Exploring the Potential of Lupin Fermentation with Exopolysaccharide-Producing Lactic Acid Bacteria to Enhance Techno-Functional Properties

Abstract

Lupin (Lupinus spp.), a legume known for its high protein content, holds great promise as a sustainable protein source to meet future global demands. Despite its nutritional benefits, including substantial dietary fibre and bioactive compounds, lupin remains underutilised in human diets due to several techno-functional and sensory limitations. This review delves into the techno-functional limitations of lupin, which include poor foaming capacity, low water and oil absorption, inadequate emulsification properties, and poor solubility. Lupin’s techno-functional limits are tied to the compact, heat-stable nature of its conglutin storage proteins and high insoluble fibre content. While research has been conducted on fermenting other legumes such as soybeans, chickpeas, peas, and lentils with Exopolysaccharide (EPS) producing bacteria, its application to lupin remains largely unexplored. Crucially, this work is one of the first reviews to exclusively link lupin’s unique protein and fibre structure with the specific polymer chemistry of bacterial EPS as a targeted modification strategy. Current research findings suggest that EPS-producing Lactic Acid Bacteria (LAB) fermentation can significantly improve the techno-functional properties of legumes, indicating strong potential for similar benefits with lupin. The analysis highlights various studies demonstrating the ability of EPS-producing LAB to improve water retention, emulsification, and overall palatability of legume-based products. Furthermore, it emphasises the need for continued research in the realm of fermentation with EPS-producing bacteria to enhance the utilisation of lupin in food applications. By addressing these challenges, fermented lupin could become a more appealing and nutritious option, contributing significantly to global food security and nutrition.

1. Introduction

Several lupin species are cultivated for human consumption, including white lupin (L. albus), also known as Egyptian lupin or tremoço; narrow-leaved lupin (L. angustifolius), also called Australian sweet lupin or blue lupin; yellow lupin (L. luteus), another commercially important Mediterranean species; and Andean lupin (L. mutabilis), also known as tarwi or chocho [1,2,3]. This genus of flowering plants in the legume family, Lupinus spp., has been recognised as a promising contributor to solving the growing global demand for sustainable protein sources. The seeds of lupin are particularly notable for their high protein content, ranging from 33% to 47% on a dry weight basis [3,4], positioning lupin as an appealing alternative to traditional protein crops like soybeans. Beyond its remarkable protein profile, lupin is also a rich source of dietary fibre, which constitutes 40% of the kernel weight in white lupin, and it also boasts a lower glycaemic index [4]. Moreover, lupins contain substantial amounts of vitamins and minerals, enhancing their reputation as a nutritious option for human consumption [4,5].

Lupin deserves a central role in future protein supply because it combines exceptionally high protein and fibre content with low fat and carbohydrates, making it a nutrient-dense, plant-based protein alternative that is well suited to growing global demand for sustainable foods [6,7,8,9]. Moreover, lupin cultivation offers important environmental advantages: it thrives in poor soils, requires relatively low water and fertiliser inputs, fixes atmospheric nitrogen in soil, improving fertility and reducing the need for synthetic fertilisers, and presents a climate-smart legume that is ideal for sustainable agriculture [4,10]. From a human-health perspective, consumption of lupin (especially whole lupin) has been associated with improved markers of cardiometabolic health, including better glycaemic control, lower blood pressure, reduced cholesterol levels, and increased satiety benefits that support its inclusion in diets aimed at preventing chronic diseases [11,12]. Given the growing urgency to diversify plant-protein sources beyond a few dominant crops (like soybean), expanding lupin utilisation represents a strategic opportunity to build a more resilient, nutritious, and environmentally balanced global food system.

Furthermore, lupin also compares favourably with other widely consumed legumes such as chickpea, lentil, common bean, fava bean, pea, and soybean, exhibiting a markedly distinct composition that further underscores its potential as a good protein source (

Table 1. Nutritional profile of some legumes; data are expressed per 100 g edible portion, dry matter basis (Adapted from FAO and USDA data bases for pulses) [13,14]. Entries in bold are based on data from Johnson, Clements [9].

Legume	Total Protein (g)	Carbohydrate (g)	Total Fat (g)	Total Dietary Fibre (g)	Ash (g)
Chickpea (Cicer arietinum)	19.9	52.4	5.5	19.0	3.2
Common bean (Phaseolus vulgaris)	23.4	45.4	1.7	25.3	4.3
Fava bean (Vicia fava)	27.2	46.5	2.3	20.8	3.1
Lentil (Lens culinaris)	27.0	49.6	1.7	19.0	3.0
Lupin (Lupinus spp.)	34.0	9.5	7.2	46.6	2.6
	39.6–42.4	7.3–11.1	7.1–8.8	37.5–40.2	2.8–3.8
Pea (Pisum sativum)	26.4	43.3	2.3	25.0	3.0
Soybean (Glycine max)	36.5	30.2	19.9	9.3	4.9

). Whereas chickpea, lentil, and pea typically contain 19.9–27 [13] g protein per 100 g, lupin provides substantially higher levels at 34.0 g/100 g or higher depending on cultivar, while also delivering a higher dietary fibre content (46.6 g/100 g) [13]. Lupin’s exceptionally low carbohydrate content (9.5 g/100 g) sets it apart even further, contrasting sharply with the much higher carbohydrate levels of chickpea (52.4 g/100 g), lentil (49.6 g/100 g), common bean (45.4 g/100 g), fava bean (46.5 g/100 g), and pea (43.3 g/100 g) [13]. Interestingly, lupin has comparable protein content with soybean while soybean has a considerably higher fat content (19.9 g/100 g) and much lower fibre (9.3 g/100 g) than lupin, reinforcing lupin’s uniquely advantageous nutritional profile [9,13].

Despite these benefits, lupins remain underutilised in human diets. This underutilisation is primarily due to various techno-functional and sensory limitations that hinder their widespread acceptance and application in food products [15].

Techno-functional properties are critical for food processing and product development. Lupin proteins exhibit poor foaming capacity and stability, which restricts their use in products that require aeration, such as bakery products and confectionery [16,17,18]. Additionally, lupin flour and protein isolates have lower water absorption capacities compared to other legumes, affecting texture and moisture retention attributes [16,19]. Furthermore, lupin proteins show poor thickening, inadequate gelation, and poor emulsification properties, which are essential for the texture and stability of various food formulations [16,20]. These limitations significantly reduce the versatility and applicability of lupin in the food industry.

Moreover, Lupin’s sensory attributes, including taste, aroma, and mouthfeel, present significant challenges [15]. The presence of bitter-tasting alkaloids, although reduced in modern sweet lupin varieties, impacts the flavour profile of lupin products [21]. In addition, lupin often exhibits a beany flavour, which can be off-putting to consumers [22]. This flavour is particularly pronounced in lupin flour and protein isolates, making it challenging to incorporate lupin into palatable food products. Furthermore, certain nutritional limitations of lupin, while less pronounced than its functional and sensory challenges, are also worth mentioning. Lupin protein, although present at high levels, lacks certain essential amino acids, particularly methionine and cysteine [4,23]. These deficiencies can affect the nutritional completeness of lupin-based diets. Additionally, lupin contains allergens and anti-nutritional factors such as protease inhibitors and phytic acid, which can reduce nutrient bioavailability [4,24]. Although these compounds are present at lower levels compared to other legumes, they still affect lupin’s nutritional value [7].

Microbial fermentation is a potential method to overcome these limitations. Specifically, fermentation with lactic acid bacteria (LAB) can improve sensory attributes, enhance protein digestibility, and reduce anti-nutritional factors and allergens [25,26] and can be potentially used to improve the palatability and nutritional quality of lupins, as illustrated in Figure 1. Fermentation also has the potential to improve techno-functional attributes, thereby enhancing the functional application of lupins in food formulations [27,28].

Fermentation using Exopolysaccharide (EPS) producing bacteria can improve the water absorption, gelation, properties, texture, viscosity, and stability of lupin-based products [26,31,32].

Fermentation using EPS-producing Lactic Acid Bacteria (LAB) is distinctive because, unlike conventional fermentation primarily driven by acid-induced protein aggregation and denaturation, these microorganisms redirect metabolic energy toward synthesising and secreting complex exopolysaccharide polymers that can potentially improve the techno-functional properties of the substrate [26,33]. The polymers (EPSs) act as natural hydrocolloids [34], forming a highly viscous, water-binding network that chemically and physically interacts with the food matrix [35], providing textural improvements (e.g., enhanced water retention and creaminess) [36,37] as a distinct, additive modification to the changes caused by acid and proteolysis.

This review aims to provide a comprehensive analysis of the advantages and techno-functional challenges associated with lupin as a food ingredient. Additionally, it will examine the impact of fermentation with EPS-producing LAB on the techno-functional properties of legumes by summarising existing research in the field. Given the limited research on the fermentation of lupin, particularly with EPS-producing LAB, the review will draw on studies involving other legumes to suggest the potential applicability to lupin. It will also propose future research directions to optimise the use of lupin in food products. Through this analysis, the review seeks to contribute to the ongoing efforts to promote and increase the utilisation of sustainable and nutritious food sources that can meet the dietary needs of a growing global population.

2. Nutritional Profile and Benefits of Lupin

The nutritional composition of edible Lupinus species shows notable variation among species. L. luteus and L. mutabilis contain the highest protein contents (41.36 and 44.74 g/100 g DM, respectively), while L. mutabilis stands out for its higher crude fat (14.07 g/100 g DM) [38]. This section provides detailed nutritional composition of lupin seeds, compares them with other legume protein sources, outlines their health benefits, and highlights their environmental and agricultural advantages.

2.1. Nutritional Composition

Protein content of lupin is on par with that of soybean, making lupin one of the richest plant-based protein source [39]. Compared to other legumes, such as lentils, peas, chickpeas, and fava beans (Table 1), lupin has a significantly higher protein content, making it a strong candidate as a protein source [4]. Several studies have highlighted that lupin exhibits a high protein digestibility, ranging from 79 to 91% [7,40]. The protein composition of lupin includes two major storage proteins: 35–37% 11S legumin-like lupin globulin (α-conglutin) and 44–45% 7S vicilin-like lupin globulin (β-conglutin), as well as two minor groups: 4–5% 7S globulins (γ-conglutin) and 10–12% 2S sulphur-rich albumins (δ-conglutin) [4,41]. The proportions of these proteins are crucial in determining their functional properties. For instance, the ratio of albumin to globulin and legumin to vicilin significantly impacts their behaviour due to the unique characteristics of each protein [42,43]. Legumin and vicilin exhibit high solubility at alkaline pH and minimal solubility at their isoelectric points, whereas albumin remains soluble across a wide pH range, thus affecting functional properties including solubility, swelling, foaming, and gelling [4,42,43]. Specifically, their compact globular structure of lupin proteins and their tendency for intramolecular disulphide crosslinking [44] are the underlying structural reasons for the poor gelation and swelling capacity observed in lupin protein products [45,46,47]. The quality of lupin proteins is quite good, featuring most of the essential amino acids, though it is relatively lower in sulphur-containing amino acids which can be mitigated by combining lupin with other protein sources that are rich in these amino acids [5].

In addition to protein, lupin seeds are rich in both insoluble and soluble dietary fibre, with a significantly higher content (46.6 g/100 g) compared to other legumes such as soybeans (9.3 g/100 g) and lentils (19 g/100 g) [29,48]. Techno-functional properties such as water binding, solubility, and viscosity are dependent on the level of cellulose versus non-cellulose polysaccharides [29], hence why these properties are generally poorer in lupin compared to other legumes. However, the kernel of lupin mainly consists of the more soluble polysaccharide fraction compared to hull [29], with a wider range of different types of polysaccharides such as cellulose, pectin substances, and non-starch and non-cellulose glucans [29].

Lupin seeds are also a good source of essential fatty acids, particularly polyunsaturated fats, which are beneficial for cardiovascular health. The fat content in lupin is around 9.5 g/100 g with a high amount of unsaturated fatty acids [49]. Moreover, lupin seeds are rich in minerals such as magnesium (1.2–2.2 g/kg), potassium (8.6–11.1 g/kg), calcium (2.1–4.7 g/kg), sodium (0.1–0.2 g/kg), and vitamins including vitamin E, with the main isomer of γ-tocopherol and various B vitamins [4,50]. These nutrients play crucial roles in maintaining overall health and well-being.

2.2. Health Benefits

Lupin seeds confer numerous health benefits due to their unique nutrient profile. The high dietary fibre content in lupin promotes digestive health by enhancing bowel regularity and preventing constipation [51]. Fibre also contributes to a lower glycaemic index, which helps in managing blood sugar levels and reducing the risk of type 2 diabetes, weight management, and in maintaining a healthy gut microbiome [50,52].

The presence of bioactive compounds such as antioxidants in lupin seeds further enhances their health benefits [52]. Antioxidants help in neutralising harmful free radicals in the body, thereby reducing oxidative stress and lowering the risk of chronic diseases including certain types of cancers [53]. The specific bioactive compounds found in lupin, including phenolic acids and flavonoids, have been linked to anti-inflammatory and anti-carcinogenic properties [54]. Lupin seeds are also beneficial for cardiovascular health. The essential fatty acids, particularly omega-3 and omega-6 fatty acids, play a crucial role in maintaining heart health by reducing cholesterol levels and improving blood lipid profiles [7,50].

2.3. Environmental and Agricultural Benefits

Beyond their nutritional attributes, lupin seeds offer significant environmental and agricultural benefits. Lupin plants are known for their ability to fix atmospheric nitrogen into the soil through a symbiotic relationship with nitrogen-fixing bacteria [55,56]. This process enriches the soil with nitrogen, reducing the need for synthetic fertilisers, which are often associated with environmental pollution and greenhouse gas emissions [57]. As such, lupin cultivation can improve soil fertility and promote sustainable agricultural practices. Lupin crops are also relatively resilient and can be grown in a variety of soil types and climatic conditions, including poor soils where other crops might struggle [58]. This adaptability makes lupin a valuable crop in regions with less fertile soil, contributing to food security in diverse agricultural landscapes. Moreover, lupin’s relatively low water requirement compared to other protein crops like soybeans makes it a more sustainable option in water-scarce regions [58,59]. The reduced need for irrigation not only conserves water resources but also lowers the overall environmental footprint of lupin cultivation.

3. Techno-Functional Properties of Lupin

The techno-functional properties of food ingredients are crucial in determining their applicability and performance in various food products. For lupin flour to be effectively utilised in the food industry, it must meet certain functional requirements such as appropriate solubility, water absorption, gelation, emulsification, and foaming properties, which must match the requirements of different food products. However, lupin exhibits several limitations in these aspects, which pose significant challenges to its widespread use. This section examines the major techno-functional properties of lupin and highlights the limitations that need to be addressed to enhance its utility as a food ingredient.

3.1. Solubility

Protein solubility is a critical functional property that affects the behaviour of proteins in food systems. Solubility influences the ease with which proteins can be incorporated into various food matrices, impacting the texture, appearance, and mouthfeel of the final product [60]. Lupin proteins exhibit a similar bell-type solubility curve as other legume proteins, with reasonably high solubility at pH lower than the isoelectric point (<pH 4.0), very low solubility at pH ~4.0 to 5.0), and increasing solubility with increase in pH above pH 5.0 to maximum solubility alkaline pH of 8.0 to 9.0. Domínguez, Bermúdez [61] measured the solubility of lupin protein isolate produced using an optimised alkaline extraction–isoelectric precipitation method as a function of pH. They reported ~60% solubility at pH 3.0, which decreased to ~6% and lower at pH 4.0 to 5.0, increased to ~80% at pH 7.0 and further increased to a maximum of ~92% at pH 8.0 to 9.0. Similarly, Chukwuejim and Aluko [62] reported ~72% and ~44% solubilities at pH 3.0, for white and blue lupin isolates, respectively, which decreased to ~20% and 0% for both lupin proteins at pH 4.0 and pH 5.0 respectively. Further increase in pH to 7.0 resulted in ~80% solubility, which increased to ~90% at pH 8.0 for both lupin varieties. The solubility profile for soybean protein isolate prepared using the same alkaline solubilization and isoelectric precipitation method was similar to the lupin protein isolates, with ~60% solubility at pH 3.0, decrease to ~4% at pH 4.0 to 5.0 and increase to ~85% at pH 7.0 and decrease to 70% at pH 8.0 [62]. Relatively lower solubility values at different pH were reported by Hojilla-Evangelista, Sessa [18], who compared the impact of extraction methods on the solubilities of lupin and soy proteins at different pH. The solubility of lupin protein isolated using alkaline extraction and isoelectric precipitation ranged from ~6% at pH 3.0, decreased to the lowest value of ~4% at pH 4.0, and gradually increased at pH from 5.5 to 7.0 to a maximum of ~55%. Lupin protein extracted using ultrafiltration and diafiltration showed slightly higher solubility ranging from ~19% at pH 3.0, decreasing down to ~10% at pH 4.0, and increased to 65% at pH 7.0. Soy protein isolates produced using the alkaline extraction and isoelectric precipitation approach showed similar pH profile but lower solubility of ~45% at pH 7.0 compared to lupin protein, although that substantially increased to 100% at pH 11.0, which the authors cautioned against as it could be due to extensive proteolysis and disaggregation. The soy protein isolates produced using ultrafiltration and diafiltration exhibited similar solubility of around 65% as lupin protein at pH 7.0, with no further increase with increase in pH [18]. Much lower protein solubility of around 9% at neutral pH was reported for lupin flour by Raikos, Neacsu [16], which was slightly lower than other legumes such as green pea and fava bean (with solubility around 10–12% at the same pH). In this case lupin flour was used in the study instead of lupin protein isolate, whereby the protein is in its native state interlinked with other molecules, which could have resulted in low protein solubility.

Factors such as pH, ionic strength, and temperature can significantly affect the solubility of proteins in general including lupin proteins [60,63,64]. The higher solubility of lupin proteins of >90% at alkaline pH (pH 8.0 to 9.0) is attributed to the strong electrostatic repulsion between negatively charged protein molecules, which promotes solubilization. However, such alkaline conditions are not typical of food products and can compromise their sensory attributes [61,65]. High solubility of lupin proteins under a wider range of conditions is essential for their broader application, which is feasible by optimising protein extraction and isolation conditions as well as physicochemical properties such as ionic strength through appropriate formulation. The protein solubility of lupin seed was determined as a function of pH and ionic strength in an experiment conducted by El-Adawy, Rahma [66]. When the soluble protein content was measured as a function of ionic strength, it was found that both bitter and sweet lupin flour had the highest soluble protein concentration, around 80–85%, at a NaCl concentration of 1 M. This concentration gradually decreased as the ionic strength increased to a NaCl concentration of 1.6 M [66].

3.2. Rheological Properties

Rheological properties of lupin proteins vary based on amino acid composition and side chain length, and structure of starch, which confer specific functional characteristics [67,68]. Lupin flours exhibit lower viscosity compared to starch-rich flours, as indicated by a study involving nine lupin cultivars, with viscosity values ranging from 863 mPa.s⁻¹ to 1593 mPa.s⁻¹ [69]. Since high viscosity in typical flours relies heavily on the swelling and gelatinisation of starch granules, mediated by the molecular structure and leaching of amylose and amylopectin, the lack of these components in lupins prevents the formation of highly viscous gels [69]. Lupin protein and fibre hydration behaviour, particularly soluble fibre interaction with water molecules, further contribute to the observed viscosity differences with lower viscosity [29].

3.3. Water Absorption and Oil Absorption Capacity

Lupin flour and protein isolates generally exhibit relatively lower water absorption capacity (WAC) compared to other legume flours or protein isolates. For example, the water absorption capacity of lupin protein was reported to be around 33%, which is lower than chickpea at 42% and broad bean at 44% [70]. This limitation can affect dough formation in bakery products as well as the juiciness and texture of meat analogues [71]. The lower WAC of lupin proteins is likely due to their unique protein structure, which may not interact with water molecules as effectively as proteins in other legumes, possibly due to higher surface hydrophobicity [72]. Improving the WAC of lupin proteins could broaden their use in a variety of food products, enhancing texture and overall quality.

Oil absorption capacity (OAC) of lupin flour depends on the lipophilic nature of lupin proteins, where non-polar side chains of amino acids bind to the hydrophobic chains of fats [69]. This interaction between lupin proteins and fats determines the effectiveness of lupin flour in incorporating oils, which is essential for maintaining moisture and flavour in various food applications. In a study where lupin was used as an ingredient for bread making, it was observed that the OAC of lupin flour ranged from 1.76 (in lupin flour) to 2.8 (in lupin protein isolates) g (oil) /g (protein) [69,73]. Rodríguez-Ambriz, Martínez-Ayala [74] reported that the OAC of lupin protein isolate prepared by isoelectric precipitation was 1.7 mL/g protein, which was slightly higher than that the 1.5 mL/g protein observed for soybean protein isolate in the same study. It has to be noted that functional properties of plant proteins are also affected by the method of preparation. For instance, lupin and soybean protein extracts prepared by micellization had relatively higher OAC values of 2.2 and 2.5 mL/g protein, respectively, and in this case the OAC of soy protein isolate was relatively higher [74].

3.4. Swelling Capacity

Protein content is a determinant of swelling capacity [75]. Higher swelling capacity generally correlates with increased protein solubility and availability [75]. Moreover, the capacity of the substrate to retain water after gelatinization through breakdown of existing hydrogen bonds between starch and generation of new bonds with water also affects the swelling ability [69]. A study of nine Australian lupin cultivars reported that the swelling power of dehulled-seed flour ranged from approximately 3.37 to 4.01 g water per gram of flour, depending on the cultivar, with Rosetta exhibiting the lowest value (3.37 g/g) and Jurien the highest (4.01 g/g). Water absorption capacity (WAC) for the same flours also varied: for example, Jurien showed a WAC of 4.4 mL water per gram flour while another cultivar (WK388) exhibited a markedly lower WAC of 1.73 mL/g; underscoring substantial inter-cultivar variability in hydration behaviour [69]. Another study comparing lupin protein isolates (LPIs) and soy protein isolates (SPIs) found that LPI has a lower swelling capacity than SPI. Unlike SPI, the swelling capacity of LPI does not increase upon heating, and observational results from the study showed that LPI forms weaker, more deformable heat-induced gels and its protein particles remain relatively compact even after heating, whereas SPI particles visibly swell and contribute to stronger, more rigid gels [44]. This difference is attributed to the compact, heat-stable structure of lupin proteins, which suppresses intermolecular bonding through disulfide bridge formation and favours intramolecular crosslinking [44]. These findings further suggests that multiple factors contribute to the behaviour specific techno-functional property.

3.5. Gelation Properties

The factors that control the gel formation process include protein source, concentration, pH, ionic strength, and temperature [20]. The compact, heat-stable properties of lupin protein particles suppress protein unfolding and disulfide bond formation, leading to weak gel network formation with limited non-covalent interactions [44,76]. This is also associated with a greater tendency for intramolecular crosslinking rather than intermolecular bonding [77]. Consequently, LPI form weaker heat-induced gels, whereas SPI exhibit better gel-forming ability, as observed in the study where LPI gels were described as softer and more deformable, and LPI particles showed minimal swelling even after heating, while SPI formed visibly firmer, more structured gels due to greater particle swelling during heat treatment [44,77]. Makri, Papalamprou [78] compared the gelation properties of lupin, pea and broad bean protein isolates. The lupin gel yielded at relatively lower strain (0.32) compared to pea and broad bean protein isolates (0.38 and 0.4, respectively) but fractured at much higher strain (8.1 Pa) compared to both broad bean and pea protein isolates, which fractured at 5.5 and 5.1 Pa respectively. The hardness of the lupin gel (~400 g) was also much higher than the pea protein (~200 g) and broad bean (~25 g), whereas its cohesiveness (~0.15) was similar to that of pea protein gel (~0.15) with broad bean protein gel (0.02) showing the lowest cohesiveness. According to the authors, stronger, probably filled type gels, were formed only with the addition of the polysaccharides xanthan gum and locust bean gum and after being aged for 10 days [78], indicating the potential of in situ produced exopolysaccharides for strengthening gels formed by plant proteins including lupin.

3.6. Emulsification Properties

Emulsifying capacity is directly influenced by factors such as protein solubility, pH, protein concentration, and lipophilicity [69]. Additionally, the presence of glycinin is associated with poor emulsifying properties due to its compact structure stabilised by disulfide bonds [23]. Studies on techno-functional properties such as emulsifying properties use different methodologies and it is often difficult to compare values across different studies. However, data from comparative studies show that lupin protein has comparable emulsifying properties with other legume proteins, whereas it has inferior emulsifying properties compared to animal proteins. For instance, Ivanov and Munialo [79] compared the emulsion setting time of lupin protein isolate with that of whey protein isolate and α-lactalbumin. They observed a 5 min setting time for lupin protein compared to 2 min for whey protein isolate and α-lactalbumin [79]. A study by Jayasena, Chih [80] compared the emulsifying properties of lupin protein isolates produced by precipitation at different acidic pHs (4.0, 4.2, 4.4, 4.5, 4.6, 4.8, 5.0 and 5.5.) among themselves and with that of soy protein isolate prepared by precipitation at pH 4.5. The emulsifying activity and emulsion stability of the protein isolates were studied at pH 2.0, 4.0, 6.0, and 8.0. There was no substantial difference in the emulsifying capacities of the different lupin protein isolates. For instance, the emulsifying capacities of lupin protein isolates at pH 6.0 were between 51.2% and 53.5% for all lupin protein isolates. The emulsifying capacity of lupin protein produced at pH 4.5 was compared to that of soybean protein isolate produced under the same condition. Largely similar emulsifying capacities (53.2% to 58.8%) were observed for the two proteins at all pH values except at pH 4.0, where the emulsifying capacity of the soybean protein isolate decreased to 4.0% while that of the lupin isolate was 53.5%, which is close to values at other pHs. The authors explained that this was due to the lower solubility of soy protein at pH 4.0, which is near its isoelectric point, whereas the same is the case for lupin; its emulsifying property was not affected due to its different protein structure. With respect to emulsion stability, similar values of ~55% were observed for all the lupin protein isolates as well as the soybean protein isolate at all pHs except at pH 4.0, where the emulsion stability of the soybean protein isolate was ~4% [80]. Similarly, no significant difference was observed in emulsion stability among chickpea, broad bean and lupin protein isolates at neutral pH with 36.3%, 37.5% and 33.8% emulsion stability reported, respectively, for the three proteins [70]. In a study by Makri, Papalamprou [78], the emulsifying properties of protein isolates from lupin, pea, and broad bean were compared by measuring the oil droplet size distribution of the emulsions formed by these proteins. All the proteins performed better at pH 7.0 compared to 5.5 showing lower mean oil droplet size at pH 7.0. Among the proteins, pea protein and bean protein isolates showed slightly better emulsifying activity compared to lupin protein isolate produced using isoelectric precipitation, with mean oil droplet size of ~15 µm compared to ~17 µm with respect to lupin protein isolate. The emulsion stability, measured by changes in oil droplet size during one month storage, was similar in all cases both at pH 5.5 and 7.0, with no substantial change in oil droplet size during storage of the emulsions for one month. Interestingly, the method of protein isolation also had impact on emulsifying capacity with proteins isolated using isoelectric precipitation showing slightly better emulsifying effect, i.e., lower mean oil droplet size compared to those produced using ultrafiltration [78]. In contrast, Hojilla-Evangelista, Sessa [18] reported higher emulsifying capacity (98.7 vs. 56.0 m²/g for soybean and 71.5 versus 45.4 m²/g for lupin) and emulsion stability (23.4 versus 15 min for soybean and 25.5 versus 15 min for lupin) for lupin and soy protein isolates prepared using ultrafiltration–diafiltration compared to those prepared using acid solubilization followed by isoelectric precipitation. There was no significant difference in the emulsifying activities of lupin and soybean protein isolates prepared by isoelectric precipitation whereas the soybean protein isolate prepared by ultrafiltration–diafiltration showed significantly higher emulsifying activity compared to lupin protein isolate prepared in the same way [18]. Makri, Papalamprou [78] used alkaline extraction prior to isoelectric precipitation whereas [18] used acid solubilization followed by isoelectric precipitation, which may have resulted in different protein structures and hence different emulsifying properties vis-à-vis protein isolates prepared using ultrafiltration.

3.7. Foaming Properties

The foaming properties of food substances are influenced by factors such as pH and protein concentration, with foaming capacity and foam stability typically decreasing at the isoelectric point and increasing with higher protein concentrations [69,81,82]. In a study comparing broad bean, pea, and lupin protein isolates, both broad bean and pea protein isolates demonstrated better foaming capacity and foaming stability compared to lupin protein isolates, with the authors observing that pea protein formed the highest foam volume (~300 mL) and maintained the most stable foam over time, broad bean showed moderate (~250 mL) but consistently stable foams, whereas lupin produced the lowest initial foam height (~90 mL) and its foam collapsed more quickly, indicating poorer overall foaming performance both at pH 5.5 and pH 7.0 [78]. Alu’datt, Rababah [70] similarly observed the highest foaming stability (70.8%) for broad bean protein isolate followed by chickpea protein isolate and lupin protein isolate, which exhibited the same foam stability of 50% at neutral pH. Hojilla-Evangelista, Sessa [18] compared the foaming properties of lupin and soybean protein isolates that were prepared in two different ways (acid extraction followed by isoelectric precipitation versus ultrafiltration–diafiltration). Soybean protein isolates prepared by both isoelectric precipitation and ultrafiltration (131 mL versus 104 mL and 144 mL versus 98 mL) had higher foaming capacity compared to lupin protein isolates. The inferior foaming properties of lupin protein become starkly clear especially when its compared with dairy proteins, which are commonly used as foaming agents in bakery and other food products. Ivanov and Munialo [79] compared the foaming properties of lupin protein isolate with that of whey protein isolate and α-lactalbumin. The foaming capacity of lupin was 105% and 115% at pH 4.0 and 7.0 whereas the foaming capacities of whey protein isolate were 415% and 170% and that of α-lactalbumin were 435% and 270% at pH 4.0 and 7.0, respectively. Interestingly, the foaming capacity of lupin protein isolate did not significantly change with pH. The foam stability rates for lupin protein were 0.03 and 0.04 cm/min at pH 4.0 and 7.0, respectively, whereas 0.09 and 0.02 cm/min were observed for whey protein isolate and α-lactalbumin, respectively, at pH 4.0 and 0.03 cm/min for both at pH 7.0. In terms of foam stability at pH 7.0, whey proteins showed similar performance as the lupin protein isolate in this study [79].

Overall, method of protein isolate preparation did not have significant effect on the foaming capacity of lupin protein isolates. In terms of foam stability, soy protein isolates prepared using isoelectric precipitation and ultrafiltration showed 95% and 77.4% foam stability, respectively, whereas lupin protein isolates showed much lower corresponding stability of 16.8% and 2.6% respectively. On the other hand, foam stability was lower for samples prepared using ultrafiltration, which could be due to the lower protein content of these protein preparations [18].

For lupin proteins to serve as efficient foaming agents, they must fulfil essential criteria. This includes the ability to swiftly adsorb at the air/water interface during foaming, undergo rapid conformational changes and rearrangement at this interface, and ultimately form a cohesive viscoelastic film through intermolecular interactions [78].

Across some key techno-functional properties, including water absorption capacity, gelation, and foaming, the unifying challenge for lupin proteins stems from their low inherent flexibility and limited surface activity. This compact, heat-stable globular structure inhibits the necessary effective interaction with surrounding molecules; whether they be water, oil, or air; which, in turn, prevents the formation of cohesive, functional networks. Overcoming this fundamental structural rigidity is therefore critical to enhancing the utility of lupin, setting the stage for modification strategies utilising external agents, such as EPS.

4. Fermenting with EPS-Producing LAB as a Strategy to Overcome Techno-Functional Challenges

It is evident from the foregoing discussion that lupin exhibits poorer performance in certain techno-functional properties compared to other legumes; for example, showing weaker gelation and thickening performance than soy proteins. Therefore, there is a need to improve these properties to make it a more versatile food ingredient. Among various methods employed to address techno-functional limitations associated with plant proteins, fermentation has numerous advantages. These include energy efficiency, enrichment of nutritional value, enhancement of sensory properties, probiotic effects, potential health benefits, and extension of shelf life [25]. Additionally, fermentation has the capability to mitigate allergens such as lup a1, associated with lupin [28], and reduce anti-nutritional factors including raffinose family oligosaccharides, alkaloids, phytic acid, protease inhibitors, tannins, and saponins in lupin [83,84].

However, fermentation is not a perfect solution; depending on the choice of strain, substrate, and fermentation conditions, it may also introduce undesirable side-effects. For instance, although fermentation of lupin “milk” analogues with LAB can reduce beany or grassy flavours [85], some fermentation regimes lead to increased acidity sour and vinegary taste and the formation of volatile compounds described as mushroom, green, soil, or nutty aromas, which can reduce consumer acceptability [86]. Fermentation also reduces pH close to the isoelectric point of proteins, causing protein coagulation, decreased solubility, and reductions in emulsifying or foaming capacity [87,88]. Moreover, while fermentation can reduce anti-nutritional factors, it may also diminish certain vitamins or antioxidants, and in some cases functional attributes like protein digestibility [89,90]. Hence, while fermentation holds real promise for enhancing the techno-functional and nutritional attributes of lupin and other plant proteins, careful optimisation of strain selection, substrate preparation, and fermentation parameters are required to avoid these unintended effects.

LAB, including Lactiplantibacillus spp., Leuconostoc spp., Lactococcus spp., and Streptococcus spp., are a group of Gram-positive bacteria known for producing lactic acid as the primary fermentation product from carbohydrates [91]. Lactic acid bacterial fermentation enhances the functional properties of food systems, while contributing to improved texture, flavour, and preservation [92].

EPS-producing LAB have gathered significant attention due to their ability to improve properties such as viscosity, gelation, foaming, and emulsification, offering promising avenues for enhancing the quality of targeted products [34,93]. While fermentation itself improves some of these properties, bacterial EPS adds another layer of improvement. The EPS produced by bacteria consists of long-chain sugar molecules, including dextrans, levans, inulin, gellan, curdlan, mauran, pullulan and xanthan, with variations depending on the fermenting bacteria and fermentation conditions [94,95]. The composition, structure, and quantity of EPS can significantly impact techno-functional properties [96]. Furthermore, the type of EPS produced can determine its main application; for example, xanthan is preferred as an emulsion stabiliser, thickener, and viscosity enhancer, while gellan excels as a gelling agent, and curdlan functions effectively as a texture modifier [93,97].

Table 2. The mechanism underlying effects of bacterial fermentation and EPS for improvement of techno-functional properties.

Techno- Functional Property	Mechanism of Enhancement by Fermentation	Mechanism of Enhancement by EPS Bacterial Fermentation	Reference
Protein solubility	Microbial enzymatic activity hydrolyses complex protein structures to simpler amino acids increasing hydrophilicity.	Electrostatic interactions between EPS and proteins stabilise dispersion by influencing the charges on the protein surface.	[87,100]
Viscosity	Alters protein structure, leading to greater molecular interaction and thereby thickening the solution.	EPS acts as a thickening agent, increasing the viscosity of the solution by forming a network that impedes flow.	[101,102]
Gelation	Breakdown of proteins into smaller peptides and amino acids, producing organic acids such as lactic acid, which acidify the food matrix and alter ionic strength, all of which may promote protein interactions that form a gel network.	Gel formation by crosslinking with proteins, creating a three-dimensional network that traps water and enhances gel strength.	[103,104,105]
Foaming properties	Breakdown of proteins into peptides, enhance their ability to stabilise air–water interfaces and form stable foams through improved surface activity and intermolecular interactions.	Stabilising air bubbles in the foam, reducing coalescence and improving foaming stability and creating hydrogen bonds at the air–water interface.	[106,107]
Emulsification properties	Alter protein conformation, enhancing their ability to stabilise oil–water interfaces through improved surface activity and interactions.	Stabilising emulsions by forming a protective layer around oil droplets, preventing coalescence, droplet aggregation, and promoting dispersion in the aqueous phase.	[108,109,110]
Oil absorption capacity	Modifying the surface properties of substrates, facilitating greater oil uptake.	EPS forms a matrix that traps oil droplets, increasing their retention within the system and enhancing oil absorption capacity.	[111,112]
Water absorption capacity	Promote hydration and swelling of broken-down starch granules, thereby enhancing substrate structure and water retention.	EPS, especially hydrophilic ones absorb water molecules and form a hydrated gel network, increasing water retention and swelling capacity.	[30,113,114]

provides a summary of studies on lactic acid bacteria fermentation with EPS-producing strains and the observed effects on techno-functional properties of fermented food products. Bacterial fermentation alone improves techno-functional properties primarily through the enzymatic breakdown of substrates (proteins to peptides and amino acids, carbohydrates to sugars, fats to fatty acids and glycerol) [89,98]. This process modifies the structures of the substrates, facilitating interactions among molecules that enhance various functional properties. On the other hand, fermentation with EPS-producing bacteria amplifies this enhancement of functional properties. EPS acts as a protective mechanism in bacteria and forms matrices or networks in the food substrate that stabilise dispersions, emulsions, foams, and gels through electrostatic interactions or physical entrapment [93,99].

For the specific matrix of lupin flour, which is characterised by exceptionally high dietary fibre content (approx. 30–40%) [115] and globulin storage proteins [116,117], the detailed physicochemical mechanisms of EPS necessitates targeted research to unlock its full potential in fermentation. Lupin fibre is rich in non-cellulosic glucans (e.g., galactans, arabinogalactans) [29,118] rather than the predominantly cellulosic structures found in some other plant matrices. It is hypothesised that the typically anionic nature of many LAB-derived EPS (e.g., due to uronic acids or phosphate groups) [119] facilitates electrostatic interactions with the exposed, positively charged domains of partially hydrolysed lupin proteins/peptides at pH values below their isoelectric point (pI), enhancing emulsion and foam stability [87,120]. Furthermore, these anionic EPS are likely to physically entangle with the hydrophilic, non-cellulose glucans (e.g., pectin-like substances) [121] of the lupin fibre fraction. This physical entanglement and hydrogen bonding among EPS, protein, and fibre components generates a more robust, highly hydrated and interpenetrating polymer network that significantly increases viscosity and WHC compared to the effects of EPS in a matrix dominated by less-reactive starches or cellulose [122,123].

Table 3. Effect of EPS-producing LAB fermentation on techno-functional properties of legume substrates.

Food Substrate and End Product	EPS-Producing Bacteria and Fermentation Conditions	Changes After Fermentation	Reference
Dough from fava bean flour	Leuconostoc spp. and Weissella spp. 25% sucrose 30 °C, 24 h	Among the strains tested, Leuconostoc pseudomesenteroides DSM 20193 produced the highest amount of EPS, increasing viscosity, thickening, and gelling capacity.	[124]
Dough from soybean and fava bean flour	Leuconostoc mesenteroides DSM 20343 sucrose (5–15%) and raffinose (10%) 30 °C, 24 h	Significant increase in viscosity in both types of flours with the same sucrose content.	[125]
Soy milk from soy extract	Lactobacillus plantarum 70810 4% sucrose 37 °C, 12 h	Improve texture, flavour, viscosity, and water absorption.	[126]
Paste made from fava bean protein concentrates with addition	Weissella confusa VTT E-143403 5% sucrose 30 °C, 24 h	Improvement of viscosity and texture.	[127]
Sourdough made from chickpea	Weissella confusa Ck15 2% sucrose 35 °C, 24 h	Improvement of viscosity Increase antioxidant activity.	[128]
Frozen dough from red bean	Weissella confusa QS813 10% sucrose 30 °C, 24 h	Enhance water absorption in freezing thawing cycle of frozen red bean dough and reduce distortions and increase structural tolerance due to ice crystallisation.	[129]
Fermented soy milk made from soy milk	Leuconostoc mesenteroides 109 50 g/L sucrose 28 °C, overnight	Improvement of viscosity and texture.	[130]
Lupin protein isolates Lupin-based yoghurt	Lactobacillus plantarum TMW 1.460 and TMW 1.1468; Pediococcus pentosaceus BGT B34; Lactobacillus brevis BGT L150 pH 4.5, 14–35 h	Denser protein network formation Improved viscosity and texture.	[131]
Fermented soy milk made from soy milk	Lactococcus lactis subsp. cremoris 30 °C 1% v/v of overnight-grown Lactococcus lactis strains	Enhance texture, viscosity, and shear-stress resistance.	[132]
Fermented soy milk made from soy milk	Streptococous thermophilus strain SBC8781 37 °C, 24 h	Enhance EPS production: 200–240 mg/L; functional properties not measured in this study.	[133]

summarises earlier research findings demonstrating how fermentation by EPS-producing LAB enhances the techno-functional properties of different legume-based products. These research findings underscore the critical role of fermentation conditions and substrate selection in determining the outcomes of fermentation processes. The versatility of EPS-producing bacteria in positively modifying the rheological, sensory, and stability attributes of food products is evident, highlighting promising avenues for their application in food processing and product development. However, to attain the desired techno-functional attributes, optimisation of fermentation parameters is vital.

The studies presented in Table 3 show that fermentation parameters including bacterial strain, carbohydrate supplementation, and incubation conditions play a decisive role in determining the rheological and structural outcomes of the end products. EPS-producing strains such as Leuconostoc and Weissella have been shown to synthesise extracellular polysaccharides that significantly increase viscosity, water absorption, and textural stability in fava bean, soybean, chickpea, and red bean matrices [36,125]. Such improvements result from the formation of a viscous hydrocolloid network within the fermented matrix, which enhances water-binding, gelling, and emulsifying capacities, thereby improving product stability and sensory attributes even under stress conditions such as low temperature or non-optimal pH conditions [134,135,136,137]. These findings underline the versatility of EPS-producing LAB in tailoring the functional properties of legume derived foods, opening opportunities for broader applications in food processing and product formulation.

Although there is limited research directly addressing the fermentation of lupin with EPS-producing LAB, the compositional similarities of lupin to other legumes particularly its high protein and dietary fibre contents suggest that comparable or even enhanced functional improvements could be achieved. The rich protein matrix and relatively low starch content of lupin may provide a unique environment for EPS–protein interactions, potentially leading to stronger gel networks and improved emulsion stability. Furthermore, lupin’s lower inherent viscosity compared to soy or fava bean matrices [44,62] could make the viscosity enhancement from EPS production even more pronounced. Therefore, extrapolating from the evidence in other legumes, EPS-producing LAB fermentation could serve as an effective bioprocessing strategy to enhance the texture, water-binding capacity, and overall functionality of lupin-based products, supporting their incorporation into diverse food systems.

Furthermore, while existing studies on fermentation processes have provided valuable insights, there remains a significant gap in research, regarding the effect of fermentation by EPS-producing bacteria on the techno-functional and other quality attributes of lupin. The results from other legume substrates indicate that the process has a significant potential to address the techno-functional limitations associated with lupin while leveraging the additional benefits of fermentation. Therefore, further studies are needed to identify specific bacterial strains and fermentation conditions tailored to lupin substrates. This avenue of research could yield new insights on how fermentation and fermentation derived EPSs interact the various components of lupin and modify its structure as the basis for a fermentation-based approach for enhancing the functional properties of lupin, to enable broader acceptance and utilisation in the food industry.

5. Conclusion and Future Directions

Lupin offers a promising solution to the growing demand for sustainable and nutritious protein sources worldwide. With their high protein content, dietary fibre, essential fatty acids, vitamins, and minerals, lupins possess unique nutritional advantages. However, challenges such as poor foaming capacity, low water and oil absorption capacity, and undesirable sensory attributes like bitterness and beany flavour limit their widespread use in food products.

Fermentation with EPS-producing LAB presents a promising strategy to overcome these challenges. By enhancing techno-functional properties and modifying sensory attributes, fermentation can unlock better nutritional and functional benefits from lupin. The production of EPS during fermentation further enhances these benefits, by improving water retention, texture, mouthfeel, and overall palatability.

Moving forward, optimising the fermentation process with EPS-producing LAB is crucial to maximising the enhancement of techno-functional properties in lupin-based products. Exploring different fermentation conditions and strains of LAB, as well as addressing sensory aspects, will be essential for consumer acceptance and market success. Additionally, scaling up fermentation processes and evaluating economic viability are necessary steps for widespread adoption by the food industry.

Overall, by addressing these challenges and research gaps, fermented lupin has the potential to emerge as an appealing and nutritious plant-based ingredient in the global food market. Harnessing the benefits of fermentation can contribute to sustainable food systems and improved public health worldwide.