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DOI: 10.1039/D3FB00171G (Paper) Sustainable Food Technol., 2024, 2, 126-140

Nutritional quality analysis of high-moisture extrudates containing mixed proteins from soy and surimi

Anna Hu† a, Yujie Zhang† a, Jinchuang Zhang *a, Tongqing Li a, Zhaojun Wang b and Qiang Wang *a
aInstitute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Key Laboratory of Agro-Products Processing, Ministry of Agriculture and Rural Affairs, Beijing 100193, China. E-mail: zhangjinchuang1002@163.com; wangqiang06@caas.cn
bState Key Laboratory of Food Science and Resource, Jiangnan University, Wuxi, 214122, China

Received 25th September 2023 , Accepted 29th November 2023

First published on 1st December 2023

Abstract

High-moisture extrusion technology emerges as a prime choice for preparing alternative protein products with a meat-like texture. However, the nutritional aspects of these products, prepared from a blend of plant and animal proteins, remain unclear. This study investigated the nutritional qualities of extrudates derived from soy protein isolate (SPI) and surimi, exploring ratios ranging from 90[thin space (1/6-em)]:[thin space (1/6-em)]10 to 50[thin space (1/6-em)]:[thin space (1/6-em)]50, with varied extrusion temperature (125 °C, 135 °C and 145 °C) and moisture content (65%, 70% and 75%). Results revealed the significant role played by surimi in enhancing both amino acid and fatty acid contents in high-moisture extrudates originating from SPI and surimi. Notably, the first limiting amino acid score (AAS/MET + CYS) increased significantly from 88.82 to 109.50 as the surimi content increased from 10% to 50%. Moreover, the levels of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in the extrudates significantly increased, concurrently reducing the n-6/n-3 fatty acid ratio. At a higher moisture content (70–75%), increasing extrusion temperature bolstered the fatty acid content in the extrudates. When the SPI–surimi ratio was 90[thin space (1/6-em)]:[thin space (1/6-em)]10, the gastric digestibility of the extrudates was the highest (60.20%). Meanwhile, the highest small intestinal digestibility was 93.07% at a SPI–surimi ratio of 70[thin space (1/6-em)]:[thin space (1/6-em)]30. At lower extrusion temperatures (125–135 °C), increasing moisture content led to a notable increase in the small intestinal digestibility of the extrudates. SPI–surimi ratios and hydro-thermal combined parameters have significant effects on the in vitro digestibility of high-moisture extrudates. This study could contribute to the improvement of nutritional qualities of alternative protein products based on mixed proteins from soy and surimi.


Sustainability spotlight

According to the UN's Sustainable Development Goals (SDGs), alternative protein products containing mixed proteins from soy and surimi can contribute to sustainability in several aspects. Firstly, this study focuses on the nutrition analysis of high-moisture extruded mixed proteins from soy and surimi, including amino acid and fatty acid contents, etc., which corresponds to SDG 2, improving nutrition and promoting sustainable agriculture. Secondly, partially replacing animal protein with plant protein has the potential to mitigate chronic diseases like heart disease and diabetes, thereby contributing to the achievement of SDG 3 of healthier food and diets. Thirdly, this study is also beneficial for achieving SDG 13 for fewer greenhouse gas emissions through alternative protein product development. In conclusion, this study has a positive impact on the UN SDGs.

Introduction

The global population is projected to reach about 10 billion individuals by 2050,1 resulting in a rapid rise in the global demand for protein sources. The global demand for animal-derived meat products is expected to reach 455 million tons.2 To meet this demand, it is necessary to explore new protein sources to complement traditional ones. Alternative protein sources3 such as plant proteins (grains, legumes, tubers, and oilseeds), insect proteins, microorganisms (fungi and bacteria) and aquatic proteins (algae) are gaining attention. These sources are grown and processed in ways that reduce greenhouse gas emissions,4,5 land, and water resource wastage.6 They are already used in food, cosmetics and pharmaceuticals.1,7 Various meat-like alternative protein products have been developed from sustainable sources.8,9 Soy protein is known for its excellent gelation properties and fibrous structure formation.10–12 Surimi, derived from animals, contains unsaturated fatty acids (e.g., DHA and EPA). Mixing soy protein with surimi can create alternative protein products with comprehensive nutrition quality.13,14 These products have garnered attention, particularly in terms of their nutrition profile,14,15 including amino acids, fatty acids and digestibility.

Combining soy protein and surimi results in products with superior nutritional properties.13,16 Researchers have explored different processing methods,7,8 such as ultra-high pressure, microwave heating, 3D printing and ultrasonic technology, to enhance the quality of surimi-based products. Food extrusion technology has also been used to improve the digestibility and texture of soy protein and surimi blends.17 Kaur et al.18 showed that adjusting the ratios of surimi and wheat protein could enhance the digestibility of extrudates. Adding soy protein can increase the content of essential amino acids,19 but a higher extrusion temperature and a lower moisture content may lead to amino acid loss.20

High-moisture extrusion is a promising method for creating alternative protein products with a meat-like texture.21 One of the advantages of high-moisture extrusion is that the extrudates produced are ready-to-eat and have an improved fibrous structure.22 It is energy-efficient and environmentally sustainable,9,23 improving the digestibility of both plant and animal proteins while reducing anti-nutritional factors.24 At present, raw materials mainly consist of plant proteins such as soy protein, pea protein, and wheat gluten. Gradually, animal proteins have been added, enriching the products with a variety of nutrients, including proteins, lipids, carbohydrates, minerals, vitamins and dietary fiber.13,16,17 Extrusion can be used to imitate the texture of marine products, such as by adding surimi during extrusion.25 Altering the raw material ratio and extrusion process parameters during the high-moisture extrusion can further enhance nutritional properties.17,26 Kaur et al.18 showed that the content of essential amino acids and fatty acids can be increased as the surimi content increased. Lin et al.27 showed that the dietary fiber content of surimi and the antioxidant capacity were enhanced with the addition of wheat. Pudtikajorn et al.28 reported that the addition of surimi increased the nutritional quality of fish tofu. Sorensen et al.29 reported that a low extrusion temperature improved the digestibility of extruded feeds. Delgado et al.30 found that different extrusion temperatures, screw speeds and moisture contents changed the nutritional content of extrudates. However, the nutritional qualities of mixed proteins from soy and surimi under high-moisture extrusion conditions (moisture content ranging from 40% to 80%) remain uncertain.31

This study aims to analyze nutritional changes in extrudates through high-moisture extrusion, varying SPI–surimi ratios and extrusion parameters. It also seeks to explore the effect of SPI–surimi ratios on amino acids and fatty acids in extrudates and examine how hydrothermal parameters affect these nutritional aspects. Additionally, the digestibility of the mixed proteins from soy and surimi was investigated. These findings reveal the nutritional potential of alternative protein products with a mixture of plant and animal proteins prepared using high-moisture extrusion.

Materials and methods

Materials

Soy protein isolate (SPI) was supplied by Yihai Kerry Co., Ltd. (Shanghai, China), containing 90.81% protein (dry basis), 5.55% moisture, 0.36% fat (dry basis) and 4.67% ash content (dry basis). Surimi was purchased from Shengteng Seafood Co., Ltd. (Qingdao, China), containing 52.78% protein (dry basis), 67.97% moisture, 8.38% fat (dry basis) and 1.73% ash content (dry basis).

High-moisture extrusion experiments

Before extruding, the SPI and surimi were mixed using a mixer (JHF-20L, Zhengzhou Jinhe Machinery Manufacture Co., Ltd, China). The extrusion experiments of the SPI–surimi mixtures were carried out using a co-rotating twin-screw food extruder (FMHE36-24, FUMACH, China) with a screw diameter of 36 mm and a length/diameter ratio of 24[thin space (1/6-em)]:[thin space (1/6-em)]1. The extruder barrel was segmented into a feeding zone and five temperature-controlled zones. At the exit of the barrel, a long cylindrical cooling die with a diameter of 22 mm was attached. The extrusion conditions of different SPI–surimi ratios and hydro-thermal combined parameters were set according to Tables 1 and 2, respectively. The cooling die was kept at 50 °C controlled by the running moisture.8
Table 1 High-moisture extrusion conditions with different SPI[thin space (1/6-em)]:[thin space (1/6-em)]surimi ratios
Number SPI[thin space (1/6-em)]:[thin space (1/6-em)]surimi Extrusion temperature (°C) Moisture content (%) Screw speed (rpm) Feed rate (kg h−1)
1 90[thin space (1/6-em)]:[thin space (1/6-em)]10 135 70 210 7
2 80[thin space (1/6-em)]:[thin space (1/6-em)]20 135 70 210 7
3 70[thin space (1/6-em)]:[thin space (1/6-em)]30 135 70 210 7
4 60[thin space (1/6-em)]:[thin space (1/6-em)]40 135 70 210 7
5 50[thin space (1/6-em)]:[thin space (1/6-em)]50 135 70 210 7

Table 2 High-moisture extrusion conditions with different hydro-thermal combined parameters
Number SPI[thin space (1/6-em)]:[thin space (1/6-em)]surimi Extrusion temperature (°C) Moisture content (%) Screw speed (rpm) Feed rate (kg h−1)
1 80[thin space (1/6-em)]:[thin space (1/6-em)]20 125 65 210 7
2 80[thin space (1/6-em)]:[thin space (1/6-em)]20 125 70 210 7
3 80[thin space (1/6-em)]:[thin space (1/6-em)]20 125 75 210 7
4 80[thin space (1/6-em)]:[thin space (1/6-em)]20 135 65 210 7
5 80[thin space (1/6-em)]:[thin space (1/6-em)]20 135 70 210 7
6 80[thin space (1/6-em)]:[thin space (1/6-em)]20 135 75 210 7
7 80[thin space (1/6-em)]:[thin space (1/6-em)]20 145 65 210 7
8 80[thin space (1/6-em)]:[thin space (1/6-em)]20 145 70 210 7
9 80[thin space (1/6-em)]:[thin space (1/6-em)]20 145 75 210 7

Determination of amino acids

The amino acid score (AAS) was estimated from the amount of protein required to provide the minimal essential amino acid (EAA) pattern for adults, using the FAO/WHO (2007) reference pattern and according to the equation:32
 
image file: d3fb00171g-t1.tif(1)

The chemical score (CS) and the essential amino acid index (EAAI) were calculated by the method of the equations:33

 
image file: d3fb00171g-t2.tif(2)
 
image file: d3fb00171g-t3.tif(3)

The biological value (BV) is the ratio of the amount of nitrogen used by the human body and the amount of nitrogen absorbed by the body after protein was digested and absorbed. BV was calculated using eqn (4):34

 
BV = (1.09 × EAAI) − 11.70(4)

The nutritional index (NI) was used to comprehensively describe the protein content and amino acid composition patterns, which was calculated using eqn (5):35

 
NI = EAA × protein (g/100 g)/100(5)

Determination of fatty acids

The fatty acid profiles were analyzed in a previous study.33 The atherosclerosis index (IA) and thrombosis index (IT), used to assess the effect of fatty acids in extrudates on human cardiovascular diseases, were calculated according to eqn (6) and (7),36 respectively.
 
image file: d3fb00171g-t4.tif(6)
 
image file: d3fb00171g-t5.tif(7)

The monounsaturated fatty acid and the polyunsaturated fatty acid were denoted as MUFA and PUFA, respectively.

In vitro protein digestibility

The in vitro protein digestibility (IVPD) of the extrudates was determined according to a previous study.8 With some modifications, 0.1 g triturated extrudates were diluted with 15 mL of 0.1 M HCL and preheated at 37.5 °C for 10 min. 2 mg pepsin (Sigma-Aldrich Ltd., St Louis, USA) was added into the preheated solution and kept at 37.5 °C for 3 h. The pepsin hydrolysis was ended by adding 7.5 mL of 0.2 M NaOH. The solution was collected to analyze the gastric IVPD. The simulated intestinal digestibility started with adding 7.5 mL of 0.2 M phosphate buffer (pH 8.0) containing 4 mg trypsin (Sigma-Aldrich Ltd., St Louis, USA) into the solution of the ended pepsin hydrolysis, and then the solution was heated at 37 °C for 4 h. The trypsin hydrolysis was ended by boiling for 10 min. The final solution was collected. All of the collected solution was precipitated with isopycnic 10% trichloroacetic acid for 1 h and then centrifuged at 1000 g for 30 min.37 The liquid supernatant was collected to determine the protein content. The blank sample was prepared by treatments under the described conditions without the extrudate sample. The IVPD of the extrudates was calculated using the equation:34
 
IVPD (%) = (Ps − P0)/Pe × 100%(8)
The Ps, P0 and Pe represent the protein content of the liquid supernatant, the blank and the triturated extrudates, respectively.

Statistical analysis

Analysis of variance (ANOVA) was used to analyze all data through Statistical Product and Service Solutions software (version 26.0, SPSS Inc., Chicago, USA). Duncan's test was used to evaluate the comparisons between treatments. The statistical significance level was set at 0.05. Principal component analysis (PCA) was performed using The Unscrambler X 10.4.

Results and discussion

Amino acid evaluation

Effect of SPI–surimi ratios. As can be seen in Table 3, the amino acid content increased as the surimi content increased from 10% to 40%, which was consistent with the study of others.38 It was also found that the most abundant amino acids were Glu (190.05–222.38 mg per g protein), Asp (110.72–120.92 mg per g protein) and Leu (75.10–89.69 mg per g protein). However, the scarcest amino acids were Cys (7.76–9.00 mg per g protein), Trp (9.77–11.20 mg per g protein) and Met (11.49–16.33 mg per g protein). Aberoumand and Baesi39 and Hughes et al.40 also found that Glu, Asp and Leu took up the highest proportion of amino acids in the surimi and SPI and Cys and Met were the scarcest amino acids of SPI, suggesting that the high-moisture extrusion had no effect on the general composition of amino acids in SPI and surimi blends. Fig. 1 displays the amino acid scores (AASs) and chemical scores (CSs) of the extrudates at different SPI–surimi ratios. It indicated that Met + Cys was the first limiting amino acid, and the corresponding scores of AAS and CS were 88.82–109.50 and 55.83–68.83, respectively, which were increased as the surimi content increased from 10% to 50%. The corresponding values for Tyr + Phe were the highest in the range of 191.65–218.28 and 121.38–138.25, respectively. Hughes et al.40 found that the first limiting amino acid was Met + Cys in SPI, and Phe + Tyr got the highest AAS values. These results indicated that SPI played a role in the AAS evaluation of extrudates with different SPI–surimi ratios by high-moisture extrusion processing. At the SPI–surimi ratios of 80[thin space (1/6-em)]:[thin space (1/6-em)]20, 60[thin space (1/6-em)]:[thin space (1/6-em)]40 and 50[thin space (1/6-em)]:[thin space (1/6-em)]50, the AAS of the extrudates was more than 100, suggesting that the amino acid contents of the extrudates was much higher than those of the FAO/WHO and the extrudates at these ratios could meet the requirements of adults' body.41
Table 3 Amino acid content of the extrudates with different SPI–surimi ratiosa
Amino acids (mg per g protein) SPI–surimi ratios
90[thin space (1/6-em)]:[thin space (1/6-em)]10 80[thin space (1/6-em)]:[thin space (1/6-em)]20 70[thin space (1/6-em)]:[thin space (1/6-em)]30 60[thin space (1/6-em)]:[thin space (1/6-em)]40 50[thin space (1/6-em)]:[thin space (1/6-em)]50
a Different letters in the same row mean significant differences (p < 0.05).
Essential amino acids (EAAs) THR 22.05 ± 0.51c 32.55 ± 0.62b 22.71 ± 0.53c 35.23 ± 0.11a 23.78 ± 1.57c
VAL 39.80 ± 0.11e 46.15 ± 0.64b 41.76 ± 0.11d 50.36 ± 0.68a 44.96 ± 0.25c
MET 11.49 ± 0.13c 13.00 ± 0.01b 13.10 ± 0.28b 15.50 ± 0.52a 16.33 ± 0.48a
ILE 35.54 ± 0.27e 43.43 ± 0.45b 37.02 ± 0.16d 46.73 ± 0.30a 40.25 ± 0.61c
LEU 75.10 ± 0.30d 85.07 ± 2.02b 78.41 ± 0.26c 89.69 ± 0.66a 83.59 ± 0.93b
TRP 9.77 ± 0.25b 11.20 ± 0.36a 9.85 ± 0.13b 10.57 ± 0.52ab 10.17 ± 0.11b
PHE 43.62 ± 0.16d 45.77 ± 0.25c 46.17 ± 0.52c 48.18 ± 0.94a 47.12 ± 0.39ab
LYS 51.63 ± 0.45d 61.21 ± 1.12b 55.40 ± 0.47c 67.69 ± 0.81a 61.61 ± 0.28b
Non-essential amino acids (NEAAs) ASP 110.72 ± 0.16c 113.85 ± 1.85bc 115.81 ± 0.70b 120.92 ± 1.45a 119.93 ± 1.46a
HIS 19.55 ± 0.08d 23.60 ± 0.42b 20.25 ± 0.13d 24.49 ± 0.54a 21.23 ± 0.24c
ARG 59.54 ± 0.80c 71.98 ± 1.10a 62.50 ± 0.42bc 75.65 ± 0.81a 64.45 ± 3.37b
PRO 51.02 ± 4.12a 40.94 ± 2.45b 51.75 ± 1.24a 41.94 ± 1.03b 46.64 ± 2.65b
CYS 8.05 ± 0.05b 8.48 ± 0.66ab 8.53 ± 0.03ab 9.00 ± 0.25a 7.76 ± 0.27b
TYR 29.21 ± 0.23c 32.90 ± 0.26b 31.00 ± 0.74c 34.77 ± 0.28a 33.79 ± 1.38ab
SER 40.60 ± 1.01c 43.58 ± 0.83b 41.45 ± 0.47c 47.58 ± 0.26a 41.89 ± 0.51c
GLU 190.05 ± 0.14d 207.33 ± 1.34b 198.52 ± 1.84c 222.38 ± 2.50a 208.33 ± 1.51b
GLY 32.38 ± 0.01c 37.68 ± 0.89b 32.91 ± 0.40c 39.49 ± 0.48a 33.67 ± 0.64c
ALA 34.97 ± 0.36d 41.40 ± 1.83ab 37.07 ± 0.52cd 43.51 ± 0.37a 39.54 ± 1.46bc
Total amino acids (TAAs) 865.01 ± 46.32c 960.06 ± 14.23b 904.17 ± 4.93bc 1023.60 ± 12.01a 945.01 ± 12.05b


image file: d3fb00171g-f1.tif
Fig. 1 Amino acid scores (AASs) (a) and chemical scores (CSs) (b) of the extrudates with different SPI–surimi ratios.

In Fig. 2, the EAA/EAAI values were between 50.17% and 55.16%, which could almost reach the reference values of 60% recommended by the FAO/WHO. At a SPI–surimi ratio of 60[thin space (1/6-em)]:[thin space (1/6-em)]40, the TAA, EAA, NEAA, EAA/NEAA, EAAI and BV of the extrudates were significantly higher than those of others. In Fig. 2b, at a SPI–surimi of 80[thin space (1/6-em)]:[thin space (1/6-em)]20, the NI of the extrudates was 32.24, which was significantly higher than that of all the others. Results showed that as the surimi content increased from 10% to 50%, the extrudates were rich in various amino acids and the amino acid pattern was more balanced, especially at a SPI–surimi ratio of 60[thin space (1/6-em)]:[thin space (1/6-em)]40. Ai et al.19 also reported that fish meal from surimi can improve the balance of the amino acid pattern. When the surimi content was excessive (50%), the interactions between soy protein and surimi protein molecules became weaker, while the protein–protein interactions of surimi were enhanced, which might not be conducive to the retention of amino acids.


image file: d3fb00171g-f2.tif
Fig. 2 The amino acid content with NEAA, EAA and TAA (a) and the amino acid evaluation with EAA/NEAA, EAAI, BV and NI (b) of the extrudates with different SPI–surimi ratios, and different letters indicate significant differences (p < 0.05).

The principal component analysis (PCA) soring plot and factor loading plot can make it easier to discriminate the differences of the samples visually and help to determine the degree of contribution of the variances (PC1-75% and PC2-24%). According to Fig. 3, EAA, NEAA and TAA were significantly related to the ratio of 60[thin space (1/6-em)]:[thin space (1/6-em)]40. And the NI was critically related to the ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20.


image file: d3fb00171g-f3.tif
Fig. 3 The PCA Bi-plots of the extrudates with different SPI–surimi ratios.
Effect of hydro-thermal combined parameters. Table 4 shows the amino acid contents of the extrudates with different hydro-thermal parameters. All the extrudates were rich in Glu (186.66–217.33 mg per g protein), Asp (100.32–116.70 mg per g protein) and Leu (73.88–82.10 mg per g protein), but lack of Cys (7.78–9.45 mg per g protein). Meanwhile, at a moisture content of 75%, as the extrusion temperature increased from 125 °C to 145 °C, the TAA content decreased from 968.68 mg g−1 to 876.47 mg g−1. This might be due to the degradation of amino acids by the Maillard reaction, which was consistent with Iwe et al.42 who also found the loss of Arg (21%) and Asp (14%) as extrusion temperature increased from 135 °C to 160 °C due to the Maillard reaction.43 Csapó et al.44 also found the loss of Lys (21%) in soy protein as extrusion temperature increased from 101 °C to 220 °C. Furthermore, the TAA content was the highest (968.68 mg per g protein) at a moisture content of 75% and extrusion temperature of 125 °C, while it was the lowest (828.36 mg per g protein) at a moisture content of 70% and extrusion temperature of 135 °C, suggesting that the increasing extrusion temperature would significantly disrupt the content of amino acids in the extrudates at a higher moisture content (70–75%).
Table 4 Amino acid contents of the extrudates with different hydro-thermal parametersa
Amino acid contents (mg per g protein) Hydro-thermal parameters
125 °C-65% 135 °C-65% 145 °C-65% 125 °C-70% 135 °C-70% 145 °C-70% 125 °C-75% 135 °C-75% 145 °C-75%
a Different letters in the same row mean significant differences (p < 0.05).
EAA THR 26.70 ± 1.82bcd 28.96 ± 0.47abc 26.62 ± 1.22cd 29.14 ± 0.06abc 25.73 ± 0.76d 29.25 ± 0.18abc 31.67 ± 2.06a 29.80 ± 0.59ab 29.32 ± 2.02abc
VAL 45.55 ± 3.31ab 48.31 ± 0.12a 44.68 ± 2.23ab 46.50 ± 0.79ab 42.18 ± 1.27b 47.60 ± 0.13a 47.83 ± 0.91a 44.75 ± 0.23ab 42.93 ± 2.85b
MET 12.59 ± 0.78abc 13.41 ± 0.38a 12.41 ± 0.38abc 12.61 ± 0.07abc 11.45 ± 0.29c 12.94 ± 0.49ab 13.05 ± 0.21a 12.45 ± 0.26abc 11.8 ± 0.77bc
ILE 39.32 ± 2.63ab 41.67 ± 0.01a 38.83 ± 1.99ab 41.36 ± 0.64a 37.04 ± 1.14b 41.91 ± 0.66a 42.96 ± 1.17a 40.47 ± 0.10ab 39.50 ± 2.86ab
LEU 75.18 ± 4.54abc 79.53 ± 0.06ab 73.88 ± 3.47bc 78.84 ± 1.28ab 69.87 ± 2.42c 79.00 ± 0.49ab 82.10 ± 3.28a 78.45 ± 0.22ab 75.38 ± 5.61abc
TRP 12.06 ± 0.52ab 12.37 ± 0.69a 10.57 ± 0.40cd 11.09 ± 0.35bc 10.26 ± 0.35cd 10.92 ± 0.42bcd 11.18 ± 0.29abc 11.05 ± 0.82bcd 9.84 ± 0.30a
PHE 43.64 ± 2.79ab 46.96 ± 0.06a 43.83 ± 1.56ab 45.00 ± 0.78ab 41.55 ± 0.95b 46.76 ± 0.31a 46.52 ± 1.73ab 44.27 ± 0.82ab 42.86 ± 3.85ab
LYS 59.41 ± 4.07abc 62.89 ± 0.39a 58.31 ± 2.40abc 61.09 ± 0.95ab 54.70 ± 1.41c 62.29 ± 0.54a 63.16 ± 1.48a 59.59 ± 0.35abc 55.93 ± 4.03bc
NEAA ASP 107.57 ± 7.38abc 115.41 ± 0.94ab 105.89 ± 4.55abc 111.51 ± 1.34ab 100.32 ± 2.67c 113.71 ± 0.91ab 116.70- ± 4.18a 109.97 ± 0.47abc 104.8 ± 7.74bc
HIS 21.41 ± 1.37abc 22.47 ± 0.69ab 20.97 ± 0.82bc 22.33 ± 0.06ab 19.79 ± 0.42c 22.53 ± 0.24ab 23.19 ± 0.87a 22.17 ± 0.15ab 21.41 ± 1.51abc
ARG 68.40 ± 4.94abc 71.96 ± 1.09ab 66.79 ± 2.86abc 70.41 ± 0.48ab 63.13 ± 1.50ab 71.34 ± 0.44ab 72.59 ± 2.36a 68.86 ± 0.72abc 65.33 ± 4.43bc
PRO 30.77 ± 0.26d 33.9 ± 0.63bcd 31.11 ± 2.89cd 35.08 ± 0.3abc 30.09 ± 1.24d 34.96 ± 0.77abc 38.21 ± 2.79a 36.52 ± 0.10ab 36.52 ± 2.45ab
CYS 8.32 ± 0.30abc 8.79 ± 0.66abc 9.04 ± 0.69ab 8.90 ± 0.40abc 7.78 ± 0.18c 8.76 ± 0.16abc 9.45 ± 0.69a 8.93 ± 0.34abc 8.00 ± 0.21bc
TYR 27.72 ± 1.70bc 29.43 ± 0.17ab 27.85 ± 1.04bc 29.74 ± 0.35ab 26.47 ± 0.84c 29.79 ± 0.25ab 31.37 ± 1.36a 30.28 ± 0.23ab 29.13 ± 2.03abc
SER 39.40 ± 2.86bc 42.88 ± 0.75abc 39.20 ± 2.17bc 42.95 ± 0.18abc 37.73 ± 1.15c 43.18 ± 0.30ab 46.78 ± 2.95a 43.29 ± 2.18ab 42.15 ± 3.12abc
GLU 200.27 ± 14.25abc 214.26 ± 1.55a 197.65 ± 8.58abc 208.47 ± 2.50ab 186.66 ± 5.47c 211.89 ± 1.48ab 217.33 ± 6.60a 205.66 ± 1.90abc 192.45 ± 14.31bc
GLY 31.54 ± 2.27bc 33.85 ± 0.19ab 31.58 ± 1.07bc 34.19 ± 0.19ab 30.49 ± 1.07c 34.15 ± 0.10ab 36.06 ± 1.40a 34.12 ± 0.30ab 33.56 ± 2.21abc
ALA 34.42 ± 2.15bc 36.86 ± 0.24ab 34.46 ± 1.29bc 36.09 ± 0.31abc 33.12 ± 0.74c 37.47 ± 0.02ab 38.53 ± 1.77a 36.20 ± 0.35abc 35.55 ± 1.82abc
TAA 884.27 ± 56.83ab 943.91 ± 8357a 873.67 ± 38.8ab 925.30 ± 11.02ab 828.36 ± 20.30b 938.45 ± 5.40a 968.68 ± 35.52a 916.83 ± 8.10ab 876.47 ± 62.12ab

Table 5 shows the AAS and CS of the extrudates under different hydro-thermal parameters and the PHE + TYR of the extrudates showed the highest scores, which were 179.00–204.97 and 113.37–129.82, respectively. The AAS and CS of the amino acids were more than 100 except Met + Cys, which can be seen as the first limiting amino acid with the corresponding scores of 90.00–102.27 and 54.94–64.29, respectively. The result indicated that the extrusion parameters had no large effect on the first limiting amino acid of the SPI–surimi extrudates. At the same time, it showed that the amino acid composition of the extrudates could meet the recommended intake.41

Table 5 Amino acid scores (AASs) and chemical scores (CSs) of the extrudates with different hydro-thermal parametersa
Amino acid evaluation (scores) Hydro-thermal parameters
125 °C-65% 135 °C-65% 145 °C-65% 125 °C-70% 135 °C-70% 145 °C-70% 125 °C-75% 135 °C-75% 145 °C-75%
a Different letters in the same row mean significant differences (p < 0.05).
AAS HIS 142.72 ± 9.14abc 149.82 ± 4.61ab 139.78 ± 5.47bc 148.85 ± 0.40ab 131.95 ± 2.83c 150.20 ± 1.63ab 154.58 ± 5.82a 147.75 ± 0.99ab 142.72 ± 10.08abc
THR 116.08 ± 7.93bcd 125.89 ± 2.04abc 115.71 ± 5.32cd 126.71 ± 0.25abc 111.85 ± 3.27d 127.18 ± 0.81abc 137.68 ± 8.95a 129.55 ± 2.57ab 127.46 ± 8.74abc
LYS 132.02 ± 9.06abc 139.75 ± 0.86a 129.57 ± 5.32abc 135.75 ± 2.12ab 121.56 ± 3.15c 138.42 ± 1.19a 140.35 ± 3.30a 132.41 ± 0.76abc 124.28 ± 8.97bc
LEU 131.07 ± 8.79ab 138.90 ± 0.01a 129.43 ± 6.66ab 137.85 ± 2.16a 123.45 ± 3.78b 139.68 ± 2.21a 143.19 ± 3.87a 134.89 ± 0.33ab 131.67 ± 9.5ab
ILE 127.43 ± 7.70abc 134.8 ± 0.10ab 125.21 ± 5.88bc 133.62 ± 2.17ab 118.43 ± 4.10c 133.89 ± 0.83ab 139.15 ± 5.57a 132.97 ± 0.37ab 127.77 ± 9.52abc
MET + CYS 95.05 ± 3.54abc 100.91 ± 1.75a 97.50 ± 1.74abc 97.77 ± 0.30abc 87.41 ± 1.30c 98.64 ± 2.22ab 102.27 ± 0.95a 97.18 ± 1.21abc 90.00 ± 3.49bc
PHE + TYR 187.79 ± 11.83ab 201.03 ± 0.59a 188.63 ± 6.83ab 196.68 ± 2.95ab 179.00 ± 4.72b 201.45 ± 1.47a 204.97 ± 8.13a 196.18 ± 2.76ab 189.45 ± 15.48ab
VAL 119.80 ± 8.48ab 127.13 ± 0.32a 117.58 ± 5.73ab 122.37 ± 2.02ab 111.00 ± 3.24b 125.26 ± 0.34a 125.87 ± 2.33a 117.76 ± 0.57ab 112.97 ± 7.31b
TRP 200.93 ± 8.70ab 206.12 ± 11.38a 176.10 ± 6.72cd 184.85 ± 5.85bc 171.04 ± 5.84cd 181.97 ± 7.09bcd 186.29 ± 4.88bc 184.13 ± 13.7bcd 163.89 ± 5.02d
CS THR 66.75 ± 4.56bcd 72.38 ± 1.17abc 66.54 ± 3.06cd 72.86 ± 0.14abc 64.32 ± 1.87d 73.13 ± 0.47abc 79.17 ± 5.14a 74.50 ± 1.48ab 73.29 ± 5.02abc
LYS 108.02 ± 7.42abc 114.34 ± 0.70a 106.02 ± 4.35abc 111.07 ± 1.73ab 99.46 ± 2.57c 113.26 ± 0.97a 114.84 ± 2.69a 108.34 ± 0.63abc 101.69 ± 7.33bc
LEU 107.40 ± 6.49ab 113.62 ± 0.08a 105.54 ± 4.96cd 112.62 ± 1.82bc 99.82 ± 3.46cd 112.85 ± 0.70bcd 117.29 ± 4.69abc 112.07 ± 0.32bcd 107.69 ± 8.03d
ILE 98.30 ± 6.59abc 104.18 ± 0.01ab 97.08 ± 5.00bc 103.39 ± 1.62ab 92.59 ± 2.84c 104.76 ± 1.65ab 107.40 ± 2.91a 101.17 ± 0.24ab 98.75 ± 7.13abc
MET + CYS 59.74 ± 2.23abc 63.43 ± 1.10a 61.29 ± 1.10abc 61.46 ± 0.18abc 54.94 ± 0.82c 62.00 ± 1.40ab 64.29 ± 0.59a 61.09 ± 0.76abc 56.57 ± 2.19bc
PHE + TYR 118.93 ± 4.37ab 127.32 ± 0.65ab 119.47 ± 5.54ab 124.57 ± 2.42ab 113.37 ± 5.58b 127.58 ± 0.86ab 129.82 ± 3.81a 124.25 ± 3.02ab 119.98 ± 10.85ab
VAL 91.10 ± 6.60ab 96.61 ± 0.25a 89.37 ± 4.46ab 93.00 ± 1.58ab 84.35 ± 2.53b 95.19 ± 0.26a 95.66 ± 1.82a 89.51 ± 0.45ab 85.86 ± 5.71b
TRP 120.56 ± 5.23ab 123.67 ± 6.83a 105.66 ± 4.03cd 110.91 ± 3.51bc 102.63 ± 3.50cd 109.18 ± 4.26bcd 111.77 ± 2.93abc 110.47 ± 8.22bcd 98.34 ± 3.01d

In Fig. 4, at a moisture content of 70% and extrusion temperature of 135 °C, the TAA, EAA, NEAA, EAAI and BV of the extrudates were significantly lower, and the EAA/NEAA values were between 53.71% and 55.18%, which could reach the reference values of 60% recommended by the FAO/WHO. At a certain temperature (125–145 °C), the EAA/NEAA and NI decreased dramatically as the moisture content increased from 65% to 75%. It indicated that at a certain temperature (125–145 °C), increasing moisture content could decrease the EAA/NEAA values slightly, and the amino acid pattern of the extrudates was also changed. Zahari et al.45 found that the amino acid pattern of the extrudates was more balanced at a moisture content of 65%. In this study, when the extrusion temperature was 125 °C, as the moisture content increased from 65% to 75%, the NEAA, EAA, TAA, EAAI and BV increased remarkably. At a moisture content of 75%, when the extrusion temperature increased from 125 °C to 145 °C, NEAA, EAA, TAA, EAAI and BV decreased dramatically, indicating that higher extrusion temperature would destroy the extrudates' amino acid pattern.46 It was further shown that the amino acid content and amino acid balance of the SPI–surimi extrudates could be improved by changing the extrusion parameters.


image file: d3fb00171g-f4.tif
Fig. 4 The amino acid content with TAA, EAA and NEAA (a) and the amino acid evaluation with EAA/NEAA, EAAI, BV and NI (b) of the extrudates with different hydro-thermal parameters, and different letters indicate significant differences (p < 0.05).

Fig. 5 shows that PC1 and PC2 could explain 72% and 25% of the total variance, respectively. Moreover, the NEAA and TAA were significantly related to the hydro-thermal combination parameters of 125 °C-75%. And the NI was significantly related to the hydro-thermal combined parameters of 125 °C-65%.


image file: d3fb00171g-f5.tif
Fig. 5 The PCA Bi-plots of the amino acid evaluation of the extrudates with different hydro-thermal parameters.

Fatty acid evaluation

Effect of SPI–surimi ratios. Table 6 shows the contents of 17 fatty acids of the extrudates with different SPI–surimi ratios. The contents of palmitic acid (184.91–251.37 mg per g fat) and linoleic acid (157.18–259.25 mg per g fat) were much higher especially at a SPI–surimi ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20, and the lowest fatty acid was butyric acid (0.00–1.76 mg per g fat). It showed that the high-moisture extrusion processing had no significant effect on the most abundant fatty acids in the SPI–surimi extrudates. As the ratio of surimi increased from 10% to 50%, the eicosapentaenoic acid (EPA) of the extrudates increased significantly from 1.44 mg per g to 10.30 mg per g and the docosahexaenoic acid (DHA) content increased prominently from 6.44 mg g−1 to 41.12 mg g−1. This result was consistent with Jannat et al.47 who also found that the addition of surimi resulted in the increase of DPA and EHA, which further confirmed that the surimi enhanced the unsaturated fatty acids (UFA) of the alternative protein foods.48
Table 6 Fatty acid contents of the extrudates with different SPI–surimi ratiosa
Fatty acid contents (mg per g fat) SPI–surimi ratios
90[thin space (1/6-em)]:[thin space (1/6-em)]10 80[thin space (1/6-em)]:[thin space (1/6-em)]20 70[thin space (1/6-em)]:[thin space (1/6-em)]30 60[thin space (1/6-em)]:[thin space (1/6-em)]40 50[thin space (1/6-em)]:[thin space (1/6-em)]50
a Different letters in the same row mean significant differences (p < 0.05). SFA denotes saturated fatty acids, UFA denotes unsaturated fatty acids, MUFA denotes monounsaturated fatty acids and PUFA denotes polyunsaturated fatty acids. n-3 and n-6 denote PUFA types.
C4:0 SFA Butyric acid 0.00 ± 0.00c 1.76 ± 0.31a 1.20 ± 0.09b 1.21 ± 0.07b 0.00 ± 0.00c
C12:0 SFA Lauric acid 3.69 ± 0a 0.00 ± 0.00c 0.00 ± 0.00c 1.04 ± 0.03b 1.01 ± 0.11b
C14:0 SFA Myristic acid 3.57 ± 0.06d 6.47 ± 0.53bc 6.09 ± 0.71c 7.79 ± 0.03ab 9.18 ± 1.09a
C15:0 SFA Pentadecanoic acid 1.04 ± 0.06c 1.83 ± 0.24b 2.11 ± 0.25b 2.52 ± 0.02a 2.76 ± 0.04a
C16:0 SFA Palmitic acid 184.91 ± 3.22d 251.37 ± 4.24a 225.55 ± 2.74b 202.32 ± 0.59c 191.83 ± 11.24cd
C16:1n7 MUFA Palmitoleic acid 2.08 ± 0.02d 5.57 ± 0.57c 6.89 ± 0.62b 9.07 ± 0.37a 10.38 ± 0.74a
C17:0 SFA Pearlescent fatty acid 2.23 ± 0.06c 3.68 ± 0.29b 3.71 ± 0.05b 4.41 ± 0.17a 4.75 ± 0.28a
C18:0 SFA Stearic acid 45.61 ± 0.35c 67.08 ± 2.72a 60.58 ± 1.01b 58.13 ± 0.74b 55.79 ± 3.64b
C18:1n9c MUFA Oleic acid 64.35 ± 0.56ab 67.55 ± 4.25a 61.94 ± 1.27ab 58.05 ± 0.74b 60.26 ± 5.51ab
C18:2n6c PUFA n-6 Linoleic acid 253.4 ± 5.56ab 259.25 ± 2.36a 246.28 ± 0.95b 184.92 ± 0.67c 157.18 ± 6.87d
C20:0 SFA Arachidonic acid 1.34 ± 0.10b 1.49 ± 0.28ab 1.67 ± 0.53ab 2.10 ± 0.27ab 2.23 ± 0.25a
C18:3n3 PUFA n-3 Alpha-linolenic acid 24.56 ± 0.80a 21.73 ± 0.10b 22.70 ± 0.06b 16.02 ± 0.01c 14.66 ± 0.67d
C22:0 SFA Behenic acid 3.38 ± 0.25b 5.29 ± 0.08a 4.47 ± 0.42a 4.31 ± 0.17ab 3.24 ± 0.77b
C20:4n6 PUFA n-6 Arachidonic acid 1.17 ± 0.24d 2.58 ± 0.65c 4.61 ± 0.97b 6.85 ± 0.24a 8.02 ± 0.31a
C24:0 SFA Lignocarboxylic acid 3.52 ± 0.04bc 4.55 ± 0.71a 3.70 ± 0.15ab 3.21 ± 0.10bc 2.78 ± 0.29c
C20:5n3 PUFA n-3 EPA 1.44 ± 0.37e 4.62 ± 0.10d 6.17 ± 0.14c 9.24 ± 0.38b 10.30 ± 0.45a
C22:6n3 PUFA n-3 DHA 6.44 ± 0.07e 18.47 ± 0.57d 26.51 ± 2.04c 37.40 ± 1.26b 41.12 ± 1.62a

As can be seen in Fig. 6, at a SPI–surimi ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20, the extrudate showed the highest saturated fatty acid (SFA), unsaturated fatty acid (UFA), monounsaturated fatty acid (MUFA), polyunsaturated fatty acid (PUFA), n-6 fatty acid (n-6FA) contents and the highest IT values. Meanwhile, at a SPI–surimi ratio of 50[thin space (1/6-em)]:[thin space (1/6-em)]50, the n-6FA, UFA and PUFA contents were the lowest and the IA values were the highest. Moreover, the n-6 fatty acid content/n-3 fatty acid content (n-6/n-3) values gradually decreased as the surimi content increased from 10% to 50%, and it might be related to the increasing n-3 fatty acid content from 32.44 mg g−1 to 66.08 mg g−1, indicating the enhanced ability of extrudates to prevent chronic diseases. The above results indicated that the fatty acid levels were the highest and the antioxidant properties of extrudates increased significantly when at a SPI–surimi ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20 during the high-moisture extrusion processing.49


image file: d3fb00171g-f6.tif
Fig. 6 The fatty acid content with the SFA, UFA, PUFA, MUFA, n-3FA and n-6FA (a) and the fatty acid evaluation with n-6/n-3, IA and IT (b) of the extrudates with different SPI–surimi ratios, and different letters indicate significant differences (p < 0.05).

Fig. 7 shows that PC1 and PC2 could explain 59% and 32% of the total variance, respectively. The SFA, UFA, PUFA, MUFA and IT were significantly related to the ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20. Additionally, the n-6/n-3 was positively related to the ratio of 90[thin space (1/6-em)]:[thin space (1/6-em)]10.


image file: d3fb00171g-f7.tif
Fig. 7 The PCA Bi-plots of the fatty acid evaluation of the extrudates with different SPI–surimi ratios.
Effect of hydro-thermal combined parameters. As shown in Table 7, palmitic acid (157.20–284.01 mg per g fat) and linoleic acid (176.89–308.46 mg per g fat) accounted for the highest portion of all the extrudates; however, the butyric acid (0.00–1.50 mg per g fat) content was the lowest. At a certain extrusion temperature (125–145 °C), the EPA and DHA contents of the extrudates decreased dramatically as the moisture content increased from 65% to 75%. Čolović et al.50 also found that increasing the moisture content could lead to less fatty acid contents during the high-moisture extrusion processing because of the inactivated lipase. However, when the moisture content was at 70%, as the extrusion temperature increased from 125 °C to 145 °C, the EPA and DHA contents increased significantly.
Table 7 Fatty acid contents of the extrudates with different hydro-thermal parametersa
Fatty acid contents (mg per g fat) Hydro-thermal parameters
125 °C-65% 135 °C-65% 145 °C-65% 125 °C-70% 135 °C-70% 145 °C-70% 125 °C-75% 135 °C-75% 145 °C-75%
a Different letters in the same row mean significant differences (p < 0.05). SFA denotes saturated fatty acids, UFA denotes unsaturated fatty acids, MUFA denotes monounsaturated fatty acids and PUFA denotes polyunsaturated fatty acids. n-3 and n-6 denote PUFA types.
C4:0 SFA Butyric acid 1.19 ± 0.08b 1.2 ± 0.05b 1.15 ± 0.20b 0.95 ± 0.10b 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 0.00 ± 0.00c 1.50 ± 0.25a
C12:0 SFA Lauric acid 1.64 ± 0.03a 1.44 ± 0.58a 1.42 ± 0.49a 0.94 ± 0.14a 1.12 ± 0.42a 0.88 ± 0.04a 0.00 ± 0.00b 1.26 ± 0.14a 1.39 ± 0.29a
C14:0 SFA Myristic acid 10.33 ± 0.20a 9.80 ± 3.99ab 7.98 ± 1.51abc 5.72 ± 0.29c 6.83 ± 0.79abc 7.22 ± 1.19abc 4.64 ± 0.17c 6.29 ± 0.48bc 7.29 ± 0.59abc
C15:0 SFA Pentadecanoic acid 1.78 ± 0.22a 1.57 ± 0.23ab 1.39 ± 0.41abc 1.00 ± 0.04c 1.18 ± 0.06bc 1.09 ± 0.08bc 1.10 ± 0.10bc 1.05 ± 0.29bc 1.40 ± 0.08abc
C16:0 SFA Palmitic acid 284.01 ± 4.68a 280.80 ± 4.32a 253.63 ± 2.36b 157.20 ± 3.47f 203.92 ± 0.07d 215.78 ± 8.86c 175.73 ± 0.49e 177.10 ± 4.96e 221.47 ± 0.91c
C16:1n7 MUFA Palmitoleic acid 3.54 ± 0.16ab 3.72 ± 0.92a 3.39 ± 0.29ab 2.21 ± 0.26cd 2.69 ± 0.11bcd 2.69 ± 0.15bcd 2.16 ± 0.11d 2.37 ± 0.17cd 3.05 ± 0.10abc
C17:0 SFA Pearlescent fatty acid 3.32 ± 0.15a 3.23 ± 0.08ab 3.02 ± 0.08b 1.74 ± 0.12d 2.30 ± 0.09c 2.31 ± 0.14c 1.90 ± 0.15d 1.90 ± 0.14d 2.51 ± 0.02c
C18:0 SFA Stearic acid 75.26 ± 0.61a 72.31 ± 1.25a 67.65 ± 0.91b 39.82 ± 1.02f 51.58 ± 1.17d 53.54 ± 2.24d 43.36 ± 0.14e 44.82 ± 1.48e 57.23 ± 1.64c
C18:1n9c MUFA Oleic acid 102.09 ± 1.24a 96.35 ± 1.83b 88.85 ± 1.04c 60.37 ± 1.80f 76.52 ± 1.30e 77.16 ± 2.97de 58.43 ± 0.44f 62.00 ± 2.72f 81.34 ± 1.38d
C18:2n6c PUFA n-6 Linoleic acid 305.84 ± 4.45a 308.46 ± 9.05a 276.19 ± 8.92b 176.89 ± 0.45e 227.69 ± 4.79c 238.82 ± 3.83c 206.75 ± 0.02d 199.34 ± 10.28d 241.15 ± 3.22c
C20:0 SFA Arachidonic acid 2.14 ± 0.03a 1.81 ± 0.43abc 2.08 ± 0.44ab 0.00 ± 0.00e 1.14 ± 0.11d 1.53 ± 0.20cd 1.35 ± 0.15cd 0.00 ± 0.00e 1.59 ± 0.03bcd
C18:3n3 PUFA n-3 Alpha-linolenic acid 27.09 ± 0.28a 26.95 ± 0.87a 24.61 ± 0.98b 15.96 ± 0.17f 19.95 ± 0.51cd 20.90 ± 0.76c 18.39 ± 0.28de 17.39 ± 1.11ef 21.01 ± 0.24c
C22:0 SFA Behenic acid 5.79 ± 0.18a 5.86 ± 0.55a 4.75 ± 0.53ab 2.11 ± 0.10e 3.47 ± 0.33cd 3.89 ± 0.51bc 2.40 ± 0.03de 2.61 ± 1.07de 3.91 ± 0.06bc
C20:4n6 PUFA n-6 Arachidonic acid 2.58 ± 0.42abc 3.45 ± 0.10a 2.88 ± 0.67ab 1.90 ± 0.16bc 2.02 ± 0.33bc 2.21 ± 0.38bc 1.82 ± 0.72bc 1.59 ± 0.34c 2.47 ± 0.16abc
C24:0 SFA Lignocarboxylic acid 5.30 ± 0.45a 5.25 ± 0.12ab 4.72 ± 0.05b 2.46 ± 0.00e 3.32 ± 0.16cd 3.71 ± 0.26c 2.95 ± 0.12de 2.63 ± 0.22e 3.40 ± 0.23cd
C20:5n3 PUFA n-3 EPA 2.06 ± 0.05ab 2.21 ± 0.42a 1.82 ± 0.03ab 1.17 ± 0.29c 1.52 ± 0.05bc 1.62 ± 0.24abc 1.82 ± 0.07ab 1.51 ± 0.27bc 1.71 ± 0.28abc
C22:6n3 PUFA n-3 DHA 16.47 ± 0.06ab 16.74 ± 1.22a 15.11 ± 0.48bc 9.61 ± 0.18g 12.32 ± 0.74ef 13.48 ± 0.12de 13.17 ± 0.87def 11.67 ± 0.46f 14.37 ± 0.58cd

As shown in Fig. 8, when the extrusion temperature was constant, the six different fatty acid contents (SFA, UFA, MUFA, PUFA, n-3FA, and n-6FA) and two indicators (n-6/n-3 and IT values) both decreased dramatically as the moisture content increased from 65% to 75%. Azam et al.51 reported the effect of low moisture on the nutritional properties of the extrudates, which was positive for increasing the various fatty acids. When the moisture content was 65%, the increasing extrusion temperature could lead to less fatty acid contents. It might be caused by lipid oxidation and thermal decomposition according to a study.50 Wang et al.52 also reported that fatty acids were broken down due to the action of high temperature, high pressure and high shear. It is generally believed that fatty acids can form complexes with carbohydrates and proteins in the extrusion process.53 Interestingly, at higher moisture contents (70–75%), increasing extrusion temperature (from 125 °C to 145 °C) enhanced the fatty acid contents due to inactivation of fatty acid hydrolases.31


image file: d3fb00171g-f8.tif
Fig. 8 The fatty acid content with the SFA, UFA, PUFA, MUFA, n-3FA and n-6FA (a) and the fatty acid evaluation with n-6/n-3, IA and IT (b) of the extrudates with different hydrothermal parameters, and different letters indicate significant differences (p < 0.05).

Fig. 9 shows that PC1 and PC2 could explain 77% and 17% of the total variance, respectively. The SFA, UFA, PUFA n-3FA and n-6FA were significantly irrelated to the hydro-thermal combined parameters of 125 °C-70%. Moreover, the n-6/n-3, IA and IT were dramatically irrelated to the hydro-thermal combined parameters of 125 °C-75%.


image file: d3fb00171g-f9.tif
Fig. 9 The PCA Bi-plots of the fatty acid evaluation of the extrudates with different hydro-thermal parameters.

In vitro digestibility

Effect of SPI–surimi ratios. As can be seen in Fig. 10, as the surimi content increased from 10% to 50%, the gastric digestibility (GD) decreased from 60.20% to 24.63% firstly but then increased significantly to 53.02%. At a SPI–surimi ratio of 70[thin space (1/6-em)]:[thin space (1/6-em)]30, the GD value was the lowest, which should be considered as the turning point of the mixed protein ratios for gastric digestion. This might be related to higher gel strength at this ratio according to our previous study.8 Furthermore, the vegetable ingredients of plant-based meat significantly reduced the number of gastric parietal cells and pepsin activity.54 An enzyme activity test also confirmed that the plant-based meat significantly decreased pepsin activity but increased trypsin activity.55 Moreover, the increased surimi content could lead to an increase in chain proteins, which promoted the contact between the pepsin and binding points. In terms of small intestinal digestibility (SD), the highest SD was 93.07% at a SPI–surimi ratio of 70[thin space (1/6-em)]:[thin space (1/6-em)]30. It was perhaps related to the increase of the surimi content and the increase of the intestinal pepsin activity according to a previous study.54 The lowest SD was only 12.16% with 40% surimi addition and further research should be necessary.
image file: d3fb00171g-f10.tif
Fig. 10 Simulated gastric digestibility and small intestinal digestibility of the extrudates with different SPI–surimi ratios (a and b) and different hydrothermal parameters (c and d), and different letters indicate significant differences (p < 0.05).
Effect of hydro-thermal combined parameters. Fig. 10 shows the GD and SD of the extrudates with different hydro-thermal parameters. When the extrusion temperature was set at 125 °C, the GD increased from 12.65% to 30.18% and the SD increased from 13.67% to 35.64% as the moisture content increased from 65% to 75%. While the extrusion temperature was set at 145 °C, the GD decreased from 31.52% to 19.24% firstly and then increased to 30.41%, and the SD increased from 17.81% to 20.03% firstly and then decreased to 3.25%. It might be because most protein substances are decomposed in the stomach, and the low concentration of substrate leads to a sharp decline in the digestibility of the small intestine.55,56 At high extrusion temperature, the denaturation of protein molecules aggravated, the protein spatial structure was destroyed, the peptide chain was expanded, and the amino acid was dissociated.

Comprehensive nutritional evaluation of SPI–surimi extrudates

Effect of SPI–surimi ratios. Fig. 11 shows that PC1 and PC2 could explain 65% and 16% of the total variance, respectively. The protein content, UFA, PUFA, n-6FA, n-6/n-3 and SD were positively correlated with PC1, while others were negatively correlated with PC1. The moisture content of the extrudates (MC), FC, SD, GD and n-6/n-3 was negatively correlated with PC2, while others were positively correlated with PC2. These nutritional indicators have a large impact on the evaluation of the comprehensive nutritional quality of the SPI–surimi extrudates at different ratios. The analysis revealed that the ratio of 80[thin space (1/6-em)]:[thin space (1/6-em)]20 was in the first quadrant, mainly influenced by AC, PUFA, UFA, and n-6FA on comprehensive nutritional quality evaluation. The ratio of 60[thin space (1/6-em)]:[thin space (1/6-em)]40 was distributed in the second quadrant, mainly influenced by EAA, EAAI, AAS (Met + Cys), CS (Met + Cys), and n-3FA. The ratio of 50[thin space (1/6-em)]:[thin space (1/6-em)]50 was in the third quadrant, mainly influenced by MC, GD and FC, and the ratios of 90[thin space (1/6-em)]:[thin space (1/6-em)]10 and 70[thin space (1/6-em)]:[thin space (1/6-em)]30 were in the fourth quadrant, mainly influenced by SD and n-6/n-3.
image file: d3fb00171g-f11.tif
Fig. 11 The PCA Bi-plots of the extrudates with different SPI–surimi ratios for comprehensive nutritional evaluation.
Effect of hydro-thermal combined parameters. Fig. 12 shows that PC1 and PC2 could explain 44% and 28% of the total variance, respectively. The SD, GD, MC, EAA, EAAI, AAS (Met + Cys) and CS (Met + Cys) were negatively correlated with PC1 while others were positively correlated with PC1. The protein content, n-6/n-3, FC, GD and SD were positively correlated with PC2, while others were negatively correlated with PC2. These nutritional indicators have a large impact on the evaluation of the comprehensive nutritional quality of the SPI–surimi extrudates at different ratios. The PCA showed that 135 °C-70% and 145 °C-75% were in the first quadrant and mainly influenced by the protein content and n-6/n-3. 125 °C-70% and 135 °C-75% were distributed in the second quadrant and were mainly influenced by SD and GD. 145 °C-70% and 125 °C-75% were in the third quadrant and mainly influenced by EAA, EAAI, AAS (Met + Cys) and CS (Met + Cys). 125 °C-65% and 135 °C-65% were in the fourth quadrant and mainly influenced by PUFA, UFA, n-6FA and n-3FA.
image file: d3fb00171g-f12.tif
Fig. 12 The PCA Bi-plots of the extrudates with different hydro-thermal parameters for comprehensive nutritional evaluation.

Conclusions

When the surimi content increased from 10% to 50%, the AAS significantly increased from 88.82 to 109.50. Furthermore, the EPA and DHA levels in the extrudates increased notably, going from 1.44 mg g−1 to 10.30 mg g−1 and from 6.44 mg g−1 to 41.22 mg g−1, respectively. These findings suggest that surimi plays a crucial role in improving both amino acid and fatty acid contents in high-moisture extrudates derived from SPI and surimi. Additionally, when the moisture content reached 75%, elevating the extrusion temperature from 125 °C to 145 °C resulted in a significant decrease in the essential amino acid content. In a certain extrusion temperature range (125–145 °C), the EPA and DHA contents of the extrudates decreased substantially as the moisture content increased from 65% to 75%. It was found that higher extrusion temperature and increased moisture content disrupted the amino acid patterns in the extrudates, while simultaneously enhancing certain fatty acid levels. Conversely, a lower extrusion temperature (125 °C) and lower moisture content (65%) contributed to higher EPA and DHA levels. During the high-moisture processing, with an SPI–surimi ratio of 70[thin space (1/6-em)]:[thin space (1/6-em)]30, the lowest GD was 24.63%, while the highest SD reached 93.07%. Higher moisture levels (70% and 75%) were associated with greater SD, and increasing the temperature at a lower moisture content (60%) or increasing moisture content at a lower temperature (125 °C) leads to an obvious increase in GD during high-moisture extrusion processing.

Author contributions

Anna Hu: investigation, validation, formal analysis, and writing – original draft. Yujie Zhang: methodology, investigation, data curation, and writing – original draft. Jinchuang Zhang: conceptualization, methodology, formal analysis, writing – review & editing, and supervision. Tongqing Li: visualization and validation. Zhaojun Wang: writing – review & editing and supervision. Qiang Wang: funding acquisition and supervision.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This research was supported by the National Key Research and Development Plan of China (2021YFC2101402), the Young Elite Scientist Sponsorship Program by CAST (YESS20220162), the Science and Technology Innovation Project of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-Q2022-IFST-05), and the earmarked fund for China Agriculture Research System (CARS-13).

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Footnote

† Equal contribution.
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