Accepted Manuscript

Prediction and modelling of microbial growth in minimally processed fresh-cut

apples packaged in a modified atmosphere: A review

Predrag Putnik, Shahin Roohinejad, Ralf Greiner, Daniel Granato, Alaa El-Din A.
Bekhit, Danijela Bursać Kovačević

PII: S0956-7135(17)30261-X

DOI: 10.1016/j.foodcont.2017.05.018

Reference: JFCO 5624

To appear in: Food Control

Received Date: 02 February 2017

Revised Date: 05 May 2017

Accepted Date: 13 May 2017

Please cite this article as: Predrag Putnik, Shahin Roohinejad, Ralf Greiner, Daniel Granato, Alaa
El-Din A. Bekhit, Danijela Bursać Kovačević, Prediction and modelling of microbial growth in
minimally processed fresh-cut apples packaged in a modified atmosphere: A review, Food Control
(2017), doi: 10.1016/j.foodcont.2017.05.018

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 ACCEPTED MANUSCRIPT

1 Prediction and modelling of microbial growth in minimally processed fresh-cut apples

2 packaged in a modified atmosphere: A review

4 Predrag Putnik a*, Shahin Roohinejad b,c; Ralf Greiner b; Daniel Granato d;

5 Alaa El-Din A. Bekhit e; Danijela Bursać Kovačevića*

7 a Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000

8 Zagreb, Croatia; b Department of Food Technology and Bioprocess Engineering, Max Rubner-

9 Institut, Federal Research Institute of Nutrition and Food, Haid-und-Neu-Straße 9, 76131

10 Karlsruhe, Germany; c Burn and Wound Healing Research Center, Division of Food and

11 Nutrition, Shiraz University of Medical Sciences, Shiraz, Iran; d Department of Food

12 Engineering, State University of Ponta Grossa, Av. Carlos Cavalcanti, 4748, 84030-900, Ponta

13 Grossa, Brazil; e Department of Food Science, University of Otago, PO Box 56, Dunedin 9054,

14 New Zealand

15

16 * Corresponding authors:

17 Predrag Putnik, PhD

18 Email: pputnik@alumni.uconn.edu

19 Tel: +385(1)4605-036

20

21 Danijela Bursać Kovačević, PhD

22 Email: dbursac@pbf.hr

23 Tel: +385(1)4605-036

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24 Abstract

25 Minimally processed fresh-cut (MPFC) fruit products are an important source of functional

26 ingredients, and increased consumers’ demand boosted an expansion of this segment of food

27 industry. Apples (Malus sp.) are commonly cultivated fruits and processed to MPFC products.

28 Their low shelf life is the main obstacle for gaining maximum nutritive and economic benefits.

29 Enzymatic, metabolic, and other physiological processes induce changes that render the product

30 spoiled and decrease the storage and marketability life of MPFC fruits. Such spoilage can be

31 controlled by modified atmosphere packaging (MAP), but intrinsic influences of numerous MAP

32 factors are not clearly elucidated. Microbial growth adds another layer of complexity to MAP.

33 Mathematical modeling can successfully predict microbial growth and spoilage, hence saving

34 production cost and provide better control over the production chain. In this sense, this review

35 provides an overview of MAP’s factors that influence the quality of stored MPFC products.

36 Focus was placed on apples and development of mathematical models that can be useful for

37 prediction of their spoilage during storage and subsequently their shelf-life.

38

39 Keywords: minimally processed fresh-cut apple browning; modified atmosphere; respiration;

40 mathematical modeling; spoilage

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41 1. Introduction

42 Minimally processed fresh-cut fruit products (MPFC) usually contain apples (Malus domestica)

43 that are one of the world’s most cultivated fruits conveniently enjoyed by consumers (Putnik &

44 Bursać Kovačevć, 2017). Aside from convenience, apples are valued for having various

45 functional properties, and their consumption is recommended in official dietary reports as a part

46 of an increased recommended daily consumption of fruits and vegetables (Perez-Escamilla &

47 Putnik, 2007; Putnik, Bursać Kovačević, Herceg, & Levaj, 2016a). MPFC apples make a

48 significant portion of international food markets with market share account to multibillion dollar

49 in sales (Nicola, Tibaldi, & Fontana, 2009; Wang, Feng, & Luo, 2007).

50 The key challenge with MPFC foods is their short storage life that is usually no longer than two

51 weeks (Anese, Lanciotti, Gardini, & Lagazio, 2012; Montero-Calderón & Milagro Cerdas-

52 Araya, 2011; Putnik, Bursać Kovačević, Herceg, et al., 2016a). The minimal processing tends to

53 cause physiological damages to fruits and induces various types of enzymatic and microbial

54 changes. These changes are related to the promotion of endogenous production of ethylene,

55 oxidation of polyphenols, and increased microbial activity that leads to surface browning, loss of

56 structural integrity of the plant tissue, and subsequent loss of nutritive and sensorial properties

57 (Putnik, Bursać Kovačević, Herceg, et al., 2016a). The short shelf life of MPFC products and the

58 onset of spoilage can quickly negatively affect the economic profits obtained from this segment

59 of industrial processing (Pristijono, Wills, & Golding, 2006). Certain strategies can sustain the

60 natural quality of fruits, such preservation, processing and packaging (Rocculi, et al., 2012). To

61 achieve this goal, appropriate food engineering factors that are capable of retaining the maximum

62 nutritive and microbiological quality need to be considered and implemented in this type of food

63 products (Oms-Oliu & Soliva-Fortuny, 2011; Putnik, Bursać Kovačević, Herceg, et al., 2016a).

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64 Modified atmosphere packaging (MAP) is a method that has been used to control the microbial

65 spoilage in MPFC fruits and vegetables. However, to extend the shelf life of MPFC products and

66 control the microbial growth using MAP, different factors need to be clearly elucidated. In order

67 to achieve this, mathematical modeling has been suggested as a potential tool for prediction of

68 microbial growth and spoilage of ready-to-eat-products packaged under MAP. The objective of

69 this review is to examine critically the spoilage and modeling methods used for the prediction of

70 microbial growth in MPFC apples packaged under MAP.

71

72 2. Spoilage of minimally processed fresh-cut fruits

73 Spoilage in MPFC fruits can be roughly divided into two broad categories, non-microbial and

74 microbial spoilage (Putnik & Bursać Kovačevć, 2017). One of the first signs of spoilage in

75 MPFC apples is the occurrence of superficial brown pigmentation on the surface. This type of

76 non-microbial spoilage is not hazardous to human health and occurs very early during storage of

77 MPFC fruits (Granato, Santos, Maciel, & Nunes, 2016; Putnik, Bursać Kovačević, Herceg, &

78 Levaj, 2017), and is one of the leading limitations that will avert customers from purchase. This

79 does not come as surprise, as it is a well-known fact that color is one of the first attributes that

80 customers consider when buying a fresh product (Putnik, et al., 2017).

81 Browning may result from enzymatic or non-enzymatic reactions (Corzo-Martinez, Corzo,

82 Villamiel, & del Castillo, 2012). Enzyme(s) responsible for browning are polyphenol oxidases

83 (PPO), which are most active between pH 5 and pH 7. Their main substrates are phenolic

84 compounds that are converted to brown polymeric pigments (from quinones to melanins) leading

85 to the majority of browning in MPFC products. It is believed that the biological origin of

86 enzymatic browning is rooted as physiological response of the fruits to a mechanical damage to

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87 prevent microbial intruders from penetrating into the internal portion of plant’s tissue. Melanins

88 form impermeable microbial barrier that will ensure natural defense against intruders (Alzetta,

89 2014). Non-enzymatic browning can occur from the oxidation of ascorbic acid that is naturally

90 present in apples (Corzo-Martinez, et al., 2012; Cropotova, Tylewicz, Cocci, Romani, & Dalla

91 Rosa, 2016). It was reported that maximum browning rate due to this reaction was achieved at

92 pH=4, in which darker polymers are formed from dehydroascorbic acid being bound to amino

93 acids (Corzo-Martinez, et al., 2012).

94 Various approaches could be used to prevent surface discoloration, such as using: (i) anti-

95 browning solutions (for example Ca-ascorbate and citric acid) (Putnik, et al., 2017); (ii) UV-light

96 and edible coatings (Chen, Hu, He, Jiang, & Zhang, 2016; Lante, Tinello, & Nicoletto, 2016;

97 Liu, et al., 2016); (iii) aqueous solutions of L-arginine (Wills & Li, 2016); and (iv) advanced

98 technologies such as ultrasound, pulsed light or cold plasma (Jang & Moon, 2011; Moreira,

99 Álvarez, Martín-Belloso, & Soliva-Fortuny, 2017; Putnik, Bursać Kovačević, Herceg, et al.,

100 2016a; Putnik, et al., 2017; Ramazzina, et al., 2016).

101 Besides the browning effects caused by various processing approaches, browning is associated

102 with the apple cultivar. A recent study from our research group investigated the browning in

103 seven apple cultivars as well as the application of anti-browning agents and the results revealed

104 that Cripps Pink and Golden Delicious were the best cultivars suitable for MPFC apple products

105 and Ca-ascorbate was the best anti-browning agent (Putnik, et al., 2017). Ca-ascorbate treatment

106 resulted in the highest sensory evaluation scores regardless the inclusion of ultrasound (Putnik, et

107 al., 2017). Ultrasound is frequently used with anti-browning solutions to facilitate better access

108 of the treatment solutions to internal tissue of the apple and to reduce the activity of

109 monophenolase (Jang & Moon, 2011). Positive synergistic influence of ascorbic and citric acids

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110 with ultrasound was reported, but not with Ca-ascorbate (Putnik, Bursać Kovačević, Herceg, et

111 al., 2016a).

112 The influence of maturity, modified atmosphere (MA) and anti-browning solutions on textural

113 changes was evaluated over shelf-life of fresh-cut apples by instrumental mechanical-acoustic

114 test (Cortellino, Piazza, Spinelli, Torricelli, & Rizzolo, 2017). The authors revealed that the

115 acoustic evaluation approach is useful to complement mechanical results by monitoring texture

116 and crispness. The results showed that both anti-browning treatments and MA did not affect

117 apple texture. Furthermore, the authors suggested that the use of time-resolved reflectance

118 spectroscopy (TRS) to sort the fruits into homogeneous maturity classes is important to achieve

119 fresh-cut apples with homogeneous texture characteristics.

120 The use of pulsed light (PL) to extend the shelf-life of pectin-coated fresh-cut apples was

121 investigated by Moreira, Álvarez, Martín-Belloso, & Soliva-Fortuny (2017). The authors found

122 that dipping in ascorbic acid/calcium chloride solutions before coating with pectin and PL

123 treatments was effective in minimizing browning and softening of apples over 14 days of

124 storage. In addition, this preservation approach based on combination of both technologies (e.g.

125 hurdle technology) led to a significant reduction in spoilage microorganisms.

126 Cold plasma technology showed great potential for prevention of superficial browning in fresh-

127 cut apples (Tappi, et al., 2014). Apples were treated with dielectric barrier discharge atmospheric

128 gas plasma for 10, 20 and 30 min. Best results (65% reduction in browning compared to the

129 control samples) were obtained with 30 min treatment. Extending treatment time decreased the

130 polyphenol oxidase content by up to 42%. The results demonstrated that fresh cut apples treated

131 with plasma had lower metabolic activity than non-treated samples (Tappi, et al., 2014). The

132 effect of plasma processed on polyphenol oxidase and peroxidase enzymes present in apples and

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133 potatoes was further documented in recent studies. The cut apple tissue treated with plasma for

134 10 min had 62% less polyphenol oxidase activity, while the peroxidase activity was reduced for

135 about 65% (Bußler, Ehlbeck, & Schlüter, 2017). Additionally, the effect of cold plasma

136 treatment on the functional properties of fresh-cut apples cv. Pink Lady® through different in-

137 vitro and ex-vivo tests was monitored (Ramazzina, et al., 2016). Results revealed that exposure to

138 gas plasma does not to generate chemical species in apples. Plasma treatment caused a slight

139 reduction in the antioxidant capacity (up to 10%).

140 3. Influence of spoilage on nutritive value in minimally processed fresh-cut apples

141 Microbial growth is the main limitation of shelf life during the storage of MPFC fruits (Ragaert,

142 Jacxsens, Vandekinderen, Baert, & Devlieghere, 2011). A number of intolerable microorganisms

143 whose presence results in off-flavors measures microbial spoilage in foods. However, tolerance

144 to the number of such microbes varies with different cultural backgrounds and legal

145 requirements (Benner, 2014). In the European Union (EU), MPFC products must be free from

146 Salmonella sp, Escherichia coli, and Listeria monocytogenes (Comission, 2005). Some of the EU

147 members states (e.g. Croatia) recommend to the food business operators (FBOs) further testing

148 of these products for the presence of Staphylococcus aureus, sulfite-reducing Clostridium,

149 Enterobacteriaceae species (EBac), aerobic mesophilic bacteria (AMB), yeasts, and molds

150 (Benussi, et al., 2011). Albeit, EBac and AMB are ordinarily exist in food production and they

151 are not generally dangerous to human health, they are the main sources of spoilage in MPFC

152 products. The negative impact of EBac and AMB increases with their growth in foods as

153 measured in CFU/g of product (Benner, 2014). Industrial control of EBac and AMB in foods is

154 regulated by ISO standards (ISO 4833-2:2013, 2013; ISO 21528-2:2004, 2004). Croatia, as one

155 of the EU member states, recommended microbial limits (M) to the FBOs for spoiled MPFC

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156 products of M≤103 cfu/g for EBac and M>105 cfu/g for AMB (Benussi, et al., 2011). Microbial

157 spoilage can be successfully retarded by modified atmosphere packaging (MAP). The MAP can

158 preserve the food quality, restrain microbial growth, and extend shelf-life of fresh products

159 (Caleb, Mahajan, Al-Said, & Opara, 2013).

160 Presence of spoilage bacteria has a negative impact on nutrition of foods. Microbes negatively

161 influence the contents of amino acids, carbohydrates, vitamins and minerals (Genta & Heluane,

162 2001). Due to their reported health benefits, polyphenolic content is commonly used to evaluate

163 the nutritive value in fruits (Bursać Kovačević, et al., 2016; Bursać Kovačević, Putnik, Dragović-

164 Uzelac, Vahčić, et al., 2015; Granato, et al., 2016; Poojary, et al., 2017). This group of

165 biologically active compounds (BAC) has antiproliferative, anticancer, anti-inflammatory,

166 antioxidant, and antimicrobial properties (Forbes-Hernandez, et al., 2014). Main polyphenols

167 found in apple are: chlorogenic acids, coumaric acids, and flavonoids; including (-)-epicatechin,

168 phloretin, and quercetin (Alzetta, 2014; Granato, Karnopp, & van Ruth, 2015).

169 Current literature lacks information on relationships among endogenous biochemical processes,

170 processing, packaging, nutritive value, and spoilage of fruits (Caleb, et al., 2013). Research

171 associated with polyphenols in fruits documented the effect of (non)thermal processing on their

172 stability (Bursać Kovačević, Putnik, Dragović-Uzelac, Pedisić, et al., 2015; Putnik, Bursać

173 Kovačević, Penic, & Dragovic-Uzelac, 2015), while few studies are dedicated to the assessment

174 of the stability of these BACs in minimally processed products under MAP.

175 For example, the influence of MAP on the contents of lycopene, ascorbic acid, and total phenols

176 of tomato fruits (Domínguez, Lafuente, Hernández-Muñoz, & Gavara, 2016). Results from that

177 study confirmed positive effects of MAP on total phenolic content in tomatoes during storage. A

178 possible explanation for that effect could be attributed to increased phenylalanine ammonia-lyase

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179 (PAL) enzyme activity because of higher CO2 concentration in MAP. PAL is a wound-induced

180 enzyme that increases phenolic compounds concentration after cell damage such as cutting or

181 slicing. PAL activity has been proposed as a possible index for determining potential surface

182 browning of minimally processed products (Cantos, Espín, & Tomás-Barberán, 2001).

183 However, there are also some reports that documented the influence of ultrasound technology, as

184 a processing aid, in minimal processed apples (Putnik, et al., 2017). This technology has the

185 ability to preserve the natural characteristics of fresh fruits (aroma, color and nutritive content),

186 and facilitate better access of anti-browning solutions to the interior of the product (apple), hence

187 better prevention of browning (Nowacka, Tylewicz, Laghi, Dalla Rosa, & Witrowa-Rajchert,

188 2014; Tylewicz, Romani, Widell, & Galindo, 2013).

189 Only one recent study reported the association of MAP, microbiology, processing, respiration,

190 and browning with changes in polyphenol content in MPFC apples (Putnik, Bursać Kovačević,

191 Herceg, Pavkov, et al., 2016). Polyphenols in Cripps Pink and Golden Delicious cultivars were

192 determined using HPLC-DAD. The main bioactive compounds reported in this study were;

193 coumaric acid, chlorogenic acid, quercetin, (-)-epicatechin, and phloridzin. Cripps Pink had

194 higher polyphenol content and antioxidant capacity than Golden Delicious. Treatment of both

195 cultivars with anti-browning solutions (Ca-ascorbate, and a mixtures of ascorbic and citric acids)

196 improved the overall antioxidant capacity. This was expected as Ca-ascorbate, and mixture of

197 ascorbic and citric acids are known antioxidants. Microbial growth was increased with storage

198 and was inversely associated with the quantities of epicatechin. Similarly, increased bacterial

199 count (CFU/g) of spoilage microbes (AMB and EBac) was statistically associated with decreased

200 antioxidant capacities. Positive correlation between microbial growth of Lactobacillus brevis and

201 L. plantarum with quercetin content was already observed by (Alzetta, 2014). In contrast,

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202 catechol was associated with decreased bacterial activity while chlorogenic acid had no

203 association with their count.

204 4. Control of spoilage by minimally processed fresh-cut fruits

205 Microbial growth and their inactivation in MPFC fruit products are dependent on numerous

206 factors, such as technology parameters for processing, packaging, and storage conditions.

207 Commonly, these factors are tailor-made for particular microorganism(s) and relevant to physical

208 production environment that they are subjected to. Sources of contamination originate from

209 naturally-occurring microbes from the surface of the fruits, water, cross-contamination from

210 industrial environment and equipment (USDA, 2014), and their levels are commonly expressed

211 by total bacterial count (Qadri, Yousuf, & Srivastava, 2015). Good Manufacturing Practice

212 (GMP) and Hazard Analysis and Critical Control Points (HACCP) are common approaches that

213 are employed to eliminate or control microbial growth in MPFC products (James & Ngarmsak,

214 2011). These approaches can be part of regular processing schemes where bacterial presence can

215 be additionally reduced by MAP (Caleb, Opara, & Witthuhn, 2012).

216 Listeria monocytogenes, Salmonella sp., Sulfite-reducing Clostridium, Escherichia coli, EBac ,

217 AMB, Staphylococcus aureus, yeasts, and molds are microorganisms whose growth and

218 inactivation are important in the manufacturing and stability of MPFC products (Comission,

219 2005). Besides packaging (active/MAP), the most common approaches that food industry

220 employs during processing to control these microorganisms include: (i) biocontrol; (ii) washing

221 and sanitizing with different solutions; and (iii) the use of edible coatings.

222 Biocontrol is the approach that restrains bacterial growth in food using microbial antagonists

223 (Qadri, et al., 2015). It has been reported that pathogens growth in MPFC apples, represented by

224 the Escherichia coli (O157:H7), Salmonella, and Listeria innocua, can be effectively inhibited

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225 by certain types of pseudomonads, such as L-59-66 of Pseudomonas syringae (Alegre, et al.,

226 2013). Another report showed that on the same medium Listeria monocytogenes or Salmonella

227 enterica serovar Poona were successfully inactivated by Gluconobacter asaii (T1-D1), Candida

228 sp. (T4-E4), Discosphaerina fagi (ST1-C9), and Metschnikowia pulcherrima (T1-E2)

229 (Leverentz, et al., 2006). Lactic acid bacteria showed promising results for suppression of

230 Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella Typhimurium,

231 and Staphylococcus aureus in Golden Delicious cultivars (Trias, Baneras, Badosa, &

232 Montesinos, 2008).

233 Washing and sanitization are widely used to increase food safety (Zhang & Yang, 2017) by

234 reducing microbial load in MPFC products (Gil, Selma, Lopez-Galvez, & Allende, 2009).

235 Literature reports that this critical approach has a tangible benefits in reducing pathogens and

236 spoilage microorganisms in MPFC food production (Alegre, Abadias, Anguera, Oliveira, &

237 Vinas, 2010; Alegre, et al., 2013; Allende, Selma, Lopez-Galvez, Villaescusa, & Gil, 2008).

238 There are number of potential disinfectants that are available for washing and sanitization.

239 Hypochlorite, chlorine dioxide, and hydrogen peroxide are just a few of many that are reported

240 for this purpose (Gil, et al., 2009). These compounds will have various efficacies and impacts on

241 the sensory attributes, such as color, of the treated products. For example, the use of hydrogen

242 peroxide could induce oxidative reactions since it is an oxidizing agent.

243 Edible coatings have been reported to successfully decrease the load of psychrotrophic bacteria,

244 coliforms, yeasts, and molds (Qadri, et al., 2015). The coating is specifically engineered from

245 various chemical compounds and can be safely consumed in MPFC products. Currently,

246 intensive research is directed towards identifying ideal but harmless edible coating materials.

247 These coatings can be engineered from organic acids, esters, polypeptides, nitrites, sulphites,

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248 various bacteriocins, and plant essential oils (Franssen & Krochta, 2003). Besides being one of

249 the active components in edible coatings, plant oils and their extracts can be used as sole agents

250 for inhibition of various microbes (Bhargava, Conti, da Rocha, & Zhang, 2015; Oms-Oliu, et al.,

251 2010). Cinnamon bark, commonly consumed spice with apples and their products, reduced the

252 growth of Escherichia coli O157:H7 and Listeria innocua when added to anti-browning solution

253 (Muthuswamy, Rupasinghe, & Stratton, 2008). Vanilin is another commonly used spice that

254 repressed the growth of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes,

255 Salmonella enterica subsp. enterica serovar Newport, Candida albicans, Lactobacillus casei,

256 Penicillum expansum, and Saccharomyces cerevisiae (Rupasinghe, Boulter-Bitzer, Ahn, &

257 Odumeru, 2006).

258 The use of natural antimicrobials for dipping, such as citron essential oil, hexanal, 2-(E)-hexenal,

259 citral and carvacrol, alone or in combination, could strongly extend the shelf life of fresh-cut

260 apples packaged in modified atmosphere (Siroli, et al., 2014). In addition to their antimicrobial

261 effect, these compounds had positive effect on several quality parameters, such as color, texture

262 and retention of volatile organic compounds in the fruit (Siroli, et al., 2014).

263 Because of the increased nutritional trends that promote consumption of healthy and less

264 processed foods with least number of chemical additives, it is very likely that the use of essential

265 oils and their extracts in MPFC production will add extra value to minimally processed food

266 products. One good example of this observation is the addition of sage and rosemary extracts in

267 meat products aiming to replace synthetic antioxidants and preservatives (Putnik, Bursać

268 Kovačević, Penić, Fegeš, & Dragović-Uzelac, 2016).

269 Current data suggests that the use of natural antimicrobials in combination with pulsed light (PL)

270 developed efficient hurdle treatment(s) for microbial control of fresh-cut produce. The

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271 application of PL in combination with malic acid (MA) dip could be successfully applied for

272 efficient control of L. innocua and E. coli on the surface of fresh-cut avocado, watermelon and

273 mushrooms for a minimum of two weeks of storage (Ramos-Villarroel, Martín-Belloso, &

274 Soliva-Fortuny, 2015).

275 The application of novel technologies, such as atmospheric cold plasma, for decontamination of

276 foods revealed promising results in terms of antimicrobial efficacy against Escherichia coli,

277 Salmonella enterica Typhimurium and Listeria monocytogenes inoculated on cherry tomatoes

278 and strawberries (Ziuzina, Patil, Cullen, Keener, & Bourke, 2014). Additionaly, high pressure

279 processing (HPP) was successfully applied for reduction of non-O157 Shiga toxin-producing E.

280 coli (STEC) in fresh strawberry puree (Hsu, Sheen, Sites, Huang, & Wu, 2014). Results from

281 this study showed that the HPP conditions should include pressures greater than 350 MPa for at

282 least 5 min to eliminate efficiently the STECs in fresh strawberry puree or in similar high acidic

283 fruit products (e.g. pH < 4.6).

284 5. Control of spoilage by modified atmosphere packaging of minimally processed fresh-cut

285 apples

286 To satisfy the increasing demands of consumers’ for high-grade MPFC products, number of

287 relevant factors need to be considered. From the manufacturing perspective, finest products

288 require high-quality raw materials, stable and well-organized distribution and cooling conditions,

289 all with strong marketing logistics (Artes, Gomez, & Artes-Hernandez, 2007).

290 Retardation of spoilage in MPFC products is usually achieved by MAP that is typically

291 combined with refrigeration (Heinrich, Zunabovic, Nehm, Bergmair, & Kneifel, 2016; Li, et al.,

292 2011; Montero-Calderon, Rojas-Grau, & Martin-Belloso, 2008).

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293 Altering content of atmospheric gases within the package, caused by selective film, will reduce

294 respiration rates of produce and delay biochemical processes that lead to spoilage. Tissue

295 respiration in the produce consists of biological pathways where organic macromolecules are

296 lysed to simpler species with consumption of O2 and formation of CO2 (Putnik & Bursać

297 Kovačevć, 2017). Selective packaging films have different permeability’s for atmospheric gasses

298 such as O2, CO2 and N2 that are frequently used in the MAP. Modified atmosphere composed of

299 2-5 kPa O2 and 3-10 kPa CO2 will have a tendency towards extending the length of storage of

300 MPFC products by inhibiting superficial browning and biological aging. CO2 is fat and water-

301 soluble gas so it can be applied to a number of foods with various compositions; also, it is

302 commercially and economically available. Changes of CO2-solubility can be easily altered by

303 changes in temperature, which might be practical for different types of produce. CO2 is an

304 antimicrobial agent, suitable for the control of microbial growth on fresh produce. For instance,

305 CO2 dissolved in water forms carbonic acid that will cause a drop in the pH of the product

306 surface, which will cause growth reduction of various pathogens and spoilage microorganisms.

307 In the lag phase, a low pH in the medium impedes maximum microbial growth rate and their

308 population densities (Devlieghere & Debevere, 2000). Effectiveness of CO2 as an antimicrobial

309 agent is significantly dependent on temperature, type of microorganism, growth phase, water-

310 activity, and chemical composition of produce (Devlieghere & Debevere, 2000).

311 Selective packaging barrier serves as a “passive pump” that will hinder anaerobic respiration by

312 increasing volumetric concentration of CO2 within the package, and decreasing the concentration

313 of O2. Engineering parameters that are used to control ratios of volumetric concentration of

314 gasses within the package are: (i) permeability of packaging material, and (ii) respiration rates of

315 the produce (Putnik, Bursać Kovačević, Herceg, et al., 2016a). For MAP systems, the following

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316 mathematical equations are used to predict performance of the packaging (Putnik, Bursać

317 Kovačević, Herceg, & Levaj, 2016b):

318

kPO2
× A×
y
out
O2 - yOin2 - V  dyO2 
×f

x 100 100  dt 
319 RO2 = (4)
M

Vf  dyCO2
×
 kPCO2
- × A×
in
yCO 2

out
- yCO 2


100  dt  x 100
320 RCO2 = (5)
M

M
321 V f = VTOTAL - (6)
Mρ

322 A= length × width (7)

323 where “R” are respiration rates in cm3 kg-1 * day-1 * atm-1; “kP/x=P” is permeance of the

324 packaging material at STP in cm3 m–2 day–1 atm–1; “STP” represents standard temperature and

325 pressure; “A” is package surface area in m2; “M” is mass of packaged apple in kg; “x” is film

326 thickness in cm; “y” volumetric concentration in % v/v O2 and CO2; “Mρ” is apples’ density;

327 “VTOTAL” is a total volume within the package in cm3; and “Vf” is free volume within the

328 package in cm3.

329 Although the bacterial load within the package can be decreased by manipulating the

330 concentrations of gases within the package, still it is not clear how each atmospheric gas interacts

331 with respiration parameters (Caleb, et al., 2013). Among others, microbial growth in MAP is

332 influenced by storage time and temperature, volumetric concentration of modified atmosphere

333 gases, package dimensions, and mass of the produce (Putnik, Bursać Kovačević, Herceg, et al.,

334 2016b). It was reported that the storage time of MPFC apples was extended with MAP at low

335 temperatures by reducing their respiratory and metabolic rates (Table 1). A combination of anti-

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336 browning solutions and MAP prolonged the storage of the apples until 12 days (Kader, 1986;

337 Waghmare, Mahajan, & Annapure, 2013). Applying MAP with low storage temperatures in

338 MPFC food production will impede aerobic spoilage and decrease bacterial load. The levels of E.

339 coli, Salmonella and L. innocua were controlled in apples packaged in MAP at 5°C for 30 days

340 (Alegre, et al., 2010). Nonetheless, one important disadvantage remains, as these storage

341 conditions may foster the growth of dangerous pathogens (Oliveira, et al., 2010). For example,

342 C. botulinum Type E, L. monocytogenes, Y. enterocolitica, Vibrio parahaemolyticus.

343 enterotoxigenic E. coli, Bacillus cereus, and Aeromonas hydrophila are hazardous food-borne

344 microorganisms capable of growing at temperatures equal or lower than 5°C. Additionally,

345 Salmonella sp. grows at 7°C and Staphylococcus aureus grows just above the refrigeration

346 temperatures at 6°C and forms toxins at 10°C. Therefore, it is very important to control the

347 growth of these microbes during production to avoid spread of potential harmful and costly food

348 outbreaks.

349 Modified atmosphere packaging experiments that were done under industrial environment

350 showed that the application of either Ca-ascorbate or mixture of ascorbic and citric acid as anti-

351 browning agents effectively prevented browning on Cripps Pink and Golden Delicious apples.

352 Additionally, it was reported that the MPFC apples in MAP can resist microbial spoilage up to

353 9.88 days based on EBac growth, while they are considered spoilt, almost as twice as fast (6.47

354 days), based on the growth of AMB. Their maximum growth rates for EBac and AMB were

355 0.25 and 0.46 log CFU/g*day, respectively, which represents 1:1.84 ratio, or roughly two-fold

356 increase for AMB (Putnik, Bursać Kovačević, Herceg, Roohinejad, et al., 2016). Mathematical

357 models that are able to predict spoilage of MPFC apples are reported by (Putnik, Bursać

358 Kovačević, Herceg, et al., 2016a).

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359

360 6. Mathematical modeling of spoilage of minimally processed fresh-cut apples in modified

361 atmosphere packaging

362 Design of experiments (DOE) and industrial modeling are regularly used to decrease the number

363 of experimental runs and lower the costs of research and development (Karnopp, Oliveira, de

364 Andrade, Postingher, & Granato, 2017; Putnik, Bursać Kovačević, & Dragović-Uzelac, 2016a,

365 2016b). Well-prepared and executed DOE will result in confident statistic relations that can

366 explain, predict, and optimize industrial production (Hacke, et al., 2016). Mathematical models

367 used for MAP usually target: extension of storage, control of microbiology, respiration

368 physiology, postharvest conditions, and preservation of quality. Modeling can be used to

369 simulate various packaging possibilities and designs (Belay, Caleb, & Opara, 2016).

370 Outcomes of respiration rates are usually targeted by modeling, as they are crucial for MAP but

371 intricate to assess due to a large number of complicated relationships. Respiration rates are

372 influenced by the volumetric concentration of gases, size of the package, mass of the fruits, and

373 type of the permeable film (Fonseca, Oliveira, & Brecht, 2002). Influences of O2 and CO2 in the

374 produce respiration process are quantified by semi-fundamental and fundamental mathematical

375 modeling (Torrieri, et al., 2009). Length of storage, respiration rates, and volumetric

376 concentration of packaging are the most important parameters for the MPFC production because

377 these factors are related to aerobic respiration (Putnik, Bursać Kovačević, Herceg, et al., 2016b).

378 Michaelis–Menten model is a semi-fundamental approach that relies on enzyme kinetics and

379 represents a simplified approach of the apple respiration (Fagundes, Augusto, Carciofi, & A.,

380 2013). Fundamental mathematical modeling offers accuracy, flexibility, tailor-made approach,

381 that incorporates a larger number of production parameters in a single equation. The major

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382 difficulty for this type of modeling is its complexity and need for in-depth background with

383 various statistical tests (Putnik, Bursać Kovačević, Herceg, et al., 2016a).

384 The most available predicative mathematical models for estimation of storage have been

385 developed under isothermal conditions, and must be validated when they are used with non-

386 isothermal cold chain conditions. A recent study that developed predictive models for shelf-life

387 estimation of MPFC salads and examined their applicability under both, non-isothermal and

388 temperature conditions was reported by (Tsironi, et al., 2017). The microbial growth in that study

389 used Baranyi Growth model, while the deterioration constant employed Arrhenius equation.

390 Integrated effects of temperature on product quality were also calculated by Arrhenius model.

391 Developed shelf-life models gave satisfactory results by confirming their applicability in

392 estimating quality deterioration during the supply chain. Another study reported predictive

393 kinetic models for monitoring the effect of storage temperature on the microbial spoilage and

394 quality parameters in MPFC dandelion’s salad during storage (Dermesonluoglu, et al., 2016).

395 The results of the non-isothermal experiment showed the potential of the mathematical models

396 for prediction of quality in fresh-cut salad under dynamic conditions, where the shelf-life was

397 calculated based on microbial growth, texture, and sensory evaluation.

398 Up to present, application of only few functional mathematical models has been reported under

399 industrial production of MPFC food products, such as commercial “Pack-in-MAP©” (Mahajan,

400 Oliveira, Montanez, & Frias, 2007) and the freely available “Anti-browning Apple Calculator -

401 C.A.P.P.A.B.L.E. ©” (Pizent, Putnik, Bursać Kovačević, & Herceg, 2015).

402 Pack-in-MAP© is an online application that contains a large number of mathematical calculators

403 integrated to graphical user interface that can assist in the production of MPFC produce

404 preserved by MAP. This application offers a selection of product type, storage parameters, mass

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405 of produce, and shape/size of a package (Mahajan, et al., 2007). Unfortunately, the website with

406 this application seemed inactive at the time of writing this review.

407 Another available application is Anti-browning Apple Calculator - C.A.P.P.A.B.L.E. © that is

408 focused on production of MPFC apples. This website offers estimation of microbial and non-

409 microbial spoilage through selection of a high number of MPFC industrial parameters. Estimates

410 are calculated from equations obtained by fundamental mathematical modeling. Non-microbial

411 spoilage is estimated from changes in browning, soluble solids contents, and pH over the storage

412 time for seven MPFC apple cultivars, whether or not they were treated with anti-browning

413 agents. Projections are calculated for 11 different agents and from initial CIELab color

414 parameters. Further, it is possible to compare the levels of browning in MPFC apples in modified

415 vs. normal atmosphere by observing joint use of MAP, anti-browning treatments and their initial

416 CIELab parameters. The models are capable of calculating: browning in apples stored for a

417 selected number of days and packaged in MAP from respiration rates; packaging size; selective

418 packaging film’s permeability; mass of packaged apple; type of cultivar; anti-browning

419 treatment, and the content of gases of the modified atmosphere.

420 Models for microbial spoilage can calculate the length of storage in days that MPFC apples can

421 spend on the market in reference to some legal standard(s). Equations calculate estimates from

422 type of cultivar, anti-browning agent, use of advanced processing technology, mean change of

423 color, pH, soluble solids content (SSC), sensory evaluation, initial levels of contamination, and

424 type of reference spoilage microorganism (Figure 1). Models that can predict microbial growth

425 on apples under the modified atmosphere for AMB and EBac are built-in to include customary

426 requirements for the length of storage, SSC, CIELab status, apple respiration, packaging size,

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427 selective packaging film permeances, mass of the apple, and the content of the modified

428 atmosphere (Figure 2).

429 Tools offered by C.A.P.P.A.B.L.E. © can improve control of apple spoilage, help with

430 optimization of production, extension of storage time, engineering quality foods, and foster

431 economic profitability for MPFC apples. External validity of the models may be decreased due

432 to a lower number of tested cultivars. Even though there is a very little biological variance

433 among apple cultivars, that might bias physiological (respiratory) pathways, nevertheless,

434 models should be verified with different apple cultivars. Detailed explanations and mechanisms

435 of the entire research are available elsewhere (Putnik, Bursać Kovačević, Herceg, et al., 2016a,

436 2016b; Putnik, et al., 2017).

437

438 7. Conclusions

439 Consumers’ preferences for MPFC foods and constant recommendations from scientific

440 communities will probably increase the production of MPFC in the future. However, these

441 products have short storage time, and tendency for spoilage, both microbiologically and non-

442 microbiologically. Non-microbial spoilage can be reduced by the use of anti-browning agents

443 and minimal processing while microbial spoilage can be deferred with modified atmosphere

444 packaging and edible coatings. One of the best-rated cultivars for MPFC productions are Golden

445 Delicious, and Cripps Pink dipped in Ca-ascorbate and stored in modified atmosphere packaging.

446 Longest time that such apples can spend on the market without microbial spoilage was 9.88 days

447 as measured by the Enterobacteriaceae, and 6.47 days Aerobic mesophilic bacteria (AMB).

448 C.A.P.P.A.B.L.E. ©, a mathematical modeling portal for spoilage in MPFC produce, can be

449 successfully applied for simultaneous consideration of a large number of industrial factors that

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450 will save time and production resources. This will promote the making of high-quality safe

451 products and increase economic profitability. Future research should be focused on development

452 of the models that are fully able to mimic analytical monitoring of microbial growth in MPFC

453 foods, so they can predict critical points and improve safety of HACCP procedures. Indeed, it

454 would be prudent to embed such models to user-friendly online applications, so that they can be

455 readily available to food science professionals without the need for in-depth statistical or

456 computer skills.

457

458 Acknowledgements

459 Shahin Roohinejad would like to acknowledge the Alexander von Humboldt Foundation,

460 Germany for his postdoctoral research fellowship award. Daniel Granato acknowledges CNPq

461 for a productivity grant (process). All authors declare that they have no conflict of interest. This

462 article does not contain any studies with human or animal subjects.

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723

724 Figure captions:

725 Figure 1. Calculating the length of shelf-life

726 Figure 2. Microbial growth calculation under modified atmosphere

27
1 Table 1. Average values for microbial growth (adopted from Putnik, Bursać Kovačević, Herceg, et al., 2016b*)
2
Independent variables n Log cfu/g AMB Log cfu/g EBac R O2 R CO2
Apple Cultivar p=0.81‡ p=0.19‡ p=0.86‡ p=0.96‡
Cripps Pink 15 5.4±0.1a 2.3±0.1a 23.0±1.3a 1.2±0.4a
Golden Delicious 15 5.4±0.1a 2.2±0.1a 22.7±1.3a 1.2±0.4a
Treatment p=0.32‡ p=0.85‡ p=0.71‡ p=0.32‡
No treatment 6 5.3±0.2a 2.3±0.1a 24.3±2.1a 0.7±0.6a
Ascorbic a. + Citric a. 6 5.4±0.2 2.3±0.1a 23.3±2.1a 1.7±0.6a
Ca-ascorbate 6 5.6±0.2a 2.2±0.1a 23.9±2.1a 0.7±0.6a
USND (3min) + Ca-ascorbate 6 5.8±0.2a 2.3±0.1a 20.7±2.1a 2.0±0.6a
USND (3min) + Ascorbic a. + Citric a. 6 5.3±0.2 2.2±0.1a 21.9±2.1a 0.8±0.6a
Shelf-life in days p≤0.01† p≤0.01† p≤0.01† p≤0.01†
1 10 2.8±0.1a 1.0±0.1a 16.2±1.6a 2.6±0.4a
7 10 4.7±0.1b 1.6±0.1b 28.3±1.6b 3.9±0.4b
14 10 8.8±0.1c 4.2±0.1c 23.9±1.6c 2.3±0.4c
Apple Cultivar by Treatment p=0.99‡ p=0.95‡ p=0.21‡ p=0.75‡
Cripps Pink, Ca-ascorbate 3 5.6±0.2a 2.2±0.2a 20.7±2.9a 0.2±0.8a
Cripps Pink, No treatment 3 5.3±0.2a 2.4±0.2a 27.4±2.9a 1.1±0.8a
Cripps Pink, USND (3min) + Ca-ascorbate 3 5.6±0.2a 2.4±0.2a 18.8±2.9a 2.3±0.8a
Cripps Pink, USND (3min) + Ascorbic a. + Citric a. 3 5.3±0.2a 2.3±0.2a 23.9±2.9a 0.9±0.8a
Cripps Pink, Ascorbic a. + Citric a. 3 5.3±0.2a 2.5±0.2a 24.0±2.9a 1.3±0.8a
Golden Delicious, Ca-ascorbate 3 5.5±0.2a 2.2±0.2a 27.1±2.9a 1.2±0.8a
Golden Delicious, No treatment 3 5.3±0.2a 2.3±0.2a 21.1±2.9a 0.2±0.8a
Golden Delicious, USND (3min) + Ca-ascorbate 3 5.7±0.2a 2.2±0.2a 22.5±2.9a 1.8±0.8a
Golden Delicious, USND (3min) + ascorbic a. + citric a. 3 5.3±0.2a 2.1±0.2a 19.9±2.9a 0.7±0.8a
Golden Delicious, ascorbic a. + citric a. 3 5.3±0.2a 2.2±0.2a 22.6±2.9a 2.0±0.8a
3 *Results are expressed as mean±standard error.
4 Values represented with different letters are statistically different marginal means at p≤0.05.
5 †statistically significant variable at p≤0.05.

6 ‡ statistically insignificant variable at p≤0.05.