Development of Cookies Enriched with Quinoa (Chenopodium quinoa) and Native Collagen from Pota (Dosidicus gigas) Nape ^†

Abstract

The giant squid (Dosidicus gigas) is a marine product from the Pacific Ocean. Its by-products can be used to obtain bioactive products such as collagen, proteins, and others. This work aimed to develop cookies enriched with Quinoa (Chenopodium quinoa) and native collagen from pota nape high in protein content, minerals, and antioxidants. Four formulations (4, 8, 12 and 16% collagen) were developed and compared with the control sample. The results showed higher protein (11.7 ± 0.3–20.8 ± 0.4%) content, lower moisture (4.7 ± 0.1–5.6 ± 0.2%), higher ash (3.0 ± 0.1–3.83 ± 0.09%), lower fat (15.29 ± 0.05–15.8 ± 0.1), and lower carbohydrate (53.89 ± 1.05–65.39 ± 0.82%) content than the control sample. Also, the cookies showed a significant content of polyphenols (618 ± 24–934 ± 23 µg gallic acid equivalent (GAE)/g), antioxidant activity (8182 ± 59–8369 ± 73 µg trolox/g) and in vitro digestibility (70.8 ± 0.1–73.6 ± 0.5%) than the control sample. The cookies also had a high mineral content: calcium (3893 ± 19 mg/kg), potassium (3222 ± 16 mg/kg), and magnesium (2108 ± 11 mg/kg). In addition, the cookies presented an adequate balance of amino acids, principally of aspartic acid, glutamic acid, serine, glycine, threonine, arginine, alanine, proline, valine, phenylalanine, and leucine. The cookies complied with the Peruvian legislation of the Healthy Law about the promotion of healthy eating for children and adolescents and with the microbiological requirements. Finally, the cookies showed a sensory acceptance of 77.8% and a shelf life of 184 days determined by the Rancimat method. The native collagen from pota nape could be used with quinoa flour to develop functional foods to help reduce child malnutrition.

1. Introduction

Cookies are considered snacks and are popular among consumers because of their size, shape, high digestibility, high energy value, relatively low production costs, and extended shelf life [1]. At present, wheat flour is being replaced by Andean pseudocereals because of its higher protein content, improving the nutritional quality of processed products [2].

The giant squid fishery is one of the most important in Peru, with a significant increase of 46.1%. However, only 50–60% of this marine resource is harvested; its by-products could be used to obtain high-value-added bioactive products such as gelatin, collagen, chitin, protein concentrates, and essential fatty acids, among others [3]. Collagen is a protein source with functional peptides with biological activity and important health benefits such as the recovery of cartilage tissues, tendons, ligaments, and reduction in joint pain. It is also used for its techno-functional properties in food development due to its high-water absorption capacity, ability to texturize, thicken and form gels [3].

Quinoa (Chenopodium quinoa) is a grain native to South America, with more than 250 species whose world production is led by Peru, Bolivia and Ecuador [2]. Seven-seed flour is a mixture of oat (Avena sativa L.), corn (Zea mays), fava bean (Vicia faba), pea (Pisum sativum), cocoa (Theobroma cacao), quinoa (Chenopodium quinoa), and maca (Lepidium meyenii) flours, which is high in protein, amino acid and essential fatty acid, vitamin and mineral content [4]. Sacha inchi oil (Plukenetia huayllabambana) contains polyunsaturated fatty acids (58%) with a high content of linolenic acid (55%). Its consumption is of great importance for health to prevent cardiovascular diseases, reduce low-density lipoproteins (LDL) or cholesterol and triglycerides [5].

The objective of this research was to develop cookies with native collagen from pota nape to evaluate the effect of collagen on the proximal composition, total polyphenols, antioxidant activity (DPPH), amino acid analysis, in vitro digestibility, sensory evaluation, microbiological analysis, health law, shelf life, and mineral content.

2. Materials and Methods

2.1. Raw Materials

Native collagen from pota (Dosidicus gigas) nape was extracted with NaOH 0.15 N for 30 h, then dried by infrared (IRC D18, Irconfort, Seville, Spain) at 60 °C, ground (Grindomix GM200, Restch, Haan, Germany) and stored in aluminized bags until further use [6]. The ingredients used were native collagen from pota nap, flour from seven seeds (oats, corn, beans, broad beans, peas, cacao, quinoa, and maca Lepidium meyenii), quinoa flour, cornstarch, panela, baking powder, salt, egg, vanilla essence, sacha inchi (Plukenetia huayllabambana) oil, and corn oil. All ingredients were purchased at the market in Lima, Peru. The sacha inchi seeds were collected in the province of Rodríguez de Mendoza, Amazon region, Peru. Sacha inchi oil was cold-pressed at the Functional Food Laboratory of the University of Lima, Peru, and stored at 4 °C in a dark flask.

2.2. Amino Acid Profile

The amino acid profile was estimated according to Chasquibol et al. [7]. Amino acids were determined in the acid hydrolysate by high-performance liquid chromatography (Acquity Arc, Waters, Milford, MA, USA) using D, L-α-aminobutyric acid as internal standard and a 300 mm × 3.9 mm reversed-phase column (Nova Pack C18 4 µm; Waters, Milford, MA, USA), and the resultant peaks were analyzed with Empower 3 software (Waters, Santa Clara, CA, USA). The calibration curves for each amino acid were developed using a mix of the amino acid standards at the same hydrolysis conditions of the samples (Merck, Madrid, Spain). Furthermore, tryptophan content was assessed according to the method described by Yust et al. [8]. Analyzes were performed in triplicate and presented as mean values.

2.3. Cookies Formulations

The cookies were formulated according to the essential amino acid composition of seven seeds (oats, corn, beans, lima beans, peas, cocoa, quinoa, and maca), seed flour, quinoa flour, and squid nape collagen (

Table 1. Cookies formulations with native collagen protein from pota (Dosidicus gigas) nape.

Ingredients	C	F1	F2	F3	F4
Seven-seed flour (g)	4	4	4	4	4
Quinoa flour (g)	17.6	15.6	13.6	11.6	9.6
Cornstarch (g)	6.5	6.5	6.5	6.5	6.5
Panela (g)	8	8	8	8	8
Baking Powder(g)	0.4	0.4	0.4	0.4	0.4
Salt (g)	0.4	0.4	0.4	0.4	0.4
Eggs (g)	6.5	6.5	6.5	6.5	6.5
Vainilla Essence (g)	0.6	0.6	0.6	0.6	0.6
Corn Oil (g)	3.2	3.2	3.2	3.2	3.2
Sacha Inchi oil(g)	2.8	2.8	2.8	2.8	2.8
Native collagen from pota nape (g)	0	2	4	6	8

C: Control, F1: Formulation 1, F2: Formulation 2, F3: Formulation 3, F4: Formulation 4.

). The powdered ingredients, including collagen, were mixed with the egg and vanilla essence for 10 min in the blender (FPSTSMPL1-053, Oster, Guandong, China). Then, corn and sacha inchi (Plukenetia huayllabambana) oil were added and mixed for 5 min. The cookie dough was divided into portions of 12.0 ± 0.5 g and shaped into circular cookies approximately 6 mm thick and 5 cm in diameter. The samples were baked for 13 min at 155 °C in a convection oven (HEB60R, Imaco, Guandong, China), cooled and stored in glass jars until further use.

2.4. Cookie Characterization

2.4.1. Proximal Composition

The proximal composition was carried out according to Chasquibol et al. [7]. The moisture content was determined at 110 °C to constant weight. The total protein content was determined as % nitrogen X 6.25 using a Kjeldahl analyzer (UDK 139, VELP, Usmate Velate, Italy). The ash content was determined by incineration at 550 °C for 72 h, in a muffle furnace. The fat content was determined with hexane for 4 h. Analyzes were performed in triplicate and presented as mean values.

2.4.2. Total Phenolic Content

The total phenolic content (TPC) was determined by Folin–Ciocalteau method [7] at 760 nm using a spectrophotometer (1280 UV–Vis Spectrophotometer Shimadzu, Kyoto, Japan). The results were expressed as µg of gallic acid equivalent (GAE)/g cookie. A calibration curve of 0.8–18 mg GAE/L was carried out. Analyzes were performed in triplicate and presented as mean values.

2.4.3. Antioxidant Activity

The antioxidant activity was determined by the DPPH method [7] with some modifications at 517 nm by spectrometry (1280 UV–Vis Spectrophotometer Shimadzu, Kyoto, Japan). The results were expressed as µg Trolox/g cookie. A calibration curve of 1.5–100 mg Trolox/L was carried out. Analyzes were performed in triplicate and presented as mean values.

2.4.4. Amino Acid Profile

The amino acid profile was determined according to Item 2.2.

2.4.5. In Vitro Digestibility (IVPD)

In vitro protein digestibility was measured according to Tapia et al. [9].

2.4.6. Microbiological Analysis

The cookies were analyzed microbiologically to stablish the microbiological criteria of sanitary quality and safety for food and beverages for human consumption [7].

2.4.7. Determination of Na, Total Sugar, Saturated Fat, and Trans-Fat

The concentrations of Na, total sugar, saturated fat, and trans fat were determined by official methods. Analyzes were performed in triplicate and presented as mean values.

2.4.8. Determination of Mineral Content

The concentrations of Ca, Cu, Fe, K, Mg, Na, P, Zn were determined by atomic absorption spectrophotometry (Nexion 350x, Perkin Elmer, MA, USA) according to Chasquibol et al. [7]. The results were expressed in mg/kg. Analyzes were performed in triplicate and presented as mean values.

2.4.9. Lifetime Analysis

The Rancimat method was determined at temperatures of 100, 110, and 120 °C [5].

2.4.10. Sensory Evaluation

The sensory evaluation test was performed with 30 panelists using a 9-point hedonic scale from 1 (I dislike it very much) to 9 (I like it very much) with a mean value of 5 (I neither like it nor dislike it) [7].

2.4.11. Statistical Analysis

Results were expressed as mean ± standard deviation. All measurements were determined in duplicate or triplicate. Analysis of variance (ANOVA) was used to analyze data acquired at a 95% significance level with Minitab 19.0 Software (Minitab Inc., State College, Palo Alto, CA, USA).

3. Results and Discussion

Table 2. Proximal composition, total polyphenol, antioxidant activity, and in vitro digestibility of cookies with native collagen protein from pota (Dosidicus gigas) nape.

Proximal Composition	C	F1	F2	F3	F4
Moisture (%)	4.43 ± 0.08 d	4.70 ± 0.14 c	4.90 ± 0.13 bc	5.05 ± 0.06 b	5.63 ± 0.15 a
Ash (%)	2.96 ± 0.01 c	2.96 ± 0.14 c	3.09 ± 0.00 c	3.30 ± 0.05 b	3.83 ± 0.09 a
Fat (%)	14.60 ± 0.02 c	15.29 ± 0.05 b	15.38 ± 0.08 b	15.78 ± 0.03 a	15.83 ± 0.10 a
Protein (%)	8.33 ± 0.00 e	11.66 ± 0.25 d	15.77 ± 0.14 c	18.57 ± 0.44 b	20.82 ± 0.40 a
Carbohydrates (%)	69.69 ± 0.66 a	65.39 ± 0.82 b	60.86 ± 0.49 c	57.30 ± 0.82 d	53.89 ± 1.05 e
Total polyphenol (µg GAE/ g)	1174.21 ± 53.78 a	934.52 ± 23 b	956.18 ± 48.23 b	873.11 ± 78.08 b	618.61 ± 24.06 c
Antioxidant Activity DPPH (µg trolox/g)	8525.97 ± 207.98 a	8181.79 ± 59.49 a	8323.34 ± 109.55 a	8420.88 ± 200.11 a	8368.92 ± 73.02 a
In vitro digestibility (%)	72.54 ± 0.51 ab	70.82 ± 0.13 b	74.62 ± 0.90 a	72.99 ± 0.13 a	73.62 ± 0.51 a

Results are expressed as means ± SD (n = 3). ^a,b,c,d,e values in the same row with different letters differing significantly when p < 0.05.

shows the proximal composition of the cookies. The moisture (4.7 ± 0.1–5.6 ± 0.2%) and ash content (3.0 ± 0.1–3.83 ± 0.09%) of the four formulations did not present statistically significant differences to the control sample (4.43 ± 0.08%), (2.96 ± 0.01%). The fat (15.29 ± 0.05–15.8 ± 0.1%) content did not present statistically significant differences to the control sample (14.60 ± 0.02%) besides the contribution of sacha inchi (Plukenetia huayllabambana) oil [5].

The protein (11.7 ± 0.3–20.8 ± 0.4%) content in the cookie formulations was statistically higher than the control sample (8.3 ± 0.0%) due to the contribution of native collagen protein from the pota (Dosidicus gigas) nape. Formulation F4 was selected because of its high protein (20.82 ± 0.40%) content, and according to Codex Alimentarius CAC/GL 23-1997 it could be declared as a protein-rich food for children under 5 years of age.

The total polyphenol content of formulations with collagen, (618 ± 24 µg GAE/g to 956 ± 48 µg GAE/g) was statistically lower than the control sample (1174 ± 53 µg GAE/g). This is due to high content of quinoa in the control sample, which contains a high amount of polyphenols (1089–1399 µg GAE/g) [2].

The antioxidant activity of formulations (8181 ± 59 µg trolox/g–8421 ± 200 µg trolox/g), did not present statistically significant differences with the control sample (8525 ± 207 µg trolox/g). The antioxidant contribution is due from quinoa (7000–15,000 µg trolox/g) [2].

In vitro digestibility (%) of formulations (70.8 ± 0.1% to 74.6 ± 0.9%), did not present statistically significant differences to control sample (72.54 ± 0.51%). This high digestibility is due to the high er digestibility (83.530 ± 0.007%) of quinoa protein.

The amino acid composition of cookies showed a balanced profile according to the FAO/WHO recommendations (

Table 3. Amino acid profile of cookies with native collagen protein from pota (Dosidicus gigas) nape.

Amino Acid	mg Amino Acid/g Protein
	C	F1	F2	F3	F4	FAO
Aspartic Acid	84.03 ± 0.83 a	83.33 ± 15.28 a	83.27 ± 35.13 a	71.41 ± 2.54 a	81.37 ± 3.04 a
Glutamic Acid	142.62 ± 5.38 a	131.73 ± 18.35 a	136.40 ± 55.34 a	109.75 ± 4.84 a	120.75 ± 5.16 a
Serine	46.07 ± 3.75 b	56.88 ± 1.42 ab	68.35 ± 25.19 ab	64.90 ± 4.56 ab	80.28 ± 1.17 a
Histidine	25.98 ± 1.15 a	25.93 ± 0.88 a	24.39 ± 6.75 a	21.49 ± 6.87 a	23.75 ± 0.12 a	15
Glycine	57.39 ± 4.12 b	115.97 ± 18.31 ab	125.54 ± 56.86 ab	126.21 ± 1.97 ab	158.45 ± 9.03 a
Threonine	40.74 ± 1.67 a	41.26 ± 3.61 a	41.15 ± 18.12 a	34.60 ± 1.87 a	39.5 ± 1.39 a	23
Arginine	62.11 ± 3.7 a	72.13 ± 4.64 a	76.36 ± 26.65 a	63.65 ± 6.54 a	76.94 ± 4.97 a
Alanine	49.33 ± 1.55 a	57.88 ± 8.23 a	59.30 ± 28.05 a	52.06 ± 1.78 a	60.85 ± 3.15 a
Proline	71.97 ± 41.97 a	108.67 ± 106.99 a	138.91 ± 3.06 a	113.93 ± 4.25 a	130.39 ± 70.83 a
Tyrosine	20.4 ± 0.07 a	17.95 ± 1.25 a	18.00 ± 5.88 a	13.83 ± 3.61 a	15.39 ± 0.45 a	38 *
Valine	46.04 ± 0.15 a	41.31 ± 4.61 a	39.23 ± 15.25 a	30.11 ± 2.56 a	33.04 ± 0.96 a	39
Methionine	0.93 ± 0.19 b	2.79 ± 1.38 ab	5.93 ± 1.31 a	4.02 ± 2.35 ab	3.03 ± 1.57 ab	22 **
Cysteine	6.3 ± 0.07 a	4.38 ± 0.22 ab	3.81 ± 1.61 b	2.12 ± 1.15 bc	1.01 ± 0.03 c
Isoleucine	38.2 ± 0.93 a	33.68 ± 3.14 a	31.70 ± 12.45 a	25.21 ± 5.42 a	27.24 ± 0.93 a	30
Phenylalanine	62.42 ± 1.28 a	55.08 ± 5.32 a	50.34 ± 19.48 a	40.05 ± 2.15 a	43.98 ± 1.46 a
Leucine	50.49 ± 1.1 a	42.03 ± 3.58 a	41.56 ± 16.67 a	31.90 ± 3.41 a	32.66 ± 2.28 a	59
Lysine	43.56 ± 0.81 a	35.77 ± 2.87 ab	34.93 ± 12.22 ab	24.16 ± 2.14 b	23.9 ± 0.55 b	45
Tryptophan	8.05 ± 0.63 a	5.55 ± 0.99 ab	4.63 ± 1.06 b	4.14 ± 1.51 b	3.76 ± 0.61 b	6
Hydroxyproline	Nd	21.57 ± 1.34 d	31.90 ± 2.21 c	40.68 ± 1.72 b	48.32 ± 1.24 a

* Tyrosine + Phenylalanine, ** Methionine +Cysteine, Nd: Not determined. Results are expressed as means ± SD (n = 3). ^a,b,c,d values in the same column with different letters differ significantly when p < 0.05.

). The essential amino acids found were histidine (21 ± 6–25 ± 1 mg/g protein), threonine (34 ± 2–41 ± 4 mg/g protein), tyrosine (14 ± 4–20.40 ± 0.07 mg/g protein), phenylalanine (40 ± 2–62 ± 1 mg/g protein), valine (30 ± 3–46 ± 0.2 mg/g protein), isoleucine (25 ± 5–38.2 ± 0.9 mg/g protein), and tryptophan (3.8 ± 0.6–8.1 ± 0.6 mg/g protein). The cookie formulations with collagen showed a high hydroxyproline content (21 ± 1–48 ± 1 mg/g protein). According to Paul et al. [3], the daily intake of collagen is 2.5–15 g per day, and a 50 g package of Formulations F1 to F4 is within this range.

Figure 1 shows the radial graph of the sensory evaluation (color, taste, texture, smell) of the developed cookie formulations, with a positive sensory appreciation equivalent to 77.78% positivity.

The results of microbiological analysis for Formulation F4 (

Table 4. Microbiological analysis, sodium, total sugar, saturated fat, and trans-fat content of formulation F4 with native collagen protein from pota (Dosidicus gigas) nape.

Analysis	Result	Maximum According to Standard
*Bacillus cereus Numbering (CFU/g)	<100	102 *
E. coli Numbering (MPN/g)	<3	20 *
Fungi: Molds Numbering (CFU/g)	<10	103 *
Salmonella Detection	Absence	Absence *
Staphylococcus aureus Numbering (CFU/g)	<10	102 *
Total sugars (g/100g)	18.67 ± 1.49	10 **
Sodium (mg/100 g)	343.98 ± 2.75	400 **
Saturated fats (g/100 g)	2.13 ± 0.17	4 **
Trans fats (g/100 g)	<0.01 ± 0.001	5 **
Life time (days)	184	60 ***

Results are expressed as mean ± SD (n = 3). * Ministerial Resolution 591-2008-MINSA-Peru. Sanitary standard that establishes the microbiological criteria of sanitary quality and safety for food and beverages for human consumption. CFU: Colony Forming Units. MPN: Most Probable Number. ** Law No. 30021 Law on the Promotion of Healthy Eating for Children and Adolescents, regulated in Supreme Decree No. 017-2017-SA-Peru. *** De Magalhães et al. [1].

) were evaluated with the maximum values allowed according to Ministerial Resolution 591-2008-MINSA-Peru [7] and complied with the established requirement.

The sodium (343 ± 3 mg/100 g) content, saturated fat (2.1 ± 0.1 g/100 g), and trans fat (<0.01 ± 0.001 g/100 g fat) content for Formulation F4 (Table 4) are within the limits established by Peruvian legislation; however, the total sugar (18 ± 1 g/100 g) content is higher due to the high sugar (3–8 g/100 g) content present in quinoa [2].

The developed cookies (F4) presented a shelf life of 184 days by the Rancimat method [5]. According to De Magalhães et al. [1], the cookies lose their physicochemical properties after 60 days of storage due to moisture absorption and decrease in organoleptic quality in the developed product.

Formulation F4 showed important quantity of minerals (

Table 5. Mineral analysis of Formulation F4 with native collagen protein from pota (Dosidicus gigas) nape.

Mineral/Metal	Result
Calcium (mg/kg)	3892.75 ± 194.64
Potassium (mg/kg)	3222.35 ± 161.12
Magnesium (mg/kg)	2108.15 ± 105.41
Zinc (mg/kg)	45.32 ± 2.27
Iron (mg/kg)	38.81 ± 1.94
Cupper (mg/kg)	3.31 ± 0.17
Phosphorus (mg/kg)	<20

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

): calcium (3892 ± 194 mg/kg), potassium (3222 ± 161 mg/kg), magnesium (2108 ± 105 mg/kg), zinc (45 ± 2 mg/kg), copper (3.3 ± 0.2 mg/kg), and iron (39 ± 2 mg/kg). According to the daily requirements for children under 5 years of National Institute of Health [10], Formulation F4 covers about 19.46% of daily calcium requirement, 81.08% of magnesium, 7.01% of potassium, and 19.41% of iron.

The developed cookies had better nutritional characteristics than commercial cookies. So, the cookie developed could contribute to the reduction in malnutrition in our country.

4. Conclusions

The native collagen from the nape of the squid (Dosidicus gigas) can be used to develop functional foods such as cookies. Formulation F4 presented high protein, in vitro digestibility, total polyphenol, and antioxidant activity. Also, Formulation F4 contains high hydroxyproline content and a balanced amino acid profile according to FAO. Formulation F4 had high sensory acceptance and complied with microbiological and health law requirements according to Peruvian legislation. In addition, Formulation F4 had a long shelf life with significant content of calcium, potassium, and magnesium.