Studies on the Mechanism of Algal Blue Protein and Its Hydrolysate to Promote the Regeneration of Maize Straight Branched Chain Starch

 Abstract: Alginin is a protein in Spirulina that has various health care functions for the human body, and it was initially found to promote the regeneration of corn starch. In order to further explore the mechanism of corn starch regrowth, 1% and 10% alginate and its hydrolysate were added to corn straight-chain, branched-chain and mixed starch, and their effects on starch regrowth were determined. The mechanism of alginate and its hydrolysate on corn starch regrowth was analyzed by X-ray diffraction, differential scanning calorimetry, infrared and solid-state nuclear magnetic analysis.

 

The results showed that the addition of 1.0% phycocyanin had no effect on the regrowth rate of straight-chain starch and increased the regrowth rate of branched-chain starch by 61.4%, while the addition of 10% phycocyanin increased the regrowth rate of straight-chain starch by 60.4% and that of branched-chain starch by 69.6%. The low level of algin hydrolyzed peptide (1.0%) had no significant effect on the regeneration rate of maize straight-chain starch, but increased the regeneration rate of branched-chain starch by 28.1%, while the high level of hydrolyzed peptide (10.0%) increased the regeneration rate of maize straight-chain starch by 184.7%, and that of maize branched-chain starch by 47.0%. The UV-visible scan showed that hydrolysis by alkaline protease could expose the phycocyanin in the center of the phycocyanin structure, and the X-ray results showed that the mixing of phycocyanin and maize branched-chain starch produced a sharp diffraction peaks with the diffraction angles of 2θ of 16.44º and 16.60º, respectively.

 

Differential thermal results showed that after mixing alginate with corn branched starch and alginate hydrolysate with corn straight-chain starch, the resulting regenerated starch lost the crystallization peak and the only recrystallization peak appeared. The results of infrared and 13C NMR showed that phycocyanin formed hydrogen bond with the aldehyde group at the reducing end of maize branched starch through the amino group of arginine, and the hydrophobic amino acid in phycocyanin drove away the water molecules at the end of the side chain of maize branched starch, which accelerated the formation of the hydrogen bond at the end of the branched starch, and increased the regeneration rate; and the hydrolyzed algalactosine hydrolysate had a cysteine sulfhydryl group and the aldehyde group at the reducing end of maize branched starch, and the regenerated starch had an increased crystallization rate, while the cysteine sulfhydryl group and maize straight-chained starch had a decreased crystallization peak. The cysteine sulfhydryl group and the aldehyde group at the reducing end of corn straight-chain starch in alginate hydrolysate formed hydrogen bonds during the mixing and regeneration process with straight-chain starch, and the molecular flinging unraveled the double helix of straight-chain starch, which greatly facilitated the formation of hydrogen bonds between straight-chain starch of maize, and improved its regeneration rate. This study provides a new technology to improve the regeneration rate of corn starch.

 

As a kind of dietary fiber, regenerated resistant starch has the efficacy of laxative, controlling body mass, assisting in the control of diabetes mellitus, and improving human immunity by promoting the propagation of lactic acid bacteria in the cecum [1]. However, the low yield is a bottleneck for its industrialization. In order to improve the preparation rate of regenerated starch, researchers at home and abroad have done a lot of research on the concentration of starch milk, storage temperature and time, drying and extrusion process, microwave and ultrasonic treatment, enzyme treatment, and addition of biomacromolecules and organic substances. Mass fraction of 1% and 10% of buckwheat starch milk at 0 ℃ storage regrowth rate is high, reaching more than 30.0%, and 5%, 15% and 20% concentration of starch regrowth rate is high at 2 and 6 ℃ storage [2].

 

Under the condition of frozen storage at -18 ℃, the concentration of glutinous rice starch increased from 1% to 25%, and the minimum regrowth rate of glutinous rice starch increased from 4.5% to 15.7% in 1 d of freezing; 10%~15% of glutinous rice starch emulsion was stored for 7 d under this condition, and the regrowth rate of glutinous rice starch would be greater than 40.0% [3]. At vacuum drying temperature of 30 ℃, vacuum degree of 0.08 MPa, aging 98 h, the regrowth rate of sweet potato starch reached 20.9% [4]. Extrusion treatment and the number of times can also affect the regeneration rate. Repeated extrusion treatment can increase the regeneration rate of corn starch to more than 40% [5], and three times repeated regeneration and high and low temperature cyclic aging can also obviously promote the regeneration of starch [6-7]. Ultrasonic treatment can increase the regeneration rate of sweet potato starch by 2.28 times [8], and the combined treatment of electrolysis and microwave can increase the regeneration rate of sweet potato starch by 1 times [9]. Prunase and high-pressure moist heat treatment increased the regeneration rate of black bean starch to 41.3% [10], while medium-temperature α-amylase treatment increased the regeneration rate of sweet potato starch by 1.68 times [11].

 

Because pullulanase and amylase break the side chain of starch and produce more straight-chain starch, which increases the nucleation and crystallization rate of starch regeneration starch [12]. According to the theory of crystal formation, the addition of crystalline seeds can also increase the production of regenerated starch, and the group once used oxalic acid to erode potato regenerated starch to prepare crystalline seeds to promote the regeneration of maize starch, which increased the regeneration rate of maize starch from 7.37% to 11.46% [13]. Although the above physical methods and chemical enzyme methods can improve the preparation rate of regenerated starch to a certain extent, it is not more than 45%, and the competitiveness of large-scale production is not strong. Therefore, the search for more effective and quicker methods to improve starch regrowth rate and related mechanisms has been one of the research hotspots in this field. In recent years, some organic macromolecule polysaccharides and proteins can have a significant effect on the regrowth rate by adding them to starch.

 

Luo et al.[14] found that the addition of inulin at a starch level of 5%-7.5% promoted the regrowth of branched-chain starch from maize, and the group also found that the addition of alcohol-soluble proteins increased the regrowth rate of maize starch from 9.4% to 29.3% in a previous study[15] . Recently, the group further proposed a possible structure of the regenerated starch containing alcohol-soluble proteins by analyzing NMR and IR [16]. In order to search for organic macromolecules that can improve the starch regrowth rate more effectively, the group tried to use alginate and its hydrolysate to promote the regrowth of corn starch and found that both alginate and its hydrolysate can promote the regrowth of maize branched-chain starch, and the addition of alginate hydrolysate with a mass fraction of starch of 10.0% can increase the regrowth rate of maize branched-chain starch to more than 50%. Through solid-state NMR and infrared analyses, the possible mechanisms of algin and its hydrolysate in promoting the regrowth of corn branched-chain starch were proposed.

 

1 Materials and Methods

1.1 Materials and Instruments

Corn starch and spirulina (Cheng Haihu brand) were sold commercially; PBS (phosphate buffer saline) was purchased from Shanghai Haring Biotechnology Co. The high temperature amylase, lipase and alkaline protease were supplied by Tianjin NOAO Technology Development Co.

 

YXQG02 Portable Electrothermal Pressure Steam Sterilizer, Shandong Ander Medical Technology Co. Bio-Rad FES135 infrared spectrophotometer, Bio-Rad, USA; Shimadzu UV-2450/2550 ultraviolet-visible spectrophotometer; Differential Scanning Calorimetry Analyzer DSC204C (Netzach, Germany); X-ray diffractometer D8 ADVANCE (Bruker AX S, Germany); Varian Unity 300 MHz nuclear magnetic resonance spectrometer (Varian, USA). (Varian, USA).

 

1.2 Test methods

1.2.1 Extraction of algal blue protein

The extraction of algal blue protein was referred to the experimental method of the group[17] . 1) Cell crushing: 400 g of Spirulina powder was dissolved in 4 000 mL of 10 mmol/L phosphate buffer solution (0.01 mmol/L pH 6.8 PBS buffer) for 4 h, and then frozen and thawed between -20 and 4 ℃ for 4 times, and each time after thawing, ultrasonic assisted crushing was used with the power of 400 W. Ultrasonic 6 s interval 15 s, ultrasonic 60 times, and the power of 400 W was used for 6 times. After each thawing, the sample was broken by ultrasonic wave with a power of 400 W. The sample was ultrasonicated for 6 s with an interval of 15 s, and the number of ultrasonic waves was 60. Then centrifuge the sample at 10 000 r/min and 4 ℃ for 30 min, discard the precipitate and take the supernatant. 2) Salt analysis: After centrifugation, slowly add (NH4)2SO4 into the blue supernatant sample at 4 ℃ until the concentration of 28% saturated, and then continue to stir the sample to make the (NH4)2SO4 dissolve fully, and then leave it at 4 ℃ for the rest of the day.

 

The supernatant was collected by freezing centrifugation at 10 000 r/min at 4 ℃ for 30 min to remove a small amount of heterogeneous proteins, and then precipitated by 55% ammonium saturated sulfate. The supernatant is then precipitated by 55% saturated ammonium sulfate, and then centrifuged at 10 000 r/min for 30 min after 4 h at 4 ℃ to collect the blue protein precipitate. 3) Dialysis: The precipitate obtained from the salting-out is collected with PBS solution, and then put into a dialysis bag to dialyze out the (NH4)2SO4 salts (the molecular weight cut-off of 8~14 kD). The dialysis bag containing the sample was dialyzed in 0.01 mol/L PBS buffer with pH 6.8, and the buffer was changed several times, and the end point of dialysis was detected by BaCl2 without precipitation. After dialysis, the obtained sample could be concentrated with polyethylene glycol, freeze-dried and stored at 4 ℃. After the lyophilized alginate was diluted, the absorbance values at 620 and 280 nm were determined, and the purity was expressed by the ratio of absorbance A620/A280, and the purity of the alginate prepared by this method was 1.35.

 

1.2.2 Alginate hydrolysis

The purified algal blue protein was hydrolyzed by alkaline protease to prepare algal blue protein hydrolysate, under the conditions of alkaline protease hydrolysis: the pH value of protein solution was 8.0, the enzyme digestion temperature was 40 ℃, the enzyme digestion time was 60 min, and the enzyme addition amount was 0.1% of the protein mass. The hydrolysate after enzymatic hydrolysis was freeze-dried and prepared for use.

 

1.2.3 Corn Gibbon Starch Preparation

The straight branched starch of corn was prepared according to the method of the research group [18]. After 10%-15% corn starch was dispersed with water, the starch was stirred and pasted in a water bath at 95 ℃ for 2 h until transparent, and then further pasted at high pressure (0.2 MPa) at 120 ℃ for 40 min. Cooled down to ambient temperature and then put in the refrigerator for 2 d at 4 ℃ for aging, and then 0.6 mL of high-temperature amylase enzyme was added in every 100 mL of starch milk, and the precipitate was obtained by centrifugation and washing to obtain the regenerated starch. Dissolve the regenerated starch with 4 mmol/L KOH and then add 3 times the volume of n-butanol, stir well and then centrifuge to separate the precipitate, which is corn straight-chain starch. Add 1 volume of ethanol to the supernatant to obtain the precipitate, which is corn branched-chain starch. The obtained maize branched-chain starch was washed with water after removing fat and protein according to the method in [19] to become purified maize branched-chain starch. The maize branched amylopectin for regeneration test was not dried, and was stored in the form of wet starch in the refrigerator at 4 ℃. 10 g of wet starch was taken and dried to obtain the dry-wet ratio of starch.

 

1.2.4 Co-regeneration of maize straight and branched starch with phycocyanin and its hydrolysates

The algal blue protein and its hydrolysate were mixed with corn straight-chain and branched-chain starch (wet state) at 1.0% and 10.0% of the mass ratio of the dried starch, respectively, and 20 mL of distilled water was added to the mixture. After cooling, the mixture was aged in a refrigerator at 4 ℃ for 48 h. The regeneration rate was determined by the method of literature [15].

 

1.2.5 Test methods

To explore the mechanism of phycocyanin and its hydrolysate in promoting the regeneration of branched starch from maize, for the determination of UV-visible absorption, infrared, solid-state NMR, and X-ray diffraction, phycocyanin was used in its pure form (98% purity of Xi'an Minglang Biotechnology Co., Ltd.), and the amount of added phycocyanin was 30% of the amount of starch. UV-Vis absorption of algal blue protein and its hydrolysate: dilute the algal blue protein and its hydrolysate with distilled water, then leave it for 30 min, and then measure the maximum absorption wavelength with Shimadzu UV-2450/2550 UV-Vis Spectrophotometer; X-ray diffraction: put the powdered sample into the D8 ADVANCE X-ray diffractometer (Bruker AX S, Germany), and then measure the maximum absorption wavelength using Cu-targeted KT rays (0.0%). X-ray diffraction was performed by placing the powdered sample into a D8 ADVANCE X-ray diffractometer (Bruker AX S, Germany) and irradiated with KT rays (0.154 nm) on a Cu target at a tube voltage of 40 kV, a tube current of 40 mA and a scanning speed of 0.1 (°)/s.

 

Infrared (IR) analysis: Samples were pressed with spectroscopically pure KBr, and starch IR absorption was determined at 27 °C using an IR spectrophotometer, Bio-Rad FES135, with a scanning range of 4,000-400 cm-1; solid-state NMR analysis: Protein or starch samples were placed in a sealed PENCIL-type (5-mm) zirconia rotor, and the resonance frequency was 75 kHz vs. 90 on a Varian Unity 300 MHz NMR spectrometer with a pulse width of 3.4 μs. Solid-state NMR analysis: Protein or starch samples were placed in a sealed PENCIL-type (5-mm) zirconia rotor on a Varian Unity 300 MHz NMR spectrometer at a resonance frequency of 75 kHz, corresponding to 90 μs, with a pulse width of 3.4 μs. A 4-mm dual-resonance HX CP/MAS (cross-polarization/magic-angle-swinging) probe was used, and the speed of the magic-angle-swinging (MAS) was controlled automatically by a rotational speed cabinet in the range of 9-12 kHz. Differential Scanning Calorimetry Analysis: The DSC204C Differential Scanning Calorimetry Analyzer (Netzach, Germany) was used for the analysis and testing, and the warming rate was 0.02 ℃/s.

 

1.3 Statistical processing

Variables were expressed as mean ± standard deviation ( ± s), using F-test. SPSS software was used for analysis.

 

2 Results and analysis

2.1 Effects of algin and its hydrolysate on the regrowth of branched-chain starch in maize Table 1 shows the effects of algin and its hydrolysate on the regrowth of branched-chain starch in maize.

Table 1 shows that the addition of 1.0% phycocyanin had no effect on the regrowth rate of straight starch. As can be seen from Table 1, the addition of 1.0% alginate had no effect on the regrowth rate of straight-chain starch, while the addition of 10% showed a more significant promotion effect, which increased the regrowth rate from 27.0% to 43.3% of the control, an increase of 60.4%, and the branched-chain amylopectin increased by 69.6%; the addition of 1% alginate had a significant effect on the regrowth of branched-chain amylopectin, which increased the rate from 26.7% to 43.1%, an increase of 61.4%. The addition of 1% alginate blue protein had an obvious effect on the regeneration of branched starch, with the regeneration rate increasing from 26.7% to 43.1%, an increase of 61.4%, while the regeneration rate of branched starch did not increase significantly when the amount of addition was increased from 1% to 10%, and only increased from 43.1% to 45.3%, an increase of 5.1%.

 

For algin hydrolyzed peptide, the low dosage (1.0%) had no significant effect on the regeneration rate of maize straight-chain, but showed a promotion effect on the regeneration of branched-chain starch (increased by 28.1%), and the high dosage (10.0%) showed a very strong effect on the promotion of maize straight-chain starch, with the rate of regeneration increasing by 184.7%, from 27.0% to 76.9%, while the rate of regeneration of branched-chain starch increased by only 47%, from 26.7% to 39.2%. The regeneration rate of corn branched chain starch increased from 26.7% to 39.2%, which was only increased by 47%.

Therefore, in the case of maize straight-chain starch, the regrowth rate increased with the addition of algin or hydrolyzed peptide. However, in the case of maize branched starch, only a low level of addition showed its effective promotion effect, and the effect of increasing the regrowth rate by increasing the proportion of added starch was not significant. This may be due to the different hydrogen bonding positions between maize branched starch and phycocyanin or peptide.

 

2.2 UV-Vis Scanning Maximum Absorption Analysis

Figure 1 shows the UV-visible maximum absorption spectra of phycocyanin and its hydrolysates. As shown in Fig. 1, the maximum UV-visible absorption wavelengths of phycocyanin were 258.0 and 616.0 nm, and there were no absorption peaks of phycobiliprotein near 374 and 340 nm, which was similar to that reported in the literature [20-21]. The UV-visible maximum absorption wavelengths of the hydrolysate of phycocyanin were 258.0, 346.0 and 613.5 nm, and the characteristic absorption of phycobilins appeared, which indicated that the hydrolysis by alkaline protease exposed phycocyanin at the center of the structure of phycocyanin.

 

2.3 X-ray Diffraction Analysis

Figure 2 shows the X-ray diffractograms of the starch obtained by mixing maize straight-chain and branched-chain starches with phycocyanin and its hydrolysate before and after regrowth. From Fig. 2a, it can be seen that the phycocyanin used in the experiment is not a single crystal, and the 2θ diffraction angles are 9.52, 21.34, 31.52, 45.22, which is different from the non-crystalline diffraction of phycocyanin in kudzu rice reported in the literature (2θ diffraction angles of 16.66, 20.68, 23.70, 29.06, 30.12, 33.66, 37.90, 40.32, 57.54) [2.2.2, 2.4.2, 2.6, 30.12, 33.66, 37.90, 40.32, 57.54]. , 40.32., 57.54.) are different [22].

 

From Fig. 2b, it can be seen that after hydrolysis of phycocyanin, two facets with 2θ diffraction angles of 31.52 and 45.22 are missing, while one facet with 2θ diffraction angle of 8.78 is added. In other words, the number of facets with small spacing decreases and the number of facets with large spacing increases. As shown in Figs. 2c and 2d, the 2θ diffraction angles of the straight-chain starch were 16.76 and 22.16, and the 2θ diffraction angles of the branched-chain starch were 17.22 and 19.28, which were basically the same as that in the previous study of the research group [23]. As shown in Fig. 2e, after the regeneration of corn starch mixed with phycocyanin, the sharp peaks with diffraction angle 2θ of 22.16 disappeared, and the diffraction peaks with diffraction angle 2θ of 19.36 and 23.52 increased, but there are many derivative peaks around these peaks, and the peak shapes are not sharp.

 

As can be seen from Fig. 2f, the peak with diffraction angle 2θ of 16.44 becomes sharper after the regeneration of maize branched starch and phycocyanin hydrolysate, indicating that phycocyanin hydrolysate accelerates the aggregation of maize branched starch on the crystalline surface, and thus accelerates the regeneration speed greatly. As can be seen from Fig. 2 g, the peak with diffraction angle 2θ of 16.60 becomes sharper after the regeneration of corn branched starch mixed with phycocyanin, indicating that phycocyanin accelerates the aggregation of corn branched backstarch on this crystal surface and accelerates the formation of crystal surface. According to the decrease of the sharp diffraction angle of the samples (2f, 2g), it can be seen that the mechanism of the two substances promoting the regeneration of corn branched amylopectin may be related to the increase of the spacing of the facets with the 2θ diffraction angle of 16.4 to 16.7 after the addition of the two substances. In Figs. 2e and 2h, the regeneration rate of starch after mixing phycocyanin with straight-chain starch and phycocyanin hydrolysate with branched-chain starch did not increase much, and the 2θ diffraction peaks did not show obvious sharp peaks, which further indicates that the appearance of sharp diffraction peaks is correlated with the increase of starch regeneration rate.

 

2.4 Differential Scanning Calorimetry

Fig. 3 Differential scanning calorimeter (DSC) of the starch obtained by mixing corn straight-chain and branched-chain starch with phycocyanin and its hydrolysate before and after regrowth. As shown in Fig. 3, there were two crystal melting peaks (134.40 and 140.69 ℃) for phycocyanin (Fig. 3a), and one main peak (112.86 ℃) and three small peaks for phycocyanin hydrolysate. There were three crystal melting peaks for maize straight-chain amylopectin (89.61, 95.48, 109.74 ℃) and two crystal melting peaks for maize branched-chain amylopectin (108.97, 143.14 ℃).

 

The melting temperatures of these crystals were lower than those of sweet potato tapioca starch [24], which may be related to the fact that the average chain length of maize tapioca starch is shorter than that of sweet potato tapioca starch. The low temperature is the melting temperature of the crystals and the high temperature is the melting temperature of the recrystallized starch [25]. From Figures 3a, 3c, 3d, 3e and 3g, it can be seen that the melting temperature of straight-chain regenerated starch increased from 109.74 to 126.93 ℃ after alginate was added to corn straight-chain branched starch, and the melting temperature of branched-chain regenerated starch decreased from 143.14 to 139.74 ℃, which indicated that alginate participated in the regeneration of straight-chain branched starch of maize, and the molecular length of the molecules involved in regeneration was between 1.5 and 2.8 mm, and the molecular length of the molecules involved in regeneration was between 2.6 and 2.6 mm, and the melting temperature was between 3.6 and 4.6 mm. The chain length of alginate molecules involved in regeneration was between that of straight-chained and branched maize starch.

 

It is worth noting that after the mixing of alginate and corn branched starch, there is only amylopectin recrystallization peak but no crystal peak, and the temperature of this peak is close to that of alginate recrystallization peak. The temperature of this peak is close to that of the recrystallization peak of phycocyanin. This indicates that the number of molecules with chain length close to that of phycocyanin in corn branched starch is large, and it is easier for these molecules to form hydrogen bonds with each other, which will produce a new kind of recycled functional starch containing phycocyanin. As shown in Figs. 3b, 3c, 3d, 3f and 3h, the melting temperature of the straight-chain retentate increased from 109.74 to 118.37 ℃ and that of the branched-chain retentate decreased from 143.14 to 130.02 ℃ after the addition of phycocyanin hydrolysate to maize branched starch.

 

Similar to the results of alginate-promoted maize branched-chain starch, in the Differential Scanning Calorimetry (DSC) of alginate hydrolysate-promoted maize straight-chain starch regeneration (Fig. 3f), the melting peaks of the crystals of straight-chain regenerated starch disappeared, and there was only a recrystallization peak at 118.37 ℃, which is close to that of the recrystallization peaks of the alginate hydrolysate. The other two low-temperature peaks should be the different molecular crystal peaks of phycocyanin hydrolysate, which are not related to the straight-chain starch. From the differential melting temperature, it can be seen that phycocyanin and its hydrolysate promote the regeneration of branched-chain starch and straight-chain starch because this combination can unravel the double helix of branched-chain starch and create convenient conditions for the formation of hydrogen bonds between branched-chain starch, which will accelerate the rate of formation of starch crystals and greatly increase the regeneration rate of starch.

 

2.5 Infrared spectral analysis

Figure 4 shows the infrared spectra of the starch obtained by mixing maize straight-chain and branched-chain starch with phycocyanin and its hydrolysate before and after regeneration, the absorption peaks around 3,282-3,315 cm-1 are the stretching vibration of O-H of the starch molecule or the N-H of the protein molecule, and the sharp peaks around 2,930 cm-1 are the absorption peaks of the asymmetric stretching vibration of the C-H bond of the starch or the protein methylene group, the absorption peaks at 1,644.1 cm-1 in Figs. 4e, 4f, 4g, and 4h are the absorption peaks of H-O-H of the water in starch. The absorption peaks at 1,644.1 cm-1 in Figs. 4e, 4f, 4g, and 4h are the bending vibrational absorption peaks of the H-O-H of water in starch, and the vibrational absorption peaks of the C-O in the C-O-C of the starch structure at 1,006.7 cm-1 [26-27]. In Figs. 4a and 4b, the telescopic vibration of C=O in the amide I bond of alginate hydrolysate is shown at 1,651.2 cm-1 [20]. In Figs. 4a and 4b, the C-H shear bending vibration of methyl group is shown at 1 408.8 and 1 411.6 cm-1 , and the C-O-H bending vibration is shown at 993-1 003 cm-1 [28]. The weakening of the intensity of the former peaks indicates that the peptide chains in the alginate hydrolysates are shortened, and the methyl group of the alginate that is involved in hydrogen bonding has been dissociated, and the decreasing of the intensity of the latter peaks may be related to the breakage of the connection between amino acids containing hydroxyl groups and polysaccharides. The decrease in intensity of the latter peak may be related to the interruption of the connection between the hydroxyl-containing amino acid and polysaccharide.

 

In Figs. 4a and 4b, the wave number at 1,538~1,550 cm-1 represents the combination of C-N stretching vibration of amide Ⅱ bond and N-H deformation vibration[28] , and the intensity of the peak there decreased after hydrolysis of phycocyanin, which was caused by the amide bond breaking during the hydrolysis by protease. In Figs. 4e and 4g, after the regeneration of alginate and maize straight-chain starch, the wave number of maize regenerated straight-chain starch changed from 3,285.3 to 3,268.2 cm-1, and that of branched-chain starch from 3,315.3 to 3,282.5 cm-1, and the decrease in the infrared wave number here represented the formation of hydrogen bond [28], and the decrease in wave number of branched-chain starch here may be related to the sulfhydryl groups in the alginate, and the reducing-end aldehyde group of branched-chain starch. The decrease in the wave number of branched starch may be related to the participation of sulfhydryl groups in phycocyanin and aldehyde groups at the reducing end of branched starch in the formation of hydrogen bonds. The absorption peaks of amide Ⅰ and Ⅱ can be observed in these two figures, indicating that the carbonyl group and amino group in phycocyanin did not participate in the formation of hydrogen bond in the process of starch regeneration.

 

In Figs. 4f and 4h, after mixing algal blue protein hydrolysate with maize branched starch, the wave number of maize regenerated straight-chain starch shifted from 3 285.3 to 3 282.5 cm-1, and branched starch shifted from 3 315.3 to 3 288.5 cm-1. According to the results in Table 1, the phycocyanin hydrolysate promoted the regeneration of maize straight-chain starch obviously, while the infrared wave number near 3 285.0 cm-1 did not change much, indicating that the driving force for regeneration came from non-hydroxyl groups, such as amino groups or sulfhydryl groups. The infrared wave number near 3 285.0 cm-1 did not change much, indicating that the driving force for regrowth was from non-hydroxyl groups such as amino or sulfhydryl groups. The disappearance of the infrared peaks corresponding to the amide I and II bonds in Fig. 4f (black arrows) confirms that these carbonyls and amino groups are involved in the formation of hydrogen bonds, and the C=O and C-N stretching vibrations have disappeared. Summarizing the infrared analysis, the reason for the promotion of maize branched starch by phycocyanin may be the formation of hydrogen bond between the sulfhydryl group in phycocyanin and the aldehyde group at the reducing end of branched starch; the reason for the promotion of maize straight-chain starch by phycocyanin hydrolysis may be the formation of hydrogen bond between the carbonyl group and amino group of the hydrolyzed peptide and the hydroxyl group of straight-chain starch.

 

2.6 13C solid-state NMR analysis

Figure 5 shows the 13C solid-state NMR spectra of the starch obtained from corn straight-chain and branched-chain starches mixed with alginate and its hydrolysate before and after regrowth. According to the literature [29], the main amino acids contained in phycocyanin are Arg, Asp, Ala, Cys, His, Thr, Glu, Leu, Ser, Val, Pro, Ile, Trp, Phe, Lys, Tyr, Met, and Gly, and the chemical shifts of 176.7 and 176.6 ppm in Figures 5 a and 5 b represent the amide bond. 156.9 ppm represents Arg, 129.6 ppm represents Arg, and 156.9 ppm represents Arg. The chemical shifts 176.7 and 176.6 ppm in Figs. 5a and 5b represent amide bonds, 156.9 ppm represents Arg, 129.6 and 128.5 ppm represent Phe, and 115.6 ppm may be the chemical shifts of Tyr C3 or His C4 [30-33], but the intensity of the peak near 139 ppm of Tyr is very weak, which indicates that the content of this amino acid is relatively small, and therefore, the chemical shifts there are only produced by His. After the hydrolysis of phycocyanin by protease, the peaks were cleaved to 118.1 and 114.8 ppm, which indicated that the amide bond of the protein was broken at the position of His linkage during the process of hydrolysis. In Figs. 5 a and 5 b, the chemical shifts at 92.9 and 93.7 ppm are for polysaccharide C1 atoms, 73.0 and 72.7 ppm are for polysaccharide C2-5 atoms, and 60.6 and 60.8 ppm are for C6 atoms [34].

 

The intensity of the polysaccharide peak was slightly enhanced after hydrolysis of phycocyanin (the most obvious change of the C6 peak at 60.8 ppm), indicating that phycocyanin may be bound to algal polysaccharides, and the hydrolysis process of protease interrupted the connection between the amino acids in the protein and the C6 of polysaccharides, so that the C6 of more polysaccharides showed chemical shifts. In Figs. 5 a and 5 b, the β-folded structural degree of Glu does not appear to be shifted by 53.5 and 54.0 ppm [35], which indicates that phycocyanin and its hydrolysate mainly exist in the form of α-helical structure. The chemical shifts of 24.9 and 25.3 ppm in Figs. 5 a and 5 b represent the -CH3 of Leu [35], and the chemical shifts here did not change much before and after the hydrolysis of phaeocyanin, which suggests that the amide bond with leucine may not be broken during the hydrolysis by protease. The chemical shifts of 16.4 and 17.1 ppm in Figs. 5a and 5b represent the -CH3 of Ala/Val/Thr[32] , which changed significantly before and after hydrolysis of phycocyanin, suggesting that the amide bond of these amino acids may have been broken, and the resonance of the methyl carbon may have been changed.

 

The peptides containing these amino acids may be linked with polysaccharides in phycocyanin to enhance the hydrophobicity of the polysaccharides after enzymatic digestion. As shown in Figs. 5 e and 5 g, the chemical shift of C1 of straight-chain starch shifted from 100.3 ppm to 102.4 and 100.3 ppm, and that of branched-chain starch shifted from 103.0 and 100.5 ppm to 103.2 ppm. According to the literature [33-36], the double-peak chemical shift of regenerated starch represents the presence of single-helix crystals of starch or amorphous starch. According to the literature [33-36], the double peak of chemical shift here means that there are single helix crystals of starch or the starch is in amorphous state. The addition of phycocyanin caused the formation of single helix crystals of maize straight-chain regenerated starch or the carbon atoms at the reducing end of the starch were changed from crystalline to amorphous state, and caused the maize branched-chain starch to change from amorphous state to crystalline state, which is in line with the results of the regeneration rate data in Table 1. The addition of phycocyanin had little effect on the chemical shifts of other carbon atoms of maize branched starch.

 

As can be seen from Figs. 5f and 5h, the chemical shifts of all carbon atoms of straight-chain starch remained basically unchanged after the regrowth of maize branched starch by mixing alginate hydrolysate and branched starch, and the chemical shift related to the amorphous region of starch decreased from 82.5 to 81.4 ppm [35], suggesting that the promotion of the regrowth of maize straight-chain starch by the hydrolysate of phycocyanin is probably related to the change of the amorphous region. The changes in chemical shifts of branched starch in this area were basically the same as those of added phycocyanin, which indicated that it was difficult to see the reason for the difference in the effects of phycocyanin and its hydrolysate on the regrowth of branched starch in the NMR. The 172-174 ppm in Figs. 5 e, 5 f, 5 g, and 5 h represent amide bonds, indicating that phycocyanin and its hydrolysate are indeed present in these samples, but the signals of the other groups are weak due to the sensitivity of solid-state NMR diffraction or the interaction between the protein and the starch in the regeneration process.

 

2.7 Mechanism of algal blue protein and its hydrolysate in promoting the regeneration of maize straight and branched starch Based on the infrared and nuclear magnetic analyses of the regenerated starch obtained from algal blue protein and its hydrolysate before and after mixing with maize straight and branched starch, the possible pathways of the regeneration mechanism have been hypothesized (Fig. 6). In Fig. 6a, phycocyanin was decomposed into different peptides and polysaccharides under the action of alkaline protease, among which the polysaccharide C6 molecule also contained hydrophilic amino acids, such as Thr, Cys, and Arg, and hydrophobic amino acid peptides, such as Val and Leu, etc. The hydrolysis of phycocyanin and its hydrolyzed products with corn starch was analyzed by IR and NMR analysis.

 

For maize branched starch, when alginate is mixed with it (Figure 6c), hydrogen bond is formed between the ammonia group of Arg in alginate and the aldehyde group at the reducing end of maize branched starch, which weakens the speed of movement of branched starch, and in the process of slow flinging, the double helix at the end of the branched starch is unraveled, and the hydroxyl groups of different molecules form hydrogen bonds to form retrogradation starch; as the glycoproteins in alginate are connected with the proteins, they do not dissolve into the solution and increase the viscosity of solution, which does not influence the unraveling of branched starch. Since the glycoprotein in alginate is attached to the protein, it does not dissolve into the solution and increase the viscosity of the solution, which does not affect the unrotation of branched starch. However, when alginate hydrolysate mixed with corn branched-chain starch (Figure 6e), hydrolysate containing Arg polypeptide molecular weight is small, chain length is short, arginine and branched-chain starch after the formation of hydrogen bonds, the molecular weight of the branched-chain starch in the movement of the process of the polypeptide can be pulled swaying left and right, coupled with the hydrolysate in the polysaccharides dissolved to increase the viscosity of solution, the maize branched-chain starch can not be unspinned, can not provide better conditions for the regeneration of branched-chain starch, so it does not promote branched-chain starch. The terminal double helix of maize branched starch cannot be unspooled, which cannot provide better conditions for regeneration, so it does not promote the regeneration of branched starch.

 

For maize straight-chain starch, its small molecular weight, many reducing ends and fast moving speed can lead to the formation of weak hydrogen bonds between the reducing end and the sulfhydryl group of cysteine in phycocyanin (Fig. 6b), and the fast moving speed of straight-chain starch prevents the deconvolution of the double helix after it binds to phycocyanin and fails to promote its regeneration. However, the molecular weight of alginate hydrolysate is small, and after the formation of hydrogen bond with one chain in the double helix of straight-chain starch (as shown in Fig. 6d, the hydrogen bond is formed between the reducing end of straight-chain starch and arginine in alginate hydrolysate), the two double helices will easily be unspinned when straight-chain starch is in motion, so that the hydroxyl groups on the double helices of starch are exposed, and the hydrogen bond between the different chains can be formed more easily, which greatly promotes the regeneration of straight-chain starch. Therefore, the formation of hydrogen bonds at the reducing end of starch can obviously promote its regeneration.

 

3 CONCLUSIONS

Phycocyanin can promote the regeneration of branched-chain starch from maize, and the hydrolysate of phycocyanin can promote the regeneration of straight-chain starch from maize. The cysteine sulfhydryl group in the hydrolysate of phycocyanin may form a hydrogen bond with the aldehyde group at the reducing end of maize straight-chain starch during the mixing and regeneration process with straight-chain starch, and the molecular flinging will make the double helix of straight-chain starch unraveled, which greatly facilitates the formation of hydrogen bonds between straight-chain starch of maize and raises the regeneration rate of straight-chain starch of maize. The hydrophobic amino acid in phycocyanin can be used to drive away the water molecules at the end of the side chain of corn branched starch, which can lead to the deconvolution of the double helix at the end of the branched starch, accelerate the formation of inter-chain hydrogen bond at the end of the branched starch, and increase its regeneration rate. The formation of hydrogen bonds at the reducing end of starch can significantly promote its regrowth.

 

The results showed that the addition of 1.0% algin had no effect on the regrowth rate of straight-chain starch and increased the regrowth rate of branched-chain starch by 61.4%, while the addition of 10% algin increased the regrowth rate of straight-chain starch by 60.4% and that of branched-chain starch by 69.6%. The low addition of hydrolyzed peptide of algin (1.0%) had no significant effect on the regrowth rate of maize straight-chain starch, but increased the regrowth rate of branched-chain starch by 28.1%, while the high addition of hydrolyzed peptide (10.0%) increased the regrowth rate of maize straight-chain starch by 184.7% and that of branched-chain starch by 47.0%.

The discovery of algal blue protein and its hydrolysate to promote the regeneration of branched-chain starch from maize opens up a new field of functional protein intervention in starch regeneration, which is a new way for the development of multifunctional health food and the broadening of the application fields of algal blue protein and maize starch.

 

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