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Water-based oligochitosan and nanowhisker chitosan as potential food preser-vatives for shelf-life extension of minced pork
Patomporn Chantarasataporn, Preenapha Tepkasikul, Yuthana Kingcha,Rangrong Yoksan, Rath Pichyangkura, Wonnop Visessanguan, SuwabunChirachanchai
PII: S0308-8146(14)00404-XDOI: http://dx.doi.org/10.1016/j.foodchem.2014.03.019Reference: FOCH 15548
To appear in: Food Chemistry
Received Date: 30 October 2013Revised Date: 29 January 2014Accepted Date: 5 March 2014
Please cite this article as: Chantarasataporn, P., Tepkasikul, P., Kingcha, Y., Yoksan, R., Pichyangkura, R.,Visessanguan, W., Chirachanchai, S., Water-based oligochitosan and nanowhisker chitosan as potential foodpreservatives for shelf-life extension of minced pork, Food Chemistry (2014), doi: http://dx.doi.org/10.1016/j.foodchem.2014.03.019
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1
Water-based oligochitosan and nanowhisker chitosan as potential food
preservatives for shelf-life extension of minced pork
Submitted to
Food Chemistry
Patomporn Chantarasataporna, Preenapha Tepkasikul
d, Yuthana Kingcha
d, Rangrong
Yoksane, Rath Pichyangkuraf, Wonnop Visessanguan**,d, Suwabun Chirachanchai*,a,b,c
aThe Petroleum and Petrochemical College, Chulalongkorn University, Soi Chula12, Phyathai
Rd., Pathumwan, Bangkok 10330, Thailand
b Center for Petroleum, Petrochemicals, and Advanced Materials, Chulalongkorn University,
Bangkok, 10330, Thailand cCenter of Innovative Nanotechnology, Chulalongkorn University, Bangkok, 10330, Thailand
dNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science
and Technology Development Agency (NSTDA), 113 Thailand Science Park, Phaholyothin
Rd., Klong Nueng, Klong Luang, Pathumthani 12120, Thailand eDepartment of Packaging and Materials Technology, Faculty of Agro-Industry, Kasetsart
University, Bangkok 10900, Thailand fDepartment of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,
Thailand
* To whom correspondence should be addressed: Tel. +66(2) 218-4134, Fax: +66(2) 215-
4459, E-mail: [email protected] **
To whom correspondence should be addressed: Tel +66(2) 564 -6700 Ext 3747, Fax:
+66(2) 564 6707, E-mail: [email protected]
2
ABSTRACT
Water-based chitosans in the forms of oligochitosan (OligoCS) and nanowhisker
chitosan (CSWK) are proposed as a novel food preservative based on a minced pork model
study. The high surface area with a positive charge over the neutral pH range (pH 5-pH 8) of
OligoCS and CSWK lead to an inhibition against Gram-positive (S. aureus, L.
monocytogenes, and B. cereus) and Gram-negative microbes (S. enteritidis and E. coli
O157:H7). In the minced pork model, OligoCS effectively performs a food preservative for
shelf-life extension as clarified from the retardation of microbial growth, biogenic amine
formation and lipid oxidation during the storage. OligoCS maintains almost all myosin heavy
chain protein degradation as observed in the electrophoresis. The present work points out that
water-based chitosan with its unique morphology not only significantly inhibits antimicrobial
activity but also maintains the meat quality with an extension of shelf-life, and thus has the
potential to be used as a food preservative.
KEYWORDS: oligochitosan; chitosan whisker; antimicrobial effect; shelf-life; minced pork,
food preservative
3
1. Introduction
Chitosan is a derivative of chitin, a naturally abundant polymer, with a structure of 1
(1, 4)-linked 2- amino-2-deoxy-β-D-glucose and 2-acetamido-2-deoxy-β-D-glucose. Chitosan 2
has important functional groups, such as amino groups at the C-2 position and primary and 3
secondary hydroxyl groups at the C-3 and C-6 position (Sudarshan, Hoover, & Knorr, 1992). 4
Chitosan exhibits antimicrobial properties via the interaction between positive charges of the 5
amino group (NH3+) and negative charges on the microorganism cell membrane. Chitosan is 6
also nontoxic, biodegradable, biocompatible and generally regarded as a safe (GRAS) 7
compound (Seiichi, Yoshiaki, Masayoshi, Norio, & Shin-Ichiro, 1994) 8
Chitosan, especially chitosan /acetic acid solution, is one of the most promising 9
natural food preservative candidates for fruit, juices, bread and meat (No, Meyers, 10
Prinyawiwatkul, & Xu, 2007). It is important to note that the use of chitosan for muscle food, 11
which is susceptible to deterioration due to its high protein content, and saturated and 12
polyunsaturated fatty acid content, might be a good method when considering the value-13
added application. The main cause of food deterioration in meat is microbial spoilage and 14
lipid oxidation. Various types of food additives have been developed to improve the quality 15
or extend the shelf-life of muscle foods. Typical synthetic additives are sodium nitrite, propyl 16
gallate and butylated hydroxytoluene (Weiss, Gibis, Schuh, & Salminen, 2010). Safe and 17
fresh food without the use of chemical preservatives has become a significant consumer 18
demand. Natural additives for food preservation are expected to substitute synthetic additives. 19
Beverlya, Janes, Prinyawiwatkula and No (2008) reported that high molecular weight 20
chitosan (1106 kDa) and low molecular weight chitosan (470 kDa) in acetic acid or lactic acid 21
solution reduced Listeria monocytogenes and extended the shelf-life of beef. 22
Chitosan can be only dissolved in acidic solvents, such as acetic acid, succinic acid 23
and lactic acid, due to its poor solubility at above pH 6.5 (Fan, Du, Zhang, Yang, Zhou, & 24
4
Kennedy, 2006). Thus, the use of acidic chitosan in foods might affect the odour, taste and 25
acid degradation. Water-soluble chitosan, e.g., quaternized carboxymethyl chitosan (Sun, Du, 26
Fan, Chen, & Yang, 2006), is an alternative choice but the chemicals and organic solvents 27
used during its derivatization are potential causes for concern. Chitosan in solid forms, such 28
as films hydrogels, and microspheres (including nanoparticles), is expected to have 29
antimicrobial activity through its surface area (Ye, Leung, Xin, Kwong, Lee, & Li, 2005; 30
Zhao, Mitomo, Zhai, Yoshii, Nagasawa, & Kume, 2003). Many attempts to modify chitosan, 31
in the forms of flakes and particles, for better dispersion ability and increased surface 32
reactivity have been investigated. For example, chitosan microspheres grafted with oleoyl 33
groups dispersed in nutrient broth were reported as having anti E. coli activity (Kong, Chen, 34
Liu, Liu, Meng, & Yu, 2008). N-trimethyl chitosan nanoparticles produced by polyelectrolyte 35
complexation and ion gelation methods showed strong antibacterial inhibition (Sadeghi, 36
Dorkoosh, Avadi, Saadat, Rafiee-Tehrani, & Junginger, 2008). 37
Chitin in whisker form, which is a uniform rigid rod–like nanocrystal prepared from 38
chitin powder, is a potential material due to its high surface area and accessibility at nano-39
sized dimensions (Nair & Dufresne, 2003). Our group succeeded in preparing chitosan 40
whiskers (CSWK) via a simple and effective one-pot deacetylation of chitin whiskers. The 41
CSWK exhibits excellent active surface and allows for the effective chemical fuctionalization 42
(Chatrabhuti & Chirachanchai, 2013; Phongying, Aiba, & Chirachanchai, 2007). Fibrous 43
branching whiskers containing a uniform fine nanofibre with 200-560 nm in length and 5-10 44
nm in diameter (L/D ratio is as high as 20-50) showed high colloidal stability in water for a 45
number of months. As well as CSWK, our group has also succeeded in preparing 46
oligochitosan (OligoCS) by enzymatic degradation to obtain clusters with individual 47
nanoparticles with the size of ~100-300 nm. 48
5
It is important to note that both CSWK and OligoCS were prepared in water without 49
chemical modification or the use of organic solvents, and that the materials are stable in water 50
as a colloidal solution. Based on the abovementioned points, it can be expected that CSWK 51
and OligoCS might be a suitable food safety preservative for processed meats, such as 52
sausages and minced pork. The present work, therefore, focuses on the use of CSWK and 53
OligoCS as a food preservative and the use of minced pork as a model food. The different 54
types of chitosan, i.e. CSWK and OligoCS not only allowed for an understanding of how the 55
morphology plays an important role on food preservation but also clarified the mechanism 56
related to the antimicrobial activity and the shelf-life extension. 57
58
Materials and methods 59
2.1. Materials 60
CSWK (91 % DD, –Mw of 1.37 × 10
5 Da) was prepared from chitin flakes, which 61
were also obtained from Seafresh Co., Ltd., as previously reported (Phongying, Aiba, & 62
Chirachanchai, 2006). OligoCS (91 % DD, –Mw of 1.0 × 104 Da) was a gift from Chitin-63
chitosan Biomaterial Research Center, Chulalongkorn University, Thailand. It was prepared 64
from chitosan treated with chitosanase (Bacillus. sp.PP8). Plate count agar (PCA), deMan 65
Rogosa Sharpe agar (MRS) and xylose lysine deoxycholate agar (XLD) were purchased from 66
Difco® Laboratories (CA, USA). MacCONKEY agar was purchased from Oxoid (Basingtoke, 67
UK). Baird Parker agar and egg yolk tellurite were purchased from Merck (Darmstadt, 68
Germany). Minisart RC4 filters were purchased from Sartorius (Goettingen, Germany). 69
6
2.2. Instruments and equipment 70
The structural characterization of CSWK and OligoCS was carried out by Fourier 71
transform infrared (FTIR) spectra (Bruker Equinox 55, Ettlingen, Germany) with 32 scans at 72
a resolution of 4 cm–1
in a frequency range of 4000–400 cm-1
and using 1H nuclear 73
nuclear magnetic resonance (NMR) spectra in CD3COOD/D2O (BrukerAvance 500 MHz 74
NMR spectrometer, Ettlingen, Germany) at room temperature (Supplementary data). The 75
%DD values were calculated from 1H-NMR. The morphology and size of CSWK and 76
OligoCS were observed by an H-7650 Hitachi transmission electron microscope (TEM) at an 77
acceleration voltage of 100 kV. Zeta potentials (ζ) of CSWK and OligoCS dispersed in 78
deionized water (0.5 mg/ml) and adjusted pH by using HCl and NaOH were determined at 25 79
°C by a Malvern Zetasizer Nano Series (Malvern Instruments Co., Ltd, Worcestershire, UK) 80
with a detection angle of 17°. Brunauer–Emmett–Teller (BET) measurements of CSWK and 81
OligoCS were determined using an Autosorp-1 gas sorption system (Quantachrome 82
Corporation, Florida, USA). The samples were preheated and degassed in nitrogen for 3-4 h 83
at 150 °C before measuring the adsorbate under liquid nitrogen. Thiobarbituric acid reactive 84
substances (TBARS) were analyzed using a U-3000 spectrophotometer (Hitachi, Tokyo, 85
Japan). An Alliance 2690 high performance liquid chromatography (HPLC) with a photo 86
diode array detector model 996 (Waters, MA, USA) was used to quantify the biogenic amine. 87
2.3. Sample preparation and measurements 88
2.3.1. Evaluation of the antibacterial activity in vitro 89
Staphylococcus aureus ATCC 6538, Listeria monocytogenes ATCC 19115, 90
Bacillus cereus C113, Salmonella enteritidis DMST 1706 and Escherichia coli O157:H7 91
DMST 12743 were used as the test organisms. The organisms were grown in tryptic soy broth 92
(TSB, Merck, Germany) at 37 °C for 24 h with shaking (200 rpm). Each culture was 93
harvested by centrifugation (7500 g for 10 min at 4 °C) and the resulting pellets were washed 94
7
twice and resuspended in sterile 0.1% (w/v) peptone solution. Before testing, the 95
concentration of each bacterium was adjusted to 107 CFU/ml with sterile peptone solution. 96
CSWK solution (1000 mg/ml) was prepared in distilled water, while OligoCS 97
solution (1000 mg/ml) was first prepared with 1% potassium lactate (pH 3.6) then adjusted to 98
pH 6.0 with 10% NaOH solution. The minimal inhibition concentration (MIC) of CSWK and 99
of OligoCS was determined by testing the sensitivity to a wide range of bacterial indicators 100
according to Qi, Xu, Jiang, Hu, and Zou (2004). A 96-well plate containing two-fold serial 101
dilutions of chitosan samples with a known amount of both chitosan samples. All of the 102
samples were inoculated under aseptic conditions with 50 µl of the inoculums of bacteria and 103
incubated at 37 °C for 24 h. The changes in cells number of each indicator tested were 104
measured using a micro-plate reader at a wavelength of 600 nm. The control only contained 105
nutrient broth and 1% potassium lactate without OligoCS. The MIC50 was defined as the 106
concentration (ppm) of the chitosan samples which gave a 50% growth inhibition. 107
2.3.2. Minced pork treatment 108
OligoCS were dialyzed 5 times in distilled water before freeze-drying to obtain 109
materials in a fine particulate form. The fresh minced pork samples were collected from a 110
local supermarket (Bangkok, Thailand) and were kept at 0 °C in an ice box for 1 h before 111
mixing with OligoCS, at a concentration of 0.2 % and 0.4 % (w/w). Each sample after 112
treating was stored in polyethylene bag at refrigerated temperature (4 °C) until analysis. 113
2.3.3. Microbiological evaluation 114
To determine the bacteria that were naturally present, each treated sample (10 g) 115
was aseptically homogenized with 90 ml of 0.1% sterile peptone water for 2 min with a 116
Seward stomacher lab blender (West Sussex, England). The homogenates were prepared for 117
ten-fold serial dilutions and spread on a specific medium agar under aerobic conditions. 118
Lactic acid bacteria counts (LAB) were estimated on MRS agar after incubating at 30 °C for 119
8
72 h. Total Enterobacteriaceae counts were enumerated using MacCONKEY agar after 120
incubating at 37 °C for 48 h. Presumptive Salmonella spp., which has red colonies with a 121
black centre, were enumerated using XLD agar. The incubation time was 48 h at 37 °C. Other 122
microbial growths on the XLD agar were simplified by recording as the total XLD count. For 123
total Staphylococcus counts, Baird Parker agar supplement with egg yolk tellurite was used. 124
Colonies were counted after 48 h at 37 °C. Total viable bacteria counts (TVB) were 125
enumerated using PCA agar. The plates were incubated at 37 °C for 48 h. All experiments 126
were repeated three times with two replications per experiment. The average of the results 127
were reported in log-scale of colony-forming units (log CFU/g). 128
2.3.4. Biogenic amine determination 129
Meat samples were prepared and biogenic amines were determined by using a 130
method modified from Tosukhowong, Visessanguan, Pumpuang, Tepkasikul, Panya, and 131
Valyasevi (2011). The biogenic amine index (BAI) was calculated using Eq. (1). 132
133
BAI = C(cadaverine) + C(putrescine) + C(tyramine) + C(histamine) (1) 134
Here C is the concentration of each amine (mg/kg). 135
136
The BAI of the fresh meat was less than 5 mg/kg, and within the acceptable range of 5-20 137
mg/kg. The BAI of the low quality meat was above 20 mg/kg, less than the level to be 138
considered spoilage (above 50 mg/kg) (Hernández-Jover, Izquierdo-Pulido, Veciana-Nogués, 139
& Vidal-Carou, 1996). 140
2.3.5. Lipid oxidation 141
Lipid oxidation was measured by thiobarbituric acid reactive substances (TBARS) 142
as previously reported (Nirmal & Benjakul, 2010). In brief, the sample (1 g) was 143
homogenized with a mixed solution (5 ml) of 0.25 M hydrochloric solution containing 0.375 144
9
% (w/v) thiobarbituric acid and 15 % (w/v) trichloroacetic acid. The mixture was heated in a 145
water bath at 100 °C for 10 min, and cooled in iced water. The sample mixture was 146
centrifuged (5785 rpm for 20 min) and analyzed with an UV- spectrophotometer at 532 nm 147
using a standard curve of malonaldehyde (0-2 ppm). TBARS was expressed in unit of mg 148
malondialdehyde/kg sample (mg MDA / kg meat). 149
2.3.6. Electrophoretic study of myofibrillar proteins 150
Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was 151
applied to detect changes in the meat proteins.(Laemmli, 1970) The minced pork (3 g) was 152
mixed with SDS solution (27 ml, 5 % (w/v)) and homogenized at 13,500 rpm for 2 min. The 153
homogenate was immersed in water at 85 °C for 1 h. The sample suspension was centrifuged 154
at 5411 rpm for 20 min and the insoluble content was removed. The solution obtained was 155
loaded 40 µg/ lane onto 5 % (v/v) polyacrylamide separating gel and 4 % (v/v) stacking gel. 156
The gel was subjected to electrophoresis at 20 mA by a Mini-Protein II Cell apparatus. The 157
gel was stained with coomassie brilliant blue R-250 (0.02 % w/v) in an aqueous solution 158
containing methanol (50 % v/v) and glacial acetic acid (7.5 % v/v). The gel was destained 159
with destaining aqueous solution I [methanol (50 % v/v) and acetic acid (7.5% v/v)], followed 160
by destaining aqueous solution II [methanol (5 % v/v) and glacial acetic acid (7.5 % v/v)]. A 161
wide-range molecular weight protein marker (from 25 to 200 kDa) was used to evaluate the 162
molecular weight of the proteins. 163
The total protein samples were prepared with or without using β-mercaptoethanol 164
(reducing agent) and disulfide-crosslinked myosin heavy chain (MHC) at 200 kDa which 165
appeared at the top position on SDS-PAGE was specifically considered (Lund, Lametsch, 166
Hviid, Jensen, & Skibsted, 2007). 167
10
2.3.7. Statistical analysis 168
The results were averaged from triplicates, and the statistical significance was 169
determined by using an SPSS 11.0 (for Windows, SPSS Inc., Chicago, Ill., USA). The data 170
were subjected to analysis of variance (ANOVA) and the mean comparison was carried out 171
using Duncan’s multiple range test (DMRT) (Steel & Torries, 1980). 172
173
3. Results and discussion 174
3.1. Morphology, zeta potential and surface area of CSWK and OligoCS 175
TEM was used to observe the morphology of CSWK and OligoCS in the solid state. 176
Fig. 1a shows OligoCS as aggregates of individual nanoparticles (∼ 100-300 nm) and CSWK 177
as a fibrous network structure (200-560 nm in length, 5-10 nm in diameter and L/D ratio ∼20-178
50). This implied that the CSWK and OligoCS are the potential materials in nanometer size. 179
This lead us to an expectation that CSWK and OligoCS allowed good accessibility to the 180
functional groups along the surface. 181
In order to use chitosan as an antimicrobial additive, one of the important factors to 182
consider was the positive charge on the amino groups along the chitosan chain. The charge is 183
significant when the amino groups are protonated, as seen in the case of chitosan under acidic 184
conditions. The positive charge, therefore, strongly depended on the amount of amino groups, 185
in other words, the %DD. The zeta potential measurement allowed for the determination of 186
the chitosan charge when the chitosan was in solution. It should be pointed out that the zeta 187
potential also reflected particle stability as a consequence of the electrostatic repulsion 188
between the chitosan particles. Moreover, the zeta potential is also useful to evaluate the ease 189
of complexation in vivo with the negative charge on the cell membranes of bacteria (Kong, 190
Chen, Xing, & Park, 2010). 191
11
Here, the zeta potential of each condition was measured to trace the positive charge 192
of CSWK and OligoCS. Fig. 1b shows the overall zeta potential of CSWK and OligoCS at 193
variable pHs. From pH 2 to pH 4, CSWK showed a significantly higher zeta potential (in the 194
range of +40 to +50 mV) than OligoCS (+20 mV). The zeta potential of CSWK significantly 195
decreased from pH 6 and became lower than OligoCS under basic conditions. This indicated 196
that CSWK could maintain its positive charge and stability in a lower pH range. Above pH 7, 197
the surface charge of CSWK exhibited a negative charge (∼ -18 mV), which implied the 198
maintaining of particle stability under negatively repulsive charges, whereas that of OligoCS 199
showed a very slight negative charge (∼ -5 mV), indicating the tendency of aggregation and 200
this can also be observed by the naked eye. This might be due to the fibrous morphology of 201
CSWK, which enhanced the particle stability in the higher pH compared to OligoCS. Based 202
on the zeta potential results, it was clear that the optimal conditions for CSWK and OligoCS 203
are below pH 5, and below pH 7, respectively. 204
In this work, CSWK and OligoCS were freeze-dried before use. As shown in Fig. 1c, 205
the dried CSWK was a finely agglomerated fibrous material, whereas OligoCS was a fine 206
powder. BET was applied to determine the surface area, which indicated the effective contact 207
area between the materials and microbes. OligoCS (~45 m2/g) showed higher surface area 208
values than CSWK (~29 m2/g) (Fig. 1c). The higher the surface area, the higher accessibility 209
to the microbes and this might play an important role in inducing intracellular component 210
leakage. 211
3.2. Evaluation of the antibacterial activity in vitro 212
CSWK and OligoCS had a wide activity range, and was inhibitory against Gram-213
positive (S. aureus, L. monocytogenes, B. cereus) and Gram-negative (S. enteritidis and E. 214
coli O157:H7) food-borne pathogenic bacteria (Fig. 2a and b, respectively). The antibacterial 215
activity increased as the concentration of CSWK and OligoCS increased. However, OligoCS 216
12
exhibited a wider spectrum of activity and stronger antibacterial activity than CSWK. These 217
results showed that CSWK exhibited the most pronounced antibacterial activity against S. 218
enteritidis (MIC50 value< 1.7 ppm), followed by L. monocytogenes (MIC50 value< 54.2 ppm). 219
While the effect of CSWK on the antibacterial activity against S. aureus, B. cereus, and E. 220
coli O157:H7 was less pronounced. In contrast, OligoCS showed antibacterial activity against 221
not only S. enteritidis (MIC50 value< 6.8 ppm) but also L. monocytogenes (MIC50 value< 6.8 222
ppm), S. aureus (MIC50 value< 13.5 ppm) and E. coli O157:H7 (MIC50 value< 217 ppm). It is 223
possible that the OligoCS allowed the most significant microbes accessibility compared to 224
CSWK. This might be related to the high surface area of OligoCS, which allowed for the 225
effective binding to microbial cell. This evidence also coincides with other reports (Ing, Zin, 226
Sarwar, & Katas, 2012; Qi, Xu, Jiang, Hu, & Zou, 2004), which demonstrated that chitosan 227
nanoparticles with a high surface area effectively performed a higher affinity to the surface of 228
bacterial cells and disrupted the membrane. Here, OligoCS was chosen for further 229
investigation in the meat model due to its wider spectrum in inhibition of both Gram-positive 230
and Gram-negative bacteria. 231
3.3. Optimal concentration of OligoCS for the antimicrobial activity in meat model 232
A number of in vivo studies on the antimicrobial activity of chitosan were reported 233
against a wide variety of microorganisms. Chitosan powder 1 % (w/w) in pork sausage has 234
been shown to be active against LAB growth, with about 1.5 log CFU/g reduction in the pork 235
as reported by Soultos, Tzikas, Abrahim, Georgantelis, and Ambrosiadis (2008). The 236
inhibitory effect of 0.1 % chitosan containing mint extract in glacial acetic acid was also 237
demonstrated in minced lamb meat, by suppressing a 2 log cycle of S.aureus (Kanatt, 238
Chander, & Sharma, 2008). Present results are in general agreement with Soultos, Tzikas, 239
Abrahim, Georgantelis, and Ambrosiadis (2008), who studied a reduction of 1.1 log CFU/g in 240
Enterobacteriaceae growth in pork sausage treated with 1 % (w/w) chitosan after 7 storage 241
13
days at 4 °C. Also, the effect of an antimicrobial film containing 2.1 % chitosan lactate 242
additive against S. enteritidis in red meat was reported (S.-i. Park, Marsh, & Dawson, 2010). 243
To study the antimicrobial effect on natural flora, meat samples were treated with 244
OligoCS. Fig. 3a shows the microbial activity based on LAB at 4 °C for 7 storage days. The 245
control sample showed a significant increase in the LAB counts from 5.5 log CFU/g to 6.5 246
log CFU/g. The treatments with OligoCS, at the concentrations of 0.2 % and 0.4 %, show the 247
reduction of LAB, compared to the control. It was expected that as the storage time increased, 248
the LAB counts for all the treatments become higher. However, the system containing 0.4 % 249
OligoCS showed the least LAB counts. This implied that OligoCS played a role in inhibiting 250
LAB. 251
The Staphylococcus growth counts were also inhibited by the presence of chitosan, 252
as shown in Fig. 3b. No change in the Staphylococcus counts were observed in the control 253
during storage (~ 4 log CFU/g), and the counts naturally decrease at the end of storage. The 254
Staphylococcus counts were significantly reduced, by about 0.5 log CFU/g and 1 log CFU/g 255
when the meat sample was treated with OligoCS 0.2 % and 0.4 %, respectively (p<0.05). 256
The antimicrobial effect of OligoCS was also investigated on Enterobacteriaceae, a 257
Gram-negative organism, which is usually found in the intestines of humans and other 258
animals. For total Enterobacteriaceae counts, the control sample exhibited a significant 259
increase in the count from 5 log CFU/g to 6 log CFU/g during storage (p<0.05), as shown in 260
Fig. 3c. Throughout the storage period, Enterobacteriaceae counts were significantly 261
inhibited by around 1 and 2 log CFU/g for the sample containing 0.2 % and 0.4 % OligoCS 262
(p<0.05), respectively. 263
The Salmonella spp. were traced by using selective cultivation on XLD agar, as it 264
has a shiny black centre in the colony (Fig. 3d). In the control conditions, Salmonella spp. 265
was at the level of 2-3 log CFU/g during 7 days of storage. On the first day, the Salmonella 266
14
spp. growth was ~ 2 log CFU/g for the sample treated with 0.2 % OligoC, whereas it is found 267
to be not detected for the sample treated with 0.4 % OligoCS. After 2 days of storage, the 268
Salmonella spp. growth in the sample containing 0.2 % OligoCS was not observed. The result 269
reconfirmed the efficacy of OligoCS on Salmonella spp. inhibition as previously observed in 270
vitro. 271
For the inhibitions of TVB (Fig. 3e), at the initial storage day (day 0), the bacterial 272
count of the control sample is approximately 5.5 log CFU/g. When the OligoCS concentration 273
was increased to 0.4 %, a decrease in the TVB was observed (~ 1 log CFU/g, p<0.05). When 274
the concentration was increased to 0.4 %, a decrease in TVB, between 15-20 %, was observed 275
after 1 day of storage. 276
It should be pointed out that the OligoCS activity was based on the solid state 277
without the use of carboxylic acid solvents. The results also showed the inhibition of Gram-278
negative bacteria to be greater than the Gram-positives. It should be noted that the pH value 279
of the meat after 1 day of storage was about pH 5.8-6.1. At that pH, OligoCS showed a more 280
positive zeta potential than CSWK. Although more investigation is needed, the function 281
might relate to the positive charge on chitosan effectively binding with the negative charge on 282
the surface of the Gram-negative bacteria as reported by Chung, Su, Chen, Jia, Wang, Wu, et 283
al. (2004). Various mechanisms related to antimicrobial action of chitosan have been 284
proposed. Kong, Chen, Xing and Park (2010) reported that the electrostatic interaction at the 285
interfacial contact area between the positive charges on the chitosan and the negative charges 286
of the phospholipids on the cytoplasmic microbial cell-membrane might be the key 287
mechanism, which leads to the leakage of proteinaceous and other intracellular constituents. 288
In this case, the performance of OligoCS might be similar. This can be confirmed from the 289
positively charged OligoCS as shown in Fig. 1b. 290
3.4. Effect of OligoCS on Biogenic Amine Index of meat 291
15
Biogenic amines (BAs) are nitrogenous compounds produced in high protein foods. 292
BAs in food are significant due to their toxicological effect on the nervous, blood and 293
intestinal systems (Jooten, 1988). Generally, most BAs are produced by naturally occurring 294
decarboxylases of microbial origin (Chander, Batish, Babu, & Singh, 1989). Therefore, BAs 295
are one of the most important indicators of microbial spoilage (Yano, Kataho, Watanabe, 296
Nakamura, & Asano, 1995). Their structures can be aliphatic (e.g. putrescine, cadaverine, 297
spermine and spermidine), heterocyclic (e.g. histamine and tryptamine) or aromatic (e.g. 298
tyramine and phenylethylamine) (Santos, 1996). 299
Although the presence of BAs in food is not an absolute criterion for the growth of 300
spoilage microorganisms, since there might be cases where BAs are produced by other 301
microorganisms, the BAs value (or BA index, BAI) is a good indicator to evaluate food 302
spoilage (Hernández-Jover, Izquierdo-Pulido, Veciana-Nogués, & Vidal-Carou, 1996). 303
It should be pointed out that biogenic amine components, e.g., putrescine and 304
histamine, were not found in all samples. In other words, only cadaverine, tyramine, 305
spermidine and spermine were found to be varied during the first two days at 4 °C (data not 306
shown). It was found that spermine was the major biogenic amine in all samples (~31-35 307
mg/kg) after 1 day of storage. During storage, both the cadaverine and tyramine levels 308
significantly increased (p<0.05), whereas spermidine levels exhibited the similar 309
concentration (p > 0.05). In order to evaluate the quality of the meat, the BAI levels as a 310
function of time during 2 days of storage were plotted (Fig. 4). On day 1, the samples treated 311
with 0.1% OligoCS showed the BAI levels to be almost at the limit value (50 mg/kg). When 312
the concentrations were increased to 0.2 % and 0.4 %, the BAIs were within the acceptable 313
range. This implies the optimal concentration to control the BAI values. At day 2, all the 314
BAIs reached the spoilage limit, although 0.2 % and 0.4 % OligoCS showed a value below 50 315
mg/kg, indicating its potential application. This implied that OligoCS have a certain ability to 316
16
retard the production of biogenic amines. The high BAI might come from the fact that the 317
major biogenic amines (cadaverine and tyramine) were related to the presence of LAB and 318
Enterobacteriaceae (Tosukhowong, Visessanguan, Pumpuang, Tepkasikul, Panya, & 319
Valyasevi, 2011). 320
3.5. Lipid oxidation 321
Lipid oxidation is one of the factors related to the deterioration in food quality, e.g., 322
undesirable rancid off-flavours and poisoning. In addition, lipids are easily oxidized in the 323
presence of light, heat and enzymes. Normally, lipid oxidation can be found in meat products 324
under storage (Hansen, Juncher, Henckel, Karlsson, Bertelsen, & Skibsted, 2004). Free 325
radical products in lipid oxidation occur by the attack of oxygen at the double bond in fatty 326
acids. Primary lipid oxidation products, hydroperoxides, continue the polymeric secondary 327
oxidation to aldehyde, ketone and alcohol compounds. TBARS are a good measurement for 328
secondary lipid oxidation products (Kamal-Eldin, Mäkinen, & Lampi, 2003) 329
The TBARS values of minced pork treated with OligoCS at different concentrations 330
are shown in Fig. 5. Initially, all the samples showed TBARS at approximately 5 - 7 mg 331
MDA/kg of meat. After 7 days of storage, the TBARS in the control sample were 332
significantly increased to be 10-12 mg MDA/kg meat, whereas the samples treated with 333
OligoCS were at ∼ 6 mg MDA/kg meat (p<0.05). However, an increase in the concentration 334
of OligoCS did not exhibit a significant reduction in the TBARS (p>0.05). 335
The discussion here might relate to the previous reports as follows. Kanatt, Rao, 336
Chawla, and Sharma (2013) reported that a chitosan (2 g/100 ml) coating in meat products 337
inhibited lipid oxidation compared to the untreated samples. Furthermore, chitosan might act 338
as an oxygen barrier to retard lipid oxidation in pink salmon fillets during frozen storage 339
(Sathivel, 2005). OligoCS in nano-particulate form with a high surface area might allow the 340
reaction between amino group and free radicals (R· , RO·, and ROO· groups) to form stable 341
17
macromolecule radicals as reported by P. J. Park, Je, and Kim (2004). This would result in a 342
reduction in lipid oxidation. 343
3.6. Electrophoretic study of myofibrillar proteins 344
Not only lipid oxidation but also protein oxidation might be a main cause of food 345
deterioration. Protein oxidation in muscle meat results in the loss of functionality and water-346
holding capacity (WHC), including changes in stability (Decker, Faustman, & Lopez-Bote, 347
2000). Davies and Dean (2003) reported that lipid and protein oxidation occur via free radical 348
chain reactions. The consequences of protein oxidation are peptide bond scissions, changes to 349
the amino acid side chain, and intermolecular cross-linked proteins. The protein oxidation via 350
protein-crosslinking can occur by several reactions, such as oxidation of cysteine thiol groups 351
to form disulfide bonds and the formation of dityrosine bond through the loss of a tyrosine 352
group (Lund, Lametsch, Hviid, Jensen, & Skibsted, 2007). 353
Here, SDS-PAGE of the total protein extractions from minced pork in the presence 354
and the absence of the disulfide bond breaking agent, β-mercaptoethanol, were compared 355
(Fig. 6). This technique was applied to observe the major component of myofibrillar protein 356
as MHC. Fig. 6a shows that the condition without any reducing agent gave a protein pattern 357
at the top of the gel, representing a high molecular weight above 200 kDa. The pattern 358
indicates a formation of cross-linked MHC band via disulfide or other chemical bonds, such 359
as ditryptophan and dityrosine. The control sample showed a higher cross-linked MHC band 360
intensity than the samples treated with OligoCS from 0 to 3 days. After 7 days, the control 361
sample exhibited a significant decrease in the band intensity of cross-linked MHC and MHC, 362
and at that time new lower molecular weight protein bands were observed in the 100 to 110 363
kDa range. This indicated that oxidation caused MHC degradation into smaller bands. 364
However, no changes in the protein patterns were found in all samples treated with OligoCS. 365
18
In the presence of a reducing agent (disulfide bond breaking agent) (Fig. 6b), the 366
cross-linked MHC bands above 200 kDa are not observed, indicating that the bands above 367
200 kDa in Fig. 6a are the crosslink protein with disulfide bonds. Nevertheless, at the end of 7 368
days of storage, the decrease in the band intensity of MHC and the new lower band were 369
significantly observed in the control sample, confirming the fragmentation of MHC. It should 370
be noted that the degradation of MHC occurred by endogenous and microbial proteinases, as 371
reported by Martinaud, Mercier, Marinova, Tassy, Gatellier, and Renerre (1997). This implies 372
the effect of OligoCS to prevent protein oxidation in meat, due to their role in inhibiting 373
microbial growth. Moreover, OligoCS is considered to be a potential lipid-derivative radicals 374
scavenger. 375
Sensory quality (odour and taste) is one of the important factors that should be 376
concerned in food applications. The effect of water-based chitosan food additives on sensory 377
quality is under investigation. 378
379
4. Conclusions 380
CSWK and OligoCS obtained from acid/base treatment and enzymatic degradation 381
without the uses of organic solvents or chemical reagents were examined as potential food 382
safety preservatives. The treatment of minced pork with OligoCS concentration 0.4 % (w/w) 383
brought in 20-30% reduction of LAB, Staphylococcus, Enterobacteriaceae and Salmonella 384
spp. compared to the food without any treatment. Furthermore, OligoCS showed a 385
suppression in the BAI values on the second day and retarded both the lipid oxidation level 386
and protein oxidation over seven days of storage at 4 °C. The present work shows how water-387
based chitosan in nanometer sizes can act as a food preservative for the extension of shelf-388
life. The main factors are suspected to relate to (i) positively charged chitosan, (ii) the high 389
surface area for effective assessment, and (iii) the efficient reaction between the free amino 390
19
groups along chitosan and free radicals, including radical scavengers during meat spoilage to 391
retard the lipid and protein oxidation. 392
393
394
Acknowledgements 395
The authors thank the National Research Council of Thailand for research funding. 396
One of the authors, P. Chantarasataporn, would like to acknowledge the National Center of 397
Excellence for Petroleum, Petrochemicals and Advanced Materials, Chulalongkorn 398
University for a scholarship. One of the authors, S. Chirachanchai, would like to express his 399
appreciation to Chulalongkorn University Centenary Academic Development Project under 400
Center of Innovative Nanotechnology. Professor Robert G Gilbert (University of Queensland, 401
Australia) is thanked for his checking of the manuscript. 402
403
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517
25
FIGURE CAPTIONS 518
Fig. 1. TEM micrographs (a); Zeta (ζ) potential of CSWK (∆) and OligoCS (○) (b); and 519
surface area (c). 520
Fig. 2. Antibacterial activity of CSWK (a) and OligoCS (b). Inhibition was determined by 521
two-fold serial broth dilution (Qi et al., 2004) and optical density method. SA: S. aureus 522
ATCC 6538 (■); LM: L. monocytogenes ATCC 19115 (○); BC: B. cereus C113 (▲); SE: S. 523
enteritidis DMST 1706 (●); and EC: E. coli O157:H7 DMST 12743 (♦). ATCC: American 524
Type Culture Collection, Rockville, MD; DMST: Department of Medical Sciences, Ministry 525
of Public Health, Thailand. 526
Fig. 3. Microbial counts log CFU/g of minced pork with different concentrations of OligoCS 527
at 4 °C during storage day as determined on LAB (a); total Staphylococcus counts(b); total 528
Enterobacteriaceae counts (c); Salmonella spp. counts (d); and TVB (e). (–□–) control; 529
samples treated with (–○–) 0.2 % (w/w); (–∆–) 0.4 % (w/w) OligoCS; and (- - -) below 530
detectable level of bacteria. 531
Fig. 4. BAI of minced pork treated with OligoCS at various concentrations during storage at 532
4 °C, (■) 1day, and (□) 2 days. Values are mean ± S.D. from triplicate determinations. The 533
lower-case letters indicate significant differences (p<0.05) within the same storage day. 534
Fig. 5. TBARS values of minced pork with different concentrations of OligoCS at 4 °C 535
during storage. (–□–) control without treatment; samples treated with (–○–) 0.2%; and (–∆–) 536
0.4% OligoCS. 537
Fig. 6. SDS-PAGE patterns of protein extracted from minced pork sample during storage at 4 538
°C. Electrophoresis gel was performed under absence of β-mercaptoethanol (a); and presence 539
of β-mercaptoethanol (b). Lane 0: protein marker; Lanes 1-3: control samples at 0, 3, and 7 540
days; Lanes 4-6: OligoCS 0.2 % at 0, 3, and 7 days; Lanes 7-9: OligoCS 0.4 % at 0, 3, and 7 541
days; MHC: myosin heavy chain. 542
543
26
FIGURE LEGENDS 544
545
546
547
548
549
550
551
552
553
554
555
556
557
558
559
560
561
562
563
564
565
566
Fig. 1. TEM micrographs (a); Zeta (ζ) potential of CSWK (∆) and OligoCS (○) (b); and 567
surface area (c). 568
27
569
570
571
572
573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
Fig. 2. Antibacterial activity of CSWK (a) and OligoCS (b). Inhibition was determined by 588
two-fold serial broth dilution (Qi et al., 2004) and optical density method. SA: S. aureus 589
ATCC 6538 (■); LM: L. monocytogenes ATCC 19115 (○); BC: B. cereus C113 (▲); SE: S. 590
enteritidis DMST 1706 (●); and EC: E. coli O157:H7 DMST 12743 (♦). ATCC: American 591
Type Culture Collection, Rockville, MD; DMST: Department of Medical Sciences, Ministry 592
of Public Health, Thailand. 593
28
594
595
29
596
Fig. 3. Microbial counts log CFU/g of minced pork with different concentrations of OligoCS 597
at 4 °C during storage day as determined on LAB (a); total Staphylococcus counts(b); total 598
Enterobacteriaceae counts (c); Salmonella spp. counts (d); and TVB (e). (–□–) control; 599
samples treated with (–○–) 0.2 % (w/w); (–∆–) 0.4 % (w/w) OligoCS; and (- - -) below 600
detectable level of bacteria. 601
. 602
603
604
605
606
607
608
609
610
611
612
613
614
615
616
617
618
619
620
30
621
622
623
624
625
626
627
628
629
630
631
632
633
634
635
636
637
638
639
640
641
642
643
31
Fig. 4. BAI of minced pork treated with OligoCS at various concentrations during storage at 644
4 °C, (■) 1day, and (□) 2 days. Values are mean ± S.D. from triplicate determinations. The 645
lower-case letters indicate significant differences (p<0.05) within the same storage day. 646
647
648
649
650
651
652
653
654
655
656
657
658
659
660
661
662
663
664
665
666
667
668
32
Fig. 5. TBARS values of minced pork with different concentrations of OligoCS at 4 °C 669
during storage. (–□–) control without treatment; samples treated with (–○–) 0.2%; and (–∆–) 670
0.4% OligoCS. 671
672 673
674 675
676 677
678 679
680
681
682
683
684
685
686
687
688
689
690
691
692 693
694 695
696 697
698 699
700
701
702
703
704
705
706
707
708
709
Fig. 6. SDS-PAGE patterns of protein extracted from minced pork sample during storage at 4 710
°C. Electrophoresis gel was performed under absence of β-mercaptoethanol (a); and presence 711
of β-mercaptoethanol (b). Lane 0: protein marker; Lanes 1-3: control samples at 0, 3, and 7 712
33
days; Lanes 4-6: OligoCS 0.2 % at 0, 3, and 7 days; Lanes 7-9: OligoCS 0.4 % at 0, 3, and 7 713
days; MHC: myosin heavy chain. 714
715
34
716
717
35
718
719
36
720
37
721
722
38
723
724
39
725
726
40
Highlights 727
• Nanowhisker (CSWK) and oligochitosan (OligoCS) are novel water-based food 728
additives. 729
• OligoCS exhibits inhibition against both Gram-positive and Gram-negative food borne 730
pathogen. 731
• OligoCS (0.4 % w/w) retards
biogenic amines index, lipid and protein oxidation in 732
meat product. 733
734
