An Examination of the Bacterial Population and Their Lactate Dehydrogenase Activities in Cheddar...
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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 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 mold-like 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, CO 2 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 casein-bound calcium was the major source of calcium for crystal formation. They also noted that crystal formation was enhanced when CO 2 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 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, R-Biopharm AG, Germany) P-029 Table 3. Mean DL-lactic acid (g/L) of identified bacterial isolates from a variety of cheeses with the calcium lactate crystal defect. Bacterial Isolates 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 Table 2. Identity of bacterial isolates from a variety of cheeses with and without the calcium lactate crystal defect. Bacterial Isolate Number Identified Cheeses with CLC Defect Lactobacillus curvatus subsp. curvatus 40 Yes Lactobacillus casei 12/13 Yes/No Lactobacillus paracasei 5/7 Yes/No Lactobacillus coryniformis 3 Yes Table 1. Universal primers for PCR used for 16S ribosomal RNA BLAST species identification. UF2 5’-AGAGTTTGATCCTGGCTCAG-3’ UR523 5’-GCTGGCACGTAGTTAGCC-3’ 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. 1 CGCAGCAACG CTGGCGGCGT GCCTAATACA TGCAAGTCGA ACGCACTNTC 51 GTTAGATTGA AGAAGCTTGC TTCTGATTGA TAACATTTGA GTGAGTGGCG 101 GACGGGTGAG TAACACGTGG GTAACCTGCC CTAAAGTGGG GGATAACATT 151 TGGAAACAGA TGCTAATACC GCATAAAACC TAGCACCGCA TGGTGCAAGG 201 TTGAAAGATG GTTTCGGCTA TCACTTTAGG ATGGACCCGC GGTGCATTAG 251 TTAGTTGGTG AGGTAAAGGC TCACCAAGAC CGTGATGCAT AGCCGACCTG 301 AGAGGGTAAT CGGCCACACT GGGACTGAGA CACGGCCCAG ACTCCTACGG 351 GAGGCAGCAG TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA 401 CGCCGCGTGA GTGAAGAAGG TTTTCGGATC GTAAAACTCT GTTGTTGGAG 451 AAGAACGTAT TTGATAGTAA CTGATCAGGT AGTGACGGTA TCCAACCAGA 501 AAGCCACGGC Figure 2. Sequence of 16S Ribosomal RNA PCR Product of Bacterial Isolate LB2 Identified as Lactobacillus curvatus subsp. curvatus. Conclusions 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. Figure 3. Electropherogram of 16S Ribosomal RNA PCR Product of Bacterial Isolate LB2 Identified as Lactobacillus curvatus subsp. curvatus. Figure 1. Cheddar cheese (6 months old) with calcium lactate crystal defect. 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
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Transcript of An Examination of the Bacterial Population and Their Lactate Dehydrogenase Activities in Cheddar...
An Examination of the Bacterial Population and Their Lactate Dehydrogenase Activities in Cheddar Cheese with Calcium Lactate Crystal
DefectBy: 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
AbstractCalcium 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.
IntroductionThe 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 mold-like 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 casein-bound 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, R-Biopharm AG, Germany)
P-029
Table 3. Mean DL-lactic acid (g/L) of identified bacterial isolates from a variety of
cheeses with the calcium lactate crystal defect.
Bacterial IsolatesMean 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
Table 2. Identity of bacterial isolates from a variety of cheeses with and without the
calcium lactate crystal defect.
Bacterial IsolateNumber
IdentifiedCheeses with CLC Defect
Lactobacillus curvatus subsp. curvatus
40 Yes
Lactobacillus casei 12/13 Yes/No
Lactobacillus paracasei 5/7 Yes/No
Lactobacillus coryniformis 3 Yes
Table 1. Universal primers for PCR used for 16S ribosomal RNA
BLAST species identification.
UF2 5’-AGAGTTTGATCCTGGCTCAG-3’
UR523 5’-GCTGGCACGTAGTTAGCC-3’
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.
1 CGCAGCAACG CTGGCGGCGT GCCTAATACA TGCAAGTCGA ACGCACTNTC51 GTTAGATTGA AGAAGCTTGC TTCTGATTGA TAACATTTGA GTGAGTGGCG
101 GACGGGTGAG TAACACGTGG GTAACCTGCC CTAAAGTGGG GGATAACATT
151 TGGAAACAGA TGCTAATACC GCATAAAACC TAGCACCGCA TGGTGCAAGG
201 TTGAAAGATG GTTTCGGCTA TCACTTTAGG ATGGACCCGC GGTGCATTAG
251 TTAGTTGGTG AGGTAAAGGC TCACCAAGAC CGTGATGCAT AGCCGACCTG
301 AGAGGGTAAT CGGCCACACT GGGACTGAGA CACGGCCCAG ACTCCTACGG
351 GAGGCAGCAG TAGGGAATCT TCCACAATGG ACGAAAGTCT GATGGAGCAA
401 CGCCGCGTGA GTGAAGAAGG TTTTCGGATC GTAAAACTCT GTTGTTGGAG
451 AAGAACGTAT TTGATAGTAA CTGATCAGGT AGTGACGGTA TCCAACCAGA
501 AAGCCACGGC
Figure 2. Sequence of 16S Ribosomal RNA PCR Product of Bacterial Isolate LB2 Identified as Lactobacillus curvatus subsp. curvatus.
ConclusionsThe 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.
Figure 3. Electropherogram of 16S Ribosomal RNA PCR Product of Bacterial Isolate LB2 Identified as Lactobacillus curvatus subsp. curvatus.
Figure 1. Cheddar cheese (6 months old) with calcium lactate crystal defect.
AcknowledgmentsThis 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
