Note: Descriptions are shown in the official language in which they were submitted.
CA 02594937 2007-07-23
PRODUCTION OF PROBIOTIC BACTERIA USING MAPLE SAP
Field of the Invention
The present invention relates to biotechnology, in particular to methods for
producing probiotic bacteria and products of probiotic bacteria.
Background of the Invention
Maple syrup is one of the hallmark products of Canada. About 84% of the
world's production of maple syrup is made in Canada and more than 93% of it
originates from the province of Quebec. Since 1999, a dramatic saturation of
the
markets has led to a large inventory surplus. At the end of the 2004 season,
the
volume of bulk inventories exceeded 60 million pounds. The Federation des
Producteurs Acericoles du Quebec is addressing the surplus problem by
exploring
new markets.
Maple sap, the sap of the maple tree, can be considered as a ready-to-use,
sugar-rich (mainly sucrose), renewable feedstock having a potential to sustain
the
growth of a large variety of microorganisms. In commonly owned United States
patent application USSN 11/715,944 field March 9, 2007, the feasibility of
growing
large amounts of the polyhydroxyalcanoate-producing bacterium, Alcaligenes
latus, was demonstrated.
According to the definition of the United Nations Food and Agricultural
Organization and the World Health Organization, probiotics are "live
microorganisms which, when administrated in adequate amounts, confer a health
benefit on the host". There is already an important market for probiotics as
food
supplements for the benefit of human and animal health. In a colloquium of the
American Academy for Microbiology held in Baltimore in November 2005,
participants with expertise in microbiology, medicine, nutrition, immunology,
animal sciences and other relevant field listed the following examples where
probiotics use benefit human and animal health: treating diarrhea caused by
Rotavirus in children, treating irritable bowel syndrome, treating bladder
cancer,
treating urogenital infections, treating Clostridium difficile infections, and
treating
atopic eczema.
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Prior art processes for the production of probiotics using various carbon
and nitrogen sources, e.g. sugarcane, are well known and many companies
already sell probiotic products. However, there remains a need in the art for
improved methods of producing probiotic bacteria.
Summary of the Invention
Here we disclose the use of maple sap or down-graded maple syrup as a
carbon and energy source for the production of probiotic bacteria. The use of
maple sap or down-graded maple syrup unexpectedly leads to an improvement in
growth of probiotic bacteria and an improvement in the yield of products
produced
by the bacteria.
Thus, there is provided a method of growing probiotic bacteria comprising
contacting the probiotic bacteria with maple sap, down-graded maple syrup or a
mixture thereof.
There is further provided a method of producing lactic acid comprising
contacting probiotic Lactobacillus bacteria with maple sap, or down-graded
maple
syrup or a mixture thereof.
Probiotic bacteria are preferably bacteria, or mixtures of bacteria, of genus
Lactobacillus, for example L. acidophilus, L. brevis, , L. buchneri, L. casei,
L.
curvatus, L. delbrueckii, L. fermentum, L. helveticus, L. plantarum, L.
reuteri, L.
rhamnosus, L. sakei and L. salivarius. L. acidophilus, L. casei and L.
helveticus
are of particular note.
Maple sap-based culture medium in accordance with the present invention
allows good growth of probiotic bacterial strains used in the production of
commercial probiotic products. Maple sap-based medium surprisingly provides
better growth yield than a sucrose-based medium. Growth yield on maple sap-
based medium may be, for example, at least 1.5 times greater than growth yield
on sucrose-based medium. In some embodiments, improvements in growth yield
of at least 2 times greater, or at least 3 times greater, or at least 4 times
greater
may be realized. In other embodiments, improvement in growth yield may be 2 to
4 times greater.
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Enhanced growth yield on maple sap-based medium may also lead to
greater production of probiotic products, for example lactic acid, anti-
oxidants
and/or other biologically active components that trigger the growth of
probiotic
bacteria. In some embodiments, production of lactic acid (or lactate) may be
at
least 1.5 times greater, or at least 2 times greater, in a maple sap-based
medium
than in a sucrose-based medium.
Advantageously, the maple sap may be formulated into a medium
containing growth supplements for the probiotic bacteria. Growth supplements
may include, for example, a yeast extract, salts, a nitrogen source, vitamins
or
mixtures thereof. Salts include, for example, potassium dihydrogen phosphate
(KH2PO4), dipotassium hydrogen phosphate (K2HPO4), manganese sulfate
(MnSO4), magnesium sulfate (MgSO4), ferrous sulfate (FeSO4), etc. Nitrogen
sources include, for example, proteins from vegetal sources (e.g. soy, pea,
etc.) or
animal sources (e.g. milk proteins such as casein). Because there is an
increasing demand for animal-free products in the food market (since the
emergence of mad cow disease and increasing food allergies toward bovine
proteins), the nitrogen source is preferably protein from vegetal sources.
Vitamins
include, for example, mevalonic acid, ascorbic acid, etc. Growth supplements
may be used in any effective concentration, for example, in a range of from
about
0.01 % to about 100% of the concentration of the maple sap based on weight.
Incubation or fermentation of the bacteria in the medium may be conducted
at a temperature in a range of from about 30 C to about 45 C, preferably 36-43
C.
Anaerobic or aerobic conditions may be used, preferably anaerobic conditions.
Cultures may be static or agitated, preferably static.
Methods of the present invention are particularly useful for commercial
scale production of probiotic bacteria or products from probiotic bacteria.
Commercial scale production is preferably carried out in bioreactors where the
bacteria are fermented in the maple sap-based medium.
Further features of the invention will be described or will become apparent
in the course of the following detailed description.
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Brief Description of the Drawings
In order that the invention may be more clearly understood, embodiments
thereof will now be described in detail by way of example, with reference to
the
accompanying drawings, in which:
Fig. 1 is a graph of ODsoonm vs. time (hours) depicting growth of probiotic
bacteria in maple sap-based medium at 37 C under static condition;
Fig. 2 is a graph of viable cell count (colony forming units (cfu)) at 0 hours
and 16 hours for the growth of probiotic bacteria in maple sap-based medium at
37 C under static condition;
Fig. 3 is a graph of viable cell count (colony forming units (cfu)) at 16
hours
for the growth of probiotic bacteria in maple sap-based medium and sucrose-
based medium at 37 C under static condition; and,
Fig. 4 is a graph of lactate produced (ppm) after 16 hours for the growth of
probiotic bacteria in maple sap-based medium and sucrose-based medium at
37 C under static condition.
Description of Preferred Embodiments
Example 1: Growth in maple sap-based medium supplemented with soy drink
Filtered-sterilized maple sap (50 mL) at pH 7.0 having about 16,000 ppm
sucrose, about 400 ppm glucose and 400 ppm fructose was inoculated with 0.1
mL of commercial Bio-K+ product, which contains high amounts of probiotic
fermentive bacteria, in this case, two lactobacilli, L. casei and L.
acidophilus.
Although maple sap is a good carbon source, it has a low carbon to nitrogen
(C/N)
ratio, therefore it was supplemented with either ammonium sulfate (2 mM) or a
commercial soy drink, UHT, (30% (v/v) soy drink was added to 70% (v/v) maple
sap) as a source of nitrogen. The cultures were incubated at 30 C for two days
under anaerobic conditions (in closed serum bottles with the headspace flushed
with argon).
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In the non-inoculated controls, no bacterial growth (i.e. no turbidity) was
observed in any of the conditions mentioned. The non-supplemented maple sap
and the maple sap supplemented with ammonium sulfate only led to poor
bacterial growth. However, the aerobic culture composed of maple sap (70%
(v/v))
and soy drink (30% (v/v)) led to the growth of an important bacterial
concentration
(approx. 4.55x109 bacteria/mL using viable counts). After two days of
incubation,
more than 60% (10,000 ppm) of the initial amount of sucrose was consumed and
significant concentrations of lactate and acetate were observed (10,320 ppm
and
333 ppm, respectively). The amount of sucrose, glucose and fructose remaining
after two days of incubation was 6009 ppm, 3603 ppm and 1302 ppm,
respectively. The pH of the culture medium, initially at 7.0, decreased to 4.
These data indicate that probiotic species can be grown on maple sap
when supplemented with a substrate rich in nitrogen, such as soy drink.
Materials and Methods for Examples 2 and 3:
Bacterial strains: Mixed culture BioK+ containing L. acidophilus and L.
casei is commercially available from Bio-K Plus (Laval, Quebec, Canada). L.
acidophilus was isolated from BioK+. L. rhamnosus was isolated from the
commercial white cheese Damablanc (Damafro, St-Damase, Quebec, Canada).
Lactobacillus acidophilus R0240 and Lactobacillus helveticus R0052 were
provided by Dr. Thomas Tompkins (Lallemand Inc., Montreal, Quebec, Canada).
Media: Maple sap was obtained in March 2007 from the Centre
d'Experimentation et de Transfert Technologique Acericole (CETTA,
Pohenegamook, Quebec). The maple sap contained 23 g/L of sucrose and the
pH was neutral. For Examples 2 and 3 below, maple sap (diluted at 20 g/L) was
supplemented with yeast extract (5 g/L), OxoidTM veggietone pea (20 g/L) (a
nitrogen source), K2HPO4 (2 g/L), MnSOa (0.2 g/L) and MgSO4 (0.05 g/L). A 20
g/L commercial sucrose-based medium, was similarly prepared.
Growth Conditions: Strains were first revived into OxoidT"" MRS medium
overnight. Then, they were pre-cultured overnight in either the maple sap-
based
or the sucrose-based medium. This latter pre-culture was used to inoculate 20
mL
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of maple sap or sucrose-based medium. Cultures were incubated at 37 C under
static conditions in a 20 ml vial.
Analysis: During the course of the incubation, samples were taken for the
analysis/determination of: optical density at 600 nm (ODsoonm), viable counts
(colony forming unit (cfu) method), pH, lactic acid concentration (HPLC) and
sucrose concentration (HPLC).
Example 2: Growth in maple sap-based medium
As shown in Fig. 1, the maple sap-based culture medium composed of
maple sap from CETTA, veggietones pea, yeast extract, K2HP04, MnSO4 and
MgSO4 supported good growth (final ODsoonm around 6.0 reached between 18 and
hours of incubation) of three of the five strains tested. Based on these
results,
the viable counts were determined for the three best growers (L. helveticus
R0052, L. acidophilus from BioK+, and the commercial mixed culture of BioK+).
Fig. 2 shows the cfu counts obtained after 16 hours of incubation in the
15 maple sap-based medium. While L. acidophilus from BioK+ and L. helveticus
R0052 grew to 6.0 x 108 cfu/mL, the BioK+ mixture grew to 1.5 x 109 cfu/mL.
This
latter cell concentration represents the targeted concentration for industrial
production. CLT is a control with no sugar source.
Example 3: Comparison of maple sap-based and sucrose-based media
20 Fig. 3 shows that the use of maple sap-based medium improves production
of probiotic bacteria in comparison to sucrose-based medium. In particular,
the
viable counts of L. acidophilus from BioK+ and L. helveticus R0052 were 5
times
higher when maple sap was used as a basis for the preparation of the culture
medium. The media were composed of the respective sugar sources together
with veggietones pea, yeast extract, K2HPO4, MnSO4 and MgSO4. CLT is a
control with no sugar source.
This "maple effect" was also observed in the production of lactic acid, as
shown in Fig 4. BioK+ mixture was the best producer with 15 g/L of lactic acid
after 16 hours of fermentation. The production of lactic acid by the two other
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strains (L. acidophilus from BioK+ and L. helveticus R0052) was around 3.5 g/L
in
the sucrose-based medium. However, the production was more than two-fold
higher (10 g/L) after fermentation in the maple sap-based medium (Fig. 4).
References:
Woodward J. and On M. 1998. Enzymatic Conversion of Sucrose to
Hydrogen. Biotechnology Progress, 14 (6), 897-902.
Morin, A., Heckert, M., Poitras, E., Leblanc, D., Brion, F., and Moresoli, C.
1995. Exopolysaccharide production on low-grade maple sap by Enterobacter
agglomerans grown in small scale bioreactors. Journal of Applied Bacteriology
79:30-37.
Other advantages that are inherent to the structure are obvious to one
skilled in the art. The embodiments are described herein illustratively and
are not
meant to limit the scope of the invention as claimed. Variations of the
foregoing
embodiments will be evident to a person of ordinary skill and are intended by
the
inventor to be encompassed by the following claims.
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