Use of microencapsulated starter cultures by spray drying in coffee under self-induced anaerobiosis fermentation (SIAF).

Using starter culture in liquid form is not economically viable in the coffee fermentation process. This work aimed to compare the fermentative performances of fresh and microencapsulated yeasts in coffee under self-induced anaerobic fermentation (SIAF). The inoculum permanence was monitored, and sugars, alcohols, acids, and volatile compounds were analyzed by chromatography. In addition, sensory analysis was performed on roasted beans. After 180 h of fermentation in the natural process, microencapsulated Torulaspora delbrueckii (MT) (7.97 × 107 cells/g) showed a higher population thanfresh Torulaspora delbrueckii (FT) (1.76 × 107 cells/g). The same acids and volatile compounds were detected in coffees with fresh and microencapsulated yeast. However, the yeast state influenced the concentration of the compounds. In pulped coffee, the coffee inoculated withmicroencapsulated Saccharomyces cerevisiae (MS) obtained the highest concentration of alcohols, esters, pyrazines, and others compared with fresh Saccharomyces cerevisiae (FS), with an increase of up to 47%. Furthermore, the coffee inoculated with MT obtained the highest concentration in almost all chemical classes in both processes compared with FT. These differences ranged up to 55%. Regarding sensory analysis, coffees inoculated with MS showed dominant notes of fruity, caramel, and nuts in the natural process. Otherwise, in pulped process, coffees inoculated with MT showed caramel, honey, and nuts. Therefore, the microencapsulated yeasts were metabolically active and may be considered with commercial potential. Considering the parameters analyzed, the most suitable yeast for natural and pulped processing would be MS and MT, respectively.

Importance of micronutrients and organic nitrogen in fermentations with Torulaspora delbrueckii and Saccharomyces cerevisiae.

The current use of non-Saccharomyces yeasts in mixed fermentations increases the relevance of the interactions between yeast species. In this work, the interactions between Saccharomyces cerevisiae and Torulaspora delbrueckii were analyzed. For this purpose, fermentations with and without contact between strains of those yeast species were performed in synthetic must. Fermentation kinetics, yeast growth and dynamics were measured over time. Additionally, the effects of nitrogen and other nutrient supplementations on the mixed fermentations were determined. Our results showed that S. cerevisiae did not always dominate the sequential fermentations, and experiments without yeast contact (in which T. delbrueckii cells were removed from the medium before inoculating S. cerevisiae at 48 h) resulted in stuck fermentations except when the inoculum size was increased (from 2 × 106 to 108 cells/mL) or there was a supplementation of thiamine, zinc and amino acids at the same concentration as initially found in the synthetic must. Our findings highlight the importance of inoculum size and ensuring the availability of enough micronutrients for all yeast species, especially in sequential fermentations.

Microencapsulation of epiphytic coffee yeasts by spray drying using different wall materials: Implementation in coffee medium.

The storage of microorganisms in liquid form is the main drawback of commercializing epiphytic coffee yeasts. This work aimed to evaluate the fermentative performance of microencapsulated yeasts by spray drying in a coffee peel and pulp media (CPM). The yeasts, Saccharomyces cerevisiae CCMA 0543, Torulaspora delbrueckii CCMA 0684, and Meyerozyma caribbica CCMA 1738, were microencapsulated using maltodextrin DE10 (MD), high maltose (MA), and whey powder (WP) as wall materials. A Central Composite Rotational Design (CCRD) was used to investigate the effect of operating parameters on the microcapsules' cell viability, drying yield, and water activity. Yeasts reached cell viability and drying yields above 90 and 50 %, respectively. WP maintained the cell viability of the three yeasts over 90 days of storage at room temperature (25 °C) and was selected as a wall material for the three yeasts. M. caribbica showed to be more sensitive to spray drying and less resistant to storage. Some differences were found in the fermentation of the CPM medium, but the microencapsulated yeasts maintained their biotechnological characteristics. Therefore, the microencapsulation of epiphytic coffee yeasts by spray drying was promising to be used in the coffee fermentation process.

Microencapsulation by spray drying of coffee epiphytic yeasts Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684.

The objective of this work was to evaluate the microencapsulation feasibility of Saccharomyces cerevisiae CCMA 0543 and Torulaspora delbrueckii CCMA 0684 in three different compositions of wall material by spray-dryer. The yeasts (109 CFU mL-1) were microencapsulated separately using maltodextrin (15%), maltodextrin (15%) with sucrose (2%), or maltose (2%) as wall material. The viability was evaluated for 6 months at two different temperatures (7 and 25 °C). The yield, cell viability after spray drying, and characterization of the microcapsules were performed. Results indicate that cell viability ranged between 94.06 and 97.97%. After 6 months, both yeasts stored at 7 °C and 25 °C presented 107 and 102 CFU mL-1, respectively. Regarding Fourier-transform infrared spectroscopy analysis, all microencapsulated yeasts presented typical spectra footprints of maltodextrin. After 6 months of storage, S. cerevisiae CCMA 0543 obtained a 10.8% increase in cell viability using maltodextrin with maltose as wall material compared to maltodextrin and maltodextrin with sucrose. However, T. delbrueckii CCMA 0684 obtained a 13.5% increase in cell viability using only maltodextrin. The study showed that maltodextrin as a wall material was efficient in the microencapsulation of yeasts. It is possible to assume that maltose incorporation increased the cell viability of S. cerevisiae CCMA 0543 during storage.

Coinoculation of lactic acid bacteria and yeasts increases the quality of wet fermented Arabica coffee.

Wet coffee fermentation is widely used in coffee-producing regions such as Colombia and Hawaii, but it is not widespread in Brazil. This study aimed to evaluate inoculating the lactic acid bacteria Leuconostoc mesenteroides CCMA1105 and Lactiplantibacillus plantarum CCMA 1065 and the yeasts Saccharomyces cerevisiae CCMA0543 and Torulaspora delbrueckii CCMA0684 as starter cultures on wet coffee fermentation using the SIAF method (self-induced anaerobiosis fermentation). The microbial activity resulted in high consumption of the carbohydrates glucose (98.6%), fructose (97.6%), and sucrose (100%), in addition to the production of lactic and acetic acids, impacting the final quality of the beverage. A total of 108 volatile compounds belonging to 17 classes were identified in the green and roasted coffee samples, including 2,3-butanediol produced by lactic acid bacteria, contributing to coffee's aromatic profile. The final scores for the coffees from the different fermentations ranged from 79.0 to 83.25. The inoculated fermentations were classified as specialty according to the Specialty Coffee Association. Therefore, whole coffee fruit processed via wet using SIAF method and yeast and lactic acid bacteria starter is an alternative for improving wet fermented coffee quality and obtaining coffee beverages with a different sensory profile.

Characterization of bioactive, chemical, and sensory compounds from fermented coffees with different yeasts species.

Selected yeasts for coffee fermentation are correlated with changes in chemical compounds and beverage sensory characteristics. This work aimed to evaluate the chemical and sensory modifications of coffee fermented with one yeast (Saccharomyces cerevisiae CCMA 0543, Candida parapsilosis CCMA 0544, or Torulaspora delbrueckii CCMA 0684) and in co-inoculation (from two to two and the three together) by dry processing. Real-time PCR analyzes, total phenolic content and antioxidant activity (DPPH, ABTS, and FRAP), liquid and gas chromatography, and sensory analysis were performed. Caparaó coffees showed a higher C. parapsilosis (6.14 Log cell.g-1) population followed by S. cerevisiae (5.85 Log cell.g-1) and T. delbrueckii (4.64 Log cell.g-1). The total phenolic content has a strong and positive correlation with the fermentation time and the roasted beans and a moderate and positive correlation with DPPH, FRAP, and ABTS. Coffee inoculated with T. delbrueckii reduced caffeine concentration during the fermentation process. In co-cultivation, the trigonelline concentration showed the most significant decrease (around 4 mg.g-1) when inoculated with S. cerevisiae and T. delbrueckii. Detection of some organic acids and volatile compounds during fermentation may indicate that the starter cultures used different metabolic routes. All co-inoculation treatments presented the best sensory scores (>86 points). In the inoculated fermentation, fruity, citric, molasses, freshness, and wine notes appeared. The co-inoculated treatment with S. cerevisiae CCMA 0543, C. parapsilosis CCMA 0544, and T. delbrueckii CCMA 0684 was the best, considering the diversity of sensory notes descriptors and the final concentration of organic acids.

Co-inoculation of yeasts starters: A strategy to improve quality of low altitude Arabica coffee.

The study aimed to improve the quality of dry-processed coffee grown at low altitudes through yeast inoculation, using three species (Saccharomyces cerevisiae CCMA 0543, Torulaspora delbrueckii CCMA 0684, and Candida parapsilosis CCMA 0544) singly and with co-inoculation for fermentation. Important chemical compounds and groups were analyzed by liquid and gas chromatography and Fourier-transform infrared spectroscopy (FTIR). The inoculated coffees with yeast populations around 106 cell/g obtained the highest scores, and the co-inoculation with C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684 had the highest score in the sensory analysis (85). Different descriptors were observed in each treatment, and body, flavor, balance, and aftertaste are strongly related to C. parapsilosis CCMA 0544. The fermentation process improved the quality of low-altitude coffees, and the combination of non-Saccharomyces yeasts (C. parapsilosis CCMA 0544 and T. delbrueckii CCMA 0684) was the most indicated as starter cultures.

The impact of mixed amino acids supplementation on Torulaspora delbrueckii growth and volatile compound modulation in soy whey alcohol fermentation.

Soy (tofu) whey is a liquid side stream generated from tofu production and is often discarded as waste after it is generated. Direct disposal of soy whey can result in environmental issue in the long run. Soy whey has been previously successfully fermented using different types of wine yeasts, but the yeast available nitrogen (YAN) was found to be deficient. In this study, the soy whey YAN was estimated to be approximately 45.9 mg N/L. A mixture of four amino acids (valine, leucine, isoleucine and phenylalanine) was added into soy whey at a total concentration of +40, +80, +120 and +160 mg N/L and fermented with Torulaspora delbrueckii Biodiva for a period of 10 days. Increasing amino acid supplementation did not affect the yeast cell growth, but it sped up the sugar utilization proportionally. Increasing amino acid supplementation resulted in lower organic acid production and higher glycerol production. Amino acid supplementation also enhances the production rate of higher alcohols; increasing amount of higher alcohols and their respective esters were obtained with increasing amount of amino acid supplementation. However, higher levels of amino acid supplementation (particularly at +160 mg N/L sample) resulted in higher residual nitrogen contents which may lead to microbial instability. Supplementation of 120 mg N/L of amino acids was found to be the optimum concentration to enhance the metabolism of the yeast without leaving a high residual amino acid content. Therefore, with proper control of the amino acid addition dosage, the usage of mixed amino acid supplementation may be a strategy to regulate the fermentation kinetics and volatile compound modulation in soy whey alcohol fermentation.

Fermentation characteristics of four non-Saccharomyces yeasts in green tea slurry.

The fermentation characteristics of non-Saccharomyces yeasts (Pichia kluyveri FrootZen, Torulaspora delbrueckii Prelude, Williopsis saturnus var. mrakii NCYC2251 and Torulaspora delbrueckii Biodiva) were evaluated in green tea slurry fermentation. Each yeast showed different fermentation performances: strains Prelude and Biodiva utilized sucrose faster than the other two yeasts; strain NCYC2251 was the only species that metabolized xylose. Strain FrootZen increased the caffeine content significantly and strain Prelude showed the opposite trend, both at a statistical level, while theanine contents in four samples were relatively stable. Biodiva and FrootZen significantly improved polyphenols content and the oxygen radical absorbance capacity of fermented teas. Some endogenous volatiles such as ketones, lactones and aldehydes decreased to lower or undetected levels, but one of the key tea aroma compounds methyl salicylate increased by 34-fold and 100-fold in P. kluyveri and W. saturnus samples respectively. Therefore, green tea fermentation by appropriate non-Saccharomyces yeasts can enhance its antioxidant capacity and alter the aroma compound profile.

Influence of fermentation conditions on the sensorial quality of coffee inoculated with yeast.

Fermentation is one of the post-harvest steps that influence coffee quality. This work evaluated the effect of Saccharomyces cerevisiae (CCMA 0543) and Torulaspora delbrueckii (CCMA 0684) inoculation on the quality of natural and pulped natural processed coffee in different producing regions. Yeast populations were assessed by the real-time polymerase chain reaction. Volatile and nonvolatile compounds were evaluated by gas chromatography-mass spectrometry and high-performance liquid chromatography, respectively. S. cerevisiae was predominant during spontaneous (average of 4 log cells/g) and inoculated (average of 7 log cells/g) fermentations in both processes. T. delbrueckii showed a similar population (3.79 log cells/g) in all assays. Glucose and fructose were the most detected sugars in coffee beans. Succinic acid was found at the end of the fermentative process. The lactic acid concentration was inversely proportional to ethanol concentration. Pyrrole and furan, which are volatile groups, allowed to differentiate the coffee processing methods. Yeast inoculation modified the sensorial profile and increased the coffee beverage scores by up to 5 points. S. cerevisiae inoculation was most suitable for pulped natural coffee, and T. delbrueckii inoculation showed the best performance in natural coffee.

Understanding the interaction of isoleucine paired with other amino acids in soy whey alcohol fermentation using Torulaspora delbrueckii.

Soy (tofu) whey is a liquid by-product generated from tofu (soybean curd) production and it is often discarded off as a waste liquid by the tofu manufacturers. Previous studies have demonstrated that soy whey can be biotransformed into a soy alcoholic beverage by using Saccharomyces and non-Saccharomyces yeasts even though soy whey is low in yeast assimilable nitrogen (YAN) content. In this study, the initial YAN of the soy whey was estimated to be 46.6 mg N/L and Torulaspora delbrueckii Biodiva was used to ferment soy whey supplemented with either isoleucine only or isoleucine paired with valine, leucine or phenylalanine (each amino acid supplemented at a dosage of 30 mg N/L). Amino acid supplementation was found to enhance sugar utilization by the yeast, which led to higher ethanol production (7.49% v/v in control versus 8.35-8.80% v/v in supplemented samples). Samples supplemented with isoleucine only experienced slower sugar utilization during the fermentation as compared to the paired amino acid samples, but the yeast was still able to utilize the sugar to low levels at the end of the fermentation. The presence of leucine supplementation counteracted the "inhibition" induced by the presence of isoleucine at the first day of the fermentation. Amino acid supplementation slowed down glutamic acid utilization and resulted in higher levels of residual glutamic acid and alanine. Amino acid supplementation increased the corresponding fusel alcohol production and the presence of other amino acids reduced the active amyl alcohol production. Therefore, interactions between amino acids can impact the metabolism of the yeast as well as the flavor modulation during soy whey fermentation.

Effect of single amino acid addition on growth kinetics and flavor modulation by Torulaspora delbrueckii in soy (tofu) whey alcoholic beverage fermentation.

Soy (tofu) whey is a by-product commonly disposed of by tofu manufacturers around the world. Due to its nutritious nature, direct disposal of soy whey into the sewage can result in a detrimental impact on the environment in the long-run. In this study, soy whey supplemented with four individual amino acids (valine, leucine, isoleucine and phenylalanine) equivalent to 120 mg N/L was fermented with a yeast Torulaspora delbrueckii Biodiva. The supplementation of an individual amino acid resulted in faster sugar utilization and lower levels of residual sugar than the unsupplemented whey but did not result in a significantly higher amount of ethanol (7-8% v/v) at the end of fermentation. Isoleucine supplementation resulted in a slightly slower initial yeast growth rate when compared to the control while the other three amino acids had identical yeast growth kinetics to the control. Isoleucine supplementation also resulted in slower sugar utilization during the first four days. Therefore, isoleucine is least preferred by the yeast. The supplementation of amino acids resulted in greater formation of higher alcohols and their corresponding alcohol-derived esters. Overall, the supplementation of a single amino acid enhanced sugar utilization and impacted flavor compounds of the resultant soy whey alcoholic beverage.

Organic acids produced during fermentation and sensory perception in specialty coffee using yeast starter culture.

Volatile and non-volatile compounds in coffee directly affect the beverage's quality. This study aimed to demonstrate how the organic acids and volatile profiles were impacted by coffee fermentation using four starter cultures (Meyerozyma caribbica (CCMA 0198), Saccharomyces cerevisiae (CCMA 0543), Candida parapsilosis (CCMA0544), and Torulaspora delbrueckii (CCMA 0684)) inoculated in two varieties of coffee (Bourbon Amarelo and Canário Amarelo) using natural and pulped natural processing methods and sensory perception. Real-time PCR (qPCR) was used to verify the dynamic behavior of yeast populations. Organic acids were detected using high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) was used to detected volatile compounds. Sensory analysis was performed on the roasted coffee. Citric, malic, succinic, lactic, oxalic, isobutyric, and propionic acids and 105 volatile compounds were detected. At the beginning of fermentation, treatments with natural processing presented higher number of volatiles compounds. After fermentation, the main compounds groups were acids, alcohols, and aldehydes. The perception of sensory attribute (fruity, nutty, cocoa) varied with the coffee variety, type of processing, and type of inoculum. The use of yeasts is an alternative for sensorial differentiation of coffee variety Canário Amarelo and Bourbon Amarelo. The stainless-steel containers showed good results for coffee fermentation.

Volatile composition of bilberry wines fermented with non-Saccharomyces and Saccharomyces yeasts in pure, sequential and simultaneous inoculations.

Bilberry (Vaccinium myrtillus L.) juice was fermented with Torulaspora delbrueckii (TD291 and TD70526) and Schizosaccharomyces pombe (SP3796 and SP70572) in pure fermentation as well as in sequential and simultaneous inoculations with Saccharomyces cerevisiae 1116 (SC1116). Altogether, 56 volatile compounds were identified and semi-quantified with HS-SPME-GC/MS in bilberry products. Yeast fermentation prominently enhanced the aroma complexity of bilberry with a sharp increase in alcohols, esters, aldehydes, and acetals. Compared to S. cerevisiae, T. delbrueckii produced less ethanol but more fusel alcohols that potentially enhance "alcohol" and "nail polish" odors in TD70526 and less "fruity" esters in TD291. SP70572 resulted in high productions of undesirable compounds of acetoin and acetaldehyde but a low content of higher alcohols and esters, SP3796 produced a high content of fatty acid ethyl esters and acetoin. In comparison with monoculture of non-Saccharomyces yeast, sequential and simultaneous cultures of S. pombe and S. cerevisiae significantly decreased the content of acetoin while increased the relative level of esters; sequential cultures of T. delbrueckii and S. cerevisiae remarkably increased the concentration of acetaldehyde; simultaneous inoculations of S. cerevisiae with TD70526 and TD291 significantly decreased the content of fusel alcohols and increased the content of esters, respectively. The findings suggested that non-Saccharomyces yeasts possess the potential to affect and modulate the aromatic profile of fermented bilberry products. Sequential and simultaneous inoculations with S. pombe strains and S. cerevisiae as well as simultaneous fermentation using T. delbrueckii strains and S. cerevisiae are optimal strategies to positively influence the aroma profile of bilberry wines.

Effect of selenium on growth and antioxidant enzyme activities of wine related yeasts.

The use of supplements in the diet is a common practice to address nutritional deficiencies. Selenium is an essential micronutrient with an antioxidant and anti-carcinogenic role in human and animal health. There is increasing interest in developing nutritional supplements such as yeast cells enriched with selenium. The possibility of producing beverages, namely wine, with selenium-enriched yeasts, led us to investigate the selenium tolerance of six wine related yeasts. The production of such cells may hamper selenium toxicity problems. Above certain concentrations selenium can be toxic inducing oxidative stress and yeast species can show different tolerance. This work aimed at studying selenium tolerance of a diversity of wine related yeasts, thus antioxidant response mechanisms with different concentrations of sodium selenite were evaluated. Viability assays demonstrated that the yeast Torulaspora delbrueckii showed the highest tolerance for the tested levels of 100 µg mL(-1) of sodium selenite. The evaluation of antioxidative enzyme activities showed the best performance for concentrations of 250 and 100 µg mL(-1), respectively for the yeast species Saccharomyces cerevisiae and Hanseniaspora guilliermondii. These results encourage future studies on the possibility to use pre-enriched yeast cells as selenium supplement in wine production.

Winemaking and bioprocesses strongly shaped the genetic diversity of the ubiquitous yeast Torulaspora delbrueckii.

The yeast Torulaspora delbrueckii is associated with several human activities including oenology, bakery, distillery, dairy industry, etc. In addition to its biotechnological applications, T. delbrueckii is frequently isolated in natural environments (plant, soil, insect). T. delbrueckii is thus a remarkable ubiquitous yeast species with both wild and anthropic habitats, and appears to be a perfect yeast model to search for evidence of human domestication. For that purpose, we developed eight microsatellite markers that were used for the genotyping of 110 strains from various substrates and geographical origins. Microsatellite analysis showed four genetic clusters: two groups contained most nature strains from Old World and Americas respectively, and two clusters were associated with winemaking and other bioprocesses. Analysis of molecular variance (AMOVA) confirmed that human activities significantly shaped the genetic variability of T. delbrueckii species. Natural isolates are differentiated on the basis of geographical localisation, as expected for wild population. The domestication of T. delbrueckii probably dates back to the Roman Empire for winemaking (∼ 1900 years ago), and to the Neolithic era for bioprocesses (∼ 4000 years ago). Microsatellite analysis also provided valuable data regarding the life-cycle of the species, suggesting a mostly diploid homothallic life. In addition to population genetics and ecological studies, the microsatellite tool will be particularly useful for further biotechnological development of T. delbrueckii strains for winemaking and other bioprocesses.

Occurrence of Yeasts in Cultivated Soils in El-Minia City, Egypt

Two-hundred two yeast strains were isolated from rhizosphere (87 strains) and nonrhizosphere (115 strains) areas of potato, maize, vegetable marrow, and cabbage plants. On the basis of 26 morphological and physiological properties, the isolated yeast strains were assigned to 9 genera and 15 species. Trichosporon beigelii, Kluyveromyces marxianus and Torulaspora delbrueckii were the dominant species. Cryptococcus humicolus and Candida tropicalis were represented by considerable numbers of strains. Of low occurrence were Saccharomyces cerevisiae and Candida blankii. Other yeast species were represented by single or two strains. Total counts of yeast cells per gram dry soil ranged from 1.1x10(3) to 6.6x10(3) in soil samples of rhizosphere areas and from 6.5x10(2) to 5.6x10(3) in soil samples of nonrhizosphere areas. Types of the tested plants affected not only the total counts of yeast cells but also spectra of yeast species. Relationships of age of potato plant, moisture contents of soil samples, and its pH values and total counts of yeast cells were discussed.