Yufei Hua

Heat-induced inactivation mechanisms of Kunitz trypsin inhibitor and Bowman-Birk inhibitor in soymilk processing

Trypsin inhibitor activity (TIA) is an important antinutritional factor in soymilk. In this study, the effects of NaCl preaddition on TIA and the heat-induced TIA inactivation mechanisms were examined. The results showed that Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) contributed 74% and 26% to raw soymilk TIA, respectively. The heat-induced quick KTI incorporation into protein aggregates was the reason for its quick TIA inactivation. The heat-induced slow cleavage of one BBI peptide bond was the reason for its slow TIA inactivation. Heat-induced protein aggregate formation had little effect on BBI inactivation owing to the fact that BBI and its degradation product tended to remain in the supernatant (197,000g, 1h) in all conditions used in this study. NaCl could accelerate the KTI incorporation into protein aggregates and the cleavage of one BBI peptide bond, which supplied a simple and quick method for low TIA soymilk processing.

Continuous hydrolysis of modified wheat gluten in an enzymatic membrane reactor.

BACKGROUND The low solubility of wheat gluten is one of the major limitations to its use in food processing, and enzymatic hydrolysis has been found to be an effective way to prepare more soluble bioactive peptides from gluten. The aim of this study was to prepare bioactive peptides from modified wheat gluten (MWG) in a continuous enzymatic membrane reactor (EMR) that allowed rapid separation of low-molecular-weight peptides from hydrolysates, thus avoiding the disadvantages of batch reaction such as inefficient use of enzymes, inconsistent products due to batch-to-batch variation, substrate-product inhibition, low productivity and excessive hydrolysis. RESULTS Wheat gluten was modified to decrease its lipid and starch contents in order to prevent membrane fouling. The optimal working conditions for Alcalase to hydrolyse MWG in the EMR were a substrate concentration of 20 g L(-1) , an enzyme/substrate ratio of 0.03, an operating pressure of 0.04 MPa, a temperature of 40 °C and a pH of 9. The operating stability of the EMR (including residual enzyme activity, productivity and capacity) was high. The permeate fractions showed antioxidant activities that were mostly due to low-molecular-weight peptides. A simple theoretical kinetic model was successfully applied to the enzymatic hydrolysis of MWG in the EMR. CONCLUSION Modification of wheat gluten made the continuous enzymatic membrane reaction more efficient and the EMR proved to be an effective means of producing peptides with particular properties and bioactivities. The permeate fractions (mainly < 1000 Da) were homogeneous and stable and also showed strong antioxidant activities.

The characterization of soybean oil body integral oleosin isoforms and the effects of alkaline pH on them.

Oil body, an organelle in seed cell (naturally pre-emulsified oil), has great potentials to be used in food, cosmetics, pharmaceutical and other applications requiring stable oil-in-water emulsions. Researchers have tried to extract oil body by alkaline buffers, which are beneficial for removing contaminated proteins. But it is not clear whether alkaline buffers could remove oil body integral proteins (mainly oleosins), which could keep oil body integrity and stability. In this study, seven oleosin isoforms were identified for soybean oil body (three isoforms, 24 kDa; three isoforms, 18 kDa; one isoform, 16kDa). Oleosins were not glycoproteins and 24 kDa oleosin isoforms possessed less thiol groups than 18 kDa ones. It was found that alkaline pH not only removed contaminated proteins but also oleosins, and more and more oleosins were removed with increasing alkaline pH.

The integral and extrinsic bioactive proteins in the aqueous extracted soybean oil bodies.

Soybean oil bodies (OBs), naturally pre-emulsified soybean oil, have been examined by many researchers owing to their great potential utilizations in food, cosmetics, pharmaceutical, and other applications requiring stable oil-in-water emulsions. This study was the first time to confirm that lectin, Gly m Bd 28K (Bd 28K, one soybean allergenic protein), Kunitz trypsin inhibitor (KTI), and Bowman-Birk inhibitor (BBI) were not contained in the extracted soybean OBs even by neutral pH aqueous extraction. It was clarified that the well-known Gly m Bd 30K (Bd 30K), another soybean allergenic protein, was strongly bound to soybean OBs through a disulfide bond with 24 kDa oleosin. One steroleosin isoform (41 kDa) and two caleosin isoforms (27 kDa, 29 kDa), the integral bioactive proteins, were confirmed for the first time in soybean OBs, and a considerable amount of calcium, necessary for the biological activities of caleosin, was strongly bound to OBs. Unexpectedly, it was found that 24 kDa and 18 kDa oleosins could be hydrolyzed by an unknown soybean endoprotease in the extracted soybean OBs, which might give some hints for improving the enzyme-assisted aqueous extraction processing of soybean free oil.

Oleosins (24 and 18 kDa) are hydrolyzed not only in extracted soybean oil bodies but also in soybean germination.

After oil bodies (OBs) were extracted from ungerminated soybean by pH 6.8 extraction, it was found that 24 and 18 kDa oleosins were hydrolyzed in the extracted OBs, which contained many OB extrinsic proteins (i.e., lipoxygenase, β-conglycinin, γ-conglycinin, β-amylase, glycinin, Gly m Bd 30K (Bd 30K), and P34 probable thiol protease (P34)) as well as OB intrinsic proteins. In this study, some properties (specificity, optimal pH and temperature) of the proteases of 24 and 18 kDa oleosins and the oleosin hydrolysis in soybean germination were examined, and the high relationship between Bd 30K/P34 and the proteases was also discussed. The results showed (1) the proteases were OB extrinsic proteins, which had high specificity to hydrolyze 24 and 18 kDa oleosins, and cleaved the specific peptide bonds to form limited hydrolyzed products; (2) 24 and 18 kDa oleosins were not hydrolyzed in the absence of Bd 30K and P34 (or some Tricine-SDS-PAGE undetectable proteins); (3) the protease of 24 kDa oleosin had strong resistance to alkaline pH while that of 18 kDa oleosin had weak resistance to alkaline pH, and Bd 30K and P34, resolved into two spots on two-dimensional electrophoresis gel, also showed the same trend; (4) 16 kDa oleosin as well as 24 and 18 kDa oleosins were hydrolyzed in soybean germination, and Bd 30K and P34 were always contained in the extracted OBs from germinated soybean even when all oleosins were hydrolyzed; (5) the optimal temperature and pH of the proteases were respectively determined as in the ranges of 35-50 °C and pH 6.0-6.5, while 60 °C or pH 11.0 could denature them.

The heat-induced protein aggregate correlated with trypsin inhibitor inactivation in soymilk processing.

Kunitz trypsin inhibitor (KTI) and Bowman-Birk inhibitor (BBI) have trypsin inhibitor activities (TIA), which could cause pancreatic disease if at a high level. It is not clear why some KTI and BBI lose TIA and some does not in the soymilk processing. This would be examined in this study. TIA assay showed residual TIA was decreased with elevated temperature and TIA was decreased quickly in the beginning and then slowly in boiling water bath. Interestingly, ultracentrifugation showed low residual TIA soymilk had more precipitate than high residual TIA soymilk and soymilk TIA loss had a high correlation coefficient (R(2) > 0.9) with precipitate amount. In addition, the TIAs of floating, supernatant, and precipitate obtained by ultracentrifugation were assayed and >80% residual TIA was concentrated in the supernatant. Tricine-SDS-PAGE showed KTI in supernatant was mainly a noncovalent bound form which might exist as itself and/or incorporated into a small protein aggregate, while KTI in precipitate was incorporated into a protein aggregate by disulfide and/or noncovalent bonds. Chymotrypsin inhibitor activity (CIA) assay showed about 89% of the original CIA remained after 100 °C for 15 min. Ultracentrifugation showed that >90% residual CIA was concentrated in supernatant. Tricine-SDS-PAGE showed soymilk (100 °C, 15 min) BBI mainly existed in supernatant but not in precipitate. It was considered that BBI tended to exist as itself with its natural conformation. Thus, it was suggested residual TIA was mainly from the free BBI and TIA inactivation was mainly from KTI incorporation into protein aggregate. This study is meaningful for a new strategy for low TIA soymilk manufacture based on the consideration of promoting protein aggregate formation.

Macronutrients and micronutrients of soybean oil bodies extracted at different pH.

UNLABELLED In this study, the macronutrients and micronutrients of pH 6.8, 8.0, 9.5, and 11.0 extracted soybean oil bodies (OBs) were examined, revealing that soybean OBs might be used as a natural carrier for bioactive components (unsaturated fatty acids, phospholipid, tocopherol, and phytosterol). pH 6.8 extracted OBs (dry basis) contained 85.88% neutral lipid, 8.18% protein, and 5.85% polar lipid (mainly phospholipid) by gravimetric analysis. The percentage of neutral lipid was increased, while those of protein and polar lipid were decreased with increasing pH. Tocopherol (about 75 mg/100 g neutral lipid of OBs) was not affected, while phytosterol was decreased (136 to 110 mg/100 g neutral lipid of OBs) with increasing pH. The detectable total monosaccaride (galactosamine, glucosamine, and glucose) content of extracted OBs was low and also decreased (35.80 to 6.13 mg/100 g neutral lipid of OBs) with increasing pH. The protein of extracted OBs had higher percentage of essential amino acids than soybean protein isolate with tryptophan and methionine as limited amino acids. The fatty acid composition of extracted OBs was rich in linoleic acid (about 59%), oleic acid (about 20%), and α-linolenic acid (about 7%). PRACTICAL APPLICATION Oil bodies (OBs) from soybean and other plant seeds are greatly examined owing to their potential utilizations in food ingredients. The determination of its macronutrients and micronutrients would be very meaningful for its efficient utilization in the future.

Soybean whey protein/chitosan complex behavior and selective recovery of kunitz trypsin inhibitor.

Proteins in soybean whey were separated by Tricine-SDS-PAGE and identified by MALDI-TOF/TOF-MS. In addition to β-amylase, soybean agglutinin (SBA), and Kunitz trypsin inhibitor (KTI), a 12 kDa band was found to have an amino acid sequence similar to that of Bowman-Birk protease inhibitor (BBI) and showed both trypsin and chymotrypsin inhibitor activities. The complex behavior of soybean whey proteins (SWP) with chitosan (Ch) as a function of pH and protein to polysaccharide ratio (RSWP/Ch) was studied by turbidimetric titration and SDS-PAGE. During pH titration, the ratio of zeta potentials (absolute values) for proteins to chitosan (|ZSWP|/ZCh) at the initial point of phase separation (pHφ1) was equal to the reciprocal of their mass ratio (SWP/Ch), revealing that the electric neutrality conditions were fulfilled. The maximum protein recovery (32%) was obtained at RSWP/Ch = 4:1 and pH 6.3, whereas at RSWP/Ch = 20:1 and pH 5.5, chitosan consumption was the lowest (0.196 g Ch/g recovered proteins). In the protein-chitosan complex, KTI and the 12 kDa protein were higher in content than SBA and β-amylase. However, if soybean whey was precentrifuged to remove aggregated proteins and interacted with chitosan at the conditions of SWP/Ch = 100:1, pH 4.8, and low ionic strength, KTI was found to be selectively complexed. After removal of chitosan at pH 10, a high-purity KTI (90% by SEC-HPLC) could be obtained.

Release behavior of non-network proteins and its relationship to the structure of heat-induced soy protein gels.

Heat-induced soy protein gels were prepared by heating protein solutions at 12%, 15% ,or 18% for 0.5, 1.0, or 2.0 h. The release of non-network proteins from gel slices was conducted in 10 mM pH 7.0 sodium phosphate buffer. SDS-PAGE and diagonal electrophoresis demonstrated that the released proteins consisted of undenatured AB subunits and denatured proteins including monomers of A polypeptides, disulfide bond linked dimers, trimers, and polymers of A polypeptides, and an unidentified 15 kDa protein. SEC-HPLC analysis of non-network proteins revealed three major protein peaks, with molecular weights of approximately 253.9, 44.8, and 9.7 kDa. The experimental data showed that the time-dependent release of the three fractions from soy protein gels fit Ficks second law. An increasing protein concentration or heating time resulted in a decrease in diffusion coefficients of non-network proteins. A power law expression was used to describe the relationship between non-network protein diffusion coefficient and molecular weight, for which the exponent (α) shifted to higher value with an increase in protein concentration or heating time, indicating that a more compact gel structure was formed.

Solubilization of proteins in extracted oil bodies by SDS: A simple and efficient protein sample preparation method for Tricine–SDS–PAGE

A simple and efficient method for preparing Tricine-SDS-PAGE protein sample of extracted oil bodies (OBs) was supplied: OB suspension was vortexed with SDS buffer (pH 6.8) for 2 min at room temperature with SDS/protein of 1.52/1(w/w), which could be analyzed by Tricine-SDS-PAGE after simple treatments (dilution and 2-mercaptoethanol). At SDS/protein of 1.52/1, about 95% of proteins in soybean OB suspension were solubilized, whereas residual 5% of proteins were weakly bound to SDS-destroyed OBs; proteins in destroyed OBs might be further solubilized by SDS in the gel and cathode buffer of Tricine-SDS-PAGE, causing about 99% of proteins in soybean OB suspension recover on Tricine-SDS-PAGE gel, which was better than acetone (89%) and diethyl ether (96%) harvested protein samples. Higher or lower SDS/protein was unbeneficial for protein solubilization from OBs. Additionally, the above method was also better than organic solvent method for peanut, sesame, and rapeseed OB suspensions.