A Proposal on

Preparation And Quality Evaluation of Sorghum Jand by Using Defined

Fermentation Starter Under Solid and Semi-solid State Fermentation

Submitted By
SAURAV SHRESTHA
B. Tech (Food) 4th Year
02/ 069-73

Supervisor: Prof. Dr. Dhan Br. Karki

Submitted To:
DEPARTMENT OF FOOD TECHNOLOGY
CENTRAL CAMPUS OF TECHNOLOGY
INSTITUTE OF SCIENCE AND TECHNOLOGY
TRIBHUVAN UNIVERSITY
HATTISAR, DHARAN
2016
 Table Of Contents
1.Introduction .............................................................................................................................. 1
1.1 Background ........................................................................................................................ 1
1.2Statement of the problem .................................................................................................... 2
1.3 Objectives .......................................................................................................................... 3
1.3.1 General objective ......................................................................................................... 3
1.3.2 Specific objectives ....................................................................................................... 3
1.4 Significance of the study .................................................................................................... 3
1.5 Limitation of work ............................................................................................................. 4
2.Materials and Methods .......................................................................................................... 5
2.1 Materials Required ............................................................................................................. 5
2.1.1 Glass wares and equipments ........................................................................................ 5
2.1.2 Chemicals Required ..................................................................................................... 5
2.2 Methods Required .............................................................................................................. 5
2.2.1 Materials ...................................................................................................................... 5
2.2.2 Fermentation of Sorghum ............................................................................................ 5
2.2.3 Preparation of sorghum Jand ....................................................................................... 6
2.3 Analytical procedure .......................................................................................................... 6
2.3.1 Total Soluble Solid ...................................................................................................... 6
2.3.2 Alcohol Content ........................................................................................................... 6
2.3.3 Acidity ......................................................................................................................... 6
2.3.4 Methanol Content ........................................................................................................ 7
2.3.5 Aldehyde Content ........................................................................................................ 7
2.3.6 Ester Content ............................................................................................................... 7
2.4 Sensory evaluation ............................................................................................................. 7
2.5 Statistical analysis .............................................................................................................. 7
2.6 General method of preparation .......................................................................................... 8
3. Work plan of the study .......................................................................................................... 9
4.References…………………………………………………………………………………..11

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 Part I
Introduction

1.1 Background

Food fermentation is regarded as one of the oldest methods of food processing and
preservation. More than anything else, man has known the use of microbes for the preparation
of food products for thousands of years and all over the world a wide range of fermented foods
and beverages contributed significantly to the diets of many people (Achi, 2005). Alcoholic
beverages have played an important role in human spiritual and cultural life both in Eastern
and Western societies. Unlike in Europe and Middle East, where indigenous alcoholic
beverages are produced primarily from fruit, alcoholic beverages in the Asia-Pacific region are
produced from cereals and serve as an important source of nutrients. Beverages vary from
crystal clear (e.g. Sake) to turbid thick products e.g. Takju in Korea (Yoon, 1993). Indigenous
alcoholic beverages not only add nutrients to the local diet, but also play an important part in
the local custom. Traditional alcoholic beverages are offered to perform the religious practice
to pray family God.

Jand is a Nepalese indigenous fermented beverage prepared by solid-state fermentation of

starchy materials, Kodo using Murcha (a traditional fermentation starter). It is one of the
socially and culturally accepted mild alcoholic beverages and is presumably nutritionally
superior to other alcoholic beverages, although its exact nutritional status is still unexplored.
The term Murcha is a Nepali word and the different ethnic communities of the region call it by
their own dialect such as Khesung by Limbu, Bharama by Tamang, Bopkha by Rai and
Buth/Thanbum by Lepcha (Karki and Kharel, 2007).

Sorghum is considered the fifth most important cereal crop in the world behind wheat, rice,
maize, and barley (ICRISAT, 1996). Sorghum is Africa’s second most important cereal in
terms of both area harvested and annual production. According to the latest global statistics
(FAO 2008), Africa contributed over 60% to the total land area dedicated to cultivation of
sorghum. Starch from sorghum has practically the same properties as the starch from corn, and
the two are used almost interchangeably. Hence, sorghum can be a good alternative for the
production of starch (Marshall, 1969).

1.2 Statement of the problem

Several traditional food fermentations e.g. Nigeriam Ogi, Japanese Sake, have been
upgraded to high technology production system because of the strong research on traditional
fermentation technology. But the production of indigenous fermented foods is still largely a
traditional family art done in homes in a crude manner consequently the production has not
increased substantially more than a cottage industry (Odufa, 1985). Although the technology
of Jand making has been practiced since antiquity, its production is still limited to home scale
in Nepal. It has many shortcomings as a result of which the final product quality is the mercy
of the Murcha and environmental conditions used during processing. It has a great potential of
being commercialized, both in domestic and foreign markets, provided its quality (safety and
nutritional value) is enhanced, which calls for a sound research and development work. Much
literature is available on the traditional alcoholic fermentation of starchy raw materials under
solid-state conditions using both traditional as well as defined fermentation starters (starters
prepared by using pure microbial cultures isolated from traditional starters (Subba, 1985;
Verma, 1991) but information regarding semi-solid fermentation is scanty (Cai and Nip, 1990;
Dung et al., 2007).

Furthermore, the inferior nutritional quality and low palatability of sorghum has
discouraged the farmers to cultivate sorghum (Salunkhe, 1984). The inaccessibility of
amylolytic enzymes to starch and presence of tannins and trypsin inhibitors have made them
more difficult to digest. According to FAO (1995), the use of sorghum for feed has been the
driving force behind increasing its global production and trade.

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1.3 Objectives

1.3.1 General objective

The general objective of my dissertation work is to investigate the effect of semi-solid

fermentation on the quality of sorghum Jand using defined fermentation starter prepared from
R. oryzae and S. cerevisiae and to investigate the potentiality of the domestic use of sorghum
organoleptically and with nutritional aspect.

1.3.2 Specific objectives

 Isolation of yeasts and molds from murcha.

 Selection of mold based on their saccharification activity.
 Preparation of koji from selected molds.
 Preparation of murcha from pure culture.
 Production of jand from sorghum (Sorghum bicolor L.Moench).
 The quality evaluation of produced jand.

1.4 Significance of the study

In Nepal, all traditional cereal-based alcoholic beverages are produced under solid-state
fermentation and, hence this work will be undertaken aimed at investigating the effect of semi-
solid fermentation on the quality of sorghum Jand using defined fermentation starter prepared
from R. oryzae and S. cerevisiae. Furthermore, the work may be fruitful in value addition of
sorghum grains and encourage in its cultivation.

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1.5 Limitation of work

 Only a single mold will be isolated.

 The isolated organism will not be studied in detail.
 Optimization of fermentation parameters like temperature and pH will not be studied.
 Shelf life of the product will not be studied.
 Effect of fermentation containers on variation will not be studied.

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 Part II

Materials and Methods

2.1 Materials Required

Raw materials: Sorghum of sweet variety, fresh murcha, molasses, wheat bran, rice flour

2.1.1 Glass wares and equipments

Tray for solid state fermentation, incubator, autoclave, petri-plates, beakers, flame, glass and
plastic containers, muslin cloth

2.1.2 Chemicals Required

Molasses, MYGP agar, 10% citric acid solution, sodium hydroxide

2.2 Methods Required

2.2.1 Materials

Sorghum (sweet variety) will be collected from local market of Dhankuta. Fermentation
starter will be prepared using yeast (Saccharomyces cerevisiae) and mold (Rhizopus oryzae) to
be isolated from traditional starter (Murcha) in MYGP agar. Yeast will be grown in molasses
broth of 6ºBx TSS for 3 days at 27 ºC and will be kept in refrigerator until use. Mold bran will
be prepared by inoculating isolated mold in sterile wheat bran, incubating for 5 days at 30 ºC,
drying at 24 ºC for 12hr and then will be packed in sterile glass bottle. Yeast (from molasses
broth) and mold (from mold bran) will be added to sterile rice flour, thoroughly mixed with
the addition of required amount of sterile distilled water and the dough will be divided into
small balls. The balls will be flattened, will be placed in a sterile petri plate, incubating at 30ºC
for 3 days and drying at 40 ºC for 24hr.

2.2.2 Fermentation of Sorghum

Sorghum (white variety) will be cleaned, dehusked, winnowed and will be washed with
water. The sorghum will be steeped in water for two hours, will be washed again, will be

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cooked for 20min at 121 ºC, and will be cooled to room temperature. Defined fermentation
starter (prepared from rice using pure cultures of S. cerevisiae and R. oryzae)will be mixed to
the cooked sorghum at the rate of 1% (m/m), and will be kept for 2 days at 30ºC for bio-mass
development (aerobic fermentation). For solid-state fermentation, the biomass developed
sorghum will be tightly packed into plastic containers whereas for semi-solid state
fermentations, sorghum will be filled into plastic containers and previously boiled and cooled
water will be added to the containers at the rate of 50% and 100% (v/m) for semi-solid1 and
semi-solid2 fermentations respectively. All containers will be then capped tightly and will be
kept at 28±1 ºC for 15 days for alcoholic fermentation.

2.2.3 Preparation of sorghum Jand

For the preparation of sorghum Jand, 1.5 parts (by vol) of previously boiled and cooled
water will be added to each part (by wt) of biomass developed sorghum used for alcoholic
fermentation. In the case of semi-solid fermented millet, the volume of water added previously
during the start of alcoholic fermentations will be subtracted from that of total volume of water
to be added. The mixture will be gently macerated, will be allowed to stand for 20 min and
will be strained through muslin cloth with gentle pressing. The Jand (millet beers) so obtained
will be subjected to chemical and organoleptic analyses.

2.3 Analytical procedure

2.3.1 Total Soluble Solid

Soluble solids will be determined (°Brix) in an Abbé refractometer according to (AOAC,
2000) with diluted sample 1:5 (w:v).

2.3.2 Alcohol Content

Alcohol content of the samples will be determined as per the method described in
(Pearson,1981) and the values will be expressed in percentage (v/v).

2.3.3 Acidity
The acidity will be determined by neutralization with NaOH 0.1 N, and the results will be
expressed in citric acid percentage (AOAC, 2000).

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2.3.4 Methanol Content
Methanol contents of the sample will be determined as per the method described by Martin
(1979) and the values will be expressed in gram per litre.

2.3.5 Aldehyde Content

Aldehyde content will be determined as per the method described in Nepal standard for
country spirits (1984) and the value will be expressed in milligram per litre as acetaldehyde.

2.3.6 Ester Content

Aldehyde content will be determined as per the method described in Nepal standard for
country spirits (1984) and the value will be expressed in milligram per litre as ethyl acetate.

2.4 Sensory evaluation

Sensory evaluation of the sorghum brew (Jand) will be performed using Hedonic Rating Test
as per Ranganna (1986).

2.5 Statistical analysis

Genstat Release 12.1 edition will be used as statistical software for the calculation of data.The
means of chemical parameters, triplicate and sensory will be compared using least significant
difference (LSD,5%)

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2.6 General method of preparation
Traditional Murcha

Sorghum collection Isolation and identification of isolated mold in sterile

wheat bran

Cleaning/dehusking/winnowing
Yeast propagation in
Innoculation of
molasses broth of 6ºBrix
Washing with water isolated mold in
TSS for 3 days at 27ºC
sterile wheat bran

Steeping in water for 2 hrs.

Incubate for 5
Keeping in refrigerator till
days at 30 ºC
Coking for 20 min. at 21 ºC use

Drying at 24 ºC for
Cooled to room temperature 12 hrs.

Keeping at 30ºC for 2 days

Packed in sterile
(aerobic fermentation)
glass bottle

Sterile rice flour

Solid state Semi-solid state
Fermentation fermentation Starter culture in
the form of cake

Incubate at 30ºC for 3 days

Keeping at 28ºC for 15 days for alcohol
Fermentation Drying at 40ºC for 24 hr

Jand preparation

Chemical and organoleptic analysis

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3. Work plan of the study

Day-1

 Spot culturing of murcha grains in MYGP agar and keeping in incubator for 3 days
 Maintain pH and TSS of molasses to grow yeast by murcha powder for 3 days

Day-2 and Day-3: -

Day-4

 Screening and isolation of mold for 4 days in MYGP agar until dense colonies with
profuse spores to be seen.
 Spread plating on 5-6 MYGP plates for isolation of yeast for 2 days.
 Performing liquefaction test for 10 days.

Day-5: -

Day-6 : Sub-culturing of isolated yeast and incubation for 2 days.

Day-7 : -

Day-8
 Preparation of mold bran by inoculating isolated mold in sterile wheat bran for 5 days.
 Negative staining for identification of fermentative (non-oxidative) yeast.
 Propagation of yeast in molasses in controlled environment for 5 days.

Day-9,10,11,12 : Sample collection (Sorghum of sweet variety) and to be tested in future

Day-13

Yeast and mold bran to be added to sterile rice flour and dough to be divided into small balls
and flattened with incubation for 3 days.

Day-14 and 15 : Proximate analysis of sorghum grains

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Day-16

 Drying of balls upto 12 hrs.

 Priliminary treatment and steeping of sorghum in water for 2 hrs and cooking for 20
mins.
 Mixing of fermentation starter
 2 days of aerobic fermentation
 Incubation for 15 days for alcoholic fermentation

Day 17 to 32 : Proximate analysis of sorghum grains.

Day 33-37 : Chemical and sensory analysis of the jand.

From Day -38 to further 2 months is estimated for report writing.

Duration of the study : November 20, 2016 to February 2 , 2017

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4. References
Achi O.K. (2005). The potential for upgrading traditional fermented food through
biotechnology. African J Biotenhnol., 4(5): 375-380.

Yoon S. S. (1993). ChenMin Yo Sul: A Translation of Chi-Min-Yao-Shu in Korea.

MinEumSa, Seoul, Korea. In:Fermented Cereals: A Global Perspective.

FAO Agricultural Services Bulletin No. 38. Food and Agriculture Organization of the United
Nations, Rome(1999), pp 74.

Karki D. B. and Kharel G. P. (2007). Fermentation starters used in the preparation of

traditional cereal based alcoholic beverages. Foodwave, NEFSTA 4: 7-8.

ICRISAT (International Crop Research Institute for Semi-Arid Tropics) (1996). The world
sorghum and millets: Facts and, trends and outlook. Patancheru, India, pp-1-25.
FAO (2008). FAOSTAT. Available at: http://faostat.fao.org [Accessed on 21 September,
2016].
Marshall, J. T. (1969). Development of a simplified process for obtaining starch from grain
sorghum. Thesis submitted to Graduate Faculty, Texas Technical College.USA.
Odufa S. A. (1985). African fermented foods. In: Wood, B.J.B (Ed) Microbiology of
Fermented Foods, Vol.2. Elsevier Appl. Sci. Publ., London, pp 155-191.

Verma S. (1991). Study on the preparation and efficacy of starter cake prepared from A.
oryzae and S. Sake using rice as binder. B. Tech (Food) Dissertation, Central
Campus of Technology, T.U., Nepal.

Subba C. (1985). Raksi production from finger millet (Kodo) by traditional method. B. Tech
(Food) Dissertation,Central Campus of Technolo, T.U., Nepal.

Dung N. T. P., Rombouts F. M. and Nout M. J. R. (2007).Characteristics of some traditional

Vietnamese starchbased rice wine traditional fermentation starter (Meu).LWT, 40:
130-135.

Cai T. and Nip W-K. (1990). Biochemical changes in the development of alcoholic

Salunkhe, D.K. (1984). Sorghum Grain Quality: Problems and prospects. In “Nutrional and
processing quality of sorghum” (D.K. Salunkhe, J.K. Chavan and S.J. Jadhav,
eds),pp. 1-20. Oxford and IBH Publishing Co., India.

Ranganna S. (1986). Handbook of Analysis and Quality Control of Fruits and Vegetable
Products, 2nd Edn. Tata McGraw-Hill Publishing Company, Ltd., New
Delhi,India.

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