The Value-adding Potential of Prebiotic Components of Australian Honey RIRDC Publication No. 09/0179
The Value-adding Potential of Prebiotic Components of Australian Honey
By Patricia L. Conway, Rosie Stern and Lai Tran
January 2010 RIRDC Publication No 09/179 RIRDC Project No. PRJ-000041
© 2010 Rural Industries Research and Development Corporation. All rights reserved. ISBN 1 74151 975 6 ISSN 1440-6845 The Value-adding Potential of Prebiotic Components of Australian Honey Publication No. 09/179 Project No. PRJ-000041 The information contained in this publication is intended for general use to assist public knowledge and discussion and to help improve the development of sustainable regions. You must not rely on any information contained in this publication without taking specialist advice relevant to your particular circumstances. While reasonable care has been taken in preparing this publication to ensure that information is true and correct, the Commonwealth of Australia gives no assurance as to the accuracy of any information in this publication. The Commonwealth of Australia, the Rural Industries Research and Development Corporation (RIRDC), the authors or contributors expressly disclaim, to the maximum extent permitted by law, all responsibility and liability to any person, arising directly or indirectly from any act or omission, or for any consequences of any such act or omission, made in reliance on the contents of this publication, whether or not caused by any negligence on the part of the Commonwealth of Australia, RIRDC, the authors or contributors. The Commonwealth of Australia does not necessarily endorse the views in this publication. This publication is copyright. Apart from any use as permitted under the Copyright Act 1968, all other rights are reserved. However, wide dissemination is encouraged. Requests and inquiries concerning reproduction and rights should be addressed to the RIRDC Publications Manager on phone 02 6271 4165. Researcher Contact Details Patricia L Conway MSc, PhD, MASM; Rosie Stern, MSc; Lai Tran PhD. CMB, School of Biotechnology and Biomolecular Sciences, The University of New South Wales SYDNEY, NSW 2052 Phone: (02) 9385 1593 Fax: (02) 9385 1285 Email:
[email protected] In submitting this report, the researcher has agreed to RIRDC publishing this material in its edited form. RIRDC Contact Details Rural Industries Research and Development Corporation Level 2, 15 National Circuit BARTON ACT 2600 PO Box 4776 KINGSTON ACT 2604 Phone: Fax: Email: Web:
02 6271 4100 02 6271 4199
[email protected]. http://www.rirdc.gov.au
Electronically published by RIRDC in January 2010 Print-on-demand by Union Offset Printing, Canberra at www.rirdc.gov.au or phone 1300 634 313
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Foreword There is emerging interest in studying the potential health benefits of eating honey. The objective of research reported here was to show that Australian honeys have properties that infer their consumption could deliver benefits beyond basic nutritional requirements. In particular, this research has shown that a number of Australian honeys can function in prebiotic roles, which means that they can promote the growth of beneficial microbes commonly found in the human intestine. Because it is known that maintenance of a healthy intestinal microflora can assist the immune system, as well as general bodily function, it can be suggested that consumption of honey has potential to promote overall human health and well-being. Australian honeys originate from the unique combinations of floral species and local environments that exist in Australia. The specific properties of these honeys can thus be expected to be highly distinctive, so enhancing their desirability and marketability. At market level, these qualities and attributes are likely to maximise product value. On a broader scale, scientific demonstration that use of honey can produce dietary and general health benefits will support beekeeping and honey industries in general, as well as assisting overall maintenance of human health. The global functional food market is one of the most rapidly expanding food industries. The findings from this research give the Australian honeybee industry scope to enter that market. Further R&D investment may also enable key honey constituents to be identified and adopted for specificallytargeted purposes. This work was funded with funds provided by the Australian honey industry and the Australian Government. This report, an addition to RIRDC’s diverse range of over 1900 research publications, forms part of our Honeybee R&D Program, which aims to improve the productivity and profitability of the Australian beekeeping industry. Most of RIRDC’s publications are available for viewing, downloading or purchasing online at www.rirdc.gov.au. Purchases can also be made by phoning 1300 634 313.
Peter O’Brien Managing Director Rural Industries Research and Development Corporation
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Acknowledgments The authors are extremely grateful to the Honeybee R&D Advisory Committee for their valuable comments, and in particular, many thanks to Bruce White for all his assistance. In addition, thanks go to all the beekeepers who supplied honey samples.
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Contents Foreword ...............................................................................................................................................iii Acknowledgments................................................................................................................................. iv Executive Summary............................................................................................................................viii 1.
Introduction .............................................................................................................................. 1 1.1
Value-adding to honey ........................................................................................................... 1
1.2
Prebiotics................................................................................................................................ 1 1.2.1 Description of prebiotics ............................................................................................... 1 1.2.2 Health benefits of prebiotics.......................................................................................... 2 1.2.3 Commercially available prebiotics ................................................................................ 3 1.2.4 Mechanism of action of prebiotics................................................................................. 3 1.2.5 Qualitative and quantitative assessment of prebiotics ................................................... 3 1.3 Honey as a prebiotic............................................................................................................... 4 1.4
Characteristics of honey......................................................................................................... 5 1.4.1 Description of honey ..................................................................................................... 5 1.4.2 Physical characteristics of honey .................................................................................. 6 1.4.3 Composition of honey ................................................................................................... 7 1.4.4 Functional aspects of honey .......................................................................................... 8 1.5 Structure of the Australian honeybee industry..................................................................... 10 1.6
Summary .............................................................................................................................. 11
2.
Objectives ................................................................................................................................ 13
3.
Methodology............................................................................................................................ 13 3.1
Approach.............................................................................................................................. 13
3.2
Honey samples ..................................................................................................................... 13
3.3
Honey oligosaccharides ....................................................................................................... 13
3.4
Bacterial cultures and incubation conditions ....................................................................... 14
3.5
Growth studies with pure cultures........................................................................................ 14
3.6
Measurement of prebiotic index in intestinal microcosms................................................... 14
4.
Results...................................................................................................................................... 15 4.1
Physical characteristics of honey samples ........................................................................... 15
4.2
Effect of honey on growth of probiotic bacteria .................................................................. 15 4.2.1 Comparison of honeys with glucose and fructose....................................................... 15 4.2.2 Comparison of honey with sucrose and a commercial prebiotic................................. 18 4.3 Effect of honey on growth of pathogenic bacteria ............................................................... 20 4.4
Effect of honey oligosaccharides on growth of probiotic bacteria....................................... 21
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4.5
The prebiotic potential of honey measured using intestinal microcosms ............................ 21 4.5.1 Effect of natural honeys on levels of potentially ‘good’ and ‘bad’ bacteria. .............. 21 4.5.2 Prebiotic index of honeys and honey oligosaccharides............................................... 22
5.
Implications............................................................................................................................. 24
6.
Recommendations................................................................................................................... 25
Appendix .............................................................................................................................................. 26 References ............................................................................................................................................ 28
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Tables Table 1.1
Properties, composition and method of manufacture of common prebiotics...................... 3
Table 1.2
Medicinal attributes of honey reported by the American National Honey Board in 2007. 9
Table 1.3
The % of total Australian production by state and the average production per beehive in 2000 (RIRDC 2007). ................................................................................................... 10
Table 1.4
Dominant floral species supporting the Australian honey industry by state..................... 12
Table 4.1
Physical characteristics of the various honey samples tested. .......................................... 16
Table 4.2
Growth of probiotic cultures in honey or glucose. ........................................................... 18
Figures Figure 4.1
Comparative growth of beneficial bacteria in the presence of honey.............................. 17
Figure 4.2
Growth of Bifidobacterium lactis and Lactobacillus plantarum in broth with honey, sucrose, inulin or no added carbohydrate. ....................................................................... 19
Figure 4.3
Growth of pathogenic bacterial species using honey as the sole carbohydrate source.... 20
Figure 4.4
Effect of honey oligosaccharides on growth of L. acidophilus. ...................................... 21
Figure 4.5
Effect of honey on growth of lactobacilli and coliform bacteria in intestinal microcosms . ......................................................................................................................................... 22
Figure 4.6
The prebiotic index (PI) of natural honeys (see Table 4.1), inulin and sucrose, as measured in a simulated intestinal microcosm. ............................................................... 23
Figure 4.7
The prebiotic index (PI) of honey-derived oligosaccharides (see Table 4.1), inulin and sucrose, as measured in a simulated intestinal microcosm. ............................................. 23
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Executive Summary Research reported here explores the potential for consumption of Australian honeys to deliver health benefits that extend beyond routine nutritional requirements. This report provides data which support the proposition that some Australian honeys possess prebiotic properties, and which indicate the importance and likely value of further investigation of honey as a functional food. Background The value of Australian honey could be increased by evidence of beneficial properties beyond its basic nutritional qualities (including its important glycaemic index properties), and its already described therapeutic and antibacterial activities. It has been predicted that the complex saccharides in honey could be used by beneficial bacteria in the large intestine, which in turn would be likely to promote good gastrointestinal health and general bodily function. It is known that improvement in the composition of intestinal microbial flora can assist immune modulation in other parts of the body, thus suggesting that consumption of honey could have potential to improve and promote overall human health and well-being. Since the composition of honey varies with the floral species of origin, local climate, and procedures used for harvesting and storage, Australian honeys can be expected to be unique. It was therefore of interest to investigate the potential of Australian honeys for use as natural functional foods, and as sources of new functional food ingredients. Aims and objectives The objective of this project was to investigate the prebiotic characteristics of Australian honeys and their potential for improving human gastrointestinal health, to provide data that could support increased use of honey by consumers and the food manufacturing industry. Methods Honeys from each state of Australia were sourced and the physical characteristics of each honey sample recorded. All honey samples were tested for their capacity to promote the growth of pure cultures of known beneficial bacteria currently used as probiotics. The effect on growth was quantified, and the five honeys which were most efficient for all cultures tested were selected for further study. The effect of honeys on the growth of cultures of known bacterial pathogens was also studied. In order to simulate the inclusion of honey in the diet and its effect on indigenous microbes in the intestine, in vitro microcosms simulating intestinal conditions were used. Results from the microcosm trials were used to calculate the prebiotic index (PI) of each honey. Because honey contains both simple and complex sugars, tests were carried out using complete natural honeys as well as honeys pre-treated to remove simple sugars. The latter approach effectively simulated the potential effects of honey after absorption of simple sugars in the small intestine. Results and key findings This study has demonstrated that Australian honeys possess prebiotic potential, based on observations that growth of beneficial bacteria such as lactobacilli and bifidobacteria was promoted when honey was added as the sole carbohydrate source. Since honey is a mix of complex and simple sugars, with the latter being rapidly absorbed by the body when honey is ingested, it was important to examine whether the complex sugars alone could also promote the beneficial bacteria. It was shown that the complex sugars in honey could also promote the growth of beneficial bacteria. In order to simulate the situation in the body, microcosms were set up in the laboratory to mimic the human intestinal
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environment. When natural honeys were added to these microcosms, it was shown that beneficial bacteria were enhanced in number, less-desirable bacteria were reduced in number, and that the complex sugars in the honeys contributed to the enhancement of the beneficial populations. It was therefore concluded that the Australian honeys have prebiotic potential, and noted that the effect varied between honeys of different floral species of origin. Implications for stakeholders The evidence that Australian honeys have prebiotic activity has potential to open an exclusive market opportunity. Currently, honey competes with sugar as a sweetening agent in the food industry. Sugar is rapidly absorbed in the small intestine and provides no benefit to intestinal microbes and overall gut health. In contrast, honey contains both simple sugars and more complex sugars, the latter of which are not degraded by host enzymes and reach the large intestine, where they are available to beneficial microbes. The market value of prebiotics for food and beverages for Europe in 2008 was €295.5 million per annum (p.a.), and is predicted to reach €766.9 million p.a. by 2015 in Europe alone, with overall required volumes of more than 200,000 tonnes p.a., and an overall compound growth rate of 14% p.a. Honey requires no further purification, and represents one of the few naturally-available prebiotics. Based on current prebiotic prices in Europe, honey could achieve sale prices more than twice those in the current commodity market if sold as a prebiotic material. While further studies are expected to provide additional in vivo-based evidence of the prebiotic capacities of Australian honey, results to date provide conclusive evidence that when the complex sugars in honey are delivered to populations of intestinal microbes, a beneficial profile of microbes can develop. This profile is consistent with those that have been associated with health benefits in clinical studies. This finding makes it potentially attractive to develop healthier beverages and confectionary which use honey as the sweetening agent. Recommendations Data provided in this report provide evidence which the honey industry can potentially use to expand its markets. These findings could, however, be strengthened by additional studies. Completion of in vivo studies which confirm the data from laboratory tests would deliver additional evidence and further support use of Australian honeys as prebiotics and components of functional foods. Other work could be undertaken to include analysis of the impact(s) of factors such as floral species and local environmental conditions of production, and processing and storage procedures, on the prebiotic capacity. In addition, the particular complex sugars that contribute to the prebiotic capacity could be identified. Availability of a quantitative assessment of the prebiotic index (Roberfroid, 2007) as used here, will allow comparison of batches and standardisation of prebiotic honey with guaranteed activity. Potentially one could envisage the emergence of ‘designer honeys’ targeting specific health applications by blending honeys from various floral sources. Public promotion of these and similar findings will maximise knowledge of the results, and of the potential benefits of Australian honeys for human health.
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1. Introduction 1.1
Value-adding to honey
There are a number of different definitions of ‘value-adding’. In general, value-adding means taking a raw material and processing it or adding something to change it into a saleable item that will be purchased by a different or expanded group of customers. Honey has recently been rediscovered as a ‘natural’ food ingredient whose use can enhance the market value of various processed food products, including baked goods and confectionary products (Krell, 1996). Adoption of new applications of honey and its constituent components can value-add to honey, and thereby extend its market potential.
1.2 1.2.1
Prebiotics Description of prebiotics
Gibson and Roberfroid (1995) coined the term ‘prebiotics’, and defined it as a “non-digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improving host health.” These workers subsequently refined the definition, stating that “a prebiotic is a selectively fermented ingredient that allows specific changes, both in the composition and/or activity in the gastrointestinal microbiota that confers benefits upon host well-being and health” (Gibson et al., 2004). Other researchers have been more specific, and have proposed that “a prebiotic is a carbohydrate that results in changes in the numbers of key bacterial genera in the colon, i.e. bifidobacteria, bacteroides, lactobacilli and clostridia” (Palframan et al., 2003). Essentially, prebiotics are carbohydrates that are not digested and absorbed by the host, and which therefore reach the large intestine where they are utilised by beneficial bacteria. Prebiotics can even be co-administered with beneficial bacteria (which are referred to as ‘probiotics’) so as to achieve added health benefits. The combination of prebiotics with beneficial bacteria (probiotics) is referred to as synbiotics. The benefits associated with probiotics and prebiotics are strain- and compound-specific, respectively, and efficacy needs to be proven for the specific dose combination present in the material consumed. An FAO (Food and Agriculture Organization of the United Nations) Technical Meeting in 2007 reviewed prebiotics and the various definitions of the term. The FAO found the past definitions of prebiotics to be restrictive in their applicability for target sites outside the gastrointestinal tract, and considered that the definitions also necessitated a single mechanism of action activity in the gastrointestinal microbiota (FAO, 2007). Consequently, FAO defined a ‘prebiotic’ as “a non-viable food component that confers a health benefit on the host associated with modulation of the microbiota.” The meanings of the terms used in this definition were specified as follows:
‘component’: not an organism or drug; a substance that can be characterised chemically, and which, in most cases, will be a food grade component;
‘health benefit’: measurable and not due to absorption of the component into the bloodstream or due to the component acting alone, and over-riding any adverse effects;
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‘modulation’ – demonstration that the sole presence of the component and the formulation in which it is delivered change the composition or activities of the microbiota in the target host. Mechanisms of modulation might include fermentation, receptor blockage or other actions.
The component to which the claim of prebiotic status is attributed must be characterised in terms of:
source, origin and purity,
chemical composition and structure, and
vehicle, concentration and amount in which it is to be delivered to the host.
In addition, and at a minimum, there is a requirement for evidence of a correlation between the measurable physiological outcomes and modulation of the micro-biota at a specific site such as the gastrointestinal tract or the skin. Prebiotics are considered to be part of the broader ‘functional food’ group. Functional foods are foods that provide health benefits beyond basic nutrition. Food Standards Australia New Zealand (FSANZ) describes functional foods as being “similar in appearance to conventional foods and intended to be consumed as part of a normal diet, but modified to serve physiological roles beyond the provision of simple nutrient requirements.” Prebiotics are also part of a new category of foods called ‘novel foods’. In Australia, FSANZ has defined novel foods as foods that “are non-traditional foods with characteristics that require an assessment of public health and safety considerations.” Novel foods have the characteristic of a degree of uncertainty of the food safety of the food. For this reason all novel foods in Australia are assessed rigorously before being allowed to be sold in Australia and New Zealand (FSANZ, 2008). Australia and New Zealand are currently preparing a new food standard which will allow health claims to be made for functional foods. A health claim can refer to the presence of a nutrient or substance in a food and to its effect on a health function. Manufacturers of such foods must use either the FSANZ Model List of pre-approved statements, provide suitable scientific texts or dietary guidelines to support their claim(s), or must hold scientific evidence to substantiate such claims and produce this evidence, on request, for enforcement agencies (FSANZ, 2008). Health claims are potentially powerful tools for marketing functional foods since a health claim will help to explain to the consumer the health benefit of the food ingredient (Williams & Ghosh, 2008).
1.2.2
Health benefits of prebiotics
Worldwide, there has been a growing awareness of the relationship between diet and health that has led to an increasing demand for food products that support health beyond simply providing basic nutrition. Incorporation of prebiotics and probiotics into foods can yield health benefits in the gastrointestinal tract and other parts of the body that are linked via the immune system. Prebiotics have been shown to both promote the growth of healthy bacteria in the large bowel and to prevent the symptoms associated with bowel disorders, including irritable bowel disease and irregular bowel function. More recently prebiotics, in particular fructooligosaccharides (FOS) and galacto-oligosaccharides (GOS), have been reported to have significant health benefits in relation to anti-cancer properties, and influence on mineral absorption, lipid metabolism, and anti-inflammatory effects (Macfarlane et al., 2007).
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1.2.3
Commercially available prebiotics
Prebiotics are usually polysaccharides or oligosaccharides (International Food Information Council [IFIC], 2006). Commonly used prebiotics include inulin, fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), soya-oligosaccharides, xylo-oligosaccharides, pyrodextrins, isomalto-oligosaccharides and lactulose. Other prebiotics include pecticoligosaccharides, lactosucrose, sugar alcohols, gluco-oligosaccharides, levans, resistant starch, xylosaccharides and soy-oligosaccharides (FAO, 2007). Macfarlane et al. (2006) summarised the properties, composition and method of manufacture of common prebiotics, as shown in Table 1.1. Table 1.1: Properties, composition and method of manufacture of common prebiotics
Name
Composition*
Method of Manufacture
Inulin
Beta(2-1) fructans
Extraction from chicory root
Fructo–oligosaccharides
Beta(2-1) fructans
Transfructosylation from sucrose, or hydrolysis of chicory inulin
Galacto–oligosaccharides
Oligo-galactose (85%) with some glucose and lactose
Produced from lactose by betagalactosidase.
Soya–oligosaccharides
Mixture of raffinose (F-Gal-G) and stachyose (F-Gal-Gal-G)
Extracted from soya bean whey
Xylo–oligosaccharides
Beta(1-4)–linked xylose
Enzymic hydrolysis of xylan
Pydrodextrins
Mixture of glucose containing oligosaccharides
Isomalto – oligosaccharides
Alpha(1-4) glucose and branched alpha(1-6) glucose
pyrolysis of potato or maize starch Transgalactosylation of maltose
* F, fructose; Gal, galactose; G, glucose
1.2.4
Mechanism of action of prebiotics
Prebiotics have been shown to be neither hydrolysed nor absorbed in the upper part of the gastrointestinal tract. Once the prebiotic enters the lower part of the gastrointestinal tract the prebiotic selectively stimulates the growth and/or activity of desirable bacteria in the colon. There are a number of desirable bacteria in the colon including species of lactobacillus and bifidobacteria which have been linked to a number of health benefits both within the digestive tract and in other organs of the body. Researchers have suggested that these bacteria protect the host by competing with bacterial or fungal pathogens for available nutrients and space and modulating the immune system. In addition, it has been reported that some short chain fatty acids (SCFA) including acetic, propionic and butyric acids are released during the fermentation of the prebiotic, and can serve as an energy source for the mucosal cells (Haddadin et al., 2007).
1.2.5
Qualitative and quantitative assessment of prebiotics
Roberfroid (2007) stated that for a material to be considered to be a prebiotic it should meet terms of a qualitative assessment of particular characteristics. This assessment requires the property of non-digestibility, which includes resistance to gastric acidity, hydrolysis by mammalian enzymes and gastrointestinal absorption. In addition there is a requirement for
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capacity for fermentation by intestinal microbes, and selective stimulation of growth and/or activity of intestinal bacteria. When reviewing the existing methodologies for the qualitative assessment for classification of a prebiotic, only two dietary non-digestible oligosaccharides fulfilled all the required criteria for classification as a prebiotic. Roberfroid (2007) further proposed use of a quantitative score, termed the Prebiotic Index (PI), as a simple method to assess whether a food ingredient is a prebiotic. The PI was originally developed by Palframen et al. (2003) to assess the functionality of the material of interest. Those researchers defined the PI as the increase in bifidobacteria (expressed as the absolute number of new cfu/g of faeces) divided by the daily dose (in grams) of prebiotic ingested. Roberfroid (2007) subsequently defined the PI as [(Bif/Total) – (Bac/Total) + (Lac/Total) – (Clos/Total)], where ‘Bif’ is the number of bifidobacteria at sample time/numbers at inoculation, ‘Bac’ is the number of bacteroides at sample time/numbers at inoculation, ‘Lac’ is the number of lactobacilli at sample time /numbers at inoculation, ‘Clos’ is the number of clostridia at sample time/numbers at inoculation, and ‘Total’ is the total bacterial numbers at sample time/numbers at inoculation.
1.3
Honey as a prebiotic
Honey is of interest as a prebiotic material because it contains many oligosaccharides and low molecular weight polysaccharides likely to resist degradation by host enzymes, and thus be available as a nutrient source for the microflora in the large bowel. In a recent review of functional foods honey was listed as a source of a functional component in the class/component ‘prebiotics’ (IFIC, 2006). Various researchers have investigated the prebiotic effects of honey. Reports to date describe laboratory studies, ie in vitro tests, but no publications of human clinical studies. Using pure cultures of bacteria, Ustunol (2007) reported that US honey enhanced the growth, activity and viability of commercial strains of bifidobacteria, which are typically used as in the manufacture of fermented dairy products. The reported prebiotic effect was strain-specific. That author also reported a synergistic effect for the carbohydrate components of honey in promoting growth and activity of bifidobacteria, on the basis that tests showed honey was more effective than an artificial combination of the purified major saccharides components of honey. Another interpretation of this result is, however, that the honey tested contained additional saccharides that were more effective at promoting the growth of bifidobacteria than those tested by the author. The reported effect of honey on the growth and activity of intestinal bifidobacteria was similar to that of commercial oligosaccharides (FOS, GOS, and inulin). This research provided promising results with respect to the growth-promoting and prebiotic activity of honey on bifidobacteria, but did not include a study of the effects on lessdesirable intestinal bacteria, nor attempt to quantify the prebiotic potential. Consequently, additional data are required before the anticipated benefits identified in this study can be reliably estimated. The effect of American honeys from sourwood, alfalfa and sage on the growth and activity of five strains of human intestinal bifidobacteria was studied by Shin at al. (2005). All three honeys enhanced growth and activity of the five bacterial species. In addition, the bifidobacteria inhibited the growth of some intestinal microflora. Haddadin et al. (2007) investigated the effect of three different honeys from Jordan on the growth and SCFA secretions of two intestinal bacteria, Bifidobacterium infantis and Lactobacillus acidophilus, and found that all three honeys increased cell counts and levels of SCFA production. In addition, they found that different strains of bifidobacteria responded in a specific way to the addition of a given type of honey to the growth medium.
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It has also been established that different honeys contain source-specific oligosaccharides. For example, Weston and Brocklebank (1999) showed that a New Zealand honey contained isomaltose and melezitose, while others reported the presence of raffinose in Italian honey (Oddo et al., 1995). In order to apply the results from laboratory studies to quantify the prebiotic potential of honey, Sanz et al. (2005) studied the effect of honey oligosaccharides upon the growth of faecal bacteria. Before the oligosaccharides were added to cultures of the faecal bacteria, the monosaccharides in the honey (which included the glucose and fructose components, were removed to avoid any influence on bacterial populations in the fermentations. This procedure simulated what would have happened in the body, where the monosaccharides are digested and absorbed in the small intestine and only the oligosaccharides pass into the large intestine to serve as a potential food source for the indigenous bacteria. Monosaccharides were removed by three different methods: nanofiltration, yeast treatment (Saccharomyces cerevisiae) and absorption onto activated charcoal. The researchers used the Prebiotic Index (PI) to compare the growth of beneficial faecal bacteria including bifidobacteria, lactobacilli and eubacteria, as well as of less desirable ones such as clostridia and bacteroides. The honey-derived samples were also compared to fructooligosaccharide (FOS), a commercial prebiotic). FOS was found to have the highest PI value (6.89); the three honey-derived oligosaccharide fractions returned similar PI values, with the highest being for the charcoal fraction (4.24), which contained the greatest oligosaccharide content. In addition, levels of the short chain fatty acids (SCFA) lactic acid and acetic acid were measured. Natural honey, FOS and the charcoal-derived honey oligosaccharides showed the highest lactic acid values, while the charcoal fraction showed the highest acetic acid value. Those workers concluded that the tested honeys contained oligosaccharides which would function well as prebiotics.
1.4 1.4.1
Characteristics of honey Description of honey
Honey is the natural sweet substance produced by honeybees from the nectar of blossoms or from the excretions of plant-sucking insects living on parts of plants, which the honeybees collect, transform by combining with specific substances of their own, store and leave in the honeycomb to ripen and mature (Codex, 2001). Honey is a complex mixture of substances, and different samples present great variations in composition and characteristics on the basis of their geographical and botanical origins. Honey's main features depend on the floral origin or the nectar source foraged by the honeybees. The composition and quality of honey also depends on environmental and other factors associated with production, such as weather, humidity inside the hive, nectar conditions, and treatment of honey during extraction and storage (Tchoumboue et al., 2007). Honey consists essentially of different sugars, predominantly fructose and glucose, as well as other substances such as organic acids, enzymes and solid particles derived from the process of honey collection. The colour of honey varies from nearly colourless to dark brown. The consistency can be fluid, viscous or partly to entirely crystallised. The flavour and aroma vary, but are considered to be derived from the plant origin (Australian Food Standards, 2000; Codex, 2001). The Australian honey industry has developed specifications to allow honey packers to use standard terms to describe the qualities of honey (see, e.g., Wescobee, 2000; Capilano, 2008), as presented in Appendix A.
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1.4.2
Physical characteristics of honey
1.4.2.1
Acidity and pH
Honey typically has a pH in the range of 3.3–5.6. The natural acidity of honey inhibits growth of many pathogenic bacteria whose minimum tolerated pH is in the range of 4.0–4.5. 1.4.2.2
Colour and flavour
The colour of honey varies from clear and colourless (like water) to dark amber or black. Colour varies with the product’s botanical origin, age and storage conditions. Less common honey colours include bright yellow (sunflower), reddish undertones (chestnut), greyish (eucalyptus) and greenish (honeydew). Once crystallised, honey turns lighter in colour because the glucose crystals are white. Honey colours in Australia are given in units of millimetres on a ‘Pfund’ scale, which is an optical density reading used in international honey trade (Krell, 1996). Variations in honey flavours reflect the local Australian flora. Honey flavours, colours and uses of some important Australian honeys are described below:
1.4.2.3
Yellow Box: a mellow flavour with an attractive golden colour, suitable for a wide range of uses including sweetening of tea or coffee. Slow to granulate. Leatherwood: a strongly flavoured honey, deep golden in colour with thick consistency. Red Gum: a stronger flavoured honey with darker, dense-golden colour. Ironbark: mild in flavour and light in colour; used for baking and as a sweetener in beverages. Blue Gum: a milder honey, similar in taste to ironbark honey, usually straw-coloured and dense consistency, with an uplifting flavour; can be used as a sweetener in tea or coffee. Stringybark: a darker honey, often used for glazes and marinades. White Clover: a light-coloured, mild-flavoured honey suitable for many uses. Yapunyah: a pale and clear honey with a delicate aroma. Crystallisation
Honey can present in a semi-solid state known as crystallised honey. This can occur naturally when glucose spontaneously precipitates out of a super-saturated honey solution. This supersaturated state occurs because of the high concentration of sugars (greater than 70%) relative to the water content (often less than 20%). Glucose tends to precipitate out of the solution, and the solution then reverts to the more stable saturated state (American National Honey Board, 2007a). Deliberate, controlled crystallisation of honey can produce creamed honey. Creamed honey is made by a technique called “seeding”, in which a small quantity of already crystallised honey is added to a liquid honey blend. The addition of the crystallised honey to the liquid honey accelerates the natural tendency of the liquid honey to crystallise. The seeded honey is then packed into jars, and held in controlled storage conditions to allow the honey to fully crystallise before being quality checked and released for sale (Capilano, 2008). Crystallisation of honey can be a serious problem for processing since it limits the flow of unprocessed honey out of storage containers. In industry settings crystallised honey is heated, but during heating hydroxylmethylfurfural (HMF) is formed. HMF is a major honey quality factor and is an indicator of honey freshness and exposure to heating. In fresh honeys there is a minimal amount of HMF but levels increase upon storage, depending on the pH of honey and on the storage temperature. Honey is sometimes also heat treated to prevent unwanted
6
fermentation by osmophilic yeasts. A common method of treatment is heating to 71oC for four minutes, followed by rapid cooling through a heat exchange unit. The colour, flavour and aroma of honey may be altered by heat treatments. 1.4.2.4
Moisture
Australian honeys contain 15–19% moisture, so that their water activity (Aw) is low (0.5–0.6), and there is very little moisture to support the growth of bacteria and yeast. The moisture level of honey is a critical factor, since it affects the capacity for storage. Honeys with greater than 19% moisture may support growth of micro-organisms, which can ferment and spoil the honey (American National Honey Board, 2007a). The moisture level of honey is measured as the soluble solids, and is determined by measuring the refractive index with a refractometer. 1.4.2.5
Viscosity
Freshly extracted honey is a viscous fluid. Its viscosity depends on a large variety of factors, and varies with its composition and water content. Viscosity is an important technical parameter for honey processing, because viscosity affects honey flow during extraction, pumping, settling, filtration, mixing and bottling. Raising the temperature of honey lowers the viscosity. This phenomenon is exploited during industrial honey processing (Krell, 1996).
1.4.3
Composition of honey
1.4.3.1
Carbohydrates
Honey is a supersaturated sugar solution with approximately 17% water. Fructose is the predominant sugar, with concentrations ranging from 36-50%, followed by glucose (28–36%). Carbohydrate composition and content of honey is variable and is dependant on the floral source of the honey. The carbohydrate composition of honey has been extensively studied using techniques including gas chromatography and high performance liquid chromatography. It has, however, been difficult to account for all carbohydrates in honey because of the lack of commercial standards and the low amounts of some of the compounds in honey (Morales et al., 2006). Disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides and other oligosaccharides are present in much smaller quantities than fructose and glucose, (American National Honey Board, 2007a). Morales et al. (2007) reported that 90% of the carbohydrates in honey are the monosacccharides glucose and fructose, and that the remaining carbohydrates comprise some 16 disaccarides and 8-12 trisaccharides. Darcy et al. (1999) reported that the disaccharides in honey include sucrose, maltose, isomaltose, nigerose, turanose, maltulose, leucrose, kojibiose, neotrehalase, gentiobiose, laminaribiose and isomaltulose. Morales et al. (2007) reported that New Zealand honeydew honeys contain three tetrasaccharides (maltotetraose, alpha-panasylD-fructofuranoside and alpha-maltosyl-D-fructofuranoside), two pentasaccharides and one hexasaccharide. Shin et al. (2005) reported that the oligosaccharides in honey are derived from the action of honeybee alpha-D-glucosidase which catalyses the transfer of alpha-Dglucopyranosyl groups from sucrose to an acceptor carbohydrate, and which thus results in formation of fructooligosaccharides and a variety of other oligosaccharides in varying amounts. Using an activated charcoal method of oligosaccharide extraction, Morales et al. (2007) found variations in the higher oliogosaccharide compositions of ten different honeys. 1.4.3.2
Enzymes
Honey contains small amounts of enzymes that are introduced into honey by bees during their honey production process. Common enzymes in honey are diastase (amylase), invertase (alpha-glucosidase) and glucose oxidase. Other enzymes such as catalase and acid phosphatase are present in lesser amounts. Glucose oxidase converts glucose in the presence of water and oxygen to gluconic acid and
7
hydrogen peroxide. The resulting acidity and hydrogen peroxide preserves and sterilises the honey during the ripening process. Full-strength honey has negligible amounts of hydrogen peroxide and active glucose oxidase (American National Honey Board, 2007a). Diastase activity is a quality factor, influenced by honey storage and heating and is thus an indicator of honey freshness. Diastase activity usually diminishes during storage (Bogdanov et al., 2001).
1.4.4
Functional aspects of honey
1.4.4.1
Preservative and antioxidant effects
Honey can act as a natural food preservative. Honey has been shown to be able to reduce enzymatic browning in fruits and vegetables, and prevent lipid oxidation in meats. The antibacterial activity of the honeys has been attributed to hydrogen peroxide generation. Various flavenoids and phytochemicals have been identified in honeys; these include caffeic acid, ferulic acid and other organic acids that may serve as sources of dietary antioxidants. The amount and type of antioxidant compounds depends upon the floral source and variety of the honey. In general, darker honeys have been shown to be higher in antioxidant content than lighter honeys (American National Honey Board, 2007b). 1.4.4.2
Health and medicinal attributes
Some honey is marketed on the basis of its medicinal properties. In the last 20 years there has been a broad resurgence of interest in the medicinal properties of honey, and, in particular, its antimicrobial and wound healing properties (American National Honey Board, 2007b). In Australia researchers from University of Sydney have found that some local honeys, such as those from the Jelly Bush (Leptospermum species), possess antibacterial and antiinflammatory properties. In 1997 Jelly Bush honey became the first and only honey currently registered as a therapeutic agent in Australia (Reeves et al., 2005). The trade name for this honey is “Medihoney”, and a number of products using this honey are registered with the Australian Therapeutic Goods Administration (TGA) for wound healing (Blair, 2008; Medihoney, 2009). Elsewhere, honey is also listed for use as an antiseptic dressing to promote healing of wounds, burns and skin ulcers, a topical antibacterial agent for the treatment of acne and other skin infections, a topical antibacterial and moisturising agent for the treatment of atopic eczema, a topical antifungal agent for the treatment of tinea, an antiseptic salve for conjunctivitis and blepharitis, an antibacterial agent and rehydrating agent for the treatment of gastroenteritis, and an antibacterial agent and healing-promoting agent for the treatment of dyspepsia and peptic ulcers (American National Honey Board, 2007b). Honey has been promoted as a healthy sweetener alternative to table sugar for persons with diabetes, on the basis that honey has a lower Glycaemic Index (GI; Stern, 1999). The GI parameter is a physiologically based measure that is used to classify carbohydrate foods according to their blood glucose-raising potential. In general, ‘high’ GI foods are those with high carbohydrate content, and are foods that are rapidly digested. Foods with a GI value of 55 or less are currently considered as being ‘low’ GI foods. Foods with a GI value of 56-69 are said to have an ‘intermediate’ or ‘moderate’ GI rating, and foods with a GI value of 70 or more are considered to be ‘high’ GI foods. Various Australian honey varieties have been shown to possess low GI values. These varieties include Yellow Box, Stringybark, Red Gum, Ironbark, and Yapunyah honeys (Arcot et al., 2005). In contrast, US-based researchers examined the GI’s of four US honeys with varying fructose/glucose ratios and reported that the average GI factor for those honeys was 72.6, with no significant differences between the tested varieties (American National Honey Board, 2007b). In 2007, the American National Honey Board summarised research in the areas of health and
8
medicinal attributes of honey; these findings are summarised in Table 1.2 of this report. Table 1.2:
Medicinal attributes of honey reported by the American National Honey Board in 2007.
Attribute Antimicrobial activity
Key Properties • • • • •
Promotion of wound healing
• • • •
•
Value in Health and Disease
Low water activity High osmotic pressure Hydrogen peroxide production High acidity Non-peroxidal components
• Management of infections
Antimicrobial activity Stimulation of immune system High viscosity and osmolarity Altered metabolism of glucose and amino acids by infecting bacteria and new tissue Antioxidant activity
•
Wound dressing for infected ulcers, surgical wounds, pressure sores and burns
such as gastritis, candidosis, tinea, wounds and opthalmologic disorders • Management of food pathogens and spoilage microorganisms • Potential anti-carcinogenicity via suppression of oral pathogens
Antioxidant activity
•
Presence of phenolic compounds, ascorbic acid, enzymes (glucose oxidase, catalase and peroxidase)
• •
Food preservation Potential protection against cellular damage from free radicals
Energy source
•
Concentrate source of fructose, glucose and other di-, tri- and oligosaccharides
• •
Athletic performance Can be used as part of a diabetic meal plan due to low GI
Prebiotic
•
Presence of a variety of oligosaccharides that can promote the growth and activity of bifidobacteria
•
Incorporation in fermented milk products with bifidobacteria to improve gastrointestinal health
9
1.5
Structure of the Australian honeybee industry
There are approximately 10,000 registered beekeepers (apiarists) in Australia, operating around 605,000 hives (RIRDC, 2007). Approximately 75% of the registered beekeepers are amateur beekeepers, most of whom own less than 11 hives. Beekeepers who own over 500 hives are termed professional beekeepers or commercial apiarists; commercial beekeepers commonly operate between 500 and 3,000 hives each (Reeves et al., 2005). In New South Wales only 4.6% of all registered beekeepers are commercial apiarists, but these beekeepers account for 54% of all hives registered in this state. Similar patterns of ownership and effort exist in all Australian states. Table 1.3 presents the % of total Australian production attributable to each State and the average production per beehive, for the period ending 30 June 2000. Table 1.3: The % of total Australian production by state and the average production per beehive in 2000 (RIRDC 2007).
State
% of national honey production
Average production per productive hive (kg)
New South Wales
41.0
77.9
Queensland
9.7
56.6
South Australia
14.0
83.7
Tasmania
4.4
80.3
Victoria
23.0
91.6
West Australia
7.5
99.6
Beekeeping activity is dependent on availability of floral sources, since bees forage for nectar to produce honey at the hive. Eucalypts are a dominant floral source for bees in Australia. Most floral sources for honey production are in the temperate areas of Australia, which includes southern Queensland, coastal New South Wales, central Victoria, coastal areas of South Australia and West Australia and parts of Tasmania. The Northern Territory has some honey floral sources but production is low. Total honey production varies year-by-year, and is influenced by factors such as prevailing weather conditions. Floods, droughts and bushfires have been reported to be gradually decreasing access of beekeepers to floral sources, and varying and increasingly strict regulatory arrangements for access to public lands have been established by the states. In addition diseases such as ‘Dieback’ have affected a number of species of eucalypt, further depleting the number of trees available as sources of nectar for bees (RIRDC, 2007). Continued land clearing, especially on privately-owned land, also reduces access of beekeepers to floral sources. Each Australian state has its own suite of floral species of particular importance to the industry in that state; dominant species are listed in Table 1.4 (Reeves et al., 2005; RIRDC, 2007; White, 2008). The Australian honeybee industry also produces a number of products other than liquid honey, including creamed honey, beeswax, queen bees, package bees, pollen, propolis, honeycomb and pollination services. These other products contribute approximately $15 million to the honeybee industry yearly. Australia has about 102,000 hives used for paid pollination, and the value of paid pollination services has been estimated to be around $3.3 million per year (Reeves et al., 2005). There has been increasing demand for these services in recent years due
10
to the increased size of the horticulture industry and changing land management practices. Stone fruit and almond growers are the largest users of pollination services, most of which are located in Victoria and South Australia.
1.6
Summary
In summary, it is apparent that the value of Australian honey could be increased by availability of data demonstrating associated health benefits beyond those already shown for therapeutic and anti-bacterial properties. It would seem that the complex saccharide composition of honey could assist in the promotion of good intestinal health and general well-being by being selectively used by beneficial bacteria in the intestine. Such improvement in microflora composition could, in turn, provide improved immune modulation at other sites in the body. Overall, therefore, consumption of honey might be expected to have potential to improve and promote general human health and well-being. Since the composition of honey varies with the floral species of origin, local climate, and procedures used for harvesting and storage, Australian honeys can be expected to be unique in character. It was therefore of interest to investigate the potential of Australian honeys for use as natural functional foods, and as sources of new food ingredients.
11
Table 1.4 Dominant floral species supporting the Australian honey industry by state
New South Wales
Victoria
Queensland
South Australia
Western Australia
Tasmania
Northern Territory
Paterson's curse
Grey Box
Blue Gum
Mallee - Coastal - White
Weeping Box
Leatherwood
Weeping Box
White Clover
Red Gum
Paperbark Tea Tree
Blue Gum
Spermacoce breviflora (herb)
Clover
Red-bud Mallee
Grey Ironbark
Red Ironbark
Brush Box
Lucerne
Red-bud Mallee
Blue Gum
Woolybutt
Box - Yellow - White - Black
Yellow Box
Yellow Box
Salvation Jane Woolybutt
Blackberry
Stringybark
Stringybark - Red - White - Yellow
Clover
Yapunyah
Tea tree
Stringybark
Paperbark
Canola
Banksia
Ironbark - Blue Top - Narrow Leaf - Gray
Canola
Paperbark - Broad leaved - Silver leaved
Salmon Gum
Coolibah
Messmate
Coolibah
Red Gum
Jarrah
Silver-leaved Paperbark
Banksia
White Mallee
Turnipweed
Clover
River Red Gum
River Red Gum
Featherbush
Yellow Stringybark Gum
Spotted Gum
Red Mallee
Ironwood
Ironwood
Gum - Spotted - Red River
Canola
Clover
Orange
Northern Grey Box
Northern Grey Box
Mugga Ironbark
Black Box
Brown Box
Almond Prunus
Red & White Mahogany
Paterson's Curse
Stringybark
Brown Stringybark
Pilliga and Mallee Box
Tea Tree Hill Gum (Melaleuca & Leptospermum)
Bloodwood Corymbia
Red Stringy bark
White Mahogany Hill/Pink Gum
Yapunyah
Blue gum
Grey Box
Manna gum
Caley's Ironbark
Broad-leaved Paperbark
Grey Box
Sugar Gum
12
2.
Objectives
The objective of this work was to investigate Australian honey varieties for prebiotic characteristics and potential for improving human gastrointestinal health. It was considered that demonstration that Australian honeys could preferentially enhance the growth of beneficial bacteria would predict health benefits from consumption, and would be valuable for promoting the use of honey directly, and as a valuable functional food ingredient.
3.
Methodology
3.1
Approach
The study sourced honey samples from each state of Australia and measured their physical characteristics. Sample sizes were sufficient to allow all experiments to be done with the same sample batch. All honey samples were tested for the capacity to promote the growth of known benefical bacteria currently used commerically as probiotics. The effect on growth was quantified, and the five honeys which were most effective in supporting growth of beneficial species were selected for further study. The effect of these honeys on the growth of known pathogenic bacterial species was also studied. In order to simulate the inclusion of honey in the diet and its effect on indigenous microbes in the intestine, in vitro microcosms simulating intestinal conditions were used. Results from the microcosm studies were used to develop a prebiotic index (PI) estimate for each sample. Because honey contains both saccharides and dissacharides as well as longer chain oligosaccharides and polysaccharides, tests were carried out using natural honey as well as honey that had been pretreated to remove the simple sugars, leaving only the oligosaccharides and polysaccahrides. This treatment effectively simulated the selective absorption of the saccharides and dissacharides in the human small intestine.
3.2
Honey samples
Honey samples were sourced from various small and large honey suppliers from throughout Australia. Information about the climatic conditions during honey production, as well as time and temperature of extraction and storage of the honey was obtained and recorded. All honey samples were stored in sealed containers at ambient temperature (20-25oC). The sample colour was recorded and quantified using the Pfund scale of optical density, and water activity of all honeys was measured. In addition, the pH values of the five honeys selected for further study were measured using pH paper.
3.3
Honey oligosaccharides
Oligosaccharides in honey were obtained from the honeys using an activated charcoal treatment (Sanz et al., 2005) and filter-sterilized prior to use as a sole carbohydrate source in the growth medium at 5% (final concentration).
13
3.4
Bacterial cultures and incubation conditions
Pure cultures of the following Bifidobacterium and Lactobacillus species/strains were obtained from the University of New South Wales (UNSW) CMB culture collection for probiotics: Lactobacillus acidophilus L10, Lactobacillus paracasei L26, Bifidobacterium lactis C10, Lactobacillus rhamnosus C20, and Lactobacillus plantarum C29. Working stocks of the cultures were prepared in MRS broth (for lactobacilli) or TryptoneYeast extract broth (for bifidobacteria) from freeze dried powder, and stocks were stored with 30% glycerol at -20oC. Experiments used freshly grown cells prepared by initially growing a primary culture from the working stock, and then a secondary culture to ensure satisfactory resuscitation. A new secondary stock was prepared for each independent experiment. Pure culture of bacterial pathogens were obtained from UNSW stock cultures. Bacteroides fragilis and Clostridium perfringens were grown in Wilkins-Chalgren Anaerobe (WCA) broth with added 3% glucose. Cultures of Enterococcus faecalis and E. coli were grown in nutrient base (NB) broth with added glucose (5%). All pathogens were grown in an anaerobic chamber at 37°C for 24 hours.
3.5
Growth studies with pure cultures
Growth studies were carried out using nutrient basal medium without carbohydrate prepared according to Sanz et al. (2005) and supplemented with 5% final concentration honey. Honey solutions were prepared and filter-sterilized according to Haddadin et al. (2007). The Lactobacillus and Bifidobacterium pure cultures were inoculated to give a concentration of log 7 cells per ml (cfu/ml). Growth was monitored by measuring optical density (OD 610nm). Probiotic cultures were prepared anaerobically using an anaerobe chamber. Subsequently, the growth of selected probiotic strains was studied in medium with added oligosaccharides from the various honey samples instead of complete (‘natural’) honey. Controls for the growth studies included glucose, fructose and sucrose used at 5% concentration. The honey samples that showed the greatest enhancement of growth of probiotic cultures were chosen for testing for their effects on pathogenic species. Honey solutions were prepared and added at 5% in the growth media (WCA or NB) as the sole carbohydrate source. The cultures were grown at 37°C in an anaerobic chamber for 24 hours. The growth of pathogens in the presence of glucose or honey was expressed as the optical density (OD) of the culture, as measured with a spectrophotometer at a wavelength 610 nm. All growth tests were done in duplicate.
3.6
Measurement of prebiotic index in intestinal microcosms
In order to study what happens to the honey when ingested, intestinal microcosms were prepared according to the method of Rang et al. (1996). These workers have reported good correlation between results using this type of microcosm and those obtained from in vivo studies in mice. In the current project, human faecal samples were used to assess the prebiotic index of honey using the microcosms. The microcosms were established using freshly voided human faecal cultures diluted with WCA broth containing the honey sample, and incubated anaerobically at 37oC. Routine samples were taken for enumeration of the major bacterial groups using selective media and plate count techniques (Sanz et al., 2005). The prebiotic index was calculated according to the method of Palframen et al. (2003), thus allowing characteristics of individual honey samples to be compared quantitatively. Control cultures used glucose, fructose or sucrose as added
14
nutrient sources. Microcosms were also established using honey oligosaccharides prepared according to Sanz et al. (2005), and results were compared to control cultures in which inulin was used as the added nutrient source. A negative control with no added saccharides was also included. After quantification of the major bacterial groups, the prebiotic index was calculated. The findings reported here represent the average of duplicate growth assays.
4.
Results
4.1
Physical characteristics of honey samples
The sources and floral types of origin of the 18 honey samples in the study are listed in Table 4.1, together with their recorded physical characteristics. Sample colour was qualitatively assessed, and optical density was also measured to provide a Pfund value. Sample pH values were measured for honeys selected for more detailed examination. The water activity of all honeys was observed to be low, in the range 0.25 to 0.42 (data not shown).
4.2
Effect of honey on growth of probiotic bacteria
4.2.1
Comparison of honeys with glucose and fructose
Honeys were used as a sole carbohydrate source for growing probiotic species of bacteria, and their nutritive capabilities were compared to those of glucose and fructose. Six honeys effectively promoted growth of all of the probiotic species tested. Those honeys were:
Honey 2:
Leatherwood from Tasmania;
Honey 5:
Banksia from NSW;
Honey 7:
Bees Creek Woolybutt from South Darwin, NT;
Honey 9:
Grey Ironbark from NSW;
Honey 14: Mugga Ironbark from NSW, and
Honey 17: Yellow Stringybark from Victoria.
As shown in Table 4.2, there was substantial variation between the nutritive capacities of the various honey samples for the different probiotic cultures tested (Table 4.2). Overall, honey H14 showed a good prebiotic effect for four probiotic cultures, namely, Bifidobacterium lactis, Lactobacillus rhamnosus, L. plantarum and L. acidophilus, but not for L. paracasei. Honey H2 was the next best, showing good prebiotic effects on 3 probiotic cultures, namely, L. rhamnosus, L. acidophilus and L. paracasei. Honeys H5, H7, H9 and H17 showed prebiotic effect on at least one probiotic culture. In contrast Honey H15 performed approximately only as well as glucose for L. rhamnosus, L. plantarum and L. acidophilus. Honey 11 also performed poorly.
15
Table 4.1
Physical characteristics of the various honey samples tested.
Sample No
Floral Variety
State
Pfund Value (OD)
Colour
1
Yapunyah
Qld
22
extra light amber
2
Leatherwood
Tas
84
light amber
3
Lucerne
SA
66
amber
4
Yellow Box
NSW
36
extra light amber
5
Banksia
NSW
110
dark amber
6
Red Gum
WA
70
amber
7
Woolybutt
NT
91
medium amber
8
Meadow
Tas
57
light amber
9
Ironbark
NSW
32
extra light amber
10
Jarrah
WA
107
dark amber
11
Featherbush
NSW
125
dark amber
12
Coolabah (commercial)
NSW
45
golden
13
Black Box (commercial)
NSW
59
light amber
3.82
Mugga Ironbark NSW (2 years old, commercial)
57
light amber
3.78
14
pH
3.80 3.77
3.68 3.87
15
Yellow Box
Vic
32
extra light amber
16
Ironbark
Vic
66
amber
17
Yellow Stringybark
Vic
77
amber
3.76
18
Canola
NSW
63
light amber
4.04
16
Figure 4.1
Comparative growth of beneficial bacteria in the presence of honey. Growth in the presence of honey (Samples H1-H17) is compared to growth with single sugar sources (Glu, Fru) or in cultures with no added carbohydrate (NB).
Prebiotic Effect of Australia Honeys on the Growth of Probiotic Cultures Bifidobacterium lactislactis (pink(pink bar);bar); Lactobacillus rhamnosus (yellow); L. plantarum (blue); L. acidophilus (red);(red); Bifidobacterium Lactobacillus rhamnosus (yellow); L. plantarum (blue); L. acidophilus L. paracasei (green). NB: no added sugars; Glu: added glucose; Fru: added fructose; H1-H17: honeys (see(see Table 4.1). L. paracasei (green). NB: no added sugars; Glu: added glucose; Fru: added fructose; H1-H17: honeys Table 4.1).
17
O p tical D en sity (610n m )
DR10
DR20
299v
L10
L26
0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 NB Glu Fru H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17
Carbohydrate Sources
Table 4.2
Growth of probiotic cultures in honey or glucose.
_____________________________________________________________ Probiotic species
Growth increase with honey as compared to glucose
_____________________________________________________________ Bifidobacterium infantis
1.00 to 2.75-fold
Lactobacillus acidophilus
1.00 to 1.90-fold
Lactobacillus rhamnosus
1.36 to 2.50-fold
Lactobacillus paracasei
1.60 to 4.15-fold
Lactobacillus plantarum
1.30 to 2.50-fold
______________________________________________________________
4.2.2
Comparison of honey with sucrose and a commercial prebiotic
To further explore the nutritive capabilities of natural honey as compared to other carbohydrate sources, two of the probiotic species (Bifidobacterium lactis and Lactobacillus plantarum) were grown in broth containing 5% of either sucrose or inulin (a commercially available prebiotic) or honey. Broth with no added sugar was used as the control. As shown in Figure 4.2, each honey promoted growth of the two prebiotic species to a greater degree than either sucrose or inulin.
18
Figure 4.2
Growth of Bifidobacterium lactis and Lactobacillus plantarum in broth with honey, sucrose, inulin or no added carbohydrate.
Bifidobacterium
L.plantarum
1.2
O D (610nm )
1 0.8 0.6 0.4 0.2 0 No sugar
Sucrose
Inulin
Honey 2
Honey 5
Honey 7
Honey 9 Honey 14
Growth is expressed as optical density (OD) at 610 nm. Honeys tested are described in Table 4.1.
19
4.3
Effect of honey on growth of pathogenic bacteria
Cultures of four species of pathogenic bacteria were grown in media containing honey or glucose for 24 hours at 37°C in an anaerobic chamber. Growth was determined by optical densitometry at a wavelength of 610 nm. The results showed that the presence of all tested honeys could suppress by 2.6-fold the growth of Bacteroides fragilis and by 1.3-fold the growth of Clostridium perfringens (Figure 4.3). In contrast, most of the honeys seemed to stimulate the growth of E. coli and Enterococcus faecalis, especially Honey 5, which increased the growth by 1.6-fold for E. coli and 2.6-fold for Enterococcus faecalis, as compared to glucose (Figure 4.3) when in pure culture. It is interesting to note that the honeys inhibited the growth of the anaerobic bacteria but not the aerobic coliforms and enterococci. It was therefore considered important to also test the effect of honeys on mixtures of beneficial and potentially pathogenic bacteria, as found in the intestine. Such studies are reported in Section 4.5.2. Figure 4.3 Growth of pathogenic bacterial species using honey as the sole carbohydrate source.
Optical Density (610nm)
Prebiotic effect of Australia Honeys on the growth of Bacteroides and Clostridia No sugar Glucose Honey 2 Honey 5 Honey 7 Honey 9 Honey 14 Honey 17
1 0.8 0.6 0.4 0.2 0 Bacteroides
Clostridia
Prebiotic effect of Australia Honeys on the growth of E.coli and Enterococci Optical Density (610nm)
No sugar 1.4
Glucose
1.2
Honey 2
1
Honey 5
0.8
Honey 7
0.6 0.4
Honey 9
0.2
Honey 14
0
Honey 17 E.coli
Enterococci
20
4.4
Effect of honey oligosaccharides on growth of probiotic bacteria
Oligosaccharides extracted from high-performing honey samples (H2, 5, 7, 9, 14 and 17; see Section 4.2.1) were tested as sole carbohydrate sources for growing probiotic cultures. Their effects were compared against growth in carbohydrate-free medium (‘C’: negative control), and cultures containing added glucose (Glu) and fructose (Fru). (It should be noted, however, that orally-ingested glucose, fructose or sucrose will be absorbed in the small intestine, so that no additional carbohydrate will reach the microbes in the large intestine. Hence the most appropriate comparison of the effects of the oligosaccharides is with the negative control culture, i.e., no added carbohydrate). All available species of probiotic bacteria were tested, and shown to be able to utilise the honey-derived oligosaccharides. As shown in Figure 4.4, when compared to the negative control, growth of Lactobacillus acidophilus was substantially promoted by the honey-derived components. All other prebiotic species showed similar growth profiles to that of L. acidophilus. Figure 4.4 Effect of honey oligosaccharides on growth of L. acidophilus. 0.3
0.25
0.2
0.15
OD 0.1
0.05
0 C
Glu
Fru
H2
H5
H7
H9
H14
4.5
The prebiotic potential of honey measured using intestinal microcosms
4.5.1
Effect of natural honeys on levels of potentially ‘good’ and ‘bad’ bacteria.
Intestinal microcosms were used to simulate the oral ingestion of honey. Two complete (‘natural’) honeys (Honeys 4 and 5, Table 4.1) were used as the sole carbon source in the microcosms, and the growth of lactobacilli (‘good’ bacteria) and enterics (potentially ‘bad’ bacteria) that were naturally occurring in the intestinal material were enumerated after fermentation with the addition of the honey. The results for each of the honeys are presented in Figure 4.5, and compared to a negative control (no added sugar) and positive controls (added glucose or fructose). The results were in general agreement with the levels of glucose and fructose known to occur in the honeys, with the Yellow Box honey (‘Honey A’; known to be high in glucose) stimulating growth of coliforms (as for the added-glucose control), and the banksia honey (‘Honey B’; known to be high in fructose) promoting the growth of lactobacilli,
21
as for the added-fructose control. The glucose and fructose levels of Honeys 4 and 5 were supplied with the honey (B. White, pers. comm..). Figure 4.5 Effect of honey on growth of lactobacilli and coliform bacteria in intestinal microcosms.
6
2 1.8
5
1.6 1.4
4
1.2 3
1 0.8
2
0.6 0.4
1
0.2 0
0 No sugar
Fructose
Glucose
Honey A
Honey B
Fructose
Lactobacilli (cfu/ml x 108)
Glucose
Honey A
Honey B
Coliforms (cfu/ml x 106)
Results are expressed as colony forming units (cfu) per ml. Honey A, Yellow Box; Honey B, Banksia.
4.5.2
Prebiotic index of honeys and honey oligosaccharides
The prebiotic effects of different honey samples were compared to inulin and sucrose (5% final concentration) using intestinal microcosms inoculated with human faecal material, and hence containing the full complex microbial community of the human intestine. Results from these studies were expressed as a Prebiotic Index (PI) for each substance (Figure 4.6). PI values were calculated on the basis of population numbers of bifidobacteria, clostridia, total lactobacilli and total anaerobes, enumerated before and after growth at 37oC under anaerobic conditions, using the method of Palframen et al. (2003). All tested honeys showed higher PI values than either inulin or sucrose. It is noted, however, that it is possible that this outcome reflects a synergistic effect of the simple and complex sugars present in the honey, which differs from the test conditions for sugar or inulin alone. Honey-derived oligosaccharides prepared by removing the simple sugars from honey samples H7 and H9 were also tested in the microcosms, and compared with inulin and sucrose used at the same total carbohydrate concentration. As shown in Figure 4.7, the oligosaccharide preparations derived from the two honeys had a PI value almost as high as for inulin alone. This study demonstrated that at least some Australian honeys possess prebiotic potential, since the growth of beneficial bacteria such as lactobacilli and bifidobacteria could be promoted when honey was added as the sole carbohydrate source. It was also shown that the complex sugars in honey alone could also promote the growth of beneficial bacteria in microcosm simulating the human large intestine.
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Figure 4.6 The prebiotic index (PI) of natural honeys (see Table 4.1), inulin and sucrose, as measured in a simulated intestinal microcosm.
PI index 450 400 350 300 250 200 150 100 50 0 Inulin
Sucrose
No sugar
Honey 2
Honey 5
Honey 7
Honey 9
Honey 14
‘No sugar’: negative control Figure 4.7 The prebiotic index (PI) of honey-derived oligosaccharides (see Table 4.1), inulin and sucrose, as measured in a simulated intestinal microcosm.
PI index 50 40 30 20 10 0 Inulin
No sugar
Oligosaccharides from honey 7
‘No sugar’: negative control
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Oligosaccharides from honey 9
5.
Implications
Evidence that some Australian honeys have prebiotic capabilities presents an important market opportunity for Australian honey industry. The market value of prebiotics for food and beverages for Europe in 2008 was €295.5 million, which is far in excess of previous predictions (Frost 2009). In 2003, the prebiotic market was valued at €87 million and predicted to reach €179.7 million by 2010 (Confectionary News, 2003). At that time market size was increasing at a rate of 9.7% per annum. It is now predicted that by 2015 the market will be valued reach €766.9 million in Europe alone, with overall volumes of 204,895 tonnes and an overall compound growth rate of 14% per year. In anticipation of this growth, a major supplier of prebiotics invested €165 million in 2004 in a second plant for production of prebiotics (O’Rafti, 2004). Currently, honey competes with sugar (largely sucrose) for use as a sweetener in the food industry. It is known that simple sugars (such as sucrose) are rapidly absorbed in the human small intestine, and are therefore not available to the microbes in the lower intestine. In contrast, honey contains both simple and complex sugars, the latter of which are not degraded by host enzymes or absorbed in the small intestine, and which are thus available to microbial populations in the large intestine. Work presented here has demonstrated that complex sugars in honey can function as prebiotic substances, and potentially enhance human health and general well-being. Honey represents one of the few naturally available prebiotic substances, requiring no further purification or processing before use. At present the Australian honeybee industry produces about 30,000 tonnes of honey per year. This production contributes an estimated gross value of production of around $75-80 million per year. Based on current prebiotic sales in Europe, honeys with proven prebiotic capacity could attain prices considerably higher than the current market price for honey. Since the composition of each different honey is influenced by factors such as the floral species of origin, and local environmental factors and processing procedures, Australian honeys can be considered to be unique. While on-going studies will provide further evidence of the prebiotic capacities of Australian honeys, the results provided here demonstrate that when the complex sugars in Australian honey are delivered to intestinal microbes, beneficial species can be selectively advantaged. The microbial profiles from such trials are consistent with those observed to be associated with health benefits in human clinical studies. While previous research has shown that beneficial microbes in pure culture can utilise natural honey, studies described here have extended that work to demonstrate an effect on a complex microbial population, as found in the human large intestine. In addition, these findings show that the complex sugars in Australian honeys confer benefits which are not realised from simple sugars such as sucrose. This finding suggests that healthier confectionary could be developed through use of honey rather than sucrose, as a sweetening agent.
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6.
Recommendations
The completion of in vivo studies to complement the current in vitro work will provide additional evidence of the functionality of Australian honeys as prebiotics, and provide support for their use as functional foods, and as a healthy sweetener alternative. Because the composition of honey is variable, and influenced by many factors such as the floral species of origin, the local environment, and the honey processing and storage procedures, additional investigations should include an analysis of the impact of the role(s) of these factors on the prebiotic capacity of honeys. Potentially one could envisage the emergence of designer honeys targeting specific applications by blending various floral varieties. In addition, efforts should be made to identify the specific complex sugars that contribute to the prebiotic capacities of honey, and to foster the development of rapid and reliable assays to demonstrate their presence and quantify their relative concentrations.
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Appendix Appendix A 1.
Physical characteristics Appearance
Honey is a viscous liquid, clear to slightly turbid and free from sediment and crystallisation.
Aroma
Honey has a pleasant characteristic aroma, free from foreign odours.
Flavour
Honey has a pleasant, characteristic, sweet flavour, sufficiently strong but not distinctive.
Colour
Honey colour varies and is determined by the Pfund Scale. This is an optical density reading used in international honey trade. Colours are as follow:
Freezing Point
2.
Specifications of honey
Colour
Pfund Scale
White Extra Light Amber (or Golden) Light Amber (or Amber) Medium Amber (or Dark)
0 – 34 mm 35 – 50 mm 51 – 85 mm 86–100 mm
(15% soln)
1.42oC – 1.53oC
(68% soln)
-5.78oC
Moisture
15 – 19%, or not more than 20%
Specific Gravity (‘density’)
1.423
Specific Heat
2.26 kJ/(kg.K) (17% moisture at 200C)
Thermal Conductivity
0.536 W/(m.K) (17% moisture at 210C)
Viscosity
70 – 175 Poise
Water Activity (Aw)
0.5 – 0.6
(17% moisture at 200C)
(17% moisture 250C)
Composition Apparent Reducing Sugar
not less than 65%
Apparent Sucrose
5 - 15% depending on floral source
Sugars
Nitrogen
Fructose Glucose Sucrose 0.05 – 0.38%
Ash (mineral content)
0.04 – 0.6%
36 – 50% 28 – 36% 0.8 – 5%
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3.
pH
3.3 – 5.6
Acid
0.57% (mainly gluconic)
Free Acid
9 – 40 meqiv/kg
Water Insoluble Solids
