English abstract
Honey is the natural sweet substance produced by honeybees. It is a very important energy source. Since adulteration of honey is frequently encountered, its quality must be controlled analytically with the aim of guaranteeing that it is genuine and protect the consumer from commercial exploitation. Besides, there is a need to establish the geographical limits of production to assure the quality. In Northern Oman, bees produce true Acacia honey from Acacia tortilis. This native wild plant is one of the most common trees throughout the country, abundant in the plains, wadies and foothills. It's flowering and fruiting period is from April to June, hence it is an important source for summer honey in Oman. Acacia honey is very popular locally and called as Sumr. It is characterized by its dark color, thick texture and stronge aroma and taste. It has many medicinal values. Moreover, in the market there is a 'white acacia' honey which is produced from the nectar of Robinia pseudoacaia flowers. This is an American species and does not grow in Oman. Robinia honey is actually a false acacia honey. It is characterized by its light amber color and distinct flavor, and fragrance due to its floral source. The commercial name of this honey as 'Acacia honey' confuses the consumer. Because of the significant importance of Acacia honey in Oman and neighboring regions, the present study was undertaken to evaluate and characterize Omani Acacia honey by using botanical, molecular, physicochemical, antioxidant and antifungal analysis. This will help in controlling the quality to identify spurious, adulterated, non Omani samples and protect its production zones. In addition Omani Acacia honey will be differentiated from Robinia (false acacia) honey, The analyses were carried out on 200 summer honey samples from Apis mellifera and Apis florea colonies collected from nine governorates of Oman during the period of 2009 - 2012. The analysis was also done on 18 Robinia honeys purchased from Omani market which were imported from six countries. These samples were labeled commercially as Acacia. This research is divided into four studies as follows:
Study 1: Botanical, geographical and molecular analysis of Acacia and Robinia honeys Acetolysed pollen from honey samples were studied and photographed using light microscope (LM) and scanning electron microscope (SEM). For molecular studies, pollen DNA from each sample was subjected to parallel pyrosequencing for DNA barcoding analysis using universal primers to study the species diversity and geographical origins of the honey samples. Analysis was also done with the imported false acacia (Robinia) honey samples for comparison. To establish a standard pollen concentration value for "pure Acacia honey", Lycopodium spore counting technique was used with the coefficient formulas to assess the quality of the honey samples. From 200 honey samples analyzed, 80 were unifloral with predominant (> 45%) Acacia tortilis pollen while out of 18 commercial Robinia honeys only 14 were authentically Robinia. Pollen concentration results showed 64% of the Omani Acacia and 79% of Robinia were classified within the normal category (Group II). This indicates that they are produced from normal floral source without addition of pollen or external feeding. From the pyrosequencing run of 12 Acacia honey samples, 678461 molecular sequences and 69 molecular taxonomic units (MOTUS) were obtained. Sixty of the MOTUs were from melliferous plants and 9 were from non-melliferous plants. This analysis showed that the bee flora of Acacia honey consisted mostly the herbaceous plants such as ompositae, Convolvulus spp., Dipterygium glaucum, Fagonia spp., Tephrosia villosa, Zygophyllum spp. and a few tree and shrub species such as Anacardiaceae type, Conocarpus erectus, Madicago sativa, Rhamnaceae type etc. The molecular analysis results showed a wide diversity in bee forage plants and incorporate both wild and cultivated plant taxa for the production of Acacia honey. This indicated the adequate floral sources in the country that can add additional nutritional value to this type of honey.
Study 2: Physicochemical properties of Acacia and Robinia honeys The following physicochemical properties of Acacia and Robinia honey samples were studied: pH, moisture content, free acidity, electrical conductivity, diastase activity and invertase activity were analyzed. Acacia honey samples showed the mean of pH 4.83, moisture content 16.75%, electrical conductivity (EC) 1.82 mS/cm, free acidity 95.30 meq/kg, diastase activity 12.00 DN and invertase activity 141.07 U/kg. These values are on par with the permitted specifications except electrical conductivity and free acidity, which were elevated. The EC of Acacia honey samples were found to correlate significantly and positively with pH, moisture content, free acidity, diastase and invertase activities. This may point out at the influence of these parameters in raising the EC of this honey indicating the richness of Acacia honey with mineral and organic acid contents. This can increase its nutritional and medicinal values. Robinia honey samples showed the mean of pH 3.80, moisture content 17.41%, EC 0.33 mS/cm, free acidity 8.03 meq/kg, diastase activity 8.00 DN and invertase activity 17.53 U/kg. These values met the national and international standards. This satisfies the consumer to deal with this product safely. The values of these parameters were higher in Acacia honey samples than Robinia. This may be attributed to the higher quality of the honey. This study showed a serious need to construct database and standards for the Arab peninsula regional honeys because the current standards were formulated based on analyses of honey from temperate or European countries.
Study 3: Antioxidant properties of Acacia and Robinia honeys The color, proline content, total phenolic content, and free radical scavenging activities (DPPH) were recorded for the honey samples using spectrophotometer. Dark amber color was dominant in the 79% of Acacia honey samples whereas 50% of Robinia showed extra white color. In Acacia honey samples the mean values of color Pfund was 133.41 mm, color intensity ABS 560 nm 0.21AU, proline content 91.03 mg/100 g, total phenolic content 45.20 mg GAE/100 g, RSA value 59.39% and IC50 16.07 mg/mL. In Robinia honey samples the mean values of color Pfund 23.11 mm, color intensity ABS 560 nm 0.04 AU, proline content 22.22 mg/100 g, total phenolic content 2.87 mg GAE/100g, free radical scavenging value 51.00% and IC50 112.27 mg/mL. The antioxidant parameter values of Acacia honey were compared with Robinia honey as well as the buckwheat and manuka, citrus and cotton honeys. It was concluded that sample set of 80 Acacia honeys is adequate to make the generalization that these types of honeys exhibited overall better antioxidant properties which may reflect its potential of therapeutic activity. It was also observed that the dark color Acacia honeys presented the highest phenolic content than the light Robinia honey samples. Accordingly, the color intensity is a good indicator of the total phenolic content which is the antioxidant factor in honey. Thus the color intensity and phenolic compounds can be considered a significant determinant of the antioxidant capacity of honey. This fact was also supported by the strong correlation of DPPH scavenging values with IC50 values, color intensity Abs 560 nm, Pfund values and phenolic contents in the Acacia honey samples. It can be concluded that dark Acacia honey samples possess higher antioxidant activity than the light Robinia honey samples. The study recommends labeling Acacia for Acacia spp. honeys and Robinia for Robinia pseudacacia honey to differentiate between them in both scientific and public communications.
Study 4: Effect of Omani Ziziphus spina-christi and Acacia tortilis honey samples on the growth and aflatoxin B1 production of Aspergillus parasiticus Aflatoxins are the most serious carcinogenic, hepatotoxic, teratogenic and mutagenic secondary metabolites which adversely affect human and animal health. This study was designed to evaluate the in vitro inhibitory effect of different concentrations of Ziziphus and Acacia honeys on the growth and aflatoxins production by A. parasiticus by using VICAM technique. This study was divided to four parts. In the 1° part, A. parasiticus was inoculated in seven concentrations (1,5, 7, 10, 15, 20 and 25g/100 mL H20) of each of Ziziphus and Acacia honeys. The results showed that the myceliai dry weights significantly increased by 44.81% - 71.70% in Ziziphus cultures and 49.55% - 76.50% in Acacia cultures. Both honey types did not inhibit the fungal growth. In the 214 part of the study it was found that the Ziziphus honey caused 6.10% - 92.34% inhibition of aflatoxin secretion. Therefore, this type of honey can be recommended as a biopreservative to enhance the shelf life of food and feed products, On the other hand the level of aflatoxin increased in Acacia honey cultures from 12.96% - 18.97%. No correlation was found between the amount of aflatoxin and the honey concentration. In the 3rd part of the study, none of the Ziziphus and Acacia honey solutions (15 g/100 mL H2O) detoxified pure aqueous aflatoxin B1. In the 4" part of the study, it was observed that the controls in honey culture that were not inoculated with A. parasiticus showed growth of yeast and different species of phycomycetes. In addition, different amounts of aflatoxin were recorded in all cultures. This showed that aflatoxin producers can grow and produce toxin in honey
