Research Article Received: 15 November 2013
Revised: 31 March 2014
Accepted article published: 16 April 2014
Published online in Wiley Online Library: 21 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6693
Effects of Fab′ fragments of specific egg yolk antibody (IgY-Fab′) against Shewanella putrefaciens on the preservation of refrigerated turbot Qian Zhang, Hong Lin, Jianxin Sui, Jingxue Wang and Limin Cao* Abstract BACKGROUND: In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab′ ) is now considered a promising method for improving the efficiency of such natural antimicrobial agents. RESULTS: The antimicrobial activity of IgY-Fab′ against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab′ showed a much greater ability to damage cell membranes than the whole IgY. CONCLUSION: Compared to whole IgY with the same molarity, IgY-Fab′ demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry Keywords: IgY-Fab′ ; refrigerated turbot; preservation; antimicrobial activity
INTRODUCTION
136
Shewanella putrefaciens has been identified as the specific spoilage organism for many marine fish during aerobic chilling storage.1,2 In our previous studies, the antimicrobial activity of the specific IgY against S. putrefaciens for refrigerated flatfishes has been confirmed, which indicated its potential as a natural antimicrobial agent for seafood preservation.3 However, there are still limitations for its actual application in food industry; for example, the relatively high concentration (100 mg mL−1 or above) that is required.3 Moreover, the possible allergenicity of the Fc fragment in IgY for some consumers should also be considered.4,5 Now there is an emerging trend to use the antigen-binding fragment plus hinge region (IgY-Fab′ ) to solve the problems mentioned above. Usually, the IgY-Fab′ is prepared by pepsin digestion of IgY, and therefore contains the Fab fragment as well as some of the hinge regions.6 IgY-Fab′ has shown several advantages over the whole IgY:6 – 10 a lower molecular weight (approximately 25% of the whole antibody), lower allergenicity due to the removal of Fc fragments, more antigen binding activity, and better stability. IgY-Fab′ is also considered more convenient for genetic modification and practical applications.11 In our previous work, the active fragment from IgY against S. putrefaciens was produced, and identified as IgY-Fab′ based on its molecular weight, its binding affinity J Sci Food Agric 2015; 95: 136–140
and its growth inhibitory ability to the antigen bacteria.12 Here the antimicrobial activity of the IgY-Fab′ was further validated and evaluated using refrigerated turbot as samples, and the detailed interaction between IgY-Fab′ and S. putrefaciens was investigated. Its potential for food bio-preservation is also discussed.
MATERIALS AND METHODS Materials and reagents The S. putrefaciens strain was CCTCC AB 2010225 from the China Center for Type Culture Collection. Chicken IgY against S. putrefaciens (P-IgY) and its Fab′ fragments (IgY-Fab′ ) were prepared as in our previous work.12 The IgY prepared from unimmunised chicken was used as a negative control (N-IgY). The protein concentration of IgY and IgY-Fab′ was determined by the Coomassie brilliant blue method. The alkaline phosphatase kit was purchased from
∗
Correspondence to: Limin Cao, Food Safety Laboratory, Ocean University of China, 5# Yushan Road, Qingdao 266003, People’s Republic of China. E-mail: [email protected] Food Safety Laboratory, Ocean University of China, Qingdao 266003, People’s Republic of China
www.soci.org
© 2014 Society of Chemical Industry
IgY-Fab’ for preservation of refrigerated turbot
www.soci.org
Table 1. Preparation of IgY solutions Solution Blank control N-IgY P-IgY IgY-Fab′
Concentrationa (mg mL−1 ) 0 100 100 25
a
Concentration refers to IgY or IgY-Fab′ , as appropriate, and is given as the protein content, in mg mL−1 .
Nanjing Jiancheng Bio-engineering Institute (Nanjiang, China). All other chemicals were of analytical grade unless otherwise stated. All centrifugations were performed with a centrifuge BR-4i (Jouan, Saint-Herblain, France). Turbot (Scophthalmus maximus) was purchased alive from Mykal Supermarket in Qingdao, China. The fish were killed and washed under sterile conditions, and muscles were cut into pieces of 2 cm × 2 cm × 1 cm (length × width × thickness). Each piece was about 5 g in weight. Nutrient broth was purchased from Beijing Land Bridge Technology Co. Ltd (Beijing, China). Preservation of fish samples The freeze-dried P-IgY, IgY-Fab′ and N-IgY were diluted with sterilised saline to reach a same molarity (Table 1), and then filtered through 0.22 μm micro-filters. The fish pieces were randomly divided into four groups: three groups were immersed in P-IgY, IgY-Fab′ and N-IgY for 30 min, and drained for 15 min, respectively, while the fourth group was treated with sterilised saline as the blank control. Then the pieces were individually packed into aseptic plastic bags and stored at 4 ± 1∘ C for 15 days. Every 3 days, three fish pieces were randomly taken from each group for microbiological, chemical and sensory evaluation.
J Sci Food Agric 2015; 95: 136–140
Statistical analysis All experiments were performed at least in duplicate, and data were expressed as the mean ± SEM, unless indicated otherwise. The Student’s t-test was carried out to determine the significant difference (P = 0.05) among different groups.
RESULTS AND DISCUSSION Microbiological changes of refrigerated fish pieces The initial TVC of the fish remained below 3.77 log CFU g−1 in the first 6 days (Fig. 1). After 9 days the TVC value of P-IgY- and IgY-Fab′ -treated samples were shown to be significantly lower than that of controls (P < 0.05). The maximum permissible limit of TVC in fish has been set to be around 7.0 (log CFU g−1 ) by the International Commission on Microbiological Specifications for Foods (ICMSF).17 According to the criteria, the shelf life of blank controls and P-IgY treated samples was estimated to be about 13 and 15 days, respectively. For IgY-Fab′ -treated samples, the shelf life was estimated to be about 17–18 days if the TVC curve was prolonged.
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Analysis of fish samples For microbial analysis, a piece (5 g) was transferred to 45 mL of sterilised NaCl solution (9 g L−1 ) and homogenised in a mortar. Total viable counts (TVC) and the number of S. putrefaciens were determined by the spread plate method using nutrient agar (containing 10 g peptone, 3 g beef power, 5 g NaCl and 15 g agar in 1 L of the product, bought from Beijing Land Bridge Technology) as the medium. The plates were incubated at 25∘ C for 24 h. S. putrefaciens was simply identified and counted according to its morphological characteristics: deep yellow, neat edge and with a humped shape.3 For each sample three of the ten-fold serial dilutions were chosen for bacterial count, and each dilution was performed in duplicate. For determination of the total volatile basic nitrogen (TVB-N), two fish pieces from each group were blended with 45 mL of perchlorate solutions (0.6 mol L−1 ), respectively. The mixture was filtered and TVB-N was determined referring to the Chinese Industrial Standard (SC/T 3032-2007).13 Each sample was tested three times. The sensory quality of fish pieces was evaluated at each sampling time (days 0, 3, 6, 9, 12, 15) by an eight-member trained panel.14 Panellists were asked to score odour, colour and texture of pieces using a 0–10 acceptability scale where a score of 10 was defined as the best. The detailed standards are described in Table 2. Average scores were calculated by removing the highest and the lowest points, and then making an average of the remaining scores.
Analysis of the physiological properties of Shewanella putrefaciens treated with IgY The cell-surface hydrophobicity was determined by the Microbial Adhesion to Hydrocarbons (MATH).15 S. putrefaciens at the logarithmic phase was inoculated into nutrient broth (containing 5 mg mL−1 IgY-Fab′ or 20 mg mL−1 IgY, with the same molar concentration) to reach a cell concentration of 103 CFU mL−1 . The suspension was incubated at 25∘ C for 8 h, and centrifuged at 3300 × g for 10 min. The cells were collected and washed with sterilised saline three times, and diluted in saline to reach an absorbance value of 0.1 ± 0.02 at 600 nm (A0 ). Then 200 𝜇L xylene was added to 1 mL of the suspension. After shaking for 60 s and standing for 60 min, the water phase of the mixture was taken and its absorbance at 600 nm (A1 ) was determined.15 The same experiment was performed with nutrient broth without any anti-bacterial agent as the blank control. The hydrophobicity rate (R, %) of the cells was calculated as follows: R = [(A0 − A1 )/A0 ] × 100. For analysis of the damage to cells, the freeze-dried IgY-Fab′ was diluted in HEPES-Na (5 mmol L−1 , pH 7.0), and then mixed with suspension of S. putrefaciens at the logarithmic phase. The final concentration of IgY-Fab′ and bacteria cell was 5 mg mL−1 (protein content) and 103 CFU mL−1 , respectively. The mixture was incubated at 25∘ C for 24 h under continuous shaking, followed by centrifugation (4∘ C, 3300 × g for 10 min). The supernatant was collected for further analysis. The same experiment performed with P-IgY (protein content, 20 mg mL−1 ) or N-IgY (protein content, 20 mg mL−1 ) was used as the positive control or negative control, respectively. The sample treated with only HEPES-Na was used as the blank control. The alkaline phosphatase (AKP) activity of the supernatant was determined using a commercial kit according to the manufacturer’s instructions, and the final absorbance value at 520 nm was measured to represent the AKP activity.15 For analysis of the leakage of nucleic acids, trichloroacetic acid was added to the prepared supernatant (up to 5% of trichloroacetic acid in the solution), shaken, and then centrifuged at 3300 × g for 5 min. The supernatant was collected after centrifugation and the performance was repeated until the absorbance value at 595 nm was determined ≤ 0.001 after its reaction with Coomassie brilliant blue,16 and then the absorbance value at 260 nm was determined.
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Q Zhang et al.
Table 2. Sensory evaluation form Score Item
10
8
Odour
Fish inherent odour, and no peculiar smell
Colour
Bright and shiny at the muscle section
Texture Compact, good flexibility, no juice
6
4
Some fish inherent With a little peculiar smell odour, and no peculiar smell Normal colour and shiny Little dim colour and no at the muscle section bright at muscle section Compact, apparent Slightly loose, low flexibility after flexibility after pressing, no juice pressing, little juice
9
2
Slight rotten fish/ammonia smell
Strong rotten fish/ammonia smell
Dim colour and no bright at muscle section
Yellow in muscle and dim at muscle section
Loose, low flexibility, obvious juice
Loose, no flexibility, juice exuded obviously
9
a
a
8 7
b
8
c
7
a b
6
b
a 5
a b a
4 3
c d
b
a a b
d
Blank Negative IgY Fab'
Log cfu/mL
log cfu/g
a 6
a
5
a
4 a
3
b b 2 0
3
6
9
12
15
In the presence of P-IgY and IgY-Fab′ , similar growth inhibition was also observed for S. putrefaciens (Fig. 2). Compared to the whole P-IgY, the IgY-Fab′ exhibited better antibacterial efficiency at the end of preservation (day 15), maybe due to the difference in structural properties. For example, the lower molecular weight of IgY-Fab′ may make it easier to interact with the target bacteria in comparison to the whole IgY.
138
Biochemical changes of refrigerated fish pieces After 6 days of storage, P-IgY-treated groups and IgY-Fab′ -treated groups exhibited much lower TVB-N values than the blank control and N-IgY treated groups (P < 0.05) (Fig. 3). The acceptable TVB-N value in aquatic products usually ranges between 20 mg 100 g−1 and 40 mg 100 g−1 , and 30 mg 100 g−1 is usually set as the end of shelf life.18,19 Therefore the shelf life of blank and N-IgY-treated group was estimated to be about 11 and 13 days, respectively. For the P-IgY- or IgY-Fab′ -treated samples, the shelf life could be estimated to be about 17–18 days if the TVB-N curve is prolonged. Such a result accords well with that of the microbial analysis.
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b b
b
c
Blank Negative IgY Fab'
3
6
9
12
15
Days
Days Figure 1. Total bacterial count in turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
b
a a b
c
c
2 0
a
a
Figure 2. The growth of Shewanella putrefaciens in turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
Sensory evaluation of refrigerated fish pieces After 12 days of storage, the sensory properties of the IgY-Fab′ -treated group and IgY-treated group were significantly better than those of blank controls (P < 0.05) (Fig. 4). It was noted that the scores of the IgY-Fab′ -treated group were relatively low at the initial phase of the preservation (up to day 6). Although detailed reasons are still unknown, the residual smells of pepsin and the influence of other protein fragments from the enzymatic degradation of the IgY may contribute to the result. The effect of IgY-Fab′ on the physiological properties of antigen bacteria Until now the detailed mechanism for the enhanced efficiency of IgY-Fab′ to inhibit the growth of antigen bacteria has been unclear. Here the effect of the IgY-Fab′ on the cell wall, the membrane and the hydrophobicity of S. putrefaciens was investigated to provide more information. A suitable level of hydrophobicity is essential for the adhesion ability of cells, and therefore considered very important for the nutrient exchange and growth of bacteria.20,21 After the treatment with the specific IgY and IgY-Fab′ , the hydrophobicity at the surface of the bacteria was significantly decreased in comparison to that of controls (P < 0.05) (Fig. 5). This result has not been indicated by previous studies. Now it still seemed very far to reach a
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 136–140
IgY-Fab’ for preservation of refrigerated turbot
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60 55 50
a
40
40
b
35
a
30
c
a
b c c
25 b b
20 15
d Blank Negative IgY Fab'
b
10 5
Hydrophoby Rate(%)
TVB-N (mg/mL)
45
0 0
3
6
9
12
30
20
10
15
Days Figure 3. Total volatile basic nitrogen (TVB-N) values of turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
Blank
Negative IgY
IgY
Fab'
Figure 5. The effect of different IgY agents on the hydrophobicity of Shewanella putrefaciens cells.
12 10
1.0
a
6
a a a
a
b
b c c
4
a a
0.8
b b Blank Negative IgY Fab'
2 0 0
3
6
9 Days
12
Absorbance at 570nm
b b b
8 Scores
0
0.6
0.4
15 0.2
Figure 4. Sensory scores of turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
0.0 Blank
J Sci Food Agric 2015; 95: 136–140
Negative IgY
IgY
Fab'
Figure 6. The activity of alkaline phosphatase in reacting solutions after IgY–Shewanella putrefaciens interaction.
Though the IgY-Fab′ did not demonstrate the same effect on hydrophobicity and cell walls as that of whole IgY, it exhibited much greater ability to damage cell membranes, which could be evidenced by the increased leakage of nucleic acids. Such a difference may be reasonably attributed to the relatively much lower molecular weight of IgY-Fab′ , which was evaluated to be only about 25% of the whole IgY and therefore could greatly enhance its ability to pass through cell walls and therefore affect the internal structure and related physiological activities of the bacteria. Of course, other structural properties of IgY-Fab′ should not be neglected. For example, the difference in electronic properties and the sensitivity to ions and proteases in fish samples, may also contribute to the relatively higher anti-bacterial efficiency of the IgY-Fab′ compared to the whole IgY.
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comprehensive explanation, but the changes in electronic properties and topological structures22 at the cell surface after the IgY (or IgY-Fab′ )–bacteria interaction were assumed to be closely related to the decreased hydrophobicity investigated here. Similarly to many other bio-preservatives, such as nisin and protamine,23,24 the anti-bacterial efficiency of specific IgY was also attributed to its ability to destroy cell structures.25 – 28 Here the idea was further confirmed by our investigation on the interaction between S. putrefaciens and different IgY agents. In comparison to blank and negative controls, the specific IgY or IgY-Fab′ resulted in significantly higher alkaline phosphatase (AKP) activity after the reaction with bacteria suspension (P < 0.05) (Fig. 6), which indicated serious damage to cell walls. But unlike previous studies which mainly focused on the interaction between IgY and its corresponding epitopes on cell walls; here the damage to cell membranes was also demonstrated by the significant leakage of nucleic acids after the reactions (absorbance at 260 nm, Table 3). The accurate information on the IgY–membrane interaction is still unclear, and it seems a very interesting problem for further studies.
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Table 3. Optical density at 260 nm of bacterial supernatant treated by IgY and IgY-Fab′ Result no. 1 2 3 Average
Blank
IgY
Negative IgY
9
IgY-Fab′
1.534 2.344 1.892 2.706 1.544 2.348 1.885 2.678 1.531 2.289 1.776 2.879 1.537 ± 0.007 2.327 ± 0.033 1.851 ± 0.065 2.754 ± 0.109
10 11 12
CONCLUSIONS Based on microbial, biological and chemical tests, the efficiency of the specific IgY-Fab′ to prolong the shelf life of refrigerated turbot could be concluded. In comparison to the whole P-IgY, the IgY-Fab′ demonstrated better antimicrobial activity and more durable efficiency. Though accurate reasons are still unknown, such a result was indicated to be closely related to its structural properties (such as the much lower molecular weight), which may enhance the ability to influence physiological activities of antigen bacteria, especially the property or/and structure of membranes. These results allow us to suggest a promising potential of the IgY-Fab′ for bio-preservation of aquatic products.
13 14 15 16 17 18
ACKNOWLEDGEMENT
19
This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-50) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1188).
20 21
REFERENCES 1 Yang X, Xu Z and Xiao L, Specific spoilage organisms from aquatic product and prediction & prolongation of the shelf life. J Fisheries China 28:106–111 (2004). 2 Xu Y, Lin H and Zhang Q, Characterization of chicken egg yolk antibodies against main spoilage organisms in Paralichthys olivaceus during chilled storage. Food Sci 31:109–113 (2010). 3 Xu Y, Lin H and Sui J, Effects of specific egg yolk antibody (IgY) on the quality and shelf life of refrigerated Paralichthys olivaceus. J Sci Food Agric 92:1267–1272 (2012). 4 Silva WD and Tambourgi DV, IgY: A promising antibody for use in immunodiagnostic and in immunotherapy. Vet Immunol Immunopathol 135:173–180 (2010). 5 Chen D and Sun S, Preparation and some properties of egg yolk immunoglobulinY (IgY) against influenza virus. Chem Ind Eng 20:448–452 (2003). 6 Akita EM, Chicken Egg Yolk Immunoglobulin Y and Fragments: Purification, Allergenicity and their Neutralization of Escherichia Coli Heat Labile Toxin. The University of British Columbia, Vancouver (2005). 7 Camenisch G, Tini M and Chilov D, General applicability of chicken egg yolk antibodies: The performance of IgY immunoglobulins raised against the hypoxia-inducible factor l alpha. FASEB J 13:81–88 (1999). 8 Akita EM, Li-Chan E and Nakai S, Neutralization of enterotoxigenic escherichia coli heat-labile toxin by chicken egg yolk
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28
Q Zhang et al. immunoglobulin Y and its antigen-binding fragments. Food Agric Immunol 10:161–172 (1998). Zhang Y, Experimental Study of the Effect of Fab′ Against Lipopolysaccharide on Preventing Intestinal Damage During the Early Stage in Severely Burned Mice. The Third Military Medical University, Beijing (2008). Zou Q, Li M and Yang S, Preparation and identification of Fab′ fragments from chicken egg yolk immunoglobulin Y. J Med Postgrad 23:133–136 (2010). Wang Y, Research process of genetic engineering antibody. Chin J Immunol 15:193–195 (1995). Zhang Q, Lin H and Dang X, Production and antibacterial activity evaluation of fragments from chicken egg yolk immunoglobulin against Shewanella putrefaciens. Sci Technol Food Ind 34:75–82 (2013). Chinese Industrial Standard SC/T 3032-2007. Determination of total volatile basic nitrogen in fishery products. Association of Official Analytical Chemists, Official Methods of Analysis, 17th edition. AOAC, Washington, DC (2000). Fan HJ, In vitro antibacterial activity of the coelomic fluid and deoxyribonuclease of the Eisenia foetida earthworm. Shanxi Medical University, Taiyuan (2008). Wang XP and Xing SL. Determination of protein quantitation using the method of Coomassie brilliant blue. Tianjin Chemical Industry 3:40–42 (2009) Sallam KI, Antimicrobial and antioxidant effects of sodium acetate sodium lactate and sodium citrate in refrigerated sliced salmon. Food Control 18:566–575 (2007). Harpaz S, Glatman L and Drabkin V, Effects of herbal essential oils used to extend the shelf-life of freshwater reared Asian sea bass fish (Lates calcarifer). Food Protect 66:410–417 (2003). Metin S, Erkan N, Varlik C and Aran N, Extension of shelf-life of chub mackerel (Scomber japonicus Houttuyn 1780) treated with lactic acid. Eur Food Res Technol 213:174–177 (2001). Zhao Q, Zhang JY and Chen LZ, Cell-surface hydrophobicity and degradation characteristics of hydrophobic hydrocarbon degrading bacteria. Environ Sci 26:132–136 (2005). Naito Y, Tohda H and Okuda K, Adherence and hydrophobicity of invasion and noninvasive strains of Porphyromonas gingivalis. Oral Microbiol Immunol 8:195–198 (1993). Huang Y, Ma T and Gu XB, The lipopeptides effect on the surface hydrophobicity and adhension of the bacteria. Acta Scientiarum Naturalium Universitatis Nankaiensis 5:74–78 (2006). Cotter PD, Hill C and Ross RP, Bacteriocins, developing innate immunity for food. Microbiology (Nat Rev) 10:777–778 (2005). Johansen C, Verheul A and Gram L, Protamine-induced permeabilization of cell envelopes of gram-positive and gram-negative bacteria. Appl Environ Microbiol 62:1058–1064 (1996). Lee EN, Sunwoo HH and Menninen K, In vitro studies of chicken egg yolk antibody (IgY) against Salmonella enteritidis and Salmonella typhimurium. Poultry Sci 81:632–641 (2002). Fujibayashi T, Nakamura M and Tominaga A, Effects of IgY against Candida albicans and Candida spp.: adherence and biofilm formation. Jpn J Infect Dis 62:337–442 (2009). Wiedemann V, Linckh E and Kuhlmann R, Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases: In vivo studies on protective effects against Escherichia coli diarrhoea in pigs. Vet Med 39:283–291 (1991). Marquardt RR, Jin LZ and Kim J-W, Passive protective effect of egg-yolk antibodies against enterotoxigenic Escherichia coli K88+ infection in neonatal and early-weaned piglets. FEMS Immunol Med Microbiol 23:283–288 (1999).
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© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 136–140
Revised: 31 March 2014
Accepted article published: 16 April 2014
Published online in Wiley Online Library: 21 May 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6693
Effects of Fab′ fragments of specific egg yolk antibody (IgY-Fab′) against Shewanella putrefaciens on the preservation of refrigerated turbot Qian Zhang, Hong Lin, Jianxin Sui, Jingxue Wang and Limin Cao* Abstract BACKGROUND: In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab′ ) is now considered a promising method for improving the efficiency of such natural antimicrobial agents. RESULTS: The antimicrobial activity of IgY-Fab′ against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab′ showed a much greater ability to damage cell membranes than the whole IgY. CONCLUSION: Compared to whole IgY with the same molarity, IgY-Fab′ demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry Keywords: IgY-Fab′ ; refrigerated turbot; preservation; antimicrobial activity
INTRODUCTION
136
Shewanella putrefaciens has been identified as the specific spoilage organism for many marine fish during aerobic chilling storage.1,2 In our previous studies, the antimicrobial activity of the specific IgY against S. putrefaciens for refrigerated flatfishes has been confirmed, which indicated its potential as a natural antimicrobial agent for seafood preservation.3 However, there are still limitations for its actual application in food industry; for example, the relatively high concentration (100 mg mL−1 or above) that is required.3 Moreover, the possible allergenicity of the Fc fragment in IgY for some consumers should also be considered.4,5 Now there is an emerging trend to use the antigen-binding fragment plus hinge region (IgY-Fab′ ) to solve the problems mentioned above. Usually, the IgY-Fab′ is prepared by pepsin digestion of IgY, and therefore contains the Fab fragment as well as some of the hinge regions.6 IgY-Fab′ has shown several advantages over the whole IgY:6 – 10 a lower molecular weight (approximately 25% of the whole antibody), lower allergenicity due to the removal of Fc fragments, more antigen binding activity, and better stability. IgY-Fab′ is also considered more convenient for genetic modification and practical applications.11 In our previous work, the active fragment from IgY against S. putrefaciens was produced, and identified as IgY-Fab′ based on its molecular weight, its binding affinity J Sci Food Agric 2015; 95: 136–140
and its growth inhibitory ability to the antigen bacteria.12 Here the antimicrobial activity of the IgY-Fab′ was further validated and evaluated using refrigerated turbot as samples, and the detailed interaction between IgY-Fab′ and S. putrefaciens was investigated. Its potential for food bio-preservation is also discussed.
MATERIALS AND METHODS Materials and reagents The S. putrefaciens strain was CCTCC AB 2010225 from the China Center for Type Culture Collection. Chicken IgY against S. putrefaciens (P-IgY) and its Fab′ fragments (IgY-Fab′ ) were prepared as in our previous work.12 The IgY prepared from unimmunised chicken was used as a negative control (N-IgY). The protein concentration of IgY and IgY-Fab′ was determined by the Coomassie brilliant blue method. The alkaline phosphatase kit was purchased from
∗
Correspondence to: Limin Cao, Food Safety Laboratory, Ocean University of China, 5# Yushan Road, Qingdao 266003, People’s Republic of China. E-mail: [email protected] Food Safety Laboratory, Ocean University of China, Qingdao 266003, People’s Republic of China
www.soci.org
© 2014 Society of Chemical Industry
IgY-Fab’ for preservation of refrigerated turbot
www.soci.org
Table 1. Preparation of IgY solutions Solution Blank control N-IgY P-IgY IgY-Fab′
Concentrationa (mg mL−1 ) 0 100 100 25
a
Concentration refers to IgY or IgY-Fab′ , as appropriate, and is given as the protein content, in mg mL−1 .
Nanjing Jiancheng Bio-engineering Institute (Nanjiang, China). All other chemicals were of analytical grade unless otherwise stated. All centrifugations were performed with a centrifuge BR-4i (Jouan, Saint-Herblain, France). Turbot (Scophthalmus maximus) was purchased alive from Mykal Supermarket in Qingdao, China. The fish were killed and washed under sterile conditions, and muscles were cut into pieces of 2 cm × 2 cm × 1 cm (length × width × thickness). Each piece was about 5 g in weight. Nutrient broth was purchased from Beijing Land Bridge Technology Co. Ltd (Beijing, China). Preservation of fish samples The freeze-dried P-IgY, IgY-Fab′ and N-IgY were diluted with sterilised saline to reach a same molarity (Table 1), and then filtered through 0.22 μm micro-filters. The fish pieces were randomly divided into four groups: three groups were immersed in P-IgY, IgY-Fab′ and N-IgY for 30 min, and drained for 15 min, respectively, while the fourth group was treated with sterilised saline as the blank control. Then the pieces were individually packed into aseptic plastic bags and stored at 4 ± 1∘ C for 15 days. Every 3 days, three fish pieces were randomly taken from each group for microbiological, chemical and sensory evaluation.
J Sci Food Agric 2015; 95: 136–140
Statistical analysis All experiments were performed at least in duplicate, and data were expressed as the mean ± SEM, unless indicated otherwise. The Student’s t-test was carried out to determine the significant difference (P = 0.05) among different groups.
RESULTS AND DISCUSSION Microbiological changes of refrigerated fish pieces The initial TVC of the fish remained below 3.77 log CFU g−1 in the first 6 days (Fig. 1). After 9 days the TVC value of P-IgY- and IgY-Fab′ -treated samples were shown to be significantly lower than that of controls (P < 0.05). The maximum permissible limit of TVC in fish has been set to be around 7.0 (log CFU g−1 ) by the International Commission on Microbiological Specifications for Foods (ICMSF).17 According to the criteria, the shelf life of blank controls and P-IgY treated samples was estimated to be about 13 and 15 days, respectively. For IgY-Fab′ -treated samples, the shelf life was estimated to be about 17–18 days if the TVC curve was prolonged.
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Analysis of fish samples For microbial analysis, a piece (5 g) was transferred to 45 mL of sterilised NaCl solution (9 g L−1 ) and homogenised in a mortar. Total viable counts (TVC) and the number of S. putrefaciens were determined by the spread plate method using nutrient agar (containing 10 g peptone, 3 g beef power, 5 g NaCl and 15 g agar in 1 L of the product, bought from Beijing Land Bridge Technology) as the medium. The plates were incubated at 25∘ C for 24 h. S. putrefaciens was simply identified and counted according to its morphological characteristics: deep yellow, neat edge and with a humped shape.3 For each sample three of the ten-fold serial dilutions were chosen for bacterial count, and each dilution was performed in duplicate. For determination of the total volatile basic nitrogen (TVB-N), two fish pieces from each group were blended with 45 mL of perchlorate solutions (0.6 mol L−1 ), respectively. The mixture was filtered and TVB-N was determined referring to the Chinese Industrial Standard (SC/T 3032-2007).13 Each sample was tested three times. The sensory quality of fish pieces was evaluated at each sampling time (days 0, 3, 6, 9, 12, 15) by an eight-member trained panel.14 Panellists were asked to score odour, colour and texture of pieces using a 0–10 acceptability scale where a score of 10 was defined as the best. The detailed standards are described in Table 2. Average scores were calculated by removing the highest and the lowest points, and then making an average of the remaining scores.
Analysis of the physiological properties of Shewanella putrefaciens treated with IgY The cell-surface hydrophobicity was determined by the Microbial Adhesion to Hydrocarbons (MATH).15 S. putrefaciens at the logarithmic phase was inoculated into nutrient broth (containing 5 mg mL−1 IgY-Fab′ or 20 mg mL−1 IgY, with the same molar concentration) to reach a cell concentration of 103 CFU mL−1 . The suspension was incubated at 25∘ C for 8 h, and centrifuged at 3300 × g for 10 min. The cells were collected and washed with sterilised saline three times, and diluted in saline to reach an absorbance value of 0.1 ± 0.02 at 600 nm (A0 ). Then 200 𝜇L xylene was added to 1 mL of the suspension. After shaking for 60 s and standing for 60 min, the water phase of the mixture was taken and its absorbance at 600 nm (A1 ) was determined.15 The same experiment was performed with nutrient broth without any anti-bacterial agent as the blank control. The hydrophobicity rate (R, %) of the cells was calculated as follows: R = [(A0 − A1 )/A0 ] × 100. For analysis of the damage to cells, the freeze-dried IgY-Fab′ was diluted in HEPES-Na (5 mmol L−1 , pH 7.0), and then mixed with suspension of S. putrefaciens at the logarithmic phase. The final concentration of IgY-Fab′ and bacteria cell was 5 mg mL−1 (protein content) and 103 CFU mL−1 , respectively. The mixture was incubated at 25∘ C for 24 h under continuous shaking, followed by centrifugation (4∘ C, 3300 × g for 10 min). The supernatant was collected for further analysis. The same experiment performed with P-IgY (protein content, 20 mg mL−1 ) or N-IgY (protein content, 20 mg mL−1 ) was used as the positive control or negative control, respectively. The sample treated with only HEPES-Na was used as the blank control. The alkaline phosphatase (AKP) activity of the supernatant was determined using a commercial kit according to the manufacturer’s instructions, and the final absorbance value at 520 nm was measured to represent the AKP activity.15 For analysis of the leakage of nucleic acids, trichloroacetic acid was added to the prepared supernatant (up to 5% of trichloroacetic acid in the solution), shaken, and then centrifuged at 3300 × g for 5 min. The supernatant was collected after centrifugation and the performance was repeated until the absorbance value at 595 nm was determined ≤ 0.001 after its reaction with Coomassie brilliant blue,16 and then the absorbance value at 260 nm was determined.
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Q Zhang et al.
Table 2. Sensory evaluation form Score Item
10
8
Odour
Fish inherent odour, and no peculiar smell
Colour
Bright and shiny at the muscle section
Texture Compact, good flexibility, no juice
6
4
Some fish inherent With a little peculiar smell odour, and no peculiar smell Normal colour and shiny Little dim colour and no at the muscle section bright at muscle section Compact, apparent Slightly loose, low flexibility after flexibility after pressing, no juice pressing, little juice
9
2
Slight rotten fish/ammonia smell
Strong rotten fish/ammonia smell
Dim colour and no bright at muscle section
Yellow in muscle and dim at muscle section
Loose, low flexibility, obvious juice
Loose, no flexibility, juice exuded obviously
9
a
a
8 7
b
8
c
7
a b
6
b
a 5
a b a
4 3
c d
b
a a b
d
Blank Negative IgY Fab'
Log cfu/mL
log cfu/g
a 6
a
5
a
4 a
3
b b 2 0
3
6
9
12
15
In the presence of P-IgY and IgY-Fab′ , similar growth inhibition was also observed for S. putrefaciens (Fig. 2). Compared to the whole P-IgY, the IgY-Fab′ exhibited better antibacterial efficiency at the end of preservation (day 15), maybe due to the difference in structural properties. For example, the lower molecular weight of IgY-Fab′ may make it easier to interact with the target bacteria in comparison to the whole IgY.
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Biochemical changes of refrigerated fish pieces After 6 days of storage, P-IgY-treated groups and IgY-Fab′ -treated groups exhibited much lower TVB-N values than the blank control and N-IgY treated groups (P < 0.05) (Fig. 3). The acceptable TVB-N value in aquatic products usually ranges between 20 mg 100 g−1 and 40 mg 100 g−1 , and 30 mg 100 g−1 is usually set as the end of shelf life.18,19 Therefore the shelf life of blank and N-IgY-treated group was estimated to be about 11 and 13 days, respectively. For the P-IgY- or IgY-Fab′ -treated samples, the shelf life could be estimated to be about 17–18 days if the TVB-N curve is prolonged. Such a result accords well with that of the microbial analysis.
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b b
b
c
Blank Negative IgY Fab'
3
6
9
12
15
Days
Days Figure 1. Total bacterial count in turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
b
a a b
c
c
2 0
a
a
Figure 2. The growth of Shewanella putrefaciens in turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
Sensory evaluation of refrigerated fish pieces After 12 days of storage, the sensory properties of the IgY-Fab′ -treated group and IgY-treated group were significantly better than those of blank controls (P < 0.05) (Fig. 4). It was noted that the scores of the IgY-Fab′ -treated group were relatively low at the initial phase of the preservation (up to day 6). Although detailed reasons are still unknown, the residual smells of pepsin and the influence of other protein fragments from the enzymatic degradation of the IgY may contribute to the result. The effect of IgY-Fab′ on the physiological properties of antigen bacteria Until now the detailed mechanism for the enhanced efficiency of IgY-Fab′ to inhibit the growth of antigen bacteria has been unclear. Here the effect of the IgY-Fab′ on the cell wall, the membrane and the hydrophobicity of S. putrefaciens was investigated to provide more information. A suitable level of hydrophobicity is essential for the adhesion ability of cells, and therefore considered very important for the nutrient exchange and growth of bacteria.20,21 After the treatment with the specific IgY and IgY-Fab′ , the hydrophobicity at the surface of the bacteria was significantly decreased in comparison to that of controls (P < 0.05) (Fig. 5). This result has not been indicated by previous studies. Now it still seemed very far to reach a
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J Sci Food Agric 2015; 95: 136–140
IgY-Fab’ for preservation of refrigerated turbot
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60 55 50
a
40
40
b
35
a
30
c
a
b c c
25 b b
20 15
d Blank Negative IgY Fab'
b
10 5
Hydrophoby Rate(%)
TVB-N (mg/mL)
45
0 0
3
6
9
12
30
20
10
15
Days Figure 3. Total volatile basic nitrogen (TVB-N) values of turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
Blank
Negative IgY
IgY
Fab'
Figure 5. The effect of different IgY agents on the hydrophobicity of Shewanella putrefaciens cells.
12 10
1.0
a
6
a a a
a
b
b c c
4
a a
0.8
b b Blank Negative IgY Fab'
2 0 0
3
6
9 Days
12
Absorbance at 570nm
b b b
8 Scores
0
0.6
0.4
15 0.2
Figure 4. Sensory scores of turbot samples during chilling storage, in which a, b, c and d indicate the differences among different groups based on the Student’s t-test.
0.0 Blank
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Negative IgY
IgY
Fab'
Figure 6. The activity of alkaline phosphatase in reacting solutions after IgY–Shewanella putrefaciens interaction.
Though the IgY-Fab′ did not demonstrate the same effect on hydrophobicity and cell walls as that of whole IgY, it exhibited much greater ability to damage cell membranes, which could be evidenced by the increased leakage of nucleic acids. Such a difference may be reasonably attributed to the relatively much lower molecular weight of IgY-Fab′ , which was evaluated to be only about 25% of the whole IgY and therefore could greatly enhance its ability to pass through cell walls and therefore affect the internal structure and related physiological activities of the bacteria. Of course, other structural properties of IgY-Fab′ should not be neglected. For example, the difference in electronic properties and the sensitivity to ions and proteases in fish samples, may also contribute to the relatively higher anti-bacterial efficiency of the IgY-Fab′ compared to the whole IgY.
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comprehensive explanation, but the changes in electronic properties and topological structures22 at the cell surface after the IgY (or IgY-Fab′ )–bacteria interaction were assumed to be closely related to the decreased hydrophobicity investigated here. Similarly to many other bio-preservatives, such as nisin and protamine,23,24 the anti-bacterial efficiency of specific IgY was also attributed to its ability to destroy cell structures.25 – 28 Here the idea was further confirmed by our investigation on the interaction between S. putrefaciens and different IgY agents. In comparison to blank and negative controls, the specific IgY or IgY-Fab′ resulted in significantly higher alkaline phosphatase (AKP) activity after the reaction with bacteria suspension (P < 0.05) (Fig. 6), which indicated serious damage to cell walls. But unlike previous studies which mainly focused on the interaction between IgY and its corresponding epitopes on cell walls; here the damage to cell membranes was also demonstrated by the significant leakage of nucleic acids after the reactions (absorbance at 260 nm, Table 3). The accurate information on the IgY–membrane interaction is still unclear, and it seems a very interesting problem for further studies.
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Table 3. Optical density at 260 nm of bacterial supernatant treated by IgY and IgY-Fab′ Result no. 1 2 3 Average
Blank
IgY
Negative IgY
9
IgY-Fab′
1.534 2.344 1.892 2.706 1.544 2.348 1.885 2.678 1.531 2.289 1.776 2.879 1.537 ± 0.007 2.327 ± 0.033 1.851 ± 0.065 2.754 ± 0.109
10 11 12
CONCLUSIONS Based on microbial, biological and chemical tests, the efficiency of the specific IgY-Fab′ to prolong the shelf life of refrigerated turbot could be concluded. In comparison to the whole P-IgY, the IgY-Fab′ demonstrated better antimicrobial activity and more durable efficiency. Though accurate reasons are still unknown, such a result was indicated to be closely related to its structural properties (such as the much lower molecular weight), which may enhance the ability to influence physiological activities of antigen bacteria, especially the property or/and structure of membranes. These results allow us to suggest a promising potential of the IgY-Fab′ for bio-preservation of aquatic products.
13 14 15 16 17 18
ACKNOWLEDGEMENT
19
This work was supported by the Earmarked Fund for China Agriculture Research System (CARS-50) and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1188).
20 21
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