SHAH, Nimish, Harshadrai (Hindustan Unilever Ltd, Research Centre64 Main Road,Whitefield, Bangalore 6, 560 06, IN)
UNILEVER PLC (a company registered in England and Wales, under company no. of Unilever House100 Victoria Embankment, London Greater London EC4Y 0DY, 41424, GB)
HINDUSTAN UNILEVER LIMITED (Hindustan Lever House, 165/166 Backbay ReclamationMaharashtra, Mumbai 0, 400 02, IN)
NIRMALA, Ronnie (Hindustan Unilever Ltd, Research Centre64 Main Road,Whitefield, Bangalore 6, 560 06, IN)
SHAH, Nimish, Harshadrai (Hindustan Unilever Ltd, Research Centre64 Main Road,Whitefield, Bangalore 6, 560 06, IN)
1 . A preservative composition comprising 0.01 to 2% by weight diacetyl and 0.01 to 1 % by weight cinnamic aldehyde.
2. A preservative composition as claimed in claim 1 comprising from 0.02 to 0.1 % by weight diacetyl.
3. A preservative composition as claimed in claim 1 or claim 2 comprising 0.01 to 0.5% by weight cinnamic aldehyde.
4. A preservative composition as claimed in any one of the preceding claims
characterized in that pH of the preservative composition is greater than 3.
5. A preservative composition as claimed in any one of the preceding claims
characterized in that pH of the preservative composition is greater than 4.2.
6. A preservative composition as claimed in one of the preceding claims comprising at least 5% by weight water.
7. A preservative composition as claimed in any one of the preceding claims wherein the preservative composition comprises at least 5% by weight fat.
8. A preservative composition as claimed in claim 1 wherein said preservation
composition is a food composition
9. A preservative composition as claimed in claim 1 wherein said preservation
composition is a cosmetic composition.
This invention relates to a preservative composition. It particularly relates to the preservative food compositions and preservative cosmetic compositions.
BACKGROUND OF THE INVENTION
Multiple-use products such as spreads, margarine etc are typically consumed over several days. Such products, particularly during prolonged use over several days, are likely to be contaminated by contact with hands of the user or contacts with other food materials, or objects such as spoon or knife or due to ariborned contamination during use. One of the problems of multiple-use products is to ensure microbial safety of foods during use, particularly during prolonged use over several days. The problem of preservation during prolonged use for multiple-use products is also a common problem in the area of cosmetic compositions such as shampoos, creams and lotions, and cleaning compositions such as body wash, hand wash and shower gel compositions.
Typically water or aqueous phase in case of emulsions is subjected to Pasteurization during manufacture before packing. However, spores of pathogens like Bacillus and mould spores can survive, and in fact can germinate during Pasteurization, thus posing a risk to consumer safety later on when such products are exposed to conditions that are favourable to growth of bacteria.
Potassium sorbate is typically used in spreads as preservative. Commonly used food preservatives such as potassium sorbate, although efficacious against yeast and mould, are relatively ineffective against bacterial pathogens belonging to Enterobacteriaceae,
Staphylococcus, Listeria, and Bacillus species. Furthermore, sorbates and benzoates are relatively less efficacious when the spreads have relatively higher pH. Furthermore, preservatives like potassium sorbate act as a fungistatic agent, but have relartively low fungicidal activity.
Present inventors have surprisingly found that inclusion of mixture of diacetyl with cinnamic aldehyde in certain range imparts relatively high antimicrobial efficacy to the preservative compositions. In particular, such compositions have relatively high antibacterial efficacy against both gram positive and gram negative bacteria over prolonged use over several weeks, and also have efficacy against spoilage agents such as yeast and mould.
SUMMARY OF THE INVENTION
According to the present invention there is provided a preservative composition comprising 0.01 to 2% by weight diacetyl and 0.01 to 1 % by weight cinnamic aldehyde.
DETAILED DESCRIPTION OF THE INVENTION Diacetyl
The term "diacetyl" as used herein means 2,3-butanedione.
The preservative composition comprises 0.01 to 2% by weight, preferably 0.02 to 1 % by weight, more preferably 0.02 to 0.5%, and most preferably from 0.03 to 0.1 % by weight diacetyl.
The preservative composition comprises 0.01 to 1 % by weight, preferably 0.01 to 0.5% by weight, more preferably 0.02 to 0.1 %, and most preferably from 0.02 to 0.08 % by weight cinaamic aldehyde.
The preservative composition may be a food composition, a cosmetic composition or a cleaning composition.
The term "food composition" as used herein means an edible composition.
The term "cosmetic composition" as used herein means any composition used for topical application.
The term "cleaning composition" as used herein means any composition used for cleaning a substrate. The substrate may be skin, fabric or hard surface such as metal or plastic.
According to one preferred aspect the preservative composition is a cosmetic or a cleaning composition, more preferably a cosmetic composition.
According to another preferred aspect the preservative composition is a food composition.
It is particularly preferred that the preservative composition is a food composition Preferably the preservative composition is in form of an emulsion. The emulsion may be water in oil emulsion or oil in water emulsion.
Preferred preservative food compositions include spreads, and the spreads preferably include margarine, mayonnaise and butter.
The preservative composition comprises preferably greater than 5%, more preferably greater than 15%, and most preferably greater than 30% by weight water. It is particularly preferred that the preservative composition comprises greater than 40% water. The preservative composition may comprise preferably less than 99% by weight, more preferably less than 90% by weight, most preferably less than 80% by weight water. The preservative composition comprising preferred amounts of water are particularly prone to spoilage due to yeast/mould and to the contamination by pathogenic gram negative and/or gram positive bacteria.
Preferably, the preservative composition comprises at least 5% by weight fat. It is particularly preferred that the preservative food compositions comprise fat.
The preservative composition may comprise up to 90% by weight fat. The preservative composition comprises preferably from 10 to 70 wt% more preferably from 20 to 60 wt%, most preferably from 30 to 40 wt% fat. The fat consists predominantly of triglycerides. The fat can be a single fat or a combination of fats.
The fat or fat blend may comprise vegetable or animal fats which may be hydrogenated, interesterified or fractionated and combinations thereof. Suitable animal fats may consist of butterfat or tallow. Suitable vegetable fats can for example be selected from the group comprising bean oil, sunflower oil, palm kernel oil, coconut oil, palm oil, rapeseed oil, cotton seed oil, maize oil, or their fractions, or a combination thereof. Interesterified fat blends of these fats or optionally with other fats are also encompassed in the invention. It is preferred that the fat comprises poly unsaturated fatty acids (PUFA). Preferred PUFA may be selected from the group comprising linoleic acid, linolenic acid, alpha linolenic acid (ALA) docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), eicosapentaenoic acid (EPA), eicosatetraenoic acid, and stearidonic acid. Preferred PUFA are omega-6 or omega-3 fatty acids. Other preferred PUFA are long-chain PUFA (LC-PUFA). Most preferred PUFA are ALA, DHA and EPA. In a preferred embodiment at least 20 wt% of the fat are PUFA, more preferred at least 30 wt% and even more preferred more than 40 wt% and most preferred more than 50 wt% of the fat is polyunsaturated.
Preferably at least 2 wt% of the fat are omega-3 fatty acids, more preferably at least 5 wt%, most preferably at least 10 wt%. In a preferred embodiment the composition comprises 2-40 wt%, even more preferred 5-30 wt%, and most preferred 7-20 wt% omega-3 fatty acids on fat.
Without wishing to be limited by theory, it is believed that the flavour profile of diacetyl blends better with preservative compositions comprising fats and therefore such compositions are likely to provide better antimicrobial efficacy with relatively low impact on the sensory properties. It is particularly preferred that the preservative composition comprise fat when the preservative composition is a food composition. pH of the composition
The preservative composition is preferably characterized in that pH of the preservative composition is greater than 3. The pH of the preservative composition is preferably greater than 4, more preferably greater than 4.2, and most preferably greater than 4.8. Without wishing to be limited by theory, it is believed that the conventional preservative agents such as potassium sorbate or sodium benzoate which are acid salts provide antimicrobial activity only in dissociated acid form, i.e. at a pH lower than pKa of the corresponding acid. Thus benzoates are not likely to be effective antimicrobial agents when the preservative
composition has pH greater than 4.2 (corresponding to pKa of the benzoic acid). Similarly, sorbates are not likely to be effective antimicrobial agents when the preservative composition has pH greater than 4.8 (corresponding to pKa of the sorbic acid). The pH of the preservative composition is preferably less than 9, more preferably less than 8, most preferably less than 7.5. It is particularly preferred that pH of the preservative composition is less than or equal to 7.0. EXAMPLES
The invention will now be demonstrated with examples. The examples are for purpose of illustration only and do not limit the scope of claims in any manner. FOOD COMPOSITIONS
Determination of Minimum Inhibitory Concentration (MIC):
The antimicrobial effectiveness of a compound is often described in terms of its minimum inhibitory concentration (MIC), the lowest concentration of the compound capable of inhibiting the growth of the challenging organisms. Agar plate method was used for MIC determination, where in agar plates containing known concentration of antimicrobial are inoculated with challenge microorganisms, growth/ no growth is observed visually at the end of incubation.
A broad spectrum of organisms representing the vegetative pathogens, spore formers and spoilage organism relevant to foods (especially those relevant for dressings and spreads) were used for the studies. These organisms were product isolates and were pooled into 6 groups, Gram negative bacteria- Enterobacteriaceae, Gram positive bacteria- Listeria, Staphylococcus aureus, Bacillus and aerobic spore formers- Bacillus, Spoilage
microorganisms- Yeast and Mould. Each group consisted of a cocktail of individual cultures as tabulated below.
Table 1 : Microorganism mixtures used for testing
Cultures were maintained in glycerol and a fresh culture was prepared for each assay. The cultures were grown overnight in broth, Tryptic Soy Broth (TSB) for bacteria and Yeast Peptone Broth (YPD) for yeast. The optical density (OD) of the overnight broth was adjusted to achieve a cell count of ~ 8 log-io units for bacteria and ~ 7log-i 0 units for yeast. An equal amount of individual culture suspensions were pooled together to get the cocktail inoculum for that particular set of organism.
Mould inoculum was prepared by growing mould cultures on Potato Dextrose Agar (PDA) plates at 25 deg C for 5-7 days till good sporulation was achieved. The spores were harvested using 0.1 % Tween 20. The harvested spore suspension was counted using a haemocytometer and inoculum adjusted to give a spore count of -7 log-io units. An equal amount of individual culture suspensions were pooled together to get the cocktail inoculum. Assay mediae Tryptic Soya Agar (TSA) was used for bacterial cocktails and Potato Dextrose Agar (PDA) for Yeast and Mould cocktails. These were commercial media sourced from Difco.
- To a tube containing 20 ml of molten agar (50- 55°C), test antimicrobial was added to achieve the required concentration. The contents were mixed in a vortex and poured into the plate.
- The plates were left to dry for 24hours. Inoculation and incubation of agar plates
- The plates were marked into required no. of zones (e.g. TSA plates were divided into five zones for the four bacterial cocktails and bacillus spores) and inoculate the agar plate by spot inoculation with 10 μΙ of ~ 10 6 cfu/ml of bacterial and bacillus spore cocktails and ~ 10 5 cfu/ml yeast or mould cocktail.
- Incubate the bacterial plates for 2 days at 30°C and yeast and moulds at 25 °C and evaluate growth / no growth by visual inspection at least once a day for 5 days. Growth (G) indicates that the composition does not have antimicrobial efficacy whilst "No Growth" (NG) indicates that the composition has antimicrobial efficacy. The results are tabulated below
Table 2: Antimicrobial efficacy in MIC assay
The results indicate that compositions comprising diacetyl and cinnamic aldehyde at selective levels provide surprising benefit in inhibiting growth of bacteria, yeast and mould.
Furthermore, these compositions show efficacy against a broad spectrum of pathogens including gram positive (L, S, and B) as well as gram negative (E) bacteria.
Efficacy of cinnamic aldehyde in comparison with other essential oils
Experiments were carried out to determine antimicrobial efficacy of compositions comprising 0.05% by weight diacetyl and 0.025% by weight an essential oil. The results are tabulated below. The result for composition comprising 0.05% by weight diacetyl and 0.025% by weight cinnamic aldehyde are reproduced for convenience. Table 3: Comparison of antimicrobial efficacy of essential oils
The results indicate that diacetyl in combination with essential oils other than cinnamic aldehyde does not provide synergistic benefit in inhibiting growth of various bacterial, yeast and mould.
Determination of in-vitro time kill kinetics:
ln-vitro time kill studies were based on the NCCLS standard: National committee for clinical laboratory standards. (1992). Methods for determining Bactericidal activity of Antimicrobial Agents: M26-T. NCCLS, Villanova, PA. Determination of the killing of a bacterial isolate over time by one or more antimicrobial agents under carefully controlled conditions is known as the time- kill method. This is a broth based method where the rate of killing of a fixed inoculum is determined by sampling a control (organisms, no antimicrobial active) and antimicrobial agent containing medium at intervals ( 0, 24, 48 hours etc) and determining the survivor colony count (CFU per milliliter) by plating each sample into a agar plate. The kill curves are constructed by plotting the cfu/ml surviving at each time point in the presence and absence of the antimicrobial agent. When used to determine the additive or synergistic effect between two (or more) antimicrobial agents, kill curves are constructed for a fixed concentration of each antimicrobial agent alone and in combination and are compared to that of the control (no active) at each time point. Organisms Used: The cocktails used were same as those used for the MIC assay, except that for moulds, only spores were used. All strains used were food product isolates from contaminated dressings and spread products. Media used: Commercial media, sourced from Difco were used in the experimentation. The media composition is given in Annex 3.
1 . Tryptic Soy Broth (TSB)
2. Yeast Peptone Dextrose Broth (YPD)
3. Tryptic Soya Agar (TSA)
4. Potato Dextrose Agar (PDA)
5. Normal saline for dilution was prepared using 0.85% sodium chloride.
6. 0.1 % Tween 20 in sterile distilled water.
1 . Diacetyl (2,3-Butanedione) >/= 99.4%
2. Potassium sorbate: Commercial grade, 99.8% purity
3. Cinnamic aldehyde: 99.2% purity
Preparation of Bacterial and Yeast inoculum: Individual bacterial cultures for each cocktail were grown in broth overnight at 30 deg C and their optical density adjusted to a standard concentration * .
An equal amount of individual culture suspensions were pooled together to get the cocktail inoculum for that particular set of organism. Preparation of Mould inoculum: Individual mould cultures were grown on Potato Dextrose Agar (PDA) plates and incubated at 25 deg C for 5-7 days till complete sporulation. The spores were harvested using 0.1 % Tween 20. The harvested spore suspension was counted using a haemocytometer and inoculum adjusted to give a spore count of 7 log-io units.
An equal amount of individual culture suspensions were pooled together to get the cocktail inoculum. Procedure:
To flasks containing 100ml of broth (TSB for bacteria and YPD for yeast and fungi) actives were added at the concentration mentioned below:
1 . Control (no antimicrobial)
2. 0.3% Potassium sorbate
3. 0.05% Diacetyl
4. 0.025% Cinnamic aldehyde
5. 0.05% Diacetyl + 0.025% Cinnamic aldehyde
1 ml of bacterial inoculum cocktail was added to each of the above flasks and mixed well to give a final bacterial concentration of 1 -5 * 10 6 cfu/ml. An aliquot of broth was taken from each flask to determine the initial inoculum by plating on TSA at zero hour and incubating at 30 deg C. The test samples were subsequently tested at 24 hour, 48 hour, 7 day, 14 day, 21 day and 28 day for survivors. Post sampling, the flasks were returned to the incubator. The plates were incubated at 30 deg C for 48 hours and counted.
The flasks which were found to be heavily turbid on visual observation were sampled for counts and subsequently discarded. The non-turbid flasks were followed in time till 28 days. Similar procedure was followed for yeast and mould cocktails also, except that the media used was Yeast peptone Broth and the samples were plated on Potato Dextrose Agar. The final yeast and mould concentration in the flasks was 1 -5 * 10 Λ 5 cfu/ml.
Results: The counts obtained for each test sample were plotted against respective time points to obtain a measure of the kill kinetics.
Procedure: Stock solutions of the microorganisms for this study were grown and used to prepare the challenge suspensions. The suspensions were adjusted to a standard
concentration and a consistent amount of the suspension was exposed to the test product at 1 day, 2 day, 7 day, 14 day, 21 day and 28 days. At the end of each exposure time, a measured amount of the test sample was removed and neutralized. The final neutralized mixture was plated on the appropriate solid culture media and incubated at the temperature appropriate for the microorganism type. Following incubation the colony counts were determined. These results reported as the Iog10 reductions from the initial population for each challenge microorganism cocktail versus each test sample were calculated. Optical density of bacteria adjusted to give a 8 log i 0 cfu per ml, and that of yeast to 7 logi 0 cfu per ml.
Table 4a: Enterobacteriaceae cocktail
Composition (%) Day 0 Day 1 Day 2 Day 7
Diacetyl 0.05% 9.50E+06 2.33E+06 1.41 E+06 7.90E+08
Cinnamic aldehyde 0.025% 1.00E+07 5.90E+08
Diacetyl 0.05 + Cinnamic aldehyde
0.025% 1.41 E+07 3.07E+06 4.30E+04 1.00E+01
Potassium sorbate 0.3% 1.05E+07 2.38E+08
No active 9.80E+06 1.60E+09
Table 4b: S. aureus cocktail
Composition (%) Day 0 Day 1 Day 2 Day 7
Diacetyl 0.05% 4.07E+06 1.79E+05 5.80E+07 2.05E+08
Cinnamic aldehyde 0.025% 3.18E+06 9.90E+05 3.70E+04 1.22E+07
Diacetyl 0.05 + Cinnamic aldehyde
0.025% 3.50E+06 3.77E+05 7.40E+03 1.00E+01
Potassium sorbate 0.3% 2.71 E+06 8.70E+08 9.70E+08
No active 2.67E+06 8.80E+08 8.70E+08
Table 4c: Yeast Cocktail
Table 4d: Mould spore Cocktail
From the results, it is clear that the composition comprising diacetyl and cinnamic aldehyde efficaciously reduces the concentration of microorganisms over time.
Effect of pH of the composition
All the above results (Tables 2, 3 and 4a-d were for the compositions which had pH of 6.5. The experiments with the Yeast cocktail were repeated along with another set of experiments at a composition pH of 4.0, the pH being adjusted by adding citric acid (10% solution) to the compositions. The results are tabulated below.
Table 4e: Yeast Cocktail: Effect of pH of the composition
From the results, it is apparent that conventional preservative like potassium sorbate, although somewhat efficacious when the composition of pH is 4, has far less efficacy when the composition of pH is 6.5. On the other hand, the composition according to the present invention is highly efficacious across a range of pH of the compositions.
Methods: Sensory Assessment
KISSAN® Real Vegetarian Mayonnaise (Hindustan Unilever Limited, India) was used in the experiments. Mayonnaise was incorporated with five different levels of Diacetyl and Cinnamic aldehyde combinations. These were coded and presented to a panel of seven panellists. The test samples were compared with the control product which was the normal formulation without test additives and panellist were asked to score the test samples as acceptable or unacceptable with respect to the control in terms of odour, taste and mouthfeel (Acceptable: at par with the control, Unacceptable: Strange sensorial as compared to the control).
The acceptability score was expressed as a ratio of number of panellists that find the product to be acceptable to the total number of panellists. Thus the higher ratio indicates better acceptability Table 5: Sensory evaluation of mayonnaise
Composition Acceptability score
Diacetyl 0.05% + Cinnamic aldehyde 0.005% 5/7
Diacetyl 0.1 % + Cinnamic aldehyde 0.05% 5/7
Diacetyl 0.05% + Cinnamic aldehyde 0.025% 5/7
Diacetyl 0.5% + Cinnamic aldehyde 0.1 % 2/7
Diacetyl 1.0% + Cinnamic aldehyde 0.5% 0/7 From the above results it is clear that the mayonnaise compositions comprising up to 0.5% diacetyl and up to 0.1 % cinnamic aldehyde have acceptable sensorials. COSMETIC COMPOSITIONS
MIC assay described earlier was used to evaluate efficacy against various microorganisms that can potentially cause skin and/or respiratory infections. All the tests were at pH of 7.
Table 6: Antimicrobial efficacy in MIC assay: skin/respiratory infection agents
The results indicate that the compositions according to the present invention are efficacious in inhibiting growth of microorganisms that can potentially cause skin and/or respiratory infections
Sensory Evaluation of cosmetic compositions comprising Diacetyl and Cinnamic aldehyde.
Diacetyl (0.05%) and cinnamic aldehyde (0.025%) were added to commercially available cosmetic compositions, and these compositions were evaluated by a group comprising 5 panellists for sensory perception, in particular for smell and tactile feel. The results are tabulated below Table 7: Sensory Evaluation of cosmetic compositions comprising Diacetyl and Cinnamic aldehyde
From the results, it is clear that the cosmetic compositions according the present invention provide antimicrobial efficacy without compromising sensory and tactile properties
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