8/13/2019 Khalil, 1998

1/4

702 JOURNAL OF FOOD SCIENCEVolume 63, No. 4, 1998

MICROBIOLOGY

Alginate Encapsulated BifidobacteriaSurvival in Mayonnaise

ALI H. KHALIL and ESAM H. MANSOUR

Authors Khalil and Mansour are affiliated with the Food

Science & Technology Dept., Faculty of Agriculture,Menofiya Univ., Shibin El-Kom, Egypt. Direct inquir-ies to Dr. A. H. Khalil.

ABSTRACT

Bifidobacterium bifidumand Bifidobacterium infantiswere added to mayonnaiseas either free cells or encapsulated. The survival of bifidobacteria and their effecton mayonnaise quality were evaluated. The viability of free cells disappearedafter 2 wk; however, encapsulated B. bifidumsurvived well for 12 and B. infantisfor 8 wk. Mayonnaise containing encapsulated bifidobacteria had lower total bac-terial counts compared to other treatments. Yeast and mold started to appearafter 12 wk of storage in mayonnaise containing encapsulated bifidobacteria andafter 6 wk with other treatments (control, cells free of bifidobacteria). Mayonnaisecontaining encapsulated bifidobacteria had higher titratable acidity and lowerTBA values than other treatments. Sensory properties of mayonnaise were im-proved by the addition of encapsulated bifidobacteria.

Key Words: survival, alginate encapsulated, bifidobacteria, mayonnaise

The egg yolks were mixed with all dry in-

gredients in a Kitchen Aid mixture (Oster,

Model 97226H, Milwaukee, WI) for 1 min

at speed 4. Oil and vinegar were added alter-

nately during a 10 min mix at speed 6. The

mixture was then pasteurized at 70C for 10

min and cooled.

The prepared mayonnaise was divided

into 5 portions for treatments. One portion

was left without bacterial inoculation as a

control. Two portions were inoculated sepa-

rately with free and encapsulated cells ofBi-

fidobacterium bifidumDI to a final cell con-centrations of about 9.2 106CFU/g. The

other two portions were inoculated with free

and encapsulated cells of Bifidobacterium

infantis4038 to a final cell concentrations of

about 8.9 106CFU/g. The resultant may-

onnaise samples were transferred to sterile

screw capped plastic jars under aseptic con-

ditions and stored at 5C for 16 wk.

Samples from each treatment were taken

at specified time intervals throughout stor-

age for bacteriological, chemical and senso-

ry evaluation. The experiment was done in

triplicate and two determinations were con-

ducted per replicate.

Microbiological analysis

Bacterial enumeration. Bifidobacteri-

um counts were enumerated on modified

MRS (Ventling and Mistry, 1993) with NPNL

(Neomycine sulphate, Paromomycine sul-

phate, Nalidixic acid and Lithium chloride)

solution (Samona and Robinson, 1991) un-

der anaerobic conditions using the BBL gas

pak (BBL, Cockeysville, MD). The encap-

sulated cells of Bifidobacteria were released

from the beads according to the procedure of

Sheu et al. (1993) by mixing mayonnaise

sample with phosphate buffer (1M, pH 7.5).Total bacterial counts were enumerated on

standard plate count agar (Marth, 1978)

which was incubated at 30C for 72h. Yeast

and mold counts were enumerated on potato

dextrose agar (Difco, 1984) which was incu-

bated at 25C for 96h.

Physical and chemical analysis

Titratable acidity was determined by ti-

tration of 10g mayonnaise to pH 8.1 with

0.1N NaOH using a Digital pH meter (Jen-

way, Model 3020, Dunmow, Essex, UK) and

results were converted to percentage of ace-

tic acid. The pH value was determined using

10% dispersion of mayonnaise in distilled

water using a Jenway Digital pH meter (Mod-

el 3020). Thiobarbituric acid (TBA) value

INTRODUCTIONTHEHEALTHANDNUTRITIONALBENEFITS

ascribed to bifidobacteria are many and var-

ied including maintenance of a healthy in-

testinal flora (Okamura et al., 1986), synthe-

sis of B complex vitamins and absorption of

calcium (Deguchi et al., 1985), amelioration

of diarrhea or constipation (Tojo et al., 1987),

antimicrobial production (Kang et al., 1989),

and immunity activation (Yamazaki et al.,

1991). Bifidobacteria also lower the levels

of faecal bacterial enzymes, responsible forcatalyzing the conversion of carcinogenic

amines (Tsuyuki, et al., 1991), improve lac-

tose utilization by lactose malabsorbers

(Hughes and Hoover, 1995) and reduce se-

rum cholesterol levels (Tahri et al., 1995).

Such beneficial microorganisms have

been added to various food stuffs to create

functional foods or nutraceuticals. Poten-

tial applications of culture blends contain-

ing bifidobacteria include sour cream, but-

termilk, yogurt, powdered milk, spreads, fro-

zen desserts and fruit juices. Mayonnaise may

also be suitable for incorporation of bifido-

bacteria.

The shelf-life of mayonnaise is mainly

affected by growth of aerobic sporeformers,

yeasts and occasionally molds (Smittle and

Flowers, 1982). The concentration of acetic

acid and low pH (3.64.6) of mayonnaise

have been reported to be the major inhibito-

ry substances against microorganisms (Rad-

ford and Board, 1993). The growth of bifi-

dobacteria is considerably retarded at pH

5.0 (Lankaputhra et al., 1996). Bifidobac-

teria must be alive in mayonnaise to provide

benefits in the intestinal tract. Encapsulation

may enhance the survival of bifidobacteria.

Therefore our study was undertaken to in-

corporate bifidobacteria in mayonnaise as en-

capsulated cells. Our objectives were to de-

termine the survival of encapsulated and free

cells of bifidobacteria in mayonnaise during

refrigerated storage (~5C) and to evaluate

their effects on any microbiological changes

and mayonnaise quality.

MATERIALS & METHODS

Preparation of encapsulated

culture

Bifidobacterium bifidumDI (Diversitech

Inc., Gainesville, Fl) andBifidobacterium in-

fantis4038 (Dr. M. El-Soda, Dairy Sci. Dept.

Fac. of Agric., Alex. Univ., Egypt) were

grown at 37C for 24h in modified MRS

(MRS 0.05% L-cystein HCl) broth (Dif-

co, Detroit, MI). Culture cells were harvest-

ed by centrifugation (3000 g, 10 min),

washed and resuspended in sterile saline so-

lution to a final concentration of 10% (w/v)

culture concentrates. Calcium alginate beads

of immobilized cells were prepared accord-

ing to the procedure of Sheu and Marshall

(1993) by mixing 10 mL of each culture con-

centrate with 40 mL sterilized 3% (w/v) so-

dium alginate (BDH Chemical LTD, Poole,

England). Calcium alginate beads were

formed by dropwise addition of 0.05 M cal-

cium chloride. The beads were harvested by

gentle centrifugation (350 g, 10 min) and

washed with sterile distilled water.

Preparation of mayonnaise

Mayonnaise was prepared using the fol-

lowing formula: corn oil (70%), fresh egg

yolk from infertile eggs (12%), distilled whitevinegar (10%), salt (2%), sugar (1.7%), skim

milk powder (3.3%) and dry mustard (1%).

8/13/2019 Khalil, 1998

2/4

Volume 63, No. 4, 1998JOURNAL OF FOOD SCIENCE 703

was determined as described by Li Hsieh and

Regenstein (1991).

Sensory evaluation

Sensory evaluation of the mayonnaise

was conducted throughout the refrigerated

storage. Evaluations were made by eight

trained staff members from the Univ. ofMenofiya, Shibin El-Kom, Egypt. Selection

of panelists was based on participant inter-

est, taste and flavor acuity and ability to un-

derstand test procedures. Panelists were

trained in two 6h sessions prior to evalua-

tion to be familiar with attributes of mayon-

naise samples under study and scaling pro-

cedures. Instruction score sheets for evalu-

ating samples were given to panelists. They

were provided with a set of five randomly

coded samples, i.e., control mayonnaise

(without bifidobacteria), and mayonnaise

containing either free or encapsulated cells

of B. bifidum and B. infantis. General at-tribute ranking evaluations were made in in-

dividual sensory evaluation booths under flu-

orescent light at ambient temperature

(~25C). Panelists were instructed to rinse

their mouth with water before starting and

between sample evaluations. Four sensory

attributes were evaluated (flavor, texture,

color and overall palatability) using an 8-

point hedonic scale where 8excellent and

1extremely poor. Accuracy and precision

were evaluated statistically.

Statistical analysis

Data of titratable acidity, pH values, thio-barbituric acid values and sensory properties

were analyzed using a 5 (treatments) 11

(storage periods) 3 (replications) com-

pletely randomized factorial design (Mont-

gomery, 1984). An analysis of variance (SAS

Institute, Inc., 1988) was conducted. When

a significant main effect was detected, the

means were separated with the Student-New-

man-Keuls test. The predetermined accept-

able level of probability was 5% (P0.05)

for all comparisons.

RESULTS & DISCUSSION

Survival of bifidobacteria

The viable counts of free cells ofB. bifi-

dumandB. infantisdecreased markedly af-

ter 1 wk of refrigerated storage reaching 4.1

104and 3.2 104CFU/g, respectively.

No viable cells were recovered after 2 wk

(Table 1). These decreases might be attribut-

ed to the bactericidal activity of acetic acid

(vinegar) in mayonnaise (Collins,1985; Lock

and Board, 1994). The viability of encapsu-

lated B. bifidum and B. infantisdecreased

slightly after 2 wk to 8.6 105and 6.5

105CFU/g, respectively. The viable count,

remained almost stable until 10 and 8 wk

reaching 6.9 105and 5.2 105CFU/g

for B. bifidumand B. infantis respectively.

The high rate of survival of the encapsulated

cells resulted from the protection by the alg-

inate. Sheu et al. (1993) reported that calci-

um alginate could provide good protection

(90%) for lactobacilli in frozen ice milk. Theviability of encapsulated bifidobacteria de-

creased slightly during the next 2 wk of stor-

age (after 12 and 10 wk for B. bifidumand

B. infantis, respectively) and continued to

decline until the end of storage reaching 9.8

102and 4.1 102 CFU/g, respectively.

This reduction might be due to the release of

bifidobacteria into mayonnaise as alginate

beads are partially degraded and consequent-

ly the free cells were influenced by the bac-

tericidal effect of acetic acid.

Note that mayonnaise containing encap-

sulated bifidobacteria had high numbers of

viable bifidobacteria, after 12 (1.2

105

) and8 (5.2 105) wk forB. bifidumandB. in-

fantis, respectively. These were higher than

the minimum levels (105to 106/g) needed at

consumption of the product to achieve the

beneficial effects of bifidobacteria (Ishiba-

shi and Shimamura, 1993).

Total bacterial, yeasts and molds

counts

Total bacterial counts of mayonnaise con-

taining encapsulated bifidobacteria decreased

as the storage period progressed to 10 wk for

B. bifidum and 8 wk for B. infantis, then

slightly increased for both strains until the

end of storage (Table 2). The presence of vi-

able bifidobacteria in mayonnaise and thus

their ability to produce antimicrobial sub-

stances (Kang et al., 1989 and Kebary, 1995)

might explain the decline of total bacterial

count. However the reduction in viable bifi-

dobacteria at late storage was associated with

the increase in total bacterial count. Control

mayonnaise and samples containing free cells

of bifidobacteria showed slight decrease in

total bacterial count for 2 wk, then gradual

increases were observed till the end of stor-

age. Mayonnaise containing encapsulated bi-

fidobacteria was free from yeasts and molds

for 10 wk, while control mayonnaise and thatcontaining free cells had detectable yeast and

molds after 6 wk on (Table 3).

Physical and chemical analysis

Titratable acidity of mayonnaise was not

affected (P0.05) by storage period. How-

ever the addition of encapsulated bifidobac-teria to mayonnaise resulted in higher (P

0.05) titratable acidity than control and may-

onnaise containing free cells of bifidobacte-

ria (Table 4 and 5). The increase in titratable

acidity could be attributed to acid produc-

tion by surviving bifidobacteria. Kebary

(1996) reported an increase in acidity of fro-

zen yogurt manufactured with Bifidobacte-

rium bifidum. Changes in pH of mayonnaise

treatments throughout refrigerated storage

were not significant. The pH value of may-

onnaise containing encapsulated bifidobac-

teria was lower (P0.05) than other treat-

ments.TBA values of all mayonnaise treatments

were increased (P0.05) as the storage peri-

od progressed (Fig. 1). These increases could

be attributed to the oxidation of polyunsatu-

rated fatty acids in oil. Li Hsieh and Regen-

stein (1991) reported that the shelf-life of fish

oil mayonnaise and soy oil mayonnaise was

limited by lipid oxidation as the product con-

tained unsaturated fats and oxygen.

Mayonnaise containing encapsulated bi-

fidobacteria had lower (P0.05) TBA val-

ues than those of controls and samples con-

taining free cells of bifidobacteria at any cor-

responding time. TBA values of mayonnaise

containing encapsulated bifidobacteria were

almost stable during the first 8 wk of refrig-

erated storage followed by a slight increase

until the end of storage. The increase of TBA

values at the end of storage might be due to

the increase of lipolytic activity as a result

of bacteria, yeast and mold growth (Tables 2

and 3) which encourage lipid oxidation.

Sensory evaluation

Scores of flavor were not significantly

(P0.05) affected by storage period, where-

as color, texture and overall palatability were

affected (P0.05) (Table 6). Mayonnaise

containing encapsulated bifidobacteria hadhigher (P0.05) scores for all tested senso-

ry properties than controls and samples con-

Table 1Survival of free and encapsulated bifidobacteria during refrigerated storage ofmayonnaisea

Viable count of bifidobacteria (CFU/g)

Storage Bifidobacterium bifidum Bifidobacterium infantis period(wk) Control Free cells Encapsulated Free cells Encapsulated

0 0b 9.20.25106 9.20.23106 8.90.26106 8.90.23106

1 0 4.10.11104 2.10.10106 3.20.12104 1.00.11106

2 0 0 8.60.28105 0 6.50.22105

3 0 0 8.50.27105 0 6.20.24105

4 0 0 8.10.26105 0 5.90.22105

6 0 0 7.80.27105 0 5.70.17105

8 0 0 7.50.25105 0 5.20.16105

10 0 0 6.90.22105 0 2.80.13104

12 0 0 1.20.09105 0 1.10.10104

14 0 0 7.60.24103 0 6.80.21102

16 0 0 9.80.26102 0 4.10.15102

aEach value in the table is the mean standard deviation of three trials.bLess than 10 CFU/g.

8/13/2019 Khalil, 1998

3/4

704 JOURNAL OF FOOD SCIENCEVolume 63, No. 4, 1998

Encapsulated Bifidobacteria in Mayonnaise . . .

taining free cells. Kebary (1996) reported that

the flavor of frozen yogurt made withB. bi-

fidumwas improved due to the increase in

diacetyl and acetyl methyl carbinol. The low

color scores of control and mayonnaise con-

taining free cells of bifidobacteria might be

due to the color changes associated with ox-

idative rancidity of the oil during storage

(Fig. 1). Li Hsieh and Regenstein (1991) ob-

served a brown discoloration in fish oil may-

onnaise due to the formation of secondary

oxidation products such as aldehydes which

presumably participated in browning reac-

tions. We have also found that texture of

mayonnaise containing calcium alginate

without bifidobacteria improved (unpub-

lished data). The improved texture in may-

onnaise containing encapsulated bifidobac-

teria could be attributed to the production of

exopolysaccharide by bifidobacteria and/or

to the presence of calcium alginate. Cerning

et al. (1986) and Roberts et al. (1995) report-

ed that exopolysaccharide produced byBifi-

dobacterium longumandLactobacillus bul-

garicusis important in the dairy industry to

ensure texture improvement, increased vis-

cosity and enhanced smoothness of mouth-

feel. Although TBA values were increased

in control mayonnaise and samples contain-

ing free cells of bifidobacteria during refrig-erated storage, panelists did not report any

oxidized off-flavor.

Table 2Total bacterial counts of mayonnaise containing free and encapsulated bifido-bacteria during refrigerated storagea

Total bacterial counts (CFU/g)

Storage Bifidobacterium bifidum Bifidobacterium infantis period(wk) Control Free cells Encapsulated Free Cells Encapsulated

0 9.40.26102 9.2 0.25102 9.3 0.28102 9.4 0.27102 9.30.27102

1 6.40.23102 6.30.24102 4.10.18102 6.50.23102 4.00.18102

2 6.10.19102 6.20.24102 3.80.14102 6.00.21102 3.90.12102

3 6.90.22102 6.70.20102 3.70.14102 6.90.19102 3.80.13102

4 9.10.28102 9.00.27102 3.40.12102 9.20.26102 3.60.11102

6 1.90.09103 1.70.10103 3.10.13102 1.80.08103 3.30.15102

8 4.80.19103 2.60.12103 3.10.14102 2.70.15103 3.30.17102

10 6.60.22103 6.10.23103 3.00.11102 6.60.24103 3.90.13102

12 9.60.26103 9.50.28103 8.10.25102 9.80.27103 9.70.25102

14 1.20.07104 9.60.28103 1.10.08103 9.70.26103 1.90.11103

16 3.10.14104 2.50.12104 2.10.13103 2.10.12104 2.40.09103

aEach value in the table is the mean standard deviation of three trials.

Table 3Yeast and mold counts of mayonnaise containing free and encapsulated bifido-bacteria during refrigerated storagea

Yeast and mold counts (CFU/g)

Storage Bifidobacterium bifidum Bifidobacterium infantis period

(wk) Control Free cells Encapsulated Free cells Encapsulated

0 1 2 3 4 6 1.10.07102 1.00.08102 1.10.05102 8 1.20.06102 1.10.07102 1.20.05102

10 3.70.11102 3.70.13102 3.80.12102 12 8.60.26102 8.50.28102 1.00.05102 8.30.27102 1.20.07102

14 2.10.08103 2.30.09103 1.30.07102 2.00.07103 1.60.06102

16 3.20.11103 3.10.12103 1.60.08102 3.00.11103 1.80.10102

aEach value in the table is the mean standard deviation of three trials.

Table 4Titratable acidity and pH changesof mayonnaise containing free and encap-sulated bifidobacteria during refrigeratedstoragea

Storage Titratable pHperiod acidity

(wk) (%)

0 0.239 4.42

1 0.240 4.412 0.240 4.413 0.239 4.414 0.238 4.406 0.240 4.418 0.241 4.41

10 0.241 4.4212 0.241 4.4014 0.238 4.4116 0.238 4.42

LSD 0.003 0.02

aMeans in the same column are not significanlty different(P>0.05).

Table 5Titratable acidity and pH changesof mayonnaise containing free and encap-

sulated bifidobacteria during refrigeratedstorage

Treatment Titratable pHacidity

(%)

Control 0.235a 4.46b

Free cells of B. bifidum 0.236a 4.45b

Encapsulated cells of B. bifidum 0.245b 4.35a

Free cells of B. infantis 0.236a 4.45b

Encapsulated cells of B. bifidum 0.244b 4.35a

LSD 0.002 0.01

abMeans in the same column with different letters aresignificantly different P 0.05).

Fig. 1TBA values of mayonnaise containing free and encapsulated bifidobacteria duringrefrigerated storage. T = control; T1 = Free cell of Bif idobacterium bif idum; *T2 = encapsulatedBifidobacterium bifidum; T3 = Free cell ofBifidobacterium infantis;and T4 = encapsulatedBifidobacterium infantis.

CONCLUSIONA GOODQUALITYMAYONNAISECOULDBE

manufactured by incorporating encapsulat-

ed bifidobacteria in calcium alginate beads.

Calcium alginate provided an excellent pro-

tection of bifidobacteria cells from the bac-

tericidal effects of vinegar. Incorporating

encapsulated bifidobacteria in mayonnaise

reduced the total bacterial count, inhibited

the growth of yeasts and molds up to the 10th

8/13/2019 Khalil, 1998

4/4

Volume 63, No. 4, 1998JOURNAL OF FOOD SCIENCE 705

Table 6Sensory properties of mayonnaise containing free and encapsulated bifidobacteria during refrigerated storage

Sensory properties Storage period (wk) LSD Treatments LSD

Control B. bifidum B. infantis

0 1 2 3 4 6 8 10 12 14 16 Free Cape Free Cape

Flavor 7.0a 7.0a 7.0a 7.4a 7.4a 7.4a 6.6a 6.6a 6.6a 6.4a 6.4a 0.7 6.6a 6.5a 7.5b 6.5a 7.3b 0.4Color 7.0d 7.0d 6.8d 6.4cd 6.4cd 5.8bc 5.8BC 5.2ab 5.0a 4.8a 4.8a 0.7 5.3a 5.4a 6.8b 5.4a 6.7b 0.5Texture 7.0b 7.0b 7.4b 6.8ab 6.8ab 6.8ab 6.2a 6.2a 6.2a 6.2a 6.2a 0.6 5.8a 5.8a 7.8b 5.8a 7.8b 0.4

Overall palatability 7.0b 7.0b 6.4ab 6.4ab 6.4ab 6.8b 6.8b 6.2ab 5.8a 5.8a 5.8a 0.8 5.8a 5.8a 7.3b 5.8a 7.3b 0.4adMeans in the same row with different letters are significantly different (P 0.05).eEncapsulated

week, prevented any increase in TBA values

and improved sensory properties. Mayon-

naise containing encapsulatedB. bifidumhad

high levels of viable bifidobacteria up to the

12th week andB. infantisup to the 8th week

of refrigerated storage (105106 CFU/g).

They could thus be a good source for pro-

viding a live beneficial organism to consum-

ers.

REFERENCESCerning, J., Bouillanne, C., Desmazeaud, M.J., and

Landon, M. 1986. Isolation and characterization ofexocellular polysaccharide produced by Lactobacil-lus bulgaricus. Biotechnology Letters 8: 625-628.

Collins, M.A. 1985. Effects of pH and acidulate type onthe survival of some food poisoning bacteria in may-onnaise. Microbiologie - Aliments - Nutrition 3: 215-221.

Deguchi, Y., Morishita, T., and Mutai, M. 1985. Com-parative studies on synthesis of water-soluble vitaminsamong human species of bifidobacteria. Agri. Biol.Chem. 49(1): 13-19.

Difco 1984.Manual of Dehydrated Culture Media andReagents for Microbiology,10th ed. Difco Laborato-ries Inc. Detroit, MI.

Hughes, D.B. and Hoover, D.G. 1995. Viability and en-zymatic activity of bifidobacteria in milk. J. Dairy Sci.78(2): 268-276.

Ishibashi, N. and Shimamura, S. 1993. Bifidobacteria:Research and development in Japan. Food Tech. 6: 126-135.

Kang, K.H., Shin, H.J., Park, Y.H., and Lee, T.S. 1989.

Studies on antibacterial substances produced by Lacticacid bacteria: Purification and some properties of anti-bacterial substance Bifilong produced byBifidobac-terium longum. Korean J. Dairy Sci. 11(3): 204-216.

Kebary, K.M.K. 1995. Production, partial purificationand stability of antimicrobial substances produced byBifidobacterium bifidum DI. Egypt. J. Dairy Sci. 23(2): 151-166.

Kebary, K.M.K. 1996. Viability ofBifidobacterium bifi-dum and its effect on quality of frozen Zabady. FoodResearch International 29(5-6): 431-437.

Lankaputhra, W.E.V. Shah, N.P., and Britz, M.L. 1996.Survival of bifidobacteria during refrigerated storagein the presence of acid and hydrogen peroxide. Milch-wissenschaft 51(2): 65-69.

Li Hsieh, Y.T. and Regenstein, J.M. 1991. Factors af-fecting quality of fish oil mayonnaise. J. Food Sci.56(5): 1298-1301.

Lock, J.L. and Board, R.G. 1994. The fate ofSalmonellaenteritidis PT4 in deliberately infected commercialmayonnaise. Food Microbiology 11: 499-504.

Marth, E.H. 1978. Standard Methods for the Examina-tion of Dairy Products, 14th ed. Am. Publ. Health As-soc. Washington, DC.

Montgomery, D.C. 1984. Experiments to compare sev-eral treatments: The analysis of variance. In Designand Analysis of Experiments, 2nd ed. p. 43-80. JohnWiley Book Co, New York.

Okamura, N., Nakaya, R. Yokota, H., Yanai, N., andKawashima, T. 1986. Interaction of Shigella with bifi-dobacteria. Bifidobacteria Microflora 5(1): 51-55.

Radford, S.A. and Board, R.G. 1993. Review: Fate ofpathogenes in home-made mayonnaise and related

products. Food Microbiology 10: 269-278.Roberts, C.M., Fett, W.F., Osman, S.F., Wijey, C.,

OConnor, J.V., and Hoover, D.G. 1995. Exopolysac-charide production by Bifidobacterium longum BB-79. J. Applied Bacteriology 78: 463-468.

Samona, A. and Robinson, R.K. 1991. Enumeration ofbifidobacteria in dairy products. J. Soc. Dairy Tech.44: 64-66.

SAS Institute, Inc. 1988. SAS Users Guide: Basic Sta-tistical analysis. SAS Institute, Inc., Cary, NC.

Sheu, T.Y. and Marshall, R.T. 1993. Microentrapment oflactobacilli in calcium alginate gels. J. Food Sci. 54(3):557-561.

Sheu, T.Y., Marshall, R.T., and Heymann, H. 1993. Im-proving survival of culture bacteria in frozen dessertsby microentrapment. J. Dairy Sci. 76(7): 1902-1907.

Smittle, R.B. and Flowers, R.S. 1982. Acid tolerant mi-croorganisms involved in the spoilage of salad dress-ings. J. Food Protect. 45: 977-983.

Tahri, K., Crociani, J., Ballongue, J., and Schneider, F.1995. Effects of three strains of bifidobacteria on cho-lesterol. Letters in Applied Microbiology 21: 149-151.

Tojo, M., Oikawa, T., Morikawa, T. Yamashita, N. , Iwa-ta, S., Satoh, Y., Hanada, J., and Tanaka, R. 1987. Theeffect of Bifid obacterium breve administration onCampylobacter enteritis. Acta Paediatr. Jpn. 29: 160-167.

Tsuyuki, S. Yamazaki, S, Akashiba, H. Kamimura, H.Sekine, K, Toida, T. Saito, M. Kawashima, T., andUeda, K. 1991. Tumor-suppressive effect of a cell wallpreparation, WPG, from Bifidobacterium infantis ingermfree and flora-bearing mice. Bifidobacteria Mi-croflora 10(1): 43-52.

Ventling, B.L. and Mistry, V.V. 1993. Growth character-istics of bifidobacteria in ultrafiltered milk. J. DairySci. 76(4): 962-971.

Yamazaki, S., Tsuyuki, S., Akashiba, H., Kamimura, H.,Kimura, M., Kawashima, T., and Ueda, K. 1991. Im-

mune response ofBifidobacterium - monoassociatedmice. Bifidobacteria Microflora 10(1): 19-31.

Ms received 7/18/97; revised 11/12/97; accepted 12/18/97.