Got Bugs in Your Kefir?

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got bugs in your kefir? JULIENNE HENRIE INTRODUCTION undergraduate researcher What is Kefir ? RESULTS [email protected] Kefir is a type of fermented milk that is cultured with Kefir grains. blast kefir brand probiotics listed on label probiotics detected our cultures “CAN ACTIVE PROBIOTICS BE EXTRACTED FROM KEFIR Kefir grains make up the probiotic culture used to produce Kefir. e-values These cauliflower-like grains contain mostly lactobacilli bacteria PRODUCTS FOUND AT SUPERMARKETS?” and yeasts held together by Kefiran, an exopolysaccharide structure. (Korsak et al. 2014). Kefir grains are Mesophilic. Mesophilic star- • Streptococcus thermophilus ters are cultured at room temperature, which allows the probiotics • Bifidobacterium animalis BB-12 • Lactobacillus acidophilus 2e-73 “WHICH LACTOBACILLI SPECIES IS DOMINANT FOR • Lactobacillus acidophilus LA-5 (Dominant Species) to remain active when consumed. Properties of mesophilic starters • Lactobacillus paracasei • Streptococcus anginosus 2e-73 also give Kefir the liquid consistency it is known for. (Sarah, 2016). • Propionibacterium freudenreichii EACH KEFIR PRODUCT TESTED?” Kefir originated in the Caucasus Mountains thousands of years ago, Dahlicious Lassi MRS MRS the name coming from the Turkish work Keyif meaning “good fee- (Sample #1) Dilute ling’. Kefir is well known for its health benefits, which can be attri- DISCUSSION • Bifidobacterium lactis Hypothesis # 1 and Hypothesis # 2 cannot be rejected. All seven buted to its probiotics. Probiotics are foods that contain beneficial • S. thermophilus, bacteria and yeasts for the human body. (Otles & Cagindi, 2003). • L. casei Kefir products do contain viable probiotics which can be extracted • L. rhamnosus • L. acidophilus • Lactobacillus Fermentum 8e-73 and cultured successfully. Also, a successful barcode can be obtained • L. delbrueckll subsp. lactis (Dominant Species) Kefir as a product • L. delbrueckll subsp. bulcaricus • Lactobacillus brevis 5e-70 from extracting DNA from the dominant Lactobacillus species of • Lactococcus lactis subsp. cremoris • Lactobacillus ingluviei 5e-70 Rising awareness in American culture for maintaining good health • Lactococcus lactis subsp. lactis each brand. The lowest e-value found on the BLAST database deter- • 5e-70 • Lactococcus lactis subsp. lacctis biovar Lactobacillus plantarum is now driving up the demand for the Probiotic Food and Beve- diacetylactis mined the dominant Lactobacillus species of each brand. This study rages Market. (“Probiotics Market Analysis”, 2015). In a highly com- Green Valley Organics • Leuconostoc mesenteroides subsp. MRS MRS cremoris Dilute found that the majority of Kefir brands, Green Valley Organics, Helios petitive consumer Probiotic Food and Beverages Market, produ- (Sample #2) Greek, Lifeway, Redwood Hill Farm, and Wallaby, presented with a cers are constantly challenged to keep the quality of their product • B. lactis dominant Lactobacillus species that was not found on the product’s high while maintaining an affordable price for their product. In the • Lb. lactis • Lactobacillus Zeae 1e-75 United States Kefir is marketed as a yogurt-like probiotic beverage. • S. florentinus (Dominant Species) label. The Kefir brands, Dahlicious Lassi, and Maple Hill were the only • S. diacetylactis • Lactobacillus casei 1e-75 • Lb. acidophilus • Lactobacillus paracasei 5e-75 Therefore, the quality of Kefir can be determined by the viability of • B. bacterium longum Kefir brands to advertise the dominant species of lactobacilli on their • Lactobacillus paracasei 5e-75 its probiotics and how effective they are at delivering health bene- • Lb. casei product’s label. Confusion in the taxonomic name of the probiotics • Lb. reuteri SUBSP. paracasei fits to the human body. (Kaufman, 2015). This study aims to disco- • Lb.plantarum • Lactobacillus paracasei 5e-75 could be the result of mislabeling. Lactobacillus zeae was previously ver if Kefir products, bought at a supermarket, can produce enough • Lb. rhamnosus SUBP. tolerans • B. bacterium breve • Lactobacillus rhamnosus 5e-75 MRS MRS classified as Lactobacillus rhamnosis or Lactobacillus casei. (Dicks et Helios Greek • Leuconostoc cremoris viable probiotics to be extracted and cultured. If successful, can a (Sample #3) Dilute dominant lactobacilli species be determined for each Kefir product al.,1996). Companies could still be using the old taxonomic names tested, using standard barcoding methods. • B. lactis for the labeling of their probiotics for marketing purposes. A phy- • Lb. lactis • Lactobacillus Zeae 1e-75 logenetic tree of the dominant species along with similar Lactoba- • S. florentinus (Dominant Species) • S. diacetylactis • Lactobacillus casei 1e-75 cillus species shows the similarity in ancestry. Contamination of the • Lb. acidophilus • Lactobacillus paracasei 5e-75 • B. bacterium longum • Lactobacillus paracasei 5e-75 Kefir grains by the dominant lactobacillus species could also lead to HYPOTHESIS • Lb. casei • Lb. reuteri SUBSP. paracasei mislabeling of Kefir products. This study only targeted lactobacilli • Lb.plantarum • Lactobacillus paracasei 5e-75 Hypotheses # 1 • Lb. rhamnosus SUBP. tolerans species of bacteria and favored aerobic conditions of growth the- • B. bacterium breve • Lactobacillus rhamnosus 5e-75 MRS MRS Lifeway • Leuconostoc cremoris If seven selected Kefir products are advertised to contain health be- (Sample #4) Dilute refore a bias exists showing only the dominant Lactobacilli species neficial probiotics, then these probiotics can be extracted from the in the Kefir product.

Kefir products and cultured onto agar plates containing an MRS • Streptococcus thermophiles medium. • L. delbrueckii Subsp. bulgaricus • Lactobacillus Casei 2e-69 • L. rhamnosus (Dominant Species) • L. casei • Lactobacillus rhamnosus 2e-69 FUTURE STUDIES • L. lactis subsp. lactis • Lactobacillus zeae 6e-69 Psychobiotics studies the ability of probiotics to produce and deliver Hypotheses # 2 • L. lactis subsp. lactis biovar. • Lactobacillus paracasei 8e-68 diacetylactis SUBSP. paracasei If probiotics from seven selected Kefir brands are successfully • Leuconostoc cremoris neuroactive substances, such as GABA, to the human body. (Dinan • 8e-68 • Bifidobacterium lactis Lactobacillus paracasei cultured onto plates containing an MRS medium, then the domi- • L. lactis subsp. cremoris SUBP. tolerans et al., 2013). GABA is an important inhibitory neurotransmitter in the MRS MRS nant lactobacilli species of each brand of Kefir can be determined Maple Hill • L. acidophilus mammalian central nervous system. The metabolic process of GABA (Sample #5) Dilute using standard barcoding methods. produces energy for the brain. GABA also controls, “the regulation of • Bifidobacterium lactis • S. thermophilus • Lactobacillus Delbrueckii 5e-75 sleep-awake cycle, motor activity, vascular tone, as well as in main- • L. casei SUBSP. Indicus • L. rhamnosus (Dominant Species) taining a high seizure threshold, memory formation and cognition”. • L. acidophilus • Lactobacillus delbrueckii 5e-75 • L. delbrueckll Subsp. lactis (Yunes et al.,2016). Lactobacillus and Bifidobacterium are both being METHODS 5e-75 • L. delbrueckll Subsp. bulgaricus • Lactobacillus delbrueckii • Lactococcus lactis Subsp. cremoris SUBSP. bulgaricus studied for their efficiency at producing GABA. (Yunes et al., 2016). Experimental Design • Lactococcus lactis Subsp. lactis • Lactobacillus delbrueckii 5e-75 • Lactococcus lactis Subsp. lactis biovar SUBSP. lactis Both probiotics can be found in Kefir. If human derived Lactobacillus The following brands of Kefir were chosen: Dahlicious Lassi, Green Diacetylactis • Lactobacillus delbrueckii 5e-75 and Bifidobacterium strains are found to produce GABA then Kefir Redwood Hill Farm • Leuconostoc mesenteroides Subp. SUBSP. sunkii MRS MRS Valley Organics, Helios Organic Greek, Wallaby, Maple Hill Creame- cremoris (Sample #6) Dilute producers can improve the quality of their Kefir by adding these ry, Redwood Hill Farm, and Lifeway. These products were chosen • Streptococcus thermophiles strains of probiotics to their products. based on the availability of Kefir brands at local supermarkets. • Propionibacterium freudenreichii subsp. Shermanii • Lactobacillus delbrueckii subsp. Protocols Bulgaricus • Lactobacillus Zeae 3e-77 • Lactobacillus casei (Dominant Species) REFERENCES • Lactobacillus acidophilus Samples were obtained from all seven brands of Kefir. Samples • Lactobacillus casei 1e-75 Dicks, L. et al. Reclassification of Lactobacillus casei subsp. casei ATCC 393 and Lactobacillus rhamnosis ATCC • Bifidobacterium lactis • Lactobacillus paracasei 1e-75 15820 as Lactobacillus zeae were cultured with an MRS medium, which favors the growth of • Lactobacillus rhamnosus nom. rev., Designation of ATCC 334 as the Neotype of L.casei subsp. casei, and rejection of the Name Lacto- • Lactococcus lactis subsp. lactis • Lactobacillus paracasei 1e-75 bacillus paracassei. Lactobacilli bacteria. A 1/10 dilution “MRS Medium” and 1/100 di- • Lactococcus lactis subsp. cremoris SUBSP. paracasei lution “MRS Dilute Medium” were prepared for each of the seven • Lactobacillus paracasei subsp. • Lactobacillus paracasei 1e-75 International Journal of Systematic Biotechnology. vol.46 no. 1, 1996, pp.337-340. Print. paracasei SUBP. tolerans Dinan et al. Psychobiotics: A Novel Class of Psychotropic. Biological Psychiatry, vol. 74 no. 10, 2013, pp.720-726. Kefir samples, a total of fourteen plates. Samples were incubated Wallaby • Lactococcus lactis subsp. lactis biovar. • Lactobacillus rhamnosus 1e-75 MRS MRS Print. (Sample #7) Diacetylactis Dilute F ierer et al. Assessment of Soil Microbial Community Structure by Use of Taxon-Specific Quantitative PCR at 37oC for 24 hours. Colonies were extracted from each plate by • Leuconostoc mesenteroides subsp. Assays. Applied Environmental scraping a t-cross shape on the agar plate with a heat wire loop. Cremoris Microbiology, vol.71 no.7, 2005, pp.4117-4120. Print. • Lactobacillus delbrueckii subsp. lactis Kaufman, Alexander. “People Are Going Crazy For A Fermented Milk Drink No One Can Pronounce.” The Huf- The MRS medium” sample was combined with the “MRS medium fington Post. dilute” sample to create one sample for each kefir brand, a total Thehuffingtonpost.com inc., 9 Jan. 2015. Web. 27 Oct. 2016. CHROMATOGRAPHS Korsak, N. et al. Short communication: evaluation of the microbiota of kefir samples using metagenetic of seven samples. These samples were then used for DNA extrac- analysis targeting the 16S and 26S Sample # 1 Sample # 4 tion. DNA extraction of the samples was done using SurePrep Soil ribosomal DNA fragments. Journal of Dairy Science, vol. 98 no. 6, 2015, pp.3684-3689. Print. Otles & Cagindi. Kefir: A Probiotic Dairy- Composition, Nutritional and Therapeutic Aspects. Pakistan Journal DNA Isolation Kit. A Quant-iT kit was used to quantify the DNA and of Nutrition, vol. 2 no. 2, 2003, check if the DNA extraction worked. The primers used for PCR am- pp.54-59. Print. Sample # 2 Sample # 5 “Probiotics Market Analysis By Application (Probiotic Foods & Beverages (Dairy Products, Non-Dairy Products, plification are EUB 338, the forward primer, and EUB 518, the reverse Cereals, Baked Food, primer, these bacterial primers amplify the 16s rRNA gene. To purify Fermented Meat Products, Dry Food), Probiotic Dietary Supplements (Food Supplements, Nutritional Sup- the DNA, an ExoSap Treatment was used to remove primers and plements, Specialty Nutrients, Infant Formula), Animal Feed Probiotics), By End-Use (Human Probiotics, Sample # 3 Sample # 6 Animal Probiotics) And Segment Forecast To 2024.” Grand View Research. Grand View Research inc, Sep. unincorporated nucleotides. Then the Utah State University Geno- 2016. Web. Accessed 27 Jan. 2016. Sarah. “Why Kefir is Healthier than Yogurt.” The Healthy Home Economist. Austus Media, http://www. mics Laboratory could produce a barcode. The barcode was analy- thehealthyhomeeconomist.com/kefir- zed using BLAST database. The seven species were then put into healthier-than-yogurt. Accessed 27 Jan. 2016. Sample # 7 Yunes et al. GABA production and Structure of gadB/ gadC genes in Lactobacillus and Bifidobacterium strains PhyloT database to create a phylogenetic tree. PHYLOGENETIC TREE OF DOMINANT BACTERIA from human microbiota. Anaerobe, vol. 42, 2016, pp.197-204. Print.

ACKNOWLEDGEMENTS :

BONNIE WARING PAUL WOLF DENNIS WELKER assistant professor professor & associate professor & director of [email protected] department head undergraduate studies [email protected] [email protected]