Post on 25-Apr-2023
Inhibition of Growth of Pathogenic Bacteria in Raw Milk byLegume Protein Esters
SAMIR MAHGOUB1 ALI OSMAN2 AND MAHMOUD SITOHY2
1Microbiology Department and 2Biochemistry Department Faculty of Agriculture Zagazig University Zagazig 44511 Egypt
MS 11-065 Received 7 February 2011Accepted 26 April 2011
ABSTRACT
Protein isolates from soybean and chickpea as well as their methylated esters were tested for their inhibitory action against
the propagation of pathogenic bacteria in raw milk during its storage either at room temperature or under refrigeration Raw milk
was inoculated with a mixed culture of Listeria monocytogenes Scott A and Salmonella enterica serovar Enteritidis strain PT4 at
ca 2 log CFU ml21 Aerobic plate count coliform count and presumptive E coli in raw milk treated with esterified legume
proteins were inhibited by 2 to 3 log relative to a control after 6 to 8 days of storage at 4uC At room temperature bacterial
populations (aerobic plate count coliform count and presumptive E coli) in raw milk treated with esterified legume proteins
were inhibited by ca 15 to 16 log relative to the control after 12 h Supplementation of raw milk with esterified soybean protein
could significantly inhibit the counts of the two inoculated pathogens (L monocytogenes Scott A and Salmonella Enteritidis
PT4) which were initially inoculated at ca 2 log CFU ml21 by ca 24 log and 16 log CFU ml21 respectively on day 8 of
storage under cold conditions Corresponding reductions amounting to 27 and 18 log CFU ml21 were observed after 12 h of
storage at room temperature Supplementation of raw milk with esterified soybean protein (05) reduced the maximum level of
titratable acidity to 021 and maintained the pH level at 64 after 8 days of storage under cold conditions as compared with 4 days
for untreated raw milk Similar results were observed when raw milk was stored at room temperature for 10 h
Raw milk often contains microorganisms that may
cause foodborne diseases (1 19) Bacterial contamination of
raw milk can originate from air milking equipment feed
soil feces and grass (12) Cowrsquos or buffalorsquos udders and
hides and milking utensils may carry predominantly
psychrotrophic bacteria along with others including Myco-bacterium Listeria Salmonella Campylobacter Staphylo-coccus aureus Escherichia coli O157H7 and Clostridiumspecies (7 25) The occurrence of Listeria spp in raw milk
may be due to fecal (16) or environmental contamination
during milking storage and transport (8)Pathogens that have been involved in foodborne
outbreaks associated with the consumption of raw milk
include L monocytogenes Salmonella Campylobacter Saureus Bacillus cereus Staphylococcus pyogenes E coliO157H7 and Clostridium botulinum (10 21) The presence
of these pathogenic bacteria in milk is a major public health
concern especially for those individuals who still drink raw
milk (30) several outbreaks and cases of food poisoning
have been associated with the consumption of contaminated
dairy products (21)Foodborne pathogens have been isolated from raw milk
and raw milkndashbased dairy products in locations worldwide
Listeria spp were isolated from 139 (675) of 2060 raw milk
samples collected from central India of which 105 samples
(51) were positive for L monocytogenes (23) About 362
of 774 raw milk samples in Spain (14) and 348 of 1300 raw
milk samples in the United States (31) were also reported to be
positive for L monocytogenes In Malaysia 90 of raw milk
samples were contaminated with coliform bacteria and 65
were positive for E coli 14 and 19 of total samples were
positive for Salmonella and L monocytogenes respectively
Similarly enterotoxigenic E coli was found in 16 samples
collected in Zimbabwe (15) Salmonella and Shigella were also
detected in Burkina Faso (32)The presence of pathogenic bacteria in raw milk even
at low levels can pose a health risk (20) so their control is
essential Different approaches have been investigated to
enhance the preservation of raw milk eg using thiocya-
nate (15 mgliter) and H2O2 (10 mgliter) to activate the
natural lactoperoxidase system to enable raw milk preser-
vation at low or high temperature (17) The lactoperoxidase
system was also used in combination with microfiltration to
enhance the safety of raw milk (3) These treatments may
result in potential hazards and interactions with milk
constituents Nitrogen gas was also proposed to control
psychrotrophs and mesophiles in raw milk (27) but this
approach requires special equipment and facilities The
efficacy of certain lactic acid bacteria to counteract the
proliferation of pathogenic E coli and Salmonella Enter-
itidis in raw milk was also investigated (26) This approach
while reportedly effective changes the raw milk into an
acidified and fermented product On the other hand Author for correspondence Tel (z20)552287567 Fax (z20)55
2372518 E-mail mzsitohyhotmailcom
1475
Journal of Food Protection Vol 74 No 9 2011 Pages 1475ndash1481doi1043150362-028XJFP-11-065Copyright G International Association for Food Protection
esterified legume proteins were observed to exert antimi-
crobial action against both gram-negative and gram-positive
bacteria due to their molecular intensified positive charge
(33) In the present work we evaluated the use of these
esterified legume proteins to prevent the propagation of
pathogenic bacteria in raw milk during its storage either at
room temperature or under refrigeration without changing
its neutral status
MATERIALS AND METHODS
Materials Soybean (Glycine max L) and chickpea (Cicerarietinum L) seeds were purchased from a local market in the city
of Zagazig Sharkia Egypt Raw buffalorsquos milk was collected from
a private farm located in Sharkia governorate Egypt and delivered
to the microbiology laboratory Faculty of Agriculture Zagazig
University Egypt in a closed glass container and was used in
experiments within 1 h after milking L monocytogenes Scott A
and Salmonella enterica serovar Enteritidis PT4 strains were
kindly provided by Professor John Nychas Department of Food
Science and Technology Laboratory of Food Microbiology and
Biotechnology Agricultural University of Athens Greece
Protein isolation Soybean and chickpea seeds were ground to
pass through a 1-mm2 sieve and the resulting powder was defatted
using a mixed solvent of chloroform-methanol (31 volvol) for 8 h
Protein was extracted by dispersing 5 (wtvol) defatted soybean or
chickpea flour in water adjusting the pH to 9 with 01 N NaOH at
room temperature and recovering the soluble protein by centrifuga-
tion for 15 min at 2000 | g The extract was adjusted to pH 45 with
1 N HCl to precipitate protein which was centrifuged at 2000 | gfor 15 min washed with distilled water dispersed in a limited volume
of distilled water (adjusting the pH to 75) dialyzed overnight against
distilled water and lyophilized (22) The total nitrogen was
determined in soybean protein isolate (SP) and chickpea protein
isolate (CP) using the micro-Kjeldahl method according to the
American Association of Cereal Chemists (2) and multiplied by the
conversion factor 625 to determine the protein content
Protein esterification Protein was esterified with methanol
alcohol in the presence of a 501 molar ratio of hydrochloric acid
carboxyl group according to the procedure of Sitohy et al (34) The
resultant modified proteins were denoted MSP (methylated soybean
protein) and MCP (methylated chickpea protein) The esterification
extent of the modified protein was quantified by color reaction with
hydroxylamine hydrochloride (9 18)
Pathogen inoculation in raw milk The cultures of Lmonocytogenes Scott A and Salmonella Enteritidis PT4 were
activated by three successive transfers in tryptic soy broth (Difco
Laboratories) at 37uC for 24 h Cells were harvested by
centrifugation (10000 | g for 10 min at 4uC) washed three
times and resuspended in Ringerrsquos solution (Lab M Bury UK) to
a final volume of 10 ml A final inoculum was prepared by serially
diluting in Ringerrsquos solution to reach a final level of 5 log CFU
ml21 as determined by optical density at 600 nm and confirmed by
plate counting on selective media Aliquots (02 ml) of the two
pathogens were inoculated into three sterile screw-cap bottles
containing raw milk alone or as supplemented with SP CP MSP
or MCP so that the final count of each was ca 2 log CFU ml21
Appropriate precautions were taken such as disinfecting all
surfaces and tools used with 70 ethanol Before and after use
pipettes and similar tools were left overnight under laminar flow
exposed to UV illumination After experimental manipulation and
determination all inoculated plates tips and bottles were
autoclaved at 121uC for 30 min before being submitted into
regulated disposal
Storage under refrigeration conditions The raw milk
delivered immediately after milking was maintained at 4uC for 1 h
and then divided into 200-ml portions and transferred in 15 sterile
screw-cap bottles All bottles were inoculated equally with a mixed
culture of L monocytogenes Scott A and Salmonella Enteritidis
PT4 so that the final count of each was ca 2 log CFU ml21 (223
log L monocytogenes Scott A and 208 log Salmonella Enteritidis
PT4) and then were divided into five groups of three bottles each
The first group was not treated and served as a control The second
and third groups received SP and CP (05 wtvol) respectively
The fourth and fifth groups received MSP and MCP (05 wt
vol) respectively All bottles were kept at 4uC for 10 days samples
were withdrawn from them under aseptic conditions every 2 days
for microbiological assay and acidity measurement
Storage at room temperature The design of this experiment
was similar to that with storage at cold conditions except that the
15 bottles were kept at room temperature for 24 h and samples
were withdrawn from them under aseptic conditions after 0 6 12
and 24 h for microbiological assay and acidity measurement
Room temperature was adjusted to 23uC using the Split Air
Conditioner lsquolsquoUnion Airrsquorsquo and followed up hourly using a Taylor
digital indoor thermometer
Microbial enumeration The samples (5 ml) were transferred
aseptically to a stomacher bag containing 95 ml of peptone saline
diluent (10 g peptone and 85 g sodium chloride in 1 liter of
distilled water) at room temperature and homogenized for 60 s A
decimal solution in peptone saline diluent was prepared and
duplicate 1-ml or 01-ml samples of appropriate dilutions were
poured or spread on nonselective or selective agar plates
Determinations were carried out for different bacterial counts
using different specific selective media (24) as follows aerobic
plate count (APC) on plate count agar (Merck Darmstadt
Germany) and incubated at 25uC for 72 h lactic acid bacteria on
de Man Rogosa Sharpe (Biolife Milan Italy) overlain with 5 ml of
the same medium and incubated at 25uC for 72 h total coliform on
MacConkey agar (Mast Group Merseyside UK) with a double
layer of the same medium incubated at 37uC for 24 h E coli on
tryptone bile X-glucuronide agar (Lab Lancashire UK) incubated
at 37uC for 24 h L monocytogenes on polymyxinndashacriflavinndash
lithium chloridendashceftazidimendashaesculinndashmannitol (PALCAM) agar
(Biolife) after incubation for 24 h at 37uC and confirmation
according to the guidelines of ISO 11290 (4) Salmonella on
xylose lysine deoxycholate (Merck) for 24 h at 37uC and
confirmation according to the guidelines of ISO 6579 (5)Populations of bacteria shown are means of two replicates and
converted to log CFU per milliliter
Determination of titratable acidity and pH The titratable
acidity of raw milk was assessed every 2 days for the samples
preserved under refrigeration (0 to 10 days) and every 6 h for the
samples preserved at room temperature (0 to 24 h) and expressed
as lactic acid (percentage) according to the standard methods (6)Milk pH was assessed for the same samples by pH meter (pH 211
Hanna Instruments Inc Woonsocket RI)
Statistical analysis Three replicates of all experiments were
performed and results were expressed as the mean plus the
standard error Data were statistically analyzed using analysis of
1476 MAHGOUB ET AL J Food Prot Vol 74 No 9
variance with the general linear models procedure (SAS version
91 SAS Institute Inc Cary NC) Least significant differences
were used to separate means at P 005
RESULTS AND DISCUSSION
Chemical and functional characterization Protein
content in SP and CP was nearly in the same range 91 and
92 respectively After methylation with methanol in the
presence of 50 mol of hydrochloric acidndash1 mol of
carboxylic group esterification extents of the two proteins
reached 80 and 83 for MSP and MCP respectively The
two modified proteins showed a higher isoelectric point of
8 which referred to the increase in net positive charge of the
esterified proteins (33)Toxicological studies of the modified proteins (MSP
and MCP) have been conducted using albino rats to
investigate acute and subacute toxicity (Detailed results
will be published in a journal concerned with toxicological
studies) Generally it was concluded that esterified legume
proteins (MSP and MCP) are free from toxicological
hazards (data not shown) Accordingly the possible
application of these proteins in milk preservation will not
be precluded by potential toxic hazards
On the other hand to manufacture yogurt these two
modified proteins were experimentally added to raw milk at
05 before the addition of commercial starter at 2 and
incubation at 42uC The coagulum formation (physical
appearance) and pH development of these preparations were
compared against control milk (no added MSP or MCP) In
all preparations the coagulum formation took the same time
ca 6 h and the pH in all cases was about 46 In spite of the
expected antibacterial action of the modified proteins lactic
acid bacteria developed normally to attain the level of
acidity required for the coagulum formation The absence of
antibacterial action against lactic acid bacteria may be due to
the high level of these bacteria at time zero of fermentation
(ca 7 log CFU ml21 in accordance with (13)) for which
the concentration of modified protein used (05) was not
sufficient to induce inhibitory action
Storage at refrigeration conditions bacterialcounts In a primary experiment different concentrations
of esterified legume proteins (01 05 and 1) were added
to raw milk to test their effect on the proliferation of APC
results indicated a concentration-dependent inhibitory
effect except that there was no significant difference
between the 05 and 1 levels So in the current
experiment 05 was uniquely applied to follow the
associated changes in details The results presented in
Figure 1 show the microbiological changes in the levels of
APC coliform count (CC) and presumptive E coli (EC) in
raw milk preserved at 4uC for 10 days as supplemented with
native (SP CP) or modified legume proteins (MSP MCP) at
05 (wtvol) Observably APC and CC increased in
control raw milk with time starting directly from the second
day of storage (ie there was no lag phase) Adding the
modified proteins (MSP or MCP) to raw milk resulted in a
4- to 6-day initial phase characterized by very little growth
of APC and CC This phase was further extended to 8 days
with EC Alternatively a significant reducing action was
observed on all bacterial counts (P 005) with the highest
magnitude recorded after 6 to 8 days of storage The
maximum inhibiting action reached a value in the range 2 to
3 log CFU ml21 in the three bacterial counts after 6 to 8 days
of storage at refrigeration conditions There was no
difference between the actions of the two modified proteins
so the action is specifically due to the chemical modifica-
tion which occurs to the same extent in the two proteins
Nonsupplemented raw milk reached the prespoilage
level 5 log APC (35) after 2 days of storage compared with
6 days for the MSP- or MCP-supplemented raw milk
samples This delay in the spoilage emergence is apparently
due to the antimicrobial action of the esterified protein
FIGURE 1 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept under refrigeration conditions for different periods (0 to 10 days)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1477
which is mainly due to its chemical modification
Esterification blocks the negatively charged carboxyl groups
on the protein molecules turning the net charge to positive
(33) The modified proteins may interact with negatively
charged components of the bacterial cell membranes (2829) by virtue of their enhanced positive charges causing
membrane disruption and subsequent bacterial inhibition
Pathogenic bacteria The data presented in Figure 2
trace the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 10 days of storage at 4uC In nontreated raw milk the
two microorganisms reached maximum levels on day 8 Lmonocytogenes reached a maximum of 606 iexcl 003 log
CFU ml21 as compared with 345 iexcl 004 log for
Salmonella Enteritidis on day 8 of storage suggesting that
L monocytogenes had higher viability because it is a
psychrotrophic bacterium Over the same period of time in
raw milk supplemented with esterified soybean protein
(MSP) the counts of the two pathogens were significantly (P 005) inhibited to the level of 360 iexcl 011 and 192 iexcl
011 log CFU ml21 with corresponding magnitudes of
reduction equivalent to ca 24 and 16 log CFU ml21
respectively After 8 to 10 days of storage under cold
conditions the level of Salmonella Enteritidis in the MSP-
supplemented raw milk was statistically similar to the
starting inoculum level suggesting a strong bacteriostatic
effect of the esterified soybean protein Supplementation
with native SP did not significantly (P 005) reduce the
proliferation of the two pathogens whereas supplementation
with the MCP exerted an effect similar to that of MSP (data
not shown) So the antimicrobial action noticed with MSP
and MCP is exclusively due to the modification process
The antimicrobial action of MSP and MCP is due to the
esterification process that endows the protein with con-
densed positive charges and renders it more reactive with
the microorganisms (33) Results indicate that modified
proteins have general antibacterial effects against both
spoilage and pathogenic bacteria This endowed potentiality
can help maintain the hygienic quality of raw milk during its
storage under cold conditions
Acidity development The changes in pH values and
titratable acidity of treated and untreated raw milk samples
during 10 days of storage at 4uC are shown in Figure 3 The
titratable acidity of untreated raw milk increased gradually
with time to reach a high level (027 iexcl 0008) by day 8
Supplementation of raw milk with MSP (05) has
significantly (P 005) and considerably reduced this
maximum level to a level of 021 iexcl 0008 over the same
period this is still within acceptable levels and indicates a
slower rate of acidity development than in the untreated raw
milk
Concomitantly the pH value gradually decreased in all
stored milk samples but at a higher rate in nontreated milk
Nontreated raw milk reached a value of ca 64 at 4 days of
storage as compared with 8 days in MSP-supplemented
milk under the same refrigeration conditions ie MSP
prolonged the period within which milk pH was at an
acceptable level MCP supplementation had a similar
magnitude of effect whereas the native forms of both
proteins were not associated with similar activities In
conclusion it can be stated that raw milk supplementation
FIGURE 2 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with native (SP) or esterified soybean protein (MSP)at 05 (wtvol) and kept under refrigeration conditions fordifferent periods (0 to 10 days)
FIGURE 3 Development of titratable acidity and pH in raw milk(C) during 10 days of storage under refrigeration conditions assupplemented with 05 (wtvol) esterified soybean protein (MSP)
1478 MAHGOUB ET AL J Food Prot Vol 74 No 9
with esterified legume proteins attenuated the development
of acidity and slowed the reduction rate of pH as a result of
their antimicrobial action which is normally associated with
reduced bacterial acid production
Rapid development of acidity in raw milk is apparently
due to uncontrolled microbial contamination with conse-
quent acid production (11) The attenuating effect on acidity
development is apparently due to the supplemented
esterified legume proteins which have been proved to exert
antimicrobial action (33) and accords with the microbio-
logical analytical results (Fig 1) So it can be concluded
that supplementation of raw buffalorsquos milk with esterified
legume proteins at a low level (05) can control the
development of titratable acidity in milk and thus prolong its
shelf life
Storage at room temperature Raw milk was stored at
room temperature (20 to 25uC) to simulate the conditions
under which raw milk may be handled transferred or
processed under artisanal conditions The study lasted only
24 h because keeping raw milk at such high temperature was
expected to cause accelerated rates of microbial proliferation
and associated acid development The three parameters
followed under the previous section were also used to evaluate
the microbiological and chemical status of the raw milk
Bacterial counts The results presented in Figure 4
show rapid microbiological changes in the levels of APC
CC and EC in raw milk stored at room temperature (20 to
25uC) for 24 h The APC in nontreated raw milk reached a
maximum (896 iexcl 003 log CFU ml21) after 12 h at which
time the MSP- or MCP-supplemented milk was inhibited by
about 15 log Native proteins (SP and CP) did not show any
significant reduction (P 005) in the APC when compared
with the control There was no difference between the
actions of the two modified proteins confirming the
previous conclusion that the antimicrobial action is
specifically due to the chemical modification
Nonsupplemented raw milk stored at room temperature
reached the prespoilage level of APC (ca 5 log) after 4 h
compared with about 7 h for MSP- or MCP-supplemented
raw milk The extension of the shelf life of raw milk at room
temperature with supplementation is valuable for transpor-
tation handling and processing under safe conditions Of
course subjecting milk to any additional refrigeration
treatment will further extend this period
The extent of the antimicrobial inhibitory action of
MSP and MCP on the CC and EC was nearly in the same
range (15 to 16 log) Generally the extent of the
antimicrobial inhibition in raw milk by esterified legume
proteins under room temperature is less than that observed
under refrigeration conditions Relatively high temperatures
may stimulate bacterial growth too much for it to be easily
counteracted by the applied substance whereas relatively
low temperatures may exert a bacteriostatic effect So it is
highly recommended that some cooling treatment be applied
to accentuate the action of the antimicrobial agents and
avoid potential contamination
This delay in spoilage emergence is apparently due to
the antimicrobial action of the esterified protein as
previously explained but it can be added that this
antimicrobial action acts at low (4uC) and at relatively high
(20 to 25uC) temperature storage conditions
Pathogenic bacteria The data presented in Figure 5
depict the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 24 h of storage at room temperature (20 to 25uC) In
nontreated raw milk the two pathogenic microorganisms
reached maximum levels after 12 h of storage at room
temperature Supplementing raw milk with MSP could
significantly (P 005) inhibit the counts of the two
pathogens L monocytogenes and Salmonella Enteritidis
counts were reduced by 27 and 18 log CFU ml21 after 12 h
FIGURE 4 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept at room temperature for different periods (0 to 24 h)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1479
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
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Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
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1998 Quantitative risk assessment of human listeriosis from
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Esterification of food proteins characterization of the derivatives by a
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652
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milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
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of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
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25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
esterified legume proteins were observed to exert antimi-
crobial action against both gram-negative and gram-positive
bacteria due to their molecular intensified positive charge
(33) In the present work we evaluated the use of these
esterified legume proteins to prevent the propagation of
pathogenic bacteria in raw milk during its storage either at
room temperature or under refrigeration without changing
its neutral status
MATERIALS AND METHODS
Materials Soybean (Glycine max L) and chickpea (Cicerarietinum L) seeds were purchased from a local market in the city
of Zagazig Sharkia Egypt Raw buffalorsquos milk was collected from
a private farm located in Sharkia governorate Egypt and delivered
to the microbiology laboratory Faculty of Agriculture Zagazig
University Egypt in a closed glass container and was used in
experiments within 1 h after milking L monocytogenes Scott A
and Salmonella enterica serovar Enteritidis PT4 strains were
kindly provided by Professor John Nychas Department of Food
Science and Technology Laboratory of Food Microbiology and
Biotechnology Agricultural University of Athens Greece
Protein isolation Soybean and chickpea seeds were ground to
pass through a 1-mm2 sieve and the resulting powder was defatted
using a mixed solvent of chloroform-methanol (31 volvol) for 8 h
Protein was extracted by dispersing 5 (wtvol) defatted soybean or
chickpea flour in water adjusting the pH to 9 with 01 N NaOH at
room temperature and recovering the soluble protein by centrifuga-
tion for 15 min at 2000 | g The extract was adjusted to pH 45 with
1 N HCl to precipitate protein which was centrifuged at 2000 | gfor 15 min washed with distilled water dispersed in a limited volume
of distilled water (adjusting the pH to 75) dialyzed overnight against
distilled water and lyophilized (22) The total nitrogen was
determined in soybean protein isolate (SP) and chickpea protein
isolate (CP) using the micro-Kjeldahl method according to the
American Association of Cereal Chemists (2) and multiplied by the
conversion factor 625 to determine the protein content
Protein esterification Protein was esterified with methanol
alcohol in the presence of a 501 molar ratio of hydrochloric acid
carboxyl group according to the procedure of Sitohy et al (34) The
resultant modified proteins were denoted MSP (methylated soybean
protein) and MCP (methylated chickpea protein) The esterification
extent of the modified protein was quantified by color reaction with
hydroxylamine hydrochloride (9 18)
Pathogen inoculation in raw milk The cultures of Lmonocytogenes Scott A and Salmonella Enteritidis PT4 were
activated by three successive transfers in tryptic soy broth (Difco
Laboratories) at 37uC for 24 h Cells were harvested by
centrifugation (10000 | g for 10 min at 4uC) washed three
times and resuspended in Ringerrsquos solution (Lab M Bury UK) to
a final volume of 10 ml A final inoculum was prepared by serially
diluting in Ringerrsquos solution to reach a final level of 5 log CFU
ml21 as determined by optical density at 600 nm and confirmed by
plate counting on selective media Aliquots (02 ml) of the two
pathogens were inoculated into three sterile screw-cap bottles
containing raw milk alone or as supplemented with SP CP MSP
or MCP so that the final count of each was ca 2 log CFU ml21
Appropriate precautions were taken such as disinfecting all
surfaces and tools used with 70 ethanol Before and after use
pipettes and similar tools were left overnight under laminar flow
exposed to UV illumination After experimental manipulation and
determination all inoculated plates tips and bottles were
autoclaved at 121uC for 30 min before being submitted into
regulated disposal
Storage under refrigeration conditions The raw milk
delivered immediately after milking was maintained at 4uC for 1 h
and then divided into 200-ml portions and transferred in 15 sterile
screw-cap bottles All bottles were inoculated equally with a mixed
culture of L monocytogenes Scott A and Salmonella Enteritidis
PT4 so that the final count of each was ca 2 log CFU ml21 (223
log L monocytogenes Scott A and 208 log Salmonella Enteritidis
PT4) and then were divided into five groups of three bottles each
The first group was not treated and served as a control The second
and third groups received SP and CP (05 wtvol) respectively
The fourth and fifth groups received MSP and MCP (05 wt
vol) respectively All bottles were kept at 4uC for 10 days samples
were withdrawn from them under aseptic conditions every 2 days
for microbiological assay and acidity measurement
Storage at room temperature The design of this experiment
was similar to that with storage at cold conditions except that the
15 bottles were kept at room temperature for 24 h and samples
were withdrawn from them under aseptic conditions after 0 6 12
and 24 h for microbiological assay and acidity measurement
Room temperature was adjusted to 23uC using the Split Air
Conditioner lsquolsquoUnion Airrsquorsquo and followed up hourly using a Taylor
digital indoor thermometer
Microbial enumeration The samples (5 ml) were transferred
aseptically to a stomacher bag containing 95 ml of peptone saline
diluent (10 g peptone and 85 g sodium chloride in 1 liter of
distilled water) at room temperature and homogenized for 60 s A
decimal solution in peptone saline diluent was prepared and
duplicate 1-ml or 01-ml samples of appropriate dilutions were
poured or spread on nonselective or selective agar plates
Determinations were carried out for different bacterial counts
using different specific selective media (24) as follows aerobic
plate count (APC) on plate count agar (Merck Darmstadt
Germany) and incubated at 25uC for 72 h lactic acid bacteria on
de Man Rogosa Sharpe (Biolife Milan Italy) overlain with 5 ml of
the same medium and incubated at 25uC for 72 h total coliform on
MacConkey agar (Mast Group Merseyside UK) with a double
layer of the same medium incubated at 37uC for 24 h E coli on
tryptone bile X-glucuronide agar (Lab Lancashire UK) incubated
at 37uC for 24 h L monocytogenes on polymyxinndashacriflavinndash
lithium chloridendashceftazidimendashaesculinndashmannitol (PALCAM) agar
(Biolife) after incubation for 24 h at 37uC and confirmation
according to the guidelines of ISO 11290 (4) Salmonella on
xylose lysine deoxycholate (Merck) for 24 h at 37uC and
confirmation according to the guidelines of ISO 6579 (5)Populations of bacteria shown are means of two replicates and
converted to log CFU per milliliter
Determination of titratable acidity and pH The titratable
acidity of raw milk was assessed every 2 days for the samples
preserved under refrigeration (0 to 10 days) and every 6 h for the
samples preserved at room temperature (0 to 24 h) and expressed
as lactic acid (percentage) according to the standard methods (6)Milk pH was assessed for the same samples by pH meter (pH 211
Hanna Instruments Inc Woonsocket RI)
Statistical analysis Three replicates of all experiments were
performed and results were expressed as the mean plus the
standard error Data were statistically analyzed using analysis of
1476 MAHGOUB ET AL J Food Prot Vol 74 No 9
variance with the general linear models procedure (SAS version
91 SAS Institute Inc Cary NC) Least significant differences
were used to separate means at P 005
RESULTS AND DISCUSSION
Chemical and functional characterization Protein
content in SP and CP was nearly in the same range 91 and
92 respectively After methylation with methanol in the
presence of 50 mol of hydrochloric acidndash1 mol of
carboxylic group esterification extents of the two proteins
reached 80 and 83 for MSP and MCP respectively The
two modified proteins showed a higher isoelectric point of
8 which referred to the increase in net positive charge of the
esterified proteins (33)Toxicological studies of the modified proteins (MSP
and MCP) have been conducted using albino rats to
investigate acute and subacute toxicity (Detailed results
will be published in a journal concerned with toxicological
studies) Generally it was concluded that esterified legume
proteins (MSP and MCP) are free from toxicological
hazards (data not shown) Accordingly the possible
application of these proteins in milk preservation will not
be precluded by potential toxic hazards
On the other hand to manufacture yogurt these two
modified proteins were experimentally added to raw milk at
05 before the addition of commercial starter at 2 and
incubation at 42uC The coagulum formation (physical
appearance) and pH development of these preparations were
compared against control milk (no added MSP or MCP) In
all preparations the coagulum formation took the same time
ca 6 h and the pH in all cases was about 46 In spite of the
expected antibacterial action of the modified proteins lactic
acid bacteria developed normally to attain the level of
acidity required for the coagulum formation The absence of
antibacterial action against lactic acid bacteria may be due to
the high level of these bacteria at time zero of fermentation
(ca 7 log CFU ml21 in accordance with (13)) for which
the concentration of modified protein used (05) was not
sufficient to induce inhibitory action
Storage at refrigeration conditions bacterialcounts In a primary experiment different concentrations
of esterified legume proteins (01 05 and 1) were added
to raw milk to test their effect on the proliferation of APC
results indicated a concentration-dependent inhibitory
effect except that there was no significant difference
between the 05 and 1 levels So in the current
experiment 05 was uniquely applied to follow the
associated changes in details The results presented in
Figure 1 show the microbiological changes in the levels of
APC coliform count (CC) and presumptive E coli (EC) in
raw milk preserved at 4uC for 10 days as supplemented with
native (SP CP) or modified legume proteins (MSP MCP) at
05 (wtvol) Observably APC and CC increased in
control raw milk with time starting directly from the second
day of storage (ie there was no lag phase) Adding the
modified proteins (MSP or MCP) to raw milk resulted in a
4- to 6-day initial phase characterized by very little growth
of APC and CC This phase was further extended to 8 days
with EC Alternatively a significant reducing action was
observed on all bacterial counts (P 005) with the highest
magnitude recorded after 6 to 8 days of storage The
maximum inhibiting action reached a value in the range 2 to
3 log CFU ml21 in the three bacterial counts after 6 to 8 days
of storage at refrigeration conditions There was no
difference between the actions of the two modified proteins
so the action is specifically due to the chemical modifica-
tion which occurs to the same extent in the two proteins
Nonsupplemented raw milk reached the prespoilage
level 5 log APC (35) after 2 days of storage compared with
6 days for the MSP- or MCP-supplemented raw milk
samples This delay in the spoilage emergence is apparently
due to the antimicrobial action of the esterified protein
FIGURE 1 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept under refrigeration conditions for different periods (0 to 10 days)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1477
which is mainly due to its chemical modification
Esterification blocks the negatively charged carboxyl groups
on the protein molecules turning the net charge to positive
(33) The modified proteins may interact with negatively
charged components of the bacterial cell membranes (2829) by virtue of their enhanced positive charges causing
membrane disruption and subsequent bacterial inhibition
Pathogenic bacteria The data presented in Figure 2
trace the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 10 days of storage at 4uC In nontreated raw milk the
two microorganisms reached maximum levels on day 8 Lmonocytogenes reached a maximum of 606 iexcl 003 log
CFU ml21 as compared with 345 iexcl 004 log for
Salmonella Enteritidis on day 8 of storage suggesting that
L monocytogenes had higher viability because it is a
psychrotrophic bacterium Over the same period of time in
raw milk supplemented with esterified soybean protein
(MSP) the counts of the two pathogens were significantly (P 005) inhibited to the level of 360 iexcl 011 and 192 iexcl
011 log CFU ml21 with corresponding magnitudes of
reduction equivalent to ca 24 and 16 log CFU ml21
respectively After 8 to 10 days of storage under cold
conditions the level of Salmonella Enteritidis in the MSP-
supplemented raw milk was statistically similar to the
starting inoculum level suggesting a strong bacteriostatic
effect of the esterified soybean protein Supplementation
with native SP did not significantly (P 005) reduce the
proliferation of the two pathogens whereas supplementation
with the MCP exerted an effect similar to that of MSP (data
not shown) So the antimicrobial action noticed with MSP
and MCP is exclusively due to the modification process
The antimicrobial action of MSP and MCP is due to the
esterification process that endows the protein with con-
densed positive charges and renders it more reactive with
the microorganisms (33) Results indicate that modified
proteins have general antibacterial effects against both
spoilage and pathogenic bacteria This endowed potentiality
can help maintain the hygienic quality of raw milk during its
storage under cold conditions
Acidity development The changes in pH values and
titratable acidity of treated and untreated raw milk samples
during 10 days of storage at 4uC are shown in Figure 3 The
titratable acidity of untreated raw milk increased gradually
with time to reach a high level (027 iexcl 0008) by day 8
Supplementation of raw milk with MSP (05) has
significantly (P 005) and considerably reduced this
maximum level to a level of 021 iexcl 0008 over the same
period this is still within acceptable levels and indicates a
slower rate of acidity development than in the untreated raw
milk
Concomitantly the pH value gradually decreased in all
stored milk samples but at a higher rate in nontreated milk
Nontreated raw milk reached a value of ca 64 at 4 days of
storage as compared with 8 days in MSP-supplemented
milk under the same refrigeration conditions ie MSP
prolonged the period within which milk pH was at an
acceptable level MCP supplementation had a similar
magnitude of effect whereas the native forms of both
proteins were not associated with similar activities In
conclusion it can be stated that raw milk supplementation
FIGURE 2 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with native (SP) or esterified soybean protein (MSP)at 05 (wtvol) and kept under refrigeration conditions fordifferent periods (0 to 10 days)
FIGURE 3 Development of titratable acidity and pH in raw milk(C) during 10 days of storage under refrigeration conditions assupplemented with 05 (wtvol) esterified soybean protein (MSP)
1478 MAHGOUB ET AL J Food Prot Vol 74 No 9
with esterified legume proteins attenuated the development
of acidity and slowed the reduction rate of pH as a result of
their antimicrobial action which is normally associated with
reduced bacterial acid production
Rapid development of acidity in raw milk is apparently
due to uncontrolled microbial contamination with conse-
quent acid production (11) The attenuating effect on acidity
development is apparently due to the supplemented
esterified legume proteins which have been proved to exert
antimicrobial action (33) and accords with the microbio-
logical analytical results (Fig 1) So it can be concluded
that supplementation of raw buffalorsquos milk with esterified
legume proteins at a low level (05) can control the
development of titratable acidity in milk and thus prolong its
shelf life
Storage at room temperature Raw milk was stored at
room temperature (20 to 25uC) to simulate the conditions
under which raw milk may be handled transferred or
processed under artisanal conditions The study lasted only
24 h because keeping raw milk at such high temperature was
expected to cause accelerated rates of microbial proliferation
and associated acid development The three parameters
followed under the previous section were also used to evaluate
the microbiological and chemical status of the raw milk
Bacterial counts The results presented in Figure 4
show rapid microbiological changes in the levels of APC
CC and EC in raw milk stored at room temperature (20 to
25uC) for 24 h The APC in nontreated raw milk reached a
maximum (896 iexcl 003 log CFU ml21) after 12 h at which
time the MSP- or MCP-supplemented milk was inhibited by
about 15 log Native proteins (SP and CP) did not show any
significant reduction (P 005) in the APC when compared
with the control There was no difference between the
actions of the two modified proteins confirming the
previous conclusion that the antimicrobial action is
specifically due to the chemical modification
Nonsupplemented raw milk stored at room temperature
reached the prespoilage level of APC (ca 5 log) after 4 h
compared with about 7 h for MSP- or MCP-supplemented
raw milk The extension of the shelf life of raw milk at room
temperature with supplementation is valuable for transpor-
tation handling and processing under safe conditions Of
course subjecting milk to any additional refrigeration
treatment will further extend this period
The extent of the antimicrobial inhibitory action of
MSP and MCP on the CC and EC was nearly in the same
range (15 to 16 log) Generally the extent of the
antimicrobial inhibition in raw milk by esterified legume
proteins under room temperature is less than that observed
under refrigeration conditions Relatively high temperatures
may stimulate bacterial growth too much for it to be easily
counteracted by the applied substance whereas relatively
low temperatures may exert a bacteriostatic effect So it is
highly recommended that some cooling treatment be applied
to accentuate the action of the antimicrobial agents and
avoid potential contamination
This delay in spoilage emergence is apparently due to
the antimicrobial action of the esterified protein as
previously explained but it can be added that this
antimicrobial action acts at low (4uC) and at relatively high
(20 to 25uC) temperature storage conditions
Pathogenic bacteria The data presented in Figure 5
depict the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 24 h of storage at room temperature (20 to 25uC) In
nontreated raw milk the two pathogenic microorganisms
reached maximum levels after 12 h of storage at room
temperature Supplementing raw milk with MSP could
significantly (P 005) inhibit the counts of the two
pathogens L monocytogenes and Salmonella Enteritidis
counts were reduced by 27 and 18 log CFU ml21 after 12 h
FIGURE 4 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept at room temperature for different periods (0 to 24 h)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1479
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
REFERENCES
1 Adesiyun A A L Webb and S Rahman 1995 Microbiological
quality of raw cow milk at collection centers in Trinidad J Food
Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
variance with the general linear models procedure (SAS version
91 SAS Institute Inc Cary NC) Least significant differences
were used to separate means at P 005
RESULTS AND DISCUSSION
Chemical and functional characterization Protein
content in SP and CP was nearly in the same range 91 and
92 respectively After methylation with methanol in the
presence of 50 mol of hydrochloric acidndash1 mol of
carboxylic group esterification extents of the two proteins
reached 80 and 83 for MSP and MCP respectively The
two modified proteins showed a higher isoelectric point of
8 which referred to the increase in net positive charge of the
esterified proteins (33)Toxicological studies of the modified proteins (MSP
and MCP) have been conducted using albino rats to
investigate acute and subacute toxicity (Detailed results
will be published in a journal concerned with toxicological
studies) Generally it was concluded that esterified legume
proteins (MSP and MCP) are free from toxicological
hazards (data not shown) Accordingly the possible
application of these proteins in milk preservation will not
be precluded by potential toxic hazards
On the other hand to manufacture yogurt these two
modified proteins were experimentally added to raw milk at
05 before the addition of commercial starter at 2 and
incubation at 42uC The coagulum formation (physical
appearance) and pH development of these preparations were
compared against control milk (no added MSP or MCP) In
all preparations the coagulum formation took the same time
ca 6 h and the pH in all cases was about 46 In spite of the
expected antibacterial action of the modified proteins lactic
acid bacteria developed normally to attain the level of
acidity required for the coagulum formation The absence of
antibacterial action against lactic acid bacteria may be due to
the high level of these bacteria at time zero of fermentation
(ca 7 log CFU ml21 in accordance with (13)) for which
the concentration of modified protein used (05) was not
sufficient to induce inhibitory action
Storage at refrigeration conditions bacterialcounts In a primary experiment different concentrations
of esterified legume proteins (01 05 and 1) were added
to raw milk to test their effect on the proliferation of APC
results indicated a concentration-dependent inhibitory
effect except that there was no significant difference
between the 05 and 1 levels So in the current
experiment 05 was uniquely applied to follow the
associated changes in details The results presented in
Figure 1 show the microbiological changes in the levels of
APC coliform count (CC) and presumptive E coli (EC) in
raw milk preserved at 4uC for 10 days as supplemented with
native (SP CP) or modified legume proteins (MSP MCP) at
05 (wtvol) Observably APC and CC increased in
control raw milk with time starting directly from the second
day of storage (ie there was no lag phase) Adding the
modified proteins (MSP or MCP) to raw milk resulted in a
4- to 6-day initial phase characterized by very little growth
of APC and CC This phase was further extended to 8 days
with EC Alternatively a significant reducing action was
observed on all bacterial counts (P 005) with the highest
magnitude recorded after 6 to 8 days of storage The
maximum inhibiting action reached a value in the range 2 to
3 log CFU ml21 in the three bacterial counts after 6 to 8 days
of storage at refrigeration conditions There was no
difference between the actions of the two modified proteins
so the action is specifically due to the chemical modifica-
tion which occurs to the same extent in the two proteins
Nonsupplemented raw milk reached the prespoilage
level 5 log APC (35) after 2 days of storage compared with
6 days for the MSP- or MCP-supplemented raw milk
samples This delay in the spoilage emergence is apparently
due to the antimicrobial action of the esterified protein
FIGURE 1 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept under refrigeration conditions for different periods (0 to 10 days)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1477
which is mainly due to its chemical modification
Esterification blocks the negatively charged carboxyl groups
on the protein molecules turning the net charge to positive
(33) The modified proteins may interact with negatively
charged components of the bacterial cell membranes (2829) by virtue of their enhanced positive charges causing
membrane disruption and subsequent bacterial inhibition
Pathogenic bacteria The data presented in Figure 2
trace the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 10 days of storage at 4uC In nontreated raw milk the
two microorganisms reached maximum levels on day 8 Lmonocytogenes reached a maximum of 606 iexcl 003 log
CFU ml21 as compared with 345 iexcl 004 log for
Salmonella Enteritidis on day 8 of storage suggesting that
L monocytogenes had higher viability because it is a
psychrotrophic bacterium Over the same period of time in
raw milk supplemented with esterified soybean protein
(MSP) the counts of the two pathogens were significantly (P 005) inhibited to the level of 360 iexcl 011 and 192 iexcl
011 log CFU ml21 with corresponding magnitudes of
reduction equivalent to ca 24 and 16 log CFU ml21
respectively After 8 to 10 days of storage under cold
conditions the level of Salmonella Enteritidis in the MSP-
supplemented raw milk was statistically similar to the
starting inoculum level suggesting a strong bacteriostatic
effect of the esterified soybean protein Supplementation
with native SP did not significantly (P 005) reduce the
proliferation of the two pathogens whereas supplementation
with the MCP exerted an effect similar to that of MSP (data
not shown) So the antimicrobial action noticed with MSP
and MCP is exclusively due to the modification process
The antimicrobial action of MSP and MCP is due to the
esterification process that endows the protein with con-
densed positive charges and renders it more reactive with
the microorganisms (33) Results indicate that modified
proteins have general antibacterial effects against both
spoilage and pathogenic bacteria This endowed potentiality
can help maintain the hygienic quality of raw milk during its
storage under cold conditions
Acidity development The changes in pH values and
titratable acidity of treated and untreated raw milk samples
during 10 days of storage at 4uC are shown in Figure 3 The
titratable acidity of untreated raw milk increased gradually
with time to reach a high level (027 iexcl 0008) by day 8
Supplementation of raw milk with MSP (05) has
significantly (P 005) and considerably reduced this
maximum level to a level of 021 iexcl 0008 over the same
period this is still within acceptable levels and indicates a
slower rate of acidity development than in the untreated raw
milk
Concomitantly the pH value gradually decreased in all
stored milk samples but at a higher rate in nontreated milk
Nontreated raw milk reached a value of ca 64 at 4 days of
storage as compared with 8 days in MSP-supplemented
milk under the same refrigeration conditions ie MSP
prolonged the period within which milk pH was at an
acceptable level MCP supplementation had a similar
magnitude of effect whereas the native forms of both
proteins were not associated with similar activities In
conclusion it can be stated that raw milk supplementation
FIGURE 2 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with native (SP) or esterified soybean protein (MSP)at 05 (wtvol) and kept under refrigeration conditions fordifferent periods (0 to 10 days)
FIGURE 3 Development of titratable acidity and pH in raw milk(C) during 10 days of storage under refrigeration conditions assupplemented with 05 (wtvol) esterified soybean protein (MSP)
1478 MAHGOUB ET AL J Food Prot Vol 74 No 9
with esterified legume proteins attenuated the development
of acidity and slowed the reduction rate of pH as a result of
their antimicrobial action which is normally associated with
reduced bacterial acid production
Rapid development of acidity in raw milk is apparently
due to uncontrolled microbial contamination with conse-
quent acid production (11) The attenuating effect on acidity
development is apparently due to the supplemented
esterified legume proteins which have been proved to exert
antimicrobial action (33) and accords with the microbio-
logical analytical results (Fig 1) So it can be concluded
that supplementation of raw buffalorsquos milk with esterified
legume proteins at a low level (05) can control the
development of titratable acidity in milk and thus prolong its
shelf life
Storage at room temperature Raw milk was stored at
room temperature (20 to 25uC) to simulate the conditions
under which raw milk may be handled transferred or
processed under artisanal conditions The study lasted only
24 h because keeping raw milk at such high temperature was
expected to cause accelerated rates of microbial proliferation
and associated acid development The three parameters
followed under the previous section were also used to evaluate
the microbiological and chemical status of the raw milk
Bacterial counts The results presented in Figure 4
show rapid microbiological changes in the levels of APC
CC and EC in raw milk stored at room temperature (20 to
25uC) for 24 h The APC in nontreated raw milk reached a
maximum (896 iexcl 003 log CFU ml21) after 12 h at which
time the MSP- or MCP-supplemented milk was inhibited by
about 15 log Native proteins (SP and CP) did not show any
significant reduction (P 005) in the APC when compared
with the control There was no difference between the
actions of the two modified proteins confirming the
previous conclusion that the antimicrobial action is
specifically due to the chemical modification
Nonsupplemented raw milk stored at room temperature
reached the prespoilage level of APC (ca 5 log) after 4 h
compared with about 7 h for MSP- or MCP-supplemented
raw milk The extension of the shelf life of raw milk at room
temperature with supplementation is valuable for transpor-
tation handling and processing under safe conditions Of
course subjecting milk to any additional refrigeration
treatment will further extend this period
The extent of the antimicrobial inhibitory action of
MSP and MCP on the CC and EC was nearly in the same
range (15 to 16 log) Generally the extent of the
antimicrobial inhibition in raw milk by esterified legume
proteins under room temperature is less than that observed
under refrigeration conditions Relatively high temperatures
may stimulate bacterial growth too much for it to be easily
counteracted by the applied substance whereas relatively
low temperatures may exert a bacteriostatic effect So it is
highly recommended that some cooling treatment be applied
to accentuate the action of the antimicrobial agents and
avoid potential contamination
This delay in spoilage emergence is apparently due to
the antimicrobial action of the esterified protein as
previously explained but it can be added that this
antimicrobial action acts at low (4uC) and at relatively high
(20 to 25uC) temperature storage conditions
Pathogenic bacteria The data presented in Figure 5
depict the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 24 h of storage at room temperature (20 to 25uC) In
nontreated raw milk the two pathogenic microorganisms
reached maximum levels after 12 h of storage at room
temperature Supplementing raw milk with MSP could
significantly (P 005) inhibit the counts of the two
pathogens L monocytogenes and Salmonella Enteritidis
counts were reduced by 27 and 18 log CFU ml21 after 12 h
FIGURE 4 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept at room temperature for different periods (0 to 24 h)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1479
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
REFERENCES
1 Adesiyun A A L Webb and S Rahman 1995 Microbiological
quality of raw cow milk at collection centers in Trinidad J Food
Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
which is mainly due to its chemical modification
Esterification blocks the negatively charged carboxyl groups
on the protein molecules turning the net charge to positive
(33) The modified proteins may interact with negatively
charged components of the bacterial cell membranes (2829) by virtue of their enhanced positive charges causing
membrane disruption and subsequent bacterial inhibition
Pathogenic bacteria The data presented in Figure 2
trace the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 10 days of storage at 4uC In nontreated raw milk the
two microorganisms reached maximum levels on day 8 Lmonocytogenes reached a maximum of 606 iexcl 003 log
CFU ml21 as compared with 345 iexcl 004 log for
Salmonella Enteritidis on day 8 of storage suggesting that
L monocytogenes had higher viability because it is a
psychrotrophic bacterium Over the same period of time in
raw milk supplemented with esterified soybean protein
(MSP) the counts of the two pathogens were significantly (P 005) inhibited to the level of 360 iexcl 011 and 192 iexcl
011 log CFU ml21 with corresponding magnitudes of
reduction equivalent to ca 24 and 16 log CFU ml21
respectively After 8 to 10 days of storage under cold
conditions the level of Salmonella Enteritidis in the MSP-
supplemented raw milk was statistically similar to the
starting inoculum level suggesting a strong bacteriostatic
effect of the esterified soybean protein Supplementation
with native SP did not significantly (P 005) reduce the
proliferation of the two pathogens whereas supplementation
with the MCP exerted an effect similar to that of MSP (data
not shown) So the antimicrobial action noticed with MSP
and MCP is exclusively due to the modification process
The antimicrobial action of MSP and MCP is due to the
esterification process that endows the protein with con-
densed positive charges and renders it more reactive with
the microorganisms (33) Results indicate that modified
proteins have general antibacterial effects against both
spoilage and pathogenic bacteria This endowed potentiality
can help maintain the hygienic quality of raw milk during its
storage under cold conditions
Acidity development The changes in pH values and
titratable acidity of treated and untreated raw milk samples
during 10 days of storage at 4uC are shown in Figure 3 The
titratable acidity of untreated raw milk increased gradually
with time to reach a high level (027 iexcl 0008) by day 8
Supplementation of raw milk with MSP (05) has
significantly (P 005) and considerably reduced this
maximum level to a level of 021 iexcl 0008 over the same
period this is still within acceptable levels and indicates a
slower rate of acidity development than in the untreated raw
milk
Concomitantly the pH value gradually decreased in all
stored milk samples but at a higher rate in nontreated milk
Nontreated raw milk reached a value of ca 64 at 4 days of
storage as compared with 8 days in MSP-supplemented
milk under the same refrigeration conditions ie MSP
prolonged the period within which milk pH was at an
acceptable level MCP supplementation had a similar
magnitude of effect whereas the native forms of both
proteins were not associated with similar activities In
conclusion it can be stated that raw milk supplementation
FIGURE 2 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with native (SP) or esterified soybean protein (MSP)at 05 (wtvol) and kept under refrigeration conditions fordifferent periods (0 to 10 days)
FIGURE 3 Development of titratable acidity and pH in raw milk(C) during 10 days of storage under refrigeration conditions assupplemented with 05 (wtvol) esterified soybean protein (MSP)
1478 MAHGOUB ET AL J Food Prot Vol 74 No 9
with esterified legume proteins attenuated the development
of acidity and slowed the reduction rate of pH as a result of
their antimicrobial action which is normally associated with
reduced bacterial acid production
Rapid development of acidity in raw milk is apparently
due to uncontrolled microbial contamination with conse-
quent acid production (11) The attenuating effect on acidity
development is apparently due to the supplemented
esterified legume proteins which have been proved to exert
antimicrobial action (33) and accords with the microbio-
logical analytical results (Fig 1) So it can be concluded
that supplementation of raw buffalorsquos milk with esterified
legume proteins at a low level (05) can control the
development of titratable acidity in milk and thus prolong its
shelf life
Storage at room temperature Raw milk was stored at
room temperature (20 to 25uC) to simulate the conditions
under which raw milk may be handled transferred or
processed under artisanal conditions The study lasted only
24 h because keeping raw milk at such high temperature was
expected to cause accelerated rates of microbial proliferation
and associated acid development The three parameters
followed under the previous section were also used to evaluate
the microbiological and chemical status of the raw milk
Bacterial counts The results presented in Figure 4
show rapid microbiological changes in the levels of APC
CC and EC in raw milk stored at room temperature (20 to
25uC) for 24 h The APC in nontreated raw milk reached a
maximum (896 iexcl 003 log CFU ml21) after 12 h at which
time the MSP- or MCP-supplemented milk was inhibited by
about 15 log Native proteins (SP and CP) did not show any
significant reduction (P 005) in the APC when compared
with the control There was no difference between the
actions of the two modified proteins confirming the
previous conclusion that the antimicrobial action is
specifically due to the chemical modification
Nonsupplemented raw milk stored at room temperature
reached the prespoilage level of APC (ca 5 log) after 4 h
compared with about 7 h for MSP- or MCP-supplemented
raw milk The extension of the shelf life of raw milk at room
temperature with supplementation is valuable for transpor-
tation handling and processing under safe conditions Of
course subjecting milk to any additional refrigeration
treatment will further extend this period
The extent of the antimicrobial inhibitory action of
MSP and MCP on the CC and EC was nearly in the same
range (15 to 16 log) Generally the extent of the
antimicrobial inhibition in raw milk by esterified legume
proteins under room temperature is less than that observed
under refrigeration conditions Relatively high temperatures
may stimulate bacterial growth too much for it to be easily
counteracted by the applied substance whereas relatively
low temperatures may exert a bacteriostatic effect So it is
highly recommended that some cooling treatment be applied
to accentuate the action of the antimicrobial agents and
avoid potential contamination
This delay in spoilage emergence is apparently due to
the antimicrobial action of the esterified protein as
previously explained but it can be added that this
antimicrobial action acts at low (4uC) and at relatively high
(20 to 25uC) temperature storage conditions
Pathogenic bacteria The data presented in Figure 5
depict the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 24 h of storage at room temperature (20 to 25uC) In
nontreated raw milk the two pathogenic microorganisms
reached maximum levels after 12 h of storage at room
temperature Supplementing raw milk with MSP could
significantly (P 005) inhibit the counts of the two
pathogens L monocytogenes and Salmonella Enteritidis
counts were reduced by 27 and 18 log CFU ml21 after 12 h
FIGURE 4 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept at room temperature for different periods (0 to 24 h)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1479
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
REFERENCES
1 Adesiyun A A L Webb and S Rahman 1995 Microbiological
quality of raw cow milk at collection centers in Trinidad J Food
Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
with esterified legume proteins attenuated the development
of acidity and slowed the reduction rate of pH as a result of
their antimicrobial action which is normally associated with
reduced bacterial acid production
Rapid development of acidity in raw milk is apparently
due to uncontrolled microbial contamination with conse-
quent acid production (11) The attenuating effect on acidity
development is apparently due to the supplemented
esterified legume proteins which have been proved to exert
antimicrobial action (33) and accords with the microbio-
logical analytical results (Fig 1) So it can be concluded
that supplementation of raw buffalorsquos milk with esterified
legume proteins at a low level (05) can control the
development of titratable acidity in milk and thus prolong its
shelf life
Storage at room temperature Raw milk was stored at
room temperature (20 to 25uC) to simulate the conditions
under which raw milk may be handled transferred or
processed under artisanal conditions The study lasted only
24 h because keeping raw milk at such high temperature was
expected to cause accelerated rates of microbial proliferation
and associated acid development The three parameters
followed under the previous section were also used to evaluate
the microbiological and chemical status of the raw milk
Bacterial counts The results presented in Figure 4
show rapid microbiological changes in the levels of APC
CC and EC in raw milk stored at room temperature (20 to
25uC) for 24 h The APC in nontreated raw milk reached a
maximum (896 iexcl 003 log CFU ml21) after 12 h at which
time the MSP- or MCP-supplemented milk was inhibited by
about 15 log Native proteins (SP and CP) did not show any
significant reduction (P 005) in the APC when compared
with the control There was no difference between the
actions of the two modified proteins confirming the
previous conclusion that the antimicrobial action is
specifically due to the chemical modification
Nonsupplemented raw milk stored at room temperature
reached the prespoilage level of APC (ca 5 log) after 4 h
compared with about 7 h for MSP- or MCP-supplemented
raw milk The extension of the shelf life of raw milk at room
temperature with supplementation is valuable for transpor-
tation handling and processing under safe conditions Of
course subjecting milk to any additional refrigeration
treatment will further extend this period
The extent of the antimicrobial inhibitory action of
MSP and MCP on the CC and EC was nearly in the same
range (15 to 16 log) Generally the extent of the
antimicrobial inhibition in raw milk by esterified legume
proteins under room temperature is less than that observed
under refrigeration conditions Relatively high temperatures
may stimulate bacterial growth too much for it to be easily
counteracted by the applied substance whereas relatively
low temperatures may exert a bacteriostatic effect So it is
highly recommended that some cooling treatment be applied
to accentuate the action of the antimicrobial agents and
avoid potential contamination
This delay in spoilage emergence is apparently due to
the antimicrobial action of the esterified protein as
previously explained but it can be added that this
antimicrobial action acts at low (4uC) and at relatively high
(20 to 25uC) temperature storage conditions
Pathogenic bacteria The data presented in Figure 5
depict the changes in the level of L monocytogenes and
Salmonella Enteritidis in raw milk as affected by the
presence of methylated soybean at a low level (05)
during 24 h of storage at room temperature (20 to 25uC) In
nontreated raw milk the two pathogenic microorganisms
reached maximum levels after 12 h of storage at room
temperature Supplementing raw milk with MSP could
significantly (P 005) inhibit the counts of the two
pathogens L monocytogenes and Salmonella Enteritidis
counts were reduced by 27 and 18 log CFU ml21 after 12 h
FIGURE 4 Aerobic plate count (APC) coliform count (CC) and presumptive E coli (EC) in raw milk as supplemented with native oresterified legume proteins (05 wtvol) and kept at room temperature for different periods (0 to 24 h)
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1479
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
REFERENCES
1 Adesiyun A A L Webb and S Rahman 1995 Microbiological
quality of raw cow milk at collection centers in Trinidad J Food
Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
of storage at room temperature as compared with the
control Moreover the level of Salmonella Enteritidis was
reduced to a level even lower than the starting level after 12
and 24 h of storage at room temperature Raw milk
supplementation with MCP gave a similar magnitude of
effect to that of MSP (data not shown) which supports the
idea that esterification is the generator of the antimicrobial
action either at low or high temperature In conclusion the
antimicrobial action of the esterified proteins was also
effective against the pathogenic bacteria when milk was
stored at relatively higher temperatures
Acidity development Titratable acidity and pH
changes in raw milk treated with MSP and stored at room
temperature (20 to 25uC) for 24 h are shown in Figure 6
The titratable acidity of untreated raw milk increased
gradually with time to reach a maximum level (072 iexcl
002) at the end of the storage period (24 h) Supplemen-
tation of raw milk with MSP (05) has significantly (P
005) and considerably reduced the maximum level of
titratable acidity to 034 iexcl 001 over the same period The
prespoilage level of titratable acidity (021) was reached
after a very short period ca 5 h for control raw milk as
compared with 10 h for MSP-supplemented milk thus
MSP could limit the development of titratable acidity even
in raw milk stored at a relatively high temperature and
thereby extend its shelf life
On the other hand the pH value was gradually decreased
in nontreated raw milk to a minimum of 405 iexcl 005 after
24 h as compared with 51 iexcl 006 for MSP-supplemented
raw milk A prespoilage pH level of 64 was reached for
nontreated milk in 4 h as compared with 10 h for MSP-
supplemented raw milk under storage at room temperature
Similar results were obtained with MCP (data not shown) So
supplementation of raw milk with esterified legume proteins
can delay the pH drop in conjunction with attenuated
development of acidity and thus extend the period of raw
milk acceptability This action may be a result of the
inhibitory action of the esterified proteins against the
contaminating bacteria which are usually associated with
acid production activity It can therefore be concluded that
supplementation of raw buffalorsquos milk with esterified legume
proteins at a low level (05) can control the development of
titratable acidity and pH drop at room temperature
Finally it can be stated that modified legume proteins
have general antibacterial properties against both spoilage
and pathogenic bacteria in raw milk preserved either under
refrigeration or at room temperature This endowed
potentiality can help keep a good hygienic quality of raw
milk during its storage under refrigeration or at room
temperature Supplementation of raw buffalorsquos milk with
esterified legume proteins at a low level (05) can
simultaneously control the development of titratable acidity
and counteract the drop in pH either under cold or room-
temperature conditions MSP or MCP supplementation
extended the shelf life of raw milk preserved under cold
conditions by 6 days and of raw milk preserved at room
temperature by more than 7 h which is valuable for safe
transportation handling and processing
REFERENCES
1 Adesiyun A A L Webb and S Rahman 1995 Microbiological
quality of raw cow milk at collection centers in Trinidad J Food
Prot 58139ndash146
FIGURE 5 Changes in the counts of inoculated pathogens (L
monocytogenes and Salmonella Enteritidis) in raw milk (C) assupplemented with 05 (wtvol) native (SP) or esterified soybeanprotein (MSP) and kept at room temperature for different periods(0 to 24 h) FIGURE 6 Development of titratable acidity and pH in raw milk
(C) during 24 h of storage at room temperature as supplementedwith 05 (wtvol) esterified soybean (MSP) protein
1480 MAHGOUB ET AL J Food Prot Vol 74 No 9
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481
2 American Association of Cereal Chemists 2000 Crude proteinmdash
micro-Kjeldahl method AACC method 46-13 In Approved methods
of the AACC 10th ed vol 2 AACC St Paul MN
3 Amornkul Y and D R Henning 2007 Utilization of microfiltration
or lactoperoxidase system or both for manufacture of Cheddar cheese
from raw milk J Dairy Sci 904988ndash5000
4 Anonymous 1998 Microbiology of food and animal feeding stuffs
Horizontal methods for the detection and enumeration of Listeria
monocytogenes Part 2 enumeration method ISO 11290 Interna-
tional Organization for Standardization Geneva
5 Anonymous 1991 Microbiologymdashgeneral guidance on methods for
the detection of Salmonella 2nd ed rev ISO 6579 International
Organization for Standardization Geneva
6 AOAC International 1997 Official methods of milk analysis 16th
ed 3rd rev AOAC Arlington VA
7 Barbuddhe S B S P Chaudhari and S V S Malik 2002 The
occurrence of pathogenic Listeria monocytogenes and antibodies
against listeriolysin O in buffaloes J Vet Med B 49181ndash184
8 Bemrah N M Sanaa M H Cassin M W Griffiths and O Cerf
1998 Quantitative risk assessment of human listeriosis from
consumption of soft cheese made from raw milk Prev Vet Med
37129ndash145
9 Bertrand-Harb C J-M Chobert E Dufour and T Haertle 1991
Esterification of food proteins characterization of the derivatives by a
colorimetric method and by electrophoresis Sci Aliments 11641ndash
652
10 Boor K J 1997 Pathogenic microorganisms of concern to the dairy
industry Dairy Food Environ Sanit 17714ndash717
11 Champagne C P R R Laing D Roy A A Mafu and M W
Griffiths 1994 Psychrotrophs in dairy products their effects and
their control Crit Rev Food Sci Nutr 341ndash30
12 Coorevits A V De Jonghe J Vandroemme R Reekmans J
Heyrman W Messens P De Vos and M Heyndrickx 2008
Comparative analysis of the diversity of aerobic-spore-forming
bacteria in raw milk from organic and conventional dairy farms
Syst Appl Microbiol 31126ndash140
13 Dave R I and N P Shah 1997 Viability of yoghurt and probiotic
bacteria in yoghurts made from commercial starter cultures Int Dairy
J 731ndash41
14 Gaya P C Saralegui M Medina and M Nunez 1996 Occurrence
of Listeria monocytogenes and other Listeria spp in raw caprine
milk J Dairy Sci 791936ndash1941
15 Gran H M A Wetlesen A N Mutukumira G Rukure and J A
Narvhus 2003 Occurrence of pathogenic bacteria in raw milk
cultured pasteurised milk and naturally soured milk produced at
small-scale dairies in Zimbabwe Food Control 14539ndash544
16 Griffiths M W 1989 Listeria monocytogenes its importance in
dairy industry J Sci Food Agric 47133ndash158
17 Haddadin M S S A Ibrahim and R K Robinson 1996
Preservation of raw milk by activation of the natural lactoperoxidase
systems Food Control 71149ndash152
18 Halpin M I and T Richardson 1985 Elected functionality changes
of Beta-lacto globulin upon esterification of side chain carboxyl
groups J Dairy Sci 683189ndash3198
19 Headrick M L S Korangy N H Bean F J Angulo S F Altekruse
M E Potter and K C Klontz 1998 The epidemiology of raw milkndash
associated foodborne disease outbreaks reported in the United States
1973 through 1992 Am J Public Health 881219ndash1221
20 Hof H 2003 History and epidemiology of listeriosis FEMS
Immunol Med Microbiol 35199ndash202
21 Jay J M M J Loessner and D A Golden 2005 Modern food
microbiology 7th ed Aspen Publishers Gaithersburg MD
22 Johnson E A and J Brekke 1983 Functional properties of acylated
pea protein isolates J Food Sci 48722ndash725
23 Kalorey D R S R Warke N V Kurkure D B Rawool and S B
Barbuddhe 2008 Listeria species in bovine raw milk a large survey
of Central India Food Control 19109ndash112
24 Lee P S 2009 Quantitation of microorganisms chap 2 In E
Goldman and L H Green (ed) Practical handbook of microbiology
2nd ed CRC Press Boca Raton FL
25 Marth E H and J L Steele 2001 Applied dairy microbiology 2nd
ed Marcel Dekker New York
26 Mufandaedza J B C Viljoen S B Feresu and T H Gadaga
2006 Antimicrobial properties of lactic acid bacteria and yeast-LAB
cultures isolated from traditional fermented milk against pathogenic
Escherichia coli and Salmonella enteritidis strains Int J Food
Microbiol 108147ndash152
27 Munsch-Alatossava P O Gursoy and T Alatossava 2010
Potential of nitrogen gas (N2) to control psychrotrophs and
mesophiles in raw milk Microbiol Res 165122ndash132
28 Pan Y B Shiell J Wan M J Coventry H Roginski and A Lee
2007 The antimicrobial activity and molecular characterization of
amidated bovine lactoferrin Int Dairy J 17606ndash616
29 Pan Y J Wan H Roginski A Lee B Shiell and W P Michalski
2005 Effect of chemical modification on anti-microbial and anti-viral
properties of milk proteins Aust J Dairy Technol 60149ndash151
30 Ryser E T 1998 Public health concerns p 263ndash403 In E H Marth
and J L Steele (ed) Applied dairy microbiology Marcel Dekker
New York
31 Ryser E T and E H Marth 1991 Listeria listeriosis and food
safety Marcel Dekker New York
32 Savadogo A C A T Ouattara P W Savadogo A S Ouattara N
Barro and AS Traore 2004 Microorganisms involved in Fulani
traditional fermented milk in Burkina Faso Pak J Nutr 3134ndash139
33 Sitohy M and A Osman 2010 Antimicrobial activity of native and
esterified legume proteins against gram negative and gram positive
bacteria Food Chem 12066ndash73
34 Sitohy M J-M Chobert and T Haertle 2000 Study of factors
influencing protein esterification reaction using b-lactoglobulin as a
model J Food Biochem 24381ndash398
35 US Department of Health and Human Services Public Health
Services 2009 Pasteurized milk ordinance US Food and Drug
Administration Washington DC
J Food Prot Vol 74 No 9 INHIBITING BACTERIAL PROPAGATION IN RAW MILK 1481