PROTEASES RECOVERY FROM SURIMI WASH WATER: EFFECT OF

MOLECULAR WEIGHT CUT-OFF

PROJECT PROPOSAL

FINAL YEAR PROJECT 2012/2013

UNIVERSITI MALAYSIA TERENGGANU

ZAIFAH BT CHE WIL

UK21695

SARJANA MUDA TEKNOLOGI (ALAM SEKITAR)

2012/2013

ASSOC. PROF. DR. NORA’AINI BT ALI

ii

TABLE OF CONTENTS

LIST OF FIGURES iii

LIST OF ABBREVIATIONS iv

ABSTRACT v

ABSTRAK vi

CHAPTER 1 INTRODUCTION 1

1.1 Research background 1

1.2 Problem statement 2

1.3 Aims 3

1.4 Objectives 3

1.5 Scope of studies 4

CHAPTER 2 LITERATURE REVIEW 5

2.1 Ultrafiltration method by Ohmic heating 5

2.2 Method of Ultrafiltration membrane 6

CHAPTER 3 METHODOLOGY 7

3.1 Preparation of Surimi Wash Water 7

3.2 Pre-treatment with Acid 7

3.3 Treatment with acid and heat 8

3.4 Concentration by ultrafiltration 8

3.5 Determination of protease activity 8

3.5.1 Alkaline Protease 8

3.5.1 Acidic protease 9

CHAPTER 4 EXPECTED RESULTS 10

REFERENCES 11

APPENDIX 13

iii

LIST OF FIGURES

Figure 1: Process preparation of surimi wash water 7

Figure 2: Effect of membrane size on protease purity 10

iv

LIST OF ABBREVIATIONS

MWCO Molecular Weight Cut Off

NSBM Nilai Sekatan Berat Molekul

PVDF Polyvinylidenefluoride

UF Ultrafiltration

v

ABSTRACT

Protease is an enzyme that has a high commercial value and has been widely used in

various industrial fields. Protease recovery from surimi wash water can be done with

ultra filtration method. The main objective of this study is to identify the effects of

different molecular weight cut off (MWCO) towards the quantity of protease

recovery. Swai fish or Pangasius Sutchi is used in process of preparing surimi wash

water. Study will be conducted in two stages: the method for protease content before

and after pre-treatment as well as methods to determine the appropriate MWCO

protease maximum revenue. Four types of membranes made from the same material

and are produced by the same manufacturer but has a different molecular weight cut

off will be used to determine the best quantity of protease. Yield protease content of

each type of membrane will determine the number of levels required to produce the

maximum protease recovery value. It is expected that value of the lowest molecular

weight cut off is ideally used for protease recovery in surimi wash water. While only

two screening stages are sufficient enough to get the good quality and quantity of

protease. Utilization of protease recovered from surimi wash water would be a means

to efficiently use raw materials and significantly reduce the burden of wastewater

treatment.

vi

ABSTRAK

Protease merupakan sejenis enzim yang memiliki nilai komersial yang tinggi dan

telah digunakan secara meluas dalam pelbagai bidang industri. Pemulihan protease

daripada air basuhan surimi boleh dilakukan dengan kaedah penuras ultra. Objektif

utama kajian ini ialah untuk mengenalpasti kesan nilai sekatan berat molekul (NSBM)

yang berlainan terhadap kuantiti pemulihan protease. Ikan patin digunakan dalam

proses penyediaan air basuhan surimi. Kajian dilakukan melalui dua peringkat iaitu

kaedah untuk mendapatkan kandungan protease sebelum dan selepas pra-olahan dan

juga kaedah untuk menentukan NSBM yang sesuai dalam perolehan protease yang

maksimum. Empat jenis membran yang diperbuat daripada bahan yang sama dan

dihasilkan oleh pengeluar yang sama tetapi mempunyai nilai sekatan berat molekul

yang berbeza digunakan untuk menentukan kuantiti hasil protease yang terbaik. Hasil

kandungan protease daripada setiap jenis membran akan menentukan bilangan tahap

tapisan yang diperlukan untuk menghasilkan nilai perolehan protease yang

maksimum. Dijangkakan nilai sekatan berat molekul yang terkecil adalah sangat ideal

digunakan untuk pemulihan protease dalam air basuhan surimi. Manakala hanya dua

tahap tapisan sudah memadai untuk mendapatkan hasil protease yang berkualiti.

Penggunaan kembali protease yang telah dipulihkan adalah langkah terbaik dalam

pengendalian bahan mentah secara cekap di samping boleh mengurangkan beban

rawatan air sisa.

1

CHAPTER 1

INTRODUCTION

1.1 Research Background

Surimi is a processed white fish which is flavored and colored to mimic seafood. It

has been introduced in Malaysia in early 80s. Since then, the valuable fish minced has

become popular and be a part of food processing industries. Surimi acts as a raw food

ingredient in producing an artificial seafood product such as crab stick, lobster and

shrimp. The process of making surimi through heading, gutting and minching the fish

requires large volume of water to remove the unnecessary compound in the fish flesh.

At least two times of washing process is required to produce a tasteless and odorless

surimi. Washing removes compounds such as sarcoplasmic proteins, inorganic salts,

low-molecular weight substances, lipids, and blood components (Christina, 2000). As

a result of washing, approximately 40–50 g/100 g of minced fish solids containing

primarily water-soluble proteins are lost in the entire process. Thus, 40–50 g/100 g of

the product that is considered waste has the potential for recovery (Bourtooma, 2009).

According to Rahman (2000), it has been estimated that more than 70 % of protein

losses occurred during the first washing operation in surimi manufacture. In recent

years, membrane filtration has become a good option invented to recover proteins and

other material from wastewater. Membrane process is a technique that permits

concentration and separation without use of heat and has emerged as one of the fastest

growing process in recovery of valuable materials from industrial wastewater today.

UF involves the pressure-driven separation of materials from water using a membrane

pore size of approximately 0.002 to 0.1 microns, an MWCO of approximately 10,000

to 100,000 daltons, and an operating pressure of approximately 200 to 700 kPa (30 to

100 psi). UF membranes can be fabricated essentially in one of two forms: tubular or

2

flat-sheet. UF membranes are usually characterized by a molecular weight cutoff

(MWCO) rather than by a particular pore size. The definition of MWCO generally

used is the molecular weight of globular protein that is 90 % retained by the

membrane, given in Daltons or gram-molecular weight. There has been increased

interest in utilizing seafood processing waste due to limited biological resources and

increased environmental concerns. Attention has been given to protease due to their

essential role in food processing, biotechnology and other industries (Benjakul, 1997).

Today, proteases account for approximately 40% of the total enzyme sales in various

industrial market sectors, such as detergent, food, pharmaceutical, leather,

diagnostics, waste management and silver recovery (Gupta, 2002). This dominance of

proteases in the industrial market is expected to increase further by the year 2005

(Godfrey and West 1996). This project will propose a way in determining the effect of

protease recovery of surimi wash water using different membrane molecular weight

cut off (MWCO) value. Four different MWCO which are 25kDa, 50kDa, 100kDa and

200kDa will be use for this research.

1.2 Problem Statement

Waste water treatment in the seafood industry is a very complicated issue raising

concerns from environmental regulatory agencies, law makers, the general public, and

seafood processors. The real focus of the issue is to reduce environmental pollution

and also to recover valuable protease from surimi waste water. Lin (2005) reported

140,000 MT of surimi generates approximately 2.8 million liters of wastewater. For

every 1 kg of fish processed into frozen surimi, approximately 5.7 L of waste water

was generated. To process 132 kg of fish, total waste water generated was 750 m3 per

day, of which 63% (473 m3) was generated in the washing-dewatering processes

(Huang, 1997). The discharging from extensive using of water in surimi processing

causes pollution in water stream. The water left over from the manufacture of surimi

is characterized as waste water. It is composed of many water-soluble substances,

fats, and suspended particles. Lin (1995) pointed out that the wastewater from the first

washing or rinsing by fresh water in surimi production contains the highest

concentrations of protein, non-protein nitrogen, fat, and ash, besides a strong fishy

odour, although it constitutes only 1.5% of the total processing water. Recycling of

processing water is gaining in importance to surimi processors due to rising utility

3

costs, limited water resources, and pollution problems associated to disposal.

Environmental regulations require that manufacturers treat this water before returning

it to the environment. Surimi wash water contains high amounts of valuable

component such as proteins and enzymes. Proteases represent one of the three largest

groups of industrial enzymes and find application in detergents, leather industry, food

industry, pharmaceutical industry and bioremediation processes (Gupta, 2005). As

demand in the market of protease is high especially for industrial use, so protease

recovery will help to reduce the dependency on the use of raw materials. Maximum

utilization of wastes from fisheries has been strongly encouraged due to limited

biological resources and increased environmental concerns. Recovering protease from

surimi wash water is not only lightens the burden of waste water treatment but it is

also brings an economic value by reducing its dependence on raw materials.

1.3 Aim

This research aims to study the effect of different membrane molecular weight cut off

(MWCO) towards protease recovery for surimi wash water.

1.4 Objective

This project will mainly focus on the following objectives:

1. To determine acidic and alkaline protease content before and after pretreatment

using pH 6.

2. To identify the effect of different membrane molecular weight cut-off (MWCO)

for surimi wash water treatment in order to recover high yield protease content

1.5 Scope Of Study

Scope of study include several thing below

1. Type of sampling

The fish will be used in this experiments is swai fish (Pangasius Sutchi) obtained

from the local fish market. 10 kilograms of fish will be processed to produce surimi

wash water.

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2. In-situ analysis

This research will be conducted at Makmal Penyelidikan Siswazah, Fakulti Sains Dan

Teknologi, Universiti Malaysia Terengganu.

3. Laboratory analysis

Four types of commercial Polyvinylidenefluoride (PVDF) Tubular membrane made

from the same material and are produced by the same manufacturer but has a different

molecular weight cut off (25kDa, 50kDa, 100kDa and 200kDa) will be use in this

study. This research only focused on the method of treatment with acid and heat in a

process of recovering protease from surimi wash water.

5

CHAPTER 2

LITERATURE REVIEW

2.1 Ultrafiltration method by Ohmic heating

Several attempts were made to recover protease from surimi wash water. Based on

research done by Kanjanapongkul (2008), Ohmic heating is used to recover protease

from waste water. Ohmic heating is a method to heat materials by the passage of

electrical energy through them. Typically, the substance is placed in a cell that is

electrically connected to the power supply. The power supply will generate electric

field strength (EFS) across the cell. After heating, the protein becomes coagulated and

could be easily removed from the liquid phase. Benjakul (1997) also have done the

same method. His research focused on recovering protease by ultrafiltration using

rapid heat application as a pretreatment. Benjakul determine that application of heat

remove 33% protein and 92.1% total suspended solid from surimi wash water. The

advantages of using ohmic heating are: it requires a simple system and low capital

investment; it offers clean technology since no chemical additives are used in the

process; it is a highly energy-efficient method. However, difficulties have been faced

by Benjakul with protease losses may have also resulted from using a membrane with

MWCO 30kDa.

2.2 Method of Ultrafiltration membrane

Systematic studies of membrane phenomena can be traced to eighteen century, and

large numbers of work were reported and focused on UF membrane performance in

application of various wastes under different operation condition. According to

M.DeWitt (2000), enzyme recovery usually involves a combination of techniques

6

such as centrifugation, ammonium sulfate fractionation, dialysis, ultrafiltration,

diafiltration, preparative electrophoresis and chromatography such as affinity, anion

exchange, and gel. Lin and others (1995) used micro and ultrafiltration to recover

proteins and recycle water from the commercial production of surimi. Ultrafiltration

works by passing low molecular weight products such as lactose, salts, water while

retaining high molecular weight products such as protein. Morissey (2000) reported

that ultrfiltration without pretreatment is not practical. Permeation of protease was

greater with heat and acid treated samples than with acid only treated samples.

Ultrafiltration concentration of protease following pretreatment with acid and heat

resulted in yields of 80% with 50 kD and 50% with 100 kD molecular weight cut-off

membranes (M.DeWitt, 2000). Optimum expression of protease activity in acid plus

heat-treated samples occurred with HCl at pH ~5-6 and L-ascorbic acid at pH ~6. In

addition, preliminary analysis to test the permeation of protease from heat-treated

samples through a 100 kD membrane had indicated that 75% of the enzymes

remained in the retentate. Pretreatment of wash water effectively reduced interfering

proteins and endogenous inhibitors of the protease targeted for recovery. Pretreatment

significantly reduced 35-205 kD proteins in surimi wash water. Attempts to increase

protease purity by ultrafiltration or microfiltration with 300 kD, 1000 kD, and 0.3 µm

polyethersulfone membranes were not successful. However, concentration of protease

was successful utilizing 30 kD and 50 kD ultrafiltration membranes. Maximum

recovery of protease in the concentrate occurred with 30 kD and 50 kD membranes.

As a result, it has been prove in this study that combined heat and acid treatment may

be more effective in removing protein from the wash water, than either alone.

7

CHAPTER 3

METHODOLOGY

3.1 Preparation of surimi wash water

Figure 1: Process of preparation of surimi wash water

3.2 Pre-treatment with acid

Surimi wash water (20.0 g) will be treated with hydrochloric acid (HCl) by dropwise

adjustment of pH with 0.1M solution of the acid. Target pH is 6. All preparation will

be doing in duplicate. Then, acid treated sample will be centrifuged at 3000rpm for 6

min, 40oC. Supernatant will be collected using filtration. Then it is collect to store at -

20oC.

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3.3 Treatment with acid and heat

Heat-treatment was optimized in preliminary studies for a standard volume of wash

water. Optimum activity was expressed when a 10 mL aliquot was placed in a 50 mL

erlenmeyer capped with aluminum foil and placed in a 60oC water bath for 10 min.

Acid treated supematants were therefore heated as described, rapidly cooled in an ice

bath, and re-centrifuged at SOOOxg for 6 min. Supernatant was collefcted and stored

at -80oC until further analysis could be performed.

3.4 Concentration by ultrafiltration

Aliquots of wash water (25 mL) will be acidified to 0.1% L-ascorbic acid. Treated

wash water then will be centrifuged at 3000 xg. The supernatant will be collected and

a 10 mL aliquot then heat in a water bath at 60oC for 10 min. Samples is let to rapidly

cooled using an ice water bath and re-centrifuged (3000 xg, 4° C, 6 min.). A 15 g

aliquot of supernatant then will be transferred (in duplicate) into pre-weighed

centrifugal units with molecular weight cut-off at 25 kDa, 50 kDa, 100 kDa and

200kDa. Centrifugal units will be assembled and centrifuged at 5000 xg for 30 min., 1

hr., or 1.5 hr. Both permeate (filtrate) and retentate (concentrate) will be collected and

stored at -80oC until further analysis for total protein and protease (Z-Phe-Arg-NMec)

activity.

3.5 Determination of Protease Activity

There have two methods to determine proteases activity. The method is alkaline

protease and acidic proteases.

3.5.1 Alkaline Proteases

Protease activity will be determined by using alkaline proteases method. Firstly,

stock solution at 0.9 ml will be prepared by dissolving 2% hemoglobin in 0.01M

HCl. After that, a solution of haemoglobin will be added with 0.1 ml of enzyme

and incubates at 37oC for 30 minutes. Then, adding of 0.4 ml of TCA (20% w/v)

and centrifuge for 5 minute and the speed is 12000 rpm. Finally, the solution will

9

be analyses with UV-VIS spectrophotometer with 280nm wavelength. The value of

absorbent will be appeared and form the results obtain, the rejection percentage

then can be calculated.

3.5.2 Acidic Proteases

Protease activity will be determined using acidic proteases method. Firstly, stock

solution at 0.9 ml of azocasein will be prepared by dissolving 3mg/ml in 0.05M of

Tris-HCl with pH 8.0. Azocasein solution then will be added with 0.1 ml of

enzyme and incubates at 37oC for 30 minutes. Next, adding of 0.4 ml of TCA (20%

w/v) and centrifuge for 5 minute and the speed is 12000 rpm. Finally, the solution

will be analyses with UV-VIS spectrophotometer with 410nm wavelength.

10

CHAPTER 4

EXPECTED RESULT

According to the study conducted by M.DeWitt (2000), less protease has been

recovered in the retentate with the 100kDa than the 50kDa membrane. Concentration

of protease using 50kDa ultrafiltration membrane is successful in recovering about

80% of original protease activity.

Based on the result from the previous research, it is expected that the maximum

protease will be recovered in the retentate of the lower molecular cut off membrane.

Therefore, two screening stages of ultrafiltration process are enough to recover the

total protease from surimi wash water (25kDa and 50kDa).

Figure 2: Effect of membrane size on protease purity (Source: C.A.M. DeWitt, M.T. Morrissey. Bioresource Technology 81 (2002) 241-247)

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REFERENCES

Christina A. Mireles DeWitt 2000. Recovery and Utilization of Catheptic Proteases

from Surimi Wash Water. PhD thesis. Dept Food Sci and Technol, Oregon State

University, Corvallis, OR.

Christina A. Mireles DeWitt, Michael T. Morrissey (2001). Parameters For The

Recovery Proteases From Surimi Wash Water. Bioresource Technology 81 (2002)

241-247.

Godfrey T, West S (1996) Introduction to industrial enzymology. In: Godfrey T, West

S (eds) Industrial enzymology, 2nd edn. Macmillan Press, London, pp 1–8.

Gupta, R.; Beg, Q.K. And Lorenz, P. Bacterial alkaline proteases: molecular

approaches and industrial applications. Applied Microbiology and Biotechnology ,

April 2002, vol. 59, no. 1, p.15-32.

Kobsak Kanjanapongkul, Tipaporn Yoovidhya, Suvit Tia and Pisit Wongsa-Ngasri3

Songklanakarin. Protein removal from fish mince washwater using ohmic heating. J.

Sci. Technol.30 (2008), 413-419.

Lihan Huang 1997. Application of Membrane Filtration to Recover Solids from

Protein Solutions. PhD thesis. Dept Food Sci and Technol, Oregon State University,

Corvallis, OR.

Lin, T. M., Park, J.W., & Morrissey, M. T. (1995). Recovered protein and

reconditioned water from surimi processing waste. Journal of Food Science, 50, 4–9.

Potential Economic Benefits from Microbial Enzyme – Proteases. Biotech Articles

http://www.biotecharticles.com.

12

R. Gupta, Q.K. Beg, P. Lorenz. Bacterial alkaline proteases: molecular approaches

and industrial applications. Appl Microbiol Biotechnol (2002) 59:15–32.

S. Benjakul, T.A. Seymour, M.T. Morrissey, and H. An. Recovery of Proteinase from

Pacific Whiting Surimi Wash Water. Journal of Food Biochemistry Food & Nutrition

Press, Inc., TrumbuU, CT.

Soottawat Benjakul April 17, 1997. Utilization of Wastes from Pacific Whiting

Surimi Manufacturing: Proteinases and Protein Hydrolvsate. PhD thesis. Dept Food

Sci and Technol, Oregon State University, Corvallis, OR.

T. Bourtooma, M.S. Chinnan, P. Jantawat, R. Sanguandeekul LWT. Recovery and

characterization of proteins precipitated from surimi wash-water. Food Science and

Technology 42 (2009) 599–605.

Wu, T.Y., Mohammad, A.W., Anuar, N. and Rahman, R.A. Potential use of

nanofiltration membrane in treatment of wastewater from fish and surimi industries.

Songklanakarin J. Sci. Technol., 2002, 24(Suppl.) : 977-987.

Zhou, Nina August 2010. Parametric Study of Ultrafiltration Membrane System &

Development of Fouling Control Mechanism. M.S.E. thesis, Purdue University.

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Appendix 1

The budget

Item Description Cost

PVDF tubular

membrane

Four type of

membrane with

different MWCO is

required

4 x RM250 = RM1000

Swai fish 10kg will be

purchased from

local fish market

RM5/kg

RM5 x 10kg = RM50

Chemical