Extraction of Collagen Through Fish Waste Fermentation Optimization and Its Cytotoxic Studies on HaCaT Cell Line

More than 800 million tons of fish are utilizing in a year and 25-30% become waste. The waste amount is beneficial source for extraction of collagen. But procedure of extraction is still to be optimized. The current study was designed to extract collagen from fish through fish waste fermentation under various conditions. For collagen extraction we did lactic acid fermentation in which yogurt and Dough bacteria was added with fish sample and placed it in incubator at 30ºC for one month. In yogurt and dough culture have at least 10 type of lactic acid bacterial species. After every week we check PH of each sample and take soup of that sample. After centrifugation of that sample’s TCA (Trichloroacetic acid) precipitation was done of yogurt and dough sample and kept at -20ºC. then did SDS-PAGE) using 6% resolving and 5% stacking gel of already stored sample. Then HaCaT cells (1 × 104 cells/well) were cultured in 96-well flat-bottom culture plates and treated with appropriate doses of FFCP (fermented fish collagen peptide) for 24 and 48 hours.The SDS-PAGE revealed that the collagen protein of fish had doublet pattern for α1 and α2 chains at corresponding to 145 kDa and 132 kDa respectively. The density for α1 twice as compared to α2. Our result agrees that The fish collagen consists mostly of α-chain as well as little amount of inter and intra molecular cross-linked components of α-chains; b (dimmer) and c (trimer). Different biochemical tests were done for identification of lactic acid bacteria catalase positive, citrate positive, urease negative. Our fermented fish collagen and peptides mixture were test for cellular cytotoxicity and proliferative effect on HaCaT cells. Current result shows that fermented extracted collagen are nontoxic and induce the proliferation of HaCaT cells. Current study is supported by various studies that revealed the medical application of fish extracted collagen as underline.

method and have short time interval but this process is very expensive. The chemical procedure comprises of the pretreatment use which is performed before the extraction of collagen may be extracted utilizing an acidic or basic process, that differs with respect to raw material origin. For the purpose of removing non collagenous substances and to get more yields pretreatment method can be utilized (Schmidt et al., 2016). For the purpose of collagen extraction, the most commonly used methods are based on the solubility of collagen in neutral saline solutions, acidic solutions and acidic solutions with added enzymes (Schmidt et al., 2016). Many known methods for extracting collagen, each method has its own limitations. Many efforts are focused to obtain collagen with high purity, high yield, reserved structural integrity and its unique properties including gel formation capabilities, water retention and thermal stability (Schmidt et al., 2016).
The acid base simplified method of collagen extraction.

1.2.1: Pre-Treatment:
Because of naturally cross-linking collagen in the connective tissues of animals' dissolves very slow, even in hot water. Small amount of chemical treatment is also needed to break crosslinks before extraction. (Schrieber and Gareis, 2007). In the end, the acids that is diluted and employed bases and collagen that is exposed to semi hydrolysis, that keeps the chains of collagen unbroken while crosslinks are cut into pieces. In pretreatment acidic form the waste materials are immersed in acidic solution till entry of solution by the substance. At a controlled temperature as the solution enters the skin structure it swells to two or three times of its actual volume and the breaking of the non-covalent inter and intra-molecular bonds occurs (Ledward, 2000). Acidic process is more suitable for more fragile raw materials with less intertwined collagen fibers, for example fish skins and porcine (Fernandes de Almeida et al., 2012). This procedure is used to thicker substances which need most energetic entry through the use treatment agents, like, bovine ossein or shavings (Ledward, 2000). Sodium hydroxide and calcium hydroxide are commonly utilized for the purpose of pre-treatment, however sodium hydroxide is more suitable for pre-treatment of the skins because it causes significant swelling, which promotes collagen extraction by increasing exchange rate of the mass in the tissue matrix (Liu et al., 2015).

1.2.2: Chemical hydrolysis
Organic acids like citric acid, lactic acid and acetic acid and inorganic acids like hydrochloric acid are used for acid hydrolysis. Though, organic acids have observed more effective compare to inorganic acids (Liu et al., 2015;Skierka and Sadowska, 2007). The non-cross linked can be dissolve by organic acids and also the inter-strand cross-liked in collagen can be break by it; which the very high solubility of collagen at the time of extraction process (Liu et al., 2015). Hence for the extraction of collagen acid solution, particularly acetic acid is used commonly. The pretreatment materials has to be added to the acid solution in order to extract acid soluble collagen, those could be 0.5 M acetic acid and should be hold under continuously stirring at 4C° for 24-72 hours depending on the type of raw material (Kaewdang et al., 2014;Liu et al., 2015;.
Filtering process is needed to isolate supernatant from the collagen after the extraction process, which has state of liquid. After that the filtrate is precipitated with NaCl to get the collagen powder.
Centrifugation has to be used to collect the precipitate and later on it could be re-dissolve in minimum volume of 0.5M acetic acid and then dialyzed in 0.1 acetic acid for 48 hours, and then distilled water are applied for 2 days, with replacement of the solution on average every 12 hours (Schmidt et al., 2016).

1.2.3: Enzymatic hydrolysis:
There is strong intermolecular covalent bond in telopeptide region of triple helix structure in collagen, which cannot be sliced by using only acetic acid which specifies enzymes like pepsin from fishes, mammalian, trypsin, papain, alkaline protease, bromelain collagenase etc. which is added to remove non-helical structures by increasing collagen solubility. Out of all these, most utilized enzyme for marine collagen extraction is pepsin (Ehrlich et al., 2010;Silva et al., 2014).
The triple helix structure is maintained by whole isolated collagen, specially collagen type I having same shape and amino acids arrangement.
The triple helix structure maintains in all extracted collagen, as the amino acid profile and similar morphology of type I collagen. Chemical analysis shows less points of interest as compared to spectral analysis of enzymatic analysis, e.g. specificity, degree control of hydrolysis, low level of actions, and in last hydrolysate lower salt contents. Additionally, enzymes have vey less use and there is no need to remove these enzymes from medium (Zavareze et al., 2009).

1.3: CONSTITUENTS OF COLLAGEN
Main protein present in skin and other connective tissue, which is present in different forms in all tissues of multicellular organisms. It performs different roles based on its position (Schmidt et al., 2016). From different species of animals, we can extract collagen and it is commonly from meat by-products. Bones, skin, tendons, and cartilage are important sources of collagen. Several other studies suggest that collagen can also be extracted from sources other than animals like birds and fish as substitute to bovine collagen from that has risk of bovine spongiform encephalopathy (Kaewdang et al., 2014;Wang et al., 2014).
The sharing of structure, composition and molar mass, and the consequent active structures and characteristics of collagen, rely on processing situations of that substances from which it is taken and the enzyme specificity used in the process of extraction. (Schmidt et al., 2016). Therefore, it's important to keep in limit process of extraction for every raw substance to get good reward and high collagen characteristics for the preferred use. Collagen can be used as industrial application because it is one of the best biomaterial due to its broad variety (Schmidt et al., 2016). In food industry collagen and gelatin has very high demand due to high content of protein and their handy stuffs, like gel formation, formation of stabilize emulsion ability and water absorption capacity.
Collagen can be used as transporter for drugs, genes and proteins and also used for human skin, blood vessels and ligaments as substitute (Gómez-Guillén et al., 2011;Kim and Mendis, 2006).
Collagen protein is present in every multicellular animals (Schmidt et al., 2016). In vertebrates and invertebrates' collagen is an essential factor in the support structures. In mammals' collagen found most abundantly resulting of about 25% of the total weight of all protein (Badii and Howell, 2006;Schmidt et al., 2016), and it is the essential protein of cartilage, bones, tendons tissues and skin particularly. Collagen in fishes and poultry play the same role as an invertebrates and is the essential constituent of the body wall (Badii and Howell, 2006).
In mammals have many type of collagen which contain typically of about 11% alanine (Ala) 21% proline (Pro), 35% glycine (Gly) and hydroxyproline. In collagen the amino acid sequence is usually repetitive triple peptide unit (Gly-X-Y), in which X is mostly Pro and Y is mostly Hyp (Schmidt et al., 2016). Until now about twenty-nine (29) type of collagen have been stated, which are placed based on their structures (Schmidt et al., 2016).

1.4: STRUCTURE OF COLLAGEN
Members of every collagen family have same right-handed triple helix which have characteristics of three alpha chain as shown in figure 1 (Lethias et al., 2010) The three identical polypeptide chain (homotrimers) may be creates the right handed triple helix structurally, as in collagen type (II, III, VII, VIII, X) and many others. Though mostly rich collagen types like (I, IV, V, VI, IX, and XI) are heteromers, which are right handed triple helix formed by two or more different heteromeric chains. An extensive left-handed helix which is created by each three α-chain and has a pitch of eighteen (18) amino acids per turn (Fallas et al., 2009). The right-handed super helix which are formed by three α-chain have one staggered residue arranged and adjacent chain as a common central axis in a right-handed style around by supercoil to make the triple helix structure.
The glycine characterizes the triple helical collagenous domain in every 3 rd point of polypeptide chain which make a (Gly-X-Y)n duplicate structure. The alpha chain assembled around central point in a way that all glycine residue is positioned in the center of the triple helix, while other amino acids larger chains are located in the outer side. This allowed the peptide chain fold strongly down the central point of the molecule while the steric clashes in the center of the assembly are been avoiding.
The proline and hydroxyproline frequently filled the X and Y positions. Depend on collagen type, some lysine and proline residue are altered by post translational enzymatic hydroxylation. For the development of intramolecular hydrogen bonds, the 4-hydroxyproline contents are vital which also helps in the conformation's stability of the triple helical. Between different types of collagen, the length of triple helical parts is different (Fallas et al., 2009;Gelse et al., 2003;Gordon and Hahn, 2010). In fibril forming collagen (I, II, III), with exemption of collagen type XXIV and XXVII, the (Gly-X-Y) replicate in the helix is the main three-layered helical domain. The length of fibril forming collagen are about 1000 amino acids residues and have perfect structure of Gly-X-Y triplet. For non-fibril forming collagen, in the triple helices have at least one interruption (Gordon and Hahn, 2010). N-propetide ore N-peptide is the fibrillar collagen end amino acids. Normally it is consisting of about one small triple helical domain called minor helix. After the formation of major triple helix, the carboxyl and amino ends are processed. The processed molecule is ranged in a procedure of a quarter stagger in the increasing fibril. Fibrils of type I and type II collagens nucleate respectively by type V and XI collagens (Gordon and Hahn, 2010;Kadler et al., 2008). The N-peptide of collagen V and XI regulate the fibril diameter which are retained after processing (Birk, 2001;Gordon and Hahn, 2010).

1.5: COMPOSITION OF COLLAGEN
Collagen peptides of fish have particular composition of amino acids with great content of proline, hydroxyproline and glycine. By ingestion of fish collagen, hydroxyproline peptides are not fully absorbed to free amino acid and in blood it can be detected. In the skin, bones and joints these hydroxyproline stimulate the cells and synthesis collagen by cell growth and activation (Subhan et al., 2015). On age collagen type III intensely depends; mostly young age skin has about 50% collagen type III but as skin but with age collagen type III percentage can be fallen from 5 to 10%.
In animal kingdom there are about twenty (20) different type of collagen. Every mature collagen consists of three polypeptide chain called α chain that might be different or same and are arranged in triple helical structure (Bella 2016). Every α chain can be identified by its nomenclature: αn(N)p, n is the identification number of α chain, p is the number of polypeptide and N is the number of collagen.
Among organs collagen distribution are different and with type the length of collagen varies. Each polypeptide is 300 nm long (corresponding to about 1000 AA residues) in type I, II, III collagens (Burgeson and Morris, 1987;CHU et al., 1987). E.g. in type I collagen each of the two identical α1(I) chain contain 1056 AA residues. Collagen molecules pack together side by side in fibrous collagen through an interchain cross-link between allysine residues formed by the extracellular lysyl oxidase (Bella, 2016).

1.6: SOURCES OF COLLAGEN
The main industrial sources for collagen production are Bovine and porcine skins. In 1930, porcine skin was the first raw material used for collagen production and nowadays porcine skin is still the most significant raw material for large scale industrial production of collagen (Gómez-Guillén et al., 2011). It has been reported that collagen of porcine skin has bovine spongiform encephalopathy (BSE), while collagen of fish has such chances of pathogen like BSE (Yamaguchi, 2002).Other than this, Jews and Muslims not accepting any food products related to pigs and Hindus not accepting or consume food products related to cow (Karim and Bhat, 2009b).
By-product from the slaughter of pork and beef are the key sources for collagen extraction (Ferreira da Silva and Barretto Penna, 2012;Jia et al., 2010). Different by products already have been studied including tendon and the Achilles , bovine bones (Paschalis et al., 2001), pericardium (Santos et al., 2013) and inner layer of bovine skin(de Moraes and Cunha, 2013), porcine lung (Lin et al., 2011) and porcine skin (Yang and Shu, 2014). Nowadays it is examined that by product of fish is alternate source for extraction of collagen (Kaewdang et al., 2014;Mahboob, 2015;Muralidharan et al., 2013;Ninan et al., 2014;Tang et al., 2015;Wang et al., 2014). Bovine spongiform encephalopathy (BSE) belongs to family of disease known as transmissible spongiform encephalopathies (TSE), which affected by the increasing of pathological prion protein (PrPSc) in central nervous system (CNS) and brain that affect bovines of adult.
Thus, many researches have keen interest to find an alternative collagen source. For now, collagen of fish waste was found to have the most relevant features to collagen of mammals. Hence, it's important to draw the industry's attention to marine collagen (Kim and Mendis, 2006;. therefore, many types of species of fish were examined as alternate source of collagen. Studies of all fish showed different denaturation temperature features. Due to this interest among research communities had raised in optimizing the yields and extraction condition and also characterizing the final collagen. These collagens were generally extracted from fish bone, scales and skin residues. Alaska Pollock, Atlantic salmon and Cod are living in cold water and were researched on their functional and physio-chemical properties. Species living in cold water showed significantly low denaturation temperatures (around 4-17 °C) while tropical species denaturation temperature is (around 18-29 °C). sub-tropical species were red Tilapia, channel catfish, yellowfin Tina, skate or grass carp, Nile perch (Gómez-Guillén et al., 2011;Karim and Bhat, 2009b).
In this type of situation collagen of fish are best as it has no risk of transmission of disease, no religious barriers and have high availability. The key difference between animal collagen and fish collagen are high essential amino acids, high biological value and low concentration of hydroxyproline. the fish fins that is waste products of canned tuna processing are high quality collagen source extracted but with low yields (Aewsiri et al., 2008)., Such as bigeye snapper (Kittiphattanabawon et al., 2005;Nalinanon et al., 2007), carp (Duan et al., 2009), threadfin bream (Nalinanon et al., 2008), yellowfin tuna (Woo et al., 2008) etc. have been characterized and extracted. Therefore, more importance has been given to alternate sources of collagen especially from fish bone, skin and scale (Nalinanon et al., 2007;Skierka and Sadowska, 2007;Wang et al., 2007).
Fish offal like bones, skin, fins and scales has gain more importance as alternate source of collagen (Gómez-Guillén et al., 2002). From the discarded portion of fish wastes fish collagen can be produced like fins, skin and scales which are rich sources of collagen (Dun et al., 2008).

1.7: BIOFERMENTATION
Bio fermentation is a natural procedure that inevitably disturb the food supply of human around the world is fermentation. The word fermentation is a Latin word which mean fervere means to boil, which indicate the presence of yeast action on extract of salted grains or fruits during alcoholic beverages production. For microbiologist fermentation word mean any process which are used for production of different products of mass culture of microorganisms. On the other hand, for a biochemist, fermentation means a process which generating energy and organic compound reacts as both electron donor and acceptor. Fermentation occur in absence of oxygen in which energy are produced. (Stanbury et al., 2013). Foods which are produce after the action of microorganism or enzymes are fermented foods due to which significant modification of food and desirable biochemical changes occur. (Campbell-Platt, 1987 (Scott and Sullivan, 2008).
Dried foods are counted as all our foods, which are mainly used for meat, milk, soybean, fish and plant products for commercial production. Acute food production is highest in Europe, Africa and North America. America in the south, large amounts of drinks and products of milk are fermented.
fermented dairy products in the middle east are important and in the Indian subcontinent grain are fingers in high amount. In Southeast and Eastern Asia, grapes and fish are the most importantly consumed nutrition, both in the source of protein. In these areas, as well as the use of cereals, can be cumulative with fingers that produce soybean in soya and rice or yoghurt production with soybean in wheat. While many yeast food items are used where they are made, there are quantities in alcohol trade, spirits and wine through growing things, and for soy sauce and for fault (Campbell-Platt, 1994).

1.8: LACTIC ACID FERMENTATION
Lactic acid bacteria are non-spore forming or coconut, Gram-positive, growing, catalase negative lacking a hem source, generally sessile and rarely nitrate reducer. Glucose has used by them for fermentation and might be also homofermentative, about 85% lactic acid could be produce by them from glucose or heterofermentive generating CO2, ethanol and acetic acid or lactic acid in the equimolar volume from lactic acid (Dworkin, 2006). Genera lactobacillus strains lactococcus, enterococcus, Aerococcus, Tetragenococcus, Vagococcus, Camobacterium, Pisciglobus created Ilactic acid; several lactobacillus strains and Leuconostoc created d-lactic acid whereas many other lactobacillus, pedicoccus and Weissella strains created dl-lactic acid (König and Fröhlich, 2017;Tanasupawat et al., 2011).

1.8.1: Condition for lactic acid bacteria (LAB) fermentation:
The most suitable environment for lactic acid bacteria (LAB) are lower ethanol contents, lower optimum PH, optimum temperature, and enough fermentable sugar contents. Furthermore, both LAB and yeast are capable to survive under less dissolved oxygen (DO) things due to which condition over turns a growth of aerobic general bacteria (Inoue et al., 2013)

1.9: FISH FERMENTATION
The products of marine finfish and fresh water, shellfish and crustaceans can be denoted by fermented fish products which are results due to the joint action of fish enzyme and bacterial enzyme with salt to carry out fermentation and avoid putrefaction (Ruddle and Ishige, 2010;Saisithi, 1994). Textural change produced by the enzymes and helped in flavor production whereas bacteria involved flavor for fish fermentation and developed the aroma (Beddows, 1998). Area which has seasonal plenty of local fish, preservation of fish occurs in those area. Fish species of fresh water certainly found in native hydrological systems have been fermented. First time the process of fermentation was developed amongst sitting farmers and anywhere salt is obtained simply. Fermentation of fish has advanced in local regions of southeast and continental East Asia (Ruddle and Ishige, 2010). In East Asia a widespread range of fermented-fish has created that an exacting grouping by product variety should be restricted to separate states. Method of preparation and end product is key cause for its classification.
fish biomass Effective utilization (fish waste and by-catch fish) is vital. Biomass of fish has recognized rich in different nutritional elements like lipids, minerals and proteins (Council, 2002).
Adding with this biomass of fish can easily ruin due to high nutritional content and high moisture.
Generally, biomass of fish is preventing from spoil by heat drying and allowed it for utilization.
Lactic acid bacteria which are usually found in raw material is used for the biological fermentation of fish waste. It could be carried out by chemical acidification using organic or inorganic acid, or introduced as starter culture (Raa et al., 1982).
Fish fermentation wastes by means of starter cultures with mixture of Lactic acid bacteria (LAB) and yeasts was observed by Faid et al. By biological fermentation fish wastes is processed using a combine starter cultures of Saccharomyces species. and Lactobacillus plantarum to change wastes of fish into stable feed ingredient (Faid et al., 1997). Process of producing of starter of sake ( Usually basic amino acids rich protein contains more nitrogen.

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properties as well as solubility of protein can be changed by denaturants. Protein analysis is complex because some foods components contains similar physical and chemical properties.
Nitrogen which are non-protein could come from amino acids, nucleic acids, peptides, amino sugars, porphyrin, phospholipids, and some vitamins, uric acid, urea, ammonium ions, and alkaloids. That's why organic nitrogen in foods would signify nitrogen initially from proteins and have low extent from all other organic nitrogen which contain non-protein substances. On methodology dependent other main food components, containing carbohydrates and lipids can affect physically with analysis of food protein.

1.11: OBJECTIVES
• The study will focus to identify the bacteria involved in fish waste fermentation, and the optimization of identified bacteria for production and purification of Type-I collagen.
• To investigate the cytotoxic effect of collagen on HaCaT culture cell line.

2.1: Study Area
The current study was designed from July 2017 to August 2018 at Department of Microbiology, Hazara university, Mansehra, KP, Pakistan.

2.2: Study purpose:
Study purpose was to do fermentation of fish through lactic acid bacteria for collagen type I extraction.

2.3: Specimens Collection and Storage
Siran River is one of the best fishing site and marketing place in District Mansehra that is situated near Hazara University. Fresh fish were collected from the Siran Fishing market in sterile bags and transported instantly to central laboratory at Department of Microbiology, Hazara University Mansehra. The specimens were washed three times and then kept at 4ºC for 24 hours for further processing. o Then autoclaved at 121ºC for 15 minutes.
o The media was preserved on room temperature at appropriate environment until further use.

2.5: Preparation of Agar Plates:
• 100 ml of already prepared HS media was poured into flask • 6 g of agar was added into the media • The media was heated on heat stirrer up to gently mixing of agar with media • The media was remained to cool to be handled easily • The media was divided into equal concentrations on 10 different sterilized petri plates • The petri plates were stored at 4 ºC.

2.5.1: Culturing on Petri Plates
➢ The petri plates were transferred to laminar flow unite.
➢ Dry dough was diluted in distilled water.
➢ The diluted dough was streaked on two different petri plates containing HS agar media through sterile swab.
➢ Similarly, yogurt was also streaked on two different HS agar petri plates.
➢ The plates were then incubated for 24 hours at 30 ºC.

2.6: Specimen Processing
The fish specimens were cut into 1-2 cm pieces and put into three different fermentation experimental media and a control medium in four different flasks (flask 1-4). Flask 1 contained HS media along with Dough culture, flask 2 contained HS media and Yogurt, flask 3 contained only HS media and flask 4 contained only distilled water (control medium). The flasks were then placed in incubator at 37 ºC. The samples (soup specimens) were taken on weekly bases from the incubated flasks.

2.7.1: Trichloroacetic acid (TCA) Precipitation
10ml of soup specimen was taken in four different 50ml tubes from the flasks on weekly bases e.g.
day 0, day 7, day 14, day 21 and day 28. One each week, the pH of the flasks was observed and noted carefully. The specimen was centrifuged on 4 ºC at 4500 rpm for 10 minutes on ultracentrifuge. After ultra-centrifugation, 3ml of soup was taken and transferred in to 15ml tubes. Then these tubes were centrifuged at 2000 rpm for 5 minutes on ordinary centrifuge. Then from each tube, 900µl of soup was transferred to 12 different 1.5ml Eppendorf tubes (3 tubes of each sample) and 100 µl of TCA was added to each tube and mixed constantly. The Eppendorf tubes were then kept on -20 ºC for 20 minutes.
After 20 minutes of freezing, the samples were centrifuged at 12,000 rpm for 10 minutes on Microspin. After centrifugation, the soup was discarded and 1ml of 80% cold Acetone was added to each tube and centrifuged again at 10,000 rpm for 3 minutes on Microspin. The Acetone was discarded after centrifugation and 1ml of fresh Acetone was added and centrifuged again. This process was repeated 2-3 times. After centrifugation, the Eppendorf tubes were put up side down for drying. After drying, 100 µl of distilled water was added and preserved at -20 ºC for further processing.

Methodology
The collagen type 1 was hydrolyzed by SDS-PAGE (sodium-dodecyl sulfate polyacrylamide gel electrophoresis) with 6% resolving gel. The gel was then kept for 45 minutes. The gel was pipetted with isopropanol to avoid oxygen inhibition of the polymerization reaction and to make the surface uniform. Then the 5% stacking gel was loaded over the resolving gel and kept for 30 minutes.
Prior to electrophoresis, 100 µl of SDS buffer (denaturing buffer) was loaded in to it and heat shock of 95 ºC for 5 minutes was given to the samples to convert the complex protein structure to linear form. Then Gyangnam protein marker and 10 µl of each sample was loaded on gel wells.
The gel tray was placed in Glycine buffer and run for 25 minutes of at 100 volts. After 25 minutes, the electric voltage was increased to 200 volts to achieve the perfect bands on resolving gel. Once the electrophoresis was completed, the gel was dyed with Coomassie Blue Brilliant G-250 Dye and incubated in shaking incubator for 24 hours at 30 ºC. then the samples were washed with acetic acid and methanol and photographed subsequently. Collagen type 1 and its hydrolyzed form were observed clearly.

2.8: Cell Culture and Reagents
HaCaT cells, spontaneously immortalized human epidermal keratinocytes, were purchased from CLS Cell Line Service and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 IU/mL penicillin, and 100 mg/mL streptomycin in a humidified 5% atmosphere at 37°C. Sub-confluent cells were harvested with trypsin-EDTA and used for further experiments.

2.8.1: Cell cytotoxicity Assay
HaCaT cells (1 × 10 4 cells/well) were cultured in 96-well flat-bottom culture plates and treated with appropriate doses of FFCP for 24 and 48 hours. Cell viability was determined using the colorimetric WST-1 conversion assay using EZ-Cytox assay kit and as per manufacturer's instructions and the percent cell viability was calculated. Experiments were performed in triplicates.

2.9: Biochemical tests for identification of Bacterial species used in fermentation
Bacterial culture is commonly identified by different biochemical tests. These biochemical tests are applied for the identification of enzymes, proteins or peptides based on biochemical activities of these enzymes and peptides. These biochemical tests give an accurate result is short time. Some biochemical tests are used in this study to identify the bacteria. Some biochemical tests used here are; oxidase test, catalase test and urease test.

2.9.1: Catalase test
This test is commonly used for the identification of an enzyme catalase secreted by many bacterial species during the growth. Some of the bacterial species produce this enzyme to catalase hydrogen per oxide to oxygen and water molecule. Bacillus species produce catalase enzyme which convert H2O2 to oxygen and water molecule. When bubbles are produced in the solution, then the bacterial sample is indicated as catalase positive.

Principle
The enzyme converts hydrogen per oxide to oxygen and water molecule. Bacterial culture to be tested is brought in contact with the hydrogen per oxide in a sterile test tube to check out the catalase activity of the culture. Whenever a bacterial culture is catalase positive it produces oxygen in the form of bubbles in the test tube. Which indicates that the culture is catalase positive. But one thing should be noticed that the culture to be tested should be fresh and should be 24 hours old.

Reagents
Hydrogen per oxide (3%), is used for the identification of catalase enzyme.
About 2-3 drops of H2O2 is poured into an autoclaved test tube. 2-4 similar culture colonies are picked though an autoclaved cotton stick and are mixed with the solution present in the test tube.
The catalase positive bacterial culture releases an enzyme, catalase enzyme, which converts hydrogen per oxide to water molecule and oxygen in the form of bubbles. The bubbles are examined by naked eyes with in few seconds.

2.9.2: Urease test
Some bacterial species hydrolyze urea and convert it in to constituents with in short of the time, like bacillus species. This test is used routinely as a diagnostic laboratory mostly.

Principle
Some bacterial culture produces an enzyme which may able to hydrolyze urea into carbon dioxide and ammonia in the presence of water molecules. The combination of these sub constituents, water molecules, ammonia and carbon dioxide results into ammonium carbonate. The presence of ammonium carbonate turns the culture medium into alkaline and turns the color of phenol red (an indicator) from yellow to bright pink color.

Procedure
The medium is prepared and autoclaved at 121ºC for 15 minutes. After, the medium is filled into the autoclaved test tubes. The broth medium in the test tube is inoculated with the help of sterilized wire loop. Pure culture is inoculated into the test tubes. The slant of each test tube is inoculated with the sample culture. After, the capes of the test tubes were kept at loose condition and the samples were incubated at 35ºC for about 20-24 hours. When the color of the medium turns to pink color, means alkaline condition.

2.9.3: TSI (triple sugar iron):
It is a microbiological test to test capability of microorganism's ability that can ferment sugar and can produce H2S (hydrogen sulfide). It is a test that has triple sugar (glucose, sucrose, lactose) and iron.
Phenol red: for identification of acidification (it is red in alkaline condition and in acidic collection it is yellow.
It also has peptone as a source of nitrogen because in aerobic condition peptone when utilize it produce ammonia.
Iron: indicator of hydrogen sulfide.

Procedure:
1. bacterial colony was picked with a well sterilized wire lope.
2. Inoculate TSI agar in the test tube and made slants of TSI medium.
3. the cap was covered loosely and incubate it at 35ºC for 24 hours.

2.9.4: Indole test
A test performed to observe the trait of an organism which can use and split tryptophan amino acid and form the indole compound.
An enzyme tryphtophanase hydrolyzed tryptophan to create three possible final products, indole is one of them. Kovac's or Ehrlich's reagent are comprised of 4(p)-dimethylamino benzaldehyde, which react with indole and produce red color compound which is a sign of indole production.
Two methods are in used; • A test detect the very active creating indole organism called spot indole test.
• Passive creating indole organism are detected by conventional tube method which need require overnight incubation.

Methods
Broth culture was inoculated to the tryptophan broth or the isolated colony of tested organism was emulsifying in tryptophan broth. Then it is incubated at 37ºC for 24-28 hours, and then 0.5ml of kovac's reagent was added to the broth culture.

2.9.5: Citrate test:
The citrate agar is used to check the ability of microorganisms that can use citrate as a sole of energy.
Medium contain (NH4)2PO4 (inorganic ammonium salt) as a source of nitrogen and citrate as carbon source. Bacteria which can grow in citrate agar media produce an enzyme called citrate permease which has the ability to convert citrate to pyruvate. 2. Placed that citrate slants in incubator at 35ºC for 3 days 3. Observed a blue color which was changed from green due to alkalanization that means test is positive.

2.9.6: Methyl red test:
In methyl red test detects that microorganisms which can perform mixed acid fermentation after supply of glucose.

Media and Reagents:
Methyl red solution, 0.02% 0.1 gm of methyl red which is already dissolved in three hundred (300 ml) of ethyl alcohol, 95 percent.
Stored at 4ºC in a bottle.

Procedure:
1. before inoculation media was allowed to adjust at room temperature.
2. 24 hour cultured bacteria was taken and inoculate in medium.
4. Added 3 drops of methyl red indicator to already incubated media. Colonies from these cultured petri plates were taken and added to fermented flasks which has already fish pieces and HS media.

Fermentation:
Fish pieces along with HS culture media and dough, yogurt culture taken in 150 ml flasks and kept in incubator at 30ºc.

3.1: Collagen Extraction Optimization:
For the extraction of fish collagen, preliminary studies were carried out for the identification of suitable extraction parameters. The samples were set at 37ºC and the changing pH was carefully observed and noted down for four weeks. Table 3. Firstly, samples from all four flasks were centrifuge at 4500rpm for 10 minutes at 4ºc and then take soup and again centrifuge in Microspin at 10000rpm for 3 minutes.

3.2: Gel Electrophoresis
The extracted collagen protein profile was observed with commercially available sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE revealed that the collagen protein of fish had doublet pattern for α1 and α2 chains at corresponding to 145kDa and 132kDa respectively. The density for α1 twice as compared to α2.
The fish collagen consists mostly of α-chain as well as little amount of inter and intra molecular cross-linked components of α-chains; b (dimmer) and c (trimer). This type of dimmer and trimer were seen in collagen of bigeye snapper (Kittiphattanabawon et al., 2005), back drum sea bream, sheephead sea bream (Ogawa et al., 2003), and ocellate puffer fish (Nagai et al.,2002). That's why

A B
it's proved that lycine derived cross link was present in the soluble collagen fraction of scales (Kimura et al.,1991).

Biochemical tests:
Catalase test: In catalase test four slides were taken and bacterial smear was formed. One drop of H2O2 was added. Bubbles were noticed for the results.

Urease test Figure 3-8: Urease negative test
In urease test urease agar was used for this test. Slants were prepared and yougurt cultures were streaked on them. These slants were incubated for 24 hours. No Color change was observed after 24 hours.

Tsi (triple sugar iron agar) test: Figure 3-9: Tsi (triple sugar iron agar) positive
• In TSI test TSI slants were formed by using TSI agar and cultures were streaked on them.
After 24 hours' incubation it was observed for color change. Cell Culturing: Cell proliferation of HaCaT cell after treatment with fermented fish collagen peptides

DISCUSSION
Greek terms "kolla" means gum and "gen" means producing, have given the word collagen (Muralidharan et al., 2013). Connective tissue has collagen as the most vital protein, and all multicellular species possess it in several forms (Schmidt et al., 2016). Bone, skin, cartilage, smooth muscle and basal lamina has collagen, which is composed of a family of proteins. ECM and connective tissue possess collagen, the vital fibrous glycoprotein which hold the structural integrity of these tissues (Sandhu et al., 2012). In human recently 28 different proteins have recognized as collagen (Vrana et al., 2008). As collagen is a vital protein and has properties like physical and chemical properties, non-toxic and biocompatible, that's why used in cosmetic, pharmaceutical industry, biomedical materials and as food additives in food industry (Tylingo et al., 2016). Old age is main reason for the deficiency of collagen in our body which lead to the looseness of skin and other organs (Sibilla et al., 2015). To fulfill the deficiency of collagen in our body, currently collagen has been isolated from the marine source. It can be used for medicine purpose and 3D biomedical engineering too (Choi and Kim, 2015). Some religious issues, primarily in Islam and Judaism and the outbreak of bovine spongiform encephalopathy usually known as mad cow resulted the non-use of mammalian collagen. Fish collagen has been substituted of mammalian collagen as a source of collagen proteins (Mahboob, 2015). Skin, bones, fines and scales of fishes such jellyfishes, squids, sponges, octopuses, fish offal and cuttlefishes are used as source of collagen proteins (Tamilmozhi et al., 2013). Successful isolation of collagen has several applications in cosmetics, food science and biomedicine (Berillis, 2015).
The current study was designed to extract collagen from fish through fish waste fermentation under various conditions. The fish waste treated with yogurt and dough culture was analysed by SDS-PAGE (sodium-dodecyl sulfate polyacrylamide gel electrophoresis) using 6% resolving and 5% stacking gel. The collagen optimization and its cytotoxic studies were performed using HaCaT cell-lines.
It is well known that different strains of enterobacteriaceae and lactic acid Bacteria mostly spoil different food types. 0,12-15. although mostly studies on just one or two type of foods. in this study we also discuss lactic acid bacteria are most dominant bacteria which can spoil food waste at temperature between 28ºC and 35ºC. LAB is well known bacteria which can play an essential role in food preservation and fermentation by lowering PH and reproduce bacteriocins which can stop the production of different pathogenic microorganisms.so in our study that's why we used dough and yogurt bacteria which is safe to use and also anti pathogenic bacteria. So the presence of LAB indicates change in PH So with increase in lacto bacillus PH increase (Newton and Gill, 1978). In our study we observe PH on weekly basis so in first week have much change in PH but with time the PH become stable. This might recognize high oil content and low PH cause by fish waste in relative anaerobic condition.
According to our result lacto bacillus isolation from yogurt and dough was more as compared to cheese. In yogurt and dough have at least 10 type of lactic acid bacteria yogurt and cheese has much importance bio medically as it is safe to use and its non-pathogenic. In cheese have dominant species of lactobacillus that is get from raw dairy products because these bacteria can grow in harsh environment (Torres-Llanez et al., 2006). But according to our results about 10 different LAB strains from dough and yogurt was isolated. Decrease in different things like loss of probiotic growth in yogurt like presence of hydrogen, low PH, increase in acidity etc.
So all of the above studies are same to our study. Results of our study showed that Bacillus subtilis was one of the common specie isolated from yogurt and dough culture fermentation, although many studies are not same to our study.
The SDS-PAGE revealed that the collagen protein of fish had doublet pattern for α1 and α2 chains at corresponding to 145 kDa and 132 kDa respectively. The density for α1 twice as compared to α2. Our result agrees that The fish collagen consists mostly of α-chain as well as little amount of inter and intra molecular cross-linked components of α-chains; b (dimmer) and c (trimer). This type of dimmer and trimer were seen in collagen of big eye snapper (Kittiphattanabawon et al., 2005), back drum sea bream, sheep head sea bream (Pati et al., 2010), and ocellate puffer fish (Nagai et al., 2002). That's why it's proved that lysine derived cross link was present in the soluble collagen fraction of scales (Pati et al., 2010).
In general, temperature, pH, extraction time and the acid influence affect the yield of collagen.
Raise in temperature and increase in time improved the yield of collagen, until it reached to optimum level of 37° (Gudmundsson and Hafsteinsson, 1997). We optimized the production of fish waste fermentation using different temperature form 30ºC to 37º C. On the other hand, declining of yield production starts after optimum conditions as the collagen is thermos-stable and denatures easily at room temperatures due to its chemical structure (Pang, 2016). The conformation of collagen may alter with high temperature for longer time that could reduce the solubility of collagen. Thus, collagen yield declines with rising temperature and lengthening the time. The amino acids composition of the fish collagen varies between species. The collagen with a lesser hydroxyproline amount exhibits a lesser thermal stability as compared to its higher amount in collagen (Karim and Bhat, 2009a).
Moreover, the pH concentration also contributes the collagen's extractability. pH can control the protein's charge density that modify the structure and electrostatic interaction of the protein (Shen et al., 2008). In current study, the pH for each sample was observed on weekly bases (see table 3).
Our fermented fish collagen and peptides mixture were test for cellular cytotoxicity and proliferative effect on HaCaT cells. Our result shows that fermented extracted collagen are nontoxic and induce the proliferation of HaCaT cells. Our results are supported by various studies that revealed the medical application of fish extracted collagen as underline.
Similarly, fish derived collagen peptides were also determined to promote HUVEC proliferation and inhibited IL-6, IL-8, and TNF-α production in lipopolysaccharide-stimulated HUVECs (Zhang et al., 2013). Recently, the marine collagen gained considerable attention because of their safety (Ólafsdóttir, 2003), and that become a key player in diverse biological properties, including antioxidant, anti-tumor , anti-hypertensive ), neuroprotective (Wong et al., 2015), anti-skin aging and epiphyseal growth-promoting (Saito et al., 2010), wound healing, and osteogenic and endothelial differentiation-promoting effects in rat bone marrow mesenchymal stem cells (Cavaliere et al., 2015).