A Comparative Study On The Peptidase System And Debittering Mechanism Among A.elegans, A.oryzae And R.oligosporus

Proteolytic enzyme treatment has been commonly used to improve the process,functional, and nutritional properties of food proteins. However, a prevailing problem in theiruse has been that many proteins yielded bitter-tasting peptides during the hydrolysis process.A. elegans, A. oryzae and R. oligosporus are widely applied in the soybean fermentationindustry, they all have powerful ability to secret many kinds of peptidase, and contributeexcellent flavor to the final products. The aim of this study was to explore endopeptidase andexopeptidase of three mold species frequently utilized in soybean products, i.e. A. elegans, A.oryzae and R. oligosporus, determine their component characteristics via different kinds ofartificially synthesized substrates, and analyze the key enzymes with debittering potency onAlcalse soybean hydrolysates at different pHs. Main results are as follows:The culture condition and the properties of proteases from A. elegans, A. oryzae, and R.oligosporus and the properties of the protease system were compared firstly in this research.The culture condition and properties of proteases from different molds showed distinctivecharacteristics: The protease system from A. elegans consist of acid, neutral and alkalineprotease, but has much lower activity than which from A. oryzae, the yield of acid protease isrelatively higher in neutral and slightly acid media, but the yield of neutral and alkalineprotease is nearly the same when cultured in acid and basic media at 28oC, the proteasesystem from A. elegans is active in the range of pH 5.0⁹.0, stable in pH5.0⁷.0 and the mostactive in pH 5.0⁶.0. The protease system from A. oryzae consist of acid, neutral and alkalineprotease, and has the highest activity among the proteases system from three molds, the yieldof acid protease, neutral protease and alkaline protease from A. oryzae is higher when culturedin acid, neutral and alkaline medium at 28³2oC respectively, the protease system of A.oryzae has fairly strong activities in the range of pH 5.0⁹.0, and is stable between pH3.0⁴.0 and pH 6.0⁸.0; the protease system from R. oligosporus mainly consists of acidprotease with highest level, the enzyme yield is relatively higher when cultured in acidmedium of pH2.5⁴.0, at 32oC, the enzyme system is most active among pH 3.0 6.0 andstable around pH5.0⁶.0.Carboxypeptidase activity from A. elegans bran koji was investigated via absorbance at507 nm after being stained by Cd-nihydrin solution, with calibration curve A, which wasmade by a set of known concentration standard leucine （mmol/L）, calibration B, made bythree sets of known concentration standard leucine solutions （mmol/L） with the addition ofthree concentrations inactive crude enzyme extracts, and calibration C, made by three sets of known concentration standard leucine solutions （mmol/L） with the addition of threeconcentrations crude enzyme extracts. The results indicated that both the calibration curve Band the calibration curve C were capable to meet the demand of precision and sensitivity ofcarboxypeptidase activity determination for there was no difference in significant enzymeactivity between the calibration B and calibration C if the proper dilute multiple was used,while the enzyme activities were always overestimated when calibration curve A was used,even though the inactive crude enzyme extract was intentionally chosen as the control. It wasconcluded that the addition of crude enzyme extracts to the calibration was necessary toeliminate the interference of free amino acids and related compounds presented in crudeenzyme extract.The characteristics of exopeptidase from the 3 molds on different kinds of syntheticsubstrates were also explored. The result indicated that carboxypeptidases in A. elegans and R.oligosporus were of much higher activity under neutral condition than acidic condition, and ofgreat preference to the hydrophobic synthetic substrates with Leu, Phe and Tyr at C-terminal,and Z-Phe-Leu was their commol/Lon best substrate; however, carboxypeptidase in A. oryzaewere of much higher activity on substrates under acidic condition than under neutral condition,and its best substrate was Z-Glu-Tyr. The aminopeptidases, dipeptidyl-peptidase, dipeptidasesand tripeptidase in A. elegans and R. oligosporus all had a distinct pH preference to neutralcondition than acidic condition. Leu-ρNA, Gly-Leu-Phe both were the best substrate ofaminopeptidase and tripeptidase in A. elegans and R. oligosporus respectively. Leucineaminopeptidase activity in A. elegans was almost the same as that from A. oryzae, and bothwere at least 2.5 times lower than leucine aminopeptidase in R. oligosporus. However,dipeptidase from R. oligosporus had no power to catalyze the substrate Leu-Tyr, but showedhighest enzyme activity for substrat Trp-Leu.Proteolysis of soy protein isolates（SPI） at different pHs by crude extracts in A. elegans, R.oligosporus and A. oryzae was investigated. Under acidic condition,β-conglycinin faction andglycinin faction both were easily hydrolyzed within three hours by three mold extracts, at thesame time, amount of free amino acids, mainly hydrophobic amino acids, such as leucine,phenylalanine and tyrosine, released much less than which under neutral and alkali condition.A. elegans expressed preference to basic units of glycinin under alkalic condition. Nobitterness was produced during each hydrolysis process.Bitter pepitde solution, obtained by hydrolyzing soy protein isolates with Alcalase, wastreated with extracts from A. elegans, R. oligosporus and A. oryzae. These three molds wereall efficient tools to decrease the bitterness of bitter peptides, because of their powerful exopeptidase activities. A. elegans and R. oligosporus peptidases liberated least free aminoacid （consisted of 73% hydrophobic amino acid） under acidic condition, displaying goodapplication prospect in protein hydrolyzate industry.Studies on leucine carboxypeptidase characteristics of extract solution produced by A.elegans 3.2778 in solid state fermentation of wheat bran were conducted, with the syntheticsubstrate N-terminal blocked peptide Z-Phe-Leu and Cd-ninhydrin method. The enzyme yieldis relatively higher when cultured under 32oC for 60h. Leucine carboxypeptidase activity wasobserved at pH 6.6 and 40 oC respectively. The enzyme actively exists between pH 5.0 and 9.5but unstable above 45 oC. It was activated by Mg²⁺ at 0.99 mmol/L and 9.10mmol/L, however,partially inhibited by Ca²⁺, Zn²⁺, Ba²⁺ and Mn²⁺ at two concentrations. Leucinecarboxypeptidase activity was completely inhibited by 1,10- phenanthroline, as well as PMSFand DFP.