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Manuscript_f5322185b9fc8cb9acde2c23b9df4416

1 Effects of tyndallization temperature on the sterility and quality of

2 kamaboko

4 Sumate KERATIMANOCH a ,Kigen TAKAHASHI a , Takashi KUDA a , Emiko

5 OKAZAKI a , Jie-Ting GENG a ,Kazufumi OSAKO* , a

7 Department of Food Science and Technology, Tokyo University of Marine Science

8 and Technology, 4-5-7, Konan, Minato-ku, Tokyo, 108-8477, Japan

10 *To whom correspondence should be addressed.

11 Phone: +81-3-5463-0620; Fax: +81-3-5463-0620;

12 e-mail: osako@kaiyodai.ac.jp

13

14 Present address:

15 a: Department of Food Science and Technology, Tokyo University of Marine

16 Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo, 108-8477, Japan,

17

18 Mailing address:

19 Kazufumi Osako,

20 Tokyo University of Marine Science and Technology, 4-5-7, Konan, Minato-ku, Tokyo,

21 108-8477, Japan.

22 Phone: +81-3-5463-0620; Fax: +81-3-5463-0620;

23 e-mail: osako@kaiyodai.ac.jp

24

25

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© 2021 published by Elsevier. This manuscript is made available under the Elsevier user license
https://www.elsevier.com/open-access/userlicense/1.0/
26 ABSTRACT

27 In this study, kamaboko gels were tyndallized at various temperatures and

28 sterilization efficiency and impact on quality parameters were assessed. The

29 microbiological, physical, and chemical properties of kamaboko gels were

30 determined throughout the tyndallization process. Superior sterilization efficiency

31 was achieved by tyndallization at a higher temperature; and the combination of

32 heat-induced germination and thermal inactivation of spores was proposed as the

33 main reason. The process had minimal effect on the color of gels. While tyndallized

34 gels heated at 80 °C possessed superior physical properties, all gels showed

35 impaired quality with the progress of heating cycles. Sodium dodecyl

36 sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the cause of

37 alterations in quality differed depending on the processing temperature. This study

38 suggests that the sterility of products could be improved by increasing the

39 processing temperature, time or number of heating cycle.

40

41

42 Key words: Tyndallization, kamaboko, induced germination, sterilization, seafood

43

44

45

46

47

48

49

50

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51 1. INTRODUCTION

52 Many food manufacturers aim to maximize the shelf-stability of their products in

53 order to facilitate storage and distribution. Thus, commercial sterilization of

54 products is necessary. There are various methods to sterilize food, but the most

55 common method currently used in the food industry is thermal sterilization (Gould,

56 2006). Thermal sterilization involves the application of heat treatment, raising the

57 product temperature to more than 110 °C for a predetermined period, to ensure the

58 complete elimination of target microorganisms, usually thermoresistant bacterial

59 spores. While this process is common, it can be cost-prohibitive to small

60 manufacturers, as it is relatively expensive in terms of equipment, operation, and

61 packaging costs and requires large production volumes to be economically viable.

62 Tyndallization, also known as fractional sterilization, intermittent sterilization,

63 germination-inactivation, or induced germination, is an alternative sterilization

64 method. This process involves heating the substance for multiple times with a

65 resting period between cycles (Tyndall, 1877). In classic tyndallization, heating

66 process is applied three times (Brown, Wiles, & Prentice, 1979; Kim et al., 2012).

67 The principle of this sterilization method is to eliminate microbes while they are in

68 the vegetative form. The application of heat destroys the vegetative cells of bacteria,

69 and the used temperature does not need to exceed 100 °C. This is comparable to

70 pasteurization which thermoresistant spores can withstand this process. The spores

71 are then allowed to germinate during the storage period and are killed by the next

72 heat application. Since vegetative cells are much easier to kill than endospore

73 counterpart (Brown, 2000), the use of a pressurized system to achieved high

74 temperature can be omitted, substantially decreasing the investment and operation

75 costs. In addition, by using a lower processing temperature, the product quality, i.e.,

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 76 texture, flavor, appearance, and nutrition, is less affected by heat damage

77 (Barbosa-Cánovas, Medina-Meza, Candoǧan, & Bermúdez-Aguirre, 2014).

78 Therefore, tyndallization has potential as an alternative sterilization method that is

79 more affordable for small manufacturers and better at preserving product quality.

80 Spore germination, together with hermetically sealed packaging, plays a crucial

81 role in effective tyndallization. Although some types of spores can germinate

82 spontaneously, they typically germinate in response to germinants, which can be

83 nutrient or non-nutrient, in the environment (Setlow, 2003). Examples of nutrient

84 germinants are single amino acids, sugars, purine nucleosides, or mixtures of

85 asparagine, glucose, fructose, and potassium ions (AGFK); while non-nutrient

86 germinants are lysozyme, calcium dipicolinate, application of high pressure, salts,

87 and cationic surfactants etc. In addition, germination rates can be improved by

88 exposing bacterial spores to sublethal heat (Luu et al., 2015; Zhang & Mathys, 2019).

89 However, excessive heating can lead to sub-optimal germination rates in some cases

90 (Ghosh, Zhang, Li, & Setlow, 2009; Krawczyk et al., 2017). With these facts known,

91 it is possible to exploit the various relevant factors to maximize the effectiveness of

92 tyndallization.

93 Surimi is a seafood product that can be made from many kinds of fish, with

94 Alaska Pollock being the most common (Park, 2005). Typically, the manufacturing

95 process includes the additional of sucrose as the cryoprotectant. Thus, kamaboko, a

96 product made from surimi, contains nutrient suitable for spore germination.

97 Moreover, intensive heat application has detrimental effects on quality parameters

98 such as gel strength and water holding capacity (Zhang, Xue, Xu, Li, & Xue, 2013;

99 Zhang, Xue, Li, Wang, & Xue, 2015). For these reasons, sterilization by

100 tyndallization could be more suitable to maintain the quality of kamaboko gel.

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101 Furthermore, there have been many studies focusing on sterilization by means of

102 germination induction strategies. While these processes have proven effective in

103 improving inactivation of bacterial spores, not all were capable of achieving

104 complete sterility (Mesquita, Teixeira, & Brandao, 1998; Brown et al., 1979; Cho,

105 Yousef, & Sastry, 1999; Kim et al., 2012; Loøvdal, Hovda, Granum, & Rosnes, 2011).

106 In addition, these studies involved only liquid broth or liquid food models. The

107 present study aimed to investigate the effectiveness of tyndallization at various

108 temperatures for sterilizing kamaboko gel and its impact on product quality. It is

109 expected that this study will provide new perspectives on the utilization of this

110 process for semi-solid foods and expand knowledge of the application of

111 tyndallization.

112

113 2. MATERIALS AND METHODS

114 2.1. Spore preparation

115 The spore preparation method was slightly adapted from Soni, Oey, Silcock, &

116 Bremer (2018). Firstly, Bacillus subtilis strain ATCC 6633 was obtained from

117 National Institute of Technology and Evaluation Biological Resource Center (Tokyo,

118 Japan) in dried form which was rehydrated and then plated with revival kit

119 (rehydration fluid 702, growth media 802) from FUJIFLIM Wako Chemicals Corp.

120 (Osaka, Japan). After incubation at 30 °C for 48 hours, a loopful of culture was

121 inoculated into 100 mL tryptic soy broth supplied with MnSO 4 0.4 g/L and incubated

122 at 30 °C in the shaker (BW201, Yamato Scientific Co. Ltd., Tokyo, Japan) at 100

123 rpm for 5 days. Next, the spores were harvested by centrifugation at 8,000 g 4 °C for

124 15 minutes and the precipitated bacteria cells were washed three times with sterile

125 ion exchanged water (IEW). The cells from multiple flasks were combined and

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126 resuspended in 30 mL sterile IEW. Finally, the suspension was heated in 80 °C water

127 bath for 15 minutes to kill the remaining vegetative cells then stored at 4 °C until

128 use. The spore count of the suspension was determined with the method explained

129 later.

130

131 2.2. Kamaboko gel preparation and inoculation

132 FA grade frozen surimi (Trident Seafoods Corporation, Seattle, USA) was thawed

133 overnight at 4 °C prior to use. Surimi was chopped into smaller cubes before mixing

134 with IEW at 1500 rpm for 1.5 minutes (UMC-5, Stephan Machinery Corp., Hamelin,

135 Germany). Next, NaCl was added to the mixture and grinded for 1.5 minutes

136 followed by addition of B. subtilis spore suspension and mixed for 1 more minute.

137 Kamaboko gel was formulated to have final moisture content of 80 % with 2 %

138 (w/w) NaCl and approximately 6 log CFU/g spore count. The vacuum pressure was

139 kept lower than 90 kPa during every mixing step. About 30 g of surimi mixture was

140 then extruded into 23 mm diameter plastic tubes and double sealed on both ends.

141 Afterwards, they were heated for 30 minutes in 40 °C water bath. These kamaboko

142 gels were considered as control sample and proceeded to tyndallize immediately.

143

144 2.3. Tyndallization procedure

145 Kamaboko gels were subjected to tyndallization using 60, 70, 80, and 90 °C heat

146 treatment. This was done by immersing prepared kamaboko gel under hot water bath

147 (BZ100D, Yamato Scientific Co. Ltd., Tokyo, Japan) at designated temperatures.

148 The temperature at the geometric center of kamaboko gels was monitored by

149 thermocouples (RTR-52A, T & D Corp., Nagano, Japan). Once the core temperature

150 of kamaboko gels reached the process temperature, gels were kept immersed for 10

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151 more minutes before cooling with running tap water until their temperatures were ≤

152 40 °C. The heat-treated kamaboko gels were incubated at 30 °C for 24 hours before

153 the next heating cycle. The whole process was repeated two more times. Kamaboko

154 gels were sampled before and after each heating session for microbiological

155 enumeration and sampled after each heating session for other measurements.

156

157 2.4. Total bacteria count (TBC) and spore count (SC)

158 Twenty g of sample was aseptically homogenized with 180 mL sterilized

159 phosphate buffer saline (As One Corp., Osaka, Japan) in sterile stomacher bag (As

160 One Corp., Osaka, Japan) with Polytron homogenizer (PT10-35GT, Kinematica AG,

161 Luzern, Switzerland) at 6,000 rpm for 30 seconds under ice slurry. The homogenate

162 was homogenized again for 2 minutes using stomacher (Basic, IUL S.A., Barcelona,

163 Spain) to ensure homogeneity. One mL of diluent from the proper dilution with

164 sterilized phosphate buffer saline were inoculated on Compact Dry TC plate (Nissui

165 Pharmaceutical Co. Ltd., Irabaki, Japan) for determining TBC. For SC, the diluents

166 were heated in sterile eppendorf tube at 80 °C for 15 minutes prior to inoculation

167 (Kim et al., 2014). The inoculated plates were incubated at 30 °C for 48 hours and

168 the forming colonies were enumerated. The number of colony forming unit per 1 g of

169 sample were calculated and converted into log CFU/g.

170

171 2.5. Color and whiteness

172 The color of the kamaboko gels was measured in CIELAB color space (L*, a*,

173 b*) using colorimeter (CR13, Konica Minolta, Osaka, Japan). The whiteness index

174 were calculated by using the obtained color parameters using the following equation

175 (Klomklao, Benjakul, Kishimura, Osako, & Simpson, 2016):

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176 Whiteness index = 100 – [(100-L*) 2 + a* 2 + b* 2 ] 1 / 2

177

178 2.6. pH

179 pH of kamaboko gels were measured according to method of Paludan-Müller,

180 Henrik Huss, & Gram (1999). The samples were homogenized with the same weight

181 of IEW. The pH of the homogenates was then determined by using pH meter

182 (LAQUA F-72, Horiba Ltd., Kyoto, Japan)

183

184 2.7. Expressible moisture content (EMC)

185 To determine the EMC, the procedure slightly adapted from Jia et al. (2018) was

186 carried out. An approximately 2 mm thick of kamaboko gel slice was weighed (W i )

187 and then force of 10 N was applied to kamaboko slice with two pieces of filter

188 papers on top and two on the bottom for 20 seconds using creep meter (RE-3305,

189 Yamaden Ltd., Tokyo, Japan). The weight of compressed kamaboko slice was

190 obtained (W f ). EMC was calculated as follow:

191

192 EMC (%) = (W i – W f )/W i × 100%

193

194 2.8. Textural properties

195 Texture profile analysis (TPA) (Jia et al., 2018) and puncture test (Jia et al., 2019)

196 were performed to assess the textural properties of the kamaboko gels. The

197 rheometer RE2-33005B (Yamaden Ltd., Tokyo, Japan) was used for both tests. For

198 TPA, samples with height of 25 mm were compressed by 30 mm diameter

199 compression platen at the speed of 1 mm/s and target stress of 30%. Hardness (N),

200 cohesiveness, adhesiveness (J/m 3 ), gumminess (N), and springiness were obtained as

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201 texture parameters.

202 The same sample height and compression speed were used for puncture test.

203 However, 5 mm spherical plunger was used instead. The plunger moved toward the

204 sample until past the gel breakage and the penetration force at breakage was taken as

205 breaking force (N). Breaking deformation (%) was calculated as the percentage of

206 plunger distance at breakage to the initial height of kamaboko gel.

207

208 2.9. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and

209 quantification of Myosin heavy chain (MHC) and actin band intensity

210 The SDS-PAGE pattern of kamaboko gels was analyzed using the technique

211 described by Jia et al. (2019) with minor alterations. Briefly, 0.5 g of samples were

212 homogenized with 2% SDS (w/v), 8 M urea, 20 mM Tris-HCl (8.8) with addition of

213 β-mercaptoethanol at 2 %(v/v), heated in boiling water for 2 minutes, and then

214 shaken overnight. The concentrations of protein in the solutions before and after

215 centrifugation at 10,000 g for 20 minutes according to the Lowry method (Lowry,

216 Rosebrough, Farr, & Randall, 1951) were compared to determine the solubility of

217 samples in percentage. Then, the dissolved samples containing 10 μg of protein were

218 then loaded into each well of gradient 5 – 20 % polyacrylamide gel (e-PAGEL, Atto

219 Co., Ltd., Tokyo, Japan). Electrophoresis was performed under 20 mA, 250 V, and

220 10 W condition. The polyacrylamide gel was stained with 0.05 % (w/v) Coomassie

221 Brilliant Blue R-250 in 3:1:6 volume ratio methanol: acetic acid: IEW and

222 subsequently destained with 3:1:6 volume ratio methanol : acetic acid : IEW.

223 Molecular weight maker ranging 10 – 200 kDa (PageRuler, Thermo Fisher Scientific

224 Inc., Tokyo, Japan) was used as a reference. The images of the gels were taken and

225 processed by CS Analyzer 3.0 software (Atto Co., Ltd., Tokyo, Japan) to quantify

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226 the band intensity of MHC and actin band. The values were expressed as relative

227 band intensity (%) which was calculated by comparing the intensity of the interested

228 band of each sample to the same protein band of control sample in percentage.

229

230 2.10. Statistical analysis

231 All experiments were conducted at least in quadruplicate. The data was expressed

232 as means ± standard deviations. The significance differences of the results were

233 tested at significance level of 0.05 using Duncan test by EXCEL XLSTAT 2019

234 (Addinsoft Inc., New York, USA).

235

236 3. RESULTS AND DISCUSSION

237 3.1. Total bacterial count and spore count

238 Microbiological enumeration results are shown in Figure 1. The initial TBC and

239 SC of kamaboko gel were 5.87 ± 0.15 and 4.815 ± 0.22 log CFU/g, respectively. The

240 1 log difference between the initial TBC and SC indicates spore germination during

241 kamaboko gel preparation. It is known that spores can respond to nutrient

242 availability promptly and germinate; however, this varies greatly among individual

243 spores and some spores can remain dormant (Zhang & Mathys, 2019).

244 As expected, SC prior to heat treatment was close to TBC after heat treatment of

245 the same heating cycle for the 60, 70, and 80 °C treatments. Although the

246 thermotolerance of bacterial spores can vary depending on the media, these heat

247 treatment temperatures are not considered capable of inactivating B. subtilis spores

248 (Montville, Dengrove, De Siano, Bonnet, & Schaffner, 2005). In the case of 90 °C, a

249 3 log reduction of SC was observed, which is reasonable since decimal reduction of

250 B. subtilis spores at 90 °C was reported to range between 4.2-12.3 minutes

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251 (Montville et al., 2005).

252 The effect of heat-induced germination was observed, i.e., as the processing

253 temperature increased, SC decreased. Before the second heat treatment, SCs in

254 samples were 1.38 ± 0.94, 1.59 ± 0.12, 0.83 ± 0.57, and 0 log CFU/g for 60, 70, 80,

255 and 90 °C treatments, respectively. The trend continued with subsequent heating

256 cycles, and by the end of the process, no spores were detected in the kamaboko gels

257 obtained from 90 and 80 °C tyndallization, while SCs in the 70 and 60 °C

258 tyndallized gels were 0.74 ± 0.84 and 1.15 ± 0.17 log CFU/g. In addition, the

259 difference in TBC and SC after heat treatment indicates that spore germination was

260 initiated during the cooling process since these heating conditions should be able to

261 eliminate all vegetative cells and TBC and SC would be identical if the bacterial

262 spores remained ungerminated. In general, a higher temperature leads to a higher

263 germination rate (Ghosh et al., 2009; Krawczyk et al., 2017) and the results

264 supported this trend. This effect can be most clearly seen when comparing 60 and

265 70 °C processes where the spores were least affected by thermal destruction.

266 Although a dramatic decrease in SC was observed in the first cycle of 60 °C

267 tyndallization, SC remained unchanged even with two subsequent heating cycles.

268 When the process temperature was increased to 70 °C, after the second heat

269 treatment, more spores were germinated, resulting in a lower SC before the third

270 cycle of tyndallization. The decreasing values of TBC and SC after the first heat

271 treatment when the process temperature was increased from 60 °C to 70 and 80 °C

272 could be another indication of higher heat-induced germination by increasing

273 process temperature. Nevertheless, Brown et al. (1979) reported the ineffectiveness

274 of tyndallization for milk products, which contradicts the result of this study,

275 despite the similar heating condition and the same incubation time. The

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276 contradictory results may be due to differences in the nutrients present in the raw

277 material. Ultimately, in this study, sterility was achieved through 90 °C

278 tyndallization only; however, it should be noted that this was a result of the

279 combined effects of thermal destruction and induced germination. For processing at

280 80 °C, although some bacteria survived, it is highly likely that an additional heating

281 cycle would have resulted in complete destruction of bacteria, since the number of

282 spores in the kamaboko gels in the last processing cycle was at an undetectable

283 level.

284

285 3.2. Kamaboko gel color and whiteness

286 The appearance of kamaboko gels changed from translucent grayish to opaque

287 white after heat treatment, resulting in significant increases (p < 0.05) of L*, b*, and

288 whiteness (Table 1). In contrast, a* of kamaboko gels was unaffected by processing.

289 The yellowness indicator, b*, was found to be highest at the third cycle of 70 °C,

290 followed by 60 °C at the same heating cycle. It is possible that at these

291 temperatures heat stable proteases had not been inactivated, resulting in increased

292 amino acid concentration (Park, 2005) and, hence, a higher degree of Maillard

293 reaction. The L* and whiteness had the same increasing trend with increasing

294 processing temperature. Higher temperatures result in a higher degree of

295 denaturation, which contributes to the kamaboko gel whiteness (Benjakul,

296 Visessanguan, & Srivilai, 2001). However, there was no significant difference of L*

297 and whiteness at the same processing temperature (p ≥ 0.05). This suggests that the

298 main factor affecting whiteness was the processing temperature, not the consecutive

299 application of heat.

300

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301 3.3. pH

302 The pH of kamaboko gels ranged between 6.79 – 6.91. Although some statistically

303 significant differences were observed (p < 0.05), the magnitude of the changes was

304 small with no clear trend despite the presence of microbiological activity during the

305 incubation period.

306

307 3.4. Expressible moisture content

308 Before heat treatment, the EMC of kamaboko gel was 4.03 % (Table 1) and this

309 content increased as the tyndallization process progressed. EMC showed the greatest

310 change when the kamaboko gel was exposed to 60 °C processing followed by 70 and

311 90 °C processing, which produced similar results. The 80 °C process had the least

312 effect on EMC of kamaboko gel, and there was no significant difference between the

313 control sample and kamaboko gel at the end of the process (p ≥ 0.05). EMC is an

314 important indicator of kamaboko gel quality; the lower the EMC, the better the gel

315 quality. The cause of gel quality deterioration was likely different for the different

316 processing temperatures. Modori, a gel weakening phenomenon caused by native

317 enzymes in surimi, can occur during the making of kamaboko gel (at 60 °C) and

318 decreases the water holding capacity (Hu et al., 2012; Klomklao et al., 2016; Okita

319 et al., 2021). Therefore, protease activity was undoubtedly the cause of the increase

320 in EMC at the above-mentioned temperature. Likewise, some proteases in surimi

321 remain active at 70 °C (Park, 2005); however, as this is further from the optimum

322 temperature of enzymes, EMC was less affected. At 80 and 90 °C, non-enzymatic

323 mechanisms likely contributed to the observed change since enzymes should have

324 already been heat-inactivated. The use of higher temperatures for kamaboko gel

325 production has been shown to cause greater alterations in the secondary structure of

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326 proteins (Uddin, Okazaki, Ahmad, Fukuda, & Tanaka, 2005). Subsequently, the

327 three-dimensional gel network is altered and the mobility of water in the gel

328 structure increases (Kong et al., 2016; L. Zhang et al., 2013); hence, there was an

329 increase in EMC.

330

331 3.5. Textural properties

332 The textural properties of tyndallized kamaboko gels were assessed by TPA and

333 the puncture test, as shown in Table 2. After the first heating cycle, kamaboko gels

334 went through textural transformation from soft and flexible to harder and more rigid

335 gels, as reflected by the increase in hardness and decrease in cohesiveness and

336 breaking deformation. Afterwards, most TPA parameters showed no significant

337 difference (p ≥ 0.05) except for at 60 °C, where the gels exhibited obvious

338 degradation by the reasons discussed earlier. For the other conditions, however, the

339 most noticeable feature was the difference in cohesiveness, which represents the

340 food’s resistance to the bite (Trinh & Glasgow, 2012). The heat-treated gel with the

341 highest cohesiveness was obtained from 90, 80, and 70 °C treatments in declining

342 order. The cohesiveness remained the same within the processing temperature

343 regardless of the number of heating cycles. In contrast, the 80 °C tyndallized gel

344 exhibited the highest breaking force and breaking deformation. This suggests that

345 the process had different effects on the compression force and penetration force. In

346 addition, the parameters from the puncture test were well inversely related to EMC.

347 The number of heating cycles is suggested to affect the quality of gels

348 similarly to the prolongation of processing time. Certainly, more heating cycles at

349 60 and 70 °C allow more time for proteases to react with proteins and degrade the

350 quality of protein gels. While outside the active range of proteases, excessive

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351 temperatures as well as processing time can have adverse effects on the textural

352 properties of kamaboko gel by decreasing gel strength and elasticity (Shie & Park,

353 1999). The effect of the number of heating cycles on breaking force was the most

354 pronounced in tbhis experiment, as significant decreases (p < 0.05) were detected

355 within the same processing temperature (Table 2). Other parameters, such as EMC

356 and breaking deformation, also exhibited changing trends. However, it is unclear

357 whether the quality of a gel processed for 3 cycles of 10 minutes each can be

358 compared with a gel processed continuously for 30 minutes at the same temperature.

359

360 3.6. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantification

361 of myosin heavy chain and actin

362 The SDS-PAGE analysis (Figure 2) revealed 2 major protein bands above 200 and

363 40 kDa, which are assumed to be MHC and actin, respectively (Zhang et al., 2013).

364 Differences in the protein pattern were observed for the different processing

365 temperatures. The kamaboko gel protein pattern shifted towards the lower molecular

366 weight region after each 60 °C heating cycle. By the end of the process, the relative

367 band intensity of MHC decreased to 28.7 %. For the 70 and 90 °C tyndallized

368 kamaboko gels, which had similar changes in terms of EMC, breaking force, and

369 breaking deformation, similar decreases in relative MHC band intensity were

370 observed. Nevertheless, the overall SDS-PAGE patterns differed. Pattern changes

371 at 70°C were similar to those at 60 °C but were lesser in magnitude. On the other

372 hand, the lower molecular weight regions under the MHC band (approximately 150

373 kDa) of the 90 °C tyndallized gel did not gradually intensify as observed in the

374 70 °C process. In fact, the high molecular weight region (> 200 kDa) became more

375 intense after treatment at 90 °C, which signified protein polymerization. Thus, it is

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376 plausible that the observed changes in quality following the 70 and 90 °C processes

377 were likely attributable to different mechanisms. Kamaboko proteins were least

378 affected by processing at 80 °C, as can be seen from the largely unchanged protein

379 pattern. Processing appeared to have little impact on actin, with its bands not visibly

380 altered in all treatments.

381 Although myosin is an important factor in gel properties, it is not the sole

382 determinant present in food matrices (Barrera, Ramírez, González-Cabriales, &

383 Vázquez, 2002). However, degradation of MHC can be an indicator of gel quality, as

384 MHC has a major role in forming gel networks, contributing to the textural

385 properties and water-holding capacity (Núñez-Flores, Cando, Borderías, & Moreno,

386 2018). By employing a processing temperature under 70 °C, the gel network was

387 susceptible to protein degradation by endogenous proteases. In contrast, at higher

388 temperatures, protein polymerization can occur, leading to disruption of the gel

389 network (Kong et al., 2016). Additionally, the high solubility (> 95%) of all samples

390 in SDS solution (data not shown) suggests that the interactions involved in

391 polymerization were mostly non-disulfide covalent bond. This indicates how protein

392 polymerization weakened the mechanical properties of gels subjected to

393 tyndallization at 90 °C. Thus, tyndallization at 80 °C is sufficient to inactivate

394 enzymes while minimizing protein polymerization.

395

396 4. CONCLUSION

397 While vegetative bacterial cells can be easily eliminated at relatively low

398 temperatures, the use of higher temperatures can induce bacterial endospores to

399 germinate at a higher rate. Furthermore, higher processing temperatures can inactivate

400 the remaining dormant spores which is significant when the majority of endospores

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401 have been germinated. From a quality standpoint, the observed deterioration in quality

402 was mainly attributed to enzymatic reactions when the heat treatment temperature is

403 not sufficient to inactivate the enzymes, as well as protein polymerization with

404 excessive heat application. Thus, it is necessary to consider the presence of enzymes in

405 the raw materials when applying this technique to other products. In this study,

406 complete sterility could only be achieved by tyndallization at 90 °C; however,

407 kamaboko gel with the highest quality was obtained from the 80 °C process. Food

408 sterility is paramount in producing shelf-stable products, and hence a higher

409 processing temperature is preferred. Nevertheless, it is anticipated that the sterility of

410 products can be improved with additional processing time or heating cycles.

411

412 Acknowledgement

413 This research was made possible by funding from the Japanese Ministry of

414 Education, Culture, Sports, Science and Technology (MEXT).

415

416 Conflicts of interest

417 There are no conflicts of interest to declare.

418

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527 FIGURE C A P T I O N S

528 Figure 1. Total bacterial count (TBC) and spore count (SC) of kamaboko gels during

529 each cycle of tyndallization using 60 °C (a), 70 °C (b), 80°C (c), and 90 °C (d) as the

530 processing temperatures.

531

532 Figure 2. The SDS-PAGE pattern of kamaboko gels at different stages of

533 tyndallization (a) and relative band intensity of MHC (b) and actin (c).

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534 Table 1 Gel color, whiteness index, pH, and expressible moisture content (EMC) of kamaboko gels during tyndallization at various

535 temperatures
Process
Heating cycle Whiteness
temperature L* a* N S b* pH EMC (%)
(time) index
(°C)
control 0 60.1±0.4 a -4.0±0.5 -2.7±0.3 a 59.8±0.4 a 6.79±0.03 a 4.03±0.18 a
60 1 74.6±0.3 b -3.7±0.1 1.9±0.2 b c 74.3±0.3 b c 6.83±0.02 a b 4.60±0.52 a b c
2 74.5±0.4 b c -3.7±0.3 2.2±0.2 c d e 74.1±0.3 b 6.81±0.04 a 13.41±1.50 d
3 74.7±0.2 b c d -3.7±0.3 2.5±0.2 e 74.3±0.2 b c 6.82±0.03 a b 26.07±0.79 e
70 1 75.0±0.6 b c d -3.7±0.3 2.2±0.4 c d e 74.7±0.6 b c d 6.89±0.07 b c 4.92±0.25 a b c
2 74.8±0.3 c d e -3.6±0.2 2.4±0.3 d e 74.4±0.3 b c d 6.91±0.08 c 5.19±0.20 b c
3 75.2±0.5 d e f -3.7±0.5 2.9±0.3 f 74.8±0.5 c d e 6.80±0.02 a 5.27±0.27 b c
80 1 75.3±0.3 d e f -3.5±0.3 2.0±0.2 b c d 75.0±0.3 d e f 6.89±0.02 b c 4.23±0.25 a
2 75.3±0.3 d e f -3.6±0.1 2.1±0.2 b c d 75.0±0.3 d e f 6.84±0.03 a b c 4.30±0.30 a
3 75.9±0.4 f -3.7±0.1 2.1±0.1 c d 75.5±0.4 f 6.83±0.04 a b 4.42±0.37 a b
90 1 75.7±0.4 e f -4.0±0.6 1.7±0.3 b 75.3±0.4 e f 6.86±0.05 a b c 4.78±0.27 a b c
2 75.3±0.4 d e f -3.6±0.1 2.0±0.1 b c d 74.9±0.3 d e f 6.89±0.02 b c 4.81±0.19 a b c
3 75.9±0.4 f -3.9±0.3 2.0±0.2 b c d 75.5±0.3 f 6.90±0.04 c 5.34±0.29 c

536 Data are presented as mean ± SD. Different superscripts within the same column indicate significant difference (P < 0.05)

537 NS = not significant

538

539

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540 Table 2 Textural properties of kamaboko gels during tyndallization at various temperatures

Process Heating Breaking

Hardness Adhesiveness Gumminess Breaking force
temperature cycle Cohesiveness Springiness deformation
(N) (J/m^3) N S (N) (N)
(°C) (time) (%)
control 0 3.80±0.34 a 0.90±0.01 g 47.55±9.73 3.44±0.31 a 0.95±0.01 c d e 3.52±0.11 f g 77.05±1.24 f
60 1 7.03±0.65 c d 0.83±0.01 c d 34.26±12.68 5.83±0.59 c 0.93±0.02 b c d 3.42±0.17 e f g 62.25±2.72 d e
2 6.34±0.53 c 0.77±0.01 b 41.51±18.11 4.88±0.34 b 0.92±0.01 b 1.01±0.09 b 28.55±5.10 b
3 5.22±0.34 b 0.68±0.02 a 37.61±9.28 3.55±0.30 a 0.88±0.01 a 0.54±0.03 a 16.65±0.47 a
70 1 7.29±0.47 c d 0.82±0.01 c 33.69±7.50 5.97±0.30 c 0.93±0.01 b c 3.24±0.05 d e f 60.18±3.77 c d
2 7.22±0.39 c d 0.82±0.01 c 20.35±3.99 5.89±0.28 c 0.93±0.02 b c d e 3.00±0.45 c d 54.58±6.62 c
cd c c bcde cd
3 7.18±0.63 0.82±0.01 32.29±6.94 5.85±0.45 0.93±0.01 2.73±0.19 53.88±4.73 c
80 1 7.08±1.18 c d 0.85±0.01 e f 32.96±7.71 6.03±0.93 c 0.95±0.01 e f 4.23±0.18 i 67.13±2.76 e
2 7.74±0.58 d 0.84±0.00 d e 32.00±6.28 6.49±0.48 c 0.95±0.01 d e f 3.87±0.20 h 62.55±3.31 d e
3 7.35±0.90 c d 0.84±0.00 d e 26.24±6.08 6.17±0.74 c 0.94±0.01 b c d e 3.90±0.38 h 61.83±2.65 d e
90 1 6.68±0.85 c d 0.86±0.01 f 38.19±8.88 5.73±0.66 c 0.97±0.02 f 3.59±0.09 g h 59.03±5.11 c d
2 6.78±0.49 c d 0.85±0.01 f 34.96±11.02 5.78±0.40 c 0.94±0.01 b c d e 3.15±0.22 d e 57.65±4.15 c d
3 6.87±0.51 c d 0.85±0.01 e f 32.28±10.99 5.82±0.41 c 0.93±0.01 b c d 2.77±0.21 c 55.45±1.56 c

541 Data are presented as mean ± SD. Different superscripts within the same column indicate significant difference (P < 0.05)

542 NS = not significant

543

544

545

546

24
Fig. 1 Keratimanoch et al.
Fig. 2 Keratimanoch et al.