Progress 11/06/14 to 09/30/19
Outputs
Target Audience:Farmers, food manufacturers, feed manufacturer, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?Over the duration of the project, the PD and Co-PD visited poi and taro producers and farms that used taro skins to feed animals on Oahu, Maui, and the Big Island. Findings from this project were shared with communities of interest via two articles published online via CTAHR Extension Communications titled "Microbial population in fermented cooked taro skins" in 2015 and "Poi: a superfood, native tradition, nature's gift" in 2018. To disseminate the information, the PD presented a poster titled "Probiotic foods: natto, yogurt, kimchee, and poi" at the Maui Fair in 2016. The results were also shared with interested food and health professionals via one presentation at the American Society forNutrition annual meeting in 2019 and three posters at the International Association for Food Protection annual meetings in 2015, 2016 and 2018. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Lactic acid bacteria (LAB) in fermented taro skins and poi have the potential to improve intestinal microbial balance and provide significant health benefits (probiotic) to humans and animals. We identified dominant bacterial species present in fermented taro skins by 16S rRNA gene sequencing and determined their antimicrobial potential against pathogenic bacteria. During fermentation, the LAB count of cooked taro skins increased by 5.7 log CFU/g in 34 h. All tested samples showed a dominance of LAB in the microbial populations, with Leuconostoc mesenteroides being the most frequently isolated species (75%). These LAB species showed varying antimicrobial activities against Salmonella Typhimurium and Listeria monocytogenes. The antimicrobial properties of certain LAB supernatants retained after being neutralized but reduced by proteinase. The physiological characteristics and potential health benefits of the isolated LAB were determined.Representative LAB strains were tested for their acid and bile tolerance and antimicrobial activity. The production of bacteriocins by the LAB isolates was also explored. 159 isolates belonging to 11 different LAB species were obtained, of which 40% were Leuconostoc mesenteroides. Four LAB isolates exhibited high tolerance to low pH and bile. Salmonella Typhimurium and Listeria monocytogenes died in their culture supernatants within 2 h and 8 h, respectively. One isolate #137 Lactococcus lactis was identified as a bacteriocin producer. Its genomic DNA carries two bacteriocin structural genes NisZ and Bac147.The anti-Listeria bacteriocins produced by the strain were heat-stable but sensitive to proteinase treatments. These results suggest both organic acids and bacteriocins may account for the antimicrobial activity of the LAB isolates. Conventional methods for isolating bacteriocin-producers are tedious and inefficient. We optimized medium composition of the deferred antagonism assay, which can simultaneously screen multiple LAB isolates for bacteriocin producing capability. The effects of different media, buffering salts, initial medium pH, and supplements were assessed with both bacteriocin producing and bacteriocin non-producing LAB strains. Tested bacteriocin-producers generated significantly larger inhibition zones in Elliker agar than M17 agar. The buffering salts did not significantly affect the size of inhibition zones. Inhibition zones caused by tested bacteriocin producing LAB enlarged with higher initial medium pH and the medium supplement Tween 80. Therefore, the Elliker agar with buffering salts, Na2HPO4 and NaH2PO4, and 1% Tween 80 at pH 6.9 was the most appropriate bottom media in deferred antagonism assay. This improved assay can enhance the possibility of isolating bacteriocin producing LAB from food. We also optimized the production of bacteriocins by LAB in liquid culture media. The influence of different broth, initial pH, and supplements on their antimicrobial activity was determined. Lactococcus lactis strains produced more bacteriocins in MRS broth than in M17 broth and Elliker broth. In comparison, the type of culture medium did not significantly affect bacteriocin production by Lactobacillus plantarum. Higher initial medium pH increased the bacteriocin production. The bacteriocin units of all tested LAB strains at pH 6.9 were more than three-fold higher than at pH 5.5. As for the supplements, 1% Tween 80 effectively increased bacteriocin production by Lactococcus lactis strains, and 1% ethanol showed remarkable enhancement in bacteriocin production by the Lactobacillus plantarum. Finally, the prebiotic potential of taro was assessed with probiotic LAB in in vitro human digestion system. Prebiotics are carbohydrates that are indigestible by the digestive tract, which can selectively stimulate probiotic growth and/or activities in the colon. Four probiotic Lactobacillus species were individually paired with 2% (w/v) taro. The pairings were subjected to an in vitro human digestive tract simulation of the mouth, stomach, and intestinal conditions to assess the fate of tested probiotics. Furthermore, an auto-agglutination assay was conducted to evaluate the effect of taro on self-agglutination of the individual probiotics. The Caco-2 cell line was used to determine whether taro could influence the ability of tested probiotics to adhere to human intestinal epithelial cells. Results indicated that L. acidophilus, L. paracasei, and L. plantarum experienced greater growth in the simulated intestinal tract when paired with taro than with inulin or glucose. In addition, L. paracasei showed strong self-agglutination ability and had the greatest adherence percentage to Caco-2 cells. In conclusion, taro is a strain-specific potential prebiotic that may be utilized to aid the modulation of a healthy gut microbiota.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Saxby, S., Li, Y., Lee, C.N., and Kim, Y.S. 2019. Assessing the prebiotic potential of taro (Colocasia esculenta) with probiotic Lactobacillus species in an in vitro human digestion system. Current Developments in Nutrition https://doi.org/10.1093/cdn/nzz040.P20-022-19
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Farmers, food manufacturers, feed manufacturer, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?The results have been shared with interested farmers and poi manufacturers. A trifold brochure titled "Poia superfood: native tradition, nature's gift" was published online via CTAHR Extension Communications. What do you plan to do during the next reporting period to accomplish the goals?Characterize isolated lactic acid bacteria strains and determine their probiotic potential.
Impacts
What was accomplished under these goals?
To optimize the production of bacteriocins by lactic acid bacteria (LAB), the influence of different liquid culture media (MRS, M17, and Elliker broth) at different initial pH (5.5-6.9) with 1% Tween 80 and/or 1% ethanol on their antimicrobial activity was determined. Fresh cultures of five selected LAB strains were centrifuged. Then the supernatants were neutralized to pH 5.8 and filter-sterilized. The antimicrobial activity of bacteriocins produced by the strains was quantified using a micro-titer plate assay with Listeria monocytogenes as the target bacteria. Growth inhibition of Listeria monocytogenes was measured spectrophotometrically at 600 nm. One bacteriocin unit (BU) was defined as the amount of bacteriocin which inhibited the growth of Listeria monocytogenes by 50% under standard assay conditions. Lactococcus lactis strains produced more bacteriocins in MRS broth than in M17 broth and Elliker broth. In comparison, the type of culture medium did not significantly affect bacteriocin production by Lactobacillus plantarum. Higher initial medium pH increased the bacteriocin production. The bacteriocin units of all tested LAB strains at pH 6.9 were more than three-fold higher than at pH 5.5. As for the supplements, 1% Tween 80 effectively increased bacteriocin production by Lactococcus lactis strains, and 1% ethanol showed remarkable enhancement in bacteriocin production by the Lactobacillus plantarum. Bacteriocins produced by LAB may be utilized as part of the hurdle technology to improve the quality and safety of food.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2018
Citation:
Zhan, Z., Dong, J., Nyean, L., and Li, Y. 2018. Isolation of bacteriocin-producing lactic acid bacteria from fermented foods using improved deferred antagonism assay. Journal of Food Protection 81S:297.
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:Farmers, food manufacturers, feed manufacturer, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Bacteriocins are antimicrobial peptides or proteins produced by bacteria. Lactic acid bacteria (LAB) are common bacteriocin-producers and often used in the production of fermented food. Bacteriocins can inhibit certain bacteria causing food spoilage or foodborne disease. Conventional methods for isolating bacteriocin-producers are tedious and have low efficiency. This study aimed to optimize the medium composition of deferred antagonism assay, which can simultaneously screen multiple LAB isolates for bacteriocin producing capability. Three types of media (de Man,RogosaandSharpe [MRS] agar, M17 agar, and Elliker agar) with two types of buffering salts (disodium-β-glycerophosphate and the combination of Na2HPO4 and NaH2PO4) at different initial medium pH (5.5-6.9) were tested with known LAB strains via deferred antagonism assay. Tween 80 and ethanol were added at 1% to the isolation media to assess their effect on bacteriocin production. Both bacteriocin producing and bacteriocin non-producing LAB formed inhibition zones on MRS agar with the two types of buffering salts. There was no inhibition zone caused by bacteriocin non-producers in other two types of media. Tested bacteriocin-producers generated significantly larger inhibition zones in Elliker agar than M17 agar. The buffering salts did not significantly affect the size of inhibition zones. But disodium-β-glycerophosphate was reported to inhibit the growth of Lactobacillus bulgaricus. Inhibition zones caused by tested bacteriocin producing LAB enlarged with higher initial medium pH and the medium supplement Tween 80. Therefore, the Elliker agar with buffering salts, Na2HPO4 and NaH2PO4, and 1% Tween 80 at pH 6.9 was the most appropriate bottom medium in deferred antagonism assay. This improved assay can enhance the possibility of isolating bacteriocin producing LAB from food.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2017
Citation:
Z. Zhan, J. Dong, C.N. Lee, and Y. Li. 2017. Isolation of bacteriocin-producing lactic acid bacteria from fermented foods using an improved deferred antagonism assay. University of Hawaii College of Tropical Agriculture and Human Resources Student Research Symposium. Honolulu, HI.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:Farmers, feed manufacturers, food manufacturers, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?They were presented at the 2016 International Association of Food Protection Annual Meeting in St. Louis, Missouri. What do you plan to do during the next reporting period to accomplish the goals?We will determine the effect of selected probiotic LAB on controlled fermentation of taro skins.
Impacts
What was accomplished under these goals?
Salmonella and Listeria have caused a large number of foodborne illnesses and great economic loss in food and animal production. Piglets recover faster from gastrointestinal illness when fed fermented taro skins. Our previous work shows that lactic acid bacteria (LAB) dominate the microbial population of the taro skins. This study aimed to determine the physiological characteristics and potential health benefits of the isolated LAB. Cooked taro skins were obtained from four poi manufacturers in Hawaii. After natural fermentation, LAB isolates were obtained on MRS agar and identified by 16S rDNA gene sequencing. Representative LAB strains were tested for their acid and bile tolerance and antimicrobial activity. We also explored the production of bacteriocins by the LAB isolates. 159 isolates belonging to 11 different LAB species were obtained, of which 40% were Leuconostoc mesenteroides. Four LAB isolates exhibited high tolerance to low pH and bile. Salmonella Typhimurium and Listeria monocytogenes died in their culture supernatants within 2 h and 8 h, respectively. Proteinase K treatment reduced the inhibition zone of certain supernatants against L. monocytogenes. After neutralization, only the supernatant of isolate #137 Lactococcus lactis still retained the bactericidal activity against L. monocytogenes. In PCR test, two bacteriocin structural genes were amplified by primers NisZ and Bac147 from its genomic DNA. #137 is the first Lactococcus lactis strain reported to carry these two bacteriocin genes simultaneously. The anti-Listeria bacteriocins produced by the strain were heat-stable but sensitive to proteinase K and pronase E treatments. Both organic acids and bacteriocins may account for the antimicrobial activity of the LAB isolates. These isolates could potentially be used as probiotics to fight bacterial infections and confer other health benefits in humans and animals. Bacteriocins produced by the isolates would offer a promising biocontrol tool for the inhibition of pathogenic bacteria in food. During the past year, the PD and Co-PD Dr. CN Lee visited poi and taro producers on Oahu and the Big Island and disseminated findings from this research.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2016
Citation:
Li, Y., Li, Q., Lee, C.N., and Dunn, M.A. 2016. Probiotic potential of lactic acid bacteria isolated from fermented taro skins. Journal of Food Protection 79S: 136-137.
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2015
Citation:
Li, Q. 2015. Novel antimicrobial activity of lactic acid bacteria isolated from taro peels. University of Hawaii at Manoa Master Thesis.
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Progress 11/06/14 to 09/30/15
Outputs
Target Audience:Farmers, feed manufacturers, food manufacturers, and consumers. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?They were presented at the 2015 International Association of Food Protection Annual Meeting in Portland, Oregon. What do you plan to do during the next reporting period to accomplish the goals?We plan to determine the acid and bile tolerance of lactic acid bacteria isolates and explore their production of bacteriocins.
Impacts
What was accomplished under these goals?
Taro is the staple of native Hawaiian diet and an important part of Polynesian culture. Pigs recover from gastrointestinal illness when fed cooked taro skins. It was hypothesized that lactic acid bacteria (LAB) may be present in the taro skins and responsible for the recovery. This study aimed to identify dominant bacterial species present in fermented taro skins and determine their antimicrobial potential against diarrhea-causing bacteria. Three samples of cooked taro skins were obtained from a taro processing facility and incubated at 21°C. Aerobic plate count and LAB count of the sample were determined at 0, 24, and 34 h. Selected bacterial isolates were identified by sequencing the 16S rRNA gene. Culture supernatants of representative LAB isolates were evaluated on their bactericidal properties against Salmonella Typhimurium and Listeria monocytogenes. Moreover, the supernatants were neutralized with NaOH or treated with proteinase K before being further assessed on their antimicrobial activity. During fermentation, the LAB count of cooked taro skins increased from 3.0 log CFU/g to 8.7 log CFU/g in 34 h. All samples showed a dominance of LAB in the microbial populations. LAB constituted 91% of 37 sequenced isolates. Leuconostoc mesenteroides was the dominant LAB species (75%), with Lactococcus lactis and Weissella soli also present. These LAB species showed varying antimicrobial activity against tested diarrhea-causing bacteria. S. Typhimurium died in culture supernatants of L. mesenteroides and W. soli within 2 h; L. monocytogenes died in a culture supernatant of L. mesenteroides within 8 h. Compared with control, the supernatants still showed antimicrobial effects after being neutralized. The proteinase treatment reduced the inhibition zones of the supernatants against the tested pathogens. This study indicates the presence of beneficial LAB in fermented taro skins. The LAB species could potentially be utilized to enhance food safety and human health.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2015
Citation:
Li, Q., Ishimoto, J., Yoshiota, J., Lee, C.N. and Li, Y. 2015. Antimicrobial activity of lactic acid bacteria isolated from fermented taro skins against Salmonella Typhimurium and Listeria monocytogenes. Journal of Food Protection 78: S260
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