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Functionalities of Food Additives and

Enzymes in Muscle Foods: A

Technological Perspective

Youling L. Xiong, PhD, ProfessorFellow of IFT, ACS AGFD, IAFoST

Scientific Editor, Journal of Food Science

Department of Animal and Food Sciences

University of Kentucky

Bertebos Conference on Food

Additives and Enzymes

August 29-30, 2016

Falkenberg, Sweden

Food additive (FDA):

“Any substance that is reasonably expected to become a component of food.”

Production, processing, treatment, packaging, transportation or storage of food.

Excludes GRAS ingredients

Purposes1) To maintain or improve safety and freshness

2) To improve or maintain nutritional value

3) To improve taste, texture and appearance

4) To aid in processing

“Additive” vs. “Ingredient”

Difference is mainly in the quantity used in any given formulation

Food additives are minor ingredients incorporated into foods to affect their properties

Additives:Key to the success of processed meats

Flavor

Color (appearance)

Texture

Mouthfeel

Products yield (water-binding)

Product stability and shelf-life (antioxidant)

Variety of products

Food safety

In the U.S. meat industry,

Brand Name!

Beef pastrami

Salami

Roast beef

Ingredients/Materials

1. Meat materials

2. Salt

3. Phosphates

4. Sweeteners

5. Alkalis/acids

6. Antioxidants

7. Nitrite/nitrate

8. Binders

9. Flavorings/seasoning

10. Casings

11. Enzymes

12. Antimicrobials

“functional”

Meat Products

QualitySafety

Organoleptic Nutritive/health

Color Texture Juiciness StabilityFlavor

Ingredients

processing

Animal Production

Feed additives

Meat Ingredients/Additives

1. Meat quality

“Micro” molecular chemicals

• Salts

• Phosphates

• Nitrite

• Antioxidants (ascorbate, tocopherols, BHA…)

• Antimicrobials

“Macro” molecular chemicals

• Non-muscle proteins

• Protein hydrolysates

• Enzymes

• Polysaccharides

Meat Ingredients/Additives

2. Meat safety

① Organic acids Citric, lactic, malic, benzoic acid

② Salt of organic acids Sodium lactate, diacetate, citrate,

③ Bacteriocins Nisin, pediocin

④ Acidified sodium chlorite

⑤ Lauric Arginate

⑥ Lactoferrin

⑦ Spices/herbs

Antimicrobials

1. Salts

Monovalent chlorides NaCl, KCl, LiCl

Divalent cations CaCl2, MgCl2

Salts (e.g., NaCl)

Main function is to solubilize/extract myofibrillar

proteins

Solubility gelation, emulsification, meat binding, water

holding… texture, tenderness, juiciness, product

stability….

Critical concentration (without phosphate): ~2% NaCl

(0.5 M)

Fat

Protein membrane

(NaCl, mol/l)

0.0001 0.001 0.01 0.1 1 10

So

lub

ilit

y (

%)

0

20

40

60

80

100

Extensively washedhighly diluted

Fresh meat

Salted meat

Xiong, Y.L. 2004. Encyclopedia of Meat Sciences, Jensen, W.K., Devine, C., and

Dikeman, M. (Eds.), pp. 218-225. Elsevier, Oxford, UK.

NaCl

Fresh meat

Salted meat (2% NaCl)

─NH3+

Cl-─NH3

+

Cl-

Cl-

Cl-

─NH3+/Cl-

─NH3+/─COO-

─NH3+

─COO-

Wate

r B

ind

ing

4.0 4.5 5.0 5.5 6.0

pH of Meat

─NH3+

+

+

__

0

0

Xiong, Y.L. 2004. Encyclopedia of Meat Sciences, Jensen, W.K., Devine, C., and Dikeman, M.

(Eds.), pp. 218-225. Elsevier, Oxford, UK.

H

H

O d–d+

KCl LiCl CaCl2 MgCl2 Salt substitute blend

• KCl/K-lactate/glycine• NaCl/KCl/CaCl2• KCl/tartaric acid/citric acid/sucrose• Transglutaminase/caseinate/KCl/dietary fiber• NaCl/KCl/MgSO4/lysine HCl

Replacement of all or part of NaClwith other chloride salts

Flavor enhancersIMP Yeast extractMSG Protein hydrolysates

Masking agentsNeutraFresSaltTrim

What About Flavor?

“Umami”

Salt replacers:Calcium chloride Magnesium chlorideMagnesium sulphatePotassium chloridePotassium iodine (Iodized salt)

Salt taste enhancers:L-ArginineCalcium diglutamateDipotassium inosinate Inosine monophosphate LactatePotassium guanylate L-lysine Mono potassium glutamate ThrehaloseL-ornithineDried bonito aroma extract(not heated)

Options of Taste

Sulfur-containing compounds, pyrazines,

alcohols, and phenols

2. Phosphates

Myofibrils

Muscle Proteins

Swelling

DissolutionAggregation

Denaturation

Gel network

Product texture,

WHC, fat binding

Phosphates Processing factors:

pH, salt, other ingredients

time, temperature

Phosphates

&

Processing

factors

Hydration

Water binding

Juiciness,

tenderness

① Extract protein

② Increase water holding

Salt

Phos

High

LowXiong, Y.L. 1999. Quality Attributes of Muscle Foods, pp. 319-334.

Kluwer Academic/Plenum Publishers, New York.

Types of phosphate

0.2–0.5%

Mono: Orthophosphate, P (1, 2, 3 Na+)

Di: Pyrophosphate, PP (1, 2, 3, 4 Na+)

Tri: Tripolyphosphate, TPP (1, 2, 3, 4, 5 Na+)

Meta: Hexametaphosphate, HMP

P

O

O

O

O P

O

O

O

O P

O

O

O

P

O

O

O

O P

O

O

O

P

O

O

O

P

O

O

O

OP

O

O

P

O

OO

P O

O

O

O

PO

O O

P O

O

O

O

PO

O

Orthophosphate Pyrophosphate Tripolyphosphate Hexametaphosphate

(TPP)(PP)(P) (HMP)

AI

Z M

Xiong, Y.L. 2000. Food Proteins: Processing Applications, Nakai, S.

and Modler, H.W. (Eds.), pp. 89-145. Wiley-VCH, New York.

Actomyosin X-bridge

③ Bind meat together

through gelationHMMLMM

S1S2

Rod1

HMMLMM

S1S2

Rod1

HMMLMM

S1S2

Rod1

HMMLMM

S1S2

Rod1

35-45°C 45-55°C >55°C

LMM HMM

Rod

Structured beef steak

Chopping/milling

15~25°C

Cooking

25 → 75°C

Fat

native myosin

myosin aggregate

Fat

Fat

Fat

Fat

Fat

④ Promote Emulsion

Theno and Schmidt (1982)

3. Nitrites (NO2–)/Nitrates (NO3

–)

4 Main functions

1) Color

2) Antimicrobial (botulinum)

3) Cured favor

4) Antioxidant

Nitric oxide (NO) is the responsible agent

Nitrates allowed for dry cured meats only

NaNO2 + H2O → HNO2 + NaOH

3 HNO2 → HNO3 + 2 NO + H2O

Salami

Ham

“No-Nitrate/Nitrite Added” Meat Curing

Idea: use nitrate or nitrite-containing

natural sources to replace pure nitrate or

nitriteRaddish juice, cabbage juice, celery powder,

spinach extract, sea salt…..

Contain 100-2,000 ppm

Products must be labeled with the statement “No

tritrate or nitrite added except that that found in

radish juices or (other)

Manufacturing Options

A) Addition of natural nitrate source (e.g., celery juice powder) combined with cultures

Staphylococcus carnosusNitrate (NO3

–) Nitrite (NO2–)

Lactobaccilus fementumNitrite (NO2

–) Nitric oxide (NO)

108 CFU/g L. fermentum application level replicates nitrite-cured sausage (60 ppm nitrite) in pink color *Risk: Nitrite level not controllable

B) Pre-fermentation to prepare nitrite

Pre-fermentation

Ferment natural nitrate source (e.g., celery juice, cherry juice)

Staphylococcus carnosusNitrate (NO3

–) Nitrite (NO2–)

Apply at desirable nitrite concentration levels with other cure adjuncts

Antioxidants derived from juicesSebranek and Bacus, 2007

4. Enzymes

-Gln-C-NH2 + H2N-Lys

-Gln-C-NH-Lys-

O

ONH3

Ajinomoto Activa TG

a. Microbial Transglutaminase (MTGase)

Streptomyces mobaraensis

Ramirez-Suarez, J.C., Xiong, Y.L., and Wang, B. (2001). J.

Muscle Foods 12:85-96.

MTGase

b. Glucose Oxidase (GO)

Wang, Xiong, etc., 2016. JAFC, In review

Protein gel

Wang, Xiong, etc., 2016. JAFC, In review

Wang and Xiong (unpublished)

c. Lipases

Di- Mono-

c. Lipases

Control

Lipase a Lipase b

• Flavor profile• Processing time

5. Antioxidants

Singlet oxygen (O2●)

Superoxide radical (O2●-)

Hydroxyl radical (OH●)

Ferryl radical (FeOH3+ or FeO2+)

Perferryl radical (Fe2+-O2●-)

H2O2-activated metmyoglobin

Lipoxygenase

Retard rancidity

Tocopherols (a, b, g, d)

Rosemary extracts and other

plant extracts (tea, licorice,…)

BHA, BHT, PG, TBHQ

Ascorbate (erythorbate)

Citrate

Protein hydrolysates (peptides)

Oxidants present in meats Anti-oxidants added

Antioxidants

Lipid soluble

Water soluble

Tocopherols

Rosemary extracts

Other phenolic extracts

BHA, BHT, PG, TBHQ

Ascorbate (erythorbate)

Citrate

Protein hydrolysates (peptides)

Legal levels of antioxidants

Strict regulation on synthetic antioxidants

- BHA, BHT, TBHQ, PG

In fresh sausage, one antioxidant is allowed at

0.01% of fat content

Combination of antioxidants are allowed at 0.02%

of fat content

For natural antioxidants, more tolerance

*Inhibition of lipid oxidation and

rancidity of cooked pork patties

by licorice phenolic extract

Jiang et al., 2013. J. Food Sci.

Antioxidant Peptides/Protein Hydrolysates

Source Sequence Reference

β-Conglycinin (soy) Leu-Leu-Pro-His-His Chen et al. (2005)

Albumin (rice) Asp-His-His-Gln Wei et al. (2007)

Yellowfish sole

(fermented)

Arg-Pro-Asp-Phe-Asp-Leu-Glu-Pro-Pro-Tyr Jun et al. (2004)

Mussel (fermented) His-Phe-Gly-Asp-Pro-Phe-His Rajapaske et al.

(2005)

Milk (fermented) Val-Leu-Pro-Val-Pro-Gln-Lys Rival et al. (2000)

β-Lactoglobulin (milk) Trp-Tyr-Ser-Leu-Ala-Met-Ala Hernandez-Ledesma

et al. (2007)

Potato protein Mixed peptides (hydrolysates) Wang and Xiong

(2005)

Whey His, Lys, Val, Leu, Ile Pena-Ramos et al.

(2004)

Buckwheat protein Trp-Pro-Leu, Val-Pro-Trp, and Val-Phe-Pro-

Trp, Pro-Trp

Ma et al. (2009)

Storage Time (day)

0 1 2 3 4 5 6 7 8

Pero

xid

e V

alu

e (

me

q/k

g)

0

2

4

6

8

10

12

14

Control

Nonhydrolyzed protein

Hydrolyzed protein

Inhibition of Lipid Oxidation by Potato Peptides in

Cooked Meat:

Lipid-H Lipid-OOH off-flavorX

Wang and Xiong, 2006

Inhibition of Lipid Oxidation by Potato Peptides

in Cooked Pork SausageInhibition of Lipid Oxidation in Cooked Pork Emulsions

By Hydrolyzed Potato Protein

Storage Time (Day)

0 1 3 7

TB

AR

S (

mg

/kg

)

0

2

4

6

8

10

12

14

15% fat

+2.5% HPP

30% fat

+2.5% HPP

by 50-80%

Nieto et al., 2010

Applications of Antioxidative Peptides and

Protein Hydrolysates

Comminuted meat

Hamburgers

Dry sausages

Meat Product

Safety

1. Chemical

2. Microbiological

a) Antioxidant strategies to

mitigate chemical hazards

- Inhibit toxins and carcinogens produced

during processing and cooking

“Processed meat was classified as

carcinogenic to humans (Group

1)”

Meat and meat product toxicity:

Lipid oxidation products (HNE, MDA)

Protein-derived hazards (HAA, PAH)

Nitrosamines (nitrite or nitrate-cured meat )

Carbohydrate-derived toxic compounds (AGE)

Natural Antioxidant Strategies?

4-hydroxy-2-nonenal

(HNE)

Malondialdehyde

(MDA)

Antioxidants inhibit nitrosamine production in nitrite-

cured meats

Table 2 NDEA (ng/mL) in the mixtures of sarcoplasmic (SP) and myofibrillar (MP) protein fractions with

nitrite (pH 6.0) after 1 h cooking at 80 °C

Storage

time (day)

NDEA (ng/mL) in SP1 NDEA (ng/mL) in MP1

Control Tocopherol

(700 ppm)

Tea PE

(300 ppm) Control

Tocopherol

(700 ppm)

Tea PE

(300 ppm)

0 19.8±0.8Ad 19.5±1.2Ac 19.5±0.4Ad 67.8±4.6Ad 64.9±3.4Ac 47.6±1.8Bd

4 72.2±3.8Ac 67.0±3.9Ab 48.5±5.0Bc 88.4±7.1Ac 86.6±7.0ABb 74.8±6.4Bc

7 136.7±4.4Ab 111.9±6.6Ba 76.6±6.8Cb 147.0±8.9Ab 123.8±2.4Ba 103.6±2.8Cb

10 171.0±2.7Aa 119.8±1.5Ba 87.7±3.6Ca 182.8±4.7Aa 129.2±6.7Ba 119.4±4.3Ba

NDEA: N-nitrosodiethylamine

Yang et al., 2014

Correlation between protein/lipid oxidation and

nitrosamine production

Protein oxidation nitrosamines

Lipid oxidation nitrosamines

• A cyclic compound with at least one nitrogen within a ring structure is called a

heterocyclic aromatic amine (HAA); If more than 2 fused benzene rings, then

polycyclin aromatic hydrocarbon (PAH)

• Most HAAs are generated from the reaction of free amino acids, creatinine, and

hexoses. PAH at pyrrolic temperatures

Antioxidants inhibit protein-based toxin formation in cooked

meats

Heterocyclic aromatic amines (HAAs)

“thermic HAAs,” IQ type (imidazoquinoxaline); formed between 100 and 300 °C

“pyrolytic HAAs,” or non-IQ type, formed at higher temperatures, above 300 °C

More than 20 HAAs have been identified in cooked meats

Keating and Bogen, 2004

PhIP in grilled meats

Concentrations of PhIP increase with cooking time

2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)

Vegas, 2012

Oxidation

56

Meat product Toxin formed Inhibitory antioxidant Responsible

agent

Reference

Fried beef

patties

HAAs (MeIQx

and PhIP)

rosemary, turmeric,

fingerroot, galangal,

cherry, vitamin E

polyphenols Balogh et al. (2000);

Britt et al. (1998);

Puangsombat (2011)

Fried meatballs HAAs (IQ,

MeIQx, PhIP)

Black pepper polyphenols Oz & Kaya (2011)

Cooked beef HAAs Green tea extract Catechin Weisburger et al.

(2002)

Beef juice HAAs (IQ,

MeIQx)

Tomato Carotenoid

Quercetin

Vitaglione et al.

(2002)

Cooked meat ALEs (MDA)

oxidized

cholesterol

ginkgo biloba extract polyphenols Kobus-Cisowska et al.

(2014)

Cooked salted

pork patties

MDA Carnosine Carnosine Decker and Crum

(1992, 1993)

Cooked cured

meat extracts

Nitrosamine

(NDEA)

α-Tocopherol, green tea

extract

Phenolics Yang et al. (2013)

Examples of natural antioxidants that inhibit toxins and carcinogens formation in cooked and processed meat

Proposed pathway for PhIP formation from phenylacetaldehyde,

creatinine, ammonia, and formaldehyde.

Zamora et al., 2014

Proposed mechanism for the inhibition of HAA formation in cooked meat by phenolic

antioxidants. For illustration, gallic acid and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

(PhIP) are used. (Based on Zomora et al., 2014)

Jiang and Xiong, 2016

a) Binding

(stabilize reactant or

intermediate)

b) Radical scavenging (remove ROS)

c) Detoxifying

(activate defense

mechanism)

Feed rats 2% tea extract for 2 wk before

and 2 wk during PhIP dosing (100 mg/kg)

*Conclusion: Phenolics from teas markedly enhance the

expression of enzymes that metabolize PhIP (detoxify!)

Carter et al., 2007.Comparison of White Tea, Green Tea, Epigallocatechin-3-

Gallate, and Caffeine as Inhibitors of PhIP-Induced Colonic Aberrant Crypts.

Nutrition and Cancer, 58, 60–65.

b) Additive strategies to

mitigate biological threats

- Microbial

For detail, see Jiang and Xiong, 2015. Crit. Rev. Food Sci. Nutr. 55:1886-1901

Ready-To-Eat (RTE) Meat

RTE

1. RTE: what is it?

2. Food safety concern

3. Measures to guard safety

◦ Role of USDA FSIS

◦ Post-lethality processing technology

◦ Formulation technology

◦ Packaging technology

RTE meat and poultry

“Deli meats”Bologna

Wiener

Frankfurter (hotdog)

Ham

Roast beef

Turkey ham

Chicken loaf

Salami

Fermented sausage

RTE Meats

“Ready-to-eat ”

Cooked at low to intermediate

temperatures

◦ 65 to 80oC

◦ Pasteurization

◦ Salmonella, E. coli (O157:H7), Campylobacter,

Clostridium, Listeria monocytogenes

◦ Re-packaged

◦ Require refrigeration

RTE meats

“Ready-to-eat ”

20 to 50% of Americans

consume RTE meats on a

given day.

Similar in many other

countries (Australia,

England, …).

RTE product safety

**Listeria monocytogenes!

Decontamination Technologies

1) Post-lethality (in-package) thermal processing

2) Non-thermal processing

3) Antimicrobial additives

4) Antimicrobial packaging

Antimicrobial additives

Growth Inhibitors

Inhibit L. monocytogenes

Growth is limited to < 2 logs over the

duration of the product’s shelf life

Listeriostatic

Antimicrobial agents

① Organic acids Citric, lactic, malic, benzoic acid

② Salt of organic acids Sodium lactate, diacetate, citrate,

③ Bacteriocins Nisin, pediocin

④ Acidified sodium chlorite

⑤ Lauric Arginate

⑥ Lactoferrin

⑦ Spices/herbs

Organic acids

Reduce the pH of products

Inhibit growth of microorganisms

◦ Membrane

◦ Intracelluar

Dip or spray acids on products

Slices in lactic acid and benzoic acid

Salts of organic acids

Formulated into products

4.8% sodium and/or potassium lactate and 0.25% sodium diacetate approved as antimicrobial agents in meat product formulation.

Synergistic effect of SL and SDA against L. monocytogenes in RTE meat products.

Sodium diacetateSodium lactate

NaO CH3

O O

Sodium lactate (SL) + Sodium

diacetate (SDA)

2.5% SL + 0.1% SDA prevent growth of L. monocytogenes in turkey slurries for 42 days at 4 oC (Schlyter et al., 1993).

1% SL + 0.1% SDA inhibit L. monocytogenes in smoked

wieners for 60 days at 4.5 oC (Glass et al., 2002).

1.6% SL + 0.1% SDA prevent growth of L. monocytogenes in

ham cured ham slices (Glass et al., 2007).

Mode of action: Reduces water activity; acidifies cytoplasm;

interferes with metabolic activity (De Wit and Rombouts,

1990; Ita and Hutkins, 1991; Brul and Coote, 1999)

Bacteriocins

Nisin

Lactococcus lactis

34 amino acids

Bacteriocins

Pediocin

Pediococcus acidilactici

44 amino acids

(N)Lys-Tyr-Tyr-Gly-Asn-Gly-Val-Thr-Cys-Gly-Lys-His-Ser-Cys-Ser-Val-Asp-Trp-Gly-Lys

Asn-Gly-Gln-His-Gly-Gly-Thr-Ala-Trp-Ala-Met-Ala-Gly-Asn-Asn-Ile-Ile-Cys-Thr-Thr-Ala

His-Lys-Cys-Thr-Gly-Gly-His-Gln-Gly-Asn-His-Lys-Cys(C)

+ +++

+

+

+

+ + +

Mode of action

Inactivates L. monocytogenes on RTE meats

a. Disruption of cell membrane (phospholipids)

b. Dissipation of proton motive force

- Energy produced by moving protons across mitochondria membrane:

ΔG = RT ln(H+in/H+out)

Spices

Garlic, sage, cloves, cassia, oregano,

pimento, thyme, rosemary, scutellaria, and

forsythia suspensa

Inhibit L. monocytogenes

Gram+ pathogens are more sensitive

Essential oils of oregano and garlic inhibit

L. monocytogenes when added to edible

films

Inhibition of L. monocytogenes

on ham slices in vacuum at 4oC

Storage time (day)

L.

monocyto

genes (

lg C

FU

/g)

0 7 14 21 28

4

5

6

7

8

9

10

Storage time (day)

Mesophili

c a

ero

bic

bacte

ria (

lg C

FU

/g)

0 7 14 21 28

0

2

4

6

8

10

Storage time (day)

Lactic a

cid

bacte

ria (

lg C

FU

/g)

a

b

c

0 7 14 21 28

4

5

6

7

8

9control

2.5 mg/mL mixed spice extract

5.0 mg/mL mixed spice extract

10.0 mg/mL mixed spice extract

a

Rosemary + licorice (1:1)

Zhang, Kong, Xiong, Sun, 2009. Meat Sci.

Eugenol

Caffeic acidCatechol

Glabrene

Liquiritigenin

O

OHO

OHO

OHO

OH

H

O O

OHHO

H

O O

OHHO

Glabridin

Catechin

Quercetin

Mode of action:Binds membrane proteins and metabolites

Active packaging

Chitosan-coated plastic films containing nisin (500 IU/cm2), sodium lactate (0.01 g/cm2), sodium diacetate (0.0025 g/cm2), potassium sorbate (0.003 g/cm2), or sodium benzoate (0.001 g/cm2)

◦ Completely inhibit L. monocytogenes

on cooked ham stored at 4 oC for

12 weeks (Ye et al., 2008)

Polylactic acid-based film incorporated with 5–15% lactic acid and 10–30% sodium lactate

◦ Completely inhibits L. monocytogenes

(Theinsathid et al., 2011)

Plastic bags coated with a pediocin powder

◦ Completely inhibits L. monocytogenes on cooked ham stored at 4 oC for 12 weeks (Ming et al., 1997)

Meat Products

QualitySafety

Ingredients

processing

Animal Production

Feed additives

Summary

Thank you!

AcknowledgmentsoGraduate students

o Postdoctorals

oCollaborators

Various food

companies