products such as spaghetti with tomato sauce, tomatoes, noodles/vegetables ... sive numbers of organisms in this area may result in leaker ... taken aseptically from five different bags or barrels per ... Homogenize 200 g of mushrooms in a sterile blender jar. .... Lee, Y.-E., M. K. Jain, C. Lee, S. E. Lowe, and J. G. Zeikus. 1993.
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CHAPTER 27
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Thermophilic Anaerobic Sporeformers Elena Enache and Richard Podolak Updated January 2013
27.1
and high pressure (500 to 700 MPa). A severe PATP treatment of 121uC and 700 MPA for 1 min was needed for complete inactivation of spores of this organism.1 Because of their high heat resistance, the spores of T. thermosaccharolyticum are expected to survive a typical thermal process in canned food; however, they rarely spoil foods processed above 121uC, if properly cooled after processing and stored below 35uC. Only when the finished product is improperly cooled or is held for extended periods at elevated temperatures do the thermophilic anaerobes express themselves. Vacuum loss and decrease in pH value are the main characteristics observed in processed food spoiled by anaerobic thermophiles.5,7 The optimum growth temperature of these organisms is 55uC to 68uC. They seldom grow at temperatures below 32uC but can produce spoilage in 14 days at 37uC if the spores are first germinated at a higher temperature. They have an optimum for growth of pH 6.2 to 7.2 but grow readily in products having a pH of 4.7 or higher. They have been responsible on occasion for spoilage in tomato products at pH values of 4.1 to 4.5.7,21 Ingredients such as sugar, dehydrated milk, starch, flour, cereals, soy protein, and alimentary pastes have been found to be the predominant sources of thermophilic anaerobes. These organisms occur widely in the soil and therefore are found on raw materials, such as mushrooms and onion products that have a history of contact with the soil.8 Excessive populations of thermophilic anaerobes can develop in ingredients such as chicken stock, beef extract, or yeast hydrolysate if an incubation period in the thermophilic temperature range is provided during concentration or hydrolysis steps. The thermophilic anaerobes do not multiply on equipment and handling systems unless an anaerobic environment containing nutrients and moisture at an elevated temperature is provided.2,10 The organism has also been observed to grow well in the exit and cooling leg (55uC area) of hydrostatic cookers, if the water is contaminated with food. Accumulation of excessive numbers of organisms in this area may result in leaker type spoilage of canned foods if the containers are held at elevated temperatures. Thermophilic spore buildup in processing equipment can be avoided through thorough
INTRODUCTION
The thermophilic anaerobes that do not produce hydrogen sulfide have been responsible for the spoilage of canned products such as spaghetti with tomato sauce, tomatoes, noodles/vegetables, sweet potatoes, pumpkin, green beans, mushrooms, asparagus, vegetable soup, and dog food.7,21 Phylogeny-based detection and identification methods using 16S rRNA and DNA sequencing comparison resulted in reclassification of certain species that had been assigned to newly emerged genera. The genus Clostridium had undergone a major revision, and five new genera and 11 new species combinations were proposed.6 As a result of reclassification, Clostridium thermosaccharolyticum, known as the causative factor of spoilage in the swelling cans of certain underprocessed foods, has been renamed Thermoanaerobacterium thermosaccharolyticum and included in genus Thermoanaerobacterium, Thermoanaerobacterales Family III.6,7,14,15 The type species of this group is Thermoanaerobacterium thermosaccharolyticum.22 These organisms are obligately anaerobic, strongly saccharolytic, and produce acetic acid, butyric acid, and lactic acid along with large quantities of gas, mostly carbon dioxide and hydrogen from glucose, lactose, sucrose, salicin, and starch.18 Proteins are not hydrolyzed, and nitrites are not produced from nitrates.16 Vegetative cells are long, slender, straight, or slightly curved, often weakly staining, Gram-negative rods. Spores are terminal and swollen. Neither toxins nor infections are produced, and, therefore, the organisms are of spoilage but not of public health significance. One of the noticeable characteristics of these organisms is the heat resistance exhibited by their spores. It is not unusual for the spores to have D values at 121uC of 3 to 4 min or higher. Their z value (slope of the thermal death time curve) is about 6uC to 7uC. Thus, the organisms can have extreme resistance in the 105uC to 113uC range; moreover, the greatest D121uC reported for T. thermosaccharolyticum were of 68 and 195 min.2,5,23 The highly heatresistant spores of T. thermosaccharolyticum also demonstrated enhanced resistance to pressure-assisted thermal processing (PATP), a thermal process that combines heat | 1 |
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Compendium of Methods for the Microbiological Examination of Foods
sanitation of tanks, blanchers, and washers on a daily basis.2,3,10,20
27.2
GENERAL CONSIDERATIONS
Methods outlined in this chapter are dictated by the fact that T. thermosaccharolyticum is a thermophilic, obligately anaerobic sporeformer. Although the primary objective is to limit the number of spores in ingredients used in canned foods, limiting the hot hold time of sensitive products/ ingredients is important as well. The recommended substrate for recovery and growth of non-hydrogen sulfide-producing thermophilic anaerobes is PE-2 medium.11,17 The medium should be supplemented to contain 0.3% yeast extract for detection of severely heatstressed spores. The AOAC International detection procedure for thermophilic anaerobes not producing hydrogen sulfide specifies liver broth as the medium of choice,4 but experience indicates that non-commercially prepared liver broth is difficult to make and is a potential source of metabolic inhibitors, including antibiotics, without offering any increased sensitivity of detection.
27.3
EQUIPMENT, MATERIALS, AND SOLUTIONS
27.31
Equipment
Media
Liver broth PE-2 Vaspar 2% Agar
27.4
Spoiled canned food suspected of thermophilic growth should not be refrigerated or frozen because vegetative cells of thermophilic anaerobes usually die under refrigeration or can be severely affected by freezing. Spores are not generally produced in the canned food but could be produced under some circumstances, such as improper holding before processing.
27.5
PRECAUTIONS
Every precaution should be taken to ensure that the ingredients of the detection medium are free from growth inhibitors. For example, peas should be obtained free of pesticides. As an added precaution, each new lot of ingredients should be incorporated into the medium and tested for growth inhibitors with a known suspension of a thermophilic anaerobe. These precautions will help to eliminate or minimize the occurrence of false negatives. The detection procedures described in Section 27.5 are not truly quantitative. The objective in surveying ingredients is to detect spores in a known quantity of the ingredient rather than to achieve absolute quantitation. It is important that in the preparation of PE-2 the dried peas be soaked in the peptone solution 1 hr before autoclaving, to ensure the proper sterilizing effect. Repeated steaming of unused tubes of medium does not reduce its effectiveness as a substrate for the thermophilic anaerobes.
PROCEDURE
The following procedures apply for the detection of spores only rather than of spores and vegetative cells. If the heating step is omitted, vegetative cells can be propagated by these procedures.
27.51
Culture Medium
Unless freshly prepared medium is used, previously sterilized tubes should be subjected to flowing steam for 20 min to exhaust oxygen and cooled to 55uC before use. After inoculation, tubes are stratified with 3 mL of sterile 2% agar or Vaspar that is allowed to solidify at room temperature before tubes are preheated to 55uC and incubated at that temperature.4 As a safety precaution, venting caps are recommended on tubes because of the abundant gas production by the organism of interest.
27.52
Blender Incubator that will maintain a uniform temperature of 55uC ¡ 2uC Microscope with 1,0006 oil immersion objective Pipettes with 10, 1.0, and 0.1 mL capacity, wide-bore pipettes 18 6 150 mm tubes with venting caps
27.32
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Sampling
27.521 Ingredients Samples of dry ingredients should consist of 225 g (0.5 lb) taken aseptically from five different bags or barrels per shipment or lot-for-lot sizes of 50 or fewer containers, from 10% of the containers for lot sizes 50 to 100, and from a number of containers equal to the square root of the lot size for shipments with greater than 100 containers. Liquid sugar should be sampled by drawing five 200 to 250 ml (6 to 8 oz) portions per tank during transfer or at the refinery during the tank filling operation. Samples should be placed in sterile, sealed containers. If preliminary analyses indicate considerable variability in a lot, the number of samples should be increased.4,16,13 27.522 Equipment and Systems The thermophilic anaerobes will not generally develop on equipment unless elevated temperatures are provided in a relatively microaerophilic environment containing nutrients. Accumulated food materials in such locations should be sampled with a sterile spatula or similar device and placed in sterile, sealed containers, and the analysis should be conducted as soon as possible. Examination of food materials before and after exposure to processing equipment will help to reveal the contamination level of the equipment. 27.523 Product in Process A 200 g sample of product in process should be obtained periodically to monitor the system. Sample timing should be arranged to coincide with the introduction of a new batch of ingredients or a shutdown that may have permitted an incubation period. The samples should be cooled by placing them at room temperature and conducting the analysis as soon as possible once the temperature is reached. Refrigeration is not recommended. The need for
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| Thermophilic Anaerobic Sporeformers
sampling will be dictated by considerations such as product spoilage and the temperature stresses the product is expected to be subjected to during storage and transit.
27.524 Finished Product for Routine Quality Analysis Representative containers of finished product should be obtained to reflect the condition of the entire population of containers in a production period. The need for sampling will be dictated by considerations such as the previous record of the product with respect to thermophilic spoilage and the temperature stresses to which the product is expected to be subjected during transit and storage. The number of containers sampled should be of the order of one per thousand containers produced. If immediate post-process cooling to 40uC to 43uC is not achievable, monitoring of surviving thermophiles becomes extremely important. Incubate the finished product at 55uC for 5 to 7 days.2,3,10,20 27.53
Enumerating
27.531 Dry Sugar and Powdered Milk4 Place 20 g of sample in a sterile flask and add sterile distilled water to a final volume of 100 mL. Aseptically stir or swirl to dissolve the sample and bring the contents of the flask to a boil rapidly. Boil for 5 min, cool by placing the flask in cold water, and bring the volume back to 100 mL with sterile distilled water. Divide 20 mL of boiled solution equally among six freshly exhausted tubes of PE-2 medium. Stratify each tube with 3 mL of sterile 2% agar or Vaspar, allow the agar to solidify, preheat the tubes to 55uC, and incubate at 55uC for 72 hr. 27.532 Liquid Sugar Place a sample containing the equivalent of 20 g of dry sugar, determined on the basis of degree Brix (29.411 g of 68u Brix liquid sugar is equivalent to 20 g of dry sugar) in a sterile flask and proceed as for dry sugar. 27.533 Fresh Mushrooms Homogenize 200 g of mushrooms in a sterile blender jar. Blend the diced sample until the pieces are finely chopped. Frequent shaking of the jar is essential to ensure proper blending. Place 20 g of blended sample in a sterile flask and proceed as for dry sugar. 27.534 Starches and Flours19 Place 20 g of sample in a sterile flask containing a few sterile glass beads and add sterile distilled water to a final volume of 100 mL. Shake well to obtain a uniform suspension. Divide 20 mL of the suspension equally among six freshly exhausted tubes of PE-2 medium. Spin three tubes at a time in the hands immediately after adding the sample. Place the tubes in a boiling water bath and continue to spin the tubes for the first 5 min of heating. Continue heating for an additional 10 min, then remove the tubes and place them in cold water. Stratify the tubes with 3 mL of sterile 2% agar or Vaspar, allow the agar or Vaspar to solidify, preheat the tubes to 55uC, and incubate at 55uC for 72 hr.4
27.535 Cereals and Alimentary Pastes19 Place 50 g of well-mixed sample into a sterile blender jar and add 200 mL of sterile distilled water. Blend for 3 min to obtain a uniform suspension. Proceed as for starches and flours. For calculations assume that 10 mL of the blended materials contain 2 g of the original sample. 27.536 Product in Process Place 100 g of product in a sterile blender jar and blend for 3 min. Distribute 20 ml or 20 g of the blended sample equally among six freshly exhausted tubes of PE-2 medium and proceed as for starches and flours. 27.537 Finished Product Representative samples of finished canned product should be incubated at 55uC for 5 to 7 days and observed daily for evidence of loss of vacuum or container distortion. Samples that show signs of spoilage such as gas formation should be removed from incubation and opened aseptically. Three grams of the contents should be placed in each of two tubes of freshly exhausted PE-2 medium by means of a widebore pipette. Smears of the product should be made for morphological confirmation. The conditions necessary for preventing laboratory contamination when subculturing cans of finished product are detailed in the literature.9 27.538 Spore Suspensions When spore suspensions are prepared for thermal inactivation studies, a greater degree of quantitation is desirable than is practiced for ingredients or finished product. In this case, 10 mL of the desired dilution of the spore suspension are placed in an 18 6 150 mm screw-cap tube and immersed in boiling water for 8 min, followed by rapid cooling in ice water. A conventional five-tube most probable number (MPN) dilution series of the boiled suspension is prepared in freshly exhausted PE-2 medium. The inoculated tubes are treated as for dry sugar, and the population of the original spore suspension is computed from MPN tables. 27.6
INTERPRETATION
Tubes of PE-2 medium positive for growth of nonhydrogen sulfide-producing thermophilic anaerobes show gas production with the peas rising to the top of the liquid medium. Thermophilic flat sour bacteria may change the color from purple to yellow without gas (Chapter Thermophilic Flat Sour Spore Formers).
27.61
N
N
Ingredients
For canners’ use. Spores of non-hydrogen sulfideproducing thermophilic anaerobes should not be found in more than 60% of the samples tested or in more than 66% of the tubes for any single sample.16 Use of ingredients meeting this standard will minimize the possibility of spoilage in the finished product. Canned foods with a pH below 4.0 are not susceptible to spoilage by thermophilic anaerobes. For other use. The presence of excessive numbers of spores of thermophilic anaerobes that do not produce hydrogen sulfide in ingredients for use other than in canned products is of little significance unless a | 3
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Compendium of Methods for the Microbiological Examination of Foods
thermophilic incubation period is provided during processing. In such a case, the number of vegetative cells present after a processing step is important and should be determined as outlined above (Section 27.538), but omitting the boiling step.3
27.62
Equipment and Systems
The presence of detectable levels of spores of non-hydrogen sulfide-producing thermophilic anaerobes on equipment and systems suggests that equipment is in need of thorough cleaning and sanitation, or growth is occurring, or both. If proper sanitation is practiced, and if the systems are properly designed, spore buildup should not occur.
27.63
Product in Process
Excessive numbers of vegetative cells or spores in the product in process, prepared from ingredients meeting the requirements of ingredients for canners’ use and for other use, suggest that multiplication is occurring during one or more of the manufacturing steps. The manufacturing sequence should be sampled and the point of increase in the microbial population determined. Remedial steps should be taken immediately. The presence of vegetative cells suggests that sporulation can and will occur.3
27.64
Finished Products
The presence of low numbers of spores of the nonhydrogen sulfide-producing thermophilic anaerobes in processed canned foods is not unusual. The organisms possess extreme resistance to the thermal process provided in many commercial processes.23 An attempt to eliminate the spores by increased thermal treatments may endanger the quality and nutritional and functional integrity of many products. If the cooling of processed cans to a center-can temperature of 43uC or less is effected immediately and the cans are stored at temperatures below 35uC, remote from heating ducts and other sources of heat, the presence of spores of thermophilic anaerobes is of no consequence.2,3,10 However, with such a presence, the potential for spoilage exists if temperature abuse of the cans occurs; therefore, this situation should be avoided through the use of meticulously selected ingredients that are carefully handled throughout the production sequence. If thermophilic anaerobes are present, the importance of efficient cooling followed by storage below 35uC cannot be overemphasized. The presence of detectable thermophilic anaerobes in canned foods destined for hot-vend service or tropical distribution constitutes an unacceptable spoilage hazard. The situation must be overcome by the use of thermophilefree ingredients or by increasing the thermal process.
27.7
REFERENCES
1. Ahn, J., V. M. Balasubramaniam, and A. E. Yousef. 2007. Inactivation kinetics of selected aerobic and anaerobic bacterial spores by pressure-assisted thermal processing. Intern. J. Food Microbiol. 113:321-329. 2. Ashton, D. H. 1981. Thermophilic organisms involved in food spoilage: thermophilic anaerobes not producing hydrogen sulfide. J. Food Prot. 44:146.
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3. Ashton, D., and T. Bernard. 2001. Thermophilic anaerobic sporformers. In: F. P. Downes and K. Ito (Editors). Compendium of methods for the microbiological examination of foods. American Public Health Association, Washington, D.C., 249-252. 4. AOAC International. 2005. Official method 972.45. Thermophilic bacterial spores in sugars. Accessed 21 December, 2010. http://www.eoma.aoac.org/gateway/ readFile.asp?id5972_45.pdf 5. Brown, K. L. 2000. Control of bacterial spores. Brit. Med. Bull. 56:158–171. Accessed December 18, 2012. http://bmb. oxfordjournals.org/content/56/1/158.full.pdf. 6. Collins, M. D., P. A. Willems, J. J. Cordoba, J. FernandezGarayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow. 1994. The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Intern. J. System. Bacteriol. 44:812-826. 7. Dotzauer, C., M. A. Ehrmann, and R. F. Vogel. 2002. Occurrence and detection of Thermobacterium in canned food. Food Technol. Biotechnol. 40:21-26. 8. Erkmen, O., and A. O. Barazi. 2011. Kinetics of microbial inactivation. In: D.-W. Sun (Editor). Handbook of food safety engineering. Wiley Publishers, Oxford, U.K. Accessed December 3, 2012. http://books.google.com/ books?id5XjTWjykzLUYC&pg5PT193&lpg5PT193&dq5c+. +thermosaccharolyticum+in+soy+protein&source5bl&ots5 oSwEXLA7CF&sig5NAKsBs--khCAI2Zh3El3ocYDA0M& hl5en&sa5X&ei5BNvQUKSjL4bh0wG-44CADA&sqi52& ved50CDUQ6AEwAQ. 9. Evancho, G. M., D. H. Ashton, and E. J. Briskey. 1973. Conditions necessary for sterility testing of heat-processed canned foods. J. Food Sci. 38:185. 10. Evancho, G. M., S. Tortorelli, and V. N. Scott. 2009. Microbiological spoilage of canned foods. In: W. H. Sperber and M. P. Doyle (Editors). Compendium of the microbiological spoilage of foods and beverages. Springer, New York, New York, 185-222. 11. Folinazzo, J. F., and V. S. Troy. 1954. A simple bacteriological medium for the growth and isolation of spoilage organisms from canned foods. Food Technol. 8:280. 12. Holmes, A. W., and P. S. Nicholes. 1952. A simple method for the determination of Clostridium thermosaccharolyticum in sugar. Proc. Amer. Soc. Sugar Beet Tech. Proceedings of the seventh General Meeting. Salt Lake City, Utah. February 5–8, 1952. Accessed January 2, 2013 http://www.sugarresearch. library.qut.edu.au/5/4/Proceedings_American_Society_of_ Sugar_Beet_Technologists_1952.pdf. 13. ICMSF (International Commission on Microbiological Specifications for Foods). 2002. Microorganisms in foods 7: microbiological testing in food safety management. Springer, New York, New York. 14. Jay, J. M. 2003. A review of recent taxonomic changes in seven genera of bacteria commonly found in foods. J. Food Prot. 66:1304-1309. 15. Lee, Y.-E., M. K. Jain, C. Lee, S. E. Lowe, and J. G. Zeikus. 1993. Taxonomic distinction of saccharolytic thermophilic anaerobes: description of Thermoanaerobacterium xylanolyticum gen. nov. , sp. nov., and Thermoanaerobacterium saccharolyticum gen. nov., sp. nov.; reclassification of Thermoanaerobium brockii, Clostridium thermosulfurogenes, and Clostridium thermohydrosulfiricum ElO0-69 as Thermoanaerobacter brockii comb. nov., Thermoanaerobacterium thermosulfurigenes comb. nov., and Thermoanaerobacter thermohydrosulfuricus comb. nov., respectively; and transfer of Clostridium thermohydrosulfuricum 39E to Thermoanaerobacter ethanolicus. Intern. J. System. Bacteriol. 43:41-51. 16. NCA Research Laboratories. 1968. Laboratory manual for food canners and processors. vol. 1. National Canners
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Association (now National Food Processors Association), Avi Publishing Co., Westport, Conn., 104. Ogunrinola, O. A., C. G. Edwards, and P. M. Davidson. 1997. Evaluation of four pea (Pisum sativum) cultivars in PE-2 medium for the MPN enumeration of anaerobic sporeforming organisms. J. Food Prot. 60:1574-1576. Pirone, G., L. LaPietra, M. Impembo, M. Longo, and G. Squitieri. 2005. Characterization of microbial spoilage in tomato products: gas-producing anaerobic thermophilic bacteria. Industria Conserve. 80:33-51. Powers, E. M. 1973. Microbiological requirements and methodology for food in military and federal specifications. Tech. Rep. 73-33-FL. U.S. Army Natick Lab., Natick, Mass. Ray, B. 2004. Important facts in microbial food spoilage (pp. 257-267) and Spoilage of specific food groups (pp. 269-288).
In: Fundamental food microbiology. 3rd ed. CRC Press, Boca Raton, Fla. 21. Rhoads, A. T., and C. B. Denny. 1964. Spoilage potentialities of thermophilic anaerobes. Research Rep. No. 3–64. National Canners Association (now National Food Processors Association), Washington, D.C. 22. Stumbo, C. R. 1973. Thermobacteriology in food processing. 2nd ed. Academic Press, New York. 23. Xezones, H., J. L. Segmiller, and I. J. Hutchings. 1965. Processing requirements for a heat tolerant anaerobe. Food Technol. 19:1001. Authors of the 4th edition version of this chapter: David Ashton and Dane T. Bernard.
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