Full Paper: PRODUCTION OF LEUCAENA SEED PROTEIN CONCENTRATE BY USING HYDROLASE ENZYMES FROM THE RUMEN OF SHEEP

PRODUCTION OF LEUCAENA SEED  PROTEIN CONCENTRATE BY USING HYDROLASE  ENZYMES FROM THE RUMEN OF SHEEP


Dedin F Rosida¹⁾, Yulistiani R ¹⁾,  Ardiani W ²⁾

1) Faculty of Industrial Technology - UPN "Veteran" Surabaya- East Java, Indonesia,
2) Food Technology of  Studies Program UPN "Veteran" Surabaya- East Java, Indonesia,

Email: rosy.upnsby@gmail.com

Abstract

.
          Protein concentrate has protein content of at least 50-70%. Protein concentrate was made by removing the non-protein components such as fats, carbohydrates, minerals, and water. protein concentrate can be obtained by hydrolyzing with an enzyme. This research used sellulose enzyme  which was derived from sheep rumen fluid to separate the protein with cellulose component. in rumen fluid of sheep there are also many other enzymes. The purpose of this research was to produced protein concentrate which is expected to be applied to food products. This study used two factors. The first factor, rumen enzyme concentration: 0%, 40%, 80% and 100% and the second factor was incubation long: 24 h and 48 h. The results showed that leucaena seed protein concentrate  produced the protein that contained 56.30 % db, and it had functional properties of oil absorption capacity of 1.58 mL/g, power foam 11.56%, on the concentration of sheep rumen fluid enzyme (10 ml / 100gr) and long incubation of 24 h, whereas the concentration of enzyme treatment of sheep rumen fluid (10 mL/100g) and incubation time of 48 h resulted in the best functional properties on water absorption of 3.42 mL/g, the density of the Kamba 0.56 g/mL, power emulsion 52.75%, 6.39% moisture content.

Keywords: protein concentrate, Leucaena, enzym of sheep rumen, functional properties
                   

Introduction

Proteins are essential food components because they are a source of amino acids needed for growth and maintenance and provide functional properties to foods. Commercially available protein foods are obtained from a range of animal and plant sources and are used as functional ingredients (PERIAGO et al.1998). Due to the increasing costs and limited supplies of animal proteins, and since vegetable protein is the most abundant source of protein on the Earth. Leucaena is a type of shrub of the tribe Fabaceae (Leguminosae, legumes), which is often used in reforestation or erosion prevention. Leucaena seeds bitter and neutral. This plant is a laxative urine (diuretic) and intestinal worms. In addition to containing mimosin, leukanin, leukanol, and protein, the leaves of this plant also contain alkaloids, saponins, flavonoids, tannins, protein, fat, calcium, phosphorus, iron, and vitamins (A, B1, and C). Leucaena  is a type of plant that can grow and thrive in tropical regions regular rainfall, even it can be to survive in areas that are dry or barren and less rainfall, such as Indonesia. Leucaena seeds contain protein 30-40%, 6.13% fat, 9.32% crude fiber, mineral water 0.26% and 35.72% with a minimum mimosine content.

Protein is an essential substance needed by animals and humans. One way of fulfilling the needs of the protein is  making food products that has a high protein content, one of them protein concentrates. This protein concentrates for many food products derived from soy protein, therefore soybean demand is getting increased. Protein concentrate is a product of protein concentrates that has a protein content of at least 50-70%. Protein concentrate is produced by removing the nonprotein components such as fats, carbohydrates, minerals, and water, this makes the protein content of the product is higher than the original raw material (Amoo et al. 2006).

The method of making a protein concentrate can be done by acid extraction, alkaline extraction and enzyme hydrolysis. Theoretically protein hydrolysis method is the most efficient use of the enzyme, because enzymes produce peptides that are less complex and easily broken. Besides hydrolysis enzyme hydrolyzate which can produce changes and damage to avoid non hydrolytic products (Johnson and Peterson 1974). According to Haslaniza et al. (2010), the hydrolysis of proteins are proteins that undergo hydrolytic degradation by acid, base, or proteolytic enzymes that produce products such as amino acids and peptides. The use of enzymes in hydrolyzing proteins is considered to be the most secure and profitable. Using enzyme hydrolysis takes place specifically, the hydrolysis process is extensively able to influence the formation of peptides and amino acids. Through the process of hydrolysis expected functional characteristics of protein modification process is also influenced by the level of hydrophobicity of non-polar part in the protein chain, the degree of hydrolysis and the type of proteolytic enzymes used (Shahidi and Botta 1994 ).

Functionality, applied to food ingredients, is defined as any property on which the utility of those foods depends (Cepeda et al.1998). Functional properties of protein concentrates, among others: water absorption (WHC), oil absorption, activity and emulsion stability, capacity and foam stability. Sheep rumen fluid is one source of alternative materials which is cheap and it can be used easily as a source of hydrolase enzymes (Moharrey and Das, 2002).  Including amylase, protease, lipase and cellulase. These enzymes include cellulase enzymes that degrade substrates that cellulase, hemicellulase / xylose is hemicellulase / xylanase, amylase starch is, pectin is pectinase, lipid / fat are lipases, proteases and protein are others (Fitriliyani, un publised). Compared to acid or alkali hydrolysis, enzymatic hydrolysis of protein, using selective proteases, provides more moderate conditions of the process and few or no undesirable side reactions or products. In addition, the final hydrolysate after neutralization contains less salts and the functionality of the final product can be controlled by selection of specific enzymes and reaction factors (Chiang et al. 1999; Madsen et al. 1997; Darwicz et al. 2000). The functional properties of hydrolysed proteins are governed to a large extent by their molecular size and their hydrophobia (Turgeon et al.1992).

Methodology
Sheep rumen fluid enzyme
Sheep rumen fluid was centrifuged at 3000 rpm for 10 minutes. The addition of 35% ammonium sulfate, stirred with a magnetic stirrer for 25 min and incubation for 24 hours at temperature 4 ^oC. filtrate centrifuged at 3000 rpm for 15 minutes. Results centrifugation filtered and added phosphate buffer pH 7 concentration of 0.05 M with a ratio 10: 1.

Leucaena seed protein concentrate
Leucaena seed flour  was made suspension (1:10) and made adjusting the pH to 7.0 with NaOH 0.1 N. Then the suspension was heated at suhu100 ° C for 5 minutes. The addition of rumen fluid crude enzyme extract (0, 2, 4, 6, C for 24 and 48 hours.°8,10 mL / 100 g) and incubated at 70 ^oC. Results centrifuged at 3000 rpm with warm water. The precipitate obtained was dried with a dryer cabinet ontemperature 45 oC

Proteolytic Enzyme Activity
5% skim milk solution included in the respective 4 erlenmeyer  of 40 mL. Enzymes with a concentration of 20%, 30% and 40% respectively as entered on 3 erlenmeyer and incubated at 37 ° C for 1 hour. Remaining erlenmeyer incubated at room temperature with the addition of 10 mL of distilled water (control). Then the sample was taken 10 mL of the sample and coupled with 2 mL of formaldehyde, pp indicator 3 drops, and titrated with 0.1 N NaOH

                                             % N = (ts-tb) mL x N NaOH x 14.008 x FP x 100%
                                                                    Sample weight (g) x 1000
Specification:
                ts = sample titration
                tb = blank titration
                sample weight = weight in grams of skim milk
                FP = dilution factor,
               
Analysis of Water Absorption (Lin et al, 1974)
A total of 1 g of sample and 10 mL of distilled water for 2 minutes vortex, chilled for 1 hour at room temperature and then centrifuged at 3000 rpm for 25 minutes. Filtrate volume was measured. Water absorption was calculated by the following equation:

Water absorption (mL water / g sample) = 10 mL - vol filtrate (mL)
                                                                       W sample (g)

Analysis of Oil Absorption (Soluski and Fleming, 1977)
A total of 0.5 g of sample plus 3 mL of soybean oil vortex for 2 minutes and then allowed to stand at room temperature. The mixture was centrifuged at 3000 rpm for 25 minutes. Supernatant was poured into 10 mL measuring cup and observed free oil volume oil absorption measurements performed two repetitions with the following equation:

Oil absorption capacity (mL oil / g sample) = 3 mL of oil - filtrate
                                                                         W sample (g)

Preparation and HPLC Analysis of Amino Acid in Leucaena Protein  Concentrate

0.1 g sample is taken and added 2 mL of Performat vortex. the mixture was hydrolyzed for 16 hours at a temperature of 0 ° C. Then added 0.3 mL of 48% HBr and incubated at a temperature of 0 ° C for 15 minutes. The solution was dried in a rotary evaporator at 60 ° C. The filtrate was pipetted 500 µL and added 40 µm AABA and aquabidest 460 µl. Furthermore, a solution of 10 µl pipette and added 70 µl AccQ-Fluor Borate and vortex. Fluor  AReagent  20  µl vortex taken and then allowed to stand 1 '. Reagents were incubated at 55 ° C for 10 '. Samples prepared injected into the HPLC. HPLC conditions : colom AccQtag column (3.9 x 150 mm), temperature 37 ^oC, mobile phase acetonitril 60% - Accq Tag Eluent A, Gradient system, flow rate 1.0 mL/min, detector Fluorescense, excitation 250 nm, emission395 nm and 5 µl  injection volume.

Result and Discussion

Enzymes are biokatalisator that can increase the rate of a chemical reaction without being reacted or without changing the balance of the reaction. Protease is an enzyme that is included in the main class of hydrolases group because this enzyme is able to catalyze peptide chain termination with the help of water and produce different end products.

The rumen is a source of enzymes that degrade polysaccharides and proteins. Sheep rumen fluid is a cheap source that can be used as a hydrolase enzymes (Moharrey and Das 2001). These enzymes include proteases /deaminase which digest protein peptides, amylase digestion of starch, cellulose-digesting sellulae, hemisellulase (xylanase) digesting the hemicellulose (xylan), fat-digesting lipase, phytase, and others (Kung 2000). In addition to containing enzymes, paunch manure also contains vitamins, minerals and amino acids.

 Polysaccharides are hydrolyzed in the rumen due to the synergistic effect and interaction of complex microorganisms, mainly producing cellulase and xillanase. Hydrolyzed by the enzyme protease protein produced in the rumen microbes. Rumen microbes secrete digestive enzymes into the rumen fluid to help degrade food particles.

Proteolytic activity of the protease enzyme is a protease enzyme's ability to hydrolyze proteins cluster into groups of amino acids that is much simpler. Dedin et al (2013), the activity of proteolytic enzyme bromelain from pineapple peel waste at a concentration of 20% -40% was 0.5743% - 1.1206%. Proteolytic activity of the protease enzyme sheep rumen fluid at a concentration of 20-40% can be seen in Figure 1

Figure 1. Proteolytic activity of sheep rumen  enzymes

In Figure 1 above shows that the higher concentration of enzyme caused the higher proteolitik activity. On the use of enzymes 20-40% was obtained proteolytic activity 1.42% - 2.690%. Low proteolytic activity was found in 20% enzyme concentration, which can be hydrolyzed protein of 1.42% and the highest proteolytic activity was found in 40% concentration of 2.690%. Activity of proteolytic enzyme was obtained the greater when compared to the activity of proteolytic enzyme bromelain from pineapple skin waste. Dedin et al (2013) showed that the hydrolyzed protein 0.5743% on enzyme concentration as low as 20% and 1.1206% at the highest enzyme concentration of 40%. Budiansyah research results (2011), it is known that the activity of the protease enzyme local cattle 7.3 ± 3.5 units / ml were measured at pH 7.0. Sheep rumen fluid protease enzyme is an enzyme produced by bacteria that have less activity when the enzyme is no longer contains the cells of bacteria and protozoa. The statement was reinforced by the opinions Moharrery and Das, (2002).

Protein

The value of leucaena seeds protein concentrate produced ranged between 56.02% -56.30% db. At the 24-hour incubation period using sheep rumen fluid enzyme concentration of 4 mL / 100 g provides the most optimal levels of protein, whereas the incubation period of 48 hours the concentration of enzyme 10 mL / 100 g which produce optimal levels of protein. Protein hydrolysis process can be influenced by the presence of phytic acid content of Leucaena seeds can effected on protease enzymes work. The statement was reinforced by the opinion Ravindran, et. al (2000), that the protease enzyme activity decreased due to the phytic acid binding protein, as well as phytic acid insoluble in neutral pH.

Sheep rumen fluid is a enzyme source of cheap can comprises amilase and cellulase. The activity of both enzymes is competing with the protease enzyme that Leucaena seed protein was hydrolyzed less optimal. Cellulase enzyme is an enzyme complex (multi-component) which consists of several enzymes that work gradually or jointly decompose cellulose into D-glucose (Kim et al. 1994 in Fitriliyani 2010). There are four main groups of enzymes that make cellulose based substrate of each enzyme, namely: First; Endo β (1-4) glukonase (β 1-4 Dglukanohidrolase, EC 3.2.1.4), Cxselulase, glikolisik bond hydrolyze β (1-4) at random. This enzyme does not attack but hydrolyze cellobiose selodekstrin. This enzyme is also active attack cellulose which has been substituted for example carboxymethyl. Second; The enzyme β (1-4) D-glucan selobiohidrolase (EC 3.2.1.91), Cl attacking non-reducing ends of cellulose chains and produce cellobiose. These enzymes can attack selodekstrin but do not attack the cellulose which has been substituted and can not hydrolyze cellobiose. Third; β (1-4) D-glucan glukohidrolase (EC .2.1.74), attacked the non-reducing ends of cellulose chains and produce glucose. This enzyme attack selooligosakarida and CMC. While the fourth is a β (1-4) glucosidase or β (1-4) D-glucoside glukohidrolase (EC 3.2.1.21), hydrolyze cellobiose and short chain selooligosakarida and glucose.

Figure 2. Protein content of Leucaena protein concentrate on incubation 24 h

Figure 3. Protein content of Leucaena protein concentrate on incubation 48 h

During hydrolysis occurs conversion proteins that are insoluble nitrogen into compounds that are soluble, further breaks down into compounds that are simpler, such as peptides, amino acids and ammonia. Haslaniza et al. (2010) stated that the concentration of proteolytic enzymes that increasing the hydrolysis process will lead to an increase in dissolved nitrogen content. Amino acid composition of leucaena seed concentrate consists of amino acids polar and non-polar so this will affect the solubility in water and oil.

Table 1. amino acid Composition of Leucaena Seed Protein Concentrate

Amino acid	Result (%)
L-Aspartic	3.99
L-Glutamic	-
L-Asparagine	4.73
L-Histidine	2.43
L-serine	0.11
L-Glutamin	0.91
L-Threonin	3.60
L-Glycine	2.17
L-Arginia	1.32
L-Alanine	2.78
L-Tyrosine	1.50
L-Thryp+L-Methionine	3.39
L-Valine	1.03
L-Phenylalanine	-
L-Isoleucine	0.95
L-leucine	2.96
L-Lycine	3.25

Figure 4. Amino acid Chromatogram of Leucaena Seed Protein Concentrate

Enzyme activity in the rumen is very closely related to the diversity of microorganisms in the rumen. Rumen microbes can be divided into 3 main groups of bacteria, protozoa and fungi (Czerkawski, 1986).  Hungate (1966) there are several types of bacteria in the rumen, including:

a.     Cellulose-digesting bacteria (Bakteroidessuccinogenes, Ruminococcus flafaciens, Ruminococcus albus, Butyrifibriofibrisolvens).

b.    Hemicellulose digestive bacteria (Butyrivibrio fibrisolvens, Bacteroides ruminocola, Ruminococcus sp.)

c.     Starch-digesting bacteria (Bacteroides ammylophilus, Streptococcus bovis, Succinnimonas amylolitica)

d.    Sugar-digesting bacteria (Triponema bryantii, Lactobacillus ruminus)

e.     Protein-digesting bacteria (Clostridium sporogenus, Bacillus licheniformis).

Water Absorption

Water absorption of leucaena seed protein concentrate produced at the value of 2.50 to 3.70 mL / g. The highest water absorption was obtained in the treatment of enzyme concentration addition of 10 mL / 100 g was equal to 3.42 mL / g and low water absorption in the treatment without the addition of enzyme concentration of (control)  sheep rumen fluid was 3.18 mL / g. Increasing sheep rumen fluid enzyme concentration and the longer the incubation period resulted in increasing water absorption. It was also an impact on the resulting color difference, the longer the incubation period causes discoloration more dark. Polar proteins molecules composition affected the binding of water. This statement is reinforced by the opinions Suwarno, (2003) which states that the binding of water depends on the composition and conformation of the molecule - a protein molecule. The interaction between water and hydrophilic groups of the side chains of proteins can occur through hydrogen bonds. The amount of water that can be retained by the protein depends on the amino acid composition, surface hydrophobicity, and processing. The amount of water that is bound to be increased if the polarity of the protein increases. Water absorption of leucaena seed protein concentrate was still in the range of soy protein concentrate. Kinsella, (1979) absorption from 2.40 to 3.40 g of soy protein concentrate H2O / g solid.

 Figure 5. Leucaena seed protein concentrate incubated 24 h (A1B6) and 48 h (A2B6)

 Figure 6. Water absorption of Leucaena seed protein concentrate 

Improved water absorption leucaena seed protein concentrate line with the length of time of incubation. Water absorption of Protein concentrate is affected by the amount of polar amino acids in proteins. The statement was reinforced by the opinions suwarno, (2003) which states that water absorption related to the amount of polar amino acid groups present in the protein molecule. Polar amino acid groups, such as hydroxyl, amino, carboxyl, and sulfihidril provide hydrophilic properties of the protein molecule that can absorb and bind water.

Water holding capacity is the ability to retain water against gravity, and includes bound water, hydrodynamic water, capillary water and physically entrapped water (Moure et al., 2006). The amount of water associated to proteins is closely related with its amino acids profile and increases with the number of charged residues (Kuntz and Kauzmann, 1974), conformation,hydrophobicity, pH, temperature, ionic strength and protein concentration (Damodaran, 1997;Kinsella, 1979).

Protein solubility is influenced by the hydrophilicity/hydrophobicity balance, which depends on the amino acid composition, particularly at the protein surface (Moure et al.,2006). The presence of a low number of hydrophobic residues; the elevated charge and the electrostatic repulsion and ionic hydration occurring at pH above and below the isoelectric pH favour higher solubility. Protein solubility is also influenced by production method and in particular by denaturation due to alterations in the hydrophobicity/hydrophilicity ratio of the surface. A highly soluble protein is required in order to obtain optimum functionality required in gelation, solubility, emulsifying acitivity, foaming and lipoxygenase activity (Riaz, 2006). Soluble protein preparations are easier to incorporate in food systems, unlike those with low solubility indices which have limited functional properties and more limited

food uses.

Bulk Density

The average bulk density of leucaena seed protein concentrate produced ranged from 0.52 to 0.66 g / mL. was generally associated with a higher water absorption cohesiveness, mainly due to the liquid bridges between the particles. Therefore, food powder which is hygroscopic at high water content causes a decrease in bulk density (Wirakartakusumah et al 1992).

 Figure 7. Bulk Density of Leucaena seed protein concentrate 

Bulk density data indicated that the longer the incubation time did not significantly affect the bulk density reduction. At the time of treatment 24 h incubation bulk density leucaena seed protein concentrate 0.56 g / mL and a slight decline in long treatment incubation of 48 hours is 0:55 g / mL. Bulk density values of various foods in powder form between 0.3 to 0.8 g / ml. Bulk density of food powder particles depending on the nature of the food material. The statement reinforced Wirakartakusumah opinion. et al, (1992) which states that the bulk density of food in powder form depending on the influence of factors such as intensity interconnected attractive forces between the particles, particle size and number of related points.

Oil absorption capacity

The average oil absorption of leucaena seed protein concentrate produced ranged from 0.9 to 1.65 mL / g. The highest oil absorption capacity was obtained in the treatment of enzyme concentration of 10 mL / 100 g was equal to (1.65 ml / g) and low oil absorption in the treatment without the use of enzymes sheep rumen fluid is 0.9 mL / g. Oil absorption capacity of leucaena seed protein concentrate was still in the range of soy protein concentrate (1.33 to 1.54 ml / g, Kinsella, 1979). Increased absorption of the oil that was in line with increasing enzyme concentration sheep rumen fluid used depends on the presence of a protein that has a lipolytic structure. At the amino acid composition of leucaena seeds protein concentrates contained amino acids lipolytic types (non-polar amino acids), such as glycine, alanine, tryptophan, valine, leucine (Table 1). This statement is supported by the opinion (Lin et al, 1974) which states that the oil absorption capacity of a protein depends on its structure. Structures that are lipolytic with nonpolar protein content of branches more dominant, contributing to the increase in oil absorption capacity.

 Figure 8. Oil absorption of Leucaena seed protein concentrate

In Figure 7 shows that the longer the incubation time increased absorption of oil Leucaena seed protein concentrate. Suwarno, (2003) which states that the denaturation of the protein can increase the protein's ability to bind fat due to the opening of the protein structure that describes the amino acids that are non-polar. According to Zayas, (1997) in Hapsari, (2009) stated that the oil absorption capacity of a protein is affected by sources of protein, the protein particle size, shape processing, other additives, temperature, and degree of protein denaturation.

Conclusion

proteolytic enzyme Activity of sheep rumen fluid obtained was greater when compared to the proteolytic enzyme activity of bromelain from pineapple skin waste and can hydrolyze leucaena seeds proteins  was optimal at a concentration of 4 mL / 100 g on  incubation time 24 h. Amino acid composition of Leucaena seed protein concentrate enough complete to produce nutrition source and had the functional properties of water and oil absorption increased with sheep rumen fluid enzyme concentration increasing  

ACKNOWLEDGEMENTS
This research was funded by the Directorate General of Higher Education, Ministry of Education and Culture through the research competitive of national strategy (STRANAS) 2014-2014

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