Cryoprotective Activity and Action Mechanism of Antifreeze Peptides

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Bioactive Constituents, Metabolites, and Functions

Cryoprotective activity and action mechanism of antifreeze peptides obtained from tilapia scales on Streptococcus thermophilus during cold stress Chen Xu, jinhong wu, Ling Li, and ShaoYun Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06514 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 30, 2019

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Journal of Agricultural and Food Chemistry

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Running title: A novel marine biological antifreeze peptide

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Cryoprotective activity and action mechanism of antifreeze peptides

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obtained from tilapia scales on Streptococcus thermophilus during

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cold stress

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Xu Chen†,‡,#, Jinhong Wu§,#, Ling Li‡, Shaoyun Wang‡,*

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†

College of Chemical Engineering, ‡College of Biological Science and Technology,

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Fuzhou University, Fuzhou, Fujian 350108, China

10

§

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Shanghai Jiao Tong University, Shanghai 200240, China

Department of Food Science and Engineering, School of Agriculture and Biology,

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ACS Paragon Plus Environment


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ABSTRACT: Cold stress adversely affects cell viability and acidification, and new

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cryoprotective methods continue to be needed in cold-chain food industry. Given

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this, we investigated the cryoprotective effects and action mechanism of antifreeze

15

peptides obtained from tilapia scales (TSAPP) on Streptococcus thermophilus during

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cold stress. Our results showed that the molecular weight of TSAPP ranged from

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180 to 2,000 Da and its thermal hysteresis activity was 0.29 °C. Growth of S.

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thermophilus was improved after treatment with TSAPP (1 mg/mL) under cold

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stress. This growth was notable when compared with the effects of other

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cryoprotectants. Furthermore, TSAPP improved the metabolic activity of S.

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thermophilus during cold stress. TSAPP likely offered its cellular protection by

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maintaining cell membrane fluidity through hydrogen bonding of the phospholipid

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bilayer. These results indicate that TSAPP has potential as a novel biological peptide

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material with cryoprotective activity for future use in probiotic or other processed

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food applications.

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KEYWORDS: antifreeze peptides, cryoprotective activity, mechanism of action,

27

Streptococcus thermophilus, cold stress

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2


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■ INTRODUCTION

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Antifreeze proteins (AFPs) are ice-structuring proteins and also known as thermal

31

hysteresis proteins. These proteins have many properties, including thermal

32

hysteresis ability (THA), which selectively depresses the freezing point of a solution

33

relative to its melting point.1 They can also inhibit the growth of crystals by binding

34

to the surface of ice crystals2, inhibit ice recrystallization (IRI) by maintaining ice

35

crystals in small shapes within a frozen sample3, and decrease cellular injury.4 The

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wide-ranging abilities of AFPs have large, potential applications in many areas,

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including in living cells, tissues, and organs as well as in crops and frozen foods.5-7

38

Lactic acid bacteria (LAB) ferment carbohydrates and produce large amounts of

39

lactic acid. Of these, S. thermophilus are widely used as a starter due to their

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nutrition, flavor, and fermentative ability in probiotic products and fermented foods.

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Freeze-drying and subsequent frozen storage is generally a useful, effective, and

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promising method to maintain the cellular stability of LAB as well as their attendant

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technological properties. However, these techniques have several undesired side

44

effects, including damaging cellular structures, deteriorating cellular viability, and

45

attenuating the fermentation performance of LAB.8,9

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Various physico-chemical events occur when cells are treated with cold stress.

47

The cellular membrane is sensitive to low temperatures and is the primary target of

48

damage, and the membrane lipid phase transition as well as membrane fluidity may

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also decline. Furthermore, loss of membrane integrity, membrane permeabilization,

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and leakage of intracellular contents are related to membrane damage after cold 3



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stress. This damage can be prevented to some extent by adding cryoprotectants.

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Good cryoprotectants should have similar characteristics, including a wide range of

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sources, low cost, hypothermic protection, and similar properties to fermented

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food.10

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Past work has shown that collagen peptides could exert a direct protective effect

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on living organisms. This is due to their range of molecular weight at inhibiting

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recrystallization of an ice cream mix as well as their solubility in water.1,11 A

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repetitive, tripeptide sequence is the most prominent characteristic of collagen

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peptides and is depicted as -(Gly-Z-X)n-. In this form, X is any amino acid residue

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and Z is always occupied by Pro or Hyp.12 Importantly, Lee and colleagues

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confirmed that Ala/Thr/Ser residues contribute to the cryoprotective ability of ice-

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structuring proteins.13

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Given this, we predicted that antifreeze peptides were obtainable from collagen

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hydrolysate and would exhibit protective abilities at low temperatures. Tilapia scales

65

are a by-product of food processing and are rich in collagen. Given this, the

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objective of this study was two-fold: (1) to evaluate their cryoprotective activity and

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(2) to investigate possible mechanisms of action of antifreeze peptides obtained

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from tilapia scales (TSAPP) on S. thermophilus.

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■ MATERIALS AND METHODS

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Materials. S. thermophilus used in the experiment was obtained from Fuzhou

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University (Fuzhou, China). The culture medium was M17 broth obtained from

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Qingdao Hope Bio-Technology Co., Ltd (Tsingtao, China). Tilapia scales were 4


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purchased from Yuanshui Food Co., Ltd (Xiamen, China). Trypsin was purchased

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from Notlas biotechnology Co., Ltd (Beijing, China). All other chemicals and

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reagents were of analytical grade and obtained from Sinopharm Chemical Reagent

76

Co., Ltd (Shanghai, China).

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Preparation of Antifreeze Peptides. Tilapia scales were dried and crushed and

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the powder mixed with deionized water at a substrate concentration of 4% (w/v).

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The mixture was stirred ultrasonically for 90 min at 228 W and then heated to

80

121 °C for 60 min to denature any native proteins. After cooling, the mixture was

81

hydrolyzed for 6.35 h using 7.30 % trypsin (w/w) at pH 7.40 and 50 °C. The mixture

82

was then heated in a boiling water bath for 10 min to inactivate the enzyme. Finally,

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supernatant fractions were centrifuged at 10,000 rpm for 10 min and then freeze-

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dried. These prepared antifreeze peptides obtained from Tilapia scales were

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lyophilized and are identified in this study as TSAPP.

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Determination of Molecular Weight Distribution. The molecular mass

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distribution of TSAPP was analyzed using high-performance liquid chromatography

88

(HPLC) with a Waters™650E Advanced Protein Purification System (Waters

89

Corporation, Milford, MA, USA). 1

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Analysis of amino acid composition. The freeze-dried samples of TSAPP were

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hydrolyzed at 110 °C for 24 h using 6 M of HCl, and the amino acid composition of

92

TSAPP was analyzed with a High Speed Amino Acid Analyzer Model L-8900

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(Hitachi, Japan).

94

Thermal Hysteresis Activity Measurement. TSAPP thermal hysteresis activity 5



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was determined using a Netzsch 204 F1 Differential Scanning Calorimetry (DSC)

96

device equipped with Proteus Thermal Analysis software (Netzsch. GmbH, Selb,

97

Germany) and according to the detailed procedures previously described by Wu et

98

al.14 Bovine serum albumin (BSA) was used as for the control.

99

Cryoprotective Measurement. The cryoprotective activity of TSAPP on S.

100

thermophilus was determined using our previously described protocol.3 Briefly, two

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separate 50 µL of a 10-3-fold gradient dilution cell solution was used, one from

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before freezing and one from after freezing. Solutions were separately incubated in

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4 mL of M17 broth for 7 h on a shaker rotating at 180 rpm at 37°C. Resulting

104

bacteria concentration was determined by measuring absorbance at 600 nm using a

105

UV-Vis spectrophotometer (UV-2600, UNICO Instrument Co. Ltd., Shanghai,

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China). S. thermophilus survival rate was defined as the ratio (expressed as a

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percentage) of the absorbance of active cells before and after freezing. The

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concentration of TSAPP used in the cryoprotective measurement was 1 mg/mL.

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The commercial cryoprotectants included sucrose, skim milk at 1 mg/mL, 20%

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glycerol (v/v) was used as the positive controls, and 0.9% saline (w/v) was used as

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a negative control.

112

Acidifying Activity Measurement. Bacterial acidifying activity was evaluated

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according to the previously published method of Meneghel et al.15 with minor

114

modifications. Briefly, 500 mL of skim milk medium consisting of 85 g/L powder

115

was sub-packed into 4 mL test tubes. All media were sterilized at 110 °C for 20 min.

116

After thawing, 50 μL of 10-3 fold gradient dilution bacterial culture was added to 4 6


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mL of skim milk medium and incubated for 18 h at 37 °C on a shaker rotating at 180

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rpm. The pH was continuously monitored every 1 h until the end of the acidification

119

measurement.

120 121

Effect of Antifreeze Peptides on the Intracellular Metabolism of S. thermophilus.

122

β-galactosidase and Lactate Dehydrogenase Measurements. Preparation of cell-

123

free extracts (CFEs) was conducted according to the previously published method of

124

Wang et al.2 Briefly, β-galactosidase activity was determined by measuring the

125

absorbance at 420 nm after generation of yellow o-nitrophenol (ONP).16 The

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reaction mixture consisted of CFEs (1 mL), 20 mM ONPG (1 mL) in 0.1 M PBS

127

buffer (pH 7.0) and 0.5 M Na2CO3 (3 mL) to a final volume of 5 mL. The reaction’s

128

absorbance was then measured at 420 nm. One unit of β-galactosidase activity was

129

defined as 1 μmoL of ONP liberated from ONPG per min.

130

The activity of LDH was determined by measuring the decrease in absorption at

131

340 nm due to the oxidation of nicotinamide adenine dinucleotide (NADH)

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following reduction of pyruvate to lactate at 340 nm.17 The reaction mixture

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consisted of 30 mM pyruvate, 0.2 M Tris-HCl buffer (pH 7.3) and 6.6 mM NADH.

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The reaction was initiated by adding 0.1 mL of CFEs and was followed by

135

measuring the decrease in absorbance at 340 nm.

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Soluble Sugar Content Measurement. Soluble sugar content was determined by

137

the anthrone reagent method.18 Briefly, standard sucrose solution curves for different

138

sucrose concentrations (0, 20, 40, 60, 80 and 100 μg/L) were prepared. The standard 7



139

curve was linear over the tested time period and the reaction mixture consisted of

140

CFEs (0.5 mL), distilled water (1.5 mL), 20 mg/mL anthrone (in ethyl acetate, 0.5

141

mL), and concentrated sulfuric acid (5 mL). The resulting mixture was then added to

142

20 mL test tubes. Racks were transferred to a boiling water bath for 1 min.

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Depending on the sugar concentration present, solution color changed from green to

144

green-blue and then to dark-blue. In absence of sugar, sample color remained a

145

transparent yellow. The solution was allowed to cool and its absorbance was

146

measured at 630 nm. The CFEs soluble sugar content was determined using the

147

generated standard curve.

148

Metabolic Activity Analysis. Metabolic activity was analyzed using the

149

reduction of the artificial electron acceptor INT to the visible intracellular form of

150

INT-formazan. After thawing, cells were washed twice and resuspended in 50 mM

151

potassium phosphate buffer (pH 7.4). A solution of 4 mM 2-(4-iodophenyl)-3-(4-

152

nitrophenyl)-5-phenyltetrazo-lium chloride (INT; ACROS) was subsequently added

153

to the cell suspension to a final concentration of 2 mM. Then bacterial suspension

154

was incubated at 37°C for 2 h. The reduction of colorless INT to red formazan was

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determined by measuring the absorbance at 584 nm.

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Cell Membrane Permeability. A sample of cell suspension (5 mL, 109

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CFU/mL) was centrifuged at 6,000 rpm for 10 min, after which cell pellets were

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washed twice in sterile water to remove any growth medium constituents. An equal

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volume of protective agent was then added, with saline used as the negative control.

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Aliquots (5 mL) of each bacterial suspension were frozen at -20 °C for 24 h. Two 8


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freeze-thaw cycles were conducted at 2 h intervals from the start time. Thawed cells

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were then centrifuged at 6,000 rpm for 10 min at 4 °C. The supernatant fractions

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were collected and the contents of extracellular proteins and nucleic acid were

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determined using Bradford’s previously published method.19

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Scanning Electron Microscopy (SEM). Cells were frozen at -80 °C for 2 h

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after the cold pretreatment with or without TSAPP for 24 h. Cells were then freeze-

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dried using a labconco freeze dryer (Changliu Scientific Instruments, Beijing,

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China) at 45 °C for 36 h under 10 Pa of pressure. A small amount of platinum was

169

introduced to the samples to avoid charging effects when undergoing microscopy.

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Microscopy was performed using a Nova Nano SEM 230 scanning electron

171

microscope (FEI Company, Hillsboro, OR, USA).

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Cell

Membrane

Fluidity

and

Membrane

Electrical

Potential.

S.

173

thermophilus in the mid-log phase was harvested using centrifugation (6,000 rpm,

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10 min) and washed twice with sterile water. An equal volume of cryoprotectant was

175

added to suspend the cells, which were then treated with cold stress. After thawing,

176

samples were washed twice with 50 mM sterile PBS (pH 7.4).

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Cell membrane fluidity was determined using the fluorescent dye DPH. Briefly,

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DPH was added to a cell suspension before and after cold stress to a final

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concentration of 4 μM. DPH was then allowed to incubate with the suspension at

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37 °C for 30 min. After washing twice with PBS, cells were resuspended in PBS and

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then prepared for F-4500 fluorescence spectrophotometry (Hitachi, Tokyo, Japan)

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using excitation and emission wavelengths of 350 nm and 425 nm, respectively. 9



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The cell membrane electrical potential was determined using the cell-permeant

184

lipophilic cationic dye RH123.20 The fluorescence intensity of RH123 was recorded

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with a spectrofluorometer at excitation and emission wavelengths of 480 nm and

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530 nm, respectively.

187

Fourier-Transform Infrared Spectroscopy (FTIR). TSAPP-cell membrane

188

interactions were conducted using soybean lecithin as a model system. The treated

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group consisted of soybean lecithin and TSAPP, with soybean lecithin used as a

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control. Samples were dissolved in 50 mM PBS (pH 7.4), and vortexed for 3 min to

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prepare the model cell membrane. After freeze-drying, samples were evaluated

192

using the Platinum ATR accessory with Bruker FTIR Tensor 27 Hyperion

193

equipment (Bruker, Karlsruhe, Germany). The spectra of each sample were

194

collected within the wavenumber range of 4000-400 cm-1 and the spectral data were

195

analyzed using OMNIC 8.2 software (Thermo Nicolet Co., Madison, WI, USA).

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Nuclear Magnetic Resonance (NMR) Micro-imaging Analysis. The melting

197

rate of the frozen solution was determined using NMR micro-imaging and was

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based on the published method of Li et al.3 Briefly, 1.0 mg/mL and 5.0 mg/mL of

199

TSAPP were obtained after dissolving samples in distilled water. 20% glycerol and

200

distilled water were used as positive and negative controls, respectively.

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Data Analyses. All experiments were conducted using at least three replicate

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experiments. Data analyses were performed using SPSS 17.0 (SPSS, Chicago, IL,

203

USA). All data are reported as mean ± standard deviation of 3-4 independent

204

experiments. Statistical significance was determined using Duncan’s multiple range 10


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tests with P< 0.05 used as the threshold for statistical significance.

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■ RESULTS AND DISCUSSION

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TSAPP Physico-chemical Properties. The molecular mass distribution of TSAPP

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as determined by HPLC is shown in Figure 1A. TSAPP was rich in short peptides of

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molecular weights between 180 to 2,000 Da; collectively, these peptides comprised

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89% of TSAPP (Figure 1B). These results suggest that TSAPP consisted mainly of

211

oligopeptides with 2-10 amino acids. Previous research has shown that short

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peptides either adsorb onto the surface of ice crystals or inhibit the process of ice

213

crystallization, thus playing an important role in protection from hypothermia.21

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Furthermore, the amino acid composition of TSAPP was analyzed by Amino Acid

215

Analyzer, and the results were given in Table 1. Results showed that TSAPP was

216

rich in glycine (24.41%), proline (12.80%), glutamic (12.43%), alanine (10.46%),

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which had been reported to be relative with the ice affinity and cryoprotective

218

activity of antifreeze proteins.2,3,11

219

DSC curves for the freezing and partial melting processes of BSA and TSAPP

220

solutions at varying hold temperatures (Th) are shown in Figures 1C and 1D. When

221

compared with the DSC data for the BSA control, the TSAPP exothermic peak

222

occurred later. The thermal hysteresis activity (THA) of TSAPP increased from

223

0.20 °C to 0.29 °C as Th increased from 0.07 °C to 0.37 °C. This finding suggests

224

that TSAPP has some thermal hysteresis properties.

225

Moreover, there was a negative, linear correlation between the thermal

226

hysteresis activity and the logarithm of the fraction of ice crystals in a given 11



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sample.22-24 As shown in Table 2, the fraction of ice nuclei (Φ) in the BSA control

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decreased from 98.17% to 90.59% as Th increased from 0.07 °C to 0.37 °C.

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However, there were less ice nuclei (Φ) in the TSAPP solution (78.81% to 12.40%)

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and higher THA values were observed. Past work has shown similar results for both

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ice-binding collagen peptides and ice-binding sericin peptides, which both had

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higher THA values when compared with BSA controls.12,14

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TSAPP Cryoprotective Activity. Although freezing and freeze-drying are

234

widely used for the long-term preservation of lactic acid bacteria, these techniques

235

have several undesirable side effects including the loss of viability and increased

236

acidification activity.25,26 After freezing stress, the relationship between cell growth

237

and acid production after a 7 h incubation is shown in Figure 2A. After TSAPP

238

addition, both bacterial growth rate and acid production were significantly higher

239

than those of either commercial cryoprotectants or saline. Moreover, the

240

acidification

241

cryoprotectants tested at -20 °C for 24 h after cold stress. These results are shown in

242

Figure 2B. With increasing incubation time, the TSAPP group had significantly

243

lower pH when compared with that of either the commercial cryoprotectants groups

244

or the saline group.

changes

showed

remarkable

differences

with

the

various

245

Bacterial growth after freezing in the presence of various cryoprotectantsis

246

shown in Figure 2C. After cryopreservation, the latent period of the bacteria in the

247

saline group was significantly longer (6 h). The logarithmic and stable phases also

248

lagged. Taken together and when compared with other commercial cryoprotectants, 12


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TSAPP had a significant cryoprotectant effect on S. thermophilus growth after 24 h

250

of freezing stress. These results show that when compared with fresh, unfrozen cells,

251

the TSAPP group exhibited only minor losses in acidification activity and growth.

252

Figure 2D illustrates the growth stability of S. thermophilus with various

253

cryoprotectants at different freezing times. With increasing freezing times, the

254

number of cells in the saline group decreased rapidly and reached an absorption

255

value at 600 nm (OD600) of 0.025 after 10 d of frozen treatment. The initial OD600 of

256

both the skim milk and sucrose groups were very high and both had sharp decreases

257

as freezing time increased. The OD600 of the glycerol group was reduced to 0.555

258

after three days of frozen treatment, after which the OD600 exhibited a slow decrease.

259

The OD600 of the TSAPP group was the highest, which validated our initial findings

260

that TSAPP had greater antifreeze activity than commercial cryoprotectants.

261

Changes in Cell Metabolic Activity. β-galactosidase catalyzes the hydrolysis

262

of galactoside bonds, which is the key enzyme used by lactic acid bacteria for their

263

probiotic effects. Given this, we next investigated the effect of cryoprotectants on

264

the activity of β-galactosidase. These results are shown in Figure 3A. Differences in

265

the activity of β-galactosidase were observed after different cryoprotectant

266

treatment. In the presence of TSAPP, sucrose, skim milk or glycerol, the β-

267

galactosidase activities were 16.20±0.22 U/mL, 11.72±0.37 U/mL, 15.02±0.14

268

U/mL and 17.14±0.32 U/mL, respectively. β-galactosidase activity was only 10.09

269

U/mL in the saline group. These results suggest that the β-galactosidase activity was

270

reduced due to the freezing damage. However, enzyme activity is protected by the 13



271

action of TSAPP, which further underscores its protective potential during

272

hypothermia.

273

LDH is a key enzyme in the metabolism of LAB, which can catalyze the

274

reduction of pyruvate to lactic acid. The activity of LDH also reflects the acid-

275

producing capacity of the strain and is known to be particularly sensitive during the

276

freezing process.27 Given this, we next determined the effects of cryoprotectants on

277

LDH activity (Figure 3A). LDH activity was impaired by saline treatment and was

278

approximately 1.58±0.53 U/mL. However, LDH activities improved in the presence

279

of both TSAPP and commercial cryoprotectants. Critically, TSAPP was better able

280

to maintain greater LDH activity than the other tested cryoprotectants. Specifically,

281

TSAPP maintained LDH activity at 14.29±0.64 U/mL, which was 8.9-fold higher

282

than that of the saline control group. Collectively, these results indicate that TSAPP

283

is an effective cryoprotectant that can prevent loss of LDH activity during cold

284

stress.

285

Cells continuously accumulate soluble sugar to maintain their osmotic pressure,

286

prevent excessive water loss, and adapt to low temperature environments; notably,

287

these same rules seem to apply to bacteria. The intracellular sugars of S.

288

thermophilus that had been subjected to cold stress are shown in Figure 3B. When

289

compared with the saline control group, the accumulation of intracellular soluble

290

sugars was increased slightly in the various cryoprotectant groups. Specifically, the

291

soluble sugar content in the saline group was 37.65±2.98 μg/mL, while that in the

292

TSAPP group reached the maximum observed level of 54.55±1.38 μg/mL. 14


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Moreover, the soluble sugar contents in the sucrose, skim milk, and glycerol groups

294

were 43.73±0.16 μg/mL, 46.81±1.87 μg/mL, and 49.58±0.40 μg/mL, respectively.

295

These were all slightly lower when compared to the level observed in the TSAPP

296

group. Taken together, these findings indicate that TSAPP stimulates cells to

297

accumulate soluble sugar to resist freezing.

298

INT is an artificial electron acceptor that can be reduced by bacteria to visible,

299

intracellular deposits of INT-formazan. These deposits can then be used to evaluate

300

cellular dehydrogenase activity.28 The metabolic activity of frozen S. thermophilus

301

under cold stress is shown in Figure 3C. When compared with fresh, unfrozen cells,

302

cellular metabolic activity decreased to different degrees after freezing. However,

303

the metabolic activity in the saline group was significantly decreased (P < 0.05)

304

when compared with the activities of the various cryoprotectant groups, all of which

305

were approximately 27.22±1.50%. S. thermophilus metabolic activity was

306

maintained at 56.07±0.25% (TSAPP), 35.91±2.16% (sucrose), 53.75±0.14% (skim

307

milk), and 79.05±4.25% (glycerol) when cells were exposed to freezing. Taken

308

together, these results suggest that TSAPP slowed the decline of cellular metabolism

309

and protected cellular viability. Similarly, Doikham soybean flour (SBF) has also

310

been shown to maintain the metabolic activity of freeze-dried cells.16

311

Cell membrane permeability. Extracellular proteins and nucleic acids can be

312

used as indicators for microbial damage.29 Using this approach, we determined the

313

leakage concentration of extracellular nucleic acids and proteins of S. thermophilus.

314

These results are shown in Figure 4. After freezing, the leakage concentration of 15



315

nucleic acids and proteins in the saline group reached the maximum observed levels,

316

which were 21.45 mg/mL and 46.48 μg/mL, respectively. These results indicated

317

that without the addition of a protective agent, refrigeration caused damage to the

318

cellular membrane and enhanced membrane permeability. Consequently, this

319

resulted in the leakage of intracellular nucleic acids and proteins to the outside.

320

Comparatively, the leakage concentration of extracellular nucleic acids and proteins

321

in cells treated with one of the tested cryoprotective agents were significantly lower

322

than those in the negative control group (P< 0.05). Importantly, the extracellular

323

nucleic acid and protein levels in the TSAPP-treated group were the lowest.

324

Collectively, these results indicate that TSAPP has a markedly positive effect in

325

protecting cell membrane permeability and preventing leakage of intracellular

326

substances during freezing stress.

327

The cell membrane is very sensitive to temperature changes and is one of the

328

primary targets of damage during the freezing process. To this end, Wang et al.2

329

reported that antifreeze peptides obtained from pigskin collagen hydrolysate may be

330

largely soluble under fully hydrated conditions. This occurs by partitioning into

331

membranes upon the removal of water, which prevents the leakage of intracellular

332

proteins and nucleic acids in S. thermophilus after freeze-drying. Given this, one

333

could infer that TSAPP is an antifreeze agent that operates through the combined

334

effect of hydrogen bonding between TSAPP and the phospholipid bilayer.

335

Collectively, this results in the maintenance of membrane stability.

336

Morphological characterization. SEM was performed better understand the 16


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effect of TSAPP on freezing-induced changes to the cellular membrane and

338

investigate a possible interaction between the two. SEM images showed that cells

339

without a protective agent were exposed to the air surface and were also clustered

340

together without clear boundaries (Figures 5A1, 5A2). One explanation could be

341

that freezing and/or freeze-drying resulted in cell membrane rupture. This caused the

342

release of intracellular materials and their deposit on the surface of cells to form a

343

continuous substrate.

344

Our results also revealed that cell membrane of the untreated group had serious

345

ruptures and cellular deformation (Figure 5A3). After treatment with 1 mg/mL

346

TSAPP, cells were entrapped in a glassy network and covered by thin layers of the

347

protective glassy matrix formed by TSAPP (Figures 5B1, 5B2). Cells also had a

348

normal, smooth, and round surface and did not exhibit the release of intracellular

349

components; these cells also had observable boundaries between cells (Figure 5B3).

350

These results were partially confirmed with those of previous studies.3 Moreover,

351

these results indicated that TSAPP effectively maintained the permeability barrier

352

and structural integrity of the cell membrane and prevented cellular leakage.

353

Changes in Cell Membrane Properties. DPH is a low molecular mass (232.32

354

Da) fluorescent probe that has strong lipophilic properties. It is used to embed in

355

lipid bilayers and is a useful indicator of membrane fluidity. More specifically, Cell

356

membrane is negatively correlated with the fluorescence intensity of the probe and

357

increased fluorescence intensity indicates the decline of cell membrane fluidity.30,31

358

As shown in Figure 6A, the DPH probe fluorescence intensity of S. thermophilus is 17



359

shown under cold stress. When compared with fresh, unfrozen cells, the cell

360

membrane fluidity was significantly decreased after freezing. However, the saline

361

control group had the most dramatic reduction in membrane fluidity when compared

362

with the other groups and had a fluorescence intensity of 187.71±4.10. In contrast,

363

the fluorescence intensity of the TSAPP-treated group was significantly lower than

364

that of the sucrose, skim milk, or saline groups. Collectively, these results indicate

365

that TSAPP maintains cell membrane fluidity better when compared with

366

other commercially available cryoprotectants.

367

RH123 is a type of lipid cationic fluorescence probe that relies on the

368

transmembrane potential of the cell matrix. To this end, cellular membrane potential

369

is positively correlated with the probe’s fluorescence intensity. Figure 6B shows the

370

RH123 probe fluorescence intensity of S. thermophilus with various cryoprotectants

371

under cold stress. When compared with fresh, unfrozen cells, cellular membrane

372

potential was significantly increased after freezing. The fluorescence intensity of the

373

probe in the saline group reached the highest observed level; comparatively,

374

fluorescence intensity decreased rapidly after the application of cryoprotective

375

agents. Under freezing stress, the fluorescence absorption intensity of the glycerol

376

and TSAPP groups were both at minimum observed levels and there were no

377

significant differences between them.

378

When taken together, these results showed that cells underwent varying degrees

379

of hyperpolarization. Moreover, this hyperpolarization was most notable in the

380

saline control group. Cellular membrane hyperpolarization plays an important role 18


Page 18 of 51

Page 19 of 51


381

in the signal transduction of cells. Moreover, the more significant the

382

hyperpolarization, the more serious the cold stress. The results presented here

383

suggest that TSAPP successfully reduces cell membrane potential, which positively

384

affects the metabolic activity of cells after cold stress.

385

FTIR. As shown in Figure 7, the interaction between TSAPP and soy lecithin

386

were also explored. The position of the C═O stretching band of the membrane

387

interface was from approximately 1750 cm-1 to 1650 cm-1 and the position of the

388

symmetric or asymmetric P=O stretching band in the phospholipid head was from

389

approximately 1260 cm-1 to 1050 cm-1.32 The FTIR spectra of the TSAPP-treated

390

group had some differences when compared with the untreated group. The FTIR

391

spectrum of freeze-dried soybean lecithin had distinct bands in a three-spectrum

392

region. These bands were 1653.56 cm-1, 1240.07 cm-1, and 1043.21 cm-1, which

393

corresponded to the soybean lecithin C═O stretching band, symmetric P=O

394

stretching band, and asymmetric P=O stretching band. With the addition of TSAPP,

395

the absorption peaks at 1653.56 cm-1, 1240.07 cm-1, and 1043.21 cm-1 for soybean

396

lecithin gradually decreased, with weakened absorption intensities. Moreover, the

397

absorption peaks were shifted to 1650.42 cm-1, 1241.98 cm-1,1046.12 cm-1 (soybean

398

lecithin: TSAPP = 1:1) and 1650.50 cm-1, 1241.96 cm-1, and 1077.95 cm-1 (soybean

399

lecithin: TSAPP = 1:5 ).

400

The presence of TSAPP altered the spectrum, which could be explained by the

401

interaction between the polar heads of lecithin and TSAPP. This interaction was

402

likely to result in hydrogen bonding.33 Normally, the cell membrane surface contains 19



403

a lot of water. Given this, the phospholipid polar head in the cell membrane has a

404

degree of hydration via the hydrogen bonding between water and the phospholipid

405

polar heads. This hydrogen bonding helps maintain the integrity of the biofilm

406

structure and its corresponding function.34 However, in the process of freezing or

407

freeze-drying, the phospholipid bilayer changes from a fluid and disordered liquid-

408

crystalline phase to a rigid and ordered gel phase. Under normal conditions, this

409

change results in significant damage to the cell membrane.15 The protective ability of

410

TSAPP may be due to its replacing the position of the water in the biofilm structure.

411

The hydrogen bonds between the -OH of TSAPP and the phospholipid head groups

412

would prevent the phospholipid heads from approaching each other, reduce the

413

phospholipid phase transition temperature, and stabilize membrane structure and

414

function. If true, this mechanism would be similar to the protective effect of sucrose

415

on cellular membranes.35

416

NMR Imaging. We next used examined proton NMR images from transverse

417

sections of frozen aqueous solutions containing TSAPP (1.0 and 5.0 mg/mL) at

418

various melting times (Figure 8). Water and 20% glycerol were used as negative and

419

positive controls, respectively. The distribution of water in frozen aqueous solutions

420

was distinctly different with increased melting time. NMR detected the spatial

421

distribution of mobile water as positive proton signals, while solid ice showed no

422

signal.36 The bright yellow along the edges of the image represented areas of mobile

423

hydrogen atoms, while the blue in the center indicated solid ice.

424

At the start of the melting process, some mobile water signals occurred in 20% 20


Page 20 of 51

Page 21 of 51


425

glycerol, but only faint signals were detected in other samples. After 30 min of

426

melting, 20% glycerol had a very strong mobile water signal, as the sample was

427

almost entirely melted. Meanwhile, clearer mobile water signals were observed in

428

the 1.0 mg/mL of TSAPP than when compared with the 5.0 mg/mL of TSAPP and

429

H2O. As melting time increased from 45 min to 60 min, both the 20% glycerol and

430

TSAPP groups were all bright yellow with little evidence of solid ice.

431

These results suggest that TSAPP has a stronger bulk melting ability for frozen

432

solutions when compared with sterile water. Moreover, the melting rate of low

433

concentration TSAPP was faster than that of high concentration TSAPP. However,

434

bulk melting of 20% glycerol was the strongest in these samples during the same

435

melting time. Moreover, Li et al.3 suggested that the addition of sericin peptides to a

436

frozen solution led to reductions in melting temperature and melting time when

437

compared with a control solution. Furthermore, the quicker melting of frozen AFP

438

solutions was expected to shorten the duration of freezing time for organisms

439

throughout the winter. This would thus help prevent tissue destruction.37 When taken

440

together, these findings indicate that TSAPP induces ice melting at lower

441

temperatures and in a shorter amount of time to reduce cellular freezing damage.

442

All in all, TSAPP had a molecular weight between 180 and 2000 Da and exhibited

443

a higher thermal hysteresis at 0.29 °C. Meanwhile, TSAPP rich in glycine, proline,

444

glutamic and alanine, which have been associated with the cryoprotective activity of

445

ice-structuring proteins. TSAPP significantly improved the viability, growth, and

446

acid-producing ability of S. thermophilus cells after cold stress. Moreover, TSAPP 21



447

significantly inhibited the leakage of intracellular proteins and nucleic acids,

448

prevented the decrease of β-galactosidase and LDH activities, facilitated the

449

accumulation of soluble sugar, and reduced cellular membrane hyperpolarization.

450

Collectively, these findings indicate that TSAPP protected cellular membrane

451

integrity and avoided damage to its permeability under cold stress. The possible

452

TSAPP protective mechanism of action might be through interactions with

453

membrane phospholipids and surrounding cells in a glassy matrix. Finally, TSAPP

454

induced ice to melt at lower temperature and in a shorter amount of time. These

455

results highlight the potential usefulness of TSAPP as a new cryoprotective agent in

456

probiotics or other processed foods to reduce the damage caused by frozen

457

conditions and temperature fluctuation.

458

■ AUTHOR INFORMATION

459

Corresponding Author

460

*Tel: +86-591-22866375, Fax: +86-591-22866278, E-mail address:

461

[email protected].

462

#:

463

Notes

464

The authors declare no competing ﬁnancial interest.

465

■ ACKNOWLEDGEMENTS

466

This work was supported by National Natural Science Foundation of China (No.

467

31571779 and 31471623).

468

■ REFERENCES

These two authors are co-first authors who are contributed equal to this paper.

22


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antifreeze peptides from fish gelatin with hypothermia protection activity. Appl.

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antifreeze

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(3) Li, L.; Wu, J. H.; Zhang, L.; Chen, X.; Wu, Y.; Liu, J. H.; Geng, X. Q.; Wang, Z.

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W.; Wang, S. Y. Investigation of the physiochemical properties, cryoprotective

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activity and possible action mechanisms of sericin peptides derived from membrane

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separation. LWT - Food. Sci. Technol. 2017, 77, 532-541.

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(4) Boonsupthip, W.; Lee, T. C. Application of antifreeze protein for food

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preservation: effect of Type III antifreeze protein for preservation of gel‐forming

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of frozen and chilled actomyosin. J. Food Sci. 2003, 68, 1804-1809.

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(5) Lei, P.; Xu, Z.; Ding, Y.; Tang, B.; Zhang, Y.; Li, H.; Feng, X.; Xu, H. Effect of

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poly (γ-glutamic acid) on the physiological responses and calcium signaling of rape

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seedlings (Brassica napus L.) under cold stress. J. Agric. Food Chem. 2015, 63,

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(6) Rubinsky, B.; Devries, A. L.; Arav, A., Interaction of thermal hysteresis proteins

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drying, and cold treatment. Appl. Environ. Microbiol. 2010, 76, 2989-2996.

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of the Lactobacillus delbrueckii subsp. bulgaricus membrane during cold and

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osmotic stress and its relevance for cryopreservation. Appl. Microbiol. Biotechnol.

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2017, 101, 1427-1441.

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galactosidase activity of yogurt cultures in skim milk 1,2. J. Dairy Sci. 1989, 72,

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freeze drying on the metabolic viability of lactic acid bacteria. Sci. Technol. Food

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Indus. 2011, 32, 203-209.

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lactic acid bacteria, antioxidant activity and in vitro inhibition of angiotensin-I-

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Converting enzyme of Lycium barbarum yogurt. Arab. J. Sci Eng. 2014, 39, 5355-

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microgram quantities of protein utilizing the principle of protein-dye binding. Anal.

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Biochem. 2003, 72, 248-254.

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antimicrobial peptide LHP7 from lactoferricin, HP and plectasin on Staphylococcus

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(22) Zhang, C.; Zhao, X. Y.; Yue, M. A.; Zhang, H.; Yao, H. Y. Determination of 25



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thermal hysteresis activity of antifreeze protein by differential scanning calorimetry.

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Technol. 2016, 9, 1746-1755.

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fraction and dilution factor on the antifreeze activity in the hemolymph of the

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cerambycid beetle Rhagium inquisitor. Cryobiology 2002, 44, 132-141.

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(25) Beal, C.; Corrieu, G. Viability and acidification activity of pure and mixed

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starters of Streptococcus salivarius ssp. thermophilus 404 and Lactobacillus

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Food. Sci. Technol. 1994, 27, 86-92.

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preservation of Lactobacillus acidophilus in saccharide matrices. Cryobiology 2000,

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1985, 22, 446-456.

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(28) Norton, J. M., & Firestone, M. K. . Metabolic status of bacteria and fungi in the

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glycoproteins: relationship between molecular weight, thermal hysteresis and the

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Biochem. Physiol. B: Biochem. Mol. Biol. 2001, 128, 265-273.

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(30) Hwang, B.; Cho, J.; Hwang, I. S.; Jin, H. G.; Woo, E. R.; Lee, D. G. Antifungal

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activity of lariciresinol derived from Sambucus williamsii and their membrane-

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Microbiol. Methods 2003, 52, 149-182.

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(33) Leslie, S. B.; Israeli, E.; Lighthart, B.; Crowe, J. H.; Crowe, L. M. Trehalose

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571

Appl. Environ. Microbiol. 1995, 61, 3592-3597.

572

(34) Zhang, Y. H.; Huo, G. C.; Guo, L. Study on the cryoprotective Mechanism of

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trehalose for lactic acid bacteria druing freeze drying. Food Ferment Ind. 2007, 33,

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(35) Allison, S. D.; Chang, B.; Randolph, T. W.; Carpenter, J. F. Hydrogen bonding

576

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577

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578

(36) Moudrakovski, I. L.; Ratcliffe, C. I.; Mclaurin, G. E.; Benoit Simard, A.; 27



579

Ripmeester, J. A. Hydrate layers on ice particles and superheated ice: a 1H NMR

580

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581

(37) Ba, Y.; Mao, Y.; Galdino, L.; Günsen, Z. Effects of a type I antifreeze protein

582

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583

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584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 28


Page 28 of 51

Page 29 of 51


601

Figure Captions

602

Figure 1.

603

TSAPP physiochemical properties. (A) and (B) TSAPP molecular mass distribution

604

as determined by HPLC; (C) and (D) TSAPP thermal hysteresis activity (THA)

605

when compared with BSA negative control at different holding temperatures of

606

0.07 °C and 0.37 °C.

607 608

Figure 2.

609

Relationship between growth and acid production of S. thermophilus after freezing.

610

(A) Differences in OD600 and acid production of S. thermophilus after 7 h

611

incubation. (B) Acid production of S. thermophilus. (C) Growth curve of S.

612

thermophilus. (D) S. thermophilus growth with various cryoprotectants at different

613

freezing times.

614 615

Figure 3.

616

Intracellular metabolism of S. thermophilus after freezing. (A) Effect of various

617

cryoprotectants on the activity of β-galactosidase and LDH activities. (B) Effect of

618

various cryoprotectants on intracellular sugar accumulation. (C) S. thermophilus

619

metabolic

620

letters indicate significant differences (P < 0.05).

activity

with

various

cryoprotectants.

621 622

Figure 4. 29


Columns

with

different


623

Effect of cryoprotectants on concentration of extracellular nucleic acids and proteins

624

from frozen S. thermophilus. Letter of A, B, C, D represent the significant

625

differences of extracellular nucleic acids (P < 0.05); letter of a, b, c, d and e

626

represent the significant differences of extracellular proteins (P < 0.05).

627 628

Figure 5.

629

Representative scanning electron micrographs of S. thermophilus after freezing. (A)

630

Control and (B) TSAPP groups. Control groups show collapsed morphology at

631

5,000-fold (A1), 20,000-fold (A2) and 50,000-fold (A3) magnifications,

632

respectively. TSAPP groups show plump cell profile at 5,000-fold (B1), 20,000-fold

633

(B2) and 50,000-fold (B3), respectively.

634 635

Figure 6.

636

TSAPP effect on cellular membrane properties. (A) DPH fluorescence intensity of S.

637

thermophilus with various cryoprotectants at different freezing times; (B)

638

Fluorescence intensity of cell membrane potential of S. thermophilus with various

639

cryoprotectants after different freezing times.

640 641

Figure 7.

642

FTIR spectra of soybean lecithin and the mixture of soybean lecithin and TSAPP.

643 644

Figure 8. 30


Page 30 of 51

Page 31 of 51


645

Time-dependent NMR microimaging of frozen aqueous solutions during melting as

646

labeled in the bottom right frame. Shown are solutions containing TSAPP

647

concentrations of 1.0 mg/mL (identified as 3 in the figure) or 5.0 mg/mL (identified

648

as 4) when compared with a water negative control (identified as 1) and 20%

649

glycerol positive control (identified as 2). Blue color represents solid ice; bright

650

yellow represents areas of high spatial distribution of mobile water.

651

31



652

Page 32 of 51

Table 1 Amino acid compositions of TSAPP Amino acids

Contents (%)

Amino acids

Contents (%)

Aspartic

6.38

Isoleucine

1.93

Threonine

2.98

Leucine

3.28

Serine

3.09

Histidine

1.47

Glutamic

12.43

Phenylalanine

2.61

Glycine

24.41

Arginine

8.65

Alanine

10.46

Methionine

2.08

Valine

3.00

Lysine

3.29

Proline

12.80

Tyrosine and cysteine

1.14

653

32


Page 33 of 51


Table 2 The hold temperature (Th), onset temperature (T0), percent ice and THA of BSA and TSAPP Sample

△Hm j/g

△Hf j/g

Φ (%)

Th/oC

T0/oC

THA

-237.81

-4.36

98.17

0.07

0.03

0.04

-237.81

-22.37

90.59

0.37

0.23

0.14

-224.68

-47.61

78.81

0.07

-0.13

0.20

-224.68

-196.82

12.40

0.37

0.08

0.29

BSA

TSAPP

33



Figure 1.

34


Page 34 of 51

Page 35 of 51


Figure 2.

35



Figure 3.

36


Page 36 of 51

Page 37 of 51


Figure 4.

Figure 5.

37



38


Page 38 of 51

Page 39 of 51


Figure 6.

39



Figure 7.

40


Page 40 of 51

Page 41 of 51


Figure 8.

41



Graphic for table of contents

42


Page 42 of 51

Page 43 of 51


Figu ure 1.



Figu ure 2.


Page 44 of 51

Page 45 of 51


Figu ure 3.



Figure 4.


Page 46 of 51

Page 47 of 51


Figu ure 5.



Figu ure 6.


Page 48 of 51

Page 49 of 51


Figu ure 7.



Figu ure 8.


Page 50 of 51

Page 51 of 51


Graphic for table of contents


Cryoprotective Activity and Action Mechanism of Antifreeze Peptides

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