Available online at ScienceDirect. Agriculture and Agricultural Science Procedia 2 ( 2014 )

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1 Available online at ScienceDirect Agriculture and Agricultural Science Procedia 2 ( 2014 ) ST26943, 2nd International Conference on Agricultural and Food Engineering, CAFEi2014 Total Phenolic Contents and Colour Intensity of Malaysian Honeys from the Apis spp. and Trigona spp. Bees Siok Peng Kek a, Nyuk Ling Chin a, *, Yus Aniza Yusof a, Sheau Wei Tan b, Lee Suan Chua c a Department of Process and Food Engineering, Faculty of Engineering, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia b Laboratory of Vaccines and Immunotherapeutics, Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang, Selangor, Malaysia c Metabolites Profiling Laboratory, Institute of Bioproduct Development, Universiti Teknology Malaysia, UTM Skudai, Johor Bahru, Johor, Malaysia Abstract The total phenolic content and colour intensity of the five common Malaysian honeys by the Apis spp. or Trigona spp. were studied following a modified Folin-Ciocalteu method and absorbance measurements, respectively. The total phenolic content and the colour intensity of honeys from the two types of bees were statistically different with P < The Trigona spp. honey gave higher total phenolic content with average mg GAE/kg than the Apis spp. honey at average mg GAE/kg. The total phenolic content of Malaysian honeys has good correlation with its colour intensity at R 2 = The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of the Scientific Committee of CAFEi2014. Peer-review under responsibility of the Scientific Committee of CAFEi2014 Keywords: Stingless bee honey; total phenolic content; colour intesity 1. Introduction Bees, the highly beneficial insect species from the family of Apidae, are important for pollinating crops, producing beneficial honey and other medicinal products, and sustaining world s natural life cycle. The honey bees are bees of the single genus Apis from the tribe of Apini, where the most popular subspecies is Apis mellifera. * Corresponding author. Tel.: ; fax: address: chinnl@upm.edu.my The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license ( Peer-review under responsibility of the Scientific Committee of CAFEi2014 doi: /j.aaspro

2 Siok Peng Kek et al. / Agriculture and Agricultural Science Procedia 2 ( 2014 ) Honeys derived from Apis mellifera are mainly produced and distributed in Europe and Asia (Guerrini et al., 2009); while other subspecies like Apis dorsata, Apis cerana, and Apis florea are found in Southern and Southeastern Asia. Unlike the Apis bees, the stingless bees are from the tribe of Meliponini have more than one type of genera, i.e. Melipona, Scaptotrigona, and Trigona. The stingless bees are native to tropical and subtropical parts of the world such as Central and South America, Africa, Asia, and northern Australia (Boorn et al., 2010). The Trigona spp., known as the Kelulut is the stingless bees species found in Malaysia. These bees produce Kelulut honey, a multifloral honey which is stored in clusters of small resin pots near the extremities of their nests while honeys from Apis spp. are stored in hexagonal-shaped honey combs. The Kelulut honey has been reported to have qualitatively antibacterial excellent potency (Zainol et al., 2013), which is useful medically and therapeutically. Biluca et al. (2014) stated that honey from stingless bees has a distinct taste and aroma, more fluid in texture, and undergoes slow crystallisation. The distribution of stingless bee honeys in the world market however is limited as compared to honeys from the Apis mellifera owing to a limited supply, a lower shelf life, and also lack of an institutional quality standard due to less knowledge available on the product (Guerrini et al., 2009). Honey is rich in phenolic compounds as it is food collected by the bees from the plants. The total phenolic content in honey is strongly correlated with its antioxidant activity (Beretta et al., 2005; Bertoncelj et al., 2007; Meda et al., 2005), thus can be used as a reliable parameter to indicate antioxidant activities in honey. The measurement of total phenolic content using the Folin-Ciocalteu method is good as it is sensitive and includes the polyphenol entitles and other electron-donating antioxidants such as ascorbic acid and vitamin E (Beretta et al., 2005). Colour intensity with measurement at absorbance of 450 nm is a parameter to reflect directly the wide range of observed honey colour. It is used to define colour intensity of a 50% honey solution (w/v) for estimating contribution of coloured phytochemicals on overall antioxidant capacity of honey (Khalil et al., 2011b). Studies reported that phytochemicals like the carotenoids have maximum absorption at 450 nm (Mendiola al., 2008) and have single oxygen quenching activities which recognised as common hydrophilic and lipophilic antioxidants (Prior et al., 2005). The objective in this research was to evaluate, compare and correlate the total phenolic content and colour intensity of Kelulut honey from the Trigona spp. with other honeys like the Tualang, Gelam, Pineapple and Borneo from the Apis spp.. Nomenclature GAE mau R 2 r gallic acid equivalent milli-absorbance unit regression coefficient Pearson correlation coefficient 2. Materials and methods 2.1. Materials Five types of Malaysian honey samples representing the Apis spp. honey or Trigona spp. honey were selected. The Apis spp. honeys consisted of the Tualang (Koompassia excelsa), Gelam (Melaleuca cajuputi), Pineapple (Ananas comosus), and Borneo. Both, the Tualang and Gelam honeys were produced by Apis dorsata which were harvested in Tasik Pedu, Kedah and Merchang, Terengganu, respectively. The Pineapple honey from Apis mellifera was collected in Rengit, Johor while the Borneo honey produced by Apis cerana from the nectar mainly from Acacia mangium trees and flowers in Kudat, Sabah. The Kelulut honey harvested by the Trigona spp. was supplied by a local bee honey collector from the forest in Perak where the nectar source is mainly from the Acacia mangium trees and flowers. Two batches of each Apis spp. honeys and three batches of Trigona spp. honeys were collected from different seasons across a period of January 2013 to March These Malaysian honeys were also compared with two randomly selected commercial honeys obtained from a local supermarket.

3 152 Siok Peng Kek et al. / Agriculture and Agricultural Science Procedia 2 ( 2014 ) Determination of total phenolic content The total phenolic content was determined by the spectrophotometric Folin-Ciocalteu method as described by Singleton et al. (1999) with minor modification. Each 1 g of honey sample was diluted to 20 ml with distilled water. 1 ml of honey solution was then mixed with 5 ml of 0.2 N Folin-Ciocalteu reagents. After incubation for 5 minutes, 4 ml of 75% w/v aqueous sodium carbonate solution was added and the mixture was incubated at room temperature for 2 hours. The absorbance of the reaction mixture was measured at 765 nm against a distilled water blank using the UV-VIS spectrophotometer (Ultrospec 3100 pro, Amersham Biosciences, USA). Gallic acid was used to produce the standard calibration curve with concentration from 5 to 100 μg/ml. The total phenolic content was expressed in mg of gallic acid equivalent (GAE) per kg of honey. Determinations were done in triplicates Determination of colour intensity The colour intensity was measured following method of Beretta et al. (2005). A 50% (w/v) honey solution was prepared with warm water at 45 to 50 C and filtered to remove any coarse particles. The net absorbance was determined as the difference between the absorbance at 450 nm and 720 nm using the UV-VIS spectrophotometer. The measurements were performed in triplicates for each honey sample and the results were expressed as mau Statistical analysis One-way analysis of variance (ANOVA) followed by Tukey s multiple-comparisons test was used to determine the significant differences between means where different letters were used to indicate significant difference at P < Correlation was established using Pearson correlation coefficient, r for the relationship between the total phenolic content and the colour intensity. All the analyses were performed using Minitab Statistical Software (Version 16, Minitab Inc., USA). All values reported were mean ± standard error. 3. Results and discussion The measurement of total phenolic content is considered as a simple and fast method to evaluate total phenol in complex matrix like honey (Chua et al., 2013). Fig. 1(a) shows that the total phenolic content varied significantly among the honey samples with P < The difference in total phenolic content is because of its dependence upon floral source of honey and its collection region (Khalil et al., 2011a). In general, these tested Malaysian honeys show higher values of total phenolic content than the commercial honey products chosen. The Kelulut honeys, in particular the A and B samples contained the highest values of phenolic content of ± 0.6 mg GAE/kg and ± 2.1 mg GAE/kg, respectively among the honey samples. There were some differences between the two different batches of all honeys from the Apis group, i.e. Tualang, Gelam, Pineapple and Borneo even though they had been sourced from the same region. This is expected as properties and composition of honey is affected greatly by various factors including its nectar source, collection season, mode of storage, and harvest technology and conditions (Kaskoniene and Venskutonis, 2010). The total phenolic contents of the Tualang, Pineapple and Borneo honeys measured in this study were 589.2, 602.4, and mg GAE/kg, which were 2 to 2.5 times higher than those reported by Moniruzzaman et al. (2013a) at 352.7, 226.3, and mg GAE/kg, respectively. In comparison to honeys from other countries, the total phenolic content of Malaysian Apis spp. honeys were of similar level to the Portuguse honeys ranging from to mg GAE/kg (Ferreira et al., 2009) and the Cuban honeys ranging from to mg GAE/kg (Alvarez-Suarez et al., 2010). The Kelulut honeys thus, recorded the highest total phenolic contents generally. Colour intensity of honey is a reliable parameter that indicates the presence of pigments which has antioxidant activities such as carotenoids and flavonoids (Moniruzzaman et al., 2013b). Saxena et al. (2010) reported that colour intensity has positive correlations of with antioxidant activities in honey samples with measurements of ferric reducing antioxidant potential (FRAP), ascorbic acid equivalent antioxidant content (AEAC), and percentage 2,2-diphenyl-1-picryl-hydrazyl (DPPH) scavenging activity. Fig. 1(b) shows that the colour intensity of all honey samples ranged from mau to 1480 mau. The highest colour intensity was Kelulut B which suggested

4 Siok Peng Kek et al. / Agriculture and Agricultural Science Procedia 2 ( 2014 ) potential of high antioxidant. The differences in colour intensity of the two batches of honey could be due to the specific contaminating pigments arising from handling, processing, and storage, or from biochemical reactions during honey maturation (Beretta et al., 2005). These reported colour intensity values were similar to the Romanian and Indian honeys which were in the range of 210 to 1228 mau and 524 to 1678 mau, respectively (Cimpoiu et al., 2013; Saxena et al., 2010). Fig. 1. (a) Total phenolic content and (b) colour intensity of different honey samples. In comparing the honey samples classified as those from the Apis spp. and Trigona spp., Fig. 2 shows that there are significant differences between them in terms of total phenolic content and the colour intensity. The Trigona spp. had a higher value of total phenolic content by 33% and colour intensity by 111% when compared to the Apis spp. honey samples. Moniruzzaman et al. (2013b) reported that the sourwood honey with higher phenolic content also had higher colour intensity. Fig. 3 shows a good correlation between total phenolic content with colour intensity of

5 154 Siok Peng Kek et al. / Agriculture and Agricultural Science Procedia 2 ( 2014 ) honey with high regression coefficient, R 2 of 0.826, and Pearson correlation coefficient, r of (P < ). This positive correlation indicates that the total phenolic content of honey increases with its colour intensity. Similar results were also reported by Bertoncelj et al. (2007) for Italian honeys with r = and Moniruzzaman et al. (2013b) in Malaysian honeys with r = Fig. 2. Comparison between Malaysian honeys classified as honey from Apis spp. (n = 24) and Trigona spp. (n = 9) with commercial honeys chosen (n = 6). 4. Conclusions Fig. 3. Correlation between total phenolic content and colour intensity of Malaysian honey samples. The Kelulut honey from the Trigona spp. has higher levels of phenolic content and colour intensity than honeys from the Apis spp.. The strong and positive correlation observed between total phenolic content and colour intensity of honey samples suggests that honey with darker colour contains higher total phenolic content. Colour measurement may be used as an indicator of total phenolic content in Malaysian honeys.

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