CA2594937A1 - Production of probiotic bacteria using maple sap - Google Patents
Production of probiotic bacteria using maple sap Download PDFInfo
- Publication number
- CA2594937A1 CA2594937A1 CA002594937A CA2594937A CA2594937A1 CA 2594937 A1 CA2594937 A1 CA 2594937A1 CA 002594937 A CA002594937 A CA 002594937A CA 2594937 A CA2594937 A CA 2594937A CA 2594937 A1 CA2594937 A1 CA 2594937A1
- Authority
- CA
- Canada
- Prior art keywords
- bacteria
- sucrose
- maple
- growth
- based medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241000208140 Acer Species 0.000 title claims abstract description 68
- 241000894006 Bacteria Species 0.000 title claims abstract description 57
- 239000006041 probiotic Substances 0.000 title claims abstract description 47
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 47
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 43
- 238000004519 manufacturing process Methods 0.000 title abstract description 16
- 241000186660 Lactobacillus Species 0.000 claims abstract description 8
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 30
- 229930006000 Sucrose Natural products 0.000 claims description 30
- 239000005720 sucrose Substances 0.000 claims description 30
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 25
- 239000000203 mixture Substances 0.000 claims description 18
- 239000006188 syrup Substances 0.000 claims description 16
- 235000020357 syrup Nutrition 0.000 claims description 16
- 240000001046 Lactobacillus acidophilus Species 0.000 claims description 14
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- 239000004310 lactic acid Substances 0.000 claims description 13
- 235000014655 lactic acid Nutrition 0.000 claims description 13
- 239000013589 supplement Substances 0.000 claims description 7
- 244000199866 Lactobacillus casei Species 0.000 claims description 6
- 240000002605 Lactobacillus helveticus Species 0.000 claims description 6
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 claims description 5
- 229940039695 lactobacillus acidophilus Drugs 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 108090000623 proteins and genes Proteins 0.000 claims description 5
- 229940039696 lactobacillus Drugs 0.000 claims description 3
- 235000013967 Lactobacillus helveticus Nutrition 0.000 claims 4
- 229940054346 lactobacillus helveticus Drugs 0.000 claims 4
- 235000013958 Lactobacillus casei Nutrition 0.000 claims 2
- 235000021120 animal protein Nutrition 0.000 claims 2
- 229940017800 lactobacillus casei Drugs 0.000 claims 2
- 230000006872 improvement Effects 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 5
- 229910052799 carbon Inorganic materials 0.000 abstract description 5
- 239000002609 medium Substances 0.000 description 26
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 10
- 239000000047 product Substances 0.000 description 9
- 230000003068 static effect Effects 0.000 description 7
- 238000011534 incubation Methods 0.000 description 6
- 240000000696 Lactobacillus helveticus R0052 Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 5
- 235000019341 magnesium sulphate Nutrition 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 4
- 229940041514 candida albicans extract Drugs 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 239000012138 yeast extract Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000001332 colony forming effect Effects 0.000 description 3
- 235000019797 dipotassium phosphate Nutrition 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 3
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- KJTLQQUUPVSXIM-ZCFIWIBFSA-N (R)-mevalonic acid Chemical compound OCC[C@](O)(C)CC(O)=O KJTLQQUUPVSXIM-ZCFIWIBFSA-N 0.000 description 1
- 244000046151 Acer negundo Species 0.000 description 1
- 235000004422 Acer negundo Nutrition 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 241000193033 Azohydromonas lata Species 0.000 description 1
- 206010005003 Bladder cancer Diseases 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 208000037384 Clostridium Infections Diseases 0.000 description 1
- KJTLQQUUPVSXIM-UHFFFAOYSA-N DL-mevalonic acid Natural products OCCC(O)(C)CC(O)=O KJTLQQUUPVSXIM-UHFFFAOYSA-N 0.000 description 1
- 206010012438 Dermatitis atopic Diseases 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 229920002444 Exopolysaccharide Polymers 0.000 description 1
- 208000004262 Food Hypersensitivity Diseases 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 241000186679 Lactobacillus buchneri Species 0.000 description 1
- 241001134659 Lactobacillus curvatus Species 0.000 description 1
- 241000186673 Lactobacillus delbrueckii Species 0.000 description 1
- 241000186840 Lactobacillus fermentum Species 0.000 description 1
- 235000005877 Lactobacillus helveticus R0052 Nutrition 0.000 description 1
- 240000006024 Lactobacillus plantarum Species 0.000 description 1
- 241000186604 Lactobacillus reuteri Species 0.000 description 1
- 241000186612 Lactobacillus sakei Species 0.000 description 1
- 241000186869 Lactobacillus salivarius Species 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 241000588912 Pantoea agglomerans Species 0.000 description 1
- 241000702670 Rotavirus Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 201000008937 atopic dermatitis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000020932 food allergy Nutrition 0.000 description 1
- 230000007407 health benefit Effects 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000002551 irritable bowel syndrome Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
Use of maple sap as a carbon and energy source for the production of probiotic bacteria (e.g. Lactobacilli spp.) unexpectedly leads to an improvement in growth of the probiotic bacteria and an improvement in the yield of products produced by the bacteria.
Description
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.
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.
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.
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.
2 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.
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.
3 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).
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).
4 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
(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
5 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
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
6 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.
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.
7
Claims (34)
1. Method of growing probiotic bacteria comprising contacting the probiotic bacteria with maple sap, down-graded maple syrup or a mixture thereof.
2. Method of producing lactic acid comprising contacting probiotic Lactobacillus bacteria with maple sap, down-graded maple syrup or a mixture thereof.
3. Method according to claim 2, wherein the lactic acid is produced in an amount at least 1.5 times greater than in a sucrose-based medium.
4. Method according to claim 2, wherein the lactic acid is produced in an amount at least 2 times greater than in a sucrose-based medium.
5. Method according to claim 1, wherein the probiotic bacteria comprise bacteria from genus Lactobacillus.
6. Method according to claim 5, wherein the probiotic bacteria have a growth yield at least 1.5 times greater than growth yield on a sucrose-based medium.
7. Method according to claim 5, wherein the probiotic bacteria have a growth yield at least 2 times greater than growth yield on a sucrose-based medium.
8. Method according to claim 5, wherein the probiotic bacteria have a growth yield at least 4 times greater than growth yield on a sucrose-based medium.
9. Method according to claim 5, wherein the probiotic bacteria have a growth yield 2 to 4 times greater than growth yield on a sucrose-based medium.
10. Method according to any one of claims 6 to 9, wherein the bacteria comprise Lactobacillus acidophilus or Lactobacillus helveticus.
11. Method according to any one of claims 1 to 4, wherein the bacteria comprise Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus or any mixture thereof.
12. Method according to any one of claims 1 to 11, wherein the maple sap, down-graded maple syrup or mixture thereof is formulated into a medium comprising one or more growth supplements for the bacteria.
13. Method according to claim 12, wherein the growth supplement comprises a nitrogen source usable by the bacteria.
14. Method according to claim 13, wherein the nitrogen source comprises vegetal or animal protein.
15. Method according to claim 13, wherein the nitrogen source comprises vegetal protein.
16. Method according to any one of claims 1 to 15, wherein the bacteria are incubated in the maple sap, down-graded maple syrup or mixture thereof at a temperature in a range from about 30°C to about 45°C.
17. Method according to any one of claims 1 to 16, wherein the bacteria are incubated in the maple sap, down-graded maple syrup or mixture thereof in a bioreactor.
18. Use of maple sap, down-graded maple syrup or a mixture thereof for growing probiotic bacteria.
19. Use of maple sap, down-graded maple syrup or a mixture thereof for producing lactic acid from probiotic Lactobacillus bacteria.
20. Use according to claim 19, wherein the lactic acid is produced in an amount at least 1.5 times greater than in a sucrose-based medium.
21. Use according to claim 19, wherein the lactic acid is produced in an amount at least 2 times greater than in a sucrose-based medium.
22. Use according to claim 18, wherein the probiotic bacteria comprise bacteria from genus Lactobacillus.
23. Use according to claim 22, wherein the probiotic bacteria have a growth yield at least 1.5 times greater than growth yield on a sucrose-based medium.
24. Use according to claim 22, wherein the probiotic bacteria have a growth yield at least 2 times greater than growth yield on a sucrose-based medium.
25. Use according to claim 22, wherein the probiotic bacteria have a growth yield at least 4 times greater than growth yield on a sucrose-based medium.
26. Use according to claim 22, wherein the probiotic bacteria have a growth yield 2 to 4 times greater than growth yield on a sucrose-based medium.
27. Use according to any one of claims 18 to 21, wherein the bacteria comprise Lactobacillus acidophilus or Lactobacillus helveticus.
28. Use according to any one of claims 23 to 26, wherein the bacteria comprise Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus helveticus or any mixture thereof.
29. Use according to any one of claims 18 to 28, wherein the maple sap, down-graded maple syrup or mixture thereof is formulated into a medium comprising one or more growth supplements for the bacteria.
30. Use according to claim 29, wherein the growth supplement comprises a nitrogen source usable by the bacteria.
31. Use according to claim 30, wherein the nitrogen source comprises vegetal or animal protein.
32. Use according to claim 30, wherein the nitrogen source comprises vegetal protein.
33. Use according to any one of claims 18 to 32, wherein the bacteria are incubated in the maple sap, down-graded maple syrup or mixture thereof at a temperature in a range from about 30°C to about 45°C.
34. Use according to any one of claims 18 to 33, wherein the bacteria are incubated in the maple sap, down-graded maple syrup or mixture thereof in a bioreactor.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US83327706P | 2006-07-26 | 2006-07-26 | |
US60/833,277 | 2006-07-26 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2594937A1 true CA2594937A1 (en) | 2008-01-26 |
Family
ID=38973749
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002594937A Abandoned CA2594937A1 (en) | 2006-07-26 | 2007-07-23 | Production of probiotic bacteria using maple sap |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA2594937A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010099617A1 (en) * | 2009-03-06 | 2010-09-10 | Fédération Des Producteurs Acéricoles Du Québec | Symbiotic maple product compositions and methods |
-
2007
- 2007-07-23 CA CA002594937A patent/CA2594937A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2010099617A1 (en) * | 2009-03-06 | 2010-09-10 | Fédération Des Producteurs Acéricoles Du Québec | Symbiotic maple product compositions and methods |
JP2012519659A (en) * | 2009-03-06 | 2012-08-30 | フェデラシオン デ プロダクテュール アセリコール デュ ケベック | Compositions and methods for producing symbiotic maple |
US8551543B2 (en) | 2009-03-06 | 2013-10-08 | Fédération Des Producteurs Acéricoles Du Québec | Symbiotic maple product compositions and methods |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Hayek et al. | Cultivation media for lactic acid bacteria used in dairy products | |
Dhakal et al. | Production of GABA (γ-aminobutyric acid) by microorganisms: a review | |
Gudiña et al. | Biosurfactant‐producing lactobacilli: screening, production profiles, and effect of medium composition | |
US20210395677A1 (en) | Microorganism-Derived Protein Hydrolysates and Methods of Preparation and Use Thereof | |
CN101589829B (en) | Probiotics fermented beverage and production method thereof | |
US20090186397A1 (en) | Product | |
CN113736716B (en) | Lactobacillus paracasei, compound biological leavening agent for yellow storage and yellow storage method | |
JP2013048586A (en) | Lactic bacterium separated from traditional fermented food in ishikawa prefecture, cultured product thereof, and utilization thereof | |
CN1236681C (en) | Method for preparing lactobacillus dry-powder product | |
Bevilacqua et al. | Combined effects of pH, yeast extract, carbohydrates and di-ammonium hydrogen citrate on the biomass production and acidifying ability of a probiotic Lactobacillus plantarum strain, isolated from table olives, in a batch system | |
US20080026441A1 (en) | Production of probiotic bacteria using maple sap | |
Prasirtsak et al. | Characterization of D-lactic acid, spore-forming bacteria and Terrilactibacillus laevilacticus SK5-6 as potential industrial strains | |
CN106399155A (en) | Bacillus licheniformis fermentation culture medium | |
CN110607253B (en) | Streptococcus thermophilus and proliferation culture method and application thereof | |
Papizadeh et al. | Using various approaches of design of experiments for high cell density production of the functionally probiotic Lactobacillus plantarum strain RPR42 in a cane molasses-based medium | |
CN114958655B (en) | Clostridium Ding Suanxing produced from pit mud produced by white spirit brewing and application thereof | |
JP3763005B2 (en) | Bacteria strains with phenotypes closely related to Lactobacillus, culture methods and uses | |
CA2594937A1 (en) | Production of probiotic bacteria using maple sap | |
Abbas Mohammed et al. | Production and optimization of hyaluronic acid extracted from Streptococcus thermophilus isolates | |
CN103667107A (en) | Enterococcus faecium strain capable of producing L-lactic acid | |
Kantha et al. | Synergistic growth of lactic acid bacteria and photosynthetic bacteria for possible use as a bio-fertilizer | |
Toptaş et al. | Lactic acid production by Lactobacillus brevis isolated from oral microbiota | |
Patel et al. | Production of single cell protein from mix fruits waste using Lactobacillus | |
Maulidiyah et al. | Tofu Whey-Based Media for Probiotic Lactiplantibacillus plantarum D4 as a Halal Starter Culture | |
Zhao et al. | Probiotic characterization of Bacillus smithii: Research advances, concerns, and prospective trends |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FZDE | Dead |