28 Biogeneration of Aromas Downloaded from pubs.acs.org by UNIV OF PITTSBURGH on 05/05/16. For personal use only.
Production of a Romano Cheese Flavor by Enzymic Modification of Butterfat 1
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Kuo-Chung M . Lee, Huang Shi , An-Shun Huang , James T. Carlin , Chi-Tang Ho, and Stephen S. Chang Department of Food Science, Cook College, New Jersey Agricultural Experiment Station, Rutgers University-The State University of New Jersey, New Brunswick, NJ 08903
A Romano cheese-like aroma was produced from a butterfat emulsion by treating it with a crude enzyme mixture isolated from Candida rugosa. The emulsion consisted of 20% butterfat and 1.5% Tween 80 in a buffer solution. The treated emulsion was held at 37°C for three hours and then aged at room temperature for three days to develop the cheese-like flavor. The volatile flavor components were isolated from both the enzyme modified butterfat (EMB) and a commercial sample of Romano cheese. The flavor isolates were separated into acidic and nonacidic fractions and analyzed by gas chromatography-mass spectrometry. The results showed good correlation between the acidic fractions of the two samples. The acidic fractions contained similar relative concentrations of eight short-chain fatty acids (C2 - C 1 0 ) . Methyl ketones and esters were major components in the nonacidic fraction of the EMB. The characteristic flavor of various cheeses is primarily due to the enzymatic action of microbial flora contained in the curd. Enzymes extracted from these microorganisms and reacted with corresponding substrates may also produce a specific cheese flavor. The flavor produced may be economical and could be classified as "natural". Several varieties of the popular Italian cheeses owe their characteristic flavor to the action of lipolytic enzymes. Romano is a very hard, ripened cheese. Originally, it was made from ewe's milk; it is now also made from cow's and goat's milk. The sharp, peppery-like flavor, traditionally termed "piquant", results from extensive lipolysis ( 1). Long and Harper (2) and Arnold et al. (3) Current address: Scientific Research Institute of Fermentation Industry, Ministry of Light Industry, Beijing, People's Republic of China 2 Current address: Nabisco Brands, Inc., Morristown, NJ 07960 3 Current address: Thomas J. Lipton, Inc., Englewood Cliffs, NJ 07632 1
0097-6156/ 86/ 0317-0370506.00/ 0 © 1986 American Chemical Society
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reported that the development of a desirable, c h a r a c t e r i s t i c Romano cheese flavor i s related to the short-chain fatty acids, e s p e c i a l l y butanoic acid. The production of butanoic acid c l o s e l y p a r a l l e l e d the development of flavor. The lipase system which f a i l e d to produce a high concentration of butanoic acid also f a i l e d to produce the desirable Romano cheese flavor. Crude rennet pastes or dried glandular enzymes of suckling young mammals are used for coagulation of milk i n the production of I t a l i a n cheeses. The l i p o l y t i c enzyme responsible for the production of the c h a r a c t e r i s t i c "piquant" flavor i s a pregastric esterase in the rennet. Rennets from microorganisms, such as Aspergillus niger and _A. oryzae, also have been used i n making I t a l i a n cheeses. A microbial esterase, Mucor miehei esterase, has been extensively studied for flavor development i n cheese and was found to produce flavor notes resembling those of Fontina and Romano cheeses ( 40 · The technology has been developed for production of flavor systems v i a controlled enzyme modification of butterfat (EMB). Lipases and esterases from various sources are used (_5). Nelson (6^) described the e s s e n t i a l steps for producing lipolyzed butterfat products. Arnold et a l . (_3) published a comprehensive review on the application of l i p o l y t i c enzymes. The purpose of this study i s to investigate the production of a Romano cheese-like flavor by enzyme modification of butterfat. Candida rugosa was selected for enzyme modification of butterfat since i t possesses a high lipase a c t i v i t y . Materials and Methods Enzyme Preparation. A crude enzyme mixture was prepared from the fermentate of Candida rugosa (American Type Culture C o l l e c t i o n , ATCC No. 14830. The C^. rugosa was revived and cultivated i n YM Agar slants at 24°C for one week. The growth on one YM Agar slant was transferred a s e p t i c a l l y to a two-liter flask containing 400 ml steri l i z e d fermentation medium consisting of 2.0% defatted soyflour, 1.0% potato starch, 1.0% maltose, 0.5% K 2 H P O 4 , 0.1% MgS04'7H 0 and 0.1% (NH4)2S04. Fermentation was carried out i n an incubator shaken at 200 rpm and 25°C for 20 hours. The fermented broth was f i l t e r e d through several layers of cheesecloth and was centrifuged at 600xG for 10 minutes to obtain the c e l l - f r e e supernatant. The supernatant was precipitated with cold acetone and separated by u l t r a c e n t r i f u g a t i o n at 25,000xG for 2 hours. The precipitate was then freeze-dried. The enzyme powder obtained was assayed to determine the optimum pH and temperature for fatty acids production (7). A 20% butterfat emulsion was used as the enzyme substrate instead of an o l i v e o i l emulsion. The optimum pH for fatty acid production was 7.7. The optimum temperature was 37°C. 2
Flavor Development. The crude enzyme mixture isolated from C^. rugosa was allowed to react with a butterfat emulsion consisting of 20% butterfat and 1.5% Tween 80 (Polyoxyethylenesorbitan monooleate) in a 0.1 M sodium phosphate buffer solution. Butterfat was prepared from Land 0'Lakes unsalted butter (Arden H i l l s , MN). Butter was melted and washed with hot water (50°C) i n a separatory funnel
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several times u n t i l the aqueous phase became c l e a r . The washed but t e r f a t was centrifuged at 600xG for 10 minutes to separate and re move water. The emulsion (pH 7.7) was prepared by passing the i n gredients through a hand homogenizer at least twice. Enzyme powder consisting of 1.5% of the emulsion was dissolved into the buffer solution and homogenized with the butterfat emulsion. It was incu bated at 37°C for three hours. This resulted i n the development of a cheese-like flavor. The emulsion was then aged for three days at room temperature to complete the flavor development. The aroma and flavor-by-mouth of the EMB were evaluated by a panel of five trained f l a v o r i s t s . A neat sample of the EMB was evaluated for aroma only. The aroma and flavor-by-mouth of the sam ple were evaluated i n a 0.5% NaCl solution at a l e v e l of 0.6%. Panelists independently provided a l i s t of product descriptions. The panel summarized i t s results i n a concensus discussion following the evaluation. Isolation of V o l a t i l e Flavor Compounds from the EMB Sample, Romano Cheese and Butterfat. The v o l a t i l e flavor compounds were isolated from the EMB sample, a commercial sample of Romano cheese and a butterfat control sample by vacuum steam d i s t i l l a t i o n . Vola t i l e s were isolated from 2.5L EMB i n five batch i s o l a t i o n s . The EMB was mixed i n a Waring blender prior to each i s o l a t i o n . Romano cheese was obtained from a commercial source ( S t e l l a Romano cheese, Universal Foods Corp., Milwaukee, Wl). V o l a t i l e s were isolated from 700 gm Romano cheese i n five batch i s o l a t i o n s . One hundred and for ty grams of cheese were cut into pieces for each i s o l a t i o n and s l u r r i e d with 360 ml 0.1% sodium phosphate buffer solution i n a blender. V o l a t i l e s were also isolated from 500 ml of butterfat emulsion con t r o l sample (20% b u t t e r f a t ) . The s l u r r y samples of EMB, Romano cheese and butterfat were vacuum d i s t i l l e d at 50°C for 8 hours. The v o l a t i l e s were condensed in a series of cold traps cooled with a dry ice-acetone s l u r r y . The condensates collected i n the traps were combined, saturated with sodium chloride and extracted with ethyl ether. The ether extracts were separated into acidic and nonacidic fractions by extraction with a 10% aqueous sodium carbonate solu t i o n . The ether solutions of a c i d i c and nonacidic fractions were dried over anhydrous sodium sulfate and subjected to a preliminary concentration using a 30-plate Oldershaw d i s t i l l a t i o n column. The ether extracts from both fractions were concentrated to f i n a l v o l umes of 5 ml using a spinning band d i s t i l l a t i o n apparatus. GC-MS Analysis and I d e n t i f i c a t i o n . A portion of the acidic frac tions were quantitatively converted to methyl esters using a B F 3 methanol reagent. The procedure used was that of Metcalfe and Schmitz (8). The e s t e r i f i e d a c i d i c fractions were analyzed using a coupled gas chromatograph-mass spectrometer (GC-MS) system consist ing of a Varian Moduline 2700 gas chromatograph equipped with a flame ionization detector interfaced by a jet separator to a Du Pont Instruments Model 21-490 mass spectrometer. A 10 f t χ 1/8 i n ο d stainless steel column packed with 10% s t a b i l i z e d DEGS on 80/ 100 mesh Chromosorb W AW DWCS was used. The He flow rate was 30 ml/min. The column temperature was held 5 minutes at 30°C, then
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programmed a t 4°C/min t o a f i n a l temperature o f 200°C. Mass s p e c t r a were o b t a i n e d a t 70eV and a s o u r c e temperature o f 250°C. The gas chromatograms g e n e r a t e d from t h e GC-MS a n a l y s e s were r e c o r d e d and i n t e g r a t e d u s i n g a H e w l e t t - P a c k a r d 5840A GC t e r m i n a l . The n o n a c i d i c f r a c t i o n s were a n a l y z e d u s i n g a c o u p l e d GC-MS system. The system c o n s i s t e d o f a H e w l e t t - P a c k a r d 5840A gas c h r o matograph and a H e w l e t t - P a c k a r d 5985 mass s p e c t r o m e t e r . A J&W f u s e d s i l i c a c a p i l l a r y column (30m χ 0.25mm)coated w i t h DB-1 ( m e t h y l s i l i c o n ) was used f o r t h e a n a l y s i s . The column was h e l d f o r 2 min at 40°C and then temperature programmed t o 250°C a t 4°C/min. Mass s p e c t r a were o b t a i n e d a t 70eV and a s o u r c e temperature o f 200°C. Compounds were i d e n t i f i e d by comparing t h e mass s p e c t r a ob t a i n e d w i t h those o f p u b l i s h e d r e f e r e n c e s p e c t r a . Approximate r e l a t i v e p e r c e n t a g e s o f t h e compounds i d e n t i f i e d i n t h e a c i d i c f r a c t i o n s were c a l c u l a t e d based on t h e c o n c e n t r a t i o n o f t h e most abundant com pound i d e n t i f i e d . R e s u l t s and D i s c u s s i o n A c r u d e enzyme m i x t u r e was i s o l a t e d from t h e f e r m e n t a t e o f C a n d i d a r u g o s a (ATCC No. 14,830), which i s r e p o r t e d t o produce h i g h a c t i v i t y l i p a s e s ( 9 ) . The enzyme m i x t u r e was added t o a 20% b u t t e r f a t emul sion. A c h e e s e - l i k e f l a v o r d e v e l o p e d a f t e r 3 hours o f i n c u b a t i o n a t 37°C. A d e s i r a b l e Romano cheese note d e v e l o p e d a f t e r c o n t i n u e d i n c u b a t i o n a t room temperature f o r t h r e e days. N e l s o n (6^) s t u d i e d l i p o l y z e d b u t t e r f a t f l a v o r and c o n c l u d e d t h a t t h e s u r f a c e a c t i v e c h a r a c t e r i s t i c s o f b o t h f a t t y a c i d s and mono- and d i g l y c e r i d e s were i m p o r t a n t i n t h e l i p o l y z e d system. A tempering p e r i o d o f hours o r even days was u s u a l l y r e q u i r e d t o e s t a b l i s h e q u i l i b r i u m a t t h e i n t e r f a c e o f aqueous and f a t phases. He p o i n t e d out t h a t t h e l i p o l y z e d f l a v o r appeared t o i n t e n s i f y as t h e e q u i l i b r a t i o n proceeded and t h a t t h i s i n t e n s i f i c a t i o n was sometimes m i s t a k e n f o r r e s i d u a l lipolytic activity. Sensory E v a l u a t i o n s . Summary d e s c r i p t i o n s o f t h e aroma and f l a v o r by-mouth o f t h e EMB were e s t a b l i s h e d by a p a n e l o f f i v e t r a i n e d flavorists. The aroma o f t h e neat sample was d e s c r i b e d as s t r o n g cheesy, s t r o n g Romano c h e e s e - l i k e w i t h s l i g h t m i l k y , creamy, k e t o n i c and soapy n o t e s . The aroma o f t h e sample i n a 0.5% NaCl s o l u t i o n a t a l e v e l o f 0.6% was d e s c r i b e d as soapy and m i l k y w i t h s l i g h t Romano c h e e s e - l i k e , a c i d and k e t o n i c n o t e s . The f l a v o r - b y - m o u t h o f t h e sample i n t h e NaCl s o l u t i o n was d e s c r i b e d as c o n t a i n i n g s l i g h t sharp cheese, b i t t e r and waxy n o t e s . Tween 80 has an a s t r i n g e n t and b i t t e r t a s t e ( 1 0 ) . However a r e l a t e d study showed t h a t t h e b i t t e r n o t e i n t h e EMB was n o t due t o the Tween 80. Gum A r a b i c , which does n o t have a b i t t e r t a s t e ( 1 0 ) was used t o r e p l a c e Tween 80 as t h e e m u l s i f i e r . The sample p r o duced s t i l l had t h e b i t t e r and soapy n o t e . A b i t t e r , soapy c h a r a c t e r i s g e n e r a l l y found i n l i p o l y z e d p r o d u c t s (11) and may be r e l a t e d to t h e mono- and d i g l y c e r i d e s formed d u r i n g l i p o l y s i s . A c i d i c Components. The Romano c h e e s e - l i k e f l a v o r o f t h e enzymem o d i f i e d b u t t e r f a t l e d t o a study o f i t s v o l a t i l e f l a v o r compounds. T a b l e I l i s t s t h e compounds i d e n t i f i e d , t h e i r a b s o l u t e c o n c e n t r a -
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tions and the approximate r e l a t i v e percentages at which they were present based on the concentration of butanoic acid i n each sample of EMB and commercial Romano cheese. The composition of the samples i s very similar. Both contain eight η-fatty acids ( C - 10^* ^ addition, sorbic acid, a preservative, was present i n the commercial product. The quantity of the a c i d i c components isolated from the v o l a t i l e s of the EMB sample was more than three times greater than that of the commercial Romano cheese. Harper (12) reported that butanoic acid and other higher fatty acids may be related to the intensity and character of Romano cheese f l a vor. c
n
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2
Nonacidic Components. Figure 1 shows the t o t a l ion chromatograms obtained from the nonacidic fractions of the flavor i s o l a t e s . A t o t a l of 22 compounds were i d e n t i f i e d i n the EMB and 12 i n the Romano cheese. Table II l i s t s the v o l a t i l e flavor compounds i d e n t i f i e d i n the nonacidic f r a c t i o n s . The odd-numbered ketones i d e n t i f i e d i n both samples are common ly found i n mold-ripened cheeses, such as Blue cheese, and are re sponsible for t h e i r c h a r a c t e r i s t i c aroma. Large quantities of these compounds also have been found i n nonmold-ripened cheeses, such as Cheddar, Swiss and Romano. These compounds may arise by 3-oxidation of the appropriate fatty acids (14), or by decomposition of β-keto acids. A series of ethyl esters of fatty acids, from butanoate to tetradecanoate, were i d e n t i f i e d i n the EMB. Two esters, ethyl octanoate and ethyl nonanoate, were found i n Romano cheese. Esters are important flavor compounds i n cheeses; however, a high concen t r a t i o n of esters may cause a " f r u i t y " defect i n cheese flavor, γ- and 6-dodecalactone were i d e n t i f i e d i n the EMB sample as well as in Romano cheese. Lactones are well distributed i n food f l a v o r s . In cheese, a series of γ- and 6-lactones have been i d e n t i f i e d (11). p-Nonylphenol was i d e n t i f i e d i n the EMB sample. Phenolic com pounds usually cause a "phenolic" defect i n Gouda cheese, and could be generated from certain variants of l a c t o b a c i l l i . However, i t i s possible that phenolic compounds also play a role i n cheese flavor. Phenol and p-cresol were found i n Cheddar cheese and were included with guaiacol i n a synthetic Cheddar cheese flavor formulation (15). 2-Phenylethanol was i d e n t i f i e d i n the EMB sample and has a rose l i k e odor and taste. I t can be produced through fermentation and i s present i n many foods including wine (16) and bread (17). n-Hexylfuran was i d e n t i f i e d i n the EMB sample and may arise from a l i p i d source. 2-Ethyl-4,5-dimethyloxazole was also i d e n t i fied i n this sample. This compound has been previously i d e n t i f i e d only i n food systems subjected to heat treatment. Benzaldehyde and phenylacetaldehyde were i d e n t i f i e d i n Romano cheese. These two compounds also have been found i n Cheddar, Swiss and Blue cheeses. Benzaldehyde has a powerful, sweet, almond-like odor and exists i n many foods. Phenylacetaldehyde has a strong, sweet f l o r a l , penetrating aroma and i s found i n many cooked foods. In dairy products, phenylacetaldehyde probably i s formed from phenylalanine i n milk protein through enzymatic transamination, f o l lowed by decarboxylation. This compound also contributes to the malty defect of cultured milk products (18).
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Table I. V o l a t i l e Flavor Compounds Identified i n the A c i d i c Fractions of Enzyme Modified Butterfat (EMB), Romano Cheese and Untreated Butterfat*
Fatty Acid
Acetic Propanoic Butanoic Pentanoic Hexanoic Heptanoic Octanoic Decanoic
Absolute Concentration (mg/kg of f a t ) Relative Concentration (%) Untreated EMB Romano Untreated EMB Romano Butterfat Butterfat 1.3 3.7 2620.0 13.4 743.6 2.6 41.9 0.3
3.1 0.8 896.0 4.5 285.2 2.0 26.6 0.1
-
56.5
-
18.8
-
10.8 0.2
0.05 0.14 100.00 0.51 28.38 0.10 1.60 0.01
0.35 0.09 100.00 0.50 31.83 0.22 2.97
