WO2024201521A1 - Milieu à base d'eau de noix de coco tendre pour la levure de boulanger et la production de bioéthanol - Google Patents

Milieu à base d'eau de noix de coco tendre pour la levure de boulanger et la production de bioéthanol Download PDF

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WO2024201521A1
WO2024201521A1 PCT/IN2024/050326 IN2024050326W WO2024201521A1 WO 2024201521 A1 WO2024201521 A1 WO 2024201521A1 IN 2024050326 W IN2024050326 W IN 2024050326W WO 2024201521 A1 WO2024201521 A1 WO 2024201521A1
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yeast
tcw
culture medium
coconut water
concentration
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Muniasamy NEERATHILINGAM
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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; 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • 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; 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
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present invention generally relates to tender coconut water (TCW)-based media for growing yeast such as baker’s yeast (Saccharomyces cerevisiae). More specifically, the present invention relates to a method of producing optimized TCW media for production of high biomass of yeast (such as Saccharomyces cerevisiae) for baking and bioethanol production.
  • TCW tender coconut water
  • the lifestyle of humans is changing worldwide with the increasing consumption of fast food that includes ready to use (RTU), ready to eat (RTE) food, and bakery products.
  • RTU ready to use
  • RTE ready to eat
  • bakery products the most essential raw material is baker’s yeast (at least 2%).
  • Baker’s yeast is a commercial preparation consisting of dried cells of one or more strains of the fungus Saccharomyces cerevisiae.
  • the baker’s yeast has an extensive history of use in the area of food processing as a leavening agent in baking bread as a fermenter of alcoholic beverages and wine production. Saccharomyces cerevisiae plays a vital role during the fermentation step to get a preferred shape, texture and quality of the product.
  • the quality of the raw material utilized should be good, and it depends on the growth media, optimum temperature, and the conversion process from wet to dry form.
  • yeast in food technology, as well as in human nutrition, as an alternative source of protein to overcome the demands in a world of low agricultural production and rapidly increasing population, makes the production of food-grade yeast quite important (Bekatorou et al. 2006).
  • Saccharomyces uvarum Saccharomyces cerevisiae. Saccharomyces cerevisiae is the cheapest available strain used for bio-ethanol production (Bhadana and Chauhan 2016). Saccharomyces cerevisiae, in the presence of oxygen converts sugar into alcohol and carbon dioxide.
  • Industrial bioethanol production plants employ mainly two types of primary feedstocks, viz., starch from cereal crops and juice or molasses from sugar crops (Mojovic et al. 2006; Wilkie et al. 2000). About 60% of the global ethanol is produced from sugar crops, while the remaining 40% is produced from starchy grains (Salassi 2007).
  • Ethanol is primarily produced by the petrochemical process or by the yeast fermentation. Saccharomyces cerevisiae is capable of very rapid rates of ethanol production under optimal conditions (Dombek and Ingram 1986).
  • the main disadvantage in using sugars as feedstock is low storability and microbial decomposition (Dodie et al. 2009), whereas downstream processing of starchy grains as a feedstock is too expensive (Azhar et al. 2017).
  • Tender coconut water is a pure colorless liquid inside the coconut palm, which is nutrient-rich and a good source of vitamins, minerals, antioxidants, amino acids, enzymes and growth factors.
  • Yeast is a microorganism that is commercially grown using synthetic growth media like YEPD, MPYA, SYPA, etc. Further, byproducts like molasses (cane, beet) or fruit wastes are commonly used as a carbon source for large scale yeast production. Yeast used for the production of bakery products may have contamination of heavy metals due to the use of waste by products.
  • molasses contains some of the heavy metals like Al, As, Cu, Fe, Mn and Zn at a concentration of 0.54, 0.24, 8.7, 0.35, 11.1 and 19.7 pg/g, respectively (Teclu et al. 2009).
  • An ideal alternative can be the utilization of a natural source that is free of chemicals and has natural nutrients required for the optimum growth of microorganisms.
  • TCW has been used earlier as a media for the growth of bacteria (Oloke and Glick 2006; Sekar et al. 2013), mushroom, and also yeast (Sekar et al. 2013). It acts as a good carbon source for the organisms as it is rich in nutrients and also provides a good yield compared to the synthetic media.
  • Yeast is used in starter cultures for the production of various types of fermented foods like bread, sourdoughs, fermented meat, vegetable products, etc. (Kandasamy et al. 2018).
  • the present disclosure relates to a culture medium comprising tender coconut water, a nitrogen source, and a carbon source, optionally along with magnesium salt.
  • the culture medium comprises a nitrogen source at a concentration of 25mM to 50mM, and a carbon source at a concentration of 0 to 10% (v/v).
  • the culture medium comprises tender coconut water at a percentage of 25% to 100% (v/v).
  • the carbon source is selected from a group comprising, glycerol, glucose, fructose and sucrose or any combination thereof.
  • the glycerol is at a concentration selected from 0%, 0.1%, 0.5%, 1%, 5%, or 10% (v/v).
  • the carbon source selected from the group comprising glucose, fructose and sucrose or any combination thereof is at a concentration selected from 0%, 0.1%, 0.5%, 1%, 5%, or 10% (w/v).
  • the nitrogen source is selected from a group comprising ammonium sulphate, ammonium acetate, ammonium carbonate, ammonium chloride and amino acid mix or any combination thereof.
  • the culture medium comprises tender coconut water at a concentration of 25mM ammonium sulphate, glucose at 1% (w/v), sucrose at 1% (w/v), glycerol at 1.25% (v/v) from 80% (v/v) of stock solution, and fructose at 1% (w/v).
  • the culture medium comprises tender coconut water at a concentration of 25mM ammonium sulphate, glucose at 1% (w/v), sucrose at 1% (w/v), glycerol at 2.5% (v/v) from 80% (v/v) of stock solution, and fructose at 1% (w/v).
  • the culture medium comprises magnesium salt at a concentration of 2mM, wherein the magnesium salt is selected from a group comprising magnesium sulphate, magnesium chloride and magnesium carbonate.
  • the culture medium enhances the growth of yeast.
  • the fold increase in yeast growth in tender coconut water- based culture medium when compared to the yeast growth in YEPD (yeast extract, peptone, dextrose) medium is found to be 6 times.
  • the culture medium is prepared by: a) extracting tender coconut water from tender coconuts; b) filter sterilizing the extracted tender coconut water; c) taking the filtered sterilized tender coconut water at percentages ranging from 25% to 100%; d) adding a nitrogen source at a concentration ranging from 25mM to 50mM; and e) adding a carbon source at a concentration ranging from 0-10%.
  • the present disclosure relates to a method of culturing yeast comprising steps of: a) adding warm water to yeast to obtain activated yeast; b) serially diluting the activated yeast in saline, wherein the serial dilution factors are 10’ 1 to 10’ 5 ; c) plating inoculums from each of the serial dilutions in tender coconut water agar (TCW agar) medium taken in five culture plates respectively labelled from 10' 1 to 10' 5 , to obtain TCW agar plates; d) incubating the TCW agar plates at 30°C for 48 hours to obtain yeast colonies on the plates; e) inoculating a single colony from the 10' 3 dilution TCW agar plate into a medium comprising tender coconut water and ammonium sulphate at a concentration of 25 mM to obtain a pre-inoculum; f) incubating the pre-inoculum at 30°C at 200 rpm for 24 hours; g
  • the TCW agar medium comprises tender coconut water and 4% of agar, wherein the tender coconut water and agar are at a ratio of 1 : 1.
  • the present disclosure relates to a method for producing ethanol from an inoculum of yeast cultured in a culture medium comprising tender coconut water, a nitrogen source, and a carbon source, optionally along with magnesium salt comprising steps of: a) inoculating 20% (v/v) of inoculum of yeast in 100 ml of the medium as claimed in claim 1 to obtain the inoculated medium; b) incubating the inoculated medium at 30°C at 200 rpm for an incubation period ranging from 4 to 16 hours to get samples; c) collecting samples after incubation; d) centrifuging the samples at 4500 rpm, at 4°C, for 40minutes; e) obtaining cell-free supematent comprising ethanol; and f) storing the cell-free supernatent at
  • Yeast inoculum size has a significant effect on ethanol production (Turhan et al. 2010).
  • different inoculum sizes i.e., 1%, 5%, 10% and 20% (v/v) are used, and it was observed that the amount of ethanol produced gradually increased with the increase in inoculum size.
  • FIG.l illustrates the optical densities recorded for the combination A comprising sub-sets Al to A4.
  • FIG. 2 illustrates the optical densities recorded for the combination B comprising sub-sets Bl to B4.
  • FIG. 3 illustrates the optical densities recorded for the combination C comprising sub-sets Cl to C4.
  • FIG. 4 illustrates the optical densities recorded for the combination D comprising sub-sets DI to D4.
  • FIG. 5 illustrates the optical densities recorded for the combination E comprising sub-sets El to E4.
  • FIG. 6 illustrates the optical densities recorded for the combination F comprising sub-sets Fl to F4.
  • FIG. 7 illustrates the optical densities recorded for the combination G comprising sub-sets G1
  • FIG. 8 illustrates the optical densities recorded for the combination H comprising sub-sets Hl to H4.
  • FIG. 9 illustrates the optical densities recorded for the combination I comprising sub-sets II to 14.
  • FIG. 10 illustrates a comparison of the growth of yeast in the present Tender coconut waterbased media versus growth of yeast in YEPD (yeast extract, peptone, dextrose) media and Molasses.
  • FIG. 11 illustrates the optical densities recorded for the combination (i) comprising 6 sub-sets for 0, 0.1%, 0.5%, 1.0%, 5.0% and 10.0 % (v/v) glycerol with 25mM Ammonium sulphate.
  • FIG. 12 illustrates the optical densities recorded for the combination (ii) comprising 6 sub-sets for 0, 0.1%, 0.5%, 1.0%, 5.0% and 10.0 % (v/v) glycerol with 25mM Ammonium sulphate and 2mM Magnesium sulphate.
  • FIG. 13 illustrates the optical densities recorded for the combination (iii) comprising 6 sub-sets for 0, 0.1%, 0.5%, 1.0%, 5.0% and 10.0 % (v/v) glycerol with 50mM Ammonium sulphate.
  • FIG. 14 illustrates the optical densities recorded for the combination (iv) comprising 6 sub-sets for 0, 0.1%, 0.5%, 1.0%, 5.0% and 10.0 % (v/v) glycerol with 50mM Ammonium sulphate and 2mM Magnesium sulphate.
  • FIG. 15 illustrates a comparison of the growth of baker’s yeast in the present Tender coconut water- based media versus growth of yeast in YEPD (yeast extract, peptone, dextrose) media, performed at a large scale (i.e. 100 ml culture in 500 ml flask).
  • YEPD yeast extract, peptone, dextrose
  • FIG. 16 illustrates sensory analysis of bread prepared using baker’s yeast grown in optimised TCW media and YPD media.
  • FIG. 17 illustrates GC-MS data for determination of ethanol.
  • FIG. 18 illustrates the production of ethanol at periodic intervals of time (at 0 th , 4 th , 8 th , 12 th and 16 th hour) for yeast inoculum size of 1%, 5%, 10%, and 20% (v/v).
  • FIG. 19a illustrates a bar-chart indicating the fold changes occurring at 16 hours with the sub sets of the combinations A to I.
  • FIG. 19b illustrates a bar-chart indicating the fold changes occurring at 20 hours with the sub sets of the combinations A to I.
  • FIG. 19c illustrates a bar-chart indicating the fold changes occurring at 24 hours with the sub sets of the combinations A to I.
  • FIG. 19d illustrates a bar-chart indicating the fold changes occurring at 28 hours with the sub sets of the combinations A to I.
  • FIG. 19e illustrates a radar chart plotted by merging the fold change values for the subsets of the combinations A to I, with the time points (i.e. 16, 20, 24, and 28 hours) together.
  • the primary objective of the present invention is to produce an organic media, particularly a tender coconut water-based medium.
  • Another objective of the present invention is to culture high quantity of healthy yeast cells in an organic way (free from animal-based media or vegan method) using tender coconut waterbased medium.
  • the organic tender coconut water-based culture medium of the present disclosure comprises sugars which are nutritious for yeast. It has been investigated in the present disclosure how carbon sources or sugars, when provided at the right combination, amount or at the optimum concentration in the culture medium, result in better and faster growth of yeast cells.
  • Another objective of the present invention is to prepare bread using yeast cultured in an organic way.
  • Yet another objective of the present invention is to produce ethanol using the tender coconut water-based medium.
  • the present disclosure relates to a culture medium comprising tender coconut water, a nitrogen source, and a carbon source, optionally along with magnesium salt.
  • the culture medium comprises a nitrogen source at a concentration of 25mM to 50mM, and a carbon source at a concentration of 0 to 10% (v/v).
  • the culture medium comprises tender coconut water at a percentage of 25% to 100% (v/v).
  • the carbon source is selected from a group comprising, glycerol, glucose, fructose and sucrose or any combination thereof.
  • the glycerol is at a concentration selected from 0%, 0.1%, 0.5%, 1%, 5%, or 10% (v/v).
  • the carbon source selected from the group comprising glucose, fructose and sucrose or any combination thereof is at a concentration selected from 0%, 0.1%, 0.5%, 1%, 5%, or 10% (w/v).
  • the nitrogen source is selected from a group comprising ammonium sulphate, ammonium acetate, ammonium carbonate, ammonium chloride and amino acid mix or any combination thereof.
  • the culture medium comprises tender coconut water at a concentration of 25mM ammonium sulphate, glucose at 1% (w/v), sucrose at 1% (w/v), glycerol at 1.25% (v/v) from 80% (v/v) of stock solution, and fructose at 1% (w/v).
  • the culture medium comprises tender coconut water at a concentration of 25mM ammonium sulphate, glucose at 1% (w/v), sucrose at 1% (w/v), glycerol at 2.5% (v/v) from 80% (v/v) of stock solution, and fructose at 1% (w/v).
  • the culture medium comprises magnesium salt at a concentration of 2mM, wherein the magnesium salt is selected from a group comprising magnesium sulphate, magnesium chloride and magnesium carbonate.
  • the culture medium enhances the growth of yeast.
  • the culture medium is prepared by: a) extracting tender coconut water from tender coconuts; b) filter sterilizing the extracted tender coconut water; c) taking the filtered sterilized tender coconut water at percentages ranging from 25% to 100%; d) adding a nitrogen source at a concentration ranging from 25mM to 50mM; and e) adding a carbon source at a concentration ranging from 0-10%.
  • step b) filter sterilizing the extracted tender coconut water is performed using 11pm Whatman filter paper followed by 0.22pm PVDF Merck Millipore filter membrane; or any sequential filtration process can be followed for reducing the cost involved in the use of 0.22 pm PVDF.
  • the present disclosure relates to a method of culturing yeast comprising steps of: a) adding warm water to yeast to obtain activated yeast; b) serially diluting the activated yeast in saline, wherein the serial dilution factors are 10' 1 to 10’ 5 ; c) plating inoculums from each of the serial dilutions in tender coconut water agar (TCW agar) medium taken in five culture plates respectively labelled from 10' 1 to 10' 5 , to obtain TCW agar plates; d) incubating the TCW agar plates at 30°C for 48 hours to obtain yeast colonies on the plates; e) inoculating a single colony from thelO' 3 dilution TCW agar plate into a medium comprising tender coconut water and ammonium sulphate at a concentration of 25 mM to obtain a preinoculum; f) incubating the pre-inoculum at 30°C at 200 rpm for 24 hours; g
  • the TCW agar medium comprises tender coconut water and 4% of agar, wherein the tender coconut water and agar are at a ratio of 1:1.
  • the present disclosure relates to a method for producing ethanol from an inoculum of yeast cultured in a culture medium comprising tender coconut water, a nitrogen source, and a carbon source, optionally along with magnesium salt comprising steps of: a) inoculating 20% (v/v) of inoculum of yeast in 100 ml of the medium as claimed in claim 1 to obtain the inoculated medium; b) incubating the inoculated medium at 30°C at 200 rpm for an incubation period ranging from 4 to 16 hours to get samples; c) collecting samples after incubation; d) centrifuging the samples at 4500 rpm, at 4°C, for 40minutes; e) obtaining cell-free supernatant comprising ethanol; and f) storing the cell-free supernatant at -20°C.
  • the cell free supernatant collected at step f) was used for further studies.
  • the cell free supernatant collected at step f) was subjected to gas chromatography (GC) to determine the ethanol content.
  • GC gas chromatography
  • the supernatant was collected and added with 1% internal standard (ethanol) and injected into the GC.
  • Example 1 Culturing of yeast in growth media comprising tender coconut water, nitrogen source and various combinations of carbon sources.
  • Tender coconut water contains reducing sugars like fructose and glucose, and nonreducing sugars like sucrose.
  • TCW Tender coconut water
  • simple sugars like sucrose, fructose, and glucose (w/v) are included in addition to the naturally present carbon sources in TCW, this ensures the availability of adequate nutrients for the growth of the yeast.
  • nine types of the media are prepared with different ratios of tender coconut water (for example, 25%, 50%, 75% and 100% (v/v)) to which sugars like glucose, sucrose and fructose are added in various combinations. In all these combinations ammonium sulphate is employed as the source of nitrogen.
  • the purpose of testing of various combinations to check for yeast growth is to identify the best combination that yields more biomass of yeast and reduces the cost of the growth media.
  • Tender coconuts are procured from farm/vendor (for the present experiment, the tender coconuts were purchased from a street-side vendor in Bengaluru, India). The fruits are cut open to extract the water.
  • the extracted tender coconut water (TCW) is filter sterilized using 11 pm Whatman filter paper followed by 0.22pm PVDF Merck Millipore filter membrane. The two-membrane system is used to avoid clogging of the 0.22pm PVDF membrane hence, reduce frequent change of this membrane.
  • the filtered TCW is stored at 4°C until further use.
  • Ammonium sulphate is prepared as a stock solution by weighing 132.14 g of ammonium sulphate and dissolving it in 1000ml of distilled/MilliQ water.
  • Glycerol; 80% (v/v) glycerol is prepared by taking 80 ml of 100% glycerol and adding into 20 ml of distilled/MilliQ water. It is autoclaved at 120°C, 15psi, for 30 minutes.
  • the above reagents are made for generating the combinations labelled A, B, C, D, E, F, G, H and I (see Table 1 below).
  • these combinations there are sub-sets prepared with an intention to optimize the TCW usage.
  • sub-sets labelled as 1, 2, 3, 4 are prepared for each of the combinations (A-I).
  • the subsets 1, 2, 3, and 4 represent 25%, 50%, 75% and 100% of TCW respectively.
  • the efficiency of the media composition(s) was calculated by difference in the optical density (OD) between ‘test’ and ‘control’ (14).
  • ‘test’ refers to the combinations of the media and the ‘control’ is TCW (100%) with 25mM ammonium sulphate.
  • Yeast Sacharomyces cerevisiae
  • Moments TM is taken for preparing glycerol stock.
  • yeast is activated by the addition of 1 ml of warm water (40-45°C) to 1.5 ml micro centrifuge tube with a pinch of dry yeast from the package followed by swirling the tube upside down 3-4 times.
  • the activated yeast is serially diluted from 10 to 10 ' 5 .
  • the process of serial dilution involves taking five 1.5ml tubes and labelling the tubes from 10 to 10 " 5 , adding 900 pl of culture media to all tubes, followed by adding 100 pl from the activated yeast tube to the first tube (i.e.
  • YEPDA yeast extract peptone dextrose agar
  • a single colony from the 10 ' 3 dilution plate was inoculated into 5 ml of YEPD and incubated overnight, at 30° C, 200 rpm, and then in a cryotube 500 pl of 30% (v/v) glycerol is added to 500 pl of yeast cells followed by immediate freezing using liquid nitrogen. These are thereafter stored at -80 °C at to obtain glycerol stock.
  • a loopful of yeast from the glycerol stock culture is inoculated in 10 ml of TCW comprising 25mM Ammonium sulphate (i.e, 250pL from IM stock solution) and incubated for 48 hours at 30°C, with shaking at the speed of 200 RPM.
  • TCW comprising 25mM Ammonium sulphate (i.e, 250pL from IM stock solution)
  • Tender coconut water is taken in four different dilutions (25%, 50%, 75%, and 100%).
  • the mixtures prepared in the step 3 are filtered through 0.22pm filter and the filtrates are labelled respectively (all the steps were performed inside Laminar air flow).
  • the filtrates are taken in well plates (a 24 well plate format is used for the experiment) Thereafter, the glycerol is added to the respective wells individually.
  • the 24 well plate is incubated at 30°C, with shaking at a speed of 200 RPM.
  • Optical density is measured at 600mm on the 16 th , 20 th , 24 th and 28 th hour.
  • a standard graph of OD versus Time is plotted (Fig. 1 to 9). It is observed that the maximum optical density of cells is usually obtained between 20- 28 hours as per the repeated and routine of the experiments with TCW.
  • optical density data of these combinations are compared to that of the ‘control’ - a combination (14) comprising 100% (v/v) tender coconut water containing 25mM ammonium sulphate. This is to know if these combinations can match or can be more efficient than the ‘control’.
  • the sets (Al, A2, A3 and A4) in combination A containing all the components i.e., glucose, fructose, sucrose and glycerol significantly show higher growth than the other sets of combinations.
  • the maximum fold change obtained was 1.835 and 1.787 times at 28 th and 24 th in A3 respectively when compared to the control 14.
  • the maximum observed OD in control (14) is 3.9 at 28 th hour which is lesser than the OD obtained in combination A2, A3, A4, B3, B4.
  • the yellow line indicates the border line indicating the fold change is higher than the control if the bar crosses the line.
  • the fold change was calculated by using the maximum OD obtained at control 14 where 100% (v/v) of TCW with 25mM ammonium sulphate was used. Also, a radar chart was plotted by merging the fold change values with all-time points together (Fig. 19e).
  • yeast growth is also compared with commercially available culture media like YEPD (Yeast Extract, Peptone and Dextrose) and molasses. It is found that the control combination (14) is more efficient and gives more yield (Fig. 10). Large culture volumes (100ml) of 4% (v/v) molasses, YEPD and TCW are prepared. The maximum OD of 26 was seen at 24 th hour in TCW with 25mM ammonium sulphate. The procedure for comparison of yeast growth in TCW and YEPD is explained in greater detail in the below example.
  • the present TCW media is found to lower the cost of growth media, as it involves addition of simple sugars, it boosts yield quickly, and, most importantly, facilitates a clean, vegan method of culturing yeast. By employing this media and technique, the quality of baker's yeast's is also improved.
  • TCW was supplied by a shop from a local market in Mysore district, Karnataka, India. To prepare a culture medium, the exocarp (outer coat) of coconut was wiped with 70% ethanol, and the TCW was dispensed in a sterile beaker inside the laminar air flow chamber. TCW was then transferred to 50ml sterile falcon tubes and centrifuged at 4000 to 5000 rpm (4-6°C) for 20 to 40 minutes to separate suspended solids. The supernatant was filtered through a 0.22pm filter membrane (Millex filter units- Millipore) and stored at 4°C until use.
  • a 0.22pm filter membrane Millex filter units- Millipore
  • Yeast extract, peptone, and dextrose (YEPD) and agar (YEPDA) media are commonly used for culturing yeast.
  • the ingredients of YEPDA are 1% (w/v) yeast extract, 2% (w/v) Peptone, 2% (w/v) Dextrose and 2% (w/v) agar.
  • this media is used for comparison with TCW agar (TCW-agar) media.
  • TCW-A is prepared by take 4% of agar and adding it into TCW, wherein the agar and TCW are at a ratio of 1:1, and pouring this mixture into petri dish.
  • Saccharomyces cerevisiae was activated by the addition of 1 ml of warm water (40-45°C) to 1.5 ml microcentrifuge tube with a pinch of dry yeast followed by swirling the tube upside down 3-4 times.
  • the activated yeast is serially diluted from 10 to 10 " 5 and plated on TCW-agar and YEPDA.
  • the process of serial dilution involves taking five 1.5ml tubes and labelling the tubes from 10 -1 to 10 -5, adding 900 pl of media to all tubes, followed by adding 100 pl of activated yeast tube to the first tube (i.e. labelled as 10 dilution) and mixing the contents. Further, 100 pl of inoculum from the first tube is added to the second tube (i.e.
  • the growth of yeast in TCW was increased by the addition of supplementary sources, viz., ammonium sulphate (25Mm) and sterile glycerol (0.1%) as a source of nitrogen and carbon, respectively.
  • IM of ammonium sulphate was used as stock. This was prepared by adding 13.2g of ammonium sulphate in 100ml of MilliQ water/distilled water. From this stock solution 250 pl is used for supplementing 10ml of TCW.
  • Glycerol is prepared as 10% stock solution, i.e. by mixing 10ml of glycerol in 90ml water. For 5ml of TCW, 10 pl of this glycerol is added as a supplement.
  • a single colony from the TCW-agar plate labelled with the dilution factor 10' 3 was inoculated into of lOmL filter- sterilized TCW having 25mM ammonium sulphate only to obtain a preinoculum.
  • a colony from the YEPDA plate labelled with the dilution factor 10' 3 was inoculated into lOmL YEPD.
  • These two pre-inoculums were incubated overnight at 30°C at 200 rpm.
  • the 1% pre-inoculum was sub cultured into a 250 ml conical flask with a working volume of 50 ml TCW with supplements and into 50 ml YEPD.
  • the harvested cells were dried at 50-55° C and made into fine granules.
  • the loaf volume of bread mainly depends on the quality and activity level of the yeast.
  • Baker’s yeast grown in TCW media along with commercially available baker’s yeast were used for baking.
  • the formulation used for the preparation of bread is mentioned in Table 1.
  • the preparation involves the following steps:
  • Flour was added to the Hobart mixer for two minutes. Activated yeast was added, followed by sugar and salt water. According to the consistency to form a dough, the remaining water was added. The mixer was stopped once the dough turned elastic. Dough was mixed thoroughly on a clean floor, then dusted with flour, and finally kept in a bowl for fermentation. Fermentation 1: The bowl covered with wet cloth was kept inside the fermentation cabinet for 90 minutes at 30°C. Remix: Dough was mixed by hand until the size of the dough reduced to its original size.
  • Fermentation 2 Again, the bowl was kept inside the fermentation cabinet for 25 minutes.
  • Sheeting and molding The fermented dough was stretched and compressed into an elongated shape to fit into a baking pan.
  • ethanol production For ethanol production, four 250 ml flasks with a working volume of 100 ml TCW (supplemented with 25m M ammonium sulphate and 0.1% glycerol) were used. 1%, 5%, 10% and 20% (v/v) of the pre-inoculum (i.e. the filter-sterilized TCW to which a single colony from TCW-A plate was inoculated, mentioned above) was added to the respective conical flasks, and incubation was carried at 30° C with 200 rpm (in duplicate).
  • TCW supplied with 25m M ammonium sulphate and 0.1% glycerol
  • GC-MS showed 100% abundance of base peak at 31 m/z (CH2OH + ) along with peak at 45 corresponding to CH3CH2O + and molecular ion peak ethanol at 46 (C2HsOH + ). No other major peak was observed which confirms sample has only ethanol as a major by product (Fig. 17).
  • the culture was grown till 16 hours for ethanol production.
  • Samples were collected every 4 hours, centrifuged and the supernatant with internal standard (IS) was injected into gas chromatography (GC). Quantity of the ethanol was estimated by a standard curve drawn using known increasing concentration of EtOH (0.5%, 1%, 1.5%, 2% and 2.5%) with IS. Standard curve was drawn using ratio of ethanol area to IS area. Ethanol was quantified and found to be 20g/L by calculating the ratio of sample to the straight-line equation.
  • Standard preparation Analytical grade (AR) ethanol was used at an increasing concentration from 0.5%, 1%, 1.5% to 2% with 1% (v/v) of butanol as an internal standard.
  • Test preparation 200p l of cell-free samples were taken with 1% (v/v) of an internal standard. All the test samples and standards were injected using GC syringe (Hamilton) and the data was obtained by calculating the retention time. These selected samples were injected into GCMS for ethanol confirmation.
  • the OD of YEPD was found to be 3.05 at 36 th hour and the OD of TCW was found to be 18.9 at 48 th hour.
  • the fold increase in yeast growth in TCW based culture medium when compared to the yeast growth in YEPD is 6 times.
  • TCW is a naturally available media that is also considered as a vegan media - free from animal sources like beef extract and peptone. Peptones are generally prepared from animal sources (Jayathilakan et al. 2012). TCW is rich in nutrients that support the growth of microbes. In an embodiment of the present invention the TCW media is optimized with the carbon and nitrogen sources for S. cerevisiae as listed in Table 5 with a combination of A, B, C and D.
  • Table 5 TCW- Optimization with various nitrogen (25mM and 50mM ammonium sulphate), carbon (0.1, 0.5, 1.0, 5.0 and 10.0 % (v/v) glycerol) source and 2mM magnesium sulphate
  • S. cerevisiae was grown and compared in yeast specific media YEPD and in the TCW with optimized nitrogen (25mM ammonium sulphate) and carbon source (0.1% glycerol). It was observed that the maximum growth in YEPD was ODeoo nm 3.05 at 36 th hour and in TCW, ODeoonm 18.9 at 48 th hour (Fig.15). This result shows TCW as a better media for higher used for baking were the same in the test and control.
  • the test and control were subjected to sensory evaluation by the Hedonic 5 scale sensory analysis method. This method is a 5-scale sensory method that includes parameters like appearance, aroma, texture, taste and overall acceptance. These parameters will be scored by people while tasting the product.
  • TCW supplemented with 25 mM ammonium sulphate and combination of carbon sources (glucose, fructose and sucrose) collectively gave the highest biomass at an economical cost. This composition also gave significantly higher biomass than the commercially available YEPD media and molasses.
  • TCW grown yeast yielded bread that is superior in sensory parameters as well as suitable for ethanol production. Finally, it is concluded that TCW can be employed as a natural, inexpensive and efficient growth media for the production of yeast for the preparation of bread and ethanol.

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Abstract

La levure de boulanger, Saccharomyces cerevisiae, est l'ingrédient principal utilisé pour les industries de cuisson, la qualité et la quantité de levure de boulanger dépendent de la qualité du milieu utilisé. Des milieux de noix de coco tendre (TCW) sont utilisés en tant que source de milieu nutritionnel pour la production de levure de boulanger. La présente invention concerne un procédé de production de milieux TCW optimisés par supplémentation en carbone et en source d'azote pour produire une bonne qualité et une bonne quantité de levure de boulanger pour la cuisson et la production d'éthanol. Des milieux TCW optimisés complétés par des sources de carbone telles que le glycérol, le glucose, le saccharose et le fructose et la source d'azote tels que le sulfate d'ammonium donnent une bonne quantité de cellules à 135 heures pour la préparation du pain et la production d'éthanol. La quantité maximale d'éthanol a été obtenue avec un inoculum corroboré de 10 % et 20 % (v/v). La croissance de levure s'est avérée être améliorée dans le milieu à base de TCW par comparaison avec des milieux YEPD et des mélasses.
PCT/IN2024/050326 2023-03-27 2024-03-27 Milieu à base d'eau de noix de coco tendre pour la levure de boulanger et la production de bioéthanol Ceased WO2024201521A1 (fr)

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Citations (1)

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Publication number Priority date Publication date Assignee Title
WO2014125429A1 (fr) * 2013-02-13 2014-08-21 Centre For Cellular And Molecular Platforms Milieu de culture pour la croissance de protéines recombinantes

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014125429A1 (fr) * 2013-02-13 2014-08-21 Centre For Cellular And Molecular Platforms Milieu de culture pour la croissance de protéines recombinantes

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NARENDRAKUMAR SEKAR ET AL.: "Tender coconut water an economical growth medium for the production of recombinant proteins in Escherichia coli", BMC BIOTECHNOL, vol. 13, 2013, pages 70, XP021159488, DOI: 10.1186/1472-6750-13-70 *
SATHEESH NEELA, PRASAD NBL: "Production of fermented coconut water beverages", ASIAN JOURNAL OF FOOD AND AGRO-INDUSTRY, NCE OF SONGKLA UNIVERSITY * FACULTY OF AGRO-INDUSTRY, TH, 1 January 2013 (2013-01-01), TH , XP093219302, ISSN: 1906-3040 *

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