阅读说明：本技术 一种利用长双歧杆菌制备发酵饲料的方法 (Method for preparing fermented feed by using bifidobacterium longum ) 是由 翟齐啸 陈卫 田丰伟 乔楠桢 于雷雷 赵建新 张灏 于 2020-05-20 设计创作，主要内容包括：本发明涉及一种利用长双歧杆菌制备发酵饲料的方法,属于生物技术领域以及发酵技术领域。本发明提供了一种以农作物为原料制备发酵饲料的方法,此方法通过将保藏编号为GDMCC No.60926的长双歧杆菌(Bifidobacterium longum)接种至含有农作物和/或农作物废弃物的发酵原料中进行发酵以得到发酵饲料；利用此方法制备得到的发酵饲料营养丰富、不易腐败且安全性很高,具体体现在：利用此方法制备得到发酵饲料中霉菌的含量仅有0.27×10<Sup>5</Sup>CFU/g,并且,于30℃下放置15d后,利用此方法制备得到发酵饲料中霉菌的含量为0。(The invention relates to a method for preparing fermented feed by utilizing bifidobacterium longum, belonging to the technical field of biology and fermentation. The invention provides a method for preparing fermented feed by using crops as raw materials, which comprises the steps of inoculating Bifidobacterium longum (Bifidobacterium longum) with the preservation number of GDMCC No.60926 into fermented raw materials containing the crops and/or crop wastes for fermentation to obtain the fermented feed; the fermented feed prepared by the method is nutritiousIs rich and not easy to decay, and has high safety, and the content of mould in the fermented feed prepared by the method is only 0.27 × 10 5 CFU/g, and after being placed at 30 ℃ for 15 days, the content of the mould in the fermented feed prepared by the method is 0.)

1. A method for preparing fermented feed by using Bifidobacterium longum comprises inoculating Bifidobacterium longum to fermented raw material containing crops and/or crop wastes, and fermenting to obtain fermented feed; the deposit number of the Bifidobacterium longum (Bifidobacterium longum) is GDMCCNo.60926.

2. The method for preparing fermented feed using bifidobacterium longum according to claim 1, wherein the bifidobacterium longum is inoculated into the fermentation raw material in the form of seed liquid; the volume of the seed liquid accounts for 0.5-2% of the total volume of the fermentation raw materials.

3. The method for preparing fermented feed using bifidobacterium longum according to claim 1 or 2, wherein the water content of the fermentation raw material is 55 to 65%.

4. The method for preparing fermented feed using Bifidobacterium longum according to claim 1 to 3, wherein the fermentation temperature is 25 to 35 ℃ and the fermentation time is 2 to 5 days.

5. The method of any one of claims 1-4, wherein the fermentation process is anaerobic.

6. The method for preparing fermented feed using Bifidobacterium longum according to any of claims 1 to 5, wherein the crop plants are fruits, vegetables, oil crops and/or food crops; the crop waste is fruit waste, vegetable waste, oil crop waste and/or grain crop waste.

7. The method for preparing fermented feed by using bifidobacterium longum according to claim 6, wherein the fruit waste is apple pomace, blueberry pomace, mulberry pomace and/or grape pomace; the vegetable waste is carrot residue, manioc residue and/or hawthorn fruit residue; the oil crop waste is soybean meal, cottonseed meal, peanut meal, sesame meal, sunflower seed meal and/or rapeseed meal.

8. The method for preparing fermented feed using Bifidobacterium longum according to any of claims 1 to 7, wherein the method comprises inoculating Bifidobacterium longum to a fermentation material comprising apple pomace, soybean meal and water, and fermenting to obtain fermented feed; in the fermentation raw material, the mass ratio of the apple pomace to the bean pulp is 16-20: 1-6.

9. A fermented feed prepared by the method of any one of claims 1 to 8.

10. Use of the method of any one of claims 1-8 for the preparation of fermented feed.

Technical Field

The invention relates to a method for preparing fermented feed by utilizing bifidobacterium longum, belonging to the technical field of biology and fermentation.

Background

However, in the crop processing process, a large amount of wastes are generated which are difficult to avoid, for example, apple pomace is generated in apple concentrated juice production, blueberry pomace is generated in blueberry juice production, mulberry pomace is generated in mulberry juice production, grape pomace and other fruit wastes are generated in grape juice production, hawthorn pomace is generated in hawthorn extract production, cassava pomace is generated in starch production, carrot pomace and other vegetable wastes are generated in carrot production, soybean meal is generated in soybean oil production, sesame meal is generated in sesame oil production, sunflower seed meal and other oil crop wastes are generated in sunflower seed oil production, and therefore, the crop wastes are also discharged in China. The problems of effective utilization and treatment of the crop wastes also become a great problem in testing the crop processing industry in China.

Currently, there have been attempts to achieve effective utilization and disposal of crop wastes by preparing them into feeds. For example, Wuzhengke et al prepares the crop wastes into high protein fermented feed by mixed bacteria solid state fermentation (specifically, see references: Wuzhengke, Liu Guo Hua, Li Yang, etc.. the process optimization of the mixed bacteria solid state fermented rapeseed meal [ J ]. Chinese agricultural science, 2019,24: 4603-; a method for preparing fermented fruit residue feed from crop wastes by using an anaerobic fermentation method in a Hao forest and the like (see a reference document: the Hao forest, Zhouzing, Yuyuan good and the like; mulberry residue nutrient component analysis and fruit residue feed fermentation process research [ J ]. silkworm industry science, 2019,4: 563-one 568); plum-north et al prepared crop wastes into fermented feeds through lactobacillus fermentation (see specifically references: plum-north, plum permanence, yellow tolerance rise, etc.. manioc waste biofermentation feeds development design [ J ] light industry science and technology, 2019,11: 30-31).

However, since agricultural wastes are rich in nutrients such as amino acids and water, and are easily attacked by filamentous fungi, filamentous fungi such as penicillium expansum, aspergillus niger, penicillium roqueforti, penicillium digitatum and the like (which are mainly responsible for crop spoilage by erosion of leaves, fruits and the like of crops and further cause storage diseases of crops, and which generate toxins such as patulin, citrinin and the like, which remain in crops and further enter human bodies through food chains and are potentially harmful) are one of the causes of crop spoilage, and therefore, even when sterilized, feeds prepared from crops as raw materials are easily spoiled during storage.

To solve the problem, the inventors of the Pinus massoniana and the like try to add a preservative into the feed prepared by using crops as raw materials to prolong the shelf life (the specific references can be found in Pinus massoniana and Wangwang. the influence of different mildewcides on the storage quality of the granulated feed [ J ]. the feed industry, 2019,40(9):38-44), but the addition of a large amount of the preservative has the problems of food safety, and the chemical additives have various limitations such as high cost, low return and adverse environmental protection (the specific references can be found in Yuzheng, Wuyingchao, Xijiayu and the like; the mildewcides dehydroacetic acid sodium dehydroacetate causes the rat bleeding test [ J ]. the animal medical progress, 2018,39(1):73-78 and the reference can be found in Kong Xueyang, Han Shumin, Lijinku and the like; the application of the mildewcides in the feed [ J ]. the feed science, 2019,40 (3-52), and insufficient addition of the preservative results in poor preservative effect.

Therefore, it is urgently required to find a method for preventing the deterioration of feed prepared from crops as raw materials with high safety and good effect.

Disclosure of Invention

[ problem ] to

The invention aims to provide a method for preventing feed prepared by using crops as raw materials from being rotten, which has high safety and good effect.

[ solution ]

The technical problem to be solved by the invention is to provide a method for preparing fermented feed by utilizing Bifidobacterium longum, which comprises the steps of inoculating Bifidobacterium longum (Bifidobacterium longum) into a fermentation raw material containing crops and/or crop wastes for fermentation to obtain the fermented feed; the Bifidobacterium longum (Bifidobacterium longum) has a deposit number of GDMCC No. 60926.

In one embodiment of the present invention, the Bifidobacterium longum (Bifidobacterium longum) is inoculated into a fermentation raw material in the form of a seed liquid; the volume of the seed liquid accounts for 0.5-2% of the total volume of the fermentation raw materials.

In one embodiment of the present invention, the Bifidobacterium longum (Bifidobacterium longum) is inoculated into a fermentation raw material in the form of a seed liquid; the volume of the seed liquid accounts for 1 percent of the total volume of the fermentation raw materials.

In one embodiment of the invention, the water content of the fermentation raw material is 55-65%.

In one embodiment of the invention, the fermentation feedstock has a moisture content of 60%.

In one embodiment of the present invention, the fermentation temperature is 25-35 ℃ and the fermentation time is 2-5 days.

In one embodiment of the invention, the temperature of the fermentation is 30 ℃ and the time is 3 d.

In one embodiment of the invention, the fermentation process is kept anaerobic.

In one embodiment of the invention, the crop is a fruit, vegetable, oil and/or food crop; the crop waste is fruit waste, vegetable waste, oil crop waste and/or grain crop waste.

In one embodiment of the invention, the fruit waste is apple pomace, blueberry pomace, mulberry pomace and/or grape pomace; the vegetable waste is carrot residue, manioc residue and/or hawthorn fruit residue; the oil crop waste is soybean meal, cottonseed meal, peanut meal, sesame meal, sunflower seed meal and/or rapeseed meal.

In one embodiment of the present invention, the method comprises inoculating bifidobacterium longum (bifidobacterium longum) into a fermentation raw material consisting of apple pomace, bean pulp and water to perform fermentation to obtain a fermented feed; in the fermentation raw material, the mass ratio of the apple pomace to the bean pulp is 16-20: 1-6.

In one embodiment of the invention, the mass ratio of the apple pomace to the soybean meal is 18: 1.

In one embodiment of the invention, the apple pomace has a particle size of 250 μm.

In one embodiment of the present invention, the particle size of the soybean meal is 250 μm.

The invention also provides the fermented feed prepared by the method.

The invention also provides application of the method in preparing fermented feed.

[ advantageous effects ]

The invention provides a method for preparing fermented feed by using crops as raw materials, which comprises the steps of inoculating Bifidobacterium longum (Bifidobacterium longum) with the preservation number of GDMCC No.60926 into fermented raw materials containing the crops and/or crop wastes for fermentation to obtain the fermented feed; the fermented feed prepared by the method is rich in nutrition, not easy to decay and high in safety, and is specifically represented by the following steps:

(1) in the fermented feed prepared by the method, the content of crude protein is up to 23.45 percent, the content of crude fiber is as low as 28.33 percent, the content of crude fat is up to 10.74 percent, and the content of total amino acid is up to 6.19 percent;

(2) the content of mould in the fermented feed prepared by the method is only 0.27 × 10⁵CFU/g, and after being placed for 15 days at the temperature of 30 ℃, the content of the mould in the fermented feed prepared by the method is 0;

(3) the fermentation strain used in the method is Bifidobacterium longum (Bifidobacterium longum), which is one of probiotics, and the Bifidobacterium longum is currently incorporated into a strain list available for food issued by the ministry of health, so that no potential safety hazard is brought to human bodies.

Biological material preservation

The Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is preserved in Guangdong province microorganism strain preservation center in 2019 at 09 month 12, and the preservation number is GDMCC No.60926, and the preservation address is No. 59 building 5 of Michelia Tokoro No. 100 of Guangzhou city.

Drawings

FIG. 1: effect of different concentrations of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the growth of Penicillium expansum mycelium.

FIG. 2: effect of different concentrations of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the amount of patulin synthesis.

FIG. 3: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on penicillium expansum patA-F gene expression

FIG. 4: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant treated in different ways on the growth of Penicillium expansum mycelium.

Detailed Description

The invention is further elucidated with reference to a specific embodiment and a drawing.

Penicillium expansum referred to in the following examples was purchased from China center for Industrial culture Collection of microorganisms with product number CICC 40658; the aspergillus niger related in the following examples is purchased from China center for culture Collection of industrial microorganisms, and the product number is CICC 2089; the Penicillium roqueforti related in the following examples is purchased from China center for culture Collection of industrial microorganisms with product number CICC 40663; penicillium digitatum referred to in the examples below was purchased from North Naita and has product number BNCC 336887; the apple pomace referred to in the following examples was purchased from Hengxing fruit juice Co., Ltd, Mei county, province, Shaanxi; the soybean meal referred to in the following examples was purchased from Hualong feed Co., Ltd, Fujian province.

The detection methods referred to in the following examples are as follows:

the water content detection method comprises the following steps: the sample is placed in an oven at 105 ℃ and dried to constant weight by adopting an oven drying method for determination, and the weight loss of the sample represents the moisture quality.

The crude protein detection method comprises the following steps: and (3) determining the content of crude protein in the sample by adopting a Kjeldahl method.

The crude fiber detection method comprises the following steps: the national standard GB/T6434-2006 'determination-filtration method for crude fiber in feed' is adopted.

The crude fat detection method comprises the following steps: the national standard GB/T6433-.

The total amino acid detection method comprises the following steps: the total amino acid content in the sample was determined by High Performance Liquid Chromatography (HPLC).

The pH detection method comprises the following steps: measured with a pH meter.

The organic acid detection method comprises the following steps: high performance liquid chromatography (reference: Xixia, screening of excellent lactic acid bacteria for silage and fermentation test research of apple pomace [ D ]. northwest agriculture and forestry science and technology university, 2014) is adopted.

The mould content detection method comprises the following steps: PDA plate dilution culture method (reference: Xixia, screening of excellent lactobacillus for silage and fermentation test research of apple pomace [ D ]. northwest agriculture and forestry science and technology university, 2014) is adopted.

The media involved in the following examples are as follows:

mrss solid medium: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5g of magnesium sulfate heptahydrate, 2g of diammonium citrate, 1g of cysteine hydrochloride and 18g of agar powder are added into 1L of distilled water, and the pH value is 6.2-6.5.

mrss liquid medium: adding 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5g of magnesium sulfate heptahydrate, 2g of diammonium citrate and 1g of cysteine hydrochloride into 1L of distilled water, wherein the pH value is 6.2-6.5.

PDA culture medium: 20g of glucose and 18g of agar were added to 1L of potato juice, and the pH was adjusted to the natural pH.