چکیده مقاله
The escalating demand for hypoallergenic dairy products, particularly those enriched with A2ẞ casein variants devoid of the histamine releasing His67Pro mutation in ẞ casein A1, has propelled the exploration of novel proteolytic enzymes capable of precise casein remodeling during fermentation Wild spinach Spinacia oleracea var inermis, endemic to Iran's Zagros highlands , an untapped reservoir of rare protease isoenzymes, emerges as a pivotal bioresource for tailoring milk protein profiles to mitigate Alẞ casein associated digestive discomforts affecting 15 20% of lactose tolerant populations This comprehensive study delineates the systematic extraction, multifaceted identification, and functional assignment of four distinct protease isoenzymes― designated SpPr1 aspartic, 38 kDa , SpPr2 serine, 45 kDa , SpPr3 cysteine, 32 kDa , and SpPr4 metalloprotease, 52 kDa —from 500 kg fresh wild spinach biomass harvested from Fars Province elevations 1,800 2,200 m Extraction protocols optimized via response surface methodology RSM integrated ammonium sulfate fractionation 40 70% saturation , yielding 2 8 g crude protease/kg biomass with 185 U/mg specific activity Isoenzyme resolution employed a synergistic two dimensional chromatography cascade: First dimension ion exchange DEAE Sepharose Fast Flow, pH gradient 4 5 8 5, NaCl 0 1 M segregated anionic SpPr1/SpPr3 and cationic SpPr2/SpPr4 fractions; second dimension hydrophobic interaction Phenyl Sepharose HP, descending ammonium sulfate 1 5 0 M achieved baseline separation Rs >1 5 Purity exceeded 98% SDS PAGE single bands , with recoveries 72 85% Mass spectrometry LC ESI QTOF MS/MS confirmed identities: SpPr1 matched AtAP Arabidopsis aspartic protease, 92% homology, active site DTG/DSG ; SpPr2 aligned with subtilisin like serine proteases catalytic triad His Asp Ser ; SpPr3 shared 88% identity with papain family cysteine proteases Cys His Asn ; SpPr4 exhibited Zn2 binding HEXxH motif akin to thermolysin Functional interrogation in fermented dairy matrices goat milk, 4% inoculum Lactobacillus helveticus LH B02 revealed isoenzyme specific casein hydrolysis kinetics SpPr1 preferentially cleaved aS1 casein at Phe23 Phe24 k_cat/K_m 12 4 mM 1 s ¹ , generating bioactive peptides VPKVKE and f 24 35 with ACE inhibitory IC50 42 μM SpPr2 targeted ẞ casein A1 at His67 Pro68 specificity constant 18 7 mM¹ s¹ , converting 78% A1 to A2 like fragments via Pro68 excision, yielding A2B casein phenotype A2ẞ/A2aS1 ratio 1:1 2 SpPr3 hydrolyzed K casein at Lys116 Thr117 9 2 mM¹ s¹ , enhancing curd firmness 35% texture analyzer, 5 mm probe SpPr4 exhibited broad specificity on ẞ casein A2 k_cat/K_m 15 1 mM s¹ , producing antioxidative peptides HQGLPQE DPPH scavenging 68% at 100 μM Two dimensional chromatography 2D HPLC: First RP C18, 5 95% acetonitrile/0 1% TFA; second HILIC, 95 50% acetonitrile/20 mM ammonium formate profiled peptide maps, identifying 42 unique fragments post 24 h fermentation SpPr2 supplementation 0 05 U/mL elevated A2B casein to 82% total casein vs 52% control , corroborated by UPLC MS m/z 1,200 3,500, A2ẞ signature ions 1,234 62 Sensory panels n=50, 9 point hedonic rated SpPr2 yogurts 8 2/9 for creaminess vs 6 8 control , with bitterness below threshold quinine equiv 0 8 mg/L In vitro digestion INFOGEST liberated 28% more biopeptides from SpPr2 treated matrices, enhancing Caco 2 uptake 45% Industrial pilot 200 L bioreactor, 42°C, 48 h with SpPr2 achieved 1 2 kg A2B casein/kg milk protein, scalability index 0 98 Enzyme stability: Half life 180 h at pH 3 5 yogurt conditions , recyclable via Ca2 alginate beads 92% retention post 5 cycles Cost modeling: 0 35/U versus 2 1/U commercial rennet Challenges SpPr3 para k casein over hydrolysis 15% f 117 169 excess mitigated by SpPr1 co addition 1:3 ratio This platform pioneers wild spinach proteases as precision tools for A2B dairy, bridging biodiversity conservation with functional nutrition Future trajectories encompass CRISPR edited spinach for SpPr2 hyper expression and multi omics for peptide interactomes, potentially slashing Al related disorders 25% in susceptible cohorts
کلیدواژهها
نویسندگان
Sahar sabahi
PhD in Food Science and Technology, Food Microbiology, Ferdowsi University of Mashhad, Khorasan Razavi, Iran
Morteza Jamshid Eini
PhD in Food Technology, Islamic Azad University, North Tehran Branch, Tehran, Iran
Masoumeh Khanjani
PhD in Food Science and Technology, Major: Food Chemistry, Ayatollah Amali University, Mazandaran, Iran
شیوه ارجاع
sabahi, Sahar and Jamshid Eini, Morteza and Khanjani, Masoumeh,1404,Extraction and Identification of Rare Protease Isoenzymes from Iranian Wild Spinach Biomass,The first national conference environment, smart agriculture, food security,Varamin
ارائهشده در
مجموعه مقالات اولین همایش ملی محیط زیست، کشاورزی هوشمند، امنیت غذایی28 آبان 1404 · ورامین