*Article* **Comprehensive SPME-GC-MS Analysis of VOC Profiles Obtained Following High-Temperature Heating of Pork Back Fat with Varying Boar Taint Intensities**

**Clément Burgeon 1,\*, Alice Markey 1, Marc Debliquy 2, Driss Lahem 3, Justine Rodriguez 2, Ahmadou Ly <sup>3</sup> and Marie-Laure Fauconnier <sup>1</sup>**


**Abstract:** Boar taint detection is a major concern for the pork industry. Currently, this taint is mainly detected through a sensory evaluation. However, little is known about the entire volatile organic compounds (VOCs) profile perceived by the assessor. Additionally, many research groups are working on the development of new rapid and reliable detection methods, which include the VOCs sensor-based methods. The latter are susceptible to sensor poisoning by interfering molecules produced during high-temperature heating of fat. Analyzing the VOC profiles obtained by solid phase microextraction gas chromatography–mass spectrometry (SPME-GC-MS) after incubation at 150 and 180 ◦C helps in the comprehension of the environment in which boar taint is perceived. Many similarities were observed between these temperatures; both profiles were rich in carboxylic acids and aldehydes. Through a principal component analysis (PCA) and analyses of variance (ANOVAs), differences were highlighted. Aldehydes such as (*E*,*E*)-nona-2,4-dienal exhibited higher concentrations at 150 ◦C, while heating at 180 ◦C resulted in significantly higher concentrations in fatty acids, several amide derivatives, and squalene. These differences stress the need for standardized parameters for sensory evaluation. Lastly, skatole and androstenone, the main compounds involved in boar taint, were perceived in the headspace at these temperatures but remained low (below 1 ppm). Higher temperature should be investigated to increase headspace concentrations provided that rigorous analyses of total VOC profiles are performed.

**Keywords:** back fat; boar taint; entire male pig; GC-MS; lipid oxidation; meat quality; pork meat; SPME; VOC

#### **1. Introduction**

Nowadays, a top priority for the pork industry is being able to correctly discriminate tainted from untainted boar carcasses. In fact, boar taint is a strong and unpleasant smell found in the meat of some uncastrated male pigs. This smell appears upon cooking of boar tainted meat and is due to the release of a complex mixture of molecules. The major molecules responsible for this smell are the steroid androstenone (5-α-androst-16-en-3-one) and the tryptophan metabolite skatole (3-methylindole), which are well-known for their urine and fecal smell, respectively [1,2].

To prevent the development of such molecules, surgical castration without pain relief has often been used worldwide given that it is a fast, cheap, and handy castration technique for farmers. However, this practice has been criticized for the pain and stress that it inflicts to piglets. Hence, alternatives to surgical castration have been suggested and

**Citation:** Burgeon, C.; Markey, A.; Debliquy, M.; Lahem, D.; Rodriguez, J.; Ly, A.; Fauconnier, M.-L. Comprehensive SPME-GC-MS Analysis of VOC Profiles Obtained Following High-Temperature Heating of Pork Back Fat with Varying Boar Taint Intensities. *Foods* **2021**, *10*, 1311. https://doi.org/10.3390/foods10061311

Academic Editor: Paulo Eduardo Sichetti Munekata

Received: 21 May 2021 Accepted: 1 June 2021 Published: 7 June 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

are now being promoted [3]. Out of all, two castration techniques appear more realistic: immunocastration (i.e., testicular functions are deactivated through the neutralization of the hypothalamic–pituitary–gonadal axis hormones [4]) and rearing of entire males. Whether it is to discriminate tainted uncastrated male pigs or simply to ensure that immunocastration has functioned correctly, the detection of tainted carcasses is an essential step in the slaughtering process.

Currently, many research studies are taking place to develop new detection methods that are ideally low cost (less than 1.30 euro/analysis), fast (less than 10 s/analysis), 100% specific and sensitive (no false negatives and no false positives), and automated [5]. These criteria are essential for methods to be used on the slaughter line.

Several detection principles have been investigated throughout the years [6]. Mass spectrometry-based methods have recently been examined and have shown interesting results. Rapid evaporative ionization mass spectrometry (REIMS) provided highly accurate classification of tainted and untainted samples at a fast speed and has shown its potential to be used for online applications given its hand-held sampling tool and estimated low cost [7]. Laser diode thermal desorption–tandem mass spectrometry (LDTD-MS/MS) has also been thoroughly investigated [8–10]. This method achieved good validation criteria, fast analysis (once sample preparation has been performed, analysis in itself takes less than 10 s/sample), and is currently being tested in a Danish slaughterhouse [11]. However, both methods would require substantial investment (expensive instruments and need for skilled staff), which could lead to reluctance in their application.

Other methods recently tested and presenting lower investment cost are devices based on Raman spectroscopy [12,13] and a new specific sensor system based on screen-printed carbon electrodes [14,15]. Additionally, these techniques are easy to use given the handheld measuring tool. However, both still need further validation given high prediction errors for Raman spectroscopy and the absence of real slaughterhouse testing with the sensor system.

The rapid detection of boar taint through volatile organic compounds (VOCs) detection has also been widely studied. Some researchers have tried using gas chromatography mass spectrometry (GC-MS) for this purpose [16,17]. However, the high initial investment and long (i.e., minimum 3.5 min [16]) analysis time remained two main drawbacks of GC-MS methods.

Boar taint detection through the use of e-noses has been extensively studied some years ago [18–22]. An e-nose is composed of an array of sensors for which a response is induced when gases, and in this case VOCs, are perceived at their surface. In a recent review by Burgeon et al. (2021) [6], the great potential of new sensor material for skatole and androstenone has been discussed, and this review concluded that sensor-based methods might be a solution for the rapid slaughterhouse detection of boar taint provided that it is able to detect low headspace concentrations of skatole and androstenone in a VOCsrich environment. This working environment is due to the extraction conditions used to volatilize skatole and androstenone.

In fact, skatole and androstenone are lipophilic molecules with low vapor pressure (7.3 × <sup>10</sup>−<sup>4</sup> kPa and 1.3 × <sup>10</sup>−<sup>6</sup> kPa at 25 ◦C, respectively); hence, fat must be heated at high temperatures to allow the volatilization of these molecules. This heating leads to the release of a variety of molecules. Most of these molecules are products of lipids degradation (oxidation of fatty acids starting at 70 ◦C [23]). Lipids can oxidize in three main ways: autoxidation, enzymatic-catalyzed oxidation, and photo-oxidation. However, the most probable oxidation mechanism during fat heating remains autoxidation where the unsaturated fatty acids react with oxygen, which is activated by temperature in this case, to produce free radicals. These free radicals are unstable and therefore decompose to form various molecules, including acids, alcohols, esters, ketones hydrocarbons, and aldehydes. The latter are present in significant quantities in products that underwent oxidation processes [24].

Such a VOCs-rich environment can quickly lead to sensor poisoning, i.e., binding of VOCs to the sensor's surface, and in turn lead to temporal sensor drift. Such drift is defined as the gradual deviation of the sensor's response when exposed to the same molecule in the same environment [25]. Understanding the VOCs environment in which the volatilization of skatole and androstenone takes place is primordial, as this could help in creating new drift-reduction solutions, which are physical solutions (such as filters) aiming to reduce interfering VOCs present in the headspace but also creating more robust drift correction models taking such environments into account.

Until now, none of the above-mentioned methods have stood out compared to the others, and that is why current slaughterhouse boar taint detection is still performed either through a colorimetric method [26] or mainly by sensory evaluation [27].

Hence, the objective of this research was to examine elevated temperature VOC profiles to facilitate new sensor development, gain the understanding of VOCs perceived during boar taint sensory evaluations, and lastly help in understanding which VOCs perceived by the consumers during the cooking of pork meat are lipid-derived. The 150 and 180 ◦C temperatures were used in the current study, as they are frequently encountered for sensory evaluation in the frame of boar taint detection [28–32] and appear in the range of temperatures used for cooking by consumers [33].

Rius et al. (2005) [34] have already analyzed VOCs produced when heating fat at a temperature of 120 ◦C. However, only back fat with low concentrations in skatole and androstenone was analyzed, and comparisons of heating temperatures were not performed.

To the best of our knowledge, our study is the first providing a thorough understanding and comparisons of VOC profiles obtained following the heating of sow fat as well as tainted and untainted boar fat at two elevated temperatures (150 and 180 ◦C) and sampling and analysis by solid phase microextraction gas chromatography–mass spectrometry (SPME-GC-MS).

#### **2. Materials and Methods**

#### *2.1. Samples*

Sow back fat (*n* = 6), tainted (*n* = 7) and untainted boar fat (*n* = 7) were collected from a local slaughterhouse. Sow fat was randomly selected. Tainted and untainted boar fat, on the other hand, were selected after these had been checked for boar taint by a trained assessor through an online human nose detection method (soldering iron). The collected samples were frozen at −20 ◦C at the slaughterhouse, transported in a cooler, and stored again at −20 ◦C. The presence or absence of boar taint was confirmed through the quantification of skatole and androstenone in fat by high-performance liquid chromatography fluorescence detection (HPLC-FD), which is described later in this section.

#### *2.2. Chemicals*

Methanol (CAS n◦ 67-56-1, HPLC grade, Sigma-Aldrich, Darmstadt, Germany), dansylhydrazine (CAS n◦ 33008-06-9, Sigma-Aldrich, Darmstadt, Germany ), boron trifluoride (BF3) at 20% in methanol *v/v* (CAS n◦ 373-57-9, VWR, Darmstadt, Germany), phosphoric acid (H3PO4) (CAS n◦ 7664-38-2, Sigma-Aldrich, Darmstadt, Germany), acetonitrile (CAS n◦ 75-05-8, HPLC grade, Supelco, Darmstadt, Germany), tetrahydrofuran (CAS n◦ 109-99-9, HPLC grade, Supelco, Darmstadt, Germany), liquid nitrogen (CAS n◦ 7727-37-9, Nippon Gases, Schoten, Belgium), 2,3-dimethylindole (CAS n◦91-55-4, Sigma Aldrich, Darmstadt, Germany), skatole (CAS n◦ 83-34-1, Sigma Aldrich, Darmstadt, Germany), and androstenone (CAS n◦ 18339-16-7, Sigma Aldrich, Darmstadt, Germany ) were used in this experiment.

#### *2.3. Skatole and Androstenone Quantification in Back Fat*

This analysis allowed quantifying the skatole and androstenone content in both tainted and untainted boar fat samples. Boar fat is considered tainted if skatole concentrations are above the thresholds of 200 ng g−<sup>1</sup> of fat and/or above 1000 ng g−<sup>1</sup> for androstenone. These thresholds were selected given that the commonly accepted threshold generally range from 200 to 250 ng g−<sup>1</sup> of fat for skatole and 500 to 1000 ng g−<sup>1</sup> for androstenone [35]. Quantification of these molecules in back fat was performed on the basis of a method by Hansen-Moller (1994) [36], which consists of a methanolic extraction of the molecules, derivatization of androstenone, and analysis by high-performance liquid chromatography fluorescence detection (HPLC-FD). This protocol was slightly adapted as described in this section.

#### 2.3.1. Extraction of Androstenone and Skatole

Two mL of methanol was added to 0.50 g of back fat cut into pieces (0.5 cm square). The sample was homogenized by an Ultra-Turrax T25 (Janke & Kunkel, Straufen, Germany) for 30 s at 13,500 rpm. Then, 500 μL of methanol was added, and the sample was homogenized again for 30 s with the Ultra-Turrax; finally, 500 μL of methanol was added and homogenized for 1 min with the Ultra-Turrax. The sample was ultrasonicated for 5 min and placed in an ice bath for 15 min before centrifugation at 7700 rpm at 4 ◦C. Then, the supernatant was passed through a 0.45 μm filter paper (Whatmann, Darmstadt, Germany), and 140 μL was put in vial for analysis.

#### 2.3.2. Derivatization

The autosampler was programmed to mix 30 μL of 2% dansylhydrazine in methanol, 4.4 μL of water, and 10 μL of 20% *v/v* BF3 with 140 μL of methanolic extract. A reaction time of 5 min was observed; then, 20 μL of the incubated sample was injected into HPLC.

#### 2.3.3. High-Performance Liquid Chromatography Fluorescence Detection (HPLC-FD)

The analysis was performed by HPLC (1260 Infinity, Agilent Technologies, Santa Clara, CA, USA) with a kinetex column EVO C18 100 A (150 × 3.0 mm × 5 μm, Phenomenex, Utrecht, Belgium) and a precolumn AJO-9297, EVO C18 (Phenomenex, Utrecht, Belgium). The solutions for the mobile phase are prepared as follows: (A) H3PO4/deionized water (1:1000 *v/v*); (B) acetonitrile; (C) THF/deionized water (99:1 *v/v*). The elution gradient profile runs as presented in Table 1. The mobile phase was pumped at a flow rate of 0.5 mL min−<sup>1</sup> throughout the process.

**Table 1.** Elution gradient for the separation of skatole and androstenone on an High-Performance Liquid Chromatography Fluorescence Detection (HPLC) system.


The detection with a fluorescence detector (FD) (Agilent Infinity 1260) was performed with an excitation wavelength of 285 nm and emission wavelength of 340 nm for skatole and 346 nm for excitation and 521 nm for emission of androstenone. The wavelength change takes place after 12 min of elution.

#### 2.3.4. Quantification of Skatole and Androstenone

Quantification of skatole and androstenone was made possible with matrix-matched calibration curves. These were prepared with sow fat (very low concentrations in skatole and absence of androstenone) that had been previously spiked with standards solutions. Calibrations curves were prepared for concentrations ranging from 45 to 500 ng/g for skatole and from 240 to 5000 ng/g for androstenone.

#### *2.4. Analysis of VOCs Found in the Headspace of Heated Back Fat Samples*

VOC profiles were established following 6 different analyses (i.e., 6 modalities): heating of sow fat at 150 ◦C, untainted boar fat at 150 ◦C, tainted boar fat at 150 ◦C, sow fat at 180 ◦C, untainted boar fat at 180 ◦C, and tainted boar fat at 180 ◦C. The analyses were performed as described in this section.

#### 2.4.1. Sample Preparation

First, 2.5 g of back fat was cut and then cooled by adding liquid nitrogen (−196 ◦C). The sample is ground for 5 s with an A11 basic IKA analytical grinder. Before recovering 1.0 g of sample in a vial, liquid nitrogen is added to the sample to freeze it. The sample is stored at −20 ◦C until analysis.

#### 2.4.2. SPME-GC-MS VOCs Analysis

Before proceeding to the headspace solid phase microextraction GC-MS analysis (HS-SPME-GC-MS), 1 μL of 2,3-dimethylindole at 125 μg mL−<sup>1</sup> in methanol is added on the inside of the 20 mL vial, which is immediately sealed with a magnetic screw cap with a PTFE septum (Sigma-Aldrich, Darmstadt, Germany).

Incubation of the sample takes place at 150 ◦C (for the first analysis) or 180 ◦C (for the second analysis) for 20 min in a heated agitator (Gerstel, Mülheim an der Ruhr, Germany). Then, sampling of VOCs was achieved with a divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm) SPME fiber (Supelco, Darmstadt, Germany) through a 5 min exposition in the headspace. The vials were shaken at 250 rpm (agitator on/off time: 10 s/1 s) during incubation and extraction. Desorption of the extracted and captured VOCs takes place for 2 min. Injection was performed in splitless mode at 270 ◦C. The fiber was conditioned for 20 min at injection temperature. Analyses were performed by GC-MS (7890A-5975C, Agilent Technologies, Santa Clara, CA, USA ) equipped with an HP-5 MS capillary column (30 m × 250 μm × 0.25 μm, Agilent Technologies, Santa Clara, CA, USA). Helium was used as a carrier gas at a flow rate of 1.2 mL/min. The oven temperature program was as follows: starting at 40 ◦C with a hold for 3 min; then, there is an increase of 5 ◦C/min up to 300 ◦C with a hold for 5 min. The mass spectrometer was set to have a temperature of 230 ◦C at the ion source and 150 ◦C at the quadrupole. The mass spectrometer was programmed with a SCAN/SIM acquisition mode. In SIM mode, the targeted ions were (quantitative ions in bold): 77, 103, and **130** for skatole; 130 and **144** for 2,3-dimethyl-indole; and lastly, 239, 257, and **272** for androstenone. The SIM mode allowed for semi-quantification of skatole and androstenone in the headspace using the following formula:

Target (ppb) = (area target quant. ion/area I.S. quant. ion) × mass of I.S. × (1/vial volume) × correction factor. (1)

The correction factor corresponds to 2.5 for skatole and 1/34 for androstenone; IS corresponds to the internal standard, i.e., 2,3-dimethylindole in this case.

In SCAN mode, mass spectra were scanned from 35 to 500 amu. Then, component identification was performed by comparison of the obtained spectra with mass spectra in a reference database (NIST17). Additionally, experimental retention indices (RI) were calculated following the injection of a mixture of n-alkanes C8-C30 (Sigma Aldrich, Darmstadt, Germany) under the same chromatographic conditions as those previously mentioned. This allowed the comparison of these RI to literature RI. Lastly, pure standards were injected for skatole (CAS n◦ 83-34-1, Sigma Aldrich) and androstenone (CAS n◦ 18339-16-7, Sigma Aldrich, Darmstadt, Germany) to ensure identification [37–39].

#### *2.5. Data Analysis*

General VOC profiles were established through a chromatographic deconvolution process (Agilent MassHunter Unknowns Analysis), and chromatographic areas were obtained for each VOC. Then, these results were used in two different ways. In the first case, they were first reported as a percentage of the total chromatographic area to allow a general analysis. General linear models (GLMs) were performed on these data to validate some observations. Fat type was used as a fixed factor and incubation temperature was used as a covariate for GLM. In the second case, the chromatographic area data were massnormalized, auto-scaled, and log-transformed (generalized logarithm transformation) to obtain a distribution of the variables closer to normal and make results more comparable. Then, a principal component analysis (PCA) as well as a heatmap were generated with these normalized data. One-way analyses of variance (ANOVAs) were performed on the normalized data of the top 25 contributors to the differences observed. The PCA and heatmap were carried out on metaboanalyst [40]. Pearson correlation coefficients were determined for the skatole and androstenone headspace and content concentrations data. These coefficients as well as GLMs mentioned earlier were established with the Minitab 19 software (Minitab Inc., State College, PA, USA). Headspace/content correlation plots for skatole and androstenone were performed on Excel (Microsoft Office 2016).

#### **3. Results and Discussion**

In this section, results concerning the analysis of VOC profiles obtained with the high incubation temperatures used, i.e., 150 and 180 ◦C, will first be examined. Fat can be heated even more for boar taint detection; however, lipid oxidation occurs at a greater extent in this case. Therefore, in this research, 150 and 180 ◦C were studied, as it seemed to be a compromise between high temperature for the extraction of skatole and androstenone and minimization of lipid oxidation and the creation of degradation products, which could potentially interfere with the detection of boar taint compounds and saturate the sensors in the case of e-noses. Thus, the detection of skatole and androstenone in the headspace will be examined in the next section.

#### *3.1. VOC Profiles Generated through High-Temperature Incubation of Fat*

#### 3.1.1. General Understanding of the Generated VOC Profiles

A total of 48 compounds were correctly identified overall in fat samples regardless of their taint (Table 2). The profiles are composed of a large diversity of molecules including, amongst others, alcohols, aldehydes, furanes, and pyridine derivatives. Although some common characteristics are observed between the six different types of profiles obtained, some differences are also observed. These mainly exist between the heating temperatures rather than between the fat types.




#### *Foods* **2021** , *10*, 1311

**Table 2.** *Cont.*


**Table 2.** *Cont.*

In fact, it can be observed from Table 2 that the major group of compounds identified is not the same at 150 and 180 ◦C. Aldehydes are the most abundant at 150 ◦C ranging from 40.09% of the total profile for tainted fats to 55.00% for sow fats compared to much lower percentages of aldehydes at 180 ◦C, ranging from 19.09% for untainted fat to 26.81% for tainted fats (effect of temperature: *p*-value < 0.05). Amongst these aldehydes, some are present in much greater quantities compared to others. These include (*E*)-Dec-2-enal, (*E*)-Undec-2-enal, (*E*,*E*)-Hepta-2,4-dienal, and (*E*,*E*)-Deca-2,4-dienal, the latter accounting for up to 16.34% of the total profile in the case of sow fat.

On the other hand, the fatty acids group is the most present at 180 ◦C, making up 52.88% to 63.14% of the total profile at this temperature. Three fatty acids stand out: octadec-9-enoic acid (up to 31.93% of the total profile), hexadecanoic acid (up to 15.85%), and lastly, octadecanoic acid (up to 20.7%). Finding these three molecules as the most abundant fatty acids is in accordance with what has been found by Zhao et al. (2017) [41], who analyzed VOCs of stewed pork broth by solvent-assisted flavor evaporation (SAFE) combined with GC-MS.

Additionally, observing octadec-9-enoic acid and hexadecanoic acid as two of the three major fatty acids in the VOCs profile corresponds to the actual fatty acids content of back fat. In fact, several studies have analyzed the fatty acid composition of back fat and have found that the most abundant was octadec-9-enoic acid followed by hexadecanoic acid [42,43]. The hydrolysis of triglycerides into free fatty acids (FFAs) and glycerol is controlled by two main lipolytic enzymes: adipose triacylglycerol lipase (ATGL) regulating the hydrolysis of triacylglycerols into diacylglycerols and FFAs and hormone-sensitive lipase (HSL) regulating that of diacylglycerols into monoacylglycerols, FFAs, and glycerols [44]. Therefore, this explains the presence of FFAs in back fat.

Regarding their presence in the headspace, one must remember that such long-chain fatty acids possess low vapor pressures (e.g., octadec-9-enoic acid has a vapor pressure of 5.46 × <sup>10</sup>−<sup>7</sup> mm Hg at 25 ◦C [45]); therefore, greater incubation temperatures lead to greater headspace concentrations of these FFAs. With temperatures increasing from 150 to 180 ◦C, it can be seen from the data that the total acids found in the headspace increase for all three fat types (*p* < 0.05).

Serra et al. (2004) [42] and Rius et al. (2005) [34] who have also analyzed VOCs obtained following incubation of fat observed that aldehydes were the most abundant class of molecules, making up respectively 37.1% and 69.61% of the total VOC profiles. However, lower incubation temperatures (60 and 120 ◦C) were used in their study, which could explain the smaller volatilization of FFAs and hence the smaller relative abundance of these in their VOC profiles. Seeing that the total aldehydes percentage in the 180 ◦C profiles is lower is simply due to the fact that more volatiles are being released at 180 ◦C compared to 150 ◦C.

As observed in Table 2, the majority of aldehydes present are unsaturated, which is explained by the higher proportions of unsaturated fatty acids than saturated fatty acids in pork back fat [46]. The most abundant aldehydes are (*E*,*E*)-deca-2,4-dienal and (*E*,*E*)-hepta-2,4-dienal, which are VOCs produced following the oxidation of linoleic acid and linolenic acid, respectively, and which are known to have a fatty and fried smell [24,41,47,48]. Benzaldehyde has also been found to originate from linolenic acid degradation [49].

In smaller proportions are ketones and alcohol. The alcohols detected at these temperatures correspond to those that have been found by Rius et al. (2005) [34] at 120 ◦C. On the other hand, two of the three ketones (pentadecan-2-one and heptadecan-2-one) observed in our study have not been observed by the latter. However, Zhao et al. (2017) [41] have found pentadecan-2-one as part of the VOCs found in pork broth.

Furans have also been found in the profiles. Furans are well-known to be responsible for the characteristic odor of fried foodstuffs. These molecules are found in a multitude of food products, including meat products [50,51].

#### 3.1.2. Understanding the Differences between the VOC Profiles Generated

Principal component analysis (PCA) was used to better visualize existing differences or groupings between the samples analyzed. Figure 1 represents a PCA scores plot of the first two principal components (PCs) of the VOC profiles dataset. Therefore, in this PCA scores plot, each sample analyzed is represented based on their respective VOC profiles. The first principal component (PC 1) explains 22.4% of the variation in the dataset, while the second principal component (PC 2) explains 7.2% of the variation. In this figure, the samples that are close to each other have similar VOC profiles. Therefore, the clear overlap of sow fat, tainted boar fat, and untainted boar fat respectively at 150 and 180 ◦C suggests that no net distinction is perceived between the VOC profiles of these three fat types. However, although a slight overlap is observed between the samples at 150 ◦C and those at 180 ◦C, a separation exists between the VOC profiles obtained following fat incubation at 150 and 180 ◦C. This suggests, as expected, that temperature has an impact on the generated VOCs. The molecules majorly responsible for the differences observed between the temperatures are described later in this section (Figure 2).

**Figure 1.** Principal component analysis (PCA) scores plot of component area normalized data of VOC profiles. Red and green dots indicate VOC profiles obtained for sow fat heated at 150 ◦C (*n* = 6) and 180 ◦C (*n* = 6), dark blue and light blue represent VOC profiles for tainted boar fat heated at 150 ◦C (*n* = 7) and 180 ◦C (*n* = 7); lastly, pink and yellow dots represent untainted fats at 150 ◦C (*n* = 7) and 180 ◦C (*n* = 7) respectively.

**Figure 2.** Heatmap generated with normalized data for the top 25 molecules responsible for the differences between the profiles. Each column corresponds to a studied modality. Red and green squares indicate VOC profiles obtained for sow fat heated at 150 and 180 ◦C, dark blue and light blue represent VOC profiles for tainted boar fat heated at 150 and 180 ◦C, and lastly, pink and yellow dots represent untainted fats at 150 and 180 ◦C, respectively. Results of ANOVAs are represented after the molecule name: NS indicates a *p*-value > 0.05 while \*, \*\*, \*\*\* indicate *p*-values < 0.05, <0.01, and <0.001 respectively.

As a reminder, the general VOC profiles have been established based on an untargeted approach following the detection of molecules in SCAN mode. Hence, skatole and androstenone semi-quantified following SIM mode detection (addressed in the next section) have not been included in the PCA data. Several other molecules have been suggested in the literature as responsible for boar taint. These include indole, 4-phenyl but-3-en-2 one, styrene, 1,4-dichlorobenzene, 2-aminoacetophenone, 5-α-androst-16-en-3-α-ol, and 5-α-androst-16-en-3-β-ol [2,34,52,53]. However, these molecules were not observed in the SCAN data, and no targeted approach (such as the use of the SIM mode) was used to attempt to detect them. Hence, this partially explains the overlapping of tainted and untainted fats. Additionally, although these molecules are not detected here in SCAN mode due to very low analytical concentrations, these still impact sensory evaluation as they may be detected by the human. The concept of odor activity values (OAVs) is very important in such analysis. This one considers the concentration of a compound in the food matrix and its odor threshold. OAV values greater than 1 are considered to be main contributors to the total flavor [54,55]. The OAV in fat of several molecules introduced above have been studied by Gerlach et al. (2018) [56]. For example, they have found that androstenone has an OAV of 25 and skatole has an OAV of 40 in boar fat. On the other hand, (*E*,*E*)-deca-2,4-dienal, the most present aldehyde in our study, only had an OAV of 1. This suggests that although this molecule is present in high concentrations in our study (Table 2), it only minorly impacts sensory evaluation compared to boar taint compounds.

The interpretation of the molecules responsible for the difference between the two temperatures is eased through the elaboration of a heatmap (Figure 2). The level of significance of the differences observed can be observed after the molecule name. From the latter, it appears that significant differences exist for 23 of the 25 molecules most responsible for the differences perceived.

As mentioned earlier and as confirmed by this figure, it can be noticed that overall, the differences mainly reside between profiles at the different temperatures. The VOCs' intensities are very different from one temperature to another and imply that assessors performing sensory evaluation at different temperature are not confined to the same working environment. This could lead to different results for the same sample. This stresses the importance of standardizing sensory evaluation protocols, from the training of the assessors to the evaluation per se performed in the slaughterhouse [27,57,58].

Some molecules are present in significantly higher concentrations in the headspace of fat heated at 150 ◦C compared to 180 ◦C. For example, this is the case for the aldehydes such as (*E*)-non-2-enal, (*E*)-undec-2-enal, and (*E*,*E*)-nona-2,4-dienal. As mentioned earlier, these molecules are secondary oxidation products of fatty acids. In meat, these molecules can further react. For example, the molecule (*E*,*E*)-deca-2,4-dienal can react with ammonia to produce 2-pentyl-pyridine [46]. Ammonia usually originates from the Strecker degradation of cysteine, which is an amino acid frequently found in the meat [59]. Careful attention was paid when sampling the fat before homogenization; however, the potential traces of muscle (Longissimus dorsi) in the sample cannot be excluded. The acceleration of such a reaction at high temperatures could explain the smaller headspace concentrations of (*E*,*E*)-deca-2,4-dienal in samples at 180 ◦C (Figure 2).

To the exception of octadec-9-enamide in the specific case of sow fat, the fatty amides, hexadecanamide, octadecanamide, and octadec-9-enamide, which are present in the 180 ◦C profiles, are simply absent from the profiles at 150 ◦C. Such amides have been obtained in several studies on the pyrolysis of meat products for waste management [60,61], hence demonstrating the implication of high temperatures in their production. These molecules are not simple degradation products of fatty acids and suggest once again the presence of small concentrations of proteins in the fat sample [60].

Another molecule present only in profiles at 180 ◦C is squalene. This one also has a low vapor pressure (6.3 × <sup>10</sup>−<sup>6</sup> mmHg at 25 ◦C), which explains its presence only at the higher temperature. Finding squalene in the three types of fats at this temperature is explained by the fact that squalene affects cholesterol production, which in turn affects the production of the steroid pregnenolone. All steroids and hence both androgens and estrogens in male and female pigs are produced starting from pregnenolone [53].

#### *3.2. Detection of Skatole and Androstenone in the Headspace of Tainted and Untainted Boar Fat*

Detection of the ions used for both qualification and semi-quantification of skatole and androstenone in boar fat was possible at both temperatures (Figure 3).

**Figure 3.** Detection of ions in selection ion monitoring mode (SIM mode) for samples incubated at 150 ◦C. Quantitative ions for semi-quantification of (**a**) skatole (*m/z* 130, peak at Rt = 23.096 min) and (**b**) androstenone (*m/z* 272, peak at Rt = 41.233 min).

Additionally, positive correlation coefficients higher than 0.77 are observed between the content (Table S1 for skatole and androstenone back fat content) and headspace concentrations of skatole and androstenone at both 150 and 180 ◦C (Figure 4).

**Figure 4.** Correlation plots between headspace concentrations (ppb) and content concentrations (ng/g) of skatole (**a**) and androstenone (**b**) respectively at 150 ◦C (in blue, *n* = 7) and 180 ◦C (in orange, *n* = 7). For the content, values below the linearity range were set at 0 arbitrarily. Pearson correlation coefficients as well as their significance levels are represented on the graph. \*\*, and \*\*\* indicate *p*-values < 0.01, and <0.001, respectively.

It can be observed from the skatole correlation plots that trends between content and emissions are similar at 150 and 180 ◦C, which therefore suggests that the skatole extraction yield and subsequently the concentrations perceived in the headspace are the same at these temperatures. To increase the headspace concentrations in skatole, several solutions exist. Amongst these solutions, a large increase in temperature, a reduction of the headspace volume, or simply the heating of bigger samples (that hence have a greater absolute quantity in skatole) could be considered. However, as mentioned earlier, in both cases, the VOCs profile will be rich in many other molecules, which might affect the response of the boar taint detection method used (saturation of the assessor's nose in the case of sensory evaluation and sensor drift for sensor-based methods).

Different results appear for androstenone correlation plots. In fact, it can be seen from Figure 4b that more androstenone is emitted with increasing temperature. This can in part be explained by the fact that androstenone has a lower vapor pressure and hence a smaller tendency to volatilize compared to skatole, thus leading to better androstenone extractions at higher temperatures.

Low headspace concentrations (maximum below 250 ppb for skatole and 700 ppb for androstenone) could in part be explained by the strong matrix effects observed with boar fat. Given the lipophilic character of skatole and androstenone, an efficient extraction process is often used prior to analytical determinations of skatole and androstenone. Sample preparation usually begins with a heating or homogenization step followed by an extraction and purification step prior to analysis. Additionally, measurements are often performed based on liquefied fat from which connective tissues have been removed (only 60% of the adipose tissue is constituted of fat per se) [62]. Various methods have been developed in the last decade to quantify skatole and androstenone content based on headspace analysis. As it is the case in our study, these researchers have only incubated fat at high temperatures prior to quantification of the boar taint compound. However, to compensate for matrix effects and subsequent low analyte extraction, internal standards were spiked directly in the liquefied fat [63,64]. This procedure was not performed in our study, as we wanted to determine real headspace concentrations of skatole and androstenone, justifying the injection of the internal standard 2,3-dimethylindole directly in the headspace of the vial. It is important to note that what is perceived by the sensory assessor in the slaughterhouse or

by the consumer when eating pork are compounds that are present in gaseous form in the headspace. In fact, VOCs can reach the nasal cavity either through orthonasal olfaction (direct inhalation in front of the nose) or through the throat while chewing (retronasal olfaction) [65].

The positive and significant Pearson correlation coefficients between the headspace concentration and the content for skatole and androstenone indicate that what is found in the headspace at both temperatures is a good representation of boar taint in the fat. Although extraction is similar for skatole at 150 and 180 ◦C (typical cooking temperatures of pork), the greater headspace concentrations for androstenone at 180 ◦C imply that higher temperatures allow a better representation of boar taint. One must remember that boar taint is a complex smell composed of a large variety of molecules. Only 33% of boar taint is explained by skatole alone, while 50% of the taint is explained by the combination of skatole and androstenone [66]. Whether it is for sensory evaluation or for sensor-based methods focusing on the detection of skatole and androstenone, using higher temperatures to detect greater amounts of androstenone should allow a better visualization of boar taint as a whole. Lastly, the headspace concentrations are low for sensor-based methods (which often operate in up to the ppm [67]) and hence emphasize the importance of testing even higher temperatures.

#### **4. Conclusions**

To the best of our knowledge, this study is the first analyzing VOCs emitted by back fat samples when heated at elevated temperatures. The aim of this study was to perform a general analysis of VOC profiles obtained with fat presenting different boar taint intensities, but being an exploratory study, it did not intend to rigorously compare the impact of different taint combinations on the emitted VOCs. As a reminder, the comprehension of the VOC profiles at these typical cooking temperatures was primordial to understand what composes the exact smell perceived during the sensory evaluation of boar taint at these temperatures and secondly understand whether VOCs sensor-based methods for boar taint detection at these temperatures can be developed.

Great differences were observed between the VOC profiles depending on the incubation temperature. Different VOC profiles might result in differences in classification of the same tainted and untainted fats when heated at different temperatures. Therefore, this stresses the need to develop and use a standardized method for the sensory evaluation of boar taint.

VOCs sensors for skatole and androstenone detection could be developed for incubation temperatures of 150 and 180 ◦C given that both molecules are found in the headspace. However, the low headspace concentration observed for both these molecules should encourage further research into higher incubation temperatures. Analyses of the general VOCs headspace should always complement research into skatole and androstenone detection as the complexity of the VOCs profile might increase with temperature. The impact of fatty acids and aldehydes (as these are the most abundant in the VOC profiles at both temperatures) should be tested on sensor material to determine the rate at which sensor drift occurs to elaborate more robust drift correction algorithms and finally determine after how many analyses the sensors should be disposed of. Solutions to reduce the development of products from lipid oxidation, such as working in a closed and controlled environment, should be further looked into for sensor development.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/foods10061311/s1, Table S1: Quantification of skatole and androstenone in fat determined by HPLC-FD. The mention < LR indicates that the content is below the linearity range (45 to 500 ng/g for skatole and 240 to 5000 ng/g for androstenone).

**Author Contributions:** Conceptualization, C.B., A.M. and M.-L.F.; methodology, C.B., A.M. and M.-L.F.; software, C.B., A.M. and M.-L.F.; validation, C.B., A.M., M.D., D.L., J.R., A.L. and M.-L.F.; formal analysis, C.B., A.M. and M.-L.F.; investigation, C.B. and A.M.; resources, C.B., A.M. and

M.-L.F.; data curation, C.B., A.M.; writing—original draft preparation, C.B., A.M.; writing—review and editing, M.D., D.L., J.R., A.L. and M.-L.F.; visualization, C.B. and A.M.; supervision, M.-L.F.; project administration, M.-L.F., D.L. and M.D.; funding acquisition, M.-L.F. and M.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the European Regional Development Fund (ERDF) and the Walloon Region of Belgium, through the Interreg V France-Wallonie-Vlaanderen program, under the PATHACOV project (No. 1.1.297); and the Micro+ project co-funded by the ERDF and Wallonia, Belgium (No. 675781-642409). This article was written within the framework of the AGROSENSOR project, which is part of the "Pole de compétitivité WAGRALIM," and was financially supported by the "Service public de Wallonie" (SPW).

**Data Availability Statement:** Data will be available upon request from the corresponding author.

**Acknowledgments:** We would like to thank Franck Michels, Thomas Bertrand and Danny Trisman for their technical contribution in this work.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Quality Characteristics of Beef Patties Prepared with Octenyl-Succinylated (Osan) Starch**

**Mohamed F. Eshag Osman, Abdellatif A. Mohamed \*, Mohammed S. Alamri, Isam Ali Mohamed Ahmed, Shahzad Hussain, Mohamed I. Ibraheem and Akram A. Qasem**

> Department of Food Science and Nutrition, King Saud University, Riyadh 1145, Saudi Arabia; moh.fareed77@yahoo.com (M.F.E.O.); msalamri@ksu.edu.sa (M.S.A.); iali@ksu.edu.sa (I.A.M.A.); shhussain@ksu.edu.sa (S.H.); mfadol@ksu.edu.sa (M.I.I.); aqasem@ksu.edu.sa (A.A.Q.) **\*** Correspondence: abdmohamed@ksu.edu.sa

**Abstract:** Octenyl-succinylated corn starch (Osan) was used to improve the physicochemical properties of ground beef patties. The study involved incorporation of 5 and 15% Osan and storage for 30 or 60 days at −20 ◦C. The tested parameters included cooking loss, microstructure image, firmness, color, and sensory evaluation of the prepared patties. Along with Osan, native corn starch was used as control and considered the patties with added animal fat. The data showed that Osan reduced the cooking loss and dimensional shrinkage significantly (*p* < 0.05), whereas the moisture retention, firmness and color of beef patties were improved. The sensory evaluation indicated enhanced tenderness and juiciness without significant alteration of flavor, color, and overall acceptability of the cooked patties. Microstructure images of cooked patties indicated uniform/cohesive structures with small pore size of patties shaped with Osan. Obviously, good storability of the uncooked patties was reflected on the physiochemical, textural, color, and sensory evaluation of the cooked patties, which points to the benefit of using Osan in frozen patties and signifies possible use in the meat industry. The overall sensory acceptability scores were given to cooked patties containing Osan starch as well as the native starch, whereas 15% animal fat was favored too.

**Keywords:** beef patties; corn starch; Osan; tenderness; cooking loss

#### **1. Introduction**

Meat patties are considered the most popular ready to eat food, due to its desirable sensory and mouth feel. This qualifies its consumption to be considered as a good part of human diet in the past few decades, in addition to its nutritional value which includes essential amino acids, vitamins, and minerals [1]. In processed meat, animal fat is a principal ingredient, found in lumps and plays a central functional and sensory role such as a binding and flavoring agent [2]. However, fat content is a critical obstacle that needs to be addressed especially at high levels. Specifically, fat has been associated with chronic deceases, such as obesity and high cholesterol which leads to hypertension and cardiovascular diseases [3]. Therefore, reducing fat turned into a current trend for the meat-processing industry to meet the consumer demands [4]. Heretofore, countless efforts have been devoted to address this concern in terms of preparing healthy products without reducing the characteristics of the final full fat product such as the physical appearance and sensory qualities. Carbohydrate-based fat replacers, such as inulin, gums, cellulose derivatives, and starches are wildly considered as fat replacer and/or fat substitute applied in low fat meat products [5,6], because of its abundancy, superior functionality, and cost-effectiveness [7,8]. Among these carbohydrates, starch exhibited extremely good functional properties improving viscosity, solubility, and water-holding capacity, as well as, adaptability [9].

Because of its poor water solubility and high retrogradation, native starches utilization is limited. This presented the need for physical, chemical, or enzymatic modifications of

**Citation:** Eshag Osman, M.F.; Mohamed, A.A.; Alamri, M.S.; Ahmed, I.A.M.; Hussain, S.; Ibraheem, M.I.; Qasem, A.A. Quality Characteristics of Beef Patties Prepared with Octenyl-Succinylated (Osan) Starch. *Foods* **2021**, *10*, 1157. https://doi.org/ 10.3390/foods10061157

Academic Editor: Paulo Eduardo Sichetti Munekata

Received: 27 April 2021 Accepted: 19 May 2021 Published: 21 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

native starches so as to expand its utilization [10]. Starch modification can improve its functionality such as viscosity, become more tolerant to various processing conditions such as extreme pH, high temperatures, and shear. For instance, chemically modified starches exhibit significant change in its functional properties compared to physical or enzyme modifications. One of the common chemically modified starches is octenyl-succinylation (Osan) which accrues by esterification of some OH groups by octenyl molecules [11]. To overcome the hydrophilic nature of the abundant hydroxyl groups, various starch modifications by introduction of hydrophobic moiety have been reported. Therefore, amphiphilic modification of starch is one of the methods widely used to improve their hydrophobicity, because amphiphilic starches have a wide range of applications, mainly in emulsification and encapsulation. The amphiphilicity of octenyl succinic anhydride (OSA) modified starch is improved due to the introduction of dual functional hydrophilic and hydrophobic groups [12]. The obtained Osan starch was utilized in various application ranging from pharmaceutical to food products, such as, puddings, sauces, and baby foods [13]. The use of sodium octenyl succinate starch in a methacrylate/polysaccharides blends, introduced good flowability, surface-active, smoother particle surface, and lowviscosity to spray dried emulsion of the blends [14]. In addition, Osan starch has been used as a fat substitute because it enhances the firmness and high palatability of some meat products [15]. In contrast, octenyl-modified waxy maize starch was used successfully in low fat mayonnaise at substitution level up to 75%, where the product exhibited great sensory quality such as texture and aroma [13]. In baked products, Osan starch improved dough machinability and handling as well as the loaf volume [16].

The objective of this work is to compare the quality characteristics of beef patties prepared with starch to those with animal fat. Therefore, ocetinyle succenylated and native corn starch as fat replacement in beef patties was utilized. This work includes the effect of storability at −20 ◦C on the quality characteristics of the patties (microstructure, cooking properties, moisture retention, texture, color, and sensory characteristics of the produced patties).

#### **2. Materials and Methods**

#### *2.1. Materials*

Lean beef meat and animal fat (Beef) were purchased from a central meat market (Al-Taamir Market, Riyadh, Saudi Arabia). Ground lean meats were selected from animals of the same age, breed, and feeding protocol (aldanon farm). While, fresh fat was obtained from Riyadh slaughterhouse (Riyadh, Saudi Arabia). Corn starch was purchased from Middle East Food Solutions Company (Riyadh, Saudi Arabia). Black paper, white paper, onion powder, garlic powder, were purchased from Panda Retail Company (Riyadh, Saudi Arabia). Analytical grade reagents, HCL, 2-Octen-1-yl succinic anhydride, NaOH were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

#### *2.2. Methods*

#### 2.2.1. Octenyl-Succinylation of Corn Starch (Osan)

Osan corn starch was synthesized via esterification according to the method of [17] with slight modification. Starch, (500 g) was suspended in 1125 mL of distill water. The pH of the slurry was adjusted to 8.5–9 using 1% NAOH, followed by the addition of Osan (4% based on starch dry weight). The 2-Octen-1-yl succinic anhydride was added slowly with continuous agitation at 35 ◦C, while maintaining the pH around 8.5–9.0 the reaction was allowed to continue for one hour and the final pH was adjusted to 6.5 using 1.0 N HCL. The obtained mixture was centrifuged at (4000× *g*) for 10 min, washed twice with distill water and once with acetone. The product was dried at room temperature for two days, ground, sieved through 250 μm sieve and stored for further use. This product is considered as food grade according to the followed preparation method.

#### 2.2.2. Fourier Transform Infrared Spectroscopy (FT-IR)

The stretching vibrational mode of the functional group (Osan) on the modified starch was detected by FTIR spectrophotometer (Bruker, ALPHA, Hanau Germany). One gram of dry sample was placed on the FT-IR cell and scanned between 4000 and 500 cm<sup>−</sup>1.

#### 2.2.3. Preparation and Processing of Beef Patties

Beef patties were prepared and processed as described by Alejandre et al. [18] with slight modification. Round beef cut was sliced to small pieces and the intramuscular fat was removed. The obtained lean meat was ground to passed through a 5-mm plate meat grinder EMG-1600R ELEKTA ltd, Elekta (Hong Kong, China). Simultaneously, the beef caul fat was melted for 5 min in the microwave oven (sharp, 1200 W output, (Osaka, Japan). Six formulations with the following common ingredients g/100 g addition based on ground lean meat weight (100 g), where 1.0 g of the following ingredients were added salt, hot pepper, white pepper, dry powdered garlic, dry onion powder, and 0.35 g of vinegar, in addition to 20 g of ice water. The experimental design included two treatments and three subsamples; two levels of caul fat, two levels of native corn starch, and two levels of Osan corn starch, hence, the one level included 5% and the second was 15%. Samples with caul fat are considered the control and the melted caul fat was added to the spiced lean ground beef drop-wise with constant hand mixing, but in the starch-containing formulation the caul fat was totally replaced by modified or native starch which was added in small amounts while mixing using a Stephan UM 12 mixer (Stephan U. Sohner GmbH and Co., Gackenbach, Germany). Patties (100 g) were prepared using a patty-making machine (Expro. Co., Shanghai, China). The compressed patties, 100 mm diameter and 10 mm thick, were packaged in vacuumed plastic bags and stored at −20 ◦C for 0, 30, and 60 days. Before analysis, frozen un-cooked patties were placed at room temperature for 1.5 h. The raw patties were cooked in two steps; the first step was to prepare precooked patties by steaming for 20 min to stabilize the diameter, whereas the second step was the final cooking using the electric hot plate (Stilfer model, 0040, Genova, Italy) for a total of 10 min with 5 min on each side at 180 ± 1 ◦C. The internal temperature of the patty was 75 ◦C measured at the geometrical center of the patties using digital thermocouple probe (Ecoscan Temp JKT, Eutech instruments, Pte Ltd., Keppel Bay, Harbour Front, Singapore) The sensory evaluation of the cooked products was carried out directly after cooking.

#### 2.2.4. Scanning Electron Microscopy (SEM)

Microstructures of the obtained cooked patties was examined using JEOL-6360A SEM (Jeol Ltd., Tokyo, Japan). Samples were cut into small pieces 5 × 5 × 1 mm, mounted on the pin stubs using copper tape before coating with gold using an automated sputter coater JFC-1600 Auto Fine Coater (Jeol Ltd., Tokyo, Japan) for 5 min at 2.5 kV operation energy. Subsequently, four fields of each sample were spotted and the selected images were captured at magnifications ranging from 100× to 1000×.

### 2.2.5. Measurement of Cooking Parameters

Cooking Loss

The cooking loss of patties was determined by weighing before and after cooking as recommended by Hollenbeck et al. [19] using the following equation:

Cooking loss = ((un − cooked patties weight) − (cooked patties weight))/(un − cooked patties weight) ∗ 100

#### Moisture Content

The moisture contents of un-cooked and cooked patties were determined based on the AOAC Method 950.46 [20].

Moisture Retention

The moisture retention, was determined as the amount of moisture retained in the cooked product per 100 g of raw sample. This value was calculated according to the following equation described by [21,22], where the weight of the patties was recorded before and after cooking, and the cooking yield was calculated by dividing the weight of cooked patties by the weight of uncooked patties and expressed in as reported by [23].

$$\text{Moisture return} = \text{cooling yield} \times \frac{\text{moisture in cooled patients}}{\text{moisture in un-cooked patients}}$$

$$\text{or } \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad \dots \quad}$$

Cooking yield = weight before cooking − cooked weight

Patties Diameter, Thickness, and Shrinkage

Change in beef patties' diameter was determined before and after cooking using Digital Electric Caliper (Pen Tools Co., Maplewood, NJ, USA) by employing the following equations.

$$\text{Diameter} = \frac{(\text{un-cooked parties diameter}) - (\text{cooked patients diameter})}{\text{un-cooked patients diameter}} \times 100$$

$$\text{Thickness} = \frac{(\text{un-cooked patients thickness}) - (\text{cooked patients thickness})}{\text{un-cooked patients thickness}} \times 100$$

$$\text{Dimensional shrinkage} = \frac{(\text{Raw thickness} - \text{cooled thickness}) + (\text{Raw diameter} - \text{cooled diameter})}{\text{Raw thickness} - \text{raw diameter}} \times 100$$

#### Firmness

The firmness of cooked beef patties was determined using a texture analyzer (TA XT Express, Micro Systems Ltd., Surrey, UK). Samples (60 mm diameter and 10 mm thickness) were pressed using aluminum cylinder probe (SMS P/20 mm diameter, TA XT Plus Micro Systems Ltd., Surrey, UK) operated at 1 mm/s. Samples were compressed to 8 mm distance with 10% strain, where the needed force is expressed in Newton (N). The shear force corresponds to maximum peak force, expressed in Newton (N). The test was performed at room temperature (25 ± 1 ◦C).

#### pH

The pH was measured using a portable pH-meter (Model pH 211, Hanna Instruments, Woonsocket, RI, USA) by injecting the probe in 25 g of meat patty and held for 10 s to obtain the pH value.

#### Surface Color Measurement

The surface color characteristics of un-cooked and cooked patties were determined after the specified storage time (0, 30, or 60) days. The measurements included, lightness (L\*), redness (a\*), and yellowness (b\*), assessed using a portable colorimeter (Konica Minolta, CR-400-Japan; Measuring aperture: 8 mm; Illuminant: CIE D65; Observer angle: CIE 2◦ Standard Observer). Five color measurements were done, where each patty was separated into four quarters one measurement on the surface of each quarter was taken and the fifth was done in the middle.

#### 2.2.6. Sensory Evaluation of Meat Patties

The sensory test was performed using a 9 points hedonic test (Affective Tests), which includes scale from 1 (dislike extremely) to 9 (like extremely) in a single session. This test is useful for evaluating the acceptance of new products. The sensory evaluation team included trained students and King Saud University staff average age between 22 and

60 years old. The panelists were trained to be able to evaluate the sensory properties of patties including overall product acceptability according to method of [24]. After training, 13 panelists were selected based on their ability and sensitivity to point out differences between the parameters. Cooked patties were tested on a 9-points scale method. The test was conducted in a designated sensory evaluation laboratory with appropriate setting such as partitioned cabinets and individual lightning at 20 ± 2.0 ◦C. Six treatments with two levels of fat content (control), native or modified starch were evaluated. Patties were cooked as described above and cut into triangles (25 × 20 mm) and served warm to the participants. Water and mint were also provided to neutralize the flavor between samples [25]. The expert panelists were asked to evaluate the color, flavor, tenderness, juiciness, and overall acceptability.

#### 2.2.7. Statistical Analysis

The statistical analysis was carried out using the Tukey HSD test (Statistic 10 Data analysis software, Inc., Chicago, IL, USA) at (*p* ≤ 0.05). The significance level of the analysis of variance (ANOVA) was applied to observe the differences. All measurements were done in triplicate.

#### **3. Results and Discussion**

#### *3.1. Fourier Transform Infrared Spectroscopy (FT-IR)*

FT-IR profile is shown in Figure 1. In general, the highly intense peaks noticed around 3430 cm−<sup>1</sup> were ascribed to (O-H) characteristics stretching vibration of amylose or amylopectin, while peaks around 2930 and 1645 cm−<sup>1</sup> are attributed to C–H stretching and to the tightly bound water present in the starch, respectively [26]. In addition, the peak at 1020 cm−<sup>1</sup> was originated from the C–O stretching vibration of glucose monomer (Garcia and Grossmann 2014). Two new peaks emerged after modification. Evidently, the region between 1720 and 1570 cm−<sup>1</sup> is considered a finger print for the main functional groups of the octenyl-succinylated (Osan) corn starch [27]. The new peak at 1571 cm−<sup>1</sup> emerged after OSA modification was ascribed to the asymmetric stretching vibration of carboxylate RCOO–, whereas the other new peak at 1725 cm−<sup>1</sup> was observed, which can be attributed to the characteristic C=O stretching vibration of an ester carbonyl group [28].

**Figure 1.** FT-IR spectra of (**a**) Osan-corn starch and (**b**) native corn starch.

#### *3.2. Scanning Electron Microscopy (SEM)*

The gel network of patties with 5% addition of either fat or starch, was loose and irregular, because large holes emerged within the structure compared to the 15% (Figure 2). Nonetheless, the gel network structure of the 15% Osan starch was more compact and dense. Furthermore, smaller holes were observed in the surface of the patties with native starch and the holes were more obvious with the increase in the amount of native starch in the samples. Moreno et al. [29] reported better surface structure using a muscle homogenizer than samples with added sodium alginate as a cold gelation technology. Tseng et al. [30] reported dense SEM images of meat balls treated with TGase enzyme compared to untreated samples which indicates rise in the formation of intermolecular ε (γ-glutamyl)-lisil cross-links due to the action of the enzyme. In this study, starch generated a dense network by absorbing the excess water released by the meat during cooking which leads to swelling and closing of the gaps created during cooking. Therefore, smoother denser surface was formed in the presence of starch and more so Osan starch compared to the control. Other researchers used plant material rich in hydrofoils reported improved microstructure of meat and homogenous network [31].

**Figure 2.** Scanning electron microscope (SEM) images of cooked patties containing animal fat, native corn starch and Osan corn starch (**a1**) control, (**a2**) native starch, and (**a3**) Osam starch 15% addition; (**b1**) control, (**b2**) native starch, and (**b3**) Osan 5% addition.

#### *3.3. Physical Properties of Beef Patties*

#### 3.3.1. Cooking Properties

The cooking properties of the patties prepared with Osan-corn starch, native corn, and control are presented in Table 1. The addition of Osan-corn starch at 5 or 15%, significantly (*p* ≤ 0.05) altered the cooking loss, moisture retention, thickness, diameter, and dimensional shrinkage of the patties. These parameters were compared to the raw patties. The data presented here are in agreement with the literature reports on the effectivity of corn starch to retain moisture of the cooked bologna [10,32,33]. The firmness of patties prepared with native starch was significantly higher than Osan starch which can be attributed to the amylose retrogradation which is less in Osan starch, thereby, patties with more starch exhibited firmer texture (Table 1) [34]. The firmness of the control was much higher at 5% fat content compared to 15% indicating softer texture due to higher fat content but, it remains significantly higher than those with either type of starch, especially Osan starch. This could be accredited to the incapacity of fat to retain moisture during cooking compared to the starch, which is in line with the water retention property difference of the patties stated in Table 1 [35]. The pH values of the patties were stable throughout the process, therefore, the use of starch did not affect the pH of the patties during storage or after cooking. The percent diameter reduction after cooking was 18.3 for the 5% fat content (control) and 23.5 for the 15% fat content, whereas for the Osan starch patties it was 7.7 and 8.6%, while the native starch exhibited reduction as 13.4 and 16.45%, respectively. This shows significantly lower diameter reduction of Osan starch patties compared to the control and the native starch. Thereby, the stability of the patties network structure due to Osan starch swelling and the formation of a semi solid gel that stabilized the diameter, is evident (Table 1). This can be attributed to the amphiphilic (hydrophilic-hydrophobic nature) property of the Osan starch. The control exhibited the greatest diameter loss after cooking by virtue of increased animal fat content. Park et al. [36] reported diameter and thickness reduction for pork patties decreased with increase in the content of black rice powder (rice powder is about 75% starch). The dimensional shrinkage followed the same trend as the diameter reduction. Consequently, the score of the cooking loss, moisture retention, diameter reduction, and dimensional reduction favored Osan over native starch. Cornejo-Ramírez et al. [37] reported that water absorption, swelling power, and viscosity of Osan starch is superior to native starch. The high moisture retention of Osan starch lead to highly viscous gel with emulsifying power capable of holding fat and water and form a gel with little un-noticeable pores (space within the structure) and improve the sensory characteristics of the patties [33]. On the other hand, the control beef patties exhibited poor binding, limited protein network structure, or entrapments of ingredients as shown by the sizable pores in the protein network. These results are in agreement with [38–41]. The thickness of the control increased at higher fat content, but with native starch, significantly lower thickness was observed at high starch content. Osan starch patties exhibited significantly higher thickness compared to the control and the native starch, since the thickness was almost twice as much (Table 1).


**Table 1.** The effect of 5 and 15% native, octenyl-succinylated (Osan) corn starch, and 5% animal fat on the physiochemical characteristics of beef patties.

<sup>1</sup> The statistical analysis was done separately for the 5% and for the 15%; <sup>2</sup> M.C.U = moisture content of uncooked patties. <sup>3</sup> M.C.C = moisture content of cooked patties. <sup>4</sup> M.R = moisture retention; <sup>5</sup> diameter reduction; <sup>6</sup> D.S = dimensional shrinkage. <sup>7</sup> control = animal fat; <sup>8</sup> thickness increase, a–c Values followed by different letters within each row are significantly different (*<sup>p</sup>* <sup>≤</sup> 0.05).

#### 3.3.2. Effect of Storage on the Cooking Properties

The effect of storage on cooking loss, firmness, and other cooking properties is presented in Table 2. Although longer storage time significantly increased the tested parameter of patties containing 5% starch or animal fat, Osan starch performed better than the control or the native starch under the same storage conditions. The storability of patties significantly enhanced when 15% of starch or animal fat were added. Osan starch improved all tested parameters at 15% addition compared to 5%. The thickness of the control remained the same at 5% addition regardless of the storage time, but either of the starches reduced the thickness at longer storage time, significantly (Table 2). Nonetheless, Osan starch increased the thickness, significantly, compared to the control or the native starch especially after 0 or 30-day storage (Table 2). The same trend was observed for the 15% addition, where the thickness decreased after longer storage time for all samples, but the drop was less for Osan starch (Table 3). The firmness of all three patties increased after longer storage time regardless of the added fat or starch level. The increase in the firmness after longer storage time can be attributed to the moisture loss, however, Osan starch exhibited the least increase in firmness. Yang et al. [42] reported the addition of 4% modified waxy maize starch to low-fat frankfurters leading to reduced moisture loss by up to 7.2%. However, Claus and Hunt [43] also reported that, modified waxy maize starch applied to low-fat bologna was more effective in controlling moisture losses relative to native wheat starch, which is consistent with a very low retrogradation of waxy starch [34,35]. There are conflicting reports regarding the effect of starch on the cooking loss of meat products, especially for low muscle products such as frankfurters and bologna [33]. Cooking loss increase was observed for all samples at longer storage time, but Osan cooking loss was much less than the other treatments (Tables 2 and 3).


**Table 2.** Effect of 5% octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the physiochemical characteristics of meat patties after storage at −20 ◦C for 0, 30, and 60 days.

<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* ≤ 0.05); <sup>2</sup> diameter reduction; <sup>3</sup> D.S = dimensional shrinkage; <sup>4</sup> thickness increase; <sup>5</sup> firmness in Newton; <sup>6</sup> control = animal fat.


**Table 3.** Effect of 15% octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the physiochemical characteristics of meat patties after storage at −20 ◦C for 0, 30, and 60 days.

<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* ≤ 0.05); <sup>2</sup> diameter reduction; <sup>3</sup> D.S = dimensional shrinkage; <sup>4</sup> thickness increase; <sup>5</sup> firmness in Newton; <sup>6</sup> control = animal fat.

#### 3.3.3. Sensory Attributes of Cooked Beef Meat Patties

The sensory evaluation results are presented in Table 4. The incorporation of native or Osan starches at 5% did not have any significant effect on the juiciness, flavor, color, or acceptability of the patties, but the tenderness was improved significantly (*p* ≤ 0.05). The addition of 15% of either starches significantly improved the juiciness, flavor and, the overall acceptability of the patties, whereas the tenderness and the color were slightly improved more with Osan starch. After storage, the sensory evaluation showed superior performance of Osan over the control and the native starch, whereas longer storage appeared to have negative effect on the parameter of samples containing native starch (Table 5). Once again, samples with 15% Osan starch scored higher than those with 5%. Nonetheless, storage at −20 ◦C for 60 days appeared to have limited effect on the overall acceptability of the patties, but Osan starch patties scored higher. Higher starch content facilitates stable protein-starch matrices, where hydrogen and covalent bonding and charge-charge interactions occur [44].

**Table 4.** The effect of different levels of octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the sensory characteristics of meat patties. The test was based on a 9 points hedonic.


<sup>1</sup> The statistical analysis was done separately for the 5% and for the 15%; (a–c) values followed by different letters within each row and addition percent are significantly different (*<sup>p</sup>* ≤ 0.05); <sup>2</sup> control = animal fat; the statistical analysis was done for the 5%, separate from the 15%.

**Table 5.** Effects of different levels of octenyl succinylated (Osan) corn starch, native starch, and animal fat on the sensory characteristics of meat patties after storage at −20 for different days.


<sup>1</sup> Control = animal fat; (a–c) values followed by different letters within each row and addition percent are significantly different (*<sup>p</sup>* ≤ 0.05); the statistical analysis was done for the 5% separate from the 15%.

#### 3.3.4. Color of Raw and Cooked Patties

Consumer acceptability of meat products is dependent on its color because it is indicative of freshness. The effect of starch on the surface color of beef patties is presented in Table 6. The control sample exhibited the lowest redness (a\*) values compared to the native and Osan starches, regardless of the added amount, but Osan starch had higher a\* value. The incorporation of plant-based material was reported to increase the a\* value of cooked meat [45]. Higher a\* values of Osan starch-containing patties compared to control and the native starch indicate color stabilization, since the reduction in a\* values is suggestive of myoglobin oxidation and the formation of net myoglobin [46]. Other researchers reported increase in meat redness after incorporation of potato starches, whereas cassava starch reduced the a\*. Therefore, the type of starch as well as the amount of the added starch can be considered as factors that affect beef patties redness. This is obvious on the magnitude of the effect of native or Osan starch on the a\* of the patties (Table 6). The amount of the incorporated Osan (5 or 15%) had slight effect on a\* (Table 6). The redness of the control increased as a function of the added amount, but it increased significantly after storage for 60 days (Table 7). Sample with Osan had the highest a\* as a function of storage time (60 days). The lightness (l\*) of the samples and the control stayed almost the same at 5% incorporation, but at 15% Osan starch exhibited significantly higher l\* value. The higher lightness could be attributed to the dilution of the color by the added starch. Reports in the literature mentioned reduction or stability of the l\* based on the incorporated material into the patties [47]. No reduction in lightness was observed after storage for 30 or 60 days for either 5 or 15% incorporation. The yellowness (b\*) of the control increased compared to the starch-containing samples where Osan exhibited the most b\* value, but after storage for 60 days a drop in b\* values was observed (Table 7).

**Table 6.** The effect of octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the color characteristics of un-cooked meat patties after 6 h.


<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* <sup>≤</sup> 0.05); <sup>2</sup> control = animal fat. <sup>3</sup> L\* = lightness, <sup>4</sup> a\* = redness and <sup>5</sup> b\* = yellowness; the statistical analysis was done for the 5% separate from the 15%.

The color of the cooked patties 6 h after preparation is presented in Table 8. The redness (a\*) of cooked patties showed no significant difference between the control at 5% addition and Osan, but Osan starch exhibited significantly higher a\* at 15% addition, whereas native starch reduced the redness. The lightness (l\*) and yellowness (b\*) of cooked patties was reduced in the presence of native or Osan starches. The effect of storage time on the color of the cooked patties is listed in Table 9. The redness (a\*) of the cooked control did not change significantly after storage at either of the fat additions, whereas Osan starch increased the redness of the cooked patties compared to the control or native starch through storage time, especially after 60 days. Samples exhibited an increase in lightness and yellowness after longer storage time regardless of the amount of the added starch or animal fat (Table 9). The ratio of a\*/b\* is often used to describe color quality. The redness of the control was significantly reduced by the addition of 15% animal fat compared to 5%, but native starch maintained similar color for both additions. The addition of 5% Osan starch significantly reduced the yellowness which is obvious on the a\*/b\* value (Table 6). Therefore, animal fat added at 15% and Osan starch at 5% had the most improvement on the color of the cooked patties, which could be interpreted as stabilizing effect of Osan starch. This led to the higher redness, which indicates myoglobin color stability. Reports in

the literature showed how the addition of processed plant leaf materials can reduce the a\*/b\* of beef patties which means lower redness [48].

**Table 7.** The effect of octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the color characteristics of un-cooked meat patties at −20 for different days.


<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* <sup>≤</sup> 0.05); <sup>2</sup> control = animal fat. <sup>3</sup> L\* = lightness, <sup>4</sup> a\* = redness and <sup>5</sup> b\* = yellowness; the statistical analysis was done for the 5% separate from the 15%.

**Table 8.** The effect of octenyl-succinylated (Osan) corn starch, native starch, and animal fat on the color characteristics of cooked meat patties after 6 h.


<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* <sup>≤</sup> 0.05); <sup>2</sup> control = animal fat. <sup>3</sup> L\* = lightness, <sup>4</sup> a\* = redness and <sup>5</sup> b\* = yellowness; the statistical analysis was done for the 5% separate from the 15%.

**Table 9.** The effect of octenyl-succinylated (Osan) corn starch, native starch and animal fat on the color characteristics of cooked meat patties at −20 for different days.


<sup>1</sup> Values followed by different letters (a–c) within each row are significantly different (*<sup>p</sup>* <sup>≤</sup> 0.05); <sup>2</sup> control = animal fat. <sup>3</sup> L\* = lightness, <sup>4</sup> a\* = redness and <sup>5</sup> b\* = yellowness; the statistical analysis was done for the 5% separate from the 15%.

#### **4. Conclusions**

In this study we examined the quality parameters of patties prepared from ground beef and processed with the addition of Osan starch consequently revealing varying positive changes in the quality properties of the final product. The addition of Osan starch did not alter the pH level or the organoleptic standards of the product. The most notable change was manifested by the increase in textural and microstructural properties and the significant improvement of the cooking characteristics such as: yield, moisture retention, patties redness, thickness, and decrease in cooking loss. Scanning electron microscope (SEM) images of the samples confirmed the rise of intermolecular interaction between the proteins and the Osan starch, which resulted in small pores on the surface of the patties.

**Author Contributions:** Conceptualization, A.A.M., M.S.A. and I.A.; methodology, M.F.E.O.; software, M.F.E.O.; validation, I.A.M.A., and A.A.M.; formal analysis, S.H. and M.I.I.; investigation, S.H.; resources, M.I.I.; data curation, A.A.Q.; writing—original draft preparation, A.A.M.; writing—review and editing, A.A.M.; visualization, S.H.; supervision, A.A.M.; project administration, A.A.M.; funding acquisition, A.A.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** Deanship of Scientific Research at King Saud University for funding this work through research group no RGP-114.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** Data will be available upon request from the corresponding author.

**Acknowledgments:** The authors extend their appreciation to the Deanship of Scientific Research at King Saud University for funding this work through research group no RGP-114.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


## *Article* **Low-Voltage Electrical Stimulation of Beef Carcasses Slows Carcass Chilling Rate and Improves Steak Color**

**Christina Bakker, Keith Underwood, Judson Kyle Grubbs \* and Amanda Blair**

Department of Animal Science, South Dakota State University, Brookings, SD 57007, USA; Christina.Bakker@sdstate.edu (C.B.); keith.underwood@sdstate.edu (K.U.); amanda.blair@sdstate.edu (A.B.) **\*** Correspondence: judson.grubbs@sdstate.edu

**Abstract:** Electrical stimulation (ES) is used in beef slaughter plants to improve tenderness; however, varying levels of low-voltage ES have not been well characterized. The objective was to evaluate the influence of two levels of low-voltage ES on temperature decline, pH, glycolytic potential, and meat quality. Forty-two beef carcasses were chosen from a commercial packing facility. One side of each carcass received either 40 or 80 volts of ES for 60 s at 45 min postmortem. The paired side of each carcass did not receive ES (Control). Temperature loggers were placed in the sirloin of 12 carcasses to record temperature decline. Longissimus muscle pH was measured at 1, 12, and 24 h, and 3 d postmortem. Strip steaks were fabricated for determination of meat quality. A treatment by time interaction was observed for carcass temperature decline (*p* < 0.001) where ES sides stayed warmer longer than Control sides. A treatment by time interaction was observed for pH decline with Control sides exhibiting an increased pH at 1 h postmortem (*p* < 0.001). Instrumental color values were increased for ES compared to Control sides (*p* < 0.001). These results indicate ES slows carcass temperature decline, hastens initial pH decline, and improves instrumental color. Similar results were observed between the ES treatments indicating either ES level may be used to achieve similar quality characteristics.

**Keywords:** beef; electrical stimulation; glycolytic potential; quality; temperature decline

#### **1. Introduction**

Electrical stimulation (ES) is a postmortem intervention utilized to enhance beef quality traits including color, tenderness, and flavor. Electrical stimulation is proposed to improve tenderness by reducing cold shortening [1], disrupting muscle structure [2], and increasing proteolytic activity [3]. Extra low-voltage ES is used on beef carcasses to facilitate the removal of blood from carcasses shortly after exsanguination, while low- and high-voltage ES is used to improve the tenderness and color of beef [4–6]. However, there are discrepancies among reports regarding the influence of varying levels of ES on beef quality traits. In a review by Adeyemi and Sazili [7], these discrepancies caused by varying levels of ES on beef quality are highlighted, with some authors reporting positive effects including improvements in tenderness and lean maturity, some reporting negative effects such as reduced color stability and water holding capacity, and others reporting no effect of ES on meat quality, thus concluding the need to further study the effective application of this technology. Throughout the beef industry in the United States, few plants utilize ES in the same manner. Some plants utilize extra low-voltage ES to facilitate blood removal, others apply low- or high-voltage ES to improve tenderness and lean maturity scores, some apply different ES voltage levels throughout the slaughter process, and yet others do not use ES at all. Thus, additional research is necessary to optimize ES applications to ensure beneficial effects are captured and deleterious effects are minimized. Therefore, the objective of this study was to evaluate the influence of two levels of low-voltage electrical stimulation applied at 45 min postmortem on temperature decline, muscle pH, instrumental color, glycolytic potential, and instrumental tenderness. We hypothesized the

**Citation:** Bakker, C.; Underwood, K.; Grubbs, J.K.; Blair, A. Low-Voltage Electrical Stimulation of Beef Carcasses Slows Carcass Chilling Rate and Improves Steak Color. *Foods* **2021**, *10*, 1065. https://doi.org/10.3390/ foods10051065

Academic Editor: Paulo Eduardo Sichetti Munekata

Received: 1 April 2021 Accepted: 8 May 2021 Published: 12 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

ES treatments would increase carcass temperature, decrease muscle pH, increase glycolytic potential, improve tenderness, and increase instrumental L\* and a\* values compared to the non-stimulated sides, with the 80 V ES treatment having a greater impact on these traits than the 40 V treatment.

#### **2. Materials and Methods**

#### *2.1. Carcass Selection and Electrical Stimulation Treatments*

Cattle were shipped from feedlots to a commercial slaughter facility and held in lairage following normal plant operating guidelines and United States Department of Agriculture Food Safety Inspection Service regulations for beef slaughter. Source and history of the cattle is unknown. Carcasses (n = 42) were selected for comparison in this study. Three collections were conducted throughout the course of the production day (11 carcasses at 0900 h, 16 carcasses at 1200 h, and 15 carcasses at 1500 h). Carcasses were harvested using standard industry methods. Prior to chilling, paired sides were identified to compare the influence of 2 levels of ES. The left side of the carcasses were subjected to one of two ES treatments, (1) 80 V (ES80; n = 20) and (2) 40 V (ES40; n = 22), 45 min after exsanguination. The right side of each carcass was used as an unstimulated control. For both ES40 and ES80 treatments, the ES was administered through the carcass trolly as it moved over a section of electrically charged rail. Electrical stimulation was applied over a 60 s period where the carcasses received a 4 s pulse of electricity with approximately 2 s between each pulse. The remaining side of each carcass served as a negative control and did not receive ES (Control; n = 42).

#### *2.2. Carcass Temperature and pH*

Following application of ES treatments, all carcasses were placed on the same rail in a cooler set to hold at approximately 3 ◦C for 48 h. Carcass temperature decline was monitored from the timepoint the carcasses entered the cooler on paired sides by inserting a temperature probe (Temprecord Multitrip, Sensitech Inc. Beverly, MA, USA) into the sirloin of both sides of the first 4 carcasses selected at each of the 3 collection time points. Once the cooler was filled with carcasses, the spray chill system was activated to spray water for 1 min every 15 min for 24 h. Upon completion of the 48-h holding period, carcasses were ribbed and allowed to bloom for approximately 30 min before standard carcass data were collected. Longissimus muscle pH was recorded on the medial side of the muscle at the 12th rib position at 1 h postmortem, at the 11th rib at 12 h postmortem, and at the 10th rib at 24 h postmortem to establish a pH decline through the completion of rigor mortis. The pH recordings were taken at different locations on the muscle to avoid influence on pH by utilizing the same probe site.

#### *2.3. Carcass Characteristics and Sample Collection*

Fat thickness at the 12th rib (BF) and ribeye area (REA) were measured on both sides of each carcass by South Dakota State University (SDSU) personnel. Fat thickness at the 12th rib and REA measurements of the two sides were averaged and used to calculate USDA Yield Grade. Hot carcass weight (HCW) was recorded from each side and added together for a total hot carcass weight for the carcass. Boneless striploins (IMPS #180) were collected, transported under refrigerated conditions to SDSU, and fabricated into 2.54 cm steaks. Steaks were fabricated in a set order. The first anterior steak was immediately frozen, 3 d postmortem, and used for glycolytic potential (GP) analysis. The second through fourth steaks were aged for 3, 7, or 14 d, respectively, and utilized for Warner–Bratzler shear force (WBSF) and cook loss determination. The second anterior steak was also used to measure ultimate pH, 3 d postmortem. The seventh steak was used to evaluate instrumental lean color for each loin.

#### *2.4. Glycolytic Potential*

Glycolytic potential was determined as described by McKeith et al. [8] with minor modifications. Briefly, steaks designated for GP analysis were minced, snap frozen in liquid nitrogen and powdered using a Waring commercial blender (Model 51BL32, Waring Products Division, New Hartford, CT, USA) to produce a homogenous sample. Three g of powdered sample was weighed into a 50 mL plastic conical tube, allowed to thaw, and then homogenized for 75 s in 0.6 N perchloric acid. Samples were then digested using amyloglucosidase and 5.4 N potassium hydroxide and incubated for 3 h, inverting the tubes every 20 min to mix. Upon completion of the incubation step, 3N perchloric acid was added and samples were centrifuged at 4.4 ◦C for 5 min at 10,000× *g*. Supernatant was collected and stored for analysis. Glucose levels were determined using a glucose assay kit (Glucose (HK) Assay Kit GAHK20, Millipore-Sigma, St. Louis, MO, USA) and absorbance was read at 340 nm (SpectraMax 190, Molecular Devices, San Jose, CA, USA). Lactate levels were determined by adding NAD+ in a glycine buffer to sample aliquots to form NADH. Samples were then read at 340 nm (SpectraMax 190, Molecular Devices, San Jose, CA, USA). Glycolytic potential of each sample was then calculated with the following equation: GP = 2(Glucose absorbance \* 111.882) + (Lactate absorbance \* 173.22).

#### *2.5. Warner–Bratzler Shear Force and Cook Loss*

Steaks utilized for WBSF were thawed at approximately 4 ◦C for 24 h prior to cooking. Steaks were cooked on a clamshell grill (George Foreman Indoor/Outdoor Grill model GGR62, Lake Forest, IL, USA) to an internal peak temperature of 71 ◦C as indicated by a temperature probe inserted to the geometric center of the steak (Atkins AquaTuff NSF Series Model 351, Middlefield, CT, USA). Steaks were then stored at approximately 4 ◦C overnight. Four h prior to evaluating shear force values, steaks were placed at room temperature and allowed to equilibrate. Six cores were removed parallel to the direction of the muscle fibers and then sheared once using a Warner–Bratzler shear machine (G-R Electric Manufacturing Company, Manhattan, KS, USA) equipped with a BFG 500 N basic force gauge (Mecmesin Ltd., West Sussex, UK) and peak shear force was recorded for each core. An average shear force value was calculated and recorded for each steak.

Cook loss was determined on steaks designated for WBSF. Raw steak weight was recorded with a balance (MWP, Cas Corporation, Seoul, South Korea) and after cooking, steaks were allowed to equilibrate to room temperature and weighed again. Cook loss was determined using the following equation: cook loss % = ((raw weight − cooked weight)/raw weight) × 100.

#### *2.6. Instrumental Color*

Steaks designated for color determination were allowed to bloom for 30 min prior to evaluation. L\*, a\*, and b\* values were recorded at two locations (medial portion of the steak and lateral portion of the steak) using a handheld colorimeter (Chroma Meta CR-410, Konica Minolta, Ramsey, NJ, USA) equipped with a 50 mm aperture, 0◦ viewing angle, 2◦ standard observer, pulsed xenon lamp light source, and calibrated with a white tile (L\* = 97.38, a\* = 0.06, b\* = 1.82). Measurements were averaged between both locations for each steak.

#### *2.7. Statistical Analysis*

The experiment utilized both sides of 42 carcasses in a completely randomized design. The data analysis was conducted using the MIXED model of SAS software (SAS Institute Inc., Cary, NC, USA) with fixed effect of treatment, random effect of carcass, and Toeplitz covariate structure. Hot carcass weights for both sides of each carcass were added together, and REA and BF measurements were averaged between sides. As HCW from both sides are needed to calculate USDA yield grades, carcass data were analyzed by ES treatment with data reported as ES40 or ES80 treatments. Contrast statements were used to compare Control vs. ES40 and ES80 sides (Control vs. ES), and ES40 vs. ES80 (ES Level). Peak

internal cooking temperature was used as a covariate for cook loss and WBSF data. Temperature decline, WBSF, cook loss, and pH were considered repeated measures. Interactions of treatment and time were evaluated where appropriate and are reported when significant. Significance was determined when *p* < 0.05.

#### **3. Results**

#### *3.1. Carcass Characteristics*

Carcass characteristics are reported in Table 1. Hot carcass weight did not differ between ES40 and ES80 treatments (*p* = 0.7200). No differences were observed in REA (*p* = 0.6172). Fat thickness measured at the 12th rib was similar between the two treatments (*p* = 0.9482). The lack of differences in HCW, REA, and BF contributed to the absence of differences in overall USDA yield grade (*p* = 0.5000). The absence of differences in carcass characteristics between ES treatments indicates that carcass characteristics likely did not impact carcass chilling or meat quality data.

**Table 1.** Least square means for carcass characteristics of carcasses subjected to 40 or 80 V of electrical stimulation for 60 s in 4 s on, 2 s off intervals prior to chilling.


<sup>1</sup> ES40 = 40 V of electrical stimulation, ES80 = 80 V of electrical stimulation. <sup>3</sup> Standard error of means. <sup>4</sup> Carcass data measured between the 12th and 13th rib according to USDA standards. <sup>5</sup> USDA Yield Grade.

#### *3.2. Carcass Temperature and PH*

An ES by chilling time interaction was observed for temperature decline (*p* < 0.0001; Figure 1). Sides treated with ES prior to chilling had similar temperatures to non stimulated sides at the onset of chilling. By 30 min of chilling, ES sides had increased temperatures compared to sides that did not receive ES, regardless of ES level. This difference persisted until 24 h postmortem when temperature data loggers were removed from the carcasses. No differences in temperature between ES treatments were observed at any time point (*p* > 0.05).

**Figure 1.** Temperature decline of carcass submitted to low-voltage electrical stimulation (ES) prior to chilling. Data are depicted as least square means ± SEM. Treatments are as follows: Control = no ES, ES40 = 40 V of ES, ES80 = 80 V of ES. Electrical stimulation was applied for 60 s in 4 s on, 2 s off intervals.

An ES treatment by chilling time interaction was observed for pH decline (*p* < 0.0001; Figure 2). At 1 h postmortem, the ES80 carcasses achieved the lowest pH, ES40 intermediate, and Control sustaining the highest pH value. The pH values measured at 12 and 24 h postmortem, as well as ultimate pH, did not differ among treatments (*p* > 0.05).

**Figure 2.** pH decline in beef carcasses subjected to low-voltage electrical stimulation prior to chilling. Data are depicted as least square means ± SEM. Treatments are as follows: Control = no ES, ES40 = 40 V of ES, ES80 = 80 V of ES. Electrical stimulation was applied for 60 s in 4 s on, 2 s off intervals. Measurements were taken at 1, 12, and 24 h postmortem in addition to ultimate pH. a–f Means with different subscripts differ (*p* < 0.05).

#### *3.3. Glycolytic Potential*

Glucose, lactate, and GP data are reported in Table 2. Glucose concentration did not differ between Control and ES sides (*p* = 0.5825) or between ES treatments (*p* = 0.7308). Additionally, no differences were observed between Control and ES sides (*p* = 0.9557) or between ES levels (*p* = 0.5655) for lactate concentration. Unsurprisingly, based on glucose and lactate results, GP did not differ between Control and ES sides (*p* = 0.6760), or between ES treatments (*p* = 0.5784).

**Table 2.** Warner–Bratzler shear force (WBSF), cook loss, glucose, lactate, and glycolytic potential (GP) of beef carcasses subjected to low-voltage electrical stimulation for 60 s in 4 s on, 2 s off intervals prior to chilling 1,2.


<sup>1</sup> Least square means ± standard error of means. <sup>2</sup> No interaction was observed for aging day and electrical stimulation treatment (effect of aging day is reported in Table 3). <sup>3</sup> Carcasses subjected to 0 (Control), 40 (ES40), or 80 (ES80) V of electrical stimulation. <sup>4</sup> Control vs. ES contrast statement compares Control carcasses vs. 40 and 80 V treatments. <sup>5</sup> ES Level contrast statement compares 40 vs. 80 V treatments

**Table 3.** Least square means for Warner–Bratzler Shear Force (WBSF) and cook loss values of beef steaks aged 3, 7, or 14 d <sup>1</sup> (n = 42/day).


<sup>1</sup> No interaction was observed for aging day and electrical stimulation treatment (effect of electrical stimulation treatment is reported in Table 2). <sup>2</sup> Standard error of means. a,b Means with different subscripts indicate a difference within row (*p* < 0.05).

#### *3.4. Warner–Bratzler Shear Force and Cook Loss*

Steaks from ES sides exhibited decreased shear force values compared to the Control sides (*p* < 0.0220; Table 2). However, when evaluating WBSF data between ES treatments, no differences were observed (*p* = 0.7332). Moreover, an aging day effect was observed for WBSF. Steaks aged 7 d had a greater shear force value compared to steaks aged for 3 or 14 d postmortem (*p* = 0.0021; Table 3).

The percentage of weight lost during cooking did not differ between Control and EStreated sides (*p* = 0.3753; Table 2) nor were differences observed between sides treated with different ES levels (*p* = 0.8536). An aging day effect was observed for cook loss (*p* = 0.0127; Table 3) with steaks aged 3 d demonstrating less cook loss than steaks aged 7 or 14 d.

#### *3.5. Instrumental Color*

Steaks from ES sides were lighter (*p* < 0.0001; Table 4), redder (*p* < 0.0001; Table 4), and more yellow (*p* < 0.0001; Table 4) than control steaks. No differences were observed between ES treatments for lightness (*p* = 0.4582), redness (*p* = 0.9460), or yellowness (*p* = 0.7079).

**Table 4.** Instrumental color values of longissimus muscle from beef carcasses subjected to low-voltage electrical stimulation for 60 s in 4 s on, 2 s off intervals prior to chilling 1.


<sup>1</sup> Least square means ± standard error of means. <sup>2</sup> Carcasses subjected to 0 (Control), 40 (ES40), or 80 (ES80) V of electrical stimulation. <sup>3</sup> Control vs. ES contrast statement compares Control carcasses vs. 40 and 80 V treatments. <sup>4</sup> ES Level contrast statement compares 40 vs. 80 V treatments

#### **4. Discussion**

Previous research has shown temperature decline trends similar to the current data with ES reported to increase carcass temperature. Bowker et al. [9] measured the temperature decline of the longissimus dorsi in pigs electrically stimulated (six pulses, 60 Hz, 500 V, 1 s on and 2 s off) at 3 min postmortem, and observed an increase in temperature of ES-treated carcasses over the monitoring duration of 56 min. In both cases, the increase in temperature was likely caused by the heat generated by the muscle contractions caused by the ES treatment [9]. Conversely, Wiklund et al. [10] evaluated the temperature decline in the longissimus muscle of red deer carcasses stimulated with 90–95 V of ES for 55 s at the time of exsanguination, and found no differences compared to non-stimulated carcasses. Additionally, Kim et al. [11] evaluated the impact of low-voltage ES (100 V for 30 s) 90 min after exsanguination of beef carcasses, and also observed no differences in the temperature decline of the longissimus dorsi compared to non-stimulated sides. The conflicting results of Wiklund et al. [10] and Kim et al. [11] compared to the current study could be due to differences in species (beef vs. red deer) or time post exsanguination of the stimulation.

Electrical stimulation can cause an increase in the rate of postmortem muscle pH decline by increasing metabolic activity. McKenna et al. [12] observed differences in early pH measurements with ES sides showing decreased pH values compared to non-stimulated sides until 6 h postmortem when pH was similar, until cessation of pH measurements at 24 h postmortem. Moreover, Nichols and Cross [13] noted a similar trend in pH with ES sides displaying a rapid pH decline in the first 6 h postmortem. Kim et al. [11] noted a more dramatic decrease in longissimus muscle pH decline, with non-stimulated sides displaying an increased pH until 24 h postmortem. The rapid pH decline observed was likely caused by the increase in postmortem glycolysis, which resulted in a buildup of lactic acid in muscle at a faster rate than would occur without ES, but resulted in similar ultimate pH values [14,15].

Similar to the current study, Ding et al. [16] observed no differences in glucose or GP values for bison meat from carcasses stimulated with 400 V of ES compared to a non-stimulated control. Conversely, Ding et al. [16] did observe a difference in lactate concentrations; however, the samples were taken from carcasses prior to chilling and rigor mortis. The lack of differences in GP observed in this study was ideal as we could conclude the animals used in this study were at similar metabolic states prior to slaughter. Further, we can conclude that pre-harvest handling did not impact the ability of carcasses in this study to experience a normal rigor processes, such as pH decline, and the differences in pH observed in the present study were likely the result of the ES treatments.

There are several mechanisms by which ES is proposed to improve tenderness [17]. It has been reported that ES disrupts muscle structure at the Z-disk and I-band, causes formation of contraction nodes, and disrupts the integrity of the sarcoplasmic reticulum causing minor separation of myofibrils [2,18,19]. Electrical stimulation has also been proposed to inhibit cold shortening by preventing the temperature of the carcass from declining too rapidly [1,20]. Others hypothesize that improvements in tenderness following ES is caused by the activation of lysosomal enzymes and increased proteolysis while carcass temperature is still increased [2,3,10]. However, some studies have found little or no effects of ES on beef tenderness. Discrepancies between studies could be related to the level of voltage applied, duration of stimulation, or timing of ES after exsanguination [11,21]. However, most studies agree that tenderness development is a complex process that likely involves more than one of the previously discussed mechanisms [2,7,14].

It is unclear why steaks aged for 7 d had increased shear force values compared to steaks aged 3 d, when most normal aging curves would show a decrease in WBSF value as aging day increased during the first few weeks of aging. The steaks utilized for shear force were taken consecutively from the anterior end of the strip loin. Previous research suggests that steaks from those locations should have similar shear force values, likely eliminating the impact of steak location on tenderness [22,23]. The WBSF values for each aging period are below the established threshold for tenderness (4.6 kg) as perceived by consumers as outlined by Shackelford et al. [24]. Additionally, the difference among days is within the 0.5 kg of force described by Miller et al. [25] as the difference in shear force detectable by consumers preparing steak in their own home, indicating that the differences in shear force based on aging day are likely not detectable by the average consumer.

Cook loss was not impacted by treatment in the current study. These data are similar to the impact of high-voltage ES on cook loss of beef steaks [5,12] or bison steaks [16]. Additionally, Wiklund et al. [10] observed no effect of ES on drip loss of steaks from red deer. However, when evaluating treatment of beef carcasses with 100 V of ES at 1 h postmortem Savell et al. [26] observed increased cook loss for ES vs. control carcasses. However, cook loss in the current study was impacted by aging day. Similar results were observed by Shanks et al. [27] when evaluating cook loss over 35 d postmortem. Increases in cook loss over time may be the result of damage to cellular membranes, which would enable a greater amount of water to leak out of the muscle during cooking [27].

Steaks from ES sides in the current study were lighter, more red, and more yellow than steaks from Control sides. Similar results were observed in beef [5,6,26,28] and in bison [16]. The increased color values can be attributed to the increased oxygen permeability of the meat as a result of the damaged muscle fibers. Weakened muscle structure caused by the intense contractions that occurred during the ES treatment can allow oxygen to penetrate deeper into the muscles, resulting in a thicker layer of oxymyoglobin formation compared to non-stimulated carcasses [6,29,30].

#### **5. Conclusions**

The goal of this study was to evaluate the effects of varying levels of low-voltage ES on beef quality traits. Collectively, these results demonstrate that low-voltage ES can be an effective means to improve the tenderness and instrumental color scores of beef carcasses without increasing cook loss, potentially improving consumer satisfaction. Within this study the only differences observed between the ES40 and ES80 treatments were the early postmortem pH levels. Thus, beef processing facilities that implement low-voltage ES immediately before carcass chilling may be able to reduce the ES voltage levels to 40 V without detrimentally impacting the meat quality characteristics expected with increased voltages. Additionally, these data show that the desired appearance and palatability benefits of high-voltage ES may be attainable using decreased voltages.

**Author Contributions:** Conceptualization, C.B., J.K.G., K.U. and A.B.; methodology, C.B. and A.B.; validation, C.B., J.K.G., K.U. and A.B.; formal analysis, C.B. and A.B.; investigation, C.B., J.K.G., K.U. and A.B.; resources, J.K.G., K.U. and A.B.; data curation, C.B., J.K.G., K.U. and A.B.; writing—original draft preparation, C.B.; writing—review and editing, C.B., J.K.G., K.U. and A.B.; visualization, C.B. and A.B.; supervision, A.B.; project administration, A.B.; funding acquisition, J.K.G., K.U. and A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by state and federal funds appropriated to South Dakota State University including support from the U.S. Department of Agriculture National Institute of Food and Agriculture, through the Hatch Act (Accession number 1005460).

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy restrictions.

**Acknowledgments:** The authors express their gratitude to DemKota Ranch Beef (Aberdeen, SD) for their assistance in data collection and support of this project.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

