Sustainable Prebiotic Dessert with Sericin Produced by Bombyx mori Worms

Abstract

(1) Background: The processing of silk threads secreted by the silkworm Bombyx mori leads to large amounts of sericin as textile waste. Its biochemical and medical properties open a new perspective for its use in the food industry and its authorization as a new food ingredient in the European Union, by the European Commission and the European Food Safety Authority. (2) Methods: Experimental and physico–chemical analyzes were carried out to obtain a sustainable prebiotic dessert with low energy value, containing Sericin produced by Bombyx Mori, which does not contain sugar and can be consumed both by people who want a healthy diet and people who need food for special nutritional conditions, such as diabetes, dysphagia or inflammatory diseases. (3) Results: The sustainable jelly was made from depectinized apple juice, pectin, sericin, lactoferrin, stevia and pectin. This dessert is natural and can be certified organic and HALAL. (4) Conclusions: Sericin can be used in the manufacture of a very wide range of foods. Being a glycoprotein, it can also be used to obtain jelling foods with low energy value, including foods for patients with dysphagia. This represents an important sustainable resource of essential amino acids for the normal homeostasis of the human body.

1. Introduction

Sericulture is an agricultural field that deals with the rearing of Bombyx mori (B. mori) silkworms. Through the endocrine system, worms secrete sericin and fibroin. Sericin protects fibroin from the action of ultraviolet (UV) rays, relative air humidity and external mechanical and biological factors. B. mori feed on leaves of trees of the genus Morus, family Moraceae [1] due to the composition in macro- and micronutrients: carbohydrates, proteins, lipids, vitamin C, nitrogen, phosphorus, potassium, calcium, sulfur, magnesium, iron, zinc, manganese, boron, copper, molybdenum, nickel, lead, lithium, titanium and organic acids (citric and malic) [1]. According to Thaipitakwong et al. [1], the protein content found in mulberry leaves is significantly higher than that in other green leafy vegetables. Also, mulberry leaves contain iminosaccharide alkaloids (e.g., 1–deoxynojirimycin in the amount of 1389–3482 mg/g, regardless of the variety), which have an inhibitory effect on the alpha–glucosidase enzyme and lead to a blood sugar-lowering action and improving insulin resistance. Other actions of mulberry leaves are antihyperlipidemic, antiobesogen, antihypertensive, antioxidant, anti-inflammatory, antiatherosclerotic and cardioprotective.

B. mori secretes silk threads composed of sericin and fibroin until the fifth larval stage, when the cocoon is still formed and the labial gland, also called the silk gland, hypertrophies. According to Kunz et al. [2], this gland is exocrine and comprises three regions: the anterior silk gland, the middle silk gland and the posterior silk gland (which secretes fibroin). The middle silk gland is subdivided into four areas (anterior, anterior–middle, posterior–middle and posterior). Each zone secretes a different type of sericin, and the posterior zone secretes the inner layer of sericin, which rapidly accumulates around the fibroin. The silk thread, which contains two layers of fibroin, is protected from external environmental and biological factors (bacteria, insects, birds) by three layers of sericin. A silk thread is between 900 and 1500 m long. To obtain the silk threads used, the cocoons are dried and processed by the textile industry. The processing consists in removing the outer layer formed by sericin. In the textile industry, this process is called degumming. This is based on the water solubility of sericin. Degumming by heat or pressure has the advantage of obtaining impurity-free sericin [2].

It is estimated that the processing of 400,000 tons of dry cocoons results in 50,000 tons of sericin, which is discharged with wastewater and thus ends up in the environment. Sericin represents about 15–30% of the weight of the cocoon. [2,3]. The removal and use of sericin has a strong economic, social and environmental impact. The white mulberry (Morus alba), black mulberry (M. nigra) and red mulberry (M. rubra) species are cultivated in Romania. In the period between 1970 and 1989, sericulture was very well-developed, but after the Revolution of 1989, this field of agriculture was destroyed. The authorization of sericin as a new food ingredient in the European Union (EU) may represent a new beginning in the re-establishment of intensive cultivation of trees of the genus Morus, family Moraceae and the pre-processing of silk threads and the obtaining of textile products (e.g., clothing and domestic textiles (such as bed linen)) from silk threads (fibroin) and the extraction of sericin and its further use. In this way, upstream and downstream jobs will be ensured, contributing to the national and European economies.

Due to its biocompatibility, silk thread (fibroin) has been successfully used in sutures [2]. In vivo and in vitro analyzes have demonstrated the safety of sericin in the fields of medicine, pharmacology, microbiology (as a supplement to culture media), cosmetics and the food industry. In medicine, sericin has applications in the following fields: anti-microbial, UV protective, anti-aging, antioxidant, hepato-protective, anti-inflammatory, anti-cancer, antiviral and wound healing [3]. In the pharmaceutical field, the latest innovations use sericin to obtain drugs in the form of nanoparticles, micelles, films and hydrogels. Sericin is associated with engineered nanoparticles (ENPs) for cutting-edge therapeutic applications [3]. Examples of sericin-based nanoformulations for biological applications: sericin and chitosan for pH-dependent release of doxorubicin and increased cellular uptake in acidic tumor environment (pH 6), sericin nanoparticles and resveratrol for significant inhibition of cancer cell growth (human colorectal adenocarcinoma, Caco2), while no cytotoxicity was observed in normal skin fibroblasts (CRL-2522), sericin-cholesterol-folate and iodide nanomics IR-780 for cancer cells (human papillomavirus-related endocervical adenocarcinoma, BGC-823) containing folate receptors [3].

Sericin is also used to obtain natural and synthetic polymers, such as chitosan, alginate, gelatin and methacrylate for the development of materials with medical applications (proliferation of fibroblasts, human medical dressings and equivalent to artificial skin). Sericin hydrogels are designed for functional skin regeneration and inflammation reduction due to various molecular mechanisms, stimulation of angiogenesis and healing process by vascular endothelial growth factor TGF-β1 and β3, accumulation of mesenchymal stem cells at the damaged site [3]. In humans, sericin induces apoptosis and acts as an antioxidant. Preclinical studies on mice have shown that feeding them with sericin for 4 weeks stimulates lymphocyte proliferation, activates NK cells (natural killers) and decreases the secretion of inflammatory cytokines (TNF-α, IFN-c and IL-10) [4]. Through in vitro studies, Niu et al. [5] demonstrated the anticancer effect of sericin, in the case of triple-negative breast cancer (TNBC). It suppressed the proliferation of cancer cells, induced cell cycle arrest and promoted cell apoptosis. The mechanism is partly explained by the down-regulation of the PI3K/Akt signaling pathway.

Kunz et al. [6] provided evidence that sericin introduced into the diet of mice, which were fed with a high-fat diet for two weeks, restored jejunal morphometry and increased fecal lipid excretion. Oral administration of sericin at doses of 2.5–5% of the daily feed ration of type 2 diabetic rats for four weeks reduced blood glucose by more than 60% compared to that of the untreated group [7]. Due to the fact the condition of the mouse embryo culture is critical to the work of transgenic, reproductive and developmental biology laboratories, Banafshi et al. [8] investigated the effect of sericin as an embryo culture medium supplement on in vitro maturation, in vitro fertilization and preimplantation embryo development in mice. The conclusion of the study was that the addition of 1% sericin to mouse embryo cultures improves the maturation rate in vivo and has a positive effect on the development of embryos derived from in vivo maturation. Also preclinically, the potential of sericin microparticles coated with metal–organic networks based on tannic acid/ferric iron (Fe³⁺) and loaded with doxorubicin was demonstrated in the treatment of metastatic lung cancer [9].

The research carried out by Aznar-Cervantes et al. [10] provides evidence that sericin, fibroin and silkworm powder, introduced into the diet, have a hypoglycemic effect [10].

Through in vivo research in Sprague-Dawley rats, Liu et al. [10,11] showed that sericin protects the kidney and prevents diabetes-induced damage by downregulating the expression levels of MKK6, p-p38MAPK, NF-κB, IL-1β, IL-6, NLRP3 and caspase-1 and inhibits the activation of renal p38MAPK signaling and NLRP3-associated inflammasome. Guo et al. [11,12] provided evidence that sericin has the effect of inhibiting MKN45 cell proliferation by regulating cellular autophagy in human gastric cancer. Rujimongkon et al. [12,13] studied the therapeutic mechanism of sericin (2.5%, 5% and 10%) in the treatment of psoriasis, in Sprague-Dawley rats (female), compared with two standard drugs with betamethasone and calcitriol. The best result was obtained after 7 days of topical treatment, at the 10% dose. The effect was similar to that of betamethasone treatment. Another study showed the effectiveness of topical treatment of psoriasis with a cream containing 8% sericin for 15 days.

According to other authors [12,13], the effect of treatment with sericin cream is similar to the mechanism of the action of calcitriol, which inhibits the expression of CeC cells motif chemokine 20 (CCL2) and leads to the reduction of inflammation and the production of Th-17 and IL-17A, by modulating the JAKSTAT signaling pathway. Deenonpoe et al. [13,14] showed that sericin, combined with naringin, down-regulates pro-inflammatory cytokines (TNF-α, IL-6, IL-23 and IL-12p40) and suggested that sericin may be used clinically in other inflammation-related diseases. Sericin down-regulated STAT3 expression and up-regulated STAT5b expression. High level of STAT5 is correlated with decreased number of Th17 cells. Qi et al. [14,15] developed a new type of composite scaffold with sericin and graphene oxide as a biometric extracellular matrix and provided histological, functional and molecular evidence demonstrating that the sericin scaffold effectively, structurally and functionally repairs calvarial bone.

Sericin biofilm has antibacterial activity (e.g., Staphylococcus aureus), stimulates bone cell proliferation and treats bone infections [15,16]. Sericin administered in the feed of mice and rats given analgesics (atropine and morphine) treated constipation occurring as an adverse reaction to opiates [16,17,18].

Constipation is a chronic non-communicable disease with an increasing prevalence. The causes of its occurrence are multifactorial, such as improper lifestyle and diet. The treatment of constipation with sericin opens new opportunities for the food industry.

Regarding the use of sericin in the food industry, the only information found so far is on granted patents. The World Intellectual Property Organization (WIPO) database was used when searching for this information. In conformity to these, sericin is used in 56 patents: drinking water and food—JP2000184868 [18,19], health food—CN107080260 [19,20], silkworm sericin drinks—CN103126028 [20,21], inhibitor of bitter and astringent taste for drinks or food—JP2012217442 [21,22], composition with hypotensive action—JP2004269395 [22,23], method of making soy sauce using silk amino acid solution—KR1020010018963 [23,24], rose flavoring and method of its preparation—CN105524762 [24,25], low-sugar prebiotic candies and their preparation method—CN103918853 [25,26]. The composition of these candies consists of freeze-dried active probiotic powder, kiwi pulp, sericin, xylitol, passion flower pulp, skimmed milk powder, microcrystalline cellulose, tea powder and polyethylene glycol. These candies are recommended for diabetics [25,26].

Another sericin patent is patent number KR1020050015178. It protects a jelly or drinks composition that contains sericin extracted from silkworm cocoons as an effective ingredient. The ingredients of this invention are 10 to 50% by weight of sericin extracted from silkworm cocoons, and an auxiliary material containing gelatin or agar [26,27]. Sericin also has technological potential in the bakery field, where the composition and process for obtaining flour assortments from various kinds of cereal and functional products with the addition of sericin, as an unconventional ingredient, are patented. The patent number is RO133023 [27,28].

One of the important directions of preventive and therapeutic medicine is presented by the use of prebiotics. They have positive effects on the intestinal microflora, metabolism, normal homeostasis of glucose and insulin resistance, improvement of the lipid profile, prevention of obesity and associated chronic diseases, and prevention of the risk of cancer [29,30].

The main objective of this work was the development of a low-energy, sustainable, sericin-containing prebiotic dessert produced by B. mori that does not contain sugar and can be consumed by both people who want a healthy diet or who need foods for special nutritional conditions (diabetes, dysphagia and inflammatory diseases).

As a dessert, we set out to develop a jelly, according to the national and European legislation in force, amended and supplemented (Order for the approval of the Norms regarding the nature, content, manufacture, labeling of jams, jellies, fruit marmalades, sweetened chestnut puree and plum marmalade intended for human consumption) [31], Directive 2001/113/EC on fruit jams, jellies and marmalades, as well as sweetened chestnut puree intended for human consumption [32] and Regulation (EC) No. 1333/2008 on food additives [33].

According to Directive 2001/113/EC [32], the Products referred to must have a soluble dry matter content of 60% or more as determined by refractometer, except for those products in which the sugars have been totally or partially replaced with sweeteners. From the point of view of the ingredients of the sustainable and prebiotic dessert, we have proposed that it does not contain synthetic chemicals, preservatives and any type of sugars from those provided by the applicable legislation for this type of food (sugars in accordance with Directive 2001/111/EC, fructose syrup, sugars extracted from fruit and unrefined sugar) or coconut sugar, even if it has a lower glycemic index compared to other types of sugar.

The ingredients we want our dessert to contain are sustainable and can be certified organic or HALAL. This category includes depectinized apple juice, inulin, pectin, sericin produced by B. mori, bovine lactoferrin and stevia.

Sericin can be used in the manufacture of a very wide range of foods. Being a glycoprotein, it can also be used to obtain gelling foods with low energy value, including foods for patients with dysphagia. This represents an important sustainable resource of essential amino acids for the normal homeostasis of the human body [2,21,22,23,24,25,26].

2. Materials and Methods

Sustainable Dietary Dessert with Sericin Produced by Bombyx Mori worms is a low-energy, sustainable, prebiotic dessert containing sericin produced by B. mori and free of sugar, synthetic substances and preservatives. It can be consumed by people who want a healthy diet, as well as people who need food for special nutritional conditions, such as diabetes, dysphagia and inflammatory diseases. To obtain this food product, we used the raw materials presented in

Table 1. The raw materials used to obtain the durable and sustainable dietary dessert with sericin produced by Bombyx mori worms.

The Raw Material	Identification Data	How to Get It
Sericin	Batch: S1911251	From the supplier 1
Bovine lactoferrin	Batch: 107CLXP	From the supplier 2
Chicory inulin 3	Batch: RHBGD1BGD1	Purchased from local trade 4
Citrus pectin	Batch: 5999884818779	Purchased from local trade 4
Stevia	Batch: 8	Purchased from local trade 5
Apple juice (depectinized)	Batch: Bm 095-22	Purchased from local trade 6
Agar—agar	Batch: 210040401	Purchased from local trade 7
Lemon	-	Purchased from local trade

¹ Sollice Biotech, France, ² KUK Romania, ³ cicory—Cichorium intybus, ⁴ Importer/distributor: Adams Vision SRL Targu Mures—Romania, ⁵ Packaged by Sly Nutritia SRL Romania, ⁶ Manufacturer S.C. Pombis S.A. Romania, ⁷ Manufacturer Biovegan GmbH Germany.

.

As can be seen from Table 1, sericin and lactoferrin cannot be purchased from local/national trade, i.e., from shops. These can be purchased as free samples or against cost, from the mentioned distributors, or other distributors, or other manufacturers (sericin from Asia, and bovine lactoferrin from the European Union).

Although Directive 2001/113/CE [32] does not provide apple juice to obtain jellies, it was chosen for this research for the following reasons: the apple culture (Malus domestica) is common in many countries; it is resistant to climate change; fruits (apples) are available throughout all year long; and unlike other fruits that are imported and stored in bulk, they are affordable for the general population. This crop can be certified organic.

In Romania there are many vegetable farms that have ecological certification for apples. Organic growers in Romania can be identified in the database of the Ministry of Agriculture, section “Agriculture”, subsection “Organic Agriculture”, subsection “Certified organic operators in 2021”, respectively, the section “Producers”:

https://www.madr.ro/agricultura-ecologica/operatorii-certificati-in-agricultura-ecologica-2021.html (accessed on 1 September 2022)

Other reasons why apple juice was chosen are the following: the powdered mixture consisting of pectin, bovine lactoferrin, sericin, inulin and stevia can be packed and marketed in stores for the general population. Packaging can be done in individual or collective packaging (e.g., 3 × 1). The premix weight of an individual package represents the dose required for a certain volume of depectinized apple juice. Depectinized apple juice was chosen because apples contain pectin, the resulting juices may contain variable amounts of pectin, and to eliminate possible quality variables (pH and Brix).

Before carrying out the experimental samples, all raw materials were analyzed from a sensory point of view (organoleoptic—appearance, color, smell, and taste) and physico–chemical (pH and °Brix (refractometric soluble dry matter)). To carry out the analyzes and experimental samples, the following measuring and monitoring equipment were used: electronic balance with two decimal places, “Digital scale”, capacity 500 g/0.01 g, pH-meter “Adwa” with measurement range 1–14, manufacturer ADWA kft, probe thermometer, special for the food industry “Checktemp °C”, manufacturer HANNA instruments, electronic refractometer HI 96801, with measuring range 0–85% Brix, manufacturer HANNA instruments and electric hob with homogenizer and re—“Nachita” rotation and temperature adjustment, model no. 690/1. Various glassware laboratory equipment were also used, such as Erlenmayer beakers, Berzelius beakers, graduated cylinders, Petri dishes and “Tefal” jelly mold. For pH measurement, the pH meter was balanced with special buffer solutions for pH 4.01 +/− 0.1 and pH 7.01 +/− 0.1. For all raw materials, three measurements/chemical parameters were made, and the final result represents their arithmetic mean.

The methods of obtaining the samples of jellies were those specific to this type of food: dosing of powdered raw materials and their homogenization, separate dosing of depectinized apple juice, over which the powdered mixture was added. The newly formed mixture (apple juice and powdered mixture) was vigorously homogenized continuously and progressively heated up to 60 °C. After reaching this temperature, lemon juice was added, homogenization was continued for 1 min, maintaining the temperature at 60 °C, after which the finished product was poured into silicone molds and kept in the refrigerator for 24 h, for the formation of bonds between pectin and the two glycoproteins (sericin and lactoferrin), inulin and stevia. The final composition can be poured into any container. We used maggots to see how the gel forms.

The depectinization of apple juice was verified by making a mixture of apple juice: ethyl alcohol of agricultural origin, with an alcohol concentration of 96%, in a ratio of 1:2. If the apple juice had been pectinized, then the pectin would have precipitated. To equate the volume with the gravimetric weight, based on the density formula ρ = m/V, the density of the apple juice used was determined. This is 1.04, which means that we can approximate that 1 mL of apple juice corresponds to one gram of juice.

The experimental plan for obtaining the prebiotic dessert with sericin produced by Bombyx mori silkworms:

Option 1 (Recipe 1): pectin 5 g, bovine lactoferrin4 2 g, sericin 1 g, stevia 1 g, inulin 15 g;

Option 2 (Recipe 2): agar—agar 1.75 g, bovine lactoferrin 1 g, sericin 1 g, stevia 1 g, inulin 15 g;

Option 3 (Recipe 3): pectin 5 g, bovine lactoferrin 2 g, sericin 1 g, stevia 1 g, inulin 15 g, calcium lactate 2 g;

Option 4 (Recipe 4): pectin 5 g, bovine lactoferrin 13 g, sericin 1 g, stevia 1 g, inulin 15 g.

All samples according to recipes 1–4 will be mixed with 200 g of depectinized apple juice.

The stages of the technological flow are the following:

1. Dosage of powdery raw materials (e.g., pectin/agar–agar, lactoferrin, sericin, stevia, inulin) and their homogenization;

2. Quantitative dosing (in grams) of depectinized apple juice;

3. Add the powdered mixture, obtained in step 1, over the apple juice;

4. The entire mixture is homogenized slowly, until the powdery mixture obtained in step 1 is completely dissolved;

5. The mixture obtained in step 4 is heated to a temperature of 65 degrees Celsius for 2 min;

6. Add the citric acid solution (citric acid and water, ratio 1:1) and homogenize until the entire mixture begins to gel;

7. Dosage of the final mixture in molds (jelly molds).

8. Rapid cooling (introduction to refrigeration (e.g., refrigerator));

9. Keeping the mold with dessert, in the refrigerator, for 24 h, until the pectin network is formed—the glycosidic groups from glycoproteins (sericin and lactoferrin).

10. Removing the jellies from the mold and eating them.

For the jellies to form more quickly, the empty molds should be kept refrigerated or frozen. After filling them with the prebiotic dessert, they can be frozen for 3–4 h.

3. Results

The results of the sensory (organoleptic) analyzes of the raw materials are presented in

Table 2. The results of the sensory (organoleptic) analyzes of the raw materials used.

The Raw Material	Sensory Analyses	Physico–Chemical Analyses
Sericin (10% 1 solution, 20 °C)	Appearance: Fine powder 2 Color: Light yellow Smell/Taste: Pleasant, specific	Brix: 11.7 pH: 4.45
Sericin (2% 3 solution, 20 °C)	Idem	Brix: 2.43 pH: 5.83
Lactoferrin (2% solution, 20 °C)	Appearance: Fine powder Color: Light pink; Smell/Taste: Pleasant, specific	Brix: 2.43 pH: 6.23
Chicory inulin (5% solution, 20 °C)	Appearance: Fine powder White color Smell/Taste: Pleasant, specific	Brix: 4.73 pH: 6.61
Pectin (2% solution, 20 °C)	Appearance: Fine powder Color: Yellow-white Smell/Taste: Pleasant, specific	Brix: 1.9 pH: 4.49
Agar—agar (1.5% solution, 20 °C)	Appearance: Fine powder Color: Yellow-white Smell/Taste: Pleasant, specific	Brix: 0.0 pH: 6.46
Stevia (1% solution, 20 °C)	Appearance: Crystals Color: Colorless Smell/Taste: Pleasant, specific	Brix: 0.9 pH: 6.48
Depectinized apple juice (100% juice, 20 °C)	Appearance: Homogeneous liquid Color: Brown Smell/Taste: Pleasant, specific	Brix: 14.26 pH: 3.68

¹ Sericin (10% solution)—according to the Analysis Bulletin received from the supplier, ² Fine, homogeneous powder, without agglomerations and foreign particles, ³ Sericin (2% solution)—to compare the results obtained with those of the 2% bovine lactoferrin solution.

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Of the two glycoproteins used in this research, only bovine lactoferrin is legally regulated in the EU, through the Commission’s Implementing Decision of 22 November 2012 authorizing the introduction of bovine lactoferrin into the market [33]. The supplier of the bovine lactoferrin sample, KUK Romania is the representative of Romania, of the lactoferrin producer mentioned in the previously mentioned Decision, Fries-land Campina (formerly known as DMV International).

The authorization of sericin as a novel food ingredient in the EU is done according to Regulation (EU) 2015/2283 of the European Parliament and of the Council on novel foods [34]. According to this Regulation, in addition to other technical and scientific requirements, the applicant must submit proposals regarding the conditions of intended use and specific labeling requirements that do not mislead the consumer or a verifiable justification that these elements are not necessary. Our research and the one patented under the number RO133023 [28] may contribute to the approval of sericin by the European Commission (EC) and the European Food Safety Authority (EFSA), as well as to other preclinical and clinical research.

The recipes applied are presented in

Table 3. The recipes applied in this research.

Recipe 11	Recipe 2 1	Recipe 3
Pectin 5 g Bovine lactoferrin 4 2 g	Agar—agar 2 1.75 g Bovine lactoferrin 1 g	Pectin 5 g Bovine lactoferrin 2 g
Sericin 1 g Stevia 1 g Inulin 15 g	Sericin 1 g Stevia 1 g Inulin 15 g	Sericin 1 g Stevia 1 g Inulin 15 g
Total weight: 24 g	Total weight: 19.75	Calcium lactate 3 2 g Total weight: 26 g
Recipe 4	Recipe 5	Recipe 6
Pectin 5 g Bovine lactoferrin 5 3 g	-	-
Sericin 1 g Stevia 1 g Inulin 15 g	-	-
Total weight: 24 g	-	-

¹ In recipes 1 and 2, 0.5 g of lemon juice was added as an acidifier and to achieve gelling, ² According to the manufacturer’s recommendations: 3.5 g per 500 mL liquid, ³ Calcium lactate was added to check the gelling power of the same recipe, compared to lemon juice. ⁴ The amount of 2 g of bovine lactoferrin is in accordance with the EU Decision [30], for the range of cakes and patisserie products, because jellies can also be used in this field: maximum 1000 mg lactoferrin/100 g product. ⁵ The amount of lactoferrin corresponds to the maximum dose provided in the EU Directive [30], for food products for special medical purposes: maximum 3 g/day.

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The amount of powdered mixture (premix) for each recipe was for 200 mL of apple juice for recipes 1.3 and 250 mL of apple juice for recipe 2 and 4. The amount of 15 g of inulin was added for the proven prebiotic effect, and relief of constipation [34] after daily consumption for 4 weeks.

The working method is the one presented in the Section 2.

Regarding the gelling ability, the results are as follows: recipes 1 and 4 worked best (Figure 1), and recipes 2 and 3 did not gel.

The jellies in Figure 1 were kept in the refrigerator for 8 h.

The sensory characteristics of the jellies obtained according to recipes 1 and 4 are: appearance—gelatinous mass with a soft consistency, color—light brown, opalescent, smell/taste—pleasant, specific to apple juice. The opalescence is imparted by pectin.

Physico–chemical parameters for the jellies obtained based on recipes 1 and 4 are presented in

Table 4. Characteristics physico-chemical parameters for jellies required based on recipes 1 and 4.

Recipe 1	Recipe 4
Brix: 25.8	Brix: 26.2
pH:5.55	pH:5.68

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From Table 4 it can be seen that the values of the two parameters are very close. Since the pH is between 5.55 and 5.68, it means that the jellies obtained are weakly acidic, towards neutral. Considering that no recipe contains preservatives or other synthetic substances, in order not to be contaminated with yeasts and molds, the jellies must be kept in the refrigerator. Considering that they contain pectin and do not contain sugar, to obtain more jellies hard, a larger amount of lemon juice can be added and kept in the refrigerator for 24 h, for the formation of bonds between pectin and glycosidic groups in glycoproteins (sericin and lactoferrin). For people suffering from dysphagia or other pathologies, including dental ones, we do not recommend adding calcium, because the jellies will be hard.

The nutritional composition of the two jelly recipes was shown in

Table 5. Average nutritional values for jellies obtained based on recipes 1 and 4.

Recipe 1 1	Recipe 4 2
Energy value: 615 KJ/147 Kcal	Energy value: 757 KJ/181 Kcal
Fats: 0.39	Fats: 0.82
of which saturated fatty acids: 0.39	of which saturated fatty acids: 0.82
Carbohydrates: 33.28	Carbohydrates: 40.28
of which sugars: 24.32	of which sugars: 29.32
Polyol: 0.98	Polyol: 0.98
Proteins: 1	Proteins: 1.15
Fiber: 14.5	Fiber: 14.75
Salt: 0.375	Salt: 0.375

¹ For 224 g/day, ² For 274 g/day.

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4. Discussion

As can be seen from the state of knowledge and the presented patents, the dessert developed by us was not made until now. The fact that the product developed by us is a powdered premix and can be used in the preparation of other food products (e.g., jellies) and by end consumers at home makes this product have industrial applicability and high sales potential. All the ingredients of the sericin dessert contribute to the normal activity of the intestine, and through the content of inulin, it is prebiotic, through a greater abundance of Bifidobacterium spp [34].

There are numerous preclinical studies demonstrating the effect of inulin consumption: reduction of weight gain and liver weight and plasma triglycerides [32,33,34,35], reduction of hepatotoxicity mediated by the dioxin-like pollutant PCB 126 and intestinal dysbiosis in Ldlr-deficient hyperlipidemic mice [35,36], reduction cholesterol [37], prevention of inflammatory bowel disease, by modulating the trophic functions of the flora, increasing indigenous Lactobacilli and/or Bifidobacteria by inducing the colonic production of short-chain fatty acids (SCFA), reducing mucosal damage and reducing mucosal inflammation [38]. Randomized double-blind clinical trials have shown the role of inulin in increasing fermentation in patients with type 2 diabetes [39] and in improving insulin control [40].

The non-caloric sweetener stevia (derived from Rubus suavissimus) has antioxidant properties and contributes to the neutralization of free radicals, has antidiabetic, antihypertensive activity, reducing weight gain in obesity, total cholesterol, triglycerides and low-density lipoprotein concentrations and increasing high-density lipoprotein high, increase in renal plasma flow, systemic and renal vasodilatation, arterial hypotension, diuresis, improvement of some biochemical parameters in patients with chronic kidney disease [41,42,43].

Lactoferrin has antioxidant, prebiotic, antiviral, antimicrobial and anti-inflammatory activity acting on interleukins 1, 4, 6, 10 (IL-1, IL-4, IL-6, IL-10, IL-12), tumor necrosis factor (TNF-α) and Th1-type cytokines, such as IFN-γ and IL-12 [44,45,46].

Sericin has an antioxidant, anti-inflammatory, scarring action, anti-cancer, anti-obesity, low cholesterol effect, treating constipation as an adverse reaction to opiates.

Citrus pectin prevents isoproterenol-induced cardiac hypertrophy associated with p38 signaling and the TLR4/JAK/STAT3 pathway [47], prebiotic [48] and antihypertensive action [49].

The premix designed by us and the dietetic and sustainable dessert with sericin produced by Bombyx mori worms can be certified organic and HALAL and is easy to be prepared and consumed, including by people with dysphagia or dental diseases. The dessert is prebiotic, rich in fiber and low in energy value. Due to the health benefits, the efficacy of the premix and the dessert (jelly) can be studied preclinically prospectively, for the treatment of diabetes, obesity, cholesterol-lowering, against cancer, inflammatory dermatological diseases (e.g., atopic dermatitis, psoriasis) or any other chronic inflammatory disease. The premix researched by us can also be used to prepare ice cream or other functional foods with essential amino acid content [49,50]. The present research may contribute to obtaining a favorable scientific opinion from EFSA and EC for the authorization of sericin as a new food ingredient in the EU, from the point of view of applications.

The preclinical verification of the effectiveness of the anti-inflammatory and constipation treatment effect will be carried out within a funded project.

5. Conclusions

Sericin, secreted by Bombyx mori silkworms, is a glycoprotein with multiple health benefits and can be used in medicine, the pharmaceutical industry or in the food industry to obtain foods with low energy value, including desserts, sauces, bakery products, patisserie, confectionery and ice cream.

Our research presents a therapeutic solution in preventive and therapeutic medicine. This research has the following disadvantages: due to the fact that the premix developed by us is natural and does not contain preservatives and calcium lactate, the jellies obtained have a short shelf life and low mechanical resistance. For the authorization of sericin as a new food in the European Union by EFSA, additional research is needed.