NANOPROBIOTICS - AN INNOVATIVE TREND IN PROBIOTIC WORLD

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NANOPROBIOTICS - AN INNOVATIVE TREND IN PROBIOTIC WORLD Bhargavi B*1, Ramachandra B*2, Pushpa B.P*3, Prabha R*4 *1M.

Tech Scholar, Dept. of Dairy Microbiology, DSC, KVAFSU, Bengaluru, Karnataka, India.

*2Assistant

Professor, Dept. of Dairy Microbiology, DSC, KVAFSU, Bengaluru, Karnataka, India.

*3Assistant

Professor, Dept. of Dairy Chemistry, DSC, KVAFSU, Bengaluru, Karnataka, India.

*4Associate

Professor and Head, Dept. of Dairy Microbiology, DSC, KVAFSU, Bengaluru, Karnataka, India.

ABSTRACT Microorganisms are extremely small unicellular living things that are visible under microscope. Microbes are present everywhere including human body, some are beneficial termed probiotics while some are harmful called pathogens involved in causing various diseases. Human gastrointestinal (GI) tract has probiotics that extend therapeutic benefits. Nanoparticles with probiotics are called nanoprobiotics. Nanoprobiotics is a field that focuses on the application of nanoscience into the probiotic related world. Microorganisms used as probiotics are Lactobacillus spp, Bifidobacterium spp, Saccharomyces boulardii. Encapsulation has become an effective way to improve survival of probiotic organisms in GI tract by acting as a protective transporter for deliverance at a targeted level. The available technologies employed to encapsulate probiotic cells are micro and nanoencapsulation techniques which use sodium alginate, polyvinyl alcohol, pectin etc., as encapsulating material. Nanoencapsulation is a process of entrapping of active ingredients in nanometer (10-1000 nm) sized capsules. It is a technique which appears to be a promising alternative approach, has a potential to improve bioavailability, enhances controlled release and enables an accurate targeting of the probiotics in a greater amount than microencapsulation techniques, because of the large surface area and enhanced preservation against heat treatment. Electrospinning is a well-known nanoencapsulation technique, which is efficient and cost-effective for fabrication of fiber mats in nanosize for encapsulation of probiotics.

Keywords: Nanoprobiotics, GI Tract, Encapsulation, Nanoencapsulation, Electrospinning, Nanofibers.

I.

INTRODUCTION

Microorganisms are extremely small unicellular living things that are visible under microscope such as bacteria, archea, yeast, mold, protozoa, algae. They are ubiquitous in nature present in human body. They are of commensals, beneficial and pathogens. Foods plays an important role in maintaining the balance of human GI tract. Food components prebiotics act as food for good bacteria termed as probiotics. Most of the probiotics belongs to LAB such as. Lactobacillus sp, Lactococcus sp, Streptococcus thermophilus etc. It has been recommended that food containing probiotic bacteria should be in the range of 10 8 -109 cfu/g right before ingestion to ensure that sufficient therapeutic minimum of 106 -107 cfu/g could reach the colon [1]. Antibiotic therapy kills the good bacteria hence balancing of probiotics in human body is necessary. When probiotics are taken orally reaches gastro-intestinal tract but survive in low numbers because of acid and bile conditions in GI tract. Necessity is the encapsulation of probiotics using the nanoparticles termed as nanoprobiotics.

Nanotechnology is a fast-rising industry which exploits the benefits in nano scale dimensions and characteristics for application in the macro world. Probiotics are defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.” [2]. The size of nanoprobiotics ranges from 0.5– 0.8 to 2 - 9µ. Nanofibers are produced by the electrospinning process whose diameter is less than 1000 nm used as encapsulating material. Nanoprobiotics is a field that focuses on the application of nanoscience into the probiotic related world which increases the viability and releases the probiotics at target sites [3].

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LITERATURE REVIEW

Microorganisms Used as Probiotics Bifidobacterium species (LA +AA)

Non lactic acid producing bacteria

Lactobacillus acidophilus

Bifidobacterium adolescentis

Escherichia coli Nissle

Lactobacillus casei

Bifidobacterium animalis

Lactobacillus reuteri

Bifidobacterium bifidum

Lactobacillus johnsonii

Bifidobacterium longum

Lactobacillus crispatus

Bifidobacterium breve

Lactobacillus rhamnosus GG

Bifidobacterium infantis

Lactic acid producing bacteria

Propionibacterium freudenreichii

Yeast

References

Saccharomyces boulardii [4]

Enterococcus faecalis Enterococcus faecium

Bifidobacterium lactis

Bacillus coagulans Characteristics & Therapeutic Benefits of Probiotics Probiotic Strain Characteristics: Human origin, Acid and bile stability, Adherence to human intestinal cells, Persistence in human intestinal tract, Production of antimicrobial substances, Antagonism against pathogenic bacteria, Safety in food, clinically validated and documented health effects Probiotics Therapeutic benefits: Control of intestinal pathogens, Help in lactose use in Lactose intolerants, Boost up the host immune response, Reduction in serum cholesterol concentration, Anticarcinogenic activity [5].

Definition of Nanofibers An emerging technology is the production of nanofiber. The word “nano” comes from Greek and means “dwarf”.

A nanometer is a thousandth of a thousandth of a thousandth of a meter (10-9 m). One nanometer is about 60,000 times smaller than a human hair in diameter, or the size of a virus. These fibers have diameters of less than 1000 nm, produced by the electrospinning process. Different materials such as inulin, pectin, alginate are used to make nanofibers which improves the viability of probiotics [6].

Encapsulation of Probiotics Encapsulation is a “process by which probiotic is coated with, or entrapped within, food grade viscous material”. Encapsulation protects and maintains the viability and efficacy of probiotics in the food products and GI tract, promotes the controlled release and optimize the delivery at site of action and prevent probiotics from multiplying in food otherwise causes change in sensory characteristics. Protective shell of encapsulate should be of food grade, GRAS status, Biodegradable and should form barrier between internal phase and surroundings [7].

Materials used for Encapsulation Materials

Examples

References

Plant Based Starch and their derivatives Plant exudates and extracts www.irjmets.com

Amylose, amylopectin, dextrins, maltodextrins, cellulose and gluten Gum Arabic, galactomannans, pectins and soluble soybean polysaccharides @International Research Journal of Modernization in Engineering, Technology and Science

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[1]

Chitosan (crab - Callinectes sapidus) Microbial Based

Bacteria

Dextran (Leuconostoc spp.), xanthan (Xanthomonas campestris) and gellan (Spingomonas spp.)

Yeast

Chitosan (Saccharomyces cerevisiae)

Algae

Carrageenan (Chondrus crispus – Red alga) and alginate (Macrocystis pyrifera - Brown alga) Animal Based

Proteins

Casein, whey proteins and gelatin

Lipid materials

Fatty acids and fatty alcohols, waxes (beeswax), glycerides and phospholipids Synthetic materials

Other Synthetic materials

PVA, PVP, Paraffin (GRAS)

Microencapsulation Microencapsulation is the process of applying a shell to sensitive probiotic to protect them from their external environment and the probiotic cells are retained within an encapsulating matrix. It is a “process in which the probiotic cells are incorporated into an encapsulating matrix or membrane that can protect the cells from degradation by the damaging factors in the environment and release at controlled rates under particular conditions.” It protects the probiotics in food processing from high temperatures, oxygen stress and pH changes. After ingestion, probiotics encounter various types of harsh environmental conditions in Stomach (acid) and in small intestine (bile &enzymes). Probiotics size ranges from 1 to 10 μm, Microgels used for encapsulation of probiotics whose diameters ranges from 1 to 1000 μm [8].

III.

METHODS OF MICROENCAPSULATION

The encapsulation techniques applied to probiotics includes emulsion, extrusion and spray drying [9].

Emulsion: Polymers used for coating oil-in-water emulsion droplets along with core material and homogenized well before chemically hardened to form a shell. Bead size with a range of 25-35 μm.

Extrusion: Dispersion of active material in a molten carrier solution and forcing of the mixture into a dehydrating liquid. Bead size ranges from 0.3-3mm depends on distance between syringe and hardening solution (cacl2).

Spray drying: Automize the infeed dispersion of the active and core mix and further dehydration of the automized particles. Inlet temperature – (126-154°C) and Outlet temperature – (73-87°C). Particle size is less than 40 μm.

Encapsulated Lactobacillus acidophilus and Bifidobacterium bifidum by Emulsion & Extrusion Techniques Lactobacillus acidophilus and Bifidobacterium bifidum are encapsulated in sodium alginate by emulsion and extrusion techniques. The viability of microencapsulated Lactobacillus acidophilus using emulsion technique at 10°C for 90 days shown the final count of 9.20 log10 cells /g for which initial count was 9.50 log10 cells /g where as for Bifidobacterium bifidum was 9.21 log10 cells /g for which initial count was 9.45 log10 cells /g and microencapsulated Lactobacillus acidophilus using extrusion technique at 10°C for 90 days shown the final count of 7.17 log10 cells /g for which initial count was 7.32 log10 cells /g where as for Bifidobacterium bifidum was 7.11 log10 cells /g for which initial count was 7.25 log10 cells /g [10].

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Nanoencapsulation

Particle size - Microns

Particle size – Nanometer

Lower surface area

Higher surface area

Increased shelf life

Enhanced shelf life

Reach targets

Precised targeting

References

Probiotics

[11]

Nanofiber

IV.

NANOENCAPSULATION OF PROBIOTICS

Nanoencapsulation is a “Process of entrapping of probiotics in nanometer (10-1000nm) sized particles”.

Nanoencapsulation is a technique which appears to be a promising alternative approach, has a potential to improve bioavailability, enhances controlled release, and enables an accurate targeting of the live organisms in a greater amount than microencapsulation techniques. In addition, nanoencapsulation brings forward some benefits such as increased immobilization efficacy because of the large surface area and enhanced preservation against heat treatment as a result of decreased thermal conductivity with volume. There are two methods of nanoencapsulation of probiotics - Electrospraying and Electrospinning [12].

V.

ELECTROSPRAYING & ELECTROSPINNING

Principle of Electrospraying & Electrospinning: Electrospraying and Electrospinning methods works under the same principle of Electrohydrodynamic processes. Both methods utilize electrically charged jet of polymer solution for production of fibers or particles in micron, submicron and nanoscale. These methods are facile, cost effective and flexible [13].

Methods of Nanoencapsulation of Probiotics Electrospraying: Electrospraying is a process, in which polymer liquids in small capillary needles as subjected to sufficiently high voltage, the polymer liquid at the mouth of the small needles is charged then a columbic repulsion force is generated, then the polymer liquid is deformed to form Taylor cone. When the electrostatic repulsion overcomes the surface tension of Taylor cone, fine uniform liquid droplets are atomized and ejected from the conetip. Electrospraying Forms automized droplets used for encapsulating of probiotic cultures by protein or polysaccharides as entrapping materials [14].

Electrospinning: Electrospinning is a nanofiber fabrication technique that applies high voltage to draw a polymer solution from a spinneret tip to grounded collector. Upon charging, a jet of polymer solution emerges www.irjmets.com

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Generally, for electrospinning flow rates (0.4 to 1.6 ml/h), voltage values (15 to 27 kV) and distances between flat collector and Taylor cone (6 to 15 cm) are maintained. Electrically charged jet of polymer solutions (Ex: chitosan, sodium alginate) forms a nanoscale fiber of particles [14].

Electrospinning Vs Electrospraying Process and equipment are same for both electrospinning & Electrospraying  Higher viscosity Solutions are used to produce Fibers  Lower Viscosity Solutions are used to produce Particles or Powders  Attachment of probiotics in both the processes depends upon electrostatic force, negative charge of bacteria and viscous nature of material used [15].

Fibers and Particles - Possible Diameter is 50nm to 1μm

Fibers

Particles

Preparation of Nano Lactobacillus acidophilus Lb. acidophilus grown in sterile MRS broth (incubation 370C/24 h) collected by centrifugation at 10000 rpm for 15 min at 4 °C, washed twice and resuspended in PBS (pH 7.4). Okara and PVA stirred at 100 °C using a magnetic stirrer (600 rpm), until complete dissolution. Okara (soluble fraction of Soya pulp) 7%, PVA 8%, 86% water and Lb. acidophilus 2% was taken and subjected to electrospinning (25 °C). The prepared spinning solutions were inserted in a 1 ml syringe with needle of 0.5 mm diameter, used flow rate of 0.2–0.5ml/h, voltage of1.1kV and distance between electrode tip & collector disc is 12 cm at room temperature with humidity of 50%. The nanofibers were gathered by a rotating mandrel collector then vacuum Packed and stored at 4°C for later usage [16].

Results And Findings SEM (Scanned electron microscopy) Image

Spun nanofiber obtained has diameter of 465 nm with 7 % moisture www.irjmets.com

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e-ISSN: 2582-5208 International Research Journal of Modernization in Engineering Technology and Science ( Peer-Reviewed, Open Access, Fully Refereed International Journal ) Volume:03/Issue:11/November-2021 Impact Factor- 6.752 www.irjmets.com In vitro viability of strain after encapsulation process  74 % Viability rate noticed after 21 days when stored at 5 °C  Initial count was 9.58 log cfu/g after electrospinning the final count observed was 7.13 log cfu/g which shows a slight decrease in the probiotic viability [16].

Nanoencapsulation of Bifidobacterium sp A. Preparation of polymer solutions All the solutions and the glassware were autoclaved at 121 °C for 15 min to achieve sterility. CS solution (1 wt%) was provided by adding an appropriate amount of its powder into acetic acid (0.5 M) over a period of 5-6 hours on a magnetic stirrer (600 rpm). PVA solution (15 wt%) was prepared by dissolving an adequate amount of PVA powder in sterile deionized water, at 90 ºC, under stirring at 3 h. Then, the mixture CS/PVA (10/90, w/w) was obtained by blending polymers and stirring the polymer solution for 6 h to acquire a uniform blend [3].

B. Preparation of bifidobacterial cell suspensions Lyophilized cells of B. animalis subsp. lactis Bb12 was grown in sterile MRS broth and agar medium and incubated under anaerobic conditions for 24 hrs at 37 °C. Then, the cells were collected by centrifugation (6000 rpm, 20 min), washed twice with PBS (pH 7.4), and re-suspended in skimmed milk to nearly 3 × 109 cell/ml [3].

C. Electrospinning of bacterial suspensions The prepared spinning solutions were inserted in a 10 mL syringe fitted with a 23-gauge steel needle. The electrospinning process used a flow rate of 0.1 ml/h, a positive high voltage of 18 kV, and tip-to-collector distance (TCD) of 15 cm. The nanofibers were gathered by a rotating mandrel collector, which was covered with an aluminum foil [3].

Results and Findings SEM (Scanned electron microscopy) Images

b.

a.

Average Diameter of Nanofibers a. 139.06 ± 75.02nm b. 217.56 ± 62.74nm The diameter of strain loaded nanofiber is greater than the free nanofiber, the diameter increased to around 217 nm, revealing that the strain could be successfully encapsulated within the nanofiber mats [3].

In vitro viability of strain after encapsulation process State of strain Viability

Viability rate (%)

Free strain (log cfu/g)

Encapsulated strain (log cfu/g)

9.47

8.40

88.70

The resistance of the strain to electrospinning process during encapsulation was revealed by in vitro viability tests. The viability of the Bifidobacterium sp cells was established after encapsulation. Above table shows that the viability rate after the electrospinning process was 88.70%, which shows a slight decrease in the probiotic viability [3].

Preparation of Nano Lactobacillus paracasei KS-199 Results and Findings Optical micrographs of free strain and encapsulated strains

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(A)

free and (B) encapsulated strain (strain-loaded nanofiber mats)

Fig.(A) optical micrographs of free strain, clearly showing the rod-shape morphology of the strain.

Fig.(B)shows the morphology of the encapsulated strains, in other words, that of the strain-loaded nanofiber mats [12].

SEM Image of Blank & Strain Loaded Nanofiber Mats

Average Diameter of Nanofibers A. 305 ± 29nm (blank nanofiber) B.842 ± 72nm (strain-loaded nanofiber) Figures shows the SEM images of blank and strain-loaded nanofiber mats along with their average diameter distributions. It shows that the nanofibers mats fabricated from PVA/SA mix had uniform, well-defined, smooth and bead-free structure. Majority of fiber mats were in the size of between 170–360 nm with an average size of 305 nm in diameter. On the other hand, the incorporation of L. paracasei into the spin solutions yielded in beaded fiber structure (Fig. B). The diameter increased to around 842 nm, revealing that the strain could be successfully encapsulated within the nanofiber mats [12].

In-vitro viability of Probiotic Lactobacillus paracasei KS-199 after Nanoencapsulation Process.

State of strain Viability

Viability rate (%)

Free strain (log cfu/g)

Encapsulated strain (log cfu/g)

9.98 ± 0.85

8.57±0.69

85.87±0.78

The resistance of the strain to electrospinning process during encapsulation was revealed by in vitro viability tests based on plate counting technique. For this purpose, the viability of the L. paracasei cells was established after encapsulation. Above table shows that the viability rate after the electrospinning process was 85.87%, which shows a slight decrease in the probiotic viability [12].

Viability of free and nanoencapsulated strains in simulated gastrointestinal conditions State of strain Encapsulated strain

Free strain Before exposure to Viability

GJ/BS (log cfu/g)

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After Exposure To GJ/BS (log

Redution Viability Viability Exposure Exposure In in viability rate rate (%) to GJ/BS to GJ/BS viability (%) (log cfu/g) (log (log (log cfu/g) Reduction

cfu/g)

Before

After

cfu/g)

cfu/g)

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6.28±0.4

64.1±17

3.51±0.3

8.51±0.7 6.02±0.36

70.8±2.

5

2.49±0.3

The viability rate of encapsulated strain is 70.8% greater than free strain which is 64.1%, clearly showing that alginate-based nanoencapsulation considerably improved the survival percentage of L. paracasei KS-199 following exposure to the simulated gastric juice. There was a reduction in viabilities of 3.51 and 2.49 log cfu/g for the free and encapsulated cells, respectively after the exposure of gastric digestion, which demonstrates that it was possible to increase the viability of the cells by around 1 log [12].

Synbiotic (prebiotic + probiotic) nanoencapsulation [Inulin + Lactobacillus fermentum] PVA solution (20% -w/v), Sodium alginate (2% - w/v), Inulin (15% -POS) and Lb. fermentum was dissolved in sterile distilled water at 6:1:2:1 ratio and subjected to electrospinning process (25°C) The prepared spinning solutions was fed at the rate of 1.5 ml/h at a tip-to-target distance of 10 cm, Voltage of 20 kV and ~50% RH. The electrospun nanofiber mats were collected from the aluminum foil [17].

Results and Findings SEM (Scanned electron microscopy) Image

Entrapment of bacteria increased fibre size up to 400 nm with encapsulation efficacy of 78 % In vitro viability of strain after encapsulation process Count (logcfu/g)

Conditions

Conditions

Initial count

9.00 Survivability (GI in-vitro)

Free cells

3.78

Nanoencapsulated

7.90

Storage Temp. (0C)

Free cells

Nanoencapsulated

4

2.80

8.00

-18

2.10

7.60

The nanoencapsulated strain has showed the higher viability of 7.90 in GI whereas free cells showed the lesser viability of 3.78 because of high acid and bile conditions, The nanoencapsulated strain stored at 40C has showed more viability of 8.00 than free cells i.e., 2.80 and also at -18 0C nanoencapsulated strain has higher viability of 7.60 than free cells which was 2.10 [17].

Release of Probiotics from Nanofiber after Ingestion

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When probiotic encapsulated nanofiber taken orally, reaches stomach, encapsulated probiotic travels safely past the stomach i.e., nanofiber protects the probiotic from acid conditions (pH-1.8-5.0) and is released in the small intestine (pH – 6.5-7.5). The probiotic growth and bacterial multiplication take place throughout the intestinal tract. The degraded nanofiber (encapsulating material) acts as prebiotic in colon [11].

VI.

CONCLUSION

Probiotics provides the therapeutic benefits, But in food processing because of harsh conditions such as heat treatment and storage temperatures, decreases the viability of probiotics and also in GI tract because of acid & bile conditions, affects the viability of probiotics and stops them from reaching to target site therefore action on probiotic is needed. Best method is the nanoencapsulation which protects and helps in release of probiotics at targeted site. Selection of good encapsulating material & techniques are optimized. Proof of high viability nano encapsulated probiotics was observed. Nanotechnology techniques have improved the viability and survivability in all the probiotics which makes nanoencapsulation a thrust area.

VII.

REFERENCES

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[2]

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Mojaveri, S.J., Hosseini, S.F. and Gharsallaoui, A., 2020. Viability improvement of Bifidobacterium animalis Bb12 by encapsulation in chitosan/poly(vinyl alcohol) hybrid electrospun fiber mats.

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Yilmaz, M.T., Edu, M.K., Taylana, O., Karakasb, Y. and Dertlib, E., 2020. An alternative way to encapsulate probiotics within electrospun alginate nanofibers as monitored under simulated gastrointestinal conditions and in kefir. DOI: https:// doi.org/ 10.1016/j.carbpol.2020.116447.

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