Selective chromogenic medium

Abstract

The invention deals with chromogenic media which are suitable for the selective growth and identification of one or more species of yeast. The subject of the invention is the method that enables us to identify and determine the cell count of Brettanomyces/Dekkera and Zygosaccharomyces yeasts. Besides, the subjects of invention are also the use of the method in wine and/or food industry and the stocks for conducting the experiment.

Claims

1. A chromogenic medium suitable for the selective growth and detection of one or more yeast(s) from a yeast of the Brettanomyces and/or Dekkera genera, a yeast of the Zygosaccharomyces genus, and/or a yeast of the Lachancea genus, said medium comprising at least a nutrient suitable for the feeding and/or growing of said one or more yeast(s) to be detected, an agent capable of inhibiting the growth of Saccharomyces species, and a chromogenic stain, wherein the chromogenic stain is the combination of the following dyes, said combination being chromatic under visible light: multiple type(s) of substituted and/or unsubstituted bis-3,7 diaminophenothiazines comprising a mixture of methylene blue and Azure B, and one or multiple type(s) of substituted fluorescein(s), wherein the substituted fluorescein is selected from the group consisting of eosin Y, eosin B and any mixture thereof.

2. The selective and chromogenic medium according to claim 1, wherein said chromogenic stain comprises the mixture of methylene blue and Azure B, and of Eosin Y.

3. The selective and chromogenic medium according to claim 2 wherein said chromogenic stain is Azure II eosinate.

4. The selective and chromogenic medium according to claim 1, wherein said chromogenic stain is a mixture of the bis-3,7 diaminophenothiazines consisting of a mixture of methylene blue and Azure B and the one or multiple type(s) of substituted fluorescein(s) in the ratio of from 1.5:1 to 1:1.

5. The selective and chromogenic medium according to claim 1, comprising a gelling agent and being formulated as one of the following forms: solid powder, gelled culture medium, wherein the medium is in a pre-prepared, ready to use form.

6. The chromogenic medium of claim 1 which is a culture medium for said one or more yeast(s) to be detected.

7. The chromogenic medium of claim 1, wherein said agent is at a concentration which does not inhibit growth the one or more yeast(s) to be detected.

8. The chromogenic medium of claim 1, wherein the nutrient in the medium is capable of feeding said one or more yeast(s) to be detected.

9. The chromogenic medium of claim 1 further comprising water.

10. The chromogenic medium of claim 1, wherein the one or more yeast(s) to be detected is/are capable of naturally growing in said medium.

11. The chromogenic medium of claim 1 further comprising glucose, yeast nitrogen base and agar.

12. The chromogenic medium of claim 1, wherein said agent is a chemotherapeutic agent or an antibiotic.

13. A method for the detection or determination of one or more yeast species selected from the group as follows: yeasts of the Brettanomyces and/or Dekkera genera, yeasts of the Zygosaccharomyces genus, and yeasts of the Lachancea genus, from a foodstuff, a food processing intermediate and/or a food raw material, said method comprising the steps of providing the medium of claim 1, obtaining a sample from a foodstuff, a food processing intermediate and/or a food raw material, preparing the sample to add to the medium, optionally filtering and/or concentrating and/or enriching the sample in microorganisms, plating the sample or suitable part thereof on the medium, incubating the medium under conditions suitable for the culturing of said yeast species until colonies are obtained, detecting said yeast species by the discolouration of said colonies.

14. The method of claim 13, wherein said yeast is selected from a yeast of the Brettanomyces and/or Dekkera genera, a yeast species of the Zygosaccharomyces genus and a yeast species of the Lachancea genus, and when the colony is pink, it is considered to be the detection of a yeast of the Brettanomyces and/or Dekkera genera, and/or when the colony is blue, it is considered to be the detection of a yeast species of the Zygosaccharomyces genus, and/or when the colony is greenish blue with a pink edge, it is considered to be the detection of a yeast species of the Lachancea genus.

15. The method according to claim 13, wherein the number of detected colonies is determined relative to the amount of the volume or weight of the original sample, so that the method is quantitative.

16. The method according to claim 13, wherein the colonies and the number of the colonies are evaluated under UV light, by fluorescence.

17. The method according to claim 13, wherein the foodstuff prepared by fermentation, is beer or wine, the food processing intermediate is malt, must, fermenting malt or must, and/or the food raw material is grape.

18. The method according to claim 13, wherein the culture medium suitable for the culturing of said yeast is provided in a pre-prepared, ready to use solid or gel form.

19. Kit for use in the method according to claim 13 containing the medium and means of preparing a gel culture medium thereof.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Identification of wild yeasts along winemaking (noble) yeasts on selective medium

(2) The patterns assigned to sequence numbers are the following:

(3) 1: Dekkera bruxellensis CBS 73; 2: Pichia membranifaciens var. membranifaciens CBS 191; 3: Zygosaccharomyces bailii var. bailii CBS 4688; 4: Zygosaccharomyces bailii var. bailii CBS 4689; 5: Zygosaccharomyces mellis CBS 684; 6: Zygosaccharomyces rouxii CBS 441; 7: Lachancea fermentati CBS 707; 8: Issatchekia orientalis CBS 6799; 9: Brettanomyces custersianus CBS 4805; 10: Saccharomyces cerevisiae T-158C; 11: Saccharomyces cerevisiae S6; 12: Schizosaccharomyces pombe

(4) FIG. 2: colonies of Brettanomyces from a vinous media, on a selective, coloured medium

DETAILED DESCRIPTION OF THE INVENTION

(5) The present inventors unexpectedly found during the creation of the invention that if azure-II-eosinate or other chromogenic paint that contains chemically similarly structured molecules is added to a selective medium—which is suitable for growing yeast, but blocking the growth of Saccharomyces—then in the medium we get, we can identify Brettanomyces species among the appearing colonies, with the help of azure-II-eosinate.

(6) Quite surprisingly, the present inventors observed the following:

(7) 1. On this medium, the Brettanomyces colonies are painted pink, which are visible for the eyes

(8) 2. The pink colonies are fluorescent in ultraviolet light, which confirms the separation of the species.

(9) With the help of this method, the Brettanomyces/Dekkera colonies can be easily distinguished from other wild yeasts that are able to grow on a selective medium, by looking at them. The identification that is based on visibility, not only makes the identification process much easier, but it also does not require experience and scent samples, furthermore, it makes scenting samples unnecessary, which bears the possibility of spreading the infection. The reason why it is more specific to Brettanomyces/Dekkera species than other methods, is that it is able to identify other yeast species that are not winemaking (noble) yeasts, which (only) causes a problem in case of sweet wines with a higher residue of sugar.

(10) We have also run the experiments using different azure and eosin dyes. All of the stains'colours changed with the increase of pH. Yet, these stains did not provide the previously experienced colour reaction and were not appropriate to distinguish the species of Brettanomyces/Dekkera from other, examined yeasts.

(11) According to the invention, for getting the desired results, we need substituted or unsubstituted bis-3,7-diamino-phenothiazines together with the substituted derivatives of fluorescein.

(12) There was a medium stain that was known before with the same components, eosin and methylene blue, but it was not used for identifying yeast contamination. For example, the United Kingdom publication no. GB1248197 [ABBOTT LAB (US), “Diagnostic method and apparatus for the detection of bacteria”] discloses an eosin methylene blue agar medium that contains lactose, however, identifying the yeasts in the reference are not based on eosin methylene blue medium. For our understanding, previously they used the azure-II-eosin for other purposes, mainly for colouring tissue samples.

(13) According to the Japanese disclosure document no. JP56106588A they used eosin-Y (0.5 g, 7.23×10.sup.−4 mol) and methylene blue (0.065 g, 2.03×10.sup.−4 mol) in one medium only, however, the document does not provide any information about the different discolouration of the medium and/or the colonies as a result of growing different types of yeasts.

(14) The chromogenic stain to be used according to the invention is therefore the combination of at least one type of substituted or unsubstituted bis-3,7 diaminophenothiazine stain and at least one type of substituted fluorescein stain. Preferably, the combination of the substituted or unsubstituted bis-3,7 diaminophenothiazine stain of formula I herebelow and the substituted fluorescein stain of formula II herebelow.

(15) The chemical structure of the bis-3,7 diaminophenothiazine stain of formula I is

(16) ##STR00003##

(17) wherein

(18) R1, R2, R3 and R4 are independently H, methyl or ethyl, preferably H or methyl,

(19) Q1, Q2, Q3 and Q4 are independently H, C1-4 alkyl, halogen, pseudohalogen, —NO or —NO.sub.2, preferably H, methyl or ethyl, preferably H or methyl, most preferably H.

(20) Preferably, the bis-3,7 diaminophenothiazine stain of formula I is present in a cationic form, such as a salt formed with an anion. The anion is preferably a halide ion, most preferably chloride ion. According to a variation, the anion is formed by the substituted fluorescent stain of formula II.

(21) The chemical structure of the substituted fluorescent stain of formula II is

(22) ##STR00004##

(23) wherein R1, R2, R3 és R4 are independently halogen, pseudohalogen, —NO or —NO.sub.2,

(24) Q1 and Q2 are H, C1-4 alkyl, C1-4 alkoxy, halogen, pseudohalogen, —NO or —NO.sub.2, 5- or 6-member heterocycle or Q1 and Q2 together form a 5- or 6-member heterocycle, in which case Q1 and Q2 are situated on adjacent C atoms.

(25) Preferably, R1 and R4 are halogen, more preferably Br or —NO.sub.2.

(26) Preferably, R2 and R3 are halogen, more preferably Br.

(27) Preferably, Q1′ is H, methyl or halogen and Q2 is H.

(28) Preferably, the substituted fluorescein stain of formula II is used in an anionic form, preferably in the form of a salt formed with a cation. Preferably, the cation is sodium ion, potassium ion or ammonium ion. According to a further preferred variation, the cation is formed by the bis-3,7 diaminophenothiazine stain of formula I.

(29) Preferably, the chromogenic medium of the invention comprises the at least one type of substituted or unsubstituted bis-3,7 diaminophenothiazine stain and the at least one type of substituted fluorescein stain in essentially identical molar amounts, i.e. the amount of the at least one type of substituted or unsubstituted bis-3,7 diaminophenothiazine stain and the amount of the at least one type of substituted fluorescein stain in the culture medium are at most 50% or 30%, preferably at most 20% or 10% different relative to the component that is present in smaller amount, that is, the ratio of the molar amounts is from 1.5:1 to 1:1 or from 1.3:1 to 1:1, preferably from 1.2:1 to 1:1 or from 1.1:1 to 1:1, most preferably the rate of the molar amounts is 1:1 or vice versa.

(30) Most preferably, the culture medium of the invention contains multiple types of substituted or unsubstituted bis-3,7 aminophenothiazine stains, in the general formula I of which R1, R2, R3 and R4 are H, methyl or ethyl so as that the substituents R1, R2, R3 and R4 are different in the different stains (R1, R2, R3 and R4 may not be identical). Most preferably, R1, R2, R3 and R4 are H or methyl and the bis-3,7 diaminophenothiazine component stains are different in the degrees of methylation.

(31) Accordingly and preferably, methylene blue and the demethylated intermediers thereof or the mixture thereof may be used in the stain, selected from

(32) a 3,7-bis(dimethylamino)-phenothiazin-5-ium salt, preferably acetate or chloride (methylene blue),

(33) a N-methyl,N′,N′-dimethylphenothiazin-5-ium-3,7-diamine salt, preferably acetate or chloride (Azure B),

(34) a N′,N′-dimethylphenothiazin-5-ium-3,7-diamine salt, preferably acetate or chloride (Azure A: CAS 531 533)

(35) a N-methylphenothiazin-5-ium-3,7-diamine salt, preferably acetate or chloride (Azure C),

(36) a phenotiazin-5-ium-3,7-diamine salt, preferably chloride or acetate (thionine).

(37) Most preferably the mixture of Azure B and methylene blue is present in the stain.

(38) Chemical formula of methylene blue:

(39) ##STR00005##

(40) Similarly, the substituted fluoresceins may be of one or more types. Most preferably, an eosin stain or a mixture of eosin stains is used, which may preferably comprise for example Eosin B or Eosin Y.

(41) ##STR00006##

(42) The eosin stain is preferably Eosin B or Eosin Y, the formulas of which are, respectively

(43) ##STR00007##

(44) Most preferably, the stain used according to the invention is Azure II eosinate. Azure II eosinate (CAS 53092-85-6) is a mixture of methylene blue and Azure B in the ratio of 1:1 and of eosin Y. Azure II eosinate is available from various manufacturers (such as Fluka, Sinopharm, CN és Nile Chemicals, Ind.).

(45) It is apparent for the skilled artisan that further substituted variants or salts of the stains of the inventions may be used, provided they are chromatic and the colour changes in the presence of yeast.

(46) Considering growth media, any growth medium being suitable for culturing yeasts and containing at least an agent which inhibits the growth of Saccharomyces strains can be used, e.g. growth media disclosed in the background of the invention.

(47) Based on the above, the invention concerns a method for selective culturing of Brettanomyces/Dekkera yeasts and differential staining of their colonies, where a growth medium suitable for culturing yeast cells is prepared, which is made selective by the addition of an appropriate chemotherapeutic agent or antibiotic inhibiting the growth of Saccharomyces strains and by the addition of chromogenic stain of the invention.

(48) The culture medium may be of varied composition. Theoretically, any growth medium suitable for culturing yeasts is appropriate and known by a person skilled in the art. The growth medium preferably contains ingredients selected from the following group: sugar, e.g. glucose; aminoacid- or peptid-containing extract or hydrolizate, such as pepton, yeast extract, “yeast nitrogen base” or “yeast carbon base; geling agent, e.g. agar; and optionally salt. Highly preferably, the growth medium comprises glucose, yeast nitrogen base and agar.

(49) Additionally, the growth medium also contains substances inhibiting the reproduction of microbes having a role in the normal or healthy fermentation of foodstuff. Provided the foodstuff in which the detection method is performed is a foodstuff prepared by fermentation, the growth-inhibiting substance feasibly prevents the growing of microorganisms performing the natural fermentation of the foodstuff, e.g. it blocks the growing of noble yeast. It is obvious for a person skilled in the art that the growth inhibitor should be applied at least in such a concentration which is already sufficient enough to block the growth of such microorganisms. At the same time the inhibitor concentration may have an upper threshold not to inhibit the growth of yeasts, the presence of which is desired to be tested. Preferably, the foodstuff is a foodstuff fermented by Saccharomyces species, such as beer, wine or other yeast containing product or intermediate, and the agent inhibiting the growth of Saccharomyces strains is an appropriate chemotherapeutic agent or antibiotic, e.g. cycloheximide applied in a concentration, e.g. of 0.5-50 μg/ml, preferably 1-20 μg/ml, particularly preferably 2-10 μg/ml, highly preferably something like 5 μg/ml, thereby the selectivity of the growth medium is enhanced or it is made selective.

(50) The sample may be any sample used in the production of such foodstuff, e.g. a sample taken from devices used in the process or a sample drawn from the liquid used for cleaning the devices.

(51) The prepared culture medium is brought to a form suitable for sample application. According to a certain variation a gel is prepared and a plate is poured into, e.g., a Petri dish. Alternatively, any other solid (e.g., in a form of gel) culture medium can be applied where the sample can be plated and the progeny (e.g. colonies) of a single cell can be separated.

(52) In addition to Petri dishes any other culturing container having large surface can be preferably used, where the sample can be spread on the surface of medium formed in it, and it can be closed (e.g., has a lid) and in which the microorganism colonies can be detected and feasibly visualized. It is preferable for the culturing container to be made of glass or plastic, more preferably plastic, and preferably it has a transparent lid.

(53) Kolle dishes or Roux flaks may also be used, they also have large surfaces but the sample should be introduced into the dish through a small opening and performing uniform plating also presents difficulties. Then the opening should be closed in a way that allows some aeration but the sample does not get uncontaminated.

(54) Consequently, according to the invention, sterilisable culturing dishes with lid may be used, in which samples to be tested can be plated on a large surface.

(55) From the samples (e.g., water used for washing barrels or other surfaces) or from their suitable dilutions a predetermined amount is plated on the surface of culture media then they are incubated at 10-37° C., preferably at 20-30° C., particularly preferably at room temperature for about 5-20 days, preferably for 8-16 days, and highly preferably for 10-14 days. It is obvious for a person skilled in the art that the incubation time is necessarily longer at lower temperatures.

(56) On culture medium prepared according to the invention, Saccharomyces yeasts stop growing, and the colour of Brettanomyces/Dekkera colonies become pink and they can be discriminated from microorganisms which are not harmful or just slightly harmful to the wine. The results are evaluated visually. In the event of sample application, the result can be made quantitative by giving the number of cultivable yeast cells per 1 ml.

(57) Detection sensitivity of Brettanomyces/Dekkera yeasts may be enhanced by filtering a higher amount of wine through membranes with 0.45 μm or 0.22 μm pore size, then by placing the membrane on the surface of the culture medium. In this case it should be ensured that no air bubbles are present between the membrane and the agar surface.

(58) Furthermore, the invention relates to culture media for performing the above method where the medium is in a powder or in a ready-to-use form, and also to the kits containing them and other components necessary for performing the examination (e.g. sample application devices) and the user instructions as well. Preferably, the reagent kit of the invention comprises the culture medium necessary for performing the method of the invention in the form and amount pre-weighed for each test and in a form poured into plastic Petri dishes in advance.

(59) The method developed by us is cheap and it does not require special instrumentation and easy to perform by anyone. The procedure requires no sterile laboratory conditions and only little attention is to be paid to ensure that the right sample is placed on the surface of the culture medium. The culture medium contains components easily available. In addition to components used for growth, the medium contains antibiotics inhibiting the growth of yeasts, e.g.—other culture media similar to selective Brettanomyces—cycloheximide as well. This antibiotic is used for the identification of different species in yeast diagnostics. In the concentration used by the inventors, it prevents the growth of most yeasts playing a role in wine-making (e.g. Saccharomyces) while this concentration is still tolerated by the species causing the degradation of wine.

(60) The present invention is further illustrated, but not limited by the following examples.

EXAMPLES

(61) In the following examples, unless indicated otherwise, the following concentrations and compositions were applied. Composition of medium that was suitable for growing yeast cells was the following: 1% glucose, 0.67% “yeast nitrogen base”, 2% agar, which was made selectively by using 5 μg/ml cycloheximide as an antibiotics for blocking the growth of Saccharomyces strains. Azure-II-eosinate was used in a 30 μg/ml concentration.

Example 1: Identifying Brettanomyces/Dekkera Species from Must

(62) We make a 10 scale dilution sequence in 5 steps from destilled water that we gained from must. From each dilution we streak 50 μl onto the surface of the selective, chromogen medium in the Petri dishes. We make the grafting in three parallel running measurements. We incubate the Petri dishes between 20-25° C. for 10-14 days. The pink colonies that appear on the surface of the medium after the incubation time is over imply the Brettanomyces/Dekkera infection. We choose the dishes in which we can easily identify the number of colonies. If we multiply the number of colonies by twenty, plus the value of the dilution we get the plate count of the Brettanomyces/Dekkera of the must applied to 1 ml.

(63) A positive dish can be examined under UV light as well. The fluorescence of the colonies confirms the obtained results.

Example 2: Identifying Brettanomyces/Dekkera Species from Bottled Wine

(64) We shake up the wine before taking a sample, then we filtrate 500 ml of it through a membrane filter with 0.45 μm pore diameter. We place the membrane filter on the surface of the selective chromogenic medium in the Petri dish, in a way that it fits properly (there should be no air bubble between them). The Petri dishes are incubated on 20-25° C. for 10-14 days.

Example 3: Identifying Brettanomyces/Dekkera Species From Red Wine Stored in Barrels

(65) We centrifugate 50 ml from the red wine in the barrel (3000 rpm, 10 min, Hereus Multifuge 3S). We suspend the pellet in 1 ml destilled water. From the suspension we streak 100 μm on the surface of the selective chromogenic medium in the Petri dish. The Petri dishes are incubated on 20-25° C. for 10-14 days.

Example 4: Identifying Brettanomyces/Dekkera Species from Barrels

(66) After washing the barrels, we filtrate 500 ml from the wash water through a membrane filter with 0.45 μm pore diameter. We place the membrane filter on the surface of the selective chromogenic medium in the Petri dish, in a way that it fits properly (there should be no air bubble between them). The Petri dishes are incubated on 20-25° C. for 10-14 days.

Example 5: Identifying Brettanomyces/Dekkera Species from Grapes

(67) We gently shake the grapes that are soaked in destilled water for an hour on room temperature. Meanwhile the cells from the grapes are being washed in the water. After this, we pour out the water from the grapes and filtrate it through a membrane filter with 0.45 μm pore diameter. We place the membrane filter on the surface of the selective chromogenic medium in the Petri dish, in a way that it fits properly (there should be no air bubble between them). The Petri dishes are incubated on 20-25° C. for 10-14 days.

Example 6: Identifying Zygosaccharomyces bailii from Bottled Sweet Wines

(68) We filtrate 500 ml from the bottled sweet wine through a membrane filter with 0.45 μm pore diameter. We place the membrane filter on the surface of the selective chromogenic medium in the Petri dish, in a way that it fits properly (there should be no air bubble between them). The Petri dishes are incubated on 30° C. for 10-14 days. The appearing blue colonies show a positive result.

Example 7: Identifying the Level of Infectivity of Collective Strains of Brettanomyces/Dekkera, Zygosaccharomyces Bailii and Lachancea fermentatii

(69) We suspend 1 loop from the culture in 5 ml destilled water. From the suspension we streak it on the surface of the differentiating medium with the loop. The Petri dishes are incubated on 20-25° C. for 10-14 days. The appearing blue Zygosaccharomyces bailii colonies, the pink Brettanomyces/Dekkera, and the greenish blue Lachancea fermentatii with the pink edge indicate infection.

Example 8: Recovering Pure Culture of Brettanomyces/Dekkera Strains in Culture Collection

(70) We suspend 1 loop of the infected culture in 5 ml destilled water. With the loop we streak on the differentiating medium from the suspension. The Petri dishes are incubated on 20-25° C. for 10-14 days.

(71) We make a suspension from the appearing pink colonies (1 loop/5 ml sterile destilled water), and from the suspension we streak on the differentiating medium with a loop. The Petri dishes are incubated on 20-25° C. for 10 days. If we do not observe other colonies apart from the pink ones, we can be ascertained about the purity of the culture.

Example 9: Testing Other Azure and Eosin Stains (Reference Example)

(72) We conducted the experiment according to example X. with the following stains as well:

(73) Stain

(74) Azure A (Azure A chloride)

(75) Azure B

(76) Azure II

(77) Eosin B

(78) Eosin Y

(79) The colour of the applied stains change in each case with the increase of pH. The stains were not suitable for clearly separating the species of Brettanomyces/Dekkera from the other, examined yeasts.

Example 10: Testing Media

(80) We conducted the experiment according to example X with the following media:

(81) YPD (1% glucose, 1% pepton, 0.5% yeast extract, 2% agar)

(82) YNB (1% glucose, 0.67% yeast nitrogen base, 2% agar)

(83) YCB (0.5% ammonium sulfate, 1.17% yeast carbon base, 2% agar)

(84) We experienced the most contrasted pink/blue discolouration on YNB medium in case of the azure II-eosin.

INDUSTRIAL APPLICABILITY

(85) The process and the media specified by the invention can be advantageously applied in the first place to monitor cell counts of Brettanomyces/Dekkera, identify or exclude their proliferation, as well as to detect for a hygienic purpose Brettanomyces/Dekkera yeasts responsible for the deterioration of wines and provisions in case of utensils used for storage with which they can get directly into contact. The usage of the medium makes the early identification of the growth of Brettanomyces/Dekkera yeasts—that can trigger the deterioration of food and wine—possible, as well as verifying the effect of the treatments that are aimed at avoiding deterioration. The method can identify other microorganisms, such as yeast species belonging to the Zygosaccharomyce genus and Lachancea genus.

(86) An advantage of this method is that it makes it possible to easily identify the colonies of Brettanomyces/Dekkera by looking at them, furthermore, colonies of yeast species belonging to the Zygosaccharomyces and Lachancea genus can be distinguished from the colonies, other species and wild yeasts that are able to grow on a selective medium. The identification that is based on visibility, not only makes the identification process much easier, but it also does not require experience and scent samples, furthermore, it makes scenting samples unnecessary, which bears the possibility of spreading the infection. The reason why it is more specific to Brettanomyces/Dekkera species than other methods, is that it is able to identify other yeast species that are not winemaking (noble) yeasts, which causes a problem in case of sweet wines with a higher residue of sugar.

(87) The method that we developed is cheap, it does not require special instruments, anyone can carry it out. Circumstances of a sterile laboratory are not necessary, it only requires minimal attention to put the right sample onto the surface of the medium. The medium contains easily accessible components.

REFERENCES

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