What Safety Assessments Do You Need For Novel Sourced Food Proteins?

Safety first is a key mantra for anybody launching a new food into the market place. It’s especially pertinent in this day and age for novel-sourced food proteins because consumers are seeking a much wider variety of environmentally sustainable, better-for-you meat-alternative ingredients to satisfy their increasingly healthier lifestyle.

Any new ingredient needs to be evaluated first off, for safety. The conclusion then is for this new protein to be deemed safe under the conditions of intended use. It must satisfy regulatory and manufacturing compliance for any regulatory body. But what are the steps needed to conclude safety for a protein source such as insect larvae or a new plant protein food?

This blog article will cover different aspects by which novel protein ingredients ought to be evaluated. We will explore and establish the unique specification and contaminant requirements to potential variations in the studies necessary to conclude that the novel protein source is safe as when part of the diet. A discussion on the development of new protein sources, potential risks, and methods to decrease potential concerns will be emphasized.

Exciting times ahead then as novel sourced food proteins enter the marketplace. However, to get a successful launch also means a considerable amount of safety assessment is required before any business can seriously launch their novel protein into the ingredients marketplace. Safety assessments are needed for insect proteins, cultured meat and now for any new plant-based foods especially. 

At this moment in time in the USA, we know that regulations from the FDA and other regulatory bodies, state that all food ingredients must be concluded as safe prior to their addition to foods generally. Any business in this industry is legally required to conduct an assessment of safety under the intended conditions of use. Finally, not only the source of the  novel protein or the protein product itself, but the manufacturing process must be assessed as it affects the overall safety of the new protein-based ingredient.

As a final point to this article’s introduction, novel sources of protein are potentially having an adverse impact on the climate. We know that increasing our protein intake from other sources means we must as consumers be fully cognisant of the social and environmental impact our decisions are having in choosing them. Some of the animal-based proteins can be replaced with new protein, plant-based sources but we don’t know yet how far this can be taken. One of the considerations in the safety assessment now is the holistic impact that a new plant protein might have when it replaces another. 

Characterisation of the Food Ingredient

We ask ourselves at first, what are the components necessary for the evaluation of new ingredients. These are especially the new protein-based ingredients which also has to include now, characterization of the end product of that ingredient. 

Consider the case where a new protein source has been sourced from a microalgae or a microbe. Regulatory bodies like the FDA or EFSA want to understand the source of that protein, what that new microbe or microalgae happens to be. So, the source of the raw ingredient must be firmly stated as well as any modifications and chemical changes that have occurred to that material. A typical chemical modification could be some external hydrolysis of the protein that improves its bioavailability prior to consumption. Such changes can have an impact on the safety of that ingredient.

We must define the manufacturing process as it will be evaluated by the regulatory body and check there has been a safe use of any technical manufacturing additives including processing aids. All these processing materials such as enzymes and antifoams need to be safe and suitable for use in a food environment or in food process development.

A complete understanding based on analysis of the whole ingredient is needed and not just the active protein component. When setting the specifications for any the new ingredient you must know and show you have such specifications that ‘define’ the ingredient and that your product being created is also reproducible from one batch to the next. Finally, characterization must also include a stability analysis meaning that the new ingredient must be stable over its lifetime.

General Composition

The components then needed to evaluate a potential new protein are built on the General composition or identity as it might be known. So:

• All the components for the final ingredient are known to virtually 100% especially the defining constituents. 
• The protein, fat, carbohydrate and ash contents are defined
• The constituents are defined as in what component is the reason to add this ingredient to food. For example, are there some specific amino acids that you would like to have increased in the diet that are necessary for growth or perform some other defined function. That implies a full analysis of the different amino acids.

Other specifications are strictly safety-related and include safety parameters and identifying characteristics:-

• heavy metal content such as cadmium, lead, zinc, arsenic, copper and mercury
• key presence of microbials and that includes total colony counts or specific microorganisms such as Salmonella and Escherichia coli. It means that the manufacturing process must be evaluated and the end products checked  to the point where there is no step in the process that allows for microbial growth
• A pesticide and toxin analysis is needed although it depends on the raw materials used
• What are the value components i.e. the specific amino acids, fatty acid and vitamins. In this day and age we are trying to upcycle ingredients using the complete raw materials in production and manufacturing of commodity products to get more value out of them. For example, if the grain from wheat has been used, but not the straw component, and so someone may come up with a way of using that waste straw to produce a protein-based product. That straw could have pesticide residues that have to be measured and accounted for especially ensuring they are not in the final product.

Production and Stability Assessments Help Define a Potential New Protein Source

The manufacturing process is critical to understanding possible issues. Regulators like to see the following:

• a diagram which shows how the ingredient is produced. Consider a microalgal-derived protein; is it a closed growing system? An open pond system for microalgal growth and wild-harvest to packaging. The FDA wants to see the whole process from growth to its packaging because all these steps impact safety. Lets say the protein contains lots of fat so packaging must be such that there is limited diffusion into or out of that fat of harmful contaminants being absorbed from the packaging.
• minimization of potential contaminants during growth
• temperature
• packaging
• documenting critical control points – to minimise potential for contamination during processing. A key feature of any HACCP study.

Stability (ambient and accelerated) of identifying characteristics; isolated and when in food or feed. The FDA also needs to know how stable the ingredient is over shelf-life. Not just the neat substance or the product being produced and that its stable on the shelf but as its being produced it does not change especially when it is added as an ingredient to beverages, bars etc. or other end consumer products. Need to analyse for the following:

• protein
• specific fatty acids to show no rancidity is occurring
• vitamins
• reasons for addition to food

Some shelf-life testing includes accelerated stability studies but the best are usually those at ambient or raised temperatures. The FDA is most interested in performance of these key aspects if they relate to the performance of the products or the claims to be made.

So as part of the assessment of a novel food ingredient which is protein based, the proteins contained in the ingredient in presentations  and dossiers will cover safety elements in different ways such as a:

• history of safe use of the protein being consumed. It could have been in a different product of course, come from a different raw material but this is from a new raw source for that protein.
• bioinformatics analysis to analyse the actual proteins being produced
• mode of action
• in vitro digestability studies to show it will be hydrolysed in the gut producing safe breakdown products such as amino acids as with any other protein.
• dietary intake including how it will be digested.

A key feature is that protein toxins that have been identified in plants include antifungal proteins and lectins. These may have been carried over from the raw source and must be proven to be present in safe enough levels or completely absent. Consider the protein from wheat straw because it could carry through antifungal proteins from the wheat stems that are carried into the final product. Need to have a good look at the overall process.

The Issue of Amino Acid Imbalances

Tryptophan consumption in weaned pigs positively affects intestinal morphology and tight junction proteins.  For example,  if you increase other amino acids at the expense of tryptophan, a tryptophan deficiency means decreased growth and reduced protein breakdown in low protein diets (Humbert et al., 2001). Pigs are good models for human guts and a good way to evaluate what might happen in a nutritional sense if a protein is taking up the majoity of the diet.

5-hydroxytryptophan (5-HTP) is a precursor of serotonin and melatonin. A 5-HTP over dose of >23.6 mg/kg bw means excessive concentrations of serotonin at target cells (GI, CNS, cardiovascular and respiratory systems) (Gwaltney-Brant et al., 2000). The effects can be very severe. It can induce a serotonin-like syndrome in dogs (Hopkins et al., 2017) such as seizures, depression, tremors, ataxia, vomiting, diarrhea, hyperthermia, transient blindness and even death. These clinical signs and can develop within 4 hours after ingestion and last up to 36 hours. It implies that you have to look at the ratio of different amino acids in a new protein to be clear about possible issues in humans.

Defining traits of source organisms

A series of questions asked by the FDA and other regulatory bodies include the following:

• is the protein microbially sourced or enzyme-based?
• is the species or strain well defined and characterised and is it deposited in a an international repository?
• is is a native species or genetically modified?
• Has it been derived via selective pressure versus CRISPR?
• If modified, are there insertion or deletion elements that contain antibiotic resistance genes?
• Is there up-regulation or deletion of native genes?
• Will the protein sourced microbe be viable in the final product?
• What is the potential to produce toxins and are there any indications of toxin-producing gene sequences found?
• Does the genus contain pathogenic species?
• Is there potential for translocation of genetically modified DNA?

Genetic Engineering

There is potential for genetically engineered proteins to become potential toxicants. Any safety assessments need to consider this possibility or at least make reference to this aspect.

The following factors are considered:

1. The source of the gene will contribute to the safety assessment. For that protein, is the gene sequence stable?
2. The similarity of the amino acid sequence of the protein of interest to that of known toxins
3. Is their stability of the protein to digestion by pepsin in an invitro digestibility assay e.g. pepsin digestion assay? It means that it would not break down in the stomach for example if not open to digestion.

Pepsin digestion – a reliable technique to check digestibility of proteins in the gut and to see what amino acids and peptides are generated.

• evaluation of protein fragments via simulated gastric fluid (SGF) on SDS-PAGE protein gels. Evaluate if proteins are readily degraded to small Molecular Weight fragments 10 kD).
• All these evaluated on a case by case basis.

Fermentation By-Products

Fermentation may have modified by-products as well as the bacterially derived protein of choice. The fermentation may be creating metabolites that are potentially unsafe for consumers (human and animal). The FDA expects a safety assessment of by-products from genetically engineered microbes.

The media components must all be food grade and or safe and suitable for their intended use. They too require separate safety assessments. It means anything that is involved in the production of the final protein products must be checked out.

Is their worker safety? allergenic potential? A need for an environmental assessment.

Anti-Nutrients Contained Within Nutrient-Based Ingredients

Nutrients are essential for growth, reproduction and maintenance when consumed within necessary levels. Certain nutrients, when consumed in excess, are toxic to companion animals. We mentioned 5-HTP when consumed in excess.

Plants both commonly consumed and novel produce substances which are anti-nutrients to defend against fungi, bacteria, insects etc., which may inhibit nutrient uptake by mammals generally.

Inclusion of novel ingredients to pet food may make a diet that is compliant with nutrition profiles, but it is what is unknown in those ingredients that could adversely impact digestion of the protein and adversely affect nutrient absorption, leading to toxicity and so decrease overall nutrient value.

Trypsin Inhibitors, Lectins and Tannins

• present at high levels in raw, unprocessed legume seeds
• Pigs: reduces ileal digestability of protein when Phaseolus beans included in diet (Huisman, 1991).
• Not known if or how much trypsin inhibitors, lectins and tannins affect dogs and cats
• Adequate heating and cooking significantly reduces levels (Shi et al., 2017).

Thiamine antagonists: Plant-derived thiamine-binding proteins inhibit thiamine bioavailability.

• oxythiamine: formed from histamine in strongly acidic media (acid protein hydrolysates)
• pyrithiamine: inhibits phosporylation of thiamine.

Induces gastric complications such as gastric ulcers, impairs glucose absorption, increased emptying time, leading to decreased food intake (Velisek, 2014).

Characterization or evaluating the production organism and manufacturing process especially of its novel.

Intake levels also critically impact the overall safety of any new proteins compared to overall protein intake. This means how much is to be consumed in the diet.

Intake is really key to understanding a novel proteins performance.

The target or lifestage must be defined as in what is the target population in general but also the impact of the food on different life stages.  How much food substance is to be added to diet? It means determination of the Estimated Daily Intake (EDI). Will the protein-based ingredient enter the bloodstream intact?

If your protein product is planned to be 50% of all protein intake in a person’s diet, it could replace other key nutrients which might not be wholly desirable.

There is replacement of other nutrients which means possibly an increase in some nutrients over others.

What about tolerance issues and amino-acid imbalances as already mentioned?

• Anti-nutritive components in fermentation by-product? Non-starch polysaccharides (NSP) may adversely affect digestion.
• beta-glucans, lectin protease inhibitors
• affect digestibility of other nutrients such as increased viscosity.

Finally, typical studies that are used to determine the safety of a novel ingredient.

A number of safety studies may be required to justify support with the FDA that they will typically want to see when supporting a food additive safety regulation, a GRAS determination to conclude safety or a petition submitted to EFSA or another international regulatory body. Some examples of typical toxicity studies include:

Repeat-dose/dietary toxicity studies in rodents

• conducted according to FDA standards or OECD protocol so they are acceptable to the regulatory authorities. They must have the right endpoints and analyses to understand and conclude the safety of the ingredient. – typically 90 days in length
• 14-day palatability study prior to main study
• safety-related endpoint parameters should be included
• test substance should meet commercial specifications.

Genotoxicity Studies

A study checking a proteins impact on the genetic code. It could be a component from the overall product hat is the culprit though. Let’s say the product is 50% protein but the other 50% may be the actual factor inducing genotoxicty.  The FDA is very keen to see this does not happen.

• typically both in vitro and in vivo genotoxicity studies
• conducted according to FDA/OECD protocols

Assessment of potential allergens may be necessary.

Conclusion

In conclusion you need to look at the novel protein as a whole entity. The critical components for evaluating a potential new protein food ingredient include

• documentation that a safe and consistent ingredient is produced so you can present the evidence it is safe and that there is consistency in its production.
• characterization of the protein source whether it’s from an organism or plant
• characterization of the final ingredient to be commercialised
• genetic modification that may have been done to increase protein content, it means too, increased scrutiny of genetic sequences and downstream events
• viability of a microbe in final product (microbial-based) increases complexity of safety evaluation – there is potential too for environmental assessment.
• safety studies conducted in vitro or in vivo i.e. an appropriate lifestage of the target companion animal.