阅读说明：本技术 含有解脂亚罗酵母的饲料、其制备方法及其应用 (Feed containing yarrowia lipolytica, preparation method and application thereof ) 是由 胡鹏 肖浩 石文静 于 2019-03-13 设计创作，主要内容包括：本发明涉及含有解脂亚罗酵母的饲料、其制备方法及其应用。本发明首次揭示一种新型的可应用于动物饲料的发酵产物,其主要通过解脂耶罗酵母在适当条件下发酵后获得,其具有良好的营养组成。本发明还揭示了所述发酵产物的制备方法及应用。(The invention relates to a feed containing yarrowia lipolytica, a preparation method and application thereof. The present invention discloses for the first time a novel fermentation product applicable to animal feed, which is obtained mainly by fermentation of yarrowia lipolytica under appropriate conditions, which has a good nutritional composition. The invention also discloses a preparation method and application of the fermentation product.)

1. A method of making a yarrowia lipolytica fermentation product, comprising:

(1) fermenting and culturing yarrowia lipolytica, and adding a carbon source substrate into a fermentation system in the fermentation process; wherein, the carbon source substrate in the step (a) is a grease substrate, and the total amount of fatty acid is controlled to be 0.05-2% (w/V); or, (b) the carbon source substrate is a substrate for generating acetate, and the concentration of acetate ions is controlled to be 0.4-2 g/L;

(2) after fermentation, the fermentation precipitate is collected and dried to obtain the yarrowia lipolytica fermentation product.

2. The method of claim 1, wherein in (a), the total amount of fatty acids is controlled to be 0.08% to 1%; preferably 0.1% -0.8%; or (b), controlling the concentration of acetate at 1 +/-0.5 g/L; preferably 1 + -0.3 g/L.

3. The method of claim 1, wherein the initial medium comprises, in the fermentative culture of yarrowia lipolytica: yeast extract, magnesium sulfate, ammonium sulfate, potassium dihydrogen phosphate and/or dipotassium hydrogen phosphate; preferably, the fertilizer also comprises trace elements; and/or

The substrate for generating acetate is sodium acetate, ammonium acetate and acetic acid.

4. The method of claim 1, wherein the yarrowia lipolytica fermentation product comprises a product selected from the group consisting of: wet yeast obtained by partially dehydrating yarrowia lipolytica fermentation liquor, yeast powder obtained by drying and dehydrating, or yeast meal obtained by extracting oil.

5. The method of claim 1, wherein in (a), the pH is maintained at 7.5 ± 0.3; preferably, 7.5 ± 0.2 or 7.5 ± 0.1; or in (b), the pH is maintained at 6.5. + -. 0.3; preferably 6.5 + -0.2 or 6.5 + -0.1.

6. A yarrowia lipolytica fermentation product produced by the method of any one of claims 1-5; preferably, the yarrowia lipolytica fermentation product comprises a product selected from the group consisting of: wet yeast obtained by partially dehydrating yarrowia lipolytica fermentation liquor, yeast powder obtained by drying and dehydrating, or yeast meal obtained by extracting oil.

7. Use of the yarrowia lipolytica fermentation product of claim 6 for the preparation of an animal feed or feed additive.

8. An animal feed or feed additive comprising the yarrowia lipolytica fermentation product of claim 6, and a dietetically or feedetically acceptable mix; preferably, the dietetic or feedlot acceptable mix comprises a material selected from the group consisting of: corn flour, pig feed premix, corn, bran and soybean meal.

9. A method of preparing an animal feed, the method comprising:

(i) mixing corn flour and feed premix;

(ii) mixing a yarrowia lipolytica fermentation product produced by the method of any one of claims 1-5 with the product mixture of (i);

(iii) (iii) mixing the mixed product of (ii) with a roughage.

10. The method according to claim 9, wherein in (i), the corn meal and the feed premix are mixed according to the weight ratio of (1-10): 1, preferably according to (2-8): 1, more preferably according to (3-7): 1 or (4-6): 1; or

(ii) In (i), the yarrowia lipolytica fermentation product and the mixed product of (i) are mixed in a weight ratio of (1-5) to (1-5), preferably (1-3) to (1-3), more preferably (1-3) to (1-3) or (1-2) to (1-2); or

(iii) In (iii), the mixed product of (ii) is mixed with the roughage in a weight ratio of (2-15): 1, preferably (4-10): 1, more preferably (5-8): 1 or (6-7): 1.

Technical Field

The invention belongs to the field of animal feed, and particularly relates to feed containing yarrowia lipolytica, a preparation method and application thereof.

Background

Animal feed is indispensable in animal husbandry production, and animal feed with good safety is one of the factors for promoting the benign development of animal husbandry production. Animal feed typically includes protein feed, energy feed, roughage, greenfeed, silage, mineral feed, and feed additives.

Animal feeds can be classified into: complete feed, concentrated feed and premix. The complete feed is a mixed batch consisting of four parts, namely protein feed, energy feed, coarse feed and an additive, is a granular feed which is mainly granulated by machine processing and sold on the market, and is an expanded granular feed, and can be directly used for feeding objects. The concentrated feed is prepared by premixing a protein raw material and an additive, and energy needs to be supplemented when feeding, so that the concentrated feed has the advantage of convenient use, is suitable for large-scale farmers, particularly farmers who own energy feeds such as corn and the like, and has at least 50 concentrated feeds in the market at present. The premix is an important component of a complete formula feed, and is an intermediate type formula feed product prepared by uniformly mixing one or more trace components (including various trace mineral elements, various vitamins, synthetic amino acids, certain medicaments and other additives) with a diluent or a mixture according to a required proportion.

With the improvement of living standard of people, the meat quality of some farm animals (such as pigs and the like) is required to be higher and higher, and the demand in the aspect can be solved by improving the formula of the feed, so that the unbalanced and reasonable nutrition supply is improved for the animals.

There are currently some animal feeds in the art that are formulated with yeast. However, yeasts commonly used in feeds in the market at present are mainly saccharomyces cerevisiae, pichia pastoris, candida utilis, phaffia rhodozyma and the like, and thalli of the yeasts can contain abundant proteins and essential amino acids, but the contents of fat and essential fatty acid are obviously insufficient, so that fish meal, grease and the like are further added when the yeasts are used as the feeds to ensure the nutritional requirements of feeding animals, the addition increases the feeding cost and limits the large-scale application of the yeasts.

There is therefore a need in the art to develop improved feeds, to provide more nutritionally complete feeds,

disclosure of Invention

The invention aims to provide a feed containing yarrowia lipolytica, a preparation method and application thereof.

In a first aspect of the invention, there is provided a method of making a yarrowia lipolytica fermentation product, the method comprising:

(1) fermenting and culturing yarrowia lipolytica, and adding a carbon source substrate into a fermentation system in the fermentation process; wherein, the carbon source substrate in the step (a) is a grease substrate, and the total amount of fatty acid is controlled to be 0.05-2% (w/V); or, (b) the carbon source substrate is a substrate for generating acetate, and the concentration of acetate ions is controlled to be 0.4-2 g/L;

(2) after fermentation, the fermentation precipitate is collected and dried to obtain the yarrowia lipolytica fermentation product.

In a preferred embodiment, in (a), the total amount of fatty acid is controlled to be 0.08-1%; preferably 0.1 to 0.8 percent.

In another preferred embodiment, (b) the acetate concentration is controlled to be 1 +/-0.5 g/L; preferably 1 + -0.3 g/L.

In another preferred embodiment, when yarrowia lipolytica is fermentatively cultured, the initial medium comprises: yeast extract (or yeast extract), magnesium sulfate, ammonium sulfate, potassium dihydrogen phosphate and/or dipotassium hydrogen phosphate; preferably, the fertilizer also comprises trace elements; and/or said acetate producing substrate is sodium acetate, ammonium acetate, acetic acid.

In another preferred embodiment, the compound comprises: 0.5-5 g/L of yeast extract (or yeast extract), 0.5-5 g/L of magnesium sulfate, 1-30 g/L of ammonium sulfate, 1-5 g/L of monopotassium phosphate and/or 1-5 g/L of dipotassium phosphate.

In another preferred embodiment, the trace elements include: ferric chloride, manganese sulfate, zinc sulfate, copper sulfate and molybdenum sulfate.

In another preferred embodiment, the yarrowia lipolytica is yarrowia lipolytica CICC 32291.

In another preferred embodiment, the fermentation product of yarrowia lipolytica comprises a product selected from the group consisting of: wet yeast obtained by partially dehydrating yarrowia lipolytica fermentation liquor, yeast powder obtained by drying and dehydrating, or yeast meal obtained by extracting oil.

In another preferred embodiment, (a) the pH is maintained at 7.5 ± 0.3; preferably, 7.5 + -0.2 or 7.5 + -0.1.

In another preferred embodiment, (b) the pH is maintained at 6.5 ± 0.3; preferably 6.5 + -0.2 or 6.5 + -0.1.

In another aspect of the invention, there is provided a yarrowia lipolytica fermentation product produced by the method described above; preferably, the yarrowia lipolytica fermentation product comprises a product selected from the group consisting of: wet yeast obtained by partially dehydrating yarrowia lipolytica fermentation liquor, yeast powder obtained by drying and dehydrating, or yeast meal obtained by extracting oil.

In a preferred embodiment, the yarrowia lipolytica fermentation product is rich in protein, fat, yeast polysaccharide and amino acids, wherein lysine, methionine and threonine are particularly abundant.

In another preferred embodiment, the yarrowia lipolytica fermentation product is rich in fatty acids, wherein the content of unsaturated fatty acids such as oleic acid and linoleic acid is particularly rich.

In another preferred embodiment, the fermentation duration is 24-120 hours, such as 36, 48, 60, 72, 84, 96, 108 hours, etc.

In another aspect of the invention, there is provided the use of said yarrowia lipolytica fermentation product for the preparation of an animal feed or feed additive.

In another aspect of the invention, there is provided an animal feed or feed additive comprising said yarrowia lipolytica fermentation product, and a dietetically or feedetically acceptable mix; preferably, the dietetic or feedlot acceptable mix includes (but is not limited to) materials selected from the group consisting of: corn flour, pig feed premix, corn, bran and soybean meal.

In another aspect of the present invention, there is provided a method of preparing an animal feed, the method comprising: (i) mixing corn flour and feed premix; (ii) mixing the yarrowia lipolytica fermentation product obtained by said method with the product mixture of (i); (iii) (iii) mixing the mixed product of (ii) with a roughage.

In a preferred embodiment, (i) when the corn flour and the feed premix are mixed, the corn flour and the feed premix are mixed according to the weight ratio of (1-10): 1, preferably according to (2-8): 1, and more preferably according to (3-7): 1 or (4-6): 1.

In another preferred embodiment, (ii) the yarrowia lipolytica fermentation product is mixed with the mixed product of (i) at a weight ratio of (1-5) to (1-5), preferably at a weight ratio of (1-3) to (1-3), more preferably at a weight ratio of (1-3) to (1-3) or at a weight ratio of (1-2) to (1-2).

In another preferred embodiment, (iii), the mixed product of (ii) is mixed with the roughage according to the weight ratio of (2-15): 1, preferably (4-10): 1, more preferably (5-8): 1 or (6-7): 1.

In another preferred embodiment, the roughage includes, but is not limited to: corn and bran.

In another preferred embodiment, the feed premix includes, but is not limited to: commercially available premixes for pigs, cattle, chickens, fish, etc.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Detailed Description

The present inventors have made intensive studies to disclose for the first time a novel fermentation product applicable to animal feeds, which is obtained mainly by fermenting Yarrowia Lipolytica (Yarrowia Lipolytica or saccharomyces Lipolytica) under appropriate conditions, and which has a good nutritional composition.

Based on the novel findings of the present invention, there is provided a method for producing a fermentation product of yarrowia lipolytica, comprising: (1) fermenting and culturing yarrowia lipolytica, and adding a carbon source substrate into a fermentation system in the fermentation process; wherein the carbon source substrate is a grease substrate, and the total amount of fatty acid is controlled to be 0.05-2%; or the carbon source substrate is a substrate for generating acetate, and the concentration of the acetate is controlled to be 0.4-2 g/L; (2) after fermentation, the fermentation precipitate is collected and dried to obtain the yarrowia lipolytica fermentation product.

The invention determines an ideal method for fermenting by using yarrowia lipolytica, and in a preferred embodiment of the invention, the yarrowia lipolytica is a strain with the preservation number CICC32291 in China center for Industrial culture Collection of microorganisms, and a fermentation product of the yarrowia lipolytica has good nutritional composition and is suitable for preparing feeds.

Feeding a carbon source substrate after the fermentation is started, wherein the carbon source substrate can be: organic acid or organic acid salt, volatile fatty acid or fatty acid salt, alcohols such as methanol and ethanol, aldehydes such as formaldehyde and acetaldehyde, glycerin, vegetable oil, animal oil, waste kitchen waste, petroleum, mineral oil, olefin and the like. Some substrates that are discarded or difficult to utilize by conventional bacteria may also be used.

In a preferred embodiment of the present invention, the carbon source substrate is a lipid substrate or an acetate-producing substrate. When the oil substrate is used for fermentation, the total amount of fatty acid is preferably controlled to be 0.05-2%, more preferably 0.08-1%, and further preferably 0.1-0.6%. When the acetate substrate is used for fermentation, the concentration of acetate is preferably controlled to be 1-2 g/L, and more preferably, the concentration of acetate is controlled to be 1 +/-0.5 g/L; further, it may be 1. + -. 0.3g/L or 1. + -. 0.2 g/L.

As a preferred mode of the invention, the yarrowia lipolytica fermentation product comprises but is not limited to: wet yeast obtained by partially dehydrating yarrowia lipolytica fermentation liquor, yeast powder obtained by drying and dehydrating, or yeast meal obtained by extracting oil. The yeast powder is preferably obtained after dehydration by drying from the viewpoint of easy storage or processing.

At the beginning of the fermentation, the fermenter is filled with the initial fermentation medium. The initial fermentation medium may be a conventional yeast medium suitable for yeast growth, capable of providing the necessary components for growth of the strain. In a preferred embodiment of the present invention, when yarrowia lipolytica is fermentatively cultured, the initial medium comprises: yeast extract (or yeast extract), magnesium sulfate, ammonium sulfate, potassium dihydrogen phosphate and/or dipotassium hydrogen phosphate; preferably, the fertilizer also comprises trace elements. For example, the medium may be a formulation as shown in Table 1, but is not limited thereto.

TABLE 1

In a preferred embodiment of the present invention, trace elements are further added to the initial fermentation medium, preferably the trace elements include: ferric chloride, manganese sulfate, zinc sulfate, copper sulfate and molybdenum sulfate. For example, the trace elements may be the formulations shown in table 2, but are not limited thereto.

TABLE 2

	General scope	Preferred range	Preferred range
				Ferric chloride	0.002～0.05g/L	0.005～0.04g/L	0.008～0.02g/L
Manganese sulfate	0.005～0.1g/L	0.008～0.08g/L	0.01～0.04g/L
				Zinc sulfate	0.005～0.1g/L	0.008～0.08g/L	0.01～0.04g/L
Copper sulfate	0.5～10mg/L	0.8～8mg/L	1～4mg/L
				Molybdenum sulfate	0.5～10mg/L	0.8～8mg/L	1～4mg/L

In the method, the fermentation duration is 24-120 hours, such as 36, 48, 60, 72, 84, 96, 108 hours and the like.

In the present invention, the pH varies depending on the substrate, and the pH is adjusted depending on the use of the substrate on the basis of the optimum of the strain itself. When the substrate is grease, the pH is properly increased for better mutual solubility under weak alkalinity.

The invention also includes the fermentation product obtained by fermentation using the above-described method. The fermentation product has rich protein, fat, zymosan, amino acids and essential amino acids, and especially rich lysine, methionine and threonine. Meanwhile, the fermentation product is rich in fatty acid, wherein the unsaturated fatty acid such as oleic acid and linoleic acid is particularly rich.

In the invention, the enrichment is obvious and has the characteristics of statistical significance, and generally means that the content of corresponding nutrient components is more than 5 percent of that of similar products sold in the market; more preferably more than 10%, such as more than 15%, 20%, 30%, 50%, 70%, 90% or more.

The concentrated fermentation product has a high content of active ingredients, such as 30-100 wt%, while the diluted fermentation product and the practically used fermentation product have a low content of active ingredients, typically 0.00005-30 wt%. In addition, other suitable trace elements, stabilizers, binders, dispersants, emulsifiers, penetrants, solvents, fillers, and other common ingredients may be included.

The fermentation product can be used as a feed additive for feeding animals or as concentrated feed after being processed properly; alternatively, it can be further mixed with a mixture or excipient acceptable in dietetics or feedology to prepare an animal feed. The animal feed is rich in nutritional ingredients. Such as concentration, drying, etc.

Accordingly, the present invention also provides an animal feed or feed additive comprising an effective amount of the fermentation product as hereinbefore described, together with a sitologically or feedstuffs (including fertilizer or agro-pharmaceutical) acceptable mix.

In the present invention, the term "comprising" means that various ingredients can be applied together in the mixture or fermentation product of the present invention. Thus, the terms "consisting essentially of and" consisting of are encompassed by the term "comprising.

In the context of the present invention, a "dietetic or feedstuffs-acceptable" ingredient is a substance which is suitable for dietetic or feedstuffs use without undue adverse side effects (such as toxicity, irritation and allergic response) to the animal, i.e. with a reasonable benefit/risk ratio.

In the present invention, some "dietetic or feedlot acceptable mixes" are exemplified by, but not limited to: corn flour, pig feed premix, corn, bran, soybean meal and the like.

The present invention also provides a method of preparing an animal feed, the method comprising: (i) mixing corn flour and feed premix; (ii) mixing the yarrowia lipolytica fermentation product obtained by the preparation method with the mixed product of (i); and (iii) mixing the mixed product of (ii) with roughage.

The fermentation product of the invention, animal feed containing the fermentation product and a dietetically or dietetically acceptable mix, can also be contained in a package (e.g., a box or a bag) for use in production or feed supply.

The invention has the beneficial effects that:

the explanation of the present invention reveals that Yarrowia lipolytica (Yarrowia lipolytica) is a yeast rich in proteins and essential amino acids, as well as fats and essential fatty acids, and the content of proteins and essential amino acids in the fermentation product is comparable to that of the yeast species commonly used in feeds on the market at present, but the content of fats and essential fatty acids, especially unsaturated fatty acids, is far more than that.

The method of the invention can utilize production raw materials with wide sources and low price, and can use waste or difficult-to-utilize organic acid or organic acid salt, volatile fatty acid or fatty acid salt, alcohols such as methanol and ethanol, aldehydes such as formaldehyde and acetaldehyde, glycerol, vegetable oil, animal oil, waste kitchen waste, petroleum, mineral oil, olefin and the like besides using conventional fermentation substrates such as glucose, xylose, cellulose and the like as substrates for fermentation. Has great potential for feed application.

Meanwhile, the feed provided by the invention can provide nutrient substances required by feeding animals, can regulate intestines and stomach of the animals and enhance immunity of the animals, is a natural component, cannot cause tolerance to the animals, and has the advantage that other feed components are incomparable.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.