improved isolation of bioactive components of bovine colostrum using cross-flow microfiltration

ORIGINALRESEARCH Improved isolation of bioactive components of bovine

colostrum using cross-flow microfiltration

THOMAS GOSCH,* SILVIA APPRICH, WOLFGANG KNEIFEL andSENAD NOVALINDepartment of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse18, A-1190, Vienna, Austria

Bovine colostrum contains bioactive components such as growth factors, immunoglobulins and anti-microbial factors. As conventional heat treatment methods inactivate these valuable components,cross-flow microfiltration (MF) seems to be a promising option for the processing of bovine colos-trum. A series of cross-flow MF experiments with tubular ceramic membranes of various pore sizesand geometries were conducted. MF with pore sizes of 0.8 and 1.4 lm resulted in a 5.4- and 3.5-log reduction of the microbial content, respectively. Applying 0.14- and 0.2-lm membranes lead toa permeate that was almost free from micro-organisms and casein. However, the maximum trans-mission of whey protein into the permeate was only 33%.

Keywords Colostrum, Microfiltration, Ceramic membrane, Bacteria removal, Casein separation.

INTRODUCTION

Colostrum is the highly concentrated, nutrient-rich secretion produced in the mammary glandsof all mammals before and immediately afterparturition. Besides containing all nutrients thatoccur in regular milk, colostrum additionallycontains a vast range of bioactive componentssuch as growth factors (Gauthier et al. 2006),immunoglobulins and antimicrobial factors, forexample, lactoferrin (Pakkanen and Aalto 1997).Bovine colostrum is exceptionally rich in immu-noglobulin G (IgG), which can be explained bythe fact that in cattle and other ruminant species,IgG is not transferred to the foetus via the pla-centa. Instead, the suckling newborn gains pas-sive immunity by the uptake of colostral IgGthrough the intestine (Pakkanen and Aalto1997).Because dairy cows usually produce greater

quantities of colostrum than the calf needs, sur-plus bovine colostrum is an interesting raw mate-rial for the production of animal feed as well asfor nutraceuticals and dietary supplements forhuman use. Uruakpa et al. (2002) give a detailedreview of potential health benefits of bovinecolostrum. As this review states, colostrum is theonly known natural source of two importantgrowth factor classes: transforming growth factor

b (TGF-b) and insulin-like growth factors 1 and 2(IGF-1, IGF-2). Colostrum is considered as a pos-sible remedy for many types of gastrointestinaldisorders and infections (Playford et al. 2000).Itmay help athletes during intense exercise (March-bank et al. 2011). One study even suggests a sig-nificant preventive effect against seasonalinfluenza (Cesarone et al. 2007).The bacteriological quality of bovine raw

colostrum is usually very poor, with plate countsoften exceeding 106 colony forming units(CFU)/mL. The probable presence of pathogensis a serious risk when human consumption ofbovine colostrum is considered (Houser et al.2008). Therefore, the collection and especiallythe processing of bovine colostrum for humanuse must provide maximum safety. Heat treat-ment procedures, used for pathogen eliminationin conventional milk processing, degrade valu-able proteins such as IgG (Dominguez et al.1997; McMartin et al. 2006; Elizondo-Salazaret al. 2010) and should not be applied to colos-trum if a high-quality product is desired.Microfiltration (MF), as it is well known from

conventional dairy applications, allows a consid-erable reduction in the microbial content atmoderate temperatures, resulting in even longershelf life than pasteurisation can provide (Branset al. 2004). Another advantageous aspect of

*Author forcorrespondence. E-mail:[email protected]

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doi: 10.1111/1471-0307.12027

microfiltration is the fact that intact somatic cells and bacte-ria are removed while heat treatment leaves behind cell deb-ris, such as thermoduric proteases, which affects the qualityof the final product (Saboyainsta and Maubois 2000).Typi-cally, ceramic membranes with a pore size of 1.4 lm areapplied in cross-flow microfiltration for bacteria and sporereduction in dairy processing. Membranes with pore sizes of0.1–0.2 lm are used to concentrate micellar casein fromskim milk. The composition of the resulting permeate, or‘milk serum’, is similar to sweet whey except that it is clearand virtually free from micro-organisms (Saboyainsta andMaubois 2000).Separating casein from colostrum seems to be reasonable

because the most valuable compounds can be found in theserum fraction. In contrast, there is only little evidence forexceptional properties of colostral casein (Brody 2000).Theparticle-free serum can easily be subject to sterile filtrationthrough dead-end filters to obtain a safe and stable product.While casein separation by acid or rennet precipitationmight lead to protein degradation, this does not occur whenMF is employed. However, it is well known that the forma-tion of a casein surface layer in cross-flow MF of milk leadsto partial retention of valuable serum proteins (Le Berre andDaufin 1998).There are several studies that describe the application of

MF to colostrum whey (Ulber et al. 2001; Elfstrand et al.2002; Wu and Xu 2009), but only one study describes thatmicrofiltration is directly applied to skimmed colostrum(Piot et al. 2004). This study proposes a process includingcasein separation by 0.1-lm MF and extensive diafiltrationfor the preparation of serocolostrum and reports an overallIgG recovery of 64%. Except for one patent (Mortensen2003) that claims to provide 99.9 % microbial reduction by1.4-lm MF of defatted bovine colostrum, no conclusiveinformation on the feasibility of a MF process, which sepa-rates microbes but leaves casein in the product, could befound.Lactoferrin (LF), due to its alkaline pH, is positively

charged near neutral pH and is thought to interact with neg-atively charged casein micelles (Croguennec et al. 2012),which hinders its permeation in membrane filtration. Severalpapers report poor transmission of LF during MF ofskimmed milk (Le Berre and Daufin 1998; Jost et al. 1999)and sweet whey (Ulber et al. 2001). As protein size andcharge are important factors influencing permeation in mem-brane filtration, high concentrations of IgG and LF in thepermeate would suggest that most other serum proteins alsopass the membrane in satisfying amounts.The objective of this work was to determine whether mem-

brane microfiltration can be applied to bovine colostrum forreduction in microbial content as it is applied in conventionalmilk processing. Standard industrial-scale ceramic membraneelements with various pore sizes and channel diameters wereutilised in cross-flow microfiltration experiments to examine

flux performance as well as the retention of micro-organismsand proteins. Additionally, comparative experiments withskimmed raw milk were performed. Bovine IgG and lactofer-rin (LF) were chosen as indicator substances. IgG, with amass of about 150 kDa, is one of the largest solute proteins inmilk and colostrum.

MATERIALS AND METHODS

Raw materialFrozen raw colostrum was provided by OCS ColostrumVitaplus GmbH, W€orgl, Austria. For each run, 14 kg of fro-zen raw colostrum was thawed in a jacketed tank at 35–40 °C. Fat was separated on an Elecrem 1 cream separator(Elecrem SA, Fresnes, France). The cream fraction, about0.9–1.2 kg, was discarded. 12 kg of the skimmed colostrumwas used in the filtration experiments, and the rest was dis-carded. For the comparative experiments, 80 kg of raw milkwas defatted as described above, and the skimmed milk wasstored at 4 °C and used within three days. In one experi-ment, 6 kg of the skimmed colostrum was diluted by adding6 kg of a saline solution (KCl 0.03 M and NaCl 0.02 M inRO water) as described by Piot et al. (2004).

Microfiltration apparatus and methodFigure 1 shows the experimental MF apparatus. 12 kg ofthe feed material was filled into the jacketed feed tank andheated to 30 °C. This temperature was chosen as a compro-mise between flux performance and protein preservationbecause preliminary tests showed that operating tempera-tures below 30 °C result in insufficient permeate flux. Thecentrifugal pump was started and adjusted to a retentateflow rate of 3.2 m³/h, which corresponds to a cross-flowvelocity of 4.0 m/s in the employed filter elements. Duringthe filtration runs, the flow rate dropped slightly as the vis-cosity of the retentate increased. This was compensated byincreasing the pump speed to maintain a cross-flow velocityof 4.0 m/s. Temperature and retentate flow rate were keptconstant over the filtration run. Transmembrane pressure(TMP) was calculated as TMP = (Pin + Pout)/2. Pin andPout are the pressures at the inlet and the outlet of the mem-brane module, respectively. The permeate flux J (L/m2/h)was determined gravimetrically in time intervals of 5–15 min and calculated as J=Dm/(A9Dt9q), where Dm isthe increase in permeate mass (kg) in the time interval Dt(h), A is the membrane surface area (m²) of the employedmembrane element and q is the density of the permeate at30 °C. In the runs with the 0.8- and 1.4-lm membranes, fil-tration was stopped when 7.0 kg of permeate was obtained;in case of 0.14 and 0.2 lm, filtration ended at 6.0 kg per-meate mass. Samples were taken from feed, permeate andretentate and stored at �30 °C prior to analysis.Tubular ceramic ISOFLUX® membrane elements were

provided by TAMI Germany. The separating layer of these

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membrane elements features a thickness gradient that allowsa uniform permeate flux. Skrzypek and Burger give detailedinformation about the ISOFLUX® concept and applicationsin the dairy industry (Skrzypek and Burger 2010). Theapplied membrane elements and their properties are listed inTable 1.

Analytical determinationsThe total viable count (TVC) was determined according to‘Amtliche Sammlung von Untersuchungsverfahren L01.00-5§ 35 LMBG’. Plate count agar (PCA) with 0.1 % skimmedmilk powder was used as substrate. The dilution mediumwas K2HPO4 (20 g/mL, pH 8.4) according to ISO8261:2001. Poured plates were incubated at 30 °C for 72 h.Dry matter content was determined according to IMV Stan-dard No. 15 (DIN 1038). Raw protein content was deter-mined according to DIN 10334, nonprotein nitrogenaccording to DIN 8968-4:2001/IDF 20 and whey protein/

casein according to IMV/FIL/IDF Standard 29. IgG and LFcontents were determined using sandwich ELISA test kitsprovided by Bethyl Laboratories, USA (E10-118, E10-126).The transmission (Tr) of components was calculated as

Tr = CP / CF 9 100, where CP and CF are the concentra-tions of the particular component in the permeate and thefeed, respectively.

RESULTS AND DISCUSSION

Results for 0.8- and 1.4-lm microfiltrationAs shown in Figure 2, the behaviour of the permeate fluxover time varied strongly, depending on pore size andfeed material. Compared with the 1.4-lm membrane, theaverage flux of the 0.8-lm membrane was about 80 %lower with skimmed colostrum. The same result wasobtained for skimmed milk. With 0.8-lm MF of skimmedcolostrum, the average permeate flux was only about10 L/m2/h.The results regarding the retention of bacteria by MF with

1.4 lm and 0.8 lm are shown in Table 2. Microfiltration ofskimmed colostrum with the 1.4-lm membrane led to a 3.5-log reduction in TVC, which means that at an initial TVCof 1.7 9 107 CFU/mL, 4.6 9 103 CFU/mL remains in thepermeate. As expected, bacteria removal using the 0.8-lmmembrane was more efficient, resulting in a TVC smallerthan 100 CFU/mL (5.4-log reduction). However, it shouldbe emphasised that as this permeate might still containpathogens, it cannot be considered safe enough for humanconsumption without further treatment. As additional treat-ment, application of mild heat or high pressure can be con-sidered, even though these methods are known to inactivatevaluable proteins to a certain extent (Trujillo et al. 2007).

Figure 2 Permeate flux vs time in experiments with 0.8 lm and 1.4 lmpore size, 6.0 mm channel diameter, Transmembrane pressure (TMP)ranged from 0.6–0.7 bar at a cross flow velocity of 4.0 m/s, temperaturewas maintained at 30 � 1 °C. Final permeate mass was 7.0 kg.

Figure 1 Microfiltration (MF) apparatus.

Table 1 List of applied ISOFLUX® membrane filter elements, theelements are 1178 mm long, and the outside diameter is 25 mm

TypeNo. ofchannels

Channelhydraulicdiameter (mm)

Membranesurfacearea(m²)

Applied poresizes (lm)

Daisy 8 6 0.2 1.4, 0.8, 0.2, 0.14Sunflower 23 3.5 0.35 0.14

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The retentates from 0.8-lm and 1.4-lm MF showed highmicrobial counts (6.8 9 107 and 3 9 108 CFU/mL, respec-tively).Transmission of casein (85%) and whey protein (87%)

was satisfactory when the 1.4-lm membrane was applied,while only 53 % of casein passed through the 0.8-lm mem-brane. The considerable retention of casein, resulting inblockage of the membrane pores, is most probably the causeof the low permeate flux and the lower whey protein trans-mission. It has to be noted that the transmission of lactofer-rin was remarkably low in case of skimmed colostrum. Thetransmission of all compounds was generally better in theexperiments with skimmed milk. Because 1.4-lm MF ofskimmed milk ran only for 10 min, which is probably tooshort to achieve steady state, the results of this run shouldbe treated with caution.

Results for 0.14- and 0.2-lm microfiltrationIn contrast to the MF runs with larger pore sizes, Figure 3shows that the permeate fluxes fall considerably with time

during 0.14- and 0.2-lm MF. This is mainly caused by theincreasing concentration of the retentate. As shown inTable 3, in case of skimmed colostrum, the total solids con-tent increases from approximately 16 % at the beginning to24 % at the end of the run. In a lesser degree, this increasealso occurs during skimmed milk MF. As expected, the per-meate flux was generally lower in case of skimmed colos-trum than with skimmed milk. Astonishing, however, is thedifference in flux behaviour between skimmed milk andskimmed colostrum when the membrane element with the3.5-mm channel diameter was applied. In case of skimmedmilk, the flux starts at a very high level and drops quicklyin a steep curve but remains well above the flux with the6.0-mm channels. With skimmed colostrum, on the otherhand, the flux decreases in a smooth curve. Moreover, apartfrom the initial phase, the flux behaviour is almost identicalwith the 6.0-mm channel diameter membrane elementdespite the higher TMP. The higher TMP at the same flowvelocity is caused by the higher flow resistance of the ele-ment with the 3.5-mm channels. Due to a sudden increase

Table 2 Retention of bacteria and transmission of proteins in the microfiltration experiments with 1.4 μm and 0.8 μm pore size

TVC (CFU/mL)Totalsolids (%)

Totalprotein (%)

Casein(%)

Wheyprotein (%)

Lactoferrin(lg/mL)

IgG(mg/mL)

1,4-lmMicrofiltration

Skimmed colostrum 1.7 9 107 16.5 11.5 5.1 6.4 220 34.1Retentate 6.9 9 107 15.5 10.9 5.2 5.7 n.d. n.d.Permeate 4.6 9 103 14.1 9.9 4.3 5.6 120 29.8Transmission ofcomponent (%)

85 86 85 87 55 87

Decimal reduction ofmicrobial content

3.5

Skimmed raw milk 1.9 9 104 9.2 3.6 2.8 0.7 160 0.3Retentate 4.9 9 104 8.5 3.4 2.6 0.7 n.d. n.d.Permeate <100 8.4 3.3 2.6 0.7 170 0.2Transmission ofcomponent (%)

91 93 92 >99 >99 68


>2.3

0,8-lmMicrofiltration

Skimmed colostrum 2.6 9 107 17.3 12 5.4 6.6 190 37.0Retentate 3.0 9 108 20.4 14.7 7.4 7.3 n.d. n.d.Permeate <100 12.6 8.3 2.9 5.4 110 28.6Transmission ofcomponent (%)

73 69 53 81 58 77


>5.4

Skimmed raw milk 1.9 9 104 9.2 3.6 2.8 0.7 160 0.3Retentate 7.5 9 104 8.9 3.8 3.0 0.8 n.d. n.d.Permeate <100 7.6 2.8 2.1 0.6 150 0.2Transmission ofcomponent (%)

83 78 74 91 94 79


>2.3

TVC, the total viable count; n.d. = not determined, Bold values indicate transmissions of components in % or the decimal reduction of

microorganisms.


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in TMP (>1.5 bar), filtration of skimmed colostrum with the3.5-mm channels was discontinued at 5.8 kg permeate massand not at 6.0 kg as in the other runs. The slight rise in per-meate flux at the end of the three lower curves can beexplained by the fact that the TMP increased towards theend of the filtration runs. Viscosity increases with the con-centration of protein and causes a higher pressure as theflow rate of the retentate was maintained constant. In termsof flux performance, the 0.14-lm and the 0.2-lm membranecan be considered as practically equal. Dilution of skimmedcolostrum (1:2) resulted in an improvement in flux of morethan 100 % under the same processing conditions. Thisseems to be in accordance with the film model, which canusually be applied in protein concentration (Cheryan 1998).The film model describes the relation between permeate fluxand feed concentration as J = k 9 ln (CG/CF), where k (m/s) is the mass transfer coefficient, CG is the gelification con-centration and CF is the feed concentration. Assuming a CG

of 22 % (Chiang and Cheryan 1987) and that k remainsunchanged, reducing the protein concentration of the feed

Table 3 Transmission of proteins in the MF runs with 0.14 μm and 0.2 μm pore size

Totalsolids (%)

Totalprotein (%)

Casein(%)

Wheyprotein (%)

Lactoferrin(lg/mL)

IgG(mg/mL)

0,14-lm Microfiltration6-mm channel diameter

Skimmed colostrum 16.1 11.3 5 6.4 180 29.7Retentate 24.2 19.4 9.6 9.8 n.d. n.d.Permeate 5.7 1.7 <0.1 1.7 40 4.7Transmission of component (%) 35 15 <2 27 22 16Diluted skimmed colostrum (1:2) 8.2 5.5 2.4 3.1 110 19.4Retentate 12.7 9.9 5.1 4.8 n.d. n.d.Permeate 2.8 0.8 <0.1 0.8 10 3.4Transmission of component (%) 34 15 <2 26 9 18Skimmed raw milk 9.2 3.6 2.8 0.7 160 0.3Retentate 11.0 5.6 4.7 0.9 n.d. n.d.Permeate 5.3 0.5 <0.1 0.5 40 0.3Transmission of component (%) 58 14 <4 71 25 >99

0,14-lm Microfiltration3,5-mm channel diameter

Skimmed colostrum 16.9 11.3 5.1 6.3 150 47.7Retentate 24.0 18.7 9.7 9.0 n.d. n.d.Permeate 6.3 2.1 <0.1 2.1 40 11.9Transmission of component (%) 37 19 <2 33 27 25Skimmed raw milk 9.2 3.6 2.8 0.7 160 0.3Retentate 11.3 5.9 4.9 1.0 n.d. n.d.Permeate 5.2 0.5 <0.1 0.5 40 0.4Transmission of component (%) 57 14 <4 71 25 >99

0,2-lm Microfiltration6-mm channel diameter

Skimmed colostrum 16.4 11.7 5.2 6.4 170 30.6Retentate 23.4 19.2 9.4 9.8 n.d. n.d.Permeate 5.2 1.4 <0.1 1.4 30 3.2Transmission of component (%) 32 12 <2 22 18 11Skimmed raw milk 9.2 3.6 2.8 0.7 160 0.3Retentate 11.4 5.9 4.9 1.0 n.d. n.d.Permeate 5.3 0.5 <0.1 0.5 50 0.3Transmission of component (%) 58 14 <4 71 31 >99

MF, microfiltration; n.d. = not determined, Bold values indicate transmissions of components in % or the decimal reduction of microorganisms.

Figure 3 Permeate flux vs time in the experiments with 0.14 lm and0.2 lm pore size. If not indicated otherwise, the channel diameter was6.0 mm; cross-flow velocity was maintained at 4.0 m/s; Transmembranepressure (TMP) ranged from 0.8 bar to 0.9 bar with 0.6 mm channeldiameter and from 1.0 bar to 1.2 bar with 3.5 mm channel diameter, tem-perature was maintained at 30 � 1 °C. Final permeate mass was 6.0 kg.


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from 11.3 % to 5.5 % should lead to a 108 % increase influx.The permeate from all MF runs with 0.14-lm and 0.2-lm

pore size was clear, contained only traces of casein and wasalmost free from micro-organisms (< 10 CFU/mL). The re-tentates showed high levels of microbial content (up to109 CFU/mL).The transmission of whey proteins was notsatisfactory as can be seen in Table 3. Apparently, the smalldifference in pore size (0.14 lm vs 0.2 lm) has no signifi-cant impact. Choosing the smaller channel diameter(3.5 mm), which also results in a higher TMP, leads to cer-tain increase in transmission of whey protein, lactoferrinand IgG.While dilution (1:2) considerably improved the flux, no

such effect was observed regarding the transmission ofproteins. Extensive diafiltration with a subsequent concen-tration step, as demonstrated by Piotet al. (Piot et al.2004), could enhance serum protein yields considerablybut also results in a much higher technical expenditure.With the exception of lactoferrin, the transmission of pro-teins was generally better during the filtration of skimmedmilk. When comparing the results from the colostrum andmilk filtration, it has to be kept in mind that the rawcolostrum was frozen and thawed before the experiments,while the raw milk was fresh. Freezing and thawing canlead to protein denaturation, and it might affect flux andprotein transmission in filtration experiments (Rice et al.2009).The residual microbial content is a serious concern. To

address this problem, additional treatment, for example, bymild heat or high pressure, can be considered. By applica-tion of 0.14-lm and 0.2-lm microfiltration, casein is sepa-rated and the permeate is virtually free from particles andmicro-organisms. It could easily be sterilised using commer-cially available sterile filters. Nonetheless, the substantialloss of valuable compounds is not acceptable. Under thesecircumstances, diafiltration could improve the serum proteinyield.

CONCLUSION

Although there are still some drawbacks that have to bedealt with, it has to be emphasised that cross-flow micro-filtration is one of the very few methods that allows a con-siderable reduction in the microbial content of heat-sensitive materials, such as bovine colostrum. Regardingthe concentrations of valuable substances such as IgG andlactoferrin, microfiltration with 1.4 and 0.8 lm provided aproduct that is close to the original raw material. In con-clusion, it has to be said that experiences from conven-tional milk microfiltration cannot be applied to bovinecolostrum easily, and more research needs to be carriedout regarding the retention of pathogens and the recoveryof bioactives.

ACKNOWLEDGEMENTS

The authors want to thank the Austrian Research PromotionAgency (FFG) and Thomas Osl (OCS Colostrum Vita-plusGmbH, W€orgl) for the financial support of this work.

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improved isolation of bioactive components of bovine colostrum using cross-flow microfiltration

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