Bacillus cereus and application thereof in fermenting apple pomace

Abstract

The invention relates to bacillus cereus and application thereof in fermenting apple pomace. The strain name is bacillus cereus LYZYH-2, the preservation number is CGMCC No.36191, and the bacillus cereus LYZYH-2 is preserved in China General Microbiological Culture Collection Center (CGMCC) on November 13, 2025, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, China. The bacillus cereus provided by the invention can be applied to apple pomace fermentation and has the advantage 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 cereus in fermented apple pomace. Background Technology

[0002] Bacillus cereus is a Bacillus strain that presents both application value and biosafety risks, with potential applications in biomedicine, feed industry, and environmental remediation. However, the selection of different strains, cultivation methods, and production processes has a crucial impact on the types of enzymes produced, enzyme activity, and the safety of metabolic byproducts, thus limiting its further application in high-requirement fields such as food. Currently, enzyme production using Bacillus cereus suffers from insufficient enzyme specificity, unstable target enzyme yield, and a tendency to generate pathogenic enterotoxins.

[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] While the existing Bacillus cereus disclosed in CN107893039B contains neutral endopeptidase, it is primarily used in waste paper manufacturing. It has poor environmental adaptability to apple pomace and is not suitable for apple pomace treatment. Furthermore, this bacterium, after undergoing radiation mutagenesis combined with gene recombination technology, is not suitable for use in the food, feed, and other fields. Summary of the Invention

[0005] In view of the problems existing in the prior art, the present invention provides an application of Bacillus cereus 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 cereus, Bacillus cereus LYZYH-2, with accession number CGMCC No.36191.

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

[0008] This invention provides the application of the above-mentioned Bacillus cereus and inoculant in fermented apple pomace.

[0009] This invention provides a method for fermenting apple pomace, comprising the following steps: fermenting apple pomace using the above-mentioned Bacillus cereus and inoculant.

[0010] Furthermore, Bacillus cereus LYZYH-2 was used to ferment apple pomace at an inoculum size of 1-5%, a temperature of 32-42℃, a culture medium pH of 6-8, and a culture time of 6-48 hours.

[0011] Furthermore, Bacillus cereus LYZYH-2 was used to ferment apple pomace at an inoculum size of 3%, a temperature of 37°C, a culture medium pH of 7, and a culture time of 24 h. Through process optimization, the pectin degradation rate of apple pomace reached 46.18% under the optimal process conditions. Compared with other Bacillus cereus strains, the Bacillus cereus LYZYH-2 provided by this invention has an excellent pectin degradation rate in apple pomace fermentation. Attached Figure Description

[0012] Figure 1 Phylogenetic tree of the LYZYH-2 16S rRNA gene sequence.

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

[0014] Figure 3 The growth curve of LYZYH-2.

[0015] Figure 4 The results of the LYZYH-2 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] LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, natural pH, prepared with water.

[0018] 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.

[0019] In the embodiments, unless otherwise specified, all methods used were 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 were all conventional products that could be purchased from legitimate channels or prepared according to conventional methods in this field.

[0020] Unless otherwise specified, the solutions of this invention are prepared using water as the solvent.

[0021] The following is a description through specific embodiments. Example 1

[0022] In February 2025, using sterile forceps in a clean bench, 5 g of internal rotten samples were collected by peeling the skin off rotten apples and placing them in 45 mL of sterile water, then thoroughly mixing. After thorough mixing, the mixture was incubated in an 80℃ 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 diluted 10-fold, and serially diluted to obtain 10⁻³-10⁻⁷ dilutions. 50 μL of each 10⁻³-10⁻⁷ rumen dilution was plated onto LB agar plates and incubated upside down at 37℃ for 24 h. After incubation, a single colony was picked and streaked. The target strain was obtained using the above method and named LYZYH-2. Example 2

[0023] 1. 16S rRNA gene analysis of strain LYZYH-2 The 16S rRNA gene sequence was determined using universal primers 27F and 1492R for bacterial 16S rRNA, yielding a 1500bp gene fragment.

[0024] 27F: AGTTTGATCMTGGCTCAG; 1492R: GGTTACCTTGTTACGACTT.

[0025] The 16S rRNA gene sequence of strain LYZYH-2 is as follows:

[0026] Comparison with known strains using the NCBI website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) revealed that strain LYZYH-2 showed the highest similarity to known strains Bacillus cereus ATCC 14579 and Bacillus cereus strain JCM 2152. A phylogenetic tree of strain LYZYH-2 and related taxa based on 16S rRNA gene sequences was constructed, as follows... Figure 1 As shown, LYZYH-2 was ultimately identified as Bacillus cereus.

[0027] 3. Colony morphology characteristics of strain LYZYH-2 like Figure 2 As shown in the figure, after 18 h of incubation at 37°C on LB medium, the colonies of bacteria LYZYH-2 were milky white, with a rough surface, irregular shape, and wrinkles.

[0028] 4. Physiological and biochemical characteristics of strain LYZYH-2 The fatty acid composition of strain GLY6-6 was detected using a rapid identification system for microbial fatty acids (MIDI). The results showed that the main fatty acids of strain GLY6-6 were C15:0 anteiso, C15:0 iso, and C17:0 iso, with contents of 20.34%, 21.18%, and 15.27%, respectively; the minor saturated fatty acids were C16:0 and C14:0 iso, with contents of 8.49% and 5.35%, respectively.

[0029] Analysis using API 50CH revealed the following positive results: D-glucose, D-fructose, D-galactose, glucosamine, ribose, maltose, sucrose, trehalose, raffinose, starch, glycogen, dextrin, glycerol, salicin, and arbutin. Negative results included D-xylose, L-arabinose, rhamnose, cellobiose, lactose, mannitol, sorbitol, cellulose, inulin, melitriose, xylitol, fucose, and arabinitol.

[0030] 5. Measure the growth curve The strain LYZYH-2 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 the OD600 was measured using a microplate reader. Growth curves were plotted. The experimental results are as follows: Figure 3 As shown, LYZYH-2 reached its first plateau at 14 h and continued to grow at 16 h. Example 3

[0031] Fermentation of apple pomace using LYZYH-2: Inoculate apple pomace with LYZYH-2 bacterial solution at an inoculation rate of 6% (i.e., 6 mL of LYZYH-2 bacterial solution per 100 g of apple pomace), ferment at 37℃, and ferment for 72 h.

[0032] The pectin content in fermentation products was determined by the carbazole colorimetric method (GB / T10742—2008).

[0033] Experimental results showed that fermenting apple pomace with LYZYH-2 bacterial solution significantly reduced the pectin content in apple pomace, with a pectin degradation rate of 46.18%. CN107893039B discloses a Bacillus cereus species containing neutral endopeptidase, but it is mainly used in waste paper production. Its environmental adaptability to apple pomace is poor, far less than the pectin degradation rate of LYZYH-2 in apple pomace according to this application.

[0034] 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 cereus, characterized in that, The strain name is Bacillus cereus ( Bacillus cereus LYZYH-2, with accession number CGMCC No.36191.

2. A microbial agent, characterized in that, Includes the Bacillus cereus of claim 1 or the fermentation broth of the Bacillus cereus of claim 1.

3. A method for fermenting apple pomace, characterized in that, Includes the following steps: Apple pomace is fermented using the Bacillus cereus of claim 1 and the inoculum of claim 2.

4. The method according to claim 3, characterized in that, The fermentation conditions were: Bacillus cereus LYZYH-2 inoculum amount 1-5%, temperature 32-42℃, culture medium pH 6-8, and culture time 6-48h.

5. The application of Bacillus cereus in fermented apple pomace, wherein the Bacillus cereus is the Bacillus cereus of claim 1.

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