This research was carried out to determine the fructooligosaccharides content in local honey samples, namely the wild Malaysian Tualang honey and common wild honey obtained from Tapah, Perak and a commercial Tualang honey. Local wild honeys were found to contain a higher concentration of fructooligosaccharides (FOS) compared to the commercial Tualang honey. The FOS quantified from local wild honeys was inulobiose, kestose and nystose. Nystoses were found at a very low amount in the commercial Tualang honey. The effects of honey on the growth of Bifidobacterium longum BB 536 were investigated. Both wild and commercial honey samples including FOS standard were found to support the growth of B. longum. The pH value of the skim milk + honey inoculated with the probiotic strain decreases as expected. Addition of honey was found to support the growth of B. longum BB 536

Abdullah Sani, Norrakiah

Mohd Som, Mohd Nordin

Soon, Jan Mei

Universiti Putra Malaysia Institutional Repository

© All Rights Reserved*Corresponding author. Email: norra@ukm.myTel: +603 8921 4053; Fax: +603 8921 3232International Food Research Journal 17: 557-561 (2010)1Jan Mei, S., 2Mohd Nordin, M. S. and 3,*Norrakiah, A. S.1Faculty of Agro Industry and Natural Resources, Universiti Malaysia Kelantan, 16100, Pengkalan Chepa, Kelantan, Malaysia2Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia3School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, Bangi, Selangor, MalaysiaIntroductionHoney was one of the man’s earliest foods, as honeybees were producing it long before man appeared on earth.  Honey remains the only sweetener that can be stored and used precisely as it is produced by nature (Doner, 2000).  Honey is primarily a carbohydrate material, where fructose and glucose account for over 85% of its solids (NHB, 1996).  Another 10% of honey solids include at least 25 other more complex sugars ranging from di- to oligosaccharides (Doner, 2000).  This group of oligosaccharides are of interest because, they are neither hydrolyzed nor absorbed in the upper part of the gastrointestinal tract, and may beneficially affect the health of the consumer by selectively stimulating the growth or and/or activity of desirable bacteria in the colon (Ustunol, 2000; Cummings et al., 2001). Fructooligosaccharides (FOS) are short-chain sugars that occur naturally and have dietary benefits for humans.  They are widely distributed in nature and are a natural part of the human diet (Hogarth et al., 2000).  Non-digestible oligosaccharides also occur naturally in foods such as fruits, vegetables, milk and honey (Tannock, 1999).  The ability of honey to enhance the growth and activity of bifidobacteria in milk and intestinal bifidobacteria in vitro were reported and suggested that this may be due to the unique carbohydrate composition and complex mixture of oligosaccharides present in honey (Chick et al., 2001; Ustunol and Gandhi, 2001; Kajiwara et al., 2002).  Bifidobacteria spp. including Bifidobacterium longum are probiotics; living organisms that affect the host in a beneficial manner (Montrose and Floch, 2005).   Materials and MethodsTwo types of wild honey known as wild Tualang and common wild honey were obtained from Tapah, Perak.  A local commercial Tualang honey was obtained from a supermarket. All chemicals and reagents used were of analytical grade.Pre-treatment of honeyThe removal of monosaccharrides and disaccharides and the recovery of Fructooligosaccharides in honey and effects of honey on growth of Bifidobacterium longum BB 536Abstract: This research was carried out to determine the fructooligosaccharides content in local honey samples, namely the wild Malaysian Tualang honey and common wild honey obtained from Tapah, Perak and a commercial Tualang honey.  Local wild honeys were found to contain a higher concentration of fructooligosaccharides (FOS) compared to the commercial Tualang honey. The FOS quantified from local wild honeys was inulobiose, kestose and nystose. Nystoses were found at a very low amount in the commercial Tualang honey.  The effects of honey on the growth of Bifidobacterium longum BB 536 were investigated.  Both wild and commercial honey samples including FOS standard were found to support the growth of B. longum.  The pH value of the skim milk + honey inoculated with the probiotic strain decreases as expected.  Addition of honey was found to support the growth of B. longum BB 536.Keywords: Fructooligosaccharides, honey, probiotic, Bifidobacterium longum558 Jan Mei, S., Mohd Nordin, M. S. and Norrakiah, A. S.International Food Research Journal 17: 557-561fructooligosaccharides (FOS) from honey were according to Morales et al. (2006). A 0.5 g of honey were mixed with 3 g of activated charcoal and were dissolved in ethanol:water (90:10, v/v) solution.  The mixture was stirred for 30 min. at room temperature, filtered through Whatman paper 1 and then washed with 25 ml of ethanol:water (90:10, v/v).  The FOS absorbed by the activated charcoal were dissolved using a water:ethanol (50:50, v/v) solution and then filtered. The filtrate was concentrated at 30°C using rotary evaporator.  The extract was then filtered through 0.22 µm membrane filter for HPLC analysis.Determination of fructooligosaccharides The determination of fructooligosaccharides was carried out according to Da Costa Leite et al. (2001).  Beneo P95 (fructooligosaccharides) was used as the FOS standard. Analyses were carried out in a Waters 717 Plus Autosampler HPLC system and an evaporative light-scattering detector (ELSD). Separation of fructooligosaccharides was on a Water Spherisorb 5µm NH2 (250 x 4.6 mm) column using acetonitrile:water (85:15, v/v) as mobile phase and flow rate of 1 ml/min.  Quantification was obtained by peak height comparison with the standard FOS. Statistical analysis of data was performed by one way analysis of variance using SPSS 11.0.Determination of Bifidobacterium longum BB 536 growth A 12% (w/v) of reconstituted skim milk was prepared and mixed with 5% each of honey and FOS standard.  Skimmed milk without honey served as the control.  Both sample and control were pasteurized at 70°C for 15 minutes in a water bath and then cooled to 37°C before inoculation (Chick et al., 2001).  An initial 5% (105 CFU/ml) inoculum of B. longum BB 536 was inoculated into the milk and incubated anaerobically at 37°C for 24 hr.  Appropriate dilutions of the fermented milk was then plated on MRS agar (Oxoid) and incubated at 37°C for 48 hr anaerobically to determine the number of colonies.  Anaerobic condition was generated by Gas Generating Kit CO2 BR39 (Oxoid, England).  pH was taken after 0 and 24 hour of incubation using a calibrated pH meter (HANNA, USA).  The B. longum BB 536 strain was a gift from Universiti Putra Malaysia and was stored at 4°C on slants of MRS agar before inoculation. Results and DiscussionFructooligosaccharides in honeyThe Beneo P95 (fructooligosaccharides) standard was analyzed using HPLC to determine the FOS peaks. The following figure shows the chromatographic profile of the Beneo P95 (fructooligosaccharides) standard (Figure 1) and a chromatogram of the FOS in the common wild honey (Figure 2). The fructooligosaccharides identified in the honey samples based on retention time were inulobiose (F2), kestose (DP3) and nystose (DP4).  The FOS contents are shown in Table 1.  Fructofuranosil-nystose (DP5) was not detected in all the honey samples.  Ruiz-Matute et al. (2007) were among the first researchers to identify the presence of inulobiose (0.93 – 6.14 mg/g) in honey. This FOS was also detected in the wild Tualang honey at 0.018 mg/g.  Inulobiose may be formed in honey via the β-transfructosylation reaction from invertase which are present in pollens (Ruiz-Matute et al., 2007).  Kestose were determined in the common wild honey at 0.023 mg/g.  The formation of kestose in honey is similar to inulobiose. It could also be due to the trans-D-fructosylation activity on sucrose by α-glucosidase enzyme secreted by honey bees or even yeasts (Siddiqui, 1970). Doner (1991) reported that if 1-kestose was found in honey, it could be caused by the presence of yeasts. Doner (2000) stated that the formations of more than 25 types of oligosaccharides in honey from the three main nectar sugars (glucose, fructose and sucrose) are very complex.  The catalysis activities caused by enzymes are believed to be responsible for the formation of these oligosaccharides.  The enzymes in honey are secreted by honey bees to assist in the ripening process of nectar to honey. Effect of honey on Bifidobacterium longum BB 536The wild and commercial honeys were found to support the growth of B. longum in skimmed milk as shown in Table 2.  The addition of 5% honey to the skimmed milk did not show any inhibitory effect on the growth of the probiotic strain.  The growth of B. longum in skimmed milk mixed with wild Tualang and common wild honey shows a significant increase from 5.5 to 7.0 log10 CFU/ml cycle and 5.4 to 7.1 log10 CFU/ml cycle respectively after 24 hr incubation. Meanwhile the probiotic inoculated in skimmed milk with commercial Tualang honey increased more than 3.0 log10 CFU/ml cycle (p<0.05).  It is also noted that wild honeys may contain antibacterial components.  Čurda and Plocková (1995) suggested that honey obtained from different floral sources shows inhibitory effects on the growth of lactic acid Fructooligosaccharides in honey and effects of honey on growth of Bifidobacterium longum BB 536 559International Food Research Journal 17: 557-561Figure 1. Chromatographic profile of Beneo P95 (fructooligosaccharides) standard obtained by HPLC-ELSD under elution conditions Absorbance  Absorbance  Retention time  Retention time Figure 2. Chromatographic profile of fructooligosaccharides in common wild honeyFOS Wild Tualang Common wild honeyCommercial TualangInulobiose (DP2) 0.018 ND NDKestose (DP3) ND 0.023 NDNystose (DP4) 0.004 0.035 0.001Total fructans 0.022 0.058 0.001Skimmed milk with honey/FOS 0 hr 24 hrWild Tualang 5.5b ± 0.02 7.0c ± 0.01Common wild honey 5.4c ± 0.03 7.1c ± 0.02Commercial Tualang 5.3d ± 0.04 8.7b ± 0.05Fructooligosaccharides 5.7a ± 0.01 8.8a ± 0.01Control (skimmed milk only) 5.8a ± 0.07 7.0c ± 0.14Table 1. Fructooligosaccharides content in honeys (mg/g)ND: Not detectedTable 2. Growth of Bifidobacterium longum BB 536 in skimmed milk with honey and FOS (log10 CFU/ml) at 37°Ca-d Means in the same column with different letters are significantly different at p < 0.05.560 Jan Mei, S., Mohd Nordin, M. S. and Norrakiah, A. S.International Food Research Journal 17: 557-561bacteria.  Some of the inhibitory effects could be due to the high sugar content which reduces the Aw for microbial growth and the presence of organic acids and hydrogen peroxide (Mundo et al., 2004). Even though the total fructans were found to be higher in both the wild honeys, the commercial honey supported the probiotic growth very well.  This may indicate the presence of other oligosaccharides in the honey which were not determined in this study that have prebiotic activity.  Weston and Brocklebank (1999) also reported that New Zealand honey contains certain oligosaccharides like isomaltose and melezitose. The pH value of the skimmed milk inoculated with B. longum, decreased after 24hr incubation (Table 3).  The fermented milk with honey showed a significant pH decrease compared to the control.  The pH of the fermented milk with Tualang honey and with common wild honey decreased from 6.3 to 4.6 and 6.5 to 4.6 respectively within 24hr of incubation. This is because bifidobacteria produce 3 mol acetic acid and 2 mol lactic acid for each two mol glucose (Scardovi, 1986).  The production of these two organic acids and a lower pH value in the colon are among the probiotic factors contributing to the well-being of the host (Crittenden, 1999).  ConclusionsFOS was detected in the local honey but in low amounts ranging from 0.001 to 0.035 mg/g.  Results showed that the local honey supported the growth of B. longum BB 536 and may have the potential as another prebiotic source.AcknowledgementsWe are grateful to Dr. Maria Luz Sanz from the Instituto de Química Orgánica General (CSIC), Spain for her technical advises, Beneo-Orafti for sponsoring the Beneo P95 Fructooligosaccharides standard and Professor Dr Mohd Yazid Abdul Manap from Universiti Putra Malaysia for sponsoring the B. longum BB 536 strain. ReferencesChick, H., Shin, H.S. and Ustunol, Z. 2001. Growth and acetic production by lactic acid bacteria and bifidobacteria grown in skim milk containing honey. Journal of Food Science 66: 478-481.Crittenden, R. 1999. Prebiotics. In Tannock, J. (Ed). Probiotics: A critical review. Norfolk: Horizon Scientific Press.Cummings, J.H., Macfarlane, G.T. and Englyst, H.N. 2001. Prebiotic digestion and fermentation. American Journal of Clinical Nutrition 73 (suppl): 415-420.Čurda, L. and Plocková, M. 1995. Impedance measurement of growth of lactic acid bacteria in dairy cultures with honey addition. International Dairy Journal 5: 727-733.Da Costa Leite, J.M., Trugo, L.C., Costa, L.S.M., Quinteiro, L.M.C., Barth, O.M., Dutra, V.M.L. and De Maria, C.A.B. 2000. Determination of oligosaccharides in Brazilian honeys of different botanical origin. Food Chemistry 70: 93-98.Doner, L.W. 1991. The enzymes of honey. In Fox, P.F. (Ed). Food Enzymology, p. 143-161. Vol. 2. London: Elsevier Applied Science.Doner, L.W. 2000. Honey. In Caballero, B., Trugo, L. and Finglas, P.M. (Eds). Encyclopedia of Food Sciences and Nutrition, p. 3125-3130. Vol. 5. Oxford: Academic Press.Hogarth, A.J.C.L., Hunter, D.E., Jacobs, W.A., Garleb, K.A. and Wolf, B.W. 2000. Ion chromatographic determination of three fructooligosaccharide oligomers in prepared and preserved foods. Journal of Agricultural and Food Chemistry 48: 5326-5330.Internet: National Honey Board (NHB) 1996. Honey information kit of the food and beverage industries. Downloaded from http://www.honeylocator.com on 23/1/2008.Internet: Ustunol, Z. 2000.  The effect of honey on the growth of Bifidobacteria. Downloaded from http://www.honey.com/pressrm/research/bifido.html on 28/2/2008.Kajiwara, S., Gandhi, H. and Ustunol, Z. 2002. Effect of honey on the growth of and acid production by human intestinal Bifidobacterium spp.: An in vitro comparison with commercial oligosaccharides and inulin. Journal Skimmed milk with honey/FOS 0 hr 24 hrWild Tualang 6.3c ± 0.01 4.6c ± 0.01Common wild honey 6.5a ± 0.01 4.6c ± 0.00Commercial Tualang 6.5a ± 0.01 4.3d ± 0.01Fructooligosaccharides 6.5a ± 0.01 5.2a ± 0.01Control 6.4b ± 0.01 5.2b ± 0.01Table 3. pH value of skimmed milk with honey/FOS inoculated with B. longuma-d Means in the same column with different letters are significantly different at p < 0.05.Fructooligosaccharides in honey and effects of honey on growth of Bifidobacterium longum BB 536 561International Food Research Journal 17: 557-561of Food Protection 65: 214-218.Montrose, D. C. and Floch, M. H. 2005. Probiotics used in human studies. Journal of Clinical Gastroenterology 39: 469-484. Morales, V., Sanz, M.L., Olano, A. and Corzo, N. 2006. Rapid separation on activated charcoal of high oligosaccharides in honey.  Chromatograpahia 64(3/4): 233-238.Mundo, M. A., Padilla-Zakour, O. I. and Worobo, R. W. 2004. Growth inhibition of foodborne pathogens and food spoilage organisms by selected raw honeys. International Journal of Food Microbiology 97: 1-8.Ruiz-Matute, A.I., Sanz, M.L. and Martinez-Castro, I. 2007. Use of gas chromatography-mass spectrometry for identification of a new disaccharide in honey. Journal of Chromatography A 1157: 480-483.Tannock, G. (Ed). 1999. Probiotics: A Critical Review. Norfolk, England: Horizon Scientific Press.Ustunol, Z. and Gandhi, H. 2001. Growth and viability of commercial Bifidobacterium spp. on honey sweetened skim milk. Journal of Food Protection 64: 1775-1779.Weston, R.J. and Brocklebank, L.K. 1999. The oligosaccharide composition of some New Zealand honeys. Food Chemistry 64: 33-37.

Fructooligosaccharides in honey and effects of honey on growth of Bifidobacterium longum BB 536

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Fructooligosaccharides in honey and effects of honey on growth of Bifidobacterium longum BB 536

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