CN110628654B

CN110628654B - Saccharomyces cerevisiae capable of being used for feed and preparation method and application thereof

Info

Publication number: CN110628654B
Application number: CN201910989530.6A
Authority: CN
Inventors: 汪志明; 陆姝欢; 熊淑婷; 余超; 李翔宇
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: 2021-11-19
Anticipated expiration: 2039-10-17
Also published as: CN110628654A

Abstract

The invention relates to the field of feed preparation, in particular to saccharomyces cerevisiae capable of being used for feed and a preparation method and application thereof. The cell membrane of the saccharomyces cerevisiae is complete, and the cell wall is partially destroyed. The preparation method comprises the following steps: after the fermentation of the saccharomyces cerevisiae is finished, 0.01-1% of enzyme preparation is added for acting for 1-8 hours by taking the total volume of the fermentation liquor as the reference; the enzyme preparation is one or a mixture of beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, alpha-1, 6 glycosidase, cellulase, alkaline protease, chitinase and helicase. The invention provides a 'semi-wall-broken' saccharomyces cerevisiae for feed, and provides a specific enzymolysis wall-breaking method for the saccharomyces cerevisiae, which can effectively prevent the destruction of intracellular effective substances, and simultaneously, effective components of thalli can be released from openings after entering animal bodies, thereby being beneficial to the rapid absorption of animals.

Description

Saccharomyces cerevisiae capable of being used for feed and preparation method and application thereof

Technical Field

The invention relates to the field of feed preparation, in particular to saccharomyces cerevisiae capable of being used for feed and a preparation method and application thereof.

Background

Saccharomyces cerevisiae is a unicellular microorganism, belongs to fungi, has strong acid resistance, and is a facultative anaerobe. Active dry yeast, yeast culture, yeast extract, yeast cell wall and the like are all feed additives allowed to be used in feed additive variety catalog (2008) of Ministry of agriculture in China. The yeast product can effectively improve the balance of the flora in the digestive tract of livestock and poultry, enhance the immunity of organisms and provide rich nutrient substances, thereby achieving multiple effects of preventing and treating digestive tract diseases, promoting growth and the like, has the characteristics of no toxicity, no side effect, no pollution, no drug resistance and the like, is a green feed additive, and has wide research and application in the feed industry.

In the field, a plurality of researches exist for transferring homologous or heterologous genes into saccharomyces cerevisiae so that the saccharomyces cerevisiae can be fermented to generate required components, for example, the carotenoid is generated by recombining a saccharomyces cerevisiae strain, and then the saccharomyces cerevisiae strain is added into feed, so that the immune function of animals can be improved, the animals can be protected to effectively prevent and inhibit diseases, the use amount of antibiotics is replaced or reduced, and a series of problems of food safety and the like caused by the feed safety problem are reduced.

However, in our studies, it was found that when the recombinant Saccharomyces cerevisiae dry bacteria is made into feed without any treatment, the required active ingredients are difficult to release in animals (such as chicken and duck) due to the thick cell wall of the yeast, resulting in low absorption efficiency of the animals. However, when the feed is prepared by breaking the walls by the traditional method, most of the required active ingredients are unstable or easily oxidized, and active substances in the dried thalli after the wall breaking are exposed in the environment, so that the conditions are extensive in the feed preparation process and the storage and transportation process, a large amount of the active substances are oxidized, damaged or deteriorated, the cost loss is caused, and hidden troubles are brought to the feed safety.

Disclosure of Invention

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

In order to achieve the above technical objects, the present invention provides, in a first aspect, a saccharomyces cerevisiae useful for feed, which has intact cell membranes and partially destroyed cell walls.

When the thalli of the saccharomyces cerevisiae is in the state, the oxidation of contained effective components can be effectively prevented, and meanwhile, when the saccharomyces cerevisiae is used as a feed, cell membranes can be dissolved in a digestive environment after the thalli enter an animal body, so that the effective components can be released smoothly, and the rapid absorption of the animal is facilitated.

The invention further provides a preparation method of the saccharomyces cerevisiae for feed, which can be used for preparing the saccharomyces cerevisiae and comprises the following steps: after the fermentation of the saccharomyces cerevisiae is finished, 0.01-1% of enzyme preparation is added for acting for 1-8 hours by taking the total volume of the fermentation liquor as the reference;

preferably, the enzyme preparation is one or a mixture of beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, alpha-1, 6 glycosidase, cellulase, alkaline protease, chitinase and helicase.

The invention discovers that the degree of cell wall dissolution of saccharomyces cerevisiae 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 and even generates a plurality of open pores, but can still retain the complete or most complete cell structure is achieved.

Preferably, the concentration of the saccharomyces cerevisiae in the fermentation liquor is 60 g/L-120 g/L.

Preferably, the desired components are produced by Saccharomyces cerevisiae by transformation with the desired gene segments.

In some embodiments, the desired gene segment is introduced by homologous recombination to produce the desired component in Saccharomyces cerevisiae.

Preferably, the desired ingredient is a carotenoid; further preferred is lycopene or beta-carotene.

Carotenoid, especially lycopene and beta-carotene, can be used as animal feed, and has effects of preventing and treating cancer of livestock and fowl. And the influence of potassium, magnesium, calcium and zinc ions on the carotenoid is small, so that the lycopene does not generate antagonism with the common feed additive. At present, in the field of eggs, consumers and producers prefer the quality of being more red than the color of egg yolk, particularly in the field of duck eggs, most of the conventional coloring agents are chemical synthetic pigments, which are frequently exposed to safety problems, and the carotenoid is used as a natural pigment, so that the color is full and natural, 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 the chemical synthetic pigments can be reduced. Based on the situation, the feed additive has great requirements and applications in feed, but the stability is poor, and factors such as oxygen, light, metal ion pH and the like can influence the feed additive, and when the method disclosed by the invention is used, the stability of the feed additive can be effectively ensured while the animal can quickly absorb the feed additive.

Preferably, the preservation number of the saccharomyces cerevisiae is CGMCC No. 13013.

Preferably, the preservation number of the saccharomyces cerevisiae is CGMCC No. 10753.

Compared with the common saccharomyces cerevisiae, the cell wall of the saccharomyces cerevisiae after homologous recombination is greatly changed, because the carotenoid is a fat-soluble nutrient and is easily enriched on the cell membrane, after the content of the carotenoid is increased in the later fermentation period, the cell membrane and the cell wall are pressed by crystals, and the cell wall is reinforced in the recombination process in order to protect the cell membrane and intracellular carotenoid products, the cell wall breaking difficulty is different compared with the common saccharomyces cerevisiae.

The carotenoid content of the two Saccharomyces cerevisiae is 2-7%.

In order to further enable the saccharomyces cerevisiae to reach the condition that partial cell wall damage even holes occur, but the cell membrane is complete as much as possible, the enzymolysis condition is further optimized, and the following preferred scheme is obtained:

preferably, the enzyme preparation is one or a mixture of beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, alpha-1, 6-glycosidase and cellulase; can degrade components such as cell wall polysaccharide and the like to achieve the effect of breaking cell walls.

Preferably, the enzyme preparation is a mixture of beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase and cellulase;

more preferably, the beta-1, 3-glucanase, the beta-1, 6-glucanase, the beta-1, 4-glucanase and the cellulase are in a mass ratio of 3-5: 0.5-1: 3-5.

More preferably, the beta-1, 3-glucanase, the beta-1, 6-glucanase, the beta-1, 4-glucanase and the cellulase are in a mass ratio of 5:1:0.5: 3.5.

Preferably, when the enzyme preparation is compounded according to the proportion, the adding amount is 0.1-0.6%, and the acting time is 1.5-5 h.

Preferably, the enzymatic hydrolysis is carried out at a pH of 4 to 6.

Preferably, citric acid or hydrochloric acid is used to adjust the pH.

Preferably, the enzymatic hydrolysis is carried out at 40-70 ℃.

Preferably, the above enzymatic reactions are carried out in combination with stirring.

Preferably, the fermentation broth is kept at a pH of 8-10 for 0.5-2h before enzymatic hydrolysis.

This operation may be carried out before the end of the fermentation or after the end of the fermentation. When the operation is matched with the enzymolysis step, the enzymolysis efficiency can be accelerated under the condition of not influencing the final effect.

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 cell wall breaking is dried by spray drying or filtration of the cells followed by vacuum drying or freeze drying.

When the method is used for preparing lycopene, the content of lycopene in dried thallus obtained by drying is generally between 2-7%.

The invention further provides the saccharomyces cerevisiae prepared by the method.

The invention further provides application of the saccharomyces cerevisiae 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 saccharomyces cerevisiae.

The invention further provides a feed containing the saccharomyces cerevisiae.

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

Other components in the feed can be common daily ration feed components such as corn, bean pulp, alfalfa, oat grass and the like, can be distinguished according to different animals, and are not further limited herein.

The invention has the following beneficial effects:

the invention provides a 'semi-wall-broken' saccharomyces cerevisiae for feed, and provides a specific enzymolysis wall-breaking method for saccharomyces cerevisiae, which can control the effect that the cell wall is only partially destroyed, but can still retain the complete or most complete cell structure. Especially, after condition optimization, the saccharomyces cerevisiae is further subjected to 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.

When the effective components in the saccharomyces cerevisiae are carotenoids such as lycopene and beta-carotene, the saccharomyces cerevisiae obtained by the invention is used as a feed additive and is applied to livestock and poultry breeding together with other daily ration components as a feed, so that the anti-inflammatory and antioxidant capacities of the bred livestock and poultry can be improved, the immunity of the bred livestock and poultry can be improved, the yolk degree of the bred livestock and poultry can be deepened aiming at the poultry animals such as laying hens, ducks, geese and the like, and the nutritional and economic values of the bred livestock and poultry can be improved.

Drawings

FIG. 1 shows semi-wall-broken Saccharomyces cerevisiae;

FIG. 2 shows the Saccharomyces cerevisiae after complete wall breaking.

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 and comparative examples, the following embodiments all use Saccharomyces cerevisiae with a preservation number of CGMCC NO.13013, which is a recombinant yeast strain capable of producing lycopene.

The fermentation process of the saccharomyces cerevisiae is as follows:

activating strains: after the strain is streaked on a slant culture medium, the strain is activated and cultured for 24-36h in a constant temperature incubator at 30 ℃.

Seed culture: and transferring the activated and cultured strain into a seed culture medium, culturing at 30 ℃ and at the rotation speed of 250rpm for 12-16 h, and taking the cultured strain as a seed solution.

Fermentation: inoculating the activated seed liquid into a fermentation culture medium, wherein the inoculation amount is 5% (v/v), the culture temperature is 30 ℃, the rotation speed is 250rpm, and the culture is carried out for 120 h.

Wherein, slant culture medium (g/L): glucose 20, yeast extract 10, peptone 20, agar 20, sterilizing at 121 ℃ for 20 min.

Seed medium (g/L): glucose 20, yeast extract 10 and peptone 20, and sterilizing at 121 ℃ for 20 min.

Fermentation medium (g/L): glucose 40, yeast extract 10, peptone 20, sterilizing at 121 deg.C for 20 min.

Example 1

After the fermentation is finished, adjusting the pH value of the fermentation liquor to 6 by using citric acid, adjusting the temperature to 50 ℃, adding 0.3 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and reacting for 2.5 hours under stirring;

the enzyme preparation is beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, and the mass ratio of cellulase is 5:1:0.5: 3.5;

and after the enzymolysis reaction is finished, spray drying is adopted to obtain dry thalli.

Example 2

After fermentation is finished, adjusting the pH value of the fermentation liquor to 6 by using citric acid, adjusting the temperature to 50 ℃, adding 0.3 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and reacting for 3 hours under stirring;

the enzyme preparation is beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, and the mass ratio of cellulase is 3:1:1: 5;

Example 3

After the fermentation is finished, adjusting the pH value of the fermentation liquor to 6 by using citric acid, adjusting the temperature to 50 ℃, adding 0.3 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and acting for 5 hours under stirring;

the enzyme preparation is beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase, and the mass ratio of cellulase is 1:1:3: 6;

Example 4

After the fermentation is finished, adjusting the pH value of the fermentation liquor to 5 by using citric acid, adjusting the temperature to 55 ℃, adding 0.6 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and reacting for 1.5 hours under stirring;

Example 5

After fermentation is finished, adjusting the pH value of the fermentation liquor to 5 by using citric acid, adjusting the temperature to 60 ℃, adding 0.01 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and reacting for 8 hours under stirring;

Example 6

After fermentation is finished, firstly regulating the pH value of the fermentation liquor to be 8, stirring for 1 hour, then regulating the pH value of the fermentation liquor to be 6 by using citric acid, regulating the temperature to be 50 ℃, adding 0.1 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and acting for 1.5 hours under stirring;

Example 7

After fermentation is finished, adjusting the pH value of fermentation liquor to 5 by using citric acid, adjusting the temperature to 60 ℃, adding 1 percent of enzyme preparation by taking the total volume of the fermentation liquor as a reference, and acting for 1 hour under stirring;

Comparative example 1

This comparative example differs from example 1 in that: and directly spray-drying the fermented saccharomyces cerevisiae liquid to obtain complete dry thalli.

Comparative example 2

This comparative example differs from example 1 in that: the saccharomyces cerevisiae obtained by fermentation adopts a mechanical wall breaking method to ensure that the grain diameter is less than 100 mu m so as to thoroughly destroy the cell wall of the saccharomyces cerevisiae.

Test example 1

In this test example, the yeast cells obtained in example 1 and comparative example 2 were observed under a microscope to obtain micrographs of half-wall-broken cells (FIG. 1) and completely-wall-broken cells (FIG. 2). As can be seen from fig. 1-2, after the partial wall-breaking treatment in example 1, lycopene contained in saccharomyces cerevisiae still remains in the cell and is red (dark after being converted into black and white figures), while lycopene in the completely wall-broken saccharomyces cerevisiae mostly flows out of the cell and is exposed, and most of lycopene in the cell is white.

Test example 2 stability of lycopene in bacterial cell

In this test example, the stability test was carried out on the yeast cells obtained in examples 1 to 7 and comparative examples 1 to 2 by the following specific procedures: sealing with aluminum foil bag, standing at 37 deg.C for 2 months, and measuring the change of lycopene content before and after sample. The detection method of lycopene in the thallus comprises the following steps:

firstly, accurately weighing 20-25mg of dry thallus sample into a centrifuge tube.

② adding tetrahydrofuran into the centrifuge tube.

Thirdly, setting the frequency of the rapid grinding instrument at 70Hz for 90s, and grinding for 3 times (paying attention to the fact that the arrangement positions of centrifugal tubes are symmetrically arranged, and a knob on the grinding instrument is screwed and covered well).

Fourthly, 5mL of supernatant is sucked and transferred to a 25mL volumetric flask.

Fifthly, adding 3mL of tetrahydrofuran into the centrifuge tube, and repeating the step (c) until no obvious red color exists in the zirconia beads. The extracted solution was transferred to a volumetric flask (cells that were not disrupted after grinding settled rapidly and extraction could be continued without centrifugation).

Sixthly, sucking 1mL of solution, adding the solution into a new 25mL volumetric flask, fixing the volume of tetrahydrofuran to a scale, detecting the absorbance on a machine, and adjusting the dilution times to ensure that the absorbance is between 0.2 and 1.2.

Setting the absorbance: 472.

the standard curve is that y is 2.1211x +0.0153, wherein x is the value of ultraviolet detection absorbance, and y is the concentration of lycopene in the sample in the cuvette and is measured in mg/L.

And (4) calculating a result:

the total lycopene content is in ω and the values are in% calculated according to the following formula, the calculation results are shown in table 1:

in the formula:

m is sample mass in mg;

c is the concentration of lycopene in the sample in the cuvette, unit mg/L;

a is sample dilution factor.

TABLE 1 lycopene content variation

As can be seen from Table 1, the lycopene content of comparative example 2 after complete wall-breaking changed the most, while the oxidation rate of lycopene in examples 1-7 was within 5% after the semi-wall-breaking treatment by the method of the present invention, which was significantly lower than that of comparative example 2. When the treatment condition is controlled in a better range, the oxidation rate can reach the same level as that of the dry thallus without wall breaking.

Test example 3 animal absorption test

In the experimental example, the saccharomyces cerevisiae contains lycopene, and the lycopene is absorbed and converted in the animal body by taking a laying hen as an example, and the most intuitive way is to express the coloring degree by measuring the color of the yolk. When the absorption conversion rate is low, the color value of the egg yolk is correspondingly reduced; when the absorption conversion is high, the degree of coloration of the egg yolk increases, and the corresponding color value increases. The test example was carried out as follows:

(1) grouping of laying hens

Selecting 230 days old, good body condition and similar weight Jingfen No.1 commercial laying hens, randomly dividing into 10 groups, each group having 3 repetitions, each repetition having 9 chickens. The control group was fed with a basal diet (corn-soybean meal type), and the other 9 groups were the treatment groups of examples 1 to 7 and comparative examples 1 to 2, respectively, and were fed with dry bacteria added in a ratio of 20mg/kg of lycopene in the diet.

(2) Egg duck grouping

Commercial egg ducks of 200 days old, good in body condition and similar in weight are selected and randomly divided into 7 groups, each group is divided into 3 repetitions, and each repetition comprises 15 ducks. The control group was fed with a basal diet (corn-soybean meal type), and the other 6 groups were the treatment groups of example 1/5/6/7 and comparative examples 1-2, respectively, and were fed with dry bacteria added at a ratio of 30mg/kg of lycopene in the diet.

(3) Feeding management

Three layers of the plants are cultivated in cages, and the illumination is 16L: 8D. Free drinking water and free intake. The ambient temperature is 20-27 ℃.

The pre-test period is 2 weeks, the eggs and duck eggs laid by each group are collected within 3 days after the pre-test period, the color of the yolk is measured by an egg quality index analyzer (Orka, Israel), and the average value is taken.

The egg assay results are shown in table 2 below.

TABLE 2

The duck egg assay results are shown in table 3 below.

TABLE 3

As is clear from the data in tables 2 and 3, the dry microbial cells prepared by the method of the present invention had a difference in the degree of coloration of 1 or more from those of comparative example 1 and the control, and a difference in coloration was recognized by the naked eye. The absorption degree of the half wall-broken dry thalli in the egg-laying layer and the egg-laying duck body is nearly equivalent to that of the completely wall-broken dry thalli in the color aspect under the better condition.

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 many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A preparation method of saccharomyces cerevisiae capable of being used for feed is characterized by comprising the following steps:

the saccharomyces cerevisiae has a preservation number of CGMCC No.13013 and/or CGMCC No. 10753;

after the fermentation of the saccharomyces cerevisiae is finished, 0.1-1% of enzyme preparation is added by taking the total volume of the fermentation liquor as the reference, and the fermentation is carried out for 1.5-5h under the conditions that the pH is 4-6 and the temperature is 40-70 ℃;

before enzymolysis, the fermentation liquor is kept for 0.5 to 2 hours under the condition that the pH value is 8 to 10;

the enzyme preparation is a mixture of beta-1, 3-glucanase, beta-1, 6-glucanase, beta-1, 4-glucanase and cellulase in a mass ratio of 3-5: 0.5-1: 3-5;

the concentration of the saccharomyces cerevisiae in the fermentation liquor is 60 g/L-120 g/L.

2. Saccharomyces cerevisiae produced by the method of claim 1.

3. Use of the saccharomyces cerevisiae yeast of claim 2 in animal farming.

4. Use of the saccharomyces cerevisiae according to claim 2 for the preparation of animal medicine.

5. Use of the saccharomyces cerevisiae yeast of claim 2 in feed.

6. A feed additive characterized by comprising the Saccharomyces cerevisiae according to claim 2.

7. A feed comprising the Saccharomyces cerevisiae according to claim 2.

8. The feed of claim 7, wherein the saccharomyces cerevisiae is added in an amount of 60-1000 mg/kg of ration.