Effect of processing treatments on physiochemical properties of two varieties of soybean and its characterization for preparation of hawaijar

Abstract

Hawaijar is a viscous, fermented soybean-based delicacy with a unique flavor, traditionally made in Manipur, India. The physicochemical and rheological properties of hawaijar were examined at various processing stages in this study, which found that fermentation significantly enhanced the nutritional value of soybeans. Fermented soybeans (hawaijar) exhibited the highest phenolic and flavonoid contents with big seed soybean having higher phenolic and flavonoid contents than small seed soybean. Additionally, fermentation led to a 89.2% and 91.17% reduction in phytic acid and 65% and 52.98% reduction in tannin content in small and big seeds, respectively. The hardness of soybean decreased with each processing step, and a soft, sticky texture was observed in the fermented product. These results indicate that the fermentation process improves the nutritional content of soybeans, rendering hawaijar a nutritious fermented product.

Introduction

Soybean (Glycine max [L.] Merrill) holds immense global significance as one of the most nutritious crops, accounting for approximately one-quarter of global edible oil output and constituting a key source of protein concentrate for livestock feed, contributing to about two-thirds of the global supply¹. The United States, Brazil, and Argentina currently dominate global production, often referred to as the “Big 3” in global soybean production². Soybean protein is acknowledged as a complete protein owing to its balanced amino acid arrangement. The seed coat contains an abundance of diverse bioactive compounds that exhibit potent antioxidative, tumor-suppressing, and cancer-protective characteristics³. Nutritionally, soybeans are rich in carbohydrates (30%), proteins (32.1–39.8%), fats (10.8–19.6%), dietary fibers (21.77–30.31%), and minerals (3.93–6.15%), including phosphorus, iron, potassium, sodium, zinc, copper, and manganese. Various soybean cultivars are also known to have high levels of vitamin B, including vitamin B₁, B₂, B₃, B₅, and B₆⁴. Soybeans possess numerous nutritional advantages, yet they also contain anti-nutritional components, such as trypsin inhibitors, phytic acid, oligosaccharides, and oxalates, which may potentially pose risks to human health. These compounds, particularly trypsin inhibitors, can disrupt protein metabolism and potentially lead to digestive discomfort, flatulence, nausea, and other health issues if not removed. Fortunately, processing methods such as cooking effectively eliminate or reduce these harmful elements, enhancing soybean digestibility^5,6.

Soaking soybeans before cooking or processing softens them, reduces cooking time and helps remove antinutrients like phytic acid, which may hinder the uptake of minerals like iron and zinc. By allowing phytic acid to leach out, soaking improves the bioavailability of these essential minerals⁷. Fermentation is a traditional method that transforms soybeans into various fermented products with enhanced flavor, aroma, texture, and nutritional value. It reduces antinutrient levels, including phytic acid, further improving mineral absorption and overall nutritional quality⁸. Common fermented products include douchi (fermented black beans), dajiang (soybean paste), sufu, soy sauce, tempeh, miso, natto which are widely produced across Asia. In India, fermented soybeans are popular in northeastern states, known as kinema in Sikkim, hawaijar in Manipur, bekang in Mizoram, tungrymbai in Meghalaya, and akhoni in Nagaland^9,10.

The preparation of fermented products like hawaijar involves key processing steps such as soaking, cooking, and fermentation, each of which influences the final product’s physicochemical and rheological properties. Despite its cultural significance and nutritional potential, there is limited scientific understanding of how these stages impact the characteristics of hawaijar. To tackle this research gap, the study focuses on how soaking, cooking, and fermentation affect the proximate composition, rheological behavior, total phenolic and flavonoid content, tannins, phytic acid, and color strength in two soybean varieties: small seeds and big seeds. The selection of these two varieties was based on their size differences, which may affect moisture absorption, texture, and nutritional content during processing. By comparing these two varieties, the study also seeks to highlight the comparative advantages of hawaijar fermentation in enhancing the nutritional value and textural transformation of soybeans, offering valuable insights into the dynamics of this traditional fermentation process. This study also provides an insight into the fermentation of soybeans based on its nutritional value.

Material and methods

Procurement of raw materials

Two distinct soybean seed varieties, encompassing big and small seeds, were obtained from the All India Coordinated Research Project on Soybean at the Central Agricultural University’s Imphal Centre in Manipur, India, as well as the local market in Imphal, Manipur, India, respectively. These seeds were subsequently stored in airtight containers for future use. The study utilized analytical-grade chemicals and standards throughout.

Processing treatments of soybean grains

The processing treatment carried out in this study includes soaking, cooking and fermentation. The manually cleaned soybean grains of both varieties (500 g each) were soaked overnight at 37 °C, cooked at 100 °C for 2 h or until soft and fermented at 38 °C for 72 h. After each step of processing soaking, cooking and fermenting, sample (~ 150 g each) was removed for physicochemical analyses. The rheological analysis was conducted by making paste of the processed seeds whereas for other chemical analyses, the seeds were oven-dried at 40 °C for 24 h. Subsequently, the dry seeds were milled to achieve a fine powder, which was used for further analyses. To analyze the effect of processing treatments, these results were compared with those of raw seed grains.

Proximate analysis of soybean grains

The moisture, fat, ash, protein, and carbohydrate contents of raw, soaked, cooked, and fermented soybean were determined using standard analytical procedures outlined by AOAC¹¹.

The moisture level of soybean flour was quantified applying the hot air oven drying technique. Ash content of soybean flour, 5 g of the powdered was weighed into a silica dish. The sample was initially heated on a heating mantle to evaporate moisture and thoroughly char the organic matter. Subsequently, the sample was incinerated at 550 °C in a muffle furnace for 6 h to get the ash. The Kjeldahl method was utilized to estimate the total protein of the soybean flour. Crude fat content was determined by Soxhlet extraction, carbohydrate content by anthrone method and crude fiber was calculated by difference method.

Phytochemical analyses

Soybean extract preparation

The extract of soybean flour was prepared by adding 1 g soybean flour to 10 mL methanol and sonicating the mixture for 20 min. The extract was adjusted to a final volume of 10 mL by methanol. Every sample (raw, soaked, cooked and fermented soybean) were extracted and stored in amber glass bottles at 4 ± 1 °C until further analysis.

Total phenolic content

A modified approach based on the method developed by Ainsworth and Gillespie¹² was employed to determine the total phenolic content of raw and processed soybean samples. Briefly, equal volumes (0.5 mL each) of the sample extract and 0.5 N Folin-Ciocalteu reagent were mixed together and permitted to incubate at ambient temperature for 5 min. Following this, 2.0 mL of saturated sodium carbonate was added, followed by dilution to a total volume of 10 mL. The mixture was incubated at room temperature for 30 min, after which absorbance was measured at 765 nm. Gallic acid was used as the standard, and the total phenolic content was expressed in mg gallic acid equivalents per gram of sample (mg GAE/g).

Total flavonoid content

Flavonoid content was assessed through the aluminium chloride colorimetric technique, with quercetin as the standard, according to the method outlined by Ayele et al.¹³. In this procedure, 1 mL of soybean extract was added to 4 mL of distilled water in a flask. After 5 min, a volume of 0.3 mL each of 5% sodium nitrite and 10% aluminium chloride was added in sequence. Following this, 2 mL of 1 M NaOH was added, and the solution was kept at room temperature for 6 min. The volume was adjusted to 10 mL with distilled water, and absorbance was measured at 510 nm. Total flavonoid content was calculated using a standard quercetin calibration curve and expressed as mg quercetin equivalents per gram of sample (mg QE/g).

Total tannin content

Tannins, phenolic compounds exceeding 500 Da, hinder protein digestion by causing precipitation and reducing bioavailability of essential amino acids. Each extraction involved a 1h shaking period at 25 °C followed by centrifugation at 3000 g for 15 min. The combined extracts were then reacted with a vanillin reagent (a 1:1 mixture of 10 g/L vanillin in MeOH and 80 mL concentrated HCl per litter of MeOH). After a 20 min incubation at 30 °C, the absorbance was measured at 500 nm using a UV–vis spectrophotometer. The tannic acid content was assessed thereafter using a tannic acid standard curve and quantified as tannic acid equivalents.

Phytic acid content

The method of Wheeler and Ferrel¹⁴ was employed to estimate phytic acid content. In brief, 3 g of the sample was extracted with 50 mL of 3% trichloroacetic acid by shaking at 25 °C for 30 min. After centrifugation at 10,000 × g for 10 min, 10 mL of the supernatant was mixed with 4 mL of ferric chloride solution and boiled for 45 min. After cooling, the mixture was centrifuged again at 10,000 × g for 10 min. The precipitate was treated with 3 mL of 15 M sodium hydroxide, producing a red complex. This complex was dissolved in hot 32 M nitric acid and filtered. The iron content in the filtrate was quantified spectrophotometrically at 480 nm after reaction with 15 M potassium thiocyanate. A ferric nitrate calibration curve was used for quantification. Phytate phosphorus was calculated based on a 4:6 molar ratio of iron to phosphorus, and phytic acid content was estimated by assuming a phosphorus content of 0.282 g per gram of phytic acid.

Characterization of processed soybean

Texture profile analysis

HD plus Texture Analyzer (Stable Microsystems, Godalming, UK) with a 5 mm diameter P/5 probe was employed to assess soybean seed texture. Fifteen measurements per seed samples were performed using a two-cycle compression test. Seeds were positioned centrally beneath the probe on a base plate. Texture Profile Analysis was performed using a pre-test speed of 1 mm/s, test speed of 0.5 mm/s, post-test speed of 5 mm/s, 60% strain, a 5s holding time, and a trigger force of 5 g, with results expressed as mean ± standard deviation.

Rheological properties

Rheological properties were analysed using an rheometer (AntonPaar MCR101, Austria) with a parallel plate (25 mm diameter, 1 mm gap) configuration. Raw grains were pulverized and mixed with water (1/3 w/v), while processed grains were mashed. Paraffin oil was applied to the plate after keeping sample to prevent sample dehydration. An amplitude sweep (0.1–100 Hz) determined the linear viscoelastic region. A frequency sweep (0.1–100 rad/s) at 25 °C and 0.1% strain measured storage modulus, loss modulus (G"), and viscosity. A temperature sweep (25–95 °C at 5 °C/min) also recorded G’, G", and viscosity as functions of temperature and time¹⁵.

FTIR analysis

FTIR spectra analysis of raw and processed (soaked, cooked, fermented) soybean samples were carried out using a Bruker ALPHA spectrometer (Laser class 1, Germany) with a platinum ATR attachment. Powdered, moisture-free samples were mixed with KBr (1:100 ratio) and pressed into pellets. Spectra were recorded in absorbance mode across the 4000–400 cm⁻¹ range.

Zeta-potential

Zeta potential of soybean samples were measured using a Malvern Nano particle size analyser (Malvern Instruments, UK) at 25 °C. Samples were diluted 50-fold with HPLC water¹⁶.

Statistical analysis

Data are presented as mean ± standard deviation from triplicate measurements. Error bars in bar charts indicate percentage variation. Duncan’s multiple range test (p ≤ 0.05) was used to determine significant differences between means using the trial version of OriginLab.

(https://www.originlab.com/try).

Results and discussion

Proximate analysis of soybean grains

Studying the proximate composition of soybeans during each processing step for the preparation of hawaijar is crucial for understanding how different processing methods affect the nutritional value and quality of hawaijar. The proximate composition of the differently processed soybean samples is presented in Table 1. Moisture content of raw soybeans were 7.25 and 8.63% (db), which increased significantly during soaking, cooking, and fermentation due to the absorption of water during hydration and thermal treatment. Fermented soybean had the highest moisture, 274.12% in small seed and 270.16% in big seed on dry weight basis. The crude protein content of small and big raw seeds are 39.17 and 38.53% respectively, slightly but insignificantly increased upon soaking, while it decreased upon cooking. The reduction in the protein content during cooking is likely due to the leaching of soluble protein components in the cooking medium^17–19. Conversely, protein content increased during fermentation, 43.84% in small seeds and 42.22% in big seeds likely due to the breakdown of proteins by protease enzyme produced by microorganisms that hydrolyze complex proteins into peptides and free amino acids²⁰. The fermentation process likely enhanced the release of nitrogen-containing compounds, such as free amino acids and peptides, which are more readily measured as protein by the Kjeldahl method. Therefore, the increase in protein content reflects increased nitrogen availability due to protein hydrolysis, even though the total mass of protein may not have changed significantly. This increase in protein content aligns with studies showing higher nitrogen content in fermented products²¹ likely due to enzymatic breakdown of proteins into more digestible forms, thus improving nutritional quality. A similar trend of decline in protein content during soaking and heat treatment and increase of protein content in short-time fermented soybean product (cheonggukjang) was reported by Piao and Eun²².

Table 1.

Proximate composition of raw and processed soybean grains.

Sample	Moisture (%)	Fat (% db)	Ash (% db)	Protein (% db)	Fiber (% db)	Carbohydrate (% db)
RS	7.25 ± 0.22c	11.50 ± 0.35e	5.92 ± 0.18a	39.17 ± 1.22b	10.05 ± 0.31b	26.11 ± 0.81a
RB	8.63 ± 0.26c	13.98 ± 0.43 cd	5.88 ± 0.18a	38.53 ± 1.07b	11.21 ± 0.35a	25.76 ± 0.80a
SS	154.26 ± 4.78b	15.25 ± 0.47b	5.34 ± 0.16b	39.20 ± 1.21b	9.78 ± 0.21b	23.42 ± 0.73b
SB	162.20 ± 5.03b	16.88 ± 0.52a	5.42 ± 0.17b	38.70 ± 1.20b	9.13 ± 0.19c	22.87 ± 0.71b
CS	271.10 ± 8.40a	14.07 ± 0.44c	3.42 ± 0.11e	36.33 ± 1.13c	8.19 ± 0.56d	17.98 ± 0.56d
CB	265.75 ± 8.24a	13.211 ± 0.41d	3.27 ± 0.10e	36.88 ± 1.14c	9.39 ± 0.60c	17.25 ± 0.53d
FS	274.12 ± 8.50a	14.99 ± 0.46b	4.13 ± 0.13c	43.84 ± 1.34a	7.52 ± 0.42e	19.52 ± 0.42c
FB	270.16 ± 9.14a	15.14 ± 0.47b	3.87 ± 0.12d	42.22 ± 1.31a	9.62 ± 0.51b	19.15 ± 0.37c

Values are represented as mean ± S.D., Mean value with different subscripts are significantly different (p ≤ 0.05).

RS raw small, RB raw big, SS soaked small, SB soaked big, CS cooked small, CB cooked big, FS fermented small, FB fermented big.

Subscripts a, b, c, and d, represent the effect of different process variables.

The fat content of the soybean grains varied across the different processing treatments. Raw big soybeans (RB) exhibited the higher fat content (13.98% db) than raw small soybeans (RS) at 11.50% db. Soaked, cooked and fermented soybeans had higher fat content than raw soybean in both small and big sized samples. The increased fat content of soaked and cooked samples (Table 1) is likely due to the loss of solids in the soaking and cooking medium²³. Fat content in the fermented soybean-hawaijar were 14.99 and 15.14% in small and big seeds respectively. Higher fat content is likely due to the the degradative properties of microbial enzymes produced during fermentation by Bacillus subtilis²⁴. Similar results were reported during koji fermentation by Zou et al.²⁵ and suggested that lipoproteins got hydrolyzed to release fatty acid and decrease of dry mass of samples due to microbial growth and enzymolysis.

The ash content, which represents the mineral content, showed significant variation among the different samples. Raw soybeans (RS and RB) had higher ash content (5.92% and 5.88% db, respectively), but the ash content reduced in processed soybeans, particularly in the cooked (CS and CB) and fermented (FS and FB) grains. The decrease in ash content could be related to the leaching of water-soluble minerals during soaking and cooking, and fermentation might lead to some mineral degradation or redistribution within the grains²⁶.

Crude fiber content was highest in raw soybeans (10.05% db in small and 11.21% db in RB), with a gradual decrease observed in soaked and cooked grains in both the varieties. The fermented soybeans (FS and FB) exhibited the lowest fiber content (7.52% and 9.62% db). Reduction in the crude fiber content in processed samples could be attributed to the breakdown or solubilization of soluble fibers, enzymatic activity of the fermenting microorganisms and hydrolysis by carbohydrate-splitting enzymes from B. subtilis during soaking, cooking and fermenting²¹.

Carbohydrate content in soybeans was highest in the raw samples (26.11% and 25.76% db for RS and RB) and decreased progressively with processing. Decline in carbohydrate content especially the polysaccharide during fermentation of soybean was also observed by Chen et al.²⁷. This decrease in carbohydrate content could be due to the hydrolysis of complex carbohydrates during soaking, cooking, and fermentation, with enzymes breaking down polysaccharides into simpler sugars that may be more readily metabolized or consumed by microorganisms during fermentation²⁸.

Chemical analyses

Total phenolic and total flavonoid content

Fermentation of soybeans increased the total phenolic and total flavonoid content while soaking and cooking reduced the same in both small and big varieties. The highest phenolic (6.17 mg GAE/g) and total flavonoid content (4.88 mg QE/g) was observed in fermented big seed soybean while cooked small seed soybean and cooked big seed soybean samples contains the least total phenolics (4.45 mg GAE/g) and flavonoids (2.96 mg QE/g) content respectively. It can be observed from the plots in Fig. 1 that the total phenolic content and total flavonoid content initially decreased insignificantly during soaking and cooking treatments but a significant increase in these values was observed upon fermentation of soybean grains. Piao and Eun²² also reported that soaked and steamed soybeans had lower TPC than raw soybeans. This could be attributed to the fact that during soaking for prolonged hours, the phenolic compounds leach out in the solvent and during cooking at high temperatures, some of the heat labile compounds are degraded leading to lesser phenolic and flavonoid content in the soaked and cooked samples. Decline in the phenolic content of boiled soybeans was observed by Chen et al.²⁹. In another study, Mhada et al.³⁰ observed decline in total phenolic content during cooking (boiling and steaming) of quinoa seeds. Similar trend of increased TPC and TFC were observed in the study conducted by Chapagain³¹ in fermenting black and yellow seeds soybean for 40 h. During fermentation microorganisms naturally present or added to the soybean grains produce enzymes that can break down complex compounds. These enzymes, such as glucosidases and esterases, can help release phenolic and flavonoid compounds that are bound to sugar or esterified. Enzymatic activity during fermentation can lead to the liberation of these compounds, resulting in an increase in their content. Further, fermentation can lead to the conversion of precursor compounds into phenolic and flavonoid compounds. For example, isoflavone glucosides naturally present in soybean grains can be enzymatically transformed into their aglycone forms, which have higher bioactivity and antioxidant potential. Fermentation can disrupt the complex structures of soybean grains, such as cell walls and protein-carbohydrate complexes, thereby facilitating the release of phenolic and flavonoid compounds that were previously inaccessible³².

Fig. 1.

(a) Total phenolic content (TPC), total flavonoid content (TFC) and (b) Phytic acid and tannic acid content of raw and processed soybean grains. Note: RS: raw small; RB: raw big; SS: soaked small; SB: soaked big; CS: cooked small; CB: cooked big; FS: fermented small; FB: fermented big.

Phytic Acid and Tannin Content

It has been observed that antinutrients- phytate and tannin present in soybean decreased (Fig. 1) gradually with each processing step in the preparation of hawaijar. Phytate content in raw soybean samples were 48.16, and 37.97 mg/L in small and big seeds. Upon soaking, phytate content decreased to 29.74 and 28.76 mg/L with a reduction of 38.23 and 24.24%, respectively. Higher reduction in phytic acid content were observed in cooking and fermenting soybeans with highest reduction of 91.17% to 3.3 mg/L in fermentation of big seed soybean. Similarly, tannin content of raw soybean, 67.03 and 79.93 mg/L in small and big seed reduced to less than half in fermented hawaijar i.e., 23.46 and 37.58 mg/L, with a percent reduction of 89.2% and 91.17% in small and big seeds, respectively. Chauhan et al.³ reported reduction in phytate and tannin content of black soybean flour in soaking, germinating and natural fermentation process. Reduction in the antinutrients content during soaking can be attributed to the fact that some of these anti-nutritional factors leach out of the grains to the soaking water, further processing such as cooking, and fermentation degrades these compounds and hence make the grains safer to consume. The duration of soaking can influence the extent of phytic acid and tannic acid reduction, with longer soaking times generally leading to greater reduction. High temperatures and prolonged cooking times are generally associated with greater antinutrients reduction. During fermentation, microbes and enzymes break down tannic acid and other compounds, resulting in decreased tannic acid levels. During soaking, cooking and fermentation of soybean grains, Egounlety and Aworh³³ observed significant effect of processing on the phytic acid and tannin content of soybean. They observed 34.65% increase in the phytate content on soaking, 9% reduction during cooking and 30.7% reduction on fermenting soybean, and also a 54.6% reduction in tannin content upon soaking of soybean grains. Another study reported a 27.9% reduction in phytic acid content after 6 h soaking and 36% reduction after 24 h soaking of Mucana flagellipes³¹. Sharma et al.³⁵ also reported a reduction in tannin content and phytic acid content in soaked, cooked and fermented soybeans. They cited that reduction in the tannin content during fermentation might be caused by the activity of polyphenol oxidase due to growth of microbiota. They also suggested that reduction in phytic acid content might be due to phytase activity shown by B. subtilis. The phytic acid content in legume grains decreased drastically upon sequential soaking and cooking treatment³⁶. Fermentation reduced the anti-nutritional factors such as cyanide, tannins, phytate, oxalate and saponins³⁷.

Texture profile analysis

The values of hardness, adhesiveness, resilience, cohesion, springiness, gumminess, and chewiness as obtained from texture profile analyses of raw, soaked, cooked and fermented soybean seeds have been presented in Table 2. Hardness of raw soybean seeds were 111.93 N and 109.25 N in small and big seed varieties. It was observed that the hardness of the seeds decreased as the processing treatment progressed from soaking to cooking to fermentation in both varieties. Microbial hydrolytic enzyme activity and increased moisture content are attributed to the reduction in hardness of soybean³⁸. Similarly, Deshpande and Bal³⁹ reported effect of soaking time and temperature on textural properties of soybean that with increase in soaking time as well as temperature, the hardness of grains decreased. Highest degrees of adhesion or stickiness were observed in small seed fermented soybean (− 8.35 g.sec) with higher negative value followed by big seed fermented seeds (− 4.92 g.sec). Further, it can be observed that cohesiveness increased upon fermentation with higher cohesiveness in big seed fermented soybean indicating stickiness in hawaijar, which is a desired texture of good quality hawaijar⁴⁰. Other textural properties- resilience, springiness, gumminess and chewiness significantly decreased with processing with the lowest values observed in fermented product. YeXing et al.⁴¹ in their study on texture of Shuidouchi, a Chinese traditional fermented soybean product, observed that fermentation process consistently changes the hardness and chewiness. They also reported that highest decrease in hardness occurred during the soaking and cooking processes. Proteolytic and microbial activity influenced the texture of processed and fermented soybean⁴².

Table 2.

Texture profile analysis of raw and processed soybean grains.

Sample name	Hardness (N)	Adhesiveness (g.sec)	Resilience (%)	Cohesion	Springiness (%)	Gumminess	Chewiness
RS	111.93 ± 2.44a	0.7 ± 2.30a	1.01 ± 0.46e	0.02 ± 0.01f	15.25 ± 4.74d	279.79 ± 157.99b	41.97 ± 22.18b
RB	109.25 ± 7.14b	0.18 ± 1.28b	2.88 ± 0.71a	0.07 ± 0.02c	10.66 ± 2.14f	774.02 ± 138.03a	83.14 ± 25.78a
SS	38.83 ± 3.69c	− 0.48 ± 0.54c	0.77 ± 0.18f	0.03 ± 0.01e	20.99 ± 4.68b	104.87 ± 16.92d	21.67 ± 4.39d
SB	34.18 ± 0.77d	− 0.9 ± 2.15d	1.31 ± 1.55d	0.04 ± 0.04d	25.94 ± 9.14a	128.01 ± 138.84c	32.72 ± 38.45c
CS	1.65 ± 0.27e	− 3.56 ± 1.09e	1.72 ± 1.36c	0.07 ± 0.04c	13.95 ± 4.39e	11.24 ± 4.99e	1.72 ± 1.41f
CB	1.1 ± 0.25f	− 4.39 ± 2.71g	1.79 ± 0.39b	0.08 ± 0.02b	19.52 ± 6.18c	8.91 ± 2.74f	1.82 ± 1.04e
FS	0.67 ± 0.12g	− 8.35 ± 4.55h	0.01 ± 0g	0.04 ± 0.01d	0.12 ± 0.03h	2.93 ± 1.09g	0.38 ± 0.19h
FB	0.27 ± 0.03h	− 4.92 ± 1.97f	0.01 ± 0g	0.09 ± 0.03a	0.18 ± 0.04g	2.56 ± 0.81h	0.48 ± 0.25g

RS raw small, RB raw big, SS soaked small, SB soaked big, CS cooked small, CB cooked big, FS fermented small, FB fermented big. Values are presented as mean ± S.D., Mean value with different subscripts are significantly different (p ≤ 0.05). Subscripts a, b, c, and d represent the effect of different process variables.

Rheological properties

Understanding the rheological properties, the viscoelastic behavior of soybean for the preparation of hawaijar is crucial for controlling its texture and sensory properties. Storage modulus measures elastic behavior, representing the energy stored during shear deformation. Loss modulus (G") measures viscous behavior, representing the energy dissipated during shear (^43,44. It can be observed from the plots in Fig. 2(a) and (b) that the loss modulus and storage modulus decreased with an increase in strain, it typically indicates a behavior known as strain softening. This means that the material’s ability to dissipate energy and dampen vibrations decreases as it undergoes deformations. The decreasing loss modulus suggests that the material becomes less resistant to cyclic loading or deformation, potentially leading to a reduction in its overall mechanical performance. The strain softening is often associated with structural changes or damage mechanisms within the material. These structural changes may include the alignment or reorientation of molecular chains, the breaking of intermolecular bonds, or the occurrence of microcracks or fractures. It can also be observed from the plots that the G′ is greater than G″ for all the samples, indicating a predominance of elastic behavior of soybean samples. These findings align with those of Singh et al.⁴³ in rice flour batter used for gluten-free muffin preparation and Giri et al.⁴⁴ in soy spreads. In addition, a marginal rise in the loss and storage moduli is evident with an increase in angular frequency, which represents the frequency-dependent viscoelastic behavior of soybean pastes Fig. 2c and d. The increase in the storage modulus suggests that the material becomes stiffer and more resistant to deformation as the angular frequency increases. Similarly, the increase in the loss modulus signifies the viscous response of the material, representing its capacity to dissipate energy and exhibit damping characteristics becomes more prominent at higher frequencies. It has been noted that G″ was lower than G′ which suggested that within the examined frequency range, soybean samples behaved predominantly like a solid. Consistent findings were noted by Mir et al.¹⁵ for both album and quinoa protein isolates.

Fig. 2.

Effect of different processing techniques on rheological properties of soybean: (a) Storage modulus as a function of strain; (b) Loss modulus as a function of strain; (c) Storage modulus as a function of angular frequency; (d) Loss modulus as a function of angular frequency; (e) Viscosity as a function of angular frequency; and (f) Viscosity as a function of temperature. Note: RS: raw small; RB: raw big; SS: soaked small; SB: soaked big; CS: cooked small; CB: cooked big; FS: fermented small; FB: fermented big.

Further the decrease in viscosity with increasing angular frequency as well as temperature represents shear thinning behavior of soybean pastes Fig. 2e and f. The reduction in viscosity with increasing temperature can be clarified by the increased thermal energy that leads to the weakening of intermolecular forces and enhanced molecular mobility. This results in decreased viscosity due to reduced resistance to flow. Similarly, as the shear rate rises, the fluid experiences higher deformation rates, causing the structural elements to align or break down, resulting in reduced viscosity. This behavior allows the fluid to flow more easily under higher shear rates.

FTIR analysis

The FTIR analyses of soybean powders obtained after different processing treatments is presented in Fig. 3.

Fig. 3.

FTIR plot of soybean grain powders obtained after different treatments. Note: RS: raw small; RB: raw big; SS: soaked small; SB: soaked big; CS: cooked small; CB: cooked big; FS: fermented small; FB: fermented big.

The FTIR spectroscopy is primarily used to extract information about the functional groups and chemical bonds present in the sample. The polypeptides and proteins in soybean flour can be characterized by nine characteristic IR absorption bands, i.e., amide A, B, and amide I–VII⁴⁵. The presence of Amides such as Amide I (C=O Stretching), Amide II (N–H bending and C-N stretching), amide III, and amide A, can be confirmed by the peaks around 1600–1800 cm⁻¹, 1470–1570 cm⁻¹, 1250–1350 cm^–1, and 3300–3500 cm^–1 respectively⁴⁶. Alterations in the 1500–500 cm^–1 region indicate the differences in protein side chains and in free amino acid composition, probably due to different processing steps. The presence of carbohydrates in soybean flour can be confirmed by the peaks around 1300–1430 cm^–1(cellulose), 1605, 1460, 1251 cm^–1 (hemicelluloses), and 1599, 1467, 1429 cm^–1 (lignins)⁴⁷. Minor absorbance peaks between 2700 and 3500 cm⁻¹ resemble to hydrogen bonding, N–H stretching, and C–H bending of amide bonds⁴⁸. There is a change in the peaks and patterns located around 2900 cm⁻¹, attributed to (C-H) stretching and 1500 cm⁻¹, often associated with aromatic ring vibrations, amide II bands in proteins and to N–O stretching in nitro compounds can be seen in the soaked samples which is heightened in the cooked and fermented soybean samples. There was a recorded C-H stretches, which are correlated with uneven packing of hydrocarbons, are near to the 2922 and 2850 cm⁻¹. The change in the peaks after soaking, cooking and fermenting process is indicative of a change in the chemical structure. A comparison of the FTIR spectra of raw and processed soybean showed some frequency and intensity differences, which indicates some alterations in the protein and carbohydrate conformation.

Zeta potential of soybean flours

Zeta potential values of soybean flours subjected to various processing treatments, revealing a progressive decrease in zeta potential with each treatment are shown in Table 3. Zeta potential of the cooked and fermented soybean varieties showed negative value with big seed soybean variety showing higher zeta potential. This can be due to the fact that processing alters the pH of the grains. The raw grains have close to neutral or alkaline pH, upon soaking the pH decreases slightly owing to leakage of compounds in soaked water. The pH of the cooked grains is further reduced, and the fermented grains have the least pH due to the formation of organic acids through fermentation. Wang et al.¹⁶ observed that with upsurge in pH from 6 to 8 the magnitude of charge of soy protein emulsions reduced from − 49.30 mV to − 35.00 mV. High absolute values of the zeta potential indicate a repulsive electrostatic force between the molecules, which is related to the key property of an aggregation-resistant suspension. Therefore, it can be inferred that the increase in electrostatic repulsive forces between the molecules will promote higher solubility and other functional properties relating to solubility⁴⁹.

Table 3.

Zeta-potential and conductivity of treated and untreated soybean flours.

Sample	Zeta (mV)	Conductivity (mS/cm)
RS	10.7 ± 3.79	0.415
RB	14.2 ± 2.95	0.493
SS	10.3 ± 3.15	0.182
SB	3.57 ± 2.96	0.127
CS	− 0.863 ± 3.67	0.189
CB	− 5.78 ± 4.44	0.211
FS	− 25 ± 6.5	0.133
FB	− 27.3 ± 6.42	0.129

RS raw small, RB raw big, SS soaked small, SB soaked big, CS cooked small, CB cooked big, FS fermented small, FB fermented big. Values are presented as mean ± S.D

Conclusion

It can be concluded that fermentation is an efficient technique for the development of nutrient rich hawaijar. The processing treatments reduced the anti-nutrient compounds and thus improved the nutrient availability of the product. The characterization of the product was studied in terms of rheological properties and texture profile analysis indicated that processed and fermented soybean had better textural attributes. It was also observed that the size of the grain also influences the effect of processing treatments on the physicochemical and textural properties of the grains. Higher hardness and adhesiveness in small seed fermented soybeans indicated more desirable texture with increased stickiness. It has been observed that the big seed soybean variety showed a higher decline in phytic acid content while small seed had the highest reduction in tannin content. The nutritional profile, in terms of proximate and phenolic composition, of both the seeds were comparable. Further, studies on storage of hawaijar and development of efficient packaging and drying techniques for the preservation of hawaijar are needed.

Author contributions

B.T. conceived the presented work, got funding, B.K. planned the experiments, S.T. verified the experimental methods methods, K.B. and S.K. conducted the experiments, T.B. and S.K. wrote the manuscript, N.J. conducted data analysis. All authors reviewed the manuscript.

Funding

Department of Scientific and Industrial Research (DSIR), Ministry of Science and Technology, Government of India, TDUPW-11011/11/2021-IRD-DSIR.

Data availability

Data supporting the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Competing Interests

The authors declare no competing interests.