WO2021096428A1 - Souche isolée de bacillus subtilis, ses utilisations et procédés utilisant ladite souche isolée - Google Patents

Souche isolée de bacillus subtilis, ses utilisations et procédés utilisant ladite souche isolée Download PDF

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Publication number
WO2021096428A1
WO2021096428A1 PCT/SG2020/050655 SG2020050655W WO2021096428A1 WO 2021096428 A1 WO2021096428 A1 WO 2021096428A1 SG 2020050655 W SG2020050655 W SG 2020050655W WO 2021096428 A1 WO2021096428 A1 WO 2021096428A1
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WO
WIPO (PCT)
Prior art keywords
raw material
plant raw
cell
spent
bacillus subtilis
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PCT/SG2020/050655
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English (en)
Inventor
Qingxin Li
Jinchuan Wu
Jeroen MULLER
Edwin ANANTA
Christina VAFEIADI
Original Assignee
Agency For Science, Technology And Research
Société des Produits Nestlé S.A.
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Application filed by Agency For Science, Technology And Research, Société des Produits Nestlé S.A. filed Critical Agency For Science, Technology And Research
Publication of WO2021096428A1 publication Critical patent/WO2021096428A1/fr

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    • 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
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
    • A23L2/52—Adding ingredients
    • A23L2/56—Flavouring or bittering agents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/20—Malt products
    • A23L7/25—Fermentation of cereal malt or of cereal by malting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/021—Preparation of other alcoholic beverages by fermentation of botanical family Poaceae, e.g. wheat, millet, sorghum, barley, rye, or corn
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus

Definitions

  • the present invention generally relates to an isolated strain of Bacillus subtilis.
  • the present invention also relates to uses of the isolated strain and a process for hydrolysing a plant raw material, producing a non-alcoholic malted beverage or producing an alcoholic beverage using the isolated strain.
  • a process for hydrolysing a plant raw material comprising the step of contacting a plant raw material with an isolated strain as defined herein, a cell culture as defined herein or a cell-free supernatant as defined herein.
  • a process for producing a non-alcoholic malted beverage comprising the step of mixing a hydrolysed plant raw material with a beverage ingredient.
  • a process for producing an alcoholic beverage comprising the step of alcoholic fermenting a hydrolysed plant raw material.
  • isolated when referring to the bacterial strain is to be interpreted broadly to mean that the bacterial strain was obtained and separated from its native environment in order to obtain a substantially pure culture of the bacterial strain. When the bacterial strain is in the substantially pure culture, this may mean that the bacterial strain is substantially free of other species of organisms.
  • the bacterial strain may then be stored in the form of a culture of a single species of organism. When the bacterial strain is used, the bacterial strain may then be in a mixture with other species.
  • the isolated strain of Bacillus subtilis may be Bacillus subtillis Bl-4 deposited with the depository agency Collection Nationale de Cultures de Microorganismes (CNCM) under deposit number CNCM 1-5356, which was deposited on 27 September 2018. This corresponds to the identification reference of the depositor of NCC-2956.
  • the isolated strain of Bacillus subtilis may be Bacillus subtillis WCB-2 deposited with the depository agency CNCM under deposit number CNCM 1-5357, which was deposited on 27 September 2018. This corresponds to the identification reference of the depositor of NCC- 2957.
  • the isolated strain may be a mutant of the Bacillus subtillis Bl-4 as deposited above.
  • the isolated strain may be a mutant of the Bacillus subtillis WCB-2 as deposited above.
  • the isolated strain in a growth medium or culture broth may form a cell culture.
  • the cell culture may comprise an isolated bacterial strain (or mutant thereof) as defined above in a growth medium or culture broth.
  • the cell culture may contain a mixture of the above bacterial strains or mutants thereof or may be a mixture of the mutants of the above bacterial strains.
  • the protease may be bacillopeptidase F, cell wall-associated protease, minor extracellular protease vpr, aminopeptidase YwaD, subtilisin E, subtilisin amylosacchariticus, putative aminopeptidase YtoP, putative aminopeptidase YsdC or extracellular metalloprotease.
  • the protease may be selected from the group consisting of subtilisin E, extracellular protease vpr, bacillolysin, and transpeptidase.
  • the subtilisin E may have the accession number P04189, while the extracellular protease vpr may have the accession number P29141.
  • the contacting step may also be known as a solubilizing step in order to break down the biological macromolecules present in the plant raw material into their respective monomers.
  • the plant raw material contain proteins or carbohydrates
  • the proteins or enzymes secreted by or extracted from the above isolated bacterial strain(s) may solubilize or break down the protein or carbohydrates into their respective amino acid monomers or monosaccharide monomers.
  • the solubilization efficiency may be defined as (W 0 -W)/W 0 x 100%, where W 0 refers to the dry weight of the plant raw material before solubilization and W refers to the dry weight of the solubilized plant raw material.
  • the solubilization efficiency may be more than the amount of protein present in the plant raw material before solubilization due to the presence of the carbohydrases which aid in solubilizing the carbohydrates present in the plant raw material in addition to the proteins present in the plant raw material (which are solubilized by the proteases).
  • the process may further comprise a step of (d) drying the hydrolysed plant raw material or the concentrated hydrolysed plant raw material.
  • the drying step may be undertaken before the mixing step (b).
  • FIG. 5 is a pie diagram showing the percentages of the various enzyme components in the enzyme supernatant of Bacillus subtilis Bl-4.
  • Spent barley grain B was selected for isolation of the microorganism and for further testing.
  • Japanese white cream waffle sticks (1 to 3 g, obtained from Bourbon Corporation of Japan) was first inoculated in 30 mL of medium 1 as above at pH 7.4. The mixture was incubated at 30°C with shaking atl80 rpm overnight. The overnight culture was spread on agar plates made from medium 1 with 1.5% agar. Single colonies were then inoculated on skim milk agars. Presence of a clear zone around the colony was used as an indication of protease - producing colony. c) Strains identification
  • Fig. 1(a) The pH profiles of Bacillus subtilis Bl-4 and Bacillus subtilis ATCC21556 is shown in Fig. 1(a).
  • Fig. 1(b) For the temperature profiling, the same assay as for the pH profiling was used but at a fixed pH of 7 with two varying temperature values of 50°C and 55°C.
  • Fig. 1(b) thus show the temperature profile at 50°C while Fig. 1(c) shows the temperature profile at 55°C.
  • the solubilization was then carried out at a temperature of 50°C using the same food-grade Nutrient-NaCl medium (as provided above) at a pH of 7.0 and at varying periods of 1 hour, 1.5 hours and 2 hours.
  • the result of this is shown in Fig. 3. From Fig. 3, it can be seen that the enzyme solution of Bacillus subtilis Bl-4 could well solubilize the wet cereal spent barley grain B. The differences in the solubilization of the spent barley grain B at 1 hour, 1.5 hours and 2 hours were not significant, and the highest solubilization of 28.5% was achieved at 2 hours.
  • Bacillus subtilis Bl-4 secreted not only proteases but also other enzymes.
  • Fig. 9 shows the solubilization result of barly spent grant using commercial carboydrase cocktails. As can be seen, the solubilization of such commercial enzyme cocktails are not as good as using the bacterial strains provided herein.

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

Abstract

L'invention concerne une souche isolée de Bacillus subtilis choisie dans le groupe constitué par Bacillus subtilis B1-4, numéro de dépôt CNCM 1-5356, Bacillus subtilis WCB-2, numéro de dépôt CNCM 1-5357, et une souche mutante associée. L'invention concerne également des utilisations de la souche isolée et des utilisations du surnageant acellulaire produit par les souches susmentionnées. Plus particulièrement, les utilisations concernent un procédé d'hydrolyse d'une matière première végétale, de production d'une boisson maltée non alcoolisée, ou de production d'une boisson alcoolisée.
PCT/SG2020/050655 2019-11-14 2020-11-13 Souche isolée de bacillus subtilis, ses utilisations et procédés utilisant ladite souche isolée WO2021096428A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SG10201910683U 2019-11-14
SG10201910683UA SG10201910683UA (en) 2019-11-14 2019-11-14 An isolated bacillus subtilis strain, uses thereof and processes using said isolated strain

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WO2021096428A1 true WO2021096428A1 (fr) 2021-05-20

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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2189268A1 (fr) * 1996-10-30 1998-04-30 Stephen J. Davids Procede de fabrication d'une boisson alcoolique ayant les caracteristiques essentielles de la biere d'orge, a partir d'un mout a base de pommes de terre
CN100593571C (zh) * 2005-04-26 2010-03-10 清华大学 一种提高微生物抗逆性的方法
WO2012078148A1 (fr) * 2010-12-08 2012-06-14 Nestec S.A. Boissons prêtes à boire comprenant des céréales entières hydrolysées
CN103555640A (zh) * 2013-11-18 2014-02-05 山东蔚蓝生物科技有限公司 一种枯草芽孢杆菌及其在畜禽养殖中的应用
CN105838529A (zh) * 2016-06-14 2016-08-10 西华大学 一种桑葚青稞啤酒的酿制方法
WO2016142278A1 (fr) * 2015-03-06 2016-09-15 Nestec S.A. Boissons prêtes à boire de longue conservation contenant des grains entiers hydrolysés, et système de stabilisation
WO2017048636A1 (fr) * 2015-09-14 2017-03-23 Agri-King, Inc. Bactéries et enzymes produites à partir de celles-ci et leurs procédés d'utilisation
CN109430634A (zh) * 2018-09-06 2019-03-08 哈尔滨伟平科技开发有限公司 一种发酵饮料的制作方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2189268A1 (fr) * 1996-10-30 1998-04-30 Stephen J. Davids Procede de fabrication d'une boisson alcoolique ayant les caracteristiques essentielles de la biere d'orge, a partir d'un mout a base de pommes de terre
CN100593571C (zh) * 2005-04-26 2010-03-10 清华大学 一种提高微生物抗逆性的方法
WO2012078148A1 (fr) * 2010-12-08 2012-06-14 Nestec S.A. Boissons prêtes à boire comprenant des céréales entières hydrolysées
CN103555640A (zh) * 2013-11-18 2014-02-05 山东蔚蓝生物科技有限公司 一种枯草芽孢杆菌及其在畜禽养殖中的应用
WO2016142278A1 (fr) * 2015-03-06 2016-09-15 Nestec S.A. Boissons prêtes à boire de longue conservation contenant des grains entiers hydrolysés, et système de stabilisation
WO2017048636A1 (fr) * 2015-09-14 2017-03-23 Agri-King, Inc. Bactéries et enzymes produites à partir de celles-ci et leurs procédés d'utilisation
CN105838529A (zh) * 2016-06-14 2016-08-10 西华大学 一种桑葚青稞啤酒的酿制方法
CN109430634A (zh) * 2018-09-06 2019-03-08 哈尔滨伟平科技开发有限公司 一种发酵饮料的制作方法

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