Consumption of Lysozyme-Rich Milk Can Alter Microbial Fecal
Elizabeth A. Maga,aPrerak T. Desai,b* Bart C. Weimer,bNguyet Dao,bDietmar Kültz,aand James D. Murraya,b
Departments of Animal Scienceaand Population Health and Reproduction,bUniversity of California, Davis, Davis, California, USA
munitymemberdiversitywaschangedsignificantlyuponconsumptionoflysozymemilk.Levelsof Firmicutes (Clostridia)de-
clinedwhereasthoseof Bacteroidetes increasedovertimeinresponsetotheconsumptionoflysozyme-richmilk.The
that promote health and combat infection. One of the main non-
is a naturally occurring antimicrobial enzyme found in the tears,
saliva, and milk of all mammals that lyses a specific link in the
peptidoglycan layer of bacterial cell walls, resulting in cell lysis
(33). Along with lactoferrin and secretory IgA, lysozyme contrib-
utes to the nonspecific immunity associated with milk consump-
tion. A number of epidemiological studies have documented the
benefits of human milk, including advantages in general health,
growth, and development and protection against a number of
acute and chronic diseases in infancy and beyond (16, 22). In
establishment of a beneficial gut microbiota. Breast-fed infants
have been shown to have a more healthy and simple gut microbi-
ota consisting mainly of bifidobacteria, lactobacilli, and staphylo-
with coliforms, clostridia, enterococci, streptococci, and bacte-
roides all being prevalent in the gut (1, 15, 34). In addition to
human milk oligosaccharides and other bioactive compounds,
another reason for the growth of fewer facultative anaerobes in
breast-fed infants is thought to be the presence of antimicrobial
factors such as lysozyme in human milk (18, 34).
The composition and function of the intestinal microbiota are
just beginning to be defined and ascertained, but it is widely ac-
cepted that it plays a role in both health and disease (41). Due to
the purported role of human milk in gut microbiota community
formation, a source of milk rich in lysozyme may shift the micro-
goat milk is readily available, it is low in key health-promoting
uman milk not only provides the newborn with all the nutri-
tion it needs to grow and develop but also provides factors
?g/ml) and 1,600 (0.25 ?g/ml) times less lysozyme, respectively
lysozyme (HLZ) in their milk at 68% the level normally found in
human milk (29) with the goal of incorporating the beneficial
protective properties of human milk into readily available live-
of milk from HLZ-transgenic goats by animal models results in
the modulation of Escherichia coli and total coliform levels in the
small intestine as determined using standard culture techniques
and histological and cytokine changes coupled with increases in
serum metabolite makers indicative of improved gastrointestinal
(GI) health (6, 10).
In order to determine if HLZ-rich milk can modulate gut mi-
crobiota composition, this study conducted feeding trials in
young pigs followed by a more in-depth fecal microbiota assess-
ment using 16S rRNA clone libraries and the G2 Phylochip. The
pig was selected as a model organism since it represents a mono-
gastric animal with GI anatomy, function, and metabolic regula-
tion similar to those of humans (2). The use of pigs as a relevant
Received 23 March 2012 Accepted 25 May 2012
Published ahead of print 29 June 2012
Address correspondence to Elizabeth A. Maga, email@example.com.
*Present address: Prerak T. Desai, Vaccine Research Institute, San Diego, California,
Supplemental material for this article may be found at http://aem.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
September 2012 Volume 78 Number 17 Applied and Environmental Microbiologyp. 6153–6160aem.asm.org
human medical model is well documented (27). Furthermore,
pigs and humans share similarities in GI microbial diversity (20)
states, and a variety of nutritional intervention approaches. This
study found that HLZ milk modulated GI microbiota by increas-
ing the ratio of beneficial bacteria and decreasing disease-causing
MATERIALS AND METHODS
Milk and milk analysis. Production and characterization of the HLZ-
transgenic line has been previously described (28, 29). This line of trans-
genic goats produces active HLZ in their milk at an average level of 270
?g/ml without disrupting the gross composition of the milk in terms of
total fat and protein content (29). For the feeding to pigs, milk was col-
genic and nontransgenic control goats from the University of California
(UC) Davis dairy goat herd. Milk was pooled into respective containers,
pasteurized to 74°C, and stored at 4°C prior to feeding to animals. Milk
ence and quantity of HLZ. Milk was also collected from a total of six
individual HLZ-transgenic and equal numbers of age- and breed-
matched nontransgenic control animals for a more in-depth analysis us-
ing two-dimensional (2-D) gel analysis coupled with mass spectrometry.
Milk was collected at peak lactation (2 months) from two transgenic an-
imals each in their 1st, 2nd, and 3rd lactations and equal numbers of
breed- and parity-matched nontransgenic controls. A total of 200 ?g of
protein from each milk sample was subjected to 2-D gel analysis followed
(MALDI-TOF/TOF) mass spectrometry as previously described (32).
Briefly, separation in the first dimension by isoelectric point was carried
5.6; 11 cm in length) followed by separation in the second dimension by
size on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gels (Protean Xi; Bio-Rad, Hercules, CA). Gels were then
stained in Coomassie blue and scanned, and proteins were quantified
(Decodon GmbH, Greifswald, Germany). A total of 29 individual spots
were then extracted from each of three gels containing milk from trans-
genic goats in their 2nd and 3rd lactation and their three corresponding
control gels and processed using a Montage In-Gel Digest Zip kit (Milli-
pore, Billerica, MA) followed by MALDI-TOF/TOF mass spectrometry.
The same 29 spots were chosen on each of the six gels. Protein identifica-
tion and annotation were carried out using GPS Explorer software with
the Mascot search algorithm and DeNovo Explorer modules included in
the 4700 Explorer software (Applied Biosystems, Foster City, CA). All
of age and housed together, receiving the same treatment and diet until 8
weeks of age, at which time they were placed into groups of two in con-
nected environmental chambers. All pigs had ad libitum access to a stan-
dard ration of dry feed (UC Davis Pig Starter Diet; Associated Feed, Tur-
each pen received 500 ml of pasteurized nontransgenic control goat milk
twice daily delivered through a lixit container for a period of 2 days in
order for the animals to become accustomed to consuming milk. Each
animal was then placed into an individual pen, with four animals receiv-
pasteurized milk from HLZ-transgenic goats for a period of 14 days. Pigs
milk. Each animal was dosed with 250 ml of its respective milk allotment
daily for the remaining 6 days. Fresh feces samples were collected from
each pair of animals before any milk was given (day 1) and then from
individual animals at the end of the adjustment period and before the
milk type was switched (day 3), 24 h after the start of HLZ milk ad-
ministration (day 4), and then every other day for the remainder of the
trial. After 14 days of milk dosing, the animals were subjected to nec-
and E. coli counts using Petrifilm count plates (3M, St. Paul, MN) as
previously described (5).
16S rRNA gene sequencing. Fecal samples from days 1 (no milk), 3
ples were stored at 4°C for no longer than 4 days prior to bacterial DNA
extraction using a QIAmp DNA stool kit with the protocol for pathogen
detection (Qiagen, Valencia, CA). Primers 27F (5=-AGAGTTTGATCCT
GGCTCAG-3=) and 1392R (5=-GACGGGCGGTGTGTAC-3=) were used
to amplify the 16S rRNA gene sequence (21). PCRs were performed as
recommended by Polz and Cavanaugh (39) to reduce bias in amplifica-
95°C for 5 min, 20 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1.5
min, and 1 cycle of 10 min at 72°C. Resulting PCR products were ligated
into StrataClone PCR cloning kit vector (Agilent Technologies, Santa
per sample were grown in 96-well plates in LB freezing media, and DNA
using primer 1392R and a BigDye Terminator v3.1 cycle sequencing kit
(Applied Biosystems, Foster City, CA).
Resulting DNA sequences were subjected to base-calling by PHRED
ison to 16S rRNA gene sequences from the Ribosomal Database Project
reads of greater than 400 bp were used. Clones with more than 97% se-
assigned to a phylum using the Classifier software available at http://rdp
.cme.msu.edu/classifier/classifier.jsp (49), which assigns an OTU se-
quence to a phylum using a naïve Bayesian rRNA classifier trained on the
known type strain 16S sequences. Complete 16S rRNA libraries for each
feeding group were compared to each other using the Student t test and
the Library Compare software available at http://rdp.cme.msu.edu
/comparison/comp.jsp (49), which estimates the likelihood that the fre-
quency of membership in a given taxon is the same for the two libraries.
The percentage of a phylum in one library was considered significantly
different (P ? 0.05) from the percentage in another library if the two
statistical methods (Student’s t test and Compare) were in agreement. As
one control-fed pig was found to be an outlier via G2 Phylochip analysis,
this pig was not used in the analysis of the 16S rRNA data.
on the endpoint samples (day 17) using the G2 Phylochip as described by
Parnell et al. (37, 38). Briefly, the ribosomal 16S gene was amplified by
PCR utilizing bacterium-specific primers (F [5=-AGAGTTTGATCCTGG
and labeled DNA (4 ?g) was hybridized to the G2 Phylochip for 16 h at
following the standard Affymetrix protocol at the Center for Integrated
BioSystems, Utah State University (Logan, UT). Raw .cel files were nor-
malized using RMA (17). The presence or absence of each OTU was de-
termined using Phylotrac (www.phylotrac.org). An OTU was considered
present if 90% of the probes in the OTU probe set had a ratio of PM/
MM ? 1.3. OTUs called present in at least one of the samples were in-
cluded in further analysis. Hierarchical clustering of data was done using
HCE 3.5 (42). Principal component analysis was done using TMEV 4.6.1
ent between the control and treatment groups. Phylogenetic groups were
on the Phylochip. Statistical overrepresentation of the OTUs due to the
Maga et al.
aem.asm.org Applied and Environmental Microbiology
treatment within specific phylogenetic groups was estimated using GSEA
obtained from Greengenes (12). The 16S sequences were aligned using
MUSCLE (14), maximum-likelihood trees were built using RAxML (43),
and iTOL ver. 2.1.1 (24) was used to project log2ratios of data from the
treatment versus control groups onto the 16S trees.
Milk analysis. The overall protein composition of milk from
HLZ-transgenic goats was not statistically different from that of
milk from nontransgenic control goats. A total of 136 protein
their locations and intensities were not significantly different be-
tween transgenic and control samples (Table 1; see also Fig. S1 in
the supplemental material). Furthermore, 23 of the 29 spots cho-
sen for mass spectrometry analysis were identified and all corre-
sponded to proteins normally found in milk (Table 1). Pigs con-
suming HLZ milk for 14 days had significantly lower numbers of
coliforms in the duodenum than did control-fed pigs (P ? 0.019;
Table 2). The numbers of coliforms and E. coli in the ileum were
lower but not significantly different in HLZ-fed pigs (P ? 0.487
and P ? 0.332, respectively).
file determined using 16S rRNA gene sequencing showed that all
animals started with similar fecal microbial populations, as no
significant differences in phyla among animals at day 1 and day 3
receiving HLZ milk had an overrepresentation of Bacteroidetes
approaching significance (P ? 0.07) and significant underrepre-
Bacteroidales were present in a significantly greater proportion in
HLZ-fed animals (P ? 0.035), with a significantly higher repre-
0.033; Table 4).
The consumption of control milk during the adjustment pe-
riod promoted the growth of members of Proteobacteria (P ?
time, levels of Bacteroidetes steadily increased and were accompa-
nied by a steady decrease in levels of Firmicutes in HLZ-fed pigs
There were significant differences in the levels of Firmicutes in
significantly different at day 17 (P ? 0.038). On day 6, HLZ-fed
than did control-fed pigs (P ? 0.001, Table 4). Within the Firmi-
HLZ-fed pigs and were significantly different from those in con-
TABLE 1 Identification and quantification of proteins in HLZ and control milk
Gel Protein identification
Spot density (% vol)
Control (n ? 6)HLZ (n ? 6)P
Serum albumin precursor
?s1-Casein A short form
0.681 ? 0.133
1.492 ? 0.367
17.278 ? 0.979
19.646 ? 1.461
4.001 ? 0.822
5.829 ? 0.216
1.600 ? 0.426
5.208 ? 0.504
2.317 ? 0.539
2.904 ? 0.205
1.642 ? 0.263
8.761 ? 0.464
0.715 ? 0.274
0.871 ? 0.277
12.180 ? 0.719
14.202 ? 0.753
9.162 ? 0.543
7.574 ? 0.413
2.099 ? 0.192
4.080 ? 0.388
4.507 ? 0.583
17.566 ? 1.001
1.422 ? 0.236
0.728 ? 0.135
1.357 ? 0.277
17.192 ? 0.950
20.338 ? 1.345
4.476 ? 0.897
5.655 ? 0.509
1.342 ? 0.434
5.152 ? 0.463
2.423 ? 0.232
2.975 ? 0.220
1.363 ? 0.165
9.148 ? 0.294
0.587 ? 0.228
1.083 ? 0.325
12.066 ? 0.692
14.198 ? 1.007
9.306 ? 0.660
7.393 ? 0.321
2.158 ? 0.283
4.110 ? 0.495
4.671 ? 0.426
18.033 ? 2.158
1.809 ? 0.285
TABLE 2 Total coliform and E. coli counts in intestinal segments at
Segment and organism(s)
log CFU/g ? SD for indicated pig group
Control fed (n ? 3)HLZ fed (n ? 4)
1.93 ? 0.81
0.25 ? 0.5a
3.66 ? 1.46
3.66 ? 1.46
3.01 ? 0.82
2.36 ? 1.65
aHLZ-fed pigs significantly different from controls (P ? 0.019).
Lysozyme in Milk Can Change Gut Microbiota
September 2012 Volume 78 Number 17aem.asm.org 6155
trol-fed pigs at both day 6 (P ? 0.005) and day 17 (P ? 0.045),
with more fluctuation in the control-fed animals (Fig. 1B and
granulum and Anaerovibrio genera in HLZ-fed animals at day 17
(Table 4). Levels of Bacilli were steadily maintained in HLZ-fed
these differences were not statistically different (P ? 0.43 at day 6
and P ? 0.682 at day 17) (Fig. 1C).
G2 Phylochip analysis. All microbes and trends found in the
clone library of the endpoint (day 17) were also found to be pres-
ent using the G2 Phylochip; plus, many additional OTUs were
observed. The Phylochip analysis identified a total of 500 OTUs
across seven samples (18 phyla, 38 classes, 64 orders, and 91 fam-
ilies) compared to 13 phyla, 18 classes, 17 orders and 32 families
detected with the clone libraries. The total fecal community pop-
ulations of all animals consuming HLZ milk for 14 days were
similar to each other, as were the populations of bacteria in the
feces of three of the four control-fed animals (Fig. 2). Using prin-
cipal component analysis (PCA), the six samples were segregated
into two distinct clusters. Based on the PCA, one of the control
samples (Con-1 in Fig. 2) was a clear outlier and hence was re-
moved from further statistical analysis. A total of 113 OTUs were
significantly different (q [adjusted P value taking into consider-
groups (see Fig. S2 in the supplemental material). Figure 3 sum-
marizes the differences at the family level, which had at least two
OTUs that were significantly different between the treatment and
control groups (total of 64 significant OTUs). Among all groups,
Clostridales were statistically underrepresented in HLZ-fed pigs
pigs (q ? 0.1) compared to pigs fed control milk. Interestingly,
beneficial microbes such as Bifidobacteriaceae (2 OTUs) and Lac-
tobacillaceae (2 OTUs) were also enriched upon consumption of
HLZ milk whereas detrimental microbes such as Streptococcaceae
(1 OTU) and Campylobacterales (2 OTUs) were depleted with
consumption of HLZ milk (see Table S1 in the supplemental ma-
Human milk contains numerous bioactive components that can
used a model system of pigs and transgenic goat milk containing
HLZ to examine the effects of consumption of this antimicrobial
milk component on beneficial microbe enrichment versus detri-
mental microbe reduction in the gut microbial community. We
tered gut microbiota populations and shifted the microbial pop-
ulation to those microbes associated with activities beneficial for
sition using 2-D gels indicated that there were no off-target pro-
teins being produced and no endogenous proteins whose
production was being diminished by the expression of HLZ and
HLZ. Moreover, human milk oligosaccharides which are known
modulators of intestinal microbiota are not found in the same
diversity or quantity in goat milk (9), demonstrating that HLZ
alone could contribute this important function to the milk of
Microbial abundance increases along the length of the diges-
TABLE 3 Fecal microbial profile after 14 days of consuming HLZ or
% of clones in indicated pig groupa
(n ? 3; 356 clones)
(n ? 4; 532 clones)P
aData represent the percentages of clones assigned to a phylum using 16S rRNA gene
sequencing. Clone values in column headings represent the number of sequences in
bHLZ-fed pigs significantly different from controls (P ? 0.05).
ing are shown. HLZ, pigs fed milk from HLZ-transgenic animals; Cont, pigs
fed milk from nontransgenic control animals. *, significantly different (P ?
Maga et al.
aem.asm.org Applied and Environmental Microbiology
the colon, and therefore the microbiota of feces is distinct from
Using standard culturing techniques, total coliform and E. coli
counts in the duodenum and ileum of HLZ-fed pigs tended to be
lower than in control-fed pigs, similar to results seen when start-
ing with 14-day-old pigs (5, 30). These results indicate the repro-
ducibility of the effect of HLZ milk in differently aged animals at
samples, the overall level of Proteobacteria was not significantly
lower in HLZ-fed animals than in controls (5.6% versus 7.3%)
and there were no differences in E. coli levels in the feces of HLZ-
fed and control-fed animals, likely a difference in sample location
(small intestine versus feces). Proteobacteria are a common com-
ponent of the distal gut, and in vitro work has demonstrated that
HLZ milk acts in a bactericidal fashion toward E. coli (31).
The predominant phyla present in the feces of both feeding
groups of pigs at all time points were the Firmicutes and Bacte-
studying the GI microbiome and the consequences of microbiota
manipulation. The microbial populations were not statistically
different between individuals at the start of the trial (day 1, no
milk) at the phylum level, indicating that all pigs were starting
with similar fecal microbiotas. It should be noted that while sam-
ples were prepared after storage at 4°C, which has been shown to
have an impact on detected microbial diversity (36), all samples
were subjected to similar storage conditions, thus allowing direct
comparison between samples. After the 3-day period of adjust-
TABLE 4 Significant differences in bacterial genera by 16S rRNA gene sequencing after 2 and 14 days of HLZ milk consumption
Phylum ClassOrder Family GenusP
% of total clones in
indicated pig group
Control fedHLZ fed
Mollicutes AnaeroplasmatalesAnaeroplasmataceaeAnaeroplasma 0.000103
FIG 2 Projection of samples on the first two principal component axes based
on the PCA of OTU abundance from the Phylochip analysis. HLZ, pigs fed
milk from HLZ-transgenic animals; Con, pigs fed milk from nontransgenic
Lysozyme in Milk Can Change Gut Microbiota
September 2012 Volume 78 Number 17aem.asm.org 6157
ment to consumption of milk, there were significantly more Pro-
altered the fecal microbiota, all animals started with similar mi-
sequent changes seen were due to direct effects of HLZ milk and
consumption of milk commenced was maintained over all later
time points in both feeding groups. Succinivibrio commonly in-
habit the rumen of dairy animals to aid in digestion, and thus,
consumption of goat milk in general increased levels of this mi-
vibrio already present or by reducing the levels of other microbes,
thereby allowing more growth of Succinivibrio. Using 16S rRNA
gene sequencing of clone libraries, no Succinivibrio bacteria were
found to be present in the milk itself (data not shown).
Previous work has demonstrated that a majority (95%) of the
were seen here, as after 14 days of milk consumption, 91% of the
Firmicutes in control-fed animals were Clostridia. In HLZ-fed an-
imals, levels of Clostridia were reduced from 92.4% (day 2) to
89.5% of total Firmicutes at day 17. While Firmicutes are impor-
tant for supplying energy in the form of short-chain fatty acids, a
skewed ratio of Firmicutes to Bacteroidetes has been implicated in
in Firmicutes has been associated with an increased ability to ex-
tract energy from the diet and/or promote the deposition of this
energy in the form of fat (19, 45). After 14 days of HLZ milk
was underrepresented and that of Bacteroidetes overrepresented
compared to animals consuming control goat milk. The gut sur-
face area of the animals used in this study was investigated previ-
ously and was found to be related to an improved absorptive ca-
longer villi and had a significantly thinner lamina propria in the
duodenum (10). A 20% increase in the proportional representa-
tion of Bacteroidetes in fecal microbiota in humans has been cor-
related with a decrease in nutrient absorption (19). Here we ob-
served a more modest increase in fecal Bacteroidetes content
(6.2%) upon consumption of HLZ milk that was related to
changes in cellular ultrastructure in the small intestine. While the
energy content of the feces was not determined in this study, the
resulting histological changes represent an intestinal epithelium
plying Firmicutes. Combined, these results suggest that microbi-
remains to be determined what other metabolic and functional
changes occur in response to HLZ-induced modulation of gut
One important issue regarding gut microbiota communities is
their stability over time (3). Animals fed control milk were more
prone to fluctuation in the relative proportions of phyla present
after the 3-day adjustment period. The significant increase in Te-
nericutes at day 6 in control-fed animals was related to one indi-
vidual; however, the proportions of rest of the phyla were more
consistent between individuals of the same feeding group. Gut
people (11). The greater fecal community member stability seen
HLZ acting to quiet challenges to the gut, the short interval be-
tween sample analysis, or a brief environmental perturbation, al-
environment. Changes in diet have been shown to rapidly induce
changes in fecal microbiota (within 3 to 4 days), after which the
microbiota was maintained for several weeks until the diet was
altered again (48). Here, the introduction of milk altered fecal
microbiota in a similarly short time frame; however, the changes
were not static. The changes being seen in both feeding groups
modulate community member diversity in a similar pattern over
Overall, the G2 Phylochip analysis supported our findings ob-
tained with 16S rRNA gene sequencing and offered more details.
Both methods identified Bacteroidetes and Firmicutes as the main
in HLZ-fed animals compared to control-fed animals. Within
phyla and families, levels of community members were both in-
creasing and decreasing in response to HLZ milk consumption,
with the exception of Bacteroidetes, where levels of all members
were elevated compared to the results from control-fed animals.
Greater variability within phyla has been reported in previous
studies (13); however, HLZ milk was also able to change the bac-
terial abundance at the phylum level.
Lysozyme-rich milk was capable of significantly altering com-
munity member diversity in a fashion that allowed identification
of the feeding group based on the resulting fecal microbiota.
17 as determined by Phylochip analysis. The numbers beside the heat map represent the OTUs that were significantly different in each family.
Maga et al.
aem.asm.org Applied and Environmental Microbiology
are associated with improved gut health. The more comprehen-
sive data generated with the Phylochip demonstrated that, com-
fed HLZ-rich milk more closely resembled that of human infants
tobacillacea, both biomarkers of increased gut and host health
of the numbers clostridia and Streptococcaceae, which are compo-
nents of the fecal microbiota of infants fed formula that lacks
lysozyme, as well as by decreased levels of disease-related bacteria
such as Mycobacteriaceae and Campylobacterales. It is likely that
the antimicrobial action and/or cationic properties of HLZ were
contributing to the selection of these beneficial bacteria by pre-
venting the growth of others. Taken as a whole, the data suggest
that addition of HLZ alone to milk is able to modulate intestinal
Untangling the complex interactions of nutrient, host, and
the hypothesis that milk components actively modified the gut
microbiome, resulting in community membership changes to in-
crease levels of beneficial microbes and reduce levels of undesir-
able community members. They also support the use of pigs as a
model animal for gut microbiota research and, along with the use
of HLZ-rich milk, offer an approach not only for the study of the
the direct manipulation of gut microbiota with the potential to
improve GI disorders, including diarrhea and inflammatory
bowel diseases such as Crohn’s disease and colitis.
We thank the UC Davis College of Agricultural and Environmental Sci-
ences Genomics Facility for carrying out template preparation and se-
quencing reactions for the 16S rRNA gene sequencing. We kindly thank
Jan Carlson and Kent Parker of the UC Davis Goat and Swine Facilities,
for animal feeding and sample collection.
This project was supported in part by the Biotechnology Risk Assess-
ment Program competitive grant 2008-33522-04842 from the USDA Na-
tional Institute of Food and Agriculture (NIFA).
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