Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
School of Nutrition and Food Sciences
Non Technical Summary
The leaky gut condition is like having very tiny holes in the gut, putting the sewage back into the body. Symptoms of leaky gut syndrome include bloating, food sensitivities, fatigue, digestive issues and skin problems. Diseases and other conditions associated with leaky gut include Celiac Disease, Type 1 Diabetes, Chron's Disease, Irritable Bowel Syndrome, Celiac Sprue, Systemic Lupus Erythematosus, Chronic Fatigue Syndrome, Food Allergies, Multiple Sclerosis and Fibromyalgia. Several ingredients have been identified to help treat leaky gut. It is not known how these ingredients influence key quality characteristics of frozen and fermented dairy foods. It is also not known how these ingredients influence probiotic characteristics of probiotic bacteria. Hence, this project proposes 8 well referenced ingredients / additives, known to treat leaky gut, on the physico-chemical, sensory and microbiological characteristics of frozen and fermented dairy foods and on the acid tolerance, bile tolerance and protease activities of probiotic bacteria. The results obtained from this proposed project has the potential to help develop future novel foods targeted to prevent leaky gut, enhance gut health and overall health of people in Louisiana, nationally and around the world.
Animal Health Component
70%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
1. To elucidate the effect of various ingredients that prevent leaky gut on the a) physico-chemical, b) sensory and c) microbiological characteristics, of (i) frozen and (ii) fermented dairy products.2. To determine the a) acid tolerances, b) bile tolerances and c) protease activities of probiotics as influenced by the ingredients that help treat leaky gut.
Project Methods
i. Yogurt / fermented milk manufacture: Yogurt / fermented milk will be manufactured according to Aryana (2003) or Haque and Aryana (2002). Treatments will consist of following ingredients and control which will have none of these ingredients. Ingredients to be used will be L-glutamine (0, 2.5, 5g), Quercetin (0, 250, 500mg), Slippery Elm Bark (0, 75, 150 mg), Marshmallow root (0, 480, 960mg), N-acetyl-D-glucosamine (NAG) (0, 75, 150 mg), Licorice root (0, 75, 150 mg), Maitake Mushrooms (0, 15, 30 mg) and Zinc Orotate (0, 25, 50 mg). These ingredients will be obtained directly from their suppliers in their commercially available powder form. These ingredients will be incorporated individually per 3 cups of 8 fluid oz. each cup, i.e. in total, per 711 mL of yogurt mix. Yogurt mixes will be homogenized, batch pasteurized, tempered to 40? and yogurt culture (Lactobacillus bulgaricus and Streptococcus thermophilus) will be added. Attributes studied will be syneresis, viscosity, titratable acidity, pH, and color (L*, a*, b* C* h*) as described in Aryana (2003). Data will be acquired weekly over 1.5 months of refrigerated storage.ii. Ice cream manufacture: Fat-free, no sugar added ice creams will be manufactured according to Aryana and Summers (2006). Treatments will be the ingredients as described above. These ingredients will be incorporated individually per 3 cups of 8 fluid oz. each cup, i.e. in total, per 711 mL of ice cream mix. Mixes will be homogenized, vat pasteurized, cooled and aged. Prior to freezing, vanilla flavor will be added. Attributes to be studied will be viscosity, pH, color, meltdown, probiotic bacterial counts and sensory flavor, body and texture which will be determined according to Aryana (2003) and Farooq and Haque (1997). Data will be acquired monthly over 1.5 years of frozen storage.iii. Microbial enumerations: a. Streptococcus thermophilus counts will be determined using Streptococcus thermophilus (ST) agar prepared according to Muramalla and Aryana (2011) and Dave and Shah (1996). The ST agar will be autoclaved at 121°C for 15 min. Plating and incubation of petriplates will be conducted as discussed in "culture growth" section below, and the colonies will be counted after the incubation period.b. Lactobacillus bulgaricus counts will be enumerated using Lactobacilli MRS agar prepared according to manufacturer's directions (DifcoTM, Dickinson and Company, Sparks, MD). The procedure will be carried out according to Muramalla and Aryana (2011) and Tharmaraj and Shah (2003). 55g of MRS broth powder and 15g of agar powder will be dissolved in 1L of distilled water. A solution of 1 N HCl will be added to adjust the pH to 5.2±0.1. This mixture will be heated to boiling, and will be autoclaved at 121°C for 15 min. Plating and incubation of petriplates will be conducted as discussed in "culture growth" section below, and the colonies will be counted after the incubation period.c. Lactobacillus acidophilus counts will be enumerated using MRS-sorbitol agar according to Olson and Aryana (2008), Dave and Shah (1996) and Tharmaraj and Shah (2003). MRS base medium without dextrose will be made and autoclaved at 121°C for 15 min. Then, a 10% (w/v) sorbitol solution will be prepared and filter sterilized. Appropriate amount of sorbitol will be added to the MRS base medium to form a 10% sorbitol solution and 90% MRS base medium mixture immediately before pouring the plates (Olson and Aryana, 2008; Dave & Shah, 1996; Tharmaraj & Shah, 2003). Plating and incubation of petriplates will be conducted as discussed in "culture growth" section below, and the colonies will be counted after the incubation period.d. Bifidobacteria counts will be determined using MRS NNLP agar according to Tharmaraj and Shah (2003) and Laroia and Martin (1991). The MRS NNLP agar will contain 52 g MRS broth, 15 g agar, 15 mg nalidixic acid, 100 mg neomycine sulfate, 3 g lithium chloride, and 200 mg paromomycine sulfate. The MRS broth and agar will be autoclaved, and the nalidixic acid, neomycine sulfate, lithium chloride and paromomycine sulfate will be filter sterilized. The filter-sterilized NNLP will be aseptically incorporated into the autoclaved MRS base. Filter-sterilized L-cysteine-HCl (0.05% final concentration) will also be added. Plates will be incubated anaerobically at 37° C for 72 h.e. Culture growth will be determined using the pour plate technique. Yogurt / fermented milk / ice cream samples of 11g will be pipetted into 99ml dilution bottles with 0.1% peptone water. Serial dilutions in peptone water from 10e-1 to 10e-10 will be prepared. Dilutions will be plated on Streptococcus thermophilus agar, MRS-sorbitol agar, and Lactobacilli MRS agar in duplicate. Pour plates will be incubated anaerobically at 37°C for 48h or Lactobacillus acidophilus, aerobicallyat 37°C for 24h for Streptococcusthermophilus, anaerobically at 43°C for 72h for Lactobacillus bulgaricus and anaerobically at 37° C for 72 h for Bifidobacterium bifidus.iv. Probiotic characteristic determinationsa. Acid tolerance of probiotics will be determined according to Muramalla and Aryana (2011), and Pereira and Gibson (2002) with slight modification. Modification will be ingredients used as described above. The pH of broths will be adjusted to 2.00. Broths containing the microorganisms will then be transferred into these acidified broths viz. acidified Lactobacilli MRS broth for L. acidophilus and acidified MRS NNLP broth for Bifidobacterium bifidus. Inoculated acidified broths will then be incubated anaerobically at 37? for two hours. Serial dilutions will be plated 0, 20, 40, 60, 80, 100, and 120 min. during 2 hours of incubated acid exposure using pour plate technique. Plating and incubation of petriplates will be done according to type of bacteria as discussed in "culture growth" above and colonies will be counted after incubation period.b. Bile tolerance of probiotics will be determined according to Muramalla and Aryana (2011), and Pereira and Gibson (2002) with slight modification. Modification will be the ingredients used as described above. Broths containing oxgall and sodium thioglycolate will then be prepared using Lactobacillus MRS broth and MRS NNLP broth individually and supplementing them with oxgall (0.3% w/v) and sodium thioglycolate (0.2% w/v). Inoculated broths containing Oxgall will then be incubated anaerobically at 37? for ten hours. Inoculated broth containing Oxgall will be serially diluted and plated in duplicate every hour over the incubated 10 hours using pour plating method. Plating and incubation of petriplates will be done according to the type of bacteria as discussed in "culture growth" above and colonies will be counted after incubation period.c. Protease activity of probiotics will be determined by o-phthaldialdehyde (OPA) spectrophotometric method as described by Muramalla and Aryana (2011) and Oberg et al., (1991) with slight modification. Modification will be ingredients used as described above. Broths containing microorganisms will be transferred into (10% [v/v]) sterile skim milk and will be incubated at 40ºC for 0, 12 and 24 hours. After incubation, 2.5 ml from each sample will be mixed with 1 ml distilled water individually and will be transferred into each of the test tubes containing 5 ml of 0.75N trichloroacetic acid (TCA) and the test tubes will be vortexed. Duplicate aliquots from each TCA filtrate will be analyzed by OPA testing using a spectrophotometer. 150 µl of each TCA filtrate will be mixed with 3 ml of OPA reagent in a 3 ml cuvette, and the absorbance at 340 nm will be read.v. Statistical analyses:Product manufacture and all experiments will be replicated three times. Data will be analyzed using Proc Mixed and Proc Multitest of the Statistical Analysis Systems. Differences of least-squares means will be used to determine significant differences at P<0.05.