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Research Reports |
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* Center for Veterinary Medicine, US Food and Drug Administration, Rockville, MD 20855; Department of Environmental Science and Technology, Department of Cell Biology and Molecular Genetics, and Department of Animal and Avian Sciences, University of Maryland, College Park 20740
Correspondence: 2 Corresponding author: Phares.Okelo{at}fda.hhs.gov
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SUMMARY |
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 TOP SUMMARY DESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Key Words: Bacillus stearothermophilus • Salmonella Typhimurium • spore • surrogate • feed • extrusion • sterilization • animal feed
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DESCRIPTION OF PROBLEM |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Bacteria and molds such as Salmonella, Bacillus spp., Listeria, pathogenic Escherichia coli, spore-forming clostridia, Aspergillus spp., and Mucor spp. often contaminate grain, feed ingredients, and animal feed. When these microorganisms are transmitted to livestock through the consumption of contaminated feedstuffs [2], they may ultimately affect animal health, the human food supply, or both [3]. To eliminate or minimize the incidence of pathogens such as Salmonella from poultry flocks, it is preferable to raise poultry on Salmonella-free feed [4]. Combinations of extrusion or pelleting conditions used in the manufacture of feed vary widely and are often not carefully controlled; the moisture content of feed required to produce a desired pellet quality varies with the feed formulation [5].
Surrogate organisms may be used to evaluate the effects and microbial responses to processing treatments such as heat pasteurization and extrusion cooking by introducing them into or onto a food product as an inoculum [1]. Salmonella is a representative of the mesophilic group of pathogenic organisms that contaminate feed, whereas Bacillus stearothermophilus, a representative of thermophiles, is a food spoilage organism that grows well at temperatures between 40 and 70° C [6]. Supplementation of culture media with divalent cations affects the heat resistance of bacterial spores. Russell [7] reviewed the work of several investigators and concluded that, with the exception of high concentrations (close to 0.1%), inclusion of manganese sulfate in culture media for the production of B. stearothermophilus spores does not have a major effect on the heat resistance of the spores. Chemically defined media can be used to stimulate production of B. stearothermophilus spores with reproducible heat resistance [8]. Mayou and Jezeski [9] produced spores of B. stearothermophilus by inoculating plates of nutrient agar supplemented with 40 ppm of manganese sulfate with vegetative cells of the organism and incubating at 55° C for 72 h. Because spores of B. stearothermophilus can germinate under optimal temperature conditions to cause problems such as flat-sour spoilage of food [10], they are an ideal agent for evaluating the stability of processed feed. The objective of this study was to evaluate the suitability of B. stearothermophilus as a surrogate for feedborne pathogens in studies to predict optimal extruder settings that would provide the greatest inactivation of pathogens during feed extrusion. The optimal extruder settings were predicted on the basis of the central composite statistical design for 3 parameters, namely, extruder barrel exit temperature (T), mash feed moisture content (Mc) and retention time (Rt) of extrudate in the extruder barrel.
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MATERIALS AND METHODS |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Experimental Design
Five levels of each of the 3 variables (Rt, T, and Mc), coded as – 
, – 1, 0, 1, and , respectively, were chosen on the basis of preliminary test results that were used to determine the possible ranges of each of the variables [11] ( = 1.682, Table 1
). The study was replicated 3 times, with each replicate consisting of 15 different combinations of levels of the 3 independent extrusion variables. Okelo et al. [12] described a detailed statistical procedure for the study.
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Dry Chalk Inoculum of Salmonella Typhimurium.
Nutrient broth (NB) [15] was used to propagate Salmonella Typhimurium. Dry chalk inoculum was prepared by submerging blocks of chalk [16] for 12 h in NB culture containing approximately 1 x 108 colony forming units (cfu)/mL of Salmonella Typhimurium, drying the chalk blocks in an incubator set to 37° C to their original dry weight, and then pulverizing the dried chalk in a sterile laminar flow hood with a mortar and pestle to obtain a powdered inoculum of approximately 1 x 107 cfu/g of chalk.
Enumeration of Salmonella Typhimurium in Dry Chalk Inoculum.
Enumeration of viable bacterial cells in the dry chalk inoculum was performed according to the standard, approved methods of the American Association of Cereal Chemists procedures [17]. Briefly, the initial 10– 1 dilution was prepared by weighing out 10 g of dry chalk inoculum in a sterile sampling container and adding NB to make 100 g of suspension. To make the 10– 2 dilution, 1 mL of the 10– 1 dilution was transferred to a 16 x 100 mm glass test tube containing 9 mL of NB. Subsequent 10-fold dilutions were obtained similarly by transferring 1 mL of a dilution to dilution blanks consisting of 9 mL of NB in test tubes. One hundred microliters of appropriate dilutions were plated on Mac-Conkey agar [15] plates emended with 100 ppm of nalidixic acid. Each batch of MacConkey agar plates prepared was tested for its selectivity by using the test organism and a negative control (a strain of Salmonella Typhimurium susceptible to nalidixic acid).
Dry Feed Inoculum of Salmonella Typhimurium.
One kilogram of dry feed was treated with 15 mL of NB culture of Salmonella Typhimurium, approximately 1 x 108 cfu/mL, previously incubated at 37° C for 24 h. The inoculated dry feed was then shaken vigorously by hand in 30-cm arcs for 5 min in a sterile stomacher bag to achieve an even distribution of Salmonella Typhimurium in the feed matrix. This procedure was repeated twice to ensure an even distribution of Salmonella Typhimurium in the dry feed inoculum.
Enumeration of Salmonella Typhimurium in Control and Inoculated Feed.
The following procedure was used to enumerate Salmonella Typhimurium [18, 19]:
Propagation and Harvesting of B. stearothermophilus Spores.
An important facet of this study was the incorporation of an organism that would not be completely inactivated by the extrusion conditions expected when using the Extru-Tech E325 [20] single-screw extruder to measure survivability of a mesophile (Salmonella Typhimurium) versus a thermophile (Bacillus stearothermophilus ATCC 12980 [21]), thus providing a measure of the upper level of sterilization potential of the system. Other indigenous acid-producing thermophiles in the feed were enumerated. Bacillus stearothermophilus was selected for the extrusion studies because its spores are among the most heat-resistant described [22]. Additionally, B. stearothermophilus spores have been used as a standard indicator in inoculated experimental pack studies to assess the effectiveness of sterilization through the autoclaving procedure [23]. Dehydrated pellets of B. stearothermophilus were maintained at 4° C in sealed vials until used. A vial containing the dehydrated vegetative cells (pellet) of B. stearothermophilus 12980 was disinfected with alcohol-dampened gauze and the seal was then removed. From a 16 x 100 mm glass test tube containing 6 mL of NB, 1 mL of NB was aseptically pipetted and transferred to the vial to rehydrate the pellet. The rehydrated pellet was aseptically transferred into the test tube containing NB and the mixture vortexed to thoroughly mix the suspension. Plates of trypticase soy agar (TSA) were streaked with the spore suspension by using a sterile loop. The plates were inverted and incubated at 55° C for 24 h.
Bacillus stearothermophilus vegetative cells and spores were propagated on plates of TSA [24] supplemented with MnSO4 (100 mg/L), to promote sporulation, by aseptically inoculating the plates with pure isolates of B. stearothermophilus. The plates were incubated at 55° C for 5 d, sealed with Parafilm to minimize drying of the media, and then further incubated at room temperature for an extra 5 d. Growth on each TSA plate was scraped from the agar surface with a sterile loop and aseptically transferred into a centrifuge tube containing 10 mL of sterile water. The mixture was vortexed thoroughly to obtain a suspension of B. stearothermophilus vegetative cells and spores.
Liquid Inoculum of B. stearothermophilus Spores.
A suspension of vegetative cells and spores was prepared by aseptically transferring the growth of B. stearothermophilus from an agar plate into 5 mL of deionized water in a 10-mL screw-capped centrifuge tube and vortexing thoroughly (5 to 10 s) to mix. Spores were recovered from the suspension by sequential centrifugation at 1,675 x g for 10 min, 1,106 x g for 15 min, and 708 x g for 15 min, respectively. After each centrifugation step, the supernatant containing vegetative cells was discarded, the pellet consisting of spores was resuspended in 5 mL of deionized water, and the suspension was vortexed for 5 to 10 s. The pellet phase of the separated mix containing the spores was examined. Spore purification by centrifugation and washing was then verified by examination of a malachite green spore stain at 1,000 x with a light microscope. An estimate of spore concentration in the final suspension was made by the direct microscopic count method with a Petroff-Hauser counting chamber [25].
Enumeration of B. stearothermophilus.
Briefly, the following procedure was used to enumerate spores. The sample was diluted so that the concentration of bacterial spores would equal 5 to 15 spores in each small square in the grid of the counting chamber, then added to the counting chamber by using a pipette, and allowed to settle for approximately 5 min before placing the counting chamber on the stage of the light microscope. Manual counts were made at 630 x magnification. The counts obtained were used to calculate spore density in the suspension [26]. A sufficient number of squares to give a total count of approximately 600 spores were counted to provide the most accurate estimation of the total spore population. The dilution factor was obtained by using the volumes of sample and diluent [27]. The lower and upper limits of the number of spores in a sample that could be detected and adequately counted by the direct microscopic count method using the Petroff-Hausser counting chamber were determined on the basis of the counting chamber specifications and the spore sample dilution factor. The lower and upper limits were based on the minimum and maximum number, respectively, of spores per small square that could be counted accurately. The volume of each small square was 2.5 x 10–7 mL [28, 29].
Enumeration of B. stearothermophilus Spores Suspended in Deionized Water.
Dilution blanks consisting of 9 mL of deionized water were prepared in test tubes. One milliliter of the initial suspension was aseptically transferred to the first dilution blank to make the 10–1 dilution. After thoroughly vortexing the 10–1 dilution, 1 mL was transferred to the next dilution blank by using a separate sterile pipette tip to make the 10–2 dilution. Subsequent decimal dilutions were prepared by using the serial dilution technique, transferring 1-mL quantities into dilution blanks with a separate sterile pipette tip for each dilution. After agitation of the spore sample, 1-mL volumes of appropriate dilutions containing spores sufficient to yield 25 to 250 colonies in each of 4 plates poured were transferred to 250-mL Erlenmeyer flasks containing 100 mL of sterile dextrose tryptone agar (DTA) maintained in a liquid state at 50 to 60° C. Samples were then heat-shocked at 100° C for 15 min in a custom-made steam chamber to stimulate germination. After heat-shocking, samples were cooled rapidly in a cold water bath to 50 to 60° C. Samples were then equally distributed into 4 sterile Petri plates under a laminar flow biohazard hood.
Preparation of Dry Feed Inoculum of B. stearothermophilus.
To prepare the feed inoculum, 10 mL of B. stearothermophilus vegetative cells and spore suspension containing approximately [3 ± 0.02] x 106 spores/20 mL was pi-petted and aseptically added to 1 kg of the standardized feed formulation in a sterile 3-L polyethylene container with a tight-fitting lid. The container lid was replaced and its contents were vigorously shaken for 5 min in a 30-cm arc to ensure even distribution of spores in the feed matrix. Feed inoculum was then refrigerated at 4° C until used.
Feed Inoculation with Dry Feed Inoculum of B. stearothermophilus.
Briefly, 10-kg samples of the standardized feed formulation was inoculated with appropriate quantities of the dry feed inoculum of B. stearothermophilus [30], and then predetermined quantities of tap water for each target mash feed moisture content that was selected on the basis of the central composite statistical design were added to the feed. The feed-inoculum-water mixtures were agitated in a paddle mixer for 15 min to ensure even distribution of spores of B. stearothermophilus in the test feed samples. The 10-kg test feed samples were then extruded at predetermined extruder barrel exit temperature and retention times, respectively, as described previously [8].
Spiking Feed with Dry Feed Inoculum of B. stearothermophilus.
Estimation of the dry feed inoculum of B. stearothermophilus required in 10-kg batches of mash feed (MMF) to elevate the density of thermophilic bacterial spores beyond background levels in mash feed (cfuD) to approximately 2.91 x 104 spores/g of feed was based on the following estimates: the background level of thermophilic bacterial spores in control mash feed (cfuMF) was 5.00 x 102 spores/g of feed and that in dry feed inoculum (cfuFI) was approximately 1.15 x 105 spores/g of feed. An inoculum size of 3.32 kg was obtained [30].
Enumeration of Bacterial Spores in Control and Inoculated Feed.
Spores of B. stearothermophilus were artificially added to mash feed possibly containing indigenous acid-producing, thermophilic microorganisms to ensure a total feed spore density (cfu/g of feed) such that a detectable level of viable and culturable spores would be left in the feed after extrusion cooking under the conditions used in this study.
Briefly, 20 g of each feed sample was weighed in a sterile sampling container set on a portable benchtop weighing scale. Deionized water was then added until the scale read 100 g. The container lid was tightly replaced and the suspension was vigorously shaken for 10 s in a 30-cm arc to obtain a uniform suspension of feed in water. Serial dilutions were prepared from this initial 1:5 dilution by aseptically transferring 10-mL quantities of suspension into 90-mL dilution blanks consisting of deionized water in 250-mL sterile polyethylene sampling containers with lids. While the suspension was agitated, 20 mL of the feed suspension was pipetted with a large bore pipette into a 250-mL Erlenmeyer flask containing 100 mL of sterile DTA maintained in a liquid state at 50 to 60° C. The flask was placed in the steam cabinet and heat-shocked at 100° C for 15 min. A thermometer was set in a 250-mL flask containing 100 mL of DTA as a temperature control. After heat-shocking, samples were cooled rapidly in a cold water bath to 50 to 60° C, after which the entire mixture was equally distributed into 5 sterile Petri plates under a laminar flow biohazard hood. A similar temperature-control flask was exposed to the same heating and cooling conditions as the test sample to monitor the heat-shocking and cooling steps of the procedures. Plates were allowed to dry partially covered for 45 min to 1 h at room temperature under the laminar flow biohazard hood and were then incubated at 60 to 65° C for 24 h. Colonies were counted between 16 and 24 h later by using a colony counter.
Determination of B. stearothermophilus Spore Density on Spore Enumeration Media.
Spore density of B. stearothermophilus in feed was determined by calculation [31].
Limits of Detection of B. stearothermophilus Spores in Feed by Culture.
The lower limit of detection was based on the lowest number of bacterial colonies per plate (25 cfu) that would accurately reflect the number of cfu in the original suspension [18]. For example, suppose that a 20-g feed sample yielded 25 cfu on each of the 5 plates poured. The total number of cfu would be 125 cfu/20 g of feed, which would be rounded to 1.3 x 102 cfu/20 g of feed (2 significant figures). Similarly, the upper limit of detection was based on the greatest number of bacterial colonies per plate (250 cfu) that would accurately reflect the number of cfu in the original suspension [18]. For a 20-g feed sample, if 250 cfu was counted on each of the 5 plates poured, the total would be 1,250 cfu/20 g of feed, and rounded would be 1.3 x 103 cfu/20 g of feed. The lower and upper limits of detection of spores in feed by culture method were estimated by using these data [32]).
Accuracy and Validity of Spore Enumeration Method
Spores of all indigenous acid-producing, thermophilic organisms present in the feed and of artificially inoculated B. stearothermophilus were recovered by using the procedures described below. The test bacillus was differentiated from indigenous thermophilic bacilli by comparing the spore densities in control (noninoculated feed samples) test feed samples with inoculated test feed samples. The accuracy of the method used to recover and enumerate the vegetative form of the bacterial spores originally added to the test feed was expressed as the ratio of the observed spore density (cfu/g of feed) to the predicted spore density (cfu/g of feed) of B. stearothermophilus based on the quantity of feed inoculum added to the test feed [33]. It measured the ability of the method to detect the test bacillus in the mash feed sample. Validity is a measure of the ability of the enumeration method to correctly recover the targeted test bacillus and can be expressed as the ratio of the density of the test bacillus recovered to the total number (B. stearothermophilus and other thermophilic bacilli) of bacilli recovered from the mash feed sample by using the enumeration media [34].
Feed Mash Moisture Content Control
To obtain feed of the desired moisture content, a predetermined amount of tap water was added to 10 kg of feed mash and the feed was then mixed for 10 min by using a paddle mixer to achieve an even distribution of moisture in the feed. Briefly, a mixing container was tared on a weighing balance, and 10 kg of dry feed and a previously determined amount of water were then added. The amount of tap water, MW, required in 10 kg (MAF) of dry feed was calculated by using a formula derived previously [35]. This method of feed moisture regulation provided for a more precise control of the feed moisture content than the regulation of feed moisture content by addition of water and steam at the extruder barrel.
Mash Feed Moisture Content Measurement.
Following mixing of 10-kg batches of ambient feed with predetermined amounts of water, two 100-g feed samples were extracted from the surface and the center of the 10-kg batches of mash feed for moisture content analysis. The batches of mash feed used for moisture feed content evaluation were not inoculated with B. stearothermophilus spores. The approved methods of the American Association of Cereal Chemists were used in this measurement [36, 37].
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RESULTS AND DISCUSSION |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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Dry Chalk and Dry Feed Inocula of Salmonella Typhimurium.
The Salmonella Typhimurium was recovered on nalidixic acid-amended MacConkey agar (100 ppm) from feed samples inoculated with dry chalk inoculum of Salmonella Typhimurium, but not from control feed samples before extrusion. Using the same selective media, we recovered no detectable cells of Salmonella Typhimurium nalr from the extrudate when feed with 245 g of moisture/kg of mash feed (wet basis) was extruded for 3 s at the lowest extruder barrel temperature of 82° C. Two temperature settings in the midrange of the extruder barrel exit temperatures (77 to 110° C) were selected to assess rapidly whether Salmonella Typhimurium cells would withstand extrusion conditions over the whole range of extrusion temperatures. No detectable cells of the spiked Salmonella were recovered under the more stringent extrusion conditions tested, as shown in Table 2. We concluded that Salmonella Typhimurium was not an appropriate monitor for exploring extrusion conditions that optimized bacterial inactivation over the permissible ranges of the Extru-Tech E325 extruder. In seeking an organism with greater thermal resistance than Salmonella Typhimurium, B. stearothermophilus was selected.
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) of the test organism before extrusion than dry chalk inoculum, indicating that dry feed inoculum was more suitable than dry chalk inoculum for spiking test feed in these studies. No detectable cells of the test organism were recovered from extrudate after extrusion under Rt of 7 s, T of 83 to 103° C for mash feed, and Mc of 285 g of moisture/kg of mash feed. This result was similar to results obtained previously in which feed inoculated with dry chalk inoculum of Salmonella Typhimurium and extruded showed no detectable cells of the test organism.
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. Approximately 2.33 x 106 spores/20 g of feed were obtained. We concluded that B. stearothermophilus was a more suitable test organism than Salmonella Typhimurium for investigating the optimal feed extrusion conditions that maximized spore inactivation with the Extru-Tech E325 single-screw extruder.
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). This was to be expected because the culture method recovered only the viable spores, whereas the direct microscopic method of spore enumeration represented all spores in the sample, that is, viable and culturable spores as well as viable but non-culturable spores. The difference in spore densities obtained by the 2 methods might also be attributed to inherent random errors of the 2 methods.
Stability of B. stearothermophilus Spores in Refrigerated Dry Feed Inoculum.
The densities of viable B. stearothermophilus spores in the dry feed inoculum at different ages during the 8-wk study period were not statistically different and they remained above the level that is required in dry feed inoculum when the inoculum is used to increase the density of bacterial spores n test feed batches beyond background levels. An average spore density of 1.23 x 106 cfu/20 g of feed inoculum was obtained.
Use of Deionized Versus Tap Water to Modify Moisture Content of Inoculated Feed.
We concluded that the use of tap water to modify the moisture content of the standardized test feed manually did not significantly affect the spore count of the artificially inoculated B. stearothermophilus spores in the 10-kg batches of test mash feed.
Limits of Detection of Spores in Feed Samples by Direct Microscopic Count and Culture Methods
The lower and upper limits of detection for the direct microscopic spore count method were 4.4 x 108 and 1.2 x 109 spores per 20 mL of suspension, respectively. The feed inoculum sample mean spore density was 7.19 x 108, with an SD of 1.5 x 108 spores/20 mL of suspension. The observed spore density was within the limits of detection, indicating that the enumeration method was suitable for estimating the density of thermophilic bacillus spores in the feed inoculum.
The lower and upper limits of detection of the culture method were 6.25 x 103 and 6.25 x 106 spores/20 g of feed, respectively. The mean spore density obtained with the culture method was 1.00 x 104 spores/20 g of feed and 3.43 x 105 spores/20 g of feed for the control and inoculated feed samples, respectively. The observed spore densities for the control and inoculated feed samples lay within the lower and upper detection limits of the culture method of spore enumeration for bacillus. This result indicates that the culture method used was suitable for quantifying the number of culturable bacillus spores used as an alternative monitor of sterilization efficiency in this extrusion-processing study involving inoculated standardized feed.
Accuracy and Validity of the Method of Detecting B. stearothermophilus Spores in Inocula and Feed Samples
Table 5 shows a summary of estimates of accuracy and validity of the method of detecting the vegetative form of spores of B. stearothermophilus in inoculated feed samples. These data indicate that the culture method of enumerating spores in control and inoculated feed was suitable for this purpose.
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CONCLUSIONS AND APPLICATIONS |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES AND NOTES |
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TOPSUMMARYDESCRIPTION OF PROBLEMMATERIALS AND METHODSRESULTS AND DISCUSSIONCONCLUSIONS AND APPLICATIONSREFERENCES AND NOTES |
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= (2n)1/4 where = units, of additional factor, along the coordinates in the central composite statistical design space and n = number of independent variables.
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