A fermented feed based on citrus pulp and a method for its preparation

Abstract

The present application relates to the technical field of feed for animal feeding, and particularly discloses a fermentation feed based on citrus peel residues and a preparation method thereof.The preparation steps of the fermentation feed include: S1 mixing citrus peel residues, carriers and urea to obtain a solid culture medium; and S2 inoculating activated compound strains into the solid culture medium to perform fermentation, thereby obtaining the fermentation feed based on citrus peel residues.Compared with the prior art, the fermentation feed prepared by the present application has the advantages of high nutritional value and good palatability, can significantly promote the growth performance, intestinal health and body immunity of animals, can shorten the fermentation period, improve the fermentation efficiency and resource utilization rate, and further improve the economic benefits.

Description

Technical Field

[0001] The present invention relates to the technical field of feedstuffs specially suitable for animals, and in particular to a fermented feed based on citrus peel residue and a preparation method thereof. Background Art

[0002] Citrus is one of the world's most important fruits, characterized by widespread cultivation and high yield. Cultivated in over 140 countries and regions worldwide, citrus production exceeds 100 million tons annually. Citrus is primarily consumed fresh, as a juice, and canned fruit. The processing of citrus peel produces a significant amount of citrus pulp, which accounts for approximately 50% of the fruit's weight. Currently, in my country, most citrus peel is treated as waste, a significant waste of resources. Untreated peel easily contaminates soil and water bodies upon decay, significantly impacting the environment and ecological balance. Therefore, the issue of citrus peel has become a major obstacle to the development of the citrus industry, necessitating the search for a green and efficient treatment method. Research has found that citrus peel contains numerous nutrients and is easily degraded by microorganisms. Combined with microbial fermentation technology, it can be used as a fermentation substrate to produce single-cell protein feed for animal consumption. This method offers advantages such as rapid production, high yield, nutrient-rich nutrition, minimal infrastructure requirements, and ease of production. This method not only alleviates food and environmental pressures, but also truly achieves green and sustainable development.

[0003] Chinese Patent 202111088521.3 discloses a method for preparing a citrus residue-based fermentation preparation, which comprises: uniformly mixing citrus residue with auxiliary materials to obtain a solid culture medium; providing fermentation bacteria and activating and culturing them; inoculating the activated and cultured fermentation bacteria into the solid culture medium for fermentation and culturing, thereby obtaining a finished product of a citrus residue-based fermentation preparation; wherein the auxiliary materials are rice bran and / or wheat bran, and the fermentation bacteria are composed of Bacillus licheniformis, Portuguese Corynespora yeast, and Aspergillus niger. Correspondingly, the invention also discloses a citrus residue-based fermentation preparation prepared by the above-mentioned preparation method and its application. The implementation of this invention can effectively increase the content of flavonoids, organic acids, and amino acids in citrus residue-based fermentation preparations and improve their nutritional value.

[0004] Chinese Patent No. 201610559322.9 discloses a method for preparing bio-feed from citrus pulp through solid-state enzymatic fermentation, as well as its product and application. The method comprises the following steps: 1) Using distiller's grains as carrier I, the distiller's grains are mixed with a complex enzyme, labeled "Mixture I," comprising pectinase, glucanase, and cellulase; 2) Using wheat bran as carrier II, the wheat bran is mixed with a complex bacterial solution, containing Candida tropicalis, Bacillus subtilis, and Lactobacillus brevis, and brown sugar or molasses is added, labeled "Mixture II"; 3) Mixing Mixture I from step 1) and Mixture II from step 2) with citrus pulp, labeled "Mixture III," adjusting the moisture content of Mixture III to 65-70%, and then anaerobically fermenting Mixture III at a starting temperature of 30-40°C for 5-21 days to obtain the citrus pulp bio-feed. The bio-feed prepared by this invention has enhanced nutritional value and improved palatability. Replacing a portion of the basal diet with citrus pulp bio-feed for growing pigs can improve their performance.

[0005] Existing technologies mostly use solid-state fermentation to treat citrus peel residue. Solid-state fermentation is a process that is easy to control, consumes low energy, is simple to operate, produces high fermentation yields, requires a relatively low sterile environment, and is less likely to cause large-scale contamination. However, during the fermentation process, the solid components in the culture medium gradually decompose, causing the matrix to agglomerate, resulting in low porosity and limited mass and heat transfer, which significantly affects fermentation efficiency. Summary of the Invention

[0006] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is a fermented feed based on citrus peel residue and a preparation method thereof.

[0007] Solid-state fermentation refers to that the culture medium used for fermentation is solid-state, contains abundant water but almost does not have the mode of utilizing microbial fermentation of free-flowing dynamic water. The fermentation matrix is ​​not only the growth place of microorganisms, but also can provide various nutritions for microbial growth. Compared with other fermentation modes, solid-state fermentation method has the advantages that raw materials are extensive and easy to get, and process is simple, the productive rate of product is high, environmental pollution is small, and cost is relatively low. Shortcoming is that water activity (aw) is low, product is difficult to purify, biomass estimation is difficult and easily infected with miscellaneous bacteria, mechanization degree is low, fermentation time is long, etc. When carrying out solid-state fermentation, often need to add wheat bran etc. as matrix carrier, but along with the carrying out of fermentation process, culture medium decomposes gradually, matrix reunites, and porosity reduces, causes mass transfer and heat transfer to be limited. In addition, impurities in culture medium easily sneak into fermentation extract, are unfavorable for the separation and purification of fermentation product.

[0008] The present invention uses 4,4'-methylenebis(phenyl isocyanate) and 1,7-heptanediol as raw materials to prepare an inert polyurethane carrier. Its structure is not easily altered by microorganisms or metabolites during microbial growth, and it can also overcome the limitations of microbial aggregation and heat and mass transfer after fermentation. After the bacterial strain is fixed to the carrier, other impurities can be prevented from mixing into the fermented product. The carrier's specific surface area provides a stable interface for oxygen transfer, thereby improving aeration conditions, shortening the citrus peel residue fermentation cycle, and increasing product yield. In addition, the carrier's high porosity, low density, relatively high water absorption rate, and sufficient pore size provide an environment for fungal growth and also help to complete fermentation as quickly as possible. Furthermore, the carrier has low density, good mechanical resistance, and a certain strength. It maintains its shape during mixing, resists shear forces without damaging the support, protects microorganisms from shear impact, and has no side effects on microbial growth and product production. The present invention produces feed by mixing the composite bacterial strain, carrier, and citrus peel residue and fermenting them. This not only reduces ecological pollution and avoids resource waste, but also has high application value.

[0009] To achieve the above object, the present invention provides a method for preparing a fermented feed based on citrus peel residue, comprising the following steps:

[0010] S1: mixing citrus peel residue, a carrier, and urea to obtain a solid culture medium;

[0011] S2 activates the composite bacterial strain and inoculates it onto a solid culture medium for fermentation, thereby obtaining a fermented feed based on citrus peel residue.

[0012] The preparation method of the carrier comprises the following steps:

[0013] 45-85 parts by weight of 4,4'-methylenebis(phenyl isocyanate) and 10-25 parts by weight of 1,7-heptanediol are added to 50-100 parts by weight of dimethyl sulfoxide, heated to 90-120°C and stirred for 1-3 hours. After stirring, the mixture is cooled to room temperature and then added to a mixture of ether and acetone (volume ratio = 1:1). A precipitate is formed, which is filtered. The residue is washed with a mixture of ether and acetone and dialyzed (Mw < 3000Da) for 2-3 days. After dialysis, it is dried to obtain the product.

[0014] Furthermore, the mass ratio of the citrus peel residue to the carrier and urea is 70-80:10-20:1-5.

[0015] Furthermore, the water content of the solid culture medium is 40-60%.

[0016] Furthermore, the composite strain is a mixture of two or more of Trichoderma viride, Geotrichum candidum, Candida tropicalis, Aspergillus usamii, and Aspergillus niger.

[0017] Furthermore, the inoculation amount of the composite bacteria is 5-20wt%, and the number of viable bacteria is ≥1×10 8 The fermentation is aerobic fermentation, the temperature is 20-35℃, and the fermentation time is 2-7 days.

[0018] The present invention also provides a fermented feed based on citrus peel residue, which is prepared by the above method.

[0019] Beneficial effects of the present invention:

[0020] 1. Compared with the prior art, the present invention uses 4,4'-methylenebis(phenyl isocyanate) and 1,7-heptanediol as raw materials to prepare a polyurethane inert carrier. Its structure is not easily changed by microorganisms or metabolites during microbial growth, and it can also overcome the limitations of agglomeration and heat and mass transfer after microbial fermentation. After the bacteria are fixed on the carrier, other impurities can be prevented from mixing into the fermentation product. The specific surface area of ​​the carrier provides a stable interface for oxygen transfer, thereby improving aeration conditions, shortening the fermentation cycle of citrus peel residue, and increasing product yield.

[0021] 2. The present invention obtains feed by mixing composite bacteria, carriers and citrus peel residues and then fermenting them, which not only reduces ecological pollution and avoids waste of resources, but also the prepared feed has high application value. DETAILED DESCRIPTION

[0022] Trichoderma viride, CGMCC3.1584; Aspergillus usamii, CGMCC3.7010; and Aspergillus niger, CGMCC3.15663, were all purchased from the China General Microbial Culture Collection. Fungi, a general term for filamentous fungi, such as Trichoderma viride, Geotrichum candidum, Aspergillus usamii, and Aspergillus niger, secrete abundant enzymes such as amylase, cellulase, pectinase, protease, and phytase. These enzymes can decompose high-molecular compounds like starch and cellulose in raw materials into monosaccharides, which are needed for microbial growth. Molds also contain high levels of bacterial protein, making them widely used in protein feed production.

[0023] Geotrichum candidum, CGMCC2.1547; and Candida tropicalis, CGMCC2.3967, were purchased from the China General Microbial Culture Collection. Yeast is the common name for single-celled eukaryotic microorganisms that reproduce asexually and sexually. Asexual reproduction includes budding and fission, with budding being the most common. Yeast has a unique color, aroma, and flavor, and is highly palatable. During fermentation, yeast is highly acid-tolerant, suitable for low-pH cultivation, is less susceptible to contamination, and has a high recovery rate. Yeast strains suitable for protein feed production include Saccharomyces cerevisiae, Candida tropicalis, Pseudomonas aeruginosa, and Geotrichum candidum. These strains secrete a variety of hydrolytic enzymes with activity levels as high as 50-60%, are rich in B vitamins, and produce bioactive substances that promote cell division, providing nutritional benefits, disease resistance, and growth promotion.

[0024] Ordinary agar slant culture medium, pH 8.0-8.2, product number: C015203, Shanghai Aiyan Biotechnology Co., Ltd.

[0025] Malt extract agar medium, product number: HS-M1413002, Shanghai Hongshun Biotechnology.

[0026] Comparative Example 1

[0027] A method for preparing fermented feed based on citrus peel residue comprises the following steps:

[0028] S1: Mix 75 g of citrus peel residue, 15 g of bran, and 3 g of urea to obtain a solid culture medium, and add water to adjust the water content of the solid culture medium to 50%;

[0029] S2: Geotrichum candidum is inoculated on a common agar slant culture medium and activated at 50°C for 18 hours, then a single colony is picked and inoculated on a malt agar medium, cultured at 50°C for 120 hours, and the bacteria on the surface of the culture medium are washed with sterile water to obtain a Geotrichum candidum liquid; Candida tropicalis is inoculated on a common agar slant culture medium and cultured at 30°C for 48 hours for activation, and then inoculated on a malt agar medium at 30°C for 48 hours to obtain a Candida tropicalis liquid; Aspergillus usami is inoculated on a common agar slant culture medium and activated at 28°C for 30 hours, and then inoculated on a Czapek medium at 28°C for 90 hours to obtain an Aspergillus usami liquid; the inoculation amount of Geotrichum candidum, Candida tropicalis, and Aspergillus usami is 1:1:1, the activated strains are mixed and inoculated on a solid culture medium at 20wt% for fermentation, the fermentation temperature is 28°C, and the fermentation is carried out for 4 days to obtain a fermented feed based on citrus peel residue.

[0030] Comparative Example 2

[0031] The same as control example 1, the only difference is that the fermentation time is 2 days.

[0032] Example 1

[0033] A method for preparing fermented feed based on citrus peel residue comprises the following steps:

[0034] S1: Mix 75 g of citrus peel residue, 15 g of carrier, and 3 g of urea to obtain a solid culture medium, and add water to adjust the water content of the solid culture medium to 50%;

[0035] S2: Geotrichum candidum is inoculated into ordinary agar slant medium at 50°C for activation for 18 hours, and then a single colony is picked and inoculated into malt extract agar medium, cultured at 50°C for 120 hours, and the bacteria on the surface of the culture medium are washed with sterile water to obtain a Geotrichum candidum liquid; Candida tropicalis is inoculated into ordinary agar slant medium at 30°C for activation for 48 hours, and then inoculated into malt extract agar medium at 30°C for culture for 18 hours to obtain a Candida tropicalis liquid; Aspergillus usami is inoculated into ordinary agar slant medium at 28°C for activation for 30 hours, and then inoculated into Czapek medium at 28°C for culture for 90 hours to obtain an Aspergillus usami liquid; the inoculation amount of Geotrichum candidum, Candida tropicalis, and Aspergillus usami is 1:1:1, the activated strains are mixed and inoculated into a solid culture medium at 20wt% for fermentation, the fermentation temperature is 28°C, and the fermentation is carried out for 2 days to obtain a fermented feed based on citrus peel residue.

[0036] The preparation method of the carrier comprises the following steps:

[0037] 50 g of 4,4'-methylenebis(phenyl isocyanate) and 17 g of 1,7-heptanediol were added to 80 mL of dimethyl sulfoxide, heated to 110°C and stirred for 2 h. After the stirring was completed, the mixture was cooled to room temperature and then added to a mixture of ether and acetone (volume ratio = 1:1). A precipitate appeared, which was filtered. The residue was washed with a mixture of ether and acetone and dialyzed (Mw < 3000 Da) for 3 days. After the dialysis was completed, it was dried to obtain the product.

[0038] Example 2

[0039] The same as Example 1, the only difference is that the strains are Geotrichum candidum and Aspergillus usami, and the inoculation ratio is 1:1.

[0040] Example 3

[0041] The same as Example 1, the only difference is that the strains are Candida tropicalis and Aspergillus usami, and the inoculation amount is 1:1.

[0042] Example 4

[0043] The same as Example 1, the only difference is that the strains are Geotrichum candidum and Candida tropicalis, and the inoculation ratio is 1:1.

[0044] Example 5

[0045] The same as Example 1, the only difference is that the strains are Geotrichum candidum, Trichoderma viride and Candida tropicalis, and the inoculation amount is 1:1:1.

[0046] The activation culture of Trichoderma viride is to inoculate Trichoderma viride into potato culture medium (PDA culture medium) and culture it at 26°C for 7 days to obtain Trichoderma viride liquid.

[0047] Example 6

[0048] The same as Example 1, the only difference is that the strains are Geotrichum candidum, Aspergillus niger and Candida tropicalis, and the inoculation ratio is 1:1:1.

[0049] The activated culture of Aspergillus niger is to inoculate Aspergillus niger into ordinary agar slant medium for activation at 30°C for 48 hours and then inoculate into Czapek medium at 30°C for 18 hours to obtain Aspergillus niger liquid.

[0050] Test Example 1

[0051] The nutritional components of the fermented feeds of the control example and the example were tested using the following methods: moisture (GB / T 6435-2014, "Determination of Moisture in Feeds"), crude protein (Kjeldahl nitrogen analyzer), crude fiber (GB / T 6438-2007, "Determination of Crude Ash in Feeds"), crude fat (GB / T 6433-2006, "Determination of Crude Fat in Feeds"), total flavonoids (UV-visible spectrophotometry), and hesperidin (high-performance liquid chromatography). The results are shown in Table 1.

[0052] Table 1

[0053]

[0054]

[0055] From the comparison between Control Example 1 and Control Example 2, it can be found that when the fermentation time is only 2 days, the fermentation is obviously not complete, while Example 1 can achieve better results than Control Example 1 only by fermentation. This may be because the carrier in Example 1 has a small particle size and a large specific surface area, which has a better fixation effect on microorganisms. Its large specific surface area is conducive to the transport of oxygen, and is therefore more conducive to fermentation, thereby shortening the fermentation time and improving product quality. In addition, the high porosity of the carrier can also absorb sufficient water to ensure the continuous fermentation. The presence of pores can also protect the growth of microorganisms from being affected, overcome the limitations of agglomeration and heat and mass transfer, and prevent other impurities from mixing into the fermented product, thereby ensuring the quality of the feed. Compared with Examples 2 to 6, the feed prepared in Example 1 has higher crude protein and crude fat contents, and the contents of other components are also mostly improved, so that the nutritional value of the fermented feed of citrus peel residue reaches a higher level. This may be because molds are more beneficial to the decomposition of polysaccharides such as cellulose, and the various enzymes secreted by Geotrichum candidum and Aspergillus usami increase the hydrolysis of organic matter in citrus peel residue and can release substrates that can be used by tropical yeast. The synergistic effect between the strains improves the nutritional value of citrus residue.

[0056] Test Example 2

[0057] The fermented feed prepared in Example 1 was used to evaluate the feeding value of pig feed. The experiment was divided into two treatments, a blank control group and an experimental group, with 5 replicates for each treatment and 8 pigs for each replicate. The blank group was fed with basic feed, and the experimental group replaced 25% (air-dried matter basis) of fermented feed with citrus residue in the basic diet of the pigs. The groups were fully mixed and then fed. During the growing pig breeding process, the pigs were free to eat and drink water. After 56 days of feeding, the growth performance of the pigs was measured and the average value was taken to obtain the data shown in Table 2. Average daily weight gain = (final weight (kg) - initial weight (kg)) / number of experimental days (d), average daily feed intake = total feed consumption (kg) / (number of experimental days (d), feed-to-weight ratio = feed consumption during the trial period (kg) / pig weight gain (kg).

[0058] Table 2 Effects of fermented feed on growth performance of growing pigs

[0059]

[0060] Citrus pulp is rich in nutrients, some of which are active ingredients, such as citrus flavonoids, polyphenols, and hesperetin, which have multiple effects such as growth promotion, anti-oxidation, anti-inflammatory and antibacterial. Using citrus pulp as a raw material for livestock and poultry feed can not only reduce environmental pollution, but also promote animal production performance, intestinal health, and immunity, and make rational use of resources. Fermenting citrus pulp can not only extend the shelf life of citrus pulp, but also improve the palatability of citrus pulp through fermentation, which is beneficial to the utilization of nutrients by livestock and poultry and enhances the digestibility of organic matter. Feeding fresh citrus pulp biological feed can significantly affect the production performance of growing pigs, and the feed weight is significantly reduced compared with the control group.

[0061] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for preparing fermented feed based on citrus peel residue, characterized in that: The steps include: S1: mixing citrus peel residue, a carrier, and urea to obtain a solid culture medium; S2 activates the composite bacterial strain and inoculates it onto a solid culture medium for fermentation, thereby obtaining a fermented feed based on citrus peel residue; The preparation method of the carrier comprises the following steps: 45-85 parts by weight of 4,4'-methylenebis(phenyl isocyanate) and 10-25 parts by weight of 1,7-heptanediol are added to 50-100 parts by weight of dimethyl sulfoxide, heated to 90-120°C and stirred for 1-3 hours. After stirring, the mixture is cooled to room temperature and then added to a mixture of diethyl ether and acetone in a volume ratio of 1:

1. A precipitate is formed, which is filtered. The residue is washed with a mixture of diethyl ether and acetone and dialyzed for 2-3 days. After dialysis, the residue is dried to obtain the product. The composite strain is formed by mixing Geotrichum candidum, Candida tropicalis and Aspergillus usamii, and the inoculation amounts of the Geotrichum candidum, Candida tropicalis and Aspergillus usamii are 1:1:

1.

2. The method for preparing the feed according to claim 1, wherein The mass ratio of the citrus peel residue to the carrier and urea is 70-80:10-20:1-5.

3. The method for preparing the feed according to claim 1, wherein The water content of the solid culture medium is 40-60%.

4. The method for preparing the feed according to claim 1, wherein The inoculation amount of the composite bacteria is 5-20wt%, and the number of viable bacteria is ≥1×10 8 indivual.

5. The method for preparing the feed according to claim 1, wherein The fermentation is aerobic fermentation, the temperature is 20~35℃, and the fermentation time is 2~7 days.

6. A fermented feed based on citrus peel residue, characterized in that: Prepared by the method according to any one of claims 1 to 5.

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