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2-2014 9.03biological and Microbiological Evaluation of Aquafeeds and feedstuffs-MBT PDF
2-2014 9.03biological and Microbiological Evaluation of Aquafeeds and feedstuffs-MBT PDF
D
Scientist
Aquaculture Department
Southeast Asian Fisheries Development Center
Tigbauan, Iloilo 5021 PHILIPPINES
*Lecture notes submitted to Technology and Information Division for the Training Course on
Feed Formulation and Feed Evaluation for Aquaculture Species, September 1-5, 2014
Introduction
the use of quality feeds is important in the success of
any aquaculture venture since 50-60% of the cost of production is
mainly attributed to feeds.
feed quality is highly dependent on the quality of raw material
and the processing technique.
formulated feed that makes use of low quality raw materials
will not give the fish farmer any significant benefit. Feedstuffs and
finished feeds should, therefore, undergo the process of evaluation
and quality control in order to produce high quality feed.
3. Microbiological
- involves the use of microorganisms to evaluate the
quality of the feed or the feed ingredient.
4. Biological
- involves actual feeding experiment. Gives an
accurate estimate of feed utilization
Advantages
In vivo approaches
On-farm experiment
Reliable
Growth/digestibility
Reliable
Disadvantages
Costly; Experimental
designs limitations
Costly; limited accessi
bility
Lab approaches
In vitro assays
Proximate analyses
Fast; reliable
Analytical
errors/limitations
3. Microbiological Evaluation
a method of feed/feed ingredients evaluation that
3. Microbiological Evaluation
Amino acid composition
this method is valuable in analyzing mixtures
of amino acids because of the speed and
reproducibility of results obtained.
a nutrient medium which contains all the
essential compounds needed for the growth
of a particular microorganism except the
amino acid to be assayed is prepared.
Lactobacilli
3. Microbiological Evaluation
the addition of this amino acid results in the growth of
the microorganism in proportion to the amount of amino acid
added. Culture tubes are set up and graded amounts of the
unknown are added to a series of tubes.
standards are set at the same time with graded amounts of the
pure amino acid. The unknown can be compared with standards by
measuring the rate of growth of the microorganism.
with organisms that form acid such as Lactobacilli , titration of
the acid formed can be used as a measure of the number of cells
present. Pure cultures need to be used in this
kind of evaluation.
3. Microbiological Evaluation
a. Determining amino acid composition
rapid development of microbiological methods for
3. Microbiological evaluation
Procedure:
Feed samples obtained by random sampling of the feeds
and feed ingredients (e. g. corn, corn gluten meal, meat
and bone scraps, soybean oil, soybean meal, alfalfa leaf
meal)are finely ground prior to assay to assure
homogeneity.
L. 'arabinosus is used as the test organism for amino
acid determinations.
Pure amino acid standards are used to eliminate
possible source of error.
3. Microbiological Determination
Express amino acid values on the basis of 10% activity for the 1-
used as standards.
3. Microbiological Evaluation
Amino acid content of rations determined by actual
Valine
Isoleucine
Phenylalanine
1.17
1.38
1.27
1.42
1.06
1.10
1.10
1.21
1.07
1.14
1.14
1.19
Basal Medium
Culture and
suspension media
In preparing
medium for given
assay, omit
particular amino
acid to be assayed
from medium
Agar culture
medium
Liquid culture
medium
Liquid suspension
medium
Streptococcus
faecalis
Lactobacillus
plantarum
Pediococcus
cerevisiae
Inoculum
Suspend cells in 10 ml
suspension medium
Assay
Using working standard solutions, test solutions,
and basal medium (omitting amino acid being
Microbiological Method of
Feed Evaluation
Vitamin Composition:
strains of microbes.
culture medium.
Incubate 6-24 h at any selected temperature (30-40C) held constant to
within 0.5C.
Store in dark room at 10C
3. Centrifuge
culture
6. Resuspend cells
in 10 ml sterile
0.9% NaCl soln
4. decant
7. Dilute aliquot
with the same soln
to give T equivalent
to that for dried cell
weight of 0.5- 0.75
mg/tube
Read against
suspension
medium set
at 100% T. cell
suspension so
obtained is
inoculum
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
and 40C held constant
to within 0.5C
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,
respectively, of standard solution.
1.Dilute basal
medium stock
solution with
aqueous solution
containing 0.2 g
niacin/ml
Inoculum
1. Transfer lactobacillus
plantarum to sterile
tube containing 10 ml
liquid culture medium
2. Incubate 6-24 h
(between 30 and 40 C
2. Add 10 ml of diluted
medium to test tubes
3. Centrifuge culture
and decant
supernatant
5. Resuspend cells in
0.9% NaCl soln
6. Cell suspension so
obtained is inoculum
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
and 40C held constant
to within 0.5C
niacin.
Multiply the value by 0.992 if potency is to be expressed as
niacinamide.
folic acid.
Refer to AOAC Official method (2000) 944.12 for the
Test Solution
Refer to AOAC (2000)Official Method 944.12 for the preparation of test
solution.
Perform assay using standard solution, test solution, basal medium stock
solution and inoculum.
Meticulously
cleanse by suitable
means followed by
heating 1-2 h at
250C
Incubate between 30
and 40C held constant
to within 0.5C
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,
respectively, of standard solution.
Calculations
For each level of test solution used, calculate vitamin
3. Microbiological evaluation
Animal feeds
Aquatic animals reared intensively require large amounts of plant or
3. Microbiological Evaluation
What is salmonella?
Salmonella is bacteria that can cause a gastrointestinal
3. Microbiological Evaluation
Salmonella may be found in feeds and feed
3.Microbiological evaluation
Code of practice has been issued for the control of
3. Microbiological evaluation
The sample should be tested on the day of receipt or on the 1st
3.Microbiological evaluation
Example of Contaminated Experimental feed
Microbiological Parameter
Control Feed
Experimental Feed
2.0 x 10-3
6.24 x 10 4
110
>240
4.9 x 102
3.8 x 102
4. E. coli (MPN/g)
Absent
Absent
5. Salmonella/25g
Absent
Absent
6. S. aureus
Absent
Absent
3.Microbiological evaluation
Microbiological analyses in feeds and ingredients
Salmonella
Total plate count
Mould*
Yeast*
Coliform bacteria
Bacillus cereus*
E. Coli
Staphylococcus aureus*
3.Microbiological evaluation
Microbiological analyses in feeds and ingredients
a. Salmonella
Procedures in Salmonella determination:
1. Fish meal
a. Aseptically weigh 25g sample into sterile blending
container.
b. Add 225ml sterile lactose broth and blend mixture for
2 minutes.
3. Microbiological evaluation
Microbiological analyses in feeds and ingredients
c. Aseptically transfer homogenized mixture to sterile
wide-mouth, screw cap jar (500 ml) or other
appropriate container and let stand for 60 min. at room
temperature with jar securely capped. (If mixture is
powder, blending may be omitted)
d. If samples are in powder form, add lactose broth and
mix thoroughly for 60 min. at room temperature.
3. Microbiological evaluation
Microbiological analyses for feeds and ingredients
e. Mix well by swirling and determine pH with test
paper. Adjust pH, if necessary to 6.8.
f. Add up to 2.25 ml steamed (15 min) Tergitol Anionic
7 or Triton X-100 and mix well. (actual quantity needed will
depend on the composition of the raw material).
g. Loosen jar caps turn and incubate sample
mixtures for 24H at 350C.
h. Tighten lid and gently shake incubated sample
3. Microbiological Evaluation
Microbiological analyses of feeds and ingredients
3.Microbiological evaluation
Microbiological analyses in feeds and ingredients
a. Staphylococcus aureus
Procedures in S. aureus determination:
1.
2.
3.
4.
5.
3. Microbiological Evaluation
Plate Count Agar (PCA), also called Standard
3. Microbiological Evaluation
Three methods of how to calculate the amount of bacteria in a plate.
The dilution table is as follows:
Unknown
Volume Transferred
Dilution A
1 ml
999
1000
Dilution B
1 ml
99
100
Dilution C
1 ml
9
10
100 l of Dilution D is used to inoculate two nutrient agar plates. After
incubation the plates show 30 and 31 colonies of bacteria. The average
number of bacteria colonies is 31.
3. Microbiological Evaluation
METHOD 1.
Express the dilution and the inoculation in scientific format. (This
value is found by dividing the Volume Transferred by the Total
Volume.)
Dilution A
1x10-3
Dilution B
1x10-2
Dilution C
1x10-1
Inoculation
1x10-1
Calculate the amount of bacteria in the original solution by:
original solution.
The plate count simulation coefficient is always expressed as a
3. Microbiological Evaluation
Method 2
1000 / 1
100 (Dilution B) =
100 / 1
10 (Dilution C) =
10 / 1
10 (inoculation of 100 or 0.1ml) = 10
31 (colonies counted on plate)
300,000,000 = 3x108 = no. of bacteria in the original solution
3. Microbiological Evaluation
Method 3
3. Microbiological Evaluation
To compute for colony plate counts???
Problem:
3. Microbiological Evaluation
The three plates illustrated below are the results of incubating agar plates
overnight after applying 0.1ml from one of the tubes in a bacterial dilution
series. Each plate used a different starting culture.
Using the colony counts and dilution factors given, work out what the original
overnight culture densities were (in bacteria/ml).
3. Microbiological Evaluation
Answer
A: 80 cfu/0.1 ml x 10 x 10 x 10 = 800,000 cfu/ml or 0.8 x
10^6
B: 46/0.1 ml x 10^5 = 46,000,000 cfu/ml or 46 x 10^6
C: 147 cfu/0.1 ml x 10^4 = 14,700,000 cfu/ml or 14.7 x
10^6
Note: cfu means colony forming unit =a single-celled
bacterium.
Colony Counter
3.Microbiological evaluation
Example of Contaminated Experimental feed
Microbiological Parameter
Control Feed
Experimental Feed
2.0 x 10-3
6.24 x 10 4
110
>240
4.9 x 102
3.8 x 102
4. E. coli (MPN/g)
Absent
Absent
5. Salmonella/25g
Absent
Absent
6. S. aureus
Absent
Absent
4. Biological Evaluation
does not provide any information about chemical composition
but offers a more accurate estimate of nutritional value and the
efficiency to produce growth and maintain a healthy organism.
live organisms are utilized to conduct well-designed feeding
trials to evaluate the specific effect of a particular nutrient or feed
formulation.
provides information that ascertain the true value of the
feedstuff to the organism.
4. Biological Evaluation
1. Feeding Experiment
feeding trials are conducted to determine the performance
of a complete diet or a particular ingredient of interest.
is usually done in tanks, in ponds, or in cages using fish as
test animals. In a laboratory experiment, environmental
conditions are easily kept constant.
4. Biological Evaluation
measure of a performance such as growth, survival, feed
utilization is used to evaluate the adequacy of an ingredient or
the feed.
more a specific measures of performance such as the
retention or loss of a specific nutrient in the body of the
organism, shifts in enzyme activity or the ability of the
organism to survive a specific environmental challenge. (e.g.
shifts in temperature and salinity and exposure to pathogenic
organisms are needed.
feeding trials should be conducted under strict experimental
conditions, which include environmental monitoring, adequate
replication, and the manipulation of only one or a few
variables at a time.
4. Biological Evaluation
Parameters to be monitored in a feeding
experiment:
Growth
Efficiency of feed utilization
Digestibility of nutrients
Efficiency of protein utilization
Survival rate
Body composition of fish samples
Biological parameters
Histological changes in tissues
Clinical signs
Reproductive performance
4. Biological Evaluation
Two most common measures of response to a particular ingredient or
feed are the following:
1. GROWTH
- measured as function of weight, length, or specific nutrient
gain (e.g. protein).
- increases in weight gain through muscle growth and
deposition of specific
biochemical components such as
proteins or lipids.
- weight gain caused by excessive deposition of lipid in the
adipose tissue is undesirable because it decreases yield and
may adversely influence shelf life, resulting in human health
concerns.
- when a diet is evaluated by means of a growth trial, the
performance parameter measured, e.g. growth as weight gain
should be complemented by an analysis of the proximate
composition of the carcass of the organism prior to and
following feed administration.
4. Biological Evaluation
2. FEED UTILIZATION
- describes to what extent food eaten by the organism is actually
converted into growth.
- particularly critical when comparing the economic cost of feeds
and their potential for polluting the culture environment.
- since the method of feeding will influence the degree of feed
utilization, the type of feeding strategy should be welldocumented in terms of ration size (restricted versus excess) as
well as the number of feedings per day.
4. Biological Evaluation
In carrying out a feeding experiment, the following factors
have to be considered:
a. the objective of the study must be clearly defined
b. experimental treatments and statistical design
appropriate to the objective of the experiment must
be carefully selected.
4. Biological Evaluation
e. duration of feeding experiment should be long enough
4. Biological Evaluation
Length of time required to conduct a feeding trial is
4. Biological Evaluation
Although purified diets produce slower growth,
4. Biological Evaluation
Types of feeding include the following:
1. Restrictive ration- where feed is offered based on a
fixed rate of the animal body weight below satiation.
2. In excess- where feed is offered in a fixed rate that is in
excess of what the animals can consume.
3. Apparent satiation- where food is offered during a
specific period of time until the test animal stop
consuming feed.
o Generally food should be offered to a semi-continous
4. Biological Evaluation
Other parameters to be monitored in a feeding
experiment:
1. Digestibility
-a measure of biological availability of nutrients or energy in
the ingredient whereas absorption refers to actual uptake .
- methods commonly used for digestibility are the following:
a. Apparent digestibility
-the most direct method to estimate digestibility
which involves feeding a specific amount of an experimental
diet and recording the quantity of feed consumed and feces
produced.
- amount of a particular nutrient in the feces is
subtracted from the initial quantity in the test feed, the
difference represents the amount of nutrient absorbed by the
animal
4. Biological Evaluation
Techniques used to approximate commercial
conditions.
Tank studies-where outdoor tanks in which natural
productivity is allowed to become established are used
as replicates.
2. Cage studies where cages are either floated in the
pond or a fixed in the pond bottom are used as
replicates.
3. Pond studies where small ponds are used as
replicates.
1.
4. Biological Evaluation
4. Biological Evaluation
b. True Digestibility
- estimates of endogenous fecal excretions can be obtained
by feeding a diet that does not contain the nutrient being
tested and then determining the amount of nutrient in the
feces.
Dry Matter
Crude protein
91
96
55
16
80
96
85
92
70
85
78
89
Soybean meal,dehulled
74
89
Processing conditions
ADC
DM
CP
Feather meal
1
82
81
84
87
125-1350C,20-30 min,17-34kPa
61
83
1330C,30-40 min,54kPa
72
88
92
96
82
82
Blood meal
4. Biological Evaluation
c. Inert Markers
- less time consuming method to obtain estimates of
digestibility.
- animals are fed a diet that contains an inert indigestible
marker such as chromic oxide (0.5-1% for several days).
The quantity of the nutrient of interest relative to the inert
marker can be determined in the feed and feces.
-% digestibility is calculated as follows:
4. Biological Evaluation
Problems and sources of error with digestibility measurements:
1. Digestibility values can vary relative to factors such as the ff.:
a. size and age of the animal
b. type of feed processing and processing conditions,
c. environmental parameters
d. interactions with other ingredients and or nutrients in the
diet
e. method of collecting the feces
f. type of inert marker used
g. leaching of nutrients
( The inert marker method is the easiest and best to determine the apparent
digestibility of an ingredient and its associated nutrients quickly).
4. Biological Evaluation
d. Tracer Studies
- radio and stable isotopes are used as tracers to track either ingestion,
digestion and assimilation of dietary nutrients. These isotopes are added
to the diet and utilized as tracer by determining the amount deposited in
the animal tissue.
- difficulties such as the possible loss and recycling of the tracer through
metabolism are associated with these technique. Once the tracer is absorb
it can be utilized for synthesis of new tissue or metabolized and excreted as
waste. Solution to these problem is to use a twin tracer technique to
account for labelled nutrient losses.
e. In vitro Digestibility
- this technique rely on the use of digestive enzyme extracted from the
organisms under study or commercial enzyme.
4. Biological Evaluation
- enzymes are added to a sample of ingredient being tested and
digestion is measured in vitro by the following methods:
1.
2.
4. Biological Evaluation
Ways of evaluating the availability of specific nutrient in the diet.
1.
Nutrient retention or deposition
2.
Electrical conductivity
-an alternative method based on the different electrical properties of
lipids and water can be used.
- application of an electro-magnetic field and measuring the
different electrical conductivity that can measure the amount of lipid
and water in alive organism.
4. Biological Evaluation
Method to asses the quality of proteins in the feed
1. Protein efficiency ratio- assess the protein quality by
comparing different protein sources in terms of fish
weight gain per unit of protein fed.
4. Biological Evaluation
2. Apparent net protein utilization (ANPU)- measurements of
the protein content of the test animal at the start and end of
the experiment, combine with an estimate of the digestibility
value of protein of interest (digestibility coefficient) can be
used to estimate ANPU.
4. Biological Evaluation
4. Biological Value (BV) measures the nutrient excretion
during a period of time. For e.g. All N2 excreted in the
feces, urine, and gills is measured and compared to the
total nitrogen fed.
4. Biological Evaluation
Alternative measurements to determine nutritional status of
small size organisms (larvae)
1.
4. Biological Evaluation
2. Challenge test for the larvae- animals are exposed to
Digestibility of nutrients
Survival Rate
Summary
The choice of high quality feedstuff for incorporation into aqua
an effective feed.
Types of Feed
1. dry meals/semi-moist pellet/wet diet
10% M.C.
30-45%M.C.
45-70% M.C.
2. crumbles
larval feed
3. hard pellets