Lithium Chloride-Sodium Propionate Agar for the Enumeration of Bifidobacteria in Fermented Dairy Products LUCIANE LAPIERRE, P. UNDELAND, and L. J. COX Quality and Safety Assurance Department Nestec Ltd. Nestle Research Center Vers-Chez-les-Blanc, Switzedand ABSTRACT
animals and humans. They are thought to play a major role in ControLling the pH of the large Lithium chloride-sodium propionate intestine through the production of lactic and agar has been developed for the enumeracetic acids. They m also thought to have ation of bifidobacteria in fermented dairy anticarcinogenic (7) and anticholesterolemic products. The medium contains lithium (6) properties. These organisms have recently chloride and sodium propionate to inhibit become popular in various fermented dairy the growth of other lactic acid bacteria products because of their potential health Pure cultures of bifidobacteria, lactoba(4). benefits cilli, and streptococci were tested for Neomycin-paromomycin-nalidixicacid-lithgrowth in this medium. With one exium chloride (NPNL) agar (11) is considered ception, all bifidobacteria were able to to be the reference medium for the isolation of grow in this medium and in a nonselecbifidobacteria from fermented dairy products. tive agar with a difference not exceeding Bifidobacteria grow well in this medium, and .4 log units. However, none of the lacto“ L shows good discrimination against bacilli tested and only one strain each of other organisms found in dajl products. HowStreptococcus salivanus ssp. thermophiever, it is time-consuming to prepare, because lus and L.actococcus Iactis ssp. ventoris it contains many ingredients, some of which g e w in lithium chloridesodium propiomust be filter-sterilized. nate agar. In those cases, the numbers of This study was thus undertaken to develop colonies were lower in lithium chloridea simple medium for the selective routine sodium propionate agar by 1.26 and 2.51 enumeration of bifidobacteria from fermented log units, respectively, compared with a milk products. The liver-cystine-lactose (LCL) nonselective agar. Bifidobacteria were agar of Blaurock (2) was chosen as a basis also selectively isolated from all ferbecause it contains the most important elemented milks and cheeses analyzed. ments for the growth of bifidobacteria. This (Key words: enumeration, bifidobacterLCL agar, containiig lactose as the carbohyia, fermented dairy products, lithium drate source and liver infusion and peptone as chloride-sodium propionate agar) nitrogen sources, was originally used for the Abbreviation key: LCL = livercystine isolation of bifidobacteria from infant stools. lactose, LP = lithium chloridesodium propio- However, LCL agar contains no substance innate, NPNL = neomycin-pammomycin- hibitory to other lactic acid bacteria, and it is nalidixic acid-lithium chloride, TJA = tomato therefore unsuitable for the enumeration of juice agar, TS = tryptone-salt. bifidobacteria from fermented dajr products containing lactobacilli, streptomxi, or lactococci. INTRODUCTION Bifidobacteria are anaerobic, Gram-positive bacteria that naturally inhabit the gut of
MATERIALS AND METHODS
Media Received June 28, 1991. Accepted January 14, 1992. 1992 J Dairy Sci 75:1192-1196
The LCL (2) and NPNL agars (1 l), used for the comparative enumeration of bifidobacteria
1192
1193
ENUMERATION OF BJFIDOBACTERIA
TABLE! 1. Composition of liver-lactose broth.' Composition
Manufacture?
Amomt
Liver infusion Lactose Bacto-peptone Sodium chloride Distilled water
D i f ~oM9-17-7 Merck 7657 D i f ~0118-01-8 Merck 6404
35 8 10 B 10 g 2g lo00 ml
'AU ingredients are dissolved in water, and the broth is then autoclaved. 2Manufacturers: Difco, Detroit, MI and Me&, Die&= Switzerland.
from commercial samples, were prepared according to the authors' instructions. Tomato juice agar (TJA) (Oxoid CM 113, Oxoid Ltd, Basingstoke, Hampshire, England), a nonselective medium in which bifidobacteria grow well, MRS agar (Oxoid CM 361), and M17 agar (1856-17,Difco Laboratories, Detroit, MI) with .5% lactose were prepared following the suppliers' instructions. AU media were sterilized at 121'C for 15 min.
Samples
Fermented dairy products, described as containing bifidobacteria in addition sometimes to Lactobacillrcs acidophilus, were purchased from French and Swiss supermarkets. They were stored at 5 'C prior to examination and analyzed before the end of their shelf-life. Portions of 10 g of each product were mixed with 90 ml of tryptone-salt (TS)broth (tryptone, Oxoid L42, 1 g/Lplus sodium chloride, 8.5 g/L)by gently swirling the bottles to avoid oxygen incorporation. Portions of cheeses were Llthlum Chloride-Sodlum Proplonate Agar homogenized for about 1 min in a stomacher. Mixtures of lithium chloride and sodium Further dilutions were made in tubes of TS propionate (LP)at concentrations of 1:1, 2:1, broth, and appropriate dilutions were incorpo2:2, 2:3, 2.53, 3:3, 3:6, and 5:6 g/L, respec- rated into pour plates of the appropriate media. tively, were added to LCL agar before sterilization. All ingredients of LP agar were disBacterial Strains solved in water by boiling and then were sterilized at 121'C for 15 min. The pH of the Pure strains of Bifidobucferium spp. were medium was adjusted to 6.7 f .2 with W obtained fmm the American Type Culture Collection, fmm Chr. Hansen's Laboratory (Le NaOH.
TABLE 2. W t x t of different concentrations of lithium chloride and sodium propionate on the growth of bifidobacteria from fermented dairy products.' Samoles
1:l ~~
Set yogurt A2 set yogurt g2 set yogurt ~3
B
set yogurt D~
B
E3 F3
C B A
Set yogurt Set yogurt
Set yogurt
d
A A
Lithium chloridesodium propionate 21 1:2 2:2 A B A A A A A A A A B A C B B B B A A A A
addition to her-cystine-lactose agar, g/L 23 253 3:3 3:6 A A A A A A A A A A A A A A A A C A A A A A A A A
5:6 -
-
-
'A = Only Midobacteria growing in the plates, B = a mixtare of bifidobacteria and streptococci growing in the plates, C = plates overgrown by streptococci. - = no growth. All ingredients are combined before autoclaving. products from France. 3prodaas from Switzerland.
Journal of Dairy Science Vol. 75, No. 5, 1992
1194
LAPIERRE ET AI..
TABLE 3. Composition of lithium chloridesodium propionate
(LP) agar.'
Amount
Conmosition
Difm 0269-17-7 Muck 7657 Difm 0118-01-8 MadE 6404 Mack 5679 Plulca 81962
Liver infosion
Lactose Bacto-peptone Sodium chloride Lithium chloride Sodium propionate Agar nurnber 1 Distilled water ~~~~
Oxoid L11 ~
'AU
~
~
~
~
~
~
ingredients of the mcdium art dissolved in distilled water, and the pH is adjusted to 6.7 f 2 with 1N NaOH; the
wata is then boiled before autoclaving. 2Manufacturers: Difm, Detroit. h4t Mack,Dietikon, Switzerland; Pluka, Buchs, Swtizerland; and Oxoid, Basingstoke, Hampshire, Eogland.
Moulin d' Aulnay, 9 1292 Arpajon, France), and tococci) for 48 h, and plates of NPNL agar from the Nestec collection. They belonged to were incubated anaerobically at 37'C for 72 h. the species Bifihbacterium bifidum, Bifidobacteriwn infantis, and Bifidobacteriwn RESULTS AND DISCUSSION longum. Lactobacilli were obtained from the Lithium chloride is a substance commonly American Type Culture Collection, from the National Collection of Food Bacteria, from used for the selective isoIation of bifidobacChr. Hansen's, and from Nestec. They be- teria (8, 10, 11). Although lithium chloride is longed to the species Lactobacillus delbrueckii commonly used in microbiology, its mechassp. bulgaricus and L. acidophilus. The cocci nism of action on bacterial cells is still poorly used were obtained from our own culture col- understood. The main effects of lithium chlolection and belonged to the species Streptococcus salivarim ssp. themphilus and Lactococcus lactis ssp. cremoris. Inocula
Strains of bifidobacteria were subcultured in liver-lactose broth (Table l), which is derived from Lp agar in which all strains of bifidobacteria grow well, and strains of lactic acid bacteria were subcultured in skim milk with .l% of yeast extract. Bifidobacteria and lactobacilli were incubated anaerobically (GasPack Plus, BBL, Cockeysville, MD) at 37'C for 24 h, streptococci were incubated aerobically at 37'C for about 4 h, and lactococci wen incubated overnight at 30'C. Further dilutions were made in TS broth, and appropriate dilutions were counted by the pour-plate method in petri dishes. Incubation Condltlons
Plates of LP agar, LCL agar, and TJA were incubated anaerobically at W C for 48 h. Plates of MRS agar were incubated anaerobically at 37'C (streptococci) and at 30'C (lacJournal of Dairy Science Vol. 75, No. 5, 1992
TABLE 4. Emuneration of bifidobacteria from some mmmercial products in lithium chloride-sodium propionate (LP) agar and neomycin-pmmomycin-aalidixic acidlithium chloride ("L)agar.
Smles
Lp
"L'
set yogurt ~2
7.54 6.11 6.93 7.26 5.08 5.65 7.83 7.63 7.40 6.40 6.94 7.04
7.63 5.76 6.43 6.60 6.75
Set yogurt B~ Set yogurt cj set yogurt d
E3 set Yogurt F3 Set yogurt
Set Yogrtrt Set yogmt H2 set yogurt 12
Stimd yogurt J3 Stimd yo$" K2 cheese L
-oog W g ) 5.46
7.61 7.04 6.57 6.68 6.68 6.28
'AU ingredients of tlae "L agar are dissolved in distilled water, and the pH is adjusted to 7.2 with 1N NaOH; the water is then boiled before autoclaving. *~odnasfrom prance. 3~oductsfrom ~ w i t z a ~ d .
1195
J3WMERATION OF BTPIDOBACCERU
TABLE 5. Enmtzation of bifidobacleria and lactic acid bacteria m lithiam chloridesodiumpropionate (Le) agar and in optimal growU~media (control).
0%
-
du/g) TJA~ 8.32 6.5 1 8.72 9.36 9.23 7.62 8.63 923 8.95 9.32 MRS 8.30 7.% 8.26 8.78 8.98 8.41 8.00 8.43 7.48
Bifidobactenum bificim Bijidoimcterium breve B$dobacteriurn infaris B. infontis Bijidobacterium longwn B. longum B. longum B. longum B. longum Bijidobncterium sp.
11863 15700 15697 Bil 15707 15708 B118 B 120 B 122 Bb12
ATCC ATCC ATCC NESTEC ATCC ATCC NESTEC NESTEC NESTEC Hansel3
Lactobacillus acidophilus L. acidophilus L. acidophilus L. aclzophilw L. acidophilus L. acidophilus L. acidophilus L. acidophilus L. acidophilus Lactobncillus delbrueckii ssp. bulgaricus L. & l h k i i ssp. bulgaricus L. delbruekii ssp. bulgaricus Streptococcus salivarius ssp. thmnophilus S. salivarius ssp. thennophilw S. saliva& ssp. thennophilus S. salivarius ssp. thennophilus Lilctocmcus lactis ssp. cremonk Lc. lactis ssp. cremoris Lc. lactis SSD. cremoris
521 4356
ATCC ATCC ATCC NCPB NCFB Hansen NESTEC NESTEC NESTEC
animals and humans. They are thought to play a major role in ControLling the pH of the large Lithium chloride-sodium propionate intestine through the production of lactic and agar has been developed for the enumeracetic acids. They m also thought to have ation of bifidobacteria in fermented dairy anticarcinogenic (7) and anticholesterolemic products. The medium contains lithium (6) properties. These organisms have recently chloride and sodium propionate to inhibit become popular in various fermented dairy the growth of other lactic acid bacteria products because of their potential health Pure cultures of bifidobacteria, lactoba(4). benefits cilli, and streptococci were tested for Neomycin-paromomycin-nalidixicacid-lithgrowth in this medium. With one exium chloride (NPNL) agar (11) is considered ception, all bifidobacteria were able to to be the reference medium for the isolation of grow in this medium and in a nonselecbifidobacteria from fermented dairy products. tive agar with a difference not exceeding Bifidobacteria grow well in this medium, and .4 log units. However, none of the lacto“ L shows good discrimination against bacilli tested and only one strain each of other organisms found in dajl products. HowStreptococcus salivanus ssp. thermophiever, it is time-consuming to prepare, because lus and L.actococcus Iactis ssp. ventoris it contains many ingredients, some of which g e w in lithium chloridesodium propiomust be filter-sterilized. nate agar. In those cases, the numbers of This study was thus undertaken to develop colonies were lower in lithium chloridea simple medium for the selective routine sodium propionate agar by 1.26 and 2.51 enumeration of bifidobacteria from fermented log units, respectively, compared with a milk products. The liver-cystine-lactose (LCL) nonselective agar. Bifidobacteria were agar of Blaurock (2) was chosen as a basis also selectively isolated from all ferbecause it contains the most important elemented milks and cheeses analyzed. ments for the growth of bifidobacteria. This (Key words: enumeration, bifidobacterLCL agar, containiig lactose as the carbohyia, fermented dairy products, lithium drate source and liver infusion and peptone as chloride-sodium propionate agar) nitrogen sources, was originally used for the Abbreviation key: LCL = livercystine isolation of bifidobacteria from infant stools. lactose, LP = lithium chloridesodium propio- However, LCL agar contains no substance innate, NPNL = neomycin-pammomycin- hibitory to other lactic acid bacteria, and it is nalidixic acid-lithium chloride, TJA = tomato therefore unsuitable for the enumeration of juice agar, TS = tryptone-salt. bifidobacteria from fermented dajr products containing lactobacilli, streptomxi, or lactococci. INTRODUCTION Bifidobacteria are anaerobic, Gram-positive bacteria that naturally inhabit the gut of
MATERIALS AND METHODS
Media Received June 28, 1991. Accepted January 14, 1992. 1992 J Dairy Sci 75:1192-1196
The LCL (2) and NPNL agars (1 l), used for the comparative enumeration of bifidobacteria
1192
1193
ENUMERATION OF BJFIDOBACTERIA
TABLE! 1. Composition of liver-lactose broth.' Composition
Manufacture?
Amomt
Liver infusion Lactose Bacto-peptone Sodium chloride Distilled water
D i f ~oM9-17-7 Merck 7657 D i f ~0118-01-8 Merck 6404
35 8 10 B 10 g 2g lo00 ml
'AU ingredients are dissolved in water, and the broth is then autoclaved. 2Manufacturers: Difco, Detroit, MI and Me&, Die&= Switzerland.
from commercial samples, were prepared according to the authors' instructions. Tomato juice agar (TJA) (Oxoid CM 113, Oxoid Ltd, Basingstoke, Hampshire, England), a nonselective medium in which bifidobacteria grow well, MRS agar (Oxoid CM 361), and M17 agar (1856-17,Difco Laboratories, Detroit, MI) with .5% lactose were prepared following the suppliers' instructions. AU media were sterilized at 121'C for 15 min.
Samples
Fermented dairy products, described as containing bifidobacteria in addition sometimes to Lactobacillrcs acidophilus, were purchased from French and Swiss supermarkets. They were stored at 5 'C prior to examination and analyzed before the end of their shelf-life. Portions of 10 g of each product were mixed with 90 ml of tryptone-salt (TS)broth (tryptone, Oxoid L42, 1 g/Lplus sodium chloride, 8.5 g/L)by gently swirling the bottles to avoid oxygen incorporation. Portions of cheeses were Llthlum Chloride-Sodlum Proplonate Agar homogenized for about 1 min in a stomacher. Mixtures of lithium chloride and sodium Further dilutions were made in tubes of TS propionate (LP)at concentrations of 1:1, 2:1, broth, and appropriate dilutions were incorpo2:2, 2:3, 2.53, 3:3, 3:6, and 5:6 g/L, respec- rated into pour plates of the appropriate media. tively, were added to LCL agar before sterilization. All ingredients of LP agar were disBacterial Strains solved in water by boiling and then were sterilized at 121'C for 15 min. The pH of the Pure strains of Bifidobucferium spp. were medium was adjusted to 6.7 f .2 with W obtained fmm the American Type Culture Collection, fmm Chr. Hansen's Laboratory (Le NaOH.
TABLE 2. W t x t of different concentrations of lithium chloride and sodium propionate on the growth of bifidobacteria from fermented dairy products.' Samoles
1:l ~~
Set yogurt A2 set yogurt g2 set yogurt ~3
B
set yogurt D~
B
E3 F3
C B A
Set yogurt Set yogurt
Set yogurt
d
A A
Lithium chloridesodium propionate 21 1:2 2:2 A B A A A A A A A A B A C B B B B A A A A
addition to her-cystine-lactose agar, g/L 23 253 3:3 3:6 A A A A A A A A A A A A A A A A C A A A A A A A A
5:6 -
-
-
'A = Only Midobacteria growing in the plates, B = a mixtare of bifidobacteria and streptococci growing in the plates, C = plates overgrown by streptococci. - = no growth. All ingredients are combined before autoclaving. products from France. 3prodaas from Switzerland.
Journal of Dairy Science Vol. 75, No. 5, 1992
1194
LAPIERRE ET AI..
TABLE 3. Composition of lithium chloridesodium propionate
(LP) agar.'
Amount
Conmosition
Difm 0269-17-7 Muck 7657 Difm 0118-01-8 MadE 6404 Mack 5679 Plulca 81962
Liver infosion
Lactose Bacto-peptone Sodium chloride Lithium chloride Sodium propionate Agar nurnber 1 Distilled water ~~~~
Oxoid L11 ~
'AU
~
~
~
~
~
~
ingredients of the mcdium art dissolved in distilled water, and the pH is adjusted to 6.7 f 2 with 1N NaOH; the
wata is then boiled before autoclaving. 2Manufacturers: Difm, Detroit. h4t Mack,Dietikon, Switzerland; Pluka, Buchs, Swtizerland; and Oxoid, Basingstoke, Hampshire, Eogland.
Moulin d' Aulnay, 9 1292 Arpajon, France), and tococci) for 48 h, and plates of NPNL agar from the Nestec collection. They belonged to were incubated anaerobically at 37'C for 72 h. the species Bifihbacterium bifidum, Bifidobacteriwn infantis, and Bifidobacteriwn RESULTS AND DISCUSSION longum. Lactobacilli were obtained from the Lithium chloride is a substance commonly American Type Culture Collection, from the National Collection of Food Bacteria, from used for the selective isoIation of bifidobacChr. Hansen's, and from Nestec. They be- teria (8, 10, 11). Although lithium chloride is longed to the species Lactobacillus delbrueckii commonly used in microbiology, its mechassp. bulgaricus and L. acidophilus. The cocci nism of action on bacterial cells is still poorly used were obtained from our own culture col- understood. The main effects of lithium chlolection and belonged to the species Streptococcus salivarim ssp. themphilus and Lactococcus lactis ssp. cremoris. Inocula
Strains of bifidobacteria were subcultured in liver-lactose broth (Table l), which is derived from Lp agar in which all strains of bifidobacteria grow well, and strains of lactic acid bacteria were subcultured in skim milk with .l% of yeast extract. Bifidobacteria and lactobacilli were incubated anaerobically (GasPack Plus, BBL, Cockeysville, MD) at 37'C for 24 h, streptococci were incubated aerobically at 37'C for about 4 h, and lactococci wen incubated overnight at 30'C. Further dilutions were made in TS broth, and appropriate dilutions were counted by the pour-plate method in petri dishes. Incubation Condltlons
Plates of LP agar, LCL agar, and TJA were incubated anaerobically at W C for 48 h. Plates of MRS agar were incubated anaerobically at 37'C (streptococci) and at 30'C (lacJournal of Dairy Science Vol. 75, No. 5, 1992
TABLE 4. Emuneration of bifidobacteria from some mmmercial products in lithium chloride-sodium propionate (LP) agar and neomycin-pmmomycin-aalidixic acidlithium chloride ("L)agar.
Smles
Lp
"L'
set yogurt ~2
7.54 6.11 6.93 7.26 5.08 5.65 7.83 7.63 7.40 6.40 6.94 7.04
7.63 5.76 6.43 6.60 6.75
Set yogurt B~ Set yogurt cj set yogurt d
E3 set Yogurt F3 Set yogurt
Set Yogrtrt Set yogmt H2 set yogurt 12
Stimd yogurt J3 Stimd yo$" K2 cheese L
-oog W g ) 5.46
7.61 7.04 6.57 6.68 6.68 6.28
'AU ingredients of tlae "L agar are dissolved in distilled water, and the pH is adjusted to 7.2 with 1N NaOH; the water is then boiled before autoclaving. *~odnasfrom prance. 3~oductsfrom ~ w i t z a ~ d .
1195
J3WMERATION OF BTPIDOBACCERU
TABLE 5. Enmtzation of bifidobacleria and lactic acid bacteria m lithiam chloridesodiumpropionate (Le) agar and in optimal growU~media (control).
0%
-
du/g) TJA~ 8.32 6.5 1 8.72 9.36 9.23 7.62 8.63 923 8.95 9.32 MRS 8.30 7.% 8.26 8.78 8.98 8.41 8.00 8.43 7.48
Bifidobactenum bificim Bijidoimcterium breve B$dobacteriurn infaris B. infontis Bijidobacterium longwn B. longum B. longum B. longum B. longum Bijidobncterium sp.
11863 15700 15697 Bil 15707 15708 B118 B 120 B 122 Bb12
ATCC ATCC ATCC NESTEC ATCC ATCC NESTEC NESTEC NESTEC Hansel3
Lactobacillus acidophilus L. acidophilus L. acidophilus L. aclzophilw L. acidophilus L. acidophilus L. acidophilus L. acidophilus L. acidophilus Lactobncillus delbrueckii ssp. bulgaricus L. & l h k i i ssp. bulgaricus L. delbruekii ssp. bulgaricus Streptococcus salivarius ssp. thmnophilus S. salivarius ssp. thennophilw S. saliva& ssp. thennophilus S. salivarius ssp. thennophilus Lilctocmcus lactis ssp. cremonk Lc. lactis ssp. cremoris Lc. lactis SSD. cremoris
521 4356
ATCC ATCC ATCC NCPB NCFB Hansen NESTEC NESTEC NESTEC
