Inhibition of growth of pathogenic bacteria in raw milk by legume protein esters

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)


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


depict the changes in the level of L monocytogenes and



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)


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


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


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


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

























































































































milk





























shelf life






















































































































REFERENCES



Prot 58139ndash146


























37129ndash145




652













J 731ndash41


















































New York







































































































































milk





























shelf life






















































































































REFERENCES



Prot 58139ndash146


























37129ndash145




652













J 731ndash41


















































New York






























shelf life






















































































































REFERENCES



Prot 58139ndash146


























37129ndash145




652













J 731ndash41


















































New York










































































REFERENCES



Prot 58139ndash146


























37129ndash145




652













J 731ndash41


















































New York





































37129ndash145




652













J 731ndash41


















































New York
















Inhibition of growth of pathogenic bacteria in raw milk by legume protein esters

Documents

Transcript of Inhibition of growth of pathogenic bacteria in raw milk by legume protein esters