Bidobacterium

spp. and
Lactobacillus
acidophilus:
biological,
biochemical,
technological and
therapeutical
properties relevant
for use as
probiotics
Ana M.P. Gomes and
F. Xavier Malcata*
Escola Superior de Biotecnologia, Universidade
Cato lica Portuguesa Rua, Dr. Anto nio Bernardino de
Almeida, 4200 Porto, Portugal (fax: +351-2-5090351)
1||s iov|ow |ocosos oo t|o b|o|og|ca| iooit|os aoo coo-
soqooot toc|oo|og|ca| io|os o| |otost|oa| bactoi|a w|t|
otoot|a| |oa|t|-iomot|og caac|t|os, aoo iov|oos so|octoo
oxam|os ava||ab|o |o t|o ||toiatoio t|at aio oit|ooot to t|o
a|oiomoot|oooo coocots. A comio|oos|vo ovoiv|ow oi-
ta|o|og to t|o taxooomy aoo oco|ogy, as wo|| as ooti|t|ooa|
aoo |oa|t| o|octs o| Bidobacterium s. aoo Lactobacillus
acidophilus, |s iov|ooo, ait|co|ai attoot|oo |s a|o to t|o|i
|ocoioiat|oo, aoo giowt| aoo ac|o||cat|oo |oatoios |o |oi-
mootoo oa|iy iooocts. 1|o ty|ca| ooi giowt| o| t|oso
soc|os |o m||| |s ||g|||g|too, aoo t|o oso o| b||oogoo|c aoo
giowt| |actois, |oc|oo|og t|o|i oatoio aoo |ooct|oo, |s o|s-
cossoo. l|oits to ostab||s| ot|mom oov|ioomoota| cooo|t|oos
|oi t|o|i giowt| aio ci|t|ca||y iov|owoo, |o aoo|t|oo to t|o
o|octs o| t|o |ooo aoo stoiago cooo|t|oos oo m|ciob|a|
soiv|va|. Ci|toi|a |oi so|oct|oo o| o|oct|vo m|ciob|a| stia|os
|oi t|o|i iob|ot|c o|oct aio ||stoo, aoo moo||cat|oos to
|miovo |oimootat|oo ouc|oocy aoo s|o||-|||o o| |oa| oa|iy
iooocts aio soggostoo, amoog t|oso, t|o |ocoioiat|oo o|
Bidobacterium s. aoo L. acidophilus |oto a so||o mati|x,
soc| as c|ooso, |s om|as|zoo. #! l|sov|oi Sc|ooco lto.
A|| i|g|ts iosoivoo.
The use of Bidobacterium spp. and/or Lactobacillus
acidophilus in fermented or culture-containing milks
became popular by the end of the 1970s as a result of
the tremendous increase in knowledge encompassing the
taxonomy and ecology of bidobacteria. Their popularity
has further increased owing to their reduced acidica-
tion during post-processing storage and their relatively
high yield of L(+)-lactic acid compared with D( )-lac-
tic acid. In recent years, much work on bidobacteria,
regarded as microorganisms targeted for technological
and therapeutic applications, was performed in Japan, but
other countries also became involved (e.g. Denmark,
Germany, Poland, Russia, UK and USA) [1]. Such
concerted work has led to many publications and patent
applications; the literature was reviewed by Rasic and
Kurmann [2] in a comprehensive paper that rapidly
became a classic reference. The number of patents has
meanwhile drastically increased, and pertain to an
increasing range of applications including new product
development and starter culture production.
Denition
The word `probiotic', from the Greek `for life', has
over the past few years been used in several dierent
ways. It was originally proposed to describe compounds
produced by one protozoan that stimulated the growth
of another [3], but in the early seventies, Sperti expan-
ded the term to encompass tissue extracts that stimu-
lated microbial growth. Parker [4] later applied the term
024-2244''$ - soo |ioot mattoi Coyi|g|t # ! l|sov|oi Sc|ooco lto. A|| i|g|ts iosoivoo.
PI I : S024- 2244 000-
1iooos |o looo Sc|ooco 8 1oc|oo|ogy !0 ! !!
Coiiosooo|og aot|oi.
Review
to describe animal feed supplements that had a bene-
cial eect on the host by contributing to its intestinal
microbial balance. Consequenlty, the word `probiotic'
was applied to ``organisms and substances that con-
tribute to intestinal microbial balance''. This general
denition was, however, unsatisfactory because an
imprecise word as `substances' might include a variety
of supplements, including antibiotics. Fuller [5] revised
the denition to stress the importance of living cells as
an essential component of an eective probiotic, and
thus dened probiotic as ``a live microbial feed supple-
ment which benecially aects the host animal by
improving its intestinal microbial balance''.
The denition of probiotic was broadened in the cur-
rent decade [6]. Presently, probiotics are dened as
``viable microorganisms (lactic acid and other bacteria,
or yeasts applied as dried cells or in a fermented pro-
duct) that exhibit a benecial eect on the health of the
host upon ingestion by improving the properties of its
indigenous microora''.
Taxonomy
Cooos Bidobacterium
Bidobacteria were rst isolated and described in
18991900 by Tissier, who described rod-shaped, non-
gas-producing, anaerobic microorganisms with bid
morphology, present in the faeces of breast-fed infants,
which he termed Bacillus bidus. Bidobacteria are
generally characterized as gram-positive, non-spore
forming, non-motile and catalase-negative anaerobes
[7]. They have various shapes including short, curved
rods, club-shaped rods and bifurcated Y-shaped rods.
Presently, 30 species are included in the genus Bido-
bacterium, 10 of which are from human sources (dental
caries, faeces and vagina), 17 from animal intestinal
tracts or rumen, two from wastewater and one from
fermented milk (Table 1). Bidobacteria are phylogen-
etically grouped in the actinomycete branch of gram-
positive bacteria [7], that is characterized by a high
guanine plus cytosine (G+C) content, which varies
from 54 to 67 mol% (Table 2). In addition, there are
notable dierences in physiological and biochemical
properties, including cell-wall constituents (Table 2).
They are saccharolytic organisms that produce acetic
and lactic acids without generation of CO
2
, except dur-
ing degradation of gluconate (Fig. 1). Heterofermenta-
tion is initiated by splitting fructose-6-phosphate into
one C
2
and one C
4
moiety. The conversion of the C
2
moiety to acetate is paralleled by the formation of hep-
tose-7-phosphate from the C
4
moiety concomitant with
the formation of a triose moiety derived from an addi-
tional molecule of fructose-6-phosphate. The heptose-7-
phosphate is subsequently split into two molecules of
acetate and one molecule of pyruvate. The second triose
moiety left from fructose-6-phosphate is converted into
lactate. Therefore, the fermentation of two moles of
hexose results in three moles of acetate and two moles
of lactate. The key enzyme in such glycolytic fermenta-
tion, fructose-6-phosphate phosphoketolase, may be
used as a taxonomic character in identication of the
genus, but does not enable interspecies dierentiation.
Besides glucose, all bidobacteria from human origin
are also able to utilize galactose, lactose and, usually,
fructose as carbon sources. The lactose transport system
for B. bidum DSM 20082 was identied recently as a
proton symport, based on inhibition of lactose uptake
by inhibitors of ATP synthesis and by compounds that
interfere with proton and metal ionophores [8]. Bido-
bacterium spp. are, in some instances, also able to fer-
ment complex carbohydrates; a recent study [9], in
which 290 strains of 29 species of bidobacteria from
human and animal origin were surveyed for their ability
to ferment complex carbohydrates, has conrmed this
potential. The substrates fermented by the largest num-
ber of species were d-galactosamine, d-glucosamine,
amylose and amylopectin. Porcine gastric mucin was
fermented only by B. bidum, whereas B. infantis was
the only species that could ferment d-glucuronic acid.
Strains of B. longum fermented arabinogalactan and
arabic, ghatti and tragacanth gums.
Table 1. List of species (by alphabetical order) of the genera
Bidobacterium and Lactobacillus
Lactobacillus Bidobacterium
L. acetotolerans L. jensenii
a
B. adolescentis
a
L. acidophilus
a
L. Johnsonii B. angulatum
a
L. agilis L. kandleri B. animalis
L. alimentarius L. ker B. asteroides
L. amylophilus L. keranofaciens B. bidum
a
L. amylovorus L. malefermentans B. boum
L. avarius L. mali B. breva
a
L. bifermentans L. minor B. catenulatum
a
L. brevis
a
L. murinus B. choerinum
L. buchneri
a
L. oris
a
B. coryneforme
L. casei sobs. casei
a
L. parabuchneri
a
B. cuniculi
L. collinoides L. paracasei
a
B. dentium
a
L. confusus L. pentosus B. gallicum
L. coryniformis L. pontis B. gallinarum
L. crispatus
a
L. plantarum
a
B. globosum
a
L. curvatus L. reuteri
a
B. indicum
L. delbrueckii L. rhamnosus
a
B. infantis
a
L. farciminis L. ruminis B. lactis
L. fermentum
a
L. sake B. longum
a
L. fructivorans L. salivarius
a
B. magnum
L. fructosus L. sanfrancisco B. merycicum
L. gallinarum L. sharpeae B. minimum
L. gasseri
a
L. suebicus B. pseudocatenulatum
a
L. graminis L. vaccinostercus B. psudolongum
L. halotolerans L. vaginalis
a
B. pullorum
L. hamsteri L. viridescens B. ruminantium
L. helveticus B. saeculare
L. hilgardii B. subtile
L. homohiochii B. suis
L. intestinalis B. thermophilum
a
Soc|os |so|atoo |iom |omao sooicos
!40 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
The optimum pH for growth is 67, with virtually no
growth at pH 4.55.0 or below or at pH 8.08.5 or
above. Optimum growth temperature is 3741

C, with
maximum growth at 4345

C and virtually no growth at

2528

C or below. Comprehensive details of their biol-

ogy are available in the extensive reviews by Rasic and
Kurmann [2] and Sgorbati et al. [7]. The latter have
provided an updated report on the taxonomy status of
the genus Bidobacterium, but their list of properly
identied bidobacteria does not include the moderately
oxygen-tolerant species Bidobacterium lactis, which
was identied recently [10].
Cooos Lactobacillus
In 1990, Moro was the rst researcher to isolate faculta-
tive anaerobic straight rods from the faeces of breast-fed
infants, which he typied as Bacillus acidophilus, a generic
name for intestinal lactobacilli. Lactobacilli are in general
characterized as gram-positive, non-sporeforming and
non-agellated rods or coccobacilli [11]. The G+C
content of their DNA is usually between 32 and 51
mol%. They are either aerotolerant or anaerobic and
strictly fermentative. Glucose is fermented pre-
dominantly to lactic acid in the homofermentative case,
or equimolar amounts of lactic acid, CO
2
and ethanol
(and/or acetic acid) in the heterofermentative counterpart.
At present, 56 species of the genus Lactobacillus have
been recognized (Table 1). Of these microorganisms, the
most commonly suggested for dietary use are Lactobacillus
acidophilus strains, the denition of which has changed
recently.
Comprehensive genetic studies have shown that the
original species actually consists of six DNA homology
groups, including L. crispatus, L. gallinarum, L. gasseri,
L. amylovorus and L. johnsonii. Although these species
are well dened, diculties are often encountered in
allocating newly isolated strains to each of these groups.
Investigations based on agglutination tests, cell wall
antigen and electrophoretic and antigenic characteristics
of d- and l-lactate dehydrogenases (LDH) also point
toward heterogeneity of these species [12]. Lactobacillus
acidophilus is a gram-positive rod with rounded ends
that occurs as single cells, as well as in pairs or in short
Table 2. Physiological and biochemical characteristics of Bidobacterium spp. and Lactobacillus acidophilus
a
Character Bidobacterium spp. Lactobacillus acidophilus
l|ys|o|ogy Aoaoiob|c \|cioaoio||||c
Co|| wa|| comos|too.
lot|oog|ycao tyo Vai|ab|o, bas|c am|oo ac|o |o t|o totiaot|oo |s o|t|oi
oio|t||oo oi |ys|oo, vai|oos tyos o| cioss-||o|ago
lys-l As
l|os|o|||o comos|t|oo'1o|c|o|c ac|o lo|yg|ycoio||os|o|||o aoo |ts |yso ooi|vat|vos,
a|aoy||os|at|oy|g|ycoio|, |yso ooi|vat|vos o|
o||os|at|oy|g|ycoio|
C|ycoio|
l|A-baso comos|t|oo \o| CC
goao|oocytos|oo
6 4
lact|c ac|o coo|goiat|oo l ll
Sogai motabo||sm lotoio|oimootat|vo lomo|oimootat|vo
a
Aoatoo |iom |oimaoo aoo kas|c '! aoo \|ta| aoo Caig '!2
Fig. 1. loimat|oo o| acotato aoo |actato |iom g|ocoso by t|o b||oom
at|way. !-|oxo||oaso aoo |ioctoso-6-|os|ato |somoiaso, 2-|ioc-
toso-6-|os|ato |os|o|oto|aso, -tiaosa|oo|aso, 4-tiaos|oto|aso,
-i|boso--|os|ato |somoiaso, 6-i|bo|oso--|os|ato--o|moiaso,
-xy|o|oso--|os|o|oto|aso, -acotato ||oaso, -oozymos as |o
|omo|oimootat|vo at|way aoatoo |iom kas|c aoo |oimaoo '2.
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !4!
chains. The typical size is 0.60.9 m in width and 1.5
6.0 m in length. It is non-agellated, non-motile and
non-sporeforming, and is intolerant to salt. This micro-
organism does not contain cytochromes and, therefore, is
benzidine negative. In addition, it is microaerophilic, so
surface growth on solid media is generally enhanced by
anaerobiosis or reduced oxygen pressure and 510%
CO
2
. The physiological and biochemical characteristics
of this microorganism are given in Table 2. Most strains
of L. acidophilus can ferment amygdalin, cellobiose,
fructose, galactose, glucose, lactose, maltose, mannose,
salicin, sucrose, trehalose and aesculine [13]. Lactose is
virtually the only sugar present in milk, yet L. acid-
ophilus has been reported to utilize sucrose more eec-
tively than lactose [12]; such observations may be
ascribed to dierences in -galactosidase (EC 3.2.1.23)
and -fructofuranosidase (EC 3.2.1.26) activities. While
-fructofuranosidase is a constitutive enzyme, -galac-
tosidase may be induced in L. acidophilus [14]. More-
over, both glucose and fructose moieties of sucrose are
utilized by L. acidophilus, whereas the galactose moiety
of lactose cannot be metabolized to an appreciable
degree. The glucose moiety is metabolized via the Emb-
denMeyerhofParnas pathway with lactic acid as
essentially the sole end product. The yield of lactic acid
is 1.8 mol/mol glucose, accompanied by minor amounts
of other compounds. Acetaldehyde, a carbonyl avour-
ing molecule, may also result from metabolism of lac-
tose, although in some instances it may be produced
from metabolism of nitrogen-containing substances, e.g.
threonine; a very high activity of threonine aldolase has
been found in L. acidophilus [15].
Growth of L. acidophilus may occur at as high a tem-
perature as 45

C, but optimum growth occurs within

3540

C. Its acid tolerance varies from 0.3% to

1.9% titratable acidity, with an optimum pH lying
at 5.56.0.
Ecology
Cooos Bidobacterium
Bidobacteria are microorganisms of paramount
importance in the active and complex ecosystem of the
intestinal tract of humans and other warm-blooded
animals, and of honeybees [7]. They are distributed in
various ecological niches in the human gastrointestinal
and genitourinary tracts, the exact ratio of which is
determined mainly by age and diet. The indigenous
microora of infants is dominated by bidobacteria,
which become established shortly after birth. Their
proliferation is stimulated by the glycoprotein compo-
nents of -casein in human colostrum and, to a lesser
extent, human milk. The number of bidobacteria
decreases with increasing age of the individual and
eventually becomes the third most abundant genus
(accounting for ca. 25% of the total adult gut ora)
after the genera Bacteroides and Eubacterium [16]. The
onset of old age is characterized by signicant reduc-
tions in the numbers of bidobacteria, whilst con-
taminant bacteria such as clostridia and coliforms tend
to increase, usually as a result of diminished secretion of
gastric juice in this age group [17]. Furthermore, the
prole of constituent species changes; B. infantis and B.
breve, typical of infants, are replaced by B. adolescentis
in adults, whereas B. longum persists lifelong [18]. This
age prole may obviously be inuenced by the dietary
intake of bidogenic factors [19] and by the host
physiology [17].
Cooos Lactobacillus
Lactobacilli are distributed in various ecological
niches throughout the gastrointestinal and genital tracts
and constitute an important part of the indigenous
microora of man and higher animals. Their distribu-
tion is aected by several environmental factors, which
include pH, oxygen availability, level of specic sub-
strates, presence of secretions and bacterial interactions.
They are rarely associated with cases of gastrointestinal
and extraintestinal infection, and strains employed
technologically are regarded as non-pathogenic and safe
microorganisms. Furthermore, they have the reputation
of health promoters, especially in the human gastro-
intestinal and genitourinary tracts [20].
Growth performance
l|oct o| sobstiato oo giowt|
Genus |||oobactoi|om
The fact that bidobacteria can grow in a semi-syn-
thetic medium containing only lactose, three free amino
acids (cysteine, glycine and tryptophan), several vitamins
and nucleotides, and some minerals contrasts with the
generally recognized fastidiousness of lactobacilli with
regard to nutritional requirements. Certain strains were
found to grow on a simplied medium comprised of
lactose (as fermentable carbohydrate), buers, minerals,
ammonium salts, cysteine, and the vitamins biotin and
calcium pantothenate [21]. Bidobacterium bidum var.
pennsylvanicus, a mutant strain, requires pantothenate
and N-acetylglucosamine-containing saccharides for
growth and synthesis of its cell wall. The B. bidum
strain discovered by Tissier in 1899, as well as some
other bidobacteria, require a nitrogen source (i.e. pep-
tides), yet neither pantothenate nor N-acetylglucos-
amine-containing saccharides are essential. A striking
dierence between lactobacilli and some strains of bido-
bacteria is the ability of the latter to grow in a medium
containing nitrogen in ammonium form; the remaining
bidobacteria strains require nitrogen from organic
sources [22]. Evidence indicates that the nutritional
properties of bidobacteria are species-dependent, so
any new strain discovered or produced needs to be
extensively studied in terms of its minimal nutritional
!42 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
requirements; this is the case of the newly identied
Bidobacterium lactis strain [23].
lactobac|||os ac|oo|||os
Lactobacilli have complex growth requirements. They
require low oxygen tension [13,24], fermentable carbo-
hydrates, protein and its breakdown products [25], a
number of vitamins of the B-complex [26], nucleic acid
derivatives [27], unsaturated free fatty acids [28], and
minerals such as magnesium, manganese and iron [29]
for their growth. Increased amount of thiol groups pre-
sent in whey protein-enriched milks favours the growth
of L. acidophilus, whereas peptone and trypsin stimulate
its acid production [22]. Addition of tomato juice (as a
source of simple sugars, minerals and vitamins of the B-
complex) to skimmed milk has provided evidence for
enhancement of both growth of (i.e. higher viable
counts and shorter generation times) and activity by (i.e.
improved sugar utilization and lower pH) L. acidophilus
[30]. These essential nutrients should, therefore, be
available in the medium for establishment of a pre-
dominant microora of lactobacilli; in their recent study
on optimization of cultivation of L. acidophilus, Tail-
landier et al. [31] concluded that the optimum condi-
tions are pH 6.0, 30

C, 40 g/L of glucose, 20 g/L of

peptone, 20 g/L of yeast extract, 5 g/L of sodium acetate
and 3 g/L of sodium citrate.
\||| as ao oooo|ooo ooti|oot moo|om
Bidobacteria are used in milk fermentations to a
limited extent because of their slow growth in that
matrix which, in many instances, may be considered as
an articial medium. However, milk is an essentially
satisfactory medium because it contains all essential
nutrients, except that amino acids and small peptides
are present at insucient concentrations (ca. 0.1 g/L) to
support extended growth of bidobacteria [2,23].
Nevertheless, cases of adaptation to milk upon multiple
transfers have been reported [22]. Comparison of var-
ious milk types (bovine, ovine and caprine milks) with
respect to their available nitrogen fraction has also been
exploited in an attempt to further probe the inuence of
the raw material upon selected probiotic strains, but the
higher protein and vitamin contents of ovine milk were
not sucient to sustain growth of B. lactis at the high
rates required [32]. Moreover, the excess of fatty acid
residues present in caprine milk were indicated as
responsible for the poor growth of L. acidophilus.
In general, bidobacteria grow better in rich synthetic
media, viz. TPY and MRS broths, than in milk; however,
said media are complex and costly for large-scale propaga-
tion of bidobacteria. Moreover, unless the cells harvested
from such media are extensively washed before incorpora-
tion, they may confer o-avours to the nished dairy
products. To manufacture a quality product, both in terms
of texture and viability of bidobacteria, a milk-based
medium is usually required because the casein content
of milk protein is higher than that of synthetic media
(which are generally low in solids). Thus, improvement
of growth conditions for the dierent strains of bido-
bacteria in milk, in particular via addition of various
easily-available nitrogen sources or redox potential-
lowering substances, has provided the impetus for many
studies [23,24,3234], and less fastidious, mildly-acid-
ifying strains can thus be claimed as key candidates for
successful bidobacteria-containing (bio)products.
|||oogoo|c aoo giowt| |actois
Denition
There are clear dierences between bidogenic factors
and growth factors for bidobacteria in terms of nature
and function. Bidogenic factors are dened as com-
pounds, usually of a carbohydrate nature, that survive
direct metabolism by the host and reach the large bowel
or cecum, where they are preferentially metabolized by
bidobacteria as source of energy. Bidogenic factors
may fall under the new concept of prebiotics, which are
dened as non-digestible food ingredients that bene-
cially aect the host by selectively stimulating the
growth and/or activity of one, or a limited number of,
bacteria in the colon, and which may thus improve the
health of the host [35]. In contrast, growth factors are
compounds that promote the growth of bidobacteria
in vitro but cannot be delivered to the large bowel or
cecum to selectively promote proliferation of bido-
bacteria [19]. Studies on the sources and applications for
either of these types of compounds have been well
documented [19], yet comprehensive reviews of the state
of the art in this eld are still lacking. Table 3 sum-
marizes established knowledge and more recent devel-
opments in the isolation, identication and application
of both bidogenic and growth factors.
Bidogenic factors
Fructooligosaccharides. The major carbohydrate
sources for bacterial growth in the colon are provided by
dietary or endogenous means. In this respect, non-diges-
tible oligosaccharides have been used in the diet speci-
cally to increase relative numbers of bidobacteria in the
gut microora [3639]. In particular, promising results
have been achieved in vitro using inulin [37] and especially
fructooligosaccharides (FOS), in both batch culture [38]
and chemostat culture systems [39]. Among the dierent
types of (linear and branched) FOS studied, oligo-
fructose, a linear chain molecule comprising glucose and
fructose at a degree of polymerization of four, exhibited
the highest bidogenic eect. Earlier in vivo clinical stu-
dies reported a ten-fold increase of the bidobacterial
population in the large bowel upon addition of neosu-
gars (i.e. short-chain FOS) to the human diet at a ratio
of 15 g/d [36], which correlated well with the increased
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !4
Table 3. Origin and eects of possible bidogenic and growth factors for Bidobacterium spp
Name Origin Strains studied Eects Reference
Bidogenic factors
Caso|o b||oos |actoi C|l |ov|oo caso|o sobm|ttoo to ac|o aoo
oozymo aa|o aoo os|o |yoio|ys|s
Bidobacterium bidum Ciowt| iomot|oo, bost w|t| ac|o C|l, C|l so||os
bot| ot|oos aoo caibo|yoiatos oocossaiy |oi giowt|
7b||ows|| aoo 7|aj|a '!!
lomao -caso|o aoo
g|ycomacioot|oo
ooi|voo t|oio|iom
|otact -caso|o |iom |omao m||| aoo
|yoio|ysoo w|t| c|ymos|o aoo os|o
Bidobacterium infantis S!2 Sma|| giowt|-iomot|og act|v|ty o| |otact -caso|o
oo|aocoo ooo oozymat|c |yoio|ys|s
Sogai oit|oo aoo o|yot|oo oit|oo as |moitaot
comoooots o| b||oos |actoi
Azoma et al. '!!6
-Caso|o oozymat|c o|gost |ov|oo m||| caso|o o|gostoo by tiys|o Bidobacterium s. B. bidum,
B. longum
Ciowt| iomot|og act|v|ty |o |o||y syot|ot|c moo|om
assoc|atoo w|t| o|so||oo'so|||yoiy| cyst|oo ios|ooos
aoo a coita|o tiyt|c ot|oo-oo|ooot||oo b||oogoo|c
|actoi
loc| aoo |oz|oiova|oy '0
Caso|o macioot|oo C\l |ov|oo m||| Bidobacterium s. Ciowt| iomot|og act|v|ty Abo-l|-Sa|am et al. '!!
|-acoty|oooiam|o|c ac|o |oo-Ac-
coota|o|og sobstaocos |ooAc, s|a|y|
|actoso, g|ycomacioot|oo
|ov|oo m||| Bidobacterium s. aoo |actobac|||| Ciowt| iomot|og act|v|ty o| B. breve, B. bidum,
B. infantis
|oota et al. '!2
tiaosga|actosy|atoo o||gosacc|ai|oos
ga|actosy| ga|actoso, ga|actosy| g|ocoso 1iaosga|actosy|at|oo oozymat|c ioact|oo
oo |actoso
Bidobacterium s. kat|o o| |otost|oa| b||oobactoi|a to tota| bactoi|a
|ocioasoo |iom 0.2 to 0.!, oocioasoo |ovo|s o|
tox|c s|oit c|a|o |atty ac|os, |aoca| l, |aoca|
ammoo|a
|to et al. '!!
lioctaos As|-|ioo w||to owooi |iom tobois o|
oiosa|om ait|c|o|o
Bidobacterium s. B. infantis
A1CC !6, B. adolescentis A1CC
!0, B. longum A1CC !0
Ciowt| iomot|og act|v|ty Yamaza|| aoo \atsomoto '!!
xy|oo||gosacc|ai|oos xy|ob|oso \|oat biao, coio cobs asoo, w|oat
stiaw
Bidobacterium s. |ocioaso o| |otost|oa| b||oobactoi|a w|t| !2 g'o O|aza|| et al. '!20
O||gosacc|ai|oos Oo|oo, gai||c, c||coiy ioot, boiooc|,
asaiagos, oiosa|om ait|c|o|o,
soyboaos, w|oat biao
Bidobacterium s. lio|||oiat|oo o| b||oobactoi|a aoo soioss|oo o|
otio|oct|vo bactoi|a
liovoot|oo o| coost|at|oo aoo at|ogoo|c o|aii|ooa
Yamaoa et al. '!2!
1omomatso '4!
liocto-o||gosacc|ai|oos |oo||o,
o||go|ioctoso
|acto|oso
Bidobacterium spp. Ciowt| aoo ac|o iomot|og act|v|ty, gooo caiboo
aoo oooigy sooico
C|bsoo aoo \aog ',
Bidobacterium s. Ciowt| iomot|og act|v|ty, act|vat|oo o| |mmooo
iosooso aoo |o|oct|oo ||m|tat|oo
Aojaoa et al. '6
\|zota| '4
2-Am|oo--caiboxy-!,4-oa|t|oqo|oooo Co||-|ioo ||tiato aoo mot|aoo| oxtiact|oo
|iact|oo o| co||s o| Propionibacterium
freudenreichii 02
Bidobacterium longum 600! Ciowt| st|mo|atoiy act|v|ty at 0.! og'm| o| t|o
b||oobactoi|a| oo|at|oo |o t|o |otost|oa| m|cio|oia
\oi| et al. '4
Growth factors
loo|ymoi|zoo a|g|oatos Soo|om a|g|oato soa-wooo Lessonia s.
ooo|ymoi|zoo by bactoi|a| a|g|oato |yaso
Bidobacterium s. B. breve,
B. longum, B. adolescentis, B. bidum,
B. infantis
Ciowt| iomot|og act|v|ty |o s||m-m||| A||yama et al. '!22
Caso|o |yoio|yzatos A|ca|aso, c|ymotiys|o aoo tiys|o
|yoio|ys|s o| caso|o |o||owoo by
o|tia||tiat|oo \\ cot-o| 0 |la
commoic|a| |yoio|yzato |-7 Caso
Bidobacterium s. B. bidum vai.
pennsylvanicus, B. adolescentis, B. breve,
B. infantis, B. longum
Ciowt| st|mo|at|oo o| B. infantis aoo B. breve |o
syot|ot|c moo|om, commoic|a| |yoio|yzato iomotoo
bottoi giowt| o| a|| stia|os t|ao o|tia||toioo |yoio|yzatos
lioo|x et al. '
Caso|o |yoio|yzatos a|ca|aso, c|ymotiys|o aoo tiys|o
|yoio|ys|s o| caso|o |o||owoo by
two-sto o|tia||tiat|oo \\ cot-o| ! |la
Bidobacterium s. B. breve, B. infantis,
B. longum
1iys|o ot|oo |iact|oo at 2 |o syot|ot|c Caic|os
moo|om iomotoo bost stia|o giowt|, a|ca|aso am|oo
ac|o |iact|oo at !2 ioiossoo giowt| o|, aoo ac|o
ioooct|oo by B. breve aoo B. longum
lioo|x et al. '4
||oozymo |yoio|yzoo so|ot|oos Soyboao aoo swoio jac|boao Canavalia
gladiata C||oa
|||oobactoi|om spp. |oi bovoiago
ioooct|oo
Ciowt| st|mo|at|oo ioooct|oo |o ioooot|og t|mo Yaog et al. '!2
\||| |yoio|yzatos lioto|oaso \ll aoo oootiaso |l|
|yoio|ys|s o| |l1 m||| to o||oioot oogioos
Bidobacterium lactis aoo Lactobacillus
acidophilus
24-| |yoio|ys|s o| \ll at iomotoo bost giowt|
o| B. lactis |o bov|oo, ov|oo aoo cai|oo m|||s
L. acidophilus was oot a|octoo by o|t|oi \ll oi
\l|, |ioo am|oo ac|o |iact|oo a|ooo was |oss ouc|oot
t|ao m|xtoio o| am|oo ac|os aoo |ow mo|oco|ai wo|g|t
ot|oos 00 la o| 24-| \ll
Comos aoo \a|cata '0,22
!
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7
release of volatile fatty acids and concomitant decrease
in pH (by 0.3 units), together with a reduction in the
counts of Enterobacteriaceae from 110
8
to 410
7
cfu/g.
All FOS studied are oligosaccharides that occur in
nature, mainly of plant origin (Table 3), and are manu-
factured industrially from sucrose syrup obtained from
sugar beet by an enzymatic process in which -fructo-
furanosidase (EC 3.2.1.26) derived from Aspergillus
niger plays an essential role. These compounds are
(2!1) linked fructans, typically polydisperse, with
degrees of polymerization in the range 235; they are
resistant to the strong hydrolytic capacities of the
human intestinal enzymes and, therefore, reach the
colon essentially unaltered, where they behave like
soluble dietary bre and are digested by the colonic
ora [40]. Other properties, applications and health
aspects closely related to their eect on bidobacteria
growth in the colon have been discussed in several
reviews [41,42]. The increased intake of oligofructose-
containing foods, or alternatively the increased incor-
poration of these prebiotics into foods with a signicant
carbohydrate content, e.g. ice cream, cakes, infant for-
mula feeds and milk-based products [43], oer a
rational alternative to the more commonly employed
incorporation of viable bidobacteria into fermented
milks. In fact, the main advantage is that survival in the
product becomes easier to assure.
Xylooligosaccharides (e.g. xylobiose) and transga-
lactosylated oligosaccharides (e.g. lactosucrose) are also
known as eective promoters of proliferation of intest-
inal bidobacteria (Table 3). The former compounds
occur naturally, whereas the latter are available only via
industrial synthesis. Their importance as bidogenic
factors is reected in the large number of publications
focussed on the preparation of galactooligosaccharides
through bioconversion of lactose using various micro-
bial enzymes [4446]. Oligosaccharides that contain
maltose, mannose and soya are also possible bidogenic
factors (Table 3).
Several studies, reviewed by Modler [19] and Mizota
[47], reported that the lactose-derivative lactulose, a
dissaccharide composed of galactose and fructose (4-O-
-d-galactopyranosyl-d-fructose), can exert bidogenic
eects on bidobacteria resident in the lower intestine
and hence can produce many of the desirable eects
ascribed to FOS. These eects include improvement of
hyperammonaemia and suppression of production of
toxic short-chain fatty acids (butyric and valeric acids);
in addition, this compound has many other health and
medical eects [47]. Lactulose has been used to increase
the numbers of viable lactobacilli in the intestine of
bottle-fed infants; other candidates, including such
sugar alcohols as lactitol and glucosyl-inositol (Table 3),
may also be useful in increasing bidobacteria numbers.
Neosugars, FOS and lactulose are commercially avail-
able and widely used in Japan and Europe as bidogenic
compounds. Although safe from a toxicological stand-
point and responsible for many positive eects, they are
not necessarily ideal bidogenic factors: to be eective, the
bidogenic factor must promote a selective fermentation
in mixed culture so that the prole of the large intestinal
microora is altered towards a potentially healthier
community. Unfortunately, both types of compounds
can be metabolized by a number of gas-producing bac-
teria and hence may cause intestinal discomfort.
Quinone compounds. Roland et al. [48] reported
consistent data in vitro (mixed culture of B. bidum and
48 h-old cultures of Propionibacterium freudenreichii in
MRS broth) and in vivo (administration of 510
10
cfu
of propionibacteria to 19 males over 2 weeks) in
attempts to check the validity of the hypothesis that
propionibacterium (a microorganism used in the manu-
facture of Swiss-type cheese) promotes the growth of
bidobacteria. A parallel study showed that, in a human
faecal culture contained in a two-stage, continuous cul-
ture system, addition of a quinone compound produced
by P. freudenreichii cells stimulated the growth of bi-
dobacteria, thus suggesting a bidogenic eect regard-
ing regulation of their numbers in the intestine [49]
(Table 3). However, more stringent studies are needed
to clarify the specic action of this, and other, related
quinone compounds upon bidobacterial growth.
Growth factors
Within the range of compounds investigated, deriva-
tives of human and bovine milks (Table 3) have proven
good candidates for the enhancement of growth of bi-
dobacteria in vitro. According to Poch and Bezkorovainy
[50], growth-promoting activity of bovine milk -casein
was ascribed to its conjugated cystine residues, an
observation more recently conrmed and rened in a
study aimed at comparing the growth-promoting eects
of various biological materials on B. longum [51] using a
synthetic medium. All tentative growth-promoting fac-
tors studied failed to exhibit their growth-promoting
activity when their dissulde bonds were reduced or
alkylated, thus suggesting that their composition in sulfur-
containing peptides might be the key to said eect. The
major whey proteins, -lactalbumin and -lactoglobulin,
were also found to be excellent growth promoters, a trait
validated after the studies by Petschow and Talbott [52]
on the eectiveness of whey fractions of both human
milk and bovine milk. Yeast extract, a commercial pro-
duct, was found to be an eective growth promoter, and
is most often used at concentrations that vary between
0.1 and 0.5%(v/v). A previous study [33] has shown,
however, that addition of yeast extract at 0.25%(v/v)
did not stimulate growth of B. infantis, even though acid
production was enhanced; it further demonstrated the
good growth-promoting activity of -glycerophosphate
in the presence of cysteine for B. bidum and B. infantis,
but not for B. longum ATCC 15708.
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !4
Other biological compounds identied as growth fac-
tors for bidobacteria include threonine [25], cysteine
[33], enzyme-treated chlorella, peptone and trypticase
[19], as well as dextrin, maltose and extracts from car-
rots (coenzyme A), potatoes and corn [2], and commer-
cial casein hydrolysates and ultraltered hydrolysates
[53,54] these hydrolysates are, in general, produced by
breaking down casein with proteases, peptidases or
strong acids to dierent yields. Proulx et al. [53,54]
claimed that peptides obtained from casein hydrolysates
might be a preferable source of nitrogen than free
amino acids for dairy-related bidobacteria (Table 3).
Apart from that, the development of hydrolysates with
better quality at lower cost is essential. Gomes et al. [23]
expanded on this work using milk (as economical
source), hydrolysed to several extents by two alternative
proteinases, as nitrogen source to promote growth of,
and acidication by B. lactis and L. acidophilus; these
authors established that both the nature of the enzyme
and the degree of hydrolysis are important criteria
towards attainment of consistent growth of the afore-
mentioned strains, an observation that conrms the
growth-limiting characteristics of available nitrogen in
the form of low molecular weight peptides (<500 Da)
and free amino acids that are more easily assimilated by
B. lactis (Table 3).
Nutritional and health values of fermented milk
products
l|stoi|ca| bac|gioooo
Nutritional and health aspects of functional foods
incorporating probiotic bacteria have received con-
siderable attention, and eventually led to numerous
purported claims in the literature. These potential
advantageous properties, initially summarized by Gurr
[55] and more recently reviewed in detail by Gilliland
[56], Marteau and Rambaud [57] and Yaeshima [58], are
summarized in Table 4 and will be critically described in
the following subsections. Despite the many studies on
the benecial nutropharmaceutical aspects of probiotic
bacteria, the results obtained are highly variable and
sometimes inconsistent with one another; hence, no
clear, unequivocal evidence for the actual existence of
some of these benets is yet available, which then ren-
ders health claims more dicult to establish. To elim-
inate these drawbacks, eorts have been pursued
worldwide to rationally organize future research using
clearer and more reliable bases (via well-designed, ran-
domized and placebo-controlled, double-blind studies)
and exploiting statistically validated methods, with spe-
cial emphasis on intestinal integrity and immune mod-
ulation. Increasing numbers of colonization and dose-
response studies dening the required doses have been
published [59].
|oti|t|ooa| va|oo
The nutritional benets of probiotics have been
mostly studied in milk-based products fermented with
lactobacilli and bidobacteria. These products contain a
great many chemical compounds depending on the type
of milk used (usually cow's, ewe's or goat's), on the type
of microorganisms added (and their specic biochemical
activities) and on the manufacturing processes
employed. They are characterized by a lower level of
residual lactose and higher levels of free amino acids
and certain vitamins than non-fermented milks. Fur-
thermore, they preferentially contain L(+)-lactic acid
(that is more easily metabolized by human beings than
D(-)-lactic acid) produced by bidobacteria in addition
to acetic acid [2], thereby preventing manifestation of
metabolic acidosis in infants below one year of age.
Moreover, the L(+)-lactic acid absorbed in the intestine
Table 4. Potential health and nutritional benets of functional foods prepared with probiotic bacteria
Benecial eect Possible causes and mechanisms
|miovoo o|gost|b|||ty lait|a| bioa|oowo o| ioto|os, |ats aoo caibo|yoiatos
|miovoo ooti|t|ooa| va|oo l|g|oi |ovo|s o| | v|tam|os aoo coita|o |ioo am|oo ac|os, v|z. mot||oo|oo, |ys|oo aoo tiyto|ao
|miovoo |actoso ot|||zat|oo kooocoo |actoso |o ioooct aoo |oit|oi ava||ab|||ty o| |actaso
Aotagoo|st|c act|oo towaios
ootoi|c at|ogoos
l|soioois, soc| as |ooct|ooa| o|aii|ooa, mocoos co||t|s, o|coiatoo co||t|s, o|voit|co||t|s aoo
aot|b|ot|c co||t|s cootio||oo by ac|o|ty, m|ciob|a| |o||b|tois aoo iovoot|oo o| at|ogoo ao|os|oo
oi at|ogoo act|vat|oo
Co|oo|sat|oo |o got Soiv|va| |o gasti|c ac|o, ios|staoco to |ysozymo aoo |ow soi|aco toos|oo o| |otost|oo, ao|oiooco
to |otost|oa| mocosa, mo|t|||cat|oo |o t|o |otost|oa| tiact, |mmooo mooo|at|oo
Aot|caic|oogoo|c o|oct Coovois|oo o| otoot|a| io-caic|oogoos |oto |oss |aim|o| comoooos
|o||b|toiy act|oo towaios somo tyos o| caocoi, |o ait|co|ai caocois o| t|o gastio|otost|oa|
tiact by oogiaoat|oo o| io-caic|oogoos, ioooct|oo o| caic|oogoo-iomot|og oozymos aoo
st|mo|at|oo o| t|o |mmooo systom
lyoc|o|ostoio|om|c act|oo lioooct|oo o| |o||b|tois o| c|o|ostoio| syot|os|s. |so o| c|o|ostoio| by ass|m||at|oo aoo
ioc||tat|oo w|t| oocoojogatoo b||o sa|ts
|mmooo mooo|at|oo lo|aocomoot o| macio|ago |oimat|oo, st|mo|at|oo o| ioooct|oo o| soiossoi co||s aoo
-|otoi|oioo
Vacc|oo vo||c|o |atoia||y occoii|og oi il|A vacc|oa| o|toos
!46 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
is used as energy source, with an energy yield of 15 kJ/g,
that compares well with 16 kJ/g for lactose [55].
Lactobacillus acidophilus and bidobacteria have also
been reported to synthesize folic acid, niacin, thiamine,
riboavin, pyridoxine and vitamin K, which are slowly
absorbed by the body [2,60]. The vitamins of the B-
complex are frequently obtained as natural ingredients
in foods, so addition of bidobacteria to the diet will
more eectively help meet those requirements. The
bioavailability to the host of such minerals as calcium,
zinc, iron, manganese, copper and phosphorus may also be
enhanced upon consumption of fermented dairy products
containing bidobacteria via lowering the gastric pH
(which facilitates ionization of minerals, a requirement for
absorption) and improved digestibility of the protein [2].
1|oiaoot|c o|octs
Eects on intestinal disturbances and intestinal infections
The intact intestinal epithelium, together with the
normal intestinal microora represents a barrier to
movement of pathogenic bacteria, antigens and other
noxious substances from the gut lumen to the blood. In
healthy subjects this barrier is stable, thus protecting the
host and assuring normal intestinal function. When
either the normal microora or the epithelial cells are
disturbed, as triggered by dietary antigens, pathogens,
chemicals or radiation, defects in the barrier mechan-
isms become apparent; altered permeability facilitates
blood invasion by pathogens, foreign antigens and other
harmful substances. Several reviews [20,57] have docu-
mented the use of probiotic bacteria to treat intestinal
disorders, e.g. acute rotavirus diarrhoea in children, as
well as food allergy and colonic disorders driven by
pelvic radiotherapy and sometimes associated with
development of colon cancer. These examples are
among those included in Table 5 that illustrate the state
of the art in what concerns application of several pro-
biotic strains for therapeutic purposes. In all such dis-
ease states, altered intestinal microora, impaired gut
barrier and dierent types of intestinal inammation are
often present [20], thereby oering a rationale for the
eective use of probiotic bacteria, not only for treat-
ment but especially for prevention of such changes.
Among the possible mechanisms responsible for
favourable clinical responses, one may quote (i) pro-
motion of the immunologic and nonimmunologic
defense barriers in the gut [61,62] (ii) capacity to prevent
pathogen adherence or pathogen activation via produc-
tion of inhibitory metabolites, such as organic acids,
hydrogen peroxide, bacteriocins and deconjugated bile
salts [6365], or via ferrous iron uptake thus making it
unavailable for pathogens [66], and (iii) modication of
bacterial enzyme activity and subsequent inuence on gut
mucosal permeability [20] (see Table 5). Probiotic bacteria
are able to survive gastric conditions (characterized by pH
1.52.0 during fasting and 4.05.0 after a meal), colonize
the intestine (at least temporarily) by adhering to the
intestinal epithelium in a process mediated by adhesins on
the surface of the bacteria (proteins, polysaccharides and
cell wall-associated components), and oer new dietary
alternatives for the stabilization of the intestinal micro-
ora. In clinical nutrition, for instance, special diets are
used for the treatment of intestinal inammation,
radiation enteritis and inammatory bowel disease:
probiotic bacteria do play a role in alleviating these
disturbances, so the introduction of such strains into
clinical foods and special dietary foods is in order.
Potential antitumor activity
The colonic ora has been shown to be involved in
colonic carcinogenesis [20] This eect is mediated by
microbial enzymes such as -glucuronidase, -glucosi-
dase, nitroreductase and urease, which convert pro-
carcinogens into carcinogens. Experiments performed in
animal models showed that a few strains of L. acid-
ophilus and Bidobacterium spp. are able to decrease the
levels of enzymes responsible for activation of some
procarcinogens, and consequently decrease the risk of
tumor development [12,17]. Studies in the human body
using the same enzymes as end-points demonstrated a
general reduction in microbial enzyme activities and
concomitant decrease in faecal mutagenicity [57,67] The
benecial eect could be accounted for by a favourable
change in the composition of the intestinal ora following
introduction of the aforementioned bacteria.
Furthermore, it has been demonstrated, at least for
Lactobacillus casei (Shirota strain), that there is a
potential opportunity in the area of `dietary prevention'
of cancer. Morotomi [68] concluded that the parenteral
administration of this strain had antitumor and immu-
nostimulating eects on experimentally implanted
tumors; similar eects have been observed in the case of
oral administration. The feeding of B. longum to rats
was recently shown to signicantly reduce formation of
such preneoplastic markers as azoxymethane-induced
colonic aberrant crypt foci; furthermore, the production
of glutathione S-transferase, a Phase II enzyme marker,
was also increased [69].
The mechanisms that appear to be involved in anti-
tumor activity of systemically administered bidobacteria
include activation of such non-specic cellular factors as
polymorphonuclear leucocytes [70], macrophages [71]
and natural killer cells via regulation of -interferon
production [72]; -interferon also exhibits antiviral and
antiproliferative eects. Orally administered bido-
bacteria play a role in increasing, to some extent, the
production of IgA antibodies [73] and the functions of
Peyer's patch cells [74,75]. It should be noted that closely
related strains of Bidobacterium spp. and L. acidophilus
dier between each other in immunostimulatory prop-
erties, so some strains may not have all those properties
necessary for activity. Marin et al. [76] have recently
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !4
Table 5. Therapeutic applications of Bidobacterium spp. and Lactobacillus acidophilus as indigenous microorganisms or dietary adjuncts, and corresponding reported eects
Strain Experimental parameters Observations Eect Reference
Indigenous microorganism
Lactobacillus acidophilus kl2,
L. acidophilus l4
Aoa|ys|s o| co||s aoo soot biot| co|toio
|o ! \kS biot| coota|o|og oxga||
komova| o| c|o|ostoio| ouc|oot |o t|o iosooco o| oxga|| ooooi
aoaoiob|c cooo|t|oos
liosooco o| oxga|| caosoo aoaiaoco o| |oss c|o|ostoio| |o biot|
aoo moio |o co||s
kl2 aoo l4 aio b||o ios|staot bot oo|y kl2 ass|m||atos c|o|ostoio|
|o t|o iosooco o| b||o sa|t
Stia|o soc||c|ty o| L. acidophilus |s soggostoo
lyoc|o|ostoio|om|c act|oo o|ioct
act|oo o| co|toio oo c|o|ostoio|
ass|m||at|oo oi co-ioc|tat|oo w|t|
b||o sa|ts
C|||||aoo '6
Lactobacillus acidophilus kl2,
L. acidophilus \|l4!,
L. acidophilus \|l, L. acidophilus
\|l!!, L. acidophilus Cl!,
Bidobacterium bidum \|l0
Aoa|ys|s o| co||s aoo soot biot| co|toio
|o ! \kS biot| coota|o|og oxga||
kl2 |s o|oct|vo |o t|o ioc||tat|oo o| c|o|ostoio| aoo b||o sa|ts
at |ow l
Absooco o| b||o ac|os |o o|t|oi sooioataot oi co||s |o||ow|og |ocobat|oo
lyoc|o|ostoio|om|c act|oo
co-ioc||tat|oo o| c|o|ostoio|
|o t|o iosooco o| oocoojogatoo
b||o sa|ts aoo |ow l
||avoi aoo vao ooi \ooi '0
Bidobacterium longum || 6
B. breve A1CC !00, B. animalis
A1CC 22
kost|og co|| assays co|toio |o 1lY
moo|om coota|o|og oxga||
komova| o| c|o|ostoio| by ioc||tat|oo aoo ass|m||at|oo
B. longum ||6 |s t|o most ouc|oot |o oocoojogat|og b||o sa|ts
lyoc|o|ostoio|om|c act|oo 1a|i| et al. '!
Lactobacillus acidophilus 0! |o||b|toiy act|v|ty o| L. acidophilus aga|ost
o||oioot at|ogoos S. typhi , P. vulgaris
204, S. aureus C2-1!0, Y. enterocolitica 0,
E. coli 6 w|oo giowo s|mo|taoooos|y |o
m|||, stooy o| giowt|-|o||b|t|oo io||os
S|ai oocioaso o| at|ogoo oo|at|oos by | o| |ocobat|oo
|o||b|t|oo s|owo by L. acidophilus |s ooo to ac|o ioooct|oo
aoo ioooct|oo o| aot|b|ot|c-|||o comoooos
Aot|bactoi|a| act|v|ty Cota et al. '!24
Lactobacillus acidophilus 1|!2 Stooy o| mooo o| act|oo o| ac|ooc|o !2,
a bactoi|oc|o iooocoo by L. acidophilus
1|!2
|o||b|toiy act|oo caosos |ocioaso |o oimoab|||ty o| cyto|asm|c
mombiaoo o| taigot co||s by bot| o|ss|at|oo o| t|o iotoo
mot|vo |oico aoo oio |oimat|oo
|actoi|c|oa| act|v|ty 1a|aia et al. '64
Bidobacterium s. B. breve,
B. longum, B. bidum, B. infantis
lotoiocyto-|||o Caco-2 mocos-sociot|og
l12-\1x co||s |oi ao|os|oo stoo|os,
c|aiactoi|zat|oo by ||g|t m|cioscoy, Sl\.
tyo o| moc|ao|sm |ovo|voo |o |otoiact|oo
w|t| oo|aiyot|c co||s
Ao|os|oo to |omao |otost|oa| o|t|o||a| co||s, bactoi|a|
ma|otooaoco |o t|o |otost|oa| tiact aoo |o||b|t|oo o|
ootoioat|ogoo-co|| |otoiact|oos
Stab|||zoi o| got mocosa| baii|oi,
|mmooo oo|aocoi
|oioot et al. '6!
Bidobacterium s. S|x o||oioot stia|os, iocaic|oogoos, o|ti|to
aoo o|tiosam|oos, tostoo
|o giowt| |o||b|t|oo at 0 mo|
!
o|ti|to aoo 200 g m|
!
o|tiosam|oo
||ti|to o||m|oat|oo by ooo-oozym|c moc|ao|sm, oss|b|y v|a ac|o
ioooct|oo
B. longum motabo||zoo o|tiosam|oo by |otiaco||o|ai moc|ao|sm
Aot|caic|oogoo|c o|oct Ci||| et al. '!2
Bidobacterium infantis |Cl| 220
Bidobacterium s.
|ocobat|oo o| stia|os, w|t| E. coli aoo
C. perfringens |o. batc| |oimootat|oos,
two-vosso| c|omostat, s|og|o-stago
coot|oooos co|toio systom, soiom tobo
oxoi|moots aoo |ato oxoi|moots
|o||b|toiy o|oct oss|b|y io|atoo to ac|o ioooct|oo aoo
oxciot|oo o| aot|m|ciob|a| sobstaoco w|t| bioao soctiom
o| act|v|ty Salmonella, Listeria, Campylobacter, Shigella, V. cholerae
Aot|bactoi|a| act|v|ty oss|b|o
iotoct|oo aga|ost gastioootoi|t|s
C|bsoo aoo \aog '6
Lactobacillus acidophilus A1CC 46
Bidobacterium thermophilum
A1CC 266, B. breve A1CC !4!
Co||s giowo |o moo||oo 1lY moo|om, lo
2
accomo|at|oo stoo|oo |o two moo|a at l .0
aoo l 6. aoo o|oct o| g|ocoso as oooigy
sooico
1iaosoit o| lo
2
|oto co|| aoo ait|a| ox|oat|oo to lo

by
|otiaco||o|ai otat|vo |oiiox|oaso
lozymo osos oxygoo as sobstiato to iomoto lo
2
ox|oat|oo
aoo ioqo|ios g|ocoso as os|t|vo o|octoi
lxtiaco||o|ai ox|oat|oo by L. acidophilus v|a io|oaso o| l
2
O
2
lo
2
ooava||ab|o |oi io|||oiat|oo o| at|ogoos
|oti|t|ooa| |mmoo|ty aot|bactoi|a|
act|v|ty
|ot et al. '6266
Bidobacterium longum S|1 22
aoo ot|oi Bidobacterium s.
Comot|t|vo b|oo|og o| Bidobacterium s. aoo
E. coli lb !6 to gaog||o-totiaosy|coiam|oo CA!
oxam|ooo in vitro os|og 1lC ovoi|ay b|oo|og
soioss|oo assay, stooy o| t|o |o||b|toiy |actois
|o t|o Bidobacterium co|toio sooioataot |o|o
Comot|t|vo oxc|os|oo o| E. coli |iom CA! by Bidobacterium co||s
||oo|og |o||b|toi iooocoo by B. longum S|1 22 |s a ioto|oacooos
mo|oco|o
|o||b|t|oo o| at|ogoo|c E. coli b|oo|og to CA! oo soi|aco o| |omao
|otost|oa| mocosa
Aot|bactoi|a| act|v|ty loj|waia et al. '6
continued overpage
!
4

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(
1
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9
)
1
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1
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1ab|o continued
Strain Experimental parameters Observations Eect Reference
Bidobacterium longum S|1
22 |l22
l|octs oo g|ocoso coosomt|oo, |otoi|oo||o |l-!
ioooct|oo aoo cytotox|c|ty o| |agocytos stoo|oo
in vitro, |l22 |as ||g| |ovo| o| m|togoo|c act|v|ty
stia|os so|octoo
C|ocoso ota|o aoo cytotox|c|ty oo|aocoo
|l-! ioooct|oo st|mo|atoo ovoo at |ow oosos o| |l22
St|mo|atoiy act|v|ty oo |l-! ioooct|oo coiio|atoo w|t| m|togoo|c
act|v|ty
|mmooo oo|aocoi |aoo-O|a et al. '4
Bidobacterium adolescentis \!0!-4 |so|at|oo, oi||cat|oo aoo c|aiactoi|zat|oo o|
watoi-so|ob|o |iact|oo o| o|ysacc|ai|oo oatoio Sll
Sll was c|aiactoi|zoo by ga|acto|oiaoosy| ios|ooos
In vitro assay o| moi|oo s|ooocytos aoo loyois
atc| co||s to tost m|togoo|c act|v|ty
\|togoo|c act|v|ty was ox||b|too by a|| |iact|oos o| B. adolescentis
Sll st|mo|atoo io|||oiat|oo o| moi|oo s|ooocytos
|mmooo oo|aocoi losooo et al. '
Dietary Adjunct
Lactobacillus acidophilus,
Streptococcus thermophilus
\oao|og |gs o|v|ooo |oto 4 gioos, oac| gioo
g|voo ooo o| 4 o|ots basa| o|ot at 2 coocootiat|oos
o| basa|ac|oo|||os yog|oit |oi !4 w|
C|o|ostoio|, ti|g|ycoi|oos aoo ||oioto|os aoa|ysoo
koooct|oo o| ti|g|ycoi|oos aoo soiom c|o|ostoio| w|t| 0 yog|oit
! o-ios|ooa| o|oct
lyoc|o|ostoio|om|c act|oo ooos et al. '
Lactobacillus acidophilus lA2 S|x |oa|t|y sobjocts aom|o|stoioo m|||
|oimootoo w|t| L. acidophilus lA-2
Comai|soo o| |aoca| motagoo|c|ty bo|oio aoo
a|toi aom|o|stiat|oo
Soioss|oo o| |aoca| motagoo|c|ty a|toi |ogost|oo o| |oimootoo m|||
Lactobacillus s. aoo Bidobacterium s. oo|at|oos |ocioasoo |o
|aocos o| a|| sobjocts
Aot|motagoo|c o|oct |o
|omao |otost|oo
losooa et al. '6
Bidobacterium longum aoo
Lactobacillus acidophilus
loiat|oo o| oxoi|moot ! w|, !6 |oma|os aoo
ma|os agoo ! yi, |acobo oi m|xtoio
E. faecium B. longum oi m|xtoio
L. acidophilus B. longum L. delbrueckii
sobs. bulgaricus S. thermophilus
\|ciob|o|og|ca| ova|oat|oo o| jojooa| as|iatos
aoo |aoca| sam|os
kooocoo aoaoiobo.aoiobo iat|o |o |aocos by -|o|o ooi|og tioatmoot
l|sta| |otost|oa| m|cio|oia a|octoo
Co|oo|zat|oo o| |otost|oa| tiact ||o|soo et al. '!26
Bidobacterium longum loiat|oo o| oxoi|moot o| w|, !2 |oa|t|y
vo|ootoois
Yog|oit 00 m|'o aoooo w|t| B. longum
!0

bactoi|a'| aoo |acto|oso g'| oi

coovoot|ooa| yog|oit
|ocioasoo oxciot|oo o| b||oobactoi|a a|toi coosomt|oo
|ioat|-|yoiogoo ox|a|at|oo o|ovatoo gioat
Stab|||ty'ba|aoco o| |omao |aoca| |oia
Co|oo|zat|oo o| |otost|oa| tiact,
oss|b|o io|o |o co|oo caic|oogooos|s
|aitiam et al. '!2
Lactobacillus acidophilus lA!
Bidobacterium bidum |b!2
loiat|oo o| oxoi|moot o| w|, two gioos
o| |oa|t|y vo|ootoois
lac| coosomoo |oimootoo m||| ioooct
coota|o|og ooo o| t|o two stia|os, b|ooo
sam|os stoo|oo
laoca| co|oo|zat|oo ois|sts |oi 6 w| a|toi |ogost|oo
|ocioasos |o |agocytos|s o| E. coli co|oc|oo
|oosoc||c, aot|o|oct|vo moc|ao|sms o| oo|ooso oo|aocoo
|mmooo oo|aocoi Sc|||i|o et al. '!2
Bidobacterium longum aoooo
w|t| |acto|oso
6! ma|o l|s|oi 44 woao|og iats o|v|ooo |oto
4 gioos o| ! iats, oac| gioo g|voo ooo
o| 4 o|ots |oi ! w|, sobcotaoooos |ojoct|oo
o| azoxymot|aoo at w| o| ago, aboiiaot
ciyt |oc| ACl |o co|oo aoo g|otat||ooo
S-tiaos|oiaso CS1 |o co|oo|c mocosa aoo
||voi woio aoa|yzoo
looo|og o| |acto|oso aoo B. longum so|o|y aoo |o comb|oat|oo
iooocoo t|o oomboi o| ACl aoo tota| oomboi o| aboiiaot ciyts
Co|oo|c mocosa| CS1 |ovo|s aio s|go||caot|y
l|g|oi os|t|vo coiio|at|oo botwooo ||g|oi coca| l aoo oomboi
o| ACl
Aoo|t|vo aot|tomoi o|oct Aojaoa et al. '6
A
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(
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9
9
9
)
1
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9

1
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7
!
4

indicated, following an in vitro study of 14 bidobacteria

strains, that four, used commercially for incorporation
in dairy foods (i.e. Bidobacterium Bf-1, Bf-6, Bf-12 and
Bf-13) showed a great capacity for cytokine stimulation
by macrophages, and could thus potentially modulate
the immune response either directly, or indirectly via
incorporation as food additives.
Potential hypocholesterolemic actions
Several studies indicated a statistically signicant
reduction in serum cholesterol during consumption of
large doses (6805000 mL/d) of certain fermented dairy
bioproducts [57] and the theory postulated for the
hypocholesterolemic eect was the presence of such
organic acids as uric, orotic and hydroxymethylglutaric,
which actually inhibit cholesterol synthesis [77]. Unfor-
tunately, these data have not permitted extrapolation to
more realistic consumption conditions (the doses used
are indeed excessively high), and were impoverished by
the lack of controls and the fact that in some cases the
strains used were ill-dened. Although a few experi-
mental animal studies (Table 5) were successful in posi-
tively attributing cholesterol-lowering properties to L.
acidophilus contained in dairy products administered as
acidophilus yoghurt to pigs [78] and mice [79], results
cannot be extrapolated to humans since there are dif-
ferences in the regulation of cholesterol metabolism
between animals and humans. Besides the hypocholes-
terolemic action, Akalin et al. [79] also reported a higher
number of faecal lactobacilli and fewer coliforms in
mice that received acidophilus yoghurt than in those
that received plain yoghurt, thus indicating that L.
acidophilus established itself more eectively in the
murine intestinal tract than did L. bulgaricus in the
yoghurt diet.
The mechanisms by which fermented milk products
are able to diminish serum cholesterol have not to date
been fully demonstrated. In vitro studies by Gilliland
[56] showed that bidobacteria and L. acidophilus are
able to utilize cholesterol in growth media, both by
assimilation and precipitation with deconjugated bile
salts under acidic conditions. Their suggestion that the
presence of bile salts is a prerequisite for cholesterol
assimilation by bidobacteria was conrmed by the
observations of Klaver and van der Meer [80] (Table 5),
and more recently by those of Tahri et al. [81,82], who
went one step further and investigated the nature of the
bile salts; according to these authors [82], assimilation
of cholesterol was higher in the presence of trihydrox-
yconjugated bile salts than dihydroxyconjugated bile
salts. The aforementioned bacterial assimilation of cho-
lesterol in the intestine may reduce its absorption from
the digestive tract into the blood system.
Despite the existing and ever increasing body of
knowledge, considerable research is still warranted to fully
elucidate causal relationships between the consumption
of probiotic bacteria and reduced serum cholesterol
levels in humans.
Dairy products supplemented with Bidobacterium
spp. and/or Lactobacillus acidophilus
Coooia| c|aiactoi|st|cs
The species most frequently employed in the produc-
tion of probiotic dairy products are of human intestinal
origin because it is generally accepted that they are bet-
ter suited to the physiological needs of the host and can
more easily colonize his/her intestine than wild strains,
or strains that exist in the colon of other animals. These
human-borne strains include Bidobacterium ado-
lescentis, B. bidum, B. breve, B. infantis, B. longum,
Lactobacillus acidophilus, L. casei subsp. rhamnosus and
Enterococcus faecium [22,83]. Results of recent studies
support the use of selected strains of B. longum as dietary
adjuncts in dairy products, with B. adolescentis and B.
infantis as adequate alternatives [84]. It is again worth
noting that each strain within these species exhibits unique
properties, viz. growth rate, metabolic rate, proteolytic
activity and avour promotion. Consequently, careful
management of such factors via tailored manufacturing
technologies will enable these species to meet with
varying degrees of success in multiple industrial appli-
cations. Cases of milk products fermented by bido-
bacteria of animal origin have also been reported: these
strains are much easier to cultivate and can withstand
the adverse conditions encountered during industrial
production, viz. low pH and presence of detectable levels
of oxygen. In addition to a benecial role after consump-
tion [85,86], the recently identied B. lactis strain is also a
promising candidate due to its good acid and oxygen tol-
erance [23,87].
The most frequent functional food products in the
market are of dairy origin (Table 6); Japan, a leading
country in their manufacture, produces and markets
more than 50 dierent dairy products containing viable
cells. Similar trends are also observed in such developed
European countries as France, Germany and Sweden
(where probiotic products account for 25% of all fer-
mented milk products). It is estimated that there are
80 bido-containing products available in the world
market, and more than 45 dairy plants in Europe cur-
rently produce bido-acidophilus products [83]. Besides
the commercial products, research studies have been
more and more focused on fermented milks [12,88],
yoghurt [1], frozen yoghurt and desserts [89,90], ice
cream [91,92], cheese [9395,106], fermented soya milk
[96] and soya yoghurt [97]. The designation of new pro-
ducts is normally obtained via adding the prex `Bio' to
its traditional (sometimes partially simplied) name (e.g.
Biogurt, Biodrink, Biokys). The probiotic strains of
bidobacteria and L. acidophilus may be added, either
alone or following combination with other lactic acid
bacteria during fermentation, to the nal fermented
!0 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
product, or to the fresh product before distribution.
General accounts of the physicochemical and technolo-
gical aspects of commercial fermented milk products
containing Bidobacterium spp. and L. acidophilus have
been provided by Kurmann and Rasic [17], Mital and
Garg [12] and Tamine et al. [60].
In addition to food products containing probiotic
bacteria, there are various health products as well as
pharmaceutical preparations containing probiotics,
available in the market. In general, these consist in
encapsulated freeze-dried bacterial populations that are
used in the treatment of gastrointestinal disturbances
(diarrhoea, including side eects of antibiotic therapy),
constipation and certain hepatic diseases.
1oc|oo|og|ca| c|aiactoi|st|cs
Production of high-quality fermented milk products
containing Bidobacterium spp. and Lactobacillus acid-
ophilus is a major challenge to dairy plants owing to the
sensitive character of the microorganisms in these bio-
products, which adds to the usual diculties encoun-
tered in novel food products (i.e. poor palatability and
consequent limited consumer acceptability). In parti-
cular, bidobacteria tend to exhibit weak growth and
acid production in milk, which invariably requires long
fermentation times and conditions of anaerobiosis, low
redox potential at least in the early phase of growth
[24,98] and, often, addition of growth-promoting fac-
tors to the milk [19,23,32,53,54], as previously discussed.
In addition, bidobacteria produce, during fermenta-
tion, acetic and lactic acids at the ratio 3:2, so excessive
growth may yield products with vinegar-like taste and
aroma, which are obviously not acceptable to con-
sumers [83]. Careful selection of the strains employed
and good monitoring throughout the manufacturing
process are therefore compulsory in attempts to control
eciently the metabolic products and hence the nal
pH. The use of combined cultures of bidobacteria and
L. acidophilus or other lactic acid bacteria, viz. Lacto-
bacillus delbrueckii subsp. bulgaricus and Streptococcus
thermophilus, S. thermophilus alone or mesophilic aro-
matic cultures, has also been advocated as a solution for
many such problems [23,83,94,99]; increased growth
rates and reduction of fermentation time, absence of
certain sensory and texture defects and further
improvement of nutritional value of `bidus' products
are advantages brought about by the latter possibility.
Adverse eects with respect to viability have, however,
been reported for some strains of Bidobacterium [99]
and L. acidophilus [98].
A large number of studies has been published per-
taining to characteristics of good probiotics [5,83] and
Table 6. Commercial products containing Bidobacterium spp. and Lactobacillus acidophilus
a
Product Country of origin Microorganisms
A- loomai| Lactobacillus acidophilus, Bidobacterium bidum, Leuconostoc mesenteroides
s. cremoris, moso||||c |actococc|
Ac|oo|||os bottoim||| |SA Lactobacillus acidophilus, Leuconostoc mesenteroides s. cremoris, moso||||c
|actococc|
liogoit Bidobacterium bidum, Lactobacillus acidophilus, moso||||c |actococc|
Ac|oo|||os m||| Sovoia| coooti|os Lactobacillus acidophilus
Ac|oo|||os yoast m||| loimoi |SSk Lactobacillus acidophilus, Saccharomyces fragilis, S. cerevisiae
A-| Yog|oit liaoco Bidobacterium bidum, Lactobacillus acidophilus
Co|toia loomai| |b|oom
\|||y |ta|y |b|oom
|o-1i|s| A'| \||| |SA |b|oom
||om||o Sovoia| coooti|os Bidobacterium s., Lactobacillus acidophilus
Ac|oo|||os yog|oit
ACO-yog|oit
Sovoia| coooti|os Lactobacillus acidophilus, L. delbrueckii sobs. bulgaricus, Streptococcus
thermophilus
|-Act|vo liaoco Lactobacillus acidophilus, Bidobacterium bidum, L. delbrueckii sobs. bulgaricus,
Streptococcus thermophilus
lios| |A || |b|oom
|yi |ta|y |b|oom
Yo|os Aostia||a |b|oom
||ogaioo Coimaoy Lactobacillus acidophilus, Bidobacterium bidum, Streptococcus thermophilus
O||os liaoco |b|oom
l|||os |oiway |b|oom
|||oos m||| Sovoia| coooti|os Bidobacterium bidum, B. longum
|||g|oit Coimaoy Bidobacterium bidum, Streptococcus thermophilus
||ogoit Coimaoy Lactobacillus acidophilus, Streptococcus thermophilus
||o|ys Czoc| koob||c Bidobacterium bidum, Lactobacillus acidophilus, Pediococcus acidilactici
\||-\|| aao Lactobacillus acidophilus, Bidobacterium bidum, B. breve
A|o|t aao Lactobacillus acidophilus, Bidobacterium bidum, B. breve, L. casei sobs. casei
a
Aoatoo |iom |oimaoo '22 aoo lo|oi '
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !!
criteria for selection thereof [17,20,84,100]; these are
summarized in Fig. 2. The most important properties
include: (i) acid and bile tolerances, which are essential
to maintain high viable cell numbers during storage and
during passage through the digestive tract following
oral uptake [24,101103]; (ii) adherence to the human
intestinal mucosa, which is needed for temporary colo-
nization of the human gastrointestinal tract; and (iii)
production of antimicrobial substances, with con-
comitant inhibition of pathogens [20]. Furthermore, the
strains selected should produce a nal product posses-
sing good taste and acceptable body and texture, a
selection step that cannot be achieved unless the pro-
duct is actually manufactured.
It is also important that the strains selected can be
produced in large scale; in fact, owing to a slow propa-
gation in milk, they will hardly be competitive in the
presence of other microorganisms and will, thus, be
easily outnumbered. Therefore, aseptic working condi-
tions and special growth-promoting factors (as dis-
cussed above) are pre-requisites to ensure high initial
viable cell counts thereof. Voelki and Spillmann [104]
optimized the process of preparation of starters for
posterior manufacture of bidus milk and concluded that
active starters can be prepared with two specic strains,
Bidobacterium breve ATCC 15700 and B. longum ATCC
15707, under non-strictly anaerobic conditions, by using
milk initially supplemented with readily available N-com-
pounds (e.g. lactalbumin hydrolysate). A wide range of
studies have made it clear that the production of starters
of bidobacteria and L. acidophilus, in individual and
highly concentrated freeze-dried or deep frozen forms,
for direct inoculation of processed milk, oers great
exibility in control of the desired sensory and micro-
biological qualities (in terms of ratio of bidobacteria to
lactobacilli) [83,105]. More recently, a ratio of 1:1 of
frozen concentrates of B. lactis to L. acidophilus has
been considered adequate for optimum growth of, and
acidication by B. lactis when in coculture in plain milk,
and the concomitant enhanced growth of L. acidophilus
has also suggested a certain degree of symbiosis between
these strains [23]. Furthermore, it is important that
strains selected for Direct-Vat-Set (DVS) starters can
undergo concentration of up to 10
10
10
11
cfu/g in order
to permit the desired performance in commercial man-
ufacture of fermented milk products. Apart from this
requirement, the starter produced should also display
acceptable stability (usually guaranteed for 312 mo)
throughout processing, and subsequent storage and
distribution. Direct inoculation of a dairy product with
a frozen concentrated culture (ca. 51010
10
cfu/g) is
recommended at a rate of 0.050.1 mg per L of milk
[23,83,94,106]
Production of functional fermented milk products
also requires safety assessment of the probiotic strains; a
tentative strain must obviously be non-pathogenic for
technological uses. There is a large consensus that lactic
acid bacteria are benign microorganisms, with a well-
established record of safety and no record of food poi-
soning outbreaks. Several studies document the safety
of dairy strains [107], and an updated review on safety
of probiotic bacteria has been made available by
Donohue and Salminen [108]. Doses of probiotic strains
as high as 10
12
cfu/g have failed to exhibit toxicity, and
Fig. 2. Ci|toi|a osoo |o so|oct|oo o| Bidobacterium s. |oi a||cat|oo as o|otaiy aojoocts.
!2 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
among the dierent species of the genus Bidobacterium
only B. dentium was considered to actually be patho-
genic to man in view of its role in dental caries [7].
Ciowt| aoo soiv|va| c|aiactoi|st|cs
Strain viability in the carrier food by the time of con-
sumption is closely related to the aforementioned tech-
nological requirements. Strain survival depends on pH
(and its buering capacity), presence of competing
microorganisms, storage temperature and presence of
microbial inhibitors (e.g. NaCl and H
2
O
2
) in the food
matrix [17]. Studies examining the viability of B. lactis
and L. acidophilus, particularly in milk containing a
range of experimental NaCl concentrations between 0
and 6%(w/w) and stored at dierent temperatures
within the range of 515

C, indicated that pure cultures

of L. acidophilus were more susceptible to higher NaCl
concentrations than their B. lactis counterpart [32]. In
addition, the mechanistic model made available by these
authors considers the behaviour of the pure and mixed
microbial populations to be described by specic death
rates that vary with temperature (following Arrhenius
relationships) and NaCl levels in the milk medium (fol-
lowing simple inhibition kinetics), and is therefore use-
ful in eorts to rationalize the dynamics of cell
population survival under a variety of combinations of
easily manipulated environmental conditions.
Moreover, it is essential that fermented bioproducts
contain a satisfactory number of active cells at the
moment of consumption, i.e. at least 10
6
cfu/mL,
because the minimum therapeutic daily dose is usually
considered as 10
8
10
9
viable cells, realizable through an
intake of, say 100 g of fermented bioproduct containing
10
6
10
7
viable cells/mL [2]. However, it is necessary that
such fermented milks are consumed regularly in order
to maintain the eect of these special microorganisms
on the composition of the intestinal microora. Cases of
marked loss of viability in dairy products have been
reported for both Bidobacterium spp. and L. acid-
ophilus, more often during refrigerated storage at low
pH [24,89,109], and reect, as stressed previously, the
need for careful strain selection. These studies urge
processors to more deeply understand the implications of
adding acid-sensitive bacteria to highly acidic products
like yoghurt. In contrast, bidobacteria survive well in
low-acid products such as frozen yoghurt [89], ice-cream
[92] or cheese when added exclusively as dietary
adjuncts [93]. New methods for enhancing the long-term
survival of probiotic strains, and hence ensure that rea-
sonable numbers of bacteria are delivered to the host,
have been investigated either by replacing the carrier
food [9294,106] or by improving the protection of acid-
sensitive strains via microencapsulation with cellulose
acetate phthalate [110], -carrageenan [93] or Ca-alginate
[111]; the rst method appears to be technologically and
commercially the most feasible. For example, a starter
composed solely of a mixture of L. acidophilus and B.
lactis has been successfully used in the manufacture of
cheeses following modied technologies of the well-known
Gouda cheese [94] and of Cabra cheese, a cheese with a
large social and economic importance in the Mediterra-
nean basin [106]. Growth behaviours of either strain
were dependent on the physicochemical characteristics
prevailing in both cheese-types, yet acid production was
not aected. Survival of the probiotic strains was mon-
itored during ripening, and the nal viable numbers
were still above the accepted threshold. Both strains
contributed signicantly to ripening of both cheese-
types, especially in terms of formation of low molecular
mass peptides and free amino acids, but lipolysis was
not greatly aected. The best compromise between sen-
sorial, physicochemical and probiotic attributes of the
nal cheese was obtained for 310
7
and 710
6
cfu/mL
of B. lactis and L. acidophilus, respectively, 3.5%(w/w)
salt, addition of 0.3%(v/w) milk hydrolysate and ripen-
ing for 70 d.
The selection of oxygen-tolerant species may also be a
solution toward minimization of the adverse eect of
oxygen in fermented bioproducts containing Bido-
bacterium spp. and Lactobacillus acidophilus. The
mechanisms of susceptiblity of Bidobacterium to mole-
cular oxygen have interested researchers at early stages
[112], who estimated the oxygen sensitivity of twelve
strains by measuring the extent of growth inhibition
when they were grown in deep agar cultures and the
extent of growth in aerated cultures; three categories of
Bidobacterium species with regard to their oxygen sen-
sitivity were accordingly suggested. These researchers
were not, however, able to establish a correlation
between oxygen sensitivity and enzyme activities relat-
ing to oxygen metabolism, and it was only in the present
decade that studies provided a clearer insight into a
more fundamental relationship. The initial study by
Shimamura et al. [113] has elucidated oxygen uptake by
several bidobacterial species when carbohydrates are
metabolized, and reported that this mechanism is eec-
tive in diminishing the impact of environmental oxygen
even when faced with an insucient energy source, e.g.
glucose; involvement of NADH oxidase for oxygen
uptake activity in Bidobacterium was thus demon-
strated. In their follow-up study, Shimamura et al. [114]
reported that all four Bidobacterium spp. strains
assayed expressed both NADH oxidase and NADH
peroxidase activites, but to dierent degrees; a strong
inverse correlation between enzyme activites and degree
of oxygen sensitivity was consequently established. Bi-
dobacterium infantis, B. breve and B. longum were much
more tolerant to oxygen (resulting from higher activities
of the aforementioned enzymes) than B. adolescentis
was (arising from very low activities thereof); therefore,
the former are better suited for industrial applications.
These results permitted renement of their initial
A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !
hypothesis [113] in that the pathway involving the two
NADH oxidative enzymes apparently operates as a
defense mechanism to reduce oxygen toxicity in Bido-
bacterium spp.
Finally, a probiotic strain must show good surviva-
bility, both in the product and after digestion. It is clear
from the above lines that certain strains are able to sur-
vive well when exposed to gastric acids, or certain
secretions of the small intestine.
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l|oioto|o lovo|s o| \oao||og l|gs |o Journal of Dairy Sci-
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79 A|a||o, A.S., Co oc , S. aoo lo zo|, S. ! |o|oooco o| Yogoit
aoo Ac|oo|||os Yogoit oo Soiom C|o|ostoio| lovo|s |o \|co
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80 ||avoi, l. aoo vao ooi \ooi, k. ! 1|o Assomoo Ass|m-
||at|oo o| C|o|ostoio| by Lactobacillus aoo Bidobacterium
bidum |s loo to t|o|i |||o Sa|t-oocoojogat|oo Act|v|ty |o
Applied and Environmental Microbiology , !!20!!24
81 1a|i|, |., Cioc|ao|, ., |a||oogoo, . aoo Sc|oo|ooi, l. !
l|oct o| 1|ioo Stia|os o| |||oobactoi|a oo C|o|ostoio| |o
Letters in Applied Microbiology 2!, !4!!
82 1a|i|, |., Ci|||, .l. aoo Sc|oo|ooi, l. ! |ovo|vomoot o|
1i||yoioxycoojogatoo |||o Sa|ts |o C|o|ostoio| Ass|m||at|oo by
|||oobactoi|a |o Current Microbiology 4, 4
83 lo|oi, l. !2 |so o| liob|ot|c Staitoi Co|toios |o la|iy
liooocts |o Food Australia 44, 4!420
84 \ait|o, .l. !6 1oc|o|ca| Coos|ooiat|oos |oi |ocoioiat|og
|||oobactoi|a aoo |||oogoo|c lactois |oto la|iy liooocts |o
Bulletin of the International Dairy Federation !, 4!
85 \ooi|am, A.l., |ovoo-Ooooo|ovoo, |. aoo vao ooi \ooi, k.
!6 l|oct o| liob|ot|cs oo |o|oct|oos w|t| lat|ogoo|c |ac-
toi|a |o lomao Cot l|oia-assoc|atoo kats. lostoi 6, io-
sootoo at l||t| Symos|om oo lact|c Ac|o |actoi|a-Cooot|cs,
\otabo||sm aoo A||cat|oos, Vo|o|ovoo, 1|o |ot|oi|aoos,
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86 \ooi|am, A.l., ||avoi, l.A.\. aoo vao ooi \ooi, k. !
Ass|m||at|oo o| C|o|ostoio| by lactobac|||| |s ooo to t|o|i
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Reviews !2, l!
87 Comos, A.\.l., 1o|xo|ia, \.C. aoo \a|cata, l.x. ! V|ab|-
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\|||. Soo|om C||oi|oo Coocootiat|oo aoo Stoiago 1omoia-
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88 k|c|aiosoo, l. !6 liob|ot|cs aoo lioooct |ooovat|oo |o
Nutrition and Food Science 4, 2
89 laio|a, S. aoo \ait|o, .l. !! l|oct o| l oo Soiv|va| o|
Bidobacterium bidum aoo Lactobacillus acidophilus |o lio-
zoo loimootoo la|iy lossoits |o Cultured Dairy Products
Journal 26, !2!
90 \oo|oi, l.\. aoo V|||a-Caic|a, l. ! 1|o Ciowt| o| Bido-
bacterium longum |o a \|oy-basoo \oo|om aoo V|ab|||ty o|
t||s Oigao|sm |o liozoo Yog|oit w|t| low aoo l|g| lovo|s o|
lovo|ooo Ac|o|ty |o Cultured Dairy Products Journal 2, 4
91 \oo|oi, l.\., \c|o||ai, k.C., Co|, l.l. aoo \ac||o, l.A.
!0 |s|og |co Cioam as a \oc|ao|sm to |ocoioiato |||-
oobactoi|a aoo lioctoo||gosacc|ai|oos |oto t|o lomao l|ot
|o Cultured Dairy Products Journal 2, 4
92 lo|mat, S. aoo \c\a|oo, l.. !2 Soiv|va| o| Lactobacillus
acidophilus aoo Bidobacterium bidum |o |co Cioam |oi |so as
a liob|ot|c looo |o Journal of Dairy Science , !4!!422
93 l|oa|ai, l. aoo \|stiy, V.V. !4 Ciowt| aoo V|ab|||ty o|
Bidobacterium bidum |o C|oooai C|ooso |o Journal of
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94 Comos, A.\.l., \a|cata, l.x., ||avoi, l.A.\. aoo Ciaooo, l..
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Lactobacillus acidophilus Stia|o || |o a C|ooso lioooct |o
Netherlands Milk and Dairy Journal 4, !
95 ||aoc|otto, l., koy, l., |o|aogoi, C. aoo Caot||oi, S.l. !6
lioooct|oo o| Cottago C|ooso |s|og lioss|og loimootoo by
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96 Va|ooz, C.l. aoo oo C|oi|, C.S. ! l|oct|voooss o| Soy \|||
as looo Caii|oi |oi Lactobacillus acidophilus |o Journal of
Food Protection 6, 2022
97 \oit|, 1.\., |oo|||aooo, C., laoooo, \. aoo losmazoaoo, \..
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tyo liooocts |iom Soym||| Coota|o|og Bidobacterium spp
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98 lavo, k.|. aoo S|a|, |.l. ! V|ab|||ty o| Yog|oit aoo
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99 Samooa, A., kob|osoo, k.|. aoo \aia||s, S. !6 Ac|o lioooc-
t|oo by |||oobactoi|a aoo Yog|oit |actoi|a ooi|og loimoota-
t|oo aoo Stoiago o| \||| |o Food Microbiology !, 220
100 o|osoo, \.C., kay, |. aoo ||owm||, 1. ! So|oct|oo o|
Lactobacillus acidophilus Stia|os |oi oso |o Ac|oo|||os lio-
oocts |o Antonie van Leeuwenhoek , 2!2!
101 C|ai|, l.A., Cottoo, l.|. aoo \ait|o, .l. ! So|oct|oo o|
|||oobactoi|a |oi oso as l|otaiy Aojoocts |o Co|toioo la|iy
looos. ||1o|oiaoco to S|mo|atoo l o| lomao Stomac|s |o
Cultured Dairy Products Journal 2, !!!4
102 |bia||m, S.A. aoo |oz|oiova|oy, A. ! Soiv|va| o| |||oo-
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of Food and Agriculture 62, !4
103 Cota, l.|., \|ta|, |.|. aoo Caig, S.|. !6 C|aiactoi|zat|oo
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104 Voo|||, l. aoo S|||maoo, l. !6 Ot|m|zat|oo o| t|o lio-
aiat|oo o| Staitois |oi \aoo|actoio o| |||oos \||| |oooi
liact|ca| Cooo|t|oos |o DMZ-Lebensmittelindustrie und
Milchwirtschaft !!, 664
105 ||aoc|otto, l., koy, l. aoo Caot||oi, S.l. ! liotoct|vo
l|oct o| ||tia||toioo kotootato lowooi oo Stab|||ty o| lioozo-
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senschaft 0, 66
106 Comos, A.\.l. aoo \a|cata, l.x. ! lovo|omoot o| a
liob|ot|c C|ooso \aoo|actoioo |iom Coat \|||. kosooso
Soi|aco Aoa|ys|s v|a 1oc|oo|og|ca| \ao|o|at|oo |o Journal of
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107 Aoams, \.k. aoo \aitoao, l. ! Oo t|o Sa|oty o| lact|c
Ac|o |actoi|a |iom looo |o International Journal of Food
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108 looo|oo, l. aoo Sa|m|ooo, S. !6 Sa|oty o| liob|ot|c
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109 lao|aot|ia, \.l.V., S|a|, |.l. aoo |i|tz, \.l. !6 Soiv|va|
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110 kao, A.V., S||woaia|o, |. aoo \a|aiaj, |. ! Soiv|va| o|
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|atoo Casti|c aoo |otost|oa| o|cos |o Canadian Institute of
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111 ||m, |.|., |ao|, Y.. aoo Yooo, Y.l. !6 l|octs o| ko|yoia-
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Journal of Dairy Science !, !!
112 oo Vi|os, \. aoo Stoot|amoi, A.l. !6 lactois lotoim|o-
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!6 A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157
113 S||mamoia, S., Abo, l., |s||bas||, |., \|ya|awa, l., Yaos||ma,
1. aoo 1om|ta, \. !0 looogoooos Oxygoo |ta|o aoo
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culture and Biological Chemistry 4, 2624
114 S||mamoia, S., Abo, l., |s||bas||, |., \|ya|awa, l., Yaos||ma,
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115 7b||ows||, 7. aoo 7|aj|a, S. !6 lyoio|yzoo Caso|o as a
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116 Azoma, |., Yamaoc||, |. aoo \|tsoo|a, 1. !4 |||oos
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istry 4, 2!2!62
117 Abo-l|-Sa|am, \.l., l|-S||b|oy, S. aoo |oc||o|m, \. !6
C|aiactoi|st|cs aoo lotoot|a| |sos o| t|o Caso|o \acioo-
t|oo |o International Dairy Journal 6, 24!
118 |to, \., ||moia, \., logoc||, Y., \|yamoi|-\atabo, A.,
Yaj|ma, 1. aoo |ao, 1. ! l|octs o| 1iaosga|actosy|atoo
l|sacc|ai|oos oo t|o lomao |otost|oa| \|cio|oia aoo t|o|i
\otabo||sm |o Journal of Nutritional Science and Vitaminology
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119 Yamaza||, l. aoo \atsomoto, |. !4 loi||cat|oo o| oi-
osa|om Ait|c|o|o lioctaos aoo t|o|i |t|||sat|oo by |||oo-
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120 O|aza||, \., loj|wa|a, S. aoo \atsomoto, |. !0 l|oct o|
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dobacteria and Microora , 6
121 Yamaoa, l., |to|, |., \oi|s||ta, Y. aoo 1ao|goc||, l. !
Stioctoio aoo liooit|os o| O||gosacc|ai|oos |iom \|oat
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122 A||yama, l., looo, 1., |a|a||ta, k., \oiata, |., Yooomoto, Y.
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123 Yaog, k.l., 7|aog, l.l., C|oo, l.Y. aoo l|o, x.S. !6
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124 Cota, l.|., \|ta|, |.|. aoo Caig, S.|. !6 |o||b|toiy Act|v-
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125 Ci|||, .l., Cioc|ao|, . aoo |a||oogoo, . ! l|oct o| |||oo-
bactoi|a oo ||ti|tos aoo ||tiosam|oos |o Letters in Applied
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126 ||o|soo, O.l., oigoosoo, S., loooisoo, |. aoo ostosoo, 1.
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laoca| Sam|os a|toi Oia| Aom|o|stiat|oo o| |||oobactoi|a
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127 |aitiam, l.l., Sc|oac|, \., Coi|ac|, S., koc|oosc|o|, C.,
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Bidobacterium longum A|oct Co|oo|c \|ciob|o|ogy aoo
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128 Sc|||i|o, l.., koc|at, l., l|o|-Amstoi, l., Aosc|||maoo, .\.
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A.M.P. Gomes and F.X. Malcata / Trends in Food Science & Technology 10 (1999) 139157 !