Caracterización, determinación de vitaminas hidrosolubles y actividad antioxidante de mieles monoflorales de Castilla-La Mancha
AuthorsLeón Ruiz, Virginia
IdentifiersPermanent link (URI): http://hdl.handle.net/10017/20310
AffiliationUniversidad de Alcalá. Departamento de Química Analítica, Química Física e Ingeniería Química
Nutrición, bromatología y toxicología
Description / Notes
Texto en español y resumen en inglés
The professional level of the beekeeping sector and quality control that the honey must pass prior to marketing are increasing. Even so, considering that the botanical origin and geographical area of production are important factors in the composition of honey, the knowledge of the properties and nutritional value of this food is still scarce. The aim of this research is to determine the characteristics and certain minority components, with a high nutritional value and importance for health, of honey produced in the Spanish region of Castilla-La Mancha, one of its areas, "La Alcarria", being well known for its rosemary and lavender honeys, with Protected Denomination of Origin (PDO). This will allow answering the new requirements imposed by the food industry and consumers in improving quality control and labelling. Until now, except for honey in the area of "La Alcarria", the rest of the beekeeping production in the region of Castilla-La Mancha has barely been studied. In order to have a greater knowledge of the honey produced in this region, a study on botanical identification, physico-chemical, nutritional and therapeutic properties of various monofloral honeys produced in this region has been performed, selecting those most representative and with continuous production. First, the physico-chemical, palynological and sensorial characteristics of each of the selected monofloral honeys are described. These characteristics are specific to each variety of honey and, in many cases, also of the production zone. Honey types studied, not described to date, are chestnut produced in "Sierra de San Vicente" (Toledo), echium of Toledo and Ciudad Real, oak honeydew from Guadalajara, and thyme honey from the area of "La Alcarria" (Guadalajara), not included in the PDO. Likewise, data of the characteristics of each monofloral honey produced in the area of "La Alcarria" with PDO, rosemary and lavender, are provided. It is important to remark that the specification of the PDO only shows the overall limits of the quality parameters estimated for packing any type of honeys commercialized under this indication of quality. With individualized data provided in this research work, the particular characteristics of rosemary and lavender honeys are better defined, specifying further quality. The analysis of physico-chemical parameters, pH, electrical conductivity, HMF, humidity, free acidity and colour, have been made using the methods established by the International Commission of Honey (IHC). The major sugars, fructose, sucrose and glucose, have been analyzed by a chromatographic method with the procedure used in the Laboratory of Honey of "Centro Agrario de Marchamalo" (Guadalajara). With the aim of studying the nutritional properties of honey from the region of Castilla-La Mancha, the amounts of several water-soluble vitamins, ascorbic acid (vitamin C) and various vitamins of B group, thiamine (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B3H), nicotinamide (vitamin B3N), pantothenic acid (vitamin B5) and pyridoxine (vitamin B6), have been determined. The concentration of vitamin C has been determined by a reverse phase high performance liquid chromatography, RP-HPLC method, in isocratic mode with a mobile phase of 0.05% H2SO4/water at pH = 2.5. The accuracy of the method was carried out by comparison with those obtained by an AOAC official volumetric method. It has been observed that all honeys studied contain vitamin C, and it should be noted the high content found in thyme honey much higher than in the rest of honeys studied, indicating that this chemical compound can be considered as a chemical marker of this variety of honey. In addition, a stability study of the vitamin C was carried out in honey with different storage temperatures during one year. This compound is stable in the product at freezing (-180C) and refrigeration (40C). At room temperature, the vitamin C content decreases about 73% of its original content. Using a linear discriminant analysis, LDA, the data obtained from the physicochemical parameters, major sugars and vitamin C, have differentiated a 78% of the various honey samples according to their botanical origin, with only four control parameters: electrical conductivity, fructose, glucose and vitamin C. For the analysis of water-soluble B vitamins, two reverse phase HPLC methods in isocratic mode have been developed, one of them using a conventional hidroorganic mobile phase and another one using a mobile phase, which contains a surfactant. The vitamins detection has been carried out with two detectors: ultraviolet (UV) and fluorescence (FL). The use of both detectors is useful because the riboflavin (vitamin B2) and pyridoxine (vitamin B6) vitamins have native fluorescence, which provides greater selectivity and sensitivity to the analysis. The rest of vitamins, thiamine (vitamin B1), nicotinic acid (vitamin B3H), nicotinamide (vitamin B3N), and pantothenic acid (vitamin B5) have been determined with UV detection. One of the developed methods is based in the use of a mobile binary phase H2SO4 0,01%/methanol 2% in water (pH=3.5) and a temperature of 25°C, achieving the separation of thiamine, nicotinic acid, nicotinamide, pantothenic acid and pyridoxine vitamins. Vitamin B2 not is eluted with this mobile phase. Due to this reason, a second method has been developed in which a surfactant is added to the previous mobile phase. A cationic surfactant, hexadecyltrimethylammonium bromide (CTAB) and another non ionic, polyoxyethylene-(23)-lauryl ether (Brij 35) have been tested and the surfactant concentration has been modified above and below its critical micellar concentration, with different organic modifiers in different proportions (methanol, ethanol, n-propanol and n-butanol). The separation has been carried out with a mobile phase H2SO4 0.01%/CTAB 0.01M/methanol 2%, to pH=2.75 and a temperature of 25°C. Finally, five honeys of each botanical type have been analyzed. In most of the honey samples nicotinic acid (vitamin B3H) and nicotinamide (vitamin B3N) were found. Vitamin B1 was quantified in most of the samples except in the rosemary honeys. Vitamin B2 has been detected in the chestnut honey and honeydew, concurring with the fact that they are the darkest honeys. Vitamin B5 has been detected in very few samples. With regards to vitamin B6, it was detected in more than half of the analyzed samples, but the concentration in which it appeared was so low that it was not possible to quantify it. The functional properties of the honeys, antioxidant and antibacterial activity have been evaluated. Although both of them are well known and widely studied, their determination is interesting due to the variability of this property with the botanical and geographic origin. Another therapeutic property, barely studied in honeys, the antihypertensive activity, was also studied. It is known that many types of honey have levels of antioxidants similar to fruit and vegetables. In order to evaluate the levels of antioxidants parameters in the honeys, antioxidant activity obtained by DPPH method and the concentration of total polyphenols, in addition to previously mentioned vitamin C, have been analyzed. Remarkable levels of antioxidant activity were obtained in the honeys of thyme, due fundamentally to their high concentration in vitamin C, although it is also important its high content in total polyphenols. Chestnut honey has special importance for its high antioxidant activity, due to the high content of total polyphenols. For the rest of honeys, the levels of antioxidant activity obtained have lower values. The antibacterial activity was evaluated by means of a method of agar in plate with a gram-positive bacterium, Staphylococcus aureus 25923 ATCC and another gram-negative, Escherichia coli ATCC 25922. Although the sensitivity of both bacteria against the different honeys is not equal, chestnut honey is always the one of greater potential, while the rest barely presents any antibacterial effect. It only appears complete inhibition of the bacterial growth with the chestnut honeys. Although hydrogen peroxide is the main bactericidal agent present in honey, the study was extended, carrying out a test with catalase, enzyme that H2O2 degrades, with the purpose of knowing another component to attribute it the greater bactericidal effect of the chestnut honey. Against E. coli the antibacterial activity disappears, which means that this activity is only due to H2O2. However, versus S. aureus the disappearance is not complete. Something more exists aside from H2O2, that it inhibits the growth of S. aureus. When the concentration of the lysozyme enzyme in all the types of honeys is measured, it is observed that the chestnut honeys contain ten times more enzyme than the rest of honeys, partly explaining the greater antibacterial activity of this honey. In order to finalize the study of functional properties of these monofloral honeys of "Castilla-La Mancha", an in vitro study of the antihypertensive activity was made. For this study, the inhibition activity of the Angiotensin I-converting enzyme (ACE) was evaluated. This is the first time that this property is investigated in several honey types. The obtained results have been positive: all the analyzed samples presented inhibition of the enzyme, being greater than 50% in the most of the chestnut honeys. The IC50 of this enzyme, for the honey of greater antihypertensive activity, has been determined giving a high value (80 mg of honey/mL). Comparing to other products of recognized antihypertensive activity, these values indicate that it is not a powerful inhibitor of the ACE, nonetheless, discovering that the honey has intrinsic antihypertensive activity is an outstanding fact.