Development of High-Moisture Meat Analogues with Hemp and Soy Protein Using Extrusion Cooking

Article Development of High-Moisture Meat Analogues with Hemp and Soy Protein Using Extrusion Cooking

Izalin Zahari 1,* , Ferawati Ferawati 2, Amanda Helstad 1, Cecilia Ahlström 1, Karolina Östbring 1 , Marilyn Rayner 1 and Jeanette K. Purhagen 1

1 Department of Food Technology Engineering and Nutrition, Lund University, Naturvetarvägen 12, 223 62 Lund, Sweden; [email protected] (A.H.); [email protected] (C.A.); [email protected] (K.Ö.); [email protected] (M.R.); [email protected] (J.K.P.) 2 Department of Chemistry and Biomedical Sciences, Linnaeus University, 392 31 Kalmar, Sweden; [email protected] * Correspondence: [email protected]

Received: 30 April 2020; Accepted: 5 June 2020; Published: 11 June 2020

Abstract: The interest in plant-based products is growing in Western countries, mostly due to health and environmental issues that arise from the consumption and production of animal-based food products. Many vegan products today are made from soy, but drawbacks include the challenges of cultivating soy in colder climates such as northern Europe. Therefore, the present study investigates whether industrial hemp (Cannabis sativa) could substitute soy in the production of high moisture meat analogues (HMMA). A twin screw co-rotating extruder was used to investigate to what extent hemp protein concentrate (HPC) could replace soy protein isolate (SPI) in HMMAs. The substitution levels of HPC were 20 wt%, 40 wt% and 60 wt%. Pasting properties and melting temperature of the protein powders were characterized by Rapid Visco Analyzer (RVA) and Diﬀerential Scanning Calorimeter (DSC), respectively and the produced HMMA was analysed by determining the texture and colour attributes. The results showed that it is possible to extrude a mixture with up to 60% HPC. HPC absorbed less water and needed a higher denaturing temperature compared to SPI. Increasing the moisture content by 5% would have resulted in a reduction of hardness and chewiness. The lightness (L* value) was found to be signiﬁcantly higher in SPI product and decreased in the mixture with higher HPC (p < 0.05).

Keywords: high-moisture meat analogue; extrusion; hemp seed concentrate; soy protein isolate

1. Introduction The Western diet includes a high proportion of products from animal origins, and the high consumption of meat is a subject of various debates on its impact on human health and the environment [1]. High consumption of meat is linked to an increased risk of cardiovascular disease, type 2 diabetes, and diﬀerent types of cancer [1]. Moreover, livestock production is responsible for 14.5% of global greenhouse gas emissions [2]. A shift towards a more plant-based diet is suggested to be one possible solution to reduce climate impact and create a healthier lifestyle [3,4]. As a consequence, there is a tremendous market growth in the plant-based food sector, driven by a growing number of consumers adopting a vegan, vegetarian, or ﬂexitarian lifestyle [5]. Plant-based alternative products range from dairy analogues to meat substitutes. The meat substitutes market is still relatively small, but is projected to increase in the future. Europe leads the meat substitutes market globally, with 40% of the total market [5], which is predicted to reach €2.4 billion in 2025 as compared to €1.5 billion in 2018 [5]. The market is also expected to increase in

Foods 2020, 9, 772; doi:10.3390/foods9060772 www.mdpi.com/journal/foods Foods 2020, 9, 772 2 of 13 Foods 2020, 9, x FOR PEER REVIEW 2 of 14 vegetableother regions, protein such are typical as North meat America substitutes and Asia on the [5]. market. Tofu, tempeh, Another seitan, type mycoprotein,of meat substitute, and texturized high- moisturevegetable meat protein analogue are (HMMA), typical meat is not substitutes widely av onailable the market.on the market Another yet; typethus, ofHMMA meat substitute,will be thehigh-moisture focus of the present meat analoguestudy. HMMA (HMMA), has a is desira not widelyble chewy available meat-like on the texture, market which yet; thus, together HMMA with will appropriatebe the focus flavouring of the present ingredients study. ca HMMAn offset has the a consumption desirable chewy of meat. meat-like texture, which together with appropriateThe vast majority flavouring of meat ingredients analogue can products offset the on consumption the European of market meat. are made from imported soybean,The while vast only majority a small of meat proportion analogue is productsmade from on other the European plant protein market sources are made such from as pea imported or mushroom.soybean, This while import only aof small raw material proportion could is madepotentially from reduce other plant the sustai proteinnable sources benefits such of asplant- pea or basedmushroom. products. This Therefore, import ofother raw crop materials that could could potentially be cultivated reduce over the a range sustainable of latitudes, benefits such of plant-basedas seeds fromproducts. industrial Therefore, hemp (Cannabis other crops sativa) that should could be beinvestigated. cultivated Optimization over a range of the latitudes, extrusion such process as seeds in orderfrom industrialto improve hemp the texture (Cannabis of sativaHMMA) should is one be of investigated. the most challenging Optimization aspects of thewhen extrusion it comes process to extrusionin order cooking. to improve There the have texture been of many HMMA estab islished one of extrusion the most challengingprocesses and aspects patents when for itHMMA comes to productionextrusion using cooking. soybean There [6–11], have but been only many a few established studies have extrusion been carried processes out on and formulations patents for based HMMA on productionplant proteins using other soybean than soy [6– 11[12–14].], but only a few studies have been carried out on formulations based onThe plant hemp proteins plant other has multifaceted than soy [12 –benefits14]. in many production sectors [15]. In Europe, hemp is mainly usedThe hempas a source plant of has fibre multifaceted for biomass, benefits textile, in and many the production rope industry sectors [15]. [However,15]. In Europe, there hempis an is increasingmainly usedinterest as ain source using of locally fibre for cultivated biomass, hemp textile, seeds and theas a rope raw industry material [15 for]. However,food production. there is an Hempincreasing seeds, the interest by-product in using from locally the cultivated fibre use for hemp the seedsconstruction as a raw industry, material forare foodrich in production. fats (25–35%, Hemp mostlyseeds, unsaturated the by-product fatty from acids), the proteins fibre use (20–25%), for the construction dietary fibre industry, (10–15%), are richvitamins, in fats and (25–35%, minerals mostly [16].unsaturated fatty acids), proteins (20–25%), dietary fibre (10–15%), vitamins, and minerals [16]. ProteinProtein is isthe the most most importantimportant ingredientingredient in thein the production production of HMMA of HMMA using extrusionusing extrusion technology. technology.During extrusion, During extrusion, protein is protein denatured, is denatured, unfolded, unfolded, realigned realigned and cross-linked, and cross-linked, due to the due shear, to the heat, shear,pressure heat, andpressure cooling and (Figure cooling1) achieved(Figure 1) in achiev the extrudered in the and extruder cooling and die, cooling respectively die, respectively [ 17]. The final [17].texture The final of HMMA texture is influencedof HMMA byis diinfluencedfferent types by ofdifferent protein bonds.types of Several protei factorsn bonds. determine Several thefactors protein determineinteractions the protein such as interactions protein type, such pre-treatment as protein type, and extrusionpre-treatment parameters and extrusion [17]. parameters [17].

FigureFigure 1. Changes 1. Changes in protein in protein conformation conformation during during extr extrusionusion of HMMA. of HMMA. Modified Modiﬁed illustration illustration adopted adopted fromfrom Zhang Zhang et al. et [17]. al. [17 ]. Previous works have shown that hemp seed protein in particular, has a high protein quality Previous works have shown that hemp seed protein in particular, has a high protein quality and and functionality [16,18,19]. However, no study uses hemp seed protein as a raw material for meat functionality [16,18,19]. However, no study uses hemp seed protein as a raw material for meat analogue production. Thus, this study was aimed to investigate the maximum ratio substitution of analogue production. Thus, this study was aimed to investigate the maximum ratio substitution of soy protein by hemp protein in the formulation of HMMA, as well as to study the characteristics of soy protein by hemp protein in the formulation of HMMA, as well as to study the characteristics of the HMMAs produced. the HMMAs produced. 2. Materials and Methods 2. Materials and Methods Soy protein isolate, SPI (91% protein, 1% carbohydrate, 3% fat, wet basis) and hemp protein Soy protein isolate, SPI (91% protein, 1% carbohydrate, 3% fat, wet basis) and hemp protein concentrate, HPC (71% protein, 15% carbohydrate, 7% fat, wet basis), were obtained from Bulk Powders concentrate, HPC (71% protein, 15% carbohydrate, 7% fat, wet basis), were obtained from Bulk Company and Natures Organic Food Company, Sweden, respectively (Figure2). Mixtures of hemp Powders Company and Natures Organic Food Company, Sweden, respectively (Figure 2). Mixtures of hemp protein and soy protein (HPC:SPI) were prepared the same day as the extrusion trials

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Foods 2020, 9, x FOR PEER REVIEW 3 of 14 protein and soy protein (HPC:SPI) were prepared the same day as the extrusion trials according to theaccording formulations to the formulations (20:80, 40:60, (20:80, 60:40) where40:60, 60:40) the numbers where the represents numbers wt% represents of each proteinwt% of each source. protein source.

Figure 2. SoySoy protein isolate (left); and hemp protein concentrate (right) powder. 2.1. Moisture Content 2.1. Moisture Content Moisture content of SPI and HPC powder sampled from packages as received from the suppliers, Moisture content of SPI and HPC powder sampled from packages as received from the and the HMMA produced by extrusion were analysed. Three to five grams of sample was transferred suppliers, and the HMMA produced by extrusion were analysed. Three to five grams of sample was into a metal container and put in an oven at 103 ◦C for at least 16 h until it reached constant weight using transferred into a metal container and put in an oven at 103 °C for at least 16 h until it reached constant modification of AACC method 44-15A [20]. From the moisture content results of the raw materials, weight using modification of AACC method 44-15A [20]. From the moisture content results of the the mixtures’ moisture contents were calculated (Table1). The moisture contents were important for raw materials, the mixtures’ moisture contents were calculated (Table 1). The moisture contents were the setting of solid and liquid dosing in the extruder software later to get the target moisture content for important for the setting of solid and liquid dosing in the extruder software later to get the target the final product. Extruded samples were collected immediately after the extrusion and were allowed moisture content for the final product. Extruded samples were collected immediately after the to cool to room temperature where after the samples were sealed in plastic bags and placed in a freezer extrusion and were allowed to cool to room temperature where after the samples were sealed in ( 18 ◦C). The samples were thawed and cut into small pieces before the measurement using the same plastic− bags and placed in a freezer (−18 °C). The samples were thawed and cut into small pieces procedure for all samples. before the measurement using the same procedure for all samples. Table 1. Moisture content data for the raw materials of the mixtures. Table 1. Moisture content data for the raw materials of the mixtures. Raw Material Moisture Content (%) Raw Material Moisture Content (%) 100% SPI 6.39 0.03 100% SPI 6.39± ± 0.03 100% HPC 8.02 0.07 100% HPC 8.02± ± 0.07 Mixtures (HPC:SPI) (%) Calculated Moisture Content (%) Mixtures (HPC:SPI) (%) Calculated Moisture Content (%) 20:80 6.726.72 40:60 7.04 40:6060:40 7.377.04 60:40 7.37

2.2. Viscosity 2.2. Viscosity The viscosity of both raw materials and mixtures of powders was measured using the standard The viscosity of both raw materials and mixtures of powders was measured using the standard AACC method 76-21 STD 1 [21] in the Rapid Visco Analyzer 4800 (Perten Instruments, Perkin Elmer, AACC method 76-21 STD 1 [21] in the Rapid Visco Analyzer 4800 (Perten Instruments, Perkin Elmer, NSW, Australia). In brief, 3.50 g of sample was mixed with water (26.66 g) on 14% moisture basis as NSW, Australia). In brief, 3.50 g of sample was mixed with water (26.66 g) on 14% moisture basis as recommended by the manufacturer’s instruction. The samples were heated to 50 C and stirred under recommended by the manufacturer’s instruction. The samples were heated to 50 ◦°C and stirred under a constant shear rate at 960 rpm for 10 s. The slurry was held at 50 C for 50 s and then heated up to a constant shear rate at 960 rpm for 10 s. The slurry was held at 50 ◦°C for 50 s and then heated up to 130 C, with temperature increasing at 12 C/min. It was held at 130 C for 2.5 min, and ﬁnally cooled 130 ◦°C, with temperature increasing at 12 ◦°C/min. It was held at 130 ◦°C for 2.5 min, and finally cooled to 50 C. The pasting properties of each raw material and mixtures was measured at least in duplicate. to 50 °C.◦ The pasting properties of each raw material and mixtures was measured at least in duplicate. 2.3. Thermal Properties 2.3. Thermal Properties The thermal properties of the raw materials were determined using a Diﬀerential Scanning The thermal properties of the raw materials were determined using a Differential Scanning Calorimetry (DSC) instrument (Seiko Instruments Inc.-EXSTAR6000 DSC, Shizuoka, Japan). Calorimetry (DSC) instrument (Seiko Instruments Inc.-EXSTAR6000 DSC, Shizuoka, Japan). The The instrument was calibrated with indium using an empty pan as a reference. 2 mg of protein powder instrument was calibrated with indium using an empty pan as a reference. 2 mg of protein powder was weighed into coated aluminium pan and hermetically sealed. Thereafter, three times the sample was weighed into coated aluminium pan and hermetically sealed. Thereafter, three times the sample weight of MilliQ water was added. The pan was sealed and heated from 25 °C to 160 °C at a rate of 10 °C/min. Each sample was measured in triplicate and the data was stored and analysed using the

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weight of MilliQ water was added. The pan was sealed and heated from 25 ◦C to 160 ◦C at a rate of 10 ◦C/min. Each sample was measured in triplicate and the data was stored and analysed using the DSC software (SII EXSTAR6000 Muse, Shizuoka, Japan). The melting temperature and the enthalpies were calculated from the thermograms based on the dry weight of the samples.

2.4. High-Moisture Extrusion Processing HMMA products were processed using a laboratory, co-rotating twin-screw extruder (KETSE 20/40D, Brabender GmbH & Co.KG, Duisburg, Germany) using a fixed screw configuration (Table A1 in AppendixA) with a screw diameter of 20 mm. The extruder has four di fferent temperature zones in the barrel which were maintained during the experiments at 40–60–80–100 ◦C. In the beginning, the chosen parameters considered in this study were concentration of HPC (0%, 20%, 40%, 60%), target moisture content (65%, 70%, 75%), temperature (40–120 ◦C) and screw speed (300–800 rpm). However, from our preliminary experiments, it was found that the screw speed of 800 rpm produced the most acceptable texture of HMMA products for all HPC mixtures formulations whereas for SPI alone, we were only able to extrude soy protein using 500 rpm at 70% and 75% of target moisture content. Thus, this experiment was carried out using eleven formulations as summarized in Table2. The solid dosing (0.8–1.1 kg/h) and liquid dosing (1.88–2.36 kg/h) was calculated from the moisture content of the raw materials, while the target moisture content of the product and the total mass flow (3 kg/h) was set using the complementary software from Brabender GmbH & Co.KG. The cooked mixture was pushed through a long flat shaped cooling die with dimensions of 25 7 300 mm (W H L) which was × × × × cooled by a stream of water from the outside. The cooling process increases the viscosity of the mixture, thus inducing shear that generates a layered and fibrous structure. Extruded products were manually cut at the end of the cooling die and were allowed to cool at room temperature before the material was transferred to a sealed plastic bag where it was kept frozen at 18 C prior to further analysis. − ◦ Table 2. Extrusion parameters involved in the screening and selected formulations of HMMA.

Screw Speed Target Moisture Temperature ( C) Formulation ◦ Selected Trial (rpm) Content (%) (Zone 1–2–3–4) 0% HPC 70 √ 500 40–60–80–100 (100% SPI) 75 √ 65 √ 800 70 √ 75 √ 20% HPC 40–60–80–100 75 600 80 400 75 300 75 65 √ 40% HPC 800 70 40–60–80–100 √ 75 √ 60 62.5 65 40–60–80–100 √ 60% HPC 800 70 √ 75 √ 65 60–80–100–120

2.5. Texture Properties Texture proﬁle analysis (TPA), cutting test, and tensile strength were carried out to evaluate the overall texture of the extruded products. The measurements were performed using a texture Foods 2020, 9, 772 5 of 13

Foods 2020, 9, x FOR PEER REVIEW 5 of 14 analyser (TVT-300XP, Perten Instruments AB, Hägersten, Sweden) [13,22]. For TPA, samples size of 2 2 cm with 7 mm thickness were compressed by 2 mm using a cylindrical probe (Ø 18 mm). Hardness, springiness,× resilience and chewiness of the samples were determined. Hardness is a measurement of springiness,how hard the resilience product and is and chewiness can be ofdetermined the samples by were the maximum determined. force Hardness of the isfirst a measurement compression of [23].how Springiness hard the product shows ishow and canwell be the determined product byretu therns maximum to its original forceof structure the ﬁrst compressionafter the first [23 ]. compression,Springiness while shows resilience how well describes the product how returns well the toits product original regains structure its afteroriginal the height ﬁrst compression, after the compressionwhile resilience [23]. describesChewiness how shows well how the productmuch work regains is needed its original to chew height the after product the compression to achieve the [23 ]. textureChewiness that is shows suitable how for much ingestion work [24]. is needed Other topa chewrameters the productsuch as cutting to achieve and the tensile texture strength that is could suitable befor linked ingestion to the [24 quality]. Other of parameters meat, for instance, such as cutting describing and tensilethe tenderness strength could[25]. A be minimum linked to theof three quality measurementsof meat, for instance,for each describingsample was the performed. tenderness For [25 the]. Acutting minimum test, oftwo three types measurements of cutting strength for each weresample measured: was performed. transversal For and the cuttinglongitudinal. test, two Samples types of of cutting 2 × 2 strength cm (transversal) were measured: and 5 transversal × 2 cm and longitudinal. Samples of 2 2 cm (transversal) and 5 2 cm (longitudinal) with 7 mm thickness (longitudinal) with 7 mm thicknes×s were cut 5 mm deep using× a knife blade (height 117 mm) at a speedwere of cut 2 5mm/s. mmdeep Transversal using a knifecutting blade was (height done in 117 the mm) direction at a speed of the of 2width mm/ s.of Transversal the samplecutting whereas was longitudinaldone in the cutting direction was of thedone width in the of the direction sample of whereas the length longitudinal of the sample cutting was(Figure done 3). in For the directiontensile of the length of the sample (Figure3). For tensile strength, a sample of 2 12 cm was prepared. strength, a sample of 2 × 12 cm was prepared. The sample was attached to the× rig and stretched 30 mmThe at samplea speed was of 1 attachedmm/s and to the the break rig and force stretched (g) was 30 measured. mm at a speed of 1 mm/s and the break force (g) was measured.

Figure 3. Transversal and longitudinal cutting directions of samples. Figure 3. Transversal and longitudinal cutting directions of samples. 2.6. Colour Determination and Appearance of HMMA 2.6. Colour Determination and Appearance of HMMA The colour of the produced HMMA products was measured using a colorimeter (Konica MinoltaThe colour CR-400, of the Osaka, produced Japan). HMMA The calibration products was was measured performed using with a colorimeter a white calibration (Konica tileMinolta before CR-400,the measurement. Osaka, Japan). The The CIE-Lab calibration parameters was wereperformed expressed with as a valuewhite of calibrationL* (lightness), tile a*before(redness the or measurement.greenness) and Theb* CIE-Lab(yellowness parameters or blueness). were Theexpressed measurements as a value were of performedL* (lightness), in triplicate a* (redness for eachor greenness)sample at and randomly b* (yellowness selected or locations. blueness). For The the measurements appearance, images were wereperformed acquired in triplicate by a rigged for cameraeach sample(Nikon at D3300,randomly AF-P selected DX 18-55 locations./3.5-5.6G, For Nikon the appearance, Corporation, images Tokyo, were Japan) acquired in a photoby a rigged box with camera black (Nikonwalls andD3300, four AF-P light DX sources 18-55/3.5-5.6G, pointed to theNikon sample Corporation, to make high Tokyo, quality Japan) pictures in a photo of the HMMAbox with products. black walls and four light sources pointed to the sample to make high quality pictures of the HMMA products.2.7. Statistical Analysis All experimental work was carried out on freshly prepared samples, in at least duplicates, and 2.7.reported Statistical as Analysis means standard deviations of the measurements. All analyses were performed using ± MicrosoftAll experimental Office Excel work 2010 was and carried Minitab out 16.2.1on freshly statistical prepared software samples, (Minitab in at Inc.,least Pennsylvania,duplicates, and PA, reportedUSA). Tukey´sas means test ± wasstandard used deviations to identify theof the diff measurements.erences in each All treatment analyses at pwere< 0.05. performed using Microsoft Office Excel 2010 and Minitab 16.2.1 statistical software (Minitab Inc., Pennsylvania, PA, 3. Results and Discussion USA). Tukey´s test was used to identify the differences in each treatment at p < 0.05. 3.1. Moisture Content 3. Results and Discussion The moisture content of all HMMA products was analysed to investigate if it corresponded to 3.1.the Moisture target moisture Content content with the solid and liquid dosing. It was found that the differences between the target and measured moisture content varied between 4.38% to 4.76%. The measured moisture The moisture content of all HMMA products was analysed− to investigate if it corresponded to content of 0% HPC (100% SPI) sample differed more (74.38 and 76.06) than the targeted moisture value the target moisture content with the solid and liquid dosing. It was found that the differences between (70% and 75%), respectively, whereas other mixture samples with HPC differed less than the targeted the target and measured moisture content varied between −4.38% to 4.76%. The measured moisture moisture values. The 20% HPC sample had the most exact value compared with the target, followed content of 0% HPC (100% SPI) sample differed more (74.38 and 76.06) than the targeted moisture value (70% and 75%), respectively, whereas other mixture samples with HPC differed less than the targeted moisture values. The 20% HPC sample had the most exact value compared with the target,

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Foods 2020, 9, x FOR PEER REVIEW 6 of 14 by 40% and 60%. This result proved that there was no significant variation between them based on followed by 40% and 60%. This result proved that there was no significant variation between them the accuracy of the method. The variations in moisture content could possibly be due to an uneven based on the accuracy of the method. The variations in moisture content could possibly be due to an flow rate of powder from the hopper during the extrusion process. uneven flow rate of powder from the hopper during the extrusion process. 3.2. Pasting Properties 3.2. Pasting Properties The Rapid Visco Analyzer (RVA) was used to measure several parameters related to the pasting propertiesThe Rapid of the Visco raw Analyzer materials (RVA) used. Obviouswas used di toff measerencesure were several found parameters between related the samples to the whenpasting it propertiescomes to their of the behaviour raw materials during used. heating Obvious and cooling differences in an excess were offound water. between From Figure the samples4, the SPI when (grey it comescurve) showedto their behaviour the highest during peak viscosity heating amongand cooling all samples in an whichexcess indicatesof water. a From good Figure paste texture 4, the andSPI (greyhigh water-bindingcurve) showed capacity. the highest This characteristicpeak viscosity could among provide all samples excellent which texture indicates which isa desirablegood paste in textureHMMA and products high water-binding [26]. Despite that, capacity. HPC aloneThis charac showedteristic a rather could inconsistent provide excellent behaviour texture which which was also is desirablefound in thein HMMA 40% (purple products curve) [26]. and Despite 60% (black that, curve) HPC HPC-SPIalone showed mixtures. a rather At the inconsistent end of the behaviour analysis, it whichwas observed was also that found the in HPC the sample40% (purple formed curve) spheres and inside60% (black the container curve) HPC-SPI which mightmixtures. alter At the the result. end ofThe the spheres analysis, hinder it was the observed paddle that in thethe HPC rotation samp causingle formed this spheres inconsistence inside the behaviour. containerAccording which might to altersome the researchers, result. The the spheres interaction hinder of the starch paddle and in fatty the rotation acids may causing also contributethis inconsistence to a new behaviour. complex, Accordingthus affecting to some some researchers, modification the and interaction texture of stability starch and of the fatty final acids product may also as previously contribute reportedto a new complex,by others thus [27,28 affecting]. Pasting some temperature modification is the and minimum texture temperaturestability of the required final product to cook as a sample previously [28], reported by others [27,28]. Pasting temperature is the minimum temperature required to cook a and it was found to vary from 53.8 ◦C to 90.8 ◦C for HPC-SPI mixtures compared to the SPI sample sample [28], and it was found to vary from 53.8 °C to 90.8 °C for HPC-SPI mixtures compared to the (50.2 ◦C). This indicates that cold swelling takes place directly for pure SPI and 20% HPC mixture SPI(pink sample curve). (50.2 SPI °C). and This the indicates 20% HPC that mixture cold swelling required takes the shortest place directly time to for reach pure maximum SPI and 20% viscosity HPC mixture(0.4 min (pink and 0.3 curve). min, SPI respectively). and the 20% It wasHPC noted mixture that required for the samplesthe shortest with time higher to reach degree maximum of hemp viscositysubstitution, (0.4 amin longer and time0.3 min, was respectively). required (8.2 It min was for noted 100% that HPC). for the This samples means with that HPChigher needs degree more of hemptime and substitution, higher temperatures a longer time to was bind required water and (8.2 denature min for 100% before HPC). reaching This themeans maximum that HPC viscosity needs morecompared time toand the higher SPI. The temperatur maximumes viscosity to bind was water reached and duedenature to the reductionbefore reaching of protein the solubility maximum as viscositya result of compared denaturation to the of SPI. native The proteins maximum [13]. viscos Overall,ity was these reached results indicatedue to the that reduction the substitution of protein of solubilityHPC had aas readily a result observable of denaturation influence of onnative pasting proteins behaviour. [13]. Overall, these results indicate that the substitution of HPC had a readily observable influence on pasting behaviour.

Figure 4. RVA curves for the raw materials and mixtures. (100% SPI: grey, 100% HPC: blue, 20% HPC: Figurepink, 40% 4. RVA HPC: curves purple, for 60% the raw HPC: materials black.). and mixtures. (100% SPI: grey, 100% HPC: blue, 20% HPC: pink, 40% HPC: purple, 60% HPC: black.). 3.3. Thermal Properties 3.3. ThermalDifferent Properties melting temperatures were observed for SPI and HPC in the DSC analysis, due to differentDifferent protein melting contents. temperatures There were were one observed to four endothermic for SPI and peaksHPC in identified the DSCin analysis, HPC, whereas due to different protein contents. There were one to four endothermic peaks identified in HPC, whereas three to five endothermic peaks were observed in the SPI. The proteins in the HPC had overall higher

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Foodsthree 2020 to ﬁve, 9, x endothermicFOR PEER REVIEW peaks were observed in the SPI. The proteins in the HPC had overall higher7 of 14 melting temperatures (90.3–123.1 ◦C) as compared with SPI (54.6–107.0 ◦C) (Figure5). This indicates melting temperatures (90.3–123.1 °C) as compared with SPI (54.6–107.0 °C) (Figure 5). This indicates that the cooking zone in the extruder with the highest temperature should be set to at least 123.1 ◦C if thatall proteins the cooking in the zone HPC in should the extruder denature with during the high extrusion.est temperature One main peakshould was be detectedset to at inleast HPC 123.1 which °C probablyif all proteins corresponds in the HPC to Edestin, should a denature storage protein during in extrusion. the hemp One seed. main It was peak found was in detected all samples in HPC with whichhigh enthalpies probably (5.53–7.25 corresponds mJ/mg) to Edestin, [16,19]. Fora storage SPI, peaks protein for 7 in S andthe 11hemp S globulins seed. It were was identiﬁed, found in butall withsamples relatively with high lower enthalpies enthalpies (5.53–7.25 (0.17–2.00 mJ/mg) and 0.57–0.90 [19,16]. For mJ /SPI,mg, peaks respectively) for 7 S and [29 ].11 S globulins were identified, but with relatively lower enthalpies (0.17–2.00 and 0.57–0.90 mJ/mg, respectively) [29].

12.00 -3 .7 0 0 2.76mJ/mg

10.00 2.00mJ/mg -3 .8 0 0 54.6Cel 8.00 -3.794mW n 0.32mJ/mg 74.6Cel 6.00 -3 .9 0 0 -3.857mW

DSC mW DSC 0.57mJ/mg

85.3Cel mW /mi DDSC 0.31mJ/mg -3.911mW 4.00

-4 .0 0 0 97.3Cel 107.0Cel -3.981mW -3.995mW 2.00

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40.0 50.0 60.0 70.0 80.0 90.0 100.0 110.0 120.0 Temp Cel

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12.00 -3 .5 0 0 6.96mJ/mg

10.00 n

-3 .6 0 0 8.00 0.41mJ/mg DSC mW DSC

0.69mJ/mg mW /mi DDSC 1.19mJ/mg 6.00

-3 .7 0 0 102.6Cel -3.675mW 123.1Cel 4.00 -3.698mW 90.5Cel 109.5Cel -3.728mW -3.725mW 2.00 -3 .8 0 0

0.00

60.0 70.0 80.0 90.0 100.0 110.0 120.0 130.0 Temp Cel

(b)

Figure 5. DSC thermograms for ( a) SPI; and ( b)HPC. 3.4. Texture Properties 3.4. Texture Properties Samples from all HMMA formulations were analysed in terms of texture profiles. All HMMA Samples from all HMMA formulations were analysed in terms of texture profiles. All HMMA made from 100% SPI showed no significant differences (p < 0.05) for hardness, springiness, and made from 100% SPI showed no significant differences (p < 0.05) for hardness, springiness, and chewiness at 70% and 75% target moisture content, except for the resilience, longitudinal cutting and tensile strength (Table 3 and Figure 6). Whereas, the substitution of SPI with different percentage of HPC had no significant effect (p < 0.05) on the springiness of HMMA at all targeted moisture contents.

Foods 2020, 9, 772 8 of 13 chewiness at 70% and 75% target moisture content, except for the resilience, longitudinal cutting and tensile strength (Table3 and Figure6). Whereas, the substitution of SPI with di fferent percentage of HPC had no significant effect (p < 0.05) on the springiness of HMMA at all targeted moisture contents. The resilience value ranged from the lowest (0.48) in 100% SPI at 75% moisture content to the highest (1.66) in 40% HPC with 70% moisture content. This means that HMMA made from 40% HPC and 70% moisture content had the highest elasticity when compared with other samples. Furthermore, springiness and resilience value of 40% and 60% HPC were found to not correlate with the moisture content, which may be due to the higher number of air cells in meat analogue structures, thus leading to lower integrity and difference distribution of fibrous texture [22]. It also might be due to the uneven flow rate from the hopper, which could have led to incompletely homogenous product during protein texturization. On the other hand, the hardness and chewiness of HMMA made from HPC were strongly affected by the target moisture content. Increasing the moisture content by 5% would have resulted in significant reduction (p < 0.05) of hardness and chewiness. This observation is in agreement with others who reported an increase in hardness and chewiness value as the moisture content decreased in HMMA made from soy protein [7]. Important to note is that the HMMA made from 60% HPC with target moisture content of 75% had a very soft and brittle texture and therefore no texture analysis could be performed on those samples.

Table 3. Texture proﬁle analysis result of HMMA at diﬀerent formulations and target moisture content 1.

Moisture Formula Hardness (g) Springiness Resilience Chewiness (g) Content (%) 0% HPC 70 527 33 aC 0.99 0.01 aA 0.51 0.01 aB 459 34 aC ± ± ± ± (100% SPI) 75 545 66 aB 0.99 0.00 aA 0.48 0.01 bB 465 44 aC ± ± ± ± 65 3444 186 aA 0.88 0.14 aA 1.59 0.88 aA 2944 123 aA ± ± ± ± 20% HPC 70 1911 18 bA 0.95 0.06 aA 0.55 0.02 aB 1630 19 bA ± ± ± ± 75 798 21 cA 0.96 0.05 aA 0.52 0.01 aA 670 8 cB ± ± ± ± 65 3275 76 aA 0.91 0.14 aA 0.57 0.03 bA 2692 71 aB ± ± ± ± 40% HPC 70 1700 66 bB 1.19 0.12 aA 1.66 0.26 aA 1435 44 bB ± ± ± ± 75 851 42 cA 0.97 0.04 aA 0.51 0.04 bAB 721 29 cA ± ± ± ± 65 2640 84 aB 1.02 0.19 aA 1.20 0.60 aA 2128 34 aC ± ± ± ± 60% HPC 70 1719 116b bB 0.93 0.04 aA 0.52 0.07 aB 1410 59 bB ± ± ± ± 75 Too soft and brittle to measure 1 All values are presented as mean standard deviation. Different lowercase letters indicate a significant difference (Tukey’s test, p < 0.05) between diff±erent target moisture at the same HMMA formulation, and different uppercase letters indicate a significant difference between different HMMA formulations at the same target moisture content.

The results for the transversal cutting depended on where on the sample the cut was performed due to the structure of the sample. The longitudinal cut gave a more complete overview of the fibre structure because the cutting was performed over the fibres rather than cutting between the fibres as for the transversal cutting. The Tukey´s test indicated that moisture content had a significant effect (p < 0.05) on transversal and longitudinal cutting strength as well as the tensile strength values (Figure6). An increase in moisture content resulted in HMMA products that were easier to cut (cutting strength) and break (tensile strength). The same effect of moisture increase had been reported before which resulted in a reduction of cutting force of HMMA made from lupin protein isolate [30]. Several authors have also argued that higher moisture content affects the viscosity and temperature of the mixture in the extruder, which might lead to incomplete denaturation and texturizing of the protein [7,30]. Almost all texture properties of the HMMA samples made from 100% SPI were the lowest when compared to other samples. Foods 2020, 9, 772 9 of 13 Foods 2020, 9, x FOR PEER REVIEW 9 of 14

(a)

(b)

(c)

FigureFigure 6. 6. (a()a Transversal) Transversal cutting cutting strength; strength; (b) ( blongitudinal) longitudinal cutting cutting strength; strength; (c) (tensilec) tensile strength strength of of differentdifferent HMMA HMMA formulations formulations at ateach each target target moistu moisturere contents. contents. Bars Bars represent represent the the mean mean ± standardstandard ± deviationdeviation of of triplicate measurements.measurements. Di Differentfferent lowercase lowercase letters letters indicate indicate significant significant differences differences (Tukey’s (Tukey’stest, p < test,0.05) p within< 0.05) thewithin same the HMMA same HMMA formulation. formulation. Different Different uppercase uppercase letters indicate letters indicate a significant a significantdifference difference between di betweenfferent HMMA different formulations. HMMA formulations. Bars without Bars standard without deviation standard represent deviation only representone measurement. only one measurement.

Foods 2020, 9, 772 10 of 13

3.5. Colour Determination and Appearance of HMMA Colour measurement of all samples was performed, and L*, a*, and b* values are reported in Table4. In general, it can be seen in Figure2 that the original colour of the SPI powder is darker than the HPC powder. However, after the extrusion process, the HMMA produced from SPI became lighter and higher substitution levels of HPC produced HMMA product with darker colour. The colour changes occurred due to several reactions during the extrusion process, including the Maillard reaction, caramelization, hydrolysis, and a nonenzymatic reaction, such as the degradation of pigments [31]. The lightness (L* value) was found to be significantly higher in SPI product and decreased with higher substitution of HPC (p < 0.05). The same trend was observed for a* and b* values, where samples with a higher substitution of hemp protein gave more redness and less yellowness compared to 100% soy. It can also be seen that at all of the substitution levels of HPC:SPI mixtures, the L* values of the product at 70% and 75% of moisture content were not statistically different (lowercase letters) (p < 0.05). On the other hand, when analysing each formulation (20%, 40% and 60% HPC), most of the results show significant differences between the moisture content for all the colour values, except for a* and b* values of 60% and 20% mixtures, respectively.

Table 4. Result of L*, a* and b* using colorimeter with a short description of each product 1.

Sample Colorimeter Moisture Content Visual Appearance L* a* b* (%) 0% HPC (100% SPI) 70 61.67 0.25 A 0.24 0.07 A 13.48 0.43 A Light colour, compact ± − ± ± 75 62.55 0.21 A 0.29 0.19 A 13.00 0.53 A Light colour, compact ± − ± ± 20% HPC 65 46.19 0.68 aB 0.84 0.08 aB 8.45 0.23 aB Compact ± ± ± 70 46.84 0.68 bC 0.69 0.15 bC 9.20 0.42 bB Compact ± ± ± 75 48.87 0.29 bC 0.30 0.09 bC 8.78 0.14 abB Less compact ± ± ± 40% HPC 65 45.92 1.30 aC 1.64 0.35 aD 9.22 1.02 aB Compact, dark spot ± ± ± 70 48.65 0.52 bB 1.73 0.11 aD 10.90 0.45 aC Compact ± ± ± 75 50.96 0.78 bD 0.95 0.15 bC 10.34 0.40 aC Pale, less compact ± ± ± 60% HPC 65 45.43 0.33 aC 2.34 0.20 aE 7.98 0.72 aB Less compact ± ± ± 70 50.42 0.56 bBD 2.29 0.04 aE 10.11 0.47 bB Less compact ± ± ± 75 49.78 1.01 bD 2.23 0.15 aE 9.56 0.41 bC Came out foamy ± ± ± 1 All L*, a*, b* values are presented as mean standard deviation. Different lowercase letters indicate a significant difference (p < 0.05) between different moisture± content with the same HPC content. Different uppercase letters indicate a significant difference between all HMMA formulations including SPI.

In terms of the appearance of the product, it can be seen from Figure7 that the meat analogue from pure SPI was more compact, with a clear fibrous structure as compared with the other products. This may be due to the higher protein composition in SPI (90% compared to 70% in HPC) which could create the protein cross-linking reaction and formation of disulphide bonds during the extrusion process [7]. High content of methionine and cystine residues may also resulted in increasing the formation of covalent disulphide bonds between individual molecules, thus increasing the extent of aggregation [19]. While with the addition of HPC, with less protein content and higher content of other components such as carbohydrate and fat could affect the internal reaction of the melt in the extruder, resulting in a soft and less compact product [32,33]. It was found that the reordering of the segments of fibres and other ingredients in the starch matrix and complex formulation also resulted in different effects which could modify the physical properties of extruded products [32]. Figure7 presents the di fferent extruded products from each formulation at 65%, 70% and 75% moisture content. The formulation Foods 2020, 9, 772 11 of 13

Foods 2020, 9, x FOR PEER REVIEW 11 of 14 withcontent. 60% HPC The formulation produced a with meat 60% analogue HPC produced with dark a meat spots analogue and liquid with phasedark spots separation and liquid with phase foam coveringseparation the surface, with foam which covering might the be surface, due to the which undenatured might be due protein to the of undenatured HPC. As can beprotein seen of from HPC. DSC andAs RVA can results,be seen itfrom is recommended DSC and RVA to results, increase it cookingis recommended temperature to increase and retention cooking time temperature as hemp and needs higherretention temperature time as hemp to induce needs protein higher denaturation.temperature to Ininduce addition, protein hemp denaturation. protein probably In addition, needs hemp more kneadingprotein elementsprobably inneeds the more screw kneading conﬁguration elements in order in the to screw increase configuration the retention in order time to and increase to develop the a moreretention laminar time ﬁbre and structure.to develop a more laminar fibre structure.

Figure 7. Images of extruded meat analogues with different formulations: (a–c) 20%, 40%, 60% HPC at 65%Figure target 7. moisture; Images of( dextruded–g) 0%, meat 20%, analogue 40%, 60%s HPCwith different at 70% target formulations: moisture; (a (–hc)– k20%,) 0%, 40%, 20%, 60% 40%, HPC 60% at 65% target moisture; (d–g) 0%, 20%, 40%, 60% HPC at 70% target moisture; (h–k) 0%, 20%, 40%, HPC at 75% target moisture. 60% HPC at 75% target moisture. 4. Conclusions 4. Conclusions Based on this study, it was possible to substitute soy protein with hemp protein in the meat analogueBased formulation. on this study, Hemp it was protein possible content to substi (20%,tute 40%, soy 60%)protein at with the dihempfferent protein targeted in the moisture meat contentsanalogue (65%, formulation. 70%, 75%) Hemp and a protein screw speed content of 800(20% rpm, 40%, had 60%) a pronounced at the different effect targeted on the texturemoisture and contents (65%, 70%, 75%) and a screw speed of 800 rpm had a pronounced effect on the texture and colour of the final product. HPC could therefore be a promising novel material to be included into colour of the final product. HPC could therefore be a promising novel material to be included into extruded products and this study shows that the resulting meat analogue gave a comparable texture extruded products and this study shows that the resulting meat analogue gave a comparable texture to SPI alone, and that soy protein could be substituted by hemp protein by up to 60%. More studies to SPI alone, and that soy protein could be substituted by hemp protein by up to 60%. More studies are needed in order to fully understand the complex extrusion process and the formulation matrix are needed in order to fully understand the complex extrusion process and the formulation matrix includingincluding the the nutritional nutritional composition composition and and consumer consumer acceptanceacceptance towards the the final final product. product.

AuthorAuthor Contributions: Contributions:Methodology, Methodology, formal formal analysis, analysis, investigationinvestigation an andd first ﬁrst draft draft preparation, preparation, I.Z., I.Z., F.F., F.F., A.H., A.H., andand C.A.; C.A.; writing writing manuscript, manuscript, I.Z.; I.Z.; supervision supervision and and writing—review writing—review and and editing, editing, K.Ö.,K.Ö., M.R.,M.R., and J.K.P.J.P. All All authors authors havehave read read and and agreed agreed to to the the published published version version of of the the manuscript. manuscript.

Funding:Funding:This This research research was was funded funded by by Malaysian Malaysian AgriculturalAgricultural Research and and Development Development Institute Institute (MARDI), (MARDI), (20190815).(20190815). Conﬂicts of Interest: The authors declare no conﬂict of interest.

Foods 2020, 9, 772 12 of 13

Appendix A

Table A1. Screw conﬁguration used for extrusion of HMMA in this study 1.

Screw Conﬁguration for Meat Analogue Product 1. SE-30/30A 18. SE-20/20R 2. SE-30/30R 19. SE-20/20R 3. SE-30/30R 20. SE-20/20R 4. SE-30/30R 21. KBW-45/5/20/R 5. SE-30/30R 22. KBW-45/5/30/R 6. SE-20/20R 23. SE-20/10L 7. SE-20/20R 24. SE-30/30R 8. SE-20/20R 25. SE-20/10L 9. SE-20/20R 26. SE-30/30R 10. SE-30/30R 27. SE-20/10L 11. SE-30/30R 28. KBW-45/5/30/R 12. SE-20/20R 29. KBW-45/5/20/L 13. KBW-45/5/20/R 30. SE-30/15R 14. SE-30/30R 31. SE-20/20R 15. SE-30/30R 32. SE-20/20R 16. SE-30/30R 33. SE-20/20R 17. SE-20/20R 34. SE-30/30R 1 SE—screw element; KBW—kneading block element.

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