WO2024228613A1 - Formulation for a probiotic stable at baking temperatures and encapsulation of the probiotic to ensure the life of same in shelf-stable foods and the viability thereof in consumption - Google Patents

Abstract

Described is an invention for scalable and affordable encapsulation that allows probiotics to survive baking processes, minimising as far as possible the overaddition needed to achieve the CFUs necessary for the corresponding declaration.

Description

FORMULATION OF A PROBIOTIC STABLE AT BAKING TEMPERATURES AND ENCAPSULATION OF THE SAME TO ENSURE ITS SHELF LIFE IN FOODS UNTIL ITS VIABILITY IN CONSUMPTION

FIELD OF INVENTION

The global market for functional foods is expanding every day, due to the growing consumer demand due to greater consumer awareness of including healthy foods that offer health benefits in their daily diet.

The global functional products market is worth 139.2 billion dollars, which for Mexico is equivalent to 5% of the world value, with a constant annual growth rate of 3.9%. The global probiotics market represents 52.9 billion dollars, in Mexico, it is worth 0.04 billion dollars, with an annual growth rate of 7%.

However, North America, especially the United States, has seen accelerated growth in this market where 3% of new food and beverage products contain probiotics.

BACKGROUND OF THE INVENTION

Probiotics are often found in dairy products, but are increasingly being incorporated into other foods such as juices, cereals, bars, etc. Most probiotics are heat sensitive, so their survival during thermal processes is a major obstacle. The heat generated in baking processes can cause significant losses during the processing and storage of bread. In addition, probiotics must survive and grow inside the host to exert their beneficial health effects. They must be metabolically stable and active, survive acidic conditions in the stomach, and reach the intestine in optimal quantities. Likewise, factors such as pH, presence of oxygen, temperature, and humidity in the product must be ideal so as not to affect their growth and survival.

Encapsulation of nutraceuticals, including probiotics, is a strategy that allows a functional ingredient to be given stability under specific conditions. This technology solves some problems in the process, because it captures the main ingredient as a central core and covers it with an inert or protective layer that improves stability during passage through the gastrointestinal tract. In the case of probiotics for use in bakery products, it is necessary to preserve viability. of probiotics at baking temperatures, it is also advisable to stabilize them so that they can reach the human intestine in a viable state, where they can exert their beneficial action on health.

Nowadays, encapsulation is an important alternative for the implementation of probiotics in foods, to allow them to arrive alive to the site where they will exert their action, protecting them from environmental conditions (heat, light, air, humidity).

There are different methods of encapsulation of bioactive compounds such as emulsion, liposome formation, electrospinning, and spray drying. Most of them involve the use of solvents or technologies that are difficult to scale in the industry (Ayala-Fuentes et al., 2021). However, spray drying is a scalable technique at an industrial level, fast, with low operating costs and that does not require the use of solvents and does not generate pollutants, therefore, it is defined as sustainable.

Patent Application MX/a/2016/006536; Process for the production and formulation of acidified fermented beverages based on strains with probiotic activity of lactobacillus paracasei tolerans pfthl-10 and pfthl-22, from juices and honeys of agave atrovirens. This document describes the process of transformation of honeys or syrups and agave sap from two different bacterial cultures belonging to the species Lactobacillus paracasei tolerans. The microorganisms mentioned have probiotic activity in vitro, since they produce bacteriocins and organic acids produced in the fermentation of honeys and agave juices; which translates into the inhibition of the growth of pathogenic and toxigenic enterobacteria. During fermentation, the strains of bacteria object of the invention rapidly consume the monosaccharides and some oligosaccharides that are available, while secreting pleasant flavors and aromas. At the end of a fermentation process of between 48 and 120 hours; and a period of stabilization of the product at refrigeration temperature for 2 to 21 days, an acidulated drink is obtained that contains the probiotic microorganisms themselves, as well as oligofructans and polymerized fructans. This product does not contain alcohol, has a very low content of glycemic sugars, and is free of animal fats, cholesterol, or allergens derived from milk-derived proteins. It is therefore a functional drink, containing probiotic microorganisms; prebiotic oligosaccharides; and short-chain organic acids.

Unlike the present invention, this document does not speak of high temperatures to preserve the properties of a probiotic, the process of transforming honey or syrups and agave sap from two different bacterial cultures belonging to the lactobacillus species is not extrapolable in any way. It forms the process that we have because it is a fermentation process and has nothing to do with heat or drying of the bacteria.

International Application MX/a/2014/012235; use of lactic acid bacteria to prepare fermented food products with increased natural sweetness.

This paper describes the purpose of providing an alternative to the addition of sweeteners to fermented milk products to achieve the desired sweet taste without the added calories. Furthermore, it would be very advantageous to establish a method to reduce lactose in fermented milk products to a level that is acceptable to consumers who are lactose intolerant. The above problems were solved by providing mutant strains of Streptococcus thermophilus and mutant strains of Lactobacillus delbrueckii subsp. bulgaricus that excrete glucose into milk when the milk is inoculated and fermented with such strains of Streptococcus thermophilus and strains of Lactobacillus delbrueckii subsp. bulgaricus. Thus, the present invention relates to strains of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus which secrete glucose into the dairy substrate during fermentation, as well as to mixed cultures comprising the Streptococcus thermophilus strains and the Lactobacillus delbrueckii subsp. bulgaricus strains, to starter cultures comprising the strains and to dairy products made from the cultures. The present method also relates to the use of the strains to decrease the lactose content of a fermented food product and to enhance the growth of the probiotic BB-12(r).

This document does not speak of high temperatures to preserve the properties of a probiotic, this is still a fermentation process. In our process there is no fermentation of grocery stores and fermented products are not used for their desired sweet taste without added calories and instead this technology does not affect the taste of the final product.

Publication: KR2021181788A; Title: A non-stop production process to improve freeze-drying survival, heat tolerance, storage stability and digestive stability of probiotics using spontaneous matrix encapsulation technique.

This paper describes a method for preparing alginate-hydrogel encapsulated probiotics, it involves (a) culturing probiotics in a medium containing alginic acid salts and salts to form hydrogels by combining with alginic acid to spontaneously generate alginate-hydrogels simultaneously with proliferation of probiotics, and (b) recovering the encapsulated probiotics by the spontaneously produced alginate hydrogel. The encapsulated probiotics are encapsulated in an alginate hydrogel. The encapsulated probiotics are of the type matrix. The medium in step (a) further comprises an encapsulation enhancer wherein the encapsulation enhancer is selected from starch, crystalline cellulose, chitosan, carboxymethylcellulose (CMC) and skimmed milk powder. The probiotics are Lactobacillus sp., Bifidobacterium sp., Streptococcus sp., Lactococcus sp., Enterococcus sp., Leuconostoc sp., Pediococcus sp. and Weissella sp.

Composition: The salt of alginic acid is 0.1-40 g/L, and the salt for forming a hydrogel by binding with alginic acid is contained in the medium in an amount of 0.5-10 g/L. The encapsulation enhancer is contained in the medium in an amount of 1-20 g/L. Preferred method: Step (a) involves dissociating the cation of the salt that binds to alginic acid to form a hydrogel by the acid generated by the culture of the probiotics, and spontaneous binding of the dissociated cation and alginic acid. The method further involves (c) freeze-drying the encapsulated probiotics with the recovered alginate hydrogel.

This document in particular mentions an encapsulation where the enhancer is chosen from starch, which may be relevant for a thermostable stabilized probiotic.

This particular document does mention temperatures and their tolerance to them. However, in the present invention a hydrogel with a composition of alginate and a complex carbohydrate is not used. In the present invention a hybrid solid carbohydrate-protein nanoparticle is used. The formulation is different in the type of encapsulant and therefore its application. Ours is for use in food and all its ingredients are used in food and especially in bakery products.

Publication: KR2006130968A; Title: Probiotic Bifidobacterium Bourn R5 strain with excellent acid tolerance, bile acid tolerance, oxygen tolerance and heat stability and probiotic composition comprising the same or its culture medium.

This paper describes a probiotic Bifidobacterium bourn R5 strain and a probiotic composition comprising the same strain are provided to inhibit the growth of various intestinal pathogenic bacteria effectively, and improve the acid tolerance, bile acid tolerance, oxygen tolerance and heat stability of the strain.

The probiotic Bifidobacterium bourn R5 strain (KCCM 10669P) capable of inhibiting the growth of intestinal pathogenic bacteria is provided, where the Bifidobacterium bourn R5 strain (KCCM 10669P) is isolated from the contents in the first stomach of a cow; and the intestinal pathogenic bacteria is Salmonella gallinarum, Salmonella enteritidis, Salmonella typhimurium, Staphylococcus aureus, Enterotoxigenic Escherichia coli, Listeria monocytogenes, Campylobacter jejuni or Clostridium perfringens. The probiotic has a composition comprising the probiotic Bifidobacterium boumR5 strain (KCCM 10669P) and its culture medium, where the composition It is used as food additives. The composition is heat-dried or freeze-dried, and the amount of probiotic Bifidobacterium boumR5 strain (KCCM10669P) is 105 to 1012 cfu/g based on the total weight of the composition.

This document deals with an isolated and modified strain. However, in the present invention no strain is used and we do not modify the strains themselves, it is a coating material.

Publication: KR201696641 A, Title: Lactobacillus fermentum pl9119 with biofunctional activities and high heat stability as a probiotic without antibiotic resistance. This paper describes a novel strain of Lactobacillus fermentum PL9119 used in composition for intestinal disorders, and is used in healthy and functional foods such as milk, yogurt, soy milk, meat products, sausage, bread, beverages, capsules or stick packs. Lactobacillus fermentum PL9119 strain comprises a 16S rRNA nucleotide sequence having 648 nucleobases, given in the specification, where Lactobacillus fermentum strain PL9119 is deposited in Korean Collection for Type Cultures (KCTC 13038BP). the strain is selected from the group consisting of strain resistant to ampicillin, gentamicin, erythromycin, clindamycin, tetracycline, chloramphenicol, vancomycin, synercid, quinupristin, daifo pristin or a combination, where the antibiotic agent is not resistant to linezolid or rifampicin group which is free from the risk of resistance metastasis. The strain or culture solution in the prophylactic composition is excellent in acid resistance and bile resistance. The strain is selected from the group consisting of Escherichia coli O157:H7 strain preserved in American Type Culture Collection (ATCC No: 43894), Salmonella typhimurium preserved in Canadian Centre for Agri-Food Research in Health and Medicine (CCARM No: 8001), Salmonella enteritidis preserved in Canadian Centre for Agri-Food Research in Health and Medicine (CCARM No: 8010), Enterococcus thecalis preserved in American Type Culture Collection (ATCC No: 29212), Staphylococcus aureus preserved in Canadian Centre for Agri-Food Research in Health and Medicine (CCARM No: 0045) and Listeria monocytogenes preserved in American Type Culture Collection (ATCC No: 19113C3a). Functional health food promotes gut health activity through inhibition of food poisoning bacterial growth and lipase production.

Unlike what has been described in the present invention, this strain is not used and the strains themselves are not modified; it is a coating material.

Publication: BR1120133284A; Title: Procedure for the preparation of heat and moisture resistant probiotic bacteria in the form of stabilized probiotic granules for a liquid-based food product, process ... preparation of a liquid-based food product, stabilized probiotic granules for mixing into a liquid-based food product, and, a food product.

This document provides thermally processed or thermally processable healthy food products that beneficially affect the consumer's intestinal microbial balance.

Food products are particularly liquid-based products comprising a probiotic component capable of withstanding heat and moisture.

The process (I) for preparing heat and moisture resistant probiotic bacteria in the form of stabilized probiotic granules for a liquid-based food product comprises: (i) preparing core granules containing probiotic bacteria, substrate and optionally other food grade ingredients; (ii) optionally coating the core granules with an inner layer to obtain sealed core granules (A); (iii) optionally coating (A) with an outer layer comprising a thermosensitive gel-forming polymer to obtain protected core granules (B); and optionally (iv) coating (B) by outer layer comprising a water-soluble polymer.

The granules comprise a core of probiotic bacteria in a substrate or mixed with a substrate, and at least one inner layer, an outermost layer or an outer layer comprising a water-soluble polymer or an erodible polymer. The substrate comprises; a component comprising bacteria supplement, stabilizer, filler and/or binder; or a prebiotic saccharide.

This document deals with a coating gel, the formation process is different because it is liquid based, the size of the final result is different, ours reaches up to 4 microns only.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the spray drying scheme. The main parts that make up a spray drying equipment are a container where the solution to be spray dried is located, in the drying chamber there is the atomizing nozzle where the solution is sprayed, very close to this nozzle there is a hot air outlet that will dry the small drops formed by the solution, finally in the drying chamber at the end the dry product is obtained, generally with a particle diameter similar to the opening of the atomizing nozzle.

Figure 2 shows the morphology of the probiotics used. On the left, Bacillus clausii is a sporulated and circular microorganism smaller than 1 micrometer, which is very small compared to the second microorganism used. On the right is Lactobacillus acidophilus, the second t-microorganism used, which is significantly larger and has a rod-shaped shape and an average size of 2 micrometers.

Figure 3 shows the optimization of Bacillus clausii encapsulation. The optimal point was determined at 35% Inu lin, 60% pea protein, 5% maltodextrin and an initial probiotic concentration in the order of 4x1011 CFU.

Figure 4 shows the optimization of Lactobacillus acidophilus encapsulation. The optimal point was determined at 30% inulin, 40% pea protein, 25% maltodextrin and an initial probiotic concentration of 4x108 CFU.

Figure 5. Optimal formulations for probiotics These formulations were obtained through 2 experimental designs using spray separation technology. The integration of the two experimental designs served as a basis for achieving an optimal formulation to propose a formulation for the combination of both probiotics in a single microencapsulation.

Figure 6 shows images of the morphology of the microencapsulates obtained by scanning electron microscopy (SEM). Scanning electron microscopy (SEM) of the microencapsulates obtained, a) the encapsulation of Basillus clausii 3.53x1010 CFU/g of encapsulation is observed, b) the encapsulation of Lactobacillus acidophilus 1.22x108 CFU/g of encapsulation is observed, and c) the encapsulation of 2 probiotics containing Lactobacillus acidophilus in 6.69X108 CFU/g of encapsulation and Basillus clausii in 2.24X101 OCFU/g of encapsulation.

Figure 7 shows the scale-up of microencapsulation production. The process was scaled from 500 mL of solution, where 4 to 5 were obtained, to a 5 L process, where approximately 150 g were obtained per run of each microencapsulation.

Figure 8 shows the prototypes of microbreading with encapsulations generated by the direct method. From left to right, the control bread, the bread with microencapsulation from Spain (positive control), encapsulation with L. acidophilus, encapsulation with B. clausii and the encapsulation with the 2 probiotics (L. acidophilus and B. clausii) are observed.

Figure 9. 460g bread prototypes with encapsulations generated by the sponge method. From left to right, we can see the bread with the 2 probiotics (L. acidophilus and B. clausii), the bread with microencapsulated B. clausii, the bread with microencapsulated L. acidophilus and finally the control bread.

Figure 10, describes the in vitro digestion process.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES

Encapsulation of nutraceuticals, including probiotics, is a strategy This technology allows a functional ingredient to be conferred stability under specific conditions. This technology solves some problems in the process, because it captures the main ingredient as a central core and covers it with an inert or protective layer that improves stability during passage through the gastrointestinal tract (Vasishtha, 2005). In the case of probiotics for use in bakery products, it is necessary to preserve the viability of the probiotics at baking temperatures, and it is also advisable to stabilize them so that they can reach the human intestine in a viable state, where they can exert their beneficial action on health. Currently, encapsulation is an important alternative for the implementation of probiotics in foods, to allow them to reach the site where they will exert their action alive, protecting them from environmental conditions.

There are different methods for encapsulating bioactive compounds, such as emulsion, liposome formation, electrospinning, and spray drying. Most of them involve the use of solvents or technologies that are difficult to scale in the industry (Ayala-Fuentes et al., 2021). However, spray drying is a scalable, fast, low-cost industrial technique that does not require the use of solvents and does not generate pollutants, therefore, it is defined as sustainable.

Spray drying is a technology widely used in the food industry to produce powdered ingredients such as instant coffee, baby milk formulas, among others. The advantages of spray drying are that it is a very robust and scalable technique and that very good yields are obtained. Figure 1 presents a diagram explaining the main components of the technology.

The main objective was to develop a scalable and affordable encapsulation process that allows probiotics to survive our baking processes, minimizing as much as possible the over addition necessary to achieve the CFU necessary to make the corresponding claim.

The following stages were considered for the implementation of the process:

1) Development of Functional Ingredient. Generate a Box-Behnken experimental design, because it reduces the number of runs to optimize the formulation, which helps us validate the microencapsulation of probiotics already reported, to maintain viability at high temperatures (170°-210° C). 24 runs of experiments must be performed to complete the experimental design and optimize the response variables: Size, viability and loading efficiency.

2) Generation of a baking prototype by Micro-baking. Evaluate the behavior of the optimized formulation in baking, carrying out 16 experiments that allow us to determine the necessary dose to ensure the desired concentration of probiotics. Finally, it is planned to produce a commercial-sized loaf for sensory analysis at the company.

3) Shelf life evaluation of developed bread. Test 3 shelf life conditions, considering 3 temperatures 4 ^° C, 25° C and 40° C, at a relative humidity (60%) to quantify the shelf life of the final product and the viability of probiotics in CFU/g.

4) In vitro digestion evaluation of the developed bread. Evaluate the quantity of viable microorganisms by performing an in vitro digestion test to determine the quantity of viable microorganisms that would potentially reach the human intestine.

STAGE 1: Functional Ingredient Development.

It was proposed to generate an experimental design (Box-Behnken) which is a spherical and rotating response surface, which includes a central point and midpoints between the corners, circumscribed on a sphere, because it reduces the number of runs to optimize the formulation, which helps us validate the microencapsulation of probiotics already reported, to preserve viability at high temperatures (170 ^or - 210 ° C). 24 runs of experiments must be performed to complete the experimental design and optimize the response variables: Size, viability and loading efficiency.

First, 3 proofs of concept were carried out to determine that through spray drying it was possible to obtain an encapsulation of 2 probiotics with different morphological characteristics (Figure 2).

These strains were chosen for their functional properties. Lactobacillus acidophilus has been widely reported to maintain and restore the health of the microbiota in the digestive system, support the immune system, and inhibit different pathogenic bacteria. As for Bacillus clausii, its properties, in addition to maintaining and restoring the microbiota of the digestive system and supporting the immune system, have been reported to maintain intestinal permeability, and therefore improve the absorption of nutrients. It also reduces the sensation of bloating, flatulence, diarrhea or constipation and relieves gastrointestinal pain and inflammation.

Subsequently, 2 experimental designs were carried out for the optimization of 2 probiotics, a sporulated one -Bacillus clausii- and a lactobacillus -Lactobacillus acidophilus-. In these experimental designs, the concentration of inulin and the vegetable protein used were varied, and the response variable was the viability of the organisms (Table 1 and Table 2).

Table 1. Experimental design performed for Bacillus clausii*.

*The factors that varied were A: % Inulin, and B: % Pea protein, the response variable was the viability obtained at the end of spray drying in Colony Forming Units per gram (CFU/g)

Table 2. Experimental design performed for Lactobacillus acidophilus*

*The factors that varied were A: % Inulin, and B: % Pea protein, the response variable was the viability obtained at the end of spray drying in Colony Forming Units per gram (CFU/g)

The optimization of both experimental designs was carried out through a surface analysis in the Design-Expert® software. The conditions were obtained optimal for both experimental designs (Figure 3 and Figure 4). Under these conditions, the optimal formulation for both probiotics was established.

Figure 3. Optimization of encapsulation of Bacillus clausii. The optimal point was determined at 35% inulin, 60% protein, thus obtaining the optimal formulation parameters for each of the microorganisms and a formulation was proposed for the encapsulation of both probiotics in a single microencapsulated ingredient obtained by spray drying (Figure 5).

At this stage, 3 encapsulates were obtained with temperature stability characteristics, whose microorganism concentration is:

1. Basillus clausii 3.53x1010 CFU/g of encapsulation

2. Lactobacillus acidophilus 1.22x108 CFU/g encapsulated

3. Encapsulated with 2 probiotics has Lactobacillus acidophilus in 6.69X108 CFU/g of encapsulation and Basillus clausii in 2.24X1010 CFU/g of encapsulation.

Figure 6. Scanning electron microscopy (SEM) of microencapsulates obtained, a) the encapsulation of Basillus clausii 3.53x1010 CFU/g of encapsulate is observed, b) the encapsulation of Lactobacillus acidophilus 1.22x108 CFU/g of encapsulate is observed, and in c) the encapsulation of 2 probiotics containing Lactobacillus acidophilus in 6.69X108 CFU/g of encapsulate and Basillus clausii in 2.24X1010 CFU/g of encapsulate.

The production of these encapsulations was scaled from a device that produced 4 to 5 grams per run (laboratory scale) to one that produced approximately 150g per run (pilot). With the same characteristics (Figure 7). This shows that the formulation is robust and can be scaled up, obtaining a similar viability.

STAGE 2: Generation of baking prototype by Micro-baking.

At this stage, the optimised formulation's behaviour in baking was evaluated by carrying out 16 experiments to determine the dose required to ensure the desired concentration of probiotics. Finally, the company planned to make a commercial-sized loaf for sensory analysis.

The three encapsulated products were tested in triplicate on boxed bread using the recipe provided by the BIMBO® team at a dose of 4 g/100 g of dry products. Breads were obtained with sensory characteristics equal to those of the control bread (Figure 8) and with viability of microorganisms at a concentration higher than 1x106 CFU/g, which is required to say that a food is probiotic (Table 3).

Table 3. Results in micro-breading tests at doses of 4 g/100 g

* In the Spanish encapsulation, the concentration necessary to be a 1x106 probiotic bread could not be quantified

Figure 8. Prototypes of microbreading with encapsulations generated by the direct method. From left to right, the control bread, the bread with microencapsulation from Spain (positive control), encapsulation with L. acidophilus, encapsulation with B. clausii and the encapsulation with the 2 probiotics (L. acidophilus and B. clausii) are observed.

The concentration of microencapsulated per 100 g of bread was increased to determine the effective dose. It was concluded that 4 g/100 g to 10 g/1 OOg is a sufficient dose to obtain cell viability above 1x106 CFU/g of bread as suggested by Mexican Standards.

También se realizaron panes de tamaño comercial de 460g, por el método de esponja. En estos prototipos se observaron diferencia en el volumen del pan. Sin embargo, estos fueron atribuidos a los tiempos de fermentación utilizados y el porcentaje de agua que se agregó a la receta (Figura 9). Table 4. Results in micro-breading tests at doses of 10g/100g

Commercial-sized loaves of 460 g were also made using the sponge method. In these prototypes, differences in the volume of the loaf were observed. However, these were attributed to the fermentation times used and the percentage of water added to the recipe (Figure 9).

These commercial prototypes, together with samples of each microencapsulation sufficient to make commercial breads, were delivered to BIMBO® so that they could be tested with its bread-making methodology.

pH measurements were taken on the prototype bread and no changes were observed due to the incorporation of any of the microencapsulants. Likewise, although a greater fermentation was observed in the breads added with Lactobacillus acidophilus, it cannot be determined that it is constant, this is attributed precisely to the encapsulation of the microorganism.

Based on these tests on micro-baking and commercial bread, it is concluded that the highest concentration of microorganisms is found in the central part of the bread crumb. In addition, the dose to obtain a concentration higher than 1x106 CFU/g must be at least 4g/100g of solid ingredients in the bread, and that with up to 10g/1 OOg of solid ingredients, concentrations higher than 1x108 CFU/g can be obtained in the bread crumb.

STAGE 3: Evaluation of shelf life of developed bread.

It was proposed to test 3 shelf life conditions, considering 3 temperatures and a relative humidity to quantify the shelf life of the final product and the viability of the probiotics in CFU/g.

Temperatures of 37° C, 20° C and 4 ^o C were tested at a relative humidity of 60% and it was determined that the shelf life of the product, using the preservatives indicated by BIMBO®, has a shelf life of more than 30 days at 37° C and increases to 30 days at 20° C and at 4 ^o C it has been monitored for at least 30 days with no apparent effect on sensory properties, no fungal growth and with viability greater than 1x108 CFU/g. The shelf life is similar in the control breads and the breads with probiotics added. Annex 1 shows the observation tables for each of the temperatures tested.

STAGE 4: In vitro digestion evaluation of the developed bread.

It was proposed to evaluate the amount of viable microorganisms by performing an in vitro digestion test to determine the amount of viable microorganisms that would potentially reach the human intestine.

The samples were digested in triplicate. One gram of each sample was subjected to:

• Digestion with porcine amylase at pH 4 for 20 minutes

• Digestion with porcine pepsin at pH 2 for 120 minutes

• Digestion with porcine pancreatin at pH 9.1 for 120 minutes

After digestion, the cell viability of the samples was measured, obtaining the following results shown in Table 5.

It can be observed that the concentration obtained in the bread was sufficient to be declared as a probiotic bread. Regarding digestion, no additional protection was observed by the encapsulation provided by AB-life Spain compared to the encapsulation obtained at the Tecnológico de Monterrey® to protect a Lactobacillus microorganism.

In the case of the microencapsulates obtained at the Tecnológico de Monterrey®, it is observed that the microorganism that best resisted in-vitro digestion was the spore-forming microorganism, although Lactobacilus acidophilus managed to survive in a concentration of 9.0x101 CFU per gram.

Table 5. Results in micro-breading tests at doses of 10 g/100 g with in-vitro digestion

The in-vitro digestion was replicated and the results are shown in Table 6. As can be seen, the best encapsulation is the double probiotic in a single encapsulation, which had the best performance not only in baking, but also in the in-vitro digestion conditions.

Table 6. Results in micro-breading tests at doses of 10 g/100 g with in-vitro digestion, Replica 1 and 2.

Optimal formulations were obtained for microencapsulation

Basillus clausii with 35% Inulin, 60% pea protein, 5% maltodextrin and an initial concentration of probiotics in the order of 4x1011 CFU.

Lactobacillus acidophilus with 30% inulin, 40% pea protein, 25% maltodextrin and an initial concentration of probiotics in the order of 4x108 CFU.

Both probiotics with 30% Inulin, 40% pea protein, 25% maltodextrin and an initial concentration of probiotics in the order of 4x1011 CFU for Basillus clausii and 4x108 CFU for Lactobacillus acidophilus.

These formulations could be scaled to produce from 4 g up to 150 g per run on spray drying equipment.

Due to the size of the microorganisms, it is determined that it is possible to change the microorganism to be encapsulated within the size range of those tested, due to the size of the encapsulated microorganisms that ranged from circular spore-forming microorganisms (smaller than one micrometer) to bacilli (approximately 2 micrometers) with the formulations obtained.

The optimal dose for incorporating bread is between 4g and 10g per 100g of dry ingredients for making boxed bread, to obtain a viability of between 1x106 and 1x108 for each microorganism.

It is possible to add it to white bread as well as whole wheat bread, obtaining sufficient viability of the microorganisms.

Boxed bread does not change its sensory properties, nor its shelf life when tested at 3 different temperatures.

The formulation that obtained the best performance in the baking process and in vitro digestion according to probiotic viability was the double probiotic encapsulation. In-vitro digestion results.

In-vito digestion 1 gram sample.

In-vito digestion 1 gram sample.

Claims

1. The formulation of a stable probiotic at baking temperatures encapsulated for food, the formulation consists of: a Bacillus clausii probiotic, where 35% inulin, 60% by microencapsulation is obtained by spray dryer, where the probiotic Basillus clausii with 35% inulin, 60% pea protein, 5% maltodextrin and an initial concentration of probiotics in the order of 4x1011 CFU, and an encapsulated Basillus clausii 3.53x1010 CFU / g is obtained that resists baking temperatures of 170 ° -210 °. 2. The encapsulated probiotic of claim 1, wherein the encapsulation of the microorganism determines that it is possible to change the microorganism to be encapsulated within the size range of those tested, this due to the size of the encapsulated microorganisms that ranged from circular sporulated (less than one micrometer) to bacilli (approximately 2 micrometers) with the formulations obtained. 3. The encapsulated probiotic of claim 1, wherein the formulation can be scaled to produce from 4 g up to 150 g per run in spray drying equipment. 4. The encapsulated probiotic of claim 1, wherein the optimal dose for incorporation into bread is between 4g to 10g per 100g of the dry ingredients for making boxed bread, to obtain a viability of each microorganism of between 1x106 to 1x108. 5. The formulation of a stable probiotic at encapsulated baking temperatures for food, the formulation consists of: a Lactobacillus acidophilus probiotic 1.22x108 CFU/g is obtained by spray drying, in a Lactobacillus acidophilus probiotic with 30% Inulin, 40% pea protein, 25% maltodextrin and an initial concentration of probiotics in the order of 4x108 CFU resistant to baking temperatures of 170°-210°. 6. The encapsulated probiotic of claim 5, wherein the encapsulation of the microorganism is determined that it is possible to change the microorganism to be encapsulated within the size range of those tested, this due to the size of the microorganisms. encapsulated that ranged from circular spore-forming (smaller than one micrometer) to bacilli (approximately 2 micrometers) with the formulations obtained. 7. The encapsulated probiotic of claim 5, wherein the formulation can be scaled to produce from 4 g up to 150 g per run in spray drying equipment. 8. The encapsulated probiotic of claim 5, wherein the optimal dose for incorporation into bread is between 4g to 10g per 100g of the dry ingredients for making boxed bread, to obtain a viability of each microorganism of between 1x106 to 1x108. 9. The encapsulated probiotic of claim 1 and 5, wherein the encapsulation consists of 2 probiotics containing Lactobacillus acidophilus in 6.69X108 CFU/g of encapsulation and Basillus clausii in 2.24X1010 CFU/g of encapsulation.