WO2016038348A1

WO2016038348A1 - A method of analysing the microscopic content of honey

Info

Publication number: WO2016038348A1
Application number: PCT/GB2015/052583
Authority: WO
Inventors: Jonah Chitolie
Original assignee: Geotechniquesresearch Limited
Priority date: 2014-09-08
Filing date: 2015-09-08
Publication date: 2016-03-17
Also published as: GB2564514A; GB201805634D0; WO2017042568A1; GB201415818D0; GB2529893A

Abstract

A method of analysing the microscopic content of honey and comprising subjecting honey sample to a filtration process to separate therefrom the pollen an microorganisms therein, thereafter determining the identity and quantity of the chytrids and the pollen.

Description

A method of analysing the microscopic content of honey Field of the Invention

The present invention relates to an apparatus and method for the content determination and quantification and quality control of honey.

Background to the Invention

It is of course well known that bees collect pollen while also collecting nectar, and that their transfer of pollen to other plants in the same species serves the purposes of plant propagation. It has more recently been realized that the bees obtain their nutrients from the pollen load they bring back to their h ive. It has been thought that in order to consume the pollen the bees break down its outer wall (sporopollenin) mechanically.

There is an international standard tech nique used to extract pollen from honey, namely Acetolysis. In th is technique concentrated Acetic and Sulphuric acids and Acetic anhyd ride are used . The technique has accordingly to be carried out by an experienced operator in a laboratory fume cupboard. It has however been found by comparative study that Acetolysis destroys some fungal spores and pollen undergoing breakdown .

In an alternative method zinc bromide is used to concentrate the pollen which then floats to the surface of the solution, where they can be manually removed . However zinc bromide is h ighly toxic and requires safe disposal. Another known method for separating pollen from honey is centrifuging, for which a sample of honey is first diluted with water. This risks damaging the pollen quite apart from being to some extent uncertain. In particular the use of a centrifuge leads to a non-detection of bisaccate pollen, derived from conifers and which now appears to be important in bee nutrition.

South Korean Patent Specification 101289352 describes a filter unit for removing beeswax from honey, the unit comprising a bank of filters in successive order.

There is accordingly a need for a process for accurate identification and quantification of organisms in a honey sample, preferably a process which can be carried out in the field and safely.

In articles in the British Journal "Beecraft" for February, March, April and May 2014 the present inventor has set out hypotheses related to bee nutrition and health. The present invention is the result of further research.

Summary of the Invention

The present inventor has now discovered that bees rely upon a group of fungi known as Chytrids to breakdown the outer and inner walls of pollen grains. Chytrids are ubiquitous and may be collected during foraging for pollen and plant sugars. This is a particular interesting. This is a particularly interesting discovery since many apiarists try to ensure that their hives are free of fungi.

It has also now been observed that bees collect the pollen from certain herbal remedy plants, such for example as St John's Wort (Hypericum perforatum) which contains Hypericin which appears to confer health benefits upon the bees. It has further now been noted that certain pollens are beneficial to the immune system of bees and, it is believed, to that of humans also.

It is also well known to ensure that bees visit certain plants, for example by placing their hives where particular plants are grown, so that the resulting honey is sold as flavoured, for example by lavender or pine, so far as is known substantially exclusively.

Speculation concerning adulteration of honey with syrup is an issue which occasionally arises. Accordingly there has arisen a need to be able to determine whether a container, for example a jar of honey has been correctly labelled.

According to the present invention, in order to ensure both thriving bees and honeys which confer particular nutritional and/or medicinal benefit to human consumers a honey sample is subjected to a filtration process to separate therefrom types of pollen therein, thereafter determining the identity and quantity of different types of pollen.

The process may also separate microbiota associated with pollen from the honey. Microbiota is found in the bee gut, in pollen stores and in honey itself.

The results of this analysis enable the assurance, by appropriate planting or hive placement, of bee health and also the selection of honeys which contain specific nutrients and or medicinal elements suitable for human preference, health and consumption.

In the filtration of pollen from a honey sample a nylon filter may be used. Such a filter readily concentrates allows a sample for microscope analysis. Typically, pollen in the UK with which we may be interested are sized 15 to 35 microns and the ideal filter size is 10 microns. In the tropics the pollen of interest are usually smaller and a filter size of 5 microns is advised. Pondlife pollen is apt to be considerably larger and a filter size of 30 microns is advised. Such a filter has been found to damage neither pollen nor chytrids.

To effect filtration the honey may be diluted with water, preferably deionized or distilled. 200ml to 250ml of water may be added per 5ml to 10ml of honey.

Preferably 200ml of water is added per 10ml of honey. The honey may be at a temperature of from 20°C to 26°C. At lower temperatures the honey is more viscous and difficult to handle. Preferably the temperature of the honey is 20°C. The water may be at a temperature of from 30°C to 38°C. Preferably the temperature of the water is 35°C. Particularly for field use a filter apparatus may be provided comprising a filter holder arranged for holding, removably, a filter, and a feeder portion constructed to permit a quantity of honey to be poured therein onto the filter. Preferably the apparatus has a vessel constructed for the retention of filtrate in a filtrate chamber and a cap so that it can be supplied to an apiarist and stored clean inside. The apparatus may be part of a kit including a pack of disposable filters, a holder for storing a loaded filter for sending to a laboratory or for mounting material pipetted from the filter in a slide with an appropriate holder for conveying to a laboratory. In the latter instance the kit may include a suitable pipette. Vibration means may be included for hastening and ensuring complete filtration. In the case of sending a loaded filter to a laboratory it may be preferred to cover the loaded filter with for cellophane or clingfilm as quickly after the filtration as possible to avoid atmospheric contamination. Thus there can be a kit which contains all that is needed to extract pollen from honey bee products namely pollen loads, pollen stores and honey. The kit may include the filter apparatus, filters, stain, slides and coverslips and miscellaneous items and instructions required to process honey bee products to producing microscopic slides containing pollen and other microscopic organisms. Comparing pollen found with illustrations of pollen from guide books or a website can lead the user to identify plants which honey bees are foraging from and are important to the nutrition of bee colonies.

It will be appreciated that such a kit may constitute a "citizen apparatus" suitable for use in schools and colleges and not mere for the analysis of honey.

The pollen determination and quantification step may also be effected electronically.

Data obtained from individuals and groups can be collated from different geographical areas giving an overall picture to the distribution of honey bees in relation to plant habitats. This can contribute to understanding issues concerning bee decline in some areas. Alternatively, and particularly in the context of quality control it may be preferred that the apiarist is simply supplied with sealable packs, preferably with labels upon which can be written origin and date, even sample tubes such as are employed in medicine, wherewith a honey sample may be sent to a central laboratory.

The apparatus may thus comprise a removable perforated support shelf which functions as a support for the filter, the outer edge when compressed against the ledge of the base forming an effective seal to prevent loss of microscopic particles. It may also support the filter and prevent the filter material from being stretched by the mass of liquid inside the apparatus. The perforated shelf is also preferably rigid so that when agitated or vibrated by, for example, an electric toothbrush motor the microscopic particles are kept in suspension and the filter prevented from blockage thus maximising the filtration efficiency of the apparatus. The filter holder may comprise a somewhat rigid divider in the form of a holed region bounded by a sealing portion whereby a seal can be formed between the filtrate chamber and the feeder portion. The filtrate chamber may accordingly have a ledge constructed so that the filter holder and the filter are mountable thereon. The feeder portion may be constructed to screw into the filtrate chamber and thus also to retain the filter holder and filter in place. The ledge may have a land at the outer

circumference thereof, the land having a depth equal to that of the filter holder plus the filter, plus any sealing washer provided. The filter holder and filter are just smaller in diameter than the inner diameter of the land, the land being arranged so that the feeder member can abut thereon and not stress the filter.

It will be appreciated that leakage of liquid around the filter can render the results unreliable. Typical filters having pore sizes appropriate to the present invention are quite flimsy and standard methods for holding them to a feeder holder risk them being folded, thus providing an unwanted filter by-pass path.

The filter apparatus as such is preferably arranged to be capable of cleaning, possibly sterylising, for reuse.

The method of separating pollen and other organisms from honey may accordingly comprise:

i) diluting the honey with water as set out above;

ii) pouring the diluted honey into a filter apparatus of the type described above; and

iii) recovering pollen and other matter, including micro-organisms, from the filter. As set out above the filter residue may be lifted, for example by pipetting, from the filter and transferred to a microscope slide to enable analysis of the residue.

The results enable the determination of appropriate environments for bee hives and, by repetition of the process, quality control of the honey and bee health. The following uses for the invention have been identified!

1. Pollen load analysis

Using the technique of the present invention pollen can also be identified from pollen loads collected by bees as such, that is not, or not yet, contained in honey, including pollen stores in beehives. Presently beekeepers estimate what pollen is being collected by bees by the colour of the pollen load. This is highly ambiguous and inaccurate. Through this technique it has been found that fungal spores from rusts are also collected by bees throughout the year in significant quantities together with pollen from conifers, (Bisaccate pollen). Both fungal spores and bisaccate pollen would seem to play a sign ificant part in bee nutrition but have received little attention hitherto. It has been commonly thought that honey bees are dependent on flowering plants on ly. The technique of the present invention enables the recovery of both pollen and fungal spores in pollen loads.

I n

tn ι ico tho tor n in i 10 nf tho nrocont nn a nnl lon Inarl a c ci irh t o load may firstly be broken up by mechan ical pressure then by the addition of an appropriate quantity of deion ized or distilled water, the loaded water then being filtered as per the present invention, and the filter resid ue being examined for example microscopically.

Microbiota are a commun ity of microorganismis wh ich exist in an environment or niche governed by particular conditions. Generally made up of bacteria and fungi which are non-pathogenic (not causing d iseases) and are symbiotic to the host environ ment or niche. Th ree areas wh ich are of interest in bee n utrition are:

i) Bee gut containing microbiota assisting in digestion;

ii) Pollen store where pollen are undergoing breakdown;

iii) Honey mainly associated with pollen breakdown.

The technique of the present invention recovers pollen at d ifferent stages of breakdown . It has in that process been observed that a type of fungi (Chytrids) are sometimes found growing on pollen and these break down the outer walls of pollen th us to enable bees to d igest and obtain benefit from pollen content. Bees cannot digest pollen alone as they simply lack the enzymes req uired for the breakdown of pollen walls. The outer wall of pollen is, incidentally, composed of sporopollenin. There is an inner wall comprising cellulose, hemicellulose and pectin . Pollen core, which the bees require, comprises nucleic acids, proteins and fatty acids. 2. Bee nutrient and medication

The present invention leads to an accurate determination of the ideal food for bees. Hitherto, apiarists concerned to feed their bees when natural conditions are adverse or just to supplement or enhance their diet have fed their bees a fructose solution. A 5 typical commercial example is a compound of natural beet extract with molasses and sodium ortho hydroxyl benzoate.

It has been found using this method that bees gather fungal spores in significant amounts also that most pollen collected are from plants that have medicinal

0 properties. A bee feed can be developed based on fungi (such as Quorn) fortified with compounds from plants that are medicinal. Medicinal feeds can be used to fight viral and bacterial infections as well as to supply the bee colony with essential nutrients for the development of healthy bees. Using the same principle natural plant substances that deters parasites can alleviate bee colonies of parasitic mites such as Varroa. Such5 medicinal feeds can be given to colonies any time when needed, throughout the

foraging season. Since such feeds need have no sugars they will not affect the flavour of honey produced.

In honey it is understood that the process of pollen breakdown is attenuated exceptΠ

- » rh■■w y trirl c s ro arti la l■l■w y i■n■■ rnntart †ho nrt llon a nrl thara ic a n arlon i l ata water supply within which the breakdown can occur. An adequate water supply is greater than 17.1% by volume of the honey. This means that in order for a bee to derive benefit from honey in the hive there must be a supply of water. A shallow, gently sloping walled container in the hive may enable provision such a water supply5 when it is otherwise short.

By analysing the pollen stores an assessment can be made of the different plants which are visited by bees during a short period of foraging. This can lead to identification of foraging areas relative to the hive. These areas are therefore important to bee nutrition.

Honey contains pollen which has been collected from plants and which have both a nutritional and medicinal value to honey bees. Most types of pollen collected also comprise medicinal compounds which confer a health benefit to developing bees and adult bees. Many pollens contain substances which benefit not just the health and fitness of bees but also of humans. By identifying and counting the types of pollen found in honey a pollen profile can be produced which will indicate plants which bees have been collecting nectar and pollen from and therefore what medicinal compounds can be found in different types of honey. Having determined such profiles it becomes possible to control honey content to at least some extent, to market honeys with a label setting out the content and to control the accuracy of such labels.

Honey falls into two general categories. These are Honeydew honey and Blossom honeys. Honey can further be categorised by three criteria using the technique of the present invention to extract pollen from honey. These criteria are:

i) the amount of pollen per volume of honey. It has been found that honeydew honey has a lower pollen content per volume of honey than blossom honeys

ii) the amount of fungal elements (fungal spores and hyphae) and yeast in honey. Honeydew honey tends to contain greater amounts of fungal elements than blossom honey whereas yeast found in honey is confined almost exclusively to nectar and blossom honey;

iii) the percentage ratio of tree and shrub pollen to herb pollen. Honey containing a very large percentage of herb pollen is more likely to be that of a blossom honey. By counting and recording the types of pollen, fungal elements and yeast and plotting the relative percentage in a pie chart an indication of honey type can be instantly assessed. 3. Honey Competitions

In judging honey in competitions the main criteria used are:

i) Colour;

ii) Aroma;

iii) Flavour;

iv) Viscosity

Mostly these criteria are subjective. A pie chart containing information of the pollen and yeast and fungal element contents may on the other hand indicate the source of the honey and authenticity. Honey which has low pollen per volume may have been adulterated with syrup in order to make the honey taste sweeter. Honey which contains poNen from a different geographical area may indicate addition of another honey blended to make the honey taste and look better. Honey which has more than expected fungal elements and which therefore indicate a honeydew source but taste very sweet may contain sugar substitutes such as sweeteners.

Thus furthermore prize winning honey when analysed for types of pollen may give an indication as to what plants are important to producing honey of a high quality.

4. Ecological assessment

The extraction of pollen from honey enables a record of the plants honey bees foraged from during a season. Types of plants indicate different types of plant habitats. By recording the pollen types it is possible to investigate the area around the beehive using a mapping software for example apple maps. A circular area of 2km radius to the hive can be drawn and within the circle an estimate of likely areas of foraging can be assessed. Habitats such as woodlands, fields, marsh, roadside/railway/motorway verges, gardens, riverside and lake habitats would indicate areas of foraging based on pollen types found in honey.

Comparing pollen types from honey collected in different years in an area can indicate changes in plant habitats over time. Changes in pollen types found in honey over time can give an indication as to how an area has developed and the impact on ecology. Pollen types which no longer or hardly appear compared to previous years reflect a loss of plant habitat. Pollen types which are newly recorded may indicate a change of plant habitats or the introduction of plants by gardeners or a change in land use, for example new type of crops being farmed in an area. An increase in honeydew pollen may coincide with urbanisation of an area. Such changes in environment may require an apiarist either to relabel his honey or to replace, in the vicinity of the hives, lost plant species.

5. Delivery of good microbiota to the gut system

The method of the present invention has also led to the observation that

microbiota in honey is inactive. This may be due to low water content and high concentrations of sugars. Having a healthy and diverse gut microbiota can contribute to human physiology, metabolism, nutrition and immune function. Delivery of good bacteria or good gut microbiota can be made by mixing good bacteria and/or gut microbiota with honey. Preparations formed into pills or capsules can be taken orally enabling delivery to the intestine. Taken regularly over a period of time these will enable healthy and diverse gut microbiota to develop which contribute to overall health and fitness. The present invention accordingly relates to an apparatus and method for separating pollen and micro-organisms from pollen loads, pollen stores and honey. This has lead to a greater understanding of specific plants which are important for nutrition, health and development of honey bees, breakdown of pollen by chytrid fungi and microbiota, understanding compounds found in specific pollen relating to bee health and development, identification of fungal spores collected by bees, analysis and classification of honey from pollen, fungal element and yeast contents, estimation of changes to local ecology over time using pollen analysis of honey. From such studies it may be possible to formulate new bee feeds based on specific compounds found in pollen to deter bee parasites and alleviate viral and bacterial diseases.

The method presently used to extract pollen from honey is called the Acetolysis method which was developed and introduced for pollen analysis by Gunner Erdtman in 1954. The technique is confined to laboratories and carried out by experienced technicians as it involves using hazardous chemicals. It is known that the technique destroys pollen material except that of the outer wall of pollen which is composed of sporopollenin. Acetolysis method has formally recognised as the standard method used for study of pollen from honey (Melissopalynology) by the International Commission for Bee Botany in 1970.

The new method developed presented here is very different from Acetolysis. The method avoids the use of hazardous chemicals and therefore can be carried out outside a laboratory by anyone wishing to study pollen and other micro-organisms. All pollen material are preserved with related micro-organisms including fungi. The method can also be carried out in a short period of time taking under one hour to complete a microscope slide for microscopic analysis.

The Novel method for recovering pollen and associated micro-organisms from pollen loads, pollen stores and honey

A pollen load or a sample from pollen stores, taken from the bee hive, is dissolved in warm (30°C) deionised water. lOg of honey is diluted with 100ml of warm (30°C) deionised water.

The supporting perforated shelf is vibrated using an electric toothbrush mechanism. The liquid leaving the apparatus is allowed to flow to waste.

More water is added to wash clean pollen and other associated micro-organisms. The residue on the filter is poured from the apparatus into a glass beaker. A stain, normally Safranin, is added for pollen.

The pollen is again washed through the apparatus with water until excess stain is removed.

A microscope slide of the residue is made and analysed using a compound microscope.

This method is valuable in understanding honey bee nutrition.

It is well known that honey bees forage for pollen and nectar from flowering plants, what is not weN known is that honey bees also collect significant amounts of fungal spores from plant rusts, pollen from coniferous plants and significant amount of plant sugars from honeydew. Honey bees collect pollen and fungal spores and form pollen loads which are transported back to the hive and deposited in pollen stores. Within the pollen stores pollen undergoes breakdown by microbiota and chytrid fungi.

Chytrid fungi gradually breakdown the outer wall of pollen which is made from sporopollenin, in doing so release p-coumaric acid wh ich is known to boost honey bees immune system. When bees feed on pollen, from pollen stores, it is in a ready state of breakdown. Compounds contained with in the pollen as well as from pollen wall can be d igested or fed to developing larvae.

Applications 1) Processing and analysis of pollen loads

Presently beekeepers estimate what pollen is being collected by bees by the colour of the pollen load . Th is is h ighly ambiguous. The techn ique enables the recovery of pollen and fungal spores in pollen loads. The identification of pollen lead to the identification of plant species foraged by honey bees. This can reveal plants which are of importance to honey bees some of which are not common ly associated with honey bees, and also plants which have been introduced by horticulturalists which have some n utritional value to honey bees.

It has been found using th is technique that some pollen loads are made up of fungal spores collected from rust growing on specific plants. Fungal spores contain a similar percentage of proteins found in pollen. The type of fungal spores are normally plant specific and therefore identification of fungal spores can indicate types of plants which are important to bees.

2) Processing and analysis of pollen stores

Pollen stores have not been studied for types of pollen and fungal spores that are stored and undergoing breakdown by microbiota. By sampling and using the techn ique to process from the pollen stores, an assessment can be made as to the different plants which are visited by bees during the a short period of foraging. This can lead to identification of foraging areas relative to the hive. These areas contain plant habitats which are important to honey bees when foraging.

3) Processing and analysis of Honey Honey contains a significant and diverse amounts of pollen which has been collected throughout the period when honey bees forage. Honey also contains significant amount of fungal elements such as fungal hyphae and yeast. Using the method it is possible to recovery and concentrate pollen, fungal elements and yeast.

3.1) Health assessment Honey contains pollen which has been collected from plants. This has a nutritional value to honey bees. Most types of pollen collected also have medicinal compounds which conveys a health benefit to developing bees and adult bees. Many plants which bees visit contain substances which are beneficial the health and fitness of bees may also have health benefits to humans. By identifying and counting the types of pollen found in honey, a pollen profile can be produced which would indicate plants which bees have been collecting nectar and pollen from and therefore what type of

medicinal compounds that can be found in different types of honey.

3.2) Classification of honey types

Honey falls into two general categories:

Honeydew honey and Blossom honey.

Honey can be categorised by three criteria using this technique to extract pollen, fungai elements and yeast from honey;

1. The amount of pollen per volume of honey. It has been found that honeydew honey have a less pollen per volume of honey than blossom honey.

2. The amount of fungal elements (fungal spores and hyphae) and yeast in honey. Honeydew honey are associated with greater amounts of fungal elements whereas yeast found in honey is associated to nectar and blossom honey.

3. The percentage of tree and shrub pollen to herb pollen, honey containing a very large percentage of herb pollen would be more likely to be that of a blossom honey.

By assessing the amount of pollen in a given volume of honey and counting the recording the types of pollen, fungal elements and yeast and plotting the relative percentage in a pie chart an indication of honey type can be instantly assessed.

3.3) Judging honey T o ma in r ritoria ι i corl tn _j i i ιΗσο hnnow are

Vicrnc itv/

Most criteria are subjective. A pie chart containing information of the pollen and yeast and fungal element contents may indicate the source of the honey and authenticity. Honey which has low pollen per volume may be adulterated with syrup in order to make the honey taste sweeter. Honey which contains pollen from a different geographical area may indicate addition of another honey, blended to make the honey taste and look better. Honey which have greater fungal elements which indicate a honeydew source but taste very sweet may contain sugar substitutes such as sweeteners.

Additionally prize winning unadulterated honey when analysed for types of pollen content may give an indication as to what plants are important to producing honey of a high uality.

3.4) Ecological assessment

Using the technique the type of pollen extracted from honey can be regarded as a record of plants honey bees foraged from during a season. Types of plants indicate different types of plant habitats. By recording the pollen types it is possible to investigate the area around the bee hive using a mapping software for example apple maps. A circular area of 2km diameter to the hive can be drawn and within the circle an estimate of likely areas of foraging can be assessed. Habitats such as woodlands, fields, marsh, roadside/railway/motorway verges, gardens, riverside and lake habitats would indicate areas of foraging based on pollen types found in honey.

3.5) Recording ecological change due to urbanisation and development

Comparing poiien types from honey collected in different years in an area can indicate changes in plant habitats over time, Changes in pollen types found in honey over time can give an indication as to how an area is developed and the impact on ecology. Pollen types which no longer appear or is significantly lower than in previous years reflect in a loss of plant habitat and pollen types which are newly recorded may indicate a change of plant habitats or introduction of plants by gardeners or a change in land use for example new type of crops farmed in an area. An increase in honeydew pollen may coincide with urbanisation of an area.

4) Study and analysis of microbiota breaking down pollen

Using this method it has been observed, through microscopic study, that pollen extracted are sometimes have the surface covered with a gel like substance. When left in water over a two day period a large number of micro-organisms, microbiota, are seen over and surround pollen grains. Some pollen grains have closely developed hyphae growing on the surface and transparent spherical bodies. These are thought to be Chytrid fungi which is breaking down the outer wall of pollen grains. The method and apparatus can be used to establish the mechanism by which pollen is broken down.

Other applications

5) Development of medicinal bee feeds

It has been found using this method that bees gather fungal spores in significant amounts also that most pollen collected are from plants that have medicinal properties. A bee feed can be developed based on fungi (such as Quorn) fortified with natural compounds from plants that are medicinal. Medicinal feeds can be used to fight viral and bacterial infections as well as to supply the bee colony with essential nutrients for the development of healthy bees. Using the same principle natural plant substances that deters parasites can alleviate bee colonies of parasitic mites such as Varroa. Such medicinal feeds can be given to colonies when needed, throughout the foraging season since feeds have no sugars it will not affect the flavour of honey produced.

6) Concentration and study of microscopic plankton from natural water bodies

Natural water bodies which include Ponds, Lakes, Rivers, Estuaries and Marine waters contain microscopic plankton community which is a source of food for other life forms. The apparatus can be used to filter a volume of water capturing microscopic plankton in water. A slide of water can be examined under a microscope to reveal micro-organisms living in natural water bodies. Plankton communities are affected by pollution, environmental conditions and climate change. By comparing season variation and annual variation it is possible to chart changes occurring due to pollution e.g. sewage, fertilisers, detergents, environmental changes, e.g. acid rain and climate change e.g. temperature changes over a period of time e.g. 10 years.

7) Delivery of good microbiota to the human gut system The method has also led to the observation that microbiota in honey is inactive, this may be due to low water content and high concentrations of sugars. From this observation healthy human gut microbiota can be stored in honey for a long period of time.

Having a healthy and diverse gut microbiota can contribute to our physiology, metabolism, nutrition and immune function. Delivery of good bacteria or good gut microbiota can be made by mixing good bacteria and/or gut microbiota with honey which has had honey bee microbiota removed. Time release capsules of honey containing healthy microbiota will enable healthy microbiota to be release into the intestine. Taken regularly over a period of time will enable a healthy and diverse gut microbiota to develop which contributes to overall health and fitness.

Description of Preferred Embodiments

For a better understanding of the present invention and to show how the same may be carried into effect, reference will now be made, by way of example, to

accompanying drawings, in which:

Figure 1 is an exploded view of one embodiment of an apparatus in accordance with the present invention;

Figure 2 is a side view, partly in cross section, of the apparatus shown in Figure 1 duly assembled;

Figure 3 is a plan view looking down on a component of the apparatus shown in Figure 1;

Figure 4 is an enlarged view of a region of Figure 2, depicting the apparatus in use; and Figure 5 is an exploded view of an alternative embodiment of an apparatus in accordance with the present invention.

Referring to Figure 1, there is shown an apparatus for extracting pollen and other organisms from honey, which apparatus is generally identified by reference numeral 101.

The apparatus 101 comprises a feeder member 102 which screws into a filtrate chamber 103, between which a filter 104 and a perforate support shelf 105 are held fast in place. The feeder member 102 comprises a cylindrical wall 106 provided with external threads 107 at a iower portion thereof. The filtrate chamber 103 comprises a cylindrical wall 108 provided with internal threads 109 at an upper portion of the cylindrical wall 108, to accommodate the external threads 107. The filtrate chamber 103 further comprises an inwardly projecting ledge 110. A land (not shown) is formed above the ledge adjacent the filtrate chamberwall and arranged forthe feeder member to abut on the land when fully screwed in. The filter holder and filterfit within this land. The land has a depth equal to that of the filter holder and filter and thus prevents excessive tightening of the feeder member onto the filter.

In assembly the perforate support shelf 105 is placed into the filtrate chamber 103 so that it rests upon the ledge 110. The filter 104 sits on top of the perforate support shelf 105, so their perimeters are aligned. The external threads 107 and the internal threads 109 are aligned and the feeder member 102 is then screwed tight into the filtrate chamber 103. This is depicted in Figure 2, which shows the alignment of the filtrate chamber 103, perforate support shelf 105, filter 104, and feeder member 102 of the apparatus 101. Referring to Figure 3, the perforate support shelf 105, comprises a holed region 111 in the inner portion of the perforate support shelf 105 and an annular sealing portion 112 at an outer region of the perforate support shelf 105.

In use, referring to Figure 4, honey (at about 20°C) is diluted with warm water (at about 35°C) in the ratio 1ι20 by volume. The resulting solution is poured through the hollow center of the feeder member 102, which is surrounded by cylindrical wall 106 and onto the filter 104. The solution passes through the spaces in the filter 104 and then through the larger holes in the holed region 111 of the perforate support shelf 105. The liquid solution that passes through the filter 104 and the support shelf 105 is collected (not shown) for disposal or further processing.

Pollen and other microorganisms are retained on the surface of the filter 104 (which typically has a 5 micron to 20 micron pore size and preferably a 10 micron pore size), as a result of being larger than the fine spaces. (A few pollens have a diameter of around 3 microns. However, as a practical matter the inventor has found that a 5 micron screen does retain a significant portion of such small pollen particles.) The deposits, primarily pollen and other microorganisms including microbiota and fungal spores together with any impurities, can then be easily removed from the filter 105 and collected for further processing or analysis.

The feeder member 102 and filtrate chamber 103 are made of Nylon and are rigid enough to retain the filter 104, perforate support shelf 105 and the fluid and are preferably hard enough to withstand prolonged and frequent use. Other plastics materials, for example polypropylene, can also be used.

Referring to Figure 5, there is shown an alternative embodiment of an apparatus also for extracting pollen and microorganisms from honey, which is generally identified by reference numeral 201. The alternative apparatus 201 similarly comprises a feeder member 202 which screws into a filtrate chamber 203, between which a seal ring 213, a filter 204 and a uniform perforate support shelf 214 are held fast in place. The filtrate chamber 202 comprises a cylindrical wall 206 provided with external threads 207 at a lower portion of the cylindrical wall 206 according to Figure 5. The filtrate chamber 203 comprises a cylindrical base wall 20S provided with interna! threads 209 at an upper portion of the wall 208, to accommodate the external threads 207. The filtrate chamber 203 further comprises an inwardly projecting ledge 210.

It is noted that the perforate support shelf 214 has holes to its edge and no seal attached thereto. In assembly the holed support shelf 214 is placed upon the lip 210. The filter 204 sits on top of the perforate support shelf 214 and the seal ring 213 sits on top of the filter 204, so that all three of the circular perimeters are aligned. The external threads 207 and the internal threads 209 are aligned and the feeder member 202 is then screwed tight into the filtrate chamber 203.

The alternative apparatus 201 is used in the same way as the apparatus 101 shown in Figure 1.

Referring to the preferred embodiment (Figures 1-4), the filter 104 is a 10 micron pore Nylon filter which is sufficiently fine to remove the pollen and other organisms, including microbiota, from the honey. The perforate support shelf 5 is a resilient plastics material (e.g. Nylon) and sufficiently rigid to support the filter 104 and inhibit it stretching under the weight of the honey. This can result in an increase in filter pore size which is undesirable as pollen and microorganisms can be lost through the pores. The holed region 111 and annular sealing portion 112 are a single piece of material, with the holed region 111 providing support of the perforate support shelf 105 to the filter 104 and the annular portion 112 providing a perimeter to the perforate support shelf 105 that can be compressed when the body 102 is screwed into the filtrate chamber 103. This compression allows a tight seal to be made between the filter 104, body 102 and filtrate chamber 103, in order to inhibit any fluid from becoming trapped in the boundary. The arrangement of the external threads 107 and internal threads 109 are so that the feeder member 102 and the filtrate chamber 103 can be screwed tight together. A land (not shown) is formed above the ledge adjacent the filtrate chamber wall and arranged for the feeder member to abut on the land when fully screwed in. The filter holder and filter fit within this land. The land has a depth equal to that of the filter holder and filter and thus prevents excessive tightening of the feeder member onto the filter.

The order of the components of apparatus 101 are so that the filter 104 is in direct contact with the annular sealing portion 112 (Figure 1) or seal ring 213 (Figure 5) and that a seal will form due to the compression of annular sealing portion 112 or seal ring 213 from the force of screwing the body 102 into the filtrate chamber 103, with the edges of the filter 104 also being compressed. Additionally the perforate support shelf 5 is underneath and in direct contact with the filter 104 in order to provide support against the weight of the honey.

In the preferred embodiment, with reference to Figure 1, the outer diameter of the feeder member 102 is 100mm, the inner diameter is 90mm, the height of the feeder member 102 is 55m and the external threads 107 are 10mm in height from the bottom of the wall 106 of filtrate chamber 103.

The outer diameter of the filtrate chamber 103 is 130mm, the inner diameter is 100mm, the height of the 103 is 14mm and the internal threads 109 are 8mm in depth from the top of the filtrate chamber 103 (as seen in Figure 1). The ledge 110 is 6mm in height from the bottom of the filtrate chamber 103 and inwardly extends 5mm from the inside of the cylindrical wall 108 to the center of the filtrate chamber 103.

The filter 104 is 97mm in diameter and has a pore size of 10 microns. The perforate support shelf 105 is 97mm in total d iameter, with the holed region being 60 mm in diameter. The holes in the holed region 111 are 10 mm in diameter, providing a functional filter area of about 2430 mm² compared to the total area of the filter at 6360 mm².

In the alternative embodiment (Figure 5), the differing features have dimensions such that the seal ring 213 is 97 mm in external diameter and 90 mm in internal diameter.

The uniform perforate support shelf 214 is 97 mm in diameter.

In one separation process, 10 ml of raw honey at 20°C were diluted with 200 ml of deionised water at 35¾C. The resulting solution took of the order of 30 seconds to pass through the apparatus 101.

In an embodiment where analysis of the filter residue is likely to occur at a station remote from the filtration, when the filtration is complete the cap is removed and a film placed over the filter. The apparatus is then dismantled and the filter placed in an appropriate holder for transmission to an analysis site.

Ajto rn atjuo jy tho j jto r residue is pipetted to a microscope slide, after which the apparatus is dismantled, the filter disposed of and the apparatus is cleaned. At the analysis site the filter residue is examined, the various organisms being identified and quantified. Various modifications to the embodiment described are envisaged, for example the annular sealing portion 112 does not necessarily have to be an integral part of the perforate support shelf 105, and could be separate from each other. An alternative combination and order of filters, supports and seal rings could be used. Another form of seal could be employed, such as an oil based substance to repel the solution and inhibiting it from entering the external threads 107, internal threads 109 and ledge 110. A built in seal could be an integral part of the filtrate chamber 103 or feeder member 102 of the apparatus 101. The components of apparatus 101 could be adhered together, creating a seal and inhibiting the components being separated from each other. An alternative means for providing support to the filter could be in place on the perforate support shelf 105, such as wires or support struts. The filter could be constructed or attached to the feeder member 102 and filtrate chamber 103 to sufficiently support itself so that an additional form of support is not required. The ledge 110 could support the filter 104 or a seal directly or it could be omitted from the filtrate chamber entirely. The pore size of the filter 104 can be larger or smaller than the preferred size, consistent with the size of the pollen to be removed. The holes in the holed region 111 of the perforate support shelf 105 can be larger or smaller than the preferred size. The holes of the perforate support shelf 105 can be arranged in an orderly manner or randomly across the area of the holed region 111. An alternative method for securing the feeder member 102 into the filtrate chamber 103 can include a slot and lock or bayonet arrangement or a combination of either the feeder member or the filtrate chamber being tapered and fitting into the corresponding part.

101. 201. Apparatus

102. 202. Feeder member

103. 203. filtrate chamber

104. 204. Filter

105. 205. Perforate support shelf

106. 206. Cylindrical wall

107. 207. External threads

108. 208. Cylindrical wall

109. 209. Internal threads

110. 210. Ledge

111. 211. Holed region

112. 212. Annular sealing portion

213. Seal ring

214. Uniform perforate support shelf

Claims

1. A method of analysing the microscopic content of honey and comprising subjecting a honey sample to a filtration process to separate therefrom the pollen and microorganisms therein, thereafter determining the identity and quantity of the chytrids and the pollen.

2. A method as claimed in claim 1 and wherein the process also separates microbiota from the honey.

3. A method as claimed in claim 1 or claim 2 and employing filtration.

4. A method as claimed in claim 3 and employing a nylon filter.

5. A method as claimed in claim 3 or claim 4 and wherein the filter has a pore size of about 10 microns.

6. A method as claimed in claim 3 or claim 4 and wherein the filter has a pore size of about 5 microns.

7. A method as claimed in claim 3 or claim 4 and wherein the filter has a pore size of about 30 microns.

8. A method as claimed in any one of the preceding claims and comprising diluting the honey sample with water prior to filtration.

9. A method as claimed in Claim 8 and wherein 200mi to 250mi of water is added per 5ml to 10ml of honey.

10. A method according to Claim 8 or claim 9 and wherein 200ml of water is added per 10ml of honey.

11. A method as claimed in any one of claims 8 to 10 and wherein the water is at a temperature of from 30°C to 38°C.

12. A method as claimed in any one of claims 8 to 11 and wherein the water is deionized water.

13. A method as claimed in anyone of the preceding Claims and wherein the honey sample is at a temperature of from 20°C to 26°C.

14. A method as claimed in Claim 12 and wherein said temperature is 20°C.

15. A method according to Claim 11, wherein said temperature is 35°C.

16. A method as claimed in any one of the preceding claims and comprising vibrating the filter.

17. A method as claimed in any one of the preceding claims and comprising pipetting the filter residue on to a microscope slide.

18. An apparatus for separating pollen and other microorganisms from honey, the apparatus comprising:

i) a substantially rigid filter support arranged for supporting a filter;

ii) a feeder member arranged to receive a quantity of a honey solution for filtration;

the filter holder being attachable to the feeder member in such a manner as to grip a filter therebetween.

19. An apparatus as claimed in Claim 18 and wherein the filter support comprises a perforated region bounded by a sealing portion.

20. An apparatus as claimed in Claim 19 and wherein the sealing portion is annular.

21. An apparatus as claimed in any one of Claims 18 to 20 and wherein the filtrate chamber has a ledge and the filter support and the filter are mountable thereon.

22. An apparatus as claimed in claim 21 and wherein the ledge has a land at its outer perimeter arranged for the snug reception of the filter holder and a filter.

23. An apparatus as claimed in any one of Claims 18 to 22 and comprising also a filtrate chamber.

24. An apparatus as claimed in claim 23 and wherein the feeder member and the filtrate chamber are screw-threaded ly connectable.

portion of the filter support is compressible.

An apparatus as claimed in any one of claims 18 to 25 and comprising vibration

27. A kit of parts for analyzing the content of a honey sample and comprising apparatus as claimed in any one of claims 18 to 26 and also a filter holder for holding a filter carrying filter residue and for the conveyance thereof to a microscope.

28. A kit of parts for analyzing the content of a honey sample and comprising apparatus as claimed in any one of claims 18 to 26 and also a pipette and a stock of microscope slides.

29. A kit of parts for analyzing the content of a honey sample and comprising apparatus as claimed ciaim 28 and aiso a stock of microscope siide holders.

30. A kit of parts as claimed in claim 28 or claim 29 and comprising also a stock of ionized water.

31. A kit of parts as claimed in any one of claims 28 to 30 and comprising aiso a jug with scales marked thereon for measuring the quantity of honey and water.

32. A kit of parts as claimed in any one of claims 28 to 31 and comprising also a thermometer.

33. A method as claimed in claim 1 and substantially as hereinbefore described with reference to the accompanying drawings

34. An apparatus as claimed in claim 18 and substantially as hereinbefore described with reference to the accompanying drawings.

35. A kit of parts substantially as hereinbefore described with reference to the accompanying drawings.