2018 年 24 巻 2 号 p. 289-298
Hydrostatic pressure treatment of food has attracted attention because it can inactivate microorganisms without heating. To understand the changes in microbial proliferation, we selected Escherichia coli, Bacillus cereus and Saccharomyces cerevisiae. The generation time of each microorganism was determined based on the number of viable cells before and after application of pressure of 20 to 50 MPa for 6 to 24 hours at 10 to 45°C. The pressure values at which the proliferation of microorganisms stopped were obtained under the respective temperatures. Hydrostatic pressure (50 MPa or less) was applied to unpasteurized sake and fruit and vegetable juices with the goal of reducing the number of viable cells and bacteria, thereby extending the use-by date. A relatively low pressure was found to lengthen the time food could be stored at low cost using pressure-storage. Our findings are considered applicable to food industry practices.
Food preservation is an important concern in our lives. To date, people have used various techniques to preserve food, and have made a great effort to prevent foods from deteriorating and spoiling due to microbial growth. In particular, heat treatment has been widely used to preserve food because it is a simple and effective method of inactivating microorganisms. However, due to the recent increasing interest in health consciousness, mainly among advanced countries, special attention has been paid to the quality and safety of food, and the negative effect of excessive heating on the taste and flavor of food. Solving these problems has become another challenge (Rastogi et al., 2010; Ohlsson, 1994; Miyamoto, 2009; Cardello et al., 2007). Furthermore, there is increasing demand for pre-cut uncooked vegetables and semi-prepared food materials. Therefore, the development of a non-thermal treatment capable of minimizing the proliferation of microorganisms and extending the use-by date for food materials has become necessary.
Hayashi proposed that hydrostatic pressure can improve the preservability of food products while maintaining the original taste and flavor, and suggested the use of high-pressure treatment for preserving food products (Hayashi, 1989). On the other hand, Sonoike reviewed scientific articles published during the past 100 years concerning the sterilization of microorganisms by high-pressure treatment, and concluded that it was very difficult to kill heat-resistant spores by the application of high pressure (Sonoike, 1997). Our previous study used two typical heat-resistant spores, Bacillus cereus and B. subtilis, to demonstrate the reduction in their viable counts following application of a high-pressure treatment of 200 MPa, to lower the heat resistance of the spores, and subsequent heat treatment at about 100°C (Ohara et al., 2015; Ogino et al., 2015).
It is known that the generation time (or doubling time) of microorganisms during the exponential phase of growth is affected by temperature (Ingraham, 1958). However, there are few reports clarifying the effects of pressure on generation time. We therefore decided to investigate the rate of microbial proliferation with respect to generation time, and attempted microbial control using hydrostatic pressure of 50 MPa or less, in order to promote this food preservation method in the food industry. A lower pressure is more attractive for smaller food companies that may want to use this technology. For this study, we selected three kinds of microorganisms: Escherichia coli as an indicator of food sanitation, Bacillus cereus as an organism involved in the deterioration and spoilage of food, and Saccharomyces cerevisiae as a fermentation organism. The generation time of each microorganism was determined based on the number of viable cells following the application of pressure of 20 to 50 MPa for 6 to 24 hours at 10 to 45°C. The changes in microbial growth and the maximum pressure values at which the proliferation stopped were obtained at temperatures between 10 to 45°C. Then, to demonstrate the applicability of these findings, we aimed to extend the use-by date of unpasteurized sake and fruit and vegetable juices by application of hydrostatic pressure.
Determination of generation time The generation time (D) of each microorganism after pressure application for n hours was determined in accordance with the following formula (Maier, 2008):
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The pressure holding time (n) was set to the period of time that it was expected to take for a cell of every kind of microorganism to be divided into at least two cells, in reference to the generation time under 0.1 MPa.
Microbial and food samples The three kinds of microorganisms: Escherichia coli NBRC3301, Bacillus cereus NBRC13494 and Saccharomyces cerevisiae NBRC1136 were obtained from the National Institute of Technology and Evaluation and used for our experiments. In addition, a commercially available unpasteurized sake (“Yuki,” 180 mL, Maruichi Ltd., Ojiya, Japan) was used for the test. In addition, apples, cherry tomatoes, carrots and spinach, all produced in Japan, were bought and prepared into four kinds of juices.
Preparation of E. coli suspension Two hundred µL of a rehydration fluid (with a formulation of 10 g/L hipolypepton, 2 g/L yeast extract, 1 g/L magnesium sulfate heptahydrate, pH 7.0) was added to a dried specimen of E. coli in an ampule (103 cfu/ampule), and the fluid was allowed to stand for several minutes. The resultant fluid was inoculated into LB medium (with a formulation of 5 g/L yeast extract, 10 g/L tryptone and 5 g/L sodium chloride), followed by shaking culture at 120 rpm for 12 hours at 30°C using a shaking bath (SB-20; Iuchi Seieido Co., Ltd., Osaka, Japan). One platinum inoculation loop of the culture liquid was transferred to and spread onto an LB plate, followed by incubation at 35°C for 24 hours. A single colony appearing on the plate was inoculated into LB liquid medium, followed by shaking culture at 120 rpm overnight at 37°C. Next, 0.3 mL of the culture liquid was centrifuged at 6500 × g for one minute at room temperature. The resultant pellet was suspended in a 67 mM phosphate buffer solution. After these washing steps were repeated twice, a bacterial suspension of 108 cfu/mL was prepared using the phosphate buffer solution. The suspension thus obtained was refrigerated at 4°C until use.
Preparation of Bacillus cereus suspension The spores were suspended in a nutrient liquid medium (with a formulation of 0.10 g/100 mL D(+)-glucose, 0.50 g/100 mL polypeptone and 0.25 g/100 mL yeast extract), followed by shaking culture for 3 hours at 35°C. After confirming the change from spores to vegetative cells, the suspension was diluted to a bacterial concentration of 102 cfu/mL. The suspension of B. cereus spores was prepared in accordance with the spore culture method (Shibata et al., 1995) described in “Experiment Manual of Spores.” The bacterial fluid was spread onto a plate of standard agar medium (Pearlcore plate count agar; Eiken Chemical Co., Ltd., Tokyo, Japan) using a bacterial cell spreader, and then incubated at 35°C for 7 days. The resultant culture liquid was collected and suspended in a phosphate buffer solution (67 mM, pH 7.0). The suspension was centrifuged at 900 × g for 20 minutes at 20°C using a centrifugal separator (H-100B; Kokusan Co., Ltd., Tokyo, Japan). The above phosphate buffer solution was added to the spore sediment in the form of a pellet, which was again centrifuged. These washing steps were repeated three times, and then 80°C heat treatment was conducted for 20 minutes to kill the vegetative cells. The spore suspension was adjusted to a concentration of 109 cfu/ mL by the addition of the phosphate buffer solution, and then tightly sealed in a sterilized soft resin bag and frozen at −80°C until use.
With respect to B. cereus, the generation times of vegetative cells and spores were separately determined at the optimum growth temperature of 35°C, as the sensitivity to pressure was considered to differ between vegetative cells and spores. The above-mentioned nutrient liquid medium was used in both cases.
Preparation of Saccharomyces cerevisiae suspension Two hundred µL of YM liquid medium (Difco Laboratories, Detroit, Michigan, USA) was added to a dried specimen of Saccharomyces cerevisiae in an ampule (103 cfu/ampule), and the fluid was allowed to stand for several minutes. The resultant fluid was inoculated into fresh YM liquid medium, followed by shaking culture at 120 rpm overnight at 28°C. One platinum inoculation loop of the culture liquid was transferred to and spread onto a YM plate, followed by incubation at 28°C for three days. A single colony appearing on the plate was inoculated into YM liquid medium, followed by shaking culture under the same conditions as above. Next, 0.3 mL of the culture liquid was centrifuged at 6500 × g for one minute at room temperature. The resultant pellet was again suspended in a 67 mM phosphate buffer solution. After these washing steps were repeated twice, a suspension was finally prepared using 1.0 mL of phosphate buffer solution for a bacterial concentration of 107 cfu/mL. The suspension thus obtained was refrigerated at 4°C until use.
Apparatus for hydrostatic pressure treatment A laboratory-scale pressurization vessel made by Echigo Seika, Co., Ltd., Nagaoka, Japan (with a maximum pressure of 100 MPa, internal dimensions of 100 (dia.) × 200 mm, and a capacity of about 1.5 L) was used, with a water-pressured manual pump (WP-1B; Riken Kiki Co., Ltd., Tokyo, Japan) and temperature controller (NCB-2400; Tokyo Rikakikai Co., Ltd., Tokyo, Japan) connected to the pressurization vessel.
Hydrostatic pressure treatment and determination of viable cell counts E. coli, the vegetative cells and spores of B. cereus, and S. cerevisiae were separately suspended in a 67 mM phosphate buffer solution. The E. coli suspension was inoculated into LB medium; the suspension of B. cereus vegetative cells into nutrient liquid medium; the suspension of B. cereus spores into trypticase-soy broth (BD, France); and the S. cerevisiae suspension into YM medium. Each kind of culture liquid was separated into 2-mL aliquots of 102 to 103 cfu/mL, which were tightly sealed in plastic pouches. These pouches were individually stored under the predetermined conditions (0.1–50 MPa, 10–45°C, 6–24 hours) using the apparatus for hydrostatic pressure treatment. Following storage, each culture liquid was appropriately diluted with a 67 mM phosphate buffer solution, and a 1-mL sample dilution was pipetted into duplicate Petri dishes. E. coli was cultured by the pour plate method using desoxycholate medium (Pearlcore desoxycholate agar; Eiken Chemical Co., Ltd.). The B. cereus vegetative cells and spores and S. cerevisiae were cultured by the pour plate method using standard agar medium. Cultures of E. coli and B. cereus were incubated at 35°C for 24 hours. Viable counts of B. cereus spores were determined after the germination of spores to vegetative cells. The S. cerevisiae culture was incubated at 28°C for 72 hours. The number of colonies appearing after the given incubation time was counted. This measurement was repeated three times or more (n = 3 to 15) for each condition to obtain average viable cell counts.
Determination of viable cell counts of S. cerevisiae in unpasteurized sake and viable bacterial counts in fruit and vegetable juices The preliminary check confirmed the absence of Lactobacillus in the unpasteurized sake. The presence of moromi (i.e., sake mash before refinement) is thought to impede the accurate determination of viable cell counts of S. cerevisiae in unpasteurized sake after incubation. Therefore, the unpasteurized sake was allowed to stand to precipitate the moromi, and the resultant supernatant liquid was subjected to the test. The supernatant (2 mL) was sealed in a soft resin bag and stored under pressure (i.e., under pressure-storage condition) at 50 MPa, 4°C for 48 hours and one week, and at 50 MPa, 20°C for 15 hours and one week. After completion of pressure-storage, the supernatant was diluted and incubated in the same manner as above. Then, based on the number of colonies appearing, the viable cell counts of S. cerevisiae were obtained. For another test, each of the fruits and vegetables was ground in a mixer (IK-8210; Izumi Products Company Co., Ltd., Matsumoto, Japan) with the same mass of water to obtain a smoothie-like juice. The obtained samples were tightly sealed in soft resin bags and subjected to pressure-storage of 50 MPa at 20°C for 30 days. Control samples were stored under 0.1 MPa. Viable cell counts were determined on the 1st day, 3rd day, 7th day, 15th day and 30th day.
Statistical nalysis The generation time was expressed as the mean ± standard deviation (SD). An unpaired Student's t-test was used to compare the generation time obtained after pressure-storage with that obtained under 0.1 MPa. The levels of significance were P < 0.05 and P < 0.01.
Generation time after hydrostatic pressure treatment Generally, generation time is defined as the rate of exponential growth of microorganisms in the exponential growth phase. In this study, however, we used the generation time as a practical concept for evaluating the pressure-dependent characteristics of microorganisms with respect to growth. Generation time commonly focuses on the exponential growth phase of microorganisms, while generation time used herein was regarded as the period of time required for the number of each microorganism to double, regardless of the microbial growth phase. That is because the application of pressure was considered to inhibit the growth of microorganisms and promote their damage and destruction whether in the exponential growth phase or not, and the growth curve depicted under the pressure was considered to differ from that under atmospheric pressure. In addition, spores coexist with the vegetative cells of spore-forming bacteria in the natural world, and the B. cereus spores used in this study were not in the exponential growth phase immediately following germination. By determining the above-mentioned generation time, we discovered a specific pressure value where the growth of each microorganism was limited. Furthermore, when the changes in generation time of the microorganisms were viewed from pressure as a factor in combination with other factors (such as temperature and pressure holding time), the microorganisms were observed to have characteristic pressure-dependent attributes, which were previously unknown. The generation time used herein was calculated in accordance with the above-mentioned formula Eq. 1. When the number of microbial cells was less than the initial number of cells, that is, lnN-lnN0 < 0, the resultant value D became negative (D < 0). In such a case, the generation time was expressed as infinity (∞) in this study. Figure 1 shows the changes in generation times of the microorganisms after application of hydrostatic pressure at 20°C for 15 hours. The generation times of the three kinds of microorganisms became longer as the pressure level increased. The hydrostatic pressure of 40 MPa caused the generation time of E. coli to extend about 21 times longer than that obtained under 0.1 MPa. The generation time of B. cereus spores became infinite and growth stopped by the application of 30 MPa. The generation time of S. cerevisiae became infinite and growth stopped by the application of 40 MPa.
Changes in generation time of E. coli, B. cereus spores and S. cerevisiae determined after application of hydrostatic pressure at 20°C for 15 hours.
The vertical bar shows standard deviation of each value. Data are expressed as mean ± standard deviation (SD). n = 3–11. * P < 0.05; ** P < 0.01 vs. 0.1 MPa (unpaired Student's t-test). Dashed-dotted line means predictive value.
Figure 2 shows the changes in generation time of the microorganisms after application of hydrostatic pressure at 35°C for 6 hours. The generation times of the three kinds of microorganisms became longer as the pressure level increased, similar to the results shown in Fig. 1 at a temperature of 20°C. The generation times of B. cereus spores and S. cerevisiae became infinite and growth stopped by the application of 40 MPa.
Changes in generation time of E. coli, B. cereus spores and S. cerevisiae determined after application of hydrostatic pressure at 35°Cfor 6 hours.
The vertical bar shows standard deviation of each value. Data are expressed as mean ± standard deviation (SD). n = 3–15. * P < 0.05; ** P < 0.01 vs. 0.1 MPa (unpaired Student's t-test). Dashed-dotted line means predictive value.
Table 1 shows the generation times and viable counts after application of hydrostatic pressure at 10°C for 24 hours, and 45°C for 6 hours. At a temperature of 10°C, the generation times of the three microorganisms drastically extended and finally became infinite at pressure values lower than those found at 20°C and 35°C, as shown in Figs. 1 and 2, respectively. The viable counts of B. cereus spores were found to decrease under every pressure condition at 10°C. The reason for this was assumed to be that a temperature of 10°C has an inhibiting effect on microbial growth. At a temperature of 45°C, on the other hand, the generation time of E. coli became infinite under every pressure condition; the growth of B. cereus stopped at 30 MPa; and S. cerevisiae was considered incapable of growth because 45°C exceeds that allowing it to proliferate.
| Orgamism | Temperature (°C) | Pressure (MPa) | Holding time (h) | Generation time (min) | Viable count (Log10CFU/mL) | |
|---|---|---|---|---|---|---|
| Initial (No) After hydrostatic pressure treatment (N) | ||||||
| E. coli | 10 | 0.1 | 24 | 1847.0 ± 110.4 | 4.0 × 102 | 6.5 × 102 |
| 20 | 3418.2 ± 489.9 | 4.1 × 102 | 5.5 × 102 | |||
| 30 | ∞ | 4.4 × 102 | 2.9 × 102 | |||
| 45 | 0.1 | 6 | ∞ | 4.3 × 102 | 3.5 × 102 | |
| 20 | ∞ | 3.9 × 102 | 9.4 × 101 | |||
| 30 | ∞ | 5.7 × 102 | 3.9 × 102 | |||
| 40 | ∞ | 4.0 × 102 | 3.3 × 102 | |||
| B. cereus | 10 | 0.1 | 24 | ∞ | 4.1 × 102 | 2.7 × 102 |
| 20 | ∞ | 4.8 × 102 | 1.1 × 102 | |||
| 30 | ∞ | 3.5 × 102 | 4.0 × 101 | |||
| 45 | 0.1 | 6 | 100.3 ± 21.3 | 1.6 × 103 | 3.3 × 104 | |
| 20 | 168.7 ± 86.2* | 3.9 × 102 | 4.4 × 103 | |||
| 30 | ∞ | 4.0 × 102 | 3.3 × 101 | |||
| 40 | ∞ | 1.7 × 103 | 3.0 × 101 | |||
| S. cerevisiae | 10 | 0.1 | 24 | 8082.9 ± 888.4 | 4.5 × 103 | 5.1 × 103 |
| 20 | ∞ | 1.7 × 103 | 1.7 × 103 | |||
| 30 | ∞ | 1.1 × 103 | 7.1 × 102 | |||
| 45 | 0.1 | 6 | ∞ | 1.6 × 103 | 0 | |
| 20 | ∞ | 4.4 × 103 | 4.5 × 101 | |||
| 30 | ∞ | 2.2 × 102 | 1.0 × 100 | |||
| 40 | ∞ | 8.5 × 101 | 0.4 × 100 | |||
Values of generation times are mean ± SD. n = 3–14.
The vegetative cells and spores of B. cereus are known to differ in their sensitivity to pressure. Thus, the generation times of the vegetative cells and spores were compared. Figure 3 shows the changes in the generation times of B. cereus vegetative cells and spores after application of hydrostatic pressure at 35°C for 6 hours. The generation times of both B. cereus vegetative cells and spores became longer as the pressure level increased, and reached infinity at 40 MPa. It was confirmed that the pressure value at which the generation time of the spores began to increase was around 20 MPa, which was lower than that in vegetative cells. Vegetative cells were observed together with spores within the pressure range from around 20 to 30 MPa. Once B. cereus spores germinated, the resultant vegetative cells were controlled under the pressure of 20 to 40 MPa, thereby extending the generation time. Then, the generation time reached infinity at a pressure value of 40 MPa, as mentioned above. In other words, the spores were not inhibited from germinating and continued to change to vegetative cells before the applied pressure reached 40 MPa. The generation time curve of the spores, therefore, merged with the generation time curve of the vegetative cells at 40 MPa.
Changes in generation time of B. cereus vegetative cells and spores determined after application of hydrostatic pressure at 35°C for 6 hours.
Based on the generation times of E. coli, B. cereus vegetative cells and spores, and S. cerevisiae treated with pressures below 50 MPa for several hours, we determined the specific pressure values capable of inhibiting the microbial growth corresponding to the respective temperature ranges. Namely, it was expected that the appropriate combination of the factors of pressure, temperature and pressure holding time could extend the generation time to infinity. The feasibility of sterilization without resorting to heat was therefore suggested. By evaluating the change in generation time in series rather than determining the change in viable counts, it was observed that the generation time approaches infinity as the pressure level increases up to a value where the growth of each microorganism is limited. Construction of an Arrhenius plot can estimate the change in growth with a high degree of reliability. Not all microorganisms living in foods are in the exponential growth phase. According to this study, however, microbial metabolism can be estimated based on the generation time of the microorganism, regardless of the phase of the growth curve which it is in. The generation time of microorganisms can therefore be used as a practical guide for food storage.
Change in viable count after hydrostatic pressure treatment The pressure value at which microbial growth stops can be read from the graph showing the change in generation time. However, it is difficult to read the change in viable count directly from the generation time. In the food manufacturing field, it is essential to fully determine the number of microorganisms in food. Therefore, we investigated the change in the number of surviving cells of the microorganisms. Figure 4 shows the changes in viable counts of E. coli, B. cereus spores and S. cerevisiae after hydrostatic pressure treatment, expressed as the survival ratio of cells. Fig. 4(A) shows a pressure of 20–30 MPa applied for 24 hours at 10°C, Fig. 4(B) shows a pressure of 20–40 MPa for 15 hours at 20°C, Fig. 4(C) shows a pressure of 20–50 MPa for 6 hours at 35°C, and Fig. 4(D) shows a pressure of 20–40 MPa for 6 hours at 45°C. As shown in Fig. 4, the growth rates of all three kinds of microorganisms decreased with increasing pressure value. In Fig. 4(A), the N/N0 of E. coli was lowered to less than 1.0 at 30 MPa, which suggested that the viable cells would be further reduced if E. coli was stored under 30 MPa or more for 24 hours or more. In Fig. 4(B), the pressure used for this test was increased up to 40 MPa to determine the pressure value where S. cerevisiae growth was inhibited in regards to the commercialization of unpasteurized sake. The N/N0 value fell below 1.0 at 40 MPa. In the pressure-storage test of unpasteurized sake, to be described later, a higher hydrostatic pressure, i.e., 50 MPa was chosen in the interest of enhanced security. As shown in Fig. 4(C), a drastic decrease in the growth rates of E. coli and B. cereus spores was respectively observed within the pressure range from 20 to 40 MPa. Although these microorganisms should typically be metabolically active at 35°C, growth inhibition was considered to result from inhibited cell division following pressure application. In Fig. 4(D), the survival ratio of E. coli cells after application of pressure of 30–40 MPa was higher than at 20 MPa pressure. It was assumed that the pressure acted to protect the cells from heat sterilization (ZoBell et al., 1962). Judging from the results of Fig. 4 as a whole, the conditions capable of reducing the N/N0 of E. coli below 1.0, that is, the combination of 30 MPa, 10°C and 24 hours, were considered to be optimal pressure-storage conditions for E. coli reduction.
Ratio of the viable counts to the initial counts of E. coli, B. cereus spores and S. cerevisiae determined after application of hydrostatic pressure.
Hydrostatic pressure treatment at 10°C, 20 to 30 MPa for 24 hours (A), 20°C, 20 to 40 MPa for 15 hours (B), 35°C, 20 to 50 MPa for 6 hours (C) and 45°C, 20 to 40 MPa for 6 hours (D). N: mean of number of microorganisms after n hours, N0: mean of initial number of microorganisms. n = 5–15.
As shown in Table 1, the viable counts of B. cereus spores decreased up to one order after hydrostatic pressure treatment of 40 MPa at 45°C for 6 hours. We speculated that prolongation of the pressure-holding time would further reduce B. cereus spores. Then, the number of surviving spores was determined after application of pressure for 6 and 24 hours (Fig. 5). We considered the results of Fig. 5 to be applicable to industrial fields; thus, we expressed the data as “mean ± 3SD,” which has been adopted as a reliable quality control standard in a wide range of industries. It was confirmed that the number of surviving B. cereus spores decreased to undetectable levels after application of 40 MPa at 45°C for 24 hours. The results indicate that B. cereus can be completely eliminated by appropriately combining the conditions of pressure, temperature and pressure holding time.
Changes in the viable number of B. cereus spores at 45°C under pressurization at 40 MPa for 6 and 24 hours.
Data are expressed as mean ± 3SD (standard deviation). ** P < 0.01 vs. control (unpaired Student's t-test). n = 6.
It is impossible to sterilize heat-resistant spores at normal room temperature (Sonoike, 1997). We conducted the experiments in light of the above finding, and demonstrated that the heat-resistant spores were eliminated, with the viable cell counts being reduced by the application of pressure ranging from 20 to 50 MPa at a temperature from 10 to 45°C for 6 to 24 hours. To inactivate spores, many reports have proposed subjecting the spores to heat treatment of 50°C or more or pressure treatment of 60 MPa or more to induce germination, thereafter inactivating the spores during the process of germination. However, those methods are not practical because simultaneous germination cannot be ensured. Most of those reports dealt with B. subtilis as the heat-resistant spore sample. For B. cereus spores, the National Agriculture and Food Research Organization (NARO) showed in a joint research project that the germination ratio of B. cereus spores remarkably increased after treatment of 40 to 50°C, 600 to 1000 atm., and 30 to 60 minutes (i). In contrast, the pressures (50 MPa or less) we selected were lower than those in the above joint research; and the temperatures (10, 20 and 35°C) we considered available in light of the results of Fig. 4 were lower than the temperatures (40 to 50°C) used in the joint research. Furthermore, our experiments confirmed that the generation time of B. cereus spores became infinite and their growth stopped after application of 30 MPa at 20°C (Fig. 1) or 40 MPa at 35°C (Fig. 2). The temperatures (20°C, 35°C) used in our experiments were also lower than 40°C and the pressure (30, 40 MPa) used was also lower than 600 atm. In consideration of the above, we concluded that B. cereus spore germination was not induced by any of the pressure values in our experimental system. As shown in Figs. 1 and 2, the pressure value at which the growth of B. cereus stopped was found to be lower than the pressure values at which the growth of the non-spore forming E. coli and S. cerevisiae stopped. Then, it is considered that the microbial generation time is controlled by a mechanism quite different from the conventional thinking. To be more specific, it is known that application of pressure of about 50 MPa induces monomer depolymerization in the cytoskeleton of microorganisms (Sato et al., 2002), which has an inhibiting effect on microbial growth and contributes largely to cell death (Furukawa, 2008). In this phenomenon, protein denatured by the pressure treatment is incorporated in the normal protein molecule. Even with the application of a pressure of just 30 MPa, the above phenomenon has been confirmed. The eukaryotic cell suffers cell division inhibition and develops morphological abnormalities, thereby delaying microbial growth and decreasing heat resistance, finally resulting in complete cell death. The mechanism by which the viable cells of microorganisms are reduced and the generation time is therefore extended following pressure treatment of just 50 MPa or less should be discussed from the viewpoint of the biologically and chemically destructive effects of pressure treatment.
Viable counts of S. cerevisiae in unpasteurized sake after pressure-storage Unpasteurized sake is characterized by its fresh flavor and taste. However, in the absence of pasteurization, the yeast present in the unpasteurized sake induces secondary fermentation in the bottle, resulting in quality deterioration during storage at room temperature. Then, in consideration of the results from Fig. 4(B), we attempted to reduce the viable counts of S. cerevisiae and extend the shelf life of unpasteurized sake samples by pressure treatment of 50 MPa. Figure 6 shows the changes in the viable counts of S. cerevisiae in the unpasteurized sake during pressure-storage under hydrostatic pressure of 50 MPa. After pressure-storage at 4°C for one week, the viable counts of S. cerevisiae decreased by two orders as compared with that initially. After pressure-storage at 20°C for one week, the viable counts of S. cerevisiae decreased by three orders as compared to that initially. These results showed that pressure-storage at 50 MPa for one week reduced the number of S. cerevisiae present in the unpasteurized sake. This pressure-storage method is especially suitable for brewed foods that benefit from a period of rest after manufacturing.
The viable cell count of S. cerevisiae in unpasteurized sake under pressurization at 50 MPa.
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: 0.1 MPa, ■: 50 MPa.
Viable counts in fruit and vegetable juices after pressure-storage The fruit and vegetable juices contain a variety of microorganisms derived from the respective raw materials. We attempted to extend the shelf life of apple, tomato, carrot and spinach juices. After pressure-storage under hydrostatic pressure of 50 MPa at 20°C for 30 days, changes in viable bacterial counts in the fruit and vegetable juices were determined (Fig. 7). The same juices stored at 0.1 MPa and 20°C all showed an increase in viable bacterial counts and spoiled shortly after storage. On the seventh day, the viable counts of any of the control juices exceeded eight orders; thus, we stopped measurements in light of the commercial value. In contrast, the viable counts in the fruit and vegetable juices stored at 50 MPa and 20°C for 30 days gradually decreased and were finally reduced by two or three orders. The viable bacterial counts were expected to further decrease under additional pressure-storage. With respect to the change in color, pressure-storage did not cause color changes in the apple or tomato juices. The carrot juice was slightly tinged with black. The spinach juice was slightly tinged with yellow, with the initial green color becoming lighter. However, the juices subjected to pressure-storage maintained their fresh color for longer and had a superior fresh taste compared to samples subjected to heat treatment. Pressure-storage has been shown to control viable bacterial counts over a long period of time; however, changes in taste, color, flavor, texture and the like, which depend on the enzyme (s) derived from the food material, has been an ongoing problem. Further, in the course of long-term storage, it is important to take measures to prevent oxidation, fading from exposure to light, and destruction of nutrients by endogenous enzymes. In view of the above, storage at low temperatures was considered to be preferable. Another issue is the ability of a container to release ethylene gas, carbon dioxide or ammonia; however, this issue will be discussed at another time. In view of microbial control, practical use of pressure-storage at 50 MPa was considered to be advantageous, since bacterial growth in fruit and vegetable juices can be inhibited to extend the shelf life.
Viable counts in fruit and vegetable juice under pressurization at 50 MPa at 20°C for 30 days.
Findings from this study and tasks for the future To date, treatment aimed at the sterilization of food products has been conducted under conditions of 200 to 600 MPa for 5 to 30 minutes. Relatively high pressure has been used to shorten the treatment time, thereby increasing productivity. However, the compression energy transmitted by hydrostatic pressure of 1000 MPa is as small as 11 cal/g (0.2 kcal/mol). Therefore, when compared with heat treatment, pressure treatment needs more time to change the item being treated. The Arrhenius equation suggests that the energy obtained by subtracting the activation energy peculiar to a microorganism from the sum of the applied thermal energy and the pressure energy is involved in protein denaturation and microbial inactivation. The findings obtained from this study support the above-mentioned theory. The findings of this study indicate that the pressure-energy induced denaturation of proteins, resulting in microbial cell death. To explain the effect of pressure, it will be necessary to discuss it from both the viewpoint of the energy transmitted by compression and the activation energy. Application of low pressure over a long period of time, as used in our experiments, is considered to be advantageous, because the pressure energy can occupy a larger fraction of the total energy to be transmitted. The use of low pressure can drastically curtail equipment costs, thereby promoting the industrial use of pressure treatment. Furthermore, the advantage of pressure treatment, that is, reduction in microbial growth, can create new systems based on pressure treatment for the brewing industry. Once the process of maturation or fermentation is conducted at low temperatures, the resultant food can be stored for a long time, preventing enzymatic deterioration and reducing microbial growth. As shown in Figs. 6 and 7, the change in viable cell counts in the food during pressure-storage varies depending on the food composition, pH and other conditions. For the practical use of pressure-storage for food preservation, therefore, it is important to confirm the increase/ decrease in the number of microorganisms in a given food and to choose the appropriate treatment conditions.
This study demonstrated that the application of 50 MPa or less of pressure to food for a long time period makes it possible to control the generation time of microorganisms in food. Thus, viable counts can be reduced by this non-thermal treatment, thereby extending the shelf life of food. In addition, this study showed that pressure-storage at 35°C or less can be practically applied to reduce viable counts, instead of cold storage. More specifically, our experiments demonstrated that S. cerevisiae present in unpasteurized sake and microorganisms in fruit and vegetable juices can be practically reduced by pressure-storage. Owing to the merits of pressure compared heat, the energy of pressure can be transmitted to the contents in a container more quickly and more uniformly. Pressure-storage is therefore more energy efficient than cold-storage, thereby drastically reducing operational costs. This study presents a new method for controlling microorganisms based on pressure, and provides valuable information that is applicable to global issues such as food shortages and commercial food waste.
Acknowledgements We would like to thank the Echigo Seika Co., Ltd. contributors: Mr. Atsushi Kobayashi (New Business Development) for providing valuable suggestions, and Ms. Eri Ohara (New Business Development) and Mr. Yuta Kazama and Mr. Yuya Imai (Research Institute) for their generous cooperation in our experiments. We would also like to express our deep gratitude to Dr. Akira Yamazaki, visiting professor at Niigata University of Pharmacy and Applied Life Sciences, for giving us suggestions on the research theme.
