JFS M: Food Microbiology and Safety

High Hydrostatic Pressure

Pasteurization of Red Wine
CHULKYOON MOK, KI-TAE SONG, YOUNG-SEO PARK, SANGBIN LIM, ROGER RUAN, AND PAUL CHEN

ABSTRACT: Pasteurization of low-alcohol wine using a high hydrostatic pressure (HHP) process was studied. A total
of 10 mL grape wine sealed in a nylon/LLDPE bag was placed inside the HHP chamber. The pressure applied to
the treatment chamber was maintained at 1000 to 3500 atm for 0 to 30 min. The effects of HHP treatments on the
physiochemical properties (alcohol, pH, acidity, total sugar) and microbes (aerobic bacteria, yeast, and lactic acid
bacteria) were examined. The HHP treatments had little impact on the physiochemical properties. The pasteurization
effect of the HHP treatments increased with treatment pressure and time. A total of 2 different stages in the microbial
inactivation were noticed when the 1st-order reaction model was used to fit the inactivation data. The inactivation
rate was higher in the initial stages than in the later stages, suggesting that might be 2 different groups of the
microorganisms, a more HHP-susceptible group and a less HHP-susceptible group.
Keywords: high hydrostatic pressure, microbes, pasteurization, wine, yeast

Introduction as Acetobacter and lactic acid bacteria. Since wine’s flavor, taste, and

M icrobial growth can be inhibited by various physical treat-

ments such as dehydration, cold storage, or freezing. Other
physical treatments, such as heating, UV or ionizing radiation, high
hydrostatic pressure (HHP), pulsed electric fields (PEF), oscillat-
ing magnetic fields (OMF), or photodynamic process, could in-
activate or kill microorganisms, many of which have been used
commercially.
During the last decade, the HHP process, among many other
physical processes, attracted a great deal of interests as promising
technologies for food processing and preservation and also for creat-
ing new types of food product (Mertens and Knorr 1992). Under equi-
librium conditions, a process associated with a decrease in volume is
favored by pressure and vice versa according to Le Chatelier’s princi-
ple (Butz and Tauscher 1998). In HHP, pressure can be applied at am-
bient or moderate temperatures and is transferred throughout food
instantly and uniformly. Therefore HHP treatment is independent of
food size and geometry. HHP has been used for microbial inactiva-
color are very sensitive to heat, a traditional thermal treatment is
generally undesirable (Mermelstein 1998). Therefore, a nonthermal
preservation technology may be the method of choice for wines.
Filtration methods have been adopted for the removal of the mi-
croorganisms in wines, but it was found that their capabilities of
removing microorganisms were not sufficient to obtain the shelf-
stable wines (Puig and others 2003).
HHP affects only noncovalent bonds of chemicals. Therefore
color, nutrients, and flavors in foods, which are mainly covalent
compounds, will be largely retained after HHP treatment (Cheftel
1992). Since wine is very sensitive to heat and is usually packaged in
bulk for aging, the HHP treatment is very suitable for wine preserva-
tion. The antiseptic effect of the HHP in wine was observed first by
Delfini and others (1994). Later, Puig and others (2003) found that
the applying 500 MPa pressure for 5 min resulted in a 99.99% reduc-
tion in the initial microbial population without resulting in changes
in the chemical or organoleptic properties of the wine. The works of

M: Food Microbiology & Safety

tion (Rademacher and others 1998), protein denaturation (Hinrichs the previous researchers are, however, rather qualitative. The objec-
and Kessler 1998), enzyme inactivation (Morild 1981; Mertens and tives of this research were to evaluate the feasibility of using HHP to
Knorr 1992; Indrawati and others 1998; Rademacher and others pasteurize low-alcohol wines and to study the quantitative kinetics
1998), and gelation (Johnston and others 1998). Microbial inactiva- of the microbial inactivation during the HHP process.
tion by HHP is probably due to numerous factors, including protein
conformation changes and membrane perturbation, leading to cell Material and Methods
leakage (Lopez-Caballero and others 1999).
Wine is one of the oldest fermented beverages in human history. Materials
Wine contains 9% to 13% alcohol. Wines containing low alcohol are Grapes (Campbell Early) grown in Yeongdong, Chungcheongbuk-
apt to spoilage and develop sourness and off-flavor if not pasteurized do, Korea in 2000 were used for the wine making. The yeasts used
properly. The causes of quality changes in wine during storage have were Saccharomyces cerevisiae KCCM 12224. The yeasts were culti-
been attributed to the growth of undesirable microorganisms such vated for 12 h at 25 ◦ C in YM broth before use.

MS 20060006 Submitted 7/17/2006, Accepted 7/20/2006. Authors Mok, Song,
and Park are with Dept. of Food and Bioengineering, Kyungwon Univ., San
65 Bokjeong-dong, Sujeong-gu, Seongnam, 461–701, Korea. Author Lim is
with Dept. of Food Science and Technology, Cheju National Univ., Cheju,
690–756, Korea. Author Ruan is with Dept. Bioproducts and Biosystems En-
gineering and Dept. Food Science and Nutrition, Univ. of Minnesota, St.
Paul, MN 55108. He is also a Yangtz scholar distinguished guest professor,
Nanchang Univ., China. Author Chen is with Dept. Bioproducts and Biosys-
tems Engineering, Univ. of Minnesota, St. Paul, MN 55108. Direct inquiries
to author Mok (E-mail: mokck@kyungwon.ac.kr).
Wine preparation
Grapes were crushed and added to 100 ppm K 2 S 2 O 5 and filtered to
collect the juice. Sucrose was added to the filtered juice to adjust the
total soluble solids to 24 ◦ Brix. The cultivated yeast was inoculated to
the grape juice at 0.5% (v/v) level and fermented at 25 ◦ C for 2 wk, and
then aged at 15 ◦ C for 14 wk. The principal properties of wine before
the HHP treatments are shown in Table 1. The analytical methods
are as follows.


C 2006 Institute of Food Technologists Vol. 71, Nr. 8, 2006—JOURNAL OF FOOD SCIENCE M265
doi: 10.1111/j.1750-3841.2006.00145.x
Further reproduction without permission is prohibited
 Pasteurization of red wine . . .

The pH of wine was measured on a pH meter (Model 340, Corning, the taste, the mouth-feel, and the overall quality using 10 trained
Inc., Corning, N.Y., U.S.A.). The titratable acidity was measured by sensory panels.
titrating 10 mL of sample with 0.1N NaOH using 1% phenolphthalein
as an indicator. The titratable acidity was then calculated by the Results and Discussion
following equation:
Microbial changes in wine during fermentation
Titratable acidity (% tartaric acid) and aging
0.1NNaOH consumed (mL) × factor × 0.0075 The changes in microorganisms in wine during the fermenta-
= tion for 14 d are shown in Figure 1. The numbers of the yeasts and
sample volume (mL)
the bacteria increased logarithmically with the fermentation time.
The maximum numbers in the yeasts and the bacteria were ob-
Sugar content was determined using the phenol-H 2 SO 4 method
served after 10 d. The numbers of the microorganisms remained un-
(Dubois 1956). Samples were diluted with distilled water. The diluted
changed afterward because of the inhibitory action of the produced
sample (0.5 mL) was transferred into a test tube and 0.5 mL of 5%
alcohol. The maximum numbers of the microorganisms were 3.0 ×
phenol solution was added. Then concentrated H 2 SO 4 was added
107 CFU/mL for the yeast and 2.8 × 107 CFU/mL for the bacteria.
and the solution sat for 20 min. The absorbance was measured on a
The wine was aged for 14 wk after the 14-d fermentation. The
spectrophotometer (UV-1201, Shimadzu Corporation, Kyoto, Japan)
changes in microbial counts during the aging are shown in Figure
at 470 nm. The sugar content was calculated using the standard
2. The numbers of both yeasts and aerobic bacteria decreased con-
curve made with glucose.
tinuously. This may be because the decreased sugars and increased
The alcohol content of the wine was measured by using a hydro-
alcohol slowed down the metabolism by microorganisms during the
metric method (Nielsen 1994). The grape wine was centrifuged for
aging stage.
10 min at 20000 × g and 100 mL of the supernatant was transferred
The microbial counts of the wine prior to the HHP treatments
to a rotary evaporator. The supernatant was evaporated at 65 ◦ C un-
were at the levels of 105 CFU/mL for aerobic bacteria, yeasts, and
der vacuum. The vapor from the evaporator was condensed until
lactic acid bacteria as shown in Table 2.
approximately 70 mL of the condensate was collected. The conden-
sate was then diluted back to the original volume (100 mL) with
distilled water and the alcohol content was measured with an al- 8
cohol hydrometer (Model 08285-00, Cole-Parmer Instrument Co.,
Vernon Hills, Ill., U.S.A.). 7

6
HHP treatment
Grape wine after 14-wk aging was treated with a high-pressure 5
Log N (CFU/mL)

food processing system (MFP-7000, Mitsubishi Heavy Industries

4
Ltd., Tokyo, Japan). Grape wine of 10 mL was packaged and vac-
uum sealed in a nylon/LLDPE bag using a vacuum sealer (Dongbang 3
Machinery, Seoul, Korea). The bags were placed inside the HHP
chamber (inside dimensions: 60 mm × 200H mm, pressure medium: 2

distilled water) and the yoke was set securely on the top of the treat- 1 Total aerobes Yeasts
ment chamber. The pressure was increased to preset levels ranging
from 1000 atm to 3500 atm and maintained for a certain period of 0
0 2 4 6 8 10 12 14
time. After the treatment, the pressure was released and the bags
were recovered. All the treatments were carried at 25 ◦ C.
Fermentation time (day)
M: Food Microbiology & Safety

Figure 1 --- Changes in microbial counts of grape wine dur-

Microbiological counts ing fermentation
The numbers of the viable cells in the grape wine were enumer-
ated by the pour-plate colony count method (Harrigan 1998). Total 8
aerobic bacteria were grown on plate count agar (Difco Co.), yeasts
were on YM agar, and the lactic acid bacteria were on Rogosa SL agar 7
of pH 5.5 (Difco Co.). The incubation conditions for the enumera-
6
tion were 48 h at 37 ◦ C for the total aerobic bacteria, 3 to 4 d at 25 ◦ C
Log N (CFU/mL)

for yeasts, and 48 to 72 h at 37 ◦ C for the lactic acid bacteria. 5

Sensory evaluation 4
The sensory properties of wine samples were evaluated on
3
9-point hedonic scales (Land and Shepherd 1984) for the aroma,
2
Total aerobes Yeasts
Table 1 --- Physicochemical properties of grape wine be-
fore high-pressure treatment 1

Properties Contents 0
0 2 4 6 8 10 12 14
Alcohol (%) 9.0
pH 3.27 Aging time (week)
Acidity (%) 0.668
Figure 2 --- Changes in microbial counts of grape wine dur-
Total sugar (%) 0.851
ing aging

M266 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 8, 2006 URLs and E-mail addresses are active links at www.ift.org
Pasteurization of red wine . . .

Inactivation of microorganisms in wine by HHP the later stages. The results indicated that there might be 2 different
treatments groups of the microorganisms, a more HHP-susceptible group and
The inactivation of aerobic bacteria in wine by HHP was as shown a less HHP-susceptible group. The more susceptible group was in-
in Figure 3. The inactivation effect by HHP was very obvious at pres- activated in very short treatment time and decreased rapidly during
sures higher than 2500 atm. The original microbial count, 4.15 ×
105 CFU/mL, decreased to 2.41 × 103 CFU/mL after 5 min treat-
ment at 2500 atm and to 1.17 × 103 CFU/mL, 7.10 × 102 CFU/mL,
2.00 × 102 CFU/mL after 10, 20, and 30 min treatments, respec-
tively. At pressures higher than 3000 atm, the inactivation effects
became extraordinary, with the initial microbial count reduced to 6
2.78 × 102 CFU/mL after 5 min and 6.60 × 101 CFU/mL after 10 min
treatment at 3000 atm. The aerobic bacteria decreased below the de- 5
tection limits after 20 min treatment at 3000 atm and after 10 min at

Log N (CFU/ml)
3500 atm. Puig and others (2003) also reported that HHP treatment 4
at 5000 atm for 5 min resulted in a 99.99% reduction in bacterial
3
count without resulting in changes in the chemical or organoleptic
1000
properties of the wine. 2 1500
The yeasts were also killed at the pressures above 3000 atm as 2000
Pressure
shown in Figure 4. The initial yeast count in the grape wine was 1 2500
(atm)
2.87 × 105 CFU/mL (5 logs), which decreased to 2.89 × 102 CFU/mL 3000
0
(2 logs) after 10 min treatment, to 1.41 × 10 CFU/mL (1 log) after
1
0 3500
5 10 20
20 min treatment, and to null after 30 min treatment at 3000 atm. 30
The lactic acid bacteria were decreased below the detection limits Time (min)
either after 20 min treatment at 3000 atm or 5 min treatment at 3500
atm, as shown in Figure 5. Hara and others (1990) also found that Figure 4 --- Changes in yeast of grape wine during high-
the yeasts and the lactic acid bacteria in sake (rice wine) were killed pressure treatment
completely at pressures above 3000 atm.

Kinetics of microbial inactivation by HHP

The pasteurization effect of the HHP treatments for red wine in-
creased with treatment pressure and time. When the 1st-order ki- 6
netic model was applied to the microbial inactivation data, 2 dif-
ferent stages in the microbial inactivation were present as shown in 5
Figure 6. The inactivation rate was higher in the initial stages than in
Log N (CFU/ml)

4
Table 2 --- Microbial counts of grape wine before high-
3
pressure treatment
1000
Microorganisms Counts (CFU/mL) 2 1500
2000
Total aerobes 4.15 × 105 Pressure
1 2500
Yeast 2.87 × 105 (atm)
3000
Lactic acid bacteria 2.86 × 105 0

M: Food Microbiology & Safety

3500
0 5 10 20 30
Time (min)
Figure 5 --- Changes in lactic acid bacteria of grape wine
during high-pressure treatment
6

5 0
Log N (CFU/ml)

4
–0.2
Log (N/No)

3 k1
1000 –0.4
2 1500
2000
Pressure k2
1 2500 –0.6
(atm)
3000 1500atm
0 3500
0 5 –0.8
10 20 0 5 10 15 20 25 30 35
30
Time (min) Treatment time (min)

Figure 3 --- Changes in aerobic bacteria of grape wine dur- Figure 6 --- Typical high-pressure sterilization kinetics of
ing high-pressure treatment wine at 15000 atm

URLs and E-mail addresses are active links at www.ift.org Vol. 71, Nr. 8, 2006—JOURNAL OF FOOD SCIENCE M267
 Pasteurization of red wine . . .

the initial HHP treatment. The less susceptible group required more
treatment time before they showed obvious decease in viable cells.
From a biphasic curve (Figure 6), 2 HHP inactivation rate con-
stants denoted by k 1 and k 2 , respectively, can be calculated through
linear regression analysis. The values of k 1 and k 2 for different mi-
croorganisms at different pressures are listed in Table 3.
Both k 1 and k 2 increased with pressure, and varied with different
types of microorganisms. Aerobic bacteria showed higher k 1 than
yeasts and lactic acid bacteria, and the trend was more apparent at
low pressures. The results suggest that the critical pressure for the
inactivation was lower for the aerobic bacteria than the yeast and the
lactic acid bacteria. On the other hand, yeasts showed the highest
k 2 at pressures under 2000 atm, while at pressure above 2000 atm
lactic acid bacteria showed higher k 2 than the yeasts and aerobic
bacteria. It should be noted that there was only 1 stage in HHP pas-
teurization at a high pressure such as 3500 atm, which meant that
the pressure-resistant microorganisms might also inactivated easily
when pressure exceeded 3500 atm.
The relationships between the treatment pressure and the inacti-
vation rate constants k 1 and k 2 are shown in Figure 7 and 8, respec-
tively. When the values of k 1 and k 2 were plotted on a semilogarith-
mic paper, the linear relationships were observed, indicating the
k 1 and k 2 increased exponentially with increasing pressure. Brna
and others (1998) also reported that there was a linear relationship
between log(k) and pressures in the inactivation of Cryptococcus
laurentii during the HHP pasteurization of strawberry puree. The
regression equations (ln k 1 (or k 2 ) = A + B. P) of the lines are listed
in Table 4.
The parameter B in the regression equations indicates the degree
of dependence of the inactivation rate constants on pressure. For k 1 ,
the B values were 0.0006, 0.0011, and 0.0010 for the aerobic bacteria,
the yeasts, and the lactic acid bacteria, respectively. The smallest
value for the aerobic bacteria indicates that the inactivation rate
constant in the initial stage of HHP treatment for the aerobic bacteria
was less sensitive than the yeasts and the lactic acid bacteria. For k 2 ,
on the other hand, the lactic acid bacteria had the greatest value of
the parameter B, 0.0024, indicating that inactivation rate constant
for the lactic acid bacteria was the most pressure sensitive in the
2nd stage in HHP pasteurization. The yeast had the smallest value
of the parameter B, 0.0010, indicating that the HHP inactivation of
the yeast was the least pressure sensitive.
Sensory properties of HHP-treated wine
The sensory properties of HHP-treated wine sample at 3500 atm
for 10 min were compared with the untreated one. There were no dif-
ferences in the aroma, taste, mouth-feel, and overall sensory quality
between the HHP-treated sample and the -untreated sample show-
ing the P-values higher than 0.05 as indicated in Table 5. The results
confirmed that the HHP process produced aseptic wine without
lowering its sensory quality.
Conclusions
L ow-alcohol grape wines were prepared for this study. The wine
samples were aged for 14 d before the HHP treatments. The
HHP treatments varied in pressure level and holding time. For each
HHP treatment, 10 mL grape wine sealed in a nylon/LLDPE bag was

Table 3 --- High-pressure sterilization rate constants of

grape wine
(unit: min−1 ) 1
Total aerobes Yeast Lactic acid bacteria Total aerobes
Pressure
(atm) k1 k2 k1 k2 k1 k2 0 Yeast
Lactic acid bacteria
1000 0.4782 0.0134 0.1465 0.0636 0.1532 0.0140
1500 0.5400 0.0173 0.2212 0.0553 0.3441 0.0141 -1
2000 0.6655 0.0533 0.3360 0.0694 0.5372 0.0404
ln k2

2500 1.0299 0.0917 0.7336 0.0971 0.7033 0.1370 -2

3000 1.4619 0.2876 1.1634 0.2778 1.2911 0.5226
3500 1.9693 N/Aa 2.1555 N/Aa N/Aa N/A∗
a
-3
Not applicable since sterilization was completed within 1st 5 min.
M: Food Microbiology & Safety

-4
1
Totalaerobes -5
0.5 Yeast
0 1000 2000 3000 4000
Lactic acid bacteria
0 Pressure (atm)
-0.5
ln k1

Figure 8 --- Semilogarithmic plot of reduction rate constant

(k 2 ) of microorganisms in wine compared with hydrostatic
-1 pressure

-1.5 Table 4 --- Parameters for regression equationa of steriliza-

tion rate constant of grape wine with respect to pressure
-2
k1 k2
-2.5 Microorganism A B R2 A B R2
0 1000 2000 3000 4000 Total aerobe −1.4626 0.0006 0.9735 −6.6949 0.0018 0.9829
Yeast −3.1209 0.0011 0.9913 −4.6241 0.0010 0.8769
Pressure (atm) Lactic acid −2.7233 0.0010 0.9707 −7.9534 0.0024 0.9972
bacteria
Figure 7 --- Semilogarithmic plot of reduction rate constant
(k 1 ) of microorganisms in wine compared with hydrostatic a
ln k 1 (or k 2 ) = A + B. P, where k 1 , k 2 = sterilization rate constant at 1st and
pressure 2nd stages, respectively (min−1 ), and P = pressure (atm).

M268 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 8, 2006 URLs and E-mail addresses are active links at www.ift.org
Pasteurization of red wine . . .
Table 5 --- Effect of high-pressure treatment on sensory
properties of grape wine
Meana
Sensory properties Control HHP treated P-value
Aroma 5.3 4.9 0.6151
Taste 3.5 4.0 0.6162
Mouth-feel 4.2 3.9 0.6747
Overall 4.4 4.1 0.7012
a
N = 10.
placed inside the HHP treatment chamber and treated at pressure
ranging from 1000 to 3500 atm for 0 to 30 min. The effects of HHP
treatments on the physiochemical properties (alcohol, pH, acidity,
total sugar) and microbes (aerobic bacteria, yeast, and lactic acid
bacteria) were quantified. The results showed that the physiochem-
ical properties exhibited little change during the HHP treatments.
The microbial inactivation by the HHP treatments increased with
treatment pressure and time. The kinetic study showed that there
were 2 different stages on the microbial inactivation curves when the
1st-order reaction model was used to fit the inactivation data. The
inactivation rates for the 2 stages were determined for aerobic bac-
teria, yeast, and lactic acid bacteria, respectively. The inactivation
rate was generally higher in the initial stages than in the later stages,
suggesting that might be 2 different groups of the microorganisms,
a more HHP-susceptible group and a less HHP-susceptible group. It
was also noticed that aerobic bacteria were more susceptible to the
HHP treatments than yeast and lactic acid bacteria in the wine sam-
ples. Our study demonstrated that HHP has the potential of being
used for nonthermal pasteurization of low-alcohol wines or other
beverages. More studies on the microbial inactivation kinetics and
impact on sensory quality by HHP are under way.
Acknowledgment
This publication is a part of results of a research project (Project
Nr 98-0402-01-01-3) supported by Korea Science and Engineering
Foundation (KOSEF). The authors appreciate the KOSEF for support
URLs and E-mail addresses are active links at www.ift.org
and Pulmuone Co. Ltd. for allowing the use of the high-pressure food
processing system.
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