Thermo-stable strains, products and methods thereof
10058577 ยท 2018-08-28
Assignee
Inventors
- Desiraju Aditya (Mysore, IN)
- Desiraju Shrilakshmi (Mysore, IN)
- Sharieff Irfanulla (Mysore, IN)
- Prakash Abhilash (Mysore, IN)
Cpc classification
A23V2002/00
HUMAN NECESSITIES
A23F3/16
HUMAN NECESSITIES
A23L33/135
HUMAN NECESSITIES
International classification
A23G1/42
HUMAN NECESSITIES
Abstract
The present disclosure relates to thermo-stable/heat stable strain(s) of micro-organisms, obtaining such thermo-stable strains and incorporating them into food products. These strains are able to survive high temperatures for longer periods of time and thus food products containing such strains are therapeutically effective and beneficial for general health.
Claims
1. A food product comprising a thermostable Lactobacillus microorganism, wherein the thermostable microorganism is selected from the group consisting of Lactobacillus plantarum with ATCC SD No. 6863 comprising genomic sequence set forth in SEQ ID No.1 and Lactobacillus acidophilus with ATCC SD No. 6864 comprising genomic sequence set forth in SEQ ID No.2.
2. A method of preparing a food product, said method comprising combining a thermostable strain selected from the group consisting of Lactobacillus plantarum with ATCC SD No. 6863 comprising genomic sequence set forth in SEQ ID No.1 and Lactobacillus acidophilus with ATCC SD No. 6864 comprising genomic sequence set forth in SEQ ID No.2; with a component of the food product during or after preparation of the food product.
3. The food product as claimed in claim 1, wherein the food product is selected from the group consisting of beverage, yogurt, dairy product, nectar, fruit juice, energy drink, bakery food, chocolate, cereal and soup.
4. The food product as claimed in claim 1, wherein the thermostable microorganism is viable at temperature ranging from about 25 C. to 250 C.
5. A thermostable Lactobacillus microorganism selected from the group consisting of Lactobacillus plantarum with ATCC SD No. 6863 comprising genomic sequence set forth in SEQ ID No.1 and Lactobacillus acidophilus with ATCC SD No. 6864 comprising genomic sequence set forth in SEQ ID No.2.
6. The thermostable microorganism as claimed in claim 5, wherein the thermostable microorganism includes the Lactobacillus plantarum with ATCC SD No. 6863.
7. The thermostable microorganism as claimed in claim 5, wherein the thermostable microorganism includes the Lactobacillus acidophilus with ATCC SD No. 6864.
8. The thermostable microorganism as claimed in claim 5, wherein the thermostable microorganism is viable at temperature ranging from about 25 C. to 250 C.
9. The method as claimed in claim 2, wherein the food product is selected from the group consisting of beverage, yogurt, dairy product, nectar, fruit juice, energy drink, bakery food, chocolate, cereal and soup.
10. The method as claimed in claim 2, wherein the thermostable microorganism is viable at temperature ranging from about 25 C. to 250 C.
11. The method as claimed in claim 2, wherein the thermostable microorganism includes the Lactobacillus plantarum with ATCC SD No. 6863.
12. The method as claimed in claim 2, wherein the thermostable microorganism includes the Lactobacillus acidophilus with ATCC SD No. 6864.
Description
BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
(1) In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:
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DETAILED DESCRIPTION OF THE DISCLOSURE
(17) The present disclosure relates to a food product comprising a thermostable Lactobacillus microorganism, wherein the thermostable microorganism is selected from group comprising Lactobacillus plantarum with ATCC SD No. 6863 and Lactobacillus acidophilus with ATCC SD No. 6864.
(18) The present disclosure also relates to a method of preparing a food product, said method comprising act of combining a thermostable strain selected from group Lactobacillus plantarum with ATCC SD No. 6863 and Lactobacillus acidophilus with ATCC SD No. 6864; with component of the food product during or after preparation of the food product.
(19) In an embodiment of the present disclosure, the food product is selected from group comprising beverage, yogurt, dairy product, nectar, fruit juice, energy drink, bakery food, chocolate, cereal and soup.
(20) In another embodiment of the present disclosure, the thermostable microorganism is viable at temperature ranging from about 25 C. to 250 C.
(21) The present disclosure also relates to a thermostable Lactobacillus microorganism selected from group comprising Lactobacillus plantarum with ATCC SD No. 6863 and Lactobacillus acidophilus with ATCC SD No. 6864.
(22) In an embodiment of the present disclosure, the Lactobacillus plantarum has genomic sequence set forth in SEQ ID No.1.
(23) In another embodiment of the present disclosure, the Lactobacillus acidophilus has genomic sequence set forth in SEQ ID No.2.
(24) In yet another embodiment of the present disclosure, the thermostable microorganism is viable at temperature ranging from about 25 C. to 250 C.
(25) The present disclosure also relates to a thermostable microorganism for use in managing a disorder in a subject, wherein the microorganism is selected from group comprising Lactobacillus plantarum with ATCC SD No. 6863 and Lactobacillus acidophilus with ATCC SD No. 6864.
(26) The present disclosure also relates to a method of managing a disorder in a subject, wherein the method comprises step of administering to the subject, microorganism selected from group comprising Lactobacillus plantarum with ATCC SD No. 6863 and Lactobacillus acidophilus with ATCC SD No. 6864.
(27) In an embodiment of the present disclosure, the disorder is selected from group comprising diarrhea, antibiotic-associated diarrhea, irritable bowel syndrome, constipation, lactose intolerance, vaginal infection, intestinal infection, inflammatory bowel disease, and combinations thereof.
(28) In another embodiment of the present disclosure, the microorganism is in free form or form of food product.
(29) In yet another embodiment of the present disclosure, the subject is selected from group comprising animal and human being.
(30) The present disclosure relates to thermo-stable strain(s) of micro-organism(s).
(31) In an embodiment of the present disclosure, the micro-organism(s) is from Lactobacillus species.
(32) In an embodiment of the present disclosure, the thermo-stable strain(s) of the present disclosure have a tolerance to high temperatures.
(33) In the present disclosure, the term thermostable is intended to convey that the strains are able to survive at high temperatures for a long period of time.
(34) In embodiments of the present disclosure, the strains are viable, tolerant and resistant to high temperatures.
(35) In embodiments of the present disclosure, the thermostable strains are isolated thermostable strains.
(36) In embodiments of the present disclosure, the thermostable strains depict viability ranging from 1% to 99%.
(37) In embodiments of the present disclosure, the viability of the strains is dependent on environmental factors such as pH, temperature and time period.
(38) In embodiments of the present disclosure, viability of the thermostable strains is equal to and greater than 1.0-1.510.sup.9 colony forming units (cfu) is considered to be satisfactory for use in a food product.
(39) It is to be noted that the number of cfu's in a product differs depending on the mode of products. For example, for tablets/sachets/capsules, it is expressed per tablet/sachet/capsule, whereas, in food example like chocolate/muffin/cookie/bread it is expressed based on the serving.
(40) Probiotic dosing varies depending on the product and specific indication. No consensus exists about the minimum number of microorganisms that must be ingested to obtain a beneficial effect. Typically, a probiotic should contain several billion microorganisms to increase the likelihood of adequate gut colonization. For Lactobacilli, typical doses used range from 1-20 billion colony-forming units per day.
(41) In embodiments of the present disclosure, the thermo-stable strain is mixed or combined with a food component during the preparation of the food product.
(42) In embodiments of the present disclosure, the thermo-stable strain is mixed or combined with the food product after preparation of the food product.
(43) In an embodiment of the present disclosure, 1.00 g of a spray dried powdered sample of Lactobacillus acidophilus TSP-La1 contains a total viable cell count of 82.0 billion cfu/gm.
(44) In an embodiment of the present disclosure, 1.00 g of a spray dried powdered sample of Lactobacillus plantarum TSP-Lp1 contains a total viable cell count of 248.25 billion cfu/gm.
(45) In embodiments, the temperature ranges from about 25 C. to about 250 C.
(46) In embodiments of the present disclosure, the free form of the strains of the disclosure is selected from group comprising spray dried capsule, powder, tablet, sachet, ez melt, and chewable tablet.
(47) In the form of a food product, the spray dried powder is used in making different foods example Gummies, chocolate, cookies, muffins, bread, etc.
(48) In particular embodiments of the present disclosure, the strains have the ability to perform their functions in different food products even at higher temperatures, which makes them therapeutically effective.
(49) Lactobacillus acidophilus is a naturally occurring beneficial bacterium which supports the health of the intestinal tract. It maintains a healthy microflora by protecting the body against an overgrowth of harmful bacteria, helps improve digestion, protects against gastrointestinal upsets, abdominal pains, constipation and antibiotic-induced diarrhea.
(50) Lactobacillus plantarum can live in the stomach for a considerable period. It continuously fights disease and prevents entry of pathogenic microorganisms. It also improves digestive health, helps treat Irritable Bowel Syndrome (IBS), and prevents food allergies.
(51) In the present disclosure, the term CFU relates to colony forming units.
(52) In the present disclosure, the term MIC relates to Minimum Inhibitory Concentration.
(53) In the present disclosure, the term TVCC relates to Total Viable Cell Count.
(54) In the present disclosure, the term GYEA relates to Glucose Yeast Extract Agar.
(55) In the present disclosure, the term PBS relates to Phosphate Buffered Saline.
(56) In the present disclosure, the term beverage includesSoup, Tea, Coffee, Energy drinks, and fruit juices.
(57) In the present disclosure, the term bakery food includes Bread, Muffins, and Cookies.
(58) The temperature-stable probiotic strains, of Lactobacillus plantarum (TSP-Lp1) & Lactobacillus acidophilus (TSP-La1) have the ability to withstand harsh manufacturing processes, especially required during making Food/Beverage and in the Pharma industries. Most of the probiotics currently available in the market cannot survive a harsh environment observed during manufacturing and acidic environment of the stomach. These currently available probiotics are enteric coated to withstand harsh conditions. Probiotics that fail to reach intestinal tract alive are not likely to provide digestive and immune support. In the table provided below, the strains of the present disclosure are compared with commercial strains and their advantageous features are highlighted.
(59) TABLE-US-00001 TABLE 1 Triphase Commercial Strain Name Parameters Strains strains Lactobacillus acidophillus High Temperature Yes No (TSP-La1) stability ATCC-SD 6864 Non-GMO Yes Yes Enteric Coating Not required Required Refrigeration/ Not required Required Cold storage Lactobacillus plantarum High Temperature Yes No (TSP-Lp1) stability ATCC-SD 6863 Non-GMO Yes Yes Enteric Coating Not required Required Refrigeration/ Not required Required Cold storage
(60) In the present disclosure, the term managing or management or manage includes therapeutic and prophylactic activities. It includes treatment and healing of a disease or disorder, or ill effects or side effects of the disease or the disorder. The term also includes prevention of further progress of the disease or disorder, and prevention of further progress of ill effects or side effects of the disease or the disorder. It further includes maintenance of the optimum state in an individual.
(61) In the present disclosure, it is within the knowledge of the person skilled in the art to determine a suitable dosage of the strains and dosage form for administering to a particular subject.
(62) In another embodiment of the present disclosure, the thermo-stable Lactobacillus strain is selected from a group comprising Lactobacillus plantarum and Lactobacillus acidophilus
(63) In yet another embodiment of the present disclosure, the thermo-stable Lactobacillus strain is Lactobacillus plantarum TSP-Lp1.
(64) The parent strains for the present disclosure have been procured form Microbial Type Culture Collection (MTCC).
(65) In yet another embodiment of the present disclosure, the thermo-stable Lactobacillus plantarum is Lactobacillus plantarum TSP-Lp1.
(66) The Lactobacillus plantarum strain of the present disclosure is also referred to as LP throughout the present disclosure.
(67) In still another embodiment of the present disclosure, the thermo-stable Lactobacillus acidophilus is Lactobacillus acidophilus TSP-La1.
(68) The Lactobacillus acidophilus strain of the present disclosure is also referred to as La throughout the present disclosure.
(69) The novel strains of the present disclosure have been deposited in American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va., 20110 USA on Feb. 17, 2015. The strains have been allotted accession numbers as follows: Lactobacillus plantarum SD 6863 (SD-Safe Deposit) Lactobacillus acidophilus SD 6864 (SD-Safe Deposit)
(70) In yet another embodiment, the thermo-stable strain(s) of the present disclosure are able to withstand temperature ranging from room temperature i.e. about 25 C. to about 250 C.
(71) The present disclosure relates to food product comprising thermo-stable strain(s) of micro-organism(s).
(72) The present disclosure relates to probiotic food products comprising thermo-stable strain(s) of micro-organism(s).
(73) In an embodiment of the present disclosure, micro-organism(s) is from Lactobacillus species. In an embodiment of the present disclosure, the food or food product contains micro-organism(s) from Lactobacillus species.
(74) In another embodiment of the present disclosure, the Lactobacillus strain(s) in food product/probiotic food products is selected from a group comprising Lactobacillus plantarum and Lactobacillus acidophilus and combinations thereof.
(75) Food products according to the present disclosure may comprise one or more strains selected from the above defined strains. Other mixtures or single thermostable strains are used advantageously within the scope of the present disclosure.
(76) These novel strains survive long periods of storage in room temperature and are endurable. Thus, food products containing the strains of the present disclosure have a long shelf life and are easily included in different food products.
(77) In yet another embodiment, the probiotic food products of the present disclosure comprising thermo-stable strain(s) of Lactobacillus provide for enhanced nutrition and help in eliminating disorders relating to gut, the disorder being selected from a group comprising but not limiting to irritable bowel syndrome, different forms of diarrhea, irritable bowel disorder, constipation, lactose intolerance and any combinations thereof and vaginal infections.
(78) In still another embodiment, the food product is selected from a group comprising but not limiting to, beverage, yogurt, dairy product, nectar, fruit juice, energy drink, bakery food, chocolate, cereal, soup and combinations thereof.
(79) In still another embodiment, the food product having heat stable or thermostable strain(s) of micro-organism(s) is referred to as probiotic food products within the ambit of the present disclosure.
(80) In still another embodiment, the present disclosure provides probiotic food products which involve heating at high temperatures, especially those required to make hot beverages like soup, tea, coffee, chocolates, cereals and other food products as the probiotic strains of the present disclosure are able to withstand high temperatures.
(81) In still another embodiment, the thermo-stable strain(s) in the food product of the present disclosure is able to withstand high temperature ranging from room temperature of about 25 C. to about 250 C.
(82) In yet another embodiment, the probiotic food products of the present disclosure eliminate the requirement of cold storage and thus are economically beneficial to the end user.
(83) In yet another embodiment, the food products of the present disclosure having heat stable strain(s) of the present disclosure when heated at a high temperature are not affected in terms of taste or efficacy of the probiotic culture.
(84) The present disclosure relates to a process of obtaining heat stable strain(s) of micro-organism(s) comprising act of fermentation and spray drying.
(85) The present disclosure also relates to a process of manufacturing a food product comprising thermo-stable strain(s) of micro-organism(s), wherein the process comprises act of mixing the thermo-stable strain(s) with at least one of the components of the food product at any time during preparation of the food product
(86) In an embodiment of the present disclosure, the probiotic food of the present disclosure has various advantages which are listed below: Able to survive the passage through the digestive system. Able to attach to the intestinal epithelia and colonise effectively and multiply. Able to maintain good viability. Able to utilise the nutrients and substrates in a normal diet. Non-pathogenic and non-toxic. Capable of exerting a beneficial effect on the host. Stability of desired characteristics during processing, storage and transportation. Anti-inflammatory, anti-mutagenic and immuno-stimulatory in nature.
(87) Thus, the present disclosure provides heat stable/thermo-stable microbial strain(s) capable of surviving high temperatures, wherein these strain(s) incorporated in foods serve as probiotic foods and manage diseases/disorders.
(88) Throughout the specification, the food product comprising thermo-stable microbial strain(s) of the disclosure is also alternatively referred to as Probiotic food or Probiotic food product and the terms have the same meaning and scope.
(89) Throughout the specification, the thermo-stable microbial strain(s) of the disclosure are alternatively referred to as heat stable microbial strain(s), heat stable/thermo-stable microbial strain(s).
(90) The present disclosure is further elaborated with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner.
EXAMPLES
Example 1: Method for Producing Probiotics i.e. Heat Stable Strains of Lactobacillus plantarum
(91) The various steps involved in the production of probiotics are provided below briefly:
(92) Fermentation/Perfusion Technology:
(93) The mother strain of Lactobacillus spp. is grown in media at optimized temperatures. The thermo-stable Lactobacillus species of the present disclosure are manufactured using a fermentation procedure by employing perfusion mode. The perfusion technology improves the cell densities during fermentation.
(94) Spray Drying:
(95) This step is carried out in order to obtain powdered form of probiotics, wherein the medium containing the strains is sprinkled on to the walls of the dryer and dried by subjecting them to a temperature of about 90 C.-120 C. The down streaming process for the Lactobacillus species is completed with a custom built spray drier wherein the microbial strains/probiotics are powdered.
(96) Testing (QA/QC):
(97) A complete microbiological analysis is performed for viable count colony forming units/g. of Lactobacillus spp on the spray dried product.
Example 2: Method of Formulating Food Products Containing Lactobacillus Heat Stable Strains
(98) The flow chart for the manufacture of probiotic food of the present disclosure is provided in
(99) As observed, the probiotic culture is taken in an inoculum vial and then added to a shake flask. The medium prepared is added into the Seed fermenter along with the probiotic strain from the shake flask. The entire culture is then transferred into a Production fermenter and allowed to grow. The cells of the culture are then filtered or centrifuged, and spray dried or vacuum dried to obtain the cells in powder form. This is followed by Formulation of the probiotic, after which the product is packed and labeled.
(100) After preparation of the probiotic strains, they are formulated into the food items in order to produce various food products such as but not limiting to beverages, yogurt, dairy products, nectars, fruit juices, energy drinks, bakery food, chocolates, cereals, soup and combinations thereof.
(101) Probiotic Gummies Preparation:
(102) Ingredients for Batch Size of 230 gm: Phase AWater36 gm and Gelatin21.0 gm Phase BCorn syrup66.4 gm, Water66.3 gm and Sugar90.2 gm Phase CCitric acid0.9 gm, Colour Red #401.2 gm and Mixed berry flavour0.9 gm Lactobacillus acidophilus3.0 gm (From 1.510.sup.9) Lactobacillus plantarum0.28 gm (From 1.910.sup.11)
(103) Temperature: 70 C.
(104) Method of Preparation: 1. The ingredient of Phase A is weighed and is mixed and is kept at room temperature for 10-15 minutes. 2. The ingredients of the Phase B are weighed and dissolved. The phase B ingredients are kept for boiling until the mixture formed is thick and viscous. 3. Then ingredients of the Phase C is weighed & dissolved. 4. Then ingredients of Phase A and B are mixed uniformly and is added to ingredients of Phase C and mixed for 2-3 minutes. 5. When the temperature is around 70 C., the probiotics (Lactobacillus acidophilus/Lactobacillus plantarum) is weighed as per requirement (500 million cfu/gummy) is added and is mixed uniformly. 6. It is then poured into the moulds and kept for refrigeration for 45 minutes, for the gummies to set. 7. After 45 minutes the gummies are subjected to conditioning for 24 hours. The gummy samples are taken up for total viable count analysis to check viability of probiotic.
(105) Probiotic Soup Preparation:
(106) Ingredients for Batch Size of 100 ml: Knorr Soup (Ready-made)7.5 gm Lactobacillus acidophilus0.75 gm (From 1.610.sup.9) Lactobacillus plantarum0.065 gm (From 2.4810.sup.11)
(107) Temperature: 55 C.
(108) Method of Preparation: 1. A known amount (7.5 gm) of the Knorr soup is mixed with water (100 ml) so that there is no formation of lumps. 2. It is placed on a stove and brought to boil while stirring continuously, then simmered for 3 minutes. 3. After 3 minutes the soup temperature is brought down to 55.0 and maintained. 4. When the soup temperature is at 55 C., the probiotic (Lactobacillus acidophilus/Lactobacillus plantarum) is weighed as per requirement (160 million cfu/serving), added and mixed thoroughly. 5. The soup temperature is maintained at 55.0 and samples of the soup are drawn at 5th, 10.sup.th & 15th minute respectively. 6. Then the samples of different time intervals are taken up for Total viable count analysis to check the percentage of viable probiotic.
(109) Probiotic Chocolate Preparation:
(110) Ingredients Batch Size of 25 g: Wilton Candy Melts (Readymade chocolates chips)25 gm Lactobacillus acidophilus1.5 gm (From 1.610.sup.9) Lactobacillus plantarum0.13 gm (From 1.910.sup.11)
(111) Temperature: 70 C.
(112) Method of Preparation: 1. The pre-weighed chocolates chips are subjected to heating in the oven at 70.0 for 1 to 2 minutes until the chocolate chips melt. 2. Once the chocolate chips are melted and temperature is around 70 C., the probiotic (Lactobacillus acidophilus/Lactobacillus plantarum) is weighed as per requirement (100 million cfu/chocolate) is added and mixed thoroughly for 2 minutes to get a uniform distribution. 3. Then it is refrigerated for 20 minutes for the chocolate to set and then taken up for Total viable count analysis to check the percentage of viable probiotic.
(113) Probiotic Bread Preparation:
(114) Ingredients for Batch Size of 20 gm: All-purpose flour10.0 gm Baking soda0.4 gm Salt0.1 gm Butter1.23 gm Milk8.08 gm Lactobacillus acidophilus0.7 gm (From 1.4410.sup.11) Lactobacillus plantarum0.52 gm (From 2.3810.sup.11)
(115) Temperature: 240 C.
(116) Baking Time: 10 minutes
(117) Method of Preparation:
(118) 1. The bread dough is prepared using all-purpose flour, baking soda, salt, butter, milk and probiotic strain (Lactobacillus acidophilus/Lactobacillus plantarum) is added to it.
(119) 2. Initially the dough is prepared by adding part amount of all-purpose flour, milk and part amount of probiotic (L. acidophilus/L. plantarum) and mixed thoroughly for 10 to 15 minutes.
(120) 3. The dough is kept at room temperature for 42 hours and the remaining probiotic strain (L. acidophilus/L. plantarum) is kept for rehydration for 42 hours at room temperature.
(121) 4. After 42 hours the remaining ingredients like (Butter, milk) are mixed with the dough and kneaded for 15-20 minutes till it is not moist and sticky.
(122) 5. Then the dough is greased & kept for proofing for 2 hours at room temperature.
(123) 6. Then the dough is baked at 240.0 for 10 minutes.
(124) 7. After this, the bread is cooled for 10-15 minutes.
(125) 8. Then the bread is taken up for total viable cell count analysis to check the viability of probiotic.
(126) Probiotic Muffin Preparation:
(127) Ingredients for Batch Size of 20 gm: All-purpose flour4.82 gm Baking powder0.329 gm Sugar2.85 gm Unsalted butter4.82 gm Egg4.3 gm Dark chocolate chips2.85 gm Lactobacillus acidophilus1.42 gm (From 1.410.sup.11) Lactobacillus plantarum0.572 gm (From 1.910.sup.11)
(128) Temperature: 160 C.
(129) Baking Time: 15 minutes
(130) Method of Preparation: 1. The probiotics (Lactobacillus acidophilus/Lactobacillus plantarum) powder is suspended in 0.9% NaCl overnight (15-17 hours). 2. The muffin batter is prepared using unsalted butter, sugar and egg is beaten together for uniform mixing, to this the overnight probiotic (Lactobacillus acidophilus/Lactobacillus plantarum) powder is added to it. 3. The dry ingredients (All-purpose flour, baking powder, sugar are then weighed and are added to the liquid batter and mixed thoroughly for 5-10 minutes. 4. The muffin mould is then greased and the batter is kept for proofing for 1 hour at room temperature. 5. The oven is kept for pre-heating and the muffin batter is baked at 160.0 for 15 minutes. 6. After baking, the muffin is cooled for 5-10 minutes at room temperature. 7. Then the muffin is taken up for total viable count analysis to check the viability of probiotic.
(131) Probiotic Tea Powder:
(132) Ingredients: Twining's Tea & Brooke Bond2.0 gm Lactobacillus acidophilus0.1682 gm (From 1.4410.sup.11) Lactobacillus plantarum0.001 gm (From 1.910.sup.11)
(133) Temperature: 50 C., 60 C. & 70 C.
(134) Method of Preparation: 1. The pre-weighed tea bag is taken and is brewed at 100.0 for 2 minutes. 2. The temperature of tea solution is then brought down to 50 C., 60 C. and 70 C. and maintained. 3. Then the probiotic (Lactobacillus acidophilus/Lactobacillus plantarum) is weighed as per requirement (100 million cfu/tea bag) is introduced in the tea solution and mixed. 1. The tea solution temperature is maintained at 50 C., 60 C. and 70 C., samples of the tea solution are drawn at 1st minutes, 3rd minutes, 5th minutes, 7th minute and 10th minute respectively. 2. Then the samples of different time intervals are taken up for Total viable count analysis to check the percentage of viable probiotic.
(135) Probiotic Cookie:
(136) Ingredients for Batch Size of 15 gm: All-purpose flour7.51 gm Baking powder0.237 gm Sugar2.37 gm Unsalted butter2.68 gm Milk1.426 gm Salt0.01 gm Vanilla essence0.118 gm Lactobacillus acidophilus0.51 gm (From 1.4410.sup.11) Lactobacillus plantarum0.55 gm (From 2.3810.sup.11)
(137) Temperature: 180 C.
(138) Baking Time: 12 minutes
(139) Method of Preparation: 1. The probiotics (Lactobacillus acidophilus/Lactobacillus plantarum) powder is suspended in 0.9% NaCl overnight (15-17 hours) 2. The dry ingredients (All-purpose flour, salt, baking powder are then weighed and mixed thoroughly for 5-10 minutes. 3. Then butter is taken in a beaker & granulated sugar is added and beaten till it becomes fluffy after approximately 2-3 minutes. 4. Then to this the overnight probiotic solution (Lactobacillus acidophilus/Lactobacillus plantarum) pre-weighed as per requirement (5.0 billion cfu/cookie) is added and is mixed uniformly. 5. To this vanilla extract & milk is added and then all the ingredients are whisked together for 5-10 minutes. 6. Then the cookie dough is flattered on a parchment paper & chilled in refrigerator (4-8 C.) for 1 hour 7. After 1 hour, the oven is kept for pre-heating and the cookie dough is baked at 180.0 for 12 minutes. 8. After baking for 12 minutes the cookie is cooled for 5-10 minutes at room temperature. 9. Then the cookie is taken up for total viable count analysis to check the viability of probiotic.
Example 3: Heat Stability of Thermostable Strains
Example 3A
(140) The stability of the heat stable probiotic strain Lactobacillus plantarum is determined in this example.
(141) The stability of Lactobacillus plantarum at 90 C., 140 C. &160 C. is checked for different time intervals in a Hot Air Oven. The results are depicted in the table below.
(142) TABLE-US-00002 TABLE 2A Sl TVSC % of No. Time Interval (billion/g) Viability Initial assay 6.20 100% 1 90 C. for 30 sec 6.10 98.3% 2 90 C. for 60 sec 6.02 97.0% 3 90 C. for 120 sec 5.90 95.1%
(143) It is derived from the table above and
(144) TABLE-US-00003 TABLE 2B Sl TVSC % of No. Time Interval (billion/g) Viability Initial assay 6.20 100% 1 140 C. for 30 sec 6.10 98.3% 2 140 C. for 60 sec 5.8 93.5% 3 140 C. for 120 sec 5.5 88.7%
(145) It is derived from the table above and
(146) TABLE-US-00004 TABLE 2C Sl TVSC % of No. Time Interval (billion/g) Viability Initial assay 6.20 100% 1 160 C. for 2 min 3.20 52.4% 2 160 C. for 5 min 0.68 million 11.0% 3 160 C. for 10 min 0.0% 0.00%
(147) It is derived from the table above and
Example 3B
(148) The stability of the heat stable probiotic strain Lactobacillus acidophilus TSP-La-1 is determined in this example.
(149) The stability of Lactobacillus acidophilus at 90 C., 140 C. &160 C. is checked for different time intervals in a Hot Air Oven. The results are depicted in the table below.
(150) TABLE-US-00005 TABLE 2D TVCC Sl (billion % of No. Time Interval cfu/gm) Viability Initial assay 2.16 100% 1 90 C. for 30 sec 2.10 97.2% 2 90 C. for 60 sec 1.98 91.6% 3 90 C. for 120 sec 1.75 81.0%
(151) Conclusion:
(152) The above data and
(153) TABLE-US-00006 TABLE 2E TVCC Sl (billion % of No. Time Interval cfu/g) Viability Initial assay 2.16 100% 1 140 C. for 30 sec 2.08 96.2% 2 140 C. for 60 sec 1.90 89.3% 3 140 C. for 120 sec 1.55 71.7%
(154) Conclusion:
(155) The above data and
(156) TABLE-US-00007 TABLE 2F TVCC Sl (billion % of No. Time Interval cfu/gm) Viability Initial assay 2.1 100% 1 160 C. for 2 min 0.86 4.09% 2 160 C. for 5 min 0.0 0.0% 3 160 C. for 10 min 0.0 0.0%
(157) Conclusion:
(158) The above data and
Example 3C
(159) During the preparation of Probiotic gummies, chocolates and soup, the probiotic strains are subjected to high temperatures where the probiotic strains are present/added. The heat stability exhibited by these probiotic strains in the food products are determined by checking their viability.
(160) As shown in Table 3A below and
(161) TABLE-US-00008 TABLE 3A TVCC (Million Sl cfu/ % of No. Time Interval gummy) Viability Initial Assay 500 100% 1 70 C. for 1 min 485 97% 2 70 C. for 2 minutes 475 95% 3 70 C. for 3 minutes 460 92%
(162) As shown in Table 3B below and
(163) TABLE-US-00009 TABLE 3B TVCC Sl (Million cfu/ % of No. Time Interval chocolate) Viability Initial Assay 150 100% 1 55 C. for 1 min 140 93.3% 2 55 C. for 2 minutes 125 83.3% 3 55 C. for 3 minutes 102 68.0%
(164) The above results depict that the Lactobacillus acidophilus is stable up to 83% and viable at 55 C. for up to 2 minutes and is 68.0% viable at 55 C. up to 3 minutes.
(165) As shown in Table 3C below and
(166) TABLE-US-00010 TABLE 3C TVCC % Sl (Million of No. Time Interval cfu/ml) Viability Initial Assay 160 100% 1 55 C. for 5 min 91.6/ml 57.2 2 55 C. for 10 minutes 42.3/ml 26.4 3 55 C. for 15 minutes 0.94/ml 0.59
(167) The same experiment as above is performed by introducing Lactobacillus plantarum strains into soup and checking the viability at 55 C. and 75 C., where the viability is found to be good, as shown in Table 3D.
(168) TABLE-US-00011 TABLE 3D TVCC % Sl (Million of No. Time Interval cfu/ml) Viability Initial Assay 160 100% 1 55 C. for 5 min 7.8 4.87% 2 55 C. for 10 minutes 3.7 2.31% 3 55 C. for 15 minutes 0.27 0.16%
Example 3D
(169) Lactobacillus plantarum (TSP-Lp1) strain and Lactobacillus acidophilus TSP-La1 strain is incorporated into cookie dough, cookies are baked and their stability is checked at 180 C. The results are provided in Tables 4A and 4B below.
(170) TABLE-US-00012 TABLE 4A Lactobacillus acidophilus TSP-La1 TVCC (Billion/ % of Time Interval cookie) Viability Intial assay 5.0 100% 1 180 C. for 12 min 2.76 55%
(171) TABLE-US-00013 TABLE 4B Lactobacillus plantarum (TSP-Lp1) TVCC (Billion/ % of Time Interval cookie) Viability Intial Assay 5.0 100% 1 180 C. for 12 min 1.85 37%
(172) It is derived from Tables 4A and 4B, and
Example 4: Heat Stability of Thermostable Strains in Muffins Food Product
(173) The probiotic muffins are prepared by adding the Lactobacillus acidophilus TSP-La1 and Lactobacillus plantarum TSP-Lp1 strains to them during their preparation. The muffins are baked in an oven at 160 C. for 15 minutes.
(174) TABLE-US-00014 TABLE 5A TVCC (Billion % of Time Interval cfu/muffin) Viability Initial assay 10.0 100% 1 160 C. for 15 min 7.5 75%
(175) TABLE-US-00015 TABLE 5B TVSC % of Time Interval (Billion cfu/muffin) Viability Initial assay 5.0 100% 1 160 C. for 15 min 3.85 77%
(176) It is observed from Tables 5A-5B and
Example 5A: Heat Stability of Lactobacillus acidophilus in Bread Food Product
(177) This study is conducted to check the stability of Lactobacillus acidophilus in bread which is baked in oven at temperatures above 200 C. The Table No. 6 as depicted below showcases that the Lactobacillus acidophilus strains of the present disclosure are stable at temperatures as high as 240 C. for a period of about 10 minutes.
(178) TABLE-US-00016 TABLE 6 TVCC (Billion % of Time Interval cfu/bread) Viability Initial assay 5.0 100% 1 240 C. for 10 min 3.0 60%
(179) Thus, it is clear that the Lactobacillus acidophilus strains of the present disclosure are stable in extremely high temperatures like 240 C. with a viability of 60% and thereby provide for efficient heat stable strains.
(180) The above data in the table and
Example 5B: Heat Stability of Lactobacillus plantarum in Bread Food Product
(181) This study is conducted to check the stability of Lactobacillus plantarum in bread which is baked in oven at temperatures above 200 C. This study showcases that the Lactobacillus plantarum strains of the present disclosure are stable at temperatures as high as 240 C. for a period of about 10 minutes.
(182) TABLE-US-00017 TABLE 7 TVCC % of Time Interval (Billion/bread) Viability Initial assay 5.0 100% 1 240 C. for 10 min 0.5 10%
Example 6A: Heat Stability of Lactobacillus acidophilus in Tea at 50 C. and 60 C. for Different Time Intervals
(183) It is stated in the prior art that Lactobacillus species are not viable in liquid medium. However, when the Lactobacillus acidophilus TSP-La1 of the present disclosure is formulated into a tea product (Twining's Tea), which is later suspended in water while brewing the tea, significant amount of viability of about 39% is observed at 50 C. and 60 C. for up to 7 minutes.
(184) TABLE-US-00018 TABLE 8A Sl TVCC No. Time Interval (million cfu/tea bag) % of Viability Initial Count 100 million 100% 1 50 C. for 1 min 65.6 million 65.6% 2 50 C. for 3 min 50.0 million 50.0% 3 50 C. for 5 min 48.0 million 48.0% 4 50 C. for 7 min 39.0 million 39.0% 5 50 C. for 10 min 1.0 million 1.0%
(185) TABLE-US-00019 TABLE 8B Sl TVCC (million No. Time Interval cfu/tea bag) % of Viability Initial Count 100 million 100% 1 60 C. for 1 min 43.0 million 43.0% 2 60 C. for 3 min 28.36 million 28.36% 3 60 C. for 5 min 25.60 million 25.60% 4 60 C. for 7 min 1.41 million 1.41% 5 60 C. for 10 min 0.84 million 0.84%
(186) From the above data in the tables and from
Example 6B: Heat Stability of Lactobacillus plantarum in Tea at 50 C. and 60 C. for Different Time Intervals
(187) It is stated in the prior art that Lactobacillus species are not viable in liquid medium. However, when the Lactobacillus plantarum TSP-Lp1 of the present disclosure is formulated into a tea product (Twining's Tea), which is later suspended in water while brewing the tea, significant amount of viability is observed at 50 C. and 60 C.
(188) TABLE-US-00020 TABLE 9A TVCC Sl No. Time Interval (million cfu/tea bags) % of Viability Initial Count 100 million 100% 1 60 C. for 1 min 29.0 million 29.0% 2 60 C. for 3 min 26.0 million 26.0% 3 60 C. for 5 min 4.1 million 4.1% 4 60 C. for 7 min 0.0 million 0.0% 5 60 C. for 10 min 0.0 million 0.0%
(189) TABLE-US-00021 TABLE 9B TVCC Sl No. Time Interval (million cfu/tea bags) % of Viability Initial Count 100 million 100% 1 60 C. for 1 min 17.5 million 17.5% 2 60 C. for 3 min 2.75 million 2.75% 3 60 C. for 5 min 0.0036 million 0.0036% 4 60 C. for 7 min 0.0 million 0.0% 5 60 C. for 10 min 0.0 million 0.0%
Example 7: Acid Tolerance of Lactobacillus acidophilus at Different pH and Time Intervals
(190) The Lactobacillus acidophilus strains are subjected to extreme pH conditions within the range of 1.5 to 3.0 to ascertain that the food products containing these strains are able to survive the low acidity of the stomach during digestion. Total plate counts for Lactobacillus acidophilus on GYEA agars at different pH values of 1.5, 3.0 and 7.2 (control) over 1.5 hour intervals. The table no. 10 below depicts the plate viability of LA strains when subjected to pH 1.5, 2.5 3.0 and 7.2 for 0.5, 1.5 and 3 hours. The period of 0 hour is the time when the cultures are plated immediately for assay upon being exposed to PBS with different pH values.
(191) TABLE-US-00022 TABLE 10 Total plate counts (log.sub.9 CFU/mL) % of % of % of Strain pH 0 hr Viability 1.5 hr Viability 3.0 hr Viability Lactobacillus 1.5 0.39 32.23% 0.0% 0.0% acidophilus 3.0 0.99 81.61% 0.95 78.5% 0.92 76.03% TSP La-1(TPPL- 7.2 1.21 .sup.100% 1.05 86.77% 1.0 82.64% ACDHS100)
(192) It is observed from the table above and
(193) Thus, the thermo-stable strains of the present disclosure are suitable for being incorporated into food products and act beneficially in the environment of the stomach.
Example 8: Acid Tolerance of Lactobacillus plantarum at Different pH and Time Intervals
(194) The same acid tolerance test as provided in Example 7 is conducted with Lactobacillus plantarum TSP-Lp1 and similar results are observed. The table no. 11 below indicates the viability % of the LP strains subjected to the low pH usually present in the stomach. The period of 0 hour is the time when the cultures are plated immediately for assay upon being exposed to PBS with different pH value.
(195) TABLE-US-00023 TABLE 11 Total plate counts (log.sub.9 CFU/mL) % of % of % of Strain pH 0 hr Viability 1.5 hr Viability 3.0 hr Viability Lactobacillus 1.5 0.76 47.20% 0.0% 0.0 0.0% plantarum 2.5 0.84 52.17% 0.19 11.8% 8 cfu/ml 0.004% TSP-Lp1 3.0 1.20 74.50% 0.58 36.02% 0.11 6.83% 7.2 1.61 .sup.100% 1.20 74.50% 1.03 63.90%
(196) From the above results in the table and
(197) Thus, the thermo-stable strains of the present disclosure are suitable for being incorporated into food products and act beneficially in the environment of the stomach.
Example 9: Antibiotic Susceptibility Profiles of Lactobacillus acidophilus by the Disk Diffusion Method
(198) In order to ascertain the sensitivity of the thermostable strains in the food products towards antibiotics, Antibiotic susceptibility profile for Lactobacillus acidophilus is created by disk diffusion method. The susceptibility criteria used is established by Charteris et al. The Lactobacillus acidophilus is grown in Man Rogosa Sharpe Agar (MRSA).
(199) Lactobacillus MRS Agar M641Method of Preparation
(200) Lactobacillus MRS Agar is recommended for cultivation of all Lactobacillus species.
(201) Composition
(202) TABLE-US-00024 Ingredients Gms/Litre Proteose peptone 10.000 Beef extract 10.000 Yeast extract 5.000 Dextrose 20.000 Poly sorbate 80 1.000 Ammonium citrate 2.000 Sodium acetate 5.000 Magnesium sulphate 0.100 Manganese sulphate 0.050 Di potassium phosphate 2.000 Agar 12.000 Final pH (at 25 C.) 6.5 0.2
(203) 67.15 grams is suspended in 1000 ml distilled water and heated to boiling to dissolve the medium completely. It is sterilized by autoclaving at 15 lbs pressure (121 C.) for 20 minutes, mixed well and poured into sterile Petri plates.
(204) The table below depicts the results carried out for a list of common antibiotics. The concentration of the tested antibiotics has been included depending upon their minimum inhibitory concentrations.
(205) TABLE-US-00025 TABLE 12 MIC Resistant/ Sl No Antibiotic Concentration Results Sensitive STRAIN: Lactobacillus acidophilus TSP-La1 01. Ampicillin 10 g 10 mm Resistant 02. Amoxicillin 30 g 32 mm Sensitive 03. Ciprofloxacin 10 g 22 mm Sensitive 04. Cephalothin 30 g 25 mm Sensitive 05. Chloramphenicol 30 g 29 mm Sensitive 06. Clindamycin 2 g 31 mm Sensitive 07. Erythromycin 15 g 14 mm Resistant 08. Gentamycin 10 g 10 mm Resistant 09. Kanamycin 30 g No Zone Resistant 10. Methicillin 5 g 30 mm Sensitive 11. Oxacillin 1 g No Zone Resistant 12. Rifampicin 5 g 35 mm Sensitive 13. Streptomycin 10 g 18 mm Sensitive 14. Tetracycline 30 g 11 mm Resistant 15. Vancomycin 30 g No Zone Resistant
(206) It is seen from the table above that the Lactobacillus acidophilus strain of the present disclosure is sensitive to Amoxicillin, Ciprofloxacin, Cephalothin, Chloramphenicol, Clindamycin, Methicillin, Rifampicin and Streptomycin and is resistant to Ampicillin, Erythromycin, Gentamycin, Kanamycin, Oxacillin, Tetracycline and Vancomycin.
Example 10: Antibiotic Susceptibility Profiles of Lactobacillus plantarum by the Disk Diffusion Method
(207) The same set of study as the previous example is conducted using Lactobacillus plantarum strain of the present disclosure and the results for the study are depicted in Table No. 13 as below. The susceptibility criteria used is established by Charteris et al. The Lactobacillus acidophilus is grown in Man Rogosa Sharpe Agar,
(208) TABLE-US-00026 TABLE 13 MIC Resistant/ Sl No Antibiotic Concentration Results Sensitive STRAIN: Lactobacillus plantarum TSP-Lp1 01. Ampicillin 10 g No Zone Resistant 02. Amoxicillin 30 g 39 mm Sensitive 03. Ciprofloxacin 10 g 31 mm Sensitive 04. Cephalothin 30 g 23 mm Sensitive 05. Chloramphenicol 30 g 31 mm Sensitive 06. Clindamycin 2 g 34 mm Sensitive 07. Erythromycin 15 g 14 mm Sensitive 08. Gentamycin 10 g 8 mm Resistant 09. Kanamycin 30 g No Zone Resistant 10. Methicillin 5 g 15 mm Sensitive 11. Oxacillin 1 g No Zone Resistant 12. Rifampicin 5 g 43 mm Sensitive 13. Streptomycin 10 g 20 mm Sensitive 14. Tetracycline 30 g 14 mm Sensitive 15. Vancomycin 30 g No Zone Resistant
(209) From the above table, it is observed that Lactobacillus plantarum is sensitive to Amoxicillin, Ciprofloxacin, Cephalothin, Chloramphenicol, Clindamycin, Erythromycin, Methicillin, Rifampicin, Streptomycin and Tetracycline and resistant to Ampicillin, Gentamycin, Kanamycin, Oxacillin and Vancomycin.
Example 11: Bile Tolerance of Lactobacillus acidophilus at Different Bile Concentration Values and Time Intervals
(210) The strains of Lactobacillus acidophilus TSP-La1 thermostable strain of the present disclosure are tested for their tolerance towards various concentrations to Bile in the body. Therefore, various concentrations of Bile ranging from 0.3% to 1% are subjected to the LA strains of the present disclosure as depicted in Table no. 14 below.
(211) Total plate counts for Lactobacillus acidophilus on GYEA agars at different bile concentration of 0.3%, 0.5%, 1.0% and control over 0, 3.0 and 6.0 hour intervals are provided in the table below. The period of 0 hour is the time when the cultures are plated immediately for assay upon being exposed to PBS with different Bile concentration values.
(212) It is to be noted that the concentration for Bile in the body only reaches up to a maximum of 1%.
(213) TABLE-US-00027 TABLE 14 Total plate counts (log.sub.9 CFU/mL) Bile % of % of % of Strain Conc., 0 hr Viability 3.0 hr Viability 6.0 hr Viability Lactobacillus 0.3% 1.37 94.4% 1.29 82.69% 0.75 44.6% acidophilus 0.5% 1.18 81.3% 1.02 65.38% 0.59 35.1% TSP-La1 1.0% 1.05 72.4% 0.12 7.69% 0.02 1.19% Control 1.45 100% 1.56 .sup.100% 1.68 100%
(214) From the above table and
Example 12: Bile Tolerance of Lactobacillus plantarum at Different Bile Concentration and Time Intervals
(215) The same study as described in the previous example is carried out with Lactobacillus plantarum TSP-Lp1 strain of the present disclosure, wherein the strain is subjected to different concentrations of Bile i.e. 0.3% to 1%.
(216) The table below provides the viability % of the LP strains of the present disclosure when subjected to varying concentrations of Bile. Total plate counts for Lactobacillus plantarum on GYEA agars at different bile concentration of 0.3%, 0.5%, 1.0% and control aver 0, 3.0 and 6.0 hour intervals are provided. The period of 0 hour is the time when the cultures are plated immediately for assay upon being exposed to PBS with different Bile concentration values.
(217) TABLE-US-00028 TABLE 15 Total plate counts (log.sub.9 CFU/mL) Bile % of % of % of Strain Conc., 0 hr Viability 3.0 hr Viability 6.0 hr Viability Lactobacillus 0.3% 1.80 .sup.100% 1.50 83.33% 1.06 58.33% plantarum 0.5% 1.55 86.11% 0.98 54.44% 0.71 39.44% TSP-La1(TPPL- 1.0% 1.20 66.66% 0.48 26.66% 0.09 5.0% PLTHS100) Control 1.80 .sup.100% 2.02 .sup.100% 2.31 .sup.100%
(218) From the above table and
Example 13: Tests for the Identification of Lactobacillus acidophilus
(219) In this study, identification studies are carried oust to ascertain the morphological, biochemical and physiological behavior of the strains of the present disclosure. As seen in the table below, the strain of Lactobacillus acidophilus TSP-La1 is identified as Bacillus (Rod), Gram-positive and non-motile.
(220) Gram Staining Procedure
(221) Materials Required
(222) Clean glass slides, Inoculating loop, Bunsen burner, Microscope, Immersion oil, Saline and Cultures.
(223) Reagents
(224) 1. Primary StainCrystal Violet
(225) 2. MordantGrams Iodine
(226) 3. DecolourizerEthyl Alcohol
(227) 4. Secondary StainSafranin
(228) Gram Stain Procedure
(229) 1. The slide with heat fixed smear is placed on staining tray.
(230) 2. The smear is flooded with crystal violet and kept aside for 1 minute.
(231) 3. The slide is tilted slightly and gently rinsed with tap water or distilled water using a wash bottle.
(232) 4. The smear is flooded with Gram's iodine and kept aside for 1 minute.
(233) 5. The slide is tilted slightly and gently rinsed with tap water or distilled water using a wash bottle. The smear appears as a purple circle on the slide.
(234) 6. It is decolorized using 95% ethyl alcohol or acetone. The slide is tilted slightly and the alcohol is applied drop by drop for 5 to 10 seconds until the alcohol runs almost clear.
(235) 7. The slide is immediately rinsed with water.
(236) 8. The slide is flooded with safranin to counter-stain and kept aside for 45 seconds.
(237) 9. The slide is tilted slightly and gently rinsed with tap water or distilled water using a wash bottle.
(238) 10. The slide is blotted dry with bibulous paper.
(239) 11. The smear is viewed using a light-microscope under oil-immersion.
(240) Motility Testing Procedure
(241) Soft Agar Stabbing (Tube Method)
(242) Materials Required Two test tubes with Nutrient Agar medium Inoculation Stab needle Over night grown Cultures18-24 hrs (Lactobacillus acidophilus & Lactobacillus plantarum)
(243) Procedure 1. The tubes of Nutrient Agar media are labeled with the names of the organisms. 2. The inoculating stab needle is flamed and cooled and inserted into the culture after flaming the neck of the tube. 3. The cap is removed from the tube of medium, the neck is flamed, and stabbed of the way down to the bottom. The neck of the tube is flamed again before returning the cap to the tube. 4. The tubes are incubated at 37 C. for 24 to 48 hours. 5. The cultures are examined for the presence or absence of a precipitate along the line of the stab inoculation.
(244) TABLE-US-00029 TABLE 16 PARAMETER RESULTS Lactobacillus acidophilus TSP-La 1 Morphology Bacillus (Rod) Gram Staining + Motility Non Motile Lactic Acid Production + Indole Test Catalase Test Gelatin Hydrolysis Test Starch Hydrolysis Test Sugar Fermentation Test Glucose (0.1%) +/ Dextrose (0.1%) +/ Sucrose (0.1%) +/+ FOS (0.1%) +/ Maltose (0.1%) +/ + is Positive. is Negative. +/ indicates the Production of Acid but does not produce Gas. +/+ indicates the production of both Acid and Gas.
Example 14: Tests for the Identification of Lactobacillus plantarum
(245) In this study, the same set of morphological, biological and physiological studies are conducted for the strain i.e. Lactobacillus plantarum TSP-Lp1 of the present disclosure. The table below lists the morphological and physiological characteristics of the LP strain.
(246) TABLE-US-00030 TABLE 17 PARAMETER RESULTS Lactobacillus plantarum TSP-Lp 1 Microscopy Bacillus (Rod) Gram Staining + Motility Non Motile Lactic Acid Production + Indole Test Catalase Test Gelatin Hydrolysis Test Starch Hydrolysis Test SUGAR FERMENTATION TEST Glucose (0.1%) +/ Dextrose (0.1%) +/ Sucrose (0.1%) +/ FOS (0.1%) +/ Maltose (0.1%) +/ + is Positive. is Negative. +/ indicates the Production of Acid but does not produce Gas. +/+ indicates the production of both Acid and Gas.
(247) As seen in the table above, the strain of Lactobacillus plantarum TSP-Lp1 is identified as Bacillus (Rod), Gram-positive and non-motile.
Example 15: Carbohydrate Utilization
(248) This example establishes that the thermo-stable strains of the present disclosure are able to utilize the nutrients and substrates in a normal diet.
(249) Materials Required
(250) 1. Phenol Red Carbohydrate Fermentation Broth.
(251) 2. Bacterial culture.
(252) 3. Inoculation loop.
(253) 4. Incubator (37. C).
(254) I. Preparation of Carbohydrate Fermentation Broth
(255) 1. The trypticase, Sodium chloride, and Phenol red is weighed and dissolved in 100 ml distilled water and transferred into conical flasks.
(256) 2. 0.5% to 1% of desired carbohydrate is added into all flasks.
(257) 3. Inverted Durham tubes are inserted into all tubes, and the Durham tubes should be fully filled with broth.
(258) 4. The tubes are sterilized at 121 C. for 20 minutes.
(259) 5. It is important to not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in breaking down of the molecules and form compounds with a characteristic color and flavor.
(260) 6. The sugar is transferred into screw capped tubes or fermentation tubes and labelled properly.
(261) Ingredients of the Fermentation Broth:
(262) 1. Trypticase: 1 g
(263) 2. Carbohydrate: 0.5 g (Glucose, Dextrose, Sucrose, Maltose, Fructo Oligosaccharide (FOS)
(264) 3. Sodium Chloride: 0.5 g
(265) 4. Phenol red: 0.0189 mg
(266) Autoclaved at 121.0 for 20 minutes.
(267) II. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth
(268) 1. Each labeled carbohydrate broth is aseptically inoculated with both the thermo-stable strains of the present disclosure and un-inoculated tubes as kept as control tubes.
(269) 2. The tubes are incubated at 18-24 hours at 37 C.
(270) 3. The reaction is observed and results are provided in
(271) Results of Carbohydrate Fermentation Test
(272) It is observed from
(273) 1. Acid production: Changes the medium into yellow colorthe organism ferments the given carbohydrate and produces organic acids thereby reducing the pH of the medium into acidic.
(274) 2. Acid and Gas production: Changes the medium into yellow colorthe organism ferments the given carbohydrate and produce organic acids and gas. Gas production can be detected by the presence of small bubbles in the inverted Durham tubes.
(275) 3. Absence of fermentation in Control: The broth retains the red color. The organism is not present so carbohydrate is not utilized.
(276) Thus, from this example, establishes that the thermo-stable strains of the present disclosure are able to utilize the nutrients and substrates in a normal diet and thus aids in providing general good health when consumed.
Example 16: Comparison with Commercially Available Strains
(277) A) The objective of this experiment is to establish the stability of the temperature stable Lactobacillus acidophilus TSP-La1 strain of the present disclosure in comparison to commercially available Freeze dried Lactobacillus acidophilus at 70 C. in chocolates.
(278) Temperature: 70 C.
(279) Probiotic Strain: Temperature stable Lactobacillus acidophilus TSP-La1
(280) Commercial strain: Freeze dried Lactobacillus acidophilus
(281) TABLE-US-00031 TABLE 18 LIMIT Viability (MILLION CFU/ (MILLION CFU/ Sl No. PROBIOTIC STRAIN CHOCOLATE) CHOCOLATE) 1. Lactobacillus acidophilus Not less than 100 103 TSP-La1 2. Freeze dried Lactobacillus Not less than 100 00 acidophilus
(282) B) The objective of this experiment is to establish the stability of the temperature stable Lactobacillus plantarum TSP-Lp1 strain of the present disclosure in comparison to commercially available Freeze dried Lactobacillus plantarum at 70 C. in chocolates.
(283) Temperature: 70 C.
(284) Probiotic Strain: Temperature stable Lactobacillus plantarum TSP-Lp1
(285) Commercial strain: Freeze dried Lactobacillus plantarum.
(286) TABLE-US-00032 TABLE 19 LIMIT Viability (MILLION CFU/ (MILLION CFU/ Sl No. PROBIOTIC STRAIN CHOCOLATE) CHOCOLATE) 1. Lactobacillus plantarum Not less than 100 105 TSP-Lp1 2. Freeze dried Lactobacillus Not less than 100 00 plantarum
(287) It is observed from the table above that commercial strains as well as the strains of the present disclosure are present in chocolate at an amount more than 100 million cfu/chocolate. However, after heating the chocolate to 70 C., all the cells of the commercially available strain are destroyed, whereas the viability of the thermo-stable strains of the present disclosure is high. Thus, it is concluded that the strains of the present disclosure is viable and stable at 70 C. (Temperature used to make chocolates as prototype).
Example 17: Long Term Stability Studies
(288) The objective of this example is to establish the stability of the thermo-stable strains of the present disclosure over a period of 1-9 months.
Example 17A
(289) PRODUCT: Lactobacillus acidophilus TSP-La1
(290) STABILITY STUDIES: At Room Temperature at 252 C.
(291) PERIOD OF TESTING: 1, 2, 3, 6, 9, 12, 18, and 24 Months
(292) QUANTITY: 50 gm
(293) TABLE-US-00033 TABLE 20 Specification Limits Initial 1.sup.st Month 01. Description Beige to Light Beige to Light Beige to Light Brown Powder Brown Powder Brown Powder 02. Loss on Drying NMT 5.0% 3.2% 3.6% 03. Total Yeast & NMT 100 cfu/gm <10 cfu/gm <10 cfu/gm Mold Count 04. Total Viable NLT 1.5 B cfu/gm 1.65 B cfu/gm 1.60 B cfu/gm Cell Count 05. Pathogens Should be Absent/10 gm Absent/10 gm Absent/10 gm 2.sup.nd Month 3.sup.rd Month 6.sup.th Month 9.sup.th Month 01. Beige to Light Beige to Light Beige to Light Beige to Light Brown Powder Brown Powder Brown Powder Brown Powder 02. 3.8% 4.1% 4.5% 4.79% 03. <10 cfu/gm <10 cfu/gm 10 cfu/gm 10 cfu/gm 04. 1.60 B cfu/gm 1.60 B cfu/gm 1.55 B cfu/gm 1.52 B cfu/gm 05. Absent/10 gm Absent/10 gm Absent/10 gm Absent/10 gm NLTNot less than NMTNot more than B cfu/gm: Billion cfu/gm.
(294) No significant changes are observed during the studies. Thus, it is concluded that the strain of the present disclosure Lactobacillus acidophilus TSP-La1 is stable for a period of up to 9 months at room temperature.
Example 17B
(295) In this example, probiotic gummy with strain Lactobacillus acidophilus TSP-La1 is stored at 252.0 and relative humidity of 605% relative humidity. Tests are performed for color and flavor. Assays are also performed. The tests are performed each month for a period of 6 months.
(296) Assay ATotal yeast and mould count.
(297) Assay BTotal viable cell count.
(298) The results are provided below.
(299) TABLE-US-00034 TABLE 21 Period of Assay Testing Colour Flavour A B Initial Red Mixed Berry <10 490 1.sup.st Month Red Mixed Berry <10 507 2.sup.nd Month Red Mixed Berry <10 500 3.sup.rd Month Red Mixed Berry <10 490 4.sup.th Month Red Mixed Berry <15 480 5.sup.th Month Red Mixed Berry 20 480 6.sup.th Month Red Mixed Berry 20 470 LIMITS Red Mixed Berry NMT 100 cfu/g NLT 450 Million cfu/gummy NLTNot less than RTRoom Temperature NMTNot more than
(300) No Significant change is observed during the studies. Thus, the probiotic strain containing product is stable at Room temperature condition for 6 months.
Example 18: Stability Studies in Cookies
(301) The objective of this example is to establish the stability of the thermo-stable strains of the present disclosure in cookies at 20 C. for a period of 6 days.
(302) Probiotic cookies are prepared with a blend of 2 strains Lactobacillus acidophilus TSP-La1 & Lactobacillus plantarum TSP-Lp1 of the present disclosure at 12.5 gm each Cookie. The stability studies are carried out at 202 C. and the humidity is maintained at 505%. The results are provided in the table below.
(303) TABLE-US-00035 TABLE 22 SI No Test Parameters Limits Initial Day 1 Day 2 Day 4 Day 5 Day 6 01. Total Viable Cell NLT 5.0 Billion 4.0 Billion 4.10 Billion 4.46 Billion 4.50 Billion 4.63 Billion 4.86 Billion Count cells/cookie cells/cookie cells/cookie cells/cookie cells/cookie cells/cookie cells/cookie
(304) It is concluded from the table above that the strains of the present disclosure are viable for up to 97.2% for 6 days at 20 C.
Example 19: Genome Sequence of the Thermostable Strains
(305) In this example, the 16S rRNA gene sequencing of the strains of the present disclosure is provided. The thermostable microorganism Lactobacillus plantarum has genomic sequence set forth in SEQ ID No.1. The thermostable microorganism Lactobacillus acidophilus has genomic sequence set forth in SEQ ID No.2.
(306) TABLE-US-00036 SEQIDNo.1-LactobacillusplantarumTSP-Lpl AATCATCTGTCCACCTTAAGGCGGCTGGGTTCCTAAAAGGG TTACCCCACCGACTTTGGGTGGTTACAAACTCTCATGGGT GTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACC GCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCA TGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGC TTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTT GTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAG GGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTT TGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTG GCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAAC CCAACATCTCACGACACGAGCTGACGACAACCATGCACCA CCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAG ATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGT AGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCC CCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGG GCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTA TGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACT TTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTT CGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGC TACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTT CCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTT TCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGC CCAATAAATCCGGACAACGCTTGCCACCTACGTATTACCG CGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAAA TACCGTCAATACCTGAACAGTTACTCTCAGATATGTTCTT CTTTAACAACAGAGTTTTACGAGCCGAAACCCTTCTTCAC TCACGCGGCGTTGCTCCATCAGACTTTCGTCCATTGTGGA AGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTG TCTCAGTCCCAATGTGGCCGATTACCCTCTCAGGTCGGCT ACGTATCATTGCCATGGTGAGCCGTTACCCCACCATCTAG CTAATACGCCGCGGGACCATCCAAAAGTGATAGCCGAAGC CATCTTTCAAACTCGGACCATGCGGTCCAAGTTGTTATGC GGTATTAGCATCTGTTTCCAGGTGTTATCCCCCGCTTCTG GGCAGGTTTCCCACGTGTTACTCACCAGTTCGCCACTCAC TCAAATGTAAATCATGATGCAAGCACCAATCAATACCAG AGTTCGTTCGACTTGCATGTATTAGGCACGCCGCCAGCGT TCGTCCT SEQIDNo.2-LactobacillusacidophilusTSP-La1 AGAGTTTGCAATGCCCAAAGCCGGTGGCCTAACCTTCGGC AAGGAGCCGACTAAGGCAGGGCAGATGACTGGGGTGAAGT CCTAACAAGGTAGCCGTACGAGAACCTGCGGCTGGATCAC CTCCTTTCTAAGGAAGCGAAGGATATGGAGAGTAGAAATA CTAAGAGAAGTATCCAGAGCAAGCGGAAGCACACTGAGAA ACTTTGCTTAGTTTTGAGGGTAACTCCTCAAGAGAGTTAC TACATTGAAAACTGAATATAATCCAATCAAAACACCGCCA CACTAAAGGAGAACATACTGTAGAGCGACCGATAACGAA TTCTTAATTAGGTCAAGTAGAAAAGGGCGCACGGTGAAT GCCTTGGCACTGAAAGCCGATGAAGGACGCGACTAACTAC GAAAGTCTTCGGGAGCCGTAAGTAGGCTTTGATCCGGAG GTCTCCGAATGGGGAACCCAGCATGTGCAGAATGCTATCC TTAAGTGAATACATAGCTTAAGGAGGGAACACGCAGCGA
(307) In view of the above examples and results, it is evident that the strain(s) of the present disclosure when incorporated in the food products are viable at high temperatures.
(308) Thus, the thermo-stable strain(s) of the present disclosure eliminate the requirement of cold storage and thus are economical and at the same time provide for enhanced nutrition to the consumers.
(309) The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
(310) While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.