Comparison of granulometric and textural
characteristics of in vitro food bolus of beef- and
plant-based patties

M. Giron, F. Oosterlinck, L. L Steenhorst-Slikkerveer, O. Francois, Peyron
M-A

► To cite this version:

M. Giron, F. Oosterlinck, L. L Steenhorst-Slikkerveer, O. Francois, Peyron M-A. Comparison of granulometric and textural characteristics of in vitro food bolus of beef- and plant-based patties. Food Hydrocolloids, 2025, 163, pp.111071. 10.1016/j.foodhyd.2025.111071. hal-05017819

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# Comparison of granulometric and textural characteristics of *in vitro* food bolus of beef- and plant-based patties

Giron M<sup>a</sup>, Oosterlinck F<sup>b</sup>, Steenhorst-Slikkerveer L<sup>b</sup>, François O<sup>c, D</sup>, Peyron M-A<sup>a, *, D</sup>

<figure><img src="image_3.png" /></figure>

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## ARTICLE INFO

**Keywords:**

Plant-based meat analogue

Artificial mastication

Food oral processing

Food bolus

Granulometry

Texture

## ABSTRACT

For some years now, consumers are progressively switching their diet to a more plant-based (PB) one for various reasons such as climate, environmental, and/or health concerns. One path is the one-on-one replacement of meat by PB analogues and hereto the latter need to give a similar sensory perception as traditional meat. However, current PB analogues do not yet have a comparable texture as their meat counterparts nor are they the same in terms of food oral processing (FOP). The food industry is currently working to overcome this challenge by developing new ingredients and/or processing techniques. In this study a method is developed to quantify and understand the textural and oral processing differences between beef and PB patties. This method will increase the knowledge on food bolus characteristics of plant-based products during mastication and this can be expected to provide new approaches in the development of products intended to generate similar oral sensation as that produced during consumption of meat products.

Hereto, samples of three commercially available plant-based (Beyond B, Sensational S, Ereburger E) and one beef patties (Albert Heijn's Ultimate beef burger) were masticated in the AM² masticator apparatus, which was programmed to mimic the *in vivo* patty bolus characteristics. For that, the AM² parameters were finetuned per patty type so that *in vitro* particle size distribution simulated the *in vivo* one. Then, the *in vitro* boluses of all burgers collected after 1/3rd, 2/3rd and at the end of masticatory sequences were characterised in terms of granulometry and for textural features acquired with the texture profile analysis test.

Results demonstrated that to imitate the *in vivo* bolus characteristics, specific AM2 programming parameters were required per patty, with the Beef Burger requiring more shear deformation to be disrupted. Depending on the type of patty, the boluses displayed granulometry and textural differences. Progress of fragmentation along mastication depended on the patty type. Burger S was the most fragmented and Burger B bolus contained more large particles than others. All PB patties produced softer and less chewy boluses as compared to the Beef Burger.

This method to quantify the FOP of PB patties throughout the masticatory sequence and the insights that were produced are undoubtedly interesting to help in the further development of PB meat substitutes intended to simulate organoleptic characteristics of traditional meat products.

## 1. Introduction

In recent years, the consumer demand for plant-based (PB) meat substitutes has been growing, mostly for ethical, environmental and/or health concerns, and this has been driving a further development of these meat alternatives (Ismail, Hwang, & Joo, 2020; McClements & Grossmann, 2021; Weinrich, 2019). To meet this growing demand as well as the consumers' expectations, agri-food companies strive to produce increasingly realistic meat substitutes. Besides the fact that

consumer perception is already a critical issue for meat products, the challenge in developing meat analogues is to mimic the visual appearance, texture, juiciness, taste and nutritional properties of the former, reflected by the significant rise in the associated literature (Bohrer, 2019; Font-i-Furnols & Guerrero, 2014; Ilić, Djekic, Tomasevic, Oosterlinck, & van den Berg, 2022; Kyriakopoulou, Dekkers, & van der Goot, 2019; Malav, Talukder, Gokulakrishnan, & Chand, 2015; Michel, Hartmann, & Siegrist, 2021; Starowicz, Kubara Poznar, & Zieliński, 2022; Tarrega, Rizo, Murciano, Laguna, & Fiszman, 2020; Troy & Kerry,

<footnote><sup>a</sup> Clermont Auvergne University UCA, INRAE, UNH, Human Nutrition Unit, F-63000, Clermont-Ferrand, France</footnote>

<footnote><sup>b</sup> DSM-Firmenich Center for Food Innovation, Delft, the Netherlands</footnote>

<footnote><sup>c</sup> Clermont Auvergne University UCA, INRAE, CROC, F-63100, Clermont-Ferrand, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: marie-agnes.peyron@inrae.fr (P. M-A).</footnote>
2010). The development of meat alternatives to replace meat in the diet depends on the combination of functional ingredients and technological processes (Kyriakopoulou, Keppler, & van der Goot, 2021). The structure of a food determines its mechanical features and therefore also its food oral processing and sensory perception. The uniqueness of meat structure is difficult to imitate and the success would depend a lot on how the plant protein source can adequately resemble meat proteins (Y. Chen Ping et al., 2022; Fiorentini, Kinchla, & Nolden, 2020; Onwezen, Bouwman, Reinders, & Dagevos, 2021; Siddiqui et al., 2022). Formulated from plant proteins (soy, pea, lupin, wheat, rapeseed ...), coloured with beet juice, thickened with egg white. methylcellulose and/or coconut oil, ...etc., the objective is to provide a product that comes as close as possible to the appearance, mouthfeel and taste of meat (Ilic, Djekic, Tomasevic, van den Berg, & Oosterlinck, 2023).

For meat and meat products, texture has long been reported as an important driver of consumer preference. Meat texture is as a multi-parameter sensory attribute and it is particularly important to consider the time-dependent evolution of the sensory properties throughout the in-mouth processing (Feng, Blank, & Liu, 2022; Fonti-Furnols & Guerrero, 2014). The meat bolus is formed thanks to a combination of a disruption of the meat architecture and incorporation of saliva, both being significantly influenced by the initial meat features (Mioche, Bourdiol, & Monier, 2003; Pematilleke, Kaur, Adhikari, & Torley, 2020; Yven, Culioli, & Mioche, 2005). This food transformation, so-called bolus formation, is governed by the structural and physicochemical parameters of the food and could be accountable for the perception in a dynamic way (Devezeaux De Lavergne, van de Velde, & Stieger, 2017; Koç, Vinyard, Essick, & Foegeding, 2013; Pascua, Koç, & Foegeding, 2013). For meat analogues, an unsatisfactory quality in terms of texture has often been reported (Michel et al., 2021) Because of the aforementioned influence of the food bolus formation, an efficient way to address the gap between analogues and traditional meat products would be to better understand the links between food structure, the complex structural arrangement, its breakdown during mastication and ultimately its perception (Wilkinson, Dijksterhuis, & Minekus, 2000). As a first step, a better understanding and quantification of what happens during mastication is key.

The primary role of mastication is to transform solid foods into a food bolus that can be swallowed easily, safely and without pain as well as to help begin the process of digestion. This is achieved through a set of processes described as Food Oral Processing (FOP) (Chen, 2009) which combines the mechanical reduction of food into smaller fragments, lubrication by saliva to form a cohesive and plastic food bolus that can be swallowed without food particles escaping into the respiratory tract, and the action/pressure of the tongue. In fact, the combination of in-mouth breakdown and saliva impregnation of the food fragments during FOP is highly dependent both on food characteristics and oral physiology, and consequently always a key issue in sensory perception (J. Chen, 2014; Guo, 2021; Koç et al., 2013). As already well described for meat and meat products, FOP and characteristics of the resulting bolus are impacted by the type of meat, technological processing and/or cooking (Ilić et al., 2022; Mioche et al., 2003; Pematilleke et al., 2020, 2021; Yven et al., 2005). These factors have been shown as impactful for plant-based alternatives as well (Ilic et al., 2022). Considering the recent growth in PB meat alternatives development, only limited data is available on the mechanisms of their in-mouth transformation, such as fragmentation, juice release, saliva impregnation, etc ..., elicited during masticatory sequence and bolus formation for these meat analogues (Ilić et al., 2022). Some recent studies have begun to address these issues by exploring either the oral processing elicited for this kind of new products, and/or some bolus characteristics such as juiciness (Ilic et al., 2023; Oppen & Weiss, 2023; Y. Zhang, Brouwer, Sala, Scholten, & Stieger, 2024). These very preliminary works confirmed for these products the well-known role of the food structure and oral processing in governing perception of texture and other food characteristics during its oral transformation (Devezeaux De Lavergne, van de Velde, & Stieger,

2017; Guo, 2021; Koç et al., 2013; Pascua et al., 2013; Stokes, Boehm, & Baier, 2013; Çakır et al., 2012).

Thus, a better understanding on sensory perception and appreciation is possible only with more knowledge on what happens in mouth during formation of bolus of these new products.

The main qualitative differences observed between the food boluses from real meat and PB analogues were illustrated for the global progress of material disruption during the *in vivo* masticatory sequence and estimated granulometric features of the bolus (Ilić et al., 2022).

Based on these considerations and to provide more knowledge on FOP of selected meat analogues compared to a beef patty, this study was conducted to describe the characteristics of boluses collected at different time-points of a normal *in vitro* masticatory sequence (performed by the AM² masticator apparatus) of samples of three plant-based and one beef patties. As a first step the AM² parameters were defined per patty so that the swallowable *in vitro* bolus granulometry mimicked the *in vivo* one. Then the *in vitro* boluses were characterised with granulometry and texture measurements in order to outline differences and similarities between the bolus evolution of PB meat analogues and beef patty during mastication.

## 2. Materials and methods

### 2.1. Patty samples and cooking

Fresh commercial patties purchased in the local supermarket of Delft (The Netherlands) and frozen in the lab were used in the present study: a Beef Burger (AH Excellent Ultimate Beef Burger®, Albert Heijn) and three meat analogue patties (Sensational™ Burger, Garden Gourmet; Beyond Burger®, Beyond Meat; Ereburger, De Vegetarische Slager). For practical reasons, the different patties used in this work were assigned the word ‘burger’ coupled with their trade name. The main ingredient was soy, pea and a mix of soy-wheat proteins for the Sensational Burger (Burger S), the Beyond Burger (Burger B), and the Ereburger (Burger E), respectively. Before each experiment, one patty was defrosted for 24 h at 4 °C. Then, the patty was grilled using an induction frying pan on a medium heat after 2 min of pre-warming. The patty was turned every 3 min until the core temperature had reached 74 °C. A probe thermometer was inserted into the core of the patty for the entire duration of the cooking process for the temperature control. Immediately after cooking, the patty was cut in “pie” portions (= standardized isosceles triangle samples with long sides about 3.5–4 cm and short side of 1.5 cm approx.) weighing 5.80 ± 0.04 g and used for *in vivo* and *in vitro* mastication. A total of 480 samples for the 4 patties were used for masticatory sequences and bolus characterization.

### 2.2. Masticatory experiments

The complete characterization of boluses of beef patty and PB meat analogues was obtained *in vitro* using the AM² masticator apparatus which was designed and validated for producing *in vitro* food boluses presenting granulometric properties resembling to those observed in food boluses collected after normal *in vivo* mastication (Mishellany-Dutour et al., 2011; Woda et al., 2010). As a first step, the specific programming of the apparatus was determined for each patty type so that the particle size distribution (PSD) of the boluses produced *in vitro* with the masticator corresponded with that observed *in vivo* at the end of mastication. Hereto, ten young healthy adults, members of the laboratory, with good oral health and accustomed in performing masticatory experiment gave their informed consent to perform masticatory sequences of plant-based and meat patties. Each individual masticated a patty sample (weighing 5.80 ± 0.04 g as described above) as naturally as possible and expectorated the bolus at the moment of the desire of swallowing was felt. The mouth was rinsed with 20 mL of water to recover the residual particles. The number of masticatory cycles required to reach swallowing desire was counted and the masticatory
sequence timed by the experimenter. Immediately after its collection, the food boluses were characterised for granulometric properties. Saliva incorporation was estimated by subtracting the patty sample mass to the bolus mass. Masticatory sequence and bolus collection were performed twice by each of the ten subjects. The mean PSD curves (n = 2x10 = 20) obtained for the boluses collected *in vivo* in place of swallowing served as the reference curve for AM² masticator programming.

During the programming step of the apparatus, the translation movement, which ensures the approaching movement of the mobile jaw to the fixed jaw (simulating the vertical movement of the mandible during closing phase of the masticatory cycle until food crushing) was kept constant for all patties, and rotation speed and rotation angle were adjusted to make the *in vitro* PSD correspond to the *in vivo* one.

The jaw rotation, as specified by rotation angle, partly determines the quantity of shearing applied to the food sample and the gathering of food fragments to place them for the next masticatory cycle and favouring their coating with liquid (as the tongue does in the mouth). The apparatus was run after the sample was placed in the masticatory chamber. After mastication, the bolus particles were thoroughly recovered and analysed by sieving. AM² programming was then progressively adjusted, until *in vivo* and *in vitro* PSD curves graphically overlapped (and superimposition statistically confirmed). The same strategy was applied to determine the mastication programming of each patty type. The final AM² programming for the *in vitro* mastication was validated for each patty with 10 replicates of bolus granulometric measurements.

For texture characterization of boluses collected at the end of the masticatory sequence, boluses were produced with the AM² masticator using the specific program for each type of patty. To characterize the changes in granulometric and rheological bolus characteristics, inter-mediary boluses were collected at 1/3rd and 2/3rd of the complete masticatory sequence. The number of masticatory cycles used for these masticatory time-points was obtained for each patty by dividing by 3 the mean number of cycles realized for the *in vitro* complete masticatory sequence. After collection, intermediate boluses were immediately analysed either for granulometric or texture measurements.

## 2.3. Granulometric analysis of food boluses

Particle size measurements were performed by the sieving method. The recovered *in vivo* or *in vitro* boluses were washed on a nylon cloth with water for particle spreading, and saliva elimination in the case of *in vivo* boluses, and left for 25-35 min (depending on the patty) at 37 °C in a ventilated oven. This step was intended to eliminate surface moisture but not to dehydrate the bolus. The 'dried' bolus was poured onto a stack of nine sieves with apertures of 7.1, 6.3, 4, 2.5, 2, 1.4, 1, 0.8 and 0.4 mm and sieved using an automatic vibrating sieve shaker (Retsch, AS 200 Digit cA) programmed to vibrate at an amplitude of 1.7 mm for 3 min. The particles retained on each sieve were weighed. The results were expressed as the cumulative curves using the mass percentage of the particles falling through each sieve. From each curve, the median particle size (d50), defined as the aperture of a theoretical sieve through which 50% of the food bolus mass can pass, was determined. Ten *in vitro* boluses collected at each of the three time-points of the masticatory sequence (1/3rd, 2/3rd and swallowing) were analysed and treated as replicates for each patty.

## 2.4. Textural analysis of food bolus

The textural properties of food boluses produced by the AM² were measured using the Texture Profile Analysis (TPA) test and TrapeziumX software (Shimadzu EZ-LX apparatus; Japan; software v.1.5.6). To check bolus geometry prior to testing, the bolus was carefully positioned to match the cylindrical shape of the container (35 mm diameter). It was then subjected to two successive compression cycles performed at 65% deformation of the initial bolus height and at a constant displacement rate of 50 mm min⁻¹ for the flat piston head (28 mm diameter) as

previously described (Blanquet-Diot, François, Denis, Hennequin, & Peyron, 2021). The thickness of the sample was 11.9 ± 0.2 mm (n = 120 boluses for 4 patty types, 3 time-points and 10 replicates of each). Hardness, adhesiveness, cohesiveness, springiness, gumminess and chewiness values were extracted from each TPA curve (Godschalk-Broers, Sala, & Scholten, 2022; Pons & Fiszman, 1996) and used for comparison of textural characteristics of bolus of the different patties. Ten *in vitro* boluses for each masticatory time-point and for each patty were used as replicates.

## 2.5. Statistical analyses

All the data were expressed as mean ± standard deviation (SD). Error bars are shown on Fig. 1 and omitted on curves of other figures for readability. Statistical analyses were performed using SPSS software (v29, IBM SPSS Statistics, USA). The absence of significant difference between the *in vivo* and *in vitro* PSD curves, step validating the AM² programming, was verified by ANOVA in a repeated-measures General Linear Model (GLM). The existence of significant differences, between the food boluses collected at different time-points of the sequence or between the four patties, was checked by several one-factor ANOVAs for all the variables measured: number of chewing cycles, duration and frequency of mastication, quantity of saliva incorporated, median particle size (d50) and texture analysis parameters (hardness, adhesiveness, cohesiveness, springiness, gumminess and chewiness). The existence of a significant difference between the PSD curves was tested by ANOVA in a repeated-measures General Linear Model (GLM), with the *within-subject* factor being the different sieves and particle mass in each sieve being the measure (*between-subject* factor). No significance between two PSD curves in this repeated-measures ANOVA model corresponds to a non-significant difference between the two curves. In all cases, the explanatory factors tested were the three time-points of the masticatory sequence (1/3rd, 2/3rd and swallowing) and the type of patty chewed. When a significant difference was observed (p ≤ 0.05), Tukey's *post-hoc* test was applied to identify where were the differences.

# 3. Results

## 3.1. Programming of AM² apparatus regarding bolus particle size at swallowing

*In vivo* masticatory sequences differed in terms of number of masticatory cycles, sequence duration and mastication frequency, the Beef Burger being masticated longer (with 26 ± 5 cycles in 18.7 ± 4.95 s) and other patties rather shorter (12.35 ± 5.28 s, 12.05 ± 3.73 s, and 13.50 ± 4.55 s for Burger S, B and E, respectively). The Burger S was masticated with a higher masticatory frequency than others. The *in vivo* food boluses were significantly different in terms of granulometry with a mean median particle size (d50) varying between 5.1 ± 0.4 mm for the Beef Burger and 4.3 ± 0.7 mm for the Burger S (Table 1). The *in vivo* Beef Burger bolus incorporated more saliva (25.53 % of bolus mass) compared to the percentage of saliva impregnation in other patties boluses (20.68 %, 18.67 % and 22.26 %, for Burger S, B and E, respectively), but this might be linked to the fact that the Beef Burger was masticated longer. Relative to the number of masticatory cycles realized until swallowing, the percentage of saliva incorporated in the boluses was 0.96, 1.28, 1.02 and 1.16 % per cycle for Beef, S, B and E burger boluses, respectively, and was always greater for boluses collected at the beginning (1/3) of the masticatory sequence with 1.53, 1.75, 1.82 and 2.21% of saliva, respectively.

In order to replicate the *in vivo* PSD data, the AM² apparatus was programmed to perform 26, 16, 18 or 20 masticatory cycles for the Beef Burger and Burgers S, B and E, respectively (Table 1). The programming for each of the four patties were adjusted by changing the values of displacement of the mobile jaw in rotation and ensuring the specific shear adjusted for each patty. The main parameter that was changed to
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Bolus granulometry validating the AM² masticator apparatus programming. Mean curves of particle size distribution of food boluses of Beef Burger [A], Burger S [B], Burger B [C] and Burger E [D] collected at the end of *in vivo* (dotted curves) and *in vitro* (solid curves) masticatory sequences. Curves are expressed as mean cumulative percentages of particle mass passing through each sieve (±SD; n = 20 for *in vivo* and n = 10 for *in vitro* conditions). Comparisons of these distribution curves were used to validate the programming of the AM² apparatus for further bolus production. Mean median particle size values (d50) were indicated on *in vivo* mean curves. No significant difference was observed between *in vivo* and *in vitro* bolus particle size distribution for each burger (ANOVA repeated-measures GLM, p > 0.05).</figcaption></figure>

**Table 1**

Data used for programming the AM² masticator apparatus. Number of masticatory cycles, either measured *in vivo* (n = 20) or applied *in vitro* on the AM² masticator apparatus to produce a food bolus ready for swallowing for Beef and plant-based burgers (Burgers S, B and E; n = 20). AM² jaw parameters selected for final programming and the median particle size (d50) of *in vivo* and *in vitro* bolus (once the programming were validated against *in vivo* particle size distribution) were given for each burger. Significance was obtained with ANOVA testing the existence of differences between burger bolus for the number of masticatory cycles (n = 20) and d50 (n = 20 *in vivo* and n = 10 *in vitro*). Different letters in a line indicate significant differences in values between burgers.

<table><thead><tr><th></th><th></th><th></th><th>Beef Burger</th><th>Burger S</th><th>Burger B</th><th>Burger E</th><th>significance</th></tr></thead><tbody><tr><td rowspan="2">masticatory sequence</td><td rowspan="2">number of cycles</td><td>in vivo</td><td>26 ± 5 a</td><td>16 ± 6 b</td><td>18 ± 4 b</td><td>20 ± 6 a</td><td rowspan="2">F=12.9 p&lt;0.001</td></tr><tr><td>in vitro</td><td>26</td><td>16</td><td>18</td><td>20</td></tr><tr><td rowspan="2">AM2 jaw movement</td><td rowspan="2">rotation speed (°/s)</td><td>in vivo</td><td>80</td><td>80</td><td>100</td><td>100</td><td rowspan="2">–</td></tr><tr><td>angle stroke (°)</td><td>90</td><td>70</td><td>50</td><td>90</td></tr><tr><td rowspan="2">bolus characteristic</td><td rowspan="2">median particle size (mm; d50)</td><td>in vivo</td><td>5.1 ± 0.4 b</td><td>4.3 ± 0.7 c</td><td>5.3 ± 0.4 a</td><td>4.9 ± 0.6 b</td><td rowspan="2">F=15.6 p&lt;0.001<br>F=44.5 p&lt;0.001</td></tr><tr><td>in vitro</td><td>5.0 ± 0.4 b</td><td>4.1 ± 0.2 c</td><td>5.3 ± 0.3 a</td><td>4.9 ± 0.3 b</td></tr></tbody></table>

ensure the *in vivo/in vitro* adjustment was the “angle stroke” which is influences the amount of shearing contact applied on the sample. These values were specifically set up at 90°, 70°, 50° and 90°, for the Beef Burger, Burgers S, B and E, respectively (Table 1). The validity of the

program parameters of Table 1 were validated since the *in vivo* and *in vitro* particle size distribution curves were superimposed (Fig. 1; p > 0.05). Compared to *in vivo* data, very similar mean d50 values were extracted from *in vitro* individual PSD curves of each patty (Table 1)
confirming the validity of the artificial masticatory program in pro-
ducing similar boluses. *In vivo* mean d50 values were indicated as
illustrative examples on Fig. 1.

Compared to PB patties, significantly more masticatory cycles were needed to form a swallowable bolus of Beef Burger (F = 12.9; p < 0.001; Table 1). The Burger S bolus was characterized by a significantly greater proportion of small particles compared to the Beef Burger and the two other PB patties (F = 15.6; p < 0.001; Table 1) while its mastication needed the least number of masticatory cycles (Table 1).

3.2. Bolus granulometry

**3.2.1. Changes in bolus PSD throughout mastication**

The analysis of the PSD observed for bolus collected at the three
time-points of the *in vitro* masticatory sequence gives insight into the
progressive changes of the fragmentation of the patty matrices (Fig. 2).

For all the 4 patty types considered, the fragmentation of the patty
matrix is observed to be progressive during the masticatory sequence.
Nevertheless, this fragmentation followed significantly different varia-
tions depending on the patty type (Fig. 2). The Beef Burger bolus was
only partially fragmented after 1/3rd of masticatory sequence but
almost ready for swallowing after 2/3rd of the masticatory sequence
(Fig. 2A; F = 109.7, p < 0.001). Most of the fragmentation of the Burger
S matrix was completed during the last part of the masticatory sequence
(F = 435, p < 0.001; Fig. 2B). There was no significant difference be-
tween the three curves obtained for the Burger B (p > 0.05) suggesting
that the food bolus is mainly fragmented in the first third of the chewing
sequence and early ready for swallowing (Fig. 2C). Finally, the Burger E
was characterized by a gradual fragmentation along the masticatory
sequence (Fig. 2D; F = 25.3, p < 0.001). That both the Beef Burger and
Burger B bolus PSD remains nearly constant for the last 1/3rd of the
matication suggests that, driven by the physics of fracture propagation,

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Progressive fragmentation of *in vitro* bolus of each burger. Mean curves of particle size distribution of *in vitro* food boluses of Beef Burger [A], Burger S [B], Burger B [C] and Burger E [D], collected at three different time-points of the complete masticatory sequence: after 1/3rd (dotted curves, square), after 2/3rd (dotted curves, triangle) and at the end of the complete masticatory sequence (swallowing time; solid curves, filled round). Curves are expressed as mean cumulative percentages of particle mass passing through each sieve (±SD; n = 10 in each series). Different small letters in a graph indicate significant differences between the curves (ANOVA repeated-measures GLM and Tukey's post-hoc test, p ≤ 0.05), ns: not significant.</figcaption></figure>
the applied deformation is no longer sufficient to fragment these patties
further, and that *in vivo* the fact that these boluses are not swallowed
upon attaining this final PSD is linked to the need for additional lubri-
cation of the boluses, something which warrants further study.

3.2.2. Granulometry of bolus burger throughout mastication

After 1/3rd of the complete masticatory sequence, PSD curves ob-
tained for the 4 patties bolus were significantly different (Fig. 3A; F =
14.8, p < 0.001). Early in the masticatory sequence (1/3rd), Beef Burger
and Burger S bolus presented the same granulometric profile (p > 0.05)
with a great proportion of large particles, differing from the bolus
granulometry of Burger B and Burger E which were both more frag-
mented at this moment of the masticatory sequence. PSD characterizing
the bolus after 2/3rd of the complete masticatory sequence highlighted
that the bolus of Burger S was very different from others and contained a
high proportion of very large particles (~70% were larger than 7 mm;
Fig. 3B; F = 70.6, p < 0.001), while its was composed of significantly

much more small particles than others at the moment it could be swallowed (Fig. 3C; p < 0.001).

At the moment of swallowing, boluses from the 4 burgers were significantly different (F = 39, p < 0.001). It was the bolus of Burger B that was composed with the larger particles (p = 0.002). The PSD characterising the swallowable bolus was significantly different between the 3 plant-based patties, the PSD of Burger E bolus being similar to that of Beef Burger (p > 0.05). This was confirmed by the mean in vitro d50 values (Fig. 3D and Table 1; F = 44.5, p < 0.001). The mean d50 values of Beef Burger and Burger E bolus were not significantly different (Fig. 3D; p > 0.05) but both significantly differed from bolus of Burger S on a side with a smaller mean value and Burger B on the other side with a greater mean value (p < 0.001).

As noted above, the bolus of Burger B could be swallowed with a
greater proportion of large particles compared to other patties, and the
swallowable boluses of Beef Burger and Burger E presented a similar
median particle size (p > 0.05).

<figure><img src="image_6.png" /><figcaption>Fig. 3. Comparison of bolus granulometry at each masticatory time-point. Mean particle size distribution curves of *in vitro* food boluses of Beef Burger, Burger S, Burger B and Burger E collected at three different times during the *in vitro* masticatory sequence: [A] after 1/3rd of the complete sequence, [B] after 2/3rd of the complete sequence and [C] at the end of the masticatory sequence (swallowing point); and [D] mean median particle size values (d50) extracted from individual curves of bolus of each burger (±SD; n = 10). Curves are expressed as cumulative percentages of particle mass passing through each sieve (n = 10 in each series). Different small letters indicate significant differences between curves (ANOVA repeated-measures and Tukey's *post-hoc* test, p ≤ 0.05) or between d50 values (ANOVA and Tukey's *post-hoc* test, p ≤ 0.05).</figcaption></figure>
## 3.3. Textural characteristics of burgers bolus

### 3.3.1. Changes in bolus texture throughout mastication

During the masticatory sequence, texture characteristics of the boluses evolved in different directions depending on the patty but few significant differences were observed (Fig. 4). Bolus hardness did not significantly change throughout the masticatory sequence in most cases (p > 0.05), except for Burger S which showed a significant small decrease at the end of masticatory sequence (Table 2 and Fig. 4A). Bolus adhesiveness increased with mastication progress but significantly for bolus of Burger E only (F = 13.7, p < 0.001; Fig. 4B). The most important change in adhesiveness of this bolus appeared in the last part of the masticatory sequence (Fig. 4B and Table 2). The Beef Burger was significantly more elastic at the end of mastication compared to the 2/

3rd of the masticatory sequence (F = 5.9, p = 0.023; Fig. 4C and Table 2) and the values of chewiness therefore also significantly increased (F = 5.7, p = 0.026; Fig. 4F and Table 2; chewiness being hardness x cohesiveness x springiness). The progress of mastication had the opposite effect on Burger B since the bolus significantly lost springiness (F = 6.7, p = 0.02; Fig. 4C and Table 2) and chewiness (F = 5.0, p = 0.012; Fig. 4F and Table 2) but again during the last third of the masticatory sequence. Finally, cohesiveness decreased along the masticatory sequence for the Burgers S (F = 4.7, p = 0.021; Fig. 4D) and E (F = 5.1, p = 0.01), with a significant impact on gumminess for Burger S (F = 5.7, p = 0.009; Fig. 4E) but not for Burger E (p > 0.05).

### 3.3.2. Textural characteristics of bolus at the end of mastication

At the swallowing step, bolus hardness, adhesiveness, springiness,

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Bolus textural features. Textural characteristics of *in vitro* food boluses from Beef Burger, Burger S, Burger B and Burger E collected at the beginning of mastication (1/3rd of sequence, white bars), after 2/3rd (grey bars) and at the end of masticatory sequence (swallowing; black bars). Hardness [A; in Newton], adhesiveness [B; in N.mm], springiness [C; unitless], cohesiveness [D; unitless], gumminess [E; in N] and chewiness [F; in N] were obtained from the Texture Profile Analysis test (TPA) performed at 65% deformation of the bolus (n = 10 in each series). \*: p < 0.05; \*: p < 0.01; \*: p < 0.001 (ANOVA and Tukey's *post-hoc* test, p < 0.05). Different small letters in a graph indicate the existence of significant differences between characteristics of bolus from different patties at swallowing (p < 0.05). *p* values are indicated in Table 2.</figcaption></figure>
<caption>**Table 2**</caption>

Statistical significance between data obtained with ANOVAs for *in vitro* bolus textural characteristics at the different time-points, 1/3, 2/3 and end (swallowing) of the masticatory sequence for the 4 burger types. Data refers to mean values presented on Fig. 4. Significance are given by the *p* values, the ‘t’ indicating a tendency towards significance and NS no significance.

<table><thead><tr><th rowspan="2"></th><th rowspan="2">masticatory sequence time-points comparison</th><th colspan="6">bolus textural characteristics</th></tr><tr><th>hardness</th><th>adhesiveness</th><th>springiness</th><th>cohesiveness</th><th>gumminess</th><th>chewiness</th></tr></thead><tbody><tr><td><strong>Beef Burger</strong></td><td>1/3 vs 2/3 2/3 vs swallowing 1/3 vs swallowing</td><td>NS NS NS</td><td>NS NS NS</td><td>NS p = 0.011 p = 0.023</td><td>NS NS NS</td><td>NS NS NS</td><td>NS p = 0.013 p = 0.026</td></tr><tr><td><strong>Burger S</strong></td><td>1/3 vs 2/3 2/3 vs swallowing 1/3 vs swallowing</td><td>NS p = 0.03 NS</td><td>NS NS NS</td><td>NS t: p = 0.064 NS</td><td>t: p = 0.062 NS p = 0.021</td><td>NS t: p = 0.059 p = 0.009</td><td>NS NS NS</td></tr><tr><td><strong>Burger B</strong></td><td>1/3 vs 2/3 2/3 vs swallowing 1/3 vs swallowing</td><td>NS NS NS</td><td>NS NS NS</td><td>NS p = 0.006 p = 0.02</td><td>NS NS NS</td><td>NS NS NS</td><td>NS NS p = 0.012</td></tr><tr><td><strong>Burger E</strong></td><td>1/3 vs 2/3 2/3 vs swallowing 1/3 vs swallowing</td><td>NS NS NS</td><td>NS p &lt; 0.001 p &lt; 0.001</td><td>p = 0.023 NS NS</td><td>NS NS p = 0.01</td><td>NS NS NS</td><td>p = 0.003 NS NS</td></tr></tbody></table>

cohesiveness, gumminess and chewiness (F = 32.5, p < 0.001) were significantly different between the patties (F = 25.9, p < 0.001; F = 11.3, p < 0.001; F = 3.2, p = 0.035; F = 40.6, p < 0.001; F = 35.9, p < 0.001; F = 32.5, p < 0.001, respectively; Figs. 4 and 5). Except for adhesiveness, values of bolus characteristics were always greater for the Beef Burger (Fig. 5). More specifically, the bolus of Beef Burger was harder than the bolus of the Burgers S and B (p < 0.001) and to a lesser extent than Burger E (p = 0.048). The swallowable bolus of Burger S had the lowest hardness value and there was no significant difference between the bolus of Burgers B and E (p > 0.05). The results for cohesiveness and gumminess followed a similar profile with the highest value for the bolus of Beef Burger, followed by bolus of Burgers B, E and finally Burger S (p < 0.001). The Beef Burger had the lowest bolus adhesiveness value, statistically different from the other samples (p = 0.007) and this parameter was similar between the three PB patties (p > 0.05). For the chewiness, once again the Beef Burger bolus presented the highest value and the Burger S bolus the lowest value. All the patties bolus presented similar springiness (p > 0.05). To resume, the bolus of Beef Burger was characterized with TPA parameters clearly differing from that obtained

for the PB analogues, harder, more elastic, cohesive and gummy, and less adhesive, relatively opposite to Burger S bolus, and forming a statistically separate group (p < 0.001), except for springiness which is relatively little impacted by the type of burger (Fig. 4). The swallowable bolus of PB patties was generally softer, chewy and gummy, less recoverable more adhesive than the bolus of Beef Burger (Fig. 5).

Interestingly, there was a significant positive correlation between the median particle size (d50) and hardness (r = 0.37; p = 0.018), cohesiveness (r = 0.41; p = 0.009) and logically with gumminess (which is hardness x cohesiveness; r = 0.36; p = 0.025) of the swallowable bolus of all patties. Indeed, the more the food bolus is composed of tiny particles, the more the bolus is soft, disintegrated and requires less energy to break.

## 4. Discussion

This *in vitro* work provided interesting insights regarding the evolution of the bolus features, throughout mastication until the moment of swallowing. It shows that this evolution depended on the beef- or PB meat substitute characteristics. Despite recent works describing some specific aspects of the FOP of PB and beef patties (Ilić et al., 2022; Y. Zhang, Brouwer, et al., 2024; Y. Zhang, Sala, Scholten, & Stieger, 2024), there is only limited understanding on the mechanisms of oral behaviour of this kind of products and the AM² device permitted to overcome several obstacles associated with the *in vivo* study of FOP (Peyron & Woda, 2016). As for many other food materials, it produced *in vitro* burger bolus in the selected and permanent control of oral conditions, while guaranteeing similar physical properties compared to the *in vivo* bolus (Mishellany-Dutour et al., 2011). Moreover, it simplified the collection of the whole bolus during the masticatory sequence for extensive analysis in a routine bolus production (Peyron et al., 2019). AM² implementation provided more information on the bolus characteristics and their comparison. Such a comparative analysis gives quantitative insights that potentially help in optimizing the PB meat substitutes composition to best mimic real meat products. Additionally, the difference in AM² programming parameters gave further insight in the textural differences between the different patties types, quantifying to a certain extent the different chewing activity required to masticate the different samples, with the Beef Burger requiring more shear to be applied. Interestingly, the Beef Burger and the Burger B had reached their maximum amount of fragmentation after already 2/3rd of the mastication sequence, suggesting that *in vivo* further mastication is linked to improving the lubrication of the bolus, which warrants further work.

The different patty materials tested clearly displayed distinct patterns in terms of granulometry and textural characteristics both for the evolution of the bolus throughout the masticatory sequence and for the bolus ready for swallowing. Observed *in vivo*, saliva incorporation in the bolus was also progressively increased along the masticatory sequence, with a larger proportion for the Beef Burger in link with the longer masticatory sequence. Nevertheless, when expressed as the number of

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Burger bolus characteristics. Kiviat plot schematically presenting the different granulometric and textural bolus characteristics extracted from particle size distribution curves (PSD) and from Texture Profile Analysis (TPA test performed at 65% deformation) of food bolus collected at the end of *in vitro* mastication (swallowing) of the four burgers. Values of median particle size, adhesiveness, springiness and cohesiveness were multiplied by a coefficient to display all variables on the same graph scale. Significant differences between burger bolus are detailed on Figs. 3 and 4.</figcaption></figure>
masticatory cycles realized for each period of the masticatory sequence, saliva was mainly incorporated during the early period (first third) of mastication for all patties and in greater proportion for the Burger S and lower for the Beef Burger. In terms of granulometry, the boluses of the three PB patties were different, PSD of bolus of Burger E collected at swallowing being very similar to that of Beef Burger. The most different PSD was observed for the bolus of Burger S which contained a greater proportion of small particles than all other bolus. Textural characteristics of the bolus ready for swallowing of the three PB patties were globally different from that of the Beef Burger, the former being softer, more adhesive, less recoverable and less gummy and chewy than the Beef Burger bolus. The PB meat analogues boluses displayed opposite variation to that of Beef Burger for springiness, which increased along the masticatory sequence while it decreased for PB meat analogues. The bolus of Burger E was comparable to that of Burger B for all textural variables. At the moment of swallowing, the Burger S bolus was the most fragmented, adhesive, softened and the less recoverable, gummy and chewy. In short, the swallowable Burger S bolus was the most different to the Beef one. Boluses of Burgers B and E presented intermediate profiles between S and Beef burgers. This experimental approach, where boluses were produced in AM² without saliva addition, depicted the textural characterization of boluses, permitting the analysis of breakage mechanisms specifically driven by structure of the matrices without saliva interference. Obviously, saliva impregnation during mastication likely plays an important role especially in terms of juiciness perception (Y. Zhang, Brouwer, et al., 2024; Y. Zhang, Sala, et al., 2024). What was interesting was the good correspondence of textural characteristics of bolus with data obtained for the corresponding patties previously described by Ilic et al. (Ilic et al., 2023). Indeed, the Beef Burger was shown by these authors to be harder, less adhesive, more springy, recoverable and chewy as it was described in the present work for the corresponding bolus. Despite several differences in tools and experimental approach, some correspondence on the effect of the progress of the masticatory sequence can be drawn with results from a recent work conducted by Zhang et al. on the same kind of products (Y. Zhang, Sala, et al., 2024). Obviously, a progressive oral breakdown of the matrices was described both for beef and PB patties. These authors also observed that juiciness, differing with cooking temperature, was not linked with changes in PB bolus properties while fluid expelled from Beef Burger bolus varied (Y. Zhang, Sala, et al., 2024) without large variations in oral processing (Y. Zhang, Brouwer, et al., 2024).

The different programming specifically adjusted to the fragmentation mode of each matrix structure gives further insight for an in-depth analysis of the mechanical behaviour during food fragmentation. While an angle stroke of 50° was used to breakdown the Burger B sample, a greater angle stroke value was selected for the Beef Burger suggesting that it probably required more applied shear deformation than the Burger B, Burger S set up value for angle stroke indicating an intermed diary condition. The similar angle stroke value set up for Beef Burger and Burger E must be considered in association with the higher rotation speed value used to masticate the Burger E, indicating a shorter time of contact, thus less applied shear force, between the teeth and sample for the latter. During the masticatory movements, the oral perception of the food characteristics drives the implementation of the best ratio of compression and shear stresses for an optimal food matrix fragmentation. From a physiological point of view, this objective is satisfied by a good combination of jaw pattern which is adapted to produce the expected compression/shear constraints during mastication. Agrawal's team suggested that the mechanical properties of foods influence the degree of lateral movement of the mandible while the vertical amplitude (not translated on AM² device) could more reflect the external surface properties (Agrawal, Lucas, & Bruce, 2000). Indeed, larger lateral displacement of the mandible during the masticatory cycle was gener ally observed for meat products (Kohyama & Mioche, 2004; Komiyama et al., 2003; Lepetit & Culiol, 1994). Jaw lateral movements are prob ably linked to meat structure and especially to fibre length, and

consequently count in adjustment of mastication to meat product features (Oppen, Attig, Weiss, & Krupitzer, 2023; Oppen, Young, Piepho, & Weiss, 2023). Therefore, and even if shearing is a complex rheological operation and difficult to interpret, these fibrous foods are logically subjected to more shear oral forces, as it was probably the case for the Beef Burger in the present work.

Differences in the profile of the structure disruption were also distinguishable all along the bolus formation and for all patties. Ilic et al. already evoked that the breakdown of plant-based patties was faster than of the Beef Burger (Ilić et al., 2022). Fragmentation of Burger E bolus was gradual along the masticatory sequence while the final granulometry was almost reach during the early phase of the masticatory sequence for the Burger B bolus, at the half for the Beef Burger and at the last stage of mastication for the Burger S bolus. With the present study, a deeper description of the kinetic of disruption of these patties was proposed, but as Ilic et al. (2022) already suggested, these different fragmentation profiles and rheological evolution throughout FOP could be attributable to their composition and recipe. Indeed, Burger S, containing soy proteins, and Burger E, containing a mix of soy and wheat proteins, resulted in bolus with different granulometric and textural behaviours evidently due to different initial matrix structures.

An increasing number of protein sources have been explored to produce meat analogues, often used as mix of various origins such as soy, wheat, pea and fungi proteins (Bohrer, 2019; Feng et al., 2022). The protein source is highly relevant in acting as a structural network and conditioning the textural perception. Current research in the development of meat analogues focuses on understanding the relationship between structure and the corresponding functionalities of PB products (Tyndall, Maloney, Cole, Hazell, & Augustin, 2024). But clearly more knowledge on the FOP mechanisms and bolus features, on which textural perception are based, would undoubtedly provide interesting guidelines to obtain a better structure and texture imitation of meat.

The approach developed in this work, using the AM² masticator to gain information about bolus properties during mastication, could help speed up the development of PB meat analogues with similar texture to real meat as is requested by consumers. Thanks to a routine use in bolus production in adapted oral conditions allowing segmentation of the masticatory sequence, the AM2 in vitro approach provides the possibility to evaluate the FOP characteristics of different formulations and recipes adjustments to resemble meat patty. Since consumers expect meat analogues with the typical meat sensory characteristics, the sensory perception during oral processing is a critical factor strongly marked by its dynamic character that could be addressed in vivo for the main sensory attributes only at the end of the development process. For all foods, the first bite being usually associated with hardness, the first perception is generally related to textural properties (Saint-Eve, Panouillé, Capitaine, Déléris, & Souchon, 2015), thus hardness of meat analogues could be one of the challenges to address apart from the fundamental fibrous structure which is another important attribute to impart to the PB meat substitutes.

The difficulty in studying the dynamic FOP or mechanisms of oral perception is partly due to their complex nature, which renders the study of the food bolus so interesting to evaluate how its formation is dependent on the interaction between oral activity/perception and food structure. Bolus features contain information about FOP operation responsible of its formation in link with initial food structure. The AM² masticator is a promising approach to gain more information about the bolus evolution of PB meat substitutes during FOP.

## 5. Conclusion

Thanks to the ability of the AM² masticatory apparatus to produce beef- and PB patty boluses similar to *in vivo* ones, this *in vitro* work gave valuable insights, which can be used to aid the development of PB meat substitutes including a quantification of what happens in the mouth during oral processing and the characteristics of boluses (Boehm,
Nicholson, & Baier, 2023; Feng et al., 2022; Ilić et al., 2022; Kołodziejczak, Onopiuł, Szpicer, & Poltorak, 2022; Siddiqui et al., 2022; X. Zhang et al., 2022). The different values, set up for the programming of each patty according to physical features, can be linked to the mechanisms of in-mouth matrix fragmentation, suggesting that greater shearing forces are required for the Beef Burger, for example. Dynamic analysis of bolus characteristics during its formation highlighted differences depending on the patty and on their structure/composition. These differences would have an impact on textural perception which is obviously continuous during mastication (Szczesniak, 2002). The developed methodology and generated insights can further help the sector in evaluating different approaches if the objective is to produce a product for which the in-mouth perception resembles even more that of meat.

### CRediT authorship contribution statement

**Giron M:** Writing – original draft, Investigation, Formal analysis.
**Oosterlinck F:** Writing – original draft, Funding acquisition, Conceptualization.
**Steenhorst-Slikkerveer L:** Formal analysis.
**François O:** Methodology.
**Peyron M-A:** Writing – review & editing, Supervision, Funding acquisition, Conceptualization.

### Funding sources

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

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