LWT - Food Science and Technology 48 (2012) 330e333

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LWT - Food Science and Technology

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Research note

Inactivation of Lactobacillus fructivorans suspended in various buffer solutions

by low-pressure CO2 microbubbles
F. Kobayashi a, *, H. Ikeura b, S. Odake a, S. Tanimoto c, Y. Hayata b
a
Faculty of Applied Life Science, Nippon Veterinary and Life Science University, 2-27-5, Sakai, Musashino, Tokyo 180-0022, Japan
b
School of Agriculture, Meiji University, Kawasaki, Kanagawa 214-8571, Japan
c
Faculty of Human Culture and Science, Prefectural University of Hiroshima, Hiroshima 734-8558, Japan

a r t i c l e i n f o a b s t r a c t

Article history: The inactivation of Lactobacillus fructivorans suspended in various buffer solutions by low-pressure CO2
Received 26 September 2011 microbubbles (MB-CO2) was investigated. The number of surviving L. fructivorans cells suspended in
Received in revised form 0.1 mol/L acetic acid/0.1 mol/L sodium acetate buffer at pH 4 was decreased by 4-log cycles by MB-CO2 at
13 February 2012
40
C and 2.0 MPa for 60 min, whereas there were no reductions in the numbers of L. fructivorans cells
Accepted 13 April 2012
suspended in 0.1 mol/L citric acid (CA)/0.1 mol/L sodium citrate (NaC) buffer at pH 4, 0.1 mol/L
CA/0.2 mol/L disodium hydrogen phosphate (NaP) buffer at pH 4, or 0.1 mol/L CA/0.2 mol/L dipotassium
Keywords:
hydrogen phosphate buffer at pH 4 by MB-CO2 under the same conditions. However, the inactivation of
Buffer capacity
CO2 microbubbles
L. fructivorans cells by MB-CO2 was similar in 0.01 mol/L CA/0.01 mol/L NaC buffer, 0.001 mol/L
Inactivation CA/0.002 mol/L NaP buffer, and deionized water. Furthermore, the inactivating effect of MB-CO2 tended
Lactobacillus fructivorans to increase with decreasing buffer pH.
Acids Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction conditions (10e30 MPa) are necessary to generate sufficient SC-

Sake is a traditional alcoholic beverage of Japan. Alcohol-philic
and alcohol-resistant lactic acid bacteria are commonly used for
brewing unpasteurized sake, because the sake brewing process
generally utilizes an open system. However, lactic acid bacteria
degrade the quality of sake by producing white turbidity, increasing
acid content and generating off-flavor during the aging and
distribution of sake. Unpasteurized sake is generally subjected to
heat treatment (65e70
C) to prevent the growth of microorgan-
isms, although heat treatment often causes undesirable changes in
the quality of sake. Therefore, new techniques for inactivating
microorganisms without affecting sake quality are desired by the
sake brewing industry.
Non-thermal processes using supercritical carbon dioxide
(SC-CO2) have been widely studied as alternative processes for food
pasteurization, because heat may cause undesirable changes in the
taste and flavor of food (Ballestra, Da Silva, & Cuq, 1996; Garcia-
Gonzalez et al., 2007; Gunes, Blum, & Hotchkiss, 2006; Haas
et al., 1989; Lin, Yang, & Chen, 1992, 1993). High-pressure
CO2 and effectively inactivate microorganisms. However, the
heavy-duty equipment required for this purpose is prohibitively
expensive from a practical viewpoint. Recently, we developed
a pasteurization technique utilizing CO2 microbubbles (MB-CO2) at
a pressure lower than 2.0 MPa, which is a pressure that has no
bactericidal effects (Oulé, Tano, Bernier, & Arul, 2006), and reported
the inactivation of Escherichia coli and Saccharomyces cerevisiae by
MB-CO2 treatment (Kobayashi, Hayata, Ikeura, Muto, & Osajima,
2010; Kobayashi et al., 2009). Pressurized CO2 efficiency was
influenced by various food components and food properties
(Garcia-Gonzalez et al., 2009). Many components in sake, including
sugars and acids, possess buffer capacity. Lactobacillus fructivorans
is a lactic acid bacterium that degrades the quality of sake (Nagatani
& Kikuchi, 1971). Therefore, this study aimed to investigate the
effect of MB-CO2 on the inactivation of L. fructivorans suspended in
various buffer solutions.
2. Materials and methods
2.1. Microbial culture

L. fructivorans s36, one of the most heat-resistant lactic acid

* Corresponding author. Tel.: þ81 422 51 6121; fax: þ81 422 51 9884.
E-mail addresses: fkoba@nvlu.ac.jp (F. Kobayashi), hikeura@isc.meiji.ac.jp
(H. Ikeura), odake@nvlu.ac.jp (S. Odake), s-tanimoto@pu-hiroshima.ac.jp
(S. Tanimoto), yhayata@isc.meiji.ac.jp (Y. Hayata).
bacterial strains (Nagatani & Kikuchi, 1971), was obtained from the
National Research Institute of Brewing (Higashi-Hiroshima, Japan).
The L. fructivorans suspension was prepared using a previously

0023-6438/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2012.04.011
 F. Kobayashi et al. / LWT - Food Science and Technology 48 (2012) 330e333 331

reported method (Tanimoto et al., 2007). L. fructivorans was inoc-
ulated into test tubes containing 10 mL of SI medium (The Brewing
Society of Japan, Tokyo, Japan) (Sugama & Iguchi, 1970) with 10 mL/
100 mL ethanol and incubated at 30
C for 7 days. Next, 0.5 mL of
the culture was transferred into test tubes containing 10 mL of SI
medium with 15 mL/100 mL ethanol and incubated at 30
C for 7
days. The cells were then collected by centrifugation (at 4
C,
8000  g, 10 min) and re-suspended in 10 mL of sterile physio-
logical saline.
2.2. Preparation of buffer solutions
The following buffer solutions were prepared:
2.6. Measurement of pH values
The pH values of sample solutions before and after MB-CO2
treatment were determined with a pH meter (pH meter F-13,
Horiba Ltd., Kyoto, Japan). The pH values of sample solutions were
quickly measured after decompression to atmosphere pressure.
2.7. Statistical analysis
All experiments were performed in triplicate. The data are
presented as means with standard errors of the results of triplicate
experiments. The significant difference was analyzed using the
TukeyeKramer method at p < 0.05.

(a) 0.1 mol/L acetic acid (AA)/0.1 mol/L sodium acetate (NaA)
3. Results and discussion
buffer solution at pH 4.00
(b) 0.1, 0.01, or 0.001 mol/L citric acid (CA)/0.2, 0.02, or 0.002 mol/L
The inactivation of L. fructivorans suspended in various buffer
disodium hydrogen phosphate (NaP) buffer solution at pH 3.00,
solutions by MB-CO2 treatment is shown in Fig. 1. There were no
4.00, or 5.00
reductions in the numbers of surviving L. fructivorans cells in
(c) 0.1 mol/L CA/0.2 mol/L dipotassium hydrogen phosphate (KP)
0.1 mol/L CA/0.2 mol/L NaP, 0.1 mol/L CA/0.2 mol/L KP and 0.1 mol/L
buffer solution at pH 4
CA/0.1 mol/L NaC buffer solutions by MB-CO2 treatment at 40
C
(d) 0.1, 0.01, or 0.001 mol/L CA/0.1, 0.01, or 0.001 mol/L sodium
and 2 MPa for 60 min, although the numbers of surviving
citrate (NaC) buffer solution at pH 4.00
L. fructivorans cells in 0.1 mol/L AA/0.1 mol/L NaA buffer solution
and deionized water were significantly reduced from 30- and
L. fructivorans cells were suspended in each buffer solutions
40-min treatments, respectively, and occurred 4- and 5-log cycles
containing 15 mL/100 mL ethanol at approximately 1  105 colony
reductions, respectively, after 60-min treatments. Furthermore, the
forming units (CFU)/mL.
inactivation of L. fructivorans by MB-CO2 treatment was not influ-
enced by the different cation types between Naþ and Kþ. Iwata,
2.3. MB-CO2 apparatus and procedure for L. fructivorans Akiyama, and Suzuki (1964) also reported that the buffer capacity
inactivation of sake depended on phosphoric and citric acids. In addition, Kim
et al. (2008) reported that the inactivation of Listeria mono-
The L. fructivorans inactivation apparatus was described previ- cytogenes by SC-CO2 decreased significantly when cells were
ously (Kobayashi et al., 2009, 2010). MB-CO2 treatments were
performed under the following conditions: temperature, 40
C;
pressure, 2 MPa; and CO2 flow rate, 2 L/min.

2.4. Measurement of surviving L. fructivorans cells

The number of surviving L. fructivorans cells were measured by

plating 1 mL of the sample or diluted sample on duplicate plates of
SI agar. The plates were anaerobically incubated at 30
C for 7 days.
After incubation, the plates of 30e300 CFU were chosen and
colonies were then counted. For low numbers of viable cells,
colonies in plates of no-diluted sample were counted (Kobayashi
et al., in press). The detection limit was 1 CFU/mL.

2.5. Measurements of dissolved CO2

The dissolved CO2 concentration (dCO2) in the L. fructivorans

suspension was measured as described previously (Kobayashi et al.,
2009) and expressed as the Kuenen gas absorption coefficient,
which is defined as the gas volume (mL) dissolved in 1 g of water
under standard conditions (Shimoda et al., 2001). dCO2 was
calculated using the following equation:

Fig. 1. Effect of buffer components on the inactivation of L. fructivorans by MB-CO2

dCO2 ¼ 273VCO2 ð760  Pwater Þðð273 þ tÞVwater 760dwater Þ1
where VCO2 was the volume of CO2 dissolved in the sample solution,
Pwater was the saturated water vapor pressure at any treatment
temperature (t), Vwater was the volume of the treated samples at any
treatment temperature (t), and dwater was the water density at any
treatment temperature (t).
(1) treatment. MB-CO2 treatments were performed at 40
C, 2.0 MPa and a CO2 flow rate of
2.0 L/min. The microbial survival ratio was expressed as the logarithmic (N/N0) with N
and N0 representing the counts (CFU/mL) after and before MB-CO2 treatment. All
experiments were performed in triplicate. * and ** represented a significant difference
within the same exposure time (p < 0.05). 0.1 mol/L acetic acid/0.1 mol/L sodium
acetate buffer solution at pH 4.00 (:), 0.1 mol/L citric acid/0.2 mol/L disodium
hydrogen phosphate buffer solution at pH 4.00 (A), 0.1 mol/L citric acid/0.2 mol/L
dipotassium hydrogen phosphate buffer solution at pH 4.00 (C), 0.1 mol/L citric
acid/0.1 mol/L sodium citrate buffer solution at pH 4.00 (), deionized water (-).
332 F. Kobayashi et al. / LWT - Food Science and Technology 48 (2012) 330e333

suspended in phosphate-buffered saline rather than in physiolog-

ical saline and that this hampered inactivation was because of the
buffering action of the phosphate-buffered saline, which blunted
the pH decrease that normally accompanies the dissolution of CO2.
Therefore, it was presumed that the presence of acids, especially
phosphoric and citric acids in the L. fructivorans suspension
inhibited the inactivating effect of MB-CO2 treatment.
The effect of pH of the 0.1 mol/L CA/0.1 mol/L NaP buffer solution
on the inactivation of L. fructivorans by MB-CO2 treatment is shown
in Fig. 2. L. fructivorans cells suspended in 0.1 mol/L CA/0.1 mol/L
NaP buffer solution at pH 4.00 and 5.00 were not inactivated by
MB-CO2 treatment at 40
C and 2 MPa for 60 min. However, as the
pH of the buffer solution decreased, the number of surviving
L. fructivorans cells suspended in 0.1 mol/L CA/0.1 mol/L NaP buffer
solution at pH 3.00 was significantly decreased by MB-CO2 treat-
ment. Our present results agreed with the report by Hong and Pyun
(1999) that the inactivation effect of pressurized CO2 on Lactoba-
cillus plantarum increased as initial pH of the buffer solution
decreased. The pH of buffer solution at 4.00 was not changed after
MB-CO2 treatment, while slightly changed to 3.04 and 4.97 at 3.00
and 5.00, respectively. Because the pH of sake is approximately 4.00
and the concentrations of citric acid and inorganic phosphate in
sake are 0e132 and 44e202 mg/L, respectively (Matsui & Sato,
1966; Sato, 1977), these concentrations of acids appear to have
little effect on the inactivation of L. fructivorans by MB-CO2.
The effects of various concentrations of CA/NaP and CA/NaC
buffer solutions on the inactivation of L. fructivorans by MB-CO2
treatment are shown in Fig. 3. In both 0.1 mol/L CA/0.2 mol/L NaP
and 0.1 mol/L CA/0.1 mol/L NaC buffer solutions, there were no
reductions in the numbers of surviving L. fructivorans cells by
MB-CO2 treatment at 40
C and 2 MPa for 60 min. On the other
hands, in 0.01 mol/L CA/0.02 mol/L NaP and 0.001 mol/L
CA/0.002 mol/L NaP buffer solutions, the numbers of surviving

Fig. 3. Effects of the concentrations of citric acid/disodium hydrogen phosphate and

citric acid/sodium citrate buffer solutions on the inactivation of L. fructivorans by
MB-CO2 treatment. MB-CO2 treatments were performed at 40
C, 2.0 MPa and a CO2
flow rate of 2.0 L/min. The microbial survival ratio was expressed as the logarithmic
(N/N0) with N and N0 representing the counts (CFU/mL) after and before MB-CO2
treatment. All experiments were performed in triplicate. *, ** and *** represented
a significant difference within the same exposure time (p < 0.05). (a) citric acid/
disodium hydrogen phosphate buffer solution with 0.1 and 0.2 mol/L (:), 0.01 and
0.02 mol/L (A), or 0.001 and 0.002 mol/L (C) at pH 4.00, (b) citric acid/sodium acetate
buffer solution with 0.1 and 0.1 mol/L (:), 0.01 and 0.01 mol/L (A), or 0.001 and
0.001 mol/L (C) at pH 4.00. Deionized water (-) was used as a control.

Fig. 2. Effect of pH in the citric acid/disodium hydrogen phosphate buffer solution on
the inactivation of L. fructivorans by MB-CO2 treatment. MB-CO2 treatments were
performed at 40
C, 2.0 MPa and a CO2 flow rate of 2.0 L/min. The microbial survival
ratio was expressed as the logarithmic (N/N0) with N and N0 representing the counts
(CFU/mL) after and before MB-CO2 treatment. All experiments were performed in
triplicate. * represented a significant difference within the same exposure time
(p < 0.05). The pHs of the 0.1 mol/L citric acid/0.2 mol/L disodium hydrogen phosphate
buffer solution were 3.00 (:), 4.00 (A), or 5.00, respectively (-).
L. fructivorans cells were significantly reduced from 30-min treat-
ments of MB-CO2 and 1.5- and 4-log cycle reductions were occurred
after 60-min treatments, respectively. Also, in both 0.01 mol/L
CA/0.01 mol/L NaC and 0.001 mol/L CA/0.001 mol/L NaC buffer
solutions, a significant reduction was observed from 30-min
treatments of MB-CO2 and 5-log cycle reductions were achieved
after 60-min treatment. Thus, the inactivating effect of MB-CO2
 F. Kobayashi et al. / LWT - Food Science and Technology 48 (2012) 330e333 333

Table 1 Acknowledgment
Dissolved CO2 concentrations and pHs in each buffer solution.

Buffer solution dCO2 pHs before pHs after This work was supported by Grant-in-Aid for Young Scientists
MB-CO2 MB-CO2 (B) (23780146) from Japan Society for the Promotion of Science.
treatment treatment
Deionized water 12.1  0.1ns 5.45 4.10
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