ACCELERATED KASHAR CHEESE RIPENING WITH ENCAPSULATED LIPASE AND PROTEASE ENZYMES

Headnote

ABSTRACT

In this study, lipase and protease enzymes were encapsulated in κ-carrageenan, gellan and sodium alginate by using emulsion and extrusion technique and were then added in cheese milk together with rennet. The effects of the encapsulating material and ripening period on the chemical characteristics of Kashar cheese were investigated. The study demonstrated that κ-carrageenan, gellan and sodium alginate could successfully be used as lipase and protease carrier systems to accelerate Kashar cheese ripening. Those samples treated with κ-carrageenan capsules showed the highest rate of lipolysis and proteolysis compared to those treated with the other capsules.

- Keywords: kashar cheese, enzyme encapsulation, κ-carrageenan, gellan, sodium alginate -

INTRODUCTION

After White cheese, Kashar cheese is the most commonly produced and consumed cheese in Turkey, the Balkan, Peninsula and the Mediterranean region. The main problem in manufacturing Kashar cheese is the long maturation period which increases the cost of handling significantly. Maturation is very important in developing the unique flavor, aroma and texture of the cheese before marketing. The time required to develop this characteristic flavor and texture varies from a few weeks for soft cheese up to 3 years for very hard cheese varieties (GRIPON et al, 1991). However, the long maturation period increases the price of the cheese (FOX, 1993).

Several attempts have been made to reduce the ripening period by the addition of individual and mixed lipase, protease and ß-galactosidase enzymes, some of which have been reported to halve the normal maturation period of cheese (LAW, 2001). Lipolysis and proteolysis play an important role in cheese ripening, and a large number of studies dealing with the acceleration of lipolysis and proteolysis through the addition of free lipolytic and proteolytic enzymes to either cheese milk or curd have been published (KOCAK et al., 1996; CAGLARand CAKMAKCI, 1998). The addition of free lipases has resulted in premature attack leading to excessive lipolysis and texture and flavor defects (KOCAK et al., 1996). Direct addition of protease enzyme to the cheese milk was not successful due to loss of enzymes in the whey, poor enzyme distribution, reduced yield and poor-quality cheese. Incorporation of encapsulated enzyme eliminated the problems associated with direct enzyme addition. The use of microencapsulated enzymes has been proposed to circumvent these drawbacks. Enzyme microcapsules physically separate the enzyme from the substrate in the curd and the enzyme is only released into the curd upon capsule breakdown during ripening (LAW, 2001). Milk-fat-coated capsules were first developed by MAGEE and OLSON (1981) and used to encapsulate a wide variety of substances.

Vegetable gels such as Konjac, liposomes, milk fat, some food gums and hydrophilic hydrocolloids are used for enzyme encapsulation. The use of liposomes as enzyme-encapsulating substances has some drawbacks. They may be expensive and are generally not regarded as safe and edible. A group of substances that exhibit excellent encapsulating abilities includes food gums or hydrophilic hydrocolloids. Gum capsules are easy to prepare, and gums are relatively widely available, cheap, and biologically compatible (KAILASAPAHTY and LAM, 2005). Because of the aforementioned drawbacks, the addition of enzymes directly or encapsulated in milk fat to cheese milk has not been successful.

Limited information is available on the accelerated ripening of Cheddar cheese using encapsulated enzymes (CAGLARand CAKMAKCI, 1998). We investigated food gums as an alternative to liposomes for enzyme encapsulation to accelerate cheese ripening. Three gums (?-carrageenan, gelIan and sodium alginate) were used to encapsulate enzymes for application to cheese milk. The objective of this work was to study the effect of encapsulated lipase and protease enzymes cocktails added to Kashar cheese milk on the lipolysis and proteolysis of the cheese during storage.

MATERIALS AND METHODS

Gums, enzymes and chemicals

?-carrageenan, gellan and sodium alginate gums were supplied by Sigma Chemicals (Istanbul, Turkey). The enzyme Palatase 20000 L (LUN 00217) and Flavourzyme 1,000 L were obtained from Novozymes (Istanbul, Turkey). Direct-set frozen lactic acid starter cultures (Ezal MAO 14) containing Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris were obtained from Ezal (France). Rennet (ECOREN 200) was obtained from Maysa Gida (Istanbul). All other reagents used were of analytical grade.

Preparation of gum capsules

?-carrageenan enzyme capsules were prepared by a modified method of AUDET and LACROIX (1989). Gum powder (1.5 g) was suspended in three lots of 50 mL deionized water, heated to 80°C, stirred and kept at that temperature for 20 min to completely dissolve the polymer. The solutions were cooled to 40°C and mixed with 0. 133 mL of 7.5% (w/v) solution of Palatase 20000 L and 13.3 mL of 7.5% (w/v) solution of Flavourzyme 1000 L to produce of capsules. The mixture was rapidly poured into 150 mL soybean oil containing 0.2% emulsifier in a beaker maintained at 40°C while stirring with a magnetic stirrer. The water-in-oil emulsions were cooled to 25°C to allow the gum droplets to gel. The oil phase was decanted, and the resulting capsules were harvested by centrifugation (100 g, 2 min). The gel beads were washed twice with distilled water, and the capsules were separated from the supernatant by sieving. The beads formed were hardened by soaking in 0.07% calcium chloride solution for 2 h.

Gellan capsules were prepared by dispersing 0.3 g of gellan powder in 50 mL of deionized water. The dispersion was heated to 90°C under magnetic stirring for 10 min. The solutions were cooled to 45°C and mixed with 0. 133 mL of a 7.5% (w/v) solution of Palatase 20000 L and 13.3 mL of 7.5% (w/v) solution of Flavourzyme 1000 L to produce capsules. The rest of the preparation procedure was performed as described for ?-carrageenan.

Preparation of sodium alginate capsules by using emulsion and extrusion techniques

The modified method of SHEU and MARSHALL (1993) was used. A 2% alginate mixture containing 2% Hi-maize resistant starch and 0. 133 mL of 7.5% (w/v) solution of Palatase 20000 L and 13.3 mL of 7.5% (w/v) solution of Flavourzyme 1000 L were prepared. The mixture was dropped into oil containing Tween 80 (0.02%). After the dropping was completed, the mixture was stirred vigorously until it was emulsified and appeared creamy. A solution of 0. 1 M calcium chloride was then added quickly along the side of the beaker; the phase separation of the oil/water emulsion then occurred. The mixture was left to stand for 30 min to allow the calcium-alginate beads to separate and settle at the bottom of the calcium chloride layer. The oil layer was drained, and beads were collected by low-speed centrini - gation (350 g, 15 min), washed once with 0.9% saline containing 5% glycerol, and stored at 4°C. Size separation of the beads was performed using 500 and 150 µp? steel sieves.

The extrusion technique of KRASAEKOOPT et al (2003) was used. In this study, the lipase in the 0. 133 mL of Palatase 20000 L solution and the protease in the 13.3 mL Flavourzyme 1000 L solution was mixed with 20 mL of 2% (w/v) sodium alginate solution (Sigma Aldrich Steinheim, Germany). The suspension was injected through a 0.11 mm needle into 0.05 M CaCl2. The beads were allowed to stand for 30 min to gelate, then rinsed with 0.9% saline containing 5% glycerol and subsequently kept in at 4°C.

Rates of enzyme entrapment

The efficiency of lipase and protease enzyme encapsulation for the three types of capsules were measured separately for lipase and protease according to the methods of TENG and XU (2007) and SARATH et al. (1989) respectively, and was used to express the entrapment efficiencies (EE). The rate of EE was the percentage of enzyme encapsulated (expressed as units enzyme activity) in capsules divided by the total units of enzyme in bulk solution multiplied by 100.

Capsules prepared by the addition of 0.133 mL of a 7.5% (w/v) solution of Palatase 20000 L to the encapsulant solutions were used for this purpose. The total enzyme activity was determined from a bulk solution of capsules (before separation of capsules from the un-encapsulated material). The bulk solutions (10 mL) containing K-carrageenan, gellan and sodium alginate capsules were separately dispersed in 50 mLof 0.4% trisodium citrate solutions and stirred for 30 min at room temperature (23°-24°C) until completely dissolved. Separated gum capsules were treated similarly in trisodium citrate solution.

The dissolved capsule solution was mixed with 0.5 mL stock solution (10 mM pNPP [Sigma] in ?-heptane). Approximately 30 µL ethanol (1 M) was added to the reaction mixture and incubated at 40°C at a shaking speed of 200 rpm for 5 to 30 min. After allowing the lipase in the reaction mixture to settle for 30 s, 25 µL of the clear supernatant was taken and then immediately mixed with 1 mL of 0.1 M NaOH in a 1 mL cuvette. The pNP liberated was extracted through the aqueous alkaline phase; the extraction was then analyzed at 410 nm against a blank without enzyme using a UV-visible spectrophotometer.

Proteolytic activity of the enzyme was determined as trichloroacetic acid (TCA) soluble peptides and amino acids following the precipitation of intact casein with TCA. Capsules prepared by addition of 5.0 mL of a 7.5% solution of Flavourzyme 1000 L to the encapsulant solutions were used for this purpose. The total enzyme activity was determined from a bulk solution of capsules (before separation of capsules from the un-encapsulated material). The bulk solutions (10 mL) containing k-carrageenan and gellan capsules were separately dispersed in 50 mL of 0.4% trisodium citrate solutions and stirred for 30 min at room temperature (23°-24°C) until completely dissolved. Separated gum capsules were treated similarly in trisodium citrate solution.

One unit of specific enzyme activity was defined as the increase in absorbance at 280nm across a 1 cm path length caused by a unit amount (1 mg) of enzyme (expressed as total nitrogen) under the conditions of the assay.

The diameters of 100 randomly selected beads of each treatment were measured with an eyepiece micrometer on an optical microscope at a magnification of 1 OOx.

Cheesemaking

A 7.5% (w/v) solution of Palatase M (20,000 LU/g) and a 7.5% (w/v) solution of Flavourzyme 1000 L (1,000 LAPU/g) were encapsulated in sodium alginate, ?-carrageenan and gellan gums as described above.

Cheese production was done in the Dairy Pilot Plant of the Food Engineering Department of Harran University. One hundred twenty kilograms of standardized milk was used for each batch (1 control (coded A) and 4 treatments). The fat content of the milk was standardized to 2.5%. All batches were pasteurized at 72°C for 1 min and then cooled to 34°C. Starter culture (1%) and CaCl2 (0.02%) were then added. For the experimental cheeses, enzyme capsules made of sodium alginate by emulsion techniques, sodium alginate by extrusion techniques, ?-carrageenan and gellan gums, were introduced into the cheese milk at 34°C, just before the addition of rennet, and the samples were coded B, C, D and E respectively. Stirring was continued after enzyme capsule addition up to the point of rennet addition. These quantities corresponded 0.133 mL Palatase M (20,000 LU/g) per kilogram milk fat and 13.4 mL Flavourzyme 1000 L (1,000 LAPU/g) per kilogram of cheese. When the pH of the milk reached 6.2-6.3, rennet diluted with pure water was added. Cutting was performed 30 min later. The curd was cut with a curd knife into cubes of 1 cm3. The cut curd was allowed to settle for 15 min. Cooking was performed by increasing the temperature from 34° to 40°C over 30 min. The heating rate was an increase of 1°C for every 4-5 min. At the end of cooking, a third of the whey content was drained from each batch. At the same time, the cheese curd was agitated. The cheese curd was fermented until it reached a pH level of 5.0. The remaining whey was then drained. Cheese whey was collected during the manufacturing and strained using a 120-µ?? stainless steel sieve. The capsules were collected on the sieve and re-added to the curd. The curd was hand-stretched in a 6% brine at 74°C for 2 min for all the cheeses studied. Brine was strained using a 120 µp? stainless steel sieve and the capsules were collected on the sieve and re-added to curd while the curd was kneading. The curds were placed into cylindrical stainless steel molds and turned 30 min later to provide a flat surface. All cheeses were cooled at room temperature, and the molds were removed. Then, the cheeses were allowed to gain their yellow color for 24 h at 15°±2°C.

The mass of one block of fresh Kashar cheese was approximately 600 g. The blocks of cheeses were surface-salted for 1 week and stored at 4°-6°C for 180 days. Cheese samples were taken for chemical analyses on the 1st, 15th, 30th, 60th, 90th, 120th and 150th days of storage. Cheese was manufactured in triplicate for each group.

Cheese composition

The pH of the milk (TSE, 1994) and cheeses (TSE, 1995) was measured using a digital pHmeter (model of Orion 250 A, Orion Research Inc., Boston, USA). The protein content of the milk and cheeses were determined by the Kjeldahl method (GRÍPON et ed., 1975). The total fat and dry matter contents of the cheese samples were determined using the method proposed by Gerber (TSE, 1994) and gravimetric methods, respectively. The salt content of the cheeses was determined by the Mohr titration method (AOAC, 1990).

Determination of free fatty acid analysis

The total free fatty acids (TFFAs) were the sum of the contents of butyric (C40), caproic (C60), caprylic (C80), capric (C100), lauric (C120), myristic (C140), palmitic (C16j, stearic (C180), oleic (C18:1) and linoleic (C182) acids.

Fat was extracted from cheese samples as described by GARCIA-LOPEZ et cd. (1994) and methylated according to the procedure of SUKHIJA and PALMQUIST (1988). Fatty acid methyl esters were analyzed using Gas Chromatography (Thermo Quest) equipped with a flame ionization detector (FID) and fitted with a fused silica capillary column (SP-2380, 30 m, 0.25 mm; Supelco Inc., Bellefonte, PA). The temperature of the injector and detector temperature was 250°C.

The initial oven temperature was set to 40°C for 1.0 min, and then increased to 240°C at a rate of 5°C/min. The final temperature was maintained for 10 min. Nonanoic acid was used as the internal standard. A standard fatty acid mixture containing 37 fatty acids (Sigma- Aldrich Chemicals 189-19) was used to provide standard retention times. Fatty acids were identified by comparing their retention times with those of fatty acids in standard samples. An auto system Thermoquest GC-MS equipped with a flame ionization detector (FID) was used to analyze the FFAs of the cheese samples. The carrier gas was helium, flowing at 2 mL min1. A 1 µL sample was applied with a split ratio of 1:30 into the injector.

Determination of proteolysis

Cheese protein (casein) degradation during ripening was evaluated after 1st, 15th, 30th, 60th, 90th, 120th and 150th days using by mini urea Polyacrylamide gel electrophoresis (Urea- PAGE). Electrophoresis was carried out on a vertical slab unit (Bio-Rad Laboratories, Inc. 1000 Alfred Nobel Drive, Herculus, California, USA) and the stacking gel system described by CREAMER (1991). Samples were prepared by gratting 0.5 g of each cheese into 25 mL of sample buffer (0.092 g EDTA 1.08 G Tris, 0.55 g boric acid and 36 g urea made up to 100 mL and adjusted to ? H 8.4). Each sample was centrifuged at 10,000 g for 10 min and 2 mL from the middle portion was taken. All samples were mixed with 3% each of 0. 1% (w/v) bromphenol blue solution and mercaptoethanol. 5 µL of the 2% cheese solutions were used for electrophoresis. For dying the gels were stained with Coosmassie Blue R-250 dye solution was used.

Statistical analyses

Each cheese experiment was repeated three times, and each analysis was done in duplicate. The experiment was designed according to a 5x8x3 (capsule material ? storage time ? repeation) factorial design. All statistical analyses were performed using the SPSS statistical software program (version 5.0). Statistically different groups were determined by the LSD (Least Significant Difference) test (BEK and EFE, 1995).

RESULTS AND DISCUSSION

Enzyme encapsulation

The encapsulation efficiencies of Palatase M (20,000 LU/g) in ?-carrageenan, gellan gums, sodium alginate produced by emulsion technique or sodium alginate by extrusion technique were, respectively, 58.7±0.51, 51.0±0.43, 42.50±0.36 and 53.1 ±0.35% of the initial activity (mean of 3 separate trials). Encapsulation efficiencies of Flavourzyme 1000 (1,000 LAPU/g) in ?-carragennan, gellan gums, sodium alginate by emulsion technique or sodium alginate by extrusion technique were, respectively, 54. 1±0.64, 47.3±0.56, 39.2±0.71 and 47.9±0.63% of the initial activity (mean of 3 separate trials). The encapsulation efficiencies of the four capsulants were statistically significantly different from each other (p<0.01). The ionic strength of the capsulehardening solution (calcium chloride) may have had an effect on the activity of the enzymes (KAILASAPATHY and LAM, 2005).

The beads were globular in shape. The capsule materials influenced on the size of the beads. The sodium alginate capsules by extrusion technique were bigger than the other capsules. The diameter of beads of ?-carragennan, gellan gums, sodium alginate by emulsion technique was 1.68 mm, which was significantly lower than that of sodium alginate by extrusion technique beads (1.90 mm).

Chemical composition

The gross chemical composition of the control and experimental cheeses is given in Table 1 . Significant differences were observed between the control and experimental cheeses. The cheese curds had a significantly higher moisture content and titratable acidity but lower pH contents as compared to the control (p<0.01). The high moisture content of the gum-capsule-treated cheese curds was due to the hydrophilic nature of sodium alginate, carrageenan, and gellan gums, which retained moisture in the cheeses. The protein, fat and salt contents of the experimental cheeses were close to those of the control. Similar results were reported for capsule-treated cheeses by KHEADR et cd. (2002 and 2003), KAILASAPATHY and LAM (2005).

Free fatty acid production

The free fatty acid (FFA) contents of the control and experimental cheeses were quantified throughout a 180-day of ripening period (Table 2). The production of FFAs was markedly stimulated by the introduction of encapsulated lipases and proteases. This effect was noticeable even on the day of manufacture. At that time, the experimental cheeses exhibited substantial increases in FFAs. This suggests that a portion of the enzyme is immobilized on the external capsule membrane and/or a loss of capsule integrity during cheese manufacture leads to the release of the encapsulated enzymes.

Upon aging, the amount of FFA produced increased with the ripening period; moreover, the experimental cheeses had accumulated higher quantities of individual FFAs, depending on the type of capsule, as compared to control (p<0.01) (Table 2). The higher incidence of FFAs in the ?carrageenan capsules-treated cheeses. The conditions to which cheeses are subjected, such as the presence of ions and lactic acid, appeared to influence the stability of gum capsules in the cheeses. The interaction between milk proteins and ?-carrageenan gum capsules most likely enhanced the interaction between the encapsulated enzyme and milk proteins (KAILASAPATHY and LAM, 2005). On the other hand, no report is available in the literature about the effect of the lipase and protease enzyme encapsulated in sodium alginate on cheese properties, to which a part of this work has been addressed. The lower FFA content of sodium-alginate-treated cheeses suggests that these capsules probably release their enzyme contents very slowly. As the cheeses aged, the progressive increase in the concentration of such FFAs suggested a gradual release of the encapsulated lipases.

Despite a rise in the rate of lipolysis, a limited amount of volatile fatty acids was produced. Capile acid, which was the main volatile free fatty acid, accounted for 1.53 and 2.75 to 3.08% of the TFFA, as observed in the 180-day-old control and experimental cheeses, respectively. The importance of volatile fatty acids in the development of cheese flavor has been discussed by other researchers (WOO et al., 1984), who have reported that a certain concentration of such volatile fatty acids was needed to develop a typical cheese flavor. However, a high production of these fatty acids could lead to off-flavor. Among the nonvolatile fatty acids, the amount of palmitic acid increased at the fastest rate, followed by that of oleic, stearic, myristic and lauric acids.

Proteolysis

There were significantly (p<0.01) higher levels of proteolysis in enzyme treated cheeses as compared to the control cheese (Figs. 1-5). Cheeses treated with ?-carrageenan capsules showed a high rate of increase in ß-, ocsl- casein degradation. The higher rate of ß-, «^-casein hydrolysis in the ?-carrageenan capsulestreated cheeses was probably due to the low stability of ?-carrageenan gels in solutions with acidic pH (KAILASAPATHY and LAM, 2005), similar to that observed in ripening cheese. The observed slower rate of ß- and agl-casein degradation in cheeses treated with sodium alginate capsules suggests that these capsules probably release their enzyme contents very slowly.

An increasing trend for ß- and asl -casein degradation was observed during the 180-day of ripening period for all cheeses (p<0.01). This was expected because amino groups are produced in cheese as a consequence of protein breakdown during ripening (FOX et al., 1993). Thus an increase in proteolysis in treated cheese over that of control at any given time during ripening indicates an acceleration of ripening.

CONCLUSIONS

According to the results, it was concluded that κ-carrageenan, gellan and sodium alginate could successfully be used as lipase and protease carrier systems in order to accelerate the fat and protein breakdown process during Kashar cheese ripening. The use of this system could be considered as a way to avoid the flavor defects that usually result from the addition of free lipases or proteases to either cheese milk or curd. This system was relatively shortened Kashar cheese ripening.

This study confirms that the impact of encapsulated enzymes in cheese ripening is influenced greatly by the nature of the encapsulating gums themselves. Cheeses treated with ?-carrageenan capsules showed the highest rate of lipolysis and proteolysis compared to those treated with gellan or sodium alginate capsules. Sodium alginate capsules disrupted under cheese manufacturing conditions appeared to be more suitable than gum capsules as enzyme encapsulants for use in accelerating cheese ripening. However, the easily ruptured gum capsules under cheese manufacturing conditions may lead to the rapid release of enzymes and excessive lipolysis during early ripening.

ACKNOWLEDGEMENTS

This study was supported financially by the TUBITAK (Project No: 1060409).

Authors also thank Novozymes for enzyme supplies.

References

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