CELLULAR MORPHOLOGY OF BIFIDOBACTERIA AND THER

SURVIVAL WKEN ENCAPSULATED IN CALCIUM ALGNATE BEADS

A Thesis

Present ed to

The Faculty of Graduate Studies

of

The University of Guelph

by

JANE C. ELLENTON

In partial fulfilment of requirements

for the degree of

Master of Science

January, 1998

@ J.C. EUenton, 1998

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ABSTRACT

CELLULAR MORPHOLOGY OF BIFIDOBACTERIA AND THEUX SURVIVAL WHEN ENCAPSULATED IN CALCTUM ALGINATE BEADS

Jane C. EtIenton University of Guelph, 1998

Advisor: Linda J. Harris

Cellular morpholgies and acid sensitivity of B~fidobactrrit~m W r f a , l r k , B. fot~gim,

and two strains of B. hrfidirm were evaluated. The addition of 0.0 1% CaClz to the culture

media promoted rod forms while the addition of 2% NaCl promoted branching for ail four

species. Acid sensitivity was strain related; B. irIfantis and B. longirn were less sensitive to

pH 4.3, 4.0 and 3.7 than the strains of B. btfidim. Two distinct sites of calcium alginate

beads were used to encapsulate B. infatrtis and B. lortgiim. Pre-incubation of both large

and small beads at 37OC for 18 h increased s u ~ v a l dunng subsequent refiigerated

storage. Survival of B. infartlis in pH 6.3 broth during refngerated storage was

significantiy (P 1 0.05) increased when encapsulated in large beads, while in pH 4.5 broth,

small beads significantly (P 5 0.05) enhanced suMvd compared to free cells.

Encapsulation of B. fottgrrm did not significantly (P > 0 .OS) affect suMval in either broth

when compared to fiee cells.

Acknowledgments

1 would like to express my sincere thanks to members of my advisory cornmittee.

Dr. Linda J. Harris, my imrnediate advisor, for her guidance and support corn afar and for

providing the opportunity to visit U.C. Davis in California. Dr. R. Yada's encouragement

and guidance was always timely and v e q appreciated. The concept of encapsuiation was

introduced to me by Dr. M. Griffiths, and for that, as well as his support, I am grateful.

Special thanks to Diane Wood, whose patience and cornpetence was invaluable to

me dunng the course of my research and writing of this manuscript. 1 have learned a great

deal from Diane and will always treasure her fnendship. Thanks dso to Kelley Leclair for

her fnendship, laughs and support.

I would especially like to thank my partner Sly Castaidi, whose love and suppori

kept me going through the tough times, and without whom 1 would not have enjoyed the

journey 1 wouid like to dedicate this thesis to Sly and to my father, Roy Ellenton, who

passed away during my Master's studies.

This study was funded through a gan t adrninistered by the Ontario Ministry of

Agriculture, Food and Rural AEhirs.

Table of Contents

................................................................................................................. List of Tables iv

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . List of Figures v

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 . 0 Introduction 1

................................................................................................ 2.0 Literature Review 5 ................................................................................. 2.1 Introduction 5

............................................................... 2.2 Morphology of Bifidobacteria - 6 2.3 Irnporfance of Bifidobacteria to the Food Industry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I O 2.4 Survival of Bifidobacferizrm Species in Acidic Environments . . . . . . . . . . . . 12

.............................................. 2.4.1 S u ~ v a l in Acidic Food Products 13 ........................................................ 2.4.2 S u ~ v a l in Gastnc Acidity 18

2.5 Oxygen Sensitivity of Bificioobacteriirm Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.6 Imrnobikation of Bacteria .................................................................. 20

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6.1 Encapsulation in Calcium Alginate Gels 20 ............................................... 2.6.2 Protedion of Encapsulated Ceils - 2 5

....................................................... 2.6.3 Encapsulated Bifidobacteria 30

....................................................................................... 3 . 0 Materials and Methods 31

4.0 Results 4.1 4.2

..................................................................... Strains and Maintenance 31 ................................................................ Enurneration of Viable Cells 31

Effect of Calcium and Sodium on Cellular Morphology, .............................................. Optical Density, and Viable Ce11 Counts 32

Survival of Bifldobactena During Storage Under Acidic Conditions ...................................................................... 32

...................................................................................... Encapsulation 32 ............................................... Solubilization of Beads for Enurneration 35

...... Effect of the Phosphate BuEer Step on Free Cells of Bifidobacteria 35 Effect of CaCOi and RSM Arnendments to Large Beads on Survival of B i~tfmztis .......................................................................... 35 .

.................................. S u ~ v a l of Free and Encapsulated Bifidobacteria 36 Low Temperature SEM and Phase Contrast Microscopy .................... 37 St atistical Analy sis ................................................................................ 38

39 ............................................................................................................. ........... Effect of CaClz and NaCl on Cellular Morphology and Growth 39

..................................... Effect of pH on the Sunival of Bifidobactena 46

4.3 Encapsulation Methods ..................................................................... 5 1 4.3.1 The effect of CaC03 and RSM Arnendrnents

.......................................................................... in Large Beads 51 4.3.2 Effect of t he Solubilization S tep

.................................................................. on Celi Enumeration 54 4.4 S u ~ v a l of Free and Encapsulatecl Bifidobactena . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 4.5 Microscopie Examination of Calcium Alginate Beads ........................... 65

......................................................................................................... 5 . 0 Discussion 71 5.1 Morphology ........................................................................................ 71 5.2 Acid Sensitivity ................................................................................... 74 5.3 Encapsulation ....................................................................................... 75

6.0 Conclusions ...............................................................................................

7.0 Recommendations for Future Research ......................................................

References ...........................................................................................................

Appendix ............................................................................................................. Appendix 1:

Appendix 2:

Appendix 3 :

Raw data including replicates. for suMval of free and encapsulated B . longum ATCC 15707 in MRS broth with adjusted pH treatments. stored at 4°C for 2 1 d .......................

Raw data, including replicates. for suMval of free and encapsulated B . itfar~tis ATCC 15697 in MRS broth with adjusted pH treatments. stored at 4°C for 2 1 d .............................

haiysis of variance for mean log decrease of free cells and encapsulated cells of B . infonlzs and B . Iot~gzirm . both incubated and non4ncubated. over 21 days storage at 4OC in pH 6.3 and pH 4.5 MRS broth ......................................

List of Tables

Table 2.1

Table 4.1

Table 4.2

Table 4.3

lonic polymers and possible counterions used in the preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . of ionotropic gels for encapsulation of whole cells . 2 1

Effect of calcium and sodium on optical density, ce11 nurnbers and cellular morphology for Bifidobacterium spp., incubated

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . anaerobically for 2 1 h at 3 7OC ..)O

EfTect of the phosphate buffer step on viable counts of fiee cells of . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. i17fntltis and B. ïongiïm 56

SuMval of free and encapsulated B. infântis and B. Ion- in . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MRS broth after 2 1 d aorage at 4°C .64

List of Figures

Figure 2.1

Figure 2.2

Figure 3.1

Figure 4.1

Figure 4.2

Figure 4.3

Figure 4.4

Figure 4.5

Figure 4.6

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Chernical structure of alginate 23

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tgg box" mode1 of calcium alginate gel 24

Apparatus for making large calcium alginate beads . . . . . . . . . . . . . . . . . . . . . . . . . . . .34

Phase-contrast micrograph of B. bifihm ATCC 15696. A: Rod morphology p w n in MRS broth supplemented with 0.0 1% CaC12. Bar s 4.0 Pm. B: Branched morphology grown in MRS broth supplemented with 2% NaCl. Bar .c 3.5 pm. . . . . . . . . . . . . . .

Phase-contrast micrograph of B. brfidzim ATCC 2952 1 . A: Rod morphology grown in MRS broth supplemented with 0.0 1 % CaC12. Lumpy appearance of some rods indicated by arrows. Bar = 3 .O p. B: Branched rnorphology grown in MRS broth supplemented with 2% NaCl. Bar s 8.3 pm. ..........................

Phase-contrast micrograph of B. it>Iat~tis ATCC 1 5697. A: Rod morphology grown in MRS broth supplemented with 0.0 1% CaCl*. Bar = 3.3 Pm. B: Branched morphology grown in MRS broth supplemented with 2% NaCl. Bar z 3.3 pm ...................................................................................

Phase-contrast micrograph of B. longi

Figure 4.7

Figure 4.8

Figure 4.9

Figure 4.1 0

Figure 4.1 1

Figure 4.12

Figure 4.1 3

Figure 4.14

Figure 4.1 5

Survival of B. infmtis ATCC 15697 stored at 4°C in MRS broth; pH 6.3 (@ ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (a) and pH 3.7 ( i ) . Results s h o w are the average of duplicate samples fiom each of two separate experiments. ........................................................

SuMval of B. Iotqpm ATCC 15707 stored at 4°C in MRS broth; pH 6.3 (@ ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (u) and pH 3.7 (a). Results shown are the average of duplicate sarnples fiom each

........................................................ of two separate experiments.

Typicd size and shape of calcium alginate beads. A: large beads with 0.5% Tween 80 B: phase contrast micrograph of

......................................................... srnall beads. Bar = 80 Pm.

SuMval of B. injar~tis encapsulated in large beads with RSM and CaCOl and stored at 4OC; pH 6.3 (A), pH 4.0 (B); non-amended control (a), 0.1% CaCO, (O), 1 .OYO CaCO, (O). 3% RSM (m) Results shown are the average of duplicate samples from each of two separate experiments. ........................................

SuMval of B. irfatltis as free celis (@ ), in large beads (a), and in srnd beads (A); stored for 21 days at 4°C in MRS broth with pH 6.3 (A), pH 5.5 (B), and pH 4.5 (C). Results shown are the average of two separate trials. .....................................................

SuMval of B. lot~gr~m as fkee cells (O ), in large beads (m), and in small beads (A); stored for 2 1 days at 4OC in MRS broth with pH 6.3 (A), pH 5.5 (B), and pH 4.5 (C). Results s h o w are the average of two separate trials. .....................................................

Survival of B. itfantis as free cells (@ ), in large beads (i), and in srnall beads (A); pre-incubated in MRS broth for 18 h at 37OC pnor to storage for 2 1 days at 4OC in broth with pH 6.3 (A), and pH 4.5 (B). Results shown are the average of two separate trials. .

Survival of B. longtrm as free cells (@ ), in large beads (i), and in small beads (A); pre-incubated in MRS broth for 18 h at 37°C prior to storage for 21 days at 4OC in broth with pH 6.3 (A), and pH 4.5 (B). Results shown are the average of two separate trials. ........ . 6 2

Phase-contrast micrograph of B. longztm encapsulated in small beads after21 d storageat 4OC. A: pH 6 .3 . B: p H 4 5 Bar= 14.5 p. .......... 66

Figure 4.16 Phase-contras micrograph of B. fongmr encapsulated in small beads and pre-incubated (1 8 h, 37OC). A: directly after pre-incubation B: afler pre-incubation and 21 d storage at 4OC in pH 6.3 broth. Bar = 13.0 W. ..................................................................................... 68

Figure 4.17 SEM micrograph of gel structure of large calcium alginate bead afker ......................................... 2 1 d storage in MRS broth (pH 6.3) at 4°C. 69

Figure 4.18 SEM micrograph of gel structure of large calcium alginate bead and . . . . cell of B. iongum after 21 d storage in MRS broth (pH 6 .3 ) at 4°C. .70

1 .O Introduction

Bifidobacteria are found as part of the natural microbiota of the gastrointestinal

tracts of humans and other warm-biooded animals. While the rnechanisms involved and

extent of their activity is not yet completely understood. there is increasing evidence to

suggea that bifidobactena do contribute to human health (Hughes and Hoover, 199 1 a;

Tarnime et al., 1995). These proposed health benefits have led to the inclusion of

bifidobacteria in more than 70 products world-wide, most of dairy ongin. with an

increasing consumer demand in North Amencan markets (Modler et al., 1990a; Sloan,

1994). It has been suggested, however, that the marketing push for these products has

outpaced both applied and basic research (Modler, 1994).

Early problems of characterization of bifidobacteria were the result of variable

morphologies observed with this organisrn. Bifidobacteria cm exhibit many difFerent forms

of cellular morphology, including rod, globular, bifid (Y-shaped), and highly branched

forms. These unusual shapes are often used to crudely idente and classi5 the organisms,

however, the mechanism responsible is not well understood. It has been suggested that

branched foms are likely the result of incomplete ce11 division due to a lack of specific

nutrients in the culture medium (Glick et al., 1960; Husain et al., 1972; Kojima et al.,

1 WOa).

There are some indications that morphology may affect physiology of

bifidobacteria. For example, Kojima et al. (1970a) reported that celi wd composition

differed between branched and unbranched foms. The arnount of acid produced may also

be dependent on rnorphology (Brown and Townsley, 1970; Husain et al., 1 972; Kojima et

al., 1968). Further understanding of the relationship between morphology and physiology

may aid in culture selection for suNival in food products. The first step. however, will be

to consistently produce branched or rod foms of bifidobacteria.

Branched foms of Brfdobac~eHa btfidm strains have been induced with the

addition of sodium chloride to the culture media and rod forms induced with the addition

of calcium chloride (Husain et al., 1972: Kojirna et al., 1968). However, morphology of

ot her speciedstrains have not been examined with these amendments.

Health benefits of these bifidobactena-containhg products can only be realized if

relatively large numbers of bifidobacteria are viable in the product at the time of

consumption and are able to reach and colonize or grow in the intestine once consumed.

This remains a challenge to food producers because bifidobacteria are relatively sensitive

to acidic and aerobic environments. One of the most popular products for the inclusion of

bifidobacteria is fermented dairy products, such as yogurt. The pH of fresh pguns can

Vary between 4.0 and 4.6, but c m decrease further during storage due to post-production

acidification (Rasic and Kurmann, 1978). However, most species of Brfidobacterizcm are

unable to s u ~ v e below pH 4.6 (Modler et al., 1990a). Research has shown that acid

tolerance is strain dependent, with strains of B. Iongim showing the most promise (Clark

et al., 1993; Lankaputhra et al., 1996; Martin and Chou, 1992; Roy et al., 1995). Recent

surveys of commercial yogurt products for the presence of bifidobacteria have s h o w

varied results, but most products have had low numbers or strains of animal rather than

human origin (Iwana et al., 1993; Lim et al., 1995; Micanel et al., 1997; Shah et al., 1995).

Other fermented dairy products have been suggested for the inclusion of bifidobactena

including fluid milk, fiozen yogurt, cheese, and ice cream. Besides tolerance to product

acidity, bifidobacteria must aiso survive gastnc acidity (pH 1-4) if health benefits are to be

realized from the consumption of these products.

Oxygen sensitivity is another factor afkting sunival of bifidobacteria in food

products. Although bifidobactena are anaerobic organisrns, oxygen tolerance has been

reported to be species dependent (Shimamura et al., 1992). Methods to reduce oxygen in

food products, such as glass packaging, has been suggested to improve suMval of

bifidobacteria (Dave and Shah, 1997; Ishibashi and Shimamura, 1993).

Encapsdation of bifidobacteria in calcium alginate beads may be a method to

enhance survival in food products. Alginate is non-toxic and is an approved food additive.

There are few reports describing protective effects of encapsulation of bifidobacteria in

calcium alginate with regards to pH, oxygen, and temperature. However, bifidobacteria

have been encapsulated successfully in calcium alginate for use in the continuous

fermentation of mik (Ouellette et al., 1994). Rao et al. (1989) encapsulated bifidobacteria

with celiulose acetate phthalate and observed an increase in survival compared to free cells

in a simulated gastnc environment. Buzas et ai. ( 1 989) reported that viability of

Saccharomyces cerevisiae in low pH was enhanced by alginate-encapdation compared to

fiee ceus.

Anaerobic organisms, such as bifidobacteria, rnay benefit fiom encapsulation

because of the anaerobic environment inside the gel bead. Bead diameter can affect the

diffusion of oxygen, with larger beads providing a more anaerobic environment than

smaller beads (Chang and Moo-Young, 1988; Chen and Humphrey, 1988; Ogbonna et al.,

199 1).

Protective effects of microorganisms encapsulated in calcium alginate beads

against low temperatures have also been established. For example, encapsulated

Lactobaciihs h~rigaTins were reported to better survive the freezing and fiozen storage

of ice milk (Sheu and Marshail, 1993; Sheu et al., 1993). These two studies suggest that

encapsulation may be a possible method to enhance delivery of probiotic cultures in food

products.

The objectives of this research were:

1. To develop methods to produce consiaently rod or branched forms of B. itIfatztis, B.

ior~gurn, and two strains of B. hfid(rn.

2. To develop techniques for encapsulation of bifidobactena in calcium alginate beads.

3. To investigate survival of encapsulated bifidobactena during refngerated storage in

acidic and non-acidic environment S.

2.0 Literature Review

2. I Introduction

Bifidobactena are Gram-positive, non-motile, non-sporeforming, anaerobic rods of

variable cellular rnorphology, that are found in the gut of humans and other warm-blooded

mimals. Bifidobacteria were first described in 1899 by Tissier, who introduced the narne

Baciiiirs bificha comrnim~s or simply B. bijldzis (Tissier, 1899). Since that time,

bifidobacteria have been assigned to various genera: Actinomyces, Baciiiz~s~ Bacreriocies,

Bacterit~m. Coryiwbacterizim. Lmtobaciilus. Nocarda, and Tissieria. It was not until the

8'h edition of Bergey's Manual of Determinative Bacteriology (Rogosa, 1974) that

BrfiJobacteriicm was recognized and accepted as an independent genus. There are

currently 24 species identifieci, including the following nine species found in humans: B.

brfiùkm. B. lorqpm, B. infm~tis. B. breve. B. odoiescentis. B. anguhtum. B. coteindatirm.

B. psezrducate!nnrlatum. and B. dendzrm (Biavati et al., 1992).

Application of Brfidobacterium species in foods, pharmaceuticals, and livestock

feed supplements have been developed extensively in the last decade (Modler et al.,

1990a). The increasing interest is due to the proposed health benefits of these bacteria.

However, it has been suggested that the marketing push for bifidobactena-contalliing

produas has outpaced research (Modler, 1994). Clearly there is a need for more basic

research regarding bifidobacteria characteristics and more applied research regarding

successful inclusion in products.

2.2 Morphology of bifidobacteria

Much of the early problems with classification of bifidobacteria were a result of

the varied morphologies observed during isolation and culturing. In 1899, Tissier first

described bifidobactena as curved rods and rods with ends spiit to give the appearance of

two short branches (Tiuier, 1 899). In addition to this characteristic '%ifid shape, variable

morphologies such as complex branc hing, swelling and bizarre distort ions have been

reported. Sundman and Bjorksten ( 19%) observed extreme globuiar swelling of severai

strains of bifidobacteria when grown in tomato agar, but more rod-shaped morphology

with the addition of tryptic digest of cow's milk. Although tomato agar pennitted

abundant growth, it was suggested that it was deficient in one or more substrates needed

for normal ce11 wall synthesis. These authors agreed with other researchers that in their

natural habitat, bifidobacteria exist as straight or curved rods which are sometimes bifid

(Noms et al., 1950; Poupard et al., 1973).

The cause of pleomorphism of the organism was fira addressed by Glick et al.

( 1960). They concluded that the formation of branching and swelling of Bzfidobacre~irm

bzfidttm was due to a deficiency of a, ,B -methyl-N-acetyl-D-glucosaminide, which is an

essential growth requirement and a precursor for ce11 wall synthesis. By gradually

increasing the concentration of this precursor (O to 8%), these bizarre forms were

eventually eliminated leavhg curved rods. They suggested that even the characteristic bifid

form rnight be a consequence of an inadequate supply of this factor.

The induction of pleomorphism in Lactobacilit~s bfldi~s (B. b~fidkm) was snidied

by Kojima et ai. ( 1968). They found sodium carbonate (0.2 M), sodium chloride (0.25-

0.35 M), sodium suiphate (0.20 - 0.35 M), sodium nitrate (0.20 - 0.35 M), tribasic sodium

phosphate (0.10 - O. 1 5 M), and sodium acetate (0.20 - 0.30 M) induced bifid and highly

branched forms. The branching was recognizable after 12 h of incubation and developed

extensively for a subsequent 8 to 10 h. The presence of chiondes of other univalent

cations also induced pleomorphism to various degrees: K' = Na' > m' > Li* > Rb' > Cs' This effect was not observed with divalent chlorides of ca2*. ~ a " . M ~ " , iMn2- or

zn2-.

Further investigation by Kojima et ai. ( 1970a) led to the suggestion that calcium

ions play a principal role in the prevention of pleomorphism in B. hfldztrm. The induction

of pleomorphism by NaCl (0.3 5 M) was completely inhibited by the addition of CaC12

(0 .O 1 -0.02 M). The addition of calcium-chelating agents induced the bifidhranched form

which would revert to rod forms with the addition of CaClz(0.02-0.06 M). The authors

suggested that a relationship exists between calcium ions, ce11 waU components, and the

pleomorphisrn in B. brfidzîm. Kojima et al. (1970b) also studied electron micrographs of

bifidobacteria in both sodium-induced branched forms and calcium-induced rod forms.

Cross wails were observed in the rod form but not in the branched fom. These

observations lend fixther support to the authors' view of the importance of calcium ions in

cytokinesis of bifidobacteria.

Husain et al. (1972) found that addition of sodium chloride (0.35 M) to a complex

growth medium caused multiple branching in a mucoid variant of B. brfdzm. However,

reversal of this effect by addition of calcium ions was not observed as reported by Kojima

et al. (1970a). Further, branching was not reversai with the addition of the following ceil

wall precursors: N-acetyl-D-glucosamine, a - E - diaminopimelic acid, and muramic acid. Branching could be suppressed with the addition of a mixture of four amino acids: alanine,

aspartic acid, glutamic acid and serine. These four amino acids are present in the c d walls

of rod-shaped bifidobacteria (Veerkamp et al., 1983), however, their absence has not yet

been demonstrateci in highiy branched foms.

Morphological dserences have been observed in a mucoid variant of B. bIfidzcm

when it was grown in two different media (Husain et al., 1972). When this organism was

grown in a complex medium which was only partiaily defined chernically, the organism

appeared as curved rods, some with bSd ends. In contrast, when grown in a defined

minimal medium, the cells were pleomorphic and so profusely branched that they lost dl

resemblance to rods. Both morphological foms of this organism were maintained on

repeated nibculture, indicating that branching was not a degenerative phase preceding

deat h.

Morphology, as it relates to ce11 reproduction, has been investigated more recently

Several papers by the research team of Novik & Vysotskü (1996a, b) have proposed the

cycle of development of bifidobacteria populations is as follows: transient forms (rod and

coccoid) to budding and branching rnultiseptate forms to products of multiseptate

fiagmentation. They too found the morphology dependent on the composition of the

nutrient media. However, these authors did not consider medium composition, nor

changes in the physiochernical environment, to be main determinants of the morphological

variabiiity. Instead, it was suggested that budding and branching was a genetically-

determineci process to maximize the reproductive potential of bifidobacteria. There is

evidence contrary to this statement in that other researchers have b e n able to maintain

either branched or rod shape over prolonged periods of tirne (Husain et al., 1972).

Although the mechanisms responsible for the varied morphology of bifidobacteria

are unclear, there are indications that the physiology and growth characteristics of

branched and rod morphologies are different. Kojima et ai. (1970a) found that the

compositions of sugars and amino acids in the cell wall were different in the branched and

rod forms. Glucose and the total arnount of sugars were more abundant in the branched

form but undetectable in the rod form; methionine and phenylalanine were present in the

branched but not in the rod form. Ce11 walls of the branched form also lacked a larger

molecular weight peptidoglycan which was detected in the rod form. Further, the calcium

ion content in intact cells and ceU walls was lower (82%) in the branched form than in the

rod form. Kojima et al. ( 1968) and Husain et al. ( 1972), found that acid production was

decreased when the organism was in the branched fom. Lower acid production was

accornpanied by decreased viable cell numbers compared to the rod fom. Brown and

Townsley ( 1970) observed a 60 to 225% increase in lactic acid production when the

bifidobacteria strains changed from branched to rod shape.

Variable cellular morphologies have also been observed in other Gram-positive

baciili. Growth media with deficiencies in deoxyribonucteic acid, vitamin BI2, and vitamin

B6 have resulted in filament formation in various lactobacilli species (Deibel et al., 1956;

Holden and Holrnan, 1957; Jeener and Jeener, 1952; Starner, 1979). Wright and

Klaenhammer ( i 98 1 ) observed a morphological transition fiorn filamentous to bacilloid

rods of Lactobucilhs acidophiIzis with the addition of 0.1 % of either calcium chlonde,

calcium carbonate, or calcium phosphate. The rod fom was found to be more resistant to

the fieeting process and çubsequent fiozen storage. The growth and morphology of

Lactobaczllus bzdgaricus is influenced by the calcium, magnesium, and manganese content

of the growth medium (Wright and Klaenhamrner, 1 98 1 ). Media deficient in divalent

cations would not support growth of this organism. The addition of magnesiurn resulted in

long chains of cells. W~th increasing concentrations of calcium and manganese the

morphology transfonned to short rods. Wright and Klaenhamer ( 1984) also found that the

addition of phosphate ( I or 2%) to milk growth medium produced long tangled chains of

L. bulgmiais cells. The phosphate acted as a calcium chelator, which not only affected

cellular morphology but decreased growt h and acid production of this organism.

The variable morphology of Midobacteria, as well as lactic acid bacteria, needs to

be hrther investigated. m e n morphological observations are used as an identification

tool, or shply arise as a consequence of other research objectives. This was illustrated by

Ventiing and Mistry ( 1993 ), who noted a change of morphology with their bifidobactena

cultures while studying the growth characteristics in skim and ultrafiitered milk. While rod

or Y-shapes in culture broth, the bacteria from the skim and ultrafiltered milks appeared as

short, rounded rods in chains. Undersandhg the mechanism by which morphological

changes occur and the effects that these changes have on culture performance may aid in

cuiture selection and preparation for food producers.

2.3 Importance of bifidobacteria to the food indus-

Over the past decade there has been an increase in the number of products

containing B~fdobacterium spp. (Hughes and Hoover, 199 1 a; Laroia and Martin, 1990;

07Sullivan et al., 1992; Sloan, 1994). Most are of dairy origin and include products such

as yogurt, buttemiilk, Sour cream, powdered miUc, fortined mik, cookies and &ozen

desserts (Hughes and Hoover, 199 1 b). Development and use of bzdobacteria-containing

food products is most widespread in Iapan which currently produces over 70

bifidobactena-containing products (Modler et ai., 1 99Oa). Products containing

Lactobacrlhs acidophilrrs and Blfidobacterirrm species have captured 4% of the total

fiesh rnik sales in France, with 1 1% of ail yogurt containing added bifidobacteria. They

also account for approximately 25% of the fermented milk products' market in Sweden

(Hughes and Hoover, 199 la). Probiotic yogurts represent more than 7.5% of the

Australian yogurt market (Kailasapathy and Rybka, 1997). In North America, demand for

products containing active cultures was recently cited as one of the top ten food

processing trends (Sloan, 1994).

The worldwide interest in bifidobacteria-containing produas is based on the

proposed health benefits of these "probiotic" bacteria. Havenaar and Huis in't Veld

( 1992) defined probiotics as " a mono- or mixed culture of live microorganisms which,

applied to humans or animals, affects beneficially the host by improving the properties of

the indigenous microflora". Strains of laciobacillus acidophilm and species of

BîfidobcterMn fa11 into this categoiy and have long been thought to benefit human

health. Although not al1 affects have been proven conclusively, the foilowing benefits have

been attributed to bifidobacteria: (i) maintenance of a normal intestinal rnicroflora, (ii)

improvement of lactose intolerance, (iii) stimulation of the immune syaem, (iv)

anticarcinogenic activity, (v) reduction of serum choiesterol levels, (vi) synthesis of B-

complex vitamins, (vii) cornpetitive exclusion of pathogenic bacteria (Hughes and Hoover,

1991a; O'Sullivan et al., 1992; Tamime et al., 1995).

The success of probiotic foods depends on the ability of the culture to survive until

tirne of consumption, and be able to reach and colonize or grow in the intestine once

consumed. Specificaily, it has been suggested that the following criteria be met to achieve

potential therapeutic benefits of bifidobacteria-containirig products (Sarnona and

Robinson, 1991; Tamime et al., 1995):

1. Regular consumption of the bifidobactena-containing product, and at a level of

approximately 400-500 g product/week;

2. A minimum bifidobactena population of 1 O x 1 o6 viable celldg of product at the time

of consumption; and

3. The species of Bzfidobactericcm used is of human origin, and able to withstand transit

through the upper regions of the digestive tract.

One of the greatest challenges facing food producers is maintaining viability of

bifidobactena in the food product until the time of consumption. Bifidobacteria species are

relatively intolerant to acidic environrnents, such as in yogurt (Modler et al., 1990a). In

addition, bifidobacteria are anaerobic organisms, making them more difficult to cultivate

and to ensure sunrivai in foods.

2.4 Survival of B i f z d o k ~ t ~ u r n species in acidic environments

The optimum pH for bifidobacteria is 6.5 to 7.0, with little or no growth below pH

5.0 (Scardovi, 1986). Moa species of Brfidobacterizrm will not survive below pH 4.6

(Modler et al., 1990a), however, acid tolerance appears to be strain dependent.

Lankaputhra et al. (1996) studied the survivd of nine strains of bifidobacteria in

reconstituted skirn d k media adjusted with lactic acid to pH 3.7,3.9,4.1 and 4.3. Only

three of these strains suMved well(>l o6 CNIrnl) under al1 pH conditions after six weeks

of storage at 4OC. Two of these three strains were of human origin (B. injûntis, B.

longum), while the other strain, B. psetidolongum, was of animal ongin. The viability of

the other six strains declined as the pH was decreased; d e r 18 days, < 10 CFUIrnl

remained viable at pH 3.7, and &er 36 days, 4 0 CFUIml remained viable at pH 4.3.

2.4.1 Survival in acidic food products

Since yogurt is the most common food vehicle for inclusion of bifidobacteria

research has focused on the s u ~ v a l of these organisms in this acidic product. Martin and

Chou ( 1992) studied survival of eleven strains of bifidobacteria in a low pH yogun (pH

4.1-4.2) and a hi& pH yogurt (pH 5.5-5.6) . There was a 1.4 to 4.0 log-cycle reduction

over 60 days of refngerated storage in the low pH yogurt. Although the rate of decline

varied &om strain to strain, seven of the strains exhibited a sharp decrease in nurnbers

during the fist week and al1 strains had viable counts of

strain, they found that three strains s u ~ v e d well(1 o6 to 10' CFUIg) during 35 days of

refhgerated storage. The fourth strain decreased to ~ 1 0 ' C N / g after only 5 days of

refngerated storage. The speciedstrains were not identified. In contrast, Sarnona and

Robinson ( 1994) found that cornrnerciaily available strains of bifidobacteria (B.

addescentis. B. hflakrn. B. Iongurn) ail maintained viability (> 1 o6 CFU/g) in laboratory-

produced yogurt (pH 4.6) over 21 days of refigerated storage.

Other researchers have surveyed commercialiy available yogurts claiming to

contain viable bifidobacteria. Of eight European commercial yogurts tested, only four had

viable counts of bifidobacteria > 1 o6 C R l I g (Iwana et al.. 1 993 ) . Of the remaining four,

one had a count of 5 x lo4 CFU/g and no bifidobacteria were detected in the other three

yogurts. Further, of the five bindobacteria strains isolated from the commercial yogurts,

only one was classiiïed as B. l o n p and the remairing were identified as B. m~rmalrs.

Since B. un~malis is of animal origin, it may not provide human health benefits and is not

recommended for inclusion in bifidobactena-containuig food produas.

Micane1 et al. ( 1997) investigated the viability of bifidobacteria in three commercial

yogurts sold in Australia. One sample maintained high levels (Ho6 CN/g) through two

weeks post-manufacture refiigerated storage. Another sample had lower initiai counts ( 1 o5

C m ) which declined sharply to

other three products had counts < 1 o3 CFU/g. Al1 the products showed a constant decline

during five weeks of refiigerated storage.

Reuter ( 1990) surveyed yogun products fiom the German, Japanese, and French

markets. He found that s u ~ v a l of Brfidobacterium spp. was related to strain, pH, length

of storage, and presence of other cultures. In general, as pH decreased and storage time

increased, the viability of bifidobacteria decreased. Of eight products tested within the

declared shelf life fiom the French market, al1 had counts > 1 o6 CFü/g; however, five of

the eight species were identified as B. ariirn~ks, the other three were B. fungum. Yogurts

obtained from the Japanese market had counts of bifidobactena c l o6 CW/g for three out

of six sarnples. However, the random sampling was done in 1980, and the storage time

was not equai among the products sampled.

Seven commercial Korean yogurts were examined for viable bifidobacteria at the

end of their respective declared shelf life (Lim et al., 1995). This study was included as

part of their evaluation of a selective medium for enumerating bifidobactena in fennented

products containing both lactobacilli and streptococci. Sumival of bifidobactena was

generally good; only two of the seven products had bifidobactena counts

high acid yogurts. Low acid sensitivity has hampered the success of delivenng these

probiotic cultures to consumers. Yogurt is a popular product for the inclusion of

bifidobactena due to consumer familiarity with the presence of bactena in these products.

However, other da j r products such as fiozen yogurt, ice cream, cheese, and fluid milk

have also been investigated as vehicles for delivery of probiotic bifidobactena.

The acidity of cornmercialiy produced frozen yogurt was found to Vary from pH

4.75 to 7.03 (Brown et al., 199 1). Modler and Villa-Garcia ( 1993) found poor survival of

B. l o n ~ m in hi@-acid (pH 4.47) frozen yogurt mix: after 1 1 weeks storage at -30°C, the

bifidobacteria in the hardened mix had declined three log cycles, and a Further 1.5 log

decline occurred after whipping and fieezing of the mix. They compared this treatment

with survival in a low-acid yogurt (pH 5.85) in which the bifidobactenal coums decreased

by only one log cycle in the hardened rnix after 1 1 weeks storage, with a further decrease

of one log cycle afler freezing. These results were similar to Laroia and Martin ( 199 1 )

who reported a decrease in B. bzfidm of less than one log cycle in fiozen yogurt (pH 5.6-

5 3) after 8 weeks of storage at -29OC. However, these researchers found no sumival of B.

bifid~m in a low-pH (3.9-4.6) frozen fermented dairy dessert directly &er fieezhg.

Cultured ice cream, made by fermenting (to pH 4.9) a standard ice cream mix with

L. acidophiltrs and B. bifidum, followed by freezing, has also been studied (Hekmat and

McMahon, 1992). The bifidobacteria strain survived very well over 17 weeks storage at

-29°C with only one log cycle reduction being observed. These results were comparable to

the study done by Christiansen et al. ( 1996). In this case, commercial cultured milks,

fermented with L. uczdophilt~s and B. brfichim, were added to the ice cream mix. The

resulting frozen product had a pH of 5.8. M e r 16 weeks of £?ozen storage, the viable

counts of bifidobacteria were approximately 10' CFü/g. Similar results have been found

for non-cultured ice cream, where 90% survival of bifidobacteria was achieved over 70

days fiozen storage (Modler et al., l99Ob).

Cheese is another food product that has been investigated for delivery of probiotic

bifidobacteria. Ghoddusi and Robinson ( 1996) studied the survival of B. b~fidirn and B.

adolescentis in white bnned cheese (pH of 4.6-4.9) made with either cheese cultures or

yogurt cultures. M e r 30 days storage at 14- 1 5OC, B. adolescentis decreased by 4 log

cycles in the presence of the cheese culture and 3 log cycles in the presence of the yogurt

culture to finai counts of IO' and 10' CFU/g, respectively. B. brfidkrn suMved better; in

the presence of cheese culture, lo4 CFU/g was observed and in the presence of yogurt

culture 105 C N / g was observed after 30 days storage. Factors other than acidic

conditions may have influenced these results. For example, the temperature was higher

than refrigerated temperatures (4OC) to enhance flavour development, and the salt

concentration of the brine may have affected s u ~ v a l of bifidobactena. Sanders et ai.

( 1996) observed stable populations of B. lot~gz~m in fluid milk at both 4 and 10°C over 2 1

days storage, but Dave and Shah ( 1997) noted a decrease in bifidobacteria (unidentified

strain) suMvai in yogun stored at 10°C compared to 4OC. Further, Blanchette et al.

(1995a) found that salt had a detnmentai effect on the s u ~ v a l of bifidobactena in cultured

cottage cheese dressing.

Fifieen strains of bifidobactetia were evaluated for their survival under cheese-

making conditions (Roy et al., 1995). Milk inodated with both lactococci and

bifidobacteria reached pH of 5.1-5.4 after fermentation and pH 4.94.6 &er 28 days of

storage at 4OC. Bifidobacteria counts d e r this storage penod varied fiom 1 o2 to 10'

CRlIml, with strains of B. longum showing the best sumival.

Other research indicated that bifidobacteria s u ~ v e d well in commercially

fermented milk under refngerated storage (Medina and Jordano, 1994). In this studv the

strain of bifidobacteria was not identified, however, aiter 28 days of aorage at 7°C in milk

of pH 4.3, the counts of bifidobacteria were still >106 CFUIrnl. In contrast. Berrada et al.

( 1 99 1 ) reported a decrease in bifidobacteria population in one commercially fermented

milk (pH 4.5) from 10' CFU/mi to less than lo3 CFUIml during 24 d storage at 6°C. and

no decrease ofbifidobacteria in a second milk (pH 4.4). The specific speciedstrains were

not identified.

2.4.2 Survival in gastric acidity

To justie the use of bifidobacteria as a probiotic in a food product, the organisrn

must possess the ability to s u ~ v e the acidic conditions of the human stomach. It has been

reported that tolerance of bifidobacteria to gastric acidity may be strain specific (Berrada

et al., 199 1 ). Of two commercial Brjîdobacteritim strains used in this study, one survived

with > 10' CNIml d e r 90 min exposure (37OC) to pH 3. The other strain decreased fiom

approxirnately 10' to 10' CFü/ml within 5 min exposure. Similar results were found with

an in vivo study of the sarne bifidobacteria strains. The species of bifidobactena used in

their study, were not identifiecl. In another in vitro study, Clark et al. (1993) reported that

survival of bifidobactena exposed to simuiated stomach acidity were species/strain

dependent. The survivai of four mains of bifidobacteria (B. adolescentzs, B. irzjimtis, B.

bzjkhum, and B. longum) in distilled water acidified to pH 1 .O, 2.0, and 3 .O with HCI were

evaluated over a penod of 3 hours incubation at 37OC. B. longrrm decreased less than one

log cycle at both pH 2.0 and 3 .O, and was more resistant to pH 1 .O than any of the other

strains tested. This strain was coosidered to be the best choice for use as a dietary adjunct

in dairy foods. Pochart et al. ( 1992) reported a significant decrease in numbers of

bifidobacteria exposed to pH 2.0 and no suMvai at pH 1.0 &er one hour incubation.

They did not, however, observe any significant decrease in numbers of bifidobacteria at

pH 3 .O, even after three hours of incubation. Rao et al. ( 1989) reported that when B.

pseudolongum was exposed to a simulateci gastnc environment (pH 1.33, 3 7°C) for 1

hour, none of the organisms survived.

2.5 Oxygen sensitivity of Bimbacterium species

Since bifidobacteria are anaerobic organisms, the presence of oxygen is a critical

factor for their suMval in food products. Tolerance to oxygen varies fiom strain to strain.

B. infmztis, B. breve and B. longrm were less sensitive to oxygen than B. adolescentzs

(Shimamura et al., 1992). During the production of dairy products, such as yogurt, oxygen

can become dissolved in the product. To avoid this problem, it has been recommended

that Sneptococczcs salivurius subsp. thennophiItcs be included to utilize oxygen in the

product (Shigeo et al., 1984). Oxygen pemeability of the packaging material may dso be

a factor. Dave and Shah (1997) found that the s u ~ v a l rate of bifidobactena in yogurt

packaged in g las bottles was 30070% higher compared with plastic cups. Similar results

were found by Ishibashi and Shimarnura ( 1 993), who observed a correlation between

oxygen pemeability of the package and viability of bifidobacteria during dorage.

2.6 Immobilization of bacteria

tmmobilization can be defined as any method that limits the free movernent of

cells. There are two general types of immobilization: (1) attachent and (2) entrapment.

Attachent techniques include methods where the microorganisms adhere t o a surface

through adsorption or covalent bonding, or adhere to other rnicroorganisrns through

flocculation and cross-linking of cells. Entraprnent is the physical restraint of cells within a

porous material, such as a sponge or fibrous substance, or by encapsulation within a

matrix or polymer-gel. The properiies and applications of the various methods of

irnmobilization have been addressed in several reviews (Dervakos and Webb, 199 1 ; Klein

and Vorlop, 1985; Kolot, 1988; Scott, 1987; Tarnpion and Tampion, 1987). This review

will focus on encapsulation, in particular calcium alginate gels, and applications specific to

bifidobacteria.

2.6.1 Encapsulation in calcium alginate gels

Encapsulation of viable whole celis can be accomplished with ionotropic gelation

of polymers. The most well-known exarnple is the calcium alginate gel, but other polymer-

comterion systems cm be used as shown in Table 2.1.

Formation of the gel network is based on an ionic cross-linking of polyanions or

polycations with multivdent counterions. The characteristics of the resulting gels are

dependent upon a number of factors, most importantly the properties of the carrier

material. This review wiil focus on alginate as the carrier material.

Table 2.1: Ionic polymers and possible counterions used in the preparation of ionotropic gels for encapsulation of whole cells. Adapted fiom Klein and Vorlop ( 1985).

Poly-ions Counterions

C O j Alginate Ca '-, Al '-, Co 2 - . . . Pectinate Mg2*,Ca2- . . Carboxymethylcellulose Ai 3 - . . . Carboxymethylguar gum Ca 2-, Al " . . .

Som3 Carrageenan K ', Ca " Cellulose sulphate K -

N H - 3 Chitosan Polyphosphate

Alginates are produced mainly by brown algae such as Laminaria hyperborea, L.

di@tuta and Macrocystis pyrrfra. but also by certain bacteria such as Azotobacter

vinelandji and several Psetrdomoms species (Smidsrod and Skjak-Braek, 1990). Alginates

are linear polymers of P(1.4) -D-mannuronic acid (M) and a (1,4)-L-guluronic acid (G)

monomers. Polymerized chains consist of blocks of homopolymeric regions (-M-M-, and

-G-G-) of various lengths, and regions of altemating structures (-M-G-M-G-) (Figure

2.1). Monomer composition, arrangement and chah length Vary according to the source

of the alginate.

A cross-linking network is formed when calcium ions are introduced and bond

primady with the polyguluronic acid portions of the polymer chain, forming junction

zones. This arrangement has been termed the "egg box" mode1 (Figure 2.2) (Grant et al.,

1973). In general, alginates with a high content of G blocks provide high mechanical

stability, hi& porosity and tolerance to salts and chelating agents, but alginates with a low

content of G blocks form sofler and more flexible gels (Smidsrod and Skjak-Braek, 1990).

The traditional method for bead formation, using a syhge and needle to drop the

cell-alginate dispersion into CaC12 solution, results in beads 0.5-3.5 mm in diameter (Klein

and Vorlop, 1985). Much smaller beads sizes (2 to 120 pn) have been made using special

devices or other techniques (Klein and Voriop, 1985; Sheu and Marshall, 1993; Stormo

and Crawford, 1992). Typically, the concentration of sodium alginate cm Vary between 1 -

8% (w/v) and the CaClz between 0.05 and 2.0% (w/v). In general, calcium alginate beads

have pore Nes ranging from 5 to 200 nm in diameter (Smidsrod and Skjak-Braek, 1990).

--guluronic acid - - guluronic acid -------- mannuronic acid ------ mannuronic acid ---.

Figure 2.1 : Chernical structure o f alginate

uluronate blocks /calcium

I

1 1

((2 a . . . m . a . . . a .

. e . e . e . a . . .

mannuronate blocks, alternating regions .

Figure 2.2: T g g box" mode1 of calcium alginate gel

The procedure for encapsulation of rnicroorganisms in calcium alginate beads is

relatively simple and is considered mild as it is temperature-independent (O to 80°C) and

non-toxic. Another advantage of using calcium alginate is the rwersibility of

encapsulation; alginate beads can be easily solubilized by sequestenng calcium ions by

immersion in phosphate buffer, which releases cells for fùrther study. Calcium alginate is

also relatively cheap and is GRAS (Generally Recognized As Safe) for use in food

products. There are many applications of alginate-encapsulated rnicroorganisms including

the production of pharmaceuticals, ethanol, enzymes, and ot her chemicals using

bioreactors, as weli as wastewater treatment and pollutant biodegradation (Cassidy et al.,

1996; Scott, 1987). Food and beverage applications include the use of encapsulated

microorganisms in cheese, wine, and beer production (Champagne and Cote, 1987; Scott,

1987).

2.6.2 Protection of encapsulated ceiis

The benefits of encapsulating cells in calcium alginate beads are related directly to

their end-use. For bioreactor systems, the advantages of imrnobilized celis over fiee celis

include enhanced biological stability, high biomass concentration, increased product yields,

increased product stability, and the ability to separate and reuse cells (Dervalcos and

Webb, 199 1). The benefits of encapsulating bifidobacteria are related to enhancing their

sumival in food products by providing protection against acidic and aerobic environrnents,

and low temperatures.

The acidity of the surroundhg medium can affect the calcium alginate gel and the

celis encapsulated within it. In particular, lactic acid can cause soflening and dissolution of

the beads (Roy et al., 1987). In this study, L. hehetims was encapsulated in calcium

alginate beads which were then used in a packed bed system for the production of lactic

acid fiom whey permeate. The pH of the columns ranged from 5.9 at the idet to 3 7 at the

outlet. Plugging of the columns was problematic and was attributed to sottend beads and

cell leakage. Lactic acid, dong with other cation-chelating agents such as phosphate and

citrate cause instability of the gel due to decalcification. In contrast. Martinsen et al.

(1992) found that calcium alginate gel strength was constant between pH 3 and pH 9,

however, the agent used to acidie the medium was not mentioned. Yoo et al. ( 1996)

found that replacing the calcium with barium in alginate gel systems greatly enhanced the

chernical and physical stability of the gels in phosphate and lactate solutions. Although

bariurn alginates were more resistant to chelation, it would not be acceptable for use in

food products.

Encapsulation may provide some protection to cells against acidic environrnents.

Buzas et al. ( 1989) reported îhat the fermentation capacity of Saccharomyces cerevisiar

free cells was dependent on pH with pH 4 being optimum, whereas the fermentation

capacity of alginate-encapsulated cells was independent of the hydrogen ion concentration

between pH 2.5 and 6.2. Further, viabiiity of the encapsulated cells was relatively stable

between pH 1.5 to 6 compared to a decrease in viability of free cells below pH 3.5.

Similarly, the range of pH over which encapsulated K'zcyverornyces manianus and

Z'omonas mobilzs exhibit optimal ethano1 production was reported to be broader in

cornparison to fiee cells (Bajpai and Margaritis, 1986; 1987).

Rao et al. (1 989) studied the sumival of encapsulated B. pseudolot~gm in

sirnuiated gastric and intestinal juices. in this in vitro study, the fieeze-dried bifidobacteria

was encapsulated in cellulose acetate phthalate, followed by coating the resulting

microspheres with beeswax. When subjected to 30 min of simulated gastric juices (pH

1.3 3; 3 7OC), foilowed by incubation in simulated intestinal juices (pH 7.43; 3 7OC), the

encapsulated bifidobacteria survived better ( lo6 CFUIml) than fiee cells (< 1 o3 CFU/ml).

These results are similar to those reported by Kim et al. ( 1 988) in which celis of L.

plmtmim, encapsulated in polyvinyl acetate phthalate, maintained viability dunng 6 hours

exposure to gastric acidity (pH 2) while uncoated celis decreased rapidly in viability.

Arnendments to calcium alginate formulations may provide some protection

against environmental stresson such as low pH. Skim miik powder has been successfùlly

added to bead formulations to increase s u ~ v a l of encapsulated bactena which were

subsequently air-dned or fieeze-dned (Bashan, 1986; Fages, 1990; Kearney et al., 1990).

Not only can the skim rnilk act as a dehydratiodcryoprotectant, it can also provide

nutnents to the encapsulated cells and offer additional protection to cells during

rehydration. For example, lyophilized encapsulated beads with skim miUc that contained L.

platztmwcm showed greater recovery than free cells when rehydrated in a wide range of pH

values (pH 3-7) (Keamey et al., 1990). The addition of calcium carbonate to bead

formulations has been shown to aid in maintainhg firmness of lactococci-containing

calcium alginate gels used in the production of lactic acid (Morin et al., 1992). Besides

afïecting gel integrity, calcium carbonate promotes growth of lactic acid bacteria through

its bdenng capacity and generation of COz.

Amendments such as skim miUc and calcium carbonate may aid in regulating pH

within beads in which acid is produced. Although lactic acid cari d f i s e through calcium

alginate beads, the rate of diffusion may be slow enough to create a pH gradient. Roy et

al. (1987) reported that pH inside beads containing L. helvetim may be lower than the

surrounding medium due to the lack of convection flow of medium inside the beads.

However, confimation of interna1 pH differences was not carried out. Begin et al. (1996)

also reported a difference in pH within the alginate beads containing Propior~ibacterium

shermonii, and the environment outside the beads. This dierence was not measured but

was assumed to be present due to the preferential fermentation patterns exhibited by this

bacteria and the diffusion of substrates into the gel.

Diffision of oxygen into beads can also be an issue; the utilization of encapsulated

aerobic microorganisms in bioreactions cm be hampered by the lack of oxygen in the

microenvironment of the beads. For anerobic microorganisrns, such as bifidobactena, this

can be an advantage of encapsulation. One of the moa important factors concerning

oxygen availability for encapsulated microorganisms is the bead size (Enfors and

Mattiasson, 1983). Critical bead diameter theories have been discussed in the literature

(Chang and Moo-Young, 1988; Chen and Humphrey, 1988).

Ogbonna et al. ( 199 1 ) concluded that reducing bead size to 0.2- 1.2 mm in

diameter was a more effective means of ensuring oxygen supply to encapsulated beads

than oxygenation of the bulk medium, or in situ oxygen generators incorporated in the

m a h . Stormo and Crawford (1992) prepared micro beads 2-50 pm in diarneter which

minimized oxygen limitation for Fiavobacreriwn cells. Using the rate of degradation of

pentachlorophenoi (PCP) as a measurernent of cellular activity, tbey reported that activity

of celis encapsulated in aiginate micro beads were similar to fiee cells, but cells

encapsulated in large alginate beads (2-3 mm diameter) were much less active. Similarly,

Trarnper et al. (1983) reported that efficiency of glucose oxidation by alginate-

encapsulated Glirconobacter q d m s was decreased with increasing bead diameter,

suggesting that oxygen was less available in larger beads.

Encapsulation may provide some protection to cells with regards to temperature.

Temperatures between O and 80°C do not affect the alginate pore size, which may also

provide stability with regards to diffusion (Nguyen and Luong, 1986).

Aldercreutz ( 1985) reported that oxidization rates of glycerol by alginate-

encapsulated G. oxydm~s cells was similar between 25 and 50°C, but rapidly decreased for

free cells between these temperatures. Dried cells of L. plantmm that were coated with

alginate or carboxymethylcellulose were more resistant to increases in temperature (Kim et

al., 1988). In this case, uncoated ceUs and coated cells maintained viability at 4°C over 5 1

days storage, but at 22OC the uncoated cells los viability more than 40-fold compared to

coated after 15 days storage. With increased storage temperatures (32 and 37"C), coated

celis were more stable than uncoated cells.

Low temperature stability has been reported for alginate-encapsulated lactobacilli

(Sheu and Marshall, 1993; Sheu et al., 1993). Two strains of Lactobacilltls btrlgancus

were encapsulated in calcium alginate beads with resulting mean diameters of 25-35 p.

The beads were added to ice milk mixes, fiozen in an ice crearn fieezer, and subsequently

stored at -20°C for 14 days. Approximateiy 40% more lactobacilli surviveci when

encapsulated than did fiee ceils (Sheu and Marshall, 1993). A subsequent study confirmed

the positive effect of encapsdation on swival during fieezing and frozen storage (Sheu et

al., 1993). In this study, approximately 90% of the encapsulated cells survived throughout

14 days storage whereas only 40% of the unencapsulated cells survived the fkst 2 days of

storage. These two studies are of particular importance to the advancernent of using

encapsulation for delivery of probiotics in food products.

2.6.3 Encapsuiated bifidobacteria

Very few studies have focused on encapsulation of bifidobacteria for their use in

food products. As previously mentioned, Rao et al. ( 1 989) encapsulated B. pse~rdolongzrrm

with cellulose acetate phthalate which suMved simulated gastric environment in larger

numbers than unencapsulated bacteria. The species used in this experiment, however, was

of animal origu5 and therefore, an uniikely candidate for human probiotics. ûther

encapsulation carriers such as K-carrageenan-locust bean gum have been evaluated for

encapsulation of bindobactena (Ouellette et ai., 1994). From this study, it was reported

that encapsulated B. infmtis could be used successfuily in the continuous fermentation of

mifk in a stirred tank reactor. S u ~ v a l of numbers of bifidobactena in the beads was

constant, which is not surprising considering that temperature, pH, and anaerobic

conditions were adjuaed to be optimal for bifidobacteria.

Bifldobacteria have also been encapsulated or coated, in butteroil and then used in

the production of fiozen yogurt (Modler and Villa-Garcia, 1993). The encapsulation

treatment, however, was ineffective in reducing the loss of ceils that was attributed to the

high acid environment of the yogurt (pH 4.5-5.9).

To date, no studies have evaluated survival of alginate-encapsulated bifidobacteria

under environmental stress such as low pH, temperature, and aerobic environrnents.

3.0 Materials and Methods

3.1 Strains and maintenance

The organisms used in this study were four strains originally isolated from humans:

Bifidobacteriwn brf dm ATCC 1 5696 (Amerkan Type Culture Collection, Rockville.

MD), Blfidobac~eritrrn bifiatum ATCC 2% 2 1. B$kiobactenum infantis ATCC 1 5697,

and Bijidobacterium Iongum ATCC 15707. Cuitured organisms were stored at -60°C in

Lactobacilii MRS broth (Difco Laboratones, Detroit, MQ supplemented with 15%

glycerol (Fisher Scientific, Fairlawn, No. Unless othenvise noted, pnor to each

experiment, the mains were subcultured three times (1% transfer) in 10 ml of f?esh MRS

broth supplemented with 0. 050h L-cysteine HCl (Fisher Scientific) and 0.0 1 % CaC12. Hz0

(Fisher Scientific) and incubated at 3 P C for 18-22 h under anaerobic conditions

(GasPakB 100 Anaerobic System, BBL Microbiology Systems, Becton Dickinson and

Co., Cockeysville, MD).

3.2 Enunieration of viabte ceUs

Celi counts were obtained by making appropriate serial dilutions of each sample in

0.1% stede peptone (Difco Laboratones), foliowed by plating on supplemented MRS

agar using the spiral plating rnethod (Mode1 D, Spiral Syaem Instruments, Cincinnati,

OH). Plates were incubated anaerobically for 48-72 h at 3 7°C.

3.3 Effect of calcium and sodium on cellular morphology, optical density and viable

ceU counts

Each strain was transferred (1%) into MRS broth supplemented with either 0.01%.

0.5% CaC12, 1%, 2%, or 3% NaCl (Fisher Scientific), or 0.5% CaClz plus 2% NaCI.

Following anaerobic incubation for 2 1 h at 3 P C , the optical density (OD) was determined

at 560 nm using a Novaspec II spectrophotometer (Biochrom Ltd, Cambridge, UK).

Viable cell counts were determined using the enumeration method described above.

Cellular morphology was examined using phase contrat microscopy (Nippon Kogaku

K.K.. Tokyo, JPN).

3.4 Survival of bifidobacteria during storage under acidic conditions

To evaluate the s u ~ v a l of bifidobacteria under acidic conditions, active cultures

were transferred ( 1%) into supplemented MRS broth that had been adjusted to pH 3.7,

4.0,4.3, and 4.6 with lactic acid (85%; Fisher Scientific). The control broth treatrnent was

pH 6.3. The cultures were stored at 4°C for 3 weeks. Samples were taken at O, 1, 2, 3, 4,

6,9, 13, 17 and 2 1 days and enurnerateci according to the method described above.

3.5 Encapsulation

A 50 ml sample of an 18 h culture was centrifuged (4000 x g, 10 min at 4°C;

Sorvall RT6000, Dupont) to form a pellet. The pellet was washed with sterile 0.1 %

peptone pnor to encapsdation in either large or smd beads.

Lmge Be&: The cell pellet was resuspended in 50 ml of a mixture consisthg of

reconstituted skim rnilk (RSM) (commercial grade; 12% stock solution, autoclaved at

1 16°C for 10 min), sodium alginate (Sigma Chernical Co., St. Louis, MO; 4% stock

solution, autoclaved at 12 1°C for 15 min), and Tween 80 (Fisher Scientific) with a final

concentration of 3%, 3%. and OS%, respectively. The beads were formed by dropping the

cell-alginate sluny into a 0.2 M CaC12 solution fiom a blunt-ended syringe needle (22GA

lin; Kontes, NI). A vacuum was applied to this syaem according to the method of

Cassidy et al. (1 995)(Figure 3.1 ) The beads were allowed to harden in the CaCIZ soiution

for 30-45 min before filtering and washing with sterile water.

S d Beactr: An adaptation of the method of Sheu and Marshall ( 1993) was used to

encapsulate the ceus. The ce1 pellet was resuspended in 6.25 ml of a sterile 12% solution

of RSM. Sodium alginate ( 1 8.75 ml of a 4% solution) was added to a final concentration

of 3%. An emulsion was formed by adding the ceil-alginate slurry dropwise to vegetable

oil (commercial grade canoia oil; 125 ml in a 500 ml flask) containing Tween 80 (0.2%),

which was stirred magnetically. After 10 min of stimng, calcium chloride (250 ml, 0.2M)

was added quickly down the side of the flask to break the waterloil emulsion. This mixture

continued stimng for 10-20 min. The beads were collected by gentle centrifugation (450 x

g, 10 min, 4°C). The upper oil phase and much of the water phase were siphoned off and

the beads were washed twice with sterile water.

ceii- slurry

Figure 3.1 : Apparatus for making large calcium alginate beads

3.6 Solubilization o f beads for enumeration

A known mass (approx. O 5 g) of large beads was suspended in 5 ml of sodium

phosphate buEer (lM; pH 6.61, followed by gentle sha

samples taken at O, 1, 2, 3 and 7 days, solubilized and plated according to the rnethod

described above.

3.9 Survivai of free and encapsulated bifidobacteria

Two strains of bifidobacteria (B. irzj'imtis, ATCC 15697; B. longiim. ATCC

15707) were encapsulated in large and small beads accordhg to the methods described

above. The large beads were divided into two lots. One lot was added to 50 ml

supplemented MRS broth and incubated anaerobically for 18 h at 37°C (pre-incubation

step). After incubation, the beads were divided into separate 10 ml screw top tubes with 5

ml of supplemented MRS broth which had been either adjusted to pH 4.5 with lactic acid

or control pH 6.3. The second lot was divided into separate 10 ml screw top tubes with 5

ml of supplemented MRS broth, adjusted to either pH 4.5, 5.5 with 85% lactic acid or

control pH 6.3. The small beads were also divided into two lots. The first lot was added

to 50 ml supplemented MRS broth followed by the pre-incubation step. M e r pre-

incubation, the first lot was gently centrifuged (450 x g, 10 min, 4OC) and the beads were

added to 20 ml of supplemented MRS broth with pH 4.5 or control pH 6.3. Mer mixing

well, the 20-mi simples were fùrther diMded into 1.5 mi microcentrifuge tubes. The

second lot was added to 20 ml of supplemented MRS broth with pH 6.3, 5.5, or 4.5,

mixed, then divided likewise.

As a control, free cells were also evaluated by centrifuging 50 ml of an 18 h

culture. The ce11 pellet was also divided into two lots. The fira lot was resuspended in 50

ml of supplemented MRS broth and pre-incubated as described above. M e r pre-

incubation the first lot was centrifuged to obtain a ce11 pellet and resuspended in pH 4.3 or

control broth. After mWng well, this was further divided into 1.5 ml microcentrifuge

tubes. The second lot was resuspended in 20 ml of supplemented MRS with pH 6.3, 5.5 ,

or 4.5, mixed, and was divided likewise.

Al1 of the above samples were stored at 4°C. Two samples for each treatment

were analyzed for viable ce1 counts on days 0, 2,4, 7, 1 1, 14 and 2 1 according to the

enurneration method described above.

3.10 Low temperature SEM and phase contrast microscopy

Phase contrast microscopy (Nippon Kogaku K.K., Tokyo, PN) was used to

examine the morphology of bifidobacteria and to examine the small beads using 40X and

IOOX oil immersion objective lenses; they were photographed with a mounted ocular

camera (Nikon AFX-II, Tokyo, JPN).

Scanning electron microscopy (SEM) was used to examine the intemal appearance

of the large calcium alginate beads. A single bead was mounted on a metal rivet using

Polyfreeze Q (Polysciences, hc., Warrington, PA) then plunged into iiquid propane

(- 189°C) which was cooled with liquid nitrogen. M e r fieezing, the bead was stored in

liquid nitrogen (-196OC) for up to two days. The bead was transferred to the Emscope

SP2000A Cryogenic Preparation System (Emscope Ltd., Ashford, Kent, UK), where a

fiesh sample surface was exposed by fracturing the bead. The bead was sublirnated for 40-

60 min at -80°C and sputter coated with approximately 300 A of gold. The coated bead

was transferred fiozen and under vacuum to the cold stage of the Hitachi S-570 SEM

(Hitachi Ltd., Tolqo, JPN) and scanned at a temperature < - 1 30°C.

3.1 1 Statisticd anaiyses

A randomized complete block design was used for the rnicroencapsulation

experirnent. Two sarnples per test period were taken and two complete replications of the

experiment was done; the average results were reported. Statistical analyses was carried

out using SAS (SAS Institute, Cary, NC, 1988). The general iinear mode1 was used and

cornparisons arnong treatment means were evaluated by least significant difference tests at

a significance level of a = 0.05.

4.0 Results

4.1 Effkct of CaClt and NaCI on cellular morphology and growth

Calcium and sodium chlondes were added to MRS broth pnor to anaerobic

incubation to evaluate their eEect on the cellular morphology and growth of B. brfid~rn

ATCC 1 5696, B. brfidirn ATCC 2952 1. B. injar~tis ATCC 1 5697, and B. !otgyn ATCC

15707. Optical density (OD) readings and ce11 enumeration were carried out before and

afler incubation to evaiuate cell growth. Ceiiular morphology was determined using phase

contrast rnicroscopy.

In general. rod-shaped morphologies predominated in al1 four strains incubated in

the unsupplernented MRS control and in MRS supplemented with both 0.0 1 and 0.5%

Ca& (Table 4.1 ). B. brfidtim ATCC 1 5696 appeared as smooth rods (Figure 4.1 A).

However, in the MRS control treatment for B. brfdtirn ATCC 2952 1, the rods did not

appear smooth but had knobs and thickened areas (lumpy), with many bifid ends. With the

addition of 0.01 and 0.5% calcium, these rods became smoother and more uniform (Table

4.1). The lumpy and smoother rods can be seen in Figure 4.2 A. Likewise, the extent and

presence of swollen rods observed with B. infants and B. 1011glim diminished with

addition of either level of calcium (Table 4.1 ). Typical cellular morphology for these N o

strains in 0.0 1% CaC12 can be seen in Figures 4.3 A and 4.4 A. The decreased swellhg of

the rods may account for a smaller increase in OD seen at the 0.5% CaCl, level compared

with the MRS control. However, with this concentration of calcium, a noticeable

precipitate formed in the broth which may have affected the OD readings. The addition of

either level of calcium did not affect the ce11 counts of the four strains when compared to

the MRS control.

Table 4.1: Effect of calcium and sodium on optical density, ce11 numbers and cellular morphology for B~jidobacteriiurn spp., incubated anaerobically for 2 1 h at 37°C.

~ e a n ' A OD ~ e a d A S train (560 nm) Log CFUI ml Morphology 3

l& bifdurn ATCC 15696 MRS control + 0.01 % CaClz + 0.5 % CaClz + 1% NaCl + 2% NaCI + 3% NaCl + 0.5 % CaCI2; 2% NaCl

B. bifidum ATCC 2952 1 MRS control + 0.01 % CaCI2 + 0.5 % CaCI2 + 1% NaCl + 2% NaCl + 3% NaCl + 0.5 % CaCI2; 2% NaCl

lX infuntis ATCC 15697 MRS control + 0.01 % CaClz + 0.5 % CaClz + 1% NaCI + 2% NaCl + 3% NaCl + 0.5 % CaC12; 2% NaCl

B. longum ATCC 15707 MRS control + 0.01 % CaCI2 + 0.5 % CaC12 + 1% NaCt + 2% NaCl + 3% NaCl

rods rads rods elongated r d ; bifid branched rock branched; rods

lumpy rods; bifid rads; iumpy rods rods; lumpy rock elongated lumpy rods; bifid branched; lumpy rods branchesi; lumpy rods elongated lump y rods; btfid

swollen rods; rods r d ; swollen rods rods; swollen rods elmgated rods; bifid b ranched rods rods; bifid

swollen rods; rods rods; swoiien rods rods; swollen rods r d ; thickened rods; bifid branched; elongated rods rods

+ 0.5 % CaCI2; 2% NaCl 0.3 5 -0.04 elongated rods; bifid

1 initial mean OD - final mean OD; n = 2 2 initial rnean Log CRJlml - final mean Log CFUIml; n = 2 3 observed cellular morphology listed in order of predominance 4 negative numbers indicate a decrease

Figure 4.1: Phase-contrast micrograph of B. brfidum ATCC 15696. A: Rod morphology grown in MRS broth supplemented with 0.01% CaC12. Bar = 4.0 p. B: Branched rnorphology grown in MRS broth supplemented with 2% NaCl. Bar = 3.5 p.

Figure 1.2: Phase-contrast micrograph of B. brfidim ATCC 2952 1. A: Rod morphology grown in MRS broth supplemented with 0.01% CaC12. Lurnpy appearance of some rods indicated by arrows. Bar = 3.0 pm. B: Branched morphology grown in MRS broth supplemented with 2% NaCl. Bar = 8.3 p.

Figure 4.3: Phase-contrast micrograph of B. infru2tis ATCC 15697. A: Rod morphology grown in MRS broth supplemented with 0.01% CaC12. Bar 3 .3 Pm. B: Branched morphology grown in MRS broth supplemented with 2% NaCl. Bar = 3.3 p.

Figure 4.4: Phase-contrast rnicrograph of B. fongum ATCC 15707. A: Rod morphology grown in MRS broth supplemented with 0.0 1 % CaCI2. Bar = 5.5 Pm. B: Branched morphoiogy grown in MRS broth supplemented with 2% NaCl. Bar = 4.0 Pm.

Ce11 growth for the four species, as measured by CFUId , was similar in MRS

supplemented with 1% NaCl and the MRS control (Table 4.1). The A OD, however,

decreased for both strains of B. bIfibr~rn while remaining unchanged for B. ;r>Iuntis md B.

lor~gum when compared to the MRS control. in general, at this lower concentration of

sodium chloride, there was a shifi in morphology to elongated and bifid rods. Increasing

the concentration to 2% NaCl resulted in a decrease in A OD and A ce11 numbers of al1

four strains compared to the MRS control. Although the OD did increase with incubation,

the cell numbers decreased (i.e. negative A Log CFUIml) for dl but B. brfidm ATCC

2952 1. This concentration of NaCl promoted branching in al1 four strains, although

different branching characteristics were observed between the different species (Figures

4.1 B, 4.2 B, 4.3 B, and 4.4 B). With addition of 3% NaCl to the medium, the ce11

numbers decreased by 0.95 to 4.43 log cycles with incubation (Table 4.1 ). This was not

reflected in the OD readings which remained Iargely unchanged with incubation.

Microscopic examination revealed few cells; this concentration of NaCl did not support

growth or promote branching.

A combination of calcium chloride and sodium chloride was added to the MRS

broth to assess its eEect on growth and cellular morphology . CaC12 (0.5%) and NaCl

(2.0%) added to the medium reduced but did not eliminate branching or bifid forms in any

of the four strains (Table 4.1 ). The OD did increase with incubation but the change was

substantialiy lower than the MRS control. Ceii counts were variable; there was a 0.95 log-

cycle decrease for B. bifidum ATCC 15696 and a 0.64 log-cycle increase for B. bipdum

ATCC 2952 1, but ce11 numbers of B. longzm and B. i~frmtis did not change more than

0.04 to 0.09 log cycles.

4.2 Effect of pH on the survival of baidobacteria

Overnight cultures of B. brfùi~rn ATCC 15696, B. b~@hirn ATCC 29521, B.

infa~tis ATCC 15697 and B. longulm ATCC 15707 were added to control pH 6.3 and pH

4.6, 43,4.0, and 3 7 broths and stored at 4°C for 2 1 days. The initial inoculum was 10'-

10' CFUImi. The ce11 numbers decreased by 4.7 log cycles during the 2 1 days of aorage

for B. brfidum ATCC 15696 in control broth (Figure 4.5). B. br$&rim ATCC 29521

(Figure 4.6) and B. itifm~tis ATCC 15697 (Figure 4.7) both survived weil in the control

medium, but B. ior>grrm ATCC 15707 decreased by 2.8 log cycles (Figure 4.8).

In general, as pH decreased so did the s u ~ v a l of al1 four grains. Both strains of B.

brfidum were more sensitive to acidic envkonments than the B. irzjial~tis or B. lotzgxm

strains. M e r two weeks of aorage, neither B. brficzim strains were detectabie at pH 1.6

or lower whle B. Ït,Im~tis and B. lorzprn mains remained viable at pH 4.6 for the entire

storage period. Based on these results, ody B. itzjantis and B. lot~gzm strains were

selected for fùrther study .

O -- --

O 5 10 15 20 25

Storage Time (d)

Figure 4.5: SuMval of B. bzjidum ATCC 15696 stored at 4OC in MRS broth; pH 6 .3 (a ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (O) and pH 3.7 (=). Results show are the average of duplicate samples fiom each of two separate expenment S.

- _ _ _ _ _ _ _ _ . _ - _ _ _ _ - O ----

O 5 10 15 20 25

Storage Time (d)

Figure 4.6: Survivd of B. brfdum ATCC 29521 stored at 4°C in MRS broth; pH 6 .3 (a ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (a) and pH 3.7 (i). Results shown are the average of duplicate samples from each of two separate experiments.

O ----- O 5 10 15 30 25

Storage T h e (d)

Figure 4.7: SuMvd of B. itlfanlis ATCC 15697 stored at 4°C in MRS broth; pH 6.3 (* ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (O) and pH 3.7 (a). Results shown are the average of duplicate samples from each of two separate experiment S.

Figure 4.8: S u ~ v a l of B. Iongum ATCC 15707 stored at 4°C in MRS broth; pH 6 . 3 (m ), pH 4.6 (O), pH 4.3 (A), pH 4.0 (a) and pH 3 .7 (=). Results shown are the average of duplicate samples from each of two separate experiments.

4.3 Eacapsulation rnethods

Severai experiments were carried out to detemine a reliable and consistent method

for encapsulation of bifidobacteria. The shape of the large beads was mon consistent using

a blunt-ended 22G lin. needle. Bevel-ended needles of various sizes, 2 1 G 1 '/z in., 22G 1

% in., and 23G 1 in. were also evaluated. The gauge of the needle did not affect the

average size of the beads, nor did the length of the needle. However, the shape of the

beads was more irregular with the beveled end. The blunt-ended needle was also chosen

for safety rasons.

The addition of Tween 80 (O. 5%) to the calcium alginate mixture resulted in a

more consistent bead size and an overall decrease in diarneter of the large beads. Without

Tween 80, the diameter of the beads was 2 to 4 mm, compared to 1.5 to 2 mm with

Tween 80. Typical shape and size of the resulting large beads can be seen in Figure 4.9 A.

The small beads ranged in size from approximately 40 to 600 p n and the shapes

varied fiom sphencal to pear-shaped as show in Figure 4.9 B.

4.3.1 The effect of CaC03 and RSM amendments in large beads

Calcium carbonate and reconstituted skim milk (RSM) were incorporated into

large beads with the objective of increasing the survival of bifidobacteria in low pH

environments. This experiment was carried out with B. infmtis.

At pH 6.3, the incorporation of RSM or CaC03 into the beads had Little effect on

the s u ~ v a l of the encapsulated strain (Figure 4.10 A). None of the treatments, including

the control treatment with no amendment, decreased more than one log cycle over the

seven day storage period. At pH 4.0 (Figure 4.10 B), the initial numbers of bifidobacteria

Figure 4.9: Typical size and shape of calcium alginate beads. A: large beads with 0.5% Tween 80 B: phase contrast micrograph of small beads. Bar -r 80 p.

Storage T h e (d)

5 O 1 2 3 4 5 6 7

Storage Time (d)

Figure 4.10: S u ~ v a l of B. infar>tis encapsulated in large beads with RSM and CaCO, and stored at 4°C; pH 6.3 (A), pH 4.0 (B); non-arnended control (a), 0.1% CaC03 (O), 1 .O% CaCO3 (a), 3% RSM (a). Results shown are the average of duplicate sarnples from each of two separate experirnents.

remained fairly constant for the first three days of storage with 1% CaC03 incorporated

into the beads. This was followed by a decrease of approximately one log cycle by day

seven. The 1% CaC03 treatment was more effective in rnaintaining initial numbers and in

the overall suMvd of the encapsulated bifidobacteria when cornpared with the non-

amended or the incorporation of 0.1% CaCOs or 3% RSM. However, both RSM and

0.1% CaC03 amendments did enhance s u ~ v a l when compared to the non-amended

treatment. The beads amended with RSM resulted in a 2.9 log-cycle decrease. the beads

arnended with 0.1% CaCO3 resulted in a 3.4 log-cycle decrease, while a 3.7 log-cycle

decrease occurred with the non-amended beads (Figure 4.10 B).

Although survivai was best enhanced with I % CaCO3, by the third day the pH of

the surrounding medium increased from pH 4.0 to pH 4.7 which was maintained for the

rest of the storage period (data not shown). The medium surrounding the 0.1% CaCO3

and the 3% RSM beads remained at pH 4.0 for the entire storage period. Incorporation of

3% RSM was chosen for further expenments as it provided some protection to the

bactena while maintainhg a constant pH in the surrounding environment.

4.3.2 Effcet of the solubiüzation step ou ceii enumeration

An experirnent was carrieci out to evaluate the effect of the solubilization step,

used in the enumeration of bifidobacteria encapsulated in large and small beads, on fiee

cells of B. infanhs and B. longurn. There were two factors in the solubiiization step that

may have affected the nnal counts of encapsulated bifidobactena. The first was the effect

of the phosphate b a e r itselfon the bacteria, the second was the t h e needed at room

temperature for solubilization. To evaluate the effect of these factors, free cells were

exposed to the phosphate b&er for same tirne period as that needed to soiubilize the

beads. These counts were compared to counts of fiee cells piated directly. Those cells

exposed to the phosphate bufkr step had consistently lower counts of viable cells for both

strains, however, the mean difference was only 0.35 t 0.1 log cycles for B. infantis and

0.41 f O. 14 log cycles for B. longm (Table 4.2) and was, therefore. considered negligible.

Table 4.2: Effkct of the phosphate buffer step on viable counts of free cells of B. infantis and B. iotigurn.

No buffer stepl Buffer step2 A Log CFCT/ml Trial (Log CFU/ml) (Log CFU/rnl)

B. infmtis 1 9.38 9.14 0.24 2 9.66 9.22 O. 44 3 8.74 8.36 0.38

Average 0.35 + O. IO

1