Fertech_lect 7_ingredient.pdf

ITP 321 Lecture Note 7

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Fermentation of Ingredients and F d Additives Food Additi

Lilis Nuraida and Ratih Dewanti-Hariyadi Department of Food Science and Technology Bogor Agricultural University LN-RDH/ITP/IPB

Ingredients from microorganisms microorganism s

• Yeast-derived flavouring agents • Biogum: • Xanthan • Pullulan • etc

• Vitamins • Oils LN-RDH/ITP/IPB

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Yeast-Derived Products

• Yeast Extract • Yeast Autolysates 

Contains guanosine 5’monophosphate (GMP), ionosine 5’’ monophosphate (IMP), glutamic acid

Application: – Flavoring agent in soup, sauces, gravies, i stew, snackk food f d etc. – Main component of savoury spreads: Vegemite and Marmite

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Yeast Extract Components

• Amino acids • Peptides • Nucleotides • Proteins • Carbohydrates • Vitamins Vit i • Flavor compounds LN-RDH/ITP/IPB

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Product definition

• Yeast Extract: Yeast extract comprises the water soluble components of the yeast cell, the composition of which is primarily amino-acids, peptides, carbohydrates and salts. Yeast extract is produced through the hydrolyses of peptide b d by bonds b the th naturally t ll occurring i enzymes present in edible yeast or by the addition of food grade enzymes The Food Chemical Codex

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Product definition

• Yeast Autolysates/Autolysed yeast: Autolyzed yeast is the concentrated, not extracted, partially soluble digest obtained from food-grade yeast. Solubilization is accomplished by enzyme hydrolysis or autolysis of yeast cells. Autolyzed yeast contains both soluble and insoluble components derived from the whole yeast cells. The Food Chemical Codex

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Composition Yeast Autolysate

Yeast Extract

• • •

Protein content: 50-75% Total carbohydrate: 413% Lipid content: very little

• • •

Protein content: 50-69% Total carbohydrate content: 15-25% Lipid content: 3-10%

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Preparation of Yeast Extract Requires disruption of cell walls: • Autolysis by endogenous enzymes*) • Plasmolysis: modified autolyses in the presence of accelerator, i.e. salt or organic solvent • Mechanical desruption • Hydrolysis by acid or by exogenous enzymes *)practiced in industrial scale LN-RDH/ITP/IPB

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Production Process of Yeast Extract Absent in production of yeast autolysate

Yeast cream

Clarification

Plasmolyses

Concentration

Autolyses y

Liquid: 50-65% dry matters Paste: 70-80% 70 80% dry matters

Pasteurisation

Packed in pails, drums

Spray, roller drying

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Source of yeast cream • •

Conventional: • Baker’s Yeast • Brewer’s Yeast Alternative:

• Candida utilis • Kluyveromyces marxianus

Enriched yeast extract in amino acid cystein produced through the application of GE-yeast (genetically engineered yeast) to over express and over produce the yeast cysteinrich protein metallothionein (Stam et al., 2000) LN-RDH/ITP/IPB

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Condition for Yeast Extract Production Steps Autolyses and Plasmolyses Pasteurisation

Concentration

Condition 55 oC for 24 h, pH 5.5 1st : 70oC for 15 h 2nd: 70-75oC for 2-5 h (after clarification) Temperature <55oC Adapted from Biocatalyst Ltd

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Common problems encountered in yeast extract production Problem

Solution*)

Enzyme not working

Ensure no direct heat

Low yield autolysis

Check pH and temperature of slurry at each stage Extend autolyses time Add protease

Over heat

Add protease

Clarification problems caused by insoluble glucans

Add glucanase

Burnt flavor

Keep final temperature <55oC during final evaporation Treatment with protease followed by nuclease resulted in the highest 5’-GMP *)Adapted from Biocatalyst Ltd (Chae et al., 2001) LN-RDH/ITP/IPB

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Other yeast-derived products •

•

•

Colorants • Pigmentedd yeasts suchh as Rhodotorula, h d l

Phodospondium, Cryptococcus, Sporidiobolus, Sporobolomyces, GE-Saccharomyces, Candida

Yeast Polysaccharides: • Food application and nutraceutical potential • Potential species S. S cerevisiae cerevisiae, Pichia holstii holstii,

Hansenula sp, Candidia, Rhodotorula

Yeast Enzymes

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Microbial Polymers Xanthan gum

Emulsion stabilization and suspension agent in foods

from X.

Foam stabilization in foods

camprestis

Crystallization inhibition in foods Viscosity control in oil drilling mud and inkjet printing

Bacterial

Moisture retention in wound dressings

cellulose

High strength acoustic diaphragms in sound reproductio

Hyalluronic acid

Hydrating agents in cosmetics and pharmaceuticals Replacement for synovial fluid and vitreos humor in biomedicine

Emulsan

Emulsifier and vaccine adjuvant

Curdlan

Gelling agent in foods

Gellan

Gelling agent in foods

Pullulan

Food coatings

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Advantage of Using Microorganisms for Gum Production • Produce consistent gum • Independence of plant resources • Independence of external factors • Possibility of invention of new gum with unique properties : certain rheological properties for food additives

• Consistent quality of gum • Exploration is being carried out continuously LN-RDH/ITP/IPB

Polysaccharide Producing Microbes and their Polysaccahrides Microorganisms Azotobacter vinelandii, Pseudomonas sp. Saccharomyces cerevisiae Acetobacter sp. Alcaligenes faecalis Leuconostoc mesentroides, L. dextranicum Pseudomonas elodea Aureobasidium (Pullularia) pullulans Xanthomonas sp. Alcaligenes spp. Sclerotium spp. Schizophyllum communis

Polysaccharides Alginate Baker yeast Glucan Cellulosa Curdlan Dextran Gellan Pullulan Xantan Welan Skleroglucan Skhizopilan

*Lapasin dan Sabrina (1995) LN-RDH/ITP/IPB

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Sucrose invertase

Fructose

Glucose glucokinase

Glucose 6-Phosphate

Fructokinase

phosphoglucose isomerase

Fructose 6-Phosphate

phosphomannose p p siomerase

Mannose 6-Phosphate phosphomanno mutase

Mannose 1-Phosphate GDP-mannose pyrophosphorylase

GDP-Mannose GDP-mannose dehydrogenase

GDP-Mannuronic Acid polymerase

Plymannuronic acid polymannuronic acid 5-epimerase acetyltransferase

Alginic Acid Alginic acid biosynthetic pathway in Azotobacter vinelandii LN-RDH/ITP/IPB

Xanthan gum Colonies of Xanthomonas campestris produced copious extracellular slime - a complex polysaccharide composed of more than one type of sugar (a heteropolymer) It is termed xanthan It is used as a gelling and stabilising agent in salad dressings, ice creams, toothpastes, cosmetics, water-based paints etc., paints, etc and also as a drilling lubricant in oil wells. Xanthomonas campestris pv. campestris is an agricultural and industrial important bacterium. In agriculture it is a pathogen that causes black rot on a number of crops. LN-RDH/ITP/IPB

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Xanthan Gum •

Xanthan gum is a high molecular weight exopolysaccharides with: • Cellulose backbone • Trisaccharides side chain

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Xanthan Gum

• Characteristics :

• High viscosity • Stable properties in extreme conditions • Pseudoplastic behavior

• Applied as: as:

• Stabilizing Stabilizing// Viscosifying/ Viscosifying/Emulsifyin Emulsifying/ g/ Thickening or Suspending agents agents

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Growth of X. campestris

• X. campestris is able to use glucose, sucrose, • •

and starch as carbon source but not lactose. This is due to the low level of enzyme  galactosidase. This enzyme converts lactose to galactose and glucose.

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Pullulans

• Aureobasidium pullulans produces the exopolysaccharide pullulan.

• Linear mixed linkage a-D-glucan consisting mainly of maltotriose units interconnected via -(1-6) linkage

• This polymer has unique film-forming and adhesive properties that make it useful for producing a film-wrap for foods LN-RDH/ITP/IPB

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Production of Pullulans

• •

Substrate: sugar and starch Industrial waste: potato starch waste, whey, mollases, brewery waste A. pullulans also produced other exopolysaccharides other than pullulans

•

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FACTORS INFLUENCING BIOGUM PRODUCTION ●

Nitrogen and Carbon Source

▪ Requires high C/N ratio ▪ Arthrobacter viscosus: Xylosa 3%, N 0.033% ▪ Xanthomonas: glukosa 1-5%, N 0.048%

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FACTORS INFLUENCING BIOGUM PRODUCTION pH

●

▪ Gum production is inhibited at low pH, needs buffering agents ▪ Xanthomonas: optimum pH 6-7.5 ▪ Propionibacterium: pH 6 ▪ pH optimum for biogum production may not be similar to that for biomass production ▪ Aeromonas almonicida requires excessive phosphate LN-RDH/ITP/IPB

FACTORS INFLUENCING BIOGUM PRODUCTION ● Mineral ▪ Some requires K, P, Mg, Mn dan Ca ▪ In some instance, mineral inhibits EPS production (Enterobacter agglomerans, Erwinia) ● Incubation time ▪ Production at the end of logarithmic phase ▪ Maximum when N is low ▪ At the end of fermentation, EPS may decrease due to enzymatic activity that cleaves EPS LN-RDH/ITP/IPB

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FACTORS INFLUENCING BIOGUM PRODUCTION Incubation temperature

●

▪ EPS is generally formed at suboptimal temperature when growth is stressed ▪ Xanthomonas: optimum at 28oC ▪ P. acidipropioni: 25oC ● Harvesting methods ▪ Agglutination by organic solvents (ethanol, aceton, isopropanol) LN-RDH/ITP/IPB

Production d i off Vitamins i i b by Microorganisms Microorganism s

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Vitamin B12 Production by Various Microorganisms Strain

Carbon Source

Micromonospora sp.

Glucose

Nocardia rugosa

Glucose-cane molasses

Propionibacterium reudenreichii

Glucose

Propionibacterium shermanii

Glucose

Propionibacterium vannielli

Glucose

Pseudomonas denitrificans

Beet molasses

Streptomyes olivaceus

Glucose-lactose

Mixed methanogenic bacteria

Methanol

B t i Bacterium FM-O2T FM O2T

M th Methanol l

Methanobacillus omellanskii

Methanol

Protoaminobacter ruber

Methanol

Corynebacterium and Rhodopseudomonas

n-Parafins

Nocardia gardneri

Hexadecane

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SYNTHESIS OF VITAMIN B12

Cobinamid + ATP

Cobinamid-P + ADP

Cobinamid-P + GTP

GDP-Cobinamid + PPi

GDP-Cobinamid GDP Cobinamid + α α-ribazole ribazole 5’-P 5 P

Cobalamine 5 5’-P P + GMP

Cobalamine 5’-P

Cobalamine + Pi

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Synthesis of L-Ascorbic Acid

• Ascorbic acid ((vitamin C)) is currentlyy synthesized by a very expensive process which includes: • A microbial fermentation step • A number of chemical steps

• convert glucose to ascorbic acid

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Commercial Synthesis of Ascorbic Acid

2-KLG : 2-keto-gluconic acid

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Synthesis of L-Ascorbic Acid 

Glucose  2,5-diketo-D-gluconic acid (2,5-DKG). microrganisms capable of 2,5-DKG 2 5 DKG synthesis:  Acetobacter, Gluconobacter, Erwinia





Enzyme 2,5-DKG reductase will convert 2,5-DKG  2-KLG (2-keto-L-gluconic acid). microrganisms having this enzyme include:  Corynebacteria, C b t i Brevibacterium, B ib t i Arthrobacter A th b t Last step in the process involves the conversion of: 2-keto-L-gluconic acid (2-KLG)  L-ascorbic acid.

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Microbial Synthesis of LAA • Glucose

2 52,5 5-DKG

Erwinia herbicola

• 2,5 2,5--DKG

KLG Corynebacterium sp

• Genetically enginered E. herbicola (recombinant having DKG reductase Glucose

from Corynebacterium) KLG

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Production of 2-KLG

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Production of microbial oils

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Production of GammaGamma-linolenic acid (GLA)

•

•

Produced by Mucorales • Gamma Gamma--linolenic acid is the only 18:3 structure in Mucorales and produced in higher amount than by other molds Commecial process: • UK: UK oil of javanicus ja anic s (Mucor M co javanicus ja anic s) • Japan: Mortierella isabellina.

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Production of GLA

• • •

Carbon source: glucose g • 15 to 18% carbon source is converted to oil Production of oil depend on growth rate and substrate conc concentration Oil production induced by high C/N ratio, i.e. 80:1

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Preservatives produced by l lactic i acid id bacteria: b i BACTERIOCIN

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Bacteriocins

• • •

Small proteins produced by bacteria h harm only l microbes i b that th t closely l l resemble bl the bacteria that manufactured them attack potentially fatal foodfood-poisoning germs (ie. L. monocytogenes and C. botulinum): • Disturb membrane membrane stability

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Bacteriocins: Highly Selective





Act against one target pathogen and probably will not sicken beneficial bacteria or humans Bacteriocins considered for protection of food against Listeria are useless against S l Salmonella ll and d E. E colili E.c

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Bacteriocin, Examples

   

Nisin Pediocin Lacticin 3147 Enterocins A and B

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Nisin Secreted by some Lactococcus lactis ssp. Lactis strains Interacts with phospholipids in the cytoplasmic membrane of undesirable bacteria This action disrupts membrane function, preventing bacterial growth P Prevents outgrowth h off spores b by inhibiting i hibi i the swelling process of germination

 

 

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Pediocin 



Produced by Pediococcus acidilactici, a l ti acid lactic id bacteria b t i (LAB) Together with fermentationfermentation-derived organic acids produced by LAB, pediocin attac attack ks gram--positive bacteria such as Lactobacillus gram and Listeria monocytogenes

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Lacticin 3147  



Produced byy Lactococcus lactis subsp. p Lactis Inhibits growth in all gram positive bacteria including L . monocytogenes, C. botulinum, & Staphylococcus aureus Inserts into cell membrane of sensitive cells, leading g to the loss of potassium p ions and the collapse of the membrane potential resulting in cell death

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Enterocins A and B   

Produced by Enterococcus faecium CTC492 Antilisterial Used in – fermented sausages – cooked ham – pate – minced pork – deboned chicken breasts

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