- Email: [email protected]
Authentication of origin of meat species processed under various Indian culinary procedures using DNA barcoding
Accepted Manuscript Authentication of origin of meat species processed under various Indian culinary procedures using DNA barcoding Nadeem Ahmed, Deepali Sangale, Anita Tiknaik, Bharathi Prakash, Raituja Hange, Ravindranathanpillai Sanil, Sajid Khan, Gulab Khedkar PII:
S0956-7135(18)30058-6
DOI:
10.1016/j.foodcont.2018.02.012
Reference:
JFCO 5975
To appear in:
Food Control
Received Date: 10 October 2017 Revised Date:
9 December 2017
Accepted Date: 9 February 2018
Please cite this article as: Ahmed N., Sangale D., Tiknaik A., Prakash B., Hange R., Sanil R., Khan S. & Khedkar G., Authentication of origin of meat species processed under various Indian culinary procedures using DNA barcoding, Food Control (2018), doi: 10.1016/j.foodcont.2018.02.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Authentication of origin of meat species processed under various Indian culinary procedures
2
using DNA Barcoding
3
Nadeem Ahmed*, Deepali Sangale, Anita Tiknaik, Bharathi Prakash#, Raituja Hange,
4
Ravindranathanpillai Sanil+, Sajid Khan, Gulab Khedkar◘
5 6 7 8 9 10 11 12 13 14 15 16 17 18
Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431 004, India.
19
Abstract
20
Indian food is exceptional from rest of the world not only in taste but also in culinary procedures,
21
which reflects a perfect blend of various cultures and ages. Indian culinary procedures involved in
22
preparation of Indian meat recipes, incur heavy processing and profound use of spices. In parts, the
23
authentication of processed ingredients present in the food is a major concern to ensure food safety
24
and quality as well as for certification. There is a growing demand for the enhancement of quality
25
controls, hence addressing scientific research towards the development of reliable molecular tools
26
for food traceability. Over the past decade, DNA barcoding was most commonly used molecular
27
method, which can ascertain biological specimens, and is used for the identification of both raw
28
materials and processed food. We tested the applicability of this method to authenticate variously
29
processed meat species under Indian culinary practices and revealed DNA barcoding can provide,
30
fast and reliable method for its traceability. The obtained results indicated that Indian culinary
31
practices for popular meat recipes although use considerable processing and profound spice, do not
32
interfere meat DNA quality for downstream application for species authentication using DNA
33
barcoding by COI gene. Species authenticity for geographical origin is exigent by the DNA barcoding
34
procedure. However, the pickled products are not trackable for species authentication since the
35
culinary processes involved, challenges DNA quality for further applications.
36
Keywords : DNA Barcoding, meat authentication, Indian culinary practices, Processed materials,
37
DNA degradation, PCR
RI PT
1
* Ministry of Food Processing Industries, Panchshill Marg, Siri Fort, New Delhi-110001, India. +
Wildlife forensic Laboratory, Department of Zoology, Government College, Ooty, the Nilgaris, Tamil Nadu, India.
AC C
EP
TE D
M AN U
SC
# Department of Microbiology, University College, Mangalore University, Mangalore, Karnataka, India. ◘ Corresponding Author Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431 004, India. Email: [email protected] Tel No. +91 240 2403216; Fax. +91 240 2403355
1
ACCEPTED MANUSCRIPT 38
Highlights of research •
Study conducted for traceability compliance of Indian culinary processing of meat
40
•
Culinary processing do not alter DNA quality required for traceability compliance
41
•
All samples were successful in amplifying COI gene, no evidence of PCR inhibitor
42
•
Employed samples were successful in generating full length DNA barcodes
43
•
Species authentication under pickled products failed in all preparations
RI PT
39
44 45 46
SC
47 48
M AN U
49 50 51
55 56 57 58 59 60
EP
54
AC C
53
TE D
52
61 62 63
2
ACCEPTED MANUSCRIPT 64
Authentication of origin of meat species processed under various Indian culinary procedures
65
using DNA Barcoding 1. Introduction
67
Indian food is exceptional from rest of the world not only in taste but also in culinary procedures,
68
which reflects a perfect blend of various cultures and ages. Generous use of spice is the speciality of
69
almost all types of recipes. Strong impact was made on the Indian cuisine during the Mughals era in
70
sixteenths century. Mughals’ cooking was truly based on meat, whose influence is strongest in north
71
and central India. Since then well-known Mughlai dishes have been developed into an important
72
culinary art and became part of Indian cuisine. Several recipes are derived from original Mughal
73
cooking blends and popularized throughout the world. These dishes are very popular globally through
74
Indian restaurants or are sold as ready to eat processed food in super market like, frozen, canned or
75
dehydrated food. In parts, the food prepared either in restaurants or processed in large batches in food
76
industries, food authentication is an important area in recent past (FSAI, 2013; FSSAI, 2013).
77
The consumer awareness and concern about the food they consume is considerably elevated.
78
Globally, consumers are empowered by the Court of Law to know about their food and its source. The
79
traceability is important in food authentication, which ensures the origin of food. In human
80
civilization, some societies prefer certain type of food while other food items are strictly prohibited,
81
either due to religious concern, health issues or personal preferences. Another issue associated is food
82
frauds by substituting highly valued food components with low quality ingredients of similar origin
83
(Bottero & Dalmasso, 2011; Khedkar et al., 2016). It not only defrauds the consumer but may result
84
into psychological consequences. Any case of food adulteration especially when reported by the
85
media, has a great impact on public opinion (Galimberti et al., 2013). For consumer and regulatory
86
confidence with respect to food quality and food safety along the supply chain from production,
87
processing and retailing from the point of origin to the point of sale is expected (Turci, SavoSardaro,
88
Visioli, Maestri, & Marmiroli, 2010). Therefore, the description and/or labelling of food must be
89
authentic and accurate, principally if the food has been processed removing the ability to distinguish
90
one ingredient from another.
91
Some examples of substitution of high quality materials with ones of lower value given that superior
92
produce can significantly appreciate the price difference compared with the corresponding replacing
93
ingredient. It is easy to see the commercial gains that can be made by devious food producers
94
(Ashurst & Dennis, 1996; Patel, 1994).They shall, if possible, give information on animal species,
95
origin, authenticity, composition, age and production systems (Woolfe and Primrose, 2005).
96
Consequently, it is necessary to have reliable methods, which allow fast and unequivocal information
97
related to these issues. The demand for reliable food traceability systems has addressed the scientific
98
research, hence generating different analytical approaches to this issue (Rasmussen & Morrisey, 2011;
AC C
EP
TE D
M AN U
SC
RI PT
66
3
ACCEPTED MANUSCRIPT 99
Bottero & Dalmasso, 2011; Fajardo, Gonzalez, Rojas, Garcia & Martin, 2010; Mafra, Ferreira & Oliveira, 2008; Asensio, Gonzalez, García, & Martin , 2008).
101 102 103 104 105 106 107 108 109 110
In recent past detection of species using DNA barcoding has become more popular and reliable due to use of mitochondrial DNA and universal primers in contrast to the use of species-specific PCR methods for detection of various mammalian and poultry species in meat and meat products (Sheikha, Farag, Mokhtar, Lamasudin, Isa & Mustafa, 2017; Zhong, Wang, Fang, Li & Yu, 2017; Hellberg, Hernandez & Hernandez, 2017; Arslan, Ilhak, & Calicioglu, 2006; Meyer, Candrian & Luethy, 1994). These PCR methods targets genomic as well as mitochondrial DNA for the purpose of meat species identification, even in cooked meat under different processing conditions. However, in the present study the mitochondrial DNA was used for meat species identification because of the maternal inheritance, normally only one allele exists in an individual and thus no sequence ambiguities are expected from the presence of multiple allele (Unseld, Beyermann, Brandt, & Hiesel, 1995).
111
Moreover, due to specific cooking methods, spicy taste, delicacy and attractive food presentation,
112
Indian meat recipes are in appreciation all over the world. In all these recipes, excessive use of oil,
113
herbs, spices and high processing temperature, may be considered as a limiting factor for obtaining
114
good quality DNA for food traceability (Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012).
115
Considering aforesaid important issues related to Indian meat cuisine and future implications in food
116
processing sector and meat trade, present study was planned. The overall objective was to test
117
applicability of DNA barcoding to authenticate meat species used in popular Indian culinary practices
118
for meat traceability compliances.
TE D
119
M AN U
SC
RI PT
100
2. Materials and methods
121
2.1 Meat sampling
122
For food traceability of various meat recipes, common and popular seven Indian culinary practices
123
were selected. Almost all types of meat items fall in these categories as far as food processing
124
methods are concern are shown in Fig. 1. All recipes were processed through professional chefs and
125
cooking temperature, various ingredients used in each recipe are mentioned in Table 1. From these
126
recipes, meat samples were randomly collected in triplicate and preserved in absolute ethanol for
127
further laboratory processing for traceability studies (Table S1). Untreated raw meat sample from
128
each species is included as a positive control.
129
2.2 DNA extraction, PCR and Sequencing
130
DNA was extracted from all meat items by taking 100mg of meat tissue using Wizard genomic DNA
131
purification kit (Promega, Madison, WI.) following manufacturer’s instructions. Purified DNA was
132
quantified and diluted uniformly to have a final concentration of 100ng/µL. Quality of DNA was
AC C
EP
120
4
ACCEPTED MANUSCRIPT checked on 1 % agarose gel. The primers and thermal cycling conditions for amplifying Cytrochrome
134
oxidase-I gene are mentioned in Table 2. PCR reaction composition for 25 µL volume containing
135
200 µM of each dNTP, 1.5 mM MgCl2, primers 1 pM each reverse and forward (Table 2), 1U Taq
136
Polymerase (Kapa Biosystems, USA) and 10 ng template DNA. Reactions were performed using
137
Veriti Thermal Cycler (Applied Biosystems, Foster City, USA).
138
All PCR products were visualized on 1.2% agarose gel using gel documentation system (Bio Rad,
139
USA) and photographed for further reference. Selected samples were sequenced bidirectional for
140
further analysis of species authentication (Sequencing carried out in-house at Paul Hebert Centre for
141
DNA barcoding and Biodiversity Studies facility on ABI 3130 genetic analyser).
142
2.3 Data analysis
143
For every meat recipe samples were collected in triplicate, thus a total no of 210 samples were tested.
144
The DNA concentration mentioned are the average concentration of samples tested in triplicates. The
145
Resultant COI sequences were rectified visually following base calling and aligned using Codon-code
146
aligner v 3.0 ( trial version of CodonCode Corporation, MA, USA). All sequences were deposited to
147
BOLD systems (www.boldsystems.org), and statistically analysed for species identification using
148
global alignment through hidden Markov model (HMM) embaded in species identification system of
149
BOLD (Eddy, 1998). Few sequences already available for meat species from various geographical
150
regions were downloaded from BOLD systems for analysis of possibility for geographical identity of
151
meat species.
152
3. Results
153
Altogether, ten meat commodities were collected under this study and processed for six traditional
154
Indian meat cuisines (Fig.1). All processed samples of meat were employed for DNA extraction,
155
where we obtained DNA in reasonable quantity for downstream laboratory applications as shown in
156
Table 3. To assess the effect of various processing methods on DNA, its quality was checked on 1 %
157
agarose gel (Fig. 2).
158
Further, all resultant DNA samples were employed for amplification of cytochrome oxidase -1 gene
159
for species authentication. We observed most of the samples were successfully amplified for
160
cytochrome oxidase -1 gene (Fig. 3). Moreover, the quality of DNA recovered from pickled samples
161
was poor, failed in PCR amplifications (Fig. 2; Table 3). The results for DNA quality and temperature
162
do not show any statistical correlation (P < 0.01).
163
Some of the randomly selected PCR products were bidirectional sequenced to check the DNA
164
sequence integrity. We could obtain full-length sequences of >500 bp on an average.
165
sequences were BLAST on NCBI (Geer et al., 2010) as well as BOLD data system to test the success
AC C
EP
TE D
M AN U
SC
RI PT
133
5
These
ACCEPTED MANUSCRIPT in species identification, where all our samples obtained similarity score in the range of 98-100%
167
(Table 4) (BLAST search conducted on September 1st, 2017).
168
4. Discussions
169
Over the time, health awareness has increased to several fold and consumers are curious to know the
170
accurate information about the food they consume. In order to keep them informed, regarding their
171
diet and the nature of the food they purchase, food traceability has become important issue
172
(Kang'ehte, Gathuma & Lindqvist, 1986). The reasons are numerous, but when certain individual is
173
allergic to some food commodity, for religious reason or personal preference, where fraudulent food
174
claims may deceive consumer confidence (Masiri et al., 2016, Perret, Tabin, Marcoz, Lior &
175
Cheseaux, 2011). In particular, Indian culinary procedures with heavy processing usually at high
176
temperature, limits the identity of ingredient to the consumer. Determination of the source of food and
177
food products is an analytically challenging problem and one that is currently the focus of global
178
scientific attention (Ali, Kashif, Uddin, Hashim, Mustafa, & Che Man, 2012; Novak, Grausgruber-
179
Gröger, & Lukas, 2007; Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012; Bauer, Weller, , Hammes, &
180
Hertel, 2003, Kharazmi, Bauer, Hammes, & Hertel, 2003).
181
Under this study, we evaluated the utility of DNA barcode based food authentication for Indian meat
182
cuisine for the traceability compliances. This approach is based on the analysis of the variability
183
within a standard region of the genome called “DNA barcode” (Hebert, Ratnasingham, & deWaard,
184
2003). This approach proved useful in solving taxonomic problems in several theoretical and practical
185
applications (Hollingsworth, Graham, Little, 2011; Rasmussen, Morrissey, & Hebert , 2009;
186
Valentini, Pompanon, & Taberlet, 2009; Khedkar, Jamdade, Naik, Lior, Haymer, 2014). Legitimately,
187
DNA barcoding is not completely novel, because molecular identification approaches were already in
188
use (Casiraghi, Labra, Ferri, Galimberti, & Mattia, 2010). A major challenge in the implementation of
189
these procedures is the development of suitable quantification methods for processed foods where
190
DNA is absent or substantially damaged and therefore difficult to detect and quantify (Ballari and
191
Martin, 2013). Understanding the effects of culinary processing on DNA was necessary for the
192
development/assessment of reliable methods for species authentication. This mandates rigorous
193
investigations on DNA fragmentation within processed food matrices. Our result of preprocessed
194
meat commodity and variously processed Indian meat cuisine demonstrates no substantial change in
195
DNA content (Table 3) and quality (Fig. 2). These results are in confirmation with the variously
196
reported studies (Nguyen-Hieu, Aboudharam, Drancourt, 2012; Bergerova, Hrncirova, Stankovska,
197
Lopasovska, & Siekel, 2010; Debode, Janssen & Berben, 2007; Samson, Gulli, & Marmiroli, 2010).
198
Contrast to the findings of this study, Ballari and Martin (2013) reported substantial DNA degradation
199
where DNA purified from transgenic maize and DNA amplicons were directly exposed to 100 0C.
200
Also, study on meat processing by Sakalar et al. (2012) reveals that the DNA fragment size was
AC C
EP
TE D
M AN U
SC
RI PT
166
6
ACCEPTED MANUSCRIPT progressively altered (degraded) into smaller fragments with increased duration of heating and
202
temperature. Our study indicates, natural DNA packing either in nucleus or in mitochondria, protects
203
DNA damaging from various agents like chemicals or temperature. In parts, our study demonstrated
204
that in some culinary processes like, roasting temperature reaches 1900C, still DNA quality was good
205
enough for downstream applications. Moreover, DNA quality and quantity from pickled products was
206
poor and can be correlated to high oil content in these products (~30%) which might be interfering
207
DNA extraction efficiency but use of vinegar might degrading DNA (Costa, Amaral, Fernandes,
208
Batista, Oliveira & Mafra, 2015; Ponzoni, Mastromauro, Gianì, & Breviario, 2009). In addition, under
209
pickling procedures, pH value is maintained in the range of 3- 4.0 when pickle is stable (Shukla and
210
Srivastava, 1999, Siriskar, Khedkar, & David, 2013), normally it
211
degradation, whereas, further lowered pH may degrade DNA rapidly. During pickling process, vast
212
variations in pH values were reported (Siriskar, Khedkar, & David, 2013).
213
For further analysis of recovered DNA for downstream success of PCR amplification, it was assumed
214
that several metabolites present in spice may inhibit PCR (Low and Shaw, 2018; Sahu, Thangaraj &
215
Kathiresan, 2012). As, all samples were successful with expected amplicon size, except all pickled
216
products where only primer dimers can be seen (Fig. 3), the cooking temperature and or additives
217
used in culinary procedures indicates that DNA extraction protocol is appropriate for meat traceability
218
compliances (Rastogi et al., 2004). In addition, study indicates that ingredients used under Indian
219
meat cuisine can be tracked for traceability compliances as they do not inhibit PCR for molecular
220
profiling (Schrader, Schielke, Ellerbroek & Johne, 2012; Moreira & Oliveira, 2011; Opel, Chung, &
221
McCord, 2010). In case of pickle samples, PCR amplification was not successful (Fig. 3), it implies
222
that, although DNA remains stable under high pH but extreme low pH (∼pH 3) may cause
223
depurination resulting nicks on DNA strand, leading to PCR failure (Kharazmi, Bauer, Hammes, &
224
Hertel, 2003; Bauer, 2003).
225
The results of randomly selected samples for DNA barcode to recover an average sequence length
226
over 500 base pairs (Table 4) were sufficient against the suggested sequence length of 300 bases by
227
Hird et al., (2006). Our findings are in contrast to the variously reported studies on correlation of
228
temperature and fragment length (Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012; Aslan, Hamill,
229
Sweeney, Reardon & Mullen, 2009). As stated earlier, DNA quality and quantity was not influenced
230
by the Indian meat culinary procedures and DNA remains well protected inside the protecting cover
231
of mitochondria (Foran, 2006). Advantages of using mitochondrial DNA for species authentication
232
were widely reported, as it protects DNA damage at high temperature cooking, as well as multiple
233
copies of mitochondria ensures DNA quantity and quality which was conceptually studied by Foran
234
(2006). There by indicating that the DNA barcoding method works well for the samples those are
235
obtained from various culinary practices used in Indian meat cuisine for traceability compliances.
RI PT
201
AC C
EP
TE D
M AN U
SC
has limited effect on DNA
7
ACCEPTED MANUSCRIPT Besides species authentication, the proportion of a species in the product is also important for quality
237
control, which is the limitation of DNA barcoding as traditional Sanger DNA sequencing generates a
238
sequence representing the dominant PCR product. Capturing less copy numbers template is a low
239
degree probability and multiple sequencing for same amplicon imparts heavy costs and excess time.
240
Minor products with certain degrees of mismatches to primers are missed and often not effectively
241
amplified (Sipos, Szekely, Palatinszky, Revesz, Marialigeti, & Nikolausz, 2007). Also, from BLAST
242
results, species can be identified easily (Table 4), but the geographical identity was not possible, as
243
within species differences are considered to be in the range < 3 based on evaluation of nearest related
244
species (Hebert, Cywinska, Ball, DeWaard, 2003). This within species genetic difference range is too
245
narrow to capture geographical variations and species boundary (Khedkar, Jamdade, Naik, David,
246
Haymer, 2014).
247
Thus, the study indicates that DNA being a stable molecule does not get damaged due to the
248
processing in most popular Indian culinary practices. DNA amplification was successful except
249
pickled products, reveals absence of PCR inhibitors in Indian culinary processes in spite of use of
250
ample spice. Amplified PCR products can be directly used for sequencing and for species
251
identification. However, due to narrow range COI sequence variations (< 3 %) geographical species
252
identity is in challenge. Moreover, our findings underline the recovery of poor quality DNA from
253
finished pickled meat products, which were not sufficient for PCR amplification and sequencing.
254
Additional studies are being carried out to simplify the process which can be applicable for
255
traceability compliances as well as to quantify amount of species in the Indian meat culinary product.
256
5. Conclusion
257
Certifying the genuinely of food materials used is important to ensure consumers confidence.
258
Authentication is pivotal for legal authorities to detect the ingredients in food products. The obtained
259
results indicated that Indian culinary practices for popular meat recipes although use considerable
260
processing and profound spice, do not interfere meat DNA quality for downstream application for
261
species authentication using DNA barcoding by COI gene. Species authenticity for geographical
262
origin is exigent by the DNA barcoding procedure. However, the pickled products are not trackable
263
for species authentication since the culinary processes involved, challenges DNA quality for further
264
applications.
265
Acknowledgements
266
We would like to thank RUSA, Maharashtra for financial support under R & I project to
267
Gulab Khedkar. We are thankful to Prof. Pratima Pawar, Shivaji University Kolhapur, India
268
for collecting pickle samples for this study. We would also like to express our gratitude to
269
Mrs. Manisha Varma and Prof. Vilas Gaikar, State Directorate, RUSA, Mumbai for their
AC C
EP
TE D
M AN U
SC
RI PT
236
8
ACCEPTED MANUSCRIPT 270
valuable suggestions in making this study inclusive. Also authors are grateful to staff and
271
faculty at Paul Hebert Centre for DNA Barcoding and Biodiversity Studies for their help in
272
completing this work.
273
Funding: This work was supported by the RUSA, Maharashtra under R & I project grant no.
275
RUSA/Release Order/R & I /2016-17/258 (337) Date: 26/07/2016. However, funders do not
276
have any role in study design; data collection; analysis and interpretation of data; in the
277
writing of the report; and in the decision to submit the article for publication.
RI PT
274
278
SC
279 References
281
Ali, M. E., Kashif, M., Uddin, K., Hashim, U., Mustafa, S., & Che Man, Y. B. (2012).
282
Species authentication methods in foods and feeds: the present, past, and future of Halal
283
forensics. Food Analytical Methods, 5(5), 935–955.
284 285 286
Aly Farag El Sheikha, Nur Fadhilah Khairil Mokhtar, Ceesay Amie, Dhilia Udie Lamasudin, Nurulfiza Mat Isa & Shuhaimi Mustafa (2017) Authentication technologies using DNA-based approaches for meats and halal meats determination, Food Biotechnology, 31(4): 281-315.
287
Arslan, A., Ilhak, I. O., & Calicioglu, M. (2006). Effect of method of cooking on
288
identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat
289
Science, 72, 326e330.
290
Asensio, L., González, I., García, T., & Martín R. (2008). Determination of food authenticity
291
by enzyme-linked immunosorbent assay (ELISA). Food Control, 19, 1-8.
292
Ashurt, P. R., Dennis, M. J.(1996). Food authentication. Blackie, London.
293
Aslan, O., R. M. Hamill, T. Sweeney, W. Reardon, & A.M. Mullen (2009). Integrity of
294
nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability.
295
Journal of Animal Science, 87 (1), 57-61.
296
Ballari , Rajashekhar V., & Asha Martin (2013). Assessment of DNA degradation induced by
297
thermal and UV radiation processing: Implications for quantification of genetically modified
298
organisms. Food Chemistry, 141(3), 2130-2136.
AC C
EP
TE D
M AN U
280
9
ACCEPTED MANUSCRIPT Bauer, T., P. Weller, W.P. Hammes, & C. Hertel (2003). The effect of processing parameters
300
on DNA degradation in food. European Food Research and Technology, 217 (4), 338-343.
301
Bergerova, E., Z. Hrncirova, M. Stankovska, M. Lopasovska, & P. Siekel (2010). Effect of
302
thermal treatment on the amplification and quantification of transgenic and non-transgenic
303
soybean and maize DNA. Food Analytical Methods, 3, 211-218
304
Bottero, M. T., A. Dalmasso (2011). Animal species identification in food products:
305
Evolution of biomolecular methods. Veterinary Journal, 190, 34-38.
306
Casiraghi, M., M. Labra, E. Ferri, A. Galimberti, & F. De Mattia (2010). DNA barcoding: A
307
six-question tour to improve users' awareness about the method. Briefings in Bioinformatics,
308
11, 440-453.
309
Costa, J., J.S. Amaral, T.J. Fernandes, A. Batista, M.B. Oliveira, & I. Mafra (2015). DNA
310
extraction from plant food supplements: Influence of different pharmaceutical excipients.
311
Molecular and Cellular Probes, 29 (6), 473-478.
312
Debode, F., E. Janssen, & G. Berben (2007). Physical degradation of genomic DNA of
313
soybean flours does not impair relative quantification of its transgenic content. European
314
Food Research and Technology, 226, 273-280.
315
Eddy, S. R. (1998) Profile hidden Markov models. Bioinformatics, 14: 755-763.
316
Fajardo, V., Gonzàlez, I., Rojas, M., Garcìa, T., & Martìn, R. (2010). A review of current
317
PCR-based methodologies for the authentication of meats from game animal species. Trends
318
in Food Science and Technology, 21: 408-421.
319
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994). DNA primers
320
foramplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan
321
invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.
322
Foran, D. R. (2006). Relative Degradation of Nuclear and Mitochondrial DNA: An
323
Experimental Approach. Journal of Forensic Sciences, 51, 766–770. doi:10.1111/j.1556-
324
4029.2006.00176.x
325
FSAI (The Food Safety Authority of Ireland) (2013). Survey Finds Horse DNA in Some Beef
326
Burger Products. Retrived from
AC C
EP
TE D
M AN U
SC
RI PT
299
10
ACCEPTED MANUSCRIPT https://www.fsai.ie/news_centre/press_releases/horseDNA15012013.html Accessed 12 June
328
2017.
329
FSSAI (Food Safety and Standards Authority of India) (2013). Retrived from
330
http://www.fssai.gov.in Accessed 12 June 2017
331
Galimberti, A., Mattia, F. D., Losa, A., Bruni, I., Federici, S., Casiraghi, M., Martellos, S.,
332
Labra, M. (2013). DNA barcoding as a new tool for food traceability. Food Research
333
International, 50 (1), 55-63
334
Geer, L.Y., Marchler-Bauer, A., Geer, R.C., Han, L., He, J., He, S., Liu, C., Shi, W., &
335
Bryant, S.H. (2010). The NCBI BioSystems database. Nucleic Acids Research, 38(Database
336
issue), 492-496.
337
Hebert, P.D.N., Stoeckle, M.Y.,Zemlak, T.S., & Francis, C.M. (2004). Identification of birds
338
through DNA Barcodes. PLoS Biology, 2, 1657-1663.
339
Hebert P.D.N., Cywinska A., Ball S.L., DeWaard J.R. (2003). Biological identifications
340
through DNA barcodes. Proceedings of the Royal Society B: Biological Science, 270, 313–
341
321.
342
Hebert, P.D.N., S. Ratnasingham, & J.R. deWaard (2003). Barcoding animal life:
343
Cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of
344
the Royal Society B: Biological Science, 270, S596–S599.
345
Hird, H., Chisholm, J., Sanchez, A., Hernandez, M., Goodier, R., Schneede, K., Boltz, C., &
346
Popping, B. (2006). Effect of heat and pressure processing on DNA fragmentation and
347
implications for the detection of meat using a real-time polymerase chain reaction. Food
348
Additives and Contaminants, 23(7), 645–650.
349
Hird, H., J. Chisholm, & J. Brown (2005). The detection of commercial duck species in food
350
using a single probe-multiple species-specific primer real-time PCR assay. European Food
351
Research and Technology, 221 (3), 559-563.
352
Hollingsworth, P.M., Graham S.W., & D.P. Little (2011). Choosing and using a plant DNA
353
barcode. PLoS One, 6, e19254.
354
Ivanova, N.V., Zemlak, T.S., Hanner, R.H., Hebert, P.D.N. (2007). Universal primer
355
cocktails for fish DNA barcoding. Molecular Ecology Notes , 7, 544–548.
AC C
EP
TE D
M AN U
SC
RI PT
327
11
ACCEPTED MANUSCRIPT Kang'ehte, E. K., J.M. Gathuma & K. J. Lindqvist (1986). Identification of species of origin
357
of fresh, cooked and canned meat and meat products using antisera to thermostable muscle
358
antigens by Ouchterlony`s double diffusion test. Journal of the Science of Food and
359
Agriculture, 37, 157-164.
360
Kharazmi, M., T. Bauer, W.P. Hammes, & C. Hertel (2003). Effect of food processing on the
361
fate of DNA with regard to degradation and transformation capability in Bacillus subtilis.
362
Systematic and Applied Microbiology, 26 (4), 495-501.
363
Khedkar G.D., Jamdade Rahul, Suresh Naik, David Lior, Haymer David (2014). DNA
364
barcodes for the fishes of the Narmada, one of India’s largest rivers. PLosOne, 9 (7), e101460
365
Khedkar G.D., Tiknaik A.D., Shinde R.N., Kalyankar A.D., Ron T.N., & Haymer D. (2016).
366
High rates of substitution of the native catfish Clarias batrachus by Clarias gariepinus in
367
India, Mitochondrial DNA, 27, 569-574.
368
Mafra, I., Ferreira, I., & Oliveira, M. (2008). Food authentication by PCR-based methods.
369
European Food Research and Technology. A, 227(3), 649−665.
370
Masiri, J., L. Benoit, B. Barrios-Lopez, C. Thienes, M. Meshgi, A. Agapov, et al.(2016).
371
Development and validation of a rapid test system for detection of pork meat and collagen
372
residues. Meat Science, 121, 397-402.
373
Meyer, R., Candrian, U., & Luethy, J. (1994). Detection of pork in heated meat products by
374
the polymerase chain reaction. Journal of AOAC International, 77,617e622.
375
Moreira, P.A., & D.A. Oliveira (2011). Leaf age affects the quality of DNA extracted from
376
Dimorphandramollis (Fabaceae), a tropical tree species from the Cerrado region of Brazil.
377
Genetics and Molecular Research, 10 (1), 353-358.
378
Nguyen-Hieu, T., Aboudharam, G., Drancourt, M. (2012). Heat degradation of eukaryotic
379
and bacterial DNA: an experimental model for paleomicrobiology. BMC Research Notes 5,
380
528
381
Novak J., S. Grausgruber-Gröger, & B. Lukas (2007). DNA-based authentication of plant
382
extracts. Food Research International, 40 (3), 388-392.
383
Opel, K. L., D. Chung, B.R., & McCord, B.R. (2010). A study of PCR inhibition mechanisms
384
using real time PCR. Journal of Forensic Sciences, 55 (1), 25-33.
AC C
EP
TE D
M AN U
SC
RI PT
356
12
ACCEPTED MANUSCRIPT Patel, N. P. (1994). The use of DNA fingerprinting in food analysis. Food Technology
386
International Europe, 171-174.
387
Perret, C., R. Tabin, J. P. Marcoz, J. Llor, & J. J. Cheseaux (2011). Apparent life-
388
threatening event in infants: Think about star anise intoxication! Archives de Pédiatrie, 18
389
(7), 750-753.
390
Ponzoni, E., F. Mastromauro, S. Gianì, & D. Breviario (2009). Traceability of plant diet
391
contents in raw cow milk samples. Nutrients, 1 (2), 251-262.
392
Rasmussen, R.S., M.T. Morrissey, & P.D.N. Hebert (2009). DNA barcoding of commercially
393
important salmon and trout species Oncorhynchus and Salmo) from North America. Journal
394
of Agricultural and Food Chemistry, 57, 8379-8385.
395
Rasmussen, R. S., & M. T. Morrisey (2008). DNA-based methods for the identification of
396
commercial fish and seafood species. Comprehensive Reviews in Food Science and Food
397
Safety, 7, 280-295.
398
Rastogi, G., M. Dharne, A. Bharde, V.S. Pandav, S.V. Ghumatkar, R. Krishnamurthy, et
399
al.(2004). Species determination and authentication of meat samples by mitochondrial 12S
400
rRNA gene sequence analysis and conformation-sensitive gel electrophoresis. Current
401
Science, 87 (9), 1278-1281.
402
Ratnasingham, S., & P.D.N. Hebert (2007). BOLD: the barcode of life datasystem
403
(www.barcodinglife.org). Molecular Ecology Notes, 7, 355-364.
404
Rosalee S. Hellberg, Brenda C.Hernandez, Eduardo L. Hernandez (2017) Identification of
405 406
meat and poultry species in food products using DNA barcoding, Food Control, 80: 23-28.
407
Sahu, S. K., M. Thangaraj, & K. Kathiresan (2012). DNA extraction protocol for plants with
408
high levels of secondary metabolites and polysaccharides without using liquid nitrogen and
409
phenol. ISRN Molecular Biology, 2012, 6pages.
410
Sakalar, E., M.F. Abasiyanik, E. Bektik, A. Tayyrov (2012). Effect of heat processing on
411
DNA quantification of meat species. Journal of Food Science, 77 (9), 40-44.
AC C
EP
TE D
M AN U
SC
RI PT
385
13
ACCEPTED MANUSCRIPT Samson, M. C., M. Gulli, & N. Marmiroli (2010). Quantitative detection method for
413
Roundup Ready (R) soybean in food using duplex real-time PCR MGB chemistry. Journal of
414
the Science of Food and Agriculture, 90, 1437-1444.
415
Schrader, C., A. Schielke, L. Ellerbroek, & R. Johne (2012). PCR inhibitors – Occurrence,
416
properties and removal. Journal of Applied Microbiology, 113 (5), 1014-1026.
417
Shiriskar D.A. , Khedkar, G.D., & Sudhakara, N.S. (2010). Preparation of pickled products
418
from anchovies (Stolephorus sp.) and studies on quality changes during storage. Journal of
419
Food Processing Preservation, 34, 176–190.
420
Shukla, P. K., & Srivastava, R. K. (1999). Storage stability of poultry pickle stored at room
421
temperature. Indian Journal of Poultry Science, 34, 285-288.
422
Sipos, R., A.J. Szekely, M. Palatinszky, S. Revesz, K. Marialigeti, & M. Nikolausz (2007).
423
Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA
424
gene-targetting bacterial community analysis. FEMS Microbiology Ecology, 60 (2), 341-350.
425
Siriskar, D. A., Khedkar, G. D., & Lior, D. (2013). Production of salted and pressed
426
anchovies (stolephorus sp.) and it’s quality evaluation during storage. Journal of Food
427
Science and Technology, 50(6), 1172–1178.
428
Turci, M., SavoSardaro, M. L., Visioli, G., Maestri, E., Marmiroli, N. (2010). Evaluation of
429
DNA extraction proceudres for traceability of various tomato products. Food control 21, 143-
430
149.
431
Unseld, M., Beyermann, B., Brandt, P., & Hiesel, R. (1995). Identification of the species
432
origin of highly processed meat products by mitochondrial DNA sequences. PCR Methods
433
and Application, 4, 241–243.
434
Valentini, A., Pompanon, F., & Taberlet, P., (2009). DNA barcoding for ecologists. Trends
435
Ecology and Evolution, 24, 110–117.
436
Vijayakumar, K. R., A. Martin, L.R. Gowda, & V. Prakash (2009). Detection of genetically
437
modified soya and maize: Impact of heat processing. Food Chemistry, 117, 514-521
438 439
Woolfe, M., S. Primrose (2008). Food forensics: using DNA technology to combat misdescription and fraud. Trends in Biotechnology, 22 (5), 222-226.
AC C
EP
TE D
M AN U
SC
RI PT
412
14
ACCEPTED MANUSCRIPT Yat-Tung Lo, & Pang-Chui Shaw (2018). DNA-based techniques for authentication of processed food and food supplements. Food Chemistry, 240, 767-774.
442 443
Zheng Zhang, Scott Schwartz, Lukas Wagner & Webb Miller (2000) A greedy algorithm for aligning DNA sequences, Journal of Computational Biology, 7(1-2):203-14.
444
Zhong, WenTao; Wang, FangMei; Li, BaiYu; Jiang, Wei; Yan, HengMei (2017) Research on the non-directional test in meat adulteration based on DNA barcode, Journal of Food Safety and Quality, 8(5):1547-1551.
445 446 447
RI PT
440 441
Figure captions
449
Figure 1.Flow chart of experimental set up for testing genetic traceability of Indian meat recipes
450 451
Figure 2. Qualitative analysis of the DNA obtained from pre-processed and post processed meat under various India recipes (electrophoretically resolved on 1.5 % Agarose gel)
452 453
Figure 3. Success of COI gene amplification for downstream species authentication using DNA Barcoding (electrophoretically resolved on 1.2 % Agarose gel)
M AN U
SC
448
454 455
457
TE D
456
Table 1. Various recipes, cooking conditions and ingredients used Meat Processing type
Raw meat Cooking/boiling (soups, biryani, Pulao)
Temperature (0C) Ambient/ freeze 980C
EP
Sr. no. 1 2
Curries
4
Deep fry 160-180 0C (Kebabs, Kentucky) Microwave cooking 70-75 0C (Grilling, biryani, cooking)
5
102 0C
AC C
3
6
458
Roasting (Tandoor, 120-170 0C barbeque/direct roasting on fire, etc.) 7 Pickling Ambient (fish pickle, prawn temperature 24pickle, chevon pickle, 35 0C beef pickle) * Composition of ingredients may vary as per recipe 15
Processing duration Ingredients* (in minutes) -Raw meat 30-35 Herbs, spices, oil, common salt, water and rice in case of biryani or pulao, meat 30-40 Herbs, spices, meat, oil, common salt and water 7-12 Oil, herbs, spices, meat and common salt 10-30 Herbs, spices, oil, meat, common salt, water and rice in case of biryani or pulao 10-20 Oil, herbs, spices, meat and common salt No specific duration, Herbs, spices, oil, meat, but it may fall from common salt and vinegar one week to a year’s duration.
ACCEPTED MANUSCRIPT
Table 2. PCR primers used and thermal conditions for amplification of COI gene Sequence
Initial Denaturation
LepF1_t1 VF1_t1 VF1d_t1 VF1i_t1
TGTAAAACGACGGCCAGTATTCAACCAATCATAAAGATATTGG TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATYGG TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGAIATIGG
C_VF1LFt1 (1:1:1:3) (Ivanova et al., 2006)
C_VF1LFt1 (1:1:1:3) (Ivanova et al., 2006)
Fish
BirdR1 VF2_t1 FishF2_t1 FishR2_t1 FR1d_t1
Prawn
LCO1490 HCO2198
Annealing 35 Cycles
Extension
Final Extension
94°C for 30 Sec
50°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
94°C for 30 Sec
51°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
94°C for 30 Sec
49°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
M AN U
(Chicken, duck, deshi hen)
BirdF1
BirdF1 (Hebert, et al., 2004) TTCTCCAACCACAAAGACATTGGCAC BirdR1 (Hebert, et al., 2004) ACGTGGGAGATAATTCCAAATCCTG
C_FishF1t1 (1:1) (Ivanova et al., 2006) TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC C_FishR1t1 (1:1) (Ivanova et al., 2006) CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA LCO1490 (Folmer et al., 1994) GGTCAACAAATCATAAAGATATTGG HCO2198 (Folmer et al., 1994) TAAACTTCAGGGTGACCAAAAAATCA
TE D
Poultry
CAGGAAACAGCTATGACTAAACTTCTGGATGTCCAAAAAATCA CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA
EP
LepR1_t1 VR1d_t1 VR1_t1 VR1i_t1
AC C
Lamb, Goat, Pork
16
Denaturation
RI PT
PCR primers used
SC
Meat type
94°C for 2 min
94°C for 30 Sec
49°C for 40 sec
72°C for 1 min
72°C for 10 min
RI PT
ACCEPTED MANUSCRIPT
Table 3. DNA obtained from meat samples processed under various culinary practices
Lamb Goat Cow Buffalo Deshi hen Broiler Duck Pork Fish Prawn
920.35 2102.28 1230.91 1136.07 2199.11 2139.20 891.12 1870.30 333.7 79.17
Deep fry (Kababs, Kentucky
418.12 657.21 875.61 997.82 1613.20 2142.48 2150.96 997.69 578.84 1869.84
EP
Pork Sea food
1625.07 1968.99 1787.95 1340.20 2027.62 1714.46 1055.59 1529.31 559.57 1113.12
Curries
AC C
Chicken
Cooking/ boiling (soups, biryani, Pulao)
SC
Chevon Chevon Beef Cara- Beef
Average DNA concentration (ng/µl) obtained from 50 mg tissue of processed meat
Raw meat
1344.72 780.84 803.07 917.88 654.01 676.12 1915.01 923.07 253.69 59.35
M AN U
Meat type
TE D
Meet Commodity
17
Microwave cooking (Grilling, bryani) 1508.3 1337.49 1640.57 1024.21 1053.58 1564.25 2076.76 100.20 511.23 196.60
Roasting (barbeque/ direct roasting on fire, etc.)
Pickling
391.40 291.21 276.05 447.55 1444.28 949.06 1870.77 1928.29 219.35 93.54
** 264.10 ** 280.30 ** ** ** ** 200.00 121.40
ACCEPTED MANUSCRIPT
Roasting Deep frying
Microwave grilling Pickling Species Authentication Ovis aries Species identified Representative NCBI Accession nos.
KF302440.1 MF004244.1
SC
Curries
M AN U
Cooking/Boiling
93.5 (0.00) 100 (0.00) 100 (0.00) 100 (0.00) 100 (0.00) 100 (0.00)
TE D
Raw/control
Capra hircus
Bos taurus, Bos indicus
Bubalus bubalis
Sus scrofa
Gallus gallus
KX845672.1 KX845672.1
KX845677.1 EU177861.1
KX758295.1 KT827230.1
KX982660.1
MF102289.1 MF541544.1
EP
Lamb
Sequence matched % with NCBI gene bank records (E values) Chevon Beef Cara-beef Pork Broiler Deshi Duck Fish Chicken Chicken 100 100 99.82 99.51 99.83 100 100 100 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 97.4 100 99.82 99.7 100 99.64 -100 (1.05E-126) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.4 99.64 99.82 100 99.65 99.18 -99.23 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.5 99.22 99.82 99.67 100 100 100 99.64 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.8 99.36 99.82 -100 99.05 -99.52 (0.00) (0.00) (0.00) (0.00) (0.00) (1.09E-101) 99.6 99.34 100 100 98.93 99.34 -99.82 (4.73E-150) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) No sequences were obtained due to poor quality DNA templet recovery from these products
AC C
Type of recipe
RI PT
Table 4: DNA Barcode based species identified using COI gene sequences using similarity score
18
Gallus gallus MF102289.1 MF541544.1
Prawn 99.14 (0.00) -97.64 (4.8E-140) 96.82 (0.00) 96.38 (0.00) 98.182 (4.67E-160)
Anas platyrhyncho s
Pangasianodon hypophthalmus
Fenneropenaes merguiensis, Metapenaeus
MF069251.1
KX685193.1 EF609427.1
KJ879289.1 KC409384.1 KX399431.1 KP637170.1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights of research
Study conducted for traceability compliance of Indian culinary processing of meat
•
Culinary processing do not alter DNA quality required for traceability compliance
•
All samples were successful in amplifying COI gene, no evidence of PCR inhibitor
•
Employed samples were successful in generating full length DNA barcodes
•
Species authentication under pickled products failed in all preparations
AC C
EP
TE D
M AN U
SC
RI PT
•
S0956-7135(18)30058-6
DOI:
10.1016/j.foodcont.2018.02.012
Reference:
JFCO 5975
To appear in:
Food Control
Received Date: 10 October 2017 Revised Date:
9 December 2017
Accepted Date: 9 February 2018
Please cite this article as: Ahmed N., Sangale D., Tiknaik A., Prakash B., Hange R., Sanil R., Khan S. & Khedkar G., Authentication of origin of meat species processed under various Indian culinary procedures using DNA barcoding, Food Control (2018), doi: 10.1016/j.foodcont.2018.02.012. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Authentication of origin of meat species processed under various Indian culinary procedures
2
using DNA Barcoding
3
Nadeem Ahmed*, Deepali Sangale, Anita Tiknaik, Bharathi Prakash#, Raituja Hange,
4
Ravindranathanpillai Sanil+, Sajid Khan, Gulab Khedkar◘
5 6 7 8 9 10 11 12 13 14 15 16 17 18
Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431 004, India.
19
Abstract
20
Indian food is exceptional from rest of the world not only in taste but also in culinary procedures,
21
which reflects a perfect blend of various cultures and ages. Indian culinary procedures involved in
22
preparation of Indian meat recipes, incur heavy processing and profound use of spices. In parts, the
23
authentication of processed ingredients present in the food is a major concern to ensure food safety
24
and quality as well as for certification. There is a growing demand for the enhancement of quality
25
controls, hence addressing scientific research towards the development of reliable molecular tools
26
for food traceability. Over the past decade, DNA barcoding was most commonly used molecular
27
method, which can ascertain biological specimens, and is used for the identification of both raw
28
materials and processed food. We tested the applicability of this method to authenticate variously
29
processed meat species under Indian culinary practices and revealed DNA barcoding can provide,
30
fast and reliable method for its traceability. The obtained results indicated that Indian culinary
31
practices for popular meat recipes although use considerable processing and profound spice, do not
32
interfere meat DNA quality for downstream application for species authentication using DNA
33
barcoding by COI gene. Species authenticity for geographical origin is exigent by the DNA barcoding
34
procedure. However, the pickled products are not trackable for species authentication since the
35
culinary processes involved, challenges DNA quality for further applications.
36
Keywords : DNA Barcoding, meat authentication, Indian culinary practices, Processed materials,
37
DNA degradation, PCR
RI PT
1
* Ministry of Food Processing Industries, Panchshill Marg, Siri Fort, New Delhi-110001, India. +
Wildlife forensic Laboratory, Department of Zoology, Government College, Ooty, the Nilgaris, Tamil Nadu, India.
AC C
EP
TE D
M AN U
SC
# Department of Microbiology, University College, Mangalore University, Mangalore, Karnataka, India. ◘ Corresponding Author Paul Hebert Centre for DNA Barcoding and Biodiversity Studies, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad-431 004, India. Email: [email protected] Tel No. +91 240 2403216; Fax. +91 240 2403355
1
ACCEPTED MANUSCRIPT 38
Highlights of research •
Study conducted for traceability compliance of Indian culinary processing of meat
40
•
Culinary processing do not alter DNA quality required for traceability compliance
41
•
All samples were successful in amplifying COI gene, no evidence of PCR inhibitor
42
•
Employed samples were successful in generating full length DNA barcodes
43
•
Species authentication under pickled products failed in all preparations
RI PT
39
44 45 46
SC
47 48
M AN U
49 50 51
55 56 57 58 59 60
EP
54
AC C
53
TE D
52
61 62 63
2
ACCEPTED MANUSCRIPT 64
Authentication of origin of meat species processed under various Indian culinary procedures
65
using DNA Barcoding 1. Introduction
67
Indian food is exceptional from rest of the world not only in taste but also in culinary procedures,
68
which reflects a perfect blend of various cultures and ages. Generous use of spice is the speciality of
69
almost all types of recipes. Strong impact was made on the Indian cuisine during the Mughals era in
70
sixteenths century. Mughals’ cooking was truly based on meat, whose influence is strongest in north
71
and central India. Since then well-known Mughlai dishes have been developed into an important
72
culinary art and became part of Indian cuisine. Several recipes are derived from original Mughal
73
cooking blends and popularized throughout the world. These dishes are very popular globally through
74
Indian restaurants or are sold as ready to eat processed food in super market like, frozen, canned or
75
dehydrated food. In parts, the food prepared either in restaurants or processed in large batches in food
76
industries, food authentication is an important area in recent past (FSAI, 2013; FSSAI, 2013).
77
The consumer awareness and concern about the food they consume is considerably elevated.
78
Globally, consumers are empowered by the Court of Law to know about their food and its source. The
79
traceability is important in food authentication, which ensures the origin of food. In human
80
civilization, some societies prefer certain type of food while other food items are strictly prohibited,
81
either due to religious concern, health issues or personal preferences. Another issue associated is food
82
frauds by substituting highly valued food components with low quality ingredients of similar origin
83
(Bottero & Dalmasso, 2011; Khedkar et al., 2016). It not only defrauds the consumer but may result
84
into psychological consequences. Any case of food adulteration especially when reported by the
85
media, has a great impact on public opinion (Galimberti et al., 2013). For consumer and regulatory
86
confidence with respect to food quality and food safety along the supply chain from production,
87
processing and retailing from the point of origin to the point of sale is expected (Turci, SavoSardaro,
88
Visioli, Maestri, & Marmiroli, 2010). Therefore, the description and/or labelling of food must be
89
authentic and accurate, principally if the food has been processed removing the ability to distinguish
90
one ingredient from another.
91
Some examples of substitution of high quality materials with ones of lower value given that superior
92
produce can significantly appreciate the price difference compared with the corresponding replacing
93
ingredient. It is easy to see the commercial gains that can be made by devious food producers
94
(Ashurst & Dennis, 1996; Patel, 1994).They shall, if possible, give information on animal species,
95
origin, authenticity, composition, age and production systems (Woolfe and Primrose, 2005).
96
Consequently, it is necessary to have reliable methods, which allow fast and unequivocal information
97
related to these issues. The demand for reliable food traceability systems has addressed the scientific
98
research, hence generating different analytical approaches to this issue (Rasmussen & Morrisey, 2011;
AC C
EP
TE D
M AN U
SC
RI PT
66
3
ACCEPTED MANUSCRIPT 99
Bottero & Dalmasso, 2011; Fajardo, Gonzalez, Rojas, Garcia & Martin, 2010; Mafra, Ferreira & Oliveira, 2008; Asensio, Gonzalez, García, & Martin , 2008).
101 102 103 104 105 106 107 108 109 110
In recent past detection of species using DNA barcoding has become more popular and reliable due to use of mitochondrial DNA and universal primers in contrast to the use of species-specific PCR methods for detection of various mammalian and poultry species in meat and meat products (Sheikha, Farag, Mokhtar, Lamasudin, Isa & Mustafa, 2017; Zhong, Wang, Fang, Li & Yu, 2017; Hellberg, Hernandez & Hernandez, 2017; Arslan, Ilhak, & Calicioglu, 2006; Meyer, Candrian & Luethy, 1994). These PCR methods targets genomic as well as mitochondrial DNA for the purpose of meat species identification, even in cooked meat under different processing conditions. However, in the present study the mitochondrial DNA was used for meat species identification because of the maternal inheritance, normally only one allele exists in an individual and thus no sequence ambiguities are expected from the presence of multiple allele (Unseld, Beyermann, Brandt, & Hiesel, 1995).
111
Moreover, due to specific cooking methods, spicy taste, delicacy and attractive food presentation,
112
Indian meat recipes are in appreciation all over the world. In all these recipes, excessive use of oil,
113
herbs, spices and high processing temperature, may be considered as a limiting factor for obtaining
114
good quality DNA for food traceability (Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012).
115
Considering aforesaid important issues related to Indian meat cuisine and future implications in food
116
processing sector and meat trade, present study was planned. The overall objective was to test
117
applicability of DNA barcoding to authenticate meat species used in popular Indian culinary practices
118
for meat traceability compliances.
TE D
119
M AN U
SC
RI PT
100
2. Materials and methods
121
2.1 Meat sampling
122
For food traceability of various meat recipes, common and popular seven Indian culinary practices
123
were selected. Almost all types of meat items fall in these categories as far as food processing
124
methods are concern are shown in Fig. 1. All recipes were processed through professional chefs and
125
cooking temperature, various ingredients used in each recipe are mentioned in Table 1. From these
126
recipes, meat samples were randomly collected in triplicate and preserved in absolute ethanol for
127
further laboratory processing for traceability studies (Table S1). Untreated raw meat sample from
128
each species is included as a positive control.
129
2.2 DNA extraction, PCR and Sequencing
130
DNA was extracted from all meat items by taking 100mg of meat tissue using Wizard genomic DNA
131
purification kit (Promega, Madison, WI.) following manufacturer’s instructions. Purified DNA was
132
quantified and diluted uniformly to have a final concentration of 100ng/µL. Quality of DNA was
AC C
EP
120
4
ACCEPTED MANUSCRIPT checked on 1 % agarose gel. The primers and thermal cycling conditions for amplifying Cytrochrome
134
oxidase-I gene are mentioned in Table 2. PCR reaction composition for 25 µL volume containing
135
200 µM of each dNTP, 1.5 mM MgCl2, primers 1 pM each reverse and forward (Table 2), 1U Taq
136
Polymerase (Kapa Biosystems, USA) and 10 ng template DNA. Reactions were performed using
137
Veriti Thermal Cycler (Applied Biosystems, Foster City, USA).
138
All PCR products were visualized on 1.2% agarose gel using gel documentation system (Bio Rad,
139
USA) and photographed for further reference. Selected samples were sequenced bidirectional for
140
further analysis of species authentication (Sequencing carried out in-house at Paul Hebert Centre for
141
DNA barcoding and Biodiversity Studies facility on ABI 3130 genetic analyser).
142
2.3 Data analysis
143
For every meat recipe samples were collected in triplicate, thus a total no of 210 samples were tested.
144
The DNA concentration mentioned are the average concentration of samples tested in triplicates. The
145
Resultant COI sequences were rectified visually following base calling and aligned using Codon-code
146
aligner v 3.0 ( trial version of CodonCode Corporation, MA, USA). All sequences were deposited to
147
BOLD systems (www.boldsystems.org), and statistically analysed for species identification using
148
global alignment through hidden Markov model (HMM) embaded in species identification system of
149
BOLD (Eddy, 1998). Few sequences already available for meat species from various geographical
150
regions were downloaded from BOLD systems for analysis of possibility for geographical identity of
151
meat species.
152
3. Results
153
Altogether, ten meat commodities were collected under this study and processed for six traditional
154
Indian meat cuisines (Fig.1). All processed samples of meat were employed for DNA extraction,
155
where we obtained DNA in reasonable quantity for downstream laboratory applications as shown in
156
Table 3. To assess the effect of various processing methods on DNA, its quality was checked on 1 %
157
agarose gel (Fig. 2).
158
Further, all resultant DNA samples were employed for amplification of cytochrome oxidase -1 gene
159
for species authentication. We observed most of the samples were successfully amplified for
160
cytochrome oxidase -1 gene (Fig. 3). Moreover, the quality of DNA recovered from pickled samples
161
was poor, failed in PCR amplifications (Fig. 2; Table 3). The results for DNA quality and temperature
162
do not show any statistical correlation (P < 0.01).
163
Some of the randomly selected PCR products were bidirectional sequenced to check the DNA
164
sequence integrity. We could obtain full-length sequences of >500 bp on an average.
165
sequences were BLAST on NCBI (Geer et al., 2010) as well as BOLD data system to test the success
AC C
EP
TE D
M AN U
SC
RI PT
133
5
These
ACCEPTED MANUSCRIPT in species identification, where all our samples obtained similarity score in the range of 98-100%
167
(Table 4) (BLAST search conducted on September 1st, 2017).
168
4. Discussions
169
Over the time, health awareness has increased to several fold and consumers are curious to know the
170
accurate information about the food they consume. In order to keep them informed, regarding their
171
diet and the nature of the food they purchase, food traceability has become important issue
172
(Kang'ehte, Gathuma & Lindqvist, 1986). The reasons are numerous, but when certain individual is
173
allergic to some food commodity, for religious reason or personal preference, where fraudulent food
174
claims may deceive consumer confidence (Masiri et al., 2016, Perret, Tabin, Marcoz, Lior &
175
Cheseaux, 2011). In particular, Indian culinary procedures with heavy processing usually at high
176
temperature, limits the identity of ingredient to the consumer. Determination of the source of food and
177
food products is an analytically challenging problem and one that is currently the focus of global
178
scientific attention (Ali, Kashif, Uddin, Hashim, Mustafa, & Che Man, 2012; Novak, Grausgruber-
179
Gröger, & Lukas, 2007; Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012; Bauer, Weller, , Hammes, &
180
Hertel, 2003, Kharazmi, Bauer, Hammes, & Hertel, 2003).
181
Under this study, we evaluated the utility of DNA barcode based food authentication for Indian meat
182
cuisine for the traceability compliances. This approach is based on the analysis of the variability
183
within a standard region of the genome called “DNA barcode” (Hebert, Ratnasingham, & deWaard,
184
2003). This approach proved useful in solving taxonomic problems in several theoretical and practical
185
applications (Hollingsworth, Graham, Little, 2011; Rasmussen, Morrissey, & Hebert , 2009;
186
Valentini, Pompanon, & Taberlet, 2009; Khedkar, Jamdade, Naik, Lior, Haymer, 2014). Legitimately,
187
DNA barcoding is not completely novel, because molecular identification approaches were already in
188
use (Casiraghi, Labra, Ferri, Galimberti, & Mattia, 2010). A major challenge in the implementation of
189
these procedures is the development of suitable quantification methods for processed foods where
190
DNA is absent or substantially damaged and therefore difficult to detect and quantify (Ballari and
191
Martin, 2013). Understanding the effects of culinary processing on DNA was necessary for the
192
development/assessment of reliable methods for species authentication. This mandates rigorous
193
investigations on DNA fragmentation within processed food matrices. Our result of preprocessed
194
meat commodity and variously processed Indian meat cuisine demonstrates no substantial change in
195
DNA content (Table 3) and quality (Fig. 2). These results are in confirmation with the variously
196
reported studies (Nguyen-Hieu, Aboudharam, Drancourt, 2012; Bergerova, Hrncirova, Stankovska,
197
Lopasovska, & Siekel, 2010; Debode, Janssen & Berben, 2007; Samson, Gulli, & Marmiroli, 2010).
198
Contrast to the findings of this study, Ballari and Martin (2013) reported substantial DNA degradation
199
where DNA purified from transgenic maize and DNA amplicons were directly exposed to 100 0C.
200
Also, study on meat processing by Sakalar et al. (2012) reveals that the DNA fragment size was
AC C
EP
TE D
M AN U
SC
RI PT
166
6
ACCEPTED MANUSCRIPT progressively altered (degraded) into smaller fragments with increased duration of heating and
202
temperature. Our study indicates, natural DNA packing either in nucleus or in mitochondria, protects
203
DNA damaging from various agents like chemicals or temperature. In parts, our study demonstrated
204
that in some culinary processes like, roasting temperature reaches 1900C, still DNA quality was good
205
enough for downstream applications. Moreover, DNA quality and quantity from pickled products was
206
poor and can be correlated to high oil content in these products (~30%) which might be interfering
207
DNA extraction efficiency but use of vinegar might degrading DNA (Costa, Amaral, Fernandes,
208
Batista, Oliveira & Mafra, 2015; Ponzoni, Mastromauro, Gianì, & Breviario, 2009). In addition, under
209
pickling procedures, pH value is maintained in the range of 3- 4.0 when pickle is stable (Shukla and
210
Srivastava, 1999, Siriskar, Khedkar, & David, 2013), normally it
211
degradation, whereas, further lowered pH may degrade DNA rapidly. During pickling process, vast
212
variations in pH values were reported (Siriskar, Khedkar, & David, 2013).
213
For further analysis of recovered DNA for downstream success of PCR amplification, it was assumed
214
that several metabolites present in spice may inhibit PCR (Low and Shaw, 2018; Sahu, Thangaraj &
215
Kathiresan, 2012). As, all samples were successful with expected amplicon size, except all pickled
216
products where only primer dimers can be seen (Fig. 3), the cooking temperature and or additives
217
used in culinary procedures indicates that DNA extraction protocol is appropriate for meat traceability
218
compliances (Rastogi et al., 2004). In addition, study indicates that ingredients used under Indian
219
meat cuisine can be tracked for traceability compliances as they do not inhibit PCR for molecular
220
profiling (Schrader, Schielke, Ellerbroek & Johne, 2012; Moreira & Oliveira, 2011; Opel, Chung, &
221
McCord, 2010). In case of pickle samples, PCR amplification was not successful (Fig. 3), it implies
222
that, although DNA remains stable under high pH but extreme low pH (∼pH 3) may cause
223
depurination resulting nicks on DNA strand, leading to PCR failure (Kharazmi, Bauer, Hammes, &
224
Hertel, 2003; Bauer, 2003).
225
The results of randomly selected samples for DNA barcode to recover an average sequence length
226
over 500 base pairs (Table 4) were sufficient against the suggested sequence length of 300 bases by
227
Hird et al., (2006). Our findings are in contrast to the variously reported studies on correlation of
228
temperature and fragment length (Sakalar, Abasiyanik, Bektik, & Tayyrov, 2012; Aslan, Hamill,
229
Sweeney, Reardon & Mullen, 2009). As stated earlier, DNA quality and quantity was not influenced
230
by the Indian meat culinary procedures and DNA remains well protected inside the protecting cover
231
of mitochondria (Foran, 2006). Advantages of using mitochondrial DNA for species authentication
232
were widely reported, as it protects DNA damage at high temperature cooking, as well as multiple
233
copies of mitochondria ensures DNA quantity and quality which was conceptually studied by Foran
234
(2006). There by indicating that the DNA barcoding method works well for the samples those are
235
obtained from various culinary practices used in Indian meat cuisine for traceability compliances.
RI PT
201
AC C
EP
TE D
M AN U
SC
has limited effect on DNA
7
ACCEPTED MANUSCRIPT Besides species authentication, the proportion of a species in the product is also important for quality
237
control, which is the limitation of DNA barcoding as traditional Sanger DNA sequencing generates a
238
sequence representing the dominant PCR product. Capturing less copy numbers template is a low
239
degree probability and multiple sequencing for same amplicon imparts heavy costs and excess time.
240
Minor products with certain degrees of mismatches to primers are missed and often not effectively
241
amplified (Sipos, Szekely, Palatinszky, Revesz, Marialigeti, & Nikolausz, 2007). Also, from BLAST
242
results, species can be identified easily (Table 4), but the geographical identity was not possible, as
243
within species differences are considered to be in the range < 3 based on evaluation of nearest related
244
species (Hebert, Cywinska, Ball, DeWaard, 2003). This within species genetic difference range is too
245
narrow to capture geographical variations and species boundary (Khedkar, Jamdade, Naik, David,
246
Haymer, 2014).
247
Thus, the study indicates that DNA being a stable molecule does not get damaged due to the
248
processing in most popular Indian culinary practices. DNA amplification was successful except
249
pickled products, reveals absence of PCR inhibitors in Indian culinary processes in spite of use of
250
ample spice. Amplified PCR products can be directly used for sequencing and for species
251
identification. However, due to narrow range COI sequence variations (< 3 %) geographical species
252
identity is in challenge. Moreover, our findings underline the recovery of poor quality DNA from
253
finished pickled meat products, which were not sufficient for PCR amplification and sequencing.
254
Additional studies are being carried out to simplify the process which can be applicable for
255
traceability compliances as well as to quantify amount of species in the Indian meat culinary product.
256
5. Conclusion
257
Certifying the genuinely of food materials used is important to ensure consumers confidence.
258
Authentication is pivotal for legal authorities to detect the ingredients in food products. The obtained
259
results indicated that Indian culinary practices for popular meat recipes although use considerable
260
processing and profound spice, do not interfere meat DNA quality for downstream application for
261
species authentication using DNA barcoding by COI gene. Species authenticity for geographical
262
origin is exigent by the DNA barcoding procedure. However, the pickled products are not trackable
263
for species authentication since the culinary processes involved, challenges DNA quality for further
264
applications.
265
Acknowledgements
266
We would like to thank RUSA, Maharashtra for financial support under R & I project to
267
Gulab Khedkar. We are thankful to Prof. Pratima Pawar, Shivaji University Kolhapur, India
268
for collecting pickle samples for this study. We would also like to express our gratitude to
269
Mrs. Manisha Varma and Prof. Vilas Gaikar, State Directorate, RUSA, Mumbai for their
AC C
EP
TE D
M AN U
SC
RI PT
236
8
ACCEPTED MANUSCRIPT 270
valuable suggestions in making this study inclusive. Also authors are grateful to staff and
271
faculty at Paul Hebert Centre for DNA Barcoding and Biodiversity Studies for their help in
272
completing this work.
273
Funding: This work was supported by the RUSA, Maharashtra under R & I project grant no.
275
RUSA/Release Order/R & I /2016-17/258 (337) Date: 26/07/2016. However, funders do not
276
have any role in study design; data collection; analysis and interpretation of data; in the
277
writing of the report; and in the decision to submit the article for publication.
RI PT
274
278
SC
279 References
281
Ali, M. E., Kashif, M., Uddin, K., Hashim, U., Mustafa, S., & Che Man, Y. B. (2012).
282
Species authentication methods in foods and feeds: the present, past, and future of Halal
283
forensics. Food Analytical Methods, 5(5), 935–955.
284 285 286
Aly Farag El Sheikha, Nur Fadhilah Khairil Mokhtar, Ceesay Amie, Dhilia Udie Lamasudin, Nurulfiza Mat Isa & Shuhaimi Mustafa (2017) Authentication technologies using DNA-based approaches for meats and halal meats determination, Food Biotechnology, 31(4): 281-315.
287
Arslan, A., Ilhak, I. O., & Calicioglu, M. (2006). Effect of method of cooking on
288
identification of heat processed beef using polymerase chain reaction (PCR) technique. Meat
289
Science, 72, 326e330.
290
Asensio, L., González, I., García, T., & Martín R. (2008). Determination of food authenticity
291
by enzyme-linked immunosorbent assay (ELISA). Food Control, 19, 1-8.
292
Ashurt, P. R., Dennis, M. J.(1996). Food authentication. Blackie, London.
293
Aslan, O., R. M. Hamill, T. Sweeney, W. Reardon, & A.M. Mullen (2009). Integrity of
294
nuclear genomic deoxyribonucleic acid in cooked meat: Implications for food traceability.
295
Journal of Animal Science, 87 (1), 57-61.
296
Ballari , Rajashekhar V., & Asha Martin (2013). Assessment of DNA degradation induced by
297
thermal and UV radiation processing: Implications for quantification of genetically modified
298
organisms. Food Chemistry, 141(3), 2130-2136.
AC C
EP
TE D
M AN U
280
9
ACCEPTED MANUSCRIPT Bauer, T., P. Weller, W.P. Hammes, & C. Hertel (2003). The effect of processing parameters
300
on DNA degradation in food. European Food Research and Technology, 217 (4), 338-343.
301
Bergerova, E., Z. Hrncirova, M. Stankovska, M. Lopasovska, & P. Siekel (2010). Effect of
302
thermal treatment on the amplification and quantification of transgenic and non-transgenic
303
soybean and maize DNA. Food Analytical Methods, 3, 211-218
304
Bottero, M. T., A. Dalmasso (2011). Animal species identification in food products:
305
Evolution of biomolecular methods. Veterinary Journal, 190, 34-38.
306
Casiraghi, M., M. Labra, E. Ferri, A. Galimberti, & F. De Mattia (2010). DNA barcoding: A
307
six-question tour to improve users' awareness about the method. Briefings in Bioinformatics,
308
11, 440-453.
309
Costa, J., J.S. Amaral, T.J. Fernandes, A. Batista, M.B. Oliveira, & I. Mafra (2015). DNA
310
extraction from plant food supplements: Influence of different pharmaceutical excipients.
311
Molecular and Cellular Probes, 29 (6), 473-478.
312
Debode, F., E. Janssen, & G. Berben (2007). Physical degradation of genomic DNA of
313
soybean flours does not impair relative quantification of its transgenic content. European
314
Food Research and Technology, 226, 273-280.
315
Eddy, S. R. (1998) Profile hidden Markov models. Bioinformatics, 14: 755-763.
316
Fajardo, V., Gonzàlez, I., Rojas, M., Garcìa, T., & Martìn, R. (2010). A review of current
317
PCR-based methodologies for the authentication of meats from game animal species. Trends
318
in Food Science and Technology, 21: 408-421.
319
Folmer, O., Black, M., Hoeh, W., Lutz, R., Vrijenhoek, R. (1994). DNA primers
320
foramplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan
321
invertebrates. Molecular Marine Biology and Biotechnology, 3, 294–299.
322
Foran, D. R. (2006). Relative Degradation of Nuclear and Mitochondrial DNA: An
323
Experimental Approach. Journal of Forensic Sciences, 51, 766–770. doi:10.1111/j.1556-
324
4029.2006.00176.x
325
FSAI (The Food Safety Authority of Ireland) (2013). Survey Finds Horse DNA in Some Beef
326
Burger Products. Retrived from
AC C
EP
TE D
M AN U
SC
RI PT
299
10
ACCEPTED MANUSCRIPT https://www.fsai.ie/news_centre/press_releases/horseDNA15012013.html Accessed 12 June
328
2017.
329
FSSAI (Food Safety and Standards Authority of India) (2013). Retrived from
330
http://www.fssai.gov.in Accessed 12 June 2017
331
Galimberti, A., Mattia, F. D., Losa, A., Bruni, I., Federici, S., Casiraghi, M., Martellos, S.,
332
Labra, M. (2013). DNA barcoding as a new tool for food traceability. Food Research
333
International, 50 (1), 55-63
334
Geer, L.Y., Marchler-Bauer, A., Geer, R.C., Han, L., He, J., He, S., Liu, C., Shi, W., &
335
Bryant, S.H. (2010). The NCBI BioSystems database. Nucleic Acids Research, 38(Database
336
issue), 492-496.
337
Hebert, P.D.N., Stoeckle, M.Y.,Zemlak, T.S., & Francis, C.M. (2004). Identification of birds
338
through DNA Barcodes. PLoS Biology, 2, 1657-1663.
339
Hebert P.D.N., Cywinska A., Ball S.L., DeWaard J.R. (2003). Biological identifications
340
through DNA barcodes. Proceedings of the Royal Society B: Biological Science, 270, 313–
341
321.
342
Hebert, P.D.N., S. Ratnasingham, & J.R. deWaard (2003). Barcoding animal life:
343
Cytochrome c oxidase subunit 1 divergences among closely related species. Proceedings of
344
the Royal Society B: Biological Science, 270, S596–S599.
345
Hird, H., Chisholm, J., Sanchez, A., Hernandez, M., Goodier, R., Schneede, K., Boltz, C., &
346
Popping, B. (2006). Effect of heat and pressure processing on DNA fragmentation and
347
implications for the detection of meat using a real-time polymerase chain reaction. Food
348
Additives and Contaminants, 23(7), 645–650.
349
Hird, H., J. Chisholm, & J. Brown (2005). The detection of commercial duck species in food
350
using a single probe-multiple species-specific primer real-time PCR assay. European Food
351
Research and Technology, 221 (3), 559-563.
352
Hollingsworth, P.M., Graham S.W., & D.P. Little (2011). Choosing and using a plant DNA
353
barcode. PLoS One, 6, e19254.
354
Ivanova, N.V., Zemlak, T.S., Hanner, R.H., Hebert, P.D.N. (2007). Universal primer
355
cocktails for fish DNA barcoding. Molecular Ecology Notes , 7, 544–548.
AC C
EP
TE D
M AN U
SC
RI PT
327
11
ACCEPTED MANUSCRIPT Kang'ehte, E. K., J.M. Gathuma & K. J. Lindqvist (1986). Identification of species of origin
357
of fresh, cooked and canned meat and meat products using antisera to thermostable muscle
358
antigens by Ouchterlony`s double diffusion test. Journal of the Science of Food and
359
Agriculture, 37, 157-164.
360
Kharazmi, M., T. Bauer, W.P. Hammes, & C. Hertel (2003). Effect of food processing on the
361
fate of DNA with regard to degradation and transformation capability in Bacillus subtilis.
362
Systematic and Applied Microbiology, 26 (4), 495-501.
363
Khedkar G.D., Jamdade Rahul, Suresh Naik, David Lior, Haymer David (2014). DNA
364
barcodes for the fishes of the Narmada, one of India’s largest rivers. PLosOne, 9 (7), e101460
365
Khedkar G.D., Tiknaik A.D., Shinde R.N., Kalyankar A.D., Ron T.N., & Haymer D. (2016).
366
High rates of substitution of the native catfish Clarias batrachus by Clarias gariepinus in
367
India, Mitochondrial DNA, 27, 569-574.
368
Mafra, I., Ferreira, I., & Oliveira, M. (2008). Food authentication by PCR-based methods.
369
European Food Research and Technology. A, 227(3), 649−665.
370
Masiri, J., L. Benoit, B. Barrios-Lopez, C. Thienes, M. Meshgi, A. Agapov, et al.(2016).
371
Development and validation of a rapid test system for detection of pork meat and collagen
372
residues. Meat Science, 121, 397-402.
373
Meyer, R., Candrian, U., & Luethy, J. (1994). Detection of pork in heated meat products by
374
the polymerase chain reaction. Journal of AOAC International, 77,617e622.
375
Moreira, P.A., & D.A. Oliveira (2011). Leaf age affects the quality of DNA extracted from
376
Dimorphandramollis (Fabaceae), a tropical tree species from the Cerrado region of Brazil.
377
Genetics and Molecular Research, 10 (1), 353-358.
378
Nguyen-Hieu, T., Aboudharam, G., Drancourt, M. (2012). Heat degradation of eukaryotic
379
and bacterial DNA: an experimental model for paleomicrobiology. BMC Research Notes 5,
380
528
381
Novak J., S. Grausgruber-Gröger, & B. Lukas (2007). DNA-based authentication of plant
382
extracts. Food Research International, 40 (3), 388-392.
383
Opel, K. L., D. Chung, B.R., & McCord, B.R. (2010). A study of PCR inhibition mechanisms
384
using real time PCR. Journal of Forensic Sciences, 55 (1), 25-33.
AC C
EP
TE D
M AN U
SC
RI PT
356
12
ACCEPTED MANUSCRIPT Patel, N. P. (1994). The use of DNA fingerprinting in food analysis. Food Technology
386
International Europe, 171-174.
387
Perret, C., R. Tabin, J. P. Marcoz, J. Llor, & J. J. Cheseaux (2011). Apparent life-
388
threatening event in infants: Think about star anise intoxication! Archives de Pédiatrie, 18
389
(7), 750-753.
390
Ponzoni, E., F. Mastromauro, S. Gianì, & D. Breviario (2009). Traceability of plant diet
391
contents in raw cow milk samples. Nutrients, 1 (2), 251-262.
392
Rasmussen, R.S., M.T. Morrissey, & P.D.N. Hebert (2009). DNA barcoding of commercially
393
important salmon and trout species Oncorhynchus and Salmo) from North America. Journal
394
of Agricultural and Food Chemistry, 57, 8379-8385.
395
Rasmussen, R. S., & M. T. Morrisey (2008). DNA-based methods for the identification of
396
commercial fish and seafood species. Comprehensive Reviews in Food Science and Food
397
Safety, 7, 280-295.
398
Rastogi, G., M. Dharne, A. Bharde, V.S. Pandav, S.V. Ghumatkar, R. Krishnamurthy, et
399
al.(2004). Species determination and authentication of meat samples by mitochondrial 12S
400
rRNA gene sequence analysis and conformation-sensitive gel electrophoresis. Current
401
Science, 87 (9), 1278-1281.
402
Ratnasingham, S., & P.D.N. Hebert (2007). BOLD: the barcode of life datasystem
403
(www.barcodinglife.org). Molecular Ecology Notes, 7, 355-364.
404
Rosalee S. Hellberg, Brenda C.Hernandez, Eduardo L. Hernandez (2017) Identification of
405 406
meat and poultry species in food products using DNA barcoding, Food Control, 80: 23-28.
407
Sahu, S. K., M. Thangaraj, & K. Kathiresan (2012). DNA extraction protocol for plants with
408
high levels of secondary metabolites and polysaccharides without using liquid nitrogen and
409
phenol. ISRN Molecular Biology, 2012, 6pages.
410
Sakalar, E., M.F. Abasiyanik, E. Bektik, A. Tayyrov (2012). Effect of heat processing on
411
DNA quantification of meat species. Journal of Food Science, 77 (9), 40-44.
AC C
EP
TE D
M AN U
SC
RI PT
385
13
ACCEPTED MANUSCRIPT Samson, M. C., M. Gulli, & N. Marmiroli (2010). Quantitative detection method for
413
Roundup Ready (R) soybean in food using duplex real-time PCR MGB chemistry. Journal of
414
the Science of Food and Agriculture, 90, 1437-1444.
415
Schrader, C., A. Schielke, L. Ellerbroek, & R. Johne (2012). PCR inhibitors – Occurrence,
416
properties and removal. Journal of Applied Microbiology, 113 (5), 1014-1026.
417
Shiriskar D.A. , Khedkar, G.D., & Sudhakara, N.S. (2010). Preparation of pickled products
418
from anchovies (Stolephorus sp.) and studies on quality changes during storage. Journal of
419
Food Processing Preservation, 34, 176–190.
420
Shukla, P. K., & Srivastava, R. K. (1999). Storage stability of poultry pickle stored at room
421
temperature. Indian Journal of Poultry Science, 34, 285-288.
422
Sipos, R., A.J. Szekely, M. Palatinszky, S. Revesz, K. Marialigeti, & M. Nikolausz (2007).
423
Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA
424
gene-targetting bacterial community analysis. FEMS Microbiology Ecology, 60 (2), 341-350.
425
Siriskar, D. A., Khedkar, G. D., & Lior, D. (2013). Production of salted and pressed
426
anchovies (stolephorus sp.) and it’s quality evaluation during storage. Journal of Food
427
Science and Technology, 50(6), 1172–1178.
428
Turci, M., SavoSardaro, M. L., Visioli, G., Maestri, E., Marmiroli, N. (2010). Evaluation of
429
DNA extraction proceudres for traceability of various tomato products. Food control 21, 143-
430
149.
431
Unseld, M., Beyermann, B., Brandt, P., & Hiesel, R. (1995). Identification of the species
432
origin of highly processed meat products by mitochondrial DNA sequences. PCR Methods
433
and Application, 4, 241–243.
434
Valentini, A., Pompanon, F., & Taberlet, P., (2009). DNA barcoding for ecologists. Trends
435
Ecology and Evolution, 24, 110–117.
436
Vijayakumar, K. R., A. Martin, L.R. Gowda, & V. Prakash (2009). Detection of genetically
437
modified soya and maize: Impact of heat processing. Food Chemistry, 117, 514-521
438 439
Woolfe, M., S. Primrose (2008). Food forensics: using DNA technology to combat misdescription and fraud. Trends in Biotechnology, 22 (5), 222-226.
AC C
EP
TE D
M AN U
SC
RI PT
412
14
ACCEPTED MANUSCRIPT Yat-Tung Lo, & Pang-Chui Shaw (2018). DNA-based techniques for authentication of processed food and food supplements. Food Chemistry, 240, 767-774.
442 443
Zheng Zhang, Scott Schwartz, Lukas Wagner & Webb Miller (2000) A greedy algorithm for aligning DNA sequences, Journal of Computational Biology, 7(1-2):203-14.
444
Zhong, WenTao; Wang, FangMei; Li, BaiYu; Jiang, Wei; Yan, HengMei (2017) Research on the non-directional test in meat adulteration based on DNA barcode, Journal of Food Safety and Quality, 8(5):1547-1551.
445 446 447
RI PT
440 441
Figure captions
449
Figure 1.Flow chart of experimental set up for testing genetic traceability of Indian meat recipes
450 451
Figure 2. Qualitative analysis of the DNA obtained from pre-processed and post processed meat under various India recipes (electrophoretically resolved on 1.5 % Agarose gel)
452 453
Figure 3. Success of COI gene amplification for downstream species authentication using DNA Barcoding (electrophoretically resolved on 1.2 % Agarose gel)
M AN U
SC
448
454 455
457
TE D
456
Table 1. Various recipes, cooking conditions and ingredients used Meat Processing type
Raw meat Cooking/boiling (soups, biryani, Pulao)
Temperature (0C) Ambient/ freeze 980C
EP
Sr. no. 1 2
Curries
4
Deep fry 160-180 0C (Kebabs, Kentucky) Microwave cooking 70-75 0C (Grilling, biryani, cooking)
5
102 0C
AC C
3
6
458
Roasting (Tandoor, 120-170 0C barbeque/direct roasting on fire, etc.) 7 Pickling Ambient (fish pickle, prawn temperature 24pickle, chevon pickle, 35 0C beef pickle) * Composition of ingredients may vary as per recipe 15
Processing duration Ingredients* (in minutes) -Raw meat 30-35 Herbs, spices, oil, common salt, water and rice in case of biryani or pulao, meat 30-40 Herbs, spices, meat, oil, common salt and water 7-12 Oil, herbs, spices, meat and common salt 10-30 Herbs, spices, oil, meat, common salt, water and rice in case of biryani or pulao 10-20 Oil, herbs, spices, meat and common salt No specific duration, Herbs, spices, oil, meat, but it may fall from common salt and vinegar one week to a year’s duration.
ACCEPTED MANUSCRIPT
Table 2. PCR primers used and thermal conditions for amplification of COI gene Sequence
Initial Denaturation
LepF1_t1 VF1_t1 VF1d_t1 VF1i_t1
TGTAAAACGACGGCCAGTATTCAACCAATCATAAAGATATTGG TGTAAAACGACGGCCAGTTCTCAACCAACCACAAAGACATTGG TGTAAAACGACGGCCAGTTCTCAACCAACCACAARGAYATYGG TGTAAAACGACGGCCAGTTCTCAACCAACCAIAAIGAIATIGG
C_VF1LFt1 (1:1:1:3) (Ivanova et al., 2006)
C_VF1LFt1 (1:1:1:3) (Ivanova et al., 2006)
Fish
BirdR1 VF2_t1 FishF2_t1 FishR2_t1 FR1d_t1
Prawn
LCO1490 HCO2198
Annealing 35 Cycles
Extension
Final Extension
94°C for 30 Sec
50°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
94°C for 30 Sec
51°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
94°C for 30 Sec
49°C for 40 sec
72°C for 1 min
72°C for 10 min
94°C for 2 min
M AN U
(Chicken, duck, deshi hen)
BirdF1
BirdF1 (Hebert, et al., 2004) TTCTCCAACCACAAAGACATTGGCAC BirdR1 (Hebert, et al., 2004) ACGTGGGAGATAATTCCAAATCCTG
C_FishF1t1 (1:1) (Ivanova et al., 2006) TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC C_FishR1t1 (1:1) (Ivanova et al., 2006) CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA LCO1490 (Folmer et al., 1994) GGTCAACAAATCATAAAGATATTGG HCO2198 (Folmer et al., 1994) TAAACTTCAGGGTGACCAAAAAATCA
TE D
Poultry
CAGGAAACAGCTATGACTAAACTTCTGGATGTCCAAAAAATCA CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCRAARAAYCA CAGGAAACAGCTATGACTAGACTTCTGGGTGGCCAAAGAATCA CAGGAAACAGCTATGACTAGACTTCTGGGTGICCIAAIAAICA
EP
LepR1_t1 VR1d_t1 VR1_t1 VR1i_t1
AC C
Lamb, Goat, Pork
16
Denaturation
RI PT
PCR primers used
SC
Meat type
94°C for 2 min
94°C for 30 Sec
49°C for 40 sec
72°C for 1 min
72°C for 10 min
RI PT
ACCEPTED MANUSCRIPT
Table 3. DNA obtained from meat samples processed under various culinary practices
Lamb Goat Cow Buffalo Deshi hen Broiler Duck Pork Fish Prawn
920.35 2102.28 1230.91 1136.07 2199.11 2139.20 891.12 1870.30 333.7 79.17
Deep fry (Kababs, Kentucky
418.12 657.21 875.61 997.82 1613.20 2142.48 2150.96 997.69 578.84 1869.84
EP
Pork Sea food
1625.07 1968.99 1787.95 1340.20 2027.62 1714.46 1055.59 1529.31 559.57 1113.12
Curries
AC C
Chicken
Cooking/ boiling (soups, biryani, Pulao)
SC
Chevon Chevon Beef Cara- Beef
Average DNA concentration (ng/µl) obtained from 50 mg tissue of processed meat
Raw meat
1344.72 780.84 803.07 917.88 654.01 676.12 1915.01 923.07 253.69 59.35
M AN U
Meat type
TE D
Meet Commodity
17
Microwave cooking (Grilling, bryani) 1508.3 1337.49 1640.57 1024.21 1053.58 1564.25 2076.76 100.20 511.23 196.60
Roasting (barbeque/ direct roasting on fire, etc.)
Pickling
391.40 291.21 276.05 447.55 1444.28 949.06 1870.77 1928.29 219.35 93.54
** 264.10 ** 280.30 ** ** ** ** 200.00 121.40
ACCEPTED MANUSCRIPT
Roasting Deep frying
Microwave grilling Pickling Species Authentication Ovis aries Species identified Representative NCBI Accession nos.
KF302440.1 MF004244.1
SC
Curries
M AN U
Cooking/Boiling
93.5 (0.00) 100 (0.00) 100 (0.00) 100 (0.00) 100 (0.00) 100 (0.00)
TE D
Raw/control
Capra hircus
Bos taurus, Bos indicus
Bubalus bubalis
Sus scrofa
Gallus gallus
KX845672.1 KX845672.1
KX845677.1 EU177861.1
KX758295.1 KT827230.1
KX982660.1
MF102289.1 MF541544.1
EP
Lamb
Sequence matched % with NCBI gene bank records (E values) Chevon Beef Cara-beef Pork Broiler Deshi Duck Fish Chicken Chicken 100 100 99.82 99.51 99.83 100 100 100 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 97.4 100 99.82 99.7 100 99.64 -100 (1.05E-126) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.4 99.64 99.82 100 99.65 99.18 -99.23 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.5 99.22 99.82 99.67 100 100 100 99.64 (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) 99.8 99.36 99.82 -100 99.05 -99.52 (0.00) (0.00) (0.00) (0.00) (0.00) (1.09E-101) 99.6 99.34 100 100 98.93 99.34 -99.82 (4.73E-150) (0.00) (0.00) (0.00) (0.00) (0.00) (0.00) No sequences were obtained due to poor quality DNA templet recovery from these products
AC C
Type of recipe
RI PT
Table 4: DNA Barcode based species identified using COI gene sequences using similarity score
18
Gallus gallus MF102289.1 MF541544.1
Prawn 99.14 (0.00) -97.64 (4.8E-140) 96.82 (0.00) 96.38 (0.00) 98.182 (4.67E-160)
Anas platyrhyncho s
Pangasianodon hypophthalmus
Fenneropenaes merguiensis, Metapenaeus
MF069251.1
KX685193.1 EF609427.1
KJ879289.1 KC409384.1 KX399431.1 KP637170.1
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Highlights of research
Study conducted for traceability compliance of Indian culinary processing of meat
•
Culinary processing do not alter DNA quality required for traceability compliance
•
All samples were successful in amplifying COI gene, no evidence of PCR inhibitor
•
Employed samples were successful in generating full length DNA barcodes
•
Species authentication under pickled products failed in all preparations
AC C
EP
TE D
M AN U
SC
RI PT
•