Extraction and characterization of acid-soluble and pepsin-soluble collagen from skin of loach (Misgurnus anguillicaudatus)

Accepted Manuscript Title: Extraction and characterization of acid-soluble and pepsin-soluble collagen from skin of loach (Misgurnus anguillicaudatus) Authors: Jing Wang, Xinli Pei, Haiying Liu, Dan Zhou PII: DOI: Reference:

S0141-8130(17)31956-6 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.08.046 BIOMAC 8038

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

1-6-2017 4-8-2017 6-8-2017

Please cite this article as: Jing Wang, Xinli Pei, Haiying Liu, Dan Zhou, Extraction and characterization of acid-soluble and pepsin-soluble collagen from skin of loach (Misgurnus anguillicaudatus), International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.08.046 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Extraction and characterization of acid-soluble and pepsin-soluble collagen from skin of loach (Misgurnus anguillicaudatus) Jing Wang1,2., Xinli Pei2,3., Haiying Liu*1,2., Dan Zhou3,4 1.State Key Laboratory of Food Science and Technology，Jiangnan University, Wuxi, Jiangsu, 214122, China 2. School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China 3.National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China 4. National Engineering Research Center for Functional Food, Jiangnan University Wuxi, Jiangsu, 214122, China *Corresponding author: Haiying. Liu Tel.: +86-510-85329036;

Fax: +86-510-85329036.

E-mail address: [email protected]

1

(Haiying Liu)

Graphical abstract

Abstract Acid-soluble collagen (ASC) and pepsin-soluble collagen (PSC) were extracted from loach skin. The yields of ASC and PSC were 22.42% and 27.32%, respectively. Sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and peptide mass fingerprint analysis revealed that loach skin contained type I collagen. There were 212 imino acids/1,000 residues in ASC and 193 imino acids/1,000 residues in PSC. Fourier transform infrared spectrometry analysis, UV measurements and circular dichroism confirmed that loach skin collagen had a triple helical structure. The denaturation temperatures of ASC and PSC were 36.03°C and 33.61°C, respectively. Zeta potential analysis revealed that net zero charge values of ASC and PSC were 6.42 and 6.51, respectively. Therefore, loach skin collagen may be an alternative to terrestrial mammalian collagen and may enhance the added value of this fish species. Keywords: Loach skin; Collagen; Characterization; Peptide mass fingerprint; Circular dichroism. 1. Introduction Collagen is the main structural protein of animals [1]. There are at least 29 types of collagen from different animal tissues and organs, and each collagen type has a specific protein structure and amino acid sequence [2]. Among the 29 collagen types, the most common is type I, which is widely used in food, cosmetic, biomedical, and pharmaceutical applications [3]. Collagen is mainly derived from the skin and bone of terrestrial animals, such as cattle, pigs, and chickens. However, due to the incidence of infectious diseases [4] and religious beliefs [5] , fish collagen has gradually become a topic of significant research interest. Researchers have extracted collagen from the skin, bone, scale, fin, and cartilage of fresh water and marine fish, such as deep-sea redfish [6-7], horse 2

mackerel [8], and tilapia [2]. Fish collagen has diverse biochemical properties based on the species, tissue, and environment [9]. Misgurnus is a genus of loaches that is commonly consumed in Asia, especially in China. Loach is rich in high quality protein, vitamins, minerals, and essential amino acids. In Traditional Chinese Medicine, loach is commonly referred to as “ginseng in the water” due to its nutritional and medicinal values [10]. However, research on loach is limited due to its breeding and propagation techniques [11-13]. Loach skin, which is a byproduct of processing that is rich in collagen, represents a potential source of this protein. The main objective of this study was to prepare and partly characterize loach skin collagen. This study could enhance the added value of this fish species and be friendly to environment. In addition it may provide some basic information for later researchers.

2. Materials and methods 2.1 Pretreatment of loach skin Loaches (M. anguillicaudatus) were purchased from a supermarket in Wuxi city, Jiangsu province, China. The weight of loaches (M. anguillicaudatus) was 15-20 g. Skin was manually removed using a scalpel and washed with cold water. Loach skin samples were soaked in 10% cold NaCl at a rate 1:5 (w/v) for 1 h at 4°C to remove mucus of loach skin and cut into small pieces (0.5 × 0.5 cm) with surgical scissors. Skin pieces were mixed with 0.1 mol/L NaOH at 4°C to remove non-collagenous proteins and pigments at a sample-to-solution ratio of 1:10 (w/v) for 24 h and washed with cold distilled water. The sample was defatted with hexane at a ratio of 1:10 (w/v) for 24 h at 4°C and washed with cold distilled water. Finally, the sample was lyophilized and stored at −20°C [14] . 2.2 Extraction of collagen The lyophilized skin sample was soaked in 0.5 mol/L acetic acid at a sample-tosolution ratio of 1:30 (w/v) for 24 h at 4°C and passed through two gauze layers with 16 mesh . The residue was re-extracted in 0.5 mol/L acetic acid at a ratio of 1:20 (w/v) for 24 h at 4°C and filtered under the same conditions. The supernatants were pooled and salted out by adding NaCl at a final concentration of 0.9 mol/L. The resulting precipitate (acid-soluble collagen, ASC) was collected following centrifugation at 8,000 × g for 30 min at 4°C and subsequently re-dissolved in 0.5 mol/L acetic acid. Salting out and solubilization were repeated three times. The precipitate was dialyzed against cold distilled water using a dialysis tubing with a molecular weight cut-off of 8,000 Da and subsequently lyophilized [14]. The residue was extracted with 20 volumes of 0.5 mol/L acetic acid containing 5% (w/v) pepsin (1,200 U/g, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) 3

for 24 h at 4°C. The viscous solution was centrifuged at 8,000 × g for 30 min at 4°C. Pepsin-soluble collagen (PSC) was obtained as previously described for ASC. 2.3 Yield Collagen (ASC and PSC) yield was calculated from the following equation, yield (%) = weight of dried collagen (g) × 100/weight of dry loach skin used (g) as previously reported [15] with slight modifications.. 2.4 UV measurements UV spectra of ASC and PSC were obtained using a spectrophotometer (UV-1800, SHIMADZU, Japan). ASC and PSC were dissolved in 0.05 mol/L acetic acid to a final concentration of 0.3 mg/ml. The wavelength was set at 200–400 nm, and the baseline was set with 0.05 mol/L acetic acid. 2.5 Fourier transform infrared spectroscopy (FTIR) FTIR spectra were obtained using an infrared spectrophotometer (IS10, Nicolet, USA) with an absorption mode at 4 cm−1 intervals and a scanning frequency of 16 × from 4,000 to 400 cm−1 as previously reported [16] with slight modifications. Discs contained 5 mg of ASC or PSC in approximately 500 mg KBr. 2.6 Sodium dodecyl sulfate polyacrylamide-gel electrophoresis (SDS-PAGE) SDS-PAGE was performed as reported by Laemmli [17]. ASC and PSC were dissolved in loading buffer at 10 mg/mL. The mixture was boiled for 3 min, allowed to cool, and centrifuged for 10 min at 4,000 × g. SDS-PAGE gels consisted of a 6% separating gel and a 5% concentrating gel. Electrophoresis was performed at 80 V for 20 min followed by 120 V for 90 min. After electrophoresis, the gels were stained with Coomassie brilliant blue R-250 solution for 30 min and repeatedly decolored at 37°C. Molecular mass markers (10–250 kDa, Sigma,USA) were used to estimate the molecular weights of the proteins. 2.7 Peptide mass fingerprint Electrophoresis was performed as reported in section 2.6. The gel was stained with Coomassie brilliant blue R-250 solution. The electrophoresis results revealed that collagen from loach skin consisted of one α2 subunit and two identical α1 subunits. For identification purposes, MALDI-TOF/TOF was performed as reported by Ahmad Asoodeh[18]. the subunit bands corresponding to the molecular mass of α2 were excised, transferred to an Eppendorf (EP) tube, cut into sections of approximately 1 mm3, and mixed with 300 µL of 100 mmol/mL NH4CO3 containing 30% acetonitrile. Following the removal of the supernatant, 50 µL acetonitrile and 5 µL of 5 ng/µL trypsin were added and incubated at 4°C for 30 min. Following the 30 min incubation, the residual liquid was removed, and 20 µL of 25 mmol/L NH4CO3 was added. The sample was incubated overnight at 37°C in a water bath. The enzyme solution was transferred to a new EP tube and centrifuged (5 µL remained). Two-stage mass 4

spectrometry was performed using an UltrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, USA) equipped with a Mascot sequence matching software with the NCBI Database. 2.8 Amino acid analysis Lyophilized ASC (100 mg) and PSC (100 mg) were dissolved in 8 mL of 6 mol/L HCl, and the mixture was evacuated with nitrogen, vacuum-sealed, and hydrolyzed at 110°C for 22 h. The amino acids in the hydrolysate were analyzed using an autoanalyzer (Agilent 1100, Agilent technologies co., Ltd, USA). Amino acid concentration was expressed as the number of residues per 1,000 residues. 2.9 Circular dichroism (CD) CD spectra were obtained to assess the secondary structure. CD spectra and denaturation temperature (Td) were determined using a MOS-450 CD spectrometer (Bio-Logic, France) [19]. ASC and PSC were diluted to 0.3 mg/mL with 0.05 M acetic acid. CD spectra were obtained using a 0.1 cm path-length quartz cell. Three scans were averaged at 190–250 nm. The melting curve of collagen was determined by heating the sample from 19°C to 50°C and monitoring the results at 217 nm. Td was defined as the temperature at which the change in ellipticity was 50% complete. 2.10 Zeta potential ASC and PSC were dissolved in 0.05 M acetic acid to a final concentration of 0.2 mg/mL and incubated at 4°C for 48 h. The zeta potential of the collagen samples was determined using a Nano Brook Omni zeta potential analyzer (Brookhaven, Inc., USA) as reported by Singh [16]. The pH of the samples (20 mL) was adjusted across a pH range (3–11) with 1 mol/L KOH and 1 mol/L HCl. The isoelectric point (pI) of ASC and PSC was determined from the pH value that resulted in a zero zeta potential. 2.11 Statistical analysis Data were analyzed using Microsoft Excel 2007 (Microsoft Corporation,USA). All experiments were repeated three times. Mean values with standard deviations were presented. 3. Results and discussion 3.1 ASC and PSC yield from loach skin ASC and PSC yield from loach skin was 22.42% and 27.32% (dry weight), respectively. Comparatively, the yield of fish skin collagen was 2.3% from black drum [20], 24.9% from ornate threadfin bream [21], and 27.2% from tilapia [2]. This study revealed that loach skin could be an alternative source of collagen. Compared to ASC, PSC had a higher yield. Similar results were reported from largefin longbarbel catfish skin collagen (ASC, 16.8%; PSC, 28.0%, dry weight basis) [22], balloon fish skin collagen (ASC, 4%; PSC, 19.5%, dry weight basis) [23] and squid (ASC, 56.8%; 5

PSC, 24.6%, dry weight basis) [24]. However, some fish species have a higher yield of ASC than PSC from skin, including brown banded bamboo shark (ASC, 9.38%; PSC, 8.86%, wet weight basis) [25], mackerel (ASC, 58.62%; PSC, 14.43%, wet weight basis) [26] and gaint grouper (ASC, 39.51%; PSC, 19.12%, dry weight basis) [27]. The difference in the results could be attributed to the fish species. 3.2 UV spectra We all knew that the maximum absorption wavelength of protein in the near ultraviolet region was 280 nm. The peak corresponded to the absorbance of aromatic amino acids (280 nm) such as tryptophan, tyrosine, and phenylalanine [28]. Some researchers have shown that the protein might be collagen if there was a maximum absorptions near 210-240nm. The UV absorption data of ASC and PSC were shown in Fig. 1. Both ASC and PSC just had maximum absorption peaks at 218 nm. No absorbance measurements were obtained at 280 nm due to low concentrations of aromatic amino acids in ASC and PSC. Similar findings were reported in collagen from walley pollock [29], shark skin [30], balloon fish [23], and squid [24]. 3.3 FTIR spectra The FTIR spectra of ASC and PSC are presented in Fig. 2. The main characteristic absorption peaks contained amide A, amide B, amide I, amide II, and amide III. Similar findings were reported in other fish species [1-3]. The amide A bands of ASC and PSC from loach skin were observed at 3,323 and 3,322 cm−1, respectively, and involved N–H stretching vibration. According to Doyle [31], N–H stretching vibration ranges from 3,400 to 3,440 cm−1 when the NH group of a peptide is associated with a hydrogen bond. This result revealed that the NH groups of loach skin collagen formed hydrogen bonds with carbonyl groups present in the peptide chain. The amide B bands of ASC and PSC were observed at 2,928 and 2,927 cm−1, respectively, which are associated with the asymmetrical stretching of CH2 [32]. The amide I band frequencies from 1,600 to 1,700 cm−1 are mainly associated with carbonyl group stretching vibrations and are characteristic of a secondary coil structure [33-34]. The amide I bands of ASC and PSC were observed at 1,658 and 1,657 cm−1, respectively. This observation confirmed that the hydrogen bonds between the N–H group and the carbonyl group were responsible for the triple helical structure [35]. Additionally, the results suggested that PSC had more hydrogen bonds than ASC, because of the greater non-helical portion of the telopeptides in ASC [30]. The amide II bands of ASC and PSC were observed at 1,548 and 15,46 cm−1, respectively, while the amide III bands of ASC and PSC were located at 1,238 and 1236 cm−1, respectively. The amide II bands corresponded to N-H bending vibrations, and the amide III bands represented C–H stretching [36]. The amide III band revealed the presence of a helical structure [37]. According to Plepis [38], the ratio of absorbance between amide III -1 and the 1400-1454 cm wavelength was 1.0, which revealed that the triple helical structure of collagen was intact. The IR values for ASC and PSC from loach skin (1.10 and 0.99, respectively) confirmed the presence of triple helical structures. 6

3.4 SDS-PAGE Fig. 3 shows the SDS-PAGE results of ASC and PSC from loach skin. There were two distinct α-regions (α1 and α2). The molecular masses of α1 and α2 were 127 kDa and 115 kDa, respectively. The chain intensity of α1 was twice that of α2, which revealed that collagen consisted of one α2 subunit and two identical α1 subunits, which were consistent with those of a typical structure of type I collagen, including two different kinds of α chains [39-40]. Type I collagen [α1(I)]2α2(I)] is the main collagen present in skin, bone, and scale. High molecular weight components were observed in ASC and PSC, which included β chains (dimers) and γ chains (trimers). 3.5 Protein identification For further identification, α2 bands excised from SDS-PAGE gels were analyzed by MALDI-TOF/TOF mass spectrometry. As shown in Table 1, individual ion scores > 61 indicated identity or extensive homology (p < 0.05). ASC and PSC subunit α2 scores (69 and 63, respectively) matched those of Ctenopharyngodon idella collagen type I subunit α2, which were higher than the individual ion scores. This result was similar to the collagen data reported by Zeng [41]. The molecular weight of ASC and PSC subunit α2 following trypsin addition is shown in Fig. 4a and Fig. 4b. The molecular weight of the peptide fragment was 2.166 kDa. 3.6 Amino acid composition The amino acid composition of ASC and PSC is presented in Table 2. ASC and PSC had similar amino acid profile. Additionally, the amino acid prolife obtained was similar to that of other aquatic species [4,21]. Gly was the dominant amino acid due to the presence of a characteristic repeat (Gly-X-Y)n throughout the central region of the A chain from the first 14 amino acid residues of the N-terminus and from the first 10 amino acid residues of the C-terminus [42]. The content of Gly was 316 and 331 residues/1,000 residues in ASC and PSC, respectively, which resulted in the aggregation of three helical α chains that formed the final collagen superhelix [9]. Compared to other collagen types, ASC and PSC had no cysteine, low concentrations of tyrosine, histidine, hydroxylysine, and methionine [43] and high concentrations of alanine, proline, hydroxyproline, and glutamic acid [3,6,9,44]. Imino acids play an important role in maintaining the integrity of the collagen structure. The imino acid (proline and hydroxyproline) concentrations of ASC and PSC were 212 and 193 residues/1,000 residues, respectively. Similar findings have been reported in striped catfish skin ASC [16] and cobia skin ASC [45]. The pyrrolidine rings of proline and hydroxyproline impose restrictions on the conformation of the polypeptide chain and strengthen the triple helix [21]. Moreover, imino acids affect collagen thermal stability. Pro- and Hyp-rich zones are likely to form junction zones stabilized by hydrogen bonds [1,46]. 3.7 CD 7

The CD spectrum of protein provides information on the protein secondary structure [47]. Fig. 5a represents a characteristic CD spectrum of ASC at 15°C with a positive peak at 217 nm and a negative peak at 197 nm. The CD spectrum of PSC had a similar profile to that of ASC. These unique spectra were characteristic of a triple helical structure [48-49]. CD data of ASC and PSC were similar to those of skin collagen [20] and bone collagen [19] from tilapia. The melting curves of ASC and PSC are presented in Fig. 5b. ASC and PSC had Td values of 36.03°C and 33.61°C, respectively. Td of fish collagen is lower than that of collagen from terrestrial animals, such as porcine skin and calf skin collagen [50]. The Td of ASC was higher than that of PSC. Some reported that collagen thermal stability is correlated with the number of imino residues primarily formed by pyrrolidine rings of proline and hydroxyproline and hydrogen bonds of hydroxyproline hydroxyl groups [51-52]. The imino acid contents of ASC and PSC were 212 and 193 residues/1,000 residues, respectively. 3.8. Zeta potential The ASC and PSC zeta potential values at various pH values are shown in Fig. 6. Collagen samples were positively charged at pH 3−6 and negatively charged at pH 7−11. When the positive charges were equal to the negative charges, the surface charge of collagen samples had a zero net charge (pI) at 6.42 (ASC) and 6.51 (PSC). The difference in pI values between ASC and PSC was attributed to the removal of PSC telopeptides by pepsin. Collagen in solution easily precipitates and aggregates due to hydrophobic interactions. Collagen isolated from various fish skins had different pI values, such as catfish skin collagen (4.72 and 5.43) [16], brown banded bamboo shark skin collagen (6.21 and 6.56) [25], and arabesque greenling skin collagen (6.31 and 6.38) [21] . 4. Conclusions ASC and PSC were extracted from loach skin and characterized. The temperature of denaturation of ASC was similar to that of porcine skin collagen, which suggests that loach skin collagen could be a potential substitute for porcine or calf collagens. 5. Acknowledgements This study was financially supported by the Fundamental Research Funds for the Central Universities (No. JUSRP1603XNC), the non-profit industry Scientific Research Special Fund Agreement (no. 201513003-8) from the Ministry of Finance, China, and Primary Research & Developement Plan of Jiangsu Province (no. BE2017316 and no. BE2017326).

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Caption of figures Fig. 1 UV spectra of ASC and PSC from the skin of loach Fig. 2 FTIR spectra of ASC and PSC from the skin of loach Fig. 3 Protein pattern of ASC and PSC from the skin of the loach:line1 and line 2=ASC;line 3=markers;line 4 and line 5=PSC. Fig. 4 Mass spectra of digested ASC(a) and PSC(b) subunit α Fig. 5 (a) CD spectra of ASC and PSC Melting curves of ASC and PSC.

from the skin of the loach at 15°C; (b)

Fig. 6 Zata potential of ASC and PSC from the skin of the loach at different pH levels.

(a)

12

Fig. 1

Absorbance

3

2

ASC PSC

1

0

200

250 300 Wavelength (nm)

13

350

400

Fig. 2

ASC PSC

60

Amide A

Absorbance

40

Amide I

Amide II Amide III

Amide B

20

0 4000

3000 2000 -1 Wavelength(cm )

14

1000

Fig. 3

15

Fig. 4

a

b 16

Fig. 5

ASC PSC

20 0

CD(mdeg)

-20 -40 -60 -80 -100 190

200

210 220 230 Wavelength(nm)

240

250

a

250

ASC PSC

200

CD(mdeg)

150 100 50 0 -50 -100 15

20

25

30

35

Temperature(℃) b

17

40

45

50

Fig. 6

Zata potential (mv)

30 25

ASC PSC

20 15 10 5 0 -5

3

4

5

6

7

pH

18

8

9

10

11

Table 1: Summary of MALDL-TOF/TOF mass spectrometry Protein

Protein description

Accession no.

Mass (Da)/PI

Coverage Score Peptide hits

ASC Collagen alpha 2(I), gi|301033167 127772/ subunit (Ctenopharyngodon 9.37 idella) α2

3%

69

2

PSC Collagen alpha 2(I), gi|301033167 127772/ subunit (Ctenopharyngodon 9.37 idella) α2

2%

63

1

Coverage: Matched peptide coverage of protein sequence.

19

Table 2: ASC and PSC from loach skin amino composition(residues/1000 amino acid residues) Amino acids

ASC

PSC

Asp

53

52

Glu

89

79

Ser

37

36

His

5

5

Gly

316

331

Thr

17

18

Arg

53

53

Ala

114

117

Tyr

2

3

Cys-s

0

0

Val

16

20

Met

13

15

Hys

5

7

Phe

14

14

Ile

11

11

Leu

21

21

Lys

22

25

Pro

117

113

Hyp

95

80

Total

1000

1000

Imino acid

212

193

Imino acid: Proline+Hydroxyproline. 20