Tempeh and Vital Wheat Gluten based Analog Meat Development as
Vegetarian Alternative Food
Enrico Stanin, Prita Dhyani Swamilaksita and Erry Yudhya Mulyani
Department of Nutrition, Faculty of Health and Sciences, Universitas Esa Unggul,
Jl. Arjuna Utara No.9, 11510, West Jakarta, Indonesia
Keywords: Analog Meat, Vegetarian, Tempeh, Vital Gluten Wheat, Vitamin B12.
Abstract: Vitamin B12 deficiency is one of the main problems in the vegetarian diet with prevalency from 11% to
90%. Tempeh and vital wheat gluten-based meat analog are expected to provide vitamin B12 for
vegetarians. Completely Randomized Design was used as the experimental design. The organoleptic test
was carried out by 25 semi-trained panelists and 30 consumers. The content of vitamin B12, protein, fat,
carbohydrate, moisture, ash, Total Plate Count, and also production cost was analyzed. Organoleptic test
result showed that F2 has the best acceptability with 6,67 mg/100g of vitamin B12, 9,25% protein, 2,94%
fat, 28,32% carbohydrate, 56,72% moisture. 2,69% ash, and 2,3 x 104 cfu/g in Total Plate Count. The cost
to produce one package of tempeh and vital gluten wheat-based meat analog is 9.171 rupiahs. Therefore, it
is proved that tempeh and vital wheat gluten can produce a meat analog with high vitamin B12 content that
also fulfills the minimum quality requirements in SNI 3818:2014. However, it was necessary to analyze the
storability of the product, also analyze the protein content with the Kjeldahl method and analyze the pseudo
vitamin B12 that might be contained in the product.
1 INTRODUCTION
The vegetarian diet is becoming more popular and
growing, not only globally (Figus, 2014), but also in
Indonesia (Siahaan, Nainggolan, & Lestrina, 2015).
The growing population of vegetarians may cause by
the benefits of the vegetarian diet compared to non-
vegetarian diets, such as lower triglyceride, lower
blood pressure, lower sugar blood level, and also
higher HDL (High-Density Lipoprotein) (Setiyani &
Wirawanni, 2012).
Vegetarian only eat plant-based food that is lack
of vitamin B12, because vitamin B12 only can be
found in animal-based food such as beef, so usually
vegetarian-only get their vitamin B12 intake from a
supplement (Pawlak, Lester, & Babatunde, 2014).
Therefore, vegetarians usually suffer from vitamin
B12 deficiency (Pawlak R., Is Vitamin B12
Deficiency a Risk Factor for Cardiovascular Disease
in Vegetarians? 2015). The prevalence of vitamin
B12 deficiency in vegetarians on every age group is
described as follows, 62% on pregnant women, 25-
86% on children, 21-41% on the teenager, and 11-
90% on elder (Pawlak, Parrot, Cullum-Dugan, &
Lucus, 2013). A study on the vegetarian community
in Jogjakarta, Indonesia also showed there is a
deficiency in vitamin B12, zinc, and folate acid
intake (Anggraini, Lestariana, & Susetyowati, 2015).
Vitamin B12 deficiency can cause megaloblastic
anemia that can increase the chance of premature
birth during pregnancy (Rogne, et al., 2017), and
also skin pigmentation disorder (Rzepka, et al.,
2018).
Early study found that vitamin B12 can be found
in some of the plant-based food, such as fermented
food, tea, mushroom, dried seaweed (nori) and algae
(Rizzo, 2016). Tempeh is an Indonesian soy-based
fermented food that also contained a considerable
amount of vitamin B12 (4,6 mcg/100g) (Okada,
1989). The vitamin B12 in tempeh is produced by
the activity of bacteria K. Pneumoneae (Mo, et al.,
2013). Tempeh is a very popular plant-based protein
source in Indonesia, and maybe can be made into
analog meat. Analog meat is one of the vegetarian
dishes with texture, taste, and color resembling
animal meat (Sedgwick, 2013).
The objective of this research is to develop a
tempeh and vital gluten wheat-based meat analog as
an alternative vegetarian food.
Stanin, E., Swamilaksita, P. and Mulyani, E.
Tempeh and Vital Wheat Gluten based Analog Meat Development as Vegetarian Alternative Food.
DOI: 10.5220/0009591902470256
In Proceedings of the 1st International Conference on Health (ICOH 2019), pages 247-256
ISBN: 978-989-758-454-1
Copyright
c
 2020 by SCITEPRESS – Science and Technology Publications, Lda. All rights reserved
247

2 METHODS
This research used a Completely Randomized
Design as the experimental design, with two
factorials and four levels of treatment which will be
compared to one control product. The main
ingredients in this experiment are tempeh that was
bought from a traditional market in Bekasi city, and
vital gluten wheat from an online shop “Tokopedia”.
Besides the main ingredients, side ingredients such
as mushroom Boullion “Totole”, soy sauce “ABC”,
water, instant yeast “Fermipan”, garlic powder
“Koepoe-koepoe”, and powdered pepper “Ladaku”.
And for the materials used in the chemical analysis
were obtained from the Esa Unggul University
chemistry laboratory.
2.1 Making Tempeh and Vital Gluten
Wheat Based Analog Meat
The guideline in making tempeh and vital gluten
wheat-based analog meat (will be referred to as
product) was based on the experiment by Bintanah
& Handarsari (2014) with modification. The
formulation of the product is shown in Table 1. The
ratio of vital gluten wheat and tempeh was chosen
based on the formulation with the highest
organoleptic score in an experiment by Bintanah &
Handarsari (2014). Unlike the guideline experiment,
the tempeh used in this research was fresh tempeh,
not tempeh flour to avoid vitamin B12 loss because
of excessive heat when making tempeh flour. Rice
bran flour that used in the guideline experiment also
replaced with vital gluten wheat to reach a meat-like
texture. Analog meat “Rodeo” is used as control.
Cookware like Phillips electric stove, steamer pot
set, Phillips food processor, glass bowl, plate, and
measuring spoon was used to make the product.
2.2 Product Organoleptic Test
The product was evaluated in an organoleptic test by
25 semi-trained panelists and 30 consumer panelists.
Color, texture, taste, and aroma of products were
rated using the Visual Analog Scale by semi-trained
panelist and Likert Scale by consumer panelist. The
participant for semi-trained panelists were students
of the Nutrition department in Universitas Esa
Unggul whose already passed organoleptic test
course, in healthy condition, and was asked to feast
(except for plain water) 1 hour before the test
begins. Whilst participants for consumer panelists
were people who have been on a vegetarian diet for
at least 4 months and in good health. The test was
carried out on Universitas Esa Unggul Organoleptic
Laboratory, Bekasi. Tools that are used in the
organoleptic analysis were organoleptic
questionnaire, plate, toothpick, and ballpoint.
2.3 Vitamin B12 Analysis
Analysis of Vitamin B12 levels was carried out using
HPLC (High-Performance Liquid Chromatography)
method by MBRIO Food Laboratory. HPLC with the
specification of UV detector, 265 nm wavelength,
C18 25 cm Eurosphere, 1,0 ml/minute flow speed,
Whatman no.41, and 0,45 mcg millipore shifter, also
with 20 mcg injector volume was used.
2.4 Protein Analysis
In this study, analysis of protein content was carried
out using the formol titration method, the analysis
was carried out in Universitas Esa Unggul
Chemistry Laboratorium. Tools that were used in
this analysis were PYREX® glassware, digital scale
(0,001 g accuracy), burette, Phillips electric stove,
cooking pot, tongs, pipettes, measuring tube, and
measuring glass.
2.5 Fat Analysis
The Soxhlet method was used to analyze the total fat
content of the product. The tools used in this analysis
were fat flask, desiccator, digital scale (0,001 g
accuracy), Soxhlet extraction device set, Memmert
laboratory oven, and tongs. The analysis was carried
out in Universitas Esa Unggul Chemistry Laboratory.
Table 1: Tempeh and vital gluten wheat-based analog meat formulation.
Formula
Vital
gluten
wheat (g)
Tempeh
(g)
Instant
yeast (g)
Mushroom
buillon (g)
Soy
sauce
(ml)
Garlic
powder
(g)
Powdered
pepper
(g)
Water
(ml)
F1 90 10 4 5 10 2 1 40
F2 50 50 4 5 10 2 1 40
F3 40 60 4 5 10 2 1 40
F4 30 70 4 5 10 2 1 40
ICOH 2019 - 1st International Conference on Health
248

2.6 Carbohydrate Analysis
By difference, the method was used to calculate the
carbohydrate total contained in the product. By
calculating the remaining from 100 percent after
deducted by the total of protein, fat, moisture and
ash content that have been analyzed. Carbohydrate
analysis was carried out in Universitas Esa Unggul
Chemistry Laboratory.
2.7 Moisture Analysis
To analyze water content, the gravimetric method is
used. Porcelain cup, digital scale (0,001 g accuracy),
Memmert laboratory oven, and tongs were used in
this analysis. The analysis was carried out in
Universitas Esa Unggul Chemistry Laboratory.
2.8 Ash Analysis
The gravimetric method with dry ashing was used to
analyze the ash content of the product. Ashing cup,
saucer, digital scale (0,001 g accuracy), Barnstead
Thermolyne 1300 furnace, and crucible tongs were
used in this analysis. The analysis was carried out in
Universitas Esa Unggul Chemistry Laboratory.
2.9 Total Plate Count Analysis
Calculation of the total plate count was carried out
using the pour plate method and was done in
Universitas Esa Unggul Microbiology Laboratory.
The tools that were used to analyze total plate count
are measuring glass cup (PYREX®), petri dish, and
dilution bottle.
3 RESULT AND DISCUSSION
3.1 Product Description
At the time of the first trial, tempeh was only
mashed with a food processor without any cooking
process, after which it was mixed with other
ingredients, the result was that the analog meat
product which was already cooked had a bitter
aftertaste and smelled unpleasant. In the next
experiment, the tempeh was cut square with a
thickness of 1 cm, and after that, it was steamed for
20 minutes on medium heat. After that, tempeh is
allowed to stand for 5 minutes at room temperature
to lower the temperature, then put into a food
processor until smooth. After that, tempeh is mixed
with other ingredients according to the product
manufacturing flow. After the product is cooked, the
product no longer has a bitter aftertaste and the
unpleasant aroma has diminished.
The ingredients that are mixed first are instant
yeast, garlic powder, mushroom bullion, and
powdered pepper after that add soy sauce and water.
After homogeneous, add tempeh and mix, finally add
wheat gluten flour then stir and knead until mixed (no
residue in the container). Mixing wheat gluten flour is
done at the end so that tempeh can be mixed with
gluten. Analog meat that has been cooked and cooled
has brown colors, solid, tough and slightly fibrous,
also has a slight aroma of tempeh.
3.2 Organoleptic Analysis
Organoleptic analysis in this research consisted of
two assessment results, a hedonic quality test to
determine the organoleptic characteristics of the
product, and also hedonic test to determine the level
of preference for panelists on products. There were
five product variations tested on organoleptic
analysis, namely F0 / control, F1, F2, F3, and F4.
After organoleptic analysis, the assessment of the
panelists is processed statistically with the One-Way
ANOVA test and Duncan post hoc test.
The result of the hedonic quality test and hedonic
test by semi-trained panelists also hedonic test by
consumer panelist is shown respectively in Table 2,
Table 3, and Table 4.
Table 2: Hedonic quality test score.
Parameter
Formulation
P-value
F0 F1 F2 F3 F4
Color
6,18 ± 2,47 4,30 ± 2,87 4,95 ± 2,69 4,91 ± 2,95 4,60 ± 3,03
0,172
Texture
6,92 ± 2,56
b
7,36 ± 1,71
b
6,49 ± 2,34
b
4,82 ± 2,17
a
4,01 ± 2,35
a
0,001*
Taste
3,96 ± 2,07
a
7,70 ± 1,42
c
d
7,86 ± 1,20
d
6,66 ± 2,25
b
c
6,10 ± 2,53
b
0,001*
Aroma
4,16 ± 2,18
a
5,83 ± 2,63
b
c
6,22 ± 2,19
c
5,47 ± 2,40
abc
4,54 ± 2,49
ab
0,013*
Note:
The data presented are mean ± Standard Deviation (scaled 0-10)
* There is a significant difference in these parameters with a value of P ≤ 0.05
Values followed by different superscript letters have significant differences
Tempeh and Vital Wheat Gluten based Analog Meat Development as Vegetarian Alternative Food
249

Table 3: Hedonic test score by a semi-trained panelist.
Parameter
Formulation
P-value
F0 F1 F2 F3 F4
Colo
r
5
,
08±2
,
20
a
5
,
72±1
,
85
a
6
,
91±1
,
91
b
6
,
07±1
,
89
ab
6
,
16±1
,
67
ab
0,020*
Texture
5
,
03±2
,
12
a
6
,
34±2
,
04
b
7
,
69±1
,
42
c
6
,
47±1
,
97
b
5
,
22±2
,
26
a
0,001*
Taste
2
,
97±2
,
19
a
6
,
59±2
,
17
b
c
7
,
47±1
,
43
c
6
,
68±2
,
27
b
c
5
,
75±2
,
28
b
0,001*
Aroma
3
,
73±2
,
38
a
6
,
17±2
,
41
b
7
,
10±1
,
85
b
6
,
33±2
,
34
b
4
,
74±2
,
06
a
0,001*
Note:
The data presented are mean ± Standard Deviation (scaled 0-10)
* There is a significant difference in these parameters with a value of P ≤ 0.05
Values followed by different superscript letters have significant differences
Table 4: Hedonic test score by consumer panelist.
Parameter
Formulation
P-value
F0 F1 F2 F3 F4
Color
2
,
00
a
3
,
00
b
3
,
00
b
3
,
00
ab
2
,
00
a
0,001*
Texture
3
,
00
b
3
,
00
b
3
,
00
c
3
,
00
b
2
,
00
a
0,001*
Taste
1
,
00
a
3
,
00
b
3
,
00
c
3
,
00
b
2
,
00
a
0,001*
Aroma
2
,
00
a
3
,
00
b
3
,
00
b
3
,
00
b
2
,
00
a
0,013*
Note:
The data presented are mean ± Standard Deviation (scaled 1-4)
* There is a significant difference in these parameters with a value of P ≤ 0.05
Values followed by different superscript letters have a significant difference
3.2.1 Color
In the assessment of color quality, the formula with
the highest mean value is F0 or control formula, this
is in accordance with the results of research by
Dinata (2014) which stated that from the results of
hedonic quality tests conducted on 6 analog meat
formulations, there are similarities in appearance.
When viewed from the mean values in the non-
control formulation (F1, F2, F3, and F4), the color
quality of the product is still not close to the desired
color quality (light brown). But this value is not
significant because of the results of the One-Way
ANOVA test on the color indicator state that the
value of P> α, which means there is no significant
difference in the color indicators between
formulations. This can be caused by the ratio of
tempeh to wheat gluten which is not much different
(Juliana, 2009).
On the other hand, the hedonic test results on the
color parameters show a significant difference (P-
value <α) after being tested statistically. When
referring to Table 3, it is seen that there is a
significant increase in the mean value of the color
parameters from F0 to F2, and there is no significant
difference between F2, F3 and F4. This means that
the increase in color preference is obtained from the
ratio of tempeh and wheat gluten to 50g: 50g, to the
ratio of tempeh and wheat gluten to 70g: 30g, this
can be due to the range of tempeh and wheat gluten
use which does not differ greatly between F2, F3
and F4. Significant increase from F1 to F2 can also
be caused by differences in the ratio of the use of
tempeh and wheat gluten flour which is quite far
(10g: 90g to 50g: 50g). The same is true of hedonic
assessments by consumer panelists, where there was
a significant increase from F0 to F1 and F2. This is
also supported by the research of Bintanah &
Handarsari (2014) which states that panelists rate
very fond of the color indicators of vegetarian
nugget formulations with a ratio of 40g flour: 60g
rice bran. Thus, from the results of the organoleptic
analysis of color parameters, it can be said that an
increase in the use of tempeh with the right ratio will
increase the preference of panelists to analog meat
colors, even though the panelists have not been able
to see the difference in color quality.
3.2.2 Texture
One Way ANOVA test results stated that there were
significant differences in the texture quality between
the five analog meat formulations. The highest
texture quality values are owned by F0, F1, and F2.
It can also be seen that there was a significant
decrease in texture quality from the three best
formulations to F3 and F4. This means that to obtain
a texture quality that is close to tough, we need
higher concentrations of wheat gluten flour
compared to tempeh concentrations. This is
supported by research by Mulyani, Rosida &
Rahmadani (2012) which stated that the less amount
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250

of gluten in vegetarian meatballs, the resulting
texture will be softer.
The hedonic test results by both trained panelists
and consumers showed that there was a significant
difference (P <α value) in the assessment of color
indicators, where the highest mean value was owned
by F2, this concluded that the panelists preferred the
texture of analog meat which tends to be tough as in
texture which is owned by F1 and F2. This is the
same as research conducted by Susanti, et al. (2017)
that panelists most like analogous meatball texture
which is more resilient.
3.2.3 Taste
From the taste quality indicator, there were
significant mean differences between formulas.
Formula with the highest mean is F1 and F2, while
the lowest mean is owned by F0, so it can be said
that F2 tends to have a savory taste and F0 tends to
have an unpleasant taste. If noted in Table 2 on the
taste indicator, there is a significant increase from F0
to F1 to F4, but there is also a significant decrease in
mean from F2 to F3 and F4. This concludes that the
addition of a tempeh use ratio can increase the taste
of analog meat savory, but if the use ratio is more
than 1: 1 with a wheat gluten flour, it will reduce the
savory taste and add the tempeh unpleasant taste in
analog meat. But the low quality of taste F0 is not
caused by the unpleasant taste produced by tempeh,
because in F0 (analog meat brand "rodeo") it does
not use raw materials of tempeh. This can be caused
by the use of mushroom broth in F1, F2, F3, and F3
containing MSG (Monosodium Glutamate) (Tsai,
2007), which can increase the savory taste of food
products (Thariq, Swastawati, & Surti, 2014), so that
F0 products that did not use mushroom broth has a
lower taste quality compared to other formulations.
From the hedonic assessment in Table 3 and
Table 4, it can also be seen that F0 has the lowest
taste preference compared to other formulations.
Judging from the evaluation by the semi-trained
panelists, the best taste acceptance is owned by F1,
F2, and F3, while the panelists of consumers prefer
the taste to F2 only. The difference between the
mean value of F2 with the mean values of F0 and F4
also shows that the ratio of tempeh and wheat gluten
to flour affects the acceptability of analog meat. This
is in accordance with the research of Bintanah &
Handarsari (2014), where there was a decrease in the
taste preference value in vegetarian nuggets in
formulas with the lowest tempeh concentration and
also the highest concentration.
3.2.4 Aroma
It can be seen in Table 2 that in the color indicator
there were significant differences between
formulations. The mean value of the highest aroma
quality is owned by F1, F2, and F3 with a slightly
flavorful characteristic typical of processed meat. A
significant decrease in the mean value between the
highest mean formulation with F4 states that the use
of a tempeh ratio that is too high can reduce the
quality of the aroma becomes unpleasant. Like the
taste quality, the low quality of aroma in F0 is not
caused by the use of a high tempeh ratio and causes
unpleasant aroma, but it can be caused by the fact
that the F0 product does not smell anything.
Then it can be seen also in Table 3 and Table 4
that there were significant differences in the hedonic
taste of analog meat products with the highest mean
owned by F1, F2, and F3. Furthermore, the post-hoc
test results in both assessments also showed that
there was a significant decrease in the formulation
with the highest mean on F0 and F4. So it can be
concluded from the results of hedonic and hedonic
quality assessments on analog meat taste indicators
that the appropriate use ratio of tempeh and wheat
gluten can maximize the quality value and taste of
tempeh aroma, and the ratio of using tempeh that is
too high or too low can reduce the quality and
acceptability aroma indicator of analog meat
products made from tempeh.
3.3 Selected Product
Based on the organoleptic analysis, it was concluded
that the formula with the best organoleptic value was
F2 (hereinafter referred to as the selected product/
formulation) with a ratio of tempeh to wheat gluten as
much as 50g: 50g. When viewed from its quality, the
chosen formulation has a neutral color (not pale or
light brown), a slightly tough texture, a taste that
tends to be savory and had a slightly distinctive aroma
of processed meat. Then in terms of hedonics (by
semi-trained panelists), it has a rather preferred color,
preferred texture, preferred taste, and preferred aroma.
3.3.1 Hedonic Quality
Based on Table 5 it can be concluded that the value
of P (sig) in the color and texture parameters is > α
(0.050), which means there was no difference in the
hedonic color quality values between the selected
product with control and also there is no difference
in the value of hedonic texture quality between
tempeh-based analog meat and wheat gluten flour as
Tempeh and Vital Wheat Gluten based Analog Meat Development as Vegetarian Alternative Food
251

a vegetarian alternative food with control. While the
results of the Independent t-test on the taste and
aroma parameters indicate that the P (sig) value is
smaller than α (0.050). This suggests that there is a
difference in the quality taste and aroma between a
selected product with a control product.
The mean difference column presents the mean
difference between the control product and the
selected product. In the taste parameter, there was a
difference between the control product and the
selected product, where the selected product has a
taste quality value of 3.896 ± 0.478 greater than the
control product. As for the aroma parameters, the
selected product has an aroma quality value of
2.068 ± 0.618 greater than the control product.
3.3.2 Hedonic
In both Table 6 and Table 7, there was a significant
difference between the control product and the
selected product which is marked by the value of P
(sig) <α (0.050) on every parameter (color, texture,
taste, and aroma). It also shows that all parameters
(color, texture, taste, and aroma) have negative mean
difference values, this shows that based on the
evaluation of panelists who are somewhat trained
and panelists of consumers, the selected products
have higher hedonic (preference) values compared
to control products. , each of which can be seen in
the difference in the mean column.
3.4 Nutrition Value
Nutritional analysis was carried out on selected
formulation with a ratio of tempeh to wheat gluten
50g: 50g. After the analysis of nutrients is carried
out, the results of the analysis of nutrients will be
compared with the control product (F0). Data on the
nutrient content of control products is obtained from
the nutritional value information table contained
behind the product packaging. The result of the
nutritional analysis and control product’s nutrition
value showed in Table 8.
Table 5: Independent t-test results in a hedonic quality score.
Parameter
Formulation
t df Sig (2-tailed) Mean difference
Color 1,693 48 0,097 1,236±0,730
Texture 0,618 48 0,540 0,428±0,693
Flavour -8,143 48 0,001* -3,896±0,478
Aroma -3,347 48 0,002* -2,068±0,618
Note:
* There is a significant difference in these parameters with a value of P ≤ 0.05
Mean difference column showed the mean difference between F0 to the selected formulation
Table 6: Independent t-test results in a hedonic score by a semi-trained panelist.
Parameter
Formulation
t df Sig (2-tailed) Mean difference
Colo
r
-3,131 48 0,003* -1,824±0,583
Texture -5,224 48 0,001* -2,664±0,510
Flavour -8,609 48 0,001* -4,500±0,523
Aroma -5,572 48 0,001* -3,360±0,603
Note:
* There is a significant difference in these parameters with a value of P ≤ 0.05
Mean difference column showed the mean difference between F0 to the selected formulation
Table 7: Independent t-test results in a hedonic score by consumer panelists.
Parameter
Formulation
t df Sig (2-tailed) Mean difference
Color -2,873 48 0,006* -0,600±0,209
Texture -3,319 48 0,002* -0,567±0,171
Flavour -10,518 48 0,001* -1,833±0,174
Aroma -3,479 48 0,001* -0,633±0,182
Note:
* There is a significant difference in these parameters with a value of P ≤ 0.05
Mean difference column showed the mean difference between F0 to the selected formulation
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252

Table 8: Nutrition value of selected product and control.
Parameter F0* Selected product SNI**
Vitamin B12 (mg/100g) - 6,68 -
Protein (%) 67 9,25 Min 8,0
Fat (%) 5,2 2,94 Max 10
Carbohydrate (%) 17 28,32 -
Moisture (%) - 56,72 Max 70,0
Ash (%) - 2,69 Max 3
Total Plate Count (cfu/g) -
2
,
3 x 10
4
Max 1 x 10
5
Note:
*Nutritional value table of analog meat “Rodeo”
**SNI 3818:2014
3.4.1 Vitamin B12
From the analysis of vitamin B12 in selected
products, it was found that the selected product
contained 6.67 mg / 100g of vitamin B12 or 6670
mcg / 100g of product. This amount is very high
when compared to the vitamin B12 content in
tempeh found by Yuniati & Almasyhuri (1989),
which is as much as 1.8 mcg / 100g when analyzed
by the Microbiological Assay method. When
compared with the vitamin B12 content in beef (3.17
mcg / 100g) (Bennink & Ono, 1982), as well as beef
liver (41.3 mcg / 100g) (Yuniati & Almasyhuri,
1989), analog meat products based on tempeh and
wheat gluten flour also still contains vitamin B12
which is much higher.
The production of vitamin B12 in tempeh is
caused by the bacteria K. Pneumoneae during the
fermentation process, not because of mold/yeast
(Areekul, et al., 1990), so it can be ascertained that
high levels of vitamin B12 in selected products are
not due to the use of instant yeast, This is also
supported by the results of Kustyawati's research
(2009), which states that the addition of yeast does
not play a role in the formation of vitamin B12 in
tempeh. Research conducted by Bennink & Ono
(1982) also states that the amount of vitamin B12 in
beef before and after cooking does not have a
significant difference, in fact, there should be a
possibility of a decrease of 27-33% after the beef is
cooked. A temporary assumption that researchers
can give is that tempeh used to make products
already contain high vitamin B12, which can be
caused by the high activity of the K. pneumoneae
bacteria as stated by Areekol, et al., (1990). Another
conjecture that led to the high analysis of vitamin
B12 in selected products is the presence of
pseudovitamin B12. Pseudovitamin B12 is a form of
biologically inactive vitamin B12, which can be
taken into account when analyzing vitamin B12 in
protein sources using the UHPLC (Ultra-High-
Performance Liquid Chromatography) method
(Schmidt, Call, Macheiner, & Mayer, 2019).
Therefore, it is necessary to conduct an analysis of
vitamin levels with other methods to prevent
overestimation of vitamin B12 content in analog
meat products based on tempeh and wheat gluten.
But it should also be noted that pseudovitamin B12
can increase the absorption of vitamin B12 by
intrinsic intestinal factors (Toporek, 1960).
3.4.2 Protein
Even though it meets the requirements of SNI
3818:2014, the results of the analysis of protein
content in selected products are very low, even
though the product uses the main ingredients of
protein sources, namely tempeh and gluten. This can
be seen when comparing protein levels in selected
products with "rodeo" control products. The low
level of protein analysis results in analog meat based
on tempeh and wheat gluten flour is caused by the
use of formol titration method which is not suitable
for analyzing protein content in solid and chewy
foodstuffs such as as analog meat. This can be
proven from the results of Mukhoyaroh's research
(2015) where the results of the analysis of protein
content in tempeh made with several types of
soybean only ranged between 0.01-0.72%, whereas
the protein content of tempeh according to the
Indonesian Food Composition Table (2017) is as
much as 20 8%. In contrast, the results of the
analysis of protein content by the Kjeldahl method
on vegetarian meatballs based on gluten and soy
flour are in the range of 55,634-71,596% (Mulyani,
Rosida, & Rahmadani, 2012). The process of
heating (cooking) does not cause a decrease in
protein levels, because according to research
Sundari, et al., (2015), an increase in protein levels
before and after boiling as much as 1- 3.2%.
Therefore, it can be concluded that a more suitable
method for analyzing protein content from analog
Tempeh and Vital Wheat Gluten based Analog Meat Development as Vegetarian Alternative Food
253

meat products based on tempeh and wheat gluten
flour is the Kjeldahl method, not the formol titration
method.
3.4.3 Fat
Besides fulfilling SNI quality requirements, the fat
content of selected products is lower when compared
to "rodeo" control products. This is caused by the
use of soy flour which has higher fat content
compared to tempeh (U.S. Department of
Agriculture, 2019). When compared with animal
protein sources such as beef (low fat), the selected
product has a much lower fat content (KEMENKES
RI, 2017). Besides that, tempeh contains 50,12% w /
w of linoleic acid (omega-6) unsaturated fatty acids
from the total unsaturated fatty acid content of 80%
w / w tempeh (Utari, 2010). Therefore, it can be
assumed that the fatty acids contained in selected
products are unsaturated fatty acids.
3.4.4 Carbohydrate
In SNI 3818: 2014, quality requirements for
carbohydrate content for mixed meatball products
are not regulated. But when compared to "rodeo"
control products, the selected products have higher
carbohydrate content, this is because the
carbohydrate analysis method used in this study is
by difference. The results of carbohydrate content by
the by difference method are obtained by calculating
the remaining 100% after subtracting the results of
an analysis of protein content and fat content.
Therefore, the results of the analysis of protein and
fat levels will affect the results of the analysis of
carbohydrate levels. Based on these principles and
the discussion of protein content analysis, it can be
concluded that the results of carbohydrate content
will also be affected by the method used to analyze
protein content in the product. By looking at data at
the United States Ministry of Agriculture's Food
Data Center (2019), it appears that tempeh (7.64g)
carbohydrate content is lower than soybean flour
(31.92g), thus supporting the assumption that
carbohydrate content of selected products should be
lower than with control products. This is because the
method used to analyze protein content is the formol
titration method, not the Kjeldahl method. In
addition, soy flour also has starch levels high enough
to cause high carbohydrate levels (Mulyani, Rosida,
& Rahmadani, 2012). In addition to the by
difference method, carbohydrate levels can also be
analyzed by chromatographic methods such as
HPLC or GC (Gas Chromatography) (BeMiller,
2017).
3.4.5 Moisture
In food products, water content affects the quality of
the product because it affects the acceptability,
freshness, and storability of the product (Utama &
Anjani, 2016). Therefore the "rodeo" control product
is sold in dry form to increase its shelf life. Then
when compared with the quality requirements of
SNI 3818: 2014, the water content of selected
products still meets the requirements. In addition,
the moisture content of the selected product
(56.72%) is also not much different from vegetarian
meatball products made from gluten and soy flour
(53.813%) made in the research of Mulyani, Rosida,
& Rahmadani, (2012). The amount of water content
in selected products is also caused by the absorption
of wheat gluten flour which can absorb 1.3-1.5 times
the amount (Mühlenchemie, 2006). High water
content in selected products can affect the shelf life
of the product, this can be seen from the results of
observations by researchers when the product is
stored in a chiller for 3 days, white hyphae appear,
but the appearance of hyphae (white fungus tissue in
tempeh) does not change the aroma product.
3.4.6 Ash
What is meant by ash content in food is inorganic
residues (such as minerals) that are left after the
elimination of organic substances either by oxidation
or combustion (Ismail, 2017). From the analysis of
ash content, it can be seen that the selected product
has an ash content of 2.69% which still meets the
SNI 3818: 2014 quality requirements, which is a
maximum of 3%. In a similar study, namely,
vegetarian sausage made from tempeh and oyster
mushroom, the results of the analysis of ash content
were not much different from the selected product,
which was 2.26% (Ambari, Anwar, & Damayanthi,
2014). The process of self-healing, especially
boiling can reduce ash content in tempeh by 0.54%.
In addition to minerals, heavy metal contamination
also includes inorganic compounds that can be
calculated in the analysis of ash content, so
sometimes it can be said that ash content also
reflects the amount of heavy metal contamination in
food (Halagarda, Kędzior, & Pyrzyńska, 2017).
3.4.7 Total Plate Count
The results of the analysis of the total plate count
can describe the contamination of a food product.
From the analysis of total plate numbers in selected
products, it can be said that analog meat products
made from tempeh and wheat gluten flour are safe to
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254

consume because they still meet SNI 3818: 2014
requirements. One way to prevent/slow the increase
in the number of total plates is by giving an edible
coating layer that can be done by utilizing algae
Caulerpa sp. (Mailoa, Tapotubun, & Marutty, 2017).
In addition, the drying process also affects the total
plate count (Ruga, 2011).
4 CONCLUSIONS
From the results of hedonic quality tests and hedonic
tests that have been carried out, the best formulation
in F2 is most preferred by panelists and has the best
color, texture, taste and aroma quality compared to
other formulations. The selected product has a
vitamin B12 content of 6.67 mg / 100g, 9.25%
protein, 2.94% fat, carbohydrate 28.32%, water
56.72%, ash 2.69%, the total plate count is 2.3 x
104. It is proved that tempeh and vital wheat gluten
can produce a meat analog with high vitamin B12
content that also fulfills the minimum quality
requirements in SNI 3818:2014.
In future research, we suggest to analyze the
vitamin B12 levels using Microbiological Assay
method and identify the content of Pseudovitamin
B12 in selected products. Also, protein analysis
should be using the Kjeldahl method or quantitative
analysis for carbohydrate content analysis. We also
suggest to Conduct an analysis to see the product
storage time, factors that influence and also changes
related to nutrients that occur. And lastly to
intervene by giving products to increase plasma
vitamin B12.
ACKNOWLEDGMENTS
The author declared that there is no conflict of
interest.
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