Source: TEXAS A&M UNIVERSITY submitted to
LIPOSOME ENCAPSULATION OF NATURALLY-OCCURRING FOOD ANTIMICROBIALS FOR THE INHIBITION OF FOODBORNE PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0213450
Grant No.
(N/A)
Project No.
TEX09235
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Feb 20, 2008
Project End Date
Feb 19, 2014
Grant Year
(N/A)
Project Director
Taylor, T.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Animal Science
Non Technical Summary
Encapsulation of food antimicrobials allows for application of antimicrobials into a broader range of foods as compared to applications of non-encapsulated antimicrobials, thus producing a broader market for antimicrobials and a safer food supply. The purpose of this study is to investigate the efficacy of liposome encapsulation for the entrapment and controlled delivery of the food antimicrobial nisin in food products not normally conducive to nisin application.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
50134501100100%
Knowledge Area
501 - New and Improved Food Processing Technologies;

Subject Of Investigation
3450 - Milk;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Research objective will focus on the optimization of antimicrobial encapsulation efficiency (EE) by alternative methods of antimicrobial loading into liposomal vesicles. Specifically, liposome loading via pH gradient-driven exchange mechanisms and multi-freeze/thaw cycling methodologies will be tested and EE determined via nisin bioassay. Objective II will seek to generate liposomes with broad-spectrum antimicrobial activity via modulating the lipid formulation and co-encapsulation of multiple antimicrobials, active against both Gram-negative and Gram-positive bacterial foodborne pathogens. Capsules that are wrapped in an antimicrobial polymer (chitosan) will be experimented with against model foodborne pathogens. Additionally, co-encapsulates containing both nisin and Gram-negative functional colicins (bacteriocins fermented by Escherichia coli spp) will be inoculated into fluid milk artificially contaminated with pathogens. Objectives three and four will focus on the application of antimicrobial-bearing liposomes to food commodities not previously tested, those being a pasteurized fruit juice (apple) and a ready-to-eat (RTE) processed meat product. Bacterial pathogens will be artificially inoculated in food commodities and liposome-based inhibition determined.
Project Methods
Objective 1 (TAMU): Liposomal antimicrobial loading efficiency (EE) will be determined experimentally via spectrophotometric assays by which percent reporter molecule encapsulated will be determined. Subsequent nisin bioassays will be generated whereby nisin or colicin activity as a function of loading methodology will allow for determination of optimized loading technology. Liposome formulation will be carried out according to previously reported methods. Objective 2 (TAMU; UTK): Liposomes bearing antimicrobial(s) will be formulated at Texas A&M University (TAMU) and subsequently coated with the antimicrobial polymer chitosan after shipment to University of Tennessee-Knoxville, Dept. Food Science and Technology (UTK). Physical-chemical and biophysical properties of liposomes (size, ?-potential, thermotropism, etc.) will be measured using appropriate analytical equipment. Antimicrobial capabilities of coated liposomes against model Gram-negative and Gram-positive pathogens will be determined in vitro by micro-broth dilution checkerboard assay. Cells will be incubated at optimal conditions in the presence of nutritious growth medium and liposomes and changes in experimental vessel optical density (OD) recorded and analyzed for evidence of pathogen growth, stasis, or death. Objective 3 (TAMU): Liposomes will be inoculated into ultra-high temperature (UHT) fluid skim, 1%, and whole milk; milk will be subsequently artificially contaminated with strains of the environmental Gram-positive pathogen L. monocytogenes and survivors will be enumerated on non-selective solid growth medium at different intervals of incubation at temperature-abuse conditions in order to determine the efficacy of antimicrobial encapsulate delivery into a model complex food matrix. Data will consequently be analyzed for potential inhibition of pathogens. Objective 4 (TAMU; UTK): Liposomal antimicrobial will be formulated and inoculated on to polymer casings intended for RTE pork products (e.g. frankfurters). Vesicles will be initially optimized by way of variations in lipid profiles; these optimizations will seek to determine the optimal choice of lipids so as to guarantee heat-driven swelling and rupture of liposomes for the release of entrapped antimicrobial on to the surface of encased RTE meat products. Data gathering and analysis of thermal properties of various liposome formulations will occur at UTK. Samples will be incubated under conditions of storage abuse and survivors enumerated on non-selective growth medium (solidified) and data analyzed for bacterial growth, stasis, or inhibition. Microbiological analyses will occur at TAMU.

Progress 02/20/08 to 02/19/14

Outputs
Target Audience: The primary aim of this research-focused project was the development, analysis, and application of nano-scaled liposomal particles for the delivery of traditional and natural food chemical preservatives (food antimicrobials) for the preservation of microbiological safety of processed human foods. To that end, the key targeted audiences that were reached by project investigator efforts included other food safety microbiologists and food safety specialists positioned within academic institutions within the U.S. and abroad. Additionally, food safety specialists stationed within U.S. food industries were also targeted by project efforts for the purposes of working towards the deployment of developed technologies for the preservation of food safety for U.S.-produced and processed human foods. Industry members within the U.S. dairy, produce packing, and processed meat/poultry industries were targeted by research studies, given the nature of the antimicrobials used in liposomal nanoparticles, current federal allowances/approvals for antimicrobial use in foods, and the types of microbial pathogens commonly associated with these food products (specifically bacterial foodborne pathogens associated with pasteurized fluid milks, post-harvest washed produce items, and processed RTE meat/poultry items). Targeted audiences were identified through ongoing research collaborations between project leader and other U.S. or non-U.S.-stationed scientists, interactions with scientists occurring at annual research and industry professional meetings (e.g., annual meeting of the International Association for Food Protection), and through other USDA-sponsored multi-state research projects on which the project leader participated. These activities allowed the dissemination of research findings to both academic and industry scientists through the presentation of scholarly abstracts and the publications of refereed manuscripts (see publications list), as well as through the publication of edited technical chapters (see publications list). Specific examples of targeted audience interactions with the project investigator include U.S.-located industry scientists seeking research support for the development of liposomal nanoparticles capable of delivery of bacteriophage-derived antimicrobial lytic proteins for inactivation of processed meat-contaminating Listeria and Salmonella species, the development and publication of three edited chapters in technical texts or monographs, and the identification of a U.S. company with interests in development of a liposome-delivered food spoilage indicator technology responsive to growth of food spoilage bacterial microorganisms. Other activities included the establishment of research collaborations with investigators stationed at the project investigator's institution studying the development and application of non-liposome based antimicrobial delivery technologies based on shared interests of researchers on nano-encapsulation and development of enhanced antimicrobial delivery systems for food safety protection. Two such collaborations have been established, leading to multiple refereed manuscripts being published and the development of a research collaboration that has spawned one funded USDA-NIFA AFRI research grant, multiple competitive grant submissions to USDA, NSF, and NASA, and a series of research studies being completed with food safety foci. These activities also resulted in the recruiting of two graduate degree-seeking students completing degree programs under the investigator's direction, assisting the training of new scientists (an aligned but non-targeted audience). Additionally, while tangential to the project completion, a graduate level fixed credit lecture-type course has been developed between the project investigator and a collaborating research/teaching faculty member at the investigator's institution teaching concepts and principles of food nanotechnology. This course assists graduate students interested in food processing systems to gain greater understanding and appreciation for the current state of U.S. food nanotechnology development, usage, regulatory processes, and industry involvement. The course has been completed two times as of this report, training a total of 25 graduate students. Changes/Problems: As discussed in the project accomplishments portion of this and previous annual reports, investigator-led research efforts indicated a failure of solvent evaporated, hydrated liposomes to effectively deliver high contents of food antimicrobial to food systems that resulted in significant pathogen inhibition. This led to the identification of the need for further optimization efforts as well as an expansion of the scope of the investigator's vision for antimicrobial encapsulation technologies for food safety preservation. Identified in a previous report (reporting date 9/30/2013), these findings led to the incorporation of research involving the use of food grade biopolymers for antimicrobial encapsulation and liposome encapsulation improvements. To date, efforts at seeking competitive funding for the investigation of liposome/polymer systems have been unsuccessful, though the project investigator has identified multiple research colleagues with interest in collaborative research should future grant submissions win funding support from USDA or another research sponsor. Additionally, although the originally approved project identified the goal of application of Gram-negative inhibiting antimicrobial polypeptides (colicins) for pathogen inhibition, this phase of the research was ultimately not attempted, largely a function of findings from other research studies completed under project objective 1. While colicins have been researched by both U.S. and non-U.S. scientists, they are currently not approved for direct addition to foods manufactured or imported into the U.S. by the FDA or USDA-FSIS. Hence, there is a reduced incentive for research scientists or industry members to conduct research or support research of these compounds. What opportunities for training and professional development has the project provided? Cumulatively, three graduate degree-seeking students (2 x M.S.; 1 x Ph.D.) were trained by the project investigator under the objectives of this project. Additionally, one graduate degree-seeking student (1 x Ph.D.) was trained by a collaborating investigator under a separate Hatch project, but using project investigator's laboratory and research expertise in antimicrobial encapsulation systems. This last student would represent an aligned training completed, indirectly contributing to project execution. The project investigator attended one professional workshop focusing on the use of food nanotechnologies offered by the Institute of Food Technologists held in conjunction with the IFT 2009 Annual Meeting and Food Expo. Additionally, the project investigator has provided multiple invited lectures on the use of food nanotechnologies for antimicrobial delivery into foods at international scientific meetings (see publications list for citations of presentations). Finally, through expert consulting activities, the investigator has interacted with a U.S. company producing chromagenic systems for food spoilage indication with interest in the use of liposomes for spoilage indicator utilization in food packaging. This firm has relayed interests in investigator's expertise in liposome development, and access to other researchers and laboratories, to explore the opportunities for liposomes to be produced that would allow for meat buyers to quickly visually identify acid fermentation-type spoilage by naturally contaminating microbes in events of loss of proper refrigeration storage during product holding, transportation, or retail display. How have the results been disseminated to communities of interest? The results of research findings have been communicated to other researchers and industry members through three (3) primary mechanisms. The first mechanism of results dissemination is that of publication of refereed research papers in journals. Multiple papers have been developed, submitted and published in which the project investigator has served as the senior author or co-author. These papers have also incorporated graduate degree-seeking students as co-authors, allowing for the further training of agricultural/food scientists. Secondly, through presentation of research findings at U.S. and international meetings, other scientists and researchers have been exposed to ongoing research projects/efforts, and trained students have been given opportunity to meet and engage with other scientists, which assisted one student in identifying and interviewing a researcher at another U.S. land grant institution to complete a Ph.D. degree under the researcher's supervision. Finally, through collaboration with U.S. industry members, findings have been presented in private sessions under confidentiality agreement with the investigator's state experiment station in order to discuss possible opportunities for industry sponsorship of investigator efforts/research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Completed research produced experimental data that indicated non-effective entrapment of food antimicrobials into nano-liposomes by conventional method (solvent evaporation-rehydration) based on the repeated exposure to freezing and warm temperatures following lipid hydration. These data led to the conclusion that encapsulation methods for high-efficiency entrapment of food antimicrobials required further optimization for application in food systems. This finding directly answered research objective 1 in that the optimization of encapsulated antimicrobial required further pursuit for delivery of food antimicrobials into food systems. It further impacted the development of research by exposing the need for further collaboration and studies into more effective means of processing of liposomal nanoparticles, leading the project investigator to seek out colleagues with nanoencapsulation interest/expertise. Two such researchers have been identified and researches are ongoing both on biopolymeric encapsulate systems, as well as coated/enrobed liposomes marrying biopolmyer (PLGA, cyclodextrin, PEG) to liposomes for antimicrobial entrapment. A change in knowledge was observed in year 2009 when research data resulted in researchers determining that liposome formulation containing poly-dispersed phospholipids made via freeze/thawing did not sufficiently entrap antimicrobial effectively. Those data were published (Schmidt et al. 2009; see publications list), allowing the establishment of a baseline of information from which further studies were anticipated. These data also initiated a change in actions, whereby then current methods of encapsulation were suspended/discontinued, as resulting liposomes were non-homogenous and ineffective delivery vehicles for food antimicrobial. Data allowed a change in conditions by the development of a novel system for delivering natural food antimicrobial to fluid milk, allowing enhanced control post-processing of the microbial pathogen Listeria monocytogenes. The primary outcome from experimentally gathered data from CY2010 activities was the understanding that simple liposomal nanostructures with entrapped antimicrobial presented significant inhibition for the suppression of foodborne pathogens in a fluid food with neutral or near-neutral pH conditions. Nonetheless, gathered data also indicated these structures insufficient except at conditions of refrigerated storage and/or high levels of antimicrobial application to suppress likely bacterial pathogens in the food product of interest. This led to the conclusion that further research and development of such technologies was critical to realize greater food safety protection via antimicrobialbearing nano-structures (e.g., liposomes, micelles, etc.). These data specifically indicated that the application of antimicrobial-bearing structures in combination with other preservation technologies/processes (acidification, fermentation, low temperature storage) will significantly enhance the observed antimicrobial effectiveness against contaminating pathogens such as L. monocytogenes. Further indications from un-published data indicated opportunity for the inhibition of Gram-negative enteric pathogens including Escherichia coli and Salmonella in fluid foods from the co-encapsulation of multiple antimicrobials including antimicrobial polypeptides, organic acids, and bacteriophage or phage-derived lytic proteins (i.e. endolysins). Secondly, the development of experimental data describing the interactions of differing classes of food antimicrobials led to the identification of novel combinations of antimicrobials capable of exerting synergistic inhibition of foodborne pathogens in model systems (Brandt et al. 2010, 2011; see publications list). These data may be applied to food systems in either an unencapsulated or an encapsulated state to inhibit bacterial pathogen proliferation. Other combinations of acidulants with antimicrobial polymers revealed significant inhibitory capacity, effectively halting growth of the pathogen under experimental conditions. These data enhanced the ability of investigators to control the proliferation of bacterial foodborne pathogens (particularly those with sensitivities to applied antimicrobials similar to that of L. monocytogenes) by offering new opportunities for preservative-driven inhibition via encapsulated or un-encapsulated antimicrobials in foods. Major findings from reported research in CY2010-2012 generated significant understanding of the antimicrobial mechanism of a commercially available biopreservative culture, a useful candidate for liposomal encapsulation for foods application. These data were novel, differing from previous studies in their specific identification of particular metabolites as compared to presumptive identification of antimicrobial types. These data applied by the commercial entity providing the biopreservative to assist the revision the composition of the antimicrobial system in an effort to produce a more effective food antimicrobial technology. Data gathered in collaboration with other researchers demonstrated the ability of nano-encapsulate systems with naturally occurring food antimicrobials to inhibit foodborne microbial pathogens in an in vitro environment, and subsequently were used to design studies on their utility in differing food systems (Hill et al. 2013a,b; see publications list). This collaboration has also generated a significant outcome in the form of a developing opportunity to engage in research collaboration with scientists in the nation of Brazil. Though ultimately not funded, one collaborative research grant was submitted to the Brazilian government entity EMBRAPA for the purposes of collecting data on liposomal and polymeric nanocapsules for food safety preservation purposes. This grant involved a Brazilian researcher (A. Brandelli), and two U.S. researchers (T.M. Taylor, project investigator; C. Gomes, TAMU). Findings from researches on biopolymer and surfactant-type nano-capsules (micelles, liposomes, shells) indicated maximum loading could occur from tailored systems that utilized combinations of hydrophobic antimicrobial and amphipathic encapsulating materials. Gram-negative and Gram-positive bacterial pathogens (E. coli, L. monocytogenes) were treated and were observed to be inhibited from growth by biopolymer systems in liquid media. These findings set the stage for later investigations of antimicrobial-bearing nanoencapsulates on surfaces of fresh produce (leafy greens, smooth fruit) for pathogen inhibition evaluation. Maximum additive concentration (MAC) experiments conducted in the project investigator's laboratory determined that carvacrol/Surfynol combinations represented an optimal pairing of antimicrobial and encapsulate, and that these systems were best prepared to deliver antimicrobial essential oil to plant surfaces inoculated with microbial pathogens.

Publications

  • Type: Book Chapters Status: Published Year Published: 2015 Citation: Brandelli, A. and T.M. Taylor. 2015. Nanostructured and nanoencapsulated natural antimicrobials for use in food products. Ch. 11. In: Handbook of natural antimicrobials for food safety and quality. T.M. Taylor (ed.). pp. 229-257. Woodhead Publishing: Cambridge, United Kingdom.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hill, L.E., C. Gomes, and T.M. Taylor. 2013. Characterization of beta-cyclodextrin inclusion complexes containing essential oils, trans-cinnamaldehyde, eugenol, cinnamon bark extract, and clove bud extracts for antimicrobial delivery applications. LWT - Food Science and Technology. 51(1):86-93.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Hill, L.E., C. Gomes, and T.M. Taylor. 2013. Antimicrobial efficacy of poly (DL-lactide-co-glycolide) (PLGA) nanoparticles with entrapped cinnamon bark extract against Listeria monocytogenes and Salmonella Typhimurium. Journal of Food Science. 78(4):N626-N632.


Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Key target audiences reached by efforts include other food safety specialists in the foods processing industry, food safety microbiology researchers, and researchers working in the development and application of nano-encapsulation for food antimicrobials utility. Changes/Problems: As discussed, one core change has been the incorporation of research focused on biopolymers for antimicrobial entrapment and delivery. This has led to novel data gathering and opportunities for future research in food safety protection. What opportunities for training and professional development has the project provided? Two graduate students were supervised during the reporting period, completing research-based thesis yielding degree requirements during the reporting period. How have the results been disseminated to communities of interest? See publications list. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Within the project, core objectives focused on the development of lipid-derived nano-capsules for the encapsulation and delivery of food antimicrobials into food systems to prevent growth of contaminating microbial pathogens. During the reporting period, research involving non-lipid based nano-capsules was pursued to complement understanding along with previously gained research in lipid systems (liposomes). These data resulted in publications listed on other reports, completed in collaboration with TAMU-located researchers. These data can be used to drive development of novel lipid/polymer hybrid structures that may possess greater process tolerance, protecting antimicrobials during mild processing. This would beneft the development of lipidic systems where antimicrobial protection during processing is required, and biopolymers can be tagged with pathogen-targeting antibodies that may allow resolution to project objectives describing targeting of pathogens by nano-systems.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Taylor, T.M. 2013. Improving the utility of plant antimicrobials for food safety management through encapsulation. Presented by invitation at the 15th Annual International Congress on Food Safety/30th Meeting of the National Committee on Microbiology, Hygiene and Toxicology of Food, Guadalajara, Mexico. Oct. 31-Nov. 1.
  • Type: Other Status: Published Year Published: 2013 Citation: Davidson, P.M., F.J. Critzer, and T.M. Taylor. 2013. Natural-occurring antimicrobials for minimally processed foods. Annual Reviews in Food Science and Technology. 4:163-190.


Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: Project research has shifted from liposomal-type nano-systems for antimicrobials development to more novel bio-polymer based systems for natural antimicrobials development and application to foods. Collaborative research has resulted in the development of antimicrobial efficacy data describing the inhibition of foodborne bacterial pathogens E. coli and Listeria monocytogenes using cyclodextrin and poly-lactic acid derived capsules containing the antimicrobials eugenol, cinnamic aldehyde, and natural extracts of spice plants. These assays were completed in cooperation with researchers described in Participants section. Efforts in investigator Taylor laboratory have focused on the use of surfactants to entrap plant derived antimicrobials in addition to other biopolymers. Maximum additive capacity experiments have been completed with multiple food-grade surfactants entrapping carvacrol and eugenol, determining the maximum loading capacity of capsules. Carvacrol-containing surfactant micelles formed from the surfactant Surfynol exhibited maximum loading capacity, indicating greatest potential for nano-encapsulated antimicrobials to be delivered to food surfaces and inhibit microbial pathogens. Research findings have been disseminated to stakeholders through refereed publications and abstracts presented at multiple national meetings. Presentations incorporating funded research findings have been presented internationally at conferences. Students have been trained as a result of project researches, both under my supervision and my advisement. Two rejected grant submissions were submitted using gathered data and seeking additional funding to complete research objectives and research extensions. Additional efforts have focused on use of non-pathogenic bacteria as useful for inhibiting pathogens. These organisms may also be delivered to foods in an encapsulated state. Outputs have included refereed publications and other presentations that have demonstrated the utility of selected pathogen antagonists in food safety preservation, as prerequisite for any experiments involving encapsulated pathogen antagonists. PARTICIPANTS: Graduate research assistants (2; Ph.D.) were supervised and advised for purposes of data collection and analysis towards completion of researches described in the Outputs portion. No other personnel were trained during the relevant project period. TARGET AUDIENCES: Targeted audiences included researchers in food safety, graduate and undergraduate students in Food Science, industry members interested in antimicrobials utilization and delivery to foods. PROJECT MODIFICATIONS: A significant shift in research focus from liposomes to biopolymers and micelle-type nanoparticles, as well as a focus on produce commodities, has occurred for the project. This deviation from originally-supported project proposal is the result of new collaborations with researchers conducting researches on biopolymers for antimicrobial delivery, and receipt of project funding for completion of research on the use of nano-systems to improve the safety of fresh produce. These collaborations were not originally anticipated, and the opportunity for research on nano-particle application to produce has arisen only in recent years for targeted funding sources. No other major changes have occurred.

Impacts
Findings from researches on biopolymer and surfactant-type nano-capsules has indicated maximum loading may occur from tailored systems that use optimal combinations of hydrophobic antimicrobial and amphipathic encapsulating material. Pathogens E. coli and L. monocytogenes were effectively inhibited by biopolymer type systems in liquid media. These findings have set the stage for further application to produce systems for pathogen inhibition. MAC experiments have determined that carvacrol/Surfynol combinations represent optimal pairing of antimicrobial and encapsulate, indicating that these systems will be best prepared to deliver antimicrobial essential oil to plant surfaces inoculated with microbial pathogens. Outputs of graduate students trained and efforts on development of novel antimicrobial encapsulation systems has produced impacts and outcomes by allowing focus on novel systems and collaboration with other researchers to expand capacity to develop and analyze nano-encapsulates. Collaboration involving microbiology expertise and laboratory access has allowed determination of Minimum Inhibitory Concentration (MIC) assay for nano-encapsulates, providing data useful for publication and submission in competitive grant proposals. Data on the use of some pathogen antagonizing bacteria has been of use in demonstrating the utility of these types of bacteria to inhibit pathogens on foods. These data serve as indicators of the potential for new researches involving the encapsulation of bacteria for delivery to inhibit pathogen growth on foods.

Publications

  • Calix-Lara, T.F., T. Duong, and T.M. Taylor. 2012. Addition of a surfactant to tryptic soy broth allows growth of a Lactic Acid Bacteria food antimicrobial, Escherichia coli O157:H7, and Salmonella enterica. Lett. Appl. Microbiol. 54:392-397.
  • Perez, K.L., L.M. Lucia, L. Cisneros-Zevallos, A. Castillo, and T.M. Taylor. 2012. Efficacy of antimicrobials for the disinfection of pathogen contaminated green bell pepper and of consumer cleaning methods for the decontamination of knives. Int. J. Food Microbiol. 156:76-82.
  • Davidson, P.M., T.M. Taylor, and S.E. Schmidt. 2012. Chemical antimicrobials and natural preservatives. p. 765-801 In: Doyle, M.P. and R.L. Buchanan (eds.) Food Microbiology: Fundamentals and Frontiers (4th edt.). Washington, DC: ASM Press.
  • Perez, K.L., T.M. Taylor, and P.J. Taormina. 2012. Competitive research and development for the food industry. p. 109-159 In: Taormina, P.J. (ed.) Microbiological Research and Development for the Food Industry. New York, NY: Taylor and Francis, LLC.
  • Taylor, T.M. 2012. Nanotechnology Applications for Food Processing and Food Safety. Presented in conjunction with the New Trends in Nanotechnology-International Symposium, Bogota, Colombia. Aug. 24.


Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: For the year 2011, significant outputs were achieved by the completion of publication of original data supported by project funds and in line with project objectives. In addition, a collaboration with a researcher in another department within the agency and I have initiated a collaboration investigating technologies similar to those described in the original hatch project for the preservation of food safety. One major output from this collaboration has been the elaboration of data demonstrating the antimicrobial efficacy of these technologies for the inhibition of foodborne microbial pathogens. In addition to these efforts, a second student was advised to completion of degree and one publication has been submitted from gathered data. These data result from completion of a funded project investigating a biopreservative culture for the inhibition of foodborne pathogens on produce surfaces. The pathogens E. coli O157:H7 and Salmonella were shown to be significantly inhibited on surfaces of biopreservative-inoculated spinach over 14 days of refrigerated storage. Data elaborating the production of L-lactic acid, acetic acid, hydrogen peroxide, and an antimicrobial polypeptide were also gathered, definitively showing antimicrobial compounds produced by this biopreservative culture as a result of fermentation. PARTICIPANTS: The Primary Investigator (T.M. Taylor) contributed 12 months of work/activity to the completion of activities related to the project's advancement. Oversight of research graduate assistants, technicians, and development of grant proposals necessary for sustained financial support of research activities. Lisa Lucia (Technician) contributed oversight and assistance to graduate assistants during scheduling, planning, and completion of laboratory experiments for completion of research trials. Graduate students participating on this project include Alex L. Brandt, Thelma Calix-Lara, and Keila Perez. These students participated in differing research programs under the project umbrella. Collaborating researchers in Texas AgriLife Research include Carmen Gomes, Alejandro Castillo, Luis Cisneros-Zevallos, Rhonda Miller, Steve Smith, Steve Talcott. In addition to research collaboration, scientists participated in development of other research proposals to extend findings gained from research. TARGET AUDIENCES: Laboratory instruction was used in completion of research activities. Special target audiences are not identified of special significance. No formal classroom instruction was completed. An addition to current curriculum is being developed to provide graduate students exposure to nano-scale science for application in the food industry. No extension or outreach activities were completed. PROJECT MODIFICATIONS: A significant hindrance to research goals from increased stringency in university/research station biohazard protocols has arisen, and became excessively problematic during year 2011. It is anticipated that based on recent events, that this problem may begin to subside with regards to its impact on research completion. However, final project objectives will be difficult to achieve without significant acceleration of project trials. Nonetheless, efforts are underway to achieve all major objectives in part or whole.

Impacts
Major findings from reported research generated significant understanding of the antimicrobial mechanism of a commercially available biopreservative culture. These data are the first of their kind, differing from previous studies in their specific identification of particular metabolites. These data have been used to revise the composition of the antimicrobial culture product, potentially leading to a more effective food antimicrobial product. Other data gathered in collaboration with other researchers have demonstrated the ability of nano-scale encapsulate systems with naturally occurring food antimicrobials to inhibit foodborne microbial pathogens in an in vitro environment, and will be used to design studies on their utility in differing food systems. This collaboration has also generated a significant outcome in the form of a developing opportunity to engage in research collaboration with scientists in the nation of Brazil.

Publications

  • Brandt, A.L., A. Castillo, K.B. Harris, J.T. Keeton, M.D. Hardin, and T.M. Taylor. (2011) Synergistic inhibition of Listeria monocytogenes in vitro through combination of octanoic acid with acidic calcium sulfate. Journal of Food Protection. 74(1):122-125.
  • Taylor, T.M. (2011) Development of nano-scale technologies for food safety protection. 2011 Annual Meeting of Arkansas Association of Food Protection, Fayetteville, AR, September 13-14.
  • Perez, K.L., L. Cisneros-Zevallos, A. Castillo, and M. Taylor. (2011) Effect of kitchen procedures for knife cleaning on the transfer of pathogens during in-home processing of fresh produce. 100th Annual Meeting of the International Association for Food Protection, Milwaukee, WI.


Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Output activities for calendar year 2010 include the analysis, submission and publication of experimentally gathered data detailing the antimicrobial efficacy of food-grade polypeptide antimicrobial (nisin) entrapped in liposomal nano-capsules formed from food-grade and non-food grade materials capable of inhibiting the bacterial pathogen Listeria monocytogenes in fluid milks of differing fat contents. Data validated the efficacy of such structures for the delivery of antimicrobial in a homogeneous liquid food matrix, yet also indicated significant opportunities for further research and development. One graduate research assistant was trained in the development of these systems. Subsequent to these activities, collaborations with other U.S. researchers and corporate entities have begun that will explore antimicrobial utility of these liposomal structures in other fermented dairy products and produce foods. Significant outputs have been the collaborative submission and awarding of two USDA-NIFA funded grants that incorporate liposomal, micellar, and other user-defined nano-structures for the delivery of food-grade antimicrobial compounds to the surfaces of pre-harvest and post-harvest obtained leafy greens and smooth produce commodities. These awards will support continued research into the development of food antimicrobial delivery technologies useful for protecting the microbiological safety of multiple types of food products. Significant research has also been conducted determining the opportunities for enhanced inhibition of Listeria monocytogenes via application of combined antimicrobial compounds for the preservation of food safety in differing ready-to-eat foods. These data, previously presented in conjunction with meetings of the relevant scientific organizations, were recently published in peer-reviewed scientific journals, disseminating findings to the relevant industries and scientific communities. PARTICIPANTS: PI: Thomas Matthew Taylor, Ph.D. Collaborating Organizations: Texas AgriLife Research, Texas AgriLife Extension Collaborators: Alejandro Castillo, Margaret D. Hardin, Kerri B. Harris, Jimmy T. Keeton, Luis Cisneros-Zevallos, Elsa A. Murano. Opportunities for Development: Support was provided for the training of one graduate research student. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The primary outcome from experimentally gathered data from CY2010 activities is the understanding that simple liposomal nanostructures with entrapped antimicrobial do present significant inhibition capability for the suppression of foodborne pathogens in a fluid food with neutral or near-neutral pH conditions. Nonetheless, gathered data also indicate that these structures are not sufficient except at conditions of refrigerated storage and/or high levels of antimicrobial application to suppress likely bacterial pathogens in the food product of interest. This understanding leads to the conclusion that further research and development of such technologies is necessitated to realize greater food safety protection via antimicrobial-bearing nano-structures (e.g., liposomes, micelles, etc.). These data specifically indicate that the application of antimicrobial-bearing structures in combination with other preservation technologies/processes (acidification, fermentation, low temperature storage) will significantly enhance the observed antimicrobial effectiveness against contaminating pathogens such as Listeria monocytogenes. Further indications from data not published indicate opportunity for the inhibition of Gram-negative enteric pathogens including Escherichia coli and Salmonella in fluid foods from the co-encapsulation of multiple antimicrobials including antimicrobial polypeptides, organic acids, and bacteriophage or phage-derived lytic proteins (i.e. endolysins). Secondly, the development of experimental data describing the interactions of differing classes of food antimicrobials has led to the identification of novel combinations of antimicrobials capable of exerting synergistic inhibition of foodborne pathogens, specifically Listeria monocytogenes, in model systems. These data can thus be applied to food systems in either an unencapsulated or an encapsulated state to inhibit bacterial pathogen proliferation. Specifically, combinations of nisin with the fatty acid octanoic acid or the weak organic acid lactic acid, present in commercially available antimicrobial products, were shown to exert bacteriostatic and bactericidal inhibition of L. monocytogenes in broth matrices, indicating no change in populations of the pathogen or at least a 3.0 log cycle decrease in numbers of the pathogen within 24 hr storage at physiological temperature. Other combinations of acidulants with antimicrobial polymeric substances revealed significant inhibitory capacity, effectively halting growth of the pathogen under experimental conditions. These data significant enhance the ability of scientists to control the proliferation of this other similar bacterial foodborne pathogens by offering new opportunities for preservative-driven inhibition by use of encapsulated and un-encapsulated antimicrobial delivery strategies in foods.

Publications

  • Schmidt, S.E., G. Holub, J.M. Sturino, and T.M. Taylor. 2010. Suppression of Listeria monocytogenes Scott A in fluid milk by free and liposome-entrapped nisin. Probiotics and Antimicrobial Proteins. 1(2):152-158.
  • Brandt, A.L., A. Castillo, K.B. Harris, J.T. Keeton, M.D. Hardin, and T.M. Taylor. 2010. Inhibition of Listeria monocytogenes by food antimicrobials applied singly and in combination. Journal of Food Science. 75(9):M557-M563.


Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Output activities for year 2009 include the generation and analysis of experimental data describing the optimization of encapsulation efficiency of food antimicrobial in liposomes as described in Objective A of project proposal. Data has been collected and analyzed for the determination of optimal liposome encapsulation processes. Further, data has been generated validating the antimicrobial efficacy of nisin-bearing liposomes to inhibit the foodborne pathogen Listeria monocytogenes in a complex food matrix (fluid milk) at multiple incubation temperatures. One graduate research student has been trained in the development of liposomes by multiple methods and the microbiological analyses of antimicrobial efficacy of liposomes against a microbial pathogen in a food matrix. Grant proposals describing the utilization of liposomes and requesting new processing instrumentation have been generated and submitted to appropriate funding agencies. Products include new collaboration with chemical engineer to enhance liposome design and provide expert analyses of physico-chemical characteristics of liposome capsules. PARTICIPANTS: Schmidt, Shannon E.: Graduate research assistant responsible for the primary collection and analysis of project data. Prepared all samples, gathered and recorded data. Assisted in analysis of data and preparation of all publications. Brandt, Alex, L.: Graduate research assistant responsible for assisting in the collection of experimental data and sample preparation. Mikolajzcak, Christine: Undergraduate student worker responsible for assisting with sample preparation, microbiological analyses, and decontamination of spent sample material. Sturino, Joseph M.: Interdepartmental collaborating scientist responsible for the analysis of data pertaining to antimicrobial efficacy. Provided assistance with data collection and analyses, manuscript and abstract preparation. Holub, Glenn: Intradepartmental collaborating scientist responsible for oversight of graduate student degree program completion. Additional assistance provided in the development of publications. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
A change in knowledge was observed in year 2009 when research data analysis resulted in researchers determining that liposome formulation containing poly-dispersed phospholipids made via freeze/thawing did not sufficiently entrap antimicrobial effectively. Additionally, data was analyzed indicating that current liposome formation methods are not currently sufficient to guarantee consistent antimicrobial delivery to foods and additional research is needed to enhance encapsulation and food safety applications. These data have been published, allowing the establishment of a baseline of information from which future studies will be conducted. These data have also initiated a change in actions, whereby we have discontinued current research into freeze/thaw encapsulation using current methods, as the resulting liposomes are non-homogenous and not effective delivery vehicles for food antimicrobial. Data have allowed a change in conditions by the development of a novel system for delivering natural food antimicrobial to fluid milk, allowing enhanced control post-processing of the microbial pathogen Listeria monocytogenes.

Publications

  • Schmidt, S.E., Holub, G., Sturino, J.M., and Taylor, T.M. 2009. Suppression of Listeria monocytogenes Scott A in fluid milk by free and liposome-entrapped nisin. 24th Meeting of the Association of Microbiology, Hygiene, and Safety of Foods, Mexico Association of Food Protection, Puerto Vallarta, Jalisco, Mexico (11/5-7).
  • Schmidt, S.E., Holub, G., Sturino, J.M., and Taylor, T.M. (2009). Suppression of Listeria monocytogenes Scott A in fluid milk by free and liposome-entrapped nisin. Probiotics and Antimicrobial Proteins, 1(2): 152-158.
  • Schmidt, S.E. (2009). Antimicrobial Efficacy of Liposome Encapsulated Nisin and Nisin's Inhibition Against Listeria monocytogenes in Fluid Milk at Different Storage Temperatures. M.S. Thesis: Texas A&M University, College Station, TX.


Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: One graduate student has been trained and supervised on the production and analysis of liposomes for the completion of outlined research. One technical presentation has been generated and given to members of the Department of Nutrition and Food Science at Texas A&M University detailing the formulation, testing, and application of experimental liposomes. PARTICIPANTS: One Project Director and one Graduate Research Assistant have functioned to conduct experiments and derive experimental data related to this project. Two collaborating scientists, both at Texas A&M University, have contributed to the collection of experimental data and subsequent analysis and interpretation. TARGET AUDIENCES: Experimental results will be applied primarily towards the enhancement of dairy safety, resulting in benefit to dairy products producers and consumers. Efforts are ongoing for the training of researchers competent in the encapsulation and delivery of food antimicrobials for dairy products. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Ongoing research has produced experimental data indicating the lack of efficient entrapment of food antimicrobials in liposomes using a simple, conventional method based on the repeated exposure to freezing and warm temperatures. This dataset has led to the conclusion that encapsulation methods for high-efficiency entrapment of food antimicrobials must be further analyzed and optimized for application in food systems. This finding directly impacts research objective 1 in that the optimization of encapsulated antimicrobial must be further pursued for delivery of food antimicrobials into food systems. It further impacts the development of research by exposing the need for further collaboration and studies into more effective means of processing of liposomes.

Publications

  • No publications reported this period