1. IntroductionA variety of dairy p

1. Introduction
A variety of dairy products, including milk, cheese, butter,
yogurt are consumed throughout the world. Before it is sold, milk is
treated by either low-temperature pasteurization at 63
C for
30 min or high-temperature, short-time pasteurization at 130
C
for 2 s (Furukawa, 2002). In Japan, dairy products are ordinarily
made from milk pasteurized using the low-temperature process,
which retains some enzyme activities, because unpasteurized milk
cannot be used for the production of dairy products.
Non-thermal pasteurization with pressurized CO2 has been
studied as an alternative to the process employing heat (Damar &
Balaban, 2006). Werner and Hotchkiss (2006) reported that pressurized
CO2 at 35
C, 20.7 MPa and 132 g/kg of CO2 feeding resulted
in 5.36 and 5.02-log reductions of aerobic bacteria and Pseudomonas
fluorescens added to raw milk, respectively. Erkmen (2000a,
b, 2001) reported that inactivation of Enterococcus faecalis, Brochothrix
thermosphacta, and Escherichia coli added to whole or skim
milk by pressurized CO2 was attempted, and the inactivation efficiency
in whole milk was less than that in skim milk. Hongmei,
Zhong, Liao, and Hu (2014) reported that the number of surviving
aerobic bacteria, yeast and molds, and coliform bacteria decreased
with increasing temperature, pressure, and exposure time. In
addition, there are some reports about precipitation of casein in
milk by pressurized CO2 and the characteristic of the casein, and
fractionation of a-lactalbumin and b-lactoglobulin from whey
protein concentration and whey protein isolate solution by pressurized
CO2 (Hofland, Berkoff, Witkamp, & Van der Wielen, 2003;
Hofland, Van Es, Van der Wielen, & Witkamp, 1999; Strange et al.,
1998; Tomasula et al., 1995; Bonnaille & Tomasula, 2012; Yver,
Bonnaillie, Yee, McAloon, & Tomasula, 2012). Until now, both
pasteurization and protein-denaturation by pressurized CO2 have
ever examined in parts.
We recently developed an equipment that pasteurized liquid
samples using heat and pressure after feeding microbubbled (MB)
CO2 into the liquid at a temperature lower than 10
C and a pressure
lower than the critical pressure (two-stage MBCO2), and also reported
the inactivation of some microorganisms and enzymes using
the two-stage MBCO2; the quality of sake and beer treated with
the two-stage MBCO2, and the inactivation mechanisms of MBCO2
on Saccharomyces pastorianus different from heat sterilization
(Kobayashi, Ikeura, Odake & Hayata, 2014; Kobayashi, Ikeura,
Odake, & Sakurai, 2014; Kobayashi et al., 2014; Kobayashi &
Odake, 2015). In addition, it was shown to decrease the freeamino acid contents in unpasteurized sake and unfiltered beer by
the two-stage MBCO2 in our previous study (Kobayashi, et al., 2014;
Kobayashi & Odake, 2015). However, solution containing high
protein or high fat like milk have never been treated by the twostage
MBCO2. Therefore, to expand the use of the two-stage
MBCO2 as a pasteurization technique for various foods, it is
important to apply this method on milk.
In this study, the inactivation efficiency of two-stage MBCO2 on
E. coli added to physiological saline (PS) and commercial bovine
milk (CBM) already pasteurized using a high-temperature shorttime
process was compared. E. coli was also used as test microorganisms,
because it was hygiene indicator bacterium and was often
used in inactivation experiment of pressurized CO2 (Damar &
Balaban, 2006). Furthermore, aerobic bacteria in unpasteurized
bovine milk (UBM) were also inactivated using the two-stage
MBCO2, and changes in protease-resistance of casein and free
amino acid contents in the treated milk were measured.