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7 protocols using 3 methylthio propanal

1

Flavor Compounds Identification Protocol

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2-methylbutanal, 3-methylbutanal, pentanal, isopropyl alcohol, 1-propanol, 1-butanol, hexanal, 2-methyl-1-propanol, (E)-2-methyl-2-butenal, limonene, heptenal, 2-butenal, 2-methylbutanol, 3-methylbutanol, 2-pentyl-furan, 3-octanone, 1-pentanol, 2-octanone, 4-isopropyltoluene, octanal, 1-octen-3-one, isobutyl hexanoate, nonanal, heptanoic acid ethyl ester, 1-hexanol, 3-octanol, octanoic acid ethyl ester, (E)-2-octenal, (E)-2-nonenal, P-cresyl methyl ether, 1-heptanol, 1-octen-3-ol, 1-octanol, benzaldehyde, 2-methyl-butanoic acid, 3-methyl-butanoic acid, 2-acetylthiazole, benzeneacetaldehyde, benzyl alcohol, phenylethyl alcohol, γ-nonalactone, were purchased from Alfa Aesar Corporation (Tianjin, China). Methanethiol, dimethyl sulfide, dimethyl disulfide, bis(methylthio)methane, 3-(methylthio)propanal, dimethyl sulfoxide, dipropyl trisulfide, 3-(methylthio)propanol, dimethyl sulfone, bis(2-methyl-3-furyl)disulfide were purchased from Sigma-Aldrich (St Louis, MO, USA). All of the chemical standards used above were of GC quality.
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2

Cultivation and Flavor Analysis of Aspergillus oryzae

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Soybeans, raw wheat flour and edible salt (NaCl) were purchased from a local supermarket (Zhenjiang, China, 2021). 3-Methylbutanal, ethanol, 3-methyl-1-butanol, ethyl propanoate, ethyl acetate, ethyl isovalerate, 2-phenylethanol, benzeneacetaldehyde, 3-(methylthio) propyl acetate, ethyl 3-(methylthio) propionate, methyl benzoate, (E)-β-damascenone, 2-phenylethyl acetate, 2-furanmethanol, 3-methylthio-1-propanol, 3-methylthio propanal, 2,6-dimethyl pyrazine, trimethyl pyrazine, 2-acetyl pyrroline, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) were all ordered from Sigma-Aldrich (Shanghai, China, 2021). 3-Furaldehyde, 4-ethylphenol, 4-ethylguaiacol, 4-vinylguaiacol, 3-methylbutanoic acid, acetic acid, 2-methylbutanoic acid, dimethyl trisulfide, 1-octen-3-ol and C6-C33 n-alkanes were provided by Aladdin Holdings Group (Shanghai, China, 2021). Other analytically pure reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China, 2021). A. oryzae 3.042 were obtained from the Guangdong Institute of Microbiology (Guangzhou, China, 2021).
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3

Analytical Standards for Food Aroma Profiling

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Dichloromethane and anhydrous sodium sulfate were purchased from VWR (Darmstadt, Germany) whereas sulfuric acid and sodium carbonate were from Roth (Karlsruhe, Germany). The reference substances were obtained from the following suppliers: acetic acid, 2-phenylethanol, 3-methyl-1-butanol (isoamyl alcohol), hexanoic acid, phenylacetic acid, ethyl 2-methylbutanoate, γ-decalactone, γ-undecalactone, 2-methylbutanoic acid and 3-(methylthio)-propanal (methional) were from Sigma-Aldrich (Steinheim, Germany), butanoic acid and ethyl butanoate from Fluka (Steinheim, Germany), 2-methylpropanoic acid (isobutanoic acid) from SAFC (Steinheim, Germany), and 4-hydroxy-3-methoxybenzaldehyde (vanillin) from ABCR (Karlsruhe, Germany). The isotopically labeled standards were purchased from the following suppliers given in parentheses: 3,4-2H2-3-methyl-1-butanol (2H2−isoamyl alcohol), 1,3,4,5,6-2H5-2-phenylethanol, 3-(methyl-2H3-thio)-propanal (2H3-methional), 5,5,6,6-2H4-y-decalactone, 6,7-2H2-y-undecalactone (Aromalab, Freising, Germany), 6,6,6-2H3-hexanoic acid, 3,3,3-2H3-2-methylpropanoic acid (2H3-isobutanoic acid), and 2H6-ethanol (CDN Isotopes, Pointe-Claire, Quebec, Canada), 1,2-13C2-butanoic acid, α,β-13C2–phenylacetic acid, 13C1-acetic acid, and 4-hydroxy-3-methoxy-13C6−benzaldehyde (13C6-vanillin) (Sigma-Aldrich, Steinheim, Germany).
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4

Aroma Compounds Extraction Protocol

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Yeast extract, sucrose, glucose, and medium with chloramphenicol were obtained from BTL (Łódź, Poland). Galactose was purchased from Acros Organic (Geel, Belgium). Citric acid, Na2HPO4·2H2O, and MgCl2 were obtained from POCH (Gliwice, Poland). Fructose was obtained from Chempur (Piekary Śląskie, Poland). L-Phenylalanine, lactic acid, sodium sulfate, ethyl acetate, dichloromethane, and diethyl ether were purchased from Sigma-Aldrich (Poznań, Poland). Inulin was obtained from Hortimex Plus (Konin, Poland). Spray-dried sour whey and buttermilk were purchased from Laktopol (Suwałki, Poland). The following reference aroma compounds were purchased from Sigma-Aldrich (Poznań, Poland): 2,3-butanedione, acetic acid, 3-methylbutanal, 3-methyl-1-butanol, butanoic acid, isovaleric acid, 3-(methylthio)-propanal, dimethyl trisulfide, phenylacetaldehyde, furaneol, maltol, 2-phenylethanol, ethyl furaneol, (E)-2-nonenal, and phenylacetic acid. The following stable isotopes were obtained from AromaLAB (Freising, Germany): [13C4] 2,3-butanedione, [13C1] acetic acid, [2H3] 2-methylbutanal, [2H2] 3-methyl-1-butanol, [2H7] butanoic acid, [2H2] isovaleric acid, [2H5] 3-(methylthio)-propanal, [2H6] dimethyl trisulfide, [2H5] phenylacetaldehyde, [13C2] furaneol, [2H3] maltol, [2H5] 2-phenylethanol, [2H2] (E)-2-nonenal, and [2H8] naphthalene.
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5

Synthesis and Purification of Volatile Compounds

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Isobutyl ITC 97%, 4-pentenyl ITC 95% and sec-butyl ITC 97% were purchased from ABCR (Karlsruhe, Germany), 3-sec-butyl-2-methoxypyrazine from Acros Organics (Geel, Belgium), (3S,3aS,7aR)-wine lactone >95% from Aromalab (Freising, Germany), 2-(methylthio)ethyl ITC >96% from FCH Group (Chernigiv, Ukraine), butanoic acid ≥99% from Fluka (Steinheim, Germany), (Z)-3-hexenal 50% from SAFC (Steinheim, Germany) and 3-methylbutyl ITC >98% from Santa Cruz Biotechnology (Heidelberg, Germany). Acetic acid >99%, AITC 95%, p-anisaldehyde 98%, benzyl ITC 98%, 3-hydroxy-4,5-dimethyl-2(5H)-furanone (sotolone) ≥97%, 3-isobutyl-2-methoxypyrazine ≥99%, 3-isopropyl-2-methoxypyrazine 99%, 2-methylbutanoic acid 98%, 3-methylbutanoic acid 99%, skatole 98%, 3-(methylthio)propanal 98%, 3-(methylthio)propyl ITC 98%, 1-octen-3-one 50%, 2-phenylacetaldehyde 90% and PEITC >99% were purchased from Sigma-Aldrich (Steinheim, Germany) and 3-butenyl ITC 95% from TCI Europe (Eschborn, Germany). CETP was synthesized as described in our previous study (Kroener and Buettner, 2017 (link)).
Dichloromethane (DCM), which was freshly distilled for purification prior to use, and anhydrous sodium sulfate were purchased from VWR International GmbH (Darmstadt, Germany).
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6

Aroma Compounds Sourcing for Research

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Yeast extract, sucrose, glucose, and medium
with chloramphenicol were obtained from BTL (Łódź,
Poland). Galactose was purchased from Acros Organic (Geel, Belgium).
Citric acid, Na2HPO4·2H2O, and
MgCl2 were obtained from POCH (Gliwice, Poland). l-Phenylalanine, lactic acid, sodium sulfate, dichloromethane, and
diethyl ether were purchased from Sigma-Aldrich (Poznań, Poland).
Inulin was obtained from Hortimex Plus (Konin, Poland). Spray-dried
sour and sweet whey products were purchased from Laktopol (Suwałki,
Poland). The following reference aroma compounds were purchased from
Sigma-Aldrich (Poznań, Poland): 2,3-butanedione, acetic acid,
3-methylbutanal, 3-methyl-1-butanol, butanoic acid, 3-(methylthio)-propanal,
dimethyl trisulfide, phenylacetaldehyde, 2-phenylethanol, and phenylacetic
acid. The following stable isotopes were obtained from aromaLAB (Freising,
Germany): [13C4] 2,3-butanedione, [13C1] acetic acid, [2H3] 2-methylbutanal,
[2H2] 3-methyl-1-butanol, [2H7] butanoic acid, [2H5] 3-(methylthio)-propanal,
[2H6] dimethyl trisulfide, [2H5] phenylacetaldehyde, [2H5] 2-phenylethanol,
and [2H8] naphthalene.
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7

Volatile Compound Standards for Food Analysis

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The volatile standards, i.e. phenylacetaldehyde (95%), 3-methylbutanoic acid (99%), 3-(methylthio)propanal (98%), 4-vinylguaiacol (≥98%), ethyl isobutyrate (99%), 3hydroxy-2-methyl-4-pyrone (maltol, 99%), 2,5-dimethyl-4-hydroxy-3(2H)-furanone (HDMF, ≥ 99%), 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (HEMF, mixture of isomers, 96%) and standards of n-alkanes (C6-C33, 99%) were purchased from Sigma-Aldrich (Steinheim, Germany). 3-Methyl-1-butanol (99.8%), ethyl butyrate (≥99.5%), 1-octen-3-ol (98%), 2-methylbutanoic acid (98%), 4-ethyl-2-methoxyphenol (98%), 2phenylethanol (98%), dimethyl trisulfide (98%), 3-(methylthio)-1-propanol (99%) and 2,5-dimethylpyrazine (98%) were purchased from Aladdin (Shanghai, China). In the GC/O-AT and sensory evaluation sessions, nine food-grade volatile compounds, including phenylacetaldehyde (>99%), 3-hydroxy-2-methyl-4-pyrone (>96%), 2,5dimethyl-4-hydroxy-3(2H)-furanone (>98%), 3-(methylthio)propanal (>98%), 1octen-3-ol (>96%), 3-(methylthio)-1-propanol (>99%), dimethyl trisulfide (>95%), 2,5-dimethylpyrazine (>99%), and 5-ethyl-4-hydroxy-2-methyl-3(2H)-furanone (>98%), were purchased from Huabao Co. (China). Distilled water was obtained from Watsons Co. (Guangdong, China). Food-grade sodium chloride was purchased from Xiangheng Co. (Hunan, China).
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