2015 年 21 巻 1 号 p. 23-29
Analyses of honey produced by Africanized Apis mellifera from cassava flowers were carried out to characterize this product. The results showed satisfactory values within the standards of marketing, except for the apparent sucrose for exporting and hydroxymethylfurfural in Brazil. The honey was properly described by the power law model and exhibited a shear thinning behavior. The viscosity decreased with an increase in temperature. The Arrhenius model gave a good description of temperature effect on apparent viscosity of the honey, where the activation energy determined at a shear rate of 10 s−1 was 69.35 kJ/mol. Cassava flower honey not presented quality indexes within the parameters required by current standards, because to the apparent sucrose content is above the level allowed for exportation. These values need to be observed and monitored in a way that farmers can market the honey produced by Africanized A. mellifera amid the cassava culture with physical and chemical quality.
In Brazil, beekeeping is attracting interest of many farmers and several institutions, as it is an activity in which one can achieve good economic results (Welke et al., 2008). To guarantee a product with quality on the market that is increasingly demanding, it is important to know the characteristics of honey. Thus, it is necessary to carry out the physical-chemical and rheological analysis, aiming to standardize the product, thereby obtaining subsidies to ensure the quality of honey by detecting its possible adulterations. Honey may undergo several changes of various causes. Some occur because of farmers' lack of information related to the extraction technology, the proper management, the equipment to be used and mainly for storage and conservation of the product (Melo et al., 2003).
Honey is a food product produced by honey bees from the nectar of flowers or secretions coming from live parts of plants or secretions of plant-sucking insects that lay on living parts of plants which bees collect, transform, combine with their specific substances, store and let ripen in the hive combs (Mercosul, 1999; Brazil, 2000; Codex Alimentarius, 2001). Honey has a complex system which contains sugars, organic acids, minerals, vitamins and phenolic compounds (Zalibera et al., 2008; Adetuyi et al., 2009; Babarinde et al., 2011). According to Hernández et al. (2005) honey has a very low mineral content (0.1 – 0.2% for floral honey), which varies widely depending on the botanical origin, edaphoclimatic conditions and technical extraction.
The viscosity is one of the most significant physical and sensory characteristics of honey, which affects the quality of the product (Yanniotis et al., 2006). Industrially, higher viscosities imply greater costs in centrifugation, decanting and mixing, caused by increases in the operation times and energy wastes. Honey viscosity, as well as its composition varies according to the species of producer bees. The flow properties are influenced by various factors such as composition, temperature and amount and size of crystals (Pereira et al., 2003).
Honey producers in the region of São Paulo state, Brazil, are producing honey from Africanized Apis mellifera, from boxes located in the middle of the cassava cultivation, trying to diversify the production of the property and ensure household income increase. The Brazilian production of honey in 2007 was approximately 34,747 tons and the participation of the state of São Paulo was of 2,332.19 tons (IBGE, 2009). There are few reports in literature on the production of honey from cassava flowers. Silva et al. (2001) observed that the A. mellifera species was the main pollinator of cassava flowers, being attracted by the fragrance, pollen and nectar of flowers. The regional characterization of honeys is very important, primarily cassava flower honey, because in Brazil, it is a rare product. Thus this study was carried out to obtain information to aid the characterization of the honey produced by Africanized A. mellifera from cassava flowers (Manihot esculenta Crantz).
Honey samples Honey was collected from hives of Africanized A. mellifera from cassava planting, industrial varieties, which flowered in May, in Tupã, São Paulo, Brazil. Extraction of honey was held on 17 and 18 May, 2009. After harvesting the honeycombs were centrifuged and transferred to nontoxic plastic pots, to hold 500 g. All tests were performed in triplicate with analytical grade reagents.
Physicochemical analysis The moisture of the honeys was determined with the aid of a bench refractometer according to No. 969, 38b Method (AOAC, 1997). The hydroxymethylfurfural (HMF) was determined by quantitative spectrometry with λ = 284 and 336 nm, according to no°. 980, 23 Method (AOAC, 1997). The methods used for determination of moisture and HMF are recommended by the Instruction of the Ministry of Agriculture and Supply (Brazil, 2000). The analysis of the free acidity was performed according to the AOAC no°. 962, 19 method (1997), which is based on the sample titration with 0.05 mol/L NaOH solution to reach pH 8.5. This procedure is indicated by the Technical Regulation of Identity and Quality of Honey (Brazil, 2000).
The pH determination was performed using a digital pH meter. The ash samples were obtained by calcination in muffle furnace, according to Almeida-Muradian and Bera (2008). Determinations of reducing sugars, total and apparent sucrose were performed by using the Lane-Eynon method from a stock solution of 20% (IAL, 2005). The content of insoluble solids was determined according to Almeida-Muradian and Bera (2008). The diastatic activity was performed according to Marchini et al. (2004), where the buffered solution of starch-honey is kept in a water bath (40°C, the time required for obtaining the specific end point (absorbance lower than 0.235) determined spectrophotometrically. The results were obtained on a Gothe scale.
Phenolic compounds were determined by Folin-Ciocauteau according to Meda et al. (2005) using gallic acid as standard. The determination of the honey color is based on different degrees of light absorption at wavelengths of 560 nm, using the pure glycerin as white and to classify the color of honey, it was used Pfund scale (Marchini et al., 2004).
The minerals were determined by nitroperchloric digestion according to the Malavolta et al. method (1989) and using atomic absorption spectrophotometer (Varian - mod. Spectrum A 10 Plus). The determination of phosphorus (P) was performed using a spectrophotometer UV / VIS according to Pavan et al. (1992).
Rheological measurements The rheological measurements were carried out with a concentric cylinder Brookfield rheometer (DV-III+), using a small sample adapter (13R/RP, 19.05 mm of diameter and depth of 64.77 mm) and spindle SC4-25 (11.76 mm of diameter and 33.02 mm length), (Brookfield Engineering Laboratories, MA, USA). A Tecnal thermostatic bath TE-184 (Tecnal, Piracicaba, SP, Brazil) was used to adjust the temperature of the sample in the range of 30 – 60°C. The sample was not reused after heating due to the change in rheological properties, and for each test, the filled sample cup (11.0 mL) and spindle were temperature equilibrated for about 15 min. The shear stress (τ) and shear rate (γ) data, were obtained using the Rheocalc Software (version V3.1-1, Brookfield Engineering Laboratories, MA, USA). Each experimental run to the upward curve had duration of 1 min with shear rate range from 1 to 15 s−1 and 1 min to the downward curve with shear rate range from 15 to 1 s−1. Both at decreasing and increasing shear rate, 25 points of shear stress were obtained resulting in a total of 50 points, whose average value of shear stress was taken for each shear rate. Three experimental runs were done for each material, and the resulting shear stress was the average of the three experimental values (Haminiuk et al., 2006a).
Statistical analysis The obtained data were statistically evaluated by the analysis of variance (ANOVA) and Tukey test (mean comparation), using the STATISTICA 7.1 (Stat-Soft, Tulsa, OK, USA). The experimental data were fitted according to the rheological models of power law and Herschel-Bulkley using the software Origin 7.0 (OriginLab Corporation, MA, USA) to obtain the rheological (n and K) and statistical parameters (R2 and χ2). The values of R2 and χ2 were obtained to evaluate the goodness of fit to the experimental results in the rheological models.
Physicochemical analysis The results of physical-chemical evaluations are shown in Table 1. The pH value is influenced by the nature of the pollen used and concentration of different acids in its composition (Alves et al., 2005). The legislation does not standardize the pH value for honey. According to Almeida-Muradian and Bera (2008) the optimal value of pH is between 3.3 and 4.6, and the value obtained in this work is in accordance with the range exhibited by the authors, Pontara et al. (2012) studied cassava flower honey also obtained value of pH (4.14) within this range. The pH influences the texture, stability and shelf life of the honey, as altered pH values may indicate fermentation or adulteration of the bee honey (Welke et al., 2008). According to Marchini et al. (2004), the pH is related to the rate of HMF formation in honey.
| Parameters | Values (± δ) | Brazil1 | Mercosul2 | Codex Alimentarius3 |
|---|---|---|---|---|
| pH | 3.94 ± 0.10 | - | - | - |
| Acidity (mEq/kg) | 28.83 ± 0.13 | Max. 50 | Max. 40 | Max. 50 |
| Moisture (g/100g) | 18.93 ± 0.83 | Max. 20 | Max. 20 | Max. 20 |
| Soluble Solids (°Brix) | 81.07 ± 0.08 | - | - | - |
| Reducing Sugars (g/100g) | 67.40 ± 0.05 | Min. 65 | Min. 65 | Min. 60 |
| Apparent sucrose (g/100g) | 5.26 ± 0.10 | Max. 6 | Max. 5 | Max. 5 |
| Total Sugars (g/100g) | 79.48 ± 0.06 | - | - | - |
| HMF4 (mg/kg) | 6.13 ± 0.09 | Max. 60 | Max. 40 | Max. 80 in tropical regions |
| Ash (g/100g) | 0.40 ± 0.08 | Max. 0.60 | Max. 0.60 | - |
| Insoluble solids in water (%) | 0.02 ± 0.01 | Max. 0,1 | Max. 0.10 | Max. 0.10 |
| Diastatic activity (Gothe scale) | 11.33 ± 0.55 | Min. 8 | Min. 8 | Min. 8 |
| Total phenol (mg/100g) | 17.57 ± 3.20 | - | - | - |
| Color | Dark brown | Colorless to dark brown | Colorless to dark brown | Colorless to dark brown |
The determination of the honey acidity was presented in accordance with the standards set by current norms (Brazil, 2000; Mercosul, 1999; Codex Alimentarius, 2001). According to Mendes et al. (1998), this value of acidity within the established pattern indicates the absence of undesirable fermentation in the presence of yeast. Ajlouni and Sujirapinyokul (2010) report that during the fermentation process, glucose and fructose are converted into carbon dioxide and alcohol and that alcohol is hydrolyzed in the presence of oxygen and converted into acetic acid. This fact contributes to increasing the level of free acidity of the honey. Evangelista-Rodrigues et al. (2005) report that the origin of the honey acidity is due to the variation of organic acids caused by different sources of nectar, by the action of the enzyme glucose oxidase. This enzyme generates gluconic acid, which is an acid present in greater quantities by the action of bacteria during the ripening of the honey. In smaller quantities, other acids are found such as acetic, butyric, lactic, oxalic, formic, malic, succinic, pyruvic, glycolic, citric, butyric-lactic, tartaric, maleic, pyroglutamic, alpha-ketoglutaric, 2- or 3-phosphogliceric, alpha-or beta-glycerophosphate and vinic (Racowski et al., 2007).
The moisture content is presented inside the maximum value of the norms (Brazil, 2000; Mercosul, 1999; Codex Alimentarius, 2001) (Table 1). The moisture content of the honey is influenced by the botanical origin, the climatic conditions, the harvest season and the degree of maturation of the honey, being the latter a very important parameter during the product storage. The moisture content below 21% prevents undesirable processes of honey fermentation (Sodre et al., 2007). Marchini et al. (2007) found 19.30 to 22.40% of moisture content in honeys produced from the flowers of Eucalyptus in the São Paulo state. The authors report that 19.30% of the honeys presented values higher than 20% moisture and that this fact is related to the moisture absorption during the storage. Serrano et al. (2004) found an average content of 16.63% moisture for honey from flowers of Eucalyptus, in the region of Andalusia, Spain. The determination of SS helps to obtain the moisture content, which uses the index of refraction, with the aid of the Chataway table to find the moisture content.
The soluble solids (SS) average value was 81.07 °Brix. The SS represent, in its majority, the sugars which are present. Reducing sugars reached an approximate value to the minimum established by legislation (Table 1). Bendini et al. (2008) obtained a content of 81.25 g/100 g in the honeys of the cashew flowering. The determined apparent sucrose content is in accordance with Brazilian legislation, but not with the export standards (Mercosul, 1999; Codex Alimentarius, 2001). Marchini et al. (2004) state that the sucrose represents on average 2 – 3% of carbohydrates and when it exceeds this value, it may be an indication of adulteration of the product or of an early harvest of the honey, that is to say, it is a product in which the sucrose has not been totally transformed into glucose and fructose by the action of invertase. Melo et al. (2003) report that during the storage, the honey enzymes are also responsible for the physical-chemical and nutritional transformations. In its formation process, honey contains enzymes from plants and insects: invertase, amylase (diastase), glucose oxidase, catalase and phosphatase. Invertase, embedded in nectar by the saliva of bees, transforms the sugars, especially sucrose, into glucose and fructose. Diastatic actions lead the transformation of ¾ of sucrose. Therefore, the older the honey, the less sucrose it will contain (Racowski et al., 2007).
The honey bee has a small amount of HMF, but with prolonged storage at high room temperature, this level can rise, altering the nutritional value of the product. Thus, the determination of HMF serves as an indicator of the honey quality, because when it is formed, the loss of some enzymes, eg, glucose-oxidase (Melo et al., 2003) may have occurred. The average obtained was 6.130 mg/kg indicating that the sample is consistent with the established value (Brazil, 2000; Mercosul, 1999; Codex Alimentarius, 2001). When Serrano et al. (2004) analyzed honeys of flowers of Eucalyptus and Citrus produced in Spain, they reported that these honeys had average values of 10.99 and 16.55 mg/kg, higher levels than those determined in this work.
The ash content of the honey can be an indicator of good manufacturing practices, as honey with a high percentage of ash may indicate improper handling or the presence of contaminant in the product (Almeida-Muradian and Bera, 2008). The value found in this study is 0.40% higher than that found by Sodré et al. (2007), who obtained 0.18%. Bendini et al. (2008) found averages of 0.2% in the total weight, and it ranged from 0.18 to 0.30% in honeys from cashew flowers in the Ceará state, Brazil. The content of insoluble solids in water is within the standard value (Brazil, 2000; Mercosul, 1999; Codex Alimentarius, 2001).
The insoluble solids in water are parts of bees (legs, wings etc.), wax residue, and other elements related to the honey or the process. The completion of this analysis allows detecting the impurities present in honey, which is a measure of hygienic control (Silva et al., 2006). The diastatic activity of honey is within the requesting legislation (Brazil, 2000; Mercosul, 1999; Codex Alimentarius, 2001). Mendes et al. (1998) evaluating honeys from Portugal, had results that ranged from 2 – 22 in Gothe scale. The amylase activity is usually expressed as diastase number (DN), also known as units of Gothe. A unit of Gothe is defined as being the amount of starch solution 1.0% which is hydrolyzed at 40°C for one hour by the enzyme present in 1 g of honey (Ajlouni and Sujirapinyokul, 2010).
Amylase is one of the enzymes in honey which has the function of digesting the starch molecule and it is very sensitive to heat, therefore, it is able to indicate the degree of conservation and overheating of the product (Mendes et al., 1998; Ajlouni and Sujirapinyokul, 2010). The absence of this enzyme indicates either procedures or adulteration performed, such as the use of temperatures above 60°C during processing, or addition of inverted sugar, or even improper storage conditions. Thus, the value obtained in this experiment reflects the care beekeepers have with honey production in the midst of cassava plantation.
The content of phenolic compounds was higher than the values found in literature. Adetuyi et al. (2009) obtained a value ranging from 0.75 to 2.85 mg/100 g, in honey of the Community Owo, in Ondo State, Nigeria. The color observed in the honey sample was dark amber, what might be due to the high content of minerals found (Table 2) (Gomes et al., 2010).
| Minerals | Content (mg/kg) (±δ)* |
|---|---|
| K | 586.40 ± 8.07 |
| Ca | 1004.09 ± 5.60 |
| Mg | 77.64 ± 2.68 |
| Zn | 5.90 ± 0.20 |
| Fe | < 0.01 |
| P | < 0.01 |
| Cu | < 0.01 |
Mendes et al. (1998) noticed a color ranging from colorless to dark amber, analyzing honey produced in Portugal. The color of honey varies according to their floral origin, age and its storage temperature, since overheating may darken the honey.
Santos et al. (2008), analyzing honey from the region of Bahia, Brazil, obtained the following results in mineral content: 32.00 mg/kg Ca; 15.00 mg/kg Mg; 555.00 mg/kg K; 0.4 g/kg Cu. Fe, P and Cu were not observed in the analysis by the detection limit of atomic absorption spectrophotometer used in the determination (Table 2). Hernandez et al. (2005), using the method of atomic absorption spectrophotometry found values ranging from 0.41 to 52.51 mg/kg for Fe and 0.10 to 0.44 mg/kg for Cu. Conti (2000) obtained values of 83.00 and 5.64 mg/kg Fe and Cu, respectively, in honey produced in Italy. Guler et al. (2007) evaluated honey produced in the midst of plantations of thyme, white nettle, clover and sage which resulted in 1.82 mg/kg P, a mineral not detected in cassava honey. The variation in the contents of mineral compounds reported in the literature occurs due to the different vegetation visited by bees as well as the nature of the soil of the regions.
Rheological measurements Typical flow curves for honey sample are shown in Figure 1. No significant changes were observed between the up and down curves hence average values of the rheological parameters were considered. As shown in Table 3, the honey from cassava flower exhibited a shear-thinning behavior because the values of the flow behavior index (n) were lower than 1 (n < 1) for all temperatures. In this study (considering the Power Law model) the n values varied from 0.82 to 0.98, while the value of consistency coefficient (K) ranged between 75.25 to 8.65 Pa.sn. A sharp decrease in the values of consistency coefficient (K) was observed (p ≤ 0.05). Generally, the values of flow behavior index increase with the temperature increase (Haminiuk et al. 2006a, Haminiuk et al., 2006b). Curiously, such behavior was not observed in the cassava flower honey, as observed in strawberry pulp (Bezerra et al., 2009). The same behavior (decrease in the n values) was observed in the study of Witczak et al. (2011), in which the rheological properties heather honey were evaluated. The increase in the pseudoplasticity of the samples with an increase in temperature is owing to the unique composition of the honey, which is rich in sugars. According to Haminiuk et al. (2006b) the heat treatment has a major effect on the consistency coefficient (K) of the non-Newtonian fluid food, analogous to the effect on Newtonian viscosity (η). The flow behavior index (n) is slightly affected by temperature. The temperature effect on apparent viscosity of fluid foods, at a constant shear rate, can be described by the Arrhenius equation (Haminiuk et al., 2006b), in which the apparent viscosity decreases in an exponential function with temperature. The shear rate of 10 s−1 was chosen considering that it is the speed used in the pumping (starting pump) and agitation processes.
Flow curves at different temperatures of cassava flower honey
| Temperature | Consistency coefficient K (Pa.sn) | Flow behavior index n (dimensionless) | SSR | R2 | χ2 |
|---|---|---|---|---|---|
| 30°C | 75.25 ± 1.25a | 0.98 ± 0.05a | 8.61 | 0.99 | 0.47 |
| 40°C | 43.25 ± 1.73b | 0.89 ± 0.06b | 92.23 | 0.99 | 5.12 |
| 50°C | 15.17 ± 2.1c | 0.85 ± 0.05b | 13.17 | 0.99 | 0.73 |
| 60°C | 8.65 ± 1.8d | 0.82 ± 0.08b | 5.16 | 0.99 | 0.28 |
Mean ± SD values followed by the same letter in each column are not significant different by Tukey's test. SSR - sum of squared residuals, R2 - determination coefficient, χ2 - chi-squared.
The Arrhenius model gave a good description of the temperature effect on apparent viscosity of the honey sample at a constant shear rate of 10 s−1, as it is seen in Figure 2. The activation energy (Ea) value calculated for the cassava flower honey was 69.35 kJ/mol with a determination coefficient (R2) of 0.99 and standard error of 0.15. The values of activation energy are consistent with the values available in a recent manuscript published by Haminiuk et al. (2009). Juszczak and Fortuna (2006), evaluating Polish floral honeys, obtained values of activation energy ranging from 92.34 to 105.25 kJ/mol. A higher value of activation energy means rapid change in viscosity with temperature (Steffe, 1996). Yanniotis et al. (2006) were evaluated in Greek honeys and found that the activation energy ranged from 70.8 to 96.3 kJ/mol. The authors describe the influence of the moisture and temperature on the viscosity. An activation energy is necessary for moving of a molecule, and as the temperature increases, the liquid flows more easily due to higher activation energy in high temperatures (Haminiuk et al., 2006a).
Temperature effect on apparent viscosity fitted by the power law model to the cassava flower honey
Cassava flower honey presented quality indexes within the parameters required by current standards, except for apparent sucrose content, which is above the level allowed for exportation. These values need to be observed and monitored in a way that farmers in the region of São Paulo state can market the honey produced by Africanized A. mellifera amid the cassava culture with physical and chemical quality.
The rheological behavior of cassava flower honey within temperatures of 30 – 60°C was properly described by the power law model. The honey showed shear-thinning behavior and the apparent viscosity was well fitted into the Arrhenius equation.
