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ARS Home » Southeast Area » Raleigh, North Carolina » Food Science Research » Research » Research Project #428731

Research Project: Improved Processes for the Preservation and Utilization of Vegetables, Including Cucumber, Sweetpotato, Cabbage, and Peppers to Produce Safe, High Quality Products with Reduced Energy Use and Waste

Location: Food Science Research

2017 Annual Report

Objective 1. Enable the commercialization of reduced NaCl fermentation technology by identifying suitable starter cultures, optimizing processing parameters to prevent freezing of the fruits; and develop a shelf-life model that allows processors to rapidly and accurately predict end-use quality. Subobjective 1a: Develop starter cultures for cucumber fermentations brined with calcium chloride. Subobjective 1b: Identify parameters for long term storage of fermented cucumbers brined with calcium chloride so that freezing damage of the fruits can be prevented and removal of goods from tanks is facilitated during the winter season. Subobjective 1c: Develop an accelerated shelf-life model for fermented cucumber pickles. Objective 2. Determine the factors that maximize survival of probiotic bacteria and retention or production of healthful components in pickled vegetables to facilitate the commercialization of new cucumber pickling methods for delivery of live, probiotic, lactic acid bacteria to consumers. Subobjective 2a: Determine the acid resistance and growth characteristics of probiotic lactic acid bacteria that influence their survival in acidified and fermented cucumbers. Subobjective 2b: Identify suitable methods for the incorporation of probiotic lactic acid bacteria in refrigerated cucumber pickles. Subobjective 2c: Evaluate production and/or retention of bioactive peptides in fermented cucumbers. Objective 3. Enable new commercial methods for process-ready-fermentation or acidification to convert surplus vegetables at grocery stores and farmers' markets into value-added products with enhanced probiotic content. Objective 4. Integrate sweetpotato genotypes intended for commercial processing with their potential for increased levels of bioactive compounds and high quality food products.

Consumer interest in vegetable products with enhanced health benefits will drive future developments in the fermented and acidified vegetable industry. One major limitation for expansion is the generation of chloride waste and excess water use from traditional preservation technologies that rely on high sodium chloride (NaCl) concentrations. Cucumber fermentation in calcium chloride (CaCl2) brine was developed and transferred to commercial processors as an alternative to reduce chlorides in waste waters. Long-term adoption of this reduced NaCl preservation technology requires: a) defining conditions for bulk storage in outdoor tanks to prevent freezing damage in cold climates; b) understanding the microbial ecology of low salt vegetable fermentations so that appropriate starter cultures can be defined; and c) developing a shelf-life model to accurately predict end-use quality. The ability to deliver probiotic bacteria in pickles and to adapt acidification and fermentation technologies to preserve a wide range of surplus vegetables from farms and retail markets will provide new knowledge and technology for processors to engage the growing consumer market interested in healthy eating. For sweetpotatoes, consumer demands for readily available processed products have increased in recent years. Development of new cultivars that are bred for desirable processing characteristics, low acrylamide formation and high bioactive compound content, will benefit sweetpotato processors and farmers and boost the contribution of this nutritious vegetable in the U.S. diet. The proposed research will support developments that broaden the markets for sweetpotatoes, acidified and fermented vegetables and will reduce food and processing wastes.

Progress Report
Progress was made in all 4 objectives. Objective 1, Subobjective 1a: Cucumber fermentations brined with calcium chloride salt instead of a sodium salt are undergoing commercial trials to help reduce the environmental impact of salt waste. For this new technology to succeed, it is necessary to develop starter cultures that efficiently complete a cucumber fermentation. Previous research showed that the bacterium, Lactobacillus (L.) pentosus dominates natural commercial cucumber fermentations. We aimed at selecting L. pentosus cultures able to complete a fermentation and deficient in malic acid utilization, an acid inherently present in fresh cucumbers. Inability to utilize malic acid reduces carbon dioxide (gas) production and may lessen formation of hollow cavities in whole cucumbers, a defect known as bloating. Cucumber bloating results in significant economic losses for the pickling industry. Attempts to isolate the desired L. pentosus deficient in malic acid utilization were unsuccessful, after screening close to 500 commercial cucumber fermentation tanks. This outcome indicates that malic acid utilization is critical for microbial survival and/or competitiveness in a cucumber fermentation. L. pentosus deficient cultures obtained from sauerkraut and cheddar cheese fermentations were tested in the reduced salt cucumber fermentation system with the aim of developing mixed starter cultures that can produce a minimal amount of gas and thus reduce bloating incidence. Objective 1, Subobjective 1b: Cucumbers fermented in outdoor tanks are subject to freezing during the winter in the northern U.S. Research conducted to determine insulation parameters for fermentation tanks identified tank covers and the use of the anti-freezing agent glycerin in surface cover brines as tools to minimize freezing. Evaluation of cover brine recirculation to prevent freezing suggested the need to utilize hot air to effectively increase in-tank temperatures during winter. Application of hot air for production of fermented cucumbers is a practice incompatible with the current infrastructure that would likely increase the demand for energy and thus processing cost. We have redirected this research to develop guidelines for salting that result in nominal freeze-induced damage of fermented cucumbers under conditions similar to those present during winter bulk storage. Such guidelines are expected to identify the amount of salt needed to reduce freezing-induced damage of the fruits as a function of storage temperature, so that the typical amounts of sodium chloride (NaCl) salt used in manufacturing is reduced. Objective 1, Subobjective 1c: Hamburger dill chips, the most common consumer product made from fermented cucumbers, are frequently dated with a 2-3 year shelf life based on historical industry practice, but little information is known about the changes in flavor quality that occur during long shelf storage. Development of an accelerated shelf life model for cucumber pickles would enable processors to rapidly predict the shelf-life of their products during shelf-storage. A trained sensory panel evaluated pickles prepared from cucumbers fermented in salt and in calcium chloride for reduced environmental impact for flavor attributes, including vinegar flavor, dill and oxidized off-flavor. Oxidative off-flavor increased during shelf storage at temperatures of 25, 35, 45, and 55°C. However, the relationship between temperature and formation of oxidative off-flavors was complex and may not be accurately modeled at the high temperatures. Further research is planned to determine the changes in volatile (aromatic) composition of cucumber pickles during ambient and accelerated temperature storage to identify compounds that can serve as indicators of off-flavor formation. Objective 2, Subobjectives 2a and 2b: Incorporation of probiotics or live microbes able to confer health benefits for consumers, in pickle products would offer a non-dairy, low-calorie alternative to the predominantly dairy-based probiotic food items currently available. Laboratory assays were developed to characterize probiotic lactic acid bacteria (LAB) for growth and survival in refrigerated pickle products. It was discovered that a particular LAB, Lactobacillus casei strain ATCC 393, survived for 2 months in a simulated refrigerated pickle product at levels that exceed the amount required for probiotic activity, if allowed to ferment at 30°C for 2 days prior to refrigeration. The ability of L. casei to survive in the simulated product was preliminarily attributed to acid resistance features and the ability to proliferate slowly in the vegetable system. Furthermore, direct incorporation of commercial probiotic LAB in a powdered form to simulate refrigerated pickle media and acidified cucumbers was tested. Powdered probiotic LAB cultures were viable for 1 month when a pre-conditioning step in cucumber juice at ambient temperature was applied. Survival for 2 months was achieved with pre-conditioning and amino acid supplementation. The amino acid, arginine, which is naturally present in foods, was found to be particularly useful in helping cultures survivel in simulated pickle products. This research shows that probiotic culture survival may be possible in refrigerated pickles through careful strain selection and process modifications to achieve the ability to deliver enough probiotic organisms in one serving of pickles to confer health benefits. Objective 2, Subobjective 2c: Bioactive peptides possessing therapeutic properties are well documented in foods fermented by LAB, including dairy products, sourdough, cured meats, grains and raw vegetables. However, bioactive peptides have not been previously investigated in fermented vegetables. Cucumber pickles, the most commonly consumed fermented vegetable in the U.S., are not amenable to typical analytical workflows without extensive sample preparation due to their high salt content. A novel mass spectrometry based method was employed for direct analysis of peptides in acidified and fermented cucumbers. Five peptides with known anti-hypertensive properties were identified in fermented cucumber: lysine-proline, isoleucine/leucine-proline-proline, valine-proline-proline, and arginine-tyrosine. Bioactive peptides, although present at low concentrations compared to other food constituents, are valuable functional food components due to their high potency. This is the first report of bioactive peptides in a fermented vegetable, and the first evidence of the potent and most prevalently studied anti-hypertensive peptide, isoleucine-proline-proline, in a vegetable source. Objective 3: This study aims to investigate the redirection of surplus vegetables from the US market to value added fermented or acidified foods. We have enhanced low salt cucumber fermentation cover brine formulation for environmentally friendly preservation, which is theoretically applicable to other vegetables. The brine contained calcium chloride, calcium hydroxide, potassium sorbate, vinegar and NaCl to enhance buffer capacity and suppress growth of undesirable bacteria during fermentation. We have also successfully preserved tomatoes by acidification using a low acid, low salt brine formulation containing natural preservatives. These two studies have enabled the development of a systematic approach to study the challenges and suitability of a variety of vegetables for preservation by fermentation and/or acidification. Objective 4: Sweetpotato French fries (SPFF) are growing in popularity, but limited information is available on SPFF textural properties. Sixteen sweetpotato genotypes with varying flesh color (orange, yellow, cream) were evaluated on chemical constituents in relation to SPFF textural characteristics. Peak force describing instrumental textural properties of SPFF were measured using a texture analyzer. Descriptive sensory analysis was conducted with a trained panel. SPFF produced from the 16 sweetpotato genotypes varied widely in sensory properties. Dry matter and starch contents in raw sweetpotatoes were significantly correlated with instrumental peak force and sensory hardness and crispness of SPFF. Total sugar content was correlated with sensory moistness and cohesiveness. Instrumental measurements were also correlated with sensory attributes of hardness, crispness, oiliness, moistness and cohesiveness. Therefore, dry matter, starch and sugar contents of raw sweetpotatoes and instrumental texture measurements of SPFF could be used as indicators to select sweetpotatoes genotypes for processing into SPFF. These results would be beneficial to breeding programs in developing new sweetpotatoes varieties for improved SPFF quality. In another study, effects of sweetpotato genotypes and long-term storage were evaluated for processing of fried chips especially on browning and formation of acrylamide, a known neurotoxin and potential carcinogen. Fifteen sweetpotato genotypes were cured and stored up to 10 months as commercially practiced, and samples were taken at 2-month intervals. Root samples were analyzed for sugar content and 1.5 mm thick slices were fried at 157ºC in canola oil for 3 minutes for analysis on acrylamide content and color values. Acrylamide content in fried chips varied widely among genotypes, from below 300ng/g to over 9000 ng/g. No significant changes in acrylamide formation were found in most genotypes after curing but various trends were observed during storage. Initial results indicated that color values and reducing sugar content were not correlated with acrylamide levels in fried sweetpotato chips. Analysis of amino acids, which are substrates of browning reaction leading to acrylamide formation, are on-going. Development of near-infrared spectroscopy method for rapid quantification of acrylamide and other chemical constituents in sweetpotato clones to facilitate the breeding effort is in progress.

1. Characterization of probiotic lactic acid bacteria for use in refrigerated pickle products. The development of probiotic pickle products will be facilitated by identifying probiotics that can survive in acidified vegetable products with a proposed shelf life of two or more months. ARS researchers in Raleigh, North Carolina, analyzed the growth and acid resistance of ten probiotic lactic acid bacterial cultures mostly for dairy products, including one Pediococcus and nine Lactobacilli. They found that cultures selected for acid resistance had superior long-term survival (up to two months, 4°C) without significant loss of viability in a simulated refrigerated probiotic vegetable product that included a fermentation step. These data may be useful to aid the development of a commercial process for the manufacture of probiotic pickles.

2. Direct analysis of biomolecules from high-salt fermented cucumbers using infrared matrix-assisted lased desorption electrospray ionization (IR-MALDESI). High-salt samples present a challenge to mass spectrometry analysis, particularly when electrospray ionization is used, requiring extensive sample preparation steps such as de-salting, extraction, and purification. ARS researchers in Raleigh, North Carolina, collaborated with faculty at North Carolina State University in applying a novel mass spectrometry imaging approach for direct analysis of fermented cucumbers that contained a high concentration of salt. From several hundred observed unique substances, three triterpenoid lipids produced by cucumbers; ß-sitosterol, stigmasterol, and lupeol, were identified, and the spatial distribution of the lipids in a slice of fermented cucumber were imaged. This study demonstrates the capacity of the IR-MALDESI technique to deliver confident identifications from highly complex samples in molar concentrations of salt without the need for sample preparation.

Review Publications
Medina Pradas, E., Perez Diaz, I.M., Garrido-Fernandez, A., Arroyo-Lopez, F.N. 2016. Review of vegetable fermentations with particular emphasis on processing modifications, microbial ecology, and spoilage. In: Bevilacqua, A., Rosaria Corbo, M., Sinigaglia, M., editors. The Microbiological Quality of Food. Cambridge, MA: Woodhead Publishing. p. 211-236.
Fan, S., Breidt, F., Price, R.E., Perez Diaz, I.M. 2017. Survival and growth of probiotic lactic acid bacteria in refrigerated pickle products. Journal of Food Science. 82(1):167-173.
Barkley, S.L., Schultheis, J.R., Chaudhari, S., Johanningsmeier, S.D., Jennings, K.M., Truong, V.D., Monks, D.W. 2017. Yield and consumer acceptability of ‘Evangeline’ sweetpotato for production in North Carolina. HortTechnology. 27(2):281-290.
Ekelof, M., McMurtrie, E., Nazari, M., Johanningsmeier, S.D., Muddiman, D.C. 2017. Direct analysis of triterpenes from high-salt fermented cucumbers using infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI). Journal of American Society for Mass Spectrometry. 28(2):370-375.
Perez Diaz, I.M., Hayes, J.S., Medina-Pradas, E., Anekella, K., Daughtry, K.V., Dieck, S., Levi, M., Price, R., Butz, N., Lu, Z., Azcarte-Peril, M. 2017. Reassessment of the succession of lactic acid bacteria in commercial cucumber fermentations and physiological and genomic features associated with their dominance. Food Microbiology. 63:217-227.
Franco, W., Johanningsmeier, S.D., Lu, J., Demo, J., Wilson, E., Moeller, L. 2016. Cucumber fermentation. Book Chapter. Chapter 7. p. 107-155.