Microbiological Safety of Citrus Fruit for Juice Processing

NON-TECHNICAL SUMMARY: In the last 5 years, a number of high profile cases of food-borne illness have been linked to the consumption of fruit juices, particularly apple and orange juices. In most of these cases, the juice was consumed in a fresh (non-pasteurized) form. Fresh juice is preferred by some consumers who view it as a more natural product with superior flavor characteristics. Consumers of fresh juice also use it as a convenient way to help meet government health recommendations for Americans to consume more fresh fruits and vegetables. However, it is clear that fresh juice presents a potential food safety risk, and the U.S. Food and Drug Administration (FDA), who is charged with ensuring the safety of fresh juices, has little actual data available to assess the extent of these risks. Successful completion of this project, which will extensively evaluate the microbial contamination of oranges entering a juice extraction facility over the course of 2 fruit seasons, will provide data to both fresh and processed juice producers regarding the potential for further food-borne illness associated with citrus juice. This research will directly impact an industry with almost $10 billion in consumer sales, and provide information from which science-based food safety regulations can be formed.

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APPROACH: Experimental methods aligned against each experimental objective are discussed in detail below. A preliminary schedule for all investigations is included that outlines workplans for the proposed two years. Comprehensively evaluate fruit surface microflora of juice oranges commercially used for production of unpasteurized juice. Evaluation will include both sound and culled fruit. During unloading at a commercial processing facility, fruit will be randomly selected and packed in a cooler for transport to the Citrus Research & Education Center in Lake Alfred. Fruit will be handled using latex gloves during all phases of the experiment. Samples will be pourplated with plate count agar , orange serum agar and acidified potato dextrose agar to yield total aerobic plate count, aciduric count and yeast and mold count, respectively. Fruit diameters will be measured with a micrometer so that total surface area can be estimated using the formula for the surface area of a sphere. Results will be reported on the basis of fruit surface area or per fruit. Juice analyses will identify impact of surface condition and treatment on the ultimate microbiological quality of the fruit. Infiltration studies will help identify the actual risk factors involved in potential pathogen infiltration into an individual fruit.

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PROGRESS: 2000/12 TO 2003/12<BR>
Thirty two experimental runs have been completed. Microorganisms on fruit surfaces are calculated in colony forming units per square centimeter of fruit surface. Preliminary results on a random sampling of runs show numbers of organisms on orange surfaces to be highest in the cull fruit group, 480,000 to 11,000 cfu/cm2 (avg. 120,000 cfu/cm2 ), and lowest in the washed and graded group, 5,000 to 150 cfu/cm2 (avg. 1600 cfu/cm2). Numbers of organisms on fruit surfaces in the unwashed group were between 49,000 and 500 cfu/cm2 (avg. 16000cfu/cm2). Microorganisms in juice samples were measured in colony forming units per milliliter of juice (cfu/mL). A random sampling of juice data shows results similar to that of the fruit surfaces: cull fruit has the highest concentration of microorganisms (86000 cfu/mL) and the washed fruit with the least (9 cfu/ml). Unwashed fruit samples contained approximately 25 cfu/mL microorganisms. A sampling of data on the studies of the pathogens (E. coli and Salmonella) isolated from the fruit surface wash and the juice show that E. coli is found on fruit (3%) and in the juice (2%). Salmonella is infrequently found, but our data show that it can be found in the juice when it is on the exterior of the fruit. At this time, 25 samples each of the three groups of fruit described (unwashed and ungraded, washed and graded and cull fruit) are used. Additional changes were made in the wash method and in the preliminary isolation of pathogens. A small amount of buffer added to each sample fruit in the sterile whirlpak bag was substituted for the sterile sponge procedure described in the research proposal. A portion of the buffer sample from each washed fruit was placed in pre-enrichment broth for more efficient isolation of any pathogens (e.g. E. coli and Salmonella) that might be present in small amounts. Studies on the fruit interiors (juice) have been changed slightly to allow for better processing of samples. Fruit is sanitized for 2 minutes rather than 4 and the fruit is not reamed on a reamer, but each cut half is hand-squeezed (with sterile gloves) over a sterile glass container which holds a sterile paper funnel. The pre-enrichment method for pathogen isolation is also utilized for juice studies for enhanced recovery over previous methods. Data collected to date is directed to our goals of enumerating concentrations of microorganisms on the fruit, incidences of these organisms getting into the interior of the fruit and the probability of pathogens present among these organisms. The information gathered from these studies will be significant in the development of criteria which enable processors to generate a safer product. Finally, infiltration/internalization studies outlined in the grant, in conjunction with related work conducted on other citrus and tomatoes shows that internalization of pathogenic microorganisms, while possible under severe (and unlikely) temperature gradients, is not a major mode of contamination of fresh oranges.
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IMPACT: 2000/12 TO 2003/12<BR>
The overall project goal was to quantify the baseline microbial hazards associated with citrus fruit and juice that will provide a foundation for development of risk assessments related to these products. This work was accomplished by meeting the following specific objectives: 1. Comprehensively evaluate fruit surface microflora of juice oranges commercially used for production of unpasteurized juice. Evaluations will include both sound and culled fruit. 2. Comprehensively evaluate the internal microbiology of citrus fruit and juice. 3. Investigate potential infiltration of microorganisms into the interior of citrus fruit and the effect of such infiltration on juice microflora. The data obtained from the proposed studies will assist government, academic and industry scientists and managers to assess, develop and understand the preventative control measures necessary to ensure microbiological safety of citrus juices. Measurable impacts of this research include decreased incidence of foodborne outbreaks due to fresh orange juice as a result of improved practices and sanitation with the fresh juice processing facilities. Processed juice manufactureres have used improved quality of incoming fruit microbial loads for better assessing risk in the fruit juice processing industy, which has become heavily regulated. Finally, data and information generated from this grant's activities has led to further sponsored research in this field of study, with an additional increase in the body of knowledge in the area of fruit and vegetable food safety.

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PROGRESS: 2001/10/01 TO 2002/10/01<BR>
Twenty eight experimental runs have been completed. Microorganisms on fruit surfaces are calculated in colony forming units per square centimeter of fruit surface. Preliminary results on a random sampling of runs show numbers of organisms on orange surfaces to be highest in the cull fruit group, 480,000 to 11,000 cfu/cm2 (avg. 120,000 cfu/cm2 ), and lowest in the washed and graded group, 5,000 to 150 cfu/cm2 (avg. 1600 cfu/cm2). Numbers of organisms on fruit surfaces in the unwashed group were between 49,000 and 500 cfu/cm2 (avg. 16000cfu/cm2). Microorganisms in juice samples were measured in colony forming units per milliliter of juice (cfu/mL). A random sampling of juice data shows results similar to that of the fruit surfaces: cull fruit has the highest concentration of microorganisms (86000 cfu/mL) and the washed fruit with the least (9 cfu/ml). Unwashed fruit samples contained approximately 25 cfu/mL microorganisms. A sampling of data on the studies of the pathogens (E. coli and Salmonella) isolated from the fruit surface wash and the juice show that E. coli is found on fruit (3%) and in the juice (2%). Salmonella is infrequently found, but our data show that it can be found in the juice when it is on the exterior of the fruit. At this time, 25 samples each of the three groups of fruit described (unwashed and ungraded, washed and graded and cull fruit) are used. Additional changes were made in the wash method and in the preliminary isolation of pathogens. A small amount of buffer added to each sample fruit in the sterile whirlpak bag was substituted for the sterile sponge procedure described in the research proposal. A portion of the buffer sample from each washed fruit was placed in pre-enrichment broth for more efficient isolation of any pathogens (e.g. E. coli and Salmonella) that might be present in small amounts. Studies on the fruit interiors (juice) have been changed slightly to allow for better processing of samples. Fruit is sanitized for 2 minutes rather than 4 and the fruit is not reamed on a reamer, but each cut half is hand-squeezed (with sterile gloves) over a sterile glass container which holds a sterile paper funnel. The pre-enrichment method for pathogen isolation is also utilized for juice studies for enhanced recovery over previous methods. Data collected to date is directed to our goals of enumerating concentrations of microorganisms on the fruit, incidences of these organisms getting into the interior of the fruit and the probability of pathogens present among these organisms. The information gathered from these studies will be significant in the development of criteria which enable processors to generate a safer product. These studies will continue as outlined in the grant proposal to replicate results from another harvest season. Microorganisms collected from these studies are being utilized in additional studies in our laboratories. Specifically, yeasts isolated from both fruit surfaces and interior have been used in a large scale screening for juice yeast method development. These yeasts are also undergoing identification by biochemical methods and genetic characterization in a related study.
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IMPACT: 2001/10/01 TO 2002/10/01<BR>
Data collected to date is directed to our goals of enumerating concentrations of microorganisms on the fruit, incidences of these organisms getting into the interior of the fruit and the probability of pathogens present among these organisms. The information gathered from these studies will be significant in the development of criteria which enable processors to generate a safer product. These studies will continue as outlined in the grant proposal to replicate results from another harvest season. Microorganisms collected from these studies are being utilized in additional studies in our laboratories. Specifically, yeasts isolated from both fruit surfaces and interior have been used in a large scale screening for juice yeast method development. These yeasts have been cataloged and identified by biochemical methods and genetic characterization in a related study.
<BR> <BR> PROGRESS: 2000/10/01 TO 2001/10/01

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Fourteen experimental runs have been completed. Microorganisms on fruit surfaces are calculated in colony forming units per square centimeter of fruit surface. Preliminary results on a random sampling of runs show numbers of organisms on orange surfaces to be highest in the cull fruit group, 400,000 to 11,000 cfu/cm2 (avg. 102,000 cfu/cm2 ), and lowest in the washed and graded group, 3,000 to 150 cfu/cm2 (avg. 1300 cfu/cm2). Numbers of organisms on fruit surfaces in the unwashed group were between 33,000 and 800 cfu/cm2 (avg. 16000cfu/cm2). Microorganisms in juice samples were measured in colony forming units per milliliter of juice (cfu/mL). A random sampling of juice data shows results similar to that of the fruit surfaces: cull fruit has the highest concentration of microorganisms (86000 cfu/mL) and the washed fruit with the least (9 cfu/ml). Unwashed fruit samples contained approximately 11 cfu/mL microorganisms. A sampling of data on the studies of the pathogens (E. coli and Salmonella) isolated from the fruit surface wash and the juice show that E. coli is found on fruit (3%) and in the juice (2%). Salmonella is infrequently found, but our data show that it can be found in the juice when it is on the exterior of the fruit. At this time, 25 samples each of the three groups of fruit described (unwashed and ungraded, washed and graded and cull fruit) are used. Additional changes were made in the wash method and in the preliminary isolation of pathogens. A small amount of buffer added to each sample fruit in the sterile whirlpak bag was substituted for the sterile sponge procedure described in the research proposal. A portion of the buffer sample from each washed fruit was placed in pre-enrichment broth for more efficient isolation of any pathogens (e.g. E. coli and Salmonella) that might be present in small amounts. Studies on the fruit interiors (juice) have been changed slightly to allow for better processing of samples. Fruit is sanitized for 2 minutes rather than 4 and the fruit is not reamed on a reamer, but each cut half is hand-squeezed (with sterile gloves) over a sterile glass container which holds a sterile paper funnel. The pre-enrichment method for pathogen isolation is also utilized for juice studies for enhanced recovery over previous methods. Data collected to date is directed to our goals of enumerating concentrations of microorganisms on the fruit, incidences of these organisms getting into the interior of the fruit and the probability of pathogens present among these organisms. The information gathered from these studies will be significant in the development of criteria which enable processors to generate a safer product. These studies will continue as outlined in the grant proposal to replicate results from another harvest season. Microorganisms collected from these studies are being utilized in additional studies in our laboratories. Specifically, yeasts isolated from both fruit surfaces and interior have been used in a large scale screening for juice yeast method development. These yeasts are also undergoing identification by biochemical methods and genetic characterization in a related study.
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IMPACT: 2000/10/01 TO 2001/10/01<BR>
Federal regulations require all juice processors to develop and implement HACCP as a food safety program; risk assessment and hazard analysis is an integral part of HACCP. This data will enhance the ability of citrus juice processors (responsible for about $6 million annual sales) to more accurately assess the food hazard risk associated with their major raw ingredient (i.e. fruit).