Viviana K. Rivera Flores

Under specific conditions of oxygen availability and lactose hydrolysis, Brettanomyces claussenii OYL-201 can convert glucose in dairy coproducts into ethanol, leaving the galactose almost untouched. This approach could be applied specifically to whey permeate (WP) to develop foods with low-glycemic loads and other potential health benefits. The aims of this research are to optimize the fermentation of WP by B. claussenii, to maximize the production of ethanol and galactose, and to characterize various products obtained with this approach. For this purpose, five fermentation factors were studied to determine their impacts on ethanol and galactose: temperature (20–40 °C), substrate concentration (5–15%TS), lactase enzyme/substrate ratio (0–40 IU/ g lactose), inoculation level (6–8 log cfu/mL), and time (6–30 days). Linear models, containing quadratic and interaction effects, were built for the optimization of both responses. Optimal levels were predicted for the maximum outputs of ethanol and galactose simultaneously, which utilized the following parameters: 15%TS, 37 IU / g lactose, 28 °C, 7.5 log cfu/mL, and 30 days, which together were predicted to produce 4.0%v/v ethanol and 51 g/L galactose in the final product. These parameters were applied to 18-L fermentations, and the fermentates were processed via distillation and freeze-drying. As a result, four product streams were obtained: a fermented product with 3.4%v/v ethanol and 56 g/L galactose, a 45%v/v ethanol distillate, a galactose-rich drink base (63 g/L), and a galactose-rich powder (55%w/w). These results demonstrate that it is possible to maximize the production of ethanol and galactose from WP and to develop novel, potentially functional bioproducts from this stream.

Previous studies highlighted Brettanomyces claussenii as a versatile yeast that produces ethanol or acetic acid from lactose, or selectively metabolizes glucose while leaving behind galactose, depending on different operational conditions. This flexibility enables the production of galactose-rich bioproducts from liquid dairy residues. The purpose of this study is to: (i) optimize the anaerobic fermentation of milk permeate (MP) by B. claussenii to maximize the yields of galactose and ethanol and minimize leftover glucose, and (ii) combine the optimized process with distillation and drying and characterize its multiple products. Response surface methodology was used for the optimization. Three fermentation parameters were chosen as input factors: temperature (25–35 °C), inoculation level (7.0–8.5 log cfu/mL), and time (4–40 days), with three metabolites as responses: galactose, glucose, and ethanol. The optimal combination of parameters resulted in temperature, 28 °C; inoculation level, 7.6 log cfu/mL; and time, 33 days. Under these conditions, the fermented product was predicted to have 63.6 g/L galactose, 4.0% v/v ethanol, and 0 g/L residual glucose. The optimal parameters were used to run 18 L fermentations followed by distillation and freeze-drying. As a result, four product streams were obtained and characterized for relevant physicochemical and nutritional attributes. Our results show that the partial fermentation of MP by B. claussenii can be the first step to develop lactose-free, low-in-glucose, galactose-rich bioproducts, which improve the value of this residue and broaden its applications in the food supply chain.

As the Greek-style yogurt market continues to experience prosperous growth, finding the most appropriate destination for yogurt acid whey (YAW) is still a challenge for Greek yogurt manufacturers. This study provides a direct alternative treatment of YAW by leveraging the abilities of Mucor circinelloides and Mucor genevensis to raise the pH of YAW and to produce fungal biomass with a high lipid content. Aerobic cultivations of these species were conducted in YAW, both with and without the addition of lactase, at 30 °C, and 200 rpm agitation. The density, pH, biochemical oxygen demand (BOD), biomass production, lipid content, fatty acid profile, and sugar and lactic acid concentrations were regularly measured throughout the 14-day cultivations. The data showed that M. genevensis was superior at deacidifying YAW to a pH above 6.0—the legal limit for disposing of cultured dairy waste. On the other hand, M. circinelloides generated more fungal biomass, containing up to 30% w/w of lipid with high proportions of oleic acid and γ-linolenic acid. Additionally, the treatments with lactase addition showed a significant decrease in the BOD. In conclusion, our results present a viable treatment to increase the pH of YAW and decrease its BOD, meanwhile generating fungal oils that can be further transformed into biodiesel or processed into functional foods or dietary supplements.

In Greek-style yogurt production, every kilogram of product yields 2 to 3 kg of acid whey (YAW); this coproduct’s composition and low pH pose challenges for its proper valorization and reinsertion into the food supply chain. However, 240 mL of YAW contains over 9 g of lactose and represents a good source of minerals; these traits can be leveraged to develop nutritious fermented beverages. The purpose of this study is to investigate the aerobic fermentation of dairy sugars by different yeasts by characterizing these processes and their products. This will determine whether such methods provide viable options for the production of acetic-acid-containing beverages from YAW. To achieve this, yeast nitrogen base was used to prepare four growth media formulations, each supplemented with lactose, glucose, galactose, or a 1:1 mix of glucose and galactose (GLU:GAL), and each adjusted to a pH of 4.20. Fermentations were performed by pure cultures of S. cerevisiae, K. marxianus, B. claussenii, or B. bruxellensis, and were held at 25 °C with agitation at 185 rpm. For each treatment, density, pH, and microbial enumeration were measured over time to obtain process profiles, while ethanol, organic acids, and sugars were analyzed at the beginning and the end of each fermentation via HPLC, to determine resulting products. ANOVA and Tukey’s honest significant difference test at a significance level of 0.05 were used to compare residual sugars and fermentation products. Variable rates of sugar consumption were observed for each species. In GLU:GAL, B. claussenii consumed all of the glucose, left behind most of the galactose, and produced a high concentration of acetic acid. These results suggest the potential to develop versatile processes that target glucose for acetic acid production, while leaving available galactose to confer products with prebiotic properties. The development of processes for the conversion of YAW into beverages with organic acids and other healthful components not only aligns with consumers’ demands for better-for-you products, but also promotes the valorization of this otherwise underutilized dairy coproduct.

Acid whey from Greek-style yogurt (YAW) is an underutilized byproduct and a challenge for the dairy industry. One alternative is the fermentation of YAW by yeasts such as Saccharomyces, Brettanomyces, and Kluyveromyces spp., to produce new styles of fermented beverages. Previous research in our group suggested that the sugar profiles of the dairy coproducts impacted the fermentation profiles produced by B. claussenii. The present work aims to describe the fermentation of dairy sugars by S. cerevisiae, K. marxianus, and B. claussenii, under conditions comparable to those of YAW. For this purpose, four preparations of yeast nitrogen base, each containing 40 g/L of either lactose (LAC), glucose (GLU), galactose (GAL), or a 1:1 mixture of glucose and galactose (GLU:GAL), all at pH 4.20, were used as fermentation media. The fermentation was performed independently by each organism at 25 °C under anoxic conditions, while density, pH, cell count, ethanol, and organic acids were monitored. Non-linear modeling was used to characterize density curves, and Analysis of Variance and Tukey’s Honest Significant Difference tests were used to compare fermentation products. K. marxianus and S. cerevisiae displayed rapid sugar consumption with consistent ethanol yields in all media, as opposed to B. claussenii, which showed more variable results. The latter organism exhibited what appears to be a selective glucose fermentation in GLU:GAL, which will be explored in the future. These results provide a deeper understanding of dairy sugar utilization by relevant yeasts, allowing for future work to optimize fermentations to improve value-added beverage and ingredient production from YAW.

Shelf-stable milk is consumed worldwide, and this market is expected to continue growing. One quality challenge for UHT milk is age gelation during shelf life, which is in part caused by bacterial heat-stable proteases (HSP) synthesized during the raw milk storage period before heat processing. Some Pseudomonas spp. are HSP producers, and their ability to grow well at refrigeration temperature make them important spoilage organisms for UHT processors to control. Previous studies have shown that lactose oxidase (LO), a natural and commercially available enzyme that produces hydrogen peroxide and lactobionic acid from lactose, can control bacterial growth in raw milk. In this research, we investigated the ability of LO to control HSP producer outgrowth, and thus delay age gelation in UHT milk. Six strains of Pseudomonas spp. were selected based on their ability to synthesize HSP and used as a cocktail to inoculate both raw and sterile (UHT) milk at a level of 1 x 10(5) cfu/mL. Groups were treated with and without LO, stored for 4 d at 6 degrees C, and monitored for cell count and pH. Additionally, a sample from each was tested for HSP activity via particle size analysis (average effective diameter at 90 degrees angle and 658 nm wavelength) and visual inspection on each day of the storage period. The HSP activity results were contrasted using Tukey's HSD test, which showed that in UHT milk, a LO treatment (0.12 g/L) effectively prevented gelation as compared with the control. In raw milk, however, a concentration of 0.24 g/L of LO was needed to obtain a similar effect. This test was scaled up to 19-L pilot plant batches of raw milk where they were challenged with Pseudomonas cocktail, treated with LO for 3 d, and then UHT processed. Resulting UHT milk bottles were monitored for gelation. Significant differences in particle size between the LO-treated samples and the control were observed as early as 1 mo after processing, and gelation was not detected in the LO-treated samples through 6 mo of storage. These results demonstrated that LO can be used to delay age gelation in UHT milk induced by HSP-producing Pseudomonas spp., representing an opportunity to improve quality and reduce postproduction losses in the shelf-stable milk market sector.