1 Research paper

2 Staphylococcus aureus
3 Incidence in a Tuna Factory:
4 An Industrial Study
5

7 John DeBeer,1* Javier Colley,2William Cole,3 Karen McKie,2 and Natasha Farmer2

1*
10 Chicken of the Sea International, 2150 E. Grand Avenue, El Segundo, CA 90245 USA;

2
11 Chicken of the Sea Georgia Canning, 129 N. Commerce Drive, Lyons, GA 30436;
3
12 TechniCAL, Inc., 101 Riviera Drive, Georgetown, KY 40324

13

14

15

16 *Corresponding author:

17 Phone: +1-760-670-6141: Fax: +1-858-362-9211

18 E-mail: john.debeer@thaiunion.com

pg 1 of 42
19 ABSTRACT

20 This research investigated the likely hazard of Staphylococcus aureus (S. aureus) growth

21 and enterotoxin formation in a tuna cannery which uses cleaned frozen albacore (Thunnus

22 alalunga) and skipjack (Katsuwonus pelamis) loins as the raw material. The FDA’s HACCP

23 Guidance for exposure time is 3 h if the material spends any time at over 21.1°C (70°F). This is

24 not enough time to process thawing tuna through to S. aureus inhibitory temperatures of 50°C

25 (122°F) in a retort. The S. aureus was analyzed on the tuna meat (loins) from Certificates of

26 Analysis from the supplier which sends frozen loins, as well as direct sampling at the loin

27 processing factory of thawed tuna loins, ingredients, equipment, and canned products.

28 From 2011 to 2018, of the over 28,000 frozen tuna loins tested for S. aureus by the

29 supplier, less than 0.8% tested positive, with the highest being 85 CFU/g. Of 162 loins sampled

30 at the loin processing factory in 2011, less than 23% were positive, with the highest being 70

31 CFU/g at receiving.

32 In 2018, frozen loins were processed (thawed, canned, and held in cans) for a time period

33 far longer than the FDA’s HACCP guidance recommends. Samples were collected over a total

34 exposure time of 21 h at temperatures suitable for growth of S. aureus. After thawing for 12 h at

35 20°C (68°F), the albacore loins had no S. aureus, and the maximum count for the skipjack loins

36 was 5.8 x 102 CFU/g. After a maximum of 8-h usage, the belts feeding the tuna fillers had no S.

37 aureus, and the recirculated vegetable broth had a maximum of 9.8 x 102 CFU/g. Sealed cans of

38 albacore and skipjack with vegetable broth added, held anaerobically (vacuum sealed) for 5 h

39 had a maximum of 2.2 x 102 CFU/g. The maximum temperature at which the sealed cans were

40 held was 26°C (78.8°F) in the retort area. Based on the results of this testing, the current

pg 2 of 42
41 operating limit of 3 h can be extended safely to 5 h and with a critical limit of 7 h after thawing

42 frozen tuna loins.

43 HIGHLIGHTS

44 • No S. aureus was detected in over 99% of the incoming frozen tuna loins.

45 • The highest level of S. aureus on arrival was 5.8 x 102 CFU/g.

46 • S. aureus remained low with temperatures up to 27°C for 21 h of exposure.

47 • The exposure time included thawing, processing, and waiting to be retorted.

48 • The FDA’s HACCP Guidance of 3 h should be extended to 5–7 h, based in minimal risk

49 of growth S. aureus enterotoxin.

50

51

52

pg 3 of 42
53 INTRODUCTION

54 This manuscript reports the results of an industrial study analyzing Staphylococcus aureus

55 (S. aureus) in a commercial tuna canning factory in Lyons, GA, USA. This factory exclusively

56 processes incoming frozen tuna meat (loins) into canned products. The objective of the research

57 investigation was to determine whether S. aureus will grow to an unsafe level (i.e., to sufficient

58 numbers to produce enterotoxin) under ordinary processing conditions at this factory and what

59 the time frames for this might be.

60 The frozen tuna meat processed at this factory comes from commercial tuna processors,

61 i.e., primary processors that process whole wild caught tuna. The primary processors (frozen

62 loin manufacturing factories) are located in other countries. Descriptions of preparing, thawing,

63 precooking, and cleaning whole round tuna are described in numerous papers (1, 13, 14, 15, 25,

64 26, 27). After thawing, butchering, and precooking the tuna, the primary processors clean the

65 tuna meat and pack them into vacuum sealed plastic bags which are called “tuna loins” in the

66 canned tuna industry. The tuna loins are frozen at the manufacturing plants and shipped in

67 frozen containers to the cannery. To protect the tuna meat from oxygen intrusion, the plastic

68 bags used for tuna loins have typical oxygen transmission rates of 20 cc/m2/24hr @ 1 atm &

69 23°C & 0% RH, well within a definition of a high barrier bag (16). The process for packing

70 canned product from these loins in this factory is described later.

71 S. aureus is well described in the literature, because staphylococcal enterotoxin is a safety

72 hazard particularly in prepared foods, including cooked seafood that is then handled by humans

73 (24). Twenty percent to 80% of humans may be permanent or intermittent S. aureus hosts,

74 primarily in nares and on skin (23). These persons are considered carriers of S. aureus although

75 they are not suffering from any illness or symptoms of infection (32). S. aureus has a

pg 4 of 42
76 temperature growth range of 6°C to 48°C (42.8°F-118.4°F), and the enterotoxins can form

77 between 10°C and 46°C (50°F–114.8°F) (29). The optimal temperatures for bacterial growth

78 are 35°C to 41°C (95°F-105.8°F) and for toxin formation are 34°C to 40°C (93.2°F-104°F) (29).

79 S. aureus survives freezing (22, 46).

80 Bennett et al. (6) indicate that 106 CFU/g of S. aureus is needed to produce enterotoxin.

81 According to the FDA (34), in order to produce enough enterotoxin to reach the infective dose,

82 the S. aureus population must range from 105 to 106 CFU/g. Kataoka et al. (22) indicates 106

83 CFU/g is needed for SE production in tuna meat. All of the temperature ranges are given for

84 aerobic conditions; in anaerobic conditions, the growth rate slows to about 25% to 35% of the

85 aerobic growth rate (5, 17). The enterotoxins (exoproteins), once formed, are very stable and

86 resistant to heat, cold, and stomach acids (30).

87 The pH range for S. aureus growth is 4 to 10, and the water activity (Aw) limits are 0.83

88 to .99 (29). By comparison, tuna, canned or otherwise, has a pH range of 5.6 to 6.4 and an Aw of

89 0.97 to 0.99 (22, 45); thus, tuna is well within the S. aureus pH and Aw growth ranges.

90 S. aureus is a poor competitor with other bacteria (4, 7, 19, 20, 21), so most cases of S.

91 aureus food poisoning are the result of re-contamination as a consequence of poor human

92 hygiene practices during processing and handling of ready-to-eat foods. Precooking tuna

93 eliminates most bacteria, so afterwards S. aureus could face little competition from other bacteria

94 if it contaminates the tuna meat after it has been precooked (8, 33). During the preparation of

95 frozen tuna loins in the loin manufacturing facility, the precooked tuna is extensively handled,

96 during deskinning, cleaning, and placement of cleaned loin meat into high-barrier plastic bags

97 (12).

pg 5 of 42
 98 All tuna canneries must follow Current Good Manufacturing Practices (CGMPs) (43) and

99 clean up often. In the raw, precooked, and loin processing areas, gowns or long lab coats are

100 required to cover street clothes. All factories have boot wash sanitation and hand wash stations

101 and require hair and beard coverings. The factories are careful to separate the raw fish

102 production areas from the areas with precooked fish, and those areas from the retort areas (12).

103 The workers in many factories these days use gloves while deskinning, cleaning, or processing

104 the fish, but a tuna cannery is definitely not a sterile environment. Thus, we would expect to find

105 some levels of bacteria on the food contact surfaces in a tuna factory. But, there has never been

106 a case reported of S. aureus enterotoxin poisoning from canned tuna (22, 45, 46).

107 All seafood processed or imported for sale into the United States must be prepared or

108 packaged under a Hazard Analysis and Critical Control Program (HACCP) plan (42). The latest

109 FDA guide for controlling food safety hazards in seafood is the Fish and Fishery Products

110 Hazards and Controls Guidance, a.k.a., the FDA HACCP Guidance published in 2011 (34). The

111 guidelines for processing time and temperatures for S. aureus control from table A-2 in

112 Appendix 4 of the FDA HACCP Guidance (34) are listed in Table 1. The table shows that if, as

113 in the case in all canneries, the environmental temperature ever is above 21.1°C (70°F), the time

114 limit is 3 h from first human touch after precooking, until inhibitory temperatures at the core of

115 the material in either the retort (36, 37, 38, 40) or the freezing chamber (38) are reached: the

116 inhibitory temperature in the retort is listed at 50°C (122°F) and in the freezing chamber is 10°C

117 (50°F). The FDA has indicated in the past (41) that product which has been exposed to unsafe

118 conditions that could result in the formation of S. aureus enterotoxin should be either destroyed

119 or diverted to a non-food use. Further, the FDA has stated (40) that to prevent the hazard of S.

120 aureus growth and toxin formation in a tuna product, the HACCP plan should identify the hazard

pg 6 of 42
121 and include controls to limit the exposures of the tuna above 21 °C to less than 3 hours from the

122 time the precooked fish are first handled at the skinning/cleaning operation until the packed

123 product is placed in the retort, and the cold spot of the pouches or cans reaches 50°C.

124 The guidelines in Table 1 were developed from data from The International Commission

125 on Microbiological Specifications for Foods (ICMSF) 5: Characteristics of Microbial Pathogens

126 (20). The source of these data was a personal communication from Adair (1990) for lag times

127 for S. aureus growth in steak and kidney pie, with no further citation. Although the FDA’s

128 HACCP Guidance does not specifically identify its source of these recommendations, an agency

129 response to a Freedom of Information Act request (35) stated they were based upon Table lb on

130 Page 307 of ICMSF 5 (20). The guidelines from Table 1b, p. 307 of ICMSF 5 are listed here in

131 Table 2. The number of hours to reach 106 CFU/gm based on the growth rates from ICMSF’s

132 Table 1b (20) are also listed. Other than the 12-h time frame for temperatures in the 51 - 70°F

133 (11-21°C) range, the other recommendations do not exactly correlate to ICMSF’s Table lb (20).

134 The specific reason for this industrial survey was to collect samples for S. aureus analysis

135 at various points in the process, i.e. under “real world” conditions, to determine if it is

136 reasonably likely for S. aureus to grow to sufficient numbers – at least 106 CFU per gram in tuna

137 meat (22) - to produce enterotoxin through thawing of the incoming loins, staging for filling,

138 cutting, media addition, sealing, and heating in the retort until the core product temperature of

139 50°C (122°F) is reached.

140 Based on the historical knowledge of no S. aureus enterotoxin ever having been found in

141 canned tuna, the danger of insipient spoilage from post-seamer, pre-retort lag time (dwell) time

142 raises far more concern when un-retorted canned tuna waits while a problem is fixed, or the

143 retort is filled. There is no FDA additional regulation limiting post-sealing, pre-retort lag times

pg 7 of 42
144 (9). However, the regulations for the state of California allows a 3-h lag time for tuna (10): a 2-

145 lag time is a suggested industry standard operating limit based on a USDA regulation (9).

146 Tuna cans sizes, retort heating rates and total process times. The FDA’s 3-h time

147 limit is a particular problem for packing large cans of tuna. The largest can size normally

148 processed in a tuna cannery is a 66-oz net weight can (603 by 408 or 63/16 in diameter x 4½ in

149 high). It can take up to 80 minutes for the core of the filled tuna can of this size to reach and

150 pass 50°C (122°F) during venting and heating in the retort from a 15°C (59°F) initial

151 temperature (IT) (12). The heating rate is about 26°C/h or 0.44°C/min, which is still a very fast

152 temperature change for a S. aureus bacteria and can lead to heat shock or cellular damage.

153 According to Baeza et al. (2, 3), S. aureus will suffer from heat shock if and when the

154 temperature increase exceeds 2°C/h or 0.033°C/min or less than 10% of the rate in commercial

155 retorts for the largest of cans. The heating rate in other can sizes is much faster possibly causing

156 more heat shock and damage.

157 Previous S. aureus work on tuna. In 2013 and 2015, in a series of laboratory

158 experiments, Wu and Su (45, 46) and Kataoka et al. (22), (WSK) studied S. aureus growth and

159 enterotoxin formation in tuna meat from commercially frozen tuna loins. The tuna meat

160 (albacore and skipjack) was inoculated with five strains of enterotoxin-producing S. aureus. The

161 incubation temperatures studied were 37°C (98.6°F), 27°C (80.6°F), and 21°C (69.8°F); the

162 studies were conducted under aerobic conditions.

163 In the study by Kataoka et al. (22) at 27°C (80.6°F), it took the S. aureus population 8 h

164 to increase from 103 to 106 CFU/g, 8 h for S. aureus enterotoxin (SE) to form, and also 8 h for

165 spoilage to take place. In the same study, at 21°C (69.8°F), it took the S. aureus 20 h to increase

166 by 3 log CFU/g and 14 h for SE to form. Although in Wu and Su 2014a (45), the spoilage

pg 8 of 42
167 occurred before the enterotoxin formation, that was not the case in the Kataoka et al. study where

168 the SE and off-odor detection occurred at the same time (22).

169 The S. aureus levels of the incoming raw material in one study by Wu and Su (2014a)

170 ranged from 10 - 103 CFU/g (44), but they were zero in the Kataoka et al. study (22). Low levels

171 of S. aureus were found in frozen tuna loins from other tuna loin manufacturing or processing

172 factories too; S. aureus was detected in 50 samples tested of up to 103 CFU/g in skipjack loins

173 imported into Italy in 2018 (11). In a study of tuna loins produced in the Ivory Coast, out of

174 more than 470 loins tested, none tested positive for S. aureus, although there were multiple other

175 bacterial species present (31).

176 Operational procedures in the cannery in this project. The normal processing

177 sequence in the loin processing factory discussed here is to receive the frozen tuna loins stacked

178 on pallets and unload them into a cold storage unit maintained at -20°C (-4°F) where they await

179 processing. At the time of delivery, the frozen loins are sampled for histamine, and organoleptic

180 evaluations are made. After these QC checks are passed, the loins are scheduled for production.

181 When production for the loins starts, the pallets are removed from the cold storage, and the

182 individual loin bags are placed on open wheeled-racks and pushed into Cabinplant thawing

183 chambers. The frozen loins are thawed in a controlled environment with a set point of 20°C

184 (68°F), normally for 4 h or 5 h, in steam-heated, fast re-circulating air. The target core

185 temperature of the thawed loins is 4.4°C (40°F). After thawing, they are transferred to a chill

186 room maintained at < 10°C (50°F) with a set point of 7°C (45°F) until they taken to the packing

187 lines.

pg 9 of 42
188 The albacore meat in the loin bags is graded based on color and piece size as a) complete

189 loins, b) large pieces and chunks, and c) chunks and flakes. The skipjack loins have the loins,

190 chunks, and flakes mixed in the same bag. The thawing time depends on the loin grade.

191 The tuna processing of this factory is separated into four areas: thawing chambers, chill

192 room, packing room, and pre-retort area. The temperatures in each area are: < 20°C (68°F) in

193 the thaw chambers, < 10°C (50°F) in the chill room, < 21.1°C (70°F) in the packing room, and >

194 21.1°C (70°F) in the retort area. On the packing lines, the high-barrier plastic bags containing

195 the thawed tuna meat are opened, and the loin meat is transferred into a commercial tuna filler

196 via a three-sided conveyor belt (tri-belt). The filling machine portions (cuts) the fish meat and

197 portions it into the cans, and the cans of tuna meat are transferred to liquid media addition

198 stations. There is an inspection station between the can filler and the media addition stations

199 where slack or overfilled cans are removed so the tuna meat can be quickly recycled. After the

200 liquid media (vegetable broth, brine, or edible oil) are added, a lid is placed on the can, and the

201 can is sealed with a double seam, after injecting steam into the headspace to create a partial

202 vacuum. The cans are then ink-jetted with a date code and time stamp and immediately loaded

203 into a retort basket (cart) and rolled into the retort staging area. If a retort is open, the retort cart

204 is loaded into the retort, else it waits until a retort is open for use. After the retort is filled, 12

205 carts in this factory, the doors are closed, and the retort heating cycle is started by first venting

206 the retort with live steam. The commercial sterilization time and total retort cycle including

207 come-up time depends on the starting IT, media style, and can size (18). After retorting, the cans

208 are cooled, labeled, and cased for shipment to customers.

209 In order to evaluate the risk of S. aureus in the process or product in this factory several

210 sources of information were evaluated:

pg 10 of 42
211 1) Certificates of Analysis (COAs) from the suppliers of incoming tuna loins prepared to

212 our specification;

213 2) Samples collected at this factory for S. aureus on incoming loins;

214 3) Bi-weekly canned samples from this factory analyzed for staphylococcal enterotoxin; and

215 4) The 2018 processing study described below.

216 METHODS AND MATERIALS

217 The research methodology will follow sampling of incoming loins for S. aureus, testing

218 possible growth of S. aureus during ordinary processing in the factory, and testing for

219 staphylococcal enterotoxin in canned product. There were four different groups of data reviewed

220 and analyzed for S. aureus or staphylococcal enterotoxins at the U.S. tuna cannery:

221 1. Certificates of Analysis (COAs) of incoming frozen loins from a frozen albacore and

222 light meat tuna processor. From 2011 through 2018, the U.S. cannery received routine

223 COAs of S. aureus from the supplier in Thailand; over 28,000 loins were sampled, and

224 certified results from the supplier’s lab were reported. The samples were collected from

225 frozen loins prior to shipment. The analytical procedure used AOAC method 2003.11,

226 using 3M Petrifilm Staph Express Count Plate Method.

227 2. Samples collected and tested in 2011 at the cannery. These were 162 samples of tuna

228 meat collected from the thawing area, packing lines, and just prior to retorting and sent to

229 an outside lab for analysis for S. aureus and Aerobic Plate Count (APC). The samples

230 were received from three different frozen tuna loin suppliers. These samples were

231 analyzed at JLA Laboratory in Albany, GA 31721. The analytical procedure used AOAC

232 2003.07, using 3M Petrifilm Staph Express Count Plate Method.

pg 11 of 42
233 3. Bi-weekly samples of canned products that were collected and sent for testing for

234 staphylococcal enterotoxin and other bacterial analysis, a Georgia Department of

235 Agriculture (GDA) requirement. From 2011 - 2018, >160 randomly selected canned

236 samples of albacore and light meat were sent to Michelson’s Laboratory in Commerce,

237 CA 90040, for analysis. The analytical procedure used was AOAC official method

238 2007.06 Vidas 2 test.

239 4. Samples for S. aureus and APC collected while the factory was operating normally under

240 typical operating conditions and temperatures in 2018. There were four suppliers of

241 frozen loins, from four different countries. There were four suppliers of albacore and two

242 suppliers of skipjack loins. All the tuna meat samples were collected under aerobic

243 conditions, except for the contents of the sealed cans, although the bagged loins were

244 held under anaerobic conditions until they were sampled. The microbiological samples

245 were collected on 11 separate days over 7-month period. The pattern of sample

246 collection for the loins and canned products followed the same general pattern; the fish

247 meat was held for extended periods of time, and after the sampling was completed, all the

248 remaining tuna meat was discarded. The fish was thawed twice as long as normal and,

249 for this study, the thawed tuna meat was purposely taken straight to the packing room,

250 bypassing the chill room, an unusual procedure.

251 a. Over 400 samples were collected during processing and tested for S. aureus and

252 Aerobic Plate Count (APC) activity. These included samples from the environment,

253 equipment, ingredients, and tuna meat. The loin bags were thawed, held at ambient

254 temperatures in the packing room, processed, and held at ambient temperatures of >

255 21.1°C (70°F) in the retort area for 3 h and 5 h in open or sealed cans. The exposure

256 time to these ambient temperatures was far longer than is recommended in the FDA’s

pg 12 of 42
257 HACCP Guidance (34), or the industry post-seamer, pre-retort lag times limits (9), or

258 California State regulations (10).

259 b. The samples included:

260 i. Environmental swabs (food contact surfaces) of fish tubs (totes) and packing

261 equipment,

262 ii. Water and vegetable broth,

263 iii. Thawed bags of previously frozen tuna in loin, chunk, and flake style, of both

264 albacore and skipjack species. The bags were vacuum-sealed until the samples

265 were taken, but no effort was made to keep, and transport the samples to the lab

266 anaerobically.

267 iv. Tuna from open cans, held for 3 h and 5 h.

268 v. Tuna from closed cans sealed with a double seam, held for 3 h and 5 h, under

269 vacuum (anaerobic) conditions.

270 Environmental Samples – Totes and Packer Machine Feed Conveyors. The food

271 contact surfaces were sampled at production startup (0 h), after 5 h, and after 10 h at end of

272 production day. The surfaces were swabbed using a cross-hatch technique on an area

273 approximately 10 cm x 10 cm. One swab sponge kit was used for each tote: sterile swab sponge,

274 gloves, and sample baggie kit (Nasco WhirlPak). Seven totes were separately sampled for each

275 sampling event. Two of the totes collected re-cut product, and five were from five production

276 lines. Re-cut (re-worked) product is from cans that were either under-filled or overfilled at the

277 tuna filler, and the tuna is added back to the tuna filling machine within 15 – 30 min.

pg 13 of 42
278 A tri-belt conveyor feeds the tuna into the filling machine and it was also swab sampled.

279 Three sampling kits (Nasco WhirlPak) were used on each line for each sampling event. The

280 swab kits were refrigerated overnight and shipped the next day to the Silliker lab.

281 Vegetable broth samples. Broth samples were collected at start-up (0 h), after 5 h of

282 production, and after 10 h at the end of the production day, and the broth tanks and delivery

283 system had not been drained or rinsed during the sampling period. The sample collector wore

284 sterile gloves (Kimberly-Clark 3G sterile nitrile gloves) and used a sterile scoop (Bel Art

285 Products Sterileware) to collect the broth. It was then poured in a sterile bottle (Eco-Sensa 100

286 ml sampling bottle). The broth samples were placed in a lab refrigerator overnight and shipped

287 to the lab the next day. The vegetable broth is mixed at this factory using city water. Samples of

288 city water were collected and treated in the same fashion.

289 Tuna meat samples. The loin bags’ dimensions were 28 – 30, 4.5, and 3 in (LWH), and

290 they held about 6 kg. Twelve bags per variable of tuna meat were used for the study; all bags

291 were selected randomly for sampling. All loins from a loin grade were selected from the same

292 pallet; a shipping pallet has 180 to 200 loin frozen bags. A pallet has loins from only one

293 supplier, one species, and one grade,

294 The frozen loins were thawed for either 8 h or 10 h (twice the normal time) at 20°C

295 (68°F) in a steam-heated forced-air temperature-controlled Cabinplant thawing chamber. After

296 thawing, the loins were transferred to the packing room (by-passing the chill room) and held

297 there for 2 - 3 h until they were sampled.

298 Tuna meat samples (about 300 g) were aseptically collected from each end of each bag as

299 follows:

pg 14 of 42
300 1. The bag of tuna meat was placed onto a surface which had been cleaned and sanitized

301 with Alpet D2 surface sanitizing wipes which contain isopropyl alcohol (58%), octyl

302 decyl dimethyl ammonium chloride (0.0075%), didecyl dimethyl ammonium chloride

303 (0.0045%), dioctyl dimethyl ammonium chloride (0.0030%), and other ingredients.

304 2. The exterior of tuna meat bag was sanitized with sanitizer wipes (Alpet D2 surface

305 sanitizing wipes). A sterile disposable blade was used to open the tuna meat bags (Safe

306 Shield Scalpel #14).

307 3. The sample collector used single-use sterile gloves. Another disposable sterile blade was

308 used to cut out the meat sample, and this sample was placed in a pre-labeled sterile

309 sample baggie (Whirl-Pak from Fisher Scientific) and sealed. This procedure was

310 repeated for the other end of the bag and for all the other samples; new gloves and sterile

311 blades were used with each new loin bag. The collection trays were also cleaned with

312 Alpet D2 surface sanitizing wipes.

313 4. After the samples had been collected, the baggies were placed into a lab freezer -20°C (-

314 4°F) for 1 h to chill them quickly and were then transferred to a lab refrigerator set at

315 38°F (3.3°C) (the actual range was 1.9°C - 3.1°C, 35.5°F - 37.5°F) until they were

316 shipped to the lab for analysis.

317 Canned Samples. The remaining meat from the tuna meat bags sampled was transferred to

318 a regular production line and packed with a commercial can filling machine. The packing

319 machine had been in operation for more than 5 h to simulate a usual mid-shift production

320 procedure. The packing machine was not rinsed prior to packing the sample loins. The albacore

321 flakes were packed into 5-oz net weight cans and the albacore and skipjack loins were packed

322 into 12-oz net weight cans.

pg 15 of 42
323 A randomly selected set of twenty cans filled with tuna meat was collected immediately

324 before any condiments and liquid media were added and before seaming. The open cans were

325 removed from the line by personnel wearing new non-sterile sanitary nitrile gloves. At the same

326 time, another set of forty sample cans filled with tuna meat was collected after the normal

327 condiments and packing media had been added and after they had been sealed with a double

328 seam. Vegetable broth with salt and sodium acid pyro-phosphate (SAPP) was added to the

329 canned albacore loins and albacore flakes. Vegetable broth with salt was added to the canned

330 skipjack. Oil packs were not used.

331 The open and sealed sample cans were transferred from the temperature-controlled

332 packing room with an ambient temperature of ≤21.1°C (70°F) to the retort area where the

333 temperature was >21.1°C (70°F) with a maximum temperature of 26°C (78.8°F). Half of the

334 sample cans were collected after 3 h and the remainder after 5 h. After the holding time had

335 been completed, the sample cans were placed in a lab freezer set at -20°C (-4°F) for 1 h to stop

336 possible bacterial growth. After 1 h, the sample cans were removed from the freezer and each

337 placed inside individual sterile sample baggies and placed in the lab refrigerator set at 3.3°C

338 (37.9°F) overnight prior to shipment to the Silliker lab.

339 Microbiological testing lab and shipping. The 2018 samples were submitted for

340 microbiological analysis to Silliker’s Laboratory, 2169 West Park Court, Suite G, Stone

341 Mountain, GA 30087. All samples were chilled in insulated cartons with frozen gel packs and

342 shipped by UPS, next day service. The lab is about 200 miles from this factory. The S. aureus

343 was tested for using AOAC 2003.07 method, using Coagulase Positive Staph Petrifilm. The

344 APC analysis used the AOAC official method 990.12, Aerobic Plate Count -Petrifilm. The

345 water was tested for coliforms, E. Coli, and Heterotrophic Plant Count using Standard Methods

pg 16 of 42
346 for the Examination of Water and Wastewater method reference 9221 B, 9221 F, 9215B

347 respectively.

348 Temperature Collection and Thermometer calibration. Temperatures of the tuna

349 loins were collected with hand-held Tel-Tru bi-metal stem dial thermometers. The range is -

350 40°C to 71.1°C (-40°F to 160°F), with a subdivision of 1.1°C (2°F) and an accuracy 1.1°C (±

351 2°F); the face diameter is 2.54 cm (1 in). The thermometer calibration was verified with certified

352 thermometer tester equipment set at 4.4°C (40°F) at the start of each day and prior to use.

353 Calibration settings were adjusted with a hand wrench, and all adjustments were recorded.

354 The internal loin temperature was taken from one loin in the sample (one out the group of

355 12 loins sampled). The packing room temperature was recorded at the time the loins were being

356 sampled. The retort area temperature was recorded at the start of the holding period (0 h), after 3

357 h, and after 5 h.

358 Headspace Analysis.

359 The headspace gases of 7-oz cans of tuna packed by this factory were analyzed by Silgan

360 Holdings Laboratory in Oconomowoc, WI, using a SRI86100C gas chromatograph with helium

361 as the carrier gas. Three cans were analyzed for gases, and 3 were analyzed for vacuum.

362 RESULTS

363 1. Incoming fish Certificates of Analysis (COAs). Sample results were received from

364 frozen loins from multiple species and loin grades (Table 3). Of the more than 28,000

365 samples, only 214 tested positive for S. aureus, with the highest at 85 CFU/g in 2011:

366 99.3 % tested negative for S. aureus.

pg 17 of 42
367 2. Samples collected at this US tuna factory in 2011. In 2011, thawed tuna loins were

368 sampled at the cannery for S. aureus. The results are reported in Table 4: 78% were

369 negative for S. aureus, and the highest level recorded was 360 CFU/g. Sampling was

370 discontinued because of the low levels of S. aureus.

371 3. Samples of finished products for S. aureus enterotoxin analysis. More than 160

372 samples were collected from canned tuna products of various species and types produced

373 by this factory. These samples were collected over an 8-year period and sent to an

374 independent lab for S. aureus enterotoxin analysis. None of these tested positive for S.

375 aureus enterotoxin.

376 4. Samples collected in this US tuna factory in 2018 during normal processing. This

377 study was set up to monitor whether S. aureus was a problem in ordinary tuna process

378 and whether the time limits in the FDA’s HACCP Guidance were appropriate. The

379 estimated core temperatures of thawed loins during thawing, and on arrival into the

380 packing room, and the ambient temperatures of the thawing chambers, packing room, and

381 retort areas are shown in Figure 1. The microbiological collection times and ambient

382 temperatures are shown in Table 5, and the results are shown in Table 6. Only the

383 maximum values of the S. aureus levels are shown. The results of the APC sampling are

384 shown as well: most of them were > 105 CFU/g.

385 The thawed skipjack and albacore loins were sampled 2-3 h after they entered the

386 packing room. They then waited 2-3 h and were packed in cans and held for 3 h and 5 h in the

387 retort area. The same set of loin meat was used for each group of samples on each sampling day.

388 The maximum levels in CFU/g of S. aureus held in open and sealed cans for skipjack are shown

389 in Figure 2 and for albacore are shown in Figure 3.

pg 18 of 42
390 Environmental sampling for S. aureus. The environmental sampling indicated that

391 there was no S. aureus on the totes or tri-belts into the tuna fillers.

392 Ingredient sampling. The levels of S. aureus in the vegetable broth remained low

393 throughout the day. The water was negative for coliforms (data not shown).

394 Headspace analysis. The headspace gas analysis is reported in Table 7. Oxygen was

395 very low with a maximum of 2.3% indicating that the sealed cans are an anaerobic environment.

396 DISCUSSION

397 Incoming loins sample. The sampling results for S. aureus indicates that the levels were

398 low (mostly zero, with the maximum at 5.8 x 102 CFU/g) on the incoming tuna loins throughout

399 this factory, as confirmed by the COAs on the incoming frozen loins and the data from our

400 sampling process from 2011 and 2018. Further, the results from the samples collected from the

401 2018 study, the levels, quite low at the start did not increase significantly throughout the rest of

402 the process, thus indicating that the loins were only in contact with equipment that had zero (0)

403 S. aureus levels and had low S. aureus multiplication rates at the unchilled ambient temperatures.

404 There were other bacteria present as indicated by the APC counts, so perhaps S. aureus was out-

405 competed. Nevertheless, any factory must assume S. aureus is present on the hand cleaned loins

406 considering up to 80% of people are intermittent carriers (23). Having such low levels of S.

407 aureus on the incoming loins as demonstrated in the study in this factory, however, reduces the

408 risk of growth of the microorganism during production to levels sufficient to produce

409 enterotoxin, a potential control factor which should be noted by any processor using frozen, pre-

410 cooked loins, particularly from outside sources.

pg 19 of 42
411 The risk of S. aureus growth in the high-oxygen-barrier loin bag can be assumed to be

412 minimal, because the tuna is held frozen or chilled under vacuum, low-oxygen conditions. The

413 sealed loin bags spend the thawing time below 20°C (68°F), then they are normally moved to a

414 chill room with an ambient temperature of <10°C (50°F). It should be noted again that, in this

415 reported study of S. aureus, the chill room was purposely bypassed, a situation not likely to

416 occur in actual production, unless the tuna loins are used immediately. Routine placement of the

417 loins in the chill room would be a further check on the growth of any S. aureus, but our study

418 also provides insight into the safety in plants which process pre-cooked frozen loins but do not

419 use a chill room for holding before packing. These loins were left in the thaw chambers, packing

420 room, and retort area to create abusive time at temperature conditions that exceeded worst case

421 scenarios encounter under normal processing conditions. Under normal production conditions,

422 the maximum time from seaming to start of retorting is ≤2 h.

423 None of the frozen precooked albacore loins had any S. aureus just after thawing when

424 the high-barrier plastic bags were opened, and the highest for skipjack was 5.8 x 102 CFU/g.

425 These results were after 12 h to 13 h of exposure to ambient temperatures of 20°C (68°F) and

426 <21.1°C (70°F) in the thawing area and packing room, respectively (Table 5). This is more than

427 two times the normal exposure for these loins.

428 The risk of contamination at the filler where the loin bags are opened and loaded into the

429 cans is also minimal: tri-belts tested free of S. aureus, and loins are pushed onto the tri-belts from

430 the side in a temperature-controlled room by workers wearing protective clothing, head

431 coverings, and using gloves, thus minimizing S. aureus re-introduction. The cans are quickly

432 filled and sealed, and any rework is done within 30 minutes. After the cans are sealed, they are

433 transferred to the retort staging area before loading them into the retort, so they can be fully

pg 20 of 42
434 retorted (commercially sterilized). Thus, the tuna meat is exposed to temperatures >21.1°C

435 (70°F) for no more than 2 h between vacuumized loin bags and low-oxygen sealed cans.

436 Tuna meat in open cans held for 3 h and 5 h under aerobic conditions. There was

437 some growth of S. aureus in the open cans held for 3 h and 5 h. For albacore in open cans, the

438 maximum count was 50 CFU/g after 3 h and 0 CFU/g after 5 h. For skipjack in open cans the

439 highest was 7.2 x 102 CFU/g after 3 h, and 2 x 104 CFU/g after 5 h, but any delay that long

440 would not happen in a normal operation, and, if it did, would trigger the destruction of the cans,

441 due to the potential for spoilage. There is no reason to leave tuna meat in open cans as open cans

442 are filled with tuna, media is added, and the cans are seamed (sealed) at 400 to 600 cans per

443 minute. This tuna meat was exposed to ambient temperatures of 20°C - 25°C (68°F - 77°F)

444 (Table 5) for a total of 19 - 21 h under mostly anaerobic conditions with only a few h under

445 aerobic conditions.

446 Tuna meat in seamed (sealed) cans held for 3 h or 5 h under vacuum conditions.

447 The levels of S. aureus in tuna meat held for 3 h and 5 h in the sealed cans were low: 0 in the

448 albacore and 7.2 x 102 CFU/g in the skipjack. The total exposure time was as indicated above.

449 Holding cans for 5 h after seaming that would not normally occur, and, if it did, the cans would

450 be destroyed. Holding the cans for 3 h after seaming would also be very rare. The operating

451 limitation in this factory is no more than 2 h after seaming before the product gets put on hold for

452 organoleptic investigation.

453 There was no appreciable growth in S. aureus in the tuna meat in seamed or open cans

454 held for 3 h and 5 h in a retort room with the ambient temperature ranging from 22°C to 26°C

455 (71.6°F – 78.8°F). At a temperature of 25°C (77°F) (about the retort area temperature), the

456 generation time is 1.2 h (Table 2) meaning that from a starting point of 103 CFU/g it would take

pg 21 of 42
457 approximately 12 h to reach 106 CFU/g. This factory has a limit of 2 – 3 h, which is well below

458 this 12-h time level.

459 In this factory, tuna is packed in cans with double seams for commercial sterilization, and

460 as the seam is made, steam is injected under the lid to create a low-oxygen environment in each

461 can. However, the data in Table 2 were acquired under aerobic conditions. Table B, page 304

462 from ICMSF (20) defines aerobic conditions as 5-20% dissolved oxygen (this should be % O2, as

463 dissolved O2 is measured in ppm), in determining limits for growth and enterotoxin production.

464 The O2 levels were 2.1-2.3% in cans of tuna from this facility that were analyzed for headspace

465 gas and vacuum levels. The vacuum levels were 8 and 10 inches of Hg (3.9 to 4.9 psi). The CO2

466 levels were 7.6 - 8.7%, and N2 was 83.7 - 85.8%. Such concentrations of the headspace gases

467 measured, along with the vacuums measured, indicate that once the cans are seamed any S.

468 aureus growth would be under low oxygen conditions where the growth rates for S. aureus slows

469 to 25% -35% of the aerobic growth rates (5, 17).

470 In the study by Kataoka et al. (22) at 27°C (80.6°F), it took the S. aureus population 8 h

471 to increase 3 log CFU/g and for S. aureus enterotoxin (SE) to be formed. In the same study, at

472 21°C (69.8°F), it took the S. aureus population 20 h to increase by 3 log CFU/g but 14 h for SE

473 to be formed. The study was conducted under aerobic conditions (22).

474 Testing for S. aureus enterotoxin in canned products. As part of the state of

475 Georgia’s Consumer Protection Division’s requirements, this factory sent a random can of

476 finished product for analysis on a bi-weekly basis. More than 160 cans were analyzed for

477 staphylococcal enterotoxin over a period of 8 years: none were positive.

478 CONCLUSIONS

pg 22 of 42
479 Pflug (28) stated “One of the major problems of science is assurance of the relevance of

480 the scientific laboratory experiments with the actual real-world conditions. The scientist should,

481 as part of any project, make an accurate scientific assessment of the applicability of the generated

482 data to the actual real-world conditions, rather than being allowed to proceed with an unproven

483 assumption of relevance.”

484 This industrial study took Pflug’s note of caution under advisement. The 3-h FDA

485 HACCP time limit was a S. aureus lag time developed from steak and kidney pie mix. WSK

486 (22, 45, 46) tested S. aureus on tuna meat in a laboratory setting under controlled temperature

487 and aerobic conditions. Neither is sufficient for analyzing processing of tuna in a cannery. This

488 study showed levels of S. aureus in a functioning commercial tuna canning factory, under

489 controlled but varying ambient temperature and oxygen availability conditions.

490 The HACCP regulation (42), 21CFR123, section §123.6(a) states that: “Hazard analysis.

491 Every processor shall conduct, or have conducted for it, a hazard analysis to determine whether

492 there are food safety hazards that are reasonably likely to occur for each kind of fish and fishery

493 product processed by that processor and to identify the preventive measures that the processor

494 can apply to control those hazards. Such food safety hazards can be introduced both within and

495 outside the processing plant environment, including food safety hazards that can occur before,

496 during, and after harvest. A food safety hazard that is reasonably likely to occur is one for which

497 a prudent processor would establish controls because experience, illness data, scientific reports,

498 or other information provide a basis to conclude that there is a reasonable possibility that it will

499 occur in the particular type of fish or fishery product being processed in the absence of those

500 controls.”

pg 23 of 42
501 A reminder, the formation of staphylococcal enterotoxin is the hazard: not the presence of

502 the S. aureus bacterium. The experience and illness data indicate that no staphylococcal

503 enterotoxin has ever caused an illness by the consumption of canned tuna. The scientific

504 evidence presented here indicates the formation of staphylococcal enterotoxin takes longer than

505 the current 3-h HACCP limit. Other information, from several sources from around the world,

506 indicates that the primary factories do a good job in controlling S. aureus on the frozen tuna

507 loins, so it does not grow and form enterotoxin during normal processing.

508 Recommended time limits for S. aureus control. We have seen from a review of

509 previous testing and the results of the current study that the highest level of S. aureus in the

510 incoming tuna loins was 5.8 x102 CFU/g. Further the bacteria did not grow to levels which can

511 produce enterotoxins for 8 h or more. Thus, the 3-h time limit if the product exceeds 21.1°C

512 (70°F) at any time in the process is unnecessarily restrictive and, hence, needs to be adjusted.

513 Based on these results, there does not appear to be an issue with S. aureus at any place at this

514 factory: loins entering the packing room would not reach levels sufficient to produce S. aureus

515 enterotoxin until long after 3 h has passed.

516 The time limit is especially restrictive when the 66-oz cans are processed based on the

517 FDA HACCP guidance of 3-h limit for S. aureus (12). This includes the elapsed time from

518 when the loins are thawed, canned, put into the retort, the retort door is closed and until when the

519 core temperature of the can has passed the 50°C (122°F) inhibitory temperature, as stated in the

520 FDA’s series of warning letters (36, 37, 38, 40). Based on our experiment and the work from

521 WSK (22, 45, 46), this time constraint is too restrictive for the risks involved for canneries such

522 as this using precooked frozen loins.

pg 24 of 42
523 Thawing to a uniform temperature, controlled below 20°C (68°F) is very important. The

524 thawing equipment needs to be set up and operated, to consistently deliver a relatively uniform

525 end-point thawing temperature in the loins from top to bottom on the thawing racks. Thawing to

526 too high a temperature – the entire thawing rack or, if not adequately controlled, a portion of a

527 rack – possibly followed by an excess delay time at ambient temperatures >21.1°C (> 70°F)

528 before starting the run through the fillers, increases the potential for the growth of S. aureus.

529 Thawing to too low a temperature (e.g., frozen at the core) might, depending on the speed of

530 packing from end of thawing to steam-on in the retort, adversely impact the initial temperature of

531 the scheduled process. The delay time between opening the thawed bags of loins until first can is

532 seamed is normally very short, a matter of minutes, not hours. The longest period of time after

533 thawing, barring a line breakdown, happens when filled retort baskets must wait to be loaded

534 into the retort but no more than 2 hr.

535 The purpose of this survey is to demonstrate what the “real-world” conditions are,

536 relative to the presumed hazard of formation of staphylococcal enterotoxin in a facility which

537 processes pre-cooked, cleaned, and frozen tuna loins into finished, commercially sterile canned

538 product. In this facility, the following procedures/conditions present hurdles to the growth of S.

539 aureus to a level sufficient for enterotoxin to be produced:

540 1) The high-barrier bags in which the incoming loins are packed and thawed provide a low-

541 oxygen environment, after a vacuum has been drawn and tuna meat was frozen.

542 2) The levels of S. aureus in the incoming frozen loins are low to non-existent. If any S.

543 aureus is present, the bacteria have to be activated in some fashion to awaken them from

544 their bacteriostatic state acquired during freezing (lag phase). We have incoming

545 sampling results from seven different suppliers from this study, 2011 data, and data from

pg 25 of 42
546 all of the COAs from one supplier. These suppliers are from four different countries.

547 Also cited are the studies in Italy and Africa (11, 31).

548 3) The thawing process is controlled at 20°C (68°F). The fact that the thawing process is

549 controlled at this temperature, rather than at 35-37°C (95°F - 98.6°F), means that the lag

550 phase will be extended.

551 4) The thawed racks or carts of bagged loins are normally staged in a chilled room,

552 controlled at ≤10°C (50°F), except during this test procedure.

553 5) The temperature in the packing room where filling, media addition, and seaming takes

554 place is controlled to ≤21.1°C (70°F). Under ideal constant conditions of atmosphere

555 pressure, pH, and water activity, according to Table 2 (from the ICMSF Table lb, the

556 FDA source for the 3-h limit), the lag time for S. aureus is 8 h. At 20°C (68°F), the

557 generation time (i.e., the time for a population to double) is 2.3 h. Starting with 103

558 CFU/g, it would take approximately 23 h to reach a level of 106 CFU/g, the minimum

559 level at which SE is formed in tuna meat (22).

560 Factors to consider for making a HACCP Critical Control Point and Critical Limit change

561 for this factory:

562 1) There are low levels of S. aureus on the incoming frozen loins that are thawed under

563 anaerobic conditions.

564 2) There is a fast transit time between opening the loin bags, and filling and sealing the cans,

565 creating yet another anaerobic environment.

566 3) There is an existing standard 2-h delay time rule operating limit after seaming to control

567 insipid spoilage, and a 3-h critical limit based on a California Regulation (10).

pg 26 of 42
568 4) In addition, the vacuum conditions in the sealed cans will slow S. aureus growth, so

569 including the time to get to growth inhibitory temperatures for S. aureus of 50°C (122°F)

570 in a 66-oz tuna can is, from a risk-based evaluation, unnecessary and too restrictive.

571 5) Other studies indicate that S. aureus is put under stress with fast changing environmental

572 temperatures, such as the core of the tuna can heating during venting and the initial stages

573 of retorting. This will also impede the growth rates of the S. aureus bacteria.

574 On the basis of review of the literature concerning the growth and enterotoxin production

575 by S. aureus, and the results of vendor and factory sampling of incoming frozen loins,factory

576 sampling of finished product, and in-line sampling and analysis that conservatively reflects

577 actual plant operating conditions, it appears that the formation of staphylococcal enterotoxin is a

578 hazard not likely to occur in a tuna loin processing operation such as this. That is, unless frozen,

579 precooked tuna loins used in canning are received heavily contaminated with S. aureus (greater

580 than 103 CFU/g), thawed to product temperatures of >25°C (77°F), and subjected to lengthy

581 holding times at temperatures of >25°C (77°F) prior to retorting, there is almost no likelihood of

582 staphylococcal enterotoxin being formed during the period from start of thawing until 50°C

583 (122°F) product temperature is reached in the retort. In the frozen tuna loin processing plant, the

584 tuna meat spends the majority of its time under low oxygen conditions. The loins are thawed

585 under anaerobic conditions in high-barrier bags, and the tuna meat is exposed to anaerobic

586 conditions after it is sealed in the can, being staged for retorting and being retorted. The only

587 time the tuna meat is exposed to aerobic conditions is while it is being loaded into the filling

588 machine and the media is added, a period of less than 2 h, including the product which is re-cut.

589 It is further recommended that any canned tuna processor, utilizing pre-cooked frozen

590 loins, pay special attention to the establishment of an effective data base of S. aureus counts on

pg 27 of 42
591 incoming frozen loins from each vendor. In the establishment of such a database, considerable

592 coordination needs to be affected between the vendor (supplier) of the frozen, pre-cooked loins

593 and the processor of canned product. Since the proliferation of S. aureus would indicate poor

594 sanitation in the frozen loin producing facility, a robust sanitation program is needed, focusing

595 on employee and product contact surface sanitation, from start of skinning to placement of the

596 loins in vacuum packaging. All this needs to be validated as a normal part of the Foreign

597 Supplier Verification Program (39). As part of this program, emphasis should also be placed on

598 the freezers in the loin manufacturing plant being capable of effecting a rapid drop in the bagged

599 loin temperature from 21.1°C (70°F) to below 10°C (50°F). Both benchmarks, a sanitation

600 program, and freezer capability, especially reaching 10°C (50°F) quickly, will significantly slow

601 the growth of any S. aureus present on the tuna meat (4, 7).

602 Accordingly, the authors recommend that in a facility processing canned tuna from

603 frozen loins, there should be a 5-h operating limit and a 7-h critical limit for the time from the

604 end of thawing, until start of retorting. For similar operations that utilize a chill room to

605 temporarily hold racks of loins previously at a maximum of 20°C (68°F) to await placement on

606 the conveyor belts feeding the filler, and that maintain temperatures of ≤70°F (21.1°C) in the

607 packing room, the operating and critical limits could likely, and reasonably, be extended beyond

608 the 7-h critical limit. For the foreseeable future, it is recommended that each operation that

609 packs precooked frozen loins and that utilizes control measures such as use of a chill room and a

610 processing room temperature controlled ≤21.1°C (70°F) would need to be evaluated

611 individually, from thawing to start of retorting, to extend the time beyond the 7-h critical limit

612 recommended.

613 ACKNOWLEDGMENTS

pg 28 of 42
614 This work was done in conjunction with the National Fisheries Institute Tuna Council

615 Technical Committee. William (Bill) Cole is a process authority with TechniCAL, New Orleans,

616 LA, and a former FDA process authority. We thank Dr. Mona Baumgartel for editorial

617 assistance with this manuscript. We thank Libby Davis for all of the sample collection, and

618 Bobbie Blaxton for arranging the shipments of the samples to the lab for microbiological

619 analysis. We thank Joe Vlasic of Silgan Holding for providing the analysis of the can headspace

620 gas.

pg 29 of 42
621 REFERENCES

622 1. Adams, F., F. Nolte, J. Colton, J. DeBeer, and L. Weddig. 2018. Precooking as a

623 Control for Histamine Formation during the Processing of Tuna: An Industrial Process

624 Validation. J. Food Protect. 81:444-455. https://doi.org/10.4315/0362-028X.JFP-17-

625 276

626 2. Baeza, R., C. Rossler, D. Mielnicki, M. Zamora and J. Chirife. 2009. Theoretical

627 modelling of Staphylococcus aureus growth in a cooked meat product kept at ambient

628 temperature using temperature profiles of selected Mexican cities. Ciência e Tecnologia

629 de Alimentos. 29:81-84. https://doi.org/10.1590/S0101-20612009000100013

630 3. Baeza, R., Rossler, C. E., Mielnicki, D. M., Zamora, M. C., and Chirife, J. 2007.

631 Simplified prediction of Staphylococcus aureus growth in a cooked meat product

632 exposed to changing environmental temperatures in warm climates. Rev. Arg.

633 Microbiol. 39:237-242.

634 4. Baird-Parker, T. C. 2000. Staphylococcus aureus in The Microbiological Safety and

635 Quality of Foods, Vol II. B.M. Lund, T. C. Baird-Parker, G.W. Gould (Eds). Aspen

636 Publishing Company; Gaithersburg, MD. 1317-1355.

637 5. Belay, N., and A. Rasooly, 2002. Staphylococcus aureus growth and enterotoxin A

638 production in an anaerobic environment. J. Food Prot. 65:199-204.

639 6. Bennett, R.W., J. M. Hait and S. M. Tallent. 2015. Staphylococcus aureus and

640 staphylococcal enterotoxin, Chap 39, p. 509 – 526. in Compendium of Methods for the

641 Microbiological Examination of Foods, 5th Edition (Eds: Y. Salfinger and M. L.

642 Tortorello). APHA Press, Washington, D.C.

pg 30 of 42
643 7. Bryan, F. L. 1976. Staphylococcus aureus in Food Microbiology: Public Health and

644 Spoilage Aspects. M.P. Defigueiredo and D.F. Splittstoesser (Eds.). Avi Publishing

645 Company, Inc. Westport, CT. 12-128

646 8. Bryan, F. L. 1980. Epidemiology of foodborne diseases transmitted by fish, shellfish and

647 marine crustaceans in the United States, 1970–1978. J. Food Prot. 43:859-876.

648 https://doi.org/10.4315/0362-028X-43.11.859

649 9. Cabes, S. Dwell / Lag Time TechniCAL (The Thermal Process Authority) Available at:

650 https://tcal1.zendesk.com/hc/en-us/articles/223757568-Dwell-Time-Lag-Time Accessed:

651 Oct 6, 2018.

652 10. California Code of Regulations, Title 17, Cannery Inspection Regulations, Section

653 §12979. Available at:

654 https://www.cdph.ca.gov/Programs/CEH/DFDCS/CDPH%20Document%20Library/FDB

655 /FoodSafetyProgram/Cannery/CACanneryRegulations.pdf Accessed Oct 7, 2018.

656 11. Casalinuovo, F., D. Brindisi, P. Rippa, C. Ceniti, L. Ciambrone, R. Musarella, and N.

657 Costanzo. 2018. Microbiological Quality and Safety of Skipjack Tuna Loins

658 (Katsuwonus pelamis) Intended for Canning. Mac Vet Rev. 41:33-37.

659 https://doi.org/10.1515/macvetrev-2017-0028

660 12. DeBeer, J. – personal observations – [john.debeer@thaiunion.com]

661 13. DeBeer, J., F. Nolte, and C. W. Lord. 2015. Precooking tuna: a study of the factors

662 impacting the time required for precooking. Food Prot. Trends. 35:448-460.

663 14. DeBeer, J., F. Nolte, C. W. Lord, J. Colton, and J. Colley. 2017. Sampling plans to

664 determine the minimum core temperature reached during the precooking of tuna. Food

665 Prot. Trends. 37:269–288.

pg 31 of 42
666 15. DeBeer, J., F. Nolte, C. W. Lord, J. Colton, J. Colley, and L. Weddig. 2017. A strategy

667 for controlling histamine formation at tuna precooking. Food Prot. Trends. 37:340–352.

668 16. Flair Flexible Packaging Corporation. 2018. Available at:

669 http://www.flairpackaging.com/pages/gourmet_snacks_treats_flair_flexible_packaging/re

670 sources/packaging101_sustainable/Oxygen%20Transfer%20Rate%20(OTR)/2 Accessed

671 Nov 13, 2018.

672 17. Fox, J. and D. Holtman. 1968. Effect of anaerobiosis on Staphylococcal nuclease

673 production. J. Bacteriol. 95:1548-1550.

674 18. Grocery Manufacturer’s Association. 1996. Thermal process for low-acid foods in metal

675 containers. Bulletin 26-L. 13th Edition.

676 19. Hennekinne, J., M. De Buyser and S. Dragacci. 2012. Staphylococcus aureus and its

677 food poisoning toxins: Characterization and outbreak investigation. FEMS Microbial

678 Rev. 36:815-836. https://doi.org/10.1111/j.1574-6976.2011.00311.x

679 20. International Commission on Microbiological Specifications for Foods (ICMSF) 1996.

680 Staphylococcus aureus in Microorganisms in Foods 5 – Characteristics of Microbial

681 Pathogens. Blackie Academic & Professional, London. 299-333.

682 21. Jay, J. M., M. J. Loessner, and D. A. Golden. 2005. Staphylococcal gastroenteritis in

683 Modern Food Microbiology. Springer Science and Business Media, Inc, New York, NY.

684 545-566.

685 22. Kataoka, A., E. Enache, C. Napier, M. Hayman and L. Weddig. 2016. Effect of storage

686 temperature on the outgrowth and toxin production of Staphylococcus aureus in freeze-

687 thawed precooked tuna meat. J. Food Prot. 79:620-627. https://doi.org/10.4315/0362-

688 028X.JFP-15-439

pg 32 of 42
689 23. Kluytmans, J., A. Van Belkum, and H. Verbrugh. 1997. Nasal carriage of

690 Staphylococcus aureus: epidemiology, underlying mechanisms, and associated Risks.

691 Clin. Microbiol. Rev. 10:505-520. https://doi.org/10.1128/CMR.10.3.505

692 24. Minor, T. E., and E. H. Marth. 1972. Staphylococcus Aureus and Staphylococcal Food

693 Intoxications. A Review: IV. Staphylococci in Meat, Bakery Products, and Other

694 Foods. J. Milk and Food Tech. 35:228-241. https://doi.org/10.4315/0022-2747-35.4.228

695 25. National Fisheries Institute. 2013. Canned_Tuna_HACCP - Example 1. Available at:

696 https://www.aboutseafood.com/about/canned-tuna-haccp-4/ Accessed Sep 28, 2018

697 26. National Fisheries Institute. 2013. Canned_Tuna_HACCP - Example 2. Available at:

698 https://www.aboutseafood.com/about/canned-tuna-haccp-4/ Accessed Sep 28, 2018

699 27. National Fisheries Institute. 2013. Canned_Tuna_HACCP - Example 3. Available at:

700 https://www.aboutseafood.com/about/canned-tuna-haccp-4/ Accessed Sep 28, 2018

701 28. Pflug, I. J. 2010. Science, practice and human errors in controlling Clostridium

702 botulinum in heat-preserved food in hermetic containers, J. Food Prot. 73:993-1002.

703 https://doi.org/10.4315/0362-028X-73.5.993

704 29. Schelin, J., N. Wallin-Carlquist, M. Cohn, R. Lindqvist, G. Barker and P. Radstrom.

705 2011. The formation of Staphylococcus aureus enterotoxin in food environments and

706 advances in risk assessment. Virulence 2:580-592.

707 https://doi.org/10.4161/viru.2.6.18122

708 30. Schwabe, M., S. Notermans, R. Boot, S. Tatini and J. Kramer. 1990. Inactivation of

709 staphylococcal enterotoxins by heat and reactivation by high pH treatment. Int. J. Food

710 Microbiol. 10:33-42. https://doi.org/10.1016/0168-1605(90)90005-P

pg 33 of 42
711 31. Sika, E., Y. Akeassi, T. Anoman, R. Koffi-Nevry, and H. Biego. 2014. Evaluation of

712 microbiological quality of tuna loins (Thunnus sp.) produced in Côte D’ivoire. Am. J

713 Adv. Sci. Res. 2:90-103.

714 32. U.S. Center for Disease Control. 2016. Staphylococcal food poisoning. Available at

715 https://www.cdc.gov/foodsafety/diseases/staphylococcal.html. Accessed 22 April 2018.

716 33. U.S. Food and Drug Administration. 2001. BAM: Staphylococcus aureus. Available at

717 https://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm071429.htm.

718 Accessed 22 April 2018.

719 34. U.S. Food and Drug Administration. 2011. Fish and fishery products hazards and

720 controls guidance. Available at https://www.fda.gov/downloads/Food

721 /GuidanceRegulation/UCM251970.pdf. Accessed 10 June 2018.

722 35. U.S. Food and Drug Administration. 2012. Response of 05/10/2012 to TechniCAL

723 Freedom of Information Act request dated April 3, 2012, for scientific documentation

724 supporting 12-hour limit for controlling histamine; and 3 h our limit for controlling

725 growth and toxin formation by Staphylococcus aureus.

726 36. U.S. Food and Drug Administration. 2012. Warning Letter for General Tuna

727 Corporation. Available at https://www.fdalabelcompliance.com/letters/ucm300077.

728 Accessed Dec 8, 2018.

729 37. U.S. Food and Drug Administration. 2014. Warning Letter for Southeast Asian

730 Packaging and Canning Limited. Available at; https://www.fda.gov/ICECI

731 /EnforcementActions/WarningLetters/ucm399641.htm Accessed Dec 8, 2018.

732 38. U.S. Food and Drug Administration. 2014. Warning Letter for Starkist (Ecuador)

733 Available at: https://www.fda.gov/ICECI/EnforcementActions/WarningLetters

734 /ucm425883.htm Accessed Dec 8, 2018.

pg 34 of 42
735 39. U.S. Food and Drug Administration. 2015. Foreign Supplier Verification Programs for

736 Importers of Food for Humans and Animals. Available at: https://www.fda.gov/food

737 /guidanceregulation/fsma/ucm361902.htm Accessed Dec 15, 2018.

738 40. U.S. Food and Drug Administration. 2015. Warning Letter to Procesamiento

739 Especializado De Alimentos. Available at :

740 https://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2015/ucm461662.htm.

741 Accessed: Oct 11, 2018.

742 41. U.S. Food and Drug Administration. 2016. Warning Letter to Unicord Public Company.

743 Available at:

744 https://www.fda.gov/ICECI/EnforcementActions/WarningLetters/2016/ucm487443.htm.

745 Accessed Oct 11, 2018.

746 42. U.S. Food and Drug Administration. 2017. 21CFR123 Fish and Fishery Products.

747 Available at

748 https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?CFRPart=123.

749 Accessed 10 June 2018.

750 43. U.S. Food and Drug Administration. 2018. 21CFR117. Current Good Manufacturing

751 Practice, Hazard Analysis, And Risk-Based Preventive Controls for Human Food.

752 Available at: https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm

753 ?CFRPart=117 Accessed Nov 26, 2018.

754 44. Wu, X. and Y. Su. 2013. Preliminary study of growth of Staphylococcus aureus in

755 cooked tuna meat. Unpublished progress report – Jan 17, 2013. Available in J. DeBeer’s

756 personal library.

pg 35 of 42
757 45. Wu, X. and Y. Su. 2014a. Growth of Staphylococcus aureus and enterotoxin production

758 in pre-cooked tuna meat. Food Control 42:63-70.

759 https://doi.org/10.1016/j.foodcont.2014.01.039

760 46. Wu, X. and Y. Su. 2014b. Effects of frozen storage on survival of Staphylococcus aureus

761 and enterotoxin production in precooked tuna meat. J. Food Sci. 79:M1554-M1559.

762 https://doi.org/10.1111/1750-3841.12530

763

pg 36 of 42
764 FIGURE LEGENDS

765 Figure 1 – Temperatures by the hour after the tuna loins were removed from the frozen storage.

766 Figure 2. Maximum S. aureus (CFU/g) in skipjack, hold time in loins, and open and seamed

767 (closed) cans

768 Figure 3. Maximum S. aureus (CFU/g) in albacore, hold time in loins, and open and seamed

769 (closed) cans.

770

pg 37 of 42
771 Table 1 – FDA’s HACCP Guidelines – from Appendix 4, Table A-2 (34)

Growth and Toxin Formation 45 - 50°F (7.2-10°C) 14 days

by Staphylococcus aureus 51-70°F (10.5-21.1°C) 12 h

Above 70°F (21.1°C) 3h

772

773

774 Table 2 – Guidelines from Table 1b, page 307, ICMSF 5 (20)

775 Hours to reach 106 CFU/gm starting at different concentrations of S. aureus under aerobic
776 conditions
Hours to
reach 106
Growth CFU/g Hours to reach
Rate Log starting 106 CFU/g
(Gen. time with 100 starting with
Substrate °C Lag (h) h) Parameters CFU/g 1000 CFU/g
Steak & 13 2.1 d 8.7 Aerobic 116 87
Kidney 15 12 4.9 65 49
Pie – 21 8 2.3 Starting 31 23
Pasteurize 25 2.8 1.2 at 103 16 12
d 30 3.4 37.1 min CFU/gm 8 6
37 1.9 24.6 min pH – 5.9 5 4
41.5 1.5 23.2 min Aw – 0.98 5 4
44 2.2 38.7 min 9 6
777
778

pg 38 of 42
779 Table 3 – Microbiological results from COAs from frozen loins

Negative Positive Highest

Staph. Staph. value -
Year N aureus aureus CFU/gm
2011 4,490 4,312 178 85
2012 5,265 5,265 0
2013 3,702 3,675 27 80
2014 3,606 3,606 0
2015 2,595 2,595 0
2016 3,354 3,351 3 10
2017 4,023 4,017 6 70
2018 1,687 1,687 0
Total 28,722 28,508 214 85
780 99.3% 0.7%
781 This is data from a single loin supplier
782

783 Table 4. Microbiological results from testing thawed

784 tuna loins in process for S. aureus in 2011.

Collection N <10 > 10 Highest

location CFU/g CFU/g CFU/g
Receiving 54 44 10 70

At Packing 54 42 12 360
machine
Just prior to 54 40 14 220
retorting
785
786 This data is collected from 4 loin suppliers

pg 39 of 42
 789
788
787

al Elapsed Estimated Ambient Area Vacuum Aerobic Container

r Time core temperat
temperat ure °C
ure °C
0 0 -20 20 Racking Vacuum Loin bag
0 1 -16.5 20 Thawing Vacuum Loin bag
0 2 -13 20 Thawing Vacuum Loin bag
0 3 -9.5 20 Thawing Vacuum Loin bag
0 4 -6 20 Thawing Vacuum Loin bag
0 5 -2.5 20 Thawing Vacuum Loin bag
0 6 1 20 Thawing Vacuum Loin bag
0 7 4.5 20 Thawing Vacuum Loin bag
0 8 8 20 Thawing Vacuum Loin bag
0 9 11.5 20 Thawing Vacuum Loin bag
0 10 15 21 Pack Room Vacuum Loin bag
0 11 16 21 Pack Room Vacuum Loin bag
0 12 17 21 Pack Room Vacuum Loin bag
0 13 19 21 Pack Room Aerobic Loin bag
0 14 19 21 Pack Room Aerobic Loin bag
Table 5 – Representative time and temperature sequence of loin and canned samples

0 15 19 21 Filling Aerobic Cans

0 16 19 26 Retort Area Vacuum Aerobic Cans

pg 40 of 42
0 17 22 26 Retort Area Vacuum Aerobic Cans
0 18 22 26 Retort Area Vacuum Aerobic Cans
0 19 23 26 Retort Area Vacuum Aerobic Cans
0 20 23 26 Retort Area Vacuum Aerobic Cans
0 21 23 26 Retort Area Vacuum Aerobic Cans
790 Table 6 - Results of sampling in a tuna factory for Staphylococcus aureus and APC
Maximum Maximum
Staph – Aerobic
Medium N Item Sampled Max Delay Time Coagulase Plate Count
Positive - (APC)
Petri film CFU/g
CFU/g
Enviro swab 21 Totes or tubs 8h <10 >2.5x105
Enviro swab 15 Tri-belts 8h <10 >2.5x105
Veg Broth–VB82 22 On Line <10 >2.5x105

Veg Broth-VB88 16 On Line 9.8x102 >2.5x105

Albacore 48 Loins in bags 0h <10 >2.5x105
Albacore 48 Flakes in bags 0h <10 >2.5x105
Skipjack 46 Loins in bags 0h 5.8 x102 1.2 x105
Albacore 29 Open Cans 3 h after seamer 5.0 x101 >2.5x105
Albacore 29 Open Cans 5 h after seamer <10 >2.5x105
Skipjack 10 Open Cans 3 h after seamer 7.2 x103 1.1 x105

Skipjack 10 Open Cans 5 h after seamer 2.0 x104 2.4 x105

Albacore 40 Closed Cans 3 h after seamer <10 >2.5x105

Albacore 40 Closed Cans 5 h after seamer <10 >2.5 x105

Skipjack 20 Closed Cans 3 h after seamer 4.6 x102 >2.5x105
Skipjack 20 Closed Cans 5 h after seamer 7.2 x102 >2.5x105

791

792

pg 41 of 42
793 Table 7 – Results of Headspace analysis of 7 oz cans, vacuum was 8” – 10” Hg (3.9 – 4.9 psi)

Gas % - min and max

O2 2.1 – 2.3

H2 1.3 – 2.1

N2 83.7 – 85.8

CO2 7.6 – 8.2

794

pg 42 of 42
 Figure 1

35
Anaerobic Environment Aerobic Anaerobic
Envirnment Environment
25

15 Retort area
Temperature °C

Packing room
5

-5

Estimated Core Temperature

-15 Ambient Temperature

-25
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Elapsed hours
 Figure 2

100,000

10,000
Max Staph. aureus, CFU/g

1,000

100

10

Open Sealed
1
0 1 2 3 4 5 6
Hours Hold time
 Figure 3

100,000

10,000
Max Staph. aureus, CFU/g

open closed
1,000

100

10

1
0 1 2 3 4 5 6
Hours Hold time