1. Introduction
Legumes are important dietary sources of essential nutrients for human and animal nutrition, such as protein, complex carbohydrates, vitamins, and minerals. However, they also contain non nutritional compounds (α-galactoside oligosaccharides, trypsin inhibitors, lectins, polyphenols, or phytic acid) that can interfere with the nutritional value of legumes but at the same time potentiate/increase their value as functional foods [
1]. In addition to the presence of antinutritional components, legumes and other plant-based protein sources have a lower potential to boost protein metabolism and retention rate at the whole body or muscle levels in animals or humans when compared to animal-based proteins. They also exhibit lower digestibility and incomplete profile of essential and biologically-active amino acids, such as leucine, lysine, or sulfur-containing amino acids than animal proteins, which can limit in vivo protein synthesis.
When processed, legumes increase their palatability and nutritive utilization. Processing may also contribute to enhancing the health benefits attributed to legume consumption. Soaking, cooking, germination, and fermentation are amongst the most effective and widely used processing methods, improving the nutritional [
2,
3] and functional value of legumes [
4,
5]. The development of novel legume-derived foods that exhibit high nutritional value and efficient action at promoting health has gained increasing attention in recent years. In this regard, cowpea (
Vigna unguiculata) is a widely cultivated legume in Asia, Central and South America, and Africa, where it is an important ingredient of several dishes that involve a different degree of culinary and technological processing [
6,
7]. Furthermore, Phillips et al. [
8] and Ayogu et al. [
9] discussed its potential to take part in the design and preparation of a variety of new food products, such as snacks, weaning foods, wheat-based cookies, or fortified traditional foods.
The cowpea has a substantial content of protein, complex carbohydrates, minerals, and vitamins, but also exhibits appreciable levels of phytic acid, tannins, α-galactoside oligosaccharides, and trypsin inhibitor activity that may interfere with its nutritive utilization. Fermentation of legumes contributes in developing flavor, aroma, and texture, and enhances the nutritive value by improving the density and availability of nutrients through the destruction of antinutritional factors, pre-digestion of certain food components, and synthesis of promoters for absorption [
10,
11,
12,
13,
14]. Furthermore, this biotechnological process has the capacity to cause significant modifications in legume-derived bioactive components, therefore increasing the health benefits of cowpea consumption [
4,
15]. Heat treatments are also widely used to improve the nutritional value of legumes due to its action on heat-labile non-nutritional components including proteolytic inhibitors, thus improving protein digestibility [
16,
17]. Such treatments may be combined with other technological processes, such as germination or fermentation, to potentiate their beneficial actions on nutrient composition and bioavailability [
12,
18].
To our knowledge, little is known regarding the digestibility of amino acids in cowpea and how the availability of protein and essential minerals is affected by the biotechnological processes of fermentation and its combination with other technological processing. Therefore, the objective of this study was to assess whether natural fermentation or its combination with autoclaving was able to improve nutritive utilization of protein, amino acids, and essential minerals like P, Ca, Mg, or K from cowpea. To evaluate the nutritional potential of raw and fermented cowpea protein, we used a growing rat experimental model and a well-stablished casein control reference protein with high nutritional value consistently described in the literature [
19,
20].
4. Discussion
Cowpea is one of the most important cultivated legumes, showing interesting agronomic and environmental benefits, as well as great potential as a nutritious and healthy food. Although it has been reported that cowpea has a promising nutrient composition, especially regarding protein and mineral content, few studies have focused on its in vivo digestibility, in relation to the legume potential as functional food. The aim of this study was to assess the effects of the biotechnological process of fermentation combined or not with autoclaving of Vigna unguiculata flour on the protein quality, amino acid digestibility, and bioavailability of four essential minerals using the rat as experimental model. Our results showed that cowpea provides a significant amount of digestible essential amino acids and fermentation improved their digestibility, making this legume a good source of highly available protein, although still inferior than casein. In addition, Vigna unguiculata was a good source of available P, Mg, and K and fermentation significantly enhanced the availability of P. The combination of fermentation with autoclaving did not enhance mineral availability of the fermented V unguiculata with the exception of the metabolic utilization of Ca.
The fermentation process selected for this experiment has been carried out based on the study of Doblado et al. [
12] who assayed different bean flour concentrations and fermentation times with the aim of achieving optimal fermentation conditions to improve the nutritional quality of
Vigna unguiculata. The authors reported a significant decrease in the content of antinutritional factors (TIA, inositol phosphates, and α-galactosides) and higher riboflavin content caused by fermentation, among other changes. When fermentation was combined with autoclave treatment, a further reduction in TIA was attained. Such reduction in antinutritional factor content more likely played a significant role in the higher digestibility of indispensable amino acids and P observed under the experimental conditions of the present study. Furthermore, the benefits of this same fermentation process on the functional value of
Vigna unguiculata were studied by Kapravelou et al. [
4], who reported a significant improvement in antioxidant capacity and different parameters of lipid metabolism induced by fermented beans in an in vivo rat experimental model.
The crude protein and mineral content of cowpea flours was within the range of values reported in the literature [
6,
27] for numerous cowpea cultivars. Patterns of amino acid composition were similar to those reported by previous studies in cowpea [
27,
28]. Although Granito et al. [
29] found lower protein content of the cowpea cultivar Orituco after fermentation, other authors reported no alteration of protein or mineral content in cowpea seeds [
2,
30], as in the present study. Fermentation or the combination of fermentation and autoclaving did not substantially alter the amino acid profile of cowpea, being similar to what has been reported for germinated cowpea flour [
31]. The increase of the time of thermal treatment has been reported to reduce protein content [
32] although this was not confirmed by our study.
Regarding the mineral content of the experimental
V. unguiculata diets, P and Mg were mainly provided by the cowpea flours, while only 43% of K was supplied by the flour. Nevertheless, due to the high solubility of K from legumes under similar conditions to those present in the gastrointestinal tract of monogastrics, K from cowpea was easily exchangeable with that from the mineral premix in the intestinal lumen. In contrast, close to 95% of Ca came from external sources such as CaHPO
4 or the mineral premix. Although fermentation does not cause major changes in total mineral content of legume flours, it may affect the form in which they are present, therefore affecting their availability [
33]. Specifically, it can increase the acidity of flours through the release of organic acids that may form mineral complexes and affect their solubility. In addition, fermentation process improves mineral availability by minimizing the action of non-nutritional components with known inhibitory effect on mineral digestibility including phytic acid or polyphenols [
2]. Mineral content and availability are also decreased by thermal treatment [
32], although in the present study fermentation combined with thermal treatment did not cause any further reduction. Under our experimental conditions, protein intake in growing rats fed cowpea diets was somewhat lower than in rats fed a casein diet of similar crude protein concentration. Conversely, a deep depression of protein intake in rats fed non supplemented raw cowpea for four weeks compared to a casein diet has been reported [
34]. Low dietary intake of legume-based diets may be related to the presence of antipalatable compounds (α-galactosides or tannins) and deficiencies in certain indispensable amino acids (mainly Met), minerals, and vitamins, leading to nutrient imbalance. The effect of autoclave treatment on non-palatable factors is variable [
35] although it did not seem to play a major role on dietary intake in the present study. In our experiment, cowpea fermentation and autoclaving did not alter dietary or protein intake, a finding that may indicate that cowpea cultivar used had low levels of antipalatable factors or that feeding duration was not long enough to detect subtle changes on dietary intake.
Growth of rats fed cowpea diets was adequate although lower than that of rats fed the casein diet. The satisfactory growth of the rats fed raw cowpea diet under our experimental conditions might be explained by a low amount of non-nutritional factors and the balancing of the amino acid profile due supplementation with the first limiting amino acid. In contrast, rats fed raw cowpea diets for 28 days [
34] lost weight and exhibited negative protein efficiency ratio (PER).
We studied the metabolic utilization of protein as nitrogen retention and R/A after ad libitum consumption of the experimental diets. Nitrogen retention should be considered in conjunction with other indices such as PER and FTI. Under our experimental conditions, higher N intake and digestibility of control diet might explain the differences in growth observed compared to cowpea diets. True nitrogen R/A was similar in control compared to raw cowpea diet in spite of higher N absorption and similar endogenous urinary N. However, the metabolic utilization of protein from cowpea diets was much greater than in rats fed other legumes [
36,
37], a finding that is reflected in our experiment as a greater nutritive utilization of protein and its direct use mainly for plastic growth functions as opposed to other secondary uses that finally result in a lower rate of metabolic use.
Legumes show protein digestibility ranging from 70% to 80% [
38] in accordance with the results presented herein. Nevertheless, true protein digestibility of diets formulated with cowpea [
29] and cowpea protein isolate [
39] was greater than the ones reported in the present experiment, a finding probably related to methodological differences in endogenous protein estimation, since we used a low protein diet, while the former authors used protein-free diets. High non-starch polysaccharide content of the cowpea diets used in our study (86.6 mg g
−1) may be related to their low protein digestibility, as high N excretion values are typically linked to high dietary non starch polysaccharide content in single-stomached animals [
40,
41]. Additionally, contribution of intestinal microbiota to fecal N excretion may be an additional factor to consider. It has been reported that bacterial N accounts for 50–80% of the total fecal N in fava bean or chickpea-fed rats [
42]. Indeed, total bacterial counts greatly increased in feces of rats fed cowpea diets compared to a casein diet [
43].
Amino acid digestibility of cowpea protein is usually determined by in vitro methods [
44]. However, information about in vivo amino acid digestibility of cowpea protein is scarce. In this regard, cockerels fed unsupplemented cowpea flour had similar apparent fecal digestibility of indispensable amino acids compared to the present study [
45]. Under our experimental conditions, natural fermentation improved apparent and true fecal digestibility of numerous indispensable amino acids which may be explained by diminished concentration of protease inhibitors, as it has been reported that fermentation almost eliminated trypsin inhibitor activity and lowered phytic acid and polyphenol content, main non-nutritional factors in cowpeas [
29,
46]. Heat treatment procedures have proved to be adequate methods for reducing contents or activity of several secondary plant metabolites in legumes [
47], especially those of the heat labile group (protease inhibitors and lectins). Furthermore, as shown in peas, heat treatment technologies may induce conformational changes in storage proteins, which may render them more accessible to digestive enzymes, and thus may increase amino acid digestibility. In our experiment, autoclaving of fermented cowpea flour did not further increase the digestibility of amino acid, but decreased the apparent and true digestibility of sulfur amino acids. This fact could be due to the degradation of methionine by the Maillard reactions, during food processing [
48] decreasing its availability [
49].
The improved digestibility of essential amino acids achieved by fermentation was not reflected, under the experimental conditions of the present study, in higher growth parameters, such as the weight gain, or protein nutritional indices, such as PER or FTI, compared to the raw cowpea protein. This lack of correlation can be attributed to the experimental design in which supplementation of methionine to legume diets partially hindered the benefits of fermentation in amino acid digestibility, or else to the fact that differences in amino acid digestibility among raw and fermented cowpea were not sufficiently high to result in higher growth indices, a potential limitation of our study. In addition, the specific structure of legume proteins and the presence in legume diets of non-nutritional components that may affect protein digestion and absorption may also be responsible for this different behavior, compared to the animal-derived casein control protein, in which a higher protein and amino acid digestibility led to significantly higher growth and nutritive utilization indices. Such different behavior can also be attributed to the higher content of highly available wheat starch incorporated to the casein control, compared to the cowpea diets, in which a considerable proportion of the starch was legume-derived and has been described to be less available. Finally, although the fermentation protocol was optimized to reduce the content of non-nutritional components that interfere with protein digestibility, it did not cause major improvements in the amino acid profile compared to raw cowpea protein, thus minimizing the positive effects of a greater amino acid digestibility.
Nutritive utilization of the minerals studied appeared to be affected by their concentration in the experimental diets, possible interactions with the food matrix or distinctive bioavailability regulation at the digestive and urinary level. Digestive and metabolic utilization of minerals from plant-based foods is usually affected by protein quality, the presence of dietary fiber and non-nutritional components, such as phytic acid or polyphenols that may interfere with absorption [
2]. The former inhibitory effects can be improved by biotechnological treatments like germination or fermentation that are able to generate new dietary components, such as organic acids capable of solubilizing and improving mineral absorption [
33]. In the present experiment, we tried to equilibrate the potential effect of legume dietary fiber, formulating a casein control with similar amounts of the fiber components found in
V. unguiculata. Such modifications in the casein diet resulted in a lower ratio of dietary intake/fecal excretion compared to
V. unguiculata diet, thus indicating a higher proportion of fiber in feces of casein control and lower degree of gut fermentation. Nevertheless, no apparent relationship was found between fecal weight, which is a measure of the mineral dragging action of dietary fiber, and mineral excretion. On the other hand, the amount of ingested mineral can affect its digestibility with lower digestive utilization in response to increasing intake. In addition, P and Ca bioavailability appeared to be affected by other dietary factors. In the case of Ca, despite a similar mineral source in the diet and daily intake, digestive utilization from
V. unguiculata diets was inferior to that from the casein control and did not improve as a result of fermentation process in a similar way to what has been detected in total protein. Although fermentation can reduce the amount of non-nutritional components present in legumes, such as phytic acid and polyphenols, to modify the structure of dietary fiber, and to release factors than enhance mineral bioaccessibility, such as organic acids, such changes were not sufficient to improve Ca bioavailability under our experimental conditions. An improvement regarding P availability likely related to fermentation was the reduction in phytic acid content and the release of potentially available P [
2,
50]. With regard to Mg and K,
V. unguiculata was a source of highly available minerals. In the case of Mg, digestibility was mainly affected by its high dietary intake, whereas the effect of other legume flour components, such as phytic acid or polyphenols, appeared to be minor as seen by the lack of differences between raw or fermented
V. unguiculata. The digestibility of K was extremely high under the experimental conditions of the present study, thus confirming the extraordinary potential of legumes as excellent dietary sources of this mineral with comparable availability to that of currently used dietary or pharmacological supplements. The nutritional importance of K is of outmost importance due to its participation in numerous cell functions, its protective action against kidney stone formation, and its essential role in bone health and in the regulation of blood pressure.
An interesting finding of this research is the different metabolic regulation of the minerals studied. In this regard, P metabolism was mainly regulated at the digestive level. Since Ca levels in the diet were adequate, most of absorbed P was retained in the body and the urinary reabsorption mechanisms worked very efficiently to achieve a mineral retention similar to that of the casein control diet. This finding was particularly evident in the groups fed fermented cowpea, in which the amount of mineral absorbed was enhanced by the technological treatment. On the other hand, K appeared to be regulated mainly at the urinary excretion level since nearly all the dietary ingested K was absorbed. Such urinary regulation bears two important facts: first, net retention of the mineral was similar for all treatments. Second, the higher urinary excretion of this mineral is reflected in changes in pH and urinary composition with well-known beneficial action to prevent kidney stone formation. Finally, Mg regulation appeared to take place at the digestive and urinary level to achieve similar net retention of the mineral compared to the casein control. With regard to Ca, and due to the adequate bioavailability of P from the diets, metabolic utilization of Ca was high, although not sufficient to reach the net retention values of the casein control. Such inferior retention values paralleled those of total N retention and body weight gain, and resulted in slightly lower levels of the mineral in femur bone and longissimus dorsi muscle.