Thesis Complete Paper Final Last Edited

ABSTRACT
The study was carried out to assess the influence of
different packaging materials such as PET, HDPE and glass on
the shelf life of PP-GBR beverage stored at 4°C through a
real time method.
Samples were evaluated through sensory evaluation,
physico-chemical and microbial analyses at a three day
interval for 15 days.
Based on the results, the TSS of the samples range from
10.0 + 0.0 to 8.93 + 0.05 ºBx. pH continually decreases from
6.43 + 0.05 to 4.93 + 0.05. The same trend of result for
viscosity was obtained, 2.77 + 0.05 drops to 1.83 + 0.05. %TA
results of all the samples significantly increase when it is
completely deteriorated (0.033 + 0.01 - 0.17 +0.01).
Statistical analysis revealed that there were
significant differences among the samples in terms of
physico-chemical parameters in different packaging materials
during storage period. Commented [1]: you should focus on what parameters ang
may significant difference and the storage day. please also
emphasize kung anong packaging ang different
Chi-square test revealed that samples packed in HDPE and
glass bottles were not significantly different in terms of
sensory attribute within 12 days of storage while
significantly different results were determined on the
samples from PET bottles. Microbiologically, samples from
0
PET bottles were within the standard count of yeast and molds
(2.0 log cfu/ml) during 9 days of storage, whereas samples in
HDPE and glass bottle were stable for 12 days.
Based on the results of the quality parameters
indicated, it can be concluded that glass bottles could
provide an efficient protection and longer shelf life for PP-
GBR beverage.
TABLE OF CONTENTS
CONTENTS PAGE
APPROVAL SHEET i
BIOGRAPHICAL SKETCH ii
ACKNOWLEDGEMENT iii
ABSTRACT v
TABLE OF CONTENTS vii
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF APPENDICES xi
INTRODUCTION 1
Objectives of the study 2
Significance of the study 3

Scope and Limitation of the study
Place and duration of the study
Definition of terms
REVIEW OF RELATED LITERATURE
Plant – based milk alternatives 10
1
Legume – based milk 11
Soy milk 12
Pigeon pea 14
Nutritional Value of Pigeon Pea 14
Health benefits of pigeon pea 16
Pigeon Pea Milk 17
Brown rice 18
Rice Milk 18
Germinated Brown Rice 19
Germination Conditions for Brown Rice 20
Nutritional aspects 20
Bio – active Compounds in Germinated 22
Brown Rice
Health effects and benefits of GBR 26
Blending for nutritional balance and 27
improvement in sensory acceptability
Shelf life and Direct Shelf life Test 29
Shelf life of the food 29
Direct Method 30
Factors Affecting Shelf Life 31
Product characteristics 31
Distribution and Storage 32
environment
Package Properties 33
Packaging 33
Shelf life of pasteurized milk in 35
different package
Glass Bottle 36
Plastic Bottle
High-Density Polyethylene Bottle 38
Shelf – life of different plant-based milks 39
Pasteurization 40
Effect of Storage on Sensory Characteristics 41
Standards or indices made as basis for 42
shelf life determination
METHODOLOGY 44
Materials and Methods 44

Preparation of Pigeon Pea extract 45
Germination and extraction of GBR milk 47
Processing of PP-GBR Beverage 49
2
Physico-chemical Analysis 52
pH Determination 52
Total Soluble Solids 53
Total Titratable Acidity (%TA)
53
Viscosity 53
Statistical analysis 54
Sensory Evaluation 57
Microbial Analysis 58
RESULTS AND DISCUSSION 60
SUMMARY AND CONCLUSION 72
Summary 72
Conclusion 73
Recommendation 74
REFERENCES 75
APPENDICES 81
LIST OF TABLES
Table Title Page
1 Plant-Based / Non-Dairy Milk Alternatives 10

2 Total Microbial Viable Count of the Soymilk 13
3 The dietary nutrients of pigeon pea 15
4 γ – Oryzanol content in rice grains 23
5 Shelf – life of different plant-based milk 39

alternatives in refrigeration temperature
6 Milk Pasteurization Temperature 40
7 Physico – chemical properties of soymilk 41
8 Microbiological limits for soymilk 42
9 Formulation of Pigeon pea – GBR Beverage 49
10 Sampling Plan for shelf life study of 58

PP – GBR beverage
3
11 Total Soluble Solids of PP-GBR beverage 60
during storage
12 pH of PP-GBR beverage during storage 62
13 Percent Titratable Acidity (%TA) of

63
PP-GBR beverage during storage
14 Viscosity of PP-GBR beverage during

storage 65
15 Results of Triangle test for PP-GBR 67

beverage in different packaging materials
stored at 4°C temperature
16 Yeast and mold count of PP-GBR beverage in 68

different packaging materials during storage
LIST OF FIGURES
Table Title Page
1 Matured Pigeon peas 14
2 Brown rice 18
3 Structure of GABA 22
4 Structure of Phytic acid 25
5 Process flow of Pigeon Pea Extract 46
6 Process flow of Germinated Brown Rice Flour 48
7 Process flow of GBR Extract 49
8 Process flow of Pigeon pea – GBR Beverage 51
9 Newly Process PP-GBR beverage 60
10 Trends of Total Soluble Solids of PP-GBR 61
4
beverage during storage
11 Trends of pH of PP-GBR beverage during 62

storage
12 Trend of Percent Titratable Acidity (%TA)

64
of PP-GBR beverage during storage
13 Trend of viscosity of PP-GBR beverage 66

during storage
LIST OF APPENDICES
Table Title Page
A Score sheet for PP – GBR Beverage 82

using triangle test
B Master Sheet for Sensory evaluation 83

of PP - GBR Beverage
C Raw data on sensory evaluation for PET 84

bottles
D Raw data on sensory evaluation for HDPE 86

Bottles
E Raw data on sensory evaluation for 88

Glass bottles
F Statistical Analysis for TSS of PP-GBR 90

Beverage
5
G Statistical Analysis for pH of PP-GBR 92
beverage
H Statistical Analysis for %TA of PP-GBR 94

beverage
I Statistical Analysis for Viscosity of 96

PP-GBR beverage
J Computation for Viable counts of yeast 98

and molds
INTRODUCTION
Plant-based milk alternatives serve as an inexpensive
source of protein and a good alternative to cow’s milk
especially for those people who suffer lactose intolerance
and other cow’s milk allergy related diseases (Sethi, S.et
al, 2016). Combination of two or more types of plant – based
milks are often made to have a product with high nutritive
value comparable to cow’s milk. In the US, a developed
simulated milk comprising soybean flour, sesame seed flour
and coconut meal was patented and can be suitably used for
infant consumption (Johnson and Linda 1968). Pigeon pea-
6
germinated brown rice beverage was produced by adding a
certain level of germinated brown rice extract to enhance the
nutritional content of pigeon pea milk.
Newly developed products like pigeon pea-germinated
brown rice beverage needs to undergo shelf life testing in
order to determine the “best before or expiration date” of
the product which regulatory bodies are requiring food
manufacturers to indicate on the package label including all
other labeling requirements to commercialize their food
products.
Shelf life testing estimates how long a food product can
be stored under specific storage conditions at optimum safety
and quality (IFST). It depends highly on the handling
procedure that starts from formulation, processing, packaging
and storage. Packaging provides protection and serves as the
final preservation method imputed to food. All processing
technologies applied in food will be ineffective if
improperly packaged. This study aims to determine the shelf
life of pigeon pea-germinated brown rice beverage in
different packaging materials. Sensory evaluation, physico-
chemical and microbial analyses of the product will be
conducted.
Objective of the Study
7
The general objective of this study was to determine the
influence of different packaging materials on the shelf life
of pigeon pea-germinated brown rice beverage.
Specifically, it aimed to;
1. Process pigeon pea-germinated brown rice beverage;
2. Evaluate the sensory attributes, physico-chemical
properties of the product, and microbial count during
storage as influenced by different packaging materials
namely glass, PET and HDPE bottles; and
3. Determine the shelf life of the product using direct
method.
Significance of the Study
This study was conducted to determine the
influence of different packaging material on the shelf life
of pigeon pea - germinated brown rice beverage through direct
method. The result of the study will contribute to the
commercialization of this newly developed product since
expiry or best before date is a mandatory requirement for all
types of food product in the market.
8
Commercialization of this beverage would offer the
consumers a new preference for a cheaper form of
protein source. It could be more beneficial to those people
who have lactose intolerance and other lactose related
diseases. Lastly, the utilization of pigeon pea and brown
rice would encourage higher production and cultivation of
these produce benefiting our local farmers.
Scope and Limitation of the study
The study focused on the shelf life testing of pigeon
pea – germinated brown rice beverage in different packaging
materials using direct method. Formulation and process were
patterned from previously established guidelines developed by
Espinosa, et al, unpublished. PP – GBR beverage was processed
using a 15 liter capacity pasteurizer and packed with 500 ml
of Polyethylene terephthalate (PET), High-Density
Polyethylene (HDPE), and glass bottles stored at 4°C storage
temperature. The product was evaluated every three days
interval in terms of sensory and physicochemical properties
using triangle test, and analyses on pH, Total Soluble Solids,
Titratable Acidity, and viscosity. Microbial evaluation
focused on viable count of yeast and molds.
9
Sensory attribute (presence of sour taste), and
microbial limit of 2.0 log cfu/ml (102) for yeast and molds
were used as the parameters or indicators at the end of the
shelf life study.
Place, Time and Duration of the Study
The study was conducted at the Department of Food
Science, Central Bicol State University of Agriculture, San
Jose, Pili, Camarines Sur from March 2021 to April 2021.
Definition of Terms
Best before date or Best-used-by date–is the date which
signifies the end of the period, under a stated storage
condition, beyond which certain quality attributes of the
product will be expected to deteriorate. However, after this
date the may still be satisfactory for human consumption and
shall remain marketable.
10
Bran – is the protective outer layer of the seeds of cereals
that is separated from the kernels during milling. Often used
in breakfast cereals and other products as a source of dietary
fiber.
Brown rice – refers to unpolished, where only the hull is
removed but the germ and bran are retained. It contains almost
five times the fiber and two times the iron of white rice.
Dal – split beans or seeds of pigeon pea with coat removed.
Consume Before date or Expiration date–is the date which
signifies the end of the estimated shelf-life of the product
under stated storage condition, beyond which the quality of
the product is significantly impaired. After this date the
food should not be regarded as marketable.
Degree Brix (°Bx) – is a hydrometer scale used for measuring
the sugar content of a solution at a particular temperature.
Germination – is the growth of an embryonic plant contained
within seed; it results in the formation of seedling.
Pigeon Pea – Germinated Brown Rice (PP – GBR) Beverage - is
a beverage made of 70% pigeon pea milk with 30% germinated
brown rice extract specifically prepared for human
consumption.
Rice milk – is the rice that has been soaked, ground and then
strained.
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Sensory evaluation – is "the scientific discipline used to
evoke, measure, analyze and interpret those reactions to
characteristics of foods and materials as perceived through
the senses of sight, smell, taste, touch and hearing.
Viscosity – is measured by running a 1 ml sample in a test
tube for 30 seconds and measuring the distance travelled by
the sample using a ruler that will be recorded in centimeter
cm2/s.
REVIEW OF RELATED LITERATURE
This research focuses on the shelf life testing of pigeon
pea – germinated brown rice beverage in different packaging
materials. Some related study and literature are presented in
this chapter as a basis for the conduct of the testing.
Plant – based milk alternatives
Plant – based milk alternatives are fluids that results
from breaking down (size reduction) of plant materials
(cereals, legumes, oil seeds, nuts) extracted in water and
further homogenization of fluid, results in particle size
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distribution in range of 5 – 20 micrometers which imitates
cow’s milk in appearance and consistency (Sethiet al., 2016).
According to Meagan Bridges (2018), homogenization and
thermal treatments are used to improve the suspension and
stability of the product.
Table 1. Plant-Based / Non-Dairy Milk Alternatives
Plant Type References

Milk
Alternative
Cereal-Based • Oat milk Parish C.R., et al, 2018
Sethi, S., et al, 2016
• Rice milk Munekata, P. E., et al, 2020
Parish C.R., et al, 2018
Legume-Based • Soy milk Sethi, S., et al, 2016
Makinen, O. et al, 2015
Munekata, P. E., et al, 2020
• Pea milk Makinen, O. et al, 2015
Vegetable- • Potato milk Parish C.R., et al, 2018
Based
Seed-Based • Flax milk Parish C.R., et al, 2018
• Hemp milk Sethi, S., et al, 2016
Szparaga, A., et al, 2019
• Sesame milk Sethi, S., et al, 2016
13
Nut-Based • Almond milk Sethi, S., et al, 2016
• Cashew milk Lima, J. R., et al, 2020
• Coconut milk Sethi, S., et al, 2016
Legume – based milk
Vegetables do not secrete milk like animals. However,
there are products extracted from oilseeds, pulses and
cereals that have the properties close to that of dairy milk.
The most commonly used legume in milk production is soybean
which has nutrient content close to dairy milk (Prado, et
al., 2008. Extraction of milk from vegetables is an old
technology that has been modified to produce non-dairy milk
and by-products.
Soy Milk
Soy milk was the first plant-based milk which served the
purpose of providing nutrients to the population where the
milk supply was inadequate. It was also popular amongst the
populations who are allergic to milk proteins and are lactose
intolerant(Sethi, S. et. al,2016).Likely the most
recognizable among dairy-free alternatives, soy milk is the
“original” replacement for cow’s milk, first appearing on
shelves in the U.S. in the 1950s. Made by soaking, crushing,
14
cooking, and straining soybeans, it is the only milk
alternative that naturally contains the same amount of
protein as a cup of cow’s milk - 8 grams - along with omega-
3 fatty acids and fiber. Most manufacturers also fortify soy
milk with vitamins A, D, B12, and calcium.
In addition, soy milk can be a good source of manganese
and magnesium.It is perhaps best known for being low in
calories, typically between 30 and 50 per 8 oz. cup
(unsweetened), or about one-third the calories of 2% cow’s
milk. Sweetened varieties, however, can contain up to 90
calories per cup and 16 g of sugar (Parish, C. R. & Bridges,
M. 2018).
Based on the shelf life study conducted by Ozoh and
Ibekwe (2018), locally produced soy milks were only stable
for 5 days at °4C storage temperature. Tale 3 shows the total
microbial viable count of the three different treatments of
locally produced soymilk from 0 to 7 days of storage.
Table 2. Total Microbial Viable Count of the Soymilk
Soy Milk Soy Milk with Soymilk

0 day Stored at Preservatives Stored in
Room Refrigerator
Temperature
(27°C),
Bacterial
count - - -
Fungi count - - -
15
3rd day
count 2.0 x 103 1.3 x 103 -
Fungi count 1.2 x 103
7th day
count 3.6 x 103 3.3 x 103 2.0 x 103
Fungi count 3.3 x 103 1.2 x 103 1038 x103
*(Source from Ozoh, C.N., Ibekwe, I.M. (2019). Determination of
Locally Produced Soymilk.International Journal of Science and
Research. p. 1215)
Pigeon Pea(CajanusCajan)
Figure 1.Matured Pigeon peas
Pigeon pea (CajanuscajanL.) is one of the important
perennial food legume crops incropin the semi-arid tropics
acting as an important source of protein, dietary fiber,
vitamins and mineralsandminerals. It is known to have been
16
cultivated in Egypt before 2000 B.C (Akande et al., 2010).
The crop represents about 5% of the world legume production,
with more than 70% being produced in India (Sharma et al.,
2011). Pigeon pea is considered an important legume grain for
human nutrition in many of the protein – deficient tropical
countries of the world as they serve as supplement to cereal
based diet.
Table 3. The dietary nutrients of pigeon pea
Immature Mature Dal*

Constituents
seed seed
Protein (%) 21.0 18.8 24.6
Protein digestibility (%) 66.8 58.5 60.5
Trypsin inhibitor (units mg- 2.8 9.9 13.5
1)
Starch (%) 48.4 53.0 57.6

Starch digestibility 53.0 36.2 -
Amylase inhibitor (units mg- 17.3 26.9 -
1)
Soluble sugars (%) 5.1 3.1 5.2

Flatulence factors (g 100 g- 10.3 53.5 -
1soluble sugar)
Crude fiber (%) 8.2 6.6 1.2

Fat (%) 2.3 1.9 1.6
Minerals (mg/100 g)
17
Calcium 94.6 120.8 16.3
Magnesium 113.7 122.0 78.9
Copper 1.4 1.3 1.3
Iron 4.6 3.9 2.9
Zinc 2.5 2.3 3.0
Vitamins (mg 100-1 g fresh weight of edible portions)
Carotene (Vit. A) 469 28 IU -
Thiamine (Vit. B1) 0.3 0.643 -
Riboflavin (Vit. B2) 0.3 0.187 -
Niacin 3.0 2.965 -
Ascorbic acid (Vit. C) 25.0 0.00 -
*(Table adopted from Faris, D.G., Saxena, K.B., Mazumdar, S. and
Singh, U. (1987) Vegetable Pigeonpea: A promising crop for India.
ICRISAT, Patancheru, AP. 502324, India: International Crops
Research Institute for the Semi – Arid Tropics. p. 420-423)
(*Dal – split beans with coat removed)
Nutritional Value of Pigeon Pea
Pigeon pea is a rich source of protein, carbohydrate and
certain minerals. The protein content of commonly grown
pigeon pea cultivars ranges between 17.9 and 24.3 g/100 g for
whole grain samples (Salunkhe et al., 1986).It also contains
significantly higher (about 25%) sulphur-containing amino
acids, namely methionine and cystine (Singh et al., 1990).
Pigeon pea contains high amounts of vitamin B, carotene and
ascorbic acid (Diller, et. al, 1956). These are deficient in
cereals; therefore, pigeon pea has a good supplemental value
of cereal based diet. Faris& Singh (1990) reported that pigeon
pea improves the amino acid amount of lysine in rice-and
wheat-based diets.
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Pigeon pea contains more minerals, like iron (Fe),
sulphur, calcium, potassium (K), magnesium, copper, zinc and
five times more vitamin A, water soluble vitamins (Table 3),
especially thiamine, riboflavin, niacin (Faris et al., 1987;
Saxena et al., 2010; Sinha, 1977; Singh et al. 1994), three
times more vitamin C and is also a good source of crude fiber
than ordinary peas (Foodnet, 2002).
Health benefits of pigeon pea
Besides its nutritional value, pigeon pea also possesses
various medicinal properties due to the presence of a number
of polyphenols and flavonoids. It is an integral part of
traditional folk medicine in India, China and some other
nations (Saxena et al. 2010). In India, leaves of pigeon pea
are used for curing wounds, sores, abdominal tumors and
diabetes (Odeny, 2007). Fresh seeds are used to help from
incontinence of urine in males, while immature seeds are
suggested for treatment of kidney ailments (Duke, 1981).
Scorched seeds are added to coffee to relieve from headache
and vertigo (Saxena et al., 2010).
Pigeon Pea Milk
Pea milk is one of the newer dairy-free milk alternatives
with only one major manufacturer currently in the U.S. but a
19
second one gearing up to introduce its own line soon. Made
from yellow field peas, this beverage is poised to gain more
popularity, owing largely to its naturally high protein
content (at least 7 g per oz. cup). During manufacturing,
yellow peas are milled into flour, which is then processed to
separate the protein content from the fiber and starch and
the protein is then further purified and blended together
with water and other ingredients.
This is different from most nut-based milks, in which
the protein content is removed and never reintroduced. Like
most other non-dairy alternatives, pea milk is fortified to
contain 150% more calcium than cow’s milk. Moreover, its taste
and consistency is actually very close to cow’s milk. An added
benefit, yellow peas are easy and inexpensive to grow, so pea
milk carries a much lower water footprint than almond milk
and a much smaller carbon footprint than cow’s milk.
Brown Rice
Rice(Oriza sativa L.) is a semi – aquatic, annual, and
a grass plant. It is a staple food in many parts of the world
especially in the East, South and SouthEast Asia, making it
the second-most consumed cereal grain. Bahadur (2003)
reported that it is better to eat unpolished (brown) rice,
because the outer bran layer of the rice grain, which is
20
removed during the milling process, is rich in fiber, iron,
vitamins and minerals.
Figure 2. Brown rice
Rice Milk
Rice milk is plant milk made from rice. Commercial rice
milk is typically manufactured using brown rice and brown
rice syrup, and may be sweetened using natural sweeteners,
and flavored by common ingredients such as vanilla. It is
commonly fortified with protein and micronutrients such
vitamin B12, calcium, iron, and vitamin D (Bridges, M.
2018).Rice milk is made commercially by pressing the rice
through a grinding mill, followed by filtration and blending
in water. It may be made at home using rice flour and brown
rice protein, or by boiling brown rice with a large volume of
water, blending and filtering the mixture (Nava, S. 2019).
Compared to cow’s milk, it has considerably less protein
(1 g per 8 oz. cup) and calcium. Rice milk contains a
significantly higher amount of carbohydrates (9% vs. 5%).
21
Commercial brands of rice milk are often fortified with
vitamins and minerals. Rice milk is the least allergenic among
plant milk, and may be consumed by people who are lactose
intolerant, allergic to soy or milk (USDA Nutrient database
; Bridges, M. 2018).
Germinated Brown Rice
Germinated brown rice (GBR) is different from normal
brown rice in that it has undergone the process of
germination; more specifically, the rice embryo is sprouted
under suitable environmental conditions.Germination is a
natural process occurred during growth period of seeds in
which they meet the minimum condition for growth and
development (Sangronis et al., 2006). During this period,
reserve materials are degraded, commonly used for respiration
and synthesis of new cells prior to developing embryo (Vidal-
Valverde, 2002). The process starts with the uptake of water
by the quiescent dry seed and terminates with the emergence
of the embryonic axis, usually the radical (Bewley and Black,
1994).
Germination Conditions for Brown Rice
Generally, brown rice can be germinated by soaking it in
warm water of 35–40 °C for about 10–12 h, draining water and
22
keeping in moist condition for 20–24 h, and during soaking
period, changing the water every 3–4 h to prevent fermentation
(which usually produces undesirable odor) and to maintain
consistent water temperature. The result yields a 0.5–1 mm
long sprout from the brown rice grain; at this stage nutrient
accumulation in the grain is maximum. Unlike white rice, GBR
provides more sweetness, excellent taste, has better texture
and is easier to cook. (Patil,S.B., et. al, 2011).
Nutritional aspects
Processing techniques such as boiling, roasting and
germination are means of improving the nutritional value of
foods (Nergiz & Gokgoz, 2007).GBR is evaluated as a functional
food because it is good in digestion and absorption, and
contains nutrients such as (GABA) and ferulic acid in plenty
as compared to ordinary brown rice (Patil,S.B., et. al, 2011).
Various analyses on GBR conducted indicated that during
the process of germination, nutrients in the brown rice change
drastically. Kayahara et al. (2001) showed that not only
existing nutrients are increased but new components are also
released from the inner change due to germination. The
23
nutrients which have increased significantly include GABA,
lysine, vitamin E, dietary fiber, niacin, magnesium, vitamin
B1, and vitamin B6 (Kayahara et al. 2001; Kayahara 2001). The
other nutrients that increased in GBR were inositols, ferulic
acid, phytic acid, tocotrienols, potassium, zinc, g-oryzanol,
and prolyl endopeptidase inhibitor (Kayahara and Tsukahara
2000).
In particular, the amount of GABA in GBR was noticed to
be ten times more as compared to milled white rice and two
times more than that of brown rice. Further, they found that
GBR contains less calories and sugar than that in milled rice.
Choi et al. (2006) reported that upon 24 h germination, the
increased amounts of these nutrients relative to those in the
non-GBR were 3.4 times for fructose, 2.75 times for reducing
sugars, and 7.97 times for GABA. Amongst nutrients, a
significant increase was observed in GABA contents.
Bio – active Compounds in Germinated Brown Rice
Gamma(γ)-aminobutyric acid (GABA) and its effects
The reason behind the popularity of GBR among consumers
is the significant increase in GABA, a four-carbon non-
protein amino acid, which is an inhibitory neurotransmitter
that have the following benefits: promotes fat loss by the
24
stimulation of the production of human growth hormone;
increases the sleep cycle giving deeper rest; boosts the
immune system; lowers blood pressure; inhibits development of
cancer cells; assists the treatment of anxiety disorders (Oh
and Oh 2004; Ito and Ishikawa 2004).
Figure 3. Structure of GABA
*(Source: Shelp, B. J., Brown, A. W., & McLean, M. D.

1999.Metabolism and functions of gamma-aminobutyric acid.
Trends Plant Sci. 4: 446-4452.)
This free amino acid has also effects on accelerating
metabolism in brain, preventing autonomic disorders during
presenile or menopausal period, and relieving insomnia
(Jakobs et al. 1993; Okada et al. 2000; Omori et al. 1987
cited in Komatsuzaki et al. 2003; Kayahara et al. 2001).
Further, Ito and Ishikawa (2004) suggested that GABA might
have preventive effects on Alzheimer’s disease, or help
25
lessen symptoms experienced from this disease and other
cerebralrelated disorders, such as amnesia and dementia.
Y –Oryzanol
Oryzanol is a mixture of ferulic acids esteridied with normal
sterols or triterpene alcohols, called α, β, γ – oryzanol, of
which the γ – oryzanol has been the most commonly mentioned.
The sterol components of γ – oryzanol are primarily
campesterol and sitosterol, and the triterpene alcohol
components are cycloartenol and 24-methylene cycloartenol
(Dey and Haarbone, 1991).
Table 4. γ – oryzanol content in rice grains
Sample Oryzanol content References

(mg/100 g)
Brown rice 20.1-38.8 Aguilar-Garcia et al.,
2007
31.0-62.7 Miller et al., 2003
36.2-62.7 Miller and Engel., 2006
48.2 Ohtsuboet al., 2005
50.0-72.0 Khatoonet al., 2004
Rice bran 56.0-99.0 Chotimarkornmet al., 2008
155.0-272.0 Aguilar-Garcia et al.,
2007
251.0-686.0 Bergman and Xu., 2003
278.76-283.96 Yu et al., 2007
310.1 Shin and Godber., 1996
440.0-474.0 Lloyd et al., 2000
783.2 Devi et al., 2007
Polished rice 6.1 Ohtsuboet al., 2005
7.0-12 Khatoonet al., 2004
30.44-30.89 Boonsit et al., 2006
26
*(Table adapted from Banchuen, J. 2010. Bio–active Compounds in
Germinated Brown Rice and its Application - Thesis. Prince of
Songkla University p. 15)
γ – oryzanol has been suggested to have potential
functionality such as antioxidant activity (Xu and Godber,
2001), reduction of serum cholesterol (Sasaki et al., 1990),
reduction of cholesterol absorption and decrease of early
atherosclerosis (Ronget al., 1997), inhibition on platelet
aggregation (Seetharamaiah et al., 1990), and inhibition of
tumor promotion (Yasukawa et al., 1998). Jiamyangyuen (2006)
reported that brown rice which was soaked in distilled water
for 6 hours and germinated for 24 hours contained more content
and activity of antioxidants compared to brown rice.
Ferulic acid
Ferulic acid (4-hydroxy-3methoxycinnamic acid) is a
ubiquitous phenolic compound in plant tissues. It is one of
the metabolites of biosynthesis of lignin from phenylalanine
and tyrosine in plants (Zhao and Mohadashian, 2008). Ferulic
acid is also bio-active compounds that have been studied for
their cholesterol – lowering properties as well as for their
antioxidant capacity. The antioxidant activity of ferulic
acid is primarily based on hydrogen donation from the ferulic
acid hydroxyl group (Nystrom et al., 2007).
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Phytic acid
Figure 4. Structure of Phytic acid
*(Source: Cheryan, M. 1980.Phytic acid interactions in food

systems.CRC Crit. Rev. Food Nutr. 13: 297-355)
Phytic acid (myo-inositol 1,2,3,4,5,6-hexakis
[dihydrogen phosphate]) is the principal storage form of
phosphorus in many plant tissues, especially bran and seeds.
Phytic acid found in the rice bran has shown to induce marked
grown inhibition in breast cancer cells without harming
normal cells. It has anticancer and antioxidant functions and
prevents coronary disease. It is able to prevent the building
up of superoxide, as well as to boost the immune system. This
bio – active compound has the ability to prevent colon cancer,
28
liver cancer, lung and skin cancer. It is also able to control
anemia and prevent cardiac infarction and diabetes (Kayahara,
2004).
Health effects and benefits of GBR
Nutrition of germinated grains has been studied for
decades. Kayahara and Tsukahara (2000) concluded that
continuous intake of GBR is good for preventing headache,
relieving constipation, preventing colon cancer of colon,
regulating blood sugar level and preventing heart disease.
Okada et al. (2000) reported that intake of GABA suppressed
blood pressure and improved sleeplessness, an autonomic
disorder observed during the menopausal or presenile period.
Mitaka (2000) gave a method to produce GBR, which is
easier to absorb and digest than regular brown rice. GBR helps
in preventing Alzheimer’s disease, due to its increased GABA
content. Go Grains E-News (2004) reported that GBR
significantly improved levels of spatial learning in mice.
Kayahara and Tsukahara (2000) showed that the brown rice
sprouts contain a potent inhibitor of an enzyme called
protylendopetidase, which is implicated in Alzheimer’s
disease. Ito et al. (2005) reported that intake of GBR instead
of white rice is effective for the control of postprandial
29
blood glucose concentration without increasing the insulin
secretion in subjects with hyperglycemia.
Hiroshi (2005) showed that besides containing other
useful components, GBR mainly contains two active components
viz. GABA, a neurotransmitter which is abundant in the brain
and spinal cord, and dietary fiber which activates the
peristalsis of the intestine. It also contains considerable
phytic acid with a powerful anticancer activity and a prolyl
endopeptidase activity inhibitor related to the metabolism of
peptide. Chikako et al. (2005) reported that the protease
activity in GBR was increased 1.5 times after germination.
They suggested that decrease in soluble proteins and
allergens was induced in part by proteolytic degradation and
two abundant allergens were degraded in a different manner
and probably by different protease in the grains during
germination.
Blending for nutritional balance and improvement in sensory

acceptability
Plant-based milks are different from one another with
respect to their nutritional value. Therefore, blending two
or more types of plant-based milk,to have a product with high
nutritive value comparable with cow’s milk, is an important
step of processing. Scientific literature dealing with
30
blending of plant-based milk is scarce. Deshpande et al.
(2008) used response surface methodology to optimize
chocolate flavored peanut-soy beverages on the basis of
sensory parameters and it was observed that the beverages
prepared with soy protein isolates were more acceptable than
those with soy flour. Also, soy protein isolates are expected
to increase the nutritional value.
In another study, plant-based milks were developed from
fermented and unfermented African yam bean (AYB) flours and
maize blends. The protein content of the developed milk was
comparable to that of soybean with appreciable quantities of
carbohydrate and minerals and traces of ascorbate (Nnam
1997). As soy milk contains one-fifth of calcium than cow’s
milk, deficient in amino acid methionine and is nutritionally
less acceptable than cow’s milk, US patent 3386833 was aimed
at the development of simulated milk comprising soy bean
flour, sesame seed flour and coconut meal with the objectives
to improve the nutritional value and palatability,
characterized by minimum enzyme destruction in the
preparation of powdered mixture used for the formation of the
milk, and can be suitably used for infant consumption (Johnson
and Linda 1968). The simulated milk developed had relatively
high proportion of the two essential amino acids lysine and
31
methionine, a much higher calcium content than that of cow’s
milk and conventional soy milk, approximately six times as
much iron is found in cow’s milk, the fats present principally
in unsaturated form and the final product showed long keeping
quality due to presence of natural antioxidants particularly
found in sesame seeds.
Shelf life and Direct Shelf life Test
Shelf life of the food
Institute of Food Technologist (1974) has defined shelf
life as “the period between the manufacture and the retail
purchase of a food product, during which time the product is
in a state of satisfactory quality in terms of nutritional
value, taste, texture and appearance”. This definition
overlooks the fact that the consumer may store the product at
home for some time before consuming it, yet will still want
the product to be of acceptable quality.
The Institute of Food Science and Technology (IFST) in
the United Kingdom has defined shelf life as “the period of
time during which the food product will remain safe; be
certain to retain desired sensory, chemical, physical,
microbiological and functional characteristics; and comply
32
with any label declaration of nutritional data when stored
under the recommended conditions”.
The quality of most foods and beverages decreases with
storage or holding time. For the majority of foods and
beverages in which quality decreases with time, it follows
that there will be a finite length of time before the product
becomes unacceptable. Manufacturers generally attempt to
provide the longest practicable shelf life consistent with
costs and the pattern of handling and use by distributors,
retailers, and consumers. Shelf life testing estimates the
shelf life of a food by actual determination of time to reach
an acceptable condition.The shelf life put into or open dates
are placed on the label of food and beverage product to assist
consumers when purchasing (Dethmers, 1979).
Real Time or Direct Method
Direct method is the alternative shelf life method that
can be used as a basis for shelf life estimation. Direct
method is commonly used in determining the shelf life of the
food; it involves storing the product under preselected
condition for a period of time longer than the expected shelf
life and checking the product at regular intervals to see
when it begins to spoil.
33
In determining the storage stability there are factors
that limit the shelf life of food products. These include the
physico-chemical and microbiological changes which result in
unacceptable sensory characteristics and become unsafe (Jone,
2000). Additionally, the use of effective packaging
contributes to longer shelf life. Direct method is a real-
time study that consists of storing the product under
conditions similar to its actual storage, to monitor its
evolution in regular intervals of time. The main advantage of
this method is that it creates a very accurate estimation of
the time it takes for a product to deteriorate. The shelf
life is terminated when a certain level of deterioration is
reached (BTSA, 2020).
Factors Affecting Shelf Life
Product characteristics
Product characteristics including formulation,
processing parameters and intrinsic factors (pH, water
activity, enzymes, microorganisms, and concentration of
reactive compounds) greatly affects product shelf life.On the
basis of the nature of the changes that can occur during
storage, foods can be divided into three categories—
perishable, semi-perishable, and nonperishable or shelf
stable—which translate into very short shelf life products,
34
short to medium shelf life products, and medium to long shelf
life products (Robertson, 2006). These factors can be
controlled through the selection of raw materials and
ingredients, as well as the choice of processing parameters,
proper handling during processing, packaging, and storage.
Distribution and Storage Environment
Environment to which the product is exposed during
distribution and storage (extrinsic factors) also affect the
shelf life of a food product.Extrinsic factors include
temperature, Relative Humidity, light, total and partial
pressures of different gases, and mechanical stresses,
including consumer handling. Many of these factors can affect
the rates of deteriorative reactions that occur during the
shelf life of a product.
The deterioration in product quality of packaged foods is
often closely related to the transfer of mass and heat through
the package. Packaged foods may lose or gain moisture; they
will also reflect the temperature of their environment,
because very few food packages are good insulators. Thus, the
climatic conditions (i.e., temperature and humidity) of the
distribution and storage environment have an important
influence on the rate of deterioration of packaged foods
(Robertson, 2006).
35
Package Properties
Foods can be classified according to the degree of
protection required from the package, such as maximum
moisture gain or O2 uptake. This enables calculations to be
made to determine whether a particular packaging material
would provide the barrier required to give the desired product
shelf life. Metal cans and glass containers can be regarded
as essentially impermeable to the passage of gases, odors,
and water vapor, but paper-based packaging materials can be
regarded as permeable. Plastics-based packaging materials
provide varying degrees of protection, depending largely on
the nature of the polymers used in their manufacture
(Robertson, 2006).
The properties of the package can have a significant effect
on many of the extrinsic factors, and indirectly on the rates
of the deteriorative reactions. Thus, the shelf life of a
food can be altered by changing its composition and
formulation, processing parameters, packaging system, or the
environment to which it is exposed.
Packaging
36
The packaging plays a fundamental role in
maintaining the quality and shelf life of foods. The package
is an integrated part of the preservation system and functions
as a barrier between the food and the external atmosphere.
Food packaging can retard product deterioration, retain the
beneficial effects of processing, extend shelf-life, and
maintain or increase the quality and safety of food (Marsh,
K. et. al, 2007). The package should be designed and developed
not only to hold the food product, but also to protect it and
add value to it, since its design may directly affect the
purchase decision of the consumer (Roberston, 1993).
Azeredo, Fria, and Brito (2004)& (Robertson, 2006)
consider that the protection provided by the packaging is the
factor of greatest importance, since it is directly related
with the safety of the consumer. These authors emphasized
that the package must be appropriate for each food product to
minimize undesirable changes that may affect the product
during its shelf life. The choice of packaging for foods
produced depends very much on the nature of food, method used
to preserve it and how long the food must remain in wholesome
condition (Beyer,R. 2012).
Role of Packaging in Controlling Deteriorative Reactions
37
As with pasteurized and UHT milk, packaging can directly
influence the quality of in-bottle sterilized milk by
protecting the product from light, O2, and microbial post
processing contamination during its shelf life. Finally,
packaging in combination with refrigerated storage and
dispensing protects pasteurized milk from recontamination and
provides milk with a satisfactory shelf life by controlling
the growth of total and psychotropic microorganisms
(Erickson, 1997).Packaging materials currently used for
pasteurized milk include narrowneck glass bottles and three-
and six-layer HDPE plastic bottles, PET bottles, and
pigmented plastic pouches.
Shelf life of Pasteurized milk in Different Packages
Glass Bottle
A bottle is a rigid container with a neck that is
narrower than the body and a “mouth”. By contrast, a jar or
jug has a relatively large mouth or opening which may be as
wide as the overall container. Glass is the most inert of all
packaging materials and provides ultimate protection O2,
moisture, and microorganisms.
Glass bottles need to be adequately cleaned and
sterilized before use. Sattar et al. (1983) analysis revealed
38
that the best protection against ascorbic acid degradation
was provided by amber glass, followed by cartoons, green glass
and clear glass. The same pattern was observed with respect
to the sensory quality of the milk.
Sattar et al. (1983) packaged buffalo milk in four
different containers: clear glass, green glass, and amber
glass bottles, and plastic/aluminum foil/paperboard brick-
shaped cartons. Samples were kept at 5–6°C and 16–24°C for 24
and 16 hr, respectively, under laminate fluorescent light.
Analysis revealed that the best protection against ascorbic
acid degradation was provided by amber glass, followed by
cartons, green glass, and clear glass. The same pattern was
observed with respect to the sensory quality of the milk.
Plastic Bottle
New advances in plastic packaging have led to the
development of natural polymers-based systems which show
several advantages such as biodegradability, environmental
friendliness, low cost, high efficiency as active supports,
and similar processing conditions to synthetic polymers.
39
Polyethylene terephthalate (PET) Bottle
Regarding plastic materials, PET is increasingly used in
beverage packaging for liquids such as milk or oil due to its
excellent mechanical properties, clarity, UV resistance, and
good oxygen barrier properties (Bacigalupi, C.et. al 2013).
PET is a colorless, transparent polymer that is almost
unbreakable and can be recycled well. These good properties
led to broad distribution of PET as a packaging material for
beverages. By contrast with beverage cans or cartons, the
transparency allows the beverage to be seen. The unbreakable
nature of PET is an advantage over glass bottles. Moreover
PET is distinctly lighter than glass (Welle,F. 2016).
Papachristou et al. (2006a, 2006b) evaluated PET as a
packaging material for bactofuge - pasteurized milk stored
either in the dark or under fluorescent light at 4°C for a
period of 13 days. Containers tested included (a) clear PET
+ UV absorber bottles with a transparent label, (b) clear PET
+ UV absorber bottles with a white label, and (c) clear PET
bottles, with (d) LDPE-coated paperboard cartons serving as
the commercial control sample. Results showed satisfactory
protection of milk packaged in all containers with regard to
microbial and chemical parameters assessed over the 13-day
storage period. On the basis of sensory analysis the shelf
40
life of bactofuge - pasteurized milk stored in the dark was
10–11 days for samples packaged in clear PET + UV absorber
bottles regardless of the type of label used and 9–10 days in
clear PET bottles and paperboard cartons. The shelf life of
milk stored under fluorescent light was 10–11 days for clear
PET + UV absorber bottles and paperboard cartons and 8–9 days
for clear PET bottles.
High-Density Polyethylene (HDPE) Bottle
High-Density Polyethylene products are very safe and are
not known to transmit any chemicals into foods or drinks.
HDPE products are commonly recycled. Items made from this
plastic include containers for milk and beverages. It is never
safe to reuse an HDPE bottle as a food or drink container if
it didn’t originally contain food or drink.
HDPE bottles of various capacities between 1 and 4 L are
widely used for pasteurized milk packaging in several
countries, including the United States, Canada, the United
Kingdom, and Australia. Unpigmented HDPE bottles transmit 58–
79% of the incident light in the wavelength range 350–800 nm.
Light transmission can be reduced by pigmenting HDPE with
TiO2 at 1–2%, producing an opaque bottle.
41
HDPE jugs are extrusion-blow-molded to provide a thin-
walled, lightweight, and tough container. An advantage of
this type of packaging, especially in the 2 and 4 L sizes, is
the handle on the bottle, which makes it more convenient to
hold than, for example, paperboard cartons. HDPE bottles are
used for pasteurized full-fat, semi-skimmed, and skimmed
milk.
Plant type Shelf-life References

(days)
Rice – Pigeon pea
14 Bardon, A. unpublished
Soymilk
(locally produced) 5 Ozoh, C.N. et al, 2019
Chickpea and Lupin 7 Lopes, M., et al, 2020
Coconut 14 Szparaga, A. et al, 2019
Hemp 14 Szparaga, A. et al, 2019
Almond 7 Hasan, N. A. 2012
Shelf – life of different plant-based milks
Table 5. Shelf – life of different plant-based milk

alternatives in refrigeration temperature
Based from the previously conducted studies, the shelf
life of different plant- based milk alternatives ranges from
five to 14 days without chemical preservatives added.
Pasteurization
42
Pasteurization is the process of heating a liquid
to below the boiling point to destroy microorganisms. ‘Milk
pasteurization’ refers to the process of heating every
particle of milk to a specific temperature, holding it at the
same temperature for specific duration (time) and followed by
rapid chilling to less than 7°C. Accordingly, there can be
numerous combinations of time and temperatures for heat
treatment to get the desirable microbial or chemical effects
like improvement in keeping quality, destruction of pathogens
or destruction of all microorganisms (Dhotre, A. V. 2014).
The pasteurization process is identified by its specific
temperature and time combination. The basic objective while
designing a pasteurization process can be described by the
official definition of pasteurization given by International
Dairy Federation (1980). It states that ‘Pasteurization is a
process applied to a product with the object of minimizing
possible health hazards arising from pathogenic
microorganisms associated with milk by heat treatment which
is consistent with minimal chemical, physical and
organoleptic changes in the product.
Table 6.Milk Pasteurization Temperature
Treatment Time
43
For 30 minutes (Low temperature
63°C
long time LTLT)
For 15 seconds (primary high
72°C temperature short time, HTST
method)
89°C For 0.1 second
*(Reference: Peth, J. MS. MPH, PhD (2015).
Effect of Storage on Sensory Characteristics
The change in color parameter may be due to the maillard
reaction between sugars and amino acids (Gonzales &Leeson,
2000). Pruthi, et. al, (1984) reported that, the decrease or
degradation of flavor during storage of product was result
from subjecting it to low temperature and due to heat
treatment applied during processing.
Standards or indices made as basis for shelf life

determination
Sensory attributes (Flavor changes)
Indicative spoilage of soymilk is the presence of off –
flavors that may be caused by physicochemical or microbial
44
changes. The main chemical defect is lipid peroxidation.
Unsaturated fatty acids are attacked by free radicals, which
is followed by the addition of O2, to form peroxides or
hydroperoxides (Min and Lee, 1996), resulting in sensory
changes like metallic or cardboard off-flavor. Microbial
changes leading to the formation of microbial flavor
described as acidic, bitter, or malty (Robertson, G.L. 2006).
Table 7.Physico – chemical properties of soymilk
Parameters Minimum limit Maximum limit

Ph 5.7 + 0.00 6.63 + 0.06
Total Soluble Solids 9.00 + 0.01 10.00 + 0.30
(°Brix)
Titratable acidity (g/100 0.043 + 0.00 0.077 + 0.00
g)
Viscosity (mPas) 2.00 + 0.03 3.13 + 0.58
(*Source: Food Sci. Technol, Campinas, 33(2): 387-394, April-
June 2013)
Microbial Limit
Table 8. Microbiological limits for soymilk
Microorganism Maximum Method of test

S/n
limit
I. Total plate count , cfu/ml 103 ISO 4833
II. E. coli, MPN/ml absent ISO 7251
45
III. Salmonella per 25 ml absent ISO 6579
IV. Yeast and moulds, cfu/ml 102 ISO 21527-1
*(Source: East African Standard; Soya Milk-Specification.
First edition, 2013)
METHODOLOGY
Formulations, processing, experimental design for shelf
life testing and analyses such as sensory evaluation, physic-
46
chemical characteristics and microbial examination were
detailed in this chapter.
Materials and Methods
Gathering of Raw Materials and other ingredients
Brown rice was purchased at Pecuaria Development
Corporation, Inc. (PDCI), Pecuaria, Lanipga, Bula, Camarines
Sur. An ICPL – 88039 variety of fully matured pigeon pea
beans was sourced from Mariano Marcos Memorial State
University, Ilocos Norte. Other materials for processing like
sugar and cheese cloth were purchased at Naga City. Pigeon
pea beans were sorted prior to processing, defected beans and
foreign matters were removed.
Sourcing and Preparation of Packaging Materials
500 ml packaging materials namely, PET (a), HDPE (b),
and glass (c) bottles were purchased from Megawise Packaging
Inc. at Taguig City, Metro Manila. Glass bottles were clear
and had colored metal cups, which were subjected to
sterilization using conventional methods at 80°C for 15
minutes. PET bottles were thin, clear or transparent and were
covered with plastic cups, whereas the HDPE bottle was thick
and the color white bottle covered also with plastic cups.
47
These two packaging materials were sterilized with 40 – 45°C
water.
Processing of Pigeon Pea Beverage with Germinated Brown Rice
Preparation of Pigeon Pea extract
(2) kg of pigeon pea was milled into grits, dehulled,
winnowed, and soaked in water overnight (8-12 hours) and
washed every after three hours for two to three times (Ozoh
et al., 2018). Once the bean splits can be opened easily and
are flat on the inside, this indicates that the beans can be
drained for grinding. Water was discarded; pigeon pea splits
were washed two to three times with clean water. Soaked PP
was wet milled with distilled water at 150 g: 1L ratio and
set aside.
Pigeon Pea Beans
Weighed
48
Grits
Dehulled and Winnowed
Washed
Soaked Drained
(8-12 hrs.)
Weighed
Blended/ Milled
Strained
Put on a sealed container
Figure 5. Process flow of Pigeon Pea Extract

(Espinosa, et al., 2018. The Potential 6f pigeon pea (Cajanuscajan)
beverage as an anti – diabetic functional drink IOP Conf. Ser.:
Earth Environ. Sci. 102 012054)
Preparation of germinated brown rice extract

Germination of brown rice
Rice germination was prepared by soaking brown rice in
distilled water for 5 hours 30+2°C on a jar covered with
49
stainless fine mesh. After 5 hours of soaking, the water was
drained off, wrapped with sterilized cheese cloth to retain
moisture, and left for 24 hours to germinate. The GBR was
washed and dried to <13% moisture content using a tray dryer
at 50 to 70 °C. Then milled and sealed in a clean plastic
container
Extraction of GBR milk
GBR flour was added with distilled water at 100 g: 1L
ratio to extract the milk. Sieved and set aside.
Brown Rice
Washed
Weighed
50
Soaked
(5 hours)
Germinated
Distilled Water
(24 hours at 30+2°C)
Rinsed
Dried
50 to 70 °C
Milled
Figure 6. Process flow of Germinated Brown Rice Flour
(Banchuen, J. 2010. Bio–active Compounds in Germinated Brown Rice

and its Application - Thesis. Prince of Songkla University, p. 23)
GBR flour
Addition of water
(100 g: 1L)
Sieved
51
Figure 7. Process flow of GBR Extract

(Banchuen, J. 2010. Bio–active Compounds in Germinated Brown Rice
and its Application - Thesis. Prince of Songkla University, p. 23)
Table 9.Formulation of Pigeon pea – GBR Beverage
Ingredients Percentage (%)

PP 64.4
GBR 27.6
Sugar 8
*Legend: PP – Pigeon pea ; GBR – Germinated Brown Rice
Processing of Pigeon pea – GBR Beverage
Pigeon pea milk with germinated brown rice beverage was
prepared using the formulation in table 7. (70:30) ratio of
PP milk and GBR extract were combined and mixed with sugar.
The mixture was pasteurized for 15 seconds at 72°C, packaged
in sterilized glass bottles. PP – GBR in glass bottles undergo
exhausting for 15 minutes at 80°C, while the samples in other
packaging are sealed and cooled. After cooling, samples were
stored at refrigerated (4°C) temperature.
52
Germinated Brown Rice extract Pigeon pea extract
Combined and Mixed
Add of sugar
53
Pasteurized
15 sec. at 72°C
Bottled
Exhausted
(glass bottle)
(15 min. at 80°C)
Sealed
Cooled
Stored
(4°C ref. temperature)
Figure 8. Process flow of Pigeon pea – GBR Beverage
Quality Parameters for Pigeon pea – Germinated Brown Rice

Beverage
These cover the evaluation of the different properties
of pigeon pea beverage with germinated brown rice which only
concentrates on the physico-chemical and sensory properties.
Microbial viable count will also be evaluated.
54
Physico-chemical Analysis
The pH value, total soluble solids (TSS), titratable
acidity (TA) of the product was evaluated for at least
triplicate during storage every three days interval at 0, 3,
6, 9, 12, 15… days respectively.
pH Determination
pH measures the level of acidity and alkalinity of food
to gauge its concentration of acid. It was measured using the
pH meter (Chinyere& Kenneth, 1997). Five ml of the product
sample was measured and diluted with 45 ml of distilled water.
Using a pH meter, the electrode was immersed in a buffer
solution pH 7 and 4 to calibrate first for the assurance of
accurate reading, then was washed with distilled water and
gently wiped with tissue. Finally, dip in the diluted sample
up to sufficient depth such that the pH meter should not touch
the bottom of the beaker and pH value will be determined.
Total Soluble Solids (TSS) Determination
The content of TSS in the pigeon pea beverage with
germinated brown rice was determined with the use of a hand
refractometer (Liu & Lin, 2008). A drop of sample was put on
the sensor for measurement and the refractometer reading was
recorded.
55
Total Titratable Acidity (%TA)
The titratable acidity of the product was determined
using titration with NaOH and phenolphthalein as an indicator
and was computed using the formula below.
% TA = Vol. of NaOH used x N OF NaOH X 100
Volume of sample
Viscosity
The viscosity of the product was measured using the
conventional method. One ml of sample was place in a test
tube. Allaying the test tube on its side and measuring the
distance travelled by the sample using a ruler after 30
seconds. The distance traveled was recorded in centimeter
(cm) as cited in the encyclopedia of food technology (1997)
then the value is computed as cm2/s using the formula below:
Viscosity = wt. of sample used
Length of product flow x time (sec)
Results in viscosity were converted into cubic centimeter as
an S.I unit for viscosity, using the conversion units below;
Conversion units: 1 ml/cm·s = cm3
56
Statistical Analysis of Data
All measurements were run for at least triplicate. One
- way Analysis of variance (ANOVA) was used to find a
significant difference (P<0.05) between the samples in
different packaging materials. Duncan’s New Multiple Range
Test was used to determine the significant differences among
means for different treatments.
Sensory Evaluation (Triangle Test)
This test is similar to duo-trio test except that the
reference sample is not identified and therefore not code
“R”. Since the panelists were to evaluate three samples the
probability of getting the correct answer by chance guess
alone is 33.3 % as a difference test, it only shows presence
or absence of difference and not directly and degree of
difference between samples must be small.
Three coded samples were presented to 15 trained
panelists. Two of these three samples are identical while the
third is odd or different. Each sample was coded with a three
digit number. The panelist was asked to identify the odd
sample in terms of flavor (by the presence of sourness as
indices of spoilage) and indicate his choice on the score
sheet. Each sample was tested from different packaging
57
materials. A randomized set was used in the design of the
master sheet.
Training of the Panelists
Fifteen to twenty panelists were selected from the
students of the Department of Food Science, College of
Engineering and Food science. These selected students
underwent screening. Panelists should have interest or
preferably are drinking plant based milk, available,
willingness, and intelligence.
The first step consisted of orientation and screening of
the pre-selected panelists. Then, presentation of the
triangle test method that was used for the evaluation of the
PP - GBR beverage. The next step was familiarizing the
panelists with the basic taste of the product by tasting the
PP – GBR beverage. Then, have a pre – trial sensory evaluation
of the product to select the 10 trained panelists that had
evaluated the product all throughout the duration of the
monitoring. Lastly, train the 10 remaining panelists with the
indicator of spoilage or deterioration of the product in terms
of flavor/ taste (presence of sourness) by presenting them
different food samples with a similar taste to sourness.
Statistical Analysis
58
Chi – square method was used in computing the significant
differences between the correct and incorrect identification
of the panelist as show below:
X2 = (01-E1)2 + (02-E2)2
E1 E2
Where:
01 = observed no. of correct identification
02 = observed no. of incorrect identification
E1 = expected no. of correct identification (3.33%)
Degree of freedom (df) = 1, since there are only three samples

to compared
Tabular X2 value at 1 df (P<0.05) = 3. 84
X2 = computed value
Microbial Analysis
Yeast and Molds
25 ml of the sample from different treatments was mixed
in 225 ml of distilled water giving 1:10 dilution. This was
diluted up to 10-3 ml. Dilution plated was 10-2 to triplicate
plates using Potato Dextrose Agar (PDA) for enumerating yeast
and molds plate count. Yeast and mold count expressed as
59
colony forming units per gram (CFU/g) using the same formula
shown below.
CFU/g = Ave. no. of colony x Dilution factor
Volume of Sample
Table 10. Sampling Plan for shelf life study of PP – GBR

beverage
Schedule of Sampling
Storage Amount of Sample
Interval of Monitoring
Temperature per Monitoring
Evaluation of the
product every 3 days:
(0, 3, 6, 9, 12 days
and until deteriorate)
Product stored
at 4°C
- - 25 ml
- - 500 ml (25
ml/glass)
- - 40 ml
Total 565 ml (per trt.)
Legend:
60
= Microbial analysis (Yeast and Molds)
= Sensory evaluation (Triangle test)
= Physico – chemical analysis (TSS, TA, pH, and
viscosity)
Real time Method Evaluation
Three packaging materials namely, glass, PET, and HDPE
bottles were used in shelf life testing of PP – GBR beverage.
Sensory evaluation, physico-chemical, and microbial analyses
were conducted and monitored every three days of interval
during storage. 30 bottles of 500 ml of newly processed PP –
GBR beverage were used in the study; every one bottle was
used for sensory evaluation (25 ml per glass) and one bottle
for physico-chemical (40 ml), and microbial analyses (25 ml)
for every treatment. Samples were stored at 4°C.
61
RESULTS AND DISCUSSION
The study was conducted in order to determine the
influence of different packaging materials namely, PET
bottle, HDPE, and glass bottle on the shelf life of Pigeon
pea – Germinated brown rice (PP-GBR) beverage using real time
method. Newly processed PP-GBR beverage was evaluated
through sensory evaluation, physico-chemical, and microbial
analyses every three (3) days interval during storage
monitoring.
Figure 9. Newly Process PP-GBR beverage
62
Results of Physico-chemical properties of PP – GBR Beverage
during storage
Total Soluble Solids
Table 11 shows the effect of different packaging
materials on the total soluble solids content of PP – GBR
beverage during storage. Statistical analysis revealed that
there is a significant difference in the results among the
treatments from the 12th and 15th day of monitoring. The
highest difference (1.25) was obtained on the 15th day of
monitoring between samples from glass and PET bottles, while
the lowest difference was observed between HDPE and glass
bottles (0.13). Rapid decrease of TSS was observed on the
samples from PET bottles (10.0-8.47 °Bx). Pruthi, et. al,
(1984) reported that, the decrease or degradation of flavor
during storage of product was result from subjecting it to
low temperature and due to heat treatment applied during
processing. Moreover, microbial proliferation greatly
contributed to the decreasing trend of TSS since yeasts
convert glucose into acids. Thus, PET bottles could not
provide sufficient protection to the product against
temperature reaction and microbial contamination.
Table 11. Total Soluble Solids of PP-GBR beverage during

storage
63
Treatment
Storage day T1 (PET T2 (HDPE T3 (Glass
Bottle) Bottle) Bottle)
Day 0 10.0 + 0.0b 10.0 +0.0b 10.0 + 0.0b Commented [2]: remove superscripts if no significant
difference observed
Day 3 9.83 + 0.05b 9.90 + 0.0b 9.90 + 0.0b Commented [3]: remove superscripts if no significant
difference observed
Day 6 9.67 + 0.05b 9.83 + 0.05b 9.87 + 0.05b Commented [4]: remove superscripts if no significant
difference observed
Day 9 9.47 + 0.05b 9.67 + 0.05b 9.73 + 0.05b
Commented [5]: remove superscripts if no significant
Day 12 8.93 + 0.05a 9.20 + 0.0b 9.33 + 0.05b difference observed
Day 15 8.47 + 0.05a 8.87 + 0.05b 8.93 + 0.05b

Legend: means with the same superscripts are not significant from
each other
Figure 10. Trends of Total Soluble Solids of PP-GBR

beverage in different packaging materials during storage
Total Sooluble Solids (°Bx)
PET Bottle
HDPE Bottle
Glass Bottle
Effect on pH
It was observed that the pH of all the samples in
different packaging materials gradually decreased during
64
storage period (Fig. 9). Table 12 shows how the pH of the
samples varied from different packaging materials over
storage period. Based on the statistical analysis, there is
a significant difference among the results of the treatments
from the 12th day of storage, the highest gap was determined
between the samples from PET and glass bottles which is 1.18.
Sample from PET bottles was found to be acidic having the
lowest pH value of 4.93 + 0.05 during 15th day of storage;
implication of product deterioration. Increasing population
of yeast and molds throughout the storage caused the decrease
of pH. As the storage prolongs, the product causes conversion
of glucose to acids.
Treatment
Storage day
T1 (PET T2(HDPE T3(Glass
Day 0 6.43 + 0.05b 6.43 + 0.05b 6.43 + 0.05b Commented [6]: remove superscripts since there is no
significant difference
Day 12 5.16 + 0.05a 5.73 + 0.05b 5.83 + 0.05b
Day 15 4.93 + 0.05a 5.36 + 0.05c 5.46 + 0.05c
Table 12. pH of PP-GBR beverage in different packaging
materials during storage
Legend: means with the same superscripts are not significant
from each other
Figure 11. Trends of pH of PP-GBR beverage during storage
65
PET Bottle
pH
HDPE Bottle
Glass Bottle
Titratable Acidity (%TA)
Table 13 presents the results of percent total
titratable acidity (%TA) of the samples in different
packaging materials during storage period. It can be
noticeable that the significant increaseincreased in (%TA)
was only observed during the complete deterioration phase of
the samples (0.15%). Statistical analysis revealed that there
is a significant difference among the treatments in terms of
%TA on the 12th day of storage. Highest increase was observed
on the samples from PET bottles (0.17). Whereas, samples
packed in HDPE and glass bottles remained acceptable.
Therefore, PET bottles have poorhave a poor protection
against chemical deterioration. Frazer in 1998 explained that
the gradual increaseincreased in (%TA) was caused by the
organic acid present in the productin product which was
oxidized into simpler acid.
66
Moreover, increased in (%TA) contributed to the
decreased pH and presence of sourness
perceived in sensory evaluation of the product which
indicates that the product is no longer fit for human
consumption (Nielsen, 1994).
Table 13. Percent Titratable Acidity (%TA) of PP-

GBR beverage in different packaging materials during
storage
Treatment
Storage
day
0.033 +b 0.033 +b 0.033 + b Commented [10]: remove superscripts since there is no
Day 0 significant difference
0.01 0.01 0.01
Day 3 0.040 +0.0b 0.040 +0.0b 0.033 +0.01b Commented [11]: remove superscripts since there is no
Day 12 0.13 +0.01a 0.067 +0.01b 0.060 +0.0b
Day 15 0.17 +0.01a 0.15 +0.01c 0.13 +0.01c
Increase in total titratable acidity of samples might be
due to the decrease in pH and microbial load (Khodke, S.
2015). Szaparaga, A. et. al, (2019) pointed out that the acid
content is a factor that determines food freshness, it largely
affects the color and taste of a beverage and other food
67
products. Acids significantly contribute to the control of
microfloramicro flora growth.
Figure 12. Trend of Percent Titratable Acidity

(%TA) of PP-GBR beverage during storage
%Titrable Acidity
PET Bottles
HDPE Bottles
Glass Bottles
Table 14. Viscosity of PP-GBR beverage in different

packaging materials
Treatment
Storage day
68
Day 0 2.77 + 0.05b 2.77 + 0.05b 2.77 + 0.05b Commented [14]: remove superscirpts
Day 12 2.03 + 0.05a 2.37 + 0.05b 2.47 + 0.05b

Day 15 1.83 + 0.05a 1.87 + 0.05a 1.97 + 0.05a
Viscosity
The viscosity of a food system is dependent on the volume
fraction occupied by the contributing particles in
combination with the inherent viscosity of the continuous
phase (Anema, et. al, 2014). According to McClements (2020),
plant-based milks are colloidal dispersions consisting of
particles (oil bodies, protein aggregates) that greatly
affect its viscosity. The standard for viscosity was based
from soymilk and brown rice milk which has 2.6 and 2.2 cm3
respectively. The viscosity of the PP-GBR beverage samples
from different packaging material continually decreases
during storage time (Fig. 11).
Based on the statistical analysis of viscosity within
the storage period, it has revealed that there is a
69
significant difference among the samples during the
15thduring 15th day of monitoring. Highest difference was
found between glass and PET bottlesbottle which is 1.89. Least
significant difference was observed between glass and HDPE
samples. Rapid decrease in viscosity was found on the sample
from the PETfrom PET bottle during the 12thduring 12th day of
monitoring, indication of deterioration. Hence, both HDPE and
glass bottlesbottle were effective in retaining the quality
of PP-GBR beverage in terms of viscosity during storage. The
decrease in viscosity is highly attributed to the decrease in
theon the pH value of the beverage.
Figure 13. Trend of viscosity of PP-GBR beverage during

storage
Vicosity (cm3)
PET Bottle
HDPE Bottle
Glass Bottle
Sensory evaluation
70
Three coded samples from different packaging materials
were evaluated during storage through triangle test in terms
of taste. Presence of sourness was the indicative parameter
of deterioration for the sensory attribute of PP-GBR
beverage. The presence of sourness in milk substitutes is
mostly attributed to the microbial content. Statistical
analysis using the chi-square test revealed
that there were no significant differences on the taste of
the samples packed in PET bottles within nine days of
storage, while samples from both HDPE and glass bottles
remained stable within 12 days of storage (Tab. 13). Thus,
PP-GBR beverage can be kept longer, retaining their
standard taste using HDPE and glass bottles.
Table 15. Results of Triangle test for PP-GBR beverage in

different packaging materials stored at 4°C temperature
Storage PET Bottle HDPE Bottle Glass Bottle

time
(days)
Taste(sourness) Taste(sourness) Taste(sourness)
2.44<3.84 2.44<3.84 2.44<3.84

0 NSD NSD NSD
0.79<3.84 2.44<3.84 0.79<3.84
3 NSD NSD NSD
0.05<3.84 0.79<3.84 0.79<3.84
6 NSD NSD NSD
71
1.24<3.84 0.19<3.84 0.19<3.84
9 NSD NSD NSD
2.44<3.84 1.24<3.84 1.24<3.84
12 S NSD NSD
15.02>3.84 15.02>3.84
15 - S S
Legend: NSD = Not Significantly Different
S = Significantly Different
The main chemical defect is lipid peroxidation.
Unsaturated fatty acids are attacked by free radicals, which
is followed by the addition of O2, to form peroxides or
hydroperoxides (Min and Lee, 1996), resulting in sensory
changes like metallic or cardboardcardboardy off-flavor.
Microbial changes leading to the formation of microbial
flavor described as acidic, bitter, or malty (Robertson, G.L.
2006).
Microbial Analysis
Plant-based milk being a rich source of nutrients serves
an ideal medium for growth of microorganisms, and therefore
its quality is adversely affected by the rapid growth of
microorganisms (Sethi, S. et. al, 2016). Table 14 presents
the enumeration of viable counts of yeast and molds from PP-
GBR beverage in different packaging materials during storage.
On the zero day, there was no growth of bacteria in all the
72
samples. The maximum standard of yeast and molds count for
plant-based beverages was 2.0 log cfu/ml (10-2).
Table 16. Yeast and mold count of PP-GBR beverage in

different packaging materials during storage
Storage time PET Bottle HDPE Bottle Glass Bottle

(days)
(cfu/ml) (cfu/ml) (cfu/ml)
0 - - -
3 1.52 1.34 1.04
6 1.64 1.52 1.35
9 1.82 1.74 1.64
12 2.52 1.89 1.82
15 - 2.56 2.55
BasedBase on the table, PP-GBR beverage packed in PET
bottles was acceptable within nine days of storage having
1.52 - 1.82 log cfu/ml. whereas samples in HDPE and glass
bottlesbottle were stable up to 12 days of storage. Microbial
growth is highly dependent on the pH of the sample, yeast and
molds grow in a wider pH range but preferably pH between 5
73
and 6. Hence, the increasing inhibition of yeast and molds
was due to the continual decrease in pH of the sample during
storage (Fig. 9). Therefore, based on these results HPDE and
glass bottlesbottle could provide prolonged protection from
microbial inhibition for the PP-GBR beverage.
Real time Shelf life
The storage life of PP – GBR beverage in PET bottles was
nine (9) days and 12 days in HDPE and glass bottles at
refrigeration temperature (4ºC). The indicators of product
deterioration include presence of sour taste or off-flavor
(sensory attribute), off-standard physico-chemical
properties and viable count of yeast and molds.
BasedBase on the data and results, the shelf-life PP-
GBR beverage was shorter than of the results from the previous
studies of pigeon-pea milk (Bardon, A. 2018 & Jumawan, E.
2019) which was 14 days and 18 days respectively. This may be
due to inadequate pasteurization (ineffective time -
temperature combination) that is not efficient enough to
inactivate food spoilage and pathogenic microorganisms. Aside
from eliminating microorganisms, pasteurization improved the
74
stabilization of suspended particles that produced a more
stable product (Munekata, et al, 2020).
Results of the physico-chemical analysis showed
significant differences among the samples from different
packaging materials in terms of pH during storage. Highest
difference was found on the samples from PET bottles. The
same trend of results was also observed in the viscosity
ranging from 2.77 to 1.83 cm3. Changes in Total soluble
solids of all the samples in different packaging materials
dropped from 10.0 to 8.47 °Bx due to the presence of bacteria
which converts sucrose into glucose and fructose by invertase
and finally to organic acids (Naknean, et al, 2010).
Statistical analysis revealed that there is a significant
difference among the treatments during storage time. Percent
Total titratable acidity (%TA) of all the samples was observed
to increase when it completely deteriorated. Based on the
statistical analysis for %TA, significant difference was
determined on the 12th and 15th day of storage.
In terms of microbial load, samples from PET bottles
were acceptable within 9 days of storage (1.82<2.0 log
cfu/ml), samples in both HDPE and glass bottles were stable
within 12 days having 1.89 and 1.82 log cfu/ml respectively.
Microbial food spoilage accompanies changes in the
75
concentration of metabolic substrates and products, producing
discoloration, textural changes, slime formation, and off-
flavor development (Dong Sun Lee, 2009).
SUMMARY AND CONCLUSION
Summary
The study was conducted to process and determine the
shelf life of PP-GBR beverage in different
packaging materials using real time method. 70:30 ratio of
pigeon pea milk to germinated brown rice extract was the
formulation used for shelf life testing. The newly processed
PP-GBR beverage was packaged in 500 ml PET bottle, HDPE and
glass bottles stored at refrigeration temperature (4°C). All
treatments were evaluated through sensory evaluation
(triangle test), physico-chemical analyses such as pH, TSS,
%TA and viscosity and viable count of yeast and mold every
three days interval monitoring for 15 days of storage.
Based on the results, the TSS of the samples in different
packaging materials has significant differencesdifference
76
during the 12thduring 12th and 15th day of storage. TSS on
samples from PET bottles shows rapid decrease (10.0 - 8.47)
that is due to poor protection against viable count of yeast
and molds which is responsible for the conversion of sugar to
organic acids. Statistical analysis revealed significant
differences on the result of pH from the samples in glass and
PET bottles (5.83 to 5.16) at 5% significance level within 15
days of storage. The same trend of results was found in the
viscosity from PET bottles which drops from 2.77 + 0.05 to
1.83 + 0.05. Evaluation during storage shows that as the pH
decreases; it also decreases the consistency of the beverage.
Significant variations on the Total titratable acidity (%TA)
results in all samples were only observed on the 12th day from
PET bottles, and 15th day on the samples from HDPE and glass,
as the product completely deteriorated.
Statistical analysis using the chi-squareusing chi-
square method revealed that there were no significant
differences on the taste of the samples packed in PET
bottlesbottle within nine days of storage, while samples from
both HDPE and glass bottles remained stable within 12 days
of storage. Viable counts of yeast and molds increase from
1.04 log cfu/ml to 2.55 log cfu/ml during storage. Rapid
77
proliferation of microorganismsmicroorganism was observed on
the samples from PET bottles.
Conclusion
The Pigeon pea - germinated brown rice beverage (PP-GBR)
can be produced by pasteurizing 70% of PP milk
with 30% GBR extract and sugar at 72°C FOR 15 seconds.Based
on the statistical analysis of the sensory evaluation,
physic-chemical, and microbial analyses, it was found that
PP-GBR beverage rapidly deteriorate when packed in PET
bottles.
These rates of chemical reactions are dependent on
several factors which can be controlledcontrol by packaging.
Therefore, packagingpackage plays a major role in prolonging
the storage life of every food product. From the results
gathered on the quality parameters used, it can be concluded
78
that both HDPE and glass bottle can be used and recommended
as the effective packaging for PP-GBR beverage, since it can
preserve the samples within acceptable level for 12 days of
storage at refrigeration temperature (4°C) longer than
compared to the PET bottles where product can only last for
9 days.
Recommendation
Based on the result of the study the following were
recommended;
1. It is recommended to use HDPE and glass bottles as
packaging material for PP-GBR beverage.
2. The recommended shelf life for PP-GBR beverage is 12 days
at refrigeration temperature.
2. Conduct validation using the actual serving size (350 ml)
packaging material for the product.
3. Addition of stabilizer to prevent separation and improve
product stability.
79
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86
APPENDICES
APPENDIX A
Score sheet for PP – GBR Beverage using triangle test
Name of Panelist: Date:

Judge no.:
Sample: Pigeon Pea – Germinated Brown Rice beverage
Instructions: You are presented with three coded samples.

Taste the samples from left to right. Two are identical; and
one is odd. Circle the corresponding code of the odd sample.
Rinse between samples with water.
Sample Code
87
Comments:
Thank you!
APPENDIX B
Master Sheet for Sensory evaluation of PP - GBR Beverage
1 239 541 *428
2 376 971 *324

3 *902 748 612
4 *704 826 107
5 348 *978 128
6 925 *243 343
7 374 *865 762
8 *926 549 891
9 *741 284 943
88
10 384 *152 863
Legend: * = odd sample
APPENDIX C
Raw data on sensory evaluation for PET bottles

Correct Incorrect
Days of evaluation
identification identification
0 1 9
3 2 8
6 3 7
9 5 5
12 10 0
15 10 0
X2 = (01-E1)2 + (02-E2)2
E1 E2
89
Where:
Degree of freedom (df) = 1, since there are only two samples

that are compared
X2 = computed value
3rd Day
X2 = (2-3.33)2 + (8-6.67)2
3.33 6.67
=0.53+0.26
=0.79<3.84
6th Day 9th Day
X2 = (3-3.33)2 + (7-6.67)2 X2 = (5-3.33)2 + (5-6.67)2

3.33 6.67 3.33 6.67
=0.03+0.02 =0.83+0.41
=0.05<3.84 =1.24<3.84
12th Day 15th Day
X2 = (10-3.33)2 +(10-6.67)2 X2 = (10-3.33)2 +(10-6.67)2

3.33 6.67 3.33 6.67
=13.36+1.66 =13.36+1.66
=15.02>3.84 =15.02>3.84
90
APPENDIX D
Raw data on sensory evaluation for HDPE bottles

Correct Incorrect
Days of evaluation
0 1 9
3 1 9
6 2 7
9 4 6
12 5 5
15 10 0
X2 = (01-E1)2 + (02-E2)2
E1 E2
91
Where:

that are compared
X2 = computed value
3rd Day
X2 = (1-3.33)2 + (9-6.67)2
3.33 6.67
=1.63+0.81
=2.44<3.84
6th Day 9th Day
X2 = (2-3.33)2 + (8-6.67)2 X2 = (4-3.33)2 + (6-6.67)2

3.33 6.67 3.33 6.67
=0.53+0.26 =0.13+0.06
=0.79<3.84 =0.19<3.84
12th Day 15th Day
X2 = (5-3.33)2 + (5-6.67)2 X2 = (10-3.33)2 +(10-6.67)2

3.33 6.67 3.33 6.67
=0.83+0.41 =13.36+1.66
=1.24<3.84 =15.02>3.84
92
APPENDIX E
Raw data on sensory evaluation for Glass bottles

Correct Incorrect
Days of evaluation
0 1 9
3 2 8
6 2 8
9 4 6
12 5 5
15 10 0
X2 = (01-E1)2 + (02-E2)2
E1 E2
93
Where:

that are compared
X2 = computed value
3rd Day
X2 = (2-3.33)2 + (8-6.67)2
3.33 6.67
=0.53+0.26
=0.79<3.84
6th Day 9th Day
X2 = (2-3.33)2 + (8-6.67)2 X2 = (4-3.33)2 + (6-6.67)2
3.33 6.67 3.33 6.67
=0.53+0.26 =0.13+0.06
=0.79<3.84 =0.19<3.84
12th Day 15th Day
X2 = (5-3.33)2 + (5-6.67)2 X2 = (10-3.33)2 +(10-6.67)2

3.33 6.67 3.33 6.67
=0.83+0.41 =13.36+1.66
=1.24<3.84 =15.02>3.84
94
APPENDIX F
Statistical Analysis for TSS of PP-GBR beverage during

storage
SUMMARY
Groups Count Sum Average Variance

Column 1 3 30 10 0
Column 2 3 29.63 9.876667 0.001633
Column 3 3 29.37 9.79 0.0112
Column 4 3 28.87 9.623333 0.018533
Column 5 3 27.46 9.153333 0.041633
Column 6 3 26.27 8.756667 0.062533
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 3.471733 5 0.694347 30.73842 1.92E-06 3.105875
Within Groups 0.271067 12 0.022589
Total 3.7428 17
95
Duncan’s New Multiple Range Test
Sx = 0.26
2 3
SSR 3.15 3.29
LSR 0.82 0.86
Mean A B C D E F
8.75 9.15 9.62 9.79 9.87 10
F vs A=10-8.75=.1.25>0.86 significantly difference

F vs B=10-9.15=0.85>0.82 significantly difference
F vs C=10-9.62=0.38<0.82 not significantly difference
F vs D=10-9.79=0.21<0.82 not significantly difference
F vs E=10-9.87=0130.82 not significantly difference
E vs A=9.87-8.75=1.12>0.86 significantly difference
E vs B=9.87-9. 9.15=0.72<0.82 not significantly difference
D vs A=9.79-8.75=1.04>0.86 significantly difference
APPENDIX G
Statistical Analysis for pH of PP-GBR beverage during

storage
SUMMARY
Column 1 3 19.29 6.43 0
Column 2 3 18.65 6.216667 0.005633
Column 3 3 18.09 6.03 0.01
Column 4 3 17.46 5.82 0.0073
Column 5 3 16.72 5.573333 0.130633
Column 6 3 15.75 5.25 0.0793
ANOVA
Between Groups 2.797867 5 0.559573 14.41786 0.000102 3.105875
Within Groups 0.465733 12 0.038811
Total 3.2636 17
96
Sx = 0.077
2 3
SSR 3.15 3.29
LSR 0.24 0.25
Mean A B C D E F
5.25 5.57 5.82 6.03 6.22 6.43
F vs A=6.43-5.25=1. 18>0.25 significantly difference

F vs B=6.43-5.57=0.86>0.24 significantly difference
F vs C=6.43-5.82=0.61<0.24 significantly difference
F vs D=6.43-6.03=0.4>0.24 significantly difference
F vs E=6.43-6.22 =0.21<0.24 not significantly difference
E vs A=6.22 -5.25=1.12>0.25 significantly difference
E vs B=6.22 -5.57=0.65<0.24 not significantly difference
E vs C=6.22 -5.82=0.4>0.24 significantly difference
E vs D=6.22 -6.03=0.19>0.24 significantly difference
D vs A=6.03-5.82=0.21>0.24 not significantly difference
APPENDIX H
Statistical Analysis for %TA of PP-GBR beverage during

storage
SUMMARY
Column 1 3 0.099 0.033 0
Column 2 3 0.113 0.037667 1.63E-05
Column 3 3 0.145 0.048333 1.63E-05
Column 4 3 0.159 0.053 0
Column 5 3 0.257 0.085667 0.001486
Column 6 3 0.45 0.15 0.0004
ANOVA
Between Groups 0.029379 5 0.005876 18.37141 2.98E-05 3.105875
Within Groups 0.003838 12 0.00032
97
Total 0.033217 17
Sx = 0.0103
2 3
SSR 3.15 3.29
LSR 0.32 0.34
Mean A B C D E F
0. 15 0.085 0.053 0.048 0.037 0.33
A vs F=0. 15-0.33=0.117>0.34 significantly difference

A vs E=0. 15-0.037=0.113>0.32significantly difference
A vs D=0. 15-0.048=0.102<0.32significantly difference
A vs C=0. 15-0.053=0.097>0.32 significantly difference
A vs B=0. 15-0.085=0.065<0.32 significantly difference
B vs F=6.22 -0.33=0.05>0.34 significantly difference
B vs E=6.22 -0.037=0.048<0.32 significantly difference
B vs D=6.22 -0.048=0.037>0.32 significantly difference
B vs C=6.22 -0.053=0.032=0.032
C vs F=6.03-0.33=0.02>0.34 not significantly difference
D vs F=6.03-0.33=0.01>0.32 not significantly difference
E vs F=6.03-0.33=0.004>0.32 not significantly difference
APPENDIX I
Statistical Analysis for Viscosity of PP-GBR beverage

SUMMARY
Column 1 3 8.31 2.77 0
Column 2 3 8.13 2.71 0.0012
Column 3 3 7.7 2.566667 0.010033
Column 4 3 7.36 2.453333 0.013433
Column 5 3 6.87 2.29 0.0532
Column 6 3 5.67 1.89 0.0052
ANOVA
Between Groups 1.568267 5 0.313653 22.65554 9.96E-06 3.105875
Within Groups 0.166133 12 0.013844
98
Total 1.7344 17
Sx = 0.096
2 3
SSR 3.15 3.29
LSR 0.30 0.32
Mean A B C D E F
1.89 2.27 2.45 2.57 2.71 2.97
F vs A=2.97 -1.89=1. 08>0.32 significantly difference

F vs B=2.97 -2.27=0.86>0.30significantly difference
F vs C=2.97 -2.45=0.61<0.30significantly difference
F vs D=2.97 -2.57=0.4>0.30significantly difference
F vs E=2.97-2.71=0.21<0.30 not significantly difference
E vs A=2.71-1.89=0.82>0.32significantly difference
E vs B=2.71-2.27=0.44<0. 0.30 not significantly difference
E vs C=2.71-2.45=0.26>0.30significantly difference
D vs A=2.57-2.27=0.3=0.30not significantly difference
APPENDIX J
Computation for Viable counts of yeast and molds
Ave. no. of colony x Dilution factor

cfu/ml =
Vol. of sample
Day 3
(PET) cfu/ml = 1 x 100 (HDPE) cfu/ml = 0.67 x 100

3 3
= log 33.33 = log 22.33
= 1.52 = 1.34
99
(Glass) cfu/ml = 0.33 x 100
3
= log 11.11
= 1.04
Day 6

3 3
= log 33.33 = log 22.33
= 1.64 = 1.52

3
= log 11.11
= 1.35
Day 9

3 3
= log 66.66 = log 55.56
= 1.82 = 1.74

3
= log 44.44
= 1.64
Day 12

3 3
= log 333.33 = log 77.78
100
= 2.52 = 1.89
(Glass) cfu/ml = 3 x 100

3
= log 66.66
= 1.82
Day 15
HDPE) cfu/ml = 11 x 100

3
= log 366.67
= 2.56
(Glass) cfu/ml = 10.67 x 100

3
= log 355.55
= 2.55
101

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ABSTRACT

The study was carried out to assess the influence of

different packaging materials such as PET, HDPE and glass on

the shelf life of PP-GBR beverage stored at 4°C through a

real time method.

Samples were evaluated through sensory evaluation,

physico-chemical and microbial analyses at a three day

interval for 15 days.

Based on the results, the TSS of the samples range from

10.0 + 0.0 to 8.93 + 0.05 ºBx. pH continually decreases from

6.43 + 0.05 to 4.93 + 0.05. The same trend of result for

viscosity was obtained, 2.77 + 0.05 drops to 1.83 + 0.05. %TA

results of all the samples significantly increase when it is

completely deteriorated (0.033 + 0.01 - 0.17 +0.01).

Statistical analysis revealed that there were

significant differences among the samples in terms of

physico-chemical parameters in different packaging materials

Chi-square test revealed that samples packed in HDPE and

glass bottles were not significantly different in terms of

sensory attribute within 12 days of storage while

significantly different results were determined on the

samples from PET bottles. Microbiologically, samples from

(2.0 log cfu/ml) during 9 days of storage, whereas samples in

HDPE and glass bottle were stable for 12 days.

Based on the results of the quality parameters

indicated, it can be concluded that glass bottles could

provide an efficient protection and longer shelf life for PP-

Objectives of the study 2

Significance of the study 3

REVIEW OF RELATED LITERATURE

Plant – based milk alternatives 10

Materials and Methods 44

RESULTS AND DISCUSSION 60

SUMMARY AND CONCLUSION 72

Table Title Page

1 Plant-Based / Non-Dairy Milk Alternatives 10

3 The dietary nutrients of pigeon pea 15

4 γ – Oryzanol content in rice grains 23

5 Shelf – life of different plant-based milk 39

6 Milk Pasteurization Temperature 40

7 Physico – chemical properties of soymilk 41

8 Microbiological limits for soymilk 42

9 Formulation of Pigeon pea – GBR Beverage 49

10 Sampling Plan for shelf life study of 58

12 pH of PP-GBR beverage during storage 62

13 Percent Titratable Acidity (%TA) of

14 Viscosity of PP-GBR beverage during

15 Results of Triangle test for PP-GBR 67

16 Yeast and mold count of PP-GBR beverage in 68

Table Title Page

1 Matured Pigeon peas 14

4 Structure of Phytic acid 25

5 Process flow of Pigeon Pea Extract 46

6 Process flow of Germinated Brown Rice Flour 48

7 Process flow of GBR Extract 49

8 Process flow of Pigeon pea – GBR Beverage 51

9 Newly Process PP-GBR beverage 60

10 Trends of Total Soluble Solids of PP-GBR 61

11 Trends of pH of PP-GBR beverage during 62

12 Trend of Percent Titratable Acidity (%TA)

13 Trend of viscosity of PP-GBR beverage 66

Table Title Page