Title: Novel Technologies for Probiotic Encapsulation

First Draft

Title: Novel Technologies for Probiotic Encapsulation

Abstract

Adequate probiotic microbe administration is frequently linked to the advantages of the health. These microorganisms must be able to cling to and colonize the human gastrointestinal system in order to promote good effects. They also must not be impacted by exposure to environmental elements. There are several encapsulation methods and materials available to create probiotic particles, but it's crucial that the procedure be minimum, gentle, or preventing cell losses and injuries. Maintaining the proper concentrations of these probiotics in food during processing and storage is the key problem, as inadequate amounts at the time of consumption will not have the desired health impact. Many studies have failed, due to concerns with evaluation techniques, application risk assessments, general flaws in encapsulating procedures, and inadequate consideration of the structural implications of encapsulating material. In addition, the particle qualities must be suitable for the intended use. The global market for supplementsand foods containing probiotics has been expanding significantly at the same time, and cell encapsulation is emerging as a substitute for adding probiotics to various food matrices. This review discuss the popularly used encapsulation techniques and novel methods for microencapsulation and benefits and potential applications of inserting generate particles into food matrices are updated in this study. Currently, a number of scientific studies claim that probiotics can be properly encapsulated using a variety of encapsulation processes, including extrusion, emulsion and spray drying. Although these encapsulation techniques are still frequently used to create microencapsulated probiotic cells, there is growing interest in novel approaches for microencapsulation systems like Nano, Electrospinning/Electro spun fibre, Double/Triple layered, 3D printing, and dual aerosol novel encapsulation processes, which enhance the thermal protection and viability of encapsulated cells and enable their incorporation into dairy and non-dairy food products at industrial level. Non-dairy food products can now deliver probiotics in a different way thanks to encapsulation. This study demonstrates how Nano encapsulation can be more beneficial than commonly used encapsulation techniques in terms of thermal stability, compound protection, and improved stability and survivability of encapsulated bacterial cells, and provides an alternative approach for the development of nutraceuticals and functional foods.

Keywords: Microencapsulation, Nano emulsions, Electrospinning/electro spun fibre, Double/Triple layered, 3D printing, and dual aerosol

Introduction

1.1 Probiotics: History, Definition and Characteristics:

Elie Metchnikoff, a professor at the Pasteur Institute in Paris and a Nobel Prize-winning scientist from Russia, believed that lactic acid bacteria could increase longevity and health advantages in 1907. He argued that altering the intestinal microbiota and substituting beneficial microbes for poisonous proteolytic germs like Clostridium spp. could prevent "intestinal self-poisoning" and the aging that results from it (Anukam & Reid, 2007).

The term "probiotic" was first used by Fuller (1989) to describe a food supplement made up of living microorganisms that could help the host by balancing the microbiota in the gut. Probiotics are currently defined by the Food and Agriculture Organization of the United Nations (FAO), with the backing of the World Health Organization (WHO), as bacteria and yeasts that, when given in sufficient proportions, boost the host's health (FAO/WHO, 2002). In terms of the biosafety of probiotic microorganisms, the majority of lactic acid bacteria are known to be food-grade and hardly ever linked to infections in people. Many of these bacteria are also prevalent in healthy people' gastrointestinal systems in addition to being naturally present in many popular animal and vegetable diets. However, taking into account potential threats to human health, the safety evaluation of

Probiotics produce substances with anticarcinogenic activity, such as short chain fatty acids and conjugated linoleic acid, as well as substances that inhibit the adhesion and prevalence of pathogenic microorganisms, directly altering the composition of the intestinal microbiota. They also degrade toxins and their associated receptors, modulate immune responses, enhance micronutrient absorption, and stimulate growth factors (Prakash et al., 2011, Reis et al., 2017, Markowiak and liewska, 2017).

Probiotics have been studied in the prevention of colorectal cancer (McIntosh et al., 1999; Jacouton et al., 2017; Chang et al., 2018, Heydari et al., 2019); inflammatory bowel disease (Zaylaa et al., 2018); and irritable bowel syndrome (Cremon et al., 2018); in the reduction of the risk associated with cardiovascular disease, (Liu et al., 2017), the improvement of the anti-hyperlipidemic and anti-hypercholesterolemic effects, as well as in non-alcoholic fatty liver disease (Bharti et al., 2017, Park et al., 2018) (Liang et al., 2018), respectively and other conditions. It is crucial to stress that the probiotic strain used can have different beneficial health benefits on the patient. The three most common probiotic bacteria used in human nutrition are Lactobacillus, Bifidobacterium, and Lactococcus. Also, Saccharomyces yeast speacies are also studied. (Markowiak & liewska, 2017).

According to Klein et al. (1998), many lactobacilli are used as functional foods, and Lactobacillus spp. has been designated as generally regarded as safe (Salminen et al., 1998). In order for probiotic foods to have therapeutic effects, it has been suggested that they include at least 106 live microorganisms per g or mL at the time of expiration (Capela et al., 2006; Manojlovi et al., 2010; Mokarram et al., 2009; Picot and Lacroix, 2004; Talwalkar and Kailasapathy, 2004). Probiotics of the Lactobacillus species made up the majority of the $16 billion global market for probiotic components, supplements, and foods in 2008. (Granato et al., 2010).

Fig. 1. HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0285" FAO/WHO (2002)guidelines for creating probiotics' safety assessment for human use, 2002.guidelines for establishing safety assessment of probiotics for human use.

These microorganisms must be stable against stomach acid and bile salts, have the ability to adhere to the intestinal mucosa, be able to colonize the human gastrointestinal tract, produce antimicrobial compounds, and maintain metabolic activity in the intestine in order to promote beneficial effects to the host (Collins et al., 1998, Saarela et al., 2000). Despite being widely employed, probiotic microorganisms have limitations since many bacteria that make this claim can be impacted by a variety of elements, including differences in oxygen content, the presence of hydrogen peroxide, pH, and temperature. As a result, the encapsulation has evolved into a tactic to safeguard probiotic cells and maintain their positive effects.

1.2 Benefits of Probiotic Cells and Encapsulation:

Probiotics have been linked to positive effects, including the modulation of the intestinal microbiota through the inhibition of pathogenic microorganisms, the production of anti-carcinogenic compounds, the modulation of immune responses, etc. (Prakash et al., 2011,Reis et al., 2017, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0550" Markowiak and liewska, 2017). They are known for promoting health and wellness. However, probiotic microorganisms must be able to colonize and maintain metabolic activity in the human digestive tract in order to produce positive effects. They must also survive exposure to environmental stimuli ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0205" Collins et al., 1998, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0830" Saarela et al., 2000). It is strongly advised that food products containing probiotics have a minimum level of 106 CFU per g or mL through the end of shelf life to benefit the host, according to Neffe-skoci et al. (2018).

Accordingly, cell encapsulation may decrease cell losses of encapsulated microbes in hydrocolloid matrices and increase probiotic bacteria' tolerance to harmful circumstances. ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0465" Kim et al., 2017, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0815" Rodrigues et al., 2017). Various probiotic encapsulation techniques are currently in use, wherein particles with various characteristics are generated obtained ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0165" Cavalheiro et al., 2015). Extrusion ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0475" Krasaekoopt and Watcharapoka, 2014,Rodrigues et al., 2017,Kim et al., 2017,Silva et al., 2018, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0240" Dimitrellou et al., 2019), emulsion ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b1000" Zhang et al., 2016, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0945" van der Ark et al., 2017, HYPERLINK "https://www.sciencedirect.com/science/article/pii/S0963996920307079" l "b0765" Raddatz et al., 2020). Various novel encapsulation techniques are gaining popularity and growing interest among researchers for microencapsulation are Double/Triple layered encapsulation, (Feng, K., Huang, R., Wu, R., Wei, Y., Zong, M., Linhardt, R. J., & Wu, H., 2020), 3D printing, Nano (Xu, C., Ban, Q., Wang, W., Hou, J., & Jiang, Z.2022), Electrospinning/Electro spun fibre, (Wen, P., Zong, M., Linhardt, R. J., Feng, K., & Wu, H., 2017), 3D printing (Kuo, C., Clark, S., Qin, H., & Shi, X.,2022), and dual aerosol novel (Sohail, A., Turner, M. S., Prabawati, E. K., Coombes, A. G., & Bhandari, B.,2012).

The stability of the generated particles depends on the choice of the appropriate enclosing materials. These substances must not be poisonous and must shield the encapsulated microbial cells from cell-damaging conditions. Additionally, they need to manage the release while it travels through the human stomach and intestines (Rathore et al., 2013, Chen et al., 2017). Although probiotics have been immobilized using polysaccharides, proteins, and lipids (Rajam and Anandharamakrishnan, 2015; Rodrigues et al., 2017; Arslan-Tontul and Erbas, 2017), naturally occurring water-soluble polysaccharides derived from mucilages and gums are being employed more frequently (Nami et al., 2017, Bustamante et al., 2017, Rodrigues et al., 2017, Mu et al., 2018, Rodrigues et al., 2018).

At the same time, the global market for supplements and foods containing probiotics has grown dramatically each year as a result of their potential health benefits (Grand View Research, 2019). However, producing foods with probiotic claims is difficult, particularly because probiotic cells added to meals have a hard time surviving and being active under processing, storage, distribution, and consumption circumstances (Min, Bunt, Mason, & Hussain, 2019). Parallel to this, the cell encapsulation consolidates as a substitute to increase the probiotics' survivability when introduced to various dietary matrices (Angiolillo et al., 2017, Rodrigues et al., 2018; Zanjani, Ehsani, Ghiassi Tarzi, & Sharifan, 2017; Dimitrellou et al., 2019, Cavalheiro et al., 2019, Bambace et al., 2019).

1.3 Encapsulation and Novel Techniques:

Numerous food items, including dairy products (milk, ice cream, yogurt, and cheese), soymilk, mayonnaise, meats, baby foods, confections, edible spreads, milk powders, sweets, cakes, and chewing gum, can contain probiotics. Probiotics are difficult to incorporate into fruit juices due to a number of factors, including pH, storage temperature, oxygen levels, and water activity. A microencapsulation technique was used because bacteria may need protection from acidic medium to maintain high viability. Microencapsulation is defined as a process in which solid, liquid and gaseous materials are retained within an encapsulating matrix or

Probiotics can be enclosed in gel particles using emulsion or extrusion techniques, and numerous encapsulation technologies have been developed for this purpose. Existing methods can create macro particles up to a diameter of 5 mm and micro particles up to a diameter of 200 m (Gibbs et al., 1999; Gouin, 2004; Shilpa et al., 2003; Zuidam and Shimoni, 2010); however, these methods have their own drawbacks, including high costs, a limited ability to scale up, large beads that can be detected through sensory means, difficulty in use, a The majority of encapsulation techniques produced water soluble capsules, which implies that once the core components are rehydrated in water, they are no longer encapsulated. ways for entrapping gel (Sohail, A., Turner, M. S., Prabawati, E. K., Coombes, A. G., & Bhandari, B., 2012)

Extrusion-based 3D printing is a revolutionary technique for semi-solid materials, such as gels, paste, dough, and icing, to create a culinary product with a customizable design (Sun et al., 2018). It is also possible to tailor the strain and dosage of probiotic bacteria in probiotic food products using 3D printing technology in order to meet the unique demands of different people. The features of 3D printing technology, including its ability to generate highly customizable and functional foods while producing less waste and requiring minimal facility changes, make it an excellent choice for this task. Incomplete research has been done on the use of alginate-gelatin hybrid hydrogels in the food sector because most recent studies have been devoted to determining whether it is feasible to create scaffolds for tissue engineering in order to encapsulate cells (Van Vlierberghe et al., 2011).

As a result, this review covers crucial ideas about probiotics and novel techniques of probiotic encapsulation that can improve the protection and survival of encapsulated cells. In addition, non-dairy food matrices are emphasized as additional delivery methods for probiotic cells.

Objective

To overview the popular encapsulation techniques and the novel Nano technologies for encapsulating the probiotic cells.

To overview the relevant encapsulation technique mechanism, process and its advantages

Different Novel technologies for Encapsulating probiotics

3.1 Encapsulation

Encapsulation techniques that are currently popular are extrusion, emulsification, and spray drying. However, these microencapsulation techniques have their own deficiencies, such as the using of high temperature ororganic solventmay lead to significant death of probiotic cells ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S1466856421001272?casa_token=GFgD3UyeQOMAAAAA:0jWis_IDcyoSc2HhsOpIvJiOGWcItdibJv9zqi3gkzegwIKcLeB3h-LZvfZmFBjl5TYk3DRAm2s" l "bb0045" Eratte et al., 2015; HYPERLINK "https://www.sciencedirect.com/science/article/pii/S1466856421001272?casa_token=GFgD3UyeQOMAAAAA:0jWis_IDcyoSc2HhsOpIvJiOGWcItdibJv9zqi3gkzegwIKcLeB3h-LZvfZmFBjl5TYk3DRAm2s" l "bb0090" Salar-Behzadi et al., 2013).

Extrusion Principle and Technique:

The extrusion technique, which is widely used to encapsulate bacterial cells, is straightforward, quick to use, and inexpensive in addition to being a very mild process that ensures high viability of the encased cells (Krasaekoopt et al., 2003, Rodrigues et al., 2017). In essence, this method uses microbial culture-containing hydrocolloid solutions that are extruded through a nozzle in a crosslinking solution, resulting in an immediate transition of the hydrocolloid solution to gel and the creation of beads. Commonly stable in acidic mediums, the resulting gel disintegrates in an alkaline environment.

Despite the above-mentioned ideal conditions, the technique's drawbacks include its slowness, which prevents it from being applied on a wide scale; its inability to effectively generate microspheres smaller than 500 m; and the need for low to moderate viscosity hydrocolloid solutions (Reis, Neufeld, Vilela, Ribeiro, & Veiga, 2006). The nozzle's diameter, the distance between the hydrocolloid solution's outflow and the cationic cross-linker solution, and the viscosity/flow rate of the hydrocolloid-microbial cell mixture are a few variables that can affect the size of the beads that are formed (Dong et al., 2013).

Spray Drying Principle and Technique:

The spray drying method, which is widely used in the food sector, offers low costs, quick processing, and great production. A solution containing the encapsulated chemical is typically atomized in a high-temperature gas using this approach, instantaneously generating a powder (Ray, Raychaudhuri, & Chakraborty, 2016). Different natural polymers, particularly arabic gum and starches, can be utilized to encapsulate bacterial cells using the spray drying approach because of their well-known capacity to form spherical particles after drying. However, other substances have also been utilized, including inulin, fructooligosaccharides, alginates, gums, and mucilages (Avila-Reyes et al., 2014, Arslan et al., 2015, Kingwatee et al., 2015, Rajam and Anandharamakrishnan, 2015, Sarao and Arora, 2017, Hadzieva et al., 2017, Bustamante et al., 2017)

For processes to run smoothly or to produce the best particles, crucial process variables including air flow, feed temperature, and inlet/outlet air temperature are crucial. The temperatures utilized are crucial because they can either create microsphere aggregates by slowing down water evaporation or severely impair cellular viability of the encapsulated microorganisms by damaging bacterial membrane components at high temperatures (Rathore et al., 2013). However, Martn, Lara-Villoslada, Ruiz, and Morales (2015) found that the outflow temperature had a greater impact on bacterial cell survival than the inlet temperature. Gardiner et al. (2000) used skimmed milk as an encapsulating substance to encapsulate Lactobacillus paracasei NFBC 338, and they found survival rates following spray drying that ranged from 97% at an outlet temperature of 70 to 75 C to 0% at 120 C.

Lactobacillus acidophilus La-5 was spray dried using input and outlet temperatures of 120 and 55 C, respectively, to encapsulate it in inulin. Following encapsulation, the bacterium had an 86.5% survival rate (Santos et al., 2019). Similar to this, Bifidobacterium BB-12 had an encapsulation efficiency of over 70% when it was placed in a solution of skimmed milk powder and prebiotics with inlet and outlet temperatures of 150 and 55 C (Fritzen-Freire, Prudncio, Pinto, Muoz, & Amboni, 2013).

Prebiotics, soluble fibers, gums, and mucilages were suggested as thermal defenders to be added to the encapsulating medium to reduce the deleterious effects of spray drying at high temperatures and increase the resistance and stability of the encapsulated cells (Ross et al., 2005, Rajam and Anandharamakrishnan, 2015, Santos et al., 2019). Bifidobacterium bifidum BB-02 was encapsulated in whey protein concentrate, mosquita gum, maltodextrin, and aguamiel by Rodrguez-Huezo et al. (2007) using the spray drying method, which the authors claim can cause a symbiotic effect, improving the encapsulated bacteria's resistance to environmental factors and boosting viability.

3.2 Double/Triple layered Encapsulation

Encapsulation Principle

Probiotics were encapsulated in an unique double-layered vehicle that is developed to increase their ability to survive in severe environments. Encapsulation of probiotic cells is done twice or thrice using same or different encapsulating agents to provide better protection and survivability of the cells.

Encapsulation Technique:

Layered encapsulation is a similar processing to encapsulation and it is an easy processing way to provide enhanced stability of the probiotic cells against adverse processing conditions. Feng, K., Huang, R., Wu, R., Wei, Y., Zong, M., Linhardt, R. J., & Wu, H.(2020) used this technology and developd layered encapsulated probiotic cells. After inoculation and harvesting of the lactobacillus acidophilus cells, this study has investigated the stability of the single and double layered cells under thermal stability and heat moisture treatment. Encapsulating carriers/agents used are sodium alginate and chitosan. After mixing the probiotic culture in the sodium alginate agent it is extruded to form probiotic cells in 2% calcium chloride solution and let it solidify and then it is again coated with chitosan agent. The formed celles were evaluated for the given conditions

The double layered cells has shown 22% higher thermal stability after heat moisture treatment and experienced a lower loss of viability (0.32 log CFU/mL) which was even higher in the triple layered encapsulated probiotic cells. The only disadvantage is the cell diameter is larger than single layered, which makes it a visible component when incorporated in foods.

3.3 3D printing Encapsulation

Encapsulation Principle:

Probiotics in gel matrix were effectively protected by the integrated process. A growing manufacturing technology is 3D printing. In addition to creating technology, it is also capable of creating digital models by layering adhesives like wax, powdered metal, and plastic. Currently, 3D printers are primarily used to build goods one layer at a time. (Anukiruthika, T., Moses, J., & Anandharamakrishnan, C., 2020)

Encapsulation Technique:

Based on previous studies food grade polymers like sodium alginate and type-B gelatin are used to prepare hybrid hydrogels as encapsulating agents under optimum conditions ( HYPERLINK "https://www.sciencedirect.com/science/article/pii/S002364382200010X" l "bib27" Kuo et al., 2021). Probiotic strains like bifiobacteria lactis and lactobacillus acidophillus are used to generate probiotic cells and mixed with hybrid hydrogels.

It was combined with an extrusion-based syringe (nozzle = 0.636 mm) that was powered by pressurized air and the K8200 printer (Velleman Inc., TX). The probiotic-containing hydrogel, which had been allowed to set for 24 hours, was placed inside the sterile syringe. The printing procedure was managed using Repetier-Host software V2.1.6 (Hot-World GmbH & Co. KG, Germany), which used an 8.00 mm/s printing speed and a 100 mm3/s feed rate. The geometry, which had the measurements L W H = 16 16 9.7 mm and a total of 11 layers, was constructed into the shape of a teardrop. The printing procedure used a concentric infilled pattern and might take up to 4 minutes per print. The 3D printed hydrogels underwent post-processing by lyophilisation for solidification and dehydration. Prior to being put into the freeze-dryer, all samples were frozen in a freezer (30 C). A pilot freeze-dryer (Virtis Genesis SQ freeze dryer) was used to freeze-dry materials for roughly 24 hours at a chamber pressure lower than 26.66 kPa (Kuo, C., Clark, S., Qin, H., & Shi, X.(2022).

Following the integrated process, the bacteria's viable counts were greater than 6 log CFU/g. After 8 weeks of storage, B. lactis' viability was still maintained at >6 log CFU/g. For this purpose, the formulations of 3 g/100 g A/G 1/2, 5 g/100 g A/G 1/1, and 7 g/100 g A/G 2/1 all demonstrated excellent potential. Since the alginate-gelatin hydrogel delivery model currently offers the most promise for 3D printing, the investigation focused solely on the probiotic's survivability within this system for the storage test. Although adding specific cryoprotectants to the alginate-gelatin hydrogel system may boost probiotic viability during freeze-drying, doing so may modify the extrudability and rheological qualities, which may make the 3D printing technique less suitable (Kuo, C., Clark, S., Qin, H., & Shi, X.(2022).

However, this investigation showed that both bacteria had survivability over 6 log CFU/g, indicating that the integrated approach has potential for preserving probiotic cells. Additionally, freeze-dried probiotics are better suited for long-term preservation than wet-form hydrogels (Tsen et al., 2002).

Encapsulation Advantages:

The dosage and strain of probiotic bacteria used in probiotic food products might also be tailored to each individual's needs utilizing 3D printing technology. The features of 3D printing technology, such as hyper customisation, reduced waste, and no facility change required, make it appropriate for the production of customisable foods and functional foods (Kuo, C., Clark, S., Qin, H., & Shi, X.(2022).

3.4 Electrospun Fiber Mat Encapsulation-

Significant research has recently been done on the use of electrospinning to encapsulate bioactive substances. The created nanofilm could facilitate the production of functional meals by improving the stability, encapsulation effectiveness, and oral bioavailability of bioactive chemicals. It could also enable targeted distribution and controlled release. The process for making fibres with a high surface-to-volume ratio and porosity is simple, easy, and adaptable. The absence of heat during the electrospinning process is a major benefit because it helps to preserve the structure and achieve high bioactive ingredient encapsulation efficacy during processing and storage. Bioactive substances have improved stability and functionality when they are enclosed in electrospun fibers.

Encapsulation Principle:

An innovative method of delivering bioactive substances, electrospinning offers a fresh perspective on food technology and has the potential to be commercialized soon. An efficient method for creating sub-micron or nanoscale polymer fibers is electrospinning. A continuous electrical field is used to draw a droplet of polymer solution or melt polymer into a fine fiber, which is then continuously deposited on a grounded collector (Wen, P., Zong, M., Linhardt, R. J., Feng, K., & Wu, H.(2017).

Encapsulation Technique:

Probiotics are enclosed in layered vehicle by a one-step coaxial electrospinning method. An efficient method for creating sub-micron or nanoscale polymer fibers is electrospinning Ahmed et al., 2015. A continuous electrical field is used to pull a droplet of polymer solution or melt polymer into a fine fiber, which is then constantly deposited on a grounded collector. Fig. 1 depicts schematically a typical electrospinning setup. It is made comprised of a rotating drum or plate as the grounded collector, a syringe pump with a metal needle, and a high-voltage power source Anu Bhushani and Anandharamakrishnan, 2014. A polymer melt or solution that has enough molecular entanglement for electrospinning is first extruded by a syringe pump into a droplet at the needle tip. Then, a conductor's hemispherical surface is exposed to an electric field between the needle tip and the conductor.

To create nanofibers, electrospinning is a quick, adaptable, and economical process. It is appealing because, in contrast to other encapsulation methods, it is a simple process that does not require any harsh conditions, making it appropriate for encapsulating bioactive chemicals Aytac et al., 2016c. The electrospun nanofibers have advantages such as submicron to nanoscale diameter, high surface to volume ratio, appropriate porosity, tunable fiber diameters, and customized shape. Due to these structural benefits, it has been demonstrated that encapsulating bioactive substances in electrospun fibers results in high EE, improved stability and bioavailability, tailored administration, and sustained release (Wen, P., Zong, M., Linhardt, R. J., Feng, K., & Wu, H.(2017)

Encapsulation Advantages:

Electrospinning has several advantages over other production methods, including: 1) a relatively simple and cost-effective method for producing nanofibers; 2) easy incorporation of bioactive compounds into nanofibers; 3) a reduced size requirement for bioactive compounds, allowing their incorporation into food systems without affecting product sensory qualities; and 4) the absence of heat during the electrospinning process, which can be beneficial (Wen, P., Zong, M., Linhardt, R. J., Feng, K., & Wu, H.(2017).

3.5 Aerosol Encapsulation:

Encapsulation Principle:

Sodium alginate solution and calcium chloride cross-linking solution are used in separate impinging aerosols in this continuous encapsulation method (Bhandari, 2009) to create water insoluble cross-linked alginate micro beads with an average diameter of less than 40 m. (Sohail et al., 2011).

Encapsulation Technique:

A two-fluid nozzle was used to inject an aerosol of microbial suspension in alginate solution into the top of a Plexiglass cylinder at a pump speed of 12 mL/min while using pressured air at a room temperature of 4.48 bars. Using a two-fluid nozzle and compressed air at 3.45 pressure, a counter aerosol of sterile 0.1 M CaCl2 solution was injected from the base of the vessel at a rate of 9 mL/min. The probiotic-containing alginate microbeads were removed from the vessel's base exit (Sohail et al., 2011).

(Sohail, A., Turner, M. S., Prabawati, E. K., Coombes, A. G., & Bhandari, B., 2012) Alginate microbeads with a mean size of 35 m (1040 m) were created in this study using the encapsulation approach, and they remained detectable after storage. Probiotic survival is affected by microbeads size as according to (Truelstrup et al. 2002), macro beads (1000 m) increased the coarseness of food texture but bifidobacteria encapsulated in micro beads with diameters under 100 m did not significantly improve cell survival in SGF when compared to free cells. On the other hand, (Mokarram et al., 2009) revealed that in order to achieve protection against gastro intestinal diseases, bacteria should be encapsulated within micro beads of a specific size range (50-75 m).In the future, however, covering the encapsulated beads with various coating materials, such as chitosan, poly lysine, etc. The probiotics L. rhamnosus GG and L. acidophilus NCFM are enclosed in alginate microbeads (1040 m) using same process where, L. rhamnosus exhibited comparable resistance in an environment of bile and acid when micro and macro beads were compared. L. acidophilus NCFM was more resistant to acid and bile tolerance when protected by alginate mega beads as opposed to micro beads. In both strains, chitosan coating demonstrated the best microbead survival in an acidic and bile environment (Sohail, A., Turner, M. S., Coombes, A., Bostrom, T., & Bhandari, B.2011).

Encapsulation Advantages:

Due to the fact that neither solvents nor heat are utilized in the process, it can be used to encapsulate chemicals that are sensitive to both. The method allows for freeze or spray drying and can generate enormous amounts of small beads.

Conclusion

An effective substitute to preserve the viability and stability of encapsulated cells is the encapsulation of microorganisms with probiotic claims. According to several findings, using various encapsulation methods and encapsulating materials produces favourable outcomes. To cut down on losses during particle production and application, however, technique and encapsulating material must be carefully chosen.

The double-layered vehicle has significant promise for probiotic encapsulation and boosting their resistance to hard conditions, but it doesn't have the microencapsulation effect. The double-layered encapsulated probiotic cells showed enhanced thermal stability and survivability. Probiotics with a new aerool encapsulation weren't considerably more survivable. has the ability to lessen acidity and any potential unfavorable probiotic sensory effects. The dual aerosol technique's capacity for continuous processing and scale-up makes it possible to efficiently encapsulate probiotics in very small alginate micro beads below sensory detection limits while yet providing adequate protection in bile and acid environments. Inelectrospinning-based bioactive chemical encapsulation, the created nanofilm could facilitate the production of functional meals by improving the stability, encapsulation effectiveness, and oral bioavailability of bioactive chemicals. It could also enable targeted distribution and controlled release. The dosage and strain of probiotic bacteria used in probiotic food products might also be tailored to each individual's needs utilizing 3D printing technology. The features of 3D printing technology, such as hyper customisation, reduced waste, and no facility change required, make it appropriate for the production of customisable foods and functional foods

At the same time, new products are needed to satisfy customer demand as the global market for supplements and probiotic foods expands. Therefore, in addition to the conventional dairy products, diets based on meat and vegetables have also been investigated as potential sources of encapsulated probiotics. Several studies have found that a suitable encapsulation method can transform non-dairy food products into alternative matrices for delivering probiotic cells, despite technological obstacles.

Further Investigations

The co-encapsulation technology has helped food products last longer by stabilizing probiotic bacteria and bioactive substances over a lengthy period of time. The release mechanism of these substances in a computer-simulated animal model system, as well as the use of co-microcapsules in the creation of affordable functional food products, will require further study.

The stability of LNCFM may be improved or the acidification of the aerosol-encapsulated cells may be further reduced by future coating of the encapsulated beads with different coating materials, such as chitosan, poly lysine, etc.

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