A novel image-based method for simultaneous counting of Lactobacillus and Saccharomyces in mixed culture fermentation.

Mixed microorganism cultures are prevalent in the food industry. A variety of microbiological mixtures have been used in these unique fermenting processes to create distinctive flavor profiles and potential health benefits. Mixed cultures are typically not well characterized, which may be due to the lack of simple measurement tools. Image-based cytometry systems have been employed to automatically count bacteria or yeast cells. In this work, we aim to develop a novel image cytometry method to distinguish and enumerate mixed cultures of yeast and bacteria in beer products. Cellometer X2 from Nexcelom was used to count of Lactobacillus plantarum and Saccharomyces cerevisiae in mixed cultures using fluorescent dyes and size exclusion image analysis algorithm. Three experiments were performed for validation. (1) Yeast and bacteria monoculture titration, (2) mixed culture with various ratios, and (3) monitoring a Berliner Weisse mixed culture fermentation. All experiments were validated by comparing to manual counting of yeast and bacteria colony formation. They were highly comparable with ANOVA analysis showing p-value > 0.05. Overall, the novel image cytometry method was able to distinguish and count mixed cultures consistently and accurately, which may provide better characterization of mixed culture brewing applications and produce higher quality products.

A novel image cytometry-based Lactobacillus bacterial enumeration method for the production of kettle sour beer.

The demands for a variety of craft beer flavors have been increasing in the United States. To meet this rising demand, breweries have been experimenting with kettle sour beer that utilizes lactic acid-producing bacteria for fermentation. The current standard bacterial quantification method is insufficient for rigorous quality control, thus there is a need for a better method to monitor lactobacilli concentration in a kettle sour environment. In this work, an automated Lactobacillus counting method was developed using fluorescence-based image cytometry. Three commonly used species were cultured, the concentrations were measured using image cytometry and evaluated against the standard spread-plating method. This procedure was undertaken in vitro at different dilutions and the method was repeated with two species in a kettle sour environment at different time points. Both the in vitro and fermentation experiments were repeated three times. Results demonstrated that the new method was not significantly different when compared to the standard plating method in either controlled settings or within the kettle sour fermentation. The proposed method provides a rapid tool to monitor and control lactobacilli growth in kettle sour beer production, and allows for standardization of the products due to the availability of near instantaneous information for quality control.

Physicochemical Properties and Consumer Acceptance of High-Pressure Processed, Sous Vide-Cooked Lobster Tails.

Sous vide (SV) and high-pressure processing (HPP) are promising techniques in the development of high-quality seafood products. The objectives of this study were to evaluate the impacts of HPP on the physicochemical quality and consumer acceptance of subsequently SV-cooked lobster tails. Raw shucked lobster tails were processed at 150 or 350 MPa for 5 or 10 min. Subsequently, half were SV cooked to a core temperature of 65 °C/10 min. Texture profile analysis, shear force, color, salt soluble protein content, water-holding capacity (WHC), moisture content, and weight loss were analyzed. Pressurization at 150 MPa/10 min decreased (P < 0.05) the hardness of raw lobsters compared to non-HPP-treated controls. However, 350 MPa for 5 or 10 min increased (P < 0.05) the shear force in raw and SV-cooked samples. HPP increased (P < 0.05) the L* values but did not affect moisture content, WHC, or weight loss of raw or SV-cooked lobsters. Lobsters were subjected to consumer acceptability testing using a 9-point hedonic scale. Although panelists rated the flavor, texture, and overall liking of the 350 MPa/10 min samples higher than the control and 150 MPa/10 min samples, there were no significant differences among treatment means, indicating that physicochemical changes induced by HPP did not affect consumer acceptance. In addition, approximately 84% of panelists reported that the 350 MPa product met their expectations compared to approximately 75% for the control and 150 MPa treatments. These results suggest that HPP has the potential to be applied in combination with SV cooking to produce consumer-acceptable, value-added lobster products. PRACTICAL APPLICATION: Lobsters are an expensive menu item in restaurants. However, they are susceptible to being overcooked using conventional methods, producing a tough and rubbery texture. Sous vide cooking is reported to provide evenly cooked lobsters with a succulent and juicy texture. In this study, lobsters were sous vide cooked to reach a core temperature of 65 °C, and then maintained at that temperature for 10 min. The application of moderate processing pressures to vacuum-packaged raw lobsters prior to SV cooking altered some physicochemical attributes but has the potential to increase the availability of high-quality, minimally processed seafood with good consumer acceptability.

A novel concentration and viability detection method for Brettanomyces using the Cellometer image cytometry.

Brettanomyces spp. can present unique cell morphologies comprised of excessive pseudohyphae and budding, leading to difficulties in enumerating cells. The current cell counting methods include manual counting of methylene blue-stained yeasts or measuring optical densities using a spectrophotometer. However, manual counting can be time-consuming and has high operator-dependent variations due to subjectivity. Optical density measurement can also introduce uncertainties where instead of individual cells counted, an average of a cell population is measured. In contrast, by utilizing the fluorescence capability of an image cytometer to detect acridine orange and propidium iodide viability dyes, individual cell nuclei can be counted directly in the pseudohyphae chains, which can improve the accuracy and efficiency of cell counting, as well as eliminating the subjectivity from manual counting. In this work, two experiments were performed to demonstrate the capability of Cellometer image cytometer to monitor Brettanomyces concentrations, viabilities, and budding/pseudohyphae percentages. First, a yeast propagation experiment was conducted to optimize software counting parameters for monitoring the growth of Brettanomyces clausenii, Brettanomyces bruxellensis, and Brettanomyces lambicus, which showed increasing cell concentrations, and varying pseudohyphae percentages. The pseudohyphae formed during propagation were counted either as multiple nuclei or a single multi-nuclei organism, where the results of counting the yeast as a single multi-nuclei organism were directly compared to manual counting. Second, a yeast fermentation experiment was conducted to demonstrate that the proposed image cytometric analysis method can monitor the growth pattern of B. lambicus and B. clausenii during beer fermentation. The results from both experiments displayed different growth patterns, viability, and budding/pseudohyphae percentages for each Brettanomyces species. The proposed Cellometer image cytometry method can improve efficiency and eliminate operator-dependent variations of cell counting compared with the traditional methods, which can potentially improve the quality of beverage products employing Brettanomyces yeasts.

The effectiveness of ozone and acidulant treatments in extending the refrigerated shelf life of fresh-cut potatoes.

The objective of the study was to determine the effectiveness of acidulant dip treatments (with or without aqueous ozone) to reduce enzymatic browning and to extend the shelf life of fresh-cut potato slices during refrigerated storage (4 °C) for 28 d. Potato slices subjected to aqueous ozone (2 ppm) had significantly (P≤ 0.05) higher L-values and lower a-values, but ozone did not appear to have any effect on aerobic plate counts (APCs) or polyphenol oxidase (PPO) activity. NatureSeal (NS) and sodium acid sulfate (SAS) were the most effective acidulant treatments in reducing browning (significantly [P≤ 0.05] higher L-values, lower a-values, and browning index values) regardless of ozone treatment. NS and SAS also had lower PPO activity compared to other treatments on days 0 and 28, and significantly (P≤ 0.05) lower APCs (≤2.00 log CFU/g) over refrigerated storage. Therefore, the SAS treatment was comparable to NS, a commercially available product, and showed promise as an effective antibrowning dip to reduce browning and spoilage in fresh-cut potato products. Practical Application: A 1% SAS dip treatment which included 1% citric and 1% ascorbic acid was found to be an effective antibrowning dip for fresh-cut potatoes along with NatureSeal®'s PS-10, compared to other treatments. They were both effective in maintaining low microbial counts over refrigerated storage. Additionally, aqueous ozone washes (2 ppm) showed significant benefits to reduce browning; however, ozone did not affect microbial counts or PPO enzyme activity. Therefore, the SAS treatment could have potential use in the fruit and vegetable industry to reduce browning and spoilage in fresh-cut potato products.