Transcript Lb. curvatus

An Examination of the Bacterial Population and Their Lactate Dehydrogenase Activities in
Cheddar Cheese with Calcium Lactate Crystal Defect
By: Boorus Yim, Nana Y. Farkye, Shakeel-Ur-Rehman and Ebenezar R. Vedamuthu
Dairy Products Technology Center, California Polytechnic State Univeristy, San Luis Obispo, California 93407
P-029
Abstract
Calcium lactate crystal (CLC) formation is a problem in Cheddar cheese.
This defect on the surfaces of cheese does not pose any health hazards, but may
be mistaken by consumers as mold, resulting in economic loss to the industry.
It is postulated that certain non-starter lactic acid bacteria (NSLAB) that grow
in cheese are responsible. The conversion of pyruvate to L(+) and D(-)-lactic
acid by lactate dehydrogenase (LDH) and a racemic mixture of L(+) and D(-)lactic acid may contribute to CLC formation, but the mechanism is not fully
understood. This study examined the NSLAB population in cheeses (Cheddar,
Gouda, Asiago and Parmesan) with and without the CLC defect. Seventy-nine
bacterial colonies were randomly isolated from streak plates on Rogosa SL agar
of the cheeses with CLC defect. DNA was extracted from 60 of the 79 colonies
and underwent 16S ribosomal RNA PCR amplification and sequencing with an
ABI Prism 3730 Genetic Analyzer for BLAST species identification. Forty of
the 60 isolates were identified as Lactobacillus curvatus subsp. curvatus. The
remaining isolates were predominantly identified as Lb. casei, Lb. paracasei and
Lb. coryniformis. Twenty colonies from cheeses without visible CLC defect were
identified primarily as Lb. casei and Lb. paracasei. Using a DL-Lactic Acid
enzyme assay kit, the concentration of D(-)-lactic acid was used as an indirect
measure of D-LDH activity of the bacterial isolates. Lb. curvatus had
approximately equal levels of L(+) and D(-)-lactic acid (mean = 1.60 g/L) or
predominately higher levels of D(-) than L(+)-lactic acid (3.01 g/L versus 0.16
g/L). Lb. curvatus also produced approximately 10 times more D-lactic acid
than Lb. casei or Lb. paracasei, which had little to no D-lactic acid production
(mean = 0.27 g/L). Lb. curvatus was used as an adjunct in Cheddar cheese
manufacture to confirm formation of CLC defect. Crystals were observed after
approximately 6 weeks of ripening at 6°C. Results suggests that Lb. curvatus
is the main NSLAB that produces D-LDH to give a racemic mixture of L(+) and
D(-)-lactic acid for CLC formation.
Introduction
The presence of white specks on Cheddar cheese, first reportedly
observed since the 1930s, is a problem that remains a burden on
manufacturers and consumers.
These white specks have been
repeatedly identified as calcium lactate crystals (CLC). CLC pose no
health hazards nor affect the cheese flavor, however, CLC have a moldlike appearance and detract from the appearance and product appeal.
CLC formation has been attributed to several factors (e.g., milk
composition, cheese packaging and types of bacteria present in cheese).
Recent studies have concentrated on non-starter lactic acid bacteria
(NSLAB) as the direct cause of CLC formation in Cheddar cheese.
Theoretically, CLC form when there is a racemic mixture of L(+)
and D(-)-lactic acid and free calcium in the serum of cheese. However,
the definitive cause of the formation of the crystals is not fully
understood. The processing variables on the formation of white
crystals on Cheddar cheese have been studied. Dybing, et al. (1988)
investigated the effect of different manufacturing methods, seasonal
effects, composition of cheese with and without crystalline
encrustations, packaging conditions (temperature, CO2 gas flushing
versus vacuum packaging), curing (ripening) temperatures, and length
of curing. They concluded that because faster acid production and
lower milling pH correlated with reduced crystallization, the caseinbound calcium was the major source of calcium for crystal formation.
They also noted that crystal formation was enhanced when CO2 gas
flushing was used during packaging. However, in vacuumed packaged
cheeses, the defect was lower. The presence of residual lactose in cheese
creates favorable conditions for the growth of NSLAB which convert
lactose to D(-) lactate.
Some NSLAB (e.g., Lactobacillus plantarum, Pediococci acidilactici,
Lb. curvatus) have generally implicated as the cause of CLC formation,
but the specific crystal–causing NSLAB(s) remains to be determined.
The commonality between these species is the presence of D(-) lactate
dehydrogenase (LDH) – the enzyme responsible for the production of
D(-)-lactic acid.
Agarwal et al. (2003) investigated the source and identity of
NSLAB in CLC formation by swabbing a cheese manufacturing facility,
and using the isolated organisms as adjuncts for cheesemaking. The
main isolates identified were Lb. curvatus and P. acidilactici. It was found
that cheeses aged for one month at 10°C compared to 7.2°C had higher
NSLAB counts. Cheeses with or without P. acidilactici adjunct did not
produce any crystals. However, cheese with Lb. curvatus exhibited CLC
by the end of 2 months of aging. Chou, et al., (2003) also observed a
high correlation of Lb. curvatus with CLC in cheese. The formation of
CLC was more extensive when the cheese was aged at 13°C and then
transferred to 4°C.
Although NSLAB have been linked to the development of white
crystalline defect, a more thorough examination of microbial, enzymatic
and genetic role in this phenomenon is necessary. Such a study should
incorporate procedures similar to Koch’s postulates for the
establishment for the etiology of disease. This involves isolation of
NSLAB from cheese with CLC, characterization of the bacteria,
inactivating the possible genes responsible for the enzyme production,
adding selected isolates (mutant and wild-type) to cheese milk,
duplicating the defect in resultant cheeses, and re-isolating the added
strain(s) from the cheese, thereby completing the cause and effect cycle.
The objective of this study was to determine the dominant microbial
population in a variety of cheeses with the CLC defect.
Materials and Methods
Cheddar, Parmesan and Gouda cheese samples purchased in
local supermarkets or provided by various companies with the
characteristic white haze of calcium lactate crystals on the surface were
selected (Figure 1). Standard methods were used to isolate NSLAB.
NSLAB was isolated from the cheeses (~10 g) using Rogosa SL agar and
deMan Rogosa Sharpe (MRS) agar; incubated at 37°C in an anaerobic
jar (BD anaerobic jar) for 3 days until good colony development was
obtained.
Pour–plating was done according to the procedures
recommended for cheese in the Standard Methods for the Examination
of Dairy Products (Marshall, 1996).
Individual colonies picked from the agars were isolated and
propagated in MRS broth; incubated at 37°C aerobically, and 1 mL was
transferred every 2-3 days to new MRS broth. Gram–staining,
morphology, and catalase production were conducted to provide initial
characteristics of each bacterial isolate. Specie identification of the
bacteria isolates was determined by 16S ribosomal RNA PCR
amplification using universal primers UF2 and UR523 (Table 1). The
resultant PCR product was sequenced with an ABI Prism 3730 Genetic
Analyzer and underwent BLAST species identification (Reference
here!). L(+) and D(-)-LDH activity was indirectly determined by
measuring the D(-)-lactic acid/L(+)-lactic acid concentrations produced
by double washed (sterile phosphate buffer, pH 7.2) isolated cells using
the D(-)-lactic acid/L(+)-lactic acid assay kit (Boehringer Mannheim, RBiopharm AG, Germany)
Table 1. Universal primers for PCR used for 16S ribosomal RNA
BLAST species identification.
UF2
5’-AGAGTTTGATCCTGGCTCAG-3’
UR523
5’-GCTGGCACGTAGTTAGCC-3’
1
51
101
151
201
251
301
351
401
451
501
Figure 1. Cheddar cheese (6 months old) with calcium lactate crystal defect.
CGCAGCAACG
GTTAGATTGA
GACGGGTGAG
TGGAAACAGA
TTGAAAGATG
TTAGTTGGTG
AGAGGGTAAT
GAGGCAGCAG
CGCCGCGTGA
AAGAACGTAT
AAGCCACGGC
CTGGCGGCGT
AGAAGCTTGC
TAACACGTGG
TGCTAATACC
GTTTCGGCTA
AGGTAAAGGC
CGGCCACACT
TAGGGAATCT
GTGAAGAAGG
TTGATAGTAA
GCCTAATACA
TTCTGATTGA
GTAACCTGCC
GCATAAAACC
TCACTTTAGG
TCACCAAGAC
GGGACTGAGA
TCCACAATGG
TTTTCGGATC
CTGATCAGGT
TGCAAGTCGA
TAACATTTGA
CTAAAGTGGG
TAGCACCGCA
ATGGACCCGC
CGTGATGCAT
CACGGCCCAG
ACGAAAGTCT
GTAAAACTCT
AGTGACGGTA
ACGCACTNTC
GTGAGTGGCG
GGATAACATT
TGGTGCAAGG
GGTGCATTAG
AGCCGACCTG
ACTCCTACGG
GATGGAGCAA
GTTGTTGGAG
TCCAACCAGA
Figure 2. Sequence of 16S Ribosomal RNA PCR Product of Bacterial Isolate LB2
Identified as Lactobacillus curvatus subsp. curvatus.
Results and Discussion
Figure 2 is one of many sequences of PCR products using the universal
primers and bacterial isolates to determine the species of each isolate. The 510 base
pair sequence represented in Figure 2 was identified as Lb. curvatus subsp. curvatus.
Table 2 shows the predominant bacterial composition of a variety of cheeses with
and without the CLC defect. From cheeses with the CLC defect, 40 of 60 isolates
were identified as Lb. curvatus subsp. curvatus. Of the 20 remaining isolates, 12
were identified as Lb. casei, 5 were Lb. paracasei and 3 were Lb. coryniformis. Of the
20 bacterial isolates from cheese with no visible signs of CLC defect, 13 were
identified as Lb. casei and 7 were identified as Lb. paracasei. Table 3 shows the mean
concentrations of DL-lactic acid produced by the bacterial isolates. Lb. casei and Lb.
paracasei predominantly produced more L(-)-lactic acid. Lb. curvatus subsp. curvatus
predominantly produced D(+)-lactic acid or approximately equal levels of D(-) and
L(+)-lactic acid (not shown). The isolated Lb. curvatus subsp. curvatus strains were
used as adjuncts in pilot-scale Cheddar cheese making to confirm their ability to
form CLC.
Table 2. Identity of bacterial isolates from a variety of cheeses with and without the
calcium lactate crystal defect.
Bacterial Isolate
Lactobacillus curvatus subsp. curvatus
Lactobacillus casei
Lactobacillus paracasei
Lactobacillus coryniformis
Number Identified
Cheeses with
CLC Defect
40
Yes
Conclusions
12/13
Yes/No
5/7
Yes/No
3
Yes
The dominant lactic acid bacteria in cheeses with visible signs of
CLC defect is Lactobacillus curvatus subsp. curvatus, while in cheeses
without visible signs of CLC defect are Lb. casei and Lb. paracasei. Lb.
curvatus subsp. curvatus produced approximately 10 times more D(-)lactic acid than Lb. casei and Lb. paracasei, which indicates Lb. curvatus
subsp. curvatus strains produced higher levels of D(-)-lactate
dehydrogenase than other lactic acid bacteria found in cheeses with CLC
defect.
Table 3. Mean DL-lactic acid (g/L) of identified bacterial isolates from a variety of
cheeses with the calcium lactate crystal defect.
Mean D(-)-Lactic Acid
(g/L)
Mean L(+)-Lactic Acid
(g/L)
Lactobacillus curvatus subsp.
curvatus
3.01
0.16
Lactobacillus casei
0.27
2.26
Lactobacillus paracasei
0.57
2.54
Bacterial Isolates
Figure 3. Electropherogram of 16S Ribosomal RNA PCR Product of
Bacterial Isolate LB2 Identified as Lactobacillus curvatus subsp. curvatus.
Acknowledgments
This project was funded by the California State University–Agriculture
Research Initiative (CSU/ARI).
References
• Agarwal, S., et al. (2003) Institute of Food Technologists Conference.
• Chou, Y.E., et al. (2003) J. Dairy Sci. 86:2516-2524.
• Dybing, S.T., et.al. (1988) J. Dairy Sci.. 71:1701-1710.
•Marshall