# Study of the Antibacterial Efficacy of Bainiku-Ekisu against Pathogens

Authors: Deng-Jye Yang; Hsin-Yi Chen; Shih-Chuan Liu
Journal: International Journal of Bacteriology
 (2014)

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## Abstract

The research was undertaken to determine the bacteriostatic effects of the concentrate of Japanese apricot juice (bainiku-ekisu), which is a popular health food in Taiwan and Japan, onEnterococcus faecalis ATCC 29212, Staphylococcus aureus ATCC 25923, and Escherichia coli ATCC 25922. The results show that E. faecalis, S. aureus, and E. coli could be killed or inhibited by bainiku-ekisu at concentrations between 1.0 and 10.0 mg/mL. The minimum inhibitory concentration (MIC) was 1 mg/mL for all strains, and the minimum bactericidal concentrations (MBCs) were 5, 2.5, and 2.5 mg/mL for E. faecalis, S. aureus, and E. coli, respectively. Using the growth rate to calculate the MICs and MBCs, the MICs were 1.55, 1.43, and 0.97 mg/mL, and the MBCs were 2.59, 2.63, and 2.25 mg/mL for E. faecalis, S. aureus, and E. coli, respectively. According to the D values, E. faecalis and S. aureus exhibited lower resistance than E. coli at lower bainiku-ekisu concentrations (1.0 and 2.5 mg/mL), and the resistance of these two pathogens was better than that of E. coli at higher bainiku-ekisu concentrations (5.0 and 10.0 mg/mL). The Z values of theE. faecalis,S. aureus, andE. coli strains were 3.47, 4.93, and 11.62 mg/mL, respectively.


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## Body

## 1. Introduction

Enterococcus faecalis,Staphylococcus aureus, andEscherichia coli have been recognized as important pathogens that cause human disease and regularly infect hospitalized patients.S. aureus andE. coli are also common causes of food poisoning. Antibiotics are the major tools to treat bacterial infections and cure patients. However, bacteria are highly adaptable and can develop resistance to antibiotics, yielding resistant pathogens such as methicillin-resistantS. aureus (MRSA) [1]. These antibiotic-resistant bacteria pose significant dangers to hospitalized patients because infections with resistant bacteria are difficult to treat with antibiotics. Therefore, it is important to reduce antibiotic consumption to eliminate the development of antibiotic-resistant bacteria.In clinical settings, antibacterial effects are usually quantified as the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) [2]. These methods are easy ways to determine the curative antibiotic dose; however, the dose range is wide. The use of more antibiotics might produce antibiotic-resistant bacteria. The decimal reduction time (D value) and the resistance coefficient (Z value) are widely used in thermal death time (TDT) data analysis to determine the heat resistance of the bacteria [3]. The difference between theD andZ values is dependent on concentration and temperature. TheD andZ values can be used to describe the drug resistance of bacteria, and these data can provide a more accurate estimate of the curative dose.Bainiku-ekisu is the concentrate of Japanese apricot juice. It has a pH of approximately 2.85, and its major components are citric acid and malic acid (40–53%) [4]. Bainiku-ekisu is a popular product in Taiwan and Japan because consumers believe that it has many types of physiological activities, such as antioxidant activity [5], the ability to improve blood fluidity [6], anti-cancer activity [7] and antibacterial activity [8, 9]. Fujita et al. [8] demonstrated that bainiku-ekisu exhibits strong bacteriostatic effects at a concentration less than 0.156% (w/w) against fourHelicobacter pylori stains and at a concentration less than 0.313% against sixH. pylori strains. When these tenH. pylori strains were mixed with 0.3% and 0.9% bainiku-ekisu, respectively, strong bacteriostatic effects were observed after 5 minutes. Nakajima et al. [9] also demonstrated that the urea breath test (UBT) values ofH. pylori-positive patients were decreased slightly when the patients were treated with 1% bainiku-ekisu solution for 2 weeks.According to these studies, bainiku-ekisu exhibits strong bacteriosterile effects; therefore, this product could be used to treat and prevent pathogenic bacterial infections and could reduce antibiotic consumptions. The objectives of this research were to assess the antibacterial effects of bainiku-ekisu onE. faecalis,S. aureus and,E. coli and to measure the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), decimal reduction time (D value) and resistance coefficient (Z value).


## 2. Materials and Methods

### 2.1. Preparation of Bainiku-Ekisu

Semi-ripe Japanese apricot fruits were harvested from trees and purchased from Hsinyi Hsiang, Nantou Prefecture, located in the middle part of Taiwan, during the spring of 2006. The pits were removed, and the fruit was minced. The minced fruit was filtered through cheese cloth, and the juice was boiled at temperature around 100°C for 8 hours to produce the bainiku-ekisu concentration. Before analysis, bainiku-ekisu was reconstituted to the fresh juice with de-ionized water. In the antibacterial assays, the bainiku-ekisu was diluted with de-ionized water to concentrations of 0.5, 1.0, 2.5, 5 and 10 mg/mL.


### 2.2. Determination of the Characteristics of Fresh Japanese Apricot Juice and Bainiku-Ekisu

All solvents were purchased from Merck Co. (Darmstadt, Germany), and all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The fresh Japanese apricot juice and the bainiku-ekisu were analyzed to determine the pH, °Brix, total acidity, total phenolic content and total flavonoid content. The pH values of samples were determined with a pH meter (Model SP-71, Suntex Inc., Taiwan). The °Brix was measured with a hand-held refractometer (ATAGO, Japan). The total acidity of the samples was measured according to the AOAC [10]. The total concentration of reducing sugars was determined using the dinitrosalicylic acid (DNS) method [11].The total phenolic content was determined using the method in the report of Julkunen-Tiitto [12]. A 50 μL aliquot of sample or standard solution was mixed with 1 mL of de-ionized water and 0.5 mL of Folin-Ciocalteu’s phenol reagent. The mixture was then added to 2.5 mL of 20% Na2CO3 solution and incubated at ambient temperature in the dark for 20 min. The absorbance of the mixture was measured at 735 nm (Spectro UV-Vis Auto Spectrophotometer, Labomed Inc., Culver City, CA, USA). A standard curve was constructed using gallic acid, and the total phenolic content was expressed as mg gallic acid equivalents (GAE)/g sample.The total flavonoid contents in samples were determined according to the method of Zhishen et al. [13] with some modifications. An aliquot (250 μL) of the sample or a standard solution was mixed with de-ionized water (1.25 mL) and 5% NaNO2 solution (75 μL) for 6 min. To the mixture was added 10% AlCl3·H2O solution (150 mL) and mixed for 5 min, and then 0.5 mL of 1 M NaOH solution was added. The total volume of the mixture was brought up to 2.5 mL with de-ionized water, and the absorbance at 510 nm was determined. The standard curve was constructed using (+)-catechin, and the total flavonoid content was expressed as mg catechin equivalents (CE)/g sample.


### 2.3. Determination of the Antioxidative Activities of Fresh Japanese Apricot Juice and Bainiku-Ekisu

The antioxidative activities of fresh Japanese apricot juice and bainiku-ekisu were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS.+ radical-scavenging assays, and each of these methods is described below.


#### 2.3.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical-Scavenging Effect

The DPPH radical-scavenging activity was determined using a modification of the method of Espín et al. [14]. Samples of 20 μL each were dispensed into a 96-well plate, and then 200 μL of 0.2 mM DPPH solution (prepared with methanol) was added to each well. These plates were incubated at 37°C in a dark chamber for 30 min and then put in a microplate reader (Multiskan Spectrum, Thermo, Vantaa, Finland) to measure the absorbance at 517 nm (A517). The DPPH scavenging activity was evaluated based on the percentage of DPPH radicals scavenged using the following equation:
(1)SDPPH={Sb-(Sc-Ss)}Sb×100,
where SDPPH is the DPPH radical-scavenging activity expressed as a percentage, Sb is the absorbance of the blank treatment, Sc is the absorbance of the sample, and Ss is the background absorbance of the sample.


#### 2.3.2. ABTS.+ Radical-Scavenging Effect

This assay was performed using the assay of Scalzo et al. [15] with some modifications. ABTS solution was prepared by mixing peroxidase, H2O2 and 2,2-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) in de-ionized water to yield a final peroxidase activity of 4.4 U/mL, an H2O2 concentration 100 mM, and an ABTS concentration 50 μM. This solution was incubated at 30°C in a dark chamber for 1 hour. A 30 μL aliquot of each sample was loaded onto a 96-well plate before dispensing 250 μL of ABTS solution into each well. The plate was then put into the microplate reader to measure the absorbance at 734 nm (A734) after standing for 3 min. The ABTS.+ radical-scavenging activity (SABTS) was calculated by the following equation:
(2)SABTS=[Ab-(Ac-As)]Ab×100,
where SABTS is ABTS.+ radical-scavenging activity expressed as a percentage, Ab is the absorbance of the blank treatment, Ac is the absorbance of the sample, and As is the background absorbance of the sample.


### 2.4. Measurement of the Antibacterial Effect

E. faecalis ATCC 29212 (Gram-positive),S. aureus ATCC 25923 (Gram-positive) andE. coliATCC 25922 (Gram-negative) were used to evaluate the antibacterial effects of bainiku-ekisu. These strains were incubated in Mueller Hinton broth (DIFCO, US) at 37°C. After incubation, the concentration of the bacteria was adjusted to approximately 1–5×105 CFU/mL. Different bainiku-ekisu concentrations were added to the incubated cultures. The cultures were then incubated at 37°C and sampled at intervals to count the number of bacteria. A 1 mL aliquot of the sample was inoculated onto a Mueller Hinton agar plate (DIFCO, US), and the plates were incubated in an inverted position at 37°C for 24 hours. After incubation, the number of colonies was determined.


### 2.5. Data Analyses

The antibacterial effects of bainiku-ekisu are discussed in the following sections.


#### 2.5.1. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

The determination of MIC and MBC is recommended by the NCCLS [2]. The MIC was determined as the lowest concentration of the tested compound in which the number of bacteria was not significantly different from the number of bacteria in the control after 18–24 hours of incubation at 37°C. The MBC was determined as the lowest concentration of the tested compound in which the number of bacteria was reduced by 99.9% relative to the CFU/mL of the control.


#### 2.5.2. Determination of the Decimal Reduction Time (D Value) and the Resistance Coefficient (Z Value)

TheD andZ values are widely used in thermal death time (TDT) data analyses to determine the resistance of bacteria. The decimal reduction time (D value) is calculated from the linear part of thermal death time curve and is equal to the time required to reduce the number of bacteria by a factor of 10 (i.e., one log count reduction). TheZ value is calculated from the regression of log (D) versus temperature and is equal to change in temperature necessary to reduce the decimal reduction time by a factor of 10. In the study, theZ value was calculated from the regression of log (D) versus the bainiku-ekisu concentration and was equal to the change in concentration necessary to reduce the decimal reduction time by a factor of 10.


### 2.6. Statistical Analysis

All experiments were carried out in triplicate. The data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests were used to identify significant differences between means at a significance level ofP<0.05.


## 2.1. Preparation of Bainiku-Ekisu

Semi-ripe Japanese apricot fruits were harvested from trees and purchased from Hsinyi Hsiang, Nantou Prefecture, located in the middle part of Taiwan, during the spring of 2006. The pits were removed, and the fruit was minced. The minced fruit was filtered through cheese cloth, and the juice was boiled at temperature around 100°C for 8 hours to produce the bainiku-ekisu concentration. Before analysis, bainiku-ekisu was reconstituted to the fresh juice with de-ionized water. In the antibacterial assays, the bainiku-ekisu was diluted with de-ionized water to concentrations of 0.5, 1.0, 2.5, 5 and 10 mg/mL.


## 2.2. Determination of the Characteristics of Fresh Japanese Apricot Juice and Bainiku-Ekisu

All solvents were purchased from Merck Co. (Darmstadt, Germany), and all chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The fresh Japanese apricot juice and the bainiku-ekisu were analyzed to determine the pH, °Brix, total acidity, total phenolic content and total flavonoid content. The pH values of samples were determined with a pH meter (Model SP-71, Suntex Inc., Taiwan). The °Brix was measured with a hand-held refractometer (ATAGO, Japan). The total acidity of the samples was measured according to the AOAC [10]. The total concentration of reducing sugars was determined using the dinitrosalicylic acid (DNS) method [11].The total phenolic content was determined using the method in the report of Julkunen-Tiitto [12]. A 50 μL aliquot of sample or standard solution was mixed with 1 mL of de-ionized water and 0.5 mL of Folin-Ciocalteu’s phenol reagent. The mixture was then added to 2.5 mL of 20% Na2CO3 solution and incubated at ambient temperature in the dark for 20 min. The absorbance of the mixture was measured at 735 nm (Spectro UV-Vis Auto Spectrophotometer, Labomed Inc., Culver City, CA, USA). A standard curve was constructed using gallic acid, and the total phenolic content was expressed as mg gallic acid equivalents (GAE)/g sample.The total flavonoid contents in samples were determined according to the method of Zhishen et al. [13] with some modifications. An aliquot (250 μL) of the sample or a standard solution was mixed with de-ionized water (1.25 mL) and 5% NaNO2 solution (75 μL) for 6 min. To the mixture was added 10% AlCl3·H2O solution (150 mL) and mixed for 5 min, and then 0.5 mL of 1 M NaOH solution was added. The total volume of the mixture was brought up to 2.5 mL with de-ionized water, and the absorbance at 510 nm was determined. The standard curve was constructed using (+)-catechin, and the total flavonoid content was expressed as mg catechin equivalents (CE)/g sample.


## 2.3. Determination of the Antioxidative Activities of Fresh Japanese Apricot Juice and Bainiku-Ekisu

The antioxidative activities of fresh Japanese apricot juice and bainiku-ekisu were determined using DPPH (2,2-Diphenyl-1-picrylhydrazyl) and ABTS.+ radical-scavenging assays, and each of these methods is described below.


### 2.3.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical-Scavenging Effect

The DPPH radical-scavenging activity was determined using a modification of the method of Espín et al. [14]. Samples of 20 μL each were dispensed into a 96-well plate, and then 200 μL of 0.2 mM DPPH solution (prepared with methanol) was added to each well. These plates were incubated at 37°C in a dark chamber for 30 min and then put in a microplate reader (Multiskan Spectrum, Thermo, Vantaa, Finland) to measure the absorbance at 517 nm (A517). The DPPH scavenging activity was evaluated based on the percentage of DPPH radicals scavenged using the following equation:
(1)SDPPH={Sb-(Sc-Ss)}Sb×100,
where SDPPH is the DPPH radical-scavenging activity expressed as a percentage, Sb is the absorbance of the blank treatment, Sc is the absorbance of the sample, and Ss is the background absorbance of the sample.


### 2.3.2. ABTS.+ Radical-Scavenging Effect

This assay was performed using the assay of Scalzo et al. [15] with some modifications. ABTS solution was prepared by mixing peroxidase, H2O2 and 2,2-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) in de-ionized water to yield a final peroxidase activity of 4.4 U/mL, an H2O2 concentration 100 mM, and an ABTS concentration 50 μM. This solution was incubated at 30°C in a dark chamber for 1 hour. A 30 μL aliquot of each sample was loaded onto a 96-well plate before dispensing 250 μL of ABTS solution into each well. The plate was then put into the microplate reader to measure the absorbance at 734 nm (A734) after standing for 3 min. The ABTS.+ radical-scavenging activity (SABTS) was calculated by the following equation:
(2)SABTS=[Ab-(Ac-As)]Ab×100,
where SABTS is ABTS.+ radical-scavenging activity expressed as a percentage, Ab is the absorbance of the blank treatment, Ac is the absorbance of the sample, and As is the background absorbance of the sample.


## 2.3.1. DPPH (2,2-Diphenyl-1-picrylhydrazyl) Radical-Scavenging Effect

The DPPH radical-scavenging activity was determined using a modification of the method of Espín et al. [14]. Samples of 20 μL each were dispensed into a 96-well plate, and then 200 μL of 0.2 mM DPPH solution (prepared with methanol) was added to each well. These plates were incubated at 37°C in a dark chamber for 30 min and then put in a microplate reader (Multiskan Spectrum, Thermo, Vantaa, Finland) to measure the absorbance at 517 nm (A517). The DPPH scavenging activity was evaluated based on the percentage of DPPH radicals scavenged using the following equation:
(1)SDPPH={Sb-(Sc-Ss)}Sb×100,
where SDPPH is the DPPH radical-scavenging activity expressed as a percentage, Sb is the absorbance of the blank treatment, Sc is the absorbance of the sample, and Ss is the background absorbance of the sample.


## 2.3.2. ABTS.+ Radical-Scavenging Effect

This assay was performed using the assay of Scalzo et al. [15] with some modifications. ABTS solution was prepared by mixing peroxidase, H2O2 and 2,2-azino-bis-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS) in de-ionized water to yield a final peroxidase activity of 4.4 U/mL, an H2O2 concentration 100 mM, and an ABTS concentration 50 μM. This solution was incubated at 30°C in a dark chamber for 1 hour. A 30 μL aliquot of each sample was loaded onto a 96-well plate before dispensing 250 μL of ABTS solution into each well. The plate was then put into the microplate reader to measure the absorbance at 734 nm (A734) after standing for 3 min. The ABTS.+ radical-scavenging activity (SABTS) was calculated by the following equation:
(2)SABTS=[Ab-(Ac-As)]Ab×100,
where SABTS is ABTS.+ radical-scavenging activity expressed as a percentage, Ab is the absorbance of the blank treatment, Ac is the absorbance of the sample, and As is the background absorbance of the sample.


## 2.4. Measurement of the Antibacterial Effect

E. faecalis ATCC 29212 (Gram-positive),S. aureus ATCC 25923 (Gram-positive) andE. coliATCC 25922 (Gram-negative) were used to evaluate the antibacterial effects of bainiku-ekisu. These strains were incubated in Mueller Hinton broth (DIFCO, US) at 37°C. After incubation, the concentration of the bacteria was adjusted to approximately 1–5×105 CFU/mL. Different bainiku-ekisu concentrations were added to the incubated cultures. The cultures were then incubated at 37°C and sampled at intervals to count the number of bacteria. A 1 mL aliquot of the sample was inoculated onto a Mueller Hinton agar plate (DIFCO, US), and the plates were incubated in an inverted position at 37°C for 24 hours. After incubation, the number of colonies was determined.


## 2.5. Data Analyses

The antibacterial effects of bainiku-ekisu are discussed in the following sections.


### 2.5.1. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

The determination of MIC and MBC is recommended by the NCCLS [2]. The MIC was determined as the lowest concentration of the tested compound in which the number of bacteria was not significantly different from the number of bacteria in the control after 18–24 hours of incubation at 37°C. The MBC was determined as the lowest concentration of the tested compound in which the number of bacteria was reduced by 99.9% relative to the CFU/mL of the control.


### 2.5.2. Determination of the Decimal Reduction Time (D Value) and the Resistance Coefficient (Z Value)

TheD andZ values are widely used in thermal death time (TDT) data analyses to determine the resistance of bacteria. The decimal reduction time (D value) is calculated from the linear part of thermal death time curve and is equal to the time required to reduce the number of bacteria by a factor of 10 (i.e., one log count reduction). TheZ value is calculated from the regression of log (D) versus temperature and is equal to change in temperature necessary to reduce the decimal reduction time by a factor of 10. In the study, theZ value was calculated from the regression of log (D) versus the bainiku-ekisu concentration and was equal to the change in concentration necessary to reduce the decimal reduction time by a factor of 10.


## 2.5.1. Determination of the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC)

The determination of MIC and MBC is recommended by the NCCLS [2]. The MIC was determined as the lowest concentration of the tested compound in which the number of bacteria was not significantly different from the number of bacteria in the control after 18–24 hours of incubation at 37°C. The MBC was determined as the lowest concentration of the tested compound in which the number of bacteria was reduced by 99.9% relative to the CFU/mL of the control.


## 2.5.2. Determination of the Decimal Reduction Time (D Value) and the Resistance Coefficient (Z Value)

TheD andZ values are widely used in thermal death time (TDT) data analyses to determine the resistance of bacteria. The decimal reduction time (D value) is calculated from the linear part of thermal death time curve and is equal to the time required to reduce the number of bacteria by a factor of 10 (i.e., one log count reduction). TheZ value is calculated from the regression of log (D) versus temperature and is equal to change in temperature necessary to reduce the decimal reduction time by a factor of 10. In the study, theZ value was calculated from the regression of log (D) versus the bainiku-ekisu concentration and was equal to the change in concentration necessary to reduce the decimal reduction time by a factor of 10.


## 2.6. Statistical Analysis

All experiments were carried out in triplicate. The data were subjected to analysis of variance (ANOVA), and Duncan’s multiple range tests were used to identify significant differences between means at a significance level ofP<0.05.


## 3. Results and Discussion

### 3.1. The Characteristics of Fresh Japanese Apricot Juice and Bainiku-Ekisu

To compare fresh Japanese apricot juice and bainiku-ekisu, bainiku-ekisu was diluted to the concentration of fresh juice using de-ionized water. The characteristics of fresh Japanese apricot juice and bainiku-ekisu are shown in Table1. The pH levels of fresh juice and bainiku-ekisu were lower than pH 3, indicating that the concentration process was performed under acidic conditions. The °Brix, total acidity and total amount of reducing sugars for bainiku-ekisu were significant lower than those for fresh juice. Those results may due to that the Maillard reaction, the caramelization reaction and mumefural generation might take place during juice concentration at high temperatures [6, 16].Table 1
Characteristics of fresh Japanese apricot juice and bainiku-ekisu.
Fresh juice
Bainiku-ekisu1
pH
2.67 ± 0.007b8
2.83 ± 0.02a
°Brix
4.9 ± 0.06a
4.1 ± 0.03b
Total acidity2 (g/100 mL)
2.18 ± 0.15a
1.77 ± 0.02b
Total amount of reducing sugar3 (mg/100 mL)
143.11 ± 0.14a
55.71 ± 1.34b
Total phenolic content4 (mg/100 mL)
26.44 ± 0.16b
51.00 ± 1.26a
Total flavonoid content5 (mg/100 mL)
11.39 ± 0.29b
16.60 ± 0.8a
EC50 of DPPH radical-scavenging activity6 (mL sample)
0.11 ± 0.00a
0.04 ± 0.00b
EC50 of ABTS·+ radical-scavenging activity (mL sample)
0.16 ± 0.00a
0.05 ± 0.00b
1Samples were diluted to the same concentration as fresh juice with de-ionized water.
2g citric acid/100 mL sample.
3mg glucose/100 mL sample.
4mg gallic acid equivalent/100 mL sample.
5mg catechin equivalent/100 mL sample.
6Volume of sample needed.
7Data are presented as the means ± standard deviations (n=3).
8Different letters for the individual extracts indicate that the values are significantly different (P<0.05).The total phenolic and flavonoid contents of bainiku-ekisu were higher than those of fresh juice (Table1). During heating at high temperature for a long time, phenolic compounds and flavonoids can be destroyed; however, the phenolic and flavonoid contents were found increased in this study. There are a few articles that reported the total phenolic and flavonoid contents of citrus peels increased during high temperature treatment [17–20]. Several low-molecular-weight phenolic compounds might form inCitrus unshiu peels heated at 150°C for 30 min [21]. Que et al. [19] proposed that the phenolic compounds might be formed from the precursors of phenolic molecules via non-enzymatic interconversion between phenolic molecules. The increases in the DPPH and ABTS.+ radical-scavenging activities could be due to the higher phenolic and flavonoid contents. Furthermore, the products of the Maillard reaction and the caramelization reaction could exhibit antioxidative activities [16, 22, 23]. The conversion of the EC50values for the DPPH and ABTS.+ radical-scavenging activities of bainiku-ekisu into weights of the final product yielded values of 2.61 and 3.33 mg/mL, respectively.


### 3.2. Measurement of the Antibacterial Effects

#### 3.2.1. MIC and MBC

Figure1 shows the growth patterns ofE. faecalis when treated with/without different bainiku-ekisu concentrations (0–10 mg/mL). WhenE. faecalis was incubated without bainiku-ekisu, the bacteria counts increased steadily during the 12 hours incubation. The bainiku-ekisu solutions affected the growth patterns ofE. faecalis.Schoenknecht et al. [24] described 4 types of “killing curves” in clinical research. When 5 and 10 mg/mL bainiku-ekisu solutions were used, the number of bacteria decreased to zero within 4 and 0.5 hours, respectively. The results demonstrate that bainiku-ekisu can inhibit and killE. faecalis (type D curve). The number of bacteria slightly decreased in the presence 2.5 mg/mL bainiku-ekisu. The killing type was similar to type D, but rate of the decrease was slower than that for 5 mg/mL. The killing type of 1 mg/mL bainiku-ekisu was type B, which involves a lag phase increase. The killing type of 0.5 mg/mL bainiku-ekisu was type C, which involves a decrease in the growth rate, with little effect on the lag time. Type A effect (“percentage growth inhibition”) was also observed in this study.Figure 1
Growth ofEnterococcus faecalis in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The MIC and MBC of bainiku-ekisu were 1 mg/mL and 5 mg/mL, respectively. Hoellman et al. [25] reported that the MICs of ampicillin and vancomycin for enterococci were 0.3 μg/mL and 4.2 μg/mL, respectively. The amount of bainiku-ekisu used in this study was higher than amounts of antibiotics used in their study, but bainiku-ekisu is a natural product. The antimicrobial activities (MIC and MBC) ofCyperus rotundus ethyl acetate extract forE. faecalis were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu were better than those ofCyperus rotundus ethyl acetate extract.Figure2 shows that the growth patterns ofS. aureus when incubated with bainiku-ekisu (0–10 mg/mL). WhenS. aureus was incubated without bainiku-ekisu, the number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL. The growth patterns ofS. aureus were affected when bainiku-ekisu solutions were added. When the 5 and 10 mg/mL bainiku-ekisu solutions were added, the number of bacteria decreased to zero within 24 and 4 hours, respectively. The kill rates forS. aureus were slower than those forE. faecalis. However, the 2.5 mg/mL bainiku-ekisu markedly killedS. aureus, resulting in 101 CFU/mL after incubation for 24 hours. This inhibitory activity was better than that againstE. faecalis. In contrast, the results for the treatment ofS. aureus with 0.5 and 1 mg/mL bainiku-ekisu were similar to the results for the treatment ofE. faecalis after a 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. Similarly, the MIC and MBC ofC. rotundus ethyl acetate extract forS. aureus were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstS. aureus were significant better than the effect ofCyperus rotundus ethyl acetate extract.Figure 2
Growth ofStaphylococcus aureus in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The growth patterns ofE. coli incubated with bainiku-ekisu (0–10 mg/mL) are shown in Figure 3. The number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL when incubated without bainiku-ekisu. The rate ofE. colikilling was the slowest when using 5 and 10 mg/mL bainiku-ekisu solutions. Under these conditions, the number of bacteria decreased to zero within 24 and 6 hours, respectively. For the 24 hours incubation, the effects of 2.5 mg/mL bainiku-ekisu onE. coli were similar to the effects onS. aureus; the number of bacteria was approximately 101 CFU/mL. However, the 1 mg/mL bainiku-ekisu solution could killE. coli, and the number of bacteria decreased to approximately 102 CFU/mL. The rate ofE. colikilling was the fastest for the 1 mg/mL bainiku-ekisu solution among three bacteria. In contrast, the 0.5 mg/mL bainiku-ekisu solution could not inhibit the growth ofE. coliduring the 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. The MIC and MBC ofCyperus rotundus ethyl acetate extract forE. coli were 5 and >5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstE. coli were significantly better than the effects ofC. rotundus ethyl acetate extract.Figure 3
Growth ofEscherichia coliin the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.Polyphenolic compounds are reported to play a role as antimicrobials [27–29]. Kilani-Jaziri et al. [26] reported that intermediate-polarity polyphenolic compounds (ethyl acetate extract) are active antibacterials. Bainiku-ekisu is a concentrated product that contains full polyphenolic compounds, and therefore, the antimicrobial activity of bainiku-ekisu was outstanding. Kilani et al. [30] reported that coumarin is the major antimicrobial compound in the TOF (Total Oligomers Flavonoids)-enriched extract ofC. rotundus. Further investigation is required to identify the compound(s) responsible for the antimicrobial activity of bainiku-ekisu.


#### 3.2.2. Growth Rates,D Values andZ Values

The MICs forE. faecalis,S. aureus, andE. coliwere all 1 mg/mL. However, by definition, the real MIC ofE. coli should be between 0.5 and 1 mg/mL since the numberi ofE. coli decreased to approximately 102 CFU/mL during incubation with 1 mg/mL. When using this antibiotic, the concentration would be 1 mg/mL, which is slightly high. The MBCs forS. aureus andE. coliwere the same (2.5 mg/mL), butS. aureus was more easily destroyed thanE. coliwith 2.5, 5.0 and 10.0 mg/mL bainiku-ekisu (Figures 2 and 3). The exact effective doses might not be the same forS. aureus andE. coli, thus the growth rate,D value andZ value were used to calculate MIC and MBC.The linear parts of the growth curves can be expressed with simple linear equations(R2≥0.910), where the slopes were used as the growth rates (vG) (Table 2). The positive growth rate meant that bainiku-ekisu could not kill or inhibit these stains at certain concentration, such as 0.5 mg/mL, and the negative growth rate showed the opposite tendency. MIC was defined that the lowest tested concentration in which the number of bacteria did not vary significantly during 24 hours; that is, the growth rate of the bacterium was around zero. For the MIC calculation, the linear regression of the growth rates with different bainiku-ekisu concentrations is shown in Figure 4(a) and the linear regression equations forE. faecalis,S. aureus, andE. coli were Y1=-1.007X+1.556 (R2=0.9312), Y2=-0.234X+0.3335 (R2=0.9855) and Y3=-0.0589X+0.0570 (R2=0.7767), respectively. Y1, Y2, and Y3 represent the growth rates, and X was the bainiku-ekisu concentration. When Y1, Y2, and Y3 set at zero, the calculated MICs (X) were found at 1.55, 1.43 and 0.97 mg/mL, respectively. These calculated MICs by growth rates forE. faecalis,andS. aureus were higher than the MICs by traditional values, and the calculated MIC forE. colishowed the opposite result. The MBC was determined to be the lowest tested concentration in which the number of bacteria was reduced 99.9% in CFU/mL; that is, the number of bacteria decreased to 0.1% of the control over 24 hours, and the growth rate of bacterium could be calculated as −0.125 (log⁡10-3 CFU/mL/24 hours). When the slope was −0.125 in the regression equation, the MBCs of bainiku-ekisu were 2.59, 2.63 and 2.25 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These calculated MBCs were different to the traditional MBCs.Table 2
Growth rates andD values (decimal reduction time) of Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli treated with various bainiku-ekisu concentrations during incubation.
Bainiku-ekisu concentration (mg/mL)
0.5
1.0
2.5
5.0
10
0.5
1.0
2.5
5.0
10
Strain
Growth rate
D value
v
G (Δlog⁡(CFU)/hour)
(hour)
E. faecalis
0.162
−0.035
−0.047
−2.106
−9.323
—1
28.7
21.1
0.47
0.11
S. aureus
0.188
−0.029
−0.086
−0.794
−2.049
—
34.5
11.6
1.26
0.49
E. coli
0.199
−0.070
−0.136
−0.372
−0.455
—
14.2
7.34
2.69
2.20
1Antibacterial effect is not sufficient to calculate the D value.Correlations between the bainiku-ekisu concentration and both the growth rate (a) and theD value (decimal reduction time) (b) forEnterococcus faecalis (●),Staphylococcus aureus (○), andEscherichia coli (▼) during incubation.
(a)
(b)The calculatedD values are listed at Table 2. For the 0.5 mg/mL concentration, theD values were not assessable and meaningless because the number of bacteria increased during incubation. A higherD value indicates that the bacterium has a higher level of resistance. It is interesting thatE. faecalisandS. aureus,exhibited higher levels of resistance thanE. coli did at lower bainiku-ekisu concentrations (1.0 and 2.5 mg/mL); in contrast,E. coliexhibited a higher level of resistance at higher concentrations (5.0 and 10.0 mg/mL). Figure 4(b) shows the linear regression of theD value versus the bainiku-ekisu concentration. The regression equations forE. faecalis,S. aureus, andE. coli were Y4=-0.2878X+1.7036 (R2=0.8843), Y5=-0.2029X+1.536 (R2=0.8834) and Y6=-0.086X+1.0954 (R2=0.7978), respectively. Y4, Y5, and Y6 were the logarithms of theD values, and X was the bainiku-ekisu concentration. According to the definition, theZ values can be calculated from these equations, and they were 3.47, 4.93 and 11.62 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. TheZ value ofE. coli was significantly higher than the values ofE. faecalis, andS. aureus, which indicated that the resistance ofE. coli against bainiku-ekisu was higher thanE. faecalis, andS. aureus.This result was different to the result from traditional MIC and MBC. Higher resistance ofE. coli in higher bainiku-ekisu concentrations might exist in some cases, nevertheless, it could not be found in the traditional method.When the number of bacteria decreased by 99.9% (the definition of MBC), the spent time was equal to 3 times theD value for 24 hours, so theD value was equal to 8 hours (24 hours/3). Using the regression equation (Y4, Y5, and Y6), the MBCs were determined to be 2.78, 3.12 and 2.24 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These results are similar to those obtained using the growth rate method. Therefore, the MBC could be calculated from theD value but the MIC could not be calculated fromD value. The difference between calculated values and traditional values might be that the traditional MIC and MBC values were only obtained from the results at 24 hours incubation, but the calculated values were obtained from the results during incubation. The traditional method is easy and fast and the calculated method is exact.


## 3.1. The Characteristics of Fresh Japanese Apricot Juice and Bainiku-Ekisu

To compare fresh Japanese apricot juice and bainiku-ekisu, bainiku-ekisu was diluted to the concentration of fresh juice using de-ionized water. The characteristics of fresh Japanese apricot juice and bainiku-ekisu are shown in Table1. The pH levels of fresh juice and bainiku-ekisu were lower than pH 3, indicating that the concentration process was performed under acidic conditions. The °Brix, total acidity and total amount of reducing sugars for bainiku-ekisu were significant lower than those for fresh juice. Those results may due to that the Maillard reaction, the caramelization reaction and mumefural generation might take place during juice concentration at high temperatures [6, 16].Table 1
Characteristics of fresh Japanese apricot juice and bainiku-ekisu.
Fresh juice
Bainiku-ekisu1
pH
2.67 ± 0.007b8
2.83 ± 0.02a
°Brix
4.9 ± 0.06a
4.1 ± 0.03b
Total acidity2 (g/100 mL)
2.18 ± 0.15a
1.77 ± 0.02b
Total amount of reducing sugar3 (mg/100 mL)
143.11 ± 0.14a
55.71 ± 1.34b
Total phenolic content4 (mg/100 mL)
26.44 ± 0.16b
51.00 ± 1.26a
Total flavonoid content5 (mg/100 mL)
11.39 ± 0.29b
16.60 ± 0.8a
EC50 of DPPH radical-scavenging activity6 (mL sample)
0.11 ± 0.00a
0.04 ± 0.00b
EC50 of ABTS·+ radical-scavenging activity (mL sample)
0.16 ± 0.00a
0.05 ± 0.00b
1Samples were diluted to the same concentration as fresh juice with de-ionized water.
2g citric acid/100 mL sample.
3mg glucose/100 mL sample.
4mg gallic acid equivalent/100 mL sample.
5mg catechin equivalent/100 mL sample.
6Volume of sample needed.
7Data are presented as the means ± standard deviations (n=3).
8Different letters for the individual extracts indicate that the values are significantly different (P<0.05).The total phenolic and flavonoid contents of bainiku-ekisu were higher than those of fresh juice (Table1). During heating at high temperature for a long time, phenolic compounds and flavonoids can be destroyed; however, the phenolic and flavonoid contents were found increased in this study. There are a few articles that reported the total phenolic and flavonoid contents of citrus peels increased during high temperature treatment [17–20]. Several low-molecular-weight phenolic compounds might form inCitrus unshiu peels heated at 150°C for 30 min [21]. Que et al. [19] proposed that the phenolic compounds might be formed from the precursors of phenolic molecules via non-enzymatic interconversion between phenolic molecules. The increases in the DPPH and ABTS.+ radical-scavenging activities could be due to the higher phenolic and flavonoid contents. Furthermore, the products of the Maillard reaction and the caramelization reaction could exhibit antioxidative activities [16, 22, 23]. The conversion of the EC50values for the DPPH and ABTS.+ radical-scavenging activities of bainiku-ekisu into weights of the final product yielded values of 2.61 and 3.33 mg/mL, respectively.


## 3.2. Measurement of the Antibacterial Effects

### 3.2.1. MIC and MBC

Figure1 shows the growth patterns ofE. faecalis when treated with/without different bainiku-ekisu concentrations (0–10 mg/mL). WhenE. faecalis was incubated without bainiku-ekisu, the bacteria counts increased steadily during the 12 hours incubation. The bainiku-ekisu solutions affected the growth patterns ofE. faecalis.Schoenknecht et al. [24] described 4 types of “killing curves” in clinical research. When 5 and 10 mg/mL bainiku-ekisu solutions were used, the number of bacteria decreased to zero within 4 and 0.5 hours, respectively. The results demonstrate that bainiku-ekisu can inhibit and killE. faecalis (type D curve). The number of bacteria slightly decreased in the presence 2.5 mg/mL bainiku-ekisu. The killing type was similar to type D, but rate of the decrease was slower than that for 5 mg/mL. The killing type of 1 mg/mL bainiku-ekisu was type B, which involves a lag phase increase. The killing type of 0.5 mg/mL bainiku-ekisu was type C, which involves a decrease in the growth rate, with little effect on the lag time. Type A effect (“percentage growth inhibition”) was also observed in this study.Figure 1
Growth ofEnterococcus faecalis in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The MIC and MBC of bainiku-ekisu were 1 mg/mL and 5 mg/mL, respectively. Hoellman et al. [25] reported that the MICs of ampicillin and vancomycin for enterococci were 0.3 μg/mL and 4.2 μg/mL, respectively. The amount of bainiku-ekisu used in this study was higher than amounts of antibiotics used in their study, but bainiku-ekisu is a natural product. The antimicrobial activities (MIC and MBC) ofCyperus rotundus ethyl acetate extract forE. faecalis were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu were better than those ofCyperus rotundus ethyl acetate extract.Figure2 shows that the growth patterns ofS. aureus when incubated with bainiku-ekisu (0–10 mg/mL). WhenS. aureus was incubated without bainiku-ekisu, the number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL. The growth patterns ofS. aureus were affected when bainiku-ekisu solutions were added. When the 5 and 10 mg/mL bainiku-ekisu solutions were added, the number of bacteria decreased to zero within 24 and 4 hours, respectively. The kill rates forS. aureus were slower than those forE. faecalis. However, the 2.5 mg/mL bainiku-ekisu markedly killedS. aureus, resulting in 101 CFU/mL after incubation for 24 hours. This inhibitory activity was better than that againstE. faecalis. In contrast, the results for the treatment ofS. aureus with 0.5 and 1 mg/mL bainiku-ekisu were similar to the results for the treatment ofE. faecalis after a 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. Similarly, the MIC and MBC ofC. rotundus ethyl acetate extract forS. aureus were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstS. aureus were significant better than the effect ofCyperus rotundus ethyl acetate extract.Figure 2
Growth ofStaphylococcus aureus in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The growth patterns ofE. coli incubated with bainiku-ekisu (0–10 mg/mL) are shown in Figure 3. The number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL when incubated without bainiku-ekisu. The rate ofE. colikilling was the slowest when using 5 and 10 mg/mL bainiku-ekisu solutions. Under these conditions, the number of bacteria decreased to zero within 24 and 6 hours, respectively. For the 24 hours incubation, the effects of 2.5 mg/mL bainiku-ekisu onE. coli were similar to the effects onS. aureus; the number of bacteria was approximately 101 CFU/mL. However, the 1 mg/mL bainiku-ekisu solution could killE. coli, and the number of bacteria decreased to approximately 102 CFU/mL. The rate ofE. colikilling was the fastest for the 1 mg/mL bainiku-ekisu solution among three bacteria. In contrast, the 0.5 mg/mL bainiku-ekisu solution could not inhibit the growth ofE. coliduring the 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. The MIC and MBC ofCyperus rotundus ethyl acetate extract forE. coli were 5 and >5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstE. coli were significantly better than the effects ofC. rotundus ethyl acetate extract.Figure 3
Growth ofEscherichia coliin the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.Polyphenolic compounds are reported to play a role as antimicrobials [27–29]. Kilani-Jaziri et al. [26] reported that intermediate-polarity polyphenolic compounds (ethyl acetate extract) are active antibacterials. Bainiku-ekisu is a concentrated product that contains full polyphenolic compounds, and therefore, the antimicrobial activity of bainiku-ekisu was outstanding. Kilani et al. [30] reported that coumarin is the major antimicrobial compound in the TOF (Total Oligomers Flavonoids)-enriched extract ofC. rotundus. Further investigation is required to identify the compound(s) responsible for the antimicrobial activity of bainiku-ekisu.


### 3.2.2. Growth Rates,D Values andZ Values

The MICs forE. faecalis,S. aureus, andE. coliwere all 1 mg/mL. However, by definition, the real MIC ofE. coli should be between 0.5 and 1 mg/mL since the numberi ofE. coli decreased to approximately 102 CFU/mL during incubation with 1 mg/mL. When using this antibiotic, the concentration would be 1 mg/mL, which is slightly high. The MBCs forS. aureus andE. coliwere the same (2.5 mg/mL), butS. aureus was more easily destroyed thanE. coliwith 2.5, 5.0 and 10.0 mg/mL bainiku-ekisu (Figures 2 and 3). The exact effective doses might not be the same forS. aureus andE. coli, thus the growth rate,D value andZ value were used to calculate MIC and MBC.The linear parts of the growth curves can be expressed with simple linear equations(R2≥0.910), where the slopes were used as the growth rates (vG) (Table 2). The positive growth rate meant that bainiku-ekisu could not kill or inhibit these stains at certain concentration, such as 0.5 mg/mL, and the negative growth rate showed the opposite tendency. MIC was defined that the lowest tested concentration in which the number of bacteria did not vary significantly during 24 hours; that is, the growth rate of the bacterium was around zero. For the MIC calculation, the linear regression of the growth rates with different bainiku-ekisu concentrations is shown in Figure 4(a) and the linear regression equations forE. faecalis,S. aureus, andE. coli were Y1=-1.007X+1.556 (R2=0.9312), Y2=-0.234X+0.3335 (R2=0.9855) and Y3=-0.0589X+0.0570 (R2=0.7767), respectively. Y1, Y2, and Y3 represent the growth rates, and X was the bainiku-ekisu concentration. When Y1, Y2, and Y3 set at zero, the calculated MICs (X) were found at 1.55, 1.43 and 0.97 mg/mL, respectively. These calculated MICs by growth rates forE. faecalis,andS. aureus were higher than the MICs by traditional values, and the calculated MIC forE. colishowed the opposite result. The MBC was determined to be the lowest tested concentration in which the number of bacteria was reduced 99.9% in CFU/mL; that is, the number of bacteria decreased to 0.1% of the control over 24 hours, and the growth rate of bacterium could be calculated as −0.125 (log⁡10-3 CFU/mL/24 hours). When the slope was −0.125 in the regression equation, the MBCs of bainiku-ekisu were 2.59, 2.63 and 2.25 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These calculated MBCs were different to the traditional MBCs.Table 2
Growth rates andD values (decimal reduction time) of Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli treated with various bainiku-ekisu concentrations during incubation.
Bainiku-ekisu concentration (mg/mL)
0.5
1.0
2.5
5.0
10
0.5
1.0
2.5
5.0
10
Strain
Growth rate
D value
v
G (Δlog⁡(CFU)/hour)
(hour)
E. faecalis
0.162
−0.035
−0.047
−2.106
−9.323
—1
28.7
21.1
0.47
0.11
S. aureus
0.188
−0.029
−0.086
−0.794
−2.049
—
34.5
11.6
1.26
0.49
E. coli
0.199
−0.070
−0.136
−0.372
−0.455
—
14.2
7.34
2.69
2.20
1Antibacterial effect is not sufficient to calculate the D value.Correlations between the bainiku-ekisu concentration and both the growth rate (a) and theD value (decimal reduction time) (b) forEnterococcus faecalis (●),Staphylococcus aureus (○), andEscherichia coli (▼) during incubation.
(a)
(b)The calculatedD values are listed at Table 2. For the 0.5 mg/mL concentration, theD values were not assessable and meaningless because the number of bacteria increased during incubation. A higherD value indicates that the bacterium has a higher level of resistance. It is interesting thatE. faecalisandS. aureus,exhibited higher levels of resistance thanE. coli did at lower bainiku-ekisu concentrations (1.0 and 2.5 mg/mL); in contrast,E. coliexhibited a higher level of resistance at higher concentrations (5.0 and 10.0 mg/mL). Figure 4(b) shows the linear regression of theD value versus the bainiku-ekisu concentration. The regression equations forE. faecalis,S. aureus, andE. coli were Y4=-0.2878X+1.7036 (R2=0.8843), Y5=-0.2029X+1.536 (R2=0.8834) and Y6=-0.086X+1.0954 (R2=0.7978), respectively. Y4, Y5, and Y6 were the logarithms of theD values, and X was the bainiku-ekisu concentration. According to the definition, theZ values can be calculated from these equations, and they were 3.47, 4.93 and 11.62 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. TheZ value ofE. coli was significantly higher than the values ofE. faecalis, andS. aureus, which indicated that the resistance ofE. coli against bainiku-ekisu was higher thanE. faecalis, andS. aureus.This result was different to the result from traditional MIC and MBC. Higher resistance ofE. coli in higher bainiku-ekisu concentrations might exist in some cases, nevertheless, it could not be found in the traditional method.When the number of bacteria decreased by 99.9% (the definition of MBC), the spent time was equal to 3 times theD value for 24 hours, so theD value was equal to 8 hours (24 hours/3). Using the regression equation (Y4, Y5, and Y6), the MBCs were determined to be 2.78, 3.12 and 2.24 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These results are similar to those obtained using the growth rate method. Therefore, the MBC could be calculated from theD value but the MIC could not be calculated fromD value. The difference between calculated values and traditional values might be that the traditional MIC and MBC values were only obtained from the results at 24 hours incubation, but the calculated values were obtained from the results during incubation. The traditional method is easy and fast and the calculated method is exact.


## 3.2.1. MIC and MBC

Figure1 shows the growth patterns ofE. faecalis when treated with/without different bainiku-ekisu concentrations (0–10 mg/mL). WhenE. faecalis was incubated without bainiku-ekisu, the bacteria counts increased steadily during the 12 hours incubation. The bainiku-ekisu solutions affected the growth patterns ofE. faecalis.Schoenknecht et al. [24] described 4 types of “killing curves” in clinical research. When 5 and 10 mg/mL bainiku-ekisu solutions were used, the number of bacteria decreased to zero within 4 and 0.5 hours, respectively. The results demonstrate that bainiku-ekisu can inhibit and killE. faecalis (type D curve). The number of bacteria slightly decreased in the presence 2.5 mg/mL bainiku-ekisu. The killing type was similar to type D, but rate of the decrease was slower than that for 5 mg/mL. The killing type of 1 mg/mL bainiku-ekisu was type B, which involves a lag phase increase. The killing type of 0.5 mg/mL bainiku-ekisu was type C, which involves a decrease in the growth rate, with little effect on the lag time. Type A effect (“percentage growth inhibition”) was also observed in this study.Figure 1
Growth ofEnterococcus faecalis in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The MIC and MBC of bainiku-ekisu were 1 mg/mL and 5 mg/mL, respectively. Hoellman et al. [25] reported that the MICs of ampicillin and vancomycin for enterococci were 0.3 μg/mL and 4.2 μg/mL, respectively. The amount of bainiku-ekisu used in this study was higher than amounts of antibiotics used in their study, but bainiku-ekisu is a natural product. The antimicrobial activities (MIC and MBC) ofCyperus rotundus ethyl acetate extract forE. faecalis were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu were better than those ofCyperus rotundus ethyl acetate extract.Figure2 shows that the growth patterns ofS. aureus when incubated with bainiku-ekisu (0–10 mg/mL). WhenS. aureus was incubated without bainiku-ekisu, the number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL. The growth patterns ofS. aureus were affected when bainiku-ekisu solutions were added. When the 5 and 10 mg/mL bainiku-ekisu solutions were added, the number of bacteria decreased to zero within 24 and 4 hours, respectively. The kill rates forS. aureus were slower than those forE. faecalis. However, the 2.5 mg/mL bainiku-ekisu markedly killedS. aureus, resulting in 101 CFU/mL after incubation for 24 hours. This inhibitory activity was better than that againstE. faecalis. In contrast, the results for the treatment ofS. aureus with 0.5 and 1 mg/mL bainiku-ekisu were similar to the results for the treatment ofE. faecalis after a 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. Similarly, the MIC and MBC ofC. rotundus ethyl acetate extract forS. aureus were 2.5 and 5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstS. aureus were significant better than the effect ofCyperus rotundus ethyl acetate extract.Figure 2
Growth ofStaphylococcus aureus in the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.The growth patterns ofE. coli incubated with bainiku-ekisu (0–10 mg/mL) are shown in Figure 3. The number of bacteria increased steadily during the 12 hours incubation, reaching approximately 108 CFU/mL when incubated without bainiku-ekisu. The rate ofE. colikilling was the slowest when using 5 and 10 mg/mL bainiku-ekisu solutions. Under these conditions, the number of bacteria decreased to zero within 24 and 6 hours, respectively. For the 24 hours incubation, the effects of 2.5 mg/mL bainiku-ekisu onE. coli were similar to the effects onS. aureus; the number of bacteria was approximately 101 CFU/mL. However, the 1 mg/mL bainiku-ekisu solution could killE. coli, and the number of bacteria decreased to approximately 102 CFU/mL. The rate ofE. colikilling was the fastest for the 1 mg/mL bainiku-ekisu solution among three bacteria. In contrast, the 0.5 mg/mL bainiku-ekisu solution could not inhibit the growth ofE. coliduring the 24 hours incubation. The MIC and MBC of bainiku-ekisu were 1 mg/mL and 2.5 mg/mL, respectively. The MIC and MBC ofCyperus rotundus ethyl acetate extract forE. coli were 5 and >5 mg/mL, respectively [26]. The antimicrobial activities of bainiku-ekisu againstE. coli were significantly better than the effects ofC. rotundus ethyl acetate extract.Figure 3
Growth ofEscherichia coliin the presence of various bainiku-ekisu concentrations. ●, Control (0 mg/mL); □, 0.5 mg/mL; ■, 1 mg/mL; △, 2.5 mg/mL; ▼, 5 mg/mL; ○, 10 mg/mL.Polyphenolic compounds are reported to play a role as antimicrobials [27–29]. Kilani-Jaziri et al. [26] reported that intermediate-polarity polyphenolic compounds (ethyl acetate extract) are active antibacterials. Bainiku-ekisu is a concentrated product that contains full polyphenolic compounds, and therefore, the antimicrobial activity of bainiku-ekisu was outstanding. Kilani et al. [30] reported that coumarin is the major antimicrobial compound in the TOF (Total Oligomers Flavonoids)-enriched extract ofC. rotundus. Further investigation is required to identify the compound(s) responsible for the antimicrobial activity of bainiku-ekisu.


## 3.2.2. Growth Rates,D Values andZ Values

The MICs forE. faecalis,S. aureus, andE. coliwere all 1 mg/mL. However, by definition, the real MIC ofE. coli should be between 0.5 and 1 mg/mL since the numberi ofE. coli decreased to approximately 102 CFU/mL during incubation with 1 mg/mL. When using this antibiotic, the concentration would be 1 mg/mL, which is slightly high. The MBCs forS. aureus andE. coliwere the same (2.5 mg/mL), butS. aureus was more easily destroyed thanE. coliwith 2.5, 5.0 and 10.0 mg/mL bainiku-ekisu (Figures 2 and 3). The exact effective doses might not be the same forS. aureus andE. coli, thus the growth rate,D value andZ value were used to calculate MIC and MBC.The linear parts of the growth curves can be expressed with simple linear equations(R2≥0.910), where the slopes were used as the growth rates (vG) (Table 2). The positive growth rate meant that bainiku-ekisu could not kill or inhibit these stains at certain concentration, such as 0.5 mg/mL, and the negative growth rate showed the opposite tendency. MIC was defined that the lowest tested concentration in which the number of bacteria did not vary significantly during 24 hours; that is, the growth rate of the bacterium was around zero. For the MIC calculation, the linear regression of the growth rates with different bainiku-ekisu concentrations is shown in Figure 4(a) and the linear regression equations forE. faecalis,S. aureus, andE. coli were Y1=-1.007X+1.556 (R2=0.9312), Y2=-0.234X+0.3335 (R2=0.9855) and Y3=-0.0589X+0.0570 (R2=0.7767), respectively. Y1, Y2, and Y3 represent the growth rates, and X was the bainiku-ekisu concentration. When Y1, Y2, and Y3 set at zero, the calculated MICs (X) were found at 1.55, 1.43 and 0.97 mg/mL, respectively. These calculated MICs by growth rates forE. faecalis,andS. aureus were higher than the MICs by traditional values, and the calculated MIC forE. colishowed the opposite result. The MBC was determined to be the lowest tested concentration in which the number of bacteria was reduced 99.9% in CFU/mL; that is, the number of bacteria decreased to 0.1% of the control over 24 hours, and the growth rate of bacterium could be calculated as −0.125 (log⁡10-3 CFU/mL/24 hours). When the slope was −0.125 in the regression equation, the MBCs of bainiku-ekisu were 2.59, 2.63 and 2.25 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These calculated MBCs were different to the traditional MBCs.Table 2
Growth rates andD values (decimal reduction time) of Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli treated with various bainiku-ekisu concentrations during incubation.
Bainiku-ekisu concentration (mg/mL)
0.5
1.0
2.5
5.0
10
0.5
1.0
2.5
5.0
10
Strain
Growth rate
D value
v
G (Δlog⁡(CFU)/hour)
(hour)
E. faecalis
0.162
−0.035
−0.047
−2.106
−9.323
—1
28.7
21.1
0.47
0.11
S. aureus
0.188
−0.029
−0.086
−0.794
−2.049
—
34.5
11.6
1.26
0.49
E. coli
0.199
−0.070
−0.136
−0.372
−0.455
—
14.2
7.34
2.69
2.20
1Antibacterial effect is not sufficient to calculate the D value.Correlations between the bainiku-ekisu concentration and both the growth rate (a) and theD value (decimal reduction time) (b) forEnterococcus faecalis (●),Staphylococcus aureus (○), andEscherichia coli (▼) during incubation.
(a)
(b)The calculatedD values are listed at Table 2. For the 0.5 mg/mL concentration, theD values were not assessable and meaningless because the number of bacteria increased during incubation. A higherD value indicates that the bacterium has a higher level of resistance. It is interesting thatE. faecalisandS. aureus,exhibited higher levels of resistance thanE. coli did at lower bainiku-ekisu concentrations (1.0 and 2.5 mg/mL); in contrast,E. coliexhibited a higher level of resistance at higher concentrations (5.0 and 10.0 mg/mL). Figure 4(b) shows the linear regression of theD value versus the bainiku-ekisu concentration. The regression equations forE. faecalis,S. aureus, andE. coli were Y4=-0.2878X+1.7036 (R2=0.8843), Y5=-0.2029X+1.536 (R2=0.8834) and Y6=-0.086X+1.0954 (R2=0.7978), respectively. Y4, Y5, and Y6 were the logarithms of theD values, and X was the bainiku-ekisu concentration. According to the definition, theZ values can be calculated from these equations, and they were 3.47, 4.93 and 11.62 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. TheZ value ofE. coli was significantly higher than the values ofE. faecalis, andS. aureus, which indicated that the resistance ofE. coli against bainiku-ekisu was higher thanE. faecalis, andS. aureus.This result was different to the result from traditional MIC and MBC. Higher resistance ofE. coli in higher bainiku-ekisu concentrations might exist in some cases, nevertheless, it could not be found in the traditional method.When the number of bacteria decreased by 99.9% (the definition of MBC), the spent time was equal to 3 times theD value for 24 hours, so theD value was equal to 8 hours (24 hours/3). Using the regression equation (Y4, Y5, and Y6), the MBCs were determined to be 2.78, 3.12 and 2.24 mg/mL forE. faecalis,S. aureus, andE. coli, respectively. These results are similar to those obtained using the growth rate method. Therefore, the MBC could be calculated from theD value but the MIC could not be calculated fromD value. The difference between calculated values and traditional values might be that the traditional MIC and MBC values were only obtained from the results at 24 hours incubation, but the calculated values were obtained from the results during incubation. The traditional method is easy and fast and the calculated method is exact.


## 4. Conclusion

Bainiku-ekisu could kill or inhibit the growths ofE. faecalis,S. aureus, andE. coli. Therefore, bainiku-ekisu could be used as a natural antimicrobial agent to reduce the cross-contamination risk and prevent food poisoning. The MICs and MBCs might be overestimated for pathogens, as higher antibiotic dose to patients might yield antibiotic-resistant bacteria. The determinations of growth rate,D value andZ value were used to determine the exact cured dose, but these methods are costly and time-consuming. The antibacterial activities of bainiku-ekisu will be tested against other pathogens and further studies will determine the mechanism of antibacterial action of bainiku-ekisu.



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*Source: 460395-2014-10-28.xml*