to milk.
View this table:
[in this window]
[in a new window]
|
Table 8. Sensory evaluation of cheeses manufactured with a mesophilic starter (MS), a thermophilic adjunct culture (TA), and nisin, in free form or incorporated in microparticles (MP).
|
|
There were significant (P < 0.01) correlations between flavor intensity scores and proteolysis (OPA test), hydrophilic peptides, and total free amino acids, with r values of 0.458, 0.538, and 0.518, respectively. Flavor quality scores correlated significantly (P < 0.05; r = 0.285) with proteolysis (OPA test). Similar correlations were recorded by Garde et al. (2002a).
Principal component analysis was carried out to correlate volatile compounds with sensory characteristics. Components 1 and 2 of principal component analysis explained the 31.4 and 14.5% of the variance, respectively. Ethyl butanoate, 1-propanol, 3-methylbutanal, 3-methyl-1-butanol, ethyl hexanoate, 2-pentanone, 2-methyl-1-propanol, 2-pentanol, ethanol, and flavor intensity correlated positively with component 1 (Table 9). Flavor quality and component 1 had a low correlation. 2,3-Pentanedione, 2,3-butanedione, 3-hydroxy-2-butanone, and flavor quality correlated positively with component 2. Flavor intensity and component 2 had a low correlation.
View this table:
[in this window]
[in a new window]
|
Table 9. Correlation coefficients for volatile compounds and sensory characteristics with the functions in the principal component analysis (PCA) with Varimax rotation.
|
|
|
CONCLUSIONS
|
|---|
Calcium alginate was chosen as the incorporation matrix of nisin because of its food-grade additive status, low cost, and simple incorporation process. Addition of nisin, either free or incorporated in calcium alginate microparticles, to milk promoted early lysis of thermophilic adjunct culture bacteria and increased aminopeptidase activities in cheese during the first days of ripening, but did not influence proteolysis, volatile compound profile, or sensory characteristics of cheese. The use of nisin incorporated in microparticles resulted in a higher aminopeptidase activity in 1-d-old cheese than when free nisin was used, but differences in aminopeptidase activity between cheeses made with free nisin and with nisin incorporated in microparticles disappeared during ripening. Addition of thermophilic adjunct culture did not influence proteolysis. However, it enhanced the formation of some volatiles considered to impact aroma compounds such as 3-methyl-1-butanol, diacetyl, and 2,3-pentanedione and improved flavor quality of cheese.
The performance of nisin, free or incorporated in microparticles, was under those of nisin-producing adjunct cultures used in previous works. A gradual release of the bacteriocin into the cheese matrix seems to be essential for the optimum lysis of lactic acid bacteria and the acceleration of cheese ripening. Further studies using different proportions of nisin-calcium alginate mixtures, which would favor or retard the release of nisin, might contribute to optimize the lysis of lactic acid bacteria.
|
ACKNOWLEDGEMENTS
|
|---|
We acknowledge financial support from project AGL2000-1426 of the Spanish Ministry of Science and Technology.
Received for publication January 28, 2003.
Accepted for publication May 16, 2003.
|
REFERENCES
|
|---|
Ayad, E. H. E., A. Verheul, J. T. M. Wouters, and G. Smit. 2000. Application of wild starter cultures for flavour development in pilot plant cheese making. Int. Dairy J. 10:169–179.
Benech, R. O., E. E. Kheadr, R. Laridi, C. Lacroix, and I. Fliss. 2002. Inhibition of Listeria innocua in Cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture. Appl. Environ. Microbiol. 68:3683–3690.[Abstract/Free Full Text]
Christensen, J. E., E. G. Dudley, J. A. Pederson, and J. L. Steele. 1999. Peptidases and amino acids catabolism in lactic acid bacteria. Antonie Leeuwenhoek 76:217–246.
Church, F. C., H. E. Swaisgood, D. H. Porter, and G. L. Catignani. 1983. Spectrophotometric assay using o-phtaldialdehyde for determination of proteolysis in milk and isolated milk proteins. J. Dairy Sci. 66:1219–1227.[Abstract/Free Full Text]
Degnan, A. J., N. Buyong, and J. B. Luchansky. 1993. Antilisterial activity of pediocin AcH in model food systems in the presence of an emulsifier or encapsulated within liposomes. Int. J. Food Microbiol. 18:127–138.[Medline]
Dunn, H. C., and R. C. Lindsay. 1985. Evaluation of the role of microbial Strecker-derived aroma compounds in unclean-type flavors of Cheddar cheese. J. Dairy Sci. 68:2859–2874.[Abstract/Free Full Text]
Engels, W. J. M., R. Dekker, C. De Jong, R. Neeter, and S. Visser. 1997. A comparative study of volatile compounds in the water-soluble fraction of various types of ripened cheese. Int. Dairy J. 7:255–263.
Engels, W. J. M., and S. Visser. 1996. Development of cheese flavour from peptides and amino acids by cell-free extracts of Lactococcus lactis subsp. cremoris B78 in a model system. Neth. Milk Dairy J. 50:3–17.
Fernández del Pozo, B., P. Gaya, M. Medina, M. A. Rodríguez-Marín, and M. Nuñez. 1988. Changes in chemical and rheological characteristics of La Serena ewes’ milk cheese during ripening. J. Dairy Res. 55:457–464.
Fox, P. F., and J. M. Wallace. 1997. Formation of flavor compounds in cheese. Adv. Appl. Microbiol. 45:17–85.[Medline]
Garde, S., P. Gaya, M. Medina, and M. Nuñez. 1997. Acceleration of flavour formation in cheese by a bacteriocin-producing adjunct lactic culture. Biotechnol. Lett. 19:1011–1014.
Garde, S., J. Tomillo, P. Gaya, M. Medina, and M. Nuñez. 2002a. Proteolysis in Hispánico cheese manufactured using a mesophilic starter, a thermophilic adjunct culture and bacteriocin-producing Lactococcus lactis subsp. lactis INIA 415 adjunct culture. J. Agric. Food Chem. 50:3479–3485.[Medline]
Garde, S., M. Carbonell, E. Fernández-García, M. Medina, and M. Nuñez. 2002b. Volatile compounds in Hispánico cheese manufactured using a mesophilic starter, a thermophilic adjunct culture, and a bacteriocin-producing Lactococcus lactis subsp. lactis INIA 415. J. Agric. Food Chem. 50:6752–6757.[Medline]
Gómez, M. J., S. Garde, P. Gaya, M. Medina, and M. Nuñez. 1997. Relationship between levels of hydrophobic peptides and bitterness in cheese made from pasteurized and raw milk. J. Dairy Res. 64:289–297.
Imhof, R., H. Glaettli, and J. O. Bosset. 1995. Volatile organic compounds produced by thermophilic and mesophilic single strain dairy starter cultures. Lebensm. Wiss. Technol. 28:78–86.
Krause, I., A. Bockhardt, H. Neckermann, T. Henle, and H. Klostermeyer. 1995. Simultaneous determination of amino acids and biogenic amines by reversed-phase high-performance liquid chromatography of the dabsyl derivatives. J. Chromatogr. A 715:67–79.
Kunji, E. R. S., I. Mierau, A. Hagting, B. Poolman, and W. N. Konings. 1996. The proteolytic system of lactic acid bacteria. Antonie Leeuwenhoek 70:187–221.
Lane, C. N., and P. F. Fox. 1997. Role of starter enzymes during ripening of Cheddar cheese made from pasteurised milk under controlled microbiological conditions. Int. Dairy J. 7:55–63.
Liu, H. J., Y. Chang, H. W. Yan, F. H. Yu, and X. X. Liu. 1995. Determination of amino acids in food and feed by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and reversed-phase liquid chromatographic separation. J. AOAC Int. 78:736–744.
Martínez-Cuesta, M. C., P. Fernández De Palencia, T. Requena, and C. Peláez. 1998. Enhancement of proteolysis by a Lactococcus lactis bacteriocin producer in a cheese model system. J. Agric. Food Chem. 46:3863–3867.
Martínez-Cuesta, M. C., T. Requena, and C. Peláez. 2001. Use of a bacteriocin-producing transconjugant as starter in acceleration of cheese ripening. Int. J. Food Microbiol. 70:79–88.[Medline]
Molimard, P., and H. E. Spinnler. 1996. Review: Compounds involved in the flavor of surface mold-ripened cheeses: Origins and properties. J. Dairy Sci. 79:169–184.[Abstract]
Morgan, S., R. P. Ross, and C. Hill. 1997. Increasing starter cell lysis in Cheddar cheese using a bacteriocin-producing adjunct. J. Dairy Sci. 80:1–10.[Abstract/Free Full Text]
Nuñez, M., J. Tomillo, P. Gaya, and M. Medina. 1996. Bacteriocin quantification by the critical dilution method: a comparison of arbitrary units with diameter and area zone of growth inhibition. Milchwissenschaft 51:7–10.
Oumer, A., E. Fernández-García, C. Serrano, and M. Nuñez. 2000. Flavour of Hispánico cheese manufactured with Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris as starter cultures. Milchwissenschaft 55:325–328.
Oumer, A., S. Garde, P. Gaya, M. Medina, and M. Nuñez. 2001a. The effects of cultivating lactic starter cultures with bacteriocin-producing lactic acid bacteria. J. Food Prot. 64:81–86.[Medline]
Oumer, A., P. Gaya, E. Fernández-García, R. Mariaca, S. Garde, M. Medina, and M. Nuñez. 2001b. Proteolysis and formation of volatile compounds in cheese manufactured with a bacteriocin-producing adjunct culture. J. Dairy Res. 68:117–129.[Medline]
Rul, F., and V. Monnet. 1997. Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis. J. Appl. Microbiol. 82:695–704.[Medline]
Rychlik, M., and J. O. Bosset. 2001a. Flavour and off-flavour compounds of Swiss Gruyère cheese. Evaluation of potent odorants. Int. Dairy J. 11:895–901.
Rychlik, M., and J. O. Bosset. 2001b. Flavour and off-flavour compounds of Swiss Gruyère cheese. Identification of key odorants by quantitative instrumental and sensory studies. Int. Dairy J. 11:903–910.
Rychlik, M., R. Warmke, and W. Grosch. 1997. Ripening of Emmental cheese wrapped in foil with and without addition of Lactobacillus casei subsp. casei III. Analysis of character impact flavour compounds. Lebensm. Wiss. Technol. 30:471–478.
Saboya, L. V., H. Goudédranche, J.-L. Maubois, A. L. S. Lerayer, and S. Lortal. 2001. Impact of broken cells of lactococci or propionibacteria on the ripening of Saint-Paulin UF-cheeses: Extent of proteolysis and GC-MS profiles. Lait 81:699–713.
Steel, R. G. D., and J. H. Torrie. 1980. Principles and Procedures of Statistics: A Biometrical Approach. 2nd ed. McGraw-Hill Book Co., New York, NY.
Wan, J., J. B. Gordon, K. Muirhead, M. W. Hickey, and M. J. Coventry. 1997. Incorporation of nisin in micro-particles of calcium alginate. Lett. Appl. Microbiol. 24:153–158.[Medline]
This article has been cited by other articles:
|
|
|
|
 
R. Attaie
Quantification of volatile compounds in goat milk Jack cheese using static headspace gas chromatography
J Dairy Sci,
June 1, 2009;
92(6):
2435 - 2443.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
ABSTRACT
INTRODUCTION
