Dear Label_Name,

Greetings! I hope you are well.

I am writing on behalf of an international publishing house, Science and Education Publishing, USA (http://www.sciepub.com).

Science and Education Publishing (SciEP) is a multidisciplinary open access publisher of Journals covering the fields of Science, Technology and Medicine. All SciEP publications are subject to high-quality peer review, editorial and production processes and are freely available online.

Welcome to the April, 2013 edition of SciEP newsletter, providing monthly summaries of articles on science & education related topics of interest to scientist and professionals. The mission of our journals is to provide an open access forum for publishing new findings on issues pertinent to scientific and academic field. We also invite you to submit original research papers, review articles, short reports, or case studies are considered for publication in our Multidisciplinary Journals.

American Journal of Public Health Research
DOI: 10.12691/ajphr-1-1-3

The Implementation of a Tele-Homecare System with Service Oriented Architecture and HL7 Message Transmission Standard
Ching-Sung Wang

American Journal of Medical and Biological Research
DOI: 10.12691/ajmbr-1-1-2

The Influence of Ethanol on Pyruvate Kinases Activity in Vivo, in Vitro, in Silico
Lelevich Sergey Vladimirovich, Khrustalev Vladislav Victorovich, Barkovsky Eugene Victorovich, Shedogubova Tatyana Aleksandrovna

American Journal of Medical Sciences and Medicine
DOI: 10.12691/ajmsm-1-1-1

Clinical and Histological Features in Wegener Granulomatosis
Li Cavoli Gioacchino, Passantino Rita, Bono Luisa, Tortorici Calogera, Ferrantelli Angelo, Giammarresi Carlo, Rotolo Ugo

American Journal of Infectious Diseases and Microbiology
DOI: 10.12691/ajidm-1-1-2

The Curse of Lymphatic Filariasis: Would the Continual Use of Diethylcarbamazine Eliminate this Scourge in Papua New Guinea?
Anne Lanham, Lillian Mwanri

We have released issues of some journals on February 2013.
American Journal of Public Health Research
American Journal of Medical Sciences and Medicine
American Journal of Medical and Biological Research
American Journal of Pharmacological Sciences
American Journal of Clinical Medicine Research
American Journal of Nursing Research
American Journal of Cancer Prevention
American Journal of Medicine Studies
American Journal of Infectious Diseases and Microbiology
American Journal of Cardiovascular Disease Research
American Journal of Biomedical Research
American Journal of Mechanical Engineering
American Journal of Microbiological Research
Journal of Preventive Medicine
American Journal of Food and Nutrition
American Journal of Hypertension Research
American Journal of Sports Science and Medicine
Journal of Cancer Research and Treatment
American Journal of Zoological Research
Journal of Biomedical Engineering and Technology
American Journal of Microbiological Research
Biomedical Science and Engineering

We are pleased to announce that submissions are now being accepted for peer review for all the journals across all our subject areas in SciEP.

Please submit your manuscript via our online submission system:
http://www.sciepub.com/author/signin

To unsubscribe from our mailing list, please click on the following link: unsubscribe

I am looking forward to hearing from you.

Kind regards,

David Rain
Email:join@sciepub.com
Science and Education Publishing (http://www.sciepub.com)

Evaluation of Honey Quality with Stored Time and Temperatures

Wing-Ming Chou*, Hao-Chun Liao, Yuan-Chang Yang, Chi-Chung Peng*

Department of Biotechnology, National Formosa University, Yunlin, TaiwanDepartment of Biotechnology, National Formosa University, Yunlin, Taiwan

Abstract: Honey from two sources, Bidens pilosa and Dimocarpus longan were stored at 35, 25, or 4°C under dark or light for 3-24 months. They were evaluated for the nutrients, antioxidant activities and quality parameters required in Codex Standard. Diastase activity of examined honey was reduced to less than 8 schade unit soon after storage for 3-6 months, and its acidity was increased with long storage time at high temperature. Storage of honey at 4°C significantly diminished the loss of diastase activity and maintained the proper acidity. Hydroxymethylfurfural (HMF) content retained low level as honey was stored up to two years at either 4 or 25°C, while its content was quickly increased at 35°C. More phenolic content but less flavonoid content was found in honey after long storage time. Along with longer deposit time and higher temperature, less antioxidant activities were detected in honey, including scavenging activity of DPPH and superoxide radicals. In contrast, the reducing power was increased. Storage of honey at 35°C caused the worst impact on its quality parameters of Codex Standard, while 4oC was the best storage condition to maintain most quality parameters, nutrients and antioxidant functions. No apparent difference was found between the storage conditions under dark and light.

Keywords: honey, storage, hydroxymethylfurfural, antioxidant

Received September 21, 2020; Revised October 23, 2020; Accepted October 30, 2020

Citation: Wing-Ming Chou, Hao-Chun Liao, Yuan-Chang Yang, Chi-Chung Peng, Evaluation of Honey Quality with Stored Time and Temperatures. Journal of Food and Nutrition Research, 8 (10). 591-599. January 2020. doi:10.12691/jfnr-8-10-8

1. Introduction

Since the ancient times, honey has been extensively applied in food and medical product. The floral nectar, the exudates of trees and honeydew were collected and regurgitated to produce honey by bees [1]. Honey is well known for its nutrient value, and comprises carbohydrates and water mainly. It also contains minerals, free amino acids, vitamins, proteins, and various substances including pigments, flavonoids and antibacterial factors [2,3]. At least 200 natural substances were found in honey [4]. The composition of floral honey is associated with the floral sources, along with external factors, environment, climate, season and processing [5].

Honey has showed the therapeutic potential for heart disease, cancer, cataracts, and several inflammatory diseases [6]. It is attributed to its antioxidant capacity, antimicrobial properties, and wound healing and anti-inflammatory activities [7]. Many studies showed that its antioxidant activity is related to the ﬂoral source [8]. Both enzymatic and non-enzymatic antioxidants have been found in honey, including glucose oxidase, catalase, ascorbic acid, carotenoid derivatives, organic acids, Maillard reaction products (MRP), phenolic compounds, and free amino acids [9,10]. Among them, the phenolic compounds are primarily responsible for the antioxidant capacity [11,12]. And honey flavonoids are originally from nectar, pollen or propolis. The identified flavonoids include apigenin (belongs to flavone), such as kaempferol (a flavonol), hesperetin (a flavavone) and diverse phenolic acids.

Honey is often preserved until use. To keep the freshness and antioxidant function of stored honey is critical for the retail and consumers. According to Codex Standard [13], all retail honey should have the following compositional and quality parameters: moisture content, ≤20 g/100 g; free acidity, ≤50 mequiv/ kg; diastase activity, ≥8 schade unit; hydroxymethylfurfural (HMF) content, ≤40 mg/kg (or ≤80 mg/Kg for honey from tropical ambient temperatures).

Honey has an acidic pH value, ranging from 3.5 to 4.5, owing to its organic acids. The acidity of honey is correlated with its flavor and stability against microorganisms [14]. Nevertheless, microbial alterations in honey may cause total acidity over the legal limitation [15,16,17,18]. Diastase in honey catalyzes starch into short-chain sugars, and its protein conformation may be destroyed after heating. Maillard reaction (MR), a non-enzymic browning reaction between sugars and free amino acids, occurs during prolonged storage and heating [19]. HMF is one of the major intermediate products of MR. Less diastase activity and more HMF was detected in honey after prolonged storage and heating [20,21,22,23,24].

Herein, we studied the effect of storage conditions on honeys from two floral sources, Bidens pilosa and Dimocarpus longan in Taiwan. D. longan is the main floral source of honey bee, and its fruits are edible and popular in Taiwan. B. pilosa is used as ingredient of herbal tea, as well as a folk medicine [25]. The climate in the plains of Taiwan is often between 25 and 35oC. Thus, the storage conditions of honey at 35, 25, or 4oC under dark or light for 3-24 months were evaluated. The quality parameters of honeys were examined and discussed, as well as antioxidant activities, total flavonoids and phenolic compound contents.

2. Materials and Methods

2.1. Honey Samples

B. pilosa and D. longan honey, produced by A. mellifera bees were prepared and provided by Honey Bee Town Company (Hualien, Taiwan). Each honey was divided into six equal parts, and subjected to different storage conditions, dark and light at temperature (35, 25, 4°C), followed by analyses of the physicochemical parameters and antioxidant activities.

2.2. Diastase Activity Assay

The diastase activity was measured using the Phadebas amylase test tablets purchased from Magle (Lund, Sweden), according to the International Honey Commission [26]. Diastase Activity was referred to as diastase number in the Schade scale, corresponding to the Gothe scale number, or hydrolyzed starch (g) /100 g of honey/hour at 40°C.

2.3. Total Acidity

5.0 g of examined honey was dissolved in 100 ml distilled water, and then subjected to determination of the total acidity, according to method of AOAC method 962.19 [27]. A pH meter (Mettler Toledo SevenEasy digital) was applied to measure pH value. The total acidity was then calculated and expressed as meq/kg.

2.4. Determination of HMF Content in Honey

5 g of honey sample each was diluted to 50 mL, and then filtered through a membrane (0.45 μm). Afterward, HMF content was determined by high-performance liquid chromatography (HPLC) under OD₂₈₅ detection, according to AOAC method 980.23 [28]. HPLC analysis was performed using a Waters 1525 pumping system, a Water 2489 detector, an RP-18 GP250 column (4.6 mm), and a Waters 717plus autosampler. And the isocratic mobile phase comprised 90% water, and 10% methanol at a flow rate of 0.7 mL/min. HMF content of the sample was calculated, according to the corresponding peak of HMF standard solutions. A linear relationship (R²=0.9981) between the concentration and the area of HMF peak was achieved. Each sample was performed three times, and the mean of HMF content was expressed as mg/kg.

2.5. Determination of Total Polyphenol and Total Flavonoid Contents in Honey

The total polyphenolic content of honey was measured by Folin-Ciocalteu colorimetric method using gallic acid as a calibration standard [8]. An aliquot of the honey sample solution (0.1 mL) was diluted to 5 mL with water, followed by adding Folin-Ciocalteu reagent (0.5 mL). After mixing for 3 min, 1 mL of an aqueous Na₂CO₃ solution (35 g/L) was added and mixed throughly by vortex. After placing the mixture at room temperature for 1 h, the absorbance of the mixture was measured at 725 nm against a blank.

The total ﬂavonoid content was determined using an aluminum chloride colorimetric method [8]. 0.5 ml of honey sample was mixed with 1.5 ml of 95% alcohol, 0.1 ml of 1 M potassium acetate, 0.1 ml of 10% aluminum chloride hexahydrate, and 2.8 ml of deionized water. After incubation at room temperature for 30 min, the absorbance of reaction mixture was measured at 415 nm. Quercetin was applied to obtain a standard curve (0–100 μg/ml). The total ﬂavonoid content in each honey sample was determined in triplicate.

2.6. Radical Scavenging Effect on 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radicals

DPPH assay was performed based on the method of Liu et al. [8]. 0.3 mL of honey sample was mixed with 2.4 mL of ethanol (99%) and 0.3 mL of 1.0 mM DPPH radical solution. The mixture was shaken vigorously and left to stand for 30 min in the dark, before the absorbance was measured at 517 nm. Ascorbic acid (0.1 mM and 1.0 mM) were used as positive controls. The capability of the test material to scavenge DPPH radicals was calculated as (%) = 1 - (OD₅₁₇ of the sample) / (OD₅₁₇ of the control) × 100.

2.7. Radical Scavenging Effect on Superoxide Radicals

The scavenging of superoxide anion radicals was estimated according to the method of Robak and Gryglewski [29]. The honey sample solution (500 μL) was mixed with an equal volume of 80 μM PMS, 624 μM NADH and 200μM NBT each. The mixture was shaken vigorously and left to stand for 5 min. The absorbance of reaction mixture was measured at 560 nm. Ascorbic acid (0.1 mM, 1.0 mM and 10.0 mM) were used as positive controls. The inhibition ratio was calculated as (%) = (OD560 of the control - OD560 of the sample) / (OD₅₆₀ of the control) × 100.

2.8. Reducing Power

The reducing power of honey was determined according to the method of Oyaizu [30]. The honey sample solution (2.5 mL) was mixed with an equal volume of 0.2 M sodium phosphate buffer (pH 6.6) and 1% potassium ferricyanide. The mixture was incubated at 50°C for 20 min. Afterward, an equal volume of 1% thrichloroacetic acid was added to the mixture, which was then centrifuged at 1400g for 10 min. The upper layer was mixed with distilled water and 0.1% ferric chloride at a ratio of 1:1:2, and the absorbance of the incident radiation by the solution was measured at 700 nm. The absorbance was proportional to the reducing power, and dibutyl hydroxytoluene (BHT) was used as a reference for comparison.

2.9. Statistical Analysis

All results were analyzed using the general linear model procedure available from Statistical Analysis System software (Statistical Analysis System Institute, Cary, NC). Duncan’s multiple range test [31] was used to detect differences between means of the treatments. Each experiment was conducted in triplicate. Differences between means at the 95% (p < 0.05) confidence level were considered as statistical significance.

3. Results and Discussion

3.1. Total Acidity Contents

The acidity of honeys under different storage conditions were examined, including 35, 25, or 4°C for 3-24 months under dark or light. As shown in Figure 1, the acidity of honeys from B. pilosa and D. longan increased with the duration of storage. Storage at high temperature would increase total acidity in honeys significantly. The initial acidity of B. pilosa honey was 43.06 meq./kg. After stored for 9 months at 35oC, its acidity was higher than legal limit, 50 mequiv/kg. After stored for 18 months at 25°C, its acidity was out of legal limit as well. B. pilosa honey kept at 4oC maintained its acidity in legal value up to 24 months. The initial acidity of D. longan honey was 18 meq./kg, less than B. pilosa honey. The increasing rate of acidity of honey from D. longan was faster than B. pilosa. After stored for 12 months at 35 or 25°C, its acidity was higher than legal limit. And the acidity of D. longan honey stored for 18 months at 4°C was out of legal limit. Moreover, there was no significantly difference between the conditions under dark and light.

According to the results, the storage of honey at 4°C is recommended in order to hold its proper acidity. The stored temperature and time had considerable effect on acidity, which is coincided with the studied results by Castro-Vázquez [32]. Among different honey types, the variation in acidity may attribute to the variation of organic and inorganic acids contents [33]. Nevertheless, the compounds affecting acidity of honeys from B. pilosa and D. longan remain further study.

3.2. Diastase Activity in Honey

The initial diastase activity was similar in both examined honeys, as shown in Figure 2. Prolonged storage under high temperature accelerated the loss of diastase activity in honeys. After storage for 3 months under our test conditions, diastase activity in the honeys was less than legal limit, 8 schade unit, except for D. longan honey stored at 4°C. Stored at 4°C may significantly diminish the loss of diastase activity, particularly for B. pilosa honey. There was no apparently difference between dark storage and light storage.

The diastase activity in honeys are more or less diverse, depending on the sugar content of honeys, the floral and geographical origins of honeys, the period of nectar collected, the age of bees, and the bee colony [34]. Its activity has been as an indicator to monitor whether the honeys had been kept for too long time, or its product was overheated, > 60°C [35,36].

3.3. HMF Contents

Both honeys from B. pilosa and D. longan contained a low initial HMF content (1.21 mg/kg and 2.13 mg/kg, respectively) (Figure 3). HMF was quickly generated in these honeys after storage at 35°C. HMF contents in B. pilosa honey stored at 35°C for 6 months was reached to the legal limit, 80 mg/Kg for honey from tropical ambient temperatures. After storage at 35°C for 9 months, HMF contents in B. pilosa honey was exceeding the legal limit; while HMF contents in D. longan honey was reached to the legal limit. HMF contents in both honeys, stored at 4 and 25°C for 24 months, remain quite low levels that were less than 10 mg/ml.

Figure 1. Acidity of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)

Figure 2. Diastase activity of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey

Figure 3. HMF content of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)

HMF was reported to have genotoxic effects and mutagenic potential [37]. Some studies indicated that heat treatment or storage at room temperature caused a considerable increase in HMF contents of honeys [38,39,40]. Based on our results with HMF contents, it is suggested to store honeys at 4-25°C, or consume honeys within 6 months.

3.4. Total Phenolic and Total Flavonoid Contents in Honey

The influence of storage condition on total phenolic and total flavonoid contents of B. pilosa honey and D. longan honey was examined and shown in Figure 4 and Figure 5. Total phenolic content of B. pilosa honey was increased to 2-3 folds after storage for 12 months. Higher temperature would more or less increase its phenolic content. Compared with B. pilosa honey, D. longan honey had less total phenolic compounds; however, its total phenolic content was increased to 10-18 folds after storage for 12 months. The effect of heating on phenolic content of D. longan honey was clearly observed. More phenolic content of D. longan honey was detected as stored at higher temperature.

B. pilosa honey was rich in total flavonoid compounds (initially 180µg/ml). After storage for 3 months, around half of its total flavonoid contents were reduced. Honey from D. longan had less total flavonoid compounds (initially 55 µg/ml) than B. pilosa in our previous and current studies [8]. After storage for 9 months, around half of total flavonoid contents of D. longan honey were reduced. The decay rate of total flavonoid contents from B. pilosa was much faster than D. longan in the first three months. In general, storage at 4°C under dark would be better for honeys to retain total flavonoid contents. Honeys stored under dark had slightly less change in total phenolic and total flavonoid contents, comparing to under light.

Figure 4. The total phenolic content of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)

Figure 5. The total flavonoid content of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)

3.5. Scavenging Effect of Honey upon DPPH Radicals

DPPH with stable radicals is often applied to demonstrate the radical scavenging ability of natural compounds [8]. The antioxidant potential of honey was directly proportional to the amount of phenolic acids and flavonoids present [7]. The higher DPPH radical-scavenging activity may be attributed to its higher phenolic and flavonoid content [8]. Compared to D. longan honey, B. pilosa honey initially contained more phenolic compound and flavonoid, and exhibited better DPPH radical-scavenging activity.

As shown in Figure 6, B. pilosa honey and D. longan honey lost DPPH radical-scavenging activity with the duration and temperature of storage. In the beginning, B. pilosa honey had better DPPH radical-scavenging activity than D. longan honey. B. pilosa honey still had better activity than D. longan honey after storage under the same condition for two years. At 25°C, B. pilosa honey lost 50% of activity within ~18 months; while D. longan honey lost ~50% of activity after storage for 9 months. When honeys were stored at 4°C, their decline rates of DPPH radical scavenging activity were retarded. No apparent difference was found between the storage conditions under dark and light.

3.6. Radical Scavenging Effect upon Superoxide Radicals

Superoxide radical damaging the cell and tissues could be produced via autoxidation and enzymatic reactions in our bodies [8]. NBT method was applied to examine the scavenging rate of superoxide radicals by honeys. Honeys from B. pilosa and D. longan could inhibit superoxide radical formation with an inhibition rate of 60-75% initially, but lost such ability during storage (Figure 7). B. pilosa honey lost ≧50% of the superoxide radical-scavenging ability soon after 3 months storage, while D. longan honey lost 15-40% of the ability. Unlike B. pilosa honey, such ability in D. longan honey gradually decreased with the prolong storage and high temperature. According to superoxide radical scavenging rate, it is suggested to store D. longan honey at 4°C. Regarding to prolong storage for more than 12 months, B. pilosa honey stored at 35°C under light had unusually better superoxide radical scavenging ability than those stored at other conditions.

Figure 6. The DPPH scavenging rate of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)

Figure 7. The superoxide radical scavenging rate of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) is D. longan honey; Each point is expressed as mean ± S.D. (n=3)

Figure 8. The reducing power of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) is Bidens pilosa honey; (b) is D. longan honey; Each point is expressed as mean ± S.D. (n=3)

3.7. Reducing Power of Honey

The reducing power is mainly associated with the phenolic contents in honey. The reducing power of honey increased after prolong storage (Figure 8). It may due to the amount of total phenolic contents increased in both D. longan and B. pilosa honey (Figure 4). Similar results were reported that there was good correlation between reducing power and polyphenolic contents in honey [8].

Table 1. The correlation coefficient between physicochemical properties, antioxidant capacity and storage conditions in Bidens pilosa honey

Table 2. The correlation coefficient between physicochemical properties, antioxidant capacity and storage conditions in D. longan honey

3.8. Correlation Analysis

The relationships between storage condition and the parameter analyzed in this study (total phenolic content, total flavonoid content, antioxidant activity, and physicochemical properties) are presented in Table 1 and Table 2.

As shown in Table 1, the correlation coefficient has a great difference of acidity and HMF content at different temperatures, it’s clearly indicated that HMF content in honey samples significantly correlated with storage duration, and total acidity. Storage duration showed a very strong correlation with HMF level, indicating that time and temperature are the most important factors that affect HMF formation. The other physicochemical parameters such as total acidity showed strong correlations with HMF formation. Since both experimental data and statistical analysis indicated that storage duration and total acidity levels significantly correlated with HMF formation, conducting additional tests such as measuring free acids and total acidity may provide ways to quickly assess honey quality.

A positive correlation between the reducing power, phenolic content, and storage condition (Table 1) was observed. The high correlation coefficient indicates that phenolics are one of the main components responsible for the reducing behaviour of honey but not storage conditions. A negative correlation between the DPPH scavenging activity, superoxide radical scavenging activity, total flavonoid content, and storage condition (Table 1) was observed.

The correlation coefficient indicates that flavonoids are one of the main components responsible for the proton radicals scavenging ability of honey but not storage conditions. Gheldof et al. [11] addressed that the antioxidant activity of honey contributed by phenolic compounds significantly, but in spite of this, it seems that antioxidant activity appears to be a result of the combined activity of honey phenolics, peptides, organic acids, enzymes and Maillard reaction products. The correlation coefficient of total phenolic content, total flavonoid content and antioxidant capacity showed insignificant difference with different temperature after two years storage, which may indicate the storage time was one reason of the parameters’ change.

Table 2 showed similar result with Table 1, suggesting that long term storage has similar influence on B. pilosa and D. longan honey.

4. Conclusion

Long term storage under high temperature affected the quality of honeys from both sources. Upon storage for longer time and higher temperature, acidity was increased, more total flavonoid contents and radical scavenging ability was loss in honey. B. pilosa honey had around 3.5 folds more flavonoid contents than D. longan honey initially. B. pilosa honey lost lots of flavonoid, and exhibited almost the same flavonoid contents as D. longan honey after one year storage. HMF content was quickly increased at 35°C, while it retained low level up to two years at either 4 or 25°C. According to our study, 4°C was the best storage condition to maintain most quality parameters, nutrients and antioxidant functions. It is suggested to store honey at lower temperature less than one year. Considering HMF content, honey should not be stored at temperature higher than 35°C.

Abbreviations

HMF: Hydroxymethylfurfural; DPPH: 1, 1-diphenyl-2-picryl-hydrazyl; MRP: Maillard reaction products; HPLC: high-performance liquid chromatography; NBT: nitroblue tetrazolium chloride; BHT: dibutyl hydroxytoluene;

Data Availability

All data used in the manuscript are already included in the manuscript.

Conflicts of Interest

The authors report no potential conflicts of interest.

Acknowledgments

This work was supported by a grant from the Ministry of Science and Technology, Taiwan, ROC (MOST 108-2320-B-150 -001-MY2). The authors are thankful for Mr. Jen-Chieh Li of the Honey Bee Town Co., Ltd. for preparing honey samples.

References

[1]   | Bassey, I.H. In Bee keeping, digest of selected literature on bee keeping, honey and bee wax processing; Libriservice limited: Lagos, Nigeria, 1986; pp. 22.
[2]   | Pérez, R.A. Analysis of volatiles from Spanish honeys by solid-phase microextraction and gas chromatography-mass spectrometry. J. Agri. Food Chem. 2002; 50: 2633-2637.
[3]   | Terrab, A.; Gonzále, M.M.L.; González, A.G. Characterisation of Moroccan unifloral honeys using multivariate analysis. European food research and technology. 2003; 218: 88-95
[4]   | da Silva, P.M.; Gauche, C.; Gonzaga, L.V.; Costa, A.C.O.; Fett, R. Honey: Chemical composition, stability and authenticity. Food Chemistry 2016; 196: 309-323.
[5]   | Persano, O.L.; Piro, R. Main European unifloral honeys: descriptive sheets. Apidologie. 2004; 35: 38-81.
[6]   | Hossen, M.S.; Ali, M.Y.; Jahurul, M.H.A.; Abdel-Daim, M.; Gan, S.H.; Khalil, M.I. Beneficialroles of honey polyphenols against some human degenerative diseases. Pharmacological Reports. 2017; 69: 1194-1205.
[7]   | Basualdo, C.; Sgroy, V.; Finola, M.S.; Marioli, J.M. Comparison of the antibacterial activity of honey from different provenance against bacteria usually isolated from skin wounds. Vet. Microbiol. 2007; 124: 375-381.
[8]   | Liu, J.R.; Yeh, Y.L.; Lin, T.Y.; Wang, Y.W.; Peng, C.C. Effect of floral sources on the antioxidant, antimicrobial, and anti-inflammatory activities of honeys in Taiwan. Food Chem. 2013; 139: 938-943.
[9]   | Khalil, M.I.; Sulaiman, S.A.; Boukraa, L. Antioxidant properties of honey and its role in preventing health disorder. The Open Nutraceuticals Journal. 2010; 3: 6-16.
[10]   | Al-Mamary, M.; Al-Meery, A.; Al-Habori, M. Antioxidant activities and total phenolic of different types of honey. Nutr Res. 2002, 22, 1041-1047
[11]   | Gheldof, N.; Engeseth, N.J. Antioxidant capacity of honeys from various floral sources based on the determination of oxygen radical absorbance capacity and inhibition of in vitro lipoprotein oxidation in human serum samples. J. Agri. Food Chem. 2002; 50: 3050-3055.
[12]   | Gheldof, N.; Wang, X.H.; Engeseth, N.J.; Buckwheat honey increases serum antioxidant capacity in humans. J. Agri. Food Chem. 2003; 51: 1500-1505.
[13]   | CODEX STANDARD FOR HONEY CODEX STAN 12-1981, Adopted in 1981, Revisions 1987 and 2001.
[14]   | Malika, N.; Mohammed, F.; Chakib, E. Microbiological and Physico- chemical properties of Moroccan honey. Int. J. Agr. Bio. 2005; 7: 773-776.
[15]   | Pérez-Arquillué, C.; Conchello, P.; Ariño, A.; Juan, T.; Herrera, A. Quality evaluation of Spanish rosemary (Rosmarinus officinalis) honey. Food Chem. 1994; 51: 207-210.
[16]   | Pérez-Arquillué, C.; Conchello, P.; Ariño, A.; Juan, T.; Herrera, A. Physico chemical attributes and pollen spectrum of some unifloral Spanish honeys. Food Chem. 1995; 54: 167-172.
[17]   | López, B.; Latorre, M.J.; Fernández, M.J.; García, M.A.; Herrero, C. Chemometric classification of honeys according to their type based on quality control data. Food Chem. 1996; 55: 281-287.
[18]   | Costa, L.S.M.; Albuquerque, M.L.S.; Trugo, L.C.; Quintero, L.M.C.; Barth, O.M.; Ribeiro, M.; De María, C.A.B. Determination of non-volatile compounds of different botanical origin Brazilian honeys. Food Chem. 1999; 65: 347-352.
[19]   | Lund, M.N.; Ray, C.A. Control of maillard reactions in foods: strategies and chemical mechanisms. J. Agri. Food Chem. 2017; 65: 4537-4552.
[20]   | Ramíre Cervantes, M.A.; Gonzále Novelo, S.A.; Sauri Duch, E. Efecto del tratamiento térmico temporal de la miel sobre la variación de su calidad durante el almacenamiento. Apiacta. 2000; 35: 162-170.
[21]   | Shapla, U.M.; Solayman, M.; Alam, N.; Khalil, M.I.; Gan, S.H. 5-Hydroxymethylfurfural (HMF) levels in honey and other food products: effects on bees and human health. Chem Cent J. 2018; 12: 35.
[22]   | Huidobro, J.F.; Santana, F.J.; Sanchez, M.P.; Sancho, M.T.; Muniategui, S.; Simal-Lozano, J. Diastase, invertase and β-glucosidase activities in fresh honey from north-west Spain. J. Apic. Res. 1995; 34: 39–44.
[23]   | Persano Oddo, L.; Pulcini, P.; Piazza, M.G. Invertase content of Italian unifloral honeys. Apic. Mod. 1996; 87: 25-29.
[24]   | Persano Oddo, L.; Piazza, M.G.; Pulcini, P. Invertase activity in honey. Apidologie 1999, 30, 57-65.
[25]   | Silva, F.L.; Fischer, D.C.H.; Tavares, J.F.; Silva, M.S.; de Athayde-Filho, P.F.; Barbosa-Filho, J.M. Compilation of secondary metabolites from Bidens pilosa L. Molecules 2011; 16: 1070-1102.
[26]   | Bogdanov, S. Harmonised Methods of the International Honey Commission. Swiss Bee Research Centre, FAM, Liebefeld. 2002.
[27]   | AOAC. Office methods of analysis (15th edition) Association of Official Analytical Chemist Washington, DC, USA. 1990; No 969. 12.
[28]   | AOAC. Office methods of analysis (15th edition) Association of Official Analytical Chemist Washington, DC, USA. 1990; No 980. 23.
[29]   | Rock, J.; Gryglewski, R.I. Flavonoids are scavengers of superoxide anions. Biochem. Pharma. 1988; 37: 837-841
[30]   | Oyaizu, M. Studies on products of browning reaction antioxidative activities of products of browning reaction prepared from glucoseamine. Jpn. J. Nutr. 1986; 44: 307-315.
[31]   | Montgomery, D.C. Design and analysis of experiments (8th ed.). New York, NY: John Wiley and Sons. 2012.
[32]   | Castro-Vázquez, L.; Díaz-Maroto, M.C.; González-Viňas, M.A.; Fuente, D.L.E.; Pérez-coello, M.S. Influence of storage conditions on chemical composition and sensory properties of Citrus honey. J. Agri. Food Chem. 2008; 56: 1999-2006.
[33]   | EI Sohaimy, S.A.; Masry, S.H.D.; Shehata, M.G. Physicochemical characteristics of honey from different origins. Annals of Agricultural Sciences. 2015; 60(2): 279-287.
[34]   | Oddo LP, Piazza MG, Pulcini P. Invertase activity in honey. Apidologie. 1999, 30, 57-65.
[35]   | Ahmed, M.; Djebli, N.; Aissat, S.; Khiati, B.; Meslem, A.; Bacha, S. In vitro activity of natural honey alone and in combination with curcuma starch against Rhodotorula mucilaginosa in correlation with bioactive compounds and diastase activity. Asian Pac. J. Trop. Med. 2013; 3: 816-821.
[36]   | Yücel, Y.; Sultanoglu, P. Characterization of honeys from Hatay region by their physicochemical properties combined with chemometrics. Food Biosci. 2013; 1: 16-35.
[37]   | Janzowski, C.; Glaab, V.; Samimi, E.; Schlatter, J.; Eisenbrand, G. 5- Hydroxymethylfurfural: assessment of mutagenicity, DNA-damaging potential and reactivity towards cellular glutathione. Food Chem. Toxicol. 2000; 38: 801-809.
[38]   | Mouhoubi-Tafinine, Z.; Ouchemoukh, S.; Bachir bey, M.; Louaileche, H.; Tamendjari, A. Effect of storage on hydroxymethylfurfural (HMF) and color of some Algerian honey. International Food Research Journal 2018; 25(3): 1044-1050.
[39]   | Singh, N.; Bath, P.K. Relationship between heating and hydroxymethylfurfural formation in different honey types. J. Food Sci. Technol. India. 1998; 35(2): 154-156.
[40]   | Turhan, K. Effects of Thermal Treatment and Storage on Hydroxymethylfurfural (HMF) Content and Diastase Activity of Honeys Collected from Middle Anatolia in Turkey. In Innovations in Chemical Biology.; Sener, B., Eds.; Springer: New York City, New York, 2009; pp. 233-239.