*2.3.1 Moisture*

By drying a sample in oven at 105°C until a constant weight was detected, the moisture content was examined using the following formula:

Moisture% = (initial weight-final weight x 100) /weight of the sample.

**Figure 6.** *Sample preparation.*

*Therapeutic Potential of Chocolate Coated Chinese Chestnut (A Nut Cum Fruit) DOI: http://dx.doi.org/10.5772/intechopen.108976*

#### *2.3.2 Protein*

The total nitrogen content was ascertained using the Kjeldahl method. The nitrogen percentage was first determined, and then it was multiplied by a factor of 4.86, or 4.86 x N to determine the protein content percentage (nitrogen percentage).

#### *2.3.3 Carbohydrate*

The content of the readily available carbohydrate was calculated by differences i.e., by subtracting from 100 the total of the values for moisture, protein, ash and fat (per 100gm).

#### *2.3.4 Fat*

As a crude ether extract of the dry material, fat is extracted from the sample. Firstly, the dry sample weighs about 5–10 gm into a thimble and covered with cotton. The thimble is then put in a Soxhlet Apparatus and extracted for 15 to 16 hours with anhydrous ether. After filtering the ether extract into the weighted conical flask, it was cleaned 4–5 times. After the ether has been eliminated through evaporation, the remaining material is dried in an oven at 80 to 100°C, cooled in a desiccator, and then weighed.

Fat content (g/100gm of sample) = (weight of ether extract x 100) /Weight of sample (equivalent to fresh sample taken).

#### *2.3.5 Ash*

Ash content was determined gravimetrically of the sample residue after ignition in vacuum oven at 512°C to constant weight.

Ash content (g/100 gm sample) = (Weight. of the ash x 100) /Weight. of the sample taken.

#### *2.3.6 XRD (X-ray diffraction)*

A laboratory-based method known as X-Ray Diffraction (XRD) is typically used to analyse unit cell dimensions and identify crystalline materials [24]. It is frequently used in a variety of disciplines, including engineering, geology, material science, and environmental sciences, to quickly identify unidentified crystalline substances using Bruker D8-eco Advance XRD (usually within 20 minutes).

It follows *Bragg's Law: nλ = 2d sinθ* formula.

It is necessary to use pure, finely ground, and homogenised samples to determine the bulk composition. This method can also be used for the identification of finegrained minerals, quantitative determination of modal quantities of minerals in a sample, comprehensive characterisation of crystalline materials, and identification of unit cell dimensions.

#### *2.3.7 Chocolate bloom*

When chocolate blooms, it means that the chocolate was not stored properly. However, one can also eat the bloomed chocolates. The two types of blooms that were seen in this study were:


#### **2.4 Microbiological analysis**

The dark-chocolate chestnut shells (DCC) which were stored at 7°C and 25 ± 2°C examined for the microbiological analysis. The reference sample (REF) was the marketed fruit and nut dark chocolate that was purchased from a nearby market. Samples were analysed for TPC (Total Plate Count), Salmonella spp., Escherichia coli, as well as yeast and moulds. The growth media used for the micro- organisms identification were: Nutritive Agar (for Total Plate Count), MacConkey Agar (for E. coli), Czapek Agar (for yeasts and moulds) and Salmonella-Shigella Agar, Baird-parker Agar (for Staphylococcus aureus).

For each of the experiments mentioned, about 3 g of sample was powdered and homogenised in 30 ml of broth in a sterile flask before being incubated for 24 hours at 37°C. All the samples were aseptically removed and progressive dilution of 102 decimal were prepared and later plated on appropriate media. The plates were then incubated for a further 48 hours at 37°C, during which time the colonies were manually counted using a permanent marker pen. It was shown that there were variations between the two samples at a 90% significance level [25].

#### **2.5 Organoleptic properties**

The distribution of the samples was done as follows for this purpose:


#### *2.5.1 Preparation of the 'Drinking Chestnut Chocolate Shells'*

Ingredients included: 250gm of ground Chinese chestnut, 50gm of roasted and ground flaxseeds, 2 tablespoons of ghee to roast the ingredients and give the chocolate a glossy appearance, 50gm of drinking chocolate powder, and 150gm of chocolate compound. No sugar was added before, during, or after preparation. **Figure 6** gives the step-by-step procedure for the preparation of this product. The final product was covered in glossy RED wrapping foils with the code T1 (SAMPLE-1) (**Figures 7** and **8**).

#### *2.5.2 Preparation of the 'Hershey's Chestnut Chocolate Shells'*

The ingredients included: 250 gm of ground Chinese chestnut, 50 gm of roasted and ground flaxseeds, 2 tablespoons of ghee to roast the ingredients and give the chocolate a glossy appearance, 50 gm of Hershey Chocolate Syrup, and 150 gm of chocolate compound. No sugar was added before, during or after preparation. **Figure 6** gives the step-by-step procedure for the preparation of this product. The final product was covered in glossy YELLOW wrapping foils with the code T2 (SAMPLE-2) (**Figures 8** and **9**).

*Therapeutic Potential of Chocolate Coated Chinese Chestnut (A Nut Cum Fruit) DOI: http://dx.doi.org/10.5772/intechopen.108976*

**Figure 7.** *Drinking chestnut chocolate shells.*

**Figure 8.** *Wrapped chocolates: T1 – Red foil, T2 – Yellow foil, T3 – Blue foil.*
