阅读说明：本技术 一种可用于饲料的三孢布拉霉菌及其制备方法与应用 (Blakeslea trispora for feed and preparation method and application thereof ) 是由 李翔宇 余超 熊淑婷 陆姝欢 汪志明 于 2019-10-17 设计创作，主要内容包括：本发明涉及饲料制备领域,具体涉及一种可用于饲料的三孢布拉霉菌及其制备方法与应用。所述三孢布拉霉菌的细胞膜完整,细胞壁部分破坏。制备方法包括：在发酵结束后,按30-150mg/每克生物质在发酵液中加入酶制剂,作用2-5h；所述酶制剂为选自蛋白酶、β-葡聚糖酶、蜗牛酶、纤维素酶、溶菌酶、几丁质酶一种或其混合物。本发明提供了一种可用于饲料的“半破壁”的三孢布拉霉菌,并针对三孢布拉霉菌提供了一种特定的酶解破壁的方法,可以有效防止胞内有效物质的破坏,同时菌体进入动物体内后有效成分能够顺利释出,有利于动物的快速吸收。(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 proportion of 30-150 mg/g biomass for acting for 2-5 h; the enzyme preparation is selected from 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.)

1. Blakeslea trispora for use in feed, characterized in that the cell membrane of Blakeslea trispora is intact and the cell wall is partially disrupted.

2. 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 proportion of 30-150 mg/g biomass for acting for 2-5 h.

3. The method according to claim 2, wherein the enzyme preparation is one or a mixture of protease, beta-glucanase, helicase, cellulase, lysozyme and chitinase.

4. The method according to claim 3, wherein 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.

5. The preparation method according to claim 4, wherein the enzyme preparation is added in an amount of 75-130 mg/g biomass for 3-4.5 h.

6. The preparation method according to any one of claims 2 to 5, wherein the enzymolysis temperature is 25 to 40 ℃; preferably 28-35 deg.C.

7. Blakeslea trispora produced by the process according to any one of claims 2 to 6.

8. Use of Blakeslea trispora according to claim 1 or 7 in animal farming, preferably in animal medicine or feed.

9. A feed additive comprising the Blakeslea trispora according to claim 1 or 7.

10. A feed comprising the Blakeslea trispora according to claim 1 or 7; preferably, the addition amount of the Blakeslea trispora is 60-1000 mg/kg of daily ration.

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 Blakeslea 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 be carried 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 guaranteed, the cost is improved in an intangible way, and the feed is not cost-effective for 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 animal body, so that the rapid absorption of the animal is facilitated.

The invention further provides a preparation method of Blakeslea trispora for feeds, which can be used for preparing the Blakeslea trispora, and the method 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 2-5 h.

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 degree of dissolution of the cell wall of Blakeslea trispora 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 the complete or most of the complete cell structure can still be kept 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 30-60 g/L.

The protease of the present invention includes alkaline protease, acidic protease and neutral protease, and preferably the protease of 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 3-4.5 h. Preferably, stirring operation is assisted in the enzymolysis process.

Further preferably, the mass ratio of the neutral protease to the cellulase to the lysozyme to the chitinase is 15:6:0.5: 0.2.

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 invention can only produce beta-carotene or lycopene, and can also make the thallus contain the beta-carotene and lycopene by adjusting a blocking agent.

The total content of carotenoid in the dry thalli of the 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 the animals include pig, cattle, sheep, etc., and the animals include 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 subjected to the condition that only holes appear on the wall, but the cell membrane is complete as much 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 2014379 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 is as follows:

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

Seed culture: respectively taking a shovel of positive bacteria and a shovel of negative bacteria from PDA slant culture media of positive strains and negative strains of the 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 positive strain seed liquid and negative strain seed liquid of the Blakeslea trispora.

Fermentation: the positive strain seed liquid of the Blakeslea trispora and the negative strain seed liquid of the Blakeslea trispora are uniformly mixed according to the mass ratio of 1:1 of the positive strain to the negative strain to obtain a seed liquid mixed liquid, the seed liquid mixed liquid is inoculated into a 250ml triangular flask filled with 40ml of fermentation liquid according to the inoculation amount of 10 percent (volume ratio), and the culture is carried out at the temperature of 25 ℃ and the speed of 180 rpm.

Wherein, 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 cubes, adding deionized water, boiling for 30 min, cooling, filtering with four layers of gauze, and adding glucose and agar powder into the filtered clear liquid.

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 pH 7.0.

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 pH 7.0.

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.