Pectin extraction from pumpkin with the aid of microbial enzymes

ARTICLE IN PRESS
FOOD
HYDROCOLLOIDS
Food Hydrocolloids 22 (2008) 192–195
www.elsevier.com/locate/foodhyd
Pectin extraction from pumpkin with the aid of microbial enzymes
N.M. Ptichkinaa,Ã, O.A. Markinaa, G.N. Rumyantsevab
aSaratov State Agrarian Vavilov University, 335 Sokolovaya Street, Saratov 410005, Russia
bMoscow State University of Applied Biotechnology, 33 Talalikina, Moscow 109316, Russia
Received 1 June 2006; accepted 2 April 2007
Abstract
An optimised procedure is reported for extraction of pectin from pumpkin pulp, using an enzyme preparation from Aspergillus
awamori. In contrast to pumpkin pectin obtained previously by digestion with cell-free culture medium from Bacillus polymyxa, the
material prepared in the present work forms gels with 60 wt% sucrose at pH 3, although the yield is somewhat lower (14% in comparison
with 22%). The main action of the enzyme complex from A. awamori is to degrade cellulose and other insoluble constituents of the plant
tissue, but it also has some pectinesterase activity, which could allow degree of esteri?cation (DE) to be manipulated by varying digestion
time. The time used in this investigation (3 h) gave a DE of 53%; reduction in DE at longer times should yield pectin with a higher
content of unesteri?ed galacturonate residues, capable of binding lead and other heavy-metal cations. Some possible medicinal and food
uses are suggested.
r 2007 Elsevier Ltd. All rights reserved.
Keywords: Pectin; Pumpkin; Enzymic extraction; Aspergillus awamori
1. Introduction
these raw materials enables the main pectin producers
(USA, Germany, Denmark) to plan an annual increase of
The natural polysaccharide pectin (a basic building
pectin production of approximately 3.8% (Phillips, 2000),
material for cell walls of higher plants) possesses a wide
searching for new pectin-containing raw materials is an
spectrum of functional properties. In combination with
important task of science and industry (May, 1990).
water and some other substances, it can act as a thickener,
Another urgent task is to improve pectin extraction
gelling agent, stabilizer, emulsi?er, cation-binding agent,
techniques from raw material. The current technology is
etc. (Bottger, 1990). One substance having so many sepa-
based on acidic hydrolysis and has at least two demerits:
rate properties of technological interest makes pectin a
acidic extraction does not allow pectin to be extracted fully
biopolymer especially valuable for medicine and food pro-
with no damage to its structure and it does not meet
duction. Pectin is not only an effective or even necessary
environmental safety due to acid usage. An enzyme-
additive to form the structure of food products but also has
hydrolytic technology seems environmentally safe and
medicinal bene?ts which include lowering the cholesterol
more effective in terms of pectin yield. Analysis of the
level in blood, removing heavy metal ions from the body,
scienti?c and patent literature shows that a number of
stabilizing blood pressure, and restoring intestinal func-
research centres have been conducting studies to develop a
tions (Voragen, Pilnik, Thibault, Axelos, & Renart, 1995).
biotechnological method of pectin extraction but these
The world consumption of pectin constantly grows and
works are of exploratory nature only, they do not yet
has already exceeded 20,000 tons a year. Dried lemon or
involve a wide range of researchers, and their results are far
orange peel and apple pomace are the main raw materials
from industrial application. However, the achievements of
for its production. And although the potential stock of
biotechnology encourage optimism, including the problem
under consideration.
Ã
This manifests itself, in particular, in sound ?nancing of
Corresponding author. Tel.: +007 8452 501 777.
E-mail address: bird@san.ru (N.M. Ptichkina).
studies during the last 2 years (Phillips, 2000) provided by
0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2007.04.002

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N.M. Ptichkina et al. / Food Hydrocolloids 22 (2008) 192–195
193
such world-known pectin producers as Hercules (USA),
Table 2
Herbstreit und Fox (Germany), CP Kelco (Denmark) and
Characteristics of pumpkin biopectins obtained from enzyme preparations
some others. The interest is obvious: the implementation of
Aspergillus
Basillus
Trichoderma
a biotechnological method would double (as a minimum)
awamori
macerans
viride
the pectin production and free the processing plants from
expensive works to neutralize the acidic components of the
Yield (%)
14.0
10.5
9.8
traditional technology. Thus, searching for enzymes or
Moisture content (%)
9.0
9.0
9.2
Ash residue (%)
2.9
3.0
3.0
enzyme complexes and ways of their application for pectin
pH of 1% solution
5.2
5.2
5.1
production is urgent in both theoretical and applied
Polygalacturonate (%)
64
64
63
aspects.
DE (%)
53
60
65
The present work was aimed at studying pectin extrac-
Molecular mass (kD)
45
43
42
tion from pumpkin pulp with the aid of some enzyme
Zsp
2.5
2.4
2.2
Gel strengtha (kPa)
10
10
10
preparations. It is based on the mechanism of action of
enzymic preparations and their ef?ciency revealed by one
a1 wt% pectin; 60 wt% sucrose; pH 3.0; measured by Valenta method
of the authors (Rumyntseva, 1993). She used apple pomace
(Pernas, Smidsrød, Larsen, & Haug, 1967).
as a pectin-containing raw material. However, pumpkin
pulp seems more promising for pectin production in Russia
(Ptichkina, 2000) and it would be appropriate to verify the
where mpp denotes the mass of air-dried pumpkin pulp and
hydrolyzing action of the early used enzyme preparation on
m denotes the mass of pectin obtained from it.
this non-traditional pectin-containing raw material.
As shown in Table 2, the enzyme preparation from the
microscopic fungus A. awamori gave the best results. The
technology for making this enzyme preparation was devel-
2. Experimental
oped at the Institute of Biotechnology, Moscow, and
patented (Zyeva, Pavlova, & Rumyantseva, 1993). This
Air-dry pumpkin pulp was prepared in the laboratory as
preparation is a complex one. Its main action is degradation
follows. Pumpkin (Volzhskaya Grey variety) was cut and
of cellulose (cellulase, activity 2000 IU/g), but it also has
the seeds were removed. The remainder was reduced to
some xylanase (820 IU/g), b-glucosidase (780 IU/g), endopo-
2–4 cm fragments and the juice was pressed out. The fresh
lygalacturonase (235 IU/g), and pectinesterase (105 IU/g)
pulp obtained was dried in an infrared (IR) drying box
activity.
made by NITI TESAR Inc. (Saratov, Russian Federation).
In order to ?nd optimum conditions of catalysis, the
The drying lasted 5–7 h at a temperature not higher than
extraction temperature and weight ratio of water to pulp
65 1C. This mode of drying is mild with respect to pectin—
were varied while the preparation dose in the enzyme
it does not cause decomposition.
solution (1 wt%) and the extraction duration (3 h) were
At a preliminary stage, six commercial enzyme prepara-
kept constant. As a result, a technique of pectin extraction
tions from Trichoderma viride, Bacillus macerans and
from dried pumpkin pulp with the aid of the complex
Aspergillus awamori, cultured under aerobic conditions in
A. awamori enzyme preparation under laboratory condi-
a stirred fermenter, were used. Their catalytic activity with
tions has been developed, and is described below.
respect to such polysaccharides as xylan, cellulose and
Pretreatment of raw material: Cut pumpkin pulp is dried
pectin (which are present in the vegetable tissue mainly in
by the IR method, and 50 g of the dried material is mixed
an insoluble form) and the ef?ciency of each preparation in
with distilled water (400 mL) and left to swell at room
the pectin hydrolysis–extraction process was evaluated.
temperature for 16–17 h.
The results are presented in Table 1.
The percentage yield of pectin (D) served as the criterion
of ef?ciency:
2.1. Preparation of enzyme solution
D ¼ 100ðm=mppÞ,
(1)
The complex enzyme preparation from A. awamori (1 g)
is diluted with distilled water (100 mL) and mixed using a
magnetic stirrer to obtain a homogeneous solution.
Table 1
Composition and activity (UI/g) of enzyme preparations
Aspergillus
Basillus
Trichoderma
2.2. Implementation of hydrolysis
awamori
macerans
viride
Xylanase
820
150
10000
The enzyme solution is poured into the swollen raw
Cellulase
2000
–
400
material at a weight ratio of 1:10. The concentration of
b-Glucosidase
780
–
840
enzyme preparation obtained is 2 mg/mL and its pH is 5.0.
Endopolygalacturonase
235
540
480
The vessel is placed in a thermostat for 3 h for hydrolysis at
Pectinesterase
105
37
28
45 1C, with periodic stirring.

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2.3. Removal of depectinated pulp
investigation. Data on pumpkin pectin obtained by acidic
hydrolysis (Kamnev et al., 1997; Ptichkina, Danilova,
After the 3 h hydrolysis period, the mixture is twice
Doxastakis, Kasapis, & Morris, 1994; Ptichkina, Ishin, &
?ltered through single-layer and double-layer nylon sieves
Danilova, 1996) are also given for comparison.
to remove the undissolved biomass. The ?lter is placed in a
Let us compare the present biopectin (BP-2) and the
Petri dish in a drying box, and the depectinated pulp is
earlier one (BP-1). One can see that in such properties as
dried at 63 1C for 5 h.
moisture content, ash residue, pH of a 1% solution and
PGA content, both samples are almost the same. Differ-
2.4. Storage of liquid pectin hydrolysate
ences between them are in the degree of esteri?cation,
molecular mass, gel strength, and product yield. The ‘‘old’’
After ?ltering through the double-layer nylon sieve, the
biopectin (BP-1) signi?cantly exceeds the ‘‘new’’ one (BP-2)
solution is held at 50 1C for 1 h (conditions found
in yield (22% against 14%). However, BP-2 has substan-
previously to be suf?cient to inactivate the enzyme) and
tially higher molecular mass. An important advantage of
placed in a refrigerator for storage (if needed).
BP-2 over BP-1 is its ability to form gels (BP-1 is non-
gelling).
2.4.1. Making pectin powder
Formally, BP-2 should be classi?ed as a high-methoxy
The extract, cooled to 8–10 1C, is infused into a double
pectin (DE 450%). However, in practice, only pectins
volume of ethanol as a thin jet under intense stirring. The
with DE460% are usually employed in the food industry.
mixture is put into a refrigerator for 30 min. The cooled
BP-1 and traditional pumpkin pectin meet this require-
mixture is ?ltered through a nylon sieve on a Buchner
ment. It may seem that the biopectin obtained using the
funnel. Using freeze drying, pumpkin pectin can be
enzyme preparation from A. awamori is not promising for
obtained as a powder.
use in food production. However, this would be a wrong
conclusion.
3. Comparisons, discussion and conclusions
The three following properties of BP-2 should be noted:
its ability to form a gel with 60% sucrose, solution pH close
Table 3 presents some important characteristics for
to neutral, and DEo60%. Pectins with DEo60% are
judging the quality of pectins (moisture content, ash residue,
known to bind ions of heavy and radioactive metals and to
pH of 1% solution, content of polygalacturonic acid (PGA),
remove them from the body. The corresponding quantity
degree of esteri?cation (DE), molecular mass, gel strength)
(complex-formation
ability)
has
values
of
340
and
for two samples of pumpkin biopectin (i.e. pectin extracted
78–114 mg Pb2+/g for pumpkin pectin (DE ¼ 56.5%) and
enzymatically). The ?rst, identi?ed as BP-1, was obtained
highly esteri?ed citrus pectin (DE ¼ 75–85%), respectively.
previously (Matora et al., 1995; Matora, Shkodina, &
This means that our biopectin can be recommended for
Ptichkina, 1996; Zhemerichkin & Ptichkina, 1995) using cell-
dietary nutrition for those who have to work with heavy
free culture medium from Bacillus polymyxa; the other one,
metals. The pH value close to neutral favours maintaining
identi?ed as BP-2, is the material prepared in the present
the acidic–basic equilibrium in the body. On the other hand,
the ability to form weak gels in sweet media makes it
possible to use BP-2 in confectionery and baking jelly-
Table 3
covered cakes, and in preparing fruit jelly products and soft
Characteristics of pumpkin biopectins in comparison with acidic hydro-
lysis
drinks. Such products with high values of cation complexa-
tion may be classi?ed as medicinal and prophylactic.
Acid
BP-1b
BP-2c
In the technology developed by us, the duration of
extractiona
enzyme hydrolysis was 3 h at the concentration of enzyme
Hydrolysis time (h)
2
24
3
preparation used (0.2 wt%). Since the enzymes from
Yield (%)
7.0
22.0
14.0
A. awamori include pectinesterase (105 IU/g), an increase
Moisture content (%)
9.2
9.2
9.0
in hydrolysis time and/or enzyme concentration will reduce
Ash residue (%)
3.7
5.0
5.0
the degree of esteri?cation of the pectin macromolecules (as
pH of 1% solution
3.2
5.4
5.2
pectinesterase eliminates methyl groups from them).
Polygalacturonate (%)
79
66
64
DE (%)
66
68
53
Therefore, our technology is capable of producing low-
Molecular mass (kD)
70
26
45
methoxy pumpkin biopectin with various DE values, which
Gel strengthd (kPa)
31
Non-
10
could substantially enhance the application of biopectin in
gelling
the production of food supplements and special medicinal
aExtracted using 0.1 N HCl (Ptichkina et al., 1996).
preparations.
bExtracted using cell-free culture medium from Bacillus polymyxa
(Matora, Shkodina, & Ptichkina, 1996).
Acknowledgement
cExtracted using enzyme preparation from Aspergillus awamori (this
work).
d
The authors would like to thank professor Edwin R.
1 wt% pectin; 60 wt% sucrose; pH 3.0; measured by Valenta method
(Pernas, Smidsrød, Larsen, & Haug, 1967).
Morris for fruitful discussion and valuable remarks.

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Document Outline

• Pectin extraction from pumpkin with the aid of microbial enzymes
  • Introduction
  • Experimental
    • Preparation of enzyme solution
    • Implementation of hydrolysis
    • Removal of depectinated pulp
    • Storage of liquid pectin hydrolysate
      • Making pectin powder
  • Comparisons, discussion and conclusions
  • Acknowledgement
  • References

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