BIOCHIMICAET BIOPHYSICAACTA
BBA
57
12177
THE IDENTIFICATION OF INHIBINE, THE ANTIBACTERIAL
F A C T O R I N H O N E Y , AS H Y D R O G E N P E R O X I D E A N D I T S O R I G I N
IN A HONEY GLUCOSE-OXIDASE SYSTEM
JONATHAN W. WHITE, JR., MARY H. SUBERS AND ABNER I. SCHEPARTZ
Eastern Regional Research Laboratory*, Philadelphia, Pa. ( U.S.A .)
(Received September 24th, 1962)
SUMMARY
Glucose oxidase, producing gluconic acid and hydrogen peroxide from glucose, is
demonstrated in honey. Its activity increases markedly on dilution of the honey.
Inhibine, the antibacterial material previously reported in honey, is shown to be
hydrogen peroxide produced in the inhibine assay by the natural glucose oxidase in
honey. A direct relationship is shown between inhibine number and hydrogen
peroxide production.
INTRODUCTION
Use of honey from ancient times to the present for its reputed wound-healing and
antiseptic properties has recently been reviewed by STOMFAY-STITZ1. It is well
established 2-n that natural unheated honey shows an antibacterial activity. I t was
first demonstrated b y DOLD and his colleagues 2 who included honey in a study of
materials with natural antibacterial action and described an assay procedure. Under
their conditions, activity of diluted honey was shown against 17 bacteria but not
against three molds and a yeast. The activity was heat-labile, somewhat lightsensitive, and retained b y bacterial filters. They related it to similar antibacterial
activities they reported in other materials of natural origin, which they termed
"inhibines". PRICA3 generally confirmed the findings of DOLD, Du AND DZIAO2 with
respect to honey, using a different assay procedure. He found the activity to pass
through a Seitz filter. Since that time the presence in honey of various amounts of
inhibine, as defined b y DOLD, has been reported b y several investigators. PLACHY4
discussed the known constituents of honey that might be responsible and concluded that activity was not due to the sugars, acids, nitrogen compounds, enzymes,
pH, vitamins or other known constituents. He noted that inhibine passed through
a dialysis membrane and withstood vacuum concentration, with no activity remaining
within the sac. He did not provide sufficient detail to allow repetition. DOLD AND
WITZENHAUSEN5 described an improved assay procedure using honey at 5 - 2 5 %
levels in a nutrient agar, inoculation with Staphylococcus aureus, and 24-h incubation
* Eastern Utilization Research and Development Division, Agricultural Research Service.
U.S. Department of Agriculture.
Biochim. Biophys. Acta, 73 (1963) 57-7 °
58
J. w. WHITE, M. H. SUBERS, A. 5. SCHEPARTZ
at 37 °. They included assays of 20 samples. MEIER AND FREITAG6 in a study of the
antibiotic powers of various sugars included a sample of honey and reported it to
permit bacterial growth at lO% in agar, but had heated it to 70-80 ° previous to
mixing with agar. In the DOLD procedure the m a x i m u m temperature reached b y the
honey is about 45 °. SCHULER AND VOGEL7 extracted undiluted honey with various
solvents and reported that the inhibitory substance was ether-soluble and probably
an ethereal oil. A propanol-soluble growth-promoting substance was stated to be
organic acid in nature. SCHADE, MARSH AND ECKERT8 showed that inhibine was
more heat-sensitive than honey amylase and that its level in a limited number of
honeys b y a modified DOLD assay did not correlate with amylase activity. WARNECKE
AND DUISBERG9 assayed 600 honey samples of 13I types for inhibine by the DOLD
procedure, concluding that inhibine activity and invertase activity ran parallel. A
later paper 1° applied statistical procedures to determine that the inhibine and invertase assays taken together were superior to other combined tests to determine
heating history of commercial honey. More recently STOMFAY-STITZAND KOMINOSn
repeated DOLD'S work and agreed that inhibine is heat-sensitive, is retained by
Seitz filters but not by paper, is active against both gram-positive and -negative
bacteria, and is of unknown constitution. They assayed 16 American honey samples.
Although GROSS12 reported an oxidase activity in honey his procedures were not
definitive. COCKERlz published on the enzymic production of acid in honey, but it
was later shown 14 that the drift in p H of partially neutralized honey samples, on
which he based his report, was probably due to hydrolysis of lactone material in
honey. An enzyme-producing acid in honey was reported by WHITE15 and it was
shown statistically ~s that some honey samples increase in titratable acidity in
storage and that such increase correlated well with amylase activity. It is of interest
that in 1941 GAUHE17 had demonstrated in the pharyngeal gland of the honeybee
an aerobic glucose-oxidizing enzyme that produced gluconic acid and hydrogen
peroxide. Its presence in honey was not shown. Gluconic acid was recently found 18
to be the principal acid in honey and lactone a common honey constituent14,19.
We wish to report that the acid-producing enzyme in honey is a glucose oxidase,
producing gluconic acid (gluconolactone) and hydrogen peroxide from glucose. I t is
evident that if sufficient amounts are produced under the assay conditions, hydrogen
peroxide could be the "inhibine" reported b y DOLD et al. 2. We have shown that this
is indeed the case, with all of the previously reported properties of inhibine consistent
with its identity as hydrogen peroxide produced by the glucose oxidase of honey.
This enzymic oxidation of glucose takes place very slowly in undiluted honey and
at much higher rates only as honey is diluted. Peroxide can accumulate in the
nutrient agar test plates used for inhibine assay to account for the antibacterial
activity shown in this test. The inhibine number of a honey sample is directly related
to the hydrogen peroxide concentration produced in the assay plates by the honey
enzyme system.
EXPERIMENTAL
Glucose oxidase in honey
2o0 g of an unheated Northeastern fall-flower honey (No. HS 37) were dissolved
in 200 ml 0.04 M phosphate (pH 5.5). All subsequent operations were carried out at
Biochim. Biophys. Acta, 73 (1963) 57-7 °
59
INHIBINE AND GLUCOSE OXIDASE IN HONEY
4 °. To t h e cold solution were a d d e d w i t h stirring 16oo m l cold a b s o l u t e e t h a n o l over
a p e r i o d of 20 rain. A f t e r c e n t r i f u g a t i o n a t 14oo × g for IO min, t h e p r e c i p i t a t e was
r e s u s p e n d e d in 20 m l o . o o i M p h o s p h a t e (pH 8.0) a n d d i a l y z e d a g a i n s t t h r e e 6oo-ml
p o r t i o n s of t h e buffer, t h e n c e n t r i f u g e d ; t h e 3 I - m l s u p e r n a t a n t was r e m o v e d a n d
s t o r e d u n d e r toluene. This crude p r e p a r a t i o n was f o u n d to c o n t a i n lO 4 SCHADE
units* of a m y l a s e , 515 units* of i n v e r t a s e a n d 113 units* of m a l t a s e a c t i v i t y p e r ml.
I t c o n t a i n e d 26 m g p r o t e i n p e r m l d e t e r m i n e d b y t h e m e t h o d of LOWRY et al. 21.
Production of hydrogen peroxide
H y d r o g e n p e r o x i d e can be d e t e c t e d a n d m e a s u r e d in low c o n c e n t r a t i o n b y t h e
p e r o x i d a s e - c a t a l y z e d o x i d a t i o n of s u i t a b l e c o m p o u n d s to colored products. P o r t i o n s
(0.25 ml) of t h e h o n e y e n z y m e p r e p a r a t i o n were t r e a t e d w i t h v a r i o u s c o m b i n a t i o n s
of glucose (0.33 mM), p e r o x i d a s e (Horseradish, T y p e I, S i g m a Chemical C o m p a n y ,
St. Louis, Mo.**) a n d 2 m l of a solution of 5 m g o-dianisidine ( 3 , 3 ' - d i m e t h o x y b e n zidine, P r a c t i c a l , E a s t m a n Organic Chemicals, Rochester, N.Y.) in 50 m l 0.5 M
p h o s p h a t e (pH 7.0). T a b l e I shows the a b s o r b a n c y values in I o - m m test t u b e s a t
TABLE I
PRODUCTION
OF HYDROGEN
Enzyme
(ml)
0.25
0.25*
o.oo
o.25
0.25
Glucose
(raM)
0.33
0.33
o.33
0.33
o
PEROXIDE
FROM GLUCOSE
BY HONEY
ENZYME
Absorbancy at 500 ml~
Peroxidase
yes
yes
yes
no
yes
romin
2omin
3omi~
0.433
0.208
0.o3z
o.336
o.336
O.815
I.OOO
o.21o
0.033
o.33o
o.333
o.215
o.o23
o.337
0.337
* Boiled before mixing; a light precipitate which formed was removed.
500 m/z. Color d e v e l o p m e n t occurred o n l y in t h e presence of u n h e a t e d e n z y m e ,
glucose, peroxidase, a n d dianisidine; t h u s H , 0 2 was p r o d u c e d .
P o r t i o n s of t h e e n z y m e p r e p a r a t i o n were d i l u t e d w i t h w a t e r ; I m l of i . I M
glucose a n d 5 m l of a m i x t u r e of IO m g o-dianisidine a n d 2 m g p e r o x i d a s e in IOO m l
0.22 M p h o s p h a t e (pH 6.8) was a d d e d to I m l of each e n z y m e dilution. T h e a b s o r b a n c y
of t h e t u b e s was m e a s u r e d a t 5o0 m/z b y t h e d i s p l a c e m e n t p r o c e d u r e 22 d u r i n g 2 h.
T h e r e a g e n t was c a l i b r a t e d w i t h H,02. The results are shown in Fig. I ; r e a c t i o n
r a t e is d i r e c t l y p r o p o r t i o n a l to e n z y m e concentration. F u r t h e r i n v e s t i g a t i o n of t h e
c h a r a c t e r i s t i c s o f this e n z y m e is in progress.
* One unit of invertase and maltase activity is that which causes 1% hydrolysis in 2 ml
reaction mixture in i h at pH 5.9, o.146 M substrate at 37 ° (see ref. 2o).
** Mention of trade and company names does not imply endorsement by the Department
over others of a similar nature not named.
Biochim. Biophys. Acta, 73 (1963) 57-7 °
60
J. w. WHITE, M. H. SUBERS, A. I. SCHEPARTZ
1.2
c-
1.0
:E
~ " 0.8
~ o.6
2
Q.
"D
(~0.4
"r
0.2
0
I
4
0
~
~
~&
20
Relative e n z y m e conc.
Fig. I. Production of hydrogen peroxide by crude glucose oxidase from honey. Substrate, o.161Vi
glucose in o.i6 IV[phosphate (pH 6.8).
Identification of gluconic acid
When 0.5 ml of the same enzyme preparation was mixed with 0.5 ml 60%
glucose, the p H dropped from 5.80 to 4.25 in 15o min; to 3.1o in 24 h.
To 2 test tubes were added 0.25 ml of the above enzyme preparation and 50 mg
glucose. One was heated in a boiling-water bath, cooled, and 4-/zl portions of each
were chromatographed as shown in Table II. A single spot, from the unheated sample
only, was shown b y the bromophenolblue reagent. It travelled with gluconic acid.
showing Rglueoseof O.21 in ethyl acetate-water-pyridine (5:5:2).
TABLE II
CHROMATOGRAPHY
OF REACTION
OF HONEY
ENZYME
WITH
GLUCOSE
Spot intensity
Time
( min )
io
60
i2o
245
unheated
enzyme
+
+ +
+ + +
++++
heated
enzyme
o
o
o
o
5O g of a goldenrod-aster h o n e y (No. HS 33) were diluted with 25 m l w a t e r
c o n t a i n i n g 12 m g merthiolate, placed in a I 6 - m m - d i a m e t e r dialysis t u b i n g a n d held
at 37 ° for 30 min. It was t h e n dialyzed against cold r u n n i n g t a p water for 24 h. To
the 94 m l of m a t e r i a l from the sac 14. 5 g D-glucose a n d a few crystals of t h y m o l
were added. I t was held with shaking at 3 °0 a n d t i t r a t e d three times over a 4o-h
period with 0.05 N N a O H ; 4.67 m e q u i v of acidity were produced. The solution was
passed t h r o u g h columns of Dowex-5o a n d Duolite A-4 which were washed to eliminate
glucose. The gluconic acid was r e m o v e d from the Duolite c o l u m n with 50 ml 2 N
NH4OH. This was passed t h r o u g h a n o t h e r Dowex-5o column which was t h e n washed
Biochim. Biophys. Acta, 73 (1963) 57-7 °
I N H I B I N E AND GLUCOSE OXIDASE IN HONEY
61
with water. 2-ml fractions of the effluent from this column were collected; those
more acidic than p H 4.5 (fractions 6-40 ) were combined, titrated and evaporated to
dryness. The salt was chromatographed with authentic sodium gluconate as a control
using 2 solvent systems. ( I ) B u t a n o l - e t h a n o l - w a t e r (4:i:5): isolated product,
29.6 m m ; sodium gluconate, 28.3 mm. (2) E t h y l acetate-water-formic acid 23 (IO :4:2) :
isolated product, IO3 m m ; sodium gluconate, lO4 mm.
Infrared examination showed differences between the unknown and the standard
in the 95o-I2OO-Cm-1 region, which were traced to small amounts of material
removed from the ion-exchange resin b y ammonia. By large-scale paper chromatographic purification of the unknown on washed W h a t m a n 3MM paper, these impurities were removed to produce a material with an infrared spectrum in K B r disc
completely identical with that of authentic sodium gluconate over the range of the
instrument (Perkin-Elmer Model 2I), 650-4000 cm -1.
Assay of inhibine
The assay procedure for inhibine was carried out essentially according to DOLD
AND WITZENHAUSENs with some of the modifications of SCHADE et al. s. Nutrient
agar plates were prepared at five honey levels, 1-5 ml of diluted honey per plate, as
described by SCHADE. The plates were inoculated immediately after solidifying with
o.I ml of a suspension of a 24-h culture of Staphylococcus aureus and judged after
24 h at 37 °.
Estimation of peroxide production in agar plates
By including peroxidase and dianisidine in the nutrient agar formula an agar
gel results that darkens as hydrogen peroxide is produced within the gel. Peroxide
can be estimated b y standardizing conditions and measuring absorbancy of the gel.
1.4
1.2
1.0
0
if)
0.8
~'o.6
x~ 0 . 4
0.2
%
2;0
H202 pep plate ()Jg)
.6o
6o0
Fig. 2. E s t i m a t i o n of h y d r o g e n p e r o x i d e i n n u t r i e n t a g a r p l a t e s .
Biochim. Biophys. Acta, 73 (I963) 57-7 o
62
J.W. WHITE, M. H. SUBERS, A. I. SCHEPARTZ
Double-strength nutrient agar (Baltimore Biological Laboratory, Baltimore,
Md.) was prepared. To each IOO ml while at 80-85 °, was added with stirring 60 mg
o-dianisidine in 2.5 ml 95% ethanol. Portions of 7.5 ml were placed in warm test
tubes and held at 5o°. In other test tubes were placed 6.5 ml of material to be plated
(water-H202, diluted honey, water). These were warmed to 41° and I drop of a
peroxidase solution (approx. IOO #g/ml) was added except to blanks. A tube of
honey-water-peroxidase or peroxide-water-peroxidase was added to a tube of
dianisidine agar, the solutions were mixed by pouring back and forth (nine pours),
then placed in a 9o-mm petri dish. Dishes were selected for uniformity, flatness and
freedom from etch and scratches. Before solidification all foam and bubbles on the
surface were moved to the sides of the dish. The thickness of the gel was about 1.82.0 mm. After gelation the dishes were inverted and their absorbancy determined
at 450 m# by a Photovolt Model 5oI-A spot photometer with a daylight fluorescent
light source behind opal-glass diffusion. Absorbancy was determined by placing the
closed dish, agar side on top, directly on the opal glass and centering the search head,
with a 45o-m# interference filter attached, on the plate. Blanks contained no peroxidase. Fig. 2 shows a calibration of this procedure with hydrogen peroxide. In the
calibration series, constant absorbancy was reached by the time the agar had solidified.
It remained constant within o.oi A for at least 20 h.
RESULTS AND DISCUSSION
An unheated aster-goldenrod honey (HS 33) was subjected to the described assays
for inhibine and peroxide production. It permitted growth on plate 5 and allowed
no growth on plates 1-4, thus having an inhibine number of 4. The cumulative production of H20 ~ in these plates for the initial 4 h is shown in Fig. 3. COULTHARD
et al. 24 in describing their work on identification of penicillin A or notatin as glucose
oxidase reported that S. a u r e u s inoculated into nutrient broth containing H20 ~
failed to grow within 24 h at 37 ° at o.oo1% H20 z, but grew at 0.00050/0 . They also
found that gluconic acid concentration up to 0.25% failed to inhibit growth. As
600
4x10 -3
50C
1
2 3
.~ 4 0 0
_go
a 30C
32~
0~20C
-r-
100
0
1
2
3
Incubation t i m e (h)
4
ill
Fig. 3. H y d r o g e n peroxide p r o d u c t i o n d u r i n g early p o r t i o n o f i n h i b i n e assay.
Biochim. Biophys. Acta, 73 (1963) 5 7 - 7 °
63
I N H I B I N E AND GLUCOSE OXIDASE IN HONEY
Fig. 3 shows, this level of H~O~ is reached within 4 h in plate 4 and exceeded earlier
in plates 1-3.
After 24 h incubation, the surfaces of plates i and 2 were washed with sterile
water, portions of which were inoculated into broth. Good growth was obtained
which was inoculated onto agar slants and shown to be S. aureus. Thus, under the
assay conditions, a bacteriostatic action rather than bactericidal action is evident.
COULTHARD et al. 24 working with broth assays found HzO 2 produced by glucose
oxidase in broth to be bactericidal; STOMFAY-STITZAND KOMINOS11 in reference to
honey inhibine use "bacteriostatic" in their title. PRICA8, whose paper is titled
"l~ber die bactericide Wirkung des Naturhonigs", used a broth assay for honey
inhibine and determined effectiveness by plating from it over a period of time.
Inhibine action was shown b y failure to grow within 48 h after transfer onto plates
and in the greater dilutions (I :5,I :IO), 96 h at 37 ° which were required to attain
sterility in the broth cultures.
Effect of H202 in nutrient agar on growth of S. aureus
Since the work of COULTHARD et al. was concerned with a liquid medium, it was
felt necessary to determine the effect of added peroxide in solid media. Hydrogen
peroxide was included in a series of sterile nutrient agar plates at levels of 0.00035
to o.o2~/o. They were inoculated and incubated as for the inhibine assay. Good
growth was found on the 0.0007% HeO 2 and lower plates, one colony on the o.ooi %
plate and none at o.oo17% and higher. These results are comparable with those of
COULTHARD et al.
When honey was included in the medium at the levels used in the inhibine
assay, the apparent requirement of HeO z to prevent growth was much higher, and
varied with the honey level. Table I I I shows the effect on S. aureus of H202 in the
presence of boiled honey. This protective effect is due to destruction of HzO ~ b y
honey. A semi-quantitative test for H~O2 was applied to the surface of all plates
outside the inoculated area after the incubation. Results showed that all fioneycontaining plates with no growth of S. aureus contained only small amounts (about
30 #g/plate) of HeO 2. No plates showing growth responded to the test (about 15 #g/
TABLE III
EFFECT OF HIOi AND HONEY SOLIDS ON THE GROWTH OF Staphylococcus aureus
IN PETRI
Concemra~ion of
boiled honey
(%)
20. 3
16. 5
12. 5
8. 5
4-3
o.o
PLATES
Growth at indicated concen~ragionof hydrogen peroxide
o.o~z%
_*
_"
_*
--"
--*
.
0.007%
.
~
~
+
±
--"
.
0.0035%
~
+
++
+
+
.
o.oo~7%
++
++
+++
++
+
0.0007%
++
+++
++++
++++
++++
*
+
0.00035%
+++
+++
++++
++++
++++
++
0.0%
+++
++++
++++
++++
++++
+++
" S e m i - q u a n t i t a t i v e t e s t ( p e r o x i d a s e a n d d i a n i s i d i n e ) i n d i c a t e d a b o u t 0 . 0 0 0 2 % (3o-45/~g)
H20 ~ per plate.
Biochim. Bi ophy s . Acta, 73 (I963) 5 7 - 7 °
64
J. w . WHITE, M. H. SUBERS, A. I. SCHEPARTZ
plate or less). This agreed with experience during calibration of the analytical
procedure, when it was noted that inclusion of honey in a calibration series produced
variable and much lower color development. It was shown that this did not occur
when honey, peroxidase, peroxide and dianisidine were mixed simultaneously, the
color-producing enzymic reaction being much faster than peroxide destruction by
honey.
The differences in growth shown at the 20% and 16% honey levels (Table III)
compared with the 12% levels are probably caused by the osmotic effect of the
higher sugar concentration; in the zero peroxide series growth was slightly less at
the highest boiled honey level.
Effect of destruction of evolved peroxide on inhibine values
Three inhibine assay series were run using honey HS 33. A catalase solution
(Worthington Biochemical Corp., Freehold, N.J.) was added to one at a level found
adequate by preliminary work, peroxidase and dianisidine to another, and the third
was retained as a control. All plates were immediately inoculated and incubated.
Results appear in Table IV. Destruction of H202 produced by the honey system by
TABLE IV
EFFECT
OF DESTRUCTION
IN PLATE
UPON
OF HYDROGEN
INHIBINE
ASSAY
PEROXIDE
OF HONEY
Growth of S. aureus in plate containing
Plate
No.
catalase
++
2
3
4
5
++
++
+++
+++
peroxidasedianisidine
+
neither
-
++
+++
+++
++++
++
either catalase or peroxidase permitted growth of the inoculum in all plates. It is
noteworthy that far greater amounts of catalase were required to destroy H202
evolved in the inhibine plates (as indicated by a semi-quantitative test for H20~)
than indicated by the catalase activity and total amount of H20 ~ produced. COHEN
AND HOCHSTEIN25 have recently proposed that catalase is not highly effective at
destroying physiological levels of H202; it easily deals with a given amount added
at once, but not with the same quantity added over a period of time.
Separation and recombination of the peroxide-producing system in honey
A 5o-g sample of honey (HS 33) was diluted with 25 ml water containing
12 mg merthiolate* and held 30 min at 37 ° in a closed i6-mm-diameter dialysis tube.
" P r e l i m i n a r y e x p e r i m e n t w i t h o u t t h i s s t e r i l i z a t i o n t r e a t m e n t g a v e a h e a v y o v e r g r o w t h on
t h e i n h i b i n e p l a t e s of a b a c i l l u s f r o m t h e honey.
Biochim.
Biophys. Aaa,
73 (1963) 57-7 °
65
INHIBINE AND GLUCOSE OXIDASE IN HONEY
I t was t h e n d i a l y z e d against cold r u n n i n g t a p w a t e r for 24 h, reaching a final v o l u m e
o f 98 ml. To m a i n t a i n th e v o l u m e relationships in t h e inhibine assay, t h e v o l u m e
was r e d u c e d to 85 m l b y u l t r a f i l t r a t i o n using an L K B ultrafilter (La Pine Scientific
Co., Chicago). T h e dialyzed m a t e r i a l was t h e n s u b s t i t u t e d for t h e 5 0 % h o n e y s o l u t i o n in t h e inhibine assay, w i t h t h e a d d i t i o n of glucose in a m o u n t s n o r m a l l y present
f r o m honey. Th e same series was also assayed for H202 production. Tab l e V shows
t h e results. A l t h o u g h it was r e t a r d e d at the higher concentrations, all plates showed
growth. T h e dialyzed m a t e r i a l w i t h o u t glucose showed a g r o w t h - p r o m o t i n g effect;
at t h e higher glucose c o n c e n t r a t i o n , g r o w t h was considerably reduced. Th e sugar
TABLE V
INHIBINE
A S S A Y OF D I A L Y Z E D
HONEY
Glucose added
Plate
No.
Dialyzate
(rot)
Amount
Growth
No addition
HlO ,
produced*
(g)
Growth
H~O,
produced*
Cm}
i
2
3
4
5
5,0
4.0
3.0
2.0
I.O
i.oo
6.80
o.6o
0.4o
0.20
+
+
+ +
+ +
+ + +
Ho
80
4°
21
3
6
7
I.O
o.o
i.oo
I.OO
+ + +
+ + +
20
o
O,g)
++++
+ + + +
+ + +
+ + +
" Increase in H,O, content of plate between first and second hour at 37 °.
c o n t e n t of p l at e I was 70/0 . T h e first plate of t h e inhibine series c o n t a i n i n g 2 0 .3 %
h o n e y wo u l d h a v e a b o u t 16.2 % t o t a l sugars, less t h a n h a l f of which is glucose.
Inhibine, as defined b y t h e DOLD assay procedure, does not c o m p l e t e l y ex cl u d e
t h e o s m o t i c effects o f h o n e y sugars. A n assay series was therefore r u n w i t h dialyzed
h o n e y in which a dried h o n e y was used as a glucose source i n s t e a d of glucose. D r y
TABLE VI
INHIBINE
A S S A Y OF D I A L Y Z E D
HONEY
Dry honey added
Pla~e
No.
Dialyzate
(ml)
Amount
(g)
No add~ion
H~02
Growth
I
5.o
2.45
--
2
3
4.O
1.96
--
4
5
3.o
2.o
I.O
1.47
0.98
0.49
6
7
I .o
o.o
2.45
2.45
produced"
(ug)
HsO,
Growth
135
+ + + +
--4++
83
+ + + +
17
+++
-+
27
o
+
produced*
(ug)
" Increase in H~O~ content of plate between first and second hour at 37 °.
Biochim. Biophys. A ct~8, 73 (tt)031 57-7 °
66
j. w. WHITE, M. H. SUBERS, A. I. SCHEPARTZ
honey was desirable rather than a boiled honey so that changes in volume relations
could be avoided. A dehydrated white-clover honey 26 was added in amounts duplicating the honey solids content of the series. Results are given in Table VI.
Growth promotion was again evident; honey solids alone did not retard growth, but
combination with the dialyzed material produced H20 ~ and prevented growth at the
upper three levels and retarded it at the fourth level.
That the differences shown between Tables V and VI are not due to some unrecognized co-factor in the dried honey, but only to the higher sugar concentration,
is demonstrated by Table VII. Here the conditions of Table V were repeated, except
TABLE VII
INHIBINE
ASSAY
OF
DIALYZED
HONEY
Sugars added
Plate
No.
Dialyzate
(ml)
Fructose only
Glucose andfructose
Glucose
(g)
Fructose
(g)
Growth
H20*
produced*
(m)
I
5.0
2
3
4
5
4.0
3.0
2.0
I.O
I .oo
0.80
0.60
0.40
0.20
1.45
1.16
0.87
0.58
0.29
--~_
+ + +
21o
17o
91
42
8
6
7
I .o
o.o
I .oo
I.OO
1-45
1.45
-+ +
32
o
--
Growth
Hi02
produced*
+++
3
+++
o
+++
o
(~g)
* Increase in H20 ~ content of plate between first and second hour at 37°.
/
that fructose was added to each plate in amounts equal to the solids difference
between corresponding plates in Tables V and VI. Addition of fructose, in itself
without effect on growth, prevented growth in the first three plates, as in Table VI.
The effect of dried honey addition, seen in Table VI, is therefore simply osmotic,
due to higher total sugar concentration. As indicated by plate number 6 in Tables VI
and VII, increasing the sugar content of plate 5 and retaining the enzyme level
allows increased H20 2 production and inhibits growth of S . a u r e u s .
Eight honey samples were assayed for inhibine, apparent H,O, content was
measured after I, 2 and 4 h at 37 ° in uninoculated plates. Table V I I I shows the
peroxide content of the indicating plates for each sample. Column 6 in the table
shows the inhibine number (highest-numbered plate with complete inhibition of
growth of S . a u r e u s after 24 h at 37°).
These data are plotted in Fig. 4. The line gives the H,O 2 production rate required to prevent growth of S . a u r e u s in the inhlbine assay; this is approx. 24/zg/
plate (0.00o17%) at I h, 32/,g/plate (0.00023%) at 2 h, and 56#g/plate (0.0004%) at
4 h. This line is roughly parallel to the peroxide production curves for the various honeys
in the plate indicating inhibine number. Thus measurement of peroxide content during
the early part of the incubation should enable prediction of the inhibine value;
2 h is a convenient time. One sample with an inhibine number of 2 showed no growth
Biochim. Biophys. Acta, 73 (1963) 57-7o
67
INHIBINE AND GLUCOSE OXIDASE IN HONEY
300
2 x i 0 -3
o No g r o w t h by 24 h
• G r o w t h by 24 h
200
1
9x10 -4
8
7
100
90
80
70
~
3
6
5
o
6o
5o
o
o
4
_o
~L
I1
.c_
cY
:~ 20
i
I
Ixi0 -4
Incubation time (h)
Fig. 4. Relation between early hydrogen peroxide content and 24-h growth of inoculum on inhibine assay plates.
at 24 h w h e n t h e 4-h H202 v a l u e was 48 yg, while t w o others (inhibine n u m b e r s 3
an d 4) allowed g r o w t h at 24 h at this level (49 and 51 #g). These differences are n o t
significant; f u r t h e r m o r e the higher solids level of plate 2 could reduce the H202
r e q u i r e m e n t s o m e w h a t. Use of th e a p p a r e n t peroxide c o n t e n t at 24 h does n o t give
consistent results; th e a n a l y t i c a l s y s t e m is subject to too m a n y u n k n o w n factors
o v e r this period of time.
E x a m i n a t i o n of peroxide p r o d u c t i o n rate curves for these samples showed t h a t
in some cases the r a t e per g r a m of h o n e y was at a m a x i m u m in the t h i r d or f o u r t h
TABLE VIII
RELATIONSHIP BETWEEN PEROXIDE PRODUCTION AND INHIBINE NUMBER OF HONEY
Plate allowing growth*
Sample
No.
No.
HS 33
HS 37
5
5
H S 35
M26I
M262
364
317
H S 36
5
4
I
o
4
3
Plate inhibiting growth**
Peroxide content *it
Ih
2h
4 h
o
o
16
17
38
38
o
18
2o
19
26
19
o
No. ~
Peroxide content •**
zh
2h
4 h
4
4
59
56
117
125
191
245
51
49
4
3
64
51
14°
84
278
14o
22
22
o
22
20
25
23
5
3
2
47
29
24
65
44
35
96
59
48
* All plates with larger numbers also allowed growth and had lower H~O~ content.
*" All plates with smaller numbers also inhibited growth and had higher H202 content.
"**/~g H~Oz per plate after indicated time at 37 °.
I Inhibine number.
Biochim. Biophys. Acta, 73 (1963) 57-7 o
68
J. w. WHITE, M. H. SUBERS, A. I. SCHEPARTZ
plate rather than the first, which had the highest enzyme and substrate concentration. This may have been due either to destruction of H20 e or interference with the
assay system by honey constituents at the higher levels. This does not occur when
dialyzed honey is tested with sugars, but does appear when tested with dried honey
(clover or buckwheat). Since both substrate and enzyme (honey) concentrations
vary in the series, definite conclusions regarding the causes of departures from
linearity of the rate vs. enzyme concentration curves are not made.
The peroxide production rates found under the assay conditions ranged from
less than 5 to nearly IOO #g H2OJg honey/h. In contrast, rates of acid production
during long-time storage of full-density honey 15 are equivalent to about o.oo2-o.o12 #g
H , O J g honey/h. The enzyme is thus practically inactive in undiluted honey; its
presence has been shown statistically 16. No appreciable accumulation of H20 2 in
honey takes place; an attempted analysis gave a value of 0.3 ppm. The other reaction
product, gluconolactone, does accumulate and is in equilibrium with gluconic acid
in honey. The amount of gluconic acid in a honey should give some insight into the
conditions of its ripening by the bees, since production essentially stops when full
density is attained in the stored honey. Gluconic acid is the principal acid of honey
and lactone was found in all but 2 of over 5o0 honey samples recently analyzed 19.
As expected, the lactone/free acid ratio is dependent upon the pH of the honey.
The relation between inhibine number and honey concentration in the plate
for both the SCHADE and the DOLD procedures is shown in Table IX. In the SCHADE
TABLE IX
RELATION
OF INHIBINE
NUMBER
AND HONEY" CONCENTRATION
IN TEST
PLATES
Honey concentration in plate
Inhibine
No.
5
4
3
2
I
o
GCHADE el al. 8
No growth at
No growth at
No growth at
No growth at
No growth at
Growth at
DOLD AND
WITZENHAUSEN~
(%, w/w)
(%, w#v)
(% v/v)
4.28
8.45
12.5
16.5
20.3
20.3
6.1o
11.9
17.4
22.7
27.8
27.8
5
IO
15
20
25
25
series the concentration of nutrient medium is essentially constant at 50% of the
double-strength medium; DOLD AND WITZENHAUSEN varied the amount of 3%
nutrient agar in the plates between 44 and 88%. The calculated honey contents of
the two series appears in Table IX. It is obvious that comparison of inhibine values
of various investigators must be done with caution; a honey giving an inhibine
number of o by ,~CHADE,MARSH AND ECKERT'S procedure might read 2 by the DOLD
AND WITZENHAUSEN assay. In general the SCHADE procedure would give inhibine
values one unit lower than the latter.
It is of interest to review some of the literature on inhibine in honey in the fight
of the work reported here. The heat sensitivity is of course due to its enzyme origin.
PLACHY'S4 results on dialysis can be explained as follows: his conditions (dilution
Biochim. Biophys. Acta, 73 (1963) 57-7°
I N H I B I N E AND GLUCOSE OXIDASE IN HONEY
69
in the tubing and holding at 4 o°) favored production of H~0~ which passed through
the membrane. Subsequent treatment of the material passing the membrane (vacuum
concentration at 45 °) would concentrate the peroxide so that the concentrate would
show an antibacterial activity. No details are given of the testing procedure for the
material remaining in the tubing, but it was found inactive. Although the enzyme
probably remained active, reduction of the concentration of the glucose substrate to
0.2 of its concentration could have a marked effect on H~02 production. Furthermore
unless precautions had been taken to suppress microbiological growth, severe contamination must have been present in PLACHY'S experiment.
The reported sensitivity of honey inhibine to light 2-4 is according to PRICAa
inconsistent with the possibility that inhibine is an enzyme. DUISBERG AND WARNECKE10 however have described the effect of storage of honey for 5-7 months in
clear- or dark-glass jars before a window. The degree of destruction that they reported for inhibine was quite similar to that for invertase in the same samples, as
shown in Table X.
TABLE X
EFFECT
OF LIGHT
ON INHIBINE
AND INVERTASE
IN HONEY
(II
SAMPLES)
Activity remaining
after 5 7 months*
Container
Clear glass
D a r k glass
Inhibine
(%)
Invertase
(%)
66
IOO
69
85
" D a t a from T a b l e 6 of DUISBERG AND WARNECKE 10.
Concerning the effect of filtration on inhibine, the literature is not in agreement.
DOLD et al. 2 reported that it is retained by Berkefeld and asbestos filtration; STOMFAYSTITZ AND KOMINOSn confirmed that Seitz E. K. and R. filters retained it, while
PRICA3 noted that it passed through a Seitz E. K. filter in a I :I dilution. PLACHY4
had variable results. Adsorption of high-molecular materials (anticephalin ~s, bloodcoagulation factors ~0-81, viruses z~) b y Seitz filter pads has been reported. An analyrical procedure for prothrombin and proconvertin 8° uses a Seitz filter for adsorption.
Thus, preformed H202 could pass such filters, but the enzyme Could be adsorbed.
The final inhibine assay procedure of DOLD AND WITZENHAUSEN5 requires a
2-h "drying" of the poured plates in the incubator before inoculation. This would
allow production of an appreciable amount of peroxide in the plates before inoculation, which could influence the result.
SCHADE et al. s noted a buckwheat honey of good amylase activity which showed
a very low inhibine content. Even if the glucose oxidase content of the honey were
parallel to the amylase, peroxide destruction b y other honey components, possibly
peculiar to certain honey types, could result in a low inhibine value.
Since gluconic acid has never been found in higher plants*, that occurring in
* B u c n i n a n 832-references b i b l i o g r a p h y of o r g a n i c a c i d s in h i g h e r p l a n t s 2~ n o t e s one
r e f e r e n c e to g i u c o n i c a c i d - - i n a b e e t - s u g a r f a c t o r y , w h e r e i t s a p p e a r a n c e on t h e o u t s i d e of e q u i p m e n t w a s a s c r i b e d to m i c r o b i o l o g i c a l a c t i o n on sugar.
Biochim. Biophys. Acta, 73 (1963) 57-7 °
7°
J. W. WHITE, M. H. SUBERS, A. I. SCHEPARTZ
h o n e y m u s t a r i s e e i t h e r f r o m m i c r o b i o l o g i c a l a c t i v i t y or b y a c t i o n o f t h e g l u c o s e
o x i d a s e a d d e d b y t h e h o n e y b e e . GAUHE 17 h a s s h o w n t h a t t h e p h a r y n g e a l g l a n d s o f
t h e h o n e y b e e , s o u r c e o f h o n e y i n v e r t a s e , also c o n t a i n a g l u c o s e o x i d a s e . S h e h a s
also shown that the contents of the honey sac become acid upon standing, though
she did not directly demonstrate the presence of the enzyme in honey. In considering
several possible functions of the enzyme, she noted that the prompt establishment
o f a l o w p H v a l u e w o u l d b e b e n e f i c i a l t o t h e u n r i p e h o n e y . I t w o u l d s e e m also t h a t
t h e p e r o x i d e p r o d u c e d d u r i n g r i p e n i n g s h o u l d h a v e a p r e s e r v a t i v e effect. COULTHARD
et al. 24 f o u n d p e r o x i d e m u c h m o r e e f f e c t i v e t h a n g l u c o n i c a c i d a g a i n s t S. aureus.
M o l d s a n d y e a s t s , h o w e v e r , d o n o t a p p e a r t o b e as s e n s i t i v e t o p e r o x i d e as b a c t e r i a .
ACKNOWLEDGEMENTS
W e w i s h t o e x p r e s s o u r a p p r e c i a t i o n t o A. E . WASSERMAN for v a l u a b l e a d v i c e a n d
A. R . GENNARO for t h e c u l t u r e o f S. aureus.
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