Bacillus amyloliquefaciens and application thereof in fermenting apple pomace

Abstract

The present application relates to a bacillus amyloliquefaciens and application thereof in fermenting apple pomace. The strain is named bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens ) JDFZYH-1, and the preservation number is CGMCC No. 36192. The strain was preserved in China General Microbiological Culture Collection Center (CGMCC) on November 13, 2025, and the address is No. 1, Yihuangxili, Chaoyang District, Beijing, China. The bacillus amyloliquefaciens provided in the present application can be applied to apple pomace fermentation, and has the advantages of high pectin degradation efficiency.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and particularly relates to the application of Bacillus amyloliquefaciens in fermented apple pomace. Background Technology

[0002] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens Bacillus is a Gram-positive rod-shaped bacterium in the genus Bacillus that possesses both probiotic properties and industrial application value. It is widely found in soil, on plant surfaces, and in various natural habitats, and is named for its highly efficient ability to decompose starch. This bacterium is a facultative anaerobe, capable of forming resilient endospores that can tolerate harsh environments such as high and low temperatures and acidic / alkaline conditions. On ordinary culture media, it forms rough, irregularly edged, milky-white colonies and secretes various active enzymes such as amylase, protease, and cellulase. It also produces antibacterial substances such as lipopeptides and polyketides. Its applications are very wide. In agriculture, it can inhibit the growth of plant pathogens and reduce soil-borne diseases by secreting antibacterial substances, and it can also promote crop root development and enhance the plant's resistance to stress. It is a commonly used biological control agent and biofertilizer strain. In industrial production, the amylase it secretes can be used in starch deep processing, textile desizing and other processes, while the protease can be used in feed additives, detergent production and other fields. In animal husbandry, some strains can also be added to livestock and poultry diets as probiotics to regulate the balance of animal intestinal flora and improve feed utilization. Moreover, due to its non-pathogenicity and good environmental compatibility, it has become an ideal microbial resource to replace some chemical agents and antibiotics.

[0003] Apple pomace is a major agricultural byproduct generated during apple processing (such as fresh juice, fruit wine, and canned apple production). Its main components are apple peel, core, pulp fragments, and a small amount of stem, possessing both nutritional value and potential for resource utilization. In terms of composition, apple pomace contains 15%-25% dietary fiber (including soluble pectin and insoluble cellulose and hemicellulose), and retains some unextracted pectin, polyphenols, vitamins, and minerals from the apple. It also contains 8%-12% crude protein and a small amount of fermentable sugars. Before resource utilization, apple pomace is often discarded directly or simply landfilled, resulting in resource waste and potential for environmental pollution due to fermentation and unpleasant odors. However, after processing such as drying, enzymatic hydrolysis, and microbial fermentation, apple pomace can be transformed into high-quality feed ingredients (used to improve the dietary fiber and palatability of livestock and poultry diets), making it a typical recyclable agricultural waste.

[0004] Existing technology CN118956690B discloses a strain of Bacillus amyloliquefaciens BZWQ833, its inoculum, and its application. It mentions that this bacterium is Gram-positive, capable of producing spores, and can produce proteases, cellulases, and pectinases. However, its purpose is to inhibit the growth and disease development of Streptomyces scabica, Rhizoctonia solani, Fusarium oxysporum, and Fusarium solani; it does not achieve a high pectin decomposition rate in apple pomace treatment. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an application of Bacillus amyloliquefaciens in fermented apple pomace.

[0006] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: This invention provides a Bacillus amyloliquefaciens, Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens JDFZYH-1, accession number CGMCC No. 36192, accession date: October 15, 2025.

[0007] The present invention provides a microbial agent comprising the above-mentioned Bacillus amyloliquefaciens or the fermentation broth of the above-mentioned Bacillus amyloliquefaciens.

[0008] This invention provides the application of the above-mentioned Bacillus amyloliquefaciens and bacterial agent in fermented apple pomace.

[0009] This invention provides a method for fermenting apple pomace, comprising the following steps: fermenting apple pomace using the aforementioned Bacillus amyloliquefaciens and inoculant.

[0010] Furthermore, Bacillus amyloliquefaciens JDFZYH-1 was cultured at an inoculum size of 1-5%, a temperature of 30-45℃, a culture medium pH of 6-8, and a culture time of 6-48 h.

[0011] Furthermore, when Bacillus amyloliquefaciens JDFZYH-1 was inoculated at 3%, at 37°C, at pH 7, and for 24 hours, the pectin degradation rate of apple pomace using the bacterial culture reached 46.89% under the optimal process. Attached Figure Description

[0012] Figure 1 Phylogenetic tree of JDZYH-1 16S rRNA gene sequence.

[0013] Figure 2 This is a photograph of the colony morphology of strain JDFZYH-1.

[0014] Figure 3 The growth curve of JDFZYH-1 is shown.

[0015] Figure 4The results of the JDFZYH-1 fermentation experiment on apple pomace are shown. Detailed Implementation

[0016] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0017] In this example, the apple pomace was purchased from Linyi Rongsheng Feed Co., Ltd., product number 20250912.

[0018] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, natural pH, prepared with water.

[0019] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 15 g / L, natural pH, prepared with water.

[0020] In the embodiments, unless specific techniques or conditions are specified, all methods used are conventional or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents and instruments used without specified manufacturers are all conventional products that can be purchased from legitimate channels or can be prepared according to conventional methods in this field.

[0021] Unless otherwise specified, the solutions of this invention are prepared using water as the solvent.

[0022] The following is a description through specific embodiments. Example

[0023] In February 2025, using sterile forceps in a clean bench, 5 g of internal rotten apple skin was peeled off and collected as a sample. This sample was then placed in 45 mL of sterile water and thoroughly mixed. After thorough mixing, the mixture was incubated in an 80°C water bath for 20 min to kill non-spore-forming bacteria, followed by centrifugation at 3000 r / min for 2 min at room temperature. The supernatant was then diluted 10-fold, and this serial dilution was repeated to obtain 10... -3 -10 -7 Diluent. Take 50 μL of 10... -3 -10 -7 The rumen dilution was spread onto LB agar plates and incubated upside down at 37°C for 24 h. After incubation, a single colony was picked and streaked again. The target strain was obtained using the above method and named JDFZYH-1. Example

[0024] 1. 16S rRNA gene analysis of strain JDFZYH-1 The 16S rRNA gene sequence was determined using universal primers 27F and 1492R for bacterial 16S rRNA, yielding a 1500bp gene fragment.

[0025] 27F: AGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT.

[0026] The 16S rRNA gene sequence of strain JDFZYH-1 is as follows:

[0027] A comparison with known strains using the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) revealed that strain JDFZYH-1 is comparable to known strains. Bacillus amyloliquefaciens strain MPA 1034 and Bacillus amyloliquefaciens strain NBRC 15535 showed the highest similarity. A phylogenetic tree based on the 16S rRNA gene sequence was constructed for strain JDFZYH-1 and related taxa, such as... Figure 1 As shown, JDFZYH-1 was ultimately identified as Bacillus amyloliquefaciens.

[0028] 3. Colony morphology characteristics of strain JDFZYH-1 like Figure 2 As shown, after 18 h of incubation at 37°C on LB medium, the colonies of bacteria JDFZYH-1 were milky white, round, smooth, and slightly convex.

[0029] 4. Physiological and biochemical characteristics of strain JDFZYH-1 The fatty acid composition of strain JDFZYH-1 was detected using a rapid microbial fatty acid identification system (MIDI). The results showed that the main fatty acid of strain JDFZYH-1 was C. 15:0 anteiso, C 15:0 iso, C 16:0 and C 17:0 ISO content was 29.41%, 30.15%, 6.32% and 6.59%, respectively.

[0030] Analysis using API 50CH revealed the following positive results: D-glucose, D-fructose, D-galactose, D-xylose, L-arabinose, ribose, rhamnose, glucosamine, sucrose, maltose, cellobiose, trehalose, raffinose, starch, glycogen, glycerol, dextrin, mannitol, sorbitol, salicin, and arbutin; lactose was found to be weakly positive. Negative reactions include lactose, cellulose, inulin, mesotriose, xylitol, and fucose.

[0031] 5. Measure the growth curve Strain strain JDFZYH-1 was inoculated into LB liquid medium at a 2% (v / v) inoculum and cultured at 37℃ and 200 rpm for 24 h. Samples were taken every 2 h, and OD was detected using a microplate reader. 600 And a growth curve was plotted. The experimental results are as follows: Figure 3 As shown, JDFZYH-1 reaches a plateau at 14 h.

[0032] Fermentation of apple pomace using JDFZYH-1: JDFZYH-1 bacterial solution was inoculated into apple pomace at an inoculation rate of 6% (i.e., 6 mL of JDFZYH-1 bacterial solution per 100 g of apple pomace), the fermentation temperature was 37℃, and the fermentation time was 72 h.

[0033] The pectin content in fermentation products was determined by the carbazole colorimetric method (GB / T10742—2008).

[0034] The experimental results showed that fermenting apple pomace with JDZYH-1 bacterial solution could significantly reduce the pectin content in apple pomace, with a pectin degradation rate of 46.89%.

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of Bacillus amyloliquefaciens, characterized in that, The strain is named Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) Bacillus amyloliquefaciens ) JDFZYH-1, and the preservation number is CGMCC No. 36192.

2. An inoculant characterized in that, Includes the Bacillus amyloliquefaciens of claim 1 or the fermentation broth of the Bacillus amyloliquefaciens of claim 1.

3. A method for fermenting apple pomace, characterized in that, Includes the following steps: Fermenting apple pomace using the Bacillus amyloliquefaciens of claim 1 or the inoculum of claim 2; The fermentation conditions were: 6% inoculum, 37℃ temperature, and 72h fermentation time.

4. The application of Bacillus amyloliquefaciens according to claim 1 and / or the inoculum according to claim 2 in fermented apple pomace.

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