Degree project work

Investigation of Probiotic Organogel

Formulations for use in Oral Health

Author: Elin Sonesson

Supervisors: Charlotte Gröön, Boel

Lindegård

Examiner: Kjell Edman

Semester: VT13Subject: Chemistry

Level: First cycle

Course code: 2KE01E

Nr: 2013:L5

i

Abstract The aim of the project is to investigate how a more viscous, gel like formulation can be

made of BioGaia´s Prodentis Drops, which is a probiotic product for oral use. The two

different strains of Lactobacillus reuteri that are used in the product, together called L.

reuteri Prodentis, have been clinically proven to be effective in treatment of gingivitis

and caries formation. The existing product is a highly liquid oil suspension that has been

described as too runny and difficult to get into tooth pockets and between teeth.

Therefore a gel formulation would be preferred. Pre-trials were excecuted to see what

combinations and quantities of ingredients could work. Three different formulations

using 3 %, 5 % and 7 % beeswax as thickening agent proceeded to another round of

trials, as well as one formulation where the original oil was exchanged for hydrogenated

rapeseed oil. In the beeswax formulations fumed silicon dioxide was being used as well.

Three different analyses were executed, considering bacterial survival, viscosity and

phase separation of gels. The bacterial survival proved to be acceptable in all samples

even after 14 days of incubation in 37oC. The formulation with 7 % beeswax was the

most viscous one, followed by 5 % beeswax, 3 % beeswax and the formulation with

hydrogenated oil, respectively. Phase separation could be seen in the hydrogenated oil

formulation already after seven days and even more so after 14 days. There were also

signs of separation in the formulation with 3 % beeswax after 14 days. It was concluded

that in further development of the Prodentis Drops it is recommendable to proceed with

the 5 % beeswax formulation.

Keywords Probiotics, gel formulation, Prodentis Drops, Lactobacillus reuteri, viscosity, bacterial

survival, phase separation, beeswax, silica, hydrogenated rapeseed oil

Thanks Big thanks to BioGaia for letting me be a part of this project and for all the help along

the way, and also for making me feel welcome at the office. A special thank you to

Charlotte Gröön, my supervisor at BioGaia, who has lead the project and guided me

through it.

I would also like to thank Boel Lindegård, my supervisor at Linnaeus University, for

support and helpful advise along the way.

ii

Contents

1 Sammanfattning ____________________________________________________ iii

2 Introduction _________________________________________________________ 1 2.1 Background ______________________________________________________ 1

2.1.1 Aim _________________________________________________________ 1

2.1.2 BioGaia _____________________________________________________ 1

2.1.3 Prodentis Drops _______________________________________________ 1

2.2 Lactobacillus reuteri _______________________________________________ 2 2.3 Oral health and L. reuteri ___________________________________________ 3 2.4 Rheology and viscosity _____________________________________________ 4 2.5 Organogels and fat crystallization ____________________________________ 4 2.6 Beeswax ________________________________________________________ 5

2.7 Silica ___________________________________________________________ 5 2.8 Analyses ________________________________________________________ 6

3 Material and methods _________________________________________________ 6 3.1 Pre-trials ________________________________________________________ 6 3.2 Production of gel _________________________________________________ 7

3.2.1 Wax gel _____________________________________________________ 7

3.2.2 Hydrogenated oil gel ___________________________________________ 8

3.3 Analyses ________________________________________________________ 8 3.3.1 Analysis of survival of L. reuteri __________________________________ 8

3.3.2 Analysis of rheology ___________________________________________ 9

3.3.3 Analysis of phase separation _____________________________________ 9

4 Results ______________________________________________________________ 9 4.1 Analysis of survival of L. reuteri _____________________________________ 9 4.2 Analysis of rheology ______________________________________________ 12

4.3 Analysis of phase separation _______________________________________ 14

5 Discussion __________________________________________________________ 16

6 Conclusions ________________________________________________________ 17

7 References__________________________________________________________ 17

Appendices ___________________________________________________________ I Appendix A: Mean values for analysis of survival of bacteria in different

formulations. _________________________________________________________ I

Appendix B: Raw data from viscosity analysis _____________________________ II

iii

1 Sammanfattning

Syftet med projektet är attundersöka hur man kan utveckla en mer viskös, gelliknande

formulering av BioGaias Prodentis Drops, en probiotisk produkt för förbättrad

munhälsa. Två olika stammar av Lactobacillus reuteri, tillsammans kallade L. reuteri

Prodentis, används i produkten. I kombination har de visat sig vara effektiva vid

behandling av gingivit och kariesbildning. Den befintliga produkten är en lättflytande

oljesuspension som har uppgetts vara för rinnig och svår att få in i tandfickor och

mellan tänder. Därför är en gelformulering önskvärd. Förförsök gjordes för att se vilka

ingredienskombinationer och sammansättningar som fungerade för ändamålet.

Formuleringar med 3 %, 5 % och 7 % bivax tillsatt till det ursprungliga receptet gick

vidare till riktiga försök. Det gjorde även en formulering med hydrogenerad rapsolja. I

bivaxformuleringarna tillsattes även kiseldioxid. Analyser av bakterieöverlevnad,

viskositet och fasseparering gjordes. Bakterieöverlevnaden var tillräckligt god i

samtliga prover. Formuleringen med 7 % bivax var mest viskös, följt av 5 % bivax, 3 %

bivax och formuleringen med hydrogenerad olja. Fasseparering kunde ses i

formuleringen med hydrogenerad olja redan efter 7 dagar, och ännu mer markant efter

14 dagar. Slutsatsen drogs att vid vidare utveckling av Prodentis Drops rekommenderas

det att fortsätta utveckla formuleringen med 5 % bivax.

1

2 Introduction

2.1 Background

2.1.1 Aim

The aim of the project is to investigate what formulations can be useful for making a

probiotic oil gel that can be placed on a tooth stick to be used between teeth and in tooth

pockets. Dentists have requested such product since the existing product is a highly

liquid oil formulation that is difficult to handle. Different formulations are developed

and analysed on parameters such as bacterial survival, viscosity and phase separation.

2.1.2 BioGaia

BioGaia is a health care company that develops and produces probiotic products. The

company was established in 1990 (1). In 2012 there were 108 clinical studies on

BioGaias patented Lactobacillus reuteri strains, which are part of all products sold by

the company and are proved to have probiotic qualities. In Sweden BioGaia is situated

in Stockholm and Lund.

2.1.3 Prodentis Drops

Prodentis Drops in its existing form is a probiotic product with two different freeze-

dried bacterial strains suspended in oil. It is meant to be used by dentists for patients

suffering from gingivitis, as a first step in a probiotic treatment. With a convenient gel

formulation the product could be administered locally at gingival infection sites for a

first boost. It is then thought to be complemented with either lozenges or chewing gums

with the same probiotic strains, to be taken at home twice daily. The product contains

pepper mint flavour for better taste. The oil that is being used contains equal amounts of

sunflower oil and medium-chain triglycerides, which are both stable against oxidation.

Fumed silica is added as a thickener and suspending agent. The fumed silica particles

form a network in the oil that gives the formulation a viscous and thixotropic behaviour.

Due to the higher viscosity of the formulation the freeze-dried bacteria particles

sediment very slowly. Particles that have sedimented can easily be re-suspended. The

two oils and silica are premixed for BioGaia, and the mixture is henceforth referred to

as Prodentis oil mixture. The product as of today is packed in a 10 ml amber glass bottle

with a dropper insert. The idea is that in the future it will come in a plastic tube.

The daily dose of 5 drops is equivalent to 0.16 ml and contains a minimum of 1.0E+08

colony-forming units (CFU) of each L. reuteri strain at the end of the shelf life, which is

set to 24 months when stored at room temperature. There is a loss of viability during

production and storage, which has been taken into account by adding L. reuteri in

excess. 2 % of each bacterial culture is added to the formulas in the study, equivalent to

4,6E+9 CFU L. reuteri ATCC PTA 5289 and 6,2E+9 CFU L. reuteri DSM 17938.

2

The active freeze dried bacteria used in the product is a fine grain powder consisting of

bacteria and freeze drying agents sucrose and polyphosphate. The freeze dried bacteria

stay in an inactive state as long as no moisture is added. They will remain inactive in

oil, which is why oil is being used as suspension media. The bottles are covered with

nitrogen before sealed, and a desiccant device, a strip is also used in the package to

protect the product against moisture during storage.

2.2 Lactobacillus reuteri

Probiotics have been defined as “live microorganisms which when administered in

adequate amounts confer a health benefit on the host” by FAO and WHO (2).

The entire Lactobacillus species has a long history of safe use and documented

probiotic effects. Lactobacillus reuteri are Gram-positive heterofermentatives that are

indigenous and symbiont to the gastrointestinal tracts of humans (3). There has been no

causing of disease related to the species, which is generally considered safe to use (3).

L. reuteri use various carbohydrates as sources of energy and carbon, and also need

nucleotides, amino acids and vitamins for growth (4). L. reuteri has been shown to

regulate immune cell activity and also these cells’ production of cytokines (3). The two

L. reuteri strains used in the Prodentis Drops are L. reuteri DSM 17938 and L.reuteri

ATCC PTA 5289. When used in combination they go under the name L. reuteri

Prodentis.

L. reuteri DSM 17938 is a daughter strain of L. reuteri ATCC 55730 which has been

cured from resistance to antibiotics, since there was a theoretical risk of transference of

the resistance to other bacteria. Two plasmids carrying resistance to tetracycline and

lincomycin were removed from the mother strain. In a study by Rosander et al. (5) it

was shown that the probiotic characteristics of the bacteria remained the same after the

removal of plasmids. Also, the bacterial chromosome was not modified and no

mutations induced. No risk of transference of antibiotics-resistance has been associated

with L.reuteri ATCC PTA 5289.

Both L. reuteri strains produce reuterin, which in a water solution exists as three

different compounds; 3-hydroxypropionaldehyde (3-HPA), hydrated 3-HPA and as a

dimer of 3-HPA (6). 3-HPA is produced in the presence of antagonists, through

dehydrogenation of glycerol (7). It has been proved to induce oxidative stress in several

pathogens in the intestinal tract (8).

3

Figure 1. Structural formula for 3-hydroxypropionaldehyd. Adapted from

http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=75049#itabs-2d (2013-06-26)

2.3 Oral health and L. reuteri

Gingivitis is caused by inflammatory reactions provoked by bacteria that accumulate in

crevices of the gums (9). A common method for treating gingivitis is mechanical and

chemical cleaning of teeth and gums. This method has been reported effective over a

short-time period, but only for up to 3 weeks before there are new signs of plaque and

gingivitis (9). An uncured gingivitis can develop into periodontitis (tooth loss) (9).

In a study by Twetman et al. (10) a group of forty two adults that suffered from

moderate gingivitis but were otherwise healthy were given Prodentis chewing gums to

take twice daily. One group was given only placebo gums, the second group one

placebo gum and one gum containing L. reuteri Prodentis, and the third group only the

Prodentis gums. After 14 days of treatment a significant decrease of bleeding on

probing as well as a significantly decreased volume of gingival crevicular fluid could be

seen in both groups given probiotic treatment. An increased volume of gingival

crevicular fluid is associated with inflammatory periodontal disease and a high

concentration of cytokines (10).

In caries formation a low pH generated from the production of organic acids from

dietary sugars plays a big role (11). The acidity disturbs the oral microbial homeostasis

and kills off acidophobic bacteria, which is beneficial for competitive caries generating

bacteria (11). All Lactobacillus strains are aciduric (11). In an in vitro study where L.

reuteri PTA 5289 was tested in regard to fermentation of nine different dietary sugars, it

was concluded that the strain was close to inactive in fermentation of all sugars tested

(11).

In the oral cavity L. reuteri inhibits the growth of for example Porphyromonas

gingivalis and Streptococcus mutans, which are markers for gingivitis and caries (12,

13). It has been proved that an oral intake of L. reuteri effectively colonizes the

intestine, where it maintains for at least seven days after intake. The levels of vivid

4

bacteria in fecal samples drop though after a stopped intake, and colonization is lost

after 2 months (14).

2.4 Rheology and viscosity

Rheology can be defined as the understanding of deformation and flowing properties of

matter. A fluids dynamic viscosity is its rate of resistance to an applied shear stress (15).

For a Newtonian fluid the shear rate does not affect the viscosity. For a non-Newtonian

fluid on the other hand, the viscosity depends on the rate of shear. If pseudoplastic, the

viscosity decreases with increasing shear rate, while a dilatant fluid acts the other way

around. A plastic fluid does not start flowing until the shear stress has reached a certain

lowest value. The viscosity of a non-Newtonian fluid can also be time dependant. If the

viscosity decreases with time under constant shear rate the liquid is thixotropic. The SI

unit for viscosity is Pa*s or kg*m-1

*s-1

(15).

In this study the viscosity of the different formulations are measured primarily to be

compared to each other. The viscometer used in the analyses is a Brookfield instrument

that drives a spindle through a spring (16). When the spring deflects due to resistance of

the fluid that is being analysed, it is measured by a transducer (16).

2.5 Organogels and fat crystallization

In some studies where wax has been used as gelator in oil systems, the system has been

named organogel. An organogel can be described as a bicontinous colloidal system with

a micro-heterogeneous solid in a liquid phase (17). As in other gels, this means that the

liquid phase is predominant and that there is a “continuous matrix of interconnected

material”, providing a solid character to the system (18). A colloid is a dispersion with

particles that are not visible by eye, but larger than small molecules. To be a colloid

system the particles should have a radius of approximately 10 nm to 0,1 mm (18). That

a solid is micro-heterogeneous means that the gelator, in this case beeswax, contains of

more than one type of compound.

Interaction forces, e.g. van der Waals attraction, between molecules and particles in the

colloidal system affect its stability (18). A lyophilic (i.e. solvent loving) system is more

in equilibrium and more physically stable than a lyophobic one (18). Since both solvent

and gelator in this study are lipophilic it should mean a more stable system.

The crystallization point of a fat system is the temperature at which the specific fat goes

from a liquid to a solid state. Crystallization starts when fat molecules aggregate and

form nuclei, a state called nucleation. At a next step, the crystal growth, molecules from

the liquid phase integrate with the newly formed nucleus, leading to developing and

growth of crystals. The crystals are dynamic and can transform after crystallization.

There are different molecular packing possibilities, giving different crystalline

structures among which the three most common ones are α, β’ and β. These polymorphs

all have different properties. α crystals are small and give an amorphous consistency,

while β’ crystals are bigger and give a smooth feel and β crystals are the largest ones

and contribute to a grainy feel of the fat. α is the polymorph with the highest Gibb’s free

5

energy and hence also the most unstable one. It has the lowest melting point and the

lowest nuclei formation activation energy. Often α crystals are first formed in a system

at crystallization, but at storage convert into crystals with lower molecular energy.

Another possible postcrystal event is growing of crystals because of merging or

migration (18). The morphology of the crystals formed in a gel are affected by the

cooling rate of the gel when produced (17). Fast cooling gives smaller crystals (17).

The solid content tells how much of the fat in a fat system that is in a solid state at a

specific temperature (18). A higher proportion of solid fat, i.e. more fat in crystalline

phase, gives a more viscous formulation. By adding beeswax or hydrogenated oil to the

Prodentis Drops formulation, the product will acquire higher viscosity because of the

increased melting temperatures and higher proportion of crystalline fat in these fats in

relation to the Prodentis oil mixture.

2.6 Beeswax

A wax ester by definition is an ester that derives from a long chain fatty acid and a long

chain fatty alcohol (17). A more commonly used definition of the term wax is “a

mixture of long chain apolar compounds found on the surface of plants and animals”

(19).

The beeswax used in this study is white wax, which is natural beeswax that has been

chemically bleached (20). Beeswax is approved as a food additive in the European

Union (21). It contains of a complex mixture of nonpolar compounds such as

monoesters, hydrocarbons, free fatty acids and free fatty alcohols (21). The main ester

in the wax is myricylpalmitate, a 16 carbon fatty acid esterified to a 14 carbon alcohol

(20).

The wax is produced by the worker honeybee and obtained from the honeycombs by

melting them after having removed the honey. It has a melting point of 62-65oC (20).

White beeswax is generally non-toxic and non-irritable and is often used in cosmetics

and farmaceuticals (20). In rare cases hypersensitivity has been reported because of

contamination of the wax (20).

2.7 Silica

Fumed silica is an amorphous (i.e. non-crystalline) silicon dioxide with low density,

which has been synthesized from chlorosilanes (22). At production the chlorosilane is

lead through an 1800oC flame, leading to hydrolysis and making of “primary particles”,

which are liquid drops of silicon dioxide (22). When the primary particles collide they

form aggregates in branched chains (22). The aggregates cool rapidly before they have

time to crystallize (22). When the aggregates have cooled to under 1710oC, which is the

melting point of silica, only mechanical entanglement takes place, leading to

agglomerates (22).

6

Silica is often used as viscosity raising agent. It gives thixotropic thickening to gels

(20). The polarity of the liquid affects the grade of thickening, and polar liquids

generally require more silica (20). In dietary supplement, addition of 1 % silica is

allowed (23). Fumed silica is considered non-toxic and non-irritable (20).

2.8 Analyses

When evaluating the formulations three different parameters are considered:

- Survival of bacteria in the product

- Viscosity of the different samples in relation to each other, and how the viscosity

changes under shear stress

- Separation of fat phases over time

3 Material and methods

3.1 Pre-trials

Pre-trials were made with different formulations and ingredients. Through earlier trials

it had been suggested that adding of a polar compound can give a more thickening

effect of the oil in the presence of silica. Therefore, and also to see how good the

survival of bacteria would be, attempts were made with ethanol in the formulations. The

resulting products were not as viscous as hoped for, but on the other hand a good

survival of the bacteria was seen after 24 hours of incubation at 5oC (data not included

in this study). For this project though, no further attempts with ethanol were made.

Another pre-trial was executed with glycerol added to the original formulation. To make

a stable emulsion lecithin was added as an emulsifier. In spite of the emulsifier,

immediate separation of the phases was seen, with the bacteria and glycerol in the

bottom of the beaker. It should be noted that the freeze-dried particles are soluble in

water but not in oil, hence the preference for glycerol. No further attempts were made

with glycerol.

In probiotic straws, one of BioGaia’s existing products, a partially hydrogenated canola

and rapeseed oil mixture (henceforth referred to just as hydrogenated oil) is used as

excipient for the bacteria. For practical reasons it would be an advantage to use

ingredients already in stock. In one pre-trial the oil in the original recipe of Prodentis

drops was exchanged with the hydrogenated rapeseed oil, resulting in a more viscous

formulation. The formulation passed on to the next round of trials, since it was viscous

enough to stay on a tooth stick.

Attempts were made with 1 %, 5 % and 10 % beeswax used together with the Prodentis

oil mixture to make a more viscous formulation. It was evaluated that the formula with

1 % beeswax was not viscous enough since it did not stay on a tooth stick. The formula

with 10 % beeswax was difficult to press out of a tube and considered as too firm. In the

next series of trials 3 % and 7 % beeswax were used. The formulations with 3 %, 5 %

7

and 7 % all had a satisfying texture at a first evaluation, and so they passed on to the

“real” trials.

The idea of using wax for its thickening properties first came from an article about

organogels where sugarcane and candelilla waxes were used (24). In stock at BioGaia

beeswax was available, which is why that specific wax was chosen for the trials.

3.2 Production of gel

The gel formulations were all made in batches of 600 g.

3.2.1 Wax gel

Table I. Beeswax gel recipes (g)

Beeswax Prodentis oil

mixture

DSM 17

938

ATCC PTA

5289

Mint

flavour

3 % 18 552 12 12 6

5 % 30 540 12 12 6

7 % 42 528 12 12 6

The Prodentis oil mixture contains 49,5 % sunflower oil, 49,5 % medium chain

triglycerides (MCT) (both oils are supplied from AarhusKarlshamn Sweden AB) and 1

% fumed silicon dioxide (Cab-o-Sil M5 from Cabot, GmbH, Germany). The peppermint

flavour (Kerry Ingredients and Flavours Italia S.p.A.) comes dissolved in pure vegetable

oil.

Prodentis oil mixture and peppermint flavour were weighed and stirred together by

hand. Approximately 100 ml of the mixture was transferred to another beaker. The

remaining mixture was then heated to 70oC in a water bath. The beeswax (Sigma-

Aldrich Switzerland, lot and filling code 1365352 40208217) was melted on the heating

plate and then added to the warm oil while stirring.

The freeze-dried bacterial cultures L. reuteri DSM 17 938 and L. reuteri ATCC PTA

5289 (Danisco USA Inc.) were added to the minted oil that was put to the side earlier.

Stirring was done by hand with a spoon until the mixture was smooth.

When the temperature of the wax and oil mixture had fallen to 50oC the premixed oil

with bacteria was added. Stirring was executed with a high shear mixer (Silverson

LM5) at 2500 rpm for 2 minutes.

When cooled to room temperature the product was transferred to 10 ml dropper tubes. A

desiccant strip was added to each tube. The tubes were sealed in a tube sealer.

8

3.2.2 Hydrogenated oil gel

Table II. Hydrogenated oil recipe (g)

Hydrogenated

oil

DSM 17 938 ATCC PTA

5289

Mint

flavour

552 12 12 6

Hydrogenated oil (AarhusKarlshamn Sweden AB) and peppermint flavour were

weighed and stirred together by hand. Approximately 100 ml of the mixture was

transferred to another beaker. The freeze-dried L. reuteri DSM 17 938 and L. reuteri

ATCC PTA 5289 were added and stirring was executed by hand with a spoon until the

mixture was smooth. The mixture was then poured back into the beaker with the rest of

the oil. Stirring was done by hand.

The product was transferred to 10 ml dropper tubes. A desiccant strip was added to each

tube. The tubes were sealed in a tube sealer.

3.3 Analyses

3.3.1 Analysis of survival of L. reuteri

The tubes with gel samples meant for the analysis were incubated at 5oC, 25

oC and

37oC, respectively. A first analysis was made on the day of the production of gels, and

then the procedure was repeated after 7 and 14 days of incubation.

The analyses were performed on room tempered formulations and in accordance to

quality control methods used by BioGaia (25-27). MRS broth was tempered to 42oC in a

water bath. Three MRS-cystein plates and three MRS-ampicillin plates were used for

each sample and dilution. 3.0 g of the sample was scraped out of the cut open dropper

tube using a spoon, and weighed in a Stomacher bag. With a Smart Dilutor MRS broth

was added automatically to make a dilution with the ratio 1:10. The dilution was

homogenized in a Stomacher for 1.0 min, after which it was put to rest in room

temperature for approximately 15 min. It was then homogenized for another 1.0 min in

the Stomacher.

1.0 ml of the homogenized dilution was pipetted to a Dilucup with 9.0 ml of 0.9 %

NaCl. The dilution was mixed through shaking for 10 seconds on a Dilushaker. 1.0 ml

from the Dilucup was then pipetted to a new Dilucup to make another 1:10 dilution. The

procedure was repeated until the dilution had a concentration of 10-6

times the original

sample. 100 µl of the 10-6

dilution was pipetted to each agar plate. It was spread over

the surface using approximately ten glass beads that were shaken in different directions

all over the agar surface. The beads were removed and plates were placed upside down

in an anaerobic jar with two anaerobic bags and a moist anaerobic indicator strip. The

sealed jar was incubated for 72 hours at 37oC.

9

The colonies were counted automatically in an aCOLyte instrument. For plates with less

than 50 colonies counting was done by hand.

3.3.2 Analysis of rheology

The viscosity measurings were executed using a Brookfield DV-II+ Pro Viscometer, 11

days after the production of gels. The beaker in which the samples were placed was a

100 ml glass beaker. The analyses were executed in room temperature on samples

stored in room temperature, and all samples had a temperature of between 21.4 and

22.0oC at the time of analysis. The samples, which were kept in 500 ml glass bottles,

were either poured out or scraped out into the beaker with a spoon depending on

viscosity. Visible bubbles in the samples were eliminated through hitting the beaker

against the table repeatedly. The rotational speed was set every 20 seconds, and the

viscosity in mPa*s was read 10 seconds after the speed was set. The rotation speed

program expressed as rotations per minute (rpm) was the following: 0,5; 1,0; 2,0; 2,5;

4,0; 5,0; 10; 20; 50; 100 and then decreasing back down to 0,5 in the reverse order. A

double analysis was executed on each formulation.

3.3.3 Analysis of phase separation

100 ml glass cylinders were filled with each formulation. The cylinders were sealed and

incubated at 37oC. The separation of the gels was studied visually after 7 and 14 days.

4 Results

4.1 Analysis of survival of L. reuteri

There is no fast decline in the bacterial counts in any of the formulations or incubation

temperatures studied, as can be seen in figures 2 to 5.

10

Figure 2. Number of viable DSM 17 938 and ATCC PTA 5289 cells in the formulation with 3

% beeswax at different temperatures after 0, 7 and 14 days of incubation.

Figure 3. Number of viable DSM 17 938 and ATCC PTA 5289 cells in the formulation with 5

% beeswax at different temperatures after 0, 7 and 14 days of incubation.

11

Figure 4. Number of viable DSM 17 938 and ATCC PTA 5289 cells in the formulation with 7

% beeswax at different temperatures after 0, 7 and 14 days of incubation.

Figure 5. Number of viable DSM 17 938 and ATCC PTA 5289 cells in the hydrogenated oil

formulation at different temperatures after 0, 7 and 14 days of incubation.

12

4.2 Analysis of rheology

The formulations with 5 % and 7 % beeswax were too viscous to give any values on

viscosity at the higher rotational speeds, see figures 7 and 8. It can be seen in the

formulations with 3 % and 5 % beeswax (figures 6 and 7) that the gels have thixotropic

properties, which means that the formulation with 7 % beeswax probably does as well.

The formulation with hydrogenated oil, as seen in figure 9, seems to be pseudoplastic,

since the viscosity is the same at a set rpm even after shear stress has been added to the

formulation.

When the different formulations are compared it can be seen that the most viscous

formulation is the one with 7 % beeswax, followed by 5 % beeswax, 3 % beeswax and

hydrogenated oil, respectively (see figure 10).

Figure 6. Viscosity of the formulation with 3 % beeswax under shear stress. The blue line

represents rpm 100, and the red line rpm 0.

0

50000

100000

150000

200000

250000

300000

0 20 40 60 80 100

Viscosity (mPa*s)

RPM

13

Figure 7. Viscosity of the formulation with 5 % beeswax under shear stress. The blue line

represents rpm 100, and the red line rpm 0.

Figure 8. Viscosity of the formulation with 7 % beeswax under shear stress. The blue line

represents rpm 100, and the red line rpm 0.

0

50000

100000

150000

200000

250000

300000

0 20 40 60 80 100

Viscosity (mPa*s)

RPM

0

50000

100000

150000

200000

250000

300000

0 20 40 60 80 100

Viscosity (mPa*s)

RPM

14

Figure 9. Viscosity of the hydrogenated oil formulation under shear stress. The blue line

represents rpm 100, and the red line rpm 0.

Figure 10. Viscosity of the different formulations under increasing shear stress.

4.3 Analysis of phase separation

After 7 days of incubation at 37oC, phase separation was seen only in the hydrogenated

oil sample, as can be seen in figure 11. It was uttered as clear zones in an otherwise

opaque phase, see figure 12. All samples with beeswax were homogenous by the look

of it.

0

50000

100000

150000

200000

250000

300000

0 20 40 60 80 100

Viscosity (mPa*s)

RPM

0

50000

100000

150000

200000

250000

300000

0 10 20 30 40 50 60 70 80 90 100

Viscosity (mPa*s)

RPM

S

3%

5%

7%

15

Figure 11. Phase separation of samples after 7 days of incubation at 37

oC. From left: 3 %

beeswax, 5 % beeswax, 7 % beeswax and hydrogenated oil formulations.

Figure 12. Phase separation of hydrogenated oil after a) 7 days and b) 14 days. The arrows in

the figures highlight the same clear zone but at different times, to notify that there is more phase

separation after 14 days than after 7 days.

After 14 days there was more separation in the hydrogenated oil sample, with even

bigger clear oil zones (figure 12). The sample with 3 % beeswax had a layer of

approximately 1 ml oil on top, as can be seen in figure 13. In the samples with 5 % and

7 % no separation could be detected.

16

Figure 13. Phase separation of formulation with 3 % beeswax after 14 days

5 Discussion

It is desirable with a viable bacterial count of 1.0E+08 CFU of each strain per 5 drops

(equals one portion or 0.16 ml) of the product. At a first analysis of the survival of

bacteria there was no obvious difference between the formulations. There were only

three times of analysis of each sample and hence no outliers could be excluded. To be

able to make conclusions regarding the expected shelf life of the product, five points of

analysis during 9 months are preferred so that possible outliers could be excluded. Due

to lack of time that was not possible in the present study. Since the preferred shelf life is

set to 24 months at room temperature and the analysis in this study only stretches over

14 days, more evaluations need to be done. With a confidence level of 95 % there is a

significant decrease of total L. reuteri count in the formulation with 5 % beeswax after

14 days incubation at 5oC and 37

oC. There is also a significant decrease of L. reuteri

DSM17938 after 14 days incubation at 37oC. Statistical calculations were excecuted

through a t test on only the 5 % beeswax formulation, since that is the most interesting

formulation for further development. Since the amount of PTA5289 has been calculated

from subtracting the amount of DSM17938 on the corresponding ampicillin-plates from

the total amount of bacteria on cysteine-plates, there is a measurement uncertainty of the

method. Another possible source of error is the laborant’s inexperience with the

methods used. The formulations were made by hand in small quantities which also can

give bigger variation between samples. What can be said from the results though is that

there is no substantial decline of viable bacterial count over the first two weeks from

production date. The bacterial count is still close to 1.0E+08 CFU of each strain per 5

drops in all formulations at all incubation temperatures.The desiccant strip that is

present in all dropper tubes helps keeping the water activity down which prolongs the

survival of bacteria.

From the stability analysis, where separation was stressed through a raised incubation

temperature of 37oC, it could be seen that the hydrogenated oil formulation rapidly

separated. There were also signs of separation of the formulation with 3 % beeswax

after 14 days. More wax seems to add better stability to the system, and inhibit

coalescence of the oil phase. A more viscous fat system is generally more stable against

17

phase separation (19). There will probably be no proceeding with the formulation with 3

% beeswax or the formulation with hydrogenated oil in further development of the

product.

From the viscosity analysis plus when handling the gels, it was concluded that the

formulation with 7 % beeswax was more viscous than the other formulations. It was

difficult to press out of a dropper tube, which is a necessity and a criterion for an

acceptable product. The formulation has to be easy to press out of a tube, but at the

same time viscous enough to stay on a tooth stick. Except for the formulation with 7 %

beeswax, all other formulations were easy to press out of a tube. Because of the silica

the beeswax formulations are thixotropic. The thixotrophy makes it easier to press the

gel out of a tube. When the shearing is disrupted (i.e. when the gel is out of the tube) the

viscosity will still be high enough for the gel to stay on a tooth stick.

The viscosity analysis in this study primarily is for comparison between formulations.

Before the project started the intention was that there would be bigger differences

considering ingredients in the different formulations.

6 Conclusions

Beeswax gives good thickening of the Prodentis Drops formula. In further development

of the product it is recommended to proceed with the 5 % beeswax formulation since it

has a satisfying feel, stays on a tooth stick, shows no tendency to separate and has

indication of good bacterial survival. The silica adds good thixotropic properties which

are preferred, and the product has a good taste thanks to the peppermint flavour. The

formulation with hydrogenated oil had separated already after 7 days at 37oC, and is not

recommended to proceed with in its current composition.

7 References

1. About BioGaia. BioGaia; [cited 2013 2013-05-27]; Available from:

http://www.biogaia.com/about-biogaia.

2. Guidelines for the Evaluation of Probiotics in Food - Report of a Joint

FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in

Food. London, Ontario, Canada: FAO/WHO, 2002.

3. Hemarajata P, Versalovic J. Intestinal Lactobacillus reuteri: Partners and

beneficial microbes. Old Herborn University; 2012.

4. Bergey DH, Holt JG, Sneath PHA. Bergey's Manual of systematic

bacteriology. Vol. 2. Baltimore: Williams & Wilkins; 1986. xxiii s., s. 965-1599 p.

5. Rosander A, Connolly E, Roos S. Removal of Antibiotic Resistance Gene-

Carrying Plasmids from Lactobacillus reuteri ATCC 55730 and Characterization of the

Resulting Daughter Strain, L. reuteri DSM 17938. Applied and Environmental

Microbiology. 2008;74(15):6032-40.

18

6. Sepová HK, Bilková A. Isolation and identification of new lactobacilli

from goatling stomach and investigation of reuterin production in Lactobacillus

reuteri strains. Folia Microbiologica. 2013;58(1):33-8.

7. Talarico T, L., Casas IA, Chung TC, Dobrogoszi WJ. Production and

Isolation of Reuterin, a Growth Inhibitor Produced by Lactobacillus reuteri.

Antimicrobial Agents and Chemotherapy. 1988;32(12):1854-8.

8. Vivekananda MR, Vandana KL, Bhat KG. Effect of the probiotic

Lactobacilli reuteri (Prodentis) in the management of periodontal disease: a

preliminary randomized clinical trial. Journal of Oral Microbiology. 2010;2(5344).

9. Krasse P, Carlsson B. Decreased gum bleeding and reduced gingivitis by

the probiotic Lactobacillus reuteri. Swedish Dental Journal. 2005;30:55-60.

10. Twetman S, Derawi B, Keller M. Short-term effect of chewing gums

containing probiotic Lactobacillus reuteri on the levels of inflammatory mediators in

gingival crevicularfluid. Acta Odontologica Scandinavica 2008.

11. Hedberg M, Hasslöf P. Sugar fermentation in probiotic bacteria – an in

vitro study. Oral Microbiology Immunology. 2008;23:482–5.

12. Caglar E, Kusco O, Kavaloglu S. A probiotic lozange administered

medical device and its effect on salivary mutans streptococci and lactobacilli.

International Journal of Pediatric Dentistry. 2008;18:35-9.

13. Iniesta M, Herrera D, Montero E. Probiotic effects of orally administered

Lactobacillus reuteri-containing tablets on the subgingival and salivary microbiota in

patients with gingivitis. A randomized clinical trial. Journal of Clinical Periodentology.

2012;39:736-44.

14. Wolf BW, Garleb KA, Ataya DG. Safety and Tolerance of Lactobacillus

reuteri in Healthy Adult Male Subjects. Microbial Ecology in Health and Disease.

1995;8:41-50.

15. Massey BS, Ward-Smith AJ. Mechanics of fluids. 8. ed. London: Taylor &

Francis; 2006. x, 696 s. p.

16. Brookfield DV-II+ Proprogrammable Viscometer Operating Instructions

Manual No. M/03-165. Brookfield Engineering Laboratories, Inc.

17. Hwang H-S, Kim S, Singh M, Winkler-Moser JK, Liu SX. Organogel

Formation of Soybean Oil with Waxes. Journal of the American Oil Chemists' Society.

2012;89:639-47. Epub 2011-11-27.

18. Damodaran S, Parkin KL, Fennema OR. Fennema's food chemistry. 4. ed.

Boca Raton: CRC Press/Taylor & Francis; 2008. [12], 1144 s. p.

19. Food Lipids: Chemistry, Nutrition, and Biotechnology. 1st ed. New York:

Marcel Dekker, Inc.; 1998.

20. Rowe RC, Weller PJ, Sheskey PJ. Handbook of pharmaceutical

excipients. 4. ed. London: Pharmaceutical Press; 2003. 776 s. p.

19

21. EFSA. Scientific Opinion of the Panel on Food additives, Flavourings,

Processing aids and Materials in Contact with Food (AFC) on a request from the

Commission on the safety in use of beeswax. The EFSA Journal. 2007;615:1-28.

22. Fumed Silica Interactive Learning Module. Cabot, GmbH, Germany;

[cited 2013 2013-04-17]; Available from: http://www.cabot-

corp.com/wcm/sepdown/rd/fs/fumed_silica.html.

23. Directive on food additives other than colours and sweeteners. Regulation

(EC) No 1333/2008 (1995).

24. Barbosa Rocha JC, Dutra Lopes J. Thermal and rheological properties of

organogels formed by sugarcane or candelillawax in soybean oil. Food Research

International. 2012;50:318-23.

25. Spädning och provsättning produkt innehållande L. reuteri samt

ytterligare en L. reuteri-stam. Doc. no: QCME103, ed. AC 2012-10-05 [BioGaia

internal methods].

26. Provberedning oljeblandningar. Doc. no: QCME003, ed. AE 2012-10-05

[BioGaia internal methods].

27. Koloniräkning med aCOLyte och bedömning av kolonimorfologi. Doc. no:

QCME206, ed. AD 2012-10-05 [BioGaia internal methods].

I

Appendices

Appendix A: Mean values for analysis of survival of bacteria in

different formulations.

Each value is the mean value from 6 plates. The amount of PTA5289 has been

calculated from subtracting the amount of DSM17938 on the corresponding ampicillin-

plates from the total amount of bacteria on cysteine-plates.

Table Ia: Mean values of bacterial counts in formulation with 3 % beeswax after 0, 7 and 14

days.

Table Ib: Mean values of bacterial counts in formulation with 5 % beeswax after 0, 7 and 14

days.

Table Ic: Mean values of bacterial counts in formulation with 7 % beeswax after 0, 7 and 14

days.

Table Id: Mean values of bacterial counts in formulation with hydrogenated oil after 0, 7 and 14

days.

0 7 14

3 %, 5°C, DSM17938 2,06E+08 1,02E+08 1,07E+08

3 %, 5°C, PTA5289 6,55E+07 6,90E+07 1,29E+08

3 %, 25°C, DSM17938 2,06E+08 1,04E+08 1,04E+08

3 %, 25°C, PTA5289 6,55E+07 5,55E+07 1,16E+08

3 %, 37°C, DSM17938 2,06E+08 1,27E+08 7,85E+07

3 %, 37°C, PTA5289 6,55E+07 2,90E+07 8,68E+07

0 7 14

5 %, 5°C, DSM17938 1,51E+08 1,28E+08 1,16E+08

5 %, 5°C, PTA5289 1,39E+08 6,88E+07 9,68E+07

5 %, 25°C, DSM17938 1,51E+08 9,23E+07 1,08E+08

5 %, 25°C, PTA5289 1,39E+08 4,53E+07 1,39E+08

5 %, 37°C, DSM17938 1,51E+08 8,48E+07 5,73E+07

5 %, 37°C, PTA5289 1,39E+08 4,05E+07 5,33E+07

0 7 14

7 %, 5°C, DSM17938 1,40E+08 1,46E+08 1,54E+08

7 %, 5°C, PTA5289 1,11E+08 4,15E+07 1,27E+08

7 %, 25°C, DSM17938 1,40E+08 1,14E+08 1,02E+08

7 %, 25°C, PTA5289 1,11E+08 5,33E+07 8,93E+07

7 %, 37°C, DSM17938 1,40E+08 7,75E+07 6,40E+07

7 %, 37°C, PTA5289 1,11E+08 2,50E+07 7,20E+07

0 7 14

H, 5°C, DSM17938 3,29E+08 4,07E+08 3,03E+08

H, 5°C, PTA5289 1,42E+08 1,21E+08 2,12E+08

H, 25°C, DSM17938 3,29E+08 3,79E+08 3,21E+08

H, 25°C, PTA5289 1,42E+08 1,50E+08 1,46E+08

H, 37°C, DSM17938 3,29E+08 3,04E+08 2,68E+08

H, 37°C, PTA5289 1,42E+08 1,50E+08 1,62E+08

II

Appendix B: Raw data from viscosity analysis Table IIa. Viscosity data as given from viscometer at different shear rates. The values are given

in mPa*s.

Rpm/min H1 H2 3:1 3:2 5:1 5:2 7:1 7:2

0,5

99579 148000 162000 199000 271000 324000 326000

1,0 81583 77983 106000 132000 133000 141000 191000 176000

2,0 47690 50089 82782 103000 134000 130000 157000 132000

2,5 38392 41511 71945 89261 123000 116000 140000 105000

4 25045 26844 44091 53239 87730 84931 114000 69892

5 19796 21835 35512 42951 88481 79463 118000 67745

10 12057 12957 19876 24535 49289 47910

20 7618 8158 11787 13217 27564 24805

50 4043 4403 6032 6463

11320

100 2172 2351 3413 3833

50 3731 3935 4031 4679 7884 8194

9130

20 6059 6689 7948 7858 14967 14937 24655 16227

10 10798 11937 14037 14097 26874 24655 43971 29034

5 19076 20876 25914 26034 48590 44031 74264 51129

4 23695 26094 31343 32393 57588 50889 86981 58438

2,5 36712 40311 50429 50149 85422 70065 118000 79683

2 45590 47990 59087 63586 100000 86681 132000 91480

1 76184 83392 98979 104000 118000 119000 192000 155000

0,5 124000 125000 156000 146000 157000 142000 245000 287000

Table IIb. Mean values of measured viscosity (given in mPa*s) in formulation with 3 %

beeswax.

3%

RPM η (RPM → 100) η (RPM → 0)

0,5 155000 151000

1,0 119000 101489,5

2,0 92891 61336,5

2,5 80603 50289

4 48665 31868

5 39231,5 25974

10 22205,5 14067

20 12502 7903

50 6247,5 4355

100 3623 3623

Table IIc. Mean values of measured viscosity (given in mPa*s) in formulation with 5 %

beeswax.

5%

RPM η (RPM → 100) η (RPM → 0)

0,5 235000 149500

1,0 137000 118500

2,0 132000 93340,5

III

2,5 119500 77743,5

4 86330,5 54238,5

5 83972 46310,5

10 48599,5 25764,5

20 26184,5 14952

50 11320 8039

100

Table IId. Mean values of measured viscosity (given in mPa*s) in formulation with 7 %

beeswax.

7%

RPM η (RPM → 100) η (RPM → 0)

0,5 325000 266000

1,0 183500 173500

2,0 144500 111740

2,5 122500 98841,5

4 91946 72709,5

5 92872,5 62696,5

10

36502,5

20

20441

50

9130

100

Table IIe. Mean values of measured viscosity (given in mPa*s) in formulation hydrogenated oil.

H

RPM η (RPM → 100) η (RPM → 0)

0,5 99579 124500

1,0 79783 79788

2,0 48889,5 46790

2,5 39951,5 38511,5

4 25944,5 24894,5

5 20815,5 19976

10 12507 11367,5

20 7888 6374

50 4223 3833

100 2261,5 2261,5