ANTIBACTERIAL ACTIVITY FROM GOAT’S MILK WHEY HYDROLIZED BY PROTEASE ENZYME OF BACILLUS
LINCHENIFORMIS
Sitta Fitri Rahmadhina1), Irma Isnafia Arief1*), Cahyo Budiman1)
Department of Animal Production and Technology, Faculty of Animal Science, Bogor Agricultural University, Jl. Agatis, Babakan, Kec. Dramaga, Kabupaten Bogor, Jawa Barat 16680
*Corresponding email: [email protected] Submitted 23 July 2022; Accepted 24 March 2023
ABSTRACT
Food additives had been used to prevent and inhibit the growth of microorganisms and extend shelf life of food. Based on the safety of their used, it has an impact such as microorganism resistance to synthetic antimicrobials. This encourages the development of antimicrobials derived from natural ingredients, especially from milk. The bioactive components of milk protein have not always existed in their natural state, including precursors or peptides that will only be active if they have been hydrolyzed in vitro from their natural proteins. Hydrolysis of milk protein can be carried out using proteolytic enzymes from various sources, including protease extracted from Bacillus lincheniformis. This study used different concentrations of the protease extracted from Bacillus lincheniformis (0.5%, 1% and 1.5%) to produce hydrolyzate which has antibacterial activity in terms of protein content, SDS PAGE profile and the size of the inhibition zone (disc method). The results showed that the best activity of the whey hydrolyzed on Gram-negative Escherichia coli was 1% concentration of protease enzyme. In comparison, Gram positive Staphylococcus aureus can be inhibited with 0.5% concentration of protease enzyme.
Keywords: antibacterial; protease from Bacillus lincheniformis; goat milk
INTRODUCTION
The spoilage of foodstuffs caused by the activity of microorganism both destructive and pathogen microorganism can cause foodborne ilness. A significant amount contamination of food also can cause economic loss, especially in the food industry. The use of additional material of food in the form of chemical material to prevent from growth of microorganism, spoilage and contamination as well as to extend shelf life has another impact towards the safety of users especially appear resitance towards syntetic antimicrobial. It encourages the development of antimicrobial from safetier chemical material without leaving residue. One of alternative sources of natural antimicrobials derived from milk.
Milk is a nutritious food that can be recognized through the balance of its amino acid and improves health. Protein consists of casein and whey protein. Casein is fractionated into α-, β- and k-casein, while whey protein belongs to α-lactalbumin, β- lactoglobulin, Bovine Serum Albumin (BSA) and immunoglobulin (Ig) (Heyman
& Desjeux 1992). It does not only work as nutritional intake but also as source of bioactive peptides with a wide biological function (Szwajkowska et al., 2011). Protein bioactive component of milk does not always exist in its nature. Some of them can be as precursor or peptide that only can be activated if it is hydrolyzed by in vitro from their natural protein. Hydrolysis of milk protein can be done by using proteolytic enzyme from several sources, for example animal digestive enzyme such as trypsin, chymotripsin, and pepsin, protease from plant like bromelain and papain or from
bacterial enzyme for example alkalase from Bacillus licheniformis (Korhonen &
Pihlanto 2007).
Many studies have been done about bioactive peptides from cow milk, thus it is necessary to conduct another research of bioactive peptide milk from another source.
Accroding to Kusumaningtyas et al., (2015) goat milk can be used as substitude of cow milk. Population of goats in Indonesia is big enough. It becomes the top 10 goats population on the world which is potentioal to produce milk (Aziz, 2010). Hydrolized bioactive peptide from casein of goat milk with papain enzyme produces antioxidant activity (Lestari et al., 2020). The antioxidant activity of goat's milk peptides was also found by hydrolyzing goat's milk with the protease Lactobacillus plantarum S31 in fraction 11 with a pH of 5.0 of 29%.
This fraction also has bacterial inhibitory activity Enteropathogenic Escherichia coli (EPEC K1.1), Staphylococcus aureus, and Listeria monocytogenes (Herlina et al., 2019). Peptide from whey protein of goat milk from hydrolyzing with human gastrointensial is known that it has ability to inhibit Escherichia coli K12 bacteria, Bacillus cereus RT INF01, Listeria monocytogenes and Staphylococcus aureus ATCC 25923 (Almaas et al., 2011). Other research on bioactive peptides hydrolyzed from goat milk using the bromelain enzyme is known to have antioxidant and antibacterial activity against bacteria such as E.coli, S.typhimurium and L.monocytogenes (Kusumaningtyas 2016).
In this study, enzymes derived from the bacterium Bacillus lincheniformis were used at various concentrations (0.5%, 1%, and 1.5%) to produce hydrolyzate from whey protein which has antibacterial
*Corresponding author:
Irma Isnafia Arief
Email: [email protected]
Department of Animal Production and Technology, Faculty of Animal Science, Bogor Agricultural University, Jl. Agatis, Babakan, Kec. Dramaga, Kabupaten Bogor, Jawa Barat 16680
How to cite:
Rahmadhina, S. F., Arief, I. I., & Budiman, C. (2023).
Antibacterial Activity From Goat’s Milk Whey Hydrolized by Protease Enzyme of Bacillus Lincheniformis. Jurnal Ilmu dan Teknologi Hasil Ternak (JITEK), 18 (1), 22-30
activity. Therefore, this research was conducted to determine the effect of enzyme concentration to produce hydrolyzate of goat's milk whey which has antibacterial activity.
MATERIALS AND METHODS
Materials
Materials used in this study were goat milk, pathogenic bacteria of Escherichia coli and Staphylococcus aureus, and chemicals to be analyzed. Equipments used in this research were glassware, Erlenmeyer, autoclave, Laminar Air Flow Cabinet (ESCO), High Speed Centrifuge (Hitachi), a set of SDS-PAGE, micro pipette (Gilson), hot plate, micro pipette tips (Axygen), incubator, magnetic stirrer, Bunsen, cuvette, petri dish, spectrophotometer UV- Vis, and aluminium foil.
Methods
Preparation of milk
Goat milk was processed using cold centrifuged (4 oC) at 1,280 × g for about 30 minutes to separate its fat (skimmed milk).
Casein fraction and whey from skimmed goat milk are separated based on method from Detha et al., (2013) with modification.
Skim goat milk is slowly acidified with HCl 2 N until pH 4.2. The milk is then cold centrifuged (4 oC) on 10,000 × g for about 30 minutes, thus it will separate between solid fraction (casein and insoluble components) and its supernatant (whey and water soluble fraction). The solid fraction (casein) is rinsed twice with phosphate buffer solution (0.05 M, pH 7), then it is stored in temperature -20 oC before it is used. The acid whey is neutralized (pH 7) by using NaOH 1 N, then it is centrifuged again with the same condition, thus it will produce neutral whey. After that, the whey is concentrated by using freeze dryer at temperature of -50 oC, vacuum pressure ± 22.4 Pa, for 24 hours. The whey concentrate stored at temperature -20 oC before used.
Phosphate buffer solution (0.05 M pH 7)
was used as casein solvent and whey concentrate diluent.
Hidrolysis procedure of whey with different concentrations protease (0.5 %, 1.0 % and 1.5 %)
Whey hydrolysis protein was done based on Kusumaningtyas (2016) method with modification. As much as 2 grams of freeze-dried whey was reconstituted with 100 mL buffer solution 0.05 M pH 7 and hydrolized by using protease from Bacillus lincheniformis with level 0.5 %, 1 % and 1.5
% at temperature 50 oC. The reaction was stopped by heating to a temperature 80 oC for 15 minutes. Then, each of hydrolyzed sample was centrifuged at 2000 rpm for 5 minutes. The supernatant obtained was taken for further testing.
Analysis of peptide profiles with SDS PAGE (Singh et al., 2011)
Protein profiles from whey and its hydrolyzate were visualized with sodium dodecylsulfate-polyacroilamide gel electrophoresis (SDS-PAGE) technique (Singh et al., 2011 with modification). The concentration of the gel of the retaining used was 4% (v/v), while for the gel separator was 15% (v/v). The samples were electrophoresized for 3 hours and 15 minutes with voltage 70 V and 20 mA. After the runnings finished, the gel was removed and rinsed twice with aquadeion water and then colored. Silver staining coloring technique was done based on standard Bollag’s method (Bollag et al.,1996).
Marker PM 2700 was used as standard (Smobio Technology, Inc.).
Measurement of protein levels (He, 2011) The test of protein levels was done by Bradford method. Standard curve used standard use bovine Serum Albumin. A total of 0.05 mL samples was put into reaction tubes and added 1.25 mL of aquades as well as 1.25 mL Bradford reactor. Then the solution was homogenized and incubated at room temperature for 5 minutes.
Absorbanasi was measured at wave length of 595 nm (He, 2011).
Measurement of antibacterial activity Antimicrobial activity testing was carried out by measuring the diameter of the clear zone of goat's milk whey without hydrolysis and hydrolysis treatment with different levels of enzyme administration (0.5%, 1% and 1.5%) on the growth of S.
aureus and E. coli using the disc method (Dhiman et al., 2011).
Statistical analysis
The research design was carried out using a single factor Completely Randomized Design (CRD), with treatments namely the level of the protease from Bacillus lincheniformis (0.5%, 1%, and 1.5%). The research data were then analyzed using an analysis of variance (ANOVA) one-way. P value < p 0.05 then the change is considered significant or significantly different (Steel & Torrie 1991).
RESULTS AND DISCUSSION
Goat Milk Whey Protein Content
The hydrolysis process produces whey hydrolyzates with different protein concentrations which are generally lower than whole whey isolates. It shows that the enzymatic treatment was proven to be able to hydrolyze whey protein, that shown decrease in the amount of protein on the samples given enzymatic treatment (Table
1). Table 1 shows that all hydrolyzates had protein concentrations that decreased with the addition of enzymes during hydrolysis.
Each protein concentration value that was treated with the addition of enzymes was 0.049 mg/mL for whey with added enzymes of 0.5%, 0.044 mg/mL for whey with added enzymes of 1%, and 0.027 mg/mL for whey added with enzymes of 1.5%. When compared with control whey without the addition of enzymes and no hydrolysis, control whey had the highest protein concentration of 0.345 mg/mL (Table 2).
The decrease in protein hydrolyzate concentration can be influenced by the enzymes used.
Protease from Bacillus sp. used can hydrolyze whey and produce smaller peptide fragments protease from Bacillus sp.
belongs to the serine protease group which is characterized by good activity at high temperature and pH (7.5-11). Serine protease or serine endopeptidase is an enzyme that breaks down proteins into peptides with serine as the active site of the enzyme. The protease produced by Bacillus sp. usually has a molecular weight of 25-46 kDa and can be stabilized with Ca2+ with a high isoelectric point. Soluble in detergents and remain active at pH 7.5-11 and temperature 37-70 oC (Ferrero et al., 1996;
Jellouli et al., 2011; Vijayaraghavan et al., 2014). Product specification is described in Table 1. Proteases from Bacillus sp. used can hydrolyze whey and produce smaller peptide fragments.
Table 1. Product specification
Name Protease from Bacillus lincheniformis-≥ 2.40 U/g
Storage temperature 2-8 ⸰C
Enzyme activity (AU/G;Protease units) ≥ 2.40
Product’s number P4860
The Bradford method used in this study is a method of measuring total protein concentration. In this method, Coomasie brilliant blue (CBB) will bind to proteins that have side chain amino acid residues of tryptophan, tyrosine, phenylalanine, arginine, histidine, and leucine in samples
under acidic conditions and produce a blue color (Purwanto 2014; Utami et al., 2016). ).
In the hydrolysis process, proteins are ceased to function by enzymes into peptides and amino acids. Peptides and amino acids cannot form complexes with CBB thus they do not produce a blue color (Utami et al.,
2016) which results in a decrease in the absorbance value when measured at a wavelength of 595 nm. This causes a protein concentration decrease in the hydrolyzate when compared to the unhydrolyzed whey.
Protein measurements performed using the Bradford method were only sensitive to whole proteins, while peptides (smaller proteins) were not detected. The decrease in the amount of protein using the Bradford method showed that protein that was still intact (control whey without hydrolysis) would continue to decrease when hydrolyzed because it was hydrolyzed by enzymes.
Whey Profile by SDS Electrophoresis PAGE
Milk proteins, especially casein and whey, contain optimal amounts of all the essential amino acids and are a vital source of bioactive peptides (Cozma et al., 2011).
Currently, whey protein is a new functional food source (Marshall 2004). Whey protein
is 18-20% of the total protein in milk, consisting of Lactalbumin (α-La) 20%, - Lactoglobulin (β-Lg) 50%, bovine serum albumin (BSA) 10%, and immunoglobulin (Ig) 10% (Jovanovic et al., 2007).
Figure 1 explains that in the control whey there was a protein of around 14 kDa, namely α-lactalbumin, and a protein with a molecular weight of 34.59 kDa, namely κ casein, while in the hydrolyzed whey there were no longer visible proteins with a molecular weight of 14 kDa and 34.59 kDa.
This proves that the protease from Bacillus lincheniformis has successfully hydrolyzed proteins to become peptides with a molecular weight < 5 kDa which cannot be seen in the SDS PAGE electrophoresis results. All of the hydrolysates showed that the large proteins in whey had been degraded and produced bands with a small molecular weight < 5 kDa. These results indicate that whey hydrolyzed is protein. In Table 2, the hydrolyzed whey has a protein content of 0.027-0.049 mg/mL.
Table 2. Whey protein content and hydrolyzate
Protein type Protein content (mg/mL)
WK 0.345 ± 0.111a
W 0.5% 0.049 ± 0.001b
W 1% 0.044 ± 0.003b
W 1.5% 0.027 ± 0.006c
Notes: *different letters in the same column indicate significantly different.
WK: control whey without hydrolysis, W 0.5%: whey with 0.5% addition of protease, W 1%:
whey with 1% addition of protease, W 1.5%: whey with the added protease of 1.5%
Antibacterial Activity
The antimicrobial activity test was carried out by measuring the diameter of the clear zone of the hydrolyzate against the growth of S. aureus and E. coli using the disc method (Dhiman et al., 2011).
According to Sapatnekar et al., (2010) the formation of a zone that is different from the surrounding area and the clear zone in the dish overgrown with indicator bacteria is used to see the antimicrobial activity of the hydrolyzate. The diameter of the clear zone on the growth of E. coli and S. aureus can be seen in Table 3. In Table 3 it can be seen that whey hydrolyzed with the addition of 1%
protease is effective against Escherichia coli. Meanwhile, whey which was hydrolyzed with the addition of 0.5%
protease was effective against Staphylococcus aureus.
These results indicate that certain hydrolyzates are specific to certain bacteria.
According to Mondhe et al., (2014) antibacterial peptides naturally or modified are species-specific, i.e. these peptides are only effective against certain bacteria so that peptides can kill pathogenic bacteria without having to kill normal flora. Antibacterial peptides interact with the bacterial cell membrane through the interaction of the
positive charge of the peptide with the negative charge on the surface of the bacterial cell. The next mechanism depends on the type of peptide. In general, it is possible to separate peptides that act by affecting membrane permeability and peptides that act in the cytoplasm. Peptides that work by affecting membrane
permeability can occur by making pores or by dissolving them (works like detergents) (Mihajlovic & Lazaridis 2010). Another mechanism is that antibacterial peptides enter the cytoplasm and inhibit intracellular functions such as inhibiting DNA, RNA, and enzymatic reactions without disrupting membrane stability (Jenssen et al., 2006).
Figure 1. Profile of whey with SDS PAGE electrophoresis. WK: control whey without hydrolysis, W 0.5%: whey with 0.5% addition of protease, W 1%: whey with 1%
addition of protease, W 1.5%: whey with the added protease of 1.5%
a b
Figure 2. Left (a): clear zone of inhibition E. coli Rigth (b): clear zone of inhibition S. aureus
The antibacterial test showed that whey before and after hydrolysis with the addition of enzymes at different levels (0.5%, 1%, and 1.5%) can inhibit the growth of Escherichia coli and Staphylococcus aureus. The results of antibacterial testing of goat's milk whey showed that the best activity of the hydrolyzate hydrolyzed by the
negative Escherichia coli was the hydrolyzate with an enzyme concentration of 1% which had an inhibition zone value of 8.8 mm. Peptide production by enzyme treatment is thought to produce many antimicrobial peptides (antimicrobial peptide/ AMP) which can be seen from the increase in the antibacterial ability of whey Standard Marker WK W1-1.5%
although with various abilities this is because each treatment produces a different peptide profile. The concentration of 1% is the optimum concentration to be able to inhibit Gram-negative, beyond that concentration, the treated sample will produce another type of peptide which is not sensitive to Gram-negative. And it could be that other concentrations produce peptides that do not have antibacterial properties. The sensitivity to Gram-negative is thought to be because at a concentration of 1% more hydrophilic peptides are produced. Gram- negative membrane cells are protected by an
outer membrane that is difficult to access by hydrophobic peptides. Hydrophilic peptides will penetrate more easily. While the concentration of 0.5% is the optimum concentration to inhibit Gram-positive (Staphylococcus aureus) because more hydrophobic peptides are produced at this concentration. This study also shows that a high hydrolysis rate will produce more peptides, but does not guarantee that the resulting peptides are all antibacterial.
Therefore, the optimum hydrolysis rate will be different for each peptide with the expected bioactive activity.
Table 3. Inhibitory zone diameter (mm) whey against pathogenic bacteria Bacteria Type chloramphenicol Whey treatment (mm)
WK W0.5% W1% W1.5%
E.coli 20.96 ± 1.86 7.11 ± 0.58b 6.48 ± 0.59c 8.88 ± 0.60a 7.66 ± 0.97b S.aureus 27.94 ± 1.80 6.94 ± 0.14y 8.70 ± 0.44x 7.28 ± 0.64y 6.50 ± 0.84y Remarks: *Chloramphenicol as a comparison was not included in the statistical data
analysis *a,b,c,x,y Superscripts differences appear in the same row indicate significant differences (p<0.05). WK: control whey without hydrolysis, W 0.5%:
whey with 0.5% addition of protease, W 1%: whey with 1% addition of protease, W 1.5%: whey with the added protease of 1.5%
The results showed that the diameter of the clear zone on the whey hydrolyzate against S. aureus and E. coli were included in the strong category because the value was greater than 6 mm. Clear zone diameters between 0-3 mm have weak inhibition, diameters between 3-6 mm are categorized as moderate inhibition, and diameters greater than 6 mm have strong inhibitory activity (Pan et al., 2009).
CONCLUSION
Enzymatic treatment was proven to be able to hydrolyze whey protein, as seen in the decrease in the amount of protein in the samples given the treatment. The enzyme used is a protease from Bacillus lincheniformis that can hydrolyze whey and produce smaller peptide fragments. Peptide production by enzymatic treatment produces many antimicrobial peptides (antimicrobial peptide/ AMP), which can be seen from the increase in the antibacterial ability of both
Gram-positive and negative pathogenic bacteria, from whey with varying abilities with varying abilities. This study shows that a high hydrolysis rate will certainly produce more peptides. The results showed that the best activity of the whey hydrolyzed on Gram-negative Escherichia coli was 1%
concentration of protease enzyme. In comparison, Gram positive Staphylococcus aureus can be inhibited with 0.5%
concentration of protease enzyme.
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