Antimicrobial composition

Abstract

Antimicrobial compositions comprising a terpenoid in combination with an antimicrobial agent are provided. In addition, uses of such compositions in various applications involving preventing, combating or treating microbial infections, or preventing microbial growth or establishment are provided.

Claims

1. An antimicrobial composition comprising dehydroabietic acid (DHAA) and an antimicrobial agent selected from the group consisting of vanillin, coniferaldehyde, thymol and stilbene; wherein when the antimicrobial agent is vanillin, the ratio of vanillin to DHAA is from 1:1 to 1:1024; when the antimicrobial agent is coniferaldehyde, the ratio of coniferaldehyde to DHAA is from 1:4 to 1:64; when the antimicrobial agent is thymol, the ratio of thymol to DHAA is from 1:32; and when the antimicrobial agent is stilbene, the ratio of stilbene to DHAA is 1:2.

2. A liquid formulation comprising the antimicrobial composition according to claim 1.

3. A method of preventing or inhibiting microbial colonization of an object, which method comprises contacting or coating a surface of the object with the antimicrobial composition according to claim 1.

4. An object comprising the antimicrobial composition according to claim 1.

5. The object according to claim 4, wherein the object is a medical device.

6. The object according to claim 4, wherein the medical device is selected from the group consisting of a catheter, a stent, a wound dressing, a bandage, a contraceptive device, a surgical implant, a replacement joint, contact lens, a bandage, a wound dressing, and a plaster.

7. A method for preventing or inhibiting microbial infection of a food product comprising: contacting or coating a surface of a food product with the antimicrobial composition according to claim 1.

Description

(1) For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now be made to the following Figures and Examples.

(2) FIG. 1 shows an inhibition experiment using one embodiment of the composition of the invention, comprising a combination of vanillin and DHAA against S. aureus (Gram-positive);

(3) FIG. 2 shows an inhibition experiment using a composition comprising a combination of stilbene and DHAA against S. aureus;

(4) FIG. 3 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against S. enteritidis (Gram-negative);

(5) FIG. 4 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against S. typhimurium (Gram-negative);

(6) FIG. 5 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against E. coli (Gram-negative);

(7) FIG. 6 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against Penicillium spp. (filamentous fungi);

(8) FIG. 7 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against Aspergillus V. (filamentous fungi); and

(9) FIG. 8 shows an inhibition experiment using a composition comprising a combination of vanillin and DHAA against S. cerevisiae (yeast).

EXAMPLES

(10) The inventors investigated the mechanisms by which mildly heated pine shavings become antimicrobial. As described in the Examples below, they observed that the terpenoid, dehydroabietic acid, can significantly enhance the antimicrobial activity of vanillin and other natural antimicrobial agents, such as essential oils.

Example 1—Synergistic Effect of Dehydro-Abietic Acid on the Antimicrobial Activity of Vanillin

(11) The aim of this example was to determine to what extent dehydro-abietic acid increases the antimicrobial activity of vanillin.

(12) Methodology

(13) A 2 fold dilution series of 20 mmol of vanillin was prepared in ethanol and water (80/20 v/v) giving concentrations from 20 mmol-0.00975 mmol. A 20 mmol solution of dehydroabietic acid in alcohol and water was also prepared.

(14) To compare the effect of dehydroabietic acid on the anti-microbial effect of vanillin, 10 μl of each vanillin dilution was pipetted onto three, 10 mg pieces of perlite, thus giving the same concentration per g perlite as was in the solution together with 10 μl of 20 mmol dehydroabietic acid. One set of control treatments did not receive dehydroabietic acid, another only 20 mmol dehydroabietic acid, and a third control treatment received neither vanillin nor dehydroabietic acid, only 10 μl alcohol and water at a ratio of 80/20. Once the alcohol had evaporated, all replicates received 10 μl of a suspension of Salmonella enterica containing approx 5×10.sup.6 colony forming units (cfu) per ml giving a bacterial load of approx. 5×10.sup.4 bacteria per perlite particle. Each perlite particle was incubated in an Eppendorf tube at 25° C. for 24 hours before each particle was dispersed in 1 ml of 0.25 strength Ringer's Solution. From each suspension, six 20 μl droplets were placed onto XLD agar each containing approximately 1000 bacterial cells.

(15) Results

(16) The results are shown in Table 2.

(17) TABLE-US-00003 TABLE 2 Effect of vanillin and a combination of vanillin and dehydro-abietic acid on the survival of Salmonella enterica (n = 3) Conc. of Dehydro-Abietic acid Conc. of Vanillin Average no of cfu (mmol) (mmmol) recovered per droplet 20 20 0 20 10 0 20 5 0 20 2.5 0 20 1.25 0 20 0.625 0 20 0.313 0 20 0.156 0 20 0.078 0 20 0.039 0 20 0.019 30.6 ± 0.7 20 0.001 >100 20 0 >100 0 20 >100 0 10 >100 0 5 >100 0 2.5 >100 0 1.25 >100 0 0.625 >100 0 0.313 >100 0 0.156 >100 0 0.078 >100 0 0.039 >100 0 0.019 >100 0 0.001 >100 0 0 >100
Conclusions

(18) The results presented in Table 2 show that:

(19) (i) Vanillin on its own, at concentrations of 20 mmol or less, had no inhibitory effect on Salmonella;

(20) (ii) In combination with dehydro-abietic acid, the inhibitory effect of vanillin was still significant at a concentration of 0.019 mmol. This indicates that dehydro-abietic acid increases the antimicrobial effect of vanillin at least 1000-2000 fold; and

(21) (iii) Dehydro-abietic acid at a concentration of 20 mmol had no significant anti-microbial activity.

Example 2—Effect of an Organic Acid on the Anti-Microbial Activity of Vanillin

(22) The aim of this example was to assess if the synergistic effect of dehydro-abietic acid on vanillin was specifically related to dehydro-abietic acid, or if it could be explained by a pH effect of the acid.

(23) Methodology

(24) A 2 fold dilution series of 20 mmol of vanillin was prepared in ethanol and water (80/20 v/v) giving concentrations from 20 mmol-0.078 mmol. A 20 mmol solution of citric acid in water was also prepared.

(25) To evaluate if citric acid had the same effect as dehydro-abietic acid (as described in Example 1), 10 μl of each vanillin dilution was pipetted onto three, 10 mg pieces of perlite, thus giving the same concentration per g perlite as was in the solution. Subsequently, 10 μl of 20 mmol citric acid was pipetted onto each perlite particle. One set of control treatments did not receive citric acid, the other control received neither vanillin nor citric acid, only 10 μl alcohol and water at a ratio 80/20. Once the alcohol had evaporated, all replicates received 10 μl of a suspension of Salmonella containing approx 5×10.sup.6 cfu per ml giving a bacterial load of approx. 5×10.sup.4 bacteria per perlite particle.

(26) Each perlite particle was incubated in an Eppendorf tube at 25° C. for 24 hours before each particle was dispersed in 1 ml of 0.25 strength Ringer's Solution. From each suspension, six 20 μl droplets were placed onto XLD agar each containing approx 1000 bacterial cells.

(27) Results

(28) The results are show in Table 3.

(29) TABLE-US-00004 TABLE 3 Effect of a combination of vanillin and citric acid on the survival of Salmonella enterica (n = 3) Conc. of citric acid Conc. Of Vanillin Average no. of cfu (mmol) (mmol) recovered per droplet 20 20 63.7 ± 3.3 20 10 >100 20 5 >100 20 2.5 >100 20 1.25 >100 20 0.625 >100 20 0.313 >100 20 0.156 >100 20 0.078 >100 20 0 >100 0 20 >100 0 0 >100
Conclusions

(30) The results presented in Table 3 show that:

(31) (i) Citric acid increased the effect of vanillin very slightly (2 fold maximum); and

(32) (ii) The effect of dehydro-abietic acid on the activity of vanillin cannot be attributed to a pH effect.

Example 3—Antimicrobial Activity of Vanillin in Combination with a Range of Triterpenoids

(33) The aim of Example 3 was to determine if triterpenoids, which also have a hydrophilic part and a hydrophobic part, have a similar activity as the diterpenoid, dehydroabietic acid, in relation to vanillin.

(34) Methodology

(35) A 10 fold dilution series of 20 mmol of vanillin was prepared in ethanol and water (80/20 v/v) giving concentrations from 20 mmol-0.02 mmol. 20 mmol solutions of ursolic acid, oleonic acid and betulin in alcohol were also prepared.

(36) To determine the effect of ursolic acid, oleonic acid and betulin dehydroabietic acid on the anti-microbial effect of vanillin, 10 μl of each vanillin dilution was pipetted onto three, 10 mg pieces of perlite, thus giving the same concentration per g perlite as was in the solution together with 10 μl of 20 mmol of each of the triterpenoids. One set of control treatments did not receive triterpenoids, and another only 20 mmol dehydroabietic acid. Once the alcohol had evaporated, all replicates received 10 μl of a suspension of Salmonella enterica containing approx 5×10.sup.6 colony forming units (cfu) per ml giving a bacterial load of approx. 5×10.sup.4 bacteria per perlite particle. Each perlite particle was incubated in an Eppendorf tube at 25° C. for 24 hours before each particle was dispersed in 1 ml of 0.25 strength Ringer's solution. From each suspension, six 20 μl droplets were placed onto XLD agar each containing approximately 1000 bacterial cells.

(37) Results

(38) The results are shown in Table 4.

(39) TABLE-US-00005 TABLE 4 Effect of a combination of vanillin and three triterpenoids (ursolic acid (a), oleanolic acid (b) and betulin (c)) on the survival of Salmonella enterica (n = 3) (a) Ursolic acid Conc. of ursolic acid Conc. Of Vanillin Average no. of cfu recovered per (mmol) (mmol) droplet 20 20 0 20 2 >100 20 0.2 >100 20 0.02 >100 20 0 >100 0 0 >100 0 20 >100 (b) Oleanic acid Conc. Of oleanic acid Conc. Of Vanillin Average no. of cfu recovered per (mmol) (mmol) droplet 20 20 0 20 2 50 20 0.2 >100 20 0.02 >100 20 0 >100 0 0 >100 0 20 >100 (c) Betulin Conc. Of betulin Conc. Of Vanillin Average no. of cfu recovered per (mmol) (mmol) droplet 20 20 0 20 2 >100 20 0.2 >100 20 0.02 >100 20 0 >100 0 0 >100 0 20 >100
Conclusions

(40) The results presented in Table 4 show that:

(41) (i) All triterpenoids tested increased the activity of vanillin,

(42) (ii) There was no significant difference in the increase in anti-microbial effect between the three triterpenoids, and

(43) (iii) The synergistic effect of triterpenoids in conjunction with vanillin was less than that observed with the combined use of dehydroabietic acid and vanillin. The most likely reason for this is that triterpenoids are very insoluble and may not be taken up in sufficient quantities by a microbial cell to exert a significant effect, even when in close contact.

Example 4—Anti-Microbial Effect of Heated and Non-Heated Wood Shavings Derived from Different Tree Species

(44) The aim of Example 4 was to determine the antimicrobial effects of heated and non-heated wood shavings derived from a range representative soft and hard wood species.

(45) Methodology

(46) For this experiment, shavings from trunk woods from different soft woods (Pine, Spruce, Cedar) and different hard woods (Beech, Birch, Ash, Sweet chestnut, Red oak), were taken, and the shavings from each were placed in six batches. Three of these batches were heated for 72 h at 140° C., and the other three were dried at 20° C., giving three replicates for each treatment. Subsequently, 1 g of shavings of each set was placed in a Universal bottle, and each was inoculated with 1 ml of a milky suspension (approx 10.sup.8 cfu per ml) of Salmonella enterica. Inoculated shavings were incubated at 25° C. for 20 h. Subsequently, a 10-fold dilution series (neat—10.sup.−8) was prepared from the shavings and 0.1 ml of each dilution was plated out onto XLD agar. The numbers of Salmonella colonies were counted after 36 hours incubation at 25° C.

(47) Results

(48) The results are shown in Table 5.

(49) TABLE-US-00006 TABLE 5 Recovery of Salmonella enterica (log cfu g.sup.−1 wood ± SE) from heated and non-heated wood shavings derived from a selection of soft and hard woods (n = 3). Each replicate was inoculated with approx 10.sup.8 cfu Wood type Non-Heated Heated Significance Pine (soft) 6.88 ± 0.11.sup.c 0 *** Spruce (soft) 7.56 ± 0.06.sup.d 4.50 ± 0.02 *** Cypress 5.24 ± 0.07.sup.b 0 *** (soft) Ash (hard) 7.12 ± 0.06.sup.c 0 *** Beech (hard) 6.85 ± 0.19.sup.c 0 *** Red Oak 7.22 ± 0.13.sup.c 0 *** (hard) Birch (hard) 7.32 ± 0.04.sup.cd 0 *** Eucalyptus 5.93 ± 0.07.sup.b 5.3 ± 0.06 *** Sweet 0.sup.a 0 NS chestnut (hard) Significance *** *** * Different letters indicate a significant difference between treatments
Conclusions

(50) From the data presented in Table 5, it can be concluded that: (i) There were small but significant differences in the antimicrobial properties of non-heated wood shavings. Spruce, Birch and Oak appeared to be the least antimicrobial, since each of these woods allowed the recovery of the greatest number of bacterial colonies. Cyprus and Eucalyptus appeared to be the most antimicrobial when before heat-treatment, since these two woods resulted in fewer bacterial colonies to grow; (ii) Heating resulted in a significant increase in the antimicrobial activity of all of the wood species that were tested; (iii) Heating resulted in a 3 fold increase in the antimicrobial activity in Eucalyptus, a 1000 fold increase in Spruce, and complete inhibition in each of Pine, Cyprus, Ash, Beach, Oak and Birch; and (iv) Besides vanillin, other antimicrobial substances must be present in Pine and Cedar.

Example 5—Toxicity Tests

(51) The aim of this example was to show that the combination dehydro-abietic acid with vanillin (or any other antimicrobial substance derived from wood) is safe for human consumption.

(52) Potential harm caused by a substance is determined by its potential toxicity and its exposure to target cells. Understanding of the potential pathways by which a potentially toxic substance could present itself to a human cell is therefore important for determining potential harm (risk). If it can be shown that the substance (or toxic combination of substances) is unlikely to come into contact with human target cells, then the substance must be regarded as being safe. In the case of dehydro-abietic acid and vanillin (or any other natural anti-microbial agent), there are at least four mechanisms that could prevent exposure of human cells when food coated with the product is ingested, i.e: 1) Ingestion of the composition will result in a sufficient dilution of the compounds to make them ineffective; 2) The cells of the digestive system are protected by a layer of mucous that protects the cells of the digestive system against contact with the composition; 3) One (or both) compounds are inactivated by the digestive system. This could be as a result of enzymatic activity; and 4) The compounds are not soluble enough in the stomach juices to have any impact on the cells of the digestive system.

(53) Only possibility 4 can be tested without using feeding studies.

(54) Methodology

(55) In this experiment, heated and non-heated pine shavings were used as test materials. Previous research has shown that heated pine shavings become highly antimicrobial. This effect is believed to be at least partly due to the synergistic effects that dehydro-abietic acid has on a number of mildly anti-microbial substances that are formed during mild and prolonged heating (see Examples 1 and 3). If these substances stay associated with the pine shavings and cannot be extracted using water, it can be assumed that they won't solubilise in the digestive system, and will not resulting in any exposure. Salmonella was used as the test organism because it has already been demonstrated that Salmonella cells are sensitive to the combination of abietic acid and vanillin (see Example 1).

(56) Water Extractions

(57) 6 g of material from each set of treated and non-treated pine shavings were soaked in water by adding 5 ml of sterile water to 2 g of shavings (in triplicate) using the following soaking regimes: 24 hours soaking at room temperature 24 hours soaking at 90° C. in water bath

(58) To assess if the extract was antimicrobial, the following procedure was followed: 1. Pour 1 ml of extract over 1 g shredded (autoclaved) filter paper (12 samples); 2. Use, as control, 0.25 strength Ringer's Solution (3 samples); 3. Dry samples at 50° C. overnight (or till dry); 4. Prepare a milky suspension of Salmonella in 0.25 strength Ringer's Solution and add 1 ml of this to each sample; 5. Incubate overnight at 30° C.; 6. Plate out a dilution series (10.sup.−1-10.sup.−6) for each (15×6=90 plates); 7. Incubate and enumerate countable plates; and 8. Compare inhibition against control treatment and extracts from non-heated shavings.

(59) To assess the antimicrobial activity of the extracted shavings, the following procedures were followed: 1. Dry the shavings that were extracted at 50° C. overnight (or till dry); 2. Use 2 g shredded filter paper as control (3 samples); 3. Add to each 2 g of shavings and filter paper 2 ml of Salmonella suspension; 4. Incubate at 30° C. for 24 h; 5. Plate out a dilution series (10.sup.−1-10.sup.−6) for each; 6. Incubate and enumerate countable plates; and 7. Compare inhibition against control (filter paper) and non-heated shavings.
Alcohol Extractions

(60) Take 6 g of material from each treated and non-treated batch and soak by adding 5 ml of methanol to 2 g of shavings (in triplicate) and soak for 24 hours:

(61) To assess if the extract was antimicrobial the following procedure was followed: 1. Pour 1 ml of extract over 1 g of shredded (autoclaved) filter paper (6 samples); 2. Use as control 0.25 strength Ringer's Solution (3 samples); 3. Soak three, 1 g samples with methanol (to ensure that alcohol residues don't inhibit microbial growth); 4. Dry each sample at 50° C. overnight (or till dry); 5. Prepare a milky suspension of Salmonella in 0.25 strength Ringer's Solution and add 1 ml of this to each sample; 6. Incubate overnight at 30° C.; 7. Plate out a dilution series (10.sup.−1-10.sup.−6) for each (12×6=72 plates); 8. Incubate and enumerate countable plates; and 9. Compare inhibition against controls and non-heated shavings.

(62) To asses the antimicrobial activity of the extracted shavings, the following procedures were followed: 1. Dry extracted samples at 50° C. overnight (or till dry); 2. Use 2 g shredded filter paper as control (3 samples); 3. Add to each 2 g of shavings and filter paper 2 ml of Salmonella suspension; 4. Incubate at 30° C. for 24 h; 5. Plate out a dilution series (10.sup.−1-10.sup.−6) for each (9×6=54 plates); 6. Incubate and enumerate countable plates; and 7. Compare inhibition against control (filter paper) and non-heated shavings.
Results

(63) Tables 6 and 7 show the results.

(64) TABLE-US-00007 TABLE 6 Recovery of Salmonella (log cfu g.sup.−1 ± SE) from inoculated filter paper treated with extracts from heated and non-heated pine shavings. Extracts were obtained by soaking 2 g shavings in cold water (Cold water Extract), water of 90° C. (Hot water Extract) or methanol (Alcohol Extract). Controls were not treated but received the same amount of Salmonella as the treated filter paper (n = 3). Extracts from Extracts from Treatment Heated shavings Non-heated shavings Cold water 7.54 ± 0.05 8.14 ± 0.03 ab Extract a* Hot water 7.37 ± 0.06 a 7.94 ± 0.05 bc Extract Alcohol 4.80 ± 0.11 b 7.91 ± 0.10 bc Extract Control 7.63 ± 0.10 a 7.72 ± 0.09 c Significance P < 0.001 P < 0.05 *Different letters indicate a significant difference between treatments

(65) TABLE-US-00008 TABLE 7 Recovery of Salmonella (log cfu g.sup.−1 ± SE) from inoculated, heated and non-heated pine shavings that were previously extracted by soaking 2 g shavings in cold water (Cold water Extract), water of 90° C. (Hot water Extract) or methanol (Alcohol Extract). Controls consisted of filter paper that received the same amount of Salmonella as the pine shavings (n = 3) Extracts from Extracts from Treatment Heated shavings Non-heated shavings Cold water 0.00 ± 0.00 7.63 ± 0.12 a Extract a* Hot water 0.00 ± 0.00 a 8.02 ± 0.05 a Extract Alcohol Extract 4.00 ± 0.00 b 7.06 ± 0.12 b Control 7.63 ± 0.10 c 7.72 ± 0.09 a Significance P < 0.001 P < 0.001 *Different letters indicate a significant difference between treatments
Conclusions

(66) From Tables 6 and 7 it may concluded that:

(67) (i) Both cold and hot water extracts from heated pine shavings were non-toxic to Salmonella;

(68) (ii) Microbial toxicity remained associated with pine shavings after extraction with cold or hot water;

(69) (iii) Alcohol extracts of heated pine shavings are toxic to Salmonella;

(70) (iv) Alcohol extraction removed some of the toxicity associated with heated pine shavings;

(71) (v) Dehydroabietic acid in combination with other anti-microbial substances in heated pine shavings are not soluble enough in water to cause significant toxicity to bacterial cells;

(72) (vi) It is expected that ingestion of food treated with dehydroabietic acid together with a natural anti-microbial agent, such as vanillin, will not lead to significant exposure to human cells.

Example 6—Applications of the Compositions of the Invention

(73) The inventors have applied their surprising observation that the antimicrobial activity of known antimicrobial agents such as vanillin can be dramatically improved upon combination with a terpenoid, to a wide range of applications.

(74) (i) Animal Bedding

(75) They have prepared a liquid formulation of a composition containing both dehydro-abietic acid and vanillin, and then sprayed this formulation on to some existing animal bedding, of the type used in poultry houses. The inventors found that the bedding decreased the levels of infection with Campylobacter and Salmonella, and that chickens raised on the bedding did not show any signs of bacterial infection.

(76) (ii) Mulch

(77) The inventors also produced some mulch which had been immersed in the liquid formulation containing dehydro-abietic acid and vanillin and left overnight so that a sufficient amount of the formulation had been absorbed. They then applied the mulch over a patch of soil and carried out tests to confirm that it was able to prevent soil-borne pathogens from infecting plants, fruits or vegetables. They were pleased to see that the treated mulch prevented microbial infection on strawberries that were grown in the mulch when compared to the control when normal mulch was used.

(78) (iii) A Medical Device

(79) The inventors then tested whether it was possible confer antimicrobial activity to the surface of a medical device, and used a wound dressing (i.e. a bandage) as a model. They dipped the bandage in the formulation, and left it over night for it to absorb an active amount. They found to their surprise that the bandage prevented the spread of micro-organism infection emanating from a wound underneath the bandage.

(80) (iv) A Textile or Polymer

(81) The formulation was then applied to pieces of cotton and wool, which were then used in the manufacture of articles of clothing. Also, a shoe was produced, and the inventors found that in all cases, microbial infection was prevented. In the case of the footwear, the production of bad odours was also minimised.

(82) (v) A Food Packaging Material

(83) The inventors also sprayed food packaging products, such as cardboard moulds, with the liquid formulation. Perishable foodstuffs such as fruit and vegetables were stored in the packaging material, and the inventors found that the foodstuffs did not rot as quickly as control foodstuffs that had been stored in traditional packaging materials.

(84) Summary

(85) The inventors have demonstrated that molecules with a polar, prolytic group and a rigid hydrophobic moiety can increase the antimicrobial activity of mildly anti-microbial substances that interfere with microbial cell membranes. Terpenoids, such as dehydro-abietic acid, can increase the antimicrobial activity of the antimicrobial agent, vanillin, by 1000-2000 fold. They have also shown that dehydro-abietic acid is poorly-soluble in water and that therefore water extracts from materials that contain both dehydro-abietic acid and vanillin are non-toxic to microbial cells. Instead, the antimicrobial activity remains within the treated material. Accordingly, ingestion of food treated with a terpenoid, such as dehydro-abietic acid, together with a natural anti-microbial agent, such as vanillin, will not lead to significant exposure to human cells.

Example 7—Synergistic Effect of Purified Dehydro-Abietic Acid (DHAA) on Antimicrobial Effect of Commercial Vanillin Against Salmonella enteritidis Using a Perlite Bio-Assay

(86) As described in the previous examples, significant results were obtained using either dehydro-abietic acid and/or vanillin that had been isolated from heated wood shavings. Experiments that gave up to 2000× greater antimicrobial effects of vanillin (isolated from heated wood) were conducted using a ‘perlite-bio-assay’ instead of a conventional dilution assay in broth. It could be argued that drying the combination onto perlite and calculating the concentrations based on the weight of the perlite could have biased the results.

(87) This experiment therefore assesses if the perlite bio-assay biases the results using pure DHAA that had been isolated from disproportionated rosin, and pure vanillin purchased from Sigma-Aldrich.

(88) Preparation of Stock solutions: A 20 mM stock solution of vanillin was made in acetone and diluted further by two-fold dilution to make a concentration range between 20 to 0.078 mM. Also, one 20 mM stock solution of DHAA was made in acetone.

(89) Bioassay test: The test was carried out using 1.5 ml Eppendorf tubes containing 10 mg of fine perlite. Ten μl of each concentration of vanillin was dispensed into four Eppendorf tubes to obtain four replicates of each concentration. Two of these received 10 μl of DHAA. The tubes were left in laminar flow chamber for 3 h to allow evaporation of the acetone. Subsequently, each tube received 1 μl of an overnight culture of Salmonella enteritidis. To keep the perlite moist during incubation, 10 μl of sterile R.O water was dispensed into each tube. As controls, two tubes received DHAA only and two tubes received R.O water only.

(90) The tubes were incubated overnight, and bacterial survival was quantified by plating the content of each tube onto nutrient agar for the bacterial count.

(91) Results

(92) TABLE-US-00009 TABLE 8 Effect of DHAA on vanillin antimicrobial activity against Salmonella enteritidis (CFU/tube) 10 0.313 mM 5 mM 2.5 mM 1.25 mM 0.625 mM mM Rep1(+DA) — — 305 480 TMTC TMTC Rep2(+DA — — 341 500 TMTC TMTC Rep 1 500 TMTC TMTC TMTC TMTC TMTC Rep 2 394 TMTC TMTC TMTC TMTC TMTC DHAA: TMTC R.O controls: TMTC
Conclusions

(93) In this experiment DHAA made vanillin approximately eight times more effective. The perlite bio-assay therefore clearly provides valid results on the activity of anti-microbial agents, and demonstrates synergism between the two pure compounds tested. Also, the inventors believe that there may be certain unknown factors that can further contribute to the synergistic activity of DHAA and vanillin when these compounds are isolated from heated wood shavings.

Example 8—Determination of Minimum Inhibitory Concentrations (MICs) of Dehydrozingeron and Coniferaldehyde in Combination with Vanillin and DHAA Mixtures Against Salmonella enteritidis

(94) It was noted that combining pure dehydroabietic acid (DHAA) and pure vanillin resulted in anti-microbial synergism between the two compounds (i.e. 8×), though not to such an extent as that observed when combining vanillin and DHAA isolated from heated pine wood (i.e. 2000×). Similar strong anti-microbial effects were obtained if only one of the components came from heated pine wood and the other was obtained from a commercial source in a pure form. Interestingly, anti-microbial effects obtained in this way resulted in an orange colour of the mix, suggesting that an unknown contaminant with a orange yellow colour could have been involved in augmenting the synergistic effects between vanillin and DHAA. A potential candidate molecule was thought to be coniferaldehyde which had previously been isolated from heated pine shavings.

(95) Similarly, if vanililin was not used immediately but was stored in acetone, the antimicrobial effects of combining DHAA with vanillin was largely restored. It is known that vanillin reacts with acetone to form a ketone, i.e. dehydrozingerone. It was therefore hypothesised that dehydrozingerone could also play a role in the antimicrobial effect obtained between DHAA and vanillin.

(96) Materials & Methods:

(97) Pure vanillin was obtained from Sigma-Aldrich. Dehydrozingerone was made by condensing vanillin with acetone. Pure dehydroabietic acid was isolated from disproportionated rosin.

(98) A 20 mM stock solution of vanillin was made to make the final concentrations. Also 0.1, 1, 5 and 10 mM stock solutions of dehydrozingerone and 20 mM stock solution of DHAA were made by dissolving the compounds in acetone. Stock solutions were stored at 5° C. in a fridge.

(99) In vitro bioassay: The minimum inhibiting concentration (MIC) of vanillin in combination with potential synergistic molecules was determined using a micro-broth dilution method. Six two fold dilution series of vanillin were prepared by diluting with sterile nutrient broth, using 96-well micro-titre plates in order to obtain a 2 fold dilution series of 20 mM to 0.078 mM vanillin. Four different concentrations (10 mM, 5 mM, 1 mM and 0.1 mM) of dehydrozingerone (DHZ) were made and combined with the different concentrations of vanillin by adding 100 μl of each stock solution to each well. In a separate experiment, DHZ was replaced by coniferaldehyde. Control wells were prepared by adding 100 μl acetone. Each treatment was replicated four times. The plates were kept in a laminar flow chamber for 8 hours to allow evaporation of the acetone. To determine anti-microbial activity in each well, plates were inoculated with 5 μl of a bacterial cell culture containing 10.sup.8 cfu/ml Salmonella enteritidis that had grown overnight at 25° C. After inoculation, the plates were incubated at 25° C. for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from each well was spotted onto nutrient agar. Thus, inoculated nutrient agar plates were incubated for 24-48 h and spots with no bacterial growth or just a few colonies were marked as ‘bacteriocidal’. To determine a bacteriostatic effect, a tetrazolium salt solution was added to each well to determine metabolic activity. After overnight incubation at 25° C. no change in colour was defined as a bacteriostatic effect while metabolism (no effect) was indicated as a red to purple colour.

(100) Results

(101) TABLE-US-00010 TABLE 9 Effect of combining different concentrations of coniferaldehyde and Vanillin on growth of Salmonella enteritidis. All treatments contained 20 mM dehydroabietic acid, except the control (−DHAA). (n = 4) Coniferyl aldehyde Concentration Vanillin Concentration (mM) (mM) 20 10 5 2.5 1.25 0.625 0.312 0.156 0.078 10 BC BC BC BC BC BC BC BC BC 5 BC BC BC BC BC BC BS BS BS 1 BS BS BS BS BS BS G G G 0.1 BS BS BS G G G G G G 0.0 BS BS BS G G G G G G 0.0-DHAA BS BS G G G G G G G BS: Bacteriostatic BC: Bactericidal G: Growth

(102) TABLE-US-00011 TABLE 10 Effect of combining different concentrations of Dehydrozingeron and Vanillin on growth of Salmonella enteritidis. All treatments contained 20 mM dehydroabietic acid, except the control (−DHAA). (n = 4) Dehydrozingeron Concentration Vanillin Concentration (mM) (mM) 20 10 5 2.5 1.25 0.625 0.312 0.156 0.078 10 BC BC BS BS BS BS BS BS BS 5 BS BS BS BS BS BS BS BS BS 1 BS BS BS G G G G G G 0.1 BS BS BS G G G G G G 0.0 BS BS BS G G G G G G 0.0-DHAA BS BS G G G G G G G BS: Bacteriostatic; BC: Bactericidal; G: Growth
Conclusions

(103) Coniferaldehyde is an effective anti-microbial agent that in combination with vanillin and DHAA confers strong anti-microbial effects against Salmonella enteritidis. Dehydrozingerone is a mild anti-microbial agent that in combination with vanillin and DHAA confers strong anti-microbial effects against Salmonella enteritidis. The results suggest that a combined concentration of >5 mM of vanillin and dehydrozingerone is required (in combination with DHAA) to be bacteriostatic.

Example 9—Role of Dehydroabietic Acid in Anti-Microbial Activity Between Coniferaldehyde and Vanillin

(104) In the previous experiments DHAA was added to combinations of vanillin and coniferaldehyde. To determine if DHAA is essential, a separate experiment was set up to quantify its role in the anti-microbial effect of the three substances.

(105) Materials & Methods

(106) Vanillin and coniferaldehyde were obtained from Sigma-Aldrich. Dehydroabietic acid was isolated from disproportionated rosin using column chromatography.

(107) A 20 mM stock solution of vanillin in deionised water was prepared. Stock solutions of 5 and 10 mM coniferaldehyde and 20 mM dehydroabietic acid (DHAA) were prepared in acetone. All stock solutions were stored at 5° C.

(108) In vitro bioassay: The MIC of the different treatments was determined using a micro broth dilution method. A 4 fold dilution series of vanillin was prepared by diluting the 20 mM stock solution with sterile water to obtain concentrations between 20 mM to 0.0002 mM vanillin using a 96-well micro-titre plate. Two concentrations of 5 and 10 mM coniferaldehyde were prepared and combined with the different concentrations of vanillin by adding 100 μL of each stock solution to each well. Control wells were prepared by adding 100 μL acetone. Half the wells received DHAA to a final concentration of 20 mM while the other half did not. Each treatment was replicated four times. The plates were placed in a laminar flow chamber for 8 hours to evaporate the solvent. Subsequently, each well was inoculated with 5 μl of a Salmonella enteritidis suspension containing 10.sup.8 cfu/ml that had grown overnight at 25° C. After inoculation, the plates were incubated at 25° C. for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from each well was spotted onto nutrient agar. Plates were incubated for 24-48 h and spots with no bacterial growth or just a few colonies were marked as ‘bacteriocidal’. Where possible, colonies were counted to obtain more precise quantitative data. To determine a bacteriostatic effect 50 μl tetrazolium salt solution was added to each well to determine metabolic activity. After overnight incubation at 25° C., no change in colour was defined as a bacteriostatic effect while metabolism (no effect) was indicated by a red to purple colour.

(109) Results

(110) TABLE-US-00012 TABLE 11 Anti-microbial effect of vanillin in combination with Coniferyl aldehyde (CFA) with and without 20 mM Dehydroabietic Acid (DHAA). Treatments were tested against Salmonella enteritidis. (n = 4) Dehydroabietic Coniferyl Vanillin concentration (mM) acid (DHAA) aldehyde (CFA) 20 5 1.25 0.312 0.078 0.019 0.001 0.0002 10 mM CFA BC BC BC BC BC BC BC BC 10 mM CFA + 20 BC BC BC BC BC BC BC BC mM DHAA 5 mM CFA BS BS BS BS BS BS BS BS 5 mM CFA + 20 BC BC BC BC BC BS BS BS mM DHAA 20 mM DHAA BS G G G G G G G BS: bacteriostatic; BC: bactericidal; G: growth

(111) TABLE-US-00013 TABLE 12 Colony counts obtained 96 well results plate map for 5 mM coniferaldehyde in combination with vanillin at concentration range between 20-0.00029 mM (4 down cells) and Vanillin/DHAA(4 top cells) Concentration (mM) Vanilin/Coniferylaldehyde 20/5 5/5 1.25/5 0.31/5 0.078/5 0.019/5 0.004/5 0.001/5 0.00029/5 0/5 0/0 +DHAA 0 0 0 0 2 6 11 36 34 TMTC TMTC 20 mM 0 0 0 0 1 8 18 30 30 TMTC TMTC 0 0 0 0 1 8 21 41 25 TMTC TMTC 0 0 0 0 2 8 20 35 38 TMTC TMTC 6 11 21 34 50 TMTC TMTC TMTC TMTC TMTC TMTC 8 0 25 34 52 TMTC TMTC TMTC TMTC TMTC TMTC 6 8 19 29 56 TMTC TMTC TMTC TMTC TMTC TMTC 5 19 20 30 48 TMTC TMTC TMTC TMTC TMTC TMTC
Conclusions

(112) Dehydro-abietic acid confers a synergistic effect on the activity of combinations of coniferaldehyde and vanillin. The presence of small concentrations of vanillin (at least 0.0003 mM) is required to make 5 mM coniferaldehyde bacteriocidal (if DHAA is present at 20 mM).

Example 10—Effect of Dehydrozingerone on Mixtures of Vanillin and Dehydro-Abietic Acid (DHAA) Against Salmonella enteritidis

(113) Previously, it was suggested that dehydrozingerone (formed from vanillin) could be a contributing factor explaining antimicrobial activity of heated wood shavings. In experiments where both vanillin and dehydrozingerone were used in combination with dehydro-abietic acid it was suggested that the anti-microbial activity of the two molecules was additive. Here, the inventors investigated if this was the case or if there were synergistic effects depending on the ratio of the two.

(114) Materials & Methods

(115) Vanillin was obtained from Sigma-Aldrich. Dehydrozingerone was made by condensing vanillin with acetone as follows: 0.5 g of vanillin was added to 2 ml acetone and shaken in a screw-top vial to dissolve the vanillin. After the vanillin had dissolved, 1 ml 10% (w/v) aqueous NaOH was added. The mixture was then stored at room temperature for 24 hours. Subsequently, 10 ml, 3 M aqueous HCL was added and the mixture was shaken vigorously to allow the formation of yellow crystals of dehydrozingerone. Purified dehydrozingerone was isolated by filtering the thus obtained suspension of dehydrozingerone using a Buchner funnel followed by rinsing the crystals with water. After air-drying the material, the structure of the isolated dehydrozingerone was confirmed using NMR.

(116) Preparation of Vanillin-Dehydrozingerone Solutions

(117) Solutions containing vanillin and Dehydrozingerone with the ratios (w/w), 100:0, 75:25, 50:50, 25:75, 0:100, were prepared in 5 ml of Acetone. Undiluted the combined concentration of dehydrozingerone and vanillin was 10 mM. 20 mM dehydroabietic acid was added to each treatment by adding 30.4 mg of to each ratio prior to antimicrobial testing. Control treatments consisted of DHAA alone and solvent only. The stock solutions were stored in the fridge at 5° C. for 48 hours.

(118) Assay Method

(119) The inhibitory activities of combined vanillin and dehydrozingerone solutions were determined by micro dilution broth method. 96 well plates were filled with 9.6 mL nutrition broth. In order to determine the MIC (minimum inhibitory concentration) of the samples, several dilutions of the samples were made (0, 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128) into the broth. After making the dilution series, plates were left in a laminar flow cabinet for 5 hours to allow the solvent to evaporate.

(120) Subsequently, each well was inoculated with 5 μl of a Salmonella enteritidis suspension containing 10.sup.8 cfu/ml that had grown overnight at 25° C. After inoculation, the plates were incubated at 25° C. for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from each well was spotted onto nutrient agar. Plates were incubated for 24-48 h and spots with no bacterial growth or just a few colonies were marked as ‘bacteriocidal’. Where possible, colonies were counted to obtain more precise quantitative data. To determine a bacteriostatic effect, 50 μl tetrazolium salt solution was added to each well to determine metabolic activity. After overnight incubation at 25° C. no change in colour was defined as a bacteriostatic effect while metabolism (no effect) was indicated by a red to purple colour.

(121) TABLE-US-00014 TABLE 13 Anti-microbial effect of vanillin in combination with hydrozingerone in the presence oft 20 mM Dehydro-abietic Acid (DHAA). Treatments were tested against Salmonella enteritidis. (n = 4) Ratios Va- nillin:Dehydrozingeron Serial dilutions (mM) 1 1:2 1:4 1:8 1:16 1:32 1:64 1:128 100:00  BS BS G G G G G G 75:25 BC BS BS BS BS G G G 50:50 BS BS G G G G G G 25:75 BS BS BS BS G G G G  00:100 BS BS G G G G G G BS: Bacteriostatic; BC: Bactericidal; G: Growth
Conclusions

(122) The combined effect of hydrozingerone and vanillin was not additive, but depended on the ratio between the two molecules. Best results were obtained when at a ratio of 1:3 of either molecule was used. On their own, hydrozingerone and vanillin had a similar anti-microbial activity, but combined at a ratio of 1 in 3, the surprising effect was 4 to 8 times greater than when used alone or at a 50:50 ratio.

Example 11—Effect of pH on the Solubility of Dehydro-Abietic Acid and Effect of Storage Condition of Stock Solution on Anti-Microbial Activity of Combinations of Vanillin and Dehydro-Abietic Acid Against Salmonella enteritidis

(123) It was noted that combining dehydroabietic acid and vanillin at high pH (>9) caused the combination to become less effective. It was hypothesised that this could be due to the low solubility of dehydro-abietic acid under alkaline conditions (resulting in dehydro-abietic acid falling out of solution). Most experiments described so far were conducted at pH 7 which gave good activity, but not as good as the extracts taken directly from heated wood shavings. Heated shavings have a pH of around 4. However, such a low pH could affect bacterial growth which is normally more favourable at neutral pH. To test the effect of pH while ensuring that DHAA was dissolved, two methods of dissolving the compound were used. In one, the compound was dissolved in a phosphate buffer with a pH of 4, and in the other treatment, DMSO was used with a pH of 7.

(124) The following treatments were tested: Vanillin stock solution, made 48 hours before the start of the experiment; solution kept in fridge at 5° C.; Dehydroabietic acid (DHAA) stock solution, made 48 hours before the start of the experiment; solution kept in fridge at 5° C.; Combination of Vanillin/DHAA stock solutions, made 48 hours before the start of the experiment; solution kept in fridge at 5° C.; Vanillin stock solution, made 48 hours before the start of the experiment and kept at room temperature (20° C.); DHAA stock solution, made 48 hours before the start of the experiment and kept at room temperature (20° C.); and Combination of vanillin/DHAA stock solutions, made 48 hours before the start of the experiment and kept at room temperature (20° C.).
Materials & Methods

(125) Vanillin was obtained from Sigma-Aldrich and DHAA was isolated from disproportionated rosin.

(126) Stock solution in buffer based broth at PH 4: Two batches of a 20 mM stock solution of vanillin and two batches of vanillin/DHAA (10 mM/20 mM) were made in acetone. One of each batch was kept in fridge and the other was kept at room temperature.

(127) Stock solution testing in buffer based broth at pH 7: Two batches of a 20 mM stock solution of vanillin and two batches of vanillin/DHAA (10 mM/20 mM) were made in DMSO. One of each batch was kept in fridge and the other was kept at room temperature.

(128) Bioassay test: The test was carried out using a micro-broth dilution technique. Vanillin and vanillin/DHAA stock solutions were diluted further down in a two fold dilution series to make a concentration range between 10 mM-0.625 mM. 100 μL of each concentration was added to 100 μL of nutrient broth in each well of a 96 well plate. Three replicates were made for each concentration. Plates were kept in a laminar chamber to allow evaporation of the solvent.

(129) Subsequently, each well was inoculated with 5 μl of a Salmonella enteritidis suspension containing 10.sup.8 cfu/ml that had grown overnight at 25° C. After inoculation, the plates were incubated at 25° C. for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from each well was spotted onto nutrient agar. Plates were incubated for 24-48 h and spots with no bacterial growth or just a few colonies were marked as ‘bacteriocidal’. Where possible, colonies were counted to obtain more precise quantitative data. To determine a bacteriostatic effect 50 μl tetrazolium salt solution was added to each well to determine metabolic activity. After overnight incubation at 25° C. no change in colour was defined as a bacteriostatic effect while metabolism (no effect) was indicated by a red to purple colour.

(130) TABLE-US-00015 TABLE 14 Effect of pH, and potential interaction of vanillin and dehydroabietic acid (DHAA) on the growth of Salmonella enteritidis. DHAA was either added at the beginning of the experiment (alone) or allowed to interact with vanillin for 48 h (combination) prior to the start of the experiment (n = 3) Treatment Vanillin [Mm] pH Temp [° C.] DHAA [Mm] 10 5 2.5 1.25 0.625 4 4 20 Alone BS BS BS G G 20 Combination BC BS BS BS G 0 BS G G G G 4 20 20 Alone BS BS BS BS G 20 Combination BC BS BS BS G 0 BS G G G G 7 4 20 Alone BS BS BS G G 20 Combination BC BS BS BS G 0 BS G G G G 7 20 20 Alone BS BS BS BS G 20 Combination BC BS BS BS G 0 BS G G G G BC: Bactericidal; BS: Bacteriostatic; G: Growth
Conclusions

(131) Dehydro-abietic acid increases the anti-microbial activity of vanillin at both pH 4 and pH 7 (pH itself had little effect on the survival of Salmonella). Allowing dehydro-abietic acid to interact with vanillin for 48 hours increases the anti-microbial activity of the two molecules.

Example 12—Synergistic Effect of DHAA on Coniferaldehyde, Thymol and Stilbene

(132) In the previous experiments, anti-microbial compounds were tested in conjunction with vanillin making it difficult to determine if DHAA increased the activity of just vanillin or also that of other natural antimicrobial molecules such as thymol, coniferaldehyde and stilbene. In the first test, these compounds were tested against the Gram-negative bacterium Salmonella enteritidis, and in a second test the same compounds were tested against the Gram-positive bacterium Staphylococcus aureus.

(133) Materials & Methods

(134) Thymol, Trans-stilbene and Coniferaldehyde were obtained from Sigma-Aldrich. Dehydroabietic acid was isolated from disproportionated rosin.

(135) Bioassay test: The test was carried out using a micro-broth dilution technique. DHAA (20 mM) was combined with either coniferaldehyde, thymol or (trans) stilbene and incubated for 48 h at 25° C. Control solutions were made up of coniferaldehyde, thymol and stilbene without dehydro-abietic acid. A solvent control with just acetone was also prepared. For the test, solutions were diluted in a two fold dilution series to make a concentration range between 10-0.156 mM active ingredient. Each concentration was then amended with dehydro-abietic acid to give a final concentration of 20 mM dehydro-abietic acid. 100 μL of each concentration was added to 100 μL of nutrient broth in each well of a 96 well plate. Three replicates were made for each concentration. Plates were kept in laminar chamber to allow evaporation of the solvent.

(136) Subsequently, each well was inoculated with 5 μl of a Salmonella enteritidis suspension containing 10.sup.8 cfu/ml that had grown overnight at 25° C. In the second test Staphylococcus aureus was used. After inoculation, the plates were incubated at 25° C. for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from each well was spotted onto nutrient agar. Plates were incubated for 24 h and spots with no bacterial growth or just a few colonies were marked as ‘bacteriocidal’. To determine a bacteriostatic effect 50 μl tetrazolium salt solution was added to each well to determine metabolic activity. After overnight incubation at 25° C. no change in colour was defined as a bacteriostatic effect while metabolism (no effect) was indicated by a red to purple colour.

(137) Results

(138) TABLE-US-00016 TABLE 15 Synergistic effect of DHAA on coniferaldehyde, thymol or stilbene against Salmonella enteritidis. Treatments with DHAA contained 20 mM DHAA. (n = 3) Serial dilutions (20 mM-0.156 mM) Treatments 10 5 2.5 1.25 0.625 0.313 0.156 Coniferyl aldehyde + BC BC BS BS BS BS G DHAA Coniferyl aldehyde BC BS G G G G G Thymol + BC BC BC BC BC G G DHAA Thymol BC BC BC BC G G G Stilbene + BC G G G G G G DHAA Stilbene G G G G G G G BC: Bactericidal, BS: Bacteriostatic, G: Growth
Results

(139) DHAA acid had a strong synergistic activity on coniferaldehyde, making the compound around 16 times more effective against Salmonella enteritidis. DHAA made thymol around twice as effective against Salmonella enteritidis.

Example 13—Antimicrobial Activity of Combinations of Dehydro-Abietic Acid and Vanillin Against a Variety of Micro-Organisms

(140) The inventors tested the antimicrobial activity of a number of different compounds on micro-organisms (Gram-positive and Gram-negative bacteria, filamentous fungi and yeast) using inhibition zone experiments on thin layer chromatography (TLC) paper.

(141) Materials and Methods

(142) Two test solutions were prepared, i.e.: (i) vanillin in combination with DHAA, and (ii) stilbene in combination with dehydro-abietic acid (DHAA). The activity of the solutions were then tested on several different micro-organisms, as follows.

(143) A droplet of a test solution was placed on a TLC plate. The solvent was allowed to evaporate and a suspension of test micro-organisms (Gram-positive and Gram-negative bacteria) in broth was applied evenly onto the TLC plate. The plate was incubated overnight. To visualise microbial activity, the TLC plate was sprayed with tetrazolium salt solution. Microbial activity was indicated by a red-pink colour.

(144) To show the efficacy of the solutions against filamentous fungi, the inoculated TLC plates were incubated until the fungus started to sporulate. To show efficacy against yeast, after incubation, the TLC plates were pressed against a nutrient agar plate. The plate was incubated until yeast colonies were clearly visible.

(145) Results

(146) FIGS. 1-8 show the effects of the test solutions on the growth of the organisms that were tested.

(147) Referring to FIG. 1, there is shown the activity of a combination of vanillin and DHAA on S. aureus. A clear zone of inhibition is shown in the test compared to the control whether bacterial growth is prevalent. FIG. 2 shows that a combination of stilbene and DHAA is significantly more antimicrobial against S. aureus than stilbene on its own, and the control. FIG. 3 shows that a combination of vanillin and DHAA is antimicrobial against S. enteritidis, and that these two compounds on their own are ineffective. FIG. 4 shows that a combination of vanillin and DHAA is antimicrobial against S. typhimurium.

(148) FIG. 5 shows that a combination of vanillin and DHAA is antimicrobial against E. coli. FIG. 6 shows that a combination of vanillin and DHAA is antimicrobial against the filamentous fungus, Penicillium spp. FIG. 7 shows that a combination of vanillin and DHAA is antimicrobial against Aspergillus spp. Finally, FIG. 8 shows that a combination of vanillin and DHAA is antimicrobial against the yeast, S. cerevisiae. Vanillin on its own is effective at 5 mM, but in combination with DHAA, it is effective at only 0.078 mM.