Compound enzyme and low-oxygen co-fermentation improves aroma quality and reduces irritation of cigar tobacco leaves

Combined use of compound enzymes and low-oxygen conditions is a promising approach for tobacco fermentation. Nevertheless, the regulatory mechanism of this co-fermentation system on flavor formation in cigar tobacco leaves remains unclear. This work aims to explore the effects of combined fermentation treatment on sensory properties and metabolic regulation of cigar tobacco leaves. Air-cured ‘Dexue 1’ cigar tobacco leaves were selected as experimental materials. Five treatments were set up: unfermented control (CK), conventional fermentation (T1), low-oxygen fermentation (T2), compound enzyme fermentation (T3), and combined compound enzyme and low-oxygen co-fermentation (T4). A combination of electronic nose analysis, volatile metabolomics and proteomics was used to identify key regulatory proteins and aroma components in fermented tobacco leaves. The optimal dosages were determined as 78 U/g of neutral protease, 82 U/g of glucoamylase and 59 U/g of polyphenol oxidase. All treatments enhanced the sensory quality of tobacco leaves. T4 obtained the highest overall sensory score, as it maintained an optimal balance between enriched desirable aromas and decreased irritating substances. These key proteins Nta23g19450, Nta20g06770, Nta18g02140 and Nta17g02240 modulated phenylpropanoid biosynthesis and tyrosine metabolism, thereby altering the accumulation of caramelized-sweet, woody, floral, and fruity aroma volatiles such as β-damascenone, 2-ethyl-3,5-dimethylpyrazine and geranylacetone. These changes shape the unique flavor profile of cigar tobacco leaves. This study provides a theoretical basis for promoting the fermentation quality of cigar tobacco leaves via compound enzyme and low-oxygen co-fermentation.