Gelation and Thermal Stability of Camel Milk Protein and Soy protein Blends: A Review

ABSTRACT

Camel milk and its products are vital sources of nutrition for inhabitants of arid and semi-arid nomadic pastoral regions, where camels are predominantly raised. Alternatively, the high demand for soy milk originates from its nutritional importance, catering to individuals seeking/plant-based alternatives with health and environmental benefits. Camel milk is rich in essential nutrients, contains less lactose than bovine milk, and offers numerous health benefits. Although camel milk contains higher levels of α-lactalbumin and β-casein than bovine milk, the absence of β-lactoglobulin and the presence of low amounts of κ-casein make it unstable at high temperatures, exhibiting poor gelation characteristics. Soymilk proteins exhibit unique heat stability and gelation characteristics based on processing variables such as pH, temperature, and presence of coagulants. The use of camel milk and soymilk proteins enables the attainment of high sustainability, desirable tastes, better nutrient profiles, and improved functionalities. Thus, this review discusses the thermal stability and gelation properties of camel milk proteins, soymilk proteins, and their respective mixtures. Understanding blends of soy milk and camel milk proteins is crucial for developing nutritionally balanced alternatives, benefiting both individuals seeking plant-based options and those in arid regions relying on camel milk for sustenance.

KEYWORDS:

• Camel milk proteins
• soymilk proteins
• heat treatment
• gelation

Introduction

The demand for milk and other dairy products continues to increase globally.^[1] Nonetheless, the conventional dairy industry has been associated with several environmental consequences, giving rise to a growing need for more sustainable and environmentally resilient alternatives to traditional dairy products. In this context, camel milk has gained considerable popularity worldwide, attributed to its remarkable nutritional profile, even though it is comparatively more expensive and has a lower supply compared to bovine milk. Furthermore, camels are more resilient to extreme climate conditions and require less water than cows, offering potential environmental benefits. Camels produce 3–10 liters of milk per day during the 12–18 months of lactation.^[2] Camel milk, with its composition closest to human milk, serves as a highly nutritious food source for children.^[3] It is an attractive choice for consumers due to its unique fatty properties and has been used to treat diseases such as tuberculosis, asthma, and jaundice.^[4] The protein fraction is a key component that significantly impacts the value, technology, and biological activity of camel milk.^[5] Camel milk proteins consist mainly of 75–88% casein, comprising αS1-casein, αS2-casein, β-casein, κ-casein, and 15–25% whey protein containing mainly α-lactalbumin and lactoferrin.^[1] However, despite its high utilization value, camel milk has drawbacks that limit its industrial use, such as poor gelation properties resulting from the lack of κ-casein interactions with β-lactoglobulin^[6] and a high ratio of whey protein (WP) to casein (CN), or larger size of casein micelles, leading to undesirable functional characteristics in secondary dairy products.^[7]

In recent years, high-quality vegetarian diets have become a focal point in new food development. According to Vallath et al. (2022),^[8] this type of food not only improves health but also reduces the environmental damage. Proteins are an invaluable dietary supplement in a vegetarian diet.^[8] Currently, soy is one of the most widely consumed legumes worldwide.^[9] Compared to other legumes, soy not only provides high-quality protein, fiber, vitamins, and minerals but is also a concentrated source of isoflavones.^[9,10] Natural soy protein is mainly composed of globulin and albumin, with globulin accounting for approximately 90% of the total protein content.^[11,12] Globulins are mainly composed of β-conglycinin (7S) and soy globulin (11S).^[13] According to Fernandez-Avila and Trujillo (2016),^[14] the molecular toughness and gelation properties of this globulin are poor, mainly due to its compact three-dimensional structure. In addition, an unpleasant flavor hinders its use in many food applications.

While both camel milk proteins and soymilk proteins have high nutritional value and utility, little is known about their joint action. However, some studies have shown that blending plant proteins with animal proteins is a promising means of overcoming the functional and nutritional limitations of both counterparts.^[15] For instance, mixing soymilk with bovine and/or buffalo milk showed increased sensory characteristics of yogurt^[16] while simultaneously acting as a potential probiotic carrier.^[17] In addition, blending can provide sufficient amounts of essential amino acids at a lower protein stoichiometry, thus effectively reducing calorie requirements and reducing the risk of intestinal discomfort.^[15] However, this approach is not entirely successful based on the type of plant and animal proteins involved, and there are still reports of negative sensory properties in mixed gel products (e.g., yogurt, etc.) as the vegetable protein content increases in the product.^[18]

Even though plant and animal milk blends are used in various food applications, the utilization of camel milk proteins in blends with plant proteins is far below its potential.^[6] Camel milk and soy milk have been frequently incorporated into existing processing technologies and utilized in isolation in various food systems with a variety of functional properties. However, understanding how camel and soymilk proteins behave solely under different food processing technologies is important. This is essential for understanding their potential in blends. The blends of camel milk and soy milk in secondary food applications, such as gelation processes offer substantial potential benefits. The gels formed from camel milk and soy protein blends can be used in a variety of food products ranging from yogurts and cheeses to spreads and desserts, offering manufacturers flexibility in new product development and other potential health and techno functional benefits. Utilizing camel milk, particularly in regions where it is abundantly available, along with widely cultivated soy, can lead to cost-effective production of high-protein food products. Combining camel milk proteins with soy proteins will result in a gel with a broad amino acid profile. The textural properties of gels can be finely tuned by adjusting the ratios of camel milk to soy proteins, catering to specific texture and firmness requirements in food products. Moreover, since soy proteins exhibit excellent thermal stability, they can complement the sensitive nature of camel milk proteins, enhancing the overall thermal stability of the gel. However, there is a lack of a comprehensive review in terms of camel and soy proteins in isolation and in blends and how they behave during food production. Therefore, the present review focuses on the composition, structure, and interactions within camel milk protein systems, soymilk protein systems, and their blends as affected by different processing technologies. Additionally, the article identifies potential gaps within the area.

Thermal stability and gelation mechanism in proteins

The thermal stability and gelation mechanisms of proteins play pivotal roles in defining their functional properties. A detailed understanding of these mechanisms is crucial for engineering protein-based products with specific textures and stability profiles and to optimize protein behavior for diverse applications. Thermal stability in proteins is a crucial characteristic that influences their functional behavior under varying temperature conditions. Proteins, due to their complex tertiary and quaternary structures, are sensitive to temperature changes, which can lead to unfolding or denaturation. The thermal stability of a protein is determined by the energy required to disrupt its stable conformation, which involves breaking non-covalent interactions such as hydrogen bonds, ionic bonds, and hydrophobic interactions.^[19] Consequently, these complex physicochemical transformations (protein unfolding and aggregation) occur during the thermal treatment of protein solutions, inducing gel formation, known as the gelation process.^[20] Gelation process typically involves three sequential stages: co-fusion, nucleation and growth. During heating, proteins undergo denaturation, which opens up their native, folded structures. This exposes their reactive and hydrophobic sites, enabling denatured proteins to coalesce, or co-fuse, into initial, small aggregates. These initial aggregates serve as nuclei for further protein aggregation. Nucleation is a key process that establishes the framework for the size and distribution of protein aggregates. It can occur uniformly throughout the solution (homogeneous nucleation) or be initiated by particulate impurities or surface irregularities (heterogeneous nucleation). Once nucleation has occurred, the aggregates grow by the addition of more protein molecules. As the aggregates enlarge, they eventually interconnect to form a three-dimensional network that transforms the fluid into a gel. This network traps water, air, and solutes, creating the gel’s structure.^[20,21] Several factors can influence the gelation process, including protein concentration, temperature, pH, and ionic strength. Additionally, the composition of the protein solution, whether it consists of a single protein or a combination of proteins, also influences these properties. For instance, camel milk proteins, which include both caseins and whey proteins, display a unique behavior under heat compared to more commonly studied bovine milk proteins. Due to the distinct composition of camel milk, which includes a different casein micelle structure and a higher mineral content, gelation occurs at higher temperatures. For camel milk, gelation typically initiates around 90°C. This higher gelation temperature can be attributed to the smaller and more inherently stable casein micelles present in camel milk, which resist aggregation at lower temperatures typical for other milk.^[22,23] Unlike animal milk proteins, soymilk proteins such as glycinin and β-conglycinin gel under different conditions due to their plant origin. The gelation of soy proteins generally involves the denaturation of the proteins at elevated temperatures, exposing hydrophobic groups. Subsequent aggregation through hydrophobic interactions and disulfide bonds. The process is influenced by environmental conditions such as pH and ionic strength and usually takes place within a temperature range of 75°C to 90°C. The presence of natural soy polysaccharides can modify these properties, typically by stabilizing the protein structure or enhancing gel strength.^[24,25]

Camel milk proteins

Camel milk is recognized as a high-quality protein source with exceptional nutritional value and advantageous effects on health. Among mammals, camel milk is the most comparable to human milk.^[26] The total protein content of camel milk is approximately 2.4–5.3%.^[27] Casein is the most abundant protein in camel milk, accounting for 75–87% of the total protein. Additionally, various bioactive proteins such as lactoferrin, GlyCAM-1, lactoperoxidase, lysozyme, and immunoglobulins are present. The composition of camel milk is not always stable, and factors contributing to this variation include, but are not limited to, sample size, different analytical techniques, milking management, livestock management, feeding patterns, water availability, breed of camels, geographical location, and seasonal variations.^[27,28]

Camel milk lacks β-lactoglobulin, whereas β-lactoglobulin is the predominant whey protein in bovine milk (Table 1). α-lactalbumin is the major whey protein in camel milk, accounting for 19.7% (w/w) of the total camel milk protein and 84% (w/w) of the total camel milk whey protein.^[30] It is a globular protein consisting of 123 amino acid residues that form a compact sphere containing one calcium atom per mole of the protein molecule. The protein structure is stabilized by four disulfide bonds. Calcium, as a divalent cation, plays an important role in stabilizing the spatial structure of α-lactalbumin.^[31] Thus, α-lactalbumin is present in holo (Calcium-loaded) and apo (Calcium-depleted) forms.^[32–35] The typical α-lactalbumin molecule consists of two structural domains: a large α-helix and a small β-sheet domain linked by a calcium-binding loop.^[36,37] When α-lactalbumin is folded under physiological conditions, it readily converts to a molten pellet state upon calcium loss, low pH, elevated temperatures, or disulfide bond reduction.^[37] In this respect, α-lactalbumin in molten globule form is one of the best-described folding intermediates for globular proteins.

Table 1. Major protein content of camel and bovine milk.^[29]

Protein	Camel	Bovine
Whey proteins
β-Lactoglobulin	ND	5.97 ± 0.14
α-Lactalbumin	2.01 ± 0.02	1.08 ± 0.04
Serum albumin	0.40 ± 0.01	0.36 ± 0.04
Lactoferrin	1.74 ± 0.06	0.20 ± 0.10
Caseins
α-Casein	2.89 ± 0.29	12.79 ± 2.31
β-Casein	12.78 ± 0.92	11.66 ± 0.87
κ-Casein	1.67 ± 0.01	4.39 ± 0.31

*Values (mg mL −1) are means (n = 6) and standard deviation of three independent experiments; ND, not detected.

Lactoferrin, a glycoprotein, is present in abundance compared to bovine milk. It is composed of 689 amino acids with a molecular weight of 75.3 kDa. PGRP (Peptidoglycan Recognition Protein) is also present in camel milk, absent in bovine milk. It plays a significant role in the inactivation of pathogens, with 172 amino acids and a molecular weight of 19.1 kDa. PGRP is rich in arginine amino acids and poor in lysine, showing similar properties to PGRP in human milk.^[30]

Casein is the primary component of camel milk protein, divided into four distinct types: αS1-casein, αS2-casein, β-casein, and κ-casein, with proportions of 22:9.5:65:3.5%. The predominant casein component in camel milk is β-casein. Notably, camel milk has 3 times lower content of κ-casein and 5 times lower content of α-casein compared to bovine milk. The αS1-casein from camel milk exists in two isoforms named A and B, with 207 and 215 amino acid residues, respectively, and molecular weights of ~24.5 kDa for variant A containing 5 or 6 phosphorylated serines, and ~25 kDa for variant B containing 5 or 6 phosphorylated serines.^[38] The isoelectric point is approximately 4.40.^[38] αS2-casein has 178 amino acids^[39] and a molecular weight of approximately 21.9 kDa. αS2-casein is the most hydrophilic among the other caseins.^[30]

Camel milk exhibits a high content of β-casein, with 217 amino acid residues and a molecular weight of 24.9 kDa.^[38] β-casein is more sensitive to digestive hydrolysis than α-casein.^[40] This property gives camel milk an advantage as a substitute for human milk.^[41] β-casein is the most hydrophobic of all caseins and is characterized by a very high amphipolar nature. β-casein has a hydrophobic C terminal (136–209) and a hydrophilic N terminal (1–40) with phosphorylated residues that provide a negative charge to the molecule. β-casein exhibits extreme resistance to heat due to the absence of disulfide bridges.^[30] Camel milk κ-casein has 162 amino acid residues and a molecular weight of ~18-23 kDa, as reported by Kappeler et al. (1998).^[38] κ-casein has a low calcium binding ability due to the presence of a single phosphorylation site at 149.

Casein proteins in camel milk are arranged as micelles. However, camel milk comprises larger casein micelles compared to bovine milk.^[42] Camel milk casein micelles are approximately 380 nm in diameter, whereas bovine milk has 150 nm micelles.^[43] Camel milk has a different casein micelle size distribution than bovine milk.^[42] It was found that the distribution of calcium and phosphate between micelles and serum fractions was almost similar (60–65%) to bovine milk. Camel casein micelles had higher levels of salt and greater citrate content, while bovine milk micelles had lower proportions of magnesium, phosphorus, and citrate than camel milk.^[44] This is consistent with studies attributing the larger micelle size of camel milk to the increased mineral content.^[45] There has also been speculation that the smaller micelle size of casein in bovine milk is associated with higher levels of κ-casein, which aligns with the κ-casein levels shown in Table 1.^[46]

Thermal treatment of camel milk proteins

Thermal stability of milk proteins is a critical variable and varies as a function of temperature, time of exposure and rate of heating.^[13] The presence of κ-casein and β-lactoglobulin, as well as their interactions, are believed to be critical to maintaining the thermal stability of bovine milk.^[13] Compared to bovine milk, camel milk is much less thermally stable. The thermal coagulation time of camel milk, observed at 140°C, manifested as 133.6 seconds, whereas its bovine counterpart exhibited a markedly protracted duration of 1807.4 seconds.^[47] Elagamy (2000)^[48] investigated the heat stability of whey proteins in bovine and camel milk, subjecting them to temperatures ranging from 65 to 100°C for durations spanning 10 to 30 minutes. The findings elucidated a temperature and time-dependent escalation in heat-induced changes to whey proteins, irrespective of the milk protein type. Similarly, Farah (1993)^[49] showed lower heat stability of camel milk proteins as opposed to bovine milk proteins. All these authors attributed this effect to the presence of low levels of κ casein (5% of total casein in camel milk to 14% in bovine milk) and absence of β-LG. β-lactoglobulin tends to bind to κ-casein on the surface of the casein micelles via thiol groups, as well as with other whey proteins which then increases the viscosity by a factor of 10 or more.^[50] Furthermore, κ-casein contributes significantly to system stability, as the negatively charged κ-caseins induce electrostatic repulsions among proteins, mitigating protein aggregation.^[51] Additionally, κ-casein in camel milk exhibits heightened susceptibility to heat, undergoing complete depletion at 100°C.^[44] The α-casein and β-casein of camel milk were affected by heating temperature (60–130°C) and time (10/20/30 min) but are more stable than κ-casein at the same temperature.^[44]

Similarly, Lajnaf et al. (2022)^[52] found that κ-casein, β-casein, and α-caseins remained almost intact when heated at 70°C and 80°C for 30 minutes but heating at 90°C and 100°C significantly decreased their stability. The decrease was associated with the emergence of a new peptide following heating at 90°C for 30 minutes, indicating protein degradation. Omar et al. (2018)^[29] revealed a significant decrease in casein micelle size with heating, irrespective of the temperature, although marked changes in the levels of whey proteins were observed with UHT treatment as opposed to HTST. During heat treatment, milk proteins undergo structural unfolding, exposing buried amino acid residues. The pronounced effect of heating on the hydrophobicity of camel β-casein suggests that camel β-casein has a more flexible conformation.^[44] In addition to the presence of low levels of κ-casein, the existence of high amounts of non-glycosylated κ-casein increases the stabilizing ability due to steric repulsion caused by charged sialic acid groups and increased hydrophilicity. Thus, hydrophobic interactions are the main driving force behind the coagulation of camel milk, as indicated by the large amount of β-casein in the micelles, and therefore, this is the reason for the observed decrease in thermal stability.^[50] However, these effects are a function of temperature.^[29]

In addition, this heat stability effect is a function of pH. Camel whey proteins are more sensitive to pH than bovine whey proteins and show the lowest heat stability near the isoelectric pH of proteins, which is at 4.5. The decrease in net charge, leading to a reduction in electrostatic repulsions, increases protein-protein interactions. Calorimetric studies conducted by Laleye et al. (2008)^[53] showed no significant difference in the heat stability of whey proteins between camel and bovine milk in liquid form. In contrast, the denaturation temperature of whey proteins was found to be significantly lower compared to bovine milk. For instance, bovine whey proteins showed the first denaturation at 81°C, while camel whey proteins denatured at 78°C.^[54] The latter denaturation was attributed to the mixture of α-lactalbumin (αLA) and camel serum albumin.

Another influential factor for thermal stability is the denaturation of α-lactalbumin. According to Fig. 1, when camel milk is heated at temperatures above 90°C, α-lactalbumin undergoes irreversible denaturation through the cleavage of intramolecular disulfide bridges, leading to the production of dimers and tetramers.^[31] As the temperature increases to 95°C, numerous protein aggregates are formed. The irreversibility of α-lactalbumin denaturation in this process is primarily attributed to the breakage of disulfide bonds during high-temperature heating. Free thiol groups are generated during this process. Subsequently, the free -SH group catalyzes intermolecular thiol/disulfide interchange reactions and forms soluble oligomers.^[55]

Figure 1. Influence of pH and temperature on the structure of a-lactalbumin.^[31]; *, denaturation temperature; **, aggregation temperature.

Table 2 outlines diverse studies focusing on the heat treatment of camel milk. Many studies clearly indicate that camel whey proteins exhibit a lower degree of thermal denaturation compared to bovine milk whey proteins.^[62] It was found that heating at 90°C for 5 minutes resulted in about 33% denaturation of α-lactalbumin in camel milk, whereas bovine milk experienced 95% denaturation of α-lactalbumin under similar conditions. The preservation of the secondary structure of camel α-lactalbumin during thermal denaturation is better compared to that of bovine α-lactalbumin.^[32] The absence of β-lactoglobulin in camel milk could explain the lower degree of denaturation of α-lactalbumin after heating. Essentially, β-lg in bovine milk initiates interactions with α-LA since α-LA lacks the additional free thiol group.

Table 2. Summary of literature on heat treatment experiments using camel milk.

No.	Heat treatment	Comments	References
1	60/70/80/90°C 60 min/120 min	No deposits were produced from camel milk whey heated at 60°C; heat treatment above 70°C led to severe blockage of the stainless-steel plates. α-LA and CSA (camel serum albumin) were involved in the deposition phenomenon. pH influenced the thermal behaviour of whey proteins.	^[Citation54]
2	63°C/80°C/90°C 30 min, 72°C 15s	Heat treatment had no effect on fat content or total nitrogen content, but did influence protein, ash and total solids contents. Non-protein nitrogen (NPN), non-casein nitrogen (NCN) and whey protein nitrogen (WPN) decreased progressively with increasing heat treatment, but the amount of casein and denaturation rate increased.	^[Citation56]
3	90°C 5 min	Produced the highest viscosity and lowest water separation.	^[Citation57]
4	63°C 30 min/72°C 15s/85°C 2 min	63°C: camel milk was stable in terms of pH, acidity, density, dry matter, lactose, fat, vitamin C, protein; 85°C: camel milk was unstable but fat content remained constant; 72°C: camel milk was unstable in terms of pH, acidity, dry matter, vitamin C and protein, and stable in terms of density, fat, and lactose.	^[Citation58]
5	63°C (T1) /85°C (T2) /95°C (T3) 30 min Control group 72°C 15s	Total solids, protein and fat content increased significantly with heat-treated at 85°C and 95°C for 30 minutes, while lactose content decreased with increasing time of heat-treatment. The change in apparent viscosity increased linearly with increasing time of heat treatment, and with increasing storage time.	^[Citation59]
6		Denatured α-La and camel serum albumin (CSA) tended to adhere to the thermal surface of milk processing equipment, either alone or together with κ-CN. Increasing pH was beneficial in improving the thermal stability of camel milk proteins. The addition of phosphate improved the thermal stability of camel milk.	^[Citation60]
7	80°C 60 min	The non-casein nitrogen content of camel milk decreased with increase in degree of heat treatment. Heat-treated deposits of camel milk contained 57% w/w protein content and 35% w/w mineral content but showed an amorphous structure.	^[Citation61]
8	65/75/85/100°C 10/20/30 min	The antimicrobial factor of camel milk was more resistant to high temperatures and the order of heat resistance was found to be lysozyme > lactoferrin > immunoglobulin G.	^[Citation48]
9	70/80/90/100°C 30 min	Greater foaming of camel milk proteins when increasing the temperature (up to 165%)	^[Citation33]
10	60–130°C 0/1/10/30 min	Lactoferrin, camel serum albumin and κ-casein were depleted as the temperature increased from 100°C/1 min, 110°C/30 min and 110°C/10 min respectively.	^[Citation51]
11	65°C 30 min/72°C 30 min/75°C 5min/85°C 5min/90°C 5min	With heat treatment, the gelation time and the time to reach maximum gel strength increased significantly. Heat treatment of camel milk at 72°C/30 seconds resulted in a loss of coagulation properties, i.e. very low gel strength or no coagulation within 60 minutes. The non-casein nitrogen content of camel milk decreased with increasing heat treatment temperature and the total whey protein denaturation rate increased with increasing heat treatment temperature.	^[Citation62]
12	40°C 30 min/65°C 30 min/72°C 30s/75°C 5 min/85°C 5 min/90°C 5 min	The α-lactoalbumin (α-La) denatured less in camel milk, whereas camel serum albumin denatured at higher heat treatment conditions. Gelation time and the time to reach G’max increased significantly with increasing heat treatment, while G’max decreased with increasing heating temperature.	^[Citation63]
13	65°C/85°C/100°C 15/30 min	Lactoferrin started to denature after 30 minutes of treatment at 100°C and had a good thermal stability.	^[Citation64]
14	63°C 30 min(T1)/72°C 15s(T2)/100.17°C 10 min(T3)	Different combinations of heating and time had a significant effect on the nitrogen fraction of milk, followed by the physical properties: pH, acidity, specific gravity, viscosity.	^[Citation65]
15	63°C 30 min/72°C 15s/100.5°C 10 min	Camel milk treated at 63°C for 30 minutes scored highest in terms of flavour and texture, while camel milk treated at 100.5°C for 10 minutes scored highest in terms of overall acceptability of sensory characteristics and had the longest shelf life.	^[Citation66]
16	63°C 30 min/90°C 3 min/100°C 3 min	The heat treatment process significantly increased the viscosity, total solids, ash and lactose content (p < .05).	^[Citation67,Citation68]
17	60°C/70°C/80°C/90°C 60/120 min	Denaturation of camel protein began between 70–80°C. After heating camel milk at 90°C, α-LA, CSA and κ-casein bands were reduced.	^[Citation69]
18	80°C 60 min	No camel α-LA or PGRP was detected in camel milk after heating at 80°C for 60 min, while CSA was significantly reduced.	^[Citation70]
19	room temperature (RT), moderate heating at 63°C, and at 98°C 1 h	The whey proteins present in camel milk were less affected when heated at 63°C than when heated at 98°C. Denaturation increased significantly as the temperature was increased from 63°C to 98°C.	^[Citation71]
20	72°C 15s /75°C 10 min/ 80°C 5 min/65°C 30 min	Camel whey proteins were significantly affected by heat treatment at 98°C, with some proteins disappearing completely from the gel pattern; however, whey proteins remained slightly stable at 63°C for 60 minutes. The degree of denaturation increased significantly with temperature from 63°C to 98°C.	^[Citation72]
21	70°C/90°C 30 min	Heat treatment at 70°C for 30 minutes improved foaming properties of camel acid whey. The lowest foaming and foam stability was observed at all heating temperatures, with a loss of interfacial properties of sweet camel whey due to protein denaturation.	^[Citation73]
22	70°C/90°C 30 min pH:4.3/6.5	Heat treatment at neutral pH had found to improve foaming properties, while at acidic pH (4.3) this property decreased. The foams were more stable after heat treatment at pH 4.3 than at 6.5. The foam produced by camel whey protein was found to be unstable at 20◦ C.	^[Citation74]
23	Ambient temperature/no heat treatment, 63°C 30 min/ 72°C 15s/90°C 10 min	The denaturation rate of camel milk increased with increase in heat treatment time. Heat treatment led to an increase in protein, ash and total solids while Non-protein nitrogen (NPN), non-casein nitrogen (NCN) and whey protein nitrogen (WPN) gradually decreased with increasing heat treatment	^[Citation75]
24	63/80/90/100°C 30 min/73°C 15s/90°C 10 min	Heating camel milk at 63, 80, 90 and 100°C for 30 minutes resulted in losses of approximately 27, 41, 53 and 67% of the vitamin C content respectively. Heat treatment caused negligible damage (0–7%) to riboflavin content of camel milk.	^[Citation76]
25	80°C 5 min /72°C 5 min	The degree of denaturation of camel milk at 80°C for 5 minutes ranged from 32%-35%. Camel milk IgG was a thermally unstable compound and the midpoint of denaturation of mature milk heated for 30 minutes was 67.2°C. After 30 minutes of heating at 62.3°C, the concentration of IgG in camel milk decreased.	^[Citation69]
26	140°C	Camel milk was unstable at 140°C and even the addition of formaldehyde/urea still did not improve the heat stability. The whey proteins of camel milk were more stable than those of bovine or buffalo milk. At 80°C for 30 minutes, the degree of denaturation of camel milk whey protein (32–35%) was less than that of bovine milk whey protein (70–75%).	^[Citation77]
27	75/80°C 30/60 min	In the current study, camel serum albumin was unaffected by all pasteurisation methods. When camel milk was heated at 75°C for 30 minutes, there was no change in the electrophoretic pattern of camel serum albumin. When camel milk was heated at 80°C for 60 minutes, camel serum albumin was significantly reduced. However, a complete disappearance of camel serum albumin was reported when camel milk was heated at 80°C for 30 minutes.	^[Citation78]

Another finding indicates that the content of non-casein nitrogen, non-casein nitrogen, and whey protein nitrogen in camel milk progressively decreased with increasing heat treatment. However, the quantity of caseins and the denaturation rate increased during heat treatment.^[56] The highest ash content was observed in camel milk heat-treated at 90°C for 30 minutes, followed by treatments at 80°C for 30 minutes and 72°C for 15 seconds. The lowest ash content was found in raw camel milk. Total nitrogen content in camel milk remained unaffected by heat treatment, though the non-protein nitrogen content was significantly influenced. Camel milk heat-treated at 80°C and 90°C for 30 minutes exhibited the lowest non-protein nitrogen and non-protein nitrogen/total nitrogen content (%). Similarly, the trends in non-casein nitrogen and non-casein nitrogen/total nitrogen content (%) mirrored those of non-protein nitrogen. Under different heat treatments, the whey protein nitrogen content (%) and whey protein nitrogen/total nitrogen content (%) decreased due to the denaturation of whey protein co-precipitated with casein.

During heating, camel milk lost more vitamin C compared to bovine milk due to its higher heat sensitivity.^[76] In the low-temperature long-time pasteurization method, 27% of vitamin C was lost in camel milk, while only 15% was lost when the high-temperature short-time method was applied. The high thermal sensitivity of vitamin C in camel milk is attributed to the less sensitive denaturation of whey proteins, compared to bovine milk, resulting in reduced sulfhydryl compounds. Regarding individual whey proteins, camel milk α-lactalbumin denatured to a lesser extent with increasing heat treatment, whereas camel serum albumin and lactoferrin denatured to a greater extent under increasing heat treatments.^[63]

Camel milk treated at 63°C for 30 minutes scored highest in flavor and texture, while camel milk treated at 100.5°C for 10 minutes achieved the highest overall sensory acceptability. Heat-treated camel milk consistently outperformed the control group in taste, texture, and overall acceptability. Specifically, camel milk subjected to heat treatment at 63°C for 30 minutes received the highest overall acceptability scores of 36.3, 24.4, and 7.4, compared to the control camel milk scores of 32.3, 21.4, and 6.7. Despite a decrease in color scores with heat treatment and time, no significant differences were observed between various combinations of heat treatment and time. The change in sensory characteristics was attributed to the flavor imparted by heating. Consumer preferences shifted with heat treatment, favoring milk treated at 79°C as compared to milk treated at 77°C, 82°C, and 85°C^[13] Besides improving organoleptic qualities, different combinations of temperature and time enhanced the preservation quality of camel milk. Heat-treated camel milk demonstrated prolonged shelf life at refrigerated temperatures, with milk treated at 100.5°C for 10 minutes exhibiting the longest preservation time.^[66] At 100.5°C for 10 minutes, camel milk reached its maximum shelf-life expiration after 76 hours and 42 days (at ambient and refrigerator temperatures, respectively). The combined effects of heat treatment and time not only eliminated spoilage microorganisms but also extended the shelf life of camel milk.

Most studies to date have focused on understanding the effect of heat treatment on camel milk as a whole rather than investigating individual protein fractions. Limited research has been conducted on the latter. There is a lack of comprehensive exploration into appropriate time-temperature combinations that can effectively ensure microbial safety while preserving the nutritional and sensorial qualities of camel milk proteins. Camel milk, renowned for its distinct flavor and aroma, lacks in-depth studies on the development of these characteristics during processing. Additionally, storage stability has been scarcely researched in both heat-treated and untreated camel milk under varying storage conditions. Comparative studies are notably lacking, hindering the establishment of relationships among different types of milk. This limitation prevents the utilization of protein mixes from different origins to balance the pros and cons of each milk type. Moreover, there is a notable absence of exploration into the use of novel processing technologies on camel milk proteins. In the future, there is a significant requirement for additional laboratory-scale fundamental studies that can be applied to pilot-scale production in the realm of thermal treatment research for camel milk.

Acid gelation of camel milk proteins

Acid gel products play a significant role in the food industry due to their diverse applications and benefits. Acid gels are formed when proteins coagulate in the presence of acid, resulting in a gel-like structure. The gel products within the dairy industry are important for their impact on texture, flavor, increase shelf life and nutritional value. Nevertheless, the production of gel-based products like yoghurts from camel milk is challenging due to the poor coagulation ability of camel milk. This leads to a thin consistency and a weak gel structure. The weakened firmness of camel milk coagulum is mainly attributed to the absence of interactions between k-casein and β-lactoglobulin.^[13] Moreover, higher whey protein (WP) to caseins (CN) ratio and the large size of casein micelle also contributed to the weak gel structures in camel milk.^[14] For instance, the use of conventional methods (fermenting camel milk with starter cultures such as Streptococcus thermophilus and Lactobacillus delbruckii subsp. bulgaricus (2.5%) at 37°C for 16 to 18 hours) in the production of yogurt from camel milk did not yield the intended curd structure. Instead, it resulted in fragile and heterogeneous dispersed flakes with a watery texture.^[79] Furthermore, camel milk contains several natural antimicrobial agents, including lysozyme, lactoferrin, lactoperoxidase, and immunoglobulin, which limit the growth of lactic acid bacteria.^[46] Lactoperoxidase from camel milk has been found to be bacteriostatic against Gram-positive strains and bactericidal against Gram-negative cultures.^[80] Therefore, even with enough fermenting agents (2%), camel milk yogurt acidity developed slowly.^[56]

Milk gels can be produced using acidulants such as GDL. In GDL-based acid-induced gels, colloidal calcium phosphate in milk gradually dissolves over time with the addition of GDL to camel milk, accompanied by a decrease in pH. Around pH 5.20, the colloidal calcium phosphate in the milk completely dissolves. Simultaneously, the charge on the surface of the casein micelles is reduced, disrupting the spatial and electrostatic stabilization of the milk, leading to gel formation.^[46] Due to the poor coagulation properties of camel milk, the addition of GDL ultimately results in a thin and weak gel structure. According to findings by Attia et al. (2000),^[45] in contrast to the continuous protein network observed in cow milk gels, camel milk gels at three GDL concentrations (0.8%, 1.0%, and 1.2%) consist of groups of protein flakes lacking firmness. During the acidification process, casein micelles in camel milk undergo three distinct stages based on pH levels. In the initial stage, casein micelles maintain their integrity as the pH reduces from around 6.2 to 5.5. However, significant structural loss occurs through dissociation, resulting in viscoelastic behavior within the pH range of 5.5 to 5.0. Below pH 5.0, the micellar structure collapses, forming a network of completely demineralized casein aggregates (such as groups of casein flakes) lacking firmness entirely. Surprisingly, camel milk does not exhibit an increase in viscosity during gelation.^[81] Some studies attribute these properties to the absence of β-lactoglobulin, combined with the large size of casein micelles and the low κ-casein content of camel milk, contributing to longer coagulation times.^[82] Smaller casein micelles with a greater reaction surface area have been shown to contribute to the formation of stronger gels.^[62]

Researchers have made various attempts to enhance the firmness and consistency of camel milk gels and to reduce the syneresis of the products during processing and storage (Table 3). The use of other mammalian milk such as buffalo milk, and various gelation agents such as hydrocolloids (carboxymethyl cellulose, pectin, gum acacia, arabic gum, guar gum, xanthan gum, alginate, κ-carrageenan, sodium carboxymethyl cellulose), bovine milk powders, polymerized milk proteins, stabilizers, calcium salts and modified starch are some of the strategies implemented to overcome these poor gelation characteristics (Table 3).

Table 3. Review of literature concerning gelation experiments conducted with camel milk.

No.	Gelation	Comments	References
1	90°C 15 min, YC-350 starter culture which contains Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus	The optimum mixture was determined to be an 80%:20% bovine: camel milk mixture. After 4 weeks of storage, increased dehydration and reduced water holding capacity. The structure of yoghurt was very fragile.	^[Citation83]
2	63°C/80°C/90°C 30 min, 72°C 15s, calf rennet powder	Raw camel milk had the lowest rennet clotting time, while rennet clotting time gradually increased with increasing heat treatment temperature. When CaCl2 (0–20 mg/100 ml) was added, the rennet clotting time increased with increasing temperature. However, the addition of calcium chloride reduced the rennet clotting time with all heat treatments.	^[Citation56]
3	glucono-delta-lactone (GDL) concentrations (0.8/1.0/1.2%, w/w), gelatin content (0.6/0.8/1.0%, w/w)	At 1.0% GDL, no suitable gel was formed unless additional gelatine was added. At 0.8% gelatine, camel milk gels had similar hardness, lower viscosity and rheological strength, and higher water holding capacity than milk gels. Heating at 85°C for 15–20 min and 2-stage homogenisation (150/50 bar) or a combination of them did not significantly affect the water holding capacity, hardness, viscosity, rheological strength, and microstructure of camel milk gels.	^[Citation84]
4	90°C 45 min, 1% algae agarose + 4% preheated skim milk powder + 4% GDL (whole camel milk); 1% algae agarose + 4% camel casein powder + 4% GDL (whole camel milk)	The viscosity of the gels formed from camel milk was lower and the curds were smaller than those from bovine milk; The addition of preheated skimmed milk powder significantly increased viscosity of acidic gels and reduced the hysteresis compared to the unheated control group; The addition of whey powder preheated at 80°C did not significantly increase the viscosity or reduce the hysteresis; The addition of camel cheese protein powder increased the viscosity of the gels, reduced the hysteresis and gave the gels a smooth texture.	^[Citation57]
5	40°C for 30 min, camel rennet concentration (40, 70 and 100 IMCU/L)	Better gel hardness was observed at 40 IMCU/L but led to longer gelation times. Similarly at 100 IMCU/L higher yields and quality were observed but led to lower organoleptic acceptability with a stiff texture and unpleasant taste and appearance. 70 IMCU/L gave better quality in terms of organoleptic properties of gels.	^[Citation85]
6		The growth of yoghurt cultures in camel milk was delayed due to the presence of lysozyme, which prolonged the gelation process. Heat treatment at 100°C for 30 minutes completely inactivated these anti-microbial agents in camel milk.	^[Citation41]
7	65°C/30 min, 72°C/30s, CaCl2	For raw milk conditioned to 40°C, short gelation times and high gel strengths were obtained and heat treatments of 65°C/30 min and 72°C/30s did not affect the coagulability of camel milk. However, the more intense heat treatments resulted in very low gel strengths and no gelation within 60 minutes. The gelation time of camel cheese proteins can be reduced by increasing the gelation temperature and adding 0.02% w/w of CaCl2.	^[Citation60]
8	80–85°C/10 min, 0, 0.5, 0.75, 1.0 and 1.25% gelatin	The addition of gelatin significantly reduced the dehydration effect, especially at 1% and 1.25% levels. The textural and rheological properties of camel milk yoghurt were enhanced, almost like those of commercial and bovine milk yoghurts, and a significant shift from the aqueous to the gel phase was noted. Structural (FTIR) analysis showed that the peak observed at 1516 cm-1 was more intense in the 1.25% yoghurt, followed by the 0.75% gelatin added yoghurt. Gelatin incorporation also affected the microstructural features, particularly in the 1.25% gelatin added sample, which appeared like bovine milk yoghurt. However, low levels of gelatin addition (0.75–1.0%) did not affect the physicochemical parameters of the yoghurt, but higher levels altered the taste and flavor and the yoghurt appeared firmer.	^[Citation86]
9	1: 0% gum arabic (AG), 2: 1% AG, 3: 2% AG; ferment of Streptococcus salivarius subsp.	The addition of gum arabic at a concentration of 2% increased the ability to retain water and reduced the ability to separate whey. Increasing the concentration of total solids on the control sample, the addition of gum increased the viscosity to a higher level than the control sample. The addition of gum improved the texture and organoleptic properties of camel milk yoghurt without affecting general acceptability. A concentration of 1% of camel milk yoghurt treated with gum arabic stabilised the texture of camel milk yoghurt without affecting the flavour of the product.	^[Citation87]

A study conducted by Ho et al., (2022)^[84] investigated the effects of different GDL concentrations (0.8–1.2%, w/w), gelatin content (0.6–1.0%, w/w) and processing conditions on the properties of camel milk acid gels. After 4 h of acidification, the pH of camel milk with 0.8, 1.0, and 1.2% GDL was reduced to 4.6, 4.3, and 4.1, respectively. However, none of them were formed a suitable gel for a yoghurt-like product unless gelatin was added. With the addition of 0.8% gelatin produced camel milk gels with similar hardness, lower viscosity and rheological strength, and higher water holding capacity as compared to cow milk gels. Moreover, it was found that in the same study, conventional heating (85°C/15–20 min) and homogenization (150/50 bar) or their combination like pre-treatments did not significantly affect the water holding capacity, hardness, viscosity, rheological strength, and microstructure of camel milk gels. The microstructure of all the camel milk gels showed similar structure (Fig. 2) with a coarse protein network, few linked protein aggregates, and a dense gel with small voids. Moreover, the integrity of the camel milk protein is preserved even with the heating and homogenization treatments, added gelatin dominated in the microstructure of the camel milk gels.

Figure 2. Scanning electron microscopic images of a) cow milk gel with 0.4% gelatin, b) camel milk gel with 0.8% gelatin, c) heated camel milk gel with 0.8% gelatin and d) homogenized camel milk gel with 0.8% gelatin.^[84]

Another study by Bulca et al (2019) ^[83] compared the properties of milk gels prepared from milk mixtures of cow-camel, sheep-camel and goat-camel milk at 80:20 and 70:30 ratios. Cow-camel milk at 80:20 ratio was found as the optimum ratio as it provides better gel properties, higher viscosity and the faster pH drop during yoghurt production. This can be attributed to the high level of protein hydrolysis and particles formation in camel milk during fermentation and after mixing. However, the increase in syneresis and a decrease in water holding capacity (%) with fragile gel was observed after the 4 weeks of storage. Therefore, it is important to understand the different strategies that can be employed to improve the gelation properties of camel milk gels. Blending milk has become one of the emerging approaches to optimize the properties of dairy milk and fulfill both consumer and manufacturer’s requirements. If one milk type lacks certain techno-functional or nutritional properties, these can be fulfilled by the other milk type present in the mixture.

Soymilk proteins

Soybean protein is a valuable plant protein widely used in food and nutraceuticals. In countries with high soy consumption, soybeans serve as a crucial source of protein and are present in various processed forms. Tofu, fried tofu, and natto are common processed soy foods, with tofu being the most prevalent. Additionally, condiments like soy sauce and tempeh, fermented by salt-tolerant yeast or enzymes, are widely used in Chinese households. Soymilk is a common offering in many Chinese hotels and restaurants but less so in other countries, primarily due to aldehydes like pentanal and hexanal, which are the main sources of the distinctive ‘soy flavor’ in soybeans.^[88]

The main components of soy protein are 2S, 7S, 11S, and 15S globulins.^[89] Approximately 80% of globulins consist of 11S and 7S globulins, known as glycoprotein and β-associated glycoprotein, respectively, in soybean protein.^[89] Among these, 11S globulins exhibit a tightly packed rigid hexagonal structure, while 7S globulins usually exist as trimers.^[90] The spatial structure of soy proteins is a critical determinant of their function and properties, with a predominant presence of α-helix and β-fold structures, where α-helix predominates. Moreover, environmental factors such as pH, ionic strength, and temperature influence the structure of soy proteins.

7S globulin, comprising about 30% of total soy protein, contains three main subunits stabilized by hydrogen bonds, hydrophobic interactions, and salt bridges in a trimeric structure.^[91] These subunits, α (~67 kDa), α’ (~71 kDa), and β (~50 kDa), form a trimeric glycoprotein predominantly connected by hydrogen and hydrophobic bonds rather than disulfide bonds. On the other hand, 11S globulin (320–400 kDa), a plant protein isolate, consists of different polypeptides forming hexamers with five distinct subunits, each (~60 kDa), including a basic subunit B (~20 kDa) and an acidic subunit A (~40 kDa), bound by disulfide bonds. AB subunits are believed to be connected by electrostatic and hydrogen bonds into two hexagonal rings, forming hollow cylinders.^[92]

The amino acid composition of soy protein closely resembles the ratio of amino acids required by the human body. Its main components include glutamic acid, serine, isoleucine, asparagine, and lysine. Furthermore, the amino acid composition of soy proteins is similar to that of animal protein, particularly in the content of essential amino acids such as phenylalanine, methionine, threonine, valine, isoleucine, leucine, tryptophan, and lysine. The essential amino acid composition in soy protein is as follows: cysteine = 13.1–35.1 mg/g protein, threonine = 14.9–45.2 mg/g protein, valine = 16.8–42.1 mg/g protein, isoleucine = 14.6–40.2 mg/g protein, leucine = 43.5–73.3 mg/g protein, lysine = 46.8–137.8 mg/g protein, methionine = 12.4–53.7 mg/g protein, phenylalanine = 24.9–50.2 mg/g protein, histidine = 27.3–58.7 mg/g protein.^[93] However, soy protein contains a lower level of sulfur amino acids compared to animal proteins.^[94] The amino acid composition of soy protein serves as a valuable tool for investigating its structure and functionality. Furthermore, the knowledge derived from the soy protein composition enables the design and development of improved soy protein products.

Thermal treatment of soymilk proteins

Heat treatment is an unavoidable unit operation in the processing of soy protein products, such as thermal sterilization and spray drying techniques. These operations have a large impact on the functional properties of soy protein and its products, such as solubility, emulsification, and stability.^[95,96] When the heat treatment temperature does not reach the denaturation temperature of soy protein, the spherically stable structure of the soy protein was not destroyed, thus allowing the sulfhydryl groups and other groups that can interact with each other and remain wrapped inside the molecule. Electrostatic repulsion always allows the individual protein molecules to be in a stable equilibrium position. When the heat treatment temperature reaches the denaturation temperature of soy proteins, soy proteins denature by partially unfolding to expose sulfhydryl and hydrophobic groups, further causing protein-protein interactions.^[97] The onset denaturation temperature of 7S globulin in soy protein was found to be about 63°C and the peak denaturation temperature was 68°C, while the denaturation temperatures of 11S globulin were 80°C and 88°C, respectively.^[98] Heat-induced protein denaturation in soybean isolates was followed by irreversible processes, such as aggregation, and the degree of denaturation of the 11S fraction increases with increasing heating temperature.

Some of the results of the current studies regarding heat treatment of soy proteins are presented in Table 4. Differential scanning calorimetry of soymilk showed that 11S globulins are more thermally stable than 7S globulins and are exceptionally sensitive to pH changes.^[106] During heating, thermal denaturation increased surface hydrophobicity. Consequently, surface hydrophobicity decreased with aggregate formation. Heat treatment increased the content of α-helical structures and decreased the content of β-sheet structures compared to soy isolates that had not been heat treated.^[108] This change may be caused by denaturation and aggregate formation of 7S and 11S globulins. Moreover, when the heat treatment temperature was below 90°C, the content of β-sheet structure showed a significant negative correlation with surface hydrophobicity. When heated at 70°C and 80°C, soymilk produced increased precipitation, whereas when the temperature was increased to 90°C or higher, much less precipitation was produced. This indicated that soymilk dispersion stability is related to heating temperature.^[111] The changes in pH affected thermal stability of soy protein with a 10°C decrease in denaturation temperature observed for 11S globulin when the pH increased from 7 to 11. In comparison, there was no real change in 7S globulin thermal stability when assessed within the same pH range.^[106] During pH rise, 11S globulin undergoes a conformational change, which reflected the lower synergism during denaturation. When the pH reached 11, 50% denaturation was observed. Heat treatment at 65°C led to maximum exposure of hydrophobic groups at pH 10–11.

Table 4. Summary of literature on heat treatment experiments using soymilk.

No	Heat treatment	Comments	References
1	Ohmic heating	The oil bodies were released when the soymilk was heated to 65°C or higher. This is the first step of the transformation. 7S and 11S proteins will escape from the subunits at 75°C and 80°C respectively. The subunits were surrounded by protein particles of 100 nm in size and the transformation was completed.	^[Citation99]
2	200/300MPa,55/65/75°C	UHPH at 300 MPa and an inlet temperature of 75°C enabled commercial sterile soymilk production. Compared to conventional heat treatment methods, UHPH significantly reduced the particle size of sample, resulting in a higher physical stability. Although the UHT-treated product had a lower trypsin inhibitor activity, the UHPH treatment produced a lower hydrogen peroxide index than heat-treated soymilk.	^[Citation100]
3	95°C, 10 min 8% SPI, pH 6.9	Increased in dispersion, hydrophobicity, WHC and SH-groups.	^[Citation101]
4	60–90°C	Heat had little or no effect on the nutritional value of soybeans.	^[Citation102]
5	130°C, 24 h/90-100°C	Even at neutral pH, nutritional value was compromised, with lysine and tryptophan lost during heating.	^[Citation102]
6	75/95°C, 15 min	Heating induced aggregation of oil droplets, as evidenced by an increase in particle size and bulk viscosity of emulsion, the effect being more pronounced after heating at 95°C. Soy protein isolate contained two main proteins: β-conglycinin and glycinin.	^[Citation103]
7	50/55/60/65/70/75/80/85/90/95°C, 5 min	When heated from 65°C to 75°C, some of the lipids and almost α and α′ subunits of ‘β-conglycinin’ in the particulate fraction were released and transferred to soluble fraction. When heated at 75°C, the lipids in the soluble and particulate fractions were released and transferred to the floating fraction. At 90°C, almost all lipids (neutral lipids) were transferred to the floating fraction.	^[Citation104]
8	60/65/70/75/80/85/90/95°C, 10 min	LOX and β-amylase in whey soy protein (WSP) interacted with 7S during denaturation at 65–75°C and were involved in protein particle formation. At 65–75°C, approximately 77–85% of protein particles are formed. Protein particles formed at 65–75°C account for 77–85% of all particles. The lectins found in WSP bind to protein particles at 85°C, resulting in a slight increase in protein particles.	^[Citation105]
9	67.8/70/76.5/80.5/84.5/90.2°C	11S globulins were more heat stable than 7S globulins, and the combination of pH 10–11 and heat treatment at ~65°C led to higher exposure of hydrophobic groups.	^[Citation106]
10	70°C/30 min	In the heated samples, there was an increase in hardness and chewiness due to the addition of SPI, and Raman spectroscopy analysis showed a decrease in α-helix structure and an increase in the β-sheet structure after heating.	^[Citation107]
11	70–90°C, 15/30/45/60/90 min	Thermal denaturation led to an increase in surface hydrophobicity, which decreased with aggregate formation. Compared to unheated SPI, heat treatment increased the α-helix structure content and decreased the β-sheet structure content. When the heat treatment temperature is below 90°C, the content of β-sheet structure showed a significant negative correlation with surface hydrophobicity.	^[Citation108]
12	80/90/100°C, 15/30/60/100/180 min	Heat treatment increased surface hydrophobicity induced SPI aggregation through hydrophobic interactions and disulphide bonding and formed soluble aggregates of different sizes. The fraction of large-sized aggregates (LA > 1000 kDa) gradually rose with increasing temperature (80 → 90 → 100°C), which corresponded to the decay of medium-sized aggregates (MA; 670–1000 kDa) that are initially abundant at 80°C.	^[Citation109]
13	50/60/70/80°C, 1/5/30 min; 90/100°C, 115/20-90 min	Heat treatment above 85°C produced a gradual decrease in concentration of AB-11S subunit and the two protein species at 20 and 29 kDa, and a gradual increase in concentration of A and B polypeptides of glycine, thus aggregation of AB glycine subunit of soy protein isolate required a heat treatment >85°C.	^[Citation110]
14	70/80/90/100°C	For one-step heating, heating soymilk produced high amount of sediment at 70 and 80°C and much less sediment at 90°C or higher, suggesting that soymilk dispersion stability was related to the heating temperature.	^[Citation111]
15	70/80/90/100°C	In the case of two-step heating (combination of 115°C and lower temperatures), the first step of heating at 115°C and the second step of heating soymilk at 70 or 80°C led to increased sedimentation.	^[Citation111]
16	95°C,5 min	The denaturation temperature of glycine (92°C) was approximately 20°C higher than that of β-conglycinin (71°C).	^[Citation112]
17	75°C,5 min, continue heating to 95°C for 5 min	Compared to single-step heating (95°C, 5 min), selective thermal denaturation (STD) affected the aggregation pathway of soy protein during denaturation, increasing the viscosity of soymilk by approximately 150%.	^[Citation113]
18	50/60/70/80/90°C, 10 min	Denaturation of 7S globulin occurred when blanching temperatures are 60–80°C.	^[Citation113]
19	65/80°C	The aqueous solution of soy protein isolates thickened and gels at 65°C at concentrations above 7%. In an open shallow dish, the surface of a 5% aqueous solution of soy protein isolate was heated at 80°C to produce films of 11S and 7S soy protein fractions.	^[Citation114]

Heat treatments play a pivotal role in the overall processing of soy protein products, exerting a significant influence on their functional attributes, including solubility, emulsification, and stability. The transformative effects of heat treatment on soy proteins are particularly noticeable in alterations to their structural characteristics, with a change in denaturation temperatures. These temperature changes are critical as they signify the points at which the protein undergoes structural modifications, impacting its behavior and functionality. A deeper understanding of the outcomes of heat treatment on soy proteins is essential for optimizing their utilization across diverse food processing techniques. For instance, the knowledge of denaturation temperatures allows food technologists to fine-tune the heat treatment conditions to achieve specific desired effects in the soy protein products. Whether aiming for improved gelation properties, solubility for food applications, enhanced emulsification for processed food products, or increased stability for a long-shelf-life products, tailoring the heat treatment process becomes a strategic tool.

Gelation of soymilk proteins

As with other globular proteins, denaturation is a prerequisite for soy protein gels, and after denaturation, hydrophobic groups buried within the natural state are exposed.^[88] Soy protein gelling is complex and divided into three parts: denaturation unfolding, aggregation and network fouling.^[12] Natural soy protein molecules have a spherical structure, which results in electrostatic repulsion from surface electrostatic charges, as well as sufficient water and the formation of a water film that ensures their dispersion and distribution in solution. Consequently, when the protein solution is heat-treated with a certain intensity, the covalent or secondary bonds between the protein molecules are broken, the subunits gradually de-fold, the spatial conformation of the protein is transformed and the hydrogen bonds between the peptide chains and other forces are broken resulting in a change from a coiled state to an extended state, where the functional groups in the protein are exposed. In parallel, as a result of the rupture of the aqueous membrane, different protein molecules approach one another and interact with one another, causing protein aggregates to form. Unlike the pure 11S fraction, soybean isolates do not precipitate upon heating due to aggregation, which is mainly due to the interaction between 7S and 11S, resulting in a structure that stabilises 11S or makes the dissociated basic subunit fusible.^[115] The formation of complexes between the 11S basic subunit and the dissociated 7S subunit has been demonstrated,^[116] and it has also been shown that heating can lead to dissociation of the two components to form soluble aggregates with a relative molecular weight greater than 1000 kDa.^[117] Aggregates are intermediate products in the protein gelling process and their shape, size and solubility can affect the physicochemical properties of the gel.^[118] In experiments, when the pH decreases, the gel takes longer to form and the aggregation time increases. By the time the temperature reaches its denaturation temperature, the aggregates formed will expand further, resulting in a rough gel structure.

When the soy protein content was greater than or equal to 8% and the protein solution was heated above the denaturation temperature and maintained for a period of time before cooling, the protein molecules will then be cross-linked by forces such as hydrophobic interactions and two sulphur bonds to form a thermopolymer and gel network structure, i.e. soy protein thermally induced gels.^[119–121] When the soy protein content is below the critical protein concentration of the gel, the addition of a coagulant is required to further facilitate gel formation.^[98] Current studies include salt coagulation gels by adding calcium sulphate and magnesium chloride, acid-induced gels by adding glucono-δ-lactone (GDL), and enzyme-induced gels by adding glutamine transaminase (TGase). Acid-induced gels and salt-induced gels are currently the most studied.

Chloride is used as a coagulant in a process known as salt brine induction, which is also the traditional method of making tofu in China.^[120] The disadvantage of this method is that the coagulation rate is too fast, resulting in a mesh-like gel structure that is not always satisfactory. Too rapid addition of salt brine or high temperature treated protein solutions can lead to rapid formation of protein flocculation in solution, which will cause in a dense and homogeneous gel network structure not being formed and therefore resulting lower WHC.^[122] Low solubility of sulphate coagulants, the most widely used coagulants worldwide, allows for a slower gel production rate, resulting in tight, dense gels with a high-water holding capacity. There are several explanations for salt-induced gelation’s mechanism. One explanation suggests that the addition of salt coagulant lowers the pH of the protein solution, causing it to deviate towards the isoelectric point. Concurrently, the positively charged alkali metal ions form when the salt coagulant dissolves shield some of the negative charges on the surface of soy protein, reducing electrostatic repulsion between protein molecules. In the presence of a certain concentration of salt ions in a solution, the repulsive forces between soy protein molecules and gravitational forces such as hydrophobic and hydrogen bonds are balanced, and the soy proteins can then bond together to form an ordered network.^[123] Another explanation is that the divalent cations in the salt coagulant bind to the carboxyl and imidazole groups of the soy protein molecules, thus linking the different protein molecules by bridging them together to form protein-metal ion bridges, further forming protein gels.^[124] Furthermore, salt precipitation caused by protein dehydration can also induce protein gelation.^[125]

GDL is one of the most used acid coagulants, as it induces gelation differently from salt and brine gels as it dissolves in water and hydrolyzes into gluconic acid, which gradually produces H+, neutralizing the negative charge on protein molecules and reducing electrostatic repulsion between molecules. Acid coagulants include natural coagulants such as lactic acid, acetic acid, and lemon juice. Whenever excessive amounts of GDL are added, the excess H+ causes the protein molecules to change their charge to positive, leading to a renewed electrostatic repulsion between the molecules due to the positive charge, resulting in a reduction in gel hardness and a loosening of the gel structure as a result. Inulin and honey are a novel and effective additive in acid-induced gel studies using GDL. Inulin enhanced the viscoelasticity of GDL-induced soy protein gels.^[126] When combined with heat treatment, the addition of 2% inulin decreased the soy protein gelation temperature by 2.8°C and improved the rheological parameters of tofu gel. In addition, it improved the hardness and fracture force of silky tofu formed. Gluconic acid concentration and total sugar content of honey promoted the gelation of soymilk, but soy protein aggregate locations differed depending on the species of honey.^[127]

In the literature, soymilk exhibits a high propensity to gel, forming gels under a wide range of conditions and capable of producing gels with enhanced mechanical strength or microstructure (Tables 5 and 6). Studies have shown that the amount of soy protein in the system significantly influences the gel properties.^[161] Gelation can occur with protein concentrations as low as 8%, and gels are obtained after heating at 60°C. However, this process requires a longer processing time, and the resulting gel has a fragile texture.^[101] As the protein concentration increases, the temperature at which maximum gelation is reached also increases. Simultaneously, heat treatment affects gel formation. Tofu gels prepared using the two-step heating method exhibit finer characteristics than those prepared using the one-step heating method. This results in finer gels with a more uniform lattice structure, higher elasticity, and a lower dehydration rate.^[112] In another study, the ratio of 7S to 11S in soy proteins influenced acid-induced gel properties. Gels formed from 11S globulins were stronger and more elastic than those formed from 7S globulins, primarily due to their different thermal denaturation sensitivities.^[161] It was found that both 7S and 11S effectively and almost equally increased gel strength with 20% incorporation, improving water-holding capacity (WHC). The particle size (D^[3,4]) of the gels increased with the addition of a high percentage of 7S or 11S. However, 20% and 40% of 11S significantly reduced gelation time during acidification, with 40% of 11S resulting in a coarser microstructure than the control. In contrast, 7S had no significant effect on gelation time, and gels containing 40% 7S were well-organized. Even a low concentration of 10% 7S or 11S improved the lubricity of the gels, while higher concentrations (40%) had the opposite effect.

Table 5. Overview of research findings on gelation experiments conducted with soymilk.

No.	Gelation	Comments	References
1	GDL	During two-step heating, selective thermal denaturation (STD) increased the viscosity of soymilk by 150%, apparent Young’s modulus of tofu by 20%, and reduced the hysteresis of tofu by 10%. The tofu gel network prepared by the two-step heating method was finer and more homogeneous than that prepared by the one-step heating method. Selective thermal denaturation increased soymilk viscosity and tofu elasticity and reduced tofu dehydration.	^[Citation113]
2	GDL	GDL-induced gels formed from soy protein isolate (SPI) dispersions heated at pH 6.9 had a significantly higher storage modulus, gel hardness and water holding capacity (WHC) than GDL-induced gels formed from unheated SPI. The former gels have a coarser structure and a larger pore size than the latter.	^[Citation101]
3	GDL	4% soy protein isolate (SPI) did not form gels even after prolonged heating, while 6% of SPI produced weak gels only after a second heat treatment. The critical protein concentration for thermally induced gel formation is 6–8%. The unheated 8% dispersion was unable to gel and required further heat treatment to form a gel. Nevertheless, the G”/G’ values of heated SPI were still above 0.1, indicating that these gels still fall into the category of weak gels or colloidal dispersions.	^[Citation101]
4	GDL+CaCl2	Thermal pretreatment after emulsification increased the strength of GDL and CaCl2-induced emulsion gels. In contrast, in the case of MTGase, thermal pretreatment before and/or after emulsification significantly inhibited the formation of the gel network. CaCl2-induced gels consisted mainly of granular, protein-coated oil droplets.	^[Citation128]
5	GDL+Mtase	In the case of MTGase, gel formation is only observed in unheated emulsions. However, the gel strength of the MTGase-induced emulsion gels is significantly higher than the gel strength of the GDL and CaCl2 gels. The WHC of MTGase-induced emulsion gels is much lower than other gels. Furthermore, the GDL or MTGase-induced emulsion gels exhibit a filamentous gel network.	^[Citation128]
6	GDL	The breaking stress of GDL-induced tofu gel made from low-β-conglycinin soymilk was nearly four times higher than that made from the low-glycinin soymilk. The tofu breaking stress and the soymilk storage modulus both increased with an increase in 11S/7S ratio. Storage modulus correlates well with tofu gel fracture stress.	^[Citation129]
7	GDL+MgCL2	The soymilk of low-beta-conglycinin soybeans solidified immediately after the addition of 0.3% magnesium chloride.	^[Citation129]
8	GDL/YO-MIXTM 511 LYO 375 DCU (0.00349%)	Acidification by lactic acid bacteria results in higher gel pH and lower gelation time compared to GDL-induced gels.	^[Citation130]
9	GDL	Compared to pure whey protein isolate (WPI) gels, mixed gels and pure soy protein isolate (SPI) gels showed a coarser, more porous microstructure with lower water retention capacity. Replacing WPI with SPI changed the constituent floc state, where flocs became larger and had a coarser, more elongated and more branched structure. The links between flocs were weaken.	^[Citation131]
10	GDL	Replacing WPI with SPI reduced the stiffness (lower G′) and stretchability (lower γc) of acid-induced gels in linear viscoelastic (LVE) systems, yet these hybrid gels show a relatively more elastic response and a more gradual transition to plastic behavior in non-linear viscoelastic (NLVE) systems. Replacing WPI with SPI reduced the proportion of soluble aggregates formed during the preheating step and slowed down GDL acidification by delaying the gelling point.	^[Citation131]
11	GDL	The presence of soy protein causes the milk to aggregate at higher pH values. The presence of soy shows a higher G’ value than skim milk gels, showing a larger pore size in gels containing large amounts of soy protein. The microstructure and viscoelastic properties of acidic protein gels were modified by changing the ratio of skimmed milk powder to soy protein concentrate.	^[Citation132]
12	(1) nigari-type salts (magnesium chloride, calcium chloride); (2) sulfate-type coagulants (calcium sulfate, magnesium sulfate); (3) glucono-δ-lactone (GDL); (4) acidic coagulants (acetic acid, lactic acid)	All eight coagulants tested can coagulate soymilk at the selected concentrations. The results indicated that the concentration of soymilk and the type of coagulant had a significant effect on the texture of the tofu gel. Tofu made with CaCl2 and MgCl2 is coarse, granular, and hard. In contrast, tofu made with calcium sulphate and GDL is smooth, soft, and homogeneous. Tofu produced by acidification with organic acids is less rigid and more viscous.	^[Citation133]
13	Lactic acid bacteria+MTGase	The average particle size of aggregates formed with bovine and soy-bovine milk mixture (SCMM) system was increased compared to LAB treatment. The combination of lactic acid fermentation and MTGase treatment promoted the degree of β-LG cross-linking and further reduced its potential allergenicity.	^[Citation134]
14	GDL	GDL induced a faster pH drop, resulting in earlier gelation as opposed to cultured gels. Such a difference in acidification rate, however, did not appear to affect gel strength or particle size if the pH at the end is similar. The rheological properties and particle size of GDL-induced soy gels are comparable to those of fermentated gels made with cultures that produce less exopolysaccharide (EPS) or no EPS at all.	^[Citation135]
15	Lactic acid bacteria (L. casei) /GDL/Citric acid	Acidification rate: citric acid > GDL > L. casei. The gelation point of citric acid-induced gels (CA gels) was not detected because the acidification rate was too fast. LC gels had a strong matrix, an excellent texture and WHC under external forces and were not prone to deformation. The microstructure of GDL gels were considered the densest. LC gels involved proteolytic and exopolysaccharide (EPS) production capacities.	^[Citation89]
16	Lactic acid bacteria (L. casei) /GDL/Citric acid	The release of hydrogen ions from L. casei was slower compared to GDL or citric acid. The rheological results showed that LC gels had significantly smaller G’ than GDL and CA gels. LC gels had the highest hydrolysis and peptide content after in vitro intestinal digestion. Acidification rate was a ‘critical step’ in acid-induced gels, and acid influenced the degree of rearrangement aggregated particles during early stage of gelation.	^[Citation89]
17	GDL	Gelation of soymilk occured at around pH 5.8. Neither the rate of acidification nor the protein concentration affected the pH of gelation. Gel hardness was influenced by protein concentration. Short-range interactions played a major role in formation of soymilk curd protein networks.	^[Citation136]
18	GDL	Gelation behaviour was similar for 4% and 7% protein soymilk, indicating that protein concentration has no effect on soymilk gelation properties, but protein concentration had an effect on the degree of gel structure formation, as 7% protein soymilk produced stiffer gels. Gelation of soymilk was dependent on gelation temperature, with an earlier gelation and much stiffer bonds at 7°C compared to 30°C.	^[Citation136]
19	GDL	The addition of anhydrous milk fat to soy protein produced stronger, firmer gels with apparent viscosity showing shear thinning behaviour. There was no significant difference in size of gel particles with the addition of AMF, palm stearin and soya oil. From the microscopic images, the soy protein with added soy oil showed smaller fat globules and a more compact protein network. The presence of fat as a filler affected the firmness and gelling properties of soybean isolate protein (SPI) gels. The addition of soybean oil resulted in softer SPI gels, although the particle size distribution and flow behavior did not differ significantly among AMF and soya oil.	^[Citation137]
20	GDL	Heat treatment (HT) enhanced the mechanical properties of soybean isolate protein (SPI) gels and increased the linear viscoelastic area of SPI gels. HT significantly improved SPI gel structures’ structure and strain resistance. the particle size of SPI gels was greatly reduced after HT, implying that HT inhibited protein aggregation. Overall, HT significantly altered the gel formation parameters and linear and non-linear rheological properties of acid-induced SPI gels prepared by high-pressure homogenisation, resulting in gels with better mechanical properties, extending the linear viscoelastic area of the gels, and improving gel structure and strain resistance.	^[Citation138]
21	Inulin (raftilineσ HP-gel) + GDL	During the gelation process, the rheological changes of soymilk depended on the decrease in pH (hydrolysis of GDL) and the specific temperature at which it was heated. As a result of adding inulin to tofu gel, the gelation temperature was reduced by 2.8°C and its rheological parameters were improved, as well as the hardness and fracture strength of silky tofu (texture profile analysis).	^[Citation126]
22	GDL	The ratio of glycinin/β-conglycinin had a significant effect on gel structure, with an increase in glycinin leading to an increase in gel stiffness. The type of glycinin subunits influenced soymilk aggregation. The lack of α′ subunit of β-conglycinin had a negative effect on gel hardness.	^[Citation139]
23	Citric acid + Salt	The addition of 0.10 g/100 mL KCl, 0.05 g/100 mL CaCl2 and 0.15 g/100 mL CaSO4 produced more elastic tofu with a higher water holding capacity, which maximized mechanical properties of citric acid-induced tofu. The water holding capacity of tofu induced by the presence of salts, although gel was firmer and less elastic. The maximum elasticity of citric acid-induced tofu was obtained with 0.15 g/100 ml of CaSO4.	^[Citation140]
24	Lactic acid bacteria (L. plantarum 70810) + Calcium salt	The coagulation rate among the coagulants followed the anion series: formate > acetate > chloride > lactate. After 4.5 hours of coagulation, the textural properties were related to the final viscosity of soymilk mixture, whereas the pH was not related to the coagulant behaviour. The counter ion of calcium source salt influenced the coagulation process of soymilk proteins.	^[Citation141]
25	GDL + TGase	TGase pretreatment altered physicochemical properties of GDL-consolidated tofu. In particular, short pretreatment of soymilk with TGase (0.5, 1 and 2 hours) improves gel strength, WHC and overall acceptability, unlike extensive pre-crosslinking with TGase for 3 hours, which led to a significant reduction in gel firmness and sensory scores.	^[Citation142]
26	Citric acid, Malic acid, Tartaric acid	In tofu made with 0.14 g/100 ml of organic acid, storage modulus, hardness, water holding capacity, and unfrozen water content were optimal, but degraded when more than 0.16 g/100 ml used. The tofu product showed higher water holding capacity and non-freezable water content, as well as a more compact gel microstructure. It was noteworthy that tofu cooked with citric or l-(-)-malic acid was of better quality than tofu made with tartaric acid. Tofu prepared with 0.12 and 0.16 g/100 ml citric acid was comparable in hardness, although the former was more elastic. Tartaric acid tofu had relatively poor physical properties and less stable chemical interactions. Additionally, tofu produced with 0.18 g/100 ml of organic acid was of lower quality, as indicated by its modulus of elasticity, hardness, water holding capacity, and unfrozen water content. Taken together, the quality of tofu produced with organic acids was comparable to tofu prepared with GDL	^[Citation143]
27	Citric acid + Nano Fish Bone (NFB)	As the volume ratio increased from 0% to 3%, the yield, moisture, and texture values of the tofu gels continued to increase. In contrast, the dehydration rate and whiteness decreased. Adding NFB to acid-induced tofu gels significantly improved yield, hysteresis, and texture, and calcium released from NFB improved gelation of the soy protein in two different ways. The tofu gel network became more ordered and denser	^[Citation144]
28	GDL + Honey	In some samples, aggregates formed in upper layer at higher honey concentrations, while in others, aggregates precipitated at moderate honey concentrations. Honey’s gluconic acid concentration and total sugar content triggered coagulation of soymilk, and addition of honey promoted protein aggregation in soymilk.	^[Citation127]
29	GDL + BSG Protein isolates	Gelation lags and maximum gelation rate values decreased in proportion to heat treatment intensity, resulting in better aggregation of protein protofibrils into agglomerates, which formed a percolation network as a result of supramolecular interactions. Heat treatment of BSG-PI dispersions favored separation of phases between glutenin and alkali-soluble cell wall dietary fibers. The cessation of separation of the segregated phases during acidification led to development of complex acid gels with enhanced elasticity, stiffness, and deformation elasticity.	^[Citation145]
30	GDL	Both 7S and 11S formed a continuous gel network when some of the protein in soymilk powder was replaced with 7S or 11S. The secondary component acted as a filler as the proportion of one component increases. With increasing gel viscosity and a sharp increase in particle size, 10% and 20% of 7S or 11S improved the gel’s lubricity. 7S/11S had different effects on soy pulp gel particle characteristics, but similar effects on overall gel strength. 20% of 7S or 11S had a significant improvement in gel properties, in terms of rheology, texture, WHC, and tribology.	^[Citation146]
31	GDL + Inulin	Inulin (long-chain fructose) enhanced the viscoelastic properties of GDL-induced condensed soy protein gels, where 11S globulin played a leading role in gel structure formation.	^[Citation126]
32	GDL	7S-GDL gels in the final stage of gelation showed almost the same storage and loss moduli as 11S gels when protein and GDL concentrations were the same. The stress at break and strain at break for fully gelatinised 7S gels were smaller than for 11S gels. As with the 11S-GDL system, the gelation rate increased, the gelation time decreased and the GDL concentration increased at a constant 7S concentration. As the concentration of 7S increased, the gelation time for a system consisting of a fixed ratio of 10:1 of 7S:GDL decreased. In the presence of GDL, the minimum concentration of 7S protein lower than that of 11S-GDL system.	^[Citation147]

Table 6. An overview of the heat treatment/gelation of soymilk-bovine milk blend system.

No.	Comments	References
1	In a blended system, soy and dairy proteins gel together simultaneously, resulting in a better gel structure. The blend with a total protein content of 4.5% produces a lower gel hardness than 4% skimmed milk or 5% soymilk alone.	^[Citation148]
2	Compared to gels formed from simultaneous aggregation of milk proteins and soy proteins, gels formed by milk protein aggregation before soy protein aggregation were more slippery and fatty. In comparison to gels with casein micelles followed by soy proteins, complex gels with simultaneous aggregation of casein micelles and soy proteins had a more uniform structure.	^[Citation148]
3	The homogenization of soymilk with cream, resulted in a coarser gel structure. Even when soy protein is present during homogenization, milk proteins had a high oral coverage and thickness when homogenized with cream. Nevertheless, when the sample was not homogenized or if the cream was homogenized with only soymilk, the resulting gel was watery and did not provide adequate oral coverage. A mixed protein gel containing milk proteins aggregated before soy proteins, displaying more pronounced fat-related properties, such as slip and fatness, as compared to a gel containing soy and milk proteins aggregated simultaneously.	^[Citation148]
4	A pH of 6.6 resulted in the maximum effect of soy protein on milk coagulation. Both heat-denatured and native soy proteins increased rennet’s coagulation time. It was only heat denatured soy protein that affected the final firmness of the curd.	^[Citation149]
5	To form gels from casein micelles mixed with phytoglobulins, the resulting networks tended to be weaker and nonhomogeneous, i.e. more porous, than gels consisting of only one component, especially when the casein micelles alone had a sufficient concentration and pH (i.e. pH < 6.0) to sustain the gel formation. At a pH of 7, thermally induced gels made from micellar casein and soybean isolate (1/1, w/w) exhibit lower yield stress and lower elastic and viscous moduli than gels made solely from soybean protein.	^[Citation150]
6	Gels made from milk and pea proteins (1/1, w/w) were as strong as pea protein gels and stronger than skim milk powder gels. Preheated mixtures of casein micelles, whey proteins, and soy proteins gel at higher pH in acid-induced gels, had stronger networks than when only casein micelles and soy proteins were present. A mixture of unheated globular proteins and casein micelles forms coarse, soft gels with little water retention capacity, although soy or pea proteins accelerated gelation.	^[Citation150]
7	By replacing 50 wt.% of whey protein with a pea protein fraction, gel strengths similar to pure whey protein gels were obtained. The albumin fraction of pea protein or a less pure pea protein concentrate provided greater gel strengths. By fractionating pea proteins at high temperatures or pH values far from neutral, protein solubility was reduced and preaggregation was uncontrolled, affecting gelling.	^[Citation149]
8	Milk gels exhibited macropores with interconnected protein chains, while soy gels exhibited dense protein aggregates and mixed soymilk gels exhibited aggregated protein networks.	^[Citation151]
9	With 4.5% total protein, the gel hardness was lower than with 4% protein skim milk alone or 5% protein soymilk alone. Blended systems had different microstructures from soy or milk alone, suggesting that they produced different gel structures.	^[Citation151]
10	The appearance of the soymilk-skim milk blend gel is intermediate between that of milk on its own or soymilk individually, showing a network of aggregated protein chains. These structures were more open than soymilk alone (a), but more compact than skim milk itself (b).	^[Citation151]
11	The soymilk inhibited skim milk aggregation, resulting in a decrease in storage modulus (G’), an increase in water content and curd yield, and a coarsened network. A decrease in G’ was offset by a more uniform curd distribution in soymilk modified with enzymes with molecular weights below 20 kDa. Moisture content and curd yield increased with soymilk and enzyme-modified soymilk.	^[Citation152]
12	Soymilk and enzyme-modified soymilk decreased the storage modulus of mixed gels and increased moisture content and curd yield compared to skim milk. With the addition of soymilk and enzyme-modified soymilk, the inhibition of gel formation was stronger and moisture content and curd yield are higher. Soymilk modified with enzymes contains more small molecules, which reduce the hindering effects of rennet-induced gels, increased the storage modulus of gels, and produced a more uniform curd.	^[Citation152]
13	Soymilk and TG had a synergistic effect and inhibited rennet gelation to some extent. With the increase of soymilk and TG, the elasticity index and storage modulus decreased, gelation time delayed, and curd yield and water content increased. Excess soymilk and TG did not form curd. Soymilk and TG had significant effects on the curd microstructure. Soymilk inhibits casein micelles aggregation and contributed to a coarser heterogeneous network. TG limited protein reorganization, resulting in a more homogeneous network with small pores. The use of soymilk and TG impaired both rennet-induced gelation and rennet hysteresis, resulting in increased high-moisture curd yield.Transglutaminase (TG)	^[Citation153]
14	The gels with less GDL did not form networks. The particle size distribution of the soymilk-buttermilk gels was about 3.5 μm. Sensory evaluation results were consistent with the gels’ textural and microstructure. Modulation of GDL for casein and soy protein aggregation allowed soymilk and milk to obtain novel milk gel products. These gels maintained the original soy flavor and added the milk flavor.	^[Citation154]
15	The increase in pea concentration led to higher acidity, higher hysteresis and lower firmness. From an organoleptic point of view, the starter cultures showed positive or negative effects in the case of pea protein up to 30 g/100 g total protein. Two groups with 0 g or 10 g/100 g total protein of pea protein seem to be the most similar to conventional dairy products. The third group consisted of products fermented with two starters with negative characteristics such as astringency and bitterness. The last two groups included members made from 10 g, 20 g, 30 g and 40 g pea protein/100 g total protein, with one group showing positive organoleptic characteristics.	^[Citation155]
16	When the gels were induced with GDL, the mixture showed gel sites well above the isoelectric point of milk proteins. This suggested that soy protein played a major role in network formation. The network of acidified gels had more branching and compact structure as well as denser clusters compared to acid-rennet hybrid gels.	^[Citation156]
17	When casein micelles were replaced by soy or pea proteins, the critical temperature for casein micelle gelation increased due to calcium chelation of these plant proteins. In heated mixture, casein micelles and plant proteins do not aggregate together, and each type of protein formed an independent network. For a given total protein content, mixed gels with vegetable proteins were less stiff than gels with casein micelles alone, and vice versa. There was the least stiff gel when the mixture contains about 40% soy protein and 70% pea protein.	^[Citation157]
18	MTGase (3.0 U/g protein) treatment of soymilk-bovine milk mixture (SCMM) at 43°C did not induce gelation even after 4 h of incubation. Therefore, LAB (0.025 UC/L) combined with SCMM results in denser and finer gel networks with smaller pores and a higher storage modulus (G’) than SCMM alone. In the presence of LAB and MTGase, higher polymerization rates of soymilk and milk proteins were obtained than treatment with enzymes alone.	^[Citation158]
19	In the soy-lacto-casein system, soy protein influenced gelation, although to a lesser degree than whey protein, which dominated network formation. At a critical ratio of soy/whey protein, soy protein was successfully added to the mixture without significantly altering the casein gel structure.	^[Citation159]
20	Enzyme-induced gels had higher fracture strain, a property associated with coarse aggregates. In this gel type, mixed gels show a lower storage modulus than pure pea or pure milk gels. In the acid-induced gels, the pure pea gels are much stiffer than the pure milk or mixed gels, probably because of their lower final pH. All of the studied pea gels benefit mechanically from fat addition, especially the mixed gels obtained from acid and enzyme gels.	^[Citation160]
21	All soy/milk mixtures had a network structure with large pores sizes due to soy protein. There was no difference in the pH of aggregation between heated and unheated mixtures. Because of the instability of soy protein at pH 6.0 and the presence of calcium ions, milk/soy gels have a higher aggregation pH than milk.	^[Citation132]

The process of soy protein gelation is a complex interplay of denaturation, unfolding, aggregation, and network formation. In conclusion, soy protein gelation is a versatile process influenced by various factors, offering opportunities to tailor gel properties for diverse applications. These factors can include the specific conditions of heat treatment, the presence of other ingredients, and the pH of the system. Understanding these factors is crucial for optimizing soy protein gels in the food industry and exploring innovative approaches to enhance their functional attributes. This knowledge also opens doors to innovative approaches, allowing the enhancement of soy protein gel attributes for specific functional roles in food products. Whether aiming for a firmer texture, improved water-holding capacity, or enhanced stability, a better understanding of soy protein gelation ensures food technologists craft products that align with consumer preferences and industry demands.

Blends of camel milk and soymilk proteins

Combining camel and soy milk can create a unique blend of flavors and nutritional profiles. Blending camel milk and soy milk is a straightforward process that can result in a new product with distinctive flavor profiles. This blending approach is easy and quite flexible to perform. However, the use of camel milk in blends is scarcely studied, while many studies have focused on the use of bovine milk.^{[132,149,150,152–160,162,163]} Additionally, there is no existing research that directly combines camel milk proteins and soymilk proteins. Camel milk gelation is characterized by a watery consistency and fragile structure. As texture is considered a crucial factor affecting consumer acceptability, the addition of other types of plant-based counterparts to deliver a specific type of yogurt was explored.^[164] Very few studies have been conducted on the combination of camel milk and soymilk, with no studies focusing on the proteins themselves.

It was found that the microstructure of the soymilk-bovine milk blend system is different, exhibiting an intermediate structure between the bovine milk and soymilk gels alone. This system shows a compact network structure of aggregated chains of proteins, in contrast with the two milk gels alone.^[130] The distribution of soymilk proteins within the gel and whey fraction is distinct. For instance, it is found that most of the β-conglycinin is lost in the whey, while glycinin is retained in the curd.^[149] When the pH of the system is 6.6, the thermally denatured soy protein is fibrous and adheres to the surface of the casein micelles, preventing direct contact between the casein micelles. As a result of this behavior, the rate of aggregation will be delayed, and the curd will be less firm, as the numbers and strength of the ties between casein micelles will be reduced. In conclusion, soymilk inhibits the aggregation of skim milk and leads to a decrease in storage modulus, significantly increasing water content and curd yield, and producing a rough network structure.^[152] Comparatively, camel milk proteins have larger casein micelles than milk proteins, resulting in a larger surface area. There is some evidence that heat-denatured soy protein maximizes adhesion to the surface of the casein micelles when the amount of soy protein is sufficiently high, further impeding direct contact between the casein micelles and resulting in a decrease in curd firmness. It is speculated that when soy protein is insufficient, the opposite outcome may occur.

Due to calcium chelation of plant proteins, when the casein micelles in a system are replaced with soy proteins, the critical temperature for gelation of casein micelles increases. During the heat treatment, casein micelles and soy proteins do not aggregate together in the blend system; instead, each forms an independent network structure. Consequently, as the casein micelles are replaced by soy protein in the system, the stiffness of the mixed gels is lower than the stiffness of the bovine milk system gels and the soymilk system gels individually.^[157] It is found that gel stiffness is least when the mixed system contains 40% soy protein. As shown in the sensory analysis of the gels prepared using the soymilk-bovine milk blend system, the mixed gels retain the original soy flavor of the soy product while incorporating milk flavor. In this way, it is hypothesized that the Camel milk-soymilk blend system will form a firmer curd than pure camel milk, but the network structure will be coarser with greater soy content. During the formation of the network, camel milk proteins and soy proteins form an independent network with each other.

The changes in post-acidification, viable cell counts of Lactobacillus spp, and Streptococcus thermophilus, total phenolic content, and antioxidant activity during 21 days of storage were evaluated. pH reduction was not affected, although sufficient pH reduction was obtained on the 7th day of storage with the addition of soybean. There was a 30% increase in viable cell counts of Lactobacillus spp, while no change was observed in viable cell counts of Strepto. There was a two-fold higher total phenolic content on day 0 and day 7, along with increased antioxidant activity. Another very recent study by Ali et al. (2023)^[164] investigated the addition of soy extract on total phenolic content, antioxidant capacity, acidity, degree of hydrolysis, and rheological properties within 21 days of storage at 4°C, with the addition of up to 6 g of soy protein to 100 g of the solution. In this study, two different starter cultures were investigated. The level of soy extract added played a significant role in the gelation properties after 21 days of storage. The degree of thixotropy increased, improving the texture and rheological properties of fermented camel milk. However, pH, TA, and antioxidant capacity were greatly affected by the type of culture used (Fig. 3).

Figure 3. (a) pH values, (b) Titratable Acidity, (c) Total Phenolic and (d) Degree of Hydrolysis of camel milk-soy extract yoghurt treatment^[149]; GAE = gallic acid equivalents. a–e = means with different lowercase letters at the same storage time differed significantly (P < .05). A–D = means with different uppercase letters at the same storage time differed significantly (P < .05). SL0 = camel milk only as a control; SL2 = camel milk + 2% soy protein; SL4 = camel milk + 4% soy protein; SL6 = camel milk + 6% soy protein; CH = high acidic culture; YF: low acidic culture.

Conclusion

In this paper, a comparative analysis is conducted between camel milk protein and soymilk protein regarding their composition, heat treatment, and acid gelation properties. Additionally, the study explores the characteristics of a hybrid system combining soymilk protein and bovine milk protein. The primary reasons for the suboptimal performance of camel milk in heat treatment and gelation are the absence of β-lactoglobulin and the low κ-casein content. Conversely, the denaturation of 7S and 11S globulins in soymilk proteins leads to aggregation and gelation during heat treatment. The combination of soymilk protein and bovine milk protein increases the critical temperature for casein micelles to gel, resulting in a more compact gel structure. However, the overall gel properties fall between those of soymilk gel and cow milk gel, with pH changes likely causing fragility and a rough structure with high water content.

Despite insights gained from blended soymilk protein-bovine milk protein systems, no studies have explored the heat treatment and gelation of camel milk protein-soy milk protein blends. The amount of soymilk protein in the system may impact the enhancement of camel milk protein gel structure, suggesting a potential association with soymilk protein. Thus, soymilk protein emerges as a promising avenue for future research to improve the heat treatment and gelation properties of camel milk protein. The combination of camel milk and soy milk presents an innovative blend with distinctive physicochemical properties and nutritional profiles, offering opportunities for product innovation. While limited research has been conducted on this specific blend, preliminary investigations indicate that the mixture yields a product with unique textures, flavors, and nutritional attributes. The initial results suggest that the combination of camel milk and soy milk holds potential for a product that surpasses the sum of its individual components. However, the intriguing aspect lies in the interactions between proteins in camel milk and soy milk, a phenomenon not yet fully understood by researchers. These interactions likely play a crucial role in shaping the physicochemical, functional, sensory, and nutritional characteristics of the final blend. Further studies are essential to gain deeper insights into the interactions between camel milk and soymilk proteins, unlocking the full potential of this promising blend. This involves exploring molecular-level interactions, studying the influence of processing conditions, and understanding consumer sensory preferences.

Disclosure statement

No potential conflict of interest was reported by the author(s).