Characterization of Yeasts Isolated from Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization

Whey is the main byproduct of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast population inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. PCR-RFLP assay of ribosomal ITS regions and phylogenetic analysis of 26S rDNA D1/D2 domains showed that PR NWS yeast population consists of the well-documented Kluyveromyces marxianus, as well as of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii), with multiple biotypes scored within each species as demonstrated by (GTG)5-based MSP-PCR. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation trials, one thermotolerant strain was suitable to release ethanol with a fermentation efficiency of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

Characterization of Yeasts Isolated From Parmigiano Reggiano Cheese Natural Whey Starter: From Spoilage Agents to Potential Cell Factories for Whey Valorization

Whey is the main by-product of the dairy industry and contains sugars (lactose) and proteins (especially serum proteins and, at lesser extent, residual caseins), which can be valorized by the fermentative action of yeasts. In the present study, we characterized the spoilage yeast fraction inhabiting natural whey starter (NWS), the undefined starter culture of thermophilic lactic acid bacteria used in Parmigiano Reggiano (PR) cheesemaking, and evaluated thermotolerance, mating type, and the aptitude to produce ethanol and bioactive peptides from whey lactose and proteins, respectively, in a selected pool of strains. We found that PR NWS yeast population consists of other species (Saccharomyces cerevisiae, Wickerhamiella pararugosa, and Torulaspora delbrueckii) in addition to the well-documented Kluyveromyces marxianus, with multiple biotypes scored within each species. Haploid and diploid K. marxianus strains were identified through MAT genotyping, while thermotolerance assay allowed the selection of strains suitable to grow up to 48 °C. In whey fermentation assay, one thermotolerant strain was suitable to release ethanol with yield of 86.5%, while another candidate was able to produce the highest amounts of both ethanol and bioactive peptides with potentially anti-hypertensive function. The present work demonstrated that PR NWS is a reservoir of ethanol and bioactive peptides producer yeasts, which can be exploited to valorize whey, in agreement with the principles of circularity and sustainability.

Saccharomyces Cerevisiae Strains Selected from Nature Significantly Increased the Production of 2-Phenylethanol Through Protoplast Fusion

Background: 2-Phenylethanol (2-PE) is an aromatic alcohol with rose fragrance, which is widely used as an additive in food, tobacco and daily chemical industries. Yeast is the main microorganism producing natural 2-PE, but it is limited by low production and weak tolerance. Nature and fermented products is a resource treasury of yeasts with excellent traits. Screening strains with good phenotypic traits and conducting breeding by cell fusion for genetic pyramiding is an effective way to improve strains. Results: In this study, 25 strains of 2-PE-producing yeasts were isolated from Chinese brewed samples. Three Saccharomyces cerevisiae strains with good traits in tolerance and 2-PE titre were screened out. The strain LSC-1 produces 2-PE of 3.41 g/L with an increase of 9.3% compared to the industrial strain CWY132. The strain NGER shows good tolerance to 2-PE at the concentration of 3.60 g/L in agar plate, and the thermotolerant strain S.C-1 shows growth ability at 41℃. Two rounds of protoplast fusion were performed with these three parent strains for pyramiding of traits. A fusant strain RH2-16 with high 2-PE titre and increased tolerance was obtained. Using 5g/L L-phenylalanine as the precursor substrate, the maximum titre of 2-PE produced by the RH2-16 strain through fermentation and transformation is 4.31 g/L, and the average titre is 4.04 g/L. The molar conversion rate of L-Phe reached 115% in 36 h. Compared to the parental strain LSC-1 and the industrial strain CWY132, 2-PE titre in RH2-16 increased by 26.4% and 38.1%, respectively.Conclusion: Diversified S. cerevisiae strains with different traits can be isolated from the brewing related samples. Protoplast fusion technology can effectively pyramid excellent genetic traits and breed yeast strains with significantly improved tolerance and 2-PE titre. Our research provided a breeding strategy for S. cerevisiae and a strain for industrial production of 2-PE.

Saccharomyces Cerevisiae Strains Selected From Nature Significantly Increased the Production of 2-Phenylethanol Through Protoplast Fusion

Background: 2-Phenylethanol (2-PE) is an aromatic alcohol with rose fragrance, which is widely used as an additive in food, tobacco and daily chemical industries. Yeast is the main microorganism producing natural 2-PE, but it is limited by low yield and weak tolerance. Nature and fermented products is a resource treasury of yeasts with excellent traits. Screening strains with good phenotypic traits and conducting breeding by cell fusion for genetic pyramiding is an effective way to improve strains. Results: In this study, 25 strains of 2-PE-producing yeasts were isolated from Chinese brewed samples. Three Saccharomyces cerevisiae strains with good traits in tolerance and 2-PE yield were screened out. The strain LSC-1 produces 2-PE of 3.41 g/L with an increase of 9.3% compared to the industrial strain CWY132. The strain NGER shows good tolerance to 2-PE at the concentration of 3.60 g/L in agar plate, and the thermotolerant strain S.C-1 shows growth ability at 41℃. Two rounds of protoplast fusion were performed with these three parent strains for pyramiding of traits. A fusion strain RH2-16 with high 2-PE yield and increased tolerance was obtained. Using 5 g/L L-phenylalanine as the precursor, RH2-16 produced 2-PE of 4.31 g/L through fermentation conversion and the molar conversion rate of L-Phe reached 115% in 36 h. Compared to the yield of the parental strain LSC-1 and the industrial strain CWY132, 2-PE in RH2-16 increased by 26.4% and 38.1%, respectively. Overexpression of the key enzyme genes ARO8, ARO10, and ADH2 in the Ehrlich pathway in RH2-16 did not increase 2-PE production.Conclusion: Diversified S.cerevisiae strains with different traits can be isolated from the brewing related samples. Protoplast fusion technology can effectively pyramid excellent genetic traits and breed yeast strains with significantly improved tolerance and 2-PE yield. Our research provided a breeding strategy for S.cerevisiae and a strain for industrial production of 2-PE. Overexpression of the key enzyme genes in 2-PE synthesis pathway does not necessarily improve increase production.

Physiological characterization of a new thermotolerant yeast strain isolated during Brazilian ethanol production, and its application in high-temperature fermentation

Background The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This characteristic could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. Results We identified and characterized the physiology of a new thermotolerant strain (LBGA-01) able to ferment at 40 °C, which is more resistant to stressors as sucrose, furfural and ethanol than CAT-1 industrial strain. Furthermore, this strain showed similar CAT-1 resistance to acetic acid and lactic acid, and it was also able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). Genes related to the production of proteins involved in secondary products of fermentation were also differentially regulated at 40 °C, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-coenzyme A synthetase 2 (ACS2). Fermentation tests using chemostats showed that LBGA-01 had an excellent performance in ethanol production in high temperature. Conclusion The thermotolerant LBGA-01 strain modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its resistance to high concentration of ethanol, sugar, lactic acid, acetic acid, and furfural. Results indicate that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Physiological characterization of a new thermotolerant yeast strain isolated during Brazilian ethanol production and its application in high temperature fermentation

Background The use of thermotolerant yeast strains can improve the efficiency of ethanol fermentation, allowing fermentation to occur at temperatures higher than 40 °C. This increment in temperature could benefit traditional bio-ethanol production and allow simultaneous saccharification and fermentation (SSF) of starch or lignocellulosic biomass. Results We identified and characterized the physiology of a new thermotolerant strain able to fermentate at 40 °C while producing high yields of ethanol. Our results showed that, in comparison to the industrial yeast CAT-1, our strain was more resistant to various stressors generated during the production of first- and second-generation ethanol, and it also was able to change the pattern of genes involved in sucrose assimilation (SUC2 and AGT1). The formation of secondary products of fermentation was different at 40ºC, with reduced expression of genes involved in the formation of glycerol (GPD2), acetate (ALD6 and ALD4), and acetyl-CoA (ACS2). Conclusion The LBGA-01 strain is a thermotolerant strain that modulates the production of key genes, changing metabolic pathways during high-temperature fermentation, and increasing its tolerance to the high concentration of ethanol, sugar, acetic lactic, acetic acid, furfural and HMF. This indicates that this strain can be used to improve first- and second-generation ethanol production in Brazil.

Ethanol and H2O2 stresses enhance lipid production in an oleaginous Rhodotorula toruloides thermotolerant mutant L1-1

Stress tolerance is a desired characteristic of yeast strains for industrial applications. Stress tolerance has been well described in Saccharomyces yeasts but has not yet been characterized in oleaginous Rhodotorula yeasts even though they are considered promising platforms for lipid production owing to their outstanding lipogenicity. In a previous study, the thermotolerant strain L1–1 was isolated from R. toruloides DMKU3-TK16 (formerly Rhodosporidium toruloides). In this study, we aimed to further examine the ability of this strain to tolerate other stresses and its lipid productivity under various stress conditions. We found that the L1–1 strain could tolerate not only thermal stress but also oxidative stress (ethanol and H2O2), osmotic stress (glucose) and a cell membrane disturbing reagent (DMSO). Our results also showed that the L1–1 strain exhibited enhanced ability to maintain ROS homeostasis, stronger cell wall strength and increased levels of unsaturated membrane lipids under various stresses. Moreover, we also demonstrated that ethanol-induced stress significantly increased the lipid productivity of the thermotolerant L1–1. The thermotolerant L1–1 was also found to produce a higher lipid titer under the dual ethanol-H2O2 stress than under non-stress conditions. This is the first report to indicate that ethanol stress can induce lipid production in an R. toruloides thermotolerant strain.