A Novel Ingredient for Zebrafish Diets: Single Cell Protein

Stephen A. Watts*, Robert J. Barry, Sophie Bru, Yuan Yuan,

L. Adele Fowler, Michael B. Williams,

Mickie L. Powell, Addison L. Lawrence

The University of Alabama at Birmingham

ProteinPercent

Source

MineralsCalcium

Iron

Magnesium

Manganese

Selenium

Copper

CarbohydratesComplex

Simple

Source

LipidsSaturated

Unsaturated

Marine

Plant

VitaminsC, E, A, K, D

Thiamine

Niacin

Riboflavin

Common Protein Sources in Aquafeeds

• Fish (type, meal, hydrolysate, isolate, etc.)• Casein• Soy• Squid Meal• Krill meal• Whey• Gluten• Gelatin• Feather and Poultry Meal• All vary in amino acid content and other bioactive nutrients

Fish Meal in Animal Feeds

What is Fish Meal?

•Wild-caught, fish that have been dried

and ground to be used as a feed

ingredient

• Very digestible and palatable

• Balanced amino acid, phospholipid,

and fatty acid profiles

• Excellent source of protein

• Every zebrafish diet contains fish meal

• A big problem: Global demand for fish meal protein has increased significantly yet production remains static

• Aquaculture industry accounts for ca. 60-70% of fish meal usage

• The product can be highly variable in quality

• A sustainable alternative to fish meal is necessary to meet the future demands of the aquaculture industry

Fish Meal Demand is Increasing

So what about zebrafish diets?Should we be concerned about fish meal in zebrafish diets?

Should we be concerned about sustainability?

Should we be concerned about variability of the product?

Should we be concerned about reproducibility?

Alternatives?

Fish Meal Alternatives

Source Examples Advantages Disadvantages

Plant Based Proteins Soybean mealCanola mealCorn gluten mealCottonseed meal

Generally high in protein content (40-70%) with the exception of wheat (~11%)

Deficient in one or more essential amino acids Anti-nutritional factors present in some ingredientsGrowth suppression

Animal Byproducts Feather mealPoultry byproducts Bloodmeal

High protein contentBlood meal rich in lysine

Most are deficient in lysine, methionine, and/or histidineBlood meal highly sensitive to heat damageLow in omega-3 & minerals

Trimmings Fish byproductsFish carcass meals

Best substitute for fish meal as it contains similar nutritional profiles

Unavailable in sufficient quantities to support fish meal replacement

8

Single Cell Protein (SCP)• High nutrient content and quality

• Amino acid profile similar to FM

• Cheap

• Production can be controlled

• Environmentally sustainable

Nutrient composition of the main group of microorganisms used for single cell protein production(% dry wt.)

SCP- dried cells of microorganisms such as bacteria, microalgae, or yeast

Composition Algae Yeast Bacteria

Protein 40-60 45-55 50-65

Fat 7-20 2-6 1-3

Ash 8-10 5-10 3-7

Miller and Litsky 1976

Question: Can SCP replace fish meal protein?• 35-day old zebrafish were fed a formulated diet containing

casein, soy, and fish protein hydrolysate (FPH) as the protein sources

• The fish protein hydrolysate was replaced with either a dried yeast or dried bacteria commercial product on a nitrogen basis

• Control formulated diets contained FPH; in experimental diets 50% or 100% of the FPH was replaced by the yeast or bacterial product

• Zebrafish were fed at either 10% body weight per day (satiation) or at 4% body weight per day (sub-satiation)

• All the usual environmental parameters were followed

Water Quality

Mean Max Min

Temperature (°C) 27.7 ± 0.24 28.2 27.2

Conductivity (µs/cm) 1505± 48 1617 1425

Salinity (ppt) 0.71 ± 0.027 0.80 0.70

pH 7.67 ± 0.15 7.960 7.12

Ammonia (mg/L) 0.000 ± 0.000 0.000 0.000

Nitritie (mg/L) 0.000 ± 0.000 0.000 0.000

Nitrate (mg/L) 92.5 ± 10.35 100.0 80.0

Diet Ration Survival (%) ± SEM

FPH-100 10 100.0 ± 0.00

DY-50 10 100.0 ± 0.00

DY-100 10 100.0 ± 0.00

MRD-50 10 100.0 ± 0.00

MRD-100 10 100.0 ± 0.00

FPH-100 4 100.0 ± 0.00

DY-50 4 99.0 ± 1.01

DY-100 4 100.0 ± 0.00

MRD-50 4 99.0 ± 1.19

MRD-100 4 100.0 ± 0.00

RESULTS

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0 2 4 6 8

Week

Ave

rage

Ind

ivid

ual

Wei

ght

(g)

Wet Weight (g)

FPH/100/10

DY/100/10

DY/50/10

MRD/100/10

MRD/50/10

FPH/100/4

DY/100/4

DY/50/4

MRD/100/4

MRD/50/4

Diet Ration (% body weight) Male mg (SEM) Female mg (SEM)

FPH-100 10 249 (6.8)a 325 (11.6)a

DY-50 10 256 (8.7)a 328 (21.3)a

DY-100 10 275 (8.4)ab 379 (19.2)a

MRD-50 10 309 (6.8)b 341 (14.2)a

MRD-100 10 297 (7.8)b 323 (14.0)a

FPH-100 4 182 (3.6) 187 (7.5)

DY-50 4 185 (4.4) 186 (8.2)

DY-100 4 184 (4.9) 204 (8.6)

MRD-50 4 170 (4.0) 177 (6.5)

MRD-100 4 166 (4.6) 179 (8.0)

Apparent Feed Conversion Ratio* (SEM)

Diet Ration Total FCR

FM-100 10 3.11 (0.380)

DY-50 10 3.61 (0.543)

DY-100 10 3.35 (0.976)

MRD-50 10 2.60 (0.358)

MRD-100 10 2.74 (0.317)

FM-100 4 1.34 (0.052)

DY-50 4 1.30 (0.057)

DY-100 4 1.25 (0.045)

MRD-50 4 1.40 (0.073)

MRD-100 4 1.46 (0.063)

*Grams feed proffered vs grams weigh gain

Diet Ration Mean Moisture % (SD)

FPH-100 10 71.8 (0.83)a

DY-50 10 71.8 (1.16)a

DY-100 10 71.2 (0.81)a

MRD-50 10 72.1 (1.31)ab

MRD-100 10 72.3 (0.97)ab

FPH-100 4 74.5 (0.58)cd

DY-50 4 73.6 (0.42)bc

DY-100 4 73.5 (0.34)bc

MRD-50 4 75.5 (0.31)d

MRD-100 4 74.5 (0.76)cd

BA

B

0

5

10

15

20

25

30

35

40

% L

ipid

Diet

Males

FPH/100/10 Male MRD/100/10 Male DY/100/10 Male

NSNS NS

0

5

10

15

20

25

30

35

40

% L

ipid

Diet

Females

FPH/100/10 Female MRD/100/10 Female DY/100/10 Female

Conclusions

• Growth rates equaled or exceeded FPH controls when FPH is replaced with 50 or 100% yeast or bacteria SCP

• Male zebrafish fed bacteria SCP showed an increase in lean matter weight gain and reduced adiposity in relation to those fed FPH

• Both yeast and bacterial SCP show promise as fish protein replacements, but require additional health and reproductive evaluations

• We do NOT suggest that fish meal protein should be replaced in zebrafish diets. Fish meal has proven to be an excellent protein source in zebrafish “chows”. However, SCP may have importance in “formulated reference diets” where reproducible dietary control is valued

Acknowledgements

19

❖ Meridian Biotech LLC.

❖ Members of the lab

❖ UAB Department of Biology

❖ Alabama Academy of Science

❖ NIH NORC P30DK056336

Proximates FM-100 DY-50 DY-100 MRD-50 MRD-100Moisture % 12.73 13.43 12.84 14.87 16.64

Protein % 43.4 43.3 43.4 41.2 40.8

Fat % 11.1 9.95 10.3 12.5 9.11

Fiber % 0.8 2.4 1.6 1.3 nd

Ash % 6.66 6.24 5.96 7.57 8.94

TDN % 82.2 75.1 76.5 76.2 70

sulfur % 0.53 0.53 0.56 0.49 0.44

phosphorous % 1.32 1.32 1.4 1.72 2.17

potassium % 0.92 0.75 0.75 0.95 0.98

magnesium % 0.04 0.07 0.11 0.26 0.49

calcium % 0.83 0.81 0.94 0.78 0.78

sodium % 0.84 0.71 0.72 0.73 0.64

iron ppm 93.4 150 205 129 172

manganese ppm 98.3 101 112 104 113

copper ppm 7.6 16.6 30.7 9.8 13.7

zinc ppm 39.2 53 70 71.2 105

• Funding: Meridian Biotech, LLC

NIH NORC: P30DK056336

• Special thanks to:Adele Fowler

Michael Williams

Chris Taylor

Audrey Powers

Ningxiang Zeng

Susmita Patnaik

Acknowledgements

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Single Cell Protein (SCP)

• High nutrients content and quality

• Amino acid profile similar to FM

• Cheap and Effective

• Selectable

• Environmentally sustainable

Nutrient composition of the main group of microorganisms used for single cell protein production(% dry wt.)

SCP- dried cells of microorganisms such as bacteria, microalgae, or yeast

Composition Fungi Algae Yeast Bacteria

Protein 30-45 40-60 45-55 50-65

Fat 2-8 7-20 2-6 1-3

Ash 9-14 8-10 5-10 3-7

Miller and Litsky 1976

Amino Acid Fish Meal % Yeast SCP % Bacterial SCP %

Aspartic Acid 5.8 3.61 5.16

Threonine 2.7 2.46 1.99

Serine 2.5 2.80 1.90

Glutamic Acid 8.6 11.50 8.22

Arginine 4 2.19 2.57

Glycine 4.8 2.10 3.19

Alanine 4.2 4.44 5.55

Proline 3.3 5.69 2.37

Cystine 0.6 1.13 0.35

Valine 3.2 3.02 3.53

Methionine 2.2 1.14 1.17

Isoleucine 2.7 2.35 2.89

Leucine 4.6 8.56 4.47

Tyrosine 2.1 2.70 2.10

Phenylalanine 2.5 3.49 2.80

Histidine 1.8 1.48 0.98

Lysine 5 1.36 3.75

Tryptophan 0.5 0.41 0.62

Amino Acid content of SCPs (% dry wt.)

• Single Cell Proteins (SCP) –dried cells of microorganisms such as yeast, bacteria, or microalgae

• Amino acid profile similar to fish meal

• Sustainable

Amino Acid Fish Meal % Yeast SCP % Bacterial SCP %

Cystine 0.6 1.13 0.35Valine 3.2 3.02 3.53Methionine 2.2 1.14 1.17Isoleucine 2.7 2.35 2.89Leucine 4.6 8.56 4.47Tyrosine 2.1 2.70 2.10Phenylalanine 2.5 3.49 2.80Histidine 1.8 1.48 0.98Lysine 5 1.36 3.75Arginine 4 2.19 2.57Tryptophan 0.5 0.41 0.62

Single Cell Proteins as Fish Meal Replacement

Ingredient

Z17-0201

FM-100

Z17-0202

DY-50

Z17-0203

DY-100

Z17-0204

MRD-50

Z17-0205

MRD-100casein - low trace metals 20 20 20 20 20

soy protein isolate 6.6 6.6 6.6 6.6 6.6

fish protein hydrolysate 25 12.5 12.5

SCP Sample 1 (DY) 18.5 37

SCP Sample 2 (MRD) 15.37 30.75

wheat starch 15.95 11.48 7 12.08 8.2

dextrin 5.5 5.5 5.5 5.5 5.5

menhaden fish oil (ARBP) 0 1.25 2.5 1.25 2.5

corn oil 5.55 2.77 5.3 5.05

soy lecithin (refined) 4 4 4 4 4

alginate (TIC algin 400) 5.38 5.38 5.38 5.38 5.38

vitamin mix (MP-VDFM) 4 4 4 4 4

mineral mix (BTm) 3 3 3 3 3

canthaxanthin (10%) 2.31 2.31 2.31 2.31 2.31

potassium phosphate monobasic 1.15 1.15 1.15 1.15 1.15

alpha cellulose 1.14 1.14 1.14 1.14 1.14

glucosamine 0.11 0.11 0.11 0.11 0.11

betaine 0.15 0.15 0.15 0.15 0.15

cholesterol 0.12 0.12 0.12 0.12 0.12

ascorbylpalmitate 0.04 0.04 0.04 0.04 0.04

Total 100 100 100 100 100

Calculated Energy Level (kcal) 3780 3600 3422 3624 3469

Calculated Protein Level (%) 45.07 45.06 45.05 45.06 45.06

Calculated Lipid Level (%) 12.44 12.43 12.44 12.43 12.43