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Amino Nitrogen in Fruit Juice, Juice Concentrates and Fruit Drinks Determined with 2,4,6-Trinitrobenzene-sulfonic Acid
Can. Inst. Food Sci. Technol. J. Vol. 18. No. 3. pp. 259-262. 1985
RESEARCH NOTE
Amino Nitrogen in Fruit Juice, Juice Concentrates and Fruit Drinks Determined with 2,4,6-Trinitrobenzene-sulfonic Acid T. Beveridge and J.E. Harrison Agriculture Canada Research Station Summerland, British Columbia VOH IZO (Canada)
ing the authenticity of fruit juices and fruit juice concentrates (Vandercook and Price, 1972; Hamed et al., 1974; Wrolstad et al., 1981). In addition, it is well established (Adrian, 1982) that amino compounds react with sugar carbonyls ultimately resulting in formation of brown pigments and this reaction between reducing sugars and free amino compounds in fruit juices and juice concentrates results in browning of these products during processing and storage (Cornwell and Wrolstad, 1981; Beveridge and Harrison, 1984). Knowledge of the level of amino compounds in juices and juice concentrates may prove useful in predicting browning potential and in following the course of this non-enzymatic browning. A simple method for the analysis of amino compounds in fruit juices would be desirable. Commonly, amino compounds in fruit juices are determined by formol titration or reaction with ninhydrin (Vandercook, 1977) but formol titration is somewhat non specific and lacks precision (Pomeranz and Meloan, 1978) and the ninhydrin color reaction requires expensive, unpleasant chemicals and a heating step (Blackburn, 1968; Moore, 1968). Hils (1974) described a method for amino nitrogen utilizing trinitrobenzene sulfonic acid but this work has not been widely applied. Recently, Kwan et al. (1983) describe a simple method for the determination of amino nitrogen in protein hydrolysates utilizing trinitrobenzene sulfonic acid. This paper reports the adaptation of Kwan's methodology to fruit juices and juice concentrates and its application to a number of liquid fruit products available in British Columbia.
Abstract Indices of ex amino nitrogen levels in various juices, juice concentrates and liquid fruit products were determined by a modified 2,4,6-trinitrobenzenesulfonic acid (TNBS) method. This method correlated well with the Formol titration method (r 2 = 0.835; p ~ 0.01), and was found to be as precise, more rapid, and more convenient for a large number of samples. Recoveries of glycine in spiked fruit juices were 100-104%. Relative extinction coefficients of the reaction mixtures of 14 different amino acids with TNBS ranged from 10.3-18.5. Larger variations were obtained with proline and lysine which had respective extinction coefficients of zero and 33.3. This method was clearly capable of distinguishing authentic juices from drink products; however, its ability to detect dilution or adulteration of authentic juice remains to be shown.
Resume A l'aide d'une methode modifiee a l'acide 2,4,6-trinitrobenzenesulfonique (TNBS), on a determine les indices d'azote x-amine dans une variete de jus, de jus concentres et de produits fruitiers liquides. Cette methode a bien correle (r 2 = 0.835; p ~ 0.01) avec la methode de titration au formol et dans le cas d'un grand nombre d'echantillons, elle fut egalement precise, plus rapide et plus commode. Les recuperations de glycine dans des jus de fruit akoolises furent 100-104%. Les coefficients d'extinction re1atifs des melanges de reaction de 14 acides amines differents avec TNBS ont varies entre 10.3 et 18.5. Des variations plus considerables furent obtenues avec la proline et la lysine dont les coefficients d'extinction furent respectivement zero et 33.3. Cette methode fut en mesure de distinguer clairement entre les jus authentiques et les breuvages. Toutefois, il n'a pas ete etabli si elle pouvait deceler le mouillage ou la falsification des jus authentiques.
Introduction Total amino acids, determined by formol titration, has been widely used to detect adulteration in citrus products (Vandercook, 1977). Also, it seems that fruit free amino acids are useful indicators for determin-
Materials and Methods Reagent grade chemicals and distilled water were used throughout. Juices were used as received and con-
'Contribution Number 614
Copyright
Q
1985 Canadian Institute of Food Science and Technology
259
centrates were diluted according to package directions and treated as juice. Cloudy or pulpy juices were centrifuged at 27000 g (Sorval RC-5; 10 min, 20°C) and all juices were diluted (1: 10 to 1:250) with distilled water prior to analysis. Soluble solids were determined refractrometrically on juice or diluted concentrate as *Brix. Solids in pear concentrates were determined by freeze-drying diluted aliquots. To 1 mL of diluted sample, 2 mL Na2 B40 7*IOH20 solution saturated at room temperature was added, followed by 1 mL 4mM TNBS (Trinitrobenzenesulfonic acid; Sigma Chemical Co.). After thorough mixing, yellow color was allowed to develop 1 h then 1 mL of 2.0 M NaH 2P0 4 containing 18 mM Na 2S0 3 was added. After mixing, the color was read at 410 nm on a Unicam SPI800 spectrophotometer utilizing 1 cm cells. The colors obtained were compared to those obtained from standard glycine solutions and results are reported in terms of glycine. For highly colored juices (grape and cranberry) a juice blank with TNBS omitted was prepared in addition to the normal TNBS reagent blank. All solutions were read against water and appropriate corrections made for the two blanks. The formol titration method was modified from AOAC (1975). Titration was performed with 0.5 N NaOH using a model TTT80 titrator fitted with a model PHM 82 pH meter (Radiometer, Copenhagen). The procedure was standardized against glycine and results reported in terms of glycine.
Results and Discussion Application of the TNBS method to fruit juices proved to be simple and to provide a rapid and inexpensive method to assess the levels of amino groups in fruit juices. RepeatabiIity was acceptable (Table 1), and recoveries of 0.0546 to 0.101 mM glycine added to pear juice ranged from 100 to 104 percent. Comparison of results obtained by the TNBS method with pear juice samples for which formol titration data had, been previously obtained (Beveridge and Harrison, 1984) is shown in Table 2. The two methods appear to be measuring similar components at similar levels as suggested by a significant correlation (r 2 = 0.835; P ~ 0.01) between the two methods .. This is to be expected since both formaldehyde and TNBS react with amino groups. When tested in pear juice, the precision of the TNBS procedure was better than the formol titration procedure (P ~ 0.05, F-test). However, when the variability encountered in the determination of the extinction coefficient is considered (Table 3), it appears the precision of the TNBS procedure is about equal to formol titration procedures. The TNBS method was more rapid and convenient especially when applied to large numbers of samples. Values for amino levels, expressed in terms of glycine, of several juices, juices from concentrates or juice preparations are shown in Table 1. These values should be interpreted as indices of amino nitrogen levels or as indices of free amino acid levels since all amino acids do not give the same color response in the test (Table 3, this paper; Satake et af., 1960). Also, some amino 260 / Beveridge and Harrison
Table I. Amino nitrogen levels in various juice products available in British Columbia expressed in terms of Glycine. e
Brix in Juice
mM Glycine
10.6
*10 e Brix Juice 4.41 4.50 ± 0.17 6.47 ± 0.10 3.35 ± 0.22 3.16
11.2
3.95 ± 0.42
3.53
11.6 10.8 14.8 10.8
4.55 ± 0.07 1.47 ± 0.01 6.60 2.97 ± 0.22
3.92 1.36 4.71 2.75
Cranberry Cocktail Cran apple Cocktail
13.8 17.7
0.82 ± 0.03 1.65 ± 0.00
0.59 0.93
Grape Juice (Purple) Grape Juice (White) Grape Juice (from concentrate) Grape Drink Grape Drink Grape Drink
16.5 15.9
± 1.11 ± 0.15
16.5 9.12
Product Apple Juice Apple Juice Apple Juice Apple Juice (from concentrate) Apple Juice (from concentrate) Apple Drink Apple Cot Nectar Apple-lime Juice
Orange Juice Orange Juice Orange Juice (from concentrate) Orange Drink (from concentrate) Orange Drink Orange Cot Nectar Five Alive Revive Grapefruit Juice (White) Grapefruit Juice (White) Grapefruit Juice (White from concentrate) Grapefruit Juice (Pink) Grapefruit Juice (Pink from concentrate) Grapefruit Juice (Honey Sweetened)
10.2
12.4 10.9 10.8 10.8
27.2 14.5 6.16 2.32 0.80 1.50
± ± ± ±
0.02 0.44 0.18 0.12
4.97 2.13 0.74 1.39
13.1 11.8
18.6 16.9
± 0.95 ± 1.15
14.2 14.3
9.5
14.6
± 0.78
15.2
10.4 10.4 14.2 10.4 11.6
5.60 7.60 6.57 10.7 18.9
± ± ± ±
5.38 7.31 4.63 10.3 16.3
26.1 23.5
± 0.23 ±1.3
31.8 25.5
23.3 ± 0.42 22.8 ± 0.28
22.8 25.9
9.4
25.0
± 0.00
26.6
13.4
25.1
± 0.46
18.7
8.2 9.2 10.2 8.8
0.00 0.06 0.56 0.15
Lemon Juice Lemon Juice (Fresh) Lemonade (From concentrate)
6.5 6.8
Tomato Juice Tomato Juice Tomato Juice
7.5 5.3 6.9
33.7 25.4 34.4
± 0.85 ± 0.66 ±I.I
44.9 47.9 49.8
Clamato Juice
12.0
47.6
± 4.8
39.7
Ceasar Cocktail
12.0
42.6
± 0.0
35.5
Garden Cocktail V-8 Shimato
5.5 5.3 5.1
30.6 ± 0.76 23.1 ± 0.60 37.1 ± 0.94
55.6 43.5 72.7
21.6 18.1 18.2
9.65 ± 0.0 17.6 ±0.Q7 24.5 ± 0.11
4.46 9.72 13.5
Prune Juice Prune Juice Prune Nectar
9.8
3.59 ± 1.3 16.6 ± 1.6 2.57 ± 0.04
5.52 24.4 2.62
14.1 ± 0.07 Pineaple Juice 12.3 11.5 Apricot Nectar 15.3 7.89 ± 0.06 5.15 Values of glycine are the means of four (4) determinations. Ranges shown are I standard deviation. e *lO Brix = values adjusted to a common soluble solids basis.
acids which are common in fruit juices, such as proline (Vandercook and Price, 1972; Wrolstad et al., 1981; Ooghe, 1982) do not react with 2,4,6-trinitroJ. Inst. Can. Sci. Technal. Aliment. Vol. 18. No. 3. 1985
Table 2. Amino nitrogen levels in stored pear juice concentrates determined by formol titration and with 2,4,6-Trinitrobenzen-sulfonic acid (TNBS). Storage Storage mM Glycine/lOO g Solids Concentrate Time Temperature ·Brix ·C (hr) Formol Titration TNBS Method 75.6 ± 14.5 o 80.8 ± 50 1.4 45.2 68.2 ± 31 8.9 72.6 ± 1.4 45.2 75.4 ± 3.7 o 81.6 ± 3.8 55.4 75.2 ± 15.8 31 1.7 73.7 ± 55.4 77.3 ± l.l 1.2 80.2 ± o 65.1 72.4 31 0.7 68.5 ± 65.1 75.6 ± 75.0 ± 13.0 2.8 o 72.5 31 0.8 55.0 ± 62.1 ± 72.5 66.9 ± 75.4 ± 80 o 0 2.3 45.2 4.4 2.8 71.8 ± 71.3 ± 8.6 45.2 68.4 ± 78.0 ± 5.6 0.8 55.4 o 4.4 2.8 60.3 66.1 ± 55.4 72.4 ± 72.0 ± 8.6 9.2 65.1 4.4 9.8 5.5 58.2 ± 61.6 ± 65.1 69.1 ± 65.7 ± 2.4 7.5 72.5 o 52.8 ± 45.6 ± 14.3 4.4 0.8 72.5 Assays performed in duplicate. ± Values shown are coefficient of variation (0J0) - not determined.
benzene-sulfonic acid and hence do not contribute to the total amino acid level as measured here. Another commonly occurring juice amino acid, aspartic acid, reacts more slowly than do the other amino acids tested and it was considered prudent to extend the reaction time from 30 min (Kwan et al., 1983) to 1 h to ensure complete reaction. Since the numbers obtained represent an index rather than an absolute level, there is little literature data against which the present values may be directly compared. However, Vandercook and Price (1972) report values for Orange Juice of 25.4 ± 5.9 mM based on formol titration ("Brix = 12.3 ± 0.5; ± values are standard deviations). These values are somewhat higher than those reported here while formol
Table 3 .Relative extinction coefficients at 410 nm of the reaction mixtures of several amino acids with 2,4,6-trinitrobenzenesulfonic acid. Relative Extinction Amino Acid Coefficient (ETc~) Arginine 15.0 ± 8.9 8.1 ± 10.7 Cysteine Histidine 12.3 ± 5.7 Glutamic Acid 12.5 ± 15.1 Glutamine 15.0 ± 8.7 Isoleucine 15.8 ± 9.3 Leucine 15.0 ± 11.3 Lysine 25.9 ± 7.0 Methionine 16.5 ± 6.6 Phenylalanine 14.9 ± 8.7 Proline 0.0 Threonine 15.2 ± 13.5 Tryptophane 15.7 ± 1.8 Valine 16.4 ± 11.0 Aspartic Acid 10.0 ± 14.6 Glycine 18.4 ± 4.1 ± Values are the % coefficient of variation determined on 8 replicate analysis. Can. Inst. Food Sei. Teehnol. J. Vo!. 18. No. 3. 1985
values of 16.4 to 22.7 mM reported by Vandercook (1977) for grapefruit juice are somewhat lower than values obtained by the TNBS method. Formol values for lemon juice (21.3 ± 3.3; Vandercook, 1977) are also higher than TNBS values obtained here. Total amino acid values of 17.7 mM for grapefruit and 19.8 mM for orange juice have been reported by Ooghe (1982) who also reports values of 4.3 mM for apple, 6.8 mM for white grapes and 8.7 mM for red grapes. In a study of a large number of table grapes, Kliewer (1969) reported the free amino acid content to lie between 10.4 and 64.5 mM (Kjeldahl) and to vary with grape maturity. Tomato juice contains about 39.5 mM (as glycine) while heated tomato juice contains about 23.2 mM amino acid (as glycine; Okitani et al., 1983). These latter values are in an agreeable range with present data. The method is clearly capable of distinguishing authentic juices from drink products which may contain little or no authentic fruit juice. This is evident, considering the difference between apple juice and apple drink, grape juice and grape drink and orange juice and orange drink, however, its ability to detect dilution or adulteration of authentic juice remains to be shown. As a means of following changes in amino nitrogen during processing or storage of fruit juices or concentrates, cheap, simply made reagents, combined with the lack of a heating step offer obvious advantages over the ninhydrin procedure (Cornwall and Wrolstad, 1981) while its spectrophotometric basis makes it preferable to the formol titration procedure when large numbers of samples are contemplated.
References Adrian, J. 1982. The Maillard Reaction. Handbook of Nutritive Value of Processed Food. Vol I. Food for human use. M. Recheigl, Jr. (Ed.). CRC Press Inc., Boca Raton. Florida.
Beveridge and Harrison / 261
AOAC, 1975. Official Methods of Analysis of the Association of Official Analytical Chemists. 12th ed. P.O. Box 540, Benjamin Franklin Station, Washington, D.e. 20044. Beveridge, T. and Harrison, LE. 1984. Non-enzymatic browning in pear juice concentrate during storage at high temperature. J. Food Sci. 49:1335. Blackburn, S. 1968. Amino acid determination. Methods and Techniques. pp. 69-80. Marcel Dekker Inc. New York. Cornwell, C.J. and Wrolstad, R.E. 1981. Causes of browning in pear juice concentrate during storage. J. Food Sci. 46:515. Hamed, M.O.E., EI-Wakeil, F.A., Foda, 1.0. and Heikal. 1974. Detection of accepted natural juices in carbonated beverage base and carbonated beverage prepared therefrom. Egypt. J. Food Sci. 2:41-57. Hils, A. 1974. Alpha amino nitrogen an additional criterion for the evaluation of orange juices. Fluess. Obst. 41 :6-9. Kliewer, W.M. 1969. Free amino acids and other nitrogenous substances of table grape varieties. J. Food Sci, 34:274. Kwan, K.K.H., Nakai, S. and Skura, B.J. 1983. Comparison of four methods for determining protease activity in milk. J. Food Sci. 48(5):1418. Moore, S. 1968. Amino acid analysis: Aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. BioI. Chem. 243:6281. Okitani, A., Kim, S-Y., Hayase, F., Chung, T-Y. and Kato, H. 1983. Heat-induced changes in free amino acids on manufacturing heated pulps, purees and pastes from tomatoes. J. Food Sci. 48:1366.
262 / Beveridge and Harrison
Ooghe, W. 1982. Possibilities of the Amino Acid analyser in the fruit juice analysis in Recent Developments in Food Analysis. W. Baltes, P.B. Czedik-Eysenberg and W. Pfannhauser (Eds.) p. 97-104. Verlay Chemie. Weinham. Deerfield Beach, Florida. Pomeranz, Y. and Meloan, e.E. 1978. Food Analysis: Theory and Practice (Revised ed.). p. 682. AVI. Publishing Co., Westport, Conn. Satake, K., Okuyama, T. Ohashi, M. and Shinoda, T. 1960. The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-Trinitrobenzene-l-sulfonic acid. J. Biochem. 47:654. Vandercook, C.E. and Price, R.L. 1972. The application of amino acid composition to the characterization of citrus juice. J. Food Sci. 37:384-386. Vandercook, C.E. 1977. Nitrogenous Compounds in Citrus Science and Technology, Vol. I. S. Nagy, P.E. Shaw and M.K. Velclhuis. (Eds.). p. 229-265. AVI Publishing Co., Westport, Conn. p. 229-265. Wrolstad, R.E., Cornwell, C.J., Culbertson, J.D. and Reyes, F.O.R. 1981. Establishing criteria for determining the authenticity of fruit juice concentrates in Quality of Selected Fruits and Vegetables. ACS Symposium Series 170. R. Teranishi and H. Barrera-Benitez (Eds.). American Chemical Society. Accepted April 29, 1985
J. Inst. Can. Sei. Teehnol. Aliment. Vol. 18, No. 3. 1985
RESEARCH NOTE
Amino Nitrogen in Fruit Juice, Juice Concentrates and Fruit Drinks Determined with 2,4,6-Trinitrobenzene-sulfonic Acid T. Beveridge and J.E. Harrison Agriculture Canada Research Station Summerland, British Columbia VOH IZO (Canada)
ing the authenticity of fruit juices and fruit juice concentrates (Vandercook and Price, 1972; Hamed et al., 1974; Wrolstad et al., 1981). In addition, it is well established (Adrian, 1982) that amino compounds react with sugar carbonyls ultimately resulting in formation of brown pigments and this reaction between reducing sugars and free amino compounds in fruit juices and juice concentrates results in browning of these products during processing and storage (Cornwell and Wrolstad, 1981; Beveridge and Harrison, 1984). Knowledge of the level of amino compounds in juices and juice concentrates may prove useful in predicting browning potential and in following the course of this non-enzymatic browning. A simple method for the analysis of amino compounds in fruit juices would be desirable. Commonly, amino compounds in fruit juices are determined by formol titration or reaction with ninhydrin (Vandercook, 1977) but formol titration is somewhat non specific and lacks precision (Pomeranz and Meloan, 1978) and the ninhydrin color reaction requires expensive, unpleasant chemicals and a heating step (Blackburn, 1968; Moore, 1968). Hils (1974) described a method for amino nitrogen utilizing trinitrobenzene sulfonic acid but this work has not been widely applied. Recently, Kwan et al. (1983) describe a simple method for the determination of amino nitrogen in protein hydrolysates utilizing trinitrobenzene sulfonic acid. This paper reports the adaptation of Kwan's methodology to fruit juices and juice concentrates and its application to a number of liquid fruit products available in British Columbia.
Abstract Indices of ex amino nitrogen levels in various juices, juice concentrates and liquid fruit products were determined by a modified 2,4,6-trinitrobenzenesulfonic acid (TNBS) method. This method correlated well with the Formol titration method (r 2 = 0.835; p ~ 0.01), and was found to be as precise, more rapid, and more convenient for a large number of samples. Recoveries of glycine in spiked fruit juices were 100-104%. Relative extinction coefficients of the reaction mixtures of 14 different amino acids with TNBS ranged from 10.3-18.5. Larger variations were obtained with proline and lysine which had respective extinction coefficients of zero and 33.3. This method was clearly capable of distinguishing authentic juices from drink products; however, its ability to detect dilution or adulteration of authentic juice remains to be shown.
Resume A l'aide d'une methode modifiee a l'acide 2,4,6-trinitrobenzenesulfonique (TNBS), on a determine les indices d'azote x-amine dans une variete de jus, de jus concentres et de produits fruitiers liquides. Cette methode a bien correle (r 2 = 0.835; p ~ 0.01) avec la methode de titration au formol et dans le cas d'un grand nombre d'echantillons, elle fut egalement precise, plus rapide et plus commode. Les recuperations de glycine dans des jus de fruit akoolises furent 100-104%. Les coefficients d'extinction re1atifs des melanges de reaction de 14 acides amines differents avec TNBS ont varies entre 10.3 et 18.5. Des variations plus considerables furent obtenues avec la proline et la lysine dont les coefficients d'extinction furent respectivement zero et 33.3. Cette methode fut en mesure de distinguer clairement entre les jus authentiques et les breuvages. Toutefois, il n'a pas ete etabli si elle pouvait deceler le mouillage ou la falsification des jus authentiques.
Introduction Total amino acids, determined by formol titration, has been widely used to detect adulteration in citrus products (Vandercook, 1977). Also, it seems that fruit free amino acids are useful indicators for determin-
Materials and Methods Reagent grade chemicals and distilled water were used throughout. Juices were used as received and con-
'Contribution Number 614
Copyright
Q
1985 Canadian Institute of Food Science and Technology
259
centrates were diluted according to package directions and treated as juice. Cloudy or pulpy juices were centrifuged at 27000 g (Sorval RC-5; 10 min, 20°C) and all juices were diluted (1: 10 to 1:250) with distilled water prior to analysis. Soluble solids were determined refractrometrically on juice or diluted concentrate as *Brix. Solids in pear concentrates were determined by freeze-drying diluted aliquots. To 1 mL of diluted sample, 2 mL Na2 B40 7*IOH20 solution saturated at room temperature was added, followed by 1 mL 4mM TNBS (Trinitrobenzenesulfonic acid; Sigma Chemical Co.). After thorough mixing, yellow color was allowed to develop 1 h then 1 mL of 2.0 M NaH 2P0 4 containing 18 mM Na 2S0 3 was added. After mixing, the color was read at 410 nm on a Unicam SPI800 spectrophotometer utilizing 1 cm cells. The colors obtained were compared to those obtained from standard glycine solutions and results are reported in terms of glycine. For highly colored juices (grape and cranberry) a juice blank with TNBS omitted was prepared in addition to the normal TNBS reagent blank. All solutions were read against water and appropriate corrections made for the two blanks. The formol titration method was modified from AOAC (1975). Titration was performed with 0.5 N NaOH using a model TTT80 titrator fitted with a model PHM 82 pH meter (Radiometer, Copenhagen). The procedure was standardized against glycine and results reported in terms of glycine.
Results and Discussion Application of the TNBS method to fruit juices proved to be simple and to provide a rapid and inexpensive method to assess the levels of amino groups in fruit juices. RepeatabiIity was acceptable (Table 1), and recoveries of 0.0546 to 0.101 mM glycine added to pear juice ranged from 100 to 104 percent. Comparison of results obtained by the TNBS method with pear juice samples for which formol titration data had, been previously obtained (Beveridge and Harrison, 1984) is shown in Table 2. The two methods appear to be measuring similar components at similar levels as suggested by a significant correlation (r 2 = 0.835; P ~ 0.01) between the two methods .. This is to be expected since both formaldehyde and TNBS react with amino groups. When tested in pear juice, the precision of the TNBS procedure was better than the formol titration procedure (P ~ 0.05, F-test). However, when the variability encountered in the determination of the extinction coefficient is considered (Table 3), it appears the precision of the TNBS procedure is about equal to formol titration procedures. The TNBS method was more rapid and convenient especially when applied to large numbers of samples. Values for amino levels, expressed in terms of glycine, of several juices, juices from concentrates or juice preparations are shown in Table 1. These values should be interpreted as indices of amino nitrogen levels or as indices of free amino acid levels since all amino acids do not give the same color response in the test (Table 3, this paper; Satake et af., 1960). Also, some amino 260 / Beveridge and Harrison
Table I. Amino nitrogen levels in various juice products available in British Columbia expressed in terms of Glycine. e
Brix in Juice
mM Glycine
10.6
*10 e Brix Juice 4.41 4.50 ± 0.17 6.47 ± 0.10 3.35 ± 0.22 3.16
11.2
3.95 ± 0.42
3.53
11.6 10.8 14.8 10.8
4.55 ± 0.07 1.47 ± 0.01 6.60 2.97 ± 0.22
3.92 1.36 4.71 2.75
Cranberry Cocktail Cran apple Cocktail
13.8 17.7
0.82 ± 0.03 1.65 ± 0.00
0.59 0.93
Grape Juice (Purple) Grape Juice (White) Grape Juice (from concentrate) Grape Drink Grape Drink Grape Drink
16.5 15.9
± 1.11 ± 0.15
16.5 9.12
Product Apple Juice Apple Juice Apple Juice Apple Juice (from concentrate) Apple Juice (from concentrate) Apple Drink Apple Cot Nectar Apple-lime Juice
Orange Juice Orange Juice Orange Juice (from concentrate) Orange Drink (from concentrate) Orange Drink Orange Cot Nectar Five Alive Revive Grapefruit Juice (White) Grapefruit Juice (White) Grapefruit Juice (White from concentrate) Grapefruit Juice (Pink) Grapefruit Juice (Pink from concentrate) Grapefruit Juice (Honey Sweetened)
10.2
12.4 10.9 10.8 10.8
27.2 14.5 6.16 2.32 0.80 1.50
± ± ± ±
0.02 0.44 0.18 0.12
4.97 2.13 0.74 1.39
13.1 11.8
18.6 16.9
± 0.95 ± 1.15
14.2 14.3
9.5
14.6
± 0.78
15.2
10.4 10.4 14.2 10.4 11.6
5.60 7.60 6.57 10.7 18.9
± ± ± ±
5.38 7.31 4.63 10.3 16.3
26.1 23.5
± 0.23 ±1.3
31.8 25.5
23.3 ± 0.42 22.8 ± 0.28
22.8 25.9
9.4
25.0
± 0.00
26.6
13.4
25.1
± 0.46
18.7
8.2 9.2 10.2 8.8
0.00 0.06 0.56 0.15
Lemon Juice Lemon Juice (Fresh) Lemonade (From concentrate)
6.5 6.8
Tomato Juice Tomato Juice Tomato Juice
7.5 5.3 6.9
33.7 25.4 34.4
± 0.85 ± 0.66 ±I.I
44.9 47.9 49.8
Clamato Juice
12.0
47.6
± 4.8
39.7
Ceasar Cocktail
12.0
42.6
± 0.0
35.5
Garden Cocktail V-8 Shimato
5.5 5.3 5.1
30.6 ± 0.76 23.1 ± 0.60 37.1 ± 0.94
55.6 43.5 72.7
21.6 18.1 18.2
9.65 ± 0.0 17.6 ±0.Q7 24.5 ± 0.11
4.46 9.72 13.5
Prune Juice Prune Juice Prune Nectar
9.8
3.59 ± 1.3 16.6 ± 1.6 2.57 ± 0.04
5.52 24.4 2.62
14.1 ± 0.07 Pineaple Juice 12.3 11.5 Apricot Nectar 15.3 7.89 ± 0.06 5.15 Values of glycine are the means of four (4) determinations. Ranges shown are I standard deviation. e *lO Brix = values adjusted to a common soluble solids basis.
acids which are common in fruit juices, such as proline (Vandercook and Price, 1972; Wrolstad et al., 1981; Ooghe, 1982) do not react with 2,4,6-trinitroJ. Inst. Can. Sci. Technal. Aliment. Vol. 18. No. 3. 1985
Table 2. Amino nitrogen levels in stored pear juice concentrates determined by formol titration and with 2,4,6-Trinitrobenzen-sulfonic acid (TNBS). Storage Storage mM Glycine/lOO g Solids Concentrate Time Temperature ·Brix ·C (hr) Formol Titration TNBS Method 75.6 ± 14.5 o 80.8 ± 50 1.4 45.2 68.2 ± 31 8.9 72.6 ± 1.4 45.2 75.4 ± 3.7 o 81.6 ± 3.8 55.4 75.2 ± 15.8 31 1.7 73.7 ± 55.4 77.3 ± l.l 1.2 80.2 ± o 65.1 72.4 31 0.7 68.5 ± 65.1 75.6 ± 75.0 ± 13.0 2.8 o 72.5 31 0.8 55.0 ± 62.1 ± 72.5 66.9 ± 75.4 ± 80 o 0 2.3 45.2 4.4 2.8 71.8 ± 71.3 ± 8.6 45.2 68.4 ± 78.0 ± 5.6 0.8 55.4 o 4.4 2.8 60.3 66.1 ± 55.4 72.4 ± 72.0 ± 8.6 9.2 65.1 4.4 9.8 5.5 58.2 ± 61.6 ± 65.1 69.1 ± 65.7 ± 2.4 7.5 72.5 o 52.8 ± 45.6 ± 14.3 4.4 0.8 72.5 Assays performed in duplicate. ± Values shown are coefficient of variation (0J0) - not determined.
benzene-sulfonic acid and hence do not contribute to the total amino acid level as measured here. Another commonly occurring juice amino acid, aspartic acid, reacts more slowly than do the other amino acids tested and it was considered prudent to extend the reaction time from 30 min (Kwan et al., 1983) to 1 h to ensure complete reaction. Since the numbers obtained represent an index rather than an absolute level, there is little literature data against which the present values may be directly compared. However, Vandercook and Price (1972) report values for Orange Juice of 25.4 ± 5.9 mM based on formol titration ("Brix = 12.3 ± 0.5; ± values are standard deviations). These values are somewhat higher than those reported here while formol
Table 3 .Relative extinction coefficients at 410 nm of the reaction mixtures of several amino acids with 2,4,6-trinitrobenzenesulfonic acid. Relative Extinction Amino Acid Coefficient (ETc~) Arginine 15.0 ± 8.9 8.1 ± 10.7 Cysteine Histidine 12.3 ± 5.7 Glutamic Acid 12.5 ± 15.1 Glutamine 15.0 ± 8.7 Isoleucine 15.8 ± 9.3 Leucine 15.0 ± 11.3 Lysine 25.9 ± 7.0 Methionine 16.5 ± 6.6 Phenylalanine 14.9 ± 8.7 Proline 0.0 Threonine 15.2 ± 13.5 Tryptophane 15.7 ± 1.8 Valine 16.4 ± 11.0 Aspartic Acid 10.0 ± 14.6 Glycine 18.4 ± 4.1 ± Values are the % coefficient of variation determined on 8 replicate analysis. Can. Inst. Food Sei. Teehnol. J. Vo!. 18. No. 3. 1985
values of 16.4 to 22.7 mM reported by Vandercook (1977) for grapefruit juice are somewhat lower than values obtained by the TNBS method. Formol values for lemon juice (21.3 ± 3.3; Vandercook, 1977) are also higher than TNBS values obtained here. Total amino acid values of 17.7 mM for grapefruit and 19.8 mM for orange juice have been reported by Ooghe (1982) who also reports values of 4.3 mM for apple, 6.8 mM for white grapes and 8.7 mM for red grapes. In a study of a large number of table grapes, Kliewer (1969) reported the free amino acid content to lie between 10.4 and 64.5 mM (Kjeldahl) and to vary with grape maturity. Tomato juice contains about 39.5 mM (as glycine) while heated tomato juice contains about 23.2 mM amino acid (as glycine; Okitani et al., 1983). These latter values are in an agreeable range with present data. The method is clearly capable of distinguishing authentic juices from drink products which may contain little or no authentic fruit juice. This is evident, considering the difference between apple juice and apple drink, grape juice and grape drink and orange juice and orange drink, however, its ability to detect dilution or adulteration of authentic juice remains to be shown. As a means of following changes in amino nitrogen during processing or storage of fruit juices or concentrates, cheap, simply made reagents, combined with the lack of a heating step offer obvious advantages over the ninhydrin procedure (Cornwall and Wrolstad, 1981) while its spectrophotometric basis makes it preferable to the formol titration procedure when large numbers of samples are contemplated.
References Adrian, J. 1982. The Maillard Reaction. Handbook of Nutritive Value of Processed Food. Vol I. Food for human use. M. Recheigl, Jr. (Ed.). CRC Press Inc., Boca Raton. Florida.
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AOAC, 1975. Official Methods of Analysis of the Association of Official Analytical Chemists. 12th ed. P.O. Box 540, Benjamin Franklin Station, Washington, D.e. 20044. Beveridge, T. and Harrison, LE. 1984. Non-enzymatic browning in pear juice concentrate during storage at high temperature. J. Food Sci. 49:1335. Blackburn, S. 1968. Amino acid determination. Methods and Techniques. pp. 69-80. Marcel Dekker Inc. New York. Cornwell, C.J. and Wrolstad, R.E. 1981. Causes of browning in pear juice concentrate during storage. J. Food Sci. 46:515. Hamed, M.O.E., EI-Wakeil, F.A., Foda, 1.0. and Heikal. 1974. Detection of accepted natural juices in carbonated beverage base and carbonated beverage prepared therefrom. Egypt. J. Food Sci. 2:41-57. Hils, A. 1974. Alpha amino nitrogen an additional criterion for the evaluation of orange juices. Fluess. Obst. 41 :6-9. Kliewer, W.M. 1969. Free amino acids and other nitrogenous substances of table grape varieties. J. Food Sci, 34:274. Kwan, K.K.H., Nakai, S. and Skura, B.J. 1983. Comparison of four methods for determining protease activity in milk. J. Food Sci. 48(5):1418. Moore, S. 1968. Amino acid analysis: Aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J. BioI. Chem. 243:6281. Okitani, A., Kim, S-Y., Hayase, F., Chung, T-Y. and Kato, H. 1983. Heat-induced changes in free amino acids on manufacturing heated pulps, purees and pastes from tomatoes. J. Food Sci. 48:1366.
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Ooghe, W. 1982. Possibilities of the Amino Acid analyser in the fruit juice analysis in Recent Developments in Food Analysis. W. Baltes, P.B. Czedik-Eysenberg and W. Pfannhauser (Eds.) p. 97-104. Verlay Chemie. Weinham. Deerfield Beach, Florida. Pomeranz, Y. and Meloan, e.E. 1978. Food Analysis: Theory and Practice (Revised ed.). p. 682. AVI. Publishing Co., Westport, Conn. Satake, K., Okuyama, T. Ohashi, M. and Shinoda, T. 1960. The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-Trinitrobenzene-l-sulfonic acid. J. Biochem. 47:654. Vandercook, C.E. and Price, R.L. 1972. The application of amino acid composition to the characterization of citrus juice. J. Food Sci. 37:384-386. Vandercook, C.E. 1977. Nitrogenous Compounds in Citrus Science and Technology, Vol. I. S. Nagy, P.E. Shaw and M.K. Velclhuis. (Eds.). p. 229-265. AVI Publishing Co., Westport, Conn. p. 229-265. Wrolstad, R.E., Cornwell, C.J., Culbertson, J.D. and Reyes, F.O.R. 1981. Establishing criteria for determining the authenticity of fruit juice concentrates in Quality of Selected Fruits and Vegetables. ACS Symposium Series 170. R. Teranishi and H. Barrera-Benitez (Eds.). American Chemical Society. Accepted April 29, 1985
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