International Journal of Food Science

International Journal of Food Science / 2019 / Article

Research Article | Open Access

Volume 2019 |Article ID 8013402 | 6 pages |

Enhanced Antibacterial Activity of Lactoperoxidase–Thiocyanate–Hydrogen Peroxide System in Reduced-Lactose Milk Whey

Academic Editor: Amy Simonne
Received13 Nov 2018
Revised03 Mar 2019
Accepted03 Apr 2019
Published23 Apr 2019


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 Escherichia coli in fresh bovine milk and its derivative products to determine the strength of antibacterial activity. Lactoperoxidase was purified from bovine whey using the SP Sepharose Big Beads Column. The enzymatic reaction involving lactoperoxidase, thiocyanate, and hydrogen peroxide was used to generate the antibacterial agent from LPOS. This solution was then added to milk, skimmed milk, untreated whey, reduced-LPO whey, reduced-lactose whey, and high-lactose solution containing E. coli at an initial count of 6.0 log CFU/mL. LPOS showed the greatest reduction of bacteria (1.68 ± 0.1 log CFU/mL) in the reduced-lactose whey among the products tested. This result may lead to a method for enhancement of the antimicrobial activity of LPOS in milk and derived products.

1. Introduction

Lactoperoxidase (LPO) was developed to inhibit the growth of foodborne pathogens in various foods and thus improve their shelf life [1, 2]. Lactoperoxidase derived from bovine milk has been shown to generate beneficial effects as a bactericidal and bacteriostatic agent [1, 3]. The lactoperoxidase system consists of three primary components: lactoperoxidase enzyme, thiocyanate, and hydrogen peroxide. This system generates hypothiocyanite, an active compound against Gram-positive and Gram-negative bacteria, including Escherichia coli [4, 5]. The lactoperoxidase system (LPO system) has attracted the attention of scientists as a natural biopreservative with generally recognized as safe (GRAS) status [6]. Hypothiocyanite, a product of LPOS, has been recognized as a safe antibacterial agent without negative effects on human health [7, 8].

Biopreservation using the LPO system could offer an additional hurdle to improve the shelf life of various food products such as fruit [9], chicken meat [10], duck meat [11], cheese [12], and local food products such as dangke [2, 13]. However, slight inhibition of pathogenic bacteria also appeared in fresh milk. Other researchers reported the slight reduction of below 1 log CFU/ml in fresh milk treated with the lactoperoxidase system [14]. It was understood that lactoperoxidase antimicrobial activity might be enhanced using lysozyme [2], beta carotene [15, 16], ectoine [17], alpha tocopherol [18], and chitosan [19], but it was inhibited by several compounds such as hydrogen peroxide and thiocyanate in excess amounts [2022] and indigenous milk compounds such as casein [23] and saccharides [24]. It was then presumed that the removal of casein and lactose from the milk enabled the use of lactoperoxidase to reduce the population of bacteria in fresh milk.

It was reported that lactose reduces LPO activity by 38% because the sugar molecules interact with the heme cavity of the LPO [24, 25]. The association of sugar molecules with the heme cavity physically blocked the substrate-binding site, thereby resulting in the prevention of the interaction of substrate with the heme iron [21]. This research aims to use LPOS to reduce pathogenic bacteria in milk and its derived products after removal of lactose and casein from milk. This research will provide beneficial information to apply LPOS in milk and derived products.

2. Materials and Methods

2.1. Materials

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 Escherichia coli FNCC 0009 was purchased from the Faculty of Agricultural Technology, Gadjah Mada University, Yogyakarta, Indonesia. A spectrophotometer (Mini UV-1240, Shimadzu, Japan) was used for the Bradford protein analysis and enzyme activity. Sterile syringe filters (Lot No. SF2030813) were purchased from Axiva Sichem Biotech Delhi, India. All chemicals used in this study were of analytical grade.

2.2. Preparation of Whey, Reduced-Lactose Whey, and High-Lactose Solution

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.

2.3. Purification of LPO from Whey

The procedure for immobilization of LPO from whey was conducted according to the method of previous researchers [25], with minor modifications. SP Sepharose™ Big Beads (SPBB) was used as the matrix for LPO purification from bovine whey. Whey was applied on a glass column (2 x 17 cm) filled with 17 g of SPBB. Preparation of SPBB was initiated by washing with 300 mL pure water and 300 mL of 0.1 mM phosphate buffer (PB) of pH 6.8 containing 1 M NaCl to remove unnecessary compounds. After the whey was applied to the column, the resin was washed with 100 mL 0.4 mM NaCl in 0.1 mM phosphate buffer of pH 7.0 using a fraction collector (10 mL per tube). The purity of the derived LPO was checked by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), using the method of a previous researcher [26]. The protein solution was filtered through a 0.22 μm syringe filter unit. The purified LPO was stored at –20°C. The LPO purification was done for multiple times until the band of LPO showed a clear image using the SDS-PAGE analysis.

2.4. Determination of Protein Concentration

Protein content was analyzed using the Coomassie Brilliant Blue reagent [27]. The protein standard was determined using bovine serum albumin.

2.5. Inoculum Preparation

The inoculum was prepared following the method of Lang [28] with minor modifications. Before each experiment, stock cultures of E. coli FNCC 0009 were streaked onto Nutrient Broth. Cultures were incubated at 39°C for 24 h.

2.6. Determination of LPO Activity

LPO activity was assayed using the method of Al-Baarri [24]. A 450 μl aliquot of 1.0 mM ABTS in 10 mM acetate buffer (pH 4.4) and 450 μl 0.55 mM H2O2 in pure water were poured into the cuvette. Immediately, 50 μl of LPO was added to the cuvette. The increase in absorbance at 412 nm was measured for 1 minute. One unit of LPO enzymatic activity was expressed as the amount of enzyme needed to oxidize 1 μmol ABTS min−1. The molar extinction coefficient of ABTS at 412 nm was 32,400 min−1 cm−1.

2.7. Determination of Antibacterial Activity

Antibacterial activity was measured using the method previously described by Touch [10] with modifications. The LPO system, composed of 3.0 U/ml LPO, 0.9 mM KSCN, and 0.9 mM H2O2, was incubated for 1 hour at room temperature to generate the antibacterial compound. The LPOS solution was then added to the milk and its derivative products, which were inoculated with E. coli at approximately 107 CFU/mL. Each mixture was incubated in a water bath shaker at 30°C. Controls with 0.1 mM PB of pH 7.0 instead of the milk were subjected to the same treatment as the samples. Serial dilutions in sterilized pure water were prepared to obtain countable numbers of bacteria. Counts were obtained by spreading 100 μL of each mixture onto triplicate plates of DHL. The plates were incubated at 37°C for 24 h. Colony forming units (CFU) were enumerated in plates containing 30–300 colonies, and cell concentration was expressed as log CFU/mL.

2.8. Determination of Lactose Content in Whey

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 [29]. Whey obtained by the previously mentioned method of whey purification was analyzed by means of the MilkoScan 203 and refractometer, resulting in a mean bias of 94 ± 1.92%. Lactose concentrations were determined by comparing the value obtained by the refractometer with a standard curve generated with lactose. The regression equation with R2 = 0.97 was used to determine lactose concentration.

2.9. Data Analysis

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 P values of less than 0.05.

3. Result and Discussion

3.1. Purification of LPO and Characteristics of the Purified Protein

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 [30, 31]. LPO is a glycoprotein consisting of a single polypeptide chain with a molecular weight of 78 kDa (Golhefors and Marklundi, 1975; Jacob et al., 2000). The purification process of LPO from bovine whey was conducted at 10°C to provide optimum binding of LPO to the SP Sepharose matrix [30]. Therefore, this research used SP Sepharose to bind LPO in whey.

A high peak of LPO activity was detected from fraction numbers 1–5, with values in the range of 80–93 units (Figure 1). No significant LPO activity was detected in fractions 6–9. Each fraction was then applied to SDS-PAGE to determine its purity. As a result, several bands were detected in fractions 1–3 (Figure 2). However, fractions 4 and 5 showed a single band with minor other proteins, indicating that purity of LPO was high in these fractions. Therefore, fractions 4 and 5 were mixed and their activity was calculated, obtaining 94 and 93 U/ml, respectively. Since previous application of LPO for reducing S. enteritidis only required 4.5 U/ml [16], the lactoperoxidase obtained by this purified LPO sufficiently fulfills the need for LPO application in the next set of experiments. Prior to enzyme collection in a 1.5 ml tube, the mixed fraction was sterilized using a 0.22 μm syringe filter, and then the enzyme was stored at -20°C.

3.2. Antibacterial Activity of the LPO System from Bovine Milk

This research used 7.0 ± 0.10 log CFU/ml of E. coli as the initial population. The incubation times were set to 1 and 4 hours at 30°C (Figure 3). It can be seen that LPOS remarkably reduced the population of E. coli in PB from the initial count to 5.58 ± 0.10 log CFU/mL, indicating a reduction of 1.42 ± 0.03 log CFU/mL after 4 hours of incubation. These findings imply that inhibitory effects on the antibacterial activity of LPO tend to increase with the higher duration of incubation. However, statistical analysis showed that there were no significant differences (P <0.05) in the antibacterial activity among treatments. This might be due to the high population of initial bacteria that was used in this research. The incubation time plays a remarkable role in bacterial reduction that could be seen by the increase in the antibacterial activity at 4 h of incubation. As can be seen in control, antibacterial activity showed less than 0.1 log CFU/mL in the sample with a 1-hour incubation and then elevated to 1.42±0.04 CFU/mL at 4 h incubation. Opstal [32] reported a greater reduction of E. coli by LPOS (2.2 log CFU/mL) from the initial count of 6.0 CFU/mL during 6 hours of incubation at 20°C. These differences between studies might have been due to differences in the bacterial load and incubation time.

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 [23]. It is known that bactericidal effects of OSCN compounds from LPOS are key to kill bacteria by disrupting sulfhydryl groups (-SH) on proteins from the bacterial cytoplasmic membrane [24], so the interaction between the sulfhydryl group and OSCN might be hindered, resulting in the weakening of antibacterial action. Inhibition of LPOS action could also occur due to hydrogen peroxide released from bacteria [23].

The lactose content in untreated whey was 1.82 ± 0.20%, and after dialysis, it was reduced to 0.69 ± 0.10% (Table 1). Results from the statistical study showed that the reduction exhibited no significant difference (P <0.05), but it showed 62% reduction resulting in the 2.7 times antibacterial activity enhancement of LPO from 0.62 ± 0.20 to 1.68 ± 0.10 that clearly indicated inhibition of antibacterial activity of LPOS by lactose (Figure 3). These results were corroborated by those of previous researchers [10], finding that LPOS was unable to reduce the significant amount of S. enteritidis in whole milk. Saccharides including lactose were potent inhibitors of lactoperoxidase activity and showed kinetic inhibition of 3.20 ± 0.52 [24]. Therefore, the reduction of the lactose amount in milk might increase the action of LPOS against the growth of bacteria. The inhibition of LPOS by lactose might be due to the weakening of enzymatic activity of LPO, since saccharides are a nonspecific stabilizer of protein that allows for direct interaction between carbohydrate and protein molecules through hydrogen bond formation, resulting in the reduction of enzymatic activity [33]. In addition, as reported by previous researchers [34], the carboxylic group might bind to the side chain of 2-Glu258 to form a strong hydrogen bond resulting in the inability of a natural substrate such as thiocyanate to bind to LPO.

MaterialsLactose content (%)

Untreated whey1.82±0.20
Reduced-lactose whey0.69±0.10
High-lactose solution2.05±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 [35] applied lactose reduction using lactose oxidase to generate an H2O2 compound resulting in the enhancement of antimicrobial function of LPOS; however the avoidance of lactose binding to the specific site of LPO might be required since lactose may still provide beneficial effect to the nutrient content of a dairy product. However, in order to achieve the practical application in the dairy industry, this research may provide the novelty with clear explanation that the reduction of lactose content is strongly suggested to exhibit the beneficial impact on the shelf life of dairy products.

4. Conclusion

This research indicated that LPOS had moderate antibacterial effects on E. coli in whole milk, skimmed milk, and whey. Lactose reduction from whey remarkably enhanced bactericidal activity. LPOS can effectively act as an antibacterial reagent in reduced-lactose dairy products.

Data Availability

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 and These prior studies are cited at relevant places within the text as references: [13] Rasbawati, A.N. Al-Baarri, A.M. Legowo, V.P. Bintoro, B. Dwiloka, Total bacteria and pH of dangke preserved using natural antimicrobial lactoferrin and lactoperoxidase from bovine whey, International Journal of Dairy Science, 9 pp. 116–123, 2014; [24] A.N. Al-Baarri, M. Hayashi, M. Ogawa, S. Hayakawa, Effects of mono- and di-saccharides on the antimicrobial activity of bovine lactoperoxidase system, Journal of Food Protection, 74 pp. 134–139, 2011.

Conflicts of Interest

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.


  1. C. Abbes, A. Mansouri, and A. Landoulsi, “Synergistic effect of the lactoperoxidase system and cinnamon essential oil on total flora and salmonella growth inhibition in raw milk,” Journal of Food Quality, vol. 2018, Article ID 8547954, 6 pages, 2018. View at: Publisher Site | Google Scholar
  2. A. N. Al-Baarri, A. M. Legowo, S. K. Arum, and S. Hayakawa, “Extending shelf life of indonesian soft milk cheese (dangke) by lactoperoxidase system and lysozyme,” International Journal of Food Science, vol. 2018, Article ID 4305395, 7 pages, 2018. View at: Publisher Site | Google Scholar
  3. C. Abbes, M. Ahlem, H. Mariem, and L. Ahmed, “Optimizing antimicrobial activity of the bovine lactoperoxidase system against Salmonella enterica Hadar, a causative agent of human gastroenteritis in Tunisia,” African Journal of Microbiology Research, vol. 7, no. 22, pp. 2719–2723, 2013. View at: Publisher Site | Google Scholar
  4. M. Kennedy, A.-L. O'Rourke, J. McLay, and R. Simmonds, “Use of a ground beef model to assess the effect of the lactoperoxidase system on the growth of Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus in red meat,” International Journal of Food Microbiology, vol. 57, no. 3, pp. 147–158, 2000. View at: Publisher Site | Google Scholar
  5. C. Garcia-Graells, C. Valckx, and C. W. Michiels, “Inactivation of Escherichia coli and Listeria innocua in milk by combined treatment with high hydrostatic pressure and the lactoperoxidase system,” Applied and Environmental Microbiology, vol. 66, no. 10, pp. 4173–4179, 2000. View at: Publisher Site | Google Scholar
  6. O. O. Alegbeleye, J. T. Guimarães, A. G. Cruz, and A. S. Sant'Ana, “Hazards of a ‘healthy’ trend? An appraisal of the risks of raw milk consumption and the potential of novel treatment technologies to serve as alternatives to pasteurization,” Trends in Food Science & Technology, vol. 82, pp. 148–166, 2018. View at: Publisher Site | Google Scholar
  7. F. Bafort, O. Parisi, J.-P. Perraudin, and M. H. Jijakli, “Mode of action of lactoperoxidase as related to its antimicrobial activity: a review,” Enzyme Research, vol. 2014, Article ID 517164, 13 pages, 2014. View at: Publisher Site | Google Scholar
  8. H. Neetoo and F. Mahomoodally, “Use of antimicrobial films and edible coatings incorporating chemical and biological preservatives to control growth of Listeria monocytogenes on cold smoked salmon,” BioMed Research International, vol. 2014, Article ID 534915, 10 pages, 2014. View at: Publisher Site | Google Scholar
  9. M. Cissé, J. Polidori, D. Montet, G. Loiseau, and M. N. Ducamp-Collin, “Preservation of mango quality by using functional chitosan-lactoperoxidase systems coatings,” Postharvest Biology and Technology, vol. 101, pp. 10–14, 2015. View at: Publisher Site | Google Scholar
  10. V. Touch, S. Hayakawa, S. Yamada, and S. Kaneko, “Effects of a lactoperoxidase-thiocyanate-hydrogen peroxide system on Salmonella enteritidis in animal or vegetable foods,” International Journal of Food Microbiology, vol. 93, no. 2, pp. 175–183, 2004. View at: Publisher Site | Google Scholar
  11. V. P. B. S. K. Arum, A. N. Al-Baarri, F. Wahono, and A. R. C. Utomo, “Microbiological analysis of fresh duck meat stored in lactoperoxidase system solution,” in Proceedings of the 2nd International Student Conference on Food Science and Technology: Global Insight for the Future of Food Production, November 2014. View at: Google Scholar
  12. Y. Amornkul and D. R. Henning, “Utilization of microfiltration or lactoperoxidase system or both for manufacture of Cheddar cheese from raw milk,” Journal of Dairy Science, vol. 90, no. 11, pp. 4988–5000, 2007. View at: Publisher Site | Google Scholar
  13. Rasbawati, B. Dwiloka, A. N. Al-Baarri, A. M. Legowo, and V. P. Bintoro, “Total bacteria and pH of Dangke preserved using natural antimicrobial lactoferrin and lactoperoxidase from bovine whey,” International Journal of Dairy Science, vol. 9, no. 4, pp. 116–123, 2014. View at: Publisher Site | Google Scholar
  14. V. Y. Villa, A. M. Legowo, V. P. Bintoro, and A. N. Al-Baarri, “Quality of fresh bovine milk after addition of Hypothiocyanite-rich-solution from Lactoperoxidase system,” International Journal of Dairy Science, vol. 9, no. 1, pp. 24–31, 2014. View at: Publisher Site | Google Scholar
  15. A. N. Al-Baarri, A. M. Legowo, S. Hayakawa, and M. Ogawa, “Enhancement Antimicrobial Activity of Hyphothiocyanite Using Carrot Against Staphylococcus Aureus and Escherichia Coli,” Procedia Food Science, vol. 3, pp. 473–478, 2015. View at: Publisher Site | Google Scholar
  16. M. Hayashi, S. Naknukool, S. Hayakawa, M. Ogawa, and A.-B. A. Ni'Matulah, “Enhancement of antimicrobial activity of a lactoperoxidase system by carrot extract and β-carotene,” Food Chemistry, vol. 130, no. 3, pp. 541–546, 2012. View at: Publisher Site | Google Scholar
  17. M. B. Boroujeni and H. Nayeri, “Stabilization of bovine lactoperoxidase in the presence of ectoine,” Food Chemistry, vol. 265, pp. 208–215, 2018. View at: Publisher Site | Google Scholar
  18. S. Shokri and A. Ehsani, “Efficacy of whey protein coating incorporated with lactoperoxidase and α-tocopherol in shelf life extension of Pike-Perch fillets during refrigeration,” LWT- Food Science and Technology, vol. 85, pp. 225–231, 2017. View at: Publisher Site | Google Scholar
  19. C. Mohamed, K. A. Clementine, M. Didier, L. Gérard, and D.-C. Marie Noëlle, “Antimicrobial and physical properties of edible chitosan films enhanced by lactoperoxidase system,” Food Hydrocolloids, vol. 30, no. 2, pp. 576–580, 2013. View at: Publisher Site | Google Scholar
  20. A. K. Singh, N. Singh, M. Sinha et al., “Binding modes of aromatic ligands to mammalian heme peroxidases with associated functional implications. Crystal structures of lactoperoxidase complexes with acetylsalicylic acid, salicylhydroxamic acid, and benzylhydroxamic acid,” The Journal of Biological Chemistry, vol. 284, no. 30, pp. 20311–20318, 2009. View at: Publisher Site | Google Scholar
  21. A. K. Singh, N. Singh, S. Sharma et al., “Inhibition of lactoperoxidase by its own catalytic product: Crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3-Å resolution,” Biophysical Journal, vol. 96, no. 2, pp. 646–654, 2009. View at: Publisher Site | Google Scholar
  22. A. K. Singh, R. P. Kumar, N. Pandey et al., “Mode of binding of the tuberculosis prodrug isoniazid to heme peroxidases: Binding studies and crystal structure of bovine lactoperoxidase with isoniazid at 2:7 å resolution,” The Journal of Biological Chemistry, vol. 285, no. 2, pp. 1569–1576, 2010. View at: Publisher Site | Google Scholar
  23. F. A. Fonteh, A. S. Grandison, and M. J. Lewis, “Factors affecting lactoperoxidase activity,” International Journal of Dairy Technology, vol. 58, no. 4, pp. 233–236, 2005. View at: Publisher Site | Google Scholar
  24. A. N. Al-Baarri, M. Hayashi, M. Ogawa, and S. Hayakawa, “Effects of mono-and disaccharides on the antimicrobial activity of bovine lactoperoxidase system,” Journal of Food Protection, vol. 74, no. 1, pp. 134–139, 2011. View at: Publisher Site | Google Scholar
  25. A. N. Al-Baarri, M. Ogawa, and S. Hayakawa, “Application of lactoperoxidase system using bovine whey and the effect of storage condition on lactoperoxidase activity,” International Journal of Dairy Science, vol. 6, no. 1, pp. 72–78, 2011. View at: Publisher Site | Google Scholar
  26. U. K. Laemmli, “Cleavage of structural proteins during the assembly of the head of bacteriophage T4,” Nature, vol. 227, no. 5259, pp. 680–685, 1970. View at: Publisher Site | Google Scholar
  27. M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding,” Analytical Biochemistry, vol. 72, no. 1-2, pp. 248–254, 1976. View at: Publisher Site | Google Scholar
  28. M. M. Lang, L. J. Harris, and L. R. Beuchat, “Survival and recovery of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes on lettuce and parsley as affected by method of inoculation, time between inoculation and analysis, and treatment with chlorinated water,” Journal of Food Protection, vol. 67, no. 6, pp. 1092–1103, 2004. View at: Publisher Site | Google Scholar
  29. M. Chigerwe and J. V. Hagey, “Refractometer assessment of colostral and serum IgG and milk total solids concentrations in dairy cattle,” BMC Veterinary Research, vol. 10, no. 1, p. 178, 2014. View at: Google Scholar
  30. A. Atasever, H. Ozdemir, I. Gulcin, and O. Irfan Kufrevioglu, “One-step purification of lactoperoxidase from bovine milk by affinity chromatography,” Food Chemistry, vol. 136, no. 2, pp. 864–870, 2013. View at: Publisher Site | Google Scholar
  31. L. M. Wolfson and S. S. Sumner, “Antibacterial activity of the lactoperoxidase system: a review,” Journal of Food Protection, vol. 56, no. 10, pp. 887–892, 1993. View at: Publisher Site | Google Scholar
  32. I. Van Opstal, C. F. Bagamboula, T. Theys, S. C. M. Vanmuysen, and C. W. Michiels, “Inactivation of Escherichia coli and Shigella in acidic fruit and vegetable juices by peroxidase systems,” Journal of Applied Microbiology, vol. 101, no. 1, pp. 242–250, 2006. View at: Publisher Site | Google Scholar
  33. S. Z. Shariat and Z. Aghelan, “Partial purification and biochemical characterization of peroxidase from rosemary (Rosmarinus officinalis L.) leaves,” Advanced Biomedical Research, vol. 4, no. 1, p. 159, 2015. View at: Publisher Site | Google Scholar
  34. P. K. Singh, H. V. Sirohi, N. Iqbal et al., “Structure of bovine lactoperoxidase with a partially linked heme moiety at 1.98 Å resolution,” Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, vol. 1865, no. 3, pp. 329–335, 2017. View at: Publisher Site | Google Scholar
  35. S. Lara-Aguilar and S. D. Alcaine, “Lactose oxidase: A novel activator of the lactoperoxidase system in milk for improved shelf life,” Journal of Dairy Science, vol. 102, no. 3, pp. 1933–1942, 2019. View at: Publisher Site | Google Scholar

Copyright © 2019 Ahmad Ni’matullah Al-Baarri et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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