How to microencapsulate crayfish shell astaxanthin?

 Astaxanthin, also known as astaxanthin, astaxanthin, because of its molecules contain a long chain of conjugated double bonds and unsaturated α-hydroxy ketone and has a strong antioxidant properties, so far the discovery of the strongest antioxidant capacity of the substance, the antioxidant capacity of natural VE can be up to 100 times more than [1]. Astaxanthin can effectively remove free radicals, sulfide, disulfide, with antioxidant, anti-aging, anti-tumor, improve immunity, retina protection and other functions, can be used in health food, high-grade cosmetics, pharmaceuticals and other fields. However, astaxanthin is a non-polar substance, insoluble in water, difficult to be added to water-soluble food, and astaxanthin's long conjugated unsaturated double-bond structure is very unstable, easy to be damaged by light, heat, and oxides and lose their activity [2], which greatly restricts astaxanthin's application in the food industry.

 

Microencapsulation is a method of encapsulating solid, liquid, gas and other substances in tiny closed capsules [3]. This method can not only effectively protect and isolate the substance, reduce the occurrence of oxidation reaction, passivate photosensitivity and heat sensitivity; it can also effectively shield the undesirable odor of the core material, control the release rate of the core material, change the physicochemical properties of the core material (e.g., dispersion, color, shape, and density), and improve the stability of the core material in storage [4-5].

 

At present, studies on astaxanthin basically focus on the extraction process of astaxanthin, and there are few reports on the research of astaxanthin microcapsule embedding technology. In this study, we applied β-cyclodextrin and gum arabic embedding method to make a kind of antioxidant slow-release microcapsules of astaxanthin from the shells of crayfish, and discussed the factors affecting the embedding effect in detail, which was aimed at providing a reference to solve the limitations of the application of astaxanthin in the food industry.

 

1 Materials and Methods

1.1 Test materials

Crayfish: Xuyi Longsheng Lobster Farm; astaxanthin standard: Sigma Investment Group Limited; porous starch: Liaoning Lida Biotechnology Co.

 

1.2 Instruments

WF180 Universal Pulverizer: Shanghai Optical Instrument Factory; RE-2000A Rotary Evaporator: Shanghai Yarong Biochemical Instrument Factory; SHA-C Constant Temperature Water Bath Oscillator: Shanghai Medical Instrument Factory; TDL-40C Low-speed and Large-capacity Centrifuge: Shanghai Anting Scientific Instrument Factory; TU-1900 UV-Vis Spectrophotometer: Beijing Pudian General Instrument Co. - DN Ultrasonic Cell Pulverizer: Ningbo Xinzhi Bio-technology Co., Ltd; T09-1S Magnetic Stirrer: Shanghai Silo Instrument Co., Ltd; GJB60-70 High Pressure Homogenizer: Changzhou Homogenizing Machinery Co.

 

1.3 Test methods 1.3.1 Sample treatment

Fresh crayfish were boiled in boiling water for 2 min and then cooled down naturally, peeled off the shrimp and internal organs, washed the head, tail and shell under running water, protected from light and sealed at -20 ℃ for use. Before the test, use the universal pulverizer to pulverize, sieve and then use.

 

1.3.2 Extraction of astaxanthin

Take a certain mass of shrimp shell powder, add 20 times the volume of acetone solution soaked for 2 h, ultrasonic treatment 30 min after centrifugation, take the supernatant, repeat three times, mixed supernatant, rotary evaporation at 60 ℃, dissolved in acetone and volume to 10 mL [5].

 

1.3.3 Preparation of microcapsules

(1) Preparation of aqueous phase: Weigh a certain amount of wall material, add appropriate amount of water and 0.2% Spectrum-80 mixing, magnetic stirrer constant temperature stirring for 30 min;

(2) Oil phase preparation: astaxanthin acetone solution was added into soybean oil, mixed well, nitrogen gas blew off the acetone, added 0.1 % of Tween-80, and stirred with a magnetic stirrer for 10 min [6];

(3) Microcapsule preparation: put the aqueous phase into the constant temperature magnetic stirrer and stir (speed 1 200 r/min), slowly drop the aqueous phase, then add the appropriate amount of water to adjust to the appropriate concentration, and then homogenize with high pressure homogenizer for use;

(4) Spray drying: The mixture is spray dried under the conditions of inlet temperature 185 ℃ and outlet temperature 85 ℃~95 ℃ to get astaxanthin microcapsule powder.

 

1.3.4 Influence of factors on the effect of microencapsulation

The wall material type (porous starch, β-cyclodextrin, maltodextrin, 80 % porous starch + 20 % gum arabic, 80 % β-cyclodextrin + 20 % gum arabic, 80 % maltodextrin + 20 % gum arabic), the wall material mass concentration (0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35 g/mL), the astaxanthin addition mass fraction ( 0.5 %, 1.0 %, 1.5 %, 2.0 %, 2.5 %, 3.0 %, 3.5 %), stirring time (20, 30, 40, 40, 50, 60, 70, 80 min), and stirring temperature (25, 30, 35, 40, 45, 50, 55 ℃) as factors, and microcapsule encapsulation rate as the evaluation index, to investigate the effect of the factors on the effect of astaxanthin microencapsulation.

 

1.3.5 Evaluation of microencapsulation effect 1.3.5.1 Plotting of standard curve

The absorbance was measured at 478 nm after homogeneous mixing, and the standard curve was plotted with the concentration of astaxanthin standard solution as the horizontal coordinate and the absorbance value as the vertical coordinate. The results showed that the linear equation of astaxanthin in the range of 10 μg/mL~50 μg/mL was y=0.008 6x-0.006 5, with a correlation coefficient of 0.999 5, which was in accordance with Lambert's law and could be used for the determination of astaxanthin content.

 

1.3.5.2 Determination of astaxanthin content on the surface of microcapsules

0.5 g of dried astaxanthin microcapsule powder was accurately weighed, 20 mL of n-hexane was added, and the extract was repeatedly shaken until colorless, the extract was mixed, centrifuged, evaporated by rotary evaporator, and then re-dissolved in acetone, and the absorbance was measured at 478 nm, and the astaxanthin content on the surface of the microcapsule was calculated according to the standard curve[7] .

 

1.3.5.3 Determination of microencapsulation rate

Accurately weigh 0.5 g of dried astaxanthin microcapsule powder, add 20 mL of water to stir into a homogeneous system, and then add 20 mL of acetone, repeated shaking extraction to colorless, mixed extract, anhydrous sodium sulfate dehydration, centrifugal separation, evaporated with a rotary evaporator, and re-dissolved in acetone, absorbance at 478 nm, according to the standard curve for the calculation of astaxanthin microcapsule in the total content of the microcapsules and calculated according to the following formula The total content of astaxanthin in microcapsules was calculated from the standard curve, and the embedding rate of microcapsules was calculated according to the following formula.

Microencapsulation rate/% = (1 - ) × 100

 

2 Results and analysis

2.1 Influence of wall type on the effect of microencapsulation

The type of wall material directly determines the amount of core material embedded, the slow release effect and storage stability, and also affects the solubility and flowability of the finished product [8]. Therefore, the selection of wall materials is essential to improve the efficiency of microencapsulation and to obtain microencapsulated products with superior performance. The effect of wall material type on the microencapsulation effect is shown in Fig. 1.

As shown in Fig. 1, when the three starch wall materials (porous starch, β-cyclodextrin and maltodextrin) were used individually, porous starch showed the lowest embedding rate of astaxanthin (38.81 %), while β-cyclodextrin showed the highest embedding rate (62.88 %). This may be due to the difference in molecular weight. Although porous starch has a large specific pore volume and surface area, it is still a starch with a large molecular weight; maltodextrin is a product of starch hydrolysis, with a molecular weight between 100 and 5,000, and some of them can reach more than 15,000; and β-cyclodextrin has the smallest molecular weight of the three, which is only 1,134.98. β-cyclodextrin has the largest number of molecules per unit mass of starchy wall material dissolved in water and can form microcapsules with astaxanthin, which may lead to the formation of microcapsules with astaxanthin, which may be the most effective way for the formation of microcapsules. The number of β-cyclodextrin molecules per unit mass of starch wall material dissolved in water is the largest, and the possibility of forming microcapsules with astaxanthin is also the largest[9] . In addition, β-cyclodextrin has a special structure of "inner cavity hydrophobic, outer wall hydrophilic" hollow oblique truncated cone, when water, β-cyclodextrin and non-polar astaxanthin core material are mixed and stirred, the repulsive force generated by the polar water will rapidly push the small molecules of astaxanthin into the cavity of the β-cyclodextrin cone structure, and the microcapsules will form through van der Waals' force[2-3] . 3].

 

From Fig. 1, it can also be seen that the encapsulation effect of the wall materials formed by the combination of the three starches and gum arabic is better than that of the wall materials used alone. This is mainly because of β-cyclodextrin, maltodextrin as a representative of the starch wall material is not only good water absorption, oil-absorbing capacity, easy to dry, do not absorb moisture, but also has good mechanical strength and thermal stability, dispersed in water and other solvents to maintain the structural integrity of the obvious[6] . Hydrocolloid wall materials represented by gum arabic have low viscosity, good fluidity, film-forming properties, emulsification and other characteristics [10-12], the use of the two can not only make up for the interfacial properties of starch wall materials do not have, improve the densification of the microcapsules, increase the effect of protection of the core material to reduce the high-temperature spray drying, oxygen, and external factors such as light and other astaxanthin structural changes, but also improve the amorphous state of the colloidal wall materials, and also improve the structure of the colloidal wall materials. It also improves the amorphous state of colloidal wall materials and increases the mechanical strength of microencapsulated products[6] . Comparing the six wall materials, it can be seen that β-cyclodextrin + gum arabic has the highest embedding rate, so 80% β-cyclodextrin + 20% gum arabic was chosen as the wall material for the embedding treatment. 2.2 Effect of wall materials on microencapsulation effect

The effect of wall additions on the effect of microencapsulation is shown in Fig. 2.

 

As shown in Fig. 2, when the mass concentration of the wall material was 0.05 g/mL, the embedding rate of microcapsules was only 13.83%, which was mainly due to the fact that it was difficult for astaxanthin to be completely embedded in the closed structure formed by a small amount of wall material, and astaxanthin present on the surface of the microcapsules was susceptible to the influence of high temperature and lost its activity during the spray drying process.

The embedding rate increased with increasing wall material mass concentration. When the concentration of wall material reached 0.20 g/mL, the embedding rate of microcapsules reached the maximum value (80.15 %). This is mainly due to the increase in wall material concentration, increase the collision rate of wall material and core material per unit volume, accelerating the cohesion of microcapsules; at the same time, the increase in wall material concentration makes the solution viscosity increase, and the high viscosity of the cohesive phase is more conducive to the spreading of astaxanthin on the surface, and the formation of a more stable microcapsule structure [13]; in addition, the increase in the amount of wall material also accelerated the speed of droplet film formation in the spray drying process, reducing the loss of the core material, and the embedding rate increased as well. In addition, the increase in the amount of wall material also accelerates the speed of droplet film formation during the spray drying process, which reduces the loss of core material and improves the efficiency of microcapsules.

When the amount of wall material added was greater than 0.20 g/mL, the microcapsule embedding rate decreased slowly when the wall material mass concentration was increased. This may be due to the fact that when the wall material mass concentration is 0.20 g/mL, the core material has basically saturated, if continue to increase, too much wall material through the molecular bonding aggregation makes the solution viscosity increase, which not only reduces the diffusion speed of the wall material and the core [7], but also makes the path of the core material to enter the interior of the wall material is blocked [3, 14]; In addition, too much wall material makes the droplet atomization speed droplets in the spray drying, material In addition, the excessive amount of wall material makes the droplet atomization speed decrease during spray drying, and the residence time of the material before atomization becomes longer, and the astaxanthin loss is increased by the influence of oxygen and heat [15].

 

2.3 Effect of core addition on microencapsulation effect

The effect of core material addition on the microencapsulation effect is shown in Fig. 3, from which it can be seen that when the mass fraction of core material added is in the range of 0.50 % to 2.50 %, the embedding rate of astaxanthin basically stays at a high level of about 80 %, with a small range of variation. This is mainly due to the fact that in this range, the wall material is sufficient and the concentration is appropriate, which can basically completely encapsulate astaxanthin. The slight fluctuation of the embedding rate may be due to the slight difference in drying temperature caused by the difference in the amount of water evaporated during spray drying.

When the mass fraction of astaxanthin added was greater than 2.50 %, the encapsulation rate decreased slightly with the increasing amount of astaxanthin added, which indicated that 0.2 g/mL of β-cyclodextrin+gum arabic mixture was the most suitable for microencapsulation with 2.50 % astaxanthin. Continuing to increase the astaxanthin addition would result in an excessive core-to-wall ratio and a thin and unstable microencapsulated capsule wall [13]. When the astaxanthin quality fraction was increased from 3.00 to 3.50 %, the embedding rate decreased rapidly, which may be due to the insufficient wall material, resulting in part of the astaxanthin was not embedded, and the surface astaxanthin was decomposed and transformed by the damage of spray drying. In addition, in the later spray drying process, the lower the concentration of the core material, the greater the energy consumption, so we chose to add core material in the range of 2.0% to 3.0% for the next step of the experiment.

 

2.4 Influence of stirring temperature on the effect of microencapsulation

The effect of stirring temperature on the microencapsulation effect is shown in Fig. 4, from which it can be seen that at 25 ℃, the encapsulation rate of astaxanthin is only 23.72%, which is mainly because at lower temperatures, the solubility of β-cyclodextrins in the aqueous solution is small, and even if there is the suspending effect of gum arabic, there is still a large amount of β-cyclodextrin precipitation, so that it can't provide a large number of β-cyclodextrins in molecular state and astaxanthin binding with each other; With the increase of temperature, the diffusion speed of wall and core molecules increased, and the collision rate of astaxanthin molecules with β-cyclodextrin and gum arabic molecules increased, which was favorable for the formation of microcapsules; in addition, the gradual increase of temperature also led to the decrease of the viscosity of the solution, and the permeability of astaxanthin increased, which was easy to diffuse to the inside of the β-cyclodextrin to form the microcapsule mesh structure [14]. However, when the temperature was too high, the long conjugated unsaturated double bond structure of astaxanthin was oxidized and decomposed by heat [1]; at the same time, the high temperature would also destroy the hydrophobic bond between β-cyclodextrin and astaxanthin, and release astaxanthin from the hollow structure of β-cyclodextrin [2]; in addition, when the temperature was higher than 50 ℃, part of the β-cyclodextrin appeared to be pasty, which would also lead to the destruction of the microcapsule network, and the rapid decrease of the embedding rate.

 

2.5 Effect of stirring time on the effect of microencapsulation

The effect of stirring time on the microencapsulation effect is shown in Fig. 5, which shows that the microcapsule embedding rate was very low (only 55.32%) in a short period of time (20 min). With the extension of stirring time, the microcapsule encapsulation rate increased rapidly, which may be due to the fact that the β-cyclodextrin encapsulation reaction mainly relies on the spatial resistance of molecules and the repulsive effect of water to adsorb the material, which is slow and time-consuming, while magnetic stirring can make the molecule diffusion speed increase rapidly in a short period of time. When the stirring time reached 50 min, the astaxanthin embedding rate reached the maximum value (87.89%), and at this time, the β-cyclodextrin embedding reaction was in a dynamic equilibrium state, and the reverse reaction of core elution and dissolution was basically balanced with the positive reaction of adsorption and embedding [2]. When the stirring time was delayed, the embedding rate decreased slowly, which was mainly due to the decomposition of part of the dissolved astaxanthin under the action of light, oxygen and heat [15], so a stirring time of 50 min was chosen for the subsequent test.

 

2.6 Optimization of microencapsulation preparation process

2.6.1 Response Surface Analysis Program and Results

Comparing the results of the four variable factors on the embedding rate, it can be seen that, within the range of factors, the wall material change has the greatest effect on the embedding rate (extreme deviation = 66.32%), followed by stirring temperature (extreme deviation = 64.09%), and the stirring time has the smallest effect on the embedding rate (extreme deviation = 32.57%). Therefore, the stirring time was fixed at 50 min, and the Box-Benhnken central combinatorial design principle was applied to optimize the three factors of wall material addition, core material addition and stirring temperature with astaxanthin embedding rate as the response value. The coding of factor levels is shown in Table 1, and the experimental program and results are shown in Table 2.

 

2.6.2 Response surface analysis and microencapsulation process optimization

One of the three factors X1 (wall material addition), X2 (core material addition) and X3 (stirring temperature) in the model was fixed at the zero level, and sas software was used to plot the contours and corresponding surface plots of the sub-models of the effects of the remaining two factors on the astaxanthin embedding rate, and the results were shown in Fig. 6~Fig. 8.

From Fig. 6 to Fig. 8, it can be seen that the contour plots of each factor are elliptical in shape, which indicates that all these factors promote the embedding rate at lower levels, but higher levels are not conducive to the production of microencapsulation. From the response surface plot, it can be seen that the graph is convex, which indicates that the synergistic effect of the factors is obvious, i.e., the increase of one factor promotes the effect of another factor on the embedding rate.

 

2.6.3 Determination of the optimum extraction process

The optimal conditions and optimal values were obtained by fitting the quadratic regression model with sas software, and the results are shown in Table 4. It can be seen from Table 4 that the optimal coded values of X1, X2 and X3 were -0.168 810, 0.363 710 and 0.180 659 at the maximum embedding rate, and the optimal extraction conditions were estimated as follows: wall additive 0.191 559 g/mL, core additive 2.681 855 %, stirring temperature 40.903 296 ℃, and the maximum embedding rate was 92.365974 %. 2.681 855%, stirring temperature of 40.903 296 ℃, and the maximum embedding rate of 92.365974%.

In order to verify the agreement between the estimated results and the actual situation, a validation test was conducted, and the test result was 92.89%, which was basically consistent with the estimated results. Therefore, the optimal process conditions for microencapsulation were determined as 0.19 g/mL of wall material, 2.68% of core material, and stirring temperature of 40.9 ℃, which resulted in 92.89% astaxanthin encapsulation.

 

3 Conclusion

The effects of wall material type, wall material addition, core material addition, stirring time and stirring temperature on the microencapsulation of astaxanthin were investigated by using the Box-Behnken centralized combinatorial experimental design (CCEED) and response surface analysis (RSA), and the microencapsulation process was studied in the shells of crayfish. The results showed that the optimal conditions for astaxanthin microencapsulation were 0.19 g/mL of wall material, 2.68% of core material, and 40.9 ℃ of stirring temperature, and the encapsulation rate of astaxanthin reached 92.89% under these conditions.

 

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