Microbiological Quality of Pasteurized Milk in Hawai`i - University of ...

Pacific Agriculture and Natural Resources
Microbiological Quality of Pasteurized Milk in Hawai`i
HONGFEI HE, YONG LI*, ALFRED L. CASTRO, JIN DONG and C. N. LEE*
Department of Human Nutrition, Food and Animal Sciences, 1955 East-West Road
University of Hawaii at Manoa, Hawaii 96822 USA
Abstract: coComplaints of spoilage of pasteurized milk purchased from Honolulu groceries highlighted the need to
address the emerging milk quality issues for the consumer’s consumption. The objective of this study was to
evaluate the microbiological quality of pasteurized milk available in Hawaii's market. Bacterial counts of different
types of milk samples (imported-M, locally produced-L and imported organic-O) were compared during refrigerated
storage. Imported bulk milk from mainland are re-pasteurized in Hawaii and packaged under different brands.
Hawaii locally produced milk are marketed with the "Island Fresh" label. The expiration or sell by date is based on
the pasteurization. Imported organic milk from the mainland were prepackaged and shipped by air freight. At 5d
before the expiration date, 70% of the mainland samples and 62% of the local samples had aerobic bacteria count
exceeding the regulatory limit of 20,000 cfu/ml for grade ‘A’ pasteurized milk set by the United States Food and
Drug Administration (FDA). Local raw milk samples from the farms supplying the processor were examined and
results indicated good microbiological quality. High levels of psychrotrophic bacteria were identified as a major
contributor to the pasteurized milk spoilage before the expiration date. The implications of this study were further
discussed.
Key words: pasteurized milk, raw milk, re-pasteurization, microbiological quality, shelf-life, spoilage bacteria,
psychrotrophic bacteria, post-pasteurization contamination, degradative enzymes, Pasteurized Milk Ordinance.
INTRODUCTION
Hawaii’s dairy industry faced numerous challenges in
the recent decade. Aging operators faced with higher
feed costs, fluctuations in raw milk prices and increased
environmental regulations (both at the state and federal
levels) found it easier to close operations versus
investing in costly new technologies for waste
management. Most of the dairy operations were
located in small acreage without the opportunity to
expand and were faced with increasing pressure of
housing development within a stone throw from their
farm sites. Displaced dairy operations resulted in the
need for larger volumes of milk to be imported to meet
local demands for fluid milk. Presently, there are three
types of imported milk from the U.S. mainland: 1) prepackaged
organic milk with refrigerated shipping or airfreighted;
2) ultra pasteurized milk with long shelf-life,
and 3) conventional high temperature short time
(HTST) pasteurized milk is cooled and shipped in bulk
tanks (>6,000 gallons) for the trans-ocean journey that
normally takes 5-7 days. Particularly, the conventional
pasteurized milk is held in insulated tankers without
refrigeration during transportation. Initial shipment of
bulk tankers had refrigeration but due to higher cost
and limited space for electrical terminals in a ship, this
method was changed. In Honolulu or Hilo, imported
bulk tank milk was then re-pasteurized (HTST),
packaged and placed for retail with a shelf-life of 17-19
days. The shelf-life dates back to the most recent
pasteurization (local) date.
Consumer complaints of pasteurized milk spoilage
in Honolulu prior to the printed expiration date began
in late 2002 and increased in 2003. The increased
complaints coincided with the increase of imported
milk into the market. Numerous studies have indicated
that spoilage of processed milk is primarily due to
bacterial activity that results in loss of sensory quality
(Cromie, 1991, Boor and Murphy, 2002, Hayes and
Boor, 2001). Among various microorganisms,
psychrotrophic bacteria are by far the most important
factor influencing pasteurized milk quality (Sorhaug
and Stepaniak, 1997). Psychrotrophic bacteria are a
group of microorganisms capable of growing at 7 o C
(44.6 o F) or less. These organisms are remarkable for
their rapid growth and short generation multiplication
under low temperatures; in raw milk or in pasteurized
milk under refrigeration. The extracellular proteolytic
and lipolytic enzymes produced by these bacteria can
degrade nutritional components of milk and cause
undesirable changes in sensory characteristics (Hantsis-
Zacharov and Halpern, 2007). Although most of the
psychrotrophic bacteria are destroyed by pasteurization,
high level of these organisms in raw milk may produce
sufficient enzymes that can tolerate pasteurization and
reduce processed milk quality. Furthermore, postpasteurization
contamination by psychrotrophic
bacteria present in the processing environment is
currently a detrimental factor for extending the shelflife
of HTST pasteurized milk (Eneroth et al., 2000).
This study was designed to determine the bacterial
levels of retail fluid milk produced locally versus milk
∗Corresponding author, E-mail: chinl@hawaii.edu, liyong@hawaii.edu
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Vol. 2: 20-25, 2010

Hongfei he, Yong Li, Alfred L., et al. 21
imported and re-pasteurized. The general goal was to
validate the legitimacy of the consumer complaints.
The hypothesis was that early spoilage of imported
milk was due to higher psychrotrophic bacteria count
even under proper handling.
MATERIALS AND METHODS
Sample collection
The first study was conducted to evaluate the
microbiological quality of pasteurized milk available in
Honolulu. Three types of milk with 2% fat were
selected: imported pasteurized milk from mainland and
re-pasteurized locally (M), locally produced and
pasteurized milk (L), and imported pre-packaged
organic milk (O). The study was conducted over a
period of several months (August 2006-April 2007). A
total of 45 commercial pasteurized milk samples were
randomly purchased from local grocery stores in
Honolulu. The sampling plan consisted of collecting
15 samples of each type (M, L, and O). Over 9 brands
of milk from 10 stores were sampled. Local milk
samples were identified by the smiling daisy logo -
"Island Fresh". The time taken to transport the milk
from the stores to the laboratory was less than 10 min.
Samples were purchased in the evening when the day
was cooler and the traffic was lighter.
In the second study, the microbiological quality of
local raw milk was evaluated. A total of six raw milk
samples were randomly collected from local dairy
farms and transported to the laboratory in ice in a
cooler. In the third study, the bacterial level of
pasteurized milk sold in the Big Island (Hawaii) was
examined; 5 M samples and 9 L samples from the
grocery stores in the Big Island were purchased and
transported by air to our laboratory in coolers
containing coolant packs. A fourth study was
conducted to evaluate the effect of storage temperature
on microbial population in milk. This "stress test" was
conducted 15 months later, under a separate grant.
All milk samples were stored at 4ºC in the laboratory
and handled according to Standard Methods for the
Examination of Dairy Products (Wehr and Frank,
2004).
Microbiological evaluation
At five days before expiration (-5), day of expiration
(0), and three days after expiration (+3), the samples
were analyzed for aerobic plate count (APC) and rapid
psychrotrophic count (RPC). Raw milk samples were
tested for APC and RPC within 2 h after on farm
collection. The milk samples were serially diluted in
0.1% peptone water and spread on plate count agar
(BD Diagnostic Systems, Sparks, Md.). The plates
were incubated at 35ºC for 48 h to determine APC or
at 21ºC for 48 h to determine RPC (Oliveria and
Parmelee, 1976). All HTST processed milk products
with different brands (M and L) in Hawaii were
processed by the only processing plant in Honolulu
and on the Big Island, respectively. The O type milk
tested was normally imported via air shipment from the
west coast.
Growth of bacteria under different
refrigeration temperature
In the fourth study aimed at determining the effects
of different refrigeration temperatures on bacterial
growth in pasteurized milk (12 samples) that were
randomly purchased in Honolulu were examined.
Upon receipt in the laboratory, each milk sample was
aseptically aliquoted into two sterile 50 ml bottles, one
stored at 4 ºC and the other stored at 7 ºC for shelf-life
study. At 10, 7 and 5 days before expiration, these
samples were analyzed for APC and RPC. This study
was part of a subsequent grant to perform
fingerprinting of the spoilage-causing bacteria in milk.
It was conducted 15 months later. The data fits this
presentation as it provided a more complete picture of
what is happening in milk microbial population
Statistical analysis
The APC and RPC data were transformed using
log 10CFU/ml. Differences between mean microbial
counts of M, L and O samples were analyzed at each
sampling time using multi-variate analysis of variance.
The APC and RPC data from the study of storage
temperature effects on microbial growth were analyzed
using paired t-test. All statistical analyses were
performed with SAS ® 9.1 (SAS Institute Inc.).
Results
Figure 1 showed the bacterial population of three
types of commercially available pasteurized milk (M, L,
O) in Honolulu assessed at three time points (-5, 0, and
+3) during their shelf-life. The bacteria numbers of all
O samples were below the 20,000 CFU/ml regulatory
limit for grade ‘A’ pasteurized milk (U.S.-FDA, 2005)
during the reported shelf-life period. However, at five
days before expiration day, 90% of the M samples and
80% of the L samples had APCs levels greater than the
legal limit. There was a rapid increase in APCs during
shelf-life, as illustrated by 90% of both M and L
samples with > 10 6 CFU/ml by expiration day. At this
level, the products became unacceptable due to the
growth and metabolism of bacteria in milk (Fromm
and Boor, 2004). Results also showed, for M and L
samples, RPCs was higher than APCs during the five
days before expiration day. All M and L samples had
higher bacterial levels than O samples (p

Log cfu/ml .
Log cfu/ml
22 Microbiological Quality of Pasteurized Milk in Hawai`i
10.0
8.0
M n=15
L n=15
O n=15
a
a
a
a
a a
a
a
a a
6.0
a
*
a
4.0
2.0
b
b
b
b
b
b
FDA
regulatory
limit
(4.3)
0.0
-5 0 3 -5 0 3
APC
Time (days)
*Means with no common superscripts are significantly different (P

Log cfu/ml .
Hongfei he, Yong Li, Alfred L., et al. 23
10.0
M n=5
8.0
6.0
4.0
L n=9
a*
a
a
a
a
a
a
a
a
a
a
b
FDA
regulatory
limit (4.3)
2.0
0.0
-5 0 3 -5 0 3
Time (days )
*Means with no common superscripts are significantly different (P

24 Microbiological Quality of Pasteurized Milk in Hawai`i
one way to determine the sites of contamination.
On the other hand, organic (O) milk samples in this
study demonstrated high quality performance in
microbial testing. It may be possible that the different
milk handling strategies designed for O milk
production and handling may contribute to its quality;
the chilled distribution system of O milk is the
probably primary factor that contributed to product
safety and extended shelf-life. Direct airfreight with
shorter transportation time may be a major
contributing factor for the superior microbiological
quality of O milk. It must also be noted that in some
rare occasions, O milk samples did exceed the legal
limits set by the FDA. This is probably due to poor
handling e.g. transportation from air cargo site to the
retail stores is not performed by refrigerated trucks as
has been observed in a few occasions when one walked
passed the stores.
Bulk tank milk imported into the state without
refrigeration generally requires 5-7 days of transit. The
volatile temperatures during transportation may have a
direct effect on the shelf-life, quality and potential
safety of the milk products. Increased temperatures
have been shown to encourage growth of
microorganisms and increase the activity of degradative
enzymes (Cromie, 1991, Hantsis-Zacharov and
Halpern, 2007, Sorhaug and Stepaniak, 1997).
Excessive high bacterial level may challenge the
efficacy of re-pasteurization, resulting in final products
with higher bacterial counts thus affecting their
subsequent shelf-life. The heat-stable enzymes
produced by bacteria may cause serious quality defects
of re-pasteurized milk (Sorhaug and Stepaniak, 1997).
For example, proteins present in milk can be digested
by proteases, resulting in production of bitter-tasting
peptides and curdling of the milk; Lipases are able to
degrade lipids and cause rancidity of milk (Hantsis-
Zacharov and Halpern, 2007). Unlike Honolulu where
the majority (>65%) of milk is imported, the majority
of the milk supply on the Big Island was produced
locally (>70%). This circumstance may be a possible
explanation for the differences in the rate of spoilage in
between the two counties (>80% vs 11%).
It must be noted that the Pasteurized Milk
Ordinance (PMO) allows the bulk shipping of
pasteurized milk without refrigeration. The guideline
recommends the temperature of 7.2ºC (45ºF) as the
indicator for rejecting milk. Hence, as long as the
temperature is met, the milk can be reprocessed for
fluid milk use. The current standards do not set a legal
microbial count. In addition, there is no data on the
bacterial level after 7 days in transit without
refrigeration. The state of California however prohibits
the re-pasteurization of milk for fluid milk market but
allows it for other utilization (CA FAC # 35832). Such
regulation does not exist in Hawai'i and as such no law
is violated or challenged. The PMO, when drafted and
revised, probably, did not consider the situation where
milk is being transported and held for more than five
days without refrigeration. There is also a need for
future studies to determine the microbial population of
milk following such mode of transportation and
handling. Sampling of the bulk tanks milk prior to repasteurization
would be required if the current PMO
standards is to be validated. Any such sampling would
need the cooperation of the processor and the
regulatory agency.
In a study conducted 15 months later, results
showed storage temperatures affected the final
microbial numbers in milk (Table 1). When this was
compared to the microbial level in the earlier studies,
only 8% of the samples at 4ºC and 25% of the samples
at 7ºC storage had bacterial counts higher than the
regulatory limit at 5 days prior to expiration date.
These results indicate that: a) the milk products from
the same processing plant had different quality during
the two different sampling periods and b) postpasteurization
contamination was still the likely culprit.
Previous report suggests the differences are probably
due to multiple factors existing in the processing
environment, such as pasteurization and storage
conditions, packaging equipment, and personal hygiene
(Gruetzmacher and Bradley, 1999). Nevertheless, 25%
incidence of high bacteria counts should raise concerns
given the fact that the major consumers of milk are
infants, toddlers and young children. It must also be
noted that shelf-life of milk in the retail space is not
governed by any regulations in any state. The duration
a product is on the shelf space is primarily a product
quality control set by the respective creamery.
In summary, these studies validate the increased
complaints from the consumers on milk spoilage prior
to the expiration date in Hawaii. We did not resolve
where the contamination occurs or identify the source
of the bacteria. These spoilage-related organisms can
be present in raw milk, but they also may enter milk
products during production and processing. Identifying
the spoilage organisms and their reservoirs are critical
for extending fluid milk shelf-life. In the present study,
our findings suggest that psychrotrophic bacteria were
the predominant organisms causing spoilage in Hawaii
locally processed milk and this is confirmed by the
subsequent study using molecular techniques to
identify the spoilage-related bacteria in pasteurized milk
(He et al. 2009). Since these bacteria are heat sensitive,
the contaminants could be attributed to postpasteurization
contamination. Our findings provide
important information on the quality status of locally
available pasteurized milk in retail stores and stress the
need for further studies to determine the source of the
spoilage organisms.
Acknowledgement
This study was supported via a grant provided by
Hawaii Department of Agriculture and the Hawaii
Farm Bureau Federation ORS# 2006 1665-4.
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Hongfei he, Yong Li, Alfred L., et al. 25
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