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. 2022 Nov 11;16:100508. doi: 10.1016/j.fochx.2022.100508

Transglutaminase treatment and pH shifting to manipulate physicochemical properties and formation mechanism of cubic fat substitutes

Lu Huang a, Di Zhao a, Yong Wang b, He Li a,⁎, Haochun Zhou c, Xinqi Liu a,⁎
PMCID: PMC9743292  PMID: 36519098

Highlights

  • •

    With the increase of pH, the thermal stability of cube fat substitute (CFS) increased.

  • •

    Transglutaminase induced soybean protein isolate crosslinking in CFS.

  • •

    Deacetylation of KGM under alkaline condition with dense network structure.

  • •

    CFS has low oral tribological coefficient and good lubricity.

Keywords: Cube fat substitute, Cross-linking, Deacetylation, Oral tribology

Abstract

A cube-shaped pork fat substitute (CFS) was created from soybean protein isolate (SPI), coconut oil, and konjac glucomannan (KGM). The effect of transglutaminase (TG) treatment and pH shifting on the mechanical, thermal, and sensory properties of the cube fat substitute were investigated. The sensory evaluation and oral tribological results showed that the CFS with 1 % TG at pH = 8 was the closest to natural animal fat. The TPA results showed that the hardness, cohesiveness, and chewiness gradually increased with the increasing pH. The DSC results indicated that TG treatment and higher pH levels enhanced the thermal stability of the CFS. FTIR spectroscopy confirmed that when heated in alkaline conditions, KGM deacetylated, and the strength of carbonyl group in acetyl group decreased gradually. The protein network structure was more evident after TG treatment as observed via CLSM, where the KGM molecular chains were entwined as the pH increased, forming continuous, gel networks.

Introduction

Animal protein production contributes to several global severe problems, including the high consumption of fresh water, fossil fuel and land, and greenhouse gas emissions. The relatively low efficiency of animal meat production, the negative impact of meat products on health, environmental pressure related to animal production, and animal welfare are the main driving forces of the meat substitute market (Sha & Xiong, 2020). Animal fat plays a vital role in the flavor, texture, and taste of food, and can increase the customer satisfaction and acceptance of meat-based food. However, studies have shown that excessive dietary fat intake is related to chronic diseases such as ischemic heart disease and obesity, and fat reduction is often considered a crucial strategy for producing healthier products (Jimenez-Colmenero et al., 2012, Kumar, 2021). However, simply removing fat from food may lead to a decrease in the overall consumer acceptance (Chen, She, Zhang, Wang, Zhang, & Gou, 2020). Fat substitutes can be potential solution to reduce the overall fat and calorie content of food, but still mimic the physicochemical properties of animal fats.

Soybean protein isolate (SPI) can be used to form emulsion gels to mimic animal fat tissue with excellent freeze–thaw stability and ideal rheological properties (Takamatsu, Tachibana, Matsumoto, & Abe, 2004). Konjac glucomannan, a water-soluble polysaccharide containing 1,4-linked β-d-mannopyranose and β-d-glucopyranose copolymer units, has also shown the potential to be used as animal fat substitute (Liu, Xu, Zhang, Zhou, Lyu, Zhao, et al., 2015). Moreover, KGM can also help to control obesity by delaying gastric emptying and can be used as a cholesterol-lowering drug by interfering with cholesterol transport (Johnston, Korolenko, Pirro, & Sahebkar, 2017). Therefore, KGM was used to enhance the textural and structural stability of meat products, such as konjac SPI mixture as a fat substitute in low-fat bologna sausages (Chin, Keeton, Miller, Longnecker, & Lamkey, 2000). Zhu et al. prepared coloring composite gels using KGM, SPI, and capsanthin and found that the gel strength, hardness, and chewiness of the optimally prepared coloring composite gels were significantly increased and the textural properties were improved (Zhu, Liu, Fan, Duan, Chen, & Zhang, 2021).

Transglutaminase (TG), i.e. protein-glutamine γ-glutamyltransferase, belongs to the class of transferases, has considerable potential to improve the compactness, viscosity, elasticity and water binding ability of food (Kieliszek & Misiewicz, 2014). Transglutaminase (TG) acts as a stabilizer, emulsifier, and thickener, preventing the adverse effect of gel products. It can catalyze the intramolecular or intermolecular cross-linking reaction between the protein residues in the ε-amino and γ-glutamine group (Zhang, Cui, Zhou, Wang, & Zhao, 2021), which can affect protein functions: solubility, emulsifying ability, foaming performance and gel (Giosafatto, Rigby, Wellner, Ridout, Husband, & Mackie, 2012).

Several meat products need fat with a specific size and shape, which make the unique texture and appearance of the product. For example, the fat in fermented sausages (e.g. salami) and sliced bacon necessitates a certain gel strength to ensure sufficient structural integrity and graininess, rather than fat paste (Dreher, Weissmuller, Herrmann, Terjung, Gibis, & Weiss, 2021). Dreher et al. used SPI and TG as raw materials and completely hydrogenated canola oil as a solid fat to prepare emulsified plant derived fat crystal networks to mimic animal fat. These substitutes exhibited the plasticity, elasticity, and mechanical properties of animal fat and could be used to produce meat products or meat analogs (Dreher, Blach, Terjung, Gibis, & Weiss, 2020a). Moreover, higher protein content increased the hardness and elasticity of cross-linked fat crystal networks, offsetting the plasticity of the fully hydrogenated canola oil (Dreher, Blach, Terjung, Gibis, & Weiss, 2020b). Chen et al. used KGM and carrageenan as raw materials to prepare cube fat substitutes to partially replace the pork fat in Harbin sausages. No significant differences were evident between the physicochemical properties and sensory scores of the sausages exhibiting replacement levels below 40 % (Chen, Zhao, Li, Liu, & Kong, 2021).

Current studies involving CFSs have focused on the preparation process. Although the prepared emulsion gel displays a gel structure, it exhibits high viscosity and a soft texture, making three-dimensional block formation challenging. This study used SPI, konjac, and coconut oil as raw materials to prepare CFSs simulating natural pork fat. The mechanical, thermal, oral tribological, and sensory properties of the CFSs were optimized by adjusting the pH and TG treatment, and the formation mechanism of SPI/KGM composite CFS gels was elucidated via FITR spectroscopy and confocal laser scanning microscopy (CLSM).

Materials and methods

Materials

The SPI (protein = 91.2 %, moisture = 5.1 %, on a dry basis) was purchased from the Shandong Yuwang Ecological Food Industry Co., Ltd. (Shandong, China), while the coconut oil was obtained from Henan Yuanzhuo Biotechnology Co. Ltd. The konjac powder (glucomannan = 83.6 %, moisture = 10.5 %, and ash = 3.8 %, on a dry basis) was acquired from the Hubei Consistent Biotechnology Co., Ltd. (Hubei, China), while the TG was purchased from Jiangsu Yiming Biological Co., Ltd. (Jiangsu, China). The pig back fat was obtained from a local supermarket (Meilianmei, Beijing, China). Edible sodium hydroxide (granulated alkali, purity 99 %) was acquired from Tianjin Zhonghe Shengtai Chemical Co., Ltd. (Tianjin, China), while food-grade lactic acid (80 %) was purchased from Zhengzhou Kangben Biotechnology Co., Ltd. (Henan, China). Calcofluor white, Nile Blue, and Nile Red colorants were purchased from Cypress Technology Co., Ltd. (Beijing, China).

Preparation of the protein/polysaccharide composite emulsion gel and CFS

The SPI (5 % w/w) was evenly dispersed in the cooking water at room temperature and stirred with an electronic stirrer (EUROSTAR 40 digital, IKA, Baden-Württemberg, Germany) at 3000 rpm for 4 min, after which coconut oil (10 % w/w) was added and stirred at 6000 rpm for 5 min to prepare the emulsion. Two separate sample groups were established at different pH levels (4.5, 6, 8, and 10). One group contained TG (1 % w/w), while the other did not. The samples of the enzyme supplemented group were dissolved in water, mixed thoroughly, and stirred with an electronic stirrer at 4000 rpm for 1 min. A small amount of food-grade lactic acid and edible sodium hydroxide was added to adjust the pH to 4.5, 6, 8, and 10, respectively. The konjac powder (8.5 % w/w) was then slowly added to the pH-adjusted emulsion and stirred at 3000 rpm for 3 min. The mixture was incubated in a 50 °C water bath pot (Jintan Kexi, HH-2 Water bath pot, Jintan Kexi Instrument Co., Ltd, Jiangsu, China) for 30 min. After incubation, it was poured into a baking mold with length, width, and height of 11.5 cm, 8.5 cm, and 3 cm and degassed at −0.09 MPa for 30 s (Exelway, DZ-300, Quanzhou Liding Mechanical Equipment Co., Ltd, Fujian, China). The samples were then boiled in a water bath at 95 °C for 60 min and stored in a refrigerator at 4 °C for 24 h for further analysis. The composition formula of the CFS is as follows: 5 % SPI, 10 % coconut oil, 8.5 % konjac flour, and 1 % TG (enzyme group), while the remaining volume consisted of water.

Rheological measurements of the SPI/konjac emulsion gel

The rheological behavior of SPI/konjac emulsion gel was determined using an oscillatory rheometer (DHR-1, TA Instruments, New Castle, USA). A parallel plate (40 mm diameter and 1 mm gap) measurement system equipped with a temperature control device was used. The surface of the measurement system was sandblasted to avoid wall slippage. About 4 g of the central part of the sample was placed in the center of the chassis, after which the upper plate was slowly moved down to a spacing of 1 mm between the upper and lower plates. The excess sample was scraped off with a spatula, and the edge of the protective cover was sealed with methyl silicone oil to prevent evaporation. The samples subjected to temperature scanning in the following conditions: frequency 10 rad/s−1, strain 1 %; incubation stage: 30–50 °C, heating rate: 5 °C/min, held at 50 °C for 30 min; ripening stage: 50–95 °C, heating rate: 5 °C/min, held at 95 °C for 60 min; cold storage stage: 95–4 °C, held at 4 °C for 180 min.

Differential scanning calorimetry (DSC) analysis

The thermal behavior of the samples was monitored via DSC using a previously calibrated differential scanning calorimeter (DSC-60 Plus, Shimadzu, Japan) in a temperature range of 30–150 °C. About 10 mg of the boiled samples were placed into a liquid crucible and sealed using a DSC-60A seal kit (Shimadzu, Japan). The initial temperature of the sample was 30 °C, which was increased to 150 °C at a heating rate of 10 °C/min.

Observation of the microstructure via CLSM

The images of the natural fat boiled for 30 min and CFSs were taken using CLSM (Nikon, A1+, Tokyo, Japan). Using a dissection blade, the samples were cut into small pieces with a cross-sectional area of approximately 16 mm2 and a height of 1 mm. The natural fat and CFSs were stained with 1 % (w/w) Calcofluor white, 0.2 % Nile Red, and 0.5 % Nile Blue aqueous solutions. The konjac flour was stained with Calcofluor white, while the oil was preferentially stained with Nile Red, and the protein was preferentially stained with Nile Blue. The determination was performed according to a method described by Wei et al. (Wei et al., 2021) with some modifications. The samples were placed on slides, after which 30 μL of calcium fluorescent white stain was added dropwise, followed by the addition of 10 μL Nile Blue and Nile Red after 10 min of equilibration in the dark. The slides were covered with coverslips and observed using a 20x magnification objective lens. The samples were sliced using a dissecting blade to create a smooth surface for CLSM. The excitation/emission wavelengths of the Calcofluor white, Nile Red, and Nile Blue were 355/440 nm, 488/543 nm, and 561/613 nm, respectively.

Fourier transform infrared (FTIR) spectrum analysis

The CFSs were characterized via FTIR (Perkin Elmer, Frontier, Massachusetts, US) to analyze their functional groups. The fat samples were cut into small cubes, pre-frozen at −40 °C for 48 h, freeze-dried (Marin Christ, Beta 1–8 LSC basic, Christ, Osterode, Germany) for 72 h, and ground into powder using a mortar and pestle. Next, 1–2 mg of the powder samples and 200 mg of pure KBr were finely ground, placed in a mold, and pressed into a transparent sheet using a hydraulic press. The specimens were then placed in an infrared spectrometer for testing in a wavenumber range of 4000–400 cm−1 with 32 scans and a resolution of 4 cm−1.

Texture profile analysis (TPA)

The CFS and natural fat were cut into 2 cm × 2 cm × 2 cm squares and placed on the base of the instrument. The TPA was conducted using a texture analyzer (CT3, Brookfield, Middleboro, USA). Uniaxial compression was used at a test speed of 1 mm/s to deform the sample twice to 40 % of the original height at a time interval of 5 s between the two compression cycles to determine the textural parameters, including hardness, cohesiveness, springiness, and chewiness.

Oral tribology

An oral tribology analysis was conducted using a stress-controlled rheometer to evaluate the lubricating properties of the CFS and natural fat. A tribological fixture with ball plate geometry was used for the measurement. The CFS granulums were obtained via rotation in a food processer (Vorwerk, Meishanpin multifunctional food processor TM5, Vorwerk, Wuppertal, Germany) at 6000 rpm for 2 min. Then, 3 g of the sample was added to 3 mL of artificial saliva (Source leaf, ISO/TR10271, neutral, Shanghai Yuanye Biotechnology Co., Ltd, Shanghai, China) to simulate the food state during swallowing (Huang et al., 2022). 3 M Transpore Surgical Tape 1527-2 (3M Medical, USA) was used to simulate the surface roughness and wettability of the tongue. The tape was cut into squares, attached to the rheometer base, and compacted. After each sample test, the base was cleaned with deionized water and wet wipes, dried, and the tape was replaced. To simulate the oral process, a normal force of 2 N was established. Moreover, the oral conditions were simulated at a temperature of 37 °C, while the rotational speed was increased from 0.01 mm s−1 to 100 mm s−1.

Sensory analysis

The sensory assessment consisted of 17 trained panelists who sequentially assessed 6 g of the natural fat and CFSs cooked for 30 min. A ten-point scale was used where 1 represented “dislike extremely”, and 10 denoted “like extremely”. The panelist tasted the samples without knowing the details of the samples. Salt-free bread and water were used to clean the palate between samples. The panelists evaluated juiciness, chewiness, textural state, and overall acceptability.

Color measurement

The cooked natural fat and samples were cut into 3 cm × 3 cm × 1 cm cubes, and their exterior color was determined using a colorimeter CM-3610A (Konica Minolta, Japan). Before use, the instrument was calibrated using a whiteboard (standard reference provided by Konica Minolta) and a standard blackboard. The lightness (L*), redness (a*), and yellowness (b*) values were recorded. Four measurements were performed for each sample.

Statistical analysis

All data were analyzed via one-way analysis of variance (ANOVA) and plotted using Origin2021 and SPSS software. Duncan's multiple range test was applied to separate the means of data when significant differences (P < 0.05) were observed. All measurements were repeated at least three times.

Results and discussion

Rheological analysis

Temperature scans were performed using an oscillatory rheometer to investigate the effect of TG treatment and pH changes on the rheological properties of the SPI/konjac gel network. The temperature scan was divided into three main stages, namely the incubation stage, ripening stage, and cold storage stage. Fig. 1, Fig. 2, Fig. 3 show the temperature scan storage modulus (G′) and loss modulus (G″) as temperatures functions for the SPI/konjac composite gel at different processing stages and pH values at 0 % and 1 % TG, respectively.

Fig. 1.

Fig. 1

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The temperature scanning results of the 0% (A) and 1% (B) TG samples at different pH levels during the incubation stage, ripening stage, and cold storage stage (A1, 3, 5 and B1, 3, 5 are heating stages, A2, 4, 6 and B2, 4, 6 are holding stages).

Fig. 2.

Fig. 2

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The DSC curves of the samples with different TG content (0 % and 1 %), pH levels, and natural fat (cooked for 30 min).

Fig. 3.

Fig. 3

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The CLSM images of the 0 % TG (A, B, C, D) and 1 % TG (E, F, G, H) samples with different pH levels (4.5, 6, 8, and 10) and the natural fat (cooked for 30 min) (I full channel, J protein channel alone) (red for proteins, green for lipids, and blue for polysaccharides). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

All samples exhibited gel-like characteristics (G′ > G″). As shown in Fig. 1, no significant changes were evident between the G′ and G″ at higher temperatures during the incubation stage. A slight increase occurred during the incubation holding stage at 50 °C for 30 min. The G′ and G″ of the 1 % TG samples were slightly higher than the untreated group. This may be because TG catalyzes the covalent cross-linking between SPI glutaminyl residues of the and lysine residues ε-amino groups (Koksel, Sivri, Ng, & Steffe, 2001) to form a network structure that changes the weak network of the batter into a more ordered gel network. There was no visible difference between G′ and G″ of the samples with different pH levels, which could be attributed to the dominant role of TG at this stage and the fact that the deacetylation of konjac glucan required alkaline conditions above 85 °C to proceed, while the maximum temperature of incubation was 50 °C. The sample structures remained unchanged at different pH levels.

As shown in Fig. 1, the G′ decreased significantly at higher temperatures during the ripening stage, while the G″ displayed no substantial changes. The G′ decline during heating is consistent with the findings of Jian et al. (Jian, Siu, & Wu, 2015), probably because heating weakened the inter- and intra-molecular attraction, destabilizing the gel network and destroying the hydrogen bonds. The G′ and G″ first decreased, followed by an increase during the holding stage at 95 °C for 60 min. An initial increase was evident at about 30 min of holding at 95 °C, probably due to heating in alkaline conditions, KGM deacetylation. Additionally, stronger interaction between the deacetylated konjac glucan and SPI molecules would allow the disrupted network structure to reorganize and rearrange into an ordered network structure.

As shown in Fig. 1, the G′ and G″ of all the samples increased gradually during the cold storage stage (95–4 °C), which may be due to the gradual and irreversible stabilization of the protein network. The G′ and G″ tended to increase during cooling at 4 °C, indicating further rearrangement within the network structure. This was consistent with the results obtained by Xu et al. (Xu, Guo, Li, Jiang, Zhong, & Zheng, 2021), who examined the effect of TG on SPI/polyphenol complex gels, showing that the G′ increased over time during the cooling scanning phase. Another possible reason for this was the formation of thermally irreversible gels via the heating acetylation of KGM in alkaline conditions.

DSc

The DSC curves of the samples exposed to different pH levels and TG treatments as well as the natural fat, are shown in Fig. 2. The peak temperatures of the samples with pH = 4.5 and 6 without TG differed significantly from those at pH = 8 and 10. The peak temperature gradually increased at a higher pH level, indicating a progressively improved stability. The reason may be that the KGM deacetylated via heating in alkaline conditions led to the formation of hydrogen bonds between konjac glucomannan molecules, which enhanced the intermolecular force and improved the thermal stability of CFS. This was coincident with the results acquired by Penroj, Mitchell, Hill, and Ganjanagunchorn (2005), who showed that the peak values of the k-Carrageenan and konjac glucomannan gel gradually increased at higher pH levels. The peak temperature of the TG samples with different pH levels steadily increased. This corresponded with the findings of Tang, Chen, Li, and Yang (2006), indicating that TG changed the internal structure of the CFS gel, exhibiting a thermal stability effect on the SPI components. The thermal trends of the DSC curves of the natural fat and CFSs vary substantially. This may be due to the significant differences between the water content of the two samples, which was also consistent with previous findings (Huang et al., 2022).

CLSm

CLSM is commonly used to analyze the morphology and structure of multicomponent polymers (Cui et al., 2021). In Fig. 3, the red fluorescence represents proteins, the green fluorescence denotes lipids, and the blue fluorescence signifies polysaccharides.

In Fig. 3, I and J represent the full channel map and protein channel map of the natural fat cooked for 30 min, respectively. The natural fat is consisted of a collagen network structure and more lipids on the surface, which was consistent with the results of Dreher, Blach, Terjung, Gibis, and Weiss (2020a). As shown in Fig. 3A, the pH was close to the SPI isoelectric point, while the protein molecules carried less electrostatic charge and tended to agglomerate into clumps, preventing the formation of an ordered network structure. A comparison between Fig. 3A, B, C, and D and Fig. 3E, F, G, and H showed that the red (orange) protein network structures of the TG-added samples were more pronounced, indicating that agglomeration occurred during the enzymatically induced gel formation, which was consistent with the findings of Yang, Liu, and Tang (2013). The konjac glucan was deacetylated at a higher pH to gradually reveal the KGM network structure. The KGM network structure at pH = 10 interspersed and interacted with the SPI network structure, showing a compact, dense internal network structure with uniform distribution. It could be because that KGM prevented SPI heating aggregation via long-chain polysaccharide site-blocking, allowing the formation of a continuous protein network and interacting with proteins to strengthen the gel network (Zhang, Li, Wang, Xue, & Xue, 2016). Due to the high degree of deacetylation, KGM contained more hydroxyl groups, glucose residues, and mannose residues, while a higher cross-linking with SPI residues was evident, possibly acting as a filler in the gel matrix (Zhang, Xue, Li, Wang, & Xue, 2015).

FTIr

The intermolecular interaction between SPI and KGM can be explained by studying the shape, intensity and position changes of the FTIR spectral peaks. The absorption band at 3430 cm−1 represents the conjoined hydroxyl stretching vibration absorption peak of KGM (Jian, Siu, & Wu, 2015). The stretching vibrational absorption peak of the carbonyl group in the acetyl group is represented at 1745 cm−1. As shown in Fig. 4A, the intensity of the acetyl group gradually decreased as the pH increased, indicating that KGM was deacetylated when heated in alkaline conditions, which involved substituting acetyl groups with hydroxyl groups (Yang, Yuan, Wang, Wang, Mu, Pang, et al., 2017). However, the strength of the hydroxyl group gradually decreased at higher pH levels since the hydrogen bond that maintained the tertiary SPI structure in alkaline conditions was broken. This corresponded with the results of Wang et al. (2020), who showed that the broad peak at around 3000–3700 cm−1 and the minor peaks at 2927 cm−1 exhibited the characteristic absorption peaks of hydrogen bond hydrogen bond —OH stretching, while —CH group stretching occurred between the proteins and carbohydrates. As shown in Fig. 4B, the absorption bands of the samples treated with TG displayed significant differences, especially in the acylamido I region (1700–1600 cm−1 referring to C Created by potrace 1.16, written by Peter Selinger 2001-2019 O stretching vibration), acylamino II region (1590–1500 cm−1 referring to bending vibration and C—N groups and stretching vibrations of C—N stretching), and 1300–1000 cm−1 (C—O stretching vibrations) (Wu, Liu, Liu, & Wang, 2017). Furthermore, 3330 cm−1 and 1642 cm−1 indicated the presence of the functional compounds of the protein amide groups —CONH—, which was stronger. This suggested that the TG catalyzed glutamyl and lysine cross-linking in food proteins via an acyl transfer reaction to form amide bonds, which was consistent with the CLSM findings that the protein network was more pronounced in the samples supplemented with TG.

Fig. 4.

Fig. 4

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The FITR curves of the 0% (A) and 1% (B) TG samples with different pH levels.

TPa

The textural properties of food are essential for taste quality perception by consumers. As shown in Table 1, the hardness, cohesiveness and chewiness gradually increased with increasing pH. The hardness of CFS with the addition of 1 % TG at pH = 6 and the addition of 0 % TG at pH = 8 is close to that of real pork fat. The hardness increased may be due to KGM deacetylation, transforming KGM from semi-curling to self-coiling, leading to self-aggregation and the formation of a three-dimensional network that can trap water and form a rigid, compact structure (Wang et al., 2017). The cohesiveness and chewiness increased may be due to the tight binding between protein and polysaccharide network matrix in fat substitutes, which has been reported in the emulsion stabilized by konjac glucomannan and SPI together (Liu, Lin, Shen, & Yang, 2020). Adding 1 % TG slightly improved the CFS’s cohesiveness, chewiness, and hardness. This was consistent with the findings of Forghani, Eskandari, Aminlari, and Shekarforoush (2017), who showed that adding TG to burger formulations enhanced the cohesion of meat batters and improved their firmness and chewiness. This may be attributed the fact that TG can catalyze the transfer of the γ-carboxyamide group of the glutamine residue in proteins to the ε-amino group of lysine residues. This reaction resulted in the formation of ε-(γ-glutamyl) lysine isopeptide and inter-protein covalent bonding (Mi et al., 2020), improving the texture of the samples. The pH and the addition of TG minimally affected the springiness, while no significant differences were evident between the samples, with the highest level of springiness in the pH = 10 sample. The 1 % TG, pH = 6 sample was close to natural fat in every parameter and could better simulate fat.

Table 1.

The textural results, the color parameters and sensory evaluation results of the samples with different TG content (0 % and 1 %) and different pH levels, and natural fat (cooked for 30 min).

Sample TPA
 Chroma
 Sensory evaluation
Hardness (g) Cohesiveness (-) Springiness (mm) Chewiness (mJ) L* a* b* Juiciness Chewiness Texture state Overall acceptability
TG
0 %		 pH = 4.5 536.75 ± 13.23a 0.58 ± 0.04ab 6.45 ± 0.14a 19.68 ± 1.54a 87.26 ± 0.31f 0.94 ± 0.11e 10.71 ± 0.24b 6.47 ± 1.62bc 4.65 ± 1.17e 4.35 ± 1.32c 4.29 ± 1.53d
pH = 6 579.00 ± 26.00ab 0.63 ± 0.01bd 6.42 ± 0.07a 23.00 ± 0.95ab 86.08 ± 0.42d 0.65 ± 0.03d 10.86 ± 0.09b 6.53 ± 1.28b 6.41 ± 1.12bc 6.06 ± 1.64b 5.76 ± 1.44c
pH = 8 779.25 ± 28.00c 0.82 ± 0.09abc 7.18 ± 0.51b 45.33 ± 8.16bcd 83.91 ± 0.19c 0.51 ± 0.03c 11.35 ± 0.08a 5.59 ± 1.62bcd 7.06 ± 1.34ab 6.53 ± 1.81b 6.53 ± 1.50abc
pH = 10 827.50 ± 86.55bcde 0.81 ± 0.11abc 7.33 ± 0.43b 47.68 ± 6.95 cd 82.40 ± 0.10b 0.40 ± 0.01ab 11.37 ± 0.13a 5.47 ± 1.62bcd 7.24 ± 1.20ab 6.47 ± 1.94b 5.82 ± 1.63c



TG
1 %		 pH = 4.5 568.75 ± 27.10ab 0.59 ± 0.01ae 6.14 ± 0.30a 20.23 ± 1.16ab 86.69 ± 0.39ef 0.86 ± 0.07e 9.91 ± 0.07c 6.24 ± 1.79bc 4.88 ± 0.99de 4.47 ± 1.33c 4.71 ± 1.83d
pH = 6 778.00 ± 27.78c 0.66 ± 0.01bd 6.54 ± 0.25a 32.93 ± 2.22c 85.91 ± 0.40d 0.56 ± 0.06 cd 10.22 ± 0.10c 5.35 ± 1.62 cd 5.71 ± 1.76 cd 6.41 ± 1.23b 6.35 ± 1.50bc
pH = 8 887.75 ± 25.66d 0.72 ± 0.02c 6.55 ± 0.06a 40.98 ± 2.14d 83.86 ± 0.18c 0. 49 ± 0.02bc 11.31 ± 0.34a 5.06 ± 1.30de 7.06 ± 1.48ab 7.59 ± 1.00a 7.53 ± 1.42a
pH = 10 1016.00 ± 4.97e 0.75 ± 0.05cde 7.10 ± 0.22b 51.93 ± 4.32d 81.56 ± 0.13a 0.47 ± 0.04abc 11.32 ± 0.58a 4.06 ± 1.30e 7.65 ± 1.77a 7.65 ± 1.37a 7.41 ± 1.28ab



Natural fat 701.25 ± 71.30acd 0.74 ± 0.08abc 6.58 ± 0.36a 33.70 ± 9.48 cd 86.26 ± 0.87de 0.37 ± 0.12a 11.38 ± 0.42a 8.88 ± 1.11a 7.88 ± 1.90a 7.76 ± 1.86a 7.06 ± 1.68ab
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The mean values with different letters in the same column were significantly different (TPA and Chroma: n = 4; Sensory evaluation: n = 17, p < 0.05).

Color analysis

Color is an essential indicator that can enhance the acceptability of food and increase appetite. Therefore, color is a crucial parameter during the research process of fat substitutes and meat products. The L*, a*, and b* color values are presented in Table 1. A gradual decrease was evident in the L*, b*, and a* of the CFSs at higher pH levels. Minimal differences were apparent between the TG and non-TG groups, with the group exposed to TG displaying slightly lower color values. The decrease in L* could be due to the nonenzymatic browning reaction of maltose in the commercial preparation of TG during heating, which was coincident with the results obtained by Marco and Rosell (2008), who revealed that the L* of TG-supplemented bread decreased. The yellowness value of the sample increased gradually with the increase of pH, which is consistent with the research results of Kaewprachu et al. They found that when SPI was exposed to alkaline environment, the color of the solution changed from ivory to dark yellow (Kaewprachu, Osako, Benjakul, Tongdeesoontorn, & Rawdkuen, 2016). The pH 4.5 and 6 samples were lighter in color, probably because the SPI was located at the isoelectric point and could not form an ordered network structure, rendering the color more transparent. In alkaline conditions, the deacetylation group of konjac glucomannan reaggregated, resulting in a denser network and a darker color. Study found that alkaline incubation led to the darker color of KGM gel (Yang et al., 2020). The a* and b* values of the pH 8 and 10 samples closely resembled that of the natural fat cooked for 30 min, while the pH 4.5 and 6 samples displayed similar L* values to the natural fat.

Analysis of the oral tribology

The use of texture analyzers, viscometers, and rheometers to study the physical properties of food products involves structural damage and shear deformation, which are only applicable to the textural properties directly related to phase deformation and not to sensory properties such as creaminess, slipperiness, and smoothness. The lubrication behavior between oral surfaces becomes the dominant mechanism related to food texture and taste (Prakash, Tan, & Chen, 2013). The friction curves of the CFS with different pH levels and TG treatments, as well as the natural fat cooked for 30 min, are shown in Fig. 5. The friction coefficients of all the CFSs were below 0.2, indicating good lubrication properties. The higher friction coefficient of the 1 % TG, pH = 10 sample may be due to the combination of TG-induced protein network cross-linking and KGM deacetylation with the protein network, resulting in a harder texture and increased roughness. In comparison, the 0 % TG, pH = 6 and 1 % TG, pH = 8 samples displayed lower coefficients of friction and were more successful in simulating the swallowing sensation of natural fat. The lower coefficient of friction of the natural fat may be due to the fact that natural fat contains a significant amount of liquid fat, producing more lubrication during testing. This was consistent with previous findings (Huang et al., 2022).

Fig. 5.

Fig. 5

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The friction curves of the samples with different TG content (0 % and 1 %) and different pH levels, and natural fat (cooked for 30 min).

Sensory evaluation

The sensory evaluation results of the CFS and natural fat (cooked for 30 min) at different pH levels and TG treatments are shown in Table 1. The results showed that the juiciness, chewiness, and texture state scores gradually decreased as the pH increased, while the overall acceptability of the pH = 8 samples was higher. The highest juiciness was evident in the natural fat cooked for 30 min, which could be related to the high oil content. The chewiness, texture state, and overall acceptability scores of the pH = 8 and 10 samples were similar to the natural fat scores after 30 min of cooking (except for the 1 % TG, pH = 10 CFS). The addition of TG improved the chewiness, texture state and overall acceptability of the CFS. These results were consistent with other reports that TG improved the textural and quality attributes of veggie burgers via the formation of cross-links between protein molecules, enhancing the sensory properties of the samples and improving the overall acceptability of the product (Forghani, Eskandari, Aminlari, & Shekarforoush, 2017). The overall acceptability initially increased, followed by a decrease at a higher pH level. The overall decline in the acceptability of the pH = 10 samples may be due to the increasing stiffness of the samples with the addition of TG and konjac acetylation. These results were consistent with the findings of Li, Easa, Liong, Tan, and Foo (2013), who showed that consumer preference tended to decrease as the texture of layered noodles became stiffer with increasing TG levels. The chewiness gradually increased at higher pH levels, corresponding to the textural data finding. The gradual decrease in juiciness with increasing pH may be because konjac gels exhibit better water-binding properties and release less liquid when chewed (Ruiz-Capillas, Triki, Herrero, Rodriguez-Salas, & Jimenez-Colmenero, 2012).

Conclusion

In this study, the mechanical, thermal, oral tribological, and sensory properties of SPI/KGM composite CFS were optimized by pH adjustment and TG treatment, and the formation mechanism of SPI/KGM composite CFS gels was elucidated by FITR spectroscopy and CLSM. The results showed that the hardness, cohesiveness, chewiness, and thermal stability gradually increase at higher pH and TG treatment levels. The heated acetylation of KGM under alkaline conditions cross-linked with the SPI protein network to form a compact and uniformly distributed network structure. TG catalyzed the covalent cross-linking between the SPI glutamine residues and the lysine amino groups. The FITR results indicate that the deacetylated KGM may react with the SPI via hydrogen bonding with SPI, resulting in a more stable structure. According to the results obtained via the sensory evaluation, TPA and oral tribology, the 1 % TG, pH = 8 CFS is most popular with consumers since the textural characteristics are most similar to natural fat. In addition, studies involving the sensory assessment of acceptability and oral tribology are key to successfully preparing CFSs and can provide a theoretical basis for further relevant studies. The cooking loss of the SPI/konjac CFS and its application in meat products should be further investigated in the future, while natural pigments and small nutritional molecules can also be added via microencapsulation technology to simulate the appearance of natural fat and improve the nutritional value.

Funding

The research was supported by the National Key Research and Development Program of China (2021YFC2101405), the fund of Cultivation Project of Double First-Class Disciplines of Food Science and Engineering, Beijing Technology & Business University (BTBU), and 2022 Postgraduate Research Capability Improvement Program.

CRediT authorship contribution statement

Lu Huang: Conceptualization, Methodology, Investigation, Writing – original draft. Di Zhao: Methodology, Investigation. Yong Wang: Writing – review & editing. He Li: Validation, Funding acquisition. Haochun Zhou: Formal analysis, Software. Xinqi Liu: Supervision.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: H.Z. is employed by Plant Meat (Hangzhou) Health Technology Limited Company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.fochx.2022.100508.

Contributor Information

He Li, Email: lihe@btbu.edu.cn.

Xinqi Liu, Email: liuxinqi@btbu.edu.cn.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (16.3KB, docx)

Data availability

Data will be made available on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary data 1
mmc1.docx (16.3KB, docx)

Data Availability Statement

Data will be made available on request.

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