JP2016190836A - Pharmaceutical composition for sterilization - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for sterilization having bactericidal actions to Listeria bacterium causing Listeria disease by infecting newborn or infant, preferably both of Listeria bacterium and general viable bacterium.SOLUTION: There is provided a pharmaceutical composition consisting of a combination of ferulic acid and organic acid such as acetic acid in a range for normal use in foods and having bactericidal actions to Listeria bacterium and general viable bacterium. There is provided a pharmaceutical composition having normal used amount of ferulic acid of 0.5 wt.% or less and normal used amount of organic acid of 4 wt.% or less. There is provided a pharmaceutical compound further containing glycine sodium acetate.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition having a bactericidal action against Listeria monocytogenes, desirably both Listeria monocytogenes and live bacteria.

Listeriosis is an infection caused by Listeria monocytogenes, a facultative anaerobic Gram-positive gonococcus, which is transmitted via the respiratory tract, oral, and contact via food contaminated with this bacterium. Listeriosis infects newborns and infants, causing meningitis and sepsis, and also in pregnant women and those with weakened immune function. Many cases of this food poisoning have been reported with ready-to-eat (RTE) foods that do not require heating or cooking, such as raw milk, salad, natural cheese, and coleslaw.
Acids such as rosmarinic acid have been studied as antibacterial agents or growth inhibitors against Listeria monocytogenes (Patent Document 1, etc.).
Since ferulic acid used in the present invention is obtained by purifying from rice bran etc., there is no safety problem even when used for food, and it is used as an antioxidant for food (Patent Document 1, etc.) . However, if the amount is too large, the taste of the food is affected, so the amount added to the food has an upper limit.
The present inventors have confirmed the growth inhibitory effect of Listeria monocytogenes and general viable bacteria on the combination of nisin, which is a fungicide used in ferulic acid alone and cheese, and ferulic acid (non-native). (Patent Documents 1, 2, etc.)

JP2010-37282

Food Control 33 (2013) 244-248 Food Control 47 (2015) 560-503

The present inventors have confirmed the growth inhibitory effect of Listeria monocytogenes and general viable bacteria on ferulic acid alone and the combination of nisin and ferulic acid in the normal use range (Non-Patent Documents 1 and 2). Etc.), these only showed the growth inhibitory effect of Listeria monocytogenes and general living bacteria, and did not show the bactericidal action against them (see Reference Example 1 described later).
Even if the growth of Listeria monocytogenes and general live bacteria is suppressed, these bacteria may grow again, and the food industry is expected to sterilize these bacteria.
Therefore, an object of the present invention is to provide a bactericidal pharmaceutical composition having a bactericidal action against Listeria monocytogenes, desirably both Listeria monocytogenes and general living bacteria.

As a result of studying the combination of ferulic acid and other drugs, the inventors have confirmed that bactericidal action against both Listeria monocytogenes and general living bacteria within these normal use ranges by combining ferulic acid and organic acid. As a result, the present invention has been completed.
That is, the present invention is a pharmaceutical composition having a bactericidal action against Listeria monocytogenes, or both Listeria monocytogenes and general living bacteria, comprising ferulic acid and an organic acid.
In addition, the present invention is a food to which the pharmaceutical composition is added, in particular, a food (excluding vegetables) including vegetables or vegetables.
The present invention is also a method for sterilizing Listeria monocytogenes, or both Listeria monocytogenes and general living bacteria, comprising adding ferulic acid and organic acid to food.

It is a figure which shows that the pharmaceutical composition of this invention has a bactericidal action. (A) shows bactericidal action against Listeria monocytogenes, and (b) shows bactericidal action against general living bacteria. It is a figure which shows the result of PMA-qPCR analysis when the pharmaceutical composition of this invention is added to coleslaw. It is a figure which shows the growth behavior of L. monocytogenes after adding acetic acid and ferulic acid alone or in a mixture in TSB medium. This is a citation of Fig. 1 of Non-Patent Document 1 (Food Control 33 (2013) 244-248). a shows changes in the growth of L. monocytogenes in cheese, and b shows changes in the growth of general viable bacteria in cheese. ◆ and ■ indicate “with ferulic acid”, and ◇ and □ indicate “without ferulic acid”. Error bars indicate standard deviation of n = 3.

フェルラ酸は、米ぬかを精製する等して得ることができる。また、バニリンと無水酢酸を反応させたり（パーキン反応）、コーヒー酸にo-メチルトランスフェラーゼを作用させることにより、得ることができる。 The pharmaceutical composition of the present invention comprises ferulic acid and an organic acid.
Ferulic acid is an organic compound that is present as a phytochemical in the cell wall of a plant and is represented by the following formula.

Ferulic acid can be obtained by purifying rice bran. Alternatively, it can be obtained by reacting vanillin and acetic anhydride (Parkin reaction) or by allowing o-methyltransferase to act on caffeic acid.

  The usual use amount of ferulic acid is 0.5% by weight or less, preferably 0.05 to 0.3% by weight, more preferably 0.05 to 0.15% by weight with respect to foods such as vegetables. Too much affects the taste of food.

The organic acid used in the present invention is represented by R (—COOH) _n .
In the formula, n represents an integer of 1 to 3.
R represents an n-valent hydrocarbon group having 1 to 6 carbon atoms, and this hydrocarbon group may be aliphatic or aromatic, and may contain a hydroxyl group.
Specifically, as the organic acid, acetic acid, lactic acid, malic acid, citric acid, propionic acid, sorbic acid, benzoic acid, butyric acid, succinic acid and the like generally used as food additives are preferable.
The normal amount of organic acid used is 4% by weight or less, preferably 0.02 to 4% by weight, more preferably 0.05 to 3% by weight, based on foods such as vegetables.

  In addition, the pharmaceutical composition of the present invention may be used in combination with other antibacterial agents, bactericides, and growth inhibitors. Examples of these include polylysine, protamine, nisin, sucrose fatty acid ester, glycine, sodium acetate, and the like. Can be mentioned.

Examples of the food targeted by the pharmaceutical composition of the present invention include vegetables, vegetable salad, coleslaw, eggs, salmon roe, ham, sausage, cheese, dairy products, lunch boxes, prepared dishes, among which vegetables and other vegetables Is suitable as a subject of the pharmaceutical composition of the present invention.
The addition method is not particularly limited. For example, the pharmaceutical composition of the present invention may be prepared by adding it to an aqueous solution, or may be kneaded into a food or the like as a powder.
The pharmaceutical composition of the present invention has a bactericidal action against Listeria monocytogenes, or both Listeria monocytogenes and viable bacteria.
General viable bacteria are a general term for mesophilic bacteria that are present in foods and grow on common media under aerobic conditions, and include E. coli and the like.
In addition, the bactericidal action appears immediately after use.

The following examples illustrate the invention but are not intended to limit the invention.
In the following experiment, L. monocytogenes 2-9 strain isolated from Suiko was used as Listeria.
The number of bacteria was measured using a PALCAM medium, which is a differential medium for Listeria monocytogenes. In each test group, three 25 g samples were prepared and tested, and the results were shown as mean ± standard deviation.

Example 1
Three cabbage leaves purchased at a retail store and the core of the cabbage were removed, cut into an appropriate size, washed with hot water at 55 ° C, and then crushed into approximately 5 mm squares with a food processor. Then, it was immersed in 2% saline for 1 minute and drained. Based on cabbage weight, mayonnaise (Cuppy mayonnaise manufactured by Kewpie Co., Ltd., 20% by weight vinegar acetate (containing 3wt% acetic acid) 12wt%) and ferulic acid (San-Eigen FFI Corporation) 1000ppm or 1500 ppm was added and mixed.
A sample to which ferulic acid was not added was used as a control test group (that is, mayonnaise included).
Inoculate the resulting salad with 2.5 mL of L. monocytogenes culture solution so that the final concentration is about 10 ⁵ CFU / g. Thoroughly remove the sample and the bacterial solution using a stomacher (manufactured by Seaward, 230 rpm, 30 sec). After mixing, it was stored at 10 ° C. for 5 days.
Collect each test group on the 0th, 1st, 2nd, 3rd, 4th, and 5th days of storage, perform serial dilution with physiological saline, smear the diluted solution on PALCAM agar medium, and determine the number of L. monocytogenes. It was measured.

The results are shown in FIG.
In the test plots where ferulic acid 1000 ppm and 1500 ppm were added, the bactericidal effect of L. monocytogenes in the salad was confirmed.
In the control test plot, there was no increase in the number of L. monocytogenes during the 5-day storage period, while the number of inoculated bacteria was 5.38 log CFU / g, while the number of L. monocytogenes on the fifth day of storage was 5.25 log CFU / g. Met.
In the ferulic acid-added section, a decrease in the number of L. monocytogenes was confirmed after the first day of storage, and the number of L. monocytogenes on the fifth day of storage was 3.98 log CFU / g at 1000 ppm addition and 3.74 log CFU at 1500 ppm addition / g.

Example 2
In order to observe the effect on the number of viable bacteria, a sample stored in the same manner as in Example 1 was prepared without inoculating the salad prepared in Example 1 with the bacteria. After storage, samples collected each day were serially diluted with physiological saline, and the number of viable bacteria was measured by the pour method using TSA medium.
The results are shown in FIG.
In the control test group, an increase in the number of bacteria was confirmed after the fourth day, and the number of bacteria on the fifth day of storage was 4.31 log CFU / g.
The number of bacteria on the fifth day of storage in the ferulic acid-added group was 2.32 log CFU / g when 1000 ppm was added, and 2.70 log CFU / g when 1500 ppm was added, indicating a decrease in the number of bacteria compared to the control test group. It was.

In Examples 1 and 2 (FIGS. 1 (a) and (b)), the numbers of L. monocytogenes and general viable bacteria decreased with time, indicating that the pharmaceutical composition of the present invention has a bactericidal action. Yes.
In contrast, the addition of ferulic acid alone (Reference Example 1) does not decrease the number of L. monocytogenes and general viable bacteria over time, and has an anti-growth effect but no bactericidal effect.

Example 3
Changes in the number of L. monocytogenes bacteria by quantitative real-time PCT (qPCR) using PMA (R) (Propidium Monoazide, Cosmo Bio Inc.) with 1000 ppm ferulic acid added to coleslaw (containing 0.07 wt% acetic acid) I investigated.
The results are shown in FIG.
Since PMA inhibits the PCR reaction when bound to nucleic acid, the difference between “without PMA” and “with PMA” in FIG. 2 corresponds to a dead cell whose cell membrane has been destroyed. That is, FIG. 2 shows that L. monocytogenes was sterilized by the pharmaceutical composition of the present invention.

Example 4
The following four types of media were prepared, and these were inoculated with Listeria monocytogenes (at the time of inoculation: 5.6 log CFU / mL).
(1) 2000 ppm ferulic acid was added to the TSB medium.
(2) 3000 ppm acetic acid was added to the TSB medium.
(3) 3000 ppm acetic acid and 2000 ppm ferulic acid were added to the TSB medium.
(4) TSB medium only as a control These were stored at 10 ° C. for 5 days and collected on days 0, 1, 3, and 5. The collected sample was serially diluted, and the solution was smeared on TSA medium and cultured at 30 ° C. for 48 hours, and then the number of bacteria was measured. The results are shown in FIG.
When acetic acid or ferulic acid was added alone, L. monocytogenes was found to have a growth-inhibiting effect but not a bactericidal effect (acetic acid only, day 0: 5.4 log CFU / mL, 5 days) Eye: 5.3 log CFU / mL; Ferulic acid only, Day 0: 5.5 log CFU / mL, Day 5: 6.3 log CFU / mL).
On the other hand, when acetic acid and ferulic acid were added in combination, they showed a bactericidal effect on L. monocytogenes, and the number of L. monocytogenes bacteria decreased by 1.78 log CFU / mL on day 5 (day 0: 5.3). log CFU / mL, day 5: 3.9 log CFU / mL).

Reference example 1
Although the present inventors have already added ferulic acid to cheese or the like already inoculated with L. monocytogenes (2000 ppm ferulic acid relative to cheese), they reported changes in the number of L. monocytogenes bacteria (non-patented). Reference 1: Food Control 33 (2013) 244-248), and Figure 1 is cited (Figure 4).
The change in the growth of L. monocytogenes and general living bacteria in Fig. 4 shows that the number of bacteria with "ferulic acid (◆ and ■)" is less than that without "ferulic acid (◇ and □)". Yes (◆ and ■) data does not decrease over time. This indicates that the addition of ferulic acid alone has a growth inhibitory effect but no bactericidal effect.

Claims (9)

A pharmaceutical composition comprising bactericidal action against Listeria monocytogenes, or both Listeria monocytogenes and viable bacteria, comprising ferulic acid and an organic acid. The pharmaceutical composition according to claim 1, wherein the amount of ferulic acid and organic acid used for food is within the range of normal usage. The pharmaceutical composition according to claim 2, wherein the normal use amount of ferulic acid is 0.5% by weight or less and the normal use amount of organic acid is 4% by weight or less based on the weight of the food. The pharmaceutical composition according to claim 2 or 3, wherein the food is a vegetable or a food containing vegetables (excluding vegetables). Furthermore, the pharmaceutical composition as described in any one of Claims 1-4 containing glycine sodium acetate. The foodstuff to which the pharmaceutical composition as described in any one of Claims 1-5 was added. The food according to claim 6, wherein the food is a vegetable or a food containing vegetables (excluding vegetables). A method of sterilizing Listeria monocytogenes, or both Listeria monocytogenes and live bacteria, comprising adding ferulic acid and organic acid to food. The method according to claim 8, wherein the food is a vegetable or a food containing vegetables (excluding vegetables).

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