As a kind of biopolymer, hydrolysates of fish swim bladder, safer than those of land mammals, are widely used in food, cosmetics as well as pharmaceutical and biomedical fields for their biocompatibility, biodegradability, and weak antigenicity. To enhance hydrolysate production, in this paper, the papain and alcalase hydrolysis processes of larimichthys polyactis swim bladder were optimized with orthogonal experiments. With 89.5% hydrolysate yield, the optimal processing conditions for alcalase were solid-liquid ratio of 1:30, enzyme concentration of 0.7%, and extraction time of 6 h. As for papain, under the optimal processing conditions: solid-liquid ratio of 1:20, enzyme concentration of 0.5%, and extraction time of 8 h, the hydrolysate yield was 65.1%. To obtain higher hydrolysate yields, the ultrasonic pretreatments were implemented before the optimal enzyme hydrolysis processes. With ultrasonic waves of 100 W for 50 min, the hydrolysate yields were increased 2.1% (alcalase) and 4.5% (papain), respectively. The Fourier Transform Infrared (FTIR) spectroscopic analysis revealed that the hydrolysates extracted by papain exist in triple-helical forms. The Ultra-Violet (UV) absorption spectra indicated that the aromatic amino acids in the hydrolysates had strong absorptions in the wavelength range of 240 nm–300 nm. The results of this research demonstrate that the alcalase hydrolysates have better solubility in water and the solution is more stable under ambient temperature. However, the hydrolysates extracted by papain have a gel property and are insoluble in weak acid at room temperature, which is more suitable for applications in feedstock of biomedical.
As a kind of natural water-soluble biopolymer material, collagen has been used in biomedical materials [
Swim bladder, a low fat biological material, is often used in medicine and food fields because it is especially rich in natural animal protein (generally over 80%) and amino acid [
In recent years, the enzyme used to extract collagen from swim bladder was mainly pepsin [
Since it is difficult to fully extract collagen using a single method, a combination of multiple methods is often adopted to improve the yield of collagen, such as ultrasonic assistance on enzymatic hydrolysis and acid hydrolysis [
Nonetheless, the extraction and characterization of collagen from swim bladder of
Farmed yellow croaker swim bladders (
The swim bladder was soaked in distilled water for 24 h to remove ash and grease on the surface, during which the water was changed at least 3–4 times. After drying and smashing, the swim bladder powder was obtained by screening through a 2 mm mesh sieve. According to different solid-liquid ratio (1:10, 1:20, and 1:30), the swim bladder powder and distilled water were added to three beakers, respectively. The mixture was placed in a thermostatic water bath under 60°C, adding papain of different mass ratios (0.3, 0.5 and 0.7, w/w %) and stirring continuously. The enzymatic hydrolysis reaction was carried out when the inner temperature was stable. After a certain time, the mixture was filtered by a 0.09 mm aperture screen to filter out all the insoluble materials. Filtered substance was dried under 40°C until the moisture content is 8%. Compared to papain, the only difference of alcalase extraction was that the pH value of the mixture needed to be maintained around 7.5. The other extraction conditions and methods are the same as above.
The yield of hydrolysates [
The optimized process conditions of enzymatic extraction were determined by the orthogonal experiment. Based on enzymatic hydrolysis process, the solid-liquid ratio, enzyme concentration, and extraction time were used as experimental factors. The hydrolysate yield was used as the evaluation index. The orthogonal experimental arrangements, the factor levels of solid-liquid ratio, enzyme concentration, and extraction time were shown in
Test No. | A | B | C | D | Conditions |
---|---|---|---|---|---|
Solid-liquid ratio | Amount of enzyme addition (w/w%) | Extraction time (h) | Null | ||
1 | 1(1:10) | 1(0.3) | 1(4) | 1 | A1B1C1D1 |
2 | 1(1:10) | 2(0.5) | 2(6) | 2 | A1B2C2D2 |
3 | 1(1:10) | 3(0.7) | 3(8) | 3 | A1B3C3D3 |
4 | 2(1:20) | 1(0.3) | 2(6) | 3 | A2B1C2D3 |
5 | 2(1:20) | 2(0.5) | 3(8) | 1 | A2B2C3D1 |
6 | 2(1:20) | 3(0.7) | 1(4) | 2 | A2B3C1D2 |
7 | 3(1:30) | 1(0.3) | 3(8) | 2 | A3B1C3D2 |
8 | 3(1:30) | 2(0.5) | 1(4) | 3 | A3B2C1D3 |
9 | 3(1:30) | 3(0.7) | 2(6) | 1 | A3B3C2D1 |
The Numbers after the letters represent three different levels of each factor.
On the basis of the experimental scheme of the highest hydrolysate yield, dried swim bladder powder was evenly mixed according to a certain solid-liquid ratio (papain 1:20, alcalase 1:30). Then, the mixture was pretreated in an ultrasonic cleaner (KH-100SPV, Kunshan Hechuang ultrasonic instrument Co., Kunshan, China). The effects of ultrasonic time (10, 30, 50, 70, and 90 min) and ultrasonic power (60 W, 80 W, and 100 W) on the yield were studied. Each group was repeated three times, whose data were averaged.
The FTIR spectra of hydrolysates were obtained using a FT-IR spectrometer (Frontier, PerkinElmer Inc., USA) Nicolette 6700. The samples of solid hydrolysates (ca. 1.0 mg) and potassium bromide (150 mg) were grounded in an agate mortar, which were pressed after drying. The samples were measured in the range of 4000 cm-1~450 cm-1.
The UV absorption investigations were performed with a UV-VIS spectrophotometer (UV-2600, Shimadazu, Japan). The dried extracted collagen was dissolved in 0.5 M acetic acid. To obtain papain hydrolyate solution, the mixture was placed in 50°C thermostatic water bath. The UV absorption analysis was conducted in the wavelength range between 400 nm and 190 nm.
Under the optimal process condition, papain hydrolysates of swim bladder reached a yield of 65.1% (solid-liquid ratio of 1:20, enzyme concentration of 0.5%, and extraction time of 8 h) and alcalase reached a yield of 89.5% (solid-liquid ratio of 1:30, enzyme concentration of 0.7%, and extraction time of 6 h), as shown in
Test No. | Conditions | Yield with papain (%) | Yield with alcalase (%) |
---|---|---|---|
1 | A1B1C1D1 | 53.0 | 75.2 |
2 | A1B2C2D2 | 57.3 | 79.3 |
3 | A1B3C3D3 | 58.5 | 79.8 |
4 | A2B1C2D3 | 57.6 | 86.8 |
5 | A2B2C3D1 | 65.1 | 87.6 |
6 | A2B3C1D2 | 59.4 | 87.3 |
7 | A3B1C3D2 | 54.3 | 86.2 |
8 | A3B2C1D3 | 55.7 | 87.7 |
9 | A3B3C2D1 | 58.2 | 89.5 |
The extraction temperature is 60°C.
To illustrate the influence of each factor on the response, the hydrolysate yields of yellow croaker swim bladder extracted with papain and alcalase were compared. As shown in
The effects of each factor on the hydrolysate yield using papain and alcalase are shown in
Enzyme | Source of variation | Mean square | F | Significance level |
---|---|---|---|---|
Papain | A | 20.368 | 7.599 | 0.116 |
B | 17.130 | 6.391 | 0.135 | |
C | 7.875 | 2.938 | 0.254 | |
Alcalase | A | 89.195 | 275.861 | 0.004 |
B | 6.479 | 20.039 | 0.048 | |
C | 2.443 | 7.554 | 0.117 |
On the basis of the optimum process, the effect of ultrasonic time on the yield of the papain and alcalase hydrolysates was studied under the conditions of ultrasonic power of 80 W and ultrasonic frequency of 25 kHz. As shown in
Initially, the cavitation and mechanical effects produced by ultrasonic pretreatment can not only facilitate the disruption of swim bladder tissue cells and make the collagen fibers loose or broken, but also expose the active points buried inside the protein to better response to enzymes, which contributes to the improvement of the protein hydrolysis [
The effect of ultrasonic power on the hydrolysate yield was studied under the condition of the optimal hydrolysis process for swim bladder with the ultrasonic frequency of 25 kHz and an ultrasonic time of 30 min. The yield increased with the increase of ultrasonic power in the range of 60 W–100 W, and reached the maximum at 100 W, as shown in
In summary, the optimal process for ultrasonic-assisted extraction was the ultrasonic frequency of 25 kHz, ultrasonic power of 100 W, and ultrasonic time of 50 min. The yield produced was 69.6% (papain) and 91.6% (alcalase), respectively. In comparison with enzymatic methods, the yields of papain and alcalase hydrolysate were all improved through ultrasonic pretreatment. However, the increases were not significant, which were mainly due to the low power of ultrasonic cleaner used in the experiment.
The FTIR spectra and main absorption peaks of the hydrolysates prepared by different processes were shown in
In the infrared spectrum, amide I, II, and III are frequently used as characteristic bands for the identification of secondary structures of collagen, which can reflect the skeleton structure of peptide chains [
Protein molecules contain chromophores, which can absorb a certain wavelength ultraviolet light. The peptide groups have light absorption in the range of 180 nm–230 nm. The aromatic amino acids, such as tyrosine (Tyr), tryptophan (Trp), phenylalanine (Phe) also showed absorption in this region, but the largest absorptions appear in the range of 240 nm–300 nm. In twenty kinds of basic amino acids, Tyr, Trp, and Phe usually have conjugate double bonds, all of which have absorption peaks at 275 nm, 280 nm, and 258 nm, respectively [
As can be seen from
The hydrolysate yield of alcalase hydrolysis was higher than that of papain under the same processing conditions. With ultrasonic waves of 100 W for 50 min, the hydrolysate yields were increased 2.1% (alcalase) and 4.5% (papain), respectively. During the enzymatic hydrolysis process, although the molecular structures of the hydrolysates of swim bladder undergo minor changes, the triple-helical structures still dominate due to the quantity of hydrogen bonds. The UV absorption spectrum indicates that the hydrolysates are composed of aromatic amino acids. The alcalase hydrolysates have better solubility in water and the solution is more stable under ambient temperature. However, the hydrolysates extracted by papain have a gel property and are insoluble in weak acid at room temperature, which is more suitable for applications in biomedical materials.