WO2024246879A1

WO2024246879A1 - Formulation of instant probiotic powders and their manufacturing process

Info

Publication number: WO2024246879A1
Application number: PCT/ID2023/000003
Authority: WO
Inventors: Roni Ridwan; Ki Ageng SARWONO; Rohmatussolihat; Rusli Fidriyanto; Wulansih Dwi Astuti; Ainissya FITRI; Yantyati Widyastuti
Original assignee: Badan Riset dan Inovasi Nasional
Current assignee: Badan Riset dan Inovasi Nasional
Priority date: 2023-05-31
Filing date: 2023-12-01
Publication date: 2024-12-05
Anticipated expiration: 2025-11-30

Abstract

This invention relates to instant probiotic powders and its manufacturing process. The main ingredients, Lactobacillus and Bacillus sp. are added to increase the productivity of ruminant livestock. The formulation of the instant Bacillus spp. probiotic composed of 15-20% dextrose, 48-54% of a protein powder mixture, and 23-27% of Bacillus spp. powder yields the highest population count of 5.0 x 109 CFU/ml. The confirmation test results of the formulation amounted to 10.45 ± 0.013 log 10 CFU/ml. The formulation of the instant Lactobacillus probiotic composed of 8-14% dextrose, 40-50% protein powder mixture, 40-45% probiotic powder, 1.5-2.5% amino acid mixture, and 0.5-1.5% mineral mixture yields the highest population count of 8.29 Log 10 CFU/g. The formulation of the instant Lactobacillus plantarum TSD-10 probiotic composed of 9-23% dextrose, 35-40% protein powder mixture, 36-40% probiotic powder, 1-3% of an amino acid mixture, and 0.5-1.5% of a mineral mixture yields the highest population count of lactic acid bacteria of 8.03 log 10 CFU/g.

Description

FORMULATION OF INSTANT PROBIOTIC POWDERS AND THEIR MANUFACTURING

PROCESS

Technical Field of the Invention

This invention relates to a type of instant probiotic powder, more specifically, regarding the formulation and manufacturing process of instant probiotic powders composed of dextrose, a protein powder mixture, an amino acid mixture, a mineral mixture, and powdered Lactobacillus, Lactiplantibacillus plantarum TSD 10, and Bacillus spp.

Background of the Invention

The importance of efforts to increase livestock productivity is closely related to the availability of animal protein sources for public consumption. Through the Regulation of the Minister of Agriculture No. 14/2017 concerning the Classification of Veterinary Drugs, since January 1, 2018, the government has banned Antibiotic Growth Promoters (AGP) in feed formulation mixtures. An alternative solution to this restriction is adding probiotics to animal feed as a safe form of supplement.

Probiotics are live microorganisms given to livestock to improve the microbial balance of the host’s rumen and positively influence them. The microbial balance of the rumen stimulated by probiotics improves feed efficiency, gastrointestinal tract health, and livestock productivity. One of the requirements of the microorganisms used as probiotics is the ability to live, develop, and play a positive role in the rumen and digestive tract of livestock. Feed additives in the form of probiotics are required to increase the cattle’s productivity and optimally maintain their health.

Lactic acid bacteria (LAB) and Bacillus spp. are microorganisms added to livestock feed or given directly as probiotics. LAB produces lactic acid during the fermentation process in the rumen and acts as a lactic acid provider for bacteria that use lactic acid, such as Megasphera eldenii and Selenomonas ruminantium. One of the species of lactic acid bacteria used in the production of probiotics is Lactiplanti.bacillus plantarum (L. plantarum), which is a type of facultative anaerobic bacteria that can live in various habitats in nature, such as in plants and the digestive tract of livestock. As a multifunctional probiotic product, the Bacillus spp. produces a variety of extracellular enzymes that improve feed digestibility and antimicrobial compounds to improve livestock digestion, stimulate the host animal’s immune system, and improve the feed conversion ratio and the livestock’s meat quality.

The composition of probiotic instant lactic acid bacteria powder and Bacillus spp. is made to facilitate field-scale production in various regions. The probiotic bacteria used in this invention are Lactobacillus spp., L. plantarum TSD-10, and Bacillus spp. The Lactobacillus spp. and L. plantarum TSD-10 were inoculated on MRS broth and agar media. Meanwhile, the Bacillus spp. was inoculated on nutrient broth and nutrient agar. The US Patent 2019/0053527A1 discloses a method for making probiotic powder consisting of at least one probiotic bacterium. The method consists of the following steps: a) preparing a composition of probiotic biomass consisting of at least one probiotic bacterium from its probiotic bacteria culture in nutrients containing whey, b) spray drying or freeze drying the probiotic biomass to obtain the probiotic powder. In contrast to the above invention, this invention uses bacterial cultures from the fermentation of Lactobaci11us plantarum, Bacillus spp., and a sterile soybean meal mixture that is dried in a drying cabinet at. a low temperature.

US Patent No. 6,716,446, entitled ’’Instant probiotic powder and method of manufacturing the same, " describes an instant probiotic product that contains several types of probiotic bacteria that have been dried into a powdered form and combined with maltodextrin, corn starch, and prebiotic fibre. The manufacturing process of this instant probiotic product includes fermentation, drying, and grinding processes. In contrast to the invention above, the proposed invention uses a mixture of dextrose, an amino acid mixture, a mineral mixture, and a drying process using a drying cabinet at a low temperature. U.S. Patent No. 10,470,960, titled ’’Probiotic feed supplement for animals”, describes a dry powder probiotic product for cattle containing several types of probiotic bacteria and yeasts combined with maltodextrin and prebiotics . This instant probiotic product can be given to cattle to improve their digestive tract health and reduce their risk of gastrointestinal infections. This patent also describes several methods of using this instant probiotic product in cow feed.

U.S. Patent No. 10,106,704, titled "Probiotic compositions comprising Bacillus coagulans and Bacillus subtilis,” describes a dry powder probiotic product containing Bacillus coagulans and Bacillus subtilis combined with maltodextrin and prebiotics. This instant probiotic product can be given to animals or humans to improve their digestive health and immune system. This patent also describes several methods for using these instant probiotic products in food or beverages. In contrast to the above invention, the proposed invention uses a mixture of sterile soybean meal, dextrose, the isolate of the bacteria Bacillus lichen!formis_f and a drying process using drying cabinets at a low temperature. U.S. Patent No. 9,415,104, titled "Methods and compositions for providing Bacillus-derived probiotics, " explains the composition of a Bacillus-containing probiotic purified and dried into a powdered form. The Bacillus was selected from several species, such as Bacillus subtilis, Bacillus coagulans, Bacillus clausii, Bacillus lichen!formis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, and Bacillus cereus.

U.S. Patent No. 9,492,573, ’’Methods of preparing Bacillus coagulans-containing probiotic compositions,” describes a manufacturing method for Bacillus coagulans probiotics dried into a dry powder or granule form. This method involves fermenting the bacteria in a suitable growth medium, drying the fermented bacteria, and grinding and sieving the final product.

U.S. Patent No, 10,5m50,546, titled "Compositions and methods for improving growth performance in livestock, " covers the composition of a probiotic containing Bacillus subtilis, which is dried into granules and combined with prebiotics and vitamins. This composition can be given to farm animals, such as cattle, to increase their growth and productivity.

Unlike the patent above, this proposed invention aims to provide an instant probiotic powder that can be easily manufactured in various regions. This probiotic instant powder uses the bacterial isolates of Lactobacillus, L. plantarum TSD 10, and Bacillus spp.,, which already contain the growth media for the probiotics, consisting of a blend of five powdered protein sources (palm kernel extract, soybean meal, fish meal, meat meal, and corn gluten meal) combined with pollard powder, an amino acid blend (Lysin and Methionine), dextrose, and a mineral blend of cattle feed supplements (Ca, P, K, Fe, Na, Se, Mg, Zn, Mn, 1, Co, S, and Cu), manufactured to increase the productivity of ruminant livestock.

Summary of the Invention

This invention aims to provide a formulation of an instant probiotic powder and its manufacturing process. The first aspect of this invention is the formulation of the instant probiotic powder. The instant probiotic powder in this invention includes dextrose; a protein powder mixture; an amino acid mixture; a mineral mixture; and a probiotic powder characterized by Lactobacillus , Lactiplantibacillus plantarum TSD 10, and Bacillus spp. The instant Bacillus spp.-derived probiotic powder comprises: 23-27% of a probiotic powder from Bacillus spp., 16- 20% of dextrose, and 48-54% of a protein powder mixture.

Meanwhile, the instant Lactobacillus-derived probiotic powder is composed of the following: 40-45% of a Lactobacillus-derived probiotic powder; 8-14% dextrose; 40-50% of a protein powder mixture; 1.5-2.5% of an amino acid mixture; and 0.5-1.5% of a mineral mixture. The instant L. plantarum TSD-10-derived probiotic powder has the following formulation: 36-40% of an L. plantarum TSD-l0-derived prebiotic powder; 9-23% dextrose; 35- 40% of a protein powder mixture; 1-3% of an amino acid mixture; and 0.5-1.5% of a mineral mixture. The second aspect of this invention begins with the production of liquid probiotics in optimized media based on the probiotics derived from Lactobacillus, Lactiplantibacillus plantarum TSD 10, and Bacillus spp. Then, the liquid probiotics are mixed with sterile soybean with a ratio of 1:1. Next, the mixture is dried, and a powdered protein mixture is made separately. The production of the protein mixture powder consists of the following steps: mixing several protein sources, extracting the mixture of protein sources, mixing the extract and pollard, drying the mixture, grinding the mixture, and sterilizing the resulting material.

Other ingredients, such as dextrose, mixed amino acids, and mineral mixtures, are also sterilized. Then, all the ingredients are mixed according to the formulation, including:

• an instant Bacillus spp-derived probiotic powder composed of a Bacillus spp.-derived prebiotic powder, dextrose, and a protein powder mixture;

• a Lactobacillus-derived instant probiotic powder composed of Lactobacillus-derived probiotic powder, dextrose, a protein powder mixture, an amino acid mixture, and a mineral mixture; * an L-plantarum TSD 10-derived pow’der instant probiotics composed of L. plantarum TSD 10-derived probiotic powder,

• dextrose, a protein powder mixture, an amino acid mixture, and a mineral mixture.

Brief Description of the Figures

Figure 1 shows the electrophoresis results from the PCR product for the sample sequencing for this invention.

Figure 2 show’s the medium optimization results, which indicate the different influences between the dextrose and protein mixture formulations on Bacillus spp jrowth. The different letters indicate significant differences according to this invention.

Figure 3 shows the response surface diagram of Bacillus spp. growth to variations in the percentage of dextrose, protein powder, and bacillus powder according to this invention.

Figure 4. shows the response surface diagram for the number of live bacteria in the instant Lactobacillus probiotic powder (Log 10 CFU/ml) according to this invention.

Figure 5. shows the response surface diagram for the number of live bacteria in the instant L. plantarum TSD-10 probiotic powder (Log 10 CFU/ml) according to this invention.

Figure 6. shows a flow chart of the manufacturing process of instant probiotic powder according to this invention. Description of the Invention

This invention aims to provide a formulation of an instant probiotic powder and its manufacturing process. The manufacturing process of the instant probiotic powder consists of several stages, as shown in Figure 6. The first stage is the production of liquid probiotics in an optimised medium. This liquid probiotic is mixed with the sterile soybean meal in a 1: 1 ratio, then dried in a drying cabinet at 60-70 °C for 24 hours. The next stage is the preparation of the protein powder mixture. The protein powder mixture consists of five different raw materials for feed. Each raw material makes up 5% (weight/volume) of the total protein powder mixture.The raw materials include palm kernel, soybean meal, fish meal, meat meal, and corn gluten meal. This protein mixture is obtained from a liquid extraction conducted at 100 °C for 30 minutes in water, with the ratio of the total protein source material to water being (1: 1.5) (weight/volume). The extracted liquid protein mixture is mixed with pollard powder in a 1: 1 ratio. The mixture is then dried at 60-70°C for 18-24 hours. After that, the powdered protein mixture is ground until smooth. The next step is sterilising the protein powder mixture in a drying cabinet at 100-110°C for 1 hour. Dextrose, an amino acid mixture, and a mineral mixture are sterilized through a heating process at 100-110 °C for 1 hour. The mixing of all the ingredients mentioned above is adapted to the formulation of this invention for an instant probiotic powder containing Lactobacillus spp, L. plantarum TSD 10, and Bacillus spp. Formulation of the Instant Probiotic Powder a. The Inoculation and Physiological Characterization of Bacillus SJDp* A total of 5 Bacillus species: Bacillus licheniformis, B, subtillis_r B. polymixca, B. coagulants, and B. pumillus were inoculated using the quadrant streaking method on Nutrient Agar (NA) media and incubated for 24 hours. The single colonies obtained were then re-grown in 3 ml of Nutrient Broth (NB). The genomic DNA of the liquid culture was then extracted using a modified DNA Isolation Kit. The remainder of the culture was then used as an isolation source for physiological tests, including an amylolysis test and a catalase test. The remaining culture was also grown at 55 °C and NaCl 6.5% and underwent a citric acid utilization test (Simmone citrate powder) and Gram staining. Molecular identification was performed by amplifying the culture’s genomic DNA with the primers 27F and 1492R using the Kappa KAPA2G Fast HotStart Readymix PGR Kit (Kapa Biosystems) . The amplification results were then sequenced. Based on the results shown in Table 4, all bacteria of the

Bacillus genus can produce catalase. Each Bacillus species can hydrolyse amylum, except for the bacteria B. pumillus. This ability is indicated by the formation of a clear zone on the NA amylum medium inoculated with the bacteria and given an iodine indicator. The bacteria B. subtilis, B. licheniformis, and B. polymixca showed growth in 6.5% NaCl medium. Bacteria B. coagulants, B. licheniformis, and B. polymixca also showed growth at an incubation temperature of 55^°C. Whole isolates of the 5 Bacillus tested can grow on Simmone citrate agar. However, only B. polymixca could not use citric acid. The colour changes from green to blue around the colony indicates the bacterium’s ability to utilize citric acid in this medium.

Table 1. Physiological test results of the selected 5

Bacillus spp. isolates

b. Formulation of the Optimized Growth Medium of Bacillus spp.

To obtain a medium that enabled the optimum growth of Bacillus spp> 0.5 ml of Bacillus spp. cultures were grown in NB media with several ratio variations of dextrose and protein mixture concentrations (Table 2). The bacterial growth was measured using the total plate count (TPC) method after 24 hours of incubation, with three repetitions. The data obtained were analysed statistically using a complete randomised design (BAL).

Table 2. The comparison of the several selected dextrose and protein mixtures in the formulation medium for liquid Bacillus spp.

The statistical analysis results of the effect of different protein mixtures and dextrose on Bacillus spp. growth has shown there is a difference between the ratios (F<0.001). Tukey's Test showed that the medium with a ratio of 0.5% dextrose and 2% protein mixture is the best medium formulation with a population of 6.3 x 1CP CFU/ml (Figure 2). Therefore, this selected medium formulation was used to manufacture the Bacillus spp. powder that will be used in the production of an instant Bacillus spp. probiotic powder. c. Formulation of the Instant Bacillus spp. Probiotic

The Bacillus spp. grown on the optimized medium is mixed with sterile soybean meal powder in a 1:1 ratio and dried at 60-70^uC. The population and viability of the Bacillus spp. powder obtained was then determined using the TPC method on NA medium. It was then mixed with dextrose and the protein powder mixture per Table 3. A total of 0.5-lg or 1-2% (weight/volume) of each experimental unit was grown in 50 ml of sterile water and incubated at 30°C for 24 hours. The culture's population was then determined using the TPC method. The population value was then analysed using the Mixture Design method on the software Design-Expert® (Stat-Ease Inc). The formulation for the instant Bacillus spp. probiotic powder was used directly in sterile water in a closed, sterile, and hygienic environment (1-2% by weight/volume) without adding more bacterial inoculum during fermentation.

Table 3. The formulation for the instant Bacillus spp. powder and population after activation for 24 hours

d. Formulation of the instant Lactobacillus and L. plantarum TSD 10 probiotic powder

The formulation testing was divided into two parts: the preliminary test and the confirmation test. The preliminary test was conducted by testing 16 formulations with the Mixture Design method. The confirmation test was conducted using the preliminary test results where the two treatments with the best viability were compared with two treatments with the lowest viability using One-way ANOVA. The formulation for the instant Lactobacillus the powders probiotic powder used in the preliminary tests is described in Table 4, and the formulation for the instant L. plantarum TSD-10 probiotic powder for the preliminary test is described in Table 8. The formulations of the instant Lactobacillus and L. plantarum TSD-10 probiotic powders used in the confirmation test are described in Table 11 and Table 12.

The materials used are sterilised by heating or by exposure using ultraviolet rays. The inoculants are inoculated on the MRS broth and the optimised media. The Lactobacillus probiotic powder was made by mixing fermented liquid probiotics with sterile soybean meal (1:1) and then dried in a drying cabinet for one night at 60-70°C. After drying, the probiotics are ground into a powder using a blender. The probiotic powder then underwent TPC testing to determine the number of living lactic acid bacteria on the MRS agar media. The prepared ingredients were then mixed according to a predetermined formulation.

As much as 0.5-1 g (1—2%) (weight/volume) of the mixed probiotic powder formulation was then added into a 100 ml serum bottle filled with 50 ml of sterile water. Then, the serum bottle was incubated for 24 hours at an incubator temperature of 30°C. After 24 hours, a sample of 0.5ml is taken from the serum vial and diluted gradually using 4.5 ml sterile water. The TPC with the pour plate method was conducted to count the number of live bacteria on an MRS agar. Then, the substrate was incubated for 48 hours.

In the preliminary test, the data were tested using the mixture design method. The study’s design quality was verified by conducting an analysis of variance (ANQVA) to the regression

(R) and the determination coefficient (R^) and conducting a lack of fit analysis. All calculations and graphing were conducted using the Design Expert version 11.1.2.0 application (Stat-Ease, Inc., United States of America).

Table 4. Composition of the instant Lactobacillus probiotic powder formulation

Table 5. Composition of the instant L. plantarum TSD-10

Probiotics

The results of the preliminary test were then used for the confirmation tests. The formulation used in this test was selected from the previous test with the mixture design. The two treatments with the best viability were compared to those with the lowest. The complete randomized design (RAL) was used in this test. The data were processed using the One-Way ANOVA procedure in SPSS 16.0 (Chicago, IL, USA) . An effect is considered significant if it has a probability level of P <0.05. LSD follow-up tests were also performed when the conditions were met. Formulation of the Instant Bacillus spp. Probiotic Powder

The population of Bacillus spp. (Log CFU/ ml) from instant, probiotics incubated for 24 hours ranged from 8.04 to 9.23 (Table 3). The equation model from the mixture design analysis shows a significant mathematical equation model (P = 0.042). However, it has a lack of fit that is insignificant (P = 0.3097). The equation obtained has a coefficient of 0.827 (Table 6). The results showed a strong correlation between population increase and the addition of dextrose, Bacillus spp. powder, and the protein powder mixture.

Table 6. Table of coefficients and regression analysis of equation models and their response functions

The Response Surface Diagram shows the higher the amount of protein powder mixture and Bacillus spp. powder added, the higher the Bacillus spp. growth after being activated for 24 hours (Figure 3). Table 3 shows that a formulation of 59% mixed proteins and 26% Bacillus powder added to the culture results in the highest population of Bacillus spp. after incubation. Based on the previous statistical tests, the subsequent analysis determines the formulation model for the instant Bacillus probiotic powder. The optimum formula for instant. Bacillus probiotic powder from the equation model obtained in Table 6 consists of 18.7% dextrose, 52.9% of a powdered protein mixture, and 25.4% of Bacillus spp. powder. The total population resulting from the formulation is expected to reach 9.7 log CFU/ml 5.0 x 10° CFU/ml. The confirmation test for the above formulation obtained a population of 10.45 ± 0.013 log CFU/ml.

The Formulation for Instant Lactobacillus spp. and L, plantarum TSD-10 Probiotics

The preliminary test results are shown in Table 10. The pH measurements of the instant Lactobacillus probiotic powder after 24 hours of incubation at 30°C showed that the pH was between 4.48-5.04. Meanwhile, the pH of the instant TSD-10 probiotic powder was between 4.48-4.92.

Lactic acid bacteria grow optimally at pH 5 to 6 and produce metabolite products at pH 3.5 to 4.5. Formulation pH data showed that adding an instant probiotic powder can reduce the pH of the solution. The lower pH is due to the fermentation of the ingredients contained in instant Lactobacillus and TSD-10 probiotics, such as dextrose, a protein mixture, and soybean meal in the probiotic powder. They produce a lactic acid product that will lower the pH. Lactic acid bacteria can metabolize carbohydrates into beneficial elements, especially lactic acid.

The number of live bacteria calculated using the mixture design method obtained regression coefficients and mathematical models for ANOVA (Table 8). The mathematical models for the number of live bacteria showed no significant effect, either linear, quadratic, or quartic, at the 95% probability level in both instant probiotic powders. The mathematical model formulas show that lack of fit is not significant {Lactobacillus P=0.430;

TSD-10 P = 0.620), and Pf amounted to 0770 and 0468 for

Lactobacillus and L. plantarum TSD-10, respectively. These results indicate that the model used in this study can sufficiently explain the effect of adding dextrose, probiotic powder, and protein mixture. The analysis of the response surface results (Figures 4 and 5) shows a tendency for the number of live bacteria to increase with increasing protein mixture. This result suggests that the protein mixture used serves as a source of nutrition for lactic acid bacteria in the instant probiotic powder. Based on the number of live bacteria calculations, formulations F5 and F9 on Lactobacillus and Fl and F3 on L. plantarum TSD-10 has the potential to be used as an instant probiotic powder.

The compositions of the instant Lactobacillus and L. plantarum TSD-10 probiotic formulations for the confirmation test are shown in Table 11 and Table 12. According to preliminary test results, the treatment of F5 and F9 in Lactobacillus Fl and F3 on L. plantarum TSD-10 could be used as probiotics and was used as a treatment in the confirmation test.

Fl and F3, as well as F13 and F16, were compared in the confirmation tests for instant Lactobacillus and L. plantarum TSD - 10 probiotic powder formulations consecutively. Each treatment was repeated five times. The results of the instant Lactobacillus and L. plantarum TSD-10 probiotic formulation tests conducted consecutively can be found in Table 11 and Table 12. The results showed that treatment F9 for the formulation of instant Lactobacillus probiotics has the highest number of live bacteria and pH compared to other treatments (number of live bacteria P=^:0.045; pH P<0.001). The number of live bacteria in the F9 treatment is 8.290 ± 0.049 log CFU/g and pH 4.778 ± 0.076. The Fl treatment for the formulation of instant L. plantarum TSD-10 probiotics had the highest number of live bacteria and the lowest pH compared to other treatments. The number of live bacteria in the Fl treatment was 8.030 ± 0.061 log CFU/g and a pH of 4.300 ± 0.059. This shows that the confirmation test results are in accordance with the preliminary test results.

Lactobacillus and L. plantarum TSD-10 is a lactic acid bacterium that can be used as a probiotic. Complex basal media for lactic acid bacteria such as de Man Rogosa and Sharpe (MRS), NaLa (sodium lactate), M17, and trypticase soy broth yeast, extract (TSBYE) is widely available on the market. However, they are costly. These media would be uneconomical for the probiotic industry because it uses high-cost nitrogen sources such as yeast extract, meat, extract, and peptone. The formulation test results showed that the formulated media has the potential to be a substitute for complex basal media for commercial uses as it can maintain a high number of live bacteria. The results of the mathematical models (Table 8) indicate that the best composition to obtain the highest number of bacteria in instant Lactobacillus probiotics is the F9 treatment consisting of 10% dextrose, 45% protein blend, 42% probiotic powder, 2% amino acid blend, and 1% mineral blend. According to the results, the composition to obtain the best number of live bacteria from the instant L. plantarum TSD-10 probiotic powder was the Fl treatment consisting of 21% dextrose, 37% protein mixture, 39% probiotic powder, 2% amino acid mixture, and 1% mineral mixture. Table 7. Number of live probiotic bacterial populations and pH levels after 24 hours of incubation

Table 8. Regression coefficients and mathematical models^ from ANOVA adjusted to the response function.

xl = dextrose, x2 ^:= mixed protein; x3 ^::= probiotic powder

2Significance at 5%

Table 10. Composition of the instant L. planatarum TSD-10 probiotic powder formulation for the confirmation test

Table 11. Number of live Lactobacillus bacteria from the probiotic instant powder after 24 hours of incubation

Table 12. Number of live L. plantarum TSD 10 from the instant probiotic powder after 24 hours of incubation

Claims

A formulation of an instant probiotic powder containing:

• dextrose;

• a protein powder mixture;

• an amino acid mixture;

• a mineral mixture; and

• a probiotic powder (Lactobacillus, Lactiplantibacillus plantarum TSD 10, and Bacillus spp.).

The formulation of an instant probiotic powder per Claim 1, where the instant probiotic powder is derived from Bacillus spp. is composed of:

• 23-27% of a Bacillus spp.-derived probiotic powder;

• 16-20% dextrose; and

• 48-54% of a protein powder mixture.

The formulation of an instant probiotic powder per Claim 1, where the instant probiotic powder is derived from Lactobacillus is composed of:

• 40-45% of a Lactobacillus-derived probiotic powder;

• 8-14% dextrose;

• 40-50% of a protein powder mixture;

• 1.5-2.5% of an amino acid mixture; and

• 0 5% of a mineral mixture.

The formulation of an instant probiotic powder per Claim 1, wnere the instant probiotic powder is derrved from 1. plantarum TSD - 10 is composed of:

• 36-40% of an L. plantarum TSD-10-derived probiotic powder;

• 9-23% dextrose;

• 35-40% of a protein powder mixture;

• 1-3% of an amino acid mixture; and

• 0.5-1.5% of a mineral mixture. A process for producing an instant probiotic powder that consists of the following steps: a) producing liquid probiotics derived from Lactobacillus, Lactiplantibacillus plantarum TSD 10, and Bacillus spp. in an optimised medium; b) mixing the liquid probiotics with sterile soybean meal in a 1:1 ratio; c) drying the mixture from stage (b) at 60-70 °C for 24 hours to obtain a probiotic powder; d) making a protein powder mixture through the following steps:

• mixing a set of protein sources, namely palm kernel meal, soybean meal, fish meal, meat meal, and corn gluten meal;

• extracting the mixture of protein sources at 100°C for 30 minutes in water, with a ratio of 1:1.5 (W/v) for the total mixture of proteins and water;

• mixing the protein mixture with powdered pollard in a 1:1 ratio;

• drying the mixture at 60~70°C for 18-24 hours to obtain a protein powder mixture;

• grinding the protein powder mixture;

• sterilising the protein powder mixture in a drying cabinet at 100—110°C for 1 hour;

• obtaining a protein powder mixture; e) sterilising the dextrose, amino acid mixture, and mineral mixture; f) mixing the instant probiotic powder ingredients according to the following steps:

• mixing the Bacillus spp.-derived probiotic powder, dextrose, and protein powder mixture to obtain an instant. Bacillus spp.-derived probiotic powder;

• mixing the Lactobacillus-derived probiotic powder, dextrose, protein powder mixture, amino acid mixture, and mineral mixture together to obtain an instant Lactobacillus-derived probiotic powder;

• mixing the L. plantarum TSD 10-derived probiotic powder, dextrose, protein powder mixture, amino acid mixture, and mineral mixture to obtain an instant L. plantarurn TSD 10-derived probiotic powder; and g) obtaining instant probiotic powders.