Strain Kluyveromyces Marxianus Research Articles

Engineering thermotolerant Kluyveromyces marxianus strains for resveratrol production by simultaneous saccharification and fermentation of whole slurry corn cob

The microbial biosynthesis of the antioxidant compound resveratrol offers an eco-friendly and sustainable alternative to chemical synthesis or plant extraction. Here, we showed that Kluyveromyces marxianus strains produce p-coumaric acid, a key precursor of resveratrol, with higher titres achieved under increased agitation conditions. Through further strain engineering, resveratrol production was achieved using glucose, xylose, and/or ethanol as substrates. Xylose emerged as the most favourable carbon source for resveratrol production, with ethanol supplementation during xylose culture resulting in increased resveratrol titres by limiting the accumulation of the by-product xylitol. At 37 ◦C, resveratrol production from corn cob hydrolysate and whole slurry substantially increased the yield of resveratrol per sugar, reaching titres of up to 69.26 mg/L. This work shows, for the first time, resveratrol production by K. marxianus and from corn cob whole slurry, establishing foundations for the development of an integrated sustainable process for resveratrol production from lignocellulosic materials.

Optimisation of coumaric acid production from aromatic amino acids in Kluyveromyces marxianus

Yeasts are attractive hosts for the production of heterologous products due to their genetic tractability and relative ease of growth. While the baker’s yeast Saccharomyces cerevisiae is a powerful workhorse of the biotechnology industry, the species has metabolic limitations and it is critical that we develop alternative platforms that will facilitate the development of bioprocesses that rely on sustainable feedstocks. In this study, we used synthetic biology tools to construct coumaric acid–producing strains of Kluyveromyces marxianus, a yeast whose physiological traits render it attractive for biotechnology applications. Coumaric acid is a building block in the synthesis of many different families of aromatics and is a key precursor for the synthesis of complect phenylpropanoid molecules, including many flavours and aromas. The starting point for this work was a K. marxianus chassis strain that has increased flux towards the synthesis of tyrosine and phenylalanine, the aromatic amino acids that can serve as starting points for coumaric acid synthesis. Following principles of synthetic biology, a modular approach was taken to identify the best solution to different metabolic possibilities and these were then combined in different ways. For the first step, it was established that the route from phenylalanine was superior to that from tyrosine and the combined overexpression of PlPAL, AtC4H and AtCPR1 delivered the highest yield of coumaric acid. Next, it was established that while Pdc5 and Aro10 both had phenylpyruvate decarboxylase activity, inactivation of ARO10 was sufficient to prevent flux loss in the pathway. Since phenylalanine is the starting point, efforts were made to improve efficiency of its production. It was found that glutamate was a preferred nitrogen source for coumaric acid production, and this knowledge was used to engineer a strain that overexpressed S. cerevisiae GDH1 and delivered higher yields of coumaric acid. Ultimately, this strategy led to the development of strains that has yields of up to 48mg coumaric acid /g glucose. Strains were evaluated in bioreactors to investigate the effects of different process parameters. These analyses indicated that engineered strains face some redox balance challenges and further work will be required overcome these to develop strains that can perform well under industrial conditions.

Process scale-up simulation and techno-economic assessment of ethanol fermentation from cheese whey.

Production of cheese whey in the EU exceeded 55 million tons in 2022, resulting in lactose-rich effluents that pose significant environmental challenges. To address this issue, the present study investigated cheese-whey treatment via membrane filtration and the utilization of its components as fermentation feedstock. A simulation model was developed for an industrial-scale facility located in Italy's Apulia region, designed to process 539m3/day of untreated cheese-whey. The model integrated experimental data from ethanolic fermentation using a selected strain of Kluyveromyces marxianus in lactose-supplemented media, along with relevant published data. The simulation was divided into three different sections. The first section focused on cheese-whey pretreatment through membrane filtration, enabling the recovery of 56%w/w whey protein concentrate, process water recirculation, and lactose concentration. In the second section, the recovered lactose was directed towards fermentation and downstream anhydrous ethanol production. The third section encompassed anaerobic digestion of organic residue, sludge handling, and combined heat and power production. Moreover, three different scenarios were produced based on ethanol yield on lactose (YE/L), biomass yield on lactose, and final lactose concentration in the medium. A techno-economic assessment based on the collected data was performed as well as a sensitivity analysis focused on economic parameters, encompassing considerations on cheese-whey by assessing its economical impact as a credit for the simulated facility, dictated by a gate fee, or as a cost by considering it a raw material. The techno-economic analysis revealed different minimum ethanol selling prices across the three scenarios. The best performance was obtained in the scenario presenting a YE/L = 0.45g/g, with a minimum selling price of 1.43 €/kg. Finally, sensitivity analysis highlighted the model's dependence on the price or credit associated with cheese-whey handling. This work highlighted the importance of policy implementation in this kind of study, demonstrating how a gate fee approach applied to cheese-whey procurement positively impacted the final minimum selling price for ethanol across all scenarios. Additionally, considerations should be made about the implementation of the simulated process as a plug-in addition in to existing processes dealing with dairy products or handling multiple biomasses to produce ethanol.

Single-Cell Protein and Ethanol Production of a Newly Isolated Kluyveromyces marxianus Strain through Cheese Whey Valorization.

The present work examined the production of single-cell protein (SCP) by a newly isolated strain of Kluyveromyces marxianus EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors' knowledge, this is the first report examining the ability of Kluyveromyces marxianus species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain's growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of K. marxianus strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0-100.0 g/L) led to increased ethanol production (Ethmax = 35.5 ± 0.2 g/L), suggesting that K. marxianus strain EXF-5288 is "Crabtree-positive". Batch-bioreactor trials shifted the strain's metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly, K. marxianus strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile.

Coupling thermotolerance and high production of recombinant protein by CYR1N1546K mutation via cAMP signaling cascades

In recombinant protein-producing yeast strains, cells experience high production-related stresses similar to high temperatures. It is possible to increase recombinant protein production by enhancing thermotolerance, but few studies have focused on this topic. Here we aim to identify cellular regulators that can simultaneously activate thermotolerance and high yield of recombinant protein. Through screening at 46 °C, a heat-resistant Kluyveromyces marxianus (K. marxianus) strain FDHY23 is isolated. It also exhibits enhanced recombinant protein productivity at both 30 °C and high temperatures. The CYR1N1546K mutation is identified as responsible for FDHY23’s improved phenotype, characterized by weakened adenylate cyclase activity and reduced cAMP production. Introducing this mutation into the wild-type strain greatly enhances both thermotolerance and recombinant protein yields. RNA-seq analysis reveals that under high temperature and recombinant protein production conditions, CYR1 mutation-induced reduction in cAMP levels can stimulate cells to improve its energy supply system and optimize material synthesis, meanwhile enhance stress resistance, based on the altered cAMP signaling cascades. Our study provides CYR1 mutation as a novel target to overcome the bottleneck in achieving high production of recombinant proteins under high temperature conditions, and also offers a convenient approach for high-throughput screening of recombinant proteins with high yields.

Obtention of pelleted Kluyveromyces marxianus CIDCA 9121 with immunomodulatory properties: Selection of protective agents using Plackett–Burman and Box–Behnken experimental designs

Probiotic yeasts are increasingly used in different animal production systems. Supply of feeding in the form of pellets provides productive advantages in several systems such as poultry production or aquaculture. The inclusion of viable yeast in the pellets has to overcome heat stress during palletization and also dehydration stress. We have worked with our previously characterized probiotic strain Kluyveromyces marxianus CIDCA 9121. We evaluated the resistance of this strain to a process dehydration and extrusion after pellet generation, conditions that were not analyzed for this genus of probiotic yeast. We determined the effects of different thermoprotective agents in order to maximize yeast viability over time and preservation of its probiotic traits. To this aim we used the single factor optimization method, Plackett-Burman central compound rotating design (P–B CCRD) and response surface methodology (RSM) for optimize survival conditions in a process performed at laboratory scale. We selected a combination of sucrose, maltose and maltodextrin and we have tested two different combinations of these agents in a pilot plant extrusion/dehydration process showing that one of them could increase yeast viability. Remarkably, we could observe that immunomodulatory capacity of this strain was not affected after dehydration in the presence of the different additives tested and subsequent rehydration, being the first report showing conservation of probiotic traits of K. marxianus strain upon pelleting opening new perspectives for product development.

Fractional mixture design: a novel design and methodology for analyzing and optimizing mixtures of several ingredients

Mixture designs are employed to systematically change the composition of mixtures and investigate how those changes impact their properties. However, all mixture designs currently available are impractical for analyzing mixtures with relatively large numbers of ingredients. In response, this article presents a novel solution that builds on the construction of a new experimental design called “fractional mixture design”. The design involves screening the ingredients in mixtures and enables the subsequent construction of a classical mixture design for optimizing mixtures. The design and its accompanying methodology were developed to analyze native strains found in successful spontaneous fermentations with the goal of constructing a mixed starter culture to transition from spontaneous to directed fermentation in the production of agave distillates. The results showed that a starter culture composed of the native strains Kluyveromyces marxianus, Clavispora lusitaniae, and Kluyveromyces marxianus var. drosophilarum, in respective proportions of 35%, 32%, and 33%, enabled the production of a fermented product with 2.1% alcohol and a broad profile of aromatic compounds. Hence, the results show, for the first time, a tool that addresses the technical challenge that allows studying a relatively large number of ingredients in mixtures and a two-stage sequential methodology to construct optimal mixtures.

Differences in the management of intracellular redox state between wine yeast species dictate their fermentation performances and metabolite production

The maintenance of the balance between oxidised and reduced redox cofactors is essential for the functioning of many cellular processes in all living organisms. While the electron transport chain plays a key role in maintaining this balance under respiratory conditions, its inactivity in the absence of oxygen poses a challenge that yeasts such as Saccharomyces cerevisiae overcome through the production of various metabolic end-products during alcoholic fermentation. In this study, we investigated the diversity occurring between wine yeast species in their management of redox balance and its consequences on the fermentation performances and the formation of metabolites. To this aim, we quantified the changes in NAD(H) and NADP(H) concentrations and redox status throughout the fermentation of 6 wine yeast species. While the availability of NADP and NADPH remained balanced and stable throughout the process for all the strains, important differences between species were observed in the dynamics of NAD and NADH intracellular pools. A comparative analysis of these data with the fermentation capacity and metabolic profiles of the strains revealed that Saccharomyces cerevisiae, Torulaspora delbrueckii and Lachancea thermotolerans strains were able to reoxidise NADH to NAD throughout the fermentation, mainly by the formation of glycerol. These species exhibited good fermentation capacities. Conversely, Starmerella bacillaris and Metschnikowia pulcherrima species were unable to regenerate NAD as early as one third of sugars were consumed, explaining at least in part their poor growth and fermentation performances. The Kluyveromyces marxianus strain exhibited a specific behaviour, by maintaining similar levels of NAD and NADH throughout the process. This balance between oxidised and reduced redox cofactors ensured the consumption of a large part of sugars by this species, despite a low fermentation rate. In addition, the dynamics of redox cofactors affected the production of by-products by the various strains either directly or indirectly, through the formation of precursors. Major examples are the increased formation of glycerol by S. bacillaris and M. pulcherrima strains, as a way of trying to reoxidise NADH, and the greater capacity to produce acetate and derived metabolites of yeasts capable of maintaining their redox balance. Overall, this study provided new insight into the contribution of the management of redox status to the orientation of yeast metabolism during fermentation. This information should be taken into account when developing strategies for more efficient and effective fermentation.

Snapshot of the Probiotic Potential of Kluveromyces marxianus DMKU-1042 Using a Comparative Probiogenomics Approach.

Kluyveromyces marxianus is a rapidly growing thermotolerant yeast that secretes a variety of lytic enzymes, utilizes different sugars, and produces ethanol. The probiotic potential of this yeast has not been well explored. To evaluate its probiotic potential, the yeast strain Kluyveromyces marxianus DMKU3-1042 was analyzed using next-generation sequencing technology. Analysis of the genomes showed that the yeast isolates had a GC content of 40.10-40.59%. The isolates had many genes related to glycerol and mannose metabolism, as well as genes for acetoin and butanediol metabolism, acetolactate synthase subunits, and lactic acid fermentation. The strain isolates were also found to possess genes for the synthesis of different vitamins and Coenzyme A. Genes related to heat and hyperosmotic shock tolerance, as well as protection against reactive oxygen species were also found. Additionally, the isolates contained genes for the synthesis of lysine, threonine, methionine, and cysteine, as well as genes with anticoagulation and anti-inflammatory properties. Based on our analysis, we concluded that the strain DMKU3-1042 possesses probiotic properties that make it suitable for use in food and feed supplementation.

Ethanol production from Agave salmiana leaf juices by consolidated bioprocessing comparing two strains of Kluyveromyces marxianus

The present work addressed the conversion of Agave salmiana leaf juice to ethanol by consolidated bioprocessing (CBP). CBP was tested at 30, 35, and 40 ºC and compared with a conventional fermentation previously subjecting the juices to an acid-thermal pretreatment. An FTIR-DRIFTS analysis was applied on the functional groups of the juice. Fermentation was evaluated by comparing the strains K. marxianus OFF1 and K. marxianus TX4; the latter was isolated in this work from the sap of A. salmiana. In results, the optimal condition pretreatment (H2SO4 0.65% (v/v), 15.7 min, 88 °C) increased the reducing sugars concentration up to 45.15 g/L. FTIR-DRIFTS showed higher peaks of functional groups related to fructose and glucose in the pretreated juice vs the raw. Both strains reached enzymatic activities of up to 2.26 U/mL growing in raw A. salmiana juice. Conventional fermentation showed a similar behavior with both strains, with reducing sugar consumption efficiencies of 98%, 16 g ethanol/L, corresponding to yields of 77%. CBP at different temperatures, showed significant differences, registering the highest ethanol production (22 g/L) and yield (99%) at 40 ºC. The native strain showed a fermentation behavior similar to that of K. marxianus OFF1. Theoretical annual ethanol yields obtained from the results (3828 L/ha/year) were compared to those obtained from other biomass juices such as sweet sorghum and sugarcane bagasse. CBP showed high efficiency in the hydrolysis of polysaccharides from Agave salmiana juice and their conversion to ethanol.

Bioethanol production from deproteinized cheese whey powder by local strain isolated from soured milk: influence of operating parameters

ABSTRACT In this paper, a local isolated yeast strain was studied for bioethanol production using protein-free cheese whey powder (CWP) in batch alcoholic fermentation experiments. Ethanol tolerance tests were performed with different ethanol concentrations (from 0% to 20%). The maximum ethanol tolerance obtained for our strain was 5% (v/v). The influence of initial lactose concentration, temperature and pH on fermentation kinetic was studied. For initial lactose concentration ranging from 57.5 to 260 gL−1, the highest ethanol production 34.8 gL−1 was obtained at 109 gL−1 initial lactose concentration corresponding to 0.413 gg−1 ethanol yield (YP/S); (While it was noticed less ethanol production, namely 0.178 g g−1) at a higher initial whey sugar concentration of 260 gL−1. The effect of the initial pH on the fermentation was studied at three different values: 3, 4 and 5. The ethanol production reached a maximum of 22 g L−1 with a lactose consumption of 77.62% at pH 4. However, an observed decrease occurred in ethanol production and lactose consumption at pH 3, with values of 10.34 gL-1 and 24.46%, respectively. For the fermentation conducted at the three temperatures of 37°C, 40°C and 43°C, the maximum ethanol production and lactose consumption were 34.82 gL-1 and 80.75%, respectively, achieved at 37°C. The experimental data was analyzed using the Gompertz model to obtain the following kinetic parameters: the maximum ethanol concentration (Pm) was found to be 36.97 gL-1, the maximum ethanol production rate (rp,m) was 0.402 gL−1h−1 and the lag phase (tl) was 10.8 h. The comparison of the isolated Kluyveromyces marxianus TXID4911 strain with the reference strain Kluyveromyces marxianus DSM 5421, showed a relatively close ethanol yield (YP/S) 0.415 and 0.505 gg,−1 respectively.

The interaction among Kluyveromyces marxianus G-Y4, Lacticaseibacillus paracasei GL1, and Lactobacillus helveticus SNA12 and signaling molecule AI-2 participate in the formation of biofilm

In this study, two strains of lactic acid bacteria (Lacticaseibacillus paracasei GL1 and Lactobacillus helveticus SNA12) and one yeast strain of Kluyveromyces marxianus G-Y4 (G-Y4) isolated from Tibetan kefir grains were co-cultured. It was found that the addition of G-Y4 could not only promote the growth of lactic acid bacteria, but also increase the release of metabolites (lactic acid, ethanol, and amino nitrogen). Furthermore, the addition of live cells and cell-free fermentation supernatant (CFS) of G-Y4 could increase the ability of biofilm formation. Morever, the surface characteristics results showed that the addition of G-Y4 live cells could enhance the aggregation ability and hydrophobicity of LAB. Meanwhile, adding live cells and CFS of G-Y4 could promote the release of signaling molecule AI-2 and enhance the expression of the LuxS gene related to biofilm formation. In addition, Fourier-transform infrared spectroscopy and chemical composition analysis were used to investigate the composition of the biofilm, and the results indicated that the biofilm was mainly composed of a small amount of protein but it was rich in polysaccharides including glucose, galactose, and mannose with different ratios. Finally, the formation of biofilm could delay the decline of the number of viable bacteria in storage fermented milk.

Inhibitory effects of epiphytic Kluyveromyces marxianus from Indian senna (Cassia angustifolia Vahl.) on growth and aflatoxin production of Aspergillus flavus

Aspergillus flavus infection and subsequent aflatoxin contamination are considered the major constraints in senna (Cassia angustifolia Vahl.) export. Using native epiphytic yeast to control phytopathogens is a successful strategy for managing plant diseases. In the present investigation, we exploited the antagonistic potential of epiphytic yeast isolates obtained from senna against A. flavus growth and aflatoxin B1 (AFB1) production. Four Kluyveromyces marxianus strains (YSL3, YSL16, YSP12, and YSF9) exhibited vigorous antagonistic activity with a maximum inhibition of 64 %. In vivo evaluation of senna pods showed that K. marxianus strains effectively reduced A. flavus colonization with a population range of 5.87 to 7.08 log10 CFU/g. In contrast, the untreated senna pods were found to have severe fungal colonization with a population of 7.84 log10 CFU/g. In addition, HPLC analysis showed that aflatoxin B1 in senna pods was drastically reduced upon yeast treatment up to 14 DAI. Furthermore, we demonstrated the antifungal action mechanisms of K. marxianus, such as surface colonizing ability on pods, production of antifungal volatiles (VOCs), siderophores, extracellular lytic enzymes, and cell wall binding ability to AFB1. All four strains of K. marxianus showed rapid colonization on the senna pod, and YSP12 reached the maximum population of 7.18 log10 CFU/pod at 9 days after inoculation (DAI). The exposure of A. flavus to K. marxianus VOCs significantly reduced the growth by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Scanning electron microscopic images demonstrated severe mycelial damage and hyphal deformities of A. flavus. In addition, yeast VOCs can reduce aflatoxin biosynthesis in A. flavus by up to 99 and 93.2 % at 7 and 14 DAI, respectively. Gas chromatography–mass spectrometry analysis confirmed the presence of antimicrobial compounds such as dimethyl trisulfide, ethyl acetate, ethanol, 3-methyl butanal, 2-methyl-1-butanol, and 3-methyl-1-butanol in the volatiles. K. marxianus strains produced siderophores and hydrolytic enzymes such as chitinase and β-1,3-glucanase. A higher AFB1 binding ability was observed in the heat-killed cells (47.5 to 70.65 %) than in the viable cells (43.16 to 60.98 %) of K. marxianus. The current study demonstrated that epiphytic K. marxianus isolated from senna could be a successful biocontrol source to reduce aflatoxin contamination in senna pods.

Screening of yeast co‐culture using crude hydrolysate for co‐fermentation of pentose and hexose

AbstractThe use of all sugars from biomass fractionation – that is, hexoses and pentoses – is essential to develop feasible processes in a biorefinery context. Hexoses are converted into different products by most microorganisms. The main issue is the conversion of pentoses, especially when mixed with hexoses. Improving sugar conversion in co‐fermentation is a major challenge, which was explored in this work. Co‐culture using Saccharomyces cerevisiae and evolved Kluyveromyces marxianus strains was evaluated by screening using 24 deep‐well plates. Glucose depletion and 83% of xylose conversion were achieved with supplemented hemicellulosic hydrolysate medium (medium volume corresponding to 2/5 of deep‐well volume), and initial cell concentrations of 2.5 g L‐1 for each yeast, incubated at 150 rpm. The screening strategy provided reliable data that confirmed the co‐culture as a promising approach for the co‐assimilation of biomass‐derived sugars. The importance of aeration control for effective xylose assimilation was also pointed out.

Enhancement of galactose uptake for bioethanol production from Eucheuma denticulatum hydrolysate using galactose-adapted yeasts.

Eucheuma denticulatum is a red macroalgae with a high carbohydrate content. The fermentable sugars from E. denticulatum were obtained through sequential thermal acid hydrolysis, enzymatic saccharification, and detoxification. Thermal acid hydrolysis of E. denticulatum was optimized under the condition of 10% (w/v) slurry content and 300mM HNO3 at 121℃ for 90min. The maximum monosaccharide concentration after thermal acid hydrolysis was 31.0g/L with an efficiency (ETAH) of 44.7%. By further enzymatic hydrolysis of pretreated biomass solution under 20 U/mL Cellic CTec2 at 50℃ and 160rpm for 72h, the maximum monosaccharide concentration reached 79.9g/L with an efficiency of 66.2% (ES). To remove 5-hydroxymethylfurfural (5-HMF), a fermentation inhibitor, absorption using 2% activated carbon was performed for 2min. Ethanol fermentation was performed using wild-type and high galactose-adapted strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, and Candida lusitaniae. As a result, galactose-adapted strains showed higher ethanol production than wild-type strains. Especially, the fermentation result by adaptively evolved S. cerevisiae produced the highest ethanol of 37.6g/L and with YEtOH of 0.48g/g. Moreover, the transcript level of MIG1 in the galactose-adapted strain was slightly lower than that in the wild-type strain. The application of adaptive evolution of microorganisms was efficient for bioethanol production.

Isolation, production and optimisation of beta-galactosidase by utilizing yeasts isolated from selected dairy products

β-galactosidase is an enzyme that converts lactose into glucose and galactose. It alleviates the issue associated with intolerance to lactose and pollution caused as a result of milk by-products (whey). This study aimed to isolate and select yeast strains that can produce β-galactosidase from fresh milk, yoghurt and locally made cheese. A total of 115 yeasts were isolated from the samples, 9 yeast isolates had the ability to produce β-galactosidase and three (3) were selected for further analysis. β-galactosidase producers were screened using Ortho-nitrophenyl-β-D-galactopyranoside (ONPG) assay. Kluyveromyces marxianus strain SLDY – 005 produced the highest β-galactosidase. It showed maximum enzyme activity (277U/mL) at temperature 30oC, pH 5.5, after an incubation period of 36 h. Glucose had a decrease in β-galactosidase production while yeast extract and urea were considered appropriate nitrogen sources for the best enzyme synthesis. Crude β-galactosidase produced by Kluyveromyces marxianus SLDY – 005 was purified. The partially purified enzyme after dialysis showed a specific activity of 165.12 U/mL and had a purification fold of 6.02 and yield of 45.91 %. The purified enzyme had an optimum temperature of 40oC and a pH 6.0. Kluyveromyces marxianus strain isolated from local cheese is a potential candidate for the production of βgalactosidase and it could be used for combating the problem of lactose intolerance.

Influence of Different Aggregation States on Volatile Organic Compounds Released by Dairy Kluyveromyces marxianus Strains.

Kluyveromyces marxianus has the ability to contribute to the aroma profile of foods and beverages since it is able to produce several volatile organic compounds (VOCs). In this study, 8 dairy K. marxianus strains, previously selected for their adhesion properties, were tested for VOCs production when grown in different conditions: planktonic, biofilm-detached, and MATS forming-cells. It was shown that biofilm-detached cells were mainly able to produce higher alcohols (64.57 mg/L), while esters were mainly produced by planktonic and MATS forming-cells (117.86 and 94.90 mg/L, respectively). Moreover, K. marxianus biofilm-detached cells were able to produce VOCs with flavor and odor impacts, such as ketons, phenols, and terpenes, which were not produced by planktonic cells. In addition, specific unique compounds were associated to the different conditions tested. Biofilm-detached cells were characterized by the production of 9 unique compounds, while planktonic and MATS forming-cells by 7 and 12, respectively. The obtained results should be exploited to modulate the volatilome of foods and beverages and improve the production of certain compounds at the industrial level. Further studies will be carried out to better understand the genetic mechanisms underlying the metabolic pathways activated under different conditions.

Valorisation of whey for fermented beverage production using functional starter yeast

Abstract Indigenous yeast strains Kluyveromyces marxianus (MH6), K. marxianus (CH1), and Saccharomyces cerevisiae (C1) were screened for whey beverage production. K. marxianus (MH6) showed significantly higher (P < 0.05) fermentation efficiency (15.2%) as compare to other yeast strains. The conditions optimised for whey fermentation were 16 Brix, pH 5.5, 28 °C, and 72 h without agitation. For fermented whey beverage production, fruits viz., kinnow (Daizy), guava (Allahabad safeda), and mango (Safeda) were blended with whey at different ratios viz., 80:20, 70:30, 60:40, and 50:50. All ratios showed significant differences for biochemical and sensory analysis (P < 0.05), out of which ratios 60:40, 70:30, and 60:40 for whey kinnow, whey mango, and whey guava, respectively, were selected. To enhance the flavour of whey beverage, flavouring agents (cinnamon, cardamom, fennel seeds, and apple essence) were added. A panel of judges assessed all whey beverages on a hedonic scale basis, and cardamom whey guava beverage received the highest score of 8.16. The whey beverages were stored under refrigerated conditions after pasteurisation, and the shelf life was assessed to be 15 days. This study conferred that K. marxianus held the potential for fermented whey fruit blend beverages production and these beverages could be an alternative healthy refreshing substitute for synthetic bottled fruit beverages.

Selection of Yeast and Lactic Acid Bacteria Strains, Isolated from Spontaneous Raw Milk Fermentation, for the Production of a Potential Probiotic Fermented Milk

Probiotic milk is a class of fermented milk that possesses health-promoting effects, not only due to the lactic acid bacteria (LAB) presence but potentially also to yeast activity. Hence, the aim of this work was to isolate and select yeasts from spontaneous milk fermentations to be used as inoculum, together with LAB, for manufacturing a potentially probiotic acidic low-alcohol fermented milk. Six yeast species were detected from the spontaneous milk fermentation. A screening of 13 yeast strains and 14 previously isolated LAB strains, based on the resistance to bile salts and to acidic conditions, was carried out. The best performing strains were successively tested for in vitro gastrointestinal tolerance. A strain of Kluyveromyces marxianus and a strain of Lactococcus lactis were selected for the manufacturing of two different fermented milk. The values of the main technological and microbiological parameters (pH, organic acids, ethanol, and microbial concentrations) of the experimental milk were in the range of those reported for this category of products. The evaluation of microorganism survival in fermented milk samples subjected to simulated gastrointestinal conditions highlighted a high resistance of both strains. In conclusion, the selected microbial starter culture enabled the setting up of potential probiotic fermented milk.

Spoilage Potential of Contaminating Yeast Species Kluyveromyces marxianus, Pichia kudriavzevii and Torulaspora delbrueckii during Cold Storage of Skyr.

This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.