Pulses in Dairy Alternatives

Wet milled whole pulses and pulse flours have been used as the starting material in many iterations of plant-based products. As an alternative base ingredient, pulse flours provide high protein content, nutritional density, and the ability to promote and maintain prebiotic growth and culture counts. In products such as plant-based cheese, the gelation of pulse proteins and the hydrocolloidal properties of pulse carbohydrates are beneficial contributors to final product texture.

Applications:

Plant-based milk, plant-based yogurts, plant-based cheese.

Function in Plant-Based Dairy Alternative:

Starting material source, nutritionally dense, high protein, source of fermentable nutrients, support probiotic growth, gelling ability, water retention, viscosity development.

Marketing Appeal:

Aligns with consumer trend: “plant-based".

Application: Plant-Based Beverages

SPECIFIC BENEFITS:

Alternative beverage base, nutritionally dense, high protein, prebiotic function, support probiotic growth.

Preparation

Multiple methods of preparation have been used to successfully evaluate the performance of pulses as the base ingredient in milk alternatives. The simplest processes soak the whole or split seeds and grind them under aqueous conditions or directly combine the flour with water prior to filtration. Al- Saedi et al (2020) reported a significant effect of the filtration method on the protein content of lupin-based beverages, where a higher concentration was obtained when a cheesecloth was used compared to a centrifuge. Xia et al. (2018) looked at the effects of increasing pressure and the number of passes during homogenization on the resulting quality of lupin-based beverage. High pressure homogenization increased the colloidal stability despite decreases in the viscosity of beverages that was accompanied with a reduction in the mean particle size of the system, where TEM images confirmed smaller individual fat globules under higher pressurized conditions. Increasing the pressure and the number of passes through the homogenizer also increased the lightness of beverages and decreased microbial loads, which could contribute to an extended shelf life. Overall, high pressure homogenization could serve as a successful treatment in the production of a lupin-based beverage, where increasing the number of passes at lower pressures may also be beneficial on final product quality.

Seed Pre-treatments

Other techniques such as seed pre-treatments, have also been employed and resulted in significant differences in the composition, acidity, viscosity and colour of beverages prepared from pulses (Joshi et al., 2023; Vallath et al., 2021). Vallath et al (2021) reported pre-treatment of chickpea seeds by boiling or roasting imparted a yellowish-orange and brownish colour to the beverages compared to the white control produced from raw seeds. However, beverages produced from raw chickpea resulted in the lowest rankings for overall acceptability due to a strong beany flavour. Roasting the chickpeas did help to relieve this flavour, however the boiled chickpea beverages were preferred by panellists for their mouthfeel, consistency, taste and colour. Similarly, sensory analysis of mung bean-based milks using a combination of preparation methods, including soaking, germinating and blanching, revealed that beverages prepared using blanching were significantly preferred based on colour, appearance, mouthfeel, taste, flavour and overall acceptability (Joshi et al., 2023). Rincon et al (2020) were able to successfully produce a chickpea-based beverage that was blended with coconut milk to produce a product with acceptable nutritional value and calcium content compared to cow’s milk, in which a 50/50 combination of chickpea to coconut was preferred by panellists during sensory evaluation.

Fermentation

Many studies have also experimented with the use of single or mixed-strain cultures of lactic acid bacteria to ferment the beverages produced from pulses with or without the aid of enzymatic hydrolysis. Zhang et al (2022b) indicated that enzymatic hydrolysis of fermented chickpea beverage resulted in the slight degradation of globulins, the release of free amino acids and an accompanying improvement of rheological characteristics based on the changes in protein gel network structure. With or without enzymatic hydrolysis, all of the pulses evaluated, including lentils, red bean, white kidney bean, faba bean and chickpea, proved to be acceptable medium for fermentation. However, Ziarno et al. (2020) did note differences in acidifying ability in fermented beverages produced from germinated white bean seeds, indicating that the starting culture strain or mix may be optimized depending on the substrate and desired end quality of the product. Fermentation of pulse-based beverages has been reported to alter the composition by enhancing antioxidant activity measured as increases in total phenolic content, DPPH, FRAP, ORAC, ABTS and OH- scavenging activity (Naprasert et al., 2019; Vasilean et al., 2021 and Zhang et al., 2022), decreasing B-Group vitamins (Ziarno et al., 2019; Zhang et al., 2020), decreasing oligosaccharides content (Ziarno et al., 2019), altering the fatty acid composition (Ziarno et al., 2020; Zhang et al., 2022) and decreasing the protein content with an accompanying increase in peptides and free amino acids (Zhang et al., 2022).

Sensory Properties

Using HS-SPME-GS/MS, Zhang et al. (2020) noted significant changes in the aroma compounds of chickpea beverages after fermentation, including an increase in total alcohol and ketone but a decrease in hexanol contents, the latter commonly associated with a beany flavour in pulses. However, in a separate study, sensory analysis of beverages were still not rated highly based on flavour and colour attributes, although fermentation did appear to impart a milder aroma (Ziarno et al. 2019). Interestingly, Zhang et al (2022) reported increased red and yellow colour for fermented chickpea beverages whereas Vasilean et al. (2021) noted a whitening effect in faba bean samples. When stirred with a spoon, the fermented faba bean beverages registered higher consistency and lower syneresis with a weak but compact gel structure when compared to chickpea samples, which were more fluid (Vasilean et al.,2021). A comparison of fermented chickpea milk to soymilk also indicated that chickpea beverages formed a weaker gel network than soy samples which resulted in increased syneresis despite similar acceptability scores during sensory analysis (Wang et al., 2018). It is clear that pulse type has a significant effect on the resulting quality of pulse-based beverages, and more research is required to determine the optimal processing conditions that will create a desirable end product. For example, the use of flavourings at low concentrations has been demonstrated to significantly improve the sensory acceptability of pulse-based beverages. The studied flavourings and their inclusions are as follow: 0.2% cardamom, 1g/100mL chocolate powder, 5-10g/100mL sugar, 20g/100mL fruit flavouring, 0.3% vanilla extract (Joshi et al., 2023; Naprasert et al., 2019; Rincon et al., 2020; Vallath et al., 2021).

Example Pulse-Based Beverage Formulations

Treatment	Preparation
Lupin Beverage (Al-Saedi et al., 2020)	10g of whole and dehulled, split seeds from two lupin cultivars were soaked overnight at room temperature overnight using a 1:3 ratio of seed to water. Soaked seeds were placed in 100mL of 45°C water and milled under aqueous conditions. Milk was prepared by stirring the filtrate derived from either filtering through four layers of cheesecloth or using a centrifuge at 2600gx g for 5 min.
Mung Bean Beverage (Joshi et al, 2023)	Mung bean samples were rinsed with distilled water and soaked for 16h and drained followed by an optional 12h germination step. The soaked and/or germinated seeds were dehulled, followed by an optional 3 min blanching treatment, then wet milled using a 1:6 sample to water ratio before straining through muslin cloth to create a mung bean extract. This extract was heated at 35-40°C for 5 min before the addition of 6% (w/w) sugar followed by a subsequent 5 min of heating. Samples were then pasteurized for 15 min at 72°C prior to the addition of 0.2% cardamom and mixing for 5 min. Once cooled to room temperature, sample were filtered a second time using a muslin cloth into glass bottles, sealed, and sterilised for 14 min at 121°C before cooling and storage at 4°C.
Fermented Red Bean Beverage (Naprasert et al., 2019)	0.7g of Xanthan gum was mixed with 100mL of 60°C water before further mixing with 10g red of bean flour and 10g of refined sugar. The mixture was pasteurized at 100°C for 30min before cooling to 37°C upon which samples were inoculated with single and mixed cultures at 5.52-6.00 log CFU/mL and fermented for 12h.
Chickpea & Coconut Beverage (Rincon et al., 2020)	5kg of chickpea seeds were soaked for 12h, then cooked with 2.5L of water in a pressure cooker at 120°C and 2.0 atm for 20 min. Cooked seeds were blended for 1 min using a 1:4 ratio of chickpea to water before straining with a cloth tissue. A coconut beverage was prepared in a similar manner and blended with chickpea.
Chickpea Beverage (Vallath et al., 2021)	Raw chickpea seeds were soaked for 12-16h, then washed and either left raw, cooked in a pressure cooker at 80°C for 30 min or roasted in an air oven at 80°C for 20-25min. Raw, cooked and roasted chickpeas were submerged in water (1:4 to 1:5), wet milled and the resulting slurry filtered through muslin cloth. The raw chickpea milk was subject to a final pasteurization at 60°C for 30 min.
Faba Bean and Chickpea Beverages (Vasilean et al., 2021)	200g of chickpea and faba bean seeds were washed and soaked for 15 min in 70% aqueous alcohol solution before further soaking in tap water for 12h. Samples were drained and wet milled with 750mL of tap water for 10 min before filtering through cotton gauze. The beverages obtained were hydrolyzed by adding 1g/100g starch of alpha amylose followed by heating to 70°C for 1h under continuous stirring. The temperature was then reduced to 60°C prior to the addition of 1g/100g starch amyloglucosidase then left for 1h. Inactivation of enzymes occurred by holding a temperature of 90°C for 10 min. The hydrolyzed beverages were inoculated with one of two different starter culture mixtures (Strepcococcus thermophilus mixed with Lactobacillus delbrueckii subsp. Bulgaricus OR Lactobacillus casei, Lactococcus lactis subsp. Cremoris, Lactococcus lactis, subsp. Lactis, Leuconostoc, Lactococcus lactic subsp. Biovar, and Steptocuccus thermophilus missed with diacetylactis) at 43°C for 10h until pH 4.6 prior to cooling and storage at 4°C.
Fermented Lentil Beverage (Verni et al., 2020)	Lentil seeds were suspended in water using a 1:10 dry weight to water ratio and soaked at room temperature for 16h. Soaked samples were homogenized and blended to create 20g of slurry before the addition 12.5mg and 25 mg of Papain, bromelin, Veron HPP and Veron PS enzymes for enzymatic hydrolysis conducted for 1h at 30°C. The hydrolyzed samples were filtered with cotton gauze before centrifuged at 10,000 rpm for 5 min and heated to 110°C for 10 min. Samples were then singularly inoculated with 6 log CFU/mL of L. Acidophilis ATCC 4356, L.fermentum DSM 20052, or L.paracasei subsp. Paracasei DSM 20312 for 24h at 37°C under anaerobiosis. A non-inoculated beverage was used as control.
Fermented Chickpea Beverage (Wang et al., 2018)	Chickpea seeds were soaked overnight at a ratio of 1 to 3.3 before milling with 6 parts water and filtration through cotton cheesecloth. The resulting beverage was then boiled for 10 min before cooling. Samples were then inoculated with 0.5% of yogurt starter (maltodextrin, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus) and fermented for 16h at 42°C before refrigerated storage.
Lupin Beverage (Xia et al., 2018)	Dehulled lupin flour was combined with water (1:20) and blended for 5 min before homogenized at 25MPa or using a high-pressure homogenizer at 50, 100 or 175 MPa for 1,4 or 6 passes. Samples were cooled before storage for 28 days at 4°C.
Fermented White Kidney Bean Beverage (Ziarno et al. 2019)	White kidney bean seeds were germinated for 72h at 25°C with added water to moisten the seeds every 24h. 1000g of germinated seeds were blended for 20 min, boiled and continuously stirred prior to filtering through 0.1mm sieve into 170mL jar. The jar was then sterilized at 121°C for 20 min and cooled to 45°C prior to inoculation with one of two yogurt starter culture mixes (Yo-Mix 205 LYO or FD-DVS ABY-3 Probio-Tec) and fermented at 43°C for 4h before cooling to 6°C after which 20g/100g of fruit flavouring (strawberry, peach-mango, apricot, forest fruits, banana-kiwi and red orange) were added or the beverage was left unflavoured.
Fermented White Kidney Bean Beverage (Ziarno et al., 2020)	White kidney been seeds were germinated for 72h at 25°C before mixing with water at a 1:9 ratio and blended. The homogenized samples were boiled for 1h prior to sterilization at 121°C for 15 min. 100mL samples were then inoculated at 37°C for 18h using single Lactobacillus strains to obtain cell count of 1x108 CFU/g before cooling to 5°C.
Fermented Chickpea Beverage (Zhang et al., 2022)	Chickpea seeds were soaked in water (1:5) overnight before milling in six parts water for 30 min and filtered using a gauze. The filtered samples were pasteurized by boiling for 10 min before being inoculated with 0.07% (w/c) of L. plantarum at 10 log CFU/mL and fermented for 24h at 37°C.
Fermented Chickpea Beverage (Zhang et al., 2022b)	Chickpea beverages were prepared as described above by Zhang et al. (2022) but subject to additional enzymatic hydrolysis at 50°C for 30 min that was inactivated by heating for 5min at 95°C followed with the addition of 5% (w/v) of glucose prior to incubation. Five treatments were applied: 1) without enzyme addition 2) 80 U/g protein papain 3) 60 U/g starch alpha-amylase 4) 80 U/g protein papain and 60 U/g starch alpha-amylase 5) 80 U/g protein papain and 60 U/g starch alpha-amylase with 10% (v/v) glucosidase.

Treatment	Preparation
Lupin Beverage (Al-Saedi et al., 2020)	10g of whole and dehulled, split seeds from two lupin cultivars were soaked overnight at room temperature overnight using a 1:3 ratio of seed to water. Soaked seeds were placed in 100mL of 45°C water and milled under aqueous conditions. Milk was prepared by stirring the filtrate derived from either filtering through four layers of cheesecloth or using a centrifuge at 2600gx g for 5 min.
Mung Bean Beverage (Joshi et al, 2023)	Mung bean samples were rinsed with distilled water and soaked for 16h and drained followed by an optional 12h germination step. The soaked and/or germinated seeds were dehulled, followed by an optional 3 min blanching treatment, then wet milled using a 1:6 sample to water ratio before straining through muslin cloth to create a mung bean extract. This extract was heated at 35-40°C for 5 min before the addition of 6% (w/w) sugar followed by a subsequent 5 min of heating. Samples were then pasteurized for 15 min at 72°C prior to the addition of 0.2% cardamom and mixing for 5 min. Once cooled to room temperature, sample were filtered a second time using a muslin cloth into glass bottles, sealed, and sterilised for 14 min at 121°C before cooling and storage at 4°C.
Fermented Red Bean Beverage (Naprasert et al., 2019)	0.7g of Xanthan gum was mixed with 100mL of 60°C water before further mixing with 10g red of bean flour and 10g of refined sugar. The mixture was pasteurized at 100°C for 30min before cooling to 37°C upon which samples were inoculated with single and mixed cultures at 5.52-6.00 log CFU/mL and fermented for 12h.
Chickpea & Coconut Beverage (Rincon et al., 2020)	5kg of chickpea seeds were soaked for 12h, then cooked with 2.5L of water in a pressure cooker at 120°C and 2.0 atm for 20 min. Cooked seeds were blended for 1 min using a 1:4 ratio of chickpea to water before straining with a cloth tissue. A coconut beverage was prepared in a similar manner and blended with chickpea.
Chickpea Beverage (Vallath et al., 2021)	Raw chickpea seeds were soaked for 12-16h, then washed and either left raw, cooked in a pressure cooker at 80°C for 30 min or roasted in an air oven at 80°C for 20-25min. Raw, cooked and roasted chickpeas were submerged in water (1:4 to 1:5), wet milled and the resulting slurry filtered through muslin cloth. The raw chickpea milk was subject to a final pasteurization at 60°C for 30 min.
Faba Bean and Chickpea Beverages (Vasilean et al., 2021)	200g of chickpea and faba bean seeds were washed and soaked for 15 min in 70% aqueous alcohol solution before further soaking in tap water for 12h. Samples were drained and wet milled with 750mL of tap water for 10 min before filtering through cotton gauze. The beverages obtained were hydrolyzed by adding 1g/100g starch of alpha amylose followed by heating to 70°C for 1h under continuous stirring. The temperature was then reduced to 60°C prior to the addition of 1g/100g starch amyloglucosidase then left for 1h. Inactivation of enzymes occurred by holding a temperature of 90°C for 10 min. The hydrolyzed beverages were inoculated with one of two different starter culture mixtures (Strepcococcus thermophilus mixed with Lactobacillus delbrueckii subsp. Bulgaricus OR Lactobacillus casei, Lactococcus lactis subsp. Cremoris, Lactococcus lactis, subsp. Lactis, Leuconostoc, Lactococcus lactic subsp. Biovar, and Steptocuccus thermophilus missed with diacetylactis) at 43°C for 10h until pH 4.6 prior to cooling and storage at 4°C.
Fermented Lentil Beverage (Verni et al., 2020)	Lentil seeds were suspended in water using a 1:10 dry weight to water ratio and soaked at room temperature for 16h. Soaked samples were homogenized and blended to create 20g of slurry before the addition 12.5mg and 25 mg of Papain, bromelin, Veron HPP and Veron PS enzymes for enzymatic hydrolysis conducted for 1h at 30°C. The hydrolyzed samples were filtered with cotton gauze before centrifuged at 10,000 rpm for 5 min and heated to 110°C for 10 min. Samples were then singularly inoculated with 6 log CFU/mL of L. Acidophilis ATCC 4356, L.fermentum DSM 20052, or L.paracasei subsp. Paracasei DSM 20312 for 24h at 37°C under anaerobiosis. A non-inoculated beverage was used as control.
Fermented Chickpea Beverage (Wang et al., 2018)	Chickpea seeds were soaked overnight at a ratio of 1 to 3.3 before milling with 6 parts water and filtration through cotton cheesecloth. The resulting beverage was then boiled for 10 min before cooling. Samples were then inoculated with 0.5% of yogurt starter (maltodextrin, Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus) and fermented for 16h at 42°C before refrigerated storage.
Lupin Beverage (Xia et al., 2018)	Dehulled lupin flour was combined with water (1:20) and blended for 5 min before homogenized at 25MPa or using a high-pressure homogenizer at 50, 100 or 175 MPa for 1,4 or 6 passes. Samples were cooled before storage for 28 days at 4°C.
Fermented White Kidney Bean Beverage (Ziarno et al. 2019)	White kidney bean seeds were germinated for 72h at 25°C with added water to moisten the seeds every 24h. 1000g of germinated seeds were blended for 20 min, boiled and continuously stirred prior to filtering through 0.1mm sieve into 170mL jar. The jar was then sterilized at 121°C for 20 min and cooled to 45°C prior to inoculation with one of two yogurt starter culture mixes (Yo-Mix 205 LYO or FD-DVS ABY-3 Probio-Tec) and fermented at 43°C for 4h before cooling to 6°C after which 20g/100g of fruit flavouring (strawberry, peach-mango, apricot, forest fruits, banana-kiwi and red orange) were added or the beverage was left unflavoured.
Fermented White Kidney Bean Beverage (Ziarno et al., 2020)	White kidney been seeds were germinated for 72h at 25°C before mixing with water at a 1:9 ratio and blended. The homogenized samples were boiled for 1h prior to sterilization at 121°C for 15 min. 100mL samples were then inoculated at 37°C for 18h using single Lactobacillus strains to obtain cell count of 1x108 CFU/g before cooling to 5°C.
Fermented Chickpea Beverage (Zhang et al., 2022)	Chickpea seeds were soaked in water (1:5) overnight before milling in six parts water for 30 min and filtered using a gauze. The filtered samples were pasteurized by boiling for 10 min before being inoculated with 0.07% (w/c) of L. plantarum at 10 log CFU/mL and fermented for 24h at 37°C.
Fermented Chickpea Beverage (Zhang et al., 2022b)	Chickpea beverages were prepared as described above by Zhang et al. (2022) but subject to additional enzymatic hydrolysis at 50°C for 30 min that was inactivated by heating for 5min at 95°C followed with the addition of 5% (w/v) of glucose prior to incubation. Five treatments were applied: 1) without enzyme addition 2) 80 U/g protein papain 3) 60 U/g starch alpha-amylase 4) 80 U/g protein papain and 60 U/g starch alpha-amylase 5) 80 U/g protein papain and 60 U/g starch alpha-amylase with 10% (v/v) glucosidase.

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