Characterising the Nutritional and Alkaloid Profiles of Tarwi (Lupinus mutabilis Sweet) Pods and Seeds at Different Stages of Ripening

Abstract

Tarwi (Lupinus mutabilis Sweet) is a key crop for Andean indigenous communities, offering proteins and fats. Both the pods and seeds of tarwi are consumed, either in their tender (immature) state or as dried, fully ripe seeds. Tarwi, like other Lupinus species, contains high alkaloid levels in its fruits and seeds that must be removed before consumption. This study evaluated the fat, protein, fibre, and alkaloid contents of four cultivars at five maturity stages ranging from 180 to 212 days after sowing. Samples of the pods and the seeds were analysed to determine their colour and protein, crude fibre, fat, and alkaloid contents. The results showed that while the protein concentration in the pods decreased as the fruits matured, the protein content in the seeds increased, reaching approximately 41%. Moreover, the pods exhibited a significant decrease in alcohol content, with the values dropping below 1% at the senescent (dry) stage for all the cultivars. In contrast, the alkaloid levels in the seeds remained stable from 196 days after sowing in the Guaranguito, Andean, and Ecuadorian cultivars, with concentrations around 4%. The present study showed that as the pods matured, their overall protein content decreased, while their seed protein content increased to around 41%. The alkaloid levels in the pods dropped below 1% in the dry stage, while the seed alkaloid levels remained stable at around 4% in the Guaranguito, Andean, and Ecuadorian cultivars after 196 days. However, in the Peruvian cultivar, the alkaloid content remained constant starting from 188 days after sowing, with concentrations just over 3%. This result suggests that as the pods mature, their alkaloid content decreases, while the alkaloid levels in the seeds stabilise from around 188 to 196 days after seeding. Consequently, the alkaloid contents found in the seeds likely originate from other aerial parts of the plant and are not significantly increased by the pods.

1. Introduction

The Andean legume Lupinus mutabilis, known as chocho or altramuz in Spanish and tahuri or tarwi in Quechua (from which the English name tarwi is derived), is a significant crop for the subsistence agriculture of indigenous communities in the Andes [1]. Tarwi belongs to the Fabaceae family and is an annual herbaceous plant that can reach up to 2 m in height. This plant is notable for its protein-rich seeds and ability to fix nitrogen in poor soils. The plants commonly present “V”-shaped branching, with digitate leaves featuring from five to twelve thin, oblong–lanceolate leaflets and small stipules at the base of the petiole. The inflorescence of tarwi is a terminal raceme, with flowers in whorls. This plant can produce more than 60 flowers, but not all of them will fructify. Tarwi is valued for its high protein and oil contents, containing up to 44% oil and 18% protein by dry weight, which exceed the values of other legume species [2]. In particular, the carbohydrate content is low in starch, but high in oligosaccharides such as stachyose and raffinose. Tarwi seeds are also a good source of essential amino acids, particularly lysine, dietary fibre, and healthy fatty acids [3]. These nutritional properties make tarwi an attractive food source, though this plant’s cultivation is limited to the Andean highlands, where indigenous communities grow it for their livelihoods. L. mutabilis is a traditional crop in the Ecuadorian Andes due to its hardiness, which allows it to thrive in poor soils in the harsh Andean climate [4]. While tarwi is mainly produced for human consumption, the plant is also used as livestock feed. Tarwi are colloquially called Andean soya beans because the nutritional value of tarwi seeds is similar to that of soya beans. However, tarwi seeds are difficult to use as a food crop due to the high alkaloid content in their seeds, which must be eliminated or reduced to safe concentrations before consumption. Nevertheless, this alkaloid content provides the plant with resistance to pests and microbial infections [5]. Due to its high nutritional content, tarwi has been long utilised in the agrifood industry and has great potential as a raw material. Tarwi paste is used in the preparation of different products, such as beverages, cookies, bread, and products similar to milk, while tarwi flour is used to enhance the protein content of different processed foods [6]. Fresh tarwi seeds are also used to dress salads, prepare different stews, and produce vegan burgers. In addition, fermented seeds are used in the preparation of tempeh and low-alcoholic beverages [7]. However, processing tarwi as food requires debittering due to the high alkaloid contents in their seeds. This preparation process seeks to eliminate or at least reduce the alkaloid content to safe levels. Although there are different debittering techniques, they all typically involve washing the seeds before cooking followed by successive washings until the alkaloid content is removed or reduced [8,9]. The traditional tarwi harvest begins when the stalks are dry and brittle, the leaves turn brown, and the seeds develop a whitish colour, with a moisture content of less than 14%. At this stage, the seeds have a high concentration of alkaloids such as sparteine and lupanine, as well as various compounds that act as protease inhibitors and glycosides with cyanogenic activity [10]. These compounds must be removed before the seeds can be consumed. Although harvesting at an earlier stage, known as the tender grain, is possible (i.e., when the seeds develop a lime green colour and have a higher degree of humidity), this process is unusual due to the arduous work involved in harvesting, as the pods must be collected individually. However, indigenous Andean communities do practice tarwi harvesting at this stage for specific culinary purposes.

Although L. mutabilis possesses a significant gene pool; many cultivars have a strong local character. In Ecuador, Guaranguito (Iniap 451) and Andino (Iniap 450) are the most common genotypes, while in other Andean areas such as Bolivia, the most representative genotype is Carabuco [11]. While different ecotypes exist, the Ecuadorian and Peruvian varieties are particularly notable because of their hardiness. Moreover, there is variability in the alkaloid composition and other nutritional properties of each genotype. Compositional differences can also be observed in the post-harvest processes, such as debittering and other agroindustrial practices [10]. The Guaranguito and Andino genotypes, which originated from the breeding programme of the Instituto Nacional de Investigaciones AgroPecuarias (National Institute of Agro-Livestock Research), have a growth cycle of approximately 6 months and a high alkaloid content. The cultivation of these genotypes is restricted to the provinces of Cotopaxi, Chimborazo, and Pichincha in the highlands between 2500 and 3600 m above sea level. According to the FAO data, the sowing area is close to 3500 hectares, offering yields of just over 3900 kg per hectare [12]. L. mutabilis is one of the main components in the agroecosystems of the Andean highlands, as this species is capable of providing soil with up to 500 kg of nitrogen per hectare due to its photosynthetic efficiency, its ability to solubilise P from the soil, and its ability to fix nitrogen symbolically [1,13]. Crop residues are often used as green manure, and dried stalks are used as fuel by indigenous communities due to their high calorific value. Tarwi is also used to restore soil fertility after clearing land and buildings. All these uses beyond food make tarwi a valuable crop that satisfies many basic needs of the Andean highland population [6,10].

Although numerous analyses have evaluated the contents of alkaloids, proteins, and other nutritional compounds [5], as well as the contents of anti-nutritional and nutritional compounds in lupine seeds after debittering [8,9], these studies were conducted on fully mature seeds. However, studies on the protein contents in developing seeds and pods are less numerous, with many focused on the nutritional analyses of foods prepared from immature seeds and pods [3,6,7,13]. To fill this gap, the present study evaluated the physicochemical characteristics of the pods and seeds from four tarwi cultivars at different stages of maturity. Although tarwi is consumed primarily in its dry stage, tender seeds are also used for different agroindustrial purposes. The alkaloid content of the pods and seeds in both the tender and ripe states was also evaluated.

2. Materials and Methods

2.1. Plant Material

The Andean Grains Project of the Faculty of Agricultural Sciences and Natural Resources of the Technical University of Cotopaxi provided four germplasms of Lupinus mutabilis: two improved cultivars (Iniap-450, known as Andino, and Iniap-451, known as Guaranguito) and two ecotypes, Ecuadorian and Peruvian. These four genotypes were grown on an experimental field at the Universidad Técnica de Cotopaxi at the Salache Experimental Centre, Latacunga, Ecuador, located 2770 m above sea level (latitude 0°58′60′′ W and 78°37′0′′ W) following cultural and traditional practices specific to the tarwi crop. The agroclimatic conditions included 500 mm of average annual rainfall and an average temperature of 14 °C. The four cultivars were grown in separate 100 m² plots at a density of 7.3 plants/m². Each plot was divided into 4 subplots of 180 plants, which comprised the replicates. Five stages of maturity were considered, from tender grain to dry grain, corresponding to 180, 188, 196, 204, and 212 days after sowing. In each subplot, 10 plants were selected, and 5 pods per plant were picked on each sampling date. As a result, 200 pods per cultivar were randomly collected on each day of sampling.

2.2. Analysis

To determine the colour of the pods, a Minolta tristimulus colourimeter (Minolta Camera, Osaka, Japan) was used to quantify the L, a, and b parameters of the CIELAB colour space, where L* represents the lightness from black (0) to white (100), a* represents green–red tonalities, and b* represents yellow–blue tonalities. With these parameters, the colour index (CI) was calculated, applying the relationship indicated by Jiménez et al. [14]:

$$CI={\displaystyle \frac{1000·a}{L·b}}.$$

(1)

After their colour was determined, the pods were shelled, and the colour of the seeds 139 was measured.

The alkaloid, crude fibre, fat, and protein contents were determined in both the pods and seeds. For each cultivar and replicate, the pods and the seeds pooled after the shelling of the fruits were divided into three batches that were analysed in triplicate. Crude fibre, fat, and protein were analysed following procedures 991.43, 981.10, and 9020.39 of the AOAC [15]. The total alkaloid content was evaluated according to the von Baer volumetric method [16].

2.3. Statistical Analysis

The data obtained were checked for normality using a Kolmogorov–Smirnov test. Analysis of variance (ANOVA) of the sampling time and cultivars was performed to calculate the statistical significance. If the ANOVA F test was positive, a post hoc LSD test was performed to compare the means. All statistical analyses were performed with Statgraphic Centurion XVI (STATGRAPHICS. Statpoint Technologies, Inc., Warrenton, VA, USA).

3. Results

3.1. Nutritional Content in Pods

Table 1. Fat, protein, and crude fibre contents and colour index of pods of four Lupinus mutabilis cultivars.

	Days after Sowing
Cultivar	180	188	196	204	212
	Fat (%)
Peruvian	1.27 b	1.93 a	0.57 c	1.33 b	2.00 a
Ecuadorian	1.00 b	1.67 a	0.73 c	1.00 b	1.17 b
Andino	1.50 a	1.50 a	0.50 c	0.50 c	1.00 b
Guaranguito	1.00 b	1.33 b	1.33 b	1.67 a	0.50 c
	Crude fibre (%)
Peruvian	35.67 b	40.67 a	42.33 a	43.00 a	42.67 a
Ecuadorian	34.33 b	39.33 a	40.67 a	40.33 a	41.00 a
Andino	33.50 b	42.00 a	41.50 a	40.00 a	39.50 a
Guaranguito	40.33 a	34.00 b	36.00 b	35.67 b	35.67 b
	Protein (%)
Peruvian	11.47 a	4.94 b	3.89 b	4.48 b	4.08 b
Ecuadorian	15.81 a	8.96 b	9.13 b	5.54 c	5.41 c
Andino	15.98 a	11.08 b	6.33 c	6.22 c	3.11 d
Guaranguito	13.81 a	12.83 a	7.39 b	4.66 c	3.99 c
	Colour Index
Peruvian	−30.85 d	−7.39 c	−4.04 c	5.44 b	33.60 a
Ecuadorian	−38.13 d	−7.67 c	−3.35 c	5.53 b	45.30 a
Andino	−22.52 d	−5.35 c	−3.23 c	6.02 b	39.84 a
Guaranguito	−32.81 d	−9.43 c	−4.04. c	6.86 b	38.23 a

Different lowercase letters indicate significant differences within each treatment according to LSD at p < 0.05 (n = 36).

shows the increase in the fat, crude fibre, and protein contents, as well as the colour index of the pods of the cultivars used in this trial. The Ecuadorian and Andean cultivars yielded the highest fat content at 188 days. However, the Peruvian cultivar produced the highest content at 188 and 212 days, with no significant differences between these two sampling days. Guaranguito presented the highest fat content at 204 days. Regarding the crude fibre content, three of the four cultivars followed a clear pattern. From 188 days onwards, there were no differences in fibre content between the different sampling times for the Andino, Ecuadorian, and Peruvian cultivars. However, in the Guaranguito pods, the highest fibre content was observed 180 days after sowing. From this stage onwards, the fibre content ranged between 34 and 36%, with no significant differences between the sampling times. The protein content of the pods was high 180 days after sowing, and then decreased until the harvest. However, this decrease was more pronounced in the Peruvian cultivar, whose protein content was almost three times less than those of Guaranguito or Andino at 188 days. At harvest, 212 days after sowing, the pods of the four cultivars presented similar values, ranging between 3.11% and 5.41%. The values of the colour index gradually increased in all the cultivars, changing from dark green shades at 180 days to dark brown or very dark shades at 212 days. Notably, the pods of Andino at 180 days showed a lighter green colour compared to those of the other cultivars. At harvest, 212 days after sowing, all the pods exhibited a characteristic dark brown colour due to the dry state of the pods. However, the Ecuadorian cultivar presented darker pods than those of the other varieties (Figure 1).

3.2. Nutritional Content in the Seeds

Table 2. Fat, protein, and crude fibre contents and colour index of seeds of the four Lupinus mutabilis cultivars.

	Days from Sowing
Cultivar	180	188	196	204	212
	Fat (%)
Peruvian	12.67 c	14.83 b	17.50 a	16.50 a	15.50 a
Ecuadorian	12.33 b	12.83 b	15.67 a	15.00 a	16.83 a
Andino	11.00 c	13.67 b	16.33 a	16.83 a	14.50 b
Guaranguito	15.33 a	15.67 a	14.67 a	15.33 a	14.50 a
	Crude fibre (%)
Peruvian	10.33 b	11.86 b	11.33 b	11.33 b	18.67 a
Ecuadorian	12.00 b	11.67 b	11.67 b	15.80 a	10.33 b
Andino	12.67 a	10.83 a	11.67 a	11.33 a	11.67 a
Guaranguito	11.33 b	12.00 b	15.00 a	15.80 a	10.33 b
	Protein (%)
Peruvian	33.83 b	36.95 b	39.86 a	41.80 a	40.25 a
Ecuadorian	31.89 b	36.95 b	39.86 a	41.80 a	40.25 a
Andino	37.43 b	37.63 b	40.15 a	39.33 a	39.58 a
Guaranguito	33.83 b	36.60 b	39.89 a	40.05 a	40.25 a
	Colour Index
Peruvian	−8.82 d	−6.53 d	−1.76 c	0.56 b	1.14 a
Ecuadorian	−7.98 d	−6.76 d	−1.98 c	0.54 b	2.21 a
Andino	−6.81 d	−5.12 d	0.85 c	1.23 b	3.11 a
Guaranguito	−8.10 d	−6.93 d	−1.74 c	1.36 b	2.09 a

Different lowercase letters indicate significant differences within each treatment according to LSD at p < 0.05 (n = 36).

shows the fat, protein, crude fibre, and colour contents of the Lupinus seeds as a function of the sampling time. In the seeds of the different cultivars, the fat content increased as the fruit matured, except for the Guaranguito cultivar, whose seed fat content was maintained without significant differences between the various sampling times. At the mature stage of the seeds (212 days after sowing), the fat content of the different cultivars was found to be between 14.5% and 16.83%. The crude fibre content in the Peruvian cultivar seeds increased as the seeds matured, reaching 18.67% at the end of the maturation period. The seeds of the Peruvian cultivar had the highest crude fibre content 212 days after maturity. However, this pattern was not observed in all the cultivars. In the Andino cultivar, the fibre content remained constant from the green to the mature stage. The protein content of the seeds at the end of harvest was similar in all the cultivars. Overall, from 196 days to harvest at 212 days, the protein content did not notably increase. The colour index increased gradually from light green values, ranging from −6.81 to −8.82, to whitish colours at seed maturity, as indicated by a colour index value between one and three, with Guaranguito presenting a creamy white colour.

3.3. Alkaloid Content in Pods and Seeds

Figure 2 shows the alkaloid content in the pods and the seeds of the different cultivars. The alkaloid content decreased in the pods and seeds of all the cultivars, although in the pods, the decrease was continuous and gradual until it reached its minimum value when the seeds were dry and ready to be harvested. However, the alkaloid content in the seeds dropped to the minimum values after 196 days, except in the Peruvian cultivar, in which the minimum content was observed at 188 days and remained constant until the end. In other words, while the minimum alkaloid content in the seeds was observed starting from the third sampling time in the Andino, Ecuadorian, and Guaranguito cultivars, the minimum alkaloid value was observed approximately 8 days earlier in the Peruvian cultivar.

4. Discussion

The use of L. mutabilis as a food source is disadvantageous due to its high alkaloid content, as this crop must undergo debittering processes [17]. In Andean communities, tarwi is traditionally consumed as both dry and green seeds. The pods are also consumed because tarwi is a highly valued crop due to its high levels of protein. However, debittering remains necessary for tarwi, regardless of the form of consumption. The results of our study showed that the protein content of the pods was very high at 180 days after sowing, but decreased from that point onwards. On the other hand, the seeds’ protein content increased as the pods and seeds developed and matured (Table 1 and Table 2). This result is interesting because the consumption of pods in a fresh, unripe state depends on two factors, the degree of fibrousness, which is very high in the ripe state (to the point of being inedible), and the tenderness of the grains [6]. When tarwi is consumed as a dry seed, it can be assumed that the alkaloid content is unimportant since the seeds will be debittered. However, the traditional debittering process is costly and not environmentally friendly, requires high water consumption, and never ensures complete alkaloid removal. Therefore, cultivars with a lower alkaloid content in their dry seeds, such as the Peruvian variety, represent a promising alternative (Figure 2) [9,18].

Although very few studies have examined the development of pods and seeds in Lupinus, the relevant developmental processes have been categorised into five stages [19,20,21]. Most authors distinguish between the stages based on various morphological and anatomical characteristics that correspond to the size of the seed. The developmental stages chosen in our study were determined based on the time elapsed since sowing. Five stages were selected, ranging from the initial stage for green harvesting, which is traditionally performed in Andean communities when tarwi is consumed fresh, to the dry harvesting stage. During our first stage, 180 days after sowing, the seeds already had a light green colour, the embryos were developed, and the endosperms were very tender, coinciding with stages I and II of the aforementioned division. The remaining stages generally align with stages III and IV, as the last stage corresponds to the dry grain harvest stage. As observed in stages III and IV, the seeds acquire a darker green colour and reach their maximum size. This division is important because it allows us to observe how the protein content increases as the seeds develop, reaching concentrations of approximately 40%, which is consistent with the typical values observed in L. mutabilis and other Lupinus species.

The protein in the seeds is known to be synthesised from asparagine during seed growth and development [21]. However, the pods also play an important role. The compounds produced by the pods’ senescence are translocated to the seeds, where they are reused [22,23]. This phenomenon has been observed in various legumes, such as broad beans, peas, and different Lupinus species. According to Pampana et al. [24], although legumes are nitrogen fixers, this fixation does not provide all the nitrogen necessary for seed formation, leading to active translocation from the senescent organs. This factor may explain the observed decrease in protein content in the pods as they develop. At harvest, the protein content in the seeds of the studied cultivars was similar to that found in other experiments carried out in Mexico and Poland. In a cultivar grown in Mexico, the authors reported a slightly higher protein content than that obtained in our study. Moreover, practically the same content was found in seeds from the Plant Breeding Station located in Przebędo, Poland [25,26]. The protein content in Andean lupine depends on different elements, including genetic, agronomic, and environmental factors, as indicated by Czubinski et al. [25] and Carvajal-Larenas [5], with a protein concentration of approximately 53% observed in L. mutabilis under particularly favourable conditions. However, tarwi seeds commonly have protein content similar to that found in our study. The fat content of the seeds of different Lupinus species is quite variable, ranging from approximately 4.55% in L. albus to approximately 12% in L. angustifolius [26]. However, in our experience, the seeds were found to have a significantly higher fat content between 14.5 and 16.8% (Table 2). Although this value is far from the 20% mark that can be achieved according to Carvalho et al. [27], and even further from the 24.6% mark indicated by Carvajal-Larenas et al. [5], the fat content of tarwi seeds can be significantly reduced under unfavourable conditions such as droughts, which are a crucial factor [27]. In our experiment, the crop did not suffer from a drought because appropriate cultivation practices were followed, so the genetic factors influenced the results. It should also be noted that the fat values obtained are in line with those provided by previous authors [3,10,28]. The colours of the pods in the different species of Lupinus usually evolve from green in an immature state to dark brown and even black at the time of maturity. Our cultivars followed this trend with reasonable, but small differences between the different cultivars. A similar trend was observed in the seeds, starting from a light greenish colour to a white colour at maturity. These results are consistent with those of Chalapuente-Flores et al. [11] and Guilengue et al. [29]. However, tarwi seeds notably present a wide variety of colours, and this species has more variability in the colour of its seeds than both other legumes and the other species of Lupinus [29]. Nevertheless, there is a consumer preference for the whitish colouration of tarwi seeds [4]. Given that our cultivars are the traditional ecotypes (Peruvian and Ecuadorian) or improved Andino and Guaranguito cultivars, it is understandable that their seeds acquired white tones when they reached maturity (Table 2).

The distribution patterns of alkaloids in the pods and seeds during the development of both structures led to a decrease in alkaloid content, although this decrease stabilised in the seeds at 188 or 196 days, depending on the cultivar. Virtually all studies indicate that seeds do not synthesise alkaloids [30,31,32,33]. This result suggests that seeds are a powerful storage sink and that alkaloids must be transported from the aerial parts. Theoretical analyses by Lee et al. [34] based on the composition of sap and the economy of water indicated that almost half of the alkaloids present in seeds were synthesised in situ. However, the subsequent studies have shown that practically all of the alkaloids present in the seeds are imported from the aerial parts of the plant [31,32,33]. In fact, in the very early stages of pod development, the alkaloid content of the pods was very high compared to the content found in the seeds according to analyses performed on Lupinus angustifolius [33]. It is reasonable to assume that the pods are the primary source of alkaloids in seeds. Since the analyses in our study were carried out at a later stage of development, we found that the alkaloid content was similar in the seeds and the pods, but the concentration rapidly dropped in the pods and stabilised in the seeds (Figure 2). This behaviour is common in L. angustifolius and L. albus [33,34] and in L. mutabilis, as noted by Williams and Harrison [30]. Although very few studies have focused on the transport and accumulation of alkaloids in L. mutabilis seeds, similar to most analyses of L. angustifolius and L. albus, we assumed that the behaviour would not be different in L. mutabilis given the characteristics and biological role of the alkaloids present in the seeds of the different species of Lupinus, whose main function is to protect the seeds against pathogens and predators [5]. Frick et al. [31], who studied the expression of genes of the alkaloid biosynthetic pathway quinolizidine in both vegetative tissues and seeds, showed that alkaloids are produced primarily in the leaves and the branches, and then become translocated to the seeds. Moreover, although the pods may play a role in the supply of alkaloids, this role is not exclusive, as alkaloids from the branches and the leaves also contribute to the alkaloid content of seeds. Our results show that in all the varieties, the alkaloid concentration remained stable from 188 days onwards in the Peruvian cultivar and from 196 days onwards in the rest of the cultivars (Figure 2), with a value lower than that observe at 180 days. Conversely, the content in the pods continued to decrease. Furthermore, Williams and Harrison et al. [30] found that the lupanine content decreased rapidly in the fully expanded cotyledons in three species of Lupinus. The authors, however, suggested that these changes may simply reflect the onset of the drying phase, as indicated above, and the rapid catabolism of the alkaloids.

5. Conclusions

Our results show that the protein content of tender tarwi beans is lower than that of dry tarwi beans. However, since the consumption of tender beans is traditionally accompanied by the consumption of green and immature pods, protein intake increases due to the higher protein content of the pods at the younger stages, as shown in our results. Therefore, using fruits with pods featuring a colour index of around −30 is recommended, corresponding to a dark green colour, as these pods will have a higher protein content. However, the pods of the Andean cultivar are notably lighter in colour at the same stage of maturity.

Changes in the alkaloid content of the pods and the seeds are also of interest. In the pods, a higher stage of maturity corresponds to a lower alkaloid content. However, the alkaloid content in the seeds remained constant from 188 to 196 days after sowing. This result could indicate that the pod does not play an important role as a source of alkaloids for seeds and that the alkaloids in the seeds instead come from other aerial parts.