Delving into textural, rheological, digestion, and microstructural properties of mung bean and chickpea-based sausages as a function of gluten

Abstract

Plant-based sausages (PBS) were formulated using heat-induced gelation to assess the influence of gluten in terms of structure, texture profile, rheology, digestibility, and shelf-life of PBS. The PBS formulation contained varying amounts of black chickpea flour, mung bean protein isolate, wheat gluten (WG), and fixed amounts of psyllium husk, water, and oil. WG significantly influenced the color. TPA showed that incorporation of WG increased the hardness (6501.8 ± 2.5 N), cohesiveness (0.9 ± 0.01 N), springiness (0.9 ± 0.01), and chewiness (5595.1 ± 2.1 N). The G″ and storage modulus G′ were enhanced with increasing amounts of WG. The WG prevented the secondary oxidative damage to the lipids in the PBS. Furthermore, the WG demonstrated its ability to prevent microbial load and improve shelf-life by suppressing the growth of aerobic microorganisms and coliform bacteria. The overall findings provide a fundamental understanding of the role of WG in PBS, which can be helpful in developing and tailoring PBS to meet consumer requirements.

Highlights

• •Mung bean protein isolate and wheat gluten were used as the major protein sources.
• •The formulation comprised of psyllium husk to create an emulsion-type plant-based sausage.
• •Wheat gluten played a crucial role in the techno-functional properties of the sausages.
• •Increasing amounts of wheat gluten significantly increased the hardness, cohesiveness, springiness, and chewiness.
• •Plant-based sausages with high wheat gluten demonstrated an improved shelf-life.

1. Introduction

The need for plant-based meat alternatives (PBMA) arises from several critical factors, including environmental sustainability, health benefits, and ethical considerations. As the global population grows, the demand for meat is increasing. However, traditional animal-based production is unsustainable, leading to significant pollution, greenhouse gas emissions, and biodiversity loss (da Silva et al., 2024). PBMA can be considered a sustainable protein source that can fulfil dietary requirements while reducing the environmental impact associated with meat production (da Silva et al., 2024). In terms of health, PBMA generally can be designed with an aim to have a more favorable nutritional profile. They are lower in saturated fats and higher in dietary fiber, which can improve cardiovascular health (Nagra et al., 2024). Additionally, the shift towards plant-based diets addresses ethical concerns related to animal welfare and food safety (Zor et al., 2024). However, challenges remain in achieving meat-like textures and flavors, necessitating further innovation in production techniques (Nagra et al., 2024). Overall, the transition to PBMA is essential for a sustainable and health-conscious future (Godschalk-Broers, Sala and Scholten, 2022)

The role of ingredients in formulating PBMA is multifaceted, focusing on nutritional, functional, and sensory attributes. Key ingredients include plant proteins, fibers, and bioactive compounds, which not only provide essential nutrients but also serve as technological agents like emulsifiers and thickening agents in meat analogues (da Silva et al., 2024). Wheat gluten (WG), a protein from wheat, is one of the major protein sources that was used in formulating PBMA (Sun et al., 2023). Due to glutenins and gliadins, WG naturally forms a thin protein layer and when subjected to thermomechanical treatment such as extrusion, shear force, or thermal shock, the protein layer transforms into a well-developed three-dimensional gluten network (Zhu, Zheng, Obadi, Qi, & Xu, 2023). The gluten network is mainly driven by disulfide linkages while hydrogen bonds and hydrophobic interactions further stabilize the network. Especially the disulfide linkages are determined by the ratio of glutenin and gliadin, where glutenin forms intermolecular disulfide linkages and gliadin forms intramolecular disulfide linkages (Kyriakopoulou et al., 2021). Incorporation of WG in PBMA enhances textural characteristics and water-holding capacity (WHC), which provides elasticity and chewiness- essential characteristics of meat (Lee et al., 2024; Ranjan Kumar, 2012). In addition, WG acts as a binding agent in meat analogues, helping to hold other ingredients together (Lee et al., 2024). Studies have shown that when WG is combined with other protein sources, it provides fibrous meat-like structures that resemble real meat (Peng et al., 2023; Zhang, Jia, et al., 2023; Cornet et al., 2020). However, the use of WG poses challenges for individuals with celiac disease, prompting the exploration of alternatives, which can also improve textural properties but may not fully replicate the fibrous structure provided by WG (Taghian Dinani et al., 2023). Thus, while WG is effective in enhancing PBMA, its allergenic nature necessitates the search for suitable substitutes.

One of the recent studies explored replacing WG with insoluble soy fiber and low acyl gellan gum in PBMA (Taghian Dinani et al., 2024; Nanta et al., 2021) formulated gluten-free sausages by substituting the gluten effects with various gums. Nevertheless, a subsequent study (Zhao et al., 2022) reported that gums are not considered “label-friendly ingredients” and often, consumers who look for gluten-free products look for clean labels. Psyllium husk (Plantago ovata) is one of the hydrocolloids commonly used in foods because of its metabolic and gut health benefits (Noguerol, Igual, & Pagan, 2022). This is composed mainly of cellulose, lignin, and pectin (Bartkiene et al., 2023) and approximately 65 % of total fiber content is comprised of arabinoxylan and xylopyranose containing water-soluble fiber (Fu et al., 2022). Due to its strong water holding capacity, psyllium husk forms gel like matrix in food system. This is vital when considering emulsion-type food product formation where psyllium husk can bind water and oil and stabilize the oil-water interface (Fu et al., 2022).

This study aimed at formulating psyllium husk containing emulsion-type PBMA, plant-based sausages (PBS) and to understand the interactions and crucial roles of the ingredients used for the formulation. Black chickpea flour (BCF) and mung bean protein isolate (MBPI) were used in this formulation. BCF is used as substitute for WG in many products due to the excellent water holding capacity, emulsifying properties, and gelation capacity (Javed et al., 2021; Rachwa-Rosiak et al., 2015). The WG-free PBS was formulated using BCF and MBPI. MBPI has superior quality owing to the abundance of proline, glutamic acid, arginine, leucine, and phenylalanine (Gholami & Paknahad, 2023). Despite the extensive advantages of mung bean, it remains an underutilized legume due to its challenging cooking characteristics (Wei et al., 2021). Given appropriate processing, mung beans could prove to be a valuable source of inexpensive protein for PBS. Unlike soy protein isolate, the interactions between MBPI and WG are weaker which demands additional stabilization for forming stronger gel structures in PBS. The psyllium husk was incorporated into PBS to create a stronger gel network, which is crucial for structural integrity. Using these ingredients, PBS was created by utilizing the principles of soft matter physics approach reported by Ryu et al. (2023). This method involves shear force to blend ingredients where proteins like WG can mainly transform to fibrous structure. Following high-shear application, the blend is subjected to heat-set gelation to retain the gel structure formed in the matrix. We hypothesized that a combination of thermal and mechanical treatment (thermomechanical) during this process aids in achieving meat sausage-like structure and functionality. The properties of the PBS were assessed in terms of texture, color, viscoelastic properties, digestibility, and shelf-life stability.

2. Materials and methods

Black chickpea flour (BCF), wheat gluten (WG), psyllium husk, and vegetable oil were purchased from a local grocery store (Al Ain, UAE). The mung bean protein isolate (MBPI) was purchased from ET proteins, China. Trichloro acetic acid (TCA), thiobarbituric acid (TBA), HCl, CaCl₂, NaCl, pepsin, lipase, bile salts, α-amylase were purchased from Sigma Aldrich, Co. Ltd. (Missouri, USA). The potato dextrose agar (PDA), plate count agar (PCA), and violet red bile agar (VRBA) were purchased from HiMedia (India). Bacteriological peptone and plate count agar were purchased from Acumedia LAB.

2.1. Preparation of plant-based sausages (PBS)

The ingredients selected for this study were BCF, MBPI, WG, psyllium husk, oil, and water. The initial formulation was optimized with BCF and psyllium husk, based on an ongoing study in our research laboratory with high-moisture low-temperature extrusion. For the study reported in this work, BCF was kept at 60 % and psyllium husk was increased from 5 % to 10 % gradually while the water and oil content were kept at 5 % and 10 %, respectively. However, with increasing psyllium content the mixture was dry and had aggregations when blending leading to a coarse nature. When water content was increasing twice the amount of psyllium husk (from 10 % to 20 %) a much smoother texture was obtained when blending. However, since sausages are emulsion-type materials, the oil content was increased to 10 % and the control recipe was formulated with 60 % BCF, 10 % psyllium husk, 20 % water, and 10 % oil (Table 1). Following this formula, the MBPI and WG were substituted for BCF while keeping the psyllium husk, water, and oil content constant. The PBS were prepared according to Ryu et al. (2023) with minor modifications. Briefly, the various food grade ingredients shown in Table 1 were blended in a high-speed mixing blender (Kenwood, Japan). A solution consisting of 50 mM NaCl and 10 mM CaCl₂ was added to the mix to make blended sausage mix which was then packed tightly in a sausage casing using a kitchen-scale sausage maker (Master Feng, Australia). The ends of the casings were sealed, and the PBS was placed in a closed system with boiling water to create saturated steam. Once the internal temperature of the PBS reached between 83 and 85 °C, the PBS was kept at the same condition for 30 min. Followed by which the samples were placed in an ice bath (4 °C) for another 30 min to create an irreversible heat-set gelation. The sausages were then stored at 4 °C and were used for further analysis. Table 1 shows the percentage of different ingredients used for formulation of (PBS) samples. Images of formulated PBS are displayed in Fig. 1.

Table 1.

Formulation of PBS using different ingredients.

Sample	Black chickpea flour (%)	Wheat gluten (%)	Mung bean protein isolate (%)	Psyllium husk (%)	Oil (%)	Water (%)
Control	60	–	–	15	10	20
PBS-1	20	–	40	15	10	20
PBS-2	20	10	30	15	10	20
PBS-3	20	20	20	15	10	20
PBS-4	20	30	10	15	10	20
PBS-5	20	40	–	15	10	20

Fig. 1.

PBS with varying proportions of black chickpea flour, mung bean protein isolate and wheat gluten. The detailed composition of PBS is given in Table 1.

2.2. Proximate analysis

Proximate analysis was performed to determine the moisture content (oven dry method at 105 °C until constant weight was obtained), protein content (Kjeldahl method, AOAC, 22nd edition, 2023), and fat content (Soxtec automated extraction method, AOAC, 22nd edition, 2023), and ash (AOAC, 22nd edition, 2023). The carbohydrate content was determined by subtracting the protein, ash, fat, and moisture from 100 [100 − (protein-ash-fat-moisture)].

$$\%\mathit{Ash}=\frac{\left(\mathit{ashe}\mathrm{d}\mathit{wt}-\mathit{crucible}\mathit{wt}\right)}{\left(\mathit{crucible\; and\; sample}\mathit{wt}.\right)}\mathrm{x}100$$

2.3. Texture profile analysis (TPA)

The TPA was carried out using CT3 Texture Analyzer (Brookfield Engineering Laboratories, USA). A cylindrical probe with 7 mm diameter was used at 5 mms⁻¹ test speed. The PBS were cut into 3.0 cm × 2.5 cm (W × H) and were compressed 40 % from the original height to analyse the hardness, chewiness, adhesiveness, and springiness.

2.4. Color

The color of the PBS was determined using HunterLab ColourFlex EZ spectrophotometer (Hunter Associates Laboratory, Inc., USA). The tri-stimulus parameters L*, a*, and b* that correspond to lightness, redness-greenness, and yellowness-blueness, respectively were recorded.

2.5. Viscoelastic properties

The viscoelastic properties were studied using a rheometer (HR-2, TA Instruments, USA). A parallel plate geometry with a diameter of 40.0 mm and a gap of 1.0 mm was used to measure the rheological parameters at 25 °C. A thin slice of approximately 0.5 mm thick sausage sample was placed on the surface of the Peltier plate. The storage modulus G′ and loss modulus G′′ were studied from 0.1 to 100 rads⁻¹ frequency sweep test and 1 % strain.

2.6. Digestibility study using INFOGEST 2.0 and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE)

The digestion pattern or the behavior was studied under in-vitro stimulated gastrointestinal conditions, as reported by INFOGEST 2.0 (Brodkorb et al., 2019). After performing the INFOGEST 2.0 protocol, the digested PBS were centrifuged at 10,000× rpm (revolutions per minute) for 5 min. The supernatants were collected and denatured with sample buffer at 1:1 for 5 min at 95 °C. The sample buffer contained, trisma base at pH 6.8 (6.25 % v/v), SDS (3 % w/v), β-mercaptoethanol (6.25 % v/v), glycerol (10 % w/v), and urea (18 % w/v).The denatured PBS were further centrifuged at 10,000× rpm for 2 min to remove the remains of non-denatured particles. The electrophoresis was carried out using a Mini Protein II unit (BioRad Laboratories, Inc., USA) at 50 mV per gel. The stacking and resolving gels were casted at 5 % and 12 %, respectively. Followed by electrophoresis, the gels were stained with 0.02 % (w/v) Coomassie Brilliant Blue (R-250) and destained with a solution that contained 50 % methanol (v/v) and 7.5 % (v/v) acetic acid for 20 min, followed by further detaining in 5 % methanol (v/v) and 7.5 % (v/v) acetic acid overnight.

2.7. Thiobarbituric acid-reactive substances (TBARS)

The secondary lipid oxidation products of the freshly prepared PBS and week-old PBS (stored at 4 °C) were determined using TBARS assay (Maqsood & Benjakul, 2010). Briefly, 0.5 g of PBS was chopped and mixed with 2.5 mL of TBA solution that was composed of 15 % TCA, 0.357 % TBA, and 2 % 0.25 N HCl and homogenized at 5000 rpm (IKA T25 Digital Ultra Turrax) for one minute. The homogenized solution was placed in boiling water for 10 min followed by gradual cooling to room temperature. The cooled samples were centrifuged at 10,000 rpm for 15 min and the absorbance of the supernatant was estimated at 532 nm. The TBARS were then calculated using an Malondialdehyde (MDA) standard curve using 1,1,3,3-tetramethoxypropane at a concentration between 0 and 10 ppm and the values were reported as MDA equivalents/kg sample.

2.8. Shelf-life study

The shelf-life study was done over a period of 9 days and the PBS were stored at 4 °C; the sampling was done every 3 days. Briefly, 10 g of PBS was homogenized in 90 mL of sterilized peptone water, using a stomacher rotor at 230 rpm for 2 min.

The stomacher solution was then subjected to a series of 10- fold dilutions using sterilized peptone water. PBS was analysed every three days over 9 days. The diluted PBS were then studied for total bacterial count using Plate Count Agar (37 °C for 48 h), coliform count using VRBA (37 °C for 48 h), and yeast and mold using PDA (25 °C for 5 days). The microbial counts were expressed as log CFU/mL. Since PDA was done for yeast and mold, 10 % of tartaric acid was added to prevent the growth of bacteria.

2.9. Microstructure study

Using the technique outlined by Yuliarti et al. (2021), the microstructure of the PBS was studied using a scanning electron microscope (SEM) (JEOL scanning electron microscope, model: JSM-6010PLUS/LA, Tokyo, Japan). Fresh sausages were specifically chopped into tiny pieces that were 2–3 mm thick before being hardened with liquid N2. 2.5 % glutaraldehyde was used to fix frozen samples for 12 h in 0.2 M phosphate buffer at pH 7.2. Following three successive 15-h rinses with distilled water, the samples were dehydrated in a serial ethanol solution (50 % for 15 min with two rinses, 70 % for 15 min with two rinses, 80 % for 15 min with two rinses, 90 % for 15 min with two rinses, and 100 % for 30 min). The samples were put into the SEM's vacuum chamber, and pictures were taken at a voltage of 20 kV and a magnification of 100×.

2.10. Statistical analysis

All formulation of the PBS were carried out in three batches, the experiments were conducted in triplicate, and the results were presented as average values with accompanying standard deviations. The collected data underwent a one-way analysis of variance (ANOVA) utilizing Minitab 21 software (Minitab, Inc., State College, PA, USA) The mean values were subsequently compared using Tukey's test (p < 0.05). Any discrepancies observed between the various sausage samples were deemed statistically significant at p < 0.05.

3. Results and discussion

3.1. Proximate analysis

Table 2 represents the proximate composition of PBS formulated using different levels of various ingredients. Mainly the major macromolecules including moisture, fat, and protein, and carbohydrate are discussed. The moisture content was higher and was statistically insignificant for PBS-2 to PBS-5. The control PBS without MBPI and WG and PBS-1 (gluten-free PBS) showed lowest moisture content (p > 0.05). Protein isolates possess the capability to form bonds with water, a vital factor in maintaining the moisture levels in meat analogues (Wittek et al., 2021). MBPI's water-holding capacity and WG’s network structure possibly work together to retain moisture during cooking (Peng et al., 2023), which resulted in a significant increase in moisture for PBS-2 in the presence of both MBPI and WG. However, a further increase in WG and decrease in MBPI did not significantly affect the moisture content.

Table 2.

The proximate composition of the PBS.

Sample	Moisture (%)	Protein (%)	Fat (%)	Ash (%)	Carbohydrates (%)
Control	55.312 ± 1.432b	9.35 ± 0.33d	2.387 ± 0.00252a	3.903 ± 0.219a,b	29.048 ± 1.302a
PBS-1	53.039 ± 0.929b	21.054 ± 0.182b	1.452 ± 0.283b	4.3031 ± 0.0553a,b	20.133 ± 0.725b
PBS-2	63.69 ± 1.79a	22.376 ± 0.193a	0.4976 ± 0.1016d	4.4092 ± 0.1334a	9.027 ± 1.573c
PBS-3	63.09 ± 2.84a	22.598 ± 0.236a	1.198 ± 0.346b,c	3.05 ± 0.343c	10.06 ± 2.73c
PBS-4	62.69 ± 1.8a	20.335 ± 0.198c	1.39295 ± 0.00255b	3.6828 ± 0.0682b	11.9 ± 2.06c
PBS-5	62.94 ± 1.026a	21.301 ± 0.262b	0.6958 ± 0.1722,d	2.778 ± 0.1257c	12.285 ± 0.843c

Values are expressed as mean ± SD and those with different superscripts signify that the values are significantly (p < 0.05) different among each other.

In terms of protein, control PBS had the lowest protein (9.35 ± 0.33), compared to other PBS because the protein in control PBS was exclusively due to the BCF. The protein content was statistically insignificant in PBS-1 (gluten-free PBS) and PBS-5 (MBPI-5). The highest protein was seen in PBS-2 and PBS-3 which has more MBPI and equal amounts of MBPI and WG, respectively. A further decrease in MBPI and increase in WG (PBS-4) showed a decrease in protein content. This shows higher protein is observed when a stable network is formed with specific ratios of MBPI and WG. A similar observation was reported in the presence of soy protein isolate and vital wheat gluten- that was processed using high moisture extrusion and shear cell technology (Peng et al., 2023).

Maximum fat content was found in the control PBS, which had 60 % BCP. Similar findings were reported in bread formulation when BCF was used as the major ingredient (Yaver, 2022). The addition of MBPI and reduction of BCF reduced the fat content from 2.38 % to 1.45 % which shows that the hydrophobic amino acids in MBPI would have bound to the added oil causing a decrease in fat content. The introduction of WG with a reduced MBPI showed a further reduction in fat content. WG is known to have varying permeability to fat permeability depending on the three-dimensional structure formed during processing (Gazmuri and Bouchon, 2009). It could be possible that the interaction between 30 % MBPI and 10 % WG (PBS-2) increased the permeability to added oil- reducing the fat content further. Moreover, PBS-5 with 40 % WG and no MBPI showed statistically insignificant difference in fat content- which implies that the matrix formed between PBS-2 and PBS-5 are similar.

The carbohydrate content of PBMA has been reported to range from 2.5 % to 30 % of the dry weight, contingent upon the product formulation (Singh & Sit, 2022). In this study, the control PBS with 60 % BCP showed maximum carbohydrate content (29.048 %) followed by PBS-1 with 20.133 % carbohydrates. Rest of the formulation (PBS-2 to PBS-5) showed significantly lower carbohydrate content, and the differences were statistically insignificant. This shows that BCF and MBPI were the major contributors of carbohydrates in PBS. The lower carbohydrate in gluten-containing PBS compared to gluten-free PBS explains that increased gluten levels might necessitate a reduced concentration of starchy fillers, leading to a decreased carbohydrate content (Singh & Sit, 2022).

3.2. Color attributes of the sausages

The color attributes of the PBS are tabulated in Table 3. The lightness (L*), value of gluten-free PBS (PBS-1) was significantly higher than the control PBS. This is because the raw MBPI, has a higher L* value (90.02 ± 0.05) compared to BCF (87.07 ± 0.01) and hence reducing the BCF to 20 % and increasing the MBPI to 40 % would have resulted in increased L*. Subsequently, a gradual decrease in MBPI content (PBS-2 and PBS-3) and a simultaneous increase in WG reduced the lightness. This can be attributed to the low L* value of WG (83.27) compared to MBPI and hence decreasing the MBPI and increasing the WG showed a decrease in lightness. In addition, the presence of both MBPI and WG would have created caramelization between the protein (in MBPI and WG) and starch (from BCF)-resulting in decreased L* values. However, a further increase in WG and a decrease in MBPI increased the lightness (PBS-4 and PBS-5) and the highest L* value was seen in PBS-5, which had 40 % WG and 0 % MBPI. The increase in L* in PBS-4 and PBS-5 can be correlated to the browning reaction that would primarily occur between WG and the starch in BCF. Moreover, the highest L* in PBS-5 could be due to the less browning reaction between 40 % WG and 20 % BCF; the PBS-1, which had 40 % MBPI and 20 % BCF showed less L* (70.49 ± 0.02) compared to the PBS-5 (71.41 ± 0.01), implying that the browning reaction between MBPI and BCF was high, compared to WG and BCF. Similar observations of browning and Maillard effects were reported in a study with wet noodles (Wen et al., 2023). The a* values of PBS showed positive values indicating that the PBS were on the red-color scale. Compared to the control PBS, the gluten-free PBS (PBS-1) showed a significantly lower a*. This can be due to the lower a* value of the MBPI; the a* of BCF and MBPI are 1.63 and 0.5, respectively. Furthermore, when the MBPI was reduced and WG was increased (PBS-3), a* value increased. This observation could be due to the addition of WG which has the highest a* (1.85), compared to BCF and MBPI. Furthermore, when adding more WG, there could be more available amino acid groups available to interact with the carbonyl groups present in BCF leading to an intensified Maillard browning, which resulted in higher a*. However, a further decrease in MBPI and an increase in WG showed non-uniformity and PBS-5 (40 % WG and no MBPI) showed statistically similar a* values with PBS-5 implying that the color was driven mainly by the innate a* value of WG. Moreover, in the absence of MBPI (PBS-5) there would have been lesser amino groups available for Maillard reaction (with only contribution of amino groups from WG) and lower a* value was expected for PBS-5. A possible explanation for a contradictory observation can be related to the matrix that is formed with 40 % WG (in PBS-5) would have same amount of available amino acid groups as PBS-2 for Maillard reaction to occur.

Table 3.

Color attributes of PBS.

Sample	L*	a*	b*
Control	68.71 ± 0.04c	3.13 ± 0.01d	23.3 ± 0.01c
PBS-1	70.49 ± 0.02b	2.51 ± 0.01e	23.56 ± 0.01b
PBS-2	64.50 ± 0.01e	3.85 ± 0.02b	23.18 ± 0.01d
PBS-3	63.54 ± 0.01f	3.89 ± 0.01a	21.95 ± 0.01e
PBS-4	65.92 ± 0.01d	3.54 ± 0.01c	20.87 ± 0.01f
PBS-5	71.41 ± 0.01a	3.84 ± 0.01b	25.97 ± 0.01a

Values are expressed as mean ± SD and those with different superscripts signify that the values are significantly (p < 0.05) different.

In terms of b*, the gluten-free PBS (PBS-1) showed increased b* value compared to the control PBS. The increase could be attributed to the innate yellowness (b* = 25.1) of MBPI. Comparatively, the PBS- 5, which had 40 % WG showed the highest b*. Although the BCF has a higher b* (20.27), the PBS-5 is majorly composed of WG and hence demonstrated a high b*.

3.3. Digestion of the sausages

The digestion fate of PBS was studied under stimulated gastric and intestinal condition and a qualitative assessment was conducted using SDS-PAGE (Fig. 2).

Fig. 2.

SDS PAGE of stimulated in vitro digested PBS (A) control sample; (B) gastric digestion; (C) intestinal digestion. L1-control, L2-PBS-1, L3-PBS-2, L4-PBS-3, L5-PBS-4, L6-PBS-5; (D) Appearance of digested PBS (1st row, gastric-phase digestion, 2nd row, intestinal phase digestion).

The control PBS did not show any significant protein bands (L1) because of the low-protein content in BCF. The gluten-free PBS (PBS-1) showed intense bands around 50 kDa, which corresponds to vicilin-like subunit in the MBPI. Another faint band was observed at ≈ 25 kDa, which also corresponds to vicilin-like subunit (Shrestha et al., 2023). As the MBPI content decreased, the intensity of the above-mentioned bands decreased gradually (L3-L4). In L5, a faint band around ≈ 49–50 kDa appeared and this is exclusively due to the low molecular weight glutenin, and gliadin subunits (Wan, Gritsch, Hawkesford, & Shewry, 2014). During gastric digestion and intestinal digestion, there were no specific bands that appeared in any of the PBS. Although vicilin is known to be resistant to gastric digestion (Santos-Hernández et al., 2020), unlike the other proteins, in the current study, the vicilin-like subunits disappeared. A complete digestion of all the PBS in gastric phase shows that the heat treatment (>80 °C) during processing would have denatured the protein in PBS while further denaturing with using SDS would have resulted in very small molecular weights of protein. In addition, the thermal processing of PBS would have deactivated the potential anti-nutritional factors present in plant proteins used in this study. Thus, the interference due to anti-nutritional factors during digestion can be ruled out.

The above observations provide a fundamental understanding of how gluten-free and gluten-containing PBS are digested under stimulated gastric and intestinal phases. This parameter is crucial to understanding the nutrient digestibility– especially the proteins– to predicate the dietary pattern of PBS. Wu et al. (2017) studied protein digestibility in pre-mixes, doughs, and baked breads made with four gluten-containing and four gluten-free flours. The physical and chemical changes that proteins underwent during proofing and baking were found to have an impact on protein digestibility, with digestibility generally increased after proofing and decreasing after baking. Moreover, we did not detect any notable aroma (a qualitative perception made by the researchers) of the digested PBS. This observation suggests that there were no volatile gases produced during protein digestion of PBS, which is an important aspect when developing PBMA. The plant-based proteins (peas and beans) are known to produce excessive gas during digestion (flatulence), which leads to various digestive disorders.

3.4. Texture profile analysis (TPA) of PBS

The TPA quantifies various textural attributes such as hardness, cohesiveness, gumminess, springiness, and chewiness (Table 4). The fundamental understanding of TPA of PBS will aid in tailoring different formulations to cater to the needs of individuals expectations with PBMA. Hardness pertains to the maximum resistance of the PBS during biting. Cohesiveness determines the PBS's ability to maintain its structural integrity. Springiness and resilience are defined by the PBS's capacity to recover from deformation. Gumminess encompasses both hardness and cohesiveness, and chewiness, which encompasses gumminess and springiness, are what characterizes the effort required to chew the meat products (Schreuders et al., 2021). The PBS showed significant differences in hardness ranging from 1892.2 N to 6501.8 N and chewiness ranging from 1595.6 N to 5595.1 N, compared to cohesiveness and springiness. WG acts as a binder and a structuring agent. During structuring, the thin protein layers in WG transform into a three-dimensional network during the thermomechanical process. During this transformation, WG encapsulates moisture and air to provide a meat-like structure (Zhang, Yang, et al., 2023). As the WG content increases, the high and low molecular weight glutenins get disrupted leading to gluten network. Godschalk-Broers et al., 2022 showed that elevated gluten concentrations generally exhibit increased levels of hardness, closely mimicking the texture of chicken meat. In this study, a maximum gluten network was formed in PBS-5 where the only protein source was WG, which interacted with BCF. With increasing WG and decreasing MBPI (PBS-2 to PBS-4) the hardness decreased from 3117 N to 1892.2 N. This could be due to the interaction between MBPI proteins and WG that prevented the complete formation of gluten network. The hardness was also positively correlated to both cohesiveness and chewiness where PBS-5 showed maximum chewiness.

Table 4.

Texture profile analysis of PBS.

Sample	Hardness (N)	Cohesiveness (N)	Springiness	Chewiness (N)
Control	3495.2 ± 1.5b	0.85 ± 0.05a,b	1.0 ± 0.0a	3354.7 ± 1.1a
PBS-1	3117 ± 2.0b	0.8 ± 0.0b	0.9 ± 0.0b	2392.2 ± 2.5b
PBS-2	2942.2 ± 1.4c	0.8 ± 0.0b	0.9 ± 0.0b	2227.7 ± 2.5c
PBS-3	2186.3 ± 5.0d	0.8 ± 0.0b	0.9 ± 0.0b	1661.1 ± 1.5d
PBS-4	1892.2 ± 1.5d	0.9 ± 0.0a	0.9 ± 0.0b	1595.6 ± 2.0e
PBS-5	6501.8 ± 2.5a	0.9 ± 0.0a	0.9 ± 0.0b	5595.1 ± 2.0f

Values are expressed as mean ± SD and those with different superscripts signify that the values are significantly (p < 0.05).

Cohesiveness demonstrates the ability of the PBS to withstand the deformation in oral cavity. The assessment of cohesiveness of the PBS showed higher cohesiveness for PBS-4 and PBS-5 (p > 0.05) while low cohesiveness was seen for the rest of the PBS, with statistical insignificance. The PBS-5, which had WG as the predominant protein source showing, both highest hardness and highest cohesiveness implies that the PBS with such formulation can remain intact in the oral cavity and with less to no crumbling of the texture. On the other hand, the PBS-5, which is coupled with highest hardness and highest cohesiveness reveals that this PBS can be considered a gel-like texture, which is important when forming emulsion-type food products. The other PBS with lower cohesiveness and moderate-to-low hardness demonstrates that the PBS network influenced mainly by MBPI and WG can influence the structural integrity of the PBS. Springiness is another important parameter that shows the resilience of the PBS upon deformation. In this study, we did not see significant differences in springiness of the PBS, except for control PBS, which has a significantly high springiness value. It could be because the springiness in this formulation is primarily driven by the amount of BCF, which was 60 % in the control PBS and 20 % in the rest of the PBS formulation.

3.5. Viscoelastic properties of PBS

The viscoelastic properties of PBS were quantified by analyzing two parameters, namely the elastic modulus G′, also known as the storage modulus, and the viscous modulus G″, also referred to as the loss modulus (McClements, 2023). These parameters are determined by measuring the sinusoidal deformation of the PBS, which results in the observation of characteristic curves.

The G′ and G″ were determined through the use of frequency sweeps in order to acquire additional information regarding the viscoelastic properties in response to varying frequencies. At lower amplitudes, the G′ and G″ exhibited no significant alterations, however, at higher amplitudes, the G′ and G″ intersected, resulting in a modification of the network due to the dominance of G″ over G′. Additionally, the frequency sweep measurements revealed that both the loss and storage moduli increased with the increase of frequency.

The G′ and G″ are influenced by protein composition, particularly gluten and mung bean protein, as well as interactions with black chickpea flour. Gluten forms a viscoelastic network due to its gliadin and glutenin protein components which contribute to Higher G′ (Zhang, Yang, et al., 2023). The domination of G′ indicated strong solid-like behavior. The enhanced elasticity of gluten is due to disulfide and hydrogen bonds that create a cohesive, stretchable matrix (Abedi & Pourmohammadi, 2021). Mung bean protein has lower G′ compared to gluten due to lack of disulfide cross-linking. The vicilin and legumin proteins in mung bean form softer gels which leads to weaker elasticity (Huang et al., 2024). The black chickpea flour might contribute to an increase in both G′ and G″ when combined with gluten and mung bean protein (Wang et al., 2023). The starch and fiber in black chickpea flour act as fillers, but this effect relies on the water content of the PBS formulation (Ladjevardi et al., 2024).

Throughout frequency range, G′ prevailed over G″, indicating the predominance of the PBS elastic nature (Fig. 3). The presence of protein and fiber in BCF facilitates the formation of a network that significantly improves the elasticity of the resultant PBS. It demonstrates relatively weak gel-like behavior, characterized by a storage modulus (G′) that surpasses the loss modulus (G″). Such behavior proves advantageous for PBMA, as it aids in preserving structural integrity and textural properties throughout processing and cooking (Kowalczewski et al., 2024). Gluten augments the storage modulus of meat analogues by establishing a robust and cohesive protein network (Abedi and Pourmohammadi, 2021, Dubey, Mateen and Singh, 2024). This network is essential for providing requisite elasticity, rendering the PBS firm and capable of maintaining its shape when subjected to mechanical stress. Furthermore, gluten enhances the loss modulus by imparting viscosity to the PBS. This characteristic plays a crucial role in replicating the chewiness and mouthfeel associated with authentic meat. Interestingly, this investigation has demonstrated that PBS produced with BCF and MBPI exhibited a less viscous yet more elastic response. This suggests that the applied energy was stored within the internal network rather than dissipated (Shahbazi et al., 2021).

Fig. 3.

The viscoelastic measurements of PBS with frequency sweeps from 0.1 to 100 rad s⁻¹ and 1 % linear domain.  A: Storage modulus G′ which contributes to elastic nature. B: loss modulus G″.

The utilization of suitable plant proteins, in conjunction with hydrocolloids, aims to embrace the viscoelastic characteristics, fracture mechanics, and water retention of the PBS throughout the process of oral consumption. When subjected to low rates of deformation or frequencies, the elongated chain molecules, characterized by timescales on the order of milliseconds, are capable of conforming to the relative deformation. Conversely, when exposed to high rates of elongation, the PBS undergo fracture because of topological constraints imposed by entanglements within the melted state (Joyner & Daubert, 2017).

3.6. Lipid peroxidation in PBS during storage

The secondary damage to the lipids in the PBS was studied by using MDA as the marker and is depicted in Fig. 4. During the preparations, PBS were steamed (under saturated condition) for 30 min, which could generate the secondary oxidation products, mainly MDA due to the presence of vegetable oil in the formulation and concomitantly the proteins in the matrix (MBPI and WG) can be heat denatured. The control PBS had a lower TBARS value (6.99 ± 0.225 mg MDA/ kg of the sample) compared gluten-free PBS (PBS-1), which had 40 % MBPI (7.50 ± 0.152 mg MDA/ kg of the sample). This could be related to the change in the PBS matrix. The control PBS had 60 % BCF and PBS-1 had 20 % BCF and 40 % MBPI. The denaturation of BCF protein in control PBS could have facilitated the binding of MDA to the amine groups of lysine to form a Schiff base (Zhao et al., 2022), since BCF is a rich source of lysine. This interaction would have reduced the availability of free MDA (Ma et al., 2021). In contrast, when the BCF content was reduced to 20 % and the MBPI was increased to 40 %, the predominant heat denaturation of MBPI could have hindered the binding of MDA to amine groups due to the varying degree of denatured proteins, which can cause molecular crowding. Afterward, decreasing the MBPI (30 % to 0 %) and simultaneously increasing the WG (10 % to 40 %) showed a statistically decreasing (p < 0.05) TBARS value. This could be due to the increased interaction between the WG proteins and the MDA, which reduces the availability of free MDA and hence decreases the TBARS values.

Fig. 4.

TBARS for the measurement of lipid peroxidation induced secondary lipid oxidation in PBS during storage at 4 °C for one week.

In contrast, during cold storage, the TBARS values increased for all the sausages, compared to the fresh sausages, and the increase was statistically significant (p < 0.05). It could be possible that even at lower temperatures, MDA can be generated upon storage. According to Liu, Liu, & Chen (2019), peanuts showed an increase in MDA levels after being stored at 15 °C for 80 days. Interestingly, the cold-stored control PBS showed high TBARS compared to all the other cold-stored PBS. This could be due to the unavailability of the feasible functional groups, in the cold-stored sausage, to react with MDA. This would have resulted in an increased amount of free MDA that would be distributed across the PBS matrix. When 40 % MBPI was added, the TBARS value decreased to 13.88 ± 0.22 mg MDA/ kg of the sample from 16.97 ± 0.57 mg MDA/ kg of the sample (control PBS) and this could be due to the cold denaturation of the MBPI. During cold denaturation, the exposure of lysine and histidine groups in the MBPI can interact with MDA. The two aldehyde groups in the MDA can electrophilically add to the amine groups in lysine and histidine to form a Schiff base; MBPI is high in lysine (≈62 %) and contains significant amounts of histidine (≈28 %) (Ma et al., 2021). This can reduce the availability of free MDA in the cold-stored PBS– resulting in lower TBARS values compared to the control PBS. However, the increase in WG and a further decrease in MBPI did not significantly affect the TBARS values in the cold-stored PBS. This could be due to the bioactivity of the peptides (the bioactivities were not measured in this study) generated during the thermal treatment of PBS that sequestered the free radicals from lipid oxidation.

The findings from the lipid peroxidation-induced change in the fresh and the cold-stored PBS suggest that MDA can strongly interact with WG compared to MBPI. Presence of WG in PBS is vital to reduce the secondary damage to the lipids. Therefore, incorporation of WG in PBS can reduce the free MDA content, which is usually regarded as a toxic substance to health.

3.7. Microbial analysis of PBS during storage

The shelf stability of the prepared PBS was studied in terms of microbial load considering the aerobic bacteria, yeast-mold, and coliform. The PBMA are highly susceptible to microbial contamination due to high protein and high moisture content (Tóth et al., 2021). In addition, to mimic real meat, PBMA demand more ingredients and hence the microbial load could be expected to be higher. The shelf stability of the PBMAs produced through a soft matter physics approach has not been reported in literature to date. Therefore, in this study, a common household meat storage condition, 4 °C was maintained over nine days and the microbial load was measured every three days. The change in the microbial load with time is depicted in Fig. 5.

Fig. 5.

Change in the (a) aerobic count; (b) yeast and mold; (c) coliform count to assess the microbial load of PBS stored at 4 °C for nine days.

In terms of aerobic plate count, the fresh PBS did not show any microbial growth. This could be because, during the PBS formation, the steaming process would have helped to kill any aerobic microorganism. On day 3, a statistically lower microbial load was observed for the control PBS, compared to the others. All other PBS showed higher microbial load compared to the control PBS but the microbial load among these PBS was statistically insignificant (p > 0.05). The lower aerobic plate count in the control PBS can be attributed to the lower protein and moisture content (Table 1 proximate analysis). However, irrespective of the differences in protein and moisture content in PBS-1 to PBS-5, the microbial load was statistically insignificant. This observation suggests that, during storage, the microenvironment of the PBS matrix would have changed similarly, in PBS-1 to PBS-5, and this could have created a feasible environment for the aerobic microbes to grow. The microenvironment encompasses mainly pH, oxygen level, nutrient content, and internal temperature during storage which can fluctuate over storage. On days 6 and 9, all the PBS showed a decrease in aerobic microbial load. In control PBS and gluten-free PBS (PBS-1), the decrease was not statistically significant (p > 0.05). In contrast, PBS-5 had a statistically significant decrease on day 9, compared to day 6. The decrease in microbial load can be because of the less favorable microenvironment in the PBS-5 matrix, which had WG as the major protein source. This demonstrates that the aerobic microbial load is multifactorial, and the type of protein also plays a major role in the microbial load. The finding from this study is also comparable to the aerobic count of a vegan ground meat product from German retail, where the average aerobic count was reported as 4.33 ± 1.95 log CFU/g (Kabisch, Joswig, Böhnlein, Fiedler, & Franz, 2024).

The yeast and mold were observed in the freshly prepared PBS, unlike the aerobic microorganism. This could be because the streaming of sausages did not kill the spores of yeast and mold. Post-processing contamination in meat analogs is a common issue when considering product hygiene (Thanh Hai, Minh Hien, & Viet Quang, 2024). The highest yeast and mold growth was observed in PBS-1 and PBS-3 and the growth was similar in both PBS, irrespective of high protein and moisture content in PBS-3, compared to PBS-1 (Table 1 proximate analysis). The only possible explanation could be that 40 % MBPI (in PBS-1) and equal amounts of MBPI and WG (in PBS-3) created a similar favorable environment for the spores of yeast and mold to grow. The yeast and mold growth with time did not show statistical significance in control PBS from 3rd to 9th day, with 3rd day being the lowest compared to other PBS. The highest yeast and mold growth, on day 3, was observed in PBS-2. This could be due to the incorporation of WG that created a favorable environment for the yeast and mold to grow. Furthermore, for all other PBS, except the control PBS, on days 6 and 9 the yeast and mold growth did not show a uniform trend, and this could be due to changes in the PBS matrix during storage, which is not feasible to explain.

Coliforms are naturally present in food substances but can be eliminated during processing. Andras et al. (2021) showed that raw WG had 3.3 log CFU/g coliform and when it was included in vegan meatballs, the coliform count increased to 5.46 and 7.26 log CFU/g stored at 36 and 48 h, respectively. In the present study, the coliform count was absent in the freshly prepared PBS, which could have been eliminated during the streaming of the PBS. In addition, control PBS and PBS-5 did not show any coliform load on day 3. In both these samples, MBPI was absent, and it could be inferred that MBPI is a favorable protein for the growth of coliform. On day 3, the coliform load was higher for gluten-free PBS (PBS-1) and it statistically decreased when the MBPI was decreased to 30 % and 10 % WG was added (PBS-2). A further decrease in MBPI and an increase in WG increased the coliform load (PBS-3 and PBS-4); the increase in coliform count was statistically insignificant for PBS-3 and PBS-4. The finding can be possibly related to the varying degree of interaction between MBPI and WG that created a unique matrix to influence the coliform population. On days 6 and 9, the coliform load on PBS-4 was statistically insignificant, and this could be due to two reasons: (1) the storage temperature did not favor the proliferation of the coliform; (2) no significant change took place in the PBS matrix upon storage.

3.8. Microstructure of PBS

The microstructure of meat analogues may differ due to variations in protein type and source, processing techniques, and the inclusion of supplementary ingredients. The physical attributes such as proximate composition, texture and color influence the microstructure of the meat analogues. Protein network provides the main structure-forming component of meat analogues, which contributes to strength, elasticity, and cohesiveness (Godschalk-Broers et al., 2022; Okeudo-Cogan et al., 2023). Fig. 6 shows the microstructures of different PBS samples at 100× and 500× magnification. Apparent sheet-like fiber formation was seen in control PBS and gluten-free PBS (PBS-1) at 500× magnification. The micrograph of these PBS reveals that both MBPI and BCF contributed to the fibrous structure. BCF significantly improves the microstructural characteristics of PBMA by establishing a robust protein matrix. The protein constituents present in BCF facilitate the formation of a cohesive network within the meat analogue (Mazumder, Panpipat, et al., 2023). This network is essential for ensuring structural stability and replicating the textural qualities of meat (Schreuders et al., 2021). The gelation properties inherent in BCF contribute to the formation of a stable matrix within the PBS, resulting in a fibrous structure (Younis et al., 2023). Previous investigation has postulated that the mixing of two proteins could markedly augment the development of fibrous networks in MAs (Grabowska et al., 2014). The incorporation of WG (PBS-2) showed fibrous structure but this was not as significant as control PBS and gluten-free PBS (PBS-1). The role of gluten in influencing the microstructure of PBS is vital for the attainment of a desirable product. The elasticity provided by WG aids in the development of a fibrous structure within the PBS (Dinali et al., 2024). Moreover, gluten's property of absorbing and keeping moisture significantly contributes to the moisture content in meat alternatives (Sengar et al., 2023). This moisture retention further influences the microstructural attributes by ensuring that the PBS remains moist and tender throughout the cooking process. When WG is subjected to thermomechanical process, the air can be incorporated to form more voids. But in PBS-2 less voids were seen which could be because of the interaction of MBPI and WG that prevented the complete formation of gluten network, which aligns with TPA (Table 4). A further increase in WG and decrease in MBPI showed agglomerates (PBS-3) and a hybrid of agglomerates and sheets (PBS-4). When MBPI was completely substituted with WG (PBS-5), a denser structure was observed, and this was in good agreement with TPA where PBS-5 showed high hardness and chewiness; the dense structure can be correlated to the increased hardness and chewiness.

Fig. 6.

The microstructures of different PBS at 100× and 500× magnification. A (control PBS 100×) and B (control PBS 500×), C (PBS-1100×) and D (PBS-1500×), E (PBS-2100×) and F (PBS-2500×), G (PBS-3100×) and H (PBS-3500×), I (PBS-4100×) and J (PBS-4500×), Images K (PBS-5100×) and L (PBS-5500×).

4. Conclusion

The study indicates that the amounts of the ingredients have a considerable effect on the techno-functional attributes of the PBS. The strategic replacement of gluten protein with non-gluten proteins like MBPI, in conjunction with ingredients such as BCF, can yield PBS with desirable characteristics while also highlighting areas for further research, particularly concerning lipid oxidation and microbial safety. The complexity associated with plant-based meat substitutes possessing the desired qualities renders the formulation of such products a formidable task. This difficulty arises from the diverse influences that various ingredients and processing parameters impose on the ultimate product. Furthermore, these elements interact in manners that further obscure the identification of the most favorable formulation. Consequently, achieving a product that fulfills all technological specifications becomes a challenging endeavor. Notably, within the vegan and vegetarian communities, there is an increasing inclination towards the exploration of novel products that are less similar to conventional meat items while still facilitating the preparation of traditional foods such as burgers or sausages. This transformation in consumer inclinations presents new opportunities concerning the acceptance and subsequent success of plant-derived products, even when their attributes significantly differ from those of traditional meat-like items.

5. Future prospects

The ongoing exploration of different protein origins and processing techniques is set to significantly influence the advancement of the vegan food industry, creating opportunities for the creation of more sustainable and nutritionally enhanced meat substitutes that correspond with consumer desires. More extensive investigations into the characterization of the sausages are necessary to assess the potential alterations in flavor, color, and texture throughout the storage period. For instance, pathways of protein and lipid oxidation and their roles in flavor development could be incorporated into forthcoming research endeavors.

CRediT authorship contribution statement

Nilushni Sivapragasam: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Mohammad Affan Baig: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Raouf Aslam: Writing – review & editing. Weibiao Zhou: Writing – review & editing, Project administration, Funding acquisition. Sajid Maqsood: Conceptualization, Writing – review & editing, Validation, Supervision, Resources, Project administration, Investigation, Funding acquisition.

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.