1 Introduction

Milk is a highly nutritious and complex food essential for human health. It contains various nutrients, with lactose being the primary milk sugar. Other important nutrients in milk include lipids, minerals, vitamins, essential amino acids, and proteins such as caseins and whey proteins. The consumption of milk may have played a significant role in human health throughout the history [1].

However, certain metabolic disorders and allergies, such as lactose intolerance and cow's milk protein allergy, hinder the digestion and absorption of milk for some individuals. For these conditions, eliminating milk from the diet is the primary treatment. According to Silva et al. [2], symptoms of lactose intolerance affect over 75% of the global population. Additionally, lactose malabsorption impacts an estimated 70% of people worldwide. As awareness of these conditions has increased in recent times, so has the demand for lactose-free food items that accommodate these dietary needs [3]. Mammalian milk may eventually be supplemented in everyday diets by plant-based milk alternatives (PBMAs). These alternatives, also referred to as “vegetable milk,” are derived from raw materials such as nuts, seeds, legumes, and pseudocereals. Plant-derived milk alternatives are receiving far more attention than expected, even in spite of the wide availability of dairy-based functional foods. Plant-based milk substitutes are gaining popularity for a variety of reasons, including their ability to eliminate cow’s milk allergies and their superior health benefits such as being cholesterol and lactose-free and having higher levels of phytochemicals. Due to its high protein content, phytosterols, phenolic acids, polyunsaturated fatty acids, and affordability, soybean (Glycine max) has been a widely grown crop in Asia for a very long time. In addition, it meets the nutritional needs of those who are lactose intolerant and makes a great non-dairy substitute [4].

Soybeans are a valuable legume as they contain all essential amino acids required by humans, making them a complete protein source [5]. Soybeans can be processed to produce soy milk, which can then be fermented into soy yogurt. For individuals with lactose intolerance, soy yogurt serves as a lactose-free alternative to milk. Soy yogurt is also considered superior to cow's milk yogurt due to its lower cholesterol, saturated fat, and absence of lactose. However, the presence of unfavorable off-flavors in many soy products limits their use for human consumption [6].

Most of the plant-derived milk and extracts go through controlled or natural fermentation, which increases the activity of bioactive components [7]. Yogurt is a dairy product that has undergone fermentation. It is made when lactose in milk is fermented anaerobically by certain bacteria, the majority of which are probiotics [8].

A coculture of two lactic acid bacteria (LAB) that are frequently consumed by humans, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, ferments milk to make yogurt. These two LAB species exchange metabolites and signaling molecules as part of a symbiotic or competitive relationship known as proto-cooperation. Generally, with the help of its surface PrtB protease, L. bulgaricus is assumed to breaks down casein to produce an excess of peptides and free amino acids which is utilized by S. thermophilus. On the other hand, S. thermophilus supplies formic acid, folic acid, carbon dioxide, and fatty acids, which stimulate the growth of L. bulgaricus. This proto-cooperation enhances the growth rate of each bacterium and, as a result, the fermentation rate when compared to the performance of single cultures [9].

Several fermented dairy products are made using “thermoduric” starter cultures that contain Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The two homofermentative lactic acid bacteria starting cultures are necessary for the manufacture of yoghurt and fermented milk. They play a crucial role in the development of yogurt’s organoleptic and potentially probiotic characteristics [10]. Rapid acidification, texturing abilities, particular flavor components, weak post-acidification, and health advantages are the yoghurt starter culture’s most significant fermentation performances [11,12,13]. The objective of the research is to assess the impact of storage time on fermented soghurt, focusing on changes in fat and protein biomolecules analyzing with CLSM, viscosity measurements, and particle size distributions under refrigerated conditions.

2 Materials and methods

2.1 Soybeans and microbiological cultures

Soybeans were procured from a nearby grocery store located in St. Lucia, Brisbane, Australia.

The yogurt cultures (YCX11), consisting of Lactobacillus delbrueckii ssp. bulgaricus (LB) and Streptococcus thermophilus (ST), were obtained from CHR Hansen, Denmark in a direct vat set (DVS) form. The DVS cultures were stored at – 40 °C for future utilization.

2.2 Preparation of soy milk

200 g of soybeans were soaked in 1.0 L of water for 16 h at room temperature (30 °C). Following this, 200 g of the soaked beans were fully dehulled after boiling for 15 min, then combined with 500 mL water and blended using a blender for 3 min. The mixture was filtered through muslin cloth and boiled for 5 min. Then, heat treatment was given to soymilk (SM) at 85–90 °C for 15–20 min and cooled to 30 °C (using cold water bath).

2.3 Preparation of the inoculum

For the fermentation of the heat-treated soymilk (80–85 °C for 15 min), a lyophilised yoghurt starter culture (YCX11) was used (CHR Hansen, Denmark), which was composed of mixed strains of S. thermophilus and L. bulgaricus ssp. bulgaricus in 1:1 ratio. The commercial yoghurt culture was prepared according to the instructions of the manufacturer to have sufficient number of bacterial cells (more than 107 cfu/g). The heat-treated soymilk was cooled to 42 °C using cold water bath and immediately inoculated with 1% yogurt culture and the mixture was homogenized. Further, it was incubated at 42 °C in an incubator.

2.4 Fermentation of soy milk (SM)

100 ml sterilized SM in 200 ml bottles was inoculated with 1.0% (w/v) of the pure yoghurt culture and incubated at 42 °C for 16 h. Soghurt was stored under refrigeration conditions (6–8 °C) upto 15 days. pH (Cube pH meter, Australia), acidity (% lactic acidity), Lactic acid bacteria counts (log CFU/ml), viscosity and confocal microscopy analysis were also done at 0, 2, 5, 7, 10, 15 days interval.

2.5 Soymilk analysis

Samples were analyzed for pH, acidity (% lactic acid) and viable lactic counts following the method Hati et al. [14]. The total titratable acidity was measured using a 0.1N Sodium Hydroxide (NaOH) solution. A 10 g sample of Soghurt was placed in a beaker, mixed with 10 ml of distilled water, and two drops of phenolphthalein indicator were added. Each sample was then titrated with the 0.1N NaOH solution until a light pink color appeared, indicating the endpoint. The volume of NaOH used was recorded for each sample. The percent acidity was calculated and expressed as % lactic acid:

A Soghurt sample weighing 11 g was homogenized with 99 ml of sterile distilled water. From this mixture, 1 ml was taken and thoroughly mixed with 9 ml of sterile 0.85% normal saline. Serial dilutions were performed from 10⁻1 to 10⁻⁶. Then, 1 ml of each dilution was used for plating by the pour plate method. Cultures were plated on selective agar media: de Man, Rogosa, and Sharpe (MRS) agar for Lactobacillus counts, M17 agar for Streptococcus counts, Potato Dextrose Agar (PDA) for yeast and mold counts, and Violet Red Bile Agar (VRBA) for coliform counts. Plates considered for Lactobacillus, Streptococcus and coliform counts were incubated at 37 °C for 24 h, whereas, that for yeast and mold counts were incubated at 25 °C for 48 h. The results were calculated and expressed as colony-forming units (cfu) per ml:

cfu/ml = No. of colonies x Dilution factor / volume of inoculum.

2.6 Viscosity analysis

The viscosity of soy yogurt samples was assessed following the method described by Gursoy et al. [15]. Measurements were conducted at 6–8 °C using a controlled-strain rheometer (Physica MCR 301, Anton Paar, Graz, Austria) equipped with a cup and probe type CC27SN13078. Data collected at 10 s intervals and each measurement repeated three times.

2.7 Particle size distribution

During the storage study, the particle size distribution of soghurt samples was assessed using the Malvern Mastersizer 2000 (Malvern Instruments Ltd., Worcestershire, UK). A refractive index of 1.47 was applied to the dispersed phase, representing soy milk, while the continuous phase, represented by water, was assigned a value of 1.33. Deionized water was utilized for sample preparation and adjusting obstruction to 15%. The measurements for particle size were taken at d(0.1), d(0.5), d(0.9), and D[3, 4]. The initial three values denote the population size of particles present below 10, 50, and 90% of the total particle count. D[3, 4] signifies a volume-based average of the population and is notably sensitive to the presence of significant particles. [16].

2.8 Confocal Laser Scanning Microscopy (CLSM)

The Zeiss LSM 700 Confocal Laser Scanning Microscope was used to examine the structural organization of protein and fat globules in soy yoghurt samples. The proteins were tagged using a 1% w/w Fluorescein isothiocyanate (FITC) solution dissolved in MiliQ water, then activated with laser light at a 540 nm wavelength [17]. Nile red solution (0.1% w/w in acetone) labelled the triglycerides, followed by excitation with laser light at a wavelength range of 515–530 nm [17]. For slide preparation, 100 μl of different soghurt samples were mixed with either 25 μl of FITC or 10 μl of Nile red solution using a Ratex VM1 vortexer for 5 s. The samples were then stained for 10 min. Slides measuring 26 × 76 mm (Sail Brand) were prepared by applying 10 μl of stained samples, then covered with 18 × 18 mm cover slips (Menzel Glaser). Examination of fat globules and protein molecules was performed using a magnification lens set at 63x [16].

2.9 Statistical analysis

The data were expressed as the mean ± standard error, derived from three separate experiments. Analysis of variance (ANOVA) was employed, and significant distinctions among the sample means were evaluated utilizing Duncan’s test at a 95% confidence level.

3 Results and discussion

3.1 Effect of storage time on pH and acidity of soghurt under refrigeration conditions

During the storage period, it was observed that the pH of soghurt decreased significantly from 4.90 to 4.54 with increase in storage time. But after 10 and 15 days, a small amount of pH (4.62 and 4.54) had been lowered compared with 2 to 7 days. Whereas, the acidity significantly increased from 0.337% to 0.476% during the storage time. Similarly, a small amount of acidity (0.449% and 0.476% lactic acidity) also increased after 10 and 15 days than 2 to 7 days (Table 1). During the fermentation of soymilk into soghurt, the pH drops primarily due to lactic acid production by bacteria namely Lactobacillus and Streptococcus in combination. These bacteria utilize the soy complex oligosaccharides present in soymilk and convert them into simpler compounds, which further lead to the synthesis of lactic acid. As lactic acid accumulates in the yoghurt, it lowers the pH of the mixture, creating a more acidic environment which is conducive to the growth of the bacteria and contributes to the characteristic tangy flavor of yoghurt.

Table 1 Changes of pH and acidity (% lactic acid) during storage of soghurt under refrigeration condition (6–8 °C)
Full size table

The physicochemical characteristics of yoghurt made from cow’s milk and soymilk, both separately and together, were assessed by Amrouche et al. [18]. The pH of both soymilk and cow milk dramatically dropped during fermentation, going from 6.70 ± 0.03 to 4.60 ± 0.05 and 6.56 ± 0.05 to 4.65 ± 0.03, respectively. The pH of the yoghurt made from the combination of these milks was 4.64 ± 0.01.

Utilizing commercially available starter cultures YFL-901 Streptococcus thermophilus and L. delbrueckii ssp. bulgaricus, Cui et al. [19] reported a pH reduction from 6.8 to 4.5 during the fermentation of soymilk for the manufacture of yoghurt. It was also noted that the pH rose from 4.58 ± 0.01 to 4.62 ± 0.01 while being stored under refrigeration from the second to the 28th day. Falade et al. [20] noted a significant increase (p ≤ 0.05) in the titratable acidity of plain soy and Bambara yoghurts stored at 7 °C, which were produced using starter cultures (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus), rising from 1.63–2.02% to 1.53–1.94%, respectively.

3.2 Effect of storage time on viability of yoghurt cultures of soghurt and coliform counts as well as mold counts under refrigeration conditions

During storage of soghurt under refrigeration temperatures, both the lactic cultures survived throughout the storage periods. But after 10 days and 15 days, one log counts of L. bulgaricus was reduced than 0 day. This may be due to depletion of overall nutrients needed for microbial metabolism along with the low storage temperature unfavorable for their growth in the acidic environment. In case of S. thermophilus, there was no significant difference of bacterial counts during entire storage periods compared to L. bulgaricus (Table 2). For yeast and mold counts, the stored soghurt did not show any yeast and mold counts upto 7 days. But after 10 days, one log increase of the yeast and mold counts was observed. This might have happened due to the overall drop in pH leading to the favorable conditions for its growth and metabolism. In case of coliform counts, no coliform counts were observed during the storage periods (Table 2). In a comprehensive study carried out by Ziarno et al. [21] on the stability of yoghurt-type fermented soy beverages during refrigerated storage using dairy starter cultures, it was observed that the viable LAB and Streptococcus counts decreased during the storage time. Yoghurt type beverage prepared using starter culture ABY-2 (containing L. delbrueckii, S. thermophilus, Bifidobacteria and Lactobacillus acidophilus) showed a decrease in Lactobacilli and Bifidobacteria counts from 7.9 ± 0.2 to 7.6 ± 0.2 log CFU/g and Streptococci counts from 8.6 ± 0.2 to 8.4 ± 0.2 log CFU/g. Similar results were also obtained by Behbahani et al. [22].

Table 2 Effect of storage periods on microbial viability of soghurt under refrigeration condition (6–8 °C)
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Almghawesh et al. [23] observed reduction in the starter bacteria counts in yoghurt sample C (50% cow milk + 50% soy milk) during storage study of 15 days. The bacterial counts (Lactobacillus delbrueckii subsp bulgaricus, Streptococcus thermophilus and Bifidobacterium bifidum) reduced from 8.41 ± 0.1 log cfu/g observed during 1st day of storage to 6.54 ± 0.01 log cfu/g at 15 days of storage under refrigerated condition (4 °C ± 1 °C). Falah et al. [24] noted the counts of the probiotics was approximately 6 log CFU/g during storage. Our results were in contrast with Zanganeh et al. [25] who found an increase in total viable counts with increase in storage time of lamb samples.

3.3 Changes of viscosity during storage time of soghurt under refrigeration condition

Viscosity generally provides the consistency and textural quality of the product. As the shear rate increases viscosity decreases. In the study, it was observed that viscosity had been gradually increased during the storage of the soghurt as because of the refrigeration temperature helps the proteins to absorb the free water molecules in a very smaller amount (Fig. 1). The increase in viscosity of soghurt during storage is primarily due to ongoing fermentation processes wherein bacteria utilize carbohydrates, including sugars such as sucrose and oligosaccharides and metabolize these sugars through enzymatic reactions, breaking them down into simpler molecules like glucose and fructose and produce lactic acid as a byproduct. As yoghurt is stored, the bacteria continue to metabolize sugars and produce more lactic acid. This increase in lactic acid content contributes to the thickening and increase in viscosity of the soy yoghurt. Lactic acid lowers the pH which in turn causes the proteins in the soymilk to coagulate and form a tighter network, resulting in a thicker consistency. Additionally, as yoghurt ages, moisture may gradually evaporate from the product, further concentrating its solids content and contributing to increased viscosity. Therefore, the viscosity of yoghurt typically increases with storage time due to the ongoing fermentation processes and associated changes in its chemical composition. As the shear rate increased, the viscosity of yoghurt decreased. This is a characteristic of typical non-Newtonian fluids including yoghurt. The relationship between shear rate and viscosity in yoghurt is often described as shear-thinning behaviour. Similar phenomenon was also observed by Huang et al. [26] and Xu et al. [27]. The viscosity of yoghurt initially decreased before increasing with higher soybean content. Overall, the apparent viscosity showed a decrease as the shear rate increased, indicating a non-Newtonian shear-thinning behavior. The decrease in viscosity was observed during 1 s − 1 and 30 s − 1. L. paracasei had produced larger quantity of extracellular polysaccharides hence, showed higher viscosity as compared to other cultures. Ityotagher and Julius [28] studied yoghurt produced from milk-soy flour blends for up to nineteen days and there was a decrease in viscosity of the yoghurt samples; also viscosity measures were lower when compared with plain yoghurt sample. The reduced viscosity observed in whole soybean-enriched yoghurt samples may be attributed to lower total solid contents. Falah et al. [29] observed increase in viscosity of the ice creams with an increase in inulin level in the formulations. Behbahani et al. [30] obtained mean particle size of Plantago major seed mucilage solution to be 448.56 ± 19.16(nm). Behbahani and Fooladi [31] found the particle size to be 400.32 nm in 0.1% Plantago major gum solution.

Fig. 1
figure 1

Changes of Viscosity during storage periods of soghurt under refrigeration condition

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3.4 Changes of particle size distribution during storage time of soghurt under refrigeration condition

Particle size distribution was estimated during the entire storage periods. It was observed that smaller particles were mostly found in the range of 3.0–50.0 µm while bigger particles were in the range of 57.0–910.0 µm. Maximum smaller particles were observed in control and 5 days stored soghurt. Maximum bigger particles were found in 10 days and 15 days stored soghurt (Fig. 2). During the initial time of storage, the soy yoghurt exhibited relatively uniform particle size distribution and was found homogenous throughout the matrix. With an increase in storage time the aggregation of particles occurs leading to increase in proportion of larger particles and particle size distribution became more heterogenous with wider range of particle size. This might occur due to the bacterial growth and/or enzymatic activity such as breakdown of protein or polysaccharide structures leading to changes in particle distribution.

Fig. 2
figure 2

Changes of particle size distribution during storage periods of soghurt under refrigeration condition

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Rui et al. [32] explored that fermentation affects the digestibility of soy protein wherein sample SY-6.0 of soy yoghurt, the particle size distribution of the 0.1–1 μm particle group diminished, and a new peak of around 6 μm appeared. Conversely, samples SY-5.7, 5.4, and 5.1 exhibited particle peaks around 12 μm, 20 μm, and 23 μm, respectively. Gastric digestion was observed to markedly impact the particle size distribution in all tested samples. Particularly, D[3, 4] and D[v,90] values for SY-5.4 and SY-5.1 experienced substantial reductions to 21–44% (D[3, 4]) and 11–19% (D[v,90]) relative to their respective values from the preceding stage. Kong et al. [33] assessed the sensory characteristics of soy yoghurt influenced by the addition of low acyl gellan gum. The particle size of soy yoghurt rose as the concentration of low acyl gellan gum increased, ranging from 181.66 nm to 323.69 nm. Notably, all soy yoghurt samples displayed a monomodal distribution of particle size.

3.5 Confocal Laser Scanning Microscopy (CLSM) study on the changes of fat and proteins during the storage of soy yoghurt under refrigeration conditions

In the CLSM study, we found that proteins and fats are dispersed slowly during the storage as because of the proteolytic and lipolytic enzymes produced by yoghurt cultures after 5 days of storage under refrigeration condition. These enzymes act on fats and proteins and hydrolyze them in to free fatty acids and smaller peptides respectively. But in case of soy milk, proteins are mainly coaggregated with fat globules in the cotyledons after 5 days of storage (Fig. 3). The protein particles as well as fat globules were slowly released from the cotyledons and dispersed throughout the matrix of soghurt during the storage study where the green color indicates protein molecules and red color indicates the fat. The particle size of proteins and fats reduced with increase in storage time as a result of enzymatic activities carried out by LAB. Whereas, the aggregates of protein in soymilk remained nearly stable and intact during the storage with very little dispersion. Similarly, Wang et al. [34] observed that the soy based yoghurt had the finest network indicated by presence of small black areas in CLSM as compared to reconstituted yoghurt and oat based yoghurt. Reconstituted yoghurt formed a homogenous but less dense network and the soy yoghurt formed beaded structure due to soy protein with less clusters forming fine and homogenous gel network. Ningtyas et al. [35] noted in their study that CLSM images of sogurt revealed the hydrolysis of fat globules by lipase enzymes produced by microorganisms, as well as lipase added externally during the in vitro gastric phase simulation. This process led to the release of proteins from the fat globules. Furthermore, during the enteric phase digestion, fats were solubilized while proteins were precipitated into smaller fragments.

Fig. 3
figure 3figure 3

Confocal Laser Scanning Microscopy (CLSM) study on the changes of fat and proteins during the storage of soghurt under refrigeration conditions

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Chen et al. [36] examined the structural and rheological characteristics of set-type yoghurt incorporating modified insoluble soybean fiber (ISF) derived from okara. The plain set-type yoghurt displayed interconnected protein aggregation, forming thick strands with long and narrow open water channels. Addition of 0.25–0.5% ISF resulted in formation of more homogenous network with lack of water channels. Lower ISF levels lead to formation of dense protein network whereas, higher ISF concentrations (0.75%) lead to formation of protein aggregates and self-entangled ISF lead to gel like appearances.

Yang et al. [37] studied the changes in the physicochemical, digestion and microstructural attributes of soy protein gel acidified with lactic acid bacteria (LC), glucono-δ-lactone (GDL) and citric acid (CA). Protein aggregates were observed within the characteristic gel formations depicted in confocal laser scanning microscopy (CLSM) images of gels generated through three different acidification methods. These aggregates typically formed minute structures that, at a specific length scale, coalesced into a network spanning the voids. In comparison to the gel induced by lactic acid (LC), which exhibited noticeable pore size likely attributed to the presence of EPS, the gel network resulting from GDL treatment appeared denser and more compact. Conversely, citric acid (CA) yielded a protein network with greater porosity, a porous aggregated structure with fewer connections, and larger aggregated formations, suggesting the formation of a weaker gel. This weaker gel may contribute to the reduced water-holding capacity and diminished gel hardness.

4 Conclusion

Soghurt stability under refrigeration condition was studied for 15 days. The lactic counts were not affected during the storage and coliform counts were absent during the storage. Yeast and mold counts were also absent upto 7 days. However, the product could be stored upto 7 days under refrigeration condition (6–8 °C). During CLSM study, it was observed that protein bodies are more sensitive during storage periods. In case of particle size distribution, maximum bigger particles were found in 10 days and 15 days stored soy yoghurt. Further standardization of the soy yoghurt with addition of flavor and colour is required to make it more delicious and palatability for increasing the acceptance of the consumer.