Title of Invention

"AN IMPROVED PROCESS FOR THE PREPARATION OF INOCULUM FOR FERMENTED FOODS"

Abstract This invention relates to an improved process for the preparation of inoculum for fermented foods in ready to use form. The potent microbial cultures selected in the preparation of inoculum for food fermentations are Lactobacillus brevis CFR 2134, Pediococcus pentosaceus CFR 2123 and Candida versatilis CFR 505. Cultural of microbial cultures are grown individually in rice semolina/grits with supplementation of moisture and desirable nutrients, followed by heating, addition of culture inoculum, mixing, spreading, incubation and removal of moisture. The preparation of ready-to-use inoculum is achieved by removing desirable amount of moisture from the substrate using rice flour at a level of 25 to 35% (w/w). The final ready-to-use product is a free flowing high volume with a high stability at low temperature storage.
Full Text The present invention relates to an improved process for the preparation of inoculum for fermented foods.
In the changing global situation, emphasis has been more on plant materials as human foods, hence interest in traditional fermentation technology for food processing has greatly increased. A majority of the indigenous foods of traditional nature are mainly cereal/legume-based and invariably undergo a certain degree of fermentation at some stage of the processing schedule, leading to the development of desirable attributes. Majority of these foods are fermented by desirable microorganisms like lactic acid bacteria and yeasts (starter cultures). An important role of these microbial starters is their ability to increase palatability, keeping quality, safety and nutritive value of the ingredients. These cultures act like growth promoters for a desirable and quality consistent food fermentations.
Reference may be made to the work of Varadaraj et al. (1999), wherein an improved process for the preparation of shelf stable idli batter is described using potent microbial cultures, Lactobacillus brevis CFR 2134, Pediococcus pentosaceus CFR 2123 and Candida versatilis CFR 505 either individually or in combinations (Pending Indian patent filed, 1129/DEL/99).
The production of active dried starter cultures is a key step for effective food fermentations. The drying of starter cultures has been studied by many researchers. The starter cultures can be dried in many ways like fluidized-bed drying, freeze drying, spray drying and oven drying. Reference may be made to the studies of Speckman (1975), Sawad (1989), Kels and Bont (1994), Kim and
Yoon (1995), Teixeira et al. 1995), To and Etzel (1997) and Linders et al. (1998) on the drying of lactic cultures.
Reference may be made to the work of Wan and Etzel (1995), wherein spray dried cultures are found to be most unstable even at refrigeration temperatures. A loss in viability of 86% is noticed after 3 months storage at 4°C in spray dried lactic cultures.
Reference may also be made to the work of Bannikova (1958), wherein a method is described for the production of dried starter by spray drying at 48-50°C with the addition of starch. As against the oven dried (45-50°C) cultures, the viability of spray dried cultures is superior (Sohweigart, 1971).
Reference may be made to the study of Hylman and Teply (1970), wherein newer technique of drying microbial cultures including yeasts, molds and bacteria for use in cheese making is described using fluidized-bed drier after absorption and concentration in as curd mass.
Reference may be made to the work of Sohweigart (1971), wherein freeze drying of bacterial or yeast cultures are achieved. The cultures are neutralized and frozen for 4 h at -30°C. Sublimation (removal of water from frozen mass) is carried out at 0.2 mm Hg in trays at +30°C for 20-24 hours.
The drawback of all these methods is the loss of viability of cultures either during the drying process or during storage. Besides, these drying methods involve expensive equipments and cumbersome operating procedures.
The main object of the present invention is to provide an improved process for the preparation of inoculum for fermented foods which obviates the drawbacks detailed above.
Another object of the present invention is to use an easily available substrate also used as a raw material of fermented foods for growing the microbial cultures.
Still another object of the present invention is to optimize growth conditions to achieve high cell populations.
Yet another object of the present invention is to use simpler method for preparing ready-to-use inoculum.
Still another object of the present invention is to condition the ready-to-use inoculum using non-heating approach.
Yet another object of the present invention is to preserve the ready-to-use inoculum in a viable state.
Accordingly, the present invention provides an improved process for the preparation of inoculum for fermented foods which comprises:
(a) steaming of a growth substrate selected from a cereal source such as rice semolina for a period of 20 - 50 min,
(b) enriching the selected growth substrate with an acidulant selected from citric
acid and tartaric acid at a level of 0.04-0.06 % and mineral nutrients: 1-2 % glucose : 0.005-0.007 % magnesium sulphate , and 0.011-0.013% manganese sulphate, and 58-62% water, (c) sterilizing of the enriched substrate obtained from the above at 85 - 100°C for 20 - 40 min,
(d) adding a microbial culture from the group comprising Lactobacillus brevis CFR 2134, Pediococcus pentosaceus CFR 2123 and Candida versatilis CFR 505, individually at a cell density of 2 x 108 colony forming units per gram (cfu/g), such as herein described,
(e) incubating of the enriched substrates for a period of 48 - 96 hours at a temperature of 25 - 45°C under sterile conditions,
(f) conditioning the above microbial rich substrate as obtained from the above by
the addition of sterile rice flour at a level, of 25 - 35% (w/w)., to obtain a free
flowing, high volume and ready-to-use inoculum with a high stability at low
temperatures to use of inoculum, either individually or in combinations to
achieve desirable fermentation attributes in foods.
In an embodiment of the present invention, the growth substrate used as rice semolina.
In an another embodiment of the present invention, sterilization of growth substrate - rice semolina is effected by steaming for 25 - 35 min.
In an another embodiment of the present invention, wherein sterilization of the enriched growth substrate is effected by convection heating for 25-35 min at 90-92°C.
In an another embodiment of the present invention, the potent microbial cultures may be selected from Lactobacillus brevis CFR 2134, Pediococcus pentosaceus CFR 2123 (lactic acid bacteria) and Candida versatilis CFR 505 (yeast), either individually or in combinations thereof.
In an embodiment of the present invention incubation of inoculated substrate is
effected by uniform spreading of the substrate in a layer of 3-5 cm thickness in
strile stainless steel trays of any dimension.
In an embodiment of the present invention growth of lactic acid bacteria in the
substrate spread in SS tray is achieved at incubation temperatures of preferably
35 - 40°C for 36 - 50 h under sterile conditions.
In an embodiment of the present invention growth of yeast in the substrate
spread in SS tray is achieved at incubation temperatures preferably 28 - 32°C
for 60 - 80 h under sterile conditions.
In an embodiment of the present invention conditioning of the microbial rich
substrate is effected by the addition of sterile rice flour at a level of 25 - 35%
(w/w).
In an embodiment of the present invention shelf stability of the ready-to-use
inoculum for fermented foods is achieved for 85-95 days preferable at -20 to
10°C.
In an yet another embodiment of the present invention the inoculum used
individually or in combinations result in desirable fermented foods.
The process relates to an improved means of providing inoculum in ready-to-use form for commercial use in food fermentations. The potent microbial cultures selected in the preparation of inoculum for food fermentations are Lactobacillus brevis CFR 2134, Pediococcus pentosaceus CFR 2123 and Candida versatilis CFR 505. Cultural conditions are optimized for growing the
microbial cultures individually in rice semolina/grits with supplementation of moisture and desirable nutrients, followed by heating, addition of culture inoculum, mixing, spreading, incubation and removal of moisture. The preparation of ready-to-use inoculum is achieved by removing desirable amount of moisture from the substrate using rice flour at a level of 25 to 35% (w/w). The dried substrate had a viable cell population of 1010 -1012 colony forming units per gram (CFU/g) and a moisture level of 8 to 12%. Requisite quantities of dried idli grits are packed in polyester-laminate pouches. Based on the performance of dried substrate with viable microbial cell population in bringing about accelerated fermentation of idli batter, the ready-to-use inoculum for fermented foods had a shelf life of 90 days at -20 to 10°C.
The novelty of the process is making available the inoculum for fermented foods in a ready-to-use form. The inoculum which comprise microbial cultures are made using a simple and easily affordable substrate. The growth of microbial cultures is achieved in the substrate by a series of steps which involve certain newer and simple methods as well as conditioning of the microbial rich substrate by a non-heating approach. This non-heating approach enables in retaining high cell density of viable microbial cultures, which is lacking in other drying methods. The final ready-to-use product is a free flowing high volume with a high stability at low temperature storage.
Flow sheet for the preparation of ready-to-use inoculum using lactic acid bacteria

(Fig Removed)

The following examples are given by way of illustrations of the present invention and therefore should not be construed to limit the scope of the present invention.
EXAMPLE -1
In the entire microbiological work, sterilization is achieved by autoclaving different growth and plating media, glasswares and other solutions for 20 min at 121 °C. ISOLATION OF MICROORGANISMS
Samples of idli batter in 100 g from different habitats (home-made, hotels and catering centres) were collected in sterile glass beakers. Prior to use, the glass beakers were washed well, air dried, mouth covered with aluminium foil and autoclaved at 121°C for 20 min. Sample aliquots in 11 g each were added into 99 ml of sterile 0.85% saline to get an initial 10"1 dilution. From this initial sample dilution, subsequent serial dilutions were prepared in 9 ml amounts of sterile 0.85% saline. Aliquots of appropriate sample dilutions in 1 ml were pour plated with conventional nutrient medium such as Lactobacillus deMan Rogosa Sharpe (MRS) agar and potato dextrose agar (PDA) for the enumeration and isolation of predominant genera of lactic acid bacteria and yeasts, respectively. Poured plates of MRS agar and PDA were incubated at 37°C for 48 h and 30°C for 72 h, respectively.On the basis of colony morphology, ten well separated colonies of lactic acid bacteria formed on incubated MRS agar plates were selected at random. The individual colonies were transferred aseptically into 10 ml aliquots of MRS
broth tubes. Inoculated tubes were then incubated at 37°C for 48 h. Incubated tubes showing good growth (turbidity) were stored at 6°C, with subculturing in MRS broth at regular intervals of 10 days. All the selected natural isolates were maintained in 2 sets. MRS broth cultures (20 h old at 37°C) of natural isolates of lactic acid bacteria were characterized by morphological, cultural and biochemical tests. The tests included Gram's reaction; catalase production; growth at 10, 37, 45 and 50°C; growth at pH levels of 2, 4, 6.5 and 10%; ammonia from arginine; gelatin liquefaction; starch hydrolysis; survival to heating at 65, 70 and 75°C each for 15 min; acid production from glucose, lactose, galactose, fructose, cellobiose, maltose, mannitol, melibiose, raffinose, rhamnose, sucrose, trehalose and xylose.
On the basis of colony morphology, well separated colonies of yeasts formed on PDA plates were selected at random and transferred aseptically onto PDA slants. Inoculated slants were incubated at 30°C for 72 h. Incubated tubes showing good growth were stored at 6°C with subculturing in PDA slants at regular intervals of 15 days. All the selected natural isolates were maintained in 2 sets. Isolated cultures were characterized by morphological, cultural and biochemical tests. The tests included were assimilation of carbon sources like cellobiose, erythritol, galactose, maltose, mannitol, melibiose, raffinose, trehalose and xylose; assimilation of nitrogen sources; growth at 37°C; pellicle formation in broth; resistance to 0.01% cycloheximide; glucose fermentation; splitting of urea.
The isolates identified were those of Lactobacillus brevis, Pediococcus
pentosaceus and Candida versatilis. The characteristics of these microbial
cultures are presented in Tables 1 and 2. The identified isolates were deposited
at Central Food Technological Research Institute culture collection centre,
Mysore and designated as Lactobacillus brevis CFR 2134, Pediococcus
pentosaceus CFR 2123 and Candida versatilis CFR 505. This is covered under
the pending Indian patent filed, 1129/DEL/99.
Table 1. Cultural and biochemical characteristics of natural isolates of lactic acid bacteria
(Table Removed)
+, positive for growth; -, negative for growth; ND, not determined
Isolates were negative for gelatin liquefaction and starch hydrolysis
Isolates showed no survival for heating at 65, 70 and 75°C, respectively for 15 min
Isolates were positive for acid production from glucose, lactose, galactose, fructose,
cellobiose, maltose, mannitol, melibiose, raffinose, rhamnose, sucrose, trehalose and
xylose, except for isolates 9 & 12 which were negative for acid production from lactose
Table 2. Cultural and biochemical characteristics of natural isolates of yeasts
(Table Removed)
PREPARATION OF INOCULUM
Rice semolina in 1000 g quantities were taken in clean individual glass beakers of 2000 ml. The beakers along with the semolina were kept in an autoclave and steaming was carried out without any pressure for 30 min. Sterile
water was prepared by heating requisite quantity of water in an autoclave for 20 min at 15 lb pressure. Sterile water in 600 ml quantity was added to the individual lots of steamed rice semolina in the glass beakers. The preparation of substrate and growth conditions for the microbial cultures are given in the following steps:
LACTIC ACID BACTERIA CULTURES
(i) The rice semolina mixed with water was enriched with 12 g of glucose, 0.06 g
of magnesium sulphate, 0.12 g of manganese sulphate and 0.5 g of citric acid.
(ii) The enriched substrate was subjected to heating at 90°C for 30 min in a
thermostatically controlled water bath.
(iii) The heated substrate was cooled to 30°C and inoculated with an active cell
suspension of the individual cultures of Lactobacillus brevis CFR 2134 and
Pediococcus pentosaceus CFR 2123 at a level of 2 x 107 cfu/g.
(iv) The inoculated substrate was mixed well and spread in an uniform layer of 3
cm thickness in a pre-sterilized SS tray of 60 x 48 x 8 cm (I x b x h) dimension
and covered with sterile aluminium foil.
(v) The tray was incubated at 37°C for 48 h.
(vi) After 24 h of incubation, the aluminium foil was removed and covered with a
sterile double layered muslin cloth to enable escape of moisture collected.
(vii) The viable cell population of the individual lactic cultures was determined by
plating with Lactobacillus deman Rogosa Sharpe (MRS) agar.
(viii) Sample in 11 g was added to 99 ml sterile 0.85% saline to get an initial 10-1 dilution. From this initial sample dilution, subsequent serial dilutions were prepared in sterile 0.85% saline and aliquots of appropriate sample dilutions were pour plated with a convenient nutrient medium like Lactobacillus MRS agar, (ix) Poured plates were incubated for 48 h at 37°C and colonies of bacteria formed in the medium were counted, multiplied by dilution factor and expressed as colony forming units per gram.
The viable count of lactic acid bacteria reached in the substrate was 14 x 1010cfu/g.
YEAST CULTURE
(i) The rice semolina mixed with water was enriched with 0.5 g of tartaric acid.
(ii) The enriched substrate was subjected to heating at 90°C for 30 min in a
thermostatically controlled water bath.
(iii) The heated substrate was cooled to 30°C and inoculated with an active cell
suspension of Candida versatilis CFR 505 at a level of 2 x 107 cfu/g.
(iv) The inoculated substrate was mixed well and spread in an uniform layer of 3
cm thickness in a pre-sterilized SS tray of 60 x 48 x 8 cm (I x b x h) dimension
and covered with sterile aluminium foil.
(v) The tray was incubated at 30°C for 72 h.
(vi) After 24 h of incubation, the aluminium foil was removed and covered with a
sterile double layered muslin cloth to enable escape of moisture collected.
(vii) The viable cell population of the yeast culture was determined by plating with
potato dextrose agar.
(viii) Sample in 11 g was added to 99 ml sterile 0.85% saline to get an initial 10-1
dilution. From this initial sample dilution, subsequent serial dilutions were
prepared in sterile 0.85% saline and aliquots of appropriate sample dilutions
were surface plated on pre-poured plates with a convenient nutrient medium like
potato dextrose agar.
(ix) Poured plates were incubated for 72 h at 30°C and colonies of yeast formed
in the medium were counted, multiplied by dilution factor and expressed as
colony forming units per gram.
The viable count of yeast reached in the substrate is 22 x 109 cfu/g.
A free flowing high volume ready-to-use inoculum was prepared by mixing the microbial rich rice semolina in 1000 g in the SS tray with 300 g of sterile rice flour. The sterile rice flour was prepared by autoclaving for 30 min at 15 lb pressure. The resulting material was distributed in 100 g aliquots in polylaminate pouches, sealed and stored at -20°C. The moisture content in the rice flour mixed substrate was determined in 10 g sample. The viable cell population of lactic acid bacteria and yeast was determined as described previously.
The rice flour mixed rice semolina had a viable cell population of 24 x 1012 cfu/g of lactic acid bacteria and 18 x 1010 cfu/g of yeast. The moisture content was 10%.
PERFORMANCE OF INOCULUM IN IDLI BATTER FERMENTATION
The freshly prepared ready-to-use inoculum (rice flour mixed rice semolina) was evaluated for its performance through accelerated fermentation of idli batter. Idli batter was prepared according to the conventional method. The batter in 100 g quantities was added with the individual dried substrate of Ped. pentosaceus CFR 2123, Lb. brevis CFR 2134 and C. versatilis CFR 505 in various combinations of (i) 2:5:2 g, (ii) 5:8:5 g and (iii) 8:12:8 g. The batter was mixed well and contents transferred to a clean 500 ml measuring glass cylinder and incubated for 6 h at 34°C. The increase in batter volume was recorded and compared against the natural fermentation of conventional idli batter. The idli batter characteristics are shown in Table 1.
Table 1. Performance of dried ready-to-use microbial starters in the accelerated fermentation of idli batter (6 h at 34°C)
(Table Removed)






We claim:
1. An improved process for the preparation of inoculum for fermented foods which comprises:
(a) steaming of a growth substrate selected from a cereal source such as rice
semolina for a period of 20 - 50 min,
(b) enriching the selected growth substrate with an acidulant selected from citric
acid and tartaric acid at a level of 0.04-0.06 % and mineral nutrients: 1-2 % glucose : 0.005-0.007 % magnesium sulphate , and 0.011-0.013% manganese sulphate, and 58-62% water,
(c) sterilizing of the enriched substrate obtained from the above at 85 - 100°C for
20-40 min,
(d) adding a microbial culture from the group comprising Lactobacillus brevis
CFR 2134, Pediococcus pentosaceus CFR 2123 and Candida versatilis
CFR 505, individually at a cell density of 2 x 108 colony forming units per
gram (cfu/g), such as herein described,
(e) incubating of the enriched substrates for a period of 48 - 96 hours at a
temperature of 25 - 45°C under sterile conditions,
(f) conditioning the above microbial rich substrate as obtained from the above by
the addition of sterile rice flour at a level of 25 - 35% (w/w)., to obtain a free
flowing, high volume and ready-to-use inoculum with a high stability at low
temperatures to use of inoculum, either individually or in combinations to
achieve desirable fermentation attributes in foods.
2. A process as claimed in claim 1, wherein the growth substrate used is rice
semolina.
3. A process as claimed in claims 1 & 2, wherein sterilization of growth substrate
- rice semolina is effected by steaming for 25 - 35 min.
4. A process as claimed in claims 1 to 3, wherein sterilization of the enriched
growth substrate is effected by convection heating for 25-35 min at 90-92°C.
5. A process as claimed in claims 1 to 4, wherein the potent microbial cultures
are used is selected from Lactobacillus brews CFR 2134, Pediococcus
pentosaceus CFR 2123 and Candida versatilis CFR 505, either individually or
in combinations such as herein described.
6. A process as claimed in claims 1 to 5, wherein incubation of inoculated
substrate is effected by uniform spreading of the substrate in a layer of 3-5 cm
thickness in sterile stainless steel trays of any dimension
7. A process as claimed in claims 1 to 6, wherein growth of lactic acid bacteria in
the substrate spread in SS tray is achieved at incubation temperatures of
preferably 35 - 40°C for 36 - 50 h under sterile conditions.
8. A process as claimed in claims 1 to 7, wherein growth of yeast in the substrate
spread in SS tray is achieved at incubation temperatures of 28 - 32°C for 60 - 80 h under sterile conditions.
9. A process as claimed in claims 1 to 8, wherein shelf stability of the ready-to-
use inoculum for fermented foods is achieved for 85-95 days at -20 to 10°C.
10. An improved process for the preparation of inoculum for fermented foods substantially as herein described with reference to the examples.

Documents:

352-del-2001-abstract.pdf

352-del-2001-claims-cancelled.pdf

352-DEL-2001-Claims.pdf

352-del-2001-complete specification (granted).pdf

352-del-2001-correspondence-others.pdf

352-del-2001-correspondence-po.pdf

352-del-2001-description (complete).pdf

352-del-2001-form-1.pdf

352-DEL-2001-Form-2.pdf

352-del-2001-form-4.pdf


Patent Number 195180
Indian Patent Application Number 352/DEL/2001
PG Journal Number 31/2009
Publication Date 31-Jul-2009
Grant Date
Date of Filing 27-Mar-2001
Name of Patentee COUNCIL OF SCEINTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 MANDYAM CHAKRAVARTHY VARADRAJ DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA.
2 SISTIA VENKATA NAGA VIJAYENDRA DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA
3 EDDIYA RATI RAO DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA
4 RENU AGARWAL DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA
5 MURTHAPPA SEEBANNA PRASAD DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA
6 KRISHNA NAND DEPARTMENT OF FOOD MICROBIOLOGY, CENTRAL FOOD TECHNOLOGICAL RESEARCH INSTITUTE, MYSORE 570013, INDIA
PCT International Classification Number C12N 1/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA