Application of Bacillus velezensis JWBIO-01

Application of the JWBIO-01 microbial formulation

Strain JWBIO-01 is a probiotic bacterium isolated from the intestinal tract of the speckled bamboo shark. It has been identified as Bacillus velezensis, previously known as Bacillus methylotrophicus and Bacillus amyloliquefaciens. The strain is preserved at the General Microbiological Center of the China National Committee for the Preservation of Microorganisms, with the accession number CGMCC No. 27495.

1. Strain Characteristics

This bacterium is a Gram-positive, rod-shaped organism. As shown in Figures 1–3, Bacillus velezensis possesses a rich array of enzymatic activities, including β‑xylosidase, α‑galactosidase, β‑glucosidase, β‑glucanase, and α‑glucosidase, which can help the host efficiently degrade and absorb feed, enhance feed utilization, and accelerate weight gain.

Figure: 1-1 Colony

Figure: 1-2 Morphology of the bacterial cell

Figure 1-3: Identification of Physiological and Biochemical Characteristics

1. Analysis of Antimicrobial Active Substances in Strains

Lipopeptides are amphiphilic molecules that exhibit both hydrophobic and hydrophilic properties. They primarily exert non‑specific modes of action against other bacteria, thereby avoiding the emergence of resistant strains, and hold promise as alternatives to conventional antibiotics, making them a current research and development hotspot. The major classes include surfactins, iturins, and fengycins. These compounds are environmentally friendly, characterized by low toxicity, minimal irritation, and biodegradability, and demonstrate broad biological activities, including antibacterial, antifungal, antiviral, and antitumor effects. Consequently, they find extensive applications in agriculture, food and health products, cosmetics, and pharmaceuticals.

This strain contains a wealth of antimicrobial compounds, including macromolecular antimicrobial proteins, small-molecule antimicrobial peptides, numerous enzymes, and various antimicrobial lipopeptides. Accordingly, we conducted an analytical study on one of the major classes of antimicrobial active ingredients—lipopeptides. Our findings, as illustrated in Figures 2‑2 and 2‑3, indicate that this strain is capable of producing multiple lipopeptide‑type bioactive substances, such as iturins, surfactins, fengycins, bacilysins, bacillomycins, mycosubtilins, and others.

Figure 2-1: Common Lipopeptide Genes and Primers

Figure 2-2: Lipopeptide PCR Results

Figure 2-3: Mass spectrum of lipopeptide identification

1. Antibacterial Activity Assay of the Active Substance

Analysis of Figures 3-1, 3-2, and 3-3 reveals that this strain effectively inhibits pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, and Clostridium perfringens. Furthermore, as shown in Figures 3-4 and 3-5, this strain exhibits strong antifungal activity against fungi such as Aspergillus niger and Fusarium oxysporum.

Figure 3-1: Inhibitory effect on Staphylococcus aureus

Figure 3-2: Inhibitory effect on Clostridium perfringens

Figure 3-3: Inhibitory Effect on Escherichia coli

Figure 3-4: Inhibitory effect on Fusarium oxysporum

Figure 3-5: Inhibitory effect on Aspergillus niger

1. Applications in Animal Husbandry
2. : Application in the treatment of calculi in Boer goats

The goats used in this experiment were Boer goats, a herd raised by farmers in Jiangu Town, Shou County, Anhui Province. A total of 200 goats were selected as the experimental group, and the animals received routine vaccinations. The study was conducted from October 2023 to September 2024.

Two hundred goats were randomly assigned to five pens, with 40 goats per pen. The goats in the five pens were arranged in the order of Pen 1, Pen 2, Pen 3, Pen 4, and Pen 5. All goats were fed the same forage and given drinking water containing fermented spray-dried powder at different concentrations.

Column 1 serves as the control group, while columns 2 through 5 constitute the experimental groups. Column 1 contains plain tap water with no additives; column 2 contains tap water supplemented with one part per hundred thousand of fermented spray‑dried powder (0.5 g added to 50 kg of tap water); column 3 contains tap water supplemented with two parts per hundred thousand of fermented spray‑dried powder (1 g added to 50 kg of tap water); column 4 contains tap water supplemented with four parts per hundred thousand of fermented spray‑dried powder (2 g added to 50 kg of tap water); and column 5 contains tap water supplemented with eight parts per hundred thousand of fermented spray‑dried powder (4 g added to 50 kg of tap water). Each group’s drinking water is placed in its respective column, and the animals are housed separately by column.

The number of goats diagnosed with urinary calculi and the number of deaths attributable to this condition were recorded quarterly. A total of four quarters were analyzed: October–December 2023; January–March 2024; April–June 2024; and July–September 2024.

Prevalence = (Number of sheep with calculous disease / Total number of sheep in each pen) × 100%

The incidence of stone disease = 1 − prevalence.

Mortality due to calculi = (Number of sheep that died from calculi / Total number of sheep in each pen) × 100%

Survival rate = 1 − mortality due to calculi

The statistical results are shown in the table below:

Table 4-1-1: Quarterly Incidence and Mortality of Calculi in Goats

	1	2	3	4	5
2023.10–2023.12 (Number of cases)	15	7	7	2	3
2023.10–2023.12 (Number of Deaths)	5	3	2	1	1
2024.01–2024.03 (Number of cases)	13	6	7	1	0
2024.01–2024.03 (Number of deaths)	3	3	1	0	0
2024.04–2024.06 (Number of cases)	11	6	8	0	0
2024.04–2024.06 (Number of Deaths)	5	2	1	0	0
2024.07–2024.09 (Number of cases)	9	5	2	0	1
2024.07–2024.09 (Number of deaths)	3	4	1	0	0

Table 4-1-2: Quarterly Incidence of Urolithiasis and Survival Rate in Goats

	1	2	3	4	5
2023.10–2023.12 (Incidence of Stone Disease)	63%	83%	83%	95%	93%
2023.10–2023.12 (Survival Rate)	88%	93%	95%	98%	98%
2024.01–2024.03 (Incidence of Stone Disease)	68%	85%	83%	98%	100%
2024.01–2024.03 (Survival Rate)	93%	93%	98%	100%	100%
2024.04–2024.06 (Incidence of Stone Disease)	73%	85%	80%	100%	100%
2024.04–2024.06 (Survival Rate)	88%	95%	98%	100%	100%
2024.07–2024.09 (Incidence of Stone Disease)	78%	88%	95%	100%	98%
2024.07–2024.09 (Survival Rate)	93%	90%	98%	100%	100%

The data were analyzed using Prism.

In summary, the results demonstrate that the microbial suspension of this invention effectively inhibits the proliferation of both bacteria and fungi. Moreover, when the spray‑dried fermentation powder is added to the drinking water of goats at a concentration of 40 parts per million (i.e., 2 g of the spray‑dried powder per 50 kg of tap water), it not only significantly reduces the incidence of urinary calculi in goats but also enhances their survival rate. Therefore, the spray‑dried fermentation powder of this invention holds great potential for the prevention of urinary tract stone disease in goats.

Figure 4-1-1: Sheep Farming

Figure 4-1-2: Goats do not suffer from fruit set rate.

Figure 4-1-3: Goat Survival Rate

1. : Application in the treatment of diarrhea in Boer goats

Diarrhea is a major cause of extremely high mortality in lambs; probiotic treatment was administered to diarrheal lambs. During the diarrheal episode, probiotics were given for 3 days, and oxytetracycline was administered for 3 days. Fecal samples were collected at three time points—during the diarrhea, after 3 days of treatment, and after 6 days of treatment—and subjected to sequencing. A total of three cases received probiotic treatment and three received oxytetracycline treatment; consequently, all other groups also included three samples each. Due to logistical challenges in sample collection, each group ultimately yielded only one composite sample, with the three individual samples pooled for analysis. Analysis of treatment outcomes indicated that the probiotic‑treated group performed better than the oxytetracycline‑treated group: by day 3, the feces had become semi‑liquid, and by day 6, it had returned to normal. Fecal samples were then used for gut microbiota profiling.

Group A consisted of normal adult lambs, Group B of normal neonatal lambs, Group C of diarrheal neonatal lambs treated with probiotics for 0 days, Group D of diarrheal neonatal lambs treated with probiotics for 3 days, Group E of diarrheal neonatal lambs treated with probiotics for 6 days, Group F of diarrheal neonatal lambs treated with oxytetracycline for 0 days, Group G of diarrheal neonatal lambs treated with oxytetracycline for 3 days, and Group H of diarrheal neonatal lambs treated with oxytetracycline for 6 days. Both the probiotic and oxytetracycline treatment groups effectively cured the diarrhea in the lambs.

Four fecal samples were collected from each group, pooled, and subjected to microbial community analysis. Figure 4‑2‑1 presents bar charts showing the top 10 most abundant taxa at the phylum level for each group. The results indicate that, compared with the small‑sheep group, the large‑sheep group exhibits a more balanced Firmicutes:Bacteroidota ratio and greater taxonomic richness, suggesting a more mature gut microbiota in larger sheep. In the diarrhea‑affected small‑sheep group treated with probiotics, the Firmicutes:Bacteroidota ratio progressively declined; moreover, on day 6 of probiotic treatment, group E displayed microbial diversity approaching that of normal large sheep, whereas group C (the control group of normal small sheep) showed lower diversity. These findings suggest that probiotics can help restore and enrich the gut microbiota, while tetracycline treatment produced the opposite effect, highlighting the differential impact of distinct therapeutic approaches.

At the phylum level shown in Figure 4-2-2, compared with normal lambs, normal large lambs exhibit a higher proportion of Bacteroides and UCG__005; in the small lambs, Clostridia UCG__ 014, Limosilactobacillus ( Lactobacillus reuteri probiotics ) The proportion is relatively high. In the probiotic treatment group, species richness in fecal samples from groups C, D, and E continuously increased, with UCG__005 and Bacteroides ( Bacteroides genus ) The proportion is steadily increasing; in the F, G, and H groups treated with oxytetracycline, the proportion of Streptococcus is high, while the proportion of probiotics is relatively low, resulting in lower microbial diversity.

Based on the fecal consistency shown in Figure 4-2-3, both probiotic supplementation and oxytetracycline treatment resulted in the cessation of diarrhea in the lambs.

Therefore, probiotics demonstrated superior efficacy compared to oxytetracycline in the treatment of diarrhea in lambs, and were more effective in maintaining intestinal microbial homeostasis.

A B C D E F G H

Figure 4-2-1: Microbial Distribution at the Door Level

A B C D E F G H

Figure 4-2-2: Horizontal Distribution of Microorganisms

Figure 4-2-3: Status of Sheep Feces

1. : Treatment of mastitis in lactating goats

Select 23 CMT-positive cases (SCC > 800 × 10 ⁴ /mL), and dairy goats without clinical signs of mastitis. The study included lactating goats with a lactation period of 110 ± 8 days, a milk yield of 1.2 ± 0.4 kg/day, an average body weight of 52 ± 3 kg, and a history of three lambings. All goats were sourced from the same farm and randomly assigned to three groups: a control group fed a basal diet (CS, n = 6); a group receiving the basal diet supplemented with 0.3% probiotics (BS, n = 10); and a group receiving the basal diet supplemented with 0.3% Radix Tetrastigmae (RT, n = 7). During the trial, goats were housed in separate groups and fed using neck collars. Probiotics or RT powder was mixed into the basal diet. Feeding occurred twice daily at 07:30 and 16:00, with ad libitum access to water. Milk yield, dry matter intake, and milk composition data were compared between Week 0 and Week 6 for the basal diet group (CS), the basal diet plus 0.3% probiotics group (BS), and the basal diet plus 0.3% RT group (RS). At Week 0, there were no significant differences among the three groups in milk yield, dry matter intake, or concentrations of milk protein, fat, and lactose. Supplementation with either BS or RT did not alter milk yield, dry matter intake, or the concentrations of milk protein, fat, or lactose by Week 6. However, when comparing Week 6 to Week 0 after supplementation with probiotics or RT, the SCC values in both the BS and RS groups were significantly reduced.

Current data indicate that probiotics exert a positive effect on the SCC index in goat milk without adversely affecting other milk parameters. Moreover, following probiotic supplementation, serum concentrations of immune and biochemical markers decrease, suggesting that probiotics may help control subclinical mastitis and thereby enhance the quality of goat milk.

Figure 4-3-1: Effects of probiotics on lactation performance and somatic cell count

Figure 4-3-2: Changes in Various Serum Parameters

1. : Application in sturgeon aquaculture

Two-month-old hybrid sturgeon fry (average body weight 5.0 g) were reared in indoor plastic tanks at the aquaculture farm of Yunnan Heilongjiang Sturgeon Group Co., Ltd. Four hundred fry were randomly divided into four groups, with 100 fish per group. The average stocking density was 0.5 kg/m³. From July to October 2017, under natural photoperiod conditions, the fry were fed every 3 hours. During the trial, water temperature remained between 10 and 12°C.

To prepare the experimental diets, different levels of probiotics and YLL2 were supplemented to the basal diet as follows: control group (Diet 1—control); Group 2: supplemented with 5.0 g/kg probiotics; Group 3: supplemented with 4.0 g/kg YLL2; Group 4: supplemented with 5.0 g/kg probiotics + 4.0 g/kg YLL2 (Table 1). The concentration of the probiotic used was 5.0 g/kg, 10 ⁷ (CFU/g feed). The YLL2 125 concentration (4.0 g/kg, 16.5 U/g feed) was determined based on the basal diet (13.5 g crude fat/100 g feed). At this concentration, YLL2 can hydrolyze crude lipids relatively efficiently while maintaining a comparatively low cost of use.

When probiotics are at 4×10 ⁷ At a dosage of CFU/g, lysozyme activity in skin mucus and serum, as well as serum peroxidase activity, were significantly enhanced. Current research indicates that dietary supplementation with YLL2 and probiotics can markedly improve growth performance, protein content, and serum immune responses in hybrid sturgeon. This study may offer a novel feeding strategy for supplementing fish diets with exogenous lipases and Bacillus species.

1. : Application in domestic pig farming

The domestic pigs used in this experiment were Large White pigs, raised by farmers in Jiangu Town, Shouxian County, Anhui Province. A total of 20 Large White pigs were selected as the experimental animals.

Twenty pigs were randomly assigned to two pens, with 10 pigs in each. One pen was designated CP1 (with no probiotic added to the feed), and the other was designated CPJ1 (with a probiotic supplement of 0.02% in the feed). All other rearing conditions were kept identical.

On the second day of rearing CP1 and CPJ1 under identical conditions, fecal samples were collected from both pens, and relevant microbial indicators in the manure of the Large White pigs in each pen were analyzed. The results are as follows:

Figure 4-5-2: Horizontal Distribution of Microorganisms

Figure 4-5-1: Microbial Distribution at the Door Level

The results indicate that, in the group fed probiotics, harmful bacteria—including Fusobacteriota, Propionigenium, and Vibrio—were significantly reduced, maintaining their populations at very low levels. This lowered the incidence of disease in the Large White pigs and enhanced their fattening performance.

1. : Applications in Poultry Farming

In this experiment, free-range native chickens—local breed hens—were selected from Xiaofeng Town, Anji County, Zhejiang Province, with a total of 260 hens.

Probiotics improve chickens’ digestive and absorptive capabilities, regulate intestinal microbial balance, and enhance immunity, among other functions.

The native hens were randomly divided into two groups, with 130 birds in each group. One group was raised under standard conditions, while the other was fed a diet supplemented with a probiotic at a concentration of one part per hundred thousand. The incidence of disease—including fowl plague, fowl cholera, pullorum disease, and mycoplasmosis—and egg production were monitored over a five-month period.

Table 4-6-1: Monthly Egg Production of Native Chickens Statistics on Egg Production of Native Chickens Over Five Consecutive Months

Figure 4-6-1: Monthly Egg Production Comparison for Native Chickens

Table 4-6-2: Monthly Incidence of Illness in Native Chickens

Analysis of the experimental results indicates that probiotics not only enhance poultry production performance and immune function but also reduce environmental pollution and the overuse of antibiotics.

1. : Applications in Rodent Husbandry

The experimental study utilized the Yila rabbit, a farm-raised meat rabbit breed from Xiaofeng Town, Anji County, Zhejiang Province, with a total population of 2,000 rabbits.

Because rabbits are relatively small animals with poor disease tolerance and a high incidence of illness—particularly susceptible to contagious bacterial diseases—they are prone to developing gastroenteritis.

Probiotics are live microorganisms that help maintain a balanced gut microbiota, boost immune function, and are characterized by being non‑toxic, safe and reliable, multifunctional, residue‑free, and environmentally friendly.

Six hundred and twenty-six weaned rabbits were randomly divided into two groups. One group of 300 was raised under standard conditions, while the other group of 326 was fed a diet supplemented with probiotics at a concentration of one part per hundred thousand. The incidence of disease, feed consumption, and weight gain were then recorded up to 90 days of age.

Table 4-7-1: Statistics on Incidence Rate, Feed Consumption, and Meat Gain Rate of Ira Rabbits

As shown by the above statistical results, rabbits fed a diet containing probiotics exhibit a significantly lower incidence of disease, a slight increase in feed intake, and a higher rate of weight gain, thereby generating greater profits for farmers.