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Fractionation of milk proteins on pilot scale with particular focus on β-casein
Accepted Manuscript Fractionation of milk proteins on pilot scale with particular focus on β-casein Katharina J.F. Thienel, Aline Holder, Thomas Schubert, Remko M. Boom, Jörg Hinrichs, Zeynep Atamer PII:
S0958-6946(17)30250-9
DOI:
10.1016/j.idairyj.2017.12.006
Reference:
INDA 4251
To appear in:
International Dairy Journal
Received Date: 25 June 2017 Revised Date:
15 December 2017
Accepted Date: 18 December 2017
Please cite this article as: Thienel, K.J.F., Holder, A., Schubert, T., Boom, R.M., Hinrichs, J., Atamer, Z., Fractionation of milk proteins on pilot scale with particular focus on β-casein, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2017.12.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Fractionation of milk proteins on pilot scale with particular focus on β-casein
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Katharina J. F. Thienela, Aline Holdera, Thomas Schuberta, Remko M. Boomb, Jörg Hinrichsa,
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Zeynep Atamera*
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Matter Science and Dairy Technology, Garbenstr. 21, D-70599 Stuttgart, Germany
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Wageningen, Netherlands
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Wageningen University, Laboratory of Food Process Engineering, P.O. Box 17, 6700 AA,
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Universität Hohenheim, Institute of Food Science and Biotechnology, Department of Soft
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*Corresponding author. Tel.: +49 711 45923646.
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E-mail address: [email protected] (Z. Atamer)
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ABSTRACT
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The aim of this study was to increase the yield and purity of casein fractions at pilot scale and
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determine the main process parameters influencing the isolation of β-casein, such as cold
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extraction time, separation speed. The fractions were obtained from micellar casein by means
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of selective precipitation following separation by a nozzle centrifuge. The purities, which
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were calculated based on the total casein content of the β-casein fraction, and the yields
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achieved for β-casein ranged from 68.7 to 89.6% and from 10 to 32%, respectively. For the
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other two fractions, αS- and κ-casein, the obtained purities were 61% and 43%, respectively.
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Using the proposed isolation method, high purities for the β-casein fraction were achievable;
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however, the other two fractions, αS- and κ-casein, need further improvement.
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1.
Introduction
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Interest in purified casein fractions has been growing constantly (Holder, 2014; Post,
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2011). β-casein has good emulsifying and foam-stabilising properties (Dickinson, 2003) and
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is a good raw material for generating bio-functional peptides (Korhonen, 2009; Silva &
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Malcata, 2005). αS-Casein (αS1- and αS2-casein) can also be used for structure formation
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(Huppertz, 2013) and encapsulation of hydrophobic compounds (either αS1- or αS2-casein)
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(Kessler, Menéndez-Aguirre, Hinrichs, Stubenrauch, & Weiss, 2013). κ-Casein is also a
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source of physiologically active compounds (Tolkach & Kulozik, 2005).
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However, in the field of isolating individual casein fractions, there is an important gap regarding their pilot recovery and economic value. For lab-scale experimental studies,
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chromatographically purified caseins have been obtained in small scales (Cayot,
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Courthaudon, & Lorient, 1992). For applications in food or food formulations, a pilot process
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for the production of casein fractions aiming for a high purity and yield needs to be developed
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(Shapira, Assaraf, Epstein, & Livney, 2010).
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A recently published review summarised current technologies for the isolation and
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purification of β-casein (Atamer, Post, Schubert, Holder, Boom, & Hinrichs, 2017). These
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include selective precipitation as well as membrane filtration at low temperatures. Selective
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precipitation exploits the calcium sensitivity of αS- and β-casein by adding calcium chloride at
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alkaline pH to separate these fractions from the raw material (Post, Ebert, & Hinrichs, 2009).
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The advantage of using alkaline pH is that the αS- and β-casein can sediment more readily,
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which makes their separation possible without using high-speed centrifugation (Law &
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Leaver, 2007). In the membrane processing method, milk is cooled (≤ 4 °C) prior to
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microfiltration process, since storage at low temperatures causes dissociation of β-casein from
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in the serum phase (O'Mahony, Smith, & Lucey, 2007). The separated β-casein is further
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purified using several filtration and demineralisation steps. In another recent patent, milk is
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pre-heated and subjected to a warm microfiltration, and the obtained MF retentate was cooled
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and subjected to a cold microfiltration; this method was proposed to produce β-casein-
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containing formulations and products (Christensen & Holst, 2016).
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The current study aimed to further investigate and improve the isolation method based
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on our previous studies. A detailed description of the fractionation method using micellar
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casein powder as raw material is provided and an assessment of the method was carried out.
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2.
Materials and methods
2.1.
Production of micellar casein
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Production of micellar casein concentrate was performed using microfiltration and
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diafiltration (Fig. 1) (Kersten, 2001) and subsequently the concentrate was spray dried (Post
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& Hinrichs, 2011). As ultrafiltration permeate (Fig. 1C), sweet whey ultrafiltration permeate
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powder [Bayolan PT, BMI, Landshut, Germany; dry matter 96.1%, protein 3.5%, fat < 1.0%,
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lactose 83.0–87.0%, ash 6.5–8.0% (w/w)] was dissolved in water (5.2%, w/w) and used for
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the diafiltration. As an alternative for ultrafiltration permeate powder, the obtained
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microfiltration permeate was subjected to an ultrafiltration process and used as the
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diafiltration medium.
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2.2.
Isolation of αS-, β- and κ-casein at pilot scale
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The isolation of the casein fractions was performed according to the method described by Post and Hinrichs (2011) with some modifications (Fig. 2). Micellar casein powder was
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reconstituted to a solution with 3.2 % protein content (80 L) and stirred at 30 °C for 15 min.
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The solution was alkalised to pH 11.0 using a sodium hydroxide solution (NaOH, 5 M; Merck
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KGaA, Darmstadt, Germany). A calcium chloride solution (CaCl2, 4.4 M; Merck KGaA) was
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added to a final concentration of 50 mM. After precipitation with CaCl2 the pH of the solution
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was readjusted to pH 7.0 using hydrochloric acid (HCl, 5 M; Merck KGaA). A selective
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isolation of the κ-casein from the αS- and β-casein was performed (separation 1). All
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separation steps were performed using a continuous nozzle separator (type KNA 3, Westfalia
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Seperator AG, Oelde, Deutschland).
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Afterwards, the αS- and β-casein-rich precipitate was re-suspended in demineralised
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water (total mass of up to 80 L) and cooled down to < 5 °C (cold extraction 1). The pH was
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adjusted to pH 4.6, while the suspension was stirred for 2 h to enable β-casein to dissociate
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into the serum phase. After separation step 2, the αS-casein was found to be in the precipitate
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and the majority of β-casein in the supernatant. The enriched β-casein supernatant was
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warmed up (to 35 °C for approximately 15 min) and the pH was adjusted to 4.6 to carry out a
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further separation step (separation 3) to concentrate the β-casein fraction. A final separation
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step (separation 4) was carried out to further purify and concentrate the β-casein fraction.
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After each separation, demineralised water was added to reach the initial volume of 80 L to
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achieve a sufficient medium for the next purification step.
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The pH of the κ-casein supernatant was adjusted to pH 3.8 for precipitation and was
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separated (separation 5) to remove the CaCl2-rich aqueous phase. After resuspension of the
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enriched κ-casein and adjusting the solution to pH 4.6, another separation (separation 6) was
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performed.
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treated with a 10% (w/w) sodium citrate solution to a final concentration of 5% (v/v) to
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improve the solubility of powder (Callison, 1988). The solubility of the powders (αS- and β-
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casein fractions) in different solutions (e.g., water, ultrafiltration permeate, and a calcium-
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depleted milk salts solution) as affected by temperature and pH in was investigated our
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previous study (Post, Arnold, Weiss, & Hinrichs, 2012). It was found that pH had a strong
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influence the solubility of the casein fractions. The powders were almost completely soluble
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in water at and above pH 6. The pH of the fractions was adjusted to 7.0 before spray drying.
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All isolated powders were stored in vacuum bags at 20 °C and used within 6 months.
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2.3.
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Analysis
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2.3.1. Major constituents of casein powders
The measurements of level of total protein, calcium, dry matter, lactose and fat
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contents were carried out according to the corresponding methods reported by Post et al.
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(2012).
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2.3.2. Reversed-phase high-performance liquid chromatography of casein powders and
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determination of yield and purity
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Selectively isolated casein fractions were analysed by reversed-phase high-
performance liquid chromatography (RP-HPLC) according to Post et al. (2009).
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The purity of single casein fractions (Pn-CN) was calculated on casein basis and defined
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as the ratio of the single casein fraction concentration (cn-CN, fraction) and the total casein content
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of fraction (ctotal CN, fraction) (Eq. 1).
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Pn-CN =
Cn-CN, fraction Ctotal CN, fraction
· 100 %
(1)
The yield (Yn-CN) of each casein fraction is defined as the ratio of the amount of the
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obtained casein fraction to the total amount of the protein in the micellar casein powder and
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was calculated as follows:
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Yn-CN = m
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where mtotal CN,fraction refers to the mass of the obtained n-casein fraction, mMCN to the initial
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mass of micellar casein (MCN) powder used for the isolation, while ctotal protein, MCN refers to
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the calculated protein content of the micellar casein using the measured total nitrogen content.
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mtotal CN, fraction · Pn-CN · MCN · Ctotal protein, MCN
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Results and discussion
3.1.
Micellar casein
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Micellar casein was selected as raw material, since it was shown to be better than
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caseinates regarding the obtained yield (Post & Hinrichs, 2010). The composition of this
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powder is summarised in Table 1 (Batch I). An addition, the composition of another batch
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(Batch II) is given. During its production (Fig. 1A), the ultrafiltration permeate obtained from
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the skim milk was used instead of a re-suspended ultrafiltration permeate powder for
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diafiltration process, in contrast to the process according to Kersten (2001). The casein
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content of the micellar casein powder was 72.2 ± 1.38% (w/w). These results confirm and
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extend those reported by Post et al. (2009). The lactose content (15.4 ± 0.02%, w/w) could be
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reduced, for example by changing diafiltration media, e.g., using water instead of
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ultrafiltration permeate. The casein content obtained for batch II was in the same range as the
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other 13 independent micellar casein batches (casein level range: 58.2–73.1%). No difference
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resulting from the type of diafiltration medium was observed. The microfiltration permeate
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can be reused to reduce waste water discharge and energy costs for spray drying as well as
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time.
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3.2.
Casein fractions
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The purity and yield of the β-casein fractions obtained from five independent
productions (batch A, B, C, D, and E, process volume 80 L; Fig. 2) are shown in Table 2. For
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the production of each batch, the changes in process parameters (such as separation speed,
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time of cold extraction) made during the production of each batch are also given. In the
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present study, the same parameters as used in the work of Post and Hinrichs (2011) were
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taken as a reference and the same parameters were used for the production of batch A.
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Maximum achievable theoretical yields based on the total protein content in bovine milk are 47% for αS-casein, 35% for β-casein, and 12% for κ-casein. The purities and yields
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achieved in this study were calculated using the RP-HPLC profiles; Fig. 3 shows typical RP-
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HPLC profiles obtained for micellar casein, and β-casein, αS-casein, and κ-casein from the
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casein fractionation.
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In this study, a maximum purity of 89.6% and a yield of 10.2% were achieved for β-
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casein (Table 2), marginally higher than those (purity: 85%, yield: 8%) reported by Post and
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Hinrichs (2011). However, compared with the reported purities (up to 96%) in studies using
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membrane-based approach for the fractionation (Famelart & Surel, 1994), the achieved purity
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in this study was somewhat lower. The values were found in the range from 68.7 to 89.6%
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and decreased from batch A to E, except that for batch D, which could be because of the
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higher separation rates. The yields obtained ranged between 10 and 32%. The observed
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increase in the yield may be related to the extension of the cold extraction time, which was
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concentration of β-casein in the serum phase. To show the relationship between the yields and
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purities of the presented processes, the yields were plotted against the purities (Fig. 4).
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According to Pearson’s correlation, a moderate negative linear relation between the yield and
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purity was observed (p < 0.05).
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The by-products of the fractionation process (αS- and κ-casein) obtained during the
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isolation of β-casein were also analysed to evaluate the isolation process. The purity and yield
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values obtained were 61% and 26% for αS-casein, respectively, and 43% and 4% for κ-casein,
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respectively. The content of the κ-casein in our powder was approximately 12% of the total
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casein protein; therefore, the maximum yield of κ-casein would be at this level. With a yield
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of 4%, approximately 33% of the maximum possible κ-casein was gained. The remainder of
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κ-casein was either lost during separation or found in the other fractions as impurities.
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Conclusion
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Although several processes for the isolation of β-casein have been proposed in literature, there is still a need for improvement in scalability and in the yield and purity that
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can be attained. Micellar casein was used as raw material gained by membrane filtration
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process, and obtained highly purified β-casein fractions at a pilot scale using nozzle
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separators. The process enables the production of highly purified β-casein fraction with high
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yields, and in addition it gives highly enriched soluble αS- and κ-casein fractions; however,
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the yields of these fractions are still low compared with that of β-casein. This short
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communication provides a detailed description of the isolation process for the fractions as
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well as for the production of micellar casein. Further studies will focus on application of a
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decanter centrifuge for the separation process and the parameters influencing the process,
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especially CaCl2 concentration applied for the precipitation and the pH value used during cold
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extraction.
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Acknowledgements
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This research project was partly supported by the German Ministry of Economics and
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Energy (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project
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AiF 17126 N. The authors would like to thank to Peter Rädler and the staff of the LAZBW,
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Wangen i. A., as well as to Birgit Greif, Luc Mertz, and Giovanni Migliore at the University
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of Hohenheim for their analytical and technical assistance.
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References
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Callison, O. (1988). Dispersion and neutralization of acid casein. European Patent 0289867.
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Cayot, P., Courthaudon, J. L., & Lorient, D. (1992). Purification of αS-, β-and κ-caseins by
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Christensen, J., & Holst, H. H. (2016). Method of producing beta-casein compositions. EU patent EP2947996 B1.
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edn., Vol. 1, pp. 1229–1260). New York, NY, USA: Kluwer Academic/ Plenum
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Kersten, M. (2001). Proteinfraktionierung mittels Membrantrennverfahren [Protein fractionation by means of membrane processing]. PhD Thesis, Munich, Germany
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Post, A. E. (2011). Fractionation of bovine casein and enrichment of functional casein peptides. Dissertation, Stuttgart, Germany: University of Hohenheim, Verlag Dr. Hut.
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Post, A. E., & Hinrichs, J. (2011). Large-scale isolation of food-grade β-casein. Milchwissenschaft, 66, 361–364. Post, A. E., Arnold, B., Weiss, J., & Hinrichs, J. (2012). Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of αS- and β-casein.
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Production of micellar casein powder at pilot scale (modified from Kersten, 2001). Section A, production of skim milk concentrate up to a volume concentration factor (VCF) of
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3 via microfiltration; section B, final concentration of skim milk concentrate up to VCF = 6; section C, production of diafiltration medium. Abbreviations are: MF, microfiltration;
MWCO, molecular mass cut-off; UF, ultrafiltration; ϑ, temperature; Vሶ, feed flow rate; t, time;
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∆PTM, trans membrane pressure.
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Fig. 2. Flow chart illustrating isolation of αS-, β-, and κ-casein fractions from micellar casein at pilot scale (modified from Post and Hinrichs, 2011). Abbreviations are: ϑ, temperature; Vሶ, feed flow rate; t, time; p, pressure.
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Fig. 3. Sample chromatographic profiles of micellar casein (MCN), β-casein, αS-casein, and κ-casein obtained from casein fractionation by means of selective precipitation on pilot scale.
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Fig. 4. Comparison of the β-casein yields obtained with the purities for different batches ( ,
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A–E) and for the production of Post and Hinrichs (2011) (). Sizes of the circles are given according to the cold extraction time ranging between 2 and 3.5 h. The Pearson's correlation coefficient was –0.555 (p < 0.05).
ACCEPTED MANUSCRIPT Table 1 Chemical composition of the micellar casein powders. a
Batch II
96.7 ± 0.08 2.26 ± 0.03 15.4 ± 0.02 1.5 ± 0.1 74.1 ± 0.08 72.2 ± 1.38 97.4 0.2 ± 0.1
95.5 ± 0.0 2.45 ± 0.06 13.0 ± 0.1 1.53 ± 0.02 69.7 ± 0.47 67.8 ± 1.3 97.3 0.2 ± 0.1
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Batch I
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Dry matter Calcium Lactose Fat Protein Casein Casein/protein Whey protein
Composition (%, w/w)
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Component
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For the production of batch II, microfiltration permeate obtained from skim milk was used instead of protein-reduced sweet whey powder (ref. Fig. 1C).
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2011). a Batch A Batch B
Batch C
Batch D
Batch E
Post and Hinrichs (2011)
Output parameters β-Casein purity (%) β-Casein yield (%)
89.6 10.2
82.7 16.9
68.7 22.1
74.5 17.5
77.6 32.1
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Process parameters Extraction frequency Extraction time (h) Separation frequency Separation 1 (L h-1) Separation 2 (L h-1)
1 2 3 600 1200
2 2.5 4 550 1300
2 3 4 510 1200
2 2.5 4 600 1200
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Parameters
2 3.5 4 530 1300
1 2 3 600 1200
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For the present study, the process volume was 80 L except in the case of batch D for which the process volume was 85 L. Further deviations in the process parameters of Post and Hinrichs (2011) are: ϑ < 2 °C (for Separation 1), CaCl2 addition at pH 6–6.5 (isoelectric point of κ-casein).
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S0958-6946(17)30250-9
DOI:
10.1016/j.idairyj.2017.12.006
Reference:
INDA 4251
To appear in:
International Dairy Journal
Received Date: 25 June 2017 Revised Date:
15 December 2017
Accepted Date: 18 December 2017
Please cite this article as: Thienel, K.J.F., Holder, A., Schubert, T., Boom, R.M., Hinrichs, J., Atamer, Z., Fractionation of milk proteins on pilot scale with particular focus on β-casein, International Dairy Journal (2018), doi: 10.1016/j.idairyj.2017.12.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Fractionation of milk proteins on pilot scale with particular focus on β-casein
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Katharina J. F. Thienela, Aline Holdera, Thomas Schuberta, Remko M. Boomb, Jörg Hinrichsa,
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Zeynep Atamera*
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Matter Science and Dairy Technology, Garbenstr. 21, D-70599 Stuttgart, Germany
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b
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Wageningen, Netherlands
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Wageningen University, Laboratory of Food Process Engineering, P.O. Box 17, 6700 AA,
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Universität Hohenheim, Institute of Food Science and Biotechnology, Department of Soft
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*Corresponding author. Tel.: +49 711 45923646.
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E-mail address: [email protected] (Z. Atamer)
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___________________________________________________________________________
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ABSTRACT
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The aim of this study was to increase the yield and purity of casein fractions at pilot scale and
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determine the main process parameters influencing the isolation of β-casein, such as cold
31
extraction time, separation speed. The fractions were obtained from micellar casein by means
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of selective precipitation following separation by a nozzle centrifuge. The purities, which
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were calculated based on the total casein content of the β-casein fraction, and the yields
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achieved for β-casein ranged from 68.7 to 89.6% and from 10 to 32%, respectively. For the
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other two fractions, αS- and κ-casein, the obtained purities were 61% and 43%, respectively.
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Using the proposed isolation method, high purities for the β-casein fraction were achievable;
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however, the other two fractions, αS- and κ-casein, need further improvement.
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1.
Introduction
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Interest in purified casein fractions has been growing constantly (Holder, 2014; Post,
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2011). β-casein has good emulsifying and foam-stabilising properties (Dickinson, 2003) and
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is a good raw material for generating bio-functional peptides (Korhonen, 2009; Silva &
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Malcata, 2005). αS-Casein (αS1- and αS2-casein) can also be used for structure formation
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(Huppertz, 2013) and encapsulation of hydrophobic compounds (either αS1- or αS2-casein)
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(Kessler, Menéndez-Aguirre, Hinrichs, Stubenrauch, & Weiss, 2013). κ-Casein is also a
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source of physiologically active compounds (Tolkach & Kulozik, 2005).
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However, in the field of isolating individual casein fractions, there is an important gap regarding their pilot recovery and economic value. For lab-scale experimental studies,
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chromatographically purified caseins have been obtained in small scales (Cayot,
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Courthaudon, & Lorient, 1992). For applications in food or food formulations, a pilot process
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for the production of casein fractions aiming for a high purity and yield needs to be developed
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(Shapira, Assaraf, Epstein, & Livney, 2010).
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A recently published review summarised current technologies for the isolation and
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purification of β-casein (Atamer, Post, Schubert, Holder, Boom, & Hinrichs, 2017). These
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include selective precipitation as well as membrane filtration at low temperatures. Selective
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precipitation exploits the calcium sensitivity of αS- and β-casein by adding calcium chloride at
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alkaline pH to separate these fractions from the raw material (Post, Ebert, & Hinrichs, 2009).
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The advantage of using alkaline pH is that the αS- and β-casein can sediment more readily,
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which makes their separation possible without using high-speed centrifugation (Law &
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Leaver, 2007). In the membrane processing method, milk is cooled (≤ 4 °C) prior to
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microfiltration process, since storage at low temperatures causes dissociation of β-casein from
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in the serum phase (O'Mahony, Smith, & Lucey, 2007). The separated β-casein is further
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purified using several filtration and demineralisation steps. In another recent patent, milk is
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pre-heated and subjected to a warm microfiltration, and the obtained MF retentate was cooled
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and subjected to a cold microfiltration; this method was proposed to produce β-casein-
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containing formulations and products (Christensen & Holst, 2016).
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The current study aimed to further investigate and improve the isolation method based
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on our previous studies. A detailed description of the fractionation method using micellar
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casein powder as raw material is provided and an assessment of the method was carried out.
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Materials and methods
2.1.
Production of micellar casein
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Production of micellar casein concentrate was performed using microfiltration and
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diafiltration (Fig. 1) (Kersten, 2001) and subsequently the concentrate was spray dried (Post
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& Hinrichs, 2011). As ultrafiltration permeate (Fig. 1C), sweet whey ultrafiltration permeate
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powder [Bayolan PT, BMI, Landshut, Germany; dry matter 96.1%, protein 3.5%, fat < 1.0%,
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lactose 83.0–87.0%, ash 6.5–8.0% (w/w)] was dissolved in water (5.2%, w/w) and used for
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the diafiltration. As an alternative for ultrafiltration permeate powder, the obtained
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microfiltration permeate was subjected to an ultrafiltration process and used as the
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diafiltration medium.
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2.2.
Isolation of αS-, β- and κ-casein at pilot scale
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The isolation of the casein fractions was performed according to the method described by Post and Hinrichs (2011) with some modifications (Fig. 2). Micellar casein powder was
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reconstituted to a solution with 3.2 % protein content (80 L) and stirred at 30 °C for 15 min.
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The solution was alkalised to pH 11.0 using a sodium hydroxide solution (NaOH, 5 M; Merck
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KGaA, Darmstadt, Germany). A calcium chloride solution (CaCl2, 4.4 M; Merck KGaA) was
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added to a final concentration of 50 mM. After precipitation with CaCl2 the pH of the solution
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was readjusted to pH 7.0 using hydrochloric acid (HCl, 5 M; Merck KGaA). A selective
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isolation of the κ-casein from the αS- and β-casein was performed (separation 1). All
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separation steps were performed using a continuous nozzle separator (type KNA 3, Westfalia
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Seperator AG, Oelde, Deutschland).
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Afterwards, the αS- and β-casein-rich precipitate was re-suspended in demineralised
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water (total mass of up to 80 L) and cooled down to < 5 °C (cold extraction 1). The pH was
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adjusted to pH 4.6, while the suspension was stirred for 2 h to enable β-casein to dissociate
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into the serum phase. After separation step 2, the αS-casein was found to be in the precipitate
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and the majority of β-casein in the supernatant. The enriched β-casein supernatant was
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warmed up (to 35 °C for approximately 15 min) and the pH was adjusted to 4.6 to carry out a
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further separation step (separation 3) to concentrate the β-casein fraction. A final separation
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step (separation 4) was carried out to further purify and concentrate the β-casein fraction.
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After each separation, demineralised water was added to reach the initial volume of 80 L to
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achieve a sufficient medium for the next purification step.
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The pH of the κ-casein supernatant was adjusted to pH 3.8 for precipitation and was
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separated (separation 5) to remove the CaCl2-rich aqueous phase. After resuspension of the
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enriched κ-casein and adjusting the solution to pH 4.6, another separation (separation 6) was
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performed.
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treated with a 10% (w/w) sodium citrate solution to a final concentration of 5% (v/v) to
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improve the solubility of powder (Callison, 1988). The solubility of the powders (αS- and β-
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casein fractions) in different solutions (e.g., water, ultrafiltration permeate, and a calcium-
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depleted milk salts solution) as affected by temperature and pH in was investigated our
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previous study (Post, Arnold, Weiss, & Hinrichs, 2012). It was found that pH had a strong
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influence the solubility of the casein fractions. The powders were almost completely soluble
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in water at and above pH 6. The pH of the fractions was adjusted to 7.0 before spray drying.
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All isolated powders were stored in vacuum bags at 20 °C and used within 6 months.
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2.3.
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Analysis
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2.3.1. Major constituents of casein powders
The measurements of level of total protein, calcium, dry matter, lactose and fat
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contents were carried out according to the corresponding methods reported by Post et al.
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(2012).
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2.3.2. Reversed-phase high-performance liquid chromatography of casein powders and
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determination of yield and purity
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Selectively isolated casein fractions were analysed by reversed-phase high-
performance liquid chromatography (RP-HPLC) according to Post et al. (2009).
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The purity of single casein fractions (Pn-CN) was calculated on casein basis and defined
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as the ratio of the single casein fraction concentration (cn-CN, fraction) and the total casein content
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of fraction (ctotal CN, fraction) (Eq. 1).
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Pn-CN =
Cn-CN, fraction Ctotal CN, fraction
· 100 %
(1)
The yield (Yn-CN) of each casein fraction is defined as the ratio of the amount of the
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obtained casein fraction to the total amount of the protein in the micellar casein powder and
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was calculated as follows:
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Yn-CN = m
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where mtotal CN,fraction refers to the mass of the obtained n-casein fraction, mMCN to the initial
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mass of micellar casein (MCN) powder used for the isolation, while ctotal protein, MCN refers to
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the calculated protein content of the micellar casein using the measured total nitrogen content.
100 %
(2)
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Results and discussion
3.1.
Micellar casein
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Micellar casein was selected as raw material, since it was shown to be better than
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caseinates regarding the obtained yield (Post & Hinrichs, 2010). The composition of this
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powder is summarised in Table 1 (Batch I). An addition, the composition of another batch
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(Batch II) is given. During its production (Fig. 1A), the ultrafiltration permeate obtained from
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the skim milk was used instead of a re-suspended ultrafiltration permeate powder for
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diafiltration process, in contrast to the process according to Kersten (2001). The casein
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content of the micellar casein powder was 72.2 ± 1.38% (w/w). These results confirm and
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extend those reported by Post et al. (2009). The lactose content (15.4 ± 0.02%, w/w) could be
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reduced, for example by changing diafiltration media, e.g., using water instead of
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ultrafiltration permeate. The casein content obtained for batch II was in the same range as the
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other 13 independent micellar casein batches (casein level range: 58.2–73.1%). No difference
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resulting from the type of diafiltration medium was observed. The microfiltration permeate
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can be reused to reduce waste water discharge and energy costs for spray drying as well as
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time.
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3.2.
Casein fractions
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The purity and yield of the β-casein fractions obtained from five independent
productions (batch A, B, C, D, and E, process volume 80 L; Fig. 2) are shown in Table 2. For
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the production of each batch, the changes in process parameters (such as separation speed,
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time of cold extraction) made during the production of each batch are also given. In the
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present study, the same parameters as used in the work of Post and Hinrichs (2011) were
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taken as a reference and the same parameters were used for the production of batch A.
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Maximum achievable theoretical yields based on the total protein content in bovine milk are 47% for αS-casein, 35% for β-casein, and 12% for κ-casein. The purities and yields
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achieved in this study were calculated using the RP-HPLC profiles; Fig. 3 shows typical RP-
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HPLC profiles obtained for micellar casein, and β-casein, αS-casein, and κ-casein from the
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casein fractionation.
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In this study, a maximum purity of 89.6% and a yield of 10.2% were achieved for β-
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casein (Table 2), marginally higher than those (purity: 85%, yield: 8%) reported by Post and
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Hinrichs (2011). However, compared with the reported purities (up to 96%) in studies using
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membrane-based approach for the fractionation (Famelart & Surel, 1994), the achieved purity
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in this study was somewhat lower. The values were found in the range from 68.7 to 89.6%
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and decreased from batch A to E, except that for batch D, which could be because of the
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higher separation rates. The yields obtained ranged between 10 and 32%. The observed
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increase in the yield may be related to the extension of the cold extraction time, which was
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concentration of β-casein in the serum phase. To show the relationship between the yields and
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purities of the presented processes, the yields were plotted against the purities (Fig. 4).
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According to Pearson’s correlation, a moderate negative linear relation between the yield and
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purity was observed (p < 0.05).
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The by-products of the fractionation process (αS- and κ-casein) obtained during the
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isolation of β-casein were also analysed to evaluate the isolation process. The purity and yield
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values obtained were 61% and 26% for αS-casein, respectively, and 43% and 4% for κ-casein,
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respectively. The content of the κ-casein in our powder was approximately 12% of the total
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casein protein; therefore, the maximum yield of κ-casein would be at this level. With a yield
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of 4%, approximately 33% of the maximum possible κ-casein was gained. The remainder of
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κ-casein was either lost during separation or found in the other fractions as impurities.
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Conclusion
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Although several processes for the isolation of β-casein have been proposed in literature, there is still a need for improvement in scalability and in the yield and purity that
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can be attained. Micellar casein was used as raw material gained by membrane filtration
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process, and obtained highly purified β-casein fractions at a pilot scale using nozzle
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separators. The process enables the production of highly purified β-casein fraction with high
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yields, and in addition it gives highly enriched soluble αS- and κ-casein fractions; however,
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the yields of these fractions are still low compared with that of β-casein. This short
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communication provides a detailed description of the isolation process for the fractions as
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well as for the production of micellar casein. Further studies will focus on application of a
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decanter centrifuge for the separation process and the parameters influencing the process,
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especially CaCl2 concentration applied for the precipitation and the pH value used during cold
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extraction.
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Acknowledgements
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This research project was partly supported by the German Ministry of Economics and
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Energy (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn). Project
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AiF 17126 N. The authors would like to thank to Peter Rädler and the staff of the LAZBW,
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Wangen i. A., as well as to Birgit Greif, Luc Mertz, and Giovanni Migliore at the University
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of Hohenheim for their analytical and technical assistance.
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References
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Atamer, Z., Post, A. E., Schubert, T., Holder, A., Boom, R. M., & Hinrichs, J. (2017). Bovine
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β-casein: Isolation, properties and functionality. A review. International Dairy
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Journal, 66, 115–125.
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Callison, O. (1988). Dispersion and neutralization of acid casein. European Patent 0289867.
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Cayot, P., Courthaudon, J. L., & Lorient, D. (1992). Purification of αS-, β-and κ-caseins by
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batchwise ion-exchange separation. Journal of Dairy Research, 59, 551–556.
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Christensen, J., & Holst, H. H. (2016). Method of producing beta-casein compositions. EU patent EP2947996 B1.
Dickinson, E. (2003). Interfacial, emulsifying and foaming properties of milk proteins. In P.
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F. Fox & P. L. H. McSweeney (Eds.), Advanced dairy chemistry. Vol 1. Proteins (3rd
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edn., Vol. 1, pp. 1229–1260). New York, NY, USA: Kluwer Academic/ Plenum
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Publishers. 10
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Famelart, M. H., & Surel, O. (1994). Caseinate at low temperatures: Calcium use in β-casein extraction by microfiltration. Journal of Food Science, 59, 548–553. Holder, A. (2014). Cross-flow electro membrane filtration for the fractionation of dairy-based
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functional peptides. Dissertation, Stuttgart, Germany: University of Hohenheim,
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Verlag Dr. Hut.
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Huppertz, T. (2013). Chemistry of caseins. In P. L. H. McSweeney & P. F. Fox (Eds.),
Advanced dairy chemistry. Vol. 1A. Proteins: Basic aspects (pp. 135–160). New York,
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NY, USA: Springer.
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Kersten, M. (2001). Proteinfraktionierung mittels Membrantrennverfahren [Protein fractionation by means of membrane processing]. PhD Thesis, Munich, Germany
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Technical: University of Munich.
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Korhonen, H. J. (2009). Bioactive milk proteins and peptides: From science to functional
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Casein-PE6400 mixtures: A fluorescence study. Faraday Discussions, 166, 399–416.
applications. Australian Journal of Dairy Technology, 64, 16–25. Law, A. J. R., & Leaver, J. (2007). Methods of extracting casein fractions from milk and caseinates and production of novel products. WO Patent 2003003847.
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Kessler, A., Menéndez-Aguirre, O., Hinrichs, J., Stubenrauch, C., & Weiss, J. (2013). αs-
O'Mahony, J. A., Smith, K. E., & Lucey, J. A. (2007). Purification of β-casein from milk. US Patent 20070104847 A1.
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Post, A. E. (2011). Fractionation of bovine casein and enrichment of functional casein peptides. Dissertation, Stuttgart, Germany: University of Hohenheim, Verlag Dr. Hut.
Post, A. E., & Hinrichs, J. (2010). Suitability of commercial caseinates in comparison to
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micellar casein as raw material for isolation of food-grade β-casein.
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Milchwissenschaft, 65, 195–198.
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Post, A. E., & Hinrichs, J. (2011). Large-scale isolation of food-grade β-casein. Milchwissenschaft, 66, 361–364. Post, A. E., Arnold, B., Weiss, J., & Hinrichs, J. (2012). Effect of temperature and pH on the solubility of caseins: Environmental influences on the dissociation of αS- and β-casein.
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Journal of Dairy Science, 95, 1603–1616.
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Post, A. E., Ebert, M., & Hinrichs, J. (2009). β-Casein as a bioactive precursor - processing for purification. Australian Journal of Dairy Technology, 64, 84–88.
Shapira, A., Assaraf, Y. G., Epstein, D., & Livney, Y. D. (2010). β-Casein nanoparticles as an
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oral delivery system for chemotherapeutic drugs: Impact of drug structure and
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properties on co-assembly. Pharmaceutical Research, 27, 2175–2186.
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Silva, S. V., & Malcata, F. X. (2005). Caseins as source of bioactive peptides. International Dairy Journal, 15, 1–15.
Tolkach, A., & Kulozik, U. (2005). Fractionation of whey proteins and caseinomacropeptide
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by means of enzymatic crosslinking and membrane separation techniques. Journal of
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Food Engineering, 67, 13–20.
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ACCEPTED MANUSCRIPT Figure legends
Fig. 1. Production of micellar casein powder at pilot scale (modified from Kersten, 2001). Section A, production of skim milk concentrate up to a volume concentration factor (VCF) of
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MWCO, molecular mass cut-off; UF, ultrafiltration; ϑ, temperature; Vሶ, feed flow rate; t, time;
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∆PTM, trans membrane pressure.
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Fig. 2. Flow chart illustrating isolation of αS-, β-, and κ-casein fractions from micellar casein at pilot scale (modified from Post and Hinrichs, 2011). Abbreviations are: ϑ, temperature; Vሶ, feed flow rate; t, time; p, pressure.
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Fig. 3. Sample chromatographic profiles of micellar casein (MCN), β-casein, αS-casein, and κ-casein obtained from casein fractionation by means of selective precipitation on pilot scale.
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Fig. 4. Comparison of the β-casein yields obtained with the purities for different batches ( ,
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ACCEPTED MANUSCRIPT Table 1 Chemical composition of the micellar casein powders. a
Batch II
96.7 ± 0.08 2.26 ± 0.03 15.4 ± 0.02 1.5 ± 0.1 74.1 ± 0.08 72.2 ± 1.38 97.4 0.2 ± 0.1
95.5 ± 0.0 2.45 ± 0.06 13.0 ± 0.1 1.53 ± 0.02 69.7 ± 0.47 67.8 ± 1.3 97.3 0.2 ± 0.1
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Batch I
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Composition (%, w/w)
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Component
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Batch C
Batch D
Batch E
Post and Hinrichs (2011)
Output parameters β-Casein purity (%) β-Casein yield (%)
89.6 10.2
82.7 16.9
68.7 22.1
74.5 17.5
77.6 32.1
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Process parameters Extraction frequency Extraction time (h) Separation frequency Separation 1 (L h-1) Separation 2 (L h-1)
1 2 3 600 1200
2 2.5 4 550 1300
2 3 4 510 1200
2 2.5 4 600 1200
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Parameters
2 3.5 4 530 1300
1 2 3 600 1200
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For the present study, the process volume was 80 L except in the case of batch D for which the process volume was 85 L. Further deviations in the process parameters of Post and Hinrichs (2011) are: ϑ < 2 °C (for Separation 1), CaCl2 addition at pH 6–6.5 (isoelectric point of κ-casein).
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