The Rheology, Texture, and Molecular Dynamics of Plant-Based Hot Dogs

Abstract

The rising demand for plant-based alternatives to traditional meat products has led to the development of plant-based sausages (PBSs) that closely mimic the texture and taste of their meat counterparts. This study investigates the rheological and textural properties, as well as proton molecular dynamics, of hot dog-type PBSs and batters used in their production. Various formulations were analyzed to understand how different ingredients and processing methods affect the characteristics of the final products. Our findings reveal that the incorporation of specific plant proteins and hydrocolloids significantly influences the rheological behavior and texture profile of sausages. The hardness of the samples ranged from 4.33 to 5.09 N/mm and was generally higher for the products with inorganic iron sources. Regarding the viscoelastic properties, all the samples showed larger values of the storage modulus than the loss modulus, which indicates their solid-like behavior. Additionally, the study utilized advanced proton nuclear magnetic resonance (NMR) techniques to elucidate the molecular dynamics within plant-based matrices, providing insights into water distribution and mobility. Key findings highlight the impact of different plant proteins and additives on the texture and stability of sausage analogs.

1. Introduction

Recently, the so-called convenience food has become very popular in the market [1]. At the same time, there is a trend to reduce the consumption of meat and animal products, and consumers are looking for vegan products that they can use in the traditional way they are familiar with [2]. Such products also include plant-based sausages (PBSs), which can be used both as part of the main course (e.g., a hot dog) and as a snack. Vegan food producers are making them more and more similar to their meat counterparts. However, it should be remembered that animal products are a rich source of exogenous amino acids and iron [3,4,5], while plant products are unfortunately often characterized by a lower nutritional value. Thus, designers of vegan food must remember to supplement it with nutrients deficient in a plant-based diet, such as iron, vitamin B_12, or essential unsaturated fatty acids [6,7].

The development of PBSs has seen significant progress, with innovations in ingredients and additives aimed at improving their nutritional profile, sensory attributes, and shelf life [8,9]. Recent advancements include the incorporation of a variety of ingredients and additives, which are critical for enhancing the functional properties of PBSs. A multitude of studies have investigated the role of plant proteins in vegan sausage. Soy protein isolates, pea protein, and wheat gluten are among the most common protein sources used [10,11,12]. Research has demonstrated that soy protein isolates can significantly improve the textural properties of vegetable sausages, providing a more meat-like mouthfeel [13,14]. Similarly, pea protein has been shown to enhance moisture retention and the overall juiciness of the product [15,16].

Among the new proteins gaining popularity is potato protein (PP). It is a by-product obtained from potato juice during the starch production process [17]. Research has shown that potato protein has a very high nutritional value and may also serve as a source of iron and biologically active compounds that have beneficial effects on the course of gastrointestinal diseases [18]. A study conducted in vitro demonstrated that PP exhibits significant antioxidant properties comparable to those of butylated hydroxytoluene. Additionally, TBARS (thiobarbituric acid reactive substances) assay revealed that elevated levels of patatin may help limit the oxidation of low-density lipoprotein [19]. PP has exceptional nutritional benefits and contains 19 amino acids, including lysine, methionine, threonine, and tryptophan [20]. It is highly digestible and features a well-balanced amino acid profile, making it comparable to the proteins found in eggs and milk [21]. PPs have unique physicochemical properties owing to their ability to form highly stable foams and emulsions [22]. Moreover, the use of the enzyme alcalase for the hydrolysis of PP can enhance overrun and increase oil absorption [23]. The combination of different plant proteins, including potato proteins, has been shown to enable the optimal functional properties of vegan sausages to be obtained. Products containing protein blends, such as those combining potato protein with pea or wheat gluten proteins, exhibit superior textural and functional properties compared to those formed with single-source proteins. This approach leverages the complementary functional attributes of various proteins to achieve a more favorable product profile.

In vegan sausage production, various plant-based fats are used to achieve the desired texture and flavor. Commonly used fats include coconut oil [24], which provides a solid texture and rich mouthfeel; palm oil [25], known for its neutral flavor and solid consistency, although its use can be controversial due to environmental concerns [26]; sunflower oil [27], which offers a light flavor and high smoke point; canola oil, chosen for its neutral taste and health benefits [28]; and soybean oil, valued for its mild flavor and functional properties [29]. These oils and fats are essential for replicating the texture and taste of traditional sausages while maintaining a plant-based composition. At present, there is no consensus on the optimal intake of n6 and n3 fatty acids. Recommendations differ depending on the country and even the specific diet being followed. This has led to various guidelines for different population groups. The WHO/FAO has proposed an ideal n6/n3 ratio between 5:1 and 10:1 [30]. However, national guidelines vary, with Sweden advising a 5:1 ratio, Canada suggesting a range of 4:1 to 10:1, and Japan recommending a 2:1 ratio [31]. In the case of Polish consumers, due to the specificity of the Polish diet, the recommended ratio of n6/n3 acids is 4-5:1 [32]. Overconsumption of n6 fatty acids may have detrimental health consequences as it contributes to inflammation, potentially increasing the risk of heart disease and other ailments [33]. Studies on food products have revealed a marked imbalance in the n6 to n3 ratio, with some cases showing ratios exceeding 20:1 [34,35,36]. An appropriate composition of vegetable oils allows obtaining a nutritionally beneficial n6/n3 fatty acid ratio.

Iron salts are most often used as a source of iron, but a new solution has been developed that uses ferritin, a protein that provides easily absorbable iron of plant origin [37,38]. Hydrocolloids such as guar gum, xanthan gum, and carrageenan have been employed to modify the texture and consistency of vegetable sausages [39]. Flavor profiles in PBSs are often adjusted using various natural and synthetic flavorings. Research has highlighted the use of yeast, extracts, and spices as natural flavor enhancers that can mask off-flavors associated with plant-based proteins [40,41]. Additionally, smoke flavoring has been shown to significantly impact the overall sensory appeal of sausages [16,42].

The combination of various ingredients and different technologies allows the creation of PBSs that have high nutritional value. However, all these ingredients and additives may significantly affect the physical and mechanical properties of the final products. Among the properties that have the greatest impact on further consumer acceptance is the structure of food products, which is determined by both rheological and textural properties [43,44]. Biopolymers, such as proteins and carbohydrates, included in the PBSs recipe, significantly influence these properties. Proteins increase viscosity and elasticity through gelation, forming networks that enhance firmness, cohesiveness, and mouthfeel [45,46]. Carbohydrates, including polysaccharides, act as thickening agents, exhibiting shear-thinning behavior and forming gels that stabilize emulsions and foams, thus improving smoothness, stability, crispness, and chewiness [47,48]. The interactions between proteins and carbohydrates can create synergistic effects that stabilize products or induce phase separation, impacting texture. These properties are crucial in food product development as they determine the flow characteristics, stability, and sensory attributes of the final products.

Considering the above, the aim of this research was to analyze the rheological, textural, and molecular properties of innovative PBSs and the impact of the ingredients used on their physical properties.

2. Materials and Methods

2.1. Raw Materials

Potato protein was obtained according to our own patented technology [49] for potato juice, as described in detail earlier [20]. Wheat gluten protein was purchased from Viresol (Visonta, Hungary), rice protein from Beneo (Veendam, Belgium), and pea protein from Brenntag (Kędzierzyn-Koźle, Poland). Rice oil was purchased from Kasisuri (Bangkok, Thailand) and rapeseed oil from ZT Kruszwica (Kruszwica, Poland). Inactivated yeast flakes were purchased from Naturalnie Zdrowe (Wiązowna, Poland), methylcellulose from DuPont (Wilmington, DE, USA), carrageenan from Agnex (Białystok, Poland), dried beetroot juice from Kaczmarek-Komponenty (Mrowino, Poland). Vinegar and salt were purchased from a local grocery store. Iron (II) sulfate was purchased from Alfachem (Lublin, Poland), and powdered lupine sprouts containing ferritin were grown and prepared according to the procedure described in a patent [37].

2.2. Preparation of Sausage Analogs

PBSs were prepared in accordance with the procedure described in Polish patent application No. P.445946 [50]. The primary goal of designing the PBB formulas was to ensure an adequate amount of protein in the final product (ca. 20%) with a complete amino acid profile and fat with a recommended n6/n3 fatty acid ratio of 5:1, which is suitable for Polish consumers. To achieve this, appropriate blends of plant proteins and oils have been used. The oil blend used was a mixture of rice bran and rapeseed oil in a ratio of 55:45 [32]. The method of producing the protein base for the plant-based analog of sausage consists of a mix of plant proteins: potato (PP), rice (RP), wheat (WP), and pea (PeP), in the proportions of 70%PP/15%RP/15%WP (marked as PBS1), 70%PP/15%RP/10%WP/5%PeP (PBS2), or 70%PP/20%RP/5%WP/5%PeP (PBS3) in the form of powders. These protein mixes were introduced to the mixer; then, water was added at a ratio of 1:6 (m/v) and mixed until a homogeneous suspension was obtained. The next step was to heat-treat the suspension. For this purpose, the suspension was poured into 250 mL metal molds, baked in an oven at 120 °C for 2 h, and then cooled to room temperature. Then the obtained semi-finished product was hydrated in water in a ratio of 1:2 (w/v) until the semi-finished product stopped absorbing water, then the hydrated protein base was strained, placed in a bowl cutter (Mainca CM-21, Equipamientos Cárnicos, S.L., Barcelona, Spain) equipped with three knives (bowl speed: 12 rpm, knives speed: 1800 rpm) and the remaining ingredients were added to it, according to recipes described in

Table 1. PBSs detailed compositions.

Ingredient [%]	PBS1/F	PBS2/F	PBS3/F	PBS1/IS	PBS2/IS	PBS3/IS
Hydrated protein base	58.4	58.4	58.4	58.4	58.4	58.4
Oil blend	15.9	15.9	15.9	15.9	15.9	15.9
Methylcellulose	4	4	4	4	4	4
Sausage aroma	1.2	1.2	1.2	1.2	1.2	1.2
Smoke aroma	0.2	0.2	0.2	0.2	0.2	0.2
Inactivated yeast	2.9	2.9	2.9	2.9	2.9	2.9
Dried beetroot juice	0.5	0.5	0.5	0.5	0.5	0.5
Salt	0.3	0.3	0.3	0.3	0.3	0.3
Carrageenan	1	1	1	1	1	1
Dry protein mix	3	3	3	4.5	4.5	4.5
Sprouts containing ferritin	1.5	1.5	1.5	0	0	0
Iron (II) sulfate	0	0	0	0.007	0.007	0.007
Ice water	11.1	11.1	11.1	11.093	11.093	11.093

, and the mixing process was conducted until all the ingredients were combined into a uniform mixture, with no visible differences observed for 30 s. The final temperature of the batter was below 10 °C. At this stage of production, samples of batters (hereinafter referred to as PBB) were collected, and the remaining quantity of filling was subjected to further production procedures to obtain PBSs. PBSs were formed by filling a cellulose casing 10–12 mm in diameter, transferring it to a smoking chamber, cooking it until a temperature of 71 °C was reached in the geometrical center of the PBS, cooling it by spraying it with cold water until the temperature was below 20 °C, and packing it. Variants differing in their basic protein base (PBS1-PBS3) were produced using ferrous sulfate (marked with suffix/IS) or sprout powder enriched with ferritin (/F) as an iron source. Similarly, batters before thermal treatment were marked as PBB1-PBB3.

2.3. Nutritional Value of PBSs

The protein content of the product was determined by the Kjeldahl method according to ISO 1871 [51], using a nitrogen-to-protein conversion factor of 6.25. The fat content was determined using (Soxhlet method) according to AOAC Official Method 948.22 [52]. The content of soluble fiber was determined using the AACC 32-07 method [53]. Determination of moisture was carried out in accordance with the AACC 44-19.01 method [54]. The total mineral content was determined according to the international standard ISO 763 [55] using mineralization at a temperature of 550 °C. The carbohydrate content was calculated using the following formula:

$$\mathrm{C}\mathrm{a}\mathrm{r}\mathrm{b}\mathrm{o}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{s}\left(\mathrm{\%}\right)=100-(\mathrm{P}+\mathrm{F}+\mathrm{F}\mathrm{i}+\mathrm{M}+\mathrm{W})$$

where P is the percentage of protein, F is the percentage of fat, Fi is the percentage of fiber, M is the percentage of minerals, and W is the percentage of water in the analyzed sample. The energy value (EV) was calculated using the following formula:

$$\mathrm{E}\mathrm{V}\left({\displaystyle \frac{\mathrm{k}\mathrm{c}\mathrm{a}\mathrm{l}}{100\mathrm{g}}}\right)={\displaystyle \frac{4\times \mathrm{P}+4\times \mathrm{C}+9\times \mathrm{F}+2\times \mathrm{F}\mathrm{i}}{\mathrm{W}}}$$

2.4. Texture Analysis of PBSs

The texture of PBSs (tenderness) was analyzed using the Warner–Bratzler shear test [56]. Before texture assessment, 15 cores, 30 mm in length and 18 mm in diameter, were cut from each sample. Each core was placed horizontally on the instrument table and sheared once through the center using a Warner–Bratzler shear attachment connected to a TA.XTplus Texture Analyzer (Stable Micro Systems, Godalming, UK). A 50 kg compression load cell was used, and the crosshead speed was 1 mm/s. The results recorded were the shear force (N/mm, the maximum force required to cut the sample) and the work of shear (N/mm∙s, the work performed to move the blade).

2.5. Rheological Analyses of Batters and Sausage Analogs

Rheological properties were determined using a RotoVisco1 rheometer (Haake Technik GmbH, Vreden, Germany) equipped with a PP20Ti coaxial measurement system with a plate spacing of 2 mm. After being placed on the measuring system, the samples were thermostated and relaxed at 25 °C for 3 min. The parameters were determined by measuring in the CS mode at a stress of 1000 Pa in the frequency range of 0.1–10 Hz. The obtained data were processed using RheoWin 3.61 software.

2.6. Low-Field Nuclear Magnetic Resonance (LF NMR) of Batters and Sausage Analogs

Measurements of the spin-lattice (T₁) and spin-spin (T₂) relaxation times for both plant-based batters (PBBs) and PBSs were performed using a PS15T ¹H NMR pulsed spectrometer (Ellab, Poznań, Poland) equipped with an integral temperature control system [57]. The device was operating at a frequency of 15 MHz at the temperature of 20.0 °C ± 0.5 °C. The samples of 0.4 cm³ were placed in measuring test tubes and sealed using Parafilm^®. For T₁ measurements, the inversion–recovery pulse sequence (π – TI – π/2) was used [58]. Measurements of the spin-spin (T₂) relaxation times were taken using the pulse train of the Carr-Purcell-Meiboom-Gill spin echoes (π/2 – TE − (π)_n) [59,60]. The repetition time was 10 s, and the number of spin echoes (n) amounted to 100. The values of relaxation times were used to calculate the lower (D) and upper (U) limits of mean correlation times (τ) according to the method described by Małyszek et al. [61]. This parameter describes the time for a molecule to rotate by 1 radian and can be calculated using Bloembergen–Purcell–Pound (BPP) equations [62]:

$${\mathrm{R}}_1={\displaystyle \frac{1}{{\mathrm{T}}_1}}={\displaystyle \frac{6}{20}}{\displaystyle \frac{{\mathsf{\mu }}_0^2}{16{\mathsf{\pi }}^2}}{\displaystyle \frac{{\mathsf{\gamma }}^4{\left(\frac{\mathrm{h}}{2\mathsf{\pi }}\right)}^2}{{\mathrm{r}}_0^6}}\left[{\displaystyle \frac{{\mathsf{\tau }}_{\mathrm{c}}}{1+{\left(\mathsf{\omega }{\mathsf{\tau }}_{\mathrm{c}}\right)}^2}}+{\displaystyle \frac{4{\mathsf{\tau }}_{\mathrm{c}}}{1+{\left(2\mathsf{\omega }{\mathsf{\tau }}_{\mathrm{c}}\right)}^2}}\right]$$

$${\mathrm{R}}_2={\displaystyle \frac{1}{{\mathrm{T}}_2}}={\displaystyle \frac{3}{20}}{\displaystyle \frac{{\mathsf{\mu }}_0^2}{16{\mathsf{\pi }}^2}}{\displaystyle \frac{{\mathsf{\gamma }}^4{\left(\frac{\mathrm{h}}{2\mathsf{\pi }}\right)}^2}{{\mathrm{r}}_0^6}}\left[3{\mathsf{\tau }}_{\mathrm{c}}+{\displaystyle \frac{5{\mathsf{\tau }}_{\mathrm{c}}}{1+{\left(\mathsf{\omega }{\mathsf{\tau }}_{\mathrm{c}}\right)}^2}}+{\displaystyle \frac{2{\mathsf{\tau }}_{\mathrm{c}}}{1+{\left(2\mathsf{\omega }{\mathsf{\tau }}_{\mathrm{c}}\right)}^2}}\right]$$

where: μ₀ is the permittivity of free space, γ is the magnetogyric ratio, h is the Planck constant, r₀ is the distance of the interacting nuclei, and ω is the resonance frequency (ω = 2πf, f is the Larmor frequency, the spectrometer frequency).

2.7. Statistical Analysis

The statistical analyses were performed using Statistica 13.3 (TIBCO Software Inc., Palo Alto, CA, USA). A one-way analysis of variance and Tukey’s post hoc test were performed to determine statistically homogeneous and heterogeneous subsets at α = 0.05. Principal Component Analysis (PCA) was performed using all the data obtained in the analyses. The result is presented in a two-dimensional system (score plot and the loading plot) obtained by plotting the observations and variables on the plane formed by the calculated principal components.

3. Results and Discussion

3.1. Basic Chemical Characteristics

Results of the determination of nutritional value are presented in

Table 2. Content of nutrients, as well as energy value of PBSs.

Parameter	Unit	Sample
		PBS1/F	PBS2/F	PBS3/F	PBS1/IS	PBS2/IS	PBS3/IS
Protein content	g/100 g	21.7 ± 1.0	20.7 ± 0.5	21.3 ± 0.6	19.9 ± 0.9	21.6 ± 1.4	21.2 ± 0.8
Fat content	g/100 g	15.8 ± 0.3	16.3 ± 0.1	16.8 ± 0.3	16.2 ± 0.3	16.4 ± 0.4	16.5 ± 0.5
Fiber content	g/100 g	6.1 ± 0.3	6.8 ± 0.1	6.8 ± 0.1	4.9 ± 0.3	4.9 ± 0.1	5.1 ± 0.6
Carbohydrates content	g/100 g	9.9 ± 1.3	12.3 ± 0.8	11.0 ± 0.7	13.2 ± 1.3	12.0 ± 1.8	8.4 ± 1.0
Mineral content	g/100 g	4.2 ± 0.2	4.0 ± 0.3	4.2 ± 0.0	3.4 ± 0.2	4.0 ± 0.4	4.1 ± 0.3
Energy value	kcal/100 g	297.3 ± 1.9	317.0 ± 3.3	313.4 ± 1.5	308.5 ± 2.2	302.7 ± 2.9	296.1 ± 1.4

.

3.2. Textural Properties of PBSs

In traditional meat products, stromal and myofibrillar proteins are responsible for the firmness of the products, and the use of other proteins, including plant proteins, may result in a reduction in the firmness and hardness of the product [63]. Results obtained using the Warner–Bratzler shear test (presented in

Table 3. Results of textural properties of PBSs.

Sample	Maximum Shearing Force (N/mm)	Total Work of Shearing (N/mm∙s)
PBS1/F	4.33 ± 0.19 c	46.71 ± 2.88 c
PBS2/F	4.68 ± 0.48 b	51.61 ± 2.99 b
PBS3/F	4.87 ± 0.32 a,b	55.07 ± 4.78 a,b
PBS1/IS	5.09 ± 0.33 a	60.61 ± 2.64 a
PBS2/IS	4.83 ± 0.17 b	52.95 ± 4.84 b
PBS3/IS	4.63 ± 0.27 b	47.89 ± 2.75 c

Values marked with the same superscript letter do not differ significantly p > 0.05.

), often used to test the tenderness of meat products [64], showed that both the shear force and the total work of shearing are influenced by both the protein mixture and the added iron source used. PBS1/IS and PBS3/F were characterized by the highest hardness. At the same time, the same two analyzed variants of PBS showed the highest shearing work. However, when comparing the values of the analyzed texture parameters with the meat equivalent of the analyzed product, it can be observed that most of analyzed samples (except PBS1/IS) have comparable tenderness to finely comminuted frankfurters made in a traditional way [65]. Kamani et al. [66] noticed that replacing the meat in sausages with plant proteins worsened the mechanical properties, and the hardness of the products decreased. In our research, however, appropriately selected proteins and production technology resulted in the obtained products having properties similar to traditional meat products. Importantly, when comparing the appropriate protein mixtures with different iron sources, it was noticed that in the case of PBS1 and PBS2, the use of an inorganic iron source (/IS) significantly increased the hardness, while in PBS3 the variant enriched with organic iron (PBS3/F) was characterized by a higher hardness. It can therefore be assumed that the protein mixtures used interact with ingredients derived from sprouts and iron sulfate at the molecular level, resulting in the observed changes in the texture of the products. As literature data on the interaction of iron with milk proteins indicate, it is possible to create a complex structure between proteins and iron crystals. Moreover, fats can also be cross-linked, which significantly increases the hardness of the product [67,68]. The proteins and fats used in the PBSs recipe could, therefore, be cross-linked in a similar way, but the affinity for structure formation depends on the type of proteins and the form of iron addition. In addition, divalent metals, including inorganic iron, can also stabilize and fill the voids between proteins, further hardening the structure [69]. It is worth noting that the tenderness of sausages also depends on other factors, such as the fat content or product temperature [70,71]. Incorporating plant oils into food products is crucial for their rheological characteristics and structural integrity, as well as for imparting a distinctive flavor profile. Fat enhances the texture, taste, mouthfeel, overall sense of smoothness, and visual appeal of these products [72,73]. Moreover, as literature data indicate, heating the sausage increases its tenderness [74]. Given the fact that hot dog sausages are eaten warm, the hardness of the products assessed by consumers will be lower. Therefore, it was decided to analyze both batters and finished products in terms of the changes in rheological and molecular properties using LF NMR.

3.3. Rheological Properties of PBBs and PBSs

The viscoelastic properties of batter are presented in Figure 1, whereas the corresponding data for sausages are presented in Figure 2. All analyzed samples were characterized by a dominance of storage over the loss modulus (G′ > G″) typical of solid food products. The extent of changes in the mechanical properties in the analyzed frequency range was also similar for all products. This observation was true regardless of the composition and processing, as indicated by the similar pattern of changes in the phase angle tangent (δ). However, the effect of processing can be clearly observed when the data for batters and sausages are compared. This is reflected by the increase in both moduli and, consequently, in the complex viscosity (η*) observed for the sausages versus the batters. This is consistent with the LF NMR data (see Section 3.3), especially considering the spin-lattice relaxation time, which was shorter for the sausages. These results are also in line with the stiffer texture of the processed products.

The effect of composition on the complex viscosity of the products was not direct, both in the case of PBBs and PBSs. The viscosity of the batter was similar for all formulations with the exception of PBB1/F, which showed a considerably higher value of this parameter. This was caused by both an increase in the storage and loss moduli. The other observed outlier was the PBB1/IS batter with a relatively low G″ value corresponding to the low energy used for the initiation of the flow. This phenomenon was accompanied by a lower phase angle tangent value. This indicates that the incorporation of sprouts into the formula may significantly change the viscoelastic properties of the sample; however, the course of the change is influenced by interactions with other proteins.

The specific values of the parameters describing the mechanical properties of the sausages were more diverse than those of the batters. However, the sausages did not follow the same pattern as the batters—indicating that the processing significantly changes the effectiveness of the used protein in binding water. In terms of the response to the applied stress, the investigated sausages could be divided into two groups. The samples PBS3/IS, PBS2/IS, and PBS3/F were characterized by a higher phase angle tangent, while PBS1/F, PBS2/F, and PBS1/IS showed lower values of this parameter. These differences were also noticeable in the case of other mechanical parameters, including the storage and loss moduli. The only exception was observed for PBS1/IS with the highest G′ and G″ as well as low values of the phase angle tangent.

3.4. Molecular Properties of PBBs and PBSs

The spin-lattice relaxation time reflects the relationship between the bound and bulk water fractions. However, molecular mobility is reflected in the component values of spin-spin relaxation times (T₂₁—bound, T₂₂—bulk) [58]. The analysis of PBBs and PBSs indicates that in all tested samples, the thermal treatment of batters reduces the bulk water fraction in relation to the bound water fraction. Moreover, its molecular dynamics are more limited in PBSs than in PBBs, which is related to the additional thermal processing of the product ingredients. The results of the relaxometry analysis are presented in

Table 4. LF NMR results for PBBs and PBSs.

Sample	T1 (ms)	T21 (ms)	T22 (ms)	τD (s)	τU (s)
Batters
PBB1/F	128.3 ± 0.5 d	32.4 ± 1.3 b	83.1 ± 4.7 b	5.02 × 10−9	1.38 × 10−8
PBB2/F	133.2 ± 0.7 c	34.0 ± 1.7 b	86.1 ± 6.2 b	5.04 × 10−9	1.36 × 10−8
PBB3/F	130.7 ± 0.6 d	38.0 ± 2.5 a	78.3 ± 5.4 bc	5.64 × 10−9	1.22 × 10−8
PBB1/IS	140.7 ± 0.7 b	32.2 ± 1.4 b	81.7 ± 5.2 bc	5.89 × 10−9	1.30 × 10−8
PBB2/IS	147.3 ± 0.7 a	37.2 ± 2.3 a	75.8 ± 5.6 c	6.86 × 10−9	1.38 × 10−8
PBB3/IS	140.6 ± 0.6 b	30.7 ± 1.0 c	95.1 ± 6.2 a	4.68 × 10−9	1.55 × 10−8
Sausages
PBS1/F	104.1 ± 0.6 d	30.7 ± 1.4 b	74.4 ± 3.4 a	4.25 × 10−9	1.20 × 10−8
PBS2/F	109.1 ± 0.4 c	33.5 ± 2.4 ab	75.3 ± 6.0 a	4.52 × 10−9	1.16 × 10−8
PBS3/F	107.5 ± 0.5 c	31.6 ± 3.3 ab	64.7 ± 6.6 b	5.60 × 10−9	1.21 × 10−8
PBS1/IS	119.9 ± 0.4 b	32.2 ± 2.8 ab	65.7 ± 7.3 b	5.90 × 10−9	1.29 × 10−8
PBS2/IS	123.3 ± 0.5 a	35.2 ± 3.2 a	69.0 ± 9.8 b	6.18 × 10−9	1.24 × 10−8
PBS3/IS	120.5 ± 0.7 b	28.1 ± 1.2 b	85.4 ± 9.2 a	4.31 × 10−9	1.40 × 10−8

Values marked with the same superscript letter do not differ significantly p > 0.05.

.

It was found that in all analyzed products, the addition of organic iron from legume sprouts (/F) causes a greater shortening of the spin-lattice relaxation times than the addition of iron (II) sulfate (/IS). This is due to the presence of a larger number of paramagnetic ions, which significantly shortens the interaction time of nuclear spins with the environment. The literature data confirm the effect of shortening relaxation times due to the presence of paramagnetic ions in the system [75,76]. Additionally, it should be noted that the values of the spin-lattice relaxation times are differentiated only by the content of/F or/IS. This confirms that the presence of paramagnetic ions has a greater impact on spin-lattice relaxation than the content of bound and bulk water fractions. Analysis of the short and long components of the spin-spin relaxation times (T₂) showed that the composition of protein mixtures plays a significant role in the dynamics of bound and bulk water molecules. Due to the overlapping effects of shortening relaxation times, which result from the presence of paramagnetic ions, and due to protein-water interactions significantly influencing the relaxation time, analysis of the mean correlation times was performed. This is a parameter that indicates the time that a molecule containing a proton needs to rotate by 1 radian. In the situation of the presence of two water fractions bulk and bound, the upper (τ_U) and lower (τ_D) limits of the values of these correlation times can be determined. The upper limit is assigned to molecules with the lowest possible rotation, while the lower limit reflects the highest possible rotation around hydrogen or ionic bonds [77,78]. Analyzing the changes in the τ_U and τ_D values, it can be noticed that the lower limit of the mean correlation time for batters samples has slightly higher values than for PBSs, especially for samples PBS1/F, PBS2/F, and PBS2/IS. In the PBS1/IS and PBS3/F samples, the values are the same for batters and final products, and in the PBS3/IS sample, slightly higher τ_D values are observed for the batter than for the final product. It should be stated that the thermal treatment did not change the rotation possibilities of the water molecules in PBS1/IS and PBS3/F. Similar properties are observed for τ_U in these samples.

The analysis of the absolute values of the τ limits shows that the PBS3/IS variant is characterized by the largest difference in the values of the lower and upper τ limits (Table 4); therefore, it should be concluded that this sample is characterized by the greatest freedom of water rotational movements. This may translate into poor water binding and, consequently, the greatest growth of microorganisms during the storage of the finished products. The smallest differences in the τ_D and τ_U values were recorded for PBS3/F, which can be interpreted as the lowest possibility of rotational movements [79]. Such an ordered structure can guarantee the best microbiological safety parameters for the finished product.

3.5. Statistical Considerations on the Properties of PBBs

In order to find the relationship between the analyzed parameters analyzed at the molecular level and the macroscopic properties of PBSs, principal component analysis (PCA) was performed. PCA analysis allows the identification of the variables that have the greatest impact on the characteristics of the final product, which facilitates the interpretation of the impact of the molecular properties of PBSs on the mechanical properties. Two main components were selected from the calculations, which allowed for 79.38% of the total variance of the analyzed variables to be explained. The first main component contains 50.00% of the information about the tested products represented by the variables, while the second main component contains 29.38% of the information. The relationships between the variables are shown in Figure 3A. Each vector represents one variable, and its size and direction describe the impact it has on the main components. The analysis showed that the T₁ relaxation time and the upper limit of the mean correlation time, measured by LF NMR, had the greatest influence on the tangent of the phase angle of the analyzed PBSs. However, the lower limit of the mean correlation time (τ_D) and complex viscosity (have the greatest impact on the final texture parameters of PBBs. The higher the τ_D and η* values, the greater the tenderness and total work of shearing. T₂ relaxation times (both their short and long components) do not significantly affect the overall characteristics of the PBSs. Importantly, the analysis also confirms the different properties of PBS3, both with the addition of/F and/IS, which can be observed in Figure 3B. Importantly, the additive used in this composition of base proteins significantly differentiates the properties of the final product (the samples are located in the opposite quadrants of the graph). PBS1 and PBS2 are also differentiated by the type of additive, but no differences were found between them. Therefore, the uniqueness of the PBS3 variant was confirmed. Looking at all the results obtained, it can be clearly stated that PBS3/F is the most advantageous variant of the analyzed hot-dog-type sausage analogs.

4. Limitations and Future Perspectives

As evidenced by this study, the complexity of plant-based meat analogs with desired properties makes designing such products difficult. This challenge results from the multiple effects that protein content, iron fortification, and processing conditions exert on the final product. Moreover, these factors interact in ways that complicate identification of the optimal formulation. This makes it difficult to obtain a product that meets all technological requirements regarding texture or molecular stability. Despite these challenges, our findings offer promising perspectives for future research and development in the field of PBSs. One of the general observations is the potential to create a wide variety of products with different mechanical properties and textures. This flexibility suggests that with further research and experimentation, it may be possible to fine-tune these properties to better meet consumer preferences and technological requirements.

Importantly, among vegans and vegetarians, there is a growing trend toward seeking out new products that do not resemble traditional meat products but still allow for the preparation of classic dishes like burgers or hot dogs. At the same time, those who are reducing their meat consumption (flexitarians) are also increasingly turning to these types of products. The observed changes in the analyzed PBSs indicate that these newly developed products could gain full consumer acceptance despite significant differences in the ingredients used in their production and their meat counterparts. This shift in consumer preferences opens up new possibilities regarding the acceptance and the resulting success of plant-based products, even if their characteristics diverge from traditional meat-like products. Therefore, the ongoing exploration of different protein sources and processing techniques will likely play an important role in advancements in the field of vegan food, offering a pathway to more sustainable and nutritionally rich meat alternatives that satisfy consumer expectations.

This study identified PBS3/F as the product most stable and durable product at the molecular level. It is significant for future PBSs as it indicates the possibility of including less conventional plant proteins, such as potato protein derived from waste juice and powdered sprouts enriched with ferritin, in vegan products to improve the technological features of these products. If future research becomes focused on these promising proteins, some of the current limitations can potentially be overcome, allowing the development of even more advanced and commercially viable plant-based meat alternatives

5. Conclusions

Developing an analog of a meat product with appropriate mechanical properties is a challenge for food designers. Taking into account the multidirectional effects of protein, iron enrichment, and processing, it is almost impossible to indicate the most favorable composition of PBSs only on the basis of their mechanical properties; therefore, further research is needed to determine the nutritional properties, biological activity, and sensory attractiveness. At the same time, the results indicate the possibility of obtaining a wide range of products with varying mechanical properties and, thus, texture. However, analysis of the properties at the molecular level revealed that PBS3/F is clearly the most stable and durable product. Therefore, the use of an appropriate composition of plant proteins, including potato protein from waste potato juice and powdered sprouts enriched with ferritin, allows for the production of desirable technological features.

6. Patent

The results presented in this article were used to prepare a patent application to The Patent Office of the Republic of Poland (patent application No. P.445946, dated 30 August 2023).