A process for the preparation of curd

Abstract

The present invention relates to a novel process for the preparation of milk product curd using corynebacterium glutamicum and method of preparation of bacterial strip containing corynebacterium glutamicum. Bacteria attached membranes is used for the preparation of curd. Bacterial strip containing corynebacterium glutamicum can also be used for addition of Histidine supplement during curd production.

Description

FIELD OF THE INVENTION:The present invention relates to a novel process for the preparation of a milk product, curd.Particularly, bacteria attached membranes is used for the preparation of curd. Thesemembranes can also be used for addition of different supplements such as Histidine duringcurd production.BACKGROUND THE INVENTION:Milk and other dairy products were considered as important foods as early as 4000 BC. In thewhole world it is one of the most important components of the human diet. Milk isconsidered to be the only foodstuff which contains almost all essential human nutrients.Especially it is an important source of protein, calcium, and the B-group vitamins (thiamin,riboflavin, niacin, vitamin B6, and folate), and provides vitamin A, vitamin C, magnesium,and zinc as well.The production of acid milk products has long been known in all countries. The mostordinary way to get acid curdled milk is actually spontaneous acidification. This course relieson lactic acid bacteria, naturally present in milk as adventitious contaminants, which growand produce the lactic acid required to coagulate the milk. Sometimes, the lactic acid bacteriaare accompanied by yeasts or moulds, which give special features to the fermented product.By this way, several communities around the world established their own characteristicfermented milks. In Caucasian countries milk is fermented by a co-culture of lactic acidbacteria and yeasts, named the product kefir. In Siberia and south Russia, along with alcoholthe fermentation of horse milk by yeasts and lactic acid bacteria results in koumiss. Similarlyanother alcoholic acid milk in America is mazun. Accordingly the important milk productEgypt and leben is yoghurt, in Italy gioddu and in Western Europe buttermilk are wellknown. Whereas in Scandina the well known fermented milks are viili and langfil.Various materials viz. ceramic, polymers, etc. are used for the preparation of membranes.These membranes are common in use due to its simplicity in the preparation and costeffectiveness. There are many kinds of bacteria which have been recovered from differentmembranes. The inherent characteristic of these bacteria are to attach the membranes evenwhen there are no transmembrane pressure differentials. The attachment between bacteria and2HI 1 5 - 8 7 - 2 8 1 7 17 : '.membrane occurs due to the accumulation of trace organic nutrients at the "solid-liqiiidinterface which is enough for bacteria to survive. The attachment does not involve anyparticular stereospecific macromolecular binding sites and usually, it is not substrate specific.Hence, numerous kinds of bacteria get attached to different polymeric membrane surfaces; itdoes not need any particular interaction. This is the reason bacteria get readily appended tothe membrane. Generally, various bacterial and membrane characteristics influence thisattachment phenomenon. Bacterial features that are responsible for adhesion to membraneinclude cell surface charge, cell wall hydrophobicity, cell surface structure, and the type ofextracellular polymeric substances (EPS) produced by it. The bacterial adhesion to themembrane not only depends on its nature but also on the membrane. The characteristicproperty like surface hydrophobicity, surface charge, chemical composition, and roughnessalso influences the attachment.Histidine is one of the standard amino acids in proteins, and which plays a critical role inplant growth and development. Human body cannot synthesize histidine. For that reason inhuman body it is coming as supplement. Corynebacterium glutamicum is a Gram-positive,aerobic, rod-shaped, and non-sporulating soil bacterium which is extensively used forindustrial L-Histidine production.Reference may be made to an article entitled "Interaction between dairy yeasts and lactic acidbacteria strains during milk fermentation" by P. A. Martinet al. published in Food Control,2008, 19, 62-70 reports that lactic acid bacteria species were mostly responsible for milkacidification whereas yeast strains produced most of the volatile maltic compounds.Balancing with these compounds different cheese varieties and other dairy products can beformed.Reference may be made to an article entitled 'The Use of Lactic Acid Bacteria StarterCulture in the Production of Nunu,-a Spontaneously Fermented-Milk Product in Ghana" byF. Akabanda et al. published in International Journal of Food Science, 2014, 721067-721078 reported that lactic acid bacteria isolated from Ghanaian traditional fermented milkproduct have desirable technological properties and successfully used as starter cultures forNunu fermentation.3LHI 1 3 - 8 7 - 2 0 17 T D -Reference may be made to an article entitled "Lactic acid bacteria and yeasts involved in thefermentation of amabere amaruranu, a Kenyan fermented milk" by B. Nyambane et al.published in Food Sci Nutr., 2, 692-699 reports a Microorganisms involved inamabereamaruranu fermentation were found to consist of lactic acid bacteria from the three generalLactobacillus, Leuconostoc and Streptococcus.Reference may be made to an article entitled "Enhanced bacterial affinity of PVDFmembrane: its application as improved sea water sampling tool for environmentalmonitoring" by S. B. Kumar et al. published in Environmental Science and PollutionResearch, 2017, 24, 5831-5840 reports for the preparation of PVDF membrane and checktheir check their affinity for the attachment of selected gram-positive (Bacillus subtilis) andgram-negative (Escherichia coli) bacteria.Reference may be made to an article entitled "Biofouling potentials of microporouspolysulfone membranes containing a sulfonated polyether-ethersulfone / polyethersulfoneblock copolymer: correlation of membrane surface properties with bacterial attachment" byT. Knoellet al. published in Journal of Membrane Science, 1999, 157, 117-138 reports thatenhancement of Mycobacterium attachment to the modified polysulfone membranescompared to Flavobacterium attachment which is related to the surface hydrophobicity of themodified polysulfone membranes.Reference may be made to an article entitled "The role of cell-surface interactions in bacterialinitial adhesion and consequent biofilm formation on nanofiltration / reverse osmosismembranes" by O. Habimanaet al. published in Journal of Membrane Science, 2014, 454, 82-96 reports that the interactions between bacterial cells and NF / RO membranes are importantfor biofouling.Reference may be made to US 20070184532, which reports a method for producing Lhistidineusing bacterium of Enterobacteriaceae family.Reference may be made to US3849250, which reports a process for producing L-arginine byfermentation.4tOBJECTIVE OF THE INVENTION:• The main objective of the present invention is to provide a novel process for thepreparation of curd.• Another objective of the present invention is to prepare Corynebacterium glutamicumcurd strips.• Another object of the present invention is to provide bacteria attached PVDFmembrane as a strip for the preparation of curd.• Yet another objective of the present invention is to provide a process for the additionof different important supplements released by the bacteria during curd formation.• Yet another objective of the present invention is to prepare curd like milk productcontaining histidine with the help of Corynebacterium glutamicum curd strips.BRIEF DESCRIPTION OF THE DRAWINGS:Fig. 1: Curd formation by the curd stripFig. 2: Bacterial attached membrane (Curd strip)DETAILED DESCRIPTION OF THE INVENTION:The present invention relates to a process for the- preparation of curd. The present inventionalso relates to prepare Corynebacterium glutamicum curd strips made of PVDF membrane forthe preparation of sweet, fermented milk product containing histidine. The design of thepresent invention retains the curd forming bacteria on the membrane. The membrane can thenbe directly dipped into the.pro.cessed milk for the. preparation of curd.,The simple method of _using strips for the curd preparation makes it advantageous as they are ready-to-use anytime.In another embodiment, the bacteria attached to the membrane can be of varied typedepending on the type of curd or any milk product.5rf JL l i ' - U f ~ £ ^ 1 / i / - d,In another embodiment, the strip prepared can be used for the production of any fermentedmilk product like yogurt and kefir etc.In yet another embodiment, the thickness of the membrane is between 130-135 ^m (30-35\im membrane thickness and 100 |am support thickness).In yet another embodiment, the membrane selected for the bacterial detection ispolyvinylidene fluoride (PVDF) (20 % w / w) (573 KDa, Solef, Solvay, France).In yet another embodiment, Corynebacterium glutamicum curd strip was prepared to addextra nutrient into the curd i.e. histidine.In yet another embodiment, the invention provides a process for the preparation of curdcomprising the following steps:a. providing cooled boiled milk;b. providing corynebacterium glutamicum',c. growing corynebacterium glutamicum in nutrient broth media for 20-30h attemperature 25-40°C followed by centrifugation with 3000-5000 rpm;d. washing the cells obtained in step (c ) by centrifugation with 3000-5000 rpm;e. resuspending the cells obtained in step (d) in 10% skimmed milk;f. dipping PVDF membranes into bacterial suspension obtained in step (e) for attachingthe bacteria onto the membrane;g. lyophilizing the bacterial attached membranes obtained in step (f) for 6-12 h to getcorynebacterium curd strips.h. dipping the strip obtained in step (g) into the milk obtained in step (a);6LHI I 3 - 8 7 - 2 8 1 7 1 7 :2i. incubating the liquid obtained in step (h) for 10-20 hours at a temperature rangingbetween 20-40° C to obtain curd.In yet another embodiment, the invention provides a membrane which is made up of 20%w / w PVDF polymer concentration prepared in DMF as a solvent and taking water as a nonsolventmedium.In yet another embodiment, the invention provides a PVDF polymer whose concentration isin the range of 6-24% w / w.In yet another embodiment, the invention provides a membrane wherein, the thickness ofmembrane is in the range of 30 to 50 \xm.In yet another embodiment, the invention provides a strip containing lyophilised form ofcorynebacterium prepared by the method steps 1 a-g.In yet another embodiment, the invention provides a kit for curd preparation consisting of astrip.In yet another embodiment, the invention provides a probiotic curd containingcoryne bacterium sp. prepared by using the method.EXAMPLESThe following examples are given by way of illustration therefore should not be construed tolimit the scope of the Invention.Example 1Membrane preparationMembranes were prepared using 20% w / w PVDF as a base membrane. All the membraneswere prepared by phase inversion technique. Membrane preparation involves two phases: (a)Preparation of solution and (b) Membrane Casting. Both test as well as control membrane7LHI 1 3 - 8 7 - 2 8 1 7 1 7 : 25polymers were dissolved in solvent N, N- dimethyl formamide (DMF), keeping temperatureof about 50-60 *C with constant stirring for overnight at around 100-120 rpm. The solutionwas kept overnight for the removal of air bubbles.The polymer solution was then poured onto the polyester fabric which was attached to theglass plate. Then the solution was uniformly spread over the fabric using a casting blade.After spreading of the polymer solution the whole system was immediately transfer to thenon-solvent bath containing reverse osmosis (RO) water. The membrane prepared was thenremoved from the water and allowed to air dry. Subsequently, the dried membrane was cutinto 2 x 2 cm2 pieces and autoclaved at 121° C for 15 min.Example 2Preparation of curd stripsCurd was collected from the local market for the experiment. The curd was then centrifugedat 1000 rpm. lmL of its supernatant was inoculated into Nutrient broth and kept it (onshaking) for overnight for the growth of bacteria present in the curd. The curd bacteria grownin the curd was washed with PBS (2-3 times) to remove all the traces of growth media. Thewashed bacteria were resuspended in. 10% skimmed milk (cryoprotectant) and then the sterile20% PVDF membrane was added to the prepared bacterial suspension. The bacteria wereallowed to attach to the membrane for 24 h on shaking (100 rpm) at 37°C. The bacterialattached membrane was finally lyophilized to make the curd strips.For Corynebacterium, the bacterium (ATCC 21607) was grown in same nutrient medium for28 h. The washed cells were again resuspended in 10% skimmed milk. The cut andautoclaved membranes were then kept into the prepared bacterial suspension for attachment8LHI i 3"07 - 2017 17 :2Sas mentioned above. The membranes with attached bacteria were then lyophilized for-t-hepreparation of Corynebacterium curd strips.Example 3Curd preparation with the help of prepared curd stripsFor curd the preparation, processed milk (Amul gold) was purchased from the local market.The milk was then boiled and allowed to cool.Three strips of curd bacteria and Corynebacterium were added into 30 mL of milk separatelyand incubated at room temperature. Curd formation was observed after 16 h for curd bacteriastrip and 20 h for Corynebacterium strip.Advantages of the invention:1. A simple technique for the preparation of curd.2. Various types of bacteria (depending on the requirement) can be attached on anypolymeric membrane which will also release required supplement during curdformation.3. A simple technique to prepare curd in consistent manner every time4. A cost effective curd strip which is simple to prepare and use.5. No requirement of keeping / storing the curd inocula every time.6. Spillage proof, portable curd strips.7. Ready-to-use.

Claims

claims1. A process for the preparation of curd comprising the following steps:a. providing cooled boiled milk;b. providing corynebacterium glutamicum;c. growing corynebacterium glutamicum in nutrient broth media for 20-30h at temperature 25-40°C followed by centrifugation with 3000-5000rpm;d. washing the cells obtained in step (c ) by centrifugation with 3000-5000 rpm;e. resuspending the cells obtained in step (d) in 10% skimmed milk;f. dipping PVDF membranes into bacterial suspension obtained in step(e) for attaching the bacteria onto the membrane;g. lyophilizing the bacterial attached membranes obtained in step (f) for6-12 h to get corynebacterium curd strips.h. dipping the strip obtained in step (g) into the milk obtained in step (a);i. incubating the liquid obtained in step (h) for 10-20 hours at atemperature ranging between 20-40° C to obtain curd.

2. The process as claimed in claim 1 (f) wherein the membrane is made up of 20% w / wPVDF polymer concentration prepared in DMF as a solvent and taking water as anon-solvent mediumr ._.-.- .. . .. .

3. The process as claimed in claim 1 (f) wherein the PVDF polymer concentration is inthe range of 6-24% w / w.

4. The process as claimed in claim 1 (f) wherein, the thickness of membrane is in therange of 30 to 50 urn.

5. A~strip~containing lyophilised-form of eorynebacteriumpr&pared .b.y__the_metho.d,step.s,1 a-g as claimed in claim 1.

6. A kit for curd preparation consisting of a strip as claimed in claim 5.

7. A probiotic curd containing coryne bacterium sp. prepared by using the method as8. A probiotic curd as claimed in claim 7 useful in Histidine deficiency.