Pepsin-Assisted Transglutaminase Modi fi cation of Functional Properties of a Protein Isolate Obtained from Industrial Sun fl ower Meal

The utilization of industrial sunfl ower meal to produce protein-rich products for the food industry is an alternative approach for bett er and more effi cient use of this agricultural by-product. Sunfl ower meal proteins possess specifi c functional properties, which however need improvement to broaden their potential as supplements for delivering high-quality products for human nutrition. The aim of the study is to evaluate the combined infl uence of low-degree pepsin hydrolysis and transglutaminase (TG) modifi cation on industrial sunfl ower meal protein isolate functionality at pH=2 to 10. Three TG-modifi ed pepsin hydrolysates with the degree of hydrolysis of 0.48, 0.71 and 1.72 % were produced and named TG-PH1, TG-PH2 and TG-PH3, respectively. All three TG-modifi ed pepsin hydrolysates exhibited improved solubility at pH between 3.5 and 5.5 as the highest was observed of TG-PH3 at protein isoelectric point (pI=4.5). Sunfl ower meal protein isolate and TG-modifi ed sunfl ower meal protein isolate had greater solubility than the three TG-modifi ed hydrolysates at pH<3 and >7. Signifi cant improvement of foam making capacity (p<0.05) was achieved with all three TG-modifi ed pepsin hydrolysates in the entire pH area studied. Pepsin hydrolysis of the protein isolate with the three degrees of hydrolysis did not improve foam stability. Improved thermal stability was observed with TG-PH3 up to 80 °C compared to the protein isolate (pH=7). At 90 °C, TG modifi cation of the protein isolate alone resulted in the highest thermal stability. Pepsin hydrolysis followed by a treatment with TG could be used to produce sunfl ower protein isolates with improved solubility, foam making capacity and thermal stability for use in the food industry.


Introduction
Sunfl ower is an economically important oil-bearing crop which is primarily used for production of vegetable oil.In 2012, sunfl ower oil production reached 15.22 million tonnes worldwide (1) and was further projected to increase due to enhanced consumer demand for trans-fat--free unsaturated fats (2) and its potential value as a feedstock for biodiesel generation (3).Either for food or technical purposes, the oil extraction results in a substantial quantity of sunfl ower meal, which may reach up to 30 % of the initial amount of the used sunfl ower seeds (4).Currently, this by-product is used as a protein source in the feed industry.However, its application in animal nutrition is limited due to high fi bre content (5).To avoid adverse performance eff ect, sunfl ower meal inclusion in broiler and swine diets should not exceed 16 and 20 %, respectively (4,6).The overproduction and accumulation of excessive amounts of unutilized sunfl ower meal causes higher storage or disposal expenses leading to overall decrease in net profi t margin.
The utilization of industrial sunfl ower meal for generation of protein-rich products for the food industry is an alternative approach for bett er and more effi cient use of this agricultural by-product.Sunfl ower meal proteins have a high nutritive value.They do not contain antinutritional compounds and exhibit well-balanced amino acid composition with the exception of a low level of lysine (7).Sunfl ower meal proteins possess specifi c functional properties, which however need improvement to broaden their potential as supplements for delivering high-quality products for human nutrition (8,9).
Major challenges in preparation of sunfl ower meal--derived protein products and their subsequent application in the food industry are related to the alteration of protein characteristics due to technological parameters of oil production and sunfl ower seed pretreatment.Industrial sunfl ower meal is produced aft er treatment of sunfl ower seeds with high temperature and organic solvents which, in most cases, decrease the nutritive value of sunfl ower meal proteins and reduce their functional properties (10).Enzymatic treatment of plant-derived proteins with pepsin, trypsin or Alcalase ® is a common approach aiming at improvement of functional properties (11)(12)(13).For example, Martinez еt al. (14) improved foaming capacity of sunfl ower protein by limited enzymatic digestion (degree of hydrolysis 1.5 %) with Alcalase.Karayannidou et al. (15) reported that limited proteolysis of sunfl ower protein isolate with trypsin was very effi cient in stabilizing emulsions and foam.By using a sequential two-step enzymatic digestion with chymotrypsin and carboxypeptidase, Bautista et al. (16) obtained sunfl ower protein hydrolysates with decreased allergenicity and a high Fischer ratio.
Transglutaminase (TG) catalyzes the cross-linking of proteins by formation of ε-(γ-glutamyl)-lysine bonds (17).TG is predominantly used to alter the functionality of proteins of animal origin and has a wide application in the dairy industry and meat and fi sh processing (18).However, successful application of TG to alter plant protein functionality for the production of tofu, bread and other wheat products has also been reported (19,20).Ivanova (9) demonstrated the potential of microbial TG to improve thermal stability and foam making capacity of proteins isolated from industrial sunfl ower meal.In a later study, it was observed that limited hydrolysis of sunfl ower meal protein isolate with pepsin facilitated TG reaction and resulted in approx.4-fold faster polymerization than unhydrolyzed counterpart (21).However, to the best of our knowledge, no information about the eff ects of preceding enzymatic hydrolysis and cross-linking with TG on the functional properties of industrial sunfl ower meal protein isolates is available in literature.The aim of the current study is to evaluate the combined infl uence of low-degree pepsin hydrolysis and TG modifi cation on sunfl ower protein isolate functionality in a wide pH range (from 2 to 10).Solubility, thermal stability, foam capacity and stability, and emulsifying activity and stability of three TG-modifi ed pepsin hydrolysates with low degree of hydrolysis (0.48, 0.71 and 1.72 %) were evaluated.

Materials
Sunfl ower meal was obtained from a local company (Biser Oliva, Stara Zagora, Bulgaria).It was produced after thermal treatment of sunfl ower seeds at 110 °С followed by oil extraction with hexane at 55 °С.Microbial TG (Activa ® WM) was kindly provided by Ajinomoto Co., Inc. (Tokyo, Japan) for research purposes.All reagents used in the study were of analytical grade and bought from Sigma-Aldrich (St. Louis, MO, USA).

Preparation of protein isolate
Proteins were extracted with 10 % NaCl (pH=6) and isolated by isoelectric precipitation at pH=2.5 with 6 M HCl as described by Ivanova et al. (22).The protein precipitate was collected by centrifugation at 1800×g for 15 min (MPW 251; Medical Instruments, Warsaw, Poland), washed three times with 100 mL of HCl (pH=2.5),dried by lyophilization (Lyovac GT2; Leybold Heraeus, Köln, Germany) and stored for further analyses.

Enzymatic modifi cation of protein isolate
Protein isolate (2.0 %) was dissolved in 70 mL of distilled water and hydrolyzed with pepsin (EC 3.4.23.1; 6 U/g protein) at 40 °С for 15, 45 and 120 min.The pH was maintained at 1.8 by adding 0.1 M HCl when needed.The reaction was stopped by increasing the pH to 7.5 with 1 M NaOH, aft er which TG (EC 2.3.2.13; 5 U/g protein) was added to the reaction mixture.TG modifi cation occurred for 2 h at 40 °С.Inactivation of TG was achieved by adding 1 % N-ethylmaleimide (23).
Degree of hydrolysis of pepsin proteolysates was evaluated aft er removal of unhydrolyzed protein by precipitation with 10 % trichloroacetic acid (fi nal concentration) and subsequent centrifugation for 20 min at 1800×g (MPW 251; Medical Instruments).It was calculated by the following equation: where DH is the degree of hydrolysis (%), γ α-N is the concentration of soluble α-amino nitrogen in the supernatant (mg/mL), and γ N is the concentration of total nitrogen in the sample used in the assay (mg/mL).The α-amino nitrogen in the supernatant was estimated by ninhydrin method and glycine was used to generate a standard curve (24).Total nitrogen was determined by Kjeldahl's method (25).

Solubility
Protein solubility was determined as described by González-Pérez and Vereĳ ken (7) with some modifi cations.Proteins were dispersed in water to a fi nal concentration of 4 mg/mL.The pH values were varied in the range from 2.0 to 8.5 with increments of 0.5 by using either NaOH or HCl.Aft er 2 h at room temperature, the suspension was centrifuged for 15 min at 1800×g (MPW 251; Medical Instruments).Soluble protein in the supernatant was evaluated by biuret method (26).Bovine serum albumin was used for generation of a standard curve.The protein solubility was calculated by the following equation: where PS is protein solubility (%), m ps is the mass of soluble protein in the supernatant (mg), and m tp is the mass of total protein in the used sample (mg).

Foam capacity and stability
Foam capacity and stability were determined as described by Sze-Tao and Sathe (27) with some modifi cations.An aliquot of 20 mL of protein solution (0.5 mg/mL) was whipped for 70 s in a graduated cylinder as described by Ivanova et al. (28).Foam capacity was determined by volume increase immediately aft er whipping and was calculated by the formula: /3/ where FC is foam capacity (%), V 1 is the volume of protein solution before whipping (mL) and V 2 is the volume of the solution aft er whipping (mL).The infl uence of pH on foaming properties was tested by varying the pH from 2 to 10 with increments of 2 units using NaOH or HCl.The foam stability was defi ned as the volume of the foam that remained aft er 60 min at room temperature (23 °C) and was calculated by the following equation: /4/ where FS is foam stability (%), V t60 is the volume of the foam that remained aft er 60 min (mL), and V t0 is the volume of the foam immediately aft er whipping (mL).

Emulsifying properties
Emulsifying activity and emulsion stability were determined as described by Neto et al. (29).A volume of 5 mL of protein solution (0.5 mg/mL) was homogenized with 5 mL of food-grade sunfl ower oil for 60 s at 1000 rpm by using a homogenizer (T18 Ultra Turrax Basic; IKA ® --Werke GmbH & Co.KG, Staufen, Germany).The emul-sion was centrifuged for 5 min at 1800×g (MPW 251; Medical Instruments) and the volume of the emulsifi ed layer was recorded (28).The emulsifying activity was calculated by the following equation: /5/ where EA is emulsifying activity (%), V el is the volume of the emulsifi ed layer (mL), and V T is the volume of the total content of the tube (mL).
Emulsion stability was established aft er heating at 80 °C in a water bath (WNB 29; Memmert GmbH+Co.KG, Schwabach, Germany) for 30 min.The emulsion was cooled down to room temperature (23 °C) and centrifuged at 1800×g for 5 min (MPW 251; Medical Instruments).Emulsion stability was calculated by the following equation: where ES is the emulsion stability (%), V el30 is the volume of emulsifi ed layer aft er 30 min of heating (mL), and V el0 is the volume of the emulsifi ed layer before heating (mL).NaOH or HCl was added to protein solutions to modulate the pH from 2 to 10 with increments of 2 where appropriate.

Thermal stability
Thermal stability was determined as described by Kato et al. (30).Aliquots of 5 mL of protein solutions (2 mg/mL) were adjusted to either рН=7.0 or 8.0 and were heated for 20 min at temperatures varying from 50 to 90 °C with increments of 10 °C.Aft er cooling to room temperature (23 °C), the turbidity of the solutions was measured at λ=500 nm (Spekol 11; Carl Zeiss Jena, Jena, Germany).Distilled water was used as a control.Thermal stability was calculated by the following equation: where TS is thermal stability (%), S Ti is the sample turbidity at a specifi c temperature, and S RT is the sample turbidity at room temperature (23 °C).

Statistical analysis
Data are presented as mean values of at least three independent experiments±standard deviation (S.D.).Statistical evaluation was performed by one-way analysis of variance (ANOVA) using Statgraphics Centurion statistical program (v.XVI; StatPoint Technologies, Inc., Warrenton, VA, USA).Mean diff erences were established by Fisher's least signifi cant diff erence test for paired comparison with a signifi cance level at α=0.05.

Results and Discussion
To evaluate a combined infl uence of pepsin and transglutaminase (TG) enzymatic modifi cations on protein functionality, sunfl ower meal protein isolate was initially hydrolyzed with pepsin for 15, 45 and 120 min.Respective hydrolysates, PH1, PH2 and PH3, were characterized with low degree of hydrolysis (DH), namely 0.48, 0.71 and 1.72 %.The following treatment with TG resulted in the preparation of TG-modifi ed pepsin hydrolysates, named TG-PH1, TG-PH2 and TG-PH3, respectively.Their functional properties were evaluated and compared to the functional properties of TG-modifi ed protein isolate (TG-PI) and untreated protein isolate.The design of the study is schematically presented in Fig. 1.

Solubility of sunfl ower protein isolates
Good solubility is usually required for a protein to have good functional properties (31).In our study, improved solubility at pH=3.5-5.5 was achieved with the three TG-modifi ed pepsin hydrolysates and the most pronounced eff ect was observed with TG-PH3 (Fig. 2).Approximately 3-and 3.5-fold increase in TG-PH3 solubility was observed compared to TG-PH2 and TG-PH1, respectively.Compared to protein isolate and TG-modifi ed protein isolate, the solubility of TG-PH3 increased more than 15-fold at pH= 4.5.As previously determined in our laboratory, the protein isolate was rich in sulfur-containing amino acids (amino acid score 99.14 %) (32) and, therefore, could serve as a food additive to balance these specifi c amino acids in human diets if appropriate solubility is provided (33).Limited pepsin hydrolysis of protein isolate combined with TG treatment resulted in structures with good solubility at pH=4.0-6.0.
The results implied that limited hydrolysis of the protein with pepsin decreased the infl uence of pH on protein solubility at the isoelectric point (pI=4.5).Apparently, pepsin hydrolysis of protein isolate is a prerequisite step for the following TG modifi cation to obtain more hydrophilic structures with improved solubility.In a previous study, the hydrolysis of sunfl ower meal protein isolate with pepsin followed by TG treatment resulted in the increase of the amount of 200-kDa protein fractions at the expense of fractions with higher molecular mass (21).Our results are in agreement with the results of Walsh et al. (34) showing increased solubility of TG-cross-linked products of soy protein isolate Alcalase™ hydrolysates.Flanagan and FitzGerald (35) also observed improved solubility of sodium caseinate at around pH=4.6 aft er combined enzymatic treatments with Protamex and TG compared Fig. 2. Infl uence of pH on the solubility of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively to either TG-modifi ed protein or sodium caseinate hydrolysate alone.Improved solubility of enzymatic digests of gluten by chymotrypsin, papain, pronase and pepsin followed by a TG treatment over a wide pH range was reported by Babiker et al. (36).
In general, protein solubility depends on molecular mass, overall charge and surface hydrophobicity.Higher solubility can be expected of proteins with lower molecular mass, higher molecular charge and low surface hydrophobicity.According to Hassan et al. (37), TG treatment of proteins decreases surface hydrophobicity due to partial deamination of glutamine and asparagine.In our study, the decrease in surface hydrophobicity was most probably compensated by the increase in molecular mass aft er protein polymerization and the solubility patt ern of TG--PI followed the one of untreated protein isolate (Fig. 2).Protein isolate and TG-modifi ed protein isolate, however, expressed greater solubility than TG-PH1, TG-PH2 and TG-PH3 at pH≤3 and ≥7, which determines their bett er practical application in formulations of food with highly acidic, neutral or slightly alkaline pH.

Alteration of foam capacity and stability by enzymatic modifi cation
Foam capacity followed the overall trend observed for protein solubility.Protein isolate expressed the lowest foam making capacity at around protein pI (pH=4 and 6), which is related to low molecular charge and the formation of high-molecular-mass aggregates (Fig. 3).The highest foam making capacity of protein isolate, observed at pH=2 (47.7 %), is probably due to dissociation of sunfl ower protein globulins to monomers, thus contributing to increase in foam volume (38).The modifi cation of protein isolate with TG slightly increased the foam making capacity at pH=4 and 6 which, however, did not exceed 40.0 %.Signifi cant improvement (p<0.05) was achieved with all three TG-modifi ed pepsin hydrolysates (TG-PH1, TG--PH2, TG-PH3), which resulted in foam making capacity varying from 52 to 66 % in the entire pH range studied.Although no direct comparison can be made because of diff erences in the used substrates and evaluation meth-ods, similar observations were reported by Babiker (39).The foam capacity of the soy protein and chymotrypsin--based soy protein hydrolysate aft er polymerization with TG increased from 500 to 590 μν/cm respectively, as measured by electrical conductivity.In an earlier study, Babiker et al. (36) also established an improvement of foaming properties aft er TG polymerization of gluten hydrolysates prepared with chymotrypsin, papain, pronase and pepsin.Flanagan and FitzGerald (40) reported 1433 % foam expansion of the sodium caseinate product obtained aft er hydrolysis with Bacillus proteinase and TG polymerization.
In contrast to foam making capacity, pepsin hydrolysis of the protein isolate with three degrees of hydrolysis (0.48, 0.71 and 1.72 %) did not improve the foam stability (Fig. 4).In fact, the highest degree of hydrolysis (1.72 %) gave the lowest foam stability at pH=6.Inverse relationship between the degree of hydrolysis and foam stability of protein hydrolysates was att ributed to the decrease in the amount of larger protein component required for foam stabilization (41).A similar trend was observed by Kong at al. (42) and Wouters et al. (43) while studying wheat gluten hydrolysates, and by Guan et al. (44) and Larré et al. (45) for hydrolysates of oat bran protein concentrate and rapeseed protein isolate, respectively.Our results were not in agreement with the results reported by Babiker (39), who observed increased foam stability of the chymotrypsin-digested soy protein polymerized with bacterial TG.This may be due to diff erences in the substrates used in the study as well as diff erences of the protein profi les of the hydrolysates obtained with pepsin and chymotrypsin.Jung et al. (46) revealed that porcine placenta was barely hydrolyzed by pepsin, resulting in peptides with molecular mass (M) greater than 7 kDa, while chymotrypsin produced peptides with broad ranges of M, from 1 to 20 kDa.Although high molecular complexes are necessary for stabilization of foam, excessive aggregation may impede the formation of a viscoelastic protein fi lm at the air-water boundary (47).Fig. 3. Foam capacity at diff erent pH values of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively Fig. 4. Foam stability at diff erent pH values of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively

Infl uence of enzymatic treatment on emulsifying properties
Overall, no statistically signifi cant improvement of the emulsifying properties of enzymatically modifi ed sunfl ower protein isolate was achieved compared to the unmodifi ed counterpart (Tables 1 and 2).Chobert et al. (48) suggested that low molecular mass peptides may have too low amphiphilic capacity to exhibit technologically satisfying emulsifying properties.Although small-size peptides are advantageous in the migration and interface absorption, they seem ineffi cient for stabilizing emulsions most probably due to improper unfolding and reorientation at the interface (49).Reductions in emulsifying properties of enzymatic hydrolysates of blends of groundnut fl our and sorghum meal and defatt ed groundnut fl our were reported by Ahmed and Ramanatham (50) and Subba Rau and Srinivasan (51) respectively.Our results agreed with Hu et al. (52), who did not observe any eff ect of TG on emulsifying activity of peanut protein isolate except at pH=4 when TG-modifi ed protein isolate exhibited the highest emulsifying activity (53.8 %, Table 1).

Infl uence of temperature on protein stability
Thermal stability was studied at pH=7 and 8 because of the relatively high solubility of all protein isolates at these pH values (Fig. 2) and their potential practical application in food systems.Reaction pH is an important factor infl uencing protein heat stability since it aff ects protein charge, conformation and sulfh ydryl reactivity upon aggregation (53).Up to 80 °C (pH=7), TG-PH3 exhibited lower increases in turbidity and, therefore, bett er thermal stability than that of protein isolate (Table 3).The thermal stability of TG-PH3 was bett er at 50 and 60 °C than of TG--modifi ed protein isolate.Babiker (39) reported improved thermal resistance of chymotrypsin-digested soy protein up to 60 °C aft er TG treatment.The lower-degree pepsin hydrolyses (0.48 and 0.71 %) followed by TG polymerization (TG-PH1 and TG-PH2) resulted in reduced thermal stability compared to unmodifi ed protein isolate at all studied temperatures.The reason may be the formation of less hydrophilic structures at pH=7 and 8 (Fig. 2), which in general, leads to a decrease in protein stability (54).Similar trends in thermal stability of TG-modifi ed sunfl ow er protein isolates, predigested with pepsin, were also observed at pH=8 (Table 4).
Improvement of heat stability aft er TG modifi cation of the sunfl ower protein isolate alone compared to unmodifi ed protein isolate was observed only at higher thermal treatments (70, 80 and 90 °C) at pH=7 (Table 3).Decreases in turbidity at 90 °C of pigeon pea and hyacinth bean protein isolates aft er TG treatment compared to the native  (53) reported that 10 °C diff erence in heating substantially infl uenced the degree of polymerization as the maximum level could be reached in 10 min at 85 °C (neutral pH), while up to 8 h may be needed for aggregate formation at 70 °C due to slower denaturation and diff usion rates.Most probably, neutral pH additionally contributes to the increased heat resistance of the protein aggregates via formation of disulfi de bonds, which are diminished at the expense of noncovalent associations maintaining the aggregates at higher pH values (58).In our study, at pH=8 no statistically signifi cant diff erences in ther mal stability between TG-modifi ed protein isolate and protein isolate were established (Table 4).Thermal treatment is a common approach in food processing, and knowledge on thermal stability of the protein isolates would facilitate their potential application as food ingredients.

Conclusions
Major challenges in preparation of sunfl ower meal--derived protein products and their application in the food industry are related to protein characteristics and changes due to processing parameters during oil production.This study demonstrated the application of low-degree pepsin hydrolysis combined with transglutaminase (TG) treatment for the improvement of specifi c functional properties of a protein isolate prepared from industrial sunfl ower meal.The results implied that hydrolysis of the protein with pepsin decreased the infl uence of pH on protein solubility at isoelectric point (pI=4.5).If greater solubility at pH<3 and >7 is needed, enzymatic modifi cation of protein isolate should be avoided.Modifi cation of protein isolate with TG could be useful in the improvement of foam making capacity at pH=4 and 6.The eff ect could be enhanced by a preceding pepsin hydrolysis, as evidenced by foam making capacity of TG-PH1, TG-PH2 and TG--PH3, which remained between 52 and 66 % in the entire studied pH range.Improved heat resistance of protein isolate at the lower range of temperatures studied (up to 80 °C, pH=7) could be achieved by pepsin hydrolysis of protein isolate to DH of 1.72 % and consequent TG treatment (TG-PH3), while at the higher temperatures (80 and 90 °C), TG modifi cation alone is suffi cient for improvement of this characteristic.Data obained in this study could be helpful for production of sunfl ower protein isolates with desired functional properties.Mean values in a row with the same lower case lett er do not diff er signifi cantly (p≥0.05).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively

Fig. 1 .
Fig. 1.Flow chart for comparative evaluation of functional properties of sunfl ower meal protein isolate enzymatically modifi ed with pepsin and transglutaminase.DH=degree of hydrolysis PI TG-PH1 TG-PH2 TG-PH3

Table 1 .
Emulsifying activity of sunflower protein isolate (PI) and sunflower protein pepsin digests after treatment with transglutaminase (TG) at different pH values

Table 2 .
Emulsifying stability of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG) at diff erent pH values (57)an values in a row with the same lower case lett er do not diff er signifi cantly (p≥0.05).Mean values in a column with the same capital lett er do not diff er signifi cantly (p≥0.05).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively proteins were observed by Ali et al.(55).According to Siu et al.(56)and O'Sullivan et al.(57), elevated temperatures denature proteins and facilitate TG cross-linking to form aggregates with more compact and heat-stable structures.Ryan et al.

Table 3 .
Thermal stability of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG) at pH=7 Thermal stability was evaluated as the increase of sample turbidity expressed in percentage.Mean values in a row with the same lower case lett er do not diff er signifi cantly (p≥0.05).TG-PI=TG-modifi ed protein isolate, TG-PH1, 2 and 3=TG-modifi ed pepsin hydrolysates with degree of hydrolysis (DH) of 0.48, 0.71 and 1.72 %, respectively *

Table 4 .
Thermal stability of sunfl ower protein isolate (PI) and sunfl ower protein pepsin digests aft er treatment with transglutaminase (TG) at pH=8 *Thermal stability was evaluated as the increase of sample turbidity expressed in percentage.