Milk-clotting enzymes in dairy processing

The world of enzymes for the food industry is something fascinating. The technological roles of such processing tools encompass improvement of sensory profile (flavor, color, aroma) and general quality, specific hydrolytic processes, and so forth.

High specificity, high catalytic efficiency, and the opportunity to use them at mild process conditions make them suitable tools in food production. In the past, sources for enzymes used to be animal and plant materials, today we can also rely on microbial fermentation and DNA technology to increase availability, purity, specificity, cost-effectiveness.

The dairy industry can exploit a wide range of enzymes (milk-clotting enzymes, proteases, lipases, lactases, catalases, etc.), allowing an incredibly high number of different applications, all with an ultimate goal: improving shelf-life of dairy products. Milk-clotting enzymes cover up to 60% of the market share (Kocabaş, 2022).

The reason why milk-clotting enzymes represent the largest category within the market is due to their specific application in the most relevant stage for cheese production: milk coagulation into curd. Different options are available to achieve this step:

• animal rennet, which derives from the abomasum of young ruminants through a peculiar extraction process. The specific milk-clotting enzymes are chymosin (the most important) and pepsin. In 1874, Christian Hansen made the extraction of rennet possible at industrial level;
• plant-derived coagulants, which are obtained from plant organs of different species (e.g., stigmas of thistle – Cynara cardunculus – flower used to produce the typical Italian cheese Caciofiore di Columella);
• microbial coagulants. The commercial potential of these enzymes is remarkable, as they can meet vegetarian consumers’ needs. They can derive either from wild, non-GM microbial species, like Mucor, or from GM strains. IFF Bioscience’s Marzyme® is an example of non-GM microbial-derived coagulant obtained from Rhizomucor miehei.
• fermentation-produced chymosin (FPC), produced from GM Aspergillus niger or Kluveromyces lactis, which allows to get 100% pure chymosin. FPC is obtained by fermentation conducted by a host microorganism in which the gene of chymosin is expressed, allowing to get an enzyme identical to calf-natural chymosin. Chr. Hansen’s CHY-MAX® ( niger) and DSM’s Maxiren® (K. lactis) are two examples.

Chymosin from rennet is a protease and is the most suitable enzyme for milk coagulation due to the ratio between milk-clotting and proteolytic activity. It hydrolyzes a specific bond between phenylalanine (Phe105) and methionine (Met106) in k-casein, thus leading the formation of the soft gel (curd) due to a reorganization of the casein micelles through cross-linking.

Rennet has still a large market, especially in those EU countries where PDO/PGI dairy products are produced: for example, in Italy and France, product specifications of authorized PDOs establish the use of rennet of different origin (calf rennet, kid rennet, etc.).

Plant-derived coagulants contains different types of proteolytic enzymes, which do not have the high specificity of chymosin from rennet for the Phe-Met bond in k-casein. Liburdi and colleagues (2019) studied the coagulation efficiency of three plant extracts (C. cardunculus, C. papaya, F. carica) in bovine, buffalo, goat, and sheep milk at different temperatures. The C. cardunculus extract proved to be the most eligible plant-derived coagulant for cheesemaking, as it showed similar coagulation properties to conventional calf rennet. The authors also highlighted how the excessive proteolytic activity of plant-derived coagulants can negatively affect cheese processing.

Before enzymes are placed on the market, the main concern is related to food safety. In the US, according to Title 21, enzymes may be either regulated as secondary direct food additives through premarket approval process or determined to be GRAS. The outcome depends on the intended conditions of use of the enzymes. These rules apply to enzymes obtained from both GM and non-GM microorganisms. If the US follows a more product-oriented approach, in the EU there is a specific regulation for food enzymes (Reg. EU n. 1332/2008) which focuses on the production process. The Regulation aims at harmonizing the provisions on the use of food enzymes among EU member states. Prior marketing, each food enzyme must be evaluated by EFSA for safety and then approved by the European Commission. Currently, there is not a Union list of authorized food enzymes; moreover, their placing on the market and use and of the product obtained through their use are under national legislation. In Italy, chymosin produced through DNA technology was authorized in 1991.

Choosing the right milk-clotting enzyme requires knowledge on the type of milk, the target final product, the specific processing conditions, and the differences between the solutions available on the market. As discussed above, PDO/PGI products are somehow forced by product specifications to be produced by using animal rennet, which may need to be of a specific origin (kid rennet rather than of calf origin) to lend peculiar sensory characteristics (spicy taste due the presence of lipases in kid rennet); by the way, the wide range of options permits dairy processors to meet vegetarian consumers’ expectations.

What to expect from enzyme technology? Trialing new, different coagulants may also be positive to enhance quality of the company’s portfolio and optimize processing costs and efficiency. Contact the ALPHA-SOLUTIONS team if you are interested to know more!

References

Didem Sutay Kocabaş, John Lyne, Zeynep Ustunol, Hydrolytic enzymes in the dairy industry: Applications, market and future perspectives, Trends in Food Science & Technology, Volume 119, 2022, Pages 467-475, ISSN 0924-2244, DOI: https://doi.org/10.1016/j.tifs.2021.12.013.

Katia Liburdi, Carlo Boselli, Gilberto Giangolini, Simonetta Amatiste, Marco Esti, An Evaluation of the Clotting Properties of Three Plant Rennets in the Milks of Different Animal Species. Foods 2019, 8(12), 600; DOI: https://doi.org/10.3390/foods8120600

FDA, 2010. Guidance for Industry: Recommendations for Submission of Chemical and Technological Data for Food Additive Petitions and GRAS Notices for Enzyme Preparations
https://www.fda.gov/regulatory-information/search-fda-guidance-documents/guidance-industry-recommendations-submission-chemical-and-technological-data-food-additive-petitions

EFSA, 2023. Food enzymes.
https://www.efsa.europa.eu/en/topics/topic/food-enzymes