—_— CHAPTER 2:7, |_-________ | Thermophilic Anaerobic Sporeformers Elena Enache and Richard Podolak 274 INTRODUCTION ‘he 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? Phylogeny-based detection and identification methods using 165 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 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 1.23 ‘The type species of this group is Thermoanaerobacterium ‘hermosaccharolyticum® These organisms are obligately anaerobic, strongly saccharolytic, and produce acetic acid, Dutyric acid, and lactic acid along with large quantities of 835, mostly carbon dioxide and hydrogen from glucose, lactose, sucrose, salicin, and starch.” Proteins are not hydrolyzed, and nitrites are not produced from nitrates."* Vegetative cells are long, slender, straight, or slightly curved, weakly staining, Gram-negative rods, Spores are ferminal and swollen. Neither toxins nor infections are Produced, and, therefore, the organisms are of spoilage but ot of public health significance. '¢ of the noticeable characteristics of these organisms 4s the heat resistance exhibited by their spores, It is not ‘Anusual for the spores to have D values at 121°C of 3 to 4 fut or higher. Their z value (slope of the thermal death fats CWrVe) is about 6°C to 7°C. Thus, the organisms can We extreme resistance in the 105°C to 113°C range; ‘over, the greatest Dy2y.c reported for T, thermosacchar- ution were of 68 and 195 min?” The highly heat- SReeRt spores of T. thermosaccharolyticum also demon- tated enhanced resistance to pressure-assisted thermal Processing (PATP), a thermal process that combines heat and high pressure (500-700 MPa). A severe PATP treatment of 121°C and 700 MPA for 1 min was needed for complete inactivation of spores of this organism. 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 121°C, if properly cooled after Processing and stored below 35°C. 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. ‘The optimum growth temperature of these organisms is 55°C to 68°C. They seldom grow at temperatures below 32°C but can produce spoilage in 14 days at 37°C 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 4.57" 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 Excessive populations of thermophilic anaerobes can develop in ingredients such as chicken stock, beef extract, for yeast hydrolysate if an incubation period in the thermophilic temperature range is provided during con- centration or hydrolysis steps, The thermophilic anaerobes, do not multiply on equipment ancl handling systems unless an anaerobic environment containing nutrients and mois- ture at an elevated temperature is provided" The organism has also been observed to grow well in the exit and cooling leg (85°C area) of hydrostatic cookers if the water is contaminated with food. Accumulation of exces- sive 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 335 © scanned with OKEN ScannerF Compendium of Methods forthe Microbiological Examination of Foods sanitation of tanks, blanchers, and washers on a daily basis 27.2 GENERAL CONSIDERATIONS Methods outlined in this chapter are dictated by the fact that T. thermosaecharalyticum 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 wel. ‘The recommenced substrate for recovery and growth of non-hydrogen sulfide-producing. thermophilic anaerobes is PE-2 medium.” The medium should be supplemented to contain 0.3% yeast extract for detection of severely heat- stressed spores. The AOAC International detection procedure for thermophilic anaerobes not producing hydrogen sulfide specifies liver broth as the medium of choice,’ 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 2731 Equipment * Blender * Incubator that will maintain a uniform temperature of 55°C 4 2°C + Microscope with 1,000 oil immersion objective « Pipettes with 10, 1.0, and 0.1 mL capacity, wide-bore Pipettes ‘+ 18 x 150-mm tubes with venting caps 27.32 Media iver broth © PE2 * Vaspar © 2% agar 27.4 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 2755 are not truly quantitative, The objective in surveying ingre- dients 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. 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 mediim docs not reduce its effectiveness as a substrate for the thes ‘mophilic anaerobes. ~ sled canned food suspected of therm, shoe not be refrigerated or frozen bee iis thermophilic anaerobes usually die yay" cfs can be severely affected by feeczing. got vionaly produced in the canned food gear Bene under some cicumstanes, sucht St Poialing before processing. : 27.5 PROCEDURE The following procedures apply forthe detection only rather than of spores and vegetative Pm heating step is omitted, vegetative cels can bey! by these procedures. Tipe opi le Ase let mt 2751 Culture Medium inless freshly prepared medium is used, pry Seltied tubes should be subjected to flowing se 20 min to exhaust oxygen and cooled to 55°C heft Aer inoculation, tubes are stratified with 3 moje 2% agar or Vaspar that is allowed to solidity ge temperature before tubes are preheated to Sic st incubated at that temperature AS a safety prey venting caps are recommended on tubes because st abundant gas production by the organism of intent 27.52 Sampling 27.521 Ingredients Samples of dry ingredients should consist of 25 (0S taken aseptically from five different bags or bares shipment or lot-for-lt sizes of 50 or fewer conines, re 10% of the containers for lot sizes 50 to 100 and fans number of containers equal to the square root of tee for shipments with greater than 100 contains igi sugar should be sampled by drawing five 200-o 29a (6-8 02) portions per tank during transfer ora the en during the tank filling operation. Samples shouldbe insterile, sealed containers. If preliminary analyse iick considerable variability in a lot, the numberof amps should be increased.!#*” 27.522 Equipment and Systems * The thermophilic anaerobes will not generally dvekp equipment unless elevated temperatures aze provided tt relatively microaerophilic environment containing ents. Accumulated food materials in such locos 84 be sampled with a sterile spatula or simile die Placed in sterile, sealed containers, and the analysis be conducted as soon as possible. Examination oF materials before and after exposure to pocessié ment will help to reveal the contamination level equipment. 27.523 Product in Process i A 200-g sample of produtct in process should be a periodically to monitor the system. Sample tii" "ay be arranged to coincide with the introduction & *y batch of ingredients or a shutdown that Ml i¥ Permitted an incubation period. The samples Sut Cooled by placing them at room temperature and es ing the analysis as soon as possible once the P24 i reached. Refrigeration is not recommended. 7 338 d © scanned with OKEN Scannerca aera) erie marc Sommer aaa Ta jing wil be dictated by considerations such as ann ge andthe temperature stresses the product on Sho be subjected 0 curing storage and transit sgt. Finished Product for Routine Quality Ot Fi alysis censtive containers of finished product should pesera to reflect the condition ofthe entire population eae na production perio. The need for samp: of crepe dictated by considerations such as the previ- tog wig of the product with respect to thermophil co 0d the temperature stresses to which the product oUt to be subjected during transit and storage eFetper of containers sampled should be ofthe order Te rier thousand containers produced. If immediate of gress cooling t0 40°C to 43°C is not achievable, post procof surviving thermophiles becomes extremely pontoring ian Incubate the finished product at $5°C for 5 to Phys 753 Enumerating 21531 Dry Sugar and Powdered Milk? jc 20 g of sample in a sterile flask and add sterile {iled water to a final volume of 100 mL. Aseptically stir trawitto dissolve the sample and bring the contents of the {isk toa boil rapidly. Boil for 5 min, cool by placing the ack in cold water, and bring the volume back to 100 mL. vrih sterile distilled water. Divide 20 mL. of boiled solution quily among six freshly exhausted tubes of PE2 nedium. Stratify each tube with 3 mL of sterile 2% agar «x Vaspar allow the agar to solidify, preheat the tubes to 55°C, and incubate at 55°C for 72 hr. 27532 Liquid Sugar.» Price a sample containing the equivalent of 20 g of dry sugar, determined on the basis of degree Brix (29.411 g of 68" Bx liquid sugar is equivalent to 20 g of dry sugar) in a sterile ask and proceed as for dry sugar. 21.533 Fresh Mushrooms ize 200 g of mushrooms in a sterile blender jar. Bend the diced sample until the pieces are finely chopped. Frequent shaking of the jar is essential to ensure proper Pending, Place 20 g of blended sample in a sterile flask and Proceed as for dry sugar. 27534 Starches and Flours"* act 20 g of sample in a sterile flask containing a few sass beads and add sterile distilled water to a final 9f 100 mL. Shake well to obtain a uniform #SPension. Divide 20 ml. of the suspension equally among freshly exhausted tubes of PE-2 medium. Spin three at atime in the hands immediately after adding the le: Place the tubes in a boiling water bath and tue to spin the tubes for the first § min of heating. ue heating for an additional 10 min, then remove the mL grt Place them in cold water, Stratify the tubes with sterile 2% agar or Vaspar, allow the agar or Vaspar Sully, preheat the tubes to 55°C, and incubate at 55°C 27.635 Cereals and Alimentary Pastes” Place 50 g of well-mixed sample into a sterife 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 malerials contain 2 g of the original sample. 27.536 Product in Process Place 100 g of product in a sterile blerider jar and blend for 3 niin, 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 55°C 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 wide- bore 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” 27.538 Spore Suspensions ‘When spore suspensions are prepared for thermal inactiva~ tion 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 suspen- sion are placed in an 18 x 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 non- hydrogen 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 (see the chapter “"Thermophilic Flat Sour Spore Formers”) 27.61 Ingredients © For canners’ use: Spores of non-hydrogen sulfide- producing thermophilic anaerobes should not be found jn more than 60% of the samples tested or in more than 66% of the tubes for any single sample."* Use of ingredients meeting this standard will minimize the possibility of spoilage in the finished product. Canned oods 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 oar © scanned with OKEN ScannerCompendium of Methods forthe Mercbsoge! Examination of Foods (ease Ashton, D. H. 1981. Thermophilco thermophilic incubation peri i BAIS Ms in is provided during spoilage: thermophilic: anaerobes ot ph Processing. In stich a case, the number of vegetative ide J. Food Prt Prodi We, Gulls present after a processing step is important ar Ashion, Dy and T. Benard. 2001, Themg My Should be determined a outingd seve Soeiona7a8) wrformes. In. P. Downes and kip ei sp or) oy Dut omitting the boiling step.? af Methods for the Microbiological Exam) Arran Publi Heath Asoc, Waagtin % 27.62 Equipment and Systems 4 AOAC International, 2005. Oat mete 2 TEPresence of detectable levels of spores of norhydrogen thermic bck pores in SES Foe tA Sulfde-producing thermophilic anaerobes on equipment 5 Brown, KL 20 “ele and systems suggests that equipment is in need of Collin, M.D. P. A. Willems, J. J. Cora “ Rherough cleaning and sanitation, or growth is occurring, 6 Garayzaal, P Garcia J. Ca. Hippo a Fen, or both. I proper sanitation is practiced, and if the systems 15 Thephylgeny ete genase EAP ‘ properly designed, spore buildup should not occur, new genera and elven new species conta a ‘ System. Bacteriol. 4812-826 ea 27.63 Product in Process 7, Dotaaver, C. M. A. Ehemann, and py, 1 Excessive numbers of vegetative cells or Spores in the Occurrence and detection of Thermobacterium ine My Product in process, prepared from ingredients meeting the Food Technol. Biotechnol. 40:21-26, Tequirements of ingredients for canmers’ use and for ther 8 Exkmen, O, and A. O. Barazi. 2011, King ot Re Suggest that multiplication is occurring during one ov igctaton rs DW. Sun (Er) Se Rare Of the manufacturing. steps. The manulscturing Sly ener: Wiley Publishers Oxo th the, mace should be sampled and the point of inerease i 9. Evancho, G. M. ee Se the microbial population determined. Remedial steps eda pele 3315) “TPS of heater should be taken immediately. The presence of Bir ceca ueeeT Emer Pir TFortoeli, and v. fa cel at ulation can and will occur.’ rere ae peas “a Tso tt porn cn el Sige re a i Sperber and M. F. Doyle (Edt) Comper 27.64 Finished Products Nibilogal Spoiage of Foods and Bevan ® fig, Presence of low numbers of spores of the non- New York, NY. 185.222, me Hydrogen sulfide-producing thermophilic’ anaerobes in 11, Foliazzo,]. F, and V. . Troy. 1954 A simple baci Processed canned foods is not unusual The orgesiane From cc ne, growth and isolation of spoage oe Inmany coneenglance tothe thermal proces provided fom canned foe Ferd ne 8290 RC nany commercial processes An attempt to ainisae 12 Holmes kv ee Nichols. 19524 simple mete {he spore by increased thecal testment may ers te, etmintion of Closet terse the quality and nutritional and functional integrit ‘of me RBar, Proceedings of wWenth General Meeting of pe gualty ional integrity of many Feeage et of Sugar Beet Technolog Sey Ifthe cooling of processed cans toa center-can tem; erature i gia oe SARC orlessiseffected immediately andthecasmeren, ferro ay Gonmisin oF Micabisge aac crear ha secs tng neal eth Mien na ther sources of heat, the presence of spores of thermophilic NY. 'y Management. Springe, Nor anaerobes is of no consequence.” However, with ‘such a M4. Jay, J.M. review i ins | Secscansieet nScpenetemretiesbue "aA Ari atetnennichete | Tega Our: therefore, thi situation should be seen 66:3041909 fe nah tne tse of meticulously selected ingredient tn re 15, Lee, YE, M. K. Jain we, and J. G. 28 cpt laced toughens atte La Tmonomic dnc’ flat 62a thermophilic anaerobes. are present, the importance of gen ne desctiption of Themoanaerbuterum saa efficient cooling followed by Storage below 35°C cannot be Ben. nov. sp. nov., and Thermoainerobaclerium sacha overemphasized Bri tN teasiaon of Tema ‘The presence of detectable thermophilic ete ont Clostridium thermosulfurogenes, and Clem Giuned foods destined for hotvend service or tropical {tesulricim E0069 as Thema bark distribution constitutes an unacceptable spoilage hazard, and emenaerabeclerim themesurgees hh ‘The situation must be overcome by the nacho thermophile. respecte mnrbacer Starostin eo +e ingredients or by increasin, al viz and transfer of Clostridium t 3! free ingredients or by increasing the thermal proce Base Trmeanaerobcterthanai. ner. 58 acterol. 4341-5) a ACKNOWLEDGMENT TS NCA Research Laboratories, 1968. Latest ye iti s: Davi fax “anners rs, vol. lati Fourth edition author: David Ashion and Dane. Bema, Aisin (oow Nato ed ican wi v1 Publishing yf S 0, Westport, CT. 10 st REFERENCE: 7 Opuncinola 0. A.C. Elwasds sod P.M. Devi 1. Atm, J, V. M Balsubrmariam, and A. & You. aoty ‘valuation ‘of four pea (Pisum satioun) les op Inactivation Kinetics of selected aerobic an e207 enumeration of anse eral spor BY Pesireasie) a inc] lod Met ssa al processing. 6 Squitees gle LaPletra, M. Impembo, M. Lore? ep ‘Squitieri. 2005, Charecletata an rnirobial POE a a a ee © scanned with OKEN Scanner