CN105018361B - A kind of method of saccharomyces cerevisiae high density fermentation culture - Google Patents
A kind of method of saccharomyces cerevisiae high density fermentation culture Download PDFInfo
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Abstract
The invention discloses a kind of methods of saccharomyces cerevisiae high density fermentation culture, belong to fermentation engineering field.It is production bacterial strain with the saccharomyces cerevisiae that research department is screened from beer waste yeast and spirit stillage, high density fermentation culture is carried out in 7L tanks, using the control of ventilatory capacity and stirring in two benches feeding strategy and fermentation process, final saccharomyces cerevisiae dry weight reaches 109.2g/L.The present invention realizes the high density fermentation of saccharomyces cerevisiae using the fermentation medium and supplemented medium of cheap and simple by parameters in scientific and reasonable control fermentation process, and the High Density Cultivation for saccharomyces cerevisiae in industrial production is offered reference.
Description
Technical field
The present invention relates to a kind of methods of saccharomyces cerevisiae high density fermentation culture, belong to bioengineering field.
Background technology
Saccharomyces cerevisiae (Saccharomyces cerevisiae), also known as Saccharomyces cerevisiae or budding yeast are one kind to go out
Unicellular fungi based on bud reproduction.Saccharomyces cerevisiae is the microorganism the closest with human relation, yeast industry its there is spy
Different superiority.Because saccharomyces cerevisiae growth and breeding is fast, enzymatic activity is stronger, produces the rate of albumen and bioactive substance and is
What plant cannot compare.Secondly, yeast nutrition is abundant, and protein 50%~60%, carbon hydrate are contained in every gram of dry ferment
Object 30%~35%, ribonucleic acid 4.5%~8.3%, fat 1%~5%.In addition, also containing abundant vitamin, mineral
The others nutritional ingredient such as matter, amino acid.Third, saccharomycete production are not limited by season, weather and area, and production is stablized,
Fermentation production foam is few, energy preventing from heavy metal, and environmental suitability is strong.
Saccharomyces cerevisiae now has been widely used for the fields such as food, pharmacy and feed.Active dry yeast is used in food service industry
In making bread and steamed bun；The excellent saccharomyces cerevisiae of character is used for beer brewing, white wine and grape wine；Make yeast atural spice
Product, Yeast protein peptide etc..In bio-pharmaceutical industry, waste beer yeast slurry is for converting production fructose 1,6-diphosphate and triphosphoric acid core
Glycosides；Yeast high density fermentation extracts glutathione, ergosterol isoreactivity substance.Feedstuff industry utilizes waste beer yeast slurry productive culture
The yeast extract and yeast extract of microorganism, while saccharomycete is the ideal for preparing domestic animals, fishes and shrimps, precious fur-bearing animal
Protein raw material.
Due to the extensive use of saccharomyces cerevisiae, high density fermentation is carried out to saccharomyces cerevisiae using new zymotechnique, is obtained
Low production cost, stable quality saccharomyces cerevisiae be imperative.
The high density fermentation of saccharomycete is a relative concept, refers generally in zymotic fluid yeast concentration in 30g/L or more.Battalion
It is foster supply be not suitable for, in zymotic fluid dissolve hypoxgia, produce inhibitory substance accumulation and zymotic fluid rheological properties influence
It will accumulation of the limitation yeast in zymotic fluid.
In high density fermentation, with a large amount of accumulation of thalline, fermentation broth viscosity increases considerably, and is non-Newtonian stream
The mass transfer of body, transmission and nutrients to oxygen all produces bigger effect.Change this phenomenon to need to pass through the knot for changing fermentation tank
Structure is realized.Therefore it in yeast conventional fermentation process, needs, using the feeding strategy of fining, to realize the fast fast-growing of yeast
Long and biomass accumulation.
Invention content
The object of the present invention is to provide a kind of methods for realizing saccharomyces cerevisiae high density fermentation.In earlier fermentation, utilization is molten
Changing to adjust the rate of supplemented medium for oxygen, maintains the required nutriment of thalli growth, and ferment later stage thalli growth
Slack-off, the utilization rate of glucose reduces, and gradually reduces feed rate at this time, extends fermentation period；In entire fermentation process
As saccharomyces cerevisiae metabolism liveness increases, biomass constantly accumulates, steps up stir speed (S.S.) and ventilatory capacity, can ensure foot
Enough ventilations, to realize that saccharomycete stablizes quick high density fermentation.Using purchased from Chinese industrial Microbiological Culture Collection pipe
The saccharomyces cerevisiae that the CICC numbers at reason center are 1511 or 32280 carries out high density fermentation, obtains preferable experimental result
The method, is control by stages culture medium feed rate during yeast fermented and cultured, early period no-feed supplement, in
Phase steps up feed rate, and the later stage gradually reduces feed rate, while stepping up stir speed (S.S.) and ventilatory capacity in mid-term.
The method is：(1) yeast starter liquid is accessed in fermentation tank, initial speed 400rpm, initial ventilatory capacity is
4.5L/min；(2) when glucose exhausts, dissolved oxygen is improved to 30% or more suddenly, start to be mended with the rate of 8-16mL/h
Material, and steps up feed rate, stir speed (S.S.) and ventilatory capacity, until feed rate be increased to 80-85mL/h, stir speed (S.S.) and
Ventilatory capacity is increased to the attainable maximum stir speed (S.S.) of Zymolysis Equipment institute and ventilatory capacity；(3) when yeast growth rate persistently reduces
When up to 2h or more, feed rate is gradually reduced until fermentation ends.
It is described to step up feed rate, refer to that every 4h improves 14-25mL/h in one embodiment of the invention.
Refer to fermentation in one embodiment of the invention when the yeast growth rate is persistently reduced up to 2h or more
When 28h.
The method includes the following steps in one embodiment of the invention：(1) yeast starter liquid is accessed and is sent out
In fermentation tank, initial speed 400rpm, initial ventilatory capacity is 4.5L/min, fermented and cultured 10h；(2) with 8-16mL/h's after 10h
Rate carries out feed supplement, and feed rate 14-25mL/h is improved per 4h；Stir speed (S.S.) is improved into 100rpm until reaching every 4h simultaneously
To the attainable maximum stir speed (S.S.) of Zymolysis Equipment institute, ventilatory capacity is improved into 1.5-3L/min every 4-8h and is set until reaching fermentation
The standby attainable maximal ventilatory volume of institute；(3) keep maximum stir speed (S.S.) and ventilatory capacity to ferment, when ferment 28h after, according to every
The speed of the reduction 10-20mL/h of 4h gradually reduces feed rate, and fermentation 32-36h stops fermentation.
The method, in one embodiment of the invention, specifically：(1) seed liquor is inoculated in by 10% inoculum concentration
It ferments in fermentation tank, cultivation temperature is 30 DEG C, and control pH is 5.5, initial speed 400rpm, and initial ventilatory capacity is
4.5L/min；(2) start to start feed supplement with 8mL/h rates after fermentation 10h, increase when dissolved oxygen is improved suddenly to 30% or more and mend
Rate is expected, by the raising feed rate 14-25mL/h per 4h until improving to 80-85mL/h；Stir speed (S.S.) is carried every 4h simultaneously
High 100rpm, until 26h stirrings are adjusted to 800rpm；Ventilatory capacity is improved to 6L/min after fermentation 14h, it will ventilation after the 22h that ferments
Amount is improved to 9L/min；(3) it ferments after 28h, the speed of 10-20mL/h is reduced according to every 4h reduces feed rate, and ferment 32-
36h, that is, fermentation ends.
The fermentation ends are in one embodiment of the invention that fermentation 34h terminates to ferment.
The saccharomyces cerevisiae is available from Chinese industrial Microbiological Culture Collection pipe in one embodiment of the invention
The saccharomyces cerevisiae that the CICC numbers at reason center are 1511 or 32280.The method of the present invention has universality to saccharomyces cerevisiae, not only
It is limited only to this two plants of bacterium.
Beneficial effects of the present invention：
(1) culture medium feed rate is controlled in two stages, and ferment middle changes according to dissolved oxygen carries out feed rate gradually
It improves, the fermentation later stage gradually reduces feed rate according to yeast growth rate；It is real by stepping up stir speed (S.S.) and ventilatory capacity
The growth of existing saccharomyces cerevisiae fast and stable, the conventional yeasts high density fermentation period, the present invention was using 34h generally in 40h or more
Can, fermentation period is short, improves fermentation tank utilization rate, reduces energy consumption, reduces microbiological contamination chance；
(2) fermentation control strategy is simple, by dissolved oxygen and growth rate (OD) variation come control speed of agitator, ventilatory capacity,
Feed rate does not need the devices such as other special detection devices, such as residual sugar detection, ethyl alcohol monitoring, tails assay, to hair
Ferment equipment requirement is low, and high density fermentation can be realized in the common fermentation equipment of general factory, can avoid scrap build, reduce at
This.Medium component is simple simultaneously, cheap, is conducive to control cost.
(3) method that the invention is provided has universality for saccharomyces cerevisiae, and it is reachable to obtain saccharomyces cerevisiae yield
109.2g/L。
Description of the drawings
Fig. 1 is saccharomyces cerevisiae (CICC 1511) fermentation process curve graph in embodiment 1；Wherein, ▲ be zymotic fluid ferment mistake
Concentration of glucose change curve in journey, ■ are the change curve of brewing yeast cell dry weight in fermentation process, ● it is fermentation process
Concentration of alcohol change curve in middle zymotic fluid；
Fig. 2 is saccharomyces cerevisiae (CICC 1511) fermentation process curve in embodiment 2；The fermentation of wherein different curve characterizations
The same Fig. 1 of procedure parameter；
Fig. 3 is saccharomyces cerevisiae (CICC 1511) fermentation process curve in embodiment 3；The fermentation of wherein different curve characterizations
The same Fig. 1 of procedure parameter；
Fig. 4 is the fermentation process curve of saccharomyces cerevisiae (CICC 1511) high density fermentation in embodiment 4；It is wherein different bent
Same Fig. 1 of Fermentation Process of Parameter of line characterization；
Fig. 5 is the fermentation process curve of saccharomyces cerevisiae (CICC 32280) high density fermentation in embodiment 5；It is wherein different bent
Same Fig. 1 of Fermentation Process of Parameter of line characterization.
Specific implementation mode
Slant preservation culture medium (g/L)：Glucose 20, peptone 20, yeast powder 10, agar 25.
Seed culture medium (g/L)：Glucose 20, peptone 20, yeast powder 10.
Fermentation medium (g/L)：Cerelose 65, urea 1.8, industrial corn powder 10, KH2PO410, MgSO44,
ZnSO410mg/L, FeSO412mg/L, MnSO4 2mg/L。
Supplemented medium：500g/L glucose solutions.
Embodiment 1：
Saccharomyces cerevisiae CICC 1511 is taken, seed liquor is prepared as follows：
(1) seed culture medium configuration is completed, and is sub-packed in 500mL shaking flasks, and 115 DEG C of sterilizing 20min are cooled to room temperature.
(2) it takes an environmental protection to be stored in the S. cervisiae in slant medium, is inoculated in seed culture medium, cultivation temperature is
30 DEG C, liquid amount 50mL/500mL, shaking speed 200rpm, culture is for 24 hours.
(3) use 4 kinds of different fermentation mediums and supplemented medium, medium component as follows in 7L tanks：
Fermentation medium 1：Cerelose 65g/L, urea 1.8g/L, industrial corn powder 10g/L, KH2PO410g/L,
MgSO44, ZnSO410mg/L, FeSO412mg/L, MnSO4 2mg/L。
Supplemented medium 1：500g/L glucose solutions
Fermentation medium 2：Molasses 102.5g/L, corn steep liquor 168.7g/L.
Supplemented medium (v/v) 2：Molasses 10%+ corn steep liquors 40%
Fermentation medium 3：Glucose 30g/L, yeast extract 10g/L, peptone 20g/L, MgSO4·7H2O 1g/L,
KH2PO4 5g/L。
Supplemented medium (v/v) 3：50% molasses solubles.
Fermentation medium 4：Glucose 10g/L, yeast powder 4.0g/L glycerine 20g/L, (NH4)2SO43.0g/L, KH2PO4
3.0g/L, CaCl20.3g/L, MgCl2 0.3g/L。
Supplemented medium 4：Glucose 100g/L+ glycerine 200g/L.
(4) 1511 seed liquors of saccharomyces cerevisiae CICC are inoculated in by 10% inoculum concentration in 7L fermentation tanks and are fermented, cultivated
Temperature is 30 DEG C, and control pH is 5.5, liquid amount 3L/7L, initial speed 600rpm, and initial ventilatory capacity is 6L/min.Fermentation
Fermentation starts to start feed supplement with 20mL/h rates in the process, and fermented and cultured 28h terminates fermentation, specific control parameter such as table 1.It is different
Fermentation results such as table 2 in fermentation medium.Fermentation process in the fermentation medium 1 of the present invention is as shown in Figure 1, fermentation 26h, wine brewing
Yeast cells dry weight is 44.3g/L, and fermentation period is most short in 4 kinds of culture mediums, dry cell weight highest, therefore following case study on implementation
In select fermentation medium 1.
Table 1
Table 2
Embodiment 2：
1511 seed liquors of saccharomyces cerevisiae CICC are prepared, is inoculated in 7L fermentation tanks and ferments by 10% inoculum concentration, are cultivated
Temperature is 30 DEG C, and control pH is 5.5, liquid amount 3L/7L, initial speed 600rpm, and initial ventilatory capacity is 6L/min.Fermentation
Start to start feed supplement with 8mL/h rates to 10h, the fermentation middle and later periods changes according to dissolved oxygen, if dissolved oxygen improve suddenly to 30% with
On, then slightly increase feed rate and gradually reduces feed rate when fermentation 28h growth rates persistently reduce 2h, specific control ginseng
Number such as table 3.Fermentation results are as shown in Fig. 2, fermentation 28h, brewing yeast cell dry weight are 65.7g/L.
Table 3
Embodiment 3：
1511 seed liquors of saccharomyces cerevisiae CICC are prepared, is inoculated in 7L fermentation tanks and ferments by 10% inoculum concentration, are cultivated
Temperature is 30 DEG C, and control pH is 5.5, liquid amount 3L/7L, initial speed 400rpm, and initial ventilatory capacity is 4.5L/min.Hair
Ferment starts to start feed supplement with 8mL/h rates to 10h, and the fermentation middle and later periods changes according to dissolved oxygen, if dissolved oxygen is improved suddenly to 30%
More than, then slightly increase feed rate, 28h starts constant speed feed supplement later.Fermentation 10h starts to improve stir speed (S.S.) every 4h
100rpm, until 26h stirrings are adjusted to 800rpm.Ventilatory capacity is improved to 6L/min, fermentation 26h and improves ventilatory capacity by fermentation 18h
To 9L/min, specific control parameter such as table 4.Fermentation results are as shown in figure 3, fermentation 30h, brewing yeast cell dry weight are 83.6g/
L。
Table 4
Embodiment 4：
1511 seed liquors of saccharomyces cerevisiae CICC are prepared, is inoculated in 7L fermentation tanks and ferments by 10% inoculum concentration, are cultivated
Temperature is 30 DEG C, and control pH is 5.5, liquid amount 3L/7L, initial speed 400rpm, and initial ventilatory capacity is 4.5L/min.Hair
Ferment starts to start feed supplement with 8mL/h rates to 10h, and the fermentation middle and later periods changes according to dissolved oxygen, if dissolved oxygen is improved suddenly to 30%
More than, then slightly increase feed rate and gradually reduces feed rate when fermentation 28h growth rates persistently reduce 2h.Ferment 10h
Start that stir speed (S.S.) is improved 100rpm every 4h, until 26h stirrings are adjusted to 800rpm.Fermentation 18h improves ventilatory capacity to 6L/
Min, fermentation 26h improve ventilatory capacity to 9L/min, specific control parameter such as table 5.Fermentation results as shown in figure 4, fermentation 34h,
Brewing yeast cell dry weight is 109.2g/L.
Table 5
Embodiment 5：
Prepare the seed liquor of saccharomyces cerevisiae CICC 32280, inoculation fermentation.Preliminary fermentation parameter was fermented with embodiment 4
Feed rate is according to the variation of yeast growth rate after dissolved oxygen variation and 28h, specific control parameter such as table 6, fermentation knot in journey
Fruit is as shown in figure 5, fermentation 34h, 32280 dry cell weight of saccharomyces cerevisiae are 107.8g/L.
Table 6
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not limited to the present invention, any to be familiar with this skill
The people of art can do various change and modification, therefore the protection model of the present invention without departing from the spirit and scope of the present invention
Enclosing be subject to what claims were defined.
Claims (1)
1. a kind of cultural method of saccharomyces cerevisiae high density fermentation, which is characterized in that the method is：In yeast fermented and cultured mistake
Control by stages culture medium feed rate in journey, early period no-feed supplement, mid-term steps up feed rate, and the later stage gradually reduces feed supplement
Speed, while stepping up stir speed (S.S.) and ventilatory capacity in mid-term；
The method is specifically：(1) seed liquor is inoculated in fermentation tank by 10% inoculum concentration and is fermented, cultivation temperature 30
DEG C, control pH is 5.5, initial speed 400rpm, and initial ventilatory capacity is 4.5L/min；(2) fermentation 10-14h is with 8mL/h rates
Start feed supplement, 14-18h is with 30mL/h rate feed supplements for fermentation, fermentation 18-22h with 55mL/h rate feed supplements, fermentation 22-26h with
80mL/h rate feed supplements, 26-28h is with 80mL/h rate feed supplements for fermentation；Stir speed (S.S.) is improved into 100rpm every 4h simultaneously, until
Stirring is adjusted to 800rpm after 26h；Ventilatory capacity is improved to 6L/min after fermentation 14h, improves ventilatory capacity to 9L/ after the 22h that ferments
min；(3) it ferments after 28h, reduces feed rate, fermentation 28-32h is with 65mL/h rate feed supplements, and fermentation 32-36h is with 55mL/h speed
Rate feed supplement, 36h, that is, fermentation ends of fermenting；
The culture medium that the fermented and cultured uses is：Contain per 1L：Glucose 65g, urea 1.8g, industrial corn powder 10g,
KH2PO412g, MgSO44g, ZnSO410mg, FeSO412mg, MnSO42mg；
The supplemented medium that the feed supplement uses is 500g/L glucose solutions.
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