Development of Extruded Snacks with Protein Hydrolysed from Jumbo Squid (Dosidicus gigas) by-Product and Cañihua (Chenopodium pallidicaule Aellen) ^†

Abstract

The jumbo squid fishery in Peru is the second most important after the anchovy fishery. During its manufacturing process, only 50% to 60% of the total jumbo squid is used, thereby, the residues could be used to develop functional foods. Cañihua (Chenopodium pallidicaule Aellen) is an Andean pseudocereal from the highlands of Peru characterized by its high nutritional value. This work aimed to develop extruded snacks with protein hydrolyzed (PH) from jumbo squid by-product (JSBP) due to its high protein content, low price, and high availability. Four extruded snacks with corn flour (55%), rice flour (20% to 30%) and cañihua flour (15%) were enriched with PH from JSBP (4% to 10%) and developed using a twin-screw extruder. The extruded snacks were characterized by their physical properties (density, expansion ratio, water absorption index) and shelf life. The addition of PH from JSBP increased the protein content from 11.20% to 15.39%; ash content from 1.40% to 1.66% and fat content ranged from 1.10% to 1.18% compared to the control sample, the moisture content was from 4.46% to 5.81%. Also, the extruded snacks showed high phenolic concentration, 5633 µg GAE/g snack to 7315 µg GAE/g snack, high antioxidant activity, 698 mg trolox/g snack to 1274 mg trolox/g snack, high in vitro protein digestibility, 72.58% to 74.40%, and low acid index (0.095 mg KOH/g snack to 0.105 mg KOH/g snack) and peroxide index (0.00 meq O₂/kg snack to 0.063 meq O₂/kg snack), respectively. The snacks were accepted by the panel evaluators, complied with the Peruvian standard NTP-209.226 and microbiological requirements. Therefore, these snacks can be a healthier alternative product and satisfy market trends.

1. Introduction

Commercial snacks expanded by extrusion are foods with great acceptance by the consumer; however, these snacks are high in calories, low in protein content, minerals, dietary fiber, essential amino acids, and other bioactive compounds. The extrusion technology can be used to manufacture a wide range of food products with low processing costs, continuous production, high throughputs, and better product quality with optimal energy utilization [1]. Food extrusion is a process of several unit operations as thermal and mechanical treatment by which protein and/or starch-rich ingredients are fed into an extruder barrel through the feed hopper and the screw then conveys the food by compression, mixing, shearing, kneading and high temperature to produce a variety of products [2]. Several studies have shown that is possible to develop extruded snacks with pseudocereals such as quinoa to improve their antioxidant properties and with jumbo squid mantle, due to its high protein content, low price, and high availability [3,4,5]. Canihua, (Chenopodium pallidicaule Aellen), is a nutritious grain from the South American Andean highlands, cultivated in Puno, Peru, between 3200 and 4200 amsl. The cañihua cultivars present higher protein content (up to 20% of Dry Mass (DM)) and lipids content (up to 10% DM), crude fiber, and mineral contents compared to common cereals such as rice, corn, barley, and pseudocereals such as amaranth or quinoa [6]. Jumbo squid (Dosidicus gigas), also known as Humboldt squid, is one of the largest cephalopods and lives in the eastern Pacific Ocean. It represents an important economic fishery resource in Peru and other countries. Nevertheless, only the jumbo squid mantle is marketed, thereby, large amounts (up to 60% of whole weight) of squid off-products, such as skin, heads, fins, tentacles, and guts are generated and discarded, which are rich in many nutrients (proteins, lipids, minerals, biopolymers, etc.) [7]. There are no reports of the addition of protein hydrolyzed from jumbo squid (Dosidicus gigas) By-Product in extruded foods as far as we know. The objective of this research was to develop and characterize extruded snacks with protein hydrolyzed from JSBP and cañihua (Chenopodium pallidicaule Aellen).

2. Material and Methods

2.1. Raw Material

Jumbo squid By-Products (JSBP) (Dosidicus gigas) were collected from the Sechura Bay, Piura Department-Peru. Andean pseudocereal cañihua (Chenopodium pallidicaule Aellen), corn and rice flours were obtained from the local market in Lima city. JSBP were thawed, washed, and dehydrated by an infrared dryer (IRC D18, Irconfort, Sevilla, Spain) at 60 °C for 12 h in the Functional Foods Laboratory from the Universidad de Lima-Peru, and they were ground into the food shredder (Grindomix GM200, Restch, Haan, Germany) to obtain JSBP flour. The protein hydrolysed (PH) from JSBP flour was optimized and characterized using response surface methodology with a Box-Behnken design [8]. The parameters to optimize were temperature (40 °C to 60 °C), time (1 h to 2 h), and pH (5 to 8), the enzyme used was flavouryzme and 30 runs were carried out. The optimized parameters for the extraction of PH from JSBP were at 46° C for 1 h, 40 min and pH 6.5. All the samples were kept in aluminised bags at room temperature for later analysis.

2.2. Samples Preparation

Four extruded snacks were developed with different amounts of PH from JSBP and cañihua (

Table 1. Formulations for extruded snacks (ES) with protein hydrolyzed (PH) from jumbo squid By-products (JSBP) with cañihua (Chenopodium pallidicaule Aellen).

Samples	Corn Flour (%)	Rice Flour (%)	Cañihua Flour (%)	PHJSBP (%)
Control	55	30	15	-
ES1	55	26	15	4
ES2	55	24	15	6
ES3	55	22	15	8
ES4	55	20	15	10

). The ingredients of each sample were homogenized using a planetary mixer (FPSTSMPL1-053, Oster, Guangdong, China) and they were kept in aluminized bags at room temperature.

2.3. Extrusion Process

Extrusion was performed in a twin-screw extruder scale pilot plant Model SLYE32-II (Saibainuo, China) at 10 hz of feeder speed and 400 rpm of screw speed. The extrusion process temperatures were 60, 90, 120 and 150 °C. The sample was fed to a mass flow of 200 g/min. The extruded products were stored at ambient conditions (25 °C, RH = 65%) in polyethylene bags until use. All the extrusion parameters were monitored during the process [4].

2.4. Physicochemical Characterization

2.4.1. Expansion Index (EI) and Bulk Density

The expansion index (EI) was measured by dividing the diameter of 15 extruded snacks by the diameter of the nozzle at the exit of the extruder (5 mm); the bulk density of the extrudates was determined using the seed (quinoa) displacement method [4]. The determinations were performed by triplicate.

2.4.2. Water Absorption Index (WAI)

A centrifuge tube was placed 1 g of extrudate with 50 mL of distilled water, left to stand for 30 min at room temperature and centrifuged for 15 min at 4000 rpm using a Centrifuge (HettichZentrifugen-Mikro, Germany). The WAI was the value of the weight of the gel obtained after removal of supernatant per unit weight of dry solids originals [4].

2.4.3. Proximal Composition

The moisture content of the extruded snacks was determined at 110 °C to a constant weight. The ash content was determined by ignition method (550 °C for 72 h). The fat content was determined with hexane for 9 h and the total Protein content was determined as % nitrogen × 6.25 using a Kjeldahl analyzer (UDK 139, VELP, Usmate Velate. Italy), by official methods.

2.4.4. Total Phenolic Content (TPC)

The total phenolic content (TPC) of the extruded snacks was determined by Folin–Ciocalteau method [9] at 760 nm using a spectrophotometer (UV 1280 Vis Spectrophotometer Shimadzu, Kyoto, Japan). The results were expressed as µg of gallic acid equivalent (GAE)/g snack. Analyzes were performed in triplicate and presented as mean values.

2.4.5. Antioxidant Activity

The antioxidant activity of samples was determined by the DPPH method [9] with some modifications at 517 nm by spectrometry, and ABTS free radical scavenging assay [9] at 734 nm (UV 1280 Vis Spectrophotometer Shimadzu, Kyoto, Japan). The results were expressed as µg Trolox/g snack. Analyzes were performed in triplicate and presented as mean values.

2.4.6. In Vitro Protein Digestibility (IVPD)

In vitro protein digestibility was measured by the method of Tinus et al. (2012) [10], with slight modification. The results will be expressed as:

IVPD (Digestibility (%) = 65.66 + 18.10 × (pH 0 min − pH 10 min))

2.5. Determination of Amino Acid Profile

The amino acid profile was estimated according to Alaiz et al. [8] with slight modifications. Amino acid profile were determined by HPLC (ARC, Waters, Milford, CT, USA) with an inner diameter of 150 mm × 9 mm, column C18 reverse phase and tryptophan was quantified by HPLC after basic hydrolysis. All measures were performed in duplicate.

2.6. Oxidative Stability

The oxidative stability was determined using an 892 Professional Rancimat© (Metrohm, Herisau, Switzerland) [9]. To calculate the shelf life at 25 °C, induction periods at 80, 100, and 120 °C were determined. The determinations were performed in triplicate. Also, acid index (NTP 206.013) and peroxide index (NTP 201.016), were determined to evaluate the degree of hydrolysis and the oxidation state of the samples at room temperature.

2.7. Sensory Analysis

A panel of 50 individuals (university students between 18–26 years old) evaluated the sensory attributes for appearance, flavor, texture, and overall acceptability. The test was based on a 9-point hedonic scale (1 = dislike extremely and 9 = like extremely). The panelists received random samples served in plastic cup and where coded with 5 different digits [9].

2.8. Statistical Analysis

Results were expressed as mean ± standard deviation, all measurements were conducted in triplicate, except for the measurement of oxidative stability and the evaluation of gastrointestinal simulation, which were done in duplicate. Analysis of variance (ANOVA) was used to analyze the acquired data at a 95% significance level (Minitab Inc., USA).

3. Results and Discussions

According to

Table 2. Physical characteristics of extruded snacks (ES) with protein hydrolyzed (PH) from Jumbo Squid By-products (JSBP) and cañihua (Chenopodium pallidicaule Aellen).

Sample	Expansion Index (cm/cm)	Density (g/cm3)	Diameter (mm)	WAI (g/g)
Control	2.49 ± 0.08	0.074 ± 0.005	12.47 ± 0.40	6.89 ± 0.23
ES1 (4% PHJSBP)	2.32 ± 0.05	0.064 ± 0.008	11.60 ± 0.26	6.40 ± 0.15
ES2 (6% PHJSBP)	1.83 ± 0.05	0.221 ± 0.047	9.17 ± 0.25	6.36 ± 0.28
ES3 (8% PHJSBP)	1.66 ± 0.05	0.226 ± 0.037	8.30 ± 0.26	6.23 ± 0.09
ES4 (10% PHJSBP)	1.50 ± 0.05	0.213 ± 0.034	7.50 ± 0.26	6.11 ± 0.15

Results are expressed as means ± SD (n = 3). WAI: Water Absorption Index.

, the expansion index (1.50 ± 0.05 cm/cm to 2.32 ± 0.05 cm/cm) and the diameter (7.50 ± 0.26 mm to 11.60 ± 0.26 mm) of the extruded snacks were significantly decreased when the content of PH from JSBP was increased (4% to 10%), while the density values (0.213 ± 0.034 g/cm³ to 0.064 ± 0.008 g/cm³) increased. These results were similar to those obtained by Roldan et al. [5]. Expansion indexes have an important role in the acceptability of the final product.

The WAI of extruded snacks were significantly higher (6.11 ± 0.15 g/g to 6.40 ± 0.15 g/g) than those reported by Roldan et al. [4]. The increase of protein content may have caused the reduction in the capacity of starch to bind with water [11]. The physical characteristics of extruded snacks showed slight difference compared to control sample without PH from JSBP.

The proximate composition is presented in

Table 3. Proximate composition of extruded snacks (ES) with protein hydrolyzed (PH) from Jumbo Squid By-products (JSBP) and cañihua (Chenopodium pallidicaule Aellen).

Sample	Moisture (%)	Lipids (%)	Protein (%)	Ash (%)	Carbohydrates (%)
CONTROL	6.04 ± 0.10	0.99 ± 0.01	8.35 ± 0.25	1.37 ± 0.01	83.25 ± 0.27
ES1 (4% PHJSBP)	5.81 ± 0.21	1.10 ± 0.07	11.20 ± 0.25	1.54 ± 0.01	80.35 ± 0.33
ES2 (6% PHJSBP)	5.35 ± 0.13	1.12 ± 0.01	12.27 ± 0.25	1.66 ± 0.04	79.60 ± 0.28
ES3 (8% PHJSBP)	4.85 ± 0.14	1.15 ± 0.05	14.14 ± 0.13	1.58 ± 0.06	78.28 ± 0.21
ES4 (10% PHJSBP)	4.46 ± 0.01	1.18 ± 0.04	15.39 ± 0.12	1.40 ± 0.01	77.57 ± 0.13

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

. By increasing the percentages of PH from JSBP, the protein content increased, being 15.39 ± 0.12% the highest in the ES4 sample compared to the control sample (8.35 ± 0.25%), as well as in comparison with other snacks, for example fish meal and corn grits (6.82% to 11.85%); fish powder, corn grits and rice grits (8.9% to 12.0%) [11]; and 13.34% for snacks enriched with jumbo squid mantle protein [5]. The lipid content (1.10 ± 0.07% to 1.18 ± 0.04%) and the ash content (1.40 ± 0.01% to 1.66 ± 0.04%) were higher than the control sample, meanwhile carbohydrates (80.35% to 77.57%) were lower. The moisture content is lower than 5.81 ± 0.21%, this percentage is a quality index that indicates that the product will be less susceptible to the effect of microbes, therefore, the extruded snacks with PH from JSBP will have a long-life stability.

The results of TPC, ABTS and DPPH are shown in

Table 4. Total polyphenolic content (TPC) (µg GAE/g snack), ABTS (µg Trolox/g snack), DPPH (mg Trolox/g snack) and in vitro digestibility from extruded snacks (ES) with protein hydrolyzed (PH) from Jumbo Squid By-products (JSBP) and cañihua (Chenopodium pallidicaule Aellen).

Sample	TPC (µg GAE/g Snack)	ABTS (µg Trolox/g Snack)	DPPH (µg Trolox/g Snack)	In Vitro Digestibility (%)
CONTROL	4726 ± 542	2714 ± 114	5745 ± 500	74.76 ± 0.47
ES1 (4% PHJSBP)	5633 ± 531	3599 ± 112	6198 ± 480	72.58 ± 0.51
ES2 (6% PHJSBP)	6236 ± 521	4944 ± 190	9028 ± 481	73.40 ± 0.13
ES3 (8% PHJSBP)	6734 ± 531	6338 ± 112	10,519 ± 461	73.03 ± 0.39
ES4 (10% PHJSBP)	7315 ± 521	7626 ± 110	12,739 ± 461	74.40 ± 0.51

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

. The extruded snacks showed a higher TPC (5633 ± 531 µg GAE/g snack to 7315 ± 521 µg GAE/g snack) compared with the control sample. The antioxidant activity by ABTS (3599 ± 112 µg Trolox/ g snack to 7626 ± 110 µg Trolox/ g snack) and by DPPH (5745 ± 500 µg Trolox/g snack to 12,739 ± 461 µg Trolox/g snack) were higher than the control sample.

According to Table 4, the extruded snacks presented high in vitro digestibility (72.58 ± 0.51% to 74.4 ± 0.51%) increased according to the amount of PH from JSBP. Extrusion has been reported to improve protein digestibility due to the protein denaturation and inactivation of antinutritional factors [7,11].

The amino acid composition of the extruded snacks showed a balanced profile, according to the FAO/WHO recommendations for healthy nutrition [8]. Taking into consideration the highest amino acids of ES4 (10% PHJSBP) were: aspartic acid + asparagine (83.88 ± 0.23 mg/g protein), glutamic acid + glutamine (147.67 ± 9.25 mg/g protein), glycine (52.26 ± 0.62 mg/g protein), alanine (64.32 ± 0.86 mg/g protein), valine (47.24 ± 0.46 mg/g protein), isoleucine (42.47 ± 0.45 mg/g protein), leucine (84.91 ± 0.51 mg/g protein), phenylalanine (41.72 ± 0.21 mg/g protein), and lysine (51.51 ± 0.24 mg/g protein).

The samples showed a low acid index (0.05 ± 0.00% of oleic acid) and peroxide index (0.00 ± 0.00 meq O₂/kg snack to 0.063 ± 0.01 meq O₂/kg snack), respectively. Also, the lifetime by the Rancimat© method showed an extrapolated shelf life at 25 °C of between 283 days to 630 days, which is higher than the control sample (248 days), this can be due to a high polyphenolic content and antioxidant activity from the addition of PH from JSBP.

The extruded snacks presented a light beige color with no residual smell of squid, similar results were reported by Roldan et al. [4,5]. In the sensory evaluation, consumers acceptability was considered above 5 (neither like nor dislike) in a hedonic scale from 1 to 9 [11]. Thus, an acceptability of 74% was obtained across all the samples, the characteristics of appearance, flavor, texture, and overall acceptability of the extruded snacks had an average value of 5.64, 4.98, 5.89, and 5.26, respectively. The sensory aspect would be one of the most important factors influencing eating behavior along with cost, safety, and accessibility.

4. Conclusions

The addition of PH from JSBP and cañihua improved the physical properties and increased the protein contented, phenolic concentration, antioxidant activity and in vitro protein digestibility of the extruded snacks compared with the control sample. Also, the results showed that the extruded snacks complied with the essential amino acids according to FAO / WHO. The snacks were accepted by the panel evaluators and complied with the Peruvian standard NTP-209.226, and they are much healthier than several commercially available snacks products. Therefore, the extruded snacks developed can be an alternative for the healthy food market and satisfy market trends.