Bread has become the second most widely consumed non-indigenous food products in Nigeria. To cut the nation’s expense on wheat importation and find wider utilization for the increasingly produced cassava roots in Nigeria, the Federal Government mandated the use of composite cassava wheat flour for baking by adding minimum of 10% cassava flour to wheat for a start (Shittu et al., 2007). According to Shittu et al. (2007), fresh crumb moisture, density, porosity and softness as well as the dried crumb hardness were significantly affected by both the baking temperature and time. The studies of Defloor et al. (1993, 1994, 1995) and Khalil et al. (2000) established that 10% substitution of wheat with cassava flour gave bread with quality not significantly different from 100% wheat bread. Comparative studies have shown that honey has less impact on blood sugar level because it offers low glycemic index (GI) response (Foster, 2008). Beyond many health claims and ability to mask any taste deficiency that may have resulted from ingredient interactions, inclusion of honey into bread formulation is said to offer functional benefits (Foster, 2008). Baking technology has been evolving continuously as new materials; equipment and processes are being developed (Selomulyo and Zhou, 2007). The impacts of various ingredients on sensory and nutritional quality of bread have been widely studied (Barcenas and Rosell, 2005; Plessas et al., 2005).

Pure honey has been shown to be bactericidal to many pathogenic microorganisms and the antimicrobial activity has been reported to be as a result of the presence of osmotic effect, acidity and hydrogen peroxide (Radwan et al., 1984; Jeddar, et al., 1985). The pH of honey is reported to be low enough to slow down or prevent the growth of many species of bacteria. The high sugar content of honey makes the water unavailable for microorganisms: no bacteria or fungi can grow in fully ripened honey, but the more diluted honey becomes, the more species can grow in it (Molan, 1992). It was also reported that glucose oxidase enzyme activated by dilutions of honey generates hydrogen peroxide which generally is the major antibacterial factor in honey. This enzyme is inactivated by heating honey, and by exposure to light in some honeys which contain a sensitizing factor. Some honeys also contain substances which destroy the hydrogen peroxide generated by the enzyme (Molan, 1992).

According to Beatriz et al. (2011), the water content is important for honey stability, while the acidity content of honey is a function of honey fermentation. Studies have shown that honeys from the tropics with high water content tend to ferment readily and the free acidity is increased (Sibel et al., 2010; Beatriz et al., 2011) and CA (2001) prescribes a maximum value of 50 milliequivalents of free acids per kilogram of honey. Antimicrobial effects of honey against microorganism associated with disease or infection, food spoilage, including foodborne pathogens and yeasts, have been reported and honey biolo-gical activity has been attributed not only to the high sugar concentration and hydrogen peroxide production but also to different compounds such as acids, phenolics, proteins and carbohydrates (Gheldof et al., 2002; Olasupo et al., 2003; Mundo et al., 2004; Guerrini et al., 2009; Gomes et al., 2010). However, the findings of Olaitan et al. (2007) revealed that microorganisms that survive in honey are those that withstand the concentrated sugar, acidity and other antimicrobial characteristics of honey. Stefan et al. (2008) reported that honey from different sources differs in appearance, sensory perception and composition. This suggests that their antimicrobial pro-perties/potential  may  vary.  Although, information is still scarce on utilization of honey in bread formulation developed from cassava wheat composite flour, previous study on the influence of a single type of honey on microbiological shelf stability of cassava-wheat composite bread revealed that the honey used extended the shelf life of the baked bread loaves (Adeboye et al., 2013). Therefore, this current study therefore aims at investi-gating the microbial shelf life stability of cassava-wheat composite bread baked with different types of honeys obtained from different locations in South Western Nigeria. 

The sweet cassava variety (Manihot esculenta Crantz) tubers used for the production of cassava flour in this study was harvested from the farm of Moshood Abiola Polytechnic, Abeokuta, Nigeria and the white wheat flour was obtained from honey well flour mill, Lagos, Nigeria. Other ingredients used were granulated sucrose sugar (Dangote groups (Nig) Ltd. Nigeria), Fermipon baking yeast (DSM bakery ingredient, Dordrecht-Holland) and baking fat (Pt Intibuca Sehtera, Jakarta, Indonesia). The various honey used were obtained from local bee keeping and honey production farms (Ibadan, Nigeria). The composite flour was made by mixing 10 part cassava flour with 90 part wheat flour.

Determination of the physicochemical properties of honey types

Total phenolic content

The procedure for the determination of total phenolic content was adapted from Zalibera et al. (2008) with some modifications. 50 μL of the honey or methanolic extract was added to 125 μL of Folin-Ciocalteu reagent. The mixture was sonicated for 5 min after which 625 μL of sodium carbonate was added and the absorbance was determined after 2 h at 760 nm. The results were expressed as milligram of gallic acid equivalents per kilogram of honey (mg GAE/kg).

Moisture content, total acidity and viscosity

Moisture content, total acidity and viscosity of the honey types were determined according to methods described by the AOAC (2000) (Official Methods 979.20, 969.38, 962.19).

Preparation of cassava flour

The matured cassava tubers were peeled, washed and grated in a mechanical grater and the pulp obtained was dewatered in a ‘muslin’ cloth placed in between a screw press. The pulverized cassava mash obtained was then dried in cabinet dryer (Lukas Engineering Nig. Ltd) at 70°C to a constant weight to give 4% moisture content. The dried cassava mash was milled in a hammer mill (Lukas Engr. Ltd. Lagos) and sieved with a mesh of 0.5 mm pore size and fine cassava flour obtained was stored in an airtight container.

Preparation of the recipe

The calculated weight of the sugar (sucrose) in the recipe used was substituted with the different honey types at the optimum substitution ratio of 30:70 of honey (H) to sugar (S). The six bread samples were one control with 100% sugar and five with different honey types. The ratio of honey and sugar was 7.56: 17.64 g in all samples.

Production and storage of honey-cassava wheat composite bread

The honey-cassava wheat bread was prepared using the procedure described by Shittu et al. (2007) with slight modification. The ingredients (yeast, water (at 40°C), honey and butter) were combined in a large liquid measuring cup and stirred until the yeast has dissolved and the baking fat has melted. The sugar, composite flour and salt were dry mixed in a large bowl. The yeast mixture was thoroughly incorporated into the mixture of dry ingredients. The dough obtained was then transferred into a lightly floured work surface of the kneading machine and kneaded for about 15-20 min to form smooth and elastic dough. The dough was cut into uniform sizes (300 g), placed in lightly greased pan and proofed at 30°C and 78-80% RH for 2 h then baked in an oven at 220°C for 30 min. Samples from each honey-cassava wheat bread were stored at ambient temperature in nylon 6-guage of polythene bag for 6 days during which the total viable bacteria and mould growth for each sample type were monitored

Microbiological analysis         

The microbial profile of the total aerobic bacteria and yeasts and moulds of the composite bread was determined using nutrient agar (NA) (Oxoid, Basingstoke, Hampshire, England, UK) and potatoes dextrose agar (PDA) (Merck, Darmstadt, Germany) (supplemented with 50 mg/litre of streptomycin), respectively. Ten gram (10 g) of each of the baked bread sample was aseptically homogenized with 90 mL sterile 0.1% buffered peptone water (BPW) (Merck) solution. After serial dilutions of all the samples, the appropriate dilution was spread plated and the NA (Oxoid) agar plates were incubated at 37°C for 24 h while yeast and mould plates were incubated at 25°C for 3-5 days. Samples were analysed after 4 and 6 days of storage.

Statistical analysis

Analysis of variance (ANOVA) was used to determine if physicochemical properties of honey affected the microbial shelf stability of honey cassava-wheat bread significantly at 95% confidence levels. Each experiment was repeated in triplicate and data were analysed using the Statistical Package for the Social Sciences (SPSS) 21.0 (IBM SPSS Inc., Chicago, IL) and mean separation was carried out with Duncan’s new multiple range test.

This study has demonstrated that the physicochemical properties of honey could influence the microbial shelf stability of cassava-wheat composite bread. However, it may be necessary to increase the ratio of partial substitution with concentration of the honey used in this current study in order to determine the minimum inhibitory concentration of honey in bread and to circumvent the effect of the baking temperature on the antimicrobial potential of the honeys.

The authors did not declare any conflict of interest.