AU2020103860A4 - Complex microbial agent and freeze-dried microbial agent for fermenting apple pomace and apple pomace protein feed - Google Patents
Complex microbial agent and freeze-dried microbial agent for fermenting apple pomace and apple pomace protein feed Download PDFInfo
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Abstract
ADstracy
The present invention provides a complex microbial agent and a freeze-dried microbial agent
for fermenting apple pomace and an apple pomace protein feed, and relates to the technical field of
animal fermented feed. The complex microbial agent for fermenting the apple pomace in the
present invention includes the following components in parts by volume: 29-36 parts of
Geotrichum candidum, 16-36 parts of Saccharomyces cerevisiae, 8-31 parts of Bacillus subtilis,
8-17 parts of Rhizopus oryzae, and 8-17 parts of Trichoderma viride. The effective number of live
bacteria of Geotrichum candidum, Saccharomyces cerevisiaes, Bacillus subtilis, Rhizopus oryzae
and Trichoderma viride is respectively more than or equal to 1x109 cfu/ml. The complex microbial
agent of the present invention can compensate for defects during the fermentation and
synergistically grow for fermentation. The apple pomace protein feed produced by utilizing the
complex microbial agent provided by the present invention has a crude protein content of more
than 20%, has a total content of 16 amino acids of more than 7.54g/100g, and is low in fiber
content, comprehensive in nutrients, green, safe, low in cost and high in benefit.
1
Description
ADstracy
The present invention provides a complex microbial agent and a freeze-dried microbial agent for fermenting apple pomace and an apple pomace protein feed, and relates to the technical field of animal fermented feed. The complex microbial agent for fermenting the apple pomace in the present invention includes the following components in parts by volume: 29-36 parts of Geotrichum candidum, 16-36 parts of Saccharomyces cerevisiae, 8-31 parts of Bacillus subtilis,
8-17 parts of Rhizopus oryzae, and 8-17 parts of Trichoderma viride. The effective number of live bacteria of Geotrichum candidum, Saccharomyces cerevisiaes, Bacillus subtilis, Rhizopus oryzae
and Trichoderma viride is respectively more than or equal to 1x109 cfu/ml. The complex microbial agent of the present invention can compensate for defects during the fermentation and synergistically grow for fermentation. The apple pomace protein feed produced by utilizing the complex microbial agent provided by the present invention has a crude protein content of more than 20%, has a total content of 16 amino acids of more than 7.54g/100g, and is low in fiber content, comprehensive in nutrients, green, safe, low in cost and high in benefit.
Description
Technical Field
The present invention relates to the technical field of animal fermented feed, and particularly relates to a complex microbial agent and a freeze-dried microbial agent for fermenting apple pomace and an apple pomace protein feed.
Background
China is an origin with largest planting area and largest annual output of apples in the world, accounting for 55% of the total output in the world. At present, not only the apples have been sold to domestic and foreign markets, but also apple juice has already become one of major extension products of the apple industry chain as well as one of main deep processing ways of the apples. Apple pomace is a byproduct of the apple juice. It is reported that the annual output of apple pomace produced from the production of the apple juice in apple processing in China is more than one million tons. With the rapid development of the juice processing industry, the output of the pomace is also increasing. The apple pomace is a byproduct produced from fresh apples in the production process of apple juice and contains pulp and peel, seeds and pedicel with content of 96.2%, 3.1%, and 0. 7 % respectively. The apple pomace has high water content (about more than 80%) and is rich in minerals such as calcium, phosphorus, potassium, iron and manganese as well as various vitamins, extremely low in protein content and relatively high in nitrogen-free extract content. Its main components are soluble carbohydrates, pectin, organic acids except for fatty acids, and hemicellulose. Studies have found that apple pomace is a high-fiber, low-protein, medium-energy and water-rich raw material. The content of heavy metals and pesticide residues in the apple pomace are within the range of feed hygiene standards. However, a majority of the apple pomace in China is discarded because it cannot be comprehensively utilized. Under the combined action of microorganisms in the apple pomace and the microorganisms in the environment, the apple pomace rots and deteriorates in a few days to give off a sour smell, causing serious waste of resources and environmental pollution.
Description
Summary
In view of this, the objective of the present invention is to provide a complex microbial agent and a freeze-dried microbial agent for fermenting apple pomace and an apple pomace protein feed. The apple pomace protein feed produced by utilizing the complex microbial agent provided by the present invention has a crude protein content of more than 20%, has a total content of 16 amino acids of greater than 7.54g/100g, and is low in fiber content, comprehensive in nutrients, green, safe, low in cost and high in benefit. To realize the above objective, the present invention adopts the following technical solutions: The complex microbial agent for fermenting the apple pomace provided by the present invention includes the following components in parts by volume: 29-36 parts of Geotrichum candidum, 16-36 parts of Saccharomycetes, 8-31 parts of Bacillus subtilis, 8-17 parts of Rhizopus oryzae, and 8-17 parts of Trichoderma viride. The effective number of live bacteria of Geotrichum candidum, Saccharomycetes, Bacillus subtilis, Rhizopus oryzae, and Trichoderma viride is respectively greater than or equal to 1x109 cfu/ml. The present invention also provides a freeze-dried microbial agent for fermenting apple pomace, which includes the complex microbial agent described in the above technical solution and a protective agent. A volume ratio of the protective agent to the complex microbial agent is -50: 100. Preferably, the effective number of live bacteria in the freeze-dried microbial agent is greater than or equal to 3x1010cfu/g. Preferably, the protective agent includes skimmed milk containing 5% by mass of sucrose and 3% by mass of trehalose. The present invention also provides an apple pomace protein feed, which is prepared from the following raw materials in parts by mass: 2-15 parts of seed solution prepared by the freeze-dried microbial agent described in the above technical solution, 80-82 parts of apple pomace, 14-16 parts of wheat bran, 1.4-1.6 parts of urea, 1.8-2.2 parts of ammonium sulfate, 0.08-0.12 parts of calcium carbonate and 0.08-0.12 parts of dipotassium phosphate. Preferably, a preparation method of the seed solution includes: inoculating the freeze-dried microbial agent described in the above technical solution to a wort liquid culture medium for
Description
primary culturing to obtain a first culture; and inoculating the first culture to an apple pomace culture medium for secondary culturing to obtain the seed solution. Preferably, the wort liquid culture medium adopts ddH 20 as a solvent which contains 120-140 g of the wort liquid culture medium per liter. Preferably, the apple pomace culture medium adopts ddH 20 as a solvent which contains -60 g of dry apple pomace per liter. Preferably, the temperature of the primary culturing and the secondary culturing is respectively 25-35°C; the time of the primary culturing is 16-24 h; and the time of the secondary
culturing is 48-72 h. Preferably, the effective number of live bacteria in the seed solution is more than or equal to 1xiO" cfu/g. The present invention provides a complex micribial agent for fermenting the apple pomace. The complex microbial agent includes Geotrichum candidum, Saccharomycetes, Bacillus subtilis, Rhizopus oryzae, and Trichoderma viride, which can compensate for their defects during the fermentation, and can cooperatively grow for fermentation. According to the present invention, the complex microbial agent is prepared into the freeze-dried microbial agent which has the characteristics of high microbial viability, long storage time and convenience in storage. In the apple pomace fermented by the complex microbial agent provided by the present invention, the content of crude proteins can be more than 20%, and the total content of 16 aminio acids is greater than 7.54g/100g, so that the protein content of the apple pomace can be comprehensively increased, the content of fibers can be reduced, the palatability is improved, and the application value of the apple pomace as the feed can be maximized.
Detailed Description
The present invention provides a complex micribial agent for fermenting the apple pomace, which includes the following components in parts by volume: 29-36 parts of Geotrichum candidum, 16-36 parts of Saccharomyces cerevisiae, 8-31 parts of Bacillus subtilis, 8-17 parts of Rhizopus oryzae, and 8-17 parts of Trichoderma viride. The effective number of live bacteria of Geotrichum candidum, Saccharomyces cerevisiae, Bacillus subtilis, Rhizopus oryzae and Trichoderma viride is respectively greater than or equal to 1x109 cfu/ml. The present invention does not specifically limit sources of Geotrichum candidum, Saccharomyces cerevisiae, Bacillus
Description
subtilis, Rhizopus oryzae and Trichoderma viride, which can be commercially available or can be obtained by conventional separation methods. In the present invention, the specific volume part of each component in the complex microbial agent is preferably as follows: 33.3 parts of Geotrichum candidum, 33.3 parts of Saccharomyces cerevisiae, 8.3 parts of Bacillus subtilis, 8.3 parts of Rhizopus oryzae, and 16.7 parts of Trichoderma viride. The present invention also provides a freeze-dried microbial agent for fermenting apple pomace, which includes the complex microbial agent described in the above technical solution and a protective agent. A volume ratio of the protective agent to the complex microbial agent is -50:100, preferably 35-45:100, and more preferably 40:100. The present invention does not specifically limit a preparation method of the freeze-dried microbial agent and preferably adopts a freeze drying or low-temperature sun curing method. In the present invention, the freeze-dried microbial agent is preferably prepared into freeze-dried powder. A preparation method of the freeze-dried powder is preferably as follows: the freeze-dried microbial agent is placed in a cavity of a ball mill; the cavity is vacuumized under a closing condition until the vacuum degree is -0.09 Mpa; then liquid nitrogen is injected; the mass/volume ratio of the freeze-dried microbial agent and the liquid nitrogen is 1:1; and the ball mill is started to grind the freeze-dried microbial agent into 60-mesh freeze-dried powder.
The freeze-dried powder of the present invention can be stored for 2 months at 24-26C, stored
for 1 year at 4-8 °C, and stored for 2 years at -20°C.
In the present invention, the effective number of live bacteria in the freeze-dried microbial agent is preferably greater than or equal to 3x1010 fu/g. In the present invention, the protective agent preferably includes skimmed milk containing % by mass of sucrose and 3% by mass of trehalose. The present invention does not specifically limit the source of the protective agent and can adopt the conventional commercially-available product. The present invention also provides an apple pomace protein feed, which is prepared from the following raw materials in parts by mass: 2-15 parts of seed solution prepared by the freeze-dried microbial agent described in the above technical solution, 80-82 parts of apple pomace, 14-16 parts of wheat bran, 1.4-1.6 parts of urea, 1.8-2.2 parts of ammonium sulfate, 0.08-0.12 part of calcium carbonate and 0.08-0.12 part of dipotassium hydrogen phosphate. The present invention does not specifically limit the sources of wheat bran, urea, ammonium sulfate,
Description
calcium carbonate and dipotassium hydrogen phosphate and can adopt the conventional commercially-available food-grade products. In the present invention, the specific mass parts of the raw materials of the apple pomace protein feed are preferably 2 parts of seed solution, 80.5 parts of apple pomace, 15.8 parts of wheat bran, 1.5 parts of urea, 2 parts of ammonium sulfate, 0.1 part of calcium carbonate and 0.1 part of dipotassium hydrogen phosphate. In the present invention, a preparation method of the seed solution preferably includes: the freeze-dried microbial agent described in the above technical solution is inoculated to a wort liquid culture medium for first culturing to obtain a first culture; and the first culture is inoculated to an apple pomace culture medium for second culturing to obtain the seed solution. In the present invention, the wort liquid culture medium preferably utilizes ddH 20 as a solvent which includes 120-140 g of the wort culture medium per liter, more preferably 130.1 g of the wort culture medium The present invention does not specifically limit the preparation method of the wort liquid culture medium. Preferably, the wort culture medium and ddH 20 are dissolved and sterilized at 121°C for 15 minutes. The present invention does not specifically limit the source of the wort culture medium and can adopt the conventional commercially-availableproduct. In the present invention, the temperature of the first culturing is preferably 25-35 'C and
more preferably 30 °C; and the time of the first culturing is preferably 16-24 h.
In the present invention, the apple pomace culture medium preferably utilizes ddH 20 as the solvent which includes 40-60 g of dry apple pomace per liter. The present invention does not specifically limit the preparation method of the apple pomace culture medium. Preferably, 6 g of dry apple pomace filtered by a 40-mesh screen and 100 mL of ddH 20 are dissolved and sterilized at 121°C under high pressure for 30 minutes. In the present invention, the temperature of the second culturing is preferably 25-35 °C and
more preferably 30 °C; and the time of the second culturing is preferably 48-72 h.
In the present invention, the effective number of live bacteria in the seed solution is preferably greater than or equal to x1011cfu/g. In the present invention, the preparation method of the apple pomace protein feed is preferably fermentation. The fermentation temperature is preferably 28-30 °C, and the
fermentation time is preferably 20-30 d. In the present invention, the fermented apple pomace is preferably sun-cured naturally or dried to obtain the apple pomace protein feed.
Description Technical solutions in the present invention are clearly and completely described below in combination with embodiments in the present invention. Apparently, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those ordinary skilled in the art without contributing creative effort fall within the protection scope of the present invention. Embodiment 1 Screening of strains of a complex microbial agent for fermenting apple pomace: 1.1 Activation of the strains Appearances of ampoule bottles in which the freeze-dried strains such as Geotrichum candidum, Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae, Trichoderma viride, Aspergillus niger and Aspergillus oryzae were disinfected separately; then the ampoule bottles were opened separately; culture solutions were added for dissolution; and then the culture solutions with the strains were transplanted onto corresponding slant culture media where each
strain grown, and cultured at 31°C for 24h-72h. Results showed that Geotrichum candidum,
Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae, Trichoderma viride, Aspergillus niger and Aspergillus oryzae all could grow on the slant culture media and could grow well. The slant culture media inoculated with Geotrichum candidum, Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae, Trichoderma viride, Aspergillus niger and Aspergillus oryzae were all conventional culture media for culturing the above strains. 1.2 Growth of the strains on apple pomace culture media with different contents Different strains in 1.1 were respectively scribed and inoculated onto agar plates with
different contents of apple pomace to culture at 31°C for 24-72 h. The growth conditions of all
strains on the apple pomace agar plates were observed. Experimental results showed that the Geotrichum candidum, Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae, Trichoderma viride, Aspergillus niger and Aspergillus oryzae all could grow in more than 4% of apple pomace and could grow well. 1.3 Plate dibbling of the strains The strains Geotrichum candidum, Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae,Trichodermaviride, Aspergillus niger and Aspergillus oryzae on slants were respectively scribed and inoculated onto 6% apple pomace culture media (6 g of 40-mesh dry apple pomace, 1.5 g of agar powder, and 100 mL of ddH 2 0), cultured at 121°C under high pressure for more
than 30 min, and cultured at 31°C for 72 h, and the growth conditions of all strains on the apple
t,
Description
pomace agar plates were observed. According to the growth conditions of the dibbling of the above 7 strains on the 6% apple pomace plates, bacterial rings of different sizes could be formed on the plates, and the growth was good. 1.4 The bacterial rings were stimulated by plate dibbling of the strains According to the syntrophism principle of microorganisms, the bacterial rings were stimulated by plate dibbling. Slant strains of Geotrichum candidum were washed with little sterile water and inoculated into the sterilized 6% apple pomace agar, then to-be-selected bacteria were respectively dibbled and cultured at 31°C for 72 h, and the growth conditions of the Geotrichum candidum at the plate dibbling position of each strain were observed. Results showed that Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae, Trichoderma viride, Aspergillus niger and Aspergillus oryzae could apparently stimulate the growth of the Geotrichum candidum. 1.5 Determination of a mixing ratio of strains 1.5.1 Fermentation of a single strain Seven strains were respectively inoculated into 6% apple pomace, fermented for 72 h, and dried at 65C after the fermentation was ended. The content of proteins of a fermented sample was determined. Experiment results showed that the seven strains all could ferment the apple pomace and could produce a high content of crude proteins. Black spores were easily generated on the appearance of the apple pomace fermented by Aspergillus niger, so the Aspergillus niger was removed in the later experiments. 1.5.2 Mixing ratio of two strains Every two of the seven strains were combined to form a group, and 10 pairs were selected and inoculated into 6% apple pomace separately according to a volume ratio of 1:0.2, 1:0.5, 1:1 and 2:1, fermented for 72 h, and dried at 65C after the fermentation is ended. The content of proteins of a fermented sample was determined. Results are shown in Table 1. Table 1 Results of mixing ratios of two strains
Description
Protein omtent ofdy sples irmented by difnt sauin mmbinatios No. Stns (mgg)
1:112 1:0.5 1:1 2:]
1 GRtrkhwu 8.981 8,981 9,559 9848 rcdimn+TrcxhdWraiwridt
2 Asp+gshdt 9.559 9.945 7535 10.619
3 Sacc-ts 9.559 8-595 9.359 9.366
4 Gw+rickrm 9.559 8.981 9.463 13.78 Oeofrichtm 5 cim+s cogescErevism 6.764 8.981 10.33 9.366
6 11,969 12-161 IL69 13319 cain+k~Rh-tLonus rOJZaE
7 Gor ntbtU&r dtC m +BtUwr 12,643 13.896 12.450 10A27 Bzaarluritdwf+Trtchodnta 8 14,378 [2.161 11L198 9.560
9 E4,960 &I981 I0]619 13.414 Emlnstszubtlis+Rtzopits 10 o 10.427 11487 1 L198 12.547
Mixing ratios of two strains (Geotrichum candidum was the core). It can be seen from Table 1 that the content of the proteins in the product of the apple pomace fermented by the combination of Geotrichum candidum + Aspergillus oryzae and the combination of Aspergillus oryzae + Trichoderma viride is relatively low. Therefore, Aspergillus oryzae was removed in the later experiments. 1.5.3 Mixing ratio of five strains The result of the proteins in the sample was determined by taking two strains as a combination, and 10 combinations were selected by taking Geotrichum candidum as the index bacterium. The strains were inoculated into the 6% apple pomace respectively according to different strain ratios, fermented for 72 h, and dried at 65C after the fermentation was ended. The content of proteins of the fermented sample was determined. Results are shown in Table 2. Table 2 Results of mixing ratios of five strain combinations No. Strain combinations Different strain ratios Protein content of dry samples after the fermentation (mg/g) 1 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:1:1:1 12.932 oryzae+Bacillussubtilis+Trichodermaviride 2 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:0.5:0.5:0.5 13.125 oryzae+Bacillus subtilis+Trichodermaviride 3 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:1:1:0.5 12.065 oryzae+Bacillus subtilis+Trichodermaviride
Description 4 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:1:0.5:1 12.836 otyzae+ Bacillus subtilis+Trichodermaviride Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:0.5:1:0.5 12.450 otyzae+ Bacillus subtilis+Trichodermaviride 6 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:0.5:1:1 13.029 otyzae+ Bacillus subtilis+Trichodermaviride 7 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:1:0.5:0.5 13.125 otyzae+ Bacillus subtilis+Trichodermaviride 8 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 2:2:0.5:0.5:1 15.149 otyzae+ Bacillus subtilis+Trichodermaviride 9 Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 1:0.5:0.5:1:0.2 12.607 otyzae+ Bacillus subtilis+Trichodermaviride Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus 1:0.5:0.5:1:0.1 12.354 otyzae+ Bacillus subtilis+Trichodermaviride
It can be seen from Table 2 that the content of the proteins in the dry sample fermented by the above 10 ratios of Geotrichum candidum+YSaccharomyces cerevisiae+Rhizopus oryzae+Bacillussubtilis+Trichodermaviride can be more than 12 mg/g. Embodiment 2 Inoculation quantity of the complex microbial agent for fermenting apple pomace The strains of Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus oryzae+Bacillus subtilis+Trichoderma viride were inoculated onto the wort liquid culture medium respectively according to an inoculation ratio of 2:2:0.5:0.5:1 and cultured for 16 to serve as basic seeds; then the basic seeds were inoculated into 100 g of dry apple pomace culture medium (a material-to-water ratio is 10: 8) according to the ratio, and cultured at 31°C for 48 h
to serve as primary seeds; the primary seeds were inoculated into OOg of sterilized fresh apple pomace (the content of dry matters in the apple pomace was 20% and the water content was 80%)
culture medium according to different ratios, and cultured at 31°C for 72 h, and the fermented
apple pomace was dried at 65C; and a product of the non-fermented fresh apple pomace of the
same batch dried at 65C was used as a control group. The protein content of the apple pomace
before and after the fermentation was determined by a biuret method. Results are shown in Table 3. Table 3 Impact of different inoculation quantities of the complex microbial agent on the content of fermented proteins Group Strain combination Protein content of dry samples fermented at different inoculation quantities (mg/g)
2% 3% 5% 10% 15% Experiment Geotrichum candidum+Saccharomyces 6.668 7.824 7.974 8.210 7.535 cerevisiae+Rhizopus otyzae+Bacillus
Description group subtilis+Trichoderma viride (2:2:0.5:0.5:1)
Control 5.244 group
It can be seen from Table 3 that the complex microbial agent consisting of Geotrichum candidum+Saccharomyces cerevisiae+Rhizopus oryzae+Bacillus subtilis+Trichodermaviride was inoculated into the sterilized fresh apple pomace at a ratio of 2%-15%, and the protein content of the product can be up to 6.668-8.210 mg/g. Embodiment 3 The complex microbial agent includes 29 mL of Geotrichum candidum, 16 mL of Saccharomycetes cerevisiae, 8 mL of Bacillus subtilis, 8 mL of Rhizopus oryzae and 8 mL of Trichoderma viride. The complex microbial agent was obtained by respectively inoculating Geotrichum candidum, Saccharomyces cerevisiae, Rhizopus oryzae, Bacillus subtilis and Trichoderma viride into the wort liquid culture medium at a ratio of 1%, shaking at 180 rpm and
culturing at 30 °C for 18 h and mixing 29 mL of Geotrichum candidum, 16 mL of
Saccharomyces cerevisiae, 8 mL of Bacillus subtilis, 8 mL of Rhizopus oryzae and 8 mL of Trichoderma viride. The effective number of live bacteria in the bacterial solutions of Geotrichum candidum, Saccharomyces cerevisiae, Rhizopus oryzae, Bacillus subtilis and Trichoderma viride was respectively 1x109 cfu/ml. Embodiment 4 The complex microbial agent includes 36 mL of Geotrichum candidum, 36 mL of Saccharomyces cerevisiae, 31 mL of Bacillus subtilis, 17 mL of Rhizopus oryzae and 17 mL of Trichoderma viride. The preparation method of the complex microbial agent was same with that in embodiment 3. The effective number of live bacteria in the bacterial solutions of Geotrichum candidum, Saccharomyces cerevisiae, Rhizopus oryzae, Bacillus subtilis and Trichoderma viride was respectively 2x109 cfu/ml. Embodiment 5 The complex microbial agent includes 33 mL of Geotrichum candidum, 33 mL of Saccharomyces cerevisiae, 8 mL of Bacillus subtilis, 8 mL of Rhizopus oryzae and 16 mL of Trichoderma viride. The preparation method of the complex microbial agent was same with that in embodiment 3. The effective number of live bacteria in the bacterial solutions of Geotrichum candidum, Saccharomyces cerevisiae, Rhizopus oryzae, Bacillus subtilis and Trichoderma viride was respectively 2.5x109 cfu/ml.
1iA
Description Embodiment 6 The freeze-dried microbial agent includes 33.3 mL of Geotrichum candidum, 33.3 mL of Saccharomyces cerevisiae, 8.3 mL of Bacillus subtilis, 8.3 mL of Rhizopus oryzae and 16.8 mL of Trichoderma viride and 30 mL skimmed milk containing 5% sucrose and 3% trehalose by mass. A preparation method of the freeze-dried microbial agent: Geotrichum candidum, Saccharomycetes cerevisiae, Bacillus subtilis, Rhizopus oryzae and Trichoderma viride were respectively inoculated into the wort liquid culture medium at a ratio of 1%, and cultured by shaking at 30°C and 180 rpm for 18 h; after the culture was ended, 33.3 mL of Geotrichum candidum, 33.3 mL of Saccharomycetes cerevisiae, 8.3 mL of Bacillus subtilis, 8.3 mL of Rhizopus oryzae and 16.8 mL of Trichoderma viride (the effective number of live bacteria in the culture is respectively more than 109 cfu/mL) were mixed and centrifuged at 4000 rpm for 30 min, and supernatant was removed; and 30mL of the sterilized freeze-dried protective agent (skimmed milk containing 5% sucrose and 3% trehalose) preheated at 37°C was added into bacterial sludge, uniformly and fully mixed and split charged into sterilized plates. In the split charging process, the mixture should be thermally preserved and uniformly shaken, rapidly frozen and vacuum dried. A freeze drying curve of the strains is provided by the present invention (freeze-drying curve: when the temperature was lowered to -25°C to -40°C, vacuumizing was carried out for 20-30 h, and then the temperature was increased to 15°C-25°C and kept for 4-6 h). After the freeze-vacuum drying was ended, the mixture was crushed into powder by a vacuum-nitrogen-filled grinding method, and the powder was rapidly charged into sterilized bottles or aluminum foil bags to be sealed. The freeze-dried powder prepared by the present invention was white spongy loose powder, and the effective number of live bacteria was 3x101 cfu/g. Embodiment 7 Apple pomace fermentation technological conditions: 1 Impact of different inoculation quantities on content of crude proteins in apple pomace The freeze-dried powder complex microbial agent prepared in embodiment 6 was inoculated into 100 g of apple pomace fermentation culture medium at a mass ratio of 2%, 3%, %, 8%, 10% and 15%, uniformly mixed, cultured at 30°C for 3 d, and dried at 65°C, and then the content of crude proteins was determined. Results are shown in Table 4. Table 4 Impact of different inoculation quantities of strains on content of crude proteins in the apple pomace
Description
Culture medium FemenationLClac m~y(W time h)
2% 3% 5%1 10% 15%
Du appe Pe 72 9.945 I1372 11.583 1 487 11.487
Fresh ale p- e
72 12J161 11ts72 11.969 11 101 11.990
Note: the content of crude proteins in the dry strains was 13.414 mg/g It can be seen from Table 4 that when the dry apple pomace was inoculated with 2%-15% of solid strains, and the fresh apple pomace was inoculated with 2%-15% of solid strains, the apple pomace can be well fermented, and the content of proteins in the fermented apple pomace product can be increased. 2 Impact of a material-to-water ratio on content of crude proteins in the fermented apple pomace In the dry apple pomace fermentation culture medium, the material-to-water ratio was 20%, %, 40%, 50%, 60%, 70% and 80% respectively; the activated freeze-dried powder complex microbial agent prepared in embodiment 6 was inoculated at 2%, fermented at 30°C for 72 h, and
dried at 65C; and the crude protein content of the product was determined. Results are shown in
Table 5. Table 5 Impact of a material-to-water ratio on content of crude proteins in the fermented apple pomace
quantity(M) tim ___h __
10:5 10:6 10:7 10:9 10:9 IOA10 Apple p-e
ckitue mdium 10 72 10.06 IM6 12.22 12.69 12,65 10.39
It can be seen from the results that when the material-to-water ratio was controlled between :5 and 10: 10, all strains can grow well on the apple pmace culture medium, and the apple pomace can be fermented to produce a product with relatively high content of proteins. 3 Impact of fermentation time and a thickness of a fermentation material layer on content of crude proteins in the fermented apple pomace
Description
The activated freeze-dried powder complex microbial agent prepared in embodiment 6 was inoculated into 100 g of apple pomace fermentation culture medium at 2%, and fermented at °C. Samples were collected at different fermentation time and different thicknesses of the fermentation material layer. The samples were dried at 65C, and then the content of crude proteins in the samples was determined. Results are shown in Table 6. Table 6 Impact of fermentation time on content of crude proteins in the fermented product
Thiknt~iof~It main Fermentation time (h)
72.6 2 24 4 ~~ 2cm 2 31 8.499 12.258 11.872 7.728 885
3cm 2 31 9.559 11.294 8.692 7.818 7.921
4cm 2 31 8.210 9.945 1.041 8.017 6,282
5cm 2 31 9.559 10.908 12.547 8.403 11.005
6cm 2 31 8.1 14 9.559 11487 7.824 7.632
8cm 2 31 8.306 9,270 10.041 8.403 9.077
I0 2 I1 7.632 9.692 8-939 U1,789 9.246
15cm 2 31 7.150 8.017 9463 12547 7.343
20cm 2 31 7.149 8.017 8.114 9.559 10.908
It can be seen from Table 6 that when the thickness of the material layer was 2-20 cm and the fermentation time was 24-120 h, the product with high content of proteins can be produced by fermentation. 5 Impact of fermentation process and contrast on content of crude proteins in the fermented apple pomace The complex microbial agent screened in the present invention, strains from Ningxia Muguang and Tianbao No. Ilstrains of China were respectively used as strains to ferment the dry apple pomace culture medium according to an inoculation quantity of 10%, a material-to-water ratio of 10:7, fermentation time of 72 h and a material layer thickness of 5 cm, and the fermented product was dried for determining the content of crude proteins in the sample. Results are shown in Table 7. The three strains were used to ferment the dry apple pomace by a same fermentation process, and all can increase the content of the crude proteins in the apple pomace fermented product. The experiment proved that the complex microbial agent of the present invention can
Description
significantly increase the content of the crude proteins in the fermented product, which was better than Ningxia Muguang and Tianbao No. II. Table 7 Impact of fermentation process and contrast on content of crude proteins in the fermented apple pomace
Expe group 1 Fermentation technological conditions Crude protein content of a fermentation product g/mg
Complex fermentation agent ofthe present vention e inoculation quantity i 10%, the material-to-water 25.353 r_ato 10-7 the fermentation tkne is 2 h and the Strains from NingxaMuguang kness ofthe materiallayer is 5 cm 23.052
20.353
Embodiment 8 Apple pomace protein feed 1. Seed solution for production The freeze-dried microbial agent prepared in embodiment 6 was inoculated into a wort
liquid culture medium and cultured at 30°C for 24 h to obtain a first culture; then the first culture
was inoculated into an apple pomace culture medium (a fermentation culture medium composed of 80.5 g of apple pomace, 15.8 g of wheat bran, 0.1 g of dipotassium hydrogen phosphate (K 2HP 44.3H 2 0), 0.1 g of anhydrous calcium carbonate, 1.5 g of urea, and 2.0 g of ammonium
sulfate, with a material-to-water ratio of 10:8), and cultured at 30'C for 72 h to obtain a second
culture, i.e. the seed solution for production of fermented apple pomace. 2. Production of fermented apple pomace The fermented seed solution for the production was inoculated into the fermentation culture medium consisting of 80.5 g of apple pomace, 15.8 wheat bran, 0.1 g of dipotassium hydrogen phosphate (K2 HOP 4 .3H 2 0), 0.1 g of anhydrous calcium carbonate, 1.5 g of urea and 2.0 g of ammonium sulfate at a mass ratio of 2% according to a material-to-water ratio of 10:8, uniformly stirred, then charged into a fermentation tank, and fermented for 30 days with a plastic greenhouse film, and the fermentation condition was observed every day. After the fermentation was completed, random sampling was carried out for quality inspection. The fermented apple pomace from Zhongning Ruiheng Technology Industry Co., Ltd., Muguang and Taifeng were collected as control samples and sent to Pony Testing Group Shanghai Co., Ltd. for test. The test results are shown in Table 8. Table 8 Test results of different fermented apple pomace
Description
Sample tet rents
Tat items Tet method E aA Em Smpl sApple pomace eam"l Taifar h
Crudepoem(%) (.6412-1994 11. 19A 183 &879 8
Crude fibers (%) GB/T434-2006 11.4 9A 72 64 51
Crude fat (%) G(r6433-2006 4. X.6 15.9 173 2.9
Ash content (%) GB/T6438-2007 7.4 7.8 9.7 43 22
Caleinm(%) Garr6436-2002 0.16 C.79 0.74 055 OM
Phosphorus (%) GBrT6437-2002 0.57 0.25 028 0.17 0.06
Asparaginic acid 0.73 0.75 0.85 0.67 0.32
Threonine 0.31 040 038 (126 00Mo3
Serine 035 037 (0133 20 0087
Glutamate 1.32 116 41 100 O.23
Proline 0.59 0.48 0 56 %37 0.061
16 Glycine 0.42 0.52 0.58 041 0081 amino Alanine 0.47 0.62 0.71 0.49 0.082 (g 100 9) Lysine 0.34 0.25 048 0.38 0,064
Valine (it18246-2O( 0.3 0O40 060 0.47 0.072
Methionine 0.19 0.25 0.11 0,06 0.017
Isoleudino 0.27 0.37 047 0.35 0062
Letcine 0.48 O61 U.3 0355 0.10 Tyrosine 0,22 0.28 025 0.46 0051
035 0.38 045 0.33 0076
Histidine u8 0,46 022 0s18 (064
Arginine 0.39 0.24 043 032 0,056
Total 7.94 7.54 8 56 6.20 1.51
It can be seen from Table 8 that the apple pomace protein feed of the present invention is higher than the apple pomace and the samples from Ruiheng, Muguang and Taifeng in the aspects of crude proteins and the sum of 16 amino acids, has crude protein content increased by
Description
9-20%; and the content of proteins and the sum of 16 amino acids in the fermented apple pomace are significantly increased. The crude proteins and the sum of the 16 amino acids of the apple pomace protein feed of the present invention are respectively increased by 15% and 6% compared with the apple pomace. The above only describes preferred embodiments of the present invention. It should be pointed out that various improvements and modifications may be made by those ordinary skilled in the art without departing from the concept of the present invention. These improvements and modifications shall also be regarded as the protection scope of the present invention.
1A -I-
Claims (10)
1. A complex microbial agent for fermenting apple pomace, comprising the following components in parts by volume: 29-36 parts of Geotrichum candidum, 16-36 parts of Saccharomycetes, 8-31 parts of Bacillus subtilis, 8-17 parts of Rhizopus oryzae, and 8-17 parts of Trichoderma viride, wherein the effective number of live bacteria of Geotrichum candidum, Saccharomycetes, Bacillus subtilis, Rhizopus oryzae, and Trichoderma viride is respectively greater than or equal to 1x109 cfu/ml.
2. A freeze-dried microbial agent for fermenting apple pomace, comprising the complex microbial agent of claim 1 and a protective agent, wherein a volume ratio of the protective agent to the complex microbial agent is 30-50: 100.
3. The freeze-dried microbial agent according to claim 2, wherein the effective number of live bacteria in the freeze-dried microbial agent is greater than or equal to 3x1010cfu/g.
4. The freeze-dried microbial agent according to claim 2, wherein the protective agent comprises skimmed milk containing 5% by mass of sucrose and 3% by mass of trehalose.
5. An apple pomace protein feed, which is prepared from the following raw materials in parts by mass: 2-15 parts of seed solution prepared by the freeze-dried microbial agent of any one of claims 2-4, 80-82 parts of apple pomace, 14-16 parts of wheat bran, 1.4-1.6 parts of urea, 1.8-2.2 parts of ammonium sulfate, 0.08-0.12 part of calcium carbonate and 0.08-0.12 part of dipotassium phosphate.
6. The apple pomace protein feed according to claim 5, wherein a preparation method of the seed solution comprises: inoculating the freeze-dried microbial agent of any one of claims 2-4 to a wort liquid culture medium for primary culturing to obtain a first culture; and inoculating the first culture to an apple pomace culture medium for secondary culturing to obtain the seed solution.
7. The apple pomace protein feed according to claim 6, wherein the wort liquid culture medium adopts ddH2 0 as a solvent which contains 120-140 g of the wort liquid culture medium per liter.
8. The apple pomace protein feed according to claim 6, wherein the apple pomace culture medium adopts ddH2 0 as a solvent which contains 40-60 g of dry apple pomace per liter.
9. The apple pomace protein feed according to claim 6, wherein the temperature of the primary culturing and the secondary culturing is respectively 25-35°C; the time of the primary
culturing is 16-24 h; and the time of the secondary culturing is 48-72 h.
Claims
10. The apple pomace protein feed according to claim 6, wherein the effective number of live bacteria in the seed solution is more than or equal to 1x10 cfu/g.
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