Physicochemical Evaluation of Cushuro (Nostoc sphaericum Vaucher ex Bornet & Flahault) in the Region of Moquegua for Food Purposes

The cyanobacterium Nostoc sp. contains considerable amounts of protein, iron, and calcium that could mitigate the problems of anemia and malnutrition in humans. However, the nutritional value of the edible species Nostoc sphaericum Vaucher ex Bornet & Flahault, which grows in the Moquegua region, is unknown. Descriptive research was developed, and samples were obtained from the community of Aruntaya, located in the region of Moquegua. Water samples were taken at two different points (spring and reservoir), and samples of the cyanobacteria were taken in the reservoir. The design used was completely randomized, with three repetitions. Sixteen characteristics associated with the water collected at two points were evaluated, and from the nutritional point of view, seven characteristics were evaluated in the collected algae. The physicochemical characteristics were determined using methods established in the Codex Alimentarius. For the morphological characterization at the macroscopic level, it was observed that the seaweed collected was spherical in shape, grayish-green in color, soft to the touch, and palatable. After carrying out the physicochemical and morphological characterization of the collected samples, it was verified that all were of N. sphaericum. When comparing the sixteen characteristics related to water at the two collection sites, highly significant differences (p < 0.01) were observed for most of the variables evaluated. The average data of the characteristics of the algae showed protein values of 28.18 ± 0.33%, carbohydrates of 62.07 ± 0.69%, fat of 0.71 ± 0.02%, fiber of 0.91 ± 0.02%, ash of 7.68 ± 0.10%, and moisture of 0.22 ± 0.01%. Likewise, calcium reported an average value of 377.80 ± 1.43 mg/100 g and iron of 4.76 ± 0.08 mg/100 g. High correlations (positive and negative) were obtained by evaluating seven characteristics associated with the reservoir water where the algae grew in relation to eight nutritional characteristics of the algae. In relation to the nutritional value, the amounts of protein, iron, and calcium exceed the main foods of daily intake. Therefore, it could be considered a nutritious food to combat anemia and malnutrition.


Introduction
In Peru, there is growing concern about anemia in the country's public health policies [1]. Anemia directly affects children between 6 and 35 months of age, with a national average incidence of 38.8%, with values of 48.7% in rural areas and 35.3% in urban areas [2]. macroalgae was analyzed on a dry basis. Both objectives were developed to determine the nutritional value of freshwater algae.

Collection Site
Nostoc algae samples were collected from streams (16°36′19.9″ S, 70°18′1.2″ W) located in the Aruntaya farming community at an altitude of 4434 masl in the Carumas district, Mariscal Nieto Province, Moquegua region ( Figure 1). The temperature of the water "in situ" was recorded with a basket thermometer. Subsequently, the collected biological sample (water and cyanobacteria) was transported in an ice cooler of 10 L capacity, inside which 6 gel pack bags of 0.5 L were placed to keep the temperature below 5 °C. The Nostoc was protected with reusable wet cloths of size 33.5 × 34.2 cm to keep them moist ( Figure 2B), inserting a layer of damp cloth and another layer of Nostoc until completing the total volume of the cooler. This sampling protocol allowed the samples to be transferred to distant places in a reduced space under adequate conditions for the conservation of the biological sample. The collection method of all the experimental samples was developed in the present work. The temperature of the water "in situ" was recorded with a basket thermometer. Subsequently, the collected biological sample (water and cyanobacteria) was transported in an ice cooler of 10 L capacity, inside which 6 gel pack bags of 0.5 L were placed to keep the temperature below 5 • C. The Nostoc was protected with reusable wet cloths of size 33.5 × 34.2 cm to keep them moist ( Figure 2B), inserting a layer of damp cloth and another layer of Nostoc until completing the total volume of the cooler. This sampling protocol allowed the samples to be transferred to distant places in a reduced space under adequate conditions for the conservation of the biological sample. The collection method of all the experimental samples was developed in the present work. Under laboratory conditions, the algae were initially identified as N. sphaericum (Figure 2B), according to the classification key described by Bornet and Flahault [23], which is integrated into the classification system [24]. For the identification of Nostoc sphaericum, the Geitler taxonomic key [25] and the Guiry classification system [26] were used.

Physical and Chemical Analysis of the Water Where the Collection Took Place
Water samples (1000 mL) were collected at the Aruntaya spring and 20 m from it (reservoir), all in triplicate. In the growth zone of the N. sphaericum strain, three samples were collected and sent to the LABINVSERV Research and Services Laboratory, which used the AOAC method [27] for pH analysis and the gravimetric method for dissolved solids and total hardness. Alkalinity and acidity were determined by the volumetric method, and sulfates were determined by the AOAC [27] turbidimetric method. Phosphates, ammonia nitrogen, total nitrogen, BOD5, and COD were determined by the AWWA method [28]. Likewise, the Certificaciones del Perú S.A. CERPER Testing Laboratory determined the amount of nitrites and nitrates by the anion method by ion chromatography: EPA Method 300.0 1993 [29].

Physical Analysis of Samples
The physical characteristics were estimated from three fresh samples of N. sphaericum. The moist matter moisture was estimated by protocol 930.04 [30]. The dry matter moisture of 1 kg of N. sphaericum ( Figure 2C) was characterized by the hot air oven method at 70 °C for 5 h [27]. According to the method of Torres-Maza et al. [31], the hydrocolloids were dried in a LABCONCO freeze dryer (Models 79480, Labconco Corp., Kansas City, MO,USA) at a pressure of 0.004 bar, with an initial temperature of −40 °C, and an air dryer (CORP. JARCON model SBT-10 × 10) at a constant temperature of 40 ± 0.5 °C, with a velocity or flow rate of 20 m 3 min −1 [5], to determine the humidity in an air oven. Under laboratory conditions, the algae were initially identified as N. sphaericum ( Figure 2B), according to the classification key described by Bornet and Flahault [23], which is integrated into the classification system [24]. For the identification of Nostoc sphaericum, the Geitler taxonomic key [25] and the Guiry classification system [26] were used.

Physical and Chemical Analysis of the Water Where the Collection Took Place
Water samples (1000 mL) were collected at the Aruntaya spring and 20 m from it (reservoir), all in triplicate. In the growth zone of the N. sphaericum strain, three samples were collected and sent to the LABINVSERV Research and Services Laboratory, which used the AOAC method [27] for pH analysis and the gravimetric method for dissolved solids and total hardness. Alkalinity and acidity were determined by the volumetric method, and sulfates were determined by the AOAC [27] turbidimetric method. Phosphates, ammonia nitrogen, total nitrogen, BOD5, and COD were determined by the AWWA method [28]. Likewise, the Certificaciones del Perú S.A. CERPER Testing Laboratory determined the amount of nitrites and nitrates by the anion method by ion chromatography: EPA Method 300.0 1993 [29].

Physical Analysis of Samples
The physical characteristics were estimated from three fresh samples of N. sphaericum. The moist matter moisture was estimated by protocol 930.04 [30]. The dry matter moisture of 1 kg of N. sphaericum ( Figure 2C) was characterized by the hot air oven method at 70 • C for 5 h [27]. According to the method of Torres-Maza et al. [31], the hydrocolloids were dried in a LABCONCO freeze dryer (Models 79480, Labconco Corp., Kansas City, MO, USA) at a pressure of 0.004 bar, with an initial temperature of −40 • C, and an air dryer (CORP. JARCON model SBT-10 × 10) at a constant temperature of 40 ± 0.5 • C, with a velocity or flow rate of 20 m 3 min −1 [5], to determine the humidity in an air oven. The color was determined by the spectrophotometric test method, through which the chromatic coordinates were determined, which is determined by the CIE L*a*b* model [32], and texture was determined by the sensory test method.
Cushuro samples were sent to the testing and research laboratory "Sistema de Servicios y Análisis Químicos S.A.C. (SLAB) to determine the proximate content of N. sphaericum. Proteins were determined according to the Kjeldahl method, crude fat content was obtained by the Soxhlet method, and carbohydrates, total ash, and fiber were determined according to the official method of the AOAC [27]. Finally, the amounts of iron and calcium were determined by atomic absorption, according to protocol 975.03 [30].

Experimental Design and Statistical Analysis
A completely randomized experimental design was used, with two treatments associated with algal tail points. Normality and homogeneity of variance were tested using the Kolmogorov-Smirnov test. Subsequently, analysis of variance (ANOVA) was performed, and Tukey's multiple range comparison test (p < 0.01) was used to determine differences between treatments. The experimental data were processed using the RBio program [33].

Physicochemical Analysis of Water
When verifying the results of the ANOVA (Table 1), it was observed that when comparing the two points of the algal tail, highly significant differences (p < 0.01) were obtained for most of the variables, with the exception of pH, which did not show differences (Table 1). Alkalinity, total hardness, nitrites as N, and nitrites as NO 2 did not show variations in the data collection, and for this reason, they were included in the analyses in Table 1. Under these conditions, the coefficients of variation were low, showing the precision of the data obtained for all the variables evaluated (Table 1). When the comparisons of means were carried out by Tukey's test, the results are shown in Table 2. The results show the water quality values of the Aruntaya spring water coming from the subsoil at a temperature of 11.7 • C on average, which at 20 m distance from its origin ( Figure 1) feeds the lentic body (or reservoir) where the strain of N. sphaericum was collected, and the water is at an average temperature of 10.7 • C, demonstrating a decrease in the temperature of the water during its journey to the reservoir. The water quality analysis showed that the water from the Aruntaya spring had the highest amount of sulfates, 2 mg L −1 , phosphates 0.36 mg L −1 , and nitrates 0.622 mg L −1 . Likewise, it can also be observed in Table 2 that in the reservoir, the biochemical oxygen demand (BOD5) increased by 7.00 mg L O 2 −1 , and the chemical oxygen demand (COD) increased by 22,955 mg L O 2 −1 ; similarly, ammonia nitrogen reached values of 0.15 mg L −1, and total nitrogen reached 1.58 mg L −1 . In the same way, increases are observed in pH, 0.1; conductivity, 1.1 µS cm −1 ; dissolved solids, 1.1 mg L −1 ; alkalinity (permanence invariable); and acidity, slightly increased by 0.09 mg L −1 ( Table 2). On the other hand, the water from the area where the cyanobacterium N. sphaericum grows presented lower values of sulfates (21.40 mg L −1) , phosphates (0.20 mg L −1) , and nitrates (NO 3 ) (0.04 mg L −1) , as shown in Table 2. Another outstanding characteristic of the water quality of the N. sphaericum collection area (growth area) is the low amount of Nitrospira (NO 2 ), which is less than 0.004 mg L −1 .

Physical Characteristics of N. sphaericum
After analyzing the cushuro samples at the Certified Laboratory of Sistema de Servicios y Análisis Químicos S.A.C., it was found that the fresh sample was N. sphaericum, which had an average fresh moisture content of 98.73%, while the dry sample had an average moisture content of 0.222 ± 0.005% in 100 g of dry matter. The chromatic coordinates of the CIE L*a*b* model showed color values of 92-9-89, on average, which corresponds to a grayish-green color. In terms of texture, the fresh samples were soft to touch and spherical in shape ( Figure 2B). Table 3 reports the chemical characteristics of the analyzed samples of N. sphaericum, whose average values are as follows: protein 28.18 ± 0.33%, carbohydrates 62.07 ± 0.69%, fat 0.71 ± 0.02%, fiber 0.91 ± 0.02%, ash 7.68 ± 0.10%, and moisture 0.22 ± 0.01%. Likewise, calcium had an average value of 377.80 ± 1.43 mg/100 g, and iron had an average value of 4.76 ± 0.08 mg/100 g. The chemical characteristics of the collected sample are compared with other values for the same species of cyanobacteria, described in previous studies (Table 3).  [34], (b) Chili-Rodriguez and Terrazas-Viza [12], (c) Ponce [6], (d) Sirajunnisa and Surendhiran [35], (e) Rosales-Loaiza et al. [5], (f) Torres-Maza et al. [31], (g) Celis-Plá et al. [11].

Correlations Established between Water and Chemical Characteristics of N. sphaericum
To determine the degree of correlation that exists between the variables obtained by evaluating the reservoir water and the properties of the algae produced under these conditions, an analysis of correlations between these variables was established, and a matrix of correlations was built, which is shown in Figure 3.

Correlations Established between Water and Chemical Characteristics of N. sphaericum
To determine the degree of correlation that exists between the variables obtained by evaluating the reservoir water and the properties of the algae produced under these conditions, an analysis of correlations between these variables was established, and a matrix of correlations was built, which is shown in Figure 3.

Discussion
Among the most globally distributed algae, we can find N. commune [36]. N. sphaeroides is another of the most common algae used as a dietary macroalgae for its nutritional value [37,38]. In this study, we verified the distribution of macroalgae of N. sphaericum in the Moquegua region and how water quality influences its growth and nutritional value.

Quality of the Water Where Sampling Was Performed
The main place where most algae develop naturally is in riverbeds. Knowing the quality of the water where they develop is important to establish cultivation parameters for this species. The water of the Aruntaya spring has a high amount of chemical compounds (22 mg L −1 of sulfates, 0.36 mg L −1 of phosphates, and 0.622 mg L −1 of nitrates), which are nutrients used to feed N. sphaericum. These compounds were correlated with nutritional contents of the evaluated algae, which shows the possibility of obtaining these algae in the characterized reservoir, and hence its potential use.
When we looked at the values for the sampling zone, the cyanobacteria growth area, the nutrients were significantly reduced, to values of 21.40 mg L −1 sulfates, 0.20 mg L −1 phosphates, and 0.04 mg L −1 nitrates. This result indicates that these components were absorbed by the cyanobacteria and contributed to their development, as well as that the amounts present in the water allowed adequate development of the algae under these conditions. This result corresponds to that described by Silambarasan et al. [39], who mentioned that Nostoc sp. remove TP (total phosphorus), NH 4 (ammonium), and TN (total nitrogen) from 6 to 10 days of culture, while COD (chemical oxygen demand) decreases in 10 days. The results of this research also agree with those described by Nagappan et al. [40], when expressing that Nostoc sp. is a nitrogen-fixing cyanobacterium. Khan et al. [41] found that Nostoc sp. assimilates NH 4 -N more efficiently and can integrate it in the form of amino acids, which are important for cyanobacterial growth [42]. The results show that these parameters are altered as a result of the absorption and growth capacity of the cyanobacteria, a fact confirmed by Silambarasan et al. [39]. These results also confirm that the reservoir water where the collected cyanobacteria grow has adequate characteristics to facilitate the growth of the algae.
In the sample collection area, the biochemical oxygen demand (BOD5) increased by 7.00 mg L O 2 −1 , similar to the chemical oxygen demand (COD), which increased by 22.955 mg L O 2 −1 . Variations in these parameters, according to Makki and Khudhair [43] and Soto et al. [44], mean that the organic material has increased, therefore, there is greater oxygen consumption. Similarly, ammonia nitrogen reached values of 0.15 mg L −1, and total nitrogen reached 1.58 mg L −1 , which were higher in the collection area due to the greater amount of organic material in the collection area. Another outstanding characteristic of the water quality of the N. sphaericum collection area is the low amount of Nitrospira (NO 2 ), which is less than 0.004 mg L −1 , permissible to achieve the growth of N. sphaericum [45]. From the precedents, it can be deduced that Aruntaya spring water contains the nutrients necessary for the growth of Nostoc sp. (Table 1).

Physical Characteristics of N. sphaericum
The fresh sample of N. sphaericum exhibited a grayish-green color, a soft texture to the touch, and a spherical shape, characteristics that confer a pleasant appearance, so it could possibly have a high degree of acceptability to the consumer. It is also known that species such as N. flagelliforme have been consumed in China [46] and N. commune in Peru [47] since pre-Columbian times [15], and the cyanobacterium N. sphaericum is consumed in different "gourmet" dishes internationally [48]. The values of chromatic coordinates mentioned for N. sphaericum (L* = 92, a* = −9, −14.6, b* = −89) indicate a very bright yellow-green color. If we compare these values with other colors common in nature, we could say that it resembles a color such as that of certain varieties of Golden Delicious Malus apples [49] and the dark greenish color of the nori macroalgae [50] used in making sushi. As shown by Li et al. [51], who measured the chromatic coordinates of the Golden Delicious apple, the chromatic coordinates for the fresh apple were L* = 92.46, a* = −2.55, and b* = 26.25. Likewise, Olguin-Santana and Jacobo-Velázquez [52] indicated the chromatic coordinates of fresh nori seaweed at different times of its culture. The average values reported were L* = 35.6 to 47.2, a* = −10.3 to −14.6, and b* = −11.6 to −18.6, verifying the color similarity between them.

Chemical Characteristics of N. sphaericum
It has been described that N. sphaericum is characterized by presenting molecules of high nutritional value (Table 3), such as proteins, that reach percentages of 31.23 ± 3.07% [5], calcium of 145 ± 8.80 mg/100 g [34], iron of 19.60 mg/100 g, and phosphorus of 258 mg/100 g [6]. The values reported in the present investigation were similar (protein, 28.18 ± 0.33%; carbohydrates, 62.07 ± 0.69%; fat, 0.71 ± 0.02%; fiber, 0.91 ± 0.02%; ash, 7.68 ± 0.10%; and moisture, 0.22 ± 0.01%) and show the possibility of collecting algae with excellent nutritional properties. Likewise, calcium had an average value of 377.80 ± 1.43 mg/100 g, and iron had an average value of 4.76 ± 0.08 mg/100 g. The values obtained in this study show that this cyanobacterium has high nutrient contents and that these values are highly correlated with variables associated with pond water, which means that as a whole it is possible to be used a food that can contribute to daily intake.
The carbohydrate values for N. sphaericum in the investigation were 62.07 ± 0.69%, predominant compared to the other macromolecules that form the cyanobacteria. Similar results, of 46.40 ± 0.59%, 62.4%, and 74.2% were found by Rosales-Loaiza et al. [5], Ponce [6], and Chili-Rodriguez and Terrazas-Viza [12], respectively. In relation to the degree of digestibility of carbohydrates, according to Capcha et al. [53], carbohydrates are rapidly digestible because they do not contain cellulose. Another experimental study, by Li et al. [54], used N. commune Vauch. polysaccharide fermented by Lactobacillus for therapeutic treatment of rats, achieving a reparative effect on Cd-induced renal injury, inhibiting apoptosis, and improving the composition of the intestinal microbiota. In addition, according to Guo and Li [55], it inhibits the occurrence of colon tumors in mice. Fiber, in this study, presented values ranging from 0.90% to 1.64 ± 0.57% [5,12,13], which is important for the digestion of short-chain fatty acids [56].
Likewise, in this investigation, the fiber content of N. sphaericum was 0.91 ± 0.02%; for the same species, Torres-Maza et al. [31] reported a content of 0.03 ± 0.07%, lower than the value found in this study. However, for Alegre et al. [13], the crude fiber content for Nostoc sp. was higher, reaching a value of 5.77 ± 0.11% of crude fiber. This difference in fiber content is probably due to the different environmental conditions of growth of the strain and the drying method, which were different for each investigation, and humidity reached in the dehydration of the matter. According to Castro et al. [58], the drying method and the applied temperature are inversely proportional, and they concluded that for the infrared drying of macroalgae, a temperature of 40 • C should be considered, to avoid the degradation of color and algae components.
There is also variability in the ash content. In the investigation, a value of 7.68 ± 0.10% was obtained, while in other studies, Alegre et al. [13], for the N. sphaericum strain, and Rosales-Loaiza et al. [5], for Nostoc LAUN0015, reported values of 7.77 ± 0.01% and 19.33 ± 2.76%, respectively, of ash. However, Torres-Maza et al. [31] found a value of 0.13 ± 0.01% ash for the N. sphaericum species, which is a low content and similar to most fruits and vegetables, that have an ash content between 0.3 and 1%. In general, ash accounts for less than 5% of the dry matter of food. Thus, the result obtained in the investigation is similar to the amount of ash present in vegetables [59], and shows the potential of the cyanobacteria collected under the conditions of Moquegua, Perú.
Iron is very important to mitigate anemia, and, therefore, its antecedents are addressed with greater emphasis. It is highlighted that the recommended daily amount of consumption is 8 mg of iron for men and 18 mg for women. Beef contains 2.6-3.0 mg/100 g, while liver contains 18.6 mg/100 g, legumes such as beans and lentils contain 1-2 mg/100 g, and spinach contains approximately 3.6 mg of iron/100 g [60]. On the other hand, clams are the richest source of iron at 28 mg/100 g [61], and Bhutan red rice contains approximately 7.6 mg/100 g [62]. It has been shown that iron absorption can be improved by consuming foods rich in vitamin C together with foods rich in iron [63].
Fleurence et al. [64] found that some species of macroalgae can contain up to 4.4 mg/100 g of dry weight. Another study, by Ganesan and Kumar [65], evaluated the effect of an extract of the macroalgae red seaweed Gracilaria corticata on iron deficiency anemia in rats, and the results showed that the administration of the extract significantly improved hemoglobin levels. In the investigation, it was found that the macroalga N. sphaericum reached a value of 4.76 ± 0.08 mg Fe/100 g of dry matter. Therefore, it can have effects on the prevention and treatment of iron deficiency. However, more studies are needed to determine the efficacy of macroalgae and their extracts as dietary supplements in humans.
One of the outstanding results of this research is the nutrient content of N. sphaericum, with values above the average of other important foods in the diet of most humans. These results suggest that this species of cyanobacteria could be considered a food of high nutritional value [12,53]. Research with a focus on modern medicine showed that N. sphaeroides contains a variety of essential amino acids, polysaccharides, and other bioactive substances [36], that highlights its nutritional value. Likewise, Celis-Plá et al. [11], Xu et al. [17], and Li et al. [18] concluded that N. calcícola and N. sphaeroides contain polyphenols, phycocyanin, and ascorbic acid, that have antioxidant, anti-inflammatory, and anti-infective therapeutic effects, respectively.
Another study has shown that the microalga Nostoc LAUN0015, grown with nitrogen injection, produces large amounts of biomass, reaching values of 890 µg mL −1 [5], evidencing an alternative for large-scale production; however, the authors of [5,[65][66][67][68] highlight the potential of cyanobacteria as a source of food and nutrients, as well as their importance in the fight against malnutrition and food security. These authors also point out the importance of addressing the challenges associated with the sustainable cultivation and production of these microalgae to maximize their positive impact on society and the environment. It is important to highlight that, until now, most of the studies have focused on production at the microalgal level, leaving the focus on the mass production of the species pending.
Consequently, the results obtained in the present research, and the previous accounts on water quality and proximal composition of the macroalga N. sphaericum, have promising possibilities of application, e.g., in pharmaceutical industries and possible applications in biotechnology. The verified nutritional value confirms that this cyanobacterium can contribute to the prevention of anemia, mitigate malnutrition due to food deficits, and serve as a potential food for humans and animals.

Conclusions
In conclusion, the study has managed to determine the water quality conditions necessary for the natural growth of the macroalgae N. sphaericum, which will improve the production and use of this natural resource.
On the other hand, the macroalgae N. sphaericum is a rich source of nutrients, reaching a protein content of 28.18 ± 0.33%, carbohydrates of 62.07 ± 0.69%, fat of 0.71 ± 0.02%, fiber of 0.91 ± 0.02%, ash of 7.68 ± 0.10%, moisture of 0.22 ± 0.01%, calcium of 377.80 ± 1.43 mg/100 g and iron of 4.76 ± 0.08 mg/100 g, values that demonstrate the potential of macroalgae as food for humans and animals.