Investigation On The Mechanism Of Fibrous Structure Formation During High-Moisture Extrusion For Preparing Plant Protein-Based Meat Substitutes

The rapid development of plant protein-based meat substitutes could contribute significantly to filling the gap of animal meat supply,promoting the environmental sustainability and meeting the healthy,green and nutritious dietary needs for consumers.In recent years,high-moisture extrusion has become a hot and advanced technology to prepare plant protein-based meat substitutes with rich meatlike fibrous structures at home and abroad.However,the conformational changes and interactions among main components including protein,starch and lipid and the mechanism of the fibrous structure formation during high-moisture extrusion processing are still unclear.Thus,in this study,the designed mixtures of pea protein isolate(PPI)-starch,PPI-fatty acid and PPI-starch-fatty acid were extruded at constant high-moisture conditions.The effects of the amylose/amylopectin ratio and unsaturation degree of fatty acid on the rheological properties of the raw material,the response parameters during highmoisture extrusion processing and the quality of extrudate including the texture,color and digestibility were clarified.Furthermore,the high-moisture extrusion process was divided into four main stages,namely the raw material,extruder barrel(consisting of the mixing zone and melting zone),the cooling die(consisting of the die and cooling zone)and extrudate.The dead-stop operations were carried out to collect samples from different extrusion zones to provide deep insights into protein-starch,protein-fatty acid and protein-starch-fatty acid molecular interactions and the mechanism of the fibrous structure formation during high-moisture extrusion.The main conclusions are as follows:1.The incorporation of 10% amylopectin could decrease the apparent viscosity,storage modulus(G′)and loss modulus(G′′)of PPI dispersion and reduce the specific mechanical energy(SME)significantly from 985.07 k J/kg to 936.78 k J/kg during extrusion processing.Meanwhile,the amylopectin which was uniformly embedded in the protein matrix,facilitated the formation of fibrous structures,but decreased the in vitro protein digestibility significantly from 85.45% to 76.93%.With the amylose/amylopectin ratio increasing,the apparent viscosity of the protein matrix increased significantly,the fibrous degree of the extrudates decreased gradually from 1.32 to 1.21 with forming denser inner structures,and the in vitro protein digestibility increased from 76.93% to 84.61%.Moreover,both the amylose and amylopectin addition could significantly increase the deformability of protein melt,reduce the die pressure and torque during high-moisture extrusion processing,promote the stable flow of melt in the extruder barrel,and decrease the hardness,springiness,chewiness and tensile resistence force of the extrudates.2.In the extruder barrel,the amylose entangled with unfolding protein chains through hydrogen bonds.In the die,the phase separation of amylose and protein matrix promoted the conversion of β-turn and random coil to α-helix and β-sheet structures,enhanced covalently self-aggregation of 11 S subunites to form stable aggregates,leading to dense gel-like structures.In the cooling zone and extrudate,the amylose coagulum formed steric hindrance for the protein rearrangement and crosslinking,consequently leading to a layered rough gel structure.On the other hand,the amylopectin could increase the fluidity of the protein matrix in the mixing zone and promote the unfolding of 7S vicilin and 11 S legumin in the melting zone.The phase separation of amylopectin and protein matrix in the melting zone enhanced the initial association and aggregation of proteins.In the die,the amylopectin promoted the unfolding of protein chains and significantly decreased the grafting degree from 44.34%to 40.10%,which was favorable for the protein rearrangement and “sub-layer transformation” crosslinking in the cooling zone to further promote the formation of anisotropic fibrous structures by enhancing the hydrophobic interactions.3.The addition of 5% fatty acid mixtures including stearic acid,oleic acid and linoleic acid could decrease the apparent viscosity,G′ and G′′ of PPI dispersion and delay the unfolding,association and aggregation of proteins under heating,leading to the decrease in the chewiness of extrudate,and the significant reduction of the in vitro protein digestibility from 85.45% to(70.56～78.60)%,but the increase in tensile resistant force from 0.19 kg to(0.62～0.85)kg.As the unsaturation degree of fatty acid increasing,the fatty acid mixtures showed stronger lubrication and plasticizing ability,which could significantly decrease the die pressure,torque and mass flow rate and increase the SME from 940.07 k J/kg to 1484.09 k J/kg.The fatty acid mixtures with higher unsaturation degrees tended to compete the hydrophobic groups on the proteins and inhibit the protein aggregation and cross-linking,resulting in the decrease of the fibrous degree from 1.22 to 1.08.4.In the extruder barrel,fatty acids hindered the unfolding of protein molecules and weakened the hydrophobic interactions between protein molecules in the melting zone.In the die,fatty acids stabilized the unfolding structures of proteins,significantly decreased the ratio of α-helix and β-sheet from 20.67% and 34.36% to(7.17～13.64)% and(24.54～28.07)% and increased the ratio of random coil from 22.34% to(36.06～40.90)%,and furtherly weaken the hydrogen bonds and hydrophobic interactions between protein molecules.Furthermore,the oxidation of unsaturated fatty acids would result in the formation of aggregates with higher molecular weights and rigid gel structures.From the cooling zone to the extrudate,the stearic acid enhanced the disulfide bonds and promoted the formation of layered fibrous structures while the coalescence of unsaturated fatty acids hindered the formation of continuous network and fibrous structures.For the extrudate of PPI with stearic acid,interactions between hydrophobic interactions and disulfide bonds were the dominant forces maintaining the protein structures,but the oleic acid and linoleic acid changed that to hydrophobic interactions.5.During high-moisture extrusion processing,the amylopectin and stearic acid had synergistic effect on the formation of fibrous structures in the PPI extrudate.In the extruder barrel,the amylopectin and stearic acid jointly induced the exposure of hydrophobic groups in proteins,promoted the uncoiling of protein molecules as well as the formation of more stable β-sheet and β-turn structures.In the die,the amylopectin and stearic acid weakened the hydrogen bonds,promoted the conversion of α-helix and β-sheet structures to the β-turn and random coil structures,significantly increased the ratio of random coil structures from 22.34% to 47.77%,inhibited the aggregation and cross-linking of the unfolding proteins,delayed the gel formation rate and defered the phase separation between protein and amylopectin from the melting zone to the die.In the cooling zone and extrudate,the amylopectin and stearic acid promoted the formation of fibrous structures by the “anchor orientation and flexible cross-linking”.Namely,the stearic acid hindered the refolding of protein molecular chains while the amylopectin promoted the rearrangement,aggregation and cross-linking of protein molecules.The amylopectin and stearic acid synergistically weakened the interaction forces between proteins and led to the formation of more loose and flexible structures in aggregates which was mainly maintained by hydrophobic interactions and hydrogen bonds.These loose and flexible structures enabled the aggregates to orientate along the extrusion direction and form the anisotropic fibrous structures in extrudates.