KR20240004274A - Bacterial isolates and their use in the production of fortified palm-based animal feed - Google Patents

Abstract

The present invention relates to a Bacillus capable of proliferating and secreting mannolytic enzymes to hydrolyze at least three types of mannan polymers upon contact with solvent-extracted palm kernel powder. Subtilis strain isolate DSM33646. The present invention also relates to a method of producing fortified palm-based animal feed with reduced mannan polymer content by contacting palm kernel meal with isolates of said bacterial strains.

Description

Bacterial isolates and their use in the production of fortified palm-based animal feed

The present invention is Bacillus It relates to a composition comprising a Bacillus subtilis isolate. In particular, the invention relates to the use of such compositions for producing fortified palm-based animal feed.

Oil palm ( Elaeis) guineensis Jacq.)) is a tropical crop grown mainly for palm oil production. Palm kernel cake (PKC) is a by-product of the oil palm industry and is produced in large quantities every year (Index Mundi, 2008a, 2008b). There are two main types of PKC available in the market based on different oil extraction methods (Kini et al, 2020). One is from a mechanical press or expeller, generally considered palm kernel expeller (PKE). The other is from solvent extraction and is referred to in this report as palm kernel meal (PKM). PKE and PKM can also be distinguished by oil content and physical properties. PKE typically has 8-10% residual oil from compression, whereas PKM has a lower oil content of <3% due to more efficient oil extraction by solvents (Alimon, 2004).

Both PKE and PKM (collectively referred to herein as PKC) are widely used as feed ingredients primarily due to their nutritional value for ruminants and reliable, low-cost protein and energy sources in animal feed (Zahari and Alimon, 2004). However, the inclusion of PKC is often limited due to its high fiber content and low metabolizable energy, especially in diets of monogastric animals such as poultry (Sharmila et al., 2014). PKC has been reported to contain high levels of non-starch polysaccharide (NSP), mainly in the form of poly-mannan (Dusterhoft et al., 1992). These mannan fibers are considered anti-nutritional factors in poultry and other monogastric animals (Saeed et al., 2019). Mannan, contained in large amounts in feed, increases intestinal viscosity and nutrient encapsulation, reducing the rate of nutrient hydrolysis and utilization of the diet (Choct and Annison, 1992).

To increase the economic value of PKC in feed, nutrient utilization must be optimized by increasing fiber digestibility to release nutritional compounds from the fiber matrix. Supplementation of PKC-based diets with exogenous fibrolytic enzymes, such as mannanase, resulted in inconclusive results in nutrient digestibility and animal performance (Sharmila et al., 2014; Iyayi and Davies, 2005). Discrepancies in enzyme effects may be due to differences in thermal and pH stability, capacity, or substrate specificity of different mannanases. Most of the commercial mannanases were not developed specifically for feed ingredients such as PKC.

Another way to improve nutrient digestibility in animals is to use direct-fed microorganisms (DFM). DFM is a category of probiotics used in the animal industry (Callaway and Ricke, 2011). As microbial additives to feed, probiotics can promote healthy microbial populations in the gastrointestinal tract, aiding proper digestion and nutrient absorption of feed, reducing pathogen infection and improving animal performance (Bajagai et al., 2016). A variety of microorganisms, either in the form of single species or multiple species, have been deployed as DFM in animal feed. Lactobacillus and Bifidobacterium are the two most commonly used genera (Bajagai et al., 2016). Recently, Grant et al. (2018) reviewed that spore-forming Bacillus spp. has distinct advantages over these two popular beneficial species as DFM. One of the highlighted characteristics of Bacillus spp. is their spore resistance to high temperatures of up to 113°C, which is an important characteristic for the bacteria to survive in feed processing stages. Bacillus spores can also withstand harsh stomach environments. Once entering the digestive system of animals, Bacillus can promote gut health through competitive exclusion or production of beneficial metabolites that can protect the host from various pathogens (Grant et al., 2018; Elshaghabee et al., 2017). . Additionally, among bacteria, β-mannannase was mostly produced by Gram-positive bacteria, mainly various Bacillus species (Chauhan, 2012).

It would be of interest to the feed industry if the beneficial characteristics of enzymes and probiotics could be brought together by developing a natural B. subtilis isolate capable of producing the essential mannolytic enzyme targeting PKC mannan fiber. It will be.

Listing or discussion of explicitly previously published documents in this specification should not necessarily be construed as an admission that such documents are part of the state of the art or common general knowledge.

All documents mentioned herein are incorporated herein by reference in their entirety.

Isolation and characterization of PKC-reactive B. subtilis isolates applicable to PKC-based feeds are provided herein. The isolate can proliferate and secrete mannolytic enzymes upon contact with PKC, especially on solvent-extracted palm kernel meal.

The use of PKC in animal feed has been limited, especially in monogastric animals, due to its high content of mannan. Direct supplementation of mannan-degrading enzymes with PKC-containing feeds faces several challenges, such as enzyme substrate specificity, thermal and pH stability during feed pelleting and during passage through the animal's digestive tract, respectively. The present invention identified beneficial bacterial strains/isolates capable of secreting mannolytic enzymes upon contact with high-mannan feed ingredients such as PKC. The isolate can be used for PKC fermentation/blending or as a direct-feed microorganism to be fermented/blended with PKC. Natural Bacillus Responsive to PKC and Serving as a Potential Probiotic for Animal Feed Isolation and identification of subtilis isolates are reported herein.

In one aspect of the invention, Bacillus capable of secreting mannolytic enzymes upon contact with non-starch polysaccharide feed ingredients. Subtilis strain isolate DSM33646 is provided. DSM33646 was deposited at the Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, by Wilmar International Limited, Singapore, on September 24, 2020. Non-starch polysaccharides make up 70 to 90% of plant cell walls. “Non-starch polysaccharide (NSP)” is meant to include any polymeric carbohydrate that differs in composition and structure from starch and has chemical cross-links between them, making it less digestible by animals. These include cellulose, mannan, pectin, gum, glucan, inulin, and chitin.

In another aspect of the invention, Bacillus A kit is provided that includes a composition comprising subtilis strain isolate DSM33646 and instructions for use of the kit. This set of instructions may include methods for producing enriched palm-based animal feed according to aspects of the invention, as described below.

In another aspect of the invention, an enhanced palm loaded with live probiotic bacteria and their secreted enzymes capable of hydrolyzing at least three types of mannan polymers, namely glucomannan, galactomannan and linear mannan. -A method of producing based animal feed is provided, the method comprising: (a) providing palm kernel meal; (b) Palm kernel powder was mixed with Bacillus contacting subtilis strains to form a blended or fermented product; and (c) drying the blended or fermented product, wherein Bacillus The subtilis strain is DSM33646. Preferably, the isolated Bacillus of the present invention Subtilis strains were selected for their ability to grow on solvent-extracted palm kernel flour. The terms PKE and PKM are used interchangeably in the industry, but PKE refers to mechanically extracted palm kernel cake.

Palm kernel meal (PKM) can be prepared via any suitable solvent extraction method known to those skilled in the art. Advantageously, the present invention provides unique B. subtilis strain isolates that are capable of effectively and efficiently secreting suitable enzymes for hydrolyzing the mannan polymers present in PKM.

“Blending” is meant to include any act of mixing and/or combining the various ingredients/reactants used in the manufacturing steps performed by the present invention. Such mixing or blending may include any “fermentation” action, including any decomposition (e.g., chemical decomposition) of the polymers in the PKM under any suitable conditions. These suitable conditions are described below.

“Solvent-extracted palm kernel meal” means solvent-extracted and comprising PKM with an oil content of 3% or less.

In various embodiments, the palm kernel meal has an oil content of less than 3% by weight.

In various embodiments, the method further includes adding water to step (b), wherein the ratio of palm biomass to water is about 1:1.5.

In various embodiments, Bacillus The concentration of the subtilis strain is about 6×10 ⁶ to 6×10 ¹⁰ cfu per gram of palm kernel powder.

In various embodiments, palm kernel powder is mixed with Bacillus The step of contacting the subtilis strain involves fermenting/blending the strain with the flour for about 4 to 72 hours at a temperature of about 25°C to 40°C. In various embodiments, fermentation and blending can be performed for 4 to 48 hours, or 4 to 24 hours. In various embodiments, fermentation and blending can be performed for 6 to 24 hours.

In various embodiments, fermentation/blending is performed with water and the resulting pH upon contact with the flour is 4.0 to 8.0.

In various embodiments, drying of the fermented product is performed at a temperature of about 60° C. to 80° C. until the moisture content of the fermented product is 10% or less.

Advantageously, the fermented/blended product produced by the present invention has hydrolytic activity towards non-starch polysaccharides.

In various embodiments, the palm kernel flour includes a high content of non-starch polysaccharides.

In various embodiments, the non-starch polysaccharide is glucomannan, galactomannan, or linear mannan.

The process of the present invention can be carried out as a continuous or batch-wise process.

In various embodiments, the blended/fermented product is further fermented/blended with a fresh amount of palm kernel meal in a ratio ranging from 1:1 to 1:10 before drying step (c). In various embodiments, the blended/fermented product can be further fermented/blended with water at a ratio of 1:1.5. “Fresh” PKM is meant to include solvent-extracted PKM that has not yet been exposed to B. subtilis isolates or has not yet been subjected to any enzymatic treatment, i.e., step (a) in the method steps described above.

In another aspect of the invention, an enriched palm-based animal feed is provided loaded with live probiotic bacteria capable of hydrolyzing at least three types of mannan polymers and their secreted enzymes, wherein the feed is of the present invention. It can be obtained or obtained by a method.

In one aspect of the invention, enrichment comprising contacting or treating palm biomass with an isolate of the Bacillus subtilis strain deposited at the Leibniz Institute, DSMZ-German Collection of Microorganisms and Cell Cultures GmbH and having deposit number DSM 33646 A method for manufacturing a palm-based feed is provided.

In various embodiments, strains are selected based on their ability to grow only on solvent-extracted palm biomass. This strain is included in animal feed as a probiotic. The strain is specific for palm kernel powder. The strain has the ability to decompose mannan. Fermentation is accelerated by the strain at least 4-6 hours after starting fermentation/blending.

Strains are introduced by fermentation/blending with a mixture of PKM and water in a PKM to water ratio of 1:1.5 or higher. The strain is used at 6×10 ⁸ cfu/ PKM g but the dosage can vary between 6×10 ⁶ and 6×10 ¹⁰ cfu. Other ratios may be used. Here, the moisture content was kept as low as possible because larger amounts of water increase drying costs.

In various embodiments, strains and mixtures are fermented/blended for 6 to 24 hours. To increase production, it is desirable to have the shortest possible time, in this case 6 hours. This is different from the traditional fermentation process, which generally takes about 24 hours or more.

In various embodiments, strains and mixtures are fermented/blended at temperatures ranging from 25 to 40 degrees Celsius. In various embodiments, the temperature is 37 degrees Celsius, including heat build-up in the device.

In various embodiments, the palm biomass is palm kernel meal with an oil content of less than 3%.

In various embodiments, the fortified palm-based feed is manufactured by continuous accelerated fermentation/blending in a manufacturing plant.

In various embodiments, the fortified palm-based feed is manufactured by batch accelerated fermentation/blending in a manufacturing plant.

In another aspect of the invention, a fermented/blended fortified palm-based feed of one aspect of the invention is provided.

In various embodiments, the feed is fortified with hydrolytic activity capable of hydrolyzing at least three different forms of mannan polymers.

In various embodiments, the three different forms of mannan polymers are glucomannan, galactomannan, and linear mannan.

In various embodiments, the fermented/blended fortified palm-based feed is in the form of a direct feed.

In various embodiments, the fermented/blended fortified palm-based feed is in dry form.

In another aspect of the invention, a fermented/blended fortified palm-based feed is provided in addition to one aspect of the invention.

In various embodiments, the product is further fermented/blended with fresh PKM at a ratio ranging from 1:1 to 1:10. Each round of fermentation/blending enhances DSM33646 growth, and the 1:10 ratio limit is to ensure sufficient growth of DSM33646 to reach optimal enhancement of mannan hydrolytic activity.

For water to PKM ratio, it is 1:1.5 (optimized) or another ratio.

In various embodiments, the product is characterized by hydrolysis of at least three different forms of mannan polymer.

In various embodiments, the three different forms of mannan polymers are glucomannan, galactomannan, and linear mannan.

In various embodiments, the additional fermented/blended fortified palm-based feed is in the form of direct feed.

In various embodiments, the additional fermented/blended fortified palm-based feed is in dry form.

In another aspect of the invention, there is provided a composition comprising the fermented/blended fortified palm-based feed of one aspect of the invention and the further fermented/blended fortified palm-based feed of one aspect of the invention, , where the fermentation/blending time is 2.5 to 24 hours.

In various embodiments, the fermented/blended fortified palm-based feed and the further fermented/blended fortified palm-based feed can be further fermented/blended.

In another aspect of the invention, Bacillus A fortified palm-based animal feed comprising subtilis strain isolate DSM33646 is provided. The animal feed additionally comprises solvent-extracted palm kernel meal.

In various embodiments, the feed has enzymatic hydrolytic activity of at least three different forms of mannan polymer, wherein the three different forms of mannan polymer are glucomannan, galactomannan, and/or linear mannan.

Animal feed may be in direct feed form or in dry form.

In various embodiments, the animal feed is further blended/fermented with the addition of water and palm kernel meal.

Advantageously, the present invention relates to Bacillus strains highly responsive to PKM, which are capable of secreting enzymes that degrade the mannan component of PKM in a very short period of time. Currently, Bacillus isolates are not specifically used for solvent extracted PKM (they were mostly for PKE and have not been proven to "react" with PKE either), i.e. they are very unlikely to react to PKM. No isolate from the prior art has been shown to be able to secrete mannanase to the levels we report when incubated directly with palm kernel cake, especially solvent-extracted PKM. This concerns the responsiveness of the strain to biomass. It is the “reactivity” of the isolated DSM33646 strain that enables the illustrated production of “fermented/blended” PKM, and thus differentiates the entire process from others (instead of long fermentations, the invention allows for shortened fermentation periods). .

In order that the present invention may be fully understood and easily put into practice, only preferred embodiments of the present invention will now be described by way of non-limiting examples, with reference to the accompanying exemplary drawings.

1 is an illustration summarizing a method according to an embodiment of the present invention;
Figures 2, 3 and 4 show the results obtained in Example 3;
Figure 5 shows the results obtained in Example 4;
Figure 6 shows the results obtained in Example 5; and
Figure 7 shows the results obtained in Example 6.

Figure 1 shows a general schematic diagram of carrying out a method according to an embodiment of the invention. Here, it can be seen that the fermented/blended PKM product is obtained by incubating solvent-extracted PKM with the B. subtilis isolate of the present invention, which is capable of producing the enzymes necessary to degrade various types of mannan in a short time. .

Example One

Materials and Methods

1. Samples, microorganisms and chemicals

PKM was obtained from a palm kernel solvent extraction plant located in East Java, Indonesia. Bacterial populations used to inoculate PKM were obtained from palm fruit exocarp from several oil palm plantations of Wilmar International. bacillus subtilis CK7 strain PT. Obtained from Wilmar Bernih Indonesia. Glucomannan (konjac; high viscosity) and mannan (1,4-β-D-mannan) (Megazyme, Wicklow, Ireland), locust bean gum from Ceratonia siliqua seeds (LBG), and DNS reagent components (3,5- Dinitrosalicylic acid, potassium sodium tartrate tetrahydrate, and sodium hydroxide (Sigma Chemical Co., St. Louis, MO, USA) were purchased and used.

2. Isolation and identification of mannan-reactive bacteria

1 g of PKM in 5 mL of water was inoculated with microorganisms obtained from the outer skin of palm fruit and incubated in an incubator at 37°C. Bacteria grown in PKM-only culture tubes that were more than 2 weeks old were plated on Luria-Bertani (LB) agar plates and single colonies were selected. Each bacterial colony was grown in 100 ml M9 minimal medium (LBG-medium) containing 0.5% (w/v) LBG as the sole carbon source (6.78 g/L Na ₂ HPO ₄ .7H20, 3 g/L KH ₂ PO ₄ , 1 g/L NH ₄ Cl, 0.5 g/L NaCl). The tubes were incubated at 37°C for 48 hours and the cell-free supernatants were collected with crude enzymes and assessed for their mannolytic activity by the assay described below. Genomic DNA of an isolate with high mannolytic activity was extracted (Hoffman, 2003) and used as a template for PCR for partial 16S rRNA gene amplification (~500 bp) using the following universal primers: 16s forward (5'- CCTACGGAGGCAGCAG -3′) and 16s reverse (5′-GGACTACHVGGG TWTCTAAT-3′) (Takahashi et al., 2014). Amplicons were purified and sequenced. Sequence similarity and homology analysis against the GenBank database was performed using the Basic Local Alignment Search Tool (BLASTN) of the National Center for Biotechnology Information (NCBI) ( https://www.ncbi.nlm.nih.gov/ ).

3. Measurement of mannolytic activity

The activity of the crude enzyme was determined by measuring the release of reducing sugars by the DNS method described by Miller (1959) with minor modifications using mannose as a standard. The reaction mixture contained 950 μl of 100 mM sodium acetate buffer (pH 5.0) containing 0.5% substrate (w/v) and 50 μl of crude enzyme. Absorbance at 540 nm was measured with a Libra S22 UV/Vis Spectrophotometer (Biochrom, UK).

5. PKC hydrolysis analysis

PKC was sterilized at 121°C for 15 minutes. Selected Bacillus 1 g PKC to each well of a 6-well plate. The isolate was inoculated with 6×10 ⁸ CFU of B. subtilis CK7 and 1.5 mL of water. PKC with water only was used as a negative control. PKC was then incubated at 37°C for various time points, e.g., 4 to 72 hours, or 6 to 23 hours. After incubation, PKC samples were diluted with water (1:4) and centrifuged at 12,000 rpm for 5 min to obtain cell-free supernatant as PKC crude extract for mannol analysis for LBG, konjac glucomannan, and 1,4-β-D-mannan. The decomposition activity was analyzed. All experiments were performed three times.

6. Drying of fermented products

The fermented product could be dried at a temperature range of 60°C to 80°C until the moisture content was below 10%.

The drying process may involve vacuum treatment.

If fermentation/blending is carried out on the farm and short-term storage is carried out on the farm, the fermented/blended product can be dried in the sun.

Results and Discussion

We sought to identify natural beneficial bacterial isolates that could improve the nutritional profile of PKC and be applied with PKC as direct feeding microorganisms. Most studies have largely focused on the utilization of fermentative strains obtained from cell collection centers or from strains isolated directly from milker cakes. In 2018, Virginia and others reported that mannanase-producing Bacillus was isolated from a palm oil mill area. Subtilis strain CK7 was identified. It has been shown that the coenzyme secreted by CK7 can release reducing sugars from PKC (Virginia et al., 2018). However, the reactivity of this variant when directly contacted with PKC has not been evaluated. In PKE (8-10% oil), the high oil content allows easy selection of strains that can use oil as an energy source; On the other hand, in solvent-extracted PKM (<3% oil), the oil content is reduced more, and this limited energy source allows mannan to secrete higher levels of cellulolytic enzymes to utilize the rich fiber. This made it easy to select the strains available. In the present invention, low oil PKM was used as a substrate for screening mannan-degrading bacteria. To inoculate PKM, bacterial communities from palm fruit hulls from various plantations were used. Bacterial isolates enriched by PKM incubation were further examined for their mannolytic activity. Among PKC-reactive bacterial isolates, DSM33646 was a Bacillus subtilis (98% pairwise identity when comparing the 16S sequence to the database in GenBank).

To further evaluate the reactivity of the DSM33646 isolate to PKC, the ability of the DSM33646 isolate to proliferate and secrete mannolytic enzymes upon incubation with PKC was examined and compared to that of the CK7 strain. DSM33646 and CK7 were reintroduced into PKM (6×10 ⁸ cfu/g). Water extracts of PKM inoculated with DSM33646 isolate at different fermentation/blending times (starting from 6 h after fermentation/blending) showed significant mannolytic activity (against at least three forms of mannan substrates: LBG, konjac and linear mannan). showed an increase, whereas inoculation with the CK7 strain did not show an increase in activity. The mannolytic activity of strain DSM33646 persisted and increased at longer fermentation/blending times.

In conclusion, the present invention identified B. subtilis isolates that were highly reactive to mannan-enriched PKC. This isolate could be further developed for use in the feed industry in fermentation/blending of PKC or directly included in PKC-based diets as DFM.

Example 2: PKM Process of the present invention for selecting specific strains

Bacteria associated with loose palm fruit were inoculated into PKM and water (1:5, w/w) and incubated at 37°C for >2 weeks to select PKM-specific strains. Viable bacteria were then grown and tested in culture. bacillus Subtilis strain DSM33646 was isolated from the process.

Other mannan-degrading bacteria were screened from bacterial collections or isolated directly from PKE (with 8-10% oil content), food, and the environment without undergoing the process of the present invention. Low-oil PKM serves as a specialized natural feedstock for isolating microorganisms that can efficiently use mannan fibers, as it has a significantly lower oil content than PKE, preventing the isolation of bacteria that can survive based on oil as a carbon source. Because it does. Bacillus revealed Subtilis strain CK7 was isolated from the environment and found to have the ability to decompose mannan, and was used as a control to demonstrate the inventiveness of the strain screened through the process of the present invention.

Example 3: DSM33646 fermentation/ blended PKM's loaded enzyme activity

PKM 1 g DSM33646 Bacillus subtilis or bacillus Fermented/blended with 6×10 ⁸ cfu of Subtilis CK7.

One unit (U) of enzyme activity (μmol/min) is required to release 1 μmol of mannose equivalent from 0.5% of each substrate (konjac/glucomannan, LBG/galactomannan, and linear mannan) per minute after incubation at 55°C. Defined as the amount of enzyme (normalized per gram of fermented/blended product).

Example 4: Obtained from the method of the present invention PKM fermentation/ blended to the product loaded Enzyme activity.

Examples 4 and 5 below illustrate how fermentation/blending can be scaled up while maintaining enzyme activity in the fermented/blended flour. Enzyme activity is measured as the amount of reducing sugars released in the enzyme assay per minute using enzyme harvested/recovered from fermentation/blending in the DNS assay. The analysis was performed as follows:

a. Reaction mixture: 50 ul of extracted enzyme harvested/recovered from PKM (including necessary dilutions) + 950 ul of linear mannan solution (0.5% linear mannan, 0.1 M pH 5.0 buffer)

b. Reaction conditions: 55℃, 1000 rpm, 1 hour

DNS test: 300 ul reaction mixture + 900 ul DNS solution; Heat at 100 dc for 5 minutes and measure absorbance at 540 nm.

0.5 g of the 6-hour fermented/blended product of claim 2 was further fermented/blended with PKM at a ratio of 1:10 for 17 hours. The enzyme activity loaded into 5.5 g of final product was measured. One unit (U) of enzyme activity (μmol/min) is defined as the amount of enzyme required to release 1 μmol of mannose equivalent from 0.5% linear mannan per minute after incubation at 55°C (grams of fermented/blended product) normalized to sugar). The positive control (Pos ctrl) is 0.5 g of product fermented/blended for 23 hours.

Example 5: PKM fermentation/ blended in the product loaded Enzyme activity.

1 g of the 2.5 hour fermented/blended product was further fermented/blended with PKM at a 1:4 ratio for 2.5 hours. 5 g of the fermented/blended product was further fermented/blended with PKM at a 1:9 ratio for 19 hours. The enzyme activity loaded into 50 g of the final product was measured. One unit (U) of enzyme activity (μmol/min) is defined as the amount of enzyme required to release 1 μmol of mannose equivalent from 0.5% linear mannan per minute after incubation at 55°C (grams of fermented/blended product) normalized to sugar). The positive control (Pos ctrl) is 1 g of product fermented/blended for 24 hours, and the negative control (neg ctrl) is 50 g of PKM without strain.

Example 6: Different capacities DSM33646 and Fermented together/ blended In PKM loaded Enzyme activity.

1 g of PKM was administered to various doses (6 × 10 ⁶ to 6 × 10 ¹⁰ CFU) of DSM33646 Bacillus Fermented/blended with subtilis . One unit (U) of enzyme activity (μmol/min) is defined as the amount of enzyme capable of releasing 1 μmol of mannose equivalent from 0.5% LBG/galactomannan per minute after incubation at 55°C (fermented/blended Normalized to per gram of product).

Although preferred embodiments of the present invention have been described in the foregoing description, it will be understood by those skilled in the art that many variations or modifications in design or construction details may be made without departing from the present invention.

Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures DSM33646 20200924

Claims (25)

Bacillus capable of secreting mannolytic enzymes upon contact with non-starch polysaccharide feed ingredients Subtilis ( Bacillus subtilis ) strain isolate DSM33646. Kit containing a separate DSM33646 and kit user manual. A method of producing fortified palm-based animal feed comprising the following steps:
(a) providing palm kernel meal;
(b) Palm kernel powder was mixed with Bacillus contacting a subtilis strain to form a blended or fermented product; and
(c) drying the blended or fermented product;
Bacillus here The subtilis strain is DSM33646. 4. The process according to claim 3, wherein the palm kernel meal has an oil content of less than 3% by weight. 5. The method according to claim 3 or 4, wherein the strains are selected for their ability to grow on solvent-extracted palm kernel meal. 6. The method of any one of claims 3 to 5, further comprising adding water to step (b), wherein the ratio of palm biomass to water is about 1:1.5. The method according to any one of claims 3 to 6, wherein Bacillus A method in which the concentration of the subtilis strain is about 6×10 ⁶ to 6×10 ¹⁰ cfu per 1 g of palm kernel powder. The method according to any one of claims 3 to 7, wherein the palm kernel powder is treated with Bacillus Subtilis strain comprising fermenting or blending the strain with the flour for at least about 4 to 72 hours at a temperature of about 25°C to 40°C. The method according to any one of claims 3 to 8, wherein the palm kernel powder is treated with Bacillus Subtilis strain comprising fermenting or blending the strain with the flour for at least about 4 to 48 hours at a temperature of about 25°C to 40°C. The method according to any one of claims 3 to 9, wherein the palm kernel powder is treated with Bacillus Subtilis strain comprising fermenting or blending the strain with the flour for at least about 4 to 24 hours at a temperature of about 25°C to 40°C. 7. The method of claim 6, wherein the pH of the water is 4.0 to 8.0. 12. The method of any one of claims 3 to 11, wherein the step of drying the fermented or blended product is carried out at a temperature of about 60° C. to 80° C. until the moisture content of the fermented or blended product is below 10%. How to be. 13. The process according to any one of claims 3 to 12, wherein the fermented/blended product has hydrolytic activity towards non-starch polysaccharides. 14. The method of claim 13, wherein the non-starch polysaccharide is glucomannan, galactomannan and/or linear mannan. 15. The method according to any one of claims 3 to 14, wherein the method is carried out as a continuous process. 15. The method according to any one of claims 3 to 14, wherein the method is carried out as a batch process. 17. The process according to any one of claims 3 to 16, wherein the blended or fermented product is further fermented or blended with a fresh amount of palm kernel meal in a ratio ranging from 1:1 to 1:10 prior to the drying step (c). How to do it. 18. The process according to claim 17, wherein the blended or fermented product is further fermented or blended with water in a ratio of 1:1.5. Enriched palm-based animal feed with reduced mannan polymer content obtained or obtainable by the process according to any one of claims 3 to 18. bacillus Fortified palm-based animal feed containing subtilis strain isolate DSM33646. Animal feed additionally comprising solvent-extracted palm kernel powder. A fortified palm-based animal feed with reduced mannan polymer content, wherein the feed has enzymatic hydrolytic activity of at least three different forms of mannan polymers, the three different forms of mannan polymers being glucomannan, galactomannan and/ or linear mannan, fortified palm-based animal feed. 23. The fortified palm-based animal feed according to any one of claims 19 to 22, in the form of direct feed. 24. The fortified palm-based animal feed according to any one of claims 19 to 23, in dry form. 25. The fortified palm-based animal feed according to any one of claims 19 to 24, wherein the animal feed is further blended or fermented with the addition of water and a fresh amount of palm kernel meal.