Sweet potato protein and its hydrolysates

CHAPTER 4

Sweet potato protein and its hydrolysates Tai-Hua Mu1, Miao Zhang1, Lawrence Akinola Arogundade1,2 and Nasir Mehmood Khan1,3 1

Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences; Key Laboratory of Agro-Products Processing, Ministry of Agriculture and Rural Affairs, Beijing, People’s Republic of China Chemistry Department, College of Physical Sciences, Federal University of Agriculture, Abeokuta, Nigeria 3 Department of Biotechnology, Shaheed Benazir Bhutto University, Sheringal, Pakistan 2

Overview of sweet potato protein and its hydrolysates Sweet potato protein The sweet potato ranks as the fifth highest food crop in China, which possesses 80% of the world’s total output. Sweet potato contains approximately 1.73%
9.14% of protein on a dry weight basis (FAOSTAT, 2016; Mu et al., 2009). Sweet potato protein (SPP) is mainly composed of sporamins, and the monomeric forms of sporamins A and B have similar compositions of amino acids, peptide maps, and characteristics (Maeshima et al., 1985). SPP is rich in essential amino acids and exhibits a higher nutritive value compared to most other plant proteins (FAO, 1990), but it is normally discarded as industrial waste in the process of sweet potato starch manufacturing. Therefore it would be meaningful to develop value-added products and/or functional ingredients in the food industry through the effective utilization of SPP. Nowadays the production of SPP from sweet potato starch wastewater is being given due attention in some starch industries in China for economic reasons and environmental concerns.

Sweet potato protein hydrolysates Protein hydrolysates, commonly generated from food proteins by enzymatic hydrolysis, gastrointestinal digestion, and food processing, present certain bioactivities that can be used in health care, such as antioxidant, antimicrobial, antihypertensive, immunostimulatory, antithrombotic, and antidiabetic activities (Valdez-Flores et al., 2016; Agyei et al., 2016). Antioxidant hydrolysates can scavenge free radicals and/or inhibit free Sweet Potato DOI: https://doi.org/10.1016/B978-0-12-813637-9.00004-1

© 2019 Elsevier Inc. All rights reserved.

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Sweet Potato

radical formation, which play an important role in delaying the oxidation of food systems and are essential to human health. The author team prepared sweet potato protein hydrolysates (SPPH) by different enzymes, and found that SPPH generated by enzymatic hydrolysis with Alcalase presented noteworthy antioxidant activities and great protective effects against oxidative DNA damage (Zhang et al., 2012, 2014).

Recovery and composition of sweet potato protein SPP is of a comparable or superior nutritional quality to most vegetable proteins (Mu et al., 2009), but its characteristic discoloration stigma constitutes a major setback in its utilization in the food system. This discoloration problem arises from enzymatic oxidative browning caused by polyphenol oxidase (PPO). PPO is a copper-containing enzyme that catalyzes the hydroxylation of certain phenols, especially mono- and diphenols in the o-position adjacent to an existing
OH group to odiphenols, which further oxidize to o-quinones. These o-quinones condense and react nonenzymatically with amino acids and proteins, resulting in the formation of brown/dark melanin pigments (Severini et al., 2003). These reactions are undesirable in food systems because of their negative effect on food appearance, the development of off-flavors, and losses in nutritional quality (Severini et al., 2003). In this section the use of oxidative browning inhibitors on sweet potato to prevent darkening during SPP recovery is presented for its potential utilization in the food system.

Sweet potato oxidative browning inhibition Oxidative browning inhibition carried out in sodium metabisulfite, sodium bisulfite, ascorbic acid, and citric acid (Fig. 4.1A) showed that the reductions in oxidative browning in sodium metabisulfite, citric acid, sodium bisulfite, and ascorbic acid aqueous media (0.01
0.3 mol/L) were in the ranges of 61%
85%, 78%
85%, 40%
75%, and 76%
80%, respectively. The highest sweet potato oxidative browning inhibition was observed in citric acid solution (0.01
0.1 mol/L). Phenolics in the extracts in the presence of various oxidative browning inhibitors are shown in Fig. 4.1B. The various concentrations of citric and ascorbic acid used decreased the extracted phenolics in comparison to distilled water, while bisulfites increased the phenolics in the extract at 0.01 mol/L concentration, but decreased it thereafter with further increases in the bisulfites concentration. Extracted phenolics in the presence of the different

Sweet potato protein and its hydrolysates

0.7

6

(A) Extractable phenolics (mg GAE/g-dwb)

Browning index (OD 500 nm)

0.6 0.5 0.4 0.3 0.2 0.1 0 0

0.01

0.05

0.1

0.2

Concentration of oxidative browning inhibitor (mol/L)

0.3

71

(B)

5 4 3 2 1 0 –1 0 0.01 0.05 0.1 0.2 0.3 Concentration of oxidative browning inhibitor (mol/L)

Figure 4.1 Sweet potato browning indices (A) and coextractable phenolics (B) in the presence of various browning inhibitors: (K) sodium bisulfite, (x) sodium metabisulfite, (W) ascorbic acid, and (▲) citric acid.

antibrowning inhibitors showed positive correlations with the observed oxidative browning (Fig. 4.1A). This further confirmed the claim of Severini et al. (2003) who reported that the enzymatic oxidative browning is caused by phenolic oxidation.

Sweet potato protein extractability and recovery The extractability profile of SPP in the presence of antibrowning agents is shown in Fig. 4.2A. In bisulfites aqueous media, consistent decreases in solubilized proteins were observed as the concentration of bisulfites increased, while in the presence of citric or ascorbic acid, the initial decrease in solubilized protein was followed by a slight increase as the antibrowning agent concentration increased. The pH trend of sweet potato extracts in the presence of various antibrowning agents at different concentrations (Fig. 4.2B) might account for this protein extractability profile. There was a positive correlation between extractable SPP and the extract’s pH. An increase in the antibrowning agent concentration continuously decreased the extract pH, which merely approached the isoelectric point (pH 4) in the presence of bisulfites, but in the presence of citric and ascorbic acids the extract pH was lowered beyond the isoelectric point, which would possibly be responsible for the observed increase with 0.05
0.3 mol/L citric acid and 0.1
0.3 mol/L ascorbic acid (below isoelectric point). The effectiveness of recovering sweet potato solubilized proteins from the various antibrowning aqueous media using ultrafiltration and isoelectric precipitation techniques are shown in Fig. 4.3A
D. In all cases, SPP

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120

7

(A)

100

5 80 4

pH

Extractable protein (%)

(B)

6

60

3 40 2 20

1

0

0 0

0.01

0.05

0.1

0.2

0.3

0

0.01

0.05

0.1

0.2

0.3

Concentration of oxidative browning inhibitor (mol/L) Concentration of oxidative browning inhibitor (mol/L)

Figure 4.2 Sweet potato extractable protein (A) and pH trend of sweet potato extracts (B) in various antibrowning aqueous media: (K) sodium bisulfite, (x) sodium metabisulfite, (W) ascorbic acid, and (▲) citric acid.

Figure 4.3 Sweet potato solubilized protein recovery by isoelectric precipitation (K) and ultrafiltration (x) techniques from various antibrowning aqueous media: (A) sodium metabisulfite, (B) sodium bisulfite, (C) citric acid, and (D) ascorbic acid.

recovered by ultrafiltration was significantly (P , .05) higher than that by the isoelectric precipitation technique. All the antibrowning agents considered in this study reduced the recoverable protein obtained by either ultrafiltration or isoelectric precipitation technique. This reduction

Sweet potato protein and its hydrolysates

73

in recoverable protein was however more pronounced with the isoelectric precipitation technique. The fractions of the extractable protein in the various extractants that were recovered by isoelectric precipitation from sodium metabisulfite, sodium bisulfite, ascorbic acid, and citric acid aqueous media were in the range of 0%
6.2%, 0%
40.3%, 10.6%
45.7%, and 4.2%
24.2%, respectively. However, with the ultrafiltration processing technique we recovered 89.2%
100%, 94.2%
98.7%, 76.9%
97.1%, and 49.5%
97.2% of the solubilized protein from sodium metabisulfite, sodium bisulfite, ascorbic acid, and citric acid aqueous media, respectively. Even though oxidative browning inhibition was least effective with sodium bisulfite compared with sodium metabisulfite, citric acid, and ascorbic acid, in view of the former’s higher protein extractability with relatively high protein recovery, it remains the best choice for optimum SPP yield by either isoelectric or ultrafiltration processing techniques. Sodium bisulfite (0.01 mol/L) with 40% browning inhibition and 97.9% extractable protein may be considered the most appropriate for SPP processing with a better yield and reduced oxidative browning.

Protein isolates yield, composition, and in vitro digestibility The protein recovery obtained by the membrane processing technique was more than double the recovery obtained by the isoelectric precipitation method (Table 4.1). SPP recovered by either isoelectric or ultrafiltration/diafiltration techniques (16%
51%) was a little lower than the recovery reported for some common legumes like soybean, lupin, and chickpea obtained by similar techniques (Chew et al., 2003; Papalamprou et al., 2009; Shallo et al., 2001). This might be due to SPP’s high solubility in aqueous media, since with our repeated washings and diafiltrations, more protein would have solubilized and washed off. Fewer washings could give a higher yield but the protein purity might be compromised. In addition to this, the possibility of some SPP fractions having their isoelectric point higher than pH 4 might have further affected SPP’s low recovery by isoelectric precipitation. The isoelectrically precipitated sweet potato (IPSP) protein which suffered low recovery syndrome also had the lowest purity (62.9%), while the ultrafiltration/diafiltration-processed sweet potato (UDSP) protein purity was in the range 76.0%
82.1%. The IPSP and UDSP proteins showed different chemical composition (Table 4.1). The Ca content of all the protein preparations was significantly (P , .05) higher than Zn. This might be due to Ca21 preferentially

Table 4.1 Yield and chemical composition of isoelectric and UDSP protein isolates.a Parameters

Yield (%) Purity (%dwbb) Moisture content (%) Ca (g/kg) Zn (g/kg) Total phenolics (mg GAE/g)c Tannin (mg GAE/g) Total flavonoids (mg CE/g)d Phytic acid (mg/100 g) Phytic acid-phosphorus (g/100 g) [Phy]/[Zn]f Trypsin inhibition (TIU/mg of protein)g In vitro digestibility (%)

Isoelectric precipitated protein

Membrane-processed proteins

IPSP

UDSP-4

UDSP-6

UDSP-7

16.0 62.9 6 0.2c 1.71 6 0.11c 2.31 6 0.07a 0.01 6 0.00b 3.76 6 0.03b 1.97 6 0.04b 3.48 6 0.05c 0.66 6 0.00a 0.19a 65.6a 0.034 6 0.003c 70.0 6 3.1c

41.3 76.0 6 0.2b 6.98 6 0.71a 0.29 6 0.01d 0.05 6 0.01a 4.08 6 0.07a 2.87 6 0.06a 2.98 6 0.13d NDe

0.033 6 0.004c 87.7 6 0.8a

41.3 82.0 6 0.5a 3.86 6 0.37b 0.82 6 0.01c 0.05 6 0.00a 2.06 6 0.03d 1.23 6 0.03d 4.04 6 0.10b 0.35 6 0.02b 0.10b 7.0b 0.111 6 0.011b 77.0 6 0.5b

51.3 82.1 6 0.9a 3.66 6 0.51b 1.96 6 0.09b 0.05 6 0.01a 2.87 6 0.04c 1.85 6 0.03c 4.56 6 0.31a 0.36 6 0.01b 0.10b 7.0b 0.143 6 0.012a 74.6 6 3.6bc

Mean values followed by different letter superscripts in the same row are significantly different (P , .05). dwb, dry weight basis. GAE, gallic acid equivalent. d CE, catechin equivalent. e ND, not detected. f Phytic acid (mg)/MW (molecular weight) of phytic acid: Zn (mg)/MW of Zn. g IU, trypsin inhibitor unit. a

b c

Sweet potato protein and its hydrolysates

75

binding to the protein and/or phytate content relative to Zn21. The Ca content was in the order UDSP-4 , UDSP-6 , UDSP-7 , IPSP. The increased Ca content of the UDSP proteins as pH increased might be due to the increased negative charges on the protein as the pH of processing media moves away from the isoelectric point, which in turn increased the Ca electrostatically binding to the proteins. Significantly higher phytic acid in IPSP (0.66 mg/100 g) compared to others (0
0.36 mg/100 g) might be responsible for the higher calcium content of IPSP than the other proteins. In addition, the [phytate]/[Zn] molar ratio of IPSP protein was much higher than that of UDSP (Table 4.1). Since a [phytate]/[Zn] molar ratio of 610 has been associated with good Zn bioavailability, Zn deficiency and its associated complications are not envisaged with UDSP protein formulated foods, while the reverse is the case with IPSP protein. The phenolic contents of SPP protein were in the order UDSP4 . IPSP . UDSP-7 . UDSP-6 (Table 4.1). The relatively high phenolic content of these proteins might be attributed to the oxidative browning inhibitor (0.01 mol/L NaHSO3) used, which was capable of extracting the tuberous root phenolics along with the protein. The variation of phenolic content among these proteins could be due to the various types of interaction they underwent with the protein and the caking problem associated with the ultrafiltration/diafiltration technique, particularly at pH 4 (UDSP-4). Phenolic compounds can undergo hydrogen bonding and electrostatic and hydrophobic interactions with proteins (Mondor et al., 2009). This might possibly be responsible for the high coprecipitation of phenolics by IPSP protein. The ultrafiltration/diafiltration technique produced proteins with significantly lower phenolics at pH 6 and 7 (UDSP-6 and UDSP-7). The severe caking problem associated with the ultrafiltration/diafiltration process at pH 4 might be responsible for UDSP-4 protein having the highest phenolics. Mondor et al. (2009) reported that cake formation during the diafiltration of chickpea protein also resulted in protein with high phenols. The protein deposited on the membrane could have rendered it less porous and thus retained polyphenols particles that would have permeated the membrane. The phenolic content of both IPSP and UDSP proteins was higher than that of chickpea (Mondor et al., 2009). Tannin content also followed the same trend as that observed with the total phenol. Protein
tannin complex formation has been associated with protein digestibility inhibition. This high tannin may be partly responsible for the fairly low SPP digestibility. There is, however, no significant

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Sweet Potato

(P , .05) correlation established between SPP digestibility and tannin, unlike the phytic acid content of these proteins which correlated positively with their in vitro digestibility. The total flavonoid content of these proteins was in the order UDSP-7 . UDSP-6 . IPSP . UDSP-4. The effect of pH treatment was significant (P , .05) on the total flavonoid content of ultrafiltration/diafiltration-processed proteins, that is, total flavonoids increased with pH. In view of the phytochemical antioxidative activities of these bioactives, retaining or eliminating them from protein entirely rests on the consumers’ needs, since their effectiveness in reducing cardio-cerebrovascular diseases and cancer mortality have been well established (Hertog et al., 1997). Phenolic acid and flavonoids have been reported to be the main phytochemicals responsible for the antioxidant capacity of plant foods (Sahreen et al., 2010). According to Sharififar et al. (2009), flavonoid-rich plants could be a good source of antioxidants that would help to increase the overall antioxidant capacity of an organism and protect it against lipid peroxidation; dietary intake of flavonoid-containing foods was therefore suggested to be of benefit. Trypsin inhibitory activities of IPSP and UDSP proteins (Table 4.1) were in the range of 0.03
0.14 TIU/mg. These values were much lower than the values reported for chickpea (18.88
20.03 TIU/mg) and mung bean (6.12 TIU/mg) protein obtained under similar processing conditions (El-Adaway, 2000; Mondor et al., 2009). SPP trypsin inhibitory activity was dependent on the pH rather than the processing techniques, it increased with pH, that is, UDSP4 , IPSP , UDSP-6 , UDSP-7. UDSP proteins had higher pepsin
pancreatin in vitro digestibility than those of IPSP (Table 4.1). This was in the order UDSP-4 . UDSP6 . UDSP-7 . IPSP. This trend might be a reflection of the proteins’ compositions and conformational attributes, since protein digestibility is a function of both the endogenous structural constitution and exogenous factors, like enzymatic inhibition, phytic acid, and polyphenol complexation. SPP digestibility correlated more negatively with its phytic acid content than tannin and trypsin inhibition activity, as noted earlier on.

Amino acid composition and nutritional quality The amino acid content of UDSP proteins (with the exception of glutamic acid, lysine, and arginine) was significantly higher than that of the IPSP protein isolate (P , .05, Table 4.2). This is an indication that UDSP

Table 4.2 Amino acid composition and nutritional quality of isoelectric and UDSP protein.a Amino acid

Alanine Arginine Aspartic acid Cysteinec Glutamic acid Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanined Proline Serine Threonine Tyrosine Valine

Isoelectric protein

FAO/WHO patternb

Membrane-processed protein at different pH

IPSP

UDSP-4

UDSP-6

UDSP-7

32.73 6 0.38c 33.50 6 0.06b 81.60 6 0.82c 14.72 6 0.44d (99.12) 69.88 6 0.70a 30.19 6 0.27c 12.48 6 0.24c (50.63) 32.10 6 0.19d (88.57) 49.09 6 0.38c (57.20) 37.11 6 0.30a (46.24) 13.57 6 0.15c 37.54 6 0.29c (85.70) 24.30 6 0.21d 33.33 6 0.37c 32.65 6 0.30c (71.44) 31.52 6 0.16b 42.14 6 0.28c (90.43)

36.67 6 1.38b 33.97 6 1.32b 112.93 6 5.09b 18.20 6 0.95c (143.4) 66.43 6 2.59b 33.74 6 1.16b 13.53 6 0.55b (71.21) 36.97 6 1.02c (132.04) 52.70 6 1.82b (79.85) 33.78 6 1.26b (58.24) 17.65 6 1.14b 50.06 6 2.44b (144.0) 30.38 6 0.76c 42.38 6 1.72b 42.90 6 1.56b (126.2) 40.64 6 3.78a 50.93 6 1.52b (145.5)

39.96 6 0.69a 37.02 6 0.81a 126.55 6 2.08a 22.21 6 0.11a (164.8) 70.37 6 1.13a 37.17 6 0.69a 14.81 6 0.14a (77.05) 40.65 6 0.80a (143.89) 57.91 6 1.15a (86.91) 36.00 6 0.78a (61.38) 19.68 6 0.37a 55.17 6 1.45a (155.1) 34.79 6 0.77a 47.68 6 0.60a 47.55 6 0.70a (138.6) 43.68 6 1.51a 56.70 6 1.01a (160.4)

40.10 6 0.62a 36.86 6 0.75a 127.60 6 2.22a 20.41 6 0.35b (145.0) 72.69 6 1.15a 37.38 6 0.52a 14.47 6 0.32a (63.89) 38.45 6 0.70b (118.43) 56.59 6 1.03a (72.33) 35.94 6 0.67a (52.21) 18.94 6 0.29a 52.89 6 0.94ab (129.5) 32.94 6 0.43b 48.39 6 0.76a 47.69 6 0.68a (114.4) 42.46 6 0.29a 56.74 6 0.88a (133.3)

2
5 years old

Adult

2.5

1.7

1.9

1.6

2.8

1.3

6.6

1.9

5.8

1.6

6.3

1.9

3.4

0.9

3.5

1.3 (Continued)

Table 4.2 (Continued) Amino acid

Sulfur-containing amino acid Total essential amino acid Total amino acid E/Te (%) PDCAASf (%)

Isoelectric protein

Membrane-processed protein at different pH

IPSP

UDSP-4

UDSP-6

UDSP-7

2.83 6 0.05

3.58 6 0.18

4.19 6 0.03

3.94 6 0.03

30.29 6 0.24

35.64 6 1.29

39.44 6 0.73

38.46 6 0.54

60.85 6 0.47 49.79 44.79

71.25 6 2.44 50.06 51.08

78.79 6 1.35 50.05 47.79

78.05 6 1.11 49.27 45.73

FAO/WHO patternb 2
5 years old

Adult

Mean values followed by different letter superscripts in the same row are significantly different (P , .05). Values within parentheses are the essential amino acid scores calculated as a percentage ratio of each essential amino acid in the various proteins to their respective FAO/WHO (1991) requirement. b FAO/WHO (1991) requirement for respective essential amino acids. c Cystine 1 methionine. d Phenylalanine 1 tyrosine. e Essential (E) and total (T) amino acid ratio. f Protein digestibility-corrected amino acid score. a

Sweet potato protein and its hydrolysates

79

proteins are more nutritionally viable than their IPSP counterparts. With the exception of a few cases mentioned above, the amino acid distributions in the four preparations of SPP were in the order IPSP , UDSP4 # UDSP-7 # UDSP-6. Aspartic acid was significantly higher than all other amino acid constituents in both UDSP and IPSP proteins. This was similar to the report of Purcell et al. (1978) and Mu et al. (2009). Lysine was their limiting amino acid, which is similar to the report of Mu et al. (2009). The sulfur-containing amino acid contents (methionine 1 cysteine) were 2.83, 3.59, 4.19, and 3.94 g/100 g in IPSP, UDSP-4, UDSP-6, and UDSP-7, respectively, suggesting that IPSP and UDSP proteins had significantly (P , .05) higher sulfur-containing amino acid than the requirements of FAO/WHO for preschool children (FAO/ WHO, 1990). This is contrary to the report of Purcell et al. (1978) and Bradbury et al. (1984), who found sulfur-containing amino acids to be the SPP limiting amino acids and lysine to be in abundance. This difference can be attributed to the use of sodium bisulfite as an oxidative browning inhibitor in our SPP preparations. Protein rich in sulfurcontaining amino acids was also reported for 11S-rich hemp protein isolate prepared with low concentration (0.0094 mol/L) of sodium bisulfite (Wang et al., 2008). Mu et al. (2009) also reported that SPP prepared with 1% sodium bisulfite had sulfur-containing amino acids much higher than the FAO/WHO requirements for 2
5-year-old children. This is an added advantage for this tuberous root protein over most leguminous proteins. Deficiency in sulfur-containing amino acids is a common problem with the great majority of leguminous seed proteins. Much lower sulfurcontaining amino acids contents were reported for isoelectric protein isolates from soybean (1.99 g/100 g), chickpea (2.11
2.20 g/100 g), and lupin (1.61 g/100 g), and ultrafiltration/diafiltration-processed lupin had 1.4 g/100 g (Chew et al., 2003; Wang et al., 2010). Dietary protein quality assessment is of great importance since this gives information on the protein’s ability to meet human nutritional needs. In this study, SPP qualities were assessed by considering amino acid score, E/T ratio, and protein digestibility-corrected amino acid scores (PDCAAS), which correlate more directly with human requirements (Table 4.2). The amino acid score showed that SPP from either isoelectric or ultrafiltration/diafiltration preparation contained excess isoleucine, valine, methionine, and cysteine, and sufficient phenylalanine and tyrosine in comparison with the requirements of the FAO/WHO for preschool

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children, but could only supply 58.2%
64.0%, 65.7%
78.0%, and 74.4%
87.7% of their lysine, histidine, and leucine requirements, respectively FAO/WHO (1990). However, IPSP and UDSP proteins can satisfy adults’ nutritional needs conveniently. The amino acid scores of UDSP proteins showed that they can perform better than isoelectric preparation in meeting the nutritional needs of both preschool children and adults. The E/T (%) values for these SPP preparations were well above 36%, which is considered adequate for an ideal protein (FAO/WHO, 1990). The PDCAAS values obtained were in the range 44.89%
50.08% and 162.4%
185.2% for preschool children and adults, respectively, which indicated a low protein quality for children but the reverse for adults (Sarwar and McDonough, 1990). All these indices suggest that SPPs, especially ultrafiltration/diafiltration-processed species, can be utilized as protein sources for human nutrition with little or no supplementation from other protein sources.

Gelation properties of sweet potato protein The common method of producing protein gel is by heating the protein dispersion. On heating, protein molecules are unfolded, resulting in the exposure of the
S
H
,
S
S
, and hydrophobic amino acid side chains. This is followed by the rearrangement and aggregation of functional groups, that is, hydrophobic interaction and
SH/S
S interchange reactions, culminating in gel with a three-dimensional network structure (Liu et al., 2004). A systematic study on SPP gel’s formation mechanism and mechanical and structural attributes is necessary to showcase its gelling potential and utilization as a gelling agent in the food industry. This section therefore introduces flow, viscoelastic, and gelation properties of SPP dispersions as affected by ultrafiltration and isoelectric precipitation methods, as well as the rheological and mechanical properties of the protein gels, along with the mechanism of gelation.

Rheological properties of sweet potato protein dispersions The steady shear properties of both IPSP and UDSP protein dispersions were modeled with Power law and Casson equations. The correlation coefficient (R2) values for Power law equation were between 0.84 and 0.99, while those of the Casson model were in the range 0.72
0.92 (Table 4.3). The degrees of the proteins’ pseudoplastic behavior as depicted by flow index (n) values are shown in Table 4.3. IPSP protein

Table 4.3 Sweet potato proteins' flow properties.a Protein processing condition

Protein concentration (%)

Apparent viscosity, η100 (mPa s)

Consistence coefficient, k (Pa sn)

Flow behavior index, n

Yield stress, τ o (Pa)

Activation energy (J/mol)

Ultrafiltered/diafiltered sweet potato (UDSP) protein

2

1.87 6 0.07e

0.00 6 0.00b

0.81 6 0.03a

0.00 6 0.00b

NA

4 6 8 10 2

3.29 6 0.35d 4.57 6 0.18c 6.68 6 0.29b 10.5 6 0.46a 9.41 6 0.02d

0.04 6 0.00b 0.03 6 0.00b 0.09 6 0.00b 0.27 6 0.07a 1.00 6 0.16b

0.49 6 0.04bc 0.53 6 0.08b 0.40 6 0.01cd 0.28 6 0.05d 0.03 6 0.02a

0.02 6 0.00b 0.02 6 0.01b 0.05 6 0.00b 0.20 6 0.07a 0.92 6 0.07b

NA NA NA NA 9.44 6 1.17b

4 6 8 10

24.45 6 3.18cd 31.25 6 1.06c 66.95 6 4.74b 92.50 6 9.92a

2.89 6 0.48b 5.14 6 1.49b 33.20 6 3.24a 29.65 6 4.93a

2 0.03 6 0.01a 2 0.05 6 0.00a 2 0.29 6 0.03c 2 0.16 6 0.02b

2.74 6 0.56b 4.88 6 1.39b 23.17 6 3.29a 24.45 6 4.54a

9.81 6 1.01b 10.58 6 0.54ab 14.37 6 0.92a 11.34 6 0.40ab

Isoelectric precipitated (IPSP) protein

NA, Not applicable. a Significant differences between means were assessed using the Tukey test and values in a column followed by different letters in each protein preparation are significantly (P # .05) different.

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was significantly more pseudoplastic than UDSP protein. This is an indication that UDSP protein had a better flow tendency than IPSP protein (Marcotte et al., 2001). The mechanical spectra showing the viscoelastic properties of UDSP and IPSP protein dispersions are shown in Fig. 4.4A and B. IPSP protein dispersion exhibited a “solid-like” behavior with the magnitude of its

Figure 4.4 Mechanical spectra of (A) ultrafiltrated and (B) isoelectric protein showing the variation of storage modulus, G (’); loss modulus, Gv (K); and complex viscosity, η (%) with angular frequency.

Sweet potato protein and its hydrolysates

83

storage modulus (G0 ) values consistently higher than the loss modulus (Gv) within the frequency range considered. UDSP protein had G0 values higher than Gv only at a lower angular frequency range with a crossover at 82 s21, signifying the changeover from a “solid-like” nature to a “liquid-like” behavior at higher angular frequency. Such a crossover of some polymeric systems was attributed to the relaxation time and the onset of glassy behavior (Barnes, 2000). At low frequency the stress is applied over a long timescale which allows molecular interaction, but when the frequency exceeds certain values the applied stress is of a short timescale and there is no long-term interaction between molecules, which induces a change of state (Puppo and Añón, 1999). The difference in the mechanical spectra of IPSP and UDSP proteins could be attributed to the difference in their composition and physical structure (Steffe, 1996).

Gelation properties and network formation mechanism of isoelectrically precipitated sweet potato and ultrafiltration/ diafiltration-processed sweet potato proteins The IPSP and UDSP protein concentrations of 20 and 40 g/L, respectively, were the lowest concentrations required to form a gel at pH 7. Considering the least gelling concentration (LGC) as the gelation capacity index, IPSP protein could have a greater gelling capacity than UDSP protein, since proteins with lower LGC have a greater gelling capacity. These sweet potato tuberous root proteins (IPSP and UDSP) have similar gelation capacity to African yam bean protein (Arogundade et al., 2012), but are higher than most leguminous proteins, with LGC in the range 6%
18% (w/v) (Boye et al., 2010). The network development during gelation of IPSP and UDSP protein was determined by dynamic oscillatory measurement (temperature sweep) and the thermomechanical spectra as shown in Fig. 4.5Ai and ii. The gel network development was monitored with G0 , Gv, and tan δ as functions of temperature and time. According to Sun and Arntfield (2010), G0 values are a measure of the elastic component of the gel network structure and represent the strength of the structure contributing to the gel threedimensional network, while Gv is a measure of the viscous component and represents interactions which do not contribute to the threedimensional nature of the gel network. The change in phase angle (tan δ), which relates the viscous nature to the elastic behavior of the sample, predicts the type of network formed with lower tan δ values indicating a better three-dimensional structure.

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100 14000

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90

100

90

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0.16

Tan Δ

Heating

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0.12 Cooling 0.08 20

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80

Figure 4.5 Heat-induced gelation profile of 10% (w/v) sweet potato protein (SPP) dispersion: (A) thermomechanical spectra of (i) ultrafiltrated and (ii) isoelectric SPP showing development of gel structure with time and temperature; G0 , storage modulus; Gv, loss modulus; Tgel, gelation onset temperature; and T, temperature protocol. (B) Phase angle of ultrafiltered (Bi) and isoelectric (Bii) protein during gelation process.

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On heating the UDSP protein dispersion, the initially constant G0 and Gv started to increase at 79.4°C, which signaled the transition from a liquid-like to a solid-like (sol
gel) state (inset of Fig. 4.5Ai). This can be taken as the onset of gelation (Tgel), since one of the common methods of detecting the gelling point in the absence of crossover between G0 and Gv is the temperature at which G0 increases and becomes greater than the background noise (Lamsal et al., 2007). Tgel constitutes the initial stage of denaturation of the protein which exposes the hydrophobic patches as a preparatory stage for gel formation (Sun and Arntfield, 2010). This initial increase of UDSP dispersion G0 became more pronounced as temperature increased to 95°C. Increased thermal treatment might have caused more denaturation which could have promoted the unfolding of the buried hydrophobic residue inside the protein molecules. This favors hydrophobic peptide interactions and the formation of disulfide bonds that reinforce the gel network structure. The gradual development of the reinforced network was reflected by the progressive increase in G0 as the heating temperature increased to 95°C. Heating of the IPSP protein dispersion gave a decreased G0 until about 75°C and thereafter (76.5°C) it started to increase gradually (inset of Fig. 4.5Aii). This represents its Tgel. G0 of IPSP protein further increased with increased temperature, as reported for UDSP protein dispersion, but to a lesser extent. Tgel for UDSP and IPSP protein corresponded to 27 and 25 min of heat treatment, respectively. Cooling further enhances the network structure of both proteins as indicated by the steady increase in G0 for both proteins. This may be attributed to the consolidation of attractive forces like van der Waals and hydrogen bonding between proteins on cooling (Lamsal et al., 2007). According to Speroni et al. (2009), the stability of hydrogen bonds increases with a decrease in temperature. At the completion of the heating and cooling circle, the G0 value for UDSP protein gel was almost twice that of IPSP. This could be attributed to the difference in response of the protein's tertiary conformation to thermal treatment (Yin et al., 2009). The progress in gel network development was also monitored through the phase angle (tan δ) as the thermal treatment progressed (Fig. 4.5B). The phase angle, which is a better indicator of the viscoelasticity of biopolymer gel, confirmed the increased gel network elasticity as the temperature increased to or was maintained at 95°C with a decreased tan δ. This also suggested that more cross-linked networks were formed in both proteins as heating progressed. Tan δ of the UDSP dispersion did not however decrease initially until a temperature of 75°C was

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reached, indicating that the rapid reaction leading to gelation started at this temperature (75°C).

Ultrafiltration/diafiltration-processed sweet potato and isoelectrically precipitated sweet potato protein gel dynamic rheological properties The mechanical spectra of 10% (w/v) IPSP and UDSP protein gels showed typical gel structure with G0 values of both proteins maintaining consistently higher values than the loss modulus Gv within the frequency range tested (Fig. 4.6A and B). The larger difference between the G0 and Gv values of the UDSP protein gel than that of IPSP suggested that a higher percentage of the stored energy was recovered at each shearing cycle for UDSP protein than for the IPSP protein. The plots of G0 and Gv as a function of frequency do provide information on the gel structure, and help in determining whether the gel formed from entanglement networks, covalently cross-linked materials, or physical gels (Doucet et al., 2001). Entanglement networks are usually characterized with G0 Bω2 and GvBω1 at low frequency and a crossover between G0 and Gv at high frequency; covalently cross-linked gels are frequency independent; while physical gels are slightly frequency dependent (Doucet et al., 2001; Kavanagh and Ross-Murphy, 1998). The dependence of G0 on frequency can be obtained from the logarithmic plot of G0 5 K0 (ω)z0 (log G0 5 z0 log ω 1 K) as proposed by Egelandsdal et al. (1986), with physically and covalently linked gel having z0 . 0 and z0 5 0, respectively. The slopes (z0 ) from a logarithmic plot of G0 versus a logarithm of ω for UDSP and IPSP protein gels showed that both gels had G0 that was dependent on ω, but to a minimal extent with the low z0 values of 0.11 and 0.13 for UDSP and IPSP protein gels, respectively, signifying a covalent bond gel structure with some tendency for physical interaction (Doucet et al., 2001). Since the strong dependence of G0 on frequency indicates that there is no specific interaction between molecules (weak gel) while G0 independent of frequency is an indication of a strong gel network (Doublier, 1992), IPSP and UDSP gels formed from 10% (w/v) dispersion were therefore a gel structure which fell within strong and weak gels. K0 values showed the level of molecular interaction of the gel matrix, with higher K depicting higher interaction (Kim and Yoo, 2009). Therefore there was higher molecular interaction in UDSP than IPSP protein gel.

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Figure 4.6 Dynamic viscoelastic properties sweet potato protein gels made from 10% dispersion: (A) mechanical spectra of ultrafiltered protein gel showing variation of storage modulus, G0 (’); loss modulus, Gv (K); and phase angle, tan δ (▲) with angular frequency. (B) Mechanical spectra of isoelectric protein gel showing variation of storage modulus, G0 (’); loss modulus, Gv (K); and phase angle tan δ (▲) with angular frequency.

Mechanical properties of isoelectrically precipitated sweet potato and ultrafiltration/diafiltration-processed sweet potato protein gels The mechanical parameters derived from this texture profile analysis are shown in Fig. 4.7. Even though UDSP gel had a more highly covalently

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Mechanical parameter value

4.5 a∗

4 3.5 3 2.5 2

b∗

1.5 1

b

a a

0.5

a b

a

0 Hardness

Springiness

Cohesiveness

Resilence

Parameters

Figure 4.7 Texture profile analysis parameters of ultrafiltered ( ) and isoelectric ( ) sweet potato protein gels. Significant differences between means were assessed using the Tukey test and values of the histogram under each parameter with different letter labels are significantly (P # .05) different.  Values of hardness are 3 102.

linked
S
S
structure than the IPSP gel, the force needed to attain the given deformation (hardness) in the IPSP protein gel was higher than that of UDSP. The higher hardness observed with IPSP protein gel might be attributed to its higher carbohydrate or nonprotein content than the UDSP protein gel. According to Lamsal et al. (2007), the carbohydrate content of protein might cause a higher hardness due to interactions between proteins and carbohydrate particles. Springiness, which represents the rate at which deformed material goes back to its original height following removal of the applied force, was also higher with the IPSP gel than that of UDSP, but their cohesivenesses were not significantly different.

Isoelectrically precipitated sweet potato and ultrafiltration/ diafiltration-processed sweet potato protein gels microstructure Scanning electron microscopy revealed the clear differences in the network structure of UDSP and IPSP protein gels (Fig. 4.8). The dark areas represent the pores in the gel network. UDSP gel had a dense, spherical particulate appearance, while IPSP gel had a regular and repeating finestranded gel network structure with small voids distributed throughout the matrix. IPSP protein gel showed a more cross-linked particle network

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Figure 4.8 Scanning electron micrographs of ultrafiltered (A) and isoelectric (B) protein gels.

than that of UDSP which had an amorphous matrix appearance. The heavy cross-linking observed with IPSP gel might possibly reinforce the IPSP gel network structure, which in turn may account for the higher mechanical properties observed with IPSP protein gel compared to that of UDSP protein gel.

Emulsifying properties of sweet potato protein Proteins are amphiphilic molecules that can be used as emulsifiers to stabilize emulsions (Damodaran, 1996). SPP has good emulsifying activity but low stability (Mu et al., 2009). The maximum interfacial protein concentration reported for SPP was 1.81 mg/m2 with 2% (w/v) protein concentration; however, it has a poor stability compared to legume protein (Guo and Mu, 2010). High hydrostatic pressure (HHP) is an effective tool to destroy the microorganisms in foods; compared to treatments such as pasteurization and sterilization. HHP has a less severe effect on the stability and the gelation properties of protein emulsions (Anton et al., 2001). Hence, the appropriate structure modification of an SPP emulsion by HHPs might lead to enhanced functionality, which could increase the applications of SPP emulsions in the food industry. This section therefore introduces the effects of HHP treatment on the stability, viscosity, interfacial protein concentration, and droplet sizes of SPP emulsions (1% w/v) subjected to different HHPs and at different pH values (3, 7, and 8) for a better understanding of SPP as a functional agent in the food industries.

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Emulsifying activity index and emulsifying stability index The emulsifying activity index (EAI) and emulsifying stability index (ESI) of SPP emulsions subjected to the different HHP levels are shown in Fig. 4.9A and B. Compared to the control (0.1 MPa) emulsions, the

Figure 4.9 (A) Emulsifying activity index (EAI) and (B) emulsifying stability index (ESI) of control and high-pressure-treated SPP emulsions.

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Table 4.4 Effect of high hydrostatic pressure (HHP) treatment on the different parameter of emulsions prepared at pH 3, 7, and 8. Sample

HHP (MPa)

D4,3

D3,2

Г (mg/m2)

pH 3

0.1 200 400 600 0.1 200 400 600 0.1 200 400 600

8.27 6 0.08a 5.68 6 0.08b 3.42 6 0.01c 3.42 6 0.05c 7.10 6 0.04a 3.45 6 0.01b 3.41 6 0.0bc 3.40 6 0.02c 6.20 6 0.05a 3.39 6 0.05b 3.44 6 0.06b 3.43 6 0.03b

3.92 6 0.06a 3.17 6 0.04b 2.61 6 0.01c 2.61 6 0.03c 3.57 6 0.02a 2.68 6 0.01b 2.63 6 0.00c 2.62 6 0.01c 3.45 6 0.04a 2.59 6 0.00c 2.67 6 0.00b 2.66 6 0.02b

0.85 6 0.07c 0.86 6 0.04c 1.13 6 0.03b 1.67 6 0.03a 1.49 6 0.14b 1.51 6 0.02b 2.19 6 0.15a 2.43 6 0.09a 1.96 6 0.01c 2.02 6 0.01c 2.62 6 0.01b 3.82 6 0.03a

pH 7

pH 8

D4,3 (μm), volume-weighted means diameter; D3,2 (μm), volume-surface mean diameter; Г (mg/m2), interfacial protein concentration.

HHP treatments resulted in an EAI increase in the SPP emulsions at the pH values 3, 7, and 8. When the pressure was increased to 200 MPa, the emulsions had higher EAI values at pH 3 and 8 than that at pH 7, whereas higher-pressure treatments (400 and 600 MPa) decreased the EAI values (Fig. 4.9A). In contrast, at 600 MPa the ESI values were higher at all the pH values considered. The droplet size is important for the stability of emulsions; the smallest average size of the oil droplets for all the treated emulsions was obtained at 600 MPa (Table 4.4). The results suggest that the emulsifying properties of the SPP emulsions could be improved by the HHP treatment.

Droplet size distribution The emulsions had a monomodal droplet size distribution at the different pH values (Fig. 4.10). However, the HHPs affect the droplet size distribution with an increasing volume frequency of the smaller droplet size compared to the control (0.1 MPa) emulsions. In the presence of a deflocculating agent [e.g., slowly digestible starch (SDS)], the maximum volume frequency percentages obtained at 600 MPa for the droplets were approximately 3.42, 3.40, and 3.43 mm at pH values 3, 7, and 8, respectively (Fig. 4.10). Also in the presence of this deflocculating agent, HHPtreated (200
600 MPa) SPP stabilized emulsion had decreased droplet sizes compared to those of the control (0.1 MPa) at pH 3, 7, and 8. This

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8 6

0.1 MPa 200 MPa 400 MPa 600 MPa

4 2 0 0.1

1

10 100 Particle diameter (μm)

1000

Figure 4.10 Effects of HHP on droplet size distribution (volume frequency) of SPP emulsion in pH 3 with SDS (A) and without SDS (B), 7 with SDS (C) and without SDS (D), and 8 with SDS (E) and without SDS (F) conditions in presence and absence of deflocculating agent (SDS).

corroborates the earlier observed increase in EAI of HHP-treated (200
600 MPa) SPP in comparison with those of 0.1 MPa at the various pHs. Since the higher the EAI values or the lower the emulsion droplet sizes then the higher the emulsifying properties of the protein, HHPtreated SPP gave better emulsifying properties than that of 0.1 MPa (native protein). The protein adsorbed to the interfacial film after the HHP treatment might have a sufficiently high zeta potential value, which most likely induced an effective electrostatic repulsion between the droplets. A similar behavior was observed by Le Denmat et al. (2000) in yolk emulsions at pH 3. The results suggest that a higher HHP treatment at pH 3 may enhance the stability of the emulsion. At pH 3, 7, and 8,

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HHP-treated SPP emulsions showed the same droplet size distribution with or without SDS except at 200 MPa (pH 3) and 0.1 MPa (pH 7 and 8), where an increase in emulsion droplet sizes was observed in the presence of SDS. Indeed, SDS, being a stronger surface active agent than vegetable protein, should displace the protein from the oil
water interface and thereby break down flocs (Demetriades and McClements, 2000), but the contrary was the case with SPP stabilized emulsion at 200 MPa (pH 3) and 0.1 MPa (pH 7 and 8). This could be due to formation of a mixed surfactant
polymer layer at the interface, with a diminished emulsifying capacity as compared to the pure polymer or SDS layers.

Emulsion microstructure The native SPP (0.1 MPa) emulsions at pH 3, 7, and 8 had fairly homogeneous structures (Fig. 4.11), which were in accordance with the results obtained in our previous study (Guo and Mu, 2010). The droplet sizes of SPP stabilized emulsion were larger in the continuous phase with the 0.1 MPa treatment than other HHP treatments, especially with application of 600 MPa (pH 3 and 8). Based on the microphotography, the oil

Figure 4.11 Microphotographs of freshly HHP-treated emulsions (15 min treatment). Images of control emulsions were made after 15 min of emulsion production. The length of a black bar is 20 mm.

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droplets were evenly distributed in the continuous phase, no coalescence was observed during the HHP treatments. With the exception of droplet size reduction, no change in the emulsion microstructure was detected with HHP treatment. In the oil
water emulsions, a decrease in the droplet size can affect the rheology and the shear thinning of the emulsion by increasing the apparent viscosity (Pal, 1996). HHP treatment enhanced the formation of smaller oil droplets at pH 3, which was most likely the cause of the rheological modifications and the increase in viscosity. Using microscopy we observed similar and important microstructural changes at pH 7 and 8 as the pressure was increased from 0.1 to 600 MPa (Fig. 4.11). Hydrophobic and electrostatic interactions of macromolecules (including proteins) are disrupted with high-pressure treatment. Such high-pressure-induced protein conformational changes have been associated with modifications of the food functional properties (Messens et al., 1997). In addition, the HHP-treated emulsion (600 MPa) at pH 3 showed a well-defined interfacial film around the oil droplets, whereas the pH 7 and 8 emulsions showed a microseparation of the phases, which was most likely a result of protein aggregation in the aqueous phase of the emulsion. The microphotography of the HHP-treated emulsions at each pH value also confirmed the reduction in the droplet sizes (D4,3 and D3,2; Table 4.4).

Interfacial protein concentration and composition The interfacial protein concentration (Г in mg/m2) of the HHP-treated emulsions is shown in Table 4.4. The Г for the HHP-treated emulsions at pH 3, 7, and 8 significantly increased (P , .05). The highest G values were obtained when the emulsions were treated with 600 MPa. The protein concentration at the oil
water interface was the critical factor in the stability of the emulsions because proteins can lower the interfacial tension at the oil
water interface of the droplets. This reduction in tension facilitates the formation of smaller droplets and increases their stability against coalescence by covering the droplets with a thin layer of protective coating (McClements, 2004). HHPs unfold protein structures by exposing the hydrophobic groups of the protein (Dickinson and James, 1998). Therefore one would expect that an increase in the surface hydrophobicity would be able to provide a better absorption potential at the oil
water interface. This hypothesis is consistent with the reduction in the droplet size (Table 4.4) and the increase in the Г as the pressure was

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increased from 200 to 600 MPa. Our previous study reported a saturated interfacial film at 1.81 mg/m2 of SPP, which was attributed to a closer packing of the adsorbed proteins in the monomolecular layer (Guo and Mu, 2010). However, the increase of the Г values by the HHP treatment might be a result of the protein in the aqueous phase, which is associated and/or aggregated to the previously formed monomolecular layer. This aggregation of the aqueous phase protein might affect HHP-induced unfolding, contributing to the formation of a secondary film where there are interactions between the proteins of the interfacial film and the aqueous phase. Our results agree with those of Puppo et al. (2011) who studied soybean protein emulsions. It has been reported that the net charge of the adsorbed protein layer is highly dependent on the pH (Dalgleish, 1997). At pH 3, proteins are positively charged, and this positive charge density decreases as the pH increases. In the HHP-treated emulsions, the interaction between SPP and the oil was stronger in the pH 3 emulsions, which was most likely due to a higher emulsion stability and lower value than for those in the pH 7 and 8 emulsions. The state of the adsorbed and the nonadsorbed proteins at the interface was analyzed by SDS-PAGE under nonreducing and reducing conditions (Fig. 4.12). Fig. 4.12A and B shows the electrophoretic profiles of the proteins belonging to the pH 3 SPP emulsion subjected to 200
600 MPa. Both sporamin A (31 kDa) and sporamin B (22 kDa), depending on the pressure level, were adsorbed to the interfacial layer after the HHP treatments (Fig. 4.12A). As the pressure was increased to 600 MPa, the sporamins were clearly visible on the SDS-PAGE (Fig. 4.12A, lane 5), which might be attributed to an increase in the interfacial protein concentration (Table 4.4). A similar pattern for the pH 7 emulsions was observed in the SDS-PAGE under nonreducing conditions (Fig. 4.12C). However, an interfacial layer, which was composed of higher molecular weight (MW) aggregates, was also observed in the pH 7 emulsions; this interfacial layer increased with increasing pressure. These results reveal that the HHP treatment contributed to the emulsion protein aggregation via disulfide bond formation. Upon the addition of the reducing agent (i.e., 2-mercaptoethanol), the higher MW aggregates, which were stabilized by the disulfide bonds, disappeared from the separating gel (Fig. 4.12D). The HHP treatment of the emulsions did not change and/ or displace the main electrophoretic bands of SPP. Furthermore, these electrophoretic bands were similar to those obtained in our previous study using SDS-PAGE (Guo and Mu, 2010).

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(A)

(B) 97.0 kDa 66.0 kDa

97.0 kDa 66.0 kDa 45.0 kDa Sporamin A 30.0 kDa

45.0 kDa 30.0 kDa

21.0 kDa 14.4 kDa STD 1

Sporamin B 2

3

4 5 6

7

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21.0 kDa

Sporamin

14.4 kDa STD 1

2

3

4

5

6 7

8

9

STD 1 2

3 4

5

6 7

8

9

Figure 4.12 SDS-PAGE profile of proteins adsorbed and nonadsorbed at the oil
water interface derived from pH 3 and 7 emulsions. (A) and (C) are in nonreducing (without 2-mercaptoethanol), (B) and (D) in reducing conditions (with 2-mercaptoethanol) for pH 3 and 7, respectively. In each of the figures (A
D) STD, marker standard; lane 1, native SPP; lane 2, control adsorbed SPP; lane 3, 200 MPa adsorbed SPP; lane 4, 400 MPa adsorbed SPP; lane 5, 600 MPa adsorbed SPP; lane 6, control nonadsorbed SPP; lane 7, 200 MPa nonadsorbed SPP; lane 8, 400 MPa nonadsorbed SPP; lane 9, 600 MPa nonadsorbed SPP.

Using SDS-PAGE we observed that an aggregation could affect the appearance of the adsorbed sporamin A and B proteins at pH 3 and 7 (Fig. 4.12A and C, lanes 2
5). The appearance of sporamin A and B at pH 3 could be attributed to no and/or little aggregation; the disappearance of these proteins on the gels might be a result of protein hydrolysis in the acidic conditions (Fig. 4.12A). The SDS-PAGE results clearly demonstrate that the interfacial film is a result of protein aggregations and reveal the interaction between the adsorbed protein at the oil
water interface and the aggregated protein in the aqueous phase of the emulsion formed by the HHP treatment, which increased the interfacial protein concentration as shown in Table 4.4. Under reducing conditions, all the emulsions (at the different pH values) had a similar sporamin band (25 kDa), indicating that sporamin was well adsorbed to the interfacial

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layer (Fig. 4.12B and D). These results suggest that, with the exception of the disulfide bonds, the HHP treatment of the SPP emulsions did not lead to other structural changes that could alter their electrophoretic mobility.

Rheological properties Rheological behavior The control (0.1 MPa) and the HHP-treated emulsions (pH 3) were modeled using the Herschel
Bulkley model (Table 4.5), where τ c . 0 and n , 1. The pH 7 and 8 emulsions could be described using the power law model, where τ c 5 0 and n6¼1. In the case of the pH 7 emulsions, a shear-thinning behavior was observed in the control (0.1 MPa) and the HHP-treated emulsions, where n , 1 in all the pressure treatments. However, the 200 and 400 MPa-treated emulsions at pH 8 had a Newtonian behavior with a power law model (τ c 5 0 and n6¼1); the control (0.1 MPa) and the 600 MPa-treated emulsions showed a shearthinning behavior, with τ c 5 0 and n , 1. The shear rate versus the shear
strain curves of the pH 8 emulsions for the control (0.1 MPa) and the 600 MPa-treated emulsions explained the shear-thinning behavior of the emulsions (data not shown). For oil
water emulsions, a decrease in the droplet size can affect the rheological behavior (Pal, 1996). However, the Newtonian behavior of the pH 8 emulsions subjected to 200 and 400 MPa reveals that a reduction in the droplet sizes by the HHP treatment could not be the only factor that affected the rheological behaviors; the droplet size reduction might also be attributed to the properties of the stabilizing molecules. The factors that might affect the rheological behavior of the emulsions are mainly the viscosity of the aqueous phase, the interfacial film formed at the interface, the nature of the emulsifying agent, the volume frequency distribution of the droplets, the flocculated droplet network, and the electroviscous effect (Sherman, 1995). Flow index The flow indices (n) of each emulsion with respect to HHP treatment are shown in Table 4.5. The n values showed that the SPP emulsion had pseudoplastic flow behavior. The variability associated with these n values at pH 3 and 7 was high, this could be due to the higher flocculating tendency of the emulsion at pH 3 and 7 than at pH 8 on shearing. Pseudoplastic flow behavior is the most common type of nonideal behavior exhibited by food emulsions. It manifests itself as a decrease in the

Table 4.5 Modeling of the flow curves between 10 and 600 s21 of shear stress of control (0.1 MPa) and HHP-treated (200
600 MPa) emulsions prepared with SPP at pH 3, 7, and 8.

pH 3

pH 7

pH 8

Herschel
Bulkley factors

0.1 MPa

200 MPa

400 MPa

600 MPa

k (mPa sn) τ c (mPa) N R2 η (mPa s) 125 s21 k (mPa sn) τc (mPa) N R2 η (mPa s) 125 s21 k (mPa sn) τ c (mPa) N R2 η (mPa s) 125 s21

5.35 6 0.01 71 6 0.14 0.89 6 1.79 0.9998 6 0.0009 2.8 6 0.006 1.9 6 0.003 060 0.97 6 1.94 0.9992 6 0.0003 1.9 6 0.006 1.5 6 0.02 060 0.99 6 0.032 0.9993 6 0.0006 1.83 6 0.001

3.05 6 0.006 34 6 0.07 0.93 6 1.86 0.9991 6 0.0004 2.5 6 0.005 2.05 6 0.004 060 0.98 6 0.00 0.9991 6 0.0008 2.3 6 0.007 1.7 6 0.001 060 1.02 6 0.042 0.9960 6 0.0024 1.8 6 0.005

3.95 6 0.007 39.5 6 0.080 0.90 6 1.80 0.9992 6 0.0003 2.8 6 0.006 2.45 6 0.004 060 0.93 6 1.96 0.9998 6 0.0001 2.36 6 0.007 2.9 6 0.003 060 1.01 6 0.048 0.9982 6 0.0015 1.93 6 0.008

7.35 6 0.010 66 6 0.130 0.81 6 1.600 0.9993 6 0.0002 4.1 6 0.008 5.55 6 0.010 060 0.81 6 1.63 0.9998 6 0.002 3.3 6 0.009 4.4 6 0.00 060 0.87 6 0.064 0.9975 6 0.0016 2.56 6 0.005

k, viscosity coefficient (mPa sn); τ c, yield value (mPa); N, flow index; R2, regression coefficient of sample fitting plot; η (mPa s), viscosity at shear rate of 125 s21.

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apparent viscosity of a fluid as the shear rate increases and is referred to as shear-thinning. Pseudoplasticity might occur in food emulsions because of the spatial distribution of the particles, that is, nonspherical particles might align with the flow field, and/or flocs might be deformed and disrupted (McClements, 2005). The flow index is important when evaluating the emulsion viscosity, which mainly depends on the structure of the oil droplets and their attraction and aggregation under the application of stress. Viscosity The viscosity of the control (0.1 MPa) and the HHP-treated emulsions were considered at a 125 s21 shear rate (Table 4.5). The control (0.1 MPa) emulsion at pH 3 was initially more viscous than the emulsions at pH 7 and 8. The flocculation of the oil droplets in the emulsions played a key role in their viscosity. Anton et al. (2001) observed a higher flocculation and viscosity in emulsions treated with a 600 MPa-HHP treatment and prepared with hen egg yolk at pH 7 than at pH 3. Furthermore, pH 3 emulsions with low viscosity have been shown to have Newtonian behavior after HHP treatment (Anton et al., 2001), while our present study indicated that the emulsions stabilized by SPP had non-Newtonian behaviors before and after the HHP treatments in the acidic medium (Table 4.5). The increase in viscosity at pH 3 was observed at 600 MPa, which might be a result of a reduction in the flow index. In addition, the viscosity coefficient k (mPa sn), which is an indicator of the viscous behavior, was higher for the control (0.1 MPa) and the pressuretreated pH 3 emulsions (Table 4.5). The pH 7 and 8 emulsions had an increased viscosity at the 600 MPa HHP. At these pH values the protein had an almost zero or insignificant zeta potential value, which reduced the electrostatic repulsion potential and favored the droplet
droplet attraction. These results were similar to those obtained by Dickinson and James (1998, 1999), who reported that the high-pressure treatment of emulsions stabilized by β-lactoglobulin or bovine serum albumin induced a flocculation among the oil droplets leading to an increase in the viscosity. In addition to flocculation, the increase in viscosity at pH 7 and 8 might be attributed to the formation of a strong network of the aggregated proteins during the HHP treatments (Fig. 4.12C). Furthermore, the increased viscosity at 600 MPa had a positive correlation with the flow index (Table 4.5). A low flow index value at 600 MPa correlated with high viscosity in all cases, which indicates the non-Newtonian character of the emulsions. The results suggest that most of the rheological

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behavior modification took place using the 600 MPa-HHP treatment of the SPP emulsions.

Preparation and antioxidant activity of sweet potato protein hydrolysates Diverse hydrolysates/peptides, which can be obtained by enzymatic hydrolysis via commercial protease, gastrointestinal digestion, and food processing, have displayed potential health beneficial, one of the most researched being antioxidant activity (Singh et al., 2014; MazorraManzano et al., 2017). Antioxidant peptides can act as free radical scavengers, transition metal ion chelating agents, and lipid peroxidation inhibitors; they can protect cells from damage by reactive oxygen species and also show good nutritional and functional properties (Erdmann et al., 2008; Xie et al., 2008). HHP technology was designed to help improve enzymatic hydrolysis and digestibility of protein, and enhance the bioactivities of protein hydrolysates. HHP could also change the protein conformations, enable protein molecular chain extension, which is beneficial for enzymatic reactions due to the exposed new restriction sites (Bonomi et al., 2003), and might also produce some novel peptides with special physiological function. The author team investigated the effect of HHP on the degree of hydrolysis (DH), antioxidant capacity, and MW distribution of SPPH by Alcalase, separated and subsequently identified the antioxidant peptides from SPPH, and explored their structure
activity dependences (Zhang and Mu, 2017), which are introduced here.

Degree of hydrolysis and antioxidant activity of sweet potato protein hydrolysates DHs of SPPH by Alcalase under HHP were significantly different (P , .05, Table 4.6). DHs of SPPH by Alcalase under 0.1 MPa (atmospheric pressure) for 30 and 60 min were 20.83% and 23.17%, respectively. With the increase of pressure (100
300 MPa) and hydrolysis time (30
60 min), DHs of SPPH increased significantly, reaching the highest value of 31.68% under 300 MPa for 60 min. The improvement of enzymatic hydrolysis under HHP is supposed to be the result of the exposure of new cleavage sites through protein unfolding, and the enhancement of

Table 4.6 Degree of hydrolysis (DH), antioxidant activity, and ,3 kDa fractions percentage of sweet potato protein hydrolysates (SPPH) by Alcalase under high hydrostatic pressure (HHP). HHP (MPa)

Time (min)

DH (%)

 OH scavenging activity (%)

Fe21-chelating ability (%)

ORAC (μg TE/mL)

Percentage of ,3 kDa fractions (%)

0.1

30 60 30 60 30 60 30 60

20.83 6 0.10g 23.17 6 0.00e 22.65 6 0.00f 24.56 6 0.00d 23.34 6 0.20e 25.26 6 0.20c 25.78 6 0.20d 31.68 6 0.20a

41.91 6 1.32e 43.06 6 0.69e 41.45 6 1.47e 51.57 6 0.61c 45.82 6 1.41d 56.21 6 0.69a 50.08 6 0.82c 54.93 6 1.15a

91.87 6 1.01c 91.68 6 0.83c 89.38 6 0.18d 95.91 6 0.20a 92.19 6 0.40c 96.16 6 0.09a 93.47 6 0.52b 94.24 6 0.39b

123.93 6 2.67a 114.65 6 4.86bc 111.64 6 6.14bc 117.50 6 3.71ab 113.86 6 9.67bc 106.84 6 2.22c 113.96 6 2.20bc 116.67 6 4.32ab

28.50h 41.89f 36.56g 51.18c 41.98e 62.13b 50.89d 67.66a

100 200 300

 OH scavenging activity, Fe21-chelating ability, and oxygen radical absorbance capacity (ORAC) were tested at 1.0 mg/mL; values followed by different letters in the same column are significantly different (P , .05).

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enzyme activity and/or substrate
enzyme interaction (Belloque et al., 2007; Dufour et al., 1995). Antioxidant activities of SPPH were determined using  OH scavenging activity, Fe21-chelating ability, and oxygen radical absorbance capacity (ORAC) (Table 4.6). SPPH by Alcalase under HHP (100
300 MPa) had significantly higher  OH scavenging activity and Fe21-chelating ability than those under 0.1 MPa for 30 and 60 min (P , .05). SPPH by Alcalase under 200 and 300 MPa for 60 min showed the highest  OH scavenging activities, which were 56.21% and 54.93%, respectively, while SPPH by Alcalase under 100 and 200 MPa for 60 min showed the highest Fe21chelating abilities, which were 95.91% and 96.16%, respectively, with no significant difference between them (P..05). In addition, SPPH by Alcalase under 0.1 MPa for 30 min showed the highest ORAC value (123.93 μg of TE, P , .05), which was not significantly different to the SPPH under 100 MPa (117.50 μg TE/mL) and 300 MPa (116.67 μg TE/ mL) for 60 min (P..05).

Molecular weight distribution of sweet potato protein hydrolysates MW distribution profiles of SPPH by Alcalase under HHP were shown in Fig. 4.13. Effluent less than 3 kDa was eluted after 29.49 min based on the standard curve (shown as a dotted line). MW distribution was classified into two groups .3 and ,3 kDa, and their proportions regarding the total distribution were calculated based on the peak area. Obviously high performance liquid chromatography (HPLC) profiles of SPPH by Alcalase under 0.1 MPa and different high pressures (100
300 MPa) were different from each other, showing differences in MW distribution. Compared with SPPH by Alcalase under 0.1 MPa, SPPH by Alcalase under 100
300 MPa exhibited much smaller .3 kDa peaks, of which the ,3 kDa fractions (after 29.49 min) were prominent for both 30 and 60 min, respectively (Fig. 4.13A and B). The higher the pressure (100
300 MPa) and the longer the hydrolysis time (30
60 min), the greater amount of ,3 kDa peptides fraction were exhibited in SPPH (Fig. 4.13 and Table 4.6). Peptides exhibited in ,3 kDa fractions were significantly increased from 28.50% and 41.89% under 0.1 MPa to 50.89% and 67.66% under 300 MPa for 30 and 60 min, respectively (Table 4.6, P , .05). It was reported that chickpea hydrolysates by Alcalase under HHP at 200 MPa for 20 min showed high antioxidant activity, had the largest amount of ,3 kDa peptides fraction, and were mainly ranged

Sweet potato protein and its hydrolysates

103

(A) 1900 3 kDa Hydrolysis time 30 min

A215nm

1500 1100

0.1 MPa

100 MPa

200 MPa

300 MPa

700 300 –100 0

10

20

30 40 Retention time (min)

50

60

70

(B) 1900 3 kDa

A215nm

1500

Hydrolysis time 60 min 0.1 MPa

100 MPa

200 MPa

300 MPa

1100 700 300 –100 0

10

20

30 40 Retention time (min)

50

60

70

Figure 4.13 The molecular weight (MW) distribution profiles of sweet potato protein hydrolysates (SPPH) by Alcalase under high hydrostatic pressure (HHP) using HPLC. (A) Hydrolysis time of 30 min; (B) hydrolysis time of 60 min.

from 500 to 1000 Da (Zhang et al., 2012). In addition, enzymatic hydrolysis at 300 MPa induced an absolute degradation of lentil proteins and improved the concentration of the ,3 kDa peptides with all the enzymes used (Garcia-Mora et al., 2015).

Antioxidant activity of peptides by ultrafiltration Low MW peptide fractions were mainly the contributors to antioxidant activity of hydrolysates from protein (Garcia-Mora et al., 2015). Thus SPPH produced by Alcalase under 300 MPa for 60 min was selected for further characterization with respect to its higher DH, antioxidant activities, and ,3 kDa peptides proportion. SPPH was filtered via ultrafiltration to generate peptides with MW .10 kDa (FI), 3
10 kDa (FII), and ,3 kDa (FIII), respectively. Compared with .10 and 3
10 kDa peptide fractions, the antioxidant

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activity of peptide fractions with lower MW (,3 kDa) was significantly stronger, which was in line with our result previously (Zhang et al., 2014). The  OH scavenging activities of different fractions were 13.44%, 43.76%, and 60.50%, and the Fe21-chelating abilities were 10.77%, 77.49%, and 97.58%, while the ORAC values were 58.07, 66.06, and 93.54 μg TE/mL for FI, FII, and FIII, respectively. It was evident that the fraction with the lowest MW (FIII) showed higher antioxidant activities than the other higher MW fractions (P , .05). High antioxidant activities of low MW fractions were considered to be due to the easier reaction with and more effective elimination of free radicals (Ranathunga et al., 2006).

Separation and identification of peptides The FIII fraction was chromatographically fractionated into 54 fractions by semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC) (Fig. 4.14 and Table 4.7). The low hydrophobicity peptides with low MW were eluted earlier, while high hydrophobicity peptides with high MW were eluted later (Wattanasiritham et al., 2016). ORAC values of fractions 1
54 ranged between 30.97 and 141.18 μg TE/mL. Among the 54 fractions, fraction 18 with an intermediate hydrophobicity exhibited the highest ORAC value (P , .05), which was 141.18 μg TE/mL, followed by those of fractions 31
34, 42, and 46 (119.23
127.80 μg TE/mL, Table 4.7). Based on their retention times, these fractions presented hydrophobic characteristics. It was also interesting that fractions 31 and 46 were dominant and showed high ORAC

Figure 4.14 RP-HPLC chromatography profile of active fraction FIII obtained by ultrafiltration from sweet potato protein hydrolysates (SPPH) at 300 MPa for 60 min.

Table 4.7 Oxygen radical absorbance capacity (ORAC) of RP-HPLC peaks from fraction FIII. Fraction no.

Time (min)

ORAC (μg TE/mL)

Fraction no.

Time (min)

ORAC (μg TE/mL)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

4.9
5.7 5.8
6.1 6.2
6.7 7.0
7.3 7.4
7.6 7.7
8.0 8.1
8.8 8.9
9.3 9.4
9.8 9.9
10.2 10.3
10.5 10.6
11.5 11.9
12.6 13.0
13.9 14.0
14.8 14.9
15.8 15.9
16.3 16.4
16.9 17.9
18.6 18.7
19.2 19.3
19.9 20.0
20.6 20.7
21.5 21.6
22.2 22.3
23.0 23.1
23.6 23.7
24.2

90.07 6 5.37qp 43.74 6 1.53v 45.27 6 0.11v 68.75 6 7.38tu 64.07 6 1.13u 30.97 6 1.24w 75.22 6 1.87rst 94.61 6 8.29nop 78.33 6 3.84rs 75.10 6 0.54rst 97.24 6 8.75mnop 114.83 6 4.26defg 111.93 6 6.36efgh 99.34 6 4.41lmno 101.99 6 5.26klmno 123.83.43 6 0.89bc 127.46 6 1.01b 141.18 6 10.84a 109.01 6 5.49fghijk 117.10 6 2.91cdef 109.16 6 3.68fghijk 79.34 6 6.38rs 71.44 6 4.64stu 100.13 6 1.42lmno 94.54 6 0.31nop 113.13 6 1.40efg 101.44 6 5.86klmno

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54

24.3
25.0 25.1
25.8 25.9
26.5 26.6
27.5 27.6
28.4 28.5
29.1 29.2
29.6 29.7
30.4 30.6
31.9 32.0
33.0 33.5
34.0 34.1
34.4 34.5
34.9 35.4
35.9 36.0
36.4 36.5
36.9 37.0
37.9 38.0
38.7 38.8
40.1 41.1
42.3 42.5
43.4 43.5
44.5 45.3
46.2 46.3
47.1 47.2
48.5 51.1
52.9 53.0
54.6

100.66 6 1.67klmno 88.70 6 1.63qp 103.10 6 1.19ijklmn 125.46 6 1.71bc 127.80 6 0.79b 124.51 6 2.01bc 123.90 6 3.07bc 106.85 6 1.61ghijkl 103.70 6 1.81hijklm 112.46 6 1.15efg 90.62 6 3.13qp 108.93 6 1.77fghijk 111.64 6 0.21efghi 118.31 6 1.20cde 122.59 6 2.20bcd 114.20 6 3.89defg 112.60 6 2.30efg 117.53 6 9.20cdef 119.23 6 2.13bcde 110.93 6 0.25efghij 114.25 6 0.43defg 102.43 6 0.45jklmn 77.39 6 15.05rst 63.82 6 0.53u 112.52 6 10.44efg 93.34 6 0.46op 83.43 6 10.39qr

Each peak (4.0 mL) was lyophilized and mixed with 200 μL of 75 mM phosphate buffer (pH 7.4) for the ORAC assay. Values followed by different letters in the same column are significantly different (P , .05).

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values of 125.46 and 119.23 μg TE/mL, respectively. Thus fractions 18, 31, and 46 were subsequently subjected to LC
MS/MS for peptide sequencing. As shown in Table 4.8, fractions 18, 31, and 46 were found to contain diverse peptides from sporamin A and B, with MW ranging from 600 to 2100 Da (5
19 amino acids). It was important to note that all the identified peptides belonged to sporamin A or B, and some of them belonged to both sporamins A and B. Sporamin, in its nonreduced form, exhibited two subunits of sporamins A and B, which had 219 and 216 amino acids according to the database from NCBI, respectively. In the case of fractions 18 and 31, the numbers of peptides from sporamin A were much less than those from sporamin B. While in the case of fraction 46, the numbers of peptides from sporamin A were similar to those from sporamin B. Orsini Delgado et al. (2016) found that diverse peptides were identified from gastrointestinal digestion of amaranth proteins, of which the MW ranged from 800 to 1700 Da with 7
15 amino acids, and the peptides mainly matched the sequence of 11S globulin, the subunit of amaranth proteins.

Synthesis of potential antioxidant peptides and conformation prediction Previously, researchers have evaluated the structure
antioxidant activity dependence of peptides. Hydrophobic amino acids (His, Tyr, or Pro) are present in the sequences of antioxidant peptides from soy β-conglycinin digests, and Leu and Val are located at the N-terminal domains (Chen et al., 1995). The His-His was necessary for the antioxidant activity of Leu-Leu-Pro-His-His and the Leu deletion of the N-terminal domains exhibited no significant effect on the activity, while the His deletion at the C-terminal domains decreased activity (Chen et al., 1996). One peptide with sequences of Ala-Thr-Ser-His-His from sandfish protein hydrolysates showed high antioxidant activity, which was due to the presence of His residues (Jang et al., 2016). Hernández-Ledesma et al. (2005) indicated that Trp, Tyr, and Met exhibited the strongest ORAC values followed by Cys, His, and Phe, while the others showed no detectable activity. The high antioxidant activities of Trp, Tyr, and Phe are based on their capability to donate hydrogen: Met can be oxidized to Met sulfoxide; Cys contributes sulfur to the hydrogen; His with the imidazole group has a proton-donating ability. Hernández-Ledesma et al. (2005) found that the conformation of peptides resulted in synergistic or

Table 4.8 Peptides sequences from selected fractions identified by LC
MS/MS. Fraction no.

Peptides

MW (Da)

Matched sequence in sporaminsa

18

GDEVRA

645.31

GDEVRAGE RLDSSSNE NIATNK

831.37 906.40 659.36

HDSASGQY HDSESGQY IKPTDM SDVIVS DVIVSPN ASDVIVSR SDVIVS PESTVVMPSTF PADPESTVVMPS RFNIATNK

863.34 921.34 719.35 618.32 742.38 845.46 618.32 1209.56 1244.56 962.53

KAGEFVSD

851.40

KAGEFVSDN

965.44

VVNDNL NDNLNAY SETPVLDINGDEVRAGENY YYMVSA EVRAGGNYYMVS RLAHLDTM RFNIA

672.34 822.35 2076.96 732.32 1344.61 955.49 619.34

RFNIATNK

962.53

f48
53 f46
51 f46
53 f73
80 f124
129 f121
126 f141
148 f138
145 f211
216 f84
89 f85
91 f82
89 f83
88 f105
115 f102
113 f122
129 f119
126 f151
158 f148
155 f151
159 f148
156 f164
169 f166
172 f37
55 f57
62 f50
61 f72
79 f122
126 f119
123 f122
129 f119
126

31

46

Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin

ORAC (μg TE/mL)b

A B B B A B A B B A A B B B B A B A B A B B B B A A A A B A B

123.06 6 13.13a 109.24 6 3.49b 68.47 6 2.94e

36.94 6 3.36fg 5.11 6 1.07h NA

95.74 6 9.69c 81.69 6 3.90d 117.30 6 6.02ab 30.54 6 4.55g NA

(Continued)

Table 4.8 (Continued) Fraction no.

a

Peptides

MW (Da)

Matched sequence in sporaminsa

NVNWGIKH NVNWGIQH NVNWGIQHD HDSESGQYF DSESGQYFVK HDSASGQYFLK HDSASGQYFLKAG SGQYFLK KAGEFVSDNSNQF

966.50 966.47 1081.49 1068.42 1158.52 1251.59 1379.65 841.43 1441.64

LKAGEFVSDNSNQFKIE RFHDPM RFHDPMLR RFHDPMLRT RFHDPMLRTT FVIKPT

1924.95 801.36 1086.54 1187.58 1288.63 703.43

YYIVS RSDFDNGDPITITPADPE DNGDPITITPA DNGDPITITPADPE NGDPITITPA NGDPITITPADPE NGDPITITPADPEST PITITPADPE VVNDNLNAYKIS RYYDPL RYYDPLTR

643.32 1958.88 1112.53 1453.66 997.51 1338.62 1526.71 1052.54 1348.70 825.40 1082.55

f131
138 f134
141 f134
142 f138
146 f139
148 f141
151 f141 2 153 f145
151 f151
163 f148
160 f150
166 f192
197 f192
199 f192
200 f192
201 f212
217 f209
214 f55
59 f89
106 f93
103 f93
106 f94
103 f94
106 f94
108 f97
106 f164
175 f189
194 f189
196

From National Center for Biotechnology Information (NCBI). ORAC of the synthesized peptides was tested at 0.2 mg/mL.

b

Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin Sporamin

ORAC (μg TE/mL)b

B A A B B A A A A B A A A A A A B B B B B B B B B B B B

98.87 6 0.92c 98.05 6 4.68c 76.31 6 2.85de NA 40.04 6 2.87f

4.46 6 0.94h 111.90 6 7.88b NA

113.97 6 7.10b

Sweet potato protein and its hydrolysates

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antagonist effects on their antioxidant activity compared to that of the amino acid itself. In addition, Li and Li (2013) detailed the structure
antioxidant activity dependence of peptides, showing the relationship between the antioxidant potentiality of peptides and the physicochemical characteristics ofthe C-terminal and N-terminal domains. In the case of ORAC, the importance of the positions were in the order C3 . C4 . C1 . N1. The bulky hydrophobic and polar/charged amino acids at the C-terminal domains contributed to ORAC, and those with minimal electronic property at the N-terminal domains (N1, N2) were also essential for it (Li and Li, 2013). Based on previous reports mentioned above, 20 peptides with potential antioxidant amino acids were selected and synthesized to verify their activity with an ORAC assay (Table 4.8, in bold). Peptides KAGEFVSD from fraction 31 and RFNIA, KAGEFVSDNSNQF, and RSDFDNGDPITITPADPE from fraction 46 did not present relevant activity when carried out at 0.2 mg/mL. Peptides RFNIATNK from fraction 31 and FVIKPT from fraction 46 had relatively low activity, while the other 14 peptides showed certain activity with ORAC ranging from 30.54 to 123.06 μg TE/mL (Table 4.8). Peptide HDSASGQY from fraction 18 presented the highest ORAC value, followed by peptides YYMVSA (fraction 46), RYYDPL (fraction 46), YYIVS (fraction 46), and HDSESGQY (fraction 18) (Table 4.8 and Fig. 4.14). The five peptides contained 5
8 amino acids, and had a MW range from 640 to 920 Da (Table 4.8), which was in line with the statement that short peptides with approximately 2
9 amino acids showed stronger antioxidant activity than larger polypeptides (Jeong et al., 2010), and further confirmed that most of the antioxidant peptides from food had an MW range from 500 to 1800 Da (Samaranayaka and Li-Chan, 2011). In addition, it was indicated that the stronger ORAC of short peptides might be due in part to the increased possibility to interact with and/or donate electrons to free radicals (Onuh et al., 2014). Peptide HDSASGQY showed His (positively charged, antioxidant) at the N1 position, Gly (hydrophobic) at the C3 region, and Tyr (bulky aromatic, antioxidant) at the C1 domain. However, its related peptide HDSASGQYFLK from fraction 46, containing the additional sequence of FLK at the C-terminal domain, presented much lower antioxidant potency than peptide HDSASGQY. Peptide HDSESGQY showed a little lower antioxidant activity than HDSASGQY, which might be because of the small difference at the N4 position. Peptide HDSESGQY presented

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Glu (negatively charged), while HDSASGQY had Ala (hydrophobic) in the N4 position. And it was interesting that its related peptide HDSESGQYF from fraction 46, containing the additional segment F at the C-terminal domain, also presented much lower antioxidant potency than peptide HDSESGQY. Peptide YYMVSA presented two Tyr (bulky aromatic, antioxidant) at the N1, N2 domain, and a Met (bulky hydrophobic, antioxidant) at the C4 region. Similarly, peptide YYIVS also contained two Tyr at the N1, N2 domain, and an Ile (bulky hydrophobic) at the C3 region. In addition, peptide RYYDPL presented an Arg (bulky positively charged) at the N1 domain, and two Tyr in the N2, N3 position. Obviously, His and Tyr were important to the antioxidant potency of the peptides, both in the N-terminal and C-terminal domains. And in the case of Tyr, the position inside the peptide sequence also contributed to its antioxidant activity. Due to the more active peptides being identified with 5
8 amino acids, some tridimensional conformations could be adopted to influence their activity. Conformations of peptides HDSASGQY (and HDSASGQYFLK for comparison), YYMVSA, RYYDPL, YYIVS, and HDSESGQY were carried out by PEPFOLD3 (Fig. 4.15). Peptide

Figure 4.15 Structures obtained by PEPFOLD3 for antioxidant peptides from SPPH: (A) HDSASGQY; (B) HDSASGQYFLK; (C) HDSESGQY; (D) YYMVSA; (E) YYIVS; (F) RYYDPL.

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HDSASGQY presented the most compact structure with four turns, exposing His and Tyr residues to the external medium (Fig. 4.15A). Compared with the overlapped peptide HDSASGQYFLK, His and Tyr residues were set out in the medium as well as a bulky Leu residue (Fig. 4.15B). Peptide HDSESGQY, presenting only one different amino acid from peptide HDSASGQY in the sequence, showed only three turns, with His and Tyr residues set out in the medium (Fig. 4.15C). Peptides YYMVSA, YYIVS, and RYYDPL showed only one turn (Fig. 4.15D
F). Peptide YYMVSA presented two Tyr with aromatic rings and one Met exposed to the external medium; peptide YYIVS also exposed two aromatic rings of Tyr to the external medium and a bulky hydrophobic residue Ile; while in peptide RYYDPL, a bulky positively charged Arg was located outside of the structure, with the two aromatic rings of Tyr revealed in the external medium (Fig. 4.15D, E, and F, respectively). Orsini Delgado et al. (2016) indicated that amaranth antioxidant peptides contained more than one bulky aromatic residue, and concluded that peptide AWEEREQGSR with the highest antioxidant activity presented the most compact structure with five folded turns, exposing Trp and Arg to the external medium.

Research and development trends of sweet potato protein and its hydrolysates Research on protein and its hydrolysates from different food resources has attracted widespread attention around the world. The following further studies are still necessary: (1) carrying out pilot and industrial production demonstrations of processing technology of SPP and its hydrolysates, in order to mitigate the environmental pollution and the waste of resources caused by waste liquid discharge from sweet potato starch processing, and to provide sufficient raw materials for the food industry; (2) developing the application of SPP and its hydrolysates in snack foods, in order to make full use of its gelation properties, emulsifying properties, antioxidant activity, etc.; and (3) developing the application of SPP and its hydrolysates in staple foods, in order to take full advantage of their nutritional value. In any case, sweet potatoes are a potential source of high-quality proteins and hydrolysates/ peptides, and it is necessary to conduct continuous and in-depth studies on them.

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