Study on Optimal Allocation of Water Resources in Ningdong Energy and Chemical Base

Lei FAN Zhenghong ZHAO Guangcai HOU Hui QIAN Xingping FENG Tao JIANG

Abstract［Objectives］ This study aimed to optimize the chelation process for complex microelement iron supplement derived from pig blood by response surface methodology.

［Methods］ On the basis of singlefactor test, pH value, concentration of polypeptide solution and volume ratio of polypeptide solution to FeCl2 solution were selected as influencing factors with Fe (II) chelation rate as the indicator for BoxBehnken central composite experimental design with three factors and three levels. The effects of three factors on the response value were analyzed by response surface methodology.

［Results］ The optimized chelation process for complex microelement iron supplement derived from pig blood by response surface methodology was as follows: pH 5.40, polypeptide solution concentration 2.27 %, volume ratio of polypeptide solution to FeCl2 solution 2.16∶1. Under this condition, the predictive Fe (II) chelation rate of iron supplement was 79.37%, while the actual value was 79.41%.

［Conclusions］ The optimized process may provide new thoughts for the development and utilization of complex microelement iron supplement derived from pig blood.

Key wordsResponse surface methodology; Pig blood protein; Iron supplement; Fe (II) chelation rate



Received: November 20, 2018Accepted: December 21, 2018

Supported by Youth Fund of National Natural Science Foundation of China (31801673); Talent Development Fund of Anhui Academy of Agricultural Sciences (17F1205); Youth Innovation Fund of President of Anhui Academy of Agricultural Sciences (17B1220); Team Building Project of Anhui Academy of Agricultural Sciences (18C1225); Youth Fund of Natural Science Foundation of Anhui Province (1808085QC94).

Jingjing HUANG (1988- ), female, P. R. China, assistant professor, devoted to research about meat product processing.

*Corresponding author. Ningning XIE (1984- ), male, P. R. China, associate professor, devoted to research about meat product processing. Email: ningxie512@163.com.

Iron is one of the essential microelements in human body. Irondeficiency anemia (IDA) is a common type of anemia caused by a lack of iron that affects hemoglobin synthesis. In China, IDA is a widespread nutritional anemia affecting a wide population with high incidence, which seriously affects peoples health and quality of life［1］. Currently, oral administration of iron supplement is one of the most effective ways treat IDA worldwide. Commercially available iron supplements are available in a variety of forms, including inorganic iron and organic iron［2］. The complex microelement iron supplement, with proteolytic polypeptide as the chelation substrate, is a novel functional product developed for people with IDA in China, which is characterized by high iron content, easy absorption and digestion, and high bioavailability［3-5］. Compared with amino acid iron supplement, complex microelement iron supplement is more advantageous due to low price and low production cost. In 1960, Newey et al.［6］ confirmed through experiments that small peptide chelated iron could be directly absorbed by the small intestine. The amino group, carbonyl group, carboxyl group and hydroxyl group in the peptide chain of polypeptide iron chelate can be covalently bound to iron to form stable five or sixmembered ring structure［7］. In most studies, small peptides with iron chelation activity were obtained from edible plants and animals, such as aquatic products［8-12］, beans, peanuts［13］ and rice residue［14］. Under the optimal chelation conditions, the chelation ability of rice peptide prepared by Kang et al.［14］ with ferrous chloride was 21.6 mg/g, and the yield of chelate reached 65.2%. Huo et al.［11］ found that the optimal chelation conditions for polypeptide iron chelate from hydrolysates of hairtail offcuts in Zhoushan were: ascorbic acid 0.1%, temperature 20 ℃, time 15 min, pH 7.0.

Pig blood is a byproduct in pig slaughtering and a raw material in food industry with high protein content, low sugar content, low fat content and rich microelements［15］. Pig blood contains fullprice proteins, in which proteins accounts for 4.3%［16］. In addition, pig blood contains 45 mg/100 g iron, as well as various microelements such as calcium, magnesium, zinc, copper, phosphorus, potassium, sodium, manganese and selenium［17］. In centrifuged pig blood, plasma accounts for 65%, containing globulin and albumin; blood corpuscles account for 35%, most of which are hemoglobins［18-19］. China has the largest consumption of pork in the world. In 2017, the total consumption of pork in China reached 54.87 million tons. Moreover, China also has the largest number of pigs in the world. In 2017, there were 689 million live pigs in China［20］. Thus, there is a broad prospect for development and utilization of pig blood resource industry.

In this study, pig blood was used as raw material to prepare pig blood polypeptides by alkaline protease, and the iron was separated. By singlefactor test, the optimal pH value, reaction temperature, reaction time, concentration of polypeptide solution and volume ratio of polypeptide solution to FeCl2 solution were determined. On this basis, the chelation process for complex microelement iron supplement derived from pig blood was optimized by response surface methodology, in order to prepare complex microelement iron supplement with high absorption rate and without digestive tract stimulation or side effects, which provided reference for the development and utilization of pig blood protein resources.

Materials and Methods

Materials

Experimental materials and reagents

Pig blood, Hefei Wanrun Food Co., Ltd.; hydrochloric acid, nitric acid, perchloric acid, sulfuric acid, sodium hydroxide, ferrous chloride, ammonium ferric sulfate, ascorbic acid, and ethanol were of analytical grade; alkaline protease, Hefei Bomei Biotechnology Co., Ltd.

Instruments

JA1103N electronic balance, Shanghai Minqiao Precision Scientific Instrument Co., Ltd.; PHS3C pH meter, Shanghai INESA Scientific Instrument Co., Ltd.; TGL16M centrifuge, Changsha Xiangzhi Centrifuge Instrument Co., Ltd.; RE52A rotary evaporator, Shanghai Yarong Biochemical Instrument Factory; FD1CE freeze drier, Beijing Detianyou Technology Development Co., Ltd.; 10-1 mm quartz micro cuvette, Wuxi Jinghe Optical Instrument Co., Ltd.; 1900PC UVVIS double beam spectrophotometer, Shanghai Puyuan Instrument Co., Ltd.; KT260 fully automatic Kjeldahl analyzer, Foss Analytical A/S (Denmark); HR801 enzymelabeled instrument, Shenzhen Huakerui Technology Co., Ltd.; iCE 3500 atomic absorption spectrometer, Thermo Fisher Scientific (U.S.); L8900 amino acid automatic analyzer, Hitachi Ltd. (Japan).

Methods

Chelation process

Pig blood → Centrifugalization → Spray drying → Enzymatic hydrolysis → Chelation → Complex microelement iron supplement.

Physicochemical and nutritional indicators

The moisture content and protein content in complex microelement iron supplement were determined to analyze the difference between the content of each component. Specifically, moisture content was determined by direct drying method［21］; protein content was determined by Kjeldahl method［22］; fat content was determined by Soxhlet extraction method［23］; iron content was determined by flame atomic absorption spectrometry［24］; amino acid composition was analyzed using L8900 amino acid automatic analyzer［25］.

Ionization of iron

Pig blood is rich in iron that should be ionized before chelation and could be added according to different needs. The plasma protein and hemocyte protein were separately hydrolyzed and centrifuged. The precipitate was freezedried, dissolved in dilute hydrochloric acid, adjusted to pH 4-5, and added with 3% ascorbic acid standard solution. The prepared acid hydrolysate of the precipitate was collected. In addition, acidified external iron solution (FeCl2 solution added with 3% ascorbic acid standard solution) was used as the iron source for the chelation reaction.

Preparation of pig blood polypeptide solution

A certain amount of plasma protein powder and hemocyte protein powder was separately weighed for enzymolysis. After centrifugation, the supernatant was collected for decolorization by activated carbon, suction filtration, ultrafiltration, concentration, and freezedrying to obtain polypeptide powder, which was dissolved in deionized water to prepare different concentrations of polypeptide solution before use.

Determination of Fe (II) chelation rate

Glutathione (GSH) (20 mg/ml) and EDTA (5 mg/ml) were used as the positive control; the solution containing FeCl2 and pig blood polypeptide was used as the blank control. Iron content was determined by flame atomic absorption spectrometry［24］. Fe (II) chelation rate was calculated according to the following formula:

Fe (II) chelation rate = (csample-cblank)/(ctotal-cblank)×100%

Where, csample, cblank and ctotal indicate iron content in polypeptideFe (II), pig blood polypeptide and mixture of FeCl2 and pig blood polypeptide, respectively.

Singlefactor test

The pH value, reaction temperature, reaction time, concentration of polypeptide solution and volume ratio of polypeptide solution to FeCl2 solution were selected as main single factors to investigate the effects of various factors on Fe (II) chelation rate. Each experiment was repeated three times.

Factors and levels in response surface methodology

According to BoxBehnken central composite experimental design and the results of singlefactor test, three significant factors (pH value, polypeptide solution concentration, volume ratio) in singlefactor test were optimized by response surface methodology. The factors and levels were shown in Table 1.

Table 1Factors and levels in response surface methodology

Level

Factor

pH(A)Volumeratio (B)Concentration ofpolypeptide solution (C)∥%

-1422

0533

1644

Data processing

The experiment was repeated three times. Data analysis and mapping were performed using DesignExpert 8.0.5.

Results and Analysis

Physicochemical and nutritional indicators

Physicochemical and nutritional indicators of pig blood powder and complex microelement iron supplement were shown in Table 2. The moisture content in pig blood powder was relatively high (10.06%), whereas the crude fat content was relatively low (0.41%). In complex microelement iron supplement, the moisture content was reduced to 7.66%, and the crude fat content was 1.51%. In addition, the iron content in complex microelement iron supplement reached 2 280 mg/kg, which was remarkably improved compared with that in pig blood powder.

Amino acid composition

The amino acid composition of pig blood powder and complex microelement iron supplement was shown in Table 3. Both pig blood powder and complex microelement iron supplement were rich in amino acids, in which 16 amino acids were detected, including seven essential amino acids. Tryptophan was destroyed during hydrolysis and was not detected. The total amount of essential amino acids in pig blood powder was 43.36%. In complex microelement iron supplement, the total amount of essential amino acids was slightly lower (41.99%), among which leucine accounted for the highest level (11.90%), followed by lysine (8.65%).

Table 2Physicochemical and nutritional indicators of pig blood powder and complex microelement iron supplement

MaterialMoisture content∥%Protein∥%Crude fat∥%Iron content∥mg/kg

Pig blood powder10.06±0.01587.40±0.0220.41±0.0131 550±80

Complex microelement iron supplement7.66±0.01988.30±0.0271.51±0.0222 280±110

Jingjing HUANG et al. Optimization of Chelation Process for Complex Microelement Iron Supplement Derived from Pig Blood by Response Surface Methodology

Table 3Amino acid composition analysis of pig blood powder and complex microelement iron supplement

No.Amino acidPig bloodpowder∥%Complex microelementiron supplement∥%

1Aspartic acid (Asp)12.31±0.02311.35±0.017

2Threonine (Thr)*3.72±0.0174.68±0.025

3Serine (Ser)5.11±0.0255.54±0.009

4Glutamine (Glu)10.20±0.02312.59±0.034

5Glycine (Gly)4.01±0.0083.91±0.018

6Alanine (Ala)7.90±0.0056.92±0.021

7Cysteine (Cys)1.17±0.0022.29±0.016

8Valine (Val)*8.12±0.0167.47±0.008

9Methionine (Met)*0.89±0.0031.02±0.002

10Isoleucine (Ile)*0.72±0.0021.81±0.004

11Leucine (Leu)*13.74±0.02211.90±0.012

12Tyrosine (Tyr)2.72±0.0163.60±0.003

13Phenylalanine (Phe)*7.11±0.0166.46±0.013

14Lysine (Lys)*9.06±0.0258.65±0.004

15Histidine (His)7.38±0.0115.55±0.007

16Tryptophan (Trp)*0.000.00

17Arginine (Arg)4.47±0.0054.77±0.007

Total essential amino acids43.36±0.03641.99±0.033

Essential amino acids/Totalamino acids43.36±0.02441.99±0.037

* means essential amino acids.

Singlefactor test

Effects of pH value on chelation rate

The concentration of polypeptide solution was 2% (w/w), and volume ratio of polypeptide solution to FeCl2 solution was 2∶1. A certain volume of acid hydrolysate of the precipitate was added. The reaction was performed at pH 3, 4, 5, 6 and 7 for 1 h at 30 ℃, respectively. Under different pH conditions, Fe (II) chelation rate was analyzed (Fig. 1).



Fig. 1Effects of pH value on chelation rate

As could be seen in Fig. 1, under certain conditions, when pH increased from 3 to 5, Fe (II) chelation rate was improved with the increase of pH (P<0.05), which reached the highest level at pH 5. However, as pH continued to increase, Fe (II) chelation rate slowly decreased (P<0.05), which was probably because pH greater than 5 affected the spatial conformation of pig blood polypeptide［26］ and the morphology of iron element, thus affecting the binding of iron ions to the substrate. Therefore, pH should be maintained at around 5 during the reaction.

Effects of reaction temperature on chelation rate

The concentration of polypeptide solution was 2% (w/w), and volume ratio of polypeptide solution to FeCl2 solution was 2∶1. A certain volume of acid hydrolysate of the precipitate was added. The reaction was performed at 25, 35, 45, 55 and 65 ℃ for 1 h at pH 6, respectively. Under different temperature conditions, Fe (II) chelation rate was analyzed (Fig. 2).



Fig. 2Effects of reaction temperature on chelation rate

As could be seen in Fig. 2, when the temperature varied within the range of 25-65 ℃, there was no significant changes in Fe (II) chelation rate (P>0.05). Therefore, the reaction temperature could be controlled at about 25 ℃.

Effects of reaction time on chelation rate

The concentration of polypeptide solution was 2% (w/w), and volume ratio of polypeptide solution to FeCl2 solution was 2∶1. A certain volume of acid hydrolysate of the precipitate was added. The reaction was performed at pH 6 for 15, 25, 35, 45 and 55 min at 30 ℃, respectively. Fe (II) chelation rate was analyzed (Fig. 3).

As could be seen in Fig. 3, when the reaction time varied within the range of 15-55 min, there was no significant changes in Fe (II) chelation rate (P>0.05). Therefore, the reaction time could be controlled at around 15 min in order to improve the efficiency of experiment and production.

Effects of polypeptide solution concentration on chelation rate

The concentration of polypeptide solution was 1, 2, 3, 4 and 5% (w/w), respectively. The volume ratio of polypeptide solution to FeCl2 solution was 2∶1. A certain volume of acid hydrolysate of the precipitate was added. The reaction was performed at pH 6 for 1 h at 30 ℃. Fe (II) chelation rate was analyzed under different concentrations of polypeptide solution (Fig. 4).



Fig. 3Effects of reaction time on chelation rate



Fig. 4Effects of polypeptide solution concentration on chelation rate

As could be seen in Fig. 4, under certain conditions, when the concentration of polypeptide solution increased from 1% to 3% (w/w), Fe (II) chelation rate was improved with the increase of polypeptide solution concentration (P<0.05), which reached the highest level when the concentration of polypeptide solution was 3% (w/w). However, as the concentration of polypeptide solution continued to increase, Fe (II) chelation rate slowly decreased (P<0.05). Therefore, the concentration of polypeptide solution should be controlled at about 3% (w/w).

Effects of volume ratio of polypeptide solution to FeCl2 solution on chelation rate

The concentration of polypeptide solution was 2% (w/w), and the volume ratio of polypeptide solution to FeCl2 solution was 1∶1, 2∶1, 3∶1, 4∶1 and 5∶1, respectively. A certain volume of acid hydrolysate of the precipitate was added. The reaction was performed at pH 6 for 1 h at 30 ℃. Fe (II) chelation rate was analyzed at different volume ratios (Fig. 5).

As could be seen in Fig. 5, when the volume ratio of polypeptide solution to FeCl2 solution was less than 3∶1, Fe (II) chelation rate was improved with the increase of volume ratio (P<0.05), which reached the highest level when the volume ratio was 3∶1. However, when the volume ratio of polypeptide solution to FeCl2 solution increased to 5∶1, Fe (II) chelation rate was reduced (P<0.05). Therefore, the volume ratio of polypeptide solution to FeCl2 solution should be controlled at about 3∶1.



Fig. 5Effects of volume ratio of polypeptide solution to FeCl2 solution on chelation rate

The results of singlefactor test showed that changes in pH value, concentration of polypeptide solution and volume ratio of polypeptide solution to FeCl2 solution exerted significant effects on Fe (II) chelation rate (P<0.05), whereas reaction temperature and reaction time had no significant effect on Fe (II) chelation rate (P>0.05). In the subsequent response surface experiment, pH value, polypeptide solution concentration and volume ratio of polypeptide solution to FeCl2 solution were optimized. According to the results of singlefactor test, the reaction temperature and reaction time were maintained at 25 ℃ and 15 min, respectively.

Response surface experiment

Using DesignExpert 8.0.5, a response surface experiment was performed with three factors and three levels (Table 4).

Table 4Response surface methodology for chelation of complex microelement iron supplement

No.

Factor

pH(A)Volume ratio ofpolypeptide solutionto FeCl2 solution (B)Concentrationof polypeptidesolution (C)∥%Chelationrate∥%

154476.26

253377.81

343464.27

462377.40

543261.78

653378.21

742363.48

864375.69

953377.48

1052465.50

1144365.23

1263471.52

1353377.40

1452277.65

1563274.55

1654274.75

1753375.26 

The above experimental data were subjected to quadratic multiple regression fitting. According to the results, the regression equation for Fe (II) chelation rate was as follows:

Y=-139.106 00+73.745 00A-1.116 50B+13.593 50C-0.865 00AB-1.380 00AC+3.415 00BC-6.146 00A2-0.636 00B2-3.056 00C2.

In order to verify the validity of the above regression equation, variance analysis was performed. The results were shown in Table 5.

Table 5Variance analysis of the regression equation

SourceSum of squaresDegree of freedomMean SquareFValuePValueSignificance

Model539.95959.9914.980.000 9*

A246.421246.4261.520.000 1**

B7.8017.801.950.205 5#

C15.62115.623.900.088 8#

AB2.9912.990.750.416 0#

AC7.6217.621.900.210 3#

BC46.65146.6511.650.011 2*

A2159.051159.0539.700.000 4*

B21.7011.700.430.535 2#

C239.32139.329.820.016 5*

Residual error28.0474.01

Misfit difference22.7737.595.760.061 9#

Pure error5.2741.32

Sum567.9916

**: Extremely significant differences; *: significant differences; #: no significant differences.

According to the results, the regression equation exhibited significant differences (P＜0.05), indicating that the model could explain the changes in most of the experimental condition. Moreover, the misfit difference was not significant, which further confirmed the rationality of the model. Therefore, the model could be used to analyze and predict the entire experimental result.

The effects of three factors on Fe (II) chelation rate were more complicated. Among all the linear terms of the above regression equation, the effects of various factors on the chelation rate demonstrated a descending order of A＞C＞B. Specifically, the term A exerted the extremely remarkable effect (P＜0.000 1), suggesting that pH value had a greater influence on the chelation rate; the terms A2 and C2 were quadratic terms that had remarkable effect (P＜0.05), and the term BC was the only interaction term that had remarkable effect (P＜0.05). Therefore, the effects of various factors on Fe (II) chelation rate were generally in a descending order of pH value > polypeptide solution concentration > volume ratio.

Determination of optimal chelation process

As shown in Table 6, Fe (II) chelation rate in group 4 reached the highest level. Therefore, the optimized chelation process using DesignExpert 8.0.5 was as follows: pH 5.40, polypeptide solution concentration 2.27 %, volume ratio of polypeptide solution to FeCl2 solution 2.16∶1. Under this condition, the predictive Fe (II) chelation rate of iron supplement was 79.37%.

Three parallel experiments were performed under the above optimal chelation conditions, and the experimental data were averaged. The actual Fe (II) chelation rate of iron supplement was 79.41%, which was 0.04% higher than the predicted value, indicating that the optimization result of chelation process by response surface methodology was accurate and reliable with relatively high practical value.

Table 6Optimization of chelation process by response surface methodology

No.pHVolumeratioConcentration ofpolypeptide solution∥%Chelationrate∥%

15.41 3.04 3.01 78.49

25.63 3.66 2.89 78.25

35.22 3.22 2.87 78.38

45.40 2.16 2.27 79.37

55.27 2.67 2.73 78.51

65.73 2.27 2.73 78.49

75.40 2.27 2.16 79.24

85.56 2.38 2.56 79.16

95.73 2.73 2.27 78.65

105.32 3.39 3.21 78.32

115.16 2.07 2.16 78.47

125.20 3.91 3.01 78.33

135.64 3.53 3.11 78.22

145.68 2.33 2.66 78.81

155.48 3.76 3.33 78.39

165.36 3.60 3.23 78.49

175.50 2.93 2.53 78.85

185.49 3.86 3.32 78.45

195.77 2.90 2.32 78.32

205.46 3.97 3.20 78.59

215.35 3.63 3.29 78.41

225.30 3.93 3.54 78.32

235.44 3.30 2.52 78.38

245.48 3.85 3.42 78.31

255.56 3.36 3.11 78.38

265.56 2.60 2.87 78.53

275.28 3.37 2.69 78.37

285.37 3.63 3.21 78.53

295.72 2.95 2.51 78.54

305.58 2.31 2.49 79.28

(Continued)

(Table 6)

No.pHVolumeratioConcentration ofpolypeptide solution∥%Chelationrate∥%

315.37 3.86 3.26 78.61

325.44 3.95 3.50 78.35

335.34 2.11 2.11 79.23

345.67 3.57 3.02 78.22

355.20 2.03 2.25 78.72

365.95 2.00 2.41 78.69

375.47 3.31 2.69 78.62

385.51 3.20 2.62 78.64

395.34 3.97 3.48 78.48

405.58 3.92 3.02 78.30

415.77 2.77 2.61 78.54

425.76 2.94 2.39 78.36

435.34 3.72 3.43 78.27 

Conclusion

Complex microelement iron supplement abundant in iron could be prepared by centrifugation, enzymatic hydrolysis, acid hydrolysis and chelation from pig blood. Through singlefactor test, it was found that pH value, polypeptide solution concentration and volume ratio of polypeptide solution to FeCl2 solution significantly affected Fe (II) chelation rate of iron supplement, whereas the reaction temperature and reaction time had no remarkable effect. The reaction temperature and reaction time were maintained at 25 ℃ and 15 min, respectively. The optimized chelation process for complex microelement iron supplement derived from pig blood by response surface methodology was as follows: pH 5.40, polypeptide solution concentration 2.27 %, volume ratio of polypeptide solution to FeCl2 solution 2.16∶1. Under this condition, the predictive Fe (II) chelation rate of iron supplement was 79.37%. In this study, complex microelement iron supplement was prepared with pig blood as raw material, while the product performance is yet to be verified.

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