Patent Application
20200397026
The present invention is on the field of antimicrobial active substances. In addition to novel uses of distinct antimicrobial mixtures according to the invention, the present invention also relates to preparations containing such mixtures, particularly preparations serving for nutrition or pleasure or pharmaceutical preparations, as well as methods for preparing such preparations and methods for antimicrobial treatment of preparations serving for nutrition or pleasure or pharmaceutical preparations. Further aspects of the present invention result from the subsequent descriptions and particularly the attached patent claims.
Foodstuffs, particularly with ingredients of animal origin (such as mayonnaise, minced meat and sausage products) as well as sugar containing foodstuffs (such as sweetened drinks, syrups, ketchup and dressings) are prone to microbial decay due to their richness in nutrients and high water activity coefficients (aw-value).
For stabilizing such products, typically the concept of hurdles is applied [Kramer, J., Alexander, P. (2017). Lebensmittel-Mikrobiologie. Stuttgart, publisher Eugen Ulmer. 7., completely reviewed edition.]: Raw materials with a low initial germination number are selected, the aw-value, the redox potential and the pH value are reduced, if possible, when formulating the products and when processing the products, particular attention is paid on food hygiene and good manufacturing practice. As the last hurdle, methods of preservation, such as pasteurization or the use of preservatives, are applied. However, classical methods of preservation are increasingly considered as critical by consumers. As an example, the amount of nutrients can be reduced and the taste can be impaired by forming cooked notes by pasteurization. Preservatives such as sorbic acid and benzoic acid are obtained synthetically and need to be declared as additives. Natural preservatives such as acidic acid, mustard seeds, essential oils and extracts thereof can only be applied in certain limits, as these strongly change the product in a sensorial manner.
While the growth of bacteria is strongly inhibited by a pH-value smaller than 4.5, with the exception of acidophilic bacteria such as Lactobacillus sp. or Acetobacter sp., yeasts and fungi can also grow at pH-values smaller than 3 and lead to decay. Special yeasts such as Zygosaccharomyces baili, Zygosaccharomyces lentus and Saccharomyces cerevisiae also show a tolerance towards preservatives such as sorbic acid and benzoic acid [Steels, H., James, S. A., Roberts, I. N. and Stratford, M. (1999). “Zygosaccharomyces lentus: a significant new osmophilic, preservative-resistant spoilage yeast, capable of growth at low temperature.” Journal of Applied Microbiology 87(4): 520-527. Juvonen, R., Virkajärvi, V., Priha, O. and Laitila, A. (2011) “Microbiological spoilage and safety risks in non-beer beverages.” VTT Tiedotteita—Research notes 2599]. Although the decay of a foodstuff by yeasts and fungi is the most difficult to control, it is predominantly the antibacterial effect of plant extracts which is known [Friedman, M. (2015). “Antibiotic-resistant bacteria: prevalence in food and inactivation by food-compatible compounds and plant extracts.” J Agric Food Chem 63(15): 3805-3822]. For example, herbs, spices and the essential oils and extracts obtained therefrom have a documented effect against bacteria [Weber, H. (2010). Mikrobiologie der Lebensmittel—Grundlagen. Hamburg, Behrs Verlag], however, these change the taste in an inacceptable manner in many products due to their own aroma. Only little plant extracts with an antimicrobial effect against yeasts and fungi are known and their use in foodstuff is often limited due to their own aroma, strongly colouring characteristics and low solubility.
Therefore, it was an aim and primary object of the present invention, to develop alternative formulations for prolonging the durability of a perishable foodstuff without undue technological effort, preferably by addition of plant based natural ingredients, and thereby preferably not to negatively impact or ideally even round or, respectively, improve the taste.
Additionally, the formulations to be provided should be toxicologically harmless, highly compatible, stable (particularly in typical formulations) and/or be inexpensively producible.
It is further desirable if as less of the antimicrobial active substances as possible has to be used in the respective formulations for achieving a certain antimicrobial effect.
The search for suitable substances which have one or more of the mentioned characteristics in a sufficient amount is additionally complicated for the skilled person by that no clear dependency between the chemical structure of a substance on the one side and its biological activity towards certain microorganisms (germs) as well as its stability exists. Furthermore, there is no predictable connection between the antimicrobial effect, the toxicological harmlessness and the stability of a substance.
The preferred object of the present invention was thus to provide formulations which fulfil several or preferably all of the above criteria or which have a desirable combination of the above characteristics.
The primary object is solved by the use of a mixture comprising or consisting of
According to an embodiment of the present invention, preferably the root and/or the fruiting organs of the plant or parts thereof, preferably endo-, meso-, exocarp and/or parts of the flower head are used for the one or more plant extract(s) with antimicrobial effect according to (A) and/or for the one or more fraction(s) thereof.
The substance(s) according to (B) or parts thereof may, according to an embodiment of the present invention, be present in form of an extract/in an extract or in form of a fraction thereof/in a fraction thereof, preferably selected from the group consisting of extracts (or fractions thereof) of the following plants: Artemisia xanthochroa, Artemisia dracunculus, Bauhinia manca, Chenopodium album, Chysothamnus ssp., Citrus ssp. (insbesondere Citrus sinensis, Citrus paradisi, Citrus bergamia), Dracaena cinnabaria, Herba santa, Hydrangea dulcis, Lippia dulcis, Lycoris aurea, Malus domestica, Mariscus psilostachys, Momordica grosvenorii, Piper ssp., Stevia ssp., Vanilla ssp., Zanthoxylum rubescens.
Preferred according to the invention is such a use as antimicrobial mixture, wherein component (B)
It is thus preferred according to the invention, that component (B) is present in the mixture to be used according to the invention in a total amount which is sufficient to improve the antimicrobial effectiveness of component (A) and/or to reduce, mask or modify an unpleasant taste impression of component (A) and/or to increase or modify a pleasant taste impression of component (A).
Here, the taste impressions to be reduced, masked or modified are preferably selected from the group consisting of astringent, bitter, herbaceous, metallic, sour.
The taste impressions to be increased or modified are preferably selected from the group consisting of sweet, juicy and full-bodied.
The mixtures to be used according to the invention are excellently suitable as antimicrobial mixture of active substances for prolonging the durability of perishable compositions.
Advantageously, the mixtures to be used according to the invention also allow to fulfil the initially mentioned criteria.
Although the experts have already partly considered the antimicrobial characteristics of components described herein, there was so far no indication that especially the mixtures to be used according to the invention have the effects and advantages as described herein. Particularly surprising is a significantly improved antimicrobial effect (particularly against the germs as described herein).
The invention is based on the surprising recognition that the mixtures according to the invention show a particularly advantageous antimicrobial effect, particularly against fungi or, respectively, yeasts, particularly against one or more germs selected from the group consisting of
Particularly preferred—and advantageous—is thus a use according to the invention against fungi or, respectively, yeasts, preferably against one, more or all germs selected from the group consisting of S. cerevisiae, Z. bailii, C. albicans, A. pullulans and A. brasiliensis.
It is preferred according to the invention if the extract(s) of component (A) is/are selected from the group consisting of berry extracts, preferably extracts of red berries, pomegranate extracts and herb extracts.
The extracts of component (A) can be produced from whole plants, plant parts, biotechnologically altered or unaltered plant materials, wherein methods are used which are known to a skilled person, preferably solid-liquid-extractions. Furthermore, if necessary, also enrichment and concentration of selected substances can be applied after the extraction has taken place.
For example, an extraction of dried plant parts can be applied with pure or a solvent mixture of acetone, butane, butane-1-ol, butane-2-ol, cyclohexane, dinitrogen monoxide, diethyl ether, ethyl acetate, ethanol, ethylmethylketone, hexane, carbon dioxide, methanol, methyl acetate, vegetable oils, propane, propane-1-01, propane-2-ol and/or water.
Excipients as for example surface active substances, hygroscopic substances and/or salts can be added to the extraction solvents for improving the yield.
Wet plant parts can be extracted with the aid of microwave radiation, ultrasound and pulsing electrical field.
A fractionation of the extracts (production of a fraction described herein) is preferably performed by separation of undesired side-components and/or concentration of selected substances, for example by adsorption distillation chromatographic separation, fermentation, filtration, liquid-liquid extraction, membrane filtration and/or centrifugation, preferably by chromatographic separation, liquid-liquid extraction and/or membrane filtration.
After the fractionation of the extracts, the components of the respective fractions can be determined with known methods, where appropriate. The determination of the compounds with mass spectrometry, particularly LC-MS, is a preferred method.
It is particularly preferred according to the invention, if component (A) comprises at least one, two, three or more compound(s) selected from the group consisting of catechines, chlorogenic acid, p-cumaric acid, epicatechines, ellagic acid, gallic acid, caffeic acid, sinapinic acid and cinnamic acid.
Dependent on the method of fractionantion, the ratios of the concentrations particularly of the substances catechines, chlorogenic acid, p-cumaric acid, epicatechines, ellagic acid, gallic acid, caffeic acid, sinapinic acid and/or cinnamic acid to the other components can be changed by the fractionation in a targeted way. A change is thereby preferably applied in a way in which the antimicrobial effect is improved.
In a further preferred embodiment, component (B) contains at least one, two, three or more flavouring agent(s) selected from the group consisting of eriodictyol, hesperetin, homoeriodictyol, naringenin, pellitorin and vanillic acid or consists thereof.
For mixtures to be used according to the invention preferably applies that the ratio of the total amount of component (A) to the total amount of component (B) is in a range of from 1000:1 to 1:10, preferably 100:1 to 1:10, preferably in a range of from 100:1 to 1:1, preferably 10:1 to 1:1. With regard to this feature, it preferably applies that the amount(s) of potential components which can be assigned to component (A) as well as to component (B) are assigned to the total amount of component (A).
Advantageously, the mixtures as described herein are particularly suitable for use in a preparation serving for nutrition or pleasure or a pharmaceutical preparation.
Accordingly, the present invention also relates to a preparation serving for nutrition or pleasure or a pharmaceutical preparation, comprising a mixture as described herein, wherein the mixture is present in an amount sufficient to achieve an antimicrobial effect, preferably a synergistic antimicrobial effect.
What was said above to mixtures to be preferably be used according to the invention applies accordingly for preferred embodiments of the contained mixture.
Accordingly, it is preferred if the mixture is contained in an amount sufficient for achieving an antimicrobial effect, preferably a synergistic antimicrobial effect, against one or more germs selected from the group consisting of
It is particularly preferred if the mixture is contained in an amount sufficient to achieve an antimicrobial effect, preferably a synergistic antimicrobial effect against one, more or all germs selected from the group consisting of S. cerevisiae, Z. bailii, C. albicans, A. pullulans and A. brasiliensis.
The use of mixtures to be used according to the invention in preparations according to the invention is thereby not limited. Thus, a plurality of different preparations serving for nutrition or pleasure or pharmaceutical preparations can be considered. Particularly preferably, preparations according to the invention are foodstuffs, particularly such with ingredients of animal origin (such as mayonnaise, minced meat and sausage products) as well as sugar containing foodstuff (such as sweetened drinks, syrups, ketchup and dressings).
According to a preferred embodiment of the present invention, a mixture to be used according to the invention is thus applied in a preparation which is selected from the group consisting of foodstuffs, preferably foodstuffs with ingredients of animal origin, such as mayonnaise, minced meat and sausage products, as well as sugar containing foodstuff such as sweetened drinks, syrups, ketchup, salad creams and dressings.
A mixture to be used according to the invention can be contained e.g. in a total amount of from 3 to 0.001, preferably of from 0.5 to 0.01, particularly preferably of from 0.1 to 0.01 wt.-%, related to the total weight of the preparation.
For these purposes, a mixture to be used according to the invention can be present in form of a liquid component or powder or spray-dried product in such preparations. Thus, for example, components (A) and (B) are mixed and dissolved and added in liquid or spray-dried form. Components (A) and (B) can also be used separately and in forms different from each other. Also, the preparations according to the invention preferably further consist of components typical for such preparations. As an example, solvents or carriers can be present.
The solvents can thereby be for example water, ethanol, 1,2-propanediol, triacetin, diacetin, triethyl citrate and/or glycerine.
Advantageous carriers are for example silicon dioxide (silica acid, silica gel), carbohydrates and/or carbohydrate polymers (polysaccharides), cyclodextrins, starches, degraded starches (starch hydrolysates), chemically or physically modified starches, modified celluloses, gum arabicum, ghatti-gum, tragacanth, karaya, carrageenan, guar gum, locust bean gum, alginates, pectin, inulin or xanthan gum; preferred starch hydrolysates are maltodextrins and dextrins, wherein maltodextrins with DE-values in a range of from 5 to 20 are particularly preferred. It is irrelevant which plant has originally provided the starch for producing the starch hydrolysates. Suitable and easily accessible are maize-based starches as well as starches of tapioca, rice, wheat or potatoes. The carriers can also act as flow additives such as for example silicon dioxide.
One aspect of the present invention also relates to a method for producing a preparation according to the invention, comprising or consisting of the following steps:
What was said above with regard to mixtures preferably to be used according to the invention also applies for preferred embodiments of the contained mixture. The same applies accordingly for preferred embodiments of the preparations to be produced.
Particularly preferred is also a method according to the invention for antimicrobial treatment of a preparation serving for food or pleasure, preferably as described above, or a pharmaceutical preparation, comprising or consisting of the following steps:
further preferably to achieve an antimicrobial effect, preferably for achieving a synergistic antimicrobial effect, against one, more or all germs selected from the group consisting of S. cerevisiae, Z. bailii, C. albicans, A. pullulans and A. brasiliensis.
What was said above with regard to mixtures preferably to be used according to the invention also applies for preferred embodiments of the contained mixture. The same applies accordingly for preferred embodiments of the preparations to be treated.
Subsequently, the present invention is further illustrated by means of selected examples, without limiting the subject-matter of the present invention thereon. As far as not indicated otherwise, all indications of % relate to the weight (wt.-%).
1. Punica granatum Extract (GRA):
A pomegranate skin extract is produced by that pomegranates are cut in half and are pressed and the remaining skin rests are subsequently homogenized with warm water at a temperature of 50° C. The mash is subsequently pressed. The obtained extract (filtrate) is subsequently concentrated by distillation.
The obtained, concentrated extract contains, among others, the following components:
1Determination of the surface percentage by UPLC-NQAD
For a subsequent production of single fractions of the concentrated extract, a membrane filtration may for example be applied.
For the production of the membrane filtrates from the pomegranate skin extract, a polyethylene glycol-based flat membrane with a membrane surface of 26 cm2 and a cut-off of 2500 Da was rinsed with ultrapure water at 30 bar and twice for 20 minutes. A 1% extract solution (200 g) was provided in the feed container, heated to 30° C. and impinged with a pressure of 30 bar. After throughput of 138.23 g, it was diluted with 100 g of water and filtered again. The membrane filtrates were lyophilized and a permeate with a yield of 16.8% and a retentate with a yield of 65.8% was obtained.
The obtained fractions were subsequently examined for their components by mass spectrometry. The obtained fractions contain, among others, the following non-volatile components:
2. Red Berry Extract—Mixture of Vaccinium macrocarpon, Vaccinium Angustifolium and Fragaria×anapassa (ROBE):
An extract is produced by that fruits of Vaccinium macrocarpon, Vaccinium angustifolium and Fragaria×anapassa are mixed and subsequently a filtrate is obtained after pressing. Subsequently, the filtrate (extract) is concentrated by distillation.
The obtained, concentrated extract contains, among others, the following components:
1Determination of the surface percentage by UPLC-NQAD
3. Vitis vinifera Extract (Grape Stone Extract):
An extract is produced by that, at first, grape stones are milled. Then these are extracted with water at 50° C. and filtered to obtain the extract. Subsequently, the obtained extract is concentrated by distillation.
The obtained, concentrated extract contains, among others, the following components:
1Determination of the surface percentage by UPLC-NQAD
For a subsequent production of single fractions of the concentrated extract, a membrane filtration may for example be applied.
For the production of the membrane filtrates from the grape stone extract, a polyethylene glycol-based flat membrane with a membrane surface of 26 cm2 and a cut-off of 2500 Da was rinsed with ultrapure water at 30 bar and twice for 20 minutes. A 1% extract solution (200 g) was provided in the feed container, heated to 30° C. and impinged with a pressure of 30 bar. After throughput of 138.23 g, it was diluted with 100 g of water and filtered again. The membrane filtrates were lyophilized and a permeate with a yield of 7.6% and a retentate with a yield of 58.8% was obtained.
Alternatively or additionally, a polar and a nonpolar fraction can be obtained, e.g. by means of chromatographic methods.
For the production of a polar and an unipolar fraction, a solution of 100 mg grape stone extract in 1 mL deionized water was produced and applied on the preparative HPLC column (PRP-1 column 250×21.5 mm; 10 μm particle size) which was preconditioned with water/ethanol (90/10) at a flow rate of 10 ml/minute. Deionized water and ethanol (99.5%) were used as solvent at an oven temperature (60° C. isotherm). First, it was eluted for 25 min with water/ethanol (90/10). Afterwards, the ethanol proportion was increased within ten minutes by ten percent to 20% and was kept at a constant level for 10 minutes. Subsequently, the ethanol proportion was increased to 100% within five minutes and maintained for five minutes. The polar fraction was collected at a run-time of from 10 to 36 minutes and the unipolar fraction was collected at a run-time of from 37 to 55 minutes. The yield of the polar fraction was 48% and the yield of the unipolar fraction was 28%.
The obtained fractions were subsequently examined for their components by mass spectrometry. The obtained fractions contain, among others, the following non-volatile components:
4. Examination of the Antimicrobial Effect of Different Extracts:
For the examination of the antimicrobial effect of different extracts alone, i.e. without a combination with component (B) according to the invention, exemplary extracts such as described in Examples 1 to 3 were used. For this purpose, the extracts were dissolved in citrate buffer at a pH of 3 and with 500 mg/kg. The samples were inoculated with 5.63×104 CFU/mL S. cerivisiae, 1.89×104 Z. bailii and 3.75×104 C. albicans.
S. cerivisiae
Z. bailii
C. albicans
A. pullulans
A. brasiliensis
With regard to S. cerevisiae, lower germination numbers were observed after 24 g incubation time. After 7 days, the growth ceased.
With regard to Z. bailii, lower germination numbers were observed after 7 days and starting with 14 days, no new colony formation has taken place.
C. albicals resisted the extracts over the 21 days.
A. pullulans could be reduced during the 28 days. A 100% reduction could be observed for GRA after 28 days.
A. brasiliensis could be detected with lower germination number after 28 days.
As shown by the results below (cf. Example 7), the antimicrobial effectiveness of the extracts can be improved by combination with a component (B) as described herein.
5. Further Examination on the Antimicrobial Effect of Selected Extracts and Fractions Thereof:
For further examination of the antimicrobial effect of different extracts alone, i.e. without a combination with component (B) or fractions thereof according to the invention, exemplary extracts or fractions thereof such as described in Examples 1 to 4 were used. The extracts or, respectively, fractions thereof were dissolved in citrate buffer at a pH of 5 and in a dose as indicated in the table. The samples were inoculated with 3.8×104 CFU/mL S. cerivisiae.
S. cerevisiae
S. cerevisiae
Vitis vinifera extract
Vitis vinifera
Vitis vinifera
Vitis vinifera
Vitis vinifera
Punica granatum
Punica granatum
Punica granatum
The negative control did not result in a significant reduction of the germination number.
However, the positive control with 375 mg/kg potassium sorbate also did not show a significant reduction in the germination number. The underlying reason is on the one side that in comparison to pH 3 as in application example table 2, sorbic acid is only in a small extent present in its effective non-dissociated form and the optimal growth of S. cerevisiae between pH 4 and 6 does occur at pH 5.
For the Punica granatum extract (and for the Vitis vinifera extract) no significant reduction in the germination number of S. cerevisiae can be observed. It was thus even more surprising that by fractionation of the extracts, an inhibition of the antimicrobial growth, i.e. reduction of the germination number by log 2 could be observed. Thus, the germination number was significantly reduced by addition of 200 mg/kg of the non-polar fraction of Vitis vinifera extract and by addition of 200 mg/kg Punica granatum membrane filtrate >2500 Da.
As is shown by the below results (cf. Example 7), the antimicrobial effectiveness of the extracts can be improved by combination with a component (B) as described herein.
6. Interaction of Extracts and Flavouring Agents:
The Examples show that by addition of distinct flavouring agents, the sensory impression of the product, to which the above-mentioned extracts were added, is improved while maintaining product-specific olfactory and gustatory notes. It has been found that undesired side-notes of the plant extracts such as bitter, astringent, metallic can be transferred into attributes which are sensorially perceived as positive such as more juicy, more full-bodied, sweeter and more complex when combined with the flavouring agents. The same applies accordingly to the combination of fractions of such extracts as described herein with the flavouring agents as described herein. For optimizing the desired effect, a skilled person may test and use different doses, depending on the matrix.
For a further test, still water, composed of 60 mg/kg Acesulfame-K, 60 mg/kg Sucralose, 0.15 g/100 g citric acid, 200 mg/kg GRA (Ex. 1) and 0.1 g/100 g cherry flavouring, was used. Flavouring agents and mixtures of flavouring agents with the effect described below were added and compared with the sensory profile of the basis without addition:
In the scope of a further test, the antimicrobial effect of aroma and extract combinations was examined. The following table shows the results of a germ load test, analogous to the experiments of Examples 5 and 6, with the difference that the mixture was produced with pure water at a pH of 7. For example, grape stone extract and the flavouring agent vanillic acid have a different effect against yeasts. In the combination, however, also an effect against fungi and an optimal effect against yeasts was observed. The pomegranate and red berry extract have a lower effect at pH 7 compared to pH 3 (cf. Example 5), the pomegranate extract eliminates S. cerevisiae in less than 14 days at 1000 mg/kg. In combination with Limonene-containing lemon oils, however, the effect can be increased for higher pH values.
S. cerevisiae
Z. bailii
C. albicans
A. pullulans
A. brasiliensis
1the amount of Limonene of the lemon oils is in a range of from 70% to 1%.
7. Application Examples:
The mixtures or, respectively, combinations to be used according to the invention can be applied to a plurality of preparations. For example it is referred to the following possibilities of application (instead of the exemplarily mentioned extracts, also other extracts or fractions thereof as described herein can be applied):
Punica granatum
Vaccinium macrocarpon,
Vaccinium myrtillus and
Vaccinium vitis-idaea
Vitis vinifera
7.1 Drink with Cherry Taste (Sugar-Free):
1The extract contains 8.3 g/kg Ellagic acid and 4.7 g/kg gallic acid
7.2 Drink with Cherry Taste (Sugar-Containing):
1The extract contains 8.3 g/kg Ellagic acid and 4.7 g/kg gallic acid
7.3 Beer Mixed Drink with Grapefruit Taste (<0.1 Vol. % Alc.):
1The extract contains 10.3 g/kg gallic acid
7.4 Alcohol-Free Malt Drink (<0.1 Vol. % Alc.):
1The extract contains 10.3 g/kg gallic acid
7.5 Tomato Ketchup:
1the extract contains 2.2 g/kg chlorogenic acid
7.6 Salad Cream (Approx. 55 wt. % Water):
1the extract contains 8.1 g/kg chlorogenic acid
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/079115 | 11/14/2017 | WO | 00 |
