DESC:FIELD OF THE INVENTION
The present invention relates to novel complexes of ß-glucan and Lactoferrin and its process for preparation.

BACKGROUND OF THE INVENTION
ß-glucan is a polysaccharide or a mixture of oligosaccharides of varying lengths with a basic skeleton of ß-(1?3)-glycosidic bonds which are found in the cell walls of yeast, bacteria, algae, fungi, lichens, oats and barley. They can be classified based on the source, cereal or non-cereal which is largely based on structure. ß-glucans from barely also differ structurally from those of oats origin. A similar diversity exists within ß-glucans from non-cereal sources. In general, all the ß-glucans are homo-polysaccharides and essentially, composed of glucose units linked together with a characteristic 1?3 linked backbones. The structural difference occurs at branching off this backbone, which is dictated by source. ß-glucans can be either branched or unbranched. Branching can usually occur at either the 1?4 or 1?6 position. These molecular and structural characteristics are fundamental to their activity which determine defined structure–activity relationship.

Cereal or grain derived ß-glucans usually have 1?3, 1?4 glycosidic linkages without any 1?6 bonds or branching. Non-cereal sources usually have 1?6 linked branches off the main side chain. Other glucans such as Curdlan, a glucan isolated from Agrobacterium does not contain side branching, but just has a ß-glucan backbone. Some exceptions such as Sorghum arundinaceum, an ancient cereal grain, was found to contain ß-glucans with alpha 1?4 linked D-glucopyranose residues with 1?3, 1?6 branching points. Moreover, different species of Sorghum have different structures; Sorghum bicolor contains 1?3 with 1?4 linkages.

ß-glucans can be recognized as antigens by macrophages via pathogen-associated molecular patterns, and possess the ability to activate peripheral immune cells and hence known to possess immunity-enhancing properties. Several studies, indicate that ß-glucans are effective substances that help the body in the key defense mechanisms of immunomodulation. This effect occurs through numerous mechanisms, including the stimulation of cellular and humoral immunity, control of metabolic diseases, such as diabetes, stimulation of regenerative systems, such as wound healing, attenuation of chronic fatigue and stress, cancer inhibitory stimuli, and lowering cholesterol to name a few. Oral supplementation with ß-glucan is the most used and widely studied and known to be safe in the doses 35 mg to 500 mg a day. ß-glucan from oats is acknowledged by the U.S. Food and Drug Administration as being safe and is listed as a GRAS (Generally Recognized as Safe) ingredient. It is an irreplaceable supplement for diabetics as it regulates the level of glucose in the blood by slowing down its absorption after eating. The beneficial effects also include reduction of serum cholesterol and glucose immunomodulation, antitumor activity and obesity prevention.

Topical application of ß-glucans in dermatology is another interesting segment where their pluripotent mechanisms of actions such as antioxidant, anti-inflammatory, regeneration effects, immunomodulation, radioprotection, moisturization, rejuvenation could help as complementary therapy in the management of various skin diseases. In clinical medicine, topical application of ß-glucans was successfully studied in the treatment of various skin diseases and conditions such as radiation dermatitis, venous ulcers, wound healing, solar keratosis, HPV-associated vulvar lesions and contact dermatitis.

On the other hand, Lactoferrin is a protein belonging to the transferrin family, discovered for the first time in cow milk in 1939. It is produced by the epithelial mucosa. It is present in milk and colostrum, but can also be found in tears, saliva, gastric mucosa, the spleen, lymph nodes, skin, and even white blood cells. Lactoferrin consists of about 700 amino acid residues, with a molecular weight of ~78 kDa. Bovine Lactoferrin consists of 696 amino acids, while human Lactoferrin has 691.

The saturation of Lactoferrin with iron (as one of the transferrins) significantly influences its structure. Each Lactoferrin molecule contains two N- and C-terminal lobes joined by a helix. Each lobe consists of parts N1 and N2 or C1 and C2. Both lobes possess sites for attaching sugar residues and binding iron. Each of these homologous lobes can attach one iron atom. The forms of Lactoferrin are classified according to the number of bound iron atoms. Lactoferrin which attaches iron at each of the two lobes is called holo-Lactoferrin, whereas the form that has no iron at all is known as apo-Lactoferrin. There are also intermediate forms which have iron attached at only one lobe. The number of attached iron atoms influences the structure of Lactoferrin. The more number of iron atoms, greater is the cohesive nature, and thus greater resistance to proteolysis and higher temperatures.

Owing to its iron binding capabilities, Lactoferrin has been used to treat anaemia or iron deficiency. Primary function of Lactoferrin is to bind iron at the molecular level and thereby act as a highly effective antimicrobial agent. Iron is an essential growth factor for virtually every cell and microorganism, and free iron promotes the growth of pathogens in the intestines (bacteria, fungi, and viruses), permitting invasion of the rest of the body through the intestinal walls. Lactoferrin is released by neutrophils to absorb free iron that would otherwise be available to bacteria, viruses and fungi for growth.

Anti-inflammatory, anti-infective, and immunoregulatory agents have been shown to be useful in treating infections with SARS-CoV-2. Lactoferrin exhibits all of these effects and can be included in the treatment of SARS-CoV-2 infection. Lactoferrin has been shown to exhibit activity against a wide range of viruses, including SARS-CoV, which is closely related to SARS-CoV-2. Lactoferrin can inhibit the binding of SARS-CoV-2 to host cells. Research by the New Zealand pharmaceutical producer Quantec showed that a protein complex containing Lactoferrin and lactoperoxidase, obtained from fresh pasteurized cow milk, can protect human cells against COVID-19.

Lactoferrin exhibits protective activity in obesity. Lactoferrin supplementation improves body mass index (BMI), waist–hip ratio (WHR), and fasting glucose concentration, as well as insulin sensitivity. Lactoferrin reduces the accumulation of visceral fat and suppresses appetite, which is particularly important for fighting obesity, a growing problem all over the world.

A daily intake of 250 mg of bovine Lactoferrin per day confirmed that it exerts anti-inflammatory, immunomodulatory, prebiotic efficacy and hypoglycaemic effects and improves insulin sensitivity. Bovine Lactoferrin supplementation can thus ensure better management of glycaemia rather than the use of conventional antidiabetic drugs alone. It is also used to prevent cardiovascular disease and exerts a beneficial effect on lipid metabolism by reducing the concentration of total and LDL cholesterol. The positive correlation between a higher serum concentration of bovine Lactoferrin and a lower BMI suggests that Lactoferrin can be used to prevent cardiovascular disease.

Lactoferrin also regulates the release of tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in vivo. Lactoferrin increases the number of fresh neutrophils in circulation by up to 116%. Lactoferrin has also found its application in tissue engineering.

Lactoferrin has diverse applications in the food, pharmaceutical and cosmetics industries. It is used in infant formulas, nutraceuticals, fermented milk products, processed meats, and dietary supplements. In the pharmaceutical and cosmetics industries, it appears as an ingredient in preparations supporting the treatment of herpes or skin lesions, strengthening immunity, raising iron levels, supporting the intestinal microbiota, or limiting bacterial growth. Lactoferrin promotes metabolic processes in the skin and is known to possess an anti-aging effect because of its antioxidant capacity, which scavenges free radicals. Due to its anti-bacterial activity, it is an essential ingredient in skin cleansers. Lactoferrin is also used in hairsprays for smoothing and hydrating hair. Further research is needed to extend the use of Lactoferrin in beauty products, including hair, face, make-up, and body care items. (Food Funct., 2022, 13, 11954).

Nichols & McKee, U.S. Pat. No. 4,977,137 suggests the use of milk Lactoferrin from human and other mammalian sources as a dietary ingredient or supplement. The Lactoferrin promoted growth of the gastrointestinal tract of human infants or non-human animals immediately on birth.

Tanaka et al., U.S. Pat. Nos. 5,098,722 and 5,008, 120 suggest methods of preparing iron-fortified beverages that contain a solution of purified bovine Lactoferrin and provide high bio-availability of iron.

Tomita et al., U.S. Pat. No. 5,304,633 presents fragments of milk Lactoferrin having potent antimicrobial activity.

Konig et al., U.S. Pat. No. 5,466,669 offers an immunostimulatory agent comprising a peptide derived from Lactoferrin.

Kunio et al., U.S. Pat. No. 5,576,299 suggests the use of Lactoferrin for preventing and treating the opportunistic infections that arise in immuno-compromised individuals.

Yamamoto et al., U.S. Pat. No. 5,725,864 offers the use of an iron-binding protein, of which Lactoferrin is one of several examples, for inhibiting infection or suppressing growth of human immunodeficiency virus. The protein is administered by diffusion through any of several epithelial membranes, or by injection.

Valenti & Antonini, U.S. Pat.No. 5,834,424 disclose the use of compositions containing Lactoferrin or other iron-binding proteins for treating gram positive bacterial infections.

Headon et al., PCT/US90/02356 and European Patent No. 0471 011 B1 presents the verified cDNA sequence of human Lactoferrin.

Kruzel, PCT/US91/01335 offers human Lactoferrin expressed from recombinant DNA, its method of production and purification and its use for Supplementing the diet with trace elements or as a topical antiseptic.

Kruzel et al., PCT/US95/05653 discusses the cloning, expression and uses of recombinant human Lactoferrin for retarding food spoilage, as a topical antiseptic, for inhibiting microbial growth in or on a mammal, for regulating iron levels within a mammal or for a nutritional supplement.

The strongly acidic conditions of the stomach, and the function of the proteolytic enzymes and Zymogens produced in the pancreas and acting in the intestines, are well known to inactivate and degrade the delicate structures of proteins, such as the components of the dietary supplements described here. The species-specific glycosylation of Lactoferrins from different mammalian sources may provide protection from proteolysis for Lactoferrin ingested naturally from maternal milk, and cross-species administration of Lactoferrin would be expected to be far less effective (Lonnerdal & Iyer, Annu. Rev. Nutr, 15:93-110, 1995). Even if the Lactoferrin succeeds in reaching the small intestine intact, specific Lactoferrin receptors enable human Lactoferrin to deliver iron to the mucosal cells of human small intestine, whereas bovine Lactoferrin is incapable of doing so (Cox et al., Biochim. Biophys. Acta, 558: 129-141, 1979).

The gastric survival of bovine Lactoferrin has been studied (Troost FJ, et al., J. of Nutrition, Aug; 131(8):2101 2104, 2001). Gastric survival of bovine Lactoferrin, analyzed by gel permeation chromatography under denaturing conditions, was only 62%. Buffering with a gastric pH buffer only increased gastric survival to 64%. Surface plasmon resonance analysis indicated that bovine Lactoferrin binds more strongly to salivary agglutinin, especially to high molecular mass glycoprotein, which is a component of the agglutinin (Mitoma M, et al., J. Biol. Chem., 276(21): 18060 18065, 2001). Mitoma demonstrated that the binding of bovine Lactoferrin to salivary agglutinin was thermostable, and the optimal pH for binding was 4.0. Bovine Lactoferrin binding with Salivary agglutinin in the mouth results in a more stable Lactoferrin component. Mucosal delivery in the mouth is preferred over the gastric delivery of Lactoferrin. Accordingly, gastric delivery in the form of an enteral feed preparation or the swallowing of a capsule requires approximately 150% of the amount of Lactoferrin that could be delivered directly into the mouth. Moreover, such a dietary supplement must be absorbed effectively, without the degradation of protein constituents that is associated with regular digestive processes such as the destruction of delicate immunoglobulins by acids in the stomach.

Dietary supplement compositions containing ß-glucan, Lactoferrin, Mannitol, Sorbitol, natural lemon flavor, and Silicon dioxide are disclosed by Marcus B. Gohlke in the patent US 2002/0054917 A1 where they formulated the combination as a liquid, tablet, lozenge, gum, or other acceptable forms. However, to date there are no reports of the molecular complexes of ß-glucan and Lactoferrin.

In spite of the knowledge of the beneficial properties of ß-glucan and Lactoferrin, there still remains an unrealized scope to explore the potential synergistic properties of this combination. In addition, the stable complexes of this combinations have great benefit on the delivery mechanisms that can not only load a considerable amount of bioactive Lactoferrin but also offer gastric protection.
In view of the above enormous potential of ß-glucan and Lactoferrin combination in Cosmeceutical, Pharmaceutical and Nutraceutical industries, it is desirable to develop an efficient and robust process for preparation of stable complexes of ß-glucan and Lactoferrin in high purities and yields, such that the bioavailability both the components gets enhanced.

SUMMARY OF THE INVENTION
The present invention provides novel complexes of ß-glucan and Lactoferrin and its process for preparation.

In one embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin, which comprises:
Dissolving ß-glucan and Lactoferrin in distilled water and stirring at room temperature or heating up to 50 °C. The resultant solution was subsequently lyophilized using the freeze dry system to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin, which comprises:
Dissolving the ß-glucan and Lactoferrin in distilled water and stirred at room temperature or heating upto 50 °C. Evaporated the solvent under vacuum to obtain the complex.

In another embodiment, the present invention provides a process for the preparation of novel complexes of ß-glucan and Lactoferrin, which comprises; Grinding dry or solvent assisted mixture of ß-glucan and Lactoferrin to obtained a complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin, which comprises:
Extrusion at elevated temperatures or at room temperature of ß-glucan and Lactoferrin mixture. The extruded material was observed to be the complex of ß-glucan and Lactoferrin.

FIGURES
Figure 1: IR of ß-glucan
Figure 2: IR of Lactoferrin
Figure 3: IR of Lyophilization complex of ß-glucan and Lactoferrin
Figure 4: IR of Evaporation complex of ß-glucan and Lactoferrin
Figure 5: IR of Grinding complex of ß-glucan and Lactoferrin

DETAILED DESCRIPTION OF THE INVENTION
ß-glucan will obtained from various sources like microbial, fungal, mushroom, yeast and plant sources.

Lactoferrin means Apo Lactoferrin & Halo Lactoferrin, obtained from Human, Bovine, Goat and Camel milk and synthesized (biocatalytic methods).

Accordingly, the present invention provides novel complexes and various processes in different ratios for the preparation of novel complexes of ß-glucan and Lactoferrin as shown below.

In one embodiment, the present invention provides a process for the preparation of novel complexes of ß-glucan and Lactoferrin by the drying (freeze drying) method.

In step-a, ß-glucan and Lactoferrin were dissolved in 100 mL of double distilled water and stirred at room temperature to 50 °C, preferably at room temperature.

The duration of the reaction may range from 2-14 hours, preferably for a period of 12 hours.

In step-b, the resultant solution was subsequently lyophilized using freeze dry system to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin by evaporation method.

In step-a, ß-glucan and Lactoferrin was dissolved in distilled water and stirred at room temperature to 50 °C, preferably at room temperature.

The duration of the reaction may range from 2-14 hours, preferably for a period of 12 hours.

In step-b, the mixture obtained in step-a is taken and evaporated the solvent under vacuum to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin by grinding method.

In step-a, ß-glucan and Lactoferrin was taken in a mortar and 3 to 10 drops of water was added, preferably 3 drops.

In step-b, the mixture obtained in step-a is grounded with pestle to obtain the complex.

In another embodiment, the present invention provides process for the preparation of novel complexes of ß-glucan and Lactoferrin by extrusion method.

Appropriate stoichiometric blends of ?-glucan and Lactoferrin were prepared and used in extrusion experiments. Extrusion experiments were conducted by passing the above blends through a co-rotating twin-screw extruder. Twin Screw Extrusion (TSE) parameters such as screw design, temperature, and residence time were studied in a series of experiments to evaluate conditions required for formation of ?-glucan and Lactoferrin complex. For the ?-glucan and Lactoferrin complexes, extruder temperature was performed at room temperature to 50 ? throughout the screw zones, while the volumetric feed rate was set at 5%, and screw speed was fixed at 100 rpm.

EXPERIMENTAL PORTION
The details of the invention are given in the examples provided below, which are given to illustrate the invention only and therefore should not be construed to limit the scope of the invention.

Example-1: In vitro anti-oxidant activity of the Cream product formulated with ß-glucan and Lactoferrin complex against H2O2 induced oxidative stress in Human keratinocytes (HaCaT) cell line.
The cytotoxicity of the cream product formulated with ß-glucan and Lactoferrin complex was determined in terms of percentage cell viability, and it was found to be more than 70% (75.72 ± 0.19%) at higher concentration (500µg/mL) on Human Keratinocyte (HaCaT) cell line (Table 1).

In the present study, cream product formulated with ß-glucan and Lactoferrin complex significantly increase the GPx and Catalase activities in LPS treated groups when compared with the cell control. This result indicates the modulatory effect of test substances on oxidative free radical scavenging enzymes activity (Table 2) against LPS induced pathological states in HaCaT cell lines.

Table 1: In vitro cytotoxicity of the cream product formulated with ß-glucan and Lactoferrin complex in terms of percentage cell viability against Human Keratinocyte (HaCaT) cell line by MTT assay
Test substance Concentration
(µg/mL) Percentage of cell viability after treatment (Mean ± SD)
Cream formulation formulated with ß-glucan and Lactoferrin complex 1000 38.98 ± 2.82
500 75.72 ± 0.19
250 86.33 ± 1.30

Table 2: Anti-oxidant activity of test product cream formulation formulated with ß-glucan and Lactoferrin complex in Human Keratinocyte (HaCaT) cells against LPS induced toxicity by estimation of glutathione peroxidase (GPx) and Catalase (CAT) levels.
S.No Samples GSH-Px Level
(U/mgprot) CAT Level
(U/mgprot)
1 Cell control 100.3 ± 4.1 99.8 ± 2.3
2 LPS- 1 µg/ml 67.06 ± 3.9 51.61 ± 1.6
3 Cream product formulated with ß-glucan and Lactoferrin complex 250 µg/ml 102.32 ± 1.9 117.38 ± 3.1
4 Cream product formulated with ß-glucan and Lactoferrin complex –125 µg/ml 85.33 ± 2.2 96.57 ± 2.8
5 Standard- Quercetin-250 µg/ml 107.51 ± 3.1 151.64 ± 3.1

The above result concluded that the cream product formulated with ß-glucan and Lactoferrin complex significantly increase the GPx and Catalase activities in Human Keratinocyte cells and have potent anti-oxidant activity in LPS treated groups when compared with the cell control. This result indicates the modulatory effect of cream product formulated with ß-glucan and Lactoferrin complex on oxidative free radical scavenging enzymes activity against LPS induced pathological states in HaCaT cell lines.

Example-2: Evaluation of moisturizing effect of the cream formulated with ß glucan and Lactoferrin complex by AQP-3 gene expression modulation in Human Keratinocytes.
Reverse Transcriptase-PCR experiment was performed by using gene specific primers. Quantitative RT-PCR analysis revealed the modulatory effect of test product on the mRNA expression of AQP3 gene over cell control. The gene AQP3 encodes the water channel protein aquaporin 3, water-transporting proteins, which play key role in providing proper skin hydration and maintaining water balance in the cell layers. The activity level of AQP3 in the epidermis showed to be associated with the degree of skin hydration moisturizing property.

Table 3: The quantitative expression level of AQP-3 gene normalized to GAPDH in terms of expression fold.
S.No Test sample AQP-3 Expression Fold
1 Cream product formulated with ß-glucan and Lactoferrin complex - 500µg/ml 0.93
2 Cream product formulated with ß-glucan and Lactoferrin complex - 250µg/ml 0.53
3 Standard- Hyaluronic acid
500µg/ml 1.71
4 Cell control 0.63

The mean level of AQP3 expression in cream formulated with ß-glucan and Lactoferrin complex treated cells with control cells was compared and relative fold expression was reported. Results showed that the upregulation of AQP3 gene expression in cells treated with test product and it was 0.93 fold higher than the control cells (0.63 fold), at 500 µg/mL concentration. This result indicates the AQP-3 gene modulation efficacy of cream formulated with ß-glucan and Lactoferrin complex, and there by it upsurge the ability to transport water and glycerol within the epidermis. This transported water is likely to play a role in epidermal hydration and hydrostatic pressure which maintain the skin texture by moisturizing effect.

The result concluded that the test product cream formulated with ß-glucan and Lactoferrin complex exhibited moisturizing property in Human Keratinocytes cells by modulating the AQP-3 gene expression.

Exampe-3: Evaluation of melanin inhibitory properties against UV induced melanin synthesis of cream formulated with ß glucan and Lactoferrin complex in Mouse Skin Melanoma (B16-F10) cell line.
Studies were carried out for the product formulated with ß-glucan and Lactoferrin complex at the non-toxic concentration of 500µg/mL and 250µg/mL. Exposing human skin to UVA radiation triggers reactive oxygen species -ROS generation and mediates excessive melanogenesis in skin cells leading to pigmentation. In this study, the percentage protection of ß-glucan and Lactoferrin complex against UV induced-Melanin synthesis in Human keratinocytes was evaluated (Table 4), and it revealed 44.94 ± 0.009% and 26.05 ± 0.001% melanin inhibition at 500 µg/mL and 250µg/mL concentration.

Table 4: Melanin inhibition percentage of test product against UV induced melanin synthesis in Mouse Skin Melanoma (B16-F10) cell line
S. No Name of
Test sample Test
Concentration % Melanin Inhibition
1 Cream product formulated with ß glucan and Lactoferrin complex 500µg/mL 44.94 ± 0.009
250µg/mL 26.05 ± 0.001

The test result concluded that Cream formulation formulated with ß-glucan and Lactoferrin complex exhibited with the efficacy to reduce melanin production in melanoma cells and their by reducing skin pigmentation.

Example-4: Process for preparation of novel complex of ß-glucan and Lactoferrin by freeze drying method.
Step-a:
A 5.0 grams of ß-glucan and 5.0 grams of Lactoferrin was dissolved in 100 mL of double distilled water and stirred at room temperature for 12 hours.
Step-b:
The resultant solution obtained in Step-a was subsequently lyophilized using freeze dry system to obtain the complex.

Example-5: Process for preparation of novel complex of ß-glucan and Lactoferrin by evaporation method.
Step-a:
5.0 grams of ß-glucan and 5.0 gram of Lactoferrin was dissolved in 100 mL of double distilled water and stirred it for 12 hours at room temperature.
Step-b:
The mixture obtained in Step-a is taken and evaporated the solvent under vacuum at below 40 °C to obtain the complex.

Example-6: Process for preparation of novel complex of ß-glucan and Lactoferrin by grinding method.
Step-a:
10.0 grams of ß-glucan and 10.0 grams of Lactoferrin were taken in a mortar and 3 drops of water was added.
Step-b:
The mixture obtained in Step-a is grounded for 15-20 minutes with pestle to obtain the complex.

Example-7: Process for preparation of novel complex of ß-glucan and Lactoferrin by extrusion method.
Step-a:
10.0 grams of ß-glucan and 10.0 grams of Lactoferrin were blended.
Step-b:
The extrusion experiment was conducted by passing the above blends (Step-a) through a co-rotating twin-screw extruder. The extruder temperature was performed at room temperature to 50 ? throughout the screw zones, while the volumetric feed rate was set at 5%, and screw speed was fixed at 100 rpm.
,CLAIMS:We claim

1) Novel complexes of ß-glucan and Lactoferrin and its process for preparation.

2) The process of preparation of novel complex claimed in claim 1, which comprises of:
a) dissolving ß-glucan and Lactoferrin in distilled water and stirring at room temperature or heating up to 50 °C;
b) the resultant solution was subsequently lyophilized using the freeze dry system to obtain the complex.

3) The process of preparation of novel complex claimed in claim 1, which comprises of:
a) dissolving Beta-glucan and Lactoferrin in distilled water and stirring at room temperature or heating up to 50 degrees;
b) the mixture obtained in step-a is taken and evaporated the solvent under vacuum to obtain the complex.

4) The process of preparation of novel complex claimed in claim 1, which comprises of Grinding dry or solvent assisted mixture of ß-glucan and Lactoferrin to obtain the complex.

5) The process of preparation of novel complex claimed in claim 1, which comprises of Extrusion of ß-glucan and Lactoferrin mixture. The extruded material was observed to be the complex of ß-glucan and Lactoferrin.

6) The novel complex of ß-glucan and Lactoferrin claimed in claim 1 is used for the treatment of Hydration.

7) The novel complex of ß-glucan and Lactoferrin claimed in claim 1 is used for the treatment of Anti-inflammatory diseases.

8) The novel complex of ß-glucan and Lactoferrin claimed in claim 1 is used as Antioxidant.