based on the article What happens to the microbial environment as cheeses like Gouda, Cheddar, or Parmesan are aged?

I need help finding the answer in the article
Transcribed Image Text:Starter cultures, generally comprised of lactic acid bacteria, aid in acidification and fermentation during cheese production and, in some instances, are re- placed by more competitive organisms during brining, rind development, and aging [14]. Other microorgan- isms, such as fungi, are integral to the manufacture of smear-ripened cheeses such as Reblochon and Taleggio. Metagenomics can aid in understanding the microbiota of cheese, how these organisms interact, and how the presence of certain organisms aid or hinder aspects of food quality and safety. For ex- ample, it is known that certain microbial consortia in cheese contain antilisterial properties [15-17]. Studies have assessed the microbiomes of various cheeses in- oculated with Listeria monocytogenes and determined that the pathogen was inhibited by a combination of lactic acid bacteria and Gram-positive, catalase- positive bacteria [17]. Ultimately, the identification of the microbial communities in cheese is important and must include how factors such as raw materials, aging, storage conditions, and specific product charac- teristics impact diversity. Manufacturing of cheeses is divided into several steps. The first step is the addition of starter cultures which acidify the milk during the ripening process. The milk and starter culture mixture is subsequently warmed prior to the addition of rennet, leading to curd forma- tion and whey separation. The resulting curd is cut, cooked, and placed in molds that are pressed into the desired shape. The finished cheese wheels are brined and aged depending on the cheese type being processed. Cheeses can be manufactured using either pasteurized or unpasteurized milk. The pasteurization of milk in- volves heat treatment to eliminate harmful pathogens and lower the overall microbial burden. Therefore, cheeses prepared using unpasteurized milk are generally comprised of more diverse and heterogeneous microor- ganisms. Previous research has determined that the na- tive microbiota in unpasteurized milk contributes to the sensory properties of the resulting cheese, and research suggests that these organisms aid in producing a more robust flavor [18, 19]. The microbiota in unpasteurized milk is influenced by the animal's teat canal and sur- rounding skin, the environmental conditions, seasonal- ity, pasture and grazing changes, personnel hygiene, starter culture selection, process and post-process con- tamination [14, 20-22]. For pasteurized milk, the micro- bial community is influenced by the thermoduric bacteria that survive pasteurization, and post-process contamination. The composition and distribution of microbiota in cheeses differ not only by milk type and environmental factors, but also by sampling location (i.e., core, rind) [8, 16, 21, 23]. The rind of the cheese is a more open ecosystem exposed to the environment and abiotic con- ditions and is comprised of a high diversity of organisms [21]. Aerobic bacteria, as well as yeasts and molds, dom- inate the rind of cheese. Halophiles are also present due to their ability to survive high salt concentrations that would be encountered during brining. Hygienic condi- tions during aging also play a role in shaping the micro- biota of the rind. Conversely, the core of the cheese is more anaerobic and generally exhibits a lower pH, lead- ing to less biodiversity [21]. For this reason, lactic acid bacteria, including those in the added starter cultures, are predominant in the core, often reaching population levels near 9 log CFU/g shortly after cheese manufacture [21]. These bacteria acidify the milk and hinder the growth of other less-competitive species and some spoil- age bacteria. Less dominant organisms in the core in- clude yeasts, Gram-positive catalase-positive bacteria, and enterococci. These different microenvironments en- countered throughout cheese affect the types of micro- biota present and how these organisms interact. Cheeses can be categorized on their degree of firmness of texture and are grouped as hard, semi-hard and soft. Hard and semi-hard cheeses are often aged. For aged cheeses, such as Gouda, Cheddar, and Parmesan, the length of the aging process plays a significant role in the composition and diversity of microbiota. When cheeses are aged, moisture content and water activity decrease. Due to increasingly limited nutrients during aging, some bacteria undergo autolysis, contributing cellular compo- nents, including enzymes and sugars, to the overall char- acteristics of the cheese [24]. It is known that populations of starter lactic acid bacteria are reduced but survive during aging. In the U. S., cheeses crafted using unpasteurized milk must be aged for at least 60 days at a minimum temperature of 35 °F (1.67 °C) prior to introduction into inter commerce [25]. This aging period is intended to eliminate pathogens that may have been present in the unpasteurized milk. How- ever, it has been determined that pathogens can survive past the 60 day aging process [26-28]. Understanding the microbiome of Gouda, an aged cheese, will aid in de- signing studies to reduce the risk of illness due to patho- gens from consumption of this type of cheese made using unpasteurized milk. During 1998-2015 a total of 113 outbreaks associated with cheese consumption were reported to the CDC, resulting in 2418 illnesses, 291 hospitalizations, and 18 deaths [CDC FOOD Tool, wwwn.cdc.gov/foodborneout- breaks]. Of the total number of outbreaks, 20% (n = 23) were specifically stated to be associated with cheeses made using unpasteurized milk. Gouda cheese has fre- quently been implicated in product recalls and outbreaks [29-31] attributed to various foodborne pathogens in- cluding Listeria monocytogenes and E. coli O157:H7. The
Transcribed Image Text:environment and could possibly be a post-pasteurization contaminant. Yersinia can also grow at refrigeration temperatures and could survive the cheese aging and storage process. Lastly, Weissella, a facultative anaerobic lactic acid bacteria in the family Leuconostocaceae, was also only identified in the samples taken just under the rind. Although some species of Weissella are pathogenic, some species are being studied as potential pro- and pre- biotic organisms. This organism has been previously identified in a wide range of habitats including milk and cheese rinds [59], Mexican Cotija cheese [60], and cheese whey [61]. Megasphaera, Caloramator, and Hymonella were only detected in the Gouda cheese cores, and Anoxybacillus and Yaniella were only detected in the inside samples. The core of a cheese represents an environment that is mainly anaerobic, explaining why the anaerobes Megasphaera and Caloramator and the facultative an- aerobe Hymonella were identified in this region. Inter- estingly, Megasphaera is known to be a commensal organism of ruminants and has been identified in unpasteurized ewe milk cheeses [62]. Anoxybacillus and Yaniella, which were only identified in the inside, were also only present in the cow Gouda cheese samples in this study. This is only the second report of Yaniella de- tected in a food product [23]. In addition to assessing the microflora of Gouda cheese through milk type and spatial variability, this study also examined the differences in microflora based on cheese aging length. Unidentified members of Bacillaceae, Lactococcus, Lactobacillus, and Staphylococcus dominated the populations of the unpasteurized Gouda cheeses which were aged for 2-4, 4-6, 6–9, or 12-18 months. Bacillaceae sequencing reads decreased during aging, whereas the reverse was observed for Lactococcus. Lactic acid bacteria, including those of the indigenous microbiota and the added starter cultures, typically comprise most the population of cheese during the aging process [21]. For Swiss and Emmental cheeses, thermophilic lactic acid bacteria derived from the starter culture (such as Lactoba- cillus helveticus and Streptococcus thermophilus) are the dominant organisms from the start of aging up to six months [21, 63, 64]. Mesophilic lactic acid bacteria, in- cluding Lactobacillus paracasei and L. rhamnosus also be- come dominant during aging, especially in cheeses aged for 10-30 months [65]. Interestingly, the population of Staphylococcus was not dependent on the length of aging, but rather spatial vari- ation. Most Staphylococcus in the aged Gouda was lo- cated in samples taken under the rind. For the Gouda sample aged 4-6 months, this genus comprised 81.1% in the samples taken under the rind. However, Staphylococ- cus decreased to 50.2 or 45.2% in Gouda aged for 6–9 or 12-18 months, respectively. Less than 1% of the population of the 2-4-month aged Gouda cheese sam- ples taken under the rind was Staphylococcus. The vast differences in these results are likely due to the environ- mental conditions of aging, personnel handling, and the pasteurization status of the milk used. Large populations of Staphylococcus have previously been observed on cheese rinds [23, 56], possibly due to environmental contamination. Furthermore, Staphylococcus is pre- sumed to be at a concentration of 2-3 log CFU/mL in unpasteurized milk [21]. Twenty-two and 38 genus-level identifications were ob- served in the unpasteurized Gouda cheese aged for 2-4 and 12-18 months, respectively. A total of 27 out of the 38 identifications in the older Gouda cheese were not found in the younger 2-4 months aged Gouda. Some of the genera identified in the Gouda cheese which was aged longer in- cluded Acidovorax, Ralstonia, Adhaeribacter, Devosia, Haemophilus, and Neisseria. Acidovorax and Ralstonia are both aerobic Gram-positive plant pathogens [66, 67]. Acidovorax has been previously identified as a contaminant of Italian Grana cheese [68], and Ralstonia has been de- tected in unpasteurized milk [43, 69] and can survive high salinity environments. Adhaeribacter and Devosia are both soil dwelling bacteria and have been previously identified in unpasteurized milk [44, 70]. Devosia has also been detected on cow teat skin [42]. Haemophilus and Neisseria, both genera which contain species of human pathogens, were also only detected in the Gouda cheese that was aged for 12-18 months. However, these genera have not previously been identified in dairy products. Conclusions This study assessed the metagenomics in commercial pas- teurized and unpasteurized Gouda cheeses. Overall, the Gouda cheeses assessed were comprised of the same or- ganisms although with different population levels. Some differences were observed between the pasteurized and unpasteurized Gouda cheeses, with more genus-level identifications being made for the unpasteurized cheeses. Twenty-eight bacterial genera were only observed in the goat Gouda cheese, indicating that milk source has vast implications for the resulting microbiome of Gouda cheese. Many other factors can influence the microbiome of Gouda cheese, including spatial variability and length of aging. Aerobic organisms and environmental contami- nants were generally identified in outer portions of the Gouda samples. In addition, the length of aging plays an important role in the fate of the microbiome, with an in- creased level of genus diversity being observed with Gouda cheeses which were aged for longer periods of time. Overall, these results agree with the published litera- ture on cheese microbiomes and provide valuable insights into the microbiome of Gouda cheese. Understanding the metagenomics of Gouda cheese is useful in improving

Question

based on the article What happens to the microbial environment as cheeses like Gouda, Cheddar, or Parmesan are aged?

I need help finding the answer in the article

Starter cultures, generally comprised of lactic acid
bacteria, aid in acidification and fermentation during
cheese production and, in some instances, are re-
placed by more competitive organisms during brining,
rind development, and aging [14]. Other microorgan-
isms, such as fungi, are integral to the manufacture
of smear-ripened cheeses such as Reblochon and
Taleggio. Metagenomics can aid in understanding the
microbiota of cheese, how these organisms interact,
and how the presence of certain organisms aid or
hinder aspects of food quality and safety. For ex-
ample, it is known that certain microbial consortia in
cheese contain antilisterial properties [15-17]. Studies
have assessed the microbiomes of various cheeses in-
oculated with Listeria monocytogenes and determined
that the pathogen was inhibited by a combination of
lactic acid bacteria and Gram-positive, catalase-
positive bacteria [17]. Ultimately, the identification of
the microbial communities in cheese is important and
must include how factors such as raw materials,
aging, storage conditions, and specific product charac-
teristics impact diversity.
Manufacturing of cheeses is divided into several steps.
The first step is the addition of starter cultures which
acidify the milk during the ripening process. The milk
and starter culture mixture is subsequently warmed
prior to the addition of rennet, leading to curd forma-
tion and whey separation. The resulting curd is cut,
cooked, and placed in molds that are pressed into the
desired shape. The finished cheese wheels are brined
and aged depending on the cheese type being processed.
Cheeses can be manufactured using either pasteurized
or unpasteurized milk. The pasteurization of milk in-
volves heat treatment to eliminate harmful pathogens
and lower the overall microbial burden. Therefore,
cheeses prepared using unpasteurized milk are generally
comprised of more diverse and heterogeneous microor-
ganisms. Previous research has determined that the na-
tive microbiota in unpasteurized milk contributes to the
sensory properties of the resulting cheese, and research
suggests that these organisms aid in producing a more
robust flavor [18, 19]. The microbiota in unpasteurized
milk is influenced by the animal's teat canal and sur-
rounding skin, the environmental conditions, seasonal-
ity, pasture and grazing changes, personnel hygiene,
starter culture selection, process and post-process con-
tamination [14, 20-22]. For pasteurized milk, the micro-
bial community is influenced by the thermoduric
bacteria that survive pasteurization, and post-process
contamination.
The composition and distribution of microbiota in
cheeses differ not only by milk type and environmental
factors, but also by sampling location (i.e., core, rind) [8,
16, 21, 23]. The rind of the cheese is a more open
ecosystem exposed to the environment and abiotic con-
ditions and is comprised of a high diversity of organisms
[21]. Aerobic bacteria, as well as yeasts and molds, dom-
inate the rind of cheese. Halophiles are also present due
to their ability to survive high salt concentrations that
would be encountered during brining. Hygienic condi-
tions during aging also play a role in shaping the micro-
biota of the rind. Conversely, the core of the cheese is
more anaerobic and generally exhibits a lower pH, lead-
ing to less biodiversity [21]. For this reason, lactic acid
bacteria, including those in the added starter cultures,
are predominant in the core, often reaching population
levels near 9 log CFU/g shortly after cheese manufacture
[21]. These bacteria acidify the milk and hinder the
growth of other less-competitive species and some spoil-
age bacteria. Less dominant organisms in the core in-
clude yeasts, Gram-positive catalase-positive bacteria,
and enterococci. These different microenvironments en-
countered throughout cheese affect the types of micro-
biota present and how these organisms interact.
Cheeses can be categorized on their degree of firmness
of texture and are grouped as hard, semi-hard and soft.
Hard and semi-hard cheeses are often aged. For aged
cheeses, such as Gouda, Cheddar, and Parmesan, the
length of the aging process plays a significant role in the
composition and diversity of microbiota. When cheeses
are aged, moisture content and water activity decrease.
Due to increasingly limited nutrients during aging, some
bacteria undergo autolysis, contributing cellular compo-
nents, including enzymes and sugars, to the overall char-
acteristics of the cheese [24]. It is known that
populations of starter lactic acid bacteria are reduced
but survive during aging. In the U. S., cheeses crafted
using unpasteurized milk must be aged for at least
60 days at a minimum temperature of 35 °F (1.67 °C)
prior to introduction into inter commerce [25]. This
aging period is intended to eliminate pathogens that
may have been present in the unpasteurized milk. How-
ever, it has been determined that pathogens can survive
past the 60 day aging process [26-28]. Understanding
the microbiome of Gouda, an aged cheese, will aid in de-
signing studies to reduce the risk of illness due to patho-
gens from consumption of this type of cheese made
using unpasteurized milk.
During 1998-2015 a total of 113 outbreaks associated
with cheese consumption were reported to the CDC,
resulting in 2418 illnesses, 291 hospitalizations, and 18
deaths [CDC FOOD Tool, wwwn.cdc.gov/foodborneout-
breaks]. Of the total number of outbreaks, 20% (n = 23)
were specifically stated to be associated with cheeses
made using unpasteurized milk. Gouda cheese has fre-
quently been implicated in product recalls and outbreaks
[29-31] attributed to various foodborne pathogens in-
cluding Listeria monocytogenes and E. coli O157:H7. The

environment and could possibly be a post-pasteurization
contaminant. Yersinia can also grow at refrigeration
temperatures and could survive the cheese aging and
storage process. Lastly, Weissella, a facultative anaerobic
lactic acid bacteria in the family Leuconostocaceae, was
also only identified in the samples taken just under the
rind. Although some species of Weissella are pathogenic,
some species are being studied as potential pro- and pre-
biotic organisms. This organism has been previously
identified in a wide range of habitats including milk and
cheese rinds [59], Mexican Cotija cheese [60], and
cheese whey [61].
Megasphaera, Caloramator, and Hymonella were only
detected in the Gouda cheese cores, and Anoxybacillus
and Yaniella were only detected in the inside samples.
The core of a cheese represents an environment that is
mainly anaerobic, explaining why the anaerobes
Megasphaera and Caloramator and the facultative an-
aerobe Hymonella were identified in this region. Inter-
estingly, Megasphaera is known to be a commensal
organism of ruminants and has been identified in
unpasteurized ewe milk cheeses [62]. Anoxybacillus and
Yaniella, which were only identified in the inside, were
also only present in the cow Gouda cheese samples in
this study. This is only the second report of Yaniella de-
tected in a food product [23].
In addition to assessing the microflora of Gouda cheese
through milk type and spatial variability, this study also
examined the differences in microflora based on cheese
aging length. Unidentified members of Bacillaceae,
Lactococcus, Lactobacillus, and Staphylococcus dominated
the populations of the unpasteurized Gouda cheeses
which were aged for 2-4, 4-6, 6–9, or 12-18 months.
Bacillaceae sequencing reads decreased during aging,
whereas the reverse was observed for Lactococcus. Lactic
acid bacteria, including those of the indigenous microbiota
and the added starter cultures, typically comprise most
the population of cheese during the aging process [21].
For Swiss and Emmental cheeses, thermophilic lactic acid
bacteria derived from the starter culture (such as Lactoba-
cillus helveticus and Streptococcus thermophilus) are the
dominant organisms from the start of aging up to six
months [21, 63, 64]. Mesophilic lactic acid bacteria, in-
cluding Lactobacillus paracasei and L. rhamnosus also be-
come dominant during aging, especially in cheeses aged
for 10-30 months [65].
Interestingly, the population of Staphylococcus was not
dependent on the length of aging, but rather spatial vari-
ation. Most Staphylococcus in the aged Gouda was lo-
cated in samples taken under the rind. For the Gouda
sample aged 4-6 months, this genus comprised 81.1% in
the samples taken under the rind. However, Staphylococ-
cus decreased to 50.2 or 45.2% in Gouda aged for 6–9 or
12-18 months, respectively. Less than 1% of the
population of the 2-4-month aged Gouda cheese sam-
ples taken under the rind was Staphylococcus. The vast
differences in these results are likely due to the environ-
mental conditions of aging, personnel handling, and the
pasteurization status of the milk used. Large populations
of Staphylococcus have previously been observed on
cheese rinds [23, 56], possibly due to environmental
contamination. Furthermore, Staphylococcus is pre-
sumed to be at a concentration of 2-3 log CFU/mL in
unpasteurized milk [21].
Twenty-two and 38 genus-level identifications were ob-
served in the unpasteurized Gouda cheese aged for 2-4
and 12-18 months, respectively. A total of 27 out of the 38
identifications in the older Gouda cheese were not found in
the younger 2-4 months aged Gouda. Some of the genera
identified in the Gouda cheese which was aged longer in-
cluded Acidovorax, Ralstonia, Adhaeribacter, Devosia,
Haemophilus, and Neisseria. Acidovorax and Ralstonia are
both aerobic Gram-positive plant pathogens [66, 67].
Acidovorax has been previously identified as a contaminant
of Italian Grana cheese [68], and Ralstonia has been de-
tected in unpasteurized milk [43, 69] and can survive high
salinity environments. Adhaeribacter and Devosia are both
soil dwelling bacteria and have been previously identified in
unpasteurized milk [44, 70]. Devosia has also been detected
on cow teat skin [42]. Haemophilus and Neisseria, both
genera which contain species of human pathogens, were
also only detected in the Gouda cheese that was aged for
12-18 months. However, these genera have not previously
been identified in dairy products.
Conclusions
This study assessed the metagenomics in commercial pas-
teurized and unpasteurized Gouda cheeses. Overall, the
Gouda cheeses assessed were comprised of the same or-
ganisms although with different population levels. Some
differences were observed between the pasteurized and
unpasteurized Gouda cheeses, with more genus-level
identifications being made for the unpasteurized cheeses.
Twenty-eight bacterial genera were only observed in the
goat Gouda cheese, indicating that milk source has vast
implications for the resulting microbiome of Gouda
cheese. Many other factors can influence the microbiome
of Gouda cheese, including spatial variability and length of
aging. Aerobic organisms and environmental contami-
nants were generally identified in outer portions of the
Gouda samples. In addition, the length of aging plays an
important role in the fate of the microbiome, with an in-
creased level of genus diversity being observed with
Gouda cheeses which were aged for longer periods of
time. Overall, these results agree with the published litera-
ture on cheese microbiomes and provide valuable insights
into the microbiome of Gouda cheese. Understanding the
metagenomics of Gouda cheese is useful in improving

Accepted Answer

Introduction 
Microbiota, also known as gut flora or microbiome, refers to the community of…