Reducing, Radical Scavenging, and Chelation Properties of Fermented Soy Protein Meal Hydrolysate by Lactobacillus plantarum LP6

Abstract

Fermented soybean protein meal hydrolysate (FSPMH) was fractionated by gel filtration on Sephadex G-15. The fractions obtained were subjected to various antioxidant assays, amino acid and molecular weight determinations. Among the seven fractions, the highest antioxidant activity was found in fraction F2, with significant differences (P < 0.01) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, hydroxyl radicals (^.OH) scavenging and Cu²⁺ chelating activity. Fraction F2 exhibited scavenging of DPPH (59.43%), ^.OH (72.80%) and 44.47% Cu²⁺ chelating activity. All other fractions showed variable activities in different assays. Amino acid analyses of F2 fraction with the strongest antioxidant activity also had the highest percentage of related antioxidative amino acids content (Histidine 3.46, Serine 5.78, Valine 4.08 and Lysine 11.49 g/100 g protein) compared with other six fractions. The molecular weight distribution of F2 was found to vary from 170 to 1500 Da.

Keywords:

• Lactobacillus plantarum Lp6
• Fermented soybean protein meal hydrolysate
• Antioxidant activity
• Hydrophobicity
• Molecular weight distribution

INTRODUCTION

In the past few years, there has been increasing interest in research on antioxidants such as phenolic compounds and antioxidant peptides, since they can protect the human body from free radicals and retard the progress of many chronic diseases.[1,2] There is a wide range of oxygen-free radicals and other reactive oxygen species (ROS), which include free radicals such as superoxide anion radicals (O₂ ^.-), hydroxyl radicals (HO^.) and non free-radical species such as hydrogen peroxide (H₂O₂) and singlet oxygen ( ^| O₂) which may form in the human body and in the food system. These radicals induce not only lipid peroxidation that causes deterioration of foods, but also cause oxidative damage by oxidizing biomolecules leading to cell death and tissue damage, such as atherosclerosis, cancer, emphysema, cirrhosis and arthritis[3]; because of increasing demand for natural antioxidant substances, several investigations aimed at assessing the antioxidant potential of biologically active peptides from protein hydrolysates, such as soy protein,[4–6] whey protein,[7,8] zein protein,[9,10] wheat protein,[11,12] porcine myofibrillar protein[13] have been conducted. The antioxidant properties of these hydrolysates have been ascribed to the cooperative effect of a number of properties, including their ability to scavenge free radicals, to act as metal–ion chelator, oxygen quencher or hydrogen donor, and to the possibility of preventing the penetration of lipid oxidation initiators by forming a membrane around oil droplets.[14,15]

Bioactive peptides can be released by the microbial activity of fermented food or through enzymes derived from microorganism.[16] Zhu et al.[17] reported significant findings with 2,2´-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activities, reducing power, and higher peptide of Meitauza kojis obtained through fermentation. Studies have confirmed the degradation of soybean allergens during fermentation by microbial proteolytic enzymes in soy sauce, miso, soybean ingredients, and feed-grade soybean meals.[18–20] Fermentation has the capacity to improve nutritional and functional properties compared to original products. Frias et al.[21] shows that soybean flour fermented with Lactobacillus sp. (L. plantarum) was able to further break down and use available proteins as nutrient sources.

The objective of this study was to investigate the antioxidant potential of different fermented soy protein meal hydrolysate (FSPMH) fractions by determining its reducing power, metal chelating and free radical (DPPH^. and OH^.) scavenging activities of the different fractions. Furthermore, amino acid composition and molecular weight (MW) distribution were also evaluated to determine their relationship with the antioxidation properties.

MATERIALS AND METHODS

Materials

Soybean protein meal ((SPM) commercial protein isolate) was obtained from Sun-Green Biotech Co. Ltd (Nantong, China). Trinitrobenzenesulfonic acid (TNBS), L-leucine, 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and protease were purchased from Sigma-Aldrich, Inc (St. Louis. MO); the strain Lactobacillus plantarum Lp6 was obtained from the culture collection of Jiangnan University (Wuxi, china). All other chemicals were of analytical grade.

Fermentation

The microorganism Lactobacillus plantarum Lp6 used was stored at 4^oC before its use and then cultured for 18 h at 37°C in Man-Rogosa-Shape (MRS) broth [Shensi Biotech Co. Ltd (Shanghai, China)] prior to use for fermentation. A 0.025 mL (1 × 10⁷ colony forming units cfu/g) of strain L. plantarum Lp6 was prepared in sterilized distilled water and then mixed with 25 g of soybean protein meal fortified with soluble starch (0.4 g/g SPM) and protease (0.01 g/g SPM) in polyethylene bag (140 mm x 200 mm) and vacuum sealed. Also 2 mg of disodium phosphate (Na₂HPO_4.12H₂O) was added to improve the activity of L. plantarum Lp6, and then solid-state fermentation was performed at 37°C for 72 h.

Preparation of Fermented Soy Protein Meal Hydrolysate (FSPMH) and Determination of its Degree of Hydrolysis

FSMPH was prepared according to the method described by Ye et al.[22] Five grams of fermented soy protein meal were mixed with 50 mL of distilled water, homogenized for 1 min and incubated at 37°C for 60 min. The incubated mixture was centrifuged at 9600 rpm for 2 min and the residue was washed with 20 mL distilled water, centrifuged again at the same speed and time and the supernatants combined. The supernatants containing peptides was poured out and degree of hydrolysis (DH) was determined using 0.004 mL TNBS in water as described by Adler-Nissen[23] and Spellman et al.[24] Aliquots (0.25 mL) of test or standard solutions were added to test tubes containing 2.0 mL of sodium phosphate buffer (0.2125 M, pH 8.2). TNBS reagent (2.0 mL) was then added to each tube, followed by mixing and incubation at 50°C for 30 min in a covered water bath (in order to prevent light). The reaction was stopped by the addition of 0.1 M Na₂SO₃ (2 mL) to each tube. Samples were then allowed to cool at room temperature for 30 min and absorbance was measured at 420 nm using a spectrophotometer (model 2.4, 2002 UNICO, WFZ UV−2102 Shanghai, China). L-Leucine (0–2.5 mM) was used to generate a standard curve. DH values were calculated using the following equation:

(1)

where h_tot is the total number of peptide bonds per protein equivalent; and h is the number of hydrolyzed bonds. h_tot = 7.8 mM/g for soybean protein.[25]

Gel Filtration on Sephadex G-15 of FSPMH

FSPMH (20 mg) was subsequently loaded onto Sephadex G-15 gel filtration column (45 cm × 2 cm, Amersham Pharmacia Biotech AB, Sweden) pre-equilibrated with phosphate buffered saline (pH 7.4). Sample was eluted with the same buffer at a flow-rate of 0.5 mL/min and eluted fractions (5.0 mL) were pooled after spectrophotometric measurement at 220 nm.

Reducing Power

The reducing power of FSPMH was measured according to Wu et al.[26] The sample (0, 1, 5, and 10 mg/mL) was added to 2 mL of 0.2 M phosphate buffer (pH 6.6) and 2 mL of 1% (w/v) potassium ferricyanide. The mixture was incubated at 50°C for 20 min, and then, 2 mL of 10% (w/v) trichloroacetic acid (TCA) was added. The mixture was centrifuged for 10 min at 3000 g, and 2 mL of the supernatant was mixed with 2 mL of distilled water and 0.4 mL of 0.1% (w/v) FeCl₃. After reaction for 10 min, the absorbance of the solution was read at 700 nm. Increase in the absorbance of the reaction mixture indicated increased reducing power.

DPPH Radical-Scavenging Activity

The scavenging effect of FSPMH fractions on 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical was measured according to the method of Shimada et al.[27] with little modification. Two milliliters of each sample solution (5.0 mg/mL) were added to 2 mL of 0.1 mM DPPH dissolved in 95% ethanol. The mixture was shaken and left for 30 min at room temperature, and the absorbance of resulting solution was read at 517 nm. A lower absorbance represents a higher DPPH scavenging activity. The scavenging effect was expressed as shown in the following equation:

(2)

Hydroxyl Radical-Scavenging Activity

The hydroxyl radical-scavenging assay was carried out using the method described by de Avellar et al.[28] with some modifications. Both 1,10-phenanthroline (0.75 mM) and FeSO₄ (0.75 mM) were dissolved in phosphate buffer (pH 7.4) and mixed thoroughly. H₂O₂ (0.01%) and FSPMH fractions (5.0 mg/mL) were added. The mixture was incubated at 37°C for 60 min, and the absorbance was measured at 536 nm. Results were determined using the following equation:

(3)

where A_S, absorbance of the sample; A₁, absorbance of control solution containing 1,10-phenanthroline, FeSO₄ and H₂O₂; and A₀, absorbance of blank solution containing 1,10-phenanthroline and FeSO₄.

Metal Chelating Activity

The ability of FSPMH fractions to chelate prooxidative Cu²⁺ was investigated according to the procedure of Wang and Xiong[4] with slight modifications. In the chelation test, 1 mL of 2 mM CuSO₄ was mixed with 1 mL of pyridine (pH 7.0) and 20 L of 0.1% pyrocatechol violet. After the addition of 1 mL of samples (10 mg/mL), the disappearance of the blue color, due to dissociation of Cu²⁺, was recorded by measuring the absorbance at 632 nm at 5 min of the reaction. The Cu²⁺ chelating activity of the FSPMH samples was calculated as:

(4)

Amino Acid Composition

The lyophilized hydrolysate fractions were digested with HCl (6 M) at 110°C for 24 h under nitrogen atmosphere. Reversed phase high performance liquid chromatography (RP-HPLC) analysis was carried out in an Agilent 1100 (Agilent Technologies, Palo Alto, CA, USA) assembly system after precolumn derivatization with o-phthaldialdehyde (OPA). Each sample (1 μL) was injected on a Zorbax 80 A C18 column (i.d. 4.6 × 180 mm, Agilent Technologies, Palo Alto, CA, USA) at 40°C with detection at 338 nm. Mobile phase A was 7.35 mmol/L sodium acetate/triethylamine/tetrahydrofuran (500:0.12:2.5, v/v/v), adjusted to pH 7.2 with acetic acid, while mobile phase B (pH 7.2) was 7.35 mmol/L sodium acetate/methanol/acetonitrile (1:2:2, v/v/v). The amino acid composition was expressed as g of amino acid per 100 g of protein.

Determination of Molecular Weight Distribution

The FSPMH fraction with the strongest antioxidant and free radical-scavenging activities was analyzed for MW distribution using a Waters TM 600E Advanced Protein Purification System (Waters Corporation, Milford, MA, USA). The hydrolysates were loaded onto TSK gel G2000 SWXL column (i.d. 7.8 × 300 mm, Tosoh, Tokyo, Japan), eluted with 45% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid at a flow rate of 0.5 ml/min and monitored at 220 nm. A molecular weight calibration curve was obtained from the following standards from Sigma: cytochrome C (12,500 Da), aprotinin (6500 Da), bacitracin (1450 Da), tetrapeptide GGYR (451 Da), and tripeptide GGG (189 Da). Results were processed using Millennium 32 Version 3.05 software (Waters Corporation, Milford, MA 01757, USA). 2.12.

Statistical Analysis

All experiments were conducted at least in triplicate. Analysis of variance (ANOVA) was performed and differences in mean values were evaluated by Tukey's test at P < 0.05 or P < 0.01 using SPSS version 13.0 (SPSS, Chicago, IL, USA).

RESULTS AND DISCUSSION

FSPMH Fractionation

The soybean protein meal contained 77.83% protein and equation (1) exhibited 22.3% degree of hydrolysis. Gel filtration column chromatography (Sephadex G-15) was used to fractionate FSPMH into seven fractions F1, F2, F3, F4, F5, F6, and F7 as shown in the Fig. 1. Each fraction was freeze-dried and stored at −20°C until further use.

Figure 1 Elution profile of FSPMH separated by gel filtration on Sephadex G-15.

Reducing Power

Studies showed earlier that antioxidant activity and reducing power were directly related.[29,30] The results of this research showed that the reducing power of the F2 fraction was the highest, followed by F1, F4, F3, F5, F7, and F6, respectively (Fig. 2). The presence of reducers (i.e., antioxidants) causes the reduction of the Fe³⁺/ferricyanide complex to the ferrous form.[31] The reducing power of FSPMH fractions increased with increasing concentrations. A similar observation has been reported by Li et al.[32] on chickpea protein hydrolysate (CPH).

Figure 2 Reducing power of FSPMH fractions used at different concentrations. Values are means ± standard deviation of three determinations.

DPPH Radical-Scavenging Activity

The estimation of DPPH radical scavenging activity was used for rapid antioxidant evaluation of the different fractions of FSMPH. The scavenging effects of different fractions on the DPPH radical are illustrated in Table 1. The different scavenging patterns for DPPH and hydroxyl radicals were likely related to the structure of peptides eluted at different stages of fractionation.[10] DPPH• is an oil-soluble free radical that becomes a stable product after accepting an electron or a hydrogen atom from an antioxidant. These antioxidants donate hydrogen to free radicals, leading to non-toxic species and therefore to inhibition of the propagation phase of lipid oxidation.[33] The results (Table 1) revealed that F2 at 5.0 mg/mL exhibited a significant (P < 0.01) DPPH radical-scavenging activity (59.43%), which was higher than that of alcalase-treated zein hydrolysate.[10] The other fractions also showed good DPPH radical-scavenging activities in the order of F3 > F4 > F1, whereas F5, F6 and F7 reacted poorly to this assay. The FSPMH fractions possibly contained some substrates, which were electron donors and could react with free radicals to convert them to more stable products and terminate the radical chain reaction.

Table 1 Antioxidant activity of fractionated FSPMH

Fractions	DPPH scavenging activity^a (%)	Hydroxyl-scavenging activity^a (%)	Cu^2+ chelating activity^b (%)
F1	33.10 ± 3.56b	59.77 ± 2.70cd	30.75 ± 0.66b
F2	59.43 ± 1.07c	72.80 ± 0.40e	44.47 ± 1.11d
F3	55.87 ± 1.07c	65.13 ± 2.68d	42.21 ± 1.02cd
F4	53.38 ± 0.71c	58.24 ± 0.39c	41.22 ± 0.03c
F5	21.53 ± 2.67a	44.06 ± 0.77b	29.64 ± 0.44b
F6	17.44 ± 0.36a	31.80 ± 1.53a	20.35 ± 0.89a
F7	21.00 ± 1.78a	38.70 ± 1.54b	21.24 ± 1.33a
^aScavenging activities were tested at 5 mg/ml; and ^bchelating activities were tested at 10 mg/ml. Values are means ± standard deviation of three determinations. Rows with different letters indicate statistical differences (P < 0.01).

Hydroxyl Radical-Scavenging Activity

Free radicals such as hydroxyl radical are generated from sequential reduction of oxygen during the normal course of aerobic metabolism. Overabundant radicals cause oxidative stress, which can lead to cell injury and tissue damage.[34] When a hydroxyl radical reacts with aromatic compounds, it can add on across a double bond, resulting in hydroxycyclohexadienyl radical. The values of the hydroxyl radical-scavenging abilities in Table 1 showed significant differences (P < 0.01) among the fractions at a dosage of 5 mg/mL. However, F2 exhibited the highest value (72.80%) for hydroxyl radical-scavenging activity compare to the others fractions (F1, F3, F4, F5, F6, and F7) with various values at the same concentrations (59.77, 65.13, 58.24, 44.06, 31.80, and 38.70%), respectively. A similar findings has been reported by Moure et al.[35] on soy protein fractions. The fraction (F2) can be a potential source of natural antioxidant. In addition, the incorporation of protein hydrolysate to foods could confer desirable nutritional and functional properties.[36,37]

Metal Chelating Activity

The F2 fraction exhibited significant (P < 0.01) Cu²⁺ chelating activity of 44.47% (Table 1) which was in accordance with Zhu et al.[10] findings on zein hydrolysate at a concentration of 10 mg/mL. It was reported that in metallothionein, which excludes surrounding water, would allow segments of the thiol protein to bind more Cu²⁺ than a structure that was lost.[38] It could be possible that fermentation with Lactobacillus plantarum Lp6 disrupted the spatial structure of FSPMH peptides conducive to binding and trapping of Cu²⁺, resulting in reduced chelation capability. The finding that metal chelating activity of soy protein meal enhanced after fermentation was in consistence with findings obtained by Moktan et al.[39] where the chelating ability of soybean was affected by bacillus fermentation to kinema. Peptides reported to stabilize prooxidative transitional metal ions include carnosine,[40,41] potato peptides,[4] and peptides rich in Histidine.[42]

Amino Acid Composition

The amino acid composition of these fractions revealed that they are rich in Glutamic acid, Aspartic acid, Tyrosine, Valine, Phenylalanine, Lysine, Leucine, Alanine and Proline.[5] Table 2 shows that the fraction F2 had the highest amount in Histidine (3.46), Serine (5.78), Valine (4.08), and Lysine (11.49 g/100 g protein), most of which reportedly have relation to antioxidant properties either in their free form or as residues in proteins and peptides;[8,13,43]. This probably accounted for the highest antioxidative activity exhibited by fraction F2 even though F2 did not exhibit the highest contents of total hydrophobic amino acids (THAA) and hydrophobic indices (ΔΦ) calculated according to Adler-Nissen.[25] The variations in the total hydrophobic amino acid (THAA) are shown in Table 2, where it can be seen that there was significantly different among the fractions (P < 0.05). As regards hydrophobic index (ΔΦ), the fractions also differed from each other (P < 0.05). Although the differences were significant in the case of hydrophobicity.[44,45] However fraction F2 still showed higher antioxidant properties than the other fractions (Table 1). Furthermore, our previous investigations showed significant increase in total free amino acids profile (unpublished data) after fermentation with Lactobacillus plantarum Lp6.

Table 2 Amino acid composition of FSPMH fractions separated by gel filtration on Sephadex G-15

Amino acids	F1	F2	F3	F4	F5	F6	F7
Aspartic acid	12.39d	10.69bc	9.44b	6.12a	11.53cd	17.97e	17.10e
Glutamic acid	35.63g	27.18f	18.64d	9.10a	12.59b	15.11c	19.37e
Serine	4.15b	5.78d	4.59bc	2.83a	3.82b	2.05a	5.24cd
Histidine	2.61c	3.46d	1.99b	1.14a	2.29bc	2.05b	2.34bc
Glycine	3.80a	3.75a	3.33a	3.56a	6.90b	12.15d	9.43c
Threonine	2.27a	3.53bc	3.39bc	1.96a	2.98b	2.28a	3.62c
Arginine	7.12d	8.55e	15.23f	4.29b	6.15c	6.90d	3.71a
Alanine	1.48a	3.45c	5.73e	1.30a	3.02c	4.38d	2.19b
Tyrosine	0.45a	0.48a	1.03b	7.95c	10.44d	0.34a	0.68ab
Cysteine-s	1.66f	0.38d	0.18b	0.10a	0.25c	1.92g	0.54e
Valine	2.03b	4.09f	4.02f	1.41a	2.79d	2.58c	3.17e
Methionine	0.84b	0.94c	2.29e	0.93c	7.32f	1.00d	0.08a
Phenylalanine	1.97a	2.20b	3.95c	48.13g	8.44f	8.06e	4.69d
Isoleucine	2.02a	3.25d	4.41f	2.22ab	2.92c	2.25b	3.69e
Leucine	2.45a	4.16c	14.81e	2.75a	4.13c	3.65b	7.86d
Lysine	7.50d	11.49e	3.07c	0.97a	1.55b	1.23ab	3.21c
Proline	11.66d	6.73c	3.88a	5.23b	12.84e	16.19f	13.08e
THAA (g/100g)	22.41a	24.70b	39.13e	61.99g	41.46f	38.04d	34.76c
ΔΦ (Kj/mol AAR)	9.70d	9.04b	8.92a	10.97g	9.16c	10.39f	10.02e
THAA: total hydrophobic amino acids; and ΔΦ: hydrophobic index. Values are the means of three determinations. Rows with dissimilar letters indicate statistical differences (P < 0.05).

Molecular Weight Distribution

The size of peptides is known to be a significant factor in the overall antioxidant activity of hydrolyzed proteins. Considering that F2 was found to possess the highest antioxidant activity, this fraction was therefore compared its MW distribution to other six fractions (Fig. 3). The chromatographic data indicated that this fraction was composed of MW peptides of which major peaks were located at 370–1500 Da (69.90%) and 162–370 Da (30.10%). Several studies have shown that short peptides with MW ranging from 370 to 1500 Da were responsible for the higher antioxidant activity in commercial whey protein hydrolysate in a liposome oxidizing system.[7] Wu et al.[26] reported that a peptide from mackerel protein hydrolysate with MW of approximately 1400 Da possessed stronger antioxidant activity compared to higher MW peptides. Chen et al.[46] claimed that six peptides of soybean protein hydrolysates with 5 to 16 amino acid residues were highly effective in exhibiting antioxidant activity. These findings are in agreement with observations from other studies and support the fact that functional properties of antioxidative peptides are highly influenced by their molecular mass.[35,37]

Figure 3 molecular weight distributions of the seven FSPMH fractions. (Figure provided in color online.)

CONCLUSION

Based on the findings obtained in this study, the FSPMH fractions obtained by gel filtration exhibited different antioxidative capacities and free radical scavenging activities. The higher antioxidant activities observed with the fraction F2 could be related to its molecular weight distribution of peptide mainly located at 370 to 1500 Da. Moreover the peptides size, the high percentages of antioxidative amino acid residues present, free amino acids and hydrophobicity appeared to collectively contribute to the strong bioactivities of fractionated FSPMH. Therefore, the benefits of F2 fraction could be extended beyond its higher antioxidative ability. Further investigations are undergoing on the purification of peptide isolated from the fractions as well as the mechanisms of their antioxidant activities.

ACKNOWLEDGMENTS

This research was supported by the National Natural Science Foundation of China (No.30671525), the National High Technology Research and Development Program (“863”Program) of China (No. 2007 AA10Z325), 111 project-B07029 and China Scholarship Council (CSC); Ms. Dai Hui is also gratefully acknowledged for her help in doing the experiments.