University of Chile

Faculty of Chemical and Pharmaceutical Sciences

Conservation Processes by Low Temperatures
Spring 2017

EFFECT OF CITRATE IONS ON THE

SOFTENING OF ROOT CROPS PREPARED
WITH FREEZE-THAW IMPREGNATION OF
MACERATING ENZYMES

Authors: Sayaka Nakatsu, Mitsuya Shimoda, Kenya Shibata, Ryo Kajihara,

Masako Ishihara, and Koji Sakamoto.
Authors are with Food Technology Research Center, Hiroshima Prefectural
Technology Research Inst, from Japan.
Authors Shimoda are with Dept. of Bioscience and Biotechnology, Faculty
of Agriculture, from Japan.
Nicole Prez student of Food Engineering, University of Chile
 INTRODUCTION

Vegetables Root crops Vegetables are a primary dietary source of

fiber and vitamins
Root crops are customarily eaten in East
Burdock Asia
Carrot
root They have unique flavors
They are difficult to soften even if they are
Lotus boiled for a long time or are processed by
rhizome high pressure
They are unsuitable for people who have
trouble with mastication or forming a food
Bamboo bolus
shoot
 INTRODUCTION

Freeze-thaw
impregnation
technique

Slow freezing Vacuum treatment Macerating enzymes

The intercellular and

Induces the Can enhance the
intracellular spaces of Helps to improve the
deformation and efficiency of To a fruit can increase Vegetables became
the foodstuff are pH reduction of
relaxation of the impregnation into the their yield of juice soft
expanded by the vegetables
foodstuffs food tissues
growth of ice crystals

These softened vegetables retain nutrients and flavors well

 OBJECTIVE
In this study, we investigated the
effect of citrate ions on the enzymatic
softening of root crops containing
various amounts of pectins.
The research is important in
understanding the mechanisms of
work action of citrate ions and thus
predicting whether its addition
enhances enzymatic softening of
different root crops with various
pectin contents.

Figure 1: Procedure for sample preparation.

The initial process differs according to the foodstuffs.
 MATERIALS AND METHODS
SAMPLE PREPARATION

Burdock root:
Were peeled, transversally cut into slices with a thickness of 1 cm and boiled in water for 10 min.

Carrot:
Were peeled, tangentially cut into semicircular slices with thickness of 1 cm and steamed at 100 C for 10 min.

Lotus rhizome:
Boiled, were tangentially cut into semicircular slices with a thickness of 1 cm.

Bamboo shoot:
Boiled, were tangentially cut into trapezoids (2 to 3 cm wide) with a thickness of 1 cm.

The samples were frozen at 20 C in a refrigerator-freezer

 MATERIALS AND METHODS
IMPREGNATING SOLUTIONS
Impregnating solutions were prepared by dissolving macerating
enzymes in each solution:
sodium citrate buffers (pH values; 4.0,4.5, 5.0, 5.5, 6.0, concentrations; 0.5, 5,
15, 50, 100, 150 mM)
50 mM sodium acetate buffer (pH 5.0)
50 mM sodium lactate buffer (pH 5.0)
Sodium phosphate buffers of pH 6.0 (concentrations; 50, 100, 150 mM)
Liquid seasoning (Mizkan Group Corp., Aichi, Japan).
The seasoning was pH 5.0 and contained 6.0% (w/w) saccharides and
0.9% (w/w) sodium.
In this study, a commercial preparation of macerating enzymes
(Amano Enzyme Inc., Aichi, Japan) was used. This namely contained
pectinolytic and cellulolytic enzymes from Aspergillus niger.
 MATERIALS AND METHODS
IMPREGNATING SOLUTIONS

Figure 4Firmness of burdock roots prepared with different

impregnating solutions containing the macerating enzymes.
Impregnated solutions contained 1.0% (w/v) macerating enzymes
Figure 3Mechanical properties before impregnation and impregnation rate. : With
and their pH values were 5.0. Values represent mean standard
freeze-thawing (before enzyme impregnation), :Without freezethawing
error (n = 10). Mean values with a different letter are significantly
(before enzyme impregnation). (A) Rigidity coefficient. (B) Breaking strain. (C)
different by Tukeys multiple comparison test (P < 0.05).
Impregnation rate. Rigidity coefficient and breaking strain were measured with a creep
meter (RE-33005B; Yamaden Co. Ltd.). Impregnation rate was calculated from the sodium
content introduced into the samples by the vacuum impregnation.
 MATERIALS AND METHODS
ENZYME ASSAY
The pectinolytic activity of the macerating enzymes were assayed as follows: one unit was defined as the amount
of enzymes required to reduce the viscosity of a substrate solution by half in 10 minutes at 40 C and pH 5.0. The
enzymes were dissolved with 50 mM sodium citrate buffer (pH 5.0). One milliliter of the enzyme preparation was
added to 10 mL of 1.0% (w/v) citrus pectin (Kanto Chemical Co., Ltd., Tokyo, Japan) dissolved in 50 mM sodium
citrate buffer (pH 5.0). The citrus pectin was composed of galacturonic acid (5070%, w/w) that had 3.07.0%
(w/w)methyl groups. After the enzymes reacted for 10 minutes at 40 C, the viscosity was measured using a cone-
plate viscometer (DVM-E2, Toki-Sangyo Co., Ltd., Tokyo, Japan). The cellulolytic activity of the macerating
enzymes were assayed in the same method as for the pectinolytic activity. One milliliter of the enzyme
preparation was added to 10 mL of 1.0% (w/v) sodium carboxymethylcellulose (Wako Pure Chemical Industries,
Ltd., Tokyo, Japan) dissolved in 50 mM sodium citrate buffer (pH 5.0). As a result, present macerating enzymes
had pectinolytic activity of 400 U/g and cellulolytic activity of 6300 U/g.
MATERIALS AND METHODS
ENZYME ASSAY
Figure 5Values of the
enzymatic activities in various
solutions. : Pectinolytic
activity, : Cellulolytic activity.
The pectinolytic and
cellulolytic activities of the
macerating enzymes were
determined using the assay
method described in the
enzyme assay section.
 MATERIALS AND METHODS
PROCEDURE OF FREEZE-THAW IMPREGNATION AND ENZYMATIC
REACTION
The procedure is illustrated in Figure 1.
Freeze-thawed sample was immersed into
the impregnating solution in a glass beaker
for 10 min. Then, the glass beaker was
placed in a vacuum chamber at 10 kPa for 5
min. A stainless mesh weight was placed on
the sample pieces in the beaker to prevent
them from coming up to the surface. After
the vacuum treatment, the pressure was
restored to atmospheric pressure in 10 s to
complete the impregnation process. The
sample was removed from the solution and
placed in an oven (MOV-212S; Sanyo
Electric Co., Ltd., Osaka, Japan) at 50 C
for 1 h to activate the macerating enzymes.
Afterwards, the sample was cooked in a
steam convection oven (TSCO-2EB, Tanico
Co., Ltd., Tokyo, Japan) at 100 C for 10
min to inactivate the macerating enzymes. Figure 1: Procedure for sample preparation.
The initial process differs according to the foodstuffs.
 MATERIALS AND METHODS
MEASUREMENT OF MECHANICAL PROPERTIES AND
IMPREGNATION RATE

Figure 6Firmness of burdock roots prepared with sodium citrate
buffer solution (pH 5.0) at different concentrations. Values
represent mean standard error (n = 10). : Containing
macerating enzymes (1.0% (w/v), : Without macerating
enzymes.
Figure 7Effect of pH value of impregnating sodium citrate buffer
solution on the firmness of burdock roots. Values represent mean
standard error (n = 10). : Containing macerating enzymes
(1.0% (w/v), : Without macerating enzymes. Mean values with
a different letter are significantly different by Tukeys multiple
comparison test (P < 0.05).
 CONCLUSION

Co-impregnation with citrate ions and macerating enzymes

significantly facilitated the softening of root crops

The softening effect of citrate ions was related to the amount of

pectins. For the burdock roots and the carrots which consist of
the polysaccharides whose 50% are pectins, the effect was
significant compared with the lotus rhizomes and the bamboo
shoots which include the pectins by 30% and 10%, respectively.