characterization of protease soluble collagen (psc) from

DOI:10.21776/ub.jpacr.2019.008.03.506J.PureApp.Chem.Res.,2019,8(3),245-25422December2019

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Thejournalhomepagehttp.jpacr.ub.ac.idp-ISSN:2302–4690|e-ISSN:2541–0733 ThisisanopenaccessarticledistributedunderthetermsoftheCreativeCommonsAttribution-NonCommercial4.0Internationalwhichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.(http://creativecommons.org/licenses/by-nc/4.0/)

Characterization of Protease Soluble Collagen (PSC) From Milkfish Scales (Chanos chanos)

Nia Lutfiana, Suharti, Evi Susanti*

Chemistry Department, Faculty of Mathematics and Natural Science

Universitas Negeri Malang, Indonesia

*Corresponding email: [email protected]

Received 7 Nopember 2019; Accepted 22 December 2019

ABSTRACT The aim of this study was to characterize protease soluble collagen (PSC) obtained from milkfish scales, extraction using protease from proteolytic bacteria HTcUM7.1 isolate. The characterization included Fourier Transform Infra Red (FT-IR) spectra, Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) profile, Field Emission Scanning Electron Microscopy (FESEM), denaturation temperature by Differential Scanning Calorimetric (DSC) and solubility. The resulting PSC from milkfish scales has white color, fiber with a length of about 20-60 µm, FTIR spectra and SDS-PAGE profile showed that PSC was collagen Type I and denaturation temperature was 145.48 °C, with maximum solubility at pH 1-3 and 1-2 % NaCl. Its high denaturation temperature value allows the collagen to be applied in the fields of medicines and cosmetics. Key word: protease collagen soluble, milkfish fish, scales, Chanos chanos

INTRODUCTION

Increased of collagen demand is encouraging various studies on alternative collagen sources such as fish waste [1,2,3,4,5]. This is supported by various studies which state that scales, bones, and skin of fish are good sources of collagen [6,7,8,9,10,11]. Fish scales has advantages over other fish wastes as source of collagen i.e. higher collagen levels but low lipid levels, and a greater surface area than other parts of fish waste [13]. According to [6] showed that4 0-90% of the total organic compound from fish scales is collagen. Beside, the amount of milkfish production in Indonesia in 2014 reached 631.125 tons [14] and milkfish scales are a relatively abundant waste from milkfish production and have not been widely studied as collagen source.

Collagen can be enzymatically extracted from milkfish into protease soluble collagen (PSC) using specific proteases. Some of the proteases used for PSC are porcine pepsin and papain [2, 4, 6, 8, 15]. Porcine and pepsin enzymes have the advantages of being cheap but produce haram collagen, and papain which is isolated from papaya is only easily obtained in the tropics and is influenced by seasons. In this perspective, concerted efforts are needed to seek out alternative easily obtainable proteases for use in preparing halal collagen. Proteases from proteolytic bacteria can be used to solve these problems. Previous study has been isolated 7 potential proteolytic bacteria from Tauco fermented food of soya beans (HTcUM), but only HTcUM7.1 isolate was non pathogenic and produces protease that was selective to milkfish scales [16].

J.PureApp.Chem.Res.,2019,8(3),245-25422December2019

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The diversity of molecular characteristics of collagen is influenced by the source of collagen and the method used for its extraction for example molecular differences in collagen based on the source, namely type I derived from isolation from skin, bone, tendon, ligament and corneal tissue; type II originates from cartilage tissue; type III derived from muscle, and blood, collagen and type V comes from hair, cell surfaces, and placenta [17]. In addition, the constituent amino acids also differ in each collagen source. Collagen derived from silver goldfish skin, goldfish skin, cod skin, and cowhide had differences in the types and amounts of the constituent amino acids [5]. However, molecular characteristics of PSC from milkfish fish scale include molecular weight of collagen, FTIR spectrum, denaturation temperature, solubility of the influence of pH-NaCl has not been done. In this article we report the results of the characterization of PSC from milkfish scales enzymatically using proteases from proteolytic bacteria HTcUM7.1. Based on this information it can be seen the type of collagen produced and its potential applications EXPERIMENT Chemicals and instrumentation

Chemicals used for were milkfish scales obtained from Milkfish Ponds located in Sidoarjo, proteolytic bacteria HTcUM7.1 isolate that was isolated from Surabaya tauco by Biochemistry Universitas Negeri Malang Research Grup, tofu liquid waste taken from the tofu factory at Sukun, Malang, sodium chloride (Merck), magnesium sulfate heptahydrate (Merck), ferrous sulphate (Merck), potassium dihydrogene phosphate (Merck), bovine serum albumin (sigma), tyrosine (Merck), tricloroacetic acid (Merck), acetic acid (Merck), folin ciocalteu (Merck), sodium carbonate ( Merck), sodium hydroxide (Merck), hydrochloric acid (Merck), ethanol 70% (OneMed), demineralized water (Makmur Sejati)

Instrumentation applied for the analysis were electrophoresis vertical tetracell biorad, spectrophotometer Vis Q20, Fourier Transform Infrared (FT-IR) Shimadzu IR Prestige 21, Field Emission Scanning Electron Microscopy (FESEM) Hitachi SU8220 and differential scanning calorimetric (DSC) Q20.

Procedure for milkfish scales preparation

Milkfish scales were washed with cold water, followed by boiling water until the scales do not feel smooth, produced clean white scales. Scales were dried at low temperatures and stored in the refrigerator until used.

Inoculum of HTcUM7.1 isolates and production of crude extract protease

The isolate HTcUM7.1 was inoculated in skim agar (SSA) media at room temperature for 24 hours to obtain a single colony bacterial culture by the streak inoculation method. Production of crude protease extracts was carried out by means of a single colony of pure culture bacteria inoculated into 100 mL of liquid media containing (g/L) 5.0 glucose; MgSO4.7H2O 5.0; FeSO4.7H2O 0.1; KH2PO4 5.0; and tofu liquid waste 10 % v/v adjusted to pH 7 with 0.1 M NaOH. The inoculum was incubated at 37 oC and 100 rpm. After three days, the inoculum was centrifuged at 3000 rpm for 30 minutes. The supernatant obtained was a crude extract of the protease enzyme which was then tested for its activity and concentration of the extracellular protein [18]. Extraction of Protease Collagen Soluble (PCS)

The enzymatic extraction method of collagen refers to research conducted by [2] with modifications. Five grams of fish scales were immersed in 0.1 N NaOH for 6 hours at a ratio


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of 1:10 (w/v) with stirring, the NaOH solution was replaced every 2 hours. Next the fish scales were washed with sterile distilled water until the pH approaches neutral. Fish scales were added with 0.5 M acetic acid and crude extract from HTcUM7.1 isolates in a ratio of 6: 1 (v/v), stirred for 48 hours in an ice bath. The mixture was filtered and centrifuged at 10,000 rpm at 4 ºC for 1 hour. The supernatant was precipitated with NaCl to a concentration of 2.9 M in tris buffer pH 7. The precipitation results were centrifuged at 10,000 rpm at 4 ºC for 1 hour, the resulting pellets were dialyzed in demineralized water or 24 hours in an ice bath and the pellets were lyophilized.

Profile of Field Emission Scanning Electron Microscopy (FESEM) of PCS

The sample was placed on the surface of a silicon wafer and sputter-coated with a thin film of gold to prevent charging under the electron beam, prior to examination. The analysis was executed at an accelerating voltage of 5 kV and electric current of 10 µA. Micrographs of each sample was obtained under varying magnification of 500x to 25,000x.

Infrared Fourier Transform Spectrum (FTIR) of PCS

The infrared spectrum of the collagen sample was recorded in a potassium bromide (KBr) disk with SHIMADZU's ATR-FTIR spectrophotometer. One milligram of PSC was placed at the sample site and measured at wavelengths of 500-4000 cm-1.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis profile

Determination of the molecular weight of collagen with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli [19] with modifications. The separating gel concentration was 12.5 % and stacking gel was 4 %. Collagen samples were added with loading buffer (0.06 M tris HCl pH 6.8, 2 % SDS, 10 % glycerol, 0.025 % bromophenol blue and β-mercaptoethanol) at a ratio of 4:1 (v/v) then heated for 10 minutes at 70 ºC. Inserted in electrophoresis wells (AE-6200, ATTO Corporation, Japan). Electrophoresis was carried out for 4 hours at a constant voltage of 100 V. After electrophoresis, the gel was stained with 0.1 % (w/v) coomassie blue R-250 in 45 % (v/v) methanol and acetic acid10 % (v/v). Protein marker was from Promega Broad Range. Measurement of thermal transitions based on DSC

A 5.0 mg of PSC was put into a crucial. It was further placed in the DSC sample chamber, and the heating temperature was set between 40-350 ºC. Rate increment was 10 ºC every minute. Solubility of PSC

Solubility of PSC on various pH was monitored by method of referred to [1] with modification. The PSC was dissolved in 0.5 M acetic acid with a concentration of 7 mg/mL, and the mixture was stirred and incubated for 10 minutes. Then, pH of the mixtures was adjusted to pH 1, 3, 4, 5, 7, 9, 11, 13 with NaOH or HCl 1.0 M, and was made up to 2.5 ml using distilled water. The mixture was vortexed and centrifuged at 10,000 rpm for 20 minutes at 4 ºC. Protein levels in the supernatant were determined using Lowry's method, and bovine serum albumin as a standard. The relative solubility was calculated based on the ratio of pH which gives the highest solubility.

Solubility of PSC on NaCl was monitored by the following procedure. The collagen was dissolved in 0.5 M acetic acid with a concentration of 7 mg/mL, added with NaCl to a final NaCl concentration of 1, 2, 3, 4, 5, and 6%. The mixture was stirred and incubated for


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10 minutes, followed by centrifugation at 10,000 rpm for 20 minutes at 4 ºC. Protein levels in the supernatant were determined using Lowry's method, and bovine serum albumin as a standard. Relative solubility was calculated by comparing with 0% NaCl.

RESULT AND DISCUSSION Yield of PSC from milkfish scales

Milkfish scales (Figure 1a) were extracted using protease derived from proteolytic bacteria HTcUM7 produce PSC in the form of white deposits (Figure 1b) with a yield of 166 mg PSC/g of milkfish scales or 16.6% (mg PSC/100 mg of fish scales).This result is higher than that extracted PSC from salmon skin [12] using collagenase enzyme (protease) derived from Bacillus cereus (CNA1) by only 3.7 % and from Klebsiella pneumoniae (CNL3) by 2.3 %.

Figure 1. Milkfish scales (a) and PSC (b) from milkfish scales

Figure 2. SEM Micrographs of lyophilized collagen from milkfish scales magnification 25000x

Surface morphology

The SEM image of collagen was presented in Figure 2. The surface was wrinkled, possibly because of dehydration during lyophilizing. The fibrils reconstituted by linear self-assembly of collagen molecule in vitro exhibited coarse surface and uniform length of about 20-60 µm.

a b


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SDS-PAGE Profile The electrophoresis profile of PSC from milkfish scales is similar to the collagen from

Red Sea Bream and Japanese Sea Bass, where only the two bands α1 and β appear (Figure 4). This is different from the collagen electrophoresis profile from pigs, which appear α1, α2 and β bands. Initially the type of collagen from Red Sea Bream and Japanese Sea Bass was thought to be a different type of collagen from pig collagen, but on further analysis based on amino acid composition it was shown that the three are the same collagen, type I collagen [8]. This phenomenon was likely because 2α1 and α2 chains making up the heterotrimeric in collagen have almost the same size so that only one band appears [21]. Based on this, it was suspected that PSC from milkfish scales obtained was collagen type I. This opinion was strengthened by the appearance of β bands (α chain dimers) measuring around 225 kDa, referred to [22] is a typical type I collagen electrophoresis profile, although the γ (α chain trimer) bands were not expected due to the concentration of separating gels that are too high. This caused the acrylamide pores to form very tight formation and increases resistance during electrophoresis.

Figure 2. SDS-PAGE electrophoresis profile: Standard protein (M), PSC from milkfish scales (A, B, C) using 15% separating gel with Coomassie Briliant Blue (CBB) staining

Fourier-transform Infrared Spectra (FTIR)

The FTIR spectra of PSC showed bands at 3383, 2912, 1612, 1417, and 1031 cm-1 as shown in Figure 3. According [23], these bands are refers to specific IR spectra for protein repeated unit known as Amide A, B, I, II and III (Table 1). Amide A associated with N-H stretching vibration, as find in PSC from golden goatfish scale (3294 cm-1) [10], and skin of deep-sea redfish (3328 cm-1) [13]. Amide B also related with asymmetrical CH2 [24]. The Amide B peaks of PSC was similar with that of PSC from emu skin (2.925 cm-1), thus signifying that PSC from milkfish scales were arranged in a helical form. Amide I as results of backbone C=O stretching vibration. This confirmed the formation of hydrogen bond between N-H stretch (X position of repeated unit of collagen) and C=O (Gly) of the fourth


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residue, responsible for introduction into triple helix. Amide II is responsible for the combination of the N-H in-plane bend and the stretching vibration [10].

Figure 3. Fourier-transform infrared spectra of protease soluble collagen from milkfish scales

Table 1. Characteristic infrared spectra of peptide linkage

Description Approximate frequency (cm-1)*

Experiment frequency (cm-1) Assignment

Amide A 3300 3383 N-H stretching Amide B 3100 2912 N-H stretching Amide I 1600-1690 1612 C=O stretching

Amide II 1480 -1575 1417 CN stretching, N-H bending Amide III 1229-1301 1031 CN stretching, N-H bending

*Kong & Yu (2007) [22].

Amide II of PSC was found at lower frequency suggested there was stronger hydrogen bond in PSC. The presence of an Amide III peak indicated intermolecular interaction in collagen refers to a triple helical structure or collagen. Amide III of PSC was the lowest as compared to other types of collagen sources suggested that PCS have more hydrogen bonds caused by longer α-chain in PSC of milkfish scales. This prediction was supported by larger β bands on SDS-PAGE (255 kDa) than other PSC. As a comparison, β band PSC from emu [20] and β band PSC from skin and bone of Spanish mackerel were also found to be almost the same 200 kDa [2]. DSC thermograph

The thermograph of PSC from 40 to 300 oC shows that the PSC undergoes three phase changes, namely the gelatinization phase at 80 oC, denaturation or known as the melting phase at 145.48 ºC and the decomposition phase at 193.92ºC (Figure 4). Many report showed that the thermal stability of collagen was correlated with environmental and body temperature [3]. PSC from part of deep sea fish as scale of spotted golden goatfish and swim bladders of yellow fin tuna had low melting point 41.01 and 33.92 ± 0.216 °C [7,8].


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Figure 4. The DSC thermograph of PSC of milkfish scales Most importantly, the milkfish ponds are located in tropical environments with relatively higher temperatures. So, it was expected that pond-cultivated milkfish has a high melting point, too. This in line with collagen from other tropical fish the swim bladders of cunang as much as 195.59 ºC [25] and catfish skin as much 154.47 ºC [26]. Both have same habitat with milkfish fish. The thermal stability of the triple helix in collagen is attributed to the hydrogen-bonded networks, and mediated by water molecules, which connect the hydroxyl group of hydroxyproline in one strand to the main chain amide carboxyl of another chain [27]. This statement is supported by the IR for amide III in IR spectra [22] which shifts to lower frequencies.

Figure 5. Relative solubility of PSC from milkfish scale at different pH


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Solubility Determination Effect of pH The results showed the pH affected the solubility of the PSC (Figure 5). PSC solubility is highest at pH 1-3 and decreased drastically at pH 4 on wards. This is in line with previous studies showing that collagen solubility is relatively stable at acidic pH starting from pH 1-4 and begins to decrease when pH goes neutral and alkaline [1,2,27,9]. It is suspected that the PSC has a isoelectric point (pI) of around 4 so that its solubility decreases. Solubility continues to decrease at neutral and basic pH. Solubility does not increase after pH 4 presumably because PSC has a low base of amino acid residues on the outside surface. Thus, an increase in pH beyond the pI does not change the protonated state of the PSC at pH above pH 4.

Figure 6. Relative solubility of PSC from milkfish scale at different %NaCl Effect of NaCl concentration

PSC solubility is also influenced by the concentration of NaCl (Figure 6). PSC solubility is very high at the addition of 1-2 % NaCl, slightly decreases at the addition of 3-4 % and drastically decreases at the addition of 5 % NaCl. This is in line with several previous studies [1,2,27,9]. The high solubility with the addition of 1-2 % indicates the occurrence of salting in, while the decrease in solubility at 5 % NaCl indicates the occurrence of salting out due to the addition of NaCl. This result can be used for the PSC deposition process from the solution. CONCLUSION

The resulting PSC from milkfish scales has white color, fiber with a length of about 20-60 µm, FTIR spectra and SDS-PAGE profile showed that PSC was type I of collagen and denaturation temperature 145,48 °C, with maximum solubility at pH 1-3 and 1-2 % NaCl. Based on these results it is possible to develop appropriate separation techniques and their application to products that halal collagen with high thermal stability suitable for applications in cosmetics and drugs formulations.


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ACKNOWLEDGMENT The authors would like to acknowledge the LP2M Universitas Negeri Malang that

funded this research through the PNBP Program in 2019. CONFLICT OF INTEREST

Authors declare no competing interest.

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characterization of protease soluble collagen (psc) from

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