Development of a Feed Protease

Abstract

Enzymes are naturally present in all living organisms such as microbes, plants, animals, and humans, where they are involved in the control and occurrence of all reactions related to life. For thousands of years, their activity has been used to process and improve raw materials, e.g., in cheese making. Since then the development and application of industrial enzymes have been refined significantly, not least due to extensive progress in molecular biology. At present, enzymes are used in many production processes across industries.

In the feed industry, the use of enzymes is well established. Nearly all commercial feeds contain phytase, which acts to make phytate bound phosphorus in plant feedstuffs available to swine and poultry. However, many other enzyme classes can significantly improve the utilization of feed such as xylanases, ß-glucanases, pectinases, amylases, and, last but not least, proteases. Mono-gastric animals such as swine and poultry produce digestive proteases such as pepsin, trypsin, chymotrypsin, and carboxypeptidases, and digest feed proteins to a high degree. Yet a fraction of the ingested feed protein is excreted in the feces; thus there is an opportunity for an exogenous protease to improve the utilization of protein.^1,2 The use of proteases in feed has not been explored as extensively as other enzyme classes and, in many cases, the proteases investigated have been present as part of an enzyme mixture—for example, as Aspergillus or Bacillus wild type fermentations—making it difficult to valuate precisely the effect and impact of applying a protease.³ The aim of the present work is to describe the development of a dedicated protease product that specifically targets feed proteins and to highlight the benefits that it can bring to the feed industry and to the environment.

Screening for a Feed Protease

Numerous strategies can aid in the development of new enzyme solutions, but in general, the larger the number of potential enzyme candidates to choose from the greater the probability of finding highly efficacious enzymes. Enzyme diversity can be a result of enzymes produced by natural wild type microorganisms or of “designed,” protein engineered enzyme variants. In the case of the feed protease described here, the foundation for the screening was Novozymes' large databank of isolated natural microorganisms. In the search for a new enzyme application it is of utmost importance at the initiation of the process to define as precisely as possible what the required action of the enzyme is and under what conditions the enzyme should be active. Globally, soybean meal is the main source of protein for poultry and pigs. Therefore, the first screening criterion was the ability of the protease candidates to degrade a soybean meal substrate that had already been substantially degraded by digestive proteases (pepsin and trypsin) and fiber-degrading feed enzymes (e.g., xylanase, pectinase, and ß-glucanase). The goal was to select enzymes that would work on a relevant feed substrate and, moreover, enzymes that could act above and beyond the enzymes normally present in the gastrointestinal tract. Access to the wild type microorganism collection and a relatively fast and simple first screening assay made it possible to screen a large number of diverse enzyme candidates.

While the first assay ensured high throughput, the target of the next step in the screening process was to determine if the proteases could withstand and function in the conditions of the gastro-intestinal tract. These characteristics included resistance to low stomach pH and the digestive proteases secreted in the stomach and small intestine, while maintaining activity in the pH range of 3–7. Many of the proteases tested in this screen, and also several of the proteases referred to in the literature, belong to the class of subtilisin proteases (S8 family). These have been shown to be instable at low pH.⁴ To rank the candidates according to these criteria, potential candidates were analyzed in an in vitro digestion system, which simulated a low pH stomach step in the presence of pepsin, as well as a small intestinal step, in which porcine pancreas enzymes were added to account for the digestive capacity normally present in the small intestine.

After incubation, the in vitro samples were analyzed for degree of protein hydrolyses (%DH) to assess the extent of hydrolysis by the combination of the digestive proteases and the exogenous test protease.⁵ In addition, samples were analyzed with high performance liquid chromatography (HPLC) to estimate the amount of solubilized protein and the percentage of solubilized protein that had a molecular weight lower than 1,500 Da using size-exclusion chromatography (designated as “in vitro digestible protein”).⁶ Based on the in vitro tests, a serine protease from Nocardiopsis prasina (S1 family) appeared to be the most promising candidate. In a group of the five remaining candidates garnered from in vitro screening and testing, this protease was later confirmed to be superior in animal feeding trials. Although in vitro tests are useful tools in the search for feed enzymes, the only way to establish that a candidate has the full potential to be a commercial feed additive is to test it in the animal species of interest.

Interestingly, in vitro benchmark tests with other commercial feed additives, which declare protease activity but primarily have other enzyme activities such as xylanase, showed that while these products also increased the solubility of protein, they did not increase protein hydrolysis, as did the Nocardiopsis protease. Feed additives that contain xylanases, for example, may render feed proteins more soluble by degrading the fiber matrix,⁷ but not directly target the protein. As soybean meal and corn are the most widely used feedstuffs, a mixture of these ingredients was selected as the substrate in the initial in vitro tests. These ingredients, along with a large number of different feedstuffs, were eventually found to be significantly improved by the selected protease.

Having confirmed the beneficial effect of the protease in feeding trials with broilers, subsequent research demonstrated that the protease was stable in feed and under storage and feed processing conditions. Finally, optimal production processes, including fermentation, recovery, and formulation, were established. The protease is produced by a genetically modified strain of Bacillus licheniformis that specifically expresses the serine protease from Nocardiopsis prasina in concentrated form with minimal side activities. The commercial product (RONOZYME^® ProAct) is marketed by DSM Nutritional Products (Basel, Switzerland) and contains 75,000 protease units (PROT)/g. One PROT is defined as the amount of enzyme that releases 1 μmol of p-nitroaniline from 1 μM of substrate (Suc-Ala-Ala-Pro-Phe-p-nitroaniline) per minute at pH 9.0 and 37°C.

Effects of the Protease in Animal Feeding Trials

A high number of feeding trials have confirmed that the Nocardiopsis protease enables broiler chickens to optimize their performance potential with lower feed costs. Important performance parameters in this context are increased body weight or improved feed utilization, meaning that less feed is required to attain the same body weight, which is commonly referred to as the feed conversion ratio (FCR).

In a recent study,³ the Nocardiopsis protease was added to a diet based on corn and soybean meal with a low protein content, and performance and digestibility were compared to broiler chickens fed a normal protein diet. The live weight of birds fed the low protein diet without the protease was 7.5% lower than those fed the normal protein level at day 22. When added to the low protein diets, the protease allowed birds to grow to a level equivalent to those fed the normal protein diet (Table 1). Feed conversion was also impaired in the low protein group, but this was corrected with protease dosages from 15,000 PROT/kg feed and above added to the low protein diets.

Table 1.

Live Performance of Broilers Fed Diets Supplemented with Graded Levels of the Nocardiopsis Serine Protease from Day 7 to 22.^3,a

TREATMENTS^b	NOCARDIOPSIS PROTEASE (PROT ^c/KG)	BODY WEIGHT GAIN (G)	FEED INTAKE (G)	FEED CONVERSION RATIO (G FEED/G WEIGHT GAIN)
PC	0	711.1^d	1,050	1.477^f
LP0	0	661.3^e	1,045	1.580^d
LP100	7,500	681.8^de	1,050	1.540^de
LP200	15,000	703.4^d	1,075	1.527^def
LP400	30,000	708.1^d	1,061	1.498^ef
LP800	60,000	708.0^d	1,062	1.500^ef
SEM		11.33	18.77	0.017
P-value^g		0.0201	0.0819	0.007

Values are means of seven replicates of five Ross 708 straight-run broiler chicks per treatment.

The positive control (PC) and low protein (LP) diets had 22.50% and 20.52% formulated CP, respectively. Reductions in essential amino acids in LP diets were proportional to those in CP. LP0=0 mg of protease/kg; LP100=100 mg of protease/kg; LP200=200 mg of protease/kg; LP400=400 mg of protease/kg; LP800=800 mg of protease/kg.

One PROT unit is defined as the amount of enzyme that releases 1 μmol of p-nitroaniline from 1 μM of substrate (Suc-Ala-Ala-Pro-Phe-p-nitroaniline) per minute at pH 9.0 and 37°C. Numbers in Table designate the calculated activity in the treated diets.

d-f

Means with different superscript letters differ (p<0.05) based on Tukey's honestly significant difference test.

From an ANOVA with all treatments.

Vieira and co-workers fed diets based on corn and soybean meal with varying concentrations (0; 7,500; 15,000; 30,000; 60,000; 120,000 PROT/kg) of the Nocardiopsis protease to broilers.⁸ Again, the FCR was improved (p<0.01) in a dose-responsive manner. Furthermore, birds supplemented with the enzyme had improved (p<0.05) uniformity in body weight at day 40. Improved body weight uniformity is also of value and permits ease of handling of animal flocks both at the farm level and later at the processing plant, when the animals are slaughtered. Ileal digestibility is another way to evaluate protease, since this determines the amount of nutrients absorbed in the small intestine and, hence, fully utilized by the animals. In the above trial, the ileal digestibility of methionine and histidine were improved by the protease supplementation (p<0.10), by 4% and 2%, respectively. In another study, it was shown that the addition of the protease to a standard commercial diet resulted in improved (p<0.05) ileal fat and energy digestibility by 4.5% and 5.8%, respectively.⁹ The enzyme treatment elevated crude protein digestibility by 7.3% and increased (p<0.05) ileal digestibility of methionine, cysteine, valine, and alanine by 8%. In this trial, protease supplementation improved body weight gain and FCR by 1.9% and 2.2%, respectively. The effect of the Nocardiopsis protease (15,000 PROT/kg) was also investigated in a broiler study with corn/soybean diets with different levels of crude protein and energy content.¹⁰ The protease significantly (p<0.01) improved FCR compared to the non-supplemented groups. Supplementation with the protease increased (p<0.01) ileal crude protein, fat, and gross energy digestibility by 5% when the treatments with the highest level of crude protein and energy were compared with the control. This study showed that protease supplementation significantly improved the performance of broilers raised to 42 days of age independent of the crude protein or energy level of the diet. Other studies have also demonstrated the efficacy of the protease on nutrient digestibility and performance of broilers fed a variety of commercial and experimental diets.^11

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A major benefit of including the Nocardiopsis protease into broiler diets is the improvement in the protein digestibility of a wide range of natural ingredients by 3–8%. As the utilization of the feed protein is increased, the enzyme offers the possibility to reduce the amount of protein in the feed and thereby save on expensive raw materials such as soybean meal. This translates into cost savings of €2–6 (USD2.5–7.6) per metric ton of feed and is achieved without compromising animal performance.

A European field trial performed in a commercial setting investigated the potential savings with use of the Nocardiopsis protease in broilers fed an industry standard diet based on corn, wheat, soybean meal, and rapeseed meal as core ingredients. The test diet containing the protease was re-formulated assuming a digestible amino acid level of the feed ingredients that was, on average, 4% higher than in the control diet, which allowed the feed formulation program to exchange expensive feed material with less costly products. Savings averaged €4.00 (USD5.1) per metric ton of feed, while the performance of the birds at 45 days of age was no different than for those fed the control diet.

Environmental Impacts of Using the Feed Protease

Public concerns regarding the impact of agriculture, and not least animal production, on the environment are increasing. When feed protein is not fully utilized in the animals, the undigested protein ends up in the manure and, thus in the environment. Protein in manure is converted to ammonia and nitrate. Ammonia emissions into the atmosphere cause water acidification, and nitrate eventually leaks into the water systems causing loss of biodiversity, and also poses direct risks to human health. Life cycle assessment (LCA) is a tool to quantify the environmental impacts of a certain process on the entire life cycle from cradle to grave. This includes, for example, raw material extraction, production, transport, and final disposal or recycling. One way to study the implications of an enzyme solution is to assess and compare the environmental impact of both a conventional and an enzyme-assisted solution delivering the same benefit.

Such a study was recently carried out to determine the environmental impact of using the protease in question in poultry feed.^15,16 The work was done using the principles for LCA described in ISO 14040 and 14044.^17,18. All steps in broiler production, from manufacture of feed ingredients to the broilers leaving the chicken house and including use of the manure, were taken into consideration. In this case, the conventional solution was a normal broiler diet, while the enzyme-assisted solutions were as follows: addition of protease to a diet reduced in protein content by changing the diet composition; addition of a protease to a diet without changing the diet composition and thus with a normal protein content. The common benefit or effect of the different solutions was the production of 1 metric ton of broiler (live weight). When all changes in environmental load were evaluated, the study showed significant benefits of the enzyme-assisted solutions for all the environmental impacts that were considered. Most important was the potential to reduce the pollution of water and air with nitrous compounds, which can lead to eutrophication and acidification. The largest effects were observed when the protease was used in diets to compensate for a reduced protein content.¹⁵

Conclusions

A targeted screening process based on application-relevant parameters and methods resulted in the first pure protease on the market for animal feed. The main effect in animals is that the Nocardiopsis protease can improve the protein digestibility of a wide range of natural ingredients in broiler diets by 3–8%. When the utilization of the feed protein is improved, it is possible to reduce the amount of protein in the feed and in this way save on the cost of expensive raw materials such as soybean meal without compromising animal performance. Experimental trials have shown that the cost savings can be in the range of €2–6 (USD2.5–7.6) per metric ton of feed. Moreover, a study on the environmental effects of the feed protease confirmed that it can help reduce the environmental load of broiler production. The Nocardiopsis protease represents a valid method to increase sustainability and at the same time improve profitability.

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