Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of biotechnology and, in specific, relates to a method that enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby developing cost-effective, easy-to-administer probiotic vaccines suitable for all age groups.
Background of the invention:
[0002] The development of safe and effective vaccines has played a crucial role in combating infectious diseases. However, traditional vaccination methods often rely on injections, which can be inconvenient and cause needle phobia, particularly in children. Edible vaccines offer a promising alternative, proposing the delivery of vaccines through the consumption of food items. This approach has the potential to improve vaccine acceptance, compliance, and overall immunization rates, especially in regions with limited access to healthcare facilities.

[0003] Lactobacillus, a genus of lactic acid bacteria commonly found in yogurt and fermented foods, has emerged as a promising candidate for delivering edible vaccines. The lactobacillus bacteria are generally considered safe for human consumption and have been shown to stimulate both mucosal and systemic immune responses. Additionally, the lactobacillus can be genetically modified to express specific antigens from various pathogens, essentially turning them into edible vaccine capsules.

[0004] Despite the potential of lactobacillus-based edible vaccines, there are challenges associated with transforming these bacteria. Traditional methods for transforming lactobacillus often rely on expensive commercially purchased strains. This can be a significant barrier, particularly for researchers in developing countries. Furthermore, existing transformation protocols typically involve heat shock treatments lasting several minutes, which can negatively impact the viability and efficiency of the process.

[0005] In one prior art, EP0262516A2 discloses a method for genetically transforming gram-positive lactic acid-producing bacteria with DNA. The method refers to non-proto plasted lactic acid-producing bacterial cells are genetically transformed with DNA by incubating a mixture of the cells with the DNA in a physiologically acceptable transformation solution containing a trans­formation-promoting amount of a water-soluble lower polyalkylene glycol. However, the heat-shocking process of the incubated transformation mixture is performed from 2 to 10 min at a temperature that varies between 40 °C and 45 °C.

[0006] In another prior art, CN111944837B discloses an expression vector for expressing a COVID-19 antigen and a construction method of a genetic engineering lactobacillus oral vaccine. the method takes specificity Protein (SP1) or receptor-binding domain (RBD) protein of the COVID-19 as an immunogen, fuses dendritic cell-induced peptide DCpep, and utilizes food-grade lactobacillus as an immune antigen transfer vector to finally construct the genetic engineering lactobacillus oral vaccine for expressing the COVID-19 antigen. However, the transformation of the expression vector requires 2 to 10 min at the temperature that varies between 40 °C and 45 °C. The transformation of the expression vector did not yield efficient transformation.

[0007] By addressing all the above-mentioned problems, there is a need for a method that enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby developing cost-effective, easy-to-administer probiotic vaccines suitable for all age groups. There is also a need for a method that enables transformation of Lactobacillus bacteria isolated from natural homemade yogurt, thereby reducing the expenses associated with traditional transformation techniques significantly. There is also a need for a method that creates edible vaccines, which are easily consumable and suitable for all age groups, thereby eliminating the need for painful injections and improving compliance, especially in children. There is also a need for a method that offers a more cost-effective and potentially more accessible source of lactobacillus for vaccine development.

[0008] By addressing all the above-mentioned problems, there is also a need for a method that expands the application of probiotic vaccines for a wide range of diseases, including pandemic-related viruses like COVID-19 and other pathogens such as Hepatitis A, Pneumococcal, and Diphtheria tetanus pertussis. There is also a need for a method that develops edible vaccines to target specific diseases, such as human immunodeficiency virus (HIV) and coronavirus Disease (COVID). There is also a need for a method that utilizes a shorter heat shock duration during transformation, potentially improving efficiency and bacterial viability. There is also a need for a method that ensures the transformed Lactobacillus cells are stored and preserved in normal refrigerators, thereby enhancing the practicality and accessibility of the edible vaccines.
Objectives of the invention:
[0009] The primary objective of the present invention is to provide a method that enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby developing cost-effective, easy-to-administer probiotic vaccines suitable for all age groups.

[0010] Another objective of the present invention is to provide a method that enables the natural homemade yogurt-based transformation of lactobacillus with the pUC19 plasmid by heat shock at a temperature of at least 42 °C for a time period of at least 45 sec, thereby enhancing and analyzing the growth of transformed cells in a super optimal broth with catabolite repression (SOC) medium.

[0011] Another objective of the present invention is to provide a method that enables transformation of Lactobacillus bacteria isolated from natural homemade yogurt, thereby reducing the expenses associated with traditional transformation techniques significantly.

[0012] The other objective of the present invention is to provide a method that creates edible vaccines, which are easily consumable and suitable for all age groups, thereby eliminating the need for painful injections and improving compliance, especially in children.

[0013] The other objective of the present invention is to provide a method that offers a more cost-effective and potentially more accessible source of lactobacillus for vaccine development.

[0014] Yet another objective of the present invention is to provide a method that expands the application of probiotic vaccines for a wide range of diseases, including pandemic-related viruses like COVID-19 and other pathogens such as Hepatitis A, Pneumococcal, and Diphtheria tetanus pertussis.

[0015] Further objective of the present invention is to provide a method that ensures the transformed Lactobacillus cells are stored and preserved in normal refrigerators, thereby enhancing the practicality and accessibility of the edible vaccines.
Summary of the invention:
[0016] The present disclosure proposes a method for transformation of lactobacillus isolated from natural homemade yogurt to develop edible vaccines. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0017] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method that enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby developing cost-effective, easy-to-administer probiotic vaccines suitable for all age groups.

[0018] According to one aspect, the invention provides a method for transformation of lactobacillus bacteria to develop edible vaccines. At one step, a drop of natural homemade yogurt is poured onto a MRS (de Man, Rogosa, and Sharpe) agar plate. At one step, the poured drop of the natural homemade yogurt is streaked onto the surface of the MRS (de Man, Rogosa, and Sharpe) agar plate using a sterile inoculation loop to separate individual cells, thereby incubating the MRS agar plate overnight at a temperature of 37 °C to enable gram-positive bacterial colonies to form. At one step, the gram-positive bacterial colonies are collected with the sterile inoculation loop upon forming the gram-positive bacterial colonies on the MRS agar plate. A volume of 5 ml of liquid medium is poured in a centrifuge tube. The liquid medium is MRS broth, which is utilized to enable the growth of a gram-positive bacterial mini-culture.

[0019] At one step, the collected bacterial colonies are inoculated into 5 ml of the MRS broth in the centrifuge tube, thereby incubating the centrifuge tube overnight at the temperature of 37 °C to enable the growth of a gram-positive bacterial mini-culture. At one step, gram-positive bacterial cells are prepared by using the gram-positive bacterial mini-culture to make membrane of the gram-positive bacterial cells capable of taking up exogenous DNA. In one embodiment, initially the gram-positive bacterial mini-culture is refrigerated at the temperature of 4 °C for at least 10 min. Next, the centrifuge tube with the gram-positive bacterial mini-culture is centrifuged at the speed of at least 5000 rpm for at least 10 min to pellet the bacteria, thereby discarding the MRS broth and leaving the bacterial pellet.

[0020] Next, the bacterial pellet is mixed with at least 30 ml of prechilled and sterile 0.1 M Cacl2 solution in the centrifuge tube, thereby incubating the centrifuge tube for at least 30 min to make the solution more permeable to DNA. Next, the centrifuge tube is centrifuged at the speed of 5000 rpm for 10 min, thereby discarding the Cacl2 solution to obtain the gram-positive bacterial cells. The gram-positive bacterial cells are dispersed in 1 ml of the prechilled sterile Cacl2 solution, thereby making the dispersed gram-positive bacterial cells into one or more aliquots to enhance the bacteria's ability to take up exogenous DNA.

[0021] At one step, the gram-positive bacterial cells are transformed with at least one plasmid DNA by using heat shock at the temperature of at least 42 °C for at least 45 sec to obtain the heat-shocked cells, thereby creating a thermal imbalance that encourages DNA uptake. The plasmid DNA contains POL and RBD genes from HIV and COVID-19, respectively.

[0022] At one step, a super optimal broth with catabolite repression (SOC) medium is added to the heat-shocked cells to obtain a primary mixture. The primary mixture is incubated at the temperature of 37 °C for at least 1 hr to assist the heat-shocked cells in recovering from the stress of transformation, thereby obtaining transformed lactobacillus cells that start expressing the antibiotic resistance gene. At one step, the transformed lactobacillus cells are transmitted onto one or more MRS agar plates with a selective agent. The one or more MRS agar plates are incubated overnight at the temperature of 37 °C, thereby examining the plates for colonies to determine whether the transformed lactobacillus cells are effectively incorporated with the plasmid DNA and are resistant to the selective agent. At one step, the transformed lactobacillus cells are utilized to make edible products, thereby consuming the edible products to induce an immune response in the human body and acts as an edible vaccine.

[0023] In one embodiment, the heat shock method is performed for the transformation of the gram-positive bacterial cells with at least one plasmid DNA. Initially 100 µl of the gram-positive bacterial cells of each aliquot is mixed with at least 5 µl of the plasmid DNA to obtain a primary mixture. The primary mixture is incubated on ice for at least 30 min. Next, the primary mixture is subjected to a heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain a heat shocked culture.

[0024] Next, 105 µl of the heat shock culture of the gram-positive bacterial cells is mixed with at least 1 ml of the SOC medium to obtain a secondary mixture. The secondary is incubated at the temperature of 37 °C for 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene. Next, at least 50 mcg of the ampicillin is spreading onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells in treating the viral diseases. The transformed lactobacillus cells express viral proteins, which are used to create the edible vaccines for viruses like HIV and COVID-19. The transformed lactobacillus cells are formulated into the edible vaccine suitable for oral administration.

[0025] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0026] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0027] FIG. 1 illustrates a flowchart of a method for transformation of lactobacillus bacteria to develop edible vaccines, in accordance to an exemplary embodiment of the invention.

[0028] FIGs. 2A-2B illustrate pictorial representations of a MRS agar plate with streaking of the natural homemade yogurt, in accordance to an exemplary embodiment of the invention.

[0029] FIGs. 3A-3B illustrate pictorial representations of a transformation of lactobacillus with Human Immunodeficiency Virus (HIV) vector, in accordance to an exemplary embodiment of the invention.

[0030] FIGs. 4A-4B illustrate pictorial representations of a transformation of lactobacillus with a receptor-binding domain (RBD) plasmid, in accordance to an exemplary embodiment of the invention.

[0031] FIG. 5 illustrates a pictorial representation of a transformation of lactobacillus with a pUC19 plasmid, in accordance to an exemplary embodiment of the invention.

[0032] FIGs. 6A-6B illustrate pictorial representations of gram staining of lactobacillus (gram Positive bacteria), in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0033] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0034] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method that prepares and transforms lactobacillus isolated from the natural homemade yogurt for edible vaccines.

[0035] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of a method for transformation of lactobacillus bacteria to develop edible vaccines. The method offers a more cost-effective and potentially more accessible source of lactobacillus for vaccine development. The method expands the application of probiotic vaccines for a wide range of diseases, including pandemic-related viruses like COVID-19 and other pathogens such as Hepatitis A, Pneumococcal, and Diphtheria tetanus pertussis. The method ensures the transformed Lactobacillus cells are stored and preserved in normal refrigerators, thereby enhancing the practicality and accessibility of the edible vaccines.

[0036] At step 102, a drop of natural homemade yogurt is poured onto a selective agar plate to ensure the growth of only transformed gram-positive bacterial cells. The selective agar plate is a (de Man, Rogosa, and Sharpe) MRS agar plate. At step 104, the poured drop of the natural homemade yogurt is streaked onto the surface of the MRS agar plate using a sterile inoculation loop to separate individual cells, thereby incubating the MRS agar plate overnight at a temperature of 37 °C to enable gram-positive bacterial colonies to form. At step 106, the gram-positive bacterial colonies are collected with the sterile inoculation loop upon forming the gram-positive bacterial colonies on the MRS agar plate, and at least 5 ml of liquid medium is poured in a centrifuge tube. The liquid medium is MRS broth, which is utilized to enable the growth of a gram-positive bacterial mini-culture.

[0037] At step 108, the collected bacterial colonies are inoculated with at least 5 ml of the MRS broth in the centrifuge tube, thereby incubating the centrifuge tube overnight at the temperature of 37 °C to enable the growth of a gram-positive bacterial mini-culture. At step 110, gram-positive bacterial cells are prepared by using the gram-positive bacterial mini-culture to make membrane of the gram-positive bacterial cells capable of taking up exogenous DNA. In one embodiment, initially the gram-positive bacterial mini-culture is refrigerated at the temperature of at least 4 °C for at least 10 min. Next, the centrifuge tube with the gram-positive bacterial mini-culture is centrifuged at the speed of 5000 rpm for 10 min to pellet the bacteria, thereby discarding the MRS broth and leaving the bacterial pellet.

[0038] Next, the bacterial pellet is mixed with at least 30 ml of prechilled and sterile 0.1 M Cacl2 solution in the centrifuge tube, thereby incubating the centrifuge tube for at least 30 min to make the solution more permeable to DNA. Next, the centrifuge tube is centrifuged at the speed of 5000 rpm for 10 min, thereby discarding the Cacl2 solution to obtain the gram-positive bacterial cells. The gram-positive bacterial cells are dispersed in 1 ml of the prechilled sterile Cacl2 solution, thereby making the dispersed gram-positive bacterial cells into one or more aliquots to enhance the bacteria's ability to take up exogenous DNA.

[0039] At step 112, the gram-positive bacterial cells are transformed with at least one plasmid DNA by using heat shock at the temperature of at least 42 °C for at least 45 sec to obtain the heat-shocked cells, thereby creating a thermal imbalance that encourages DNA uptake. The plasmid DNA contains genes from both HIV and COVID-19, such as the HIV POL gene and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein.

[0040] At step 116, a super optimal broth with catabolite repression (SOC) medium is added to the heat-shocked cells to obtain a primary mixture. The primary mixture is incubated at the temperature of at least 37 °C for at least 1 hr to assist the heat-shocked cells in recovering from the stress of transformation, thereby obtaining transformed lactobacillus cells that start expressing the antibiotic resistance gene. At step 118, the transformed lactobacillus cells are transmitted onto one or more MRS agar plates with a selective agent. The one or more MRS agar plates are incubated overnight at the temperature of 37 °C, thereby examining the plates for colonies to determine whether the transformed lactobacillus cells are effectively incorporated with the plasmid DNA and are resistant to the selective agent. At step 120, the transformed lactobacillus cells are utilized to make edible products, thereby consuming the edible products to induce an immune response in the human body and acts as an edible vaccine.

[0041] In one embodiment, the heat shock method is performed for the transformation of the gram-positive bacterial cells with at least one plasmid DNA. Initially the at least 100 µl of the gram-positive bacterial cells of each aliquot is added with at least 5 µl of the plasmid DNA to obtain a primary mixture. The primary mixture is incubated on ice for at least 30 min. Next, the primary mixture is subjected to a heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain a heat shock culture.

[0042] Next, at least 105 µl of the heat shock culture of the gram-positive bacterial cells is added with at least 1 ml of the SOC medium to obtain a secondary mixture. The secondary mixture is incubated at the temperature of at least 37 °C for at least 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene. Next, at least 50 mcg of the ampicillin is spreading onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells in treating the viral diseases. The transformed lactobacillus cells express viral proteins, which are used to create the edible vaccines for viruses like HIV and COVID-19. The transformed lactobacillus cells are formulated into the edible vaccine suitable for oral administration.

[0043] According to another exemplary embodiment of the invention, FIGs. 2A-2B refer to pictorial representations (200, 202) of the MRS agar plate with streaking of the natural homemade yogurt.in one embodiment herein, the MRS agar plate is a selective growth medium primarily used for the cultivation of Lactobacillus species and other lactic acid bacteria. The MRS agar plate provides a nutrient-rich environment that supports the growth of Lactobacillus bacteria, which are often used in dairy fermentation and probiotic research.

[0044] In one embodiment herein, initially a drop of the natural homemade yogurt is poured on the MRS agar plate as shown FIGs 2A and 2B. the bacteria of the natural homemade yogurt is streaked onto the surface of the MRS agar plate in a specific pattern to separate individual bacterial cells, thereby Incubating the MRS agar plate overnight at the temperature of at least 37 °C to enable the gram-positive bacterial colonies to form on the MRS agar plate.

[0045] In one embodiment herein, the MRS agar plates in FIGs 2A and 2B represent Lactobacillus transformed with one of the plasmids, such as the HIV plasmid (containing the HIV Pol gene) or the RBD plasmid (containing the receptor binding domain from the coronavirus spike protein). The growth of colonies on the MRS agar plate enable effective transformation and subsequent growth of the transformed cells.

[0046] According to another exemplary embodiment of the invention, FIGs. 3A-3B refer to pictorial representations (300, 302) of the transformation of lactobacillus with Human Immunodeficiency Virus (HIV) vector. Initially the at least 100 µl of the gram-positive bacterial cells is added with at least 5 µl of the plasmid DNA that contains genes from the HIV vector, such as the HIV gene, thereby obtaining the primary mixture related to the HIV vector. The primary mixture is incubated on the cooling member for at least 30 min. Next, the primary mixture is subjected to the heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain the heat shock culture related the HIV vector.

[0047] Next, at least 105 µl of the heat shock culture of the gram-positive bacterial cells is added with at least 1 ml of the SOC medium to obtain the secondary mixture. The secondary is incubated at the temperature of at least 37 °C for at least 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene, as shown in FIG. 3A. Next, at least 50 mcg of the ampicillin is spreading onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells in treating the HIV disease. The transformed lactobacillus cells express viral proteins, which are used to create the edible vaccines for HIV virus, as shown in FIG. 3B. The transformed lactobacillus cells are formulated into the edible vaccine suitable for oral administration.

[0048] According to another exemplary embodiment of the invention, FIGs. 4A-4B refer to pictorial representations (400, 402) of the transformation of lactobacillus with the multi-disulfide receptor-binding domain (RBD) plasmid. Initially the at least 100 µl of the gram-positive bacterial cells is added with at least 5 µl of the (RBD) plasmid DNA that contains genes from the COVID-19, such as the COVID-19 gene, thereby obtaining the primary mixture related to the COVID-19. The primary mixture is incubated on the cooling member for at least 30 min. Next, the primary mixture is subjected to the heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain the heat shock culture related the COVID-19.

[0049] Next, at least 105 µl of the heat shock culture of the gram-positive bacterial cells is added with at least 1 ml of the SOC medium to obtain the secondary mixture. The secondary is incubated at the temperature of at least 37 °C for at least 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene, as shown in FIG. 4A. Next, at least 50 mcg of the ampicillin is spreading onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells in treating the COVID-19 disease. The transformed lactobacillus cells express viral proteins, which are used to create the edible vaccines for COVID-19, as shown in FIG. 4B. The transformed lactobacillus cells are formulated into the edible vaccine suitable for oral administration.

[0050] In one embodiment herein, the receptor-binding domain (RBD) is an immunogenic fragment from a virus that binds to a specific endogenous receptor sequence to gain entry into host cells. The receptor-binding domain (RBD) of the sars-cov-2 spike protein expressed in E. coli using a Sep-tag produces antisera interacting with the mammalian cell expressed spike (S1) protein. The E. coli is group of bacteria that usually lives in human gut microbiota without hurting humans. But some strains can make humans sick with watery diarrhea or a UTI.

[0051] According to another exemplary embodiment of the invention, FIG. 5 refers to a pictorial representation 500 of a transformation of lactobacillus with a pUC19 plasmid. In one embodiment herein, the transformation is a process that introduces new genetic material into a bacterial cell, allowing it to express new traits. The pUC19 plasmid is commonly used in molecular biology for cloning and expression studies. This plasmid carries an antibiotic resistance gene (ampR) that provides a selectable marker for identifying successful transformants. The transforming lactobacillus with pUC19 plasmid showcases the capability of these bacteria to incorporate and express exogenous DNA, which is pivotal for genetic engineering applications.

[0052] Initially the at least 100 µl of the gram-positive bacterial cells is added with at least 5 µl of the pUC19 plasmid DNA, thereby obtaining the primary mixture related to the COVID-19. The primary mixture is incubated on the cooling member for at least 30 min. Next, the primary mixture is subjected to the heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the pUC19 plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain the heat shock culture related the COVID-19. Next, at least 105 µl of the heat shock culture of the gram-positive bacterial cells is added with at least 1 ml of the SOC medium to obtain the secondary mixture. The secondary is incubated at the temperature of at least 37 °C for at least 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene. Next, at least 50 mcg of the ampicillin is spreading onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells in treating the viral diseases.

[0053] In one embodiment herein, the presence of colonies on ampicillin-containing MRS agar plates indicates successful transformation. Each colony arises from a single Lactobacillus cell that has taken up and expressed the pUC19 plasmid, specifically the ampR gene, enabling it to survive the antibiotic selection. The transformation of Lactobacillus with the pUC19 plasmid successfully demonstrates the bacteria's ability to incorporate and express new genetic material. This ability is crucial for various biotechnological applications, including the development of edible vaccines, metabolic engineering, and other genetic studies.

[0054] According to another exemplary embodiment of the invention, FIGs. 6A-6B refer to pictorial representations (600, 602) of gram staining of lactobacillus (gram Positive bacteria). In one embodiment herein, the Gram staining is a differential staining technique used to classify bacteria into Gram-positive and Gram-negative groups based on the structural differences in their cell walls. The staining process highlights the thick peptidoglycan layer in Gram-positive bacteria, which retains the crystal violet dye, appearing purple under the microscope. The gram staining confirms the gram-positive nature of Lactobacillus helps in identifying and characterizing the bacteria used in the experiments.

[0055] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure a method for transformation of lactobacillus isolated from natural homemade yogurt to develop edible vaccines is disclosed. The proposed invention provides the method that enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby developing cost-effective, easy-to-administer probiotic vaccines suitable for all age groups. The method enables the natural homemade yogurt-based transformation of lactobacillus with the pUC19 plasmid by heat shock at a temperature of at least 42 °C for a time period of at least 45 sec, thereby enhancing and analyzing the growth of transformed cells in a super optimal broth with catabolite repression (SOC) medium. The method enables transformation of Lactobacillus bacteria using natural homemade yogurt, thereby reducing the expenses associated with traditional transformation techniques significantly.

[0056] The proposed invention provides the method that creates edible vaccines, which are easily consumable and suitable for all age groups, thereby eliminating the need for painful injections and improving compliance, especially in children. The method offers a more cost-effective and potentially more accessible source of lactobacillus for vaccine development. The method expands the application of probiotic vaccines for a wide range of diseases, including pandemic-related viruses like COVID-19 and other pathogens such as Hepatitis A, Pneumococcal, and Diphtheria tetanus pertussis. The method ensures the transformed Lactobacillus cells are stored and preserved in normal refrigerators, thereby enhancing the practicality and accessibility of the edible vaccines.

[0057] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I/We Claim:
1. A method (100) for transformation of lactobacillus bacteria to develop edible vaccines, comprising:
pouring a drop of natural homemade yogurt onto a selective agar plate;
streaking the poured drop of the natural homemade yogurt onto the surface of the selective agar plate using a sterile inoculation loop to separate individual cells, thereby incubating the selective agar plate overnight at a temperature of at least 37 °C to enable gram-positive bacterial colonies to form;
collecting the gram-positive bacterial colonies that are formed on the selective agar plate with the sterile inoculation loop and pouring at least 5 ml of liquid medium in a centrifuge tube;
inoculating the collected bacterial colonies into at least 5 ml of the liquid medium in the centrifuge tube, thereby incubating the centrifuge tube overnight at the temperature of 37 °C to enable the growth of a gram-positive bacterial mini-culture;
preparing gram-positive bacterial cells by using the gram-positive bacterial mini-culture to make membrane of the gram-positive bacterial cells capable of taking up exogenous DNA;
transforming the gram-positive bacterial cells with at least one plasmid DNA by using heat shock method at the temperature of at least 42 °C for at least 45 sec to obtain the heat-shocked cells, thereby creating a thermal imbalance that encourages DNA uptake;
adding a super optimal broth with catabolite repression (SOC) medium to the heat-shocked cells to obtain a primary mixture and incubate the primary mixture at the temperature of at least 37 °C for at least 1 hr to assist the heat-shocked cells in recovering from the stress of transformation, thereby obtaining transformed lactobacillus cells that start expressing the antibiotic resistance gene;
transmitting the transformed lactobacillus cells onto one or more selective agar plates with a selective agent and incubating the one or more selective agar plates overnight at the temperature of 37 °C, thereby examining the plates for colonies to determine whether the transformed lactobacillus cells are effectively incorporated with the plasmid DNA and are resistant to the selective agent; and
utilizing, the transformed lactobacillus cells to make edible products, thereby consuming the edible products to induce an immune response in the human body and acting as an edible vaccine.
2. The method (100) as claimed in claim 1, wherein the selective agar plate is a (de Man, Rogosa, and Sharpe) MRS agar plate.
3. The method (100) as claimed in claim 1, wherein the liquid medium is MRS broth, which is utilized to enable the growth of a gram-positive bacterial mini-culture.
4. The method (100) as claimed in claim 1, wherein the gram-positive bacterial cells are prepared by using the gram-positive bacterial mini-culture by:
refrigerating the gram-positive bacterial mini-culture at the temperature of at least 4 °C for at least 10 min;
centrifuging the centrifuge tube with the gram-positive bacterial mini-culture at the speed of at least 5000 rpm for at least 10 min to pellet the bacteria, thereby discarding the MRS broth and leaving the bacterial pellet;
mixing, the bacterial pellet with at least 30 ml of prechilled sterile of at least 0.1 M Cacl2 solution in the centrifuge tube, thereby incubating the centrifuge tube for at least 30 min to make the solution more permeable to DNA;
centrifuging the centrifuge tube at the speed of at least 5000 rpm for at least 10 min, thereby discarding the Cacl2 solution to obtain the gram-positive bacterial cells; and
dispersing, the gram-positive bacterial cells in at least 1 ml of the prechilled sterile Cacl2 solution, thereby making the dispersed gram-positive bacterial cells into one or more aliquots to enhance the bacteria's ability to take up exogenous DNA.
5. The method (100) as claimed in claim 1, wherein the selective agent is an ampicillin.
6. The method (100) as claimed in claim 1, wherein the heat shock method is performed by:
mixing at least 100 µl of the gram-positive bacterial cells of the each aliquot with at least 5 µl of the plasmid DNA in a container tube to obtain a primary mixture, and incubating the container tube with the primary mixture on ice for at least 30 min;
subjecting the primary mixture to a heat shock at the temperature of at least 42 °C for at least 45 sec to facilitate the uptake of the plasmid DNA by the gram-positive bacterial cells, thereby incubating the primary mixture on the cooling material at the temperature of at least 0 °C for at least 5 min to obtain a heat shocked culture;
mixing at least 105 µl of the heat shock culture of the gram-positive bacterial cells with at least 1 ml of the SOC medium to obtain a secondary mixture, and incubating the secondary mixture at the temperature of at least 37 °C for at least 1 hr to obtain the transformed cells that start expressing the antibiotic resistance gene; and
spreading at least 50 mcg of the ampicillin onto the MRS agar plate, thereby spreading at least 200 µl of the transformed cells on the MRS agar plates with the ampicillin to assess the potential of the transformed lactobacillus cells.
7. The method (100) as claimed in claim 1, wherein the plasmid DNA contains POL and RBD genes from HIV and COVID-19, respectively.
8. The method (100) as claimed in claim 1, wherein the transformed lactobacillus cells express viral proteins, which are used to create the edible vaccines for viruses like HIV and COVID-19.
9. The method (100) as claimed in claim 1, wherein the transformed lactobacillus cells are formulated into the edible vaccine suitable for oral administration.