Dairy Foods III: Microbiology
388 Genomics of Advenella, Psychrobacter and Psychroflexus
strains from the surface of Austrian artisanal hard cheeses:
insights into ripening and flavor generation. S. Schmitz-Esser*1,2,
E. Nischler2, M. Dzieciol2, E. Mann2, and M. Wagner2, 1Iowa State
University, Department of Animal Science, Ames, IA, 2University
of Veterinary Medicine Vienna, Institute for Milk Hygiene, Vienna,
Austria.
Vorarlberger Bergkäse (VB) is an artisanal raw milk washed-rind hard
cheese manufactured in Western Austria without adding external ripening cultures. The composition of the VB rind microbiota has been
described recently by our group. To learn more about the functional
contribution of abundant gram-negative bacteria to cheese ripening,
we performed genome sequencing of Advenella (Betaproteobacteria),
Psychrobacter (Gammaprotoeobacteria) and Psychroflexus (Bacteroidetes) isolated from VB rinds. Furthermore, total and species-specific
bacterial numbers during ripening were determined using quantitative
PCR (qPCR). Cheese rind samples were taken from ripening cellars
of 2 cheese producing facilities in Austria at the day of production and
after 14, 30, 90 and 180 d of ripening. Genome sequencing resulted in
17 to 27 contigs with assembly sizes of 2.7 Mbp (Psychroflexus), 3 Mbp
(Psychrobacter), and 4.3 Mbp (Advenella). Our results reveal that each
genome harbors enzymes shown to be important for cheese ripening such
as: Cystathionine/Methionine β or gamma-Lyases, many proteases and
peptidases (including proline imminopeptidases), aminotransferases, and
lipases. Thus, all 3 isolates have the potential to contribute positively to
cheese ripening. Psychrobacter and Psychroflexus increased significantly
during the first 30 d, then decreased to their initial abundance during the
rest of ripening. Advenella decreased significantly in the first month of
ripening, but increased significantly throughout ripening. In conclusion,
the 3 species quantified were stable community members throughout the
ripening process and their abundance on cheese rinds together with the
results from genome sequencing suggests an important contribution of
these bacteria to cheese ripening. Although Advenella, Psychrobacter
and Psychroflexus have been found on cheese rinds in recent studies in
various different cheeses, our knowledge about their possible contributions to cheese ripening is still limited. Here, we provide first insights
into their genomic potential with a particular focus on cheese ripening.
Key Words: cheese rind bacteria, genome
389 Lactoferrin protect Caco-2, HEK, Hep-G2 and SK-N-SH
cell lines inhibits aflatoxin-induced cytotoxicity and oxidative
DNA damage. H. Zhang1,4, N. Zheng1,2, J. Liu5, Y. N. Gao1,2, and
J. Q. Wang*1,2, 1Ministry of Agriculture-Key Laboratory of Quality
& Safety Control for Milk and Dairy Products, Institute of Animal
Science, Chinese Academy of Agricultural Sciences, Beijing, China,
2Ministry of Agriculture-Laboratory of Quality and Safety Risk
Assessment for Dairy Products, Beijing, China, 3Ministry of Agriculture-Milk and Dairy Product Inspection Center, Beijing, China, 4College of Food Science& Engineering, Ji Lin University, Chang Chun,
China, 5China National Research Institute of Food and Fermentation
Industries, Beijing, China.
Aflatoxin is a natural potent carcinogen produced by Aspergillus flavus
and A. parasiticus that can be detected in milk. It is a major cause of liver
cancer. However, the involved molecular mechanisms and intervention
methods remain largely unexplored. Lactoferrin (LF) was examined
with regard to its potential role as a scavenger against radical oxygen.
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Moreover, no one has studied whether LF would inhibit the cytotoxicity of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1). The purpose of
this study was to evaluate the AFB1 and AFM1 induced cell toxicity by
determining cell viability, membrane permeability and genotoxicity, and
then investigate the capacity of LF to protect cells against AFB1 and
AFM1. Cell viability, lactate dehydrogenase (LDH) release, TBARS
and glutathione production and DNA damage were determined. In the
Comet assay, DNA damage was quantified by assessing tail moment
(TEM; arbitrary unit) and tail length (TL; arbitrary unit), as indicators of
DNA strand breaks in single cell gel electrophoresis. Data showed that
4 µg/mL AFB1 or AFM1 could significantly inhibit Caco-2 cells, HEK
cells, Hep-G2 cells, and SK-N-SH cell growth, increase lactate dehydrogenase (LDH) and cause genetic damage (P < 0.05). In comparison,
AFB1 was found to be more toxic than AFM1 on all 4 cells especially
on Hep-G2 cells, indicating liver is more sensitive than other organ as a
an important detoxification system. All these cytotoxic outcomes might
be associated with intracellular ROS generation, leading to membrane
damage and DNA strand break. Significant reductions in cytotoxicity
and oxidative DNA damage were observed when cells were pretreated
with 10, 100 or 1000 µg/mL LF then exposed to 4 µg/mL AFB1 or
AFM1. Our data suggested that AFB1 or AFM1 induces DNA damage
in Caco-2 cells, HEK cells, Hep-G2 cells, and SK-N-SH cells, whereas
that the antioxidant activity of LF may contribute to the alleviation of
AFB1 or AFM1-induced cytotoxicity and DNA damage by reducing
oxidative stress, it proved that LF plays an active role in inhibiting the
toxicity of mycotoxins.
Key Words: aflatoxin, lactoferrin, oxidative DNA damage
390 Impact of the addition of exopolysaccharides containing
β (1→ 4), and β (1→ 3) linkages isolated from Streptococcus thermophilus into milk prior to fermentation on physical and rheological properties of fermented milk gels. S. N. Khanal*1 and J.
A. Lucey1,2, 1University of Wisconsin, Department of Food Science,
Madison, WI, 2Wisconsin Center for Dairy Research, Madison, WI.
Streptococcus thermophilus strains St-143 and DGCC7785 are known
to produce exopolysaccharides (EPS), where sugars in the repeating
units are linked mainly by β (1→ 4), and β (1→ 3) types of gylcosidic
linkages, respectively. We investigated the impact of addition of these
isolated EPS to milk before fermentation on the properties of milk gels.
Milk samples were fermented (at a similar rate of acidification) by these
strains and these EPS solutions were isolated by ultrafiltration. Skim milk
was rehydrated in these isolated EPS solutions containing 60, 120 and
200 mg of EPS/L, heated to 85°C for 30 min, cooled, and fermented at
40°C by a non-EPS producing S. thermophilus strain ST-1UWM until
pH 4.6. Milk gels formed without any EPS, and formed by the respective
EPS producing S. thermophilus strains were used as 2 types of controls.
Rheological properties of the gels during fermentation were determined
using small-strain dynamic oscillatory measurements. Yield stress and
yield strain of the gels were determined at pH 4.6. Whey separation and
permeability of the milk gels were measured at pH 4.6. The fermentation
times for all the milk samples were similar (~250 min). The pH of gelation of the milk samples ranged from pH 5.1- 5.3. The storage modulus
(G′) values at pH 4.6 of the gels made with in situ production of the β
(1→ 3) linked EPS (72 Pa), and the in situ β (1→ 4) linked EPS (35 Pa)
were significantly different from the control (non-EPS producer) (53
Pa). Addition of isolated EPS to milk did modify the rheological, whey
separation and permeability properties, however, additional of isolated
J. Dairy Sci. Vol. 100, Suppl. 2
EPS could not exactly replicate the properties of the gels where this type
of EPS was produced in situ. Other possible factors like the time during
fermentation when EPS was produced, or the distribution of EPS within
the gel network could play a role in explaining why isolated EPS could
not better replicate the physical properties of acid milk gels.
Key Words: exopolysaccharide, glycosidic linkages, rheological
properties
391 Transcriptomic analysis of high exopolysaccharide-producing dairy starter bacterium Streptococcus thermophilus ASCC
1275 in milk. Q. Wu and N. P. Shah*, The University of Hong
Kong, Pok Fu Lam Road, Hong Kong.
Streptococcus thermophilus ASCC 1275, a typical dairy starter bacterium that has been completely sequenced, could produce the highest
known amount (~1000 mg/L) of exopolysaccharide (EPS) in milk
under the optimal condition among the species of Str. thermophilus.
However, little is known about the mechanistic insights into the effects
of environmental factors such as pH, temperature and whey peptides on
its EPS production. In this study, RNA-seq was applied to this organism
cultivated in skim milk under different conditions: (1) Cd1 – pH 6.5
and 37°C; (2) Cd2 – pH 5.5 and 37°C; (3) Cd3 – pH 5.5 and 40°C; (3)
Cd4 – pH 5.5 and 37°C with whey peptide supplementation. TopHap and
Cufflinks pipelines were used for differential gene expression analysis.
Results showed that there were 526 genes that have been significantly
changed in Cd2 compared with that in Cd1; specifically, ribosomal
protein synthesis and EPS assembly genes were significantly upregulated in Cd2 which may explain the enhanced EPS production in Cd2.
As for temperature effect, only 49 genes were significantly changed in
Cd3 compared with that in Cd2; purine and lactose metabolism were
enhanced in Cd3 suggesting that more nucleotide sugars (UDP-glucose
and UDP-galactose) may have been synthesized for EPS assembly in
Cd3. Whey peptide supplementation induced the significant changes of
16 genes in Cd4 compared with that in Cd2; it was found that cysteine
and methionine metabolism and some unidentified amino acid/peptide
transporters were improved for bacterial growth fitness. This study
indicates that regulation of nucleotide sugar synthesis, EPS assembly
and bacterial fitness are responsible for an enhanced EPS production
from Str. thermophilus ASCC 1275.
392 Un-shielding biofilm forming bacteria of protective extracellular matrix provides novel mean to improve dairy products
microbial quality. M. Shemesh*1, N. Ben-Ishay1,2, D. Inbar1,3, R.
Reifen2, and D. Steinberg3, 1Department of Food Quality and Safety,
Institute for Postharvest Technology and Food Sciences, Agricultural Research Organization, Rishon LeZion, Israel, 2The Robert H.
Smith Faculty of Agriculture, Food and Environment, The Institute of
Biochemistry, Food Science and Nutrition, The Hebrew University of
Jerusalem, Rehovot, Israel, 3Biofilm Research Laboratory, Institute
of Dental Sciences, Faculty of Dental Medicine, Hebrew UniversityHadassah, Jerusalem, Israel.
Microbial damages caused by biofilm forming bacteria in the dairy
industry are a fundamental threat to safety and quality of milk products.
Bacillus species, which are common contaminants of dairy products,
can form structured multicellular communities known as biofilms on
contact surfaces as well as within the milk products themselves. We
investigated the role of extracellular matrix (EM) produced through
biofilm formation in bacterial survival during milk processing. We used
molecular genetics and food microbiology methodology to show that
EM, produced by Bacillus subtilis during biofilm formation in milk,
have a major role in bacterial survival during the milk processing.
Noticeably, the mutant strains of B. subtilis, which cannot produce EM
during biofilm formation, showed hypersensitivity to milk processing
procedures such as heat pasteurization. This finding indicates that the
EM could serve as a protective material for biofilm forming bacteria and
un-shielding it would lead to increased sensitivity of bacterial cells to
stressful environments encounter during milk processing. Consequently,
we aimed to mitigate biofilm formation using the ability of divalent
cations such as Mg2+ of blocking EM production. Our further findings
indicate that in the presence of Mg2+ bacterial cells are hypersensitive
to the heat pasteurization applied during milk processing. We therefore
suggest that un-shielding biofilm forming bacteria of protective EM
may provide novel mean to improve dairy products microbial quality.
Key Words: biofilm formation, extracellular matrix, dairy food
Key Words: Streptococcus thermophilus, exopolysaccharide, enviromental factors
J. Dairy Sci. Vol. 100, Suppl. 2
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