CN105861343A - Hansenula polymorpha for preparing high-lysine single-cell protein by using methanol and application thereof - Google Patents

Abstract

The invention relates to a Hansenula polymorpha strain for preparing high-lysine single-cell protein by using methanol and application thereof, wherein the strain is classified and named as pleomorphicHansenula (Hansenula polymorpha) (III)Hansenula polymorpha) AP23, which has been preserved in China Center for Type Culture Collection (CCTCC) at 2016, 4 and 5 days, with the preservation number: CCTCC NO: M2016173. The invention utilizes a plasma mutagenesis technology, uses methanol as a unique carbon source to screen out a strain which can produce single-cell protein rich in lysine, and further combines with methanol gradient screening to obtain the Hansenula polymorpha which efficiently utilizes the methanol. The strain can realize high-density fermentation, and methanol protein is produced by using a 5L fermentation tank, so that the final wet weight of the strain reaches 350-400 g/L, the dry weight of the methanol protein reaches 115-130 g/L, the protein content reaches 68%, and the lysine content reaches 60 mg/g. The invention has great social significance and economic value for the development of animal feed protein additives.

Description

One strain utilize methanol prepare high-lysine single cell protein polymorpha and Its application

Technical field

The present invention relates to bioengineering field, particularly one strain utilizes methanol production many rich in lysine single cell protein Shape Hansenula yeast bacterium and application thereof.

Background technology

Protein resource shortage is the key subjects that the whole world faces, and each state is all paying attention to reinforcement exploitation novel protein resource Research.Wherein, what industrialization was the fastest is, and single cell protein produces, and is considered as to solve human protein's scarcity of resources to have most One of approach of effect.

Single cell protein (single cell protein, SCP), refers to that yeast, mycete, fungus, non-pathogenic are thin Produced bacterium protein in the unicellular microorganism bodies such as bacterium, its protein content typically constitutes from the 40% ~ 60% of thalline dry. It is referred to as second filial generation single cell protein for raw material production Methanol Protein out with industrial methanol, compared with native protein, its Crude protein content, will be high than fish flour and Semen sojae atricolor more than 70%, and containing abundant essential amino acids, minerals and vitamins, Can part Peru Fish Dietary, Semen sojae atricolor, bone meal, meat and defatted milk powder, and have not against arable land, weatherproof, egg The advantages such as white matter steady quality.

From the sixties in 20th century, just there are many countries to carry out the research of Methanol Protein, and develop into worldwide Popular research topic.Wherein account for the ICI company of Yao Shu Britain of leading position, the device just having scale to be 100,000 t/a in 1980 It is constructed and put into operation.The Methanol Protein trade name " Pruteen " that ICI company produces." Pruteen " product contains the thick egg of 72% In vain, methionine and lysine content are the most close with fish flour, as the feedstuff rich in heat, vitamin, mineral and high protein Commercially sell.Nineteen eighty-three U.S. Phillips, oil company developed again Methanol Protein production new technique, improved fermentation tank Heat exchange and oxygen condition of transmitting, can produce high density Methanol Protein.Subsequently, Germany's Hoechst-Uhde, Mitsubishi gas The companies such as, Sweden Norprotein and France IFP also establish pilot-plant.Since 20 century 70s, China also begins to The research of Methanol Protein.But up to the present, the industrialization of Methanol Protein is still at an early stage in China, domestic also do not have work The Methanol Protein process units of industry, some units also stopped the research and development to Methanol Protein.

Methanol is a kind of platform chemicals, its wide material sources, and the methanol production capacity of China alreadys more than 45,000,000 t/a, and In continuing increase trend, and domestic methanol demands amount is only 10,000,000 t/a, and most methanol Business survivals are hard to carry on, are badly in need of Exploitation Downstream Products of Methanol, and with methanol for raw material production protein resource, the imbalance between supply and demand of methanol can be alleviated.At present, methanol Cheap, Methanol Protein production cost is low, replaces the albumen such as traditional fish flour and beans with Methanol Protein, it will reduce feedstuff Cost, improves feeding efficiency, accelerates the development of China feed industry, is the most also to solve the shortage of current albumen feedstuff, rely on into The effective way of this contradiction of mouth.

Methanol Protein produces bacterial strain antibacterial and yeast etc., and saccharomycetic acid resistance is the best, it is possible to reduce extensive The risk polluted during cultivation, energy high-density growth in cheap synthesis or semisynthetic medium, multiple-shaped nuohan inferior yeast is again in addition There is of a relatively high optimal growth temperature, the speed of growth can be improved, shorten fermentation time, reduce Zymolysis Equipment simultaneously Refrigeration requirement, thus beneficially large scale fermentation produces.But yeast how can be utilized to realize efficiently utilizing methanol production Also do not have been reported that rich in certain amino acid whose single cell protein.

Summary of the invention

It is inferior that the first object of the present invention is to provide a strain to utilize methanol to prepare the multiform Chinese of high-lysine single cell protein Yeast.

For realizing the first object of the present invention, the technical solution used in the present invention is as follows:

One plant height effect utilizes methanol production rich in the multiple-shaped nuohan inferior yeast of lysine single cell protein, and its Classification And Nomenclature is the multiform Chinese Inferior yeast Hansenula polymorphaAP23, its deposit number is: CCTCC NO:M 2016173.Preservation day is 2016 On April 5, in.

The screening technique of multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 of the present invention is, by the multiform Chinese Inferior yeast starting strain (ATCC34438) is after plasma mutation, using methanol as sole carbon source, utilizes high concentration methanol to put down Screen choosing obtains the bacterial strain that methanol tolerance is stronger, then obtains high content of protein and high-load lysine through shake flask fermentation screening Bacterial strain, after tame through high concentration methanol, it is thus achieved that can efficiently utilize methanol production rich in the multiform of lysine single cell protein Hansenula yeast.

The screening of multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 specifically comprises the following steps that

(1) plasma mutation: by multiple-shaped nuohan inferior yeast original strain activation culture, 100 ml triangular flask liquid amounts are 20 Ml, in 37 DEG C of shaking tables, 200 rpm shaken cultivation 24 h, obtains being in the bacterium solution of exponential phase, is diluted to by the cell of cultivation OD₆₀₀=3-4, drips on the slide glass after sterilizing cools down, dries up with filtrated air；With helium as discharge gas, make with 80-120W For radio-frequency power, using 10-30SLM as gas flow, as exposure time, bacterial strain is carried out plasma inducing using 10-1800s Become.

(2) high concentration methanol flat board primary dcreening operation: the slide glass after mutation is placed in the tool plug test tube equipped with 1-2 mL normal saline In, acutely shake, by the bacterial strain eluting on slide glass, be diluted to variable concentrations and coat the culture medium flat plate containing 500 mM methanol On, cultivate 2-3 days, pick out eugonic bacterium colony for 37 DEG C.

(3) rich in the screening of lysine single cell protein bacterial strain: choose eugonic single bacterium colony and be inoculated in respectively and comprise In 200 ml fermentation medium of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, centrifugal tropina of collecting, detection thalline The bacterial strain that the content of middle albumen and the content of lysine, selection protein content and lysine content are the highest.

(4) high concentration methanol domestication: protein content in selecting step (3) and the highest bacterial strain of lysine content continue It is seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic (log) phase, is forwarded to the selection containing 1M methanol and cultivates On base；It is forwarded on the Selective agar medium containing 1.5 M by same method afterwards, the most again bacterium solution is transferred to containing 2 M methanol In Selective agar medium, after repeating to tame 2 times, take 0.2 ml bacterium solution and be coated on the Selective agar medium flat board comprising 2 M methanol, 37 DEG C constant temperature culture, obtains multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23.

The second object of the present invention is that multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 relies ammonia at preparation height Application in acid single cell protein.The invention provides one and utilize multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23 In preparing high-lysine single cell protein, the method for application, specifically comprises the following steps that

(1) polymorpha AP23 ferments at 5 L fermentation tank middle-high densities: polymorpha AP23 ferments at 5 L Using fermentation medium to carry out tropina production in tank, wherein, fermentation medium liquid amount is 2L, 121 DEG C of sterilizing 15 min, Inoculation pipeline, ammonia and methanol feeding pipeline, sample tap pipeline, air filter are carried out sterilizing simultaneously, treat fermentation medium When being down to about 37 DEG C, regulating pH to 5.0 with ammonia, add seed liquor and ferment, fermentation temperature is 37 DEG C, controls dissolved oxygen 20%-60%, speed of agitator 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream and add methanol, monitor by gas phase Methanol concentration so that it is control between 0.5%-2%, every 8 hours sampling and measuring thalline OD₆₀₀And weight in wet base, after 96-100 hour, Fermentation ends, collects fermentation liquid.

(2) rich in the extraction of lysine Methanol Protein: the fermentation liquor that above-mentioned steps (1) is collected in 5 L fermentation tanks Centrifuge 5000r/min continuous centrifugal concentrates and obtains thalline, weighs its weight in wet base after clear water washing thalline；By collect thalline in Dry to constant mass for 55 DEG C, collect dry powder and be single-cell methanol protein.

The beneficial effects of the present invention is:

Using plasma mutation multiple-shaped nuohan inferior yeast of the present invention, utilizes methanol flat screen to select protein content and lysine content Higher bacterial strain, utilizes high concentration methanol to tame on this basis so that this bacterial strain can utilize methanol production unicellular efficiently Albumen.In 5L fermentation tank, utilize glycerol 30 g/L, methanol 500 ml/L can produce 115-130g/L tropina, Qi Zhonglai Histidine content, up to not containing methanol in 60mg/g, and the Methanol Protein dry powder obtained, is the desirable feedstock substituting forage protein, There is great social meaning and economic worth.

Biomaterial explanation

Biomaterial of the present invention, its Classification And Nomenclature is multiple-shaped nuohan inferior yeast (Hansenula polymorpha) AP23, Being preserved in China typical culture collection center (being called for short CCTCC), deposit number is CCTCC NO:M 2016173, preservation day Phase is: on April 5th, 2016, preservation address is: China. Wuhan. and Wuhan University.

Detailed description of the invention

The present invention is only described in further detail by following embodiment, but does not constitute any limitation of the invention.

Embodiment 1

This example demonstrates that the method that multiple-shaped nuohan inferior yeast original strain is carried out first step plasma mutation.

The method that multiple-shaped nuohan inferior yeast original strain carries out first step plasma mutation is as follows:

By multiple-shaped nuohan inferior yeast original strain activation culture, 100 ml triangular flask liquid amounts are 20 ml, in 37 DEG C of shaking tables 200 Rpm shaken cultivation 24 h, obtains growing bacterium solution vigorous, that thalline is sturdy；The cell taking fresh cultured is diluted to cell concentration OD₆₀₀=3-4, drips on the slide glass after sterilizing cools down, dries up with filtrated air；With helium as discharge gas, using 100W as Radio-frequency power, using 10SLM as gas flow, carries out plasma mutation as exposure time to bacterial strain using 10-1800s, lures After change, the Mycoderma on carrier is eluted, calculate survival rate.Test result indicate that 450 s are optimal mutation exposure times.

Embodiment 2

The explanation screening of this example efficiently utilizes methanol production rich in the method for the multiple-shaped nuohan inferior yeast of lysine single cell protein.

Wherein, the culture medium prescription used:

(1) seed culture medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, remaining is water.

(2) Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, chlorine Changing calcium 0.4 g/L, yeast extract 1 g/L, agar powder 1.5 g/L, remaining is water, and pH is natural.

(3) fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two Water magnesium sulfate 14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；

(4) fermentation medium 2:85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two water sulfur Acid magnesium 14.9 g/L, potassium hydroxide 4.13 g/L, methanol 20ml/L, trace element solution 4 ml/L, water 1 L, every 24h during cultivation Add 20ml/L methanol.

Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L, Sodium Molybdate Dihydrate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 G/L, biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.

Screening step is specific as follows:

(1) high concentration methanol flat board primary dcreening operation:

Slide glass after mutation in embodiment 1 is placed in equipped with in the tool plug test tube of 1-2ml normal saline, acutely shakes, by slide glass On bacterial strain eluting, be diluted on the Selective agar medium flat board that variable concentrations is coated containing 500mM methanol, 37 DEG C cultivate 3-4 days, Pick out eugonic bacterium colony.

(2) rich in the screening of lysine single cell protein bacterial strain: choose eugonic single bacterium colony and be inoculated in respectively and comprise In 200 ml fermentation medium of 500 mM methanol, 37 DEG C, 200 rpm cultivate 48h, centrifugal tropina of collecting, detection thalline The bacterial strain that the content of middle albumen and the content of lysine, selection protein content and lysine content are the highest.

(3) domestication of resisting high-concentration methanol: protein content in selecting step 2 and the highest bacterial strain of lysine content continue Continue and be seeded on the Selective agar medium containing 500 mM methanol, after it grows to logarithmic (log) phase, be forwarded to the selection training containing 1M methanol Support on base；It is forwarded on the Selective agar medium containing 1.5 M by same method afterwards, the most again bacterium solution is transferred to containing 2 M methanol Selective agar medium in, after repeating to tame 2 times, take 0.2 ml bacterium solution and be coated on the Selective agar medium flat board comprising 2 M methanol, 37 DEG C of constant temperature culture, obtain multiple-shaped nuohan inferior yeast Hansenula polymorphaAP23.

(4) shake flask fermentation screening:

Strains A P23 that step (3) is obtained and original strain access seed culture medium amplification culture, cultivation temperature 37 DEG C, 100 ML triangular flask liquid amount 20 mL, incubation time 24 h.Ferment the most in the fermentation medium, inoculum concentration 10%(v/v), fermentation Temperature 37 DEG C, 1L triangular flask liquid amount 200 mL, after cultivating 24h, stand overnight, change fermentation medium 2, fermentation time 72 h Protein content and the lysine content of each bacterial strain of rear detection are as shown in table 1:

Table 1

Embodiment 3

This example demonstrates that the hereditary stability test of the multiple-shaped nuohan inferior yeast AP23 of embodiment 2, strain passage fermenting experiment is such as Shown in lower.

Utilize shake flask fermentation, the mitotic stability of detection mutant AP23.Strains A P23 passes on fermentation test result such as table 2 Shown in:

Table 2 strains A P23 passes on fermentation test result

As can be seen from the table, passing on through 7 times, strains A P23 albumen and lysine content are stable, and mitotic stability is good.

Embodiment 4

This example demonstrates that multiple-shaped nuohan inferior yeast AP23 efficiently utilizes methanol production rich in the application of lysine single cell protein.

Wherein, the culture medium prescription used:

(1) seed slant medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, agar powder 20 g/L, its Yu Weishui.

(2) Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, chlorine Changing calcium 0.4 g/L, yeast extract 1 g/L, remaining is water, and pH is natural.

(3) fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, two Water magnesium sulfate 14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；

Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L, two water Sodium molybdate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, Biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.

Multiple-shaped nuohan inferior yeast AP23 bacterial strain is in the method for 5 L fermentation tank middle-high density fermenting and producing Methanol Proteins, and step is such as Under:

(1) cultivation of seed: multiple-shaped nuohan inferior yeast AP23 bacterial strain method of scoring is seeded on seed slant medium, 37 DEG C of trainings Support 48 hours, take 1 cultured inclined-plane seed, with 3 mL sterilized water, seed is rinsed, be seeded to comprise 200 mL choosings Select in 1 L triangular flask of culture medium, 37 DEG C, 220 rpm shaken cultivation about 24 hours to OD=9-10.

(2) 5 L fermentation tank middle-high density fermentations: multiple-shaped nuohan inferior yeast AP23 bacterial strain uses fermentation training in 5 L fermentation tanks Foster base carries out tropina production, and wherein, fermentation medium liquid amount is 2L, and 121 DEG C of sterilizing 15 min, simultaneously to inoculated tube Road, ammonia and methanol feeding pipeline, sample tap pipeline, air filter carry out sterilizing, treat that fermentation medium is down to about 37 DEG C Time, regulating pH to 5.0 with ammonia, add seed liquor and ferment, fermentation temperature is 37 DEG C, controls dissolved oxygen 20%-60%, and stirring turns Speed 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream and add methanol, monitor methanol concentration by gas phase so that it is Control between 0.5%-2%, every 8 hours sampling and measuring thalline OD₆₀₀And weight in wet base, after 96-100 hour, fermentation ends, collect Fermentation liquid.Subsequently fermentation liquor centrifuge 5000 r/min continuous centrifugal is concentrated and obtain thalline, claim after clear water washing thalline Measure its weight in wet base；The thalline collected is dried to constant mass in 55 DEG C, collects dry powder and be single-cell methanol protein.

Final thalline weight in wet base reaches 350-400 g/L, and Methanol Protein dry weight reaches 115-130 g/L, and protein content reaches To 68%, wherein lysine content is up to 60 mg/g.

Claims (7)

1. a strain utilizes methanol to prepare the polymorpha of high-lysine single cell protein, it is characterised in that Classification And Nomenclature For multiple-shaped nuohan inferior yeast (Hansenula polymorpha) AP23, it is preserved in Chinese Typical Representative culture on April 5th, 2016 Preservation center CCTCC, deposit number: CCTCC NO:M 2016173.

2. the preparation method of polymorpha described in claim 1, it is characterised in that preparation process includes using high concentration Methanol flat board primary dcreening operation, then through shake flask fermentation, after tame through high concentration methanol.

The preparation method of polymorpha the most according to claim 2, it is characterised in that described primary dcreening operation and fermentation step Middle methanol concentration is 500mM.

The preparation method of polymorpha the most according to claim 2, it is characterised in that described domestication step methanol is dense Degree graded, is finally 2 M.

5. the application in prepared by high-lysine single cell protein of the polymorpha described in claim 1.

Polymorpha application in prepared by high-lysine single cell protein, its feature the most according to claim 5 It is, comprises the steps:

(1) seed culture: by multiple-shaped nuohan inferior yeast AP23 inoculation to seed slant medium, cultivate 48 hours for 37 DEG C After, take in the triangular flask that cultured inclined-plane seed is seeded to comprise Selective agar medium, 37 DEG C, 220 rpm shaken cultivation about 24 Hour to OD=9-10；

(2) fermentation culture: multiple-shaped nuohan inferior yeast AP23 bacterial strain uses fermentation medium to carry out tropina production in fermentation tank, 121 DEG C of sterilizing 15 min, when fermentation medium is down to 37 DEG C, regulate pH to 5.0, add seed liquor and ferment, fermentation temperature Degree is 37 DEG C, controls dissolved oxygen 20%-60%, speed of agitator 250-500 rpm；After 24 hours, glycerol depletion in culture medium, start stream Add methanol, monitor methanol concentration by gas phase so that it is control between 0.5%-2%, every 8 hours sampling and measuring thalline OD₆₀₀With wet Weight, after 96-100 hour, fermentation ends, collect fermentation liquid；

(3) albumen is extracted: concentrated by fermentation liquor centrifuge 5000 r/min continuous centrifugal and obtain thalline, wash thalline with clear water Its weight in wet base of rear weighing；The thalline collected is dried to constant mass in 55 DEG C, collects dry powder.

Polymorpha application in prepared by high-lysine single cell protein, its feature the most according to claim 6 It is, uses medium component as follows:

Seed slant medium: yeast powder 10 g/L, peptone 20 g/L, glucose 20 g/L, agar powder 20 g/L, remaining For water；

Selective agar medium: methanol 500 mM, ammonium sulfate 1 g/L, potassium dihydrogen phosphate 1 g/L, magnesium sulfate 0.7 g/L, calcium chloride 0.4 G/L, yeast extract 1 g/L, remaining is water, and pH is natural；

Fermentation medium: 85% phosphoric acid 26.7 ml/L, calcium sulphate dihydrate 0.93 g/L, potassium sulfate 18.2 g/L, sulfate dihydrate magnesium 14.9 g/L, potassium hydroxide 4.13 g/L, glycerol 30 g/L, trace element solution 4 ml/L, water 1 L；

Described trace element solution is: copper sulphate pentahydrate 6 g/L, potassium iodide 0.088 g/L, manganese sulfate monohydrate 3 g/L, two water Sodium molybdate 0.2 g/L, boric acid 0.02 g/L, cobalt chloride hexahydrate 0.5 g/L, zinc chloride 20 g/L, ferrous sulfate heptahydrate 65 g/L, Biotin 0.2 g/L, mass concentration is concentrated sulphuric acid 5 ml/L of 98 %, and remaining is water.