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Article

Exploring CAZymes Differences in Pediococcus acidilactici Strain OM681363 and Lacticaseibacillus paracasei Strain ON606241 Based on Whole-Genome Sequencing

by
Miao Lin
1,*,†,
Shakib Mohamed Jama
1,†,
Zhiqiang Cheng
1,
Yujie Zong
1,
Yanjing Su
2,
Wengboyang Liu
1 and
Li Liu
1
1
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
2
Bright Farming Co., Ltd., Key Laboratory of Dairy Cattles Genetic Improvement in Southern China Ministry of Agriculture and Rural Affairs Shanghai 200436, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Fermentation 2025, 11(2), 64; https://doi.org/10.3390/fermentation11020064 (registering DOI)
Submission received: 23 November 2024 / Revised: 22 January 2025 / Accepted: 24 January 2025 / Published: 1 February 2025
(This article belongs to the Special Issue Lactic Acid Fermentation and the Colours of Biotechnology: 4th Edition)
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Abstract

:
Lactic acid bacteria (LAB) is a collective term for bacteria capable of producing lactic acid from fermentable carbohydrates. Despite their widespread presence in the gastrointestinal tracts of humans and animals, where they play important physiological roles, functional analysis of specific strains from particular sources requires further enrichment. The objective of this study was to explore the differences between Pediococcus acidilactici OM681363 and Lacticaseibacillus paracasei ON606241, both isolated from the rumen of Chinese Holstein dairy cows, using whole-genome sequencing. The results indicate that P. acidilactici OM681363 contained three CRISPR fragments and numerous enzymes involved in carbohydrate degradation. Additionally, P. acidilactici OM681363 possessed more genes related to fiber degradation, especially cellobiose, and the sole carbon source experiment also confirmed this. However, it lacked genes associated with polysaccharide lyase. In contrast, L. paracasei ON606241 was found to be more specialized in breaking down non-fiber carbohydrates, producing more acetic and lactic acids. Overall, P. acidilactici OM681363 may have a greater capacity to degrade complex carbohydrates, while L. paracasei ON606241 appears to specifically target non-fiber carbohydrates.

1. Introduction

Most lactic acid bacteria (LAB) are Gram-positive, spore-free, catalase-negative, anaerobic microorganisms with a diverse range of species. They can be isolated from various sources, including food, the digestive tract, and plants [1,2,3]. Common probiotic LAB include genera such as Lactobacillus, including species like Lactobacillus fermentum, Lactobacillus plantarum, and Lactobacillus rhamnosus. Other important genera include Lacticaseibacillus, such as Lacticaseibacillus casei and Lacticaseibacillus paracasei, and Lactococcus, which encompasses species like Enterococcus faecalis, Pediococcus pentosaceus, and Pediococcus acidilactici. The probiotic functions of LAB strains vary significantly and can influence host health and metabolism through multiple mechanisms. These include regulating gut microbiota composition and promoting the production of short-chain fatty acids (SCFAs) [4], inhibiting the adhesion of harmful bacteria to the digestive tract epithelium, and reducing inflammation [5,6]. As a result, ongoing research into the functional properties of LAB is essential for the development of probiotic resources and their precise application in promoting human health and enhancing animal production.
The metabolic products of LAB are diverse and include biologically active compounds such as organic acids, phenols, steroids and their derivatives, antimicrobial peptides, and bacteriocins. Although both Enterococcus sp. and Lacticaseibacillus casei are derived from cheese, they produce varying quantities of acetic acid and lactic acid [7]. This underscores the importance of considering the specific characteristics of individual bacterial strains for diverse applications. Pediococcus can be isolated from silage [8,9,10], with certain strains capable of decomposing phenolic aldehydes, leading to increased production of D-lactic acid. This suggests that the bacterium may contribute to the degradation of lignocellulose. Recently, the genus Lactobacillus was reclassified into 23 genera, encompassing over 250 species and 57 subspecies [11]. The probiotic characteristics of Lacticaseibacillus paracasei include modulation of gut microbiota composition and enhancement of gastrointestinal function [12].
However, LAB degrades carbohydrates, proteins, and lipids through various enzyme systems, resulting in the production of different types of metabolites [11]. The fermentation of substrates by LAB is primarily controlled by these enzyme systems and is further regulated by genetic factors, particularly those related to carbohydrate fermentation enzymes. These systems are crucial in determining the selectivity and efficiency of LAB in substrate degradation. In this study, Pediococcus acidilactici OM681363 and Lacticaseibacillus paracasei ON606241, which were isolated from the rumen of dairy cows under anaerobic conditions in our laboratory, were selected for analysis. MRS medium was used to investigate the differences in potential gene functions through whole-genome sequencing. The results of this study may provide valuable insights into the potential applications of these two strains in various biotechnological and industrial processes.

2. Materials and Methods

2.1. Strains

The isolation and identification protocol followed Hu et al. (2022) [12]. The 16S rDNA sequencing results were analyzed using BLAST on the NCBI website, and the GenBank accession numbers for P. acidilactici and L. paracasei are OM681363 and ON606241, respectively. The whole-genome sequences were submitted to the NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 23 January 2025.) under study accession number PRJNA1213984.

2.2. Biological Identification of Strains

2.2.1. Revival and Morphological Observations of Strains

The DeMan, Rogosa and Sharpe (MRS) medium (52.25 g/L) was flushed with carbon dioxide for at least 3 h and then dispensed with 10 mL each in Hungate tubes, and the tube was sealed with butyl rubber stoppers and aluminum caps. In order to remove the oxygen from the medium, the gas exchange procedure (3 cycles) involved vacuum pumping for 3 min, followed by filling with carbon dioxide for 15 s. The medium was then autoclaved at 121 °C for 15 min and cooled to room temperature. Before physical observation, the preserved strains, stored at −80 °C with glycerol (50% v/v), were revived in MRS medium at a 1% inoculum volume (v/v). The bacterial broth was shaken well and incubated at 39 °C for 16–24 h until the OD600 of the broth was approximately 1.0 (or until the logarithmic growth phase was reached). The strain broth was dipped using a 1 μL inoculation loop, streaked on MRS solid medium (agar 1%, w/v), and incubated in an anaerobic incubator at 39 °C for 24 h. The size, color, and morphology of the colonies were recorded. Single colonies were picked and enriched in MRS medium using the same procedure. The size, color, and morphology of the bacteria cell were observed using the Gram staining method.

2.2.2. Catalase Activity and Hemolysis Test

A sterile inoculating loop was used to pick a colony and place it on a glass slide; then, 3% hydrogen peroxide solution was added dropwise. The observation of bubbles within 0.5 min indicates a catalase-positive bacterium, whereas the absence of bubbles signifies a catalase-negative bacterium. Purified single colonies were picked and spot inoculated onto the blood agar plate. The colonies were then incubated at 39 °C for 48 h, and the hemolysis of the colonies was observed. Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were inoculated on the same agar culture plate as the pathogen control, and sterile water was used as a negative control.

2.2.3. Growth Curves and Gas Production Monitoring

The enriched broth was counted using a hemocytometer, Improved Neubauer (XB.K.25., 0.10 mm, 1/400 mm2). After adjusting the concentration to 3 × 109 cfu/mL, the bacteria suspension was inoculated into MRS liquid medium (1%, v/v) and incubated at 39 °C for 24 h. Growth was monitored by recording absorbance values at 600 nm at 0, 3, 6, 9, 12, 24, 36, and 60 h (OD600 value), and gas production was recorded at the same time using a digital gauge (DPG1000B15PSIG-5, CeComp Electronics Inc., Libertyville, IL, USA). The gas volume was calculated according to the following equation:
Vgas = Vj × Ppsi × 0.068004084
where Vgas is the gas volume at 39 °C, ml; Vj is the vial volume headspace of liquid, mL; and Ppsi is the pressure of the vial, psi. Three replicates were performed for each strain.

2.2.4. Chemical Parameter Determination

After 24 h, the broth was centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatant was collected and prepared for the measurement of acetic acid and lactic acid. Metaphosphoric acid was added to the supernatant (supernatant/metaphosphoric acid 5:1, metaphosphoric acid solution containing 60 mmol/L crotonic acid), and the mixed solution was refrigerated at −20 °C overnight. It was then centrifuged again (12,000 rpm, 20 min) and filtered through a 0.22 µm aqueous filter membrane for the determination of acetic acid by gas chromatography. The gas chromatography parameters were as follows: a capillary column of 30 m × 0.32 mm × 0.25 μm, a column temperature of 80 °C, a vaporization temperature of 200 °C, a hydrogen ion flame detector with a detection temperature of 200 °C, a pressure of 60 KPa, a hydrogen pressure of 50 KPa, an oxygen pressure of 50 KPa, and nitrogen as the carrier gas. The injection volume was 1.0 μL. The DL-lactic acid in the broth was determined using a colorimetric method from a kit provided by Nanjing Jiancheng Bioengineering Institutes (Nanjing, China).

2.3. Whole-Genome Sequencing

2.3.1. Sequencing Methods

Total DNA was extracted from 20 mL of enrichment culture, and the genome was sequenced using a combination of the second-generation Illumina NovaSeq 6000 platform. Quality control of the data obtained from second-generation sequencing was performed using Trimmomatic v0.36, which involved checking the base content distribution, GC content distribution, and assessing any potential AT/GC separation. Additionally, the GC distribution and average mass distribution were evaluated. The data were assembled using ABySS v2.2.0 sofeware. The assembled sequences were subjected to BLAST alignment, and an evolutionary tree was constructed using the neighbor-joining method in MEGA 11.0 software. The phylogenetic tree was visualized using Chiplot (https://www.chiplot.online/tvbot.html, accessed on 23 January 2025) [13].

2.3.2. Gene Analysis and Prediction

Coding genes were predicted using GeneMarkSv4.17. tRNA genes and rRNA genes were predicted using tRNAscan-SE v2.04, Barrnap (0.9-dev) and the Rfam database, respectively. The CRISPR finder was used to predict forward repeat sequences and spacer regions in the whole genome.

2.3.3. Gene Function Annotation

The obtained sequences were compared with the NCBI database (NR library) to provide functional annotations. Further comparisons were made with the BacMet (Antibacterial Biocide and Metal Resistance Genes) database, the TCDB (Transporter Classification Database), and the CAZy database to screen for carbohydrate metabolism genes. Protein-coding genes were analyzed against the COG (Clusters of Orthologous Groups), GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases using BLAST, with results filtered before annotating gene functions. Predicted genes were subjected to BLAST comparisons against each database (E-value ≤ 1 × 10−5), and gene functions were annotated by selecting the genes with the highest scores in the BLAST results (identity ≥ 40%, coverage ≥ 40%).

2.4. Identification of Carbohydrate Degradation Ability

2.4.1. Growth with Pure Fiber Source

Glucose, xylan, pectin, cellobiose, and sodium carboxymethyl cellulose solution (10 g/L) was prepared separately in the bottles, and the air in the solution was replaced with CO2 using the same method as above, sterilized. The carbohydrate-free basic culture medium was consisted with peptone (10 g/L), beef extract (8 g/L), yeast extract (4 g/L), and a mineral salt solution according to Menke et al. [14]. After introducing CO2 for approximately 1 h, 8 mL aliquots of carbohydrate-free basic medium were transferred into Hungate tubes. Each tube was sealed with a butyl rubber stopper and covered with an aluminum cap. The oxygen was removed from the medium using the same procure with MRS medium. On the day of inoculation, 1 mL of carbohydrate source solution and 0.1 mL of MRS-revived strain solution were added into the Hungate tube. The mixture was inoculated at 39 °C for 24 h. The OD600 at 0, 2, 3, 6, 8, 12, and 24 h was recorded to draw the plot growth curves.

2.4.2. Corn Straw Degradation Rate

Approximately 0.36 g of corn straw was weighed and placed into a nylon bag (6 cm × 3 cm, 40 μm mesh diameter). After sealing with heat, the bag was placed in a 128 mL serum bottle with 50 mL of mineral salt solution and 5 mL of each bacteria solution. The control group was balanced with 5 mL of sterile water. The bottles were sealed with butyl rubber stoppers and covered with aluminum caps. In order to remove the oxygen from the bottle, the vacuum pumping and CO2 flowing cycle was performed as above. The gas pressure at 24, 48, and 72 h was measured. The fermentation was terminated at 72 h, and the liquid samples were collected to measure concentrations of acetic acid, propionic acid, and butyric acid according to the method of Hu et al. [12]. The nylon bag was rinsed with clean water and dried at 65 °C for 48 h to calculate the degradation rate of corn straw.

2.5. Statistical Analysis

The significance of differences was analyzed using one-way ANOVA in SPSS 21.0 software, with the treatment group as the variable. For indicators not following a normal distribution, non-parametric statistical tests were performed. A p-value of <0.05 was considered statistically significant for determining differences among groups.

3. Results

3.1. Basic Biological Identification

As shown in Figure 1, the colonies of both strains exhibited similar morphologies, appearing white to light brown with predominantly rounded bumps and relatively neat edges, ranging from approximately 0.5 to 2 mm in diameter and catalase-negative. P. acidilactici OM681363 was observed as cocci, primarily in diplococci form, non-motile, and Gram-positive. L. paracasei ON606241 was characterized by its long rod-shaped cells, absence of spores, and Gram-positive nature. Neither bacterium produced colorless transparent zones on the hemolysis plate, indicating that they are hemolysis-negative (Figure 2).
As illustrated in Figure 3A, P. acidilactici OM681363 entered the log phase after 6 h of incubation, reaching the stationary phase at 12 h. In contrast, L. paracasei ON606241 entered the log phase after 9 h of incubation, reaching the stationary phase at 36 h. The gas production curves were consistent with the growth curves (Figure 3B). The gas production was increased to the maximum at 12 h for P. acidilactici OM681363 and 36 h for L. paracasei ON606241. After 24 h of incubation, P. acidilactici OM681363 and L. paracasei ON606241 produced 65.54 mmol/L and 74.94 mmol/L of acetic acid, respectively, along with 45.37 mmol/L and 65.50 mmol/L of lactic acid (Figure 3C).

3.2. Bacterial Genome-Wide Analysis

3.2.1. Basic Genome-Wide Features

The two strains were compared using the NR database. P. acidilactici OM681363 belongs to the Bacillota phylum, Lactobacillaceae family, and Pediococcus genus, with 1941 genes. L. paracasei ON606241 belongs to the Bacillota phylum, Lactobacillaceae family, and Lacticaseibacillus genus. A phylogenetic tree was constructed using MEGA 11.0, revealing that strain OM681363 shares 100% similarity with Pediococcus acidilactici NGRI 0510Q and strain ON606241 has a 99% similarity with Lacticaseibacillus paracasei R094 (Figure 4).
As shown in Table 1, the total number of sequences was 2068 and 3031, respectively, and the total lengths of the genomic fragments for the two strains were 1,793,250 bp and 2,537,370 bp, respectively. The GC content was 43.1% for P. acidilactici OM681363 and 46.9% for L. paracasei ON606241. Chromosome maps of the assembled sequences were generated using CGView software 2.0.3, as shown in Figure 5.

3.2.2. Non-Coding RNA

The tRNAs and rRNAs of the bacteria were analyzed using tRNAscan-SE and Barrnap software, respectively, while other ncRNAs were predicted by comparison with the Rfam database. The ncRNA statistics are presented in Table 2. A total of 56 tRNAs were identified in P. acidilactici OM681363, along with one copy each of 5S rRNA, 16S rRNA, and 23S rRNA. In L. paracasei ON606241, 54 tRNAs were identified, along with one copy each of 5S rRNA, 16S rRNA, and 23S rRNA.

3.2.3. CRISPR Prediction

The predicted CRISPRs in the bacterial genomes are summarized in Table 3. P. acidilactici OM681363 contained three CRISPRs, while L. paracasei ON606241 contained one CRISPR.

3.2.4. Genome-Wide Gene Prediction and Functional Annotation

The numbers of metal resistance genes were 157 and 253, respectively. The total counts of transporters classified by the Transporter Classification Database (TCDB) were 389 and 581, respectively. COG (Cluster of Orthologous Groups) function-based annotation included 1649 genes in P. acidilactici OM681363 and 2278 genes in L. paracasei ON606241. According to Gene Ontology (GO) annotation, the number of metabolic genes was 544 in P. acidilactici OM681363 and 636 in L. paracasei ON606241. Based on KEGG (Kyoto Encyclopedia of Genes and Genomes) annotation, the number of genes involved in metabolic pathways was 1108 for P. acidilactici OM681363 and 1426 for L. paracasei ON606241.
As shown in Figure 6, the genes of P. acidilactici OM681363 involved in KEGG annotation were distributed as follows: metabolism (79.80%), genetic information processing (10.5%), environmental information processing (3.51%), cellular processes (2.58%), and organismal systems (1.75%). The genes of L. paracasei ON606241 involved in KEGG annotation were distributed as follows: metabolism (79.67%), genetic information processing (10.63%), environmental information processing (6.84%), cellular processes (3.28%), and organismal systems (0.29%). In addition, the primary functions of the core gene set include global and overview maps, carbohydrate metabolism, nucleoside metabolism, and amino acid metabolism.

3.2.5. Comparison of Carbohydrate Synthesis and Degradation Enzyme

The genes related to carbohydrate synthesis and degradation were 80 enzymes in P. acidilactici OM681363 and 107 enzymes in L. paracasei ON606241. P. acidilactici OM681363 had no polysaccharide lyase (PL) genes (Figure 7), whereas L. paracasei ON606241 had two PL families, PL8 and 12. P. acidilactici OM681363 contained 23 classes of glycosyltransferases (GT), while L. paracasei ON606241 had 34. P. acidilactici OM681363 had 65 classes of glycoside hydrolases (GHs), while L. paracasei ON606241 had 45. P. acidilactici OM681363 possessed 9 carbohydrate esterases (CEs), whereas L. paracasei ON606241 had 17. There were five carbohydrate-binding modules (CBM) in P. acidilactici OM681363 and two in L. paracasei ON606241. Additionally, there were five auxiliary activities (AA) in P. acidilactici OM681363 and eight in L. paracasei ON606241.
In detail, the carbohydrate synthesis and degradation genes specific to P. acidilactici OM681363 included GT26, GH2, GH18, GH43, GH63, GH123, GH170, CE3, CE7, CE20, CBM16, CBM32, CBM56, and AA10 families (Table 4). The genes specific to L. paracasei ON606241 included PL8, PL12, GT5, GT83, GH4, GH13, GH20, GH36, GH39, GH65, GH94, GH136, CE4, CE10, CE12, AA3, and AA4 families.

3.2.6. Fibrous Carbohydrate Degradation Ability

As shown in Figure 8, the OD600 values of both strains were similar in media without and with a carbon source when xylan, pectin, and sodium carboxymethyl cellulose were used as the sole carbon sources. In contrast, a gradual increase in OD600 was observed for P. acidilactici OM681363 when glucose and cellobiose were used as sole carbon sources. The OD600 reached peak values in this experiment at the 12th hour and was even higher than that of the glucose group at 8 and 12 h.
For L. paracasei ON606241, although a gradual increase in absorbance was observed in the medium with glucose as the sole carbon source, the values were similar to those of P. acidilactici OM681363. However, in the cellobiose-added medium, the absorbance exceeded that of the control group only at the 24th hour but remained lower than that of the glucose medium.
As shown in Table 5, although the addition of the two bacterial strains did not significantly affect the 72 h dry matter degradation rate of corn straw, the substrate degradation rate in the P. acidilactici OM681363 group increased by 10.06% compared to the control group. In co-culture with non-sterilized solid substrates and culture medium, gas production gradually increased over 24, 48, and 72 h, with the P. acidilactici OM681363 group showing higher gas production than the control group.
The addition of lactic acid bacteria significantly increased the production of acetic acid, propionic acid, and butyric acid during corn straw fermentation. Notably, butyric acid production was higher with P. acidilactici OM681363 addition, while L. paracasei ON606241 exhibited higher acetic acid production (p < 0.01).

4. Discussion

In this experiment, the growth curve of P. acidilactici OM681363 was similar to that observed in Fugaban et al.’s research, entering the logarithmic phase after 3 h and reaching the stationary phase around 12 h at 37 °C [10]. The growth rate of Lactobacillus paracasei CCUG 32212 was found to be faster than those of Lactobacillus rhamnosus ATCC 7469 and Lactobacillus plantarum ATCC 1491 [15]. In MRS medium, L. rhamnosus LHL6 and LHL7 exhibits a 12 h lag phase at 37 °C [16]. The optimal growth temperature for most lactic acid bacteria is between 30 and 40 °C. P. acidilactici LC-9-1 cultured under anaerobic conditions and 39 °C exhibits good probiotic functions for poultry [17]. The strains isolated from rumen, which has a normal temperature of 39 °C, demonstrated a faster growth rate in this experiment.
LAB produces a variety of bacteriostatic substances, including organic acids, hydrogen peroxide, and bacteriocins, which inhibit the growth of spoilage bacteria and pathogenic microorganisms [18]. High lactic acid production is a key factor in ensuring the quality of silage [19], as significant lactic acid accumulation rapidly reduces the pH value, thereby inhibiting the proliferation of harmful bacteria [4]. In Xu et al.’s study, the pH of high-quality corn silage was found to be 3.92 ± 0.02, with a lactic acid concentration of 66.75 ± 1.97 g/kg of dry matter [20]. Acetic acid is known to increase the aerobic stability of silage [21], whereas propionic acid is associated with dry matter loss [22]. P. acidilactici V202 isolated from the rumen of goats has a greater ability to decrease pH compared to Enterococcus faecium strain [23]. Additionally, when lactating cows were fed corn silage containing LAB, the digestibility of the total mixed diet increased, resulting in higher milk production [24]. In this study, L. paracasei ON606241 produced more lactic acid and acetic acid than P. acidilactici OM681363 at 24 h. These results suggest that applying L. paracasei ON606241 may result in higher silage quality compared to P. acidilactici OM681363. Lactic acid bacteria produce volatile fatty acids, hydrogen, and carbon dioxide under anaerobic conditions. The amounts of these substances produced vary depending on the strain and the fermentation substrate [25].
Lactic acid bacteria isolated from the rumen and further studied for their genetic and functional differences are rarely seen in the literature. The comparative genomic analysis of 41 strains of P. acidilactici by Li et al. [26] demonstrated that all strains produce bacteriocins and can metabolize different carbohydrate sources, and all strains had no or only one CRISPR. Genome editing based on CRISPR/Cas9 is has been rapidly developing technology in recent years, which can greatly improve the accuracy of target sites in LAB [27]. However, in this study, P. acidilactici OM681363 had three CRISPRs and L. paracasei ON606241 had one CRISPR, indicating that both strains may have potential for gene engineering.
A study conducted by Niu et al. showed that the genes of P. acidilactici in KEGG annotation are involved in cellular processes (2.63%), human diseases (3.68%), environmental information processing (10.35%), genetic information processing (11.8%), and metabolism (69.84%) [28]. According to KEGG analysis, both strains in this study had a high proportion of genes involved in metabolism (79.80% and 79.67%). Notably, in the CAZymes database, the strains showed various carbohydrate-related enzymes involved in synthesis and degradation. Glycosyltransferases (GTs) facilitate the transfer of sugar moieties from activated donors to specific acceptors, such as proteins, lipids, or other polysaccharides, forming glycosidic linkages. In this study, both strains showed higher levels of GT2 and GT4 than the other CT families, which may indicate a strong function in cellulose synthase, chitin synthase, sucrose synthase, etc.
Carbohydrate esterases (CEs) catalyze the de-esterification of various carbohydrate substrates. In this experiment, both strains were found to have abundant acetyl xylan esterase [29], including CE3, CE5, and CE7 specific to P. acidilactici OM681363 and CE4 and CE12 specific to L. paracasei ON606241. In this experiment, both strains exhibited the highest numbers of GH enzymes. Glycoside hydrolases (GHs) catalyze the hydrolysis of glycosidic bonds in a wide range of carbohydrates. The GH32 family is β-D-fructofuranosidase, which functions in hydrolyzing fructans and is widely present across microbes [30]. L. paracasei ON606241 had 2 enzymes in the GH32 family, indicating this strain has a greater ability to digest fructans than P. acidilactici OM681363. Chitin, a structural polysaccharide crucial in insects and fungi, is synthesized by highly conserved enzymes. Notably, P. acidilactici OM681363 in this study not only contains chitin synthase (CT2) but also possesses unique chitinase (GH18). GH43 is a beta-xylosidase, prominently found in the ruminal Bacteroidets phylum, where it plays a key role in digesting hemicellulose [31]. In addition, in this study, P. acidilactici OM681363 was found to have specific carbohydrate-binding modules (CBMs) 16, 32, and 56. CBM16 binds to cellulose and glucomannan; CBM32 binds to galactose and lactose; CBM56 binds to beta-1,3-glucan. Auxiliary activities (AAs) include a class of redox enzymes that act on carbohydrates. AA10 proteins are copper-dependent lytic polysaccharide monooxygenases [32]. Additionally, lytic cellulose monooxygenases (C1-hydroxylating) have been shown to act on both chitin and cellulose [33]. In this experiment, P. acidilactici OM681363 did not possess polysaccharide lyase (PL), an enzyme that breaks down polysaccharides containing uronic acid. In contrast, L. paracasei ON606241 has genes encoding hyaluronate (PL8) and chondroitin (PL12), indicating its potential to degrade these carbohydrates, specifically P. acidilactici OM681363 encoding no GH13, which is the main alpha-amylase family [34]; however, L. paracasei ON606241 has eight specific GH13 genes. L. paracasei ON606241 is unique in its possession of maltose-6-phosphate glucosidase (GH4), alpha-galactosidase (GH36), cellobiose phosphorylase (GH94), cellulose dehydrogenase (AA3), and vanillyl-alcohol oxidase, respectively (AA4). Therefore, the specific presence of GH18, GH43, AA10, and CBM families suggests that P. acidilactici OM681363 may have the potential to degrade fibrous polysaccharides. In contrast, the specific presence of genes encoding the GH13 family enables L. paracasei ON606241 to break down more non-fiber polysaccharides.
The incubation trial in this study observed that P. acidilactici OM681363 grew well in cellobiose as the sole carbohydrate source and showed a higher degradation rate of corn straw, indicating that this strain has the ability to digest fiber. Wan et al. expressed Endoglucanase (EG) and cellobiohydrolase (CBH) cloned from the rumen of yak in Lactococcus lactis NZ9000, demonstrating that the genetically engineered strain decreased the neutral detergent fiber content in whole-plant corn silage [35]. This study suggests that P. acidilactici OM681363 has advantages in fermenting a wider variety of carbohydrates than L. paracasei ON606241. Although there are fewer reports indicating the role of P. acidilactici in degrading fiber-rich plants, P. acidilactici OM681363, isolated in this experiment, could be explored for its potential in plant fiber degradation in the future, such as in silage, with the aim of improving fermentation quality.

5. Conclusions

The analysis of two LAB strains isolated from the rumen revealed differences in growth, metabolite production, and whole-genome functions. After anaerobic cultivation at 39 °C for 24 h in MRS broth, P. acidilactici OM681363 grew faster; however, L. paracasei ON606241 was found to produce higher levels of lactic acid and acetic acid. The core functions of both bacterial strains are centered on carbohydrate metabolism, nucleoside metabolism, and amino acid metabolism. Compared to L. paracasei ON606241, P. acidilactici OM681363 lacks genes related to the polysaccharide lyases and the glycoside hydrolase 13 family but possesses more genes encoding glycoside hydrolases 18, and 43 and carbohydrate-binding modules. Polysaccharide degradation experiments demonstrated that P. acidilactici OM681363 can utilize cellobiose as a sole carbohydrate source and contribute to the degradation of complex carbohydrates, such as corn straw. This suggests that P. acidilactici OM681363 may have potential for reducing plant fiber fraction, while L. paracasei ON606241 may have potential for fermenting starch or other easily fermented carbohydrates in plant.

Author Contributions

Conceptualization, M.L.; Methodology, S.M.J. and Y.Z.; Formal Analysis, S.M.J., L.L. and W.L.; Data Curation, S.M.J. and Y.Z.; Writing—Original Draft Preparation, M.L., S.M.J. and Z.C.; Writing—Review and Editing, M.L. and Y.S.; Project Administration, M.L. and Y.S.; Funding Acquisition, M.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the earmarked fund for CARS (CARS-36).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article. The whole-genome sequences were submitted to the NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 23 January 2025) under study accession number: PRJNA1213984. The data that support the findings of this study are available from the corresponding author upon reasonable request.

Conflicts of Interest

Author Yanjing Su is employed by the company Bright Farming Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Peng, C.; Sun, W.; Dong, X.; Zhao, L.; Hao, J. Isolation, Identification and Utilization of Lactic Acid Bacteria from Silage in a Warm and Humid Climate Area. Sci. Rep. 2021, 11, 12586. [Google Scholar] [CrossRef]
  2. Quilodrán-Vega, S.R.; Muñoz-Flores, C.; Pino, A.; Buldres, P.; Sandoval, F.; Aguirre, A.; Portillo, B.; Parra, N.; Altamirano, C.; Albarracín, L.; et al. Isolation, Characterization, and Immunomodulatory Activity Evaluation of Probiotic Strains from Colostrum and Canine Milk. Front. Vet. Sci. 2023, 10, 1266064. [Google Scholar] [CrossRef]
  3. Keresztény, T.; Libisch, B.; Orbe, S.C.; Nagy, T.; Kerényi, Z.; Kocsis, R.; Posta, K.; Papp, P.P.; Olasz, F. Isolation and Characterization of Lactic Acid Bacteria with Probiotic Attributes from Different Parts of the Gastrointestinal Tract of Free-Living Wild Boars in Hungary. Probiot. Antimicrob. Proteins 2024, 16, 1221–1239. [Google Scholar] [CrossRef] [PubMed]
  4. Li, J.; Wang, W.; Chen, S.; Shao, T.; Tao, X.; Yuan, X. Effect of Lactic Acid Bacteria on the Fermentation Quality and Mycotoxins Concentrations of Corn Silage Infested with Mycotoxigenic Fungi. Toxins 2021, 13, 699. [Google Scholar] [CrossRef]
  5. Nishiyama, K.; Sugiyama, M.; Mukai, T. Adhesion Properties of Lactic Acid Bacteria on Intestinal Mucin. Microorganisms 2016, 4, 34. [Google Scholar] [CrossRef] [PubMed]
  6. Han, X.; Ding, S.; Ma, Y.; Fang, J.; Jiang, H.; Li, Y.; Liu, G. Lactobacillus plantarum and Lactobacillus brevis Alleviate Intestinal Inflammation and Microbial Disorder Induced by ETEC in a Murine Model. Oxid. Med. Cell. Longev. 2021, 2021, 6867962. [Google Scholar] [CrossRef] [PubMed]
  7. Silva, L.F.; Sunakozawa, T.N.; Monteiro, D.A.; Casella, T.; Conti, A.C.; Todorov, S.D.; Penna, A.L.B. Potential of Cheese-Associated Lactic Acid Bacteria to Metabolize Citrate and Produce Organic Acids and Acetoin. Metabolites 2023, 13, 1134. [Google Scholar] [CrossRef]
  8. Mansour, N.M.; Elkalla, W.S.; Ragab, Y.M.; Ramadan, M.A. Inhibition of Acetic Acid-Induced Colitis in Rats by New Pediococcus Acidilactici Strains, Vitamin Producers Recovered from Human Gut Microbiota. PLoS ONE 2021, 16, 0255092. [Google Scholar] [CrossRef] [PubMed]
  9. Fugaban, J.I.I.; Bucheli, J.E.V.; Kim, B.; Holzapfel, W.H.; Todorov, S.D. Safety and Beneficial Properties of Bacteriocinogenic Pediococcus acidilactici and Pediococcus pentosaceus Isolated from Silage. Lett. Appl. Microbiol. 2021, 73, 725–734. [Google Scholar] [CrossRef]
  10. Fugaban, J.I.I.; Bucheli, J.E.V.; Park, Y.J.; Suh, D.H.; Jung, E.S.; Ivanova, I.V.; Holzapfel, W.H.; Todorov, S.D. Antimicrobial Properties of Pediococcus acidilactici and Pediococcus pentosaceus Isolated from Silage. J. Appl. Microbiol. 2022, 132, 311–330. [Google Scholar] [CrossRef] [PubMed]
  11. Pfeiler, E.A.; Klaenhammer, T.R. The Genomics of Lactic Acid Bacteria. Trends Microbiol. 2017, 15, 546–553. [Google Scholar] [CrossRef]
  12. Hu, G.; Jiang, H.; Zong, Y.; Datsomor, O.; Kou, L.; An, Y.; Zhao, J.; Miao, L. Characterization of Lactic Acid-Producing Bacteria Isolated from Rumen: Growth, Acid and Bile Salt Tolerance, and Antimicrobial Function. Fermentation 2022, 8, 385. [Google Scholar] [CrossRef]
  13. Xie, J.; Chen, Y.; Cai, G.; Cai, R.; Hu, Z.; Wang, H. Tree Visualization By One Table (tvBOT): A Web Application for Visualizing, Modifying and Annotating Phylogenetic Trees. Nucleic Acids Res. 2023, 51, W587–W592. [Google Scholar] [CrossRef] [PubMed]
  14. Menke, K.H.; Raab, L.; Salewski, A.; Steingass, H.; Fritz, D.; Schneider, W. The Estimation of the Digestibility and Metabolizable Energy Content of Ruminant Feedingstuffs from the Gas Production When They Are Incubated with Rumen Liquor in Vitro. J. Agric. Sci. Camb. 1979, 93, 217–222. [Google Scholar] [CrossRef]
  15. Piwat, S.; Teanpaisan, R.; Dahlén, G.; Thitasomakul, S.; Douglas, C.W.I. Acid Production and Growth by Oral Lactobacillus Species in Vitro. J. Investig. Clin. Dent. 2012, 3, 56–61. [Google Scholar] [CrossRef]
  16. Kang, W.; Pan, L.; Peng, C.; Dong, L.; Cao, S.; Cheng, H.; Wang, Y.; Zhang, C.; Gu, R.; Wang, J.; et al. Isolation and Characterization of Lactic Acid Bacteria from Human Milk. J. Dairy Sci. 2020, 103, 9980–9991. [Google Scholar] [CrossRef] [PubMed]
  17. Li, C.; Wang, S.; Chen, S.; Wang, X.; Deng, X.; Liu, G.; Chang, W.; Beckers, Y.; Cai, H. Screening and Characterization of Pediococcus acidilactici LC-9-1 toward Selection as a Potential Probiotic for Poultry with Antibacterial and Antioxidative Properties. Antioxidants 2023, 12, 215. [Google Scholar] [CrossRef]
  18. Gueimonde, M.; Sánchez, B.; de Los Reyes-Gavilán, C.G.; Margolles, A. Antibiotic Resistance in Probiotic Bacteria. Front. Microbiol. 2013, 4, 202. [Google Scholar] [CrossRef]
  19. Ennahar, S.; Cai, Y.; Fujita, Y. Phylogenetic Diversity of Lactic Acid Bacteria Associated with Paddy Rice Silage as Determined by 16S Ribosomal DNA Analysis. Appl. Environ. Microbiol. 2003, 69, 444–451. [Google Scholar] [CrossRef] [PubMed]
  20. Xu, Z.; Zhang, S.; Zhang, R.; Li, S.; Kong, J. The Changes in Dominant Lactic Acid Bacteria and Their Metabolites during Corn Stover Ensiling. J. Appl. Microbiol. 2018, 125, 675–685. [Google Scholar] [CrossRef]
  21. Danner, H.; Holzer, M.; Mayrhuber, E.; Braun, R. Acetic Acid Increases Stability of Silage under Aerobic Conditions. Appl. Environ. Microbiol. 2003, 69, 562–567. [Google Scholar] [CrossRef] [PubMed]
  22. Killerby, M.A.; Almeida, S.T.R.; Hollandsworth, R.; Guimaraes, B.C.; Leon-Tinoco, A.; Perkins, L.B.; Henry, D.; Schwartz, T.J.; Romero, J.J. Effect of Chemical and Biological Preservatives and Ensiling Stage on the Dry Matter Loss, Nutritional Value, Microbial Counts, and Ruminal in Vitro Gas Production Kinetics of Wet Brewer’s Grain Silage. J. Anim. Sci. 2022, 100, skac095. [Google Scholar] [CrossRef] [PubMed]
  23. Hwanhlem, N.; Salaipeth, L.; Charoensook, R.; Kanjan, P.; Maneerat, S. Lactic Acid Bacteria from Gamecock and Goat Originating from Phitsanulok, Thailand: Isolation, Identification, Technological Properties and Probiotic Potential. J. Microbiol. Biotechnol. 2022, 32, 355–364. [Google Scholar] [CrossRef]
  24. Daniel, J.L.P.; Queiroz, O.C.M.; Arriola, K.G.; Daetz, R.; Basso, F.; Romero, J.J.; Adesogan, A.T. Effects of Homolactic Bacterial Inoculant on the Performance of Lactating Dairy Cows. J. Dairy Sci. 2018, 101, 5145–5152. [Google Scholar] [CrossRef] [PubMed]
  25. Rombouts, J.L.; Kranendonk, E.M.M.; Regueira, A.; Weissbrodt, D.G.; Kleerebezem, R.; Van Loosdrecht, M.C.M. Selecting for Lactic Acid Producing and Utilising Bacteria in Anaerobic Enrichment Cultures. Biotech Bioeng. 2020, 117, 1281–1293. [Google Scholar] [CrossRef] [PubMed]
  26. Li, Z.; Song, Q.; Wang, M.; Ren, J.; Liu, S.; Zhao, S. Comparative Genomics Analysis of Pediococcus acidilactici Species. J. Microbiol. 2021, 59, 573–583. [Google Scholar] [CrossRef] [PubMed]
  27. Mu, Y.; Zhang, C.; Li, T.; Jin, F.-J.; Sung, Y.-J.; Oh, H.-M.; Lee, H.-G.; Jin, L. Development and Applications of CRISPR/Cas9-Based Genome Editing in Lactobacillus. Int. J. Mol. Sci. 2022, 23, 12852. [Google Scholar] [CrossRef]
  28. Niu, T.; Jiang, Y.; Fan, S.; Yang, G.; Shi, C.; Ye, L.; Wang, C. Antiviral Effects of Pediococcus acidilactici Isolated from Tibetan Mushroom and Comparative Genomic Analysis. Front. Microbiol. 2022, 13, 1069981. [Google Scholar] [CrossRef]
  29. Puchart, V.; Biely, P. Microbial Xylanolytic Carbohydrate Esterases. Essays Biochem. 2023, 67, 479–491. [Google Scholar]
  30. Cheng, X.; Liu, X.; Jordan, K.W.; Yu, J.; Whitworth, R.J.; Park, Y.; Chen, M.-S. Frequent Acquisition of Glycoside Hydrolase Family 32 (GH32) Genes from Bacteria via Horizontal Gene Transfer Drives Adaptation of Invertebrates to Diverse Sources of Food and Living Habitats. Int. J. Mol. Sci. 2024, 25, 8296. [Google Scholar] [CrossRef]
  31. Xue, M.-Y. Investigation of Fiber Utilization in the Rumen of Dairy Cows Based on Metagenome-Assembled Genomes and Single-Cell RNA Sequencing. Microbiome 2022, 10, 11. [Google Scholar] [CrossRef] [PubMed]
  32. Meier, K.K.; Jones, S.M.; Kaper, T.; Hansson, H.; Koetsier, M.J.; Karkehabadi, S.; Solomon, E.I.; Sandgren, M.; Kelemen, B. Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars. Chem. Rev. 2018, 118, 2593–2635. [Google Scholar] [CrossRef] [PubMed]
  33. Forsberg, Z.; Sørlie, M.; Petrović, D.; Courtade, G.; Aachmann, F.L.; Vaaje-Kolstad, G.; Bissaro, B.; Røhr, Å.K.; Eijsink, V.G. Polysaccharide Degradation by Lytic Polysaccharide Monooxygenases. Curr. Opin. Struct. Biol. 2019, 59, 54–64. [Google Scholar] [CrossRef]
  34. Lombard, V.; Ramulu, H.; Drula, E.; Coutinho, P.M.; Henrissat, B. The Carbohydrate-Active Enzymes Database (CAZy) in 2013. Nucl. Acids Res. 2014, 42, D490–D495. [Google Scholar] [CrossRef]
  35. Wan, X.; SunKang, Y.; Chen, Y.; Zhang, Z.; Gou, H.; Xue, Y.; Wang, C.; Wei, Y.; Yang, Y. Co-Expression of Endoglucanase and Cellobiohydrolase from Yak Rumen in Lactic Acid Bacteria and Its Preliminary Application in Whole-Plant Corn Silage Fermentation. Front. Microbiol. 2024, 15, 1442797. [Google Scholar] [CrossRef] [PubMed]
Fermentation 11 00064 g001aFermentation 11 00064 g001b
Figure 1. Morphology of the two strains cultured in MRS medium. (A) Colonies of P. acidilactici OM681363 on agar plate, (B) Gram staining of P. acidilactici OM681363, (C) colonies of L. paracasei ON606241 on agar plate, (D) Gram staining of L. paracasei ON606241.
Figure 1. Morphology of the two strains cultured in MRS medium. (A) Colonies of P. acidilactici OM681363 on agar plate, (B) Gram staining of P. acidilactici OM681363, (C) colonies of L. paracasei ON606241 on agar plate, (D) Gram staining of L. paracasei ON606241.
Fermentation 11 00064 g001aFermentation 11 00064 g001b
Fermentation 11 00064 g002
Figure 2. The Hemolysis Test of P. acidilactici OM681363 and L. paracasei ON606241. The three colonies in the same row were inoculated with the same bacterium. (a) Escherichia coli ATCC 25922, (b) Staphylococcus aureus ATCC 29213, (c) P. acidilactici OM681363, (d) L. paracasei ON606241, (e) sterile water.
Figure 2. The Hemolysis Test of P. acidilactici OM681363 and L. paracasei ON606241. The three colonies in the same row were inoculated with the same bacterium. (a) Escherichia coli ATCC 25922, (b) Staphylococcus aureus ATCC 29213, (c) P. acidilactici OM681363, (d) L. paracasei ON606241, (e) sterile water.
Fermentation 11 00064 g002
Fermentation 11 00064 g003
Figure 3. The growth and organic acid production of the two strains in MRS medium. SE was used as the error bar in all three figures. * means significant (p < 0.05). (A) the OD600 value during 60 h incubation, (B) the gas production during 60 h incubation, (C) the acetic acid and lactic acid production at 24 h incubation.
Figure 3. The growth and organic acid production of the two strains in MRS medium. SE was used as the error bar in all three figures. * means significant (p < 0.05). (A) the OD600 value during 60 h incubation, (B) the gas production during 60 h incubation, (C) the acetic acid and lactic acid production at 24 h incubation.
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Fermentation 11 00064 g004
Figure 4. Neighbor-joining phylogenetic tree of P. acidilactici OM681363 and L. paracasei ON606241.
Figure 4. Neighbor-joining phylogenetic tree of P. acidilactici OM681363 and L. paracasei ON606241.
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Fermentation 11 00064 g005aFermentation 11 00064 g005b
Figure 5. Chromosome maps of the assembled sequences, drawn using CGView software. (A) P. acidilactici OM681363, (B) L. paracasei ON606241.
Figure 5. Chromosome maps of the assembled sequences, drawn using CGView software. (A) P. acidilactici OM681363, (B) L. paracasei ON606241.
Fermentation 11 00064 g005aFermentation 11 00064 g005b
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Figure 6. KEGG annotation of the two strains. (A) Metabolism, (B) genetic information processing, (C) environmental information processing, (D) cellular processes, (E) organismal system.
Figure 6. KEGG annotation of the two strains. (A) Metabolism, (B) genetic information processing, (C) environmental information processing, (D) cellular processes, (E) organismal system.
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Figure 7. The gene numbers of the two strains related to carbohydrate synthesis and degradation. PL: Polysaccharide lyases; GT: glycosytransferases; GH: glycoside hydrolases; CE: carbohydrate esterases; CBM: carbohydrate-binding modules; AA: auxiliary activities.
Figure 7. The gene numbers of the two strains related to carbohydrate synthesis and degradation. PL: Polysaccharide lyases; GT: glycosytransferases; GH: glycoside hydrolases; CE: carbohydrate esterases; CBM: carbohydrate-binding modules; AA: auxiliary activities.
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Figure 8. Growth (OD600) of the two strains cultured in different carbon source media for 24 h.
Figure 8. Growth (OD600) of the two strains cultured in different carbon source media for 24 h.
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Table 1. Basic information on genomic fragments.
Table 1. Basic information on genomic fragments.
P. acidilactici OM681363L. paracasei ON606241
Gene number20683031
Gene total length (kb)1,793,2502,537,370
GC content in gene region43.1%46.9%
Table 2. The different numbers and average length of ncRNA between the two stains.
Table 2. The different numbers and average length of ncRNA between the two stains.
ncRNA TypeNumberAverage Len (bp)Length/Genome (%)
acidilactici OM681363tRNA56750.2%
5S rRNA11150%
16S rRNA115650.07%
23S rRNA129210.14%
L. paracasei ON606241tRNA54750.13%
5S rRNA11160%
16S rRNA114670.04%
23S rRNA136520.12%
Table 3. The CRISPR positions and bp lengths of the two stains.
Table 3. The CRISPR positions and bp lengths of the two stains.
StartEndRepeatNumAverRepeatLenSpacerNumAverSpacerLen
P. acidilactici OM681363315,248319,36271237035
319,961320,5051035921
53,98955,34421362030
L. paracasei ON60624132,89734,78029362830
Table 4. Carbohydrate synthesis and degradation enzymes.
Table 4. Carbohydrate synthesis and degradation enzymes.
TypeEnzymesStrains
P. acidilactici OM681363L. paracasei ON606241
Polysaccharide lyasesPL801
PL1201
GlycosytransferasesGT2913
GT4611
GT501
GT822
GT2610
GT2710
GT2811
GT3211
GT3501
GT5123
GT8301
Glycoside hydrolasesGH165
GH230
GH312
GH401
GH1308
GH1810
GH2001
GH2310
GH2526
GH2923
GH3122
GH3202
GH3511
GH3602
GH3822
GH3901
GH4310
GH6320
GH6501
GH7321
GH7820
GH8801
GH9401
GH10913
GH12310
GH12511
GH13601
GH17020
Carbohydrate esterasesCE128
CE211
CE310
CE401
CE520
CE710
CE922
CE1004
CE1201
Carbohydrate-binding modulesCBM1610
CBM3210
CBM5062
CBM5610
Auxiliary activitiesAA111
AA302
AA401
AA622
AA712
AA1010
Table 5. Fermentation parameters in corn straws cultured with the two strains of lactic acid bacteria.
Table 5. Fermentation parameters in corn straws cultured with the two strains of lactic acid bacteria.
ItemsControlP. acidilactici OM681363L. paracasei ON606241SEp-Value
Degradation rate (72 h, %)59.6365.6358.324.430.21
Gas production (mL/g DM)
24 h35.7755.7256.774.320.06
48 h53.9488.3494.5111.990.06
72 h61.65 b119.91 a93.90 ab13.450.04
VFA production (72 h, mmol/L)
Acetic acid6.80 c12.64 b17.25 a0.58<0.01
Propionic acid0.355.508.212.040.06
Butyric acid2.67 b10.89 a3.26 b0.74<0.01
Different lowercase letters in the same row indicate significant differences (p < 0.05). VFA = volatile fatty acid, including acetic acid, propionic acid, and butyric acid. SE = standard error.
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Lin, M.; Jama, S.M.; Cheng, Z.; Zong, Y.; Su, Y.; Liu, W.; Liu, L. Exploring CAZymes Differences in Pediococcus acidilactici Strain OM681363 and Lacticaseibacillus paracasei Strain ON606241 Based on Whole-Genome Sequencing. Fermentation 2025, 11, 64. https://doi.org/10.3390/fermentation11020064

AMA Style

Lin M, Jama SM, Cheng Z, Zong Y, Su Y, Liu W, Liu L. Exploring CAZymes Differences in Pediococcus acidilactici Strain OM681363 and Lacticaseibacillus paracasei Strain ON606241 Based on Whole-Genome Sequencing. Fermentation. 2025; 11(2):64. https://doi.org/10.3390/fermentation11020064

Chicago/Turabian Style

Lin, Miao, Shakib Mohamed Jama, Zhiqiang Cheng, Yujie Zong, Yanjing Su, Wengboyang Liu, and Li Liu. 2025. "Exploring CAZymes Differences in Pediococcus acidilactici Strain OM681363 and Lacticaseibacillus paracasei Strain ON606241 Based on Whole-Genome Sequencing" Fermentation 11, no. 2: 64. https://doi.org/10.3390/fermentation11020064

APA Style

Lin, M., Jama, S. M., Cheng, Z., Zong, Y., Su, Y., Liu, W., & Liu, L. (2025). Exploring CAZymes Differences in Pediococcus acidilactici Strain OM681363 and Lacticaseibacillus paracasei Strain ON606241 Based on Whole-Genome Sequencing. Fermentation, 11(2), 64. https://doi.org/10.3390/fermentation11020064

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