Meat Science 111 (2016) 67–77

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Capillarity proposed as the predominant mechanism of water and fatstabilization in cooked comminuted meat batters

Wenjie Liu c,⁎, Tyre C. Lanier a, Jason A. Osborne b

a Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Box 7624, Raleigh, NC 27695, United Statesb Department of Statistics, North Carolina State University, Campus Box 8203, 5238 SAS Hall, Raleigh, NC 27695, United Statesc James Ford Bell Technical Center, General Mills, 9000 Plymouth Ave. N, Minneapolis, MN 55427, United States

⁎ Corresponding author.E-mail address: Wenjie1108@gmail.com (W. Liu).

http://dx.doi.org/10.1016/j.meatsci.2015.08.0180309-1740/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 4 March 2015Received in revised form 20 August 2015Accepted 24 August 2015Available online 29 August 2015

Keywords:Fat stabilizationWater holding capacityFractureMicrostructure

Fat- and nonfat-containingmeat gels structurally became coarser and porous by partial substitution ofwhey pro-tein isolate for myofibrillar protein, creating a weaker texture plus greater cook loss (CL: fat + water) and ex-pressible water (EW). Microstructure examinations revealed a tendency for fat to coalesce during cooking ofthemore coarse-structuredgels. This tendencywas unaffected by fat pre-emulsification prior to addition, arguingagainst a strong role of an interfacial protein film in stabilizing fat. Instead, a gel structurewith evenly distributedsmall pores leads to lower CL and EW, thus controlling both water- and fat- holding since fat cannot readily per-meate small water-filled hydrophilic pores. Onlywhen large pores or continuous fissures are structurally presentcan water be released, allowing liquid fat to also migrate and coalesce. This changes the current paradigm ofunderstanding regarding the mechanism of fat/water-holding in comminuted meat products: gel capillarity(gel structure), not fat emulsifying ability of protein, is the likely determining factor.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Finely comminuted meat products, such as frankfurters and lun-cheon meats, typically contain 20–30% fat and high water content aswell. An understanding of the mechanism(s) responsible for stabiliz-ing/binding fat and water is very important since retention of waterand fat within such products affects not just their texture and juicinessbut also cooking (production) yields.

Classic emulsion theory was initially proposed to explain fat holdingability during cooking, wherein solubilizedmyofibrillar proteins (MFPs)stabilize the fat/water interface of what was considered to be an emul-sion of fat in a protein solution, the fat phase becoming liquid as thetemperature is raised (Hansen, 1960; Helmer & Saffle, 1963; Jones &Mandigo, 1982; Koolmees, 1989; Gordon & Barbut, 1992). The commi-nution phase was considered important in minimizing fat particle sizeand ensuring its more even distribution according to considerations ofStokes Law (McGuire, 2009). In seeming support of this understandingof comminuted meats as emulsions, it was found that non-meat pro-teins could be used to effectively pre-emulsify fat and thereby improveproduct stability during cooking (Aoki, Shirase, Kato, &Watanabe, 1984;Zayas, 1985).

Other workers began to question this simple emulsion concept,however, and placedmore emphasis on the concept of the ‘entrapment’of fatwithin the protein gel matrix formed upon cooking (Lee, Carroll, &

Abdollahi, 1981; Brown, 1972; Hamm, 1973; Lanier, 1986; Lee, 1985).Several of these workers still however considered that the interfacialprotein film (IPF) formed byMFP on the surface of fat droplets presentsa physical barrier to fat coalescence and outward flow (Hansen, 1960;Helmer & Saffle, 1963; Baron, 1965; Borchert, Greaser, Bard, Cassens,& Briskey, 1967; Saffle, Christian, Carpenter, & Zirkle, 1967; Acton &Saffle, 1969; Jones & Mandigo, 1982; Koolmees, 1989; Gordon &Barbut, 1992). A more thick and robust IPF was thought to explainwhy fat was better entrapped in some formulations than in others(Gordon & Barbut, 1990a).

We instead now postulate that fat stability (fat holding ability, FHA)during cooking of high fat batters is directly linked to water-holdingability (WHA), the latter of which has never been directly associatedwith either fat emulsification or formation of an IPF in cookedmeat bat-ters. Rather,WHA is thought to bemediated primarily by capillary pres-sure, which itself is a function of the gel matrix structure/composition(pore size, surface properties, and dissolved solutes) according to theLaplace equation (Atkins, 1978; Stevenson, Dykstra, & Lanier, 2013;Stevenson, Liu & Lanier, 2013). Scanning electron microscopy (Gordon& Barbut, 1990b) shows that an IPF undoubtedly does exist at the fat/protein matrix interface. But if the protein matrix is mainly filled bywater, such that the protein matrix consists of a network of very smallpores exhibiting a very hydrophilic surface, then no physical entrap-ment by an IPF would be needed to retain fat droplets. Indeed the sys-tem could be considered as a reverse of that presented by a water-resistant, ‘breathable’ fabric, such as Goretex®, which does not allowwater droplets of sufficient size topass through hydrophobic pores of a

68 W. Liu et al. / Meat Science 111 (2016) 67–77

smaller size (Dobrusskin, Hannah, & Gerhard, 1991). In effect, then, theproperties of the protein gel matrix that allow it to hold water by capil-lary pressure would also suppress movement of droplets of liquid fat.Only when the gel is so poorly constituted (large pores, small contactangle of liquid with surface, etc.), such that water can flow freely fromit, would fat also likely leave the matrix. Thus with this explanation, itis logical fat and water stability should be indeed be interlinkedincooked, comminuted meat products. Brown and Toledo (1975) pre-sented clear evidence that fat andwater stability (i.e. FHA &WHA) dur-ing cooking of comminutedmeat products do in fact correspond closely.

Liu (2012) recently showed that confocal laser scanningmicroscopy(CLSM) and variable-pressure scanning electron microscopy (VP-SEM)can optimally be used in tandem to examine the microstructure ofmeat batters and gels as relating to water and fat stability duringcooking, with minimal artifacts of sample preparation. The present re-search utilized these instruments to examine the microstructuralchanges that may relate to fat- and water-holding properties of higherfat-containing batters during cooking.

2. Materials and methods

2.1. Materials

Boneless skinless chicken breast meat was purchased from a localchicken processor (Pilgrim's Pride, Sanford NC), packaged in ~3.5 kgportions in Cryovac B-series bags (Cryovac, Duncan, SC, U.S.A.) andheld frozen at −20 °C until use (b5 months). Frozen chicken wasthawed at 4 °C for 12 h before the trimming of visible fat and connectivetissue. Rendered pork fat (lard) was obtained from a local grocery(Armour brand), packaged in 9.1 kg plastic containers and kept at 4 °Cuntil used (b2 months).

2.2. Preparation of meat batters and paste

Finely comminuted, fat-containing batters were prepared by chop-ping thawed chicken breast meat (proximate analysis 23.21% protein,1.34% fat, 69.45% water) (51.17%, w/w), water (24.83%, w/w), lard(20%, w/w), salt (2%, w/w), sugar (1.7%, w/w), and tripolyphosphate(0.3%, w/w) in a Stephan UMC-5 vertical-cutter/mixer (Stephan Ma-chinery Corp., Columbus, OH) under vacuum for 6 min at 2,5000 rpm.Final chopping temperature did not exceed 10 °C. Lean, non-fat (NF)meat pastes were prepared in the same way except that fat wasdeducted from the formulation. Thus the ratios of all remaining ingredi-ents remained constant [percentages of each remaining ingredient inthe non-fat formulation would be calculated as (original ingredient% /80) × 100%]. Therefore the gel matrix portion of both batters and pasteswere identical in composition, such that the formulations differed onlyby the presence/absence of dispersed fat particles. The fat-containingbatter formulation simulated a commercial frankfurter composition;previously it was shown to produce similar gel properties to that com-mercial frankfurter when cooked by a time/temperature process sched-ule used commercially (5 to 70 °C at the rate of 0.5 °C/min) (Liu, 2012).

To lower the gelling quality (texture and WHA/FHA) of the meatportion of the formulation for contrast, whey protein isolate (WPI)(BiPRO, Davisco Foods International Inc., Eden Prairie, MN) was addedin substitution of 15% or 30% MFP, to imitate a meat block having arelatively higher proportion of sarcoplasmic (globular) protein. Thiswould be expected to decrease gel-forming ability (Burgarella, Lanier,Hamann, & Wu, 1985; Miyaguchi, Nagayama, & Tsutsumi, 2000;Asghar, Samejima, & Yasui, 1985). WPI (Ca content 0.04%, w/w),which is essentially non-gelling below 70 °C, thus served as a substitutefor sarcoplasmic meat proteins that are present at higher percentage inlower quality meats.

For each treatment (both fat- and non-fat-containing batters/pastes),samples were produced at each level of WPI substitution (0%, 15%,30%).Fat-containing batters were further divided into 2 sub-groups

(non-pre-emulsified vs. pre-emulsified) as explained in Section 2.3below. Thus the experiment overall evaluated three levels of WPIsubstitution for each of three separate treatments (pre-emulsified fat-containing batters; non-pre-emulsified fat-containing batters; non-fat-containing pastes). All preparations of batters/pastes were conductedtwice. The order of preparation of all batches of batter/or paste at eachlevel of WPI substitution (0%, 15%, 30%) was randomized.

Subsequently in this report pastes/gels made with no added fat aredesignated ‘NF’, batters/gels containing pre-emulsified fat are designat-ed as ‘PE’, and batters/gels containing non-pre-emulsified fat are desig-nated as ‘NPE’.

2.3. Pre-emulsification procedure

Considering the maximum reported thickness of IPF from transmis-sion electronmicroscopy images and theminimum fat particle size like-ly attained during chopping (Gordon & Barbut, 1991), we couldestimate, by the following calculation, that pre-emulsification of thefat using only about 20% of the total protein from the final formulationshould be more than sufficient to ensure a thick IPF around all fatparticles.

The approximate total number of fat particles, estimated as beingabout 5 μm in diameter (Van der Oord & Viser, 1973) was calculated as:

Number of fat particles per 100 gram batter ¼ Total fat volumevolume per fat particle

¼

mfat

ρfat

!

43π rfat� �3

where mfat is the mass of the fat; ρfat is the density of the fat(Charrondiere, Haytowitz, & Stadlmayr, 2012) and rfat is the radius ofthe fat particle.

If each fat particle is coated by an IPF of approximate 0.1 μm(Swasdee, Terrell, Dutson, & Lewis, 1982; Gordon & Barbut, 1990a),the total volume of IPF needed per fat particle is,

Volume of IPF per particle ¼ 43π IPF thicknessþ rfat� �3−4

3π rfat� �3

¼ 43� 3:14 0:1 μmþ5 μmð Þ3−4

3�3:14 0:1 μ5 μmð Þ3 ¼ 3:2� 10−8mL

and the total amount of protein needed to create the IPF around each fatparticle could thus be calculated as,

Protein required per 100 gram of batter for IPF formation¼ Volume of IPF � ρprotein

� �� Number of fat particles

¼ 3:2� 10−8mL � 1:35gmL

� �� 4:25� 107 ¼ 1:84g

where ρprotein is the density of the protein (Tanford, 1954).Thus,

Actual meat required per 100 gram of batter for IPF formuation

¼ Protein required per 100 gram of batter for IPF formationprotein concentration per gram of meat

¼ 1:84g23%

¼ 8g

(Protein concentration per gram of chicken breast meat; 23%: Xiong,Cantor, Pescatore, Blanchard, & Straw, 1993).

Pre-emulsions were prepared in a vacuum homogenizer by mixingtwo parts of water and two parts of partially melted(at 30 °C) lard(Hughes, Cofrades, & Troy, 1997; Miklos, Xu, & Lametsch, 2011) withone part of chicken breast meat, plus 2% NaCl, w/w, at 30,000 rpm for5 min at 25 °C. This pre-emulsion was first chilled to 5 °C, and then

69W. Liu et al. / Meat Science 111 (2016) 67–77

slowlymixed into the rest of the well-choppedmeat paste at low speedin a Stephan UMC-5 vertical-cutter/mixer (Stephan Machinery Corp.,Columbus, OH) during making of batters containing the added pre-emulsion to a final chopped temperature of 10 °C. Water, fat, meat,and salt used to make the pre-emulsion were thus subtracted fromthe total formulation used for those fat-containing formulations notcontaining a pre-emulsion, such that composition of all fat-containingformulations, +/− added pre-emulsion, would be identical.

2.4. Small strain oscillatory testing during heating and subsequent cooling

Rheological changes (G′ and phase angle) of raw, chilled pastes/batters were subsequently measured during heating and cooling in aserrated (cross-hatched) plate and plate attachment (20 mmdiameter;with 2mmgap), conducted on a constant stress, small strain rheometer(Stresstech, Rheologica Instruments AB, Lund, Sweden). An oscillationof 0.1 Hz with a resistance stress of 15 Pa was used for testing, asthis was pre-determined for both fat- and non-fat-containing gelsto be in the linear viscoelastic region (LVR). Samples were heatedat 0.5 °C/min to an endpoint of 70 °C and immediately cooled at 5°C/min to a final temperature of 5 °C. All treatments in triplicate wererandomized to minimize testing effects.

2.5. Heat processing of gels for fracture testing, cook loss, and expressiblewater measurements

Meat batters (pastes) were first vacuum-packaged in CryovacB-series bags (Cryovac, Duncan, SC, U.S.A.) with a Multivac 8941(Multivac, Allgau Germany) to remove visible air pockets. A cornerwas clipped and the bag placed into amanual sausage stuffer for extrud-ing into cellulose casings (inner diameter 1.9 cm; tied off to lengths of17.8 cm), which were then vacuum packed inside water impermeableplastic bags for subsequent heat processing in a water bath. Thesewere then cooked in a programmable water bath (Neslab InstrumentsInc., Portsmouth, NH) from 5 to 70 °C (internal temperature) atthe rate of 0.5 °C/min, followed by immediately cooling in an ice bath.Actual product temperature was confirmed with a thermocoupleinserted into the geometrical center of one stuffed casing.

2.6. Fracture testing of cooked, cooled gels

Cooked, cooled cylindrical gels (from both fat- and non-fat-containing formulations) were held overnight under refrigeration,removed from the casings and subsequently cut into specimens2.54 cm long, each end of which was glued to plastic disks (GelConsultants Inc., Raleigh N.C.) using an instant adhesive. Capstan-shaped samples were milled from each specimen to 1 cmminimum di-ameter (at the center) on a milling machine (Gel Consultants Inc., Ra-leigh NC), then wrapped in plastic wrap (to prevent moisture loss)and brought to room temperature before torsion testing. For testing,gel specimenswere vertically mounted and twisted to the point of frac-ture at 2.5 rpm on a Hamann Torsion Gelometer (Gel Consultants Inc.,Raleigh NC). Stress (kPa) and strain (dimensionless) at fracture werecalculated with the manufacturer’s software for each sample, corre-sponding to the strength and deformability of the gels, respectively(Hamann, Amato, Wu, & Foegeding, 1990). Each variable has 6 repli-cates for data analysis.

2.7. Cook loss (CL)

CLwasmeasured in triplicate by subtracting the post-cookedweightof gels from the pre-cooked weight of the batters (or pastes), expressedas a percentage of pre-cooked batter weight. Liquid release on the sur-face of cooked gels was removed before weighing.

A CEM SMART Trac (CEM, Matthews, NC) rapid moisture analysissystem was used to determine moisture content in both raw and

cooked batters/pastes. Water loss during the cooking of gels wascalculated as:

Water loss% ¼ Water content in raw− 1−cook loss%ð Þ� water content in cooked gelð Þ:

A simplifying assumption was made that CL consisted only of waterand fat; thus fat loss was calculated as:

Fat loss% ¼ Cook loss%−Water loss%:

2.8. Expressible water (EW) determination

EWof each cooked cylindrical gelwasmeasured in triplicate using themicrocentrifuge-based method of Kocher and Foegeding (1993). Thecenter of each gel was cut into 10 × 4.8 mm cylinder specimens using acork borer and these specimens were placed into themicrocentrifuge fil-tration unit which was comprised of a 2.0 mL microcentrifuge tube(Beckman Instruments, Inc., Palo Alto, California, U.S.A.) that collectedmoisture released through the bottom mesh of an inner tube containingthe specimen. Specimens were centrifuged at 152 ×g for 10 min for thistype of comminuted meat gels. EW was calculated as:

EW %ð Þ ¼ Weight of water releaseWeight of samples

� 100:

2.9. Oil migration into gels

Pasteswere prepared as in Section 2.2, with fat omitted from the for-mulation. Two pasteswere used for these experiments: either 0% or 30%added WPI in partial substitution for MF protein.

Three different approaches were used to examine whether oil couldmigratemore readily into and through a coarsermeat protein gelmatrix(30% added WPI in partial substitution for MF protein) versus a finermeshed, smaller pore size gel matrix (0% addedWPI in partial substitu-tion for MF protein):

• During cooking, no applied pressure. A small drop (0.1 mL) of oilcontaining Oil Red-O (Sigma-Aldrich Corp. St. Louis, MO), to facilitateobservation of oil migration, or Nile blue, to facilitate CLSM observa-tion of oil migration, was injected into the geometric center of glasscylinders (ϕ, 1.5 cm; h, 2.8 cm) filled with either of the two NFpastes(Fig. 1a). The cylinders were each topped with fresh paste tofully encase the injection point of the drop, such that the ends weresealed from water contact, and were then cooked in a programmablewater bath (Neslab Instruments Inc., Portsmouth, NH) heated from 5to 70 °C at the rate of 0.5 °C/min. Product temperature wasmonitoredwith a thermocouple inserted into the geometric center of a separatetube filled only with meat paste. Samples were removed from thewater bath at pre-determined end point temperatures (50, 55, 60,65, and 70 °C) and immediately placed in plastic bags, from whichheadspace air was removed under a vacuum before sealing, andthen quickly frozen at −18 °C prior to later thawing and imaging byCLSM. Other gels were cooled and sliced open for visual assessmentof oil migration away from the point of injection.

• During cooking,while pressurized oil was applied to a paste surface. In aspecially designedmetal container (Fig. 1b) a pressure of 8 PSI (not sohigh as to result in extrusion of paste from the base outlet but suffi-ciently high to create a positive oil pressure onto the gel surface)was applied to a dyed (Red-O only, since only visual examinationwas subsequently done) oil layer over a 1 cm thick raw NF meatpaste layer. During pressurized holding (2 h) the container was im-mersed in a water bath at varying temperatures (50, 60, or 70 °C).Gels were subsequently cooled and sliced open for visual assessmentof oil migration at the surface.

Fig. 1. a. Cylindrical gel injected with single oil drop, sliced after cooking to reveal injected oil drop (Red: oil). b. Diagram of pressurized metal container to assess possible oil penetrationinto NF gels while cooking (Red: oil). c. Diagram of funnel setup for suction-assisted oil permeability test. (For interpretation of the references to color in this figure legend, the reader isreferred to the web version of this article.)

70 W. Liu et al. / Meat Science 111 (2016) 67–77

• After cooking (gelation), with vacuum to assist oil migration. A cooked(at heating rate of 0.5 °C/min from 5 to 70 °C) gel disc (ϕ, 2 cm; h,2 cm) was prepared to fit tightly into a glass funnel having a fritteddisc at its base (Fig. 1c) such that no dyed (Red-O only, since only vi-sual examination was subsequently done) oil would leak around thegel sample after being layered on top of the gel sample. This also in-sured that when vacuum (30 in. Hg) was fully applied to the base ofthe gel, via the attached flask, it might draw oil into the top surfaceof the gel, as fluid in the gel was drawn through the bottom surfaceof the gel by the applied vacuum. After 10 min under a vacuum thegels were removed, sealed in plastic bags, cooled, and sliced to deter-mine the depth of oil penetration achieved.

2.10. Confocal laser scanning microscopy (CLSM)

Meat gels, held in sealed bags at 4 °C overnight, were sliced into sec-tions approximately 5 mm × 5mm× 1mm thick using a razor blade. A

0.2% solution of Nile Blue A Sulfate (MP Biomedicals, LLC; Solon, OH)fluorescent dye in de-ionized water was filtered twice using WhatmanNo. 3 (Maidstone, Kent UK) filter paper, and 20 μL of the solution waspipetted onto the cut surface of eachmeat gel slice. The dyewas allowedto absorb into the gel at room temperature for 10min. Gel sampleswerethen turned over (dyed gel surface against the coverslip) onto a single-welled slide with a #1.5 coverslip adhered to the bottom of the slide viasilicone grease. Samples were imaged on a Zeiss LSM 710 Confocal laserscanning microscope (CLSM) attached to a Zeiss Axio Observer Z1inverted microscope using 10 × 0.45 NA (Numerical Aperture) dry,20 × 0.8 NA dry and 40 × 1.2 NA water immersion objectives. A488 nm argon laser (to excite Nile Blue in the fat phase) and a 633 nmhelium neon laser (to excite Nile Blue in the protein phase) were usedsequentially to image the samples. Emission spectra were collectedfrom 500 to 650 nm for the fat phase and 650–800 nm for the proteinphase, and the resulting images were overlaid. The pinhole was opti-mized to the sizeof 1 airy unit (diameter of the airy disk produced by

Fig. 3. Fracture strain of NF, NPE, and PE gels as affected by addition of 0%, 15% and 30%whey protein isolate (WPI) in substitution of MF protein, holding moisture content con-stant. Means with the same letter are not significantly different; error bars extend ±1standard deviation.

71W. Liu et al. / Meat Science 111 (2016) 67–77

the objective) to reduce unwanted light dispersion that might blur theimage, and the zoom was maintained at 1.0000 across all images, re-gardless of z-stack position. For each gel treatment, 2 sampleswere pre-pared; 3 images were taken per sample at the 10× objective, resultingin a total of 6 images per treatment.

2.11. Variable pressure scanning electron microscopy (VP-SEM)

Cylindricalmeat gels (ϕ: 2 cm)were held at 4 °C until cut into blocksof 2 cm thick using a razor blade. To avoid the error from the surfacesmashing, the blocks were lightly frozen for 20 s on ametal stabber sur-rounding by liquid nitrogen. The internal structures of the gels were ex-posed by cutting the gels into half. The samples were mounted on acopper specimen holder and the exposed surface was observed undera SEM (S-3400, Hitachi) with a VP-mode (sample stage, b−5 °C; pres-sure, 30 Pa; accelerating voltage, 20 kV). For each gel treatment, 2 sam-ples were prepared; 3 images were taken per sample at each objective:250×; 500×; and 1000×, resulting in a total of 18 images per treatment.

2.12. Statistical analysis

Factorial effects models appropriate to the crossed 3 × 3 two-factordesign were fit using the GLM procedure of the SAS statistical softwarepackage (SAS version 8.02, SAS Institute, Cary, NC, USA). The experi-mental factors were fat composition (NF/NPE/PE) and whey proteinisolate (WPI, with levels 0%, 15% and 30%). Since, for many of the re-sponses, analysis of variance indicated interaction between the two fac-tors, simple effects were investigated by carrying out all pairwisecomparisons among the 9 factor level combinations using Tukey's hon-estly significant difference (Tukey, 1949). Pearson's correlation coeffi-cients were computed to quantify linear associations between fractureproperties, cooking stability (fat/water loss) andwater holding capacityacross the three levels ofWPI in substitution ofMFPwhile holdingmois-ture content constant.

3. Results and discussion

3.1. Fracture properties of gels

IncreasingWPI substitution for MFP significantly decreased fracturestress of gels (P b 0.05), irrespective of fat addition or pre-emulsificationof fat (Fig. 2) (Burgarella et al., 1985). As expected, fat addition also re-sulted in decreased fracture stress (gel strength), at all levels of WPIsubstitution for MFP (Niwa et al., 1989).

WPI substitution for MFP also depressed fracture strain (geldeformability) of fat containing gels (Fig. 3), but did not significantly

Fig. 2. Fracture stress (kPa) of NF, NPE, and PE cooked gels as affected by addition of 0%,15% and 30% whey protein isolate (WPI) in substitution of MF protein, holding moisturecontent constant.Means with the same letter are not significantly different; error barsextend ±1 standard deviation.

affect fracture strain of gels not containing fat (Burgarella et al., 1985).Fat addition also generally depressed fracture strain of all gels.

3.2. Small strain properties of gels

Substitution of MFP by 15% WPI dramatically decreased storagemodulus (G′; rigidity) of cooked, cooled gels regardless of fat additionor pre-emulsification (Fig. 4). Interestingly however, there was no sig-nificant change in G′ comparing 15% to 30% substitution of MFP byWPI as occurred for fracture strength (stress) of gels (Fig. 2). This great-er effect in going from 0 to 15% WPI substitution was also seen in frac-ture deformability (strain) data (Fig. 3).

Interestingly, phase angle, whichmeasures thefluidity of gels, variedmarkedly due to the addition of fat, but did not change significantlywithincreasing substitution of MFP by WPI (Fig. 4).

The addition of fat increased G′ in the absence of WPI but had only aslight or negative effect on G′ ofWPI-containing gels. Thus, while fat ad-dition decreased gel strength (Fig. 2), gel rigidity (G′) was increased.This points up the well-recognized fact that rheological measurementsat small strain do not reliably predict the fracture properties of meatgels (Hamann & Lanier, 1987). Small strain data are most useful as indi-cators of microstructural changes (Montejano, Hamann, & Lanier, 1984;Vanroon, Houben, Koolmees, & Van Vliet, 1994; Iwasaki, Washio,Yamamoto, & Nakamura, 2005).

Fig. 4. Small strain properties (G′ — storagemodulus; δ— phase angle) of NF, NPE, and PEgels immediately after cooling to 25 °C, as affected by addition of 0%, 15% and 30% wheyprotein isolate (WPI) in substitution of MF protein, holding moisture content constant.Error bars extend ±1 standard deviation.

Fig. 5. Cook loss of NF, NPE, and PE gels as affected by addition of 0%, 15% and 30% wheyprotein isolate (WPI) in substitution of MF protein, holding moisture content constant.W: Gradient Fill: CL (non-fat gels); No Fill: fat loss. Letters in the upper line are used to in-dicate significant differences in fat loss. Letters in the lower line are used to indicate signif-icant differences in water loss.Means with the same letter are not significantly different.Error bars extend ±1 standard deviation.

Table 1Sample correlation coefficients (with corresponding p-values from tests of no correlationbeneath) among fracture properties, cooking stability and water holding capacity of fat-containing cooked gels as affected by WPI substitution.

PE WPI EW WL FL CL Stress Strain

PE 1 0 −0.4563 0.0123 0.1749 0.0942 −0.1163 −0.09281 0.057 0.9612 0.4876 0.6614 0.4993 0.5905

WPI 1 0.7398 0.9621 0.913 0.9353 −0.9346 −0.73720.0004 b0.0001 b0.0001 b0.0001 b0.0001 b0.0001

EW 1 0.7575 0.7106 0.7878 −0.7403 −0.51250.0003 0.0009 0.0001 0.0004 0.0297

WL 1 0.8396 0.9179 −0.9129 −0.6393b0.0001 b0.0001 b0.0001 0.0043

FL 1 0.8811 −0.8402 −0.4709b0.0001 b0.0001 0.0486

CL 1 −0.8648 −0.5454b0.0001 0.0058

Stress 1 0.86331b0.0001

Strain 1

72 W. Liu et al. / Meat Science 111 (2016) 67–77

3.3. Water and fat holding properties

WPI partial substitution for MF protein caused significantly higherCL (water loss in non-fat gels; water + fat loss in fat-containing gels;Fig. 5) and slightly higher EW (Fig. 6). ‘Pre-emulsifying’ fat had no effectin reducing either CL or EW. This contrasts with two previous studieswhich concluded that meat batter gels made with pre-emulsified fathad less CL (Aoki et al., 1984; Zayas, 1985). These cited studies also uti-lized nonmeat proteins in the preparation of their pre-emulsions,however.

Notably, the increase in water loss during cooking, associated withincreasing partial substitution of WPI in fat-containing gels, highly cor-relatedwith the increase in fat loss (R2=0.8396, P b 0.0001; Table 1), inagreement with the finding of Brown and Toledo (1975). These CL datawere also well correlated with EW (Table 1).Water loss (WL; themajorpart of CL) had slightly higher correlation with EW (R2 = 0.7575) thandid fat loss (FL, the remainder of CL) (R2= 0.7106) (Table 1). In non-fatgels there was also significant correlation between CL and EW (R2 =0.7546, Table 2).

Fig. 6. Expressible Water (EW)% of NF, NPE, and PE cooked gels as affected by addition of0%, 15% and 30% whey protein isolate (WPI) in substitution of MF protein, holding mois-ture content constant. Means with the same letter are not significantly different. Errorbars extend ±1 standard deviation.

3.4. Microstructure

3.4.1. Confocal microscopyIn non-fat gels towhichnoWPIwas added, CSLM revealed transition

from a rather homogeneous, fine pore gel network to a more open net-work with prominent irregular cavities when WPI was added at 15%substitution of MFP (Fig. 7 A1 & A2). WhenWPI addition was increasedto 30% in substitution of MFP, the gel network appeared even moredisrupted (Fig. 7 A3). This pattern was repeated in gels containing fat(Fig. 7 B1-3, C1-3).In these gels the distributed fat droplets can also beseen to grow in size (during cooking) and become more irregular inshape as more WPI was added in substitution of MFP.

Pre-emulsification of fat with MFP resulted in little difference in mi-crostructure when comparing micrographs of gels made at identicalWPI contents containing pre-emulsified versus non-pre-emulsified fat.The raw, pre-emulsion preparation (Fig. 8), prior to its addition to alean meat paste (held three days in refrigeration before viewing)shows a fine distribution of small spherical fat droplets. So the liquidfat was indeed well distributed by the pre-emulsion process, whichshould favor formation of a strong IPF of meat protein around each fatdroplet. Yet, irrespective of whether or not the fat had been pre-emulsified, fat apparently migrated and coalesced into larger bodies incooked gels wherein 30% ofMFPwas substituted byWPI(Fig. 7 B3& C3).

It seems most likely then that movement and coalescence of the fatduring cooking can be attributed to the less homogeneous gel matrixstructure of the 30% WPI substituted gel, rather than because a less-than-optimum IPFwas formed around fat droplets in such gels. This fol-lows since an IPF, formed by pre-emulsification with MFP, should havepersisted even after its incorporation into themore open gel network of

Table 2Sample correlation coefficients (with corresponding p-values from tests of no correlationbeneath) among fracture properties, cooking stability and water holding capacity of non-fat cooked gels as affected by WPI substitution.

WPI EW WL CL Stress Strain

WPI 1 0.83549 0.88925 0.93032 −0.96706 −0.49230.005 0.0013 b0.0001 b0.0001 0.038

EW 1 0.71612 0.75456 −0.83567 −0.486250.03 0.0188 0.005 0.1844

WL 1 0.94577 −0.88395 −0.534570.0001 0.0016 0.1381

CL 1 −0.8501 −0.422110.0005 0.1717

Stress 1 0.613060.0068

Strain 1

Fig. 7. CLS micrographs of NF (A1, A2, A3),NPE (B1, B2, B3), and PE (C1, C2, C3) cooked gel structures affected by addition of 0% (A1, B1, C1), 15% (A2, B2, C2) and 30% (A3, B3, C3) wheyprotein isolate (WPI) in substitution ofMFP, holdingmoisture content constant. Red: protein phase; Green: fat phase: Scale bar: 100 μm. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

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the WPI-substituted batters/gels. At that point the IPF should haveprotected against fat migration and droplet coalescence (Gordon &Barbut, 1990a; Gordon & Barbut, 1990c). Much less likely would be anexplanation that the IPF of the fat droplets, pre-formed with onlymeat protein during the pre-emulsification stage, would later becomedisrupted by the presence of a minor amount of soluble WPI, thusallowing for fat movement and coalescence.

Concurrent with the change in gel structure caused by WPI partialsubstitution for MFP was an increase in CL (Fig. 5). It seems likely thatwater could now more easily move within such a structure of muchlower capillary pressure (inferred from the larger size of pores;Lewicki, Busk, & Labuza, 1978; Labuza & Busk, 1979). And as water

Fig. 8. Pre-emulsion prepared with functional myofibrillar protein, water and melted fat.Red: protein phase; Green: fat phase; Scale bar: 100 μm.(For interpretation of the refer-ences to color in this figure legend, the reader is referred to theweb version of this article.)

moves during cooking, liquid fat may also become more mobile withinthe gel network.

The gels containing no partial substitution of WPI for MFP exhibitedamore homogeneous gel matrixwith smaller diameter pores. Pores of aprotein matrix would have a very hydrophilic inner surface, and thecombination of small size and hydrophilic surface leads to higher capil-lary pressure (Stevenson, Dykstra & Lanier 2013). Thus they exhibitedlower CL and EW (Figs. 5,6). This same fine pore gel structure also ex-hibited less fat movement/coalescence (Fig. 7 B1 and C1). Small hydro-philic pores, filled with water held by capillary pressure, are a likelygood barrier to immobilize melted fat, irrespective of any IPF formedduring batter preparation. Lee et al. (1981) also conjectured that fatand water release during cooking of comminuted batters results whenlarge channels form within the gel matrix.

3.4.2. VP-SEMVP-SEM offers resolution of the gel matrix microstructure at much

highermagnification thanCLSMbut is likewise not prone to artifacts ne-cessitated by the extensive sample preparation steps needed for con-ventional scanning electron microscopy.

A first, and somewhat startling, observation is that clearly a proteingel matrix exists even in the raw paste/batter (Fig. 9, A1-3). Whilethis actually is not the first such recorded observation using SEM(Hermansson, 1986; Barbut & Gordon, 1996) there has been seeminglyno open discussion of this fact, especially by thosewho still regardmeatbatters as classic emulsions (McGuire, 2009; Knipe, 2015). This gelstructure, formed even at the raw batter stage, persists upon heating/cooking (Fig. 9, B-D), but is known to develop more numerous andthicker walls as proteins further denature and aggregate (Barbut,Gordon, & Smith, 1996) (Fig. 10). Therefore Stokes' considerations ofliquid fat coalescence andmovement (McGuire, 2009) could not be pos-sibly applied in such a gel matrix system (Labudde & Lanier, 1995),which is present even in the raw batter stage. Thus, the view of thesesystems as classic emulsions is not valid even at the raw/comminutedstage of processing.

For NF cooked gels, substitution of 15% and 30% WPI for MFP led tosome detectable change in pore structure (Fig. 9, B1-3), though not asextensive as was apparent atmuch lowermagnification in CLSMmicro-graphs (Fig. 7, A1-3). Using ImageJ image analysis freeware to quantify

Fig. 9.VP-SEMmicrographs of NF rawmeat paste (A1, A2, A3) andNF (B1, B2, B3), NPE (C1, C2, C3), and PE (D1, D2, D3) cooked gelmicrostructures affected by addition of 0%, 15% and30%whey protein isolate (WPI) in substitution of MF protein, holding moisture content constant. Grey: protein matrix phase; darker grey fat phase; Scale bar: 20 μm.

74 W. Liu et al. / Meat Science 111 (2016) 67–77

the diameter of pores (Stevenson, Liu, & Lanier 2013),we found that theaverage pore size increased from9.3 μmto 17.65 μmasWPI substitutionfor MFP was increased up to 30%.

It thus seemsmost likely that the greater CL and EWof gels contain-ing 15% and 30% WPI in substitution of MFP arose from the larger gelcavities apparent only at relatively low magnification (×10) by CLSM.The small differences in pore size we measured, or other non-homogeneity evident in the VP-SEM micrographs would seeminglyhave relatively minor contributions to water- and fat-holding in theface of the larger cavities in the gel structure evident only at lowermagnification.

In fat-containing gels, fat globules appear immersed within thisthree-dimensional protein network. Even at this high level ofmagnifica-tion, the effect of lowering protein quality by 30% WPI substitution forMFP is quite apparent in that fat globules have apparently coalescedand are larger than when no WPI was in the formulation (Fig. 9 C&D,1 & 3).

Fig. 10. Gel formation diagram and SEM m

In comparing Fig. 9 C 1-3 to D 1-3 there appears to be little or noeffect of PE as compared to NPE treatments in the appearance of fatglobules after cooking, as was also noted in CLSM micrographs (Fig. 7B 1-3 vs C 1-3).

3.5. Oil migration into NF pastes during and after cooking

Both visual assessment (Fig. 1a) and confocal microscopy of the in-terface of the relatively large injected oil drop and the surrounding NFcooked gel (Fig. 11) revealed no substantial penetration of oil into theprotein matrix as the gel structure was made coarser in pore structurebyWPI substitution for MFP (30%). The same held true (as judged visu-ally only; data not shown) even when gels were cooked slowly whilepressurized oil was applied to the paste surface and when fully cookedgels were subjected to a vacuum at their base with oil contacting thetop surface. Varying the cook temperature of the pressurized samples(50, 60, 70 °C) also had no effect.

icrographs of raw and cooked gels.

Fig. 11. CLSM of oil drop injected into NF gels (cooked to 70 °C) madewith either (A) 0% or (B) 30%WPI in substitution ofMFP in the formulation. Red: protein; Green: fat; Magnification:×10; Scale bar: 100 μm.The left image of eachpair of images (A, B)was located approximately 50 μmto the left of the image on the right. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)

Fig. 12. Schematic representation of capillary explanation for both fat- and water-holdingproperties of cooked, comminuted meat gels. In the circled two-dimensional cross-sectional diagramof a fat-containing gel: Grey: cross-section of protein three-dimensionalmatrix fibers; Blue: water phase; Yellow: fat/oil phase. (A) In the ‘high quality gel’(i.e., excellent fat- and water-holding ability), pores within the protein matrix arerelatively small, and thus water is held tightly by high capillary pressure. Fat dropletsare thereby also immobilized, by the relatively smaller size of pores in thematrix coupledwith their high hydrophillicity(‘reverse Gore-tex®model’), as well as by the immobiliza-tion of water by capillary pressure (no water movement to facilitate fat/oil droplet move-ment during cooking). (B) In a ‘low quality gel’ (i.e., excessive loss of water andmovement/coalescence of fat), pores within the protein matrix are relatively quite large,and thus capillary pressure is low: water can more freely flow outwards from the gel. Aswater moves (mainly during cooking), so do melted fat droplets, which can exit the gelwith the water and/or coalesce within the coarser structure of the meat gel to form fatpockets when cooled. (Illustration by Liz Bradford). (For interpretation of the referencesto color in this figure legend, the reader is referred to the web version of this article.)

75W. Liu et al. / Meat Science 111 (2016) 67–77

However, when fat was finely incorporated into batters prior tocooking, oil migration and coalescence could be detected in gels madecoarser in structure by WPI partial substitution for MFP (Figs. 7 B3,C3and 9 C3,D3). Clearly, when NF pastes/gels of correspondingly fine orcoarse structure were cooked with, or subjected to a larger body of oilby pressure or vacuum, no differences in oil penetration were notedfor the gels of differing microstructure.

We interpret these differences to arise because the very hydrophilicgel, swollenwithwater by capillary pressure, presents a formidable bar-rier for very large oil surfaces to penetrate (experiments of Section 2.9).This in effect is the reverse of the properties of the outer layer of water-resistant yet ‘breathable’ fabrics, such as Goretex®, in which a very hy-drophobic matrix of small pores blocks the entrance of large waterdroplets, yet allows smaller water vapor droplets to pass (Dobrusskinet al., 1991). When fat is however entrained into the gel matrix asvery tiny droplets (in liquid form while the product is hot), smallerthan the voids apparent by CLSM (Fig. 7 B1 & C1), these are able, uponthe movement of water through these voids during cooking, to migrateand coalesce (Fig. 12).

3.6. A capillary hypothesis of water- and fat-binding in comminutedmeat products

It seems clear from the present data that fat stability during cookingof high fat batters corresponds directly to water-holding ability, justas had been previously reported by Brown and Toledo (1975).Hermansson and Lucisano (1982) had also observed that the morefine and homogeneously constructed that a fat- containing meat gelstructure is, the less is the moisture loss and tendency towards phaseseparation (loss of liquid fat).Water holding in protein gels is likelyme-diated primarily by capillary pressure (Labuza & Busk, 1979), which inturn is a function of the gel matrix structure and composition (poresize, surface properties, and dissolved solutes) according to the Laplaceequation (Atkins, 1978; Stevenson, Dykstra & Lanier 2013).

Previous work (Gordon & Barbut, 1990a; Gordon, 1993) showedthat an IPF likely does exist at the fat/proteinmatrix interface in commi-nuted meat products. It seems quite likely also, as many workers havesuggested (Lee et al., 1981; Hermansson, 1986; Gordon & Barbut,1990c; Labudde & Lanier, 1995; Labudde & Lanier, 2011), that this IPFis basically continuouswith the surroundingprotein gelmatrix. The sur-rounding matrix is likely quite porous, but being hydrophilic, is alsocompletely filled by water. This porous, hydrophilic, and water-filledmatrix would thus have a ‘reverse Gortex®’ effect (‘reverse’ in thesense that Gortex® is a hydrophobic matrix which blocks water migra-tion,whereasmeat gels are a hydrophilicmatrixwhich blocks oilmigra-tion). Therefore, liquid fat droplets enmeshed in such amatrix could not‘wet’ the smaller hydrophilic pores of the protein matrix, even if thesewere not filled with water by capillary pressure. Whether or not anIPF would tend to physically entrap droplets of liquid fat as has beenproposed bymany, it is certain that this hydrophilic, water-swollenma-trix of small (much smaller than the fat droplets) pores indeedwould do

76 W. Liu et al. / Meat Science 111 (2016) 67–77

so, irrespective of the influence of any IPF formed at the fat/water inter-face. Thus the characteristics of the protein gel matrix system that im-part a high water holding ability to the meat gel (small, hydrophilicpores) also in turn likely would suppress movement of liquid fat. Onlywhen the gel is so poorly constituted (presence of large pores orvoids) that water can flow freely from it would fat also likely leave thematrix.

Indeed, if the IPF alone was able to hold fat even against such a poorcapillary condition that allowed water to exit the matrix, then it wouldbe expected that some cooked batterswould exude prodigious amountsof water while still retaining most or all of their fat. This has never beenreported to occur however. In the present work, attempts to protect fatfrom migration within a coarse matrix by pre-emulsification to form astrong IPF (as has been suggested a basis for fat stability; Gordon &Barbut, 1990a) completely failed in this regard.

4. Conclusions

As expected for both non-fat and fat-containing gels, fracture stresssignificantly decreased with the increasing partial substitution of WPIfor MFP (P b 0.0001), but fracture stain was insignificantly affected bythe substitution (P N 0.05). Higher CL and EW seemed to correspondto presence of a more coarse gel structure, as did fat coalesce apparentby microscopy. This occurred despite the attempt to pre-emulsify fatwith MFP and stabilize it by formation of a strong IPF. Gel matrix char-acteristics clearly play a major role in determining WHA/FHA in fat-containing gels.

Fat-loss accompanied water loss during cooking, suggesting that themechanisms of fat- and water-holding are linked. We propose that theexplanation derives from the requirement for a fine porous gel matrixcapable of holding water by capillary pressure, which in turn also pre-sents a water-swollen and quite hydrophilic barrier to liquid fat move-ment during cooking, especially when lipid droplets are larger than thefine pores. Thus in effect not only water, but fat as well, is held withinthe gel matrix due to capillary pressure.

Acknowledgments

This project was supported by National Research Initiative Grant2008-02209 from the USDA Cooperative State Research, Educationand Extension Service. We wish to thank Dr. Eva Johannes of theNorth Carolina State University Laboratory for Cellular and MolecularImaging Facility (CMIF) for her support with Confocal Laser Scanningmicroscopy experiments, as well as Dr. JoAn Hudson from AdvanceMaterials Research Laboratories (AMRL), Clemson University for her as-sistance with the VP-SEM experiments.

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