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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

2020 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
This is the final report for project 6070-41000-008-00D. Significant research results and technology transfer to pickled vegetable and sweetpotato industries was accomplished by ARS researchers at Raleigh, North Carolina, including 30 peer-reviewed journal articles, 4 book chapters, 2 review articles, 6 material transfer agreements, 29 cooperative research agreements, and 1 patent issued. Results for the life of the project for each objective and subobjective are below: Subobjective 1a: Develop starter cultures for cucumber fermentations brined with calcium chloride. A collection of 1400 bacteria, isolates from commercial pickling vessels, was developed by ARS researchers at Raleigh, North Carolina, to identify starter cultures that could be used for low salt cucumber fermentations to assure safety and performance consistency. Selected isolates, capable of fermenting cucumbers under various conditions of pH, salt content and temperature, were characterized and classified by DNA sequence analysis. Four selected bacterial cultures of Lactobacillus plantarum and the Lactobacillus pentosus species were made available for commercialization. A method to produce starter cultures that can meet kosher requirements was also developed and shared with processors. Subobjective 1b: Identified 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. Polyethylene plastic sheets, typically used for building greenhouses in the Midwest, were used by ARS researchers at Raleigh, North Carolina, to build insulating tank cover prototypes, that were tested in Michigan and North Carolina. Temperature monitoring showed the effectiveness of the approach and developed predictive heat transfer models. Insulating tank covers delayed the freezing of in-tank fermentation cover brines, but hot air was needed to prevent it during long-term storage. The addition of 15% glycerol in cover brines located at the top of the tanks, prevented freezing of top layers of the brine and facilitated the removal of the fermented stocks during the winter. A quality prediction chart for freeze and salting damage of fermented cucumbers was developed by ARS researchers at Raleigh, North Carolina. Data indicated that lower levels of sodium chloride can be used in tanks to prevent freezing damage, reducing environment problems due to disposal of waste salt. Subobjective 1c: Develop an accelerated shelf-life (ASLT) model for fermented cucumber pickles. ASLT was developed by ARS researchers at Raleigh, North Carolina, which enabled rapid and accurate prediction of the shelf-life of pickle products. A trained sensory panel found that sensory texture attributes of crispness and crunchiness were highly correlated to instrumental firmness values. This enabled the selection of an appropriate cutoff value to predict the end of shelf life using the ASLT model. Volatile compound profiles were analyzed to identify components that may be responsible for oxidized off-flavor development. Of the hundreds of components that changed during shelf storage of cucumber pickles, 20 newly identified compounds were highly associated with oxidized off-flavor development. The ASLT model enables pickle processors to rapidly predict the shelf-life of their products during shelf-storage to enhance the adoption of new, more environmentally friendly processes. Subobjective 2a: Determine the acid resistance and growth characteristics of probiotic lactic acid bacteria that influence their survival in acidified and fermented cucumbers; and 2b: Identify suitable methods for the incorporation of probiotic lactic acid bacteria in refrigerated cucumber pickles. Laboratory assays were developed by ARS researchers at Raleigh, North Carolina , to characterize probiotic lactic acid bacteria (LAB) for growth and survival in refrigerated pickle products. It was discovered by ARS researchers at Raleigh, North Carolina, that a particular LAB (Lactobacillus casei culture ATCC 393) survived for 2 months in a simulated refrigerated pickle product at levels that exceed the amount required for probiotic activity, if the pickles were allowed to ferment with the culture prior to refrigeration. Commercial probiotic LAB powders added directly into acidified cucumbers were viable for 2 months with pre-conditioning and supplementation with arginine, a naturally occurring amino acid. Probiotic pickles had higher consumer liking than the product without probiotics due to enhanced flavor. A market survey of 1,100 consumers indicated that they would prefer pickles labeled as ‘Probiotic,’ and ‘Supports digestive health’. This research by ARS researchers at Raleigh, North Carolina, demonstrated that high quality, probiotic refrigerated pickles can meet consumer preferences and be produced through careful culture selection and process modifications to deliver enough probiotic organisms in one serving of pickles to confer health benefits. Subobjective 2c: Evaluate production and/or retention of bioactive peptides in fermented cucumbers. LAB were found by ARS researchers at Raleigh, North Carolina, to produce bioactive peptides and health-promoting amino acids in addition to their primary role of acidification during vegetable fermentation. A novel mass spectrometry-based method developed for the direct analysis of peptides in fresh, acidified (vinegar added to fresh cucumbers), and fermented cucumbers identified five peptides with known anti-hypertensive properties in pickled cucumbers. Four of these peptides were uniquely present in fermented cucumbers. Another difference between acidified and fermented cucumbers (particularly those fermented in low salt brines) showed that gamma amino butyric acid (GABA) increased during fermentation. GABA is known to have anti-hypertensive and anti-anxiety effects and may improve cognitive function. This research showed that the content of health-promoting compounds is enhanced in fermented vegetables. Objective 3: Vegetables that dominate the United States market were incorporated in the development of environmentally sustainable preservation methods applicable in the reclaim of surplus volumes. Fresh corn, tomatoes, green bell peppers, green peas, green beans, green leaf lettuce and broccoli were acidified by ARS researchers at Raleigh, North Carolina, with vinegar and natural preservatives or fermented to achieve long-term microbial stability. Lactic acid producing bacteria prevailing in the fermentations were isolated and identified, and fermentations were stable for up to 14 days. For most vegetables, acidification with vinegar and natural preservatives resulted in stable brines for up to 60 days. The preservation of vegetables by fermentation or acidification can be helpful for meeting global demand for nutritious food and preventing vegetable spoilage. Objective 4: Variety selection for minimizing acrylamide in fried sweetpotato. Over 300 sweetpotato (SP) breeding lines were evaluated by ARS researchers at Raleigh, North Carolina, using wet chemistry and near infrared spectroscopy to measure beta-carotene, anthocyanins, total phenolics, asparagine and sugars in sweetpotato genotypes to facilitate breeding efforts. The quality of French fries and chips processed from over 300 new breeding lines were evaluated by ARS researchers at Raleigh, North Carolina, and at least 44 promising genotypes were selected for further evaluation by food processing companies. Fifteen SP cultivars were further evaluated by ARS researchers at Raleigh, North Carolina, for long-term storage and chip processing. Curing SP roots prior to long-term storage as commonly practiced in the United States resulted in an increase in sugars in 13 of the 15 cultivars but did not affect undesirable acrylamide formation during frying in most cases. Unlike white potato, there was no relationship between reducing sugar content or brown color development and acrylamide content in sweetpotato chips. In contrast, the contents of free amino acids, especially asparagine, was highly associated with acrylamide formation during sweetpotato chip processing. The mean acrylamide content in chips varied greatly (300 to over 9,000 parts per billion), indicating that genetic background should be carefully considered in varietal development for fried products with low acrylamide content. Sixteen sweetpotato cultivars with varying flesh color (orange, yellow, cream) and dry matter content were analyzed to determine the best characteristics for production of sweetpotato French fries (SPFF). Sugar, starch content and pasting properties, along with dry matter influenced SPFF texture. In particular, dry matter and starch contents of raw sweetpotatoes and instrumental texture measurements of SPFF could be used to screen sweetpotato types for producing high quality SPFF. These studies have identified important measurable properties of sweetpotato that may be used to develop and improve commercial products with consumer-preferred attributes.

1. Prospects for health-promoting fermented vegetables. A non-protein amino acid that may be present in both fresh and fermented vegetables, gamma-amino butyric acid (GABA), has been reported to promote anti-hypertensive and anti-anxiety effects as well as improve cognitive function. ARS researchers at Raleigh, North Carolina, optimized a mass spectrometry-based method to quantify amino acids, including health-promoting GABA, in raw, acidified, and fermented cucumbers. ARS researchers at Raleigh, North Carolina, showed that the content of several amino acids differed between acidified and fermented cucumbers, including production of GABA during cucumber fermentation. Brining in lower salt (2% sodium chloride) increased GABA formation during natural cucumber fermentation. Dill pickles that were fermented and packed in their original fermentation brines had the highest GABA content among the commercial products tested. These studies show that bioactive compounds were formed during natural, lactic acid fermentation of cucumber, and process modifications (i.e. lower salt fermentation without desalting or brine replacement) further enhances and retains the content of health promoting GABA in finished pickle products.

2. Variations in free amino acid profiles of commercial sweetpotato varieties. A new method for the determination of free amino acid profiles in sweetpotatoes was developed and validated by ARS researchers at Raleigh, North Carolina. This method was used to determine the free amino acid composition of five commercial sweetpotato cultivars. Asparagine was the most abundant free amino acid and content varied significantly among the cultivars. Fifteen other amino acids also varied among cultivars and contributed to the differentiation of the varieties based on their complete amino acid profiles. The method described herein is efficient, reliable, sensitive, and could be used for profiling free amino acid content to develop value-added sweetpotato varieties.

3. Discovering the genomic diversity of unique Lactobacillus buchneri cultures. L. buchneri, a common non-pathogenic bacterium, can cause spoilage of fermented cucumbers and other foods due to its unique metabolism. Conversely, this microbe is beneficial to stabilizing silage and has been proposed for use in several biotechnological applications. ARS researchers at Raleigh, North Carolina, in partnership with North Carolina State University, generated whole genome sequences of several unique cultures of L. buchneri and analyzed them to provide a deeper understanding of the universal and culture specific genetic characteristics of this microbial species. These newly sequenced and assembled genomes greatly expanded the number of reference genomes for this specie and were made publicly available through the National Center for Biotechnology Information Genbank repository.

4. Use of a modified Bag-in-Box technology for the fermentation of cucumbers. In a collaborative effort with Michigan State University, an ARS scientist at Raleigh, North Carolina, designed and built the first prototype for a Fermentation Purged Box to be used in the reclaiming of surplus vegetables to reduce food waste. The first Purged Box prototype was tested in-house for cucumber fermentations brined with low salt. The parameters and attributes of the fermentation microbiology and chemistry were measured and the quality of the fermented stocks assessed. Fermentation performance developed as expected and appropriate levels of microbial counts and lactic acid production were achieved. Quality aspects that need to be improved were identified, optimization strategies have been delineated and future research objectives identified for post-pandemic efforts.

5. Development of buffer capacity (BC) models for the analysis of salad dressing ingredients. The pH of most acid food products depends on undefined and complex buffering of ingredients but is critically important for regulatory purposes, product development and food safety. ARS researchers at Raleigh, North Carolina, developed a novel buffer capacity model to define the BC of ingredients in salad dressing products. Ingredients of salad dressings were titrated individually and in combination using concentrations typical of dressing products. Titration curves for ingredient solutions were used to generate BC curves. Most ingredients had very low BC values compared to the acetic acid that is typically present in salad dressing and pickled vegetable formulations. Therefore, the ingredients had little influence on the final product pH of the dressings tested. The BC data were used to quantify the effects of salad dressing ingredients on the final product pH to aid regulatory agencies and manufacturers in assessing product pH and safety.

6. Identification of bacterial viruses that aid vegetable fermentations. Lactic acid bacteria (LAB) dominate vegetable fermentations. However, rapid elimination of the non-lactic acid bacteria present on fresh vegetables may be important for the quality of the fermented pickles, in particular to reduce bloater defect (gas pockets in pickles) during fermentation. Bacterial viruses (phages) may be useful to enhance growth of LAB by helping eliminate competing bacteria. This study explored the abundance, diversity, and functional role of phages infecting undesirable bacteria in vegetable fermentations, including total of 39 phage isolates. Eight of these phage were selected by ARS researchers at Raleigh, North Carolina, for further characterization based on the ability to infect undesirable bacteria. The phage were characterized to determine basic characteristics, including protein and DNA structure as well as growth kinetics on selected bacterial hosts. The data indicate that the phage isolates may play an important role in the elimination of Gram-negative bacteria, thereby facilitating the dominance of LAB and possibly minimizing bloater defect.

7. Starter cultures that can initiate cucumber fermentations were developed. A starter culture can accelerate the conversion of sugars to acids in a fermentation while outcompeting undesired bacteria in commercial pickle production. Out of 243 bacterial isolates from commercial cucumber fermentations, ARS scientists at Raleigh, North Carolina, identified 18 cultures that were able to ferment cucumbers under various conditions of temperature, pH and salt content. Cultures that were unable to produce potentially carcinogenic biogenic amines in cucumber fermentation brines were selected for further study. These starter cultures produced exopolysaccharides or sugar chains that may serve as prebiotics, potentially feeding helpful bacteria naturally present in the human gut. The study included checks for potentially harmful antibiotic resistance traits that could be transferred to other bacteria. It was concluded by ARS researchers at Raleigh, North Carolina, that five of the selected cultures were suitable for use as starter cultures in low salt fermentations currently undergoing commercial development. One starter culture was transferred to the private sector for commercial production and was used in the USA, Germany, Mexico and Turkey for the manufacture of cucumber pickles. The availability of these starter cultures enables the continued and expanded use of the technology to manufacture pickles with low salt and minimized environmental impact.

Review Publications
Perez Diaz, I.M. 2019. Fermented vegetables as vectors for the relocation of microbial diversity from the environment to the human gut. Book Chapter. p 91-120.
Nethery, M.A., Daughtry, K.V., Henriksen, E., Johanningsmeier, S.D., Barrangou, R. 2019. Comparative genomics of eight Lactobacillus buchneri strains isolated from food spoilage. Biomed Central (BMC) Genomics. 20:902.
Lu, Z., Perez Diaz, I.M., Hayes, J., Breidt, F. 2020. Bacteriophages infecting gram-negative bacteria in a commercial cucumber fermentation. Frontiers in Microbiology. 11:1306.
Ucar, R.A., Perez Diaz, I.M., Dean, L.L. 2020. Content of xylose, trehalose and L-citrulline in cucumber fermentations and utilization of such compounds by certain lactic acid bacteria. Food Microbiology. 91:103454.
Perez Diaz, I.M., Dickey, A., Fitria, R., Ravishankar, N., Hayes, J.S., Campbell, K., Arritt, F. 2020. Modulation of the bacterial population in commercial cucumber fermentations by brining salt type. Journal of Applied Microbiology. 128(6):1678-1693.
Zhai, Y., Perez Diaz, I.M. 2020. Contribution of Leuconostocaceae to CO2-mediated bloater defect in cucumber fermentation. Food Microbiology. 91:103536.
Qiu, X., Reynolds, R., Johanningsmeier, S.D., Truong, V. 2020. Determination of free amino acids in five commercial sweetpotato cultivars by hydrophilic interaction liquid chromatography-mass spectrometry. Journal of Food Composition and Analysis. 92:103522.
Chilungo, S., Muzhingi, T., Truong, V., Allen, J. 2019. Effect of storage and packaging materials on color and carotenoid content of orange-fleshed sweetpotato flours. International Journal of Innovative Science and Research Technology. 4(9):362-369.
Chilungo, S., Muzhingi, T., Truong, V.D., Allen, J. 2019. Effect of processing and oil type on carotene bioaccessibility in traditional foods prepared with flour and puree from orange-fleshed sweetpotatoes. International Journal of Food Science and Technology. 54(6):2055-2063.
Truong, A.N., Thor, Y., Harris, K., Simunovic, J., Truong, V. 2019. Acid inhibition on polyphenol oxidase and peroxidase in processing of anthocyanin-rich juice and co-product recovery from purple-fleshed sweetpotatoes. Journal of Food Science. 84(7):1730:1736.
Franco, W., Perez Diaz, I.M., Connelly, L.E., Diaz, J. 2020. Isolation of exopolysaccharide-producing yeast and lactic acid bacteria from quinoa (Chenopodium quinoa) sourdough fermentation. Foods. 9(3):337.