PROCESS OF EXTRACTION OF LACTOPEROXIDASE PROTEIN FROM YOGURT WHEY

BTE 4418
SEPARATION PROCESS 2
SEMESTER 2, 2017/2018
TERM PAPER
TITLE: PROCESS OF EXTRACTION OF
LACTOPEROXIDASE PROTEIN FROM YOGURT WHEY
NO NAME MATRIC NO
1 AIN NUR SYAZWANI BT ABDUL LATIF 1428692
2 KU MARIAM ZAINAB BT KU ABDULLAH 1422960
3 WAN MAJIDAH BT WAN ZULKIFLI 1423928
SUBMISSION DATE : 5/5/2018

TABLE OF CONTENT
INTRODUCTION 1
MARKET SURVEY 2
PROCESS FLOW DIAGRAM 3
SECTION 1
1.1 PRE-TREATMENT 4
SECTION 2
DOWNSTREAM PROCESS
2.1 SEPARATION OF INSOLUBLES 5
2.2 ISOLATION AND PURIFICATION 7
2.3 POLISHING 9
REFERENCES 11

INTRODUCTION
Lactoperoxidase (LP) is one of the most prominent enzymes in bovine milk and
catalyses the inactivation of a wide range of micro-organisms in the lactoperoxidase system
(LP-s). Its concentration is around 30 mg/l, constituting about 0´5 % of the whey proteins (de
Wit & van Hooydonk, 1996). According to research published in the Journal of Applied
Microbiology, it has antibacterial properties that are helpful for the skin and can eliminate acne-
causing bacteria (Truthinaging.com, 2018). Lactoperoxidase is a member of the peroxidase
family, a group of natural enzymes, also widely distributed in nature and found in plants and
animals, including man. Their primary function is to catalyse the oxidation of certain
molecules, at the expense of hydrogen peroxide, in order to generate reactive products with a
wide antimicrobial activity (Kussendrager & van Hooijdonk, 2000).
The biological significance of lactoperoxidase is its involvement in the natural host
defence system against invading micro-organisms. In bovine milk it is one of the indigenous
antimicrobial agents. Next to that antiviral activity, degradation of various carcinogens and
protection of animal cells against peroxidative effects have been reported. Bovine LP consists
of a single polypeptide chain containing 612 amino acid residues. Its amino acid sequence is
known and the molecular weight is approximately 78 kDa. LP is a basic protein having a high
isoelectric point of 9´6 (Kussendrager & van Hooijdonk, 2000).
Lactoperoxidase is a very interesting natural active ingredient, can be obtained from
special milk fractions using chromatographic methods previously only with great effort in
sufficient purity (Fao.org, 2018). The availability of industrial processes, for the isolation of
LP from bovine milk and whey, has resulted in a growing interest in the valorisation of the LP-
s as a biopreservative in a wide range of products. Although a number of applications for the
LP-s are already exploited commercially, further growth in the utilisation of LP and LP-
systems, in a broad field of food and non-food products, can be expected. The natural biological
functions of the LP-s and the increasing pressure to restrict the use of chemical antimicrobials
are considered as important driving forces behind this growth.
1

MARKET SURVEY
Based on application, the lactoferrin and lactoperoxidase market is classified into food
and beverages (food supplements, infant formula, dairy products, sports nutrition and others),
pharmaceuticals (iron tablets, immune system stimulation, others), and cosmetic and hygiene
products (skin care, body care and oral hygiene) and others.High usage of lactoferrin and
lactoperoxidase as antimicrobial in food products to increase its shelf life is expected to support
the demand of antimicrobial in lactoperoxidase market during the forecast period.
Globally due to its wide usage and applications, lactoferrin and lactoperoxidase is
predicted to show a healthy growth during the forecast period. Among all the regions North
America is expected to be the major contributor in terms of revenue in lactoferrin and
lactoperoxidase market followed by Europe. Rising demand of sports and energy drinks in the
U.S is expected to support the demand of Lactoferrin and lactoperoxidase market across the
North America region. Asia Pacific is also expected to show a substantial growth in lactoferrin
and lactoperoxidase market during the forecast period. Increasing need for dietary supplements
across the region and high usage of cosmetics product in Japan, China and Southeast Asian
countries is expected to furl the market growth in the near future (Newswire.com,2018).
2

PROCESS FLOW DIAGRAM
The Figure 1.1 below consists of upstream and downstream processing which includes
centrifuge, homogenizer, fermenter, microfiltration, ion exchange chromatography, and spray
dryer. The feed that is supply to the centrifuge is 15000 L/batch. Lactoperoxidase gain is
about 809L/batch.
Figure 1.1: Process flow diagram of lactoperoxidase powder production.
3
Centrifuge
Fermenter

SECTION 1
1.1 PRE-TREATMENT
The basic procedure of making yogurt follow the steps below shown in Figure 1.1.
The first step of making yogurt is centrifugation to separate fat from milk. This
standardization process typically involves reducing the fat content and increasing the total
solids. For yogurt manufacture, the solids content of the milk is increased to 16% with 1-5%
being fat and 11-14% being solids-not-fat (SNF) through evaporation using water or adding
concentrated milk or milk powder. The second step is homogenization, a process in which the
milk is forced through small openings at a high pressure and fat globules are broken up due to
shearing forces The fat globules in milk are broken up into smaller, more consistently
dispersed particles producing a much smoother and creamier end product. Before the
fermentation process, the milk is cooled to between 109.4-114.8° F (43-46° C) and the
fermentation culture (Lactobacillus bulgaricus) is added in a concentration of about 2%. It is
held at this temperature for about 3 to 4 hours while the incubation process takes place.
During this time, the bacteria metabolizes certain compounds in the milk producing the
characteristic yogurt flavour. An important by-product of this process is lactic acid (How
Products Are Made, n.d).
Figure 1.2 : Steps of making yogurt
4
Milk Centrifugation Homogenization Fermentation Yogurt

REFERENCES
Anon, (2018). Advantages & Disadvantages of Spray Drying. [online] Available at:
http://www.ehow.co.uk/info_8666922_advantages-disadvantages-spray-drying.html
[Accessed 3 May 2018].
How yogurt is made - manufacture, making, used, composition, product, machine, Raw
Materials. Retrieved from http://www.madehow.com/Volume-4/Yogurt.html
How Whey Protein is Made: Microfiltration, Ultrafiltration, Hydrolyzed Whey. Retrieved from
http://www.bodybuildingforyou.com/protein/whey-protein-processing-2.htm
Faraji, N., Zhang, Y., & Ray, A. (2017). Optimization of Lactoperoxidase and Lactoferrin
Separation on an Ion-Exchange Chromatography Step. Separations, 4(2), 10.
https://doi.org/10.3390/separations4020010
Fao.org. (2018). Food safety and quality: Lactoperoxidase. [online] Available at:
http://www.fao.org/food/food-safety-quality/a-z-index/lactoperoxidase/en/. [Accessed 1 May
2018]
Kussendrager, K. D., & van Hooijdonk, A. C. M. (2000). Lactoperoxidase: physico-chemical
properties, occurrence, mechanism of action and applications. British Journal of Nutrition,
84(S1), 19–25. https://doi.org/10.1017/S0007114500002208
Newswire.com. (2018). Lactoferrin and Lactoperoxidase Market Share and Trends, 2015 - 2021.
[online] Available at: https://www.newswire.com/news/lactoferrin-and-lactoperoxidase-
market-share-and-trends-2015-2021 [Accessed 3 May 2018].
Sosnik, A. and Seremeta, K. (2015). Advantages and challenges of the spray-drying technology
for the production of pure drug particles and drug-loaded polymeric carriers. Advances in
Colloid and Interface Science, 223, pp.40-54.
Truthinaging.com. (2018). Lactoperoxidase (LPO). [online] Available at:
https://www.truthinaging.com/ingredients/lactoperoxidase [Accessed 1 May 2018]
11

5
SECTION 2
DOWNSTREAM PROCESS
2.1 SEPARATION OF INSOLUBLES
The three processes that is suitable for separation of whey product from yogurt are ion exchange and membrane filtrations (microfiltration
and ultrafiltration). The reason why centrifuge is not listed because the separation of whey is at large scale. Hence, centrifuge is not suitable to be
used in this process. Based on the Table 2.1, the best unit operation to separate whey from yogurt is membrane filtration. Both microfiltration and
ultrafiltration conduct the same process to separate the product. But, to choose either these two type of membrane filtration, the end product is
needed to be determined for example, if concentrated whey is needed, microfiltration is suitable meanwhile if powdered whey is needed,
ultrafiltration is the best. For this project, we need concentrated whey thus, microfiltration is chosen.
Table 2.1 : Separation of Insoluble (Bodybuildingforyou.com, n.d)
Type of separation Ion Exchange Microfiltration Ultrafiltration
Process The process is to concentrate and purify whey
protein. The protein is placed into an ion
exchange tower and undergoes a chemical
purification process. Typically, two chemicals
are used in this process: hydrochloric acid and
sodium hydroxide. However, most of the
contents are left intact. Once the protein has been
concentrated, it is then placed into a drying
tower to remove the remaining water. The final
step is to package the protein powder into
various-sized containers (e.g., 25, 50, or even up
to 1000 kilograms) and ship them to distribution
centers.
This method uses fine specialty filters to strain the protein. The
filters are called micro-filters or ultra-filters because the size of the
holes/pores of the filters is microscopic. This is a physical means of
removing the contents from the protein.

6
Applications -Purification of sugar beet
-Whey processing
-Dimineralization of slaughterhouse
-Whey processing
-Bacteria reduction
-Milk protein fractionation
-Milk fat removal
-Whey processing
-Protein concentration
-Protein standardisation
-Decalcification
-Fresh cheese
-Lactose Reduction
Advantage -Low cost
-End product has less fat and lactose
-Minimal denaturing of protein
-Separation of protein without the
use of heat or chemicals
-Contain more calcium and less
sodium
-Does not damage the many immune
boosting components (eg : alpha
lactalbumin, immunoglobulins, and
GMPs)
-The size is 0.2–2 μm which is
suitable to produce concentrated
whey
-Minimal denaturing of
protein
-Separation of protein
without the use of heat or
chemicals
-Contain more calcium and
less sodium
-Does not damage the many
immune boosting
components (eg : alpha
lactalbumin,
immunoglobulins, and
GMPs)
-The size is 1–500 μm
which is suitable to produce
powdered whey
Disadvantage -Chemicals need to be used which are
hydrochloric acid and sodium hydroxide
-Due to the used of reagents, some amino acids
are denatured :
• Glycomacropeptides (GMPs)
• Immunoglobulins (antibodies)
• Lactoferrin
-Some alpha lactalbumin
-High Cost -High Cost

7
2.2 ISOLATION AND PURIFICATION (CHROMATOGRAPHY)
Liquid chromatography processes are based on the differential affinity of various soluble molecules for specific types of component.
Currently, the isolated and purified lactoferrin and lactoperoxidase are mainly ion exchange chromatography, hydrophobic interaction
chromatography and affinity chromatography (Faraji, Zhang, & Ray, 2017). Based on these three types of chromatography ion exchange
chromatography is the most favourable because of the lower in cost of operations, large sample volume can be operated and high purity of product.
In addition, affinity chromatography is high cost and hydrophobic interaction chromatography yields are low, therefore the methods are not suitable
for large-scale industrial production.
Table 2.2: Purification by using chromatography (Faraji, Zhang, & Ray, 2017)
Type of chromatography Ion Exchange Chromatography Hydrophobic Interaction
Chromatography
Affinity Chromatography
Process The adsorption of the molecules
to the solid support is driven by
the ionic interaction between the
oppositely charged ionic groups
in the sample molecule and in the
functional ligand on the support.
By increasing the salt
concentration (generally by using
a linear salt gradient) the
molecules with the weakest ionic
interactions start to elute from
Separates protein molecules
using the properties of
hydrophobicity. In this method,
proteins containing both
hydrophilic and hydrophobic
regions are applied to an HIC
column under high salt buffer
conditions. Proteins adhere to the
hydrobhobic surface under high
salt conditions and redissolve
Configure a high concentration
solution of salt diluted protein
solution

8
the column first. The binding
capacities of ion exchange resins
are generally quite high.
into the mobile phase as the salt
concentration is reduced
Application -Analysis of amino acids
-determine the base composition
of nucleic acids
-water purification
1. -proteins seperation
2. -separation of many vitamins,
other biological amines, and
organic acids and bases
-Remove product aggregate
species
-Purification of protein
-Isolate proteins
-Enzyme-ligand
-Enzyme-cofactor
-Receptor-agonist
-Purification of monoclonal
antibodies
Advantages -Lowcost
-Large sample volume
-Easier to precisely control the
conditions for amplification
industrial production
-High purity
-High purity, without loss of
biological activity of the protein
-Specific solutes
Disadvantages - Buffer requirement -High cost, and therefore difficult
industrialization
-Sensitive to pH
-Sensitive to buffer type
-Sensitive to temperature
-Protein precipitation
-Protein loss
-Poor resolution
-Low purity of the protein

9
2.3 POLISHING (DRYING)
Drying is the last step in separation process, which is the process of thermally removing volatile (water) to yield a solid. The most common
reason for drying a biological product is that it is susceptible to chemical or physical degradation during storage in a liquid formulation.
Furthermore, drying is for convenience in the final use product. Out of this four types of dryer, spray dryer is the best dryer to dried the
lactoperoxidase solution. The reason why is because spray dryer can operate the lactoperoxidase into dried cosmetic product. Although the
operation of spray dryer is high capital and overhead costs and has maintenance issues (Anon, 2018), the production is rapid, continuous,
reproducible, single-step, and thus, scalable without major modifications, scalability and cost-effectiveness (Sosnik, A. and Seremeta, K., 2015)
Table 2.3 ; Drying process.
Types of
Dryer
Vacuum Shelf Dryers Batch Vacuum Rotary
Dryers
Freeze Dryers Spray Dryers
Process Trays filled with the
product to be dried rest on
shelves to the tray through
which heat exchange
medium is circulated.
Vacuum is applied to the
trays to allow drying to
take place at lower
temperatures.
Heat is supplied by heat
exchange medium
circulated through a
jacket on the rotating
double cone drum. The
solids are continually
tumbled by rotation of the
drum.
The product is cooled to a
low temperature to allow
complete solidification.
The pressure is reduce so
drying can occur by
sublimation. Unbound
water in drying phase
(primary drying). A higher
shelf temperature is
required to remove bound
water (secondary drying)
Transform feed in liquid
state into dried particulate
form by spraying the liquid
into a hot gas(air). 3 basic
unit used is liquid
atomization, gas droplet
mixing, and drying from
liquid droplets.
Application Pharmaceutical product Agriculture, beans and
nuts, biomass
Use for products that have
high sensitivity to heat.
Pharmaceutical, chemical,
cosmetic, food industry

10
Advantages -Drying action becomes
faster as heat is easily
transferred throughout the
body of the dryers, due to
its large surface area
-Uniform drying
-Short cycle time
-No degradation due to
over-exposure, more
temperature and time
-Dry product has a very
specific surface area
-Rapid, continuous,
reproducible, single-step, and
thus, scalable without major
modifications, scalability and
cost-effectiveness
Disadvantages -Low efficiency.
-Expensive
-Require skilled labour to
operate
-Cost of maintenance are
high
-Not suitable for tumbling
motion
-Maintaining frozen
storage is costly.
-Failure of freezing
equipment would risk the
total loss of the product
-High Capital and Overhead
Costs
-Maintenance Issues

PROCESS OF EXTRACTION OF LACTOPEROXIDASE PROTEIN FROM YOGURT WHEY

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