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Accepted Manuscript
Conjugates of α-lactalbumin, β-lactoglobulin, and lysozyme with
polysaccharides:
Characterization
and
techno-functional
properties
Igor José Boggione Santos, Héctor Luís Hernandez Hernandez,
Mariana Helena Cardoso Costa, José Antônio de Queiroz Lafetá
Júnior, Jane Sélia dos Reis Coimbra
PII:
DOI:
Reference:
S0963-9969(18)30656-2
doi:10.1016/j.foodres.2018.08.065
FRIN 7873
To appear in:
Food Research International
Received date:
Revised date:
Accepted date:
11 January 2018
9 August 2018
18 August 2018
Please cite this article as: Igor José Boggione Santos, Héctor Luís Hernandez Hernandez,
Mariana Helena Cardoso Costa, José Antônio de Queiroz Lafetá Júnior, Jane Sélia dos
Reis Coimbra , Conjugates of α-lactalbumin, β-lactoglobulin, and lysozyme with
polysaccharides: Characterization and techno-functional properties. Frin (2018),
doi:10.1016/j.foodres.2018.08.065
This is a PDF file of an unedited manuscript that has been accepted for publication. As
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ACCEPTED MANUSCRIPT
Conjugates of α-lactalbumin, β-lactoglobulin, and lysozyme with
polysaccharides: characterization and techno-functional properties
Igor José Boggione Santos1,2, Héctor Luís Hernandez Hernandez2,3, Mariana
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Helena Cardoso Costa2, José Antônio de Queiroz Lafetá Júnior2, Jane Sélia
1
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dos Reis Coimbra2, jcoimbra@ufv.br
Departamento de Química, Biotecnologia e Engenharia de Bioprocessos,
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Universidade Federal de São João del-Rei, Campus Alto Paraopeba, MG
2
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443, km 7, 36420-000 Ouro Branco, MG, Brazil
Departamento de Tecnologia de Alimentos, Universidade Federal de
Departamento de Ingeniería de Alimentos, Universidad de Córdoba, Cra 6a
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3
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Viçosa (UFV), Av.P. H. Rolfs, s/n, 36570-000 Viçosa, MG, Brazil
*
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N°- 76 - 103 Montería, Córdoba, Colombia
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Corresponding author.
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ABSTRACT
Conjugates of protein (-lactalbumin, β-lactoglobulin, and lysozyme) with
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polysaccharides (guar, locust, pectin, and carboxymethilcellulose) were
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prepared via Maillard reaction by the dry-heating method. The conjugates
grafting
degree,
sodium
dodecyl
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were characterizated by using the browning index, extent of reaction,
sulfate
–
polyacrilamide
gel
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electrophoresis, fluorescence, and circular dichroism. The emulsifying
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properties and foaming ability of the formed conjugates were also
evaluated. Conjugates with pectin and Lz-CMC system showed an increase
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in the browning index with the increase of the heating time. Circular
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dichroism and fluorescence data pointed out to conformational changes of
proteins during glycation. The lysozyme (lz) conjugates presented the
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highest degree of glycation (89.1 %). The -Lactalbumin (-la) -
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polysaccharides (PS) conjugates showed foam stability higher than the
mixture (-la + PS), the pure -la, and the conjugates of β-lactoglobulin
(β-lg) and lysozyme (lz) for all studied time (30, 60, and 120 min). The αla-carboxymethylcellulose (CMC) conjugate presented the highest value of
foaming stability (85.71). The pure β-lg shows greater foam stability and
volume than β-lg-PS conjugates and mixture (β-lg + PS). The lz conjugates
do not exihibit foam stability, except for the lz-CMC conjugate that showed
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stability up to 60 min. Furthermore, emulsion stability of the systems was
affected by sonication time. Conjugates of α-la have greatly potencial
applications as novel foaming agents in food industry.
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Keywords: emulsifying properties, glycosylation, foaming properties,
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protein, Maillard reaction.
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1. Introduction
Proteins and polysaccharides play a key role in various food products since
they confer differentiated technical and functional properties to the
products as well as they modify their nutritional properties (Alahdad,
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Ramezani, & Aminlari, 2009; Yadav, Parris, Johnston, Onwulata, & Hicks,
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2010).
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However, industrial application of proteins is limited due to their instability
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under: (1) extreme flow conditions, (2) elevated temperature, (3) presence
of organic solvents, and (4) proteolytic agents. In this context, the need
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arises to study protein-polysaccharide conjugates because these products
make possible greater stability of proteins, new properties, and expansion
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of industrial applications of proteins (J. Liu, Ru, & Ding, 2012; Oliver,
Melton, & Stanley, 2006).
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One of the effective methods to promote the conjugation of proteins with
polysaccharides is the Maillard reaction. This reaction improves the
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stability of the protein and does not require the use of chemical catalysts
(Oliver, Melton, & Stanley, 2006). However, the Maillard reaction must be
performed under controlled conditions for it does not reach the final stage
of the reaction in which there is production of colored compounds,
insoluble, and low nutritional value. Parameters controlled during Maillard
reaction to produce conjugates with improved properties include
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temperature (usually ranging from 40 to 80 °C), relative humidity (65 % or
79 %) and reaction time (depending on the type and conformation of
protein as well as the type of polysaccharide) (Jimenez-Castano, Villamiel,
& López-Fandino, 2007).
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Among the various types of proteins, whey proteins stand out for their
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excellent techno-functional and nutritional properties. Whey proteins are a
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diverse group of proteins with different structural characteristics such as αlactalbumin (α-la) and β-lactoglobulin (β-lg). These proteins, purified or
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concentrated may be used in food products to provide specific functional
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characteristics (gelling, emulsifying, foaming) and to benefit the human
health (Jimenez-Castano et al., 2007).
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Egg white is also a natural source of protein of recognized nutritional and
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biological value. Three egg white proteins present technological interest,
the lysozyme, ovalbumin, and the ovotransferrin. Lysozyme (3.5% of total
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proteins of egg white) has antibacterial properties and it is used in the
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preservation of food and in the pharmaceutical industry (Awade, Moreau,
Molle, Brulé, & Maubois, 1994; Vachier, Piot, & Awade, 1995).
Polysaccharides are high molar mass macromolecules resulting from the
condensation of a number of aldoses and ketoses molecules as well as
multiple hydroxyl groups. Polysaccharides are highly stable, safe, nontoxic, hydrophilic, biodegradable, abundant in nature, and have low cost
ACCEPTED MANUSCRIPT
processing (Z. Liu, Jiao, Wang, Zhou, & Zhang, 2008). Thus, they are
excellent ingredients for food industry.
The glycosylation of proteins via Maillard reaction is a research area with
potential for use in the food industry. Therefore, in order to add value to the
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proteins of whey and egg white, in this work not only the formation but
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also some characteristics of the conjugates of such proteins with
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polysaccharides were evaluated.
2. Materials and methods
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2.1. Materials
Powder of -lactalbumin (-la; 95% protein, 90% of which is -la) and β-
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lactoglobulin (β-lg; 95% protein, 90% of which is β-lg) were kindly
donated by Davisco Food International, Inc. (Eden Prairie, MN, USA).
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Lysozyme (lz) was purchased from Merck (Darmstadt, Germany). Guar
gum and pectin were purchased from Arinos Química (São Paulo, Brazil)
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and CPKelco (Georgia, USA), respectively. Locust bean gum and
carboxymethyl cellulose (CMC) were purchased from Vetec Química Fina
(Rio de Janeiro, Brazil). All other chemicals were of analytical reagent
grade and used without further purification. Deionized water (Millipore
Co., MA, USA) was used in all the experiments.
2.2. Formation of conjugates of protein-polysaccharides
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Conjugates of protein-polysaccharide (-la-pectin, -la-locust, -la-CMC,
-la-guar, β-lg-pectin, β-lg-locust, β-lg-CMC, β-lg-guar, lz-pectin, lzlocust, lz-CMC, lz-guar) were prepared by Maillard reaction in accordance
to Kato (2002). Protein and gum solutions were dissolved in water in a
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mass ratio of 1:1 and stirred (Magnetic stirrer Fisatom, São Paulo, Brazil)
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for 1 hour at 25 ºC. The concentrations of protein and gums were of 1
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mg.mL-1. Then, the solution was frozen at -40 C (Ultra freezer, Terroni,
Brazil) and lyophilized (Lyophilizer LS 3000, Terroni, Brazil). Upon this
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operation, 0.300 g of lyophilized mixture was incubated (Quimis Q 316M,
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Brazil) at 60 °C and 79% relative humidity in a desiccator (0.8 L approx.
volume) with saturated NaCl solution at different days (1, 2, 3, 5, 7, and 9
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days). Each system required 3 h to reach the equilibrium condition. After
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this interval, the heating time was immediately recorded, then samples
were collected during 9 days (at the days 1, 2, 3, 5, 7, and 9) and they were
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kept refrigerated (4 ºC) until analysis. Pure protein was used as control.
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2.3. Browning index determination
Browning development is an indicator of the Maillard reaction progress. It
was measured according to Martinez-Alvarenga et al. (2014). Color
changes of lyophilized conjugates under different times of heat treatment
were determined by using the browning index (BI) (Equation 1).
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(1)
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(2)
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Being x obtained by Equation 2.
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Where L, a, and b are values obtained by using the colorimeter
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(Colorimeter CR-410, Konica Minolta, USA).
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2.4. Measurement of the extent of reaction
Conjugation extent of protein with polysaccharides was measured
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according to Vigo et al. (1992) and Xu et al. (2010). Reaction between o-
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phthalaldehyde (OPA) and free –NH2 groups in proteins allow determining
the extent of reaction using a colorimetric assay. Protein solution (2
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mg·mL-1) was added to 4 mL of OPA reagent (1.40 mg·mL-1 in 0.1 mol·L-1
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Borax buffer, pH 9.85). The solution was mixed, left 3 min for incubation
at 25 ºC and its absorbance was measured at 340 nm (Spectrophotometer
Care 50 Probe, Varian, USA). A blank was prepared with water. An
analytical curve of standard lysine was built and used to determine free –
NH2 groups in proteins. The assays were carried out in triplicate.
The grafting degree (DG) was calculated according to Geng et al. (2014)
with modifications(Geng et al., 2014) by using Equation 3.
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(3)
Where A0 is the content of free amino group of the mixture protein +
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polysscharides without incubation and At is the content of free amino at the
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time of reaction.
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2.5. Circular dichroism analyses
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Circular dichroism (CD) is used to evaluate the secondary structures of
proteins in the conjugates. CD spectra were obtained at 25 ºC using a
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quartz cuvette of 1 mm (Hellma Analytics, Germany) in a Jasco J-810
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spectropolarimeter (Jasco Corporation, Japan) with a temperature controller
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Peltier-type (PFD 425S, Jasco, Japan) coupled to a thermostatic bath
(AWC 100, Julabo, Germany). Wavelength range used was of 190-260 nm,
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being the deionized water used as blank. Each spectrum of sample without
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dilution was obtained by averaging ten consecutive reading. Pure protein,
pure polysaccharide, and mixtures of protein with polysaccharides without
heat treatment were used as control. All concentrations were of 1 mg.mL-1.
2.6. Fluorescence spectroscopy analyses
Fluorimetry is used to evaluate conformationl changes of proteins in the
conjugates. Spectra were obtained at 25 ºC (9001 PolyScience, USA)
using a quartz cuvette of 10 mm (Hellma Analytics, Germany) without
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dilution of samples in the K2 spectrofluorometer (ISS, USA). Wavelength
range used was of 290-500 nm with excitation wavelength at 280 nm. Pure
protein, pure polysaccharide, and mixtures of protein with polysaccharides
2.7. Techno-functional properties of conjugates
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2.7.1. Foaming ability
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without heat treatment were used as control.
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Aliquots of 5 mL were homogenized in an ultra turrax (T25 digital, IKA,
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Germany) for 72 s at 13500 rpm. Foaming ability of the conjugates was
evaluated by measuring the total volume and the foam volume immediately
(5)
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after homogenization by Equation 5.
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Where VI(%) is the volume increase, A is the volume of protein suspension,
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conjugates or mixture of protein and polysaccharide before agitation (mL),
and B is the volume of protein suspension, , conjugates or mixture of
protein and polysaccharide after agitation (mL).
Foam stability of conjugates was measured immediately after agitation and
at 5 min intervals, until 120 min at 25 ºC. Foam stability (FS) percentage
was obtained by Equation 6.
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(6)
Where Vft is the foam volume after time t and Vf0 is the foam volume at
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time zero.
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2.7.2. Emulsifying properties
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The emulsion stability (ES) of pure protein, protein/polysaccharide
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mixture, and conjugate were determined according to Adebiyi and Aluko
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(2011) with adaptations (Adebiyi & Aluko, 2011). Emulsion of oil-in-water
was prepared mixturing 0.66 mL of sunflower oil with 5.0 mL of sample
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(pure protein, mixture or conjugate of protein, and polysaccharide). All
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concentrations were of 1 mg.mL-1. This emulsion was shaken and
submitted to homogenization (Ultra Turrax DI 25 Basic, IKA, Germany) at
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13,500 rpm for 1 min at 25 °C. Then, the sample was immediately
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sonicated using pulsed tip sonicator (Ultrason Unique, DES500, Brazil) at a
power of 300 W at different times (0, 1, 3, and 5 min). The oil droplet size
(d32) of the emulsions was measured in the Zetasaizer Nano ZS (Malvern
Instruments Inc., UK). ES was calculated according to Equation 4.
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(4)
Where d32,t is the oil droplet size at time 1, 3 or 5 min and d 32,0 is the oil
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droplet size at time zero.
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2.8. Statistical analysis
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Significant differences among means of browning index, grafting degree,
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and emulsion stability were evaluated by the Tukey HSD test with p<0.05
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using Statistica 10®. Data were expressed as mean ± standard deviation.
3. Results and discussion
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3.1. Browning index and grafting degree
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Conjugates were obtained using thermal treatment (60 oC and 79% relative
humidity). Table 1 shows the BI values of all studied conjugates. Systems
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formed with pectin and Lz-CMC system showed an increase of the brown
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color with the increase of the time of the treatment, which indicates the
progress of the Maillard reaction (Echavarría, Pagán, & Ibarz, 2012).
Similar results were reported for -la-acacia gum conjugates (de Oliveira et
al., 2015). However, α-la-CMC and β-lg-CMC systems and the conjugates
formed with guar and locust gums presented a random behavior between
the browning index and the incubation time. This fact can be explained by
the conformational change of polysaccharide and/or protein to prevent
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contact between the ε-amino groups of protein with the aldehyde group of
polysaccharides (Geng et al., 2014). Geng et al., (2014) described similar
results for CMC-OVA conjugates with degree of substitution equal to 0.81.
The extent of protein conjugation with polysaccharides by grafting degree
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(DG) was investigated since non-enzymatic browning could be associated
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to caramelization and/or Maillard reaction (Eichner, 1980; Randhir, Kwon,
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& Shetty, 2008). Results indicated that systems with: (1) higher DG were
α-la-pectin (93.28 %), β-lg-guar (values of Table 1 with the same letter “a”
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by the Tukey Test, 88.69 %), and lz-pectin (values of Table 1 with the
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same letter “a” by the Tukey Test, 89.11 %); (2) lower DG were lz-CMC
and lz-Guar; (3) substantially DG constant with the increase of incubation
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time (1 to 9 days) were β-lg-CMC, β-lg-guar, lz-pectin, lz-locust, and α-la-
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CMC; and (4) random behavior in the DG's relationship with the heating
time were β-lg-pectin, β-lg-locust, and α-la-locust. Similar behavior trends
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were reported by Geng et al. (2014) and Mu et al (2006), for the conjugates
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of CMC-OVA and β-casein-dextran, respectively, with maximum values of
DG near 14 % (Geng et al., 2014; Mu, Pan, Yao, & Jiang, 2006).
For most of the conjugates evaluated the time of 3 days was the most
appropriate for heat treatment according to the results of combining of
higher browning index and higher degree of binding with less reaction
time, according to the file made by other authors (Qiu et al., 2018).
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Therefore, the systems with the time of 3 days were chosen for the next
analyzes.
3.2. Circular dichroism spectroscopy
Circular dichroism (CD) analysis is appropriate to study the conformational
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change of the protein secondary structures. Figure 1a shows the CD spectra
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of mixtures and conjugates of β-lg with polysaccharides and of pure
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protein. Native β-lg spectrum is typical of β-sheet structure with minimum
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ellipticity around 216-218 nm (Kobayashi et al., 2001). The spectrum of
the mixture and conjugate systems showed the decrease of the negative
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peaks, which suggests the reduction of the β-sheet content with the addition
of the polysaccharide in the systems. The β-sheet reduction was higher for
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the conjugate systems, mainly for β-lg-pectin. Kobayashi et al. (2001)
described for β-lg-carboxymethyldextran conjugates the decrease of the
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peak in the presence of the polysaccharide as well a blue peak shift.
Figure 1b shows the CD spectra of mixtures and conjugates of α-la with
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polysaccharides and of pure protein. Native α-la spectrum is typical of αhelix structure with double minimum ellipticity around 208 and 222 nm
(Geng et al., 2014). The conjugates of α-la-CMC presented the highest
reduction of α-helix structures as compared with tpure protein. According
to Kelly, Jess, & Price (2005), the increase of α-helix ellipticity suggests
the increase of structural compactness. Therefore, the decrease of ellipticity
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in mixture and conjugate systems suggests structure changes for less dense
structures.
Figure 1c shows the CD spectra of mixture and conjugate systems of Lz
with polysaccharides and of pure protein. Native Lz spectrum is typical of
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α-helix structure (Takahashi, Lou, Ishii, & Hattori, 2000). The spectrum of
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the mixture and conjugate systems showed the decrease of the α-helix
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negative peaks. The highest reduction of α-helix was observed for Lz and
pectin mixture, in which a tendency to modify the α-helix structures for a
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3.3. Fluorescence analysis
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random structure was observed (Malomo & Aluko, 2015).
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The fluorescence analysis allows the study of an environment containing
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tryptophan residues. Therefore, changes in the fluorescence intensity (FI)
and peak shifts in the presence of polissacharides are valuable observations
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to study tertiary structures of proteins in conjugate systems.
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Figure 2a shows tryptophan residues of pure gums. According to Blackburn
(2004) galactomannans respond to fluorescence spectroscopy due to the
presence of glucose in their structures. Figures 2b to 2d show that FI values
in pure protein systems are higher than in mixture and conjugate systems of
proteins and polyssacharides. Therefore, it is possible to evaluate changes
of protein structures in the latter systems.
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Figure 2b shows the FI of β-lg in the mixture and conjugate systems. The
fluorescence intensity decreased in the conjugate systems when compared
with pure protein system, with the exception for β-lg-guar conjugate. The
FI of β-lg and guar mixture decreased more than the corresponding
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conjugate. In addition, a shift to shorter wavelengths (330 nm to 325 nm)
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was also observed. The displacement was subtle, suggesting that the
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protein has maintained its native form in accordance with Kobayashi et al
(2001) (Kobayashi et al., 2001). However, the FI lower suggests that
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tryptophan residue is located further inside the aggregate.
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Figure 2c shows the FI of α-la in the mixture and conjugate systems. A
peak shift from 320 to 350 nm was observed for the conjugate systems,
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except for the α-la-locust conjugate. Mixtures of α-la with CMC, guar,
locust, and conjugates of α-la-locust presented a decrease of FI when
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compared with system of pure protein. Malomo and Aluko (2015) observed
that tryptophan residues at 350-360 nm are in a polar environment and
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feature low fluorescence intensity(Malomo & Aluko, 2015).
Figure 2d shows the FI of Lz in the mixture and conjugate systems. The
increase of FI and a peak shift from 320 to 350 nm were observed.
Mixtures of Lz and pectin, Lz and locust, and Lz and guar showed higher
FI than the conjugates indicating que mixture systems promoted greater
exposure of tryptophan. Takahashi et al (2000) found for Lz-glucose stearic
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acid monoester systems that the native Lz and the conjugate presented
equal maximum value of FI without shift wavelenght.
Different spectra of circular dichroism and fluorescence of the pure protein,
mixture, and conjugate systems suggest that in the formation of the
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conjugates via glycosidic binding there were changes in the secondary and
Techno-functional
properties
protein-polyssacaharides
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conjugates
of
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3.4.
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tertiary structures of proteins.
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Proteins were analysed for changes of their techno-functional properties
profiles in conjugate systems in terms of foam and emulsifying properties.
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Table 2 shows the foam properties of the conjugates. None of the gums
show increase in volume and foam stability (data not shown).
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Conjugates of α-la with polysaccharides showed foam stability (FS) higher
than the systems of mixture and pure protein for all studied time (30, 60,
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and 120 min), despite volume increase (VI) of the pure protein have been
higher. These results confirm the results of circular dichroism
measurements, by increasing the structural compaction of the molecules,
the high adsorption could avoid the rupture of the air bubbles and this
would guarantee a high stability of the foam (Marinova et al. 2009). Haar et
al. (2011) found a higher FS for α-la-rhamnose conjugate than for pure
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protein in their studies with the conjugates of α-la with arabinose, glucose,
rhamnose monohydrate, maltotriose, maltoheptaose, and trigalacturonic
acid with up to 24 h of incubation. The other conjugates had higher
stability than the pure protein(ter Haar, Westphal, Wierenga, Schols, &
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Gruppen, 2011). The authors concluded that the FS depends on the
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incubation time and the type of saccharide, since higher the time, higher the
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glycation of protein with the saccharide. Therefore, the results obtained in
our study suggest that the incubation time was sufficient to form conjugates
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with greater FS than the pure protein.
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Pure β-lg showed lower FS and VI than the mixture and conjugate systems.
Rade-Kukic, Schmitt, & Rawel (2011) and Schmitt, Bovay, & Frossard
foaming
capacity
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reported
for
conjugates
of
β-lg-allyl
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(2005)
isothiocyanate and β-lg-acacia gum, respectively, slightly lower or about
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equal the protein alone. Different values of pH and incubation time were
tested. Thus, the pH or incubation time did not favor either the quantity or
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type of connections needed to increase the FS of the conjugates, therefore it
did not really favor the stability of the foam. Guar gum, in this study, was
the best gum in terms of FS for α-la and β-lg.
The pure Lz has a higher VI than the mixture and conjugate systems,
however the pure protein, the mixture, and the conjugates systems did not
show FS. Except the conjugate Lz-CMC showed FS up to 60 min.
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Ramezani et al. (2008) also found better FS for the conjugate Lz-casein
than for pure protein (Ramezani, Esmailpour, & Aminlari, 2008).
Table 3 shows the emulsifying properties of the conjugates in different time
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of ultrasonication.
Treatment with ultrasound has been widely used in the food industry for
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the production of stable emulsions, since this technique promotes the
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breakdown of bonds and/or decreases the size of the oil droplets
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(Bhaskaracharya, Kentish, & Ashokkumar, 2009). The decrease in size
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would promote a greater emulsion stability (Levine & Sanford, 1985).
However, our results showed that the ultrasound does not promote
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enhancement in stability of the emulsion. Thus, a difference in emulsion
4. Conclusions
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stability at different sonication times by the Tukey test was not noticed.
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Browning index, extent of reaction, degree of binding, fluorescence, and
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circular dichroism confirmed glycation of the proteins and polysaccharides
studied. For most of the conjugates evaluated the time of 3 days was the
most appropriate for heat treatment according to the results of combining of
higher browning index and higher degree of binding with less reaction
time. Conjugates of α-la with polysaccharide had higher foam stability than
pure protein and mixtures of protein and polysaccharide. Pure β-lg showed
the best results for foam stability when compared with mixtures and
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conjugates systems. Pure Lz and conjugates of lysozyme showed no
significant difference by the Tukey HSD test with p>0.05 between their
characteristics, with the exception for the Lz-CMC conjugate. Ultrasound
did not result in greater stability of the emulsion of the protein conjugates.
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In particular, it is necessary further investigation with regard to the: (i)
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development and adaptation of methods for preparing conjugates; (ii) basis
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for a better understanding of the relationship between the structures of
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proteins in the conjugate.
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Acknowledgments
The authors thank the Conselho Nacional de Pesquisa e Desenvolvimento
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Tecnológico (CNPq) and the Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG) for the financial support, and Centro Nacional
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de Pesquisa em Energia e Materiais-Laboratório Nacional de Biociências,
São Paulo Centro Nacional de Pesquisa em Energia e Materiais (CNPEM-
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LNBio) for fluorescence and circular dichroism analyses.
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http://dx.doi.org/10.1016/j.foodchem.2011.03.116
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conjugate.
Journal
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Awade, A. C., Moreau, S., Molle, D., Brulé, G., & Maubois, J. L. (1994).
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white ovomucin, ovotransferrin and ovalbumin ad characterization of
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ACCEPTED MANUSCRIPT
Figure 1. CD spectra of pure protein systems and of mixture and conjugate
systems of protein and locust, guar, CMC, and pectin gums. (a). β-lg; (b) αla; (c) Lz.
Figure 2. Emission spectra of tryptophan fluorescence of pure protein
PT
systems and of mixture and conjugate systems of protein and locust, guar,
AC
CE
PT
E
D
MA
NU
SC
RI
CMC, and pectin gums. (a) pure gums; (b) β-lg; (c) α-la; (d) Lz.
ACCEPTED MANUSCRIPT
Table 1. Browning index (BI) and degree of grafting (DG) of proteins and
polysaccharides conjugates
-la-pectin
time
-la-locust
DG
BI
-la-CMC
DG
BI
(day)
DG
BI
(%)
(%)
8.22
RI
6.68 ±
±
-
-
0.24ª
7.56
9.51 ±
±
±
±
0.49
b
13.38
30.50
MA
±
±
±
±
0.71
b
0.20b
2.00a
78.68
7.90
50.02
22.98
8.85
22.22
±
±
±
D
0.39ª
PT
E
±
6.51 ±
±
0.38ª
0.77ª
0.30ª
0.57ª
1.96ª
0.67c
0.70b
93.28
13.10
36.72
24.56
5.80
28.89
±
±
±
b
1.27ª
0.34d
1.93ª
33.48
22.57
8.09
25.90
±
±
±
0.87ª
0.27c
2.50ªb
22.47
8.16
28.34
6.80 ±
±
AC
3
5.25
3.42ª
CE
10.18
25.14
0.11ª
±
0.52
0.16ª
0.38ª
9.45 ±
b
-
9.09 ±
b
2
47.35
NU
79.85
0.56
±
0.40ª
SC
0.23ª
1
(%)
6.46
6.54 ±
-
DG
BI
(%)
0
-la-guar
PT
Heating
5.82
±
±
0.57ª
b
79.39
0.56
b
14.70
1.94
6.34 ±
5
±
±
±
±
0.63ª
c
0.50ª
0.14
15.52
80.84
8.33
0.37
7
c
0.84
bc
33.58
8.61 ±
ACCEPTED MANUSCRIPT
0.40c
0.30ª
0.33ª
3.65bc
32.09
80.17
18.57
30.38
±
±
±
2.65d
0.46ª
1.22d
β-lg-pectin
time
BI
(day)
±
±
1.06ª
0.16c
1.84ª
17.95
22.34
17.43 16.10
±
±
±
±
±
0.75c
0.63c
1.41ª
0.97d
0.06c
β-lg-CMC
DG
BI
(%)
8.14 ±
-
10.12
±
MA
-
-
±
-
0.87ª
24.51
10.22
62.98
±
±
±
±
±
±
±
±
3.48ª
0.21b
0.03ª
0.28ª
1.21ª
0.27b
0.33ª
14.99
27.20
9.34
23.61
12.64
63.37
10.99 87.34
±
±
±
±
±
±
±
±
0.42b
1.19ªb
0.40c
0.22b
0.77b
0.83ª
0.39ª
0.11bc
17.64
24.30
10.73
23,06
13.06
61.27
9.92
88.42
±
±
±
±
±
±
±
±
0.49c
0.17ª
0.50d
0.04c
1.08b
0.33ª
0.61c
0.31ªb
29.55
5.83 ±
9.91
17,34
11.72
63.83
11.91 87.94
CE
PT
E
8.09
D
0.39ª
25.28
AC
5
(%)
14.10
0.43b
3
BI
(%)
0.31ª
2
DG
9.62 ±
0.76ª
1
β-lg-guar
DG
BI
(%)
6.93
0
±
β-lg-locust
DG
0.14b
PT
±
RI
±
SC
Heating
±
NU
9
±
13.97 88.69
ACCEPTED MANUSCRIPT
0.09c
±
0.73d
Heating
±
±
0.48cd
0.12d
0.21b
3.22ª
0.75c
0.41ªb
14.10 88.34
9.73
28,57
15.44
62.53
±
±
±
±
±
±
±
±
1.15e
0.58b
0.47cd
0.02e
0.51c
0.51ª
0.51b
0.30ªb
30.42
26.00
11.93
27,63
19.78
63.60
±
±
±
±
±
0.59d
2.63ªb
1.01e
0.04f
Lz-pectin
Lz-locust
DG
MA
(day)
(%)
-
0.42ª
88.61
±
19.30
61.53
RI
±
±
0.41a
0.48b
0.74c
Lz-guar
DG
DG
BI
BI
(%)
(%)
15.38
±
14.98
-
±
-
0.45ª
1.46ªb
17.48
14.16
10.65
±
±
b
0.89ªb
0.73ª
13.38
8.22
±
±
0.96ª
3.29ªb
12.19
3.22
2.38 ±
±
±
±
d
0.59ª
AC
±
-
1.06ª
CE
14.88
±
PT
E
±
17.01
14.00 86.77
±
Lz-CMC
(%)
D
14.60
0.55d
DG
BI
PT
29.08
BI
1
±
27.00
time
0
±
SC
9
±
NU
7
±
0.44ª
0.55ª
1.87
18.47
88.70
14.64
b
3.88ª
1.21
58.10
18.47
5.37 ±
2
±
±
±
±
±
0.87
b
24.59
0.75
3
bc
0.28ª
0.54
c
bc
2.17ª
0.68
88.31
14.01
58.02
18.41 6.66 ±
ACCEPTED MANUSCRIPT
±
±
±
±
±
0.49c
0.10ª
1.11d
1.12ª
22.09
88.42
19.54
61.97
1.78c
±
±
0.57bc
1.05c
1.80b
19.52
12.60
4.19
±
±
1.13ª
1.24b
3.51 ±
5
±
±
±
±
±
bd
1.46ª
28.30
0.74
0.44ª
1.10
b
cd
0.78ª
0.49
88.98
15.14
58.87
20.78
PT
d
13.61
7.20
±
±
de
0.28ªb
0.85ªb
22.14
15.92
4.81
±
±
1.27b
0.82b
7
±
±
±
±
±
RI
1.93 ±
0.16ª
c
0.32ª
2.56ª
89.11
13.24
62.52
48.33
9
±
±
0.46ª
MA
± 1.37
±
f
0.36
c
0.50
NU
1.59
e
SC
d
1.30ª
0.72ª
4.94 ±
±
0.64
1.05
e
bc
D
Values with the same letter were different by Tukey test (p < 0.05). The
AC
CE
PT
E
measurements were done in triplicate.
ACCEPTED MANUSCRIPT
Table 2. Foaming ability of systems.
Foaming
System
Pure protein
Conjugate
Mixture
ability
80.00 ± 0.00b
FS30
3.48 ± 0.14A
50.00 ± 0.00B
0
FS60
0
37.50 ± 0.00C
0
FS120
0
18.75 ± 0.00D
0
VI
132.50 ± 3.54a
α-la-pectin
3.48 ± 0.14A
PT
E
D
FS30
FS60
CE
FS120
AC
VI
α-la-CMC
SC
NU
α-la-guar
MA
VI
PT
132.50 ± 3.54a
RI
15.00 ±
0.00c
60.00 ±
50.00 ± 0.00c
0.00b
33.33 ±
40.00 ± 0.00B
0.00C
0
30.00 ± 0.00C
0
0
0
0
85.00 ±
132.50 ± 3.54a
35.00 ± 0.00b
0.00c
52.94 ±
FS30
3.48 ± 0.14A
85.71 ± 0.00B
0.00C
29.41 ±
FS60
0
42.86 ± 0.00D
0.00E
FS120
0
28.57 ± 0.00E
17.65 ±
ACCEPTED MANUSCRIPT
0.00F
50.00 ±
VI
25.00 ± 0.00b
132.50 ± 3.54ª
0.00c
20.00 ±
50.00 ± 0.00B
FS60
0
40.00 ± 0.00D
PT
0
FS120
0
0
0
SC
3.48 ± 0.14A
137.50 ± 3.54ª
90.00 ± 0.00b
0.00c
FS30
94.17 ± 0.00A
44.44 ± 0.00BC
0
FS60
80.83 ± 0.00A
33.33 ± 0.00CD
0
FS120
68.33 ± 0.00AB 22.22 ± 0.00CDE
0
MA
NU
VI
PT
E
CE
VI
FS30
137.50 ± 3.54ª
30.00 ±
20.00 ± 0.00b
0.00bc
50.00 ±
94.17 ± 0.00A 25.00 ± 0.00BDE
AC
β-lg-pectin
0.00C
15.00 ±
D
β-lg-guar
FS30
RI
α-la-locust
0.00BCD
33.33 ±
FS60
80.83 ± 0.00A
0
0.00BDE
16.67 ±
β-lg-CMC
FS120
68.33 ± 0.00AC
0
0.00E
VI
137.50 ± 3.54ª
25.00 ± 0.00b
85.00 ±
ACCEPTED MANUSCRIPT
0.00c
94.17 ± 0.00B
40.00 ± 0.00A
0.00AC
FS60
80.83 ± 0.00B
20.00 ± 0.00AC
0
FS120
68.33 ± 0.00B
0
0
VI
137.50 ± 3.54ª
80.00 ± 0.00b
7.50 ± 0.32c
47.37 ±
SC
68.33 ± 0.00BCD
0
0
36.00 ± 0.00a
5.00 ± 0.00b
5.00 ± 0.00b
0
0
0
FS60
0
0
0
FS120
0
0
0
36.00 ± 0.00a
5.00 ± 0.00b
5.00 ± 0.00b
FS30
0
0
0
FS60
0
0
0
FS120
0
0
0
MA
FS120
PT
E
FS30
CE
VI
AC
26.32 ±
80.83 ± 0.00BC 31.25 ± 0.00DE
VI
Lz-guar
11.78CDE
FS60
D
β-lg-locust
94.17 ± 0.00AB 50.00 ± 0.00CDE
NU
FS30
PT
FS30
RI
17.65 ±
6.54E
Lz -pectin
10.00 ±
Lz-CMC
VI
36.00 ± 0.00a
15.00 ± 0.00b
0.00c
FS30
0
33.33 ± 0.00A
0
FS60
0
33.33 ± 0.00A
0
FS120
0
0
0
36.00 ± 0.00a
5.00 ± 0.00b
5.00 ± 0.00b
FS30
0
0
0
FS60
0
0
PT
0
FS120
0
RI
ACCEPTED MANUSCRIPT
0
0
VI
SC
Lz-locust
NU
VI: volume increase; FS: foam stability. VI values with the different lower-
MA
case letter in the same row were different by Tukey test (p < 0.05). FS
values with the different upper-case letter for each system were different by
CE
PT
E
D
Tukey test (p < 0.05). The measurements were done in triplicate.
AC
Table 3. Emulsifying properties of systems.
ES
Ultrasonication time (min)
Systems
1
3
5
α-la-guar
0.32 ± 0.03a
0.53 ± 0.05 a
0.30 ± 0.04 a
α-la-locust
0.25 ± 0.02 a
0.26 ± 0.03 a
0.21 ± 0.01 a
ACCEPTED MANUSCRIPT
0.20 ± 0.02 a
0.15 ± 0.03 a
0.29 ± 0.01 a
α-la-pectin
0.30 ± 0.01 a
0.31 ± 0.08 a
0.39 ± 0.01 a
β-lg-guar
0.20 ± 0.01 a
0.34 ± 0.01 a
0.45 ± 0.01 a
β-lg-locust
0.23 ± 0.01 a
0.19 ± 0.01 a
0.21 ± 0.01 a
β-lg-CMC
5.38 ± 0.11 a
8.03 ± 1.10 a
5.99 ± 1.38 a
β-lg-pectin
0.41 ± 0.02 a
0.56 ± 0.03 a
0.33 ± 0.01 a
Lz-guar
0.16 ± 0.04 a
0.07 ± 0.01 a
Lz-locust
0.23 ± 0.08 a
Lz-CMC
0.24 ± 0.03 a
Lz-pectin
0.40 ± 0.01 a
0.09 ± 0.01 a
0.27 ± 0.05 a
0.14 ± 0.08 a
0.22 ± 0.02 a
0.26 ± 0.03 a
0.48 ± 0.06 a
0.32 ± 0.08 a
MA
NU
SC
RI
PT
α-la-CMC
Values with the same letter were different by Tukey test (p < 0.05). The
AC
CE
PT
E
D
measurements were done in triplicate.
ACCEPTED MANUSCRIPT
Highlights
 Conjugates of protein and polysaccharides were obtained by
Maillard reaction.
 Circular dichroism and fluorescence show conformational changes of
PT
proteins in conjugates.
RI
 Conjugates with pectin and Lz-CMC system showed an increase in
SC
the browning index with the increase of the heating time
 Proteins presented differentiated techno-functional properties in the
AC
CE
PT
E
D
MA
NU
conjugates.
Figure 1
Figure 2
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2018, foodres, 065
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