JP3783020B2 - Bacteriostatic agent for beverage and beverage having bacteriostatic action - Google Patents

Description

  The present invention relates to a treatment method for processing and treating a bacteriostatic agent containing a predetermined polyphenol as a main component into a beverage additive, and a beverage containing a beverage additive obtained by the treatment method.

  It is well known that the French paradox phenomenon that adults (lifestyle-related diseases) such as arteriosclerosis are rare in regular drinkers of red wine, but this is caused by polyphenols that are abundant in red wine. Has been. It is also said that the disease prevention effect of this polyphenol is largely due to the antioxidant function of suppressing active oxygen in the body.

  The reason why such usefulness has been pointed out for polyphenols is due to the great attention paid to the antioxidant function of many polyphenols. And if you look only at the phenomenon of oxidation, it will also attract the attention of the beverage industry because it contributes to the deterioration of beverage quality, and polyphenol as an antioxidant is added to the beverage It has become a serious consideration.

  Under such circumstances, some polyphenols have been added to beverages as antioxidants in earnest, but this is because of deterioration of beverages due to oxidation. One of the main factors is that polyphenols themselves are naturally derived and are easily accepted by health-conscious consumers, in addition to being considered more problematic than before.

  In this way, when the functions and usefulness of polyphenols as antioxidants are well known, research on extraction raw materials, extraction methods, and application ranges based on their antioxidant functions will be promoted. became.

  However, as a result of extensive research on the polyphenols by the present inventors, polyphenols (particularly, proanthocyanidins themselves and polyphenols extracted in the vicinity of proanthocyanidins) have a bacteriostatic action. Became clear.

  Furthermore, the present inventors have also found that a certain fraction has a strong bacteriostatic effect when the polyphenol is separated by HPLC under a predetermined condition, and the bacteriostasis mainly comprising the predetermined polyphenol. The present invention relating to the agent has been completed, and a patent application has already been filed (Japanese Patent Application No. 11-336958). That is, the present inventors have already found an effective and useful bacteriostatic agent and a beverage containing the same by finding a polyphenol fraction having a more effective bacteriostatic action than before by further separation of the polyphenol fraction. -ing

  However, as a result of continuing research on the bacteriostatic agent and beverages containing the same, when the beverage to which the bacteriostatic agent is added is left for a while, precipitation occurs, and the appearance quality of the beverage is descend.

  In this case, the fact that precipitation occurs and appearance quality deteriorates is a problem that cannot be ignored in the current situation where transparent containers are the major measure. In addition, the occurrence of precipitation leads to a problem in storage stability, and ultimately it is not suitable for use as a bacteriostatic agent.

  The present invention has been made in view of the problems as described above, and its purpose is in a case where a beverage is added with a bacteriostatic agent containing a polyphenol fraction having a predetermined effective bacteriostatic action, and this is left for a while. However, it is to prevent the occurrence of precipitation.

  As a result of intensive studies conducted by the present inventors in order to achieve the above-described object, the above-mentioned bacteriostatic agent was once cooled and partially solidified, and then a precipitate formed when it was thawed. When the treatment of filtering was performed, it was found that even if the bacteriostatic agent after the treatment was added to the beverage and left for a while, precipitation did not occur, and the present invention was completed.

  That is, in the present invention, as an effective treatment method for preventing precipitation after leaving for a while, the bacteriostatic agent is frozen once and then the precipitate that is generated when it is thawed is filtered. While providing a process, the drink with the good preservability to which the bacteriostatic agent after the said process was added is provided.

  More specifically, the present invention provides the following beverages and bacteriostatic agents.

(1) Corresponds to the last peak eluted among the major peaks obtained when the polyphenol fraction obtained from fruits, fruit seeds, etc. is separated by liquid column chromatography for reverse phase separation. A processing method for processing and processing a bacteriostatic agent containing as a main ingredient a beverage additive,
A treatment method comprising the steps of dissolving the bacteriostatic agent in water-containing ethanol, thawing it after partially solidifying it by cooling, and removing the precipitate generated after the thawing by ultrafiltration .

(2) Among the major peaks obtained when the polyphenol fraction obtained from fruits, fruit seeds, etc. is separated by liquid column chromatography for reverse phase separation, the peaks disappeared by NF membrane treatment A processing method for processing and processing a bacteriostatic agent containing a part as a main component into a beverage additive,
A treatment method comprising the steps of dissolving the bacteriostatic agent in water-containing ethanol, thawing it after partially solidifying it by cooling, and removing the precipitate generated after the thawing by ultrafiltration .

(3) The peak eluted at the end of the major peaks among the peaks obtained when the polyphenol fraction obtained from fruits, fruit types, etc. is separated by liquid column chromatography for reverse phase separation And a bacteriostatic agent that contains a peak part that disappears by NF membrane treatment as a main component to a beverage additive,
A treatment method comprising the steps of dissolving the bacteriostatic agent in water-containing ethanol, thawing it after partially solidifying it by cooling, and removing the precipitate generated after the thawing by ultrafiltration .

(4) A bacteriostatic agent produced so as to contain more fractions eluted later when the polyphenol fraction obtained from fruits, fruit seeds, etc. is separated by liquid column chromatography for reverse phase separation Is a processing method when processing and processing into a beverage additive,
A treatment method comprising the steps of dissolving the bacteriostatic agent in water-containing ethanol, thawing it after partially solidifying it by cooling, and removing the precipitate generated after the thawing by ultrafiltration .

  (5) The processing method according to any one of (1) to (4), wherein the liquid column chromatography for reverse phase separation is HPLC.

(6) A processing method for processing and processing a bacteriostatic agent mainly composed of proanthocyanidins into a beverage additive,
A treatment method comprising the steps of dissolving the bacteriostatic agent in water-containing ethanol, thawing it after partially solidifying it by cooling, and removing the precipitate generated after the thawing by ultrafiltration .

  (7) The bacteriostatic agent according to any one of (1) to (5), wherein the polyphenol fraction is obtained from grape seeds.

  (8) The bacteriostatic agent according to (6) or (7), which is a bacteriostatic agent having a bacteriostatic action against heat-resistant acidophilic bacteria.

  (9) A beverage additive produced by passing through the processing method according to any one of (1) to (6).

  (10) The beverage additive according to (9), which is a beverage additive having a bacteriostatic effect on an acidic beverage.

  (11) A beverage containing a bacteriostatic effective amount of the beverage additive according to any one of (9) and (10).

[Definition of terms]
“Bacteriostatic” means that it does not have the action of killing bacteria, but has the action of suppressing the growth and proliferation of bacteria and preventing the deterioration of product beverages based on the activity of bacteria. The term “bacterial agent” means one that can exert a bacteriostatic action in a beverage without adversely affecting the flavor or the like of the beverage when added to the beverage.

  “Acid drink” is typically a fruit juice drink having a pH of about 3.0 to 3.5, but sports drinks and isotonic drinks having a pH adjusted to around 3.0 to 4.0, Includes health drinks.

  “Heat-resistant acidophilic bacteria” means heat sterilization that is commonly performed in “acidic beverages” among bacteria (acidophilic bacteria) that grow and proliferate in “acidic beverages” as described above. It means a heat-resistant material that does not die even by treatment, and is a bacteriostatic target in the beverage industry. Here, among acidophilic bacteria, heat-resistant ones (thermophilic acidophilic bacteria) form spores, but the bacteriostatic agent according to the present invention is effective in preventing proliferation due to germination of the surviving spores in the beverage. It is observed to work.

[HPLC]
“Liquid column chromatography for reverse phase separation” means column chromatography in which separation is carried out by a combination of a hydrophobic stationary phase and a hydrophilic mobile phase. A solid (usually a substance in which a hydrophobic group (hydrocarbon chain) is chemically bonded to the surface of silica gel) or a synthetic polymer having a hydrophobic surface, water or an aqueous solution as a mobile phase, or an organic solvent that is well mixed with water Use a mixture with water. A typical example of "liquid column chromatography for reverse phase separation" is "HPLC (High Performance Liquid Chromatography)" in which a silica gel bonded with octadecyl group is packed and a buffer solution is used as an eluent. This is also adopted in the examples.

[Major peak]
In the present specification, the “major peak” means a conspicuously conspicuous among the many peaks. The “bacteriostatic effective amount” is appropriately determined according to the type of polyphenol and fraction used as a bacteriostatic agent.

[Partial solidification]
“Partial solidification” means that the object does not become a completely frozen body, but forms a state in which a part is solid and part is liquid. And when water-containing ethanol is cooled, such a form will appear.

  Therefore, the filtrate obtained by dissolving the bacteriostatic agent in water-containing ethanol and removing the generated precipitate by ultrafiltration is preferably -30 ° C to -4 ° C, more preferably -20 ° C to- When it is cooled to about 7 ° C., it does not become a completely solid frozen body, but forms a thick sherbet. In this specification, this state is basically referred to as “partially solidified by cooling”.

[Thawing]
“Thawing” refers to heating a solid object to a liquid state, but in this specification, the bacteriostatic agent partially solidified by cooling is not left in the solid state. Mainly meant to be in no perfect liquid state.

[Extraction of polyphenol fraction]
Any plant such as grape and tea can be used as the raw material of the present invention as long as it contains polyphenol. As for grapes, it is also a good idea to use the seed part, especially for the seed part, since it is used as an unused by-product. In addition, the seed part contains a lot of useful polyphenols, and various studies have been made on the method for efficiently extracting the useful polyphenols, and the present inventors have also found out (see Japanese Patent Application). Hei 11-209578).

  Extraction of the polyphenol fraction is carried out using water or ethanol as a solvent. Thereafter, in order to increase the accuracy of the extract, it is also possible to appropriately add an enzyme or perform centrifugation.

[Ultrafiltration]
The “ultrafiltration” in the present invention is a kind of so-called “membrane filtration” and basically has a molecular weight of 100,000 or more, preferably a molecular weight of 80,000 or more, more preferably a molecular weight of 70,000 or more. It means ultrafiltration excluding those having a molecular weight of 50,000 or more in some cases.

  Here, when the filtration process is performed except for those having a molecular weight of 100,000 or more, the occurrence of precipitation cannot be sufficiently prevented. On the other hand, when the filtration process is performed except for a molecular weight of 20,000 or more. Therefore, most (about half) of the effective polyphenol having a bacteriostatic action is removed, and even if the occurrence of precipitation can be prevented, the adverse effects due to the filtration treatment will become apparent.

  Therefore, in order to achieve the object of the present invention, it is necessary to carry out a filtration treatment that leaves a polyphenol having a molecular weight of 20,000 or more, more preferably leaving a polyphenol having a molecular weight of 50,000 or more while removing those having a molecular weight of 100,000 or more. As such a filtration process, an ultrafiltration process is suitable.

  In addition, in this invention, when diatomaceous earth (brown plant) filtration is given instead of ultrafiltration, precipitation generation | occurrence | production after drink addition cannot be prevented.

  Here, “diatomaceous earth filtration” means a filtration treatment in which filled diatomaceous earth is used as a filtering agent, and is a method generally used in the purification filtration treatment. When diatomaceous earth treatment is performed, for example, diatomaceous earth is deposited on a glass filter, and the sample is poured from above in a state where the diatomaceous earth is sufficiently filled, and filtration is performed.

[NF membrane treatment]
“NF membrane treatment” refers to a treatment using a charged reverse osmosis membrane or a charged ultrafiltration membrane, and the nanofiltration membrane used in this treatment has a molecular weight cut off of several hundred to several thousand. It is a charged membrane and has the property of blocking electrolyte permeation.

  As described above, according to the present invention, a predetermined polyphenol is collected from a predetermined one, or a polyphenol fraction is separated by reverse phase separation liquid column chromatography, whereby a bacteriostatic agent for a naturally derived beverage is obtained. It can be manufactured and can be made into a beverage having bacteriostatic action by mixing this fraction in the beverage, but in this process, the beverage bacteriostatic agent is cooled and partially solidified By performing the process of filtering the precipitate generated later, it becomes possible to prevent the occurrence of precipitation that deteriorates the appearance quality of the beverage after the addition of the bacteriostatic agent.

  Thereby, it becomes possible to safely fill a transparent container with a beverage to which a naturally-derived polyphenol-containing beverage bacteriostatic agent is added, and the stability of the beverage is also ensured. In other words, according to the present invention, it is possible to produce a beverage additive suitable for a naturally derived polyphenol-containing beverage bacteriostatic agent, particularly a beverage additive suitable for a beverage filled in a transparent container.

[Detection of polyphenols]
Detection of polyphenols (qualitative / quantitative analysis) is performed according to a conventional method. Examples of the method for detecting polyphenols include an analysis method by liquid chromatography, an iron tartrate method, a Folin-Denis method, and the like. In the examples described later, the amount of polyphenol was all measured by the Folin-Denis method (tannic acid conversion).

  Polyphenols themselves are substances contained in almost all plants, and since there are many types of polyphenols themselves, it has been difficult to demonstrate the relationship between their components and functions from the beginning of the research. However, as research on polyphenols progresses, it has been found that a component called proanthocyanidins, which are a kind of polyphenols, has an extremely excellent antioxidant function (for example, Japanese Patent Application Laid-Open Nos. 3-2000781 and 9-212484). Currently, an extract from grape seeds having a large amount of this component is registered as one of food additives as an antioxidant (existing addition of Ministry of Health and Welfare Notification No. 120) (Listed No. 367), has been commercially produced and sold.

  Examples of commercially available polyphenols containing a large amount of proanthocyanidins include commercially available gravinol (trade name), which is preferably used in practice. Gravinol (trade name) is obtained by extracting proanthocyanidins from grape seeds using a mixed solvent of ethanol / water, and is generally sold as a product. In addition, as a bacteriostatic agent, this gravinol may be used as it is, or may be used after further separation (for example, separation by HPLC described above).

  Incidentally, proanthocyanidins, which are the main components of Gravinol (trade name), are components positioned as shown in the following table as a kind of polyphenol.

  This substance called proanthocyanidins is a condensed tannin obtained by polymerizing catechins, and is also used as a pharmaceutical product (vascular therapeutic agent) in France because of its strong antioxidant action.

  As for proanthocyanidin itself, although a patent application has been filed for a method for stabilizing the quality of coffee beverages (Japanese Patent Laid-Open No. 8-228865), it does not leave its antioxidant action, but rather its antioxidant. It is only intended to stabilize the quality based on the action.

  Commercially available Gravinol (trade name) was subjected to HPLC under the following conditions.

  As a result, a chromatogram as shown in FIG. 1 was obtained.

  Similarly, when commercially available gravinol (trade name) was subjected to HPLC under the same conditions as in Table 2, a chromatogram as shown in FIG. 2 was obtained.

  Furthermore, when a grape seed extract (Japanese Patent Application No. 11-209578) obtained by performing liquefied carbon dioxide extraction was subjected to HPLC under the same conditions as in FIG. 2, a chromatogram as shown in FIG. 3 was obtained. It was. Further, when a grape seed extract obtained by hot water extraction from grape seeds was subjected to NF membrane treatment and subjected to HPLC under the same conditions as in FIG. 2, a chromatogram as shown in FIG. 4 was obtained. It was.

  Further, the catechin obtained from tea was subjected to HPLC under the same conditions as in FIG. 2 (however, in order to slow down the elution rate, the gradient after 20 minutes (increase in the amount of acetonitrile added (ie As shown in FIG. 5, a chromatogram as shown in FIG. 5 was obtained by appropriately reducing the eluent polarity))) (see the right end of the lower part of Table 2).

  As is clear from these figures, in the HPLC chromatogram of Gravinol (trade name) shown in FIG. 1, there are three major peaks, “Peak 1”, “Peak 2” and “Peak 3”. This is the same as in FIG.

  And the peak similar to “Peak 3”, which is the last major peak eluted in this, is the one in FIG. 3 which is an HPLC chromatogram of a grape seed extract obtained by performing liquefied carbon dioxide extraction. ("Peak 3 '" in FIG. 3) is not seen in FIG. 4 where the hot water extracted is treated with an NF membrane or in FIG. 5 which is an HPLC chromatogram of catechin obtained from tea.

  The major peak that is eluted last, “Peak 3” (or “Peak 3 ′”), is thought to contain a large amount of polyphenols having a high molecular weight, and thus the substance is proanthocyanidins. Although it is predicted that it will deviate, it has not yet been confirmed. However, if it is detected by any method that at least the same amount of proanthocyanidins as the major peak eluted last is contained, it can be assumed that the bacteriostatic agent according to the present invention will be used. it can.

[Example 1]
First, commercially available Gravinol (trade name) is mixed with a general turbidity type sports beverage (fruit juice 3%, soluble solid content less than 1 Brix, pH about 3.8), turbidity type juice beverage (fruit juice less than 10%, soluble Solid content 7-8 Brix, pH about 3.0), transparent type fruit juice (less than 10% fruit juice, soluble solid content 4-5 Brix, pH about 3.2), and heat-resistant acidophilic bacteria 2 Species (DSM2498 strain (Deutsche Sammlung von Microorganismen und Zellkulturen GmbH) and ATCC49025 strain (American Type Culture Collection) were inoculated at a ratio of “1000 spores / 500 ml PET bottles” and the progress was observed. 3 and the inoculated beverage was left at 37 ° C. for 14 days.

  Table 3 below shows the results when the addition amount of the extract according to this example (converted to the concentration of the total polyphenol contained in the extract) was changed.

  In Table 3, the growth of bacteria in the product and the accompanying odors and odors are indicated as + (In Table 3, the left three of the commas indicate the DSM2498 strain for the “+” and “-” signs. The results show that the right three commas show the results for the ATCC49025 strain).

  From this result, it can be seen that commercially available gravinol (trade name) has a bacteriostatic effect regardless of the type of beverage when mixed with a beverage such that the polyphenol concentration is 10 ppm or more.

[Example 2]
Next, the bacteriostatic effect of each of the major peaks (“peak 1”, “peak 2” and “peak 3” (FIG. 1)) obtained when the polyphenol fraction is separated by HPLC as described above. I decided to compare them. For this purpose, first, gravinol was converted to a polyphenol-converted solution of 0.5%, and 36 ml thereof was separated into three fractions “peak 1”, “peak 2” and “peak 3” by HPLC. Then, each separated part was concentrated and diluted after collection to make 7.2 ml. The recovery rate by peak comparison is shown in the following table.

  Considering the recovery rate of each of the above fractions, each fraction was added to a turbid sports drink so as to correspond to the addition of 10 ppm of Gravinol (trade name) in terms of polyphenol. After filling this turbid sports drink into a 500 ml PET bottle at 85 ° C in a hot pack, inoculate the heat-resistant acidophilic bacterium (DSM2498 strain) at a ratio of “1000 spore count / 500 ml PET bottle”. Hold and cool.

  The bacteriostatic effect was confirmed by the presence or absence of bacterial growth by pour culture of 1 ml of the product solution after culturing at 35 ° C. for 14 days. Moreover, in order to further clarify which fraction has a bacteriostatic action, both single addition and mixed addition were performed.

  As a result, the following results were obtained.

  In Table 5, “+” indicates that bacteria have grown in the product. As is clear from this result, the fraction of “Peak 3” is added to the beverage in both single addition and mixed addition. It was found that the growth of the bacteria was suppressed.

  Therefore, according to Example 1 above, it is proved that gravinol (trade name) has a bacteriostatic action, and according to this Example 2, gravinol (trade name) is obtained by reverse phase liquid column chromatography. It became clear that the component which has a bacteriostatic action was contained in the fraction eluted at the end when isolate | separating.

  Therefore, the fraction eluted at the end when Gravinol (trade name) is separated by reversed-phase liquid column chromatography is removed according to a conventional method, and the portion of the fraction is added to the beverage. It is clear that fungal action can be expressed.

[Example 3]
Next, the grape extracts extracted by different extraction methods and the bacteriostatic action of other polyphenols were compared with each other. Specifically, (1) Gravinol (trade name) which is a grape seed extract extracted using a mixed solvent of water / ethanol, (2) Grape seed extract extracted with liquefied carbon dioxide, 3) Grape seed extract obtained by subjecting grape seed extract extracted with hot water to NF membrane filtration, and (4) Green tea catechin preparation, respectively. About 3.8), and thermostable acidophilic bacteria (DSM2498 strain) were inoculated there at a ratio of “1000 spores / 500 ml PET bottle”, and the course was observed. The population of each test group was 2, and the beverage inoculated with the bacteria was left at 37 ° C. for 14 days.

  Table 6 below shows the results when the addition amount of the extract according to this example (converted to the concentration of the total polyphenol contained in the extract) was changed.

  In Table 6, the growth of bacteria in the product and the accompanying odor and odor are indicated as +.

  From this result, it is understood that when the grape seed extract is filtered with an NF membrane, the bacteriostatic action disappears from the grape seed extract. As described above, this is because the peak corresponding to “Peak 3” or “Peak 3 ′” that is eluted at the end disappears through the NF membrane treatment. The fact that the “peak 3” or “peak 3 ′” portion has a bacteriostatic action is also supported by the phenomenon.

  Similarly, the reason why green tea catechin does not have a bacteriostatic action is considered to be that a peak corresponding to "peak 3" or "peak 3 '" does not exist originally.

[Example 4]
The present invention can be carried out according to the following flow using a grape seed extract (for example, Gravinol (trade name)).

  Here, in accordance with the flow shown in Table 7 above, UF20,000 ultrafiltration membrane, UF50,000 ultrafiltration membrane, and UF100 were added to 2.5% aqueous solution of gravinol in ethanol (total polyphenol about 1%). In the case where the present invention is carried out using an ultrafiltration membrane of 1,000,000, the obtained bacteriostatic agent is used as a beverage additive to make a beverage (transparent type fruit juice: less than 10% fruit juice, soluble solid content 8-9 Brix, pH about As a result of observing the occurrence of precipitation after leaving the product added to 3.9) (for about 48 to 120 hours), the following results were obtained (note that the amount added is the concentration of each treatment solution added to the beverage) ).

  As shown in this table, when the present invention was carried out using a UF 100,000 ultrafiltration membrane, precipitation was observed, but a UF20,000 ultrafiltration membrane and a UF50,000 limit. When the present invention was carried out using an outer filtration membrane, no precipitation was observed in any case.

  Next, according to the flow shown in Table 7, when the present invention was carried out using an ultrafiltration membrane of UF20,000 and an ultrafiltration membrane of UF50,000, the obtained bacteriostatic agent was used as a beverage additive. As a result of examining the expression of the bacteriostatic effect when added to a beverage (clear type fruit juice beverage: less than 10% fruit juice, soluble solid content 8-9 Brix, pH about 3.9), the following results were obtained (note that The amount added is expressed as the concentration of each treatment solution added to the beverage).

  For each treatment solution, the results of applying this to HPLC are shown in FIG. 2 (untreated), FIG. 6 (for UF50,000 ultrafiltration membrane) and FIG. 7 (UF20,000 ultrafiltration membrane). Case).

  From the results shown in Table 9 and FIGS. 2, 6 and 7, it is apparent that polyphenol loss is large when the ultrafiltration membrane of UF20,000 is used, and the bacteriostatic action is reduced.

It is a figure which shows the chromatogram obtained as a result of applying commercially available gravinol (brand name) to HPLC (refer Table 2). It is a figure which shows the chromatogram obtained as a result of applying commercially available gravinol (brand name) to HPLC under the conditions of Table 2. FIG. It is a figure which shows the chromatogram obtained as a result of having applied the grape seed extract obtained by performing liquefied carbon dioxide extraction to HPLC on the same conditions as FIG. FIG. 3 is a diagram showing a chromatogram obtained as a result of subjecting a grape seed extract obtained by hot water extraction from grape seeds to NF membrane treatment and HPLC under the same conditions as in FIG. 2. It is a figure which shows the chromatogram obtained as a result of having applied the catechin obtained from tea to HPLC on the conditions of Table 2 (the lower end of the lower part). It is a chart figure which shows the result of having applied the bacteriostatic agent which passed through partial solidification-> thawing-> filtration (in the case of an ultrafiltration membrane of UF50,000) by cooling on HPLC on the same conditions as FIG. It is a chart figure which shows the result of having applied the bacteriostatic agent which passed through partial solidification-> thawing-> filtration (in the case of a UF20,000 ultrafiltration membrane) by cooling on HPLC on the same conditions as FIG.

Claims (1)

A bacteriostatic agent mainly composed of proanthocyanidins, a processing method for processing and processing into an additive for acidic beverages having a pH of 3.0 to 4.0 ,
The bacteriostatic agent is dissolved in water-containing ethanol, and this is partially solidified by cooling to −30 ° C. to −4 ° C. and then thawed. The precipitate generated after the thaw is subjected to ultrafiltration to obtain a molecular weight of 50,000 The processing method characterized by including the process of removing the above polyphenol .

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