Diet has taken on a new importance as the result of heightened global commitment to prevention of disease, especially of the gastrointestinal tract.¹ WHO states that digestive disorders affect more than 40 per cent of the world population, and their prevalence is rising in developed as well as developing countries.^2,3 While traditional dairy based probiotics, like yogurt, have already proven beneficial, their range is limited to sensory preference and lactose intolerance.⁴ At the same time, lifestyle associated gastrointestinal problems and immune deficiencies are increasing, heavy economic charge on healthcare systems.⁵ The total market value of the global functional food was USD 177.77 billion in 2021 and is expected to witness a CAGR more than 8.5% till 2030 due to the rising demand for a range of food stuffs in the market that adds health benefits over basic nutrition.⁶ But there are limitations of current formulations to the stability and acceptability of live probiotic cultures. As such, there is an urgent need to develop functionsally effective palatable and stable probiotic carriers that fill the current therapeutic gaps in gastrointestinal health.

A robust solution in the incorporation of Lactobacillus spp. particularly Lactobacillus acidophilus and Lactobacillus rhamnosus into food matrices, as they already prove to offer a role in restoring the gut microbiota, improving immune response and preventing the growth of pathogenic organisms.^7,8 These gram positive, facultative anaerobic bacteria have a cell wall composed of a peptidoglycan heavy building, making them resistant to the harsh conditions of gastric pH and bile.⁹ The mechanism of action of their proposal is competitive exclusion of harmful microbes, production of lactic acid, and modulation of immune responses through gut associated lymphoid tissue (GALT).¹⁰ Numerous clinical studies have shown benefits such as decreased antibiotic-associated diarrhea, irritable bowel syndrome symptoms, and had reported modest lipid lowering effects. In addition, L. acidophilus represents a viable probiotic for frozen foods since it maintains viability under refrigerated and frozen conditions.¹¹ It has also been shown to promote mood and cognitive function by means of the gut-brain axis, a topic of frontier research in neurogastroenterology. This relates to its use as a bioactive agent for creating therapeutic and functional food products such as probiotic ice cream.¹²

An innovative delivery matrix for probiotic organisms is probiotic ice cream, which also encourages consumer compliance.¹³ Ice cream is different from yogurt in that the fat content and cold storage of ice cream provides such protective barrier to harsh external conditions thus enabling long term stability of live cultures.¹⁴ Due to its emulsified nature, the ice cream matrix will ensure uniform distribution of probiotics while sugars and dairy form prebiotics that support bacterial growth during storage. More recently, improving triality probiotic survival rates from freezing to gastrointestinal transit was accomplished with microencapsulation using alginate or whey protein based biopolymers.¹⁵ In addition, ice cream offers a highly acceptable sensory profile across age groups as a result of which it overcomes compliance challenges of medicinal supplements. Probiotic estimates within ice cream have been shown by studies to persist up to 20 days post manufacture with only minor decrease in colony forming units (CFU). Probicic ice cream therefore represents a technically superior, patient friendly, and scalable system of targeted delivery of beneficial microbial strains.¹⁶

The purpose of this study was to formulate and test a viable ice cream containing Lactobacillus spp. that would provide therapeutic viability during refrigerated storage. Specifically, physicochemical properties, probiotic survivability and nutritional value of the final product are assessed. This research introduces a novel indulgence in conjunction with gut health for functional delivery of probiotic in desserts.

Materials MRS broth and agar (analytical grade) were obtained from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). DNSA reagent, phenol red lactose broth, and BSA (analytical grade) were purchased from SRL Pvt. Ltd. (Mumbai, India). Hydrogen peroxide (3%) and oxidase reagent were sourced from Loba Chemie Pvt. Ltd. (Mumbai, India). Sulfuric acid (AR grade) and isoamyl alcohol were procured from Merck Life Science Pvt. Ltd. (Bangalore, India). Vanilla extract and raw honey (food grade) were locally sourced from certified vendors in Maharashtra, India. All other chemicals and reagents used were of analytical grade. Methods Sample Collection Fresh homemade curd was used as the source for probiotic strain isolation. A 10 g sample was aseptically homogenized in 90 mL of sterile normal saline (0.85% NaCl) to obtain a uniform microbial suspension. From this, 10 mL was inoculated into 90 mL of sterile MRS broth and incubated at 37 ± 1°C for 24 hours to enrich lactic acid bacteria, particularly Lactobacillus spp. All procedures were carried out under aseptic conditions using sterilized media and equipment. The enriched culture was stored at 4°C for further isolation and characterization. Isolation of Probiotic Bacteria A single colony of individual bacterial species was isolated using the quadrant streaking method following enrichment on the culture. Aerobic incubation of the plates at 37 ± 1°C was for 48 hours. Colonies were selected for morphology differentiating size, shape, margin and surface characteristics in distinct groups. Some colonies were sub cultured onto fresh MRS agar plates to obtain pure isolates. The microbiological procedures were carried out under aseptic conditions with sterilized tools and media to avoid cross contamination. Samples were preserved at 4°C and later grown pure isolates which were characterized with respect to their morphology, biochemical and functional features.17,18 Microscopic Identification The isolated bacteria were microscopically identified by Gram staining technique to study cell morphology and Gram reaction. A bacterial smear from a thin smear onto a clean glass slide was allowed to dry in air and was heat fixed or fired. The crystal violet (1 min), Gram’s iodine (1 min), safranin (30 seconds), 95% ethanol (30 seconds), and distilled water (gentle rinsing after each step) treated slide was sequentially. It was air dried and stained slide, and the stained slide was examined under oil immersion (100x) with a compound light microscope. Presumptive identification as Gram positive, rod shaped, violet appearing bacteria of the genus Lactobacillus were made and were considered suitable for further biochemical characterization.19,20 Biochemical Characterization Lactose Fermentation The ability of the isolated bacterial strain for lactose fermentation was tested on phenol red lactose broth in which an inverted Durham tube was utilized to detect the gas production. Aseptically a loopful of the pure culture was inoculated into the broth, which was incubated at 37 ± 1°C for 24 to 48 hrs. The color change of broth from red to yellow indicated a positive lactose fermentation indicating acid production; and gas bubbles in the Durham tube confirmed also gas formation with this. It confirmed the isolation of lactose fermenting Lactobacillus species.21 Catalase Test Isolated bacterial strain was used in slide test method to assess its catalase activity. Some fresh bacterial culture was transferred a sterile inoculating loop to a clean dry glass slide. In the same manner as above, 1 drop of 3% hydrogen peroxide (H₂O₂) was added to the culture on the slide and its reaction was observed almost immediately. A negative catalase reaction as evidenced by lack of bubble formation was consistent with the typical characteristics of catalase negative Lactobacillus spp., that are catalase negative facultative anaerobes.22,23 Oxidase Test Oxidase activity of the isolate was determined by the filter paper spot method. A fresh drop of oxidase reagent (1% tetra methyl p phenylenediamine dihydro chloride) was placed on a sterile filter paper strip. A small bit of the bacterial colony was applied to the reagent soaked area by a sterile wooden applicator stick. Giving the samples a dark purple coloration within 30 seconds showed a positive result. However, in this case no color change was seen, which underlines an oxidase negative reaction which is commonly Lactobacillus spp.24 Preparation of Natural Cream Cow’s milk fresh and raw was acquired and put into a sterile glass container. Gravity assisted separation of milk fat was allowed to occur at refrigeration temperature (4 ± 1°C) for a minimum of 12 hours prior to milk defatting with κ-CaSO4. At this particular time, the fat globules rose to the surface to form a separate cream layer. Once separated, the upper cream layer was skillfully and carefully skimmed off with a sterile stainless steel spatula to minimize disturbed underneath skim milk. Gently homogenized the collected cream with a sterile glass rod to do indirectly to give uniform fat distribution and smooth texture. The freshly prepared natural cream was immediately stored in an sterile airtight sterile container at 4°C and used as the primary fat source within 24 hours to prepare the probiotic ice cream base.25 Formulation of Probiotic Ice Cream Base A sterile beaker containing natural cream freshly extracted (125 mL) and whole milk (75 mL) was mixed to make base mixture of the probiotic ice cream. In this case, 20 mL pure honey was added as a natural sweetening agent and stirred thoroughly with the help of magnetic stirrer to make sure total solubilization. The mixture contained 0.5 teaspoon (about 2.5 mL) of pharmaceutical-grade pure vanilla extract to enhance flavoring. Sterile aluminum foil was used to cover the formulation and then it was subjected to cold conditioning at 4 ± 1°C for 12 h for maturation and stabilization of the structural fat globules after which it was inoculated with the probiotic. This step achieved uniform emulsification, contributed to the body and texture and was ideal for the medium in which probiotic viability can be preserved. All ingredients were pre sterilized or obtained from micro biologically safe source and mixing was done in an aseptic condition under laminar airflow chamber to avoid contamination.26 Probiotic Inoculation and Incubation After cold maturation, the ice cream base was inoculated with a cell suspension of Lactobacillus spp. prepared from actively growing cultures in MRS broth. Aseptically, the volume of about 0.1mL containing approximately 10⁸ CFU/mL was added to 100g of the prechilled base mixture under laminar airflow condition. The mixture inoculated with the probiotic cells was then gently stirred so that the probiotic cells would be distributed evenly throughout the formulation. Viability and stability of the probiotic cultures were monitored by final product transferring in to sterile airtight containers and stored at 4 ± 1°C for 20 days. To avoid stress-induced loss of viability during refrigerated shelf-life storage, no freezing step was applied. Physicochemical and microbiological evaluation of samples were periodically withdrawn at defined intervals. To prevent contamination and the integrity of probiotic viability during experimental period, all inoculation and storage procedures were performed according to aseptic conditions.27,28 Physicochemical Evaluation pH Measurement Digital pH meter (LI-120, Labindia Instruments Pvt. Ltd., India) was used to measure pH at regular time interval. The samples (10 g) are melted, and the melted samples provide the measurements for immersing the electrode directly into the sample. Before using the instrument was calibrated with standard buffer solutions (pH 4.0 and 7.0). The data were expressed as mean ± SD and performed in triplicate.29 Reducing Sugar Estimation Sugar content was reduced and determined using dinitrosalicylic acid (DNSA) method. 1 mL of melted ice cream sample was pipetted into a test tube and then 1 mL of DNSA reagent was added. The mixture was heated in boiling water bath for 10 minutes and cooled to room temperature, followed by diluting 8 mL distilled water. Absorption of sample was measured at 600 nm using UV – Visible spectrophotometer (UV 1800, Systronics, India). Built the calibration curve using glucose standards and results were presented as percent reducing sugar. All data are presented as mean ± SD with triplicates.30 Protein Estimation A boiled mixture of 1 mL of melted sample with 1 mL of DNSA reagent was used to estimate protein content. The mixture was vortexed, after which 8 mL of distilled water were added and vortexed. Absorption of reaction mixture was taken at 600 nm in a UV Visible spectrophotometer (UV 1800, Systronics, India). The amount of protein added is calculated as percentage based on a standard bovine serum albumin (BSA) curve. The reading were made triplicate and taking as a mean ± SD.31 Fat Estimation The fat content was determined by Gerber method. The standard butyrometer was adjusted to a 10 mL melted sample and 10 mL of concentrated sulfuric acid and 1 mL of isoamyl alcohol was added. Sealing of the butyrometer was done in a screw glass, later it is centrifuged at 1100 rpm for 10 minutes using Gerber centrifuge (Model: G-02, manufacturer: REMI Elektrotechnik Ltd., India). For the purposes of the present study, fat content was read directly from the butyrometer scale in millimetres and expressed as a percentage. Each measurement was carried out 3 times in triplicate and the mean ± SD was reported.32,33 Colony Forming Unit (CFU) Count The viable probiotic cell count was calculated according to the spread plate technique on MRS agar. A sterile 0.85% saline was prepared and serial ten-fold dilutions of melted ice cream sample were done by it. Appropriate dilutions of 0.1 mL aliquots were spread on MRS agar plates and incubated at 37 ± 1°C for 48 hours. Colony number were counted manually and results were expressed as colony forming units per gram (CFU/g) of sample. All the determinations were done in triplicate with mean ± SD.34