Accepted Manuscript

Milk from transgenic goat expressing human lysozyme for

recovery and treatment of gastrointestinal pathogens

Igor de S Carneiro, Jos Nilson Rodrigues de Menezes, Julyana

Almeida Maia, Andr Marrocos Miranda, Victor Bruno Soares de
Oliveira, James D. Murray, Elizabeth A. Maga, Marcelo Bertolini,
Luciana Relly Bertolini

PII: S0928-0987(17)30623-1
DOI: doi:10.1016/j.ejps.2017.11.005
Reference: PHASCI 4294
To appear in: European Journal of Pharmaceutical Sciences
Received date: 25 April 2017
Revised date: 20 October 2017
Accepted date: 4 November 2017

Please cite this article as: Igor de S Carneiro, Jos Nilson Rodrigues de Menezes,
Julyana Almeida Maia, Andr Marrocos Miranda, Victor Bruno Soares de Oliveira, James
D. Murray, Elizabeth A. Maga, Marcelo Bertolini, Luciana Relly Bertolini , Milk from
transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal
pathogens. The address for the corresponding author was captured as affiliation for all
authors. Please check if appropriate. Phasci(2017), doi:10.1016/j.ejps.2017.11.005

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 ACCEPTED MANUSCRIPT
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MILK FROM TRANSGENIC GOAT EXPRESSING HUMAN LYSOZYME FOR

RECOVERY AND TREATMENT OF GASTROINTESTINAL PATHOGENS

Corresponding author:
Luciana Relly Bertolini (L. Bertolini)
Phone number: +55 (51) 3320-3931
e-mail address: luciana.bertolini@pucrs.br

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Postal address: Biotechnology and Genetic Engineering Lab
School of Pharmacy, Pontifical Catholic University of Rio Grande do Sul (PUCRS)

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Av. Ipiranga, 6681 - Porto Alegre, RS - Brazil - 90.619-900

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Igor de S Carneiroa, Jos Nilson Rodrigues de Menezesb, Julyana Almeida
Maiac; Andr Marrocos Mirandad; Victor Bruno Soares de Oliveirae; James D.
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Murrayf; Elizabeth A. Magag; Marcelo Bertolinih; Luciana Relly Bertolinia,i
a
Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington
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Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear, Brazil. E-mail: igor_scar@hotmail.com
b
Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington
Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear, Brazil. E-mail: nilsonmenezes@unifor.br
c
Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington
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Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear, Brazil. E-mail:

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julyanaamaia@hotmail.com
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d
Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington
Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear, Brazil. E-mail:
andremarrocosmiranda@gmail.com
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e
Laboratory of Molecular Biology and Development, University of Fortaleza, Avenida Washington
Soares, 1321, Edson Queiroz, 60.811-905, Fortaleza, Cear, Brazil. E-mail:
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oliveira.bruno.fisio@gmail.com
f
Department of Animal Science and Department of Population Health and Reproduction, University of
California, Davis, CA 95616, USA. E-mail: jdmurray@ucdavis.edu
g
Department of Animal Science and Department of Population Health and Reproduction, University of
California, Davis, CA 95616, USA. E-mail: eamaga@ucdavis.edu
h
School of Veterinary Medicine, Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. E-
mail: mbertolini@ymail.com
i
Pontifical Catholic University of Rio Grande do Sul (PUCRS), Porto Alegre, RS, Brazil. E-mail:
luciana.bertolini@pucrs.br
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ABSTRACT
Lysozyme is an important non-specific immune protein in human milk, modulating the
immune response against bacterial infections. The aim of this study was to
characterize the milk of a transgenic goat expressing a recombinant human lysozyme
(rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against
pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-
transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to

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physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity
against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and

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cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence
of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The

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expression of ZO-1 and IL-6 genes were evaluated in IEC-18 to evaluate the effect of
rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical
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parameters between goat Tg and nTg milk were similar and within normal values for
human consumption, with hLZ concentrations being similar between Tg (224 g/mL)
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and human (226 g/mL) milk. The Tg milk had bactericidal activity against M. luteus,
no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E.
feacalis than controls. Better migrating parameters were observed in cells in culture
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with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted
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improved migrating parameters than controls, except for S. sonnei, with lower cell
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numbers in the presence of nTg samples and E. faecalis and S. sonnei. No

differences in ZO-1 relative expression patterns were observed in cultured cells, with
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increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg
group being similar to all groups. In conclusion, goat milk containing rhLZ
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demonstrated valid evidence for its potential use as a nutraceutical for improvement
of health and nutrition quality in humans.
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1. INTRODUCTION
Breast milk is the ideal source of nutrition for infant development, providing
nutrients and factors that promote health and fight infections. The benefits of human
milk are attributed to the antimicrobial action of milk proteins such as Lysozyme (LZ).
Such enzyme is part of the passive immunity and natural defence against Gram-
positive bacteria and, with cooperation of other factors present in milk, LZ has also
demonstrated activity against Gram-negative bacteria, viruses, parasites and fungi

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(Rollins et al., 2016; Victora et al., 2016).
Lysozyme is capable of catalyzing the hydrolysis of glycosidic bonds

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between 1-4 acetylmuramic acid and N-acetylglucosamine in the peptidoglycan layer

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of the bacterial cell walls. Its antimicrobial activity results from that cleavage, which
causes leakage of the internal components of the cells and consequent destruction of
bacteria (Norman et al., 2012). Lysozyme is a globular protein with 129 amino acid
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residues and 14.314 kDa, which, besides being present in human milk, can be found
in different concentrations in various species and secretions such as tears, sweat and
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saliva. In human milk, the typical concentration is about 200-400 g/mL, many fold
higher than the content of LZ present in the milk of goats (0.25 g/mL), cattle (0.13
g/mL) and pigs (0.065 g/mL), for instance (Cerven et al., 2008; Maga et al., 2012;
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Ning et al., 2009; Pupek et al., 2003; Yang et al., 2011).

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Considering the benefits of LZ, several research groups have developed

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genetically engineered organisms for production of recombinant human lysozyme

(rhLZ). The rhLZ expression by the transgenic brown rice has reached 0.6% of grain
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weight (Huang et al., 2002). Transgenic chickens produced an average of 29.90

g/mL in the egg white (Wang et al., 2016). The concentrations of rhLZ in the milk of
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transgenic mice varied from 250 to 710 g/mL (Maga and Murray, 1995); transgenic
cows and transgenic pigs expressed in their milk about 25.96 g/mL (Yang et al.,
2011) and 116 mg/mL (Lu et al., 2014) of rhLZ, respectively.
Milk from transgenic dairy animals expressing rhLZ has potential to prevent
and treat infant diarrhea and some other infectious diseases, also reducing in the
burden of malnutrition (Brundige et al., 2008; Cerven et al., 2008; Maga et al., 2012).
Goat rhLZ milk can indeed modulate gut microbial populations in a fashion similar to
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the human milk (Cooper et al., 2013; Maga et al., 2012). In previous studies, the fecal
microbiota analysis of young pigs fed with pasteurized milk of rhLZ transgenic goats
showed significant increase in the number of bacteria associated with gut health
(Bifidobacteriaceae and Lactobacillaceae) and decreased number of bacteria
associated with diseases (Escherichia coli, Mycobacteriaceae, Streptococcaceae
and Campylobacterales; Maga et al., 2006b, 2012). Young pigs have also presented
improved gut morphology and circulating metabolites (Cooper et al., 2011; Brundige

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et al., 2010), helping to ameliorate symptoms of diarrhoea (Cooper et al., 2013). To
further investigate and explore the benefits of human lysozyme, a line of transgenic

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dairy goats expressing rhLZ in their milk was produced in Brazil. In this study, we
aimed to validate the potential usefulness of the milk as from such transgenic line by

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evaluating the physicochemical characteristics of the milk, also identifying and
quantifying rhLZ in milk, and its enzyme activity in vitro on intestinal epithelial cell
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(IEC-18) cultures and against gastrointestinal bacterial pathogens. This represents
the first necessary step in testing the milk properties in systematic in vitro and in vivo
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studies for future potential use of the rhLZ goat milk as nutraceutic product in
humans.
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2. MATERIAL AND METHODS

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2.1 Ethics statement

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This study was conducted in compliance with guidelines of the Brazilian

National Technical Biosafety Committee (Comisso Tcnica Nacional de
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Biossegurana - CTNBio), in facilities accredited by the Biosafety Certification

Number 0294/10 and the Ethical Principles of Animal Experimentation described by
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the Brazilian College of Animal Experimentation (Colgio Brasileiro de

Experimentao Animal - COBEA, 1991). The described experiments were approved
by the Research Ethics Committee of the University of Fortaleza (process number
003/2016).
2.2 Transgenic animals
A line of transgenic goats expressing rhLZ in milk was generated by
pronuclear microinjection of one-cell stage embryos with a gene construct (23 Kb)
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consisted of the human lysozyme cDNA (540 bp) under the bovine S1-casein
promoter and 3 regulatory elements, according to Maga (2003). Milk from a female
transgenic founder (Tg) and from two non-transgenic control goats (nTg1 and nTg2)
matching the same breed, parity and stage of lactation was used throughout this
study.
2.3 Milk sample collections
Transgenic and non-transgenic goats were kept in separate stalls, and

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provided hay, mineral salt and water ad libitum, and daily exposure to sunlight. Milk
samples from the Tg goat and from the nTg1 and nTg2 goats were collected once a

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week, starting at the third week of natural lactation, and for the subsequent eight
weeks. The nTg2 goat was used only for comparisons in the physicochemical

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analyses. The collection procedure was performed manually and under aseptic
conditions, with teats sanitized and dried prior to each collection, as recommended
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by biosafety standard protocols. As for the human control milk (hM), samples were
obtained from unidentified donors, approximately at the fourth month of lactation.
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2.4 Physicochemical analyses

Upon each milk collection, during the eight-weeks period, 50 mL of fresh milk
collected from the transgenic and non-transgenic goats were subjected to physical
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and chemical analyses in triplicate. Samples from two nTg were used for substantial
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equivalence, as previously defined by the OECD (Organization for Economic

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Cooperation and Development, 1998). The amount of fat, lactose, protein and non-fat
solids, as well as the pH and the freezing point were verified in each milk sample for
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identification of potential alterations in milk composition between groups, using a milk

analyser apparatus (Lactoscan S, Milkotronic Ltd., New Zagora, Bulgaria). The lactic
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acid content was measured by titrating the milk with 0.1 N Sodium hydroxide (Dornic
solution) and the alkaline level was measured in the presence of a phenolphthalein
indicator at pH 8.0 (FAO, 2017; Salva, 2011). To analyse the specific gravity of milk,
the density was obtained following FAO recommendations (Food and Agriculture
Organization, 2012) and to determine the proximate analysis for nutrition evaluation,
the ash content, which represents the total mineral content, was obtained by total
incineration of the samples through 24-h heating in an oven at 100C, followed by 6 h
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in muffle furnace at 550C (Nielsen, 2006).

After each weekly physicochemical analyses, the remaining milk samples
were cooled down and pasteurized at 64C for 35 min. Samples were immediately
cooled to -4C, being stored at -80C and thawed only at runtime analyses.
2.5 Lysozyme identification and quantification
The rhLZ contents in milk samples were quantified by Western blot in
triplicates. The nTg, Tg and hM were initially diluted 1:1 in Laemmli buffer and

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denatured at 95C for 3 min, for separation by 15% SDS-PAGE in electrophoretic run
(90 min at 100 V). Commercial recombinant human lysozyme (L1667, Sigma-Aldrich,

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St. Louis, MO, USA) diluted at 270 g/mL was used as positive control and also to
quantify the rhLZ in milk. Proteins were transferred to nitrocellulose membrane for 2 h

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at 100 V by wet transfer in the Mini Trans-Blot Cell device (Bio-Rad, Hercules, CA,
USA). Membranes were blocked for 1 h using TBS-T with 5% non-fat dry milk (8 g/L
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NaCl, 2.42 g/L Tris, 0.05% Tween-20 detergent). Subsequently, membranes were
rinsed three times for 15 min in TBS-T, and human anti-lysozyme antibody (DAKO,
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Glostrup, Denmark) was diluted to 1:2,500 in the blocking solution for 16 h at room
temperature (RT). Incubation was then performed with alkaline phosphatase-labelled
anti-rabbit secondary antibody (Invitrogen, Carlsbad, CA, USA) diluted to 1:20,000
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in TBS-T (at RT for 1 h). Three additional 15-min washes were carried out in TBS-T,
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with the membranes then covered with the NBT/BCIP (Sigma-Aldrich). After 24 h,
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semi-quantification of rhLZ expression in milk was determined by the analysis of

digitalized and photographed gels, where the intensity of each specific western blot
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band diameter was measured using the Image J software 1.4 (NIH, Bethesda, MD,
USA) comparing to known amounts of commercial rhLZ (S-rhLZ group). Results were
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converted into numeric values referring to each measured band.

2.6 Activity test
To assess the biological activity of lysozyme in the milk produced by the
transgenic goat, we assayed its antimicrobial activity analysing the formation of
bacterial inhibitory growth zones into culture plates. Samples of Tg, nTg, hM, S-rhLZ,
and a solution identified as nTg+rhLZ (a preparation of the nTg sample added with
270 g/mL of commercial rhLZ, meant to simulate the rhLZ concentration in human
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milk) were subjected to a spot-on-the-lawn activity assay, by incubating 30 L of each

sample in a punched hole of an agarose plate incorporated with 10% Micrococcus
luteus (ATCC 00356). After the 48-h plate incubation at 37C, photographic images
were analysed and the inhibitory growth zone measurement was assessed for each
group. The assay was based on the diffusion procedure adapted from Maga et al.
(1995).
2.7 Bactericidal assay

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The ATCC (Rockville, MD, USA) strains used to investigate the bactericidal
activity of lysozyme were Shiguella sonnei (ATCC 25931), Enterococcus faecalis

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(ATCC 29212), and M. luteus (ATCC 00356). Bacteria from each strain were
cultivated at 37C for 24 h and then diluted in 0.9% saline solution to a 0.5

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concentration in the McFarland scale. Then, each bacterial suspension was diluted
1,000-fold in Luria-Bertani (LB) broth. Milk samples from the Tg, nTg, S-rhLZ and
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nTg+rhLZ groups were also diluted 20-fold in LB broth, remaining at 37C under 220
x g for 60 min. Then, 15 L were plated onto two LB-agar medium plates. After 24 h,
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the colony-forming units (CFU) were observed, counted and calculated. The
experiment was performed in triplicates.
2.8 Cell proliferation assay
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Cellular viability, migration and gene expression in presence of Tg and

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controls were determined by using rat intestinal epithelial cells (IEC-18 line, ATCC,
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Rockville, MD, USA) in culture, at passage 29, as previously described (Brito et al.,
2005). In brief, cells were cultured at 37C in 5% CO2 in DMEM (Dulbeccos modified
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Eagle medium, GIBCO, Grand Island, NY, USA) containing 10% heat-inactivated
fetal bovine serum, 50 IU/mL penicillin and 50 g/mL streptomycin (GIBCO). Milk
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samples from each group were diluted 1:5, 1:20 and 1:40 in DMEM, with phosphate-
buffered saline (PBS) used as negative control.
2.8.1 Cell viability
To determine cell viability in the presence of different milk dilutions, the MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (Sigma-Aldrich)
was used to measure the mitochondrial reductase activity in living cells (Mosmann,
1983). In brief, IEC-18 were seeded in 96-well plates in a total concentration of 4 x
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104 cells/well in 100 L of culture medium. After 24 h incubation, the media was
removed and replaced by DMEM containing PBS or each sample dilution in triplicate
(n=3). After 24, 48, and 72 h, cells were washed with PBS and then incubated for 4 h
in 100 L culture medium with 10 L MTT solution (5 mg/mL). Plates were
centrifuged at 3,000 rpm for 15 min, media containing MTT were removed by fast
inversion, and formazan crystals were diluted with acidified isopropanol solution (0.04
N HCl). Prior to reading, plates were stirred for 5 min and the absorbance was

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measured in a ELISA reader set at 575 nm.
2.8.2 Migration assay

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The ability of hrLZ-containing milk to affect the IEC-18 migration in the
presence or absence of bacteria was determined as previously described (Carvalho

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et al., 2012). Briefly, cell migration was evaluated in the presence of rhLZ in the milk.
IEC-18 were transferred to a 12-well plate at a concentration of 2.4 x 10 5 cells/well.
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After a 24 h growth period, cells were incubated with Mitomycin C (5 g/mL; Sigma-
Aldrich) for 30 min, the monolayer was scratched with the aid of a sterile blade, so
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that cells were dragged from the center to the right-side edge of the well. Culture
medium containing Mitomycin C was discarded and replaced by fresh medium with
PBS or milk dilutions (1:5, 1:20, and 1:40) from each group. After incubation for 24 h,
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wells were washed twice with PBS for observation of cell migration across the
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scratch line under an inverted microscope at 10X magnification. Cell migration

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analysis was based on the number of migrating cells, the migration distance and
growth velocity, after assessment of photographic images by using the Image J
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software.
2.8.3 Cell migration in the presence of pathogens
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The ability of the milk from transgenic or non-transgenic goats to modulate

cell migration was assessed in the presence of main intestinal pathogens associated
with stomach diseases and diarrhea Escherichia coli EPEC (ATCC 3905), Shiguella
sonnei (ATCC 25931), Enterococcus faecalis (ATCC 29212) and Staphylococcus
aureus (ATCC 6535), based on van Vuuren et al. (2015). Milk samples were diluted
at 1:20 in DMEM, with bacteria (2.5x105 CFU/mL) added to cell cultures. Following
24 h incubation, cells were washed with PBS and examined for CFU, as described
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above (Carvalho et al., 2012).

2.8.4 Gene expression
As intestinal inflammation may be associated with intestinal barrier disruption, qRT-
PCR analysis was used to evaluate the expression of genes for the tight junction
protein ZO-1 and the pro-inflammatory cytokine IL-6 in IEC-18 in culture, using -
actin as housekeeping gene for normalization. Briefly, IEC-18 were seeded into 12-
well plates until 60-70% confluence, when Tg, nTg, nTg+S-rhLZ and S-rhLZ samples

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were added in three biological replicates for each treatment, remaining in culture for
additional 24 h. Cells were washed twice with PBS and harvested by trypsinization.

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Suspended cells were washed in PBS, and then stored at -80C. Samples were
thawed and total RNA was extracted using the RNeasy spin column purification kit

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(Qiagen, Valencia, CA, USA), following the manufacturer's instructions. RNA
concentration was quantified and purity was checked by UV absorbance at 260 nm
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and 280 nm (NanoDrop 2000, Waltham, Massachusetts, USA). cDNA was
synthesized via reverse transcription PCR, using 1 g total RNA treated with DNase,
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oligo (dT) as primers, and Super Script III enzyme (Invitrogen). RT-qPCR reactions
were performed in 20 L containing 10 L Power SYBR Green PCR Master Mix 2x
(Applied Biosystem, Foster City, CA, USA), 1 L cDNA with 600 nM of gene-specific
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primers designed to assess ZO-1 and IL-6 transcript expression, and 6.6 L ultrapure
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water. Primers were synthetized by Invitrogen (So Paulo, Brazil), with nucleotide
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sequences shown in Table A.1. Amplification consisted of 5 min at 94C, followed by

40 cycles of 30 s at 94C, 30 s at the annealing temperature (60C), and 30 s at
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72C, followed by 40 cycles of 0.5C increments (10 s each) for the melting curve,
starting at 75C. Fluorescence was measured during the annealing step of each
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cycle. All amplifications were carried out using the thermocycler StepOne PlusTM
(Applied Biosystems, Foster City, CA, USA).
2.9 Statistical analysis
Data obtained from the bactericidal evaluation, cell viability and migration,
cell number, migration velocity and distance for samples and pathogens were
compared by the 2 or Fishers tests. The normality of the quantitative data was
analysed by the Kolmogorov-Smirnov test and, when required, values were
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normalized by logarithmic transformation on a base 10 and analyzed by ANOVA with

paired comparisons by the Tukeys test (GLM-Minitab, State College, PA, USA). A
simple correlation (Pearson) test was used to assess relationships between
variables. Graphic figures were produced by GraphPad Prism software (Version
5.01). Gene expression data, representative of three independent biological
replicates for each treatment, were subjected to analysis of variance, using Minitab
Statistical Software (Minitab Inc., State College, PA, USA), with means compared by

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the Tukeys test. The level of significance was 5%.
3. RESULTS

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3.1 Physicochemical analysis
Physicochemical analyses showed the mean milk parameter values for goat

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Tg milk samples to be similar to goat nTg milk throughout the eight consecutive
weeks of collection, with all parameters falling well within the required values for
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human consumption, according to minimum quality standards by MAPA (Ministrio da
Agricultura e Pecuria, Brazil, 2000). However, in spite of the normal range values,
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milk from the transgenic goat founder had significantly lower fat and higher protein
contents than milk from both non-transgenic control goats (Table A.2).
3.2 Lysozyme identification and quantification
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The rhLZ was identified by Western blot in milk samples from Tg, hM and S-
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rhLZ (Fig. A.1). After comparing each specific band intensity for each sample to the
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S-rhLZ group (270 g/mL commercial rhLZ), the mean concentration values for
human lysozyme in Tg milk was 224 g/mL. Such mean LZ value was similar to that
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found in the hM control (Table A.3).

3.3 Activity test in vitro
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The M. luteus growth inhibition zone was observed after exposure to Tg, S-
rhLZ, nTg+rhLZ and hM samples, demonstrating a growth restraining effect, whereas
no inhibition zone was observed for the nTg milk sample (Fig. A.2), as expected.
3.4 Bactericidal assay in vitro
The bactericidal activity of the Tg milk group against M. luteus was
demonstrated by a 7- to 8-fold reduction in CFU number observed after incubation in
comparison to the Negative Control and to the nTg group. Additionally, the S-rhLZ
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and nTg+rhLZ samples completely inhibited the M. luteus growth. Samples used
against S. sonnei showed no bactericidal effect, whereas E. feacalis showed a
relative sensitivity to nTg+rhLZ and a discrete sensitivity to Tg and S-rhLZ samples
when compared to the Negative Control and to the nTg Groups (Table A.4).
3.5 In vitro cell viability
The evaluation of the sample dilution effects (1:5, 1:20, and 1:40) on cell
viability showed that the 1:20 and 1:40 dilutions of nTg, S-rhLZ and nTg+rhLZ groups

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significantly reduced the viability after 24 h of exposure when compared to the other
groups. After 48 h of exposure, however, cells exposed to Tg samples at 1:5 and 1:20

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dilutions had lower viability when compared to controls and the nTg group. After 72 h,
the Tg and nTg dilutions were statistically similar between one another and between

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groups, with all dilutions used in the control group showing increased cell viability.
The rhLZ group showed a significantly decrease in cell viability when compared to
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the others group after 24, 48 and 72 h of culture (Fig. A.3).
3.6 In vitro cell migration assay
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The migration features of IEC-18 were not negatively affected by milk or the
presence of human lysozyme. An increase in number of migrating cells was observed
at the 1:20 dilution for all groups in comparison to controls. An increase in migrating
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velocity was observed at all dilutions for the nTg, Tg and S-rhLZ groups, being similar
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to controls when rhLZ is added to nTg. The migration distance was also increased at
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the 1:20 and 1:40 dilutions for the nTg and Tg groups, whereas a reduction was seen
for the nTg+rhLZ group at 1:5 and 1:40 dilutions. No differences were detected
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between dilutions within groups (Table A.5). The 1:20 dilution for the nTg and Tg
groups had significantly better migrating parameters for cells in culture (p<0.05) than
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controls.
3.6.1 In vitro cell migration assay in the presence of pathogens
In general, and compared to controls, the presence of rhLZ in culture was
favourable for in vitro cell migrating velocity and distance in the absence or presence
of E. faecalis, S. aureus and E. coli, with no positive effect observed for S. sonnei. In
the absence of selected pathogens at 1:20 dilution, the Tg groups improved the
number of migrating cells, the nTg, Tg and S-rhLZ groups improved migrating
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velocity, and all groups increased migrating distance in cells in culture when
compared to controls. In the presence of pathogens, the Tg milk, S-rhLZ and nTg-
rhLZ groups at 1:20 dilution promoted an increase in cell number (E. coli for Tg and
S-rhLZ, and S. aureus for S-rhLZ), growth migration velocity (E. faecalis, S. aureus
and E. coli) and distance (E. faecalis, S. aureus and E. coli) when compared to the
control group, except for S. sonnei (p<0.05). Conversely, nTg samples showed lower
cell numbers in the presence of E. faecalis and S. sonnei. In the presence of S.

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sonnei, S-rhLZ and nTg+rhLZ samples were associated with lower number of cells,
with lower migrating distance (p<0.05) in the presence of nTg+rhLZ samples (Table

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A.6).
3.7 Gene expression

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No differences were observed between groups for the relative expression
patterns of ZO-1 in cultured cells. However, albeit the IL-6 expression pattern was
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statistically similar between the control (PBS) and the rhLZ groups, its expression
was increased in the group of cells exposed to nTg milk samples (nTg and
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nTg+rhLZ), with the Tg group being similar to all groups (Table A.7).

4. DISCUSSION
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Goat (Capra hircus) milk contributes with approximately 2.4% of the global
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milk production, representing an important source of proteins in poor communities

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(FAO, 2013). Goat milk has more protein and mineral contents than human milk
(FAO, 2013), and the easy adaptation of goats to environments of extreme poverty
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renders this species attractive for the production of recombinant proteins and
nutraceuticals for human consumption. Consequently, boosting goat milk with human
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lysozyme may be an interesting strategy to combat undernutrition and endemic

diseases. In this study, in view of such potentiality, we report the production of a
transgenic goat lineage for expression of rhLZ in the milk and the characterization of
the milk produced by the founder animal, which is an important step in testing the
milk properties for future potential use of the rhLZ goat milk as nutraceutic product.
Most analyses and parameters evaluated in this study indicated features of great
potential for such application.
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Food composition is an issue of interest in the food industry for product

development, quality control, or regulatory purposes, to ensure quality and safety,
and the physicochemical milk analysis is an important requirement for human
consumption (Nielsen, 2006). The proximate composition of foods includes the
contents of macronutrients, specifically moisture, ash, lipid, protein, and carbohydrate
(Nielsen, 2006). Fresh milk contains the natural ability to resist to pH changes, owing
to its "natural acidity"; however, the action of bacteria that normally develop in raw

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milk produces more or less lactic acid (FAO, 2017). Physical and chemical properties
of fresh milk from the transgenic goat founder was analysed during eight weeks,

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being widely similar to those observed in non-transgenic goats, and in compliance
with the quality standards required by law for human consumption in Brazil (MAPA,

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2000). The significance of the differences in fat and protein composition observed in
the milk from the transgenic goat, although within normal values, may be related to
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the animal effect. Future studies including more animals are necessary to unravel
those findings.
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The rhLZ mean content in the milk from the transgenic goat was about 224
g/mL, which represents 56% of the typical lysozyme concentration found in human
breast milk (400 g/mL; Ning et al., 2009; Yang et al., 2011). The mean lysozyme
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concentration values in human milk varies, as it decreases from the colostrum (370
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g/mL) to the transitional milk (270 g/mL), and to the mature milk between 15 and
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28 days (240 g/mL) of lactation. However, lysozyme levels increase in mature milk
from days 29-56 (330 g/mL) up to days 57-84 (890 g/mL) of lactation (Montagne et
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al., 2001).
The rhLZ present in milk from transgenic goat has been characterized in
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previous studies as capable of modulating microbial population (Maga et al. 2006a,

b; Scharfen et al. 2007). Experiments using the pig model, where animals were fed
with transgenic goats milk containing rhLZ at 270 g/mL, have shown positive impact
on the gastrointestinal morphology, serum metabolites, lymphocyte populations, and
increased anti-inflammatory cytokine expression (Brundige et al. 2008, 2010; Cooper
et al. 2011). In fact, after lactation, the transgenic goats kept a lower level of somatic
cell counts in milk in comparison to non-transgenic goats. Such measure is used to
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monitor infection of the mammary gland, indicating a healthier udder. Such benefit is
probably associated with the antimicrobial activity of lysozyme, resulting in improved
milk safety and animal welfare (Carvalho et al., 2012; Maga et al., 2006a; 2006b).
The analysis of the antimicrobial effect of the milk showed that Tg, S-rhLZ
and nTg+rhLZ samples had antibacterial and bactericidal activity against M. luteus, a
Gram-positive strain normally used as reference organism for studies on lysozyme
activity (Diler et al., 2011). The other Gram-positive strain, E. faecalis, showed

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discreet sensitivity to the presence of Tg and S-rhLZ samples, whereas the CFU
number decreased significantly in the nTg+rhLZ group, which contained rather higher

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rhLZ amounts. The high resistance of E. faecalis to the action of lysozyme is already
well known, which allows survival of the pathogen in the mammalian host (Benachour

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et al., 2012; Varahan et al., 2013).
Despite the effective bactericidal effect against Gram-positive strains,
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lysozyme was shown effective against Gram negative strains only when associated
with other substances, such as lactoferrin (Cerven et al., 2008). Both proteins are
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present in mammalian milk, presenting synergistic antimicrobial properties.

Lactoferrin binds to lipopolysaccharides on the outer bacterial membrane, thus
contributing to membrane disruption and allowing lysozyme to have better access to
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the peptidoglycan layer in Gram negative bacteria (Leitch and Wilcox, 1999).
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Therefore, as expected for the Gram negative bacteria S. sonnei, no sensitivity was
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verified in the sole presence of lysozyme. Our results corroborate with other studies
(Masschalck et al., 2001), in which the presence of lysozyme (at concentrations of 10
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and 100 g/mL) resulted in no variation or delay in growth curves for any of the six
tested types of Gram-negative bacteria (Escherichia coli, Pseudomonas fluorescens,
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Salmonella enterica serovar Typhimurium, Salmonella enteritidis, Shigella sonnei,

and Shigella flexneri).
The potential antimicrobial effect of lysozyme, with positive modulation of the
microbial population, has been previously described by multiple studies. Moreover,
the stability of LZ to heat treatments and acidic conditions ensures integrity and
effectiveness along the gastrointestinal tract (Masschalck et al., 2001; Mcinnins et al.,
2015), which makes the transgenic milk containing rhLZ a promising nutraceutical
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product, suitable for consumption as fresh raw or pasteurized milk. Furthermore, the
rhLZ can be purified from milk for use as supplement to oral rehydration solutions. In
fact, the use of transgenic goat milk as potential nutraceutic product or as
supplement to oral rehydration therapy was reported by Carvalho et al. (2012), which
demonstrated that the presence of nutrients in culture medium, in special proteins
and fat of goat milk, can be beneficial to intestinal cell proliferation in vitro. Results in
the cell assay obtained in this study showed alterations in cell viability in the

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presence of diluted samples. After 24 h, the 1:20 dilution of Tg milk sample was
significantly similar to the control group and different from nTg, showing positive

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effect on cell migration.
Besides the nutrient source, the antimicrobial ability of the transgenic milk

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was evaluated by comparing cell migration in the presence of 1:20 sample dilutions,
24 h after the in vitro inoculation of pathogenic bacteria. Improved cell proliferation
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was observed in cultures exposed to samples from the Tg group along with E.
faecalis, S. aureus (statistically similar to S-rhLZ and nTg+rhLZ), and S. sonnei.
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Although Tg milk samples showed no bactericidal effect against E. faecalis, the

presence of milk per se was beneficial to cell proliferation. The lower cell migration
effect observed after exposure to samples from the S-rhLZ and nTg+rhLZ groups
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may be due to higher LZ protein activity in the Tg milk than in the rhLZ-supplemented
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milk, as the lysozyme produced in vivo in the milk through the mammary gland has
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been previously shown to be more potent as antimicrobial agent than the milk
supplementation with purified protein (Carvalho et al., 2012; Maga et al., 2006a).
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Tight junction proteins are mainly responsible to function as intestinal

mucosa barrier against macromolecular diffusion (Zhang and Gou, 2009). The
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presence of samples in cell culture did not alter relative gene expression pattern for
ZO-1 protein, suggesting no effect on intestinal epithelial permeability in cultured
cells. Conversely, the expression pattern of pro-inflammatory cytokine IL-6 by IEC-18
was increased in the presence of milk samples (nTg, Tg and nTg+rhLZ), which
suggests that transgenic or non-transgenic goat milk may elicit an intestinal
inflammatory process under in vitro conditions (Atreya and Neurath, 2005). However,
the in vivo analysis of gut regions (duodenum and ileum) of pigs that received an
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association of pasteurized transgenic goat milk and pasteurized non-transgenic cow

milk, with a final concentration of 135 g/mL rhLZ, showed no increase in the
expression of IL-6 (Cooper et al., 2013). More studies are needed to evaluate the in
vitro and in vivo effects of the Tg and nTG milk on the intestinal epithelium, on pro-
and anti-inflammatory molecules, and on gut permeability.
5. CONCLUSIONS
This study focused on the characterization of the goat milk from a transgenic

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line that expresses human lysozyme in the milk. The physicochemical properties of
milk samples from transgenic and non-transgenic goats were similar and in

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compliance with the minimum quality requirements for human consumption (MAPA,
2000). Active rhLZ is present in the milk of the transgenic goat line at amounts

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comparable to values found in human breast milk, also demonstrating in vitro
antibacterial and bactericidal effects. The transgenic milk did not alter or affect the in
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vitro viability, proliferation and migration of intestinal epithelial cells (IEC-18) in
culture. This study was carried out in the Brazilian Northeast region, where the
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number of infant deaths due to malnutrition and infectious diseases is high. The rhLZ
produced in milk has the potential to be used in infant formulas or in natura, as
nutraceutic product, with the nutritional and medical values comparable to human
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milk. Additional in vitro and in vivo studies using animal models are under way, which
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are absolutely necessary to be performed to ensure safety as well as the observation

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of beneficial effects with no detrimental or unintended consequences of the use of

transgenic goat milk containing rhLZ.
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ACKNOWLEDGEMENT
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This study was funded by the Studies and Projects Funding Agency
(Financiadora de Estudos e Projetos Finep) of the Ministry of Science and
Technology of Brazil, under grant number 0460.08.
CONFLICT OF INTEREST
Igor de S Carneiro, Jos Nilson Rodrigues de Menezes, Julyana Almeida
Maia, Victor Bruno Soares de Oliveira, James D. Murray, Elizabeth A. Maga, Marcelo
Bertolini and Luciana Relly Bertolini state that there are no conflicts of interest.
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Figures

Fig. A.1. Representative Western blot for the identification of the hLZ in samples of

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non-transgenic goat milk (nTg), transgenic goat milk (Tg), human breast milk (hM)
and S-rhLZ solution (270 g/mL of commercial rhLZ from Sigma-Aldrich diluted in

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saline) used as standard control. L=protein ladder (ProSieve Color Protein Maker -
Lonza).

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Fig. A.2. In vitro antimicrobial activity (linear measurement, in mm, and surface area,
in mm2, of the inhibition zone) of (nTg) non-transgenic goat milk (0,00 mm, 0,000
cm2), (Tg) transgenic goat milk (1,29 mm, 0,502 cm 2), (hM) human breast milk (2,30

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mm, 0,73 cm2), and commercial rhLZ diluted (S-rhLZ) in saline (2,39 mm, 0,830 cm2)
or (nTg-rhLZ) in nTg milk (1,51 mm, 0,670 cm2) against Micrococcus luteus strain.

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Fig. A.3. In vitro cell viability in the presence of 1:5, 1:20 and 1:40 dilutions of non-
transgenic goat milk (Tg), transgenic goat milk (nTg), human breast milk (hM), and
commercial rhLZ diluted in saline (S-rhLZ) or in nTg milk (nTg+rhLZ) exposed for 24
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a d:
(A), 48 (B) and 72 h (C) in comparison to LZ-free control sample. Columns with

different superscripts for each dilution differ (p<0.05).

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Tables
Table A.1. Sequence of PCR primers, melting temperatures (C) and GenBank

accession number for gene products analysed in cells in culture exposed to non-

transgenic goat milk (nTg), transgenic goat milk (Tg), human milk (hM), and

commercial rhLZ diluted in saline (S-rhLZ) or in nTg milk (nTg+rhLZ).

Melting GenBank

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Gene Primer sequence 5-3 temperature (C) accession

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number

F GAGGCTTCAGAACGAGGCTATTT

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ZO-1 81.7 NM_001106266.1
R CATGTCGGAGAGTAGAGGTTCGA NU
F ACCACCCACAACAGACCAGT
IL-6 77.1 NM_012589.2
R ACAGTGCATCATTCGCTGTTC
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F CCCTGGCTCCTAGCACCAT
-Actin 78.2 NM_031144.3
R GAGCCACCAATCCACACAGA
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Table A.2. Physicochemical analysis of milk samples from the transgenic founder

goat (Tg) and two non-transgenic control goats (nTg and nTg2). Results represent

mean values obtained for milk collected during eight consecutive weeks starting at

the third week of lactation.

Standards by
Tg nTg nTg2

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MAPA (2000)1

Lactic acid (%) 0.16a 0.20a 0.18a 0.13 0.18

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Density at 15 C (g/mL) 1.02a 1.01a 1.00a 1.02 1.03

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Cryoscopic constant (H) -0.55a -0.55a -0.55a -0.55 -0,585

Fat (%) 2.2a 2.7b 2.4b < 2.9

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Protein (%) 3.5a 2.9b 2.9b > 2.8
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Lactose (%) 4.5a 4.3a 4.4a > 4.3

Non-fat solids (%) 8.4a 8.2a 8.2a > 8.2

6.5a 6.4a 6.5a

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pH --*
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Ashes (%) 0.7a 0.8a 0.9a > 0,7

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a,b:
Numbers with different supercripts in the row differ (p<0.05)
1
MAPA recommendations (Ministrio da Agricultura, Pecuria e Abastecimento, Brazil).
*Not described.
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Table A.3. Quantification of recombinant human lysozyme (rhLZ) present in the

milk from the transgenic founder (Tg), at the third week of lactation, in

comparison to milk from non-transgenic goat (nTg), human breast milk (hM)

and commercial rhLZ diluted into saline solution (S-rhLZ). Results represent the

semi-quantitative conversion of the lysozyme band intensity assessed from the WB

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relative to the S-rhLZ group (270 g/mL commercial rhLZ), analysed by the Image J

software.

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Western

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nTg Tg, g/mL hM, g/mL S-rhLZ*, g/mL
Blotting
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1 - 230 226 270

2 - 221 231 270

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3 - 221 221 270

Mean 224 226 270

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*Saline solution with known amounts of commercial rhLZ (270 g/mL rhLZ).
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Table A.4. Evaluation of the bactericidal activity of non-transgenic (nTg) and

transgenic (Tg) milk samples, commercial rhLZ diluted in saline solution (S-rhLZ) or

in nTG milk (nTg+rhLZ) samples against pathogenic bacteria in culture .

CFU Control nTg Tg S-rhLZ nTg+rhLZ

M. luteus 28.0 3.2a 32.3 11.9a 4.0 1.2b 0.0 0.0c 0.0 0.0c

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S. sonnei 117.7 18.8a 140.3 11.5a 111.3 5.7a 105.3 10.3a 90.7 5.6a

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E. faecalis 165.3 14.4a 142.0 18.2a 113.7 12.8ab 110.7 14.7ab 63.3 15.0b
a,b:
Means in the row with different superscripts differ (p<0.05).

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Table A.5. Number of migrating cells, velocity and distance of cell migration in vitro in

the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples diluted at 1:5, 1:20 and 1:40.

Migration
Control Dilution nTg Tg S-rhLZ nTg+rhLZ
assay

1:5 23.3 0.9aA 23.7 1.2aA 23.7 0.3aA 22.6 1.2aA

Migrating

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22.0 0.6a 1:20 27.3 2.0bA 32.0 1.5bA 27.7 0.3bA 28.0 2.1bA
cells, n

1:40 23.3 0.7aA 25.0 1.0aA 23.7 0.7aA 23.0 0.6aA

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1:5 22.3 1.6bA 23.3 0.0bA 18.9 1.9bA 15.2 1.0aA

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Migrating
11.3 0.5a 1:20 22.2 2.5bA 24.7 2.6bA 19.0 0.0bA 17.0 1.2abA
velocity

1:40 21.7 2.6bA 23.6 2.6bA 19.32 1.3bA 15.8 1.5aA

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1:5 518.0 55.2aA 537.2 32.5aA 449.1 26.5abA 381.2 33.6bA

Migrating
471.6 12.3a 1:20 550.4 55.3bA 593.7 62.7bA 473.0 33.6aA 407.5 28.6aA
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distance

1:40 505.6 52.0bA 567.8 61.8bA 438.3 32.7abA 301.2 33.6cA

a,b:
Means in the same row with different superscripts differ (p<0.05).
A,B:
Means in the same column with different superscripts, for each migration assay, differ (p<0.05).
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Table A.6. Number of migrating cells, velocity and distance of cell migration in vitro in
the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples diluted at 1:20 and
pathogenic bacteria.

Migration
Pathogens Control nTg Tg S-rhLZ nTg+rhLZ
assay

Absent 22.0 0.6a 27.3 2.0ab 34.7 41.2b 27.7 0.3ab 28.0 2.1ab

E. feacalis 21.7 0.3ab 16.0 0.0b 23.0 3.0a 25.0 1.2a 23.3 1.2a

PT
Migrating S. aureus 21.3 0.3ab 18.0 1.2a 26.3 1.3bc 30.67 2.7c 26.7 2.3bc
cells, n

RI
E. coli 20.7 0.3a 24.3 2.4ab 28.3 1.2bc 22.7 1.5ab 29.7 0.7c

SC
S. sonnei 20.3 0.3ab 16.7 1.9b 23.3 1.8a 14.0 1.2b 12.7 3.2b

Absent 11.3 0.5a 23.2 2.3b 24.0 2.6b 21.4 1.7b 17.0 1.2ab
NU
E. feacalis 11.1 0.1a 9.3 0.0a 17.2 1.6b 19.3 0.8b 17.4 0.4b

Migrating S. aureus 11.3 0.0a 15.2 2.4ab 21.6 0.5b 22.1 1.5b 21.6 1.5b
velocity
MA

E. coli 11.0 0.1a 16.0 2.6ab 20.1 5.1b 17.5 0.8b 18.8 1.0b

S. sonnei 11.2 0.0ab 15.7 1.5a 17.7 2.1a 12.4 1.6ab 7.2 1.0b
D

Absent 271.6 12.3a 555.7 55.0b 593.8 62.7b 473.0 33.6b 407.6 28.6b
E

E. feacalis 270.8 0.5a 222.6 0.0a 412.1 39.4b 412.0 39.4b 417.6 10.1b
PT

Migrating S. aureus 270.2 0.2a 394.3 66.0ab 507.6 10.9b 530.0 36.0b 518.2 35.7b
distance
CE

E. coli 269.6 0.2a 383.6 62.1ab 410.0 60.5b 418.87 18.6b 450.8 24.4b

S. sonnei 269.2 0.2ab 402.0 37.3a 425.6 51.2a 297.6 37.9ab 173.6 23.2b
AC

a b:
Means in the same row with different superscripts differ (p<0.05).
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32

Table A.7. Relative expression levels for the ZO-1 and IL-6 genes in IEC-18 in

culture after incubation in the presence of nTg, Tg, S-rhLZ and nTg-rhLZ samples.

Gene
Control nTg Tg S-rhLZ nTg+rhLZ
Expression

a a a a a
ZO-1 1.2 0.1 0.8 0.1 0.8 0.2 1.1 0.1 0.7 0.1

PT
b a ab b a
IL-6 1.8 0.8 12.9 0.3 7.6 3.5 1.7 0.3 10.3 0.5
a,b:
Means within the same row with different superscripts differ (p<0.05).

RI
SC
NU
MA
E D
PT
CE
AC
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33

Graphical abstract

PT
RI
SC
NU
MA
DE
PT
CE
AC