WO2009051509A1 - Milieu de culture lactique pour obtenir une masse de bactéries probiotiques - Google Patents

Milieu de culture lactique pour obtenir une masse de bactéries probiotiques Download PDF

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Publication number
WO2009051509A1
WO2009051509A1 PCT/RU2007/000563 RU2007000563W WO2009051509A1 WO 2009051509 A1 WO2009051509 A1 WO 2009051509A1 RU 2007000563 W RU2007000563 W RU 2007000563W WO 2009051509 A1 WO2009051509 A1 WO 2009051509A1
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WO
WIPO (PCT)
Prior art keywords
milk
culture medium
sodium chloride
yeast
bifidobacteria
Prior art date
Application number
PCT/RU2007/000563
Other languages
English (en)
Russian (ru)
Inventor
Aleksei Vladimirovich Molokeev
Aleksandr Georgievich Kusliy
Natalia Algimantasovna Zykova
Roma Miroslavovna Ilyina
Leonid Georgievich Nikulin
Vyacheslav Vladimirovich Mironenko
Galina Vladimirovna Yasudis
Irina Aleksandrovna Guseva
Original Assignee
Obschestvo S Ogranichennoy Otvetstvennostyu 'sibbiosan'
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Obschestvo S Ogranichennoy Otvetstvennostyu 'sibbiosan' filed Critical Obschestvo S Ogranichennoy Otvetstvennostyu 'sibbiosan'
Priority to PCT/RU2007/000563 priority Critical patent/WO2009051509A1/fr
Publication of WO2009051509A1 publication Critical patent/WO2009051509A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor

Definitions

  • the invention relates to biotechnology and can be used in the microbiological, food industry and for the preparation of nutrient media for the cultivation of bifido and lactobacilli in order to obtain biomass of microorganisms and its further use in the production of starter cultures for therapeutic and prophylactic products, as well as for the production of biologically active additives (B HELL).
  • Cultivation in such an environment should contribute at least to the preservation of the probiotic properties of microorganisms.
  • a production nutrient medium must provide a high reproduction rate: for lactobacilli up to 5-7 hours and for bifidobacteria up to 18 hours, a high concentration of viable microbial cells per unit volume of the medium - at least 1-10 10 CFU / ml, high safety of viable microbial cells - not less than 70-90 days.
  • the nutritional needs of lactic acid bacteria are extremely high.
  • SUBSTITUTE SHEET (RULE 26) diverse and associated with the biochemical activity of microorganisms. As the medium is enriched with protein, the ability of bacteria to ferment lactose increases, the addition of peptones and peptides (products of protein hydrolysis) enhances the growth of lactic acid bacteria to a much greater extent than amino acids. It should be noted that the amount of amino acids required by various lactic acid bacteria is variable and depends on the needs of the strains. Bifidobacteria most require amino acids: proline, series, alanine, aspartic and glutamic acids. Possessing some proteolytic activity, bifidobacteria can, to a small extent, additionally provide their needs for nitrogenous substances.
  • Nutrient media of various compositions are used to build biomass.
  • the composition of the nutrient medium for the growth of bifidobacteria and lactobacilli is known, which is a milk hydrolyzate, into which peptone, sodium chloride, agar-agar, lactose, hydrochloric acid cysteine are added, in the following ratio
  • SUBSTITUTE SHEET (RULE 26) components (Instructions for the preparation of sour milk bifidumbacterin in dairy kitchens. - M .:
  • this composition is not an effective nutrient medium for growing bifidobacteria and lactobacilli.
  • a known nutrient medium for increasing the biomass of bifidobacteria and lactobacilli based on pea hydrolyzate in the following ratio of components RF Patent N ° 2061037, IPC C
  • Pea hydrolyzate diluted with water to an amine nitrogen content of 130-150 mg% - the rest is up to 100%.
  • the above composition has a low content of low molecular weight peptides and amino acids (amine nitrogen 130-150 mg%), therefore, it does not provide biomass production of high concentration - not more than 10 8
  • a nutrient medium is also known for the accumulation of biomass of bacteria based on whey in the following ratio of components (RF Patent JV22087532, IPC C 12 N 1/20 publ. 20.08.97 g), mass. %:
  • a medium of this composition although it gives a high growth rate, does not have enough nutrients (proteins, peptides, amino acids) for the long-term preservation of microbial cells, which is very important for drugs and dietary supplements - probiotics.
  • An additional effect of high growth indicators is a significant increase in titratable acidity, which negatively affects the organoleptic properties of the final product.
  • the corn-lactose medium (CLS) is widely known for the cultivation of bifidobacteria and lactic acid bacteria (TU 10-
  • KJIC does not contain hydrolyzed protein components of milk or yeast cells, which does not ensure the cultivation of long shelf life of the final product.
  • the presence in the medium of a non-standard component - corn extract determines the non-standard of the medium itself, as well as the unsatisfactory organoleptic properties of the obtained dietary supplements and starter cultures.
  • the closest analogue (prototype) to the claimed one is a milk nutrient medium for the production of a liquid concentrate of bifidobacteria, containing hydrochloric acid cysteine (L-cystine), D (-) - lactose, milk protein hydrolyzate and yeast autolysate and sodium chloride.
  • pancreatic hydrolyzate of milk dairy is used as a hydrolyzate of milk proteins
  • the enzymatic autolysate of yeast is used as a yeast autolysate
  • milk return normalized by dry residue is used with a total content of amine nitrogen in the nutrient medium of 260-300 mg% in the following ratio of its components, wt.% (RF patent I ° 2169763, IPC C12N1 / 20, A61K35 / 74, A23C9 / 12, publ.
  • SUBSTITUTE SHEET As an enzymatic autolysate of yeast, an autolysate with an amine nitrogen index of 350-400 mg% is used. The milk return is normalized by dry solids to a value of 13.0-14%.
  • soy isolate is additionally added to the nutrient medium in an amount by weight of 0.2-0.3%.
  • sodium citrate is additionally introduced into the nutrient medium in an amount by weight of OD -0.2%.
  • pancreatic hydrolyzate of milk protein proteins contains, along with high molecular weight and low molecular weight peptides, partially hydrolyzed casein and other components that are poorly absorbed by bacteria, and the enzymatic autolysate of yeast, along with vitamins and stimulating bacterial growth factors, contains toxic substances - products of enzymatic autolysis that are not conducive to conditions growth of bacteria (T. M. Ervolder, A. V. Gudkov and L. P. Semenova. Effect of enzymatic hydrolysates on biomass accumulation bifidobacteria. Journal of Dairy Industry, N 12, 1980, pp. 15-17) as a result of which the terms and titer of biomass of bifidobacteria and lactobacilli during storage are reduced.
  • the technical result of the claimed invention is the creation of such a nutrient medium that would allow the cultivation of both bifidobacteria and lactobacilli with longer shelf life and higher titers of bacterial biomass during storage.
  • the indicated technical result is achieved in that in a milk nutrient medium for obtaining biomass of probiotic bacteria containing a base in the form of a milk sample normalized by the dry residue, hydrochloric acid cysteine (L-cystine) or ascorbic acid, milk protein hydrolyzate and growth factors from yeast cells, sodium chloride, according to the invention, the medium additionally contains manganese sulfate MnSO 4 • 4H 2 O and magnesium sulfate MgSO 4 -7H 2 O,
  • SUBSTITUTE SHEET (RULE 26) an enzymatic hydrolyzate of whey proteins of milk is used as a hydrolyzate of milk proteins, purified yeast extract is used as growth factors from yeast cells in the following ratio of its components, wt.%: enzymatic hydrolyzate of whey proteins of milk 5.0-7.0; hydrochloric cysteine (L-cystine) or ascorbic acid 0.001-0.002; sodium chloride 0.03 - 0.05; manganese sulfate MnSO 4 • 4H 2 O 0.001-0.005 magnesium sulfate MgSO 4 -7H 2 O 0.01-0.02 purified yeast extract 1.5 - 2.0;
  • Milk return (base), normalized by dry solids - The rest is up to 100%.
  • Milk culture medium contains purified yeast extract with an amine nitrogen index of 2.1-2.8%, total protein of at least 3-4%, an enzymatic hydrolyzate of whey milk proteins ( lactopeptone) with an indicator of total protein of at least 11%, amine nitrogen of at least 4%, and milk return contains lactose in an amount of not more than 15% and is normalized by dry residue to a value of 13.0 - 14.0%.
  • the total amine nitrogen in the nutrient medium is contained in an amount of not less than 300 mg.%
  • whey proteins of milk (lactalbumin and lactoglobulin) enriches it with a mixture of high molecular weight and low molecular weight peptides, which are more effective stimulators of the growth of bifidobacteria and lactobacilli than, for example, pancreatic protein hydrolyzate (the main component of casein milk.
  • Whey proteins of milk in amino acid composition are similar to proteins of human milk. Compared to casein, they are more easily
  • SUBSTITUTE SHEET (RULE 26) undergo complete hydrolysis, contain irreplaceable sulfur-containing acids in the amount corresponding to human needs, 4 times more contain cysteine and 19 times more tryptophan and their use is more preferable in the production of probiotics for children.
  • the purified yeast extract contains B vitamins and stimulating growth factors of bifidobacteria, as well as the optimal content of amine nitrogen, which provides the most favorable growth conditions for bifidobacteria and lactobacilli, and, in comparison with the enzymatic yeast autolysate, does not contain toxic products of yeast metabolism that inhibit bacterial growth.
  • a tangible increase in the quality of the yeast extract is achieved by improving the technology for its production - the soluble state is converted to 30-50% yeast protein, the digestibility of the cell protein is increased by 8-10% due to loosening of all layers of the cell wall.
  • Yeast extract contains:
  • the components providing the reducing properties of the claimed medium are mainly cysteine or ascorbic acid, as well as the amino acids glutamine and asparagine, which are rich in purified yeast extract, which is part of the medium.
  • lactic acid bacteria need inorganic compounds, especially manganese ions and magnesium ions. Manganese inhibits cell autolysis and is necessary for
  • SUBSTITUTE SHEET (RULE 26) normal processes of fat metabolism. Mn ++ and Mg ++ remove the toxic effect of Zn 4 ⁇ if it is present in the medium.
  • the invention allows to obtain a high initial titer and increase the shelf life without inhibiting growth and increasing the active acidity of probiotic preparations obtained using the proposed nutrient medium.
  • lactopeptone an enzymatic hydrolyzate of whey milk proteins
  • skim milk and a yeast extract a lactopeptone
  • Whey proteins of milk play an important role in increasing the biomass of probiotic bacteria and shaping the quality of the final product, since they have a high content of essential and sulfur-containing amino acids.
  • Milk-based proteins (mainly casein) are characterized by high biological value and a unique set of amino acids, which helps to improve the overall balance of the amino acid composition of the final product. Milk contains a unique disaccharide - lactose, which has a beneficial effect on the composition of the intestinal normoflora.
  • milk When milk is heated, for example, during sterilization, lactose turns into lactulose, which is a powerful bifidogenic factor. Milk contains a significant amount of macrea and trace elements, vitamins. Especially important is the digestible calcium of milk, vitamins A, B 1 , B 2 , D, C, carotene.
  • Example 1 Minimum content of medium components (wt.%): Enzymatic hydrolyzate of whey milk proteins 5.0 cysteine hydrochloride (L-cystine) 0.001 sodium chloride 0.03 manganese sulfate MnSO 4 • 4H 2 O 0.001 magnesium sulfate MgSO 4 -7H 2 O 0.01
  • Example 2 The average content of medium components (wt.%): Enzymatic hydrolyzate of whey milk proteins 6.0 ascorbic acid 0.0015 sodium chloride 0.04 manganese sulfate MnSO 4 • 4H 2 O 0.003 magnesium sulfate MgSO 4 -7H 2 O 0.015 purified yeast extract 1, 7
  • Example 3 The maximum content of medium components (wt.%): Enzymatic hydrolyzate of milk whey proteins 7.0 cysteine hydrochloride (L-cystine) 0.002 sodium chloride 0.05 manganese sulfate MnSO 4 • 4H 2 O 0.005 magnesium sulfate MgSO 4 -7H 2 O 0.02 purified yeast extract 2.0
  • Example 4 The study of the duration of storage of a consortium of strains of lactobacilli in a liquid concentrate of lactobacilli
  • a significant period (up to 72 days) of storage with a titer of at least 10 9 CFU / ml is associated with ensuring the initial acidity of the drug
  • SUBSTITUTE SHEET (RULE 26) a consortium of lactobacilli not more than (120-140) ° T, obtaining the initial titer of lactobacilli (not lower than 10 10 CFU / ml) through the use of the inventive milk culture medium with the addition of lactopeptone and yeast extract.
  • Example 5 The study of the duration of storage of biomass of a consortium of strains of bifidobacteria in a liquid concentrate of bifidobacteria obtained using the inventive nutrient medium
  • SUBSTITUTE SHEET (RULE 26) A significant period (up to 72 days) of storage of a liquid concentrate of bifidobacteria with a titer of at least 10 10 CFU / ml is associated with ensuring the initial acidity of the preparation of a consortium of bifidobacteria not more than (140-145) ° T, obtaining the initial titer of bifidobacteria (not less than 10 11
  • the result of the claimed invention confirms the creation of such a nutrient medium that provides the ability to cultivate both bifidobacteria and lactobacilli with longer shelf life (up to 72 days or more) and higher titers of bacterial biomass during storage.
  • the invention can be implemented using simple processing equipment in biotechnology or the food industry.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
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  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Polymers & Plastics (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
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  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Food Science & Technology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne la préparation de milieux de culture destinés à la culture de bifidobactéries ou de lactobactéries et peut s'utiliser dans des industries microbiologique ou alimentaire. Le milieu de culture permet la culture de bifidobactéries ou de lactobactéries avec des durées de conservation plus grandes et des titres plus élevés de biomasse pendant la conservation. Le milieu de culture lactique destiné à la fabrication d'une biomasse de bactéries probiotiques comprend une base se présentant comme un lactosérum normalisé pour son résidu sec, une cystéine chlorhydrique (L-cystine) ou l'acide ascorbique, l'hydrolysat de protéines lactiques et des facteurs de croissance faits à partir de cellules de levure, du chlorate de sodium, du sulfate de manganèse MnSO4⋅4H2O et du sulfate de magnésium MgSO4⋅7H2O. On utilise en tant que hydrolysat de protéines lactiques un hydrolysat fermentatif des protéines de sérum du lait, et en tant que facteurs de croissance fabriqués à partir de cellules de levure on utilise un extrait de levure avec un rapport de ses composants, en % en masse : 5,0-7,0 d'hydrolysat de protéines de sérum du lait, 0,001-0,002 de cystéine chlorhydrique (L-cystine) ou d'acide ascorbique, 0,03-0,5 de chlorure de sodium, 0,001-0,005 de sulfate de manganèse MnSO4⋅4H2O, 0,01-0,02 de sulfate de magnésium MgSO4⋅7H2O, 1,5-2,0 d'extrait de levure purifié, le reste jusqu'à 100% étant constitué de base sous forme de lactosérum pour son résidu sec.
PCT/RU2007/000563 2007-10-15 2007-10-15 Milieu de culture lactique pour obtenir une masse de bactéries probiotiques WO2009051509A1 (fr)

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Application Number Priority Date Filing Date Title
PCT/RU2007/000563 WO2009051509A1 (fr) 2007-10-15 2007-10-15 Milieu de culture lactique pour obtenir une masse de bactéries probiotiques

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PCT/RU2007/000563 WO2009051509A1 (fr) 2007-10-15 2007-10-15 Milieu de culture lactique pour obtenir une masse de bactéries probiotiques

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2104706C1 (ru) * 1996-09-10 1998-02-20 Государственный научный центр вирусологии и биотехнологии "Вектор" Способ получения бифидосодержащих препаратов
RU2142234C1 (ru) * 1999-03-10 1999-12-10 Государственный научный центр вирусологии и биотехнологии "Вектор" Способ получения возрастоспецифичных бифидопрепаратов
RU2169763C1 (ru) * 2000-06-26 2001-06-27 Дочернее Государственное Унитарное Экспериментально-Производственное Предприятие "Вектор-Биальгам" Гнц Вб "Вектор" Молочная питательная среда для получения жидкого концентрата бифидобактерий
US6340585B1 (en) * 1999-03-23 2002-01-22 Nestec S.A. Synthetic medium for cultivating Lactobacillus and Bifidobacteria
RU2006100739A (ru) * 2006-01-10 2007-07-20 Закрытое акционерное общество "Вектор-БиАльгам" (RU) Консорциум штаммов лактобактерий и способ получения на его основе препарата, используемого в качестве биологически активной добавки или закваски для производства кисломолочных продуктов

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2104706C1 (ru) * 1996-09-10 1998-02-20 Государственный научный центр вирусологии и биотехнологии "Вектор" Способ получения бифидосодержащих препаратов
RU2142234C1 (ru) * 1999-03-10 1999-12-10 Государственный научный центр вирусологии и биотехнологии "Вектор" Способ получения возрастоспецифичных бифидопрепаратов
US6340585B1 (en) * 1999-03-23 2002-01-22 Nestec S.A. Synthetic medium for cultivating Lactobacillus and Bifidobacteria
RU2169763C1 (ru) * 2000-06-26 2001-06-27 Дочернее Государственное Унитарное Экспериментально-Производственное Предприятие "Вектор-Биальгам" Гнц Вб "Вектор" Молочная питательная среда для получения жидкого концентрата бифидобактерий
RU2006100739A (ru) * 2006-01-10 2007-07-20 Закрытое акционерное общество "Вектор-БиАльгам" (RU) Консорциум штаммов лактобактерий и способ получения на его основе препарата, используемого в качестве биологически активной добавки или закваски для производства кисломолочных продуктов

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