Formation Mechanism And Control Strategy Of Premature Yeast Flocculation Factor In The Malt

Malt is the primary ingredient in beer brewing,providing the foundation for beverage’s unique flavor and character.However,there is a class of factors in individual malts that leads to premature yeast flocculation（PYF）.PYF is defined as the phenomenon in which yeast settles out of the fermenting wort abnormally early or heavily before reaching full attenuation.This untimely or premature flocculation can lead to serious quality issues,including high residual sugars,low alcohol content and undesirable flavors and low production.Therefore,understanding the mechanism of PYF factor formation is crucial and valuable for malt and beer production enterprises to develop precise control strategies to prevent PYF occurrence at its source.This research identifies Fusarium graminearum as the main microorganism for the production of PYF factors in malt.The composition characteristics and production mechanism of PYF factor were analyzed,and antifungal substances were used to inhibit the growth of Fusarium graminearum during wheat production to eliminate PYF factor in malt.The main findings are as follows:（1）Based on PYF potential analysis of various malts,two PYF-positive malts J6 and J12were selected.The analysis of the surface fungal community structure revealed a significantly higher relative abundance of Aureobasidium,Alternaria,and Fusarium genera in these malts compared to six other PYF negative malts.Additionally,there was a significantly negative correlation with the number of yeast cells during the corresponding wort fermentation process.Ten fungal strains were isolated and identified from PYF positive malt,among which four filamentous fungi,Aureobasidium pullulans MA1,Aspergillus flavus MC1,Fusarium graminearum MH1 and Alternaria tenuissima M3-3 showed a significant impact on production of PYF factors.Fusarium graminearum MH1 exhibited the highest biomass and strongest PYF activity at malting temperature.Small-scale brewing experiments further confirmed that Fusarium graminearum-infected malt had the most pronounced flocculation effect on yeast,identifying Fusarium graminearum as the main microorganism causing the production of PYF factors in malt.（2）The polysaccharide extract obtained from barley husk fermented with Fusarium graminearum MH1 was fractionated.The monosaccharide composition,molecular weight distribution,Zeta potential and minimum concentration of each variable for triggering PYF were analyzed.The high molecular weight components were found as follows:Negative charged polysaccharides with a molecular weight between 10 and 30 k Da,arabinose/xylose ratio of 0.5 were found to induce severe PYF.Preliminary analysis of the mechanism of polysaccharide PYF factors causing premature yeast flocculation revealed that in the presence of PYF factors.The expression of yeast flocculation protein decreased compared to control group.However,there is an affinity between PYF factor and the N terminal of yeast flocculation protein（Lg-FLO1）,and yeast exhibited a higher Zeta potential compared to control group.These results indicate that negatively charged PYF factor may connect with negatively charged yeast cells through Ca²⁺bridges,breaking spatial hindrance,and subsequently leading to the specific binding of the yeast cells flocculating proteins with PYF factor mannose side chains through affinity interactions.（3）The PYF activity of the Fusarium graminearum MH1-fermented barley husk extract was evaluated at different inoculation levels.Higher inoculation levels were found to correspond to increased PYF activity in the extract.Among 214 secreted proteins of Fusarium graminearum MH1,liquid chromatography-tandem mass spectrometry analysis identified 40husk degrading enzymes,including 25 hemicellulases,eight cellulases and seven pectinases.Similar types of husk degrading enzymes were also identified from PYF positive malt husk.Correlation analysis of PYF values of Fusarium graminearum MH1-fermented barley husk extract and the variables in the simulated malting process suggested that xylanase,glucanase and cellulases involved in barley husk degradation were the main factors responsible for the formation of PYF factors.The high arabinoxylan content and high negative Zeta potential in degraded husk products were the main characteristics of PYF factors,and xylanase was confirmed as the main enzyme that causes PYF.The recombinant expression of endo-1,4-β-xylanase,which had the highest relative abundance index in the secreted proteome of Fusarium,produced PYF factors with characteristic features through husk degradation,demonstrating GH11 xylanase family of Fusarium can lead to PYF.（4）A strain of Bacillus velezensis B-3 with strong inhibitory activity against Fusarium graminearum MH1 was isolated and obtained.Its metabolites induced the contraction,distortion and limit the branching of Fusarium mycelia hyphae,resulting in an inhibition rate of 83%.Antifungal active substances were isolated from the fermentation broth of Bacillus velezensis using ammonium sulfate precipitation and semi-preparative HPLC.The minimum inhibitory concentration was determined to be 0.2 mg·m L^-1.In addition,confocal laser scanning microscopy revealed visible abnormalities in the morphology of F.graminearum hyphae,with a significant portion displaying signs of cell death.An ultra-performance liquid chromatography quadrupole time of flight mass spectrometry analysis indicated that the antifungal substances were composed of lipopeptides,such as iturin A,surfactant B and surfactant C.These lipopeptides effectively suppressed the production of PYF factors in barley husks infected by Fusarium graminearum,thereby keeping Fusarium graminearum at lower level during malting process without affecting other malt qualities.Beer fermentation experiments showed that malt infected with Fusarium graminearum exhibited the slowest fermentation rate,higher residual sugars,incomplete diacetyl reduction and lower alcohol content.After the addition of antifungal substances,the beer fermentation characteristics,conventional physical and chemical indexes and flavor substances showed no significant difference from normal wort.