The product of the lactoperoxidase system (LPOS) has been developed as a preservative agent to inhibit foodborne bacteria, but its action was, heretofore, limited to several original compounds in milk. This research was conducted to analyze the application of the lactoperoxidase system against
Lactoperoxidase (LPO) was developed to inhibit the growth of foodborne pathogens in various foods and thus improve their shelf life [
Biopreservation using the LPO system could offer an additional hurdle to improve the shelf life of various food products such as fruit [
It was reported that lactose reduces LPO activity by 38% because the sugar molecules interact with the heme cavity of the LPO [
SP Sepharose™ Big Beads (Lot No. 10081054) was purchased from GE Healthcare Bio-Sciences AB, Sweden. Microbial rennet was purchased from Prodinvest Group, Russia. Deoxycholate hydrogen sulfide lactose agar (DHL) (Lot No. 395-00461) was obtained from Shinnihonseiyaku Co., Ltd., Japan. ABTS was purchased from Wako Pure Chemical Industry, Japan. Bovine milk was freshly obtained from the experimental farm at the Faculty of Animal and Agricultural Science, Diponegoro University, Semarang, Indonesia. Culture stock of
Whey was obtained using fresh bovine milk that was treated with 0.02% (w/v) rennet. Through these treatments, 1 L of fresh bovine milk was converted into 800 mL of whey. Casein was removed using a sterile filter cloth; lactose removal of whey was carried out by dialysis. Untreated whey was dialyzed to produce reduced-lactose whey, and the solution eluted from the dialysis membrane was collected as high-lactose solution.
The procedure for immobilization of LPO from whey was conducted according to the method of previous researchers [
Protein content was analyzed using the Coomassie Brilliant Blue reagent [
The inoculum was prepared following the method of Lang [
LPO activity was assayed using the method of Al-Baarri [
Antibacterial activity was measured using the method previously described by Touch [
Lactose content in whey was determined by using a refractometer. The ability of the refractometer to provide accurate measurements was indicated by how closely the test results matched those obtained with the MilkoScan. This method was adapted from Chigerwe [
The analyses for antimicrobial activity and lactose content were carried out in triplicate from 3 independent experiments; then they were analyzed using descriptive analysis to explain their changes. Data are showed as means ± standard error of the mean. Statistical significance was calculated using the GraphPad Prism statistical software (San Diego, USA). The ANOVA analysis was used to decide the significance at
Lactoperoxidase is known as an antimicrobial agent in milk, saliva, and tears because of its inhibitory action on bacteria through the oxidation reaction involving thiocyanate and hydrogen peroxide [
A high peak of LPO activity was detected from fraction numbers 1–5, with values in the range of 80–93 units (Figure
LPO activity in nine fractions obtained from the elution of 0.4 mM NaCl in 0.1 mM phosphate buffer of pH 7.0 through column containing Sepharose™ Big Beads.
SDS-PAGE profile of nine fractions that were eluted from column packing Sepharose™ Big Beads. Lanes 1 to 8 were the samples from fractions 1 to 8.
This research used 7.0 ± 0.10 log CFU/ml of
Antibacterial effect of LPOS against
Bacterial reductions in whole milk, skimmed milk, and untreated whey were less than in the control (<1.0 log CFU/mL), possibly due to the presence of casein and lactose in milk and whey. Casein is the abundant component in milk protein that might protect substrate microorganisms from absorption of the antimicrobial component, thus weakening the inhibitory effect on bacteria [
The lactose content in untreated whey was 1.82 ± 0.20%, and after dialysis, it was reduced to 0.69 ± 0.10% (Table
Lactose content in untreated whey, reduced-lactose whey, and high-lactose solution.
Materials | Lactose content (%) |
| |
Untreated whey | 1.82±0.20 |
Reduced-lactose whey | 0.69±0.10 |
High-lactose solution | 2.05±0.30 |
Values are means ± SE (n = 5).
It was described that lactose had performed as an LPO inhibitor; therefore the lactose conversion into another compound was suggested. Previous researchers [
This research indicated that LPOS had moderate antibacterial effects on
The authors state that the data in this article were obtained as naturally as possible with the proper replication. The authors also state that the previously reported “lactoperoxidase–thiocyanate–hydrogen peroxide system” was used to support this study and is available at
The authors declare that they have no conflicts of interest regarding the publication of this paper.
Financial assistance from the Ministry of Research, Technology and Higher Education of the Republic of Indonesia’s Grant to conduct this research is gratefully acknowledged.