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Probiotic Bacillus: Fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores
Accepted Manuscript Probiotic Bacillus: Fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores
Mojtaba Jafari, Amir M. Mortazavian, Hedayat Hosseini, Fahimeh Safaei, Amin M. Khaneghah, Anderson S. Sant'Ana PII: DOI: Reference:
S0963-9969(17)30089-3 doi: 10.1016/j.foodres.2017.03.001 FRIN 6608
To appear in:
Food Research International
Received date: Revised date: Accepted date:
13 January 2017 1 March 2017 3 March 2017
Please cite this article as: Mojtaba Jafari, Amir M. Mortazavian, Hedayat Hosseini, Fahimeh Safaei, Amin M. Khaneghah, Anderson S. Sant'Ana , Probiotic Bacillus: Fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/ j.foodres.2017.03.001
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ACCEPTED MANUSCRIPT Probiotic Bacillus: fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores Mojtaba Jafari 1, Amir M. Mortazavian 1*, Hedayat Hosseini 1, Fahimeh Safaei 1, Amin M. Khaneghah2*, Anderson S. Sant'Ana2 1
Department of Food Science and Technology, Faculty of Nutrition Sciences, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2
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Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
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*Corresponding Authors: Amir M. Mortazavian E-mail address: [email protected] Postal address: 46, West Arghavan St., Farahzadi Blvd., Shahrak Qods, National Nutrition & Food Technology Research Institute, Zip Code: 1981619573, P.O. Box 19395-4741, Tehran, Iran Tel: +98 21 22357483-5; Fax: +98 21 22360660.
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Amin Mousavi Khaneghah E-mail address: [email protected] Department of Food Science, Faculty of Food Engineering, State University of Campinas (UNICAMP), Campinas, São Paulo, Rua Monteiro Lobato, 80. Caixa Postal: 6121. CEP: 13083-862. Campinas. São Paulo, Brazil. Telephone: +55(19) 3521-0091. Fax: +55(19) 3521-2153
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ACCEPTED MANUSCRIPT Abstract The current study was designed to assess the fate of probiotic strains of Bacillus coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 (as spores) during processing and refrigerated storage of cooked sausage as well as the influence of culturing conditions on the
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recovery of spores. The highest recovery of B. coagulans ATCC 31284 and B. subtilis var. Natto
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ATCC 15245 spores were obtained on trypticase soy agar (TSA) (15.22 and 15.12 log CFU /
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mL, respectively) which was carried out under conducted heat shock (68 °C, 20 min) (15.61 and 15.24 log CFU / mL, respectively). According to result; a 3- 4 log reduction in the counts of the
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spores inoculated in the raw batter of sausage after the cooking step was observed. The findings
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revealed that the counts of the spores were significantly changed after heat shock (80 °C /10 min) (P ≤ 0.05). Data indicated that the count during sausage processing and the entire cold storage
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was more than 106 CFU /g. The ability of the probiotic sporeforming bacteria for sustaining
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within the cooking process, storage stage; traits that cannot be ensured with vegetative probiotic bacteria, which proposed their capacity for usage, especially in functional cooked food products
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was demonstrated by the results of the current study.
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Keywords: Cooked sausage, Enumeration, Probiotic Bacillus, spores
Studied Chemical compounds in this article TE buffer solution (PubChem CID:16218629); TRIS.HCL (PubChem CID:93573); NaCl (PubChem CID:5234); KCl (PubChem CID:4873 ); Sodium dodecyl sulfate (PubChem CID:3423265); Egg white lysozyme (PubChem CID:71581463).
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ACCEPTED MANUSCRIPT 1.
Introduction The development and consumption of food products containing probiotics as an
increasingly global consumer trend could achieve a considerable remark in functional food market (Khan et al., 2011). The commonly accepted definition of probiotics by FAO and WHO
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can be noted as “live microorganisms while ingested in adequate amounts could offer a health
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benefit on the host” (FAO/WHO, 2002; Venema and do Carmo, 2015; Almada et al., 2015).
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Although, no agreement has been conducted for the recommended minimum level of
of 106 CFU g-
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or ml-
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ingested probiotics to provide the proposed functionality (Champagne et al., 2005), populations in the final product at the time of consumption, supposing a daily
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consumption of 100 g or mL, was established as a minimum daily therapeutic dose of probiotic cultures in processed foods (Ashraf and Shah, 2011). This will result in a dose of 108 CFU,
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which should be adequate to confer health benefits to consumers (Jayamanne and Adams,
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2006).With the application of probiotic microorganisms in foods aiming to result in a health
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benefit, the assessment of the fate of probiotic microorganisms during processing and storage is of foremost importance. The ability of probiotics in order to survive the harsh and severe
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manufacturing process, the storage of the product and finally passage through the gastrointestinal
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tract can be considered as key concerns for both manufacturing and marketing of meat products (Shah 2001; Bezkorovainy, 2001). For instance, the required microorganisms for the production of probiotic foods such as Lactobacillus and Bifidobacterium could not survive such thermal processing (cooking at approximately 75°C at the cold point) (Ruiz et al., 2011; Baka et al., 2015). Also, providing the knowledge on the best conditions for recovery and enumeration of probiotic Bacillus are crucial. Moreover, depending on the applied conditions for recovery and enumeration, the counts of probiotics can vary significantly (Ashraf and Shah, 2011). The
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ACCEPTED MANUSCRIPT application of Bacillus species’ spores as probiotic dietary supplements is expanding extensively and rather rapidly due to some health benefices such as immune stimulation, antimicrobial activities, and competitive exclusion (Spinosa et al., 2000; Patel et al., 2009; Katsutoshi et al., 2011). Among the 100 known Bacillus spp., only a few strains (including B. coagulans and B.
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subtilis var. Natto) have been approached as probiotics for human consumption (Urdaci et al.,
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2004; Nithya and Halami, 2013). Therefore, sporeforming probiotic bacteria could be considered
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as a valuable solution for overcoming the limitations related to stability of Bifidobacterium and Lactobacillus strains during processing and storage of probiotic foods (Patel et al., 2009; Endres
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et al., 2009; Cutting, 2011; Katsutoshi et al., 2011; Postollec et al., 2012).
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To the best of author's knowledge, this is the first conducted study aiming to assess the fate of probiotic Bacillus strains during cooking and storage of sausages. In addition, conditions for
Materials and Methods
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2.
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enumeration methods were assessed.
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recovery of probiotic Bacillus using different culture media, heat shock conditions, and
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2.1. Materials and chemical reagents All used chemical and reagents in analytical grade were supplied by Merck Co.
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(Darmstadt, Germany).
2.2. Bacterial spores and spore preparation Commercial probiotic sporeforming bacteria strains of B. coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 were obtained from Natures Only, INC., USA and World Intellectual Resource Co., Taiwan, respectively (Bhat et al., 2013; Azimirad and 4
ACCEPTED MANUSCRIPT Alebouyeh, 2016). In order to evaluate the purity of the strains, each one was separately streaked onto Müller-Hinton agar (Merck, Germany). Then, Gram staining and direct microscopic examinations were carried out after 7-10 days to confirm whether sporulation was completed. Further, the described method by Alebouyeh et al. (2009) was used for the preparation of spore
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suspensions. After intermittent steps of washing with sterile deionized water (SDW) and
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centrifugation (12000 × g, 15 min), the final suspensions obtained were diluted in defined
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amounts of sterile deionized water. The concentration of spores per mL of each spore suspension was verified using Neubauer chamber (hemocytometer), confirmed through plate count
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(Alebouyeh et al., 2009), the following storage at -70 °C for further analysis.
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2.3. Production of cooked sausage 2.3.1. Cooked sausage formulations
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Frozen meat packs (15% fat) were kept at 4 °C for approximately 18 h for thawing. The
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used primary formula for sausage preparation contained minced meat (40%), water (as ground ice) (22%), vegetable oil (19%), salt (NaCl) (1.5%), polyphosphate (0.35%), sodium nitrite (120
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ppm), sodium ascorbate (400 ppm), filler agents including texturized soy protein (19%), wheat
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flour, frozen garlic (1.8%), wheat starch, gluten, pasteurized whole egg (2%), sugar (1%) and
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spice mix specially prepared for this type of meat product ( ca. 1%). 2.3.2. Sausage preparation and inoculation with probiotic Bacillus In practice, the control sausage with no added probiotic sporeforming bacteria was produced as follows: the grounded minced meat was mixed with salt (NaCl), nitrite, and polyphosphate at low speed in a 10 kg bowel mini cutter device (Allen, K21 Ras 83132, Germany) for about 2 min. Then one-half of the proposed water in the formulation (as ground ice) was added and subsequently mixed thoroughly in the cutter at high speed. Consequently
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ACCEPTED MANUSCRIPT after lowering the temperature of the mixture about 1–2 °C, oil was added, and mixing was continued until reaching the temperature up to 8 °C. Afterward, the remaining water (second half) and the rest of ingredients were incorporated, respectively, thoroughly mixed and chopped until the smooth distribution of all ingredients and the emulsion was obtained (batter) (for an
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additional 3 min, approximately). In order to monitor and verify the temperature of the emulsion,
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(Kane-May, KM330, Harlow, Germany) was applied.
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which should be maintained below 12 °C during the preparation of batter, a digital thermometer
In order to the production of inoculated formulations, spores of B. coagulans ATCC 31284
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and Bacillus subtilis var. Natto ATCC 15245 were individually inoculated at the start of cutter
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step after adding the first half of water and mixed at low speed for approximately 3 min in order to obtain a proper dispersion of the spores in the batter. The added inoculum concentration to the
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bowel cutter were 12.88 and 12.96 log CFU / 10 kg (or 8.88 and 8.96 log CFU/g), respectively.
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All batches after comminution/ chopping step were filled into artificial polyamide casings (24 mm diameter) (Arta, Tabriz, Iran) using a stuffer (Handtman, Germany). Then, sausages were
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cooked in a cooking cabinet with steam at 80± 1°C (up to reaching the temperature of the
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geometric center to 75 °C) for approximately 60 min. Subsequently, the cooked sausages were cooled down to about 25 °C using cold water (12 °C) for approximately 8–10 min. Then, cooked
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sausages were stored at 4±1 °C for 45 days. For each batch, the above-described processing was repeated two times. Samples were randomly taken from each sausage batch before and immediately after the cooking step of the sausage manufacture and at 1, 15, 30, and 45 days of refrigerated storage time. In fact, sausages were sampled just after filling into casings by selecting at random three segments of sausage samples from each treatment. It should be mentioned that separate sample units were taken for each type of test.
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ACCEPTED MANUSCRIPT 2.3.3. Enumeration of probiotic Bacillus in sausage As the first part of the current study, the recovery number of B. coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 inoculated spores in sausages (raw and cooked) and during 45-day storage (d0, d1, d15, d30, d45) at 4±1 °C (cooked sausage) was determined by plate
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count on nutrient agar (Himedia, India). The nutrient agar was chosen due to the low price and
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high availability as the general medium in most of the laboratories. The enumeration was done
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before and after subjecting samples to a thermal treatment (80 °C for 10 min) (Leuschner et al., 2003). Plates were aerobically incubated at 37 °C for 2 days, and colonies grown were counted
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and the results expressed as CFU/g.
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Despite the very wide diversity of the genus, the majority of Bacillus species including the investigated strains in the current study will grow well on routine and general media such as
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nutrient agar or trypticase soy agar (Vos et al., 2009). Also, the general sporeformers can be
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found in the batter of inoculated sausage samples. So, by subtracting these counts from the initial counts of added probiotic sporeforming bacteria to the sausage samples, the actual counts of B.
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coagulans and B. subtilis spores in raw and cooked sausage can be calculated. In addition, there
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is no selective or selective differential medium for enumeration of bacteria in Bacillus genus so their counting could be based on general media. Eventually, the actual counts of Bacillus
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coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 spores supplemented in raw and cooked sausage would be obtained 2.4. Influence of culture medium on the recovery of probiotic Bacillus spores In the second part of investigation, in order to measure the recovery of the probiotic sporeforming bacteria, the recommended seven general culture media (Merck, Germany; Himedia, India; Difco, USA), among the most frequently cited literature, were approached
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ACCEPTED MANUSCRIPT (Alcock, 1984; Juneja et al., 2003; Turnbull et al., 2007; Lee et al., 2015). In practice, following the thawing process, the frozen cultures of the spores and appropriate serial decimal dilutions, enumeration of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores were carried out through spread plate technique in all chosen media, followed by incubation for 48 h
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at 37 °C.
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2.5. Influence of heat shock conditions on the recovery of probiotic Bacillus spores Finally, the heat shock (or heat-activation) treatments of the probiotic sporeforming
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bacteria, were carried out according to the suggested treatments including exposure to a
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temperature of 68 °C for 15, 20, and 30 min, and 80 °C for 10 and 15 min, respectively (Alcock, 1984; Juneja et al., 2003; Turnbull et al., 2007). The sporicidal effect of temperatures above 80
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°C (especially 90 °C) was taken into consideration (Rice et al., 2004; Turnbull et al., 2007). In
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practice, following an appropriate dilution, suspensions of B. coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 were exposed to heat (68 °C or 80 °C) in a water bath
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(Memmert, Germany) for the times mentioned above and then they were immediately cooled
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down using ice water bath. Afterward, they were spread plated using TSA (Difco, USA), following incubation at 37 °C for 48 h.
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2.6. Statistical analysis
All of the obtained data were presented as mean ± SD for each treatment. All experiments were done in replicate. Colony-forming units (CFUs) in all experiments were converted to log10 values. Statistical analysis of the data was carried out using Student’s t-test and ANOVA (oneway procedure and repeated measures), Version 20 of SPSS (SPSS Inc, Chicago, IL, USA). The
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ACCEPTED MANUSCRIPT differences among mean values were detected by the Duncan’s multiple’s range test at the significance level of P ≤ 0.05 (Granato et al., 2014; Nunes et al., 2015).
3. Results and Discussion 3.1 Effect of cooking process on probiotic Bacillus
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The results of the counts of inoculated probiotic sporeforming bacteria in raw and cooked
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sausage samples during 45 days of refrigerated (4±1 °C) storage were summarized in Table 2.
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Our findings indicate a 3- 4 log reduction in the counts of the studied spores in the raw batters of
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inoculated sausages before cooking step (80± 1°C for 10 min). It can be speculated that this significant lowering in the number of CFU of the studied Bacillus spores was related to the
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impact of sausage ingredients especially those have impact in spore germination and outgrowth (such as pH, presence of acidulants, sodium nitrite, salt, sodium ascorbate) (Bell and De Lacy,
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1984; Nagler et al., 2014). Furthermore, there was no significant decline in the number of
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Bacillus spores inoculated in cooked sausage immediately after the cooking step (d0) (from 9.82
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to 8.50 log CFU / g for B. subtilis var. Natto ATCC 15245 and from 9.26 to 8.78 log CFU / g for B. coagulans ATCC 31284) (P > 0.05) (Table 3). In another word, the recovery yield of 100%
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was obtained for Bacillus spores in the inoculated cooked sausage after the cooking step. It can
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be speculated that no virtual reduction in the number of the prepared probiotic sporeforming bacteria was primarily can be correlated to their endurance to the cooking step in the sausage manufacture. On the other hand, the cooked sausage samples inoculated with B. coagulans ATCC 31284 spores differs significantly between before and after heat shock (80 °C for 10 min) at days 0, 1 and 45, whereas the viable spore counts of B. subtilis var. Natto ATCC 15245 were significantly different after exposure to the heat shock at the entire refrigerated storage time (Table 2). Total counts of the cooked control sample were differ significantly before and after the
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ACCEPTED MANUSCRIPT heat shock at all the storage time except the first day of storage (P ≤ 0.05). Also, according to obtained results, the application of heat shock at 80 °C for 10 min seems to be unsuitable due to no rise in the viable spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 in cooked sausages (Table 3). As already known and confirmed by this study, in spite of
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the elapsed time during the shelf life and also the influence of composition and ingredients of
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cooked sausage, the spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC
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15245 in raw and cooked samples were not significantly changed within the commercial life of the product (P > 0.05) (Table 3). The probiotic sporeforming bacteria could demonstrate
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acceptable performances in comparison with traditional vegetative probiotic bacteria like
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Bifidobacterium and Lactobacillus in processed foods. In fact, they are capable of enduring high temperatures such as cooking or heating treatments and can be kept viability for extended
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periods of time (survivability during food manufacturing and storage) in comparison with the
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traditional probiotics (Spinosa et al., 2000; Urdaci et al., 2004). Based on the obtained data, in all raw and specially cooked samples, the counts of B. coagulans ATCC 31284 and B. subtilis var.
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Natto ATCC 15245 spores were higher before the heat shock (Table 2). However, the counts of
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the spores inoculated in cooked sausage samples were still above the recommended minimum
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daily therapeutic dose of spore probiotics (i.e. 106 CFU / g) during the entire refrigerated storage.
3.2. Influence of Culture medium The type and composition of media can be considered as implicated relevant parameters in germination and outgrowth, and consequently enumeration and the maximum recovery of spores. When spores are inoculated on a nutrient medium, production of a colony relies on (1) spore germination and outgrowth of a vegetative cell, and (2) endurance and proliferation of the
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ACCEPTED MANUSCRIPT vegetative cell. These two steps (phases) may require varying optimal conditions (Cook and Lund, 1962). The comparison of viable spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 after the inoculation on several selected media was reported in Table 1. According to Table 1, the order of spore counts of B. coagulans ATCC 31284 can be
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summarized as trypticase soy agar (TSA)> plate count agar (PCA)> brain heart infusion (BHI)
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agar, followed by glucose yeast extract agar and nutrient agar media. There was no statistically
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significant difference amongst the recovery in TSA for B. coagulans ATCC 31284 spore (15.22 log CFU / mL), PCA (15.18 log CFU / mL) and BHI agar (15.12 log CFU / mL). On the other
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hand, no significant difference (P > 0.05) was observed between the viable spore counts of B.
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coagulans ATCC 31284 on glucose yeast extract agar 14.40 log CFU / mL and nutrient agar 14.38 log CFU / mL. As shown in Table 1, for B. subtilis var. Natto ATCC 15245 spore,
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However, TSA medium yielded the highest viable enumeration (15.12 log CFU / mL), no
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statistically significant different at P > 0.05 was observed between LB agar (14.83 log CFU / mL), glucose yeast extract agar (14.81 log CFU / mL), which was followed by PCA (14.79 log
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CFU / mL). Also, no statistically significant differences (P > 0.05) was noted in the viable spore
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counts of B. subtilis var. Natto ATCC 15245 spore enumerated on BHI agar (14.70 log CFU / mL) and nutrient agar (14.67 log CFU / mL). However, In current study, determination of fate of
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examined strains of Bacillus by using simple media (nutrient agar) was approached, according to the reported results ,employing of more specific conditions in the case of other probiotic sporeforming bacteria can be recommended . The results clearly revealed that ingredients, pH, a salinity of culture media, and also whether or not nutritionally too weak or too rich are undoubtedly critical factors which could effect on a spore germination and outgrowth and thereby on its maximal recovery. The reason for
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ACCEPTED MANUSCRIPT the low percentage of spores giving colonies on a nutrient medium may be (a) due to notviability of a proportion of spores, (b) they may have viability but fail to germinate in the supplied growth conditions, or (c) germination may be triggered, but the environment was unfavorable and may fail to support emergence (outgrowth) and reproduction (Cook and Lund,
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1962). In spite of the very broad diversity of the genus, most Bacillus species will grow on
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conventional media such as nutrient agar or trypticase soy agar (Vos et al., 2009). In fact, it
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seems that variability on the recovery of the counts of germinated spores is a function of strain and medium. Both B. coagulans ATCC 31284 and especially B. subtilis var. Natto ATCC 15245
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are not considered as Bacillus particular species which require numerous growth factors; rather
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they grow and cultivate on routine or even minimal media such nutrient agar without the special nutritional need(s) (Vos et al., 2009). However, nutritionally rich media could provide growth
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factors which often stimulate better germination and outgrowth of the spores examined (Vos et
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al., 2009). The highest yield of TSA for B. subtilis var. Natto ATCC 15245 spore could be attributed to the composition of TSA which containing enzymatic digests of casein and soybean
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meal provides amino acids, longer-chained peptides, and other organic nitrogenous compounds
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for a variety of microorganisms like B. subtilis. However, the higher price of TSA can be accounted as an obstacle in the routine application in wide range of laboratories. In addition,
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TSA could be even a suitable medium for cultivating B. coagulans ATCC 31284 spore, although not significantly better than PCA, which in turn did not differ statistically from BHI agar (Table 1). Rich media like BHI agar may not cause more viable spore counts of B. subtilis var. Natto ATCC 15245, even though it could have the high recovery for B. coagulans ATCC 31284. LB agar is classified as a rich medium due to providing all of the required nutrients (e.g. such as peptides and peptones, vitamins, and trace elements) for reproducing of bacteria. However, it
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ACCEPTED MANUSCRIPT contains 10 g/L NaCl (Merck, Germany) which renders it as an appropriate medium for cultivating B. subtilis, not B. coagulans. B. subtilis grows well in the presence of up to 7% NaCl; some strains will tolerate 10% NaCl, whereas B. coagulans does not grow when 5% NaCl or more are present (Vos et al., 2009). Considering both PCA and nutrient agar as general culture
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media, the results indicated that PCA resulted in more viable enumeration for the examined
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spores especially for B. coagulans ATCC 31284. Although the yeast extract (in PCA) and beef
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extract (in nutrient agar) both provide sources of carbon, nitrogen and vitamins for general growth of bacteria, the yeast extract also comprises vitamins and amino acids which render the
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medium nutritious (Vos et al., 2009).
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3.3. Influence of heat shock condition
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In conducted experiment, the counts of B. subtilis var. Natto ATCC 15245 and B. coagulans ATCC 31284 spores were measured as 15.20 and 15.10 log CFU / mL in the control
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suspension (without heat shock), respectively (Table 1). However, the used heat shocking
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condition (80 °C/10 min) in the current investigation can be accounted as a common approach for general enumeration of sporeforming bacteria, this condition might not be considered as a
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valid treatment for all of the probiotic sporeforming bacteria.
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In comparison to the controls, the spore counts of B. coagulans ATCC 31284 (15.61 log CFU / mL) and B. subtilis (15.24 log CFU / mL) were highest only after a heat shock at 68 °C for 20 min. which was above the critical minimum heat activation temperature/time combinations for the spores examined. In another hand, however, a higher viable count was obtained by application of heat shock of ≥ 80 °C, as shown in Table 2, in particular for long exposure times caused lowering the counts of the studied spores (Finley and Fields, 1962; Turnbull et al., 2007), which can be called as “heat induced dormancy” (Turnbull et al., 2007). 13
ACCEPTED MANUSCRIPT The findings revealed that heat shock temperatures of B. subtilis var. Natto ATCC 15245 spore should be kept to ≤ 70 °C (e.g. 68 °C) with holding times, not beyond 15-20 min. Our results are in good agreement with the conducted studies by Turnbull et al. (2007). Furthermore, this outcome affirms that the optimal temperature and time of heat shock varies from species to
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species, though not all spores can be activated by heat.
4. Conclusion
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In order to exert the positive therapeutic effects on the health of the host, the recovery and
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survivability of the probiotic bacteria during food manufacturing and storage and until consumption is a critical criterion. The employing of Bacillus species' spores of like B.
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coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 as probiotics is a growing trend
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in the food market. The ability of the probiotic sporeforming bacteria for sustaining within the cooking process, storage stage; traits, and further proposing their capacity for usage, especially in
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functional cooked food products was demonstrated by the current study. The sequence of
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experiments in current investigation were designed in order to determine the best condition (Culture medium + heat shock conditions) to find the proper probiotics counts as well as strain-
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dependent in the case of probiotic sporeforming bacteria The findings also indicated that during sausage processing and the refrigerated storage, the viability rate of the studied Bacillus spores was higher than the minimum recommended daily therapeutic quantities. On the other hand, results suggested that heat shock conditions (species dependent) and the complexity or composition of culture medium in which spores are suspended had a profound effect on the recovery and maximizing germination rates of probiotic sporeforming bacteria. Therefore, the use of general culture medium and heat shock conditions possible to the general enumeration of 14
ACCEPTED MANUSCRIPT sporeforming bacteria is not adequate when dealing with probiotic sporeforming bacteria. Specific conditions and culture medium should be determined in a matrix-dependent approach..
Acknowledgements The authors would like to acknowledge Research Center for Gastroenterology and Liver
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Diseases, Shahid Beheshti University of Medical Sciences and Gooshtiran Company of Meat
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Products, Tehran, Iran for their significant contributions to this research. A. Mousavi Khaneghah
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likes to thank the assistance support of CNPq-TWAS Postgraduate Fellowship (Grant
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#3240274290). A. S. Sant'Ana acknowledges the financial support of "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq) (Grant #302763/2014-7).
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Conflict of interest
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Authors declare that they have no conflict of interest.
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Endres, J. R., Clewell, A., Jade, K. A., Farber, T., Hauswirth, J., & Schauss, A. G. (2009). Safety assessment of a proprietary preparation of a novel Probiotic, Bacillus coagulans, as a food ingredient. Food and Chemical Toxicology, 47, 1231-8. FAO/WHO. (2002). Guidelines for the Evaluation of Probiotics in Food. London Ontario, Canada. Finley, N., & Fields, M. L. (1962). Heat activation and heat-induced dormancy of Bacillus stearothermophilus spores. Applied Microbiology, 10, 231-6. Granato, D., de Araújo, C., Verônica, M., & Jarvis, B. (2014). Observations on the use of statistical methods in Food Science and Technology. Food Research International, 55, 137-149. Golden, D., & Arroyo-Gallyoun, L. (1997). Relationship of Frozen-Food Quality to Microbial 16
ACCEPTED MANUSCRIPT Survival. In M. Erickson, & Y. C. Hung (Eds.), Quality in Frozen Food (pp. 174-193). New York: Springer. Jayamanne, V. S., & Adams, M. R. (2006). Determination of survival, identity and stress resistance of probiotic bifidobacteria in bio-yoghurts. Letters in Applied Microbiology, 42, 189-194.
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Juneja, V. K., Novak, J. S., Huang, L., & Eblen, B. S. (2003). Increased thermotolerance of Clostridium perfringens spores following sublethal heat shock. Food Control, 14, 163168.
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Katsutoshi, A., Shinichi, M., Tadasi, H., Ichirou, T., Kazuya O., Shuji, K., Susunu, I., & Hisakazu, I. (2011). Effect of spore-bearing lactic acid-forming bacteria (Bacillus coagulans SANK 70258) administration in the intestinal environment, defecation frequency, fecal characteristics and dermal characteristics in humans and rats. Microbial Ecology in Health and Disease, 14, 4-13.
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Khan, M. I., Arshad, M. S., Anjum, F. M., Sameen, A., Aneeq-Ur, R., & Gill, W. T. (2011). Meat as a functional food with special reference to probiotic sausages. Food Research International, 44, 3125-3133.
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Lee, N-K., Son, S-H., Jeon, E. B., Jung, G. H., Lee, J-Y., & Paik, H-D. (2015). The prophylactic effect of probiotic Bacillus polyfermenticus KU3 against cancer cells. Journal of Functional Foods, 14,513-8.
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Leuschner, R. G., Bew, J., Cruz, A., Adler, A., Auclair, E., Bertin, G., et al. (2003). Enumeration of probiotic Bacilli spores in animal feed: interlaboratory study. Journal of AOAC International, 86,568-75.
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Lund, B. (2000). Freezing. In B. M. Lund, T. C. Baird-Parker, & G. W. Gould (Eds.), The microbiological safety and quality of food (pp. 122-145). Maryland: Aspen Publishers.
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Nagler, K., Setlow, P., Li Y. Q. & Moeller, R. (2014). High salinity alters the germination behavior of Bacillus subtilis spores with nutrient and non nutrient germinants. Applied and Environmental Microbiology, 80(4), 1314-21. Nithya, V., & Halami, P. (2013). Evaluation of the probiotic characteristics of Bacillus species isolated from different food sources. Annals of Microbiology, 63, 129-137. Nunes, C. A., Alvarenga, V. O., de Souza Sant'Ana, A., Santos, J. S., & Granato, D. (2015). The use of statistical software in food science and technology: Advantages, limitations, and misuses. Food Research International, 75, 270-280. Patel, A. K., Ahire, J. J., Pawar, S. P., Chaudhari, B. L., & Chincholkar, S. B. (2009).
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ACCEPTED MANUSCRIPT Comparative accounts of probiotic characteristics of Bacillus spp. Isolated from food wastes. Food Research International, 42, 505-510. Postollec, F., Mathot, A. G., Bernard, M., Divanac'h, M. L., Pavan, S., & Sohier, D. (2012). Tracking spore-forming bacteria in food: from natural biodiversity to selection by processes. International Journal of Food Microbiology, 158, 1-8.
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Rice, E. W., Rose, L. J., Johnson, C. H., Boczek, L. A., Arduino, M. J., & Reasoner, D. J. (2004). Boiling and Bacillus spores. Emerging Infectious Diseases, 10, 1887-8.
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Shah, N. P. (2001). Functional foods from probiotics and prebiotics. Food Technology, 55, 46-53.
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Spinosa, M. R., Braccini, T., Ricca, E., de Felice, M., Morelli, L., Pozzi, G., et al. (2000). On the fate of ingested Bacillus spores. Research in Microbiology, 151, 361-8. Turnbull, P. C., Frawley, D. A., & Bull, R. L. (2007). Heat activation/shock temperatures for
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Bacillus anthracis spores and the issue of spore plate counts versus true numbers of spores. Journal of Microbiological Methods, 68, 353-7.
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Urdaci, M. C., Bressollier, P., & Pinchuk, I. (2004). Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities. Journal of Clinical Gastroenterology, 38, S86-90. Venema, K., & do Carmo, A. P. (2015). Probiotics and prebiotics: current research and future trends. In K. Venema, & A. P. do Carmo (Eds.), Probiotics and prebiotics: current research and future trends (pp. 3-12). Norfolk: Caister Academic Press.
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Vos, P., Garrity, G., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F. A., et al. (2009). Bergey's manual of systematic bacteriology. New Delhi: Springer-Verlag (1450 PP.).
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Table 1- Comparison of viable counts (log CFU / mL) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores inoculated in several microbial cultures after 48 h at 37 °C and activated under different heat shock conditions a. Selected culture medium or Heat shock condition
Viable spore count
14.67±0.019c 14.79±0.019b 14.70±0.018c 15.12±0.068a 14.81±0.028b 14.83±0.017b
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14.38±0.03b 15.18±0.05a 15.12±0.06a 15.22±0.07a 14.40±0.03b NDb
Nutrient Agar PCA BHI Agar Trypticase Soy Agar Glucose Yeast Extract Agar LB Agar
B. subtilis
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B. coagulans
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Not determined
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b
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Control 15.20±0.02d 15.10±0.01b cd 68 °C for 15 min 15.25±0.02 15.11±0.02b 68 °C for 20 min 15.61±0.01a 15.24±0.02a b 68 °C for 30 min 15.51±0.02 14.92±0.03c c 80 °C for 10 min 15.29±0.01 14.46±0.05d 80 °C for 15 min 15.10±0.05e 14.51±0.07d a 3 Values were determined from serially decimally diluted samples to approximately 10 CFU / mL in sterile 0.1% peptone water and then selected media (trypticase soy agar medium for heat shock treatment) to give a statistically valid range of colonies, and independent experiments were performed in duplicate. Initial concentrations of Bacillus coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 spores were 15.39 and 15.59 log CFU / mL, respectively. Means ± SD values in the same column followed by the same letter are not significantly different (P > 0.05).
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ACCEPTED MANUSCRIPT Table 1- Viable counts (log CFU/g) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores in cooked sausage before and after the application of heat shock (80 °C / 10 min) on NA medium during 45 days of refrigerated (4 ±1 °C) storage.
Before
After
Control
Before
After
d45
8.78±0.04 Aa
8.54±0.05 Aa
8.62±0.02 Aa
8.62±0.04 Aa
8.32±0.10 Aa
7.89±0.05 Bb
7.65±0.02 Bb
8.23±0.12 Ba
8.16±0.12 Ba
8.50±0.15Aa
8.70±0.05Aa
8.77±0.02Aa
8.21±0.09Aa
8.39±0.04Aa
7.52±0.06Bb
7.78±0.03Bb
7.23±0.08Bb
7.27±0.9Bb
2.29±0.04Aa
2.31±0.03Aa
2.26±0.05Aa
2.30±0.05Aa
1.89±0.05Aa
2.27±0.11Ba
1.84±0.08Bb
1.85±0.07Bb
1.81±0.09Bb
1.78±0.05Bb
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d30
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B. subtilis
d15
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After
d1
7.80±0.03Bb
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Before
d0
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B. coagulans
Storage time (days) b
Heat shock
8.08±0.12
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Treatment a
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b
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B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores were inoculated into the sausage batters at 12.88 and 12.96 log CFU / 10Kg, respectively. B. coagulans: sausage formulation containing B. coagulans ATCC 31284; B. subtilis: sausage formulation containing B. subtilis var. Natto ATCC 15245, Control: production control without inoculation of probiotic sporeforming bacteria.
A, B
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Each value represents the mean of two independent experiments (± SD).d0: the time immediately after the manufacture in raw sausages and the time immediately after cooking in cooked sausages; d1:1 day of storage; d15: 15 days of storage; d30: 30 days of storage; d45: 45 days of storage.
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Different capital letters in the same row denote significant differences (P ≤ 0.05) among days of storage for the same treatment. a, b Different lower case letters in the same column denote significant difference (P ≤ 0.05) among treatments.
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Cooking process b
Raw
Storage time (days) c d0
d1
d15
d30
d45
9.26±0.13 Aa
9.35±0.14 Aa
9.80±0.05Aa
9.12±0.11Aa
9.07±0.10 Aa
8.78±0.04 Ba
8.54±0.05 Ba
8.62±0.02 Bb
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Treatment a
8.62±0.04 Ba
8.32±0.10 Ba
9.82±0.03 Aa
9.89±0.02 Aa
9.75±0.02Aa
9.64±0.04 Aa
9.83±0.02Aa
8.50±0.15Ba
8.70±0.05Bb
8.77±0.02Bb
8.21±0.09Bb
8.39±0.04Bb
3.20±0.03 Aa
2.70±0.03 Aa
3.00±0.07 Aa
3.10±0.04 Aa
3.00±0.09 Aa
2.31±0.03Ba
2.26±0.05Ba
2.30±0.05Ba
1.89±0.05Bb
Raw
Raw Control
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2.39±0.04Ba
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Cooked
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B. subtilis
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Cooked
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Cooked
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B. coagulans
Table 3- Viable counts (log CFU/g) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores in raw and cooked sausage. a
b
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B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores were inoculated into the sausage batters at 12.88 and 12.96 log CFU / 10Kg, respectively. B. coagulans: sausage formulation containing B. coagulans ATCC 31284; B. subtilis: sausage formulation containing B. subtilis var. Natto ATCC 15245, Control: production control without inoculation of probiotic sporeforming bacteria. Cooking process was done at 80± 1°C for approximately 60 min.
c
A, B
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Each value represents the mean of two independent experiments (± SD).d0: the time immediately after the manufacture in raw sausages and the time immediately after cooking in cooked sausages; d1:1 day of storage; d15: 15 days of storage; d30: 30 days of storage; d45: 45 days of storage. Different capital letters in the same row denote significant differences (P ≤ 0.05) among days of storage for the same treatment. a, b Different lower case letters in the same column denote significant difference (P ≤ 0.05) among treatments.
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ACCEPTED MANUSCRIPT Highlights Cooked sausage as potential carrier for the delivery of Bacillus probiotics Application of sporeforming probiotics in formulation of functional foods due to high thermal
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resistance
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Heat shock conditions and the medium as the key factors for the maximum recovery of
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Bacillus probiotic
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Mojtaba Jafari, Amir M. Mortazavian, Hedayat Hosseini, Fahimeh Safaei, Amin M. Khaneghah, Anderson S. Sant'Ana PII: DOI: Reference:
S0963-9969(17)30089-3 doi: 10.1016/j.foodres.2017.03.001 FRIN 6608
To appear in:
Food Research International
Received date: Revised date: Accepted date:
13 January 2017 1 March 2017 3 March 2017
Please cite this article as: Mojtaba Jafari, Amir M. Mortazavian, Hedayat Hosseini, Fahimeh Safaei, Amin M. Khaneghah, Anderson S. Sant'Ana , Probiotic Bacillus: Fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/ j.foodres.2017.03.001
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ACCEPTED MANUSCRIPT Probiotic Bacillus: fate during sausage processing and storage and influence of different culturing conditions on recovery of their spores Mojtaba Jafari 1, Amir M. Mortazavian 1*, Hedayat Hosseini 1, Fahimeh Safaei 1, Amin M. Khaneghah2*, Anderson S. Sant'Ana2 1
Department of Food Science and Technology, Faculty of Nutrition Sciences, National Nutrition and Food Technology Research Institute, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2
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Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), Campinas, São Paulo, Brazil
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*Corresponding Authors: Amir M. Mortazavian E-mail address: [email protected] Postal address: 46, West Arghavan St., Farahzadi Blvd., Shahrak Qods, National Nutrition & Food Technology Research Institute, Zip Code: 1981619573, P.O. Box 19395-4741, Tehran, Iran Tel: +98 21 22357483-5; Fax: +98 21 22360660.
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Amin Mousavi Khaneghah E-mail address: [email protected] Department of Food Science, Faculty of Food Engineering, State University of Campinas (UNICAMP), Campinas, São Paulo, Rua Monteiro Lobato, 80. Caixa Postal: 6121. CEP: 13083-862. Campinas. São Paulo, Brazil. Telephone: +55(19) 3521-0091. Fax: +55(19) 3521-2153
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ACCEPTED MANUSCRIPT Abstract The current study was designed to assess the fate of probiotic strains of Bacillus coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 (as spores) during processing and refrigerated storage of cooked sausage as well as the influence of culturing conditions on the
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recovery of spores. The highest recovery of B. coagulans ATCC 31284 and B. subtilis var. Natto
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ATCC 15245 spores were obtained on trypticase soy agar (TSA) (15.22 and 15.12 log CFU /
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mL, respectively) which was carried out under conducted heat shock (68 °C, 20 min) (15.61 and 15.24 log CFU / mL, respectively). According to result; a 3- 4 log reduction in the counts of the
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spores inoculated in the raw batter of sausage after the cooking step was observed. The findings
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revealed that the counts of the spores were significantly changed after heat shock (80 °C /10 min) (P ≤ 0.05). Data indicated that the count during sausage processing and the entire cold storage
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was more than 106 CFU /g. The ability of the probiotic sporeforming bacteria for sustaining
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within the cooking process, storage stage; traits that cannot be ensured with vegetative probiotic bacteria, which proposed their capacity for usage, especially in functional cooked food products
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was demonstrated by the results of the current study.
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Keywords: Cooked sausage, Enumeration, Probiotic Bacillus, spores
Studied Chemical compounds in this article TE buffer solution (PubChem CID:16218629); TRIS.HCL (PubChem CID:93573); NaCl (PubChem CID:5234); KCl (PubChem CID:4873 ); Sodium dodecyl sulfate (PubChem CID:3423265); Egg white lysozyme (PubChem CID:71581463).
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Introduction The development and consumption of food products containing probiotics as an
increasingly global consumer trend could achieve a considerable remark in functional food market (Khan et al., 2011). The commonly accepted definition of probiotics by FAO and WHO
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can be noted as “live microorganisms while ingested in adequate amounts could offer a health
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benefit on the host” (FAO/WHO, 2002; Venema and do Carmo, 2015; Almada et al., 2015).
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Although, no agreement has been conducted for the recommended minimum level of
of 106 CFU g-
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or ml-
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ingested probiotics to provide the proposed functionality (Champagne et al., 2005), populations in the final product at the time of consumption, supposing a daily
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consumption of 100 g or mL, was established as a minimum daily therapeutic dose of probiotic cultures in processed foods (Ashraf and Shah, 2011). This will result in a dose of 108 CFU,
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which should be adequate to confer health benefits to consumers (Jayamanne and Adams,
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2006).With the application of probiotic microorganisms in foods aiming to result in a health
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benefit, the assessment of the fate of probiotic microorganisms during processing and storage is of foremost importance. The ability of probiotics in order to survive the harsh and severe
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manufacturing process, the storage of the product and finally passage through the gastrointestinal
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tract can be considered as key concerns for both manufacturing and marketing of meat products (Shah 2001; Bezkorovainy, 2001). For instance, the required microorganisms for the production of probiotic foods such as Lactobacillus and Bifidobacterium could not survive such thermal processing (cooking at approximately 75°C at the cold point) (Ruiz et al., 2011; Baka et al., 2015). Also, providing the knowledge on the best conditions for recovery and enumeration of probiotic Bacillus are crucial. Moreover, depending on the applied conditions for recovery and enumeration, the counts of probiotics can vary significantly (Ashraf and Shah, 2011). The
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ACCEPTED MANUSCRIPT application of Bacillus species’ spores as probiotic dietary supplements is expanding extensively and rather rapidly due to some health benefices such as immune stimulation, antimicrobial activities, and competitive exclusion (Spinosa et al., 2000; Patel et al., 2009; Katsutoshi et al., 2011). Among the 100 known Bacillus spp., only a few strains (including B. coagulans and B.
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subtilis var. Natto) have been approached as probiotics for human consumption (Urdaci et al.,
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2004; Nithya and Halami, 2013). Therefore, sporeforming probiotic bacteria could be considered
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as a valuable solution for overcoming the limitations related to stability of Bifidobacterium and Lactobacillus strains during processing and storage of probiotic foods (Patel et al., 2009; Endres
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et al., 2009; Cutting, 2011; Katsutoshi et al., 2011; Postollec et al., 2012).
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To the best of author's knowledge, this is the first conducted study aiming to assess the fate of probiotic Bacillus strains during cooking and storage of sausages. In addition, conditions for
Materials and Methods
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2.
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enumeration methods were assessed.
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recovery of probiotic Bacillus using different culture media, heat shock conditions, and
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2.1. Materials and chemical reagents All used chemical and reagents in analytical grade were supplied by Merck Co.
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(Darmstadt, Germany).
2.2. Bacterial spores and spore preparation Commercial probiotic sporeforming bacteria strains of B. coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 were obtained from Natures Only, INC., USA and World Intellectual Resource Co., Taiwan, respectively (Bhat et al., 2013; Azimirad and 4
ACCEPTED MANUSCRIPT Alebouyeh, 2016). In order to evaluate the purity of the strains, each one was separately streaked onto Müller-Hinton agar (Merck, Germany). Then, Gram staining and direct microscopic examinations were carried out after 7-10 days to confirm whether sporulation was completed. Further, the described method by Alebouyeh et al. (2009) was used for the preparation of spore
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suspensions. After intermittent steps of washing with sterile deionized water (SDW) and
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centrifugation (12000 × g, 15 min), the final suspensions obtained were diluted in defined
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amounts of sterile deionized water. The concentration of spores per mL of each spore suspension was verified using Neubauer chamber (hemocytometer), confirmed through plate count
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(Alebouyeh et al., 2009), the following storage at -70 °C for further analysis.
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2.3. Production of cooked sausage 2.3.1. Cooked sausage formulations
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Frozen meat packs (15% fat) were kept at 4 °C for approximately 18 h for thawing. The
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used primary formula for sausage preparation contained minced meat (40%), water (as ground ice) (22%), vegetable oil (19%), salt (NaCl) (1.5%), polyphosphate (0.35%), sodium nitrite (120
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ppm), sodium ascorbate (400 ppm), filler agents including texturized soy protein (19%), wheat
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flour, frozen garlic (1.8%), wheat starch, gluten, pasteurized whole egg (2%), sugar (1%) and
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spice mix specially prepared for this type of meat product ( ca. 1%). 2.3.2. Sausage preparation and inoculation with probiotic Bacillus In practice, the control sausage with no added probiotic sporeforming bacteria was produced as follows: the grounded minced meat was mixed with salt (NaCl), nitrite, and polyphosphate at low speed in a 10 kg bowel mini cutter device (Allen, K21 Ras 83132, Germany) for about 2 min. Then one-half of the proposed water in the formulation (as ground ice) was added and subsequently mixed thoroughly in the cutter at high speed. Consequently
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additional 3 min, approximately). In order to monitor and verify the temperature of the emulsion,
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(Kane-May, KM330, Harlow, Germany) was applied.
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which should be maintained below 12 °C during the preparation of batter, a digital thermometer
In order to the production of inoculated formulations, spores of B. coagulans ATCC 31284
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and Bacillus subtilis var. Natto ATCC 15245 were individually inoculated at the start of cutter
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step after adding the first half of water and mixed at low speed for approximately 3 min in order to obtain a proper dispersion of the spores in the batter. The added inoculum concentration to the
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bowel cutter were 12.88 and 12.96 log CFU / 10 kg (or 8.88 and 8.96 log CFU/g), respectively.
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All batches after comminution/ chopping step were filled into artificial polyamide casings (24 mm diameter) (Arta, Tabriz, Iran) using a stuffer (Handtman, Germany). Then, sausages were
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cooked in a cooking cabinet with steam at 80± 1°C (up to reaching the temperature of the
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geometric center to 75 °C) for approximately 60 min. Subsequently, the cooked sausages were cooled down to about 25 °C using cold water (12 °C) for approximately 8–10 min. Then, cooked
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sausages were stored at 4±1 °C for 45 days. For each batch, the above-described processing was repeated two times. Samples were randomly taken from each sausage batch before and immediately after the cooking step of the sausage manufacture and at 1, 15, 30, and 45 days of refrigerated storage time. In fact, sausages were sampled just after filling into casings by selecting at random three segments of sausage samples from each treatment. It should be mentioned that separate sample units were taken for each type of test.
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count on nutrient agar (Himedia, India). The nutrient agar was chosen due to the low price and
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high availability as the general medium in most of the laboratories. The enumeration was done
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before and after subjecting samples to a thermal treatment (80 °C for 10 min) (Leuschner et al., 2003). Plates were aerobically incubated at 37 °C for 2 days, and colonies grown were counted
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and the results expressed as CFU/g.
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Despite the very wide diversity of the genus, the majority of Bacillus species including the investigated strains in the current study will grow well on routine and general media such as
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nutrient agar or trypticase soy agar (Vos et al., 2009). Also, the general sporeformers can be
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found in the batter of inoculated sausage samples. So, by subtracting these counts from the initial counts of added probiotic sporeforming bacteria to the sausage samples, the actual counts of B.
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coagulans and B. subtilis spores in raw and cooked sausage can be calculated. In addition, there
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is no selective or selective differential medium for enumeration of bacteria in Bacillus genus so their counting could be based on general media. Eventually, the actual counts of Bacillus
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coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 spores supplemented in raw and cooked sausage would be obtained 2.4. Influence of culture medium on the recovery of probiotic Bacillus spores In the second part of investigation, in order to measure the recovery of the probiotic sporeforming bacteria, the recommended seven general culture media (Merck, Germany; Himedia, India; Difco, USA), among the most frequently cited literature, were approached
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at 37 °C.
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2.5. Influence of heat shock conditions on the recovery of probiotic Bacillus spores Finally, the heat shock (or heat-activation) treatments of the probiotic sporeforming
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bacteria, were carried out according to the suggested treatments including exposure to a
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temperature of 68 °C for 15, 20, and 30 min, and 80 °C for 10 and 15 min, respectively (Alcock, 1984; Juneja et al., 2003; Turnbull et al., 2007). The sporicidal effect of temperatures above 80
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°C (especially 90 °C) was taken into consideration (Rice et al., 2004; Turnbull et al., 2007). In
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practice, following an appropriate dilution, suspensions of B. coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 were exposed to heat (68 °C or 80 °C) in a water bath
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(Memmert, Germany) for the times mentioned above and then they were immediately cooled
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down using ice water bath. Afterward, they were spread plated using TSA (Difco, USA), following incubation at 37 °C for 48 h.
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2.6. Statistical analysis
All of the obtained data were presented as mean ± SD for each treatment. All experiments were done in replicate. Colony-forming units (CFUs) in all experiments were converted to log10 values. Statistical analysis of the data was carried out using Student’s t-test and ANOVA (oneway procedure and repeated measures), Version 20 of SPSS (SPSS Inc, Chicago, IL, USA). The
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ACCEPTED MANUSCRIPT differences among mean values were detected by the Duncan’s multiple’s range test at the significance level of P ≤ 0.05 (Granato et al., 2014; Nunes et al., 2015).
3. Results and Discussion 3.1 Effect of cooking process on probiotic Bacillus
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The results of the counts of inoculated probiotic sporeforming bacteria in raw and cooked
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sausage samples during 45 days of refrigerated (4±1 °C) storage were summarized in Table 2.
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Our findings indicate a 3- 4 log reduction in the counts of the studied spores in the raw batters of
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inoculated sausages before cooking step (80± 1°C for 10 min). It can be speculated that this significant lowering in the number of CFU of the studied Bacillus spores was related to the
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impact of sausage ingredients especially those have impact in spore germination and outgrowth (such as pH, presence of acidulants, sodium nitrite, salt, sodium ascorbate) (Bell and De Lacy,
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1984; Nagler et al., 2014). Furthermore, there was no significant decline in the number of
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Bacillus spores inoculated in cooked sausage immediately after the cooking step (d0) (from 9.82
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to 8.50 log CFU / g for B. subtilis var. Natto ATCC 15245 and from 9.26 to 8.78 log CFU / g for B. coagulans ATCC 31284) (P > 0.05) (Table 3). In another word, the recovery yield of 100%
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was obtained for Bacillus spores in the inoculated cooked sausage after the cooking step. It can
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be speculated that no virtual reduction in the number of the prepared probiotic sporeforming bacteria was primarily can be correlated to their endurance to the cooking step in the sausage manufacture. On the other hand, the cooked sausage samples inoculated with B. coagulans ATCC 31284 spores differs significantly between before and after heat shock (80 °C for 10 min) at days 0, 1 and 45, whereas the viable spore counts of B. subtilis var. Natto ATCC 15245 were significantly different after exposure to the heat shock at the entire refrigerated storage time (Table 2). Total counts of the cooked control sample were differ significantly before and after the
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ACCEPTED MANUSCRIPT heat shock at all the storage time except the first day of storage (P ≤ 0.05). Also, according to obtained results, the application of heat shock at 80 °C for 10 min seems to be unsuitable due to no rise in the viable spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 in cooked sausages (Table 3). As already known and confirmed by this study, in spite of
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the elapsed time during the shelf life and also the influence of composition and ingredients of
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cooked sausage, the spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC
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15245 in raw and cooked samples were not significantly changed within the commercial life of the product (P > 0.05) (Table 3). The probiotic sporeforming bacteria could demonstrate
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acceptable performances in comparison with traditional vegetative probiotic bacteria like
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Bifidobacterium and Lactobacillus in processed foods. In fact, they are capable of enduring high temperatures such as cooking or heating treatments and can be kept viability for extended
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periods of time (survivability during food manufacturing and storage) in comparison with the
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traditional probiotics (Spinosa et al., 2000; Urdaci et al., 2004). Based on the obtained data, in all raw and specially cooked samples, the counts of B. coagulans ATCC 31284 and B. subtilis var.
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Natto ATCC 15245 spores were higher before the heat shock (Table 2). However, the counts of
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the spores inoculated in cooked sausage samples were still above the recommended minimum
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daily therapeutic dose of spore probiotics (i.e. 106 CFU / g) during the entire refrigerated storage.
3.2. Influence of Culture medium The type and composition of media can be considered as implicated relevant parameters in germination and outgrowth, and consequently enumeration and the maximum recovery of spores. When spores are inoculated on a nutrient medium, production of a colony relies on (1) spore germination and outgrowth of a vegetative cell, and (2) endurance and proliferation of the
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ACCEPTED MANUSCRIPT vegetative cell. These two steps (phases) may require varying optimal conditions (Cook and Lund, 1962). The comparison of viable spore counts of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 after the inoculation on several selected media was reported in Table 1. According to Table 1, the order of spore counts of B. coagulans ATCC 31284 can be
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summarized as trypticase soy agar (TSA)> plate count agar (PCA)> brain heart infusion (BHI)
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agar, followed by glucose yeast extract agar and nutrient agar media. There was no statistically
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significant difference amongst the recovery in TSA for B. coagulans ATCC 31284 spore (15.22 log CFU / mL), PCA (15.18 log CFU / mL) and BHI agar (15.12 log CFU / mL). On the other
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hand, no significant difference (P > 0.05) was observed between the viable spore counts of B.
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coagulans ATCC 31284 on glucose yeast extract agar 14.40 log CFU / mL and nutrient agar 14.38 log CFU / mL. As shown in Table 1, for B. subtilis var. Natto ATCC 15245 spore,
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However, TSA medium yielded the highest viable enumeration (15.12 log CFU / mL), no
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statistically significant different at P > 0.05 was observed between LB agar (14.83 log CFU / mL), glucose yeast extract agar (14.81 log CFU / mL), which was followed by PCA (14.79 log
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CFU / mL). Also, no statistically significant differences (P > 0.05) was noted in the viable spore
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counts of B. subtilis var. Natto ATCC 15245 spore enumerated on BHI agar (14.70 log CFU / mL) and nutrient agar (14.67 log CFU / mL). However, In current study, determination of fate of
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examined strains of Bacillus by using simple media (nutrient agar) was approached, according to the reported results ,employing of more specific conditions in the case of other probiotic sporeforming bacteria can be recommended . The results clearly revealed that ingredients, pH, a salinity of culture media, and also whether or not nutritionally too weak or too rich are undoubtedly critical factors which could effect on a spore germination and outgrowth and thereby on its maximal recovery. The reason for
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ACCEPTED MANUSCRIPT the low percentage of spores giving colonies on a nutrient medium may be (a) due to notviability of a proportion of spores, (b) they may have viability but fail to germinate in the supplied growth conditions, or (c) germination may be triggered, but the environment was unfavorable and may fail to support emergence (outgrowth) and reproduction (Cook and Lund,
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1962). In spite of the very broad diversity of the genus, most Bacillus species will grow on
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conventional media such as nutrient agar or trypticase soy agar (Vos et al., 2009). In fact, it
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seems that variability on the recovery of the counts of germinated spores is a function of strain and medium. Both B. coagulans ATCC 31284 and especially B. subtilis var. Natto ATCC 15245
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are not considered as Bacillus particular species which require numerous growth factors; rather
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they grow and cultivate on routine or even minimal media such nutrient agar without the special nutritional need(s) (Vos et al., 2009). However, nutritionally rich media could provide growth
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factors which often stimulate better germination and outgrowth of the spores examined (Vos et
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al., 2009). The highest yield of TSA for B. subtilis var. Natto ATCC 15245 spore could be attributed to the composition of TSA which containing enzymatic digests of casein and soybean
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meal provides amino acids, longer-chained peptides, and other organic nitrogenous compounds
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for a variety of microorganisms like B. subtilis. However, the higher price of TSA can be accounted as an obstacle in the routine application in wide range of laboratories. In addition,
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TSA could be even a suitable medium for cultivating B. coagulans ATCC 31284 spore, although not significantly better than PCA, which in turn did not differ statistically from BHI agar (Table 1). Rich media like BHI agar may not cause more viable spore counts of B. subtilis var. Natto ATCC 15245, even though it could have the high recovery for B. coagulans ATCC 31284. LB agar is classified as a rich medium due to providing all of the required nutrients (e.g. such as peptides and peptones, vitamins, and trace elements) for reproducing of bacteria. However, it
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ACCEPTED MANUSCRIPT contains 10 g/L NaCl (Merck, Germany) which renders it as an appropriate medium for cultivating B. subtilis, not B. coagulans. B. subtilis grows well in the presence of up to 7% NaCl; some strains will tolerate 10% NaCl, whereas B. coagulans does not grow when 5% NaCl or more are present (Vos et al., 2009). Considering both PCA and nutrient agar as general culture
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media, the results indicated that PCA resulted in more viable enumeration for the examined
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spores especially for B. coagulans ATCC 31284. Although the yeast extract (in PCA) and beef
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extract (in nutrient agar) both provide sources of carbon, nitrogen and vitamins for general growth of bacteria, the yeast extract also comprises vitamins and amino acids which render the
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medium nutritious (Vos et al., 2009).
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3.3. Influence of heat shock condition
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In conducted experiment, the counts of B. subtilis var. Natto ATCC 15245 and B. coagulans ATCC 31284 spores were measured as 15.20 and 15.10 log CFU / mL in the control
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suspension (without heat shock), respectively (Table 1). However, the used heat shocking
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condition (80 °C/10 min) in the current investigation can be accounted as a common approach for general enumeration of sporeforming bacteria, this condition might not be considered as a
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valid treatment for all of the probiotic sporeforming bacteria.
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In comparison to the controls, the spore counts of B. coagulans ATCC 31284 (15.61 log CFU / mL) and B. subtilis (15.24 log CFU / mL) were highest only after a heat shock at 68 °C for 20 min. which was above the critical minimum heat activation temperature/time combinations for the spores examined. In another hand, however, a higher viable count was obtained by application of heat shock of ≥ 80 °C, as shown in Table 2, in particular for long exposure times caused lowering the counts of the studied spores (Finley and Fields, 1962; Turnbull et al., 2007), which can be called as “heat induced dormancy” (Turnbull et al., 2007). 13
ACCEPTED MANUSCRIPT The findings revealed that heat shock temperatures of B. subtilis var. Natto ATCC 15245 spore should be kept to ≤ 70 °C (e.g. 68 °C) with holding times, not beyond 15-20 min. Our results are in good agreement with the conducted studies by Turnbull et al. (2007). Furthermore, this outcome affirms that the optimal temperature and time of heat shock varies from species to
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species, though not all spores can be activated by heat.
4. Conclusion
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In order to exert the positive therapeutic effects on the health of the host, the recovery and
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survivability of the probiotic bacteria during food manufacturing and storage and until consumption is a critical criterion. The employing of Bacillus species' spores of like B.
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coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 as probiotics is a growing trend
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in the food market. The ability of the probiotic sporeforming bacteria for sustaining within the cooking process, storage stage; traits, and further proposing their capacity for usage, especially in
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functional cooked food products was demonstrated by the current study. The sequence of
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experiments in current investigation were designed in order to determine the best condition (Culture medium + heat shock conditions) to find the proper probiotics counts as well as strain-
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dependent in the case of probiotic sporeforming bacteria The findings also indicated that during sausage processing and the refrigerated storage, the viability rate of the studied Bacillus spores was higher than the minimum recommended daily therapeutic quantities. On the other hand, results suggested that heat shock conditions (species dependent) and the complexity or composition of culture medium in which spores are suspended had a profound effect on the recovery and maximizing germination rates of probiotic sporeforming bacteria. Therefore, the use of general culture medium and heat shock conditions possible to the general enumeration of 14
ACCEPTED MANUSCRIPT sporeforming bacteria is not adequate when dealing with probiotic sporeforming bacteria. Specific conditions and culture medium should be determined in a matrix-dependent approach..
Acknowledgements The authors would like to acknowledge Research Center for Gastroenterology and Liver
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Diseases, Shahid Beheshti University of Medical Sciences and Gooshtiran Company of Meat
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Products, Tehran, Iran for their significant contributions to this research. A. Mousavi Khaneghah
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likes to thank the assistance support of CNPq-TWAS Postgraduate Fellowship (Grant
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#3240274290). A. S. Sant'Ana acknowledges the financial support of "Conselho Nacional de Desenvolvimento Científico e Tecnológico" (CNPq) (Grant #302763/2014-7).
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Conflict of interest
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Authors declare that they have no conflict of interest.
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Table 1- Comparison of viable counts (log CFU / mL) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores inoculated in several microbial cultures after 48 h at 37 °C and activated under different heat shock conditions a. Selected culture medium or Heat shock condition
Viable spore count
14.67±0.019c 14.79±0.019b 14.70±0.018c 15.12±0.068a 14.81±0.028b 14.83±0.017b
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14.38±0.03b 15.18±0.05a 15.12±0.06a 15.22±0.07a 14.40±0.03b NDb
Nutrient Agar PCA BHI Agar Trypticase Soy Agar Glucose Yeast Extract Agar LB Agar
B. subtilis
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B. coagulans
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Not determined
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b
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Control 15.20±0.02d 15.10±0.01b cd 68 °C for 15 min 15.25±0.02 15.11±0.02b 68 °C for 20 min 15.61±0.01a 15.24±0.02a b 68 °C for 30 min 15.51±0.02 14.92±0.03c c 80 °C for 10 min 15.29±0.01 14.46±0.05d 80 °C for 15 min 15.10±0.05e 14.51±0.07d a 3 Values were determined from serially decimally diluted samples to approximately 10 CFU / mL in sterile 0.1% peptone water and then selected media (trypticase soy agar medium for heat shock treatment) to give a statistically valid range of colonies, and independent experiments were performed in duplicate. Initial concentrations of Bacillus coagulans ATCC 31284 and Bacillus subtilis var. Natto ATCC 15245 spores were 15.39 and 15.59 log CFU / mL, respectively. Means ± SD values in the same column followed by the same letter are not significantly different (P > 0.05).
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ACCEPTED MANUSCRIPT Table 1- Viable counts (log CFU/g) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores in cooked sausage before and after the application of heat shock (80 °C / 10 min) on NA medium during 45 days of refrigerated (4 ±1 °C) storage.
Before
After
Control
Before
After
d45
8.78±0.04 Aa
8.54±0.05 Aa
8.62±0.02 Aa
8.62±0.04 Aa
8.32±0.10 Aa
7.89±0.05 Bb
7.65±0.02 Bb
8.23±0.12 Ba
8.16±0.12 Ba
8.50±0.15Aa
8.70±0.05Aa
8.77±0.02Aa
8.21±0.09Aa
8.39±0.04Aa
7.52±0.06Bb
7.78±0.03Bb
7.23±0.08Bb
7.27±0.9Bb
2.29±0.04Aa
2.31±0.03Aa
2.26±0.05Aa
2.30±0.05Aa
1.89±0.05Aa
2.27±0.11Ba
1.84±0.08Bb
1.85±0.07Bb
1.81±0.09Bb
1.78±0.05Bb
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d30
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B. subtilis
d15
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After
d1
7.80±0.03Bb
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Before
d0
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B. coagulans
Storage time (days) b
Heat shock
8.08±0.12
Bb
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Treatment a
a
b
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B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores were inoculated into the sausage batters at 12.88 and 12.96 log CFU / 10Kg, respectively. B. coagulans: sausage formulation containing B. coagulans ATCC 31284; B. subtilis: sausage formulation containing B. subtilis var. Natto ATCC 15245, Control: production control without inoculation of probiotic sporeforming bacteria.
A, B
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Each value represents the mean of two independent experiments (± SD).d0: the time immediately after the manufacture in raw sausages and the time immediately after cooking in cooked sausages; d1:1 day of storage; d15: 15 days of storage; d30: 30 days of storage; d45: 45 days of storage.
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Cooking process b
Raw
Storage time (days) c d0
d1
d15
d30
d45
9.26±0.13 Aa
9.35±0.14 Aa
9.80±0.05Aa
9.12±0.11Aa
9.07±0.10 Aa
8.78±0.04 Ba
8.54±0.05 Ba
8.62±0.02 Bb
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Treatment a
8.62±0.04 Ba
8.32±0.10 Ba
9.82±0.03 Aa
9.89±0.02 Aa
9.75±0.02Aa
9.64±0.04 Aa
9.83±0.02Aa
8.50±0.15Ba
8.70±0.05Bb
8.77±0.02Bb
8.21±0.09Bb
8.39±0.04Bb
3.20±0.03 Aa
2.70±0.03 Aa
3.00±0.07 Aa
3.10±0.04 Aa
3.00±0.09 Aa
2.31±0.03Ba
2.26±0.05Ba
2.30±0.05Ba
1.89±0.05Bb
Raw
Raw Control
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2.39±0.04Ba
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Table 3- Viable counts (log CFU/g) of B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores in raw and cooked sausage. a
b
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B. coagulans ATCC 31284 and B. subtilis var. Natto ATCC 15245 spores were inoculated into the sausage batters at 12.88 and 12.96 log CFU / 10Kg, respectively. B. coagulans: sausage formulation containing B. coagulans ATCC 31284; B. subtilis: sausage formulation containing B. subtilis var. Natto ATCC 15245, Control: production control without inoculation of probiotic sporeforming bacteria. Cooking process was done at 80± 1°C for approximately 60 min.
c
A, B
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Each value represents the mean of two independent experiments (± SD).d0: the time immediately after the manufacture in raw sausages and the time immediately after cooking in cooked sausages; d1:1 day of storage; d15: 15 days of storage; d30: 30 days of storage; d45: 45 days of storage. Different capital letters in the same row denote significant differences (P ≤ 0.05) among days of storage for the same treatment. a, b Different lower case letters in the same column denote significant difference (P ≤ 0.05) among treatments.
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ACCEPTED MANUSCRIPT Highlights Cooked sausage as potential carrier for the delivery of Bacillus probiotics Application of sporeforming probiotics in formulation of functional foods due to high thermal
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Heat shock conditions and the medium as the key factors for the maximum recovery of
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