Milk Lysozyme Research Articles

Comparing microbiotas of foals and their mares’ milk in the first two weeks after birth

BackgroundThe mare-foal relationship is essential for the well-being and growth of a foal. Mare’s milk provides a foal with nutrients, protective immunity, and microbes. Within the first two weeks of life, there is a risk for a foal to suffer from diarrhea, particularly “foal heat diarrhea” which happens at about the time of a mare’s estrus cycle but is more likely due to transitions in the microbiota in the foal’s gastrointestinal (GI) tract. We hypothesized that this GI microbiota transition could be caused by changes in lysozyme and microbial populations in the mare’s milk. To test this hypothesis, fifteen mare-foal pairs were followed in the first 15 days post-foaling. Every other day milk was collected from mares and rectal swabs were collected from foals. Lysozyme activity in the mare’s milk was measured using a fluorescence assay. Microbial DNA was isolated from the milk and swabs and the V4 domain of 16 S rRNA genes were PCR amplified and sequenced using Illumina MiSeq technology. Microbial populations were analyzed using DADA2 and phyloseq within R.ResultsMare’s milk lysozyme activity peaked for samples at Day 1 and levels dropped to 72.5% of Day 1 activity by Day 15; however, microbial populations in the mare’s milk did not vary significantly over the two weeks. Furthermore, levels of microbial diversity found in foal rectal swabs were initially similar to microbial diversity seen in mare’s milk; however, over the first fifteen days, diversity increased for the foal rectal swab microbiota and swab microbial populations differed from milk microbes. A transition occurred shifting from microbes from the phylum Proteobacteria early in rectal swabs to those primarily from the phyla Firmicutes and Bacteroidota after the first few days post-foaling. These phyla contained several families and genera of microbes that promote utilization of milk components in healthy gut transition. Microbial abundance levels correlated more with days post-parturition than with lysozyme activity and mare’s milk microbial populations.ConclusionsThe findings suggest that much of the microbial populations responsible for the transition of the foal’s gut comes from sources outside of mare’s milk species and levels of lysozyme activity.

DYNAMICS OF CHANGES IN MORPHOIMMUNOLOGICAL INDICATORS OF THE MAMMARY GLAND SECRETION DURING THE DEVELOPMENT OF SUBCLINICAL MASTITIS IN COWS

The article reflects the results of researches conducted on studying the changes in the morphoimmunological indicators of the mammary gland secretion in cows with subclinical mastitis in the dynamics of its development. Thus, in the initial stages of the subclinical mastitis in cows and its further development, significant changes occur in the immune status and homeostasis of sick animals, characterized by the occurrence of a strong inflammatory reaction in the mammary gland against the background of a weakening of the general nonspecific resistance in the udder of sick animals. Already on the third day of the disease, the amount of milk lysozyme was 0.974, total immunoglobulins – 2.99 g/L, circulating immune complexes – 0.163, the number of somatic cells increased by 25.7% and amounted to 2.3 ths/ml, neutrophils – 77.2%, in comparison with the first day of the disease, which indicates the activation of the protective function of the mammary gland. Thus, with the further subclinical mastitis development, significant changes occur in the immune status and homeostasis of sick animals, characterized by the occurrence of a strong inflammatory reaction in the mammary gland against the background of a weakening of the general nonspecific resistance in the udder of sick animals.

Antibacterial activity of donkey’s milk against clinical isolate of Klebsiella pneumoniae

The purpose of this study was to investigate the antibacterial activity of raw donkey milk toward the clinical isolate of Klebsiella pneumoniae at 9, 15 and 38 °C as well as to clarify the role of lysozyme, lactoferrin and calcium content in the antibacterial activity of donkey’s milk. The effects of contamination level and incubation period on this antibacterial activity were also examined. Antibacterial assays were performed by determing the bacterial count in intentionally contaminated (102, 103, 104 cfu/mL) donkey milk samples during 8 and 96 hours. Lab-on-a-chip electrophoresis and atomic absorption spectrometry were used for the determination of lysozyme, lactoferrin and calcium contents in donkey milk, respectively. The donkey milk samples showed varying degrees of antibacterial activity against the tested strain K. pneumoniae. The antibacterial synergism of lysozyme and lactoferrin was proven for this clinical strain as the samples with higher lactoferrin amount showed a stronger antibacterial activity. The correlation between calcium content and antibacterial activity of donkey milk samples was not established. Donkey milk showed stronger antibacterial potential at 15 °C compared to 9 °C, but this was limited by a higher growth rate of K. pneumoniae at 15 °C. The higher level of contamination resulted in a faster consumption of the antibacterial capacity of donkey milk. The artificial neural network model for prediction of K. pneumoniae count gave acurate fit to experimental data, showing a reasonably good (overall r2 for donkey milk was 0.986, with training error 4.67∙10-4, while r2 for nutrient broth was 0.982 and training error was 0.002).

Natural Association of Lysozyme and Casein Micelles in Human Milk.

Using reversed phase high-performance liquid chromatography with ultraviolet (UV) detection and electrospray ionization (ESI)-quadrupole time-of-flight mass spectrometry (RP-HPLC-UV-ESI-Q-TOF), the lysozyme content in the milk of 10 volunteering mothers was quantified, ranging from 29 to 96 μg/mL. Following ultracentifugation, it was found that the lysozyme in human milk, unlike other whey proteins, is mainly bound to casein micelles (ca. 75%). The enzymatic activity of human lysozyme, measured as lytic activity against cell walls of Micrococcus lysodeikticus, was similar for the micelle-bound and free protein, indicating that the micellar structure should not affect the antibacterial activity of lysozyme. The results indicate that lysozyme is an integral component of casein micelles in human milk.

Stability of lactoferrin and lysozyme in human milk at various temperatures and duration of storage

Introduction: Exclusive breastfeeding, especially in the first six months, is essential for infants as it provides nutrition and protection against various diseases. Colostrum, which is found in the first breast milk produced, contains various protective factors, such as lactoferrin and lysozyme. Human milk can be stored at room temperature, refrigerated, or kept frozen. Several factors affect the stability of the bioactive content in human milk, such as temperature and storage time. The aim of this study was to measure the stability of lactoferrin and lysozyme levels in human milk during the first six hours (h) at different temperatures and compare it with that of frozen human milk. Methods: Human milk samples were obtained from 11 breastfeeding mothers using certain criteria. The human milk was stored at room temperature and 4°C for 1, 3, and 6 h and classified as never frozen, while frozen human milk was stored at -20°C for 1, 3, and 6 days. Measurement of the lactoferrin and lysozyme levels was performed using enzyme-linked immunosorbent assay. Results: The results showed that storage at room temperature significantly reduced lactoferrin and lysozyme levels. Lactoferrin levels in frozen human milk did not significantly decrease during the first six days. Meanwhile, the lysozyme levels in frozen human milk decreased significantly. Conclusion: The levels of lactoferrin and lysozyme in frozen human milk stored for the first six days were more stable than those stored at room temperature and 4°C in the first 6 h.

An Event-Specific Real-Time PCR Method for Measuring Transgenic Lysozyme Goat Content in Trace Samples.

Lysozymes are used in sterilisation, antisepsis, dairy additives, inflammation, and cancer. One transgenic goat line expressing high levels of human lysozyme (hLZ) in goat milk has been developed in China. Herein, we established an event-specific real-time polymerase chain reaction (real-time PCR) method to detect the transgenic hLZ goat line. The developed method has high specificity, sensitivity and accuracy, and a wide quantitative dynamic range. The limit of detection and limit of quantification was 5 and 10 copies per reaction, respectively. The practical sample analysis results showed that the method could identify and quantify transgenic lysozyme content in trace samples in routine lab analyses. Furthermore, the potential applicability in risk assessment, such as molecular characterisation and gene horizontal transfer, was confirmed. We believe that this method is suitable for the detection of transgenic hLZ goat line and its derivate.

Preparation and application of lysozyme aptamer fibre for specific recognition of lysozyme in food samples

A novel aptamer solid-phase microextraction (SPME) fibre-based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) for lysozyme-specific recognition has been successfully prepared. The prepared SPME fibres were characterised by various instrumental methods, including scanning electron microscopy, X-ray photoelectron spectroscopy and Fourier-transform infrared spectrometry. The results showed that the SPME fibre exhibited a porous structure, which is beneficial for the enrichment of the aptamer. The solid-phase microextraction-high-performance liquid chromatography-fluorescence detection (SPME-HPLC-FLD) with the superior selectivity and sensitivity responded linearly to the concentration of lysozyme over the 0.025–10 mg/L range, and the limit of detection was low at 6.8 μg/L, with a linearity of 0.9995. The method showed excellent reproducibility, and the relative standard deviations (RSDs) were less than 5.5%, which facilitated the analysis. The method was applied for the analysis of lysozyme in cow milk and papaya samples, and the recoveries ranged from 86.7–93.5% with RSDs of 4.3–6.1%, which clearly state that the proposed lysozyme aptamer SPME-HPLC-FLD method is a promising analytical tool for the analysis of lysozyme in food samples.

Article Review: Camel Milk as an Amazing Remedy for Health Complications: A Review

Camel milk has unique benefits for human health. Protein is the main component which gives special properties to camel milk and effectively influences its nutritional value. Due to lack of β-lactoglobulin in the camel milk, it may be as a proper alternative for human milk. Camel milk is rich in vitamins C, manganese and iron. There are high amount of unsaturated fatty acids, immunoglobulin’s, insulin like protein and protective enzymes like lactoferrin and lysozyme in the camel milk. The lactoferrin has the effects or properties of antibacterial, antiviral and anti-inflammatorry. Also, it has been proven that camel milk has beneficial application in disorders of stomach and intestinal, food allergy, diabetes mellitus, cancer, autism, and viral hepatitis. Camel milk containing insulin like protein and may help to heal diabetes of Type 1 and 2 and gestational diabetes. It contains small size immunoglobulins which strengthens the immune system. In addition, camel milk reduces blood cholesterol amount, avoiding of psoriasis disease, healing of inflammation and improving of tuberculosis patients. Camel milk may be effective as unique miracle in many healthy issues of human and especially cardiovascular system. Therefore, it is not only food, but also it is as amazing remedy for treatment and healthy issues.

Effects of thermized donkey milk with lysozyme activity on altered gut barrier in mice exposed to water-avoidance stress

Nutrition plays a crucial role in human gut health through the improvement of gut barrier functionality. Donkey milk represents an interesting source of natural antimicrobial factors such as lysozyme. Recently, anti-inflammatory properties of donkey milk lysozyme activity were described in a mouse model of ileitis. The current increase of donkey milk consumption highlights the necessity to propose a healthy milk compliant with microbiological standards. This study aims to define a heat treatment of donkey milk, retaining its high lysozyme activity, and to evaluate its beneficial effects on a gut barrier impairment model due to chronic stress in mice. To perform this experiment, samples of raw donkey milk were collected in 15 distinct French farms. Microbiological analysis and lysozyme content and activity were evaluated for each sample. Then, several heat treatments were carried out to define a time and temperature combination that allowed for both a reduction in the number of total micro-organisms, increasing the shelf-life of the product, and preservation of lysozyme activity. The beneficial effect of heated donkey milk on the gut barrier of mice was evaluated and compared with raw donkey milk. We found that samples of raw donkey milk showed low total mesophilic microbial counts, and no pathogens were detected. Among the different heat-treatment procedures tested, a 2-min, 72°C combination was determined to be the most optimal time and temperature combination to preserve lysozyme activity and increase the shelf-life of donkey milk. Oral administration of this heat-treated donkey milk in mice counteracted chronic stress-induced intestinal damage, illustrated by gut hyper-permeability and low-grade inflammation, similar to raw donkey milk. We have demonstrated for the first time that oral intervention with donkey milk, optimally heat-treated to retain enzymatic lysozyme activity, improves intestinal barrier damage linked to psychological stress in mice.

Determination of the structural ensemble of the molten globule state of a protein by computer simulations.

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.

Donkey milk lysozyme ameliorates dextran sulfate sodium-induced colitis by improving intestinal barrier function and gut microbiota composition

Donkey milk lysozyme (DML) is a potential therapeutic agent for inflammatory bowel disease (IBD), while the mechanism remains obscure. Here, we reported that DML ameliorated weight loss, colon damage and mucosal inflammation in colitis mice. Additionally, DML increased the expression of occluding and zonula occludens-1, and reduced that of claudin-2, thus improving mechanical barrier function. It also inhibited the expression of tumour necrosis factor-α and interleukin-13, and reduced the myeloperoxidase level for strengthen of immune barrier function. DML increased gut microbiota composition diversity, promoting growth of probiotics and inhibiting pernicious bacteria, indicating that DML played an important role in microbial barrier function. The X-ray structure of DML was determined, which contained a core structure with conserved active site. The different conformation and residues of binding subsites may indicate diverse binding ability to substrates. These results suggested that oral administration of DML is a promising novel therapeutic for treatment of IBD.

Atomic Detail of Protein Folding Revealed by an Ab Initio Reappraisal of Circular Dichroism.

Circular dichroism (CD) is known to be an excellent tool for the determination of protein secondary structure due to fingerprint signatures of α and β domains. However, CD spectra are also sensitive to the 3D arrangement of the chain as a result of the excitonic nature of additional signals due to the aromatic residues. This double sensitivity, when extended to time-resolved experiments, should allow protein folding to be monitored with high spatial resolution. To date, the exploitation of this very appealing idea has been limited, due to the difficulty in relating the observed spectral evolution to specific configurations of the chain. Here, we demonstrate that the combination of atomistic molecular dynamics simulations of the folding pathways with a quantum chemical evaluation of the excitonic spectra provides the missing key. This is exemplified for the folding of canine milk lysozyme protein.

Hot topic: Holder pasteurization of human milk affects some bioactive proteins

The aim of this research was to investigate the effect of Holder pasteurization (HoP; 62.5°C, 30 min) on the protein profile and activities of glutathione peroxidase (GPx) and lysozyme (LZ) in human milk. Over 6 mo of lactation, human milk samples were analyzed before (raw) and after HoP for GPx and LZ activity and electrophoresis protein profile. Holder pasteurization reduced human milk lactoferrin, immunoglobulin fractions, and GPx activity. In addition, GPx activity, which is high in colostrum and transitional milk, was naturally reduced over the 6-mo lactation period. In contrast, HoP did not affect human milk LZ activity. Besides its critical cellular antioxidant role in protecting the organism from oxidative damage, GPx decreases the redox potential of milk, stimulating the growth of anaerobic microorganisms, such as the probiotic Bifidobacterium. Considering the role of lactoferrin in infant health, we conclude that an important part of its function has been inactivated by pasteurization. These compounds should be replaced by human milk banks after the HoP step to recover lost functionality. Otherwise, an alternative technology to HoP that better retains human milk properties should be used by milk banks to eliminate the risk of transmission of infectious agents.

Construction of a universal recombinant expression vector that regulates the expression of human lysozyme in milk

The mammary gland provides a novel method for producing recombinant proteins in milk of transgenic animals. A key component in the technology is the construction of an efficient milk expression vector. Here, we established a simple method to construct a milk expression vector, by a combination of homologous recombination and digestion-ligation. Our methodology is expected to have the advantages of both plasmid and bacterial artificial chromosome (BAC) vectors. The BAC of mouse whey acidic protein gene (<i>mWAP</i>) was modified twice by homologous recombination to produce a universal expression vector, and the human lysozyme gene (<i>hLZ</i>) was then inserted into the vector by a digestion-ligation method. The final vector containing the 8.5 kb <i>mWAP</i> 5′ promoter, 4.8 kb <i>hLZ</i> genomic DNA, and 8.0 kb <i>mWAP</i> 3′ genomic DNA was microinjected into pronuclei of fertilized mouse embryos, to successfully generate two transgenic mouse lines that expressed recombinant human lysozyme (rhLZ) in milk. The highest expression level of rhLZ was 0.45 g·L⁻¹, and rhLZ exhibited the same antibacterial activity as native hLZ. Our results have provided a simple approach to construct a universal milk expression vector, and demonstrated that the resulting vector regulates the expression of hLZ in milk.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat β-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

The Simultaneous Human FVIII/vWF Purification and Virus Inactivation Combined in Chromatographic Column

Well known that the decrease of the number of degrees of freedom prevents protein’s denaturation. In the previous investigation, we showed that the chromatographic gel binding protein can serves as an agent reducing the number of degrees of freedom of the protein. In addition, the high dynamic capacity of new developed gels provided a satisfactory retention of the peptide/protein at elevated temperature of the chromatographic process. These both factors allowed the implement of the virus inactivation for the peptide (streptokinase fragment SK1-61) and low (milk lysozyme) and an average (Fibrinogenolytic enzyme from the snake venom) molecular weight protein directly in the column during targets isolation. The goal of this study was an investigation of the possibility to save the activity of the high molecular weight complex FVIII/vWF, bound by ion exchange and affinity gels during the virus inactivation directly in the chromatographic column. Another goal was to show that the binding protein adsorbent can serves as reliable “sieve” for mechanical washing away of infecting viruses. Using various chromatographic, photometric, RT-PCR approaches it was discovered that the high dynamic capacity, which determines the high temperature-depending capacity of the adsorbent, allowed to perform the virus-inactivating complex FVIII/vWF by solvent/detergent treatment directly in the chromatographic column at the temperature 40-50°C during a long time 3-5 hrs. The FVIII biological activity was completely preserved; enveloped and non-enveloped viruses were effectively removed. The level of modeling virus elimination was sufficient for a full inactivation of viruses that allowed us to recommend this method to use in pharmaceutical industry.

Variability of lysozyme and lactoferrin bioactive protein concentrations in equine milk in relation to LYZ and LTF gene polymorphisms and expression.

Equine milk is considered to be an interesting product for human nutrition, mainly owing to its low allergenicity and significant amounts of bioactive proteins, including lysozyme (LYZ) and lactoferrin (LTF). The present study assessed the effect of genetic factors on LYZ and LTF concentration variability in mare's milk. Significant effects of horse breed and lactation stage on milk LYZ and LTF contents were observed. The highest level of LTF and the lowest concentration of LYZ were recorded for the Polish Warmblood Horse breed. The highest amounts of both proteins were found for the earliest investigated time point of lactation (5th week). Altogether 13 (nine novel) polymorphisms were found in the 5'-flanking regions of both genes, but they showed no significant relationship with milk LYZ and LTF contents. Several associations were found between selected SNPs and the LYZ gene relative transcript level. While the present study indicated the existence of intra- and interbreed variability of LYZ and LTF contents in mare's milk, this variation is rather unrelated to the 5'-flanking variants of genes encoding both proteins. This study is a good introduction for broader investigations focused on the genetic background for variability of bioactive protein contents in mare's milk. © 2016 Society of Chemical Industry.

Effects of different heat treatments on lysozyme quantity and antimicrobial activity of jenny milk

Thermal treatments are used to improve milk microbial safety, shelf life, and biological activity of some of its components. However, thermal treatments can reduce the nutritional quality of milk, affecting the molecular structure of milk proteins, such as lysozyme, which is a very important milk component due to its antimicrobial effect against gram-positive bacteria. Jenny milk is characterized by high lysozyme content. For this reason, in the last few years, it has been used as an antimicrobial additive in dairy products as an alternative to hen egg white lysozyme, which can cause allergic reactions. This study aimed to investigate the effect of pasteurization and condensation on the concentration and antimicrobial activity of lysozyme in jenny milk. Furthermore, lysozyme quantity and activity were tested in raw and pasteurized milk after condensation at 40 and 20% of the initial volume. Reversed-phase HPLC was performed under fluorescence detection to monitor lysozyme in milk samples. We evaluated the antimicrobial activity of the tested milk against Bacillus megaterium, Bacillus mojavensis, Clavibacter michiganensis, Clostridium tyrobutyricum, Xanthomonas campestris, and Escherichia coli. Condensation and pasteurization did not affect the concentration or antimicrobial activity of lysozyme in jenny milk, except for B. mojaventis, which showed resistance to lysozyme in milk samples subjected to heat treatments. Moreover, lysozyme in jenny milk showed antimicrobial activity similar to synthetic antibiotics versus some gram-positive strains and also versus the gram-negative strain X. campestris.

Lysozyme in breast milk is a selection factor for bifidobacterial colonisation in the infant intestine.

The objective of this work was to study the residential characteristics of bifidobacteria, which can be classified as either human-residential bifidobacteria (HRB) or non-HRB. We investigated the growth of different strains of HRB and non-HRB in human breast milk with the aim of understanding the mechanisms involved in the unique habitation of each taxon. The growth of 37 strains of different bifidobacterial species or subspecies in breast milk was investigated by incubating each under anaerobic conditions at 37 °C. The tolerance of each strain to either egg white or human lysozyme was compared. Among the infant-type HRB strains, all strains of Bifidobacterium longum subsp. infantis and Bifidobacterium breve grew well in breast milk, but the growth characteristics of B. longum subsp. longum and B. bifidum were strain-dependent. In contrast, the tested strains of adult-type HRB and non-HRB generally failed to grow and died after incubation in breast milk. Most infant-type HRB strains were tolerant to high concentrations of lysozyme, while adult-type HRB strains possessed intermediate tolerance to lysozyme, and non-HRB strains were susceptible to lysozymes of egg white or human origin. These data suggest that breast milk lysozyme content plays a central role in the exclusion of non-HRB, while other factors, together with lysozyme content, are involved in the growth inhibition of adult-type strains in human milk. Our results suggest that infant-type HRB strains would be suitable candidates for use as infant probiotics.

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.