FORM 2
THE PATENTS ACT, 1970
(39 of 1970) COMPLETE SPECIFICATION
(See section 10; rule 13)
1. Title of the invention. 1. Lycopene Enriched Whey Fruit Juice Health
Beverage (A functional food - to be patented) 2. Production technology of lycopene enriched health beverage (A processing technology -to be patented)
2. Applicant (s)
a)Name a) Marathwada Krishi Vidyapeeth, Parbhani
b) Nationality b) Univercity Constituted Under Maharashtra Agril.
Universities (Krishi Vidyapeeth) Act 1983 MKV
Parbhani
c) Central Administrative Building, Krishi Nagar, c)Address
Parbhani -431402 (M.S.) India
3. Preamble to the description 1. The functional beverages are presently receiving
renewed interest as per the expectations of the tenet entitled "Let Food be a Medicine And Medicine Be a Food". Presently the said tenet seems to be applicable exclusively for the functional foods. Therefore the invention to be filed for patent is innovative, need base,

consumer lifestyle stimulatory and market driven. The functional beverage as a food product and process technology developed are feasible for filing independent patents. The beverage as a functional food product has buildup its capacity to bear resistance and provide health claim against non communicable diseases like obesity, diabetes, cardiovascular diseases, cancer and inflammation. The therapeutic efficacy assessed on the basis of clinical studies recorded prominent results to provide anticipated health claims. The clinical assessment based on animal (Rabbits) study recorded positive impact and laid down a foundation stone for human clinical studies.
4. Description of the present invention
Field of Invention: Present investigation relates to functional food product
development and processing technology.
Background of the invention and prior art:
The bioactivity of lycopene is recognized on the basis of its efficacy to provide protection against cancer and other degradative diseases caused by free radicals (Levy et al. 1995). Epidemiological studies on risk assessment factor of cancer (Prostate gland, stomach and lungs) coupled with increased blood lycopene is justifiable due to its therapeutic value (Stahl and Sies 1996). Carotenoiods including lycopene possess antioxidant capabilities and could be used in preventing different diseases like cancer and hence render health benefits (Davis et al. 2003). Tomato and tomato products are considered as one of the live sources of lycopene (Sheetaletal.2007).

The lycopene has justified its status as an antioxidant scavenger on the basis of its molecular configuration and having high demand not only because of pharmaceutical value but also to enrich the food as a nutraceutical for health benefits. The stability of lycopene of processed tomato juice and paste recorded unaltered at ambient temperature for a period of 12 months (Agarwal et al. 2001). The stability of pure crystalline tomato lycopene is poor than its native form present in the pulp..
Whey, a byproduct of cheese and curd manufacturing considered as waste product. The recognition of whey as functional food with nutritional applications has elevated whey to a co-product in the manufacturing of cheese. The components of whey include beta-lactoglobulin, alpha-lactalbumin, bovine serum albumin, lactoferrin, immunoglobulins, lactoperoxidase enzymes, glycomacropeptides etc (Walzem RL et al. 2002). Whey is popular dietary protein supplement to provide immune modulation, antimicrobial activity, improved muscle strength and body composition and prevention of cardiovascular disease and osteoporosis (Keri Marshall, MS, ND 2004).
Whey proteins contain all the essential amino acids, having high concentration of branched-chain amino acids (BCAAs)- leucine, isoleucine, and valine-important factors in tissue growth and repair. Leucine is a key amino acid in protein metabolism (Anthony JC et al 2001).
Mango juice as supplementary acceptability enhancing ingredient recorded its bioactivity strength on the basis of phytochemicals present like Vitamin C, Vitamin B, and Vitamin A along with notified minerals like iron, phosphorus and calcium. Vitamin C is potent antioxidant. (Online data- Agrixpo Industries Manufactures, Importers and Exports)
In the present, investigation efforts were made to standardize processing conditions of tomato puree as a lycopene rich commercial base and to formulate whey base health beverage.
Objectives of the present Invention
1. To assess purity of lycopene by TLC and HPLC
2. To standardize synergetic health beverage
3. To assess sensorial quality parameters
4. To assess therapeutic value of standardized synergetic beverage by clinical assessment Advantages

5. Development of innovative type of speciality food product (health beverage).
6. Utilization of dairy industry by product as a low cost raw material for high value food product through techno economical mode of new product development and formulation.
7. Product development to compensate with change of life style of the consumer and provides health protection to ensure health security.
8. Commercialization of the functional foods helps in increasing GDP and acts as a driving force for modulation of agrobased industrialization.
9. It is high need of time to find a suitable solution to irradicate malnutrition, the functional food development certainly helps in elevation of malnutrition.
10. Present trend of national nutrition schemes involving supply of quality and non ready to consume food material to stake holders need to be modulated for their intentional use as a remedial source to fight against malnutrition. The development and popularization of functional foods will leave no any stone unturned to offer suitable solution to provide nutritionally rich and healthy diet.
Description of the health beverage
Lycopene Enriched Whey Fruit Juice Beverage
The standardized whey fruit juice base (M.K.V., recommendations-2006) for preparation of lycopene enriched beverage by using processed tomato puree (John Shi el al 2008 -concentrated source of lycopene) as constituent ingredient was used and efforts were made to justify its therapeutic value on the basis of clinical studies carried out at COVAS, Parbhani.
Standard recipe of lycopene enriched whey fruit juice beverage

Ingredients Standard formulation (1000 ml)
Whey (ml) 735.60
Fruit juice (ml) 100
Preservative (g) 0.6
Stabilizer (g)
Sugar (g) 60
Citric acid (g) 0.8
Whey to puree ratio 735.60:100
Lycopene (mg) 18.3

Standard recipe - constitutional ingredients justification

Sr. No. Ingredients (%) Whey fruit juice beverage (samples )

A B C D E
1 Whey (ml) 83.56 78.56 73.56 68.56 63.56
Percentage of whey ingredients in/sample A,B,C,D&E
(According to the reference) Lactoferrin-1-2
alpha-Lactalbumin-20 Bovine Serum Albumi beta Lactoglobulin -50-55 -25, Lactoperoxidase-0.50 n-5-10 Glycomacropeptides-10-15
2 Fruit juice (ml) 10 10 10 10 10
Percentage of mango juice ingredients in/sample A,B,C,D & E (According to the reference) Vitamin C-20.00 mg
Vitamin A-2 mg Phosphorus-18.00 mg Vitamin B-l. 07 mg Iron-1.60 mg Calcium-16.00mg
3 Preservative (gm) 0.06 0.06 0.06 0.06 0.06
4 Stabilizer (gm) 0.3 0.3 03 0.3 0.3
5 Sugar (gm) 06 06 06 06 06
6 Citric acid (gm) 0.08 0.08 0.08 0.08 0.08
7 Whey to puree ratio 100:0 78.76:05 73.76:10 68.76:15 63.76:20
8 Lycopene (mg) 0.0 0.91 1.83 2.74 3.66
Note: The Lycopene in the form of processed puree was fortified in whey fruit juice beverages in varying concentrations (Blank, 5 ml, 10ml, 15ml & 20ml).

5. Detailed Description of the present invention
The data on effect of extraction method on per cent yield and nature of lycopene depicted in Table 1 indicate that heat induced maceration of fleshy edible mass of tomato substantially facilitated the release of tissue entrapped lycopene representing higher per cent yield (18.30 to 18.80 mg/lOOg) coupled with specified time period (90°C for 1 h.) notifying its nature as techno-economically viable process. One hour heat induced tomato matrix effective maceration for lycopene extraction is in sole agreement with previous literature. The recorded results are feasible for specifying diversity in anatomy of tomato matrix (Shi et al. 2008.).
Table 1 Effect of maceration and time on extraction of tomato lycopene

Lycopene Source Extraction method Confirmatory Percent yield of lycopene
Tomato
(local
variety) Hot method (1 h) Cold method 18.30 to 18.80
Per cent
yield
(mg/lOOg) 700C 800C 90oC Room temperature

9.20 to 10.80 12.30 to 12.85 18.30 to 18.80 3.30 to 3.88

(Each value represents an average of 3 determinations)
The research findings on development of lycopene as a nutraceutical till to date were based on local tomato cultivars. To widen up the limitations of lycopene as a nutraceutical and as per the guidelines of third group meeting screening of lycopene rich tomato cultivars was taken as targeted objective to economize the enrichment process of food products. The tomato cultivars Pusa Ruby and Pusa Early Dwarf evaluated for lycopene content as national check (Obtained from Indian Agricultural Research Institute, New Delhi) were recorded discouraging results. The data depicted in table no. 2 represents the lycopene content of national check tomato cultivars. The relative per cent yield of extracted lycopene under similar conditions exhibited by local cultivars is comparatively higher than that of Pusa Ruby and Pusa early Dwarf cultivars.


Table 2. Effect of cultivars on lycopenc content

Sr. No. Tomato cultivars Lycopene content (mg/100g)
1 MKV, Local cultivar 18.30 to 18.80
2 Pusa Ruby 10.23 to 12.36
3 Pusa Early Dwarf 11.40 to 15.58
During later phase of academic year 2011-12 owing to the consolidated suggestions from Vth Annual group meeting need based changes with respect to contributory share of individual constituents of beverage was emphasized technologically and modulation of efficacy

was considered to notify standard lycopene RTS as synergic lycopene fruit juice RTS as health beverage. Accordingly an innovative project on Synergic efficacy of lycopene enriched whey beverage was designed and evaluated for therapeutic value on the basis of clinical studies at COVAS, Parbhani



The chromatographic data on purity of lycopene presented in fig.5 and 6 revealed that the lycopene from MAU cultivar was at par with pure lycopene (Sigma aldrich). The per cent yield of lycopene from local cultivar (18.30 to 18.80 mg/100 g) was confirmed its purity by chromatogram profile.
Table 3 Effect of concentration of tomato puree on chemical composition of beverages

Parameter Sample A Sample B Sample C Sample D Sample
E
PH 4.5 4.3 4.1 4.0 4.0
T.S.S 12.9 12.6 12.2 11.9 11.5
Acidity (%) 0.58 0.53 0.49 0.46 0.43
Ash(%) 0.43 0.40 0.36 0.33 0.29
Total sugar (%) 12.22 12.09 11.95 11.30 11.10
Reducing sugar (%) 07.87 07.68 07.12 06.54 06.0
Non-reducing sugar
(%) 4.35 4.41 4.83 4.76 5.10
Total solids (%) 14.3 13.9 13.3 12.7 12.0
Lycopene (mg) 0.0 0.91 1.83 2.74 3.66

The data on chemical composition of various beverages depicted in Table 3 indicate that the acidity of beverages exhibited slight linear decrease with decreased concentration of whey and increased concentration of puree. The meager variation in ash content of whey mango fruit juice beverages was recorded. The beverage containing 5 per cent of tomato puree reported 0.40 per cent ash content which is comparatively higher against the beverages containing 10, 15 and 20 per cent puree respectively exhibiting linear decreasing trend (Samples C, D and E). The lowest value of ash content (0.29 per cent) recorded by beverage prepared by using 20 per cent tomato puree. The total sugar content of beverage recorded pronounced variation in values. The beverage containing 20 per cent tomato puree reported lowest total sugars (11.10 per cent) as compared to other relative samples followed by D (11.30 per cent) due to successive decrease in whey concentration and increase in puree concentration.
The comparatively lowest total solids (12.0 per cent) content of beverage containing 20 per cent puree (sample E), is associated with total solids content of whey and puree. The perceptible concentration of tomato puree feasible for enrichment is self justifiable for consumer acceptability of ready to serve beverage.
The addition of excess amount of supplementary ingredient not only affects mouth feel and taste but also significantly affects techno-economical feasibility of whey beverage. The tomato puree as a supplementary ingredient recorded its optimal concentration as 10 per cent for standardizing commercially viable beverage recipe on the basis of data on sensorial quality parameters. TSS of all lycopene enriched beverages exhibiting reducing trend affected overall perception of sensorial quality parameters. pH being shelf life monitoring attribute remained unchanged during storage in all types of lycopene enriched whey mango beverages.
Table 4 Effect of storage conditions on cone, of lycopene in beverage (mg/lOOml)

Storage conditions Storage period (weeks)
0 2 4 6 8
Cold storage (5± 1°C) 1.83 1.83 1.73 1.65 1.40
Ambient storage (25±2oC) 1.83 1.70 1.42 1.10 0.80
The data presented in Table 4 summaries the potential stability of lycopene during storage tenure of 8 weeks to justify its feasibility of numerical servings of whey fruit juice beverage in coordination with threshold concentration of 40mg per day which is sufficient enough to substantially reduce low density lipo-protein (LDL) oxidation (Agarwal and Rao

1998). It appeared that oxidation of lycopene is considerably reduced during cold storage as compared to ambient conditions. The linear reduction in lycopene content of beverage recorded under both conditions may be associated with other factors like exposure to sun light, and change in pH of beverage. Cumulative degradation of lycopene is certainly associated with isomerization and oxidation of molecule due to exposure to light and temperature (Shi J et al. 2008).
Table 5 Effect of lycopene content on sensorial quality parameters of beverages

Beverages (WFJB) / lycopene (mg %) Appea
-rance Color Taste Flavor After taste Overall
acceptability
0.00 7.4 7.4 7.4 7.2 7.2 7.4
0.91 6.7 6.7 6.5 6.5 7.1 6.8
1.83 7 7 7 6.9 7.2 7.1
2.74 7 7 6.6 6.1 6.6 6.5
3.66 7 7.1 6.1 6.1 6.4 6.2
The overall acceptability of whey fruit juice base beverage (WFJB) prepared by using 10 per cent tomato puree found superior and very close to beverage free from puree as an additional ingredient on the basis of relative score of sensorial quality parameters. The concentration of puree of 15 per cent and above superimposed its effect as bitter taste. Moreover the relative score of sensorial quality parameters of whey fruit juice base beverage prepared by using 10 per cent tomato puree recorded highest on the basis of visual (attractive and eye appealing), olfactory and taste sensations.
Microbial Quality Assessment
(A Mendez-Vilas Ed. (2007) Communicating Current Research and Educational Topics and Trends in Applied Microbiology)
Table 6 Microbial quality assessment of standardized beverage
Microbial parameters Particulars of Samples

Standard Average
Total plate count 6 x l04 4x 105
Staphylococcus spp. 100 cfu/gram 24.25
E-coli 5x102 cfu/gram 3x 101
Coli forms 100 cfu/gram 1.40
Salmonella Spp. 0/25 gram ND
Yeast <10 cfu/gram 1.2
Mold <10 cfu/gram 0.5

The consumer safety of beverage was assessed on the basis of microbial analysis of selective organisms representing cumulative activity during storage at refrigeration temperature. The probable activity enhancement was reported by earlier scientists during processing and handling operations. The beverage subjected to microbial analysis recorded growth profile of selective organisms recorded in Table 6. The data on microbial analysis notified presence of staphylococcus [100 cfu/g] at par with standardized beverage as supported by Ojokoh A.O. 2006 and Joshi D. R. 2004 respectively and the cumulative growth of E-coil as a surveillance organism below 0°C compensates with earlier data of scientists. The standard Total Plate Count of organisms to the tune of 4 x 10 cfu/g recorded E-coli count as 5 xl02 cfu/g as individual standard. The beverage E-coli count recorded (3 x 101) is far below than that of standard value (5x102). This may be associated with intensive sanitation care exhibited during the preparation of beverage. The values of Yeast and Mold recorded are negotiated and Salmonella spp. was not detected. The E-coli in borne disease symptoms and their manifestation severity could be negotiated with present data on the basis of it survival predominance at lower temperature. This assessment gives future directives for microbial safety of the product.
Cost Economics

Sr. No. Ingredients Standard
formulation
(1000 ml) Cost for 1 lit.
(Rs.) Cost of one bottle (200ml) (Rs)
1. Whey (ml) 735.60 36.78 10 rupees per bottle
Serving of 5 bottles
2. Fruit juice (ml) 100 12.94

Preservative (g) 0.6 0.48 per day if continued for one week provides
4. Stabilizer (g) 3 3.75 opportunity to absorb
5. Sugar(g) 60 1.80 40 to 45mg of lycopene on the basis
6. Citric acid (g) 0.8 0.12 of 50% assimilation efficacy that compensates with threshold in vivo concentration feasible to justify therapeutic
7. Whey to puree ratio 735.60:100 2.50

8. Lycopene (mg) 18.3 —

Total cost 58.37 per lit

Large sc unit cost productio ale production may of production hen n substantially reduces ce probable cost of 50/- per lit No. of
bottles from
1 L =5 bottles
(200 ml) effect of lycopene.

Clinical Assessment: Therapeutic value
The consolidated data on clinical assessment has been protected intentionally for filing the patent on whey fruit juice health beverage.
Summery and Conclusion
The research findings of the present investigation are encouraging with reference to the earlier research workers. The heat induced maceration leading to increased lycopene content of whole tomato [18.30 mg/l00g] and standard recipe to produce lycopene, proteins and vitamins rich synergistic health beverage justifiable for its nutraceutical potential emerged out are remarkable achievement of this investigation.
The therapeutic beverage prepared by using standard whey fruit juice base and 10 per cent tomato puree reported its superiority on the basis of relative score of sensorial quality parameters and recorded 1.83 mg/100ml bioactive lycopene as a nutraceutical.
The therapeutic value of whey beverage is synergic of contributory action of predominant ingredients specially tomato lycopene. whey proteins and mango vitamin C justifying threshold concentration could be confirmed on the basis of ongoing clinical studies.
Therapeutic efficacy of health beverage
Therapeutic efficacy assessed on the basis of animal clinical study related to synergic mode action of variety ingredients (tomato lycopene, whey proteins and mango vitamin C) recorded positive impact on health claim for non communicable prevalent diseases like obesity, cardiovascular disease, cancer cell development and inflammation. The confirmation of threshold concentration requires human clinical studies and will be undertaken in due course of time.
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We claim for
1) Patent for Food Product
• Food Product - lycopene enriched whey fruit juice health beverage Standard recipe of lycopene enriched whey fruit juice beverage

Ingredients Recipe for small scale production (1000 ML) Recipe for large scale production (100 L/Kg)
Whey (ml) 735.60 73.56
Fruit juice (ml) 100 10
Preservative (g) 0.6 6
Stabilizer (g) 3 0.3
Sugar(g) 60 6
Citric acid (g) 0.8 0.08
Whey to puree ratio 735.60:100 73.56:10
Lycopene (mg) 18.3 1.83

2) Patent for processing technology
• Processing Technology - lycopene enriched whey fruit juice health beverage
Processing technology parameters
i) Whey (Dairy industry by product) : Sparklingly clear
ii) Lycopene : Tomato puri as a source
iii) Fruit Juice : Sparklingly clear
Sugar-12.2°Bx
Acidity -0.35
iv) Pasteurization temperature : 72 C for 15 m
v) Packaging : Pet bottles
vi) Storage ability : 12 months at refrigerator temperature
vii) Functionality : Resistance against non communicable
diseases (obesity, diabetics, cardiovascular
diseases, cancer)
viii) Cost per unit (200ml bottle) : Rs.10/- per bottle (200ml)
ix) Total plate count : 2 X104
x) Lycopene content : 1.83mg/100ml
Therapeutic efficacy [Clinical study base ]
• Weight Control/ modulation - Linear increase in weight during experimental
tenure
• Cholesterol reduction - Decrease in cholesterol - beneficial effect
• Increase in High Density Lipoprotein - beneficial effect
• Reduction in Triglycerides- beneficial effect