WO2020089756A1

WO2020089756A1 - Detection method for halal food and halal food additives

Info

Publication number: WO2020089756A1
Application number: PCT/IB2019/059176
Authority: WO
Inventors: Canan DOGAN; Mediha Esra ALTUNTOP YAYLA
Original assignee: Tubitak
Priority date: 2018-10-30
Filing date: 2019-10-25
Publication date: 2020-05-07

Abstract

The present invention is a molecular determination method which distinguishes between food additives produced by the processing of raw materials obtained from animals or humans, which may be controversial whether halal or not, and which distinguishes between food additives produced by fermentation using microorganisms. The sample will be identified as halal if it is detected to be produced by using microorganisms with fermentation. The analyses that are developed for the detection of microbial load in samples are not able to detect origin of microorganism in hydrolyzed and processed samples, and correct results cannot be obtained from the analysis. The present invention provides a solution for this problem. There are 1.7 billion Muslim people on the world. Because of religious requirements, Muslim population demands the food they consume to be acceptable with their belief. Halal status of animal originated food additives cannot be determined by analytical methods since how animal slaughtering process was performed cannot be ascertained. In the scope of present invention, an analytical method was developed for the detection of food additives that are produced by fermentation by the usage of microorganisms.

Description

DESCRIPTION

DETECTION METHOD FOR HALAL FOOD AND HALAL FOOD ADDITIVES Technical Field

The present invention is a molecular detection method for discrimination of food and food additives that are accepted as halal.

Prior Art

There are 1.7 billion Muslim people on earth. Muslim population consumes food that are characterized as suitable for requirements of Islam and halal. In order for a food of animal origin to be accepted as halal food, the animal species must be in accordance with the Islamic religion and the animal should be slaughtered in accordance with religious rituals. There are various methods that are used to detect the origin of non-halal animal species or other food products derived from these animals. Yet, the process of slaughtering cannot always be tracked. Therefore, it can not be determined by analytical methods whether the food additives of animal origin are halal. Food additives that are produced by using microorganisms with recombinant DNA technology are accepted as halal. The food products that are produced by these food additives are more expensive but preferred by Muslims. There is a need for an analytical method for the detection whether food additives that are produced from microorganisms by recombinant DNA technology and the foods containing these food additives are halal or not. The determination of whether food additives are halal or not can only be made by declaring all over the world. Besides, there have not been any studies to determine whether the food additives are halal or not in the prior art.

It is envisaged that the DNA of the host bacteria can be detected in a foodstuff by using molecular techniques, in order to detect whether a food additive is produced by microorganisms and/or by using recombinant DNA technologies.. However, because of their nature, foods are matrices composed of various different components and exposed to plenty of physical and chemical stresses during their production, the required sensitivity can not be reached with the classical protocols and the host microorganism DNA cannot be detected. DNA isolation methods capable of cellular DNA isolation are used in routine microorganism analysis. The principle that these methods are based on is bursting the cells and the purification of DNA by exposing them. In order to prevent false hybridizations, DNA regions with less copy number and longer base pairing are preferred during identification of the obtained chromosomal DNA, . Therefore, gene regions that are longer than 100 base pair (bp) are targeted and analyzed by PCR method. In processed food, DNA is damaged and broken. Shorter DNA regions with high copy number should be preferred when identifying DNA isolated from those samples . Primer-probe sets that needs to be designed with respect to the target DNA region must have major variances. Therefore, the analyses developed for the detection of microbial load cannot be used for origin detection in processed food.

Methods similar to the detection method described in the present invention regarding the use of molecular methods for the detection of microorganismal DNAs are available in the prior art. Detection of very short strand DNA sequences in body fluids is disclosed in US patent document number US 20100068711A1 . It is stated that when the target DNA region gets shorter, the probability for detection increases, and the detection of short strand bacterial DNA in body fluids is described by that method. However, the patent document does not mention usage of the described method for the detection in food samples with complex matrices and in food additives that are produced with halal status. In the scope of present invention, it was ascertained that short DNA extraction requires further protocols. In order to prevent breakage of DNA during DNA extraction, increased pH, increased salt concentration, heat treatment, usage at least one of ethylenediaminetetra acetic acid, guanidine-HCl, guanidinisothiocyanate, N-laurylsarcosine or sodium dodecyl sulfate chemical agents are within the scope of of this invention with its different applications.

CN 105112400A discloses the extraction of long (> 500 bp) and short (<500 bp) chain DNA molecules from blood plasma and urine for prenatal diagnosis and tumor imaging using magnetic beads and different percentages of isopropanol solutions, after removing long DNA strands, short DNA strands are isolated.

In the document WO2013192302 Al, phenol-chloroform method is used for isolation of short strand Mycobacterium tuberculosis DNA from urine, and isolated DNA is precipitated by alcohol.

A method is described in EP 2 598 655 Bl patent document in which microorganisms are detected with real-time polymerase chain reaction (RT-PCR) and to be able to obtain certain quantitative data, secondary nucleic acid is added into the reaction as a control. However, usage of short DNA sequence iss not stated in the document as the food matrices is out of the scope of the document.

In the patent application document number W02012028740A1, it is stated that host DNA residuals remain in the protein produced by recombinant DNA, and DNA from prokaryotic organisms does not need an additional resolution while eukaryotic organisms require selection of high copy number genes in order to increase sensitivity of the analysis. This patent does not consider food matrix and does not describe whether the target DNA region is short-chained.

Although bacterial DNA was detected from body fluids using short- stranded DNA sequences, there has not been any document in the prior art in which microorganismal DNAs in food products were detected using short- stranded DNA regions

Brief Description of Invention

Food additives that are produced by processing animal or human originated raw materials, which may be controversial whether halal or not can be producedusing microorganisms. If a food additive is detected to be produced by fermentation, it will be proved halal. The method for the molecular detection method developed within the scope of the present invention is aimed to reliably detect whether food additives produced using microorganisms and the foods to which these food additives are added are halal. For this purpose, gene regions shorter than 100 bp are detected with polymerase chain reaction (PCR) method.

The origin of the food additives produced by microorganisms but containing trace amounts of broken microorganismal DNA in food additives as a result of excessive hydrolysis after production can be determined with the developed method if they are added in raw material or food. DNA isolation with PCR procedure is unified systematically in the developed method .If positive result is obtained after the process, the result will be able to be confirmed by classical microorganism cultivation methods by showing the absence of live bacteria in the sample. Detailed Description of Invention

1. The present invention includes following steps: a. Food sample is dissolved in lysis buffer. The buffer contains one or more of the chemicals that are Tris-HCl, HEPES, EDTA, NaCl, SDS, DTT, b- mercaptoethanol, Guanidine thiocyanate and CTAB, Tween 20, Triton X-100 or a similar detergent agent, proteinase K or a similar enzyme; one or more incubations are possible with the aforementioned chemicals for the implementation of the invention. b. The content of lysis buffer has to contain preferably non-organic chemical solutions with no more than 8 N and no less than 5 mM ionic strength, organic chemical solutions no greater than 30% and no less than 1%. The total detergent and caotropic agent amount in the solution has to be no more than 25%.

2. The mixture is incubated at 45-80°C for 30-120 minutes or overnight.

3. DNA isolation is performed. During DNA isolation, one of the practices of the present invention includes addition of phenol:chloroform:isoamyl alcohol (25:24: 1) with a ratio of 10-250% of the total mixture and after vortexing, separating the water phase by centrifuging for 1-60 minutes at 100-50000 g. The g force applied and the duration of centrifuge may vary depending on the content of the mixture. Any g force and duration that enables the separation of the mixture is acceptable as a practice of the invention. At this point with DNA precipitation with alcohol, or DNA isolation methods with magnetic particles, or DNA isolation methods with DNA binding silica based or similar colon systems, or a similar DNA isolation method can be used for isolation of DNA in aqueous phase.

4. In order to identify the microorganismal DNA from the total isolated DNA a gene region less than 100 bp is preferably amplified with primers and amplified DNA amount is identified by probe. At this point, although for the detection of amplification of DNA, the use of the probe based on Taqman proceduce is predicted, sybrgreen or a similarchemical that emits a fluorescence radiation with double stranded DNA is able to be used for the detection of amplification of DNA in different applications of the invention.

At this stept, for the amplification of DNA, durations of incubations and cycle numbers that can vary are followed as given:

90-96°C first denaturation,

Cycles of

90-96°C - denaturation

45-70°C - annealing

70-75°C - extension The cycles set with or without the extending step is possible.

For the detection of gene region, usage of any type of PCR method and showing the presence of target DNA is under the scope of the present invention.

Example-l : Escherichia coli (E.coli) DNA was analyzed from this food additive for the detection of origin of L-cysteine that is used as a food additive and produced by fermentation.

DNA isolation:

L-cysteine food additive was dissolved in resuspension buffer (50 mM Tris-HCl, pH 8, 20 mM EDTA, 5 M Guanidinium thiocyanate, %2 Tween 20). 400 pL organic extraction buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 50 mM EDTA, %0.5 SDS), 40 pL 20% SDS, 50 pL 1 M DTT, 30 pL 20 mg/mL Proteinase K was added into the mixture.

The mixture was incubated at 65°C at least for 1 hour. Phenol: chloroform: isoamyl alcohol (25:24: 1) was added and vortexed in an equal volume. Then, it was centrifuged for 10 minutes at 12,000 rpm. Tipper phase was transferred to a new tube and DNA isolation was continued with magnetic beads.

Tepnel (Pharma Services) DNA extraction magnetic particles were used for this step. After phase separation by using phenol:chloroform:isoamyl alcohol, the upper layer was mixed with binding buffer from Tepnel kit and added into 50 pL magnetic beads and washed with isopropanol for DNA isolation. Real-time PCR Procedure:

Forward primer: 5’-CATCC ACAGAACTTTCCAG-3’

Reverse primer: 5’ -C ATTTC AC AAC ACGAGCTG-3’

TaqMan Probe: 5’ -F AM-C AGTTCCCGAAGGC ACC AATCC ATC-BHQ 1 -3’

The gene region was of 90 bp which was amplified by the given primer-probe set that targets rRNA region.

RT-PCR analysis was done by using 2X Quantitech PCR mastermix. 300 nM primer and 150 nM probe concentrations were used for the reaction. PCR protocol was performed as: 1. 95°C, 10 minutes, first denaturation,

2. 95°C, 30 seconds, denaturation,

3. 54°C, 30 seconds, annealing,

4. 72°C, 30 seconds extension. with 45 cycles.

By using this method, E.coli detected from laboratory-produced L-cysteine with a Ct value (Cycle threshold, minimum threshold value) of 32.22, commercial L-cysteine produced by fermentation from Wacker company with a Ct value of 36.91, commercial L-cysteine produced by fermentation from Sigma company with a Ct value of 36.98, negative control sample and Bacillus aerius (cross reaction control sample) did not give any Ct value.

L-cysteine food additives produced by fermentation were cultivated to agar plates by following ISO 16649-2:2001 standard and microbial growth was not detected.

Example-2:

For the detection of origin of L-cysteine that is used as a food additive and produced by fermentation, presence of E.coli DNA was analyzed from this food additive.

DNA isolation:

L-cysteine food additive was dissolved in HC1 solution. 625 pL organic extraction buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 50 mM EDTA, %0.5 SDS), 25 pL 20% SDS, 250 pL 1 M DTT, 100 pL 20 mg/mL Proteinase K was added into the mixture. The mixture was incubated at 65°C at least for 1 hour. Phenol: chloroform: isoamyl alcohol (25:24: 1) was added in an equal volume and vortexed. It was centrifuged for 10 minutes at 12,000 rpm. Same volume of cold absolute isopropanol was added to precipitate DNA. The mixture was incubated at least for 1 hour at 20°C. Then, it was centrifuged for 30 minutes at 12,000 rpm at 4°C and precipitation with 75% isopropanol. The precipitation was repeated twice. After the precipitate was dried, it was dissolved in 50 mM Tris-HCl, pH 8 and became ready to be used for PCR analysis. Real-time PCR Procedure:

Forward primer: 5’-CATCC ACAGAACTTTCCAG-3’

Reverse primer: 5’ -C ATTTC AC AAC ACGAGCTG-3’

TaqMan Probe: 5’ -F AM-C AGTTCCCGAAGGC ACC AATCC ATC-BHQ 1 -3’ The gene region was of 90 bp which was amplified by the given primer-probe set that targets rRNA region RT-PCR analysis was done by using 2X Quantitech PCR mastermix. 300 nM primer and 150 nM probe concentrations were used for the reaction. PCR protocol was performed as:

1. 95°C, 10 minutes, first denaturation, 2. 95°C, 30 seconds, denaturation,

3. 54°C, 30 seconds, annealing,

4. 72°C, 30 seconds extension with 45 cycles.

By using this method, E.coli detected from commercial L-cystei ne-HCl . H?0 produced by fermentation from Wacker company with a Ct value of 32.95, commercial L-cysteine produced by fermentation from Sigma company with a Ct value of 36.35, negative control sample and Bacillus aerius (cross reaction control sample) did not give any Ct value.

These results indicated that origin of L-cysteine that is produced by E.coli microorganism by biotechnological methods are able to be detected. L-cysteine food additives produced by fermentation were cultivated to agar plates by following ISO 16649-2:2001 standard and microbial growth was not detected.

Example-3 :

Flour that contains L-cysteine food additive that is produced by fermentation and bread that is prepared from a dough containing this flour with addition of water and salt and cooked were used for DNA isolation and detection of Escherichia coli {E.coli) DNA. DNA isolation:

Flour and bread samples that contained L-cysteine food additive were dissolved in resuspension buffer (100 mM Tris-HCl, pH 8, 10 mM EDTA, 0.5% Tween 20 (w/v)). 625 pL organic extraction buffer (50 mM Tris-HCl, pH 8, 100 mM NaCl, 50 mM EDTA, %0.5 SDS), 25 pL 20% SDS, 250 pL 1 M DTT, 100 pL 20 mg/mL Proteinase K was added into the mixture.

The mixture was incubated at 65°C at least for 1 hour. Pphenol: chloroform: isoamyl alcohol (25:24: 1) was addedin an equal volume and vortexed. It was centrifuged for 10 minutes at 12,000 rpm andcold absolute isopropanol in an equal volume was added to precipitate DNA. The mixture was incubated at least for 1 hour at 20°C. Then, it was centrifuged for 30 minutes at 12,000 rpm at 4°C and precipitation with 75% isopropanol was repeated twice. After the precipitate was dried, it was dissolved in 50 mM Tris-HCl, pH 8 and became ready to be used for PCR analysis.

Real-time PCR Procedure:

Forward primer: 5’-CATCC ACAGAACTTTCCAG-3’ Reverse primer: 5’ -C ATTTC AC AAC ACGAGCTG-3’

TaqMan Probe: 5’ -F AM-C AGTTCCCGAAGGC ACC AATCC ATC-BHQ 1 -3’

The gene region was of 90 bp which was amplified by the given primer-probe set that targets rRNA region RT-PCR analysis was done by using 2X Quantitech PCR mastermix. 300 nM primer and 150 nM probe concentrations were used for the reaction. PCR protocol was performed as:

1. 95°C, 10 minutes, first denaturation,

2. 95°C, 30 seconds, denaturation,

3. 54°C, 30 seconds, annealing,

4. 72°C, 30 seconds extension. with 45 cycles.

By using this method, E.coli detected from flour sample that contains L-cysteine produced from E.coli by fermentation and bread produced from this flour have Ct values of 35.95 and 37.35 respectively, negative control sample and Bacillus aerius (cross reaction control sample) did not give any Ct value. Flour sample that contains L-cysteine produced from E.coli by fermentation and bread produced from this flour were cultivated to agar plates by following ISO 16649-2:2001 standard and microbial growth was not detected.

Example-4: For the detection of origin of L-cysteine that is used as a food additive and produced by fermentation, E.coli screening method from literature was used for E.coli DNA detection.

For DNA isolation from the sample, Ausubel et al. protocol was followed.

(F. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.). 1997. Short protocols in molecular biology, 3rd ed., p. 2-11-2-44. John Wiley & Sons Inc., New York, N.Y.)

ECA75F/ ECR619R primer set that targetsl6S rRNA gene region was used (Sabat et al. 2000). Amplified region was 544 bp long.

(Sabat G, Rose P, Hickey W, Harkin J.M. Selective and Sensitive Method for PCR Amplification of Escherichia coli l6Sr RNA Genes in soil. Applied and Environmental Microbiology 2000; 66: 844-849)

EC A75F; 5’ -GGAAGAAGCTTGCTTCTTTGCTGAC-3’

ECR619R 5’ - AGCCCGGGGATTTC AC ATCTGACTT A-3’

In 50 pL total volume, 10X PCR buffer (100 mM Tris-HCl, pH 9, 500 mM KC1, %0. l Triton X-100) was used with 1 : 10 dilution. 200 pM four types of deoxynucleotide triphosphates, 2 mM MgCh, 0.4 pM primers, 40 pg/reaction bovine serum albumine, 1.5 U Taq DNA polymerase and sample DNA were added into mixture.

PCR protocol was performed as:

1. 94°C, 30 seconds, first denaturation,

2. 94°C, 45 seconds, denaturation, 3. 72°C, 45 seconds, annealing,

4. 72°C, 10 minutes, extension. with 40 cycles. PCR products were run in 2% agarose gel and band formations were observed. Positive control samples were observed to have correct band formations, while L-cysteine food additive that is produced from E.coli by fermentation was not observed to have any band formations, presence of E.coli DNA was not able to be shown. It was shown that PCR analysis developed for the detection of microbial load were not suitable for the analysis of food additives that are hydrolyzed.

Application of the Invention to the Industry:

Origin of L-cysteine that is used extensively in production of halal foods such as flour and pastry is skeptical for consumers. The present invention that is used for the halal origin detection in L-cysteine food additive and other additives in cooked, frozen and other food products will be easily be applied to industrial products. With the representative study, origin of food additives that are produced by microorganisms in a similar way was shown to be detected, and it was proved that food additives that frequently and extensively used in food products being halal will be able to be detected. The present invention will be typically used in origin detection during halal food certification and correct certification will be enabled.

Claims

1. A method for the detection of halal food and halal food additives comprising following steps;

a. Resuspending food sample in lysis buffer

b. Preferably transferring the aqueous phase of the mixture

c. Isolating total DNA from aqueous phase

d. Isolating the DNA of the microorganism which was used for the production of the food additive from the total isolated DNA.

2. A lysis buffer according to step a of Claim 1, characterized in that containing one or more of the chemicals that are Tris-HCl, EDTA, NaCl, SDS, DDT, b-mercaptoethanol, Guanidinium thiocyanate, CTAB, Tween 20, Triton X-100 or a similar reducing agent, SDS, or a similar detergent agent, proteinase K or a similar enzyme.

3. The method according to Claim 1 characterized in that food sample is incubated in the lysis buffer at 45-80°C for 30-120 minutes.

4. The method according to Claim 1 characterized in that transferring the aqueous phase by preferably adding phenol:chloroform:isoamyl alcohol in food sample.

5. The method according to Claim 1 or 4 characterized in that the ratio of phenol:chloroform:isoamyl alcohol mixture is 10-250% of the total mixture of the food sample.

6. The method according to Claim 1, 4 or 5 characterized in that the mixture of phenol:chloroform:isoamyl alcohol is in 25:24: 1 ratio in volume.

7. The analysis method according to Claim 1, 4, 5 or 6 characterized in that the sample is centrifuged for 1-30 minutes at 300-20000 g after alcohol addition.

8. The total DNA isolation according to step c of Claim 1 characterized in that isolating DNA in aqueous phase by using DNA precipitation with alcohol, DNA isolation methods with magnetic particles, or DNA binding silica based or similar colon systems, or a similar DNA isolation method.

9. The method according to Claim 1 or 8 characterized in that total DNA is washed with ethanol, isopropanol, any other alcohols containing 2-8 carbon, or Tris buffer, EDTA, acetate buffer or distilled water during DNA isolation method with magnetic particles.

10 The microorganismal DNA isolation according to step c of Claim 1 characterized in that amplification of a gene region preferably less than 100 bp with primers and detection of amplified DNA amount is by probe in order to detect microorganismal DNA inside total DNA.

11. The analysis method according to Claim 1 or 10 characterized in that detection of isolated microorganismal DNA by amplification with real-time PCR, isothermal PCR, nested PCR, classic PCR or a similar PCR method.

12. The analysis method according to Claim 1, 10 or 11 characterized in that using of Taqman or a similar prob system, SYBR Green or a similar fluorescence emitting chemical with double strand DNA for the detection of amplified microorganismal DNA.