Animal Feed Ingredient: Antioxidants, Preservative & acidulant

Project title: Production of Citric Acid by Fermentation and Its Application.

Time Required: 1 year
Major Raw Material: Pineapple waste, Apple Pomace, Banana Peels, Coffee Husk, Grape
Pomace, Cane Molasses, Corn Cobs
Highest Yield Reported: 603.50 mg/g, Raw material Corn Cobs (Behera Et all,2021)
Application Areas: Used as Antioxidant in Poultry, Cattle & Fish feed, Used as Acidulant in
food and beverage industry.
1.Introduction:
Microbial citric acid is an important organic acid widely used in pharmaceutical, food, feed,
beverage and cosmetics industries. Although citric acid is produced by different types of
microorganism, the filamentous fungus Aspergillus niger is the major one
Citric acid is a known Antioxidant that acts to inhibit the oxidation of other molecules by
scavenging reactive oxygen species (ROS) that are produced as by-products of normal
cellular function.
In addition, Citric acid is a common acidulant and preservative (due to lower pH most
microbes can’t be able to grow) widely used in food and beverage industry.

Stock Culture
Preparation
Of Aspergillus Niger

Culture Sporulation
on PDA slants

Spore Inoculation

Fermentation
Fermentation
Medium

Supernatant Purification
Filtration
(Citric Acid)

Residue Purified
(Fungal Biomass) Citric Acid

Fig: Flow Diagram of Citric Acid Production

2.Project Design:
2.1. Isolation & characterization Of Novel microbes:

 The fungus Aspergillus niger is a type of mould that commonly present outside of
certain foods, such as apricots, onions, grapes etc, Thus, it can be easily isolated and
cultivated in laboratory conditions.
 Aspergillus niger has a plethora of strains, so it is necessary to isolate the most
effective strain (through Primary Screening and Secondary Screening of Strain
Selection) capable of high yielding of Citric acid.
 For the characterization all biochemical test & 16s RNA sequencing followed by BLAST
on NCBI is required.
2.2. Fermentation:

 By the Isolated novel Aspergillus niger, mentioned raw material (Pineapple waste,
Apple Pomace, Banana Peels, Coffee Husk, Grape Pomace, Cane Molasses, Corn
Cobs) is fermented by both SSF & SMF method.
 Citric Acid and Residue is Separated through filtration/centrifugation, followed by
purification of citric acid.
 Citric acid assay should be done for determination of concentration.

2.3. Optimization of Citric Acid Production:

 Production of Citric Acid should be optimized in terms of following parameters -The

concentration of Carbon Sources, temperature, Additional carbon & Nitrogen source,
Phosphate source, pH, Aeration, Trace Elements.
 All parameters are tested by OVAT method (One Variable at A Time).
3. Efficacy test Method of Products:

 Antioxidant property test:

Several test available for Antioxidant property determination including- Oxygen Radical
Absorption Capacity (ORAC) test, the Hydroxyl Radical Antioxidant Capacity (HORAC)
test, the Total Peroxyl Radical Trapping Antioxidant Parameter (TRAP) test, and the
Total Oxyradical Scavenging Capacity (TOSC) test.
 PET (Preservative Efficacy Test):
Preservative efficacy test should be done as per ISO 11930 or Pharmaceutical
Guidelines for PET.
 Acidulant:
■ Form of the acid: solid or liquid – does the application limit the form of acidulant. For
example, for most of the food applications which are aqueous systems do not need to use
solid form of acids but some of them like tablets, instant beverages, powder blends, etc.
strictly require solid form of acidulant. ■ Solubility – it is important to understand how much
of acid is required to achieve expected pH level (acidity), when playing with low soluble acids.
■ Taste profile and interaction with flavours – most of acidulants have a great role in creating
the overall taste profile and have synergies with flavours and sweetening system.
4. Application:
4.1. Food and Feed Industry:

 The most common uses of citric acid are as a preservative and flavouring agent in food
and beverages.
 Citric Acid as Antioxidants prevent oxidative losses of vitamins A and E and pigmentary
(oxy- and keto-carotenoids) in stored mixed feeds. Stabilizes critical oxidation-
susceptible nutrients that are naturally present in a feed composed of several
feedstuffs so that losses are minimal from mixing and storing. In lipid peroxidation, the
unsaturated fatty acids undergo a loss of hydrogen, resulting in the formation of a free
radical at the site of unsaturation. If the feed material in which this reaction is taking
place does not contain vitamin E or some other effective antioxidant, the free radical
is quickly converted to a fatty acid peroxide free radical and finally to a fatty acid
hydroperoxide. An Antioxidant can block this peroxidation by supplying a hydrogen in
the first free radical formed, thereby reconverting it to the original fatty acid. If the
hydroperoxides are allowed to form, they continue to decompose by breaking down
into a variety of aldehydes and ketones.
4.2. As cleaning & chelating agent

Citric acid is considered as an excellent chelating agent. Limescale is removed from

boilers and evaporators with the help of citric acid. Citric acid is used for softening
water, which makes it useful in laundry detergents and soaps. It can also be used in
shampoo for washing colouring and wax from the hair.

4.3. In cosmetics

It is used widely as an acidulent in gels, creams, and liquids of all kinds.

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