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Inactivation of Lactobacillus fructivorans in physiological saline and unpasteurised sake using CO2 microbubbles at ambient temperature and low pressure

Authors


Fax: +81 422 51 9884; e-mail: fkoba@nvlu.ac.jp.

Summary

The ability of carbon dioxide microbubbles (MB-CO2) to inactivate Lactobacillus fructivorans suspended in physiological saline and unpasteurised sake at ambient temperature and a pressure lower than 2.0 MPa was investigated. The number of L. fructivorans cells in physiological saline solution containing 15% ethanol showed a 6-log reduction following MB-CO2 treatment at 40 °C and 2.0 MPa for 50 min. The effectiveness of the treatment increased concomitantly with temperature, pressure and ethanol concentration of the sample solution but was unaffected by the glucose concentration in the sample solution. Furthermore, the number of L. fructivorans cells showed a 5-log reduction in sake after MB-CO2 treatment at 40°C and 2.0 MPa for 60 min. Sensory evaluation revealed no significant difference between MB-CO2-treated sake and unpasteurised sake. These results indicated that MB-CO2 treatment was highly effective for the inactivation of L. fructivorans and might become a practical method for pasteurising sake at ambient temperature.

Introduction

Sake is a traditional alcoholic beverage in Japan. Unpasteurised sake commonly contains the Alcohol-philic and alcohol-resistant lactic acid bacteria known as hiochi bacteria because the sake brewing process is usually an open system. Lactobacillus fructivorans, L. hilgardii, L. paracasei and L. rhamnosus were detected as the hiochi bacteria at the sake breweries (Sun et al., 2007). L. fructivorans are classified into homofermentative and heterofermentative types based on the products of lactic acid fermentation. They are further classified into alcoholophilic and alcohol-tolerant bacteria (Wada & Mizoguchi, 2007). Generally, heat tolerance in the heterofermentative types is higher than that in homofermentative. During ageing and distribution, the quality degradation and spoilage of sake that produces white turbidity, increases acid contents and generates off-flavours such as diacetyl was frequently induced by the hiochi bacteria (Noshiro & Momose, 1970). The unpasteurised sake is generally subjected to heat treatments (65–70 °C) to prevent the growth of hiochi bacteria, although the heat treatment often causes undesirable changes in the quality of sake. Therefore, new techniques for inactivating the hiochi bacteria without affecting the quality of sake need to be developed.

Nonthermal processes with supercritical carbon dioxide (SC-CO2) have been widely studied as alternative processes for food pasteurisation, because heat may cause undesirable changes in the taste and flavour of the food (Haas et al., 1989; Lin et al., 1992, 1993; Ballestra et al., 1996; Gunes et al., 2006; Garcia-Gonzalez et al., 2009). Shimoda et al. (1998, 2001, 2002) reported that SC-CO2, which is bubbled by passing it through a filter (the diameter of the SC-CO2 bubbles ranged from about 220 to 400 μm), was efficient for inactivating various types of micro-organisms. We have previously reported that the ability to inactivate micro-organisms and extract volatile compounds by SC-CO2 treatment depends on the dissolved CO2 concentration and pressure, respectively (Kobayashi et al., 2006, 2007). Dissolved CO2 can easily diffuse into bacterial cells because of increased membrane permeability. It then accumulates in the cytoplasmic interior and decreases intracellular pH, thereby seriously impairing cell viability (Garcia-Gonzalez et al., 2009). Furthermore, the inactivation of micro-organisms and enzymes in unpasteurised alcoholic beverages such as sake and beer was performed by the SC-CO2 treatment (Ishikawa et al., 1995; Tanimoto et al., 2005, 2007, 2008; Dagan & Balaban, 2006). High-pressure conditions (10–30 MPa) are necessary to generate sufficient SC-CO2 and effectively inactivate micro-organisms. However, the heavy duty equipment required for this purpose is prohibitively expensive from a practical viewpoint. Recently, we developed a pasteurisation technique with CO2 microbubbles (MB-CO2) at a pressure lower than 2.0 MPa, a pressure at which no bactericidal effects are observed (Ouléet al., 2006). In addition, we reported that the inactivation of Escherichia coli and Saccharomyces cerevisiae by MB-CO2 treatment might depend on dissolved CO2 concentration (Kobayashi et al., 2009, 2010). MBs, very fine bubbles with diameters less than 50 μm, have been studied extensively in many fields. MBs can be generated by the pressurising dissolution and shear methods and are associated with characteristic actions such as high solubility, shear force, cavitation, shock waves and hydroxyl radicals that arise because of their collapse (Takahashi, 2004; Li & Tsuge, 2006; Matsuo et al., 2006; Xu & Shiina, 2007; Li et al., 2009; Tsuge et al., 2009). Therefore, the aim of this study was to investigate the effects of MB-CO2 treatment on the inactivation of L. fructivorans suspended in physiological saline and unpasteurised sake at ambient temperature and a pressure lower than 2.0 MPa.

Materials and methods

Microbial culture

Lactobacillus fructivorans s36 from the National Research Institute of Brewing (Higashi-Hiroshima, Japan) was used in the study. L. fructivorans s36 (heterofermentative type) is one of the most heat-resistant hiochi bacteria that degrades the quality of sake and can remain viable after heat treatment at 70 °C (Nagatani & Kikuchi, 1971). The L. fructivorans suspension was prepared by a previous reported method (Tanimoto et al., 2007). L. fructivorans was inoculated into test tubes containing 10 mL of S.I. medium (The Brewing Society of Japan, Tokyo, Japan) with 10% ethanol and incubated at 30 °C for 7 days. Next, 0.5 mL of the culture was transferred to test tubes containing 10 mL of S.I. medium with 15% ethanol and incubated at 30 °C for 7 days. The cells were then collected by centrifuge (4 °C, 8000 g, 10 min) and resuspended in 10 mL of sterile physiological saline.

Preparation of model solution and unpasteurised sake

Lactobacillus fructivorans cells were suspended in sterile physiological saline containing 10%, 15%, 18% and 20% ethanol (model solution) and in unpasteurised sake at approximately 1 × 106 and 1 × 105 colony forming units (CFU) mL−1, respectively. The initial concentration of L. fructivorans cells was lowest level by which the quality degradation of sake was caused (Noshiro & Momose, 1970). Unpasteurised sake, with an ethanol concentration of approximately 18%, was purchased from Nakamura Syuzou, Inc. (Akiruno, Tokyo, Japan). The number of the natural hiochi bacteria in the unpasteurised sake used in present study was approximately 1 × 102 CFU mL−1.

Apparatus and conditions of the MB-CO2 treatment

The experimental apparatus for MB-CO2 treatment used in this study has been previously reported (Kobayashi et al., 2009, 2010). The maximum relative particle diameter of MB measured using a nano particle size analyzer (SALD-7100, Shimadzu Co., Ltd., Kyoto, Japan) was about 38 μm (Fig. 1).

Figure 1.

 Size distribution of MBs generated in physiological saline containing 15% ethanol.

For the model solution, MB-CO2 treatments were performed at temperatures of 35, 40 and 45 °C; CO2 pressures of 0.0 (atmospheric pressure), 0.5, 1.0 and 2.0 MPa; ethanol concentrations of 10%, 15%, 18% and 20% in physiological saline and glucose concentrations of 0, 20, 40 and 80 g L−1. For unpasteurised sake, MB-CO2 treatments were performed at a temperature of 40 °C and CO2 pressure of 2.0 MPa.

Measurement of surviving L. fructivorans cells

The number of surviving L. fructivorans cells were measured by plating 1.0 mL of the sample or diluted sample on duplicate plates of S.I. agar. The plates were anaerobically incubated at 30 °C for 7 days. After incubation, the plates of 30–300 CFU were chosen and colonies were then counted. For low numbers of viable cells, colonies in plates of no-diluted sample were counted. The detected limit was 1.0 CFU mL−1. All experiments were performed in triplicate. The data are presented as means with standard errors of the results of triplicate experiments.

Inactivation kinetics measurement

The decimal reduction time (D value), defined as the time required for 90% inactivation of the microbial population, was calculated from negative reciprocals of the slope of the regression lines from the straight portions of the survival curves that showed rapid inactivation of the microbial population (Watanabe et al., 2003). In addition, the lag (L value) denoted the time during which the number of cells remained constant before inactivation (Ouléet al., 2006).

Measurement of the concentration of dissolved CO2

The CO2 concentration dissolved in the model solution and unpasteurised sake was measured as described in previous report (Kobayashi et al., 2009).

Sensory evaluation of sake

Sensory evaluation of sake was conducted by a panel of five experts at the Daiwa Can Company (Sagamihara, Kanagawa, Japan). The panel evaluated the flavour and taste of sake based on five levels (4, very good; 3, good; 2, normal; 1, bad; 0, very bad). Statistical analysis of the results was performed using analyses of variance (anova), and significant differences were evaluated by Student’s t-test (P < 0.05).

Results and discussion

Inactivation of L. frctivorans in model solution by MB-CO2 treatment

The effect of temperature on inactivation of L. fructivorans by MB-CO2 treatment is shown in Fig. 2. The number of surviving L. fructivorans cells was reduced by approximately 1.5-log following MB-CO2 treatment at 35 °C and 2.0 MPa for 60 min, while 6-log reductions were achieved following MB-CO2 treatment at 40 °C for 50 min and 45 °C for 30 min. L values were 40, 20 and 0 min at 35, 40 and 45 °C, respectively, and D values were 18.9, 5.19 and 3.52 min at 35, 40 and 45 °C, respectively (Table 1). These values reduced significantly with increase in temperature. This result is in accordance with the result of our previous studies indicated that the inactivation effect of MB-CO2 treatment on E. coli and S. cerevisiae increased concomitantly with treatment temperature (Kobayashi et al., 2009, 2010). On the other hand, the saturating concentration of dissolved CO2, which might be one of the key factors for inactivating micro-organisms by MB-CO2 treatment, decreased with an increase in temperature (Table 2). Dissolved CO2 could rapidly reach a high concentration using a MB generator (Kobayashi et al., 2009, 2010) and could easily diffuse into bacterial cells, resulting in a decrease in intracellular pH and cell death (Garcia-Gonzalez et al., 2009). This is believed to occur because of increased diffusibility of CO2 and fluidity of the cell membrane at higher temperatures (Haas et al., 1989). Furthermore, not only dissolved CO2 but also the characteristic action of MBs, such as shock waves generated by the collapse of MBs (Li et al., 2009; Tsuge et al., 2009), cavitation and shear force might contribute supplementarily to the inactivation of L. fructivorans by MB-CO2 treatment. It was also considered that the contacts between CO2 and the membrane of micro-organisms might be accelerated by MB-CO2.

Figure 2.

 Effect of temperature on inactivation of Lactobacillus fructivorans by MB-CO2 treatment.

Table 1.   D and L values on inactivation of Lactobacillus fructivorans in the model solution and unpasteurised sake by MB-CO2 treatment
 Treatment conditionD value (min)*L value (min)
Temperature (°C)Pressure (MPa)Ethanol concentration (%)Glucose content (g L−1)
  1. *The time required for 90% inactivation of the microbial population.

  2. The time required until the number of surviving cells decreased rapidly.

In model solution352.0150.018.940
402.0150.05.1920
452.0150.03.520
400.0150.060
400.5150.010.020
401.0150.05.1620
402.0100.05.7430
402.0180.04.0310
402.0200.03.5510
402.015206.5320
402.015408.0020
402.015806.9920
In pasteurised sake402.018 8.2620
Table 2.   The saturated concentration of dissolved CO2 in the model solution and unpasteurised sake on MB-CO2 treatment
 Treatment conditionThe saturated concentration of dissolved CO2 (mL g−1)
Temperature (°C)Pressure (MPa)Ethanol concentration (%)Glucose content (g L−1)
In model solution352.0150.013.8 ± 0.2
402.0150.012.5 ± 0.1
452.0150.011.8 ± 0.1
400.0150.01.26 ± 0.14
400.5150.05.00 ± 0.12
401.0150.07.64 ± 0.08
402.0100.012.4 ± 0.1
402.0180.012.8 ± 0.4
402.0200.012.8 ± 0.1
402.0152012.3 ± 0.2
402.0154012.4 ± 0.2
402.0158011.8 ± 0.1
In pasteurised sake402.018 12.6 ± 0.2

The effect of pressure on the inactivation of L. fructivorans by MB-CO2 treatment is shown in Fig. 3. L. fructivorans was slightly inactivated by MB-CO2 treatment at atmospheric pressure. However, a 4-log reduction in the L. fructivorans population occurred after MB-CO2 treatment at 40 °C and 0.5 MPa for 60 min, and a 6-log reduction was observed at both 1.0 and 2.0 MPa for 50 min. L values of 20 min were observed at all treatment pressure, except for atmospheric pressure, and D values obtained were 10.0, 5.16 and 5.19 min, respectively (Table 1). Thus, D values reduced with increase in pressure and were almost the same at 1.0 and 2.0 MPa. Theoretically, pressure controls both the rate of solubilisation of CO2 and its solubility in a suspending medium. In the present study, the saturating concentration of dissolved CO2 increased with the increase in pressure (Table 2). Therefore, a higher pressure enhances CO2 solubilisation to facilitate acidification of the external medium and its contact with and penetration into the cells (Hong et al., 1997).

Figure 3.

 Effect of pressure on inactivation of Lactobacillus fructivorans by MB-CO2 treatment.

The effect of ethanol concentration in the sample solution on the inactivation of L. fructivorans by MB-CO2 treatment is shown in Fig. 4. In the sample solution with 10% ethanol, a 5-log reduction in the L. fructivorans population was observed after MB-CO2 treatment at 40 °C and 2.0 MPa for 60 min. Furthermore, a 6-log reduction in the L. fructivorans population was achieved after MB-CO2 treatment at 50 and 20 min in 15% and 20% ethanol, respectively. L values were 30, 20 and 10 min at ethanol concentrations of 10%, 15% and 20%, respectively, and D values were 5.74, 5.19 and 3.55 min, respectively (Table 1). Our present results agreed with the previous report that the ability of MB-CO2 treatment to inactivate S. cerevisiae was enhanced with increasing ethanol concentration in the model solution (Kobayashi et al., 2010), although the saturating concentration of dissolved CO2 was almost the same at ethanol concentration between 10% and 20% (Table 2). The same phenomena were, also, reported in SC-CO2 inactivation (Tanimoto et al., 2007; Wu et al., 2007), but it was still not clear why SC-CO2 efficiency was enhanced by the addition of ethanol.

Figure 4.

 Effect of ethanol concentration on inactivation of Lactobacillus fructivorans by MB-CO2 treatment.

The effect of glucose concentration in the sample solution on the inactivation of L. fructivorans by MB-CO2 treatment is shown in Fig. 5. An L value of 20 min was obtained at all tested glucose concentrations. D values of 5.19, 6.53, 8.00 and 6.99 min were obtained at glucose concentrations of 0, 20, 40 and 80 g L−1, respectively (Table 1). D values increased by the addition of glucose to the sample solution; however, there was no correlation between D values and glucose concentration. The saturating concentration of dissolved CO2 also remained nearly unaffected by glucose concentration at a range from 0.0 to 80 g L−1 (Table 2). Garcia-Gonzalez et al. (2009) reported that the effect of inactivation of Pseudomonas fluorescens by pressurising CO2 treatment at 35 °C and 10.5 MPa for 20 min was almost the same as the effect at 0–26% sucrose. Therefore, we concluded that inactivation of L. fructivorans by MB-CO2 treatment was not influenced by glucose because the glucose concentration tested in the present study was less than 80 g L−1. In general, the total sugar content in sake is approximately 30–70 g L−1 (Tanimoto et al., 2007). Thus, sugar is expected to have little effect on the inactivation of micro-organisms in sake by MB-CO2 treatment.

Figure 5.

 Effect of glucose concentration on inactivation of Lactobacillus fructivorans by MB-CO2 treatment.

Inactivation of L. frctivorans in unpasteurised sake by MB-CO2 treatment

There are some reports that the inactivation effect of pressurised CO2 increases with increase in temperature and pressure (Garcia-Gonzalez et al., 2007). In the model solution, a 6-log reduction could be achieved with MB-CO2 treatment at 40 °C for both 1.0 and 2.0 MPa. It was considered that MB-CO2 treatment at 40 °C was lower temperature, energy and negative impacts than that at 45 °C. Furthermore, the contacts between CO2 and bacterial cells might be inhibited by food components such as sugar and acids, while the concentration of dissolved CO2 increased with lowering temperature and increasing pressure. Therefore, we performed MB-CO2 treatment at 40 °C and 2.0 MPa for the inactivation of L. fructivorans in unpasteurised sake. Inactivation of L. fructivorans in unpasteurised sake by MB-CO2 treatment is shown in Fig. 6. Survival curves of L. fructivorans treated with MB-CO2 showed almost the same form in the model solution and unpasteurised sake. In addition, the saturating concentration of dissolved CO2 in the model solution was similar to that in unpasteurised sake, as shown in Table 2. However, the MB-CO2 treatment was less effective in inactivating L. fructivorans in unpasteurised sake than in the model solution, indicated by the L and D values were 20 and 8.26 min for unpasteurised sake and 10 and 4.03 min for the model solution with 18% ethanol, respectively (Table 1). This was probably because of the existence of components such as sugar and acids in sake. Pressurised CO2 efficiency was influenced by different food compounds and food properties such as sucrose, gelatine, polyethylene glycol, glycerol and oleic acid (Kim et al., 2008; Garcia-Gonzalez et al., 2009).

Figure 6.

 Inactivation of Lactobacillus fructivorans in physiological saline and unpasteurised sake by MB-CO2 treatment.

The results of sensory evaluation of sake before and after MB-CO2 treatment are shown in Table 3. Scores of flavour (3.4) and taste (3.7) for unpasteurised sake were slightly higher than those (3.2 and 3.3 for flavour and taste, respectively) for MB-CO2-treated sake; however, no significant differences could be observed. Therefore, we suggested that MB-CO2 treatment at 40 °C and 2.0 MPa does not alter the properties of unpasteurised sake.

Table 3.   Sensory evaluation of sake before and after MB-CO2 treatment
 FlavourTaste
  1. nsno significant difference.

Unpasteurised sake3.4 ± 0.2ns3.7 ± 0.3ns
MB-CO2-treated sake3.2 ± 0.2ns3.3 ± 0.4ns

Conclusions

MB-CO2 treatment was effective in inactivating L. fructivorans in physiological saline, and its effectiveness increased concomitantly with temperature, pressure and ethanol concentration in the solution. In addition, the L. fructivorans population in unpasteurised sake showed a 5-log reduction following MB-CO2 treatment. Based on sensory evaluation tests, we determined that MB-CO2-treated sake was almost of the same quality as unpasteurised sake. These results suggest that MB-CO2 treatment may become a novel inactivation method for unpasteurised sake.

Acknowledgments

The research was financially supported by the Sasakawa Scientific Research Grant from The Japan Science Society. We would like to thank Shimadzu Co., Ltd., (Kyoto, Japan) for lending us a nano particle size analyzer and the National Research Institute of Brewing (Higashi-Hiroshima, Japan) for providing the cultures of L. fructivorans s36.

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