Development of a Plant-Based Beverage with Tarwi (Lupinus mutabilis) Milk, Polysaccharides from Cushuro (Nostoc sphaericum), and Blueberry Extract ^†

Abstract

Plant-based milk alternatives are a rapidly growing niche in functional beverages, driven by demand from vegans and lactose-intolerant consumers. However, commercial options often have low protein content (<1.5%) and contain additives. Tarwi, native to the Andes, is rich in protein (45%), oils, and essential nutrients, while blueberries offer flavor and health benefits. Cushuro (Nostoc sphaericum), is an Andean microalga and contains high protein content and polysaccharides (42%). The objective of this research was to develop a plant-based beverage (PBB) with tarwi (Lupinus mutabilis), polysaccharides from cushuro (PC) and blueberry extract (Vaccinium corymbosum)(BE), compared with a control sample with carboxymethyl cellulose (CMC), a commercial thickening agent. The beverage was optimized and characterized using a design of rotatable central composite of surface methodology with nine formulations and four replicates in the center point. The effects of three independent variables were examined: tarwi milk (45% to 55%) and polysaccharides from cushuro (0.05% to 0.2%). The variable blueberry extract content was used as the differential factor between these two conditions. The response variables were protein (%) content and viscosity coefficient (mPa·s). The optimized beverage showed high protein (2.72%) content, viscosity coefficient (23.05 mPa·s), °Brix (2.5), pH (4.49), and acceptable sensory attributes using a 1-to-9-point hedonic scale with 67% positive acceptance. This powdered beverage complied with the Peruvian normative NTP 203.111.2021. Thereby, the plant-based beverage could be a nutritious alternative to functional plant-based beverages.

1. Introduction

The global PBB market is estimated to be worth USD 12.1 to USD 18.5 billion by 2022, and it is projected to reach over USD 24 billion by 2025. Consumers are increasingly adopting a healthy plant-based diet, which is the reason why the food industry is orienting its production towards plant-based products as an alternative to an animal-based diet. Despite the high nutritional value of animal-based foods, the intake of fatty meats can increase the risk of non-communicable diseases (NCDs) [1]. Most vegetable drinks (almond, oat, cashew, etc.) contain less than 1.5% protein and have a low concentration of essential amino acids (35.1% ± 0.2%) [2]. Tarwi (Lupinus mutabilis) is a legume cultivated in the Andes of Peru, Bolivia, and Ecuador. It contains protein between 32% and 51.6%, a high oil (13–24%) content, and minerals such as iron, magnesium, and phosphorus [3]. Blueberries (Vaccinium corymbosum) are high in anthocyanins, which act as protective antioxidants against oxidative stress and inflammation. Cushuro (Nostoc sphaericum) is an Andean microalga that grows at more than 3000 and 4000 m.a.s.l. It is consumed for its high content of fiber, proteins, vitamins, minerals, and polysaccharide compounds [4] that can be used as stabilizers and thickening properties in the beverage and food industry.

The objective of this research was to optimize, characterize and evaluate the PBB with polysaccharide from cushuro (Nostoc sphaericum), tarwi milk (Lupinus mutabilis) and blueberry extract (Vaccinium corymbosum) with the commercial additive CMC.

2. Materials and Methods

2.1. Raw Material

Tarwi (Lupinus mutabilis) was obtained from the Collahuasi district (3310 m.a.s.l.), Recuay province, Ancash region, Peru. The tarwi seeds were placed in a container filled with water (1:2 w/v). It was soaked for 24 h, changing the water approximately every 6 h. The hydrated tarwi seeds were boiled in water for 2 to 3 h to remove the bitter alkaloids present in the seeds. Blueberry (Vaccinium corymbosum) was obtained in the local market of Lima City, Perú.

The polysaccharide from cushuro (Nostoc sphaericum) was obtained at Laboratorio de Alimentos Funcionales of the Universidad de Lima, according to Chasquibol et al. [5]. The cushuro flour was mixed with water, heated to 80 °C, and stirred for 30 min. The supernatants were filtered under vacuum using a muslin cloth. The final filtrate was concentrated with a rotary evaporator (Buchi B-100, Flawil, Switzerland) and then precipitated using isopropanol (70%). The precipitate was dried at 50 °C and stored in aluminized bags at room temperature until use.

2.2. Experimental Design

To optimize protein content (%) and to obtain a viscosity coefficient greater than 22 mPa·s, the experimental design employed a response surface methodology (RSM) with a rotating central composite design (CCD) using Minitab 19 software (USA). Two independent variables were used: X1—tarwi milk (TM) (%), and X2— polysaccharides from cushuro (PC) (%). The blueberry extract content variable was determined by the differential between the two previous factors (Table 1). The TM concentration was optimized in the range between 45% and 55% to maintain the purple color of the blueberry and avoid the white color of the tarwi milk. Additionally, the blueberry extract concentration variable was determined by the differential between the above factors (Table 1).

Table 1. Experimental design to development of plant-based beverages formulations with tarwi milk (TM), blueberry extract (BE), and polysaccharides from cushuro (PC).

Formulation	StdOrder	RunOrder	PtType	Blocks	TM (%)	PC (%)	BE (%)
F1	1	1	1	1	45.00	0.05	54.95
F2	2	2	1	1	55.00	0.05	44.95
F3	3	3	1	1	45.00	0.20	54.80
F4	4	4	1	1	55.00	0.20	44.80
F5	5	5	−1	1	42.93	0.13	56.94
F6	6	6	−1	1	57.07	0.13	42.80
F7	7	7	−1	1	50.00	0.02	49.98
F8	8	8	−1	1	50.00	0.23	49.77
F9 a	9	9	0	1	50.00	0.13	49.87
F10	10	10	0	1	50.00	0.13	49.87
F11	11	11	0	1	50.00	0.13	49.87
F12	12	12	0	1	50.00	0.13	49.87
F13	13	13	0	1	50.00	0.13	49.87

^a F9, central point worked with four repetitions. Variable blueberry extract are the difference between tarwi milk (TM) and polysaccharides from cushuro (PC).

2.3. Development of the Beverages

For the development of the plant-based beverages (PBBs), fresh tarwi was liquefied in a solid-to-water ratio 1:3 (w/v). Blueberries were heated in a beaker with water (1:3 w/v) at 80 °C for 15 min to retard all biochemical reactions produced by the polyphenol oxidase enzyme during processing. The blueberry extract was vacuum filtered and mixed with the tarwi milk and polysaccharide from cushuro. Complete homogenization was carried out in a blender at a speed of 2900 rpm for 1.5 min and the PBB was pasteurized at 80 °C for 10 min and cooled in a water bath at 20 °C. The control sample was prepared with carboxymethylcellulose (CMC) in substitution of polysaccharide from cushuro.

2.4. Proximal Composition

The ash content was measured using the ignition method at 500 °C for 22 h. The total protein content was calculated as nitrogen percentage multiplied by 6.25 using a Kjeldahl analyzer (UDK 139, VELP, Usmate Velate, Italy) according to official methods [4].

2.5. Viscosity Coefficient

The oscillatory rheological tests were determined by the methodology described by Jeong et al. [6], with some modifications to suit the experimental conditions. The measurements were obtained using a Rheometer (MCR 92 Viscometer, Anton Paar, Graz, Austria) equipped with a plate-plate geometry setup (35 mm diameter and 1 mm gap). The viscosity was determined at 25 ± 0.1 °C and applying a constant shear stress of 20 Pa.

2.6. Antioxidant Activity

The antioxidant activity of the samples was assessed using the DPPH method of Chasquibol et al. [5], described with some modifications, at 517 nm using a UV-Vis spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan). The results were expressed in µg of Trolox per gram of beverage. Each analysis was performed in triplicate, and the values are reported as means.

2.7. Determination of Amino Acid Profile

The amino acid profile was determined following the method of Alaiz et al. [7] with slight modifications. The analysis was performed using HPLC (ARC, Waters, Milford, CT, USA) equipped with a C18 reverse phase column (150 mm × 9 mm internal diameter). Tryptophan was quantified separately by HPLC after undergoing basic hydrolysis. All measurements were conducted in triplicate.

2.8. Sensory Analysis

A sensory evaluation panel composed of 40 individuals, specifically university students aged from 18 to 25, assessed the attributes of appearance, flavor, texture, and overall acceptability. This evaluation employed a 9-point hedonic scale ranging from 1 (extreme dislike) to 9 (extreme like). Panelists were provided with randomly assigned samples. The overall acceptability of the plant-based beverage was determined by calculating the average scores of all sensory attributes.

2.9. Statistical Analysis

The results were presented as mean ± standard deviation, with most measurements performed in triplicate. However, the antioxidant activity measurement was conducted in duplicate. Data were analyzed using analysis of variance (ANOVA) followed by a post hoc Tukey test to identify significant differences (p < 0.05) between the two variables, utilizing Minitab^® statistical 19 software (State College, PA, USA).

3. Results and Discussion

3.1. Design of Experiment

To optimize protein (%) content was employed the response surface experimental design (RSM) with a rotating central composite (CCD). Table 2 shows thirteen formulations with four replicates at the central point, resulting in protein (%) content and viscosity (mPa·s) coefficients. All the results showed an interaction between tarwi milk (TM), polysaccharides from cushuro (PC), and blueberry extract (BE). The protein content ranged from 1.43% to 2.80%, while the viscosity coefficient varied between 20.52 mPa·s and 41.67 mPa·s, respectively. The highest protein content was obtained for formulation F6 (2.80% ± 0.03) and the highest viscosity coefficient was observed for formulation F8 (41.67 ± 0.04 mPa·s.).

Table 2. Results of experimental design of the plant-based beverage formulations according to tarwi milk (TM), blueberry extract (BE), and polysaccharides from cushuro (PC).

Formulation	TM(%)	PC (%)	BE (%)	Protein (%)	VC (mPa·s)
F1	45.00	0.05	54.95	1.43 ± 0.04	23.32 ± 0.07
F2	55.00	0.05	44.95	2.57 ± 0.02	23.44 ± 0.09
F3	45.00	0.20	54.80	1.46 ± 0.03	37.58 ± 0.12
F4	55.00	0.20	44.80	2.49 ± 0.01	39.56 ± 0.06
F5	42.93	0.13	56.94	1.59 ± 0.02	29.05 ± 0.03
F6	57.07	0.13	42.80	2.80 ± 0.03	31.31 ± 0.05
F7	50.00	0.02	49.98	2.09 ± 0.02	20.52 ± 0.08
F8	50.00	0.23	49.77	2.08 ± 0.02	41.67 ± 0.04
F9	50.00	0.13	49.87	2.05 ± 0.01	32.56 ± 0.02
F10	50.00	0.13	49.87	2.10 ± 0.03	32.33 ± 0.01
F11	50.00	0.13	49.87	2.14 ± 0.02	31.42 ± 0.12
F12	50.00	0.13	49.87	2.16 ± 0.04	32.43 ± 0.07
F13	50.00	0.13	49.87	2.17 ± 0.02	35.30 ± 0.05

Results are expressed as means ± SD (n = 3).

Table 3 shows the regression models and adjustments of the central composite design (CCD) for the independent variables: (TM) and (PC) based on the dependent variables (protein content and viscosity coefficient). After obtaining the results of the output variables, Minitab was used to optimize the plant-based beverage formulation. The parameters were set according to the results of TM (45–55%) content and PC (0.05–0.2%) content in order not to increase the tarwi milk content more than 55%, which could interfere with the purple color of the blueberry extract. The protein content was set a maximum value of 2.8%, while the viscosity coefficient was established a maximum value of 25 mPa-s.

Table 3. Regression models of the central composite design (CCD) for independent and dependent variables evaluated in the formulations of plant-based beverages.

Variable	Equation
Protein content	y= −3.60 + 11.7 TM + 612 PC − 1.1 TM × TM − 102444 PC × PC − 733 TM × PC
	R2 = 95.85%
	p-value = 0.000
Viscosity coefficient	y = −103.7 + 490 TM + 7294 PC − 7294 PC − 492 TM × TM − 1375447 PC × PC + 12382 TM × PC
	R2 = 98.85%
	p-value = 0.000

Figure 1 shows that protein content increase in relation to the percentage of TM content. The region of greatest interest for the protein content of more than 2% was upper than 50%. The viscosity coefficient increased in direct relation to the concentration of the polysaccharide from cushuro, in the region of major interest superior to 0.01%, for a viscosity coefficient above 20 mPa-s.

Figure 1. Contour plot of the central composite design for protein content and viscosity coefficient.

Figure 2 shows the optimization of the independent variables that affect to protein content and viscosity capacity. The optimal formulation was: tarwi milk (56.98%), polysaccharide from cushuro (0.07%) and blueberry extract (42.95%), with protein content of 2.8% and viscosity coefficient of 25 mPa·s.

Figure 2. Optimization of the Box–Behnken design obtained from the Minitab 19 software.

3.2. Physicochemical Characterization

Table 4 shows the physicochemical characterization of the PBBs. The PBB with PC showed a higher protein (2.72% ± 0.12) content than the PBB with CMC (2.36 ± 0.15). Lopes et al. [8] showed protein content between 1.8% and 2.4% (w/v) in the tarwi-based beverage. On the other hand, seed-based beverages are characterized by their variable lipid (ca. 1–7%) and protein (ca. 1–5%) content [9].

Table 4. Physicochemical characterization of plant-based beverage with polysaccharide from cushuro (PBB+PC) and plant-based beverage with carboxymethyl cellulose (PBB+CMC).

Sample	Protein Content (%)	Viscosity Coefficient (mPa·s)	pH	°Brix	Ash (%)	DPPH (µg Trolox/g Beverage)
PBB + PC	2.72 ± 0.12 a	23.05 ± 0.09 a	4.49 ± 0.2 a	2.5 ± 0.24 a	0.03 ± 0.01 a	1098.98 ± 63.48 a
PBB + CMC	2.36 ± 0.15 b	21.04 ± 0.13 b	4.3 ± 0.13 a	2.3 ± 0.31 a	0.02 ± 0.02 a	746.44 ± 32.98 b

Results are expressed as means ± SD (n = 3). a and b values in the same column with different letters differ significantly when p < 0.05. PBB + PC: plant-based beverage with polysacharides from cushuro; PBB + CMC: plant-based beverage with carboxymethylcellulose.

The viscosity coefficient in the PBB with PC was much higher (23.05 ± 0.09 mPa·s) than the PBB with CMC (21.04 ± 0.13 mPa·s) (Table 4). PC is a branched hydrocolloid that can form many hydrogen bonds and greatly increase the viscosity. In addition, a significant interaction effect of PC concentration on the °Brix was observed.

The addition of hydrocolloids (PC and CMC) (0.07% w/v) and BE (42.95% w/v) also influenced pH. The pH (4.49 ± 0.2) of PBB + PC was higher than the pH (4.3 ± 0.13) of PBB + CMC. The addition of commercial CMC (0.1, 0.3 and 0.5%) to snake fruit syrup did not affect pH. However, the addition of pectin (0.2 and 0.3%) and xanthan gum (0.1 and 0.2%) to orange juice with pulp showed a significant influence on pH [10].

The DPPH results are shown in Table 4. The PBB with PC had a significantly higher DPPH (1098.98 μg GAE/g ± 63.48) value than the PBB with CMC (746.44 μg GAE/g ± 32.98). The observed difference in DPPH values was due to the contribution of antioxidants from the cushuro polysaccharide and blueberry extract.

3.3. Amino Acid Profile

According to Table 5, the relevant amino acids in the PBB with PC were lysine (58.08 ± 12.71 mg/g protein), threonine (56.15 ± 35.89 mg/g protein) and tyrosine (45.43 ± 14.59 mg/g protein). According to the protein of reference established by FAO/WHO [4], the composition of amino acids in the plant-based beverage presented a balanced profile. Walther et al. [2] reported that plant-based beverages made from oats, coconut, cashew, spelt and other ingredients had an average value of the sum of amino acids (AA) ranging from 8.1 mg AA/g protein to 11.5 mg AA/g protein. The PBB with PC had a high percentage of tryptophan, above the recommendations of the FAO (6 mg/g protein).

Table 5. Amino acid profile of plant-based beverages with polysaccharide from cushuro (PBB+PC) and plant-based beverages with carboxymethyl cellulose (PBB+CMC).

Amino Acids	PBB + PC (mg AA/g Protein)	PBB + CMC (mg AA/g Protein)	FAO/WHO (mg AA/g Protein)
Aspartic acid	24.19 ± 1.51 a	22.36 ± 1.79 a
Glutamic acid	35.14 ± 15.00 a	33.63 ± 1.10 a
Serine	44.23 ± 1.23 a	44.52 ± 3.79 a
Histidine	22.11 ± 2.00 a	33.45 ± 4.37 b	15
Glycine	20.00 ± 10.22 a	18.15 ± 2.90 a
Threonine	56.15 ± 35.89 a	55.65 ± 4.04 a	23
Arginine	16.99 ± 7.04 a	13.97 ± 1.77 a
Alanine	14.51 ± 1.82 a	16.04 ± 4.36 a
Proline	33.76 ± 21.18 a	32.85 ± 2.35 a
Tyrosine	45.43 ± 14.59 a	45.61 ± 13.43 a	38
NH3	34.30 ± 4.59 a	34.56 ± 1.54 a
Valine	32.22 ± 14.27 a	32.97 ± 12.73 a	39
Methionine	32.17 ± 13.13 a	32.16± 13.24 a	22
Cysteine	25.78 ± 17.01 a	26.71 ± 4.70 a
Isoleucine	17.14 ± 1.23 a	44.80 ± 4.24 b	30
Leucine	42.33 ± 4.69 a	28.37 ± 2.77 b	59
Phenylalanine	45.09 ± 11.18 a	35.05 ± 4.61 b
Lysine	58.08 ± 12.71 a	56.42 ± 5.90 a	45
Tryptophan	15.03 ± 3.57 a	13.26 ± 6.47 a	6

Results are expressed as means ± SD (n = 3). a and b values in the same row with different letters differ significantly when p < 0.05. Note: According to the FAO Database on Protein Quality Evaluation [4].

3.4. Sensory Analysis

A comparative analysis was performed between the sample (PBB + PC) and the control sample (PBB + CMC). Both beverage displayed a light purple coloration. (PBB + PC) attained an average consumer acceptance rate of 67%, whereas (PBB + CMC) received an average approval rating of 62%. Regarding specific sensory attributes, including appearance, colour, flavour, texture, and taste, (PBB + PC) was rated at 8.7, 7.8, 6.5, 6.7, and 7, respectively. (PBB + CMC) obtained scores of 8.5 for appearance, 7.2 for colour, 6.6 for flavour, 5.9 for texture, and 6.8 for taste.

4. Conclusions

The PBB with PC was optimized using a central composite rotational design and validated experimentally. The formulation optimized was: tarwi milk (56.98%), polysaccharide from cushuro (0.07%), and blueberry extracts (42.95%). The synergy between tarwi milk, polysaccharide from cushuro and blueberry extract provided a unique nutritional profile with high protein content, high viscosity, °Brix, antioxidant capacity and amino acid profile than control sample with CMC. The favorable sensory acceptance suggests strong potential for market success, positioning this beverage as an innovative alternative in the plant-based and functional food sector.