CN110637921B

CN110637921B - Blakeslea trispora for feed and preparation method and application thereof

Info

Publication number: CN110637921B
Application number: CN201910988763.4A
Authority: CN
Inventors: 李翔宇; 余超; 熊淑婷; 陆姝欢; 汪志明
Original assignee: Cabio Biotech Wuhan Co Ltd
Current assignee: Cabio Biotech Wuhan Co Ltd
Priority date: 2019-10-17
Filing date: 2019-10-17
Publication date: 2022-12-27
Anticipated expiration: 2039-10-17
Also published as: CN110637921A

Abstract

The invention relates to the field of feed preparation, and in particular relates to Blakeslea trispora for feed and a preparation method and application thereof. The cell membrane of the Blakeslea trispora is intact, and the cell wall is partially destroyed. The preparation method comprises the following steps: after fermentation is finished, adding an enzyme preparation into the fermentation liquor according to the ratio of 30-150 mg/g biomass for acting for 2-5h; the enzyme preparation is one or a mixture of protease, beta-glucanase, helicase, cellulase, lysozyme and chitinase. The invention provides the Blakeslea trispora which can be used for the 'semi-wall breaking' of the feed, and provides a specific enzymolysis wall breaking method for the Blakeslea trispora, so that the damage of intracellular effective substances can be effectively prevented, and meanwhile, effective components can be smoothly released after thalli enter an animal body, thereby being beneficial to the rapid absorption of the animal.

Description

Blakeslea trispora for feed and preparation method and application thereof

Technical Field

The invention relates to the field of feed preparation, and in particular relates to Blakeslea trispora for feed and a preparation method and application thereof.

Background

The carotenoid is used as animal feed, has the effects of preventing and treating and inhibiting cancers for livestock and poultry, can improve the immune function of animals, protects the animals, effectively prevents and inhibits the occurrence of diseases, can replace or reduce the use amount of antibiotics, and reduces a series of problems of food safety and the like caused by the safety problem of the feed. The carotenoid is full and natural in color, and the carotenoid added into the feed has a certain application space for improving the color of meat, so that the harm caused by using a chemically synthesized pigment can be reduced. And potassium, magnesium, calcium and zinc ions have little influence on the carotenoid, so the carotenoid does not generate antagonism with the common feed additive.

Blakeslea trispora can produce a plurality of carotenoids, especially beta-carotene and lycopene in the carotenoids have the characteristics of high oxidation resistance and in-vivo free radical scavenging, so the brassarella trispora has been widely used as a functional raw material in foods and health care products.

The Blakeslea trispora is filamentous thallus, has complex cell wall components, is difficult to release in an animal body, and causes low absorption efficiency of the animal. If the carotenoid in the mould is directly extracted to prepare the feed, or the mould is completely broken to prepare the feed, the oxidation is very easy to occur in the preparation, storage and transportation processes, nitrogen filling and vacuum pumping are needed to carry out on the product in order to protect the carotenoid from being oxidized, even if the oxidation reaction in the preparation and use processes cannot be ensured, the cost is improved virtually, and the cost is not cost-effective for the feed application.

Disclosure of Invention

In order to solve the technical problems, the invention provides the Blakeslea trispora which can improve the stability of required components and is beneficial to the absorption of animals, and the preparation method and the application thereof.

In order to achieve the technical purpose, the technical scheme of the invention is as follows:

the invention firstly provides the Blakeslea trispora which can be used for feeds, wherein the cell membrane of the Blakeslea trispora is complete, and the cell wall is partially damaged.

When the thallus of the Blakeslea trispora is in the state, the carotenoid or other effective substances contained in the thallus can be effectively prevented from being oxidized, and meanwhile, when the thallus is used as a feed, the carotenoid or other effective substances can be released from the open pores after the thallus enters the body of an animal, so that the rapid absorption of the animal is facilitated.

The invention further provides a preparation method of Blakeslea trispora for feed, which can be used for preparing the Blakeslea trispora and comprises the following steps:

after the fermentation of the Blakeslea trispora is finished, adding an enzyme preparation into the fermentation liquor according to the proportion of 30-150 mg/g biomass for acting for 2-5h.

Preferably, the enzyme preparation is selected from one or a mixture of protease, beta-glucanase, helicase, cellulase, lysozyme and chitinase.

The invention discovers that the dissolution degree of the Blakeslea trispora cell wall can be controlled by selecting the specific enzyme and further controlling the addition amount (equivalent to controlling the concentration of the enzyme during reaction) and the treatment time of the enzyme by adopting an enzymolysis wall breaking method, so that the effect that the cell wall is only partially destroyed to generate a plurality of open pores, but can still retain the complete or most complete cell structure is achieved.

In order to further ensure that the blakeslea trispora only has partial damage to cell walls but the cell membranes are complete as much as possible, the invention further optimizes the enzymolysis conditions to obtain the following preferred scheme:

preferably, the concentration of Blakeslea trispora in the fermentation broth is 30-60g/L.

The protease to be mentioned in the present invention includes alkaline protease, acidic protease and neutral protease, and preferably the protease in the present invention is neutral protease.

Preferably, the enzyme preparation is a mixture of neutral protease, cellulase, lysozyme and chitinase;

preferably, the weight ratio of the neutral protease, the cellulase, the lysozyme and the chitinase is (10-20): 5-10): 0.4-0.8): 0.1-0.5.

Preferably, when the enzyme preparation is compounded according to the proportion, the addition amount is 75-130 mg/g biomass, and the action time is 3-4.5h. Preferably, stirring operation is assisted in the enzymolysis process.

Further preferably, the mass ratio of the neutral protease, the cellulase, the lysozyme and the chitinase is 15.

Preferably, the enzymatic hydrolysis is carried out at 25-40 ℃; more preferably from 28 to 35 deg.c.

In most of the schemes in the field, the fermentation liquor needs to be dried and then used for preparing feed, and when the drying is applied to the technical scheme of the invention, good effect can be obtained according to the drying method generally used in the field.

Preferably, the fermentation broth after the wall breaking is dried by spray drying or vacuum drying after filtering the cells.

The Blakeslea trispora mentioned in the present invention can produce only beta-carotene or lycopene, or can adjust the blocking agent to make the thallus contain both beta-carotene and lycopene.

The total content of carotenoid in the dry thalli of Blakeslea trispora prepared by the method is 3-10%.

The invention further provides the Blakeslea trispora prepared by the method.

The invention further provides application of the Blakeslea trispora in animal breeding, preferably application in animal medicines or feeds.

The animals include livestock and fowl, and include pig, cattle, sheep, chicken, duck, goose, etc.

The invention further provides a feed additive containing the Blakeslea trispora.

The invention further provides a feed containing the Blakeslea trispora.

Preferably, the addition amount of the Blakeslea trispora is 60-1000 mg/kg of daily ration.

The other ingredients in the feed may be common daily ration feed ingredients such as corn, soybean meal, alfalfa, oat grass and the like, and are not further limited herein.

The invention has the following beneficial effects:

the invention provides a 'semi-wall-broken' Blakeslea trispora which can be used for feeds, and provides a specific enzymatic wall-breaking method for Blakeslea trispora, which can control the effect that the cell wall of the Blakeslea trispora is only partially broken to generate a plurality of holes, but can still retain the complete or most complete cell structure. Especially after condition optimization, the Blakeslea trispora is further allowed to reach the condition that only the wall is provided with holes but the cell membrane is as complete as possible. The preparation method can effectively prevent the effective substances in the cells from being damaged, and simultaneously the effective components of the semi-wall-broken thalli are easy to release after entering the animal body for digestion, thereby being beneficial to the quick absorption of the animal.

The Blakeslea trispora obtained by the invention can be used as a feed additive and used as a feed together with other daily ration components in livestock and poultry breeding industry, can improve the anti-inflammatory and antioxidant capacities of the bred livestock and poultry, and simultaneously improve the immunity of the bred livestock and poultry, wherein the Blakeslea trispora can deepen the yolk color of the bred livestock and poultry, such as laying hens, and improve the nutritional and economic values of the bred livestock and poultry.

Detailed Description

The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

In order to compare the effects of the different examples with those of the comparative examples, blakeslea trispora (Blakeslea trispora) BT7251 (+) with the collection number CCTCC M2014378 and Blakeslea trispora (Blakeslea trispora) BT7603 (-) with the collection number CCTCC M2014379 were used in the following embodiments.

Since the main objective of the present invention is to examine the effect of the treatment method of B.trispora on carotenoids in the treatment method and the transformation ability in animals, B.trispora in the following examples is not added with a blocking agent during fermentation, and only the application of beta-carotene is examined. It is to be noted, however, that the B.trispora product is not limited to beta-carotene in practical applications.

The fermentation process comprises the following steps:

slant culture: spore suspensions of the positive and negative Blakeslea trispora are respectively coated on a PDA slant culture medium and cultured in a constant temperature incubator at 25 ℃ for 5-7 days.

Seed culture: respectively shoveling a shovel of positive bacteria and a shovel of negative bacteria from PDA slant culture media of positive and negative strains of Blakeslea trispora by using an inoculating shovel, respectively inoculating the positive bacteria and the negative bacteria into 1000ml triangular flasks containing 150ml of seed culture media, and culturing for 48 hours at 25 ℃ under the condition of 180 revolutions per minute to obtain Blakeslea trispora positive strain seed liquid and Blakeslea trispora negative strain seed liquid.

Fermentation: uniformly mixing the positive strain seed liquid and the negative strain seed liquid of the Blakeslea trispora according to the mass ratio of 1 to 1 of the positive strain to the negative strain to obtain a seed liquid mixed liquid, inoculating the seed liquid mixed liquid into a 250ml triangular flask filled with 40ml of fermentation liquid by 10 percent (volume ratio), and culturing at 25 ℃ and 180 rpm.

Wherein, the slant culture medium (g/L): 20g/L of glucose, 25g/L of agar powder and 200g/L of peeled potatoes; the preparation method can be referred to as follows: cutting potato into 1cm blocks, adding deionized water, boiling for 30 min, cooling, filtering with four layers of gauze, collecting the filtrate, and adding glucose and agar powder.

Seed medium (g/L): 10g/L of glucose, 30g/L of corn starch, 50g/L of corn steep liquor dry powder, 1g/L of monopotassium phosphate, 0.1g/L of magnesium sulfate and pH7.0.

Fermentation medium (g/L): 20g/L of glucose, 40g/L of corn starch, 25g/L of yeast extract, 40g/L of soybean cake powder, 1g/L of monopotassium phosphate, 0.1g/L of magnesium sulfate and pH7.0.

And (3) adding nicotine with the concentration of 1mol/L into the fermentation solution after fermenting for 40 hours to ensure that the final concentration of the fermentation solution is between 0.05 and 0.2 percent.

Example 1

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to 108.5 mg/g biomass, and acting for 4 hours;

the enzyme preparation comprises neutral protease, cellulase, lysozyme and chitinase in a weight ratio of 15.5.

And after the enzymolysis wall breaking is finished, spray drying is adopted to obtain dry thalli.

Example 2

After fermentation is finished, adjusting the temperature to 40 ℃, adding an enzyme preparation into the fermentation liquor according to 75 mg/g biomass, and acting for 3 hours;

Example 3

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to 127.5 mg/g biomass, and acting for 4 hours;

the enzyme preparation comprises neutral protease, cellulase, lysozyme and chitinase in a weight ratio of 20.4.

Example 4

After the fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to the biomass per gram of 106.5mg, and acting for 4 hours;

the enzyme preparation comprises neutral protease, cellulase, lysozyme and chitinase in a weight ratio of 10.

Example 5

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to 108.5 mg/g biomass, and acting for 2 hours;

Example 6

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to 108.5 mg/g biomass, and acting for 5 hours;

Example 7

After the fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to the proportion of 118 mg/g biomass, and acting for 4.5 hours;

the enzyme preparation comprises neutral protease, cellulase, lysozyme and chitinase in a weight ratio of 18.3.

Example 8

the enzyme preparation comprises beta-glucanase, cellulase, lysozyme and chitinase in a weight ratio of 6.5.

Example 9

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to the proportion of 30 mg/g biomass, and acting for 5 hours;

Example 10

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to the proportion of 150 mg/g biomass, and acting for 2 hours;

Comparative example 1

After fermentation is finished, adjusting the temperature to 40 ℃, adding an enzyme preparation into the fermentation liquor according to the proportion of 10 mg/g biomass, acting for 6 hours, and starting stirring for 2-5 minutes every 1 hour;

Comparative example 2

After fermentation is finished, adjusting the temperature to 35 ℃, adding an enzyme preparation into the fermentation liquor according to 200 mg/g biomass, and acting for 1.5h;

Comparative example 3

This comparative example differs from example 1 in that: and directly spraying and drying the fermentation liquor after the Blakeslea trispora is fermented to obtain dry thalli.

Comparative example 4

The comparative example differs from example 1 in that: and after the Blakeslea trispora is fermented, spray drying the fermentation liquor, and breaking the walls by a dry method, wherein the particle size of the broken fermentation liquor is less than 100 mu m.

Test example 1 stability of Blakeslea trispora in cells

In this test example, the stability of beta-carotene contained in the dried cells of Blakeslea trispora obtained in examples 1 to 8 and comparative examples 1 to 4 was compared.

The stability investigation method specifically comprises the following steps: and (3) putting the dry thalli into an aluminum foil bag, sealing the aluminum foil bag at 37 ℃, and detecting the content after 6 months.

The detection method of beta-carotene in dry thallus comprises the following steps:

accurately weighing a sample, repeatedly extracting with trichloromethane after wall breaking until the sample is completely colorless, and metering volume with cyclohexane.

The absorbance was measured at 455nm using an ultraviolet spectrophotometer.

The calculation formula of the content of the beta-carotene is as follows, and the calculation results are shown in a table 1:

wherein: a-absorbance value of the test sample;

n-dilution multiple;

m-weighing the sample weight;

the percent absorption coefficient of 2500-beta-carotene in cyclohexane;

TABLE 1 variation of beta-carotene content

Group of	Initial content	Content of 3 months	Rate of change
				Example 1	5.53	5.08	8.2％
Example 2	5.47	5.02	8.3％
				Example 3	6.25	5.72	8.5％
Example 4	5.84	5.33	8.8％
				Example 5	4.95	4.53	8.5％
Example 6	5.32	4.76	10.6％
				Example 7	6.17	5.23	14.8％
Example 8	5.20	4.77	8.2％
				Example 9	5.33	4.89	8.2％
Example 10	5.45	4.78	12.3％
				Comparative example 1	5.50	4.99	9.3％
Comparative example 2	4.82	3.94	18.2％
				Comparative example 3	5.24	4.82	8.0％
Comparative example 4	5.72	4.28	25.1％

As can be seen from table 1, after 6 months, the content of β -carotene in comparative example 4 after complete wall breaking changed the most, while the oxidation rate of β -carotene in examples 1 to 10 was within 15% after half wall breaking treatment, which was significantly lower than that of comparative example 4. When the treatment conditions are controlled in a better range, the oxidation rate of the bacterial strain is equivalent to that of the dry bacterial strain without wall breaking.

Test example 2 experiment for promoting absorption in animals

(1) Grouping animals

Selecting 230-day-old Jingfen No. 1 commercial laying hens with good body conditions and similar body weights, randomly dividing the commercial laying hens into 15 groups, and repeating the groups for 3 times, wherein each group comprises 7 chickens. Wherein, the control group was fed with a basal diet (corn-soybean meal type), and the other 14 groups were the treatment groups of examples 1 to 10 and comparative examples 1 to 4, respectively, and the feed was fed with dry cells added in a ratio of 20mg/kg of β -carotene in the diet.

(2) Feeding management

Three-layer cage culture, illumination is 1698D. Water drinking and food intake are free. The ambient temperature is 20-27 ℃.

The pretest period is 2 weeks, the eggs laid by each group are collected every day within 3 days after the pretest period, the egg yolk color is measured by an egg quality index analyzer (Orka), and the average value of the results is calculated. The results of the measurements are given in Table 2 below.

TABLE 2

Group of	Yolk color
		Example 1	10.73
Example 2	10.75
		Example 3	10.02
Example 4	10.23
		Example 5	8.89
Example 6	10.90
		Example 7	10.62
Example 8	8.84
		Example 9	9.30
Example 10	10.31
		Comparative example 1	8.46
Comparative example 2	10.83
		Comparative example 3	8.14
Comparative example 4	11.32
		Control group	7.56

As can be seen from the data in Table 2, the difference between the coloration degree of Blakeslea trispora prepared by the present invention and the control group is 1 or more, and the color has changed significantly. When the dry thalli with complete wall breaking is directly used for feeding laying hens, the coloring rate of the dry thalli can reach 11.32, and the coloring rate of the dry thalli of the Blakeslea trispora treated by adopting the optimal partial wall breaking method can reach the level equivalent to the coloring rate of the dry thalli of the Blakeslea trispora.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that modifications and improvements can be made thereto without departing from the scope of the invention. Accordingly, it is intended that all such modifications and alterations be included within the scope of this invention as defined in the appended claims.

Claims

1. A preparation method of Blakeslea trispora for feed is characterized by comprising the following steps: after the fermentation of the Blakeslea trispora is finished, adding an enzyme preparation into the fermentation liquor according to the biomass of 75-130 mg/g for acting for 3-4.5h;

the enzyme preparation is a mixture of neutral protease, cellulase, lysozyme and chitinase in a weight ratio of (10-20): (5-10): (0.4-0.8): (0.1-0.5).

2. The method of claim 1, wherein the enzymatic hydrolysis temperature is 25-40 ℃.

3. The method according to claim 1, wherein the enzymolysis temperature is 28 to 35 ℃.

4. Blakeslea trispora produced by the production method according to any one of claims 1 to 3.

5. Use of Blakeslea trispora according to claim 4 for the preparation of an animal medicament.

6. Use of Blakeslea trispora according to claim 4 for the preparation of feed.

7. A feed additive characterized by containing the Blakeslea trispora of claim 4.

8. A feed comprising the Blakeslea trispora of claim 4.

9. The feed according to claim 8, wherein the Blakeslea trispora is added in an amount of 60 to 1000mg/kg of ration.