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Short communication: Effects of vacuum freeze-drying on inactivation of Cronobacter sakazakii ATCC29544 in liquid media with different initial inoculum levels

  • Author Footnotes
    1 These authors contributed to the manuscript equally.
    Rui Jiao
    Footnotes
    1 These authors contributed to the manuscript equally.
    Affiliations
    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China

    State Key Laboratory of Applied Microbiology Southern China, Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
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  • Author Footnotes
    1 These authors contributed to the manuscript equally.
    Jina Gao
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    1 These authors contributed to the manuscript equally.
    Affiliations
    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
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    1 These authors contributed to the manuscript equally.
    Xiyan Zhang
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    1 These authors contributed to the manuscript equally.
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    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
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    1 These authors contributed to the manuscript equally.
    Maofeng Zhang
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    1 These authors contributed to the manuscript equally.
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    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
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  • Jiren Chen
    Affiliations
    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China
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  • Qingping Wu
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    State Key Laboratory of Applied Microbiology Southern China, Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
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  • Jumei Zhang
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    State Key Laboratory of Applied Microbiology Southern China, Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
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  • Yingwang Ye
    Correspondence
    Corresponding author
    Affiliations
    Center of Detection and Control of Foodborne Risk Factors, School of Food Science and Engineering, Hefei University of Technology, Hefei, 230009, China

    State Key Laboratory of Applied Microbiology Southern China, Provincial Key Laboratory of Microbiology Culture Collection and Application, Guangdong Institute of Microbiology, Guangzhou, 510070, China
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    1 These authors contributed to the manuscript equally.
Open ArchivePublished:January 12, 2017DOI:https://doi.org/10.3168/jds.2016-11937

      ABSTRACT

      Vacuum freeze-drying is an important food-processing technology for valid retention of nutrients and bioactive compounds. Cronobacter sakazakii has been reported to be associated with severe infections in neonates through consumption of contaminated powdered infant formula. In this study, effects of vacuum freeze-drying treatment for 12, 24, and 36 h on inactivation of C. sakazakii with different initial inoculum levels in sterile water, tryptic soy broth (TSB), skim milk, and whole milk were determined. Results indicated that the lethality rate of C. sakazakii in each sample increased with the extension of vacuum freeze-drying time. With initial inoculum levels of 102 and 103 cfu/mL, the survival of C. sakazakii in different liquid media was significantly affected by vacuum freeze-drying for 12, 24, and 36 h. In addition, the lethality rates of C. sakazakii in whole milk, skim milk, and TSB was significantly reduced compared with those in sterile water. Furthermore, whole milk showed the strongest protective role for C. sakazakii cells, followed by skim milk and TSB medium. Using the scanning electron microscope, the intracellular damage and obvious distortion of C. sakazakii cells were observed after vacuum freeze-drying for 24 and 36 h compared with the untreated sample, and the injured cells increased with the extension of vacuum-drying time. We concluded that inactivation of vacuum freeze-drying on C. sakazakii cells is related to the food matrix, and a combination with other methods for inactivating C. sakazakii is required for ensuring microbial safety of powdered infant formula.

      Key words

      Short Communication

      Cronobacter sakazakii is considered an opportunistic food-borne pathogen involved in meningitis, septicemia, and necrotizing enterocolitis in neonates (
      • Ye Y.W.
      • Li H.
      • Wu Q.P.
      • Zhang J.M.
      • Lu Y.D.
      The Cronobacter sp. in milk and dairy products: Detection and typing.
      ). Epidemic surveys showed that powdered infant formula might play an important role in Cronobacter infections as the main transmission vehicle (
      • Biering G.
      • Karlsson S.
      • Clark N.V.C.
      • Jonsdottir K.E.
      • Ludvigsson P.
      • Steingrimsson O.
      Three cases of neonatal meningitis caused by Enterobacter sakazakii in powdered milk.
      ;
      • van Acker J.
      • De Smet F.
      • Muyldermans G.
      • Bougates A.
      • Naessens A.
      • Lauwers S.
      Outbreaks of necrotizing enterocolitis associated with Enterobacter sakazakii in powdered milk formulas.
      ). However, the true source of Cronobacter spp. in powdered infant formula is largely unknown (
      • Ye Y.W.
      • Li H.
      • Wu Q.P.
      • Zhang J.M.
      • Lu Y.D.
      The Cronobacter sp. in milk and dairy products: Detection and typing.
      ).
      In China, Cronobacter isolates in dry food samples have been described (
      • Li Y.
      • Chen Q.
      • Zhao J.
      • Jiang H.
      • Lu F.
      • Bie X.
      • Lu Z.
      Isolation, identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China.
      ;
      • Xu F.
      • Li P.
      • Ming X.
      • Yang D.
      • Xu H.
      • Wu X.
      • Shah N.P.
      • Wei H.
      Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR.
      ,
      • Xu X.
      • Wu Q.P.
      • Zhang J.M.
      • Ye Y.W.
      • Yang X.J.
      • Dong X.H.
      Occurrence and characterization of Cronobacter spp. in powdered formula from Chinese retail markets.
      ), and C. sakazakii is the most frequently isolated species within the genus in powdered formula (Xu et al., 2014). Presence of Cronobacter strains in dry food samples showed unusual abilities to survive dry stress (
      • Breeuwer P.
      • Lardeau A.
      • Peterz M.
      • Joosten H.M.
      Desiccation and heat tolerance of Enterobacter sakazakii..
      ;
      • Osaili T.
      • Forsythe S.
      Desiccation resistance and persistence of Cronobacter species in infant formula.
      ;
      • Kuo L.S.
      • Wang B.J.
      • He Y.S.
      • Weng Y.M.
      The effects of ultraviolet light irradiation and drying treatments on the survival of Cronobacter spp. (Enterobacter sakazakii) on the surfaces of stainless steel, Teflon and glass.
      ). Consequently, effects of drying technologies on inactivation of C. sakazakii is of importance for understanding the risk of contamination in powdered infant formula.
      Freeze-drying is considered a promising technology for preservation of microorganisms (
      • Miyamoto-Shinohara Y.
      • Sukenobe J.
      • Imaizumi T.
      • Nakahara T.
      Survival curves for microbial species stored by freeze-drying.
      ). Freeze-drying also can inactivate food-borne pathogens through dehydration of food samples. Vacuum freeze-drying is a complex process consisting of a freezing component as well as a vacuum-drying component (
      • Claussen I.C.
      • Ustad T.S.
      • Strmmen I.
      • Walde P.M.
      Atmospheric freeze drying—A review.
      ;
      • Shi A.M.
      • Wang L.J.
      • Li D.
      • Adhikari B.
      The effect of annealing and cryoprotectants on the properties of vacuum-freeze dried starch nanoparticles.
      ). However, scant research has focused on the effects of the vacuum freeze-drying on inactivation of C. sakazakii in liquid media.
      The present study aimed to determine the effects of vacuum freeze-drying for 12, 24, and 36 h on survival of C. sakazakii in whole milk, skim milk, tryptic soy brother (TSB), and sterile water at different initial levels (103, 102, and 101 cfu/mL). Furthermore, scanning electron microscopy was used to evaluate the morphological changes of C. sakazakii cell after vacuum freeze-drying treatment.
      Cronobacter sakazakii ATCC 29544 was inoculated into TSB (Luqiao, Beijing, China) for incubation at 37°C for 16 to 18 h. Cronobacter sakazakii suspensions at different concentrations were prepared through 10-fold dilutions, and then C. sakazakii was inoculated under sterile conditions, at 103, 102, and 101 cfu/mL into different liquid media (whole milk, skim milk, TSB, and water). Thereafter, the liquid media was frozen at −30°C for12 h, and was further transferred to a vacuum freeze-dryer (FD-1–50, Boyikang, Beijing, China) for 12, 24, and 36 h. The samples frozen at −30°C for 12 h were used as control samples.
      After vacuum freeze-drying, whole milk, skim milk, TSB, and water samples (10 mL) were diluted using sterile 0.85% NaCl (90 mL). The appropriate dilution was then spread onto the tryptic soy agar (Luqiao) and left to sit for 24 h at 37°C. Plates containing 30 to 300 colonies were selected to determine the survival of C. sakazakii in different liquid media after vacuum freeze-drying for 12, 24, and 36 h. In addition, the lethality rate was regarded as the decreasing percentage of colonies between the control (untreated) and treated samples. Each experiment was replicated 3 times.
      To determine morphological changes of C. sakazakii cells, TSB samples mixed with C. sakazakii cells after vacuum freeze-drying for 12, 24, and 36 h were reconstituted. The treated and untreated samples were then centrifuged at 3,000 × g for 10 min at room temperature and the pellets were further washed using 0.1 mol/L phosphate buffer (pH = 7.4). Thereafter, subsequent steps were completed as described in
      • Wang C.Y.
      • Huang H.W.
      • Hsu C.P.
      • Shyu Y.T.
      • Yang B.B.
      Inactivation and morphological damage of Vibrio parahaemolyticus treated with high hydrostatic pressure.
      . Finally, the morphological changes of C. sakazakii after vacuum freeze-drying were determined through scanning electron microscopy (Hitachi S4700, Tokyo, Japan).
      From Figure 1A, the survival of C. sakazakii in different liquid media at 103 cfu/mL was significantly affected (P < 0.05) by vacuum freeze-drying for 12, 24, and 36 h compared with untreated samples. With extension of treatment time, the number of colonies significantly decreased. Furthermore, compared with sterile water, whole milk had the highest protective role in inhibiting inactivation of C. sakazakii, followed by skim milk and TSB medium. A similar result was observed in liquid media with 102 cfu/mL, as shown in Figure 1B. The survival of C. sakazakii in sterile water with 101 cfu/mL was affected significantly by vacuum freeze-drying for 12, 24, and 36 h, whereas no significant changes of C. sakazakii in whole milk, skim milk, and TSB were observed (Figure 1C). Initial concentrations of cells for freeze-drying were related to the protective medium and growth phase in order to obtain the highest recovery (
      • Costa E.
      • Usall J.
      • Teixido N.
      • Garcia N.
      • Vinas I.
      Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying.
      ;
      • Corcoran B.M.
      • Ross R.P.
      • Fitzgerald G.F.
      • Stanton C.
      Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances.
      ). Survival rate of 10 species (Saccharomyces cerevisiae, Brevibacterium flavum, Brevibacterium lactofermentum, Corynebacterium acetoacidophilum, Corynebacterium gultamicum, Streptococcus mutans, Escherichia coli, Pseudomonas putida, Serratia marcescens, and Alcaligenes faecalis) after freeze-drying and long-term storage was evaluated. The results indicated that survival rates of yeast as well as gram-positive and gram-negative organisms were 10, 80, and 50%, respectively, after 10 yr of storage under a vacuum at 5°C. In addition, the survival rates of positive bacteria were higher than those of negative cells (
      • Miyamoto-Shinohara Y.
      • Imaizumi T.
      • Sukenobe J.
      • Murakami Y.
      • Kawamura S.
      • Komatsu Y.
      Survival rate of microbes after freeze-drying and long-term storage.
      ). Those authors further inferred that the excellent survival of each species after freeze-drying might be attributed to the high level of desiccation and to sealing under vacuum. The survival of microorganisms such as Escherichia (n = 1), Pseudomonas (n = 4), and Lactobacillus (n = 1) in dry conditions without a vacuum for 4 yr was not detected (
      • Antheunisse J.
      • de Bruin-Tol J.W.
      • van der Pol-Van Soest M.E.
      Survival of microorganisms after drying and storage.
      ). Pseudomonas sealed in ampoules at 6.7 Pa and stored in the dark at room temperature for 5 to 6 yr did not survive (
      • Antheunisse J.
      Viability of lyophilized microorganisms after storage.
      ), whereas Pseudomonas was able to be recovered after 10 yr of storage under a vacuum at 5°C (
      • Miyamoto-Shinohara Y.
      • Sukenobe J.
      • Imaizumi T.
      • Nakahara T.
      Survival curves for microbial species stored by freeze-drying.
      ). In Table 1, the mean lethality rates of C. sakazakii in whole milk with different initial inoculum levels after vacuum freeze-drying for 12, 24, and 36 h were 0 to 8.22, 12.5 to 59.69, and 25 to 83.36% respectively; similarly, mean lethality rates were 6.82 to 13.61, 20.45 to 63.15, and 29.54 to 84.65% in skim milk, 10.36 to 21.62, 20.45 to 68.73, and 29.54 to 85.16% in TSB, and 91.28 to 100, 99.38 to 100, and 99.82 to 100% in sterile water, respectively. The differences of lethality rates (P < 0.01) were significant between nutrients in the food matrix (whole milk, skim milk, and TSB) and oligotrophic medium (sterile water), but survival rates of C. sakazakii after vacuum freeze-drying in whole milk and skim milk were not significantly different (P > 0.05). Results indicated that the food matrix could play a protective role in inhibiting inactivation of C. sakazakii for vacuum freeze-drying. Pantoea agglomerans showed the highest viability in 10% nonfat skim milk after freeze-drying (
      • Costa E.
      • Usall J.
      • Teixido N.
      • Garcia N.
      • Vinas I.
      Effect of protective agents, rehydration media and initial cell concentration on viability of Pantoea agglomerans strain CPA-2 subjected to freeze-drying.
      ). In another paper, trehalose was found to be an effective protectant for freeze-dried and vacuum-dried samples (
      • Conrad P.B.
      • Miller D.P.
      • Cielenski P.R.
      • de Pablo J.J.
      Stabilization and preservation of Lactobacillus acidophilus in saccharide matrices.
      ).
      • Carvalho A.S.
      • Silva J.
      • Ho P.
      • Teixeira P.
      • Malcata F.X.
      • Gibbs P.
      Effects of various sugars added to growth and drying media upon thermotolerance and survival throughout storage of freeze dried Lactobacillus delbrueckii ssp. bulgaricus..
      reported that fructose and sorbitol could provide better protection than carbohydrate glucose in Lactobacillus. Using a mixture of skim milk and carbohydrate sugars, recoveries of Candida sake cell ranged from 45 to 85% (
      • Abadias M.
      • Teixido N.
      • Usall J.
      • Benabarre A.
      • Vinas I.
      Viability, efficacy, and storage stability of freeze-dried biocontrol agent Candida sake using different protective and rehydration media.
      ). Consequently, effects of the food matrix on the viability of microorganisms may be species-dependent.
      Figure thumbnail gr1
      Figure 1Treatment of vacuum freeze-drying on Cronobacter sakazakii cells in different media with different concentrations. TSB = tryptic soy broth. Data are means ± SD; asterisks represent a difference compared with time zero at *P < 0.05 and **P < 0.01.
      Table 1Lethality rates of vacuum freeze-drying treatment on Cronobacter sakazakii in different liquid media
      Intial levels (cfu/mL)Treatment time (h)Lethality rates of C. sakazakii (%, mean ± SD)
      Whole milkSkim milkTryptic soy brothSterile water
      103125.02 ± 3.89
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      9.54 ± 2.25
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      21.62 ± 3.09
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      94.97 ± 0.80
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      2459.69 ± 3.80
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      63.15 ± 3.08
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      68.73 ± 1.90
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      99.38 ± 0.10
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      3683.36 ± 1.60
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      84.56 ± 0.98
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      85.16 ± 3.39
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      99.82 ± 0.04
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      102128.22 ± 2.55
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      13.61 ± 6.11
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      18.24 ± 1.73
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      91.28 ± 2.13
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      2445.04 ± 7.99
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      47.78 ± 6.94
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      54.72 ± 4.71
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      100
      3672.80 ± 2.55
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      71.94 ± 0.69
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      75.16 ± 2.04
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      100
      101120 ± 6.0
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      6.82 ± 19.32
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      11.36 ± 7.91
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      100
      2412.5 ± 6.25
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      20.45 ± 12.5
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      20.45 ± 10.23
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      100
      3625 ± 5.0
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      29.54 ± 14.76
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      29.54 ± 12.5
      Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      100
      a–c Different lowercase superscripts in the same row indicate significant differences (P < 0.05).
      A–C Different uppercase superscripts in the same row indicate significant differences (P < 0.01).
      In Figure 2, scanning electron microscopy images show intracellular damage and distortion of C. sakazakii after vacuum freeze-drying for 24 and 36 h compared with the untreated sample. Furthermore, the injured cells and distortion of cells increased with the extension of vacuum freeze-drying time. During vacuum freeze-drying, the formation of ice crystals might have contributed to injury of cells and subsequent release of cellular contents, resulting in irreversible damages (
      • Orndorff G.R.
      • MacKenzie A.P.
      The function of the suspending medium during the freeze-drying preservation of Escherichia coli..
      ). Although the freeze-drying is an alternative method for preservation of microorganisms, freezing is also detrimental to the viability of bacterial cells after drying (
      • Morgan C.A.
      • Herman N.
      • White P.A.
      • Vesey G.
      Preservation of micro-organisms by drying; A review.
      ).
      Figure thumbnail gr2
      Figure 2Scanning electron micrographs of Cronobacter sakazakii cells after vacuum freeze-drying treatment for 0, 12, 24, and 36 h. Arrows indicate intracellular damage and distortion of C. sakazakii after vacuum freeze-drying compared with the untreated sample.
      In summary, as a potential food-engineering technology for the production of powdered milk, vacuum freeze-drying can cause loss of viability of bacterial cells but cannot thoroughly inactivate C. sakazakii within 36 h in the food matrix. Scanning electron microscopy further indicated that the inactivation of C. sakazakii after vacuum freeze-drying was involved in injured cell membranes, further release of cellular contents, and irreversible damage of partial cells. The findings here contribute to the understanding and evaluation of the risks or hazards in production of powdered food using vacuum freeze-drying technology.

      ACKNOWLEDGMENTS

      We gratefully acknowledge the financial support of the National Natural Science Foundation of China (31671951), the Anhui provincial Grand Project special of Science and Technology (15czz03109), Project of Science and Technology in Guangzhou (201604020036), and State Key Laboratory of Applied Microbiology Southern China Open Foundations, Guangdong Institute of Microbiology, Guangzhou (SKLAM004-2015).

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