Journal of Applied Bacteriology 1992,72, 327-334
Factors affecting antibacterial activity of hop compounds and their derivatives W.J. Simpson and A.R.W. Smith‘
’
Brewing Research Foundation Nutfield, Redhill, Surrey and School of Biological and Chemical Sciences, Thames Polytechnic, Woolwich UK I
I
3714/06/91: accepted 20 September 1991
antibacterial effect of weak acids derived from the hop plant (Humulus lupulus L.) increased with decreasing p H . Analysis of the minimum inhibitory concentration of such compounds against Lactobacillus brevis I F 0 3960 over p H 4 - 7 suggests that undissociated molecules were mainly responsible for inhibition of bacterial growth. T h e antibacterial activity of trans-isohumulone was ca 20 times greater than that of humulone, 11 times greater than that of colupulone and nine times greater than that of trans-humulinic acid when the degree of ionization was taken into account. Monovalent cations ( K + , N a + , NH;, R b +,Li ’) stimulated antibacterial activity of trans-isohumulone but the effect was smaller than that observed with H + . T h e response to divalent cations varied : Ca2 had little effect on antibacterial activity, whereas M g Z reduced activity. Lipid materials and P-cyclodextrin also antagonized the antibacterial action of trans-isohumulone. W . J . S I M P S O N A N D A . R . W . S M I T H . 1992. T h e
+
INTRODUCTION
Compounds derived from flowers of the hop plant (Humulus lupubs L.) contribute a desirable bitter flavour to beer. Of greatest importance is a group of resins, commonly termed a-acids, represented by humulone and its congeners (cohumulone, adhumulone, prehumulone, posthumulone). A second group of substances, commonly termed p-acids, represented by lupulone and its congeners (colupulone, adlupulone, prelupulone, postlupulone), is also found in hops. The chemistry of these compounds has been the subject of several reviews (Ashurst 1967; Stevens 1967; Verzele 1986). a-Acids are isomerized during wort boiling to form the iso-a-acids, which occur as both czs- and transisomers; the prefix refers to the orientation on the fivemembered ring of the 3-hydroxy- and 3-methyl-2-butenyl substituents (Trolle 1969). The iso-a-acids (trans-isohumulone, cis-isohumulone and their congeners transisocohumulone, cis-isocohumulone, trans-isoadhumulone, cis-isoadhumulone) are the major bitter substances in beer (Verzele 1986). During isomerization of a-acids to iso-aacids, a range of humulinic acids (e.g. cis- and transisomers, co-, ad- forms, etc.) may be formed in hydrolysis reactions. Most hop compounds and hop-derived compounds inhibit the growth of Gram-positive bacteria (Shimwell Correspondence to : W . J . Sippson, Brewing Research Foundation,N u t j e d , Redhill, Surrey RHI 4 H Y , U K .
+
1937a; Schmalreck P t al. 1975) and some inhibit growth of fungi (Mizobuchi & Sat0 1985). Several studies have highlighted differences in the antibacerial activity of such compounds (Walker & Blakebrough 1952; Hough et al. 1957; Teuber & Schmalreck 1973; Schmalreck et al. 1975; Mizobuchi & Sato 1985). In these investigations, however, the compounds used were either ill-defined or impure and/or the pH of the test medium was given scant attention or not stated. Since the materials are weak acids, this latter point is particularly important (Simon & Blackman 1949). The aim of this work was to establish the influence of the pH value of the growth medium and other variables on the antibacterial activity of hop resin materials and their derivatives.
MATERIALS AND METHODS Reagents
All chemicals except those derived from hops were obtained from Sigma (UK) or BDH (UK) and were at least of analytical reagent grade. They were used without further purification. i
Hop compounds and hop-derivedcompounds
(-)-Humdone was prepared from a commercial liquid CO, hop extract of high a-acid content by complexation
328
W . J . S l M P S O N AND A . R . W . S M I T H
with o-phenylenediamine (McGuinness 1974). Pale yellow crystals (m.p. 63°C) were obtained after regeneration of the free acid by addition of concentrated HCl and repeated recrystallization at - 20°C from cyclohexane. Colupulone was prepared from a commercial liquid CO, hop extract rich in P-acid by recrystallizing the compound several times at 4°C from petroleum ether (b.p. 60"-80°C). White crystals (m.p. 92°C) were obtained. Trans-isohumulone was prepared by photoisomerization of ( - )-humulone as described by Clarke & Hildebrand (1965). White crystals (m.p. 63°C) were obtained after several recrystallizations at - 20°C from 2, 2, 4-trimethyl-pentane. Trans-humulinic acid, prepared by alkaline hydrolysis of ( - )-humulone (Ashurst 1966), was recrystallized several times at 4°C from petroleum ether (b.p. 6O0-8O"C) to give white crystals (m.p. 92.5"C). As they are very sensitive to oxidation and hydrolysis, all compounds were stored under nitrogen at -70°C. Solutions of the compounds (in methanol) were used within 30 min of preparation. Ultraviolet (u.v.) infrared, 'H and 13C nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (both electron impact and chemical ionization), melting point and optical rotation were used to confirm the identity of the test compounds. High performance liquid chromatography (Buckee & Baker 1987; Buckee 1990), 'H and I3C N M R spectroscopy were used to assess purity. These techniques allowed detection of less than one part in 200 of related contaminant compounds. Determinationof acid dissociation constants (pKa values)
The pKa value of each of the compounds was determined in water at 25°C by a potentiometric technique. Methods and activity corrections were as described by Albert & Serjeant (1984). In brief, a known quantity of each test compound was dissolved in 100 ml of water and titrated with 10 equal portions of NaOH solution to effect complete neutralization. The p H value of the solution, determined with an accurately calibrated p H meter, was recorded after each addition of titrant and a set of nine pKa values calculated for each set of determinations. Media
MRS medium was used for the maintenance of the test organism (de Man et al. 1960). For estimation of minimum inhibitory concentration (MTC) the medium was modified by the omission of Tween 80 and the p H value was adjusted with concentrated HCl before sterilization. Tween 80 was omitted as it antagonized the antibacterial action of hop resin materials and their derivatives (see below). Throughout, this modified medium is referred to as mod. MRS.
Test organism
Lactobacillus breais I F 0 3960, obtained from the Institute of Fermentation (Osaka, Japan), was maintained on MRS agar (subcultured at three-monthly intervals) and stored at
4°C. Assay of minimum inhibitory concentration
T h e MICs of humulone, colupulone, trans-isohumulone and trans-humulinic acid were estimated by a tube dilution technique. Preliminary experiments employing doubling dilutions of each compound established the appproximate value of the M I C under each set of conditions. More precise values were then obtained with an arithmetic series. These latter experiments were designed to maintain a constant level of error (as % coefficient of variation) in each test (Kavanagh 1963). Typically 15 tubes were employed in each series, and experiments were designed so that growth of the test strain was obtained in 10 or 11 of the tubes. All tests were performed on more than one occasion with independently prepared media and test solutions as follows. T h e test inoculum was prepared by transferring a colony of Lart. brezis I F 0 3960 from M R S agar to 10 ml mod. MRS. The culture was incubated at 25°C for 3 d, aerobically without shaking and then diluted 1 : 100 in sterile deionized water. T h e viability of the cells in this diluent remained constant throughout the inoculation period. A range of concentrations of hop compounds and hop-derived compounds in mod. MRS medium was prepared in disposable plastic tubes of 7 ml capacity (Sterilin, UK). Tubes containing 2 ml of growth medium were inoculated with diluted culture (40 p1 containing 4 x lo6 organisms). As a precaution against photochemical conversion of the test compounds (Clarke & Hildebrand 1965) all tubes were incubated in darkness. Growth, after incubation at 25°C for 48 h, was assessed spectrophotometrically at 560 nm in disposable plastic microcuvettes (1 cm path length) in a Philips P U 8720 UV/vis spectrophotometer. Effect of ionic composition of test medium on MIC
The M I C of humulone colupulone, trans-isohumulone and trans-humulinic acid against Lact. brezis I F 0 3960 was determined over the p H range 4-7. T h e influence of other monovalent cations on the antibacterial activity of trans-isohumulone was also studied. Cations (K', Na', Cs', NH:, Li+, Rb+, Ca2+,MgZ+, C o 2 + , N i 2 + ) were added (as chlorides) to mod. MRS medium (pH 5.2) to.give an added concentration of 0.2 mol/l. All media (including the control) were sterilized by membrane filtration and the p H values recorded immediately before use.
HOP COMPOUNDS
Effect of potential neutralizers on MIC of trans-isohumulone
The M I C of trans-isohumulone against Lact. brevzs I F 0 3960 was determined in the presence and absence of potential neutralizers in mod. M R S at p H 5.2. Compounds tested were Tween 80 ( 5 mmol/l), Tween 80/lecithin (3 g/l; 2 g/l), a-cyclodextrin ( 5 mmol/l), P-cyclodextrin ( 5 mmol/ l), bovine serum albumin (1 g/l) and cholesterol (5 mmol/l).
RESULTS
'
Identity and purity of test compounds
All analyses were consistent with the expected identity of the test compounds; their structures are shown in Fig. .I. High performance liquid chromatography, 'H and I3C NMR spectroscopy indicated the following level of contamination of the test compounds (on a molar basis): humulone-cohumulone, loo/" ; adhumulone, 1YO; colupulone-lupulone, 10% ; trans-isohumulone-no detectable contaminants; trans-humulinic acid-8% ctshumulinic acid. T h e difficulties in separating these compounds from related congeners are well known (Verzele 1986). Acid dissociation constants
Precise estimates of pKa could not be obtained for any of the compounds. A drift in the observed pKa values was
329
observed during potentiometric titration of each compound in water. Approximate, or equilibrium (Albert & Serjeant 1984), pKa values (pKaeqUi,)for the most acidic function of each of the compounds were : humulone, 5-0; colupulone, 6.1 ; trans-isohumulone, 3-1; trans-humulinic acid, 2.7. The effect of pH on the antibacterial activity of hop compounds and their derivatives
Adequate growth of the test organism was obtained in 48 h if the initial p H value of the growth medium was between 4 and 7 (Fig. 2). T h e antibacterial activity of humulone, colupulone, trans-isohumulone and trans-humulinic acid was influenced by p H (Fig. 3). T h e change in MIC with respect to change in p H was greatest in the case of transisohumulone and trans-humulinic acid ; less for humulone, and slight for colupulone. The effect of monovalent and divalent cations on the antibacterial activity of trans-isohumulone
The antibacterial activity of trans-isohumulone was enhanced by the addition of monovalent cations (K', Na+, N H f , R b + , L i f ) (Table 1). The growth yield of control cell suspensions was not affected by the same concentration of these ions in the absence of trans-isohumulone. However, no growth was obtained in the presence of 0.2 moI Cs+/I. 2.5r
2.0
'
.*
I
0
I
It
0.5t . la1 0 3.5
4.0
V
1
1
I
I
I
4.5
5.0
5.5
6.0
6.5
, 8,
7.0 7.5
PH Fig. 2 The effect of initial pH on growth of Lactobacillus brevis 111
IV
Fig. 1 Structure of I, colupulone; 11, humulone; 111,
trans-isohumulone and IV, trans-humulinic acid
IF0 3960 in mod. MRS at 25°C. Mod. MRS medium, adjusted to a range of pH values as shown, was inoculated with Lact. brevis I F 0 3960 (8 x lo4 organisms/ml). After 48 h incubation, growth was assessed spectrophotometrically at 560 nm in a cell of 1 cm path length
330 W . J . S I M P S O N AND A . R . W . S M I T H
4'01
3.5
3.0 T 5
2.5
-
-..E 2.0 5
0
E
-
1.51.0 0.5 0.01 3.5
I
1
I
1
4-0
4-5
5.0
5.5 PH
I
6.0
I
I
6.5
7.0
., I
7-5
Fig. 3 The relationship between the initial pH value of the growth medium and the antibacterial activity of A,humulone; colupulone; A, trans-isohumulone, and 0, trans-humulinic acid. Minimum inhibitory concentrations (MICs) for each of the compounds were assessed using Lactobacillus brevis IF0 3960 (8 x lo4 organisms/ml) in mod. MRS at 25°C
Addition of each of the salts caused a change in the p H value of the growth medium. In order to allow a comparison to be made of the antibacterial activity of transisohumulone in the presence of each of the salts, the
amount of undissociated trans-isohumulone present was calculated by the method described below. T h e increase in antibacterial activity of trans-isohumulone elicited by such salts ranged from about a 1.2-fold enhancement in the case of L i + to a 3-fold enhancement in the case of R b + (Table 1). Calcium ions reduced the antibacterial activity of transisohumulone to about 3/4, but the effect of Mgz+ was more pronounced, > 4-fold reduction in activity being recorded. T h e effect of this latter ion was particularly interesting since, in the absence of trans-isohumulone, no growth of Lart. brevzs IF0 3960 took place over the 2 d incubation period. However, addition of trans-isohumulone in the range 3.75-1 1.25 ,umol/l caused a concentrationdependent stimulation of growth. Above this concentration, growth was progressively inhibited by trans-isohumulone, with an M I C of 56 pmol/l. No growth was obtained in the presence of 0.2 mol Co2+ or Ni2+/1 regardless of whether trans-isohumulone was present or not. The effect of potential neutralizers on the antibacterial activity of trans-isohumulone
Table 2 shows that cholesterol, bovine serum albumin and cr-cyclodextrin had no significant effect on the antibacterial activity of trans-isohumulone. In contrast, Tween 80, a mixture of Tween 80 and lecithin, and P-cyclodextrin all reduced antibacterial activity.
DISCUSSION Ionic composition of the growth medium
Table 1 EXect of monovalent and divalent cations on antibacterial activity of trans-isohumulone
Addition*
p H of medium?
None
5.18 5.33 5.38 5.28 5.20 5.13 4.86 4.77
.K + Rb' NH: Na+ Li Ca2 Mg2+ +
+
32 16 16 19 18 23 21 56
,
264 94 84 125 142 213 359 1172
* 0.2 mol/l added as the chloride. Prior to additions being made, the medium contained (mmol/l): K', 41; Rb',
Factors affecting antibacterial activity of hop compounds and their derivatives W.J. Simpson and A.R.W. Smith‘
’
Brewing Research Foundation Nutfield, Redhill, Surrey and School of Biological and Chemical Sciences, Thames Polytechnic, Woolwich UK I
I
3714/06/91: accepted 20 September 1991
antibacterial effect of weak acids derived from the hop plant (Humulus lupulus L.) increased with decreasing p H . Analysis of the minimum inhibitory concentration of such compounds against Lactobacillus brevis I F 0 3960 over p H 4 - 7 suggests that undissociated molecules were mainly responsible for inhibition of bacterial growth. T h e antibacterial activity of trans-isohumulone was ca 20 times greater than that of humulone, 11 times greater than that of colupulone and nine times greater than that of trans-humulinic acid when the degree of ionization was taken into account. Monovalent cations ( K + , N a + , NH;, R b +,Li ’) stimulated antibacterial activity of trans-isohumulone but the effect was smaller than that observed with H + . T h e response to divalent cations varied : Ca2 had little effect on antibacterial activity, whereas M g Z reduced activity. Lipid materials and P-cyclodextrin also antagonized the antibacterial action of trans-isohumulone. W . J . S I M P S O N A N D A . R . W . S M I T H . 1992. T h e
+
INTRODUCTION
Compounds derived from flowers of the hop plant (Humulus lupubs L.) contribute a desirable bitter flavour to beer. Of greatest importance is a group of resins, commonly termed a-acids, represented by humulone and its congeners (cohumulone, adhumulone, prehumulone, posthumulone). A second group of substances, commonly termed p-acids, represented by lupulone and its congeners (colupulone, adlupulone, prelupulone, postlupulone), is also found in hops. The chemistry of these compounds has been the subject of several reviews (Ashurst 1967; Stevens 1967; Verzele 1986). a-Acids are isomerized during wort boiling to form the iso-a-acids, which occur as both czs- and transisomers; the prefix refers to the orientation on the fivemembered ring of the 3-hydroxy- and 3-methyl-2-butenyl substituents (Trolle 1969). The iso-a-acids (trans-isohumulone, cis-isohumulone and their congeners transisocohumulone, cis-isocohumulone, trans-isoadhumulone, cis-isoadhumulone) are the major bitter substances in beer (Verzele 1986). During isomerization of a-acids to iso-aacids, a range of humulinic acids (e.g. cis- and transisomers, co-, ad- forms, etc.) may be formed in hydrolysis reactions. Most hop compounds and hop-derived compounds inhibit the growth of Gram-positive bacteria (Shimwell Correspondence to : W . J . Sippson, Brewing Research Foundation,N u t j e d , Redhill, Surrey RHI 4 H Y , U K .
+
1937a; Schmalreck P t al. 1975) and some inhibit growth of fungi (Mizobuchi & Sat0 1985). Several studies have highlighted differences in the antibacerial activity of such compounds (Walker & Blakebrough 1952; Hough et al. 1957; Teuber & Schmalreck 1973; Schmalreck et al. 1975; Mizobuchi & Sato 1985). In these investigations, however, the compounds used were either ill-defined or impure and/or the pH of the test medium was given scant attention or not stated. Since the materials are weak acids, this latter point is particularly important (Simon & Blackman 1949). The aim of this work was to establish the influence of the pH value of the growth medium and other variables on the antibacterial activity of hop resin materials and their derivatives.
MATERIALS AND METHODS Reagents
All chemicals except those derived from hops were obtained from Sigma (UK) or BDH (UK) and were at least of analytical reagent grade. They were used without further purification. i
Hop compounds and hop-derivedcompounds
(-)-Humdone was prepared from a commercial liquid CO, hop extract of high a-acid content by complexation
328
W . J . S l M P S O N AND A . R . W . S M I T H
with o-phenylenediamine (McGuinness 1974). Pale yellow crystals (m.p. 63°C) were obtained after regeneration of the free acid by addition of concentrated HCl and repeated recrystallization at - 20°C from cyclohexane. Colupulone was prepared from a commercial liquid CO, hop extract rich in P-acid by recrystallizing the compound several times at 4°C from petroleum ether (b.p. 60"-80°C). White crystals (m.p. 92°C) were obtained. Trans-isohumulone was prepared by photoisomerization of ( - )-humulone as described by Clarke & Hildebrand (1965). White crystals (m.p. 63°C) were obtained after several recrystallizations at - 20°C from 2, 2, 4-trimethyl-pentane. Trans-humulinic acid, prepared by alkaline hydrolysis of ( - )-humulone (Ashurst 1966), was recrystallized several times at 4°C from petroleum ether (b.p. 6O0-8O"C) to give white crystals (m.p. 92.5"C). As they are very sensitive to oxidation and hydrolysis, all compounds were stored under nitrogen at -70°C. Solutions of the compounds (in methanol) were used within 30 min of preparation. Ultraviolet (u.v.) infrared, 'H and 13C nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry (both electron impact and chemical ionization), melting point and optical rotation were used to confirm the identity of the test compounds. High performance liquid chromatography (Buckee & Baker 1987; Buckee 1990), 'H and I3C N M R spectroscopy were used to assess purity. These techniques allowed detection of less than one part in 200 of related contaminant compounds. Determinationof acid dissociation constants (pKa values)
The pKa value of each of the compounds was determined in water at 25°C by a potentiometric technique. Methods and activity corrections were as described by Albert & Serjeant (1984). In brief, a known quantity of each test compound was dissolved in 100 ml of water and titrated with 10 equal portions of NaOH solution to effect complete neutralization. The p H value of the solution, determined with an accurately calibrated p H meter, was recorded after each addition of titrant and a set of nine pKa values calculated for each set of determinations. Media
MRS medium was used for the maintenance of the test organism (de Man et al. 1960). For estimation of minimum inhibitory concentration (MTC) the medium was modified by the omission of Tween 80 and the p H value was adjusted with concentrated HCl before sterilization. Tween 80 was omitted as it antagonized the antibacterial action of hop resin materials and their derivatives (see below). Throughout, this modified medium is referred to as mod. MRS.
Test organism
Lactobacillus breais I F 0 3960, obtained from the Institute of Fermentation (Osaka, Japan), was maintained on MRS agar (subcultured at three-monthly intervals) and stored at
4°C. Assay of minimum inhibitory concentration
T h e MICs of humulone, colupulone, trans-isohumulone and trans-humulinic acid were estimated by a tube dilution technique. Preliminary experiments employing doubling dilutions of each compound established the appproximate value of the M I C under each set of conditions. More precise values were then obtained with an arithmetic series. These latter experiments were designed to maintain a constant level of error (as % coefficient of variation) in each test (Kavanagh 1963). Typically 15 tubes were employed in each series, and experiments were designed so that growth of the test strain was obtained in 10 or 11 of the tubes. All tests were performed on more than one occasion with independently prepared media and test solutions as follows. T h e test inoculum was prepared by transferring a colony of Lart. brezis I F 0 3960 from M R S agar to 10 ml mod. MRS. The culture was incubated at 25°C for 3 d, aerobically without shaking and then diluted 1 : 100 in sterile deionized water. T h e viability of the cells in this diluent remained constant throughout the inoculation period. A range of concentrations of hop compounds and hop-derived compounds in mod. MRS medium was prepared in disposable plastic tubes of 7 ml capacity (Sterilin, UK). Tubes containing 2 ml of growth medium were inoculated with diluted culture (40 p1 containing 4 x lo6 organisms). As a precaution against photochemical conversion of the test compounds (Clarke & Hildebrand 1965) all tubes were incubated in darkness. Growth, after incubation at 25°C for 48 h, was assessed spectrophotometrically at 560 nm in disposable plastic microcuvettes (1 cm path length) in a Philips P U 8720 UV/vis spectrophotometer. Effect of ionic composition of test medium on MIC
The M I C of humulone colupulone, trans-isohumulone and trans-humulinic acid against Lact. brezis I F 0 3960 was determined over the p H range 4-7. T h e influence of other monovalent cations on the antibacterial activity of trans-isohumulone was also studied. Cations (K', Na', Cs', NH:, Li+, Rb+, Ca2+,MgZ+, C o 2 + , N i 2 + ) were added (as chlorides) to mod. MRS medium (pH 5.2) to.give an added concentration of 0.2 mol/l. All media (including the control) were sterilized by membrane filtration and the p H values recorded immediately before use.
HOP COMPOUNDS
Effect of potential neutralizers on MIC of trans-isohumulone
The M I C of trans-isohumulone against Lact. brevzs I F 0 3960 was determined in the presence and absence of potential neutralizers in mod. M R S at p H 5.2. Compounds tested were Tween 80 ( 5 mmol/l), Tween 80/lecithin (3 g/l; 2 g/l), a-cyclodextrin ( 5 mmol/l), P-cyclodextrin ( 5 mmol/ l), bovine serum albumin (1 g/l) and cholesterol (5 mmol/l).
RESULTS
'
Identity and purity of test compounds
All analyses were consistent with the expected identity of the test compounds; their structures are shown in Fig. .I. High performance liquid chromatography, 'H and I3C NMR spectroscopy indicated the following level of contamination of the test compounds (on a molar basis): humulone-cohumulone, loo/" ; adhumulone, 1YO; colupulone-lupulone, 10% ; trans-isohumulone-no detectable contaminants; trans-humulinic acid-8% ctshumulinic acid. T h e difficulties in separating these compounds from related congeners are well known (Verzele 1986). Acid dissociation constants
Precise estimates of pKa could not be obtained for any of the compounds. A drift in the observed pKa values was
329
observed during potentiometric titration of each compound in water. Approximate, or equilibrium (Albert & Serjeant 1984), pKa values (pKaeqUi,)for the most acidic function of each of the compounds were : humulone, 5-0; colupulone, 6.1 ; trans-isohumulone, 3-1; trans-humulinic acid, 2.7. The effect of pH on the antibacterial activity of hop compounds and their derivatives
Adequate growth of the test organism was obtained in 48 h if the initial p H value of the growth medium was between 4 and 7 (Fig. 2). T h e antibacterial activity of humulone, colupulone, trans-isohumulone and trans-humulinic acid was influenced by p H (Fig. 3). T h e change in MIC with respect to change in p H was greatest in the case of transisohumulone and trans-humulinic acid ; less for humulone, and slight for colupulone. The effect of monovalent and divalent cations on the antibacterial activity of trans-isohumulone
The antibacterial activity of trans-isohumulone was enhanced by the addition of monovalent cations (K', Na+, N H f , R b + , L i f ) (Table 1). The growth yield of control cell suspensions was not affected by the same concentration of these ions in the absence of trans-isohumulone. However, no growth was obtained in the presence of 0.2 moI Cs+/I. 2.5r
2.0
'
.*
I
0
I
It
0.5t . la1 0 3.5
4.0
V
1
1
I
I
I
4.5
5.0
5.5
6.0
6.5
, 8,
7.0 7.5
PH Fig. 2 The effect of initial pH on growth of Lactobacillus brevis 111
IV
Fig. 1 Structure of I, colupulone; 11, humulone; 111,
trans-isohumulone and IV, trans-humulinic acid
IF0 3960 in mod. MRS at 25°C. Mod. MRS medium, adjusted to a range of pH values as shown, was inoculated with Lact. brevis I F 0 3960 (8 x lo4 organisms/ml). After 48 h incubation, growth was assessed spectrophotometrically at 560 nm in a cell of 1 cm path length
330 W . J . S I M P S O N AND A . R . W . S M I T H
4'01
3.5
3.0 T 5
2.5
-
-..E 2.0 5
0
E
-
1.51.0 0.5 0.01 3.5
I
1
I
1
4-0
4-5
5.0
5.5 PH
I
6.0
I
I
6.5
7.0
., I
7-5
Fig. 3 The relationship between the initial pH value of the growth medium and the antibacterial activity of A,humulone; colupulone; A, trans-isohumulone, and 0, trans-humulinic acid. Minimum inhibitory concentrations (MICs) for each of the compounds were assessed using Lactobacillus brevis IF0 3960 (8 x lo4 organisms/ml) in mod. MRS at 25°C
Addition of each of the salts caused a change in the p H value of the growth medium. In order to allow a comparison to be made of the antibacterial activity of transisohumulone in the presence of each of the salts, the
amount of undissociated trans-isohumulone present was calculated by the method described below. T h e increase in antibacterial activity of trans-isohumulone elicited by such salts ranged from about a 1.2-fold enhancement in the case of L i + to a 3-fold enhancement in the case of R b + (Table 1). Calcium ions reduced the antibacterial activity of transisohumulone to about 3/4, but the effect of Mgz+ was more pronounced, > 4-fold reduction in activity being recorded. T h e effect of this latter ion was particularly interesting since, in the absence of trans-isohumulone, no growth of Lart. brevzs IF0 3960 took place over the 2 d incubation period. However, addition of trans-isohumulone in the range 3.75-1 1.25 ,umol/l caused a concentrationdependent stimulation of growth. Above this concentration, growth was progressively inhibited by trans-isohumulone, with an M I C of 56 pmol/l. No growth was obtained in the presence of 0.2 mol Co2+ or Ni2+/1 regardless of whether trans-isohumulone was present or not. The effect of potential neutralizers on the antibacterial activity of trans-isohumulone
Table 2 shows that cholesterol, bovine serum albumin and cr-cyclodextrin had no significant effect on the antibacterial activity of trans-isohumulone. In contrast, Tween 80, a mixture of Tween 80 and lecithin, and P-cyclodextrin all reduced antibacterial activity.
DISCUSSION Ionic composition of the growth medium
Table 1 EXect of monovalent and divalent cations on antibacterial activity of trans-isohumulone
Addition*
p H of medium?
None
5.18 5.33 5.38 5.28 5.20 5.13 4.86 4.77
.K + Rb' NH: Na+ Li Ca2 Mg2+ +
+
32 16 16 19 18 23 21 56
,
264 94 84 125 142 213 359 1172
* 0.2 mol/l added as the chloride. Prior to additions being made, the medium contained (mmol/l): K', 41; Rb',
