Myrna B. Teruel, Ph. D Scientist Aquaculture Department Southeast Asian Fisheries Development Center Tigbauan, Iloilo 5021 PHILIPPINES *Lecture notes submitted to T echnology and Information Division for the Training Course on Feed Formulation and Feed Evaluation for Aquaculture Species, September 1-5, 2014
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2-2014 9.03Biological and Microbiological Evaluation of aquafeeds and feedstuffs-MBT.pdf
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7/23/2019 2-2014 9.03Biological and Microbiological Evaluation of aquafeeds and feedstuffs-MBT.pdf
defined ??? This is a step-wise process in which feeds and feed
ingredients are analyzed by a series of physical , chemical,
microbiological and biological tests, blended to produce anassessment to ensure that the final product is of highquality and that deleterious effects of the produce are avoided.
No single test will provide the necessary data for adequatefeed evaluation. Hence, a variety of evaluation/assessmentmethods are utilized to provide information required toassess quality and nutritional value of feeds and feedingredients.
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a method of feed/feed ingredients evaluation thatutilizes various microorganisms (e.g. Lactobacillusrhamnosus; Enterococcus hirae; Pediococcusacidilactica) in order to determine the nutritionalquality of a product.
3. Microbiological Evaluation
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Feed samples obtained by random sampling of the feedsand feed ingredients (e. g. corn, corn gluten meal, meatand bone scraps, soybean oil, soybean meal, alfalfa leafmeal)are finely ground prior to assay to assurehomogeneity.
L. 'arabinosus is used as the test organism for amino
acid determinations. Pure amino acid standards are used to eliminate
possible source of error.
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The amount of each amino acid contributed by the variousprotein ingredients was calculated on the basis of thepercentage of the ration in the ingredient.
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The microbial assay of vitamins is based upon thecomparison of the stimulation of growth of bacteria bymeasured concentration of vitamin with that produced by
known concentration of standard preparation of vitaminhaving known activity.
growth of microorganisms is proportional to requirement
for specific vitamin
The microbiological assay of vitamins utilizes variousstrains of microbes.
Microbiological Method of Feed
Evaluation
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1. Incubate tubes 72 h and then titrate contents of each tube with 0.1 M NaOH,using bromothymol blue indicator, or to pH 6.8 measure potentiometrically.
* Disregard results of assay if response at inoculated blank level is equivalent totitration of > 1.5 ml greater than that of uninoculated blank level.
* Response at 5.0 ml level of standard solution should be equivalent to titration of8-12 ml.
2. Determine amount of vitamin for each level of test solution by interpolationfrom standard curve.
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,respectively, of standard solution.
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Refer to AOAC Official method (2000) 944.13 for thepreparation of Basal Medium Stock Solution andNiacin Standard Solutions (stock solution,intermediate solution and working solution).
Niacin is sensitive to light and high temperature.
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Applicable only to materials containing free forms offolic acid.
Refer to AOAC Official method (2000) 944.12 for thepreparation of Basal Medium Stock Solution and Folicacid Standard Solutions (stock solution, intermediate
solution I and II and working solution).
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1. Incubate tubes 72 h and then titrate contents of each tube with 0.1 M NaOH,using bromothymol blue indicator, or to pH 6.8 measure potentiometrically.
* Disregard results of assay if response at inoculated blank level is equivalent totitration of > 1.5 ml greater than that of uninoculated blank level.
* Response at 5.0 ml level of standard solution should be equivalent to titration of8-12 ml.
2. Determine amount of vitamin for each level of test solution by interpolationfrom standard curve.
* Discard any observed titration values equivalent to < 0.5 ml or > 4.5 ml,respectively, of standard solution.
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Aquatic animals reared intensively require large amounts of plant oranimal protein in the feed. This material is prepared from meat,
offal, bones, blood or feathers, or combinations. Animalproteins often contain high level of Salmonella which dependson the initial contamination of the raw materials and on thehygiene of feed manufacture.
Animals fed with contaminated feed, often carry these salmonella in
their intestinal tracts, with no sign of illness.
Feed from infected ingredients may become contaminated duringprocessing and feed preparation (inadequate cooking andstorage procedures).
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Salmonella is bacteria that can cause a gastrointestinalinfection known as salmonellosis. Usuallysalmonellosis is referred to as "salmonella." Thisinfection can occur in humans and animals.
People become infected with salmonella by swallowing
the bacterium. This can happen when contaminatedingredient are incorporated into feed formulation.
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The sample should be tested on the day of receipt or on the 1st working day that allows the method to be completed if the test isnot begun on the day of receipt.
The sample must be stored in a refrigerator until required.
Refrigerated samples should be left at room temperature for atleast 4 h before examination.
The sample should be tested in duplicate 25 g portions forSalmonella, five 10 g portions for Enterobacteriaceae, and forrendered material derived from high-risk material duplicate 10 gportions for Clostridium perfringens
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3. Microbiological evaluation Microbiological analyses for feeds and ingredients
e. Mix well by swirling and determine pH with testpaper. Adjust pH, if necessary to 6.8.
f. Add up to 2.25 ml steamed (15 min) Tergitol Anionic7 or Triton X-100 and mix well. (actual quantity needed willdepend on the composition of the raw material).
g. Loosen jar caps ¼ turn and incubate samplemixtures for 24H at 350C.
h. Tighten lid and gently shake incubated sample
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3. Microbiological Evaluation Microbiological analyses of feeds and ingredients
i. Transfer 0.1 ml mixture to 10 ml Rappaport- Vassiliadis (RV) medium and another 1 ml mixture to10 ml tetrathionate (TT) broth.
j. Incubate RV medium for 24H at 420C (for high
microbial load) and and TT broth for 24 H at 430
C.K. Mix and streak 3 mm loopful (10 µl) incubated TTbroth on bismuth sulfite BS agar. Prepare BS platesthe day before streaking and store in dark at roomtemperature until streaked.
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Staphylococcus aureus is a gram positive coccalbacterium, that is a member of the Firmicutes, and isfrequently found in the human respiratory tract and
on the skin. It is positive for catalase and nitrate reduction.
S. aureus is not always pathogenic, but it is a commoncause of food poisoning.
S. aureus can survive from hours to weeks, or evenmonths, on dry environmental surfaces, depending onstrain.
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3. Microbiological Evaluation Method 2 Calculate the ratios of the Total Volume / VolumeTransferred and then multiply it by the colonies countedon the plate. 1,000 (Dilution A) = 1000 / 1
100 (Dilution B) = 100 / 110 (Dilution C) = 10 / 110 (inoculation of 100 or 0.1ml) = 10
31 (colonies counted on plate)300,000,000 = 3x108 = no. of bacteria in the original solution
The plate count simulation coefficient is always expressedas a whole number. The answer is 3x108 not 3.1x108.
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3. Microbiological Evaluation Method 3 -To use this method three criteria must be met:
1.the number of bacteria colonies on the plate must be between 10 and 90 2. all dilutions must be done as 1 to 9, 1 to 99 or 1 to 999 3. inoculation selected must be 100 The coefficient for this solution will be 3. Use the 10's digit from
the plate count. The exponent for this problem can be determined by counting
zeros. Count all the zeros for Dilution A, B, and C and then add 2. The
exponent for this problem is 8 (6 zeros in the dilutions +2). The total number of bacteria in the original sample is 3x108.
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3. Microbiological Evaluation To compute for colony plate counts???
Problem:
a small volume e.g. 0.1ml from one of the tubes is transferredonto an agar plate, where it is spread out over the surface.
Since each colony comes from a single starting bacterium, thenumber of colonies = the number of (living) bacteria in the 0.1mlsample that was spread on the plate. By working backwards it is
possible to calculate how many bacteria there were in theoriginal 1ml of overnight culture used to make the dilutionseries.
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The three plates illustrated below are the results of incubating agar platesovernight after applying 0.1ml from one of the tubes in a bacterial dilutionseries. Each plate used a different starting culture.
Using the colony counts and dilution factors given, work out what the originalovernight culture densities were (in bacteria/ml).
Plate A - Diluted 10-fold three times.80 colonies
Plate B - Diluted 10-fold five times.46 colonies
Plate C - Diluted 10-fold four times.147 colonies.
3. Microbiological Evaluation
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A: 80 cfu/0.1 ml x 10 x 10 x 10 = 800,000 cfu/ml or 0.8 x10^6B: 46/0.1 ml x 10^5 = 46,000,000 cfu/ml or 46 x 10^6C: 147 cfu/0.1 ml x 10^4 = 14,700,000 cfu/ml or 14.7 x10^6
Note: cfu means colony forming unit =a single-celledbacterium.
3. Microbiological Evaluation
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does not provide any information about chemical compositionbut offers a more accurate estimate of nutritional value and theefficiency to produce growth and maintain a healthy organism.
live organisms are utilized to conduct well-designed feedingtrials to evaluate the specific effect of a particular nutrient or feedformulation.
provides information that ascertain the true value of the
feedstuff to the organism.
more preferred method and the ultimate test of performance.Disadvantages of being time consuming, expensive to conduct andrequires specialized facilities for holding live animal.
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Two most common measures of response to a particular ingredient orfeed are the following:
1. GROWTH- measured as function of weight, length, or specific nutrientgain (e.g. protein).- increases in weight gain through muscle growth and
deposition of specific biochemical components such asproteins or lipids.- weight gain caused by excessive deposition of lipid in theadipose tissue is undesirable because it decreases yield andmay adversely influence shelf life, resulting in human healthconcerns.- when a diet is evaluated by means of a growth trial, theperformance parameter measured, e.g. growth as weight gainshould be complemented by an analysis of the proximatecomposition of the carcass of the organism prior to andfollowing feed administration.
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Length of time required to conduct a feeding trial isdependent on the following factors: Objectives of the experiment
Species utilized Age of the animal
Growth trial should be of sufficient duration to
produce relatively large increases in growth andstatistically significant differences between dietarytreatments. (e.g. 14-28 days for larvae, 6-8 weeks for juveniles, 14-18 weeks for larger fish.
4. Biological Evaluation
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Although purified diets produce slower growth,growth rates in the laboratory should be comparable tothose achieved under natural conditions. (e.g. ponds) where both high quality feeds and natural productivityare available as food sources.
Ingredients for making the experimental diets shouldbe well characterized so that composition is defined.
The final feed product should of suitable size andtexture for the animals to consume easily.
Animals should be fed according to a well definedfeeding ration which must be adjusted as the animalgrows.
4. Biological Evaluation
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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 inexcess of what the animals can consume.
3. Apparent satiation- where food is offered during aspecific period of time until the test animal stopconsuming feed.
o Generally food should be offered to a semi-continousbasis to larvae 4x a day for small juveniles, 2x daily forlarge juveniles and once daily to sub-adults.
4. Biological Evaluation
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Apparent Digestibility (AD)- means that the feces also contain endogenous fecalexcretion in addition to unabsorbed feed hence thedigestibility estimate can be an underestimate because
some of the nutrient present in the feces could come fromendogenously produced waste
4. Biological Evaluation
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c. Inert Markers- less time consuming method to obtain estimates ofdigestibility.- animals are fed a diet that contains an inert indigestiblemarker such as chromic oxide (0.5-1% for several days).The quantity of the nutrient of interest relative to the inertmarker can be determined in the feed and feces.
-% digestibility is calculated as follows:
4. Biological Evaluation
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- radio and stable isotopes are used as tracers to track either ingestion,digestion and assimilation of dietary nutrients. These isotopes are addedto the diet and utilized as tracer by determining the amount deposited inthe animal tissue.
- difficulties such as the possible loss and recycling of the tracer throughmetabolism are associated with these technique. Once the tracer is absorbit 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 toaccount for labelled nutrient losses.
e. In vitro Digestibility
- this technique rely on the use of digestive enzyme extracted from theorganisms under study or commercial enzyme.
4. Biological Evaluation
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- enzymes are added to a sample of ingredient being tested anddigestion is measured in vitro by the following methods:
1. pH-drop method. As proteolytic enzymes attack the peptide bondsof proteins hydrogen is released and the pH of the protein solutionreduced. The pH reduction is highly and positively correlated withthe degree of protein digestion.
2. pH-stat method. To keep digestive enzymes close to their optimalpH, NaOH is added. The amount of NaOH consume is proportionalto the degree of protein hydrolysis and is highly correlated to in vivoapparent protein digestibility.
4. Biological Evaluation
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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 thedifferent electrical conductivity that can measure the amount of lipidand water in alive organism.
4. Biological Evaluation
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2. Apparent net protein utilization (ANPU)- measurements ofthe protein content of the test animal at the start and end ofthe experiment, combine with an estimate of the digestibility value of protein of interest (digestibility coefficient) can beused to estimate ANPU.
3. True net protein utilization (TNPU) – determination is doneby feeding a protein free diet for the same length of time and
determining the change in carcass protein.
4. Biological Evaluation
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4. Biological Value (BV) – measures the nutrient excretionduring a period of time. For e.g. All N2 excreted in thefeces, urine, and gills is measured and compared to the
total nitrogen fed.
5. True biological value- an estimate of endogenous loss of
nutrient in question by feeding a N2 free diet.
4. Biological Evaluation
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Alternative measurements to determine nutritional status ofsmall size organisms (larvae)
1. RNA/DNA ratio- the quantity of ribonucleic acid (RNA),the transcriptor and translator of genetic information isdirectly proportional to protein synthesis inside the cell.
- the ratio of RNA to DNA correlates
well with growth/protein synthesis and nutritional statusof the fish. High RNA/DNA ratios indicate adequategrowth and nutritional status while low ratios indicatepoor nutritional conditions.
4. Biological Evaluation
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