Different methods of stabilizing probiotics (2023)

1. Introduction

Starter cultures form a basis in the production of fermented foods. Probiotics are the most important group of bacterial starter cultures. Commercial starter cultures were initially provided in liquid form before concentrated starter cultures were made. Advances in biotechnology later led to the use of concentrated starter cultures in frozen and lyophilized forms for direct incorporation into food formulations. Using frozen or lyophilized starter cultures eliminates on-site subculturing, reduces costs associated with mass culture production, and reduces the risk of bacteriophage infection.Desmodet al. 2002).

Very low transport and storage temperatures are the main commercial disadvantages of frozen starter cultures.Gandhi et al. 2012). In addition to the risk of thawing, high transport costs can limit the use of frozen starter cultures in distant areas or countries. Probiotic bacteria starters are generally preserved by freeze-thaw and lyophilization. Despite efficient methods, freezing and lyophilization have high manufacturing costs and high energy consumption. For this reason, alternative drying methods such as spray drying, fluidized bed drying and vacuum drying have received increasing attention.

Most vegetative forms of microorganisms are characterized by low thermostability. They show considerably high rates of death and loss of activity as a result of thermal inactivation in the temperature range of 40 to 60°C^ÖC. With regard to microbial biomass, there are certain critical water contents (depending on the property of the object), which leads to inactivation of dehydration. This can be attributed to the fact that, in the case of vegetative forms of microorganisms, water not only provides an environment for their life, but also serves as a substrate for biochemical reactions, and its removal below a certain level prevents the maintenance of functions. metabolic processes and, consequently, leads to cell death. Dehydration methods that allow maintaining the viability of microbial biomass include: lyophilization, sublimation drying, including fluidized drying using inert materials (vehicles) and spray drying (Santivarangkna et al. 2008).

Lyophilization is therefore more convenient and simpler as no freezing conditions are required during distribution. While freeze-drying is the traditional drying technique used commercially by starter culture producers, it is time consuming and more expensive than other drying methods.Fonseca et al. 2001,Ampatzoglouet al. 2010,Morganet al. 2006). Many attempts have been made to develop alternative drying methods at lower cost, and some authors have reported reasonable cell viability after drying.Tymczyszynet al. 2008).

Spray drying is considered a good long-term preservation method for probiotic cultures. Spray drying of microorganisms dates back to Rogers' 1914 study of dried lactic acid cultures. The concept of spray drying was first patented by Samuel Percy in 1872, and its industrial application in milk and detergent production began in the 1920s. large amounts of starter cultures. Since then, much research has been done on spray-drying bacteria without loss of cellular activity to overcome the difficulties associated with handling and maintaining liquid cultures.

Spray drying is a unique process in which particles are formed simultaneously with drying. It is very suitable for the continuous production of powdered, granulated or agglomerated dry solids, liquid starting materials such as pumpable solutions, emulsions and suspensions. The final spray drying product must meet precise quality standards in terms of grain size distribution, residual moisture, bulk density and grain shape. In the spray drying process, dry granulated powders are produced from a paste-like solution by atomizing the wet product at high speed and spraying the droplets into a stream of hot air, e.g. 150-200ºC. Atomized droplets have a very large surface area in the form of millions of micron-sized droplets (10-200 µm), resulting in a very short drying time when exposed to hot air in a drying chamber (Sunny-Roberts, Knorr 2009).

Spray drying involves atomizing a liquid feedstock into a jet of droplets and contacting the droplets with hot air in a drying chamber. Sprays are generated by rotating atomizers (wheels) or nozzles. Evaporation of moisture from droplets and formation of dry particles occur under controlled temperature and airflow conditions. Powder is continuously discharged from the drying chamber (Peighambardoust al. 2011).

Spray drying is a common industrial and economical method for preserving microorganisms and producing starter cultures used in the manufacture of fermented dairy products. Survival of lactic acid bacteria is an important issue when spray drying is used to produce microbial cultures. However, the biological activity of a lactic yeast, which includes cell viability and physiological state, is a criterion for evaluating yeast quality.Carvalhoet al. 2004,Anantaet al. 2005).

Both the rate of water evaporation and the temperature of droplets containing microbial cells have been shown to have a significant impact on their survival during spray drying. Since it is not yet possible to quantify changes in bacterial cells and their survival in situ during spray drying, single drop drying is used. Single drop drying, in which a single drop is suspended in air conditioning and in motion, offers the closest experimental resemblance to the spray drying environment. Drying of individual droplets can be done in a number of ways, for example (a) a single or a stream or streams of droplets can be dropped under gravity into a tower-like dryer, (b) a drop can be levitated using ultrasound or aerodynamic fields, or (c) a drop can be suspended at the end of a thin glass thread. The first two methods are not very popular because they are expensive and the heat and mass transfer rates in these environments are not close to the convection drying environment of spray drying. Li et al. (2006) investigated the inactivation kinetics of two probiotic strains (Bifidobacterium infantisES. thermophilus) at air temperature and relative humidity in the range of 70-100°C and 3.7-0.5%, respectively, using single drop drying in skimmed milk as the suspending medium. They reported that inactivation occurred mainly in the early stages of desiccation, when the evaporation rate was high. The above studies do not provide a unanimous view on whether drying speed or droplet temperature is the limiting factor in bacterial survival during drying.

Lyophilization is a preferred drying method for thermally sensitive bacteria as it maintains their survival at a reasonably high level. However, freeze-drying is a batch process with a considerably long drying time. It is also expensive due to high energy demand. For drying starter cultures, spray drying can be a viable alternative if the survival rate can be increased to make it economically attractive. Because spray drying is relatively inexpensive, energy efficient, has a high yield and is a hygienic process (Papapostolou et al. 2008,Carvalhoet al. 2004).

To minimize cell death, the effects of drying parameters (supply and exhaust air temperature, air flow rate, relative humidity, residence time, protectants) on bacterial survival and viability must be understood in minute detail. The drying process damages the cell wall and cellular components, mainly the cytoplasmic membrane and proteins, resulting in loss of survival. This cell damage leads to cell inactivation and adversely affects the productivity and properties of the dry culture, so cell damage must be minimized. Preservatives such as carbohydrates, proteins, amino acids, gums and skim milk are used to minimize bacterial inactivation during drying. It is reported that low molecular weight carbohydrates such as sugars stabilize the membrane and protein chains of cellular macromolecules in the dry state by hydrogen bonds instead of water when the water molecules are removed by desiccation. Proteins are able to form relatively stable intracellular glasses, and this allows them to be more effective than sugars as protective materials for bacterial cultures. It is reported that the combination of different protectants (eg mixtures of sugar and protein) may have a synergistic effect on cell viability rather than acting individually. Both the rate of water evaporation and the temperature of droplets containing microbial cells have been shown to have a significant impact on their survival during spray drying.

Encapsulation of probiotics is used to increase the resistance of bacteria to freezing and freeze-drying food. In most studies, probiotic bacteria were encapsulated in a gel matrix made from natural biological materials such as alginate, κ-carrageenan, and gellan/xanthan (Semenov and ai. 2010,Kanmaniet al. 2011). The core and wall solution was converted into droplets of the desired size by an extrusion process using an emulsion or by transferring organic solvents. One problem with the probiotic entrapment approach is that gel bead technologies primarily stabilize bacteria in liquid products and are difficult to grow. In order to extend shelf life, it is convenient to convert the microcapsules into a dry powder using techniques such as spray drying, freeze drying and/or fluid bed drying. Spray drying is an economical and effective technology, but it causes high mortality due to simultaneous dehydration, heat and oxygen stress on bacteria during the drying process. Lyophilization is considered one of the most suitable methods for drying biological materials and delicate foods. However, when this method is used to dry probiotic bacteria and other cells, undesirable effects occur, such as cell membrane leakage due to changes in the physical state of membrane lipids or changes in the structure of sensitive proteins in the bacterial cell. Protective solutes like cryoprotectants (saccharides and polyols) and other compatible solutes like adonitol, betaine, glycerol and skim milk have been used to increase bacterial viability and prolong their survival during freeze-drying and subsequent storage. These studies lead to the conclusion that the effect of each preservative on the viability of a specific strain of lactic acid bacteria during or after the freeze-drying process needs to be determined on a case-by-case basis (Heidebach et al. 2010,Krasaekoopt et al. 2003).

As mentioned above, dry probiotic microcapsules can be coated with an additional layer (shell) to protect the bacterial nucleus from the acidic environment of the stomach and to avoid the harmful effect of bile salts on the cell membrane. This extra wrapping can help release the bacterial nucleus to a desired location in the GIT. To be further coated, frozen bulk powders are micronised to a narrow particle distribution. This process is complex, consumes a lot of energy and reduces the viability of dry cells.

The pharmaceutical industry has recently used spray freeze-drying to produce pharmaceutical powders. This process combines the narrow article size distribution of an extruder and the lyophilization process to produce a dry powder with the desired particle size and narrow distribution. The basic principle of spray freeze-drying is to spray a solution containing dissolved/suspended material (e.g. protein) through an atomizing nozzle into a cold vapor phase of a cryogenic liquid such as liquid nitrogen so that the droplets freeze during their passage through the cold can begin to freeze the vapor phase and freeze completely on contact with the cryogenic liquid phase. The frozen drops are then dried by lyophilization (Lianet al. 2002,Gardiner and ai. 2002).

Spray-dried powders have a controlled size, larger specific surface area and better pore character than spray-dried powders. The particles retain their spherical, porous morphology and can be further coated with a food-grade enteric biological polymer designed to disintegrate at specific locations in the GIT.

Recently, this method was developed and the solution is sprayed directly into liquid nitrogen through a needle under appropriate pressure. Cooling rates in the spray freezing section depend on many factors and are therefore very difficult to estimate. However, it has been claimed that maximum freeze cooling rates in liquid nitrogen are on the order of 300 K/s, which is believed to be the upper limit of the cooling rate. To the best of our knowledge, the spray freeze-drying process has not yet been used to produce dry powder of probiotic cells.

Vacuum drying has been described as the most promising method for storing sensitive biological material due to its acceptable cost-effectiveness. However, vacuum drying conditions (time, temperature) need to be optimized to allow for the best bacterial recovery after dehydration-rehydration and to avoid cell damage.Tymczyszynet al. 2008).

It has been suggested that bacterial death results from the inactivation of critical sites in cells. Membranes, nucleic acids and certain enzymes have been identified as cellular targets for dehydration damage. It has been reported that after dehydration-rehydration, microorganisms can be recovered even if the cell membrane is damaged. Furthermore, it was also observed that an increase in the absolute value of the zeta potential can be accompanied by an increase in the latency time. Changes in this parameter were correlated with the loss of the original orientation of surface macromolecules and therefore the ability to restore surface properties after rehydration. This indicates that there are other bacterial structural parameters besides membrane integrity that influence bacterial viability after dehydration-rehydration. In this sense, the data obtained by differential scanning calorimetry show that the damage caused to membrane lipids, ribosomes and DNA is reversible, while the damage caused to proteins is not.

When using vacuum drying, it should be noted that heat stress occurs in parallel with water stress, which is likely to cause irreversible damage. For this reason, microorganisms must be exposed to high temperatures for the shortest possible time, and the right choice of dehydration times and temperatures is crucial to achieving the best vacuum drying conditions.

The challenge of making vacuum drying a widely used method for preserving microorganisms is the difficulty of defining standardized conditions that allow the comparison of results obtained in different laboratories. The reason for the difficulty is that times and temperatures for dehydration processes are related to drying conditions (i.e. exposure area, vacuum system pressure, sample weight or volume, etc.), which are often equipment dependent. used. Therefore, to make the results comparable, it is necessary to relate the experimental conditions to a parameter independent of these experimental conditions, for example, the water activity of the sample after dehydration under certain conditions.

Consequently, considering that both the drying time and the drying temperatures affect the final water activities of the samples, the definition of the drying conditions in relation to the final water activity becomes important to define parameters correlated with the state of dehydration of the samples. cells. This fact would help to achieve the best conditions for preservation processes.

Bifidobacteria benefit human health by improving the balance of the intestinal microbiota and strengthening mucosal defenses against pathogens. However, for probiotics to be therapeutically effective, it is suggested that the products contain at least 6 log CFU/g of bacteria until the end of their shelf life. Although bifidobacteria are increasingly recognized as probiotics with beneficial properties, they are also fastidious, obligate anaerobes and therefore represent a technological challenge for the food industry. storage and oxygen content.

In this context, microencapsulation of probiotic bacteria is receiving increasing attention as a method to improve the stability of probiotic organisms in functional foods. Microencapsulation can improve the survival of these microorganisms both during processing and storage and during passage through the human gastrointestinal tract. Spray drying is recognized as a microencapsulation method and has been studied as a means of stabilizing probiotic bacteria in a variety of food matrices, most commonly composed of proteins, polysaccharides, sugars and combinations thereof. The survival rate of the crop during spray drying and subsequent storage depends on several factors which may include the species and strain of the crop, the drying conditions and also the use of encapsulating agents.

Reconstituted skim milk is an encapsulating agent that has been shown to have a beneficial effect on increasing cell survival during the spray drying process. Another approach to increasing the viability of bifidobacteria is the use of prebiotics, which are non-digestible food components that positively affect the host by selectively stimulating the growth and/or activity of bacteria in the colon. Inulin is a prebiotic with a degree of polymerization (DP) between 10 and 60. It is obtained from chicory roots and consists of chains of fructose units. Oligofructose is obtained by the partial hydrolysis of inulin and therefore has a lower DP, ranging from 2 to 8. A mixture of oligofructose and inulin is known as oligofructose-enriched inulin. These prebiotics could potentially be used as carriers for spray drying and could be useful for improving the survival of probiotics during processing. However, the use of different encapsulating agents to prepare microcapsules can result in different physical properties depending on the structure and properties of each agent. (Fritzen-Freire et al. 2012).

The study was conducted to evaluate the feasibility and physical properties ofBifidobacteriumBB-12 microencapsulated by spray drying as a partial substitute for reconstituted skimmed milk (RSM) as an encapsulant with the prebiotics inulin, oligofructose and inulin enriched with oligofructose (ratio 1:1, 200 g/total concentration). Viable cell counts of microcapsules were determined during 180-day storage at 4°C and -18°C. Partial replacement of RSM by inulin and partial replacement of RSM by oligofructose-enriched inulin increased the initial number of bifidobacteria in the microcapsules. On the other hand, inulin enriched with oligofructose and microcapsules prepared with oligofructose showed better protection for bifidobacteria during storage. The use of prebiotics did not affect the morphology of the microcapsules. However, capsules made with oligofructose had a smaller particle size. The inclusion of prebiotics reduced the moisture content and water activity in the microcapsules. Microcapsules made with inulin showed the lowest dissolution in water, while microcapsules made with oligofructose were the most hygroscopic. The general color difference of the microcapsules was considered not obvious to the human eye. The results of thermal analyzes indicate an increase in the stability of microcapsules produced with prebiotics. Finally, the results showed that oligofructose-enriched inulin is the most suitable prebiotic that can be used as a partial replacement of RSM for microcapsules.BifidobacteriumBB-12 by spray drying with great potential as a functional ingredient for dairy applications. (Fritzen-Freire et al., 2012).

An ultrasonic vacuum spray dryer was used to produce a dry powder of highly viable probiotic cells. Drying was carried out in two steps: vacuum spray drying of the solution followed by fluidized bed drying of the powder. The inclusion matrix was a combination of trehalose and maltodextrin. The impact of external and internal variables on cell survival during the drying and storage process was examined. The hypothesis was that minimizing oxidative and thermal stress in the drying stages, in addition to an adequate choice of formulation, increases cell viability during drying and storage. It was concluded that the faster the inclusion matrix reaches the glassy state during the drying process, the greater the survival of the probiotic. Evaluation of the water activity and moisture limit of the glass matrix showed that maltodextrin DE5 is a better encapsulation matrix than maltodextrin DE19. Combining trehalose with maltodextrin in the encapsulation matrix resulted in a significant increase in survival of up to 70.6 ± 6.2%.

Higher temperatures used during spray drying can be harmful to bacteria. However, this is not the case for certain lactic acid bacteria. For example, similar survival rates have been obtained when freeze-drying and spray-drying concentrated cultures.Lactobacillus bulgaricus. Cell damage caused by probiotics can be reduced and viability preserved by controlling drying parameters; in particular by lowering the outlet temperature of spray dryers and incorporating suitable vehicles into the drying medium. The addition of sugars to the growth medium also affects the survival of dried probiotic preparations. The incorporation of glucose into the formulations did not appreciably affect the survival of the probiotics during drying, but had marked effects.lactobacilliGG survival during subsequent long-term storage. These results confirm those of others who also found that although LGG survival during spray drying was not significantly affected when different media (reconstituted skim milk (RSM), RSM/polydextrose, RSM-raphthylose P95) were used, the survival of affected bacteria during long-term storage. A similar result was reported where the presence of sugars (fructose, trehalose or sucrose) or sugar alcohols (inositol, sorbitol) improved survivallactobacillus plantsEL. rhamnosusduring storage but not during freeze-drying (Yang Ying et al. 2012).

The glucose-containing formulations in the study improved the storage stability of spray-dried LGG microcapsules stored under similar environmental conditions, although the glass transition temperature of these formulations was decreased compared to those of glucose-free formulations. It has been suggested that the incorporation of small sugars improves bacterial survival during desiccation, as they can replace water removed from proteins/enzymes in cells and reduce the membrane phase transition temperature. The results suggest that the effect of glucose is more significant during storage than during drying, although the glucose-containing formulations did not retain their glassy state under different storage conditions. The results of the work are consistent with those of others, showing that a glassy state during storage alone is not sufficient to stabilize dry bacterial preparations.

Preservatives that maintain the structural integrity of cell membranes, protein and enzymatic functions are needed to improve the viability during storage of dried probiotic preparations. These results suggest that a prerequisite for LGG survival in the glassy state are direct interactions between a low molecular weight sugar and cellular components, helping to preserve cellular functions during desiccation with subsequent beneficial effects on long-term storage. . Both the maintenance of a glassy state during storage and the incorporation of glucose or a low molecular weight sugar into the drying medium are necessary for optimal survival of probiotic powders during storage.Yang Ying em 2012).

We investigated the method of forming dry encapsulated probiotics using ultrasonic vacuum drying (UVSD) and a microcapsule matrix composed of maltodextrin and trehalose. The results of this study show that the use of UVSD quickly brought the matrix to a glassy state and allowed high survival of probiotic cells – 3.3 x 10⁹cfu/g TS, this was obtained with Maltodextrin DE-Trehalose (1:1) 20%g/100g matrix and 7.0 x10⁹ufc/g dm inicialL. paracaseiConcentration. MD DE5 was found to be a better encapsulation matrix than MD DE19, probably due to the fact that the DE5 matrix retained its glassy state to a greater extent._c. The addition of trehalose significantly increased the viability during drying and during storage of the dry powder. The MD DE5-trehalose (1:1) combination gave the highest survival rate (70.6 ± 6.2%). Obviously, the cells must be further protected from oxidation, as nitrogen storage was essential to achieve storage stability. (Semenov and ai. 2011)

Improved production methods of starter cultures, which are the most important element of probiotic preparations, were investigated. The objective of the presented research was to analyze changes in the viability ofLactobacilina. acidófiloEBifidobacterium bifidumafter stabilization (spray drying, lyophilization, fluidized bed drying) and storage under refrigeration for 4 months. The highest number of viable cells up to the fourth month of refrigerated storage is on the order of 10⁷cfu/g preparation were recorded for theB. bifidumDSM 20239 bacteria with N-Tack starch applied for spray drying. Fluidization drying of encapsulated bacteria allowed to obtain a preparation of a comparable number of viable bacterial cells up to the fourth month of storage with the encapsulated bacteria subjected to lyophilization, but the previous method was much shorter. The greater survivability of the encapsulatedPound. acidophilusDSM 20079 eB. bifidumDSM 20239 cells subjected to lyophilization were obtained using skim milk as a cryoprotectant. Stabilization of bacteria by microencapsulation can produce a product that is easy to store and apply to make dry food compositions.Goderska, Czarnecki 2008).