Physicochemical Properties of Stingless Bee Honey

Physicochemical Properties of Stingless Bee Honey: History

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Stingless bee honey (SLBH) has a high moisture content, making it more prone to fermentation and leading to honey spoilage. Dehydration of SLBH after harvest is needed to reduce the moisture content.

stingless bee honey
dehydrated honey
chemical composition

1. Introduction

Stingless bee honey (SLBH) is an emerging functional food due to its many health benefits. SLBH is rich in flavonoid and phenolic content, which contributes to its high antioxidant activity ^[1]. The common phenolic compounds in SLBH are the same as Apis mellifera, such as salicylic acid, p-coumaric acid, caffeic acid, chlorogenic acid, ferulic acid and quercetin ^[2]. Despite its benefits, SLBH has generally higher moisture content than Apis spp. ^[3]. A previous study showed that the moisture content in SLBH is the highest with 33.24%, compared to Apis spp. which is between 21.96–27.41% ^[4]. SLBH of Melipona spp. has a moisture content above 24.8% compared to Apis spp. with 18.6% ^[5]. A comprehensive review reported that SLBH contains more moisture (21.52–31%) than Tualang and Gelam honey (17.53–26.51%) ^[6]. Furthermore, SLBH has a high water activity of 0.76 compared to a range between 0.60–0.67 in Apis spp. ^[4].

The high moisture content in SLBH makes it more susceptible to alcoholic fermentation contributing to honey acidity ^[7]. The rapid fermentation by microorganism growth in SLBH leads to honey spoilage ^[8]. Apart from high water content, SLBH has higher free acidity, electrical conductivity and lower diastase activity compared to Apis spp. ^[3]. Hence, it is difficult for SLBH to follow the honey standard. Several studies have proposed a different standard for SLBH given the difficulty for SLBH to follow the International Honey Commission (IHC) standard ^[9].

Owing to the high water content, it is a challenge to maintain the quality of SLBH. Therefore, dehydration of SLBH upon collection is suggested to lower the moisture content. Microbial stability can be achieved through dehydration, thus, prolonging the shelf life of honey ^[8]. Additionally, lowering the moisture content will help the SLBH adhere to the standard. However, previous studies have shown that the dehydration process can reduce the phenolic content ^[10]. In addition, thermal treatment can increase hydroxymethylfurfural (HMF) content in honey ^[11]. The phenolic content is an essential source of antioxidants in honey ^[1]. Meanwhile, HMF is a potential carcinogenic and genotoxic agent ^[12]. Therefore, a suitable dehydration method is needed to obtain the maximum benefit from SLBH by reducing its moisture content without compromising its phenolic content and ensuring a safe level of HMF.

Globally, almost 500 species of SLBH are distributed in South America, Africa, Australia and Southeast Asia ^[13]. Despite the numerous species, the most commonly domesticated stingless bee honeys by beekeepers worldwide are from Melipona and Trigona genera ^[14]. To the best of the knowledge, there are limited publications on the dehydration of SLBH that provide information on the changes in its physicochemical properties. This review aims to provide an overview of the available information on the physicochemical properties of SLBH before and after the dehydration process. This will help to determine the most optimal setting and method of dehydration for SLBH without compromising its benefit. The physicochemical information retrieved includes moisture content, water activity, pH, free acidity, hydroxymethylfurfural (HMF), ash, electrical conductivity, diastase, sugar content, total soluble solids, total phenolic content and total flavonoid content.

2. Physicochemical Properties of Dehydrated Stingless Bee Honey

2.1. Moisture Content

Moisture content is the amount or percentage of water present in the honey ^[15]. Water in honey is the key factor for honey quality as it determines the ability of honey to resist spoilage by microorganism fermentation ^[16]. Previous studies presented in Table 1 showed that the percentage of moisture content of raw SLBH was between 23.9 and 40%. However, another study showed that the moisture content of raw SLBH was between 13.26 and 45.8% ^[9]. The wide range in the percentage of moisture content was due to environmental factors such as seasonal weather and humidity ^[8]. Harvest and storage conditions also influenced the moisture content in SLBH ^[9].

Table 1. Moisture content of raw and dehydrated stingless bee honey (SLBH).

Method of Dehydration		SLBH Species	T (°C)/ PL	Time	Moisture Content (%)		Water Reduction (%)	Author
Method of Dehydration		SLBH Species	T (°C)/ PL	Time	Raw	Dehydrated	Water Reduction (%)	Author
Thermal treatment		Tetragonisca angustula	52 °C	470 min	23.9	21.6 *	9.6	^[17]
		T. angustula	55 °C	170 min	23.9	22.7 *	5.0
		T. angustula	57 °C	60 min	23.9	23.3 *	2.5
		T. angustula	60 °C	22 min	23.9	23.3 *	2.5
		T. angustula	66 °C	8 min	23.9	23.3 *	2.5
		T. angustula	66 °C	3 min	23.9	23.4	2.1
		T. angustula	68 °C	1 min	23.9	23.4	2.1
		T. angustula	71 °C	24 s	23.9	23.5	1.7
		Melipona bicolor	90 °C	15–60 s	30.8	29.5 *	4.2	^[18]
		M. bicolor	95 °C	15–60 s	30.8	29.5–29.6 *	3.9–4.2	^[18]
		-	45–90 °C	30–120 min	30.93	28.8	6.9	^[19]
Thermosonication		-	45–90 °C	30–120 min	31.06	25.9	16.6	^[19]
Vacuum	Drying (5% moisture)	Heterotrigona itama	40–60 °C	-	31.9	5	84.3	^[20]
	Drying (11% moisture)	H. itama	40–60 °C	-	31.9	11	65.5
	Evaporation (11% moisture)	H. itama	40–60 °C	-	31.9	11	65.5
Freeze-drying (5% moisture)		H. itama	−54 °C	24 h	31.9	5	84.3
Microwave heating		H. itama	20 PL	15–60 s	31.47	25.24–26.46	16–20	^[21]
		H. itama	60 PL	25–60 s	31.47	15.04–20.3 *	35–52
		H. itama	100 PL	5–15 s	31.47	22.29–24.32 *	23–29
Dehumidification		H. itama	35 °C	1–2 days	31.47	17.21–17.48 *	44–45
Food dehydrator		H. itama	40 °C	36 h	40	<8	>80	^[22]
		H. itama	55 °C	18 h	40	<8	>80
		H. itama	55 °C	36 h	40	0	100
		H. itama	70 °C	18 h	40	<8	>80
		H. itama	70 °C	36 h	40	0	100
MARDI dehydrator		H. itama	30 °C	8 h	29	19	35	^[23]
Glass bottle storage		Geniotrigona thoracica	25 °C	1–10 days	26.21	25–26	0.8–4.62	^[24]
Clay pot storage	Small surface area	G. thoracica	25 °C	1–10 days	26.21	24.32 *	7.21
	Large surface area	G. thoracica	25 °C	1–10 days	26.21	23.35 *	10.9
		H. itama	25 °C	1–21 days	25.82	18.13–25.13 *	2.7–29.8	^[25]
		H. itama	35 °C	1–3 days	25.82	19.56–23.68 *	8.3–24.2	^[25]

Several studies summarized in Table 1 showed that reduction in the water content of the SLBH after harvesting could be achieved either by increasing the temperature through various dehydration methods or via passive diffusion. The temperature used in the dehydration process was between 30 and 95 °C, while the temperature for the passive diffusion method was between 25 and 35 °C. As a result, the moisture content of SLBH was reduced between 29.6 and 5% after the dehydration process using these various methods. Moisture content below 17% could prevent the fermentation process by the microorganisms ^[26].

As a conclusion, according to the data presented in Table 1, the dehydration process using thermal treatment will only cause less than a 10% reduction in water content. Meanwhile, another study showed that the thermosonication method of the dehydration process caused a 16.6% reduction in water content compared to 6.9% using the thermal method ^[19]. These findings suggest that thermosonication is the better method for the dehydration process for SLBH compared to thermal treatment. However, both methods could not reduce the moisture content below 17% (25.9% for thermosonication and 28.8% for the thermal treatment method).

A study showed that the moisture content of the SLBH was reduced from 31.9 to 11 and 5% after the dehydration process using vacuum and freeze-drying methods ^[20]. As presented in Table 1, the vacuum drying and freeze-drying at 5% moisture setting could achieve an 84.3% reduction in water content. Meanwhile, a 65.5% reduction in water content of the SLBH was observed after dehydration using vacuum drying and evaporation at 11% moisture setting. These findings suggest that both vacuum treatment and freeze-drying are the best methods in reducing the moisture content of SLBH. In addition, both methods could achieve a safe level of moisture content below 17%.

A study by Yegge et al. ^[21] showed that the dehydration process using microwave heating and dehumidification methods could reduce water content by up to 52% and 45%, respectively, as presented in Table 1. In the study, both methods could reduce almost half of the water content in raw SLBH. The microwave heating method used a power level of energy (PL) of 20, 60 and 100. However, only the microwave heating method at 60 PL for 60 s could reduce the moisture content below 17% (from 31.47 to 15.04%). Meanwhile, the dehumidification process was performed for 1 to 2 days. Therefore, microwave heating at 60 PL for 1 min was the best method for achieving the recommended moisture content level below 17%. In addition, this method was more practical because it takes less time to prepare the dehydrated SLBH.

From the data provided in Table 1, a previous study has also shown that the dehydration process of SLBH using a food dehydrator could reduce the water content of SLBH up to 80–100% ^[22]. The food dehydrator could achieve 80% water reduction at 40 °C for 36 h or at 55 °C and 70 °C for 18 h. Complete water reduction was achieved at 55 °C and 70 °C by prolonging the duration of the dehydration process to 36 h ^[22]. Another study showed that a dehydrator developed by the Malaysian Agricultural Research and Development Institute (MARDI) could reduce 35% of the water content ^[23]. However, the MARDI dehydrator set at 30 °C for 8 h was unable to reduce the moisture content below 17% ^[23]. Meanwhile, the conventional food dehydrator set between 40 and 70 °C for the duration of 18 to 36 h could achieve recommended moisture content level below 17% ^[22]. These findings suggest that a higher temperature would result in a higher reduction in moisture content.

Several studies summarized in Table 1 showed that the dehydration process of the SLBH can be performed via passive diffusion by storage in a clay pot. A study by Ghazali et al. ^[24] showed that the reduction in the moisture content was significant in the clay pot compared to the glass container. In addition, the storage in a clay pot with a larger surface area resulted in a 10.9% reduction in water content compared to a smaller clay pot with only 7.21%. This finding suggests that the larger the surface area of the container, the more effective the passive diffusion process will occur. On the other hand, the storage of SLBH at 35 °C for three days could reduce up to 24.2% of water ^[25]. Meanwhile, the storage of SLBH at room temperature (25 °C) for 21 days could reduce water content by up to 29.8% content ^[25]. These findings suggest a higher temperature would expedite the passive diffusion process. However, the dehydration process via passive diffusion requires a long duration to reduce the moisture content of the SLBH. Furthermore, the moisture content after storage in the clay pot was between 18.13 and 25.13%, which was still above the recommended moisture content level at 17%.

Various dehydration methods of SLBH can reduce moisture content depending on the temperature and duration of the dehydration process. Researchers concluded that the higher the temperature setting, the more reduction in water content. For that, researchers suggest the dehydration method of SLBH at a high temperature setting to achieve at least less than 17% moisture content to retard the fermentation process. The methods that yield low moisture contents are vacuum treatment, freeze-drying, food dehydrator and microwave heating at 60 PL. In conclusion, researchers observed that the food dehydrator is the best method because it could remove up to 80 to 100% water content, resulting in moisture content of less than 17%. However, it takes up to 18 to 36 h in duration. Therefore, microwave heating at 60 PL is the method of choice due to the short duration of 60 s with the moisture content of less than 17%. Although the vacuum treatment could reduce the moisture content to 5 and 11%, the duration of the dehydration process was not mentioned by the authors.

2.2. Water Activity

Water activity is a measurement of free unbound water that can be utilized by microorganisms for growth ^[27]. Water activity gives a better prediction of the likelihood of the fermentation process occurring compared to moisture content ^[16]. Therefore, water activity is used as an indicator of food stability, which is important for the determination of honey spoilage due to microbial growth ^[4]. Water activity (aw) is expressed in decimals and calculated from equilibrium relative humidity (ERH) divided by 100 (aw = ERH (%)/100) ^[28]. ERH is the equilibrium of humidity of the food product with its environment.

Microorganisms will not grow below a particular water activity level, which is 0.90 for bacteria and 0.70 for molds. A water activity of less than 0.6 will halt all types of microbial growth ^[27]. Hence, it is crucial to maintain the water activity of SLBH below 0.6. Water activity is strongly correlated with moisture content ^[16]. Therefore, the dehydration process of SLBH is needed to reduce moisture content and water activity in SLBH. Subsequently, the dehydration process will help to prevent the likelihood of fermentation due to the inability of the microorganism to grow in the SLBH. A previous study reported that SLBH has the highest water activity of 0.76 compared to Apis spp. and commercialized honey with water activity ranges between 0.54–0.67 ^[4]. Several studies compiled in Table 2 showed that the water activity of raw SLBH was between 0.79 and 0.807. After the dehydration process, the water activity was reduced between 0.28 and 0.785 as presented in Table 2.

Table 2. Water activity of raw and dehydrated stingless bee honey (SLBH).

Method of Dehydration		SLBH Species	T (°C)	Time	Water Activity		Water Activity Reduction (%)	Author
Method of Dehydration		SLBH Species	T (°C)	Time	Raw	Dehydrated	Water Activity Reduction (%)	Author
Thermal treatment		-	45–80	30–100 min	0.795	<0.767	<3.5	^[19]
Thermal treatment		-	90	120 min	0.795	0.767	3.5
Thermosonication		-	45–80	30–100 min	0.807	<0.743	<7.9
Thermosonication		-	90	120 min	0.807	0.743	7.9
Vacuum	Drying (5% moisture)	Heterotrigona itama	40–60	-	0.79	0.28–0.29 *	63.3–64.6	^[20]
	Drying (11% moisture)	H. itama	40–60	-	0.79	0.45–0.48 *	39.2–43
	Evaporation (11% moisture)	H. itama	40–60	-	0.79	0.47–0.5 *	36.7–40.5
Freeze-drying (5% moisture)		H. itama	−54	24 h	0.79	0.3 *	62
Food dehydrator		H. itama	40	36 h	0.788	<0.6	>23.9	^[22]
		H. itama	55	18 h	0.788	<0.6	>23.9
		H. itama	70	12 h	0.788	<0.6	>23.9
Glass bottle storage		Geniotrigona thoracica	25	1–10 days	0.8	0.782–0.785	1.9–2.25	^[24]
Clay pot storage	Small surface area	G. thoracica	25	1–10 days	0.8	0.679–0.774 *	3.3–15.1
	Large surface area	G. thoracica	25	1–10 days	0.8	0.632–0.737 *	7.9–21
		H. itama	25	1 day	0.79	0.79	-	^[25]
		H. itama	25	7–21 days	0.79	0.63–0.7 *	11.4–20.3
		H. itama	35	1–3 days	0.79	0.7–0.76 *	3.8–11.4

According to the data summarized in Table 2, thermosonication causes a 7.9% reduction in water activity compared to 3.5% for the thermal method ^[19]. This suggests that thermosonication is the better method in reducing water activity compared to thermal treatment. However, the water activity was 0.743 and 0.767 for thermosonication and thermal treatment, respectively, which was still above 0.6.

A study by Chen et al. ^[20] showed that dehydration processes using vacuum and freeze-drying methods at a 5% moisture setting were able to reduce the water activity level from 0.79 to less than 0.3, as presented in Table 2. Meanwhile, the water activity level of the vacuum drying and evaporation at an 11% moisture setting could reduce the water activity level to less than 0.5. These findings suggest that both vacuum treatment and freeze-drying methods could reduce the water activity level of SLBH to less than 0.6. It is observed that vacuum drying and freeze-drying at a 5% moisture setting was the best dehydration process for reducing the water activity level. However, the freeze-drying method at a 5% moisture setting needs 24 h to achieve a 0.3 water activity level. Meanwhile, the duration of vacuum treatment was not mentioned by the author.

A study showed that dehydration of SLBH using a food dehydrator could reduce water activity levels from 0.788 to less than 0.6 ^[22]. In the study, the water activity of less than 0.6 was achieved with 40 °C for 36 h, 55 °C for 18 h, and 70 °C for 12 h, as summarized in Table 2. The study findings showed that the dehydration process using a food dehydrator at a higher temperature will take less time to reduce the water activity level to less than 0.6.

From the data provided in Table 2, the dehydration of SLBH via passive diffusion could reduce water activity levels from a range between 0.79–0.8 to 0.63–0.785 ^[24]. The study showed that SLBH stored in a clay pot could reduce water activity up to 21% compared to storage in a glass container, which was only up to 2.25%. The surface area of the clay pot also plays an important role in the reduction in water activity. The study showed up to 21% reduction in the water activity level in a clay pot with a larger surface area compared to 15.1% for a clay pot with a smaller surface area ^[24]. Another study has also shown more reduction in water activity will be achieved in a clay pot at 35 °C compared to 25 °C ^[25]. In the study, the water activity level of the SLBH in a clay pot at 35 °C was 0.7 after three days. Meanwhile, the water activity level of the SLBH in a clay pot at 25 °C was 0.7 after seven days. Therefore, the duration of the dehydration process is shorter as the temperature setting in the clay pot storage increases. However, this passive diffusion method of the dehydration process was unable to reduce the water activity level below 0.6.

All the dehydration methods could reduce water activity levels. In addition, a higher temperature setting of the dehydration process progressively reduces the water activity level of the SLBH. The methods of dehydration that produced a water activity level below 0.6 were vacuum method, freeze-drying, and food dehydrator. The food dehydrator at 70 °C was the best method of dehydration to achieve a water activity level below 0.6 within a shorter duration. Meanwhile, the vacuum method could reduce water activity levels below 0.5, but the duration was not mentioned by the authors.

2.3. Hydroxymethylfurfural

Hydroxymethylfurfural (HMF) is a chemical compound from the furan group that indicates the freshness, overheating and ageing of honey ^[7]. HMF and diastase activity are indicators of overheating. However, HMF is a more reliable parameter for overheating compared to diastase activity ^[29]. The HMF content can provide information regarding total heat exposure to honey ^[29]. Apart from overheating, prolonged honey storage also promotes the formation of HMF by degradation of the honey sugar into HMF ^[30]. A previous study showed that raw SLBH has a lower HMF content than Apis mellifera honey because raw SLBH has higher acidity and water activity that can slow down the Maillard reaction ^[31].

Codex Alimentarius Standards (2001) has set that the HMF level should not exceed 40 mg/kg for honey, except for that from the tropical region, which should not exceed 80 mg/kg. A high concentration of HMF is potentially carcinogenic and genotoxic ^[12]. A previous study showed that the heat from thermal treatment can increase the HMF content in honey ^[11]. Therefore, the dehydration process needs to be controlled to ensure strict adherence to the maximum permitted amount of HMF. Several studies summarized in Table 3 showed that the HMF was not detected in raw SLBH, except for in a study by Syariffuddeen et al. ^[23] that reported a HMF level of 2.27 mg/kg. After the dehydration process, the HMF content either remained unchanged or increased between 2.39 and 238.18 mg/kg, as presented in Table 3.

Table 3. Hydroxymethylfurfural (HMF) content of raw and dehydrated stingless bee honey (SLBH).

Method of Dehydration	SLBH Species	T (°C)	Time	HMF (mg/kg)		Author
Method of Dehydration	SLBH Species	T (°C)	Time	Raw	Dehydrated	Author
Thermal treatment	Tetragonisca angustula	52	470 min	<LOQ	<LOQ	^[17]
	T. angustula	55	170 min	<LOQ	<LOQ
	T. angustula	57	60 min	<LOQ	<LOQ
	T. angustula	60	22 min	<LOQ	<LOQ
	T. angustula	66	8 min	<LOQ	<LOQ
	T. angustula	66	3 min	<LOQ	<LOQ
	T. angustula	68	1 min	<LOQ	<LOQ
	T. angustula	71	24 s	<LOQ	<LOQ
	Melipona bicolor	90	15–60 s	<LOQ	<LOQ	^[18]
	M. bicolor	95	15–60 s	<LOQ	<LOQ	^[18]
	-	75–95	20–60 s	<LOQ	<LOQ	^[31]
	-	75	15 min	<LOQ	<LOQ
	-	75	24 h	<LOQ	238.18
	-	45–67.5	30–75 min	0	0
	-	67.5–90	100–120 min	0	↑ up to 42.40 *	^[19]
Thermosonication	-	45–67.5	30–75 min	0	0
Thermosonication	-	67.5–90	100–120 min	0	↑ up to 62.46 *
Vacuum drying (5% moisture)	Heterotrigona itama	40	-	0	9.3 *	^[20]
	H. itama	50	-	0	10.71 *
	H. itama	60	-	0	12.18 *
Freeze-drying (5% moisture)	H. itama	−54	-	0	9.29 *
Food dehydrator	H. itama	40	18–36 h	0	0	^[22]
	H. itama	55	18 h	0	<5.81
	H. itama	55	36 h	0	5.81
	H. itama	70	18 h	0	<50
	H. itama	70	36 h	0	83.19
MARDI dehydrator	H. itama	30	8 h	2.27	2.39	^[23]

According to the data provided in Table 3, the HMF content remained below the detection level after thermal treatment. However, as the duration of thermal treatment was prolonged at 75 °C for 24 h, the HMF content increased to 238.18 mg/kg, which exceeded the standard set by Codex ^[31]. Similarly, a study by Chong et al. ^[19] showed that the HMF level remained undetected at a low temperature and short duration in the thermal treatment and thermosonication method. However, the HMF content increased as the temperature and duration of dehydration increased to 67.5–90 °C for 100–120 min. HMF level was higher after the thermosonication process (62.46 mg/kg) compared to in the thermal treatment (42.40 mg/kg). These findings suggest that the thermal treatment is the better dehydration method for SLBH compared to thermosonication. However, prolonged duration and higher temperature setting in both methods can increase HMF level above the permitted level of 40 mg/kg.

A previous study showed that HMF content increased after the dehydration process using vacuum drying and freeze-drying methods from zero to up to 12.18 mg/kg, and 9.29 mg/kg, respectively ^[20]. The HMF content increased as the temperature of vacuum drying increased, as shown in Table 3. Both dehydration methods were able to increase the HMF content even at low temperatures, as low as 40 and −54 °C. However, the increase in HMF content was far below the permitted level of 40 mg/kg.

A study by Yap and colleagues ^[22] showed that HMF content remained undetected after the dehydration process using a food dehydrator at low temperature as presented in Table 3. However, as the temperature and duration increased to 55 and 70 °C for 36 h, the HMF content increased to 5.81 and 83.19 mg/kg, respectively. These findings suggest that the HMF content increases along with the increase in temperature and duration of the dehydration process. On the other hand, the dehydration process using the MARDI dehydrator at 30 °C for 8 h showed a slight increase in HMF from 2.27 to 2.39 mg/kg ^[23]. Therefore, the settings in which HMF content remained below 40 mg/kg were a food dehydrator at 40 and 50 °C for 18 to 36 h and the MARDI dehydrator. The HMF content exceeded the permissible limit using a food dehydrator above 70 °C.

In conclusion, the HMF content increased as the temperature and duration of the dehydration process increased. These were observed in thermal treatment, thermosonication, vacuum drying and dehydration using food dehydrator methods. The dehydration method that could maintain HMF content below the permitted level of 40 mg/kg were the thermal treatment for a short duration, thermosonication at 45–67.5 °C for 30–75 min duration, vacuum drying, freeze-drying, food dehydrator at 40 and 55 °C for 18–36 h, and MARDI dehydrator. The best dehydration method was food dehydrator at 40 °C for 36 h duration setting, because the HMF content remained undetectable, although heated for many hours.

2.4. pH

Honey is naturally acidic due to the presence of organic acids ^[15]. SLBH has a mixed sweet and sour taste ^[32]. Previous studies reported that SLBH has the lowest pH of 3.04 compared to Tualang and Acacia honey, with pH of 3.63 and 3.61, respectively ^[33]. Honey becomes more acidic as a result of the fermentation process ^[15]. Therefore, the rapid susceptibility of SLBH to alcoholic fermentation will further reduce the pH level. Hence, dehydration of honey is required for water removal, which subsequently, can prevent honey fermentation. Various studies compiled in Table 4 showed that the pH of raw SLBH ranges from 3.11 to 5.18. These values were consistent with a previous study that showed that the pH level of raw SLBH ranges between 3.15 and 6.64 ^[9]. After the dehydration process, the pH value ranges between 3.11 and 5.14, as shown in Table 4.

Table 4. pH of raw and dehydrated stingless bee honey (SLBH).

Method of Dehydration		SLBH Species	T (°C)/ PL	Time	pH		Alteration (%)	Author
Method of Dehydration		SLBH Species	T (°C)/ PL	Time	Raw	Dehydrated	Alteration (%)	Author
Thermal treatment		Tetragonisca angulusta	52 °C	470 min	5.18	4.93 *	↓ 4.8	^[17]
		T. angulusta	55 °C	170 min	5.18	5 *	↓ 3.5
		T. angulusta	57 °C	60 min	5.18	5.04	Not sig.
		T. angulusta	60 °C	22 min	5.18	5.01 *	↓ 3.3
		T. angulusta	66 °C	8 min	5.18	5.08	Not sig.
		T. angulusta	66 °C	3 min	5.18	5.01 *	↓ 3.3
		T. angulusta	68 °C	1 min	5.18	5.03	Not sig.
		T. angulusta	71 °C	24 s	5.18	5.14	Not sig.
		Melipona bicolor	90 °C	15–60 s	3.25	3.25	–	^[18]
		M. bicolor	95 °C	15–60 s	3.25	3.26	Not sig.	^[18]
		-	50 °C	-	3.81	3.85	Not sig.	^[34]
		-	75 °C	-	3.81	3.89 *	↑ 2.1
		-	90 °C	-	3.81	3.96 *	↑ 3.9
Vacuum	Drying (5% moisture)	Heterotrigona itama	40–60 °C	-	3.16	3.36–3.41 *	↑ 6.3–7.9	^[20]
	Drying (11% moisture)	H. itama	40–60 °C	-	3.16	3.21–3.31 *	↑ 1.6–4.7
	Evaporation (11% moisture)	H. itama	40–60 °C	-	3.16	3.2–3.29	Not sig.
Freeze-drying (5% moisture)		H. itama	−54 °C	-	3.16	3.14	Not sig.
Microwave heating		H. itama	20 PL	15 s	3.58	3.5	Not sig.	^[21]
		H. itama	20 PL	30–60 s	3.58	3.45–3.48 *	↓ 2.8–3.6
		H. itama	60 PL	25–30 s	3.58	3.45 *	↓ 3.6
		H. itama	60 PL	60 s	3.58	3.51	Not sig.
		H. itama	100 PL	5–15 s	3.58	3.46–3.47 *	↓ 3.1–3.4
Dehumidification		H. itama	35 °C	1 day	3.58	3.54 *	↓ 1.1
Dehumidification		H. itama	35 °C	2 days	3.58	3.62	Not sig.
Glass bottle storage		Geniotrigona thoracica	25 °C	1–10 days	3.11	3.11–3.13	Not sig.	^[24]
Clay pot storage	Small surface area	G. thoracica	25 °C	1–10 days	3.11	3.12–3.16	Not sig.
	Large surface area	G. thoracica	25 °C	1–10 days	3.11	3.11	–
		H. itama	25 °C	1–7 days	3.44	3.55–3.62 *	↑ 3.2–5.2	^[25]
		H. itama	25 °C	14–21 days	3.44	3.34–3.38 *	↓ 1.7–2.9
		H. itama	35 °C	1–3 days	3.44	3.52–3.63 *	↑ 2.3–5.5

Several studies presented in Table 4 showed that the dehydration process by thermal treatment could maintain or decrease the pH level. Meanwhile, another study showed that thermal treatment at 75 °C and 90 °C could increase the pH value by 2.1% and 3.9%, respectively ^[34]. These findings suggest that the dehydration process of SLBH at a high temperature could improve the pH level, making SLBH less acidic. However, the duration was not mentioned by the authors.

The dehydration method by vacuum drying at 5% moisture content could increase pH by 7.9% compared to 4.7% in vacuum drying at 11% moisture content, as shown in Table 4 ^[20]. Meanwhile, no significant changes were observed in pH level after the dehydration process by vacuum evaporation and freeze-drying method ^[20]. Therefore, vacuum drying was the best method because it could improve the pH value.

A study by Yegge et al. ^[21] showed that the dehydration process using microwave heating decreased the pH value from 3.58 to a range between 3.45 and 3.51. This could be attributed to the microwave heating pressure that degrades the honey into organic acids, which gives rise to acidity ^[21]. The same study showed that dehydration of SLBH by the dehumidification process initially decreased pH value on day 1; subsequently, the pH value increased on day two of treatment, as presented in Table 4. However, the improvement in pH was not significant and the dehumidification process had a long duration, up to days. Hence, researchers observed that both microwave heating and dehumidification methods did not improve the pH value by much.

According to the data provided in Table 4, dehydration of SLBH via passive diffusion by a clay pot storage for 10 days and storage of SLBH in a glass container did not alter the pH value ^[24]. Both samples were stored at 25 °C. This indicates that no fermentation process occurred. In contrast, a study by Baroyi et al. ^[25] showed that storage of SLBH in a clay pot at 25 °C for seven days and 35 °C for three days increased the pH level by 5.2 and 5.5%, respectively. These findings suggest that storage of SLBH in a clay pot at a higher temperature could improve the pH level better than storage at a lower temperature. However, the pH value started to deteriorate when stored for longer than 14 days at 25 °C ^[25]. This scenario might be due to a successful dehydration process during the early days of storage that improved the pH value. However, as the duration of storage was prolonged for more than two weeks, the fermentation process started to occur, which reduced the pH level. These suggest that the passive diffusion method is able to improve the pH level during the early days of storage and cannot further improve the pH level as soon as the fermentation process has occurred. Researchers observed that the dehydration via passive diffusion method by a clay pot storage at higher temperature of 35 °C was the better method compared to other passive diffusion dehydration settings. In this setting, it could achieve the highest increase in pH, up to 5.5% within three days.

In conclusion, the dehydration process of SLBH can raise, maintain or reduce the pH level. Despite successful water removal through the dehydration process, it cannot ensure that the pH of SLBH will improve. Methods of dehydration that could increase the pH level are thermal treatment at higher temperature, vacuum drying, and passive diffusion by storage in a clay pot at 25 °C for seven days and at 35 °C for three days. The best dehydration method is vacuum drying at a 5% moisture setting because it resulted in the highest improvement in pH value, which was 7.9%.

2.5. Free Acidity

Free acidity is an indicator of organic acids in honey ^[9]. The elevation of the free acidity level indicates that the fermentation process from sugar to organic acids has occurred ^[35]. Free acidity is expressed in milliequivalents of acid per one kilogram honey (meq/kg) unit. A previous study reported that SLBH has a high free acidity level that is 2.7 times higher than Apis spp., Manuka and commercialized honey ^[4].

Earlier studies have shown that the free acidity of raw SLBH was between 23.2 and 172 meq/kg, as summarized in Table 5. Meanwhile, another study showed that the free acidity of raw SLBH ranges between 5.9 and 592 meq/kg ^[9]. The wide variations in free acidity levels are attributed to the differences in organic acids, floral and geographical origin ^[36]. Following the dehydration process, the free acidity ranges from 21.3 to 181 meq/kg, as shown in Table 5.

Table 5. Free acidity of raw and dehydrated stingless bee honey (SLBH).

Method of Dehydration		SLBH Species	T (°C)	Time	Free Acidity (meq/kg)		Alteration (%)	Author
Method of Dehydration		SLBH Species	T (°C)	Time	Raw	Dehydrated	Alteration (%)	Author
Thermal treatment		Tetragonisca angulusta	52	470 min	23.2	23.3	Not sig.	^[17]
		T. angulusta	55	170 min	23.2	21.7	Not sig.
		T. angulusta	57	60 min	23.2	21.3 *	↓ 8.2
		T. angulusta	60	22 min	23.2	21.4 *	↓ 7.8
		T. angulusta	66	8 min	23.2	21.4 *	↓ 7.8
		T. angulusta	66	3 min	23.2	21.3 *	↓ 8.2
		T. angulusta	68	1 min	23.2	23	Not sig.
		T. angulusta	71	24 s	23.2	21.3 *	↓ 8.2
		Melipona bicolor	90	15–60 s	32.9	31.4–31.5	Not sig.	^[18]
		M. bicolor	95	15 s	32.9	32.2	Not sig.
		M. bicolor	95	60 s	32.9	33.5	Not sig.
Vacuum	Drying (5% moisture)	Heterotrigona itama	40–60	-	152.5	112.5–117 *	↓ 23.3–26.2	^[20]
	Drying (11% moisture)	H. itama	40–60	-	152.5	120–132 *	↓ 13.4–21.3
	Evaporation (11% moisture)	H. itama	40–60	-	152.5	113–123.5 *	↓ 19–25.9
Freeze-drying (5% moisture)		H. itama	−54	-	152.5	150.5	Not sig.
Glass bottle storage		Geniotrigona thoracica	25	1–10 days	172	174–177	Not sig.	^[24]
Clay pot storage	Small surface area	G. thoracica	25	1–10 days	172	179–181	Not sig.
	Large surface area	G. thoracica	25	1–10 days	172	174–178	Not sig.
		H. itama	25	1–7 days	85	88–99 *	↑ 3.5–16.5	^[25]
		H. itama	25	14–21 days	85	82–83 *	↓ 2.4–3.5
		H. itama	35	1–3 days	85	89–94	Not sig.

According to the data compiled in Table 5, the thermal treatment could reduce the free acidity level. However, thermal treatment at low temperature for a long duration or at high temperature for a short duration did not affect the free acidity level in SLBH. These findings suggest that organic acids can be preserved with thermal treatment. Otherwise, the thermal treatment may reduce the organic acid content in SLBH.

A study by Chen et al. ^[20] showed that the dehydration process by vacuum methods decreased the free acidity level by up to a 26.2% reduction, as presented in Table 5. Meanwhile, the freeze-drying method did not affect the free acidity level ^[20]. The temperature setting in the vacuum methods was at 40 to 60 °C while freeze-drying was at −54 °C. Free acidity reduced in the vacuum methods because the heat from the vacuum promoted the loss of volatile organic acids by evaporation or decomposition ^[20]. Therefore, freeze-drying was the better method compared to vacuum methods given its ability to preserve the content of organic acid after the dehydration process. This was because the freeze-drying method was conducted at a very low temperature. Hence, the SLBH was not exposed to heat.

Previous studies summarized in Table 5 showed that the dehydration method by passive diffusion via storage in a clay pot could maintain the free acidity level when stored at 25 °C for 10 days and at 35 °C for three days. These findings suggest that the fermentation process did not occur when SLBH was stored at both storage conditions. In contrast, a study by Baroyi et al. ^[25] showed that storage of SLBH in a clay pot at 25 °C increased the free acidity up to 16.5% during the first seven days of storage, while it was decreased after 14–21 days of storage. These findings suggest that the fermentation process may occur as early as the first week of storage, as evidenced by an increase in the free acidity level. After 2–3 weeks of storage, the organic acid content in SLBH started to decline, evidenced by a decrease in the free acidity level. Overall, researchers observed that storage of SLBH in a clay pot resulted in various outcomes with regards to the free acidity. This might be due to the nature of the dehydration method via passive diffusion method, which took days. The prolonged storage duration increases the possibility of the fermentation process to occur.

Some dehydration methods could preserve the organic acid in SLBH by maintaining the free acidity level. However, it depends on the temperature used during the dehydration process, in which less exposure to the heat treatment preserves the free acidity. The dehydration methods that can maintain the free acidity level are thermal treatment at low temperature for a long duration or at high temperature for a short duration, freeze-drying, and passive diffusion at 35 °C for a three day storage period. Researchers observed that the best dehydration method was the freeze-drying method at a 5% moisture setting because it was performed at a low temperature. Hence, the lack of heat exposure limits the possibility of organic acid reduction in SLBH.

This entry is adapted from the peer-reviewed paper 10.3390/molecules27217243

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