Fermentation methods for producing steviol glycosides with multi-phase feeding

A technology of steviol glycosides and steviol glycosides is applied in the field of steviol glycoside compositions, and can solve the problems such as the inability to fully provide the production rate of steviol glycosides

Pending Publication Date: 2018-01-19
CARGILL INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current fermentation methods using recombinant organisms do not adequately provide the desired steviol glycoside production rates

Method used

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  • Fermentation methods for producing steviol glycosides with multi-phase feeding
  • Fermentation methods for producing steviol glycosides with multi-phase feeding
  • Fermentation methods for producing steviol glycosides with multi-phase feeding

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] Production of Reb D and Reb M in a two-stage feed process

[0113] For inoculum preparation, yeast strain B was grown in 150 mL inoculum flask medium in 1 liter shake flasks at 250 rpm and 30°C for 20-24 hours.

[0114] Table 1 Inoculation flask culture medium

[0115] components

Mode

concentration

unit

Biospringer 0251 Yeast Extract

7.5

g / L

Glucose monohydrate

C 6 h 12 o 6 *H 2 o

22.0

g / L

[0116]For fermentation, 75 mL of the inoculum culture was transferred to the initial fermentation medium as indicated in Table 2, where the initial volume was 0.75 liters (38.5% tank level). Batch-feed fermentations were performed in 2L New Brunswick BioFlo 310 fermenters. with 12% NH 4 OH controlled the fermentation at pH 5.0 and kept the temperature below 30°C throughout the process. The air flow rate was 1.75 SLPM and the stirring rate was 1200 rpm throughout the fermentation.

[0117] Glucose concentratio...

Embodiment 2

[0138] For inoculum preparation, yeast strain B was grown as described in Example 1 using the inoculation flask medium of Table 1 of Example 1. Fermentation, initial fermentation medium and fermentation feed medium were as described in Example 1.

[0139] table 5

[0140]

[0141] The stage I feed rate was kept constant and the stage II feed rate was variable but lower than that used in Example 1 . The above data show that at lower stage II feed rates, the yield increases.

Embodiment 3

[0143] For inoculum preparation, yeast strain C was grown as described in Example 1 using the inoculated flask medium of Table 1 of Example 1. Unlike Example 1, the initial fermentation medium did not contain cobalt, molybdate and borate, and only vitamins and trace minerals were added to the initial fermentation medium, but not to the fermentation feed medium.

[0144] Table 6

[0145]

[0146] An increase in yield was observed at lower feed rates compared to higher rates with faster feed (control).

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Abstract

Disclosed are methods for producing steviol glycosides, such as rebaudioside D and rebaudioside M, using engineered yeast. The methods include at least two phases: first and second phases where a glucose-containing feed composition is provided to the medium in different modes of feeding in each phase, such as variable feeding and then constant feeding. The two phase feeding can result in a growthrate that is slower in the second phase than in the first phase, and consequently increased steviol glycoside production rates, reduced fermentation times, and reduced biomass concentrations.

Description

[0001] References to Sequence Listings: [0002] This application contains references to amino acid sequences and / or nucleic acid sequences filed concurrently with this document in the form of an ASCII text file titled "CAR0212WO_Sequence_Listing.txt" created May 27, 2016 and is 92 kilobytes in size . Pursuant to 37 C.F.R. §1.52(e)(5), the Sequence Listing is hereby incorporated by reference in its entirety. [0003] Cross References to Related Applications [0004] This application claims priority to US Provisional Application No. 62 / 168,372, filed May 29, 2015, which is hereby incorporated by reference in its entirety. field of invention [0005] The present invention relates to fermentation methods for producing steviol glycosides, fermentation compositions and steviol glycoside compositions produced by fermentation. Background of the invention [0006] Sugars such as sucrose, fructose and glucose are utilized to provide a pleasant taste to beverages, foods, pharmaceut...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N1/19C12N1/21
CPCC12P15/00C12N1/185C12R2001/865C12N15/74C12N15/81C12P1/02
Inventor J·C·安德松T·L·卡尔松A·M·福斯梅尔
Owner CARGILL INC
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