Animal Feed Ingredient: Probiotics

Project title: Production of probiotics for animal feed.

Time Required: 1 year
Major Raw Material: Depend on strain, Common raw materials are as follows- soy peptone,
glucose monohydrate, yeast extract, Casein, KH2PO4, Na2HPO4, Trace elements
1.Introduction:
Direct-fed microbials, commonly known as DFM or probiotics, are live microorganisms that,
when provided in adequate amounts in the diet, can improve gut microbial balance (Fuller,
1989). Direct-fed microbials are generally categorized into: Bacillus-based, lactic acid-
producing bacteria, and yeasts (Stein and Kil, 2006).

Bacillus-based DFM are spore-forming bacteria. Spores are thermostable and survive at low
pH, which makes Bacillus-based DFM stable during feed processing and gastric
digestion. Bacillus-based DFM produce spores that germinate but do not proliferate in the
intestine, which means a constant supply of DFM is required to maintain the microbial
population. Lactic acid-producing bacteria are not spore-forming and
include Lactobacillus acidophilus, Bifidobacterium bifidum, and Enterococcus faecium. Lactic
acid-producing bacteria are able to proliferate in the intestine and sustain a microbial
population. However, survival during feed processing is of concern because lactic acid-
producing bacteria are not thermostable. Direct-fed microbials are available as a single-
species or single-strain product, but most commercial products contain more than one
species, strains, and even a combination with yeasts and prebiotics (Liao and Nyachoti, 2017).

Direct-fed microbials, similar to prebiotics, increase the beneficial gut bacterial population
mostly by increasing short chain fatty acids (SCFA) production. Short chain fatty acids lower
the pH, reduce enteric pathogens, and also stimulate intestinal cell proliferation which
maintains gut integrity. The increase in the population of beneficial bacteria also controls
enteric pathogens by competitive exclusion. However, the mode of action of DFM seems to
be even more comprehensive (Liao and Nyachoti, 2017).

Direct-fed microbials have sometimes been associated with performance improvements when
added to swine diets (Zimmermann et al., 2016). Apparently, lactic acid-producing bacteria
appear to be more beneficial for weanling pigs to help on gut microbial balance after weaning,
whereas Bacillus-based DFM seem to be more beneficial for growing-finishing pigs to
increase the digestibility of energy and nutrients in high-fiber diets (Liu et al., 2018). However,
the effects of DFM in performance are often inconsistent, probably due to the variation in
microbial strains, inclusion rate, feeding duration, as well as stage of production, health status,
and husbandry practices (Liao and Nyachoti, 2017). Thus, it is difficult to generalize in terms
of the effects of DFM on swine diets.

Abe et al. (1995) reported that performance was improved (decreased scouring and improved
growth) when probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium
pseudolongum) were fed. Timmerman et al. (2005) fed two different direct-fed microbial
formulations to 1- to 2-week-old veal calves in four different experiments. Results from all four
experiments suggested that direct-fed microbials increased growth and feed efficiency in
calves during the first two weeks. This appeared to be especially true when calves were
stressed, and disease incidence was significant. Ellinger et al. (1980) reported that feeding L.
acidophilus to calves decreased the content of faecal coliforms, which may be related to
presence of scours. Finally, Adams et al. (2008) suggested that a novel direct-fed microbial
(Propionibacterium jensenii 702) resulted in greater body-weight gain, not only during the milk
feeding period (the bacterium was added to milk), but also after weaning.
 Fig: Flow Diagram of Probiotics Production
2.Project Design:
Step 1: Strain Selection
Strain selection is the first vital step in the probiotics manufacturing process. The strain
selection solely depends on goal for creating a specific probiotics supplement and potential
health claims. Whether it is a supplement to support digestion, boost the health of immune
system, support a healthy response to occasional stress, or more.
Each strain has specific features, and they support specific benefits. Some strains support
healthy immunity, while others support digestion.
For the characterization all biochemical test & 16s RNA sequencing followed by BLAST on
NCBI is required.
Step 2: Media Formulation
Besides an effective formula with the correct doses of raw materials, selecting naturally bile
and acid-resistant strains is vital. Probiotics strains are also must be tested for intestinal
survivability. The chosen strains then undergo fermentation and stabilization.
A bio-processing lab studies of the probiotic strain should be done to check what controllable
parameters and nutrients can be optimized for growth. Optimization should be based on-
Selective Carbon Sources on Biomass Production, Effect of Aerobic or Anaerobic Incubation on
Biomass Production, temperature, Additional carbon & Nitrogen source, Phosphate source,
pH, trace element.
Once the unique combination of nutrients and process parameters are established, a large-
scale production can commence.
Step 3: Fermentation
Fermentation of probiotics possible by both Liquid state(LSF) and submerged
fermentation(SF).
The strain multiplies in the nutritious and warm ingredient bath until it reaches the desired
count (CFU – Colony-forming Units). During this process, metabolites are also formed, which
is the by-product of the bacteria's metabolism of the nutrients.
Probiotics are challenging to work with during the production process and require a high
amount of overages to ensure that each strain meets the claim mentioned on the
supplement label.
Step 4: Centrifugation
Once the cultures are ready, probiotic strains from the metabolites separated through
centrifugation. The stability of probiotics is another critical aspect that needs close attention in
the probiotics production process. Probiotic products begin to lose their stability/freshness the
moment they're packaged. Various methods are used to maintain supplement stability and
potency for long-term storage. These processes are vital and influence the probiotic strains'
viability and application suitability.
 Refrigeration - The probiotic bacteria are subjected to extremely low temperatures.
 Avoiding hot/humid environments - This step keeps the bacteria free from humidity.
The step involves a few drying methods -
(i) Freeze Drying - A more prolonged but gentler process.
(ii) Spray Drying - A shorter process characterized by higher
temperatures, but not too high for the bacteria to survive.
After these processes, the probiotic is then transformed into a dry powder.

Step 5: Blending and Bottling

The above powder contains a single strain. For multi-strain formula, other probiotic powders
are blended to form an evenly distributed, balanced mixture. Besides probiotics, other
essential ingredients can be mixed, including prebiotics, flavouring ingredients, binders to
produce alternate dosage forms, ingredients that complement the probiotic's health focus, etc.
This blend then gets ready to be presented into its final dosage forms like tablets, capsules,
and powder.
Probiotics are highly sensitive to environmental conditions such as temperature, humidity, and
light. These conditions differ from strain to strain and affect the expiration of the product.
Hence, they should be carefully packaged. They should be protected from direct sunlight, high
temperature, and moisture.

4. Test Method of Products:

 Confirmation Tests of the Isolates:

Growth in selective medium, other biochemical test Like Oxidase, catalase activity,
sugar fermentation etc, Sequencing of the PCR Products, Phylogenetic Analysis.
 In Vitro Characterization of Probiotic Properties:
Common methods for in vitro analysis of probiotic properties include- tolerance to low
pH, tolerance against bile salt, antibiotic susceptibility, antimicrobial activity, bacterial
adherence to stainless steel plates, Detection of Toxin & Virulence Gene
 In-vivo test (Rat or Mice model):
(i) In-vivo pathogenicity test
(ii) In-vivo trial for efficacy test-Performance parameters (Body weight gain, feed
intake, GIT health improvement)
5. Application:
Dietary feed Supplement for Bovine, Swine, Poultry bird and Aquatic animal for gut health
improvement, feed digestibility and immunity bosting.
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