foods

Article
The Investigation of Changes in Bacterial Community of
Pasteurized Milk during Cold Storage
Xinyi Lan 1 , Shuyan Wu 2 , Qijing Du 3,4 and Li Min 5, *

1 College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, China;
[email protected]
2 Hopkirk Research Institute, AgResearch Ltd., Palmerston North 4442, New Zealand;
[email protected]
3 Grasslands Research Centre, AgResearch Ltd., Palmerston North 4472, New Zealand; [email protected]
4 College of Food Science and Engineering, Qingdao Agricultural University, Qingdao 266109, China
5 Ministry of Agriculture Key Laboratory of Animal Nutrition and Feed Science in South China, Institute of
Animal Science, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China
* Correspondence: [email protected]

Abstract: The quality of pasteurized milk is commonly assessed through microbiological analysis,
with variations in storage conditions significantly impacting the suppression of bacterial growth
throughout the milk’s shelf life. This study investigated the dynamics of total bacterial counts (TBCs)
and bacterial community shifts in milk that underwent pasteurization at 80 ◦ C for 15 s. The milk
was subsequently stored at 4 ◦ C for varying intervals of 1, 4, 7, 10, 13, and 16 days. Culture-based
testing revealed a significant TBC increase during the storage period spanning 1 to 16 days (up
to −log10 4.2 CFU/mL at day 16). The TBC in pasteurized milk exhibited accelerated microbial
growth from day 13 onwards, ultimately peaking on day 16. Bacillus was detected through 16S
rRNA identification. Principal component analysis demonstrated a significant impact of storage time
on bacterial communities in pasteurized milk. Analysis of bacterial diversity revealed a negative
correlation between the Shannon index and the duration of pasteurized milk storage. Using high-
throughput sequencing, Streptococcus and Acinetobacter were detected as prevalent bacterial genera,
with Streptococcus dysgalactiae and Streptococcus uberis showing as dominant taxa. The presence of
Citation: Lan, X.; Wu, S.; Du, Q.; Min,
Streptococcus dysgalactiae and Streptococcus uberis in pasteurized milk might be attributed to the initial
L. The Investigation of Changes in
contamination from raw milk with mastitis. This study offers new evidence of the prevalence of
Bacterial Community of Pasteurized
bacterial community in pasteurized milk, thereby adding value to the enhancement of quality control
Milk during Cold Storage. Foods 2024,
13, 451. https://doi.org/10.3390/
and the development of strategies for reducing microbial risks.
foods13030451
Keywords: microorganism; pasteurized milk; cold storage; Bacillus; Streptococcus
Academic Editors: Bianca Castiglioni,
Pascal Degraeve and Aurelio López-
Malo

Received: 9 November 2023 1. Introduction

Revised: 25 December 2023 Dairy products are widely recognized as essential components of a healthy diet [1].
Accepted: 30 December 2023 Raw milk contains a diverse spectrum of bacterial populations including beneficial lactic
Published: 31 January 2024 acid bacteria that contribute to milk processing as well as spore-forming and psychrotrophic
bacteria associated with spoilage and potential health concerns. Microbial contamination
within the dairy chain can manifest at various stages, spanning from on-farm origins,
during transportation, to processing facilities. Consequently, both the raw and pasteurized
Copyright: © 2024 by the authors.
milk microbiota composition is profoundly shaped by numerous factors including farm
Licensee MDPI, Basel, Switzerland.
This article is an open access article
management practices, seasonal variations, hygienic protocols, and storage conditions
distributed under the terms and
spanning the entire value chain [2]. Pasteurization, a thermal treatment to eliminate
conditions of the Creative Commons pathogenic microorganisms, holds a critical role in extending milk shelf life and enhancing
Attribution (CC BY) license (https:// product quality [3,4]. In many countries, especially in the United States, consumers prefer
creativecommons.org/licenses/by/ high-temperature short-time (HTST) fluid milk to ultra-high temperature (UHT) milk with
4.0/). a longer shelf life. This, in turn, can lead to higher sales of dairy products and contribute to

Foods 2024, 13, 451. https://doi.org/10.3390/foods13030451 https://www.mdpi.com/journal/foods

Foods 2024, 13, 451 2 of 11

the reduction in food waste [5]. The dairy industry continues to face a significant challenge
in the production of pasteurized milk with an extended shelf life and enhanced quality
throughout its shelf life.
Maintaining effective temperature control throughout the entire process, from milking
to storage in the raw milk bulk tank, is of importance [6]. In some cases, cold storage
is not consistently maintained for consumer refrigerators, resulting in an increase in the
susceptibility to spoilage and the proliferation of potential pathogenic microorganisms in
stored dairy products [2]. The proper storage temperature of pasteurized milk is vital for
minimizing bacterial growth [7]. Earlier studies demonstrated the risks associated with
insufficient refrigeration conditions, which could lead to spoilage and potential pathogen
growth during storage [8]. Current studies have primarily focused on the storage tempera-
ture of raw milk and UHT milk [9,10]. A substantial proliferation of bacteria might occur
during the storage time in raw milk [9]. During the storage of UHT milk at cold storage
(3 ◦ C), notable alterations occur in the protein structure, consequently impacting the quality
of the product. These changes become more pronounced under high-temperature storage
conditions [10]. Other studies have investigated various approaches to extend the shelf-life
of HTST fluid milk [6]. A recent study indicated that storage temperature has a substan-
tially larger effect on fluid milk shelf life than HTST and suggests that abuse temperatures
(e.g., storage at 10 ◦ C) allow for the growth of Bacillus species that do not grow at lower
temperatures [11]. Limited evidence exists regarding the bacterial variation of pasteurized
milk during cold storage. Therefore, understanding the change in bacterial communities
during storage could provide new insights into the degradation of pasteurized milk.
Both microbial culture and advanced high-throughput sequencing methods have
been widely employed to identify and characterize the microorganisms present in milk [2].
Culture methods offer reliability in microbial community analysis, but they often come
with time-consuming and labor-intensive processes that might not fully capture the diverse
complexity of microbial communities. Polymerase chain reaction (PCR) cloning and the
sequencing of the 16S ribosomal RNA gene (rRNA) are also popularly applied as the
standard methods [12]. The culture method is unable to detect some challenging-to-
culture strains or those in a viable but nonculturable (VBNC) state. High-throughput
sequencing provides a more accurate method for obtaining information about microbial
colonies, enabling the detection of low-abundance microorganisms [3]. Furthermore, a more
advanced high-throughput sequencing method can generate far more reads than the culture
method and facilitate the discovery of even greater bacteria diversity [13]. This study aimed
to investigate alterations in the bacterial community and quality attributes of pasteurized
milk during storage, using these methods to identify bacteria in milk samples stored at
low temperatures. The research outcomes provide new insights into potential bacterial
risks in pasteurized milk during cold storage, while also gaining a better understanding of
variations in pasteurization safety.

2. Materials and Methods

2.1. Collection and Treatment of Milk Samples
In September 2022, a total of five factories were chosen from various regions in Henan,
China, to collect pasteurized bovine milk. In each commercial dairy industrial factory,
three bottles of pasteurized milk (80 ◦ C/15 s) were collected on five separate dates. The
collected milk was promptly transferred into 200 mL sterile bottles. All samples were
transported to the laboratory and then stored at 4 ◦ C for up to 16 days. The pasteurized 95
milk samples were taken for analysis at 1, 4, 7, 10, 13, and 16 days of storage.

2.2. Culture-Based Microbial Identification

TBC was immediately detected upon arrival at the laboratory, representing the initial
value (day 0). Milk samples were subjected to total bacterial count (TBC) detection using
culturing methods in accordance with the National Standards of the Republic of China [14].
Briefly, 25 mL of each milk sample was homogenized in 225 mL diluent solution (0.85%
Foods 2024, 13, 451 3 of 11

NaCl). A series of 10-fold dilutions were prepared and each diluent was spread on Plate
Count Agar (PCA, Beijing Land Bridge Technology Co. Ltd., Beijing, China). The plates
were incubated at 37 ◦ C for 48 h. The results are expressed as decimal logarithms of
colony-forming units per milliliter (log10 CFU/mL). Each sample and its diluents were
plate-counted in triplicate.

2.3. DNA Extraction and Pyrosequencing 16S rRNA

Fifty bacterial colonies were selected from the cultured plates for DNA extraction
and sequencing. The DNA of the bacterial colony was isolated using the PowerFood
Microbial DNA Extraction Kit (MO BIO Laboratories, Carlsbad, CA, USA) and stored at
−20 ◦ C prior to PCR. Extracted DNA was assessed by agarose gel (1%) electrophoresis
and quantified using a Nanodrop spectrometer (Thermo Scientific, Waltham, MA, USA).
DNA was diluted to a concentration of 50 ng/µL and used as a template for amplification
in the following PCRs. The 16S rRNA gene fragment was amplified by polymerase chain
reaction with a pair of universal primers 27F (5′ -AGAGTTTGATCMTGGCTCAG-3′ ) and
1492R (5′ -TACGGYTACCTTGTTACGACTT-3′ ). PCR amplification began with a 5 min
denaturing step at 94 ◦ C, followed by 30 cycles at 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 72 ◦ C
for 30 s; extension was achieved at 72 ◦ C for 15 min. The purified PCR products were
sequenced by ABI 3730XL (ThermoFisher, Waltham, MA, USA). For strain identification,
the 16S rRNA gene sequences of the isolates were blasted against the NCBI nr database.

2.4. The Examination of Bacterial Communities by High-Throughput Sequencing Analysis

Total DNA was isolated from 2 mL of pasteurized milk samples that were stored
for 16 days using the PowerFood Microbial DNA Extraction Kit (MO BIO Laboratories,
Carlsbad, CA) and stored at −20 ◦ C prior to PCR. The DNA extraction and qualification
procedures were conducted following previously described methods.
The 16S rRNA gene fragment was amplified by polymerase chain reaction with a pair
of universal primers 341F (5′ -CCTAYGGGRBGCASCAG-3′ ) and 806R (5′ -GGACTACNNG
GGTATCTAAT-3′ ). PCR amplification began with a 5 min denaturing step at 94 ◦ C, fol-
lowed by 30 cycles at 94 ◦ C for 30 s, 50 ◦ C for 30 s, and 72 ◦ C for 30 s; extension was
achieved at 72 ◦ C for 15 min [15]. PCR amplicons were extracted from 2% agarose gels
and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union, CA,
USA) according to the manufacturer’s instructions and quantified using QuantiFluor-ST
(Promega US, Madison, WI, USA). Purified amplicons were pooled in equimolar and
paired-end sequenced on an Illumina MiSeq platform according to the standard protocols.
Operational taxonomic units (OTUs) were generated by clustering at 97% sequence
identity using the UCLUST algorithm in QIIME 1.9 [16,17]. The OTUs were further assigned
to taxa using the RDP classifier [18]. The Simpson, Shannon, Chao1, and PD whole tree
index were calculated for each sample in QIIME 1.9 [2]. The weighted UniFrac distance
was used for principal coordinate analysis (PCoA) [19].

2.5. Statistical Analysis

Data are expressed as the mean standard deviation (SD) of three replicates. Significant
differences between the means of parameters were calculated with the Duncan’s multiple-
range test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA). p < 0.05 was considered
statistically significant.

3. Results
3.1. Total Bacterial Counts in Pasteurized Milk
The total bacterial count (TBC) was assessed in five pasteurized milk samples at
various storage times. As depicted in Figure 1, there was a statistically significant difference
in the total bacterial count (TBC) in pasteurized milk observed across different storage
times (p < 0.05). The TBC in pasteurized milk was 1.3 log10 CFU/mL at the sampled day,
which is below the standard limit. As per the China National Food Safety Standard, a
 3.1. Total Bacterial Counts in Pasteurized Milk
3.1. Total Bacterial Counts in Pasteurized Milk
The total bacterial count (TBC) was assessed in five pasteurized milk samples at var-
The totaltimes.
ious storage bacterial count (TBC)
As depicted was assessed
in Figure 1, there in
wasfive pasteurizedsignificant
a statistically milk samples at var-
difference
ious storage times. As depicted in Figure 1, there was a statistically
in the total bacterial count (TBC) in pasteurized milk observed across different storage significant difference
in the(p
times total bacterial
< 0.05). The TBCcount in (TBC) in pasteurized
pasteurized milk was milk observed
1.3 log10 CFU/mL across different
at the sampled storage
Foods 2024, 13, 451 4day,
of 11
times (p < 0.05). The TBC in pasteurized milk was 1.3 log10 CFU/mL
which is below the standard limit. As per the China National Food Safety Standard, at the sampled day,a
which is below
maximum of 4.2 the
log10standard
CFU/mL limit. As peracceptable
is deemed the China forNational Food milk
pasteurized Safety Standard,
[20]. The TBCa
maximum
of the samplesof 4.2
grewlog10 CFU/mL
rapidly fromisisday
deemed acceptable
13, reaching for
thefor pasteurized
highest on daymilk milk
16 <[20].
(p[20].
0.05)The TBC
maximum of 4.2 log10 CFU/mL deemed acceptable pasteurized The(Figure
TBC of
of
1). the samples grew rapidly from day 13, reaching the highest on day 16 (p < 0.05) (Figure
theFrom
samplesdaygrew
4 to rapidly
day 10, from
the TBC
day did not change
13, reaching the substantially
highest on day (p16> 0.05). As shown
(p < 0.05) (Figure in1).
1). From
Figure dayof4the
1, all to day 10, the TBC did not change notsubstantially (p > 0.05).A As shown in
From day 4 to day milk samples
10, the TBC did wenotsampled
change did exceed
substantially (p the standard.
> 0.05). As shown positive cor-
in Figure 1,
Figure 1,between
relation all of thethemilk
TBC samples
and we sampled
storage time ofdid not exceed
pasteurized theat
milk standard.
4A wasAobserved.
°Cpositive positive cor-
All
all of the milk samples we sampled did not exceed the standard. correlation
relation between the TBC andwere storage time ofaspasteurized milk16Satobserved.
4 °C was observed. All
50 selected
between thebacterial
TBC andcolonies
storage time of identified
pasteurized Bacillus 4 ◦ C was
milk atusing rRNA PCRAll sequencing.
50 selected
50 selected bacterial colonies were identified as Bacillus using 16S rRNA PCR sequencing.
bacterial colonies were identified as Bacillus using 16S rRNA PCR sequencing.

Figure 1. TBC in pasteurized milk samples during the storage time. Different superscripts in the bar
Figure
Figure
chart TBCsignificant
1.TBC
1.
indicate inpasteurized
in pasteurized milksamples
milk
differencessamples duringthe
during
(p < 0.05). thestorage
storagetime.
time.Different
Differentsuperscripts
superscriptsin
inthe
thebar
bar
chart indicate significant differences (p < 0.05).
chart indicate significant differences (p < 0.05).
3.2. Bacterial Diversity
3.2. Bacterial
3.2. BacterialDiversity
Diversity
The observed species and Shannon index data are presented in Figure 2, revealing
The observed
The observed species and
species and Shannon index index data
data are
are presented
presented inin Figure
Figure 2, 2, revealing
revealing
the significant differences duringShannon
storage time. Subsequently, the observed species in milk
thesignificant
the significantdifferences
differencesduring
during storage
storage time.
time. Subsequently,
Subsequently, the the observed
observed species species
in milk in
samples
milk reached
samples their lowest
reached their point on
lowest day on
point 16.day
Between
16. day 4 and
Between day
day 4 16, there
and day were
16, no
there
samples reached
substantial their lowest
fluctuations observed point
in on
theday 16. Between
Shannon day 4 and day 16, therestable
weremi-no
were no substantial
substantial fluctuationsfluctuations
observed observed
in the in theindex,
Shannon Shannon
index,
indicating
indicating
a relatively
index, indicating
a relatively astable
relatively
mi-
crobial diversity during
stable microbial diversitythis timeframe.
during Our investigation
this timeframe. demonstrated
Our investigation that the bacterial
demonstrated that the
crobial diversity
communities in during
milk this
samples timeframe.
displayed Our
higherinvestigation
diversity demonstrated
values during thatinitial
the the bacterial
storage
bacterial communities in milk samples displayed higher diversity values during the initial
communities
phase. in milk the
samples displayed higher diversitythevalues during the initial storage
storageFurthermore,
phase. Furthermore, negative correlation
the negative between
correlation Shannon
between index
the Shannon and the storage
index and the
phase.
time ofFurthermore,
pasteurized the negative
milk implied correlation betweeninthe Shannon index and the storage
storage
storage time of pasteurized milk aimplied
graduala gradual
decline bacterial
decline diversity
in bacterial as the
diversity as the
time of
duration pasteurized
increased. milk
These implied
findings a gradual
are valuabledecline in
evidence bacterial
for diversity
comprehending as the
the storage
dynam-
storage duration increased. These findings are valuable evidence for comprehending the
duration
ics increased.
of bacterial These findings
communities are valuable
in pasteurized milk evidence
during for comprehending the dynam-
dynamics of bacterial communities in pasteurized milk storage.
during storage.
ics of bacterial communities in pasteurized milk during storage.

Figure 2. Richness and diversity indices at different storage times from pasteurized milk. Different
superscripts in the bar chart indicate significant differences (p < 0.05).

3.3. Effect of Storage Time on the Bacterial Communities in Pasteurized Milk

The composition of the bacterial communities of pasteurized milk during storage at
4 ◦ C was assessed by principal coordinate analysis. The analysis of Bray–Curtis distances
effectively differentiated between the various milk samples. The principal coordinates 1, 2,
and 3 explained 83%, 5.93%, and 2.99% of the variation, respectively. It demonstrated a
significant shift in the bacterial composition between day 1 and day 16 (Figure 3). The results
 3.3. Effect of Storage Time on the Bacterial Communities in Pasteurized Milk
The composition of the bacterial communities of pasteurized milk during storage at
4 °C was assessed by principal coordinate analysis. The analysis of Bray–Curtis distances
Foods 2024, 13, 451 5 of 11
effectively differentiated between the various milk samples. The principal coordinates 1,
2, and 3 explained 83%, 5.93%, and 2.99% of the variation, respectively. It demonstrated a
significant shift in the bacterial composition between day 1 and day 16 (Figure 3). The
suggest that storage
results suggest time significantly
that storage influenced
time significantly the changes
influenced in bacterial
the changes communities
in bacterial commu-
within pasteurized milk.
nities within pasteurized milk.

Figure3.3. Principal
Figure Principal coordinates
coordinates analysis
analysis plot
plotof
of microbial
microbialdata
datafor
forpasteurized
pasteurizedmilk
milksamples
samplesduring
during
storage.
storage.

3.4.
3.4. The
The Composition
Composition of of Bacterial
Bacterial Community
Community
Bacterial
Bacterial diversity
diversity of of the
the samples
samples was
was evaluated,
evaluated, and
and the
the relative
relative abundance
abundance was was
characterized at the genus level (Figure 4). During the storage period,
characterized at the genus level (Figure 4). During the storage period, two genera weretwo genera were
predominant
predominant in in the microbiota. Streptococcus
themicrobiota. Streptococcus emerged
emerged as as the
the major
major genus,
genus, constituting
constituting
90% of all OTUs. Acinetobacter
90% of all OTUs. Acinetobacter was the second most dominant genus, displayingvariable
was the second most dominant genus, displaying variable
abundance
abundance as as the
thestorage
storageprogressed.
progressed. TheThe combined
combined abundance
abundance of ofother
otherundetermined
undetermined
genera
generawaswas lower
lower than
than that
that of dominant Streptococcus
of dominant Streptococcus but
but greater than Acinetobacter,
greater than Acinetobacter, show-
show-
ing a decreasing trend from day 1 to day 16. While Streptococcus and Acinetobacter
ing a decreasing trend from day 1 to day 16. While Streptococcus and Acinetobacter were were
identified
identifiedasasthe
thedominant
dominantcomponents
componentsofofthe microbiota,
the microbiota,our
ourresults revealed
results a fluctuating
revealed a fluctuat-
abundance of Acinetobacter, with a notable increase at day 10. These findings from our
ing abundance of Acinetobacter, with a notable increase at day 10. These findings from
Foods 2024, 13, x FOR PEER REVIEWstudy 6 ofour
11
highlight the dynamic nature of bacterial composition changes during the storage of
study highlight the dynamic nature of bacterial composition changes during the storage
pasteurized milk.
of pasteurized milk.

Figure 4. Relative
Figure 4. Relative abundance
abundance of
of top
top 10
10 most
most abundant
abundant bacteria
bacteria at
at genus
genus level
level in
in pasteurized
pasteurized milk
milk
samplesduring
samples duringstorage.
storage.

Regarding
Regardingthe dominant Streptococcus
the dominant Streptococcusgroup,
group,the
thespecific
specific abundances
abundances of of Streptococcus
Streptococcus
uberis and Streptococcus
uberis and Streptococcus dysgalactiae
dysgalactiae were
were further
further elucidated
elucidated (Figure
(Figure 5a).
5a). The
The evolutionary
evolutionary
history
history dendrogram
dendrogram of of Streptococcus
Streptococcus isolates
isolates was
was constructed,
constructed, illustrating their relation-
ships
ships with reference strains and other isolated species. (Figure
with reference strains and other isolated species. (Figure5b).
5b). The
The findings
findings revealed
revealed
the associated taxa, where Streptococcus dysgalactiae
the clustering of associated taxa, where Streptococcus dysgalactiae represented 84.1%,
clustering of represented 84.1%, and
and
Streptococcus uberis constituted 15.9%. The reference strains were annotated with their cor-
responding GenBank accession numbers and species names, enabling precise identifica-
tion and classification of the strains in the dendrogram.
 samples during storage.

Regarding the dominant Streptococcus group, the specific abundances of Streptococcus

uberis and Streptococcus dysgalactiae were further elucidated (Figure 5a). The evolutionary
Foods 2024, 13, 451 history dendrogram of Streptococcus isolates was constructed, illustrating their relation-
6 of 11
ships with reference strains and other isolated species. (Figure 5b). The findings revealed
the clustering of associated taxa, where Streptococcus dysgalactiae represented 84.1%, and
Streptococcus
Streptococcus uberis
uberis constituted
constituted 15.9%.
15.9%. The
Thereference
referencestrains
strainswere
wereannotated
annotatedwith
withtheir
theircor-
cor-
responding GenBank accession numbers and species names, enabling precise identifica-
responding GenBank accession numbers and species names, enabling precise identification
tion
and and classification
classification ofstrains
of the the strains indendrogram.
in the the dendrogram.

Streptococcus
Figure5.5.Streptococcus
Figure isolated
isolated from
from pasteurized
pasteurized milkmilk samples
samples during
during storage.
storage. (a) The(a) The relative
relative abun-
dance of Streptococcus
abundance uberisuberis
of Streptococcus and Streptococcus dysgalactiae
and Streptococcus during
dysgalactiae storage.
during (b) The
storage. (b) evolutionary his-
The evolutionary
tory dendrogram
history dendrogram of Streptococcus isolates.
of Streptococcus isolates.

4. Discussion
4. Discussion
4.1. Effects of Storage on Bacteria in Pasteurized Milk
4.1. Effects of Storage on Bacteria in Pasteurized Milk
Raw milk and properly pasteurized milk of high quality usually exhibit low total
Raw milk and properly pasteurized milk of high quality usually exhibit low total
bacterial counts (TBCs) [14]. The TBC serves as an effective indicator of the quality of both
bacterial counts (TBCs) [14]. The TBC serves as an effective indicator of the quality of both
raw milk and pasteurized milk throughout the processing stages. The elevated level of
raw milk and pasteurized milk throughout the processing stages. The elevated level of
microbial contamination observed in pasteurized milk can be attributed to insufficient
microbial contamination observed in pasteurized milk can be attributed to insufficient
cooling and inadequate storage conditions [21]. In this study, the TBC in pasteurized milk
cooling and inadequate storage conditions [21]. In this study, the TBC in pasteurized milk
showed an increase during storage at 4 ◦ C. Porcellato et al. [2] reported a similar impact of
showed an increase during storage at 4 °C. Porcellato et al. [2] reported a similar impact
storage time and temperature on both pasteurized full-fat milk and pasteurized carton milk
of storage time and temperature on both pasteurized full-fat milk and pasteurized carton
when stored at 8 ◦ C, but this effect was not observed in milk samples stored at 4 ◦ C. Liu
milk when stored at 8 °C, but this effect was not observed in milk samples stored at 4 °C.
et al. [9] also found that the microbiota remained stable with constant bacterial populations
Liu
when et al. [9] also
stored at 4found that the microbiota
◦ C. Another remained
study indicated that stable
the TBCwith constantrelatively
remained bacterial popu-
stable
lations
during the initial 14 days for pasteurized milk (heated at 75 C, 85 C, and 90 ◦ C)relatively
when stored at 4 °C. Another study indicated that ◦the TBC
◦ remained stored at
stable during the that
3 ◦ C, suggesting initial 14 days
lower for pasteurized
storage temperatures milk (heated
could delayatthe
75onset
°C, 85of°C, and 90 and
microbial °C)
chemical changes [11]. These varied research findings could potentially be linked to factors
such as the initial quality of the raw milk, variations in pasteurization procedures, and the
specific conditions of low-temperature milk storage [22]. Additionally, post-pasteurization
contamination (PPC) with Gram-negative bacteria is another factor leading to spoilage
and reduced shelf life in high-temperature, short-time pasteurized milk. Existing evidence
shows that approximately 50% of HTST-pasteurized fluid milk spoilage is due to PPC [20].
Psychrotolerant Gram-negative bacteria originating from PPC can grow in refrigerated
fluid milk [23]. Utilizing raw milk with shortened storage duration can help to prevent
bacterial proliferation and subsequent contamination in pasteurized milk stored at 4 ◦ C.
In this study, the detection of Bacillus bacteria in the milk samples using the culture
method aligns with many other studies demonstrating the prevalence of Bacillus in pasteur-
ized milk. Zhai et al. [24] isolated 114 Bacillus strains from 133 pasteurized milk samples.
Studies conducted by Gao et al. [25] and Zhao et al. [26] in China as well as by Porcellato
et al. [27] in Norway reported B. cereus contamination rates of 27%, 12%, and 11.5%, re-
spectively. The contamination could even be found in milk samples applied with thermal
treatment at a temperature of 85 ◦ C for a duration of 10 min, which exceeds the parameters
currently employed in commercial pasteurization processes in China [24]. A current survey
showed that 41% of the pasteurized milk exhibited Bacillus spp. under refrigerated storage
(6 ◦ C) for 21 days [28]. Bacillus, possessing enhanced heat resistance, exhibits resilience
against pasteurization and can undergo vegetative growth during refrigerated storage due
to its capacity to thrive at lower temperatures such as 6 ◦ C or below [29]. This pathogen
can lead to gastrointestinal ailments including gastric diseases, vomiting, diarrhea, and, in
Foods 2024, 13, 451 7 of 11

severe cases, fatalities. It has been reported to exhibit a high incidence of contamination in
all sorts of dairy products [26]. Considering that the D-value for spores of Bacillus strains
is near 15 min at 90 ◦ C [29], the heating condition of 85 ◦ C for 15 s used in this study was
insufficient to inactivate Bacillus spores. This could lead to their persistence during storage
at 4 ◦ C for 16 days, as observed in our samples. It is necessary to consider the detection of
Bacillus strains in raw milk, which could exist in raw milk and persist until pasteurization
or induced to regrow during pasteurization or storage. These results underscore the impor-
tance of effective microbiological control measures for Bacillus in pasteurized milk during
storage at 4 ◦ C.

4.2. Effects of Storage on the Bacterial Community Diversity of Pasteurized Milk

In this research, microbial diversity in pasteurized milk was evaluated using high-
throughput sequencing of the V3 and V4 regions of the 16S rRNA gene. The influence of
storage time on the milk bacterial community was addressed. Detected species and Shannon
were employed to determine the alpha diversity of the microbiome in the pasteurized milk.
The findings indicated that both the observed species and Shannon index values were
significantly higher on day 1 in comparison to the later storage periods. This suggests that
the abundance and diversity of bacterial species were most prominent during the initial
storage phase. The alpha diversity of the microbiome in our samples became lower as
the storage time progressed. A similar result was also reported by Liu [9], who observed
fluctuations in the microbial composition of HTST milk at room temperature storage,
displaying a decreasing alpha diversity from 4 h to 24 h. A loss of diversity could also
be seen in low-quality milk [30,31]. The current findings suggested a decreased diversity
during storage, implying that a subset of bacteria became dominant in samples [30].
The impact of different storage days on the bacterial community in pasteurized milk
was presented in the PCoA (shown in Figure 3). The correlation plots of PC1 (Figure 3)
showed a distance between samples of day 1 (on the right) and of day 16 (on the left), indi-
cating significant variations in bacterial communities within pasteurized milk throughout
the storage period. In the PC1 plot, the milk samples from day 1 and day 10 exhibited
clustering, while the samples from day 13 and day 16 clustered in both the PC1 and PC2
plots. This suggests that there were negligible differences in bacterial community compo-
sition between the samples from day 1 and day 10 as well as between the samples from
day 13 and day 16. However, the plot demonstrated a noticeable distance between the
samples from day 1 and those from day 9. Xue et al. detected differences in the milk
microbiota between pre-HTST pasteurized milk and post-HTST pasteurized milk stored
for 10 days [32]. The growth of psychrotrophic species (within the genus Pseudomonas) [33]
could be the major contributors to affect the PC1 and PC2 of the pasteurized milk samples.

4.3. The Bacterial Community Composition in Pasteurized Milk during the Storage
Our results revealed that Streptococcus was the dominant genus in milk samples during
storage, while Acinetobacter was the second major genus. A previous survey showed that
the prevalence of Streptococcus in 205 pasteurized milk from fresh milk bars was 1.5% in
China [34]. In an Australian survey, Streptococcus uberis, Streptococcus dysgalactiae, and Strep-
tococcus agalactiae were identified at proportions of 44%, 28%, and 22%, respectively [35].
Streptococcus. thermophilus was identified in soy milk and cow milk; the exopolysaccharides
produced by Streptococcus. thermophilus induced a significant reduction in IL-6 and IL-8
expressions in intestinal cells [36]. Pseudomonas and Acinetobacter were the genera with
the highest relative abundances in the bulk tank samples [37] and pasteurized milk [2].
A recent Swedish study identified Pseudomonas, Streptococcus, Acinetobacter, and Staphylo-
coccus as the top abundant bacterial genera in tank milk [38]. The variation in bacterial
community composition in raw milk could be attributed to the core microbiota present in
pasteurized milk.
In this study, there was no significant difference in the dominant genera of pasteur-
ized milk during 16 days of storage. This finding is consistent with a similar study that
Foods 2024, 13, 451 8 of 11

investigated milk microbiota over a 12-day storage period at temperatures of 5 ◦ C and

8 ◦ C [39]. Bacteria can remain active after pasteurization during prolonged storage. For
instance, microbial interactions between probiotic strains and Streptococcus thermophilus
improved the viability of microorganisms in fermented milk during a 21-day storage period
at 4 ◦ C [40]. Therefore, effective supervision and control measures for Streptococcus are
necessary to manage microbial contamination in pasteurized milk. Streptococcus spp. was
identified in all pasteurized milk samples, aligning with its categorization as a constituent
of the core microbiota in pasteurized milk. Our findings are consistent with a previous
study that reported Streptococcus spp. as one of three potential pathogens in pasteurized
milk between the northern and the western regions of China [34]. Acinetobacter spp. are
commonly found in bulk milk. The previous investigation found that 89.4% of Acinetobacter
isolates was identified in raw milk [41].
Streptococcus has a significantly detrimental effect on milk flavor and is considered a
key indicator of microbial contamination in pasteurized milk [3]. Streptococcus are major
mastitis pathogens, along with Staphylococcus aureus and coliforms [42]. Thermophilic Strep-
tococcal bacteria have exhibited resilience to the pasteurization process and the capacity to
form biofilms in dairy equipment [2]. The analysis based on functional genes identified
the presence of Streptococcus dysgalactiae and Streptococcus uberis in the pasteurized milk
samples. Streptococcus dysgalactiae carries multiple virulence determinants and resistance
genes, and can cause mastitis [43]. Streptococcus dysgalactiae infections can occur via sev-
eral routes of transmission and may persist on dairy farms for more than one year [44].
Streptococcus dysgalactiae and Streptococcus uberis likely presented in pasteurized milk due
to the fact the raw milk originates from cows suffering from mastitis, and these bacteria
exist in the raw milk. Therefore, the prevention and treatment of mastitis in dairy herds are
crucial for enhancing the safety of dairy products. Assessing the clinical pathogenicity of
Streptococcus spp. based on its contagiousness revealed substantial heterogeneity among
different strains [44]. Additionally, the prevalence of subclinical and clinical infections and
clinical manifestations collectively suggest that the virulence of Streptococcus dysgalactiae in
bovine mastitis is relatively lower compared to that of Streptococcus uberi [44]. Streptococcus
uberis, one of the most common pathogens isolated from clinical mastitis, is prevalent in
farming environments. It is regarded as a main causative agent of mastitis, leading to
significant financial losses annually within the dairy industry [45]. Streptococcus uberis is
characterized by the presence of a distinctive membrane-bound protein referred to as the
surface adhesion molecule. This protein assumes a pivotal role in facilitating the adherence
of Streptococcus uberis to bovine mammary epithelial cells through the interaction with
bovine lactoferrin [46]. In this study, the detection of Streptococcus dysgalactiae and Strepto-
coccus uberis in pasteurized milk suggests a potential association with raw milk sourced
from cows with mastitis. As the pathogenicity of this bacterium is largely attributed to their
ability to encode and produce numerous virulence factors [47], effective measures should
be taken for controlling Streptococcus dysgalactiae and Streptococcus uberi in dairy farms.

5. Conclusions
This study clarified the diversity of bacterial community in pasteurized milk stored
at 4 ◦ C. Employing both culture-based methods and high-throughput sequencing, we
identified predominating bacterial genera, with Bacillus emerging as a significant cultured
bacterium. Streptococcus and Acinetobacter dominated at the genus levels within the storage
period, while Streptococcus dysgalactiae and Streptococcus uberis stand out as key taxa. This
study offers new evidence of the prevalence of bacterial communities in pasteurized milk,
thereby adding value to the enhancement of quality control and the development of
strategies for reducing microbial risks.

Author Contributions: Conceptualization, X.L.; Data analysis, X.L. and Q.D.; Writing, X.L.; writing,
reviewing and editing, S.W.; Reviewing and editing, L.M. All authors have read and agreed to the
published version of the manuscript.
Foods 2024, 13, 451 9 of 11

Funding: This study was conducted with funding supports from the National Scientific Innovation
Strategy Project (R2017YJ-YB3006, R2018PY-QF008), the Science and Technology Innovation Pro-
gram of Hunan Province (2022RC1161), Guangdong Modern Agro-Industry Technology Research
System project (2023KJ114), the High-level Talent Research Fund of Qingdao Agricultural Univer-
sity (6631111317), and the Shandong Provincial Government-University Joint Training Program
([2021]45-2-13).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: We thank all the authors who participated in the study.
Conflicts of Interest: Author Shuyan Wu was employed by the company Hopkirk Research Institute,
AgResearch Ltd. The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential conflict of interest.

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