Monitoring and evaluation of free acidity levels in Talh honey originated from Talh tree Acacia gerrardii Benth

1 Introduction

Acacia gerrardii Benth., also locally known as “Talh”, is a native tree from the arid and semi-arid regions of Africa and West Asia (Dharani, 2007), including the Arabian Peninsula (Alqarni et al., 2011; Alqarni et al., 2015). Acacia trees are the most prevalent desert plants in Saudi Arabia, and are the main provenance of nectar and pollen for honey bee colonies (Alqarni et al., 2011). They are quite rich in proteins and minerals, and provide nectar-rich summer and subtropical bee forage in Saudi Arabia (Awad et al., 2017). Honey originating from Talh trees (A. gerrardii) is regionally called “Talh honey” in reference to its floral nectar origin. It is one of the most desirable types of honey in the Arabian Peninsula (Adgaba et al., 2017; Alqarni et al., 2017), and exhibits significant antimicrobial activity against pathogenic microorganisms (Owayss et al., 2020).

Natural honey is highly preferred in Saudi Arabia because of its unique cultural, medicinal, and nutritional values (Alqarni et al., 2016). Honey consumption in Saudi Arabia is gradually increasing (Owayss et al., 2020). According to the latest statistics, more than 20,000 tons of honey are available in the Saudi market annually. Of these, domestic production is approximately 2,500 tons, and 18,000 tons are imported from different countries to fulfil market demands (MEWA, 2018).

Honey originates from flowers or certain plant exudates and is processed by honey bees in their stomachs and hives through different physical and physiological treatments. It is a complex nutrient source composed basically of carbohydrates and water with a wide range of minerals, vitamins, enzymes, and organic acids; of these, gluconic acid is predominant in honey as a byproduct of the enzymatic activity of glucose oxidase. Organic acids also influence honey by determining its flavor and aroma (Machado De-Melo et al., 2018). The composition and characteristics of honey depend on variable factors, such as the geographical nectar source, climatic conditions, soil composition, beekeeping practices, honey processing and storage conditions (Pavlova et al., 2018). Certain physicochemical characteristics of honey, e.g., its color, taste, mineral content, pH, moisture, electrical conductivity (EC) and acidity level, are linked with its botanical origin, whereas its hydroxymethylfurfural content and purity are associated to the manufacturing process (Bogdanov et al., 2004).

Various studies have been performed on certain quality parameters of Saudi honeys (Al-Khalifa and Al-Arify, 1999; Mohammed et al., 2017). Talh honey is characterized by a distinct taste and high mineral, acid, and phenolic contents. More importantly, its free acidity levels were shown to exceed the standard permitted levels (Alqarni et al., 2014; Alqarni et al., 2016). This has led to problems in the marketing and exportation of Talh honey. Therefore, this introductory study investigated the free acidity of Talh honey at different stages, i.e., where it originates (leaves and flowers), is processed (bee crops), and is prepared (inside beehives). In recent decades, tools have been developed to assess honey quality and authenticity for consumer protection and fair producer competition (Soares et al., 2017). Quality standard limits and values are usually determined by national and international organizations based on honey composition databases. In Saudi Arabia, there is no comprehensive database characterizing Saudi Arabian honeys, and no published research has addressed this subject. If available, such data could be used to formulate an appropriate and nationally accepted honey quality standard for Talh honey, among others.

2 Materials and methods

3 Results

The physicochemical properties (FA, pH and EC) were determined in the plant leaves, flowers, crops of worker bee (honey stomachs), and the unripe and ripe honey originating from A. gerrardii.

3.1

3.1 Free acidity (FA)

The results revealed significant differences among the FA levels in all tested sources (leaves, flowers, bee crops, and unripe and ripe honey) throughout the flowering season of A. gerrardii (Fig. 1). The highest mean FA was recorded in ripe honey (77 ± 1.28 meq/kg), followed by unripe honey (72 ± 1.56 meq/kg). FA increased gradually from the lowest value in the leaves (30 ± 0.99 meq/kg) to the highest one (77 ± 1.28 meq/kg) in the ripe honey. FA decreased in the order ripe honey (77 ± 1.28 meq/kg) > unripe honey (72 ± 1.56 meq/kg) > honey bee crops (43 ± 1.80 meq/kg) > flowers (34 ± 0.92 meq/kg) > leaves (30 ± 0.99 meq/kg). Levels of FA continued to increase after honey extraction. The mean level of FA from harvested Talh honey after extraction was 82 ± 0.01 meq/kg (Table S1).

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Fig. 1

Level of physicochemical properties (mean ± SE): (A) free acidity, (B) pH and (C) electrical conductivity detected in leaves, flowers, bee crops, unripe honey and ripe honey originating from Talh trees (Acacia gerrardii). Means with the common letter in each figure are not significantly different from each other (p < 0.05, Duncan’s test).

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3.2

3.2 FA at each interval

Throughout the flowering season, the FA at two-week intervals exhibited a gradual constant increase in each tested source, with significant differences among intervals (Fig. 2). The lowest values of FA were recorded in the 1st interval of the flowering season from the leaves (22 ± 1.14 meq/kg), flowers (26.6 ± 0.29 meq/kg), bee crops (34.6 ± 2.60 meq/kg), unripe honey (54.0 ± 1.22 meq/kg) and ripe honey (68.6 ± 0.55 meq/kg), whereas the highest FA values were recorded from leaves (35 ± 0.63 meq/kg) at the 3rd interval, flowers (38.2 ± 1.32 meq/kg) at the 6th interval, bee crops (54.6 ± 1.30 meq/kg) at the 6th interval, unripe honey (77.6 ± 0.24 meq/kg) at the 4th interval, and ripe honey (84.4 ± 0.55 meq/kg) at the 6th interval (Fig. 2 and Table S2).

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Fig. 2

Determination of free acidity (mean ± SE) from A) leaves, B) flowers, D) unripe honey, C) ripe honey, and E) bee crops at fortnightly intervals. Means with the same letter in each figure are not significantly different from each other (p < 0.05, Duncan’s test).

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3.4

3.4 pH at each interval

Fig. 3 shows the pH data at fortnightly (two-week) intervals for leaves, flowers, bee crops, unripe honey, and ripe honey during the flowering season of A. gerrardii. A diversified pattern of fluctuating pH was recorded at fortnightly time intervals throughout the flowering season. There were significant differences among pH values at fortnightly intervals. Relatively low pH values were recorded from leaves (6.3 ± 0.04) at the 3rd, 5th and 6th fortnights; flowers (6.1 ± 0.04) at the 4th and 6th fortnights; bee crops (4.4 ± 0.13) at the 6th fortnight; unripe honey (4.8 ± 0.13) at the 3rd fortnight; and ripe honey (4.3 ± 0.04) at the 1st fortnight. The highest pH values (low acidity) were recorded from leaves (6.7 ± 0.04) at the 1st fortnight, flowers (6.4 ± 0.04) at the 2nd and 3rd fortnights, bee crops (5.5 ± 0.09) at the 3rd fortnight, unripe honey (5.2 ± 0.09) at the 5th fortnight and ripe honey (5.2 ± 0.09) at the 2nd fortnight (Fig. 3 and Table S3). Generally, the pH values of unripe and ripe honey samples were acidic, within the range of 4.3 ± 0.04 to 5.2 ± 0.09, which fall within the recommended limits (pH 3.4 to 6.1) for fresh honey.

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Fig. 3

Determination of pH (mean ± SE) from A) leaves, B) flowers, D) unripe honey, C) ripe honey, and E) bee crops at fortnightly intervals. Means with the same letter in each figure are not significantly different from each other (p < 0.05, Duncan’s test).

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3.6

3.6 EC at each interval

Fig. 4 shows the variable patterns of EC data at each fortnightly (two-week) interval for each tested source across the flowering season. Significant differences were observed among intervals for each tested source. Lower values of EC were recorded in the leaves (0.75 ± 0.05 mS/cm) at the 1st fortnight, flowers (0.97 ± 0.08 mS/cm) at the 5th fortnight, while bee crops (0.75 ± 0.25 mS/cm), unripe honey (1.10 ± 0.04 mS/cm) and ripe honey (0.69 ± 0.0.03 mS/cm) were recorded at the 1st fortnight. The highest EC was recorded from leaves (1.59 ± 0.04 mS/cm) at the 3rd fortnight, flowers (1.27 ± 0.02) at the 1st fortnight, however, bee crops (1.30 ± 0.17 mS/cm), unripe honey (2.01 ± 0.23 mS/cm) and ripe honey (1.75 ± 0.03 mS/cm) were recorded at the 2nd fortnights (Table S4).

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Fig. 4

Determination of EC (mean ± SE) from A) leaves, B) flowers, D) unripe honey, C) ripe honey, and E) bee crops at fortnightly intervals. Means with the same letter in each figure are not significantly different from each other (p < 0.05, Duncan’s test).

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3.7

3.7 Seasonal mean values of all three parameters

The values of FA, pH, and EC for each tested source were combined. Fig. 5 shows graphical illustration at each fortnightly interval, regardless of the source, during the season. The data analysis revealed significant differences in FA, pH, and EC over time. FA increased at each interval, with significantly lower values recorded during the early fortnights (1, 2 and 3; values of 41 ± 3.65 meq/kg, 47 ± 3.77 meq/kg and 51 ± 4.30 meq/kg, respectively) than during the last fortnights (4, 5 and 6; values of 55 ± 4.48 meq/kg, 55 ± 4.31 meq/kg, and 58 ± 4.12 meq/kg, respectively) (Table S2). For the pH values, the trend was steady, with values ranging between 5.39 ± 0.15 at the 6th interval and 5.77 ± 0.13 at the 3rd interval with insignificant differences (Table S3). For EC, the data showed a significant increase from the lowest value at the 1st interval (0.91 ± 0.07 mS/cm) to highest value 1.45 ± 0.10 mS/cm at the 2nd interval and then decreased significantly towards the end of the season (Table S4).

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Fig. 5

Seasonal data (mean ± SE) of physicochemical properties from all sources (leaves, flowers, bee crops, and unripe and ripe honey) A) free acidity, B) pH and C) electrical conductivity during the flowering season of Acacia gerrardii. Mean values with the same letter are not significantly different (p < 0.05, Duncan’s test).

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3.8

3.8 Meteorological data and correlation analyses between variables

During the flowering season of A. gerrardii, the mean temperature was 37 °C (highest 40 °C; lowest 34 °C). The mean RH was 11%, with a maximum value of 14% and a minimum value of 8.8% (Fig. 6 and Table S5). The Pearson correlation analyses (p < 0.05) were done among the meteorological data (temperature and RH) and the physicochemical properties (FA, pH and EC) (Table 1). FA had a significant positive correlation with RH (r = 0.18069), whereas EC exhibited a significant negative correlation (r = -0.29246) with temperature (Table 1(. A significant negative correlation (r = -0.70402) was recorded between FA and pH, and a significant positive correlation (r = 0.50974) between FA and EC (Table 1).

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Fig. 6

Mean ± SE of temperature and relative humidity every two weeks (fortnightly) during the flowering season of Acacia gerrardii. (mean seasonal temperature = 37 °C; mean seasonal Humidity = 11).

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Table 1 Pearson correlation coefficients (*Significance at p < 0.05). probability level.

Parameter	Correlation Coefficient values (r)
	EC	pH	FA	Temp.	RH
EC	1	−0.17321	0.50974*	−0.29246*	−0.11576
pH		1	−0.70402*	−0.02298	−0.05912
FA			1	−0.10517	0.18069*
Temperature (°C)				1	−0.22177
Relative Humidity (RH) %					1

4 Discussion

Our results confirmed that A. gerrardii honey is normally characterized by increased levels of FA than those of standards. The values of FA among all tested sources were significantly different along the pathway from leaves and flowers to bee crops and unripe and ripe honey. The FA values of Talh honey in the present study exceeded the maximum level (50 meq/kg) permitted by the (Codex Alimentarius Commission, 2019) and the EU (Council, 2002) as well as the maximum limit (40 meq/kg) adopted by the GC (Council, 2014). The levels of FA in Acacia honey recorded in this study are in line with those reported in the previous studies. (Alqarni et al., 2016) measured a wide range of FA values (12.0–134.5 meq/kg) in a variety of local and imported honey samples and found high acidity values (112–134.5 meq/kg) in Acacia honey that were over the permitted levels, and were even higher than the FA values recorded in the present study. In addition, honey imported from Tualang (Malaysia), the Black Forest (Germany), and Cotton (Egypt) also revealed high values of FA. (Al-Farsi et al., 2018) found high FA levels with an average of 84.9 meq/kg (range: 38.5 to 126 meq/kg) in 18 out of 21 Acacia honey samples obtained from various areas of Oman. (Al-Doghairi et al., 2007) found that FA ranged between 9.12 and 93.02 meq/kg in different Saudi honeys. The FA of Acacia honey was also found to be affected by altitude such as 19.8 meq/kg at 1000 m altitude to 65 meq/kg at 2200 m altitude in the Asir Mountains of Saudi Arabia (Mohammed et al., 2017). The levels of FA in honey are correlated mainly to its content of organic acids, specifically gluconic acid (Crane, 1990). Gluconic acid in honey is produced after the bee enzyme glucose oxidase acts on the glucose in nectar (White et al., 1978). FA is associated with the flower type, mineral content, harvest season, and presence of inorganic ions (Karabagias et al., 2014). The FA levels in our study increased from the leaves and flowers to the bee crops and the unripe and ripe honey and finally to the harvested honey. This increase may be due to the presence of high levels of gluconic acid in Acacia flowers, which would confirm the value of Acacia trees as a rich source of nectar (Awad et al., 2017). (White and Crane, 1975) stated that the gluconic acid levels in honey increased with the time required for the total conversion of nectar into honey.

Another possible source of the high levels of FA in Talh honey is the extrafloral nectaries (EFNs) found on each compound leaf of the Talh tree. (Phadke et al., 1970) confirmed that honey derived from EFNs may show increased FA levels. Several studies have reported that EFNs are frequently visited by honey bee workers (Alqarni et al., 2015; Alqarni et al., 2017). The presence of numerous components, such as amino acids and carbohydrates in nectar reflects the specific taste of nectar from floral and EFN nectaries and corresponds to their attractiveness to forager insects (Nicolson et al., 2007).

High FA levels that are above the permitted standards could therefore be considered a feature rather than an indicator of low quality and are related to the geographical and floral origin of the honey (Ananias et al., 2014). The adoption of national guidelines for monofloral honey quality evaluation has been thoroughly deliberated. (Thrasyvoulou et al., 2018) reported that several countries; such as Greece and Serbia, have national legislations regarding the properties of eight types of monofloral honey. Germany has laws regarding ten monofloral honeys, and Turkey manages national regulations for almost all monofloral honeys produced in the country. The botanical origin of honey is normally determined through pollen analysis (Bogdanov et al., 2004). According to (Louveaux et al., 1978); the botanical of honey was determined through its pollen content analysis. We found that 88% of the pollen examined in Talh honey was related to A. gerrardii (Talh), indicating that Talh honey tested in this study was monofloral i.e. originating primarily from A. gerrardii. Several countries have established legal pollen limits for some kinds of monofloral honey. For example, Croatia has defined 12 kinds of monofloral honey, Germany has defined nine, Serbia has defined eight, Greece has defined six, and Italy has defined two (Thrasyvoulou et al., 2018). The establishment of legal standards for the characteristics of Talh honey is crucial to overcome its ineligibility for trade due to its high FA levels. Previous reports have argued that a single parameter should not be used to disqualify honey (Welke et al., 2008).Therefore, it is very important to review the honey quality standards for local Saudi honeys, especially Talh honey. FA levels in natural Talh honey should be modulated when compared to standard quality evaluation criteria, as the existing standards could disqualify the natural Talh honey which has naturally high FA.

For pH values, the trend was similar to that for FA. The pH values in unripe and ripe honey and bee crops were more acidic (lower) than those in leaves and flowers. Thus, Talh honey originating from A. gerrardii is acidic in nature. These results are consistent with those in several previous studies (Al-Doghairi et al., 2007; Alqarni et al., 2016; Mohammed et al., 2017). The pH range recorded in the present study was within the permitted limits (3.42–6.10) of the (Codex Alimentarius Commission, 2019). An acidic pH is desirable in honey both for flavor and to resist microbial growth during the long shelf life of honey (Al-Doghairi et al., 2007; Krishnasree and Ukkuru, 2017).

The EC values (0.98–1.56 mS/cm) in all tested samples (leaves, flowers, bee crops, unripe honey, and ripe honey) exceeded permitted standards (0.8 mS/cm) (Codex Alimentarius (Commission, 2001); EU (Council, 2002); GC (Council, 2014). Previous study reported similar findings (Mohammed et al., 2017). High levels of FA correspond to high EC values, and vice versa (Krishnasree and Ukkuru, 2017); this implies the high mineral content of Talh honey, which is considered a distinctive nutritional feature (Mohammed et al., 2017). Therefore, considering high EC values in the quality evaluation criteria for Talh honey is recommended for the same reasons provided to consider high FA values in the criteria.

The seasonal variations in FA, pH, and EC were monitored at fortnightly intervals during the experimental period. A gradual, significant increase in FA and fluctuating patterns of pH and EC over time were recorded from the tested samples of leaves, flowers, bee crops and in-hive honey (unripe and ripe honey). The unique patterns of physicochemical parameters in different honeys provide a useful method for distinguishing among honeys of various geographical origins (Acquarone et al., 2007). Many biotic and abiotic factors can significantly influence the quality and quantity of nectar secretions (Adgaba et al., 2017). The temperature and humidity can affect the volume and nectar concentration (Nicolson et al., 2007). The rate of nectar secretion varies hourly and monthly, mainly due to fluctuations in weather (temperature, rainfall, relative humidity, etc.) during the plant flowering season (Alqarni, 2015); these factors may indirectly affect honey characteristics and quality. The basic properties of honey change over time due to variable aspects of the nectar, flora and season (Nicolson et al., 2007; Adgaba et al., 2017; Alqarni et al., 2017). Therefore, fluctuations in physicochemical properties such as those observed in our experiments are inevitable. Our results revealed significant positive correlations between FA and RH and between FA and EC. (Krishnasree and Ukkuru, 2017) indicated that high levels of FA were associated with high EC, and vice versa. (Živkov-Baloš et al., 2018) also found a strong positive correlation between FA and EC. Direct correlations between FA and pH (Cavia et al., 2007) and strong positive correlations between FA, EC and pH (Ratiu et al., 2020) were also reported. The different geographical and floral origins, environmental factors, and beekeeping practices over the globe may result in different trends in honey. However, for A. gerrardii (Talh) honey, it is obvious that the high levels of FA associated with high EC values are related to the nature of its botanical origin rather than to a lack of quality.

5 Conclusion

Our investigation demonstrated that free acidity during the Talh tree blooming season gradually increased from leaves and flowers to bee crops and unripe honey and reached its highest value in ripe honey. Likewise, we recorded a strong, significant connection among the free acidity, pH level, and electrical conductivity. The high FA and EC levels in Talh honey can be viewed as a feature that is related to its botanical origin rather than to a lack of quality. Further studies are warranted to fully understand the mechanism underlying the natural high free acidity in Talh trees and honey, especially in the hot and arid regions e.g. the Arabian Peninsula.