PROLINE IN ARGENTINE HONEYS J. Di Gerónimo and R. Fritz Departamento de Química. Facultad de Ciencias Exactas y Naturales. Universidad Nacional de Mar del Plata. Funes 3350. 7600. Mar del Plata. Te: 0223-4756167; Fax: 0223-4753150 e-mail: [email protected] SUMMARY Proline content in Argentine honeys has been examined taking into account that proline is a free amino acid used as an indication of quality in honeys. The methodology makes use of the coloured complex developed between proline and ninhydrin, then by the spectrophotometric method the amino acids are quantified. The concentration of proline is calculated through a calibration curve obtained with increasing quantities of standard proline, submitted to the same treatment that the sample and expressed as a proportion of the mass of honey in milligrams per kilogram. Proline content has been determined in 40 commercial samples. Content mean value was 356,1 ppm, standard deviation was 117,7, minimum and maximum values were 73,3 ppm and 577,3 ppm respectively. Keywords: honey, proline, spectrophotometric method. INTRODUCTION Honey contains a few amounts of amino acids and the proline is the most important. It represents the 50-85% in respect to the others (Konamine, 1960; Davies, 1975; Serra Bonvehi and Escola Jorda, 1997). The study of this analytical parameter has been of interest in many investigations. The analysis of proline in natural honey should point out the determination of ripeness and genuiness. Most of the proline in the honeys originates from the secretion of honeybees. (von der Ohe et al., 1991). A comparison between honey samples from A. dorsata, A. cercana and A. mellifera was done to clarify the proline origin. The values obtained were significantly different among the honey collected from the different bee species. The analytical values obtained were 875,8 323 and 610,2 ppm respectively (Joshi et al., 2000; Ihtishamulhaq, 1999). The other proportion of proline present in the honey has a vegetal origin. Contributions to the understanding of the botanical origin were carried out in different studies. A total of 59 honey samples from different botanical origin sources were analysed. Among floral honey, those from acacia had lowest proline content (Foldhazi et al., 1996 ). In another work, proline content was higher in Helianthus annus honey in comparison with Eucalyptus lanceolatus (Bath et al., 1999).This parameter could be used for the botanical origin classification as it was made by Biino in 1971. Highly sensitive and accurate HPLC methods were used for the determination of total amounts of proline and their enantiomeric ratios in different honey samples. Low concentrations of D-proline were found in honeys of different botanical and geographical origins. It is suggested that the enantiomeric ratios of amino acids could be used to test for Proceedings of the 37th International Apicultural Congress, 28 October – 1 November 2001, Durban, South Africa APIMONDIA 2001 ISBN: 0-620-27768-8 To be referenced as: Proc. 37th Int. Apic. Congr., 28 Oct – 1 Nov 2001, Durban, South Africa Produced by: Document Transformation Technologies Organised by: Conference Planners

storage effects, age, and the quality of the processing of the honey (Pawlowska and Armstrong, 1994). On the other hand, we can cite the following as some of the procedures generally used to quantify the proline: we can cite: fine layer and paper chromatography (Palma de Maldonado et al.,1982), HPLC (Evershed, 1993), enzymatic methods (Boehringer Mannheim, 1984) and the spectrophotometric method (AOAC, 1984) which we adopted in this study. In this case the determination of proline was done in the presence of the other amino acids whose quantities are negligible because they are present in a very low concentration. The aim of this work was to determine the proline concentration in honey to contribute to the knowledge of one of the parameters that can be used as a quality parameter for the samples that are consumed in our country and exported to others. MATERIALS AND METHODS Commercial Argentine heterofloral honeys purchased from commerces or donated by beekeepers have been used. These samples came from the south of Buenos Aires province, mainly from the area of Mar del Plata. The technique followed was the AOAC official procedure with the difference that the honey samples and proline standards were determined in duplicate. Preparation of the samples 2,5 g. of honey were weighted into a baker and dissolved in about 25 ml distilled water. Then the solution was transferred quantitatively to a 100 ml. volumetric flask, diluted to volume with distilled water and was very well shaken. Procedure 1 ml. of the sample solution was put in each of the two tubes, 0,5 ml. of formic acid (98100%) and 2 ml. of ninhydrin solution (3% in ethylene glycol monomethylether) were added to each tube. The tubes were capped carefully and then they were shaken vigorously. In the same way, 1 ml distilled water was added in one tube instead of in the sample solution and then the previous procedure was followed. In both cases, tubes were placed in a boiling water bath for 15 minutes and were transferred to a water bath of 22ºC for 10 minutes. 10 ml. of the 2-propanol-water solution (1:1) were added to each tube in regular intervals of time. Tubes cooled at 22ºC were removed before 35 minutes and the absorbances at 520 nm determined. Strict control of time in each step is critical. The honey colour was corrected determining the absorbance of the solution containing 1 ml of the sample solution, 2,5 ml distilled water and 10 ml. of 2-propanol . This value was substracted from the absorbance of the sample before estimated. Blancks of reactives also were also made and considered.

RESULTS The calibration curve of absorbance (A) against concentration (c) was obtained with increasing quantities of standard proline, following the same treatment that the samples. The curve C = 1668,795.A – 100,189 R = 0,99988 gave a linear response over a wide range of concentrations (Fig 1). The relative standard deviation of replicate measurements (n=16) was 12,320. The proline content was expressed as a proportion of the mass of honey in milligrams of proline per kilogram of honey. The mean value was 356 ppm, standard deviation 117,7, minimum and maximum values 73,3 and 577,3 ppm respectively. Recovery Recovery was tested by the standard addition procedure in order to demonstrate the accuracy of analysis. Known amounts of proline was added to the samples. Four determinations with two spiking levels were carried out (200 ppm and 400 ppm). Results obtained are shown in table 1. Precision Six experiments of the same sample were performed using the same reagents and apparatus to evaluate the method precision. Four known quantities of proline were used (500, 1000, 1500 and 2000 ppm) for the precision study. Results of table 1 show that a relative standard deviation (RSD) of less than 7% was obtained for all proline levels in the samples. DISCUSSION A similar study carried out in Italy in honeys from the Region of Buenos Aires present a mean value 302,5, minimum and maximum values 165 and 560 ppm respectively (del Lungo et al., 1993). The samples were from another region than the ones used in our study. Although, the results of that work are compatible with our present study, the mean and the maximum values are near, but the minimum value is minor in our case. Other investigations were carried out to contribute to know the proline content in honey, that could be used for the botanical origin classification like the studies made from 24 Spanish commercial honeys (mean value 500 ppm, standard deviation 130,5, minimum and maximum values 302 and 773 ppm respectively), and 91 Galician honeys (mean value 762 ppm, standard deviation 286, minimum and maximum values 302 and 1540 ppm respectively) (Huidobro et al., 1989). Also, 115 honeys from Basque country were analysed. The values obtained were 812 ppm ± 207,these values ranged between 340 and 1332ppm (Sancho et al., 1991). These results were similar as Huidobro found in Galician honeys. As we see, Spanish honeys have higher quantities of proline in comparison with the honey from Buenos Aires province, but they have, in general, medium and low content in comparison with the results obtained by Davies (1976) and White (1979).

In our present study, the recovery of proline was satisfactory, since it was always higher than 94%. It was ranged from 94,95 to 98,79% when the level spiking was 200 ppm and from 95,74 to 102,55 % when the level spiking was 400 ppm. The method precision evaluation showed that the results were satisfactory according to the Howirtz formula for interlaboratory (Howirtz, 1982). In agreement with the obtained values in this work, we concluded that the honeys of Buenos Aires province present certain variability in the results. It could be attributed to the different floral origin, but in general they contain important quantities of the amino acid. In the 92,5% of the cases, the proline content exceeded the minimum value (180 mg. of proline/Kg. of honey) suggested by the International Honey Commission. (Bogdanov et al., 1999).

1,4

Abs.(520 nm.)

1,2 1,0 0,8 0,6 0,4 0,2 0,0 0

200

400

600

800

1000 1200 1400 1600 1800 2000 2200

proline conc. (ppm)

Fig.1. Typical standard curve for proline. Values represents means ± sd (n=16) . 600

proline conc. (ppm)

500

400

300

200

100

0

10

20

30

samples

Fig.2. Proline concentration of forty samples from argentine honeys.

40

Table 1. Recoveries of standard addition in samples of honeys. Sample initial contents level I a mean recovery,% level II b mean recovery,% ppm 1 517,26 687,89± 23,18 97,73±6,94 866,86±59,02 95,74± 5,65 2 520,59 679,13±49,07 94,95±7,61 883,97±35,80 96,55±4,06 3 347,04 511,83±21,77 96,36±6,11 750,05±6,170 102,55±3,90 4 492,22 642,42±41,15 95,11±8,00 864,78±30,78 98,71±4,14 5 677,46 881,89 ± 76,09 98,79±9,74 1069,21±56,37 97,73±3,75 a

200 ppm. b 400 ppm.

Table 2. Precision of the method for quantification of proline in honeys. Proline conc.a X ± SD b RSD, c % 500 478,237±24,182 5,056 1000 1015,532±62,042 6,109 1500 1540,440±78,415 5,090 2000 1964,959±104,347 5,310 a

Proline concentration expressed in milligrams per kilogram. kilogram (n = 6). c Relative standard deviation.

b

Mean ± standard deviation in milligrams per

REFERENCES ASSOCIATION OF OFFICIAL ANALYTICAL CHEMIST (1984). Official Methods of Analysis. 14th Edition, Arlington, Virginia 22209, USA. BATH, P. K.; NARPINDER SINGH; SINGH, N. (1999). Food Chemistry, 67 (4), 389-397. BIINO, L. (1971). Ricerca di alcuni aminoacidi in due varietà di miele. Riv. Ital. Essence Profumi, 53 (2), 8084. BOEHRINGER MANNHEIM (1984). Análisis enzimático de alimentos. 1ª edición. Barcelona. BOGDANOV, S. (1999). Honey quality and international regulatory standards: review by the International Honey commission. Bee World, 80(2),61-69. DAVIES A.M.C. (1975). Amino acid analysis of honey from eleven countries. J. Apic. Res. 14 (1), 29-39. DAVIES A.M.C. (1976). J. Food Technol. 11, 515-523. EVERSHED, R. P. (1993). Handbook of Derivatives for Chromatography, ed. by K. Blau, J. Halket, Wiley New York. 53 pp, FOLDHAZI, G.; AMTMANN, M.; FODOR, P.; ITTZES, A. (1996). Acta Alimentaria Budapest. 25 (3), 237-256. HUIDOBRO, J. F.; SIMAL, J. Y COUSO, I. (1989). Determinación de prolina en miel. III Congreso Nacional de Apicultura. Ponencias y comunicaciones 153-160. IHTISHAMULHAQ (1999). Sarhad Journal of Agriculture. 15 (1), 61-63. JOSHI, S. R.; PECHHACKER, H.; WILLAM, A.; OHE, W. VON DER. (2000). Apidologie.31 (3), 367-375. KONAMINE, A. (1960). Amino acids in honey. Acta chem. fenn. B. 33,185-187. LUNGO, T. DEL; CIURLO, R.; BALLETTO, A.; NOVARI, G.; MALERBA, A. (1993). Proline content in honeys imported from Argentina. Industrie Alimentari. 32: 314, 349-353. OHE, W VON DER; DUSTMANN, J. H; OHE, K. VON DER. (1991). Prolin als Kriterium der Reife des Honings. Deutsche Lebensmittel Rundschau 87 (12), 383-386. PALMA DE MALDONADO, S.; FONTANARROSA, M.E. Y VIGIL, J. B. (1982). Chromatographic detection of amino acids in honeys. Rev. Fac. Ing. Quím. (Univ Nac. Litoral), 45 (1), 73-80. PAWLOWSKA, M.; ARMSTRONG, D.W. (1994). Chiriality, 6 (4), 270-276. SERRA BONVEHI, J.; ESCOLA JORDA, R. (1997). J. Agric. and Food Chem. 45 (3), 725-732. SANCHO, M. T.; MUINIATEGUI, S.; HUIDOBRO, J. F. Y SIMAL, J. (1991). Honey from the Basque Country (Spain). II: Formol number and proline content. Anales de bromatología 18 (1), 87-99. WHITE, J. W. JR. (1979). J. Assoc. Off. Anal. Chem. 62 (3), 515-526.

PROLINE IN ARGENTINE HONEYS J. Di Gerónimo, M. Eguaraz And R. Fritz Departamento De Química. Facultad De Ciencias Exactas Y Naturales. Universidad Nacional De Mar Del Plata. Funes 3350. 7600. Mar Del Plata Tel: 0223-4756167, Fax: 0223-4753150, E-Mail: [email protected] Dr. Rosalía Fritz Education 1992- 1997 1967-1970 1963-1967

Doctor of Clinical and Industrial Bacteriology University of La Plata, Buenos Aires, Argentina Clinical and Industrial Bacteriologist University of La Plata, Buenos Aires, Argentina Pharmacist University of La Plata, Buenos Aires, Argentina

Academic Experience 1967- 1978 Teaching Assistant at the Faculty of Exact Sciences and the Faculty of Veterinary Sciences. University of La Plata, Buenos Aires, Argentina 1978- 1982 Argentina.

Teaching Assistant at Engineering Faculty. Universty of Mar del Plata., Buenos Aires,

1982- 1984 Teaching Assistant at Natural and Exact Sciences Faculty. Universty of Mar del Plata, Buenos Aires, Argentina. 1985 to date Associate Professor of Bromatology and Food Microbiology at Natural and Exact Sciences Faculty. Universty of Mar del Plata., Buenos Aires, Argentina. Research Experience 1993 to date : Director of Bromatology Research Group. Chemist Department. Natural and Exact Sciences Faculty. University of Mar del Plata. International Scientific Publications: 10 Works presented in National and International Congress: 36 Publications in the last 5 years: *Saiz, A.I., Perez Borla, O. y Fritz R. 1997. "El gluten como mejorador de harinas de trigo" Alimentaria 280 : 83-87. España. *Fritz, R., Oliva, G.A. y Añón M. C. 1998. "Bacterias acidolácticas iniciadoras de la fermentación de masas panadería de harinas de trigo "Heladeria y Panadería Latinoamericana" 133: 40. Argentina *Fuselli, S. R; Casales, M. R.; Fritz, R.; Yeannes, M. I. 1998 "Isolation and characterization of micro organisms associated with marinated anchovy (E. anchoita )". Journal of Aquatic Food Product Technology 7 (3) : 29-38. EEUU. *Saiz, A.I., Perez Borla, O. y Fritz R. 1998 "Il glutine come miglioratore delle farine di frumento" Tecnica molitoria 49 (4) : 347-354 . Italia. * Saiz, A.I., Perez Borla, O.; Motta, E.L.; Fritz, R. 2000 “Características fisicoquímicas y microbiológicas de harinas y efecto de tratamientos previos a la panificación.” Alimentaria- Revista de Tecnología e Higiene de los Alimentos 313 : 51-57. España. * Saiz, A.I. ; Manrique, G. D.; Fritz, R. 2001. “Determination of benzoyl peroxide and benzoic acid levels by HPLC during wheat bleaching process” Journal of Agricultural and Food Chemistry 49 : 98-104. USA.