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International Journal of

Food Microbiology 31 (1996) IL26

Review article

Microorganisms in honey

Jill A. Snowdon”,*, Dean 0. Cliverb

“Sci ent i $c Consul t ant , Nat i onal Honey Board, 390 Lashl ey St reet , Longmont , CO 80501, USAbPopulat i on Healt h and Reproducti on, Uni versit y of Cali forni a, School of Veteri nary M edici ne, 1019

Har ing H all , Dav is, CA 95616, USA

Received 24 February 1995; revised 6 October 1995; accepted 28 November 1995

Abstract

Knowledge of the moisture and temperature conditions influencing growth of microorgan-isms in honey has long been used to control the spoilage of honey. However, the need for

additional microbiological data on honey will increase as new technologies for, and uses of

honey develop. Microorganisms in honey may influence quality or safety. Due to the natural

properties of honey and control measures in the honey industry, honey is a product with

minimal types and levels of microbes. Microbes of concern in post-harvest handling are those

that are commonly found in honey (i.e., yeasts and spore-forming bacteria), those that

indicate the sanitary or commercial quality of honey (i.e., coliforms and yeasts), and those

that under certain conditions could cause human illness.

Primary sources of microbial contamination are likely to include pollen, the digestive

tracts of honey bees, dust, air, earth and nectar, sources which are very difficult to control.

The same secondary (after-harvest) sources that influence any food product are also sourcesof contamination for honey. These include air, food handlers, cross-contamination, equip-

ment and buildings. Secondary sources of contamination are controlled by good manufactur-

ing practices.

The microbes of concern in honey are primarily yeasts and spore-forming bacteria. Total

plate counts from honey samples can vary from zero to tens of thousands per gram for no

apparent reason. Most samples of honey contain detectable levels of yeasts. Although yeast

counts in many honey samples are below 100 colony forming units per gram (cfu/g), yeasts

* Corresponding author, 2623 Poinsettia Drive, White Oak, PA 15131, USA; Tel: + 1 412 672 9706;

fax: + 1 412 672 9708.

0168-1605/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved

PI I SO168-1605(96)00970-l



2 J.A. Snowdon, D.O. Cl&r /ht. J. Food Microbiology 31 (1996) l-26

can grow in honey to very high numbers. Standard industry practices control yeast growth.

Bacterial spores, particularly those in the Bacil lus genus, are regularly found in honey. The

spores of C. botulinurn are found in a fraction of the honey samples tested ~ normally at

low levels. No vegetative forms of disease-causing bacterial species have been found in

honey. Bacteria do not replicate in honey and as such high numbers of vegetative bacteria

could indicate recent contamination from a secondary source. Certain vegetative microbescan survive in honey, at cool temperatures, for several years. However, honey has anti-micro-

bial properties that discourage the growth or persistence of many microorganisms. Typically,

honey can be expected to contain low numbers and a limited variety of microbes.

A routine microbiological examination of honey might include several different assays. A

standard plate count provides general information. Specialized tests, such as a count of

yeasts and an assay for bacterial spore-formers, may also be useful. An indicator of sanitary

quality as provided by coliform counts might be included. Additional tests, to explain

unusually high counts or address a certain problem, may be needed. The use of honey in

products that receive no or limited heat treatment may require additional tests. More

information on the source and control of microbes in honey is needed to answer the concerns

currently facing the industry.

Keywords: Bacteria; Food safety; Honey; Microorganisms; Molds; Specifications; Spoilage;

Yeasts

1. Microbial content and the quality and safety of honey

Honey, the nectar and sweet deposits from plants as gathered, modified andstored in the honeycomb by honey bees, is a popular sweetener. New technologies

and innovative uses of honey are expanding marketing opportunities. However,

new microbiological requirements pertaining to quality and safety may be associ-

ated with these opportunities. A more comprehensive understanding of honey’s

microbiological characteristics is needed as honey is used in new ways.

Honey is packaged for retail sale, for food service, and in bulk for use as an

ingredient. In the US, retail sales for direct use of honey in the home kitchen, often

as an ingredient in sauces and baked goods, total about 100 million pounds of

honey per year. Honey is also sold as a food service item to restaurants or directly

to consumers at warehouse- or ‘club’-style stores. Approximately 46 million pounds

of honey are sold this way each year in the US. About 150 million pounds of honey

per year are used as a food ingredient. A small amount of honey is used in

non-food items such as drugs, cosmetics or pet-food. These figures are estimates

provided by the National Honey Board (a research and promotion board adminis-

tered by the United States Department of Agriculture).

Current purchasing specifications for microorganisms in honey are often based

on microbial specifications for other foods and ingredients. Due to honey’s unique

properties, such as its anti-microbial activities, some of those specifications may not

be germane to honey. Purchasing specifications currently in use include a standardplate count and tests for coliform bacteria, yeasts, molds and certain pathogenic



J.A. Snowdon, D.O. Clivrr 1hr. J. Food Microbiology 31 (1996) l-26 3

bacteria such as Staphylococcus, Salmonella and Clost r id ium species. Examples of

recent microbiological specifications for honey are shown in Table 1.

Honey is essentially water (average 17.2%) suspended in fructose (average 38.4%)

and glucose (average 30.3%; White et al., 1962b). Honey also contains sucrose

(average 1.3%) and other carbohydrates (about 12X), minerals (average 0.169%)

and proteins (169 mg/lOO g; White et al., 1962b). The pH of honey ranges from 3.4

to 6.1 with an average of 3.9, while the water activity varies between 0.5 and 0.6

(White et al., 1962b). Honey has distinctive properties that inhibit or kill most

microorganisms. Hence, microbes of interest to the honey processing industry are

those that withstand the concentrated sugar, acidity and anti-microbial character of

honey. These microbes could be put in three categories: (1) microorganisms that are

commonly found in honey (certain strains of yeasts and spore-forming bacteria); (2)

microorganisms that indicate sanitary or commercial quality (coliforms or yeasts);

and (3) microorganisms that, under certain conditions (e.g. germination and growth

in a non-heat-treated food product), could cause illness. A fourth category,microorganisms that cause diseases in honey bees, is not considered in this paper.

2. Background on honey microbiology

2.1. Sour ces of m i cr obes i n honey

The following information leads to the conclusion that primary sources of

microbial contamination are likely to include pollen, the digestive tracts of honeybees, dust, air, dirt and flowers. Secondary sources of microbes in honey are likely

to be the same as for other foods.

2.1. I. Primary sources

Microbes found in comb honey are principally bacteria or yeasts and come from

the bees, the raw material (nectar) or from external sources. Bacteria and yeasts

may have different origins. Larvae may be sterile initially (White, 1921; Gilliam,

1971) but they are fed nectar and pollen by workers and are therefore subject to

inoculation by the nectar, pollen and workers’ flora before pupation.

Many microorganisms are associated with specific foods or components of the

ecosystem (Jay, 1992). Organisms found in the environment around honey (i.e.

bees, hives, pollen, flowers, soil, etc.) are also likely to occur in honey. Actinetobac-

ter , Bacil lu s, Clostr id ium , Corynebacter iu m , Pseud omonas, Psychr obacter and Vago-

coccus are bacteria commonly found in soil. Air and dust are important sources of

Baci l lus, Clostr idium and M icr ococcus species. Baci l lus and Clost r id ium species are

important bacterial contaminants of cane and beet sugars, Saccharomyces and

Toru la yeasts can be found in high-moisture sugars, and L euconostoc mesen teroi des

has been found in sugar refineries. Brochothr ix, Citrobacter, Enterobacter, Erwinia,

Fl auobacter iu m , L actobacil lu s, Lu ctococcus, L euconostoc, L ister ia and Pediococcusare found in plants and plant products.



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J.A. Snowdon, D.O. Cliver /ht. J. Food Microbiology 31 (1996) l-26 5

Sackett (1919) summarized research by others which indicated that Baci l lus,

M i crococcus and Saccharomyces species can be readily isolated from honey combs

and adult bees. Comb honey and healthy larvae contained no microbes detectable

by methods then available.

A number of microbial species were isolated from the feces of bee larvae (Gilliam

and Prest, 1987). Bac i l l us species were most prevalent, followed by gram-variable

pleomorphic bacteria. Molds, Actinomycetes, gram-negative rods (probably Enter-

obacteriaceae) and yeasts were also recovered, while Streptomyces spp. were

recovered from one larva. No anaerobic bacteria were recovered. This is in

comparison to the intestinal microflora of adult honey bees, which is dominated by

gram-variable pleomorphic bacteria of uncertain taxonomic status, Baci l lus spp,

Enterobacteriaceae, Penic i l l ium spp., Aspergi l lus spp. and sometimes yeasts (fre-

quently Torulopsis spp) (Gilliam et al., 1988).

Pollen may be the original source of microbes in the intestines of honey bees

(Gilliam et al., 1983). The honey bees appear to be seeded microbiologically by

pollen consumption and by other bees in the colony through food exchange. The

scarcity of microbes in nectar suggests that nectar plays a minor, if any, role in this

process. Root (1983) suggests that flowers and hives are more important sources of

microbes than the soil.

Aerobic spore-forming bacilli (which could be Baci l lus) were the most frequently

encountered microbes on the external surface, crop and intestine of the honey bee

(El-Leithy and El-Sibaei, 1972). The guts of larvae, pupae or newly-emerged honey

bees are often sterile; fungi and bacteria are regularly found in the intestines of

adult worker bees a few days after emergence (Gilliam, 1971). Likewise, no or

limited microbes were found in the nectar that was sampled (Gilliam et al., 1983).Bacteria belonging to the genus Baci l lus were found in the digestive tract,

hemolymph and trachea of healthy honey bees (Gilliam and Valentine, 1976).

Gilliam (1978) suggests that pollen may be the main source of the gut microflora in

worker bees since there are few or no microbes in nectar.

The intestines of bees have been found to contain: 1% yeast-shaped microbes;

29% gram-positive bacteria including Baci l l us, Bacter i di um (sic), Streptococcus and

Clost r id ium species; and 70% gram-negative or gram-variable bacteria, including

Achr omobacter , Cit r obacter , En ter obacter , Er wi n& Escher ichi a coli , Fl avobacter iu m ,

Klebsiel lrt, Proteus and Pseudomonas (Tysset and Durand, 1968; Tysset et al.,

1970a).Nectar, the body of the bee, apiary soil and honey-house air and equipment are

all considered possible sources of yeast (Crane, 1979). Troller (1979) suggests that

the ultimate reservoir for yeasts in honey may be the hive and that the worker bees

distribute the yeast to the nectar as it is collected.

Bacteria introduced to honey by the bees are probably members of the Bac i l l us

family (Tysset and Rousseau, 1981). The mold, Aspergil lus, may occasionally be

present in honey but would be found only in the dormant, spore form (which would

preclude the production of mycotoxins such as aflatoxin). The primary sources of

sugar-tolerant yeasts are flowers and soil; yeasts may be present in honey combs, air

and equipment in the honey house (Graham, 1992). Morse and Hooper (1985)maintain that yeasts are found in nectar.



6 J.A. Snowdon, D.O. Cli ver 1 mt. J . Food M icrobiology 31 (1996) l -26

Nakano et al. (1992) analyzed 273 samples of non-honey sweeteners (e.g.,

unrefined sugar and corn syrup) destined to become food for bees, for the presence

of C. botu l i nurn . Three out of 56 (5%) of the sweeteners contained botulinal spores,

while 8 of 217 (4%) of the sweeteners not intended to feed bees contained spores.

The authors suggest that the sweeteners used to feed bees may be a source of sporesin honey.

Most research on primary sources of microbes in honey is done to understand the

microbial ecology of the honey bee. A listing of the microbes reported to be

associated with bees can be seen in Table 2. It appears that pollen, not soil, is the

proximate source of microbes that ‘seed’ the intestines of bees. Although not

identical, there is a considerable overlap between the microbes that are in bees

(Table 2) and those that have been found in honey (Table 3). This suggests that

some microbes that are introduced into honey by bees do not survive in honey and

that some microbes not associated with bee intestines are introduced into honey.

Baci l l us, Clost r i d ium, and M icrococcus species are common in air and dust and inhoney. This suggests that air and dust are also sources of primary contamination in

honey. Soil and flowers may be sources of yeasts in honey. The roles of the hive and

nectar as primary sources of microbial contaminants in honey are undetermined,

while the sweeteners used to feed bees may be a source of spores in honey. More

research is needed to determine the relative importance of these various routes.

Table 2

Microbes associated with honey bees”

Bacteria Fungi

Yeasts Molds

Achromobacter

Actinomycetes

Baci l lus

Bacter id ium (sic)

CitrobacterC lost r id ium

E. co/ i

Enterobacter

E rw in i a

Flavobacterium

Klebsiella

M i crococcus

Proteus

Pseudomonas

Streptococcus

Streptomyces

Sacchar omyces

Torulopsis

Aspergi l lus

Pen ic i l l ium

“Does not include microbes that are specifically pathogenic to bees. See Shimanuki and Knox (1991).



J.A. Snowdon, D.O. Clioer i Int. J. Food Microbiology 31 (1996) l-26

Table 3

Microbes reported to be found in honey

Bacteria Fungi

Yeasts Molds

Alcali~mrs

Bucillus

Bucteridium (sic)

Bacterium (sic)

Bre&xwterium

Clostridium

Entrrohncter

Flal;ohuc,terium

Klebsiellu

Micrococcus

Neisser iciProteus

Pseudomonas

Xunthomonus

Ascosphaeru

Debaryomyces

Hansen&

Lipomyces

Nematospora

Oosporidium

Pichia

Rhodotorula

Succharomycrs

Sc/2irosacclzarumycrs~accl~aromyce.~

SchwanniomycesTrichosporun

Torulu

Torulopsis

Zy~osuc.cliuroniyces

Aspergillus

Atichiu

Bettsia u&i

Cephalosporium

Chcretomium

Coniothecium

Hormiscium

Peniciilium

Peronsporaceae

Peyroneliu

Triposporium

Uredianceae

Ustilaginaceae

2.1.2. Secondary sources

Data on secondary sources of microbes in honey are relatively sparse in the

scientific literature. A comprehensive microbiological analysis of 12 honey samples

suggested that microbial contamination is more likely to take place during and after

extraction than in the hive (Tysset et al., 1970b). Tysset and Rousseau (1981) point

out that secondary sources of contamination in honey are humans, equipment,

containers, wind, dust, insects, animals and water. They caution that microbes may

survive longer than is generally believed and that good manufacturing practices are

important for the control of microbes in all food, including honey. Yeasts have

been recovered from equipment in honey houses; contaminated equipment can

introduce yeasts into otherwise clean honey (Root, 1983). Secondary sources of

contamination are often the same among different foods. Possible routes of

transmission into extracted honey would include air (in the honey house or while

the honey was being packed), food handlers (from skin infections, sneezing or frank

fecal contamination), cross-contamination (largely from animals or animal prod-

ucts), and equipment (including residues of food and water). Floors, walls and

ceilings can also be reservoirs of microbes that enter food. Thousands of cfu of

vegetative microbes (which are not expected in honey and cannot grow in honey)

per gram might indicate recent contamination of the honey via a secondary source.

Secondary sources of contamination are controlled by standard sanitation and

good manufacturing practices. Numerous texts and manuals have been written on

this subject. These control measures have been developed by the processed food

products industry and are also applicable to products that receive little or noprocessing.



8 J.A. Snowdon, D.O. Cliver /ht. J. Food Microbiology 31 (1996) l-26

2.2. Types, l evels and persi stence of mi crobes i n honey

A list of all microbes currently reported to be in honey can be seen in Table 3.

No information is available on the presence or persistence of viruses and parasites.

2.2.1. Molds

Mold is associated with the intestinal contents of bees, the hive, and the

environment in which the bees forage. Aspergillus has been recovered from the

intestines of honey bee larvae (Haisig and Kamburov, 1966). A few scientists have

described the presence of ‘sooty molds’ (epiphytic fungi that are osmophilic) in

honeydew and honeydew honey (as cited in Crane, 1979). Few of the molds are

identified with certainty, but typical genera may include Atichia, Coniothecium,

Hormiscium, and Triposporium. Morse and Hooper (1985) report that ‘when combs

are stored in damp areas, mold often appears on the comb surface’ and that ‘honey

bees have a remarkable ability to clean and restore moldy comb’. Furthermore,they state that stored pollen can be attacked by a fungus called Bett sia alvei. Molds,

including Aspergillus, Chaetomium, Penicillium, and Peyronelia, have been isolated

from the feces of bee larvae (Gilliam and Prest, 1987) while Tysset et al. (1970b)

found Ascosphaera, Aspergillus, Cephalosporium, and Penicil l ium molds in honey.

There are few reports that quantify the levels of mold in honey. Tysset et al.

(1970a) found an average of 254/g of honey with a range from 0 to 2500/g. Piana

et al. (1991) found mold in all 50 Italian honey samples analyzed. The levels were

very low, ranging from l-43 cfu/g. Crane (1979) reported that hyphae and spores

of fungi associated with bees (the pollen mold, Bett sia alvei) and plant-pathogenic

fungi (Peronsporaceae, Uredinaceae and Ustilaginaceae) can occasionally be foundin honey sediments.

Troller (1979) lists Chryosospori um, Eurot ium , Monascus and Xeromyces bisporus

as molds that can survive environments in which the water activity ranges from 0.7

down to 0.605, respectively. None of these genera have been recovered from honey

or honey bees. This suggests that factors other than a tolerance of osmotic pressure

(i.e., low water activity) may be important in determining molds that are likely to

appear in honey.

The low mold counts reported by Piana et al. (1991) suggest that molds may

survive but do not tend to grow in honey, which is confirmed by recent experiences

in the industry. One industry report (unpublished data) recorded the absence of

mold in 40 out of 50 batches of honey, with the other 10 batches containing no

more than 15 cfu/g. High mold counts may be indicative of the recent addition of

mold, perhaps from growth in the foraging environment of the bee, in the hive, or

on processing equipment.

2.2.2. Yeasts

Yeasts can grow under acidic conditions and are not inhibited by sucrose.

Osmophilic or sugar tolerant yeasts are a problem in the honey industry, because

they can grow even at the limited level of water available in ripe honey. As a result,osmophilic yeasts readily ferment honey.



J.A. Snowdon, D.O. Cliver / Int. J. Food Microbiology 31 (1996) I --26 9

Conditions that encourage fermentation in honey include increased moisture,

moderate temperatures, granulation, a high yeast count and the presence of ash

and nitrogen (Crane, 1979). During fermentation, the yeast acts upon the sug-

ars, producing alcohol and carbon dioxide. In the presence of oxygen, the alco-

hol may be converted into acetic acid. Fermentation usually happens in

micro-environments (such as the top of a barrel of honey) where the water

content has increased. This subject is reviewed comprehensively in beekeeping

textbooks (Crane, 1979; Root, 1983) and scientific journals (Tysset and de Raut-

lin de la Roy, 1974).

Succhuromyces spp. represents the dominant yeast found in honey (Tysset and

Rousseau, 1981) but other genera have been reported. Tysset and de Rautlin

de la Roy, 1974 recovered Rhodotorula as well as Succharomyces from honey.

Furuta and Okimoto (1978) list Debnryon?yces, Hansenula, Lipomyces, Oosporid-

i um, Pi chiu, Succharomyces, Torul opsis, and Trichosporan as genera of yeast re-

covered from Japanese honey. Studies since the turn of the century, assembledby Crane (1979), report the following yeasts: Nematospora, Saccharomyces,

Schizosct ccharomy ces, Schwanni omy ces, Torul u and Zygosacchuromyces.

The concentration of yeasts is proportional to the availability of moisture

(Crane, 1979; Quilez and Barrado, 1976). Honey made from flowers in humid

regions has more yeast and can spoil in the comb (Tysset and Rousseau, 1981).

Piana et al. (1991) assayed 50 samples of Italian honey and primarily found

osmophilic yeasts in the range of l-3500 cfu/g, only 34 of the samples con-

tained osmophilic yeasts. An average of 254 cfu/g of yeasts and molds, with a

range of 0 to 2500, were found by Tysset et al., 1970b in honey. However,

there were fewer than 10 cfu/g in 64% of the samples. Combined mold and

yeast counts averaging 90/g (range O&2500) and osmophilic yeast counts averag-

ing 102/g (range 0- 10 500) were reported in a survey of 175 samples of honey

(Tysset and Rousseau, 1981). Yeast counts averaging 9 cfu/g (ranging from

O&300) were observed by Nakano and Sakaguchi (1991) during the analysis of

270 retail honey samples. A range from 0 to 10000 (with the majority between

10 and 100) was attributed to Italian scientists in the report of Tysset et al.

(1970b). It is generally accepted that the number of yeast spores in various

honeys can vary a million-fold from 1 in 10 g to 100 000/g (Graham, 1992). In

one study, osmophilic yeasts were found only in samples of honey with a water

activity higher than 0.65 (Piana et al., 1991). Assays of 320 samples of Cana-

dian honey demonstrated sugar-tolerant yeasts ranging from 0.1 to 1000000

cfu/g (Root, 1983).

As fermentation is proportional to the concentration of yeast, honey with a

very high yeast count is not likely to be palatable or marketable. Out of 50

samples of finished honey assayed by a packer in the honey industry (unpub-

lished data), only 10 contained yeasts. Although the highest count was 315

cfu/g, the median was zero, and four of these samples contained osmophilic

yeasts, ranging up to 250 cfu (average; 6 cfu/g). Because of industry control

efforts, yeast counts in finished honey are not likely to exceed a few hundredcfu/g.



10 J.A. Snowdon, D.O. Cliwr 1 ht. J. Food Microbiology 31 (1996) l-26

2.2.3. Bacteria

2.2.3.1. Species, quantity and frequency of isolation. With the exception of research

on C. botulinum, very little quantitative measurement of bacteria in honey is

reported in the scientific literature (Table 4). Nakano and Sakaguchi (1991) tested

270 honey samples (from retail supplies in Japan) and recorded a mean aerobic

plate count of 83 cfu/g. Tysset et al. (1970b) tested 14 samples of ‘freshly harvested’

French honey and found total plate counts of less than 100 cfu/g. They were unable

to detect E. coli, Streptococcus D (now Enterococcus), sulfite-reducers or Staphylo-

coccus. In 198 1, Tysset and Rousseau examined 175 samples of ‘commercial’ honey

from different geographical regions of France and found the average total plate

count to be 227 cfu/g. These figures are within the range of current industry

experience where the bacterial levels of finished honey tends to range from 1 to

5000 cfu/g, with lower numbers possible with additional treatment. Variation inbacterial numbers may be due to the type of sample (raw, finished or retail), the age

of the honey, the time of harvest and ‘the analytical technique.

Qualitative examination of the organisms recovered on total plate counts from 12

samples of French honey (Tysset et al., 1970b) found the following organisms:

Bacteridium (sic), Bacterium (sic), Bacillus, Brevibacterium, Enterobacter, Flavobac-

terium, Micrococcus, Neisseria, Pseudomonas, and Xanthomonas. The most numer-

ous isolates were members of the genus Bacillus, specifically B. cereus and B. pujilus.

Thirteen percent of the samples contained Micrococcus and Pseudomonas (which

the authors attribute to the intestines of bees). Micrococcus, Pseudomonas, and

Staphylococcus have recently been found in industrial honey in the USA (unpub-

lished data). Staphylococcus was found in 3 of 25 samples. Micrococcus was found

in 4 of 12 samples and Pseudomonas was found in 1 of 8 samples. Tysset et al.,

1970b also found Flavobacterium lactis in honey and suggested that it was probably

introduced by man via contaminated water. Vegetative microbes such as these are

likely to be introduced into honey via secondary contamination and hence may be

expected to appear in honey on a sporadic basis. The authors concluded that

‘natural honey’ contains very few types of microbes and is devoid of vegetative,

non-spore-forming bacteria.

The incidence of Bacillus and Clostridium spores in honey from processing plants

and retailers has been studied (Kokubo et al., 1984). Spores were found in 67 of the

71 (94%) of the honey samples tested at levels of lo-100 spores per gram. Most of

the spores were facultative and in the genus Bacillus. The predominant species was

B. cereus followed by B. coagulans, B. megaterium, and B. alvei. Fifty- six of 71

(79%) of the samples contained clostridial spores, with 6 samples containing C.

perfiingens. None of the honey samples in this study contained C. botulinum. B.

cereus spores were found in 24 of the 50 samples tested by Piana et al. (1991) with

counts ranging from 0.1~ 1 cfu/g. C. perfringens or C. botulinum was not recovered,

although unidentified anaerobic spores were found in 22 of the 50 samples, at levels

between 0.1 - 1 cfu/g. Spores of aerobic species were found in all samples, at levels

ranging from l-67 per gram.



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12 J.A. Snowdon, D.O. Cliuer /Ini. J. Food Microbiology 31 (1996) l-26

There are over a dozen reports in the scientific literature on the incidence of

botulinal spores in honey (Sugiyama et al., 1978; Midura et al., 1979; Flemming

and Stojanowic, 1980; Hartgen, 1980; Huhtanen et al., 1981; Kautter et al., 1982;

Stier et al., 1982; Guilfoyle and Yager, 1983; Kokubo et al., 1984; Aureli et al.,

1985; Sakaguchi et al., 1987; Hauschild et al., 1988; Nakano and Sakaguchi, 1991;

Nakano et al., 1989; and Du et al., 1991). These reports represent information on

honey from all over the world. Of the 2033 total samples tested in the studies listed

above, 104 (5.11%) contained detectable levels of botulinum spores. Many of the

studies detected no botulinum spores at all. One research group, while evaluating

the efficacy of various techniques to detect botulinum spores in honey, recorded

that 62% (15 out of 24) of their samples contained botulinum spores (Stier et al.,

1982); their work highlights variation in test results between samples and analytical

techniques. Most of the studies found botulinum spores in 5 to 15% of samples.

Typically, botulinum spores were found at levels below l/g of honey (Sugiyama et

al., 1978; Stier et al., 1982; and Nakano et al., 1989). Midura et al. (1979) assayedhoney suspected of transmitting infant botulism, and estimated (sic) the concentra-

tion of botulinum spores as being from 5 to 80 spores per gram. Nakano et al.

(1989) found that 1 of their 270 honey samples contained high levels of spores

(36660/g) with the remainder containing < l/g. The reason occasional samples of

honey contain higher than usual levels of spores is not known. It can be concluded

that botulinum spores are present in honey some of the time (about 5%) and

typically at low numbers ( < l/g). Nonetheless, honey is an inappropriate food for

infants because infants have a limited intestinal microflora and are susceptible to

developing infant botulism (Snowdon, 1991).

From 0 to 60 spores of C. botulinurn were detected, per gram of honey, in 8.5%of the 270 samples tested by Nakano et al. (1989). Spore counts were most common

in samples collected from apiaries (23%) followed by samples from drums (18%),

followed by samples from retail packages (5%). This corresponds with reports from

Sugiyama et al. (1978) who reported a higher incidence in samples taken from

apiaries than from retail supplies. Nakano et al. (1989) also observed a decrease in

viable spores after honey was stored for a year at 25°C and concluded that ‘some

processes during purification and prolonged storage contributed to the lower

incidences in marketing honey’. Huhtanen et al. (1981) sought to determine why

numbers of botulinal spores vary so greatly among honey samples. They found

spores in 3 of 20 (15%) of the bulk samples and only 4 of 60 (6.6%) of the samples

from apiaries. Counts for Bacillus spp. varied from 0 to 180 cfu/g and counts of

anaerobic microbes from 1 to 132 cfu/g. They also detected Alcaligenes, Pseu-

domonas, Flavobacterium and gram-positive asporogenic bacteria.

Huhtanen et al. (1981) inoculated bees with C. botulinurn spores and recorded the

appearance of botulinal spores in the honey in their hives. They found no evidence

that C. botuknum multiplied during honey-making and did not detect any anaerobic

organisms, such as C. botulinurn, in the digestive tracts of the bees. Hartgen (1980),

assayed 210 commercial samples of honey from retail markets in South Bavaria and

found no botulinal toxin. C. botulinurn must multiply for toxin to be produced.

Growth of C. botulinurn in ripe honey has never been demonstrated and is unlikely.



J.A. Snowdon, D.O. Clivrr / hi. J. Food Microbiology 31 (1996) l-26 13

Over 100 strains of yeasts and bacteria isolated from honey were studied by

El-Leithy and El-Sibaei (1972). Six strains of Bacillus spp. were studied further via

inoculation into different solutions of honey and water; none could multiply in

solutions containing over 50% honey or more acidic than pH 4. Only moderate

growth was reported at sugar concentrations as low as 30%. Though bacteria maybe unable to multiply in mature honey, perhaps, as has been suggested for

Clostridia, bacterial growth can occur while the nectar is ripening into honey.

2.2.3.2. Survival. Most studies on bacterial survival in honey involve the introduc-

tion of vegetative pathogenic organisms, which are not normally present in honey

(Table 5). Sackett (1919) hypothesized that bees could carry pathogenic microbes

from human excrement to honey. He demonstrated that 10 species of bacteria

(non-spore-forming intestinal bacteria) inoculated into pure honey survived only a

few hours or a few days. Solutions of less than 50% honey in water sustainedbacterial life for longer periods, but never exceeding 40 days. Sackett concluded

that ‘the probability of honey acting as a carrier of typhoid fever, dysentery and

various diarrhoeal affections is very slight’.

The survival of some gram-negative bacteria in commercial honey was studied by

Tysset and Durand (1973). These bacteria were similar to Sackett’s species and are

of interest because they may cause human illness. The investigators inoculated

‘aseptically-collected honey’ with various bacteria, held it at 20°C (68°F) and

monitored the change in bacterial numbers. Loss of bacterial viability was observed

within 8 to 34 days (dependent upon the species). In another study, Tysset and

Durand (1976) investigated the survival of similar bacteria at 10°C (50°F) in freshly

harvested honey of mixed flower sources. The bacteria survived from 6 months to

almost 2.5 years. The authors emphasized the need for vigilance not to introduce

these microbes into honey and cautioned against overestimating the antibacterial

properties of honey. In another experiment Tysset et al. (1979) reported survival

times, at 20°C (68”F), ranging from 26 to 77 days for various species of M~~obuc-

terium, including the species responsible for tuberculosis. Molan, 1992b suggests

that bacterial survival differs among different types of honey.

Staphylococcus was recovered from 3 of 12 samples of raw honey being prepared

by a US honey packer (unpublished data). No staphylococci were recovered from

13 finished samples, and in no instance was a pathogenic species (e.g. Staphylococ-

cus aureus) recovered. Staphylococcus appears to be present in honey only rarely,

appears not to survive processing, and is not likely to grow.

The survival of spore-forming bacteria found in commercial honey was studied

by Kokubo et al. (1984). B. cereus, C. perfringens, and C. botulinurn spores were

inoculated into honey and stored at 25°C (77°F) for 4 months. The spore counts

remained the same. The C. botulinum spore population in honey did not change in

over a year when stored at 4°C (39°F; Nakano et al., 1989). At 25°C (77°F)

however, the number of spores began to decrease after 100 days. No spores were

detected after 5 days of storage at 65°C (117°F). These findings indicate that sporesmay persist in honey, but do not germinate to vegetative cells that might multiply.



14 /.A. Snowdon, D.O. Cli ver / Int . J. Food M icrobi ology 31 (1996) l -26

In conclusion, spore-forming bacteria, molds and yeasts are typically found in

honey, often at low numbers, while spores can persist indefinitely. Vegetative forms

of pathogenic bacteria have never been found in honey; if introduced, they can

survive in honey for extended periods of time, particularly at cool temperatures.

Microbial survival may be influenced by the type of honey and its moisture content.

Table 5

Survival of vegetative bacteria in honey

Species Tempterature (“C) Survival time Reference

Non-spore-forming intestinal

bacteria

Edwardsiella tarda

Room temperature A few days Sackett (1919)

20 i 10 days

Erwinia amylovora 4 8 weeksEscherichia coli 20 < 10 days

M ycobacterium

chelonie 20 26 days

phlei

tuberculosis

tuberculosis bovis

tuberculosis avium

Proteus vulgaris

Pseudomonas aeruginosa

Salmonella

derby

dubl in

20 17 days

20 67 days

20 77 days

20 71 days

20 < 10 days

20 8h

10 6 months, 13 days

10

20

2 years, 4 months 12

days

enterit idis

enterit idis

typhi

typhi

typhimurium

typhimurium

Serratia marcescens

Shigella

34 days

10

20

t year, 1 t months, 5

days

26 days


20

10

30 days

2 years, 4 months, 12

days

20 18 days


Tysset and Durand

(1973)

De Wael et al. (1990)Tysset and Durand

(1973)

Tysset and Durand

(1973)

Tysset and Durand

(1973)

Tysset et al. (1979)



Tysset and Durand

(1973)

Tysset and Durand

(1973)

Tysset and Durand

(1976)

Tysset and Durand

(1976)

Tysset and Durand

(1973)

Tysset and Durand

(1976)

Tysset and Durand

(1973)

Tysset and Durand

(1976)

Tysset and Durand

(1973)

Tysset and Durand

(1976)

Tysset and Durand

(1973)

Tysset and Durand

(1976)



J.A. Snorwlon, D.O. Cliuer / Ini. J. Food Microbiology 31 (1996) I-26 15

Table 6

Factors that might contribute to the anti-microbial nature of honey

High osmotic pressure, low water activity (A,)

Low pH-acidic environment

Glucose oxidase system ~~ forms hydrogen peroxideLow protein content

High carbon to nitrogen ratio

Low redox potential (Eh), due to high content of reducing sugars

Viscosity opposes convection currents and limits dissolved oxygen

Chemical agents

Pinocembrin

Lysozyme

Acids (phenolic)

Terpenes

Benzyl alcohol

Volatile substances (perhaps phytochemicals influenced by bee enzymes)

(Molan, 1992a; and Tysset and de Rautlin de la Roy, 1974).

2.2.4. Other microbes

Microbes other than yeasts, molds and bacteria could be present in honey. Algae

may be present in honeydew honey and in ‘honey sediment’ under certain climatic

factors such as when the relative humidity is high (Crane, 1979). The skeletons of

dinoflagellates could be introduced into honey from diatomaceous-earth filters, but

are non-viable. Some human enteric viruses, such as hepatitis A, sustain dry

conditions. Human enteric viruses could be expected to persist in honey, their

numbers decreasing at rates dependent upon temperature and time. As human feces

are the exclusive source of human enteric viruses, control measures that keep

human feces out of food will also preclude the presence of human enteric viruses.

Protozoa and multicellular parasites are not likely to be transmitted through honey.

These microbes are given no other consideration in this paper due to lack of

information in the scientific literature.

2.2.5. Summury

Molds and yeasts are the only microbes that have been reported to grow in

honey. Certain bacteria will survive in honey but growth is unlikely. Vegetative

bacteria inoculated into ripe honey for experimental purposes die off within a few

weeks, unless the honey is stored at cool temperatures. The hostile conditions found

in ripe honey increases the demise of most bacteria. In practice, the spores of

Bacillus, molds and yeasts tend to be present in honey on a regular basis. Clostridial

spores (typically of species other than C. botulinum) may also be present. Reports

from industry (unpublished data) indicate that Pseudomona and Micrococcus

might also be found and that total numbers vary unpredictably. No information isavailable on viruses or parasites.



16 J.A. Snowdon, D.O. Cliver / Int. J. Food Microbiology 31 (1996) I-26

2.3. Ant i-mi crobial propert ies of honey

Honey has unique properties that render it bacteriostatic and bactericidal (Table

6). The subject of the antibacterial properties of honey has been recently and

extensively reviewed (Molan, 1992a). Limited information is available on other

anti-microbial properties (e.g., honey’s effect on viruses, fungi or parasites).

The inhibitory properties of honey were noted early in the century by Sackett

(1919) and others. The responsible agent was called inhibine, but White et al.

(1962a) later identified this factor as hydrogen peroxide. The peroxide is formed by

the glucose oxidase system, which is an enzymatic process that is active only in

unripe or diluted honey. The enzyme (glucose oxidase) is heat-labile and is

destroyed by pasteurization. The system breaks down a small amount of glucose,

producing gluconic acid and hydrogen peroxide. ‘Molds and yeasts, do not appear

to be as sensitive to peroxide as bacteria’. (White et al., 1962a).

In a study of honey from eight different floral sources, Smith et al. (1969) showed

bacteriostatic effects (at 100% concentration) against B. cereus, M i crococcus fl uuus,

and Surcina lutea. It was found that not all honey is equally effective against all

microbes all the time; one of the eight types of honey under scrutiny, for example,

did not inhibit B. subtilis.

Gram-negative bacteria inoculated into honey were found to decline rapidly

Tysset and Durand, 1973. Tysset and de Rautlin de la Roy (1974) suggested that a

variety of factors may be responsible for the antibacterial nature of honey. The low

protein content and high carbon-to-nitrogen ratio of honey are not conducive to

microbial growth, nor is the acidity of honey. The low redox potential of honey

(which is due to its high content of reducing sugars) discourages growth of moldsand aerobic bacteria, while the viscosity of honey opposes convection currents and

limits the entry of dissolved oxygen. As the osmotic pressure is high, the microbes

shrivel as water flows out of their cells into the surrounding honey.

Since honey has antibacterial activity against B. cereus, the low incidence of this

organism in foraging worker bees might be due to honey consumption by the bees

(Gilliam, 1978). Honey contains unknown factors active mainly against gram-nega-

tive bacteria and higher fungi such as Aspergillus (Radwan et al., 1984).

The antibacterial activities of a variety of honeys from New Zealand were tested

against Staphylococcus (Allen et al., 1991). Experimentation with 345 unpasteur-

ized, monofloral samples (26 floral sources) ascertained that the floral source madea difference in the antibacterial activity of the honey. Honey derived from manuka

and vipers bugloss flowers had antibacterial activity due to a non-peroxide compo-

nent. Willix et al. (1992) determined that honey with non-peroxide antibacterial

activity and honey with antibacterial activity due to hydrogen peroxide are both

effective in preventing the growth of bacteria that infect wounds. However, the

order of efficacy among the seven bacterial strains was dependent upon the nature

of the antibacterial activity in the honey. Molan (1992~) studied the same samples

of honey and showed that manuka honey has the strongest antibacterial action

against E. coli , Hel i cobacter pyl ori , Prot eus mi rabi l i s, Pseudomonas aeruginosa,

Salmonel l a t yphi mur i um, Serr at i a marcescens, S. aureus, and Streptococcus pyoge-nes.



J.A. Snowdon, D.O. Cli ver / t. J . Food M icrobiology 31 (1996) 1-26 11

An extensive review of the antibacterial properties in honey was published by

Molan (1992a). He proposed that the antibacterial properties of honey are due to

acidity; osmolarity; the conversion from glucose to hydrogen peroxide via glucose

oxidase upon dilution of the honey; and other less clearly defined factors such as

pinocembrin (an antibacterial component of honey), lysozyme, acids (phenolic and

others), perhaps terpenes and benzyl alcohol and volatile substances (perhaps

phytochemicals influenced by bee enzymes). Dozens of species of bacteria have been

found to be susceptible to honey at a wide range of concentrations. Honey’s

antibacterial factors are reported also to be effective against many fungal species,

including Aspergillus, Candida, Penicillium, and Saccharomyces. Growth of bacteria

and fungi from sewage, soil, air, and tap water has been prevented by solutions

containing as little as 25% honey, while solutions containing as little as 200/o honey

prevented the growth of airborne contaminants.

2.4. Cont rol of mi crobes

There are intrinsic properties of foods that affect microbial growth. These include

pH, moisture content, oxidation-reduction potential, nutrient content and anti-mi-

crobial constituents. As discussed above, honey has a number of inherent qualities

that make it bacteriostatic or bactericidal. Extrinsic factors that have the greatest

influence in determining the presence of foodborne organisms include storage

temperature, relative humidity of the environment and presence and concentration

of gases in the environment (Jay, 1992). Due to the natural properties of honey and

control measures in the honey industry, honey is a product with minimal types and

levels of microbes.

Honey as stored in the comb serves as an excellent model of how to preclude

microbial growth, although dormant forms of microbes may persist in the honey.

Bees produce a food for themselves that is resistant to microbial degradation, by

removing water and creating a barrier to oxygen. Similar principles can be followed

even after the honey is harvested. Packaging the product so as to exclude air and

to prevent cycles of water vaporization and condensation that can cause local

dilution of the sugar will prevent microbial growth. Controlling the moisture

content of the honey and the temperature at which honey is stored are two

examples of how industry controls the growth of microbes in honey. These

protective features vanish when honey is diluted or used as an ingredient. Indeed,

the dormant forms of microbes normally present in honey or ‘hitch-hiking’ mi-

crobes from post-harvest contamination can grow in the formulated food product.

There is little or no control over the exposure of honey to microbes before

harvest. It is not practical to try to control the quality of air, dust, earth and flowers

or the materials carried in by the bees. The conditions in the hive will have some

influence on the microbial quality of the honey. The practices that keep bees

healthy will also influence the type and number of microbes to which honey is

exposed. Moldy combs, for example, could be a source of mold spores in honey.

American foulbrood disease (a common disease of honey bees in North America)is caused by bacteria of the genus Bacil lus and could contribute to the presence of



18 J.A. Snowdon, D.O. Cliver /ht. J. Food Microbiology 31 (1996) l-26

Bacil lus spores in honey. Good beekeeping practices can generally be expected to

keep the number of microbes in honey low; but it has been suggested that using

Bacillus thuringiensis (which is harmless to honey bees) to control wax moth could

result in Bacil lus spores in honey.

Good manufacturing practices for beekeeping and for packing are described

thoroughly elsewhere (Crane, 1979; Graham, 1992). Hence, this paper will make

only brief reference to a few recent reports on the post-harvest control of microbes

in honey. It is important to (1) avoid inoculation of honey with undesirable

microbes and (2) avoid handling or storing the honey in such a way as to encourage

microbial growth. Additionally, it is possible to treat foods such as honey so as to

minimize or eliminate microbes.

Certain microbes that originate in mammals, especially man, could cause illness

if swallowed with honey or other foods. These microbes are carried with skin

infections, in nasal passages and in feces. Soil contaminated with these types of

microbes could transmit disease indirectly. Pathogenic microbes have never beenfound to occur naturally in honey and they do not survive in honey for very long

at 20°C (68°F); however, lengthy survival (Table 5) is possible if honey is stored

below 10°C (50°F). Control of these microbes can be achieved by using sanitary

practices (hand washing, avoidance of sneezing or coughing into food, etc.).

Controlling microbes from sources other than man (air, equipment, etc.) is

equally important, but often more difficult. Troller (1979) suggests limiting the

exposure of honey to the atmosphere and observing good sanitary practices in the

apiary and in the honey processing facility. He also suggests drying honey process-

ing equipment thoroughly after washing so the water activity of the honey doesn’t

increase and allow microbes to grow. Root (1983) urges strict cleanliness at the timeof extraction, and further recommends the use of vessels that are essentially sterile.

He suggests removing any traces of honey or nectar from equipment when

processing is finished, using fastidious practices during the ripening of honey to

avoid a high moisture content, and maintaining the storage temperature below 50°F

(lO”C), or heating to 140-145°F (60&63”C) for 30 min to control the growth of

undesirable spoilage microbes.

Fermentation of honey can be prevented by storage at 10°C (50°F) or below with

a relative humidity below 50% or by pasteurization (Tysset and Rousseau, 1981).

Sugar-tolerant yeasts will not grow below 11°C (52°F) or above 38°C (100°F).

Honey that contains more than 17% water is susceptible to fermentation and honey

over 19% moisture is very likely to ferment. Heating honey to 63°C (145°F) for 30

min will destroy most of the yeasts responsible for fermentation (Crane, 1979,

Graham, 1992). However, temperatures that are cool enough to deter spoilage are,

unfortunately, cool enough to prolong bacterial survival.

Current recommendations for heat-treating honey are probably based on inacti-

vating yeasts and preventing fermentation. Different time and temperature combi-

nations may be necessary to inactivate other types of microbes. Spores are

particularly difficult to inactivate in honey because extreme heat treatment destroys

the organoleptic qualities of honey. Holding food for 30 min at 63°C (145°F) is

generally considered sufficient for the removal of vegetative microbes. Commer-



J.A. Snowdon, D.O. Cliver /ht. J. Food Microhio1og.t~ 31 (1996) l-26 19

cially prepared honey is often heated to 71°C (160°F) for about 30 min; yet viable

microbes can be recovered from finished product. A given combination of time and

temperature may be less effective in honey than other foods (such as milk) due to

the absence of water.

The number of effective combinations of time and temperature to removeundesirable organisms is very large. Inactivation of microbes is often spoken of in

terms of thermal death time. Thermal death time is dependent upon: time,

temperature, concentration of microbes, form (spore or vegetative) of microbe,

stage in the microbial life cycle and health (stressed or robust) of the microbe. The

nature of the food product must also be considered, e.g., sugars might protect

microbes from inactivation. Effective thermal processes can be calculated for

various situations; the assistance of a food microbiologist or food engineer is

recommended. Aseptic packaging may avert microbiological problems after the

honey has been properly processed.

Other physical processes can kill microbes in honey. Shimanuki et al. (1984)

eliminated viable Bacillus spores from honey via two 3 Mrad doses of high velocity

electrons. Huhtanen (1991) determined the irradiation levels necessary for the

inactivation of C. botulinum and B. subtilis spores in honey; the D values ranged

from 1.91 to 12.8 kGy. Spoilage yeasts in honey are very sensitive to ultraviolet rays

(Tysset and de Rautlin de la Roy, 1974). Ten minutes at 20 cm under a germicidal

tube will inactivate microbes in the laboratory; a similar technique could be

developed for use in industry for disinfection of a thin top layer of honey.

Ultrafiltration will remove all bacteria.

Nakano et al. (1989) tried to reduce or inactivate the spores of C. botulinum in

honey. They studied heat shock, sonication, detergents, enzymes, alcohol, acids and

bases; none had a significant effect. They suggest ‘long term storage’ (presumably

about 400 days) at 25°C (77°F) or ‘mild heating’ at 65°C (149°F) for 5 days to

eliminate or reduce C. botulinum spores in honey.

Chemical disinfectants such as copper, iodine, mercury, quaternary ammonium

salts or sodium hypochlorite may be used for cleaning food processing equipment

(Tysset and de Rautlin de la Roy, 1974). Certain antibiotics - nystatin, actidione

or cycloheximide, and amphotericin B - could be used to prevent microbial

growth. Preservatives such as benzoic acid, sorbic acid, sulfites, and CO, are listedas agents that control microbes (Jay, 1992). Preservatives such as sorbic acid or

potassium sorbate and sodium or potassium benzoate are used in pie fillings, jams

and jellies and may have use in controlling microbes in honey. More research, is

definitely needed in this area.

There is increasing interest and activity in the honey industry regarding the

microbial content of honey. In the absence of information from the scientific

literature, the honey industry is starting to generate its own data. Precise or

comprehensive information is lacking, but general questions are being posed and

introductory information is being developed. As a consequence control methods are

being developed to reduce the levels of microbes in the finished product.



20 J .A. Snowdon, D.O. Cl iver / ht. J . Food M icrobiology 31 (1996) l-26

3. Analyzing honey for microbes

No special techniques for the sampling or analysis of honey, for microbes of

significance to human health or honey quality, have been developed and stan-

dardized. Standard methods for microbiological analysis can be adapted for

honey. Details of these techniques are available in standard microbiology or food

microbiology texts such as Jay (1992) or references such as the Bacteriological

Analytical Manual (1984); Official Methods of Analysis (1990); the Codex Ali-

mentarius (1992); Microorganisms in Foods (1982) and the Compendium of

Methods for the Microbiological Examination of Foods (Vanderzant and

Splittstoesser, 1992). There is little information in the scientific literature about

detecting microbes in honey. More information can be expected if research is

conducted and the results are made available.

References in the scientific literature for the detection of microbes in honey

(other than those pathogenic to bees) are primarily for the recovery of botulinalspores (Sugiyama et al., 1978; Midura et al., 1979; Flemming and Stojanowic,

1980; Hartgen, 1980; Huhtanen et al., 1981; Kautter et al., 1982; Stier et al.,

1982; Guilfoyle and Yager, 1983; Hauschild and Hilsheimer, 1983; Kokubo et al.,

1984; Aureli et al., 1985; Sakaguchi et al., 1987; Nakano and Sakaguchi, 1991;

Nakano et al., 1989; and Du et al., 1991). Others who have reported microbial

levels in honey include Tysset et al. (1970b) and Tysset and Rousseau, 1981.

Bonvehi and Jorda (1993) examined a few different methods of determining

aerobic colony counts. They maintain that a membrane filter method eliminates

‘swarming’ and is more accurate.

Standard techniques need to be adapted for use in honey. For example, tradi-tional media for detecting foodborne fungi may underestimate fungi capable of

growing at reduced water activity; some media for moderately osmophilic yeasts

and molds may also detect other types of yeasts and molds (personal communica-

tion, Dr. Larry Beuchat, University of Georgia). Research to identify optimal

analytical techniques for the recovery of yeasts and molds from honey is war-

ranted.

4. Types and levels of microbes expected in honey

4.1. Data currently available

Information on the levels of microbes in honey, as reported in the scientific

literature, is summarized in Table 4. The median value of 14 honey samples from

France, surveyed by Tysset et al. (1970b) was less than 100 cfu/g. Tysset and

Rousseau, 198 1 examined 175 samples of honey from different regions of France

and found a mean total plate count of 227 cfu/g. Thirty-five retail samples of

honey of international origin were found to contain a range of O-72 and a mean

of 24 cfu/g (Nakano and Sakaguchi, 1991). An additional 18 samples from 5 lotsof Argentinean honey (receiving additional scrutiny for high clostridial spore



J .A. Snowdon, D.O. Cli ver / ht. J . Food M icrobiology 31 (1996) 1-26 21

counts) ranged from 21-260 cfu/g. If these data, and the data in Table 4, are

representative of current product in the US, one might generally conclude that

total plate counts will vary from 10 to 10000 cfu/g of honey with low numbers

possible in the finished product.

In addition to the data on total plate counts presented above, some of thereports provide information on selected microbial species. Root (1983) recounts

a Canadian study on yeasts and molds where as few as 1 in 10 g and as many

as a million spores per gram were found among the 320 samples tested (the

fermentation status was not mentioned). In a survey of 14 samples of French

honey Tysset et al. (1970b) found an average yeast and mold count of 254

cfu/g (range o-2500). French researchers studying 175 honey samples found an

average of 90 yeast and mold cfu/g with a range of 0 to 2500 (Tysset and

Rousseau, 1981). Counts for osmophilic yeasts had a were similar, but ranged

up to 10500. An average of 9 yeast colonies per gram of honey was found in

35 retail samples tested by Nakano and Sakaguchi (1991). It appears that while

yeasts and molds can grow to high numbers in honey, microbial levels are

controlled by standard industry practices that prevent fermentation.

Bacillus spore counts reported by two research groups suggest that levels of

BacilZus spores in honey are likely to be below 200 per gram. Tysset et al.

(1970b) found less than 100 spores, and Nakano and Sakaguchi (1991) from 0

to 180 cfu/g. This is in contrast to recent industry reports (unpublished data)

which include at least one sample where counts of BacilluS were closer to

10000/g; these high counts are thought to be the exception, not the norm.

Great variation in levels of Bacillus spores among samples has also been ob-served.

Tysset et al. (1970b) also looked for spores of Clostridium, but found none in

any of their 14 samples. From 1 to 132 anaerobic microbes (likely to be

Clostridium) were found in 35 retail samples of honey (Nakano and Sakaguchi,

1991). Typically, C. botulinum spores are found at levels ( l/g of honey

(Sugiyama et al., 1978; Stier et al., 1982; and Nakano et al., 1989). In rare

instances, a sample of honey has been found to contain from 36660 C.

botulinurn spores/g (Midura et al., 1979; Nakano et al., 1989).

Information from industrial experience is increasing but is not widely avail-

able. A compilation of six different reports on total plate counts in honey from

laboratories in the US showing an overall mean value of 2270 cfu/g is summa-

rized in Table 4. No coliforms, E. coli, yeasts, molds, Staphylococcus or

Salmonellu were found in these six examples. Total plate counts of finished

products observed in informal industry reports tend to range in 104-lo5 cfu/g

of honey. Total plate counts in raw product are more variable and can reach

104-lo5 cfu/g. Additionally, specific microbes can appear in raw honey in high

numbers (104-lo5 cfu/g) on an occasional and erratic basis. Bacillus species and

Pseudomonas have both been found at high levels (thousands per gram) in raw

honey. It is unknown if the microbes originate from primary or secondarysources.



22 J.A. Snowdon, D.O. Ciiver 1 ht. J. Food Microbiology 31 (1996) l-26

4.2. Expected impact of processing upon microbes in honey

Honey prepared for table use is subjected to minimal processing. It is typically

heated to as much as 70°C (160°F) for 30 min. Strained honey is passed through a

1.50pm screen, while filtered honey is often passed through a filter with 1 pm pores.

The process of heating and filtering may reduce or eliminate many microbes.

Honey used as an ingredient is subjected to several different processes. Sold in

bulk, it has had similar treatment as honey for table use. Some honey is dried to

crystalline form before being sold as an ingredient. Additional treatment is depen-

dent upon the food with which the honey is combined. Most of the processes will

inactivate any microbes present, usually due to high heat. New products and new

processes all need to be evaluated from the view of microbiological safety.

The cereal and bakery industries are the two largest consumers of honey. Honey

is also used in condiments, salad dressing, barbecue sauce and peanut butter. Dairy,

meat, beverage, snack and candy manufacturers also use honey as an ingredient.Short summaries of the processes associated with foods where honey is used as an

ingredient are presented in Table 7. Honey is a product that is free of most

microbes, and those microbes that may be present are likely to be in very low

Table 7

Processing of foods containing honey

l BREAD: In bread, honey is added to the dough as part of the fermentation process. The dough

is held at 36°C (96’F) at 80&85’%1 humidity for IO-25 min. This is followed by baking at 199-

216°C (390&420”F) for 20 min; the internal temperature of the bread reaches 96°C (204°F).

0 CANDIES: The manufacture of honey candies usually involves blending and heat treatment.

Heat treatment may include holding at lO4- I 16°C (220&240’F) for 30-40 min. These tempera

tures will inactivate many microbes.

0 CEREAL: In some types of cereal production, honey is incorporated into the cereal and baked

at temperatures exceeding 149°C (300’F). In other types of cereal, honey is blended into a slurry

which is used to coat the cereal. The temperature of the slurry is between 82288°C (180-190°F).

The coated cereal enters a dryer for lo-30 min at 999104°C (2lO-220°F).

0 GRANOLA: In making granola, honey is mixed with butter and sweeteners and heated to 70°C

(IWF). The mixture is cooled to 45550°C (I l3- 122°F) mixed with the granola, cooled to

4.441O”C (40&50”F) and packed at about 15°C (59’F).

0 HAM: In making a honey-cured ham, the meat is injected with a solution that includes honey.

The meat is held at approximately 73°C (164°F) for 8 h. The meat is then chilled and prepared

for sale. Other meat products, such as a honey beef stick, are made with an emulsion which

contains honey. The emulsion is heated to 60°C (140°F) before it is packed into a casing. These

processes will inactivate many microbes. Fresh sausage with honey is not heated at all; it remains

refrigerated at about 3.3’C (38°F) and is intended to be cooked thoroughly before being eaten.

0 PEANUT BUTTER: Honey and other dry ingredients are added to ground peanuts in making

peanut butter. The mixture is ground, oxygen is removed and the product is heated to 88°C

(190°F). It is then cooled and packed. A number of conditions (absence of oxygen, low water

activity, heat) make microbial persistence unlikely.

0 SALAD DRESSING AND MUSTARD are generally not heat processed. Honey would be

blended in with agitation. The factors that ensure microbial stability of the dressing or mustard

(e.g. preservatives, low water activity) are not likely to be affected by the addition of honey.

(Personal communication, Jill Clark, Dutch Gold Honey, Lancaster, PA.).



J.A. Snowdon, D.O. Clicer i hr. J. Food Microbiology 31 (1996) l-26 23

numbers. Most of the processes involve heat treatments that would inactivate any

microbes contributed by honey. Control of microbial growth in products that are

not heated depends on other anti-microbial procedures or agents.

4.3. Comments on microbial speciJications

Purchase specifications for microbes in honey vary widely (Table 1). They are

often based on information pertinent to other foods. Tysset et al. (1970b) suggested

that honey is no longer marketable if it contains over 1000 yeast spores/g. The

authors do suggest that such honey could be used in products requiring high heat

(e.g. making candy). Tysset et al. (1970b) recommended measuring total counts,

fecal contamination (via testing for E. col i or Streptococcus D (sic)), and anaerobic

sulfite reducers (as an indicator of C. perf%zgens). The authors of this paper

comment on current specifications below.

(1) The standard plate count provides very general information and is useful as apoint of comparison to other data and as a general indicator of the microbial

quality of honey. Additional testing to identify the type of microbe present is

often needed in conjunction with high standard plate counts. Honey with fairly

high standard plate counts (10 000/g) could be acceptable if other microbial

criteria (e.g., indicating presence of yeast or freedom from fecal contamination)

were satisfied.

(2) Yeasts and molds can be expected to be found in honey on a regular basis, but

levels can be controlled by standard industry practices. A routine count for

yeasts and molds combined would be an economical way to indicate the quality

and condition of honey, as well as predict shelf life and spoilage potential. As

mold counts are typically low, their inclusion may not be necessary; however,

some commercial laboratories only provide the combined tests, making it more

convenient to count both types of microorganisms.

(3) Coliform counts are an indicator of sanitary practices. Since fecal contamina-

tion of honey has not been reported, and growth of any associated microbes

does not appear possible, an assay for coliforms could also be used as a general

indicator of fecal contamination as well as sanitation. As such, it could replace

tests for specific pathogens of fecal origin (such as Salmonel la).

(4) E . col i counts are a specific indicator of fecal contamination and, consequently,

sanitation relative to fecal contamination. As fecal contamination is not regu-

larly associated with honey, this assay may not be necessary.

(5) Although vegetative bacterial cells such as Salmonel la may survive in honey,

they will not multiply, and there have been no reports of their presence in

honey. Vegetative bacterial cells can be controlled by thermal processing.

Testing for Salmonel la could be applicable for those that are producing

products with no heat treatment. A general indicator of sanitation might make

this assay unnecessary.

(6) Staphylococcus is unable to reproduce in honey; hence, its toxin will not be

produced in honey. The chances of a food handler introducing Stuphylococcusinto honey are no greater than for any other food and are best controlled by



24 J.A. Snowdon, D.O. Cliuer 1 ht. J. Food Microbiology 31 (1996) 1-26

good manufacturing practices. No vegetative pathogenic species of bacteria have

ever been recovered from honey. Hence, testing for this microbe or its toxin is not

indicated.

(7) The spores of bacteria in the genera Bacillus and Clostridium can be expected to

be found in honey. Although typically found below about 200 cfu/g, reasonably

high levels (thousands per gram) of Bacillus could be present with impurity

depending upon the species and the nature of the product. If present, C. botulinum

spores are typically found at levels of < 1 cfu/g, but are infrequently found as high

as 60 cfu/g.

(8) When a particular microbe becomes a problem, additional special testing for

identification and confirmation may be necessary.

In short, one scheme for testing would be a total plate count, a count for yeasts

and molds, a coliform count and a count for total spore-forming bacteria. Addi-

tional tests ~ to explain unusually high counts, measure osmophilic microbes, or

address a certain problem - may be needed. The use of honey in products thatreceive no or limited heat treatment may require additional tests. Since the

information reported in the scientific literature is limited, these ideas should be

revised after industry data are collected. Research in the areas of microbial quality

in honey is needed to answer more of these questions with accuracy and precision.

Acknowledgements

The authors would like to thank L. Beuchat and H. Shimanuki for their reviewof this manuscript.

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