Membrane technology in prophylactic biologicals in milk

Application of Membrane Technology in Prophylactic Biologicals in
Milk
10/30/2018Membrane Technology in Dairy Processing
1
Presented by:
Parth Hirpara
Dairy Technology Dept.

What is Prophylactic Biologicals?
 Prophylactic is defined as a preventive measure.
 The word comes from the Greek for "an advance guard," an
suitable term for a measure taken to fend off a disease or
another unwanted consequence.
 A prophylactic is a medication or a treatment designed and
used to prevent a disease from occurring.
(www.medicinenet.com)
 Prophylactic biologicals can be defined as “ to prevent a
range of human ailments such as arthritis, toothache,
allergies, various kinds of viral infections such as common
cold, etc.”
2

Why prophylactic biologicals from milk &
Membrane Technology?
 Extra-nutritional role of milk proteins in maintaining the
physiological normalcy of the body at organ & cellular level
function.
 Due to side effects of synthetic drugs as milk is a natural
source.
 The physiological functions of the drug principle from milk
can be:
 Get destroyed to various extent during routine processing (Depends
upon severity of the heat treatment).
 Also influenced by environmental contaminants such as
organopesticides & insecticides.
3

How to extract this biologicals?
 Membrane processing of milk allows separation of insoluble
(fat & casein) fraction from soluble (whey proteins, Lactose,
peptides & NPN) fraction.
 Casein & fat fraction processed separately, avoiding inevitable
interaction between the casein and whey protein fractions
under the influence of heat.
 This allows reconstitution of milk with “Bio-protective factor”
intact.
 Permeate whey proteins are further UF to get highly pure
undenatured whey protein isolates, which displays
prophylactic quality.
4

5
Categories of
prophylactic
biologicals
GMP
Lf & LP
Bioactive
peptides
Phospholipids

Milk peptides with cardiovascular activity
 There is a sequence homology between γ-fibrinogen in blood & κ-casein (GMP).
 GMP:
 Inhibits platelet aggregation.
 Combine with receptor site & consequently prevents fibrinogen binding with blood
platelets (anti-thrombotic activity).
 In vitro, anti-aggrigative properties is reinforced by the presence of Lys residue
in the sequence.
 The 112-116 peptide resulting from the tryptic hydrolysis of GMP seems to be 200
fold more active than 113-116 sequence.
 Hydrolysis of angiotensin-I by ACE is the key step in the physiological regulation
of blood pressure.
 43-52 peptide of β-casein possess such activity.
 All such peptides have mol. Wt. between 30-80kDa & primarily UF is used and
further they are isolated using SDS-PAGE.
6

Biologically functional peptides derived from
milk proteins
1. Opioid agonist peptides bind to opioid receptors and
exhibits morphine like activity.
First characterized opioid agonist peptides from milk proteins
are:
β-casomorphin 7: Try-Pro-Phe-Pro-Gly-Pro-Ile (60-66th)
 β-casomorphin 5: Try-Pro-Phe-Pro-Gly (60-64th)
β-casomorphin 5 is five times more active than BCM 7..
Similar peptides are present in αs-casein (90-96th) known as αs-
casein exorphin.
They are produced by chemical synthesis (isoelectric
precipitation) or enzymatic digestion.
7

2. Enzymatic hydrolysis of casein seems to produce ACE-
inhibitory peptides.
 ACE converts angiotensin I to II; the later is very hypertensive peptide.
 This enzyme also hydrolysis bradykinin, which is a hypotensive peptide.
 Therefore ACE inhibitors are antihypertensive peptides.
 ACEI from β-casein & κ-casein are most potent inhibitor is a
decapeptide (43-52).
3. Antimicrobial peptides:
 Some fragments of β-casein gives phagocytosis stimulating peptides.
 This peptides in vitro stimulates phagocytosis activity of murine &
human macrophages & exert in vivo a protective effect against
Klebsiella Pneumonia infection of mice.
8

Bioactive components from cheese whey
 Lf & LP have very high values as fine chemicals as well as functional foods
as well as pharmacological products.
 Both Lf & LP have pI between 4.2-5.3.
 For isolation of Lf & LP the whey is exposed to certain exchanger for
selective adsorption.
 Use is made here of the net positive charge of the both Lf & LP in contrast
to the other whey proteins, which have net negative charges in this pH
range.
 By charge interaction Lf & LP molecules binds to the negatively loaded
functional groups of the cation exchanger which leads to fixation of these
molecules on the ion exchange resins while the other whey proteins pass
through it (because they are negatively charged).
9

Basic criterion for isolation of Lf & LP
 Need of particle free whey (to keep high flow rate during the
loading phase) because very high volumes of whey have to
pass the ion exchange column.
 LP in sweet whey is ~20mg/L & Lf is ~35mg/L.
 So, theoretically for 100% yield: 50m of whey has to be
treated to get 1Kg of LP and 30m for 1Kg Lf.
 The capacity of used ion exchange resin is equivalent to
adsorption of 40-45g of LP plus Lf from whey per litre of resin
can pass the ion exchange column during 15-20hr per ion
exchange cycle.
10

Pharmaceutical application of Lf
 Bio-protective nature of Lf.
 Lf in infant formulas, health foods, skin creams as
antimicrobial product.
 To treat leukemia (Cancer).
 In association with the human γ-interferon, Lf supresses easily
human monocytes or mono-blastoid cell lines.
 Lf also enhance the action of certain antibiotics which
enhances the efficacy of pharmaceutical preparations.
11

Industrial preparation of Lf & LP
Past. Sep. Whey
MF
Ion exchange elusion
UF + Ro UF + Ro
Sterile filtrate Sterile filtrate
Spray drying(Lf) Spray drying(LP)
12

Phospholipid from MFGM
 Phospholipids and glycoproteins from the MFGM affect several
cell functions, such as growth, molecular transport system,
memory processing, stress responses, and central nervous
system myelination.
 Have an important role as emulsifiers in food systems and can
be used to improve the features of bread, chocolate,
margarine and dairy products.
 Use in a delivery or carrier system for drugs and other
substances.
 By using membrane technology, 95% pure phospholipid can
be obtained.
13

Use of ultrafiltration and supercritical fluid
extraction to obtain a whey buttermilk
powder enriched in milk fat globule membrane
phospholipids
14
Marcelade Rezende Costa, Xiomara Elizabeth Elias-Argote, Rafael Jiménez-Flores and
Mirna Lúcia Gigante (2010)
ARTICLE1

Introduction
 Whey buttermilk, a by-product from whey cream processing to butter, is rich
in milk fat globule membrane (MFGM) constituents, which have technological
and potential health properties.
 Whey butter milk was concentrated by ultrafiltration (10X) and subsequently
Diafiltered (5X) (10kDa molecular mass cut off membrane) at 25oC and the
final retentate was spray dried.
 The whey butter milk powder was submitted to supercritical
extraction(350bar,50oC) using carbon dioxide.
 The membrane filtration removed most of the lactose and ash from the whey
buttermilk, and the super critical extraction extracted exclusively non-polar
lipids.
 The final powder contained 73% Protein and 21% lipids, of which 61% were
phospholipids.
 This ingredient, a phospholipids-rich dairy powder, could be used as an
emulsifier in different food systems.
15

Material & Method
 Three batches of whey cream, around 120 L each.
 The product was derived from unsalted whey obtained from renneted
cheese processing.
 The cream was processed at 12oC using a rotatory churn to obtain whey
butter and butter milk.
 Butter milk was recovered in milk cans, after butter fines were removed by
filtration through cheese cloth, and stored overnight at 4oC until
membrane filtration.
 A pilot plant scale system (R-12 model, GEA-Niro Filtration, Hudson,
WI,USA) using two spiral polymeric membranes fitted in parallel on the
module(10kDa molecular mass cut off,11.33m2 total surface area) was used
for butter milk concentration.
16

Contd…
 The process was carried out at 25oC, the transmembrane pressure was
around 6bar and feed pump was operated at 35Hz.
 The ultrafiltration(UF) was conducted until a ten-fold volumetric
concentration factor was reached.
 Diafiltration (DF) was done by adding continuously tap water at 25oC to
the feed tank to replace the removed permeate until reaching a five-fold
diafiltration factor (5XDF).
 The final retentate from all experiments were spray-dried (Niro Filter lab
Spray-drier, Hudson, WI, USA) using 35 bar of pressure, and inlet and
outlet air temperatures of 185 C and95oC, respectively, to obtain whey
butter milk powders(WBP).
 A portion of the powder obtained from each whey cream batch was
submitted to supercritical fluid extraction(SFE).
17

Contd…
 Circulated deionized water at 3oC was used for cooling different zones in the
SFE apparatus.
 Carbon dioxide tanks (50-lb) were filled.
 The system conditions were controlled manually by Windows 2000 based
software.
 Approximately135g of each sample were submitted to three extraction cycles
using the following conditions: 1500g of CO2 at a flow rate of 20g min-1,
extraction pressure of 350bar, and both extraction and collection temperature
of 50oC. The SFE trials were done in triplicate.
 The powders submitted to the SFE (SFE-WBP)as well as the WBP were stored
at10oC until further analysis.
18

Results
Composition of whey cream, Butter &
BM Composition of WBM & SFE-WBP
19

Contd…
 The permeate flux during the filtration started at 45Lh-1m-2, dropped
approximately 26% in the ultrafiltration step and increased gradually in the
diafiltration phase, reaching 36Lh-1m-2 at the end of the process.
 The average flux for the whole filtration process was 31Lh-1m-2.
 The SFE process removed approximately 34 g of lipids from each 100 g of sample
(47.26 g of lipids), using 97 g of carbon dioxide to extract each gram of this fat.
 This extraction represented a reduction of 72% in the quantity of total lipids
originally present in the whey buttermilk powder.
20

Contd…
 As expected, the extraction using
supercritical CO2 selectively removed
non-polar lipids from the matrix
powder.
 The removed fat did not contain any
polar lipids, as can be seen in the
chromatogram(lines7 and 8),
consequently their concentration
increased in the powders after the SFE
procedure (lines 4-6) when compared
with the non- treated whey butter
milk powders (lines1-3).
21

Contd…
 SFE did not influence the proportion of the phospholipids in the powder, since this
process did not remove any of them.
 However, the lipid to protein ratio was reduced from 1:1 to1:3.5 while the
phospholipid to lipid ratio increased from 1:6.6 to1:1.7.
 The whole filtration (UF/DF) and SFE processing resulted in an increase of 500% in
the phospholipid content, in comparison to the original whey buttermilk on dry
matter basis.
 The SDS-PAGE protein profiles revealed that all samples had three distinct protein
groups: milk fat globule membrane proteins, caseins and immunoglobulin G light
chains, and the main whey proteins β-Lactoglobulin and a-lactalbumin.
 The samples of whey buttermilk, whey buttermilk powder, and whey buttermilk
powder after SFE showed a lower proportion of proteins in the molecular weight
range of caseins and Ig-G light chains and a greater proportion of MFGM and whey
proteins, especially after the SFE step.
22

Downstream Processing of Bovine
Lactoferrin from Sweet Whey
23
ARTICLE1I
ULBER R., PLATE K., WEISS T., DEMMER W., BUCHHOLZ H. AND SCHEPER T. (2001)

Introduction
 Whey is a by-product from the cheese manufacturing process that is often
used to produce whey protein concentrate powders for food applications.
 Besides the major whey proteins such as Lactalbumin or BSA, minor whey
proteins are present such as lactoperoxidase and bLF.
 In addition to the well-known biological functions as an antimicrobial and
antiviral agent, bLF shows immunomodulatory functions in the host
defence system.
 For the isolation of bLF, a two-step downstream process was developed
based on membrane systems.
 The glycoprotein lactoferrin (LF) belongs to the transferrin protein family
with binding properties three times stronger to iron than to transferrin.
 LF is also particularly involved in ALZHEIMER'S disease and could serve as
an HIV prevention clinical agent.
24

Contd…
 Only minor amounts in bovine milk ranging from 20–200 μg/ml.
 Due to the basic isoelectric point (8.0–9.5) and the almost
positive charge, bLF can induce interactions with other whey and
milk proteins such as β-lactoglobulin and caseins.
 To date, minor protein components like bLF are isolated by
standard column chromatographic procedures.
 At present, Sartobind® adsorber (SARTORIUS, Göttingen,
Germany) is used for the direct removal of bLF from sweet whey.
Due to its high isoelectric point of 8.5–9.0, bLF can be bound on
the strong cationic membrane adsorber (Sartobind S®).
25

Membrane Adsorber Technique
 Membranes can be converted into efficient absorbers by attaching functional groups
to the inner surface of synthetic microporous membranes.
 Depending on the choice of the attached functional groups, various adsorber systems
can be obtained that can be used for affinity adsorption, ion exchange or immobilized
metal affinity chromatography (IMAC).
 Commercially available are membrane ion exchangers of the strongly acidic (sulfonic
acid), the strongly basic (quaternary ammonium), the weakly acidic (carboxylic acid)
and the weakly basic (diethylamine) type.
 The membrane adsorber technology is typically applied for the concentration of
proteins and monoclonal antibodies, removal of contaminants (e.g. DNA, endotoxins)
and reduction of virus content.
 Using such membrane adsorber technique requires a pre-treatment of whey to
remove the insoluble particles and lipids which otherwise will block the membrane.
 continuous crossflow filtration steps have to be performed prior to the ion exchange
step. The permeate coming from this filtration step can be loaded directly on the
membrane adsorber.
26

Major advantages of membrane adsorber
technology
 Major advantages over classical separation methods:
 Due to the membrane structure, diffusion processes do not limit the
binding of proteins; therefore, the loading and elution can be performed
at very high fluxes resulting in very short cycle times.
 The compressibility of the membrane under normal operation conditions
can be neglected, channelling cannot occur, and the pressure distribution
inside the modules is designed to have plug flow through the module
altogether leading to sharp breakthrough curves.
 Scale-up is very easy, materials and systems allow CIP and the validation
of the process is made easier due to the usage of standard products and
validation service of suppliers.
27

Scheme of the downstream process for the isolation of bLF from
sweet whey
28

Materials and Methods
1. Substances:
 Sweet cheese whey.
 Salts for sodium phosphate buffers (NaH2PO4 and Na2HPO4) and eluents (NaCl).
 Bovine lactoferrin for standard measurements.
 The matrix substance used for MALDI-MS measurements (matrix assisted laser
desorption/ ionization-mass spectrometry), α-cyano-cinnetatic acid, and the
chemicals for gel electrophoresis (sodium dodecyl sulphate,
ethylenediaminetetraacetic acid, bromphenol blue, tromethamine, sodium
thiosulfate, silver nitrate).
29

Contd… 2. Crossflow Filtration: For the crossflow filtration of cheese whey, various membranes of
different geometries and pore sizes were used as shown in Table:
30

Contd…
 The experiments were carried out in the recycled batch mode, in
which the retentate as well as the permeate was led back into the
batch reactor to keep the concentration of bLF constant over the
whole filtration time.
 To assess the cleaning effect of the filtrations, transmission
measurements were made against water at 600 nm (Uvikon 922
spectrophotometer, KONTRON INSTRUMENTS) with retentate and
permeate samples.
 Permeates showing transmission values of at least 30 did not cause
blocking effects on the ion exchange membranes anymore.
 The filtration temperature was 50 °C.
31

3. Cation-Exchange Membranes
 For the isolation of bovine lactoferrin, strongly acidic membranes of the so-
called “Sartobind ® Membrane Adsorber Factor Two Family” from
SARTORIUS, Göttingen, Germany were used.
 The reeled modules are available in various heights and layers, resulting in
membrane areas ranging from 0.1 to 8.0 m2 per module.
 The presented results were received with two 1- m2 modules (type S-10k-15-
25) in series.
 The flow rates varied between 1.0 and 3.0 l/min; the process was monitored
by an UV detector (model 662 UV Analyzer, WEDGEWOOD TECHNOL., Inc.) at
280 nm.
 Prior to use, the membranes were pre-equilibrated with a 20 mM sodium
phosphate buffer at pH 7.0; afterwards bLF and lactoperoxidase as a by-
product were eluted in a three-step sodium chloride salt gradient.
32

Contd…
4. Recovery:
 The eluates were desalted and concentrated using a “Sartocon II” plant from SARTORIUS,
Göttingen, Germany.
 A cellulose acetate membrane with an area of 0.7 m2 and a cut-off of 30 kDa was used.
 The removal of sodium chloride was monitored via conductivity measurement.
 The desalted lactoferrin solution was lyophilized.
5. Analysis:
 The quantitative determination of bLF in solution was carried out using HPLC.
 The elution gradient for the by-product lactoperoxidase was 0.5 M sodium chloride in 20 mM
sodium phosphate buffer (pH 7.0) and 2.0 M sodium chloride in 20 mM sodium phosphate
buffer (pH 7.0) for the target protein.
 For the qualitative determination of bovine lactoferrin, MALDI-MS measurements with a
Compact Maldi 3 (KRATOS ANALYTICAL) and SDS-PAGE (sodium dodecyl sulphate-
polyacrylamide gel electrophoresis) with a PhastSystemTM (PHARMACIA, Uppsala, Sweden)
were used.
33

Results and Discussion
 The process for the production of bovine lactoferrin from cheese whey is divided into three
steps: a crossflow microfiltration, the isolation via cation-exchanger membranes and
recovery.
 In the first step, the crossflow filtration, insoluble particles such as lipids, caseins and
precipitated proteins are removed from the whey. This is necessary to prevent blocking of
the cation exchange membrane modules in the isolation step.
 To obtain suitable permeates in high yields with a maximum content of the target protein, a
membrane screening was carried out (The results of this screening are shown in Table).
 The tubular modules allowed the highest permeate fluxes and constantly high permeation
rates of bovine lactoferrin to be obtained.
 Based on their geometry, tubular modules also allow extremely high retentate fluxes to be
achieved; the resulting shear rates of about 9000 1/s prevent the fouling of the membrane's
surface, which may limit flux and permeation rates.
 The pore size of 1 μm did not lead to permeates of adequate quality (transmission values of
less than 30), therefore, for the following whey filtrations a Microdyn 0.2 μm tubular module
was used.
34

Contd…
 In the second step, bLF was isolated from the permeates by using Sartobind® cation
exchanger membranes.
 The dynamic binding capacity of the cation-exchange membranes for bovine lactoferrin was
found to be 0.2 mg/cm2 (Table) independent of the flow rate.
 This allows 4 g of the target protein per process cycle to be isolated.
35

Contd…
 The dynamic binding capacity was determined at 10% breakthrough of bLF in the filtrate of
the adsorber step.
 Figure below shows the bLF breakthrough curve for the used adsorber system (two modules
S-10k-15-25 in series).
36

Contd…
 After loading and rinsing the modules with 20 mM sodium phosphate buffer (pH
7.0) to remove non-bound components, a step gradient (0.2 –1 M NaCl) was used
for the elution of bLF and the second bound protein, lactoperoxidase.
 90% of the bound lactoperoxidase was eluted from the modules at a salt
concentration of 0.2 M NaCl, together with 16% (0.64 g) of the bound lactoferrin.
 The 0.3 M NaCl fraction contained 8% of lactoperoxidase and 48% (1.92 g) of
lactoferrin.
 The 0.4 M NaCl fraction still contained rests of lactoperoxidase and 32% (1.28 g) of
the bound lactoferrin.
 At salt concentrations of more than 0.5 M NaCl, no more proteins could be detected
in the eluates.
 To get two protein fractions as pure as possible, the salt concentration of the used
eluents was optimized.
37

Contd…
 The best result was achieved with a three-step sodium chloride elution which led to a
lactoperoxidase fraction of about 85% purity (0.08 g bLF) and a lactoferrin fraction of about
95% purity (3.4 g bLF).
38

Contd…
 The content of lactoferrin in the processed cheese whey was 0.1 g/l.
 Loading the modules with a flow rate of 2 l permeate per minute it was possible to
carry out two cycles per hour with a yield of 8 g bLF.
 Several cycles were performed successively without a cleaning procedure so that
the loading step of each new process cycle could lead to a self-regeneration of the
ion-exchange groups on the surface of the modules.
 The desalting and concentration of the protein-containing eluate was carried out
by crossflow ultrafiltration.
 The collected lactoferrin-containing permeates of five process cycles of about 12 l,
were desalted within two hours.
 The loss of protein that was bound to the membrane's surface was 10%.
 By rinsing the module with distilled water after finishing the ultrafiltration, the loss
could be reduced to 6%.
39

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Membrane technology in prophylactic biologicals in milk

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