Processed Animal Proteins Research Articles

Challenges related to the application of analytical methods to control insect meals in the context of European legislation

Since their approval for use in aquaculture in 2017, processed insect proteins have been extensively studied for their nutritional quality in animal feed. This new type of meal is highly promising but requires, as for other products used in animal feed, strict sanitary control in accordance with European legislation. Within this legal framework, light microscopy and PCR remain the official methods but have some analytical limitations that other methods could overcome. This paper aims to provide an overview of the European legislation concerning use of processed insect proteins, but also to highlight the advantages and disadvantages of the official methods for their analysis. It also points out other analytical methods, which have already proved their worth for the analysis of processed animal proteins, which could be used as complementary methods.

Consumers' Preferences for Chicken Fed on Different Processed Animal Proteins: A Best-Worst Analysis in Italy.

The increase in meat consumption expected in the next decade will require more and more proteins for animal feeding. The recent amendments to the European "BSE Regulation" allow the use of insects and porcine-based meals in poultry farming, providing novel, sustainable substitutes for vegetable fodder. While the technological and nutritional properties of novel feeds containing processed animal proteins are widely recognized, far less is known about consumers' acceptance of meat produced by animals fed on animal-based meals. In the present research, a best-worst survey was applied to estimate consumers' preferences for chicken fed on plants, insects, or porcine-based meals using a sample of 205 Italian consumers. Furthermore, product price, type of farming, and "Free-from" labeling were considered in the analysis to evaluate the relative importance of feed ingredients compared to other important attributes of meats. The results show that the most relevant attributes are type of farming and "Free-from" claims, while type of feed represents the third attribute in order of importance. Notably, both insect and porcine flour are considered as negative characteristics of the product, suggesting that mandatory labeling signaling the use of these feeds would negatively impact on the value of chicken meat.

Optimization and validation of a quick and responsive LC-ESI-MS/MS method to evaluate tetracycline residues in processed animal proteins (PAPs).

Following the bovine spongiform encephalopathy (BSE) in 2001, processed animal proteins (PAPs) reintroduction is envisaged in non-ruminant feed thanks to their high protein content, easy availability and cost-effective characteristics. PAPs must be submitted to rendering practices, providing sterilization of products, under standardized conditions of temperature and pressure, according to Regulation (EC) No 142/2011. However, the chemical risk associated to these raw materials has been never evaluated. The aim of this study was to develop and validate a reliable liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the determination and quantification of tetracycline residues in PAPs at µg kg-1 level. The LC-MS/MS method performances were evaluated in terms of specificity, linearity (25-500µgkg-1), limit of quantitation (LOQ) (25µgkg-1), accuracy and precision (CV% < 25%), uncertainty, recovery (80-120%) and ruggedness. All the evaluated parameters fulfilled the analytical performance criteria, and the validated LC-MS/MS method fits for purpose as confirmatory method on the occurrence of residues (µg kg-1) of tetracyclines in PAPs. PAPs are a powerful product which could be used both as raw materials in feed and in organic fertilizer production in a circular economy context. Therefore, the lack of regulation and control over antibiotic occurrence should be implemented to avoid a misuse and an increment of antibiotic resistance pressure over the environment and to ensure safety of the feed and food chain.

Evaluating the Functional Properties of Spray-Dried Porcine Plasma in Gilthead Seabream (Sparus aurata) Fed Low Fish Meal Diets.

Blood by-products are an untapped source of high-quality ingredients for aquafeeds, containing a broad variety of cytokines, hormones, growth factors, proteins, bioactive peptides, and amino acids. The effects of the spray-dried porcine plasma (SDPP), a type of processed animal protein on several immune parameters, were evaluated in sea bream using ex vivo and in vitro assays. In this study, fish were fed with two isoproteic, isolipidic, and isoenergetic diets: control diet (7% fish meal, FM) and SDPP diet (2% FM and 5% SDPP). At the end of the 92-days trial, those fed the SDPP diet were larger in body weight (p < 0.05) without differences in feed conversion ratio (p > 0.05). The ex vivo immune stimulation of splenocytes indicated that SDPP had a beneficial effect in promoting systemic immunity, since the surface cell marker (cd4), pro- (il-1β), and anti-inflammatory (tgf-β1) cytokines, and genes involved in humoral immunity (IgM) were up-regulated. The co-culture assays of skin mucus corroborated that SDPP enhanced the antibacterial capacity of mucus against V. anguillarum. In addition, main mucus biomarkers did not show significant differences, except for cortisol levels which were lower in the SDPP diet. The present study indicated that SDPP may be considered a functional ingredient in aquafeeds formulated with low FM levels.

Effects of dietary plant and animal protein sources and replacement levels on growth and feed performance and nutritional status of market-sized turbot (Scophthalmus maximus) in RAS

One part of aquaculture sustainability is reducing the environmental footprint of aquaculture feeds. For European aquaculture, this means finding feed ingredients that are produced within the economic community, and that are not in conflict with human consumption. This is especially challenging when formulating diets for carnivorous fish such as turbot with low tolerance to fishmeal replacement that are both nutritious and economically and environmentally sustainable. Therefore, we investigated the effects of two novel and innovative feed formulation concepts on growth and feed performance and the nutritional status of market-sized turbot in a recirculating aquaculture system. In a 16-week feeding trial, 440 turbot (300 ± 9 g) were fed twice a day with a control diet (CTRL), based on a commercial formulation, and four experimental diets. The experimental diets were designed to investigate the effects of two formulations concepts based on sustainable terrestrial plant proteins (NoPAP) or processed animal proteins (PAP) and of 30% and 60% fishmeal replacement with emerging feed ingredients (fisheries by-products, insect meal and fermentation biomass). Turbot from the CTRL group had a similar growth and feed performance than fish fed the NoPAP30 formulation, with a significant decline of performance in the fish fed both PAP formulations and the NoPAP60. Comparing the two formulation concepts with each other the voluntary feed intake and protein efficiency ratio on tank basis as well as the individual weight gain and relative growth rate was significantly higher in the fish from the NoPAP groups than PAP groups. Furthermore, the apparent digestibility of nutrients and minerals was significantly reduced in the fish fed with the diets with 30% and 60% fishmeal replacement level compared to the fish from the CTRL group. In conclusion, the performance of the fish fed the NoPAP30 formulation concept highlights the potential of the used combination of sustainable ingredients, such as fisheries by-products, insect meal, microbial biomass and plant protein for turbot. Furthermore, this study shows that turbot has a higher tolerance to the incorporation of plant and insect protein than of processed animal protein.

Preliminary Data on Essential and Non-essential Element Occurrence in Processed Animal Proteins from Insects.

Insects represent a valuable and environmentally friendly protein alternative in food and feed. The Farm to Fork strategy encouraged the reintroduction of animal by-products in feed production to optimise recycling and to valorise under-used resources. In order to grant safe and valuable feed products, this study investigated the black soldier fly (BSF) (Hermetia illucens) chemical risk. Samples collected in different steps of production (8 samples of substrate for culturing, 7 samples of larvae, 15 samples of protein meal, 18 samples of spent substrate) were analysed for microessential elements (chromium, copper, iron, nickel, selenium and zinc) and inorganic contaminants (aluminium, arsenic, cadmium, lead, tin and vanadium) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Microessential elements were found in the following order: Fe > Zn > Cu > Ni > Se > Cr (mg kg-1). Non-essential element concentrations were found lower than the set limits according to the European Union Regulations. The growing demand for alternative protein sources for feed production could be partially compensated by black soldier fly (BSF) (Hermetia illucens) meal, as it appears a good source for high-quality proteins and microessential elements which play a pivotal role in animal growth. In the foreseeable future the current legislation and the official monitoring plans may be implemented and broaden, to focus and assess limits for upcoming matrices, and to ensure feed and food safety.

Development and application of criteria for classification of hydrolysed proteins in the framework of feed safety

In the view of a circular economy, there is an increasing need for (re-)using animal by-products that have a wide range of applications and sufficient safety. Hydrolysates of animal proteins (HPs) are frequently used as feed ingredients. Nevertheless, clear criteria for legal use and methods for monitoring feed applications are not available. Here, a range of methods have been used and evaluated for characterizing a set of 26 samples of hydrolysed proteins, ‘hydrolysed’ feather meals and processed animal proteins (PAPs), with verification based on an additional set of eight samples. Methods included determination of ash content, sediment (mineral fraction) content, protein content, species identity, solubility, protein solubility, size exclusion chromatography and polyacrylamide gel electrophoresis (SDS-PAGE). After a comparison of results obtained with water and SDS, water was chosen as the solvent for environmental and occupational reasons. Typical HP samples have a protein content higher than 60%, a solubility exceeding 50% and a virtual absence of a mineral fraction. The first discrimination between HPs and PAPs could be based on the absence or presence, respectively, of a mineral fraction. An approach for HP characterization is designed using a Hydrolysation Index (HI) based on the fraction of peptides smaller than 10 kDa, the solubility of the sample and the fraction of soluble proteins. A simplified version (HIs), exclusively based on the fraction of peptides smaller than 10 kDa and the solubility of the sample, shows a trend among the samples highly comparable to HI. Values for HI and HIs exceeding 60% would characterise HPs. Feather meals, which are heat treated instead of treatment by a chemical process of hydrolysation, range among the PAPs and should not be indicated as “hydrolysed.” The HIs can be used as an easy parameter for classifying HPs and for legal enforcement.

Laboratory Experience with the Microscopic Method for the Detection of Insects in Poultry Feeds.

The use of insects and their processed animal proteins (PAPs) for animal nutrition creates the need for research into methods useful for routine surveillance for their presence. The aim of this study was to evaluate a modified microscopic method for the detection of particles of insects in poultry feed. A total of 90 samples including PAP of insects (Hermetia illucens and Tenebrio molitor), poultry feeds produced with different levels (0-27%) of insect PAP content, and other poultry feeds spiked with insect PAP at 1% were investigated using a modified microscopic method with a double sedimentation protocol. Characteristic features of insects including cuticulae, muscles, bristles and tracheoles were determined in the microscopic images obtained. In all spiked samples, characteristic fragments of insects were detected. The fragments of muscle and tracheoles only indicated the presence of material from members of the insect class but could not facilitate identification of organisms to species level. The results obtained with this double sedimentation protocol for the isolation of insect PAP from feed for poultry have shown that the method can be used in routine analysis.

Towards the Identification of a Suitable Commercial Diet for Carpione (Salmo carpio, Linnaeus 1758): A Multidisciplinary Study on Fish Performances, Animal Welfare and Quality Traits.

Simple SummaryCarpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. In the present study, four different grossly isolipidic, isoproteic, and isoenergetic diets containing ingredients from different origins were used for S. carpio culture. Specifically, a diet largely based on marine ingredients, and currently used for carpione farming, was used as control. Three other diets were formulated in order to include relevant percentages of vegetable ingredients or processed animal proteins (at two different inclusion levels). After a three-month feeding trial, fish zootechnical performances, welfare, and flesh quality were evaluated through a multidisciplinary approach, including histology, gene expression, chemical analysis, and Fourier transform infrared spectroscopy (FTIR). This study provided the first insights on carpione physiological responses to different commercial dietary formulations.Carpione (Salmo carpio, Linnaeus 1758) is an endangered precious endemism of Lake Garda (Northern Italy), the largest Italian lake. To date, several bottlenecks about its culture remain unsolved, including the identification of a proper growth-out diet. The aim of the present study was to test four different grossly isolipidic, isoproteic, and isoenergetic diets in which the main ingredients had a different origin. Specifically, a diet currently used by local farmers for carpione culture, largely based on marine ingredients, was used as control (CTRL), while the other three diets were formulated by partially replacing marine ingredients with plant ones (VEG) or with different percentages of processed animal proteins (PAP1 and PAP2). The feeding trial was run in triplicate, over a three-month period. No significant differences in growth performance among the experimental groups were observed. However, remarkable histological alterations and inflammatory markers upregulation were observed in VEG group, while PAP inclusion played a role in attenuating inflammation and improving nutrient uptake. Fillet analyses highlighted significant differences in marketable traits and flesh fatty acid composition among the experimental groups, including the reduction of polyunsaturated fatty acids related to PAPs inclusion. In conclusion, PAPs used in the present study promoted S. carpio gut health and absorption capacity, while further studies are required to maintain proper quality traits of the final product.

Adult European Seabass (Dicentrarchus labrax) Perform Well on Alternative Circular-Economy-Driven Feed Formulations

There is an increasing need in the aquaculture industry for more sustainable and functional feed concepts for marine finfish. This study provides results for the effect of alternative feed formulations on health status, welfare parameters, sensory analysis, and growth performance in European seabass (Dicentrarchus labrax) over an 83-day feeding trial. Fish were fed twice a day with five experimental diets. A control diet (control) and four different alternative feed concepts rich in processed animal proteins (PAP), other alternative ingredients (NOPAP), and a positive (NOPAP+) and negative (PAP−) formulation were tested. All alternative formulations contained hydrolysates from aquaculture by-products and macroalgae. The results indicate that the alternative feed concepts are more sustainable alternatives compared with the commercial diet. Equally interesting, the alternative formulations did not affect the sensory analysis of the fillet quality or the animal welfare. These are increasingly important factors in aquaculture products and, accordingly, also in the formulation of new feeds. Feed concepts that are not only more sustainable in their production, have shorter transportation distances, recycle the resources (usage of by-products), and have no adverse effect on growth or welfare parameters are highly needed. Therefore, the experimental diets tested in this study are a win-win concept for future seabass aquaculture production.

Dioxins and polychlorinated biphenyls in insect processed animal proteins used as a feed – is there a health risk?

Insects have all the characteristics necessary to become an alternative source of protein in feed. However, as with feed materials of animal origin, insect processed animal proteins (IPAPs) can also accumulate persistent organic pollutants such as dioxins and polychlorinated biphenyls (PCBs). Therefore, the aim of the present study is to complete the information on dioxin and PCB concentrations in feeds and compound feeds containing insect protein from mealworms (Tenebrio molitor) and the black soldier flies (Hermetia illucens). For the IPAPs samples of both mealworms and the black soldier flies, polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) results ranged from 0.05 to 0.10 ng WHO-TEQ/kg feed at 12% moisture, for the total PCDDs/PCDFs/dl-PCBs ranged from 0.07 to 0.12 ng WHO-TEQ/kg feed at 12% moisture, and for non-dioxin-like polychlorinated biphenyls (ndl-PCBs) from 0.04 to 0.43 μg/kg feed at 12% moisture. Additionally, an experiment was conducted in which broilers were fed feeds containing IPAPs in an attempt to assess the risk to food consumers regarding dioxins and PCBs. Our studies showed that IPAPs from mealworms (T. molitor) and black soldier flies (H. illucens) will not pose a risk in terms of dioxins and PCBs.

Towards a circular bioeconomy: High added value protein recovery and recycling from animal processing by-products

This paper reports the studies on the recovery of proteins from animal by-products (ABPs). Specifically it proposes the use of ABPs Category 2 as secondary raw material as an alternative to ensure a sustainable production of higher added value products, mainly protein biopolymers as retanning agent for leather manufacturing and amino acid-based biostimulants, using an enzymatic process as methodological solution that has proven to be suitable for the treatment of non-tanned hides and processed animal proteins (PAPs) to obtain hydrolysed collagen with good yields and performance. The developed process has proved to allow the recovery up to 85% protein present in the treated animal by-products and it has shown itself to be a versatile process easily customisable to different kinds of ABPs. The validation test for application as biostimulant have been conducted at lab scale through cabbage germination showing an improvement of 25%. In addition, the retanning process results have been satisfactory and the retanned leathers comply with the requirements established to be used in footwear manufacturing.

Hyperspectral imaging coupled with CNN: A powerful approach for quantitative identification of feather meal and fish by-product meal adulterated in marine fishmeal

Marine fishmeal (MFM) adulterated with low-cost processed animal proteins (PAPs) such as hydrolyzed feather meal (HFM) and fish by-product meal (FBM) has frequently occurred in the Chinese trade market. This commercial fraud generates a serious threat to farmed animal health and even human food safety. This study aims to develop a rapid detection method using near-infrared hyperspectral imaging (NIR-HSI) combined with deep learning modeling for qualitative and quantitative identification of MFM adulterated with HFM, FBM, and the binary adulterant (HFM-FBM). Three convolutional neural network (CNN) architectures with optimized parameters were constructed to predict sample classes, adulterant concentration, and amino acid content of adulterated samples, respectively. Partial least squares (PLS) and support vector machine (SVM) models were compared with the proposed CNN models. The overall results showed that the CNN outperformed the PLS and SVM on both classification and regression. The six-classification accuracy obtained by the CNN was up to 99.37%, while the R2 of CNN regression prediction varied from 0.984 to 0.997. This study demonstrates that NIR-HSI coupled with CNN calibration provides a promising technique for the detection of MFM adulterated with PAPs.

Glyceroltriheptanoate (GTH) occurrence in animal by-products: a monitoring study to minimise safety-related risk of misuse

Early in this century, the crisis connected to the spread of bovine spongiform encephalopathy caused a great concern related to the use of animal by-products (ABPs). According to the Commission Regulation (EU) No 1069/2009, these materials are classified in three categories according to their related risk. In 2011 Commission Regulation (EU) No 142/2011 established that meat and bone meal (MBM) and fat deriving from ABPs not intended for human consumption (category 1 and 2) are required to be permanently marked with glyceroltriheptanoate (GTH), at a minimum concentration of 250 mg kg–1 of fat, while category 3 processed animal proteins (PAPs) must not contain this compound. PAPs are bio resources, which could be used in a renewable and regenerative way in a circular economy model for a conscious usage of raw materials. The aim of this study was to provide information on GTH occurrence in MBM and, if any, in PAPs. Samples were collected from 2017 to 2021 and analysed by GC-MS. Detected non-compliant samples were exclusively of MBM category 1 and 2, probably due to the addition of an inadequate amount of GTH during the manufacturing processes. These results highlighted the importance of National Monitoring Programs as a useful tool to minimise safety related risk due to the misuse of GTH. Thus, investigating the critical points in feed supply-chain and sharing the information on its occurrence may help to improve animal and human wellness and safety.

Sustainable fish feeds: potential of emerging protein sources in diets for juvenile turbot (Scophthalmus maximus) in RAS

In Europe, turbot aquaculture has a high potential for sustainable production, but the low tolerance to fishmeal replacement in the diet represents a big issue. Therefore, this study investigated the effects of more sustainable feed formulations on growth and feed performance, as well as nutritional status of juvenile turbot in recirculating aquaculture systems. In a 16-week feeding trial with 20 g juvenile turbot, one control diet containing traditional fishmeal, fish oil and soy products and two experimental diets where 20% of the fishmeal was replaced either with processed animal proteins (PAP) or with terrestrial plant proteins (PLANT) were tested. Irrespective of diets, growth performance was similar between groups, whereas the feed performance was significantly reduced in fish of the PAP group compared to the control. Comparing growth, feed utilisation and biochemical parameters, the results indicate that the fish fed on PAP diet had the lowest performance. Fish fed the PLANT diet had similar feed utilisation compared to the control, whereas parameters of the nutritional status, such as condition factor, hepato-somatic index and glycogen content showed reduced levels after 16 weeks. These effects in biochemical parameters are within the physiological range and therefore not the cause of negative performance. Since growth was unaffected, the lower feed performance of fish that were fed the PAP formulation might be balanced by the cost efficient formulation in comparison to the commercial and the PLANT formulations. Present study highlights the suitability of alternative food formulation for farmed fish.

Detection of Alphitobius diaperinus by Real-Time Polymerase Chain Reaction With a Single-Copy Gene Target.

Use of edible insects as an alternative source of proteins in food and feed is increasing. These last years, numerous companies in Europe have started producing insects for food and feed purposes. In the European Union, the use of edible insects for human consumption falls within Regulation (EU) No. 2015/2283 on novel foods. For feed, Commission Regulation (EU) 2017/893 authorizes seven insect species as processed animal proteins for aquaculture. Methods of authentication are required to check the conformity of the products. In this study, we propose a real-time polymerase chain reaction (PCR) method for the specific detection of the lesser mealworm (Alphitobius diaperinus), one of the species included in the shortlist of authorized insects. The selected target is the cadherin gene with a single-copy (per haploid genome) illustrated by our experimental evidence. The PCR test amplified a 134-bp fragment of the cadherin gene. The qualitative method was assessed toward several performance criteria. Specificity was checked against 54 insect species next to other animal and plant species. The sensitivity, efficiency, robustness, and transferability of the PCR assay were also successfully tested. Finally, the applicability of the test was assessed on real-life processed samples (industrial meals) of A. diaperinus. The study also showed that there seems to be a huge confusion on the correct labeling of the marketed mealworms. We did not succeed to get Alphitobius laevigatus samples. They all appeared to belong to the A. diaperinus taxon.

The consumer preferences for processed meat products based on choice brand priorities

Processed animal protein is typically preferred over fresh livestock products for consumption because it is more stable and environmentally friendly when stored over a long period. Therefore, this preliminary study aimed to determine consumer preferences for processed meat products based on consumer choice brand priorities. The consumers were asked to select three preferred processed meat product brands, including sausage, corned beef, meatballs, nuggets, and meat floss, as well as the primary reason for their selection. The study included 450 meat consumers who were selected by purposive sampling. Subsequently, descriptive statistics and Chi-Square analysis were used to determine the preferences among the three product selections. The results showed that the three main preferences of consumers in selecting processed meat products were based on taste, quality, and price in that order. Consumers preferred and selected products with superior taste and quality but were more likely to consider price as the priority decreased. Therefore, the ability to understand consumer preferences plays a key role in developing a sustainable product since cost is not considered a primary concern.

The utilisation of European processed animal proteins as safe, sustainable and circular ingredients for global aquafeeds

AbstractThe global increase in seafood demand has resulted in significant growth in aquaculture production in a wide range of aquatic species. Consequently, this has led to an industry‐wide need to find sustainable feed ingredients that would meet the nutritional requirements used in aquafeeds. The associated dependency on plant by‐products as the major alternatives has brought concerns to aquaculture through associated carbon footprint, increase deforestation and arable land use to meet the demands of plant proteins and oils, and the constraining effects of plant by‐products has on farmed aquatic animal growth and health. Animal by‐products (ABPs) are produced as a direct consequence of terrestrial animal production and the associated meat processing industries. The link between feeding meat and bone meal (MBM) and bovine spongiform encephalopathy (BSE) outbreak in Europe during the 1980s resulted in a ban of its use animal feeds. This led to a radical overhaul of the rendering industry, including the use of only low‐risk ABP and the development of rendering processes to reduce the risk of prions that causes BSE to enter the food chain. The resulting processed animal proteins (PAPs) are considered safe to be used in farmed animal feeds. This review examines how ABP production has changed due to the BSE outbreak, leading to the current commercially available PAP products for aquafeed use. We evaluated how these products can be effectively used as viable protein sources in aquaculture and examine their limitations and the potential advancements that could lead to a more circular food production system.

Processed animal proteins (PAPs) in animal nutrition: Assessment of the chemical risk of essential and non-essential elements

BackgroundProcessed animal products (PAPs) could be a great alternative to common protein supplements and represent a good example of recycling and valorization of by-products. Due to the reintroduction of certain types of PAPs in feed, a deeper knowledge of these heterogeneous matrices is needed. Thus, the aim of this study is to evaluate the levels of essential elements and inorganic contaminants in 55 PAPs considered as potential alternatives to common protein supplements. MethodsPAPs samples were analysed for essential (cobalt, nickel, chromium, copper, zinc, iron and manganese) and non-essential elements (arsenic, cadmium, lead and mercury) by Inductively Coupled Plasma Mass Spectrometer (ICP-MS), Graphite Furnace Atomization Atomic Absorption Spectrometer (GF-AAS) and dual cell Direct Mercury Analyzer spectrometer (DMA-80). ResultsEssential elements were found with the following decreasing order iron>zinc>copper>manganese>chromium>nickel>cobalt (mg kg−1). Only one sample was found non-compliant to lead concentration according to the European Union Regulation while negligible values of others non-essential elements were found. ConclusionsThis study suggests that PAPs could be a useful supplement for animal diet due to their natural content of essential elements. A careful monitoring of chemical elements should be required and eventually guidelines have to be drafted for a correct use of PAPs to ensure a safe and sustainable feed production.

Re-authorization of gelatin and collagen of ruminant origin in non-ruminant feed: a new analytical challenge for the control of the feed ban

Description of the subject. A recent revision of the feed ban provisions authorizes the use of ruminant collagen and gelatin in feed for non-ruminant farmed animals. This authorization will promote the use of former foodstuffs (FFS) in poultry and pig feed. Objectives. The study aimed to investigate how these ruminant materials could impact the capacity to detect processed animal proteins (PAP) of ruminant origin in feed using the official PCR method and to evaluate the added value of a mass spectrometry protocol in development. Method. Presence of ruminant DNA in samples of collagen hydrolysate, gelatin and FFS collected from the industry was assessed using the official DNA extraction and PCR method. This allowed to evaluate the PCR inhibition properties of gelatin and collagen. The same samples were also submitted to a mass spectrometry-based proteomics (UHPLC-MS/MS) protocol targeting ruminant proteins, including collagen, to distinguish between ruminant by-products (unauthorized or authorized). Results. The results show the complementarity of PCR and UHPLC-MS/MS approaches in the context of the use of former foodstuffs in animal feed. Their combination has allowed to evidence that the presence of ruminant DNA in samples containing FFS was more linked to the presence of milk than to the presence of gelatin. On contrary, some samples have shown an increase of the cycle threshold value (Ct) that could correspond to an inhibitory effect due to gelatin addition. Conclusions. In the context of the circular economy, FFS is an interesting source of nutriment for animal feed. However, due to the presence of dairy ingredients, interference with official methods giving false suspicion of prohibited materials is to be expected. Furthermore, a masking effect of the presence of PAP due to a PCR inhibitory effect by FFS is also possible.