WO2017012570A1

WO2017012570A1 - Bacillus coagulans, high cell density fermentation method thereof, and method for preparing spray-dried powder thereof

Info

Publication number: WO2017012570A1
Application number: PCT/CN2016/090895
Authority: WO
Inventors: 张梁
Original assignee: 江南大学
Priority date: 2015-07-22
Filing date: 2016-07-21
Publication date: 2017-01-26
Also published as: CN104974966A; AU2016297402A1; CN104974966B; AU2016297402B2

Abstract

Provided is a Bacillus coagulans strain (serial number: ZS-007) deposited in the China Center for Type Culture Collection (CCTCC) and having a deposition number of CCTCC M2015273. Also provided are a fermentation method of the Bacillus coagulans (serial number: ZS-007), and method for preparing a spray-dried powder of the Bacillus coagulans via the fermentation method.

Description

Instruction manual

Title: A Bacillus coagulans and a high-density fermentation method thereof, and a method for preparing dry powder

Technical field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bacillus coagulans and a high-density fermentation method thereof and a prepared dry powder.

Background technique

[0002] Microecological preparations are microbial agents prepared by a special process and contain microorganisms beneficial to the host. Micro-developing agents have natural and non-toxic effects and promote the health of the host, and are increasingly used in human health and animal health.

[0003] Lactic acid bacteria and Bacillus microecological preparations are the two most widely used varieties in the feed industry. The lactic acid bacteria mainly include lactic acid bacteria, bifidobacteria, etc., and the bacilli mainly include Bacillus subtilis, Bacillus licheniformis and the like.

[0004] Lactic acid fungi microecological preparations function by producing lactic acid, changing intestinal pH, and regulating the intestinal microenvironment. Such microecological preparations have poor resistance to stress and are prone to loss of activity during processing. Bacillus microecological preparations can produce spores, can withstand high temperature, stomach acid, choline and other adverse environments, and have strong resistance to stress, but they do not have the intestinal conditioning function of lactic acid bacteria.

[0005] Bacillus coagulans, Gram-positive bacteria, a lactic acid-producing Bacillus

. Bacillus coagulans is characterized by: 1. Producing lactic acid under the anaerobic environment of the intestine, forming an acidic environment, inhibiting the growth of pathogenic microorganisms, and beneficial to the health of the host; 2. It can exist in the state of spores, high temperature tolerance, gastric acid tolerance, choline resistance, environment Strong resistance to stress. It can be said that Bacillus coagulans has the advantages of both lactic acid bacteria and microbial preparations of Bacillus.

[0006] Due to its good feeding and processing properties, Bacillus coagulans has been included in the list of feed additives and has become an important member of the feed microecological preparation industry.

[0007] There has been an increasing interest in Bacillus coagulans, and a great deal of research has been done. ZL200710122111 .X published a preparation of a Bacillus coagulans feed additive with a maximum viable count of 5.0x10 9 cfu/mL. ZL 200810235512.0 announced the liquid fermenter of Bacillus coagulans CGMCC No. 2602 Art. In the IT fermenter, the total number of bacteria in the fermentation broth is 6.0x 10 ⁹ cfu/mL, and the number of spores is 5.8x10 ⁹

Cfu/mL, spore rate 96.7 <3⁄4. ZL 201110428027.7 announced a process for solid state fermentation of Bacillus coagulans CICC No.20138, the cell amount 1.16x 10 ^1Q cfu / g, Bacillus rate 77.2 <¾. ZL

201210464404.7 announced the feedback feeding culture process of Bacillus coagulans CGMCC No.6681, the highest number of live bacteria in the fermentation broth was 5.8x10 fu/mL.

[0008] Despite the extensive research work in the industry, the price of Bacillus coagulans in the market is still high, and product promotion is difficult. The reason is that the industrial fermentation level of Bacillus coagulans is low, the loss of bacteria in the processing process is high, and the content of the finished cells is low. These unfavorable factors directly lead to high product prices, affecting the widespread use of Bacillus coagulans.

technical problem

In view of the above problems in the prior art, the Applicant provides a Bacillus coagulans and a high-density fermentation method thereof, and a method for preparing a dry powder. The invention is a new burst of Bacillus coagulans, which has the characteristics of high-density fermentation and high bacterial content, is beneficial to reducing product cost, contributing to product marketing, and has positive significance for promoting healthy breeding of livestock and poultry.

Problem solution

Technical solution

[0010] The technical solution of the present invention is as follows:

[0011] The Bacillus coagulcmi) provided by the present invention has been preserved in the China Center for Type Culture Collection, Culture Collection No. CCTCC M 2015273. This is achieved by the following steps:

[0012] 1. Screening of Bacillus coagulans

[0013] Weigh 2g from the soil sample near a dairy farm in Mashan, Wuxi, Jiangsu, add 98ml of sterile physiological saline, heat treatment at 80 °C for 10 minutes, shake at 120 rpm on a 37 °C shaker for 20 min, absorb the supernatant The liquid was added to LB medium and diluted and coated for strain isolation. The species of the strain were effectively and accurately determined by means of morphological observation, physiological bioassay, and 16S rDNA analysis.

[0014] The strain numbered ZS-007 was finally obtained and identified as Bacillus coagula.

[0015] ZS-007 was deposited with the China Type Culture Collection on May 3, 2015 under the accession number CCTCC M 2015273.

[0016] 2, Bacillus coagulans lactic acid production test [0017] Bacillus coagulans ZS-007 was inoculated into an acidogenicity test medium, and statically cultured at 37 ° C to carry out a lactic acid production test. The acid-producing test medium used was: glucose 20 g/L, peptone 10 g/L, yeast powder 5 g/L, potassium dihydrogen phosphate 2 g/L, magnesium sulfate lg/L, manganese sulfate 0.5

g/L. The medium was adjusted to pH 7.0 with 10<3⁄4 NaOH and sterilized at 121 °C for 20 min.

[0018] The lactic acid content in the fermentation broth was measured by HPLC, and the results are shown in FIG. It can be seen from Fig. 1 that the lactic acid content is about 10 g/L.

[0019] 3, Bacillus coagulum high-density fermentation

(1) Activation of strains

[0021] The Bacillus coagulans strain is inoculated onto the nutrient agar slant medium, and cultured at 25-37 ° C for 24-48 hr; then the streak is transferred to the slant of the nutrient agar eggplant bottle, and cultured at 25-37 ° C 24- 48hr.

[0022] The composition of the nutrient agar slant medium and the nutrient agar eggplant bottle are: peptone 10g / L, beef cream

3 g/L, sodium chloride 5 g/L, agar 15-20 g/L, pH 7.2-7.4.

[0023] (2) Shake flask culture

[0024] The activated strain was inoculated into a shake flask and shake cultured for 18-30 hr.

[0025] Shake flask medium formula: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L,

pH 7.2-7.4. The volume of the 250 mL shake flask is 20 mL, and the volume of the 500 mL shake flask is 50 mL.

[0026] Culture conditions: shake flask speed 200 r/min, temperature 25-37 °C.

[0027] (3) Seed tank expansion

[0028] Expanded strains for later fermentation. Seed tank medium composition: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4. The seed tank has a volume of 10-1000L and a liquid volume of 50-80%.

[0029] Culture conditions: stirring speed 180-250r/min, aeration amount 0.5-1.0vvm, temperature 25-37 ° C, culture time between 18-30hr.

[0030] (4) Fermentor Fermentation

[0031] The seed liquid is connected to the fermenter with an inoculum of 0.1%-10<3⁄4, and the fermentation medium is composed of: glucose 25 g/L, peptone 15 g/L, yeast powder 10 g/L, potassium dihydrogen phosphate lg /L, magnesium sulfate 0.5 g / L, initial pH 7.0. The fermenter has a volume of l-50m ³ and a liquid loading of 50-80%.

[0032] Culture conditions: agitation speed of 100-200 r / min, aeration of 0.5-0.8 vvm, temperature of 25-37 ° C, cultured for 16-48 hr. [0033] Taking the fermentation liquid microscopic examination, when more than 90% of the cells form spores, that is, to the end of the fermentation, the final fermentation liquid is obtained.

[0034] The viable cell count and the number of spores of the final fermentation broth were measured by a plate coating method, and the spore rate was calculated. Among them, the spore rate is the ratio of the number of spores to the number of viable cells. The medium used was a nutrient agar medium: peptone 10 g/L, beef extract 3 g/L, sodium chloride 5 g/L, agar 15-20 g/L, pH 7.2-7.4. The number of live bacteria in the final fermentation broth was measured and determined by gradient dilution; the number of spores was determined, and the final fermentation broth was heat-treated at 80 ° C for 10 min, and the mixture was diluted by a gradient. The number of viable cells in the final fermentation broth was 1.5×10 ^1Q cfu/mL or more, the number of spores was 1.4 10 /!1^ or more, and the spore rate was 90.8% or more.

[0035] 4, dry powder preparation

[0036] After the final fermentation broth is centrifuged, the wet bacterial sludge is collected, and the dry bacterial powder is obtained by a spray drying process. Operating conditions are: inlet air temperature 210 °C, outlet air temperature 100 °C. The number of viable bacteria in dry powder is 8.0x10 "cfu/g or more.

[0037] The obtained dry powder can be mixed with a filler such as starch, glucose, and other standards-compliant carriers to obtain a Bacillus coagulans microecological preparation.

Advantageous effects of the invention

Beneficial effect

[0038] The beneficial technical effects of the present invention are:

[0039] The present invention employs a liquid fermentation process in which the number of viable bacteria in the fermentation broth is 1.52×10 i. Above cfu/mL, the number of spores was 1.38x10 W cfu/mL or more, and the spore rate was 90.8% or more. In the dry powder obtained by spray drying, the number of viable cells was 8.0 x 10 11 cfu/g or more. The dry powder can be mixed with a filler such as starch or glucose to obtain a coagulating microbial preparation.

[0040] The production process of the invention is simple and stable, and the number of viable bacteria in the fermentation liquid is high, which can directly reduce the application cost and is beneficial to product promotion. This has implications for the widespread use of Bacillus coagulans microecological preparations.

Brief description of the drawing

DRAWINGS

1 is a HPLC method for detecting lactic acid content in a fermentation broth of Bacillus coagulans according to the present invention.

Embodiments of the invention Example 1: Screening of Bacillus coagulans

[0043] Weigh 2g from the soil sample near a dairy farm in Mashan, Wuxi, Jiangsu Province, add 98ml of sterile physiological saline, heat treatment at 80 °C for 10 minutes, shake at 120 rpm on a 37 °C shaker for 20 min, absorb the supernatant 1 ml of the solution was added to LB medium and diluted and coated for strain isolation. The species of the strain were effectively and accurately determined by means of morphological observation, physiological and biochemical experiments, and 16S rDNA analysis.

[0044] The strain numbered ZS-007 was finally obtained and identified as Bacillus coagula.

[0045] ZS-007 was deposited with the China Type Culture Collection on May 3, 2015 under the accession number CCTCC M 2015273.

[0046]

Example 2: Preparation of lm ³ fermentor

[0048] Activation of the strain: The strain obtained in Example 1 was inoculated on a nutrient agar slant medium, cultured at 30 ° C for 24 hours, and then streaked to the slant of the nutrient agar eggplant bottle, and cultured at 30 ° C for 24 hr. The composition of nutrient agar slant medium and nutrient agar eggplant bottle are: peptone 10g/L, beef extract 3g/L, sodium chloride 5g/L, agar 15-20g/L, pH 7.2-7.4.

[0049] Shake flask culture: The activated strain was inoculated into a shake flask and shake cultured for 20 hr. Shake flask medium formula: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4, shake flask volume 50mL / 500mL triangle bottle. Culture conditions: shake flask speed 200r/min, temperature 30. C.

[0050] Fermentor Fermentation: 20 bottles of flask liquid were collected as seeds for access to the lm 3 fermentor. The lm 3 fermenter has a liquid volume of 65%. Medium composition: glucose 25 g/L, peptone 15 g/L, yeast powder 10

g/L, potassium dihydrogen phosphate l g/L, magnesium sulfate 0.5 g/L, initial pH 7.0. The cultured seeds are connected to the fermenter for fermentation. Culture conditions: stirring speed 200 r / min, aeration rate 0.8 wm, temperature 30 ° C, culture day 40-44 hr.

[0051] broth, viable cell count 1.52x 10 ^1Q cfu / g, the number of spores 1.38x10 ^1Q cfu / g, Bacillus rate 90.8 <¾.

[0052]

Example 3: Preparation of 3m ³ Fermentor

[0054] The species activation was the same as in Example 2.

[0055] Shake flask culture: The activated strain was inoculated into a shake flask and shake cultured for 20 hr. Shake flask medium formula: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4, shake flask volume is 20mL / 250mL triangle bottle. Culture conditions: shake flask speed 200r / min, temperature 30 ° C.

[0056] Seed tank expansion: Collect 10 bottles of 250 mL shake flask seed solution for seed tank inoculation. Seed tank medium composition: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4. The seed tank has a volume of 30L and a liquid volume of 80<3⁄4. Culture conditions: stirring speed 200 r / min, aeration 0.8 vvm, temperature 30 ° C, incubation time 20 hr.

[0057] Fermentor Fermentation: The liquid volume of the 3 m 3 fermenter was 70%. Medium composition: glucose 25 g / L, peptone 15 g / L, yeast powder 10 g / L, potassium dihydrogen phosphate lg / L, magnesium sulfate 0.5 g / L, initial pH 7.0. The cultured seeds are connected to a fermentor for fermentation. Culture conditions: stirring speed 150 r / min, aeration of 0.7 _V vm, temperature 35 ° C, cultured for 36-42 hr.

[0058] In the fermentation broth, the number of viable cells was 1.79× ^{10 1 ()} cfu/g, the number of spores was 1.72× ^{10 1 ()} cfu/g, and the spore rate was 96.1<3⁄4.

[0059]

Example 4: Preparation of 30m 3 fermentor and dry powder

[0061] Strain activation is the same as in Example 2

[0062] Shake flask culture: The activated strain was inoculated into a shake flask and shake cultured for 20 hr. Shake flask medium formula: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4, shake flask volume 50mL / 500mL triangle bottle. Culture conditions: shake flask speed 200r/min, temperature 30. C.

[0063] Seed tank expansion: 20 bottles of 500 mL shake flask seed solution were collected for seed tank inoculation. Seed tank medium composition: peptone 10g / L, beef extract 3g / L, sodium chloride 5g / L, pH 7.2-7.4. The seed tank has a volume of 500L and a liquid volume of 75%. Culture conditions: stirring speed 200 r / min, aeration rate 0.8 vvm, temperature 30 ° C, culture time 20 hr.

[0064] Fermentor Fermentation: The amount of liquid in the 30 m 3 fermenter is 70%. Medium composition: glucose 25 g / L, peptone 15 g / L, yeast powder 10 g / L, potassium dihydrogen phosphate lg / L, magnesium sulfate 0.5 g / L, initial pH 7.0. The cultured seeds are connected to a fermentor for fermentation. Culture conditions: stirring speed 100 r / min, aeration rate 0.6 _{v V} m, temperature 35 ° C, cultured for 40-45 hr.

[0065] In the fermentation broth, the viable cell count was 1.99× ^{10 1 (} cfu/g, the spore number was 1.92× ^{10 1 (} cfu/g, and the spore rate was 96.5<3⁄4.

[0066] The wet bacterial sludge is collected by centrifugation, and a dry bacterial powder is obtained by a spray drying process. Operating conditions are: inlet air temperature 210 °C, outlet air temperature 100 °C. The number of viable bacteria in dry powder is 8.32x10 "cfu/ga [0067]

Example 5: Preparation of dried powder

The fermentation broth of Example 4 was used to obtain a fermentation broth of Bacillus coagulans, and the wet bacterial sludge was collected by centrifugation, and a dry powder was obtained by a spray drying process. Operating conditions are: inlet air temperature 220 °C, outlet air temperature 100 °C. Number of viable bacteria in dry powder 6.54x10 "cfu/ga

[0070]

[0071] It will be apparent to those skilled in the art that the present invention may be modified or altered in light of the above description.

Print (original is in electronic form)

0-1 PCT/RO/134 Table (SAFE) on preserved microorganisms

Or description of other biological materials (PCT Article 笫 13

of two)

0-1-1 Software Version CEPCT

Version 1.01.00 MT/FOP 20140331/0.20.5.21

0-2 International Application No. PCT/CN2016/090895

0-3 File number of the applicant or agent HY0082

1 The following instructions are mentioned here with the instructions in this application.

Preserved microorganisms or other biological materials related:

1-1 Page 0015

1-2 line number: 1-2

1-3 Deposits

1-3-1 Depository Name China National Culture Collection

1-3-2 Depository Address Wuhan University, Wuhan, Hubei Province, China, 430072, Hubei

(CN).

1-3-3 Deposit Date May 03, 2015 (03.05.2015)

1-3-4 Deposit No. CCTCC M2015273

1-4 Supplementary explanation

1-5 This note is for all designated countries in the following designated countries.

1-6 Instructions submitted separately

These instructions will be subsequently submitted to the International Bureau and filled out by the receiving Office.

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Claims

Claim

[Claim 1] A Bacillus coagulant ZS-007 is deposited with the China Center for the Conservation of Traditional Cultures under the accession number CCTCC M 2015273.

[Claim 2] The high-density fermentation method of Bacillus coagulans according to claim 1, wherein the specific steps are as follows:

(1) Strain activation

The Bacillus coagulans strain was inoculated onto the nutrient agar slant medium, and cultured at 25-37 ° C for 2 4-48 hr; then the streak was transferred to the slant of the nutrient agar eggplant bottle, and cultured at 25-37 ° C for 24-48 h. The composition of nutrient agar slant medium and nutrient agar eggplant bottle are: peptone 10 g / L, beef extract 3g / L, sodium chloride 5g / L, agar 15-20g / L, pH 7.2-7.4;

(2) Shake flask culture

Inoculate the strain obtained in step (1) into a shake flask and shake culture for 18-30 hrs; shake flask culture formula: peptone 10 g/L, beef extract 3 g/L, sodium chloride

5g / L, pH 7.2-7.4; 250mL shake flask volume is 20mL, 500mL shake flask volume is 5 OmL; culture conditions: shake flask speed 200r / min, temperature 25-37 ° C;

(3) Seed tank expansion

Seed tank medium composition: peptone 10 g / L, beef extract 3g / L, sodium chloride

5g / L, pH 7.2-7.4; seed tank volume 10-1000L, liquid volume 50-80%; culture conditions: stirring speed 180-250r / min, ventilation 0.5-1.0vvm, temperature 25-37 ° C, Training day 1

8-30hr _;

(4) Fermentation tank fermentation

The seed liquid obtained in step (3) is connected to the fermenter at an inoculation amount of 0.1% to 10%. The fermentation medium consists of: glucose 25 g/L, peptone 15 g/L, yeast powder 10 g/L, dihydrogen phosphate Potassium lg/L, magnesium sulfate 0.5

g/L, initial pH7.0; fermenter volume l-50m ³ , liquid loading 50-80%; culture conditions: stirring speed 100-200r/min, aeration 0.5-0.8vvm, temperature 25-37 ° C, The culture was carried out for 16-48 hrs _{; the} fermentation broth was microscopically examined, and when more than 90% of the cells formed spores, the fermentation broth was obtained. [Claim 3] A method for preparing a dry powder according to the high-density fermentation method of Bacillus coagulans according to claim 2, characterized in that the fermentation liquid obtained in the step (4) is centrifuged, and the wet bacterial sludge is collected, using a spray. The drying process obtains dry powder; the spray drying conditions are: inlet air temperature 210 ° C, air outlet temperature 100 ° C; dry bacteria powder live bacteria number 8.0 x ^{10 11} cfu / g or more.