Saturday, November 15, 2014

Thermal Stability of Bovine Lactoferrin and its Bioactive Properties

This was my first literature review article, which I wrote for a class several years ago. 


Abstract

Lactoferrin, or lactotransferrin, is an iron-binding globular glycoprotein present in mammalian milk and other secretory fluids.  Lactoferrin is an important component of the immune system and serves a diverse range of physiological functions, including antimicrobial, antiviral, immunomodulatory, and antioxidant activities, particularly to infants feeding on milk[6].  In recent years it has become apparent that the oral administration of lactoferrin provides additional beneficial effects on the health of mammals, including antiseptic, anti-carcinogenic, and anti-inflammatory effects.  This has led to the growth of its potential for application as a food additive.  Bovine lactoferrin is produced on an industrial scale from cheese whey and skim milk.  The concern of heat stability is important when lactoferrin is intended to be used as a bioactive component in foods as it has been shown to display a range in resistance to heat, depending on its environment.  To ensure safety, pasteurization methods have been developed and their effects on the activity of lactoferrin have been studied.  This paper will review the thermal stability of lactoferrin as well as bioactive hydrolysates and nanoparticles that have potential as health promoting food additives.


Introduction

Bovine milk is a widely available source of lactoferrin (LF).  Bovine LF (bLF) occurs in the range of .1 of .3 mg/ml in normal milk and 2 to 5 mg/ml in colostrum[9].  LF is an 80-kDa glycoprotein composed of a single polypeptide chain with two lobes of equal size, each with a capacity to reversibly bind one Fe3+ ion[6].  The thermal behavior of bLF was studied in simulated milk ultrafiltrate at pH 6.7, by differential scanning calorimetry (DSC).  A single peak of denaturation was found for iron-free bLF, apolactoferrin (apo-bLF), and two peaks of denaturation for iron-saturated bLF (Fe-bLF)[9].  Protein denaturation was found to be irreversible, which was attributed to the formation of protein nanoparticles, with the size depending on temperature and duration of the thermal treatment[1]. 

It is suggested that bLF is thermostable under acidic conditions, particularly at pH 4.0[4].  LF treated at pH 2.0 and 120oC for 15 min had no iron-binding capacity and significantly decreased antigenicity but had an increase in antibacterial activity[6].  Reverse-phase high performance liquid chromatography (HPLC) fractionation of the treated bLF revealed the production of several peptides fractions having strong antibacterial activity.  It was also shown that the thermal stability of LF stabilized oil-in-water emulsions depend on pH, holding temperature, and thermal history[13].

Since thermal exposure may compromise protein functionality, the heat-induced denaturation of native bovine lactoferrin was monitored by a recently developed optical biosensor-based immunoassay.  Kinetic parameters were determined between 70oC and 90oC, which confirmed that thermal exposures typical of pasteurization were relatively benign with respect to retained conformational integrity, while higher temperature–time exposures resulted in significant loss of conformationally intact lactoferrin[3].

Resistance of bLF to thermal denaturation depends on its iron saturation, as the more compact Fe-bLF is more heat stabile than apo-bLF[12].  It has been reported that the HTST pasteurization (72°C, 15s) commonly used in the cheese-making industry induces partial denaturation of the native protein, affecting its lower unfolding transition by 40 to 50%, whereas UHT pasteurization led to a complete denaturation of the protein[9].  A similar finding was reported for human LF[15].  In a study of bLF recovery in rennet-coagulated skim milk, the extent of the recovery in the whey fraction was found to reduce as the heating temperature increased, particularly with apo-bLF[2].  In a study of pulsed electric field (PEF) treatments at various temperatures, PEF treatments did not change any properties of apo-bLF or Fe-bLF, while at higher temperatures, the bLF concentration decreased, particularly for apo-LF[11].  The thermal stability of LF is affected by environmental conditions such as pH, salts, and whey proteins[8].  LF bioactivity can also be affected by other milk proteins[10].  Therefore, the parameters of bLF denaturation must be examined under the conditions of the application of interest to ensure the preservation of bioactivity.


Potential for infant formula


It is understood that breast-fed infants have a much richer gut flora than do bottle-fed infants, particularly with Bifidobacteria and Lactobacilli.  Such flora can be attributed to an increased resistance to colonization of the digestive tract by pathogenic bacteria.  Bovine milk is particularly deficient in lactoferrin compared to human milk.  In a study in which LF was added to an infant formula fed to bottle-fed infants, levels of Bifidobacteria increased[5].  However, the levels of Bifidobacteria were not as high as breast-fed infants, and colonization took significantly longer to develop.  Developing an infant formula that resembles human milk more closely than existing formulas, while providing similar health benefits, can be by adapted from bovine milk by removing certain surplus ingredients, shown in Figure 1, and adding ingredients such as LF, vitamins and trace elements and by adapting the fat composition[7].  Such formulas have been developed and marketed in Asia but not in Europe or the Americas.  It is expected there would be a market in the U.S. if an effective formula were to be developed, which would require preserving the bioactivity of LF through minimized thermal degradation during processing.


Lactoferrin Hydrolysates

A hydrolysate of lactoferrin has also been used in infant formula.  LF treated at pH 2.0 and 120°C for 15 min and degree of hydrolysis of about 10% had no Fe-binding capacity and less antigenicity (about 10-6) than untreated LF[6].  Heat-treated LF increased in antibacterial activity, and the activity was maintained in an Fe-rich medium.  After fractionation of heat-treated LF by reverse-phase HPLC, several peptide fractions were found that had potent antibacterial activity, one being lactoferricin[14].  It is suggested that LF contains at least one bactericidal domain that is activated upon limited acid hydrolysis of the protein.  The bactericidal activity of the peptide fragments of LF was shown to have no relation to Fe-chelation, in contrast with the antibacterial behavior of native LF[6].


Effect of Low pH on Lactoferrin Thermostability

To develop a practical method for pasteurization of lactoferrin, the influence of pH on the thermal stability of bLF was studied[4].  Solutions (5%; wt/vol) of apo-bLF in distilled water were adjusted to various pH values from 2.0 to 11.0 and heated at various temperatures (80oC to 120oC) for 5 min.  The heated samples were analyzed by HPLC and assayed for iron-binding capacity, antigenic and antibacterial activity.  At acidic pH, LF in the heated samples remained soluble.  The HPLC pattern of apo-bLF treated at pH 4.0 and 100oC was similar to that of unheated apo-bLF.  It also did not show significant losses of iron-binding capacity, antigenic or antibacterial activity.  Apo-bLF treated at pH 2.0 or 3.0 and 100oC or 120°C for 5 min was apparently degraded, but the antibacterial activity was stronger than that of unheated apo-bLF, suggesting that LF fragments produced by heat under acidic conditions have antibacterial activity.  These results indicate that apo-bLF is stable at pH 4.0 and high temperatures, presenting a possible pasteurization method for bLF while preserving bioactivity.


Apo-bLF and Fe-bLF Derivatives of Lactoferrin

Heat-induced enthalpy changes in different forms of bovine lactoferrin in water were examined by differential scanning calorimetry[9].  Two thermal transitions were observed, depending on the iron-binding status of the protein.  Fe-bLF was more resistant to heat induced changes than apo-bLF.  Native LF had two transitional peaks, and pasteurization affected only the low temperature transition.  Fe-bLF revealed a single transitional peak that was resistant to pasteurization.  However, both forms were completely denatured by UHT.  The effect of pasteurization and UHT on the bacterial interaction capacity was examined in a 125I-labeled LF-binding inhibition assay.  The bacterial interaction capacity of native and Fe-bLF was unaffected by pasteurization, while UHT treatment decreased this capacity.  Both pasteurized and unheated native LF showed similar antibacterial properties.  Fe-bLF, however, did not inhibit bacterial growth, regardless of heat treatment.


The effect of heat treatments on the recovery of LF in whey coming from rennet-coagulated skim milk was studied[2].  The impact of iron saturation was also assessed using skim milk spiked with different LF iron forms.  The recovery of LF in the rennet whey fraction was determined by reverse-phase HPLC, shown in Figure 2.  One- and two-dimensional SDS PAGE analyses were performed on rennet curds to characterize LF interactions in heated milk.  The extent of LF recovered in the whey fraction was found to reduce as the heating temperature increased.  The binding of iron by LF improved its thermal stability and its recovery in the whey fraction.  The association of LF in unheated milk rennet curd involved noncovalent interactions, whereas upon heating, intermolecular disulfide linkages were also involved.  Depending on the severity of the heat treatment, LF aggregates with Cys-containing proteins through thiol/disulfide exchange reactions.  These noncovalent and covalent interactions explained the lower recovery of LF in heated milk.


Interactions of Lactoferrin with Other Milk Proteins 

The interaction of lactoferrin from human and bovine milk with the human promonocytic cell line U937 was studied[10].  Both human and bovine Fe-LF bound to the cells.  Binding of bLF was inhibited by excess bovine LF but not by human LF, suggesting that the binding mechanisms for the two proteins are different.  Binding of bovine LF was not inhibited by bovine lactoperoxidase nor by a 20-fold excess of human IgA.  Human and bovine α-lactalbumins, bovine β-lactoglobulin, and human lysozyme did not affect binding of bLF.  Samples of Fe-bLF and apo-bLF in capillary tubes were exposed to temperatures of 72°C for 20s, 85°C for 20 min or 137°C for 8s.  All heated samples inhibited binding of native Fe-bLF and apo-bLF, though to a lesser extent than the native proteins.  Both heated and native lactoferrins enhanced thymidine incorporation by U937 cells, although Fe-bLF heated at 85°C for 20 min was inhibitory.  These results suggest that heat treatment of bLF under conditions used during industrial processing does not greatly affect its ability to interact with monocytic cells and that other milk proteins generally do not affect this interaction.


Pasteurization and UHT Treatment of Lactoferrin Solutions

It has already been discussed that the degree of heat treatment affects the denaturation and bioactivity of lactoferrin.  While the bacterial interaction capacity of native and Fe-bLF was unaffected by pasteurization, UHT treatment decreased this capacity[9].  The UHT treatment denatured the protein structure and also diminished the antibacterial properties of bLF.  Pasteurization seems to be the preferred method because it did not alter either the bacterial interaction capacity or the antibacterial activity of bLF.  However, because LF has been associated with several biological functions, the effects of heat treatment on these properties should not be overlooked.

In a study of human milk, heating at 62.5oC for 5 min completely destroyed Escherichia coli and Staphylococcus aureus in inoculated samples, and heating at 56.0oC for 15 min destroyed over 99% of bacteria[15].  The mean percentage remaining activity of LF after heat treatment at 62.5oC for 30 min, 62.5oC for 5 min and 56oC for 15 min were 27%, 59% and 91% respectively.  This suggests that denaturation is temperature-time dependant and could be modeled to predict and minimize degradation while ensuring adequate microbial inactivation, aiding in the development of pasteurization methods that ensure safety without compromising desired bioactivity.


Inactivation Kinetics

It has been shown that bovine lactoferrin undergoes a two-step protein unfolding process, and the temperatures of the denaturation of the two lobes of bLF were found to be around 60 and 90oC[1].  The size of the particles formed during the thermal treatments depend on the temperature-time exposure of the heating process.  Higher temperatures resulted in a faster aggregation and the formation of larger protein particles.  The protein nanoparticles produced using a controlled thermal processing approach has been suggested as potentially useful as function ingredients in commercial products.  It is expected that the protein nanoparticles would be less reactive than native LF, but would preserve select bioactive activities and offer potential commercial application.  

A study using optical biosensors to analyze the heat denaturation (70oC – 90oC) of LF was compared to those results of HPLC[3].  Biosensor-based immunoassay was demonstrated to be a valuable technique to complement existing methods and has confirmed that, at normal physiological pH, conventional pasteurization conditions result with minimal LF denaturation, while loss of bioactivity is extensive at higher temperatures and longer exposure times.

In a kinetic approach the effect of heat treatment on LF unfolding was analyzed by DSC and aggregation, derived from radial immuno-diffusion analysis[8].  The results reveal that a kinetic approach to the heat denaturation of LF provides information that can be used to predict and control the effects of heating processes.  Because the thermal stability of LF is affected by environmental conditions such as pH, salts and protein, the kinetic parameters of the heat denaturation of LF have to be determined under the conditions of the application of interest.

Kinetic parameters for heat denaturation of LF under different conditions were determined over a temperature range 72oC – 85oC[12].  Denaturation could be described by first-order reaction kinetics.  Apo-bLF is denatured more rapidly than Fe-bLF, and both forms are more heat-sensitive when treated in milk than in phosphate buffer.  Values of change in enthalpy of activation of LF denaturation are high, which indicates that a large number of bonds are broken but not affected by the association of LF with β-lactoglobulin.  This information can be used to minimize the denaturation of LF while preserving bioactivity.


Pulsed Electric Field Treatment of Lactoferrin

Native bovine lactoferrin, apo-bLF and Fe-bLF dissolved in simulated milk ultrafiltrate were treated by pulsed electric fields[11].  Various heat treatments were conducted as comparisons.  The concentration of LF, electrophoretic mobility of the proteins, surface hydrophobicity and the release of LF-bound ferric ions to the aqueous phase of the LF preparations after the PEF or thermal treatments were determined.  PEF treatments did not affect the physicochemical properties of all forms at treatment temperatures of 50oC - 65oC.  Changes in properties during PEF treatments at higher temperatures were largely due to thermal effects.  The thermal stability of LF correlated with the level of iron saturation.  These results can be useful to develop a PEF process for microbial inactivation of LF-containing dairy products while maximizing the preservation of the heat-sensitive protein. 


Lactoferrin Stabilized Oil-in-Water Emulsions

The influence of pH (2.0 – 9.0) and thermal processing (30°C – 90°C, 20 min) on the stability of lactoferrin stabilized oil-in-water emulsions was studied[13].  At ambient temperature, the emulsions were stable to droplet aggregation at low pH (pH ≤ 6.0) but exhibited some aggregation at pH 7.0 – 9.0.  The thermal stability of the emulsions depended on pH, holding temperature, and thermal history.  LF-coated droplets were heated in distilled water, and then the pH was adjusted in the range 2.0 – 9.0.  They were highly unstable to aggregation at pH 7.0 – 8.0.  However, when the pH was altered first and then they were heated, the LF-coated droplets were highly unstable to aggregation at pH ≥ 5.0 when heated above 50°C.  These results reveal important implications for the formulation and production of emulsion-based products using lactoferrin as an emulsifier.


Conclusion

Bovine lactoferrin is used to supplement foods such as infant formula, nutritional supplements, yogurt, skim milk, drinks, and pet foods.  The expected effects of these products include anti-infection, improvement of gastrointestinal flora, immunomodulation, anti-inflammation, anticarcinogenicity, and antioxidation.  Because LF is denatured by heat treatment under certain conditions, high temperature treated milk is not suitable as a source for bioactive bLF purification.  Therefore, skim milk and whey that have not undergone rigorous heating can be sources of bLF.  There exist low temperature purification techniques that can preserve the health promoting bioactivity of bLF, with particular emphasis on infant formulas.  When selecting a source of the LF, an emphasis should be placed on selecting one which has not been treated with the high temperatures typical of conventional pasteurization techniques.  Raw milk would have no LF denaturation, and would be an ideal source; however, pasteurization is often implemented to ensure adequate microbial activation.  Choosing lower temperatures during pasteurization while applying known kinetic parameters, one can maximize and accurately predict the recovery of bioactive bLF for commercial application.


References
[1] Bengoechea C., Peinado I., McClements D., Formation of protein nanoparticles by controlled heat treatment of lactoferrin: Factors affecting particle characteristics, Food Hydrocolloids, In Press, Corrected Proof, January 2011.

[2] Brisson G., M. Britten, Y. Pouliot, Effect of Iron Saturation on the Recovery of Lactoferrin in Rennet Whey Coming from Heat-Treated Skim Milk, Journal of Dairy Science, Volume 90, Issue 6, June 2007, Pages 2655-2664.

[3] Harvey E. Indyk, Iain J. McGrail, Gaylene A. Watene, Enrico L. Filonzi, Optical biosensor analysis of the heat denaturation of bovine lactoferrin, Food Chemistry, Volume 101, Issue 2, 2007, Pages 838-844.

[4] Hiroaki Abe, Hitoshi Saito, Hiroshi Miyakawa, Yoshitaka Tamura, Seiichi Shimamura, Eiji Nagao, Mamoru Tomita, Heat Stability of Bovine Lactoferrin at Acidic pH, Journal of Dairy Science, Volume 74, Issue 1, January 1991, Pages 65-71.

[5] Hiroyuki Wakabayashi, Koji Yamauchi, Mitsunori Takase, Lactoferrin research, technology and applications, International Dairy Journal, Volume 16, Issue 11, Technological and Health Aspects of Bioactive Components of Milk, Technological and Health Aspects of Bioactive Components of Milk, November 2006, Pages 1241-1251.

[6] Hitoshi Saito, Hiroshi Miyakawa, Yoshitaka Tamura, Seiichi Shimamura, Mamoru Tomita, Potent Bactericidal Activity of Bovine Lactoferrin Hydrolysate Produced by Heat Treatment at Acidic pH, Journal of Dairy Science, Volume 74, Issue 11, November 1991, Pages 3724-3730.

[7] J.N. de Wit, Nutritional and Functional Characteristics of Whey Proteins in Food Products, Journal of Dairy Science, Volume 81, Issue 3, March 1998, Pages 597-608.

[8] Kussendrager, K. D. Effects of heat treatment on structure and iron-binding capacity of bovine lactoferrin. Pages 133–146 in IDF Bulletin: Indigenous Antimicrobial Agents of Milk: Recent Developments. International Dairy Federation, Brussels, Belgium. 1994.

[9] Marie A. Paulsson, Ulla Svensson, Alugupalli R. Kishore, A. Satyanarayan Naidu, Thermal Behavior of Bovine Lactoferrin in Water and Its Relation to Bacterial Interaction and Antibacterial Activity, Journal of Dairy Science, Volume 76, Issue 12, December 1993, Pages 3711-3720.

[10] Oria, Rosa, Maznah Ismail, Lourdes Sánchez, Miguel Calvo and Jeremy H. Brock (1993). Effect of heat treatment and other milk proteins on the interaction of lactoferrin with monocytes. Journal of Dairy Research, 60, pp 363-369.

[11] Qian Sui, Hubert Roginski, Roderick P.W. Williams, Cornelis Versteeg, Jason Wan, Effect of pulsed electric field and thermal treatment on the physicochemical properties of lactoferrin with different iron saturation levels, International Dairy Journal, Volume 20, Issue 10, October 2010, Pages 707-714.

[12] Sanchez, L., Peiro, J., Castillo, H., Perez, M., Ena, J. and Calvo, M. Kinetic Parameters for Denaturation of Bovine Milk Lactoferrin. Journal of Food Science, 57: 873–879. 1365-2621.1992.

[13] Tokle, T., McClements D. J., Physicochemical properties of lactoferrin stabilized oil-in-water emulsions: Effects of pH, salt and heating, Food Hydrocolloids, Volume 25, Issue 5, July 2011, Pages 976-982.

[14] Tomita M, Wakabayashi H, Yamauchi K, et al. Bovine lactoferrin and lactoferricin derived from milk: production and applications. Biochem Cell Biol 2002;80:109-112.

[15] Wills M. E., V. E. M. Han, D. A. Harris, J. D. Baum, Short-time low-temperature pasteurisation of human milk, Early Human Development, Volume 7, Issue 1, October 1982, Pages 71-80.


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