Role of trypsin inhibitors and their analysis

Introduction

Trypsin inhibitors are naturally occurring proteins found in many plants, particularly legumes (beans, peas, lentils) and some cereals. They play a defensive role in plants by inhibiting trypsin, a digestive enzyme in insects and animals, that helps break down proteins. In the context of food, trypsin inhibitors have a complex role, acting as both antinutrients and potentially beneficial compounds.

Role of Trypsin Inhibitors

Nutritional Impact: Trypsin inhibitors can affect protein digestion and absorption in the gastrointestinal tract. By inhibiting trypsin, they reduce the availability of amino acids from dietary proteins, potentially leading to decreased protein utilization and nutrient absorption.

Antinutritional Factors: In high concentrations, trypsin inhibitors can be considered antinutritional factors because they interfere with protein digestion and may contribute to nutritional deficiencies, particularly in diets heavily reliant on protein-rich foods.

Defense Mechanism: In plants, trypsin inhibitors serve as defense mechanisms against herbivores. By inhibiting digestive enzymes in the gut of herbivores, they can reduce the nutritional value of ingested plant proteins, making the plant less appealing as a food source.

Heat Stability: Trypsin inhibitors can withstand certain food processing methods, including heating, which means they may still be present in significant amounts in cooked foods.

Importance of testing

Analyzing trypsin inhibitors in foods is important for several reasons:

 Standards and Regulations

FSSAI

The specifications related to trypsin inhibitors as per the FSSAI standards include the following requirements for formulated supplements for children:

• Cereals used should be processed to reduce anti-nutrients like phytates, tannins, lectins, trypsins, and chymo-trypsin inhibitors that can affect protein quality and digestibility
• Legumes and pulses must contain at least 20% protein on a dry basis and should be processed to reduce anti-nutritional factors like lectins, trypsin, and chemo-trypsin inhibitors
• Oil seed flours and oil seed protein products should have reduced anti-nutritional factors and toxic substances like gossypol and urease activity
• Animal source foods used should be nutrient-dense and a source of high-quality protein
• Fats and oils added should not contain partially hydrogenated fats
• Fruits, vegetables, milk, and milk products should be included for their nutritional value
• Other ingredients like digestible carbohydrates, protein isolates, probiotics, prebiotics, vitamins, minerals, and nutrients can be added to enhance the nutritional quality of the product

https://fssai.gov.in/upload/uploadfiles/files/Draft_Notice_Comments_Supplemnts_Children_28_03_2018.pdf

EU

• The European Union (EU) regulations related to trypsin inhibitors include specific requirements for food enzymes containing trypsin. A safety evaluation was conducted for a food enzyme containing trypsin, chymotrypsin, α-amylase, and triacylglycerol lipase from porcine pancreas, ensuring compliance with EU regulations
• Additionally, the safety evaluation of mung bean protein as a novel food pursuant to Regulation (EU) 2015/2283 included data on anti-nutritional factors like trypsin inhibitors, emphasizing the need to assess the safety of novel foods in accordance with EU regulations

https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2021.6846

USFDA

The USFDA (United States Food and Drug Administration) provides specifications and standards for trypsin inhibitors, focusing on safety evaluations and inactivation methods. These regulations emphasize the importance of assessing the safety of food enzymes containing trypsin, chymotrypsin, α-amylase, and triacylglycerol lipase from porcine pancreas to ensure compliance with food safety standards

https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2021.6846

Analysis of Trypsin Inhibitors in Foods

Enzyme Activity Assays: Trypsin inhibitor activity in foods can be assessed using enzyme activity assays. These assays involve measuring the ability of a food extract to inhibit the activity of trypsin on a specific substrate.

Protein-Based Assays: Some assays use purified trypsin inhibitors as standards to quantify the amount of inhibitor present in food samples. These assays often involve protein quantification techniques such as Bradford assay or Lowry assay.

Immunological Methods: Immunoassays, such as enzyme-linked immunosorbent assay (ELISA), can be used to detect and quantify specific trypsin inhibitors in food samples using antibodies raised against known inhibitors.

Molecular Techniques: Polymerase chain reaction (PCR) and related molecular techniques can be used to detect and identify genes encoding trypsin inhibitors in food sources. This allows for the analysis of trypsin inhibitor levels at the genetic level.

Biochemical Methods: Chromatographic techniques, such as high-performance liquid chromatography (HPLC) and gas chromatography (GC), can be employed to separate and quantify trypsin inhibitors in complex food matrices.

Eurofins role in testing trypsin inhibitors

Eurofins, a global food testing company, likely offers testing services to analyze trypsin inhibitor activity in foods, although it's not explicitly mentioned in the provided FSSAI notification. Here's why:

Analytical Techniques:

• In Vitro Assays: Eurofins likely has the expertise to perform in vitro assays, a common approach for quantifying trypsin inhibitor activity. These assays might include:
• Radial Diffusion Assay: This method involves creating a well containing trypsin and a substrate (a substance trypsin breaks down) in a gel. The sample containing trypsin inhibitors is added nearby. If inhibitors are present, they will create a clear zone around the well due to inhibited trypsin activity.
• Spectrophotometric Assay: This technique utilizes a chromogenic substrate (changes color when broken down by trypsin). Eurofins can measure the rate of color change in the presence and absence of the sample. Reduced color change indicates the presence of trypsin inhibitors.
• Chromatographic Techniques: For a more detailed analysis, Eurofins might employ chromatographic techniques to separate and identify specific types of trypsin inhibitors.
• Size-Exclusion Chromatography (SEC): This method separates molecules based on size. Trypsin inhibitors can be isolated and quantified based on their elution (exit) time from the chromatography column.
• Affinity Chromatography: This technique uses a column containing immobilized antibodies that specifically bind trypsin inhibitors. The sample is passed through the column, and the captured inhibitors are then eluted and quantified.

Expertise in Food Analysis: Eurofins is a recognized leader in food testing with a broad range of capabilities. Analyzing anti-nutrients like trypsin inhibitors aligns with their expertise in food safety and quality control.

Regulatory Compliance: FSSAI emphasizes good manufacturing practices for CFOI&YI, and Eurofins likely offers services to help ensure compliance with these guidelines. Trypsin inhibitor analysis can be part of this process.

Global Reach: The FSSAI notification is an Indian regulation, but Eurofins operates globally. Their services likely encompass testing relevant to international food standards that might indirectly address trypsin inhibitors.

Quantifying Trypsin Inhibitor Activity: Eurofins likely has the expertise and equipment to measure trypsin inhibitor levels in food samples using established methods like in vitro assays or chromatographic techniques.

Supporting Process Optimization: By analyzing trypsin inhibitor content before and after processing, Eurofins can help manufacturers identify the most effective methods for reducing these inhibitors in CFOI&YI.

Ensuring Compliance: If future regulations mandate specific trypsin inhibitor limits, Eurofins can provide testing services to demonstrate compliance with these standards

References

1. Avilés-Gaxiola, S., Chuck-Hernández, C., & Serna Saldívar, S. O. (2017). Inactivation Methods of Trypsin Inhibitor in Legumes: A Review. Journal of Food Science, 83(1), 17-29. https://doi.org/10.1111/1750-3841.13985
2. Savage, G., & Morrison, S. (2002). TRYPSIN INHIBITORS. Encyclopedia of Food Sciences and Nutrition (Second Edition), 5878-5884. https://doi.org/10.1016/B0-12-227055-X/00934-2
3. https://www.coherentmarketinsights.com/market-insight/trypsin-market-4866

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