NZ623867B2 - Preservative combinations comprising propionic acid and vanillin and/or cinnamic acid - Google Patents

Abstract

The present disclosure relates to a preservative system capable of protecting against spoilage by bacteria, yeasts and moulds in carbonated and non-carbonated beverages. In accordance with the disclosure this objective is realized with a preservative system comprising a combination of propionate with vanillin and/or cinnamate. Vanillin and propionate are both known for their anti mould action. However the dosages that are needed of the separate ingredients are too high for most applications. The present inventors found that potassium cinnamate at low concentrations has a positive effect on the inhibition of moulds in combination with vanillin and propionate. Moreover, the present disclosure, using a combination of vanillin, propionate and cinnamate accomplishes complete inactivation of all moulds at manageable sensory levels. Hence the present disclosure provides a preservation system comprising combinations of propionate with vanillin and/or cinnamate as well as applications of such preservation systems in alimentary products, especially carbonated and non-carbonated beverages. h vanillin and/or cinnamate. Vanillin and propionate are both known for their anti mould action. However the dosages that are needed of the separate ingredients are too high for most applications. The present inventors found that potassium cinnamate at low concentrations has a positive effect on the inhibition of moulds in combination with vanillin and propionate. Moreover, the present disclosure, using a combination of vanillin, propionate and cinnamate accomplishes complete inactivation of all moulds at manageable sensory levels. Hence the present disclosure provides a preservation system comprising combinations of propionate with vanillin and/or cinnamate as well as applications of such preservation systems in alimentary products, especially carbonated and non-carbonated beverages.

Description

PRESERVATIVE COMBINATIONS COMPRISING PROPIONIC ACID AND VANILLIN AND/OR CINNAMIC ACID Field of the Invention The present invention relates to a preservative system with a broad spectrum of applications. In particular, the invention provides a preservative system capable of protecting against spoilage by bacteria, yeasts and moulds in carbonated and non- carbonated beverages. The invention further provides the use of the preservative system as well as to alimentary ts containing the preservative system, especially carbonated and non-carbonated beverages.

Background of the Invention ial spoilage of beverages is a significant concern in the beverage industry. ges have varying degrees of ivity to microbiological spoilage depending on intrinsic factors of the beverage such as pH, nutrient t (e.g., juice, vitamin, or micronutrient content), carbonation level, BriX, water quality (e.g., alkalinity and/or hardness), and vatives. Spoilage events occur when microorganisms are able to overcome the beverage's intrinsic factors and grow.

Microbiological ge can result from one or more yeasts, bacteria, and/or moulds. For example, yeasts and ia are capable of spoiling carbonated and non- carbonated beverages such as fruit drinks, teas, coffees, enhanced waters, etc.

The ability of yeasts and certain bacteria to grow anaerobically s their growth in carbonated beverages. Typically, bacteria tend to e off-flavors and odors with associated sedimentation. Spoilage by yeasts usually manifests itself as fermentation with gas and ethanol production, as well as sedimentation, off-flavors and odors, and loss of cloud or emulsion ity. Yeasts such as Saccharomyces, Zygosaccharomyces, Candida, Dekkera spp. and Pichia are often responsible for spoilage incidents in common beverages, both ated and non-carbonated.

Moulds are restricted to aerobic metabolism, and therefore do not usually grow in carbonated beverages. On the other hand, moulds may survive in low oxygen environments and thus can still spoil carbonated soft drinks when carbonation is diminished.

Mould spoilage of non-carbonated beverages, poses a more serious concern. Mould spoilage may be evident after mould mycelial growth, by floating globules, clumps or surface pellicles. Heat-processed ges can be classified as hot-filled and aseptically- or cold-filled. Since they are heat-processed, both groups can be spoiled by heat-resistant fungi (HRM) that produce ascospores. These ascospores not only survive the heat ent given to these beverages but also can be activated and grow during storage.

Spores of heat resistant mould spores of Byssochlamys, Neosarloria and Fusarium can survive pasteurization and may spoil non-carbonated hot-filled ts such as sport drinks and teas. Packaged waters are susceptible to growth by moulds as well. Moulds like Penicillium and Aspergillus spp. in particular cause spoilage of cold-filled beverages.

Protection against microbiological spoilage of beverages can be ed using chemical preservatives and/or processing techniques such as hot , tunnel pasteurization, ultra-high temperature treatment (UHT) or pasteurization followed by aseptic packaging, and/or pasteurization followed by chilling the beverage.

Beverages having a pH > 4.6 must be processed such that spores are destroyed using ultra-high temperatures followed by aseptic filling into packages or retorting sealed packages of product.

Beverages with a pH < 4.6 can be ally preserved, heat processed, and filled into packages such that the product is not re-contaminated. For example, process techniques such as cold filling with chemical preservatives.

Current preservation systems for acidic, shelf-stable carbonated and non-carbonated soft drinks rely on acidic vatives, especially benzoic acid, benzoates, sorbic acid, sorbates and te. These preservatives can have an impact on the flavour and the use of these preservatives is restricted in many countries. Furthermore, these acidic preservatives can not protect against mould spoilage when used in amounts that are considered acceptable e. g. in terms of organoleptic properties of the t.

It has therefore often been attempted to reduce the amount of sorbic and/or benzoic acid necessary to achieve microbial stability. One such t involves the addition of naturally occurring and less potentially toxic agents such as oils of cinnamon and thyme.

US 6,042,861 teaches the use of ic acid in the preservation of carbonated and non-carbonated tea based beverages, so as to achieve a reduction in the amount of sorbic and/or benzoic acid necessary to inhibit microbial growth. The combination of 0.40 g/l of potassium sorbate and 30 ppm cinnamic acid is tested in a still tea based beverage.

The compositions of US 6,042,861 n substantial s of an acidulant in order to achieve vation stability. In US 6,042,861 only preservation against yeast spoilage is tested. While it is demonstrated that the growth of moulds at beverage pH values is a ntial risk, protection against mould ge is not accomplished in US 6,042,861.

Such protection would require substantial or complete inactivation of mould spores.

It is an object of the present invention to solve one or more of the aforementioned shortcomings of the existing s of chemical preservation. It is a particular object of the present invention to provide a vative system with a broad spectrum of activities against bacteria, yeasts and/or , especially a system providing long-term protection against mould spoilage in carbonated as well as non-carbonated beverages.

Summary of the Invention In ance with the invention this objective is realized with a preservative system comprising a combination of propionate with vanillin and/or cinnamate.

The present inventors found that binary combinations of propionate with vanillin or propionate with cinnamate were effective in inhibiting the growth of n moulds often associated with spoilage of (non-carbonated) beverage, at dosages suitable for this kind of application.

The present inventors also observed that potassium cinnamate can be consumed by moulds like Penicillium and Aspergillus when the dosage is too low for inhibition of germination and outgrowth. Via this way potassium cinnamate is actually oxylated to styrene which is an unwanted chemical because of its unacceptable smell. Potassium cinnamate can thus not be used on its own in too low concentrations. The present inventors now found that potassium ate at low concentrations does have a positive effect on the inhibition of moulds when used in combination with propionate and in particular in combination with propionate and vanillin.

Vanillin and propionate are both known for their antimicrobial action. However the dosages that are needed of the te ingredients are much too high to be used as general crobial system in sweet applications like beverages. Moreover, a total inactivation of mould and mould spores is not accomplished with the sole ingredients. By total inactivation it is meant that it is not possible to revive any mould spores after a period of exposure to the ation of cinnamate, vanillin and propionate. Using a combination of vanillin, propionate and cinnamate total inactivation of mould spores at manageable sensory levels can be accomplished, as will be explained and demonstrated in more detail in the appending examples.

Even more ably, the inventors found that a preservative system comprising propionate, ate and vanillin resulted in protection t all moulds species .

In order to achieve these and other benefits, the preservative components only need to be used at levels well below the limits normally considered acceptable.

Hence the present invention provides for the first time a preservation system, relying on combinations of propionate with vanillin and/or cinnamate and in particular combinations of propionate, vanillin and cinnamate, as well as applications of such preservation systems in alimentary products, ally carbonated and rbonated beverages.

Detailed Description of the Invention A first aspect of this invention provides a preservative system sing (i) nic acid or a salt thereof, in combination with at least one component selected from (ii) vanillin and derivatives thereof and (iii) cinnamic acid and salts and derivatives thereof.

In a particularly preferred embodiment of the invention a preservative system as defined herein before is provided, sing (i) propionic acid or a salt thereof, in combination with (ii) vanillin or a derivative f and (iii) cinnamic acid or a salt thereof.

Propionic acid (or propanoic acid), is a well known food additive. As used herein, the term ‘propionate’ refers to any agent including the propionic acid anion and capable of ting said anion upon dissolving the agent, especially propionic acid and propionate salts. Typically, the salt can be a water soluble salt of propionic acid, such as sodium propionate, m propionate and potassium propionate. In accordance with the t ion, the preservative system preferably comprises propionic acid, sodium nate, calcium propionate, potassium propionate or a mixture of two or more of said agents.

Propionates are often used as preservatives, mainly against fungi, ally in bakery goods and in meat products against ial spoilage. Due to the strong smell, application of propionates in other products is limited. Sensory tests in model drinks have shown that the maximum acceptable amount is approximately 630 ppm.

In one preferred embodiment of this invention, the preservative system comprises propionate in an amount of less than 70 wt.% based on the total weight of the preservative system, preferably less than 60 wt.%, preferably less than 50 wt.%, preferably less than 40 wt.%, preferably less than 35 wt.%, preferably less than 30 wt.%, preferably less than 25 wt.%, preferably less than 20 wt.%, preferably less than 15 wt.%, preferably less than 12 wt.%, preferably less than 10 wt.%, based on the total weight of the preservative system.

In one preferred embodiment, the preservative system comprises propionate in an amount of more than 0.1 wt.%, based on the total weight of the preservative system, preferably more than 0.25 wt.%, preferably more than 0. 5 wt.%, preferably more than 1 wt%, preferably more than 2.5 wt%, preferably more than 4 wt%, preferably more than 5 wt%, preferably more than 6 wt%, preferably more than 7 wt%, preferably more than 8 wt%, based on the total weight of the preservative system.

As is known by those skilled in the art, nate/propionic acid can be obtained by fermentation. In the context of this invention, it is highly advantageous to employ propionate obtained by fermentation, in particular a composition obtainable by ting e, lactose or lactate, particularly lactate, using an appropriate micro-organism, such as propionic acid bacteria. Hence, in a particularly preferred embodiment of the invention the preservative system comprises a ferment or fermentation product as the source of propionate. Such a fermentation product or ferment is typically characterized by the presence of other fermentation products such as acetate and/or succinate.

Hence, in a preferred embodiment of the invention the vative ition is characterized by the presence of e, the ratio of propionate : e being more than 1, preferably more than 1.5, most ably more than 1.75. Said ratio is preferably less than 3, more preferably less than 2.5, most preferably less than 2.25. Said ratio can for example be approximately 2.

Fruit- or vegetable-based extracts or compositions can be used as substrate for fermentation, such as a melon-based extract or a composition originating from tomatoes.

Hence, in an embodiment of the invention, the preservative system comprises fermentation product or ferment obtained from fermentation of fruit- and/or vegetable-based extracts or compositions, especially melon-based abstract and/or tomato based abstracts. Such ferments or fermentation products have very favorable organoleptic profiles, which contribute positively to the taste and odor experience of food and drink products.

Raw propionate ferments lly comprise propionate in amounts of 0.02 wt% and up to 6 wt%. Raw ts based on fruit- or vegetable-based ts may contain up to 12 wt% of propionate. In an embodiment of the invention such raw ferments may be combined with the other preservative agent or agents of this invention, yielding a composition that may be used as such. Alternatively, raw nate ferments may be trated, purified, dried, etc., as described in more detail herein below, Cinnamic acid (3-phenylpropenoic acid) is well known as a food ingredient, which obtained RAS status in 1965. As will be understood by those skilled in the art, any soluble cinnamic acid salt may be used in accordance with the ion.

Typically, the cinnamate is a water soluble salt of cinnamic acid. For convenience, the term ‘cinnamate’ is used herein to refer to any substance containing the cinnamic acid anion, in particular to denote cinnamic acid and the salts thereof. Furthermore, a number of cinnamic acid derivatives are known and used in the food industry, including p- dimethylaminocinnamate, cinnamaldehyde, cinnamyl acetate, yl alcohol, cinnamyl benzoate, cinnamyl cinnamate, cinnamyl e, cinnamyl isobutyrate, cinnamyl isovalerate and cinnamyl phenylacetate, which may also be referred to herein as mate derivatives’. In accordance with the invention these derivatives are equally suitable for use in the preservative system either alone or in combination with cinnamic acid or other cinnamate salts or derivates. In a particularly preferred embodiment of the ion the preservative system comprises cinnamic acid and/or a cinnamic acid salt ed from the group of sodium cinnamate and potassium ate. In a preferred embodiment, the preservative comprises potassium cinnamate.

In one preferred embodiment, the preservative system comprises cinnamate and/or a cinnamate derivative in an amount of less than 50 wt.% based on the total weight of the preservative system, preferably less than 45 wt.%, preferably less than 40 wt.%, ably less than 35 wt.%, preferably less than 30 wt.%, preferably less than 25 wt.%, preferably less than 20 wt.%, preferably less than 15 wt.%, preferably less than 12 wt.%, preferably less than 11 wt.%, ably less than 10 wt.%, based on the total weight of the preservative system.

In one preferred ment, the vative system comprises cinnamate and/or cinnamate derivative in an amount of more than 0.1 wt.%, based on the total weight of the preservative system, preferably more than 0.25 wt.%, preferably more than 0. 5 wt.%, preferably more than 1 wt.%, preferably more than 2.5 wt%, preferably more than 4 wt.%, ably more than 5 wt%, preferably more than 6 wt.%, preferably more than 7 wt.%, preferably more than 8 wt.%, based on the total weight of the preservative system.

Vanillin (4-Hydroxymethoxybenzaldehyde) is ed as a food additive by authorities world wide. Vanillin was given FEMA-GRAS status in 1965. Derivatives of vanillin such as methyl vanillin, ethyl vanillin and vanillin 2,3-butanediol acetal may also suitably used in accordance with this invention, although the use of in is particularly preferred.

In one preferred embodiment, the preservative system comprises vanillin and/or a vanillin derivative in an amount of less than 25 wt. % based on the total weight of the preservative system, preferably less than 20 wt. %, preferably less than 15 wt. %, preferably less than 12 wt. %, preferably less than wt. %, preferably less than 10 wt. %, preferably less than 9 wt. %, preferably less than 8 wt. %, preferably less than 7 wt. %, preferably less than 6 wt. %, ably less than 5 wt. %, preferably less than 4 wt. %, preferably less than 3.5 wt. %, preferably less than 3 wt. %, based on the total weight of the preservative system.

In one preferred embodiment, the preservative system comprises vanillin and/or a vanillin derivative in an amount of more than 0.025wt. %, based on the total weight of the preservative system, preferably more than 0.05 wt. %, preferably more than 0.1 wt. %, preferably more than 0.25 wt. %, preferably more than 0.5 wt%, preferably more than 1 wt. %, preferably more than 1.5 wt%, preferably more than 2 wt. %, preferably more than 2.5 wt. %, based on the total weight of the preservative system.

In one preferred embodiment, the preservative system is characterized by a (molar) ratio of (i) propionate : (ii) in and/or derivatives thereof of less than 100 (100.01), preferably less than 40 (100.025), preferably less than 20 (100.05), preferably less than (100.067), preferably less than 10 (10:01), preferably less than 5 (10:02). In one red embodiment, the preservative system is characterized by a ratio of (i) propionate: (ii) vanillin and/or derivatives thereof of more than 0.1 (1.0:10), preferably more than 0.5 (102), preferably more than 1 (101), preferably more than 2 ), ably more than 2.5 (10:04).

In one preferred ment, the vative system is characterized by a ratio of (i) propionate : (iii) cinnamate of less than 100 (100.01), preferably less than 40 (100.025), preferably less than 20 (100.05), preferably less than 15 (100.067), preferably less than 10 (10:01), preferably less than 5 (10:02). In one preferred embodiment, the preservative system is characterized by a ratio of (i) propionate : (iii) cinnamate of more than 0.1 ), preferably more than 0.5 (102), preferably more than 1 (101), preferably more than 2 (10:05), preferably more than 2.5 (10:04).

The preservative system of the present invention can ally include other preservatives. Weak acid preservatives are preferred for this purpose. As indicated in the foregoing however, an advantage of the present invention s in the fact that the presence of other preservatives, especially synthetic preservatives such as benzoates and sorbates can be minimized or avoided altogether while achieving the desired level of microbial stability. The preservative system of the present invention typically contains no or only minor s of additional preservative agents, such as, in particular, benzoate and/or sorbate. In a red embodiment of the invention the preservative system contains less than 1 wt% of preservative agents selected from the group consisting of sorbates and tes, preferably less than 0.5 wt%, preferably less than 0.1 wt%, more preferably less than 0,05 wt%. In a particularly preferred embodiment of the invention the preservative system is essentially or completely free from preservative agents selected from the group of benzoates and sorbates.

In one embodiment of the invention the vative composition further comprises a carrier material, the choice of which will largely depend on the physical form in which the preservative system is to be provided. The carrier material is typically used in any amount required to provide a product that has the desired properties relating to production, storage and dosing.

In one embodiment of the invention, a vative system in the form of a free flowing powder or granulate, which may comprise a carrier material. In another preferred embodiment a free flowing powder is ed consisting essentially of the preservative combination. Such a free flowing powder may be obtained by combining the various components in an aqueous sion or solution followed by drying, e. g. spray-drying.

In another embodiment of the invention the preservative system is produced by drying, typically spray-drying, of an s propionate ferment, before or after ing with the other preservative agents of this invention. Such a preservative system may contain propionate in amounts of more than 30 wt%, preferably more than 40 wt%, most preferably more than 50 wt%.

In r embodiment a liquid preservative system is provided comprising solution or dispersion of the above deflned components in an aqueous phase, which for instance may be obtained by concentrating the aqueous dispersion or solution.

In another embodiment of the invention the preservative system is produced by concentrating an aqueous nate ferment before or after combining with the other preservative agents of this invention. Such a preservative system may contain propionate in amounts of more than 10 wt%, preferably more than 20 wt%, most preferably more than wt%.

As discussed herein the present preservative system is capable of preventing and/or ting the growth of, and/or killing of a micro-organism in a food system. This may be slowing or arresting a organism, or by killing the micro-organism present on contact with the present composition. In a highly preferred aspect the microbiocidal or iostatic effect is a fungicidal or fungistatic , optionally including effect against . In a preferred aspect the microbicidal or iostatic effect is in respect of an organism associated with food spoilage or food borne disease. In a preferred aspect the microbicidal or microbiostatic effect is in t of at least one, more preferably at least two, more preferably at least three organisms selected from Yeasts, especially from the species of a (e.g. C. krusei, C. ilosz's, C. ulilis, C. valida), Dekkem (e.g. D. bruxellensis), Debaryomyces (e. g. D. hansenii), Hanseniaspora (e. g. H. ) Kluyveromyces (e. g. K. locll's), Pichia (P. membranaefaciens), Rhodosporidium, Rhodotorula (Rh mucilaginosa), Saccharomyces (e. g. S. bayanus, S. boulardi, S. carlsbergensis, S. cerevisiae, S. exiguus, S. florentinus, S. unisporus), Zygosaccharonmyces (e.g. Z. rouxii, Z baili) and moulds, especially from the species of Aspergillus (e.g. A. niger, A. restrictus, A. olor, A. flavus), Byssochlamys (e.g. B. fulva, B. nivea), cillium, Eurotl'um, Fusarl'um (F. oxysporum, F. graminearum, F. solam'), Geotrichum, Mucor, Neosaflorya (e.g. N. fischeri var. fischerl), Penicillium (e.g.

P. islandl'cum, P. citrinum, P. chrysogenum, P. aurantiogriseum, P. brevicompaclum, P. camembertii, P. candidum, P. chrysogenum, P. commune, P. corylophilum, P. cyclopl'um, P. or, P. nalgiovense, P. rogueforli), Talaryomyces (e. g. T. macrosporus).

In one preferred embodiment of the ion the fiingicidal effect is in t of one or more moulds species selected from Aspergillus, Penicillium, Byssochlamys and Fusarium.

The present invention is particularly effective in preventing spoilage of ges that can be initiated by either vegetative mould hyphae or spores of moulds that are capable of germinating to a vegetative form when suspended in a beverage. Mould spores may not be inactivated by the presence of the preservative system invention, but the spores are either prohibited from germinating in the presence of the preservative system or the vegetative form of the mould that results upon ation is prohibited from growth beyond a small number of cell cycle replications. In one preferred embodiment the preservative system is capable of ntial or complete inactivation of mould spores. In an embodiment the preservative system is capable of prohibiting an increase of the mould spore count of a beverage, which typically means that the initial spore count (spores/ml) in a (test) beverage will not increase after contacting it with the preservative system of the invention. Even more ably the preservative system is capable of decreasing the mould spore count of a ge, which typically means that the initial spore count (spores/ml) in a (test) beverage will decrease after contacting it with the preservative system of the invention. Preferably the preservative system is capable of decreasing mould spore counts s / ml) by at least 10 %, more preferably at least 25 %, more preferably at least 50 %, more preferably at least 75 %, more preferably at least 85 % and most preferably at least 90 %. Preferably the mould spores selected from Aspergillus, Penicillium, Byssochlamys and Fusarium spores.

A second aspect of the invention concerns an tary product, comprising an effective amount of (i) propionic acid or a salt thereof, in combination with at least one component selected from (ii) vanillin and tives thereof and (iii) cinnamic acid and salts and derivatives f.

One preferred embodiment concerns an tary t comprising an effective amount of a combination (i) propionic acid or a salt thereof, in combination with at least one component selected from (ii) vanillin or a derivative thereof and (iii) cinnamic acid or a salt thereof.

As used herein the term ‘effective amount’ refers to an amount sufficient to preserve the product to which the present preservative system is added, i.e. to keep the product from microbial ge. As commonly understood in the art, the definitions of the terms "preserve," "preservative," and "preservation" do not provide a standard time period for how long the t to be ved is kept from spoilage, decomposition, or discoloration. The time period for "preservation" can vary greatly depending on the subject matter. As used herein, the terms rve," "preservative," and "preservation" refer to the protection against spoilage of a product that is the result of the growth of spoilage microorganisms for a period of at least 1 weeks, preferably at least 2 weeks, preferably at least 5 weeks, preferably at least 10 weeks, preferably at least 15 weeks. This period is in keeping with the time required to transport a beverage product from on of manufacture, through distribution channels, into the hand of the consumer. Typically, the product is preserved under ambient conditions, which include the full range of temperatures experienced during storage, transport, and display (e.g., 00C to 400C, 100C to 300C, 200C to 25°C) without tion to the length of exposure to any given temperature. e of spoilage is noted by absence of any evidence of growth of spoilage organisms dity, viable count, direct microscopic count or other standard methods of enumeration) and by the e of any discernable change in the product attributes that could be routinely attributed to metabolism of spoilage organisms.

As noted herein before, the present preservative system is particularly suited for beverages, including non-carbonated beverages. Hence in a preferred embodiment an alimentary product as defined above is provided, which is selected from the group consisting of beverages, more preferably from the group of still beverages. Some examples of still beverages include flavored waters, tea, coffee, nectars, mineral drinks, sports W0 58650 11 beverages, vitamin waters, juice-containing ges, punches or the concentrated forms of these beverages, In one preferred embodiment, the beverage comprises vanillin and/or vanillin derivative in an amount of less than 1000 ppm, ably less than 800 ppm, preferably less than 700 ppm, preferably less than 600 ppm, preferably less than 500 ppm, preferably less than 450 ppm, preferably less than 400 ppm, ably less than 375 ppm, preferably less than 350 ppm.

In one preferred embodiment, the beverage ses vanillin and/or vanillin derivative in an amount of more than 1 ppm, preferably more than 2.5 ppm, ably more than 5 ppm, preferably more than 10 ppm, preferably more than 25 ppm, preferably more than 50 ppm, preferably more than 100 ppm, preferably more than 150 ppm, ably more than 200 ppm, preferably more than 250 ppm, most preferably more than 275 ppm.

In one preferred embodiment, the beverage comprises cinnamate and/or cinnamate derivative in an amount of less than 1000 ppm, preferably less than 800 ppm, preferably less than 700 ppm, preferably less than 600 ppm, preferably less than 500 ppm, preferably less than 450 ppm, preferably less than 400, preferably less than 350 ppm, ppm, preferably less than 300 ppm, preferably less than 275 ppm, preferably less than 250 ppm, most preferably less than 225 ppm.

In one preferred embodiment, the beverage comprises cinnamate and/or cinnamate tive in an amount of more than 1 ppm, preferably more than 2.5 ppm, preferably more than 5 ppm, preferably more than 10 ppm, preferably more than 25 ppm, preferably more than 50 ppm, preferably more than 100 ppm, preferably more than 125 ppm, preferably more than 150 ppm, preferably more than 175 ppm.

In one red embodiment, the beverage comprises propionate in an amount of less than 2000 ppm, preferably less than 1500 ppm, preferably less than 1200 ppm, preferably less than 1100 ppm, preferably less than 1000 ppm, preferably less than 900 ppm, preferably less than 850, preferably less than 825 ppm, ppm, preferably less than 800 ppm.

In one preferred embodiment, the beverage comprises propionate in an amount of more than 1 ppm, preferably more than 2.5 ppm, preferably more than 5 ppm, preferably more than 10 ppm, preferably more than 25 ppm, preferably more than 50 ppm, preferably more than 100 ppm, preferably more than 250 ppm, preferably more than 300 ppm, W0 2013/058650 12 preferably more than 350 ppm, preferably more than 400 ppm, preferably more than 450, preferably more thab 475, most preferably more than 500 ppm.

In one embodiment a beverage as defined herein above is provided, which contains less than 50 ppm of preservative agents selected from the group of es and benzoates, ably less than 10 ppm, more preferably less than 5 ppm, more preferably less than 1 ppm, more preferably less than 0.5 ppm, more preferably less than 0.1 ppm and most preferably less than 0.05 ppm. This ensures that no negative taste effects are observed. In a particularly preferred embodiment of the invention the beverage is essentially or completely free from vative agents selected from the group of benzoates and sorbates.

In one preferred embodiment of the invention, the beverage is a non-carbonated beverage or still beverage.

Herein, the term "still beverage" is any combination of water and ingredient which is intended for human ption and which possesses no more than 0.2 volumes of carbon dioxide, as opposed to ated beverages, which typically possess a carbon dioxide tration of 0.2 s of C02 or r. The term e of C02" is understood to mean a quantity of carbon e absorbed into the liquid wherein one volume C02 is equal to 1.96 grams of carbon dioxide (CO2) per liter of product (0.0455M) at 25 °C.

Such beverages may be supplemented with flavours, sweeteners, fruit juices vitamins, nutrients, minerals, amino acids, proteins, carbohydrates, etc.

Typically, beverages according to the present invention will possess a specified range of acidity. The invention typically can function at a pH within the range of 2-7. In one preferred embodiment of the invention, the pH is at least 2, preferably at least 2.5, preferably at least 2.75, preferably at least 3, preferably at least 3.2, ably at least 3.3, preferably at least 3.4, preferably at least 3.5. In one preferred embodiment of the invention, the pH of the beverage is below 7, preferably below 6, preferably below 5.5, preferably below 5, preferably below 4.75, preferably below 4.6, preferably below 4.5. For highly acidic beverages, the invention is not limited by the type of acidulant employed in acidifying the t. Typically, in accordance with the present invention, acidulants may be inorganic acids, such as phosphoric acids, or organic acids, such as citric, malic, ascorbic, tartaric, lactic, ic, and succinic acid, c acid. The various acids can be combined with salts of the same or different acids in order to manage pH or the buffer capacity of the beverage to a specified pH or pH range. Virtually any organic acid salt can W0 2013/058650 13 be used so long as it is edible and does not provide an off-flavor. The choice of salt or salt mixture will be determined by the lity and the taste. Citrate, malate and ascorbate yield ingestible xes whose flavors are judged to be quite acceptable, particularly in fruit juice beverages. Tartaric acid is acceptable, particularly in grape juice beverages, as is lactic acid. Longer-chain fatty acids may be used but can affect flavor and water solubility.

For essentially all purposes, the malate, gluconate, citrate and ascorbate and e moieties are preferred.

Certain exemplary ments of the beverage product of the invention include juice-containing beverages and juices, or the concentrated forms of juice-containing beverages as well as beverage concentrates which contain at least about 45% by weight of juice, especially fruit or vegetable juice.

By way of example, juice can be obtained from the fruit of apple, cranberry, pear, peach, plum, apricot, nectarine, grape, cherry, currant, raspberry, goose-berry, blackberry, blueberry, strawberry, lemon, orange, grapefruit, passionfruit, mandarin, mirabelle, tomato, lettuce, celery, spinach, cabbage, watercress, dandelion, rhubarb, carrot, beet, cucumber, ple, custard- apple, coconut, pomegranate, guava, kiwi, mango, papaya, watermelon, Io han guo, cantaloupe, pineapple, banana or banana puree, lemon, mango, papaya, lime, tangerine, and es thereof.

Preferred juices are the citrus juices, and most preferred are the non-citrus juices, apple, pear, cranberry, strawberry, grape, , mango and . Any juice can be used to make the beverage of this invention. If a beverage concentrate is desired, the fruit juice is concentrated by conventional means from about 12° Brix to about 65° Brix. Beverage concentrates are usually 40° Brix or higher (about 40% to about 75% sugar ).

The ion could be used to preserve a formulation that is essentially 100% juice. The invention can be used in products containing juice wherein juice concentration is below 100%. Lowering of juice tration below 10% will typically favor the use of lowered concentrations of preservatives. In a preferred embodiment of the invention the beverage product comprises fruit juice in an amount of less than 5 % (v/v), preferably less than 4 % (v/v), preferably less than 3 % v/v, more preferably less than 2 % v/v, most ably less than 1 % v/v. In another ment of the invention the beverage product comprises fruit juice in an amount exceeding 10 % v/v, preferably in an amount exceeding 12 % v/v, more preferably in an amount exceeding 15 % v/v, most preferably in an amount exceeding 20 % v/v. Certain further examples of the ge product of the invention include tea based beverage (carbonated or non-carbonated) and flavored waters.

W0 2013/058650 14 Another exemplary embodiment of the ge product of the invention includes tea based beverages. Tea based ges typically contain the solid extracts of leaf material from Camellia sinensis, Camellia assamica, or Aspalathus is. The tea may be added to the beverage in various forms including an extract, a concentrate, a powder or as granules. Without preservation, tea acts as a nutrient that enhances the potential for microbial spoilage, at low concentrations, such as 0.01 to 3%.

Another exemplary ment of the beverage product of the invention includes flavored water. The term “flavored water” refers to a beverage essentially consisting of water with added natural or artificial flavors, herbs, and sweeteners, which may be carbonated and non-carbonated. The flavored water type beverages are usually low in calories, as compared to regular soft drinks, and are typically marketed as diet or light drinks. In many cases, flavored waters comprise fruits or fruit juices, in limited amounts, as a source of the vitamins, minerals and flavors.

Further exemplary embodiments of the beverage product of invention include sports beverages (carbonated or non-carbonated), especially electrolyte balancing sports beverages. Typical sport ges contain water, sucrose syrup, glucose-fructose syrup, and l or artificial flavors. These beverages can also contain sodium chloride, citric acid, sodium citrate, mono-potassium phosphate, as well as other natural or artificial substances which serve to replenish the balance of electrolytes lost during perspiration.

The preservation function of the t invention in beverage formulations typically is not affected by the type of sweeteners present therein. The sweetener may be any ner commonly employed for use in beverages. Sweeteners suitable for use in various embodiments of the beverages disclosed here e nutritive and tritive, natural and artificial or synthetic sweeteners. The sweetener can include a monosaccharide or a haride. Peptides possessing sweet taste are also permitted. The most commonly employed saccharides include sucrose, fructose, se, maltose and e and invert sugar. Mixtures of these sugars can be used. Other natural ydrates can be used if less or more sweetness is desired. Suitable non-nutritive sweeteners and combinations of such sweeteners include e.g. aspartame, e, and alitame, and non-peptide based sweeteners, for example, sodium saccharin, calcium saccharin, acesulfame potassium, sodium cyclamate, calcium ate, neohesperidin dihydrochalcone, and sucralose. ge products lly contain flavors of various types and nature. In general, the beverage preservative system according to the present invention is ible with beverages formulated to contain artificial flavours, natural flavors, botanical flavors, fruit W0 2013/058650 15 , aqueous essences, etc. The vation function of the present invention is typically is not affected by such components. The term "botanical flavor" refers to flavors derived from parts of a plant other than the fruit. Also ed within the term "botanical flavor" are synthetically prepared flavors made to simulate cal flavors d from natural sources. cal flavors can be derived from natural sources such as essential oils and extracts, or can be synthetically prepared. As used herein, the term "aqueous essence" refers to the water e aroma and flavor materials which are derived from fruit juices.

Beverage products typically can be fortified with added nutrients, vitamins, minerals, trace elements and the like. Such additional components lly do not affect the preservation function of the invention. Non-limiting examples of such additional components that may typically be present in the beverages of the invention include vitamins A, B1, B2, B6, B12, C, D, E, K, Biotin, Folic Acid, Pantothenic Acid, Niacin, calcium, magnesium, iron, zinc, potassium, selenium, copper, manganese, etc.

The preservative system of the invention may also function in beverage products of the meal substitute type, in which case substantial amounts of protein, carbohydrate, dietary fibers and/or lipids are typically present. Another aspect of the invention concerns the use of the preservative system of this invention, for the preservation of an alimentary product, especially a beverage as defined previously. Yet another aspect of the invention concerns a method of preserving an alimentary product, especially a beverage as defined previously, comprising the step of adding to said product the preservative system of this invention. Preferred ments concern uses and methods, wherein the preservation comprises inactivation of mould spores and/or ting mould growth. In particularly preferred embodiments the mould is a mould selected from the group consisting of Fusarium oxisporum, illus niger, clamys fuvla and Penicillium Sp. In a particularly preferred embodiment uses and methods are provided, wherein the vation comprises vation of spores and/or inhibiting growth of at least two or at least three or at least four of the above moulds. The details and preferred ments of these aspects of the invention will be readily understood by those skilled in the art based on the foregoing detailed descriptions of the preservative system and products containing them.

Thus, the invention has been described by nce to certain embodiments discussed above. It will be recognized that these embodiments are susceptible to various modifications and alternative forms well known to those of skill in the art.

W0 2013/058650 16 Many modifications in addition to those described above may be made to the structures and techniques described herein without departing from the spirit and scope of the invention. Accordingly, although c embodiments have been described, these are examples only and are not limiting upon the scope of the invention.

Furthermore, for a proper understanding of this document and in its claims, it is to be understood that the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not e the possibility that more than one of the element is present, unless the context y requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way Example: Anti-fungal combinations with preservative potential for beverages.

Introduction In this example the effect of propionate, ium cinnamate and vanillin was tested against a ion of 4 moulds that are associated with spoilage of non carbonated beverages. The combination of these three ingredients was derived from a formulation of 200ppm potassium cinnamate and 300ppm vanillin which appears to be a substitution of the benzoate /sorbate preservative system in carbonated beverages (data not shown). The addition of propionate was hypothesized to broaden the spectrum of applications to non- carbonated ges.

Besides testing the sole ingredients against the moulds the effect of combinations were tested, in all cases at ge pH. These combinations form the basis of a ial preservative system for still beverages.

The total inactivation of spores was discovered by the ion of a long term application ment. To see the long term anti microbial effect in ge application, several dosage levels of the propionate, vanillin, cinnamate formulation were tested in an apple juice based model drink. Data obtained from this study showed inhibition of wth of mould spores. Moreover, a total inactivation of mould spores have been observed in some concentrations after 3 weeks of incubation.

W0 58650 17 Methods and materials 2.1 Cultures and e conditions Table 1 shows the cultures that were used in all tests.

The cultures were grown on Malt extract agar (MEA) at pH 5.5 at 25°C. 2.2 media The medium that was used for the tests of single ients against single moulds was Malt extract agar (Oxoid) adjusted on pH 3.2-3.5. Table 2 shows the components of the model beverages as it is used in the binary, ternary and long term application tests. 2.3 Components The components that were used in the tests are mentioned in table 3. The ations that were used in the long term application tests are shown in table 4. 2. 4 Spore preparation 5ml of water comprising 0.05% tween was poured on a layer of sporulating mould.

Spores were scraped of and the liquid was transferred to a sterile tube containing 4mm glass beats. After vigorous shaking the liquid is filtered by glass wool. The filtrate was used as inoculum. 2.5 Single moulds tests t a single component 2. 5. 1 solid agar Plate preparation Each experiment existed of 10 MEA agar plates containing a successive amount of component, starting from 0ppm to the maximal used concentration.

MEA was prepared by dissolving 20g of Malt extract (Difco, England), lg of Peptone (Becton Dickinson, USA), 20g of glucose and 20g of bacteriological agar (Nol. Oxoid, d) in 11 of dematerialized water. The pH was adjusted to 3.2 by adding 5.9ml of Hcl 1M after heat sterilization.

A stock solution was made for each component. The stock was 25 times the maximal used concentration. A successive amount from 0 to 900ul of these , making the concentration range, was pipetted in 10, 50ml capped test tubes.

Subsequently, 25 ml of warm (SO-90°C) MEA agar was pipetted, using a sterile 25ml pipette, in the tubes and the tubes were vortexed slowly but vigorously. Next, the component containing agar was poured out in 9 cm Petri dishes.. Besides single W0 2013/058650 18 components t single moulds, combinations of propionate with ium cinnamate or vanillin were tested on solid agar. To prepare these binary combinations 6.25 ml of a 4 times concentrated component was mixed with the other components that was also 4 times concentrated. The mix of components were mixed with 12.5ml double concentrated MEA to reach the preferred concentrations. 2.5.2 preparation of96 ates The tests where two components or more components were combined in model drink were carried out in sterile 96-well iter plates. Sterile matrix was prepared according recipe of Nestle with sing quantities of two different inhibitors. The trations of each tor were presented in 8 equal concentration steps that ranged from O to l — 2 times the (estimated) MIC value of particular mould for a particular inhibitor resulting in 64 different media. 200 ul of each medium was transferred to a panel of a sterile 96-well microtiter plate. ted well plates were stored at 4 0C until further use. The data was obtained via optical observation of growth. 2. 6 Inoculation and incubation 2. 6.1. Agar plate experiments After the plates were dried a dot was spotted at the backside and in the centre of the petridish 0.5 ul of the mould spore solution was spotted on the agar targeting the dot in the centre. When the drop was dried in, the plates were placed bottom down in the incubator at °C.

The growth of colonies was determined by measurement of the colony diameter at different points in time. The growth curves were converted to dose response curves as described in 2.6. 2. 6.2 model drink ments Spores of all moulds were collected via the method as described in 2.3. Spores were counted using the Burker-turk method and adjusted to a log5 per ml in PFZ and mixed to a homologue suspension. This suspension was diluted 100 times for inoculation. 2. 7 Dataprocessing ofagar plate experiments The data from the agar plate experiments were obtained by measuring the colony size changes. The change in time was interpreted as growth speed and growth curves were plotted.

To convert the growth curves to dose response curves the u Max (maximum growth rate) was calculated per concentration of antimicrobial component. The curves were fitted to a Logistic Dose se (LDR) Intercept Form (Equation 8013) using Table curve 2D n 5.1 Table 1. Used organisms.

Mould acronym Reference origin Fusarl'um oxisporum Fusoxl CBS 111552 Fruit juice after Byssoaclamysfulva Bysful2 CBS 113225 Multifruit juice Penicillium Sp Pen381 AR 381 Sweet cream cheese Table 2. model drink composition.

Ingredient Amount [g] Water Taste water 949.85 Sucrose Granulated sugar — Van 40.0 Gilse Apple juice concentrate Cargill 8.30 Apple flavour Givaudan 55078-DO 0.35 Citric acid Across, M&A-021 1.50 Table 3. Used components and dosage ranges component Origin Used range (% w/v) Potassium cinnamate on product from 0- 0.018 Cinnamic acid (commerically available) and potassium hydroxide WWW 00.18 —vanillin——Rhodia 00.18 WWW 0-0045 _SodmmhenzL_Acmmrganms.Bdgnm 0-0045 W0 2013/058650 20 Table 4. Used formulations in the application tests. ations Vanillin K—cinnamate propionate Benzoate Sorbate 1 300 ppm 200 ppm 500 ppm 0 0 2 300 ppm 200 ppm 250 ppm 0 0 3 300 ppm 100 ppm 500 ppm 0 0 4 150 ppm 200 ppm 500 ppm 0 0 150 ppm 100 ppm 500 ppm 0 0 6 150 ppm 200 ppm 250 ppm 0 0 7 300 ppm 100 ppm 250 ppm 0 0 8 150 ppm 100 ppm 250 ppm 0 0 9 0 0 0 180 ppm 335 ppm 0 0 0 0 0 11 300 ppm 200 ppm 1000 ppm 0 0 12 300 ppm 200 ppm 1250 ppm 0 0 13 300 ppm 200 ppm 800 ppm 0 0 Results 3.1 Single ents on pure cultures In order to show the effect of the sole components on pure cultures of moulds some agar plate experiments were done. The results for vanillin are shown in figures 1a-c. These figures show that all moulds are sensitive to vanillin and that approximately 0.09% (900 ppm) is needed for an overall inhibition. Earlier done taste experiments showed that the maximal manageable amount of vanillin in beverages should be 300ppm.

The results for nate are shown in figure 2 (2a: Pen381 v. vanillin; 2b: Bysfu12 v. vanillin; 2c: Fusoxl v. in) and show that all moulds are sensitive to nate. Most difficult species to inhibit are Penicillium and Byssoclamys spp. Both are inhibited at approximately 0.15%. Sensory experiments of propionate in water reveal that 630ppm is the max acceptable amount. Sensory tests in model drink show that 500ppm of propionate from a melon ferment has a specific but not a negative taste impact.

The effect of potassium cinnamate is shown in figure 3 (3a: Pen381 v. propionate; 3b: Bysfu12 v. propionate; 3c: Fusoxl v. propionate)a-c. The figures show that Penicillium is not inhibited at levels up to 200ppm, which is a realistic amount for application in beverages. er, a dramatic increase of Penicillium can be observed after 100ppm.

This could be possibly explained by the consumption of potassium cinnamate of Penicillium. 3.2 Combinations tested on agar plates againstpure cultures W0 2013/058650 21 In order to reduce the amounts of the single ients the effect of combinations of ingredients was tested on the single moulds on agar plates of pH3.2. Several dosages of binary formulations of nate combined with potassium cinnamate, and propionate combined with vanillin were tested. Figure 4 (4a: Pen381 V. propionate + in, 4b: Bysfu12 V. propionate + vanillin, 4c: Fusoxl V. propionate + vanillin) show the effect of propionate combined with vanillin on the growth curve of the four moulds. The figures reveal that Penicillium is the most difficult to inhibit and that complete inhibition takes place at propionate level of 0.075% (750ppm) combined with a vanillin level of . um shows complete inhibition at all combinations.

The other combination that was tested is propionate with ium cinnamate and the figure 5 (5a: Pen381 v. propionate + cinnamate, 5b: Bysfu12 v. propionate + cinnamate, 5c: Fusoxl v. propionate + cinnamate) depict the effect of this ation on the growth of the moulds. Although small, an enhanced inhibitive effect on Penicillium (fig. 5a) is visible when ium cinnamate is combined with propionate. Again Fusarium is already completely inhibited at the lowest dosage of this combination.

The results of the combined experiment show that the amount of propionate can be reduced by two times if combined with an acceptable amount of vanillin as per sensory perspective. There is a 1/3 reduction when propionate is combined with a low amount of potassium ate. 3. 3 Combinations ofcomponents in model drink against a cocktail ds In order to have an idea of the efficacy of the combination of propionate, vanillin and potassium cinnamate in model drink, some experiments were done in microwells plates. These experiments were visually observed after 3 weeks of incubation at 20° and used as pre-application experiments in order to create an expectation for the efficacy of the components in long term application experiments. The data that is shown in table 5 below is divided in “+” and “-“. The “+” means visible growth, while the “-“ means no visible growth of the mould cocktail at the specific concentrations. The experiment shows that 350ppm of potassium cinnamate still is not sufficient for inhibition of the growth of the mould cocktail. However, combined with an amount of nearly 400 ppm of nate and 75ppm of vanillin the model drink is stable for at least 3 weeks.

Table 5 W0 2013/058650 22 proionate (I) (O-O.15% Kcinnamate O-0.03 50% (w/v) (W/V))+ 0.00 0.00 0.01 0.01 0.02 0.02 0.03 0.03 Vanillin (II) (0-0.03% 00 50 00 50 00 50 00 50 (WM) 0.0000% (1) + 0.0000% 1 1 1 1 1 1 1 1 (11) 0.012323% (1)+0.00375% (11) 0.0375% (1)+0.0075% (11) 0.0563% (1)+0.01125% (11) 0.075% (1)+0.015% (11) 0.0938% (I)+0.01875% (11) 0.1125% (I)+0.02625% (11) 0.15% (1)+0.03% (11) 3. 4 Long term application experiment A solution to control carbonated beverages has been developed based on 200ppm of ium cinnamate and 300ppm in. The spectrum of this formulation could hend moulds by addition of propionate. As mentioned in table 4 the formulations existed of 100 or 200ppm ium cinnamate, 150 or 300ppm vanillin and propionate ranging from 250ppm to 1250ppm of propionate.

The data were obtained by observation of growth or a volume of l-20 ml was filtered and successively grown on agar in case no growth was observed.

Formulations 2 to 8 all were visually spoiled within several weeks. However, the other ations, including the sorbate benzoate control, showed a quick decrease of viable counts at levels of propionate 800ppm and higher in combination with 200ppm of potassium cinnamate and 300ppm vanillin. After 3 weeks not any count was observed in the total volume of the model drink ated with 103 CFU. After 10 months still no outgrowth of moulds can be observed in the s where 800ppm of propionate was used (sample 11, 12 and 13).

W0 2013/058650 23 Conclusions: Using certain blends of the selected ents, the combinations are very effective in inactivation of mould spores. The results show that combinations can achieve complete inactivation of spores. The potassium cinnamate, vanillin and propionate as individual antimicrobials are needed in higher concentration to inhibit the mould spores The susceptibility/resistance of different moulds to different antimicrobials is different and hence multiple species of relevant mould spores were evaluated as a cocktail in model drink against the proposed combinations and the microbial efficacy has been found consistent.

A vative system was developed able to-, but without the use of- benzoate and sorbate that can be used in ated as well as non-carbonated beverages.

Claims (19)

Claims

1. Preservative system comprising (i) propionic acid or a salt thereof, in combination with (ii) vanillin or a derivative thereof, and (iii) cinnamic acid or a salt thereof.

2. Preservative system according to claim 1, comprising a combination of (i) propionic acid or a salt f, in combination with (ii) vanillin and (iii) cinnamic acid or a salt thereof.

3. Preservative composition ing to claim 1 or 2, comprising vanillin in an amount 10 within the range of 1-10 wt%.

4. vative composition according to any one of the preceding claims, comprising cinnamate in an amount within the range of 5-50 wt%. 15

5. Preservative composition according to any one of the preceding claims, comprising propionate in an amount within the range of 5-50 wt%.

6. Preservative composition according to any one of the preceding claims wherein the ratio of (i) : (ii) is within the range of 1.0 : 0.1-0.5 and/or the ratio of (i) : (iii) is within the 20 range of 1.0 : 5.

7. Preservative composition according to any one of the preceding claims, comprising a fermentation broth as the propionate source. 25

8. Preservative composition according to any one of the preceding claims, containing less than 1 wt% of preservative agents selected from the group consisting of sorbic acid and benzoic acid.

9. Alimentary t, comprising an effective amount of (i) propionic acid or a salt 30 thereof, in ation with (ii) vanillin or a derivative thereof, and (iii) cinnamic acid or a salt thereof.

10. Alimentary product according to claim 9 comprising an effective amount of a combination (i) propionic acid or a salt thereof, in combination with (ii) vanillin and (iii) cinnamic acid or a salt thereof. 5

11. Alimentary product ing to claim 9 or 10, which is selected from the group consisting of beverages.

12. Alimentary product according to any one of claims 9-11, which is selected from the group consisting of non-carbonated beverages.

13. Alimentary product according to any one of claims 9-12, comprising vanillin in an amount within the range of 200-400 ppm; cinnamate in an amount within the range of 100- 300 ppm; and propionate in an amount within the range of 500-800 ppm. 15

14. tary product according to any one of claims 9-13, n the t is a beverage having a pH of at least 2 and below 5.5.

15. Use of a vative composition according to any one of claims 1-8, for the preservation of an alimentary product; for the inactivation of mould spores in an alimentary 20 product; and/or for inhibiting mould growth in an alimentary product.

16. Use ing to claim 15, wherein the mould is selected from the group of Fusarium oxisporum, Aspergillus niger, Byssoaclamys fuvla and Penicillium sp. 25

17. Preservative system according to claim 1, substantially as herein bed or exemplified.

18. Alimentary product according to claim 9, substantially as herein described or exemplified.

19. Use according to claim 15, substantially as herein described or exemplified. WO 58650 WO 58650 growth