A protective yeast is supposed to suppress unwanted microbes—but here, it worked against one Hanseniaspora species and failed against another. The clue points not to oxygen, but to a race for nitrogen resources. Bioprotection has been proposed as a biological alternative to the antimicrobial action of sulfur dioxide, or SO2, to limit or prevent microbial alteration in grape musts.

This study analyzes the effect of a bioprotective Metschnikowia pulcherrima strain on one Hanseniaspora valbyensis strain and one Hanseniaspora uvarum strain. The central result is species-dependent: bioprotection was observed only against Hanseniaspora valbyensis at the two lowest concentrations.

At the beginning of winemaking, non-Saccharomyces yeasts are predominant in grape must, and they continue to grow until Saccharomyces cerevisiae takes over alcoholic fermentation. During this early stage, Hanseniaspora populations are frequently reported as having a negative impact on wine organoleptic properties.

Winemakers traditionally add sulfur dioxide to avoid economic losses from microbial spoilage, but consumers increasingly demand chemical-free products. The bioprotection strategy uses selected non-Saccharomyces yeasts at harvest or after pressing to protect grape must against indigenous yeasts.

A main hypothesis is nutrient competition, especially for nitrogen resources, because nitrogen is one of the most important nutrients in must. Nitrogen resources are classified as preferential, meaning consumed first, or non-preferential, meaning consumed when preferential resources are limited.

Different yeasts do not necessarily share the same preferential resources, and growth conditions can influence amino-acid consumption order and level. In oenology, nitrogen requirements have rarely been studied for non-Saccharomyces yeasts in non-Saccharomyces interactions involving bioprotection.

Interactions between bioprotective yeasts and indigenous flora had not been elucidated, creating a major gap in understanding bioprotection mechanisms. The bioprotective yeast was Metschnikowia pulcherrima MCR24, while the two apiculate yeasts were Hanseniaspora uvarum strain 3137 and Hanseniaspora valbyensis strain ScS, isolated from grape musts.

The cultures used forty milliliters of synthetic must containing three hundred milligrams of nitrogen per liter, and they were conducted for seventy-two hours at twenty degrees Celsius. Metschnikowia pulcherrima started at five times ten to the fifth colony-forming units per milliliter, while each Hanseniaspora strain was tested at five times ten to the fourth, five times ten to the fifth, and five times ten to the sixth colony-forming units per milliliter.

Each condition was performed in quadruplicate, with samples collected every three hours during the first forty-eight hours and every six hours afterward. Growth kinetics were monitored through colony enumeration on agar plates, using different media for Metschnikowia pulcherrima and the Hanseniaspora strains.

The maximal growth rate was calculated with the RStudio package called Growthrates. Amino-acid concentration was measured through High Pressure Liquid Chromatography for each single-culture replicate. Ammonium content was measured for single and co-cultures using a manual enzymatic kit.

The study measured growth kinetics for each strain in single and co-cultures at three Hanseniaspora inoculation levels. Maximal growth rate and final population were extracted and compared between single culture and co-culture to determine the interaction effect on growth.

Figure one tracks Hanseniaspora uvarum cell concentration over time at three starting concentrations: five times ten to the fourth, five times ten to the fifth, and five times ten to the sixth CFU per milliliter. Green shows the single culture, while orange shows co-culture with Metschnikowia pulcherrima; the curves and error bars represent the growth trajectories and their variability.

The authors use these profiles to extract maximal growth rate and final population, then test whether co-culture changes growth for either strain. Hanseniaspora uvarum showed no significant impact on its maximal growth rate from interaction with Metschnikowia pulcherrima, regardless of initial concentration.

Its maximal population after seventy-two hours was also similar in single and co-cultures at every initial population. In contrast, the maximal population of Metschnikowia pulcherrima was negatively impacted by Hanseniaspora uvarum, with stronger reduction at higher Hanseniaspora concentration.

Table one compares single cultures with co-cultures of Hanseniaspora uvarum strain 3137 and Metschnikowia pulcherrima MCR24 after seventy-two hours at twenty degrees Celsius. It reports both maximal growth rate and maximal population across H. uvarum starting concentrations of five times ten to the fourth, fifth, and sixth CFU per milliliter.

The statistical letters show that H. uvarum growth rate was not significantly affected, while M. pulcherrima showed a significant co-culture difference at the highest H. uvarum inoculation.

Under these conditions, Metschnikowia pulcherrima MCR24 did not protect the must by limiting Hanseniaspora uvarum growth at any initial Hanseniaspora concentration. Instead, Hanseniaspora uvarum negatively affected development of the bioprotective strain.

Figure two compares Hanseniapora valbyensis growth in single culture, shown in green, with co-culture alongside Metschnikowia pulcherrima, shown in orange, across three starting concentrations. The curves indicate that the co-culture effect depends on the initial inoculation level, with the clearest separation at five times ten to the power of four and five times ten to the power of five CFU per milliliter.

This supports the reported reductions in maximum growth rate at those conditions, while no such effect was observed at five times ten to the power of six. Hanseniaspora valbyensis maximal growth rate dropped by thirty-five percent and fifty-five percent in co-culture when its initial concentration was five times ten to the fifth and five times ten to the fourth colony-forming units per milliliter.

At five times ten to the sixth colony-forming units per milliliter, co-culture had no effect on its maximal growth rate. Its maximal population was negatively impacted at every initial concentration, with more than a one-log reduction at the lowest initial concentration.

Table two compares maximal growth rate and maximal population after seventy-two hours at twenty degrees Celsius, when the two yeast species are grown separately or together. For Hanseniaspora valbyensis, co-culture is associated with statistically distinct growth-rate values at initial populations of five times ten to the power of four and five times ten to the power of five CFU per milliliter, while Metschnikowia pulcherrima shows no such distinction in growth rate.

The table also reports population differences, including a statistically distinct co-culture value for M. pulcherrima at five times ten to the power of six CFU per milliliter. The growth results suggest that Metschnikowia pulcherrima has a bioprotective effect that depends on the Hanseniaspora species: it appeared against Hanseniaspora valbyensis but not Hanseniaspora uvarum.

The results need confirmation on other strains belonging to these species, because Hanseniaspora uvarum can remain after alcoholic fermentation even after bioprotection or sulfur dioxide addition during pre-fermentative steps. Real conditions contain more species and strains, creating considerable interaction diversity.

The study investigated nitrogen requirements to determine whether competition for nitrogen could explain the bioprotective effect, especially against Hanseniaspora valbyensis. After seventy-two hours at twenty degrees Celsius, nitrogen resources were not fully consumed by any strain or initial concentration.

Ammonium, cysteine, and glycine were not consumed, while arginine and histidine were only partially consumed. Table three reports the percentage of each amino acid and ammonium consumed after seventy-two hours at twenty degrees Celsius, across three cell densities for each Hanseniaspora strain and one condition for Metschnikowia pulcherrima.

Red bars mark resources consumed by at least fifty percent; examples include isoleucine, leucine, lysine, methionine, and valine, while ammonium consumption remains near zero across the listed conditions. The authors use this profile to investigate whether nitrogen competition could help explain the previously observed bioprotective effect.

Four amino acids— isoleucine, leucine, lysine, and tryptophan—were almost entirely consumed by all three strains in every condition. They were the first to be consumed and became limiting in all conditions, making them common preferential resources. That pattern strongly suggests possible competition for these amino-acid resources during co-cultures.

The bioprotective strain and Hanseniaspora strains had the same preferential amino-acid requirements, but different assimilation kinetics. That competition for nitrogen resources seems linked to the bioprotective effect, although other nutrient competitions may also contribute.

Oxygen was also considered as a naturally available resource in must after pressing, and its consumption could create competition between microorganisms. Hanseniaspora valbyensis had very low oxygen-consumption rates independently of inoculation rate, with a total dissolved-oxygen consumption time of four and a half hours, compared with three hours for Metschnikowia pulcherrima.

Because Hanseniaspora valbyensis required little oxygen, rapid dissolved-oxygen depletion by Metschnikowia pulcherrima in co-culture had no significant effect on its fitness. Therefore, oxygen competition was probably not responsible for limiting Hanseniaspora valbyensis growth.

Table five reports oxygen-consumption parameters for three yeast strains in single culture at twenty degrees Celsius, across specified starting concentrations. It includes maximal consumption speed, T fifty—the time to consume half the dissolved oxygen—and total consumption time, with statistical group letters marking comparisons.

The authors highlight that Hanseniaspora uvarum reached a maximal speed of seven point four one milligrams per liter per hour at five times ten to the power of six CFU per milliliter, while Metschnikowia pulcherrima MCR24 consumed all oxygen in three point sixteen hours. The metabolomic analysis focused on the Metschnikowia pulcherrima and Hanseniaspora valbyensis pair to investigate mechanisms involved in bioprotection.

Untargeted metabolomics provided a global and unbiased view of the medium's chemical composition at a specific time point. The analysis examined seventy-two-hour co-cultures at Hanseniaspora valbyensis initial concentrations of five times ten to the fourth and five times ten to the sixth colony-forming units per milliliter, together with associated single cultures.

Figure three maps the single cultures and co-cultures using principal component analysis of three thousand twenty-eight UHPLC-qToF-MS/MS features. Dimension one explains fifteen point seven percent of the variability and dimension two explains nine point five percent.

The plotted groups occupy distinct regions, with M. pulcherrima single cultures separated from H. valbyensis cultures, while the co-culture triangles show additional positioning by initial H. valbyensis concentration.

This matters because it visualizes broad metabolic differences associated with the interaction. Where bioprotection had the strongest effect, at the lowest Hanseniaspora valbyensis initial concentration, the co-culture metabolic footprint was farther from the Hanseniaspora valbyensis single culture footprint.

Where there was no bioprotective effect, at the highest initial concentration, that separation was not observed in the same way. The analysis then focused on the exo-metabolome of single cultures and co-culture at the lower Hanseniaspora valbyensis concentration. Metschnikowia pulcherrima limited Hanseniaspora valbyensis growth, but not Hanseniaspora uvarum.

Shared preferential amino acids, especially lysine and tryptophan, support competition as part of the explanation, while oxygen competition probably was not responsible.