J. Dairy Sci.
84:319–331
 American Dairy Science Association, 2001.
Invited Review: The Scientific Basis of Lactobacillus acidophilus
NCFM Functionality as a Probiotic
M. E. Sanders* and T. R. Klaenhammer†
*Dairy and Food Culture Technologies
7119 S. Glencoe Ct.
Littleton, CO 80122-2526
†Department of Food Science
North Carolina State University, Raleigh
ABSTRACT
Lactobacillus acidophilus NCFM is a probiotic strain
available in conventional foods (milk, yogurt, and tod-
dler formula) and dietary supplements. Its commercial
availability in the United States since the mid-1970s is
predicated on its safety, its amenability to commercial
manipulation, and its biochemical and physiological at-
tributes presumed to be important to human probiotic
functionality. The strain has been characterized in
vitro, in animal studies, and in humans. NCFM is the
progenitor of the strain being used for complete chromo-
some sequencing and therefore will be a cornerstone
strain for understanding the relationship between ge-
netics and probiotic functionality. Both phenotypic and
genotypic techniques have verified its taxonomic status
as a type A1 L. acidophilus strain. It adheres to Caco-
2 and mucus-secreting HT-29 cell culture systems, pro-
duces antimicrobial compounds, and is amenable to ge-
netic manipulation and directed DNA introduction.
NCFM survives gastrointestinal tract transit in both
healthy and diseased populations. NCFM inhibits aber-
rant crypt formation in mutagenized rats, indicative of
activity that could decrease the risk of colon cancer. A
blend of probiotic strains containing NCFM decreased
the incidence of pediatric diarrhea. NCFM led to a sig-
nificant decrease in levels of toxic amines in the blood
of dialysis patients with small bowel bacterial over-
growth. At adequate daily feeding levels, NCFM may
facilitate lactose digestion in lactose-intolerant sub-
jects. Further validation of the probiotic properties of
NCFM in humans and clarification of its mechanisms
of probiotic action are needed to better understand the
role this strain might play in promoting human health.
(Key words: Lactobacillus acidophilus NCFM, probi-
otic, functional food)
Received February 14, 2000.
Accepted August 31, 2000.
Corresponding author: M. E. Saunders; e-mail: mesanders@
msn.com.
319
Abbreviation key: GI = gastrointestinal, NCSU =
North Carolina State University.
INTRODUCTION
Probiotic functionality depends on the ability of a
strain to confer health advantages on a host upon oral
consumption of viable cells. Many different strains and
species of lactobacilli (Table 1) and bifidobacteria have
been used commercially as probiotics. Numerous mech-
anisms have been proposed for probiotic functionality
and many clinical end targets have been measured
(Sanders, 1999). However, it is difficult to assess the
health effects that might be expected from probiotic
consumption by the general population, since most re-
search to date has focused on animal studies, biomark-
ers, or small human study group sizes. More extensive
epidemiological evaluations will be necessary to better
study these properties. Since such intervention trials
are quite costly, it is important that strains be well
characterized prior to this use. Concomitantly, exten-
sive strain characterization is important prior to com-
mercial development or use. This has led to research
focused on probiotic strains of commercial interest. Lac-
tobacillus acidophilus NCFM is one such strain (Fig-
ure 1).
Lactobacillus acidophilus NCFM was isolated and
characterized in the food microbiology research labora-
tories at North Carolina State University (NCSU) in
Raleigh by M. Speck and S. Gilliland and was subse-
quently studied there by T. Klaenhammer. The strain
was isolated from a human source in the 1970s (Gilli-
land et al., 1975) and has since been the subject of
research at NCSU and other institutions worldwide.
This strain has been sold commercially (Rhodia, Inc.,
Madison, WI) for over 25 yr for use in the formulation
of fluid milks and yogurts containing probiotic cultures,
in dried dietary supplements, in toddler formula and in
juice. (See Appendix for description of published strain
designations for NCFM.)
This article will summarize research findings on
NCFM.
320 SANDERS AND KLAENHAMMER

Table 1. Lactobacillus species used as human probiotics. Table 2. Growth and fermentation characteristics of NCFM com-
pared with the neotype Lactobacillus acidophilus strain (Gilliland
Species and Speck, 1977b). +, growth; −, no growth.
Obligately homofermentative
L. acidophilus L. acidophilus ATCC 4356
L. crispatus Characteristic NCFM (Neotype)
L. amylovarus
L. gallinarum Growth @ 15°C − −
L. gasseri Growth @ 45°C + +
L. johnsonii Ammonia from arginine − −
L. helveticus Aesculin + +
L. delbrueckii subsp. bulgaricus Cellobiose + +
L. salivarius subsp. salivarius Galactose + +
Lactose + +
Facultatively heterofermentative Maltose + +
L. casei Mannitol − −
L. paracasei subsp. paracasei Rhamnose − −
L. paracasei subsp. tolerans Salicin + +
L. plantarum Sorbitol − −
L. rhamnosus Sucrose + +
Obligately heterofermentative Trehalose + +
L. fermentum
L. reuteri
cal to the L. acidophilus neotype strain ATCC 4356
(Gilliland and Speck, 1977b; Table 2). It is a Gram-
Taxonomy positive, homofermentative, catalase-negative rod. Its
DNA has a 38.4% GC ratio. Fermentation results in
The taxonomy of L. acidophilus has undergone sig- 34% D- and 66% L-lactic acid. The percentage of D- and
nificant revisions over the past decade, leading to the L-lactic acid of NCFM and other species of probiotic
establishment of several new species (Klaenhammer lactobacilli is shown in Table 3 (Girgis et al., 2000).
and Russell, 1999; Klein et al., 1998).NCFM has been Studies using pulsed-field gel electrophoresis have
shown by several phenotypic (Gilliland and Speck, shown that NCFM is highly related to the type L. acido-
1977b; Table 2) and genotypic (Kullen et al., 2000; Sand- philus strain, ATCC 4356 (Figure 2). Band differences
ers et. al., 1996) criteria to be a member of the type A1 between strain 4356 and NCFM are apparent, although
L. acidophilus species. Hybridization with a species- the genomic organization is predominantly the same.
specific oligonucleotide probe (5’ TCTTTCGATGCATC- This technique shows restriction nuclease digestion
CACA 3’; Pot et al., 1993) using slot blots provided patterns of total chromosomal DNA, and, as such, can
further evidence that NCFM belongs to the type A1 L. discriminate between strains. However, small differ-
acidophilus group (Sanders, et. al., 1996). Sequencing ences within the DNA sequence would not be detected
of the 16S ribosomal RNA gene of NCFM confirmed its by this technique unless they disrupt a restriction site
identity as L. acidophilus (Kullen et al., 2000). Fermen- used in the analysis.
tation and growth characteristics of NCFM are identi-
Genetic Access
The ability to introduce homologous or heterologous
DNA into commercial lactobacilli enables useful re-

Table 3. Percentage of lactic acid enantiomers formed by selected

Lactobacillus strains. Lactic acid was measured using a commercial
kit from Boehringer Mannheim (Indianapolis, IN; catalogue number
1112821).
Species Strain % D-Lactic acid % L-Lactic acid

L. acidophilus NCFM 33.8 66.2

L. acidophilus ATCC 4356 21.5 78.5
L. amylovorus ATCC 3620 56.3 43.7
L. casei ATCC 393 2.6 97.4
L. crispatus ATCC 33820 56.4 43.6
L. delbrueckii ATCC 4797 99.5 0.5
L. gallinarum ATCC 33199 39.0 61.0
L. gasseri ATCC 33323 59.1 40.9
L. helveticus NCK 936 40.5 59.5
Figure 1. Electron micrograph of Lactobacillus acidophilus L. johnsonii ATCC 33200 54.6 45.4
NCFM cells.

Journal of Dairy Science Vol. 84, No. 2, 2001

 REVIEW: L. ACIDOPHILUS NCFM PROBIOTIC ACTIVITY
Figure 2. Pulsed-field gel electrophoresis of chromosomal DNA
of NCFM (A) compared with the Lactobacillus acidophilus type strain,
ATCC4356 (B). Molecular weight markers in last lane (M) as follows:
194, 145, 97, 48.5, 23.1, 9.42, 6.55, 4.36, 2.32 kb. Unmarked lanes
contain DNA from another Lactobacillus strain.
search approaches, including strain improvement and
investigation into the genetic linkage of important com-
mercial traits. The lactobacilli have typically been re-
fractory to high efficiency DNA transfer methods, espe-
cially transformation. Lactobacillus acidophilus NCFM
was transformed with plasmid vectors with protocols
developed by Luchansky et al. (1989, 1991) and Walker
and Klaenhammer (1996), whereas transformation of
the neotype strain, L. acidophilus ATCC 4356, resulted
in significantly fewer or no transformants. Walker and
Klaenhammer (1996) demonstrated reliable introduc-
tion of DNA into NCFM and other true L. acidophilus
strains at frequencies of 2 × 104 chloramphenicol-resis-
tant transformants/µg of pGK12 DNA. The physiologi-
cal attributes of the NCFM strain enabling more effi-
321
cient transformation are not known. However, NCFM
appears to be better suited for genetic analysis than
the type strain of L. acidophilus.
Genetic studies pave the way for future enhancement
of the healthful properties of NCFM. A recent study
by Kullen and Klaenhammer (1999) identified an acid-
inducible operon (ATPase) in NCFM. This and similar
systems in other bacteria are responsible for main-
taining the acid tolerance under low pH conditions.
Understanding the regulation of this operon could pro-
vide insight into the possible benefits of delivering pro-
biotics in fermented foods and lead to improved survival
under acid conditions. Other genetic studies have fo-
cused on understanding the mechanisms of bacteriocin
production (Klaenhammer et al., 1992).
Stability
Stability of commercial probiotic strains is important
to assure that stated levels of viable cells are delivered
in probiotic products. Frozen culture concentrates of
NCFM were tested for stability during frozen storage
(Sanders et. al., 1996). (In this publication, NCFM was
coded as commercial strain LH1.) The results showed
that concentrated cultures of NCFM lost only about a
quarter of a log cycle in viability over 6 wk frozen stor-
age at −20°C. Stability in a dried format at room temper-
ature resulted in steadily decreasing viability until less
than 102 viable cells per gram were detected after 8 mo
of storage (Crowell, 1998). The stability of NCFM in
commercial products at room temperature has not
been published.
NCFM showed excellent stability in milk inoculated
with about 107 cfu/ml. After 21 d at 4°C or 10°C, levels
of NCFM were essentially unchanged (Sanders et al.,
1996). NCFM was also stable in commercially produced
fermented dairy products (Iturriria et al., 1999). After
52 d of storage at 4°C, NCFM counts in strawberry
yogurt fell only from 1.2 × 107 cfu/g to 8.7 × 106 cfu/g
and in plain yogurt from 2.4 × 107 cfu/g to 1.5 × 107 cfu/
g, reflecting minimal decreases in numbers. However,
comparative counts of NCFM on MRS agar, with and
without 0.15% bile, revealed that about 50% of the pop-
ulation was injured after 52 d of storage in plain yogurt.
In cottage cheese, counts fell from 6 × 104 cfu/g to 3 ×
104 cfu/g, a 50% drop in viable cells after 37 d at 4°C.
The losses reflected here indicate that NCFM is suitably
stable in commercial dairy products.
Stability is dependent on many factors, including
growth conditions and storage conditions (relative hu-
midity, oxygen content, stabilizers, and temperature).
The importance of growth conditions, including the
presence of calcium, in the production of stable frozen
Journal of Dairy Science Vol. 84, No. 2, 2001
322 SANDERS AND KLAENHAMMER
cultures of NCFM has been established (Klaenhammer
and Kleeman, 1981; Wright and Klaenhammer, 1981).
Antimicrobial Activity
Antimicrobial activity is thought to be an important
means for probiotic bacteria to competitively exclude
or inhibit activities of harmful or pathogenic intestinal
microbes. Antimicrobial compounds produced by probi-
otic bacteria include organic acids (lactic and acetic
acid), hydrogen peroxide (in environments in which oxy-
gen is present), diacetyl, β-hydroxypropionaldehyde
(produced by Lactobacillus reuteri) or bactericidal or
bacteriostatic peptides and proteins (DeVuyst and Van-
damme, 1994). The ability to inhibit a range of microbes
(pathogenic, spoilage, and other lactic acid bacteria) in
laboratory assays and cocultivation experiments has
been demonstrated repeatedly with many members of
lactic acid bacteria. Figure 3 shows bacteriocin activity
produced by L. acidophilus NCFM against another Lac-
tobacillus strain, Lactobacillus delbrueckii subsp. lactis
ATCC 4797 (Barefoot and Klaenhammer, 1983). What
is less clear, however, is the role that inhibitory activity
plays in vivo. A reduction in fecal pH and an increase in
short chain fatty acids has been correlated with higher
fecal counts of lactobacilli and bifidobacteria in some
feeding studies, suggesting that probiotic growth and
concomitant production of fermentative metabolites in
vivo have a measurable, physiological influence. The
role of bacteriocins in vivo is, however, less clear. Bacte-
Figure 3. Zones of inhibition produced by NCFM bacteriocin
against Lactobacillus delbrueckii subsp. lactis ATCC 4797. This bac-
teriocin also inhibited strains of Lactobacillus bulgaricus, Enterococ-
cus faecalis, Lactobacillus fermentum and Lactococcus lactis (Barefoot
and Klaenhammer, 1983).
Journal of Dairy Science Vol. 84, No. 2, 2001
riocins are proteinaceous compounds produced by bac-
teria that exhibit a bactericidal or bacteriostatic activity
against susceptible bacteria. In vitro studies have
shown that the majority of strains of Lactobacillus fre-
quently (but not always), produce bacteriocins that kill
closely related species. The roles and in vivo activities of
Lactobacillus bacteriocins remain to be demonstrated.
Antagonistic activity was produced by NCFM against
foodborne disease agents, Staphylococcus aureus, Sal-
monella typhimurium, enteropathogenic Escherichia
coli, and Clostridium perfringens (Gilliland and Speck,
1977a). This study documented an 80 to 90% inhibition
of these pathogens in broth culture under laboratory
conditions. Inhibition resulted from organic acids, hy-
drogen peroxide, and perhaps other antimicrobial
products.
L. acidophilus NCFM was found to produce a bacte-
riocin, designated lactacin B (Barefoot and Klaenham-
mer, 1983). The biochemical and genetic properties of
this bacteriocin were characterized in detail (Barefoot
and Klaenhammer, 1983; Barefoot et al., 1994; Klaen-
hammer et al., 1992; Nettles, 1992). In vitro tests of
inhibition indicated a range of activity only against
other Lactobacillus strains and Enterococcus faecalis,
not against pathogens (Barefoot and Klaenhammer,
1983). The molecular mass of this bacteriocin was deter-
mined to be 6500 and its sensitivity to proteinase K
and pronase confirmed its proteinaceous nature. It is
stable to heat (121°C, 3 min, pH 5), cold (−20°C), and
chemical chaotropic agents (ß-mercaptoethanol, 8 M
urea, and 1% SDS). Properties of this bacteriocin have
been reviewed extensively (Barefoot et al., 1994; Klaen-
hammer et al., 1992).
Adherence
Adherence and colonization are attributes considered
to be important for probiotic functionality, particularly
in roles in which intestinal enterocytes are stimulated.
Intimate, extended association with the intestinal mu-
cosa is likely to require adherence of the bacterium with
the epithelial cells, and without it, the probiotic may
not influence some important physiological parameters.
Unfortunately, in vitro studies on adherence are often
not predictive of the in vivo situation, since conditions
in vitro are so vastly different than in vivo. Further-
more, even strains isolated from human sources are
allochthonous or nonnative to consumers, and, as such,
should not be expected to behave as native strains do.
Gastrointestinal (GI) tract colonization by autochtho-
nous strains is mature and reasonably stable from early
childhood (Tannock, 1999). Results from probiotic feed-
ing studies suggest that exogenous strains, even those
demonstrating adhering capability in vitro and sup-
 REVIEW: L. ACIDOPHILUS NCFM PROBIOTIC ACTIVITY
plied in great number, generally do not persist (Alander
et al., 1999; Bouhnik, et al., 1992). Although some ad-
herence may occur, it does not appear to be at high
efficiency or accompanied by prolific colonization, as
populations of probiotics steadily drop to undetectable
levels in feces when consumption has ceased. Studies
employing biopsies of intestinal tissues are rare, but
are necessary to confirm adherence and retention at
key locations in the GI tract.
Nevertheless, the adhering capability to intestinal
cells in tissue culture is a commonly assessed attribute
for probiotic strains, and NCFM has been included in
such studies. L. acidophilus NCFM was shown to ad-
here to human fetal intestinal cells and Caco-2 cells,
and the level of attachment was enhanced in the pres-
ence of calcium ions (Greene and Klaenhammer, 1994;
Kleeman and Klaenhammer, 1982). The mucus-secret-
ing intestinal cell line, HT-29, was thought to more
closely mimic in vivo conditions, providing an improve-
ment over the nonsecreting intestinal cell lines. Later
evidence from cell culture experiments suggested that
NCFM adheres through a protein-mediated mecha-
nism, although other probiotic lactobacilli may use car-
bohydrate moieties as well. Compared with other lacto-
bacilli assayed, this adherence was high and fairly sta-
ble at physiologically relevant pH values between 6 and
8 (Greene and Klaenhammer, 1994). Using scanning
electron microscopy, Hood and Zottola (1987) showed
that NCFM did not appear to have an external polysac-
charide layer, an observation that may be important to
cell surface binding capability. The adherence of NCFM
compared with other lactobacilli is summarized in Ta-
ble 4. These results provide in vitro evidence that
NCFM can adhere to human cells, but the fate of NCFM
inside the human intestine has not been assessed.
Cholesterol
The reduction of total cholesterol or low-density lipo-
proteins found in human plasma is thought to lower
the risk of coronary heart disease. Probiotic cultures
have been suggested to play a role in reduction of these
blood lipids, although research to date is equivocal (Tay-
lor and Williams, 1998). Studies on L. acidophilus
NCFM (Gilliland et. al., 1985; Gilliland and Walker,
1990) suggested its ability to remove cholesterol from
a laboratory growth medium. NCFM was reported to
take up cholesterol in the presence of bile and in the
absence of oxygen, both conditions present in the intes-
tinal tract. Evaluation of other L. acidophilus strains
showed that cholesterol assimilation was strain-depen-
dent. The significance of these in vitro studies cannot,
however, be determined without human studies that
evaluate serum cholesterol levels in NCFM consumers.
323
Cholesterol-assimilating and isogenic, nonassimilating
strains must be compared in a clinical trial to assess
the importance of this attribute.
Thompson et al. (1982) fed 250 ml of Sweet Acido-
philus milk daily to 12 healthy subjects for a 9-wk study
period, as part of a wider study on the effects of different
dairy products on serum cholesterol. Sweet Acidophilus
is a registered trademark of the North Carolina Dairy
Foundation (NCSU, Raleigh, NC) and is used to de-
scribe low fat milk supplemented with 2 × 106 cfu/ml
NCFM cells. The test product was consumed for 3 wk
of this time. The authors concluded that among Sweet
Acidophilus milk, yogurt, and buttermilk, none had a
significant effect on serum cholesterol. The levels of
viable L. acidophilus NCFM were not reported in the
study, although typical Sweet Acidophilus milk formu-
lation levels would have provided ~2 × 108 cfu of viable
L. acidophilus per day in this study. It would be worth-
while to determine whether feeding humans higher lev-
els of NCFM would have a different effect.
Gastrointestinal Tract Influence
An important attribute for certain probiotic bacteria
functions is survival and growth in the intestinal tract.
Once in the GI tract, probiotic bacteria have the oppor-
tunity to influence the populations and activities of dif-
ferent intestinal bacteria. Therefore, demonstrating
that a probiotic can survive GI transit and influence
GI tract flora is important for establishing probiotic
characteristics.
Gilliland et al. (1978) demonstrated that levels of
lactobacilli increase significantly in the feces of human
subjects consuming nonfermented milk containing
NCFM. Because the technology was not available at
that time to specifically track the NCFM strain, GI
survival of NCFM was deduced from increases in total
fecal lactobacilli.
More recently, Crowell (1998) studied the ability to
recover NCFM from both healthy and ill individuals
consuming 2 × 1010 NCFM daily. NCFM could be iso-
lated from the feces of healthy subjects and from jejunal
contents and brushings of chronic kidney failure pa-
tients fed NCFM in capsules. (Capsules were coated
with hydroxypropylmethyl cellulose phthalate to en-
hance NCFM survival through the stomach.) To spe-
cifically identify NCFM among indigenous lactobacilli,
isolates of lactobacilli were randomly chosen and sub-
jected to pulsed field gel electrophoresis. Chromosomal
DNA patterns specific to NCFM were identified among
those isolates. NCFM was not recovered from fecal sam-
ples from any of five subjects after NCFM feeding had
been stopped for 2 wk (Figure 4). This suggests that to
achieve probiotic functionality, NCFM should be re-
Journal of Dairy Science Vol. 84, No. 2, 2001
324 SANDERS AND KLAENHAMMER

Table 4. Comparison of adherence properties of NCFM and other probiotic lactobacilli. +, adherence observed;
−, no adherence; HT29, indicates HT29 or HT29-MTX cells.
Cell line used
in adherence assay
Factors
Lactobacillus species HITH involved in
and strain s0074 Caco-2 HT29 adherence Reference
L. acidophilus + + + Protein Greene and
NCFM Klaenhammer,
1994
L. acidophilus LA + + + Protein Chauviere et al.,
L. acidophilus LB + + Secreted 1992; Coconnier
protein et al., 1992
L. acidophilus LA1 + + Secreted
protein
L. crispatus BG2FO4 + + + Protein and Greene and
carbohydrate Klaenhammer,
L. gasseri ADH + + + Carbohydrate 1994
L. rhamnosus GG + + Mack et al., 1999
L. plantarum 299 & + Mannose- Adlerberth et al.,
299V adhesion 1996
L. delbrueckii 1489 + + Greene and
Klaenhammer,
1994

Figure 4. Pulsed-field gel electrophoresis patterns of chromosomal DNA from lactobacilli isolated from healthy humans fed 2 × 1010 cfu/
day NCFM (Crowell, 1998). A, prefeeding; B, during feeding; C, 2 wk postfeeding. Arrows indicate pattern generated by NCFM.

Journal of Dairy Science Vol. 84, No. 2, 2001

 REVIEW: L. ACIDOPHILUS NCFM PROBIOTIC ACTIVITY
peatedly and regularly consumed. These results are
consistent with recovery and clearance studies per-
formed with other probiotic strains (Alander et al.,
1999; Bouhnik et al., 1992). Evidence for long-term per-
sistence is lacking for most probiotic bacteria studied.
Methods to track changes in species of fecal bacteria
have been developed (Kaplan et al., in press). Prelimi-
nary data have demonstrated the effectiveness of a
PCR-based technique, terminal restriction fragment
polymorphism for characterizing the nature of probi-
otic-induced changes in fecal microbial communities
(Kaplan et al., in press). Fecal samples from rats fed
NCFM have provided DNA-based evidence for the sur-
vival of NCFM through the rodent GI tract and for
concomitant alterations in fecal microbial communities.
The presence of a dominant fragment of L. acidophilus
DNA in rats fed NCFM, which was absent in rats in
the control group or prior to feeding, demonstrated the
usefulness of this technique for tracking NCFM. This
technique will be applicable in feeding studies with
subjects not harboring dominant levels of L. acido-
philus as autochthonous strains.
Conway et al. (1987) investigated the fate of NCFM
in low pH phosphate buffer saline and in aspirated
human gastric juice. A rapid decline in counts was ob-
served for NCFM and other lactobacilli (L. gasseri ADH
and L. bulgaricus) fed in this experimental system. A
seven-log cycle drop in counts after 1 h at pH 1 in
buffered saline was reported for NCFM. Much greater
stability was seen at pH 3 and 5 for all strains assayed.
Gastric juice at pH 1 resulted in over a three log cycle
drop after 30 min, whereas gastric juice at pH 2.5 or
buffered with skim milk to pH 3 showed longer term
survival. These results confirm that the highly acidic
environment in the stomach is harsh on the survival of
probiotic lactobacilli and supports the recommendation
that these cultures be consumed with food or milk to
enhance survival through the stomach into the intes-
tine. Enteric coating was also used to promote survival
through the stomach in a study by Simenhoff et al.
(1996).
Lactose Intolerance
The consumption of lactose by certain people can re-
sult in intestinal discomfort due to the colonic fermenta-
tion of lactose that passes through the small intestine
undigested. Fermented dairy products have been re-
peatedly shown to enhance tolerance to lactose com-
pared with unfermented products or lactose alone
(Shah, 1993). This observation has been attributed in
part to the ability of lactic acid bacteria to serve as a
source of lactase in the small intestine, contributing to
the digestion of lactose in the lactase-deficient person.
325
Studies have suggested that, in general, the yogurt
starter cultures, Streptococcus thermophilus and Lacto-
bacillus bulgaricus (organisms that do not survive in-
testinal passage) are better at decreasing breath hydro-
gen production and symptoms of lactose maldigestion
than lactobacilli of intestinal origin, even when the
studies controlled for cell count (Lin et al., 1991, 1998).
A few studies of probiotic lactobacilli have been shown
to improve lactose digestion or symptoms in lactase-
deficient individuals (Kim and Gilliland, 1983; Lin et
al 1998; Montes et al., 1995; Mustapha et al., 1997).
Total cellular lactase levels are not always predictive
of a strain’s ability to aid lactose digestion in vivo (Mus-
tapha et al., 1997). This may be because strains differ
in their ability to make lactase available in the small
intestine (lactase stability, lactose transport, and lac-
tase release) or due to the nature of cell lysis and lactase
assay. Bile sensitivity and acid tolerance seem to be
attributes important to lactase availability in vivo
(Mustapha et al., 1997).
Several in vitro evaluations of lactase levels of NCFM
have been reported. Lactase activity on a colony forming
unit-basis was reported to be 4.43 µg of σ-nitrophenol
× 10−7/cfu per minute for NCFM (Sanders et. al., 1996),
a level that was high among all probiotic lactobacilli
tested. Levels for the other probiotic lactobacilli in the
study ranged from <0.01 to 4.73 µg of σ-nitrophenol ×
10−7/cfu per minute. However, compared to levels ob-
served for all S. thermophilus strains (which ranged
from 11.8 to 189 µg of σ-nitrophenol × 10−/cfu per min-
ute), levels of lactase in probiotic lactobacilli were low.
Hughes and Hoover (1995), in a paper comparing
NCFM to some bifidobacteria strains, showed that the
lactase activity of NCFM was roughly equivalent to
Bifidobacterium bifidum, Bifidobacterium breve, and
Bifidobacterium longum strains examined. Nielsen and
Gilliland (1992) conducted some basic experiments to
purify and characterize the lactase enzyme of NCFM,
providing biochemical information on the enzyme’s size,
conditions for optimum activity, and stability to refrig-
erated storage. These data demonstrate the active lac-
tase present in NCFM.
In rats fed unfermented milk containing NCFM and
a water control, no temporal changes in lactase activity
in the stomach, small intestine, cecum, or colon were
observed, although a diet supplemented with yogurt
did improve lactase levels in the small intestine only
(Rao et al., 1991). This indicates that this milk may not
be the most advantageous formulation for targeting
improvement of lactose digestion. Both the choice of
lactic bacterium (yogurt cultures compared with L.
acidophilus) and formulation levels (~108 cfu/g in yo-
gurt compared to 2 × 106 cfu/g in unfermented, culture-
Journal of Dairy Science Vol. 84, No. 2, 2001
326 SANDERS AND KLAENHAMMER
containing milk) could be optimized for the desired
physiological effect.
Several studies have examined the effect of dairy
products containing NCFM on lactose intolerance in
human subjects. Newcomer et al. (1983) tested NCFM
added to milk at fairly low levels (2 × 106/ml) to aid the
digestion of lactose-deficient subjects, but showed no
improvement, a result also demonstrated by McDon-
ough et al. (1987) and Payne et al. (1981) using NCFM
in Sweet Acidophilus milk. In contrast, NCFM included
at high levels in milk was found to reduce lactose intol-
erance symptoms (bloating, diarrhea, gas) in 9 of 10
lactose-maldigesting children (Montes et al., 1995).
Similarly, Kim and Gilliland (1983) found that NCFM
improved lactose utilization in human subjects, al-
though other studies did not confirm this finding (Lin
et. al., 1991; McDonough et al., 1987; Newcomer et al.,
1983; Savaiano et. al., 1984).
In interpreting these results, it is important to note
that adequate levels of the culture must be consumed
for a positive effect. Some products with L. acidophilus
contain levels of the culture insufficient to help with
lactose digestion. These studies show that not all uses
of NCFM resulted in an improved digestion of lactose.
However, given in adequately high levels, some symp-
tom relief and improved digestion of lactose may occur
in lactose maldigesters when they consume NCFM. Pre-
sumably, these effects result from the bacterium’s abil-
ity to metabolize lactose during digestion and transit
through the GI tract.
Small Bowel Bacterial Overgrowth
The influence of NCFM on microflora-associated
symptoms experienced by dialysis patients with chronic
kidney disease has been studied (Simenhoff et al.,
1996). As a result of their disease, these patients suffer
from disruption of the environment of their small intes-
tine, leading to an abnormally high level of bacterial
growth in this organ. This condition is known as small
bowel bacterial overgrowth. The small bowel normally
harbors relatively few microbes. Although colonization
of the distal ileum reflects that of the cecum, the rapid
transit time and high bile concentrations generally
limit small intestinal colonization. Small bowel bacte-
rial overgrowth can be diagnosed directly by confirma-
tion of elevated microbe counts in small intestinal sam-
ples, or indirectly by determining a significant increase
in breath hydrogen 45 min after lactulose ingestion,
indicating bacterial fermentation in the small bowel.
(Colonic fermentation of this substrate occurs much
later.) Bacterial overgrowth is accompanied by produc-
tion of toxic metabolites, including nitrosodimethylam-
Journal of Dairy Science Vol. 84, No. 2, 2001
ine and dimethylamines. These compounds can be mea-
sured in the blood of the patients.
Blinded, placebo-controlled studies demonstrated
that feeding freeze-dried preparations of NCFM low-
ered the levels of dimethylamine and nitrosodimethy-
lamines in the blood of these patients (Dunn et al.,
1998a, 1998b; Simenhoff et al., 1996). In addition, the
nutritional status of the patients improved, as evi-
denced by increased calorie consumption and weight
gain (Dunn et al., 1998b). No adverse effects were ob-
served with NCFM treatment. These studies suggest
that NCFM inhibits the populations or activities of di-
methylamine- and nitrosodimethylamine-producing
overgrowth bacteria, and can positively influence colo-
nization of the small bowel.
Anti-carcinogenicity
Studies have been conducted in animal and human
systems to evaluate the control of negative activities
of human intestinal flora. Animal experiments, which
evaluate the levels or time-course of mitogen-induced
tumor development or indices of tumor development
are important since this type of evaluation can never
be conducted directly in humans. Activities of microbial
enzymes, which are thought to play a role in the conver-
sion of procarcinogens to carcinogens, have been mea-
sured in feces. These enzymes include ß-glucuronidase,
nitroreductase, and azoreductase. Studies conducted on
rats consuming a meat-based diet showed a lower inci-
dence of colon cancer after a 20-wk experimental period,
suggesting that the NCFM supplement increased the
latency period for colon cancer in experimental rats
(Goldin and Gorbach, 1980). Administration of NCFM
along with antibiotics was shown to decrease colon tu-
mors in rats (Goldin and Gorbach, 1984c). Goldin and
Gorbach (1984a) also tested the effect of feeding NCFM
on the production of free amines in the feces of rats
consuming a meat-based diet. They found that NCFM-
fed rats had a significantly lower level of free amines.
Using human subjects, Goldin and Gorbach (1984b)
found that daily consumption of milk containing NCFM
resulted in a 2- to 4-fold reduction in the activity of
these three fecal enzymes. Similar results were found
in another human study (Goldin and Gorbach, 1984c)
and also in rats (Goldin et. al., 1980).
More recently, a blinded, placebo-controlled study
was conducted to evaluate the effect of NCFM on abnor-
mal lesions in the colon of mutagenized rats (Rao et al.,
1999). This study found a 29 and 39% inhibition of these
aberrant colonic crypts in rats fed freeze-dried NCFM
at 2 and 4% of their diet, respectively.
Taken together, these results support a positive role
of NCFM in reducing potentially harmful microbial ac-
tivities in the intestine.
 REVIEW: L. ACIDOPHILUS NCFM PROBIOTIC ACTIVITY
Immune Function
It has been postulated that probiotic bacteria may
mediate antipathogenic and anticarcinogenic activities
through their ability to positively influence the mucosal
and systemic immune responses. NCFM has been
tested for this ability in two animal systems. Wagner et
al. (1997a) demonstrated that NCFM elicited antibody-
and cell-mediated responses to Candida albicans in im-
munodeficient mice, which was thought to play a role
in decreasing the severity of candidiasis. The levels of
serum IgG, IgA, and IgM were greater in euthymic
immunocompromised mice. Interestingly, the Bifido-
bacterium strain used in this study increased serum
IgG, IgA, and IgM levels in athymic and euthymic mice,
while treatment with the L. reuteri strain resulted in
early death of all athymic mice. Strain-specific re-
sponses are evident in the study of this model system.
Yogurt prepared with a yogurt culture (DPL ABY-
2C, Rhodia, Inc.) containing NCFM, S. thermophilus,
L. bulgaricus, and Bifidobacterium infantis was tested
for its effect on the mucosal and systemic IgA and IgG
response of mice immunized orally with cholera toxin
(Tejada-Simon et al., 1999). Intestinal and serum IgA
specific for cholera toxin were higher in mice fed the
yogurt prepared with S. thermophilus, L. bulgaricus,
B. infantis, and NCFM than in mice fed skim milk or
mice fed yogurt made with only S. thermophilus and
L. bulgaricus. These results suggest that a combined
culture including NCFM may increase immune re-
sponse to oral antigens, although the specific role of
NCFM, if any, in these results was not determined in
these studies.
Urogenital Applications
Urogenital infections are widespread in women.
These infections are frequently caused by Enterococcus
faecalis, Klebsiella pneumoniae, Pseudomonas aerugi-
nosa, and Escherichia coli, which originate in the intes-
tinal tract. The use of ingested or intravaginal applica-
tions of probiotic bacteria to control the incidence of
these infections has been explored. NCFM was tested
using several laboratory assays for traits thought to
be useful in helping prevent urinary and vaginal tract
infections (Reid, 2000). NCFM was suggested to pro-
duce a biosurfactant that inhibited by over 90% the
adhesion of Enterococcus faecalis 1131 to polystyrene.
NCFM was also shown to adhere to uroepithelial and
vaginal epithelial cells collected from urine of healthy
premenopausal women during midmenstrual cycle.
Furthermore, preincubation of NCFM with these same
cells with subsequent exposure to three uropathogens
(E. coli Hu734, K. pneumoniae, and P. aeruginosa AK1)
showed that NCFM competitively excluded these
327
Figure 5. Production of hydrogen peroxide by selected strains
representing Lactobacillus species (Girgis and Klaenhammer, 2000).
Arrows indicate hydrogen peroxide production by different species
as follows: A, L. amylovorus, L. gallinarum, L. acidophilus and L.
crispatus; B, L. gasseri; C, L. johnsonii; D, L. delbrueckii. Protocol
was derived from Pick (1986).
pathogens, with inhibitions of 30, 11, and 30%, respec-
tively. Because hydrogen peroxide production may play
a role in competitive exclusion of these urogenital
pathogens, levels were assessed for NCFM. NCFM was
found to produce H2O2, but at a low level relative to
varying levels found among different strains (Figure
5). NCFM characteristics found through these in vivo
tests suggest its potential application in the control of
urogenital infections.
Diarrhea
The specific effect of NCFM as a single strain on
diarrhea has not been studied. However, NCFM was
included as part of a probiotic blend tested on pediatric
populations in Mexico. Initially, tolerance to the probi-
otic blend (containing L. acidophilus NCFM, L. reuteri,
and B. infantis BBI) was tested in a placebo-controlled
study of 72 subjects 12 to 36 mo of age randomized into
four groups (Ruiz-Palacios et al., 1996a). The groups
received either placebo, or formula providing a dose of
106, 108, or 1010 cfu of probiotic culture daily. No adverse
reactions were detected over the 3-wk feeding period.
This initial evaluation was followed by investigation
into the effect of probiotic-supplemented formula on
the prevention of community-acquired diarrhea (Ruiz-
Palacios, et al., 1996b). In one study, children aged 12 to
36 mo were randomized into two groups, one receiving a
beverage containing the probiotic blend described above
at 1010 cfu/d (119 subjects completed study) and the
other receiving a control (120 subjects). Both lactobacilli
were provided in the beverage at equal levels, whereas
the Bifidobacterium strain was present at approxi-
mately 1 log cycle lower levels. Intake, tolerance, stool
Journal of Dairy Science Vol. 84, No. 2, 2001
328 SANDERS AND KLAENHAMMER
pattern, and fecal counts of lactobacilli were monitored.
A higher portion of children fed the probiotic were free
of diarrhea (90/119) than the control group (77/120),
although no differences were seen in severity of diar-
rhea. Seven cases of diarrhea were caused by rotavirus
in the control group, whereas only two cases were diag-
nosed as such among the probiotic group. In another
study, a 16-wk feeding study was conducted with chil-
dren 12 to 32 mo of age to determine the effect of two
different probiotic preparations on diarrhea (Guerrero
et al., 1996). In this study, three groups were defined
as follows: control (132 subjects), probiotic beverage
with L. reuteri, L. acidophilus NCFM, and B. infantis
BBI (130 subjects) and probiotic beverage with a differ-
ent strain of L. acidophilus and Bifidobacterium (133
subjects). The probiotic drink containing L. reuteri, L.
acidophilus NCFM, and B. infantis BBI significantly
decreased the incidence of diarrhea. Reductions in diar-
rhea in the other probiotic beverage group did not
achieve statistical significance. Diarrhea duration was
reduced in both probiotic beverage groups. These stud-
ies demonstrate that L. acidophilus NCFM is well toler-
ated among pediatric populations even in high doses.
However, the role of NCFM in reducing the risk of
diarrhea in young children cannot be specifically deter-
mined from these studies.
Competitive Colonization
The ability of NCFM to protect two different strains
of immunodeficient mice from Candida infection was
evaluated by Wagner et al. (1997a). The oral and anal
cavities of these mice were inoculated with NCFM and
then exposed to Candida albicans. Mice not colonized
with NCFM showed high levels of Candida throughout
the GI tract. Lower levels of Candida were present in
mice associated with NCFM. In addition, significantly
lower levels of lethality were seen at both 4 wk and 8- to
12-wk assessment periods. Mice of one strain associated
with NCFM showed no mortality, whereas mice from
this same strain exposed to Candida without probiotic
showed 50 and 100% mortality at these time periods,
respectively. These results clearly show the protective
effect that NCFM can have against systemic infections.
Safety
In an immunodeficient mouse model system, Wagner
et al. (1997b) evaluated the tolerance of neonatal and
adult mice to colonization by NCFM. Neither neonatal
nor adult mice suffered any mortality when colonized
with high levels of NCFM (106 to 107 cfu/g of feces in
neonates and 108 to 1010 cfu/g of feces in adults). Al-
though L. reuteri did not translocate, some mortality
Journal of Dairy Science Vol. 84, No. 2, 2001
of neonatal mice was observed with this bacterium,
suggesting the need to proceed cautiously when using
high doses of this strain in neonatal, immunocompro-
mised hosts. Furthermore, production of serum IgM
and IgG was induced by NCFM in one strain of immuno-
compromised mice, supporting a potential role for this
bacterium in immunomodulation activities.
CONCLUSIONS
In conclusion, a significant body of research aimed
at understanding L. acidophilus NCFM at microbiologi-
cal, genetic, and clinical levels has been conducted over
the past 25 yr. These studies have provided insight into
the probiotic functionality of this strain. Furthermore,
this strain has been used successfully in commercial
applications, with a minimum of technological hurdles.
Confirmation of probiotic functionality will, however,
require well-controlled clinical evaluations aimed at ap-
propriate target populations and clinical end points.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Doug DiRienzo,
Dairy Management Inc., for identifying the need for
this review. The authors thank Denise Crowell and
Hany Girgis for providing data for incorporation into
this review. Continuing support for research on Lacto-
bacillus acidophilus NCFM has come from the North
Carolina Dairy Foundation, Dairy Management Inc.
and Rhodia, Inc. (formerly Marschall Products). Sup-
port has also come from the California Dairy Research
Foundation and USDA.
Paper No. FSR-00-21 of the Journal Series of the
Department of Food Science, NCSU, Raleigh, NC
27695-7624. The use of trade names in this publication
does not imply endorsement by the North Carolina Ag-
ricultural Research Service of the products named nor
criticism of similar ones not mentioned.
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APPENDIX
Multiple strain designations appear in the literature
for NCFM or for single colony isolates of the NCFM
parent culture. The designations NCFM, N2, NCK56,
NCK45, N2 and RL8K are essentially identical strains.
The parent NCFM culture carried the NCSU laboratory
designation RL8K and was composed of rough and
smooth variants. These variants were designated
RL8K-R (bile sensitive) and RL8K-S (bile resistant)
upon isolation from the RL8K culture (Klaenhammer
and Kleeman, 1981). N2 is a smooth, bile-resistant iso-
late from NCFM selected by scientists at Marschall
Products (now Rhodia Inc.) as a bile-resistant colony,
and as this pure culture, is used as the seed for all
commercial production runs of NCFM. The mixed par-
ent culture has not been used commercially or for re-
search studies since before1975. NCK56 is a Klaenham-
mer laboratory designation for N2, and NCK45 is a
Klaenhammer laboratory designation for the parent
culture NCFM comprised of rough and smooth colony
variants NCFM. These different isolates cannot be dif-
ferentiated genetically by pulsed field gel electrophore-
sis (Walker and Klaenhammer, 1996), a technique
which provides an electrophoretic pattern of restriction
enzyme digested chromosomal DNA. A purified isolate
of the RL8K-S culture, designated ATCC700396, is un-
dergoing chromosomal DNA sequencing (Cano and Wil-
loughby. 1999; Klaenhammer, 1998). In some papers
involving NCFM, the strain is not identified or NCFM
 REVIEW: L. ACIDOPHILUS NCFM PROBIOTIC ACTIVITY
may be present only as one strain in a combination of
several lactic acid bacteria. However, the source of the
culture used is generally provided, indicating that the
strain used was L. acidophilus from Marschall, Miles,
Rĥone-Poulenc or Rhodia. These different company
names reflect changes in business structure or owner-
ship of the company marketing NCFM, but not in mar-
keting rights. In some cases, commercial cultures pro-
vided to a study were coded to keep the identity of
331
specific strains confidential. In studies conducted by
Simenhoff and colleagues on small bowel bacterial over-
growth in chronic kidney failure patients, L. acido-
philus NCFM was abbreviated LBA. Any confusion over
use of NCFM in publications reviewed for this paper
was clarified by personal communications with com-
pany representatives and researchers. NCFM is a regis-
tered trademark of the North Carolina Dairy Foun-
dation.
Journal of Dairy Science Vol. 84, No. 2, 2001