薏苡仁多肽特性.doc

Property（特性） of the Semen Coicis（薏苡仁） polypeptide（多肽）
Abstract Shelled（有壳的，脱壳的） Semen Coicis power were taken as the raw material, then determinate the content of polypeptide of the lactic acid bacteria fermentation products. Take the methods that unfermented and fermented polypeptide were purified by ultrafiltration and Sephadx G-25 gel column chromatography on glucose, and to compare the oxidation resistance of the each larger elution peak. The results show that, the income rate of the fermented polypeptide were 0.468%, they were 8 times as the blank control group, the rate of scavenging superoxide anion of fermented Semen Coicis polypeptide were 58%, the rate of scavenging of Hydroxyl radicals can achieve 83% and the reductive capacity of Fe3+ both were excel remarkably than the unfermented polypeptide.
Key words shelled Semen Coicis; lactic acid bacteria; ferment; polypeptide; oxidation resistance.
Introduction Semen Coicis ,it is a kind of the dried and riped seed of gramineous plants ,it is also known as Chinese sorghum, Job's-tears, it is commonly known as“Medicine King Rice”、“Hui Hui rice”、“Six Gorge Rice”and so on. In recent years, Scholars at home and abroad make a research into the chemical composition and pharmacological activity of Semen Coicis . Modern pharmacological research shows that , Semen Coicis polypeptide has the obvious effects of inhibition of ACE activity、antioxidant、improve immunity and so on. At present more researches are about the Semen Coicis polypeptide, mainly use the mold fermentation or enzymolysis to extract the Semen Coicis polypeptide, and at the same time make a research into its activity, yet few of determinations about comparison of antioxidant activity for the shelled Semen Coicis fermentation of lactic acid bacteria after and before. Make a comparative research of antioxidant activity between use the probiotic lactic acid bacteria to ferment the shelled Semen Coicis power into Semen Coicis polypeptide and the unfermented, for the future research in fermented foods about Semen Coicis and also provide references .
1 Materials and methods（材料和方法）
1.1 Materials and reagents（试剂） Tested lactic acid bacteria: two strains of lactic acid bacteria which were isolated from the traditional fermented food (they were preserved into the microbiology laboratory of Inner Mongolia Agricultural University College of Food science and Engineering ), shelled Semen Coicis(Japan imports), SDS, β-Mercaptoethanol ,sodium tetraborate, orthophthalaldehyde, acetocaustin, pyrogallic acid, ferrous sulfate, potassium ferricyanide and so on the reagents were analytically pure.
1.2 Instrument and quipment（仪器和设备） TU1810 UV visible spectrophotometer(Beijing PuXi general instrument Ltd. );YM50 Stainless steel vertical electric steam sterilizer(Shanghai Sanshen medical apparatus Ltd. ); High speed centrifuge(Thermo Fisher OF The United States ); Ultrafiltration centrifugal tube.
1.3 Experiment Methods 实验方法）
1.3.1 Fermentation process（发酵过程） After make the tested lactic acid bacteria Sc6-3 and NM01 activate three generations, as 3%(Sc6-3:NM01=1:2) inoculate in the 8% of Semen Coicis power culture medium, the fermented temperature is 25℃, matrix particle size is about 20 orders, pH is natural value, ferment for 72 hours, unfermentations are blank control group. After centrifugation , take the supernatant and concentrated, after ultrafiltration centrifugal filtrate, freeze-dried to get the Semen Coicis coarse polypeptide.
Technological process
Shelled Semen Coicis→Choose→Crush→Weight→

tested lactic acid bacteria→activate→tested bacteria liquid
inoculate or not inoculate ↓
↓
Water immersion→Sterilize→ Ferment supernatant→Concentrate→Ultrafiltration centrifugal filtrate and concentrate →Freeze-dried→Coarse polypeptide.
1.3.2 Determination（决定，确定） of polypeptide content（内容）
A: The reagent preparation of OPA:
Weight 40mg orthophthalaldehyde accurately and soluble in the 1ml methanol, add 25ml 100mmol∕L sodium tetraborate ,2.5ml 10% SDS and 100µLβ-Mercaptoethanol, then add water to 50ml .(Should be prepared when using)
B: Draw the standard curve:

Concentration(mmol∕L)
L-Phenylalanine standard curve
C: Determination of the content of polypeptide in the sample
150µL sample were taken into the tube , then add 3ml OPA reagent, measure time and oscillating and mixing, then put to react at room temperature for 2 minutes, after that determinate the absorbance at 340nm and correspond to the standard curve to derive the protein hydrolysis activity. When determinated it , it was easy to appear the phenomenon of absorbance baseline drift because of lamp current and light battery, so we should use the blank tube to zero frequently in order to eliminate the system error of the resulting.
Semen Coicis polypeptide income rate=M2∕M1×100%
M2=165.19×C×V×10-6=(A-b)∕a×V×165.19
Among that :
M1-the quality of shelled Semen Coicis power(g);
M2-the quality of coarse polypeptide in the culture medium(g);
C- concentration of culture medium(mmol﹒L-1) ;
V-volume of culture medium(ml);
A- absorbance;
a- the slope of the standard curve of L- phenylalanine;
b- intercept of standard curve in the vertical coordinate.
1.3.3 Purification（提纯） of Sephadex G25（交联葡聚糖凝胶“作为分子筛用于化学品的分离与提纯”25数字用来区别型号，表示介质凝胶按交联度不同，数字越小质交联度越大，分级范围越小，反之亦然） column（列、柱） chromatography（层析法，色谱法）经Sephadex G-25柱层析纯化
By natural sedimentation to fill the Sephadex G-25 gel column, and deal balance with the bidistilled water . Confect the Semen Coicis coarse polypeptide into 100mg∕ml solution, the volume is 3ml, then with bidistilled water elutied, detect the wavelength at 280nm ,3ml per tube, make the eluted liquid pipe number as the abscissa and absorbance as ordinate, draw the elution curve. Collect the larger elution peak and detect their antioxidant activity respectively.
1.3.4 Method for determination（确定） of antioxidant（抗氧化剂） activity（活性）
1.3.4.1 Method for determination of scavenging（清除） oxygen free radicals（氧自由基）
1.3.4.2 Make use of pyrogallic acid in the alkalescent environment to self oxidation and decomposition of oxygen free radicals and colored intermediates, the absorbance of colored intermediates in the value at 320nm have obvious linear relationship with the time(in 5 minutes), they can calculate the rate of removal of oxygen free radicals.
Preparation of 9mmol∕L pyrogallic acid solution by 10mmol∕L HCl solution, then preparation of 50mmol∕L Tris-HCl buffer solution(pH 8.2), take the preparation of Tris-HCl buffer solution 4.5ml and add polypeptide solution with different concentrations(with distilled water instead of polypeptide solution for blank) ,after mix them, to be with the pyrogallic acid solution in the bath at 25℃ and after insulate 20 minutes, add 1ml pyrogallic acid solution in it, pour the mixed solution into the cuvette immediately, record the absorbance every 30 seconds at 320nm and determinate for 5 minutes. Calculate the rate of polypeptide of removal of oxygen free radicals:
M= (N0-NX)∕N0 ×100%
N0 –the timerate of the blank solution absorbance
the experiment determination N0 =0.0684;
NX -the timerate of the different concentrations of the sample solution absorbance .

1.3.4.2 Method for the determination of hydroxyl radical（羟基） scavenging（清除）
ability
Sodium salicylate can effectively capture hydroxyl free radicals generated by perhydrol (H2O2) and Fe2+, and produce the colored substance, which has strong absorption at the wavelength of 510nm, at result, a negative correlation was found between absorbance value and the scavenging capacity of this substance.
0.5mL 2mmol·L-1 sodium salicylate - ethanol solution、0.5mL 9mmol·L-1 ferrous sulfate ( ferrous sulfate solution were instead by distilled water to eliminate interference) and 1.5mL different concentrations of polypeptide solution (peptide solution were instead by distilled water as the blank control)were mixed in test tube one by one ,then added 0.5mL perhydrol to start the reaction and put the tubes into 37 ℃ water bath immediately lasting 1h ,at last ,we can obtain the absorbency value at different concentrations.
Scavenging rate E of Hydroxyl Radical (·OH) was calculated as the following formula:
E＝（A0-Ax）/ A0×100%
Ax＝A1-A2
Ao: absorbance value of blank solution after the reaction;
Ax: the real absorbance eliminate interference after the reaction,
A1:absorbance value of sample solution at different concentrations;
A2: absorbance value after distilled water instead of ferrous sulfate .
1.3.4.3 Method for determination of reductive ability（还原剂能力）
1mL polypeptide solution with certain concentration、2.5ml 0.2mol•L-1、pH=6.6 phosphate buffer and 2.5ml mass fraction of 1% potassium cyanide solution were mixed into the test tubes which were put in 50 ℃ water bath lasting 20min and added 2.5 mL mass fraction 10% trichloroacetic acid solution，and then centrifuged for 10 min at a speed of 3000 r/min and took 2.5ml upper solution next ,after that added 2.5 mL distilled water and 0.5 mL mass fraction of 0.1% ferric chloride solution, at last ,determined absorbance values at the wavelength of 700 nm。
2 Result and analysis
2.1 Sephadex G-25 column chromatography purification （Sephadex G-25柱层析纯化
Unfermented and fermented polypeptides which have been pre-treated were purified by Sephadex G-25 glucose gel column chromatography. The separate effect was shown in Figure 2 and figure 3.

0
0.2
0.4
0.6
0.8
1
1.2
0
10
20
30
40
50
60
70
Tube numbers
Absorbance
Figure 2: Elution curve of unfermented sample polypeptide
With elution going, ultraviolet absorbance of separated eluate appeared two obvious elution peak at different stages, when reached to NO.30 tube, polypeptides were largely eluted which was composed by more than two components, the second peak was greater among all peaks, so NO.16-23 of tubes were collected and named group III, then concentrated and freeze-dried to use next.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0
10
20
30
40
50
60
70
Tube numbers
Absorbance

Figure 3: Elution curve of fermented sample polypeptide
The above figure showed that elution peak of this fermented polypeptide appeared later than unfermented, the first peak appeared at the NO.20 of tube, then appeared two obvious peaks, which show that the polypeptide contains at least three or more components, the first peak did not have good symmetry, The results indicated that pure water failed to achieve the ideal effect. TheNO. 21-30 of tubes are collected and named group IV, then concentrated and freeze-dried to use next.
2.2 Scavenging effect of oxygen free radical（O2-·）清除氧自由基的影响

0
10
20
30
40
50
60
70
0
2
4
6
8
10
c/(mg·mL﹣1)
Clesrance rate（%）
Fermentation polypeptide
Unfermentation polypeptide

Figure 4: Scavenging effect comparison of oxygen free radical（O2-·） of fermented and unfermented sample polypeptide
We can see from Figure 4, the polypeptides III and IV all have ability to scavenge oxygen free radical, and the IV was significantly higher than the III. When the concentration of polypeptide beyond 8 mg·mL-1, scavenging ability of III on superoxide anion radical was invariant with the increasing of concentration, but IV improved obviously. At concentration of 10 mg·mL-1, scavenging rate of IV had reached to 58% and continued to improve possibly, but III was only 36% and impossible to rise.
2.3 Scavenging effect of Hydroxyl Radical (·OH)

Clearance rate（%）
0
20
40
60
80
100
0
2
4
6
8
10
c/(mg·mL﹣1)
Fermentation polypeptide
Unfermentation polypeptide

Figure 5: Scavenging effect of comparison of hydroxyl radical (·OH) fermented and unfermented sample polypeptide
The figure 5 showed that scavenging ability of hydroxyl radical of peptides IV was obviously higher than polypeptide III. The ability of III increased significantly at concentration of 2 ~ 6 mg·mL-1and not obvious at 6 ~ 10 mg·mL-1; When the concentration of was 2 ~ 6 mg·mL-1, the ability of IV increased by a wide margin comparing with III, followed by slow but increase than III. The hydroxyl radical scavenging rate of IV was 83% but the III is only 59% at the concentration of 10 mg·mL-1 .
2.4 Determination of reductive ability还原剂能力
0
0.2
0.4
0.6
0.8
1
0
2
4
6
8
10
c/(mg·mL﹣1)
absorbance
Fermentation polypeptide
Unfermentation polypeptide

Figure 6: reductive ability of comparison of fermented and unfermented sample polypeptide
The figure 6 showed that reductive\ antioxidative ability of polypeptide III on Fe3+
ascend slowly at concentration of 2 ~ 6 mg·mL-1and increased quickly at concentration of 6 ~8, followed by changed gently, but polypeptide IV changed smoothly at 2 ~ 6 mg·mL-1 and 6~8 mg·mL-1 and the rest had a obvious rising trend ,which showed that reduction/oxidation ability of peptides IV on Fe3+ were obvious than polypeptide III with increasing of concentration.
3 conclusion

3.1 The yield of fermented polypeptide was 0.468%, which was 8 times of blank control group.
3.2 In purification experiment, polypeptides were separated initially by the centrifugal ultrafiltration and purified Sephadex G-25 column chromatography for further, then eluted by pure water, as result , unfermented sample polypeptides appeared two elution peaks, but fermented polypeptides appeared three peaks, then collect their larger elution peak III and IV respectively, the result show that the component of fermented polypeptide was much more than unfermented.
3.3 In antioxidative ability test, both fermented and unfermented polypeptide have antioxidative activity ,but scavenging ability on the different free radical has division of the strong and the weak, they have strong scavenging effects on hydroxyl radical (•OH), reducing ability also reflects on the antioxidative ability to some extent. The result show that antioxidative activity of fermented polypeptide was stronger than unfermented.

-