The plant Linum usitatissimum L., commonly known as flax plant, has been grown in Europe and Asia for as long as 10,000 years  . Five main nutritional components of flaxseed are fiber, protein, lignans, oil and vitamins as determined by gas chromatography and high-performance liquid chromatography (HPLC) techniques  . Flaxseed meal is rich in bioactive compounds and their extracts exhibit strong anti-radical activity  . Flaxseed has the highest amount of the plant lignan secoisolariciresinol diglucoside (SDG)  . Whole flaxseed contains 6.1 - 13.3 mg SDG/g flaxseed  . Bacteria in the colon convert the SDG into mammalian lignans including enterodiol and enterolactone  , which are responsible for anti-estrogenic and weak estrogenic effects in mammalian tissue  . Multiple studies have shown that flaxseed consumption leads to improvements in metabolism of glucose or lipids or both       . Supplementing biscuits with flaxseed and sidr leaf has been shown to provide various health benefits including renal-protective and immune-enhancing properties  .
An early definition of the term “probiotic” deals with one species of protozoa that produces growth-promoting factors that benefits the growth of another species  . A more recent and commonly used definition of probiotics is “live microorganisms which when administered in adequate amounts confer a health benefit on the host”  . Species of Lactobacillus and Bifidobacteria are the most common types of probiotics, but other types of probiotics also exist. L. acidophilus (first described by Ernst Moro in 1900) is a well-known probiotic that is commonly added to yogurt. L. bulgaricus (first described by Stamen Grigoroff in 1905) and S. thermophilus (first described by Sigurd Orla-Jensen in 1919) are used as yogurt starters  as they produce lactic acid and have a symbiotic relationship with each other. These yogurt starters have also been referred to as probiotics   . Some studies have shown that yogurt bacteria can survive transit through the human gastrointestinal tract since these organisms can be recovered in feces   . A summary of health benefits provided by probiotics has been given by Fijan  .
Bile tolerance refers to an in vitro test that measures the ability of a bacterial strain to survive in a media that contains bile salts with similarities to conditions within the small intestines to give an indication of the likelihood that a bacterial strain would survive in the host. It is an important selection criteria for probiotics for incorporation into foods and beverages. Sherman  mentioned the tolerance for bile in the enterococcus division. In an early attempt in classification, Wheater  classified various lactobacilli cultures into L. acidophilus, L. bulgaricus, and a third intermediate group based on many tests including tolerance to bile salt (sodium tauroglycocholate). Lankaputhra and Shah  have shown that survival of L. acidophilus in 1.0% and 1.5% bile varied substantially among different strains. Bile tolerance and metabolism of bile salts have recently been reviewed by Ayyash  .
There have been many recent attempts to improve bile tolerance including addition of various ingredients and application of various processing techniques. However, it does not appear that there are any reports examining if bile tolerances of free L. acidophilus, L. bulgaricus, and S. thermophilus can be improved by addition of flaxseed. Some studies   have shown that growth of L. acidophilus, L. bulgaricus, and S. thermophilus can be improved by addition of flaxseed. It was hypothesized that the bile tolerances of L. acidophilus, L. bulgaricus, and S. thermophilus could potentially be improved by addition of flaxseed. With hopes of also utilizing health benefits provided by flaxseed, the objective of this study was to determine if flaxseed can enhance bile tolerance of L. acidophilus LA-K, L. bulgaricus LB-12 and S. thermophilus ST-M5.
2. Materials and Methods
The following supplies were obtained for this project. L. acidophilus LA-K (a pure culture strain commonly added as a probiotic to yogurt), L. bulgaricus LB-12 (a pure culture strain commonly used as a starter culture in yogurt), and S. thermophilus ST-M5 (another pure culture strain commonly used as a starter culture in yogurt) were obtained from Chr. Hansen (Milwaukee, WI). Broths that were used included BD Difco Lactobacilli MRS broth (Becton, Dickinson and Company, Sparks, MD) and Oxoid M17 broth (Oxoid Ltd., Basingstoke, Hampshire, England). Oxgall was obtained from United States Biological (Swampscott, MA) and sodium thioglycolate (mercaptoacetic acid, sodium salt) was obtained from Acros Organics (Geel, Belgium). Flaxseed that was used in the MRS and M17 broths was obtained from a local grocery store. Agar (Acros Organics, Geel, Belgium) was used in the MRS broth and M17 broth to form MRS agar and M17 agar, respectively. Lactose (Oxoid Ltd., Basingstoke, Hampshire, England) was used in both the M17 broth and agar. BD Bacto peptone (Becton, Dickinson and Company, Sparks, MD) was used for making dilutions.
2.2. Determination of Bile Tolerance
Bile tolerance was determined according to Pereira & Gibson  with modifications. A control (no flaxseed) broth and experimental (61.85 g flaxseed/L) broth were prepared for each culture. For L. acidophilus LA-K and L. bulgaricus LB-12, MRS broths  were prepared by mixing 55 g of MRS broth, 3 g of oxgall, 2 g of sodium thioglycolate, and 1 L of distilled water. For S. thermophilus ST-M5, 37.25 g of M17 broth  , 3 g oxgall, and 950 mL of water were mixed. Also, 50 mL of a 10% lactose solution for the M17 broth was prepared. The M17 broth and the lactose solution were autoclaved separately at 121˚C for 15 min. The lactose solution was then added aseptically to the M17 broth containing oxgall after autoclaving. All broths were tempered to 37˚C before inoculation.
After inoculating the broths with the cultures forming a 10−2 dilution, broth samples were taken hourly for 8 h for dilution with 0.1% Bacto peptone and pour plating. L. acidophilus LA-K was plated in MRS agar and incubated anaerobically at 37˚C for 48 h. L. bulgaricus LB-12 was plated in MRS agar and incubated anaerobically at 37˚C for 72 h. S. thermophilus ST-M5 was plated in M17 agar containing 5% of a 10% (w/w) lactose solution and incubated aerobically at 37˚C for 24 h. The change in counts (reported as log CFUs/mL) over 8 h for the experimental broths was compared to the corresponding change in counts of the control (no flaxseed) for each culture. Three replicates were performed for both L. acidophilus LA-K and L. bulgaricus LB-12 while four replicates were performed for S. thermophilus ST-M5.
2.3. Statistical Analysis
The log plate count data were analyzed as a two factor (presence versus absence of flaxseed and time (hour) after inoculation) factorial experiment in a randomized block design using PROC MIXED in SAS 9.4 (SAS Institute Inc., Cary, NC). Slopes of regression lines of log counts as a function of time represent the net growth or decline of the bacteria over time, and a comparison of the slopes of the regression lines between the flaxseed-containing broth and the control would provide information about the effect of the presence of flaxseed on the growth of these bacteria in the presence of bile. Significant differences in slopes of regression lines were tested using a random coefficient model by examining the significance of the flaxseed (presence versus absence) and time (hours after inoculation) interaction term in the analysis of variance. Significance was set at α = 0.05.
3.1. Lactobacillus acidophilus
Figure 1 presents the log counts of L. acidophilus LA-K in MRS broths containing 0.3% oxgall and 0.2% sodium thioglycolate either in the presence or absence of flaxseed during an 8-h incubation. Although not statistically significant, log counts of L. acidophilus LA-K in the presence of flaxseed tended to be higher than in the absence of flaxseed. The slope of the regression line in the graph of log counts of L. acidophilus versus hours in the control broth was negative (−0.04109) and significant (P = 0.0124) (Table 1) indicating decreasing counts over time. Conversely, the slope of this type of graph for the flaxseed-containing
Figure 1. Log counts of L. acidophilus LA-K in MRS broths containing 0.3% oxgall and 0.2% sodium thioglycolate either in the presence or absence of flaxseed during an 8-h incubation.
broth was 0.01403, but not significant (P = 0.2030).
For L. acidophilus, the flaxseed (its presence versus its absence) and hour (after inoculation of L. acidophilus) factors were not significant in the analysis of variance (Table 2). However, the flaxseed and hour interaction was significant (P = 0.0147) indicating that the rate of change of log counts of L. acidophilus over time (hours) was significantly different for the flaxseed containing broth compared to the control broth. Therefore, the bile tolerance of L. acidophilus LA-K in MRS broth was significantly enhanced by flaxseed.
3.2. Lactobacillus bulgaricus
The log counts of L. bulgaricus LB-12 in MRS broths containing 0.3% oxgall and
Table 1. Parameter estimates for intercept and slope and corresponding P value for log counts of L. acidophilus, L. bulgaricus, and S. thermophilus enumerated hourly over 8 h in presence or absence of flaxseed.
Table 2. F values and P values for main effects (flaxseed and hour) and their interaction (flaxseed * hour) for log counts of L. acidophilus, L. bulgaricus, and S. thermophilus enumerated hourly over 8 h in presence or absence of flaxseed.
0.2% sodium thioglycolate either in the presence or absence of flaxseed during an 8-h incubation are shown in Figure 2. The slopes of the regression lines in the graph for log count of L. bulgaricus over time were not significantly different from 0 for both the broth containing flaxseed and the control broth (Table 1).
For L. bulgaricus, presence or absence of flaxseed was significant (P = 0.0201) (Table 2), and log L. bulgaricus counts in presence of flaxseed were significantly higher than in its absence. However, hour was not significant. The flaxseed and hour interaction was not significant, indicating no significant differences in the rate of change of log counts of L. bulgaricus over time (hours) for the broth containing flaxseed versus for the control broth. Therefore, the bile tolerance of L. bulgaricus LB-12 in MRS broth was not significantly enhanced by flaxseed.
3.3. Streptococcus thermophilus
Figure 3 presents log counts of S. thermophilus ST-M5 in M17 broths containing 0.5% lactose and 0.3% oxgall either in the presence or absence of flaxseed during an 8-h incubation. Similar results were obtained for S. thermophilus as
Figure 2. Log counts of L. bulgaricus LB-12 in MRS broths containing 0.3% oxgall and 0.2% sodium thioglycolate either in the presence or absence of flaxseed during an 8-h incubation.
Figure 3. Log counts of S. thermophilus ST-M5 in M17 broths containing 0.5% lactose and 0.3% oxgall either in the presence or absence of flaxseed during an 8-h incubation.
for L. bulgaricus. The slopes of the regression lines in the graph for log count of S. thermophilus over time were also not significantly different from 0 for both the broth containing flaxseed and the control broth (Table 1).
Likewise, flaxseed was significant (P = 0.0164). Log counts of S. thermophilus ST-M5 in the presence of flaxseed were higher than in the absence of flaxseed. However, hour and the flaxseed and hour interaction were not significant (Table 2). Therefore, the bile tolerance of S. thermophilus ST-M5 in M17 broth was not significantly enhanced by flaxseed.
Addition of various ingredients and application of various processing techniques have been shown to improve the bile tolerance of these microorganisms. Addition of up to 3% whey protein isolate improved the bile tolerance of pure cultures of S. thermophilus ST-M5 and L. bulgaricus LB-12 during 5 h of oxgall exposure  . Likewise, addition of 5% lactose improved the bile tolerance of pure cultures of S. thermophilus ST-M5 and L. bulgaricus LB-12 during 12 h of oxgall exposure  . Addition of inulin to yogurt milk improved the ability of L. acidophilus to tolerate bile salts compared to L. acidophilus in yogurt not containing inulin  . Encapsulating L. bulgaricus in alginate-milk microspheres allowed it to survive in 1% and 2% porcine bile salt solutions for 1 and 2 h while free L. bulgaricus did not survive  . Various low homogenization pressures ranging from 3.45 to 13.80 MPa improved bile tolerance of S. thermophilus ST-M5, and a homogenization pressure of 6.90 MPa improved bile tolerance of L. acidophilus LA-K during 10 h of exposure to 0.3% oxgall  .
The effect of flaxseed on the growth of L. acidophilus, L. bulgaricus, and S. thermophilus has also been investigated in other studies. Although the growth of L. acidophilus in MRS broth containing oxgall improved in the presence of flaxseed for 8 h in the present study, Bialasová et al.  found decreased growth of L. acidophilus, L. bulgaricus, and S. thermophilus in the presence of flaxseed meal in milk during 28 d of storage. Differences between the present results and the results from Bialasová et al.  could be due to numerous factors including different strains of microorganisms, presence of bile in the present study, different time periods, and different growth media. However, Mihoubi et al.  found consistently increased S. thermophilus and L. bulgaricus counts in yogurt supplemented with 3% ground flaxseed compared with its control during the 3.5 h fermentation time and 28 d of storage. Likewise, Mousavi et al.  found higher L. acidophilus counts in stirred yogurt during 28 d of storage when supplemented with either 2% or 4% flaxseed. Smolová et al.  reported that they were able to keep L. acidophilus counts above 107 cfu/g in UHT skim milk containing various varieties and forms of flaxseed during 28 d of 4˚C to 6˚C storage. Bustamante et al.  found that L. acidophilus that was encapsulated with flaxseed mucilage either in the presence or absence of flaxseed soluble protein maintained greater relative viability for 6 h in MRS broth containing bovine bile.
It is important to note while the dosage of flaxseed was determined by SDG content, there is no proof the beneficial effect of flaxseed on L. acidophilus is strictly due to SDG. Further studies would have to be performed to determine which component of the flaxseed is responsible for this beneficial effect.
The purpose of this study was to investigate the effect of flaxseed on the bile tolerance of L. acidophilus LA-K, L. bulgaricus LB-12 and S. thermophilus ST-M5. Whole flaxseed administered at 61.85 g/L of broth significantly increased the growth, and therefore bile tolerance, of L. acidophilus LA-K but not L. bulgaricus LB-12 and S. thermophilus ST-M5 in the presence of oxgall (3 g/L) between 0 to 8 h when compared to their control. Improving the in vitro bile tolerance of L. acidophilus would likely improve their survival in the host.
This research was supported in part by a Howard Hughes Medical Institute grant through the Undergraduate Biological Sciences Education Program to LSU.
 Herchi, W., Arráez-Román, D., Boukhchina, S., Kallel, H., Segura-Carretero, A. and Fernández-Gutierrez, A. (2012) A Review of the Methods Used in the Determination of Flaxseed Components. African Journal of Biotechnology, 11, 724-731.
 Tavarini, S., De Leo, M., Matteo, R., Lazzeri, L., Braca, A. and Angelini, A. (2021) Flaxseed and Camelina Meals as Potential Sources of Health-Beneficial Compounds. Plants, 10, 156.
 Zhang, W., Wang, X.B., Liu, Y., Tian, H.M., Flickinger, B., Empie, M.W. and Sun, S.Z. (2008) Dietary Flaxseed Lignan Extract Lowers Plasma Cholesterol and Glucose Concentrations in Hypercholesterolaemic Subjects. British Journal of Nutrition, 99, 1301-1309.
 Johnsson, P., Kamal-Eldin, A., Lundgren, L.N. and Åman, P. (2000) HPLC Method for Analysis of Secoisolariciresinol Diglucoside in Flaxseeds. Journal of Agricultural and Food Chemistry, 48, 5216-5219.
 Cunnane, S.C., Ganguli, S., Menard, C., Liede, A.C., Hamadeh, M.J., Chen, Z.-Y., Wolever, T.M.S. and Jenkins, D.J.A. (1993) High-Linolenic Acid Flaxseed (Linum usitatissimum): Some Nutritional Properties in Humans. British Journal of Nutrition, 69, 443-453.
 Pan, A., Sun, J., Chen, Y., Ye, X., Li, H., Yu, Z., Wang, Y., Gu, W., Zhang, X., Chen, X., Demark-Wahnefried, W., Liu, Y. and Lin, X. (2007) Effects of a Flaxseed-Derived Lignan Supplement in Type 2 Diabetic Patients: A Randomized, Double-Blind, Cross-Over Trial. PLoS ONE, 2, e1148.
 Yari, Z., Cheraghpour, M. and Hekmatdoost, A. (2021a) Flaxseed and/or Hesperidin Supplementation in Metabolic Syndrome: An Open-Labeled Randomized Controlled Trial. European Journal of Nutrition, 60, 287-298.
 Yari, Z., Cheraghpour, M., Alavian, S.M., Hedayati, M., Eini-Zinab, H. and Hekmatdoost, A. (2021b) The Efficacy of Flaxseed and Hesperidin on Non-Alcoholic Fatty Liver Disease: An Open-Labeled Randomized Controlled Trial. European Journal of Clinical Nutrition, 75, 99-111.
 Almehmadi, A., Lightowler, H., Chohan, M. and Clegg, M.E. (2021) The Effect of a Split Portion of Flaxseed on 24-h Blood Glucose Response. European Journal of Nutrition, 60, 1363-1373.
 El Seedy, G.M., El-Shafey, E.S. and Elsherbiny, E.S. (2021) Fortification of Biscuit with Sidr Leaf and Flaxseed Mitigates Immunosuppression and Nephrotoxicity Induced by Cyclosporine A. Journal of Food Biochemistry, 45, e13655.
 Uriot, O., Denis, S., Junjua, M., Roussel, Y., Dary-Mourot, A. and Blanquet-Diot, S. (2017) Streptococcus thermophilus: from Yogurt Starter to a New Promising Probiotic Candidate? Journal of Functional Foods, 37, 74-89.
 Mater, D.D.G., Bretigny, L., Firmesse, O., Flores, M.-J., Mogenet, A., Bresson, J.-L. and Corthier, G. (2005) Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus Survive Gastrointestinal Transit of Healthy Volunteers Consuming Yogurt. FEMS Microbiology Letters, 250, 185-187.
 Elli, M., Callegari, M.L., Ferrari, S., Bessi, E., Cattivelli, D., Soldi, S., Morelli, L., Feuillerat, N.G. and Antoine, J.-M. (2006) Survival of Yogurt Bacteria in the Human Gut. Applied and Environmental Microbiology, 72, 5113-5117.
 Fijan, S. (2014) Microorganisms with Claimed Probiotic Properties: An Overview of Recent Literature. International Journal of Environmental Research and Public Health, 11, 4745-4767.
 Ayyash, M.M., Abdalla, A.K., AlKalbani, N.S., Baig, M.A., Turner, M.S., Liu, S.-Q. and Shah, N.P. (2021) Invited Review: Characterization of New Probiotics from Dairy and Nondairy Products-Insights into Acid Tolerance, Bile Metabolism and Tolerance, and Adhesion Capability. Journal of Dairy Science. (In Press)
 Mihoubi, M., Amellal-Chibane, H., Mekimene, L., Noui, Y. and Halladj, F. (2017) Physicochemical, Microbial, and Sensory Properties of Yogurt Supplemented with Flaxseeds during Fermentation and Refrigerated Storage. Mediterranean Journal of Nutrition and Metabolism, 10, 211-221.
 Mousavi, M., Heshmati, A., Garmakhany, A.D., Vahidinia, A. and Taheri, M. (2019) Optimization of the Viability of Lactobacillus acidophilus and Physico-Chemical, Textural and Sensorial Characteristics of Flaxseed-Enriched Stirred Probiotic Yogurt by Using Response Surface Methodology. LWT-Food Science and Technology, 102, 80-88.
 Pereira, D.I.A. and Gibson, G.R. (2002) Cholesterol Assimilation by Lactic Acid Bacteria and Bifidobacteria Isolated from the Human Gut. Applied and Environmental Microbiology, 68, 4689-4693.
 Vargas, L.A., Olson, D.W. and Aryana, K.J. (2015) Whey Protein Isolate Improves Acid and Bile Tolerances of Streptococcus thermophilus ST-M5 and Lactobacillus delbrueckii ssp. bulgaricus LB-12. Journal of Dairy Science, 98, 2215-2221.
 Shi, L.-E., Li, Z.-H., Li, D.-T., Xu, M., Chen, H.-Y., Zhang, Z.-L. and Tang, Z.-X. (2013) Encapsulation of Probiotic Lactobacillus bulgaricus in Alginate-Milk Microspheres and Evaluation of the Survival in Simulated Gastrointestinal Conditions. Journal of Food Engineering, 117, 99-104.
 Muramalla, T. and Aryana, K.J. (2011) Some Low Homogenization Pressures Improve Certain Probiotic Characteristics of Yogurt Culture Bacteria and L. acidophilus LA-K. Journal of Dairy Science, 94, 3725-3738.
 Bialasová, K., Němečková, I., Kyselka, J., Štětina, J., Solichová, K. and Horáčková, S. (2018) Influence of Flaxseed Components on Fermented Dairy Product Properties. Czech Journal of Food Sciences, 36, 51-56.
 Smolová, J., Němečková, I., Klimešová, M., Švandrlík, Z., Bjelková, M., Filip, V. and Kyselka, J. (2017) Flaxseed Varieties: Composition and Influence on the Growth of Probiotic Microorganisms in Milk. Czech Journal of Food Sciences, 35, 18-23.
 Bustamante, M., Villarroel, M., Rubilar, M. and Shene, C. (2015) Lactobacillus acidophilus La-05 Encapsulated by Spray Drying: Effect of Mucilage and Protein from Flaxseed (Linum usitatissimum L.). LWT-Food Science and Technology, 62, 1162-1168.