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Vol. 69, Nr. 7, 2004—JOURNAL OF FOOD SCIENCE E303

E: Food Engineering & Physical Properties

A New Method for Characterizing Fiber Formation in Meat Analogs during High-moisture Extrusion

G. YAO, K.S. LIU, AND F. HSIEH

ABSTRACT: A noninvasive method based on fluorescence polarization spectroscopy was developed to measure the fiber formation of extruded meat analogs. Soy protein, wheat gluten, and unmodified wheat starch were mixed and extruded at high moisture conditions to form meat analogs with 60% to 72% moisture (w.b.). This newly developed method and a texture analyzer were used to analyze the fiber formation of extruded products as well as samples collected from different zones in the extruder and cooling die upon a dead-stop operation. The results indicated that the texture profile analysis could not adequately describe the fiber formation while the new method showed good agreements with results obtained from visual inspection and digital imaging of the dissected samples.

Keywords: fiber formation measurement, meat analogs, high-moisture extrusion, soy proteins, fluorescence po- larization spectroscopy

Introduction

T

here is increased interest in using twin-screw extruders to tex- turize vegetable proteins into fibrous meat alternatives under high moisture (40% to 80%) conditions (Cheftel and others 1992;

Thiebaud and others 1996; Lin and others 2000, 2002). Unlike low moisture extruded protein products, proteins extruded under high moisture conditions can have well defined fiber formations, resem- ble chicken or turkey breast meat, and therefore have enhanced visual appearance and taste sensation. The high-moisture extrusion process shows great promise for texturizing vegetable proteins that meet increasing consumer demands for healthy and tasty foods.

Many researchers have used texture profile analysis and micro- structure examination to investigate textured protein products produced under low moisture extrusion conditions (Breene and Barker 1975; Maurice and others 1976; Kazemzadeh and others 1986). Similar methods were also used for studying textured protein products extruded under high moisture conditions (Lin and others 2000, 2002). However, textural profiling and ultrastructural examination do not accurately describe textural characteristics of fibrous protein products made by high-moisture extrusion. Al- though a high-resolution camera can record the degree of fiber formation, the sample usually needs to be peeled or dissected to better reveal the fibrous structures. In addition, visual examination is subjective and does not provide a numeric index for accurate and convenient comparison among products.

Fluorescence spectroscopy–based techniques have been widely used in food science as a tool for food quality assessment (Swatland 1987; Wold and others 1999; Defour and others 2001; Skjervold and others 2003). By incorporating polarization measurement, fluorescence polarization spectroscopy has been a useful tool for molecular mobility measurement (Marangoni 1992; Lentz 1993; Wei and Herron

1993). Inoue and others (2002) used fluorescence polarization measurement to study the orientation of green fluorescence protein.

Gibson and Strauss (1989) developed a technique based on fluorescence polarization to study structural changes at the molecular levels in solid corn meal samples before and after extrusion. No information on fiber formation was derived from their measurements, however.

According to fluorescence polarization theory, when a polarized light excites the fluorescence substances in a sample, the polarization of the fluorescence light depends on the excited dipoles in the sample (Marangoni 1992; Inoue and others 2002). We hypothesize that the polarization degree of the fluorescence light can be used as an index or relative weight of structured components, which is the degree of fiber formation in products like meat analogs. The objective of this research is to develop a technique, based on fluorescence polarization spectroscopy, that can measure the degree of fiber formation objectively for products like meat analogs and to provide a numeric index for quality evaluation and comparison.

Materials and Methods

Materials

Soy protein isolate (Profam 974) was obtained from Archer Daniels Midland (Decatur, Ill., U.S.A.); wheat gluten and unmodified wheat starch (Midsol 50) was from MGP Ingredients, Inc. (Atch- ison, Kans., U.S.A.). These raw ingredients were blended at a ratio of 6:4:0.5, using an 18.9L Hobart Mixer (Hobart Corp., Troy, Ohio, U.S.A.) for 30 min to ensure the uniformity of the feeding material.

Extrusion

Extrusion was performed using a pilot-scale, co-rotating, inter- meshing, twin-screw food extruder (MPF 50/25, APV Baker Inc., Grand Rapids, Mich., U.S.A.) with a smooth barrel and a length-diameter ratio of 15:1. The clamshell style barrel is segmented into 5 temperature-controlled zones that are heated by an electric cartridge heating system and cooled with water. The barrel can be split hori- zontally and opened to enable rapid removal and cleaning of the MS 20040092 Submitted 2/17/04, Revised 4/7/04, Accepted 4/25/04. Authors

Yao and Hsieh are from Dept. of Biological Engineering, Univ. of Missouri, Columbia, MO 65211. Authors Liu and Hsieh are from Dept. of Food Sci- ence, Univ. of Missouri, Columbia, MO 65211. Direct inquiries to author Hsieh (E-mail: hsiehf@missouri.edu).

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E: Food Engineering & Physical Properties

barrel and the screws. The screws are built with screw elements and lobe-shaped paddles, which can be assembled on hexagon-shaped shafts to give different screw geometries. The screw profile com- prised of (from feed to exit): 100 mm, twin lead feed screw; 50 mm, 30°

forwarding paddles; 100 mm, single lead screw; 87.5 mm, forwarding paddles; 175 mm, single lead screw; 87.5 mm, forwarding paddles; 50 mm, 30° reversing paddles; and 100 mm, single lead screw.

A K-tron type T-35 twin screw volumetric feeder (K-tron Corp, Pit- man, N.J., U.S.A.) was used to feed the raw materials into the extruder at a feeding rate of 12 kg/h. While operating, water at ambient temperature was injected, via an inlet port, into the extruder by a posi- tive displacement pump with a 12-mm head. The inlet port was lo- cated on the top of the barrel, 0.108 m downstream from the feeding port. The pump was pre-calibrated and adjusted so that the extrudate moisture content would vary from 60% to 72%. The screw speed was set at 125 rpm. At the end of the extruder, a long cooling die was attached, with a dimension of 60 × 10 × 300 mm (W × H × L). Cold water (5 °C) was used as the cooling medium for the die. The extruder barrel temperatures were set at 25, 36, 100, 155, and 170 °C from the 1st (feeding zone) to the 5th zone, respectively. The extruder responses, including die pressure, percent torque, and product temperature before the cooling die, were recorded. Two sets of samples, 5 kg each, were collected for each treatment and immediately put into airtight plastic bags. One set was stored in a refrigerator at 4 °C and the other in a freezer at –18 °C. The refrigerated samples were used for measurement and analysis within 48 h. The frozen samples were intended to be used only as backup in case the refrigerated samples ran out, which was not the case in this study.

Dead-stop operation

One dead-stop extrusion run was conducted at the end of a run at the moisture level of 60.11%. At this moisture, products with well-defined fibrous structures were produced under the de- scribed extrusion conditions. The extrusion operation was inten- tionally shut down (dead-stop) after reaching steady state. The barrel was cooled using the maximum cooling capacity and opened immediately, and samples along the extruder barrel at each of the 5 zones and the cooling die and the extruded product, were collected. The sample from Zone 1 corresponded to the raw mixture. Zone 5 was the last zone adjacent to the cooling die.

Moisture and texture measurements

Sample moisture contents were determined by the official AOAC method, with minor modification, using a vacuum oven (AOAC 2000).

The texture profile analysis was conducted using a TA.XT2 analyzer following the method of Lin and others (2000). A cylindrical probe (25.4 mm in diameter) was used for the test, and a metal puncher was used to obtain cylindrical testing samples (about 10 mm in both diameter and thickness). Samples were compressed to 50% of their initial thickness. Five attributes were recorded: springiness, cohesiveness, gumminess, chewiness, and hardness. Data from 5 pieces of each treatment were collected and used in the analysis.

Visual examination and image recording by a digital camera

Samples were dissected by hand, peeling along the direction of fiber orientation. The dissected samples were examined visually for the degree of fiber formation. Their images were also taken by a high- resolution camera attached to a computer and recorded digitally.

Measurement by fluorescence polarization spectroscopy

The measurement setup is illustrated in Figure 1. An LED (1 mW

output, 375 nm wavelength) was used as the excitation light source.

The excitation light was collimated by a lens and polarized by a polarizer (P1). The fluorescence light from the sample passed through another polarizer P2 and was collected by a fiber probe and detected by a spectrometer (USB2000, Ocean Optics, Dunedin, Fla., U.S.A.).

A typical spectrum is shown in Figure 2. The peak at 375 nm is from the excitation light. The fluorescence emission is rather broad- band. The signal intensity at emission wavelength of 540 nm was recorded and processed in all our measurements.

The degree of polarization (P) (Kliger 1990) can be calculated as:

(1)

where I₉₀ is the fluorescence intensity measured when the polarizer P2 is at 90° with polarizer P1, and I₀ is the fluorescence intensity measured when the polarizer P2 is at 0° with polarizer P1. Two measurements are needed to calculate the P value. When the sample fiber orientation is aligned with the P1 direction, a higher P value will be obtained. During high-moisture extrusion, the sample fiber orientation is always aligned with the extrudate moving direction in the long cooling die. Therefore, the sample is mounted with its fi- bers’ orientation aligned with incident polarization direction.

Another index used in our study is the anisotropy index (N) that

Figure 1—Experimental setup for fluorescence polariza- tion measurements

Figure 2—A typical raw fluorescence spectrum from ex- truded products

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E: Food Engineering & Physical Properties

is defined as:

(2)

where P₉₀ is the polarization degree measured with the polarizer P1 at 90°, and P₀ is the fluorescence polarization degree measured with the polarizer P1 at 0°; i.e., rotating the polarization direction of the excitation light by 90°. In other words, 2 degrees of polarization values are measured with polarization direction of the excitation light parallel and perpendicular to the sample’s fiber orientation.

Results and Discussion

Extruder responses

The extruder responses during extrusion, including percent torque, die pressure, and product temperature before the cooling die, were recorded and reported in Table 1. As the moisture content in the extruded products increased, all 3 parameters increased.

This is readily comprehended because water functions as a lubri- cating agent. These extruder responses were similar to those reported by Lin and others (2000).

Fiber orientation by visual examination and digital imaging

Visual examinations were performed on peeled/dissected samples. The corresponding digital images were shown in Figures 3 and 4. All images were approximately 1.9 × 1.4 cm (W × H) in size. Among all the samples extruded under various levels of moisture contents while keeping other extrusion conditions the same, the one extruded at 60.11% (w.b.) showed the best and most well-defined fiber orientation. As the moisture content increased from 60.11% to 72.12%, the fiber formation became less defined (Figure 3).

For the samples obtained during a dead-stop run at 60.11%

moisture content, fiber formation did not occur until the last zone of the extruder barrel (Zone 5). It is interesting to note that the sample collected from the cooling die had the best fiber orientation by visual examination, even better than the extrudate shown in Figure 3a. The extended cooling after dead-stop operation might help continue the fiber formation process.

Texture profile analysis

The texture profile analysis results on extruded samples at different moisture contents are plotted in Figure 5. It can be seen that cohesiveness and springiness basically remained the same for all

the samples extruded at different moisture contents, whereas hardness, chewiness, and gumminess all decreased with increasing moisture contents of the extrudates. The textural profile data relat- ed little with real fiber formation because, based on visual examination, only samples extruded at 60.11% moisture had well-defined fiber orientation. The rest of the samples were paste-like products and lacked well-defined fibrous texture.

Fluorescence polarization

Figure 6 shows the polarization degree and the anisotropic index of extruded samples at different moisture contents. Multiple measurements at different sample sites were performed to calculate the polarization degree. The anisotropic index was calculated from 2 polarization degrees measured with 2 orthogonal incident polarization states (Eq. 2). The results indicated that the sample at 60.11%

moisture content had a much higher polarization degree and anisotropic index value than samples from higher moisture contents. This agrees very well with the imaging results (Figure 3) and visual examination. Furthermore, the anisotropic index appeared to be a more superior indicator of the fiber formation than the polarization degree.

The former showed greater differences among samples with different degrees of fiber formation and the latter showed one erroneous result: the polarization degree at 72.12% moisture was slightly higher than those at 64.31% and 66.78% moisture contents.

Measurements on samples obtained from a dead-stop operation of extrusion at 60.11% moisture were also conducted. The results clearly indicated that the cooling die sample had the best fibrous structure (Figure 7). According to the anisotropic index measurements (Figure 7b), the fiber formation was started at Zone 5, which was confirmed by the results from visual inspection and

Figure 3—Digital images of extruded products at 3 moisture contents

Table 1—The effect of moisture content on percent torque, die pressure, and product temperature before the cooling die

Moisture Die Product temp.

content Torq u e p r e s s u r e before the

(%, w.b.) (%) (kPa) cooling die (°C)

72.12 4.8 30.3 134.0

71.33 4.9 56.5 138.1

66.78 5.6 144.8 140.1

64.31 6.1 207.5 142.4

60.11 7.1 329.6 143.4

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E: Food Engineering & Physical Properties

digital imaging (Figure 4). The moisture contents of the samples were also plotted in Figure 7b. It appears that the fluorescence measurements were not affected by the sample moisture contents.

Figure 7 also confirms that the anisotropic index is a better indicator than the polarization degree. This is because the anisotropic index can compensate the effect of sample inhomogeneity. For inhomogeneous samples, there are sometimes localized irregular regions that can also produce high polarization degree values.

Samples of higher moisture content tend to be more affected by such an inhomogeneous problem as shown in Figure 6a. However, if the sample has no dominant structural orientation, such localized irregular regions have randomized distribution. If we rotate the incident polarization direction or the sample, we will obtain similar polarization degrees. In addition to being an indicator of the degree of fiber formation, the anisotropic index can also be an indicator of the sample’s inhomogeneity. When the sample is homogeneous and has a dominant structural orientation, it will have a high polarization degree and a high anisotropic index. If the sample is homogeneous and has no dominant structural orientation, it will have a low polarization degree and a low anisotropic index. If the sample is inhomogeneous and has no dominant structural orientation, it may have a moderate polarization degree with a small anisotropic index.

To verify that the new method can detect sample structure information, additional experiments were conducted. Two different samples were used. Sample-1 was extruded at 60.11% moisture content and had well-defined fiber orientation; sample-2 was extruded at 72.12% moisture content and lacked fibrous texture.

These samples were ground into pastes to destroy their fibrous structures and were measured for polarization degrees and anisotropic indices. The measured polarization degrees were 0.08 and 0.11 for ground sample-1 and sample-2, respectively; and the measured anisotropic indices were 0.1 for both ground samples. Obvi-

Figure 4—Digital images of samples collected at various zones and cooling die after a dead-stop run at 60.11% mois- ture content

ously, both polarization degree and anisotropy index of ground samples approached the values of raw material mixtures (Zone 1- 4 in Figure 7). In addition, those values of sample-1 were significant- ly less than that of the original chunk sample because the fibrous structures were destroyed. This clearly indicated that our measurements did reflect the sample structural properties.

Conclusions

F

iber formation of proteinaceous materials, such as a mixture of soy protein and wheat gluten, could be induced under high- moisture extrusion conditions. The moisture content had a pro- found effect on fiber formation. Texture profile analysis could not adequately describe the degree of fiber formation. A new method,

Figure 5—Textural profile analysis of extruded products at different moisture contents

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E: Food Engineering & Physical Properties

based on fluorescence polarization spectroscopy, was developed to assess the degree of fiber formation in extruded samples. Both polarization degree and anisotropy index can describe the degree of fiber formation of the extrudates, but the latter gave a better description and correlated better with actual fiber orientation based on visual examination and high-resolution imaging.

Acknowledgments

The authors thank Mr. Harold Huff and Mr. Jinjun Xia for their as- sistance during the experiments.

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Figure 6—(a) Polarization degree and (b) anisotropic index of samples collected from samples at various moisture contents

Figure 7—(a) Polarization degree and (b) anisotropic index of samples obtained from a dead-stop operation of extru- sion at 60.11% moisture content.