tstar proposal application template - ctahr · web viewto illustrate the health promoting...

TSTAR-Pacific FY 2011Proposal Cover Page

1. Project Title: Enhancing the Quality, Value and Safety of Fermented Taro

2. Project Director/Principal Investigator (PD) Contact Information:

Yong Li, PhDAssistant Professor University of Hawaii at Manoa Department of Human Nutrition, Food and Animal Sciences 1955 East West Rd, AgSci 216, Honolulu, HI 96822Phone: 808-956-6408Fax: 808-956-4024Email: [email protected]

3. Total Project Funding Requested: $148,570.4

Other Project Information

1. Are Human Subjects are involved? √ Yes No

If Yes, 1a. Is the IRB Review Pending? √ Yes No IRB Approval Date:

Exemption number [1-6]: Human Subjects Assurance Number:

2. Are Vertebrate Animals Used? Yes √ No

If Yes, 2a. Is the IACUC Review Pending? Yes No IACUC Approval Date: Animal Welfare Assurance Number:

3. Does project involve recombinant DNA? Yes √ No

If yes, 3a. IBC Review Pending? Yes NoDate of Review:

4. Is proprietary/privileged information included in the application? Yes √ No

5a. Does this project involve activities outside the US? Yes √ No

5b. If yes, identify countries:

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mailto:[email protected]

Table of Contents

SectionTotal # of Pages

in SectionStarts on

Page #

Proposal Cover Page 1 1

Table of Contents 1 2

Project Summary / Abstract 1 3

Project Narrative (Page limit: 10 pages max) 10 4

- Justification/Literature Review 4

- Objectives Listed 8

- Procedures 8

- Project Timetable 12

- Expected Outcomes / Impacts 12

- Plan for Outreach or Technology Transfer 13

References to Project Narrative 3 14

Key Personnel List 1 17

Project Director CV (Publications: list last 4 years, 2 page max) 2 18

Other Co-PD CVs 4 20

Current and Pending Support Forms for PDs and Co-PDs 3 24Collaborative Arrangements, Sub-Contracts (including Letters of

Support)2 27

Budget and Narrative (Year 1) 2 29

Budget and Narrative (Year 2) 2 31Cumulative Budget 1 33

Appendices 2 34

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PROJECT SUMMARY / ABSTRACT

Project Director(s) (PD):Intentionally Left Blank

PD _Yong Li_____________ Institution University of Hawaii at Manoa

CO-PD _Wayne Iwaoka________ Institution University of Hawaii at Manoa

CO-PD Michael Dunn Institution University of Hawaii at Manoa

CO-PD Institution

Project Title: Enhancing the Quality, Value and Safety of Fermented Taro

Key Words: Fermented taro, quality, value, safety, controlled fermentation, probiotic

For TSTAR Proposals Only:

Panel: __________________

For centuries, taro has provided a nutritious staple food for Hawaiians and other Polynesians throughout the Pacific. However, the last decades witnessed a continuous decrease in the number of taro farms, the amount taro planted, and the productivity of some taro varieties. In Hawaii, more than 90% of taro is processed into poi, a fermented paste from pounded, cooked taro corms. Despite being an excellent carbohydrate source, poi has displayed little commercial value outside of Hawaii. Further, the natural fermentation process may yield products with inconsistent quality or even present a safety concern. There have been several high-profile outbreaks of foodborne illness associated with poi.

This project proposes to exploit the potential of lactic acid bacteria (LAB) in poi production and preservation. LAB in naturally fermented poi will be isolated and characterized. The dominant LAB in poi that have strong fermentation capability will be employed in controlled fermentation to enhance product quality. Those with antimicrobial activity will be used to inhibit the growth of pathogenic bacteria. The isolates exhibiting health-promoting (probiotic) effects will be employed to develop functional poi. Promoting poi as a non-dairy probiotic food could raise its popularity among health conscious people and expand poi’s consumer base considerably, which would help revitalize the taro industry. The improvement in the control of pathogenic bacteria potentially present in poi would minimize the risk of poi-borne illness and protect the public’s health.

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Project Narrative

Project Title

Enhancing the Quality, Value and Safety of Fermented Taro

Introduction

Taro is an important crop native to tropical and subtropical regions around the world. In Hawaii, taro is a vital part of the nutritional, spiritual, and agricultural traditions of the Hawaiian people (Hollyer et al., 1997). The crop takes 10-14 months to mature, but the plants are generally hardy and resilient. The plant is grown primarily for its edible corm; though the leaves and stalk are consumed by certain ethnic groups. Since taro corms are bulky and eventually perishable, they have been predominantly fermented into a natural end product called “poi”. Being an excellent carbohydrate source, poi has been a traditional staple food in Hawaii. Fermentation adds significant value to the crop. Combined, the islands grow approximately 5 million pounds of raw taro, which are valued at just under $3 million annually. By contrast, poi generates revenues topping $23 million each year (Conrow, 2005). Due to urbanization, however, Hawaii lost 60 taro farms between 1994 and 2004, bringing the total down to about 130. At the same time, the amount of land planted with taro plummeted from 550 to 370 acres (Conrow, 2005). Taro production has been declining steadily from about 14 million pounds in 1950 to an average of about 5 million pounds a year from 1980 on (USDA, 2010). Despite being popular in Hawaii, taro and poi have displayed very little commercial value outside of the islands. Moreover, the natural fermentation process may yield products with inconsistent quality or even present a safety concern. There have been several outbreaks of foodborne illness associated with poi contaminated by Shigella or Staphylococcus aureus (CSPI, 2008; Khambaty, et al., 1997; Lewis et al., 1972). These pathogens have posed serious public health hazards and caused significant economic losses.

As a matter of fact, taro and poi have many unique chemical and biological properties. Taro and poi are rich in minerals. The starch in poi is composed of small granules and is extremely digestible. Poi is considered hypoallergenic and can be used as a food substitute for people allergic to dairy or cereal products (Brown and Valiere, 2004). Further, lactic acid bacteria (LAB) thrive in naturally fermented poi. They can be isolated and used as starter cultures in controlled fermentation that can generate products with reliable characteristics. Certain LAB even have positive effects on human health. Therefore, it would be highly desirable to produce poi with these health-promoting (probiotic) organisms that would make poi a functional food and expand its consumer base considerably. To fulfill its potential, it is imperative to ensure the safety of poi. LAB may produce a large variety of antimicrobial compounds and control pathogenic bacteria present in the same food ecosystems (Alakomi et al., 2000; Batish et al., 1997; Cleveland et al., 2001). Our long-term goal is to promote the taro industry in Hawaii and other tropical and subtropical regions in the U.S. by developing high quality, long shelf-life, and value-added taro products. To reach this goal, LAB in naturally fermented poi will be profiled and characterized. These LAB could play a pivotal role in improving the quality, profitability, and safety of the fermented taro.

Justification

This project will fulfill the following four TSTAR program goals.

3. Enhance the role of value-added agriculture in tropical island ecosystems;

4. Expand and diversify presently unexploited food and fiber products which have potential for

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commercial production in the U.S. tropical and subtropical regions;

5. Expand tropical and subtropical agriculture’s linkages to related industries and economic sectors;

8. Enhance the linkages of agricultural and food production and consumption by designing foods and intervention strategies that lead to healthy and productive citizens in the tropical and sub-tropical regions.

Food products containing probiotic microorganisms are gaining popularity among U.S. consumers. Data shows people would rather prevent than cure disease, are more aware of the link between health and nutrition, and would like to spend less money on health care (Naidu et al., 1999). Probiotics provide a natural, non-chemical means of supplementing human health. The global functional food market is thriving with up to a $50 billion annual share (Stanton et al., 2005). Although many probiotic foods are of dairy origin, non-dairy probiotics are becoming more common. According to Mattila-Sandholm et al. (2002), “new product categories […] will certainly be the key research and development area for future functional food markets”. Due to the dominance of functional dairy products on the market, lactose intolerant consumers, totaled about 40 millions in the U.S., have few if any options when it comes to probiotic foods. The growing market for functional foods demands variation.

Poi, a thin, pasty, highly digestible mass of taro starch, potentially represents a viable option as a non-dairy probiotic food. Because it is naturally fermented, poi may already contain probiotic organisms and would not be subject to the development problems faced by other non-dairy functional foods. Controlling factors vital to bacterial survival, such as pH, water activity, storage temperature, oxygen level, and presence of competing microorganisms, are a serious challenge to the development of most non-dairy probiotic foods whereas LAB grow and thrive in poi. To illustrate the health promoting potential of the bacteria that ferment poi, it is said that ancient Hawaiians had no word for indigestion. Poi has been used to treat infants suffering from failure-to-thrive. It has tumor specific anti-cancer properties (Brown et al., 2005a). Despite having a large market on the islands of Hawaii, poi is virtually unknown elsewhere; though many people from various cultures know taro. Isolating and characterizing the LAB in poi will increase our knowledge of a Hawaiian food that is an essential part of Polynesian heritage. Finding these organisms to be probiotic could expand the consumer base of poi considerably; increased awareness of poi could raise its popularity among health conscious people of non-Polynesian ancestry. Controlled fermentation of poi with the probiotic LAB isolates would greatly improve its product quality and market value. In 2009, Hawaii taro production is estimated 4.0 million pounds, hitting record low in last decade (USDA, 2010). Manufacturing and marketing poi as a functional food could help revitalize the taro industry, which is important in rural development, providing various jobs in planting, cleaning, sorting, processing and shipping. While functional poi is possibly lucrative, it remains susceptible to the Shigella and S. aureus contamination. To create an opportunity for poi to increase its status as a thriving local staple as well as an export commodity, it is absolutely necessary to improve the microbiological safety of poi. Controlled fermentation of poi with bacteriostatic and/or bacteriocidal LAB may inhibit Shigella and S. aureus potentially present in the raw materials and final products. This biological control is a method that takes into consideration the aroma and taste of poi and seeks to maintain the integrity and authenticity of the product. It would both ensure the safety and increase the commercial options for poi manufacturers.

This study will lay the groundwork for future animal studies and human clinical trials to further investigate the health benefits of developed functional poi. Since enhancing human health and

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wellness through food, nutrition and innovative products is one of national priorities, this funding would enhance our ability to compete for extramural grant funds.

Literature Review

Taro. Taro (Colocasia esculenta) is a member of the Araceae family, a group of rhizomatous or tuberous plants. A native to Southeast Asia, taro was brought to the Hawaiian Islands by migrating Polynesians around 450 C.E. It is the oldest cultivated crop in Hawaii and is grown throughout Polynesia, Southeastern Asia, West Africa, and Mediterranean areas (Tagodoe and Nip, 1994). The taro plant is grown primarily for its edible corm, an underground starchy stem (Huang, et al. 2000). The taro corm is low in fat, protein, and vitamins but is a good source of starch and minerals (Tagodoe and Nip, 1994). The glycemic index for taro is 46 for normal individuals and diabetic patients (Tecson-Mendoza, 2007). Hawaii taro production is estimated at 4.5 million pounds in 2006, and value of sales was estimated at $2.6 million. Taro for processing totaled 4.4 million pounds in 2006. Approximately 95% of processed taro is made into poi (USDA, 2007).

Natural fermentation of taro to poi. Poi is a fermented paste made from pounded, boiled taro corms. Although the tools used in producing poi have changed through the years, the method has essentially remained the same. The corms are washed, cooked, peeled, trimmed, ground, strained, and blended with some water to produce the final product (Moy and Nip, 1983). Poi is a staple in the traditional Hawaiian diet and has been for centuries. Early Hawaiians are estimated to have consumed up to five pounds of poi per day. As with taro, poi is low in fat and protein but high in starch. It is easily digestible due to the small starch granule size of taro. Poi is considered hypoallergenic due to its low protein content and can be used as a food substitute for people with allergies to cereals or grains (Brown and Valiere, 2004). It can also be fed to infants with allergies to milk or soymilk and has been used to treat infants suffering from failure-to-thrive (Brown and Valiere, 2004). Fresh poi is often stored for 2 to 3 days at room temperature before serving to develop a strong, sour flavor. The souring process is a natural fermentation proceeding without inoculated starter cultures. Fermentative microorganisms, which mainly consist of LAB, may originate from natural sources including the soil taro is grown in, the plant’s surface, or from the processing equipment used to manufacture poi (Huang et al., 1994). They are responsible for flavor, aroma, and textural properties in sour poi. Because the natural microbial floral in the raw materials may not always be the same, it is difficult to produce poi with consistent characteristics over a long period of time. Moreover, chances of product failure due to growth of spoilage or pathogenic organisms are high.

Microbial safety concerns. Poi is high in carbohydrates and has a water activity near 1. Although it has a final pH below 4.6, the fermentation may take several days to complete (Brown et al., 2005b; Huang et al., 1994). Therefore, poi is considered to be potentially hazardous and requires time/temperature control for safety. Unfortunately, when stored at refrigeration temperature, poi turns from a paste to a rubbery solid due to the quick retrogradation of taro starch (Jane et al., 1992). This has been deemed undesirable by consumers; consequently, poi is often stored at room temperature in the market (Huang et al., 2002). Under these conditions, harmful microorganisms contaminating the raw materials and final products may proliferate during the processing, distribution and marketing of poi. There have been several high-profile outbreaks of foodborne illness associated with poi. In 1972, there was an outbreak of at least 600 cases of Shigella sonnei infection on Maui, and poi was identified as the source (Lewis et al., 1972). An outbreak of Shigella flexneri occurring in 1996 that affected over 270 people was attributed to poi. The source was identified as a poi preparer (Khambaty, et al., 1997). Humans are the only source of Shigella, which is normally passed from one infected person to the next through direct contact or through contaminated water or food. Shigella can cause severe illnesses known as bacillary dysentery and even death for children. The infectious dose of Shigella is

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very low. Another major concern with poi is Staphylococcus aureus. This pathogen is widely distributed in nature and is commonly found in the nose, throat, hair and skin of humans and animals. The preparation of poi, generally done by hand, provides conditions for the contamination by S. aureus. Many strains of S. aureus produce highly heat-stable enterotoxins that cause food poisoning if ingested. According to the Outbreak Alert Database, two S. aureus outbreaks associated with poi were reported in Hawaii, involving a total of 14 cases (CSPI, 2008).

Controlled fermentation with LAB. Food may be controlled fermented by inoculating the starting materials with a high population of starter cultures. Controlled fermentation can not only make products of stable quality but also reduce the chance of product failure and foodborne illnesses (Ray, 2004). LAB have been used in the production of fermented foods for centuries without adverse effects on humans. They are Gram-positive, non-sporeforming bacilli or cocci. Most LAB are facultative anaerobes which can grow under both anaerobic and aerobic conditions; many are also tolerant of acidic conditions. They are often used as starter cultures to ferment dairy foods. Lactic acid fermentation is a simple and safe way of preserving foods and was common over the entire world before the Industrial Revolution (Molin, 2001). The ability of LAB to lower the pH of foods and inhibit the growth of undesirable organisms greatly enhances the quality and safety of food products.

Competitive inhibition of foodborne pathogens by LAB. LAB have historically been used to ferment vegetables and preserve them longer (Ruiz-Barba et al., 1994). The success of LAB in preventing the growth and activity of pathogenic bacteria in a large variety of foods may be due to the diversity of mechanisms that can be combined. Among the mechanisms that have been proposed to result in antagonism, the production of organic acids, bacteriocins, or hydrogen peroxide is the most commonly referred in the literature (Alakomi et al., 2000; Batish et al., 1997; Cleveland et al., 2001). A number of studies indicate the presence of LAB with antagonistic activity in vegetables and fruits (Gómez et al., 2002; Rosalia Trias et al, 2007; Wilderdyke et al., 2004). Because LAB are able to thrive in poi, competitive inhibition could provide a natural means of controlling pathogens throughout the processing and distribution.

Formulating functional foods with probiotic LAB. In addition to enhancing the microbiological safety of foods, LAB may have significant effects on human health. Living microorganisms (probiotics) can be used as supplements to restore gut health at times of illness (Salminen and Gueimonde, 2004). Probiotic organisms can have a wide range of positive effects on the host, including balancing intestinal microbiota, immune enhancement, lowering fecal enzyme activity, prevention of viral and bacterial diarrhea, treatment of lactose intolerance, reducing cystic fibrosis symptoms, reducing the reoccurrence of bladder cancer, and prevention of intestinal disturbances (Salminen and Gueimonde, 2004). They produce these benefits via a variety of mechanisms including changing the intestinal pH as well as producing desirable enzymes and antimicrobial agents (Santosa et al., 2006). Many known probiotic bacteria come from the LAB group, and most probiotics belong to the genera Lactobacillus and Bifidobacterium. A microorganism must meet many selection criteria to be considered probiotic. Probiotic bacteria must have good technological properties; they must be able to withstand manufacturing and incorporation to food products without losing viability or functionality (Mattila-Sandholm et al., 2002). They must not create unpleasant flavors or textures in the food and must be able to survive passage through the upper gastrointestinal tract (Mattila-Sandholm et al., 2002). Probiotic bacteria must be able to adhere and establish long-term presence in the gut to exert their health-promoting effects (Naidu et al., 1999). The absence of virulence factors and transmissible antibiotic resistance must also be considered when identifying probiotic bacteria (Gasser, 1994; Salyers et al., 2004).

For the most part, fermented dairy products such as yogurt and buttermilk have been used to deliver probiotic bacteria. There are few choices for those lactose-intolerant consumers. Several

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fermented non-dairy foods contain or are produced by probiotic bacteria, including kefir, yakult, miso, natto, and tempeh (Santosa et al., 2006). Unlike these foods engineered to contain probiotic bacteria, poi may naturally contain beneficial strains of LAB. Many of the problems and technical challenges with the development of non-dairy probiotics would not affect poi. Poi is an environment where bacteria thrive, and consumers rely on these bacteria to produce the desired sour flavor. Previous research indicates that poi can significantly inhibit rat YYT colon cancer cell proliferation and stimulate the immune system (Brown et al., 2005a). That probably explains why Hawaiians have a low incidence of colorectal cancer.

Few studies have been conducted on the fermentative microorganisms present in poi and these studies have yielded minimal results. Allen and Allen (1933) determined there were at least three Lactobacillus and two Lactococcus species within poi but did not endeavor to create a complete microbial profile. A more recent study found that Lactococcus lactis is the most prevalent bacteria in poi (Huang et al., 1994). The probiotic potential of LAB in poi is not yet well known. Moreover, previous microbiological analysis of poi focused on products from a single manufacturer. Since poi is naturally fermented, products from different manufacturers may be significantly different in microbial community composition due to the fact that each processing location may have unique properties and purchase taro from a variety of different suppliers.

Current work. We collected Poi from four major manufacturers in Hawaii. Three samples were collected for each brand. Bacteria, yeast and mold were enumerated using the plate count method. Bacterial DNA was extracted from each sample and subjected to the polymerase chain reaction-temporal temperature gel electrophoresis (PCR-TTGE), a molecular technique commonly used for genetic fingerprinting of microbial populations in food. Plate counting showed highly variable microbial numbers among brands, but the counts of samples from the same brand were highly correlated. The bacterial count was at least 2.8 log higher than the yeast and mold count for each brand (Appendix 1). PCR-TTGE disclosed that each poi brand had a consistent microbial community composition. Moreover, products from different poi manufacturers had distinct microbial profiles and varied dominant LAB (Appendix 2). Weissella confusa and Lactobacillus delbrueckii were dominant in brands A and B. Enterococcus faecium was dominant in brand C whereas Lactobacillus delbrueckii and Lactococcus lactis were dominant in brand D (Li et al., 2009). Finally, we have isolated 19 LAB strains from poi brand A. Enzyme analysis suggests that one of them has high activity for ß-galactosidase, an enzyme that can decrease lactose in food. This highlights the probiotic potential of LAB in poi and calls for further research.

Objectives Listed

The specific objectives of this project are as follows:

1. Isolate and identify LAB in poi;

2. Examine the probiotic nature of LAB isolated from poi;

3. Select starter cultures for controlled fermentation of poi;

4. Control Shigella and S. aureus in poi by selected LAB;

5. Produce functional poi by controlled fermentation with selected probiotic LAB.

Procedures

1. Isolate and identify LAB in poi

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Isolation of LAB in poi. Fresh poi from all six major manufacturers in Hawaii will be analyzed. Two samples of each commercial brand will be collected at least two weeks apart to make sure that different batches of poi are examined. Each sample will be homogenized and serially diluted in sterile 0.1% peptone water. Appropriate dilutions will be spread on DeMan-Rogosa-Sharpe (MRS) agar (Difco). After incubation anaerobically at 35oC for 48 h, 40 colonies will be randomly picked up for each brand, purified, and maintained in MRS broth. Fresh cultures of these pure isolates will be tested for their reaction to oxidase and catalase tests.

Identification of LAB isolated from poi. Genomic DNA will be extracted from each LAB isolate using the DNeasy Tissue Kit (Qiagen), following the protocol for Gram-positive bacteria. The purity and concentration of DNA extracts will be measured with a spectrophotometer. Randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) will be performed as described by Pulido et al. (2005) using the primer M13 (5’-gagggtggcggttct-3’). The amplification program will include a hold at 94oC for 5 min followed by 35 cycles of 94oC for 1 min, 40oC for 20 s, ramp to 72oC at 0.6oC s-1, and 72oC for 2 min. These cycles will be followed by a hold at 72oC for 5 min. RAPD-PCR products will be separated by gel electrophoresis, stained with SYBR green, and photographed. Repeated isolates will be eliminated by comparing RAPD profiles. One representative from each group of banding patterns will be identified by using PCR with 16S rRNA gene-based universal primers (forward, 5'-ggagagtttgatcctggctcag-3'; reverse, 5'-tattaccgcggctgctggcac-3') (Vliegen et al., 2006). The amplification program will include a hold at 94oC for 5 min followed by 30 cycles of 94oC for 30 s, 62oC for 30 s, and 72oC for 45 s. These cycles will be followed by a hold at 72oC for 5 min. The amplicons will be recovered and sequenced by the Biotechnology Core Facility of University of Hawaii at Manoa. Searches in the GenBank with the BLAST program will be performed to identify the closest known matches to the 16S rDNA sequences obtained.

2. Examine the probiotic nature of LAB isolated from poi

Survival under simulated human gastrointestinal conditions.

(i) Sensitivity to low pH: The LAB isolates will be tested for their ability to survive at low pH, similar to that in the human digestive tract (Prasad et al., 1999). Cultures will be grown overnight in MRS broth and adjusted to an optical density of 0.6-0.7. Cells will be harvested from the broth suspension by centrifugation and resuspended in phosphate-saline buffer (PBS). The solutions will be diluted to 5 mL with sterile phosphate-saline buffer pre-adjusted to a series of pH with HCl between 1.0 and 3.0. The solutions will be incubated at 37oC for 0, 1, 2 or 3 h, and viable organisms will be enumerated on MRS agar.

(ii) Tolerance against bile: The procedures of Klaenhammer et al. (1981) will be followed to evaluate the tolerance of the LAB isolates against bile. Cultures will be grown overnight in MRS broth and harvested by centrifugation. Pellets will be washed with PBS and resuspended in MRS broth containing bile at final concentration of 0, 0.25, 0.5, 0.75, or 1.0% (w/v). The solutions will be incubated at 37oC for 48 h, and the growth will be monitored with a spectrophotometer at OD650nm.

(iii) Resistance to phenol: Phenols, formed in the gut by bacterial deamination of some aromatic amino acids derived from dietary or endogenously produced proteins, may be bacteriostatic (Suscovic et al. 1997). The ability of the LAB isolated from poi to grow in the presence of phenol will be estimated by inoculating an overnight culture (2% of the total volume) adjusted to OD600 0.60-0.70 into 10 ml of MRS broth with or without 0.4% phenol (Sigma) as described by Xanthopoulos et al. (2000). These solutions will be incubated at 35oC for 24 h. Growth will be estimated by spread plating serial dilutions on MRS agar at 0 and 24 h. MRS plates will be incubated at 35 oC for 48 h to enumerate surviving bacterial cells.

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Enzymatic activity. Enzymatic activity of the LAB isolates will be examined using the API-ZYM System (bioMérieux), following the manufacturer’s instructions. A volume of 65 µl of McFarland standard 5 suspensions of each isolate will be deposited into each well. Bacterial activity for 19 enzymes, including alkaline phosphatase, esterase, esterase lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase, will be evaluated. Enzyme activity readings will be taken after 5 h of incubation at 35oC.

Antimicrobial potential.

(i) Production of hydrogen peroxide: The LAB isolates will be spotted on MRS agar containing 10 ml/L 2-2’ azino-bis-3 ehtylbenzthiazoline-6-sulphonic acid (ABTS) (Sigma) and 3 mg/L horseradish peroxidase (Sigma). Horseradish peroxidase will be added to autoclaved media after the temperature decreases to 50oC, as described by Marshall (1979). 5 µl aliquots of bacterial isolates adjusted to OD600 0.60-0.70 will be spotted onto the agar and incubated at 35oC for 48 h. Upon exposure to O2, spots will be examined for the formation of a violet halo.

(ii) Acid production: The LAB isolates will be inoculated into MRS broth to examine their production of acid over time. Cultures will be incubated at 35oC. The pH of the cultures will be measured with a pH meter after 0, 4, 8, 12, 24, and 48 h of incubation.

(iii) Bacteriocin production: Production of antimicrobial substances will be tested by the spot-on-a-lawn method on Brain Heart Infusion agar (Difco) buffered with 0.15 M sodium phosphate (pH 7.2) (Omar et al., 2004). The plates will be incubated at 35oC for 12 h and then overlaid with buffered soft agar previously inoculated with the indicator strains Shigella sonnei ATCC25931, Staphylococcus aureus ATCC25923, Escherichia coli O157:H7 C7927, Salmonella Typhimurium ATCC14028, or Listeria monocytogenes ScottA (all available in the PD’s culture collection). The plates will be incubated for another 12 h and then be examined for halos of inhibited growth around the spots. To test the proteinaceous nature of the inhibitors, 10 µl of trypsin solution (10 mg/ml) will be deposited on the side of each spot of bacterial growth. Then the plates will be incubated at 35oC for 3 h before being overlaid with the indicator strain as above. The absence of inhibition zones in the trypsin-spotted zone will indicate protease sensitivity.

Adhesion assay. The ability of the LAB isolates to adhere to human cells will be investigated using the Caco-2 intestinal cell line, following the procedure of Chauviere et al. (1992). Monolayers of the epithelial cells will be prepared in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal calf serum (Sigma) in 6-well tissue culture plates (Nunc) at a concentration of 105

cells/ml. The cells will be then incubated with approximately 107 CFU/ml of the isolate to be tested. After 2 h of incubation at 35oC, monolayers will be washed 5 times with PBS. The adhered bacterial cells will be detached by repeated pipetting with chilled sterile water, diluted in 0.1% peptone water, and enumerated on MRS agar (Difco).

Antibiotic resistance. Antibiotic resistance of the LAB isolates will be assessed with the Sensititre Anaerobe MIC Plate (TREK Diagnostic Systems), following the manufacturer’s instructions. The isolates will be tested for their resistance to 18 antibiotics, including Ampicillin/Sulbactam (2:1 ratio), Amoxicillin/Calvulanic Acid (2:1 ratio), Cefotetan Na, Penicillin, Imipenem, Meropenem, Clindamycin, Cefoxitin, Metronidazole, Chloramphenicol, Ampicillin, Piperacillin, Tetracycline, Mezlocillin, and Piperacillin/Tazobactam Constant 4.

3. Select starter cultures for controlled fermentation of poi

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Selection of starter cultures. The selection of starter cultures for each poi manufacturer will be mainly based on the prevalence of the LAB isolates in the product, enzymatic activity, and acid production capability. The ideal candidates should dominate the microbial community of each brand, show high activity for enzymes that hydrolyze starch or ferment simple sugars, and produce acid at high rates.

Controlled fermentation of poi. Taro corms will be obtained from local grocery stores and washed thoroughly in tap-water. The outer skin of the corm will be removed using a knife. After the corm is peeled, it will be cut into approximately 1” × 1” × 1” pieces. The pieces of corm will be steamed for 30 min. Sterilized water will be added into the pieces in a blender jar, 2:1 in ratio, and blended at high speed setting for 3 min. The selected starter cultures will be grown in MRS broth at 35oC for 24 h. The cells will be harvested, resuspended in 0.1% peptone water, and inoculated individually into the cooked taro paste to achieve a final concentration of 107 CFU/g. The control will inoculated with the same amount of 0.1% peptone water. The paste will be mixed and stored at 21oC. This temperature is chosen because poi is displayed in retail stores at room temperature. The pH of the taro paste will be measured on days 0, 1, 2, and 4. The starter cultures are expected to create a strong, sour flavor in poi quickly and consistently.

4. Control Shigella and S. aureus in poi by selected LAB

For this purpose, the LAB isolates should be able to produce hydrogen peroxide or bacteriocins. Those isolates that have a strong acid production capability will also be considered. Cooked taro paste will be prepared as described above. The selected LAB strains will be grown in MRS broth at 35oC for 24 h. The cells will be harvested and resuspended in 0.1% peptone water. S. sonnei ATCC25931 and S. aureus ATCC25923 will be grown in tryptic soy broth (Difco) at 35ºC for 16 h and serially diluted in 0.1% peptone water. Each selected LAB strain and one of the pathogenic bacteria will be inoculated into the taro paste to achieve a final concentration of 107 CFU/g and 105

CFU/g, respectively. The paste will be mixed and stored at 21oC. On days 0, 1, 2, and 4, appropriate dilutions of the paste will be spread on MRS agar to enumerate LAB. Meanwhile, xylose lysine deoxycholate agar and Salmonella-Shigella agar will be used to enumerate S. sonnei whereas Baird Parker agar will be used to enumerate S. aureus.

5. Produce functional poi by controlled fermentation with selected probiotic LAB

Selection of probiotic LAB. The ideal candidates must survive at low pH, tolerate bile, and resist phenol. These properties will ensure their viability during passage through the human gastrointestinal tract. They should also be able to colonize the gastrointestinal tract. The absence of transmissible antibiotic resistances is required. The selected strains would have antimicrobial potential or produce enzymes that may promote health. Strains exhibiting activity for β-galactosidase would be of special interest since this enzyme is involved in lactose degradation from dairy products and is of vital importance for proper utilization of the sugars found in many foods.

Production of functional poi. Cooked taro paste will be prepared as described above. The selected probiotic strains will be grown in MRS broth at 35oC for 24 h. The cells will be harvested, resuspended in 0.1% peptone water, and inoculated individually into the cooked taro paste to achieve a final concentration of 107 CFU/g. The control will be inoculated with the same amount of 0.1% peptone water. The paste will be mixed and stored at 21oC.

Microbiological, chemical and sensory analyses of functional poi. On days 0, 1, 2, and 4 during storage, the poi samples will be serially diluted in 0.1% peptone water and spread on the

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following media: (a) plate count agar incubated at 35°C for 48 h to enumerate total aerobic bacteria; (b) MRS agar (acidified to pH 5.4 with acetic acid to enhance its selectivity) incubated in an anaerobic jar at 35°C for 48 h to enumerate probiotic LAB; and (c) potato dextrose agar (acidified to pH 3.5 with tartaric acid) incubated at 21°C for 5 d to enumerate yeast and mold.

The pH of the poi samples will be measured with a pH meter. Organic acids will be extracted from the samples with 1N HCl according to the procedure of Huang et al. (1994). The changes of four organic acids (lactic, acetic, oxalic, and succinic acids) in functional poi will be determined by high performance liquid chromatography (HPLC) (Huang et al., 1994).

Functional poi samples will be evaluated and compared with the control sample by a panel of 20 assessors. Faculty, staff and student volunteers at the University of Hawaii at Manoa will be recruited for the sensory analysis. All samples will be randomized and identified by a numerical code that is unknown to panel members. The panel will use a 9-point hedonic scale sensory rating to evaluate appearance, aroma, taste, mouth feel, and overall acceptance of the poi samples (1, dislike extremely; 2, dislike very much; 3, dislike moderately; 4, dislike slightly; 5, neither like nor dislike; 6, like slightly; 7, like moderately; 8, like very much; 9, like extremely) (Peryam and Pilgrim, 1957). The sensory evaluation will be conducted in a facility designed for this purpose at the University of Hawaii at Manoa. All procedures will be reviewed by UH Institutional Review Board (IRB) and sensory evaluation activities will be contingent on approval by IRB.

Project Timetable

Procedures Year 1 Year 2

1. Isolating and identifying LAB in poi I--------I

2. Screening for probiotic LAB I------------I

3. Controlled fermentation of poi I--------I

4. Competitive inhibition of pathogens I-----------I

5. Producing and evaluating functional poi I--------------------I

Expected Outcomes/Impacts

Expected outcomes/impacts Outcome/impact indicatorsImprovement in poi quality Starter cultures are selected and used in controlled

fermentation of poi A sour flavor is produced in poi quickly and

consistentlyImprovement in poi value Probiotic LAB strains are screened out from the

natural isolates Functional poi is produced successfully and gains

sensory evaluation scores comparable to those of the un-inoculated control

Functional poi becomes popular and generates increased revenues

Improvement in poi safety Selected LAB isolates can inactivate Shigella and S. aureus in poi

The number of foodborne illness associated with poi is reduced significantly

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Data Analysis

The experiments will be performed in duplicate. Bacterial counts will be log-transformed before further analysis. Descriptive statistics including means and standard deviations will be calculated for quantitative measurements. Data will be subjected to analysis of variance (ANOVA) using SAS® 9.1 (SAS Institute Inc.) at P < 0.05. Tukey’s specific comparison test will be used to determine which particular means are significantly different.

Plan for Outreach or Technology Transfer

The selected starter cultures will be available for poi manufacturers to conduct controlled fermentation. Once a probiotic LAB strain is identified and successfully applied in controlled fermentation of functional poi, a provisional patent will be filed. The PD will make a conscientious effort to transfer the technology developed in this project to interested poi manufacturers or other parties. Upon conclusion of the project, all data will be compiled, statistically analyzed, and submitted to USDA-NIFA. A summary of our findings will be presented at the Institute of Food Technologists (IFT) Annual Meeting and submitted to peer-reviewed journals for publication.

Personnel Safety

The PD’s Food Microbiology Laboratory conforms to all the requirements of the University of Hawaii’s Environmental Health and Safety Office. It is a certified Biosafety Level-2 Laboratory with proper safety and emergency protocols in place. All personnel directly involved in this study will be required to undergo “Hazard Material Management” and “Biological Safety” training courses offered by the Environmental Health and Safety Office at UH prior to the start of the project.

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References / Works CitedAlakomi, H.L., Skyttä, E., Saarela, M., Mattila-Sandholm, T., Latva-Kala, K., and Helander, I.M., 2000. Lactic acid permeabilizes Gram-negative bacteria by disrupting the outer membrane. Applied and Environmental Microbiology 66, 2001-2005.

Allen, O. and Allen, E., 1933. The manufacture of poi from taro in Hawaii; with special emphasis on its fermentation. Hawaii Agricultural Experiment Station Bulletin No. 70.

Batish, V.K., Roy, U., Lal, R., and Grover, S., 1997. Antifungal attributes of lactic acid bacteria - a review. Critical Reviews in Biotechnology 17, 209-225.

Brown, A.C., and Valiere, A., 2004. The Medicinal Uses of Poi. Nutrition in Clinical Care 7, 69-74.

Brown, A.C., Reitzenstein, J., Liu, J., and Jadus, M., 2005a. The Anti-Cancer Effects of Poi (Colocasia esculenta) on Colonic Adenocarcinoma Cells In Vitro. Phytotherapy Research 19, 767-771.

Brown, A.C., Shovic, A. Ibrahim, S.A., Holck, P., and Huang, A., 2005b. A non-dairy probiotic’s (poi) influence on changing the gastrointestinal tract’s mictroflora environment. Alternative Therapies 11, 58-64.

Chauviere, G., Coconnier, M.H., and Servin, A.L., 1992. Adhesion of human lactobacillus acidophilus strain LB to human Caco-2 cells. Journal of General Microbiology 138, 1689-1696.

Cleveland, J., Montville, T.J., Nes, I.F., and Chikindas, M.L., 2001. Bacteriocins: safe, natural antimicrobials for food preservation. International Journal of Food Microbiology 71, 1-20.

Conrow, J. 2005. Battle in the taro patch. Available at: http://honoluluweekly.com/cover/2005/08/battle-in-the-taro-patch/.

CSPI. 2008. Outbreak alert database. Available at: http://www.cspinet.org/foodsafety/outbreak/outbreaks.php?column=pathogen&colval=Staphylococcus%20aureus&letter=p&sort=pathogen&dir=DESC&letter=p&letter=p&letter=all&sort=pathogen&dir=DESC.

Elo, S., Saxelin, M., and Salminen, S., 1991. Attachment of Lactobacillus casei strain GG human colon carcinoma cell line Caco-2: comparison with other dairy strains. Letters in Applied Microbiology 13, 154-156.

Gasser, F., 1994. Safety of lactic acid bacteria and their occurrence in human clinical infections. Bulletin De L’Institut Pasteur 92, 45-67.

Gómez, R., Muñoz, M., Ancos, B., and Cano, P., 2002. New procedure for the detection of lactic acid bacteria in vegetables producing antibacterial substances. Lebensmittel-Wissenschaft-Technologie 35, 284-288.

Hollyer, J. Teves, G, Sullivan, J., and Welch, K., 1997. Taro Mauka to Makai: a taro production and business guide for Hawaii growers. College of Tropical Agriculture and Human Resources, University of Hawaii at Manoa, Honolulu, HI.

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http://www.cspinet.org/foodsafety/outbreak/outbreaks.php?column=pathogen&colval=Staphylococcus%20aureus&letter=p&sort=pathogen&dir=DESC&letter=p&letter=p&letter=all&sort=pathogen&dir=DESC



http://honoluluweekly.com/cover/2005/08/battle-in-the-taro-patch/

Huang, A.S., Lam, S., Nakayama, T., and Lin, H., 1994. Microbiological and Chemical Changes of Poi Stored at 20oC. Journal of Agriculture and Food Chemistry 42, 45-48.

Huang, A.S., Titchenal, C.A., and Meilleur, B.A., 2000. Nutrient Composition of Taro Corms and Breadfruit. Journal of Food Composition and Analysis 13, 859-864.

Huang, A.S., Komarasamy, K., and Liu, X., 2002. Textural and sensory properties of α-amylase treated poi stored at 4oC. Journal of Food Processing Preservation 26, 1-10.

Jane, J., Shen, L., and Kasemsuwan, T., 1992. Physical and Chemical Studies of Taro Starches and Flours. Cereal Chemistry 69, 528-535.

Khambaty, F.M., Davis, M.A., and Lampel, K.A., 1997. Genetic instability of a Shigella flexneri strain responsible for a foodborne outbreak in Hawaii. Available at: http://vm.cfsan.fda.gov/~frf/forum97/97G12.htm.

Klaenhammer, T.R., and Kleeman, E.G., 1981. Growth characteristics, bile sensitivity, and freeze damage in colonial variants of Lactobacillus acidolphilus. Applied and Environmental Microbiology 41, 1461-1467.

Lewis, J.N., Loewenstein, M.S., Guthrie, L.C., and Sugi, M., 1972. Shigella sonnei outbreak on the island of Maui. American Journal of Epidemiology 96, 50-58.

Li, Y., Dong, J., Pirazzini, D., and He, H., 2009. Molecular fingerprinting of microbial populations in poi. Institute of Food Technologists Annual Meeting, paper 010-13. New Orleans, LA.

Marshall, V.M., 1979. A note on screening hydrogen peroxide producing lactic acid bacteria using a non-toxic chromogen. Journal of Applied and Environmental Bacteriology 47, 327-328.

Mattila-Sandholm, T., Myllärinen, P., Crittenden, R., Mogensen, G., Fondén, R., and Saarela, M., 2002. Technological challenges for future probiotic foods. International Dairy Journal 12, 173-182.

Molin, G., 2001. Probiotics in foods not containing milk or milk constituents, with special reference to Lactobacillus plantarum 299v1-3. American Journal of Clinical Nutrition 73(suppl), 380S-385S.

Moy, J.H. and Nip, W.-K., 1983. Processed Food. In: Wang, J.K. (Ed.), Taro, A Review of Colocasia esculenta and its Potentials. University of Hawaii Press, Honolulu, pp. 261-262.

Naidu, A.S., Bidlack, W.R., and Clemens, R.A., 1999. Probiotic Spectra of Lactic Acid Bacteria (LAB). Critical Reviews in Food Science and Nutrition 39, 13-126.

Omar, N.B., Castro, A., Lucas, R., and Galvez, A., 2004. Functional and safety aspects of Enterococci isolated from different Spanish foods. Systematic and Applied Microbiology 27, 118-130.

Peryam, D.R. and Pilgrim, F.J. 1957. Hedonic scale method of measuring food preferences. Food Technol. 11, 9-14.

Prasad, J., Gill, H., Smart, J., and Gopal, P., 1998. Selection and characterization of Lactobacillus and Bifidobacterium strains for use as probiotics. International Dairy Journal 8, 993-1002.

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http://vm.cfsan.fda.gov/~frf/forum97/97G12.htm

Pulido, R.P., Ben Omar, N., Abriouel, H., López, R.L., Martínez Cañamero, M., and Gálvez, A., 2005. Microbiological study of lactic acid fermentation of caper berries by molecular and culture-dependent methods. Applied and Environmental Microbiology 71, 7872-7879.

Ray, B. 2004. Fundamental Food Microbiology. 3rd Ed. CRC Press. Boca Raton, FL.

Rosalia Trias, Lluís Bañeras, Esther Badosa, and Emilio Montesinos., 2008. Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria, International Journal of Food Microbiology 31, 50-60.

Ruiz-Barba, J.L., Cathcart, D.P., Warner, P.J., and Jiménez-Díaz, R., 1994. Use of Lactobacillus plantarum LPCO10, a bacteriocin producer, as a starter culture in Spanish-style green olive fermentations. Applied and Environmental Microbiology 60, 2059-2064.

Salminen, S. and Gueimonde, M., 2004. Human Studies on Probiotics: What is Scientifically Proven. Journal of Food Science 69, 137-140.

Salyers, A.A., Gupta, A., and Wang, Y., 2004. Human intestinal bacteria as reservoirs for antibiotics resistance genes. Trends in Microbiology 12, 412-416.

Santosa, S., Farnworth, E., and Jones, P., 2006. Probiotics and Their Potential Health Claims. Nutrition Reviews 64, 265-274.

Stanton, C., Ross, R.P., Fitzgerald, G.F., and Van Sinderen, D., 2005. Fermented functional foods based on probiotics and their biogenic metabolites. Current Opinions in Biotechnology 16, 198-203.

Suscovic, J., Brkic, B., Matosic, S., and Maric, V., 1997. Lactobacillus acidophilus M92 as a potential probiotic strain. Milchwissenschaft 52, 430-435.

Tagodoe,A. and Nip, W.-K., 1994. Functional Properties of Raw and Precooked Taro (Colocasia esculenta) Flours. International Journal of Food Science and Technology 29, 457-462.

Tecson-Mendoza, E.M., 2007. Development of Functional Foods in the Philippines. Food Science and Technology Research 13, 179-186.

United States Department of Agriculture, 2007. National Agricultural Statistics, Hawaii Taro, February 26. Available at: http://www.nass.usda.gov/hi/vegetble/taro.pdf.

United States Department of Agriculture, 2010. National Statistics for Taro. Available at: http://www.nass.usda.gov/Statistics_by_Subject.

Vliegen, I., Jacobs, J. A., Beuken, E., Bruggeman, C. A. and Vink, C., 2006. Rapid Identification of bacteria by real-time amplification and sequencing of the 16S rRNA Gene. Journal of Microbiological Methods 66, 156-164.

Wilderdyke, MR, Smith, DA, and Brashears, MM., 2004. Isolation, identification, and selection of lactic acid bacteria from alfalfa sprout for competitive inhibition of foodborne pathogens. Journal of Food Protection 67, 947-951.

Xanthopoulos, V., Litopoulou-Tzanetaki, E., and Tzanetakis, N., 2000. Characterization of Lactobacillus isolates from infant faeces as dietary adjuncts. Food Microbiology 17, 205-215.

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http://www.nass.usda.gov/Statistics_by_Subject

Key Personnel ListThis project is a collaborative effort among three researchers in the Department of Human

Nutrition, Food and Animal Sciences. Key personnel and their specific responsibilities are as follows:

Yong Li (Food Microbiologist): has microbiological, molecular biology, and statistical expertise needed to conduct the experiments and analyze the data. During his postdoctoral training, Dr. Li was actively involved in two research projects on probiotic LAB in yogurts and soy yogurts. He has responsibility for overseeing the execution of the project and coordinating the work with Co-PDs and postdoctoral fellow. He will be responsible for supervising the postdoctoral fellow performing the experimental work and directing the writing of project reports and manuscripts.

Wayne Iwaoka (Food Chemist): has a specialty in general food analysis and HPLC. He will work closely with the PD on the chemical analysis of functional poi.

Michael Dunn (Nutritional Scientist): has expertise on nutritional biochemistry and metabolism. He has already set up the Caco-2 cell culture system in his lab. He will supervise the examination of the probiotic nature of LAB isolates from poi.

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Key Personnel Biographical Sketch / CVsYong Li, Ph.D.Assistant Professor

Department of Human Nutrition, Food and Animal SciencesCollege of Tropical Agriculture and Human Resources

University of Hawaii at Manoa

Contact Information

1955 East West Road, AgSci 216Honolulu, Hawaii 96822Phone: (808) 956-6408Fax: (808) 956-4024E-mail: [email protected]

Education

M.A. Statistics, University of Missouri, Columbia, 2005 Ph.D. Food Science, University of Missouri, Columbia, 2004 M.S. Microbiology, China Agricultural University, China, 1997 B.S. Microbiology, China Agricultural University, China, 1994

Employment

Assistant Professor, University of Hawaii, 2005-present Post-Doctoral Associate, Department of Food Science, University of Missouri, 2004-2005 Graduate Research Assistant, dual majored in Food Science and Statistics, University of Missouri,

1998-2004

Current Research Interests

Genetic fingerprinting of microbial communities in food Microbial survival and virulence during food processing Rapid detection of foodborne pathogens Probiotics and functional food Predictive microbiology

Representative Publications

Liang, N.J., Dong, J., and Li, Y. 2010. Detection of viable Salmonella in lettuce by propidium monoazide real-time PCR. Journal of Food Science (accepted)

He, H.F., Li, Y., Castro, A.L., Dong, J., and Lee, C.N. 2010. Microbiological quality of pasteurized milk in Hawaii. Pacific Agriculture and Natural Resources (accepted)

Iwaoka, W.T., Li, Y., and Rhee, W.Y. 2010. Measuring gains in critical thinking in food science and human nutrition courses: the Cornell Critical Thinking Test, problem-based learning activities, and student journal entries. Journal of Food Science Education 9:68-75

Xia, Y.Y., Li, Q.X., Gong, H.J., Li, Y., Cao, Y.S., Liu, X.L., and Li, J.Q. 2010. Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the analysis of the new fungicide 2-allylphenol in strawberry fruits. Food Chemistry 120:1178-1184

Kandukuru, P., Huang, A.S., Dong, J., Bittenbender, H.C., and Li, Y. 2009. Rapid identification

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of bacterial isolates from aqueous kava (Piper methysticum) extracts by polymerase chain reaction and DNA sequencing. Letters in Applied Microbiology 49: 764-768

Wang L.X., Li Y., and Mustapha, A. 2009. Detection of Escherichia coli O157:H7 by ethidium monoazide-multiplex real-time PCR. Journal of Applied Microbiology 107:1719-1728

He, H.F., Dong, J., Lee, C.N., and Li, Y. 2009. Molecular analysis of spoilage-related bacteria in pasteurized milk during refrigeration by polymerase chain reaction and denaturing gradient gel electrophoresis. Journal of Food Protection 72:572-577

Wang L.X., Li Y., and Mustapha, A. 2007. Rapid and simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in ground beef using real-time multiplex PCR and IMS. Journal of Food Protection 70:1366-1372

Zhu, S.S., Liu, X.L., Liu, P.F., Li, Y., Li, J.Q., Wang, H.M., Yuan, S.K., and Si, N.G. 2007. Flumorph is a novel fungicide that disrupts microfilament organization in Phytophthora melonis. Phytopathology 97: 643-649

Mustapha, A. and Li, Y. 2006. Molecular Detection of Foodborne Bacterial Pathogens. In: J.H. Maurer (ed), PCR Methods in Foods, pp. 69-90, Springer, New York

Fu, C.J., Carter, J.N., Li, Y., Porter, J.H., and Kerley, M.S. 2006. Comparison of agar plate and real-time PCR on enumeration of Lactobacillus, Clostridia, and total anaerobic bacteria in dog feces and the feasibility of bacterial DNA as a total rumen bacterial marker. Letters in Applied Microbiology 42:490-494

Courses Offered at University of Hawaii at Manoa FSHN 403. Microbiology of Foods FSHN 440. Food Safety FSHN 701. Rapid Detection and Fingerprinting of Microbes in Food

Students’ Achievements Hongfei He, 2nd Place, Food Microbiology Division John Ayres Graduate Student Paper

Competition, Institute of Food Technologists (IFT) Annual Meeting, New Orleans, 2008 David Pirazzini, 1st Place, Master Student Oral Presentation Competition, American Society for

Microbiology (ASM) Branch Meeting, Hawaii, 2008 Alfred Lee Castro, Undergraduate Summer Research Award, University Research Council,

University of Hawaii, 2008 Ningjian Liang, College (CTAHR) 2nd Place MS Student Poster Presentation, Gamma Sigma Delta

Award, CTAHR Student Research Symposium, University of Hawaii at Manoa, 2010 Alfred Lee Castro, College (CTAHR) 2nd Place Undergraduate Student Poster Presentation,

CTAHR Student Research Symposium, University of Hawaii at Manoa, 2010 Hongfei He, College (CTAHR) 1st Place MS Student Poster Presentation, CTAHR Student

Research Symposium, University of Hawaii at Manoa, 2008 David Pirazzini, College (CTAHR) 2nd Place MS Student Poster Presentation, Gamma Sigma Delta

Award, CTAHR Student Research Symposium, University of Hawaii at Manoa, 2007 David Pirazzini, Department (HNFAS) Best Graduate Student Poster Presentation, CTAHR

Student Research Symposium, University of Hawaii at Manoa, 2008 Hongfei He, Department (HNFAS) Best Graduate Student Poster Presentation, CTAHR Student

Research Symposium, University of Hawaii at Manoa, 2007

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CURRICULUM VITAE

Wayne T. IwaokaUniversity of Hawaii at ManoaHonolulu, Hawaii 96822Ph. (808) 956-6456, FAX (808) 956-4024

EDUCATIONUniversity of Hawaii at Manoa, Honolulu, HI Bachelor of Science, Soil Science, 1967University of Illinois, Urbana, IL Ph.D., Food Chemistry, 1973Massachusetts Institute of Technology Post-Doctoral Research Associate in Cambridge, MA Analytical Food Toxicology, 1973

PROFESSIONAL EXPERIENCE

Associate Researcher Dept. Human Nutrition, Food, Animal Sciences August, 2006 – University of Hawaii at Manoa present

Vice Chancellor for University of Hawaii at Manoa October, 2005 – Students (Interim) Honolulu, HI July, 2006

Associate Researcher Dept. Human Nutrition, Food, Animal Sciences June, 2000 – University of Hawaii at Manoa Sept., 2005

Associate Dean for College of Tropical Agriculture & Human Resources July, 1997 -Academic & Student University of Hawaii at Manoa May, 2000Affairs (Interim)

Associate Researcher/ Dept. of Food Science & Human Nutrition 1994-1997 University of Hawaii at Manoa

Assistant Researcher/ Dept. of Food Science & Human Nutrition 1988-1994 University of Hawaii at Manoa

Adjunct Faculty Institute for Food Science & Technology 1983-1988 College of Fisheries, University of Washington Seattle, WA

State Pesticide Pesticides Program 1985-1988Program Manager Hawaii Dept. of Agriculture

Honolulu, Hawaii

Chemistry Laboratory Laboratories Branch 1983-1985Manager Hawaii Dept. of Health

Honolulu, HI

Associate Professor Institute for Food Science & Technology 1979-1983 College of Fisheries, University of Washington Seattle, WA

Assistant Professor Institute for Food Science & Technology 1975-1979 College of Fisheries, University of Washington Seattle, WA

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Publications (last four years)Kartika, H., Shido, J., Li, Q.X., Nakamoto, S., and Iwaoka, W. 2010. Nutrient composition of Mamaki (Pipturus albidus) leaves and tea. Journal of Food Composition and Analysis. In press.

Iwaoka, W.T., Li, Y., and Rhee, W.Y. 2010. Measuring gains in critical thinking in food science and human nutrition courses: the Cornell Critical Thinking Test, problem-based learning activities, and student journal entries. Journal of Food Science Education, 9 (2), 68 - 75.

Iwaoka, W.T. and Crosetti, L. 2008. Using journals to help students learn subject matter content, develop and practice critical reasoning skills, and reflect on personal values in food science and human nutrition classes. Journal of Food Science Education, 7 (2), 19 – 29.

Iwaoka, Wayne T. and Li, Qing X. Toxicants (revised and updated). 2007 In Food Chemistry: Principles and Application. 2nd Edition. Ed. Y.H. Hui. Science Technology System. West Sacramento, CA

Kartika, Henny, Li, Qingxiao, Nakamoto, Stuart, Wall, Marisa, and Iwaoka, Wayne T. 2007. Major Phenolic Acids and Total Antioxidant Activity in Mamaki Leaves, Pipturus albidus. Journal of Food Science, 72 (9), 696-701.

Relevant publications for this research (more than 5 years old)Zhang, X., W.T. Iwaoka, A.S. Huang, S. Nakamoto, and R. Wong. 1994. Gingerol decreases after processing and storage of ginger. J. of Food Sci. 59(6):1338-1340 & 1343.

Iwaoka, W., Y. Hagi, K. Umano, and T. Shibamoto. 1994. Volatile Chemicals Identified in Fresh and Cooked Breadfruit. J. Agriculture & Food Chemistry, 42:975.

Iwaoka, W.T., X. Zhang, R.A. Hamilton, C.L. Chia, & C.S. Tang. 1993. Identification and changes of major volatile constituents of soursop (Annona muricata L.) during ripening. HortScience 28:817.

Jonas-Davis, J., J.J. Sullivan, L.L. Kentala, J. Liston, L. Wu, and W. T. Iwaoka, 1984. Semiautomated method for the analysis of PSP toxins in shellfish. J. Food Science 49 (6):1506-1509 & 1516.

Sullivan, John J., Martha G. Simon and Wayne T. Iwaoka, 1983. Comparison of HPLC and mouse bioassay methods for determining PSP toxins in shellfish. J. of Food Science 48:1312-1314.

Sullivan, John J. and Wayne T. Iwaoka, 1983. High pressure liquid chromatographic determination of the toxins associated with paralytic shellfish poisoning. J. Assoc. Official Anal. Chem. 66:297-303.

Iwaoka, Wayne T. and Cheryl A. Krone. 1982. Problems associated with the extraction of mutagens from food, in Carcinogens and Mutagens in the Environment. Vol. 1, Food Products, ed. H.F. Stich, CRC Press, Inc., Boca Raton, FL.

Hansen, T., L. C. Green, W. T. Iwaoka, and S. R. Tannenbaum, 1977. Analysis of N-nitrosoproline in raw bacon: Further evidence that nitrosoproline is not a major precursor of nitrosopyrrolidine. J. Agric. Food Chem., 25:1423.

Iwaoka, Wayne T., and Steven R. Tannenbaum, 1976. Liquid Chromatography of N-Nitrosoamino acids and their syn and anti conformers. J. Chromatography, 124:105.

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BIOGRAPHICAL SKETCHNAME Michael A. Dunn

POSITION TITLE

Associate Professor, Human NutritionEDUCATION/TRAINING (Begin with baccalaureate or other initial professionaleducation, such as nursing, and include postdoctoral training.)

INSTITUTION AND LOCATION DEGREE(if

YEAR(s) FIELD OF STUDY

The Pennsylvania State University, University B.S. 1974 BiochemistryThe Pennsylvania State University, University M.S. 1979 Animal NutritionThe Pennsylvania State University, University Ph.D. 1985 Nutritional Sciences

Positions and Honors.

1996 - present Associate Professor, Department of Human Nutrition, Food & Animal Sciences, University of Hawaii at Manoa, Honolulu, HI 96822.

1989 - 1996 Assistant Professor, Department of Food Science and Human Nutrition, University of Hawaii at Manoa, Honolulu, HI 96822.

1985 - 1989 Postdoctoral Research Associate, Department of Food Science and Human Nutrition, The University of Florida, Gainesville, FL 32611.

Member: American Society for Nutritional Sciences (ASNS). 1990-present.

Chairman: Graduate Programs in Nutrition (PhD) and Nutritional Sciences (MS), Department of Human Nutrition, Food & Animal Science University of Hawaii at Manoa. 2001-present.

Honor’s related to instruction:

2009 Recipient of Dean’s Award for Excellence in Teaching, College of Tropical Agriculture and

Human Resources, University of Hawaii at Manoa.

2010 Recipient of “Chancellor’s Citation for Meritorious Teaching”, the University of Hawaii at Manoa

Specialties by Training or Experience

Instruction: Teaching courses in nutritional biochemistry and metabolism, and diet-disease relationships at the cellular and molecular level. Research: Nutritional biochemistry and metabolism with emphasis on mineral metabolism especially aluminum and iron interactions. Effects of aluminum toxicity on iron metabolism and its relationship to disease processes (e.g. Alzheimer’s disease). Caco-

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2 cell culture based assays for food iron bioavailability. Regulation of gene expression and cellular metabolism by dietary minerals.

Selected peer-reviewed publications and abstracts

2010 Lai, J.F., J. Dobbs and M.A. Dunn. The Iron and Aluminum Content of Clams Along with Iron Bioavailability Assessed by a Caco-2 Cell Model Indicate that Clams are Questionable as a Recommended Source of Iron. (submitted to American Journal of Clinical Nutrition). 2009 Lai, J.F., J. Dobbs, M. A. Dunn, S. Tauyan, and C. A. Titchenal. Inconsistent values for iron

content complicate recommending clams as an iron source. J Food Comp and Analysis: S78-S82.2008 N.K. Bobbili, Y.S. Kim, M.A. Dunn, J. Yang, and A. Ong. Effects of maternal immunization against myostatin on postnatal growth and skeletal muscle mass of offspring in mice. Food and

Agricultural Immunology 19(2):93-106.2008 Titchenal, C. A., K. Hatfield, M. Dunn, and J. Davis. Does prior exercise affect oral glucose tolerance test results? (abstract), International Society of Sports Nutrition Conference,

June 8- 10, Las Vegas, NV.2007 Dunn, MA and H-Y Wang. Effects of aluminum on iron uptake and ferritin synthesis in

cultured human intestinal Caco-2 cells. Tenth Asian Congress of Nutrition, Sept 9-13, Taipei, Taiwan, (abstract).

2007 Miyasaka, S.C., N.V. Hue, and M.A. Dunn. Aluminum. In: A.V. Barker and D.J. Pilbeam (eds), Handbook of Plant Nutrition, CRC press, Boca Raton, FL.2001 Cox, K.A. and M.A. Dunn. Aluminum toxicity alters the regulation of calbibdin-D28k

protein and mRNA expression in chick intestine. J. Nutr.131:2007-13.

Research Support. List selected ongoing or completed research projects (federal and non-federal support). Begin with the projects that are most relevant to the research proposed in this application. Briefly indicate the overall goals of the projects and responsibilities of principal investigator identified above.

Active Grants:Principal Investigator:

USDA/HATCH “Improving health through the establishment of a relative iron-bioavailability database” Funded for 3 years for a total of $60,000. Start Date: Oct 1, 2008

Co-PIs: Drs. Joannie Dobbs, Halina Zaleski, and Yong Soo KimPrincipal Investigator:

USDA-CSREES-SRGP (TSTAR) “Identifying tropical plant-derived sources of dietary iron: linking tropical food production and consumption to consumer health. Funded for two years for a total of $67,703. Start date: 9/01/2009. Co-PI: Dr. Joannie Dobbs.

Selected Previous Grants:

Principal Investigator:Allen Foundation Inc. “Establishing an Iron-Bioavailability Database” Goal: establish an intestinal cell- culture based assay (Caco-2 cells) to determine the bioavailability of iron from foods. Funded for 2 years for $79,828. Start Date: July 1, 2005 Co-PIs: Drs. Joannie Dobbs, Yong Soo Kim, and Halina Zaleski

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CURRENT & PENDING SUPPORT

LIST SEPARATELY FOR EACH PD, co-PD and/or Key Personnel

Name: Yong Li

Instructions:Who completes this template: Each project director/principal investigator (PD/PI) and other senior personnel that the Request for Applications (RFA) specifies How this template is completed:

Record information for active and pending projects, including this proposal. All current efforts to which PD/PI(s) and other senior personnel have committed a portion of their time must be listed, whether or not salary for the

person involved is included in the budgets of the various projects. Provide analogous information for all proposed work which is being considered by, or which will be submitted in the near future to, other possible

sponsors, including other USDA programs. For concurrent projects, the percent of time committed must not exceed 100%..

Note: Concurrent submission of a proposal to other organizations will not prejudice its review by CSREES.

NAME(List/PD #1 first)

SUPPORTING AGENCY AND AGENCY ACTIVE

AWARD/PENDING PROPOSAL NUMBER

TOTAL $ AMOUNT

EFFECTIVE AND

EXPIRATION DATES

% OF TIME COMMITTED

TITLE OF PROJECT

Li, Y. and Iwaoka, W.T.

Li, Y.

Li, Y. and Huang, A.S.

Jun, S., Li, Y., and Jenkins, D.M.

Active:

NOAA

USDA-TSTAR

USDA-TSTAR

USDA Hatch Act Funds

$101,432

$108,326

$48,844

$60,000

2009-2011

2008-2011

2008-2011

2008-2011

10%

10%

5%

2%

Detection and Control of Salmonella in Ahi and Pacific White Shrimp

Survival, recovery, and quantification of target pathogenic bacteria in pineapple, guava, and orange juices.

An integrated approach for the quality improvement of guava puree and kava beverage by a non-thermal dense phase carbon dioxide pasteurization.

Detection of Escherichia coli O157:H7 using a nanoneedle probe biosensor.

Li, Y., Iwaoka, W.T. and Dunn, M.

Jun, S. and Li, Y.

Iwaoka, W.T., Nakamoto, S., Li, Q., Li, Y., and Hamasaki, R.

Pending:

USDA-TSTAR(this proposal)

USDA-TSTAR

USDA-STAR

$148,570.4 2011-2013

2011-2013

2011-2013

10%

5%

5%


Using nanoscale cantilever biosensor to detect extracellular DNA markers of foodborne pathogens in tropical fresh produce

Adding value to Hawaii tea (Camellia sinensis): Identification of minerals and major bioactive and flavor compounds in 5 promising cultivars.

*TSTAR proposals: Cost sharing not mandatory, for information purposes only.

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(COPY THIS PAGE AND USE ADDITIONAL SHEETS FOR EACH KEY PERSON)

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Name: Wayne T. Iwaoka









TOTAL $ AMOUNT

EFFECTIVE AND

EXPIRATION DATES

% OF TIME COMMITTED

TITLE OF PROJECT

Susan Miyasaka andWayne Iwaoka

Yong LiWayne Iwaoka

Active:

County of Hawaii

National Oceanic andAtmospheric Administration

$9,600

$101,432

September 1, 2010 – August 31, 2011

2009 - 2011

5%

5%

Innovative Agricultural Research to Evaluate Olive Varieties for Oil Production in Hawai`i

Detection and control of Salmonella in ahi and Pacific White shrimp

Iwaoka, Wayne,Stuart Nakamoto, Qing Li, Yong Li. & Randall Hamasaki

Li, Y., Iwaoka, W.T. and Dunn, M.

Pending:

USDA TSTAR

USDA TSTAR $148,570.4

2011-2013

2011-2013

20% Adding value to Hawaii tea (Camellia sinensis): Identification of minerals and major bioactive and flavor compounds in 5 promising cultivars.


*TSTAR proposals: Cost sharing not mandatory, for information purposes only. (COPY THIS PAGE AND USE ADDITIONAL SHEETS FOR EACH KEY PERSON)

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Name: Michael A. Dunn









TOTAL $ AMOUNT

EFFECTIVE AND

EXPIRATION DATES

% OF TIME COMMITTED

TITLE OF PROJECT

1. Michael A. Dunn (PI)

2. Michael A. Dunn (PI)

Active:

USDA/HATCH Project #: HAW00261H

Active: USDA: T-STAR Award #: 2009-34135-20060

$60,000

$67,703

Start: Oct. 1, 2008End: Sept. 30, 2011

Start: Sept 1, 2009End: Aug 31, 2011

0.20 FTE

0.20 FTE

Improving health through the development of a relative iron-bioavailability database

Identifying tropical plant-derived sources of dietary iron: linking tropical food production and consumption to consumer health.

3. Michael Dunn Co-PI PI: Rachel Novotny

4. Michael Dunn Co-PI PI: Yong Li

Pending:

USDA NIFA AFRI:

Pending:USDA: T-STAR( this proposal)

$23,864,205

$148,570.4

03/01/2011-2/30/2016

2011-2013

0.10 FTE

0.05 FTE

Child Obesity Prevention


*TSTAR proposals: Cost sharing not mandatory, for information purposes only. (COPY THIS PAGE AND USE ADDITIONAL SHEETS FOR EACH KEY PERSON)

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Collaborative Letters/Emails of Support

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Budget with Narrative (Year 1)

A. Key Person Support NOT ALLOWED FOR TSTAR

B. Other Personnel

Number of Personnel

Project Role(Post Doc, Grad Student, Undergrad Student, Secretarial, Technician, etc)

Salary Fringe Benefits Funds Requested

1 Post Doc 43,000 15,772.4 58,772.41 Undergrad Student 3,200 12.8 3,212.8

Total Salary, Wages, and Fringe Benefits 61,985.2

Narrative Description of Personnel Needed:

We plan to hire a postdoctoral fellow who will assist the PD in coordinating the project, training and supervising student help, performing experiments, analyzing data, and drafting project reports. One undergraduate student help will be employed to assist with the project by performing some of the experiments and conducting routine lab maintenance such as washing dishes, making media, etc. The student help will work 400 hours per year, and the hourly rate charged to the grant is $8/h. Fringe benefits are calculated as 36.68% for postdoctoral fellow and 0.4% for student help.

C. Equipment (List all non-expendable equipment purchases)

Equipment Item Funds

Requested1.2.3.4.5.

Total Equipment Cost 0

Narrative Description of Equipment Requested:

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D. Travel Funds Requested

1. Domestic Travel Costs2. Foreign Travel Costs

Total Travel Cost 0

Narrative Description of Travel Including Locations and Dates:

E. TRAINEE SUPPORT NOT ALLOWED FOR TSTAR GRANTS

F. Other Direct Costs

Funds Requested

1. Materials and Supplies 10,0002. Publication Costs 8003. Consultant Services4. ADP/Computer Services5. Subawards/Consortium/Contractual Costs6. Equipment or Facility Rental/User Fees7.8.

TOTAL OTHER DIRECT COSTS 10,800

Narrative Description of Materials and Supplies:

Laboratory supplies include chemicals, microbiological media, DNA extraction kit, oligonucleotide primers, PCR reagents, API-ZYM System, Sensititre Anaerobe MIC Plate, and consumable materials like paper towels, gloves, glassware, pipet tips, Petri dishes, etc. Funds are requested to cover costs of preparing and publishing 1 scientific article (approximately $100/page).

Narrative Description of Subawards/Consortium/Contractual Costs:(Note: All Subcontracts must be listed here and have their own budget forms which total to the amount requested.)

Narrative Description of any other items in Other Direct Costs:

TOTAL DIRECT COSTS REQUESTED Year 1: $72,785.2

NOTE: Indirect Costs are not allowable for TSTAR Grants according to Special Research Grant Rules under P.L. 89-106.

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Budget with Narrative (Year 2)

A. Key Person Support NOT ALLOWED FOR TSTAR

B. Other Personnel

Number of Personnel


Salary Fringe Benefits Funds Requested



Narrative Description of Personnel Needed:

We plan to hire a postdoctoral fellow who will assist the PD in coordinating the project, training and supervising student help, performing experiments, analyzing data, and drafting project reports. One undergraduate student help will be employed to assist with the project by performing some of the experiments and conducting routine lab maintenance such as washing dishes, making media, etc. The student help will work 400 hours per year, and the hourly rate charged to the grant is $8/h. Fringe benefits are calculated as 36.68% for postdoctoral fellow and 0.4% for student help.

C. Equipment (List all non-expendable equipment purchases)

Equipment Item Funds

Requested1.2.3.4.5.


Narrative Description of Equipment Requested:

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1. Domestic Travel Costs 3,0002. Foreign Travel Costs

Total Travel Cost 3,000

Narrative Description of Travel Including Locations and Dates:

Funds are requested for PD to attend the Institute of Food Technologists (IFT) Annual Meeting to present results.

E. TRAINEE SUPPORT NOT ALLOWED FOR TSTAR GRANTS

F. Other Direct Costs

Funds Requested

1. Materials and Supplies 10,0002. Publication Costs 8003. Consultant Services4. ADP/Computer Services5. Subawards/Consortium/Contractual Costs6. Equipment or Facility Rental/User Fees7.8.


Narrative Description of Materials and Supplies:

Laboratory supplies include chemicals, microbiological media, cell culture media and reagents, HPLC supplies, and consumable materials like paper towels, gloves, glassware, pipet tips, Petri dishes, etc. Funds are requested to cover costs of preparing and publishing 1 scientific article (approximately $100/page).

Narrative Description of Subawards/Consortium/Contractual Costs:(Note: All Subcontracts must be listed here and have their own budget forms which total to the amount requested.)

Narrative Description of any other items in Other Direct Costs:

TOTAL DIRECT COSTS REQUESTED Year 2: $75,785.2

NOTE: Indirect Costs are not allowable for TSTAR Grants according to Special Research Grant Rules under P.L. 89-106.

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Cumulative Budget

A. Key Person Support NOT ALLOWED FOR TSTAR SupportB. Other Personnel

Number of Personnel


Salary Fringe Benefits

Funds Requested



C. Equipment (List all non-expendable equipment purchases) Equipment Item Funds Requested

1.2.3.4.5.



1. Domestic Travel Costs 3,0002. Foreign Travel Costs

Total Travel Cost 3,000

E. TRAINEE SUPPORT NOT ALLOWED FOR TSTAR GRANTSF. Other Direct Costs

Funds Requested1. Materials and Supplies 20,0002. Publication Costs 1,6003. Consultant Services4. ADP/Computer Services5. Subawards/Consortium/Contractual Costs6. Equipment or Facility Rental/User Fees7.8.


TOTAL TSTAR GRANT REQUESTED Year 1 + Year 2: $148,570.4

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Appendix 1. The pH, bacterial count, yeast and mold count of poi

Brand pH Bacterial count(log10 CFU/g)

Yeast and mold count(log10 CFU/g)

A

B

C

D

5.44 ± 0.37

4.49 ± 0.63

6.04 ± 0.02

5.42 ± 0.14

9.34 ± 0.12

9.13 ± 0.16

6.89 ± 0.61

9.23 ± 0.28

3.97 ± 0.37

6.36 ± 1.43

1.51 ± 0.52

4.33 ± 0.14

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Appendix 2. Fingerprinting of lactic acid bacteria in poi by PCR-TGGE

TGGE profile of PCR products from the microflora of poi. Letters A, B, C, and D correspond to each respective poi manufacturer. Bands marked a through i were excised and sequenced. The identifications are reported in the table below.

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Band Closest Relative % Identity GenBank Accession No. a Weissella confusa 95 AJ508722b Lactobacillus delbrueckii 100 AB289092 c Streptococcus sp. 91 AB264619d Lactobacillus plantarum 99 AF157037e Calycanthus floridus 100 DQ629462 f Leuconostoc citreum 97 EF221781g Enterococcus faecium 91 EF373551h Lactococcus lactis 89 AY348313i Bacillus megaterium 100 EF114346

a

b

ch

A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3

g

d

ie

4

f

tstar proposal application template - ctahr · web viewto illustrate the health promoting...

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