- Email: [email protected]
Effects of microwave irradiation dose and time on Yeast ZSM-001 growth and cell membrane permeability
Accepted Manuscript Effects of microwave irradiation dose and time on Yeast ZSM-001 growth and cell membrane permeability Shu-Wei Zeng , Qi-Lin Huang , Si-Ming Zhao PII:
S0956-7135(14)00326-0
DOI:
10.1016/j.foodcont.2014.05.053
Reference:
JFCO 3886
To appear in:
Food Control
Received Date: 15 November 2013 Revised Date:
24 May 2014
Accepted Date: 31 May 2014
Please cite this article as: ZengS.-W., HuangQ.-L. & ZhaoS.-M., Effects of microwave irradiation dose and time on Yeast ZSM-001 growth and cell membrane permeability, Food Control (2014), doi: 10.1016/ j.foodcont.2014.05.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of microwave irradiation dose and time on Yeast
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ZSM-001 growth and cell membrane permeability
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Shu-Wei Zenga, Qi-Lin Huanga, *, Si-Ming Zhaoa
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Correlative Dietology, Huazhong Agricultural University, Wuhan 430070, China
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College of Food Science and Technology and MOE Key Laboratory of Environment
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* Corresponding author. Tel: +86-027-87288375; fax: +86-027-87288375; e-mail:
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[email protected]
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ACCEPTED MANUSCRIPT Abstract: To obtain a theoretical reference for microwave control of yeast used in the
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fermented products, the effects of microwave irradiation dose and time on growth and
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cell membrane permeability of Yeast ZSM-001, Brettanomyces custersii isolated from
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spontaneously fermented rice paste, were evaluated. When treated at a microwave dose
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from 1.0 to 1.6 W/g for 90 s or at the optimal dose of 1.6 W/g for duration ≤ 120 s, the
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yeast growth was enhanced according to Gompertz equation, and the yeast cells were
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almost intact with smooth surfaces observed by SEM. Meanwhile, DNA, protein and
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electrolyte leakage, as well as the fluidity of H+ and Ca2+ increased within a reversible
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range as compared with the untreated control. At a microwave dose above 2.0 W/g or
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duration over 160 s, the yeast growth rate decreased, and the cells were collapsed and
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electroporated with rough surfaces. Therefore, low-intensity microwave (dose ≤ 1.6 W/g
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or duration ≤ 120 s) favors yeast growth and induces a repairable increase in cell
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membrane permeability without cell damage. High-intensity microwave results in the
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yeast cell death mainly due to an irreversible increase of electrolyte, Ca2+ and DNA
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leakage.
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Keywords: Yeast ZSM-001; Microwave dose; Microwave time; Growth; Cell membrane
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permeability
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Chemical compounds studied in this article
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Sucrose (PubChem CID: 5988); Agar (PubChem CID: 71571511); NaCl (PubChem CID:
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5234); BCECF-acetoxymethyl (PubChem CID: 53229972); Fluo 3/AM (PubChem CID:
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5086914); Glutaraldehyde (PubChem CID: 3485); Ethanol (PubChem CID: 702);
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Platinum (PubChem CID: 23939).
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1. Introduction Yeasts are widely applied in fermented food including wine, beer, and baked foods
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with rice and flour as raw materials. In our previous work, yeast strain Brettanomyces
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custersii (ZSM-001) was successfully isolated from spontaneously fermented rice paste,
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and the yeast strain used for fermentation could ensure product quality and shorten the
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fermentation time to a certain degree, as compared with the long fermentation time
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(14~16 h) and unstable quality for the traditional fermented rice cake (Liu, 2008; Liu, Liu,
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Zhao, & Xiong, 2010). The particular processing technique will select for the growth of
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particular types of yeasts which are adapted to grow under the created condition, such as
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pH, NaCl, temperature and microwave etc (Betts, Linton, & Betteridge, 1999; Grundler,
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Keilman, Frohlich, & Srube, 1982).
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Microwaves are part of the electromagnetic spectrum and considered to be the
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irradiation ranging in frequency from 300 MHz to 300 GHz, corresponding to a
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wavelength range from 1m to 1mm (Banik, Bandyopadhyay, & Ganguly, 2003). Some
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researchers have found that subtle effects induced by low-level microwave exposure were
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not directly attributable to heat but to non-thermal effect (Belyaev, 2005; De Pomerai et
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al., 2002; Welt, Tong, Rossen, &Lund, 1994). The growth rate of yeast Sacharomyces
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cerevisiae has been reported to either increase up to 15% or decrease down to 29% by
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certain frequencies of microwave irradiation within 41.8~42.0 GHz (Grundler, Keilman,
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& Frohlich, 1977). Furthermore, several studies have indicated that the exposure of
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Escherichia coli and Bacillus subtilis cell suspensions to microwave irradiation at the
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frequency of 2.45 GHz may alter structural and functional properties of cell membranes
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& Tosoratti, 2004; Suga, Goto, & Hatakeyama, 2006; Woo, Rhee, & Park, 2000).
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Additionally, the interaction between microwave treatment and organisms has a
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resonance-like dependence on the microwave irradiation frequency, time and power
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(Chandrasekaran, Ramanathan, & Basak, 2012; Grundler et al., 1982; Wang, Wang, &
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Wang, 1996). Up to now, much work has been reported about microbial destruction
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induced by microwave irradiation in a certain frequency, time and power range (Cheng et
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al., 2013; Campanha. et al., 2007; Woo et al., 2000). However, little information is
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available on the positive response of yeast to microwave irradiation, let alone
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optimization of microwave exposure conditions (2.45 GHz, dose and time) for yeast
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growth.
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Therefore, the objective of the current study was to evaluate the effects of microwave
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treatment dose and time on the growth of Yeast ZSM-001 by measuring the growth
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curves according to Gompertz equation followed by the SEM verification. Moreover, the
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parameters of cell membrane permeability such as DNA and protein leakage, electrolyte
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leakage, and H+ and Ca2+ permeability were measured to test the effects of various
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microwave treatments on yeast cell membrane. This research explored the microwave
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environment beneficial to the growth of Yeast ZSM-001, and provides useful information
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about the microwave control in enhancing yeast growth for large-scale production of
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fermented food.
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2. Materials and methods
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2.1. Materials Yeast strain ZSM-001, Brettanomyces custersii, was isolated previously from
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spontaneously fermented rice paste as described by Liu et al. (2008) and was preserved in
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China Center for Type Culture Collection (CCTCC, No. M207150). All reagents used
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were of analytical grade. All media and reagent solutions were prepared with distilled
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water.
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2.2. Cultivation of Yeast ZSM-001
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Potato liquid medium was prepared as follows. Peeled potatoes (200 g) were cut into
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cubes, and put into distilled water at a volume ratio of 1:2, followed by heating at 100oC
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for 15 min. The potato broth was filtered through a filter cloth, and then the filtrate was
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supplemented with sucrose (20 g). Finally, the as-prepared filtrate was diluted to 1000
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mL with distilled water at the ratio of 1:4 to obtain the potato liquid medium, followed by
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autoclaving at 121oC for 30 min. Potato solid medium was prepared by adding agar (13 g)
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into the above potato liquid medium.
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Before microwave treatment, Yeast ZSM-001 preserved in CCTCC was activated on
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potato solid medium at 30oC for 48 h. After activation, three loops of Yeast ZSM-001
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were inoculated into 250 mL of potato liquid medium and incubated on a rotary shaker at
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90 rpm and 30oC for 12 h. Then the culture medium was centrifuged at 4000 rpm for 5
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min to obtain yeast slurry. Finally, the yeast slurry was suspended in a sterile 0.85% NaCl
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solution to prepare yeast suspension at a concentration of 3 wt% (1× 105 CFU/mL) for
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microwave treatment.
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2.3. Microwave treatment
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ACCEPTED MANUSCRIPT The microwave treatments of the samples were performed using a single-mode
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microwave oven (QW-15HM, Guangzhou Kewei Microwave Oven Energy, Guangzhou,
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China), with an internal capacity of 42 × 42 × 36 cm. This microwave device was mainly
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constructed from standard rectangular waveguides and a magnetron oscillator with a
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maximum continuous wave output power of 900 W at 2.45 GHz as the source of the
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microwaves. The cross section shape of microwave cavity and medium properties
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maintained the vertical axial uniformity. The temperature inside the oven was measured
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using a precise infrared temperature sensor with a resolution of 0.1oC.
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The microwave irradiation conditions and procedures were described as follows. The
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yeast suspension (100±0.1 g) poured into a 250 mL conical flask was placed in a central
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location inside the microwave oven, and exposed to microwave irradiation from 0 to 200
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s at a 10 s interval and at various doses (1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4 W/g),
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which was corresponding to the ratio between the microwave output power (W) and the
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mass (g) of the sample (Özbek, & Dadali, 2007). After irradiation, the temperature of
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yeast suspension was measured immediately with a JDDA80 point thermometer (Jingda
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Instrument Factory Co., Wuhan, China) at three different randomly selected positions so
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as to determine the standard temperature-time curve at various doses. Meanwhile, the
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typical microwave dose and time range were determined according to the normal
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temperature range for yeast growth (28~38oC). Additionally, the microwave doses and
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durations deviated from the normal conditions were also selected for evaluation of yeast
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growth.
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2.4. Determination of growth curve
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Yeast at a concentration of 3 wt% was inoculated into 97 mL of sterile potato liquid medium to prepare yeast suspension for growth curve determination. The growth curve of yeast was measured by the turbidimetric method. After 6 h
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incubation on a rotary shaker at 90 rpm and 30oC, yeast suspension was treated under
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preselected microwave conditions. The absorbance values (OD) were measured at 560
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nm every 3 h to determine the biomass of yeast (Iwashima, Kimura, & Kawasaki, 1990).
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All the experiments were made in triplicate. The yeast suspension untreated with
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microwave served as control.
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2.5. Determination of DNA and protein leakage
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The yeast suspension before and after microwave treatment was filtered, and the
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absorbances of DNA and protein in the supernatant were measured with a UV-2600
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spectrophotometer (Younikang Co., Shanghai, China) at 260 and 280 nm, respectively.
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The blank was composed of sterile 0.85% NaCl (Woo et al., 2000).
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2.6. Determination of electrolyte leakage
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Electrolyte leakage (EL) from cells treated with different microwave doses and
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durations was measured on an FE30 conductivity meter (Mettler-Toledo Instruments Co.,
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China) as described previously (Campanha et al, 2007; Soro, Djè, & Thonart, 2010).
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After cooling to room temperature, the microwave-treated yeast suspension was
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subjected to the measurement of the initial electrical conductivity (Ei). After Ei
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measurement, the microwave-treated yeast suspension was placed into a 100oC boiling
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water bath for 15 min for the measurement of the total electrical conductivity (Et). The
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initial and total electrical conductivities (Ci and Ct) of the control without microwave
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irradiation were measured under the same conditions as the microwave-treated samples.
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The electrolyte leakage was calculated as follows: EL (%) = [1 - (1 - Ei/Et) (1 - Ci/Ct)] ×100
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where Ei and Et are the initial and total electrical conductivities of the microwave-treated
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samples; Ci and Ct are the initial and total electrical conductivities of the control,
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respectively.
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2.7. Determination of H+ and Ca2+ permeability
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After microwave treatment, fluorescent probe BCECF-AM (1 mmol/L) was added
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into yeast suspension at a volume ratio of 1:300 with a final concentration of 3 µmol/L.
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Yeast suspension was cultured at 37oC for 30 min, and then centrifuged at 4000 rpm for
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5min to collect yeast slurry. Finally, the yeast slurry was washed with 0.85% NaCl and
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resuspended in 3 mL of 0.85% NaCl. The amount of H+ released after microwave
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treatment was determined according to the fluorescence intensity obtained on a
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RF-5301PC fluorospectrophotometer (Shimadzu Co, Japan) with the double-wavelength
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ratio method at an excitation wavelength of 520 and 440 nm (FIR = 520/440 nm), and an
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emission wavelength of 530 nm (Breeuwer, & Abee, 2000; Chen et al., 2009; Russell,
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Pottier, & Valenzeno, 1995). The yeast suspension supplemented with fluorescent probe
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but without microwave treatment was used as reference.
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The pretreatment for Ca2+ permeability with Fluo-3 AM as fluorescent probe was
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similar to H+ permeability. Briefly, after microwave treatment, fluorescent probe Fluo-3
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AM (1 mmol/L) was added into yeast suspension at a final concentration of 3 µmol/L,
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and cultured at 37oC for 30 min in the dark, followed by centrifugation at 5000 rpm for
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amount of Ca2+ released after microwave treatment was determined according to the
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fluorescence intensity obtained by a fluorospectrophotometer at an excitation wavelength
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of 490 nm, and the fluorescence intensity was observed at 509 nm (Russell et al., 1995;
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Zhang, Rengel, & Kuo, 1998).
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2.8. Morphology of yeast cells by scanning electron microscopy (SEM)
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The morphology of yeast cells was observed by a JSM-6390LV scanning electron
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microscope (NTC, Japan) at an accelerating voltage of 20 kV. The yeast suspension was
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centrifuged after microwave treatment, and the yeast slurry was collected and fixed by
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2.5% glutaraldehyde for 4 h. After 15 min centrifugation at 8000 rpm, the yeast slurry
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was washed twice with deionized water, and dehydrated successively with 30, 50 and
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70% ethanol. The final yeast slurry was freeze-dried and sputtered with platinum using an
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ion sputter coater (IB-5, EIKO Ltd., Tokyo, Japan). The morphology of yeast cells was
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observed and photographed with a scanning electron microscope (JSM-6390LV, JEOL
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Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV.
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2.9. Data analysis
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All treatments and assays were performed in triplicate and repeated three times. The
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data were processed by Microsoft Excel 2010. The statistical significance of differences
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for DNA, protein and electrolyte leakage, H+ and Ca2+ permeability assays were
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calculated by Statistical Analysis System Software 8.1 (SAS Institute Inc., USA).
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Difference among the control and treated samples was considered significant at p < 0.01.
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3. Results and discussion
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3.1. Determination of microwave dose and time for yeast growth Fig. 1 shows the curves of temperature vs microwave dose and time for yeast
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suspension. It was reported by Craig and Jacobsen (1984) that the normal temperature for
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yeast growth metabolism was in the range of 28~38oC, whereas the lethal temperature
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was 51oC. As shown in Fig. 1, the temperature of the suspension increased linearly with
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the time at various doses. The temperatures at microwave doses of 1.0~2.4 W/g for the
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fixed time of 90 s were within a range of 32.0 to 50.2oC including normal and lethal
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temperatures for yeast growth and exhibited a homogeneous distribution. When the
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microwave dose increased up to 2.2 W/g for 90 s, the temperature reached 47.8oC close
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to the yeast lethal temperature (51oC). When the treatment time increased up to 200 s, the
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temperatures at all microwave doses were almost all above 40oC, and deviated from the
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normal temperature range of yeast growth (28~38oC). Therefore, six microwave doses
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(1.0, 1.4, 1.6, 1.8, 2.0, and 2.2 W/g) for the fixed time of 90s, and five time points (40, 80,
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120, 160 and 200 s) at the appropriate dose of 1.6 W/g were selected to investigate the
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effects of microwave dose and time on yeast growth and cell membrane permeability.
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3.2. Growth curve of yeast
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Fig. 2 shows the growth curves of yeast estimated indirectly by OD values after
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treatment with different microwave doses and durations. OD values could reflect the
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biomass of the yeast. The yeast biomass increased gradually with the time of culture, and
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entered the logarithmic phase after ca. 6 h, at which time point the yeast was treated by
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microwave, then the stationary phase at approximate 15 h, and finally the decline phase
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microwave dose from 1.0 to 1.6 W/g for 90 s resulted in a greater increase over the
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untreated control in the yeast biomass. With a further increase up to 1.8 W/g, the biomass
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was lower than that of the untreated control and the minimum biomass occurred at a dose
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of 2.2 W/g (Fig. 2A). At the stationary phase, the yeast biomass reached the maximum
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after treatment with a microwave dose of 1.6 W/g, which was defined as the optimum
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microwave dose favoring yeast growth and cell membrane permeability. At the optimum
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microwave dose of 1.6 W/g, the growth curves exhibited a similar trend and were all
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higher than that of the untreated control with the treatment time varying among 40, 80
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and 120 s. When the exposure time exceeded 160 s, the biomass was much lower than
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that of the control (Fig. 2B).
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Fig. 2 shows that there is early adaptability to yeast growth by microwave process, so
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the Gompertz equation can be used to describe the growth characteristics of yeast as
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follows (Gompertz, 1825; Fang, Hu, Xiong, & Zhao, 2011):
N = N 0 + b0 e ( −b1e
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−b 2 t )
(1)
where N is the total increment of biomass at time t; N0 is the value related to the initial
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yeast (when t = 0, initial yeast = N0 + b0e -b1); b0 is a parameter related to the equivalent
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yeast counts (when t → ∞, the equivalent yeast count = N0 + b0); b1 is a parameter related
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to the early adaptability to growth; and b2 is a parameter related to a growth rate constant,
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and is usually a function of processing conditions such as microwave dose. It can be seen
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that the theoretical curves obtained from equation (1) gave the best fit to the experimental
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data points (solid points), and the residual sum of squares was much lower than the
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This result indicated that the accuracy of equation (1) is quite high and the growth
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characteristics of yeast could be well described by the Gompertz equation. According to
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equation (1), the data of N0, b0, b1 and b2 are obtained by a least-squares method, and
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listed in Table 1. The growth rate constant (b2) increased with the microwave dose from
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1.0 to 1.6 W/g, and reached the maximum at 1.6 W/g, followed by a drop with a further
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increase of the microwave dose. As the microwave time was prolonged at the optimum
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dose of 1.6 W/g, b2 increased up to the maximum at 120 s, followed by a decrease against
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the untreated control. The value b0 presented a similar trend to b2. The results indicated
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that microwave treatment with a low dose (≤ 1.6 W/g) and duration (≤ 120 s) has a
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positive effect on the yeast growth. According to the curve of temperature vs dose or
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duration, the temperatures were in the range of 32.0~35.9oC and 25.9~38.8oC after
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microwave treatment with a dose of 1.0~1.6 W/g for 90 s and for duration of 0~120 s at a
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dose of 1.6 W/g, both of which were almost in the normal temperature range of yeast
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growth, suggesting that the positive action on the yeast growth was due to the
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non-thermal effect rather than the thermal effect of microwaves. This result has been
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individually confirmed by Ramundo-Orlando (2010) and de Pomerai et al. (2002). It was
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well known that microwave causes thermal effect, but has non-thermal character, i.e., the
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action of irradiation does not produce essential heating for the biological system or
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destroy its structure. Low-level microwave energy produces various types of molecular
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transformations and alterations, but the energy is too weak to break down the chemical
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bonds or destroy the primary structure. Hence, low-level microwave exposure results in
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de Pomerai et al., 2002). On the other hand, microwave can inhibit yeast growth at a dose
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≥ 1.8 W/g or duration ≥ 160 s, due to heat production and a sharp increase in temperature
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that can be systemic or localized, resulting in cell death (Ramundo-Orlando, 2010).
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3.3. DNA and protein leakage
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Cellular damage causes DNA and protein leakage, so the dissolution rate of DNA and
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protein can reflect cell membrane permeability and cell growth. Fig. 3 shows the protein
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and DNA leakage of the yeast treated with various microwave doses (1.0, 1.4, 1.6, 1.8,
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2.0 and 2.2 W/g for 90 s) and durations (40, 80, 120, 160 and 200 s at 1.6 W/g). As
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microwave dose increased from 1.0 W/g to 1.8 W/g for 90 s, the absorbances of DNA and
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protein increased by 11.6~15.6% and 10.9~14.5%, respectively, as compared with the
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untreated control. However, with a further increase to 2.2 W/g for 90 s, the two
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absorbances showed an increase of 28.5% and 27.2% over the control, indicating an
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increase of cell membrane permeability. Low-intensity microwave exposure was reported
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to induce reversible externalization of phosphatidylserine (PS) molecules in the
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membrane of the exposed cells without detectable membrane damage or induction of cell
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death (Ramundo-Orlando, 2010). Hence, a slight increase in cell membrane permeability
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induced by low-dose (≤ 1.8 W/g) microwave exposure could be repaired reversibly. On
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the other hand, high-dose (≥ 2.0 W/g) microwave irradiation induced irreversible
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electroporation on the phospholipid bilayer membrane, leading to the damage of the
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membrane structure and essential leakage of cell contents (Suga et al., 2006).
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At the optimum dose of 1.6 W/g with an exposure time range of 40 to 160 s, the
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compared with the untreated control, followed by a drop (18.3% and 14.5%) at 200 s.
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This result suggested that the microwave energy accumulated by duration was strong
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enough to change the cell membrane permeability, accelerate the leakage of DNA and
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protein, and even destroy the yeast cell. When the energy accumulation reached a critical
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point, the protein and other biomacromolecules aggregated and denatured, leading to
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diffusion difficulty and a decrease in DNA and protein leakage, which is in good
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agreement with the study on high-energy pulse-electron-beam-induced molecular and
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cellular damage in Saccharomyces cerevisiae (Zhang et al., 2013).
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3.4. Electrolyte leakage
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Cellular electrolyte leakage was determined by measuring electrolyte conductivity,
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which reflects the function of cell membrane (Ovchinnikova, 1996). The effects of
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microwave treatments (at doses of 1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90s or durations
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of 40, 80, 120, 160 and 200 s at 1.6 W/g) on electrolyte leakage are shown in Fig. 4. The
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electrolyte conductivity rose slowly with the microwave dose from 1.0 to 1.8 W/g,
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followed by a dramatical rise at a dose above 1.8 W/g (Fig. 4A). At the optimum dose of
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1.6 W/g, the electrolyte conductivity increased gradually with treatment time, followed
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by a slight decrease after 160 s (Fig. 4B). The rising trend of electrolyte leakage may be
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ascribed to the acceleration of material exchange inside and outside the cells induced by
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the microwave irradiation at a dose ≤ 1.8 W/g or duration ≤ 120 s. With a further increase
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of the microwave dose (> 1.8 W/g), the structure of yeast cell membrane was damaged
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(Fig. 7), and yeast was unable to utilize the metal ions (Ca2+, Na+, and K+) due to the loss
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through the membrane, leading to a significant increase of electrolyte leakage (Woo et al.,
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2000). Cojocaru et al. (2005) proposed another explanation for the increase in electrical
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conductivity induced by high-intensity microwave irradiation: the activation of lipid
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peroxidation by metal ions led to the damage of the phospholipid membrane. Additionally,
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the cell membrane blockage caused by the aggregation and denaturation of the
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intracellular biomacromolecules resulted in a slight decrease of electrolyte leakage at 200
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s, which has been confirmed by another study in our laboratory, the microwave effect on
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Aspergillus (Fang et al., 2011).
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3.5. H+ permeability
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Intracellular pH values can reflect the intracellular H+ concentration and the cell ion
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channel. Fig. 5 shows the intracellular pH values of yeast treated with different
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microwave doses (1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90 s) and durations (40, 80, 120,
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160 and 200 s at 1.6 W/g). With an increase of microwave dose from 1.0 to 1.6 W/g for
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90 s and time from 40 to 120 s at 1.6 W/g, the internal pH values of the yeast slightly
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increased from 6.18 to 6.25, and from 6.20 to 6.48, but still remained in the normal pH
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range of 6.1~6.5, which was similar to the results reported by Peña et al. (1995) that the
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internal pH of normal yeast varied slightly even if the yeast was subjected to
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electroporation at 1500 V and environmental simulation of pH from 4.0 to 8.0. The
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low-intensity microwave irradiation (dose ≤ 1.6 W/g or duration ≤ 120 s) might induce
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the activation of cytoplasmic membrane H+-ATPase, resulting in the extrusion of H+ from
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the cytoplasm (corresponding to the increase of internal pH) and the concentration of H+
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(dose ≤ 1.6 W/g or duration ≤ 120 s) would promote nutrient intake loaded by H+
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co-transport, and accelerate the yeast metabolic activity to increase the viability, as
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indicated in the curve of growth rate (Haworth, & Fliegel, 1993; Viegas, Almeida,
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Cavaco, & Sá-Correia, 1998). Furthermore, the effects of low-level microwave on ion
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transport may be the consequence of its direct effect on membrane proteins and the
329
phospholipid domain organization (Ramundo-Orlando, 2010). However, microwave
330
treatment at a dose ≥ 2.0 W/g or time ≥ 160 s caused irreversible changes in the cell
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membrane permeability of the yeast, and even induced the cell death partly mediated by
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deactivation of the key enzyme combined with the cellular BCECF-AM probe, which
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finally led to a decrease in pH measured by the fluorescence changes of BCECF-AM
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probe (Breeuwer, & Abee, 2000; Grinstein, & Rothstein, 1986; Imai, & Ohno, 1995).
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3.6. Ca2+ permeability
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Ca2+ is closely related to cell proliferation, division, movement, secretion, metabolism
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(energy, oxygen, glucose) and many other cellular processes. The release of Ca2+ from the
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vacuole into the cell’s cytoplasm and subsequently into the extracellular medium depends
339
on the vacuole and cell membrane (Campanha et al., 2007). The effects of microwave
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doses (1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90 s) and time (40, 80, 120, 160 and 200 s at
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1.6 W/g) on Ca2+ permeability are shown in Fig. 6. The Ca2+ concentration is positively
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correlated with fluorescence intensity (Matsuoka, Kosai, Saito, Takeyama, & Suto, 2002).
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The fluorescence intensity (FI) of Ca2+ was lower than that of the untreated control within
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the dose range of 1.0~1.6 W/g, followed by an increase over the control with a further
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treatment time at the fixed dose of 1.6 W/g, the FI of Ca2+ decreased within 80 s,
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followed by a gradual increase. Moreover, the FI of Ca2+ was close to the control at 1.6
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W/g or 120 s.
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The movement of Ca2+ is strictly self-regulated and the intracellular concentration of
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Ca2+ is significantly lower than the extracellular concentration (Houchi, Yoshizumi,
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Ishimura, & Oka, 1996). Under low-intensity microwave exposure (dose ≤ 1.6 W/g or
352
duration ≤ 120 s), the effusion of a small amount of intracellular Ca2+ was beneficial to
353
maintaining the Ca2+ concentration gradient inside and outside the cell, and promoted
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yeast growth. However, microwave treatment at a dose ≥ 1.8 W/g or duration ≥ 120 s
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resulted in a small influx of Ca2+ probably due to the increase of membrane fluidity,
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thereby inducing the opening of Ca2+ channels (Bonilla, & Cunningham, 2003; Repacholi,
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1998). A further higher dose (2.2 W/g) caused a sharp decrease of cellular Ca2+
358
concentration against the untreated control, due to cell rupture and death, which was
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related to the precipitation of phosphate and the deactivation of the esterase used to
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hydrolyze fluorescence probe Fluo-3/AM into Ca2+-sensitive form of probe (Zhang et al.,
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1998).
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3.7. SEM observation
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Fig. 7 shows SEM photograph of yeast cell after microwave treatment with various
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doses and durations. It can be observed from Fig. 7 (A and a) that the untreated yeast
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cells were intact and presented a spherical, oblate or nephritic shape. After microwave
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treatment at a dose ≤ 1.6 W/g or duration ≤ 120 s, yeast cells were still complete and
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the untreated control, significant damages were detected on yeast cells with obvious
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metamorphosis at 2.2 W/g, i.e., part of the cells were collapsed and electroporated, and
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the surfaces were rough with sawtooth-shaped edges.
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The morphology of yeast cell indicated that low-intensity microwave treatment (dose
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≤ 1.6 W/g and duration ≤ 120 s) could preserve the cell structure. Nevertheless,
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high-intensity microwave exposure (dose ≥ 2.0 W/g or duration ≥ 160 s) induced the
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essential changes in structural integrity and permeability of cell membrane and cell wall
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which may have detrimental effects on the cell metabolism and lead to cell death
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(Campanha et al., 2007). SEM imaging also demonstrated the influence of microwave
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treatment dose and duration on the yeast growth and cell membrane permeability in terms
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of proteins, DNA leakage of yeast cells and the ion channels.
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4. Conclusion
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The effects of microwave exposure dose and time on Yeast ZSM-001 growth and cell
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membrane permeability were investigated. Treated with a microwave dose from 1.0 to
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1.6 W/g for 90 s or at the optimal dose of 1.6 W/g for 40~120 s, yeast growth rate was
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increased up to the maximum at 1.6 W/g for 120 s, and the yeast cells were almost
385
complete with smooth surfaces. Meanwhile, the microwave exposure at a dose ≤ 1.6 W/g
386
or duration ≤ 120 s induced a repairable increase in cell membrane permeability, together
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with an increase in DNA and protein, electrolyte leakage and transport of H+ and Ca2+
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between inside and outside cells. When the microwave dose increased up to 2.0 W/g or
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were observed in cells with rough surfaces. Hence, we conclude that microwave
391
treatment with a low dose (≤ 1.6 W/g) and duration (≤ 120 s) favors yeast growth, and
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induces a repairable increase in cell membrane permeability without cell damage.
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High-intensity microwave may lead to the yeast cell death mainly due to a significant and
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irreversible increase in electrolyte and DNA leakage, as well as H+, Ca2+ permeability.
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However, the mechanism of cell damage induced by high-intensity or long-time
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microwave irradiation remains to be elucidated at the molecular level. This research
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provides useful information about the potential control of yeast growth for large-scale
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production of fermented food.
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Acknowledgments
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This work was supported by University Agricultural Science and Technology Service
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of Ministry of Science and Technology of China (2013BAD20B00), and Application and
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Foundation Research Project of Wuhan Science and Technology Bureau of China
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(2014020101010068).
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ACCEPTED MANUSCRIPT Table Caption Table 1. Yeast growth characteristics represented by values of N0, b0, b1 and b2
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obtained from Gompertz equation.
ACCEPTED MANUSCRIPT Figure Captions Fig. 1. The curves of temperature vs microwave dose and time for yeast suspension Fig. 2. Growth curves of yeast after microwave treatment with various doses for 90 s (A)
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and for different durations at 1.6 W/g (B). Fig. 3. DNA and protein OD values of yeast cells after microwave treatment with various doses for 90 s (A) and for different durations at 1.6 W/g (B). Significant differences
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between the control and treated samples were calculated to be p < 0.01.
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Fig. 4. Electrolyte leakage of yeast cells after microwave treatment with various doses for 90 s (A) and for different durations at 1.6 W/g (B). Different letters (a~f) indicate significant differences among different microwave treatments (p< 0.01). Fig. 5. The intracellular pH values of yeast cells after microwave treatment with various
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doses for 90 s (A) and for different durations at 1.6 W/g (B). Different letters (a~g) indicate significant differences among different microwave treatments (p< 0.01). Fig. 6. The intracellular Ca2+ fluorescence intensity of yeast cells after microwave
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treatment with various doses for 90s (A) and for different durations at 1.6 W/g (B).
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Different letters (a~f) indicate significant differences among different microwave treatments (p< 0.01).
Fig. 7. SEM photographs of yeast cells treated with various microwave doses for 90 s (B~D) and at 1.6 W/g for different durations (b~d) in comparison with the untreated control (A and a).
1
ACCEPTED MANUSCRIPT Table 1 Yeast growth characteristics represented by values of N0, b0, b1 and b2 obtained from Gompertz equation Treatment condition
b0
b1
b2
0.260 0.247 0.215 0.211 0.271 0.288 0.283
1.022 1.092 1.155 1.185 0.960 0.811 0.512
28.949 28.055 27.291 30.528 30.546 32.567 24.395
0.371 0.375 0.393 0.413 0.368 0.358 0.336
0.502 0.443 0.453 0.471 0.478 0.517
1.368 1.480 1.477 1.468 1.210 0.724
21.318 18.606 19.614 21.330 17.856 17.646
0.325 0.335 0.338 0.341 0.318 0.309
Microwave time
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90 s
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Microwave dose 0 W/g 1.0 W/g 1.4 W/g 1.6 W/g 1.8 W/g 2.0 W/g 2.2 W/g Microwave time 0s 40 s 80 s 120 s 160 s 200 s
N0
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Microwave dose
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1.6 W/g
1
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80 1.0 W/g
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1.2 W/g
70
1.6 W/g
60
1.8 W/g 2.0 W/g 2.2 W/g
50
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2.4 W/g
40
30
20 0
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Temperature, ℃
1.4 W/g
50
100
Fig. 1
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150
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1.5
A
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1.1 0.9
0 W/g 1 W/g 1.4 W/g 1.6 W/g 1.8 W/g 2 W/g 2.2 W/g
0.5 0.3
6
12
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Biomass ( 560 nm)
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Growth time, h
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B
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0s 40 s 80 s 120 s 160 s 200 s
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Biomass ( 560 nm )
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Growth time, h Fig. 2
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30
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1 DNA 260 nm
A
Protein 280 nm
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OD value
0.8
0.6
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0.4
0
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0.2 0.5
1
1.5
2
2.5
Microwave dose ， W/g 1
DNA 260 nm
Protein 280 nm
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B
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OD value
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0.2
0
50
100
150
Microwave time, s Fig. 3
3
200
250
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A
85
b
80
d
75
c
c
1.6
1.8
e 70
f
65 1
1.4
2
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Electrolyte leakage,%
90
2.2
Micromave dose, W/g
B
76 74
a
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e
b
c
d
f
72
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Electrolyte leakage,%
80
70
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40
80
120
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Micromave time, s Fig. 4
4
200
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6.3
b c
PH
6.2
d
e
f
6.1
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g
6.0 1
1.4
1.6
1.8
2
2.2
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Microwave dose, W/g 6.8
B
a
6.4
c
d
e
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b
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PH
6.6
f
6.0
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40
80
120
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Micromave time, s Fig. 5
5
200
630
570
A c
a
b e
d
e
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540 510
f
480
0.0
1.0
1.4
1.6
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Fluorescence intensity ( 509 nm )
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1.8
2
2.2
B
a
b
600
c 550
d
e
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Fluorescence intensity （ 509 nm）
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Micromave dose, W/g
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f
450
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40
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Micromave time, s Fig. 6
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B 1.0 W/g
C 1.6 W/g
D 2.2 W/g
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A 0 W/g
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Highlights: Microwave dose and time on yeast are tested to provide insight in growth
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control Yeast growth is enhanced at ≤1.6W/g and ≤120s with an optimal membrane permeability
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Yeast cell death is caused at ≥2.0W/g and ≥160s by electrolyte, Ca2+ and DNA
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SEM shows intact and electroporated yeasts respectively under the two above
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S0956-7135(14)00326-0
DOI:
10.1016/j.foodcont.2014.05.053
Reference:
JFCO 3886
To appear in:
Food Control
Received Date: 15 November 2013 Revised Date:
24 May 2014
Accepted Date: 31 May 2014
Please cite this article as: ZengS.-W., HuangQ.-L. & ZhaoS.-M., Effects of microwave irradiation dose and time on Yeast ZSM-001 growth and cell membrane permeability, Food Control (2014), doi: 10.1016/ j.foodcont.2014.05.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Effects of microwave irradiation dose and time on Yeast
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ZSM-001 growth and cell membrane permeability
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Shu-Wei Zenga, Qi-Lin Huanga, *, Si-Ming Zhaoa
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a
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Correlative Dietology, Huazhong Agricultural University, Wuhan 430070, China
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College of Food Science and Technology and MOE Key Laboratory of Environment
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* Corresponding author. Tel: +86-027-87288375; fax: +86-027-87288375; e-mail:
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[email protected]
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ACCEPTED MANUSCRIPT Abstract: To obtain a theoretical reference for microwave control of yeast used in the
15
fermented products, the effects of microwave irradiation dose and time on growth and
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cell membrane permeability of Yeast ZSM-001, Brettanomyces custersii isolated from
17
spontaneously fermented rice paste, were evaluated. When treated at a microwave dose
18
from 1.0 to 1.6 W/g for 90 s or at the optimal dose of 1.6 W/g for duration ≤ 120 s, the
19
yeast growth was enhanced according to Gompertz equation, and the yeast cells were
20
almost intact with smooth surfaces observed by SEM. Meanwhile, DNA, protein and
21
electrolyte leakage, as well as the fluidity of H+ and Ca2+ increased within a reversible
22
range as compared with the untreated control. At a microwave dose above 2.0 W/g or
23
duration over 160 s, the yeast growth rate decreased, and the cells were collapsed and
24
electroporated with rough surfaces. Therefore, low-intensity microwave (dose ≤ 1.6 W/g
25
or duration ≤ 120 s) favors yeast growth and induces a repairable increase in cell
26
membrane permeability without cell damage. High-intensity microwave results in the
27
yeast cell death mainly due to an irreversible increase of electrolyte, Ca2+ and DNA
28
leakage.
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Keywords: Yeast ZSM-001; Microwave dose; Microwave time; Growth; Cell membrane
30
permeability
31
Chemical compounds studied in this article
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Sucrose (PubChem CID: 5988); Agar (PubChem CID: 71571511); NaCl (PubChem CID:
33
5234); BCECF-acetoxymethyl (PubChem CID: 53229972); Fluo 3/AM (PubChem CID:
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5086914); Glutaraldehyde (PubChem CID: 3485); Ethanol (PubChem CID: 702);
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Platinum (PubChem CID: 23939).
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1. Introduction Yeasts are widely applied in fermented food including wine, beer, and baked foods
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with rice and flour as raw materials. In our previous work, yeast strain Brettanomyces
40
custersii (ZSM-001) was successfully isolated from spontaneously fermented rice paste,
41
and the yeast strain used for fermentation could ensure product quality and shorten the
42
fermentation time to a certain degree, as compared with the long fermentation time
43
(14~16 h) and unstable quality for the traditional fermented rice cake (Liu, 2008; Liu, Liu,
44
Zhao, & Xiong, 2010). The particular processing technique will select for the growth of
45
particular types of yeasts which are adapted to grow under the created condition, such as
46
pH, NaCl, temperature and microwave etc (Betts, Linton, & Betteridge, 1999; Grundler,
47
Keilman, Frohlich, & Srube, 1982).
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Microwaves are part of the electromagnetic spectrum and considered to be the
49
irradiation ranging in frequency from 300 MHz to 300 GHz, corresponding to a
50
wavelength range from 1m to 1mm (Banik, Bandyopadhyay, & Ganguly, 2003). Some
51
researchers have found that subtle effects induced by low-level microwave exposure were
52
not directly attributable to heat but to non-thermal effect (Belyaev, 2005; De Pomerai et
53
al., 2002; Welt, Tong, Rossen, &Lund, 1994). The growth rate of yeast Sacharomyces
54
cerevisiae has been reported to either increase up to 15% or decrease down to 29% by
55
certain frequencies of microwave irradiation within 41.8~42.0 GHz (Grundler, Keilman,
56
& Frohlich, 1977). Furthermore, several studies have indicated that the exposure of
57
Escherichia coli and Bacillus subtilis cell suspensions to microwave irradiation at the
58
frequency of 2.45 GHz may alter structural and functional properties of cell membranes
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& Tosoratti, 2004; Suga, Goto, & Hatakeyama, 2006; Woo, Rhee, & Park, 2000).
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Additionally, the interaction between microwave treatment and organisms has a
62
resonance-like dependence on the microwave irradiation frequency, time and power
63
(Chandrasekaran, Ramanathan, & Basak, 2012; Grundler et al., 1982; Wang, Wang, &
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Wang, 1996). Up to now, much work has been reported about microbial destruction
65
induced by microwave irradiation in a certain frequency, time and power range (Cheng et
66
al., 2013; Campanha. et al., 2007; Woo et al., 2000). However, little information is
67
available on the positive response of yeast to microwave irradiation, let alone
68
optimization of microwave exposure conditions (2.45 GHz, dose and time) for yeast
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growth.
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Therefore, the objective of the current study was to evaluate the effects of microwave
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treatment dose and time on the growth of Yeast ZSM-001 by measuring the growth
72
curves according to Gompertz equation followed by the SEM verification. Moreover, the
73
parameters of cell membrane permeability such as DNA and protein leakage, electrolyte
74
leakage, and H+ and Ca2+ permeability were measured to test the effects of various
75
microwave treatments on yeast cell membrane. This research explored the microwave
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environment beneficial to the growth of Yeast ZSM-001, and provides useful information
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about the microwave control in enhancing yeast growth for large-scale production of
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fermented food.
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2. Materials and methods
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2.1. Materials Yeast strain ZSM-001, Brettanomyces custersii, was isolated previously from
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spontaneously fermented rice paste as described by Liu et al. (2008) and was preserved in
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China Center for Type Culture Collection (CCTCC, No. M207150). All reagents used
85
were of analytical grade. All media and reagent solutions were prepared with distilled
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water.
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2.2. Cultivation of Yeast ZSM-001
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Potato liquid medium was prepared as follows. Peeled potatoes (200 g) were cut into
89
cubes, and put into distilled water at a volume ratio of 1:2, followed by heating at 100oC
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for 15 min. The potato broth was filtered through a filter cloth, and then the filtrate was
91
supplemented with sucrose (20 g). Finally, the as-prepared filtrate was diluted to 1000
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mL with distilled water at the ratio of 1:4 to obtain the potato liquid medium, followed by
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autoclaving at 121oC for 30 min. Potato solid medium was prepared by adding agar (13 g)
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into the above potato liquid medium.
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Before microwave treatment, Yeast ZSM-001 preserved in CCTCC was activated on
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potato solid medium at 30oC for 48 h. After activation, three loops of Yeast ZSM-001
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were inoculated into 250 mL of potato liquid medium and incubated on a rotary shaker at
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90 rpm and 30oC for 12 h. Then the culture medium was centrifuged at 4000 rpm for 5
99
min to obtain yeast slurry. Finally, the yeast slurry was suspended in a sterile 0.85% NaCl
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solution to prepare yeast suspension at a concentration of 3 wt% (1× 105 CFU/mL) for
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microwave treatment.
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2.3. Microwave treatment
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ACCEPTED MANUSCRIPT The microwave treatments of the samples were performed using a single-mode
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microwave oven (QW-15HM, Guangzhou Kewei Microwave Oven Energy, Guangzhou,
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China), with an internal capacity of 42 × 42 × 36 cm. This microwave device was mainly
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constructed from standard rectangular waveguides and a magnetron oscillator with a
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maximum continuous wave output power of 900 W at 2.45 GHz as the source of the
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microwaves. The cross section shape of microwave cavity and medium properties
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maintained the vertical axial uniformity. The temperature inside the oven was measured
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using a precise infrared temperature sensor with a resolution of 0.1oC.
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The microwave irradiation conditions and procedures were described as follows. The
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yeast suspension (100±0.1 g) poured into a 250 mL conical flask was placed in a central
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location inside the microwave oven, and exposed to microwave irradiation from 0 to 200
114
s at a 10 s interval and at various doses (1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.2 and 2.4 W/g),
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which was corresponding to the ratio between the microwave output power (W) and the
116
mass (g) of the sample (Özbek, & Dadali, 2007). After irradiation, the temperature of
117
yeast suspension was measured immediately with a JDDA80 point thermometer (Jingda
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Instrument Factory Co., Wuhan, China) at three different randomly selected positions so
119
as to determine the standard temperature-time curve at various doses. Meanwhile, the
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typical microwave dose and time range were determined according to the normal
121
temperature range for yeast growth (28~38oC). Additionally, the microwave doses and
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durations deviated from the normal conditions were also selected for evaluation of yeast
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growth.
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2.4. Determination of growth curve
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Yeast at a concentration of 3 wt% was inoculated into 97 mL of sterile potato liquid medium to prepare yeast suspension for growth curve determination. The growth curve of yeast was measured by the turbidimetric method. After 6 h
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incubation on a rotary shaker at 90 rpm and 30oC, yeast suspension was treated under
129
preselected microwave conditions. The absorbance values (OD) were measured at 560
130
nm every 3 h to determine the biomass of yeast (Iwashima, Kimura, & Kawasaki, 1990).
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All the experiments were made in triplicate. The yeast suspension untreated with
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microwave served as control.
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2.5. Determination of DNA and protein leakage
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The yeast suspension before and after microwave treatment was filtered, and the
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absorbances of DNA and protein in the supernatant were measured with a UV-2600
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spectrophotometer (Younikang Co., Shanghai, China) at 260 and 280 nm, respectively.
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The blank was composed of sterile 0.85% NaCl (Woo et al., 2000).
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2.6. Determination of electrolyte leakage
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Electrolyte leakage (EL) from cells treated with different microwave doses and
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durations was measured on an FE30 conductivity meter (Mettler-Toledo Instruments Co.,
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China) as described previously (Campanha et al, 2007; Soro, Djè, & Thonart, 2010).
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After cooling to room temperature, the microwave-treated yeast suspension was
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subjected to the measurement of the initial electrical conductivity (Ei). After Ei
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measurement, the microwave-treated yeast suspension was placed into a 100oC boiling
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water bath for 15 min for the measurement of the total electrical conductivity (Et). The
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initial and total electrical conductivities (Ci and Ct) of the control without microwave
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irradiation were measured under the same conditions as the microwave-treated samples.
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The electrolyte leakage was calculated as follows: EL (%) = [1 - (1 - Ei/Et) (1 - Ci/Ct)] ×100
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where Ei and Et are the initial and total electrical conductivities of the microwave-treated
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samples; Ci and Ct are the initial and total electrical conductivities of the control,
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respectively.
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2.7. Determination of H+ and Ca2+ permeability
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After microwave treatment, fluorescent probe BCECF-AM (1 mmol/L) was added
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into yeast suspension at a volume ratio of 1:300 with a final concentration of 3 µmol/L.
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Yeast suspension was cultured at 37oC for 30 min, and then centrifuged at 4000 rpm for
157
5min to collect yeast slurry. Finally, the yeast slurry was washed with 0.85% NaCl and
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resuspended in 3 mL of 0.85% NaCl. The amount of H+ released after microwave
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treatment was determined according to the fluorescence intensity obtained on a
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RF-5301PC fluorospectrophotometer (Shimadzu Co, Japan) with the double-wavelength
161
ratio method at an excitation wavelength of 520 and 440 nm (FIR = 520/440 nm), and an
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emission wavelength of 530 nm (Breeuwer, & Abee, 2000; Chen et al., 2009; Russell,
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Pottier, & Valenzeno, 1995). The yeast suspension supplemented with fluorescent probe
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but without microwave treatment was used as reference.
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The pretreatment for Ca2+ permeability with Fluo-3 AM as fluorescent probe was
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similar to H+ permeability. Briefly, after microwave treatment, fluorescent probe Fluo-3
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AM (1 mmol/L) was added into yeast suspension at a final concentration of 3 µmol/L,
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and cultured at 37oC for 30 min in the dark, followed by centrifugation at 5000 rpm for
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amount of Ca2+ released after microwave treatment was determined according to the
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fluorescence intensity obtained by a fluorospectrophotometer at an excitation wavelength
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of 490 nm, and the fluorescence intensity was observed at 509 nm (Russell et al., 1995;
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Zhang, Rengel, & Kuo, 1998).
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2.8. Morphology of yeast cells by scanning electron microscopy (SEM)
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The morphology of yeast cells was observed by a JSM-6390LV scanning electron
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microscope (NTC, Japan) at an accelerating voltage of 20 kV. The yeast suspension was
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centrifuged after microwave treatment, and the yeast slurry was collected and fixed by
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2.5% glutaraldehyde for 4 h. After 15 min centrifugation at 8000 rpm, the yeast slurry
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was washed twice with deionized water, and dehydrated successively with 30, 50 and
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70% ethanol. The final yeast slurry was freeze-dried and sputtered with platinum using an
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ion sputter coater (IB-5, EIKO Ltd., Tokyo, Japan). The morphology of yeast cells was
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observed and photographed with a scanning electron microscope (JSM-6390LV, JEOL
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Ltd., Tokyo, Japan) at an accelerating voltage of 20 kV.
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2.9. Data analysis
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All treatments and assays were performed in triplicate and repeated three times. The
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data were processed by Microsoft Excel 2010. The statistical significance of differences
187
for DNA, protein and electrolyte leakage, H+ and Ca2+ permeability assays were
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calculated by Statistical Analysis System Software 8.1 (SAS Institute Inc., USA).
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Difference among the control and treated samples was considered significant at p < 0.01.
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3. Results and discussion
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3.1. Determination of microwave dose and time for yeast growth Fig. 1 shows the curves of temperature vs microwave dose and time for yeast
194
suspension. It was reported by Craig and Jacobsen (1984) that the normal temperature for
195
yeast growth metabolism was in the range of 28~38oC, whereas the lethal temperature
196
was 51oC. As shown in Fig. 1, the temperature of the suspension increased linearly with
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the time at various doses. The temperatures at microwave doses of 1.0~2.4 W/g for the
198
fixed time of 90 s were within a range of 32.0 to 50.2oC including normal and lethal
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temperatures for yeast growth and exhibited a homogeneous distribution. When the
200
microwave dose increased up to 2.2 W/g for 90 s, the temperature reached 47.8oC close
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to the yeast lethal temperature (51oC). When the treatment time increased up to 200 s, the
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temperatures at all microwave doses were almost all above 40oC, and deviated from the
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normal temperature range of yeast growth (28~38oC). Therefore, six microwave doses
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(1.0, 1.4, 1.6, 1.8, 2.0, and 2.2 W/g) for the fixed time of 90s, and five time points (40, 80,
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120, 160 and 200 s) at the appropriate dose of 1.6 W/g were selected to investigate the
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effects of microwave dose and time on yeast growth and cell membrane permeability.
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3.2. Growth curve of yeast
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Fig. 2 shows the growth curves of yeast estimated indirectly by OD values after
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treatment with different microwave doses and durations. OD values could reflect the
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biomass of the yeast. The yeast biomass increased gradually with the time of culture, and
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entered the logarithmic phase after ca. 6 h, at which time point the yeast was treated by
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microwave, then the stationary phase at approximate 15 h, and finally the decline phase
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microwave dose from 1.0 to 1.6 W/g for 90 s resulted in a greater increase over the
215
untreated control in the yeast biomass. With a further increase up to 1.8 W/g, the biomass
216
was lower than that of the untreated control and the minimum biomass occurred at a dose
217
of 2.2 W/g (Fig. 2A). At the stationary phase, the yeast biomass reached the maximum
218
after treatment with a microwave dose of 1.6 W/g, which was defined as the optimum
219
microwave dose favoring yeast growth and cell membrane permeability. At the optimum
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microwave dose of 1.6 W/g, the growth curves exhibited a similar trend and were all
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higher than that of the untreated control with the treatment time varying among 40, 80
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and 120 s. When the exposure time exceeded 160 s, the biomass was much lower than
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that of the control (Fig. 2B).
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Fig. 2 shows that there is early adaptability to yeast growth by microwave process, so
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the Gompertz equation can be used to describe the growth characteristics of yeast as
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follows (Gompertz, 1825; Fang, Hu, Xiong, & Zhao, 2011):
N = N 0 + b0 e ( −b1e
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−b 2 t )
(1)
where N is the total increment of biomass at time t; N0 is the value related to the initial
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yeast (when t = 0, initial yeast = N0 + b0e -b1); b0 is a parameter related to the equivalent
230
yeast counts (when t → ∞, the equivalent yeast count = N0 + b0); b1 is a parameter related
231
to the early adaptability to growth; and b2 is a parameter related to a growth rate constant,
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and is usually a function of processing conditions such as microwave dose. It can be seen
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that the theoretical curves obtained from equation (1) gave the best fit to the experimental
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data points (solid points), and the residual sum of squares was much lower than the
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This result indicated that the accuracy of equation (1) is quite high and the growth
237
characteristics of yeast could be well described by the Gompertz equation. According to
238
equation (1), the data of N0, b0, b1 and b2 are obtained by a least-squares method, and
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listed in Table 1. The growth rate constant (b2) increased with the microwave dose from
240
1.0 to 1.6 W/g, and reached the maximum at 1.6 W/g, followed by a drop with a further
241
increase of the microwave dose. As the microwave time was prolonged at the optimum
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dose of 1.6 W/g, b2 increased up to the maximum at 120 s, followed by a decrease against
243
the untreated control. The value b0 presented a similar trend to b2. The results indicated
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that microwave treatment with a low dose (≤ 1.6 W/g) and duration (≤ 120 s) has a
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positive effect on the yeast growth. According to the curve of temperature vs dose or
246
duration, the temperatures were in the range of 32.0~35.9oC and 25.9~38.8oC after
247
microwave treatment with a dose of 1.0~1.6 W/g for 90 s and for duration of 0~120 s at a
248
dose of 1.6 W/g, both of which were almost in the normal temperature range of yeast
249
growth, suggesting that the positive action on the yeast growth was due to the
250
non-thermal effect rather than the thermal effect of microwaves. This result has been
251
individually confirmed by Ramundo-Orlando (2010) and de Pomerai et al. (2002). It was
252
well known that microwave causes thermal effect, but has non-thermal character, i.e., the
253
action of irradiation does not produce essential heating for the biological system or
254
destroy its structure. Low-level microwave energy produces various types of molecular
255
transformations and alterations, but the energy is too weak to break down the chemical
256
bonds or destroy the primary structure. Hence, low-level microwave exposure results in
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de Pomerai et al., 2002). On the other hand, microwave can inhibit yeast growth at a dose
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≥ 1.8 W/g or duration ≥ 160 s, due to heat production and a sharp increase in temperature
260
that can be systemic or localized, resulting in cell death (Ramundo-Orlando, 2010).
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3.3. DNA and protein leakage
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Cellular damage causes DNA and protein leakage, so the dissolution rate of DNA and
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protein can reflect cell membrane permeability and cell growth. Fig. 3 shows the protein
264
and DNA leakage of the yeast treated with various microwave doses (1.0, 1.4, 1.6, 1.8,
265
2.0 and 2.2 W/g for 90 s) and durations (40, 80, 120, 160 and 200 s at 1.6 W/g). As
266
microwave dose increased from 1.0 W/g to 1.8 W/g for 90 s, the absorbances of DNA and
267
protein increased by 11.6~15.6% and 10.9~14.5%, respectively, as compared with the
268
untreated control. However, with a further increase to 2.2 W/g for 90 s, the two
269
absorbances showed an increase of 28.5% and 27.2% over the control, indicating an
270
increase of cell membrane permeability. Low-intensity microwave exposure was reported
271
to induce reversible externalization of phosphatidylserine (PS) molecules in the
272
membrane of the exposed cells without detectable membrane damage or induction of cell
273
death (Ramundo-Orlando, 2010). Hence, a slight increase in cell membrane permeability
274
induced by low-dose (≤ 1.8 W/g) microwave exposure could be repaired reversibly. On
275
the other hand, high-dose (≥ 2.0 W/g) microwave irradiation induced irreversible
276
electroporation on the phospholipid bilayer membrane, leading to the damage of the
277
membrane structure and essential leakage of cell contents (Suga et al., 2006).
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At the optimum dose of 1.6 W/g with an exposure time range of 40 to 160 s, the
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280
compared with the untreated control, followed by a drop (18.3% and 14.5%) at 200 s.
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This result suggested that the microwave energy accumulated by duration was strong
282
enough to change the cell membrane permeability, accelerate the leakage of DNA and
283
protein, and even destroy the yeast cell. When the energy accumulation reached a critical
284
point, the protein and other biomacromolecules aggregated and denatured, leading to
285
diffusion difficulty and a decrease in DNA and protein leakage, which is in good
286
agreement with the study on high-energy pulse-electron-beam-induced molecular and
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cellular damage in Saccharomyces cerevisiae (Zhang et al., 2013).
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3.4. Electrolyte leakage
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Cellular electrolyte leakage was determined by measuring electrolyte conductivity,
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which reflects the function of cell membrane (Ovchinnikova, 1996). The effects of
291
microwave treatments (at doses of 1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90s or durations
292
of 40, 80, 120, 160 and 200 s at 1.6 W/g) on electrolyte leakage are shown in Fig. 4. The
293
electrolyte conductivity rose slowly with the microwave dose from 1.0 to 1.8 W/g,
294
followed by a dramatical rise at a dose above 1.8 W/g (Fig. 4A). At the optimum dose of
295
1.6 W/g, the electrolyte conductivity increased gradually with treatment time, followed
296
by a slight decrease after 160 s (Fig. 4B). The rising trend of electrolyte leakage may be
297
ascribed to the acceleration of material exchange inside and outside the cells induced by
298
the microwave irradiation at a dose ≤ 1.8 W/g or duration ≤ 120 s. With a further increase
299
of the microwave dose (> 1.8 W/g), the structure of yeast cell membrane was damaged
300
(Fig. 7), and yeast was unable to utilize the metal ions (Ca2+, Na+, and K+) due to the loss
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302
through the membrane, leading to a significant increase of electrolyte leakage (Woo et al.,
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2000). Cojocaru et al. (2005) proposed another explanation for the increase in electrical
304
conductivity induced by high-intensity microwave irradiation: the activation of lipid
305
peroxidation by metal ions led to the damage of the phospholipid membrane. Additionally,
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the cell membrane blockage caused by the aggregation and denaturation of the
307
intracellular biomacromolecules resulted in a slight decrease of electrolyte leakage at 200
308
s, which has been confirmed by another study in our laboratory, the microwave effect on
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Aspergillus (Fang et al., 2011).
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3.5. H+ permeability
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Intracellular pH values can reflect the intracellular H+ concentration and the cell ion
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channel. Fig. 5 shows the intracellular pH values of yeast treated with different
313
microwave doses (1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90 s) and durations (40, 80, 120,
314
160 and 200 s at 1.6 W/g). With an increase of microwave dose from 1.0 to 1.6 W/g for
315
90 s and time from 40 to 120 s at 1.6 W/g, the internal pH values of the yeast slightly
316
increased from 6.18 to 6.25, and from 6.20 to 6.48, but still remained in the normal pH
317
range of 6.1~6.5, which was similar to the results reported by Peña et al. (1995) that the
318
internal pH of normal yeast varied slightly even if the yeast was subjected to
319
electroporation at 1500 V and environmental simulation of pH from 4.0 to 8.0. The
320
low-intensity microwave irradiation (dose ≤ 1.6 W/g or duration ≤ 120 s) might induce
321
the activation of cytoplasmic membrane H+-ATPase, resulting in the extrusion of H+ from
322
the cytoplasm (corresponding to the increase of internal pH) and the concentration of H+
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324
(dose ≤ 1.6 W/g or duration ≤ 120 s) would promote nutrient intake loaded by H+
325
co-transport, and accelerate the yeast metabolic activity to increase the viability, as
326
indicated in the curve of growth rate (Haworth, & Fliegel, 1993; Viegas, Almeida,
327
Cavaco, & Sá-Correia, 1998). Furthermore, the effects of low-level microwave on ion
328
transport may be the consequence of its direct effect on membrane proteins and the
329
phospholipid domain organization (Ramundo-Orlando, 2010). However, microwave
330
treatment at a dose ≥ 2.0 W/g or time ≥ 160 s caused irreversible changes in the cell
331
membrane permeability of the yeast, and even induced the cell death partly mediated by
332
deactivation of the key enzyme combined with the cellular BCECF-AM probe, which
333
finally led to a decrease in pH measured by the fluorescence changes of BCECF-AM
334
probe (Breeuwer, & Abee, 2000; Grinstein, & Rothstein, 1986; Imai, & Ohno, 1995).
335
3.6. Ca2+ permeability
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Ca2+ is closely related to cell proliferation, division, movement, secretion, metabolism
337
(energy, oxygen, glucose) and many other cellular processes. The release of Ca2+ from the
338
vacuole into the cell’s cytoplasm and subsequently into the extracellular medium depends
339
on the vacuole and cell membrane (Campanha et al., 2007). The effects of microwave
340
doses (1.0, 1.4, 1.6, 1.8, 2.0 and 2.2 W/g for 90 s) and time (40, 80, 120, 160 and 200 s at
341
1.6 W/g) on Ca2+ permeability are shown in Fig. 6. The Ca2+ concentration is positively
342
correlated with fluorescence intensity (Matsuoka, Kosai, Saito, Takeyama, & Suto, 2002).
343
The fluorescence intensity (FI) of Ca2+ was lower than that of the untreated control within
344
the dose range of 1.0~1.6 W/g, followed by an increase over the control with a further
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346
treatment time at the fixed dose of 1.6 W/g, the FI of Ca2+ decreased within 80 s,
347
followed by a gradual increase. Moreover, the FI of Ca2+ was close to the control at 1.6
348
W/g or 120 s.
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The movement of Ca2+ is strictly self-regulated and the intracellular concentration of
350
Ca2+ is significantly lower than the extracellular concentration (Houchi, Yoshizumi,
351
Ishimura, & Oka, 1996). Under low-intensity microwave exposure (dose ≤ 1.6 W/g or
352
duration ≤ 120 s), the effusion of a small amount of intracellular Ca2+ was beneficial to
353
maintaining the Ca2+ concentration gradient inside and outside the cell, and promoted
354
yeast growth. However, microwave treatment at a dose ≥ 1.8 W/g or duration ≥ 120 s
355
resulted in a small influx of Ca2+ probably due to the increase of membrane fluidity,
356
thereby inducing the opening of Ca2+ channels (Bonilla, & Cunningham, 2003; Repacholi,
357
1998). A further higher dose (2.2 W/g) caused a sharp decrease of cellular Ca2+
358
concentration against the untreated control, due to cell rupture and death, which was
359
related to the precipitation of phosphate and the deactivation of the esterase used to
360
hydrolyze fluorescence probe Fluo-3/AM into Ca2+-sensitive form of probe (Zhang et al.,
361
1998).
362
3.7. SEM observation
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Fig. 7 shows SEM photograph of yeast cell after microwave treatment with various
364
doses and durations. It can be observed from Fig. 7 (A and a) that the untreated yeast
365
cells were intact and presented a spherical, oblate or nephritic shape. After microwave
366
treatment at a dose ≤ 1.6 W/g or duration ≤ 120 s, yeast cells were still complete and
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368
the untreated control, significant damages were detected on yeast cells with obvious
369
metamorphosis at 2.2 W/g, i.e., part of the cells were collapsed and electroporated, and
370
the surfaces were rough with sawtooth-shaped edges.
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The morphology of yeast cell indicated that low-intensity microwave treatment (dose
372
≤ 1.6 W/g and duration ≤ 120 s) could preserve the cell structure. Nevertheless,
373
high-intensity microwave exposure (dose ≥ 2.0 W/g or duration ≥ 160 s) induced the
374
essential changes in structural integrity and permeability of cell membrane and cell wall
375
which may have detrimental effects on the cell metabolism and lead to cell death
376
(Campanha et al., 2007). SEM imaging also demonstrated the influence of microwave
377
treatment dose and duration on the yeast growth and cell membrane permeability in terms
378
of proteins, DNA leakage of yeast cells and the ion channels.
379 380
4. Conclusion
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The effects of microwave exposure dose and time on Yeast ZSM-001 growth and cell
382
membrane permeability were investigated. Treated with a microwave dose from 1.0 to
383
1.6 W/g for 90 s or at the optimal dose of 1.6 W/g for 40~120 s, yeast growth rate was
384
increased up to the maximum at 1.6 W/g for 120 s, and the yeast cells were almost
385
complete with smooth surfaces. Meanwhile, the microwave exposure at a dose ≤ 1.6 W/g
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or duration ≤ 120 s induced a repairable increase in cell membrane permeability, together
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with an increase in DNA and protein, electrolyte leakage and transport of H+ and Ca2+
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between inside and outside cells. When the microwave dose increased up to 2.0 W/g or
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were observed in cells with rough surfaces. Hence, we conclude that microwave
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treatment with a low dose (≤ 1.6 W/g) and duration (≤ 120 s) favors yeast growth, and
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induces a repairable increase in cell membrane permeability without cell damage.
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High-intensity microwave may lead to the yeast cell death mainly due to a significant and
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irreversible increase in electrolyte and DNA leakage, as well as H+, Ca2+ permeability.
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However, the mechanism of cell damage induced by high-intensity or long-time
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microwave irradiation remains to be elucidated at the molecular level. This research
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provides useful information about the potential control of yeast growth for large-scale
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production of fermented food.
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Acknowledgments
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This work was supported by University Agricultural Science and Technology Service
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of Ministry of Science and Technology of China (2013BAD20B00), and Application and
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Foundation Research Project of Wuhan Science and Technology Bureau of China
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(2014020101010068).
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ACCEPTED MANUSCRIPT Table Caption Table 1. Yeast growth characteristics represented by values of N0, b0, b1 and b2
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obtained from Gompertz equation.
ACCEPTED MANUSCRIPT Figure Captions Fig. 1. The curves of temperature vs microwave dose and time for yeast suspension Fig. 2. Growth curves of yeast after microwave treatment with various doses for 90 s (A)
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and for different durations at 1.6 W/g (B). Fig. 3. DNA and protein OD values of yeast cells after microwave treatment with various doses for 90 s (A) and for different durations at 1.6 W/g (B). Significant differences
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between the control and treated samples were calculated to be p < 0.01.
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Fig. 4. Electrolyte leakage of yeast cells after microwave treatment with various doses for 90 s (A) and for different durations at 1.6 W/g (B). Different letters (a~f) indicate significant differences among different microwave treatments (p< 0.01). Fig. 5. The intracellular pH values of yeast cells after microwave treatment with various
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doses for 90 s (A) and for different durations at 1.6 W/g (B). Different letters (a~g) indicate significant differences among different microwave treatments (p< 0.01). Fig. 6. The intracellular Ca2+ fluorescence intensity of yeast cells after microwave
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treatment with various doses for 90s (A) and for different durations at 1.6 W/g (B).
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Different letters (a~f) indicate significant differences among different microwave treatments (p< 0.01).
Fig. 7. SEM photographs of yeast cells treated with various microwave doses for 90 s (B~D) and at 1.6 W/g for different durations (b~d) in comparison with the untreated control (A and a).
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ACCEPTED MANUSCRIPT Table 1 Yeast growth characteristics represented by values of N0, b0, b1 and b2 obtained from Gompertz equation Treatment condition
b0
b1
b2
0.260 0.247 0.215 0.211 0.271 0.288 0.283
1.022 1.092 1.155 1.185 0.960 0.811 0.512
28.949 28.055 27.291 30.528 30.546 32.567 24.395
0.371 0.375 0.393 0.413 0.368 0.358 0.336
0.502 0.443 0.453 0.471 0.478 0.517
1.368 1.480 1.477 1.468 1.210 0.724
21.318 18.606 19.614 21.330 17.856 17.646
0.325 0.335 0.338 0.341 0.318 0.309
Microwave time
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90 s
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Microwave dose 0 W/g 1.0 W/g 1.4 W/g 1.6 W/g 1.8 W/g 2.0 W/g 2.2 W/g Microwave time 0s 40 s 80 s 120 s 160 s 200 s
N0
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Microwave dose
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1.6 W/g
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80 1.0 W/g
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1.2 W/g
70
1.6 W/g
60
1.8 W/g 2.0 W/g 2.2 W/g
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2.4 W/g
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20 0
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Temperature, ℃
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100
Fig. 1
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Time, s
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1.5
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1.1 0.9
0 W/g 1 W/g 1.4 W/g 1.6 W/g 1.8 W/g 2 W/g 2.2 W/g
0.5 0.3
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Biomass ( 560 nm)
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Growth time, h
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Biomass ( 560 nm )
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Growth time, h Fig. 2
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1 DNA 260 nm
A
Protein 280 nm
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OD value
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0.6
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0.2 0.5
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Microwave dose ， W/g 1
DNA 260 nm
Protein 280 nm
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Microwave time, s Fig. 3
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A
85
b
80
d
75
c
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1.6
1.8
e 70
f
65 1
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Electrolyte leakage,%
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Micromave dose, W/g
B
76 74
a
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Electrolyte leakage,%
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Micromave time, s Fig. 4
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b c
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6.0 1
1.4
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Microwave dose, W/g 6.8
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Micromave time, s Fig. 5
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630
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A c
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b e
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0.0
1.0
1.4
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Fluorescence intensity ( 509 nm )
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2.2
B
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b
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c 550
d
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Fluorescence intensity （ 509 nm）
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Micromave dose, W/g
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f
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Micromave time, s Fig. 6
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B 1.0 W/g
C 1.6 W/g
D 2.2 W/g
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A 0 W/g
a0s
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Highlights: Microwave dose and time on yeast are tested to provide insight in growth
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control Yeast growth is enhanced at ≤1.6W/g and ≤120s with an optimal membrane permeability
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Yeast cell death is caused at ≥2.0W/g and ≥160s by electrolyte, Ca2+ and DNA
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leakage
SEM shows intact and electroporated yeasts respectively under the two above
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