Bacterial Cultures were obtained from Microbiologics in the form of LYFO DISK (lyophilized bacteria pellet). The individual cultures utilized in all experiments were as follows: Lactobacillus plantarum (ATCC 8014), Lactobacillus rhamnosus (ATCC 7469), Lactobacillus casei (ATCC 334) and Streptococcus salivarius subsp. thermophilus (ATCC 19258). The Lactobacillus species were grown and maintained on MRS agar or in MRS Broth (Difco) and S. thermophilus was grown and maintained on Brain Heart Infusion (BHI) agar or broth (Difco) at 36°C and under 5% CO2. All manipulations of bacteria were performed in a biological safety cabinet to limit contamination.
Lactobacillus casei
Manufactured by American Type Culture Collection
Lactobacillus casei is a type of bacteria that is commonly used in laboratory settings. It is a gram-positive, rod-shaped bacterium that is known for its ability to ferment carbohydrates and produce lactic acid. Lactobacillus casei is often used in research and testing applications, but its core function is not elaborated upon further in this description.
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Lab products found in correlation
Lactobacillus plantarum, by American Type Culture Collection (2 mentions)
Lactobacillus rhamnosus, by American Type Culture Collection (2 mentions)
Enterococcus faecalis, by American Type Culture Collection (2 mentions)
Bacitracin, by Merck Group (1 mentions)
Streptococcus mutans, by American Type Culture Collection (1 mentions)
Fusobacterium nucleatum, by American Type Culture Collection (1 mentions)
Lactobacillus gasseri, by American Type Culture Collection (1 mentions)
Lactobacillus fermentum, by American Type Culture Collection (1 mentions)
Lactobacillus helveticus, by American Type Culture Collection (1 mentions)
Lactobacillus reuteri, by American Type Culture Collection (1 mentions)
Mrs broth, by BD (1 mentions)
Lactobacillus strains, by American Type Culture Collection (1 mentions)
S mitis, by American Type Culture Collection (1 mentions)
S sobrinus, by American Type Culture Collection (1 mentions)
S mutans, by American Type Culture Collection (1 mentions)
S sanguinis, by American Type Culture Collection (1 mentions)
S salivarius, by American Type Culture Collection (1 mentions)
Bacteroides fragilis, by American Type Culture Collection (1 mentions)
6 protocols using lactobacillus casei
1
Cultivation of Lactobacillus and Streptococcus Strains
2
Comprehensive Vitamin Biomarker Quantification
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The concentrations of total vitamin B1 in whole blood and urine were measured by the high-performance liquid chromatography (HPLC)-postlabeled fluorescence method of Kimura et al.19 (link) The concentration of total vitamin B2 in whole blood was determined by the HPLC–lumiflavin method of Ohkawa et al,20 with slight modifications. The concentration of vitamin B2 in urinary excretion was analyzed according to the method of Ohkawa et al.21 (link) Pyridoxal phosphate (a coenzyme of vitamin B6) in plasma was determined using the HPLC method.22 4-PIC, a catabolite of vitamin B6, was measured in urine by the HPLC method.23 (link) Concentrations of vitamin B12 in plasma and urine were assayed by the microbiological method with Lactobacillus delbrueckii subsp. lactis ATCC 7870.24 The total nicotinamide content in whole blood was measured by the method of Shibata et al.25 (link) The quantities of Nam, 2-Py, and 4-Py in urine were measured simultaneously by the HPLC method of Shibata et al.26 (link) The content of MNA was measured by the method of Shibata.27 Plasma and urinary folates were determined by the microbioassay method using Lactobacillus casei ATCC 2733.28 (link) Plasma and urine contents of reduced and oxidized ascorbic acid and 2,3-diketogluconic acid were determined by the HPLC method.29
3
Cultivation of Oral Bacterial Strains
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The following standard strains were used: Enterococcus faecalis (ATCC 51299), Actinomyces israelii (ATCC 12102), Lactobacillus casei (IAL#523), Streptococcus mutans (ATCC 25175), and Fusobacterium nucleatum (NCTC 11326) provided by Oswaldo Cruz Foundation—FIOCRUZ, Rio de Janeiro, Brazil. Bacterial suspensions were prepared as described by Caiaffa et al. [29 (link)] from cultures grown in Brain Heart infusion Agar—BHIA (Difco Laboratories, Kansas City, MO, USA) for E. faecalis and A. israelii, MRS Rogosa (Difco) for L. casei, Mitis Salivarius Agar (Difco) with 0.2 U/mL bacitracin (Sigma-Aldrich) for S. mutans and BHI blood agar (Difco) containing 5 mg/mL of hemin, 5 mg/mL of menadione, 0.5% yeast extract powder (YE, Difco), and 5% defibrinated sheep blood for F. nucleatum. All bacteria were grown in an incubator at 37 °C and 5% CO2, except F. nucleatum which grown in an anaerobic chamber (AnaeroGen, Oxoid, Thermo Scientific, Waltham, MA, USA).
4
Lactobacillus Strains Preparation Protocol
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The PP mixture comprised various Lactobacillus strains supplied by the American Type Culture Collection (ATCC) (Manassas, VA, USA). The strains included Lactobacillus gasseri (ATCC 33323), Lactobacillus plantarum (ATCC BAA-793), Lactobacillus reuteri (ATCC 23272), Lactobacillus helveticus (ATCC BAA-2840), Lactobacillus fermentum (ATCC 23271), Lactobacillus rhamnosus (ATCC BAA-2836), and Lactobacillus casei (ATCC BAA-2843). Following the supplier’s protocol, we cultured the strains in MRS broth (Beckton Dickinson, Sparks, MD), and preserved them as glycerol stocks at −80°C. To initiate culture growth, we prepared 5 mL of pre-warmed MRS broth with the glycerol stocks and then incubated these starter cultures at 37°C in a 5% CO2 environment for 2 h. The bacteria were cultured overnight in 1 L of MRS broth under identical conditions until they reached the logarithmic growth phase, confirmed by measuring optical density at 600 nm (OD600). After growth, the bacterial cells were collected through several centrifugation steps at 3000×g and 4°C, followed by a wash in cold PBS. We then resuspended the bacterial pellets in 10% glycerol in PBS and promptly froze them in 1 mL aliquots for future administration to mice. The viability and concentration of the bacteria were verified by serial dilution and colony-forming unit (CFU) enumeration on agar plates.
5
Antibacterial Potential of Resin Compounds
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Authentic P. elliottii (batch number 21811-09) and P. tropicalis (batch number 20511-09) resin samples were obtained from the Brazilian Association of Resinators (ARESB), located in the city of Avaré – SP, Brazil. DHA was isolated from the P. elliottii resin and was obtained by the method previously described by Leandro et al. (2014) (link) (Supplementary Figures S1 , S2 ).
The following bacteria from the American Type Culture Collection (ATCC) collection were used to conduct the antibacterial assays: S. mutans (ATCC 25175), S. mitis (ATCC 49456), S. sanguinis (ATCC 10556), S. sobrinus (ATCC 33478), S. salivarius (ATCC 25975), Lactobacillus casei (ATCC 11578), and Enterococcus faecalis (ATCC 4082). These microorganisms were maintained in a freezer at −80°C in 20% glycerol solution in the Laboratory of Applied Microbiology Research (LaPeMA) – University of Franca (Unifran).
The following bacteria from the American Type Culture Collection (ATCC) collection were used to conduct the antibacterial assays: S. mutans (ATCC 25175), S. mitis (ATCC 49456), S. sanguinis (ATCC 10556), S. sobrinus (ATCC 33478), S. salivarius (ATCC 25975), Lactobacillus casei (ATCC 11578), and Enterococcus faecalis (ATCC 4082). These microorganisms were maintained in a freezer at −80°C in 20% glycerol solution in the Laboratory of Applied Microbiology Research (LaPeMA) – University of Franca (Unifran).
6
Microbial Strain Acquisition for Research
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Blautia coccoides (JCM 1395) was provided by Japan Collection of Microorganisms, RIKEN BRC, which is participating in the National BioResource Project of the MEXT, Japan. Clostridium leptum (ATCC 29065), Bacteroides fragilis (ATCC 25285), Bifidobacterium pseudocatenulatum (ATCC 27919), Atopobium parvulum (ATCC 33793), and Lactobacillus casei (ATCC 334) were purchased from ATCC. Escherichia coli (W3110) was kindly provided by Dr. Akira Okamoto at the Aichi University of Education.
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