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Food Science and Technology


Effect of the Partial Substitution of Soy Proteins by Highly Methyl-Esterified Pectin on Chemical and
Sensory Characteristics of Sausages
C.M. Pereira, M.F. Marques, M.K. Hatano and I.A. Castro
Food Science and Technology International published online 18 October 2010
DOI: 10.1177/1082013210366888

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Effect of the Partial Substitution of Soy Proteins by Highly
Methyl-esterified Pectin on Chemical and Sensory
Characteristics of Sausages

C.M. Pereira,1 M.F. Marques,2 M.K. Hatano2 and I.A. Castro1,*

Department of Food and Experimental Nutrition, Faculty of Pharmaceutical Sciences,
University of São Paulo, Av. Lineu Prestes 580 B14, 05508-900, São Paulo, Brazil
Kienast and Kratschmer Ltda. Av. Industrial, 3331, Santo Andre´ 09080-511, Brazil

Pectin can be used as a natural emulsifier in food formulations. In this study, textured soybean protein
(TSP), used as an emulsifier in commercial sausages, was partially replaced by a mixture containing pectin
and isolated soybean proteins, which were either extruded (EXT) or not extruded (MIX), and the chemical
and sensory characteristics of samples were evaluated after 60 days of storage at 4  C. Responses such as
oxidation measured by PV and TBARS, hardness, color, pH and sensory characteristics were compared
with those of a commercial sausage (CON). The mixture containing highly methyl-esterified pectin, tex-
tured soybean proteins and isolated soybean proteins, as emulsifier agent, reduced the hardness (EXT:
21.69±0.98 and MIX: 20.17±2.76 N) and the pH (EXT: 5.46±0.03 and MIX: 5.29±0.01) of the samples
and increased the concentration of peroxides (EXT: 0.10±0.01 and MIX: 0.15±0.01 meq/kg) when com-
pared with samples formulated only with TSP (28.57±2.54 N, pH of 6.92±0.04 and PV ¼ 0.07±0.01 meq/
kg). These effects were likely caused by the anionic character of the emulsifier. However, no sensory
difference was observed between the sausages containing highly methyl-esterified pectin, textured soybean
proteins and isolated soybean proteins submitted to the extrusion process (EXT) and the control sausages,
suggesting that the formulation proposed in this study can be a potential alternative for the further
development of sausages that have functional properties or are free of artificial additives.

Key Words: oxidation, sensory, emulsion, sausage, pectin, soy proteins

INTRODUCTION (BHT) act as antioxidants to avoid lipid oxidation;

and some proteins, hydrocolloids and sodium stearoyl
Sausage is a popular meat emulsion that is widely lactylate (SSL) act as emulsifiers to inhibit phase inver-
consumed in all parts of the world. In general, sausages sion (Flores et al., 2007; Valencia et al., 2007). Meat
are vacuum packed in plastic bags and stored under emulsions, such as sausages, can be defined as a two-
refrigeration (4  C) for 60—90 days. The major factors phase system consisting of the dispersion of a solid (fine
that contribute to this short shelf-life are microbiological particles of fat) in a liquid (matrix or aqueous solution
development, sensory damage caused by water loss, dis- of salt and proteins) in which the solid is not miscible
coloration and oxidative reactions, which are responsi- (Schut, 1976).
ble for unpleasant flavors (Rubio et al., 2007; Valencia Surfactants, also known as emulsifying agents, are
et al., 2007). Usually, a series of additives is applied to amphiphilic molecules that usually are composed of a
preserve the sausage quality and security over its shelf- hydrophilic part and a hydrophobic part. They are cat-
life. Sodium nitrite or nitrate, for example, are used to egorized as nonionics (e.g., monoacylglycerol, polyox-
prevent bacterial growth; sodium erythorbate, butyl yethylene sorbitan monooleate), anionics (lactic acid
hydroxyanisole (BHA) and butylhydroxytoluene esters, phospholipids) or cationics, which are not used
in foods (Walstra, 1996). Natural emulsifiers have some
advantages over synthetic emulsifiers, such as lower tox-
*To whom correspondence should be sent icity (Rodrigues et al., 2006). In addition, natural com-
(e-mail: pounds are ‘label friendly’ (Mun et al., 2008), as they are
Received 20 July 2009; revised 20 October 2009.
described in food labels as ingredients and not as addi-
Food Sci Tech Int 2010;0(00):0001–0007 tives. Proteins are among the natural compounds able to
ß SAGE Publications 2010 exert an emulsifying effect in a food matrix (Walstra,
Los Angeles, London, New Delhi and Singapore
ISSN: 1082-0132 1996). Besides meat proteins, a variety of nonmeat ingre-
DOI: 10.1177/1082013210366888 dients have been used as fillers, binders and extenders.
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Casein, whey and soybeans are some examples of pro- Methods

tein sources applied in meat emulsion formulations that
are able to reduce interfacial tension (Benichou et al., Experimental Design
2002; Flores et al., 2007). Soy proteins are relatively
The mixture containing highly methyl-esterified
inexpensive when compared to meat and milk proteins
pectin, textured and isolated soybean proteins was pre-
and have special gelling properties that not only aid in
pared by adding the ingredients in a specific proportion
binding chunks of meat together but also bind water
(34 : 36 : 30) on a dry basis. Half part of the mixture was
(Schilling et al., 2004). Some types of carbohydrates,
submitted to an extrusion process (EXT) and half was
such as starch and soluble fibers, have also been applied
not extruded (MIX), according to the parameters previ-
as emulsifying stabilizers. Because of their high
ously described by Bueno et al. (2009). Briefly, extrusion
water-binding and gel-forming abilities and their
experiments were carried out in a laboratory single screw
unique characteristics in building texture, stability and
extruder (RXPQ Labor 24, Inbramak Ind. Maq. Ltd.,
emulsification, starch and hydrocolloids are extensively
Ribeirão Preto, Brazil). The barrel had three zones with
used as binders in meat products (Kumar and Sharma,
independent electric resistance heaters and a 3.55 : 1
2004; Andrés et al., 2006). Alginate, carrageenans, xan-
compression ratio screw. The temperature in the barrel
than gum, locust bean gum, cellulose derivates and pec-
from the inlet to the die was set at 50, 115 and 135  C,
tins are examples of hydrocolloids used in meat
with a moisture content of 15% and a screw speed rota-
emulsions (Candogan and Kolsarici, 2003; Lurueña-
tion of 263 rpm. The die diameter adopted was 4.68 mm.
Martı́nez et al., 2004). Some researchers have reported
The feed mass flow rate was kept constant at 100 g/min
a synergistic effect when emulsions are prepared by
(dry matter). The temperatures of all the sections were
mixing proteins with hydrocolloids (Benichou et al.,
set, and, once these were reached, corn grits were
extruded at a screw speed of 263 rpm to stabilize the
In a previous study, the emulsifying formation and
flow at 200 g/min before processing the soy proteins
stability in vitro of a mixture containing highly methyl-
and pectin mixture. Finally, the mixture was fed to the
esterified pectin, textured and isolated soybean proteins
extruder, and, after 5 min and stable ampere input read-
was optimized by Response Surface Methodology
ings, the samples were collected. In addition to the treat-
(Bueno et al., 2009). It was observed that pectin could
improve the emulsifying properties of the soybean pro- ments containing pectin, a control (CON) was carried
teins and that the extrusion of this mixture could add out, corresponding to a commercial product that is well
additional advantage to interfacial properties of this accepted by consumers (Table 1).
combination. Thus, the objective of this study was to
evaluate the effect of the partial replacement of textured Sausage Processing
soybean protein by the mixture containing pectin, tex-
tured and isolated soybean proteins, extruded or not, on All the sausages used in this experiment were manu-
chemical and sensory characteristics of sausages after 60 factured the same day in the pilot plant of Kienast and
days of storage at 4  C.

Table 1. Formulation of the assays (mean±SD).


Without With
Materials Ingredients (g/100 g) extrusion extrusion Control

Sausages were processed using fresh meat (lean pork Mechanically separated meat 50.000 50.000 50.000
Chicken skin 10.000 10.000 10.000
meat, pork back fat, and mechanically separated chicken
Lean pork meat (90/10) 17.000 17.000 17.000
meat) obtained from local industries. Isolated soybean Pork back fat 3.000 3.000 3.000
protein (Maisol) and textured soybean protein (NL1000) Ice 11.664 11.664 11.664
were provided by Exin Ind. Com. Ltd. (Massaranduba, Salt (NaCl) 2.000 2.000 2.000
SC, Brazil). Highly methyl-esterified pectin extracted Textured soybean protein — — 3.500
Cassava starch 2.000 2.000 2.000
from citrus peel (GrindstedÕ Pectin USP) was provided Sodium nitrate (NaNO3) 0.030 0.030 0.030
by Danisco Cultor Brasil Ltd (São Paulo, SP, Brazil). Sodium nitrite (NaNO2) 0.006 0.006 0.006
The following reagents were purchased from Sigma Tripolyphosphates 0.250 0.250 0.250
Chemical Co. (St. Louis, MO, USA): 2-thiobarbituric Sodium erythorbate 0.050 0.050 0.050
acid (TBA), 98%; butylated hydroxy toluene (BHT); tri- Spice 0.500 0.500 0.500
Emulsifier — MIX 3.500 — —
chloroacetic acid (TCA); and 1,1,3,3-tetraethoxypro- Emulsifier — EXT — 3.500 —
pane (TEP). All other reagents used were of analytical
Total 100 100 100
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Sausages Emulsified with Pectin and Soybean Proteins 3

Kratschmer Ltd. (Santo André, BR) according to the solution of iron III (FeCl3) was prepared to construct
formulation shown in Table 1. The pork meat and the the calibration curve. The peroxide value, expressed as
mechanically separated chicken meat (MSM) were milliequivalents of peroxide/kilogram of sample, was
chopped into cubes (approximately 3 cm3) and ground calculated as: PV ¼ [(AsAb)  m]/[55.84  m0  2],
with a commercial food processor (Mado New 603, where As represented the absorbance of the sample; Ab
Germany). Afterward, the mixed meat was homogenized represented the absorbance of the blank; m represented
with ice in the cutter (Kilia EX3000 RS, Germany) for the slope, which was obtained from the calibration curve
8—10 min at 12  C. Other dried ingredients were slowly (which in our experiment was 37.56); m0 represented the
added, and the mixture was homogenized for 1 min at mass in grams of the sample; and 55.84 is the atomic
500 rpm while the temperature was maintained under weight of iron.
12  C. Then, the emulsion was mechanically stuffed The secondary products of lipid oxidation were deter-
(Handtmann VF50, Germany) into cellulose-imperme- mined by TBARS (thiobarbituric acid reactive sub-
able casings (22 mm diameter). Sausages were hand- stances) according to Miller (1998). Briefly, sausage
linked at 15 cm intervals and cooked by the following samples were homogenized at 18,000 rpm in a Turratec
sequence: drying for 30 min at 65  C (open), drying for TE-102 (TecnalÕ , Piracicaba, SP). An aliquot of the
40 min at 75  C (closed), and steam cooking at 80  C to homogenate (5 g±0.5) was mixed with 45.8 mL of
an internal temperature of 72—74  C using a Bastra 10% TCA in H3PO4 and 1 mL of 0.2 mg/mL BHT.
smoke-house (model Bastramat 850, Germany). The The TCA solution volume varied according to the
temperature was monitored by a thermocouple inserted sample solution volume before addition. The extraction
into the center of the sample. Finally, samples were sample was prepared using a Turratec TE-102 homoge-
immediately chilled in an ice-water bath, peeled, nizer (TecnalÕ , Piracicaba, SP) at 18,000 rpm for 30 s.
vacuum packed (Röscherwerke Röschermatic, Samples were filtered with Whatman no. 3 paper, and an
Germany) in plastic bags (five sausages per bag), and aliquot (5 mL) of this filtrate was transferred to two
stored under refrigeration (4  C) for 60 days. Cooking tubes (A and B). After the transfer, 5 mL of 0.02 M
loss values were determined by the weight difference of TBA was added to tube A, and 5 mL of distilled water
all samples before and after cooking, expressed as a per- was added to tube B. The contents of the tubes were
centage of raw weight. This measurement was carried stored in the dark for 20 hours at room temperature.
out after the end of the heat processing without repli- The absorbance was measured at 530 nm. The results
cates, as the whole batch for each treatment was weighed were expressed as mg MDA equivalent/kg sample,
at once. using a TEP standard curve.

Chemical Composition of the Sausages Texture Profile Analysis

Moisture, proteins, lipids and ashes contents were The texture profile analysis of the sausages was per-
determined according to AOAC (1990). The samples’ formed according to Breene (1975) using a TA-XT2i
pH was measured by blending 2 g of sausage with texture analyzer (Stable Microsystems, Surrey, UK)
20 mL of distilled water for 2 min. The pH was measured with Texture Expert v.1.20 software. Six sausage sam-
using a pH meter (Quimis Q400A, Diadema, Brazil). All ples for each assay (2.0 cm in diameter) were compressed
measurements were taken in triplicate. twice with a cylindrical probe 25 mm in diameter, at a
speed of 1 mm/s, and with a level of compression of 50%
Oxidation Measurements (PV and TBARS) of the original height between flat plates. Hardness was
defined by peak force during the first compression cycle
Initially, sausage samples were submitted to lipid and was expressed in Newtons (N). Although other
extraction using the method of Bligh and Dyer (1959). parameters, such as springiness, cohesiveness, adhesive-
The determination of lipid hydroperoxides was con- ness and chewiness have been measured, only hardness is
ducted according to the method reported by Shanta discussed in this study.
and Decker (1994). Briefly, 0.2 g of the lipid sample
was mixed in a disposable glass tube with 9.8 mL chlo- Sensory Analysis
roform—methanol (7 : 3 v/v) on a vortex mixer for 3 s.
Ammonium thiocyanate solution (50 mL) was added, Twenty candidates were recruited from the University
and the sample was mixed for 3 s. Afterward, 50 mL of staff to compose a trained panel. They were first tested
iron II (FeCl2) solution was added, and the sample was on the four basic tastes (salt, sweet, sour and acid), and
mixed for 3 s. After a 5-min incubation in subdued light 15 candidates were approved for the first training step.
at room temperature, the absorbance was determined at Here, several sausage samples were presented to the
500 nm by a Shimadzu Model spectrophotometer UV panel, and five attributes were identified and discussed
1240V (Tokyo, Japan) against a blank that contained in the group: appearance, odor, color, flavor and tex-
all of the reagents except the sample. A standard ture. Individuals were trained on how to evaluate these
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five sensory characteristics using a ‘Scale Difference p < 0.05 to be significant. Sensory results were treated by
Method’ with a 9-point scale, where 1 was designed as two-way ANOVA (samples and panelists). All statistical
extremely similar to the control and 9 was extremely analyses were performed using the statistical package
different. Afterward, individuals were submitted to an Statistica (version 7.1, Statsoft South America, Tulsa,
individual sensory test and performed in individual OK, USA).
booths, where they were presented three samples (two
control samples and one sample that was different from
the control) to be compared with the control. The test RESULTS AND DISCUSSION
was repeated three times, and the discrimination capac-
ity and repeatability of each candidate was measured by
No differences were observed in protein (p ¼ 0.598)
two-way ANOVA. This procedure was performed in
and lipid (p ¼ 0.207) content among all assays
order to check the training quality. Finally, nine individ-
(Table 2). Samples containing pectin (MIX and EXT)
uals were ranked according to their scores (ratio) for
presented higher ashes content than did the control sam-
participation in the analysis, where the ratio for samples
ples (CON) (p ¼ 0.002) containing only TSP. The value
discrimination was computed as Mean Square
observed for moisture of the EXT samples (63.5%) was
(MS)samples/MSresidue, and for repeatability as
greater when compared with the MIX samples moisture
MSpanelists/MSresidue. Samples (CON, EXT and MIX)
(63.0%). Although without replicates, the cook loss of
were removed from the refrigerator (4  C), placed into
the assays containing pectin (10.7% and 9.1%) was
boiling water for 5 min, kept at room temperature for
lower than that found with the control (12.2%).
10 min, and randomly served to the tasters in a recepta-
After 60 days of storage at 4  C (Table 3), water activ-
cle identified with three digits.
ity (aw) did not differ among all samples (p ¼ 0.505).
Both assays containing pectin presented lower values
Surface Color Analysis
of hardness (p < 0.001) and pH (p < 0.001) than those
Color was measured using a Hunter Color Lab color- found in the control samples. Although the peroxide
imeter; model Color Quest XE (Hunter Associates value observed in MIX and EXT samples have been
Laboratory Inc., Reston, VA, USA). Universal higher than control samples (p ¼ 0.003), no difference
Software 4.10 was used for calibration against black was observed for TBARS among the assays
and white reference tiles with an illumination of D65 (p ¼ 0.778). Regarding the sensory aspect, MIX samples
and a 10 angle. Each slice of sausage was placed with differed from the control (p ¼ 0.038), while no difference
the central part against the aperture. In the Hunter Lab was noted by the trained panelists between EXT and
colorimeter, the color of a sample is denoted by the three CON samples. Significant changes to the color of the
dimensions L*, a* and b*, corresponding to the XYZ samples were observed after 60 days of storage at 4  C
CIE lab system. (Table 3).
Stability, cook loss, texture, palatability and cost are
Statistical Analysis the most important factors to be considered in the eval-
uation of the sausages (Flores et al., 2007). In this study,
The results were expressed as mean±SD and were the food formulations differed only in the type of ingre-
submitted to a variance homogeneity test (Hartley). dients applied as emulsifier: textured soybean protein
All measurements were performed in triplicate except (CON), highly methyl-esterified pectin, textured soy-
for cook loss experiments, which were performed with- bean proteins and isolated soybean proteins submitted
out replicates, and sensory data, which represented the to the extrusion process (EXT) and highly methyl-
mean value of nine responses. The chemical parameters esterified pectin, textured soybean proteins and isolated
of the three samples were analyzed by one-way soybean proteins not submitted to the extrusion process
ANOVA, followed by the Tukey HSD test, considering (MIX). Thus, the differences observed among the

Table 2. Chemical composition and cook loss of the assays (mean±SD).


Chemical composition (g/100 g) Without extrusion With extrusion Control P1

Moisture 63.0±0.1 a 63.5±0.0 b 63.3±0.1 ab 0.008

Protein 14.1±0.9 14.0±0.2 14.5±0.3 0.598
Lipids 16.5±0.9 13.9±2.2 12.6±1.6 0.207
Ashes 8.0±0.2 b 8.1±0.1 b 5.8±0.2 a 0.002
Cook loss % 10.7 9.1 12.2 —
Probability value obtained by one-way ANOVA. Values followed by the same letter are not statistically different (p < 0.05).

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Sausages Emulsified with Pectin and Soybean Proteins 5

samples in this study can be only attributed to the partial in sausages, which confers an anionic character to
replacement of the textured soybean proteins (TSP) by both polymers, contributing to food acidification.
pectin and isolated soybean proteins, aside from the MIX and EXT samples showed lower pH values than
effect of the extrusion on this mixture. control (p ¼ 0.019) since the first day of the experiment
Initially, the substitution of the textured soybean pro- (6.77, 6.80 and 6.92, respectively). Since the acidity alters
teins (TSP) by the mixture containing pectin, textured the protein solubility, causing chain aggregation or
soybean proteins and isolated soybean proteins, inde- repulsion as a function of their respective isoelectric
pendently of the extrusion process, reduced the hardness points (Pérez-Gago and Krochta, 1999), the reduction
and the pH of the samples. Soy proteins are, in fact, a of pH observed in the MIX and EXT samples may have
mixture of different proteins containing hydrophobic contributed to reduce the polypeptidic chain aggregation
and hydrophilic amino acids. When a protein adsorbs and increase the starch hydrolysis, promoting a reduc-
to the interface, only a fraction of hydrophobic residues tion of the sausages’ hardness.
are positioned facing the oil phase, and most protein The emulsifier containing soy proteins and pectin,
molecules are suspended in the bulk aqueous phase, submitted or not submitted to an extrusion process, oxi-
forming loops that provide steric stability dized more than the control when evaluated by PV.
(Damodaran, 2005). Among the hydrophilic amino Emulsifiers can influence the oxidation of lipid emul-
acids, soy proteins are predominately rich in negatively sions by chelating pro-oxidant metals, inactivating free
charged amino acids, such as aspartic and glutamic radicals, or forming a physical barrier at the lipid inter-
acids, which are partially responsible for their isoelectric face (Donnelly et al., 1998). Oxidation is favored by the
point around 4.5 (Damodaran, 1996). Pectins are highly increased contact of lipids with transition metals dis-
hydrophilic carbohydrates, which are soluble due to solved in the liquid phase, such as iron and copper.
electric charge repulsion. They are formed by long The presence of anionic emulsifiers around the oil drop-
chains of a(1-4) linked D-galacturonic acid units, inter- let in food emulsions containing transition metals can
rupted by the insertion of L-rhamnopyranosyl residues, increase the oxidation susceptibility via the electrostatic
whereas the side chains consist mainly of D-galactose attraction of the metals to the oil droplet surface, as
and L-arabinose (Candogam and Kolsarici, 2003; suggested by Sørensen et al. (2008). Although the sub-
Funami et al., 2007). The emulsifying properties of stitution of the TSP by the mixtures containing pectin
pectin are most likely due to the protein residues present has increase the oxidation, as measured by PV, it is
within the molecule (Leroux et al., 2003). However, the important to emphasize that the oxidation measured in
predominantly hydrophilic character of the polysaccha- all samples after 60 days by PV and TBARS showed low
ride itself promotes steric and mechanical stabilization values. Valencia et al. (2007) reported good stability for
not negligible for the emulsion (Funami et al., 2007). fermented sausages in which TBARS and PV values
High methyl pectins have more than 50% of their were lower than 0.2 mg/kg and 1.23 meq O2/kg fat,
acidic residues esterified with methyl groups (Liu respectively. In a study carried out by Thomas et al.
et al., 2007), which confers an uncharged character to (2008), sausages presenting TBARS values around
this part of the molecule and contributes to the reduc- 0.2 mg MDA/kg stored at 37  C retained good overall
tion of the strong electrostatic repulsion between the acceptability. Liu et al. (2006) reported that TBARS
chains. Soy protein and pectin are negatively charged in sausages remained below a value at which certain

Table 3. Water activity, texture, pH, peroxide value (PV), TBARs and sensory score of the assays evaluated after
60 days of storage at 4  C (mean±SD).

Parameters Without extrusion With extrusion Control P1

Water activity (aw) 0.958±0.004 0.961±0.006 0.953±0.005 0.505

Texture (Hardness, N) 20.17±2.76 a 21.69±0.98 a 28.57±2.54 b <0.001
pH 5.29±0.01 a 5.46±0.03 a 6.92±0.04 b <0.001
Peroxide value (meq LOOH/kg) 0.15±0.00 c 0.10±0.01 b 0.07±0.01 a 0.003
TBARS (mg MDA/kg) 0.88±0.04 0.83±0.05 0.86±0.08 0.778
SD-CON2 4.00±2.06 b 3.44±2.0 ab 1.78±1.09 a 0.038
l 51.9±0.9 a 53.0±0.1 a 55.2±0.5 b 0.001
a 10.7±0.3 b 8.5±0.1 a 9.2±0.3 a <0.001
b 13.0±0.2 a 14.0±0.1 b 14.5±0.5 b 0.003
Probability value obtained by one-way ANOVA. Values followed by the same letter are not statistically different (p < 0.05).
SD-CON, Scale Difference from Control. Sensory scores on a 9-point difference scale (9 ¼ extremely different from control and 1 ¼ no difference from control).
There was no difference among the panelists evaluation for all samples (p ¼ 0.095).

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