RO127522A2 - Process for microencapsulation of probiotic bacteria by drying with ultrasonic spraying - Google Patents

Abstract

The invention relates to a process for microencapsulation of probiotic bacteria through a process of drying by ultrasonic pulverization with a frequency of 120 kHz, in the drying chamber of a SonoDry750 installation, at temperatures ranging from minimum 130°C and maximum 170°C, a depression of 250 mm water gauge and a circulating air flow of maximum 80 m/h. The process ensures upper characteristics to the microencapsulated product and efficiency in using the same as addition to functional products destined to human nourishment.

Description

PROCESS FOR MICROINCAPSULATION OF PROBIOTIC BACTERIA BY DRYING WITH ULTRASONIC SPRAYING

The present invention relates to a process of microencapsulation of probiotic bacteria, based on ultrasonic spray drying. The field to which the proposal for invention is addressed is the application of biotechnologies to obtain microbiological preparations, probiotic bacteria, which are beneficial for human health. They are used in the manufacture of dairy products with functional properties and with a high degree of food safety.

Probiotic microogranisms can be defined as microorganisms that are or are introduced into the gut that have a beneficial effect on the host organism, by improving the balance of the intestinal microflora (Fuller 1991; Goldin 1998; Costin and Segal 1999; Gismondo et al. 1999). Numerous benefits to human health have been identified as being due to the consumption of probiotic products. Of these, the most frequently mentioned are the antimutagenic and anticarcinogenic effects, the anti-infectious, immunostimulatory properties, the reduction of serum cholesterol, the reduction of lactose intolerance and the improvement of the use of nutritional principles (Costin and Segal, 1999). Species of Lactobacillus acidophilus, Lactobacillus casei, Bifidobacterium bifidum, Bifidobacterium longum, Bifidobacterium patent, Bifidobacterium infantis and Bifidobacterium lactis are the most commonly used bacteria considered to be probiotics.

Research on the viability and survival of probiotic bacteria in the gastrointestinal tract and in food (especially in acidic dairy products) has shown that, in general, the viability of microorganisms dramatically decreases due to exposure to harmful environmental factors such as acids, Hydrogen ions, molecular oxygen or various antibacterial components (Dave and Shah 1997, Kailasapathy and Rybka, 1997; Hamilton-Miller et al, 1999). In addition, the beneficial effect of probiotic microorganisms occurs when they reach the large intestine in sufficient and viable numbers, after having withstood the above environmental conditions (Gilliland, 1989). The minimum number of probiotic cells (ufc / g) that must exist in the product at the time of consumption for the occurrence of a therapeutic effect has been quantified as "minimum of bio-value" (MBV) (Mortazavian et al. 2006). The International Dairy Federation (IDF) recommends that this index be higher than 10 ⁷ cfu / g, until the last validity of the probiotic food. In other countries, such as Argentina, Paraguay and Brazil the standard is at least 10 ⁶ cfu / g but refers only to bifidobacteria, and in Japan the total number of probiotic cells must be in excess of 10 ⁷ cfu / g. Also the literature records 10 ⁶ cfu / g (with strict reference to probiotic microorganisms) as the minimum value for which a product can be called probiotic, and in case reference is made only to bifidobacteria as probiotic microorganisms the minimum value recorded and accepted is of 10 ⁷ cfu / g.

In addition to the MBV index, the Dl (daily intake) index, which refers to the total daily amount of probiotic bacteria introduced consciously and voluntarily into the body, has also been defined, an index that is also determining for the effectiveness of a probiotic treatment. . The minimum value of Mr was set at 10 ⁹ viable cells per day. (Shah et al. 1995, Kurman and Rasic, 1991). The type of culture medium used in counting probiotic bacteria is also considered to be a particularly important factor in determining the viability of these cells, especially due to the different degree of selectivity of the various media used.

The loss of viability of bacteria in food (and especially in fermented pods) as well as the action of acids and bile salts in the gastrointestinal tract has led to numerous studies and researches to find new and efficient methods to improve the viability of probiotic bacteria.

Micro-encapsulation is one of the newest and most effective methods of maintaining the viability of probiotic bacteria at the present time. From a microbiological point of view, micro-encapsulation can be defined as the process of capturing / incorporating the cells of microorganisms by covering them with one or more layers of certain hydrocolloids, in order to delimit and protect the cell from the action of the external environment, in a way in which the cell can be released into the intestinal environment (Sultana et al., 2000, Krasaekoopt et al., 2003,; Picot and Lacroix, 2003).

Micro-encapsulation operation is an efficient method of increasing the viability of microorganisms and it has an important practice.

encapsulation is generally defined as an action of perfect coating of one substance into another substance that forms the outer shell, the capsule itself. Microencapsulation

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performing the micro-encapsulation operation (capsules smaller than 1 mm and reaching 2-4pm).

Therefore, the encapsulation represents a protection by covering a substance with a higher value of the capsule shell, but which, compared to this shell, is labile in nature, easily destructible under certain environmental conditions.

Towards the end of the nineteenth century a pharmacist, Upjohn, patented what we now call the dredging process. Relatively large particles of the solid substance are placed in a cylindrical-shaped mixer and a liquid solution is sprayed on top of them. In general, the liquid solution is a sugar solution in water, and the process itself involves several consecutive sprays and evaporation until the sugar coating reaches the desired characteristics. The process is still widely used today in pharmaceutical technology in order to make a sweet coating for certain medicines. In the food industry this process has expanded rapidly to various types and brands of sweets.

Since the end of the nineteenth century, various encapsulation processes have been studied by the international scientific community, starting with Upjohn's dredging, mentioning methods such as spray drying, solvent dehydration, extrusion encapsulation, fluidized bed encapsulation, extruded fluid bed , the formation of clathrates, the process of cooling spray and reaching today methods such as spray drying using ultrasound, the method used to micro-encapsulate biological materials.

Most specialists believe that pioneering micro-encapsulation was accomplished in the third decade of our century by the National Cash Register Company (Dayton, Ohio). Using the chemical process called co-preservation (phase separation), the National Cash Register has developed a method of absorbing a dye in a cross-linked gelatin coating. The process approved by them was extended to commercial scale in 1954 for the production of the first self-printing paper. In 1981, the production of self-printing paper worldwide reached already 500,000 tons per year.

The discovery of the National Cash Register and the new co-preservation process generated great interest around them in the 1950s and the encapsulation process itself began to extend to other areas such as agriculture, cosmetics, electronics and food. The co-preservation process itself has been studied and, in some areas, was considered not only applicable especially economically compared to the processes used previously. In other areas, however, the process was not applied due to the technical limitations that it proved. But this process reserves the merit of being a roadblock to the future encapsulation technologies that we apply and develop today.

For the encapsulation of living organisms, bacteria in particular, emulsification-gelling, extrusion or spray drying and, more recently, the use of fluidized bed are used as basic methods.

All these more or less advanced technical procedures must be supported by the use of polymers that are as efficient as possible and adapted to direct human consumption. Consumption safety, gelling properties as well as resistance to actions of gastric juice and bile salts are the main criteria for choosing encapsulating biopolymers.

At the moment in the first place as use for the purpose of encapsulation of living organisms is alginate (sodium or calcium). Also tested are substances such as starch, xanthan, gelane, carrageenan, gelatin, chitosan, rosemary flour, whey protein and even calcium chloride.

Within the ICA Research & Development Bucharest, microincapsulation of probiotic bacteria was performed by the classical method of gelation and simple extrusion, then by spray drying.

It was concluded that the first two encapsulation techniques (extrusion and emulsification) are not reliable for industrial applications, and the product resulting from the application of these techniques cannot be used for the intended purpose (the microcapsules obtained were uneven, very hygroscopic and very difficult to dry).

The spray drying technique has generated much better results. Depending on the encapsulating biopolymer or the mix of biopolymers, depending on their concentration and the nature of the gel generated, this technique has led to obtaining homogeneous products from a structural and compositional point of view, but also to easily reproducible results.

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The advantage of spray drying also lies in the fact that it can be easily and industrially applied.

Spray drying is probably one of the oldest encapsulation techniques and is currently the most widely used technique for preparing dry stable food additives and especially food flavorings. The flavors obtained by this process are available on the market since the 1930s. The process is economical, flexible, adaptable to industrial use, an advantage also being the fact that there is a wide range of machines specially designed for this purpose. Generally the process is carried out in three stages. An aroma, usually an oil (but water-soluble aromas may be used) is mixed with an edible polymer such as gelatin, vegetable gums, modified starch, dextrin or non-reducing proteins, usually in a ratio of 1: 4 (aroma: substances for fixing). An emulsifier is added and the mixture is homogenized to produce an oil emulsion in the water with a slight micellar structure. The emulsion is atomized and dried using one of the methods in which an aerosol is introduced into a hot air column, located in a drying chamber. The drops take on a spherical shape, with the aroma oil gravitating to the center of the drop and the aqueous solution forms the particle shell. Rapid evaporation of the water from the casing causes the temperature to remain below the boiling point of the water even in the hot air columns with particularly high temperatures. Fortunately, the exposure to high temperatures in the drying room is very short, about a few seconds. Water-soluble flavors (or other food ingredients) can also be fixed or immobilized with the aid of certain hydrocolloids, but these particles do not have a well-defined separation between the encapsulating substance and the encapsulating substance. There are mentioned some cases in which the two substances made a homogeneous mixture before drying.

The spray-dried particles usually have dimensions below 100pm, which gives them a very good solubility or dispersibility in water. If necessary, the spray-dried particles can be obtained ready to be agglomerated or granulated, as in the case of dry mixtures, where a greater uniformity of the dimensions of the component particles is required.

With the appearance of aromas dried by spray, the oxidative phenomenon was significantly reduced as well as the evaporation phenomenon of volatile substances. The coatings of polymeric colloidal nature prove to achieve a certain protection on the inner layers of the active substance. Initially, for this type of substances, the term used was captive or protected flavors. Later, in the early 1950s, the term encapsulated flavors was established.

Microincapsulation methods of probiotic bacteria

The probiotics encapsulation technology can be divided into two parts:

a) Microencapsulation of probiotics in the encapsulating solution (microencapsulation itself)

b) Drying of the wet capsules obtained, in order to obtain powder or granules.

a) Microencapsulation itself

Extrusion or emulsification techniques, also called droplet method and two-phase method respectively, are two of the basic principles of encapsulation.

Extrusion technique.

The extrusion method is the oldest and most frequently used technique for the production of hydrocolloid capsules. In general, it is a cheap and easy to use method at the laboratory level, being also the method that affects the viability of bacterial cells to a small extent. The defining terms for this method are said to be biocompatibility and flexibility (Tanaka et al., 1984, Martinsen et al. 1989). Unfortunately this technique cannot be applied in industrial production, due to the weight with which the particles are formed and the reduced working speed.

Emulsification technique

The emulsification technique has been successfully applied for microencapsulation of probiotic lactic bacteria. Unlike the extrusion technique, this technique can be easily transposed at industrial level, and the particle size is considerably smaller (25um-2 mm).

However, the method requires high costs for making good quality capsules, compared to the extrusion method due primarily to the use of an oil to form the emulsion. In this technique, a small amount of polymer-cell mixture is added to a large volume of oil (soybean oil, sunflower oil, corn or any other vegetable oil may be used). The resulting solution should then be homogenized by appropriate stirring, until a water emulsion is formed

Og-2 O 1 0 - 0 1 1 7 2 - - \ 12 h -11- 2010 in oils. For a better homogenization, emulsifiers such as Tween 80 can be added in 0.2% concentrations (recommendation made by Sheu and Marshal, 1993). Once the emulsion is formed, the soluble polymer is insolubilized by the addition of calcium chloride. Smaller sizes of water particles in the emulsion will lead to small capsule sizes obtained.

b) Drying of the wet capsules obtained, in order to obtain powder or granules

Drying of the capsules obtained for the purpose of obtaining powder or granules can be done by different methods. Of these, the most important methods are lyophilization, spray drying and fluidized bed drying. In general, the drying process itself affects to a certain extent the granules formed, reducing their viability. In the freeze-drying process the temperature affects very little the finished product, compared to the other drying processes. However, this method is expensive and difficult to adapt to industrial use.

The most efficient method for obtaining capsules was considered spray drying, as it has relatively low costs and can be used for drying large volumes of solutions. In this case, however, there is a decrease in the viability of the encapsulated microorganisms, due to the simultaneous dehydration and heating.

However, it seems that a solution for using this method is the one presented by Picot and Lacroix in 2003. The procedure was considered to be economical due to the fact that it managed to maintain the viability of the cells in a very large percentage. The method consisted of coating drops of milk fat containing probiotic bacteria lyophilized with polymers from among the whey proteins, when used with spray drying of certain emulsifiers. The particle size of the lyophilized culture is based on the particle size obtained from the drying process. For proper encapsulation, these particles must be larger than the cells (2-4pm) but smaller than the fat particles to be incorporated (10-15pm).

The patented process refers to the preparation of the micro-encapsulated solution containing the bifidobacteria and the drying of the preparation in an ultrasonic spray drying plant.

The experiments for obtaining microincapsulated probiotic bacteria (bifidobacteria) were performed on a laboratory facility, SonoDry750, where the product supply in the drying chamber is made by spraying with an ultrasonic dialysis.

Ultrasonic spray drying plant

In the case of this drying process of the obtained capsules, the temperature is the determining factor of the process, being a restrictive factor, and which had both an upper and lower limit.

The lower limit is achieved by the drying process itself, which can only be achieved completely if a minimum drying temperature is reached. This minimum temperature is determined by the analysis of the drying process and it is sufficient if the particles of the drying tower will not stick to product particles only in insignificant quantities and if the liquid phase recovery vessel, located immediately below the drying tower, is leaking. leave empty at the end of a drying process.

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The upper temperature limit is determined by analyzing the viability of the bifidobacteria in the recovered powder. Important is the relationship between the drying temperature and the viability of the bacteria, so the lowest temperature at which the drying process is performed well in the SonoDry750 dryer must be chosen.

The microcapsules of the bifidobacteria were obtained by making a preparation consisting of:

% Strength Ringer solution

11% lyophilized bacterial preparation

2% maltodextrine.

The variable factors in the experiments were: frequency of the ultrasound nozzle, inlet and outlet temperatures from the drying chamber, the optimum power applied to the ultrasonic nozzle used to supply the drying chamber, the flow of circulating air.

The choice of the optimum variant was conditioned by the optimization of the technological process for obtaining the maximum yield in the product, and by the viability of micro-encapsulated bacteria.

Example no. 1.

Frequency of the product spray spray in the drying chamber in the SonoDry750 - 60 kHz installation air inlet temperatures 90, 130, 150 °, 170 ° C.

The air outlet temperature from the inlet was automatically adjusted, depending on the product flow rate and drying speed, and was between 70 and 96 / - 5 ° C.

Preparation subjected to drying:% strength Ringer solution; 11% lyophilized bacterial preparation; 2% maltodextrin, 2% sodium alginate the optimum power applied to the ultrasonic nozzle used to supply the drying chamber with product ... 11 watts;

- the flow of circulating air in the spray drying plant, maximum, 80 m ³ / h;

pressure ... - 250mm water column

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Figure no. 3 . Diagram of the drying process in the SonoDry750 dryer, with 60kHz duct

From the diagram it can be observed that three successive drying processes were carried out, each differing from the previous one by temperature.

After the test it was concluded that the temperature of 130 ° C is not suitable, as there were difficulties in drying the product. Setting the starting temperature at 150 ° C a

CV 2 0 1 0 - 0 1 1 7 2 -2 Ί -11- 2010 led to better physical results. The preparation was drier, but a film was produced on the top of the drying tower. From the point of view of the drying process, at 170 ° C the desired results were obtained, ie a dry, non-sticky preparation. However, the viability of the bacteria after drying was lower.

Table no. 1 Viability of lactic bacteria from the product obtained by applying different drying regimes with 60 KHZ nozzle

Nr. crt. Air inlet temperature (° C) Initial viability (cfu / g) - ^v i Viability of the preparation obtained (cfu / g) - v _f Decreased viability (v / Vf) 1. 130 95 x 10 ^lu 28 x 10 ^a 34 2. 150 95 x 10 ^lu 24 x 10 ^y 39 3. 170 95x 10 ^w 23 x 10 ^a 41

The microcapsules obtained must not exceed the size of 30 micrometers, in order not to be detected by the taste buds, when introduced into different products.

The 60kHz nozzle can generate capsules below the limit size or close to 30 microns.

Although the results regarding the viability of bacteria in the case of 60 kHz nozzle use are good (table no. 1), it was found that the microencapsulation was not completely performed, as demonstrated by the gastrointestinal tract simulation test [following MRS-NNLP cultivation in anaerobiosis, none of the three showed biological activity at dilution D (-5)].

Example no. 2. Frequency of the spray nozzle of the product in the drying chamber in the SonoDry750 - 120 kHz installation air inlet temperatures 130, 150 °, 170 ° C.

The air outlet temperature from the inlet was automatically adjusted, depending on the product flow rate and drying speed, and was between 70 and 96 / - 5 ° C.

- Preparation subjected to drying:% strength Ringer solution; 11% lyophilized bacterial preparation; 2% maltodextrin, 1% sodium alginate the optimum power applied to the ultrasonic nozzle used to supply the drying chamber with product ... 11 watts;

- the flow of circulating air in the spray drying plant, maximum, 80 m ³ / h;

pressure ... - 250mm water column

- the optimum flow rate set for the 10 ml syringe pump is 4ml / min. In this case, the drying option was excluded in a minimal regime, with the air inlet temperature of 90 ° C. The flow rate of the liquid product was maintained at 4 ml / min.

The experiment in which the air temperature at the entrance to the drying chamber was 130 ° C, using an ultrasound frequency of 120 KHz, revealed an unsatisfactory drying regime, due to the product sticking on the top of the drying chamber.

The application of a drying process at which the air inlet temperature in the drying chamber was set to 150 ° C (figure no. 4) was carried out in good conditions, without the product being stuck to the dryer walls and the yield was better.

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Figure 4. Diagram of the drying process in the dryer SonoDry750, at 150 ° C and 170 ° C

In the case of the technological process variant with raising the air temperature at the entrance to the drying room at 170 ° C, the drying proceeded in very good conditions, but part of the obtained product, with very small particles, passed through the first cyclone and joined the the second cyclone separation vessel. In Figure 4. the drying cycles at 150 ° C and 170 ° C are present.

It can be observed that the drying cycle at 170 ° C is slightly more unstable than at 150 ° C. The viability of probiotic bacteria after the microincapsulation and drying process is presented in table no.2.

Table no. 2. Viability of lactic bacteria from the product obtained by applying different drying regimes with a dose of 120 KHZ

Nr. crt. Drying temperature a of the mixture (° C) Initial viability (cfu / g) - Vi Viability of the preparation obtained (cfu / g) Vf Decreased viability (v ₍ / v _f )) Yield obtained % (quantity of product dry g / 100ml liquid product) 1. 130 165 x 1O ^1U 82 x 10 ^a 20 2.21 2. 150 165 x 1O ^1U 21 x 10 ^a 78 2.93 3. 170 165 x 10 ^w 1 x 10 ^a 1650 2.88

It is thus found that the optimum temperature regime of the air used in the drying process is 150 ° C ± 10 ° C. This value ensures a satisfactory process both from the point of view of the obtained yields and from the microbiological point of view.

The optimum microincapsulation variant of probiotic bacteria by drying is the one in which an ultrasonic spray nozzle, with a frequency of 120kHz, is used to supply the product in the drying chamber. In the product subject to drying, alginate is used in proportion of 1%.

Micro-encapsulation technology

The technological process regarding the micro-encapsulation of probiotic bacteria is carried out according to the technological scheme presented in figure no. 5

The novelty of the invention proposal was to apply a drying process of microcapsules of probiotic bacteria (bifidobacteria) in a spray drying plant.

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4 -11- 2010 ultrasonic. Through this system there is also an emulsification of the biological preparation with probiotic bacteria, which results in obtaining microcapsules of small size, of approx. 30 pm. The size of the microcapsules is important when used in the manufacture of dairy products, by directly adding them to the finished product or milk to be fermented, because they do not have to be detected by the taste buds.

From the point of view of the efficiency when drying the microcapsules, the optimum temperature is between 150-170 ° C, and d.p.v. of the viability of bacteria after the drying process, the optimum temperature is in the range 130-150 ° C. The optimum frequency of the ultrasonic spray nozzle of the product in the drying chamber of the SonoDry750 is 120 kHz.

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Claims (9)

1. Process for micro-encapsulating probiotic bacteria by drying, based on the ultrasonic spray of the micro-encapsulated product entering the drying room of a SonoDry750 drying plant. 2. The dry product is obtained by micro-encapsulating probiotic bacteria (bifidobacteria) in a mixture of% strength Ringer's solution; 11-12% lyophilized bacterial preparation; 1- 2% maltodextrine, 1-2% sodium alginate. 3. The supply of micro-encapsulated probiotic bacterial product into the drying chamber of the installation is made by an ultrasonic spray nozzle with a frequency of 120 kHz. 4. The temperature limits at the entrance of the product in the drying room of the installation, according to the procedure from item 1 are of min. 130 ⁰ C and max. 170 ° C 5. The outlet temperature of the inlet air depending on the product flow rate and the drying speed, according to the procedure from item 1 is between 70 and 96 / - 5 ° C. 6. The flow of circulating air in the spray drying plant, according to the procedure from item 1 is maximum 80 m ³ / h; 7. The working pressure according to the procedure from item 1 is ... - 250mm water column 8. The process of micro-encapsulation of probiotic bacteria in an ultrasonic spray drying plant, operating at the parameters according to points 3-7, ensures a viability of the dry product between 20 and 28%. 9. The concentration of viable probiotic bacteria in the micro-encapsulated product, according to point 8, is between 10 ⁹ and 10 ¹⁰ bacteria / gram.