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USE OF RECONSTITUTED YOLK SYSTEMS TO STUDY THE GELATION MECHANISM OF FROZEN-THAWED HEN EGG YOLK Monica Primacella, Tong Wang, and Nuria C. Acevedo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04370 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 9, 2017

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Journal of Agricultural and Food Chemistry

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USE OF RECONSTITUED YOLK SYSTEMS TO STUDY THE GELATION MECHANISM OF FROZEN-THAWED HEN EGG YOLK

Monica Primacella, Tong Wang, Nuria C. Acevedo*

Department of Food Science and Human Nutrition, Iowa State University 2312 Food Sciences Building, 536 Farm House Lane, Ames, Iowa 5011, United States

*

Corresponding author

Tel.: 515-294-5962 (N. Acevedo) Fax: 515-294-8181 Email address: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: Yolk gelation upon 5-week freezing-thawing was studied in 4 recombined yolk

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systems containing different plasma and granule proportions. Fractionation for mass distribution,

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sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein distribution

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and rheological properties were explored. Results indicate that both plasma and granule

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components including LDL, HDL, and α-livetin proteins, contributed to gelation. Protein

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aggregation was reflected through large mass increase in granule fraction and appearance of a

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floating LDL layer upon fractionation of gelated yolk systems. A significant increase in gel

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strength (elastic modulus, G’) was observed with the increase of granule content. Overall, this

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study provides a better understanding of yolk gelation mechanism that may consequently lead to

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the design of innovative methods for preventing gelation. A schematic presentation of yolk

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gelation mechanism is also proposed.

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KEYWORDS: egg yolk, gelation, freezing, lipoproteins

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INTRODUCTION

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Hen egg yolk is one of the most used ingredients in many products due to its high

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nutritional value and unique functionalities. Being an excellent emulsifier, egg yolk is used

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extensively in foods, such as mayonnaise, salad dressing, and sauces. Recent statistical analysis

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showed that out of the 231 million cases of shell eggs (or 83 billion eggs) produced in U.S. in

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2015, approximately 30% underwent breaking for further processing 1. However, when egg

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yolks are frozen under -6°C and thawed, an irreversible loss of fluidity, termed gelation, occurs.

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This change is unfavorable because it reduces the yolk functionality and its ability to mix with

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other ingredients 2. Current gelation prevention practices include the additions of salt, sugar, or

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corn syrup to yolk prior to freezing 3. However, consumer’s preference for low salt and low

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sugar products may limit the range of frozen yolk application.

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Researchers have proposed many different explanations regarding the mechanism of

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gelation related to its composition. Yolk is composed of about 50% water and 50% dry matter, in

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which the dry matter could be broken down to 77-81% plasma and 19-23% granules. Plasma

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contains 85% low density lipoprotein (LDL) and 15% livetin, while granule contains 70% high

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density lipoprotein (HDL), 16% phosvitin, and 12% LDL 4. Scientists have stated no difference

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between plasma LDL and granule LDL. The most common explanation for yolk gelation is that

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there are aggregations of plasma LDLs that result from a concentration of yolk components due

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to the formation of large ice crystals during freezing 5-7. However, disagreement exists on the

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mechanism of LDL aggregation. Telis and Kiechbusch 6 proposed that dehydration of proteins

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located on the surface of LDL micelles following the breaking up of LDL micelles leads to LDL

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aggregation. Kurisaki et al. 8 suggested surface components of LDL are liberated during freeze-



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thaw, causing aggregation of the newly exposed sites. Wakamatu et al. 7 believed that LDL

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aggregates due to conformational changes, and not because of liberation of LDL components.

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Interaction of protein molecules after disruption of lecithin-protein interactions is another

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proposed LDL aggregation mechanism by Mahadevan et al. 9 and Kumar and Mahadevan 10. The

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real cause of LDL aggregation is still unclear.

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While majority of studies emphasized the role of plasma LDL in yolk gelation, only a

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few studies include the granule fraction in their work. Wakamatu et al. 7 indicated that they could

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not exclude the involvement of granular LDL (LDLg) in LDL aggregation because the lipid

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compositions of LDL and LDLg were very similar. Chang et al. 5 found that gelation was

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enhanced when granule is in the system, compared to plasma alone. They proposed that LDLg

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are released from granules disrupted during freezing, causing both LDL and LDLg to aggregate.

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Regardless of the differences in existing proposed mechanisms of gelation, most researchers

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agreed that removal of water through ice crystal formation is necessary for gelation to occur.

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Removal of water by freezing might have decreased physical distance and increased

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hydrophobic interaction which cause LDL destabilization. Studies showed that phospholipase-A

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treated LDL had inhibited gelation because they were more hydrophilic 11, 12. A phospholipase-C

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treated LDL was more lipophilic and it promoted aggregation 9. This provides some evidence

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that gelation may be due to surface hydrophobic interactions of LDL.

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The most recent study on the effect of prolonged freezing storage on egg yolk gelation

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suggested the occurrence of two-stage gelation, which involved aggregation of lipoprotein

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particles resulting from water removal during slow freezing in the first stage (d 1-28), and release

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and re-aggregation between or within the previously aggregated proteins in the second stage


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(between d 28-84) forming a stronger gel network 13. The author also suggested that

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granules/HDL particles were also involved in the aggregation, and various methods including

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evaluations of particle size, matrix mobility, protein aggregation and microstructure were able to

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show that these components played a role in gelation. However, the involvement of HDL

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proteins in gelation still needs further validation.

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The overall goal of this research is to elucidate how the different yolk fractions are

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involved in freeze-thawed yolk gelation. We hypothesize that during freezing storage, various

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types of lipoprotein particles in the plasma and granule fractions interact, resulting in gelation.

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Yolk recombined systems made with different proportions of plasma and granules were studied,

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and gel properties of these systems and compositions of their fractionated components were

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analyzed to test our hypothesis.

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MATERIALS AND METHODS Materials Fresh large Grade AA white shell eggs were obtained from farms in Ames, IA. Eggs

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were produced by Hy-Line W-36 laying hens raised in conventional cage housing, and hens’ age

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was 30-35 weeks. Eggs were stored in 4ºC refrigerator at the research laboratory for no longer

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than 7 days.

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Yolks were separated following the method by Powrie et al. 2 with modifications. Fresh

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eggs were manually broken and the yolks were carefully separated from the albumen, with the

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chalazae removed. Each yolk with intact vitelline membrane was rolled on a paper towel to

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remove any remaining albumen and chalazae adhering to the vitelline membrane. The vitelline

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membrane was pierced to collect the pure egg yolk in a beaker. Following the harvest of



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approximately 1 L of pure egg yolk solution, the yolks in the beaker were slowly stirred for

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sample homogeneity.

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Preparation of plasma and granules for recombined yolk systems

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Yolk was fractionated into plasma and granules using a modified method by McBee and

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Coterill 14. Yolk was diluted 1:1 (v/v) in deionized water and stirred until they were well-mixed.

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This dispersion was then centrifuged at 10,000 g for 45 minutes at 4ºC, and the plasma

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(supernatant) was separated from the granules (pellet). The plasma was centrifuged again using

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the same parameter for more complete separation of plasma and granules. Following the addition

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of 200 ppm sodium azide for preservation, the collected fractions were stored in capped

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containers at 4ºC refrigerator until further processing.

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Plasma ultrafiltration to remove water

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To remove the added water from centrifugation step, the plasma solution was filtered using the Minimate™ TFF System (Pall Corporation, Port Washington, NY) with a Minimate™

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Tangential Flow Filtration Capsules of 5 kD pore size. The filtration was run continuously in 4ºC

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walk-in refrigerator until plasma volume was reduced by approximately 50%. To ensure

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adequate water removal, moisture content of plasma was determined using oven drying at 110ºC

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overnight.

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Preparation of recombined yolk systems

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Four yolk systems that would mimic (1) whole egg yolk (78% plasma, 22% granules,

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db), (2) pure plasma fraction (100% plasma, db), (3) plasma mixed with 50% granule fraction

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(88% plasma, 12% granules, db), and (4) granule mixed with 50% plasma fraction (64% plasma,

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36% granules, db) were prepared by adding the filtered plasma fraction to the granule fraction


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using calculated proportions (Table 2.1). Moisture and total solid contents were kept constant

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across the four systems, 53% and 47%, respectively. Mixtures were stirred manually using

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spatula before mixed with Ultra-Turrax® T rotor stator homogenizer (Laboratory Supply

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Network, Inc., Atkinson, NH) at 8,000 RPM for 30 seconds for a more homogenous mixture.

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Yolk freezing and thawing for gel formation

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The yolk systems were divided into three batches: fresh, frozen, and frozen for rheology

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and gel measurements. For the frozen samples, 40 g of each yolk mixture was poured into a 50

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mL conical polypropylene centrifuge tube, and they were vacuum-sealed in a vacuum bag with a

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FoodSaver® V222 vacuum sealing system (SunBeam Products, Inc., Jarden Consumer Solutions,

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Boca Raton, FL) to reduce freezer burn. The vacuum-sealed bag of four centrifuge tubes

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containing the different systems was submerged in the reservoir of a Haake SC 100

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refrigerated/heated bath circulator (Thermo Fisher Scientific, Waltham, MA) filled with 1:1

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ethylene glycol:Milli-Q water at 0°C. The bath was then set to -20°C. After the samples reached

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-20°C at a cooling rate of 0.3°C/min, they were held in the -20°C bath for 3 hours before storing

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in a -20°C freezer for 5 weeks.

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Preparation of samples for rheological analysis

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Samples for rheology were prepared following a method by Au et al. 13 using a custom-

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made aluminum apparatus composed of two heat transfer blocks (23.2 cm length, 7.2 cm width,

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2.5 cm height) and an aluminum mold plate (23.2 cm length, 7.2 cm width, 3 mm height) with

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five circles of 35 mm-diameter cutouts. The heat transfer blocks were connected with plastic

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tubing positioned level to the inlet and outlet ports on Haake SC 100 refrigerated/heated water

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bath circulator (Thermo Fisher Scientific, Waltham, MA) with 1:1 ethylene glycol:Milli-Q water



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mixture circulated through the blocks. When temperature reached 0°C, a sheet of Parafilm was

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placed over the bottom block, followed by the aluminum mold plate filled with egg yolk and

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another sheet of Parafilm to cover the mold plate. The top heat transfer block was quickly placed

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over the filled mold plate, and the cooling bath was set to -20°C. After reaching -20°C at a

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cooling rate of 0.3°C/min, the yolks were held in the apparatus for another hour before the mold

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plate was removed and sealed in a vacuum bag to reduce freezer burn. The samples were then

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stored in a -20°C freezer for 5 weeks.

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Fractionation of egg yolk into plasma, granule, LDL and livetin fractions

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The four fresh and frozen-thawed yolk systems were fractionated using a method

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modified from Ulrichs and Ternes 15 and McBee and Cotterill 14, immediately for fresh yolk after

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they were recombined and five weeks for the frozen-thawed samples. Mixtures of 1:3

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yolk:deionized water were prepared and stirred until well mixed. The mixtures were again mixed

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with Rotor-Stator at 8,000 RPM for 30 seconds to improve homogenization. Then, 30 mL of

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each yolk:water mixture was aliquoted to three 50 mL conical polypropylene centrifuge tubes.

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The tubes were transferred to a FIBERLite F15-8x-50cy fixed angle rotor in a Sorvall Legend

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XT centrifuge (Thermo Fisher Scientific, Germany) and centrifuged at 15,500 g and 4°C for 1

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hour. The plasma (supernatant), granule (pellet), and LDL (floating lipidic layer from frozen

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systems) were collected and the mass was recorded.

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The collected plasma was further fractionated into LDL and livetin using a method

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modified from Ulrichs and Ternes 15. Mixtures of 3:2 plasma:1% (w/v) carboxymethyl cellulose

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solution were centrifuged with the same parameter as above. The LDL (floating lipidic layer)

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and livetin (watery fraction) were collected and the mass was recorded.


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Determination of mass balance of all fractionated yolk components

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Small amounts of all fractions were taken for moisture/solid content analysis. Samples

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were 130°C oven-dried overnight, transferred to a desiccator for 5 minutes, and measured for

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change in mass. Moisture and solid contents were calculated to allow all data to be converted to

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dry mass balance (db). Fraction distributions (%) within each system were then compared among

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yolk systems and between fresh and gelled yolk for changes in fractionation behavior caused by

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freezing and thawing.

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Protein characterization by gel electrophoresis

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Protein distributions of fresh and frozen samples of all four recombined yolk systems

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were studied using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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performed according to Bio-Rad instructions and a modified method from Laca, Paredes, and

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Díaz 16. Samples of different fractions from each system were diluted in deionized water based

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on their estimated protein content quantified using bicinchoninic acid (BCA) assay and then 1:1

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(v/v) in a mixture of 95% Bio-Rad 2x Laemmli Sample Buffer (Tris-HCl/glycerol/bromophenol

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blue) and 5% β-mercaptoethanol, to bring sample concentrations to approximately 1 µg

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protein/µL. After dilutions, samples were heated in boiling water bath for 6 minutes. Protein

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standard Precision Plus Protein™ Dual Color Standards (BioRad Laboratories, Inc., Hercules,

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CA) and samples containing fractions from the same system were loaded onto a Bio-Rad Mini-

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PROTEAN® TGX™ precast polyacrylamide gel (4–20% gel, 12-well, 20 µL) at volume of 5 µL

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for standard and 7 µL for samples, and electrophoresed at 175 V for 35 minutes using the

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standard SDS-PAGE running buffer (250 mmol Tris, 1.92 mol glycine, and 10 g SDS per L).



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The gels were fixed in a solution consisting of 40% methanol, 10% acetic acid and 50%

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deionized water for 30 minutes. After removing the solution, 50 mL Bio-Safe™ Coomassie F-250

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Stain was added to stain the gels, and gels were gently shaken for 1 hour before rinsed with

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deionized water for 30 minutes. Gels were scanned with an ImageScanner flatbed scanner

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(Amersham Pharmacia Biotech Inc., Piscataway, NJ) for quantification and kept in water for

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storage.

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For densitometry analysis, the scanned images of the gels were processed with Image

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Processing and Analysis in Java software, ImageJ (National Institutes of Health, Bethesda, MD).

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An external standard was used for calibration following the NIH optical density calibration

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procedure 17. Each lane on the gel was plotted as a density spectrum where each peak represented

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a protein band. Each protein band was compared to published literature 18 for protein

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identification and the optical density, or peak area, was determined. Density is reported in

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percent relative optical density units (%OD).

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Rheological analysis

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After five weeks of freezing, yolk discs were analyzed using an Ares-G2 rheometer (TA

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Instruments, New Castle, DE) with a set of 25 mm diameter parallel plates. Sample thawing was

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performed for one disk at a time. A polyvinyl chloride cylindrical plunger (35 mm diameter, 62

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mm height) was used to push the yolk disk out of the mold to thaw on the bottom parallel plate at

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room temperature (23ºC) for 15 minutes.

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All four yolk systems, 10 replicate discs each, were subjected to an oscillation amplitude

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sweep test. Normal force of 0.2 N was applied for all samples. Oscillation strains in the range of

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0.1-10% was applied at a frequency of 1 Hz with 41 steps (data points). The average elastic


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modulus (G’) within the linear viscoelastic region (LVR) was reported as a measure of gel

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strength. Yield stress (σ*) was obtained as the stress where a 10% reduction from the average G’

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of was achieved.

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With outliers removed, average G’ and σ* were analyzed for significant difference.

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Values outside mean±2SD range were considered as outliers. With the outliers eliminated, 9

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discs from system 1:0, 5 discs from system 1:0.5, 8 discs from system 1:1, and 9 discs from

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system 1:2 were used for data analysis.

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Statistical analysis

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Statistical analysis was performed for rheology samples (1 treatment replication, >5

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samples replications) with JMP Pro 12, statistical software from Statistical Analysis System

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(SAS) Institute Inc. (Cary, NC). One-way analysis of variance (ANOVA) tests were conducted,

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and significance of difference (p-value

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