Preservation of Raw Cow Milk by Adding Different Concentrations of Hydrogen Peroxide (H2O2)

University of Veterinary and Animal Sciences, Lahore-Pakistan.

Muhammad Usman Akram Email: mh.usman@hotmail.com Skype: mh.usman1 Website: www.facebook.com/usman101pak Mobile No.: +923217773736

Note:
I am student of B.S. (Hons.) Dairy Technology (Session 2009-13), in the University of Veterinary and Animal Sciences, Lahore-Pakistan. The purpose of making this synopsis was although not a research point of view, but to make an assignment for my department on the behalf of training for future studies. But as I end up my assignment, I felt that I have to make new discoveries about this method of chemical treatments for the prevention of milk losses.

Request:
Everyone who read this can copy this data for his own research because it was copied by different trusted websites, research papers, books, and thesis with the guide of departments professors. You people please contact to me for sharing some knowledge about dairy, so that I can assign myself for your topic or problems, also comment about it if you have some recent research or news about this. God Bless All of You.

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Introduction
Milk contains on average water, fat, protein, lactose and minerals with 87.25, 3.80, 3.50, 4.80, 0.65 % respectively (Eckles et al., 1951). Milk contains considerable amount of water soluble vitamins (Vitamin B complex and Vitamins C) and fat soluble vitamins (Vitamin A, D, E, K). Milk is also good food for the growth of different micro-organism either desirable or undesirable. On the other hand, Undesirable micro-organisms may disturb the actual flavor or texture, also may be pathogenic causing different diseases in human beings. Pakistan is on 4th number in the list of milk producing countries with the production of 47.951 million tons milk annually (Government of Pakistan, 2012). Mostly milk is predominantly produced in small batches in far flung rural areas. Due to its traditional unhygienic handling and perishable nature, mostly milk spoiled, most particularly in summer season. At rural level it is not possible to safe milk by cooling, so chemical preservative is preferably added. To prevent this spoilage, milk collectors use variety of chemicals or keep the temperature down by using ice blocks, to prevent the growth of microbes. One of the important chemical is hydrogen peroxide which is used to activate the LP-system, for the purpose to kill the spoilage causing microbes. Lactoperoxidase (LP) system is naturally present in raw milk that prevents the bacterial multiplication due to its bacteriostatic effect. By activating the LP-system, shelf life of milk can be increased for 8 to 12 hours. For this purpose, different ingredients like salt of thiocyanate or hydrogen peroxide or both in small concentrations is used (Recommended dosage 15-30 ppm for each salt). Lactoperoxidase (LP) system consists of three components; LP, thiocyanate and hydrogen peroxide. And it is active only when all of three components are present. Lactoperoxidase catalyses the oxidation of thiocyanate by hydrogen peroxides and generates an intermediate product with antibacterial properties. These products have a broad range of antimicrobial effects against bacteria, fungi and viruses (de Wit & van Hooydonk, 1996; Kussendrager & van Hooikdonk, 2000.) Objectives Raw milk preservation by use of H2O2 is done for many important purposes like; 1. To enhance the shelf life of milk for consumption without microbial contamination. 2. To minimize the processing cost by heating the milk.

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Review of Literature
Preservative effect of lacto-peroxidase system on keeping quality of raw milk is determined by addition of thiocyanate and hydrogen peroxide, while untreated milk was declared as control sample. After regular time interval, titratable acidity of both (treated and untreated samples) were calculated. The result shows that it is possible to enhance the keeping quality of raw camel milk by activating the LP system (Kamau et al., 1984). The LP system also serves as a processing aid in the production of other dairy products (Kamau et al., 1990). The lactoperoxidase (LPO) thiocyanatehydrogen peroxide system (LPO system), a naturally occurring antimicrobial system in milk and saliva, has been used not only with foods (Wolfson and Sumner, 1993). The LPO system has been recognized as critical in the dairy industry for the preservation of raw milk (Haddadin et al., 1996). Enhancement of the keeping quality of raw sheep milk was attempted by addition of H2O2 in two different concentrations (100 ppm & 400 ppm), the activation of the LP system by adding two different concentrations (ppm) in equimolar ratio of thiocynate and hydrogen peroxide and cooling. Control, hydrogen peroxide treated and LP activated samples were kept at 20oC and 35oC for 15 hours whereas the cooled milk was kept at 5oC. For same period titratable acidity test, resazurin test, rennet coagulation time and starter culture activity were determined in milk samples at 3 hours intervals. This showed significant improvement in enhancing the keeping quality of raw sheep milk by activating LP system, but cooling was the best method for the preservation of raw sheep milk (Celalettin et al., 2001). Preservation of raw milk by using different concentration of H2O2 is determined and the milk, which is used for experiment was divided into seven samples. In six samples different concentrations of H2O2 was added for preservation and remaining sample was preserved without addition of H2O2. Before preservation some physical and chemical tests from all samples were taken and similarly after preservation with regular interval of time up to spoilage. It resulted that keeping quality of milk is significantly increase with H2O2 as compared to control sample. To preserve milk up to 24 hours, 0.04-0.05% H2O2 is enough (B.K. Saha et al., 2003). Raw milk was LPS-activated by adding various concentrations (ppm) of thiocyanate and peroxide and denoted as 0:0, 7:10 ppm, 10:10 ppm and 20:20 ppm. The keeping quality of the activated milk samples was assessed by the alcohol stability and clot-on-boiling tests, pH changes and titratable acidity. The milk in all the treatments remained fresh during the first 12 hours but the control was spoiled by the 15th hour. There was a continuous drop in pH values matched by a steady rise in titratable acidity. For all parameters measured, 20:20ppm was the last treatment to spoil, suggesting that the shelf life of milk increases with increasing concentrations of thiocyanate and peroxide.With small amounts of thiocyanate (20 ppm) and peroxide (20 ppm) the shelf life of raw milk can effectively be extended under Cameroonian conditions by approximately 9 hours without refrigeration. Thus LPS-activated milk can be stored for as long 21 hours, allowing sufficient time for its appropriate disposal (Fonteh et al., 2005). Preservative nature of honey in pasteurized milk is studied due to presence of hydrogen peroxide. The preservative ability of honey was determined by monitoring the growth of bacteria in pasteurizes milk which was inoculated by nutrient broth. Samples were Page | 3

made with different concentration of honey along with control sample which was without honey. It showed that milk preserved with honey at 4oC inhibit the growth better compared to milk without honey (Krushna et al., 2007). The feasibility of hydrogen peroxide, boric acid, sodium azide, potassium sorbate, bronopol and azidiol as milk preservatives on the quality of raw milk was accessed. Treated milk samples were stored at 20oC and 4oC, while untreated milk sample was considered as control sample. Chemical and biological tests were performed just before preservation and then regularly after equal time intervals. The results showed that for preservation of raw milk, most suitable preservatives are sodium azide, bronopol and azidiol (Seskena et al., 2007). Studies carried out to develop a technique for the preservation of cow's milk in raw condition using hydrogen peroxide (H2O2) as a preservative. Fresh cows milk was collected and experiments were conducted by four treatments in order to achieve the optimum condition of storage. The treatments were with various concentration of H2O2 starting from 0.05 %, 0.1 %, 0.2 %, 0.3 %, 0.4 %, & 0.5 %. Treated milk with 0.05 % concentration of H2O2 had storage period of 20 days compared to that of the control one (5 days only) in refrigerated temperature (8C). On the other hand hydrogen peroxide treated milk (0.05 %) had a storage period of 8 hours at room temperature (28C). Results also showed that the higher concentration of H2O2 had no effect on storage period than that of control (Rokhsana et al., 2007). The effect of the Lactoperoxidase system on antimicrobial activity, alcohol test, aerobic test and lactoperoxidase activity were determined in raw cow milk. Active LP system was found to impressively enhance the keeping quality of all milk samples with decrease in microbial contamination @ 87 % (untreated milk sample) and 78% (E-Coli treated milk sample), so LP system can serve as alternative method to control the microbial growth in cow milk (Katekan et al., 2008). Although the LP system has been shown to have potential as a bio-preservative, its potency has so far been extensively investigated in only dairy and meat products. Its effectiveness against pathogens in other foods is generally unexplored.

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Materials and Methods

The experiment will be conducted at Dairy Technology Lab for undergraduate of Department of Dairy Technology, University of Veterinary and Animal Sciences, Lahore-Pakistan. Milk Source & Materials Whole raw cow milk will be collected from B-block Farms of University of Veterinary and Animal Sciences, Ravi Campus Pattoki after taking all hygienic measures. Experimental Protocol Samples, after through mixing will be divided into seven equal parts. Out of the seven parts, one part was kept as untreated milk (fresh) and six portions were treated with 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 % of H2O2. Plastic containers will be used to preserve milk. The parameters used to monitor the physical and chemical quality of milk will be determined initially just before adding hydrogen peroxide and then after adding the preservatives until the milk samples will spoil. Preservation Procedure This study was conducted over a period of three weeks. Each week a five liter cow milk sample was collected from fresh bulked milk of the dairy farm. The collected milk samples after thorough mixing were divided into three equal parts. Of the three parts, one part was kept as untreated milk (fresh) and two portions were treated with 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 % of H2O2 respectively. Plastic containers were used in milk preservation. Each of the three treatments was further divided into two equal portions. One portion from each treatment was stored at 4 C and the other portion at 20 C. The untreated milk samples (control samples) were analyzed immediately after delivery to the laboratory and then after 24 h, as the standard LVS 175:1999 requires raw milk without preservation can be used for analysis only within 36 h. Analysis of preservative-treated milk samples was conducted after 24, 48, 72 and 96 h, respectively. All parameters used to monitor the quality of milk were determined in ten replications in CRD pattern. Physical Tests Organoleptic tests will be performed visually to observe the flavor, color and texture. Specific gravity will be determined by using lactometer. Chemical Tests Fat and protein tests were made using infrared spectroscopy on a MilkoScan FT6000TM configured as a CombiFossTM 6000FC together with a FossomaticTM FC (FOSS Electric A/S, Denmark) according to ISO 9622:1999(E) and manufacturers recommended procedures (FOSS Page | 5

Electric 2005). Methylene blue reduction (MBR) test will be performed according to (Longree and Ambruster, 1996). Clot on boiling (COB) test will be performed according to (Yadav et al., 1993). Bacteriological analysis Total bacterial count (TBC) will be performed according to technique recommended by (Richarson, 1985). Psychrophic count (PC) will be performed according to technique recommended by (Biolife, 1991).

Statistical Analysis
Milk samples were taken in complete randomization design as per (Steel and Torrie, 980), to check the storage activity and treatment conditions after regular intervals of time with reference to control sample. Analysis of variance test will be done to find the statistical difference (Significant or not) between the different treatments and to make a comparison between different treatment means LSD value was calculated. Storage activity Fresh cows milk was collected and experiments were conducted by for treatments in order to achieve the optimum condition of storage. The treatments were with various concentration of H2O2 starting from 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 % respectively. Treated milk with 0.04 % concentration of H2O2 had storage period of 20 days compared to that of the control sample (5 days only) in refrigerated temperature (8C). On the other hand hydrogen peroxide treated milk (0.04 %) had a storage period of 8 hours at room temperature (28C). Treatment conditions Control and treated milk samples are subjected to different treatment conditions like change of temperature, pH, acidity etc. for the determination of potential of milk samples to denature or spoil in a pre calculated time.

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Summary
Milk is treated with different concentrations of hydrogen peroxide (0.01, 0.02, 0.03, 0.04, 0.05 and 0.06 % of H2O2), along with control sample (untreated sample), for the purpose to activate Lactoperoxidase system, which destroy the bacterial population by its antibacterial effect. Storage activity and treatment conditions were traced after regular intervals for determining quantitative and qualitative analysis with reference to control sample. Preservation effect of milk did not increased by using higher concentration; normally 0.04% H2O2 concentration is enough to preserve milk up to 24 hours. This research improves the preservation activities of cow milk in rural areas where temperature and humidity is high.

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Literature Cited
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16.Mullan, W.M.A. and Bjoerck, L. "Lactoperoxidase." In 'Determination of indigenous

antimicrobial proteins of milk' p29-31, Bulletin of the International Dairy Federation No. 284/1993, Brussels (Belgium). 17.Sofos, John N. "Naturally Occurring Antimicrobials in Food" Council for Agricultural Science and Technology (CAST), IA, USA publishing catalogue ISBN 1-887383-12-3, April 1998, 103 pp., 28.00 18.ISO 9622:1999(E). 1999. Whole Milk Determination of Milkfat, Protein and Lactose Content Guidance on the Operation of Mid-infrared Instruments. Th e International Organization for Standardization, Geneva. 27 p. 19.LVS 175:1999. 1999. Sampling of Raw Milk. LVS, Riga. 5 p. (in Latvian). 20.Kamau D.N and Kroger M. 1984 Preservation of raw milk by treatment with hydrogen peroxide and by activation of the lactoperoxidase (LP) system. Milchwissenschaft, 39 (11) 658-661. 21.International Dairy Federation 1988 Bulletin Number 234. Code of practice for the preservation of raw milk by the lactoperoxidase system. Brussels, Belgium: IDF. 22.National Institute of Statistics 2001 Cameroon statistical yearbook 2000.Yaounde, Cameroon. pp 68-69. 23.Fonteh F A 2001 Role of the lactoperoxidase system in raw milk preservation. PhD. Thesis. The University of Reading, England. pp. 206 - 208. 24.Bjorck L 1975 Antibacterial effect on the lactoperoxidase system on psychrotrophic bacteria in milk. Journal of Dairy Research, 45 109-118.

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