Progress 10/21/16 to 09/30/21
Outputs
Target Audience:Farmers, 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?The results were shared with interested food professionals via four presentations at the International Association for Food Protection annual meetings in 2019, 2020, and 2021. We also conducted applied research on establishment of ohelo plantings, essentially domestication of a wild species,with the intention of using that information in extension activities to develop a new crop. A small field was established at Volcano research station to test establishment and cultivation/crop management techniques to be later shared with potential growers, and for the field to eventually be a demonstration plot. There is variation among plants, so we also collected information to identify desirable traits and superior plants. The work relied in part on earlier experience of PIs with blueberriesfor Hawaii, and in developingohelo ai (Vaccinium reticulatum)as an ornamental potted plant. A few plants were shared with cooperators for trial planting in different locations. Unfortunately, Covid and the departure (retirement) of key personnel, plus the lack of a market for fruit, has hampered further extension-outreach work. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Listeria monocytogenes is a foodborne pathogen and causes illnesses with a high mortality rate in susceptible populations. Several dairy related outbreaks have been attributed to contamination by L. monocytogenes, which requires antimicrobial interventions to enhance the safety of these products. We evaluated the antimicrobial activity of ohelo berry (Vaccinium calycinum) against L. monocytogenes in culture media and milk products. The effect of ohelo berry juice at sub-inhibitory concentrations on the physicochemical properties, biofilm formation, and gene expression of L. monocytogenes was also investigated. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ohelo berry juice against L. monocytogenes were 12.5% and 50%, respectively. A sub-inhibitory concentration of ohelo berry juice (6.25%) significantly increased the auto-aggregation and decreased the hydrophobicity, swimming motility, and swarming motility of L. monocytogenes.Biofilm formation capability of L. monocytogenes in Mueller Hinton broth decreased significantly by 45.67% and 77% with 3.12% and 6.25% ohelo berry juice, respectively.The relative expression of genes for motility (flaA), biofilm formation and disinfectant resistance (sigB), invasion (iap), listeriolysin (hly), and phospholipase (plcA) was significantly downregulated in L. monocytogenes treated by the 6.25% juice. L. monocytogenes was significantly inhibited in whole and skim milk supplemented with 50% ohelo berry juice, regardless of the fat content. Because ohelo berry is a wild relative of cranberry, the total phenolic contents and antimicrobial properties of these two fruits were determined and compared. Crude extracts of ohelo berry and cranberry were measured for pH, phenolic content, and anthocyanin concentration. Each extract was further evaluated against Escherichia coli and Staphylococcus aureus via the agar well diffusion assay and determined for MIC and MBC. Ohelo berry extract (pH 3.23) was less acidic than cranberry extract (pH 2.78). The phenolic content of ohelo berry was about three times that of cranberry (84.6 mg/g vs 27.24 mg/g dry weight). However, the anthocyanin concentration of cranberry extract (3.99 mg/g) was double that of ohelo berry (1.91 mg/g). In the antimicrobial test, inhibition zones generated by the two berry extracts against E. coli were not significantly different. However, the inhibition zone of ohelo berry extract against S. aureus was bigger than that of cranberry extract. Moreover, cranberry extract exhibited lower MBC against E. coli, whereas ohelo berry extract had lower MBC against S. aureus. To understand the antimicrobial potential of constituents of ohelo berry, its crude extract was separated into sugar plus organic acids (F1), non-anthocyanin phenolics (F2), and anthocyanins (F3). It was found that F3 had the highest concentrations of total phenolics and anthocyanins among all three fractions. None of the fractions generated a clear inhibition zone against E. coli or Salmonella Typhimurium in agar. However, they caused a significant reduction in the counts of tested bacteria in broth at 24 h. The largest inhibition zones against S. aureus and L. monocytogenes were generated by F3, at 16.5 and 14.25 mm, respectively. The MIC of F3 against S. aureus and L. monocytogenes were 6.85 and 13.69 cyanidin-3-glucoside equivalent mg/L, respectively, which were not affected by neutralization. Besides, the MIC and MBC of F2 against S. aureus and L. monocytogenes were not affected considerably by pH neutralization.Finally, F3 was further separated into anthocyanidins and proanthocyanidins (PAC). The MIC of PAC against E. coli was determined to be 300 μg/ml, while the growth of probiotic Lactobacillus rhamnosus GG was not affected even at the highest tested PAC concentration (800 μg/ml). A sub-inhibitory concentration of PAC (100 μg/ml) significantly reduced the motility and biofilm formation capability of E. coli. Overall, ohelo berry possesses strong antimicrobial property comparable to cranberry. Both organic acids and phenolics contribute to its antimicrobial property. The berry extracts have the potential to conserve probiotic bacteria while inhibiting pathogenic bacteria. Ohelo berry may be useful as a natural antimicrobial for preserving food and enhancing human health.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Wu, B., Liu, X., Nakamoto, S.T., and Li, Y. 2021. Effect of Ohelo berry (Vaccinium calycinum) Juice on Physicochemical Properties, Biofilm Formation, and Virulence Gene Expression of Listeria Monocytogenes. Journal of Food Protection 84S:40
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Wu, B., Liu, X., Nakamoto, S.T., Wall, M., and Li, Y. 2022. Antimicrobial Activity of Ohelo Berry (Vaccinium calycinum) Juice against Listeria monocytogenes and Its Potential for Milk Preservation. Microorganisms 10:548. https://doi.org/10.3390/microorganisms10030548
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Farmers, 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?Given that research is in the early, preliminary stages, there is little to report for ohelo or coffee. The research has been informally shared with individual growers and agricultural professionals, who expressed interest in products that could utilize what are now byproducts or waste. What do you plan to do during the next reporting period to accomplish the goals?We plan to investigate the antimicrobial properties of coffee cherry extract against foodborne pathogens.
Impacts
What was accomplished under these goals?
Ohelo berry (Vaccinium calycinum) is a Hawaiian wild relative of cranberry (Vaccinium macrocarpon). The concentration of proanthocyanidins in ohelo berry is approximately twice as high as that in cranberry, which is known to have antimicrobial activities against pathogenic bacteria.This study aimed to investigate the antimicrobial, antibiofilm, and antimotility effects of ohelo berry proanthocyanidins (oPAC) on Escherichia coli O157:H7.The crude methanol extract of ohelo berry was prepared from freeze-driedoheloberries and separated using a C-18 Sep-Pak cartridge into sugarplus organic acids (F1), non-anthocyanin phenolics (F2), and anthocyanins plus proanthocyanidins(F3). F3 was further separated into oPAC and anthocyanidins using anLH-20 Sephadex column. The minimum inhibitory concentration (MIC) of oPAC was determined against E. coli O157: H7 and probiotic Lactobacillus rhamnosus GG. The antibiofilm and antimotility activities of oPAC against E. coli O157:H7 were evaluated using the standard microtiter plate assay and semisolid agar assay, respectively.The MIC of oPAC against E. coli O157:H7 was 300 µg/ml, while the growth of probiotic L. rhamnosus GG was not affected even at the highest tested oPAC concentration (800 µg/ml). A sublethal concentration of oPAC (100 µg/ml) significantly inhibited biofilm formation of E. coli O157:H7 by 62%. In addition, it significantly impaired flagella-mediated motility of E. coli O157:H7. The colony diameter was 3 mm compared to 33 mm for the untreated cells.These results indicated that proanthocyanidins in ohelo berry have the potential to conserve probiotic bacteria while inhibiting pathogenic bacteria. It may be useful as a natural antimicrobial for preserving foodand enhancing gut health.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Wu, B., Nakamoto, S.T., and Li, Y. 2020. Antimicrobial properties of proanthocyanidins in Ohelo Berry (Vaccinium calycinum) against Escherichia coli O157:H7. Journal of Food Protection 83S:212
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Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Farmers, 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?The PI and co-PI visited a farm on Big Island that plants ohelo berry. The findings from this study were shared with the farmer and other interested parties. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Ohelo berry (Vaccinium calycinum), the Hawaiian wild relative of cranberry, was suggested to have antimicrobial properties in our previous studies. It was hypothesized that the antibacterial activity of ohelo berry might be attributed to various bioactive compounds it contains. The purpose of this study was to determine which constitutes have the most potent antimicrobial effects on pathogenic bacteria. The crude extract of ohelo berry was separated using a C-18 Sep-Pak cartridge into sugar plus organic acids (F1), non-anthocyanin phenolics (F2), and anthocyanins (F3). Each fraction was analyzed for pH, °brix/acid ratio, phenolics, and anthocyanin content. The antimicrobial properties of crude extract and the three fractions at their native pH and neutral pH were evaluated against Escherichia coli O157:H7, Salmonella Typhimurium, Staphylococcus aureus, and Listeria monocytogenes via the agar well diffusion assay. Moreover, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the fractions were determined against the four bacteria. The results demonstrated that F3 contained the highest concentrations (p<0.05) of total phenolics and anthocyanins among all three fractions. None of the fractions generated clear inhibition zone against E. coli O157:H7 or S. Typhimurium in agar. However, they caused a significant reduction (p<0.05) in all tested bacteria, compared with control, in 24 h. F1 at native pH had the same MIC (1.39/0.36 °Brix/acid) and MBC (5.55/1.06 °Brix/acid) against the four bacteria, while neutralized F1 did not inhibit the growth of any tested pathogens. The largest inhibition zones against S. aureus and L. monocytogenes were generated by F3, at 16.5 and 14.25 mm, respectively. The MIC of F3 against S. aureus and L. monocytogenes were 6.85 and 13.69 cyanidin-3-glucoside equivalent mg/L, respectively, which were not affected by neutralization. Besides, the MIC and MBC of F2 against S. aureus and L. monocytogenes were not affected considerably by neutral environment. Both phenolics and organic acids contribute to the antimicrobial properties of ohelo berry. They have the potential to be used as natural antibacterial agents in the food industry.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Liu, X.H., Nakamoto, S.N. and Li, Y. 2019. Antimicrobial properties of ohelo berry (Vaccinium reticulatum) fractions: anthocyanins, non-anthocyanin phenolics, and organic acids. Journal of Food Protection 82S:180.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Liu, X.H., Nakamoto, S.N. and Li, Y. 2019. Comparison of the Antimicrobial Activities of Ohelo Berry (Vaccinium reticulatum) and Cranberry (Vaccinium macrocarpon). Journal of Food Protection 82S:180.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Farmers, 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?We plan to use extension publications, presentations/posters at coffee industry events, and/or workshops. What do you plan to do during the next reporting period to accomplish the goals?We plan to fractionate bioactive compounds in ohelo berry and determine their antimicrobial activity individually. In cooperation with our other project, we plan to use extension publications, presentations/posters at industry events, and/or workshops to disseminate information to industry and potential growers.
Impacts
What was accomplished under these goals?
Oheloberry, a wild relative of cranberry, is an endemic plant in Hawaii. Previous research indicates that bothoheloberry and cranberry are rich sources of phenolic compounds, which may hold antimicrobial potential. This study aimed to determine and compare the total phenolic contents and antimicrobial activities ofoheloberry and cranberry. Freeze driedoheloberry and cranberry powder were individually extracted with organic solvent 80% methanol with 0.5% acetic acid. The crude extracts ofoheloberry and cranberry were measured for pH, the concentration of phenolic compounds by the Folin Ciocalteu method, and the concentration of anthocyanins by the pH differential method. Each extract was further evaluated against bacteria via the agar well diffusion assay and determined for the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Two bacterial strains,Escherichia coliandStaphylococcus aureus, were used in the microbiological analysis. Both extracts ofoheloberry and cranberry were acidic, with pH 3.23 and 2.78, respectively. The concentration of total phenolics inoheloberry was about three times higher than that of cranberry (84.6 mg/g vs 27.24 mg/g dry weight). However, the anthocyanin concentration of cranberry extract (3.99 mg/g) was double that of ohelo berry (1.91 mg/g). In the antimicrobial test, cranberry extract generated bigger inhibition zones againstE. colithanoheloberry extract. But the inhibition zone ofoheloberry extract againstS. aureuswas bigger than that of cranberry extract. In the MIC and MBC analysis, the extract of cranberry only had lower MIC againstE. coli, while lower MIC and MBC were observed with the extract ofoheloberry toS. aureus. Overall,oheloberry possesses strong antimicrobial activity comparable to cranberry.
Publications
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Progress 10/21/16 to 09/30/17
Outputs
Target Audience:Farmers, 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?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We plan to test the Ohelo berry extracts against common foodborne pathogenic bacteria.
Impacts
What was accomplished under these goals?
Pathogenic bacteria and viruses present in food pose a serious public health concern. In this study we investigated the antimicrobial activity of Ohelo berry against model bacteria and bacteriophage. Juice was made from Ohelo berries and neutralized with NaOH. Both Ohelo berry juice (OJ) and neutralized Ohelo berry juice (NOJ) were serially diluted and tested against Escherichia coli and Staphylococcus aureus via agar well diffusion assay. In addition, these bacteria and bacteriophage MS2 were individually inoculated into OJ and NOJ and incubated at 35°C. Over time, the bacteria and MS2 were enumerated by plate counting and plaque assay, respectively. Finally, the growth potential of E. coli and S. aureus in nutrient broth containing 25% OJ or NOJ was evaluated at 35°C. NOJ did not generate any inhibition zones against E. coli. Its minimum inhibitory concentration against S. aureus was 12.5%. In NOJ, E. coli at 5.25 log CFU/ml reached 7.95 log CFU/ml at 24 h. S. aureus at 4.66 log CFU/ml was inactivated within 24 h. In OJ and NOJ, MS2 at 4.75 log PFU/ml reduced by 1.54 and 1.35 logs, respectively, within 1 h. At 24 h, MS2 became undetectable in OJ and reached 1.3 log PFU/ml in NOJ. Finally, E. coli and S. aureus in nutrient broth containing 25% OJ died within 24 h and 48 h, respectively. With a pH value of 3.1, Ohelo berry has high contents of total phenolics and proanthocyanidins. These results suggest that Ohelo berry juice possesses strong antimicrobial activity, which may be due to a combined effect of organic acids and phenolic constituents.
Publications
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