POSTBIO FERMENTUM WITH CHOLESTEROL REDUCTION, ANTIBACTERIAL AND ANTIBIOFILM PROPERTIES

Abstract

ANNEXUREI Title Lactocasei Postbiotic Feed Supplements for Milk Booster in Dairy Cows ABSTRACT: This study introduces a Lactocasei Postbiotic supplement containing 15 CPU of Lacticaseibacillus paracasei DDS12 (1 gms), along with Cotton seed cake (20gms), groundnut oil cake (15gms), rice husk (15gms), wheat flour (lOgms), corn flour (lOgms), ragi flour (lOgms), calcium powder (lgm), jaggery powder (8gms) . In vitro trials reveal that the supplement improves gut microbiota balance, enhances nutrient absorption, and increases immune system and milk quality and quantity. Lactocasei Postbiotic exhibits strong antibacterial properties against the pathogen, ensuring the safety of postbio feed supplements. The dry heat process enhances the postbiotic stability, bioavailability, and health benefits, ensuring a 1-year shelf life at room temperature. Additionally, it suggestively decreases the inflammation caused by the mastitis pathogen and increases the healthy udder micrometre of the cattle. Keywords: Postbiotics, Lacticaseibacillus paracasei DDS 12, Dairy cow, feed formulation, Milk compostion.

Specification

ANNEXUREI
Title
Lactocasei Postbiotic Feed Supplements for Milk Booster in Dairy Cows



Background of the present investigation
The dairy industry is vital for global agriculture and food production, providing essential nutrition for human consumption. To ensure sustainability and productivity, it's crucial to focus on dairy cows' well-being, as their health directly impacts milk production and farm efficiency [1], Antibiotic have been utilized in animal husbandry for a long time as a growth promoters, on the other hand excessive use of antibiotic alter the gut microbiota[2], Des field and Considine experiment discovered three new variants of nisin A M17Q, nisin A HTK, and nisin A T2L, which show antibacterial activity against S. aureus strains associated with bovine mastitis and reduce growth inhibition of commensal bacteria in milk, paving the way for antibiotic substitutes on farms [3]. Probiotic bacterial feed supplements have gained interest for their potential to enhance dairy cows' health and performance. Probiotics are beneficial microorganisms that, when administered in adequate amounts, confer health benefits to the host. In the context of dairy farming, probiotic bacterial feed supplements have shown promising results in improving digestive health, nutrient absorption, and overall immunity in dairy cows. These supplements, when administered in adequate amounts, have shown promising results in improving digestive health, nutrient absorption, and overall immunity in dairy cows [4]. Probiotic bacteria, such as Lactobacillus and Bifidobacterium species, contribute to the fermentation process in the rumen, promoting the breakdown of complex carbohydrates and improving the efficiency of nutrient utilization.




Postbiotics, as a new area of interest in enhancing the cattle health, they are prepared from inactivated microorganisms or their components that increases the gut microbiota. These components, such as exopolysaccharides, short-chain fatty acids, enzymes, and cell-free supernatants, regulate the host's mucosal, promote a balanced intestinal microbiota [5], improve nutrient absorption which increase the concentration of the milk fat, Colostrum,
protein, and lactose; hence, they result in increasing milk quality and improving milk flavour

[6]. It stimulates the immune system, making cattle resistant to mastitis* Postbiotics possess advantages over probiotics, such as avoiding the transmission of virulence or antibiotic resistance genes, and their ability to be maintained at room temperature for years without losing biological activity due to bacterial viability [7]. These advantages drawn the interest towards the production of Postbiotic feed supplement for dairy cows.



1. Field of present investigation

This research is pioneering in developing Lacticaseibacillus paracasei DDS12 as a postbiotic product Latocasei Postbiotic Feed Supplements for Milk Booster in Dairy Cows. This Latocasei Postbiotic is a feed supplements prepared using cereal based ingredients, which enhance the milk yield in dairy cow. It supports digestive health, enhances the natural gut flora, and prevents pathogen colonization, thus fostering an improved equilibrium of gut microbiota.

2. Description and related art

We have suggested that a Latocasei Postbiotic feed supplements, which is a milk booster for dairy cow* It is prepared by mass-producing the probiotic bacteria Lacticaseibacillus paracasei DDS12. This involves a lab-scale fermentation process followed by the preparation of postbiotics through thermal heat processing by breaking live cells.


Pure culture of Lactleaseibacillus paracasei DDS12 Evaluation of antagonist activity of L. paracasei DDS12 Pilot scale production of Probiotic Storage, viaability analysis and preparation of postbiotic.



Postbiotic feed supplement formulation using cereals grains, oil cake,

jaggery, calcium powder etc.



Latocasei Postbiotic feed supplement infers the functional benefits in Dairy

cows such as

Milk yield, Milk chemical composition, Gut micrometre and prevents udder

infections




Detailed description of present investigation

Methodology

Preparation of Postbiotics from Lacticaseibacillus paracasei DDS12

Lacticaseibacillus paracasei DDS12, an isolate from the cow teat of native breed which was collected from a velan farms at Velagoundampatti, Nammakal district, Tamilnadu, India. The culture was cultured in 250 mL Erlenmeyer flasks with 100 mL of de Man Rogosa Sharpe medium. The biomass (18.9g) was added to 1000 mL of basal medium. The volume was moved to a 170L bioreactor to produce probiotics on pilot scale. L. paracasei DDS12 biomass production was optimized in a medium with maltose, tryptone, potassium chloride, sodium acetate, K.2HPO4, and Tween 80 at pH 8, 25°C, and 200 rpm. The probiotic biomass was separated by centrifugation at 8000 rpm for 15 minutes at 4°C. The obtained biomass was dried using a tray drying process. The dried probiotic powder (1 kg) was combined with food-grade talk talc powder, which is food additive, under the conditions of good manufacturing practices (GMPs). The postbiotic product is then packed in a 1kg clean container.
Quality Control

To ensure the quality of the Latocasei Postbiotic, microbiological testing showed the presence of viable (Lacticaseibacillus paracasei DDS12) and absence of pathogenic bacteria. The moisture content was quantified and confirmed at 2% to prevent spoilage. The powder was stored at room temperature to preserve stability and potency. Additionally, the powder was packaged in sterile, airtight containers to protect it from contamination and moisture ingress.

Effect of Postbiotic feed supplement in HF dairy cows
The formulated feed was tested in healthy HF dairy cows. These cows were divided into two groups control and test groups with 3 animals in each group. The animals were coosen with the consultation of a veterinarian (Dr. S. Kathiravan) at Edappadi. Each test group cows were fed with 3 kg of Lactocasei Postbiotic feed supplement along with regular feed ( rice brawn,C04 grass, super napier etc.). Whereas the control group cows were fed only with regular feed. These experiment wsa continued for 90 days for the analysis of milk yiled, milk chemical profile and the gut microbiome were analysed for every 30 days intervals as illustrated

Functional benefits

i Increases the quantity of Milk

*k Increases the quality of Milk by raising their fat and SNF levels as illustrated in table. 1

Increases the gut Microfloraa Milk yield

•t A rational combination of LacticaseibaciUus paracasei DDS12 fortified with valuable edible products designed and developed to enhance gut microflora & to optimize the utilization of all essential nutrients from feed to boost milk yield with an improved fat and SNF.
1. Survival at GI Tract

> Survival at low pH: Lactocasei Postbiotic must survive the acidic environment of the cow stomach.

> Bile salt tolerance: Lactocasei Postbiotic must withstand bile salts in the intestine.
2. Potent Antibacterial properties

^ In vitro tests: Antibacterial property is assessed in vitro against feed supplement bacteria including Staphylococcus aureus and Pseudomonas aeruginosa

Mechanisms: Lactocasei Postbiotic can produce substance like organic acids such as short-chain fatty chain and lactic acid, hydrogen peroxide and bioactive peptides that inhibit pathogens.
4. Non-haemolytic properties

> Hemolysis assay: Lactocasei Postbiotic should exhibit a non-haemolytic pattern, which can be evaluated using blood agar plates

5. Enhanced Gut Health

> Improve gut health: Strengthen the intestinal barrier, preventing the enter of harmful substance and promote the growth of beneficial gut bacteria.




CLAIM:
Claim 1: The Lactocasei Postbiotic supplement composition comprising: A proprietary postbiotic blend with 15 billion CFU of Lacticaseibacillus paracasei DDS12(lgms) with Cotton seed cake(20gms), groundnut oil cake(15gms), rice husk(15gms), wheat flour(lOgms), com flour(lOgms), ragi flour(lOgms), calcium powder(lgm), jaggery powder(8gms)

Claim 2: The Lactocasei Postbiotic supplement composition of Claim 1, where the dry heated Lacticaseibacillus paracasei DDS12 offers beneficial metabolic by products for gut health, enhance nutritional adsorption and boost immune system ,ensuring effectiveness for up to 1 year without refrigeration.

Claim 3: The Lactocasei Postbiotic supplement composition of Claim 1, wherein the consumption of the Lacticaseibacillus paracasei DDS12 blend provides dual benefits of improved gut microbiota balance, as evidenced by invitro trials demonstrating a statistically significant reduction in gastrointestinal issues.

Claim 4: The Lactocasei Postbiotic shows strong antibacterial properties against mastitis pathogens including Staphylococcus aureus and Pseudomonas aureginosa

Claim 6: Lactocasei Postbiotic is safe for use and free from virulence factor. Hence,our bacterial isolate is a non pathogenic and beneficial bacteria. We observed gamma hemolysis and 0% RBC lysis using both qualitative and quantitative methods, confirming prevent the udder infection (Bovine Mastitis)
Claim 8: Lactocasei Postbiotic significantly increases milk yield and chemical composition in the experimental cow groups in comparison with control group cows.. It increases the level of protein, fat and lactose composition in milk.
Claim 9: Lactocasei Postbiotic decreases the usage of antibiotic, excessive antibiotic use leads to the development of antibiotic-resistant bacteria. Antibiotic residues in milk pose a health risk to consumers.


SUMMARY:

The supplement composition comprises 15 billion CFU of Lacticaseibacillus paracasei DDS12 (lgm), with Cotton seed cake (20gms), groundnut oil cake (15gms), rice husk (15gms), wheat flour (lOgms), com flour (lOgms), ragi flour (lOgms), calcium powder (lgm), jaggery powder (8gms). The supplement was prepared by inoculating a Lacticaseibacillus paracasei in basal medium undergoes pilot scale fermentation. The probiotic biomass was separated by centrifugation and dried using a tray drying process. The probiotic powder is then packed in a clean container. This Lactocasei Postbiotic supplement offers several benefits: First, it improves gut health, enhances beneficial microbe growth, and leads to increased nutrition absorption. It enhances the immune system; resistance to the pathogen enters. Improve the quantity and the quality of the milk compost. Additionally, it reduces the antibiotic use. It exhibits strong antibacterial properties against.pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. The highest MIC was recorded for Staphylococcus aureus (7.5 mg/mL) and the lowest for Pseudomonas aeruginosa (3.75 mg/mL). It also controls pathogen colonization through its antibiofilm properties. Lactocasei Postbiotic significantly increases milk yield and chemical composition in the experimental cow groups in comparision with control group cows. It increases the level of protein, fat and lactose composition in milk. Further, Lactocasei Postbiotic decreases the usage of antibiotic, excessive antibiotic use leads to the development of antibiotic-resistant bacteria. Antibiotic residues in milk pose a health risk to consumers.


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