Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
(N/A)
Non Technical Summary
Consumer interest in fermented foods is steadily increasing in the United States. A subset of these products is beverages produced with the use of yeast and bacteria together in a symbiotic mass (SCOBY). The microorganisms in SCOBY systems are very diverse and variable, which causes the products to look, smell and taste different. In addition, presence and amount of health promoting compouds may vary significantly from product to product. This project will look at the natural variation in culture composition, the factors that influence composition and their effects on finished product, with the goal of helping the industry to standardize products in order to maximize health benefits and consumer acceptability.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
This project aims to improve knowledge of various aspects of non-dairy, low alcohol fermented beverages. Using a next-generation sequencing approach to assess culture systems and beverages, we will generate more reliable, complete data regarding the composition of the products, giving insight into methods for targeting a defined finished product. Commercial, dried versions of cultures for these products are not currently available from any source. Consistency of product suffers as a result, resulting in a high potential for alienation of consumers. As part of this project, we will generate valuable information about consumers of fermented beverages, their expectations with regard to sensory and health beneficial characteristics of such products. This combination of comprehensive microbiological, biochemical, and consumer data will generate approaches for optimizing product quality and market potential.
Project Methods
Fermented beverages will be produced in non-sterile ½ gallon glass jars. Kombucha and water kefir fermentations will be monitored daily until pH reaches 3.0 and 4.0 respectively, at which point samples of culture biomass and fermentate will be taken aseptically. DNA will be extracted using the MoBio PowerFood Microbial DNA Isolation Kit. DNA extracts will be frozen at -80C until being sent for sequencing. Whole-sample DNA extracts will be used as templates for amplification of the D1-D2 region of the eukaryotic large subunit (LSU) rRNA gene, and V1-V3 region of the prokaryotic 16S rRNA gene, enabling profiling of yeast, mold and bacterial populations based on relative proportions of discrete ribotypes, following previously published protocols. Indexed and pooled amplicon libraries will be subjected to high throughput DNA sequencing on the Illumina MiSeq platform. Analysis of these datasets will give more in-depth information regarding the effects of heterogeneity among the microbial profile of SCOBYs and resulting products, and the reproducibility of kefir and kombucha produced by a given SCOBY over multiple passes or generations.Simultaneously, cultures will be grown as described to produce sufficient biomass for preservation by ultralow temperature freezing (-80°C) and lyophilization. In a subsequent set of experiments, preserved cultures will be compared side-by-side to fermentations started with "daughter cultures" collected from repeated generation cycles (generations 10 and 20 from original cultures). Fermentation efficacy and duration (to previously identified pH targets) will be monitored, and samples of finished products will be collected to compare microbial composition as previously described. These experiments will assess the feasibility of starter culture preservation and degree of community temporal drift in traditionally propagated culture systems.From the culture systems analyzed, a maximum of three from each product category will be used to investigate the effects of fermentation time and refrigerated storage on the resulting product. Extended fermentations of up to 6 days for water kefir and 30 days for kombucha will be conducted utilizing conditions previously described. Fermentate will be analyzed intermittently at predetermined sampling points for simple sugars (fructose, glucose, sucrose), organic acids (lactic, acetic and gluconic) and ethanol concentrations. pH will be measured at each sampling time. A subset of samples from duplicate fermentations will be pooled and subjected to metagenomic sequencing as previously described. These data will provide a picture of metabolite production during the course of beverage fermentation. By combining analytical data with sequencing results, the relationship between microbial community structure and finished product characteristics can be elucidated. A subset of treatments (2, 4 and 6 day water kefir fermentations and 10, 20 and 30 day kombucha fermentations) will be replicated for shelf life analysis. At the conclusion of fermentation, fermentate will be aseptically separated into sterile conical tubes and refrigerated for up to 90 days. At two week increments, samples will be analyzed by HPLC/RID as described, spectroscopy to determine turbidity and cultural enumeration of fungi, total aerobic mesophiles, and lactic acid bacteria, using established methods (29). The visual presence of culture biomass and pH will also be recorded. These data will provide insight into the stability of unpasteurized fermented beverages during storage, as well as changes to visual attributes over time.Permission to conduct research with human subjects will be obtained from the University of Maine Institutional Review Board. An internet survey of 2000 U.S. adults at least 18 years old will be conducted. Qualtrics software (Provo, UT) will be used to create and administer the survey. Survey participants will be screened for consumption of water kefir and kombucha during the previous 18 months. The survey instrument will collect demographic data and contain questions designed to quantify low-alcohol fermented beverage purchase and consumption habits and to identify major barriers to consumer acceptance of these products. Photographs of beverages with different colors, clarity, and presence of clumps and sediment will be presented to classify visual preferences of specific consumer demographic groups. For example, do some consumers like cloudy drinks with sediment because they believe the traits indicate less-processing or more health benefits?A minimum of 100 adult consumers from greater Bangor, ME will be recruited to participate in each of two acceptability tests for water kefir and kombucha, respectively. The UM Sensory Evaluation Center has 12 Hewlett Packard ElitePro tablets available to collect data. The Center has 12 partitioned cubicles constructed with controlled Daylight 65 lighting and temperature and airflow control. Questionnaires, experimental designs, and statistical analyses will be created with SIMS sensory evaluation software (v. 6.0, Sensory Computer Systems, Berkeley Heights, NJ). The standard 9-point hedonic scale (1= dislike extremely; 5 = neither like nor dislike; 9= like extremely) will be used to assess liking of appearance, color, aroma, body (viscosity), flavor, degree of carbonation, and overall acceptability of samples. Just-about-right (JAR) scales will be used to assess consumer opinions of cloudiness/clarity, sweetness, tartness/acidity, and viscosity. Demographic questions including age, gender, kombucha (or water kefir) purchase habits, and purchase intent will also be asked. No more than five samples will be served in a single test to avoid sensory fatigue; samples will be served at 5°C. In year 2 , formulations based on the overall liking and JAR results for each product category will be tested with 100 interested consumers to confirm the suitability of the formulations. The unique approach to relating chemical constituents to consumer liking can provide a framework for quality assurance recommendation for fermented drink processors.The biochemical content of these fermented beverages will be identified and quantified using high performance liquid chromatography (HPLC) and will focus on the major flavor components - acids (acetic, lactic and gluconic) and residual sugars (fructose, glucose and sucrose). Ethanol, methanol and other minor organic acids will be monitored by HPLC, as well. Turbidity will be measured with a Hanna HI847492 haze turbidity meter for beer (Woonsocket, RI); CIE L*a*b* color will be measured with a Hunter Labscan colorimeter.