2024 Volume 25 Issue 2 Pages 25-33
This study aimed to investigate the effects of soymilk ingredients on the inactivation of Lactobacillus plantarum subsp. plantarum and Lactobacillus pentosus. Pretreated bacteria were inoculated in MRS broth or soymilk and incubated at 30°C for 48 h. The cells were suspended in 0.85% NaCl or soymilk and incubated at 65°C for 60-120 s at high-pressure in the 300–450 MPa range. The logarithmic survival ratios of the lactobacilli cultured in MRS or soymilk decreased with heating time. The inactivation rate constant, k, did not change between MRS and soymilk medium by heat treatment. The difference in composition of cell membranes was also small. The survival behavior of each sample subjected to high-pressure was described by first-order kinetics. A gradual increase in k was observed at 300–450 MPa. It is found that pre-exponential factor and activation volume of the lactobacilli subjected to high-pressure treatment were related linearly. These indices did not have clear relationship with membrane composition of the lactobacilli. Thus, the different effects of heat and high-pressure stresses on cell resistance are possibly due to the incubation conditions. The results of this study indicate that the mechanism of inactivation may be essentially similar to numerous chemical processes in many microorganisms though factors for the acquisition of heat and pressure tolerance are diverse.
微生物の増殖は，温度や圧力といった生育環境から受けるストレスや培地組成などによって影響を受けることはよく知られている．加えて，食品中では，共存物質と微生物との相互作用にも影響を受ける．とくに共存物質によって微生物の膜特性が変わることで，加熱や高圧などへのストレス耐性を獲得することが報告されている．大豆系食品においては，大豆由来の脂質成分が細胞膜の組成変化に寄与し，熱および高圧ストレス耐性が向大豆の脂質成分と微生物の細胞膜との関係にはまだ不明瞭な点も多い．そこで本研究では，大豆由来成分共存下の系における微生物生産の最適な制御のための基礎的研究として，豆乳成分が共存している環境で乳酸菌を培養・懸濁し，豆乳中の成分が乳酸菌の熱および高圧ストレス応答にどのように関わっているかをMRS培地による培養と比較することで実験的に検討した．まず，MRS培地および豆乳を添加した培地で培養した乳酸菌２種（Lactobacillus plantarum subsp. plantarum （L. plantarum subsp. plantarum），Lactobacillus pentosus （L. pentosus）の膜の脂肪酸組成に着目し，膜特性を評価し環境因子の影響について検討を行い､次に温度と圧力に対する死滅挙動について評価した．
MRS培地および豆乳を培地として30°C，48時間培養した場合における乳酸菌２種の脂肪酸組成について検討した．MRS培地で培養した場合，C16:0，C18:0およびC19:0が含まれていた．一方，豆乳培地では，MRS培地と異なりC19:0が含まれていなかった．しかし，豆乳中にほとんど含まれないC14:0は，MRS培地同様に検出された．このことは，培養液の共存物質の変化によって，細胞膜の脂肪酸組成も変化したと考えられた．高温条件下の乳酸菌の死滅挙動について，MRS培地および豆乳培地で培養した乳酸菌２種を回収し，細胞を0.85% NaCl溶液もしくは豆乳に懸濁し，65°Cで加熱処理を施した．高温条件下の乳酸菌の死滅曲線は，いずれの条件においても一次反応式で記述された．熱死滅速度定数に乳酸菌の種類や培地による違いは顕著ではなく，細胞膜の脂肪酸組成と熱死滅速度定数における相関は認められなかった．乳酸菌の場合において，熱ストレスでは，豆乳を含む培地で培養した影響が現れなかったと考えられた．
300-450 MPaの高圧条件下の乳酸菌の死滅挙動について，MRS培地及び豆乳培地で培養した乳酸菌2種を回収し，細胞を0.85% NaCl溶液もしくは豆乳に懸濁し，各種圧力条件下で加圧処理を施した．高圧条件下の乳酸菌の死滅曲線は，いずれの条件においても一次反応式で記述された．乳酸菌２種において，0.85% NaCl溶液中での高圧条件下では，豆乳培地ではMRS培地と比較して耐圧性が低くなることが示された．しかし，豆乳中での高圧条件下では，豆乳培地で培養した場合においても，耐圧性が高くなる傾向が認められた．このことから，培養液の共存物質によって細胞膜の膜特性が変化し，高圧条件下での死滅挙動に影響を与えたものと考えられた．そこで，各培地条件での死滅速度定数の圧力依存性を解析した．全ての条件について，死滅速度定数の自然対数と処理圧力との間に直線関係が認められたため，前指数因子および活性化体積を算出した． L. plantarum subsp. plantarumの死滅反応において，豆乳共存下では0.85% NaCl溶液中よりも大きな体積変化が必要であることが示唆された．一方，L. pentosusの死滅反応では，反対の傾向が認められた．L. plantarum subsp. plantarumは，L. pentosusと比較して，MRS培養において活性化体積が小さく前指数因子は大きいのに対し，豆乳培養では活性化体積が大きく前指数因子は小さい傾向を示した．これらの結果から，生育する環境の影響の違いは，微生物の種類によって多様であることが示唆された．いずれの条件においても前指数因子の自然対数と活性化体積には直線関係が認められたことから微生物の種類が異なっていても死滅反応は本質的には同様の反応である可能性が考えられた.
In fermented foods that utilize microorganisms, it is necessary to construct food processes for the optimal control of microorganism growth, which is greatly influenced by environmental factors including temperature, osmotic pressure, pH, oxygen concentration, dehydration, high-pressure, and medium composition. Especially, when subjected to heat stress, changes in cell morphology [1], the expression of heat shock proteins [2] and stabilization of mRNA production [3] as well as a change in growth rate occur following a change in growth temperature. Moreover, when subjected to physicochemical stress such as high-pressure and salt concentration, changes also occur in the cell membrane such as changes in fatty acid composition, the amount of unsaturated fatty acids and the carbon chain length of fatty acids [4-8].
In addition, in the case of soy food products, the coexisting substances also contributes to microbial cell membranes under environmental stress. Regarding of soybean as an ingredient, a significant increase in the heat resistance of Clostridium sporogenes spores was observed when heated in soymilk in comparison to 0.1% peptone water [9], and the presence of Bifidobacteria in tea extract during soymilk fermentation altered the phospholipid composition of the membrane [10]. These reports indicate that the membrane characteristics of microorganisms are altered by the coexisting materials in the culture solution. As a result, changes in the characteristics of the cellular membrane influence fermentation and the subsequent disinfection process. In addition to membrane characteristics, soybean extract also affects the stress adaptation of microorganisms [11,12]. In contrast, it has been reported that phospholipids in soy milk additives protect cell membranes, resulting in improved heat and high-pressure stress tolerance [13]. Therefore, it is important to clarify the relationship between the lipid components of soy and the cell membranes of microorganisms for the optimal control of food processes using microbial production in systems with coexisting soybean-derived components.
In this study, we focused on the membrane properties of microorganisms in order to clarify the factors that contribute to the acquisition of heat and pressure stress tolerance of microorganisms in the coexistence of soybean-derived components. The fatty acid composition in Lactobacillus plantarum subsp. plantarum (L. plantarum subsp. plantarum) and Lactobacillus pentosus (L. pentosus) which are closely related and are widespread in the food industry and non-food environment were investigated to evaluate the membrane characteristics [14]. Membrane characteristics were evaluated by comparing heat and high-pressure treatments because protein denaturation is caused by heat treatment and tissue destruction is caused by high-pressure treatment. Moreover, the survival behavior of Lactobacilli cultivated in broth or soymilk under temperature and high-pressure was measured.
Two bacterial strains, L. plantarum subsp. plantarum and L. pentosus, were obtained from AKITA KONNO Co., Akita, Japan and were selected for food processing. L. plantarum subsp. plantarum was isolated from pickles and L. pentosus was isolated from silages.
Soymilk containing 11.5% w/w solids (5% proteins, 3.2% lipids, 2.8% carbohydrate, and 0.5% others) was produced by Taishi Food Inc. The initial pH of soymilk was 6.8. Soymilk used in this study was autoclaved before the experiment.
2.2 Sample preparationAfter 24 h of preculture at 30°C in MRS broth (Becton, Dickinson and Co., Franklin Lakes, NJ, USA), cells were harvested by centrifugation at 3,000 xg for 5 min. The cells were washed three times with a 0.85% (w/v) NaCl solution and inoculated into fresh 3 mL of MRS broth or 10 mL of soymilk. After 48 h of incubation at 30°C in MRS broth, the cells were harvested and washed three times as described above. The resultant cell pellets were suspended in 30 mL of 0.85% (w/v) NaCl solution. The cells in soymilk, which was clabbered by the bacterial activities, were mashed in 90 mL of a 0.85% (w/v) NaCl solution using a Stomacher Lab-Blender 400 (Seward Ltd., Worthing, UK) and Stomacher bag (AS ONE Corp., Osaka, Japan). 30mL of the resulting solution were centrifuged at 3,000 g for 5 min, and the cell pellets were washed thrice. Subsequently, the cell pellets were suspended in 30 mL of 0.85% (w/v) NaCl solution or 30 mL of soymilk. The three types of sample solutions obtained, MRS culture cells in 0.85% NaCl (MRS/NaCl), soymilk culture cells in 0.85% NaCl (SM/NaCl), and soymilk culture cells in soymilk (SM/SM) were immediately used in the following experiments.
2.3 Lipid extractionThe lipid extract from bacterial cells was prepared by a modified method described by Santivarangkna et al. [15]. The cells were mixed well with 50 mL of chloroform–methanol (1:1) containing 0.1% butylated hydroxytoluene and were ultrasonicated for 1 min and centrifuged at 8,000 g for 15 min at 20°C. The liquid phase was pooled in a 500 mL separatory funnel. One-hundred and fifty milliliters of 0.58% (w/v) NaCl solution were added to the funnel. The organic phase was then collected and evaporated. The remaining liquid was removed via purging with dry nitrogen gas.
2.4 Gas chromatographyTotal lipid extract was dissolved in 1 mL of Boron Trifluoride Methanol Complex Methanol Solution (Fujifilm Wako Pure Chemical Co., Osaka, Japan) to a concentration between 10 and 20 mg lipid/mL, and the solution was heated at 100°C for 2 min. Fatty acid methyl esters were analyzed by gas chromatography (GC-2014; Shimadzu Co., Kyoto, Japan) using a packed column (Unisole 3000 Uniport C80/100, GL Sciences Inc., Eindhoven, Netherlands; 3.1 m in length, 3.2 mm in internal diameter) and a flame ionization detector (Shimadzu Co., Kyoto, Japan). Helium was used as the carrier gas at a flow rate of 40 mL min-1. The injection volume and temperature were 5 µL and 250°C, respectively. The oven temperature program was initially set at 140°C with a temperature ramp of 4°C min-1 to 190°C, which was maintained for 17 min, followed by a second temperature ramp of 5°C min-1 to 210°C, which was maintained for 25 min; followed by a third temperature ramp of 6°C min-1 to 220°C, which was maintained for 20 min. Data acquisition and analysis were performed using the LabSolutions software (ver. 5.57; Shimadzu Co., Kyoto, Japan). The fatty acid assignments were verified by comparing the retention times with those of the standards. Fatty acid composition was calculated from the fractions of individual peaks of the total peak area and expressed as a percentage of total fatty acids.
2.5 Heat treatmentUsing a thermostatically controlled water bath (Brookfield Engineering Inc., Middleboro, USA), 2 mL of the sample solution in a test tube was heat-treated at 65°C for up to four steps, 30, 60, 90, and 120 s. The test tubes were gently shaken to maintain the samples at a constant temperature during the heat treatment. After processing, the test tubes were quickly placed in ice for cooling after each time-point.
2.6 High-pressure treatmentTwo milliliters of the sample solution was placed in a 4 x 3 cm nylon-polyethylene bag (Hiryu N-9, Asahi Kasei Pax, Tokyo, Japan) and heat sealed with a heat sealer (Fuji Impulse Co., Osaka, Japan). The bags containing the sample solution were settled in a stainless container (NET: 21 mL) connected to a high-pressure hand pump (HP-500, Syn Corporation Ltd., Kyoto, Japan), and it was subjected to a hydrostatic pressure of 300-450 MPa at 25°C. The pressure medium is water (purified water), and the holding time of high-pressure treatments were 60-180 s.
2.7 Viable cell countingSerial dilutions of the samples (6 log CFU/ml) were performed using a 0.85% (w/v) NaCl solution. Samples were inoculated onto MRS agar plates (Becton, Dickinson and Co., Franklin Lakes, USA) containing 0.5% (w/v) CaCO3. Note that the CaCO3 was added to prevent acidification of the medium and to maintain quality. After two days of incubation at 30°C, visible colonies were counted.
2.8 Kinetic analysis of high-pressure inactivationTo describe the stress tolerance of the cells, the following first-order kinetics equations were applied;
 | (1) |
 | (2) |
where N0 is the initial number of cells (CFU/mL), N is the number of survivors (CFU/mL), t is the exposure time (s), and k is the first-order inactivation rate constant (s－1).
To describe the pressure dependence of k, activation volume ΔV*(mL/mol) was calculated using the following equation:
 | (3) |
where k0 is the pre-exponential factor (s－1), P is the pressure (MPa), R is the gas constant(J/(K･mol)), and T is the absolute temperature (K).
The fatty acid composition of Lactobacilli grown in different media is shown in Table 1. The membranes of cells of both Lactobacilli in MRS broth contained many C16:0, C18:0, and C19:0 fatty acids, and their values were close to those of reference [16,17]. In soymilk incubation, the fatty acid compositions of both Lactobacilli tended to be similar to the fatty acids of soymilk, but C19:0 was not detected; the two species were similarly affected by the medium as previously reported [18]. However, because C14:0 was maintained in cell membranes at a higher concentration than in soymilk, it is possible that some fatty acids could be converted to other fatty acids in the membrane, whereas others could not.
| Fatty acid | Soymilk | % of total composition in MRS broth of: | % of total composition in soymilk of: | ||
|---|---|---|---|---|---|
| Lactobacilius plantarum subsp. plantarum |
Lactobacillus pentosus | Lactobacilius plantarum subsp. plantarum |
Lactobacillus pentosus | ||
| 12:0 | 0.2 | 0.1 | |||
| 14:0 | 0.2 | 3.3 | 3.3 | 1.9 | 1.4 |
| 16:0 | 12.9 | 14.1 | 15.1 | 16.9 | 15.8 |
| 16:1 | 4.5 | 2.9 | 0.5 | ||
| 18:0 | 4.9 | 7.8 | 6.6 | 6.4 | 7.9 |
| 18:1 | 21.4 | 30.6 | 36.2 | 12.7 | 15.2 |
| 18:2n-6 | 52.1 | 0.7 | 4.7 | 52.4 | 49.1 |
| 18:3n-3 | 7.7 | 8.3 | 7.2 | ||
| 19:0 | 20.1 | 14.8 | |||
| 22:0 | 0.3 | <0.1 | <0.1 | ||
| Unknown | 0.5 | 18.7 | 16.3 | 0.9 | 3.4 |
The survival curves of Lactobacilli, in three sets of incubation/inactivation mediums; MRS/NaCl, SM/NaCl, and SM/SM under 65°C are shown in Fig. 1. It was found that the logarithm of the survival ratio decreased with heating time for the lactobacilli. In this study, thus, the entire behavior of the survival curve under heat treatment was investigated using first-order kinetics, and k was obtained (Table 2). In MRS broth and soymilk, no difference was observed between the inactivation rate constants of L. plantarum subsp. plantarum and L. pentosus. In the presence of soymilk ingredients, both inactivation rate constants were slightly low. A previous study has reported that changes in fatty acid composition can lead to heat tolerance [19]. Thus, the amounts of C16:1 and C18:1 relative to those of C16:0 and C18:0 (the (C16:1＋C18:1)/(C16:0＋C18:0) ratio) and the inactivation rate constants were compared. However, in this study, no relationship was found between the (C16:1＋C18:1)/(C16:0＋C18:0) ratio and the inactivation rate constants. These results were different from a case of E. coli. [13], a gram-negative bacterium. On the other hand, it was reported that the medium did not affect the survival behavior of Staphylococcus aureus under heat treatment [20]. A previous study indicated that the cell membrane was easily strengthened by phospholipids in the soybean extract [13], because gram-negative bacteria such as E. coli have a thinner layer of peptidoglycan and a cell wall that includes many lipids. However, because gram-positive bacteria such as Lactobacillus or Staphylococcus aureus have a thicker layer of peptidoglycan and a cell wall that includes few lipids, it is likely that the effect of soymilk cultivation on survival is negligible under heat stress.
Survival curves of L. plantarum subsp. plantarum (a) and L. pentosus (b) under heat treatment at 65°C. Note that ‘survival ratio’ of Y-axis is N/N0, and N0 is the initial number of cells (CFU/mL), and N is the number of survivors (CFU/mL). Error bar indicates the standard deviation of ln (survival ratio) (n ＝ 3). MRS/NaCl, SM/NaCl and SM/SM indicates the set of incubation/inactivation medium, respectively. MRS is MRS medium, SM is soymilk, and NaCl is 0.85% NaCl solution.
| Microorganism | Medium (incubation) | Medium (inactivation) | Inactivation rate constant, k [s-1] |
|---|---|---|---|
| Lactobacilius plantarum subsp. plantarum | MRS | 0.85% NaCl | 0.067 |
| Soymilk | 0.85% NaCl | 0.068 | |
| Soymilk | Soymilk | 0.056 | |
| Lactobacillus pentosus | MRS | 0.85% NaCl | 0.065 |
| Soymilik | 0.85% NaCl | 0.060 | |
| Soymilk | Soymilk | 0.057 |
The survival curves of Lactobacilli in three types of medium, MRS/NaCl, SM/NaCl, and SM/SM, under high hydrostatic pressure stress are shown in Fig. 2. The entire behavior of survival curve under high-pressure treatment can be described by first-order kinetics, and k was obtained (data not shown). In both L. plantarum subsp. plantarum and L. pentosus, a tendency for inactivation at lower pressure in soymilk incubation was observed than that in MRS broth (Fig. 2(a)/(d) and (b)/(e). In addition, the tendency for the lactobacilli to become inactivate at higher pressure in the bacterial suspension with the presence of soymilk ingredients (Fig. 2(c)/(f)) was observed when compared to 0.85% NaCl (Fig. 2(b)/(e)). Carbohydrates, proteins, and lipids have protective effects that reduce the effects of high-pressure [21]. It appears that cells were altered by the incubation ingredients: the survival behavior under high-pressure stress conditions showed differences between each incubation condition.
Survival curves of L. plantarum subsp. plantarum (a)–(c) and L. pentosus (d)–(f) under high-pressure treatment in the 300-450 MPa. Note that ‘survival ratio’ of Y-axis is N/N0, and N0 is the initial number of cells (CFU/mL), and N is the number of survivors (CFU/mL). Error bar indicates the standard deviation of ln (survival ratio) (n ＝ 3). MRS/NaCl, SM/NaCl and SM/SM indicates the set of incubation/inactivation medium, respectively. MRS is MRS medium, SM is soymilk, and NaCl is 0.85% NaCl solution.
The pressure dependence for each medium and high-pressure condition was analyzed for L. plantarum subsp. plantarum and L. pentosus. The relationship between the natural logarithm of the inactivation rate constants and hydrostatic pressure is shown in Fig. 3. A linear relationship was observed between the natural logarithm of the inactivation rate constants and the hydrostatic pressure for all experimental conditions. The activation volume, (ΔV*), and the pre-exponential factor, (k0), were required by Eq. (3). Table 3 summarizes the absolute value of the activation volume, |ΔV *|, and the pre-exponential factor, k0, for each medium condition in L. plantarum subsp. plantarum and L. pentosus and compares their values with those of similar studies [22-26]. In L. plantarum subsp. plantarum, a large volume change in the presence of soymilk ingredients was needed for inactivation compared with 0.85% NaCl. However, in L. pentosus, a small volume change in the presence of soymilk ingredients was required compared to 0.85% NaCl. In MRS broth, L. plantarum subsp. plantarum showed a lower activation volume than L. pentosus. In the soymilk medium, L. plantarum subsp. plantarum showed a larger activation volume than L. pentosus. In the high-pressure treatment in soymilk, L. plantarum subsp. plantarum showed a fairly small pre-exponential factor and a large activation volume. Therefore, L. plantarum subsp. plantarum in soymilk had greater pressure resistance than under other conditions. No relationship was found between the grouping obtained based on the stress response patterns of L. plantarum, L. pentosus, and L. paraplantarum [27]. These results indicated that differences in environment varied depending on the type of bacterium. The correlation between activation volumes and the natural logarithm of the pre-exponential factors in various microorganisms and media is shown in Fig. 4. A linear relationship was observed between the natural logarithm of the pre-exponential factors and the activation volumes under all conditions (y ＝ -0.131x - 4.437, r ＝ -0.67). The pre-exponential factor (k0) includes the entropy change to the active state, whereas the activation volume (ΔV*) includes the enthalpy change. Therefore, the linear relationship between the natural logarithm of the activation energy and the activation volume observed in pressure-induced microbial inactivation can be interpreted in terms of enthalpy-entropy compensation (Meyer-Neldel rule) [28]. The inactivation of microorganisms by high-pressure treatment can be described as occurring by a mechanism common to many chemical reactions in which enthalpy-entropy compensation is observed.
Relationship between the natural logarithm of the inactivation rate constants and the high-pressure of 300-450 MPa in (a) L. plantarum subsp. plantarum and (b) L. pentosus.
| Microorganism | Medium (incubation) | Medium (inactivation) | Pre-exponential factor, k0 [s-1] | Activation volume, |ΔV*| [mL/mol] | P [MPa] | T [°C] |
|---|---|---|---|---|---|---|
| Lactobacilius plantarum subsp. plantarum | MRS | 0.85% NaCl | 9.8×10-3 | 16 | 350, 400, 450 | r.t. |
| Soymilk | 0.85% NaCl | 1.3×10-3 | 38 | 300, 325, 350 | r.t. | |
| Soymilk | Soymilk | 1.9×10-5 | 48 | 400, 425, 450 | r.t. | |
| Lactobacillus pentosus | MRS | 0.85% NaCl | 2.1×10-4 | 43 | 350, 375, 400 | r.t. |
| Soymilik | 0.85% NaCl | 4.0×10-3 | 29 | 300, 325, 350 | r.t. | |
| Soymilk | Soymilk | 4.2×10-3 | 22 | 400, 425, 450 | r.t. |
*r.t. indicates room temperature;25°C
The effect of the (C16:1＋C18:1)/(C16:0＋C18:0) ratio on the inactivation rate constants under pressure stress at 350 MPa is shown in Fig. 5. The inactivation rate constants under 350 MPa pressure decreased with increasing unsaturated fatty acid ratio. Thus, it is likely that the pressure resistance is sensitive to the nature of the cell membrane. And there was a difference in the level of high-pressure resistance in MRS broth and soymilk. In the case of E. coli, membrane fluidity is the dominant factor affecting resistance in exponential-phase cells [29]. The possibility that the soymilk component improves membrane fluidity and, consequently, high-pressure tolerance has been recognized in our studies [13].
Effect of the (C16:1＋C18:1)/(C16:0＋C18:0) ratio in the cell membrane on the inactivation rate constants in pressure stress at 350 MPa. MRS and SM is indicated as abbreviation of MRS/NaCl and SM/NaCl
On the other hand, microbial inactivation, protein denaturation, lipid phase change, and enzyme inactivation can disturb the cell morphology, genetic mechanisms, and biochemical reactions during high-pressure processing. When cell metabolism is slowed down during high-pressure treatment, the greater resistance to high-pressure may be due to the accumulation of cell components that reduce the effect of high-pressure, and certain food constituents such as proteins, carbohydrates and lipids can have a protective effect [30]. However, simultaneously, Ueno et al. suggested that the cell structure of a plant is easily damaged by high-pressure treatment [31,32]. Therefore, it was considered important to evaluate the condition of membrane cells during high-pressure treatment in terms of both protection of membrane cells and promotion of microbial growth.
Fig. 6 shows the effect of the (C16:1＋C18:1)/(C16:0＋C18:0) ratio on the pre-exponential factors and the activation volumes under high-pressure treatment. The behavior of both parameters differed from that of the inactivation rate constant. A reverse tendency between L. plantarum subsp. plantarum and L. pentosus was observed in the two types of media. The activation volume indicates the volume variation between the activated complex and the initial states of the bacterial pressure inactivation reaction. A higher volume change becomes possible due to higher cell membrane fluidity, and cell membrane injury is considered to be suppressed due to the lipid phase transition in the bacterial pressure inactivation reaction. However, because activation volume differences did not correlate with the membrane composition in this study. Thus, as reported before for E. coli. [13], the presence of substances in soymilk (i.e., phospholipids) might contribute to the acquisition of high-pressure tolerance of the microbial cell.
Effect of the (C16:1＋C18:1)/(C16:0＋C18:0) ratio on the pre-exponential factors, k0, (a) and the activation volumes, |ΔV *|, (b) under high-pressure treatment at 350MPa. MRS and SM is indicated as abbreviation of MRS/NaCl and SM/NaCl.
This study carried out to investigate the effects of soymilk ingredients on the inactivation of Lactobacillus plantarum subsp. plantarum and Lactobacillus pentosus. Pretreated bacteria were inoculated in MRS broth or soymilk and incubated at 30°C for 48 h, and the cells were suspended in 0.85% NaCl or soymilk. They were subjected to heat treatment or high-pressure treatment. Both of the behavior of the survival curve by heat and high-pressure treatments were described by first-order kinetics. Although the survival behavior of lactobacilli subjected to heat treatment did not show clear difference about the effect of culturing in a medium containing soy milk, under high-pressure conditions, a tendency toward high-pressure resistance was observed, when cultured in soymilk medium. This suggests that the coexisting substances in the culture medium changed the membrane properties of the cell membranes and affected their mortality behavior under high-pressure conditions.
The pressure dependence of the death rate constant for each culture condition was analyzed. A linear relationship was observed between the natural logarithm of the pre-exponential factor and the activation volume under all conditions, suggesting that the death reaction may be essentially the same for different types of microorganisms.
There are no conflicts of interest to declare.
Daitaro Ishikawa contributed to experiment of this study and prepared the original draft of the manuscript. Genki Onozawa, Takato Nakayama, Tomohiro Kudo and Yoshihiro Tsukada contributed to the research work. Tomoyuki Fujii conceptualized the study, contributed to the methodology, and reviewed the manuscript. The datasets generated and/or analyzed during the present study can be made available by the corresponding author upon reasonable request.
