JP5971980B2 - Methods for preventing microbial contamination of beverages - Google Patents

Description

  The present invention relates to the prevention of microbial contamination of beverages.

  For commercial beverages, it is important to prevent spoilage due to microorganisms such as yeast, bacteria, and mold, and to enable long-term storage. As typical sterilization or preservation methods, heat treatment, addition of preservatives, aseptic packaging, nitrogen-filled packaging, and the like are performed. Beverage sterilization methods and their bactericidal resistance are determined by the nature of the beverage, including the components (eg, sugar, fruit juice, alcohol, vitamins or micronutrients), carbonic acid, water quality, pH, etc. Can be affected.

  It is conventionally known that catechins have various physiological activities such as body fat burning promotion. Therefore, in recent years, the demand for catechin-containing beverages has increased with the increase in health consciousness. Catechins have antibacterial effects against various bacteria such as Escherichia coli, Salmonella bacteria, Vibrio bacteria, Pseudomonas bacteria, Staphylococcus aureus, Sebasia, lactic acid bacteria, Listeria bacteria, Bacillus bacteria, etc. 2) Beverages containing catechins, in particular, beverages containing a high amount of catechins, tend to be superior in bacteria resistance compared to other beverages.

Nonetheless, catechin-containing beverages are also at risk of spoilage. For example, since various spore-forming fungi are widely distributed in soil and the like, there is a high risk of contamination and they can survive even in harsh environments where normal bacteria are unlikely to exist. In particular, catechin-resistant spore bacteria such as Bacillus coagulans can survive and cause spoilage even in commercial catechin-rich beverages.

Saponin is a food additive mainly used as an emulsifier using plants such as kiraya, yucca, soybean, and tea as raw materials. Patent Document 3 describes that an antifungal agent containing a saponin derived from Mockkoku ( Ternstromemia gymnanthera ) and blending it into food and drink. Patent Document 4 discloses that a preservative composition containing saponin is used to inhibit and / or reduce the growth of at least one microorganism selected from mold, yeast and bacteria in beverages and / or foods. Have been described. However, the solubility of saponins is low and the amount that can be added to a beverage is limited. Furthermore, since saponin has a unique bitter taste, it is not preferable in terms of flavor to add a large amount to food or drink. Therefore, it is considered difficult to sufficiently prevent microbial contamination of food and drink with saponin alone.

  Gallic acid is a food additive mainly used as an antioxidant. A growth inhibitory effect against S. aureus has also been reported (Non-patent Document 1). Patent Documents 5 to 6 describe that the flavor and color tone of a packaged beverage containing non-polymer catechins and gallic acid in a predetermined amount are stably maintained. However, these documents do not examine the bactericidal resistance of the beverage. Patent Document 7 discloses that a composition containing catechins, gallate-type catechins, caffeine and gallic acid obtained by contacting polyvinyl pyrrolidone powder with a green tea extract has deodorant, antibacterial and antioxidant activities. Have been described. However, it is not clear whether the antibacterial effect of the composition is superior to catechin alone.

  Therefore, the effects of saponin and gallic acid on the antibacterial properties of catechin-containing beverages have not been known so far.

JP 2008-173050 A JP 2008-174487 A JP 2008-013520 A JP 2009-536822 A JP 2007-325585 A JP 2007-330253 A JP 2006-81778 A

Japanese Bacteriological Journal, 1990, 45 (2), p561-566

  The present invention provides a method for preventing microbial contamination by improving the antibacterial properties of a catechin-containing beverage and suppressing the growth of microorganisms in the beverage, and provides a catechin-containing beverage with excellent bacterial resistance. About that.

  By adding gallic acid and saponin together to a catechin-containing beverage, the present inventors can obtain synergistically high antibacterial activity, and can suppress microbial growth in the beverage even when added at a low dose, Accordingly, it has been found that the use of catechin, gallic acid and saponin in a beverage can effectively prevent microbial contamination and spoilage of the beverage.

That is, the present invention provides the following.
(1) A method for preventing microbial contamination of a beverage, comprising adding catechins, gallic acid, and saponin.
(2) The method according to (1), wherein the beverage has a catechin content of 350 ppm or more.
(3) The method according to (1) or (2), wherein the beverage has a gallic acid content of 200 to 50 ppm.
(4) The method according to any one of (1) to (3), wherein the beverage has a saponin content of 100 to 30 ppm.
(5) The method according to any one of (1) to (4), wherein the beverage has a pH of 5.0 to 8.0.
(6) The method according to any one of (1) to (5), wherein the microorganism is Bacillus coagulans .
(7) A catechin-containing beverage comprising 350 ppm or more of catechins, 200 to 50 ppm of gallic acid, and 100 to 30 ppm of saponin.
(8) The beverage according to (7), wherein the beverage has a pH of 5.0 to 8.0.
(9) A method for producing a catechin-containing beverage, wherein 200 to 50 ppm of gallic acid and 100 to 30 ppm of saponin are blended in a liquid containing 350 ppm or more of catechins.
(10) The method according to (9), wherein the pH of the beverage is adjusted to 5.0 to 8.0.

  According to the present invention, since a catechin-containing beverage having excellent bacteria resistance can be obtained, it is possible to effectively prevent microbial contamination of the catechin-containing beverage due to bacteria that have been difficult to suppress conventionally. Further, according to the present invention, since the amount of saponin added can be reduced by using it together with gallic acid, it is advantageous in that it is not necessary to limit the amount used in consideration of the solubility and bitterness of saponin.

  In the present invention, examples of catechins include non-polymer catechins and polymer catechins, and among these, non-polymer catechins are preferred. Non-polymer catechins include non-epimeric catechins such as catechin, gallocatechin, catechin gallate, and gallocatechin gallate; and epicatechins such as epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate. These may be used alone or in admixture of two or more.

As the catechins, tea or other raw material-derived products, column purified products, and chemically synthesized products may be used, but tea-derived products such as tea extracts, concentrated or purified products thereof are used. Is preferred. The tea extract can be a Camellia genus, such as C. sinensis , C.I. It can be obtained by adding water or hot water or an extraction aid to these to a tea made from assamica or their hybrids, preferably its tea leaves, and performing stirring extraction and the like. Moreover, you may use together the method of extracting in so-called non-oxidative atmosphere, ventilating inert gas, such as boiling deaeration and nitrogen gas, and removing dissolved oxygen. Examples of the extraction aid include organic acids such as sodium ascorbate or organic acid salts thereof. The tea making includes: (1) green teas such as Sencha, Bancha, Gyokuro, Tencha, Kamasencha; (2) Semi-fermented, such as iron kannon, color species, golden katsura, wushuiwacha, etc. Tea; (3) Fermented tea such as Darjeeling, Uba, and Keyman called black tea is included.

  Concentration of the tea extract can be performed by concentrating the extract, and purification of the tea extract can be performed by purification using a solvent or a column. Various forms such as a solid, an aqueous solution, and a slurry are exemplified as the form of the concentrate or purified product of the tea extract. For example, the tea extract can be prepared by a method exemplified in detail in JP-A-59-219384, JP-A-4-20589, JP-A-5-260907, JP-A-5-306279, and the like. it can. Commercial products can also be used. Examples of such commercially available products are ITO EN “Tear Franc”, Mitsui Norin “Polyphenon”, Taiyo Kagaku “Sunphenon”, Suntory “Sunulon” Or the like.

  The catechins in the tea extract are present mainly as a non-polymer and dissolved in a liquid or as a solid that is adsorbed or included in a suspension of fine tea powder. In addition, most tea catechins exist as epi-catechins, and these epi-catechins can be converted into non-epi-isomers that are stereoisomers by treatment with heat, acid, alkali, etc. it can. Accordingly, when non-epimeric catechins are used, an extract from green tea, semi-fermented tea or fermented tea or a concentrate of tea extract is made into an aqueous solution, for example, 40 to 140 ° C., 0.1 It can be obtained by heat treatment for min to 120 hours. Moreover, you may use the concentrate of the tea extract with high non-epicatechin content. They may be used alone or in combination.

  The content of catechins in the tea extract is preferably 30 to 98% by mass. The content of catechins in these total polyphenols is preferably 10% by mass or more, more preferably 20 to 70% by mass, immediately after production. As the catechins of the present invention, the tea extract and the fractions thus obtained may be used as they are, or those diluted with an appropriate solvent, diluted concentrates and dry powders. You may use what was prepared and what was prepared in the paste form. In the present invention, these tea extracts may be used in such an amount that the desired catechin content is finally achieved.

  Gallic acid is isolated by adsorbing it on a gallic acid-adsorbing resin after subjecting the gallate catechin to tannase treatment according to the method described in JP-A-2003-33157 to obtain a mixture of catechins and gallic acid. Can be prepared. Alternatively, a commercially available product (available for purchase from Wako Pure Chemical Industries, Ltd.) may be used.

  Examples of saponins used in the present invention include saponin-containing plants, such as beans such as kiraya, yucca, soybeans, peas, tea, peanuts, oats, eggplants and leeks, tomatoes, asparagus, spinach, sugar beet, A saponin-containing extract extracted from yam, blackberry, licorice, primrose root, Senegal root, gypsophila, mukuroji, horse chestnut, etc. may be used, or a commercially available saponin (available for purchase from Wako Pure Chemicals, etc.) may be used. Also good. These can be used alone or in combination, but preferably soybean saponin or tea saponin is used.

  The beverage produced by the present invention is a beverage containing catechins (catechin-containing beverage), and preferably a catechin-rich beverage containing a high concentration of catechins. The catechin-containing beverage is not particularly limited as long as it contains catechins, for example, green tea, oolong tea, tea beverages such as black tea, catechin-containing soft drinks and sports drinks, catechins as various active ingredients A functional drink etc. are mentioned.

  The catechin-containing beverage produced according to the present invention further contains gallic acid and saponin. The beverage of the present invention may be produced by adding catechins, gallic acid, and saponin to any liquid that is the raw material of the beverage, but preferably contains catechins that are the raw material of the beverage It can be produced by adding gallic acid and saponin to a liquid, for example a tea extract solution. The order of adding catechins, gallic acid, and saponin is not particularly limited.

  Moreover, the catechin-containing beverage produced according to the present invention may further contain components and additives usually used in general tea beverages and soft drinks. Examples of the component include sugars, sweeteners, amino acids, fruit juice, wort, vegetable juice, other plant extracts, and dairy products. Examples of such additives include emulsifiers, pH adjusters, antioxidants, acidulants, various organic acids, thickeners, colorants, flavors, and seasonings. Furthermore, other preservatives may be used in combination. Moreover, as long as the antibacterial effect exhibited by catechins, gallic acid, and saponins is not impaired, the beverage of the present invention can further contain other active ingredients, medicinal ingredients, cosmetic ingredients, and the like.

  What is necessary is just to manufacture the catechin containing drink of this invention according to the manufacturing method of a normal catechin containing drink except adding a gallic acid and a saponin. For example, it can be produced by adding catechins, gallic acid and saponin, and other ingredients and additives as required, in any step while following the production method of ordinary tea drinks and soft drinks. it can. The method for producing a catechin-containing beverage of the present invention can also include conventional sterilization steps such as overheating and filtration, and conventional anti-corruption treatment steps such as aseptic packaging, hermetic packaging, and gas-filled packaging.

  In the beverage of the present invention containing catechins, gallic acid, and saponin thus obtained, the growth of microorganisms is suppressed, so that microbial contamination of the beverage can be prevented. As shown in the examples below, when both gallic acid and saponin are added to a catechin-containing beverage, high microbial growth inhibition is achieved with a smaller addition amount than when either gallic acid or saponin is added alone An effect can be obtained. Such a synergistic microbial growth inhibitory effect due to the combined use of gallic acid or saponin is seen in both catechin-low beverages and catechin-rich beverages, but is more pronounced in catechin-rich beverages.

Accordingly, the content of catechins in the beverage of the present invention is preferably 350 ppm or more, more preferably 500 ppm or more, and further preferably 1000 to 2000 ppm.
Further, the content of gallic acid in the beverage of the present invention is preferably 50 ppm or more, and in the case of a beverage containing a low catechin content, for example, a general tea beverage or a soft drink having a catechin content of less than 500 ppm, it is 200 ppm or less. In the case of a high catechin content beverage with a catechin content of 500 ppm or more, it may be 200 ppm or less, preferably 150 ppm or less, more preferably 100 ppm or less from the aspect of flavor, and a high catechin content with a catechin content of 1000 ppm or more. In the case of a beverage, it may be 200 ppm or less, but is preferably 100 ppm or less, and more preferably 80 ppm or less from the aspect of flavor.
Further, the content of saponin in the beverage of the present invention is preferably 30 ppm or more, and in the case of a catechin low content beverage such as a general tea beverage or soft drink having a catechin content of less than 500 ppm, it may be 100 ppm or less, In the case of a catechin-rich beverage with a catechin content of 500 ppm or more, it may be 100 ppm or less, preferably 50 ppm or less, and in the case of a catechin-rich beverage with a catechin content of 1000 ppm or more, preferably 50 ppm or less, and 30 to 40 ppm in flavor. From the viewpoint of

  Although the pH of the drink of this invention is not specifically limited, What is necessary is just the range of pH 5.0-8.0, Preferably it is the range of pH 5.5-6.5.

Examples of microorganisms whose growth is inhibited in the beverage of the present invention include Escherichia coli, Salmonella bacteria, Vibrio bacteria, Pseudomonas bacteria, Gram negative bacteria such as Sephacia bacteria, Staphylococcus aureus, Listeria bacteria, Bacillus bacteria, Alicyclobacillus Gram-positive bacteria such as genus bacteria, yeasts such as Saccharomyces, Tigosaccharomyces, Candida and Deckella species, acidophilic bacteria such as lactic acid bacteria, Leuconostoc genus, Gluconobacter genus and Zymomonas species, and molds such as penicillin and Aspergillus . Of these, spore-forming fungi such as Alicyclobacillus and Bacillus are widely distributed in the soil and the like, and thus there is a high risk of contamination. Because it can survive in the environment, catechin-containing beverages are likely to become contaminating bacteria. Therefore, these bacteria are desirable microorganisms to be targeted for growth inhibition or contamination prevention in the present invention. Among them, Bacillus coagulans has a resistance to catechins, and thus may grow in a catechin-containing beverage and cause its spoilage. Therefore, as microorganisms desirable than should be subject to growth inhibition or pollution in the present invention, mention may be made of the Bacillus coagulans (Bacillus coagulans).

  EXAMPLES Hereinafter, an Example is shown and this invention is demonstrated more concretely.

Example 1 Antibacterial Test of Catechin-Containing Beverage (1) Preparation of Test Bacteria As a test bacterium, Bacillus coagulans known to have high catechin resistance among spore-forming bacteria: Japan Canning Association 1105, 1106, 1107, 1108, 18 strains of 1109, 1179, 1180, 1182; DSM2308, DSM2312, DSM2385, DSM2383, DSM2384, DSM2311, DSM2356, DSM2314, DSM2350; and NBRC12583 were used.
Eighteen types of Bacillus coagulans strains were aerobically cultured for 3 days at 45 ° C. on SCD agar medium (OXOID). The cultured cells were adjusted to 10 ^5-6 cfu / ml in physiological saline to obtain a sample bacterial solution.

(2) Antibacterial test-1
To examine the antibacterial effects of catechin, gallic acid and soy saponin in tea beverages, a challenge test was conducted. In the test, a commercially available tea beverage (catechin concentration 40 mg / 100 ml, pH 6.0) as a beverage having a low catechin concentration, and “Helsia Green Tea Maroyaka” (Kao) (catechin concentration 154 mg / 100 ml, pH 6.5) as a beverage having a high catechin concentration. 0) was used.
Add gallic acid (Wako Pure Chemicals) and soybean saponin (Wako Pure Chemicals) alone or in combination to the above tea beverage so as to have the concentrations shown in Table 1, stir vigorously using a magnetic stirrer, and dissolve completely. It was. Each beverage obtained is inoculated with the bacterial solution prepared in (1) so that the final concentration of the bacteria is 10 ³ cfu / ml, and stored aerobically at 45 ° C. for 14 days, after which the total bacterial concentration and The follicle behavior was evaluated by measuring the number of spores. In the measurement, the stored tea drink was vigorously stirred to homogenize the system, and then diluted with physiological saline. The total cell concentration (cfu / ml) was determined by coating the SCD agar medium (manufactured by OXOID) without culturing the diluted solution and culturing. And calculated. The number of spores was calculated according to the same procedure from the one obtained by heat-treating the diluted solution at 80 ° C. for 10 minutes and then smearing and culturing on a YSG agar medium (manufactured by Microbio). The case where growth of 1 order or more was confirmed as the total cell concentration or the number of spores was judged as “proliferation”, and the case where growth of less than 1 order or reduction or death of cells or spores was confirmed as “non-proliferation”.

The results are shown in Table 1. In the low catechin beverage, the growth inhibitory concentration (MIC) with gallic acid alone was 450 ppm, and with soybean saponin alone, growth could not be inhibited even at the solubility limit of 100 ppm. In the case of the combined use of gallic acid and soybean saponin, the fungus growth inhibitory effect was observed at 100 ppm.
On the other hand, in the high catechin beverage, the MIC with gallic acid alone was 180 ppm, and the soybean saponin alone could not inhibit growth even at the solubility limit of 100 ppm. In the case of the combined use of gallic acid and soybean saponin, bacterial growth was inhibited by the addition of 60 ppm gallic acid and 30 ppm soybean saponin. Therefore, in both the low catechin beverage and the high catechin beverage, a synergistic bacterial growth inhibitory effect by the combined use of gallic acid and soybean saponin was observed, but the effect was more remarkable in the high catechin beverage.

(3) Antibacterial test-2
Add catechin, gallic acid (Wako Pure Chemicals) and soybean saponin (Wako Pure Chemicals) alone or in combination to a commercially available blended tea beverage (pH 6.0) containing no catechins at the concentrations shown in Table 2 and completely dissolve I let you. Each obtained beverage was inoculated with the fungus in the same procedure as in (2) above, and then the total bacterial count and spore count were measured to evaluate the fate of the fungus.
The results are shown in Table 2. Even when both gallic acid and soybean saponin were added, bacterial growth was not inhibited when the catechin concentration was low. However, the higher the catechin concentration, the more effectively the bacterial growth was inhibited.

(4) Antibacterial test-3
To examine the antibacterial properties of various saponins, a challenge test was conducted. In the test, “Helicia Green Tea Maroyaka” (Kao) (catechin concentration: 154 mg / 100 ml, pH 6.0) was used as a beverage having a high catechin concentration. To the tea beverage, gallic acid (Wako Pure Chemical Industries) and tea leaf saponin were added in combination so that the concentration of gallic acid was 60 ppm and the concentration of various tea leaf saponins was 30 ppm.
The used tea leaf saponins were prepared by the following method. 1 kg of fresh tea leaves (manufactured by Kenya) was refluxed and extracted with 5 L of methanol (3 hours, 65 to 70 ° C.) and then filtered through No. 2 filter paper to obtain a methanol extract. The obtained methanol extract was concentrated under reduced pressure to obtain 385 g of a methanol extract. 200 g of the obtained methanol extract was subjected to liquid-liquid partition with 5 L of water and 5 L of ethyl acetate, and the obtained water fraction was partitioned with water-saturated n-butanol to obtain an n-butanol fraction. The n-butanol fraction was concentrated under reduced pressure to obtain 96 g of an n-butanol fraction solid. 96 g of n-butanol fraction solids were dissolved in ion-exchanged water, adsorbed on HP-20 resin (1 kg, manufactured by Mitsubishi Chemical Corporation), and 0, 20, 40, 60, 80, 99.5% ethanol each 4 L And eluted. Among the obtained fractions, an 80% ethanol-eluted fraction solid (1.0 g) was obtained as a crude tea leaf saponin fraction. Next, the 80% ethanol elution fraction (1.0 g) was adsorbed to ODS Chromatrex (100 g, manufactured by YMC), and eluted with 20, 40, 60, 80, and 100% methanol. Among the obtained fractions, an 80% methanol-eluted fraction (400 mg) was fractionated by HPLC (LC-908, manufactured by Japan Analytical Agency), and foliathasaponin II (35.2 mg), fliatheasaponin IV (30.2 mg). ), Theaaponin B1 (34.6 mg) was obtained. These purified tea leaf saponins were obtained from literature (Morikawa T., Nakamura S., Kato Y., Muraoka O., Matsuda H. and Yoshikawa M., Chem. Pharm. Bull. , 55 (2): 293-298, 2007).
Each of the obtained beverages was inoculated with the bacterial solution prepared in (1) above so that the final concentration of the bacteria was 10 ³ cfu / ml, and 25 ml was dispensed into a 25 ml glass screw cap test tube. Stored aerobically at 14 ° C. for 14 days. The total number of bacteria and the number of spores were measured over time by the same procedure as in (2) above, and the change behavior of the bacteria was evaluated.
The results are shown in Table 3. All the tea leaf saponins were effective in inhibiting fungal growth.

Claims (8)

In beverages, catechins 600ppm or more and, gallic acid 200～50Ppm, is contained and a saponin 100～30Ppm, and microbial contamination prevention method of the beverage, which comprises adjusting the pH to 5.0-8.0 . The method of claim 1, wherein Ru is contained Saponi down 1 00~ 5 0 ppm in the beverage. The method according to claim 1 or 2, wherein the microorganism is a Bacillus bacterium. The method according to claim 3 , wherein the microorganism is Bacillus coagulans. A catechin-containing beverage comprising 600 ppm or more of catechins, 200 to 50 ppm of gallic acid, and 100 to 30 ppm of saponin, and having a pH of 5.0 to 8.0 . The beverage according to claim 5, wherein the saponin content is 100 to 50 ppm. Production of a catechin-containing beverage characterized by blending 200 to 50 ppm of gallic acid and 100 to 30 ppm of saponin in a liquid containing catechins of 600 ppm or more and adjusting the pH to 5.0 to 8.0. Method. The method according to claim 7, wherein the amount of saponin is 100 to 50 ppm.