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Appl Environ Microbiol, June 1998, p. 2192-2199, Vol. 64, No. 6
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Development of a Probiotic Cheddar Cheese
Containing Human-Derived Lactobacillus paracasei
Strains
G.
Gardiner,1
R. P.
Ross,1
J. K.
Collins,2
G.
Fitzgerald,2 and
C.
Stanton1,*
Dairy Products Research Center, Moorepark,
Fermoy,1 and
Department of
Microbiology, University College Cork,2 County
Cork, Ireland
Received 10 November 1997/Accepted 10 March 1998
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ABSTRACT |
Cheddar cheese was manufactured with either Lactobacillus
salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct.
These five strains had previously been isolated from the human small
intestine and have been characterized extensively with respect to their
probiotic potential. Enumeration of these strains in mature Cheddar
cheese, however, was complicated by the presence of high numbers
(>107 CFU/g of cheese) of nonstarter lactic acid bacteria,
principally composed of lactobacilli which proliferate as the cheese
ripens. Attempts to differentiate the adjunct lactobacilli from the
nonstarter lactobacilli based on bile tolerance and growth temperature
were unsuccessful. In contrast, the randomly amplified polymorphic DNA
method allowed the generation of discrete DNA fingerprints for each
strain which were clearly distinguishable from those generated from the
natural flora of the cheeses. Using this approach, it was found that
both L. paracasei strains grew and sustained high
viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.
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INTRODUCTION |
Cheddar cheese may offer certain
advantages as a carrier of probiotic microorganisms. Having a higher pH
than the more traditional probiotic foods (e.g., yogurts and fermented
milks), it may provide a more stable milieu to support their long-term
survival. Furthermore, the matrix of the cheese and its relatively high
fat content may offer protection to probiotic bacteria during passage
through the gastrointestinal tract. However, among the most important criteria when considering Cheddar cheese as a probiotic food is that
the microorganisms be able to survive the relatively long ripening time
of at least 6 months and/or that they grow in the cheese over this
period. During the ripening period, generally performed at 2 to 16°C
(21), a number of nonpathogenic bacteria, chiefly
lactobacilli (Lactobacillus plantarum, L. casei, and L. brevis) and pediococci
(Pediococcus pentosaceus) proliferate in the maturing cheese
(5). Many of these nonstarter lactic acid bacteria (NSLAB)
have complex proteolytic systems, which have been associated with the
maturation process. Consequently, lactobacilli with defined proteolytic
systems have been deliberately added as adjuncts to cheese milk in
order to influence cheese maturation (4, 26, 27, 40). The
results of such an approach can be variable depending on the particular
strain used. At present, a number of Lactobacillus adjuncts
employed for the improvement of flavor are commercially available for
cheeses, including Cheddar.
There are relatively few reports concerning cheese as a carrier of
probiotic organisms, even though there are a small number of probiotic
cheeses currently on the market worldwide. In 1994, Dinakar and Mistry
(10) incorporated Bifidobacterium bifidum into
Cheddar cheese as a starter adjunct. This strain survived well in the
cheese and retained a viability of approximately 2 × 107 CFU/g of cheese even after 6 months of ripening,
without adversely affecting cheese flavor, texture, or appearance. This
example suggested that Cheddar could provide a suitable environment for the maintenance of probiotic organisms at high levels over long periods. In another study, bifidobacteria were used in combination with
L. acidophilus strain Ki as a starter in Gouda cheese
manufacture (12). In this case, there was a significant
effect on cheese flavor in the resultant product after 9 weeks of
ripening, possibly due to acetic acid production by the bifidobacteria.
In order to exert a probiotic effect, cultures must maintain their
viability in food products through to the time of consumption, which
for Cheddar cheese is many months after manufacture. The potential
health-promoting effects achieved by the consumption of dairy products
containing probiotic organisms, such as Lactobacillus and
Bifidobacterium spp., have resulted in intensive research efforts in recent years (for reviews, see references 11,
25, and 35). The products which have
received the most attention in this regard include fermented milks,
such as yogurt and buttermilk, as well as unfermented milks with
cultures added (3, 33, 36-38) together with frozen desserts
such as ice cream and frozen yogurt (6, 14, 23). The
importance of these probiotic-containing products, commonly regarded as
functional foods, in the maintenance of health and well-being is
becoming a key factor affecting consumer choice. This has resulted in
rapid growth and expansion of the market for such products, in addition
to increased commercial interest in exploiting their proposed healthful
attributes.
This study investigates the performance of a number of
Lactobacillus strains, including L. salivarius and L. paracasei, when employed as
adjuncts in Cheddar cheese, over 8 months of ripening. These strains
had previously been isolated from healthy human intestine and
characterized in detail with regard to their probiotic potential
(8). In this respect, these strains have been shown to be
acid and bile tolerant, adherent to human epithelial cells, and
nonpathogenic and to have desirable antibiogram profiles. A
prerequisite to the enumeration of these strains from cheese was the
development of a reliable genetic fingerprinting method to distinguish
them from the resident flora. The results demonstrate that the
probiotic L. paracasei species used in this study are particularly suitable for Cheddar cheese applications. They grow to
numbers in excess of 108 CFU/g and remain at this
level even after 8 months of ripening, while their presence has
negligible effects on cheese composition, flavor, and aroma.
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MATERIALS AND METHODS |
Bacterial strains and culture conditions.
The probiotic
Lactobacillus strains used in this study had previously been
isolated from the human gastrointestinal tract and were obtained from
University College Cork, Cork, Ireland, under a restricted materials
transfer agreement. These strains were identified as L. salivarius (subsp. salivarius) and L. paracasei (subsp. paracasei) by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (PAGE) analysis of total
cell protein (31) and were designated L. salivarius NFBC 310, NFBC 321, and NFBC 348 and L. paracasei NFBC 338 and NFBC 364. NSLAB Lactobacillus
strains (L. curvatus DPC 2042 and DPC 2081, L. plantarum DPC 2102 and DPC 2142, and L. casei subsp. casei DPC 2047 and DPC 2103) which had
previously been isolated from 8-week-old commercial Cheddar cheeses
were obtained from the culture collection of the Dairy Products
Research Centre. All Lactobacillus strains were routinely cultured in MRS broth (9) (Difco Laboratories, Detroit,
Mich.) under anaerobic conditions (anaerobic jars with Anaerocult A gas packs; Merck, Darmstadt, Germany) at 30 and 37°C for NSLAB and probiotic strains, respectively. Lactococcus lactis subsp.
cremoris strains 227 and 223, obtained from C. Hansen's
Laboratories (Little Island, Cork, Ireland) in the form of freeze-dried
pellets, were used as starters for cheesemaking. These were grown
overnight at 21°C in heat-treated (90°C for 30 min) 10% (wt/vol)
reconstituted skim milk (RSM).
Bile and temperature tolerance.
To investigate the tolerance
of both the probiotic and NSLAB Lactobacillus isolates to
bile, overnight MRS broth cultures of each of the
Lactobacillus strains were serially diluted in maximum
recovery diluent (Oxoid Ltd., Basingstoke, Hampshire, United Kingdom)
and appropriate dilutions were pour plated on MRS agar with 0, 0.1, 0.3, 0.5, 1.0, or 3.0% porcine bile (Sigma Chemical Co., Poole,
Dorset, United Kingdom). After 3 days of incubation, the plates were
examined, and where colonies were present, their numbers and sizes were
recorded. Temperature tolerance of the probiotic lactobacilli was
investigated by pour plating appropriate dilutions of overnight
cultures on LBS agar (34) (Becton Dickinson, Cockeysville,
Md.) and incubating the plates anaerobically both at 37°C (which is
the optimum temperature of growth for these strains) and at 42°C.
Colony numbers obtained after 5 days were compared. In the same way,
the temperature tolerance of these strains and NSLAB, following
isolation from Cheddar cheese, was also investigated.
Genetic fingerprinting by RAPD-PCR.
Randomly amplified
polymorphic DNA (RAPD)-PCR analysis was carried out on each of the
probiotic Lactobacillus strains and on cultures grown from
Lactobacillus colonies isolated from Cheddar cheese. Genomic
DNA was isolated from 1.5 ml of overnight MRS broth cultures by using a
modification of the method of Hoffman and Winston (13). This
procedure utilizes shearing with glass beads to lyse the cells and was
modified as outlined by Coakley et al. (7). One microliter
of the extracted DNA was used in subsequent PCR amplifications, which
were performed in a total volume of 25 µl in a Perkin-Elmer (Norwalk,
Conn.) DNA Thermal Cycler. The method was employed essentially as
described by Coakley et al. (7) and used a single primer of
arbitrary nucleotide sequence (5' ATGTAACGCC 3'), obtained
from Pharmacia Biotech (Uppsala, Sweden). DNA was amplified for 35 cycles using the following temperature profile: denaturing at 93°C
for 1 min, annealing at 36°C for 1 min, and polymerization at 72°C
for 1 min. Taq DNA polymerase (0.625 U; Bioline) was added
to the reaction mixture during the first denaturation step (hot start).
Between 5 and 10 µl of the PCR mixture was analyzed on a 1.5%
(wt/vol) agarose (Sigma) gel with ethidium bromide staining. A 100-bp
ladder (Pharmacia) was used as a molecular weight standard. Gels were
run for approximately 3 h at 100 V, and the DNA was visualized by
UV transillumination.
Cheddar cheese manufacture.
Laboratory-scale cheesemaking
trials (trials 1 and 2) were performed initially with 25 liters of
pasteurized whole milk in each cheese vat. To limit contamination with
wild lactobacilli, these cheeses were manufactured under controlled
bacteriological conditions, as described by McSweeney et al.
(28). A 1.5% inoculum of the mixed-strain starter culture
was used, and in each trial one vat (vat 1) acted as a control to which
starter only was added. To each of the experimental vats, one probiotic
Lactobacillus strain, grown overnight in 10% RSM, was added
as an adjunct to the starter culture. In trial 1, the probiotic
adjuncts L. salivarius NFBC 348 and L. paracasei NFBC 364 were added at an inoculum level of 0.1% to
vats 2 and 3, respectively. In the second trial, L. salivarius NFBC 310 (vat 2), L. salivarius NFBC
321 (vat 3), and L. paracasei NFBC 338 (vat 4) were
inoculated at a level of 0.2%. Cheddar cheeses were then manufactured
according to standard procedures as follows. Filter-sterilized rennet
(C. Hansen's Laboratories) was added at a concentration of 0.07 ml/liter 35 min after starter and adjunct addition, and the curd was
cut approximately 40 min later. Curds were cooked to 39°C, pitched at
pH 6.1, and milled at approximately pH 5.3. Salt was added at a rate of
2.8% (wt/wt), and the curds were placed in molds and pressed at
approximately 200 kPa overnight. The cheeses were removed from the
molds, vacuum packed, and ripened at 8°C for approximately 8.5 months. Subsequently, two pilot-scale cheesemaking trials (trials 3 and
4) were performed with two of the adjunct Lactobacillus
strains which were found to maintain high viability in the
laboratory-scale cheeses during ripening. In each trial, two vats, one
experimental and one control, each containing 450 liters of
standardized (fat/protein ratio = 1) pasteurized whole milk, were
used. As in the laboratory-scale trials, a 1.5% inoculum of the
starters 223 and 227 was added to each vat. In addition, in each trial
the experimental vat (vat 2) contained a 0.1% inoculum of either
L. paracasei NFBC 364 (trial 3) or NFBC 338 (trial 4)
added as a starter adjunct. The cheesemaking procedure was as
previously described for the laboratory-scale cheeses except that the
salting level was 2.7% and the curds were pressed overnight at
approximately 413 kPa.
Cheese compositional analysis.
Grated cheese samples were
analyzed in duplicate for salt content by a potentiometric method
(15), for fat content by the Gerber method (17),
for moisture content by oven-drying at 102°C (16), and for
protein content on a LECO FP-428 nitrogen determinator. The pH of a
slurry, prepared by blending 12 ml of H2O with 20 g of
grated cheese, was measured by using a standard pH meter (Radiometer,
Copenhagen, Denmark).
Bacteriological analyses of cheeses.
Viability of
lactobacilli (both probiotic adjuncts and NSLAB) in the inoculated
cheese milk and in the cheeses during ripening was determined on LBS
agar after 5 days of anaerobic incubation at 30°C, while starter
lactococci were enumerated on LM17 agar (39) after 3 days of
incubation at 30°C. Coliforms in cheese milk and cheeses were
enumerated on violet red bile agar (VRBA; Oxoid) after incubation at
37°C for 24 h. Cheeses were aseptically sampled in duplicate for
bacteriological analysis at intervals during ripening. Cheese samples
were emulsified in sterile 2% (wt/vol) trisodium citrate and diluted
in maximum-recovery diluent, and appropriate dilutions were pour
plated. After one, three, and six monthly intervals, 18 individual
Lactobacillus colonies from each cheese were randomly
selected from the LBS agar plates for RAPD-PCR analysis.
Sensory evaluation of Cheddar cheese.
Cheeses were graded
blindly after 3 and 6 months of ripening by a commercial grader from a
local cheese manufacturing plant. The cheeses were graded for the
characteristics "flavor/aroma" and "body/texture," with maximum
scores of 45 and 40, respectively. Minimum scores of 38 and 31 for
flavor/aroma and body/texture, respectively, are required for
commercial Cheddar cheese.
Assessment of proteolysis in Cheddar cheese.
Cheeses were
analyzed by urea-PAGE using the stacking gel system of Andrews
(1). Cheese samples were prepared by dispersing 10 mg of
grated cheese in 1 ml of sample buffer, and 10 µl of this sample was
applied to the gel. Sodium caseinate (5 µl) was used as a standard
for comparative purposes, and gels were stained by the direct-staining
procedure of Blakesley and Boezi (2).
Water-soluble extracts (pH 4.6) of each of the cheeses were prepared
according to the method of Kuchroo and Fox (22) and freeze-dried. The size distribution of peptides in these freeze-dried extracts was determined by size exclusion high-performance liquid chromatography (HPLC), using a TSK G2000 SW (Beckman Instruments Ltd., High Wycombe, Buckinghamshire, United Kingdom) gel permeation column (7.5 nm by 60 cm) fitted to a Waters HPLC system (Waters Chromatography Division, Milford, Mass.). The column was
eluted at a flow rate of 1 ml/min with 30% acetonitrile
containing 0.1% trifluoroacetic acid. The freeze-dried water-soluble
extracts were reconstituted (3 mg/ml) in HPLC-grade water and filtered through a Whatman 0.2-µm-pore-size filter, and 20 µl of the
filtered extract was applied to the column. Column eluates were
continually monitored at 214 nm. Data were collected by using a PC
Minichrom system (VG Data Systems, Altrircham, Cheshire, United
Kingdom), and the results were compared to a previously prepared
calibration curve.
Individual free amino acids (FAA) in the water-soluble extracts were
determined by using a Beckman System 6300 High Performance Analyzer
(Beckman Instruments Ltd.) equipped with a Beckman P-N 338052 Na+ column (12 by 0.5 cm) as described by Lynch et al.
(26). Amino acid concentrations were expressed as micrograms
per milliliter of cheese extract, which was subsequently converted to
micrograms per gram of cheese.
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RESULTS AND DISCUSSION |
Probiotic strain identification and enumeration.
A
prerequisite to the successful enumeration of added probiotic strains
is their selective enumeration from the natural, often complex
microflora found in food products. Since NSLAB can reach levels up to
107 to 108 CFU/g in cheese during ripening
(30), the present study initially focused on evaluating a
number of methods aimed at selectively distinguishing the lactobacilli
added as starter adjuncts from these NSLAB.
(i) Bile and temperature tolerance of Lactobacillus
adjuncts.
Both the probiotic adjuncts and NSLAB
Lactobacillus strains varied considerably with regard to
their ability to tolerate bile, and therefore selections based on bile
tolerance were not a useful means of distinguishing the probiotic
adjunct lactobacilli from the NSLAB lactobacilli during the ripening of
Cheddar cheese. Similarly, temperature tolerance of the two groups of
lactobacilli was found to be nonselective, although it has been shown
that NSLAB isolated from Irish Cheddar cheeses do not grow at 45°C (19) but that some of the human-derived probiotic
lactobacilli may withstand such temperatures (20).
(ii) RAPD-PCR analysis.
Consequently, the RAPD method, which
involves PCR using an arbitrary primer, was used to generate DNA
fingerprints for each of the probiotic strains. Each of the
Lactobacillus strains generated reproducible discrete DNA
fingerprints (Fig. 1) which were found to
be substantially different from those of representative NSLAB lactobacilli. Thus, the RAPD method proved to be a successful means of
identifying the probiotic organisms and demonstrated potential as a
means of selective identification of these strains from the NSLAB flora
in Cheddar cheese.
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FIG. 1.
RAPD-PCR-generated DNA fingerprints for probiotic
Lactobacillus strains L. salivarius NFBC 310 (lanes 2 and 3), L. salivarius NFBC 321 (lanes 4 and
5), L. paracasei NFBC 338 (lanes 6 and 7),
L. salivarius NFBC 348 (lanes 8 and 9), and
L. paracasei NFBC 364 (lanes 10 and 11). Lanes 1 and 12 contain 100-bp ladders.
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Incorporation of Lactobacillus species into Cheddar
cheese.
Initially, laboratory-scale cheese trials were performed
under microbiologically controlled conditions (thus limiting
development of high numbers of NSLAB during ripening) to assess the
performance of five probiotic Lactobacillus strains in
Cheddar cheese. Firstly, for inoculation purposes, the performance of
these strains in RSM was investigated. None of the strains performed
well in milk (levels of only 107 to 108 CFU/ml
were achieved), and all were subsequently found to be nonproteolytic or
only weakly proteolytic (data not shown). Thus, when 0.1 to 0.2%
inocula of these L. salivarius and L. paracasei strains were used as starter adjuncts, relatively
low levels (104 to 105 CFU/ml of milk) were
obtained in the experimental vats during cheese manufacture. All
adjunct lactobacilli were found to survive during cheesemaking, and
acid development during the process was unaffected by the presence of
these strains.
Microbiology of the Cheddar cheeses during ripening and the RAPD-PCR
profiles of
Lactobacillus isolates from the ripened cheeses
are shown in Fig.
2 to
4. It should be noted that day 0 (Fig.
2A,
3A, and
4A) represents the day of cheese manufacture, while
lanes 1 (Fig.
2C and D, Fig.
3C to E, and Fig.
4B) represent the
RAPD-PCR
profile of the probiotic strain added to the experimental
cheeses
during manufacture. Results demonstrate that cheese made
with NFBC 364 and NFBC 338
L. paracasei adjuncts (Fig.
2A and
3A)
contained high levels of these probiotic strains after 8 months
of
ripening; with final counts of 9.2 × 10
7 and 1.4 × 10
8 CFU/g achieved, respectively. This was confirmed
following comparison
of the RAPD-PCR fingerprints generated for
L. paracasei NFBC 364
and NFBC 338 (respectively, Fig.
2D and
3E, lanes 1) and those
obtained for lactobacilli isolated from
the cheeses (Fig.
2D and
3E, lanes 2 to 10 and 12 to 20) which were
found to be identical.
In contrast, although lactobacilli grew to high
levels (10
8 CFU/g) in the cheese to which strain NFBC 310 was added (Fig.
3A) and subsequently remained at this level throughout
ripening,
these lactobacilli (Fig.
3C, lanes 2 to 10 and 12 to 20) were
identified by RAPD-PCR as NSLAB. Levels of lactobacilli in cheeses
with
L. salivarius adjuncts NFBC 348 and NFBC 321 (Fig.
2A
and
3A) declined to 1.2 × 10
5 and 8.6 × 10
4 CFU/g, respectively, after 4 months of ripening,
although these
levels did increase slightly to reach final levels of
3.5 × 10
5 and 1.1 × 10
6 CFU/g,
respectively, after 8 months of ripening. Interestingly,
the genetic
fingerprints of isolates taken from each of these
cheeses after 6 months revealed that these lactobacilli were predominantly
NSLAB (Fig.
2C and
3D, respectively). Thus, the
L. salivarius strains used in this study did not maintain viability in Cheddar
cheeses during ripening. Furthermore, many of the NSLAB isolated
from
the cheeses in which the adjunct strain declined and from
the control
cheese to which no probiotic adjunct was added yielded
identical
PCR-generated DNA fingerprints (compare Fig.
3B, lanes
3 to 9 with,
Fig.
3D, lanes 12 to 18). This suggests that the
DNA was obtained from
identical strains and shows a predominance
of certain
Lactobacillus strains in the NSLAB population of these
cheeses.
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FIG. 2.
(A) Survival of lactobacilli and starter during cheese
ripening in trial 1 laboratory-scale Cheddar cheeses manufactured under
microbiologically controlled conditions. (B to D) RAPD-PCR profiles of
a representative number of Lactobacillus isolates from each
of the cheeses. Lanes 1 (C and D) show the RAPD profile of each
probiotic Lactobacillus strain added to the cheese at
manufacture, while a 100-bp ladder is shown in lane 19 (B) or 11 (C and
D) and all other lanes show RAPD profiles of Lactobacillus
isolates from 6-month (180-day)-ripened cheeses.
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FIG. 3.
(A) Survival of lactobacilli and starter during cheese
ripening in trial 2 laboratory-scale Cheddar cheeses manufactured under
microbiologically controlled conditions. (B to E) RAPD-PCR profiles of
a representative number of Lactobacillus isolates from each
of the cheeses. Lanes 1 (C to E) show the RAPD profile of each
probiotic Lactobacillus strain added to the cheese at
manufacture, while a 100-bp ladder is shown in lanes 11 (B to E) and
all other lanes show RAPD profiles of Lactobacillus isolates
from 6-month (180-day)-ripened cheeses.
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FIG. 4.
(A) Survival of lactobacilli and starter during cheese
ripening in trial 3 pilot-scale Cheddar cheeses. (B) RAPD-PCR profiles
of a representative number of Lactobacillus isolates from
vat 2 cheese to which L. paracasei NFBC 364 was added
during manufacture. Lane 1 shows the RAPD profile of the added strain.
A 100-bp ladder is shown in lane 10, while lanes 2 to 9 and 11 to 20 show RAPD profiles of Lactobacillus isolates from the
6-month (180-day)-ripened cheese.
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Subsequently, pilot-scale cheese trials were performed, in which only
the two
L. paracasei strains, NFBC 338 and NFBC 364,
that survived to high levels in the laboratory-scale trials were
incorporated into Cheddar cheese. These strains were added at
inocula
of 1.7 × 10
5 and 8.9 × 10
5 CFU/ml
of cheese milk, respectively. Thereafter, both NFBC 364
(Fig.
4A) and
NFBC 338 grew in the cheese from initial numbers
of 2.7 × 10
7 and 1.1 × 10
7 CFU/g, respectively, to
reach levels between 2.9 × 10
8 and 1.5 × 10
8 CFU/g after 3 months of ripening, and viability was
sustained
at this level for the remainder of the ripening period. These
results were confirmed for both pilot-scale trials by RAPD-PCR
of
cheese isolates as described above. Only the data for isolates
from
cheese manufactured with NFBC 364 are shown (Fig.
4B).
Taken together, the data from the laboratory- and pilot-scale cheese
trials provide molecularly based evidence for the persistence
in
Cheddar cheese of strains selected for their potential as probiotics.
In order to appreciate the beneficial effects of probiotic foods,
it
has been proposed that viable probiotic organisms should be
present at
levels of at least 10
7 viable cells per gram or milliliter
of product (
18). The probiotic-containing
cheeses developed
as part of the present study contained levels
up to 10
8
CFU/g of cheese, thus satisfying these criteria for a probiotic
food
product.
It should also be noted that lactococcal starter numbers in the control
cheeses of all trials showed a typical decline during
the ripening
period (Fig.
2A,
3A, and
4A). However, due to the
growth of
lactobacilli on the LM17 medium used to enumerate these
starter
organisms, it was possible to monitor starter organisms
only in the
cheeses to which no adjunct lactobacilli had been
added, and then it
was possible only in the early stages of ripening.
Although lactobacilli have previously been added as adjuncts to Cheddar
cheese and have subsequently been found to remain
at high levels
throughout maturation (
4,
26,
28), no definitive
identification method was used in these previous studies to
distinguish
the adjunct lactobacilli from the natural flora of the
cheese.
However, in our study RAPD-PCR analysis, when used as an
identification
method, was capable of determining that probiotic
L. paracasei strains grew and maintained high viability
(10
8 CFU/g) in cheese, while the particular
L. salivarius adjunct
strains used did not appear to be suited for
such an application.
Furthermore, in the present study, survival of
these probiotic
Lactobacillus strains at high numbers in
Cheddar cheese was achieved
with a relatively small inoculum (0.1 to
0.2%) in the cheese vat
and without altering the cheesemaking process
in any way. This
was possible because these strains were added as
starter adjuncts
and were not therefore necessary for acid production
during cheesemaking.
In a study conducted by Gomes et al.
(
12), bifidobacteria and
L. acidophilus were
used as the sole starters in Gouda cheese
manufacture, requiring
relatively large inocula (3%) of both strains
and adaptation of
cheesemaking technology. Thus, our approach
for incorporation of
probiotic organisms into Cheddar cheese offers
certain advantages to
industry; no alteration of existing cheesemaking
technology and low
cost due to the low inoculum required.
Cheese compositional analysis.
The composition of the cheese
was generally found to be within the range typical for Cheddar (Table
1). Atypical values for salt in moisture,
fat, and pH were obtained for some of the trial 1 cheeses, which
reflects the difficulties in controlling the cheesemaking parameters
(i.e., temperature) at laboratory scale. In contrast, all the
compositional analysis values obtained for the pilot-scale trials were
generally within the typical range for Cheddar. Thus, the comparable
values observed for control and experimental cheeses (Table 1) indicate
that incorporation of probiotic lactobacilli as starter adjuncts, and
their survival at high numbers, had no direct effect on cheese
composition.
Sensory evaluation.
With the exception of the control cheese
of trial 2, all cheeses could be described as commercial grade with
respect to sensory criteria, after 6 months of ripening, having
achieved minimum scores of 38 and 31 for flavor/aroma and body/texture,
respectively (Table 1). Lactobacillus adjuncts have
previously been reported to improve Cheddar cheese flavor (4, 26,
28), although in some cases they were responsible for flavor
defects (24, 32). In this study, laboratory-scale cheeses
with high levels of Lactobacillus adjuncts were found to
have flavor and texture comparable to those of control cheeses,
indicating that addition of these probiotic lactobacilli to Cheddar
cheese had no adverse effects on sensory criteria. Furthermore, when
production was repeated on a larger scale, sensory parameters remained
unaffected by the presence of high levels of these adjuncts.
Proteolysis in laboratory-scale Cheddar cheeses.
Urea-PAGE patterns of whole cheese samples after 8 months of
ripening were typical for Cheddar and did not show any differences in
the extents of primary proteolysis between the control cheeses and
those manufactured with adjunct lactobacilli (data not shown). Similarly, others (26, 28) have shown that adjunct
lactobacilli have no effect on PAGE electophoretograms, which is not
surprising, as proteolysis at this level is due to the action of
plasmin and rennet and is not influenced by the activity of cheese
flora (29). The molecular mass distribution of peptides in
water-soluble extracts from the cheeses (as measured by size exclusion
HPLC) serves as a further indication of the extent of proteolysis
in the cheeses during ripening; the greater the extent of
proteolysis, the higher the level of low-molecular-mass peptides
generated. After 6 months of ripening, these low-molecular-mass
peptides (<500 Da) were found to have accumulated to high levels in
all cheeses (data not shown). Moreover, similar levels were detected in
the control and experimental cheeses, even in those cheeses which had
high levels of survival of L. paracasei NFBC 338 and
364 adjuncts, indicating that the extent of proteolysis in the
cheeses, as demonstrated by generation of small peptides, was not
affected by adjunct addition.
However, higher levels of individual FAA were detected in some of the
cheeses made with added lactobacilli, after 6 months
of ripening (Fig.
5). Most notably, concentrations of
glutamic
acid, methionine, leucine, and lysine (trial 1) (Fig.
5A) in
addition
to valine (trial 2) (Fig.
5B) were higher in the cheeses made
with added lactobacilli than in the control cheese to which no
adjunct
had been added. This was found to be true even for the
cheeses in which
the
Lactobacillus adjuncts declined during ripening.
This
may be accounted for by the release of intracellular peptidases
as the
organisms died and lysed. Thus, in general the results
suggest that the
adjunct lactobacilli, whether they survived to
high levels or not, did
contribute to proteolysis in the cheese,
as demonstrated by increased
formation of FAA. Similarly, addition
of both lactobacilli and
bifidobacteria to Cheddar cheese has
previously been shown to increase
proteolysis at the level of
FAA formation (
4,
26,
28,
32).
In contrast, in a study
by Dinakar and Mistry (
10)
bifidobacteria were found not to
alter proteolysis in Cheddar cheese;
however, FAA were not measured
in that study.
View larger version (33K):
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|
FIG. 5.
Concentration of individual FAA in water-soluble
extracts of 6-month-old control and experimental Cheddar cheeses of
trial 1 (A) and trial 2 (B).
|
|
Conclusions.
The results of the present study demonstrate that
probiotic L. paracasei strains incorporated into
Cheddar cheese proved particularly suitable as starter adjuncts. These
strains were found to grow and proliferate to high cell numbers
in cheese over 8 months of ripening, even when added at
a relatively small inoculum. Furthermore, RAPD-PCR
proved extremely useful in distinguishing these probiotic adjuncts from
NSLAB. Moreover, the results from the control cheese suggest the
predominance of certain NSLAB strains. While proteolysis during cheese
ripening was influenced by the adjuncts at the level of FAA formation,
cheese flavor, texture, and appearance were not affected. Incorporation
of these probiotic adjuncts to Cheddar cheese, as conducted in this
study, can be achieved without alteration of the cheesemaking
technology, thus making this system attractive for commercial
exploitation. The results of the present study indicate that Cheddar
cheese offers potential as an effective vehicle for delivery of these
strains to the consumer.
| |
ACKNOWLEDGMENTS |
The technical assistance of Finbar Drinan, Helen Slattery, and
Eddie Mulholland is gratefully acknowledged. We thank Pat Fenton, Dairygold, Mitchelstown, Co. Cork, Ireland, for sensory analyses.
This work was supported by the European Research and Development Fund.
G.G. was supported by a Teagasc Walsh Fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dairy Products
Research Center, Moorepark, Fermoy, County Cork, Ireland. Phone:
353-25-42222. Fax: 353-25-42340. E-mail:
cstanton{at}dpc.teagasc.ie.
| |
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Abstract
Introduction
