Title of Invention: BEVERAGE EXHIBITING MICROBIAL GROWTH INHIBITION

Technical Field

[0001] The present invention relates to a beverage containing one or more specific compounds. More specifically, the present invention relates to a beverage exhibiting microbial growth inhibition by containing one or more specific compounds derived from natural products. And more specifically the present invention relates to a beverage exhibiting microbial growth inhibition and having a commercial value of a beverage product, such as stability of appearance.

Background Art

[0002] A preservative is an additive which improves preservability by inhibiting microbial growth responsible for the spoilage and deterioration of food and drink. Synthetic preservatives or synthetic antimicrobial agents such as sodium benzoate and potassium sorbate are usually used as a preservative for beverage. However, synthetic preservatives have been recently shown to have impacts on the gut microbiota, and the concern is the effects on health. Furthermore, when reacted with vitamin C, sodium benzoate is considered to carry a risk of generation of cancer-causing benzene. In addition to the effects on health, sodium benzoate has a distinctive astringency (Patent Literature 1) which may affect the flavor of beverage. Furthermore, potassium sorbate has the problem of low effects on inhibiting growth of specific microorganisms and a characteristic odor.

[0003] To reduce the use of such synthetic preservatives, preservatives derived from natural products have been studied (Patent Literature 2, Patent Literature 3). Compounds derived from natural products, such as chitosan, are known to have antimicrobial properties. Citation List

Patent Literature

[0004] PTL 1 : Japanese Patent Laid-Open No. 2020-171301

PTL 2: Japanese Patent Laid-Open No. H05-7482 PTL 3: Japanese Patent Laid-Open No. H02-211856

Summary of Invention

Technical Problem

[0005] Although there are some studies on preservatives derived from natural products, few of them have verified that the preservative actually inhibits microbial growth in beverage. Furthermore, fewer reports have proved that the commercial value of beverage, such as stability of appearance and flavor, is also not lost. With variety of preferences of recent consumers, types of beverage products have been diversified, and thus to respond to many different properties and flavors of a wide variety of beverage products, exploring a novel preservative which is derived from natural products and suitable for use in beverage is desired.

Solution to Problem

[0006] The present inventors have explored compounds derived from natural products suitable for use as a preservative for beverage products (microbial growth inhibitor) and as a result have found 6 compounds. The inventors have conducted a test to observe microbial growth by actually adding these compounds to commercially available beverages, and then have found that the compounds have an effect on inhibiting microbial growth. Furthermore, the present inventors have found that these compounds have stability of appearance (a property of not causing change in appearance over time), which is required for beverage products, and have little effect on the original color and/or taste of beverage, and have arrived at the present invention. More specifically, the 6 compounds are represented by formulae 1 to 6 described in the section of "DESCRIPTION OF EMBODIMENTS" below.

[0007] The present invention relates to a beverage comprising any one or more of compounds represented by formulae 1 to 6, or salts thereof. The present invention relates to a beverage comprising any one or more of compounds represented by formulae 1 to 6, or salts thereof at a total concentration of 0.1 to 250 ppm. The present invention also relates to the above beverage in which microbial growth is more inhibited when a microorganism is added to the beverage and the beverage is stored at room temperature for 2 weeks, as compared with that in a beverage with the same composition except for not containing any of the compounds represented by formulae 1 to 6 or a salt thereof. The microorganism is preferably a yeast and/or mold, and more preferably any one or more of the genus Zygosaccharomyces, the genus Saccharomyces, the genus Brettanomyces and the genus Neosartorya. Further, the present invention relates to the above beverage in which the growth of the microbial, genus Alicyclobacillus is more inhibited, as compared with that in a beverage with the same composition except for not containing any of the compounds represented by formulae 1 to 6 or a salt thereof. The present invention relates to a method for producing a beverage, comprising adding any one or more of compounds represented by formulae 1 to 6, or salts thereof. Furthermore, the present invention relates to a method for producing a beverage, comprising adding any one or more of compounds represented by formulae 1 to 6, or salts thereof at a total concentration of 0.1 to 250 ppm. The compounds represented by formulae 1 to 6 are all derived from natural products, but actual addition of the compounds to a beverage product to verify the effect on inhibiting microbial growth and commercial value of the beverage have not been reported before as far as the present inventors know.

Advantageous Effects of Invention

[0008] Since the beverage of the present invention comprises any one or more of compounds represented by formulae 1 to 6, or salts thereof, microbial growth in the beverage is inhibited, and the beverage has high preservability. All of the compounds represented by formulae 1 to 6 are derived from natural products. The beverage of the present invention comprising any one or more compounds represented by formulae 1 to 6 has the advantage of having an effect on inhibiting microbial growth in a beverage without using a synthetic preservative or with using a reduced amount of the synthetic preservative, for which a synthetic preservative such as sodium benzoate and potassium sorbate must have been used. When a synthetic preservative is used, the synthetic preservative is usually added to a beverage at about 200 to 400 ppm, but the compounds represented by formulae 1 to 6 or a salt thereof have the effect on inhibiting microbial growth even at the same or lower concentration than that of a synthetic preservative. One of the compounds represented by formulae 1 to 6 or a salt thereof may be added to a beverage, or two or more of them may be added thereto in combination. The compound to be used may be selected depending on the type and the flavor of the beverage.

[0009] The compounds represented by formulae 1 to 6 have the advantage of not reducing the commercial value of beverage (e.g., stability of appearance, color and taste) when added to the beverage. Chitosan, for example, has been known as an antimicrobial substance derived from a natural product, but is likely to cause cloudiness over time depending on the type of beverage (in other words, chitosan has slightly low stability of appearance). When the compounds of formulae 1 to 6 are used at a concentration at which they have antimicrobial effects, they sometimes have appearance stability similar to or higher than that when chitosan is used at the same concentration. Furthermore, when the compounds of formulae 1 to 6 are used at a concentration at which they have antimicrobial effects, they have little effect on the original color of the beverage (have low discoloration) or have little effect on the taste.

Description of Embodiments

[0010] The present invention relates to a beverage comprising any one or more of compounds represented by formulae 1 to 6 or salts thereof. In the following, the compound represented by formulae 1 is abbreviated as "compound 1," and the compound represented by formula 2 is abbreviated as "compound 2." All of compounds 1 to 6 are compounds derived from natural products.

[0011] Compound 1 (CAS No. 867578-35-6), compound 5 (CAS No. 1427039-33-5), and Compound 3 (CAS No. 1427028-86-1), are compounds having the following formulae 1 to 3, respectively and are compounds extracted from culture medium of Ustilago maydis, which is a type of mold growing on com and the like.

[Chemical Formula 1]

[Chemical Formula 3] formula 3

[0012] Compound 4 (C51H82O21) (CAS No. 68124-04-9) is a compound having the following formula 4 and is a compound extracted from the root of Dioscorea mexicana. [Chemical Formula 4]

[0013] Compound 5 (C46H74O16XCAS No.30994-75-3) is a compound having the following formula 5 and is a compound extracted from the leaf of Securinega leucopyrus.

[Chemical Formula 5] [0014] Compound 6 (CAS No. 168778-19-6) is a compound having the following formula 6 and is a compound extracted from the root of Agave americana.

[0015] Compounds 1 to 6 may be obtained by extraction and purification from the culture medium of microorganisms or plant described above. A commercially available product of each of the compounds may also be used and contained in the beverage of the present invention. Alternatively, each of the compounds may be synthesized by any method.

[0016] Compounds 1 to 6 may be in the form of any salt in the beverage.

[0017] (Beverage)

The beverage of the present invention comprises any one or more of the above compounds 1 to 6 or a salt thereof. One of compounds 1 to 6 or a salt thereof may be used, or two or more of them may be used in combination, depending on the type of the beverage or the desired level of the effect on microbial growth inhibition, and taking the impact on the flavor of the beverage into consideration.

[0018] The amount of each of the compounds used in the beverage is such that each of them has a microbial growth inhibitory effect in the beverage, and is not particularly limited. For example, the lower limit is preferably 0.1 ppm or more, more preferably 0.5 ppm or more, further preferably 1.0 ppm or more, still more preferably 10 ppm or more, still further preferably 20 ppm or more, and still further preferably 30 ppm or more in terms of the total concentration of compounds 1 to 6 or a salt thereof. It is preferable to set the amount of compounds 1 to 6 in the beverage as little as possible in the range in which a desired effect is obtained in order to minimize the impact on the flavor and appearance of the beverage. For example, the upper limit is preferably 400 ppm or less, more preferably 300 ppm or less, further preferably 250 ppm or less, still more preferably 125 ppm or less, still further preferably 100 ppm or less and yet more preferably 50 ppm or less.

[0019] As described later, the beverage of the present invention may be a ready-to-drink beverage or a concentrated beverage which is to be diluted to drink. In the case of a concentrated beverage, compounds 1 to 6 or a salt thereof may be used to meet the above content in the concentrated beverage in order to obtain a microbial growth inhibitory effect in a concentrated state during storage. Alternatively, to obtain a microbial growth inhibitory effect in a diluted state during storage to be prepared from the concentrated beverage, compounds 1 to 6 or a salt thereof may be contained in the concentrated beverage in an amount calculated by multiplying the above content by a predetermined degree of dilution of the beverage so as to meet the above content in a diluted state, depending on the predetermined degree of dilution of the concentrated beverage.

[0020] Conventional synthetic preservatives such as sodium benzoate and potassium sorbate are used in beverage at a concentration of usually about 200 to 400 ppm. Compounds 1 to 6 or a salt thereof may achieve a microbial growth inhibitory effect in beverage at the same or lower concentration than that of a conventional synthetic preservative. Furthermore, compounds 1 to 6 has a microbial growth inhibitory effect at the same or lower concentration than that of chitosan, which is known as an antimicrobial substance derived from a natural product, and compounds 1 to 6 are unlikely to reduce the commercial value of beverage products such as stability of appearance.

[0021] The method for measuring the content of each of the compounds in the beverage is not particularly limited. For example, a standard product of the compounds may be prepared and the content may be measured using HPLC and the like while setting conditions depending on the type of beverage. [0022] The type of beverage to which one or more of compounds 1 to 6 or a salt thereof is added is not particularly limited. Examples thereof include carbonated beverages, fruit juice beverages, vegetable juice beverages, coffee beverages, tea beverages, cocoa beverages, jelly beverages, lactic beverages, probiotic beverages, soy milk, energy drinks, sports drinks, alcoholic beverages and concentrated beverages which are to be diluted to drink. Of them, acidic beverages with a pH of 2 to 5 (preferably pH 2.5 to 4.5) are preferred as a beverage containing one or more of compounds 1 to 6 or a salt thereof since synthetic preservatives have been frequently used for them. Examples of acidic beverages include carbonated beverages, fruit juice beverages, vegetable juice beverages, probiotic beverages, energy drinks and sport drinks. The beverage may be a ready-to-drink beverage or a concentrated beverage which is to be diluted to drink. The type of fruit juice is not particularly limited and examples thereof include fruit juice of citrus fruits (lemons, grapefruit, limes, oranges, mandarin oranges, Citrus unshiu, tangor, Citrus natsudaidai, sweet summer oranges, Citrus hassaku, Citrus tamurana, Citrus depressa, Citrus sudachi, Citrus junos, Citrus sphaerocarpa, Citrus aurantium, Citrus iyo, Citrus reticulata, kumquat, Citrus sulcate, oroblanco and Citrus maxima'), blackcurrant juice, apple juice, grape juice, peach juice, tropical fruit juice (e.g., pineapples, guavas, bananas, mangoes, acerola, papayas, passion fruit and lychees), Japanese apricot juice, pear juice, apricot juice, plum juice, berry juice, kiwi fruit juice, cherry juice, chestnut juice, water melon juice, tomato juice, carrot juice, strawberry juice and melon juice.

[0023] The beverage of the present invention is preferably a beverage in which any one or more of compounds 1 to 6 or salts thereof are used instead of all or a part of a synthetic preservative which has been conventionally used in beverage. Compounds 1 to 6 or a salt thereof is preferably intentionally added to beverage separately from usual ingredients of the beverage so as to increase a microbial growth inhibitory effect in the beverage.

[0024] Usually, a beverage contains a wide variety of ingredients, and thus even a substance which has been generally known to have a growth inhibitory effect on microorganism cultured in culture medium does not necessarily have the same effect in the beverage. For example, a substance difficult to be dissolved in a beverage or a substance which reacts with other components in the beverage cannot always inhibit microbial growth in the beverage even if they are effective for microorganism in culture medium.

Furthermore, pectin and polysaccharide in fruit juice and the like, for example, may surround coexisting compounds to reduce the antimicrobial effect. To deal with this, in Examples described later, the growth inhibitory effect of compounds 1 to 6 on various microorganisms has been actually observed using existing carbonated beverages and fruit juice beverages. [0025] The beverage of the present invention is a beverage more inhibiting microbial growth after adding a microorganism thereto and storing at room temperature for 2 weeks, as compared with a beverage (comparative beverage) with the same composition except for not containing compounds 1 to 6 or a salt thereof. As used herein, the microorganism means one responsible for the deterioration and spoilage of beverage, and examples thereof include yeasts and molds. Typical examples of yeasts and molds responsible for the deterioration and spoilage include yeasts of the genus Zygosaccharomyces, the genus Saccharomyces or the genus Brettanomyces and molds of the genus Neosartorya. In the beverage of the present invention, preferably growth of one or more of those microorganisms is more inhibited as compared with the above comparative beverage. Further, as a microorganism responsible for the deterioration and spoilage of beverage, Thermo-Acidophilic Bacilli having high heat resistance in a high acidic region (for example, the genus Alicyclobacillus) is known. Preferably, the beverage of the present invention is a beverage more inhibiting growth of the Thermo- Acidophilic Bacilli, as compared with the comparative beverage as described above.

[0026] The expression "microbial growth is more inhibited" as compared with the comparative beverage means that microbial growth responsible for the deterioration and spoilage as described above is less than that in the above comparative beverage. Less microbial growth after adding the microorganism and storing at room temperature for 2 weeks than that in the comparative beverage shows that the growth of the microorganism added to the beverage has been inhibited because of the presence of compounds 1 to 6 or a salt thereof in the beverage. The procedure of "adding the microorganism and storing at room temperature for 2 weeks" is carried out for the purpose of testing microbial growth in the beverage of the present invention (test for observing microbial growth). No such microorganism is of course added to beverage to be shipped as a regular product. In the test for observing microbial growth, the amount of the microorganism added is about

10,000 cfu/ml in the case of yeast, and about 15 to 70 cfu/ml in the case of mold. The room temperature means 10 to 30°C, and preferably about 25 to 28°C. In some cases, depending on the type of the microorganism, it is more desired that a temperature other than the room temperature is used in the test. Examples of specific methods of the test for observing microbial growth include the method shown in Examples described later.

[0027] The beverage of the present invention is preferably a beverage that is not degraded or spoiled after adding a microorganism thereto and storing at room temperature for 2 weeks, because microbial growth is very little. The beverage of the present invention is more preferably a beverage in which no microorganism grows under such conditions.

[0028] Furthermore, the beverage of the present invention preferably has stability of appearance such that the commercial value of the beverage is not reduced. The stability of appearance means that the appearance of beverage is unlikely to be changed over time. For example, the stability of appearance means that the change in the appearance of beverage is small when the beverage opened is stored at room temperature for about 2 weeks.

Furthermore, the beverage of the present invention is preferably a beverage whose color and taste are not changed much from the original color and taste of the beverage (the color and taste of a beverage with the same composition except for not containing compounds 1 to 6 or a salt thereof), although the beverage contains compounds 1 to 6 or a salt thereof.

[0029] (Method of production)

The present invention also provides a method for producing a beverage, comprising adding any one or more of compounds 1 to 6 or salts thereof. The method of the present invention comprises a step of adding any one or more of compounds 1 to 6 or salts thereof in the usual steps of producing a beverage. The timing of addition of each of the compounds is not particularly limited and each of them may be added thereto at any stage in the steps of producing the beverage. For example, compounds 1 to 6 or a salt thereof may be added to and mixed with the beverage at the time of addition and mixing of usual ingredients of beverage, or compounds 1 to 6 or a salt thereof may be added thereto and mixed therewith after usual ingredients have been mixed. It is preferable that compounds 1 to 6 or a salt thereof to be added is obtained by extraction and purification of the natural product described above (e.g., plant or microbial culture medium). Alternatively, a commercially available product of each of the compounds may be used. Each of the compounds may also be synthesized by any method. Compounds 1 to 6 or a salt thereof to be added may be in the form of solid such as powder, or liquid prepared by dissolving or suspending the compound in a solvent. The method of addition of each of the compounds is not particularly limited, and a method of addition and mixing of ingredients in usual production of beverage may be used.

[0030] The amount of compounds 1 to 6 or a salt thereof to be added may be such that the compounds have a microbial growth inhibitory effect in the beverage produced, and is not particularly limited. For example, the lower limit is preferably 0.1 ppm or more, more preferably 0.5 ppm or more, further preferably 1.0 ppm or more, still more preferably 10 ppm or more, still further preferably 20 ppm or more, and still further preferably 30 ppm or more in terms of the total concentration of compounds 1 to 6 or a salt thereof. Furthermore, as described above, the upper limit is preferably 400 ppm or less, more preferably 300 ppm or less, further preferably 250 ppm or less, still more preferably 125 ppm or less, still further preferably 100 ppm or less and yet more preferably 50 ppm or less.

[0031] The type of beverage is not particularly limited. Examples of beverages are as described above. As described above, the above content may be adjusted depending on if the beverage is a ready-to-drink beverage or a concentrated beverage, and in the case of a concentrated beverage, depending on whether a microbial growth inhibitory effect in a concentrated state is intended or a microbial growth inhibitory effect in a diluted state is intended. Examples

[0032] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. In the present description, the numerical range includes the endpoints unless otherwise specified.

[0033] (Yeast growth test 1)

Using a commercially available carbonated beverage containing 1% lemon juice (containing saccharides (high fructose com syrup and sugar), lemon juice, carbonic acid, flavoring, vitamin C, acidulant, Carthamus yellow, calcium pantothenate, vitamin B6, carotene pigment) and a commercially available carbonated beverage containing 12% orange juice (containing fruit (oranges, lemons, mandarin oranges, and grapefruit), saccharides (sugar and high fructose com syrup), fruit fiber, orange peel extract, carbonic acid, flavoring, antioxidant (vitamin C)), the growth inhibitory effects of compounds 1 to 6 and comparative chitosan on various yeasts in the respective beverages were observed. The specific procedures are as follows.

[0034] The above respective beverages were opened, and the carbonated beverages were left until flat, and then the pH of the beverages was adjusted to 3.3 using aqueous sodium hydroxide. Each of compounds 1 to 6 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/mL, and the resulting solution of each of the compounds was added to the beverages so that the concentration of each of the compounds was 260.3 ppm in the beverages. For comparison, a beverage was prepared by adding an aqueous solution containing chitosan at 20 mg/mL and ascorbic acid at 20 mg/mL to the above beverages so that the concentration of chitosan was 260.3 ppm in the beverage. A 96-well microplate was prepared, and a 240-pL aliquot per well of each of the above beverages to which each of compounds 1 to 6 was added and the beverage to which chitosan was added was pipetted. [0035] A 10,000 cfu/mL solution of each of three types of yeasts (Zygosaccharomyces sp., Saccharomyces sp., Brettanomyces sp.) was prepared.

[0036] 10 pL per well of the solution of each of the yeasts was added to three wells to which the beverage containing any of compounds 1 to 6 or chitosan was added (that is, a test group of 3 wells was prepared per compound and yeast). Furthermore, 10 pL of normal saline was added to three wells out of the wells to which the beverage containing compounds 1 to 6 or chitosan was added as a blank. Then the final concentration of compounds 1 to 6 and chitosan was 250 ppm in the respective wells. Furthermore, a group was prepared by adding 10 pL of a solution of each of the yeasts to 240 pL of a beverage to which no compounds 1 to 6 or chitosan were added as a group to which no compound was added. [0037] The microplate prepared as described above was stored at 28°C for 2 weeks. After storing, yeast growth in the group to which no compound was added was recognized, and then yeast growth in the beverages to which each of the compounds was added was visually observed in comparison with the blank. More specifically, the presence or absence of precipitation due to the yeast was visually observed, and the one in which precipitation was visually observed was rated as growth observed. For each of the compounds and each of the yeasts, the number of wells in which the yeast grew out of the three wells is shown in Table 1.

[Table 1]

[0038] The results in Table 1 show that compounds 1 to 6 have a growth inhibitory effect on the yeasts as chitosan, which is a known antimicrobial, does.

[0039] (Yeast growth test 2) The same procedures as those in yeast growth test 1 were repeated except for using compounds 1 to 3 and setting the final concentration of compounds 1 to 3 in respective wells to those shown in Table 2, and the yeast growth was visually observed. For each of the compounds and each of the yeasts, the number of wells in which the yeast grew out of the three wells is shown in Table 2.

[Table 2]

[0040] The results in Table 2 show that, depending on the type of beverage, compounds 1 to 3 have a growth inhibitory effect on the yeasts, even when used at a lower concentration. [0041] (Mold growth test 1)

A commercially available beverage containing 20% apple juice (containing apples, saccharides (high fructose com syrup and sugar), acidulant, and flavoring) and a commercially available beverage containing 30% orange juice (containing fruit (oranges and mandarin oranges), saccharides (high fructose corn syrup and sugar), acidulant, flavoring, and vitamin C) were used. The growth inhibitory effects of compounds 1 to 6 as well as chitosan and potassium sorbate as comparisons on various molds in the respective beverages were observed. The specific procedures are as follows.

[0042] The above beverages were opened and the pH of the beverage was adjusted to 3.5 using aqueous sodium hydroxide. Each of compounds 1 to 6 was dissolved in DMSO at a concentration of 20 mg/mL and the resulting solution of each of the compounds was added to the beverages so that the concentration of each of the compounds was 250 ppm in the beverages. For comparison, a beverage was prepared by adding an aqueous solution containing chitosan at 20 mg/mL and ascorbic acid at 20 mg/mL to the above beverages so that the concentration of chitosan was 250 ppm in the beverage. Similarly, for comparison, a beverage in which potassium sorbate was added at the concentration of 250 ppm in the beverage was prepared. A 3-mL aliquot of each of the beverages to which each of compounds 1 to 6 was added and the beverage to which chitosan or potassium sorbate was added was pipetted to a centrifuge tube with a capacity of 15 mL.

[0043] A suspension of spores of mold (Neosartorya sp.) was added to the respective centrifuge tubes at 50 to 200 cfu/ tube.

[0044] Separately from the above, a blank to which no suspension of spores was added was prepared. Furthermore, as a group to which no compound was added, a group was prepared by adding the respective suspensions of spores of mold at 50 to 200 cfu/ tube to 3 mL of a beverage to which no compounds 1 to 6, chitosan, or potassium sorbate were added.

[0045] The centrifuge tubes prepared as described above were stored at 28°C for 8 weeks. After storing, hyphal growth of the mold in the group to which no compound was added was recognized, and then mold growth in the beverages to which each of the compounds was added was visually observed in comparison with the blank. More specifically, the presence or absence of hyphal growth of the mold was confirmed by visual observation, and the one in which hyphal growth was visually observed was rated as growth observed (+) and the one in which no hypha was observed was rated as growth not observed (-). The results are shown in Table 3.

[Table 3]

[0046] For chitosan, mold growth was found after 2 weeks at 28°C. For potassium sorbate, mold growth was found after 4 weeks. In contrast, for compounds 1 to 6, mold growth was not found even after 8 weeks at 28°C. The results show that compounds 1 to 6 are more effective in inhibiting mold growth than chitosan which has a known antimicrobial effect and potassium sorbate which is a known synthetic preservative.

[0047] (Mold growth test 2)

The same procedures as those in mold growth test 1 were repeated except for setting the concentration to be added to the respective beverages to those shown in Table 4, and the mold growth after storing at 28°C for 8 weeks was visually observed. The results are shown in Table 4.

[Table 4]

[0048] The results in Table 4 show that, depending on the type of beverage, compounds 1 to 6 have a growth inhibitory effect on the mold, even when used at a lower concentration.

[0049] (Thermo- Acidophilic Bacilli growth test)

Using a commercially available beverage containing grape juice (containing fruit juice (grapes and lemons), saccharides (fructose and sugar), salt, lemon extract, flat lemon extract, Satsuma extract, Yuzu peel, extract of dried tomato, flavoring, lactic acid, potassium chloride, antioxidant (vitamin C)), the growth inhibitory effects of compounds 1 to 6 and comparative sodium benzoate on Thermo-Acidophilic Bacilli in the beverage were observed. The specific procedures are as follows.

[0050] The above beverage was opened, and the pH of the beverage was adjusted to 3.3 using aqueous sodium hydroxide. Each of compounds 1 to 6 was dissolved in dimethyl sulfoxide (DMSO), and the resulting solution of each of the compounds was added to the beverage so that the beverage finally contained each of the compounds at the concentration shown in Table 5. For comparison, a beverage in which sodium benzoate was added at the concentration shown in Table 5 was prepared. A 3-mL aliquot of each of the beverages to which each of compounds 1 to 6 was added and the beverage to which sodium benzoate was added was pipetted to a test tube.

[0051] A 400,000 cfu/mL solution of Thermo-Acidophilic Bacilli (Alicyclobacillus acidocaldarius) was prepared.

[0052] 10 pL per tube of the solution of the bacteria was added to test tubes to which the beverage containing any of compounds 1 to 6 or sodium benzoate was added. Furthermore, as a group to which no compound was added, a group was prepared by adding the solution of the bacteria to which no compounds 1 to 6 or sodium benzoate were added.

[0053] The test tubes prepared as described above were stored at 45°C for 2 weeks.

After storing, a 100-pL aliquot was collected from each of the beverages, spread on a YSGA medium, and stored at 55°C for 3 days to culture the Thermo-Acidophilic Bacilli. After the culture, the number of colonies on the medium was counted. The number of the bacteria was calculated based on the number of the colonies. The one in which the number of the bacteria increased more than 10 times based on the number of the inoculated bacteria was rated as growth observed (+) and the one in which the increase in the number of the bacteria from the number of the inoculated bacteria was less than 10 times was rated as growth not observed (-). The results are shown in Table 5.

[Table 5]

As shown in Table 5, it was found that at least compounds 1 to 3 and 5 have an effect on inhibiting growth of Thermo-Acidophilic Bacilli. The effect on inhibiting growth of Thermo-Acidophilic Bacilli obtained by these compounds is higher than that obtained by sodium benzoate that is a known synthetic preservative.

[0054] (Test for stability of appearance 1)

Using the 2 commercially available beverages used in the yeast growth test 1 and the 2 commercially available beverages used in the mold growth test 1 , stability of appearance of compounds 1 to 6 and comparative chitosan was evaluated. The specific procedures are as follows.

[0055] The above beverages were opened. The lemon carbonated beverage and the orange carbonated beverage were left until flat, and then the pH of the beverages was adjusted to 3.3 using aqueous sodium hydroxide. Each of compounds 1 to 6 was dissolved in DMSO at a concentration of 20 mg/mL and the resulting solution of each of the compounds was added to the beverage so that the concentration of each of the compounds in the beverage was 250 ppm. For comparison, a beverage was prepared by adding an aqueous solution containing chitosan at 20 mg/mL and ascorbic acid at 20 mg/mL to the above beverages so that the concentration of chitosan was 250 ppm in the beverage. A 96-well microplate was prepared, and a 240-pL aliquot per well of each of the above beverages to which each of compounds 1 to 6 was added and the beverage to which chitosan was added was pipetted. For the beverage containing apple juice and the beverage containing orange juice, the pH of these beverages was adjusted to 3.5 using aqueous sodium hydroxide.

Similarly to the carbonated beverages, each of compounds 1 to 6 was added to the respective beverages so that the concentration of each of the compounds in the beverage was 250 ppm. For comparison, the beverage containing 250 ppm of chitosan was prepared. A 3-mL aliquot of each of the above beverage to which each of compounds 1 to 6 was added and the beverage to which chitosan was added was pipetted to a centrifuge tube with a capacity of 15 mL.

[0056] Separately from the above, as a group to which no compound was added, a beverage to which compounds 1 to 6 or chitosan was not added was prepared.

[0057] The microplate prepared as described above was stored at 28°C for 2 weeks, and the centrifuge tubes prepared as described above were stored at 28°C for 8 weeks. After storing, the appearance of the beverages to which compounds 1 to 6 or chitosan were added was scored based on the following criteria in comparison with the appearance of the group to which no compound was added.

0: No difference in appearance from that of the group to which no compound was added 1 : Slightly different from the appearance of the group to which no compound was added 2: Different from the appearance of the group to which no compound was added

3: Significantly different from the appearance of the group to which no compound was added, no commercial value of beverage product

[0058] For the above criteria, score 0 to 2 means that the beverage has qualities required for a beverage product (has commercial value of beverage) and score 3 means that the beverage does not have qualities required for a beverage product (does not have commercial value of beverage). The results are shown in Table 6.

[Table 6]

[0059] The results of Table 6 show that, depending on the type of beverages, compounds 1 to 6 keep appearance stability that does not reduce commercial value of beverage even when used at a concentration of 250 ppm.

[0060] (Test for stability of appearance 2)

The same procedures as those in test for stability of appearance 1 were repeated except for using compounds 1 to 3, lemon carbonated beverage, and orange carbonated beverage, and setting the final concentration of each compound in the respective beverage to those shown in Table 7, and the appearance of the beverages was scored. The results are shown in Table 7.

[Table 7]

[0061] As shown in Table 2, compounds 1 to 3 have an effect on inhibiting growth of yeasts in the lemon carbonated beverage at 31 ppm. The results in Table 7 show that compounds 1 to 3 keep appearance stability that does not reduce commercial value of the lemon carbonated beverage when used at such a concentration in the beverage.

[0062] Further, As shown in Tables 1 and 2, compounds 1 to 3 have an effect on inhibiting growth of yeasts in the orange carbonated beverage at 31 ppm, and compound 2 has the effect at 250 ppm. The results in Tables 6 and 7 show that compounds 1 to 3 keep appearance stability that does not reduce commercial value of the orange carbonated beverage when used at such concentrations in the beverage.

[0063] (Test for stability of appearance 3)

The same procedures as those in test for stability of appearance 1 were repeated except for using compounds 1 to 3, apple beverage, and orange beverage, and setting the final concentration of each compound in the respective beverage to those shown in Table 8, and the appearance of the beverages was scored. The results are shown in Table 8.

[Table 8]

[0064] As shown in Table 4, compounds 1 to 3 have an effect on inhibiting growth of mold in the apple and orange beverages at 31 ppm. The results in Table 8 show that compounds 1 to 3 keep appearance stability that does not reduce commercial value of the apple and orange beverages when used at such a concentration in the beverages.