Exploitation of Yeasts with Probiotic Traits for Kefir Production: Effectiveness of the Microbial Consortium

Kefir is a fermented milk made by beneficial lactic acid bacteria and yeasts inoculated as grains or free cultures. In this work, five yeast strains with probiotic aptitudes belonging to Candida zeylanoides, Yarrowia lipolytica, Kluyveromyces lactis, and Debaryomyces hansenii species were assessed in a defined consortium, in co-culture with a commercial strain of Lactobacillus casei, in order to evaluate the yeasts’ fermentation performance during kefir production, using different milks. The concentration of each yeast was modulated to obtain a stable consortium that was not negatively affected by the bacteria. Furthermore, all yeasts remained viable for five weeks at 4 °C, reaching about 8.00 Log CFU in 150 mL of kefir, a volume corresponding to a pot of a commercial product. The yeasts consortium showed a suitable fermentation performance in all milks, conferring peculiar and distinctive analytical and aromatic properties to the kefirs, confirmed by a pleasant taste. Overall, the panel test revealed that the cow’s and sheep’s kefir were more appreciated than the others; this evaluation was supported by a distinctive fermentation by-products’ content that positively influences the final aroma, conferring to the kefir exalted taste and complexity. These results allow us to propose the yeasts consortium as a versatile and promising multistarter candidate able to affect industrial kefir with both recognizable organoleptic properties and probiotic aptitudes.

Applicability of Saccharomyces cerevisiae Strains for the Production of Fruit Wines Using Cocoa Honey Complemented with Cocoa Pulp

Research background. Cocoa honey (CH) and cocoa pulp (CP) are both fruit pulps highly appreciated but, until now, CH is less processed than CP. In this work, it was investigated the applicability of strains of S. cerevisiae to ferment CH complemented with CP, to obtain fruit wines and improve CH commercialization. Experimental approach. The selection of a strain, previously isolated from cachaçaria distilleries in Brazil, took place based on its fermentation performance. The conditions for fermentation with S. cerevisiae L63 were then studied in relation to: volumetric proportion (φCH) of CH (complemented with CP), sucrose addition (γsuc), temperature (T) and inoculum size (No). The best conditions were applied in order to obtain fermentation profiles. Results and conclusions. S. cerevisiae L63 (No=107–108 cell/mL) is capable to ferment φCH of 90 and 80 % (V/V) for 24 or 48 h with γsuc of 50 and 100 g/L at T=28–30 °C resulting in wines with ethanol contents from 8 to 14 % (V/V). Additionally, the φCH=90 % (V/V) wine resulted in the lowest residual sugar concentration (<35 g/L) than the φCH=80 % (V/V) wine (~79 g/L) which could be classified as a sweet wine. In general, S. cerevisiae L63 resulted in a similar fermentation performance than a commercial strain tested, indicating its potential for fruit pulp fermentation. Novelty and scientific contribution. Therefore, S. cerevisiae L63 is capable to ferment CH complemented with CP to produce fruit wines with good commercial potentials that may also benefit small cocoa producers by presenting a product with greater added value.

Recycling and Conversion of Yeasts into Organic Nitrogen Sources for Wine Fermentation: Effects on Molecular and Sensory Attributes

Organic nitrogen plays a significant role in the fermentation performance and production of esters and higher alcohols. This study assessed the use of yeast protein hydrolysate (YPH) as a nitrogen source for grape must fermentation. In this study, we prepared an enzymatic protein hydrolysate using yeasts recovered from a previous fermentation of wine. Three treatments were performed. DAP supplementation was used as a control, while two YPH treatments were used. Low (LDH) and high degrees of hydrolysis (HDH), 3.5% and 10%, respectively, were chosen. Gas chromatography and principal component analysis indicated a significant positive influence of YPH-supplementations on the production of esters and higher alcohols. Significantly high concentrations of 3-methyl-1-penthanol, isoamyl alcohol, isobutanol, and 2-phenylethanol were observed. Significant odorant activity was obtained for 3-methyl-1-pentanol and ethyl-2-hexenoate. The use of YPH as nitrogen supplementation is justified as a recycling yeasts technique by the increase in volatile compounds.

The Effect of Sound Frequency and Intensity on Yeast Growth, Fermentation Performance and Volatile Composition of Beer

This study investigated the impact of varying sound conditions (frequency and intensity) on yeast growth, fermentation performance and production of volatile organic compounds (VOCs) in beer. Fermentations were carried out in plastic bags suspended in large water-filled containers fitted with underwater speakers. Ferments were subjected to either 200–800 or 800–2000 Hz at 124 and 140 dB @ 20 µPa. Headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) was used to identify and measure the relative abundance of the VOCs produced. Sound treatment had significant effects on the number of viable yeast cells in suspension at 10 and 24 h (p < 0.05), with control (silence) samples having the highest cell numbers. For wort gravity, there were significant differences between treatments at 24 and 48 h, with the silence control showing the lowest density before all ferments converged to the same final gravity at 140 h. A total of 33 VOCs were identified in the beer samples, including twelve esters, nine alcohols, three acids, three aldehydes, and six hop-derived compounds. Only the abundance of some alcohols showed any consistent response to the sound treatments. These results show that the application of audible sound via underwater transmission to a beer fermentation elicited limited changes to wort gravity and VOCs during fermentation.

Cellulosic ethanol production by consortia of Scheffersomyces stipitis and engineered Zymomonas mobilis

Background As one of the clean and sustainable energies, lignocellulosic ethanol has achieved much attention around the world. The production of lignocellulosic ethanol does not compete with people for food, while the consumption of ethanol could contribute to the carbon dioxide emission reduction. However, the simultaneous transformation of glucose and xylose to ethanol is one of the key technologies for attaining cost-efficient lignocellulosic ethanol production at an industrial scale. Genetic modification of strains and constructing consortia were two approaches to resolve this issue. Compared with strain improvement, the synergistic interaction of consortia in metabolic pathways should be more useful than using each one separately. Results In this study, the consortia consisting of suspended Scheffersomyces stipitis CICC1960 and Zymomonas mobilis 8b were cultivated to successfully depress carbon catabolite repression (CCR) in artificially simulated 80G40XRM. With this strategy, a 5.52% more xylose consumption and a 6.52% higher ethanol titer were achieved by the consortium, in which the inoculation ratio between S. stipitis and Z. mobilis was 1:3, compared with the Z. mobilis 8b mono-fermentation. Subsequently, one copy of the xylose metabolic genes was inserted into the Z. mobilis 8b genome to construct Z. mobilis FR2, leading to the xylose final-consumption amount and ethanol titer improvement by 15.36% and 6.81%, respectively. Finally, various corn stover hydrolysates with different sugar concentrations (glucose and xylose 60, 90, 120 g/L), were used to evaluate the fermentation performance of the consortium consisting of S. stipitis CICC1960 and Z. mobilis FR2. Fermentation results showed that a 1.56–4.59% higher ethanol titer was achieved by the consortium compared with the Z. mobilis FR2 mono-fermentation, and a 46.12–102.14% higher ethanol titer was observed in the consortium fermentation when compared with the S. stipitis CICC1960 mono-fermentation. Furthermore, qRT-PCR analysis of xylose/glucose transporter and other genes responsible for CCR explained the reason why the initial ratio inoculation of 1:3 in artificially simulated 80G40XRM had the best fermentation performance in the consortium. Conclusions The fermentation strategy used in this study, i.e., using a genetically modified consortium, had a superior performance in ethanol production, as compared with the S. stipitis CICC1960 mono-fermentation and the Z. mobilis FR2 mono-fermentation alone. This result showed that this strategy has potential for future lignocellulosic ethanol production.

Experimental Whisky Fermentations: Influence of Wort Pretreatments

In addition to ethanol yield, the production of flavour congeners during fermentation is a major consideration for Scotch whisky producers. Experimental whisky fermentations can provide useful information to the industry, and this is the focus of this paper. This study investigated the impact of wort pretreatments (boiled, autoclaved, filtered) on fermentation performance and flavour development in Scotch whisky distillates as an alternative to freezing wort for storage. Our study showed that no significant sensorial differences were detected in low wines (first distillates), while the chemical compositions showed clear changes in increased levels of esters and higher alcohols in boiled and autoclaved wort. In contrast, filtered wort comprised overall lower levels of congeners. Regarding alcohol yield, all three pretreatments resulted in decreased yields. In practice, the pretreatment of wort prior to fermentation requires additional process operations, while freezing requires large storage units. The pretreatments adopted in this study significantly influence the composition of the malt wort used for experimental whisky fermentations, and this results in a poorer fermentation performance compared with untreated wort. We recommend the use of fresh or frozen wort as the best options for small-scale fermentation trials.

‘TeeBot’: A High Throughput Robotic Fermentation and Sampling System

When fermentation research requires the comparison of many strains or conditions, the major bottleneck is a technical one. Microplate approaches are not able to produce representative fermentative performance due to their inability to truly operate anaerobically, whilst more traditional methods do not facilitate sample density sufficient to assess enough candidates to be considered even medium throughput. Two robotic platforms have been developed that address these technological shortfalls. Both are built on commercially available liquid handling platforms fitted with custom labware. Results are presented detailing fermentation performance as compared to current best practice, i.e., shake flasks fitted with airlocks and sideports. The ‘TeeBot’ is capable sampling from 96 or 384 fermentations in 100 mL or 30 mL volumes, respectively, with airlock sealing and minimal headspace. Sampling and downstream analysis are facilitated by automated liquid handling, use of 96-well sample plate format and temporary cryo-storage (<0 °C).

Lager Yeast Design Through Meiotic Segregation of a Saccharomyces cerevisiae × Saccharomyces eubayanus Hybrid

Yeasts in the lager brewing group are closely related and consequently do not exhibit significant genetic variability. Here, an artificial Saccharomyces cerevisiae × Saccharomyces eubayanus tetraploid interspecies hybrid was created by rare mating, and its ability to sporulate and produce viable gametes was exploited to generate phenotypic diversity. Four spore clones obtained from a single ascus were isolated, and their brewing-relevant phenotypes were assessed. These F1 spore clones were found to differ with respect to fermentation performance under lager brewing conditions (15°C, 15 °Plato), production of volatile aroma compounds, flocculation potential and temperature tolerance. One spore clone, selected for its rapid fermentation and acetate ester production was sporulated to produce an F2 generation, again comprised of four spore clones from a single ascus. Again, phenotypic diversity was introduced. In two of these F2 clones, the fermentation performance was maintained and acetate ester production was improved relative to the F1 parent and the original hybrid strain. Strains also performed well in comparison to a commercial lager yeast strain. Spore clones varied in ploidy and chromosome copy numbers, and faster wort fermentation was observed in strains with a higher ploidy. An F2 spore clone was also subjected to 10 consecutive wort fermentations, and single cells were isolated from the resulting yeast slurry. These isolates also exhibited variable fermentation performance and chromosome copy numbers, highlighting the instability of polyploid interspecific hybrids. These results demonstrate the value of this natural approach to increase the phenotypic diversity of lager brewing yeast strains.