CHAPTER 2
LITERATURE REVIEW
2.1 Manuka honey
Manuka honey is a monofloral honey delivered from the Manuka bush Leptospermum scoparium, has long been known as a food and beverage. Study also suggested that manuka honey has ameliorative properties in wound healing, fungal infections, ophthalmic disorders, diabetes, gastrointestinal tract disorders, skin ulcers and infectors (Medhi et al, 2008). Bees foraging and gather nectar from the manuka bush, create a outstanding and unique honey with high level of antibacterial properties, which termed as non-peroxide antibacterial (NPA) activity (Allen, Molan, & Reid, 1991). These antibacterial activity are normally rate in Unique Manuka Factor, UMF. It is used to measure the capacity of the honey to destroy the harmful virus, microorganism and bacteria. The level of UMF is equivalent to the level of non-peroxide activity.
These non-peroxide antibacterial activity is largely attributable to the presence of unusually high concentration of methylglyoxal(MGO) in manuka honey. Methylglyoxal(MGO) is a naturally occurring bioactive compound forming from a chemical conversion dihydroxyacetone(DHA), which found in high concentration in the nectar of manuka flowers (Adams, Manley-Harris, & Molan, 2009).
2.2 Enzyme activity in honey
2.2.1 Diastase
Diastase is any group of enzymes added by bees during honey production, facilitating the conversion of starch into maltose. It means any α-, β-, or γ-amylase (all of them hydrolases) that help to break down carbohydrates into simple sugars. -amylase function to catalyze the degradation of starch into a complex of the disaccharide maltose, the trisaccharide maltotriose and oligosaccharides known as dextrins, contribute to the losing of viscosity (Nikola Sakac, & Milan Sak-Bosnar, 2012). On the other hand, β-amylase catalyze the splitting of the second α-1,4 glycosidic bond from the ends of the starch chain and formed reducing sugar maltose (Laid et al., 2008). Diastase is widely recognized as important parameter for evaluating the quality and freshness of honey, due to its high sensitivity towards heat. The changing behavior of diastase activity makes it an uncertain parameter to investigate if honey has been undergoes heating (Subramanian et al., 2007 ; Gui, Nuray Sahinier & Aziz, 2005; Fallico et al., 2004).
Diastase activity is calculated as diastase number (DN) in Schade units and is defined as one diastase unit corresponds to the enzyme activity of 1 g of honey, which can hydrolyse 0.01g of starch in 1h at 40â-¦C. In bakery factory, honey was used to mix with starch containing food ingredients. It was found out that the high diastase activity in honey may contribute to a poor bread texture, therefore, the honey was mainly controlled in a low Diastase values. A major use of diastase is controlling the quality of honey. The EU(European Union) Honey Directive pointed out that a honey must meet the following standard or requirement for Schade units, before it is ready to market for human consumption,:

In general more or equal to 8 schade untis (except baker’s honey);
Honeys with low natural enzyme content (e.g. citrus honeys) and an hydroxymethylfurfural HMF content of not more than 15 mg/kg: more or equal to 3 schade units.

(M. L. Science, 2014. ; Bogdanov, S., & Martin, P., 2002)
2.3 Diastase activity of different honey source
Diastase is naturally occurring in honey, its amount depends upon floral origin and geographic area. Fresh honey samples contained diastase activity ranged from 11.2 to 45.5 DN, while for commercial samples; it ranged from 10.9 to 17.8DN only. Among fresh honeys, lowest values were found in blossom honeys(from 11.24 to 30.3DN), compound honeys they ranged from 15.9 to 40.3DN. Honeydew honeys contained the highest value of diastase enzyme (from 13.6 to 45.4DN) (L. Vorlova, & A. Pridal, 2002).

Fresh Honeys

Diastase content, DN

Blossom honey

11.24 – 30.30

Compound honey

15.90 – 40.30

Honeydew Honey

13.60- 45.40

Table 2.1 : Fresh honeys with their respective diastase content.

Honey

Honey type

Diastase number,DN (schade units)

Reference

Rape honey

Monofloral

23.09 ± 3.00

(Balkanska, R., & Ignatova, M., 2013)

Coriander honey

Monofloral

15.02 ± 1.46

(Balkanska, R., & Ignatova, M., 2013)

Clover honey

Monofloral

5.73-17.3

(Babacan, 2001).

Buckwheat honey

Monofloral

36.8

(Babacan, 2001).

Starfruit honey

Monofloral

4.00

Subramanian et al. 2007

Honeydew honey

–

18.0-23.2

(Kowalski et al., 2012)

Forest honey

Monofloral

21.8

(Kowalski et al., 2012)

Aegean honey

Monofloral

15.5

(Kowalski et al., 2012)

Sunflower honey

Monofloral

20.37 ± 3.82

(Elif et al., 2012)

–

Multifloral

26.0

(Saric et al., 2008)

–

Multifloral

38.5 ± 3.5

(Samborska, K., & Czelejewska, M., 2012)

Table 2.2 : Diastase number of different type of honeys
2.4 Thermal treatment on honey
Honey are highly concentrated solution of glucose and sucrose converted from the sucrose in nectar by invertase. A freshly extracted honey is liquid in form. However, long storage time of honey may reduced its quality by crystallization. Losing of homogeneity, changing of its appearance to waxy and opaque, and a two phases solution of crystalline and liquid can be found coexistence together in honey indicated that a honey was crystallized (Kowalski et al., 2012).

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Crystallization of honey may created difficulty to us in handling and pouring. In the worst case, when water activity is higher than its original value, part of the water release from solid phase and increase the moisture level in liquid phase. This may greatly enable the development of the present microbial floral (Tosi et al., 2008). Moreover, fermentation can be happen and produce acetic acid as a result of osmophilic flora action on glucose and fructose, releasing gaseous of carbon dioxide, formation of foam, and ethanol, which, aiding with the present oxygen (Tosi et al., 2008).
Honey processing is provided to prolong the storage time of honey in liquid phase, including liquefaction, filtering, heating under specific range of temperature, bottling, cooling and storage. Among this, thermal treatment is the important step to keep the honey in liquid state by reducing its viscosity, preventing recrystallization and destroying of micro-organisms which will contaminate honey (Tosi et al., 2008; Turhan et al., 2008). A temperature of 40-50oC or lesser is recommended to avoid heat damage of sensitive substances (Castro-Vasquez et al., 2008).
2.4.1 Transient heating stages
During transient heating, all molecules whose free energy exceeds the energy barrier undergo a complete and irreversible denaturation, according to the theory of Eyring. Treated substances may undergoes rising of temperature from initial value to a desired temperature (Tosi et al., 2004). The decrease in the diastase activity related to an increase in temperature (Tosi et al., 2008).
2.4.2 Isothermal heating stages
During isothermal heating, samples maintain at a fix temperature; for example, 60, 70, 80 and 90 °C, the number of activated molecules which could exceed the energetic barrier of the transition stage was low. All samples showed a decrease of the diastase activity at short heating times but increased when temperature increase. According result, temperature between 90 to 100 caused the irreversible of honey diastase (Tosi et al., 2008)
2.5 Effect of thermal treatment
Any thermal processing of honey may contribute to product quality degeneration. Uncontrolled heating of honey may modify the essential composition of honey, destroy thermolabile honey components, influences the parameter such as enzymatic activity and hydroxymethylfurfural (HMF) content; for example, diastase activity in honey was reduced and the level of hydroxymethylfurfural (HMF) content was increased. (Biago et al., 2004). Increasing of storage time or temperature can have a significant effect on the chemical composition and oganoleptic characteristics of honeys (Castro-Vasquez et al., 2008). Heat labile compounds may destroyed after excessive heat treatment and honey flavor was changed as a result of producing volatile compound by Maillard reaction (Wootton et al., 1978).
2.6 Variation of diastase activity(diastase number) of honey after heating
Diastase activity is sensitive to temperature and storage condition, so it is use as an indicator of freshness and controls during processing of the honey (Gui, 2005). When honey was added to the starch matrix, starch thinning and significant viscosity loss occurred after 4 hour. However, zero amylase activity and no change in viscosity when honey was heat to 76oC for 21 hour before adding to starch (Babacan, 2001).
According to a study lead by Katarzyna and Monika (2012), they found out that fresh multifloral honey has diastase activity of 39.5DN while fresh rape honey has 17.9DN without any heating process. Upon heat treatment on 50°C and 70°C, the diastase activity of multifloral honey dropped to 10.9DN and 8.3 DN after 120min. Raising the heating temperature to 90°C resulted in significant decrease of DN to 6.5 after 30 minutes heating. In the case of rape honey, it has 17.9DN when it is fresh without any heating. After 120 minute of heating at 50°C, the diastase number has decreased to 10.9. At 70°C, the diastase value (5DN) after 120 minutes was lower than the standard. Raising the temperature of heat treatment to 90°C, the diastase number had dropped below minimun level after 15minutes (Katarzyna S., & Monika C., 2012)
The relative reduction of diastase activity in fresh multifloral honey was higher than that in monofloral honey. In the study carried out by Katarzyna and Monika (2012), a closer diastase activity values were recorded after 120 min of treatment at 50 and 70C in both honeys. However, the multifloral honey contained twice higher the initial diastase value than the rape honey(monofloral honey), so the relative reduction of diastase number in multifloral honey was higher. Diastase activity of multifloral honey after 120 min at 50C was 28.3% of the initial value, while for rape honey, it was 60.9% (Katarzyna S., & Monika C., 2012)
From the study carried out by Babacan(2001), he pointed out that amylase(diastase) has heat resistance, but amylase activity was reduced by heat treatment at 85oC. According to his result, it showed that amylase activity decreased slightly when honey heat to 63oC as compare to honey under heat treatment on 85oC. More amylase activity is losing when exposure time to heat(85oC) is increased, left with dark brown honey with burnt odor. In one study of heat treatment onto polyfloral honey, the result showed that ratio of diastase activity loss is 7.9% at 50°C, 15.3% at 80°C and 48.29% at 100°C after first 30 minutes heating(Anca et al., 2011) In another way, it can be said that the diastase enzyme activity contain in honey can control using heat treatment. Increasing of temperature may results in lower diastase number of honey (Babacan, 2001)
On the other hand, some research are carried out to investigate the optimum temperature and storage time to have the least diminution of diastase in honey. For example, diastase activity of citrus Honey decrease after storage for 12 months especially at 40 oC. Diastase activity had decreased 2.3 units in citrus Honey samples stored at 10 oC, with respects to fresh samples. While for the citrus honey that stored at 20 oC for a year had a diminution of 4 diastase units (Castro-Vasquez et al., 2008). In addition, a honey storing at 20 ± 5 oC for one year had a small reduction in diastase activity for about 27% (Yilmaz H.,& Kufrevioglu I., 2001). According to this study, back then in year 1986, a similar study was carried out and researcher Thrasyvoulou(1986) has found out that 20 of his honey samples stored for one year at 25 oC has a 40% decrease in their diastase activity. Later in year 1992, Sancho et al. (1992) recorded a 33% decrease of diastase activity on 115 samples stored at 15-25 oC for one year (Yilmaz H.,& Kufrevioglu I., 2001).