Metabolite Profiling Thiab Anti-Aging Activity Ntawm Rice Koji Fermented Nrog Aspergillus Oryzae Thiab Aspergillus Cristatus: Kev Tshawb Fawb Sib Piv

Abstract:Ricekoj, siv los ua tus pib ua kom muaj txiaj ntsig zoo tshaj plaws fermentation, tsim ntau yam khoom kawg nyob ntawm cov inoculum microbes siv. Ntawm no, peb tau ua metabolite profileing raupiv movkojfermented nrog ob qho tseem ceeb filamentous fungi,Aspergillus oryzae yog qhov txawv txavthiabA. cristatus, rau 8day. Cov kev txheeb xyuas ntau yam tau pom cov qauv sib txawv ntawm thawj thiab theem nrab metabolites hauv obkoj. Rice koji fermented nrogA. oryzae(RAO) showednce -Kev ua haujlwm ntawm glucosidase thiab cov ntsiab lus ntau dua ntawm cov piam thaj derivatives ntau dua li qhov fermented nrogA. cristatus (RAC). RAC qhiatxhim kho -glucosidase kev ua haujlwm thiab nce cov ntsiab lus ntawm flavonoids thiablysophospholipids, piv rau RAO. Zuag qhia tag nrho, nyob rau theem kawg fermentation (8 hnub), lubantioxidantkev ua ub no thiab kev tiv thaiv kev laustau siab dua hauv RAC dua li hauv RAO, sib xws rau the ncemetabolitesxws liflavonoidsthiab auroglaucin derivatives hauv RAC. Qhov no sib piv metabolictxoj kev tuaj yeem siv rau hauvntau lawm optimizationthiab kev tswj xyuas zoo ntawmkojcov khoom.

Ntsiab lus: mov koji; microbe; solid-state fermentation;anti-aging nyhuv; kev ua haujlwm antioxidant

Nyem qhov no Kom Tau Txais Cov Lus Qhia Ntxiv Txog Cistanche Anti-aging Effect

1. Taw qhia

Fermentation, uas muaj keeb kwm ntau txhiab xyoo, tau raug lees paub tias yog ib txoj hauv kev txhim kho cov khoom noj khoom haus thiab bioactivities ntawm cov khoom noj, ntxiv rau kev ua thiab khaws cia lawv [1]. Rice koji yog tsim los ntawm solid-state fermentation siv steamed nplej nplej inoculated nrog microorganisms kom secrete enzymes thiab ua tau zoo metabolites. Nyob rau hauv xyoo tas los no, ntau yam kev sim los tsim cov fermentation ilv tej yam kev mob tau ua rau siab fermentation efficiency thiab zoo dua zaub mov palatability [2,3]. Vim nws qhov zoo, mov koji pom cov ntawv thov hauv kev lag luam xws li cov khoom noj fermented thiab dej haus thiab tshuaj pleev ib ce [4–6].

Reactive oxygen hom(ROS) yog tsim nyob rau hauv tej yam kev mob of oxidative kev nyuaj siabthiab yog cov los ntawm cov khoom ntawm aerobic metabolism. Cov nodawb radicalstuaj yeem ua rau muaj kev puas tsuaj ntawm biomolecules, ua rau muaj kev puas tsuaj oxidative, xws li o thiab nrawm ntawm daim tawv nqaij laus txheej txheem [7]. Txhawm rau txhim kho qhov sib npaug ntawm ROS ntau lawm thiab tshem tawm, ROS scavengers, hu ua antioxidants, ua lub luag haujlwm tseem ceeb hauv kev txo qis oxidative kev nyuaj siab thiab feem ntau tau los ntawm cov khoom ntuj tsim [8]. Cov dawb radicals no koom nrog cov txheej txheem kev laus, thiab tshem tawm lawv los ntawm kev noj cov tshuaj antioxidants los ntawm cov khoom ntuj tsim yog qhov tseem ceeb hauv kev ncua kev laus [9]. Nyob rau hauv xyoo tas los no, ntau cov kev tshawb fawb tau tshaj tawm tias mov koji tuaj yeem txhim kho cov peev txheej antioxidant ntawm cov khoom siv raw los ntawm kev txhim kho cov fermentation substrate [10,11].

Cov tawv nqaij extracellular matrix (ECM) muaj cov collagen thiab elastin fibers, uas txhawb cov elasticity ntawm daim tawv nqaij los kho thiab tswj nws tus qub zoo thiab lub xeev [12]. Kev puas tsuaj ntawm dermal ECM yog qhov qhia txog kev laus. Nws tshwm sim vim kev txhim kho ntawm collagen-degrading matrix metalloproteinase -1 (MMP-1), los yog collagenase. Yog li ntawd, kev tshawb fawb ntawm ntau yam phytochemicals uas tuaj yeem ua rau qeeb ntawm daim tawv nqaij laus los ntawm kev txhawb nqa collagen thiab elastin synthesis thiab inhibiting MMP-1 yog nce [13–16]. Seo et al. qhia tias fermented nplej bran cuam tshuam rau daim tawv nqaij fibroblast collagen, inflammatory factor (IL-a), thiab MMP-1 [17]. Yog li ntawd, ntau lub tebchaw pom nyob rau hauv mov, xws li flavonoids thiab phenolic acids muaj cov tshuaj antioxidant, thiab fermented mov koji muaj peev xwm ua kom tawv nqaij photoaging los ntawm UV hluav taws xob [18]. Aspergillus, fifilamentous fungus, yog ib qho inoculum microbe uas tsim tau ntau cov txiaj ntsig metabolites xws li cov suab thaj yooj yim, fatty acids, thiab amino acids los ntawm koji hauv Asia. Hauv particular, Aspergillus oryzae yog cov kab mob uas siv ntau tshaj plaws hauv kev tsim koji vim tias nws muaj kev ruaj ntseg thiab ntau yam enzymes, xws li amylase, protease, thiab peptidase [19].

Aspergillus cristatus yog siv hauv tshuaj yej fermentation, xws li Fuzhuan cib tshuaj yej, uas muaj probiotics thiab tiv thaiv UVB-induced photoaging [20,21]. Nws kuj tau raug tshaj tawm los txhim kho cov kev ua haujlwm antioxidant ntawm ntau yam khoom siv raw [22,23]. Tam sim no, kev siv zog tau nce siab rau kev txhim kho qhov zoo ntawm fermentation pib [4,24]. Cov kev tshawb fawb yav dhau los tau qhia txog kev sib piv cov kev tshawb fawb metabolic ntawm Aspergillus thiab Bacillus, dav siv hauv mov koji [25]. Txawm li cas los xij, muaj qhov tsis txaus ntawm cov ntaub ntawv hais txog qhov sib txawv ntawm cov metabolic ntawm tib genera tab sis txawv hom fungi. Txhawm rau xaiv cov kab mob zoo tshaj plaws uas tuaj yeem qhia hauv kev lag luam rau kev noj qab haus huv nrog kev siv nutraceutical thiab cosmeceutical, yuav tsum muaj kev nkag siab zoo ntawm cov metabolism ntawm cov inoculum microbes sib txawv los ntawm kev sib piv lawv cov bioactivity thiab metabolites.

Nyob rau hauv txoj kev tshawb no, peb profiled cov metabolites ntawm mov koji fermented nrog txawv Aspergillus spp. (A. cristatus thiab A. oryzae) nyob rau hauv cov nqe lus ntawm metabolomics los sib piv cov metabolism ntawm ob fifilamentous fungi. Peb kuj tau ntsuas kev ua enzyme, kev ua haujlwm antioxidant, thiab RNA qhia ntawm cov tawv nqaij tiv thaiv kev laus (collagen, elastin, thiab MMP-1) los sib piv ob koji. Tsis tas li ntawd, peb tau soj ntsuam kev sib raug zoo los tawm tswv yim cov neeg sib tw metabolites uas pab txhawb rau kev ua haujlwm antioxidant thiab cov tawv nqaij tiv thaiv kev laus. Kev tsom xam tag nrho ntawm MS-based metabolite profiling rau kev sib piv ob koji inoculums tsim kev sib raug zoo ntawm enzyme kev ua ub no, metabolomes, thiab bioactivities. Ntawm no, peb nthuav qhia daim ntawv qhia dav dav ntawm lub xeev metabolic tag nrho, cuam tshuam nrog bioactivities ntawm ob hom koji inoculums.

2. Cov txiaj ntsig

2.1. Metabolic Profiling rau Rice Koji Fermented nrog txawv Aspergillus spp.

Cov metabolomes sib txawv ntawm cov nplej koji cov qauv inoculated nrog A. cristatus lossis A. oryzae tau muab piv nrog kev txheeb xyuas ntau yam raws li GC-MS thiab LC-MS cov ntaub ntawv teev. Cov ntsiab lus tseem ceeb ntawm kev txheeb xyuas (PCA) cov qhab nia tau los ntawm UHPLC-LTQ-Orbitrap MS/MS thiab GC-TOF-MS qhia tag nrho qhov sib txawv ntawm 40.9 feem pua ​​(PC1, 22.01 feem pua; PC2, 18.89 feem pua) thiab 52.88 feem pua ​​(PC1, 34.70 feem pua ​​​​; PC2, 18.18 feem pua ​​, feem (Daim duab 1A, B). Ob qhov PCA cov txiaj ntsig tau qhia tias qhov pib ntawm qhov fermentation tau sib sau ua ke, tab sis yog li ntawd txawv los ntawm cov kab mob sib txawv raws li lub sijhawm sib txawv. Ib feem tsawg squares discriminant tsom xam (PLS-DA) elucidated statistical qauv tib yam rau cov metabolite tis nyob rau hauv PCA (Ntxiv daim duab S1A,B).

Raws li pom hauv PCA tau txais los ntawm UHPLC–LTQ–Orbitrap–MS/MS kev soj ntsuam (Daim duab 1A), muaj qhov sib txawv tseem ceeb nyob rau hnub yim, thiab ob qho tib si yim-hnub cov qauv tau raug rau ib qho kev ntsuam xyuas ib nrab ntawm qhov tsawg tshaj plaws square discriminant tsom xam (OPLS -DA), uas tau qhia meej meej sib cais los ntawm OPLS tivthaiv 1, suav rau 86.11 feem pua ​​​​ntawm qhov sib txawv ntawm cov ntaub ntawv (Cov duab ntxiv S1C). Cov 31 metabolites raug xaiv los ntawm UHPLC–LTQ–Orbitrap–MS/MS cov ntaub ntawv, uas yog suav hais tias yog ib tug loj contributor rau qhov tsis sib xws nyob rau hauv yim-hnub mov kojis fermented nrog ob tug sib txawv inoculum microbes raws li lawv qhov sib txawv qhov tseem ceeb ntawm qhov projection qhov tseem ceeb (VIP> 1 . Cov metabolites suav nrog 2 carboxylic acids, 5 phenolic acids, 7 flavonoids, 2 long-chain fatty acids, 11 lysophospholipids, thiab anc4 hydroquinones. Cov metabolites tau tshawb pom los ntawm kev sib piv cov ntaub ntawv luam tawm (qhov hnyav molecular, molecular mis, lub sijhawm khaws cia, cov qauv tawg, thiab UV absorbances) thiab cov ntaub ntawv los ntawm lub tsev qiv ntawv hauv tsev.

Daim duab 1. Kev txheeb xyuas cov ntsiab lus tseem ceeb (PCA) cov qhab nia los ntawm (A) UHPLC-LTO-Orbitrap-MS/MS thiab (B) GC-TOF-MSdata pawg ntawm mov koi fermented nrog Aspergillus cristatus lossis A. oryzne.(cov cim sau , A. cristatus; unfilled symbols, A. oryzne, O, 0 hnub; , , 2 hnub; V, V, 4 hnub; 6 hnub; , 8 hnub).

2.1.1. Temporal Metabolomes rau Rice Koji nrog txawv Aspergillus spp. Inoculation Raws li lub sij hawm fermentation

Cov txheej txheem metabolic ntawm mov koji nyob ntawm qhov sib txawv inoculation microbes tau sawv cev los ntawm daim ntawv qhia tshav kub kom pom cov qauv hloov pauv metabolite raws li lub sijhawm fermentation (Daim duab 2). Cov xim ntawm xiav-rau-liab gradient sawv cev rau qhov txhais tau tias cov txheeb ze txheeb ze ntawm txhua qhov metabolite nyob rau hauv txhua qhov kev sim. Cov qauv ntawm feem ntau cov metabolites nyob rau hauv mov koji fermented nrog A. cristatus (RAC) thiab A. oryzaeRAO) pom ib tug zuj zus qauv nrog lub sij hawm fermentation. Cov metabolites txuam nrog carbohydrate metabolism feem ntau sawv cev rau tus qauv nce ntxiv tshwj tsis yog cov piam thaj, xylose, sucrose, thiab maltose, uas yog cov suab thaj. Tsis tas li ntawd, phenolic acid flavonoids, thiab hydroquinone cov ntsiab lus tau txhim kho nrog lub sij hawm fermentation, tshwj tsis yog rau ferulic acid. Ntawm cov fatty acids, feem ntau cov metabolites pom ib tug zuj zus qauv thaum pimelic acid pom ib tug txo. Lysophospholipids nthuav tawm cov qauv sib txawv nrog lub sijhawm sib txawv fermentation thiab inoculation fungi.

Daim duab 2. Cov txheej txheem ntawm txoj kev metabolic thiab cov txheeb ze ntawm cov metabolites hauv mov koji fermented nrog Aspergillus cristatus lossis A. oryzae. Txoj kev tau hloov kho los ntawm Kyoto Encyclopedia of Genes thiab Genomes (KEGG) database thiab hloov kho. Cov xim squares sawv cev rau qhov hloov pauv (xiav rau liab) normalized los ntawm qhov nruab nrab ntawm tag nrho cov txiaj ntsig rau txhua qhov metabolite.

2.1.2. Cov txheeb ze tsis sib xws nyob rau theem ntawm kev ntxub ntxaug Metabolite hauv Rice Koji Fermented byA. cristatus los yog A. oryzae

Raws li pom nyob rau hauv daim duab 2, cov ntsiab lus ntawm thawj thiab theem nrab metabolites nthuav tawm cov qauv sib txawv raws li qhov sib txawv inoculation fungi. Nyob rau hauv cov ntaub ntawv ntawm cov piam thaj uas yog qhov chaw ntawm carbohydrate metabolism, cov qauv ntawm A. cristatus koji pom qhov txo qis, whereas A. ib koji pom cov qauv qis ntawm qhov pib fermentation tab sis maj mam nce mus txog qhov kawg fermentation point. Tsis tas li ntawd, cov piam thaj qab zib tau siab dua hauv RAO dua li hauv RAC. Tshwj xeeb, auroglaucin derivatives tau txhim kho qhov tseem ceeb hauv RAC vim tias lawv yog cov xim tshwj xeeb uas tsim los ntawm A. cristatus. Tsis tas li ntawd, feem ntau cov flavonoids tau nce ntau hauv RAC piv rau RAO, tshwj tsis yog rau 3,8-dimethylherbacetin. Ntawm cov phenolic acids, ferulic acid thiab benzoic acid tau nce nyob rau hauv ob qho tib si kuaj, tab sis dihydroxybenzoic acid, caffeoylquinic acid thiab vanillic acid tau nce tsuas yog hauv RAC. Lysophospholipids tau nce hauv RAC, tab sis qhov sib txawv nyiam tau pom hauv RAO. Fatty acids pom cov qauv ntawm RAO nce ntau dua li hauv AC

2.2. Kev sib piv ntawm Enzymatic Production thiab Bioactivity nyob rau hauv Rice Koji Fermented nrog txawvMicroorganisms

Txhawm rau sib piv cov phenotypes ntawm RAC thiab RAO, peb tau soj ntsuam cov kev ua enzyme thiab kev tiv thaiv kev laus ntawm cov tawv nqaij, kev ua haujlwm antioxidant, tag nrho cov ntsiab lus flavonoid (TFC), thiab tag nrho cov ntsiab lus phenolic (IPC) (Daim duab 3). Enzyme ntau lawm ntawm koji nce nrog lub sijhawm fermentation, tshwj tsis yog rau a-amylase hauv RAO. Interestingly, cov ntsiab lus a-glucosidase yog ob zaug siab dua hauv RAO dua li hauv RAC nrog 10.12 thiab 3.52 units raws li; Hauv qhov sib piv, cov ntsiab lus B-glucosidase tau nce siab dua hauv RAC dua li hauv RAO nrog 19.{{20}}5 units thiab 5.49 units raws li lub sijhawm fermentation. Kev ua haujlwm phenotype ntawm ob qho tib si koji (antioxidant kev ua haujlwm thiab cov tawv nqaij tiv thaiv kev laus) qhia tias cov nplej koji nrog A. cristatus muaj cov haujlwm antioxidant ntau dua hauv ABTS, DPPH, thiab FRAP ntawm lub sijhawm fermentation kawg (8 hnub) nrog 1. {24}}5, 0.40, 0.66 TEAC (Trolox equivalent antioxidant peev) feem. Tsis tas li ntawd, cov ntsiab lus ntawm flavonoid tau siab dua hauv RAC dua li RAO nrog 0.07 NE (naringin sib npaug) thiab 0.01 NE feem. Whereas cov ntsiab lus ntawm tag nrho cov phenol yog siab dua nyob rau hauv RAO tshaj RAC nrog 0.32 EGA (sib npaug gallic acid) thiab 0.28 EGA Raws li. Cov txiaj ntsig ntawm daim tawv nqaij tiv thaiv kev laus (elastin, collagen, thiab MMP-1) qhia lub kaus mom ntawm qhov kev txiav tawm ntawm fermentation. ACRNA qhia theem nrog 7.77 thiab 13.76 thiab qis dua tus txheeb ze MMP-1 RNA qhia theem nrog 2.35 piv rau B-actin. Lub caij no, RAO pom qhov nce ntxiv hauv RNA qhia ntawm elastin thiab collagen tom qab fermentation.

Daim duab 3. Kev sib piv ntawm cov enzyme ntau lawm (A), daim tawv nqaij tiv thaiv kev laus (B) thiab kev ua haujlwm antioxidant, tag nrho cov ntsiab lus flavonoid), thiab tag nrho cov ntsiab lus phenolic (IPC) (C) hauv mov koji fermented nrog txawv Aspergillus spp. (dub xim, A. cristatus dawb xim, A. oryzne). Kev ua haujlwm enzymatic yog a-amylase kev ua, kev ua B-glucosidase, thiab kev ua haujlwm a-glucosidase (A). Tus txheeb ze mRNA qhia theem yog ntsuas rau cov hauv qab no: collagen (COL1A1), elastin (ELN), thiab matrix metalloproteinase-1 (MMP-1) ​​(B). Cov haujlwm antioxidant uas tau piav qhia yog ABTS, DPPH radical scavenging, FRAP, tag nrho cov ntsiab lus Mavonoid, thiab tag nrho cov ntsiab lus phenolic (C). Qhov sib txawv ntawm qhov sib txawv inoculation microbes tau txheeb xyuas los ntawm t-test (* p < {{10}}.05, ** p < 0.01).

Txhawm rau txiav txim siab cov metabolites uas muaj feem cuam tshuam rau bioactivity, kev txheeb xyuas kev sib raug zoo ntawm fermented koji metabolites thiab bioactivities tau ua (Supplement.tary Figure S2). Zuag qhia tag nrho, Pearson daim ntawv qhia kev sib txheeb coefficient pom RAC ntau dua kev sib raug zoo nrog bioactivities dua RAO. Hauv RAC, organic acids, flavonoids, lysophospholipids, fatty acids, hydroquinone, qab zib derivatives pom muaj kev sib raug zoo nrog bioactivities. Rau RAO, organic acids, flavonoids thiab fatty acids, thiab qab zib derivatives qhia txog kev sib raug zoo nrog bioactivities. Cov metabolites uas muaj Pearson correlation coefficient siab dua 0.5 yog sawv cev rau hauv daim ntawv qhia network (Daim duab 4) Hauv ob qho tib si koji cov khoom, organic acids, fatty acids, flavonoids, thiab qab zib derivatives yog cov muaj peev xwm contributors ntawm bioactivities. . RNA qhia ntawm elastin yog txuam nrog metabolites ntawm RAC, whereas RNA qhia ntawm collagen yog txuam nrog metabolites ntawm RAO. Tsis tas li ntawd, TFC tau qhia txog kev sib raug zoo nrog RAC. Tsis tas li ntawd, lysophospholipids thiab hydroquinone yog cov muaj zog antioxidant kev ua rau RAC

Daim duab 4, Cov metabolites uas muaj Pearson tus nqi sib txheeb coefficient siab dua 0.5 yog sawv cev los ntawm daim ntawv qhia network hauv mov koji fermented nrog (A) Aspergillus cristatus lossis (B) A. oryzne. Cov cim lub thawv sawv cev rau bioactivities (xim grey, kev ua haujlwm antioxidant TPC thiab TFC; xim dub, tawv nqaij tiv thaiv kev laus ntawm tes) thiab cov cim xim xim qhia cov metabolites (tib series tau txawv ntawm cov xim sib txawv thiab cov duab: o, hydroquinone: , organic. acids: fatty acids, flavonoids;, lysophospholipids; o, qab zib thiab qab zib derivatives; tsis paub).

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