Enhancing the authenticity of animal by-products: harmonization of DNA extraction methods from novel ingredients

Abstract

Introduction: The increasing global pressure to explore alternative protein sources derived from animal by-products has opened-up opportunities, but it has also created the need to assess their compliance with labelling statements, to ensure consumer's trust in the composition of both feed and food products. Assessing the authenticity of highly processed animal by-products, particularly within the rapidly expanding Halal food market, presents a significant challenge due to the lack of robust and standardized methodologies. However, the success of DNA based authenticity system is highly dependent on the extracted DNA quantity, quality, and purity ratios from heterogeneous matrices. Material and methods: In this work, nine DNA extraction methods were tested on selected processed animal by-products with high-value and interest for the feed industry: meals from poultry meat, blood and feather, and hydrolysates from swine meat and bone, fish, and black soldier fly. The proposed DNA extraction methods are developed to specifically target swine-specific mitochondrial region, as a case study. Results and discussion: Both the conventional CTAB method and the commercial kits, specifically Invisorb^® Spin Tissue Mini and NucleoSpin™ Food, demonstrated superior extraction efficiency and quality ratios. Nevertheless, commercial kits enabled faster detection in comparison to the conventional methods. The absence of swine DNA was successfully validated and confirmed in all animal meals and hydrolysates that did not contain swine in their composition beforehand, demonstrating their compliance with the Halal market requirements.

Keywords: DNA extraction; PCR; animal feed ingredients; authenticity; processed by-products; swine.

FIGURE 1

Agarose gel electrophoresis (0.8%) of DNA extracted from Poultry Meat Meal (PMM), Blood (PBM) and Feather meals (FM) using nine different extraction methods: (A)-CTAB, (B)-CTAB N*, (C)-Modified Wizard-CTAB, (D)-Modified Wizard-CTAB N*, (E)-ZymoBIOMICS™ DNA Miniprep, (F)-Quick-DNA™ Miniprep Plus, (G)-Invisorb^® Spin Tissue Mini, (H)-Invisorb^® Spin Blood Mini, (I)-NucleoSpin™ Food. Lane M- 1 kb DNA ladder; Lane 1- Poultry Meat Meal 1 (PMM1); Lane 2- Poultry Meat Meal 2 (PMM2); Lane 3- Poultry Meat Meal 3 (PMM3); Lane 4- Poultry Meat Meal 4 (PMM4); Lane 5- Poultry Meat Meal 5 (PMM5); Lane 6- Poultry Blood Meal (PBM); Lane 7- Feather Meal (FM); Lane 8- swine sausage (positive control, PC); Lane 9- fennel leaf (negative control, NC).

FIGURE 2

Comparison of DNA extracts purity from 10 meals and hydrolysates derived from animal by-products and two controls samples (swine sausage as positive control and fennel leaf as negative control) employing different extraction methods. DNA extracts with a quality A260/280 nm ratio below 1.7 are colored in yellow, DNA extracts with a quality between of 1.7–2.0 are colored in green, while DNA extracts with A260/280 nm ratio above 2.0 are colored in red.

FIGURE 3

Average DNA concentrations (ng/µL ± standard deviation) of meals and hydrolysates obtained from animal by-products using conventional extraction methods and commercial kits. The data is represented as mean values for each group of matrices.

FIGURE 4

Limit of detection (LOD) of the PCR assay for the detection of swine DNA extracted from swine sausage (positive control) (A), and Meat and Bone Swine Hydrolysate (MBSH) (B). PCR products amplified with serial 10-fold dilutions of extracted DNA ranging from 2 ng (lane 1) to 2 × 10⁻⁶ ng of total DNA (lane 7). M - DNA ladder of 1 kb.

FIGURE 5

Agarose gel electrophoresis (1.5%) of PCR assay targeting the swine mitochondrial D-loop amplicon of 531 bp in meals used in aquafeed production. (A) CTAB, (B) CTAB N*, (C) Modified Wizard-CTAB, (D) Modified Wizard-CTAB N*, (E) ZymoBIOMICS™ DNA Miniprep, (F) Quick-DNA™ Miniprep Plus, (G) Invisorb^® Spin Tissue Mini, (H) Invisorb Spin Blood Mini, (I) NucleoSpin Food. Lane M- 1 kb DNA ladder; Lane 1- Poultry Meat Meal 1 (PMM1); Lane 2- Poultry Meat Meal 2 (PMM2); Lane 3- Poultry Meat Meal 3 (PMM3); Lane 4- Poultry Meat Meal 4 (PMM4); Lane 5- Poultry Meat Meal 5 (PMM5); Lane 6- Poultry Blood Meal (PBM); Lane 7- Feather Meal (FM); Lane 8- swine sausage (positive control, PC).

FIGURE 6

Agarose gel electrophoresis (1.5%) of PCR assay targeting the swine mitochondrial D-loop amplicon of 531 bp in hydrolysates used in aquafeed production. (A) CTAB, (B) CTAB N*, (C) Modified Wizard-CTAB, (D) Modified Wizard-CTAB N*, (E) ZymoBIOMICS™ DNA Miniprep, (F) Quick-DNA™ Miniprep Plus, (G) Invisorb^® Spin Tissue Mini, (H) Invisorb Spin Blood Mini, (I) NucleoSpin Food. Lane M- 1 kb DNA ladder; Lane 1- Meat and Bone Swine Hydrolysate (MBSH); Lane 2- Fish Hydrolysate (FH); Lane 3- Black soldier fly Hydrolysate (BSH); Lane 4- Swine sausage (positive control, PC). The PCR results obtained for the different hydrolysates are highlighted by the blue square.

FIGURE 7

Parameters employed to compare the nine DNA extraction methods. The color code represent the classification of each method’s outcome for each parameter: green (good), yellow (medium), or red (poor). Average Yield per extraction method: <20 ng/μL (red), 20–50 ng/μL (yellow), >50 ng/μL (green); Average A260/280 nm ratio: >2.0 (red), <1.7 (yellow), 1.7–2.0 (green); PCR amplification: successful amplification of swine DNA on samples containing swine (green), no amplification of swine DNA in swine containing samples (red); Average extraction time: >10 h (red), 1–10 h (yellow), <1 h (green).