Apart from an ubiquitous microflora, this investigation into 52 samples of honey revealed some undesirable

spore-forming bacteria, Bacillus alvei and B. larvae which are bee pathogens. Bacillus cereus can cause

spoilage and food poisoning.

It is, therefore, considered essential that every country includes microbiological standards in its Food Safety

Regulations for honey, so that the consumer is guaranteed as to the wholesomeness as well as the quality

of the product

Agostini, Franco

Deiana, Pietrino

Farris, Giovanni Antonio

Fatichenti, Fabrizio

UnissResearch

ANNAbI 
DELLA FACOLTA' DI AGRARIA DELL' UNIVERSITA' 
SASSARI 
DIRETTORE: G. RIVOIRA 
COMITATO DI REDAZIONE: M. DATTILO - S. DE MONTIS - F. F;\TICHENTI 
C. GESSA - L. IDDA - F. MARRAS - P. MELIS - A. MILELLA - A. PIETRACAPRINA 
R.PROTA-A. VODRET 
studi s.ss.r.sl 
ORGANO UFFICIALE 
DELLA SOCIETÀ SASSARESE DI SCIENZE MEDICHE E NATURALI 
:stituto di Microbiologia agraria e tecnica. Università degli Studi di Sassari 
(Direttore Pro!. F. Fatlchentl) 
G.A. FARRIS - F. FATICHENTI - P. DEIANA - F. AGOSTINI 
AEROBIC ANO ANAEROBIC SPORE-FORMING BACTERIA IN SAROINIAN HONEY· 
RIASSUNTO 
I risultati della presente indagine hanno evidenziato che nei 52 campioni di miele esaminati, ad una miero-
flora casuale ed ubiquitaria si associano altre forme a significato ben preciso. È il caso del B. alvei e del 
B. larvae, i quali possono avere effetti negativi sulla sanità delle popolazioni delle api; del B. cereus, spesso 
legato a tossinfezioni alimentari o ad alterazioni degli alimenti; del CI. beijerinckl/, responsabile di fermenta-
zioni butirrica e acetica. 
Per tali considerazioni è indispensabile che tutti i Paesi inseriscano nelle proprie legislazioni riguardanti 
il miele, anche i parametri microbiologici, al fine di garantire il consumatore sulla qualità e sanità del prodotto. 
SUMMARY 
Apart trom an ubiquitous microflora, this investigation into 52 samples of honey revealed some undeslrable 
spore-forming bacteria, Bacillus alvei and B. larvae which are bee pathogens. Baciflus cereus can cause 
spoilage and food poisoning. 
It is, therefore, considered essential that every country includes microbiological standards In its Food Safe-
ty Regulations for honey, so that the consumer is guaranteed as to the wholesomeness as well as the quali-
ty of the producI. 
INTROOUCTION 
Like ali complex biological foodstuffs, honey is subject to changes which may partly 
ìmpair its nutritional and organoleptic properties. Some of these changes occur 
• Work perfomed with Ministry of Public Educa!lon con!rlbullon (Research ProJect Inves!lga!lon m!o !he sIate 01 
apiculture in Italian Regions with the obJect of increasmg producllOn Coordlnator: Pro!. Romolo Prota, Unlverslty 
of Sassari). 
174 
simply with age, whereas others depend upon the methods used in processing and 
conditions of storage. Variations in water content and acidity are particularly im-
portant; e.g., increase in water content with decrease in acidity favours microbial 
growth. 
Within certain limits, water content variability as a hazardous factor would depend 
upon the number of microorganisms and spores present (Leuveant, 1968; Troller, 
1979), but to obviate microbi al development 20% moisture should not be excee-
ded. This safety limit does not seem to be universally respected. In Sardinia, for 
example, an examination of 26 samples showed that 10 of them had a water con-
tent of well over the 20Ofo mark, one indeed reaching 250/0 (Campus et al., 1983). 
The law passed in Italy on 22nd October 1982 (following the EEC directive to equa-
lize legislation on honey) does not cover the question of microbial content; nor does 
it deal adequately with the problem of moisture. Article Il, section 2, states ccwater 
content: not more than 21 % ; (but) for heather, clover and strawberry-tree honeys: 
not more than 23%». 
Generally, honey is not a good substrate for the development of microorganisms, 
but some, for instance osmophilic yeasts and moulds, manage to do so and cause 
spoiling (Farris et aL, 1982; Malan and Ozino-Marletto, 1974; Marletto et aL, 1975; 
Miniria, 1976; Vecchi and Zambonelli, 1979; Yamazaki et aL, 1975). Regarding the 
presence of bacteria, the literature appears to be not very extensive, being limited 
to a generi c count of some sporogenous forms, examining the survival capacities 
of certain pathogenic species (Hornitzky, 1982; Tysset and Durand, 1973; Tysset 
et al., 1979), and the detection of: the non-sporing Glucanabacter sp. and Lacta-
bacillus viridescens (Ruiz-Argueso and Rodrigues-Navarro, 1973), the aerobic spore-
forms Bacillus larvae, B. alvei, B. thuringiensis, B. subtilis, B. megaterium (Bur-
ges, 1976; Farris et aL, 1982; Tysset and Rousseau, 1981; Vecchi and Zambonelli, 
1968); and the anaerobic spore-forms Clostridium botulinum (Arno n et al., 1979; 
Bentler and Frese, 1981; Huhtanen et aL, 1981; Midura and Arnon, 1976; Sugiya-
ma et aL, 1978). As is well known, ingesting the latter with honey can give rise to 
botulism particularly in 1 to 6 month-old babies (Aureli and Accorti, 1981; Midura 
and Arnon, 1976; Pickett et al., 1976). 
Thus, microbiological as well as chemical and physical standards are necessary 
in arder to establish satisfactory food-safety regulations for commerciai honeys. 
However, a great deal more information is required on their microbiological com-
position for legislation to be adequate. It is hoped that the results reported in this 
paper will help towards this end. 
175 
MATERIALS ANO METHOOS 
The 52 samples of honey examined came from different localities in Sardinia, 34 being 
of multifloral origin and 18 unifloral'. 
Most C'f them had been stored for 1 year, 8 for 6 months and 1 for 2 years. Ali were 
taken 1 rom honeys produced in movable frame hives and which had undergone no 
commerciai processing. 
The analysis of each sample was carried out by first heating 100g in 300-ml flasks 
to 80°C and continuously agitating at this temperature for 10 min. Of each heated 
sample, 10g were then taken and dilutions prepared in sterile physiological saline so-
lution and replicated 3 times. For the Bacillus spore count, 1 mi of each dilution was 
poured into a petri-dish containing nutrient agar (Oifco); for the Clostridium spore count 
- Weinzirl method (Weinzirl, 1916) - 1 mi of each dilution was poured into a test-tube 
containing liquid RCM (Reinforced Clostridial Medium, Oxoid) and heated at 80°C 
for 2 min to melt the paraffin seal and eliminate any possible oxygen. Counts were 
effected after incubating ali the media at 35°C for 48 - 72 h, the Hoskins MPN (Most 
Probable Number) method being used for the clostridial spores (Hoskins, 1983). For 
isolating Bacillus cultures, dilutions were streaked onto nutrient-agar dishes; and for 
Clostridium onto RCM-agar dishes incubated anaerobically at 35°C (BBL Gas Pak 
System). The API 50 CHB system was used to identify Bacillus strains, and the me-
thods of Buttiaux (Buttiaux et al., 1975) for Clostridium strains. 
RESUL TS Ai'JD OISCUSSION 
Table 1 gives the number of spores found, plant origin and storage age for each of 
the honey samples, 88.4% of which contained bacterial spores. Of these, 86.5% we-
re aerobes and 19.2% anarobes. In many instances the counts were high, and even 
discounting the question of deleterious effects, it would seem that extraction and sto-
rage procedures were insufficiently circumspect, as we have often seen in many pro-
cessing centres. 
The strains isolated amounted to 187 and are reported in table 2; 162 belonged to 
the genus Bacillus and 25 to Clostridium. Six species of the former genus were clas-
sified. One of these, B. alvei (once considered the presumed agent of European foul 
brood) was present in 15.3% of the samples. B. cereus was present in 48.0% of the 
samples, and can be a serious danger in honey destined for the food or cosmetic 
, Melisso-palynological analyses ellected by Dr. Angela Melis. Apiculture Laboratory. Instltute of Agrlcultural Ento-
mology. University 01 Sassari. Italy. 
176 
Table 1 - Plant orlgln, storage age and mlcroblc count for each honey sample 
Origine fioraie, età di conservazione e conta mlcroblca per ciascun campione di miele 
Sample Plant origin Age (years) Bacillus (spores/g) Clostridium (spores/g) 
1 Multiflora 100 4 
2 Multiflora 100 
3 Arbutus unedo 2 
4 Multitlora 4 
5 Arbutus unedo 10 
6 Multiflora 100 
7 Multitlora 1 
8 Multiflora 
9 Multiflora 
10 Erica sp. 5 
11 Citrus sp. 10 
12 Multiflora 1,000 
13 Multitlora 1,000 
14 Eucaliptus sp. 1 1,000 4 
15 Arbutus unedo 0,5 10 
16 Asphodelus sp. 1 10 4 
17 Multitlora 1 10 
18 Arbutus unedo 10 
19 Multiflora 100 
20 Arbutus unedo 100 
21 Multiflora 1,000 
22 Multiflora 1 10 
23 Multiflora 1 4 
24 Multiflora 2 
25 Multiflora 100 
26 Multiflora 100 
27 Arbutus unedo 
28 Multiflora 10 8 
29 Multiflora 
30 Multiflora 
31 Multiflora 100 
32 Multiflora 10 
33 Multiflora 10 
34 Erica sp. 10 
35 Multiflora 1,000 
36 Carduus 100 
37 Asphodelus sp. 100 8 
38 Multiflora 10 
39 Multiflora 10 
40 Multiflora 
41 Multiflora 1 8 
42 Multiflora 0.5 100 2 
43 Erica sp. 0.5 2 
44 Multiflora 0.5 10 
45 Eucaliptus sp. 0.5 100 4 
46 Carduus sp. 0.5 10 
47 Asphodelus sp. 0.5 2 2 
48 Multiflora 1 100 
49 Multiflora 1 10 
50 Multiflora 0.5 10 2 
51 Multiflora 10 
52 Multiflora 100 
Tab. 2 - Stralns Isolated In each honey sample. Specie Isolate da ciascun campione di miele 
Species Sample 
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 
Genus Bacillus 
B. cereus 4 3 2 5 4 2 6 2 9 2 2 2 
B. licheniformis 
B. polimixa 
B. alvei 2 
B. macerans 2 2 2 
B. subtilis 2 4 2 2 2 2 2 2 3 
Genus Clo-
stridium 
Cf. acetobutilicum 
CI. beijerinchii 3 3 2 
(segue) Tab. 2 
--.-----~-----_.~----~-~-~-~~~~~-------~ 
Species Sample 
------ .. -
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 
~--- _._--_._~--
Genus Bacillus 
B. cereus 2 2 2 4 2 2 4 2 
B. licheniformis 1 2 3 4 
B. polimixa 3 2 
B. alvei 5 1 
B. macerans 2 2 4 2 
B. subtifis 2 3 3 
Genus CIo-
stridium 
CI. acetobutilicum 4 2 2 
CI. beijerinchii 3 2 2 2 
........ 
........ 
178 
industries. Also present were B. subtilis, B. licheniformis, B. polimixa and B. mace-
rans; with the exception of the latter, ali are producers of antibiotic substances. The 
first two are typical food bacteri a, especially sugary foodstuffs, while the latter are cha-
racteristic of decomposing vegetable matter. The spores of the last two can remain 
in honey for an indeterminate length of time, but do not cause spoiling. Three species 
of Closfridium were classified and were present in 10 of the samples, namely CI. ace-
tobuty/icum, CI. beijerinckii and CI. pasteurianum. While the effect of these species 
on honey is not known, CI. beijerinckii has been found in spoiled candied fruits; it might 
also cause anomalies in honey. 
Further experiments are envisaged to indivualize each strain and verify if they can 
develop during storage, or in conditions where processing involves Aw> 0.9. 
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Aerobic and anaerobic spore-forming bacteria in Sardinian honey.

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