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Food Composition and Chemistry


Nguyen, Jenny
jennguyen13@gmail.com
California State University, Long Beach (CSULB)
Department of Family and Consumer Science
1250 Bellflower Boulevard, Long Beach CA 90840
ABSTRACT
Experiments conducted were to test for browning enzymes in apples, to determine
presence of starch and reducing sugar in dissimilar solutions, to examine sugar content in
different beverages, and to evaluate quality of milk by evaluating smell, color, temperature, pH,
and level of lactic acid. Browning enzyme, polyphenoloxidase, was tested by placing apple slices
in different solutions to establish levels of enzymatic browning reaction of each mixture. Lugols
iodine reagent was used to distinguish starch from other polysaccharides, Benedicts reagent
detects presence of reducing sugars, and Brix hydrometer was used to determine the sugar
content of various beverages. Titration was performed to quantify the amount of lactic acid in
milk while a thermometer and a pH meter was used to determine temperature and pH level
respectively. According to the browning color grading scale from 1 (not brown) to 5 (extremely
brown), apple placed in: catechol=5, deionized distilled water=3, ascorbic acid and
ethylenediaminetetraacetetic acid (EDTA)=2, and sodium metabisulfite=1. Mass of sugar content
found in water, Sprite, grape juice, apple juice, and orange juice were 0, 35, 35, 25, and 27.5
g/250 mL respectively. Average lactic acid found in three 10 mL milk samples was 0.206%.
Volume of 0.1 N sodium hydroxide (NaOH) used in samples 1, 2, and 3 of milk samples were
2.2, 2.2, and 2.4 mL respectively. In conclusion, these tests were performed to regulate food
quality, alterations, and safety to ensure ethical practice regarding fair trade.
Key Words: Polyphenoloxidase, Lugols-Reagent, Benedicts-Reagent, Hydrometer, Titration
INTRODUCTION
Food is composed of three macronutrients: carbohydrate, protein, and fat. Carbohydrates
(comprised of carbon, hydrogen, and oxygen) are arranged in different forms and contain one to
numerous linkages (monosaccharides to polysaccharides), giving the macronutrients unique
ability to be displayed as precursors to flavors and pigments, thickening and bulking agents, and
sweeteners. Carbohydrates found in fruits and vegetables contain natural phenols, which can
change in pigment and flavor when exposed to oxygen (Engelking 2015). Starch, another
carbohydrate composed of amylose and amylopectin, contains glucose linked by alphaglucosidic bonds (Bronner 1975); it can be used to change the texture and mouthfeel of foods.
Another type of carbohydrates is reducing sugars (glucose, fructose, and maltose to name a few)
which are sugars that have a free aldehyde or ketone group (Kunz and others 2011). Many
beverages, such as wine, beer, juices, and carbonated beverages contain these category of sugars
to enhance the flavor. Protein, on the other hand, are made up of 4 structures: primary,
secondary, tertiary, and quaternary. Food products have enzymes, a quaternary structure protein
that aids in oxidation, hydroxylation, and phosphorylation reactions. These reactions occur
naturally in food but can be inhibited by its environment through denaturation (Engelking
2015). Therefore, it is crucial to have food control systems to protect the health and safety of
domestic consumers. Some food control tests can be done with Lugols iodine reagent, Benedicts

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reagent, Brix hydrometer, and titration. Evaluating compositions of food assures the safety and
quality entering the market and warrants regulation requirements. Objectives of these
experiments are to explore different components of food through various qualitative and
quantitative techniques, discover how the components of food related to food quality and safety,
and practice various laboratory apparatus.
MATERIALS AND METHODS
Part 1: Enzymatic Browning in Apples
Took 6 pieces of sliced apple and placed 1 piece into a pedri dish for each slice to be
saturated in different solutions: ddH2O, 0.2% ascorbic acid, 0.2% catechol, 0.1% EDTA, and
0.2% sodium metabisulfite. After the apple slices were covered in the solutions for 30 minutes,
observed the physical appearance of the apple, shown in Figure 1. Rated and recorded the
browning of the apple from 1 to 5 (1 meaning not brown and 5 meaning extremely brown) in
Table 1.
Part 2: Detection of Carbohydrates in Food using Qualitative/Quantitative Techniques
Identification of starch using Lugols Reagent
25 mL of water control, glucose solution, potato juice, maltose solution, table sugar
solution, and whole milk were poured into a measuring cylinder and each solution was placed
into 50 mL beakers that were labeled WC, GS, PJ, MS, SS, and M respectively with a permanent
marker. Pipetted 2 mL of each solution into test tubes labeled WC, GS, PJ, MS, SS, and M
respectively with a permanent marker and added 1 drop of Lugols iodine solution to each test
tube. Recorded observations are showcased in Table 2.
Test for Reducing Sugar using Benedicts Reagent
1 mL of water control, glucose solution, potato juice, maltose solution, table sugar
solution, and whole milk were pipetted into 6 test tubes labeled WC, GS, PJ, MS, SS, and M
respectively with a permanent marker. Then 30 mL of Benedicts solution was poured into a 50
mL beaker by using a measuring cylinder. 5 mL of Benedicts Reagent was pipetted to each test
tube labeled WC, GS, PJ, MS, SS, and M. Test tubes were placed into a beaker with water and
heated until water boiled with a Vortex device for 10 minutes. 5 minutes after the test tubes were
removed from Vortex, observations and negative or positive results of reducing sugars were
recorded in Table 3.
Determination of the sugar content of selected beverages using a hydrometer
250 mL of deionized distill water (water control) was poured into a measuring cylinder
and gently inserted the Brix hydrometer into the solution. The degree of Brix was recorded in
grams in Table 4 and repeated these steps with Sprite, grape juice, apple juice, and orange juice.
Part 3: Determination of Milk Quality Using Various Parameters
Temperature and pH of milk were evaluated by using a thermometer and pH meter
respectively as well as smell and color of the milk (recorded in Table 6 and 7). 10 mL of milk
sample, 50 mL of ddH2O, and 2 drops of Phenolphthalein were added into a volumetric flask and
titration was performed with sodium hydroxide (NaOH, 0.1N) until pink endpoint was attained.
The amount of milk in mL and NaOH, 0.1N in grams used to titrate was recorded in Table 6.
This procedure was repeated and recorded 2 more times.

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RESULTS AND DISCUSSION


Like many fruits and vegetables, apples contain phenols, chemical compounds consisting
of a hydroxyl group. When phenols are exposed to oxygen in the air or during natural chemical
reaction, called ripening, the phenols will bind to polyphenoloxidase (PPO), an enzyme that
transform the colorless phenols to yellow quinione. These quinione then become polymerize to
form reddish-brown melanoid due to oxidization (Engelking 2015). In the Enzymatic Browning
Apple experiment, shown in Figure 1, the piece of apple placed in catechol solution resulted in
extremely brown appearance while when exposed to distilled water caused moderate browning.
The enzyme, catechol oxidase, catalyzes the reaction of colorless catechol, a phenolic compound
found in small quantities in plant cells, to a yellow quinione. This compound reacts with oxygen
to form melanins. When catechol solution is added to the apple, PPO reacted due to the increased
amount. However, adding distilled water to the apple did not increase or decrease the browning.
Since water contains oxygen molecules, the PPO reacted to them to create moderately browned
apples. When it was exposed to ascorbic acid and EDTA, the apples showed slight browning.
Because of their high pH levels, ascorbic acid and EDTA inhibits the enzymatic browning
reactions by denaturing PPO and reducing the quinones back to the original phenol compounds
(Engelking 2015). The apple slice that was placed in sodium metabisulfite appeared to look the
same when it was first cut with no evidence of melanoid pigments. Sodium metabisulfite inhibits
enzymatic browning by blocking polymerization in quinones (Evans and others 2013). In
conclusion, enzymatic browning is beneficial for the developing color and flavor in dried fruits
such as figs and raisins. However, browning of fresh fruits and vegetables are undesirable and
create heavy economic losses for farmers (Engelking 2015). Sulfites have been used extensively
in the food industry to control enzymatic browning; however, there has been an effort to avoid
the use of these agents in foods due to safety, regulatory, and labeling issues. Therefore, safe
alternatives to sulfites have been developed such as ascorbic acid, which prevents or minimizes
oxidative flavor and color deterioration (Lambrecht 1995).
Starch, found in grains and vegetables, is made up of polysaccharides composed of
amylose and amylopectin which are glucose monomers with alpha (1-4) linkage that causes a
helical structure (Bronner 1975). Lugols iodine reagent is imperative to differentiate starch from
other polysaccharides by yielding a blueish-black color in the presence of starch (Bronner 1975).
When this reagent reacts to other polysaccharides and monosaccharides, there is no reaction and
the solution remains the same. Since amylose and amylopectin have shorter branches than other
polysaccharides, the iodine in the reagent will fit into the helices and form a starch-iodine
complex, causing the dark reaction (Bronner 1975). In Identification of starch using Lugols
Reagent experiment, six different solutions were used to test for the presence of starch: water,
glucose solution, potato juice, maltose solution, table sugar solution, and milk. Shown in Figure
2 and Table 2, results indicated that potato starch solution reacted with the iodine reagent,
creating a blueish-black solution, while the remaining solutions had no reaction with Lugols
reagent, indicating a yellow-brown solution. Therefore, potato starch contains amylose and
amylopectin that reacted with the iodine to create a starch-iodine complex. Some food companies
may add starch to their product to make it more viscous. When performing quality assurance, it
is important to use Lugols iodine reagent to test for starch in products to determine if the food
company is in compliant with fair trade. When adding starch to food, such as milk, it may
adversely affect the nutritional content and quality of the product. Thus, it is unethical and food
products should be tested to prevent this usage of starch.

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Monosaccharides and disaccharides that contain an aldehyde or a ketone group are


considered reducing sugars called aldose or ketose respectively (Kunz and others 2011). To test
for the presence of reducing sugars, Benedicts reagent (a blue liquid that contains Cu2+) is added
to solutions and rests in a water bath for approximately 10 minutes. When this reagent reacts
with reducing sugars, the Cu2+ will be reduced to Cu+ while the aldehyde group will be oxidized
to carboxyl acid, hence, will cause a positive result with brick-red precipitation (Kunz and others
2011). If there is a negative reaction, the solution will remain the same (aqua blue). In the Test
for Reducing Sugar using Benedicts Reagent experiment, water, glucose solution, potato juice,
maltose solution, table sugar solution, and milk were tested for presence of reducing sugars.
Shown in Figure 3 and Table 3, results indicated presence of reducing sugars in glucose solution,
maltose solution, and milk while negative reactions occurred in the remaining solutions. Glucose
solution contains glucose, a well-known reducing sugar with an aldehyde group. In Figure 3,
glucose reacted with the reagent and produced red precipitation. On the other hand, starch
reacted poorly with Benedicts reagent because of the absence of reducing sugars, thus, it
produced a negative reaction. In the next solution, maltose reacted positively with the reagent
because it contains an aldehyde group, so the solution appeared violet with red precipitation on
the bottom and top of the test tube. Table sugar, made up of fructose and glucose joined by a
glycosidic bond, did not react with the reagent because the linkage of the monosaccharides
prevents isomerization; therefore, is not a reducing sugar. Lastly, milk contains lactose, a
disaccharide made up of glucose and galactose. When oxidized, aldehyde reacts with the Cu2+
and is oxidized to a carboxylic acid, while Cu2+ is reduced to Cu+. This causes orange-red
precipitate, consequently a positive result. According to the colors developed, glucose solution
had the highest amount of reducing sugars while milk was a runner up and maltose placed third.
All in all, the properties of reducing sugars are used in the food industry, particularly for the
shelf life of beverages, mainly in wine, beer, and juice to increase the overall quality of the
product (Kunz and others 2011). Additionally, it is important to use the Benedicts reagent to test
for products that brand themselves sugar-free or lactose-free to see if these products are true
to their labels.
In the Determination of the sugar content of selected beverages using a hydrometer
experiment, Brix hydrometer was used to quantify the sugar in the juices and soda
(Dharmadhikari 1996). Brix is defined as the percentage of dissolved sugar in a water solution
on a weight for weight basis and is expressed in degrees Brix (B) (Shachman 2004). For
example, a 10B solution implies that in 100 g solution, there are 10 g of dissolved sugar. Apart
from the water, sugar is by far the largest component of naturally sweetened soft drinks and
juices, and it makes up about 13% of their content (Shachman 2004). Thus, a Brix hydrometer
reading can provide an estimation of the residual sugar content in a beverage. In this experiment,
250 mL of distilled water, Sprite, grape juice, apple juice, and orange juice were evaluated for
their sugar content, shown in Figure 4. According to the results in Table 4, mass of sugar content
(g) found in water, Sprite, grape juice, apple juice, and orange juice were 0, 35, 35, 25, and 27.5
per 250 mL respectively while density (g/mL) were 0.00, 0.14, 0.14, 0.10, and 0.11 respectively.
Sprites grape juices, apple juices, and orange juices sugar content (g) found on the nutrition
facts panel (shown in Figure 5-8 and Table 5) showed 38/350 mL (density=0.11 g/mL), 40/240
mL (density=0.17), 28/240 mL (density=0.12), and 24/240 mL (density=0.10) respectively. After
determining the density of each product, it appears that Sprite and orange juice branded their
sugar content lower than the results from the Brix hydrometer while grape and apple juices
branded their sugar content higher than results from the Brix hydrometer. However, according to

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the Food and Drug Administration (FDA), the sugar amounts displayed on the nutrition facts
panel are rounded by 0.5 g (2013), thus, can be inaccurate. In conclusion, its important to use a
hydrometer to test for quality assurance in beverages to maintain consistency; and it is needed to
continuously monitored and controlled on production line during the processes (Shachman
2004).
Manufacturing high quality milk is vital for consumers health. There are several
different parameters used to assess quality of milk: temperature, smell, color, pH, and percent of
lactic acid. Since milk is composed of 87% water, temperature will need to be monitored to
assure quality and safety of the product and prevent bacteria growth. The aroma of milk is
extremely important as well to guarantee no presence of rancidity. According to Food and
Agriculture Organization (FAO), reference values of milk has a pH level of 6.6-6.7 with acidity
of 0.16-0.18%. pH and percent of lactic acid are key indicators to determine the amount of
bacteria producing acid; the lower the pH, the higher amount of bacteria caused by the utilization
of lactose sugar. This ultimately causes protein denaturation and presence of milk curdles appear.
Results in Table 7 shows the pH levels, temperatures, smells, and colors of the 3 samples. pH
levels were in normal ranges (shown in Figure 9) with pH levels of 6 as well as normal ranges of
temperature, averaging 17.25C. The aroma of milk did not smell rancid and the color looked
standard. Results in Table 6 shows an average of 0.206% lactic acidity in 3 milk samples
completed through the process of titration with NaOH, shown in Figure 10. According to FAO,
the first clotting of milk is due to acid development which can first be seen at 0.21-0.23% lactic
acid. It is important to understand the relationship between these parameters and their normal
reference ranges; these parameters ensure safety and high quality of milk. Meeting consumer
expectations will ensure the continuation of a successful and stable dairy industry.
CONCLUSION
It is essential to understand food components and their chemical make up. Macronutrients
have many special functions in foods: they can be precursors to flavors and pigments, thickening
and bulking agents, sweeteners, and aid in natural chemical reactions. Comprehending different
chemical structures will allow food scientists to determine which tests should be achieved. These
food assessments performed require technical and analytical skills to ensure standardized quality
and safety for consumers as well as any food alterations that occurs along the process.

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REFERENCES
Bronner R. 1975. Simultaneous Demonstration of Lipids and Starch in Plant Tissues. Stain Tech
50(1): 1-4. DOI: 10.3109/10520297509117023
Dharmadhikari M. 1996. Determining Residual Sugar Using a Hydrometer. Vineyard & Vintage
View 11(6): 5.
Engelking C. 2015. Genetically Modified Non-Browning Apples Are Approved in the U.S.
Discover 26: 56-57.
Evans EC, Yakubu G, Bello T. 2013. EVALUATION OF ASCORBIC ACID AND SODIUM
METABISULPHITE AS INHIBITORS OF BROWNING IN YAM (D. rotundata) FLOUR
PROCESSING. Annals: Food Sci and Tech 14(2): 247-260.
Food and Agriculture Organization of the United Nations. Milk testing and Quality Control.
Available from: http://www.fao.org/ag/againfo/resources/documents/MPGuide/mpguide2.htm.
Accessed 2016 February 11.
Food and Drug Administration. 2013. Guidance for Industry: A Food Labeling Guide (7.
Nutrition Labeling; Questions G1 through P8). Available from:
http://www.fda.gov/Food/GuidanceRegulation/GuidanceDocumentsRegulatoryInformation/Labe
lingNutrition/ucm064894.htm. Accessed on 2016 February 21.
Kunz T, Lee V, Schiwek, V, Seewald T, Methner F-J. 2011. Glucose a Reducing Sugar?
Reducing Properties of Sugars in Beverages and Food. Brewing Sci 64: 61-67.
Lambrecht HS. 1995. Sulfite Substitutes for the Prevention of Enzymatic Browning in Foods.
American Chemical Society 600: 313-323. DOI: 10.1021/bk-1995-0600.ch024
Shachman M. 2004. Brix The Basics. In: Schachman M, editor. The Soft Drinks Companion:
A Technical Handbook for the Beverage Industry. Boca Raton: CRC Press. p 1-12.

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APPENDIX

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Figure 1: Results of slice of apple in distilled water, ascorbic acid, catechol, EDTA, and
sodium metabisulfite (from left to right) after 30 minutes.
Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 2: Results of 2 mL distilled water, glucose solution, potato juice, maltose solution,
table sugar solution, and whole milk (from left to right) with 1 drop of Lugols iodine.
Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 3: Results of 1 mL distilled water, glucose solution, potato juice, maltose solution,
table sugar solution, and whole milk (from left to right) with 5 mL of Benedicts Reagent
after heated on boiling water for 10 minutes.
Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

Figure 4: Results of hydrometer in 250 mL distilled water, Sprite, grape juice, apple juice,
and orange juice (from left to right).
Photo Courtesy of Jessica Lopez from the Food Science and Nutrition Laboratory.

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Figure 5: Nutrition Facts Panel of Sprite.


Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

Figure 6: Nutrition Facts Panel of grape juice.


Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 7: Nutrition Facts Panel of apple juice.


Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 8: Nutrition Facts Panel of orange juice.


Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 9: pH levels of sample 1, 2, and 3 of milk.


Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Figure 10: Sample 1, 2, and 3 of milk after titration with 0.1N NaOH.
Photo Courtesy of Jenny Nguyen from the Food Science and Nutrition Laboratory.

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Table 1. Enzymatic browning observation of apples


Apple
Treatment

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297

Deionized
Distilled
Water
(Control)
3

Table 2. Lugols test for Starch


Test Substance
Water Control (WC)
Glucose solution (GS)
Potato Juice (PJ)
Maltose solution (MS)
Table sugar solution (SS)
Whole milk (M)

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299

Catechol

EDTA

Sodium
Metabisulfite

Observation
Clear yellow
Clear yellow
Opaque violet
Pale yellow
Pale yellow

Result
(Positive [+] or Negative [-])
+
-

Milky white

Table 3. Observations for the Benedicts Test for Reducing Sugars


Test Substance
Observation
Result
(Positive [+] or Negative [-])
Water Control (WC)
Blue
Glucose solution (GS)
Pink/rose
+
Potato Juice (PJ)
Blue
Malt solution (MS)
Blue/violet with red particles
+
on bottom and floating on top
Sugar Solution (SS)
Blue
Milk (M)

300

Ascorbic
Acid

Orange

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Table 4: Determination of % sugar content using a hydrometer.


Test Substance
Brix measurement
Mass of sugar (g)
(B; g/100mL)
Water Control (Distilled
0
0
Water)
Sprite (Decarbonated)
14
35
Grape Juice
14
35
Apple Juice
10
25
Orange Juice
11
27.5

Table 5: Sugar content on Nutritional Facts Panel


Test Substance
Mass of sugar (g)
Water Control (Distilled
Water)
Sprite (Decarbonated)
38
Grape Juice
40
Apple Juice
28
Orange Juice
24

Table 6: Determination of TA in milk


Food Item
V1 (mL)
(Volume of 0.1N
NaOH used)
Milk #1
2.2 mL
Milk #2
2.2 mL
Milk #3
2.4 mL

Volume Used (mL)

Density (g/mL)

250

0.00

250
250
250
250

0.14
0.14
0.10
0.11

Volume (mL)
-

Density (g/mL)
-

350
240
240
240

0.11
0.17
0.12
0.10

V2 (mL)
(Volume of Sample
used)
10 mL
10 mL
10 mL

% of Lactaid Acid
0.20%
0.20%
0.22%

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Table 7: Determination of temperature, smell, color, pH in milk


Food Item
Temperature
Smell
Color
(C)
Milk #1
Not sour
Milky white
15C
Milk #2
Not sour
Milky white
17C
Milk #3
Not sour
Milky white
20C

pH
6
6
6

GRADING RUBRIC FOR LAB REPORTS (FSCI 332)


(Total 50 points)
Nguyen, Jenny
CRITERIA BEING EVALUATED
TOTAL POINTS (Pts) TO BE
AWARDED
Title: Be brief and Use Title Case
Name of Student: Last name, First name
5 Pts
Email address:
Name of Institution/Dept. attended:
Abstract: Say what was done, how it was done,
provide major NUMERICAL results and
conclusions in not more than 250 words. NO
acronyms or references should be present in the
10 Pts
abstract. Provide 5 key words, after the
abstract. *Two words are not counted as one.
Introduction: In half of a page (1) Provide a brief
background and say what is important about the
subject; and (2) State the overall objective; (3)
Cite references where appropriate in text
according to the Journal of Food Science (JFS)
guidelines. See Pg. 5 in JFS guidelines.
Materials and Methods: (1) Provide sufficient
detail so work can be repeated; (2) Use
subheads for clarity and define abbreviations and
acronyms at least once; and (3) Do not to commit
self-plagiarism by using exact wording from a
previous publication (e.g. a peers lab report or
laboratory manual/handouts). Write the
methodology in past tense. DO NOT INCLUDE a
list of materials used. These must be included in
the methodology in paragraph form.
Results and Discussion: (1) Present and
discuss results concisely, using figures and
tables as needed (but not the same information in
both figures and tables). (2) compare results to
those previously reported in other literature (i.e.,
peer reviewed articles). (3) Place figures and
tables at the end of the document, after
references in the appendix. ALL raw
calculations must be placed in the lab notebook
and not report. Only state answers in report. (4)
Discussion MUST refer to the respective tables or
figures. All tables should be labeled according to
JFS guidelines. See Pg. 4 in JFS guidelines.
Conclusion: State conclusions briefly by stating
what was observed and how the results are
applicable to industry.
References:
Refer to the Journal of Food Science (JFS)
Author Guidelines for required format of 5 or
more references. This is posted in BeachBoard.
See Pg. 5 in JFS guidelines. Include DOI#.

10 Pts

10 Pts

10 Pts

5 Pts

2 points will be deducted for each error and a score a zero (0) will be issued for any lab
report that has been plagiarized. Line numbers are to be inserted into the document at
all times.