Technical paper
Microbial Reduction and Quality Changes in Powdered White and Black Pepper by Treatment with Compressed Oxygen or Carbon Dioxide Gas
2015 Volume 21 Issue 1 Pages 51-57
Details
2015 Volume 21 Issue 1 Pages 51-57
Powdered white and black peppers were treated with high-pressure oxygen (10 MPa) or carbon dioxide (5 MPa) at 70 – 100°C to decrease the microbial populations. The degree of microbial reduction in the pepper after treatment with high-pressure gases was dependent on the temperature and duration of treatment. Mesophilic aerobic bacterial counts of less than 103 CFU/g in white and black pepper were achieved with gas pressurization at 100°C for 10 and 40 min, respectively. The reduction in the microbial population of the two types of pepper was attributed to the microbicidal effects of high-pressure gas and heat treatment. The piperine content of the pepper treated with gases decreased by approximately 10%. However, treated pepper also showed lower L* values, indicating a darker color, and qualitative alterations in volatile compounds.
White and black pepper (Piper nigrum), which possess distinctive flavors, are widely used in preparing meat and fish products as well as other prepared foods. In general, pepper is produced from whole dried fruits, which are processed through various treatments such as harvesting, threshing, and drying (Schweiggert et al., 2007). During these processes, the pepper is often contaminated with molds, yeasts, and spore-forming bacteria, which can cause foodborne illnesses in consumers and spoilage of pepper-containing foods (Emam et al., 1995; Zaied et al., 1996; Schweiggert et al., 2007). Therefore, a process to inactivate microorganisms in pepper is required. A decontamination level corresponding to a total plate count of less than 3 log colony forming units (CFU/g) is desirable in spices (Ferrentino and Spilimbergo, 2011).
Generally, thermal treatment is used to inactivate microorganisms in food; however, heat treatment results in alteration of the volatile compounds present in spices (Maarse and Nussen, 1980; Chacko et al., 1996). A high-temperature steam treatment is currently available for microbial inactivation of spices (Hamano and Nishimura, 1991; Almela et al., 2002; Lilie et al., 2007). Steam treatment for 20 s at 120°C resulted in a 3 log decrease in CFU in black pepper, and treatment at 130°C for 20 s caused no significant changes in the volatile oils (Lilie et al., 2007). However, treated pepper showed a small increase in moisture content, which would shorten its shelf life (Lilie et al., 2007). Thus, nonthermal processes, including ethylene oxide exposure, ionizing radiation, and hydrostatic pressurization (physical pressurization), have attracted much attention for the microbial inactivation of spices. Ethylene oxide has been applied to microbial inactivation of spices, significantly reducing the initial microbial load (Koizumi et al., 1964). However, the application of ethylene oxide to inactivation of microbes in food has been banned in many countries because of its carcinogenic properties (Fowles et al., 2001; Schweiggert et al., 2007). Many studies have investigated the effects of ionizing radiation on the microbiological and physiochemical qualities of spices (Emam et al., 1995; Schweiggert et al., 2007; Waje et al., 2008). Irradiation with electron beams and gamma rays (≤ 10 kGy) was effective in reducing the microbial population without affecting the color or flavor of spices, leading to the legal acceptance of irradiation in many countries (Hayashi et al., 1993; Schweiggert et al., 2007; Waje et al., 2008). Nevertheless, some consumers have reacted negatively to the use of irradiation for food. Hydrostatic pressure (liquid pressure) processing of black pepper has also been reported, but it was shown to decrease the contents of essential oils and volatile monoterpenes (Skapska et al., 2003). Moreover, a costly apparatus is required for decontamination of pepper via this technique. Overall, previous nonthermal techniques have several disadvantages ranging from toxicity, poor acceptance by consumers, and the need for expensive equipment. Hence, there is a need to develop a new method for decontaminating spices.
The use of pressurized gas has begun to attract attention for its antimicrobial action (Erkmen, 2000; Spilimbergo et al., 2003; Muramoto et al., 2004; Garcia-Gonzalez et al., 2007; Tamura, 2007; Ferrentino et al., 2009; Garcia-Gonzalez et al., 2009; Calvo and Torres, 2010; Kawachi et al., 2010; Ferrentino and Spilimbergo, 2011). Recently, we demonstrated that yeasts in sudachi juice, a citrus juice, could be inactivated with pressurized oxygen at 10 MPa and 50°C without color alteration or significant loss of vitamin C or limonene (Muramoto et al., 2004; Tamura, 2007). In contrast to ethylene oxide, oxygen, the antimicrobial agent in this technique, is not toxic to humans, even when exposure occurs via accidental leaks. Oxygen gas is also inexpensive and readily acceptable to consumers. Furthermore, the cost of high-pressure apparatus can be further reduced, because the pressure required (≤ 10 MPa) is 1 – 2 orders lower than that required for hydrostatic pressure processing. Pressurization with carbon dioxide has also been reported to be useful for inactivating microorganisms in foods (Erkmen, 2000; Garcia-Gonzalez et al., 2007; Ferrentino et al., 2009; Calvo and Torres, 2010). Further study may illuminate the potentially negative aspects of pressurization with oxygen or carbon dioxide; irrespective, this technique shows promise as an alternative method for decontamination of spices. However, the application of the gas pressurization technique has been limited to liquid foods and little work has been conducted on food powders, including white and black pepper.
To identify an effective method for decontamination of white and black pepper, we attempted to reduce the number of microbes present in the pepper by combining compression of oxygen or carbon dioxide with thermal treatment. We measured the color, piperine content, and volatile composition of the treated pepper. Very few studies have examined the antimicrobial potential of high-pressure oxygen and its interaction with food compounds in comparison to carbon dioxide. Therefore, we also aimed to bridge this data gap and clarify a direction for further research on microbial inactivation using compressed gases.
Pepper and gas pressurization apparatus Non-sterilized powdered white and black pepper were purchased from K. Kobayashi Co., Ltd., Kobe. The purchased white and black peppers were harvested in Malaysia and India, respectively. The mean water activity (aw) values for white and black pepper were 0.576 and 0.556, determined using a water activity meter (IC-500 AW-LAB; Novasina, Lachen, Switzerland). The gas pressurization apparatus was comprised of a gas cylinder, pressure control valve, pressure sensor, pressure gauge, decompression valve, stop valve, high-pressure lines, 17-mL high-pressure container, and oil bath. The purity of all gases used was greater than 99.5%. A schematic diagram of the apparatus was as described previously (Muramoto et al., 2004). Pepper was added into the sterile high-pressure container, and the air in the headspace of each container was replaced with oxygen or carbon dioxide. Gas was injected into the container via high-pressure lines until the desired pressure was reached. Unless otherwise stated, the pressure of the gas to which the pepper was exposed, as measured by a pressure sensor, was 10 and 5 MPa for oxygen and carbon dioxide, respectively. The carbon dioxide used in this experiment was in a gaseous physical state, not in a liquid or a supercritical state. After compression, the vessel containing the pepper was placed into an oil bath and maintained at the designated temperature, between 70 and 100°C. The pepper was not stirred during the microbial inactivation process. After the bacterial inactivation treatment, the vessel was cooled with ice, and decompressed to a normal pressure. Decompression was performed as slowly as possible at a rate of less than 0.6 MPa/min. Pepper exposed with oxygen was re-pressurized with nitrogen at room temperature and 10 MPa for 10 min, to reduce oxidation. We previously removed dissolved oxygen from sudachi juice using nitrogen pressurization (Muramoto et al., 2005). The same removal method was performed here, under the assumption that oxygen in contact with food powder is removed by high-pressure nitrogen.
Microbiological analysis In studies of the development of microbial inactivation methods for spices, the total mesophilic aerobic bacterial count is often used as a representative index to evaluate the degree of inactivation (Emam et al., 1995; Zhao and Cranston, 1995; Almela et al., 2002; Skapska et al., 2003; Lilie et al., 2007; Waje et al., 2008; Calvo and Torres, 2010). For determination of the total mesophilic aerobic bacterial count, 1 g of pepper was adequately diluted with phosphate-buffered saline, and mixed with standard plate count agar (Nissui Pharmaceutical Co., Ltd., Tokyo). Bacterial counts were performed after 48 h of incubation at 35°C. Aerobic spore-forming bacteria were determined in the same manner, except that the inoculum was heated at 80°C for 20 min. Coliforms were determined on desoxycholate agar (Nihon Pharmaceutical Co., Ltd., Tokyo) plates, incubated at 35°C for 20 h. Total fungi were determined on potato dextrose agar (Becton Dickinson and Co., Franklin Lakes, NJ, USA), acidified by 10% tartaric acid. Plates were counted after 5 days of incubation at 23°C.
Analysis of physicochemical properties Pressurized white pepper at 100°C for 30 min and black pepper at 100°C for 60 min were used for analysis of the physicochemical properties of the pepper. Piperine is known to be a major pungent component in pepper. The piperine content of the pepper was evaluated using the AOAC official method (AOAC, 2006). Piperine extracts were prepared by mixing 0.5 g pepper with denatured alcohol and refluxing the mixture for 1 h in the dark. The extracts were filtered, diluted, and placed in a spectrophotometer (U-2001; Hitachi, Tokyo). The piperine content was determined by measuring the absorbance at 343 nm. Although piperine isomers, such as piperettine and pyrroperine, which may be present in pepper also absorb at 343 nm (Mori et al., 1974; AOAC, 2006), the spectrophotometric method is simple to perform and has been used in various studies (Chacko et al., 1996; Skapska et al., 2003). The color values (L*, a*, and b*) of the pepper were determined using a color meter (ZE6000; Nippon Denshoku Industries Co., Ltd., Tokyo). The total color difference, expressed as 
, was obtained from the L*, a*, and b* values. Qualitative analysis of the volatile compounds present in the pepper was performed using gas chromatography (6890N; Agilent Technologies Co., Ltd., Santa Clara, CA, USA). A DB-5ms column was used (60 m × 0.32 m, 1.0 µm film thickness, Agilent Technologies Co., Ltd.), and helium gas was used as the carrier gas. The injector temperature was set at 250°C, and the oven temperature was programmed from 38 to 330°C. The splitless injection mode was used in this experiment. The sampling of headspace volatiles in vials was performed after warming the vials at 60°C for 20 min. The identification of the separated compounds was performed on a mass spectrometer (5793; Agilent Technologies Co., Ltd.) with an ionization voltage of 70 eV. The scan range was 43 – 400 m/z. The components were identified with reference to the Wiley 7n mass spectral database.
Effects of compressed oxygen or carbon dioxide treatment on microbial reduction in pepper Total aerobic bacteria and spore-forming bacteria in untreated white pepper ranged from 4.3 to 4.4 and 3.6 to 3.8 log CFU/g, respectively. No coliforms (< 30 CFU/g) and fungi (< 10 CFU/g) were detected in untreated white pepper. Figure 1 shows the total aerobic bacterial count in white pepper treated with oxygen and carbon dioxide at 70 – 100°C. To separately evaluate the effect of thermal treatment on microbial inactivation, we also performed tests without the addition of pressurized gases. When the count was below the detection level (< 100 CFU/g), we expressed the total aerobic bacterial count as 2 log CFU/g. Microbial inactivation was dependent on temperature and treatment time. All treatments under 70°C were ineffective for reducing the total mesophilic aerobic bacterial count in white pepper. However, gas pressurization treatment at 80°C reduced the microbial population in white pepper. Microbial counts in white pepper treated with gas exposure for 4 h showed a 1.7 – 2.4 log reduction whereas thermal treatment alone for the same period caused no significant decrease. Heat alone at 90°C led to a slight reduction of the microbial population, however, addition of compressed gas resulted in greater inactivation. This is likely caused by the synergistic action of heat and compressed gas. We cannot exclude the probability that high temperature promoted an increase of gas pressure. Increasing the temperature to 100°C with gas pressurization for 10 min reduced the microbial count to < 3 log CFU/g in white pepper. Pressurization at 100°C for 20 min with either oxygen or carbon dioxide was sufficient to reduce the total bacterial count to the minimum detectable level.
Comparative effects of heat alone, high-pressure oxygen, and carbon dioxide on microbial inactivation in white pepper
○, Heat alone; ▴, High-pressure oxygen; ▪, High-pressure carbon dioxide.
Untreated black pepper contained 6.1 to 6.2 log CFU/g of total aerobic bacteria, and 5.9 to 6.0 log CFU/g of spore-forming bacteria. Untreated black pepper was contaminated with no coliforms and fungi. Figure 2 shows the total bacterial counts in black pepper treated with gas compression at 80 – 100°C. Microbial inactivation of black pepper depended on temperature; however, the minimum temperature that influenced the microbial population was higher than that for white pepper. At 80°C, all methods, except for 4 h of gas pressurization, which caused a slight microbial reduction, produced no significant reduction in the bacterial count of black pepper. A substantial decrease in the bacterial population was produced when the pepper was subjected to treatment with compressed oxygen or carbon dioxide at 90°C. Although 4 h of heat inactivation alone produced < 1.0 log decrease in the bacterial population, oxygen or carbon dioxide exposure resulted in a 2.5 and 4.0 log reduction in total aerobic bacterial count, respectively. As expected, the degree of inactivation further improved when the temperature was increased to 100°C. Using oxygen or carbon dioxide pressurization, the bacterial count was reduced to < 3 log CFU/g (the recommended decontamination level for pepper) in only 40 min. Based on the data shown in Figs. 1 and 2, the superiority of oxygen over carbon dioxide compression for microbial inactivation of pepper was not obvious.
Comparative effects of heat alone, high-pressure oxygen, and carbon dioxide on microbial inactivation in black pepper
○, Heat alone; ▴, High-pressure oxygen; ▪, High-pressure carbon dioxide.
We also investigated the effects of increasing the gas pressure of oxygen on the efficacy of microbial inactivation in black pepper. An increment of oxygen pressure from 10 to 20 MPa, which also results in increased oxygen contact with food, led to greater microbial reductions. At 80°C, increasing the oxygen pressure to 20 MPa for 4 h produced no effect; the pepper showed a high level of contamination (5.2 log CFU/g). Treatment with 20 MPa oxygen at 90°C for 2 h reduced the number of viable cells from 4.8 to 3.9 log CFU/g. Treatment with 20 MPa oxygen for 4 h reduced microbial contamination below the detection limit, indicating complete decontamination. Although this experiment was not performed on white pepper, similar effects should be observed on all types of pepper. Because carbon dioxide changes to a supercritical state at 7 – 8 MPa, the effects of carbon dioxide at higher pressure were not investigated.
Effects of compressed oxygen or carbon dioxide treatment on the piperine content of pepper The piperine contents of the pepper treated with high-pressure oxygen or carbon dioxide are shown in Table 1. The piperine content of the treated pepper ranged from 88 to 96% of that in untreated pepper. The piperine content was slightly higher in pepper treated with carbon dioxide than with oxygen. However, when the results were subjected to an analysis of variance and Tukey's multiple comparison method, no significant differences (P ≥ 0.05) were found in piperine contents between methods using oxygen and carbon dioxide.
| Sample | Piperine (mg/g) | |
|---|---|---|
| White pepper | Black pepper | |
| No treatment | 125±4 | 123±4 |
| Heat (100°C) + Oxygen (10 MPa) | 114±0 | 108±4 |
| Heat (100°C) + Carbon dioxide (5 MPa) | 120±5 | 114±1 |
Effects of compressed oxygen or carbon dioxide treatment on pepper color Table 2 summarizes the color alterations observed in pepper exposed to pressurized oxygen or carbon dioxide. Color data are represented by L* (lightness), a* (redness), and b* (yellowness). Compared to untreated pepper, the parameter L* of gas-treated pepper decreased markedly, indicating a darker appearance. A slight difference in b* was observed between the treated and untreated white pepper.
| Sample | White pepper | Black pepper | ||||||
|---|---|---|---|---|---|---|---|---|
| L* | a* | b* |  |
L* | a* | b* |  |
|
| No treatment | 71.65 | 2.52 | 25.87 | 47.05 | 4.71 | 19.66 | ||
| Heat (100°C) | 63.55±0.33 | 5.05±0.09 | 29.90±0.17 | 9.41 | 37.42±0.36 | 6.23±0.04 | 20.02±0.16 | 9.76 |
| Heat (100°C) + Oxygen (10 MPa) | 62.67±0.36 | 5.24±0.13 | 30.03±0.47 | 10.27 | 38.71±0.75 | 6.93±0.19 | 21.63±0.45 | 8.88 |
| Heat (100°C) + Carbon dioxide (5 MPa) | 64.06±0.09 | 4.85±0.28 | 30.07±0.39 | 8.99 | 38.84±0.73 | 6.22±0.27 | 20.90±0.57 | 8.47 |
Values are indicated as means ± standard deviation.
Effects of compressed oxygen or carbon dioxide treatment on volatile compounds of pepper Figure 3 shows gas chromatograms of volatile compounds in untreated, oxygen-treated, and carbon dioxide-treated white pepper. Gas chromatography showed that compressed oxygen produced new aldehydes, isobutanal and 2-methylbutanal. These aldehydes were not detected after carbon dioxide pressurization. In untreated pepper, the relative absorbance ratio of β-pinene / p-cymene or sabinene / α-pinene exceeded 1, but in oxygen-treated pepper the ratio approached approximately 1. This indicates a change in the relative composition of the volatile oils, possibly due to the loss of some volatile components. The same alteration was observed in white pepper treated with carbon dioxide. Treatment with oxygen or carbon dioxide caused no apparent increases in 4-terpineol or terpinene, which have been reported to be produced by thermal and oxidative effects (Maarse and Nussen, 1980; Zhao amd Cranston, 1995; Skapska et al., 2003).
Gas chromatograms of headspace vapor from white pepper (a) untreated, (b) treated with high-pressure oxygen, and (c) treated with high-pressure carbon dioxide
1, Acetone; 2, Hexane; 3, 3-Methylbutanal; 4, α-Thujene; 5, α-Pinene; 6, Camphene; 7, Sabinene; 8, β-Pinene; 9, α-Terpinolene; 10, Phellandrene; 11, δ-3-Carene; 12, p-Cymene; 13, Limonene; 14, γ-Terpinene; 15, Linalool; 16, 4-Terpineol; 17, δ-Elemene; 18, Copaene; 19, β-Elemene; 20, trans-Caryophyllene; 21, α-Humulene; 22, δ-Cadinene; 23, Isobutanal; 24, 2-Methylbutanal.
The results for black pepper showed that oxygen or carbon dioxide compression altered the ratios of δ-3-carene / β-pinene. Black pepper treated with oxygen showed that acetaldehyde and 3-methylbutanal were increased slightly whereas the abundance of other major peaks was decreased compared with untreated pepper.
Gas chromatograms of headspace vapor from black pepper (a) untreated, (b) treated with high-pressure oxygen, and (c) treated with high-pressure carbon dioxide
1, Acetaldehyde; 2, Ethanol; 3, Acetone; 4, Isobutanal; 5, Acetic acid; 6, Hexane; 7, 3-Methylbutanal; 8, 2-Methylbutanal; 9, α-Thujene; 10, α-Pinene; 11, Camphene; 12, Sabinene; 13, β-Pinene; 14, Phellandrene; 15, δ-3-Carene; 16, α-Terpinolene; 17, p-Cymene; 18, Limonene; 19, γ-Terpinene; 20, β-Terpineol; 21, Linalool; 22, 4-Terpineol; 23, δ-Elemene; 24, α-Cubebene; 25, Copaene; 26, trans-Caryophyllene; 27, β-Elemene; 28, α-Humulene; 29, β-Bisabolene; 30, β-Selinene.
In this study, we evaluated the effectiveness of compressed oxygen or carbon dioxide bacterial inactivation for white and black pepper. Treatment of black pepper with compressed oxygen or carbon dioxide at 100°C for 40 min reduced bacterial counts to 2.0 and 2.9 log CFU/g, respectively, below the 3 log CFU/g target in the present study. Compared to other studies on decontamination of black pepper with approximately the same initial bacterial load, pressurization with carbon dioxide at 100°C for 50 min produced microbial inactivation similar to irradiation with gamma rays at 10 kGy, and the impact of gas pressurization at 100°C for 30 min was greater than liquid pressure treatment with 1000 MPa at 100°C for 30 min (Skapska et al., 2003; Waje et al., 2008). Bacterial inactivation in white and black pepper was impacted by treatment temperature, time, and gas pressure. In combination with gas pressurization, the lowest temperature that produced a bacterial reduction was 80 and 90°C for white and black pepper, respectively. The 10°C difference between the types of pepper might be due to a difference in the initial bacterial population and the solid matrix containing fat and oil. For carbon dioxide pressurization, a higher degree of inactivation was obtained with lower initial bacterial concentration (Garcia-Gonzalez et al., 2007). Increased resistance to high-pressure carbon dioxide of bacteria in an oil-containing medium was found (Garcia-Gonzalez et al., 2009). Generally, the outer skin is removed from white pepper during processing, and white pepper has a lower essential oil content than black pepper (Gopalam et al., 1991). Although we cannot exclude other reasons, the observed difference in microbial inactivation could be explained by these phenomena. Moreover, it should be noted that increasing the oxygen pressure resulted in a stronger lethal effect on bacteria. This is not surprising when we consider that the concentration of a gas increases as pressure increases. Several authors have reported that higher pressure of oxygen or carbon dioxide enhanced the rate of microbial inactivation in liquid foods (Erkmen, 2000; Muramoto et al., 2004; Garcia-Gonzalez et al., 2007; Ferrentino et al., 2009). In contrast, increasing carbon dioxide pressure as high as 20 MPa produced less than a 0.4 log reduction of total bacteria in paprika powder (Calvo and Torres, 2010). The reason for this discrepancy is unclear at present, because food type, contaminating microorganisms, and apparatus differed between these studies.
The amount of piperine in pressurized white and black pepper was approximately 90% of the amount in untreated samples. This indicates that the application of pressurized gas had little effect on the concentration of piperine in the pepper. Following physical pressurization of 1000 MPa at 100°C for 30 min, approximately 80% of the initial piperine remained in black pepper (Skapska et al., 2003). Gamma irradiation at 10 kGy decreased the piperine content of black pepper by approximately 10% (Waje et al., 2008). Since the samples used in this study are not identical to those used in other studies, conclusions can not be drawn from comparisons between gas pressurization and other methods. However, our results are useful for expanding the application of gas pressurization to food production. Color analysis indicated that pepper treated with compressed gas showed a distinct decrease in brightness. Especially for white pepper, dark color is less favorable to consumers. This alteration was also observed in samples treated with heat alone. Steam treatment at 100°C for 16 min also reduced L values in ground black pepper (Waje et al., 2008). We have investigated whether this color change may also be responsible for the absorbance of water. The water activity did not change after treatment with gas pressurization. The blackening may be attributed to the oxidation of phenols or the Maillard reaction. Gas chromatography analysis indicated that gas treatment altered the relative ratios of volatile oils, compared with untreated samples. This phenomenon may be due to the loss of some components during thermal treatment. Another study has reported that the reduction of monoterpenes, such as sabinene and phellandrene, in black pepper after high pressurization at 60 – 140°C for 30 min was temperature-dependent (Skapska et al., 2003). Thermal treatment (70°C, 15 min) has been shown to increase the relative ratios of some monoterpenes, including pinene and myrcene (Zaied et al., 1996). Air oven processing (≥ 130°C) resulted in changes in the relative composition of limonene, pinene, and sabinene (Chacko et al., 1996). In addition, decompression may have contributed to the decrease in volatile oils in the pepper. Exposure with oxygen caused the production of aldehydes in white pepper. Another researcher reported that three aldehydes, likely resulting from the oxidation of hydrocarbons, were detected in black pepper exposed to ozone (Zhao and Cranston, 1995). The detection of aldehydes in pepper, which was in contact with large amounts of oxygen during pressurization, might be explained in the same way. White and black pepper treated with compressed carbon dioxide seemed to have little contact with oxygen, and hence changes in volatile oils should be limited to those produced by thermal action and the release of carbon dioxide.
Our results are not applicable to all kinds of white and black pepper. When gas pressurization was applied to the pepper used in this study, the high temperatures and long treatment times required made it difficult to avoid altering the color and flavor. Other studies have shown that water activity may play an essential role in microbial inactivation of food powders (Zhao and Cranston, 1995; Calvo and Torres, 2010). An increase in water content reduced the heat resistance of spores and increased carbon dioxide sorption by microbial cells (Härnulv et al., 1977; Kumagai et al., 1997); however, increased water content decreases the shelf life of food. We believe that the contact between pepper and gas was insufficient to produce antimicrobial action because of the absence of stirring in the high-pressure container. A gas pressurization system equipped with stirring to facilitate gas contact with the food would be necessary for microbial inactivation of powdered pepper.
Acknowledgements The authors thank the R&D Center at Nippon Meat Packers Inc. for analyzing the volatile compounds in white and black pepper. The authors also thank K. Kobayashi Co., Ltd. for providing unsterilized pepper. This work was funded by a grant from the Yamazaki Spice Promotion Foundation of Japan.
