Capela, 2007

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Effect of homogenisation on bead size and survival of encapsulated probiotic bacteria

P. Capela a,1 , T.K.C. Hay b , N.P. Shah a, *

a School of Molecular Sciences, Victoria University, Werribee Campus, P.O. Box 14428, Melbourne, Victoria 8001, Australiab DSTO-Scottsdale, Defence Nutrition, Scottsdale, Tasmania 7260, Australia

Received 21 March 2007; accepted 14 August 2007

Abstract

This study investigated homogenisation techniques for reducing the size of calcium alginate beads during the microencapsulation of Lactobacillus acidophilus , Lactobacillus casei , Lactobacillus rhamnosus and Bidobacterium longum . Bead sizes were reduced using threehomogenisation techniques including Ultra-Turrax benchtop homogeniser, Avestin Inc. piston homogeniser and Silverson mixer for var-ious durations, number of passes and pressures. The survival of probiotic organisms during homogenisation was also investigated. Thesmallest beads (39.2 l m) were created using the Ultra-Turrax benchtop homogeniser at 13500 rpm for 4 min. There was a signicantreduction in the survival of each organism (<5.5%) using the Silverson mixer. However, homogenisation using Ultra-Turrax and AvestinInc. homogenisers resulted in better survival at 64.4% and 47.7%, respectively. Overall, homogenisation reduced size of beads containingviable probiotic organisms during microencapsulation.

2007 Elsevier Ltd. All rights reserved.

Keywords: Homogenisation; Bead size; Survival; Encapsulation; Probiotic bacteria

1. Introduction

The consumption of certain species of probiotic micro-organisms is benecial for reducing the duration and sever-ity of diarrhoeal symptoms ( Isolauri, Juntunen, Rautanen,Sillanaukee, & Koivula, 1991 ). Supplementing a diet withfood containing benecial bacteria can be used as a strat-egy for preventing diarrhea ( Oksanen et al., 1990; Shahani

& Chandan, 1979; Siitonen et al., 1990 ). Probiotics aredened as ‘living microorganisms, which upon ingestionin certain numbers, exert health benets beyond inherentbasic nutrition’ ( Guarner & Schaafsma, 1998 ). However,for obtaining health benets, a minimum of one million

probiotic organisms per gram of a food is recommended(Kurman & Rasic, 1991 ).

During processing and storage of foods, probioticmicroorganisms may be exposed to high temperatures,low pH, high osmotic pressure and high levels of oxygen(Gardiner et al., 2000; Prasad, McJarrow, & Gopal, 2003;Talwalkar & Kailasapathy, 2003 ). These factors may havedeleterious effects on probiotics. The survival of probiotic

organisms is also affected by acid encountered in the stom-ach and the bile salts in the intestine tract ( Chandramouli,Kailasapathy, Peiris, & Jones, 2004; Conway, Gorbach, &Goldin, 1987 ). Microencapsulation is the process of apply-ing a shell to sensitive microorganisms to protect themfrom their external environment.

Microencapsulation for food applications involves theuse of a non-toxic, food grade coating material, such assodium alginate. When sodium alginate is combined withcalcium chloride, a gel matrix is formed creating a shellto protect sensitive probiotic organisms ( Shah & Ravula,

0963-9969/$ - see front matter 2007 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodres.2007.08.006

* Corresponding author. Tel.: +61 3 9216 8289; fax: +61 3 9216 8284.E-mail address: [email protected] (N.P. Shah).

1 Present address: DSTO-Scottsdale, Defence Nutrition, Scottsdale,Tasmania 7260, Tasmania, Australia.

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Food Research International 40 (2007) 1261–1269

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2000). Other food-grade agents used to immobilize lacticacid bacteria include kappa-carrageenan, locust bean gum(Audet, Lacroix, & Paquin, 1992 ) and gellan-xanthan(Sun & Griffiths, 2000).

The emulsion technique of microencapsulation involvescombining a mixture of probiotic organisms and an encap-

sulating agent such as sodium alginate with vegetable oil(Capela, Hay, & Shah, 2006; Krasaekoopt, Bhandari, &Deeth, 2004 ). An alginate/water-in-oil emulsion is formedby agitating the mix, usually with a magnetic stirrer. Thealginate beads are formed by slowly adding calcium chlo-ride to the emulsion while stirring. Shah and Ravula(2000) found that calcium alginate beads formed using theemulsion technique improved the viability of probioticorganisms during processing and storage of frozen yoghurt.However, there are several disadvantages of using the emul-sion technique including a wide distribution of bead size,difficulty in automating the technique and exceedingly largebeads with diameters ranging from 200 to 1000 l m (Ponc-elet et al., 1992).

Studies by Audet, Paquin, and Lacroix (1998) andArnaud, Lacroix, and Choplin (1992) found that encapsu-lation techniques such as the emulsion technique are con-ducive to forming large beads which can inuence thetexture and mouthfeel of products such as yoghurt. Tyle(1993) found that particles that are generally soft, rounded,and up to 80 l m are not perceived as gritty, but beadsexceeding this size may be detectable in the mouth. Tech-niques employing spray guns ( Lee, Hwang, Park, & Park,2003) and air atomizers ( Kwok, Groves, & Burgess,1991) with varying nozzle sizes have been used to reduce

the bead size. The bead sizes could be reduced by using ahomogeniser during the encapsulation process to form ane water-in-oil emulsion containing small droplets.

High speed mixers are commonly used in the food indus-try for homogenising oil and aqueous phases. Duringhomogenisation, the interface between the oil and wateris disrupted causing the liquids to blend together. Variousmixing heads can be attached to high speed mixers toreduce droplet size by generating intense disruptive forces.High-pressure valve homogenisers (such as the Avestin Inc.and Manton-Gaulin APV) create small droplets by forcingliquid through a narrow valve under high-pressure. A mix-ture can be passed through a high-pressure valve homoge-niser repeatedly to achieve the desired droplet size(McClements, 1999 ).

Food emulsions containing a varied particle size distri-bution of droplets are referred to as polydisperse emulsions(McClements, 1999 ). Small particles in a microscopic poly-disperse emulsion may not be visible to the naked eye. Aparticle size distribution diagram illustrating the size rangeand population of calcium alginate beads can be generated.Particle sizing equipment, such as Mastersizer TM 2000 canbe used to measure particles ranging between 0.02 l mand 2000 l m by passing the particles through a laser beam(http://www.malvern.co.uk ). However, due to the sensitiv-

ity of a Mastersizer , probiotic organisms may be

recorded as particles which may inuence the particle sizedistribution.

As the population of probiotic organisms may beaffected during the microencapsulation process itself, it isessential to ensure that the selected microencapsulationtechnique is gentle to sensitive probiotic organisms. Some

treatments may have deleterious effects on anaerobic probi-otic organism due to the presence of oxygen, heat gener-ated during the process or mechanical shear ( Arnaud,Lacroix, Foussereau, & Choplin, 1993; Talwalkar & Kaila-sapathy, 2003 ). Individual species of probiotic organismsmay vary in sensitivity to external stresses such as thoseencountered during homogenisation. Cell wall elasticity isthought to improve resistance to mechanical stress due tovariations in cell morphology ( Schar-Zammaretti & Ubb-ink, 2003).

The effect of homogenising sodium alginate (containingprobiotic organisms) in oil using high speed mixers andhigh-pressure valve homogenisers in order to reduce thesize of calcium alginate beads has not been thoroughlyinvestigated. The aim of this study was to determine theeffect of various homogenisers on the uniformity and diam-eter of calcium alginate beads and the survival of encapsu-lated Lactobacillus acidophilus 33200, Lactobacillus casei 279, Bidobacterium longum 536 and Lactobacillus rhamno-sus GG.

2. Materials and methods

2.1. Preparation of probiotic bacteria

B. longum 536 was obtained from the Victoria Univer-sity Culture Collection (Werribee, Victoria, Australia).The organism was originally obtained from Morinaga MilkIndustry Co. Ltd. (Tokyo, Japan). L. casei 279 wasobtained from the Australian Starter Culture Collection(Werribee, Victoria, Australia). L. acidophilus 33200 wasobtained from the American Type Culture Collection(Manassas, VA, USA), and L. rhamnosus GG was isolatedfrom a commercial product. Each of the organisms wasgrown in sterile MRS (deMann, Rogosa, Sharp) broth(Oxoid Ltd., Hampshire, UK) using a 1% inoculum. B. longum 536 was supplemented with lter-sterilized 0.05%(w/v) L-cysteine Æ hydrochloride (Sigma Chemical Co., Cas-tle Hill, Sydney, Australia) to create an anaerobic environ-ment. Each culture was grown and propagated three timessuccessively at 37 C for 12–15 h for activation. Probioticorganisms were harvested by centrifuging (Sorvall RT7Newtown, CT, USA) at 1510 g at 4 C for 15 min and thecell pellet was suspended in saline solution followed bymicroencapsulation.

2.2. Microencapsulation of probiotic organism using theemulsion technique

The emulsion method of microencapsulation was used

to encapsulate probiotics ( Sheu & Marshall, 1993 ). Briey,

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120 mL of sterile 3% (v/w) sodium alginate was mixed with30 mL of suspension containing 9.0–10.0 log 10 cfu/g of combined L. acidophilus 33200, L. casei 279 B. longum536 and L. rhamnosus GG. Fifty millilitres of the suspen-sion of sodium alginate and probiotic organisms was gentlydispensed using a pipette into a beaker containing 200 mL

of sterile vegetable oil (Eta Blended Vegetable Oil, Good-man Fielder Pty. Ltd., Melbourne, Australia) and stirredat 200 rpm using a magnetic stirrer (IEC Industrial Equip-ment & Control Pty. Ltd., Melbourne, Australia). Calciumchloride (0.1 M) was gently added down the side of the bea-ker until the emulsion was broken.

2.3. Microencapsulation of L. acidophilus 33200, L. casei 279, B. longum 536 and L. rhamnosus GG using homogenisation (Ultra-Turrax benchtop, Avestin Inc. pistonor Silverson mixer)

The sodium alginate solution and probiotic organismswere combined with vegetable oil as described in Section2.2. An emulsion was formed by homogenising the mixtureat various speeds, pressures, passes and times using theUltra-Turrax benchtop (Ika Laboratory and AnalyticalEquipment, Staufen, Germany), Avestin Inc. pistonhomogeniser (Avestin, Ottawa, Canada) or Silverson mixer(Silverson Machine Ltd., Waterside, Chesham Bucks, Eng-land) as described below.

To prepare alginate beads using the Ultra-Turraxbenchtop homogeniser, an emulsion was prepared byhomogenising 200 mL mixtures (as described in Section2.2) at 8000 rpm and 13,500 rpm for 2 or 4 min. To prepare

alginate beads using the Avestin Inc. piston homogeniseran emulsion was formed by passing the mixture (asdescribed in Section 2.2) through the homogeniser 2 or 3times at 5 or 10 MPa of pressure. Calcium alginate beadswere formed by homogenising 200 mL of the mixturesusing the Silverson mixer (described in Section 2.2) for 2or 4 min. The homogenisation temperature during eachof the treatments was 21 C.

After each of the homogenisation treatments, eachemulsion was transferred into a beaker. Calcium chloride(0.1 M) was gently added to the side of the beaker to theemulsions while stirring using a magnetic stirrer at200 rpm. The mixture was stirred for 10 min to ensure thatthe emulsion was completely broken. Small calcium algi-nate microspheres were formed and subsequently measuredusing a Mastersizer (Hydro-2000G Malvern InstrumentsLimited, Worcester, UK).

2.4. Measurement of particle size of calcium alginate beads

Calcium chloride solution (0.1 M) containing 1–5 g of calcium alginate beads was passed through a MastersizerHydro-2000G to measure the size of the beads. Sampleswere added to circulating ltered water until laser obscura-tion exceeded 10%. The mean particle size was expressed asd 32 , which represented the area-volume mean diameter.

Acceptability of the bead size was also assessed by examin-ing the d (0.9) values (the diameter of bead for an observa-tion at 0.9 of the distribution).

2.5. Equations used

The area-volume mean diameter was determined usingthe following equation, in which d represented the dropletdiameter, n represented the number of droplets in a class.

d 32 ¼ P i¼1nid 3i

P i¼1nid 2i

The following equation was used to determine the sur-vival of probiotic organisms during homogenisation:

Survival ð% Þ ¼ y þ z

x 100

where x is the initial population of probiotic organismsprior to homogenisation; y is the population of probioticorganisms encapsulated within calcium alginate beads; zis the population of probiotic organisms in the externaluid surrounding the calcium alginate beads.

The following equation was used to evaluate the effi-ciency of the homogenisation treatments:

Efficiency ð% Þ ¼ y z

x 100

where x is the initial population of probiotic organismsprior to homogenisation; y is the population of probioticorganisms encapsulated within calcium alginate beads; zis the population of probiotic organisms in the external

uid surrounding the calcium alginate beads.

2.6. Statistical analysis

Data analysis was carried out using SPSS Inc. software(14.0: SPSS Inc., Chicago, IL). The signicant differencebetween means (level of signicance P = 0.05) was analysedusing one-way ANOVA. Multiple comparisons betweenmeans were analysed using Tukey’s test. The enumerationof probiotic population was independently replicated threetimes (n = 3), with two measurements per replicate.

2.7. Enumeration of L. acidophilus 33200, L. casei 279, B.longum 536 and L. rhamnosus GG within calcium alginatebeads

The entrapped probiotic organisms were released fromthe calcium alginate beads by sequestering calcium ionswith phosphate buffer at neutral pH. Once released, probi-otic organisms were selectively enumerated using the tech-niques of Tharmaraj and Shah (2003) . L. acidophilus wasselectively enumerated on MRS-sorbitol agar using anaer-obic incubation at 37 C for 72 h. L. casei was enumeratedon LC agar using anaerobic incubation at 37 C for 72 h.B. longum was selectively enumerated on MRS-NNLP

(nalidixic acid, neomycin sulphate, lithium chloride and

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paromomycin sulphate) agar incubated at 37 C for 72 hand L. rhamnosus was enumerated on MRS-vancomycinagar using anaerobic incubation at 43 C for 72 h.

3. Results and discussion

3.1. Effect of homogenisation on bead size

The effect of homogenisation on the size of calcium algi-nate beads is shown in Table 1. Each homogenisation treat-ment aided in the formation of calcium alginate beads witharea-volume mean diameters (AVMD) that were signi-cantly smaller (P < 0.05) than the control (381.2 l m).Large beads in the control samples were due to the emul-sion being created by agitation using a magnetic stirrer.Ideally, particle size should be below 80 l m, otherwisethe mouth feel of products such as yoghurt, as well as over-all acceptability of the product is affected ( Tyle, 1993).

There was no signicant reduction ( P < 0.05) in bead sizewhen the homogenisation speed was increased from8000 rpm to 13,500 rpm. Nor was there a signicant reduc-tion in particle size when the duration of homogenisation

was increased from 2 to 4 min (when using the Ultra Tur-rax). The AVMD of beads created using the Silverson mixer(41.6 l m after 2 min and 42.9 l m after 4 min) were signi-cantly smaller than those created using the Avestin Inc. pis-ton homogeniser (191.6 l m after three passes at 5 MPa).

Acceptability of calcium alginate beads were assessed by

examining d (0.9) values (which represented the diameter of the bead at the 90th percentile). The values of all beadsformed by instrumental homogenisation (209.1–519.8 l m)were signicantly smaller than the control (1312.2 l m) atd (0.9) as shown in Table 1. However, despite these obser-vations being signicantly smaller ( P < 0.05) than the con-trol, a proportion of these beads is still unacceptable forincorporation into a commercial product as many of theseparticles exceeded the 80 l m sensory perception thresholdproposed by Tyle (1993). The large differences betweenAVMD of the beads and d (0.9) values indicate an unac-ceptably wide distribution in diameters of the beads.

Particle size distributions are illustrated in Figs. 1–6.Beads which were formed using a conventional emulsion

Table 1Effect of homogenisation on the size ( l m) of calcium alginate beads usingUltra-Turrax T 25, Avestin Inc. piston or Silverson mixer

Treatment d 32 (l m) area-volume meandiameter d

d (0.9) (l m)d

Control 381.2 ce ± 29.5 1312.2b e ± 51.7Ultra-Turrax benchtop

homogeniser 8000 rpm for2 min

118.6ab ± 37.2 248.0a ± 154.3

Ultra-Turrax benchtophomogeniser 8000 rpm for4 min

82.1ab ± 15.6 506.6a ± 173.5


52.6ab ± 7.7 360.7a ± 80.9


39.2a ± 4.6 209.1a ± 4.9

Silverson Mixer for 2 min 41.6a ± 4.2 224.2a ± 50.3Silverson Mixer for 4 min 42.9a ± 4.9 290.9a ± 0.6Avestin Inc. piston

Homogeniser 5 MPa · 2passes

93.1ab ± 4.9 330.6a ± 45.0

Avestin Inc. pistonHomogeniser 5 MPa · 3passes

191.6b ± 51.9 519.8a ± 132.3


121.6ab ± 4.5 444.4a ± 82.8


102.4ab ± 11.2 296.9a ± 13.4

a,b,c Means in the same column with different superscripts are signicantlydifferent (P < 0.05).

d Results are expressed as means ± standard error of two repeatedexperiments.

e Data point is expressed as means ± standard error of four repeated

experiments.

0.01 0.1 1 10 100 1000 3000Particle size (µm)

1

2

3

4

5

6

7

8

9

10

11

12

V o l u m e

( % )

Fig. 1. Particle size distribution of calcium alginate beads prepared usingthe emulsion microencapsulating technique; –– –– Control ‘a’, -- --Control ‘b’, —— Control ‘c’, .... Control ‘d’.

0.01 0.1 10 100 1000 3000Particle size (µm)

1

2

3

4

5

6

7

8

V o l u m e

( % )

1

Fig. 2. Particle size of calcium alginate beads prepared using the Ultra-Turrax T 25 benchtop homogeniser at 8000 for 2 or 4 min; —— 8000 rpm

for 2 min, ---- 8000 rpm for 4 min.


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technique ( Fig. 1) displayed the largest span of particle size.The wide peaks in this distribution highlighted the lack of uniformity in the diameters of calcium alginate beads. Thenarrowest spans were recorded for beads produced usingthe Ultra-Turrax or Silverson mixer ( Figs. 2–4). Beadsformed using the Ultra-Turrax and Silverson mixer weresmaller than the control due to mechanical disruption of particles encountered during the emulsion stage of micro-encapsulation ( McClements, 1999). Small particle sizewas also obtained using the Avestin Inc. piston homoge-niser (Figs. 5 and 6 ). This was possibly due to the effectof high-pressure on the size of particles formed during

the emulsifying stage (as described in Section 3.2).

3.2. Effect of homogenisation on the survival of encapsulated probiotic organisms

The effect of homogenisation using Ultra-Turrax bench-top, Avestin Inc. piston or Silverson mixer on survival of L.rhamnosus GG, L. casei 279, L. acidophilus 33200, and B.longum 536 is shown in Figs. 7–10. L. rhamnosus provedto be the most robust organisms as it displayed the highestpercentage survival (64.4%) when homogenised for 2 minat 8000 rpm using the Ultra-Turrax ( Fig. 7). However,there was a signicant reduction ( P < 0.05) in the survivalof L. rhamnosus when homogenisation was increased from8000 rpm (64.4%) to 13,500 rpm (25.5%). A similar reduc-tion in the survival of L. casei was identied when thehomogenisation speed was increased ( Fig. 8). These resultssuggest a ‘mechanical shear’ effect on the survival of certainprobiotic organisms using an Ultra-Turrax homogeniser.

The survival of L. rhamnosus (Fig. 7) was signicantlylower than the control after homogenisation using the Silv-erson mixer. This treatment was the most severe as countsof all probiotic organisms were below 5.5%. The reductionin viability of probiotic organisms was possibly due to heatand mechanical shear generated during homogenisation

(Arnaud et al., 1993 ). Mechanical energy in the system

0.01 0.1 1 10 100 1000 3000

1

2

3

4

5

6

7

Particle size (µm)

V o l u m

e ( % )

Fig. 3. Particle size of calcium alginate beads prepared using the Ultra-Turrax T 25 benchtop homogeniser at 13,500 rpm for 2 or 4 min; —— 13,500 rpm for 2 min, - -- - 13,500 rpm for 4 min.

0.01 0.1 1 10 100 1000 3000Particle size (µm)

1

2

3

4

5

6

7

V o

l u m e

( % )

Fig. 4. Particle size of calcium alginate beads prepared using a SilversonMixer for 2 or 4 min, —— 2 min; ---- 4 min.

0.01 0.1 1 1000 3000Particle size (µm)

1

2

3

4

5

6

78

9

10

11

12

V o l u m e

( % )

10 100

Fig. 5. Particle size of calcium alginate beads prepared using an AvestinInc. piston homogeniser at 5 MPa after two and three passes; —— two

passes, -- - - three passes.

0.01 0.1 1 10 100 1000 3000Particle size (µm)

1

2

3

4

5

6

7

8

9

10

11

V o

l u m

e ( % )

Fig. 6. Particle size of calcium alginate beads prepared using an AvestinInc. piston homogeniser at 10 MPa after two and three passes; —— twopasses, - -- - three passes.


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Vogel, 2000). Interestingly, pre-treating a material contain-ing L. rhamnosus to mild pressure (100 MPa for 10 min)

prior to exposure to an elevated temperature (60

C) hasbeen found to improve the survival of probiotic organisms(http://www.tu-berlin.de/~foodtech/ResearchFINAL ).

Homogenisation time was set at 2 or 4 min using theUltra-Turrax and Silverson mixer to investigate whetherthere was a ‘time effect’ on the survival of the probioticorganisms ( Figs. 7–10). There was no signicant differencein populations of probiotic organisms for any of the organ-isms homogenised at 2 or 4 min, with the exception of L.acidophilus using the Ultra-Turrax, as there was no reduc-tion after initial cell death. Therefore, a ‘time effect’ wasnot observed under the conditions of this study. Overall,the Ultra-Turrax and the Avestin Inc. piston homogeniserhad the least impact upon the counts of viable probioticorganisms. The survival of probiotic organisms wasexpected to be highest for each of the controls ( Figs. 7– 10) as the solutions were not homogenised during thesetreatments. However, unexpectedly, the survival of L.casei , L. acidophilus and B. longum in control sampleswas lower than some Ultra-Turrax and Avestin Inc. pistonhomogenisation treatments.

3.3. Efficiency of homogenisation technique

Producing calcium alginate beads using homogenisation

was not highly efficient as probiotic organisms were lost to

the uid surrounding the beads. The comparative efficien-cies of the homogenisation techniques are shown in

Fig. 11. The Ultra-Turrax and Piston homogenisers werenot signicantly different ( P < 0.05) to the control. How-ever, the Silverson mixer was the least efficient process asthe efficiencies after treatments for 2 or 4 min of homogeni-sation were 79.08% and 88.88%, respectively.

4. Conclusion

It was possible to reduce the bead size below 100 l musing the Ultra-Turrax homogeniser. The Ultra-Turraxhomogeniser was the most suitable for preparing calciumalginate beads with a narrow range of diameters. The pop-ulation of L. acidophilus 33200, L. casei 279, B. longum 536and L. rhamnosus GG was not adversely affected duringhomogenisation using Ultra-Turrax benchtop and AvestinInc. piston homogenisers. Homogenisation using a Silver-son mixer had a negative impact upon probiotic popula-tions and would not be recommended for the preparationof calcium alginate beads. However, microencapsulatingprobiotic organisms using these techniques was laborious,time consuming, and produce a large amount of wasteoil. The waste oil also contained small sodium alginate/probiotic droplets that were difficult to remove by centrifu-gation. Therefore, further research is required to develop

an automated technique for the large scale production of

60

65

70

75

80

85

90

95

100

E f f i c i e n c y ( % )

All organisms

U T 8 0 0 0 r p m 2 m i n

U T 8 0 0 0 r p m 4 m i n

U T 1 3 5 0 0 r p m 2 m i n

U T 1 3 5 0 0 r p m 4 m i n

P 5 M P a -

2 p a s s e s

P 5 M P a -

3 p a s s e s

P 1 0 M P a -

2 p a s s e s

P 1 0 M P a -

3 p a s s e s

S M

2 m i n

S M

4 m i n

C o n t r o l -

N o t

h o m o g e n i s e

d

Treatment

Fig. 11. Efficiency of homogenisation process using Ultra-Turrax T 25 (UT), Avestin Inc. piston (P) and Silverson mixer (SM). (Efficiency is expressed asthe percentage of probiotic organisms retained in calcium alginate matrix and not lost to surrounding uid).


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encapsulated probiotics in calcium alginate beads (in a con-tinuous large scale operation).

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Documents

Capela, 2007