Lysozyme (Muramidase) - a natural antibacterial agent in broiler chickens diets
Lysozyme is an enzyme that has bactericidal properties, i.e. it kills the bacteria. It is contained in egg white, tears, saliva, sweat and breast milk and acts as an antibacterial agent. As an enzyme, lysozyme is a glycosidic hydrolase that catalyzes the hydrolysis of 1-4-beta bonds between N-acetyl muric acid and N-acetyl-D-glucosamine. These two compounds are the major building blocks of peptidoglycans, the polymeric compounds that build the walls of bacterial organisms.
In the search for alternatives to nutritional antibiotics as growth stimulants, in addition to probiotics, prebiotics, synbiotics, herbal extracts, etc., in the middle of 2000, special attention was paid to the use of lysozyme in the diets of suckling and weaned pigs. The studies used lysozyme derived from egg white, milk, and microbial lysozyme produced by Trichoderma reesei. The enzyme has been shown to have a positive effect on animal growth, feed utilization, piglet health and the microbiota in the gastrointestinal tract. Researchers are expanding their research into the use of lysozyme in other species and ages of animals including broiler chickens.
Three different doses of 10% lysozyme were tested in 120 broiler chickens Ross 308: 0,.70, 90 and 120 g/t feed for 35 days. The best results are achieved with a dosage of 90 g/t 10% lysozyme. At the end of the experimental period, chickens in this group had reached a live weight of 1830 g versus 1730 g for the negative control group, and feed consumption per unit gain (FCR) was 6% lower. The authors found a longer villi length (10.30 µm vs. 6.02 µm) and a greater crypt depth (1.69 µm vs. 0.86 µm) in the intestinal mucosa. The highest number of lactobacilli in the intestine has also been demonstrated in the group supplemented with 90 g/t feed 10% lysozyme (Latif et al., 2017).
Lichtenberg et al. (2017) evaluated the safety of muramidase produced by Trichoderma reesei and did not observe adverse effects in a 90-day subchronic toxicity studies in rats. Also, a 6-week broiler chicken tolerance study on one-times, five-times and ten-times the maximum recommended dose of lysozyme, showed no adverse effects.
The Food Safety Committee of the European Union also concludes that birds tolerate well the ten-fold maximum recommended dose of 45000 LSU (F)/kg feed muramidase from Trichoderma reesei DSM 32338 as a feed additive for broiler chickens and poultry species (EFSA, 2018).
Y. Xia et al. (2019) conclude that lysozyme is known to lyse intestinal pathogens in poultry and improve their growth, but it is not clear whether it can replace antibiotic growth promoters without the associated risk of antibiotic resistant bacterial strains. According to the authors, the effects on the composition, biodiversity and function of the gut microbiota in birds remain unclear. This is of crucial importance, since the gut microbiota plays a role in the assimilation of nutrients, the production of vitamins and amino acids and the prevention of colonization of pathogenic bacteria. In this regard, the authors experiment with 400 one day old chicks of the Di-Gao (China) breed, divided into 5 groups and fed with corn-soybean feed mix. The scheme of the experiment is as follows: negative control - no additives, positive control supplemented with 400 ppm flavomycin and three groups supplemented with 40, 100 or 200 ppm concentrated lysozyme of chicken egg white (≥ 90%, ≥ 40,000 units/mg protein). There was no difference in the live weight of the chickens depending on the dosage. The live weight of lysozyme-supplemented chickens was 5% lower than that of antibiotic-supplemented chickens and 5.9% higher than the negative control, but the differences were not statistically significant. Feed conversion was best in the group with the highest dosage of lysozyme, but it was also statistically unreliable. In conclusion, the authors note that the breed of chickens may be an important factor in how lysozyme affects the growth results of chickens.
Three different studies of Ross 308 broiler chickens tested different doses of muramidase from Trichoderma reesei DSM 32338. The first study: 0, 25000, 35000 and 450000 LSU (F)/kg feed; second study: 0, 25000 and 35000 LSU (F)/kg feed and third study: 0, 15000 and 25000 LSU (F)/kg feed. In all three experiments, statistically significant higher live weight was not achieved in the chickens treated with the muramidase supplement compared with the negative control group, and the differences ranged from 1.0 to 2.0%. In addition, the live weight of the chicks from the experimental groups was practically the same, i.e. variable dosages did not affect this indicator differently. However, from 2% to 3% statistically significant better feed conversion was achieved in birds receiving muramidase supplementation at the lowest recommended level of 25000 LSU (F)/kg feed (EFSA, 2018).
In the patent application for the "Method for Improving Animal Productivity" in the United States, 2016, the applicant Novozymes A/S proposes the use of animal feed comprising microbial polypeptides having lysozyme activity. Three broiler chicken experiments were annexed to the documentation and the microbial lysozyme was from Trichophaea saccata strain CBS 804.70 and Acremonium algalophilum CBS 114.92.
The experiments were conducted with one-day-old male broiler chickens from the hybrid combination Ross 308. The diet contained maize, wheat, rye and soybean meal as main components, without the addition of a coccidiostat. The combined feed was granulated at 700C and supplemented with lysozyme at doses of 0, 25, 50, 100 and 200 mg/kg feed or 10 mg/kg feed of the antibiotic avilamycin. For days 1 to 36, the addition of lysozyme did not positively affect the live weight of the chickens, despite a positive trend of 1.6% in the 25 mg/kg feed of lysozyme and avilamycin compared with the negative control group. The incorporation of microbial lysozyme improved feed conversion even at the lower dosage of 25 mg/kg feed compared to the negative control group and this effect was comparable to the antibiotic supplementation group. In the second experiment, dosages of 0, 6,25, 12,5 and 25 mg/kg feed lysozyme and 10 mg/kg feed avilamycin were administered. For the 1 to 36 day period, the dosage of 25 mg/kg feed of lysozyme improved the live weight of the chickens by 2% and the feed conversion by 2.2% compared to the negative control. The third experiment lasted 42 days and found better feed conversion and better EPEF. This is a European production efficiency factor and is a way of comparing animal productivity between farms. The indicator is complex because it includes live weight of chickens, feed conversion, health status and length of rearing.
In another experiment with 3200 one-day-old broiler chickens divided into 4 groups, the effect of the product Biozyme-S (fermentation derived lysozyme) was compared with two nutritional antibiotics - Stafac and Enramycin. The highest daily gain, the best feed conversion and the lowest mortality were achieved in the lysozyme addition group compared to the negative control group and the antibiotic addition groups.
M. Gong (2014) applied different dosages of lysozyme to the diets of broiler chickens: 0, 50, 100 and 200 ppm, and monitored the growth, feed conversion and the intestinal microbiota. All groups with the addition of lysozyme have better growth and better utilization of feed compared to the negative control group, and the zootechnical results get better, with dosage increase. The number of aerobic bacteria, coliforms, E. coli and Clostridium perfringens in the intestines of chickens was determined. It has been found that the addition of even the lowest dose of lysozyme has a positive effect on the intestinal microbiota.
The research of R. Cegielska et al. (2009) is in a slightly different direction. They aim to evaluate the effect of various lysozyme preparations on the shelf life of the breast muscle of broiler chickens stored at 3oC ± 1oC. The analysis of the obtained results showed that the surface application of the lysozyme preparations makes it possible to extend the shelf life of the chicken breast muscles by approximately a factor of 2 at the storage temperature used.
G. Zhang et al (2010) tested an antimicrobial mixture based on lysozyme by Entegard in broiler chickens infected with Eimeria maxima and Clostridium perfringens. The aim of the experiment was to investigate whether the lysozyme product will help reduce the negative effects of the infection, which include reduced growth, reduced feed consumption, impaired feed utilization, necrotic enteritis, high incidence of necrotic enteric lesions and high mortality. The effect of the Entegard supplement at 200 g/t feed was comparable to the effect of treating infected chickens with 55 g/t feed bacitracin methylene disalicylate (BMD). The weight gain of uninfected chickens was 975 g, of infected - 685 g, and of infected and treated with the lysozyme product - 872 g. The feed conversion was 1.658, 2.173 and 1.852 g, respectively. The incidence of necrotic enteric lesions was 0, 1.4 and 0.6, and the mortality rate was 0.09, 32.06 and 9.04%.
Studies in broiler chickens treated with lysozyme or lysozyme-based feed additives reveal the possibility of replacing nutritional antibiotics as productivity stimulants in birds with the natural antibacterial enzyme lysozyme, also called muramidase.